Title of Invention | A METHOD OF IDENTIFYING A COMPOUND THAT AMELIORATES PRE-ECLAMPSIA OR ECLAMPSIA AND THE IDENTIFIED COMPOUND |
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Abstract | The present invention relates to a method of identifying a compound that ameliorates pre-eclampsia or eclampsia, said method comprising contacting a cell that , expresses a sF It-I, VEGF, or PIGF nucleic acid molecule with a candidate compound, and comparing the level of expression of said nucleic acid molecule in said cell contacted by said candidate compound with the level of expression in a control cell not contacted by said candidate compound, wherein an alteration in expression of said sFlt:-l, VEGF, or PIGF nucleic acid molecule identifies said candidate compound as a compound that ameliorates pre-eclampsia or eclampsia. |
Full Text | METHODS OF DIAGNOSING AND TREATING PRE-ECLA.MPSLA OR ECLAMPSIA Field of the Invention In general, this invention relates to the detection and treatment of subjects having pre-eclampsia or eclampsia. Background of the Invention Pre-eclampsia is a syndrome of hy"pertension, edema, and proteinuria that affects 5 to 10% of pregnancies and results in substantial maternal and fetal morbidity and morta!ity\ Pre-eclampsia accounts for at least 200,000 maternal deaths worldwide per year. The sympioms of pre-eclampsia typically appear after the 20" week of pregramacy and are usually detected by routine monitoraig of the woman"s blood pressure and urme. However, these monitoring methods are ineffective for diagnosis ot the syndrome at an early stage, which could reduce the risk to the subject or developing fetus, if an. effective tteatment were ivailable. Currently their are no known cures for pre-eclampsia. Pre-eclampsia can "ary in severity from mild to life threatening. A mild form of pre-eclampsia can be treated with bed rest and trequent monitormg- For moderate to severe cases, lospitaiization is recommended and blood pressure medication or anticonvulsant nedications to prevent seizures are prescribed. If the condition becomes life ireatening to the mother or the baby the pregnancy is terminated and the baby is elivered pre-tem The proper develoment of the fetus and the placenta is mediated by several growth factors. One of these growth factors is vascular endothelial rowth factor (VEGF). VTGF is an endothelial cell-specific mitogen, an igiogenic inducer, and a mediator of vascular pemieabilin.". VEGF has also ;en shown to be important for glomendar capillary repaii". VEGF binds as a bomodimer to one of two homologous membrane-spanning tyrosine kinase receptors, the fms-hke tyrosine kinase (Flt-1) and the kinase domain receptor (KDR), which are differentially expressed in endothelial cells obtained from many different tissues. Flt-I, but act ICDR- is highly expressed by troplioblast cells which contribute to placenta! formation. Placental growth factor (PIGF) is a N"^GF family member that is also involed in placental development. FIGF is expressed by cytotrophobjasts and syncytiocropbobiasts and is capable of inducing proliferation, migration, and activation of endothelial cells. PIGF binds -as a homodimer to the F!t-1 receptor, bur not tlie KDR receptor. Both PIGF and VEGF contribute to the miiogenic activity and angiogenesis that are critical for the developing placenta. A soluble form of the Flt-l receptor (sF!t-l) was recendy identified in a cultured medium of human umbilical vein endothelial cells and in vivo expression was subsequently demonstrated in placenta! tissue. sFlt-I is a splice variant of the Flt-l receptor which lacks the transmembrane and cytoplasmic domains. sFlt-1 binds to VEGF with a high affinity but does not stmiulate mitogenesis of indothehal cells. sFlt-1 is believed to act as a "physiologic sink" to down-■egulate VEGF signaling pathways. Regulation of sFlt-l levels therefore works 0 modulate VEGF and VEGF signaling pathways. Careful regulation VEGP ,nd PIGF signaling pathways is critical for maintaining appropriate proliferation, riigration, and angiogenesis by trophobiast cells in the developing placenta, "here is a need for methods of accurately diagnosing subjects at risk for or having re-eclampsia, particularly before the onset of the most severe symptoms. A eaiment is also needed. Summary of the Invention We have discovered a means for diagnosing and effectively treating pre-;iampsia and eclampsia prior to the development of symptoms. Using gene expression analysis, we have discovered that levels of sFlt-I e markedly elevated in placental tissue samples from pregnant women suffering _ im pre-eclampsia, sFli-1 is known to antagonize VEGF and PlGF by acting as a pnysioiogic sunk" and- in pre-ectamptic or eclamptic women, sFit-i may be depleting the placenta of necessary amounts of these essential angiogenic and mitogenic factors. Excess sFlt-1 may also lead to eclampsia by disrupting the endothelial cells tiiat mamtain the blood-brain barrier and/or endothelial ceils lining the choroids plexus of the brain thus leading to cerebral edema and the seizures seen in eclampsia. In :l".e present m"vent!on, compounds that increase VEGF and PIGF levels are administered to a subject lo treat or prevent preeclampsia or eclampsia by countering the effects of elevated sFlt-l. In addition, antibodies directed to sFIt-1 are used to competitively Li±ibit binding of \"EGF or PIGF to sFlt-1. thereby increasing the levels of free VEGF and PIGF. RNA interference and antisense nucleobase oligomers are also used to decrease the levels of sFlt-l. Finally, the present invention provides for the use and monitoring of sFlt-1, VEGF, and PIGF as detection tools for early diagnosis and management of pre-eclampsia or eclampsia, or a predisposition thereto. .A.ccording!y, in one aspect, the invention provides a method of treating or preventing pre-eclampsia or eclampsia in a subject by administering to the subject a compound capable of binding to sFlt-I, where the administering is for a tune and-in an amount sufficient to ti"eat or prevent pre-eclampsia or eclampsia in a subject. In a related aspect, the invention provides a method of treating or preventing pre-eclampsia or eclampsia in a subject by administering to the subject a compound (e.g., nicotine, theophylline, adenosine. Nifedipine, Minoxidil, or Magnesium Sulfate) tliat increases the level of a growth factor capable of bmding to sFlt-1, where the administenns is for a time and in an amount sufficient to treat or prevent pre-eclampsia or eclampsia in a subject. In another related aspect, the invention provides a method of treating or preventing pre-eclampsia or eclampsia m a subject by administering a purified sFlt-1 antibody or antigsn-binding fragment thereof to the subject for a time and in an amount sufficient lo treat or prevent pre-eclampsia or eclampsia in a subject. In yet another related aspect, the invention provides a method of treating or preventing pre-eciampsia or eclampsia in a subject by administering to the subject an antisense nucleobase oligomer complementary to at least a portion of an sFlt-1 nucleic acid sequence, where the administering is sufficient to treat or prevent pre-eciampsia or eclampsia in a subject. In one embodiment, the antisense nucleobase oligomer is 8 to 30 nucleotides in length. In another related aspect, the invention provides a method of treating or preventing pre-eciampsia or eclampsia in a subject. Th-e method involves the step of administering to the subject a double stranded RNA (dsRNA) that contains at least a portion of an sFli-l nucleic acid sequence, where the administering is sufficient to treat or prevent pre-eciampsia or eclampsia in (he subject. In one embodiment, the double stranded RSA is processed into small interfering RNAs (siRNAs) 19 to 25 nucleotides in length. In various embodiments of the above aspects, the candidate compound is a growdi factor such as vascular endothelial growth factor (VEGF), including all isoforms such as VEGF189, VEGF121, or VEGF165; placental growth factor (PIGF), including all isoforms; or fragments thereof In preferred embodiments, the candidate compound is an antibody that binds sFlt-l. In other embodiments of the above aspects, the method finther involves administering to a subject an anti-hypertensive compound. In still other embodiments of tiie above aspects, the subject is a pregnant human, a post-paitum human, or a non-human (e.g., cow, a .lorse, a sheep, a pig, a goat, a dog, or a cat). In another aspect, the invention provides a method of treating or preventing pre-eciampsia or eclampsia. The method involves administering to a ;ubjectin need of such treatment an effective amount of a pharmaceutical lomposition comprising a VEGF or P!GF polypeptide. In one embodiment, the ■.omposition contains a VEGF polypeptide, hi another embodiment, the ;omposition contains a PIGF polypeptide. In a related aspect, the invention provides a method of treating or ireventing pre-eciampsia or eclampsia. This method involves administering to a ubject in need of such treatment an effective amount of a pharmaceutical composition comprising a nucleic acid molecule encoding VEGF or PIGF. In one embodiment, tlie composition contains a VEGF nucleic acid moiecuie. In another erabodiraent, the composiiion contains a PIGF nucie;c acid molecule. In another related aspect, the invention provides a method of treating or preventing pre-eclampsia or eclampsia in a subject. Tlie method involves tiie step of admmistering to the subject 2 compound (e.g., chemical compound, polypeptide, peptide, antibody, or a fragment thereof) tiiat inliibits growtli factor binding to an sFlt-1 polypeptide, where the admmistsring is sufficient to ti-eai or prevent pre-eclampsia or eclampsia in a subject. In one embodiment, the compound binds to sFlt-1 and blocks growth factor binding. In various embodiments of the above aspects, the method further involves tlie step of administering to a subject an anti-hypertensive compound (e.g.. adenosine, Nifedipine, Minoxidil, and Magnesium Sulfate). In other embodiments of the above aspects, the subject is a pregnant human, a post-parmm human, or a non-human ("e.g., a cow, a horse, a sheep, a pig, a goat, a dog, or a :aO. In another aspect, the in\"ention provides a method of diagnosing a subject is having, or havmg a propensit}" to develop, pre-eclampsia or eclampsia, the nethod involves measuring the level of sFll-l, VTGF, or PIGF poljq^eptide m a lampie fi-om the subject. In a related aspect the ln^"ention provides a method of diagnosijig a subject IS havmg, or having a propensity to develop, pre-eclampsia or eclampsia, by letemiining the levels of at least two of sFlt-1, VEGF, or PIGF polypeptide in a ample from a subject and calculating the relationship between the levels of sFlt-1 ""EGF, or PIGF using a metric, where an alteration in the subject sample relative D a reference diagnoses pre-eclampsia or eclair^psia in a subject. In one mbodiment, the metric is a pre-eclampsia anu-angiogenic mdex (PA.^): [sFlt-/VEGF - PIGF], where the PAAl is used as an indicator of anti-angiogenic ctivity. In one embodiment, z ?J\A1 greater than 20 is indicative of pre-;lampsia or eclampsia. In another embodiment, the levels of sFlt-l, VEGF, or IGF polypeptide is determined by an inmiunological assay, such as an ELIS.A.. m vanous emDOQiments ot the above aspects, the sample is a bodily fluid, such as serum or urine. In one embodiment, a level of sFIt-l greater than 2 ng/ml is indicative ofpre-eclampsia or eclampsia. In preferred embodiments of the above aspects, the level of sFIt-1 polypeptide measured is the level of free, bound, or total sFlt-1 polypeptide. In other preferred embodiments of the above aspects, the level of VEGF orPlGF is the level of &ee VEGF or PIGF. In another aspect, the invention provides a metliod of diagnosing a subject as having, or having a propensity to deveFop, pre-eclampsia or eclampsia. This method involves measuring the level of sFli-I, VEGF, or PIGF nucleic acid molecule in a sample from the subject and comparing it to a reference sample, where an alteration in the levels diagnoses pre-eclampsia or eclampsia in Ihe subject, or diagnoses a propensity to develop pre-eciampsia or eclampsia. In another aspect, the invention provides a method of diagnosing a subject as having, or having a propensity to develop, pre-eclampsia or eclampsia. This method invoJves determining tiie nucleic acid sequence of a sFU-1, VEGF, or PIGF gene in a subject and comparing it to a reference sequence, where an alteration in the subject"s nucleic acid sequence that changes the level of gene product in the subject diagnoses the subject with pre-eclampsia or eclampsia, or a propensity to develop pre-eclampsia or eclampsia. In one embodiment, the alteration is a polymorphism in the nucleic acid sequence. In various embodiments of the above aspects, the sample is a bodily fluid (e.g., urine, amniotic fluid, serum, plasma, or cerebrospinal fluid) of the subject in which the sFlt-l, VEGR, or PIGF is normally detectable. In additional embodiments, the sample is a tissue or a cell. Non-limiting examples include placental tissue or placental cells, endothelial cells, and leukocytes (e.g., monocytes). In other embodiments of the above aspects, the subject is a nonpregnant human, a pregnant human, or a post-partum human. In other embodiments of the above aspects, the subject is a non-human (e.g., a cow, a horse, a sheep, a pig, a goat, a dog, or a cat). In other embodiments of the above aspects, at least one of the levels measured 4s the level of sFlt-1 (free, bound, or :otaI). In other embodiments of the above aspects, when the level of VEGF is measured then the level of sFU-1 orPIGF is also measured. In various embodiments of the above aspects, an increase in the level of sFlt-I nucleic acid or polypeptide relative to a reference is a diagnostic indicator of pre-eciampsia or eclampsia. In other embodimencs of the above aspects, a decrease in the level of free VEGF polypeptide or VEGF nucleic acid relative to a reference is a diagnostic indicator of pre-eclai".".psia or eclampsia. In other embodiments of the above aspects, a decrease in the level of free PIGF polvpeptide or PIGF nucleic acid relative to a reference is a diagnostic indicator of pre-ec!ampsia or eclampsia. In additional embodiments of the above aspects, the levels are measured on two or more occasions and a change m the levels between the measurements is a diagnostic indicator of pre-eciampsia or eclampsia. In one prefen"ed embodiment, the level of sFlt-I increases from the fu"St measurement to the next measurement. In another preferred embodiment, the level of VEGF or PIGF decreases from the first measurement to the next measurement. In another aspect, the invention provides a diagnostic kit for tlie diagnosis of pre-eciampsia or eclampsia m a subject comprising a nucleic acid sequence, or fragment thereof, selected from the group consisting of sFlt-1, VEGF, and PIGF nucleic acid molecule, or a sequence complementan." thereto, or any combination thereof. In a preferred embodiment, the kit comprises at least two probes for the detection of an sFlt-1, ^"EGF, or PIGF nucleic acid molecule. In a related aspect, the invention provides a Idt for tlie diagnosis of preeclampsia or eclampsia in a subject comprising a means of detecting a sF!t-l, VEGF, or PIGF polypeptide, and any combination thereof. In one embodiment, the means of detecting is selected from the group consisting of an immunological assay, an enzymatic assay, and a colonmetric assay. In other embodiments of the above aspects, the kit diagnoses a propensity to develop pre-eciampsia or eclampsia in a pregnant or a non-pregnant subject. In preferred embodiments of the above aspects, the kit detects sFlt-1 or PIGF. In other preferred embodiments of the above aspects, when the kit detects VEGF then sFIt-1 or PiGF is also detected. In another aspect, the invention provides a metliod of identiiying a compound that ameliorates pre-eclampsia or eclampsia, the method involves contacting a cell that expresses a sFlt-1, VEGF, or PIGF nucleic acid molecule with a candidate compound, and comparing the level of expression of the nucleic acid molecule in the cell contacted by the candidate compotind with the level of expi"ession in a conhol cell not contacted by tlie candidate compotmd, where an alteration in expression of the sFlt-1, VEGF, or PIGF nucleic acid molecule identifies the candidate compound as a compound that ameliorates pre-eciampsia or eclampsia. In one embodiment, the alteration is a decrease in the level of sFlt-1. In other embodiments, the alteration is an increase in the level of VEGF or PIGF. In other embodiments, the alteration is m transcription or in translation. In another embodiment, when the method identifies a candidate compound that increases the expression of VEGF, the candidate compoimd also increases the expression of PIGF or decreases the expression of sFlt-1. In another aspect, the invention provides a pharmaceutical composition including a VEGF or PIGF polypeptide or portion thereof, formulated in a phannaceutically acceptable carrier... In a related aspect, the invention provides a pharmaceutical composition comprising a PIGF nucleic acid molecule, or portion thereof, formulated in a pharmaceuticaliy acceptable carrier. In one embodiment, the composition further contains a VEGF nucleic acid molecule, or portion thereof. In another aspect, the invention provides a composition comprising a purified antibody or antigen-bindmg fragment thereof that specifically binds sFlt-I. In one preferred embodiment, the antibody prevents binding of a growth factor tosFlt-l. In another embodiment, the antibody is a monocionai antibody. In otlier preferred embodiments, the antibody or antigen-binding fragment thereof is a human or humanized antibody. In other embodiments, the antibody lacks an Fc portion. In still other embodiments, the antibody is an F(ab")2, an Fab, or an Fv sn-ucture. In other embodiments, the antibody or antigen-binding fragment thereof is present m a phaniiacemically acceptable carrier. In another aspect the invention provides a meihod of identifying a compound that ameliorates pre-ec lamps ia or eclampsia. Tliis method involves contacting a cell tliat expresses an sFlt-1. V"EGF, or PIGF polypeptide witli a candidate compound, and comparmg the ieve! of expression of the polypeptide in the cell contacted by the candidare compound vvidi the level of polypeptide expression in a control cell not contacted by die candidate compound, where an alteration m the expression of the sFlt-l. V"EGF, or PIGF polypeptide identifies die candidate compound as a compound that ameliorates pre-eclampsia or eclampsia. In one embodimeni. the alteration in expression is assayed using an immunological assay, an enzymatic assa\. or an immunoassay. In one embodiment, the alteration in expression is a decrease in the level of sFlt-1. hi another embodiment, the alteration in expression is an increase in the level of VEGForPlGF. In another aspect, the invention provides a method of identifying a compound that ameliorates pre-eclampsia or eclampsia. The method involves contacting a cell that expresses an sFlt-I. VTGF. or PIGF polypeptide with a candidate compound, and comparing the biological activity of the polypeptide in the cell contacted by the candidate compound with the level of biological activity in a control cell not contacted by the candidate compound, where an increase in the biological activitj" of the sFlt-1, VEGF. or PIGF polypeptide identifies the candidate compound as a compound that ameliorates pre-eclampsia or eclampsia. In one embodiment, the increase in biolosicai activity is assayed using an immunologica! assay, an enzymatic assav, or an immunoassay. In one embodiment, the alteration in expression is a decrease in the activity of sFlt-1. In another embodiment, the alteration in expression is an increase in the activity of V^GF or PIGF. In another aspect, the in"-.-ention provides a niethod of identifying a :Qmpound that ameliorates pre-eclampsia or eclaizpsia. The method involves ietecting bindmg benveen an sFlt-1 polvpeptide a:;d a growth factor in the :iresence of a candidate compound, where a decrea-se m. the binding, relative to > - DLiding between the sFlt-1 polypeptide and the gro"^^"tii factor in the absence of the candidate compound identifies tlie candidate compound as a compound that ameliorates pre-eclampsia or eclampsia. In one embodiment, the growth factor is VEGF. In another embodiment, the growth factor is PIGF. In anoflier aspect, the invention provides a method of identifying a polypeptide, or fragment thereof, that prevents binding between an sFlt-1 polypeptide and a growth factor. The method involves detecting binding between an sFlt-1 polypeptide and a growth factor in the presence of the candidate "polypeptide, where a decrease m the bmding, relative to binding between the sFlt-1 polypeptide and the growth factor in the absence of the candidate polypeptide identifies the candidate polypeptide as a poh-peptide that prevents binding between an sFlt-1 polypeptide and a growth factor. In one embodiment, the growth factor is VEGF. In another embodiment, the growth factor is PIGF. In another aspect, the invention provides a method of identifying a compound that ameliorates pre-eclampsia or eclampsia, comprising detecting binding of an sFIt-1 polypeptide and a candidate compound, where a compound that binds tlie sFlt-1 polypeptide ameliorates pre-eclampsia or eclampsia. In a related aspect, the invention provides a compound identified according to the previous aspect, where the compound is"a polypeptide specifically binds im sFlt-1 polypeptide and prevents die sFlt-I polypeptide from binding VEGF or PIGF. In one preferred embodiment, the polypeptide is an antibody. In another preferred embodiment, the polypeptide is a &agment of sFlt-l, VEGF, or PIGF. In preferred embodiments of the above aspects, the compound tliat ameliorates pre-eclampsia or eclampsia decreases the expression levels or biological activity of sFll-1. Inprefened embodiments of the above aspects, the compound that ameliorates pre-eclampsia or eclampsia increases the expression levels or biological activity of VEGF or PlGF. For the purpose of the present invention, the following abbreviations and terms are dsfmed below. By "alteration" is meant a change (increase or decrease) in the expression levels of a gene or polypeptide as detected by standard art known methods such IS those described above. As used herein, an increase or decrease includes a 10% change in expi-ession levels, preferably 125° o change, more preferably a 40% change, and most preferably a 50% or greater change in expression levels. "Alteration" can also indicate a change (increase or decrease) in tlie biological activity of any of the pol^-peptides of the invention (e.g., sFlt-l, VEGF, orPlGF). Examples of biological activity for PIGF or \"EGF include binding to receptors as measured by immunoassays, ligand binding assays or Scatchard plot analysis, and induction of cell proUferauon or migration as measured by BrdU labeling, cell counting experiments, or quantitative assays for DNA synthesis such as ■"H-thymidine incorporation. Examples of biological activity for sFlt-l include bindmg to P!GF and VEGF as measured by immunoassays, ligand binding assays, or Scatchard plot analysis. Additional examples of biological activity for each of the polypeptides are described herein. As used herein, an increase or decrease includes a 10% change in biological activit\", preferably a 25% change, more preferably a 40% change, and most preferably a 50%) or greater change in biological activity. By "antisense nucleobase oligomer" is meant a nucleobase oligomer, regardless of length, that is complementary" to the codmg strand or mRNA of an sFlt-1 gene. By a "nucleobase oligomer" is meant a compound that includes a chain of at least eight nucieobases, preferably at least twelve, and most preferably at least sixteen bases, joined together by linkage groups. Included in this definition are natural and non-natura! oligonucleotides, both modified and uimiodified, as well as oligonucleotide mimetics such as Protein Nucleic Acids, locked nucleic acids, and arabinonucleic acids. Numerous nucieobases and Unkage groups may be employed in the nucleobase oligomers of the invention, mcluding those described in U.S. Patent Application Nos. 20030114412 and 20030114407, incorporatefi herein by reference. The nucleobase oligomer can also be targeted to the translational start and stop si:es. Preferably the antisense nucleobase oligomer comprises from about S to 30 nucleotides. The antisense nucleobase oligomer can also contain at least 40, 60, S5, 120, or more consecutive nucleotides that are complementary to sFlt-l mRNA or DNA, and may be as long as the fuli-length mRNA or gene. By "compound" is meant any small molecule chemicat compound, antibody, nucleic acid molecule, or polypeptide, or fragments thereof. By "chimeric antibody" is meant a polypeptide comprising at least the antigen-binding portion of an antibody molecule linked to at least part of another protein (typically an imraunoglobulin constant domain). By "double-stranded RNA (dsRNA)" is meant a ribonucleic acid molecule comprised of bodi a sense and an anti-sense strand. dsRNAs are typically used to mediate RNA interference. By "expression" is meant the detection of a gene or polypeptide by standard art known methods. For example, polypeptide expression is often detected by western blotting, DNA expression is often detected by Southern blotting or polymerase chain reaction (PCR), and RNA expression is often detected by northern blotting, PCR, or RNAse protection assays. By "fragment" is meant a portion of a polypeptide or nucleic acid molecule. This portion contains, preferably, at least 10%, 20%, 30%, 40%, 50%, or 60% of the entire lengdi of the reference nucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30. 40, 50, 60, 70. 80, 90, or 100, 200, 300,400, 500, 600, 700, SOO, 900, or 1000 nucleotides or amino acids. By "homologous" is meant any gene or protein sequence that bears at least 30% homology, more preferably 40%, 50%, 60%, 70%, S0%, and most preferably 90% or more homology to a known gene or protein sequence over the lengtli of liie comparison sequence. A "homologous" protein can also have at least one biological activity of the coir^arison protein. For polypeptides, the length of comparison sequences will generally be at least 16 amino acids, preferably at least 20 amino acids, more preferably at least 25 amino acids, and most preferably 35 amino acids or more. For nucleic acids, the length of comparison sequences will generally be at least 50 nucleotides, preferably at least 50 nucleotides, more preferably at least 75 nucleotides, and most preferably at least 110 nucleotides. "Homology" can also refer to a substantial similarity Detween an epitope used to generate antibodies and the prj>tein or fragment hereof to which the antibodies are directed. In this case, homology refers to a recognize tlie protein at issue. By "humanized andbody" is meant an immunoglobulin amino acid sequence variant or fragment thereof that is capable of binding to a predetemiinei aiitigeu. Ordinai"ily, the antibod;-- will contain both the light chain as well as at least the variable domain of a he^i^/y chain. The antibody also may include the CHI, Iimge, CH2, CH3, or CH4 regions of the hea\-y chain. The humanized antibody comprises a framework region (FR) having substantially the amino acid sequence of a human immunoglobulin and a compiementarity determining regioi {CDR} having substantially the amino acid sequence of a non-human immunoglobulin (the "import" sequences). Generally, a humanized antibody has one or more amino acid residues mtroduced into it from a source that is non-human. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variabl domams (Fab, Fab", F(ab")2. Fabc. Fv) in which all or substantially all of the CDF regions coirespond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human unmunoglobulin. By "complementarity deiemiimng region (CDR)" is meant the three aypervanable sequences in the variable regions within each of the immunoglobulin hght and heav7 chains. By "framework: region (FR)" is meant the sequences of amino acids ocated on either side of the three hj"pen"ariable sequences (CDR) of the mmunoglobulin light and hea\y chains. The FR and CDR regions of the humanized antibody need not correspond )recisely to the parental sequences, e.g.. the import CDR or the consensus FR nay be mutagenized by substinjtion, insertion or deletion of at least one residue ;o that the CDR or FR residue at that sire does not correspond to either the ;onsensus or the import antibody. Such mutations, however, will not be extensive. Usually, at least 75%, preferably 90%, and most preferably at least 95% of tl\e humanized antibody residues will correspond to those of the parental FR and CDR sequences. By "hybridize" is meant pair to form a double-stranded molecule between conaplementary polynucleotide sequences, or portions thereof, under various conditions of stringency. (See. e.g., Wah! and Berger (1987) Methods Enzymol. 152:399; Kmmisl, Methods Enzymol. 152:507, 19S7.) For example, stringent salt concentration will ordinarily be less than about 750 niM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and most preferably less than about 250 mM NaCI and 25 mM trisodium citrate. Lovi^ stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and most preferably at least about 50% formamide. Stringent temperature conditions will ordinarily include temperahjres of at least about SCC, morepreferably of at least about 37""C, and most preferably of at least about 42°C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed. In a preferred embodiment, hybridization will occur at 3Q°C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization will occur at 21°C m 500 mM NaCl, 50 mM trisodium cihate, 1% SDS, 35% formamide, and 100 \ig/ml denatured salmon sperm DNA (ssDNA). In a most preferred embodiment, hybridi2ation will occur at 42"C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 fig/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art. For most applications, washing steps that follow hybridization will also /ary in stringency. Wash stringency conditions can be defined by salt :oncenn-ation and by temperature. As above, wash stringency can be increased ty decreasing salt concentration or by increasing temperature. For example. stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCl and 3 mM msodium citrate, and most preferably less tiian about 15 niM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 35°C, more preferably of at least about 42°C. and mostpreferably of atleast about 6S°C. In a preferred embodiment, wash steps will occur at 25"C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 42°C in 15 mM KaCl, 1.5 mM trisodium citrate,"and 0.1% SDS. In a most preferred embodiment, wash steps will occur at 68°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations oo these conditions will be readily apparent to tliose skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196:180,1977); Grtmstein and Hogness {Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Moleciilar Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 19S7, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York. By "intrauterine growth retardation (lUGR)" is meant a syndrome resulting in a birth weight which is less that 10 percent of the predicted fetal weight for the gestational age of the fetus. The current World Health Organization criterion for low birth weight is a weight less than 2,500 gm (5 lbs. S oz.) or below the 10* percentile for gestational age according to U.S. tables of birth weight for gestational age by race, parity, and infant sex (Zhang and Bowes, Obstet G^oiecol. S6-.200-208,1995). TTieselowbirth weight babies are also referred to as "small for gestational age (SGA)". Pre-eclampsia is a condition known to be associated with lUGR or SGA. By "metric" is meant a measure. A metric may be used, for example, to compare the levels of a poiypepiide or nucleic acid molecule of interest. Exemplary metrics include, bu: are norlimited to, mathematical formulas or algorhithms, such as ratios. The metnc to be used is that which best discriminates eclampsia and a normal control subject Depending on the metric that is used, the diagnostic indicator of eclampsia or pre-«clainpsia may be significantly above or below a reference value (e.g., from a control subject not having pre-eclampsia or eclampsia). sFlt-1 level is measured by measuring tlie amount of free, bound (i.e., bound to growtli factor), or total sFlt-1 (bound -r free). VEGF or PIGF levels are determined by measuring the amount of free PIGF or free VEGF (i.e., not bound to sFU-1). One exemplary metric is [sFlt-l/(VEGF -i- PIGF)], also refen-ed to as the pre-eclampsia anti-angiogenic index (PAAI). By "pre-eclampsia anti-angiogenesis index (PAAI)" is meant the ratio of sFlt-lA""EGF + PIGF used as an indicator of anti-angiogenic activity. A PAAI greater than 20 is considered to be indicative of pre-eclampsia or risk of preeclampsia. By "operably linked" is meant that a gene and a regulatory sequence(s) are connected in such a way as to permit gene expression when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the regulatory sequence(s). By "pharmaceuticaily acceptable carrier" is meant a carrier that is physiologically acceptable to the treated mammal while retaining the dierapeutic properties of the compound wiiii which it is administered. One exemplary pharmaceuticaily acceptable carrier substance is physiological saline. Other physiologically acceptable carriers and their formulations are known to one skilled in the art and described, for example, in Remington"s Pharmaceutical Sciences, (20^" edition), ed. A. Gennaro, 2000, Lippincott, Williams & Wilkins, Philadelphia, PA. By "placental growth factor (PIGF)" is meant a mammalian growth factor thai, is homologous to the protein defmed by GenBank accession number P49763 and that has PIGF biological activity. PIGF is a glycosylated homodimer belonging to tlie VEGF family and can be found in two distinct isoforms tiirough alternative splicing mechanisms. PIGF is expressed by cyto- and syncytiotrophoblasts in the placenta and PIGF biological activities include induction of proliferation, migration, and activation of endotlteiial cells, particularly trophoblast cells. By "pre-eclampsia" is meant the multi-sysiem disorder tliat is characterized by hypertension with proteinuria or edema, or both, glomerular dysftmction, brain edema, liver edema, or coagulation abnorraalities due to pregnancy or the influence of a recent pregnancy. Pre-eclampsia generally occurs after the 20* week of gestation, Pre-eclampsia is generally defmed as some combination of the followmg symptoms: (1) a sysiolic blood pressure (BP) >I40 mmHg and a diastolic BP >90 ramHg after 20 weeks gestation (generaUy measured on two occasions, 4-16S hours apart), (2) new onset proteinuria (1+ by dipstik on urinanaysis, >300ma of protein in a 24-hour urine collection, or a single random urir\e sample having a piotevn/creatinine ratio >0.3), and (3) resolution of hypertension and proteinuria by 12 weeks postpartum. Severe preeclampsia is generally defined as (I) a diastolic BP> 110 mmHg (generally I measured on two occasions, 4-16S hours apart) or (2) proteinuria characterized by a measurement of 3.5 g or more protein in a 24-hour urine collection or two random unne specimens with at least 3+ protein by dipstick. In pre-eciaropsia, hypertension and proteinuria generally occur within seven days of each other. In severe pre-eclampsia, severe hypertension, severe proteinuria and HELLP syndrome (hemolysis, elevated liver enzymes, low platelets) or eclampsia can occur simultaneously or only one symptom at a time. Occasionally, severe preeclampsia can lead to the development of seizures. This severe form of the syndrome is referred to as "eclampsia." Eclampsia can also include dysfiinction jr damage to several organs or tissues such as the hver (e.g., hepatocellular Jamage, periportal necrosis) and the central nervous system (e.g., cerebral edema md cerebral hemorrhage). The etiology of the seizures is thought to be secondary 0 the development of cerebral edema and focal spasm of small blood vessels in he kidney. By "protein" or "polypeptide" or "polypeptide fragment" is meant any chain of more than two amino acids, regardless of post-trans lational modification (e.g., glycosyiation or phosphorylation), constituting ail or part of a naturally occurring polypeptide or peptide, or constituting a non-naturally occurring polypeptide or peptide. By "reduce or inhibit" is meant the ability to cause an overall decrease preferably of 20% or greater, more preferably of 50% or greater, and most preferably of 75% or greater, in the level of protein or nucleic acid, detected by the aforementioned assays (see "expression"), as compared to samples not treated with antisense nucleobase oligomers ot dsR^"A used for RNA interference. By "small interfering RNAs (siRNAs)" is meant an isolated dsRNA molecule, preferably greater than 10 nucleotides in length, more preferably greater than 15 nucleotides in length, and most preferably greater than 19 lucleotides in length that is used to identify the target gene or mRNA to be legraded. A range of 19-25 nucleotides is the most preferred size for siRNAs. iRNAs can also include short hairpin RNAs in which both strands of an siRNA uplex are included within a single RNA molecule. siKNA includes any fomi of sRNA (proteolytically cleaved products of larger dsRNA, partially purified NA, essentially pure RNA, synthetic RNA, recombinantly produced RNA) as ell as altered RNA that differs &om naturally occurring RNA by the addition, iletion, substimtion, and/or alteration of one or more nucleotides. Such terations can include the addition of non-nucleotide material, such as to the ,d(s) of tlie 21 to 23 nt RNA or internally (at one or more nucleotides of the ^A). In a preferred embodiment, the RNA molecules contain a 3 "hydroxyl 3up. Nucleotides in the RNA molecules of the present invention can also tnprise non-standard nucleotides, including non-naturally occurring nucleotides deoxyribonucleotides. Collectively, ail such altered RNAs are referred to as dogs of RNA. siRNAs of the present invention need only be sufficiently lilar to natural RNA that it has the ability to mediate RNA interference •JAi). As used herein, RNAi refers to the ATP-dependent targeted eleavage degradation of a specific mRNA molecule through the introduction of small interfering RNM or dsRNAs into a cell or an organism. As used herein "mediate KNAi" refers to the ability to distinguish or identify which RNAs are to be degraded. By "soluble Flt-I (sFlt-I)" (also known as s\"EGF-RI) is meant the soluble form of the Flt-1 receptor, that is homologous to tiie protem defined by GenBanlc accession number UO: 134, and that has sFlt-1 biological activity. The biological activity of an sFIt-1 polypeptide may be assayed using any standard method, for example, by assaying sFlt-1 binding to VEGF. sFlt-1 lacks the transmembrane domain and the cytoplasmic tyrosine kinase domain of the Flt-1 receptor, sFIt-1 can bmd to VEGF and PIGF bind with high affinity, but it cannot induce prohferation or angiogenesis and is therefore fimctionally different &om the Flt-I and KDR receptors. sFlt-I was initially purified fi-om human umbUical endothelial cells and later shown to be produced by trophoblast cells in vivo. As used herein, sFlt-1 includes any sFIt-l family member or isoform. By "specifically binds" is meant a compotmd or antibody which ■ecognizes and binds a polypeptide of the invention but that does not substantially ecognize and bind other molecules in a sample, for example, a biological sample, vhich naturally includes apolypeptide of the invention. In one example, an jitibody that specifically binds sFlt-I does not bind Flt-1. By "subject" is meant a mammal, including, but not limited to, a human or on-human mammal, such as a bovine, equine, canine, ovine, or feline. Included 1 this definition are pregnant, post-partum, and non-pregnant mammals. By "substantially identical"" is meant an amino acid sequence which differs iLy by conservative amino acid substitutions, for example, substitution of one nino acid for another of the same class (e.g., valine for glycine, arginine for sine, etc.) or by one or more non-conservative substitutions, deletions, or sertions located at positions of the amino acid sequence which do not destroy e function of the protein. Preferably, the amino acid sequence is at least 70%, Dre preferably at least about 80%. and most preferably at least about 90% mologous to another amino acid sequence. Methods to determine identity are" ailable in publicly available computer programs. Computer program methods to determine identity between two sequences include, but are not limited to, &e GCG program package (Devereux et al, Nucleic Acids Research \2: 387,1984), BLASTP, BLASTN, and FASTA (Altschul et al, / Mol. Biol. 215:403 (1990). The well-known Smith Waterman algorithm may also be used to determine identity. The BLAST program is publicly available from NCBI and other sources (BLASTManual, Altschul, et al., N"CBI NLM NTH, Betliesda, MD 20894; BLAST 2.0 at ht^y/w^nv.ncbi.nim.nili.gov/blasijQ. These software programs match similar sequences by assigning degrees of homology to various substitutions, deletions, and other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonme; lysine, arginine; and phenylalanine, tyrosine. By "symptoms of pre-eclampsia" is meant any of the following: (1) a systolic blood pressure (HP) >14QmmHg and a diastolic BP >90 mmHg after 20 weeks gestation, (2) new onset proteinuria (1+ by dipstifc on urinanaysis, >300mg of protein in a 24 hour urine collection, or random urine protein/creatinine ratio >0.j), and (3) resolution of hypertension and proteinuria by 12 weeks postpartum. The symptoms of pre-eclampsia can also include renal dysfunction and glomerular endotheUosis or hypenrophy. By "symptoms of eclampsia" is meant the development of any of die following symptoms due to pregnancy or the influence of a recent pregnancy: seizures, coma, thrombocytopenia, liver edema, pulmonary edema, and cerebral edema. By "therapeutic amount" is meant an amount that when adnunistered to a padent suffering &om pre-eclampsia or eclampsia is sufficient to cause a qualitative or quantitative reduction in the symptoms of pre-eclampsia or eclampsia as described herein. A "therapeutic amount" can also mean an amount that when administered to a patient suffering from pre-eclampsia or eclampsia is sufficient to cause a reduction in the expression levels of sFlt-1 or an increase in the expression levels of VEGF or PIGF as measured by the assays desciibed herein. ay ucauiig IS meant admiEistermg a compound or a pharmaceutical compositioa for prophylactic and/or therapeutic purposes. To "treat disease" or use for "therapeutic treatment" refers to administering treatment to a subject already suffering from a disease to improve tlie subject"s condition. Preferably, tlie subject is diagnosed as suffering &om pre-eclainpsia or eclampsia based on identification of any of the characteristic symptoms described below or the use of the diagnostic metliods described herein. To "prevent disease" refers to prophylactic treatment of a subject who is not yet ill, but who is susceptible to, or otherwise at risk of, developing a particular disease. Preferably a subject is determined to be at risk of developing pre-eciampsia or eclampsia using the diagnostic methods described herein. Thus, in the claims and embodiments, treating is the admmistration to a mammal either for therapeutic or prophylactic purposes. By "Irophoblast" is meant the mesectodermal cell layer covering tlie jiastocyst that erodes the uterine mucosa and through which the embryo receives lourishment from the mother; the ceils contribute to the formation of tlie )Iacenta. By "vascular endothelial growth factor (VEGF)" is meant a mammalian ;rowtii factor diat is homologous to the growth factor defmed in U.S. Patent Nos. ,332,671; 5,240,848; 5,194,596; and Chamoclc-Jones et al. (Biol. Reproduction, S: 1120-112S, 1993), and has VEGF biological acrivity. VEGF exists as a lycosylated homodimer and includes at least four different alternatively spliced loforms. The biological activity of native VEGF includes the promotion of elective growth of vascular endothelial cells or umbilical vein endothelial cells id induction of angiogenesis. .A.s used herein, VEGF includes any VEGF family lember or isoform (e.g. VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, EGF189, VEGF165, or VEGF 121). Preferably, VEGF is the VEGF121 or EGF165isofonn(Tischeretal.,j:5/o/. Chem.266, II947-11954, 1991; eufed et al. Cancer Metastasis 15:153-158,1996), which is described in U.S. itent Nos. 6,447,768; 5;2t9,739; and 5494,596, hereby incorporated by Terence. Also included are mutant forms of VEGF such as tiie KDR-selectivs ^—ww-i.., . i-ji ucaunoea in Lriiie ec ai. (./. iSioi (Jhein. 276:3222- 3230,2001). Although human. VBGF is preferred, the invention is not limited to human forms and can include other animal fonns of VEGF (e.g. mouse, rat, dog, or chicken). By "vector" is meant a DNA molecule, usually derived from a plasmid or bacteriophage, into which fragments of DNA may be inserted or cloned. A recombinant vector will contain one or more unique restriction sites, and may be capable of autonomous replication in a defined host or vehicle organism such that the cloned sequence is reproducible. A vector contains a promoter operably linked to a gene or coding region such that, upon transfection into a recipient cell, an RNA is expressed. Other features and advantages of the invention will be ^parent from the following description of die preferred embodiments thereof, and from the claims. Brief Description of the Drawings Figure 1 shows mRNA and protein expression of sFIt-1 in pre-eclanipsia. Figure lA shows mRNA expression of placental sFlt-1 from tluee patients with pre-eclampsia (PI, P3, P3) and three nomiotensive term pregnancies (Nl, N2, N3) as determined by northern blot analysis. The higher band (7.5 kb) is the full length fit-1 mRNA and the lower, more abundant band (3.4 kb) is the alternatively spliced sFlt-l mRNA GAPDH is included as a control and the arrowhead indicates 28S RNA. Patients PI and P2 had severe pre-eclampsia, whereas patient P3 had mild pre-eclampsia. Figure IB is a graph showing sFlt-1 levels in serum from patients with mild pre-eclampsia (mild PE), patients with severe pre-eclampsia (severe PE), and normotensive pregnant women at term (normal). sFlt-1 levels were measured by an ELISA performed for sFlt-1 using a conmiercially available kit (R & D Systems, Minneapohs, MN). Patients with pre-term deliveries for other reasons (pre-term) were included as additional controls to rule out gestational age specific changes. The number of patients tested is shown in parenthesis in the X axis. Samples were collected prior to delivery (t=0) and 48 hours after delivery (t=48). Figure IC is a graph showing anti-angiogenesis index ratios (P.AAI=sFIM/(VEGF + PIGF)) ratios at the time of delivery (t=0) as determined by ELISA for all the patients described in Figure IB. Figures 2A-2F are photomicrographs showing die anti-angiogenic effect of excess sFlt-I in pre-eclampsia. Endothelial tube assays were performed using serum from four nomial pregnant controls and four patients with pre-eclampsia. A representative experiment from one normal control and one patient with preeclampsia is shown. Figures 2A. 2B, and 2C show assays performed using serum from a normal patient, while Figures 2D, 2E, and 2F show assays performed using serum from a patient with pre-eclampsia. In Figure 2A, t=0 {10% serum from a normal pregnant woman at term); in Figure 2B, t="4S (10% serum from normal pregnant woman 4S hours after delivery); in Figure 2C, tr=0 + exogenous sFlt-1 (10 ng/ml); in Figure 2D, t=0 (10% serum from pre-eciamptic woman prior to dehvery); in Figure 2E, t=4S (10% serum from pre-eclamptic woman 4S hours after delivery); and in Figure 2F, t=0 + exogenous V"EGF(10 ng/ml) + PIGF (10 ng/ml). The mbe assay was quantitated and the mean tube length +/- SEM is shown in pixels at the bottom of each panel. Figures 3A and 3B are graphs showing that inhibition of VEGF and PIGF induced vasodilation of renal microvessels by sFlt-l. Figure 3A shows that liie increase in relaxation responses of rat renal arterioles to sFlt-1 (S), VEGF (V), PIGF (P) was measured at three different doses. V-i- and P+ represent /asodilatory responses of the individual reagents in the presence of sFIt-l at 100 ig/nol. All experiments were done in 6 different dissected rat renal microvessels md data is shown as mean +/- SEM. Tlie * represents statistical significance with t ngiurcs *tA ana its snow sMt-l induction of glomerular endotheiiosis. Figure 4A is photoniicrograph showing hematoxylin and eosin (H & E) staining in a capillary occlusion in tlie sFlt-1 treated animals with enlarged glomeruli and swollen cytoplasm as compared to controls. "Glomerular endotheiiosis" with bubbly cytoplasm is shown in tlie sFU-1 treated anmials on periodic acid schiff (PAS) stain. All light microscopy pictures were taken at 60X, original magnification. Figure 4B is an electron micrograph of sFlt-1 treated glomeruli that confuTOS cytoplasmic swelling of tiie endocapillary cells. The immunofluorescence (IF) for fibrin pictures were taken at 40X and the EM pictures were taken at 24-OOX, original magnification. All figures were reproduced at identical magnifications. Figures 5A-5C show sFlt-1 levels measured before and after the onset of pre-eclampsia by gestational age. Figure 5A is a graph showing the mean serum concentrations in pg/ml for nomiotensive controls (lighter Ime with open ariarigles), cases before pre-eclampsia (filled circles), and cases after pre eclampsia -"endpoint" specimens - (filled squares) within 4-5 week gestational age windows prior to onset of labor. Brackets indicate standard en"or of the mean. Asterisks indicate significant differences with respect to control specimens within the same gestational age window after logarithmic transformation: *p concentrations of sFltl in pg/nil for cases before and after the onset of pre eclampsia within intervals of weeks before pre-eclampsia. PE indicates the arillimetic mean of 43 endpoint specimens (obtained on or following onset of pre eclampsia). Mean gestational age (days) is indicated in parentheses below each time interval. The horizontal line indicates the level in the endpoint specimens. The vertical lines demarcate tlie period 5C is a graph showing the mean serum concentrations of sFlt-I in pg/ml by gestational age windo-ws for normotensive controls and cases before pre eclampsia, after excluding specimens obtained within 5 weeks of onset of pre eclampsia. There are no significant differences. " " Figures 6A-6C show the levels of PIGF before and after pre-eclampsia by gestational age. Figure 6A is a graph showing PIGF levels in all specimens obtained before labor and delivery. Brackets indicate standard error of the mean. Asterisks indicate significant differences with respect to control specimens within the same interval after logarithmic transformation: ■"*p Figure 7A and 7B show sFlt-1 and PIGF levels by pre-eclampsia status and severity. Figure 7A is a graph showing the arithmetic mean serum concentrations of sFIt-I (black bars) and PIGF (white bars) at 23-32 weeks of gestation in controls and cases (before onset of clinical disease) with mild pre-eclampsia, severe pre-eclampsia. pre-eclampsia with onset Detailed Description We have discovered that sFlt-1 levels are elevated in blood serum san^les taken from pre-eclampuc women. sFlt-I binds to "V"EGF and PIGF with high affinity and bloclcs the mitogenic and angiogenic acrivity of these growth factors. Tims, sFlt-i is an excellent diagnostic marker for pre-eclampsia and VEGF and PIGF may be used to treat pre-eciampsia. Furthermore, we have discovered therapeutic agents tliat interfere with sFlt-1 binding to purified VEGF orPlGF, or agents that increase levels of biologically active VEGF or PIGF, can be used to treat or prevent pre-eclampsia or eclampsia in a subject. Such agents include, but are not limited to, antibodies to sFlt-1, oligonucleotides for antisense or RNAi that reduce levels of sFlt-1, compounds that increase the levels of VEGF or PIGF, and small molecules that bind sFlt-1 and block the growtii factor binding site. The invention also features methods for measiiring levels of growth factors", the methods can be used as diagnostic tools for early detection of pre-eclampsia or an increased risk of developing pre-eclampsia or eclampsia. While tlie detailed description presented herein refers specifically to sFlt-1, VEGF, or PIGF, it will be clear to one skilled in the art that die detailed description can also apply to sFlM, \"EGF, or PIGF family members, isoforms, and/or variants, and to growtii factors shown to bind sFlt-1. The following examples are for the purposes of illustrating the invention, and should not be construed as limiting. Example 1. Increased levels of sFIt-1 mRNA and protein in pregnant women with pre-eclampsia. In an attempt to identify novel secreted factors playing a pathologic role in 3re-eclampsia, we performed gene expression profiling of placental tissue from Afomen with and without pre-eclampsia using Affymetrix U95A microarray ;hips. We found that the gene for sFIt-1 was upregulated in women witli pre-jclampsia. In order to confirm the upregulation of sFIi-1 in pre-eclampsia, we perfonned Northern blots to analyze the placental sFlt-1 mRNA levels (Figure ■lA) and ELISA assays to measure serum protein levels of sFlt-1 (Figure IB) in pre-eclamptic pregnant women as compared with nonnotensive pregnant women. Pre-eclarapsia was defined as (1) a systolic blood pressure (BP) >140 mmHg and a diastolic BP >90 mmHg after 20 weeks gestation, (2) new onset proteinuria (1+ by dipstik on urinanaWsis, >300mg of protein in a 24 hour urine collection, oi random urine protein/creatinine ratio >0.3, and (3) resolution of hypertension and proteinuria by 12 weeks postpartum. Patients with underlying hypertension, proteinuria, or renal disease were excluded. Patients were divided into mild and severe pre-eclampsia based on the presence or absence of nephritic range proteinvaria (>3g of protein on a 24 hour urine collection or urine protein/creatinine ratio greater than 3.0). The mean urine protein/creatinine ratios in the mild pre-eclampsia group were 0.94 +/- 0.2 and in the severe pre-eclampsia group were 7.S +/- 2.1. The mean gestational ages of the various groups were as follows: normal 38.S +/-0.2 weeks, mild pre-eclampsia 34 +/- 1.2 weeks, severe pre-eclampsia 31.3 +/-0.6 weeks, and pre-term 29.5 +/- 2.0 weeks. Placental samples were obtained immediately after delivery. Four random samples were taken from each placenta, placed in RNAlater stabilization solution (Ambion, Austin, TX) and stored at -70" C. RNA isolation was performed using Qiagen RNAeasy Maxi Kit (Qiagen, Valencia, CA). We detected an increase in both placental sFlt-1 mRNA and maternal serum sFlt-1 protein in pre-eclamptic pregnant women as compared to normotensive pregnant women. The average serum level of sFlt-I was ahnost four times higher in the severe pre-eclampsia patients as compared to norma! control pregnant women. To exclude the possibility that this effect was due to the earlier gestational age of the pre-eclamptic cases, we also measured sFlt-1 levels in gestationally matched normotensive women delivering pre-maturely for other reasons (gestational ages 23-36 weeks), and we found no significant difference in this group compared with normotensive term pregnancies. The probes used for northern blots were obtained by PCR and included a 500 bp fraament in the coding region from pUC 118 human fit-1 cDNA, and a GAPDH cDNA that was used as normalization control. In nonnal pregnancy there is a balance between pro- and anti-angiogenic factors secreted by the placenta that is necessary for adequate placental deveiopment. We hypothesized that in pre-eclampsia, increased production of sFlt-1 and decreased production of VEGF and PIGF shifts the balance in favor of anti-angiogenesis. To address the net anti-angiogenic activity we measured VEGF and PIGF serum levels and found that PIGF and VEGF serum levels were lower in patients with pre-eclampsia as compared to normal control patients (mean PIGF, 235.3 +/-45.3 pg/"ml versus 464 +/-116.6 pg/ml) as has been described (Tidweli et a!., Am. J. Obstet. Gynecol., 184:1267-1272, 2001). When we incorporated sFlt-l, VEGF and PIGF levels into an anti-angiogenic mdex, or PAAI, as an indicator of net anti-angiogenic activity, we found that we could clearly separate the pre-eclamptic &om the normal patients and that tlie PAAI seemed to correlate with severity of the pre-eclampsia (Figure IC). This PAAI can be used as diagnostic tool for the detection of pre-eclampsia in pregnant women. Example 2. Serum from women with pre-eclampsia inhibits angiogenesis in an in vitro endothelial tube assay. We hypottiesized that excess circulating.sFlt-1 in patients with preeclampsia causes endothelial dysfunction and leads to an anti-angiogenic state. To address this, we used an endothelial tube assay as an m vitro model of angiogenesis. Growth factor reduced Matrigel (7 mg/mL, Collaborative Biomedical Products, Bedford, MA) was placed in wells (100 |j.l/well) of a pre-chilled 48-well cell culture plate and mcubated at 37" C for 25-30 minutes to allow polymerization. Human umbilical vem endothelial cells (30.000 + in 300 ^LI of endothelial basal mediuft with, no serum., Clonetics, Walkersville, MD) at passages 3-5 were ti-eated with 10% patient serum, plated onto the Matrigel coated wells, and incubated at 37" C for 12-16 hours. Tube formation was then assessed through an mverted phase contrast microscope at 4X (Nikon (corporation., lokyo, Japan) and quantitatively analyzed (tube area and total length) using the Simple PCI imaging analysis software. The conditions of the tube formation assay were adjusted such that normal human umbilical vein endothelial cells form tubes only in the presence of exogenous growth factors such as VEGF. Under these conditions, we found that while serum from nonnotensive women induced endotlielial cells to form regular tube-like structures, serum from women with pre-eclampsia inhibited tube formation (Figure 2). Notably, by 48 hours post-partum this anti-angiogenic effect had disappeared suggesting tliat the inhibition of tubes noted with the serum from pre-eclampsia patients was probably due to a circulating factor released by the placenta. When sFlt-1 was added to nonnotensive serum at doses similar to those found in patients with pre-eclarapsia, tube formation did not occur, mimicking the effects seen with the serum from pre-eclamptic women. When exogenous VEGF and PIGF were added to the assay using pre-eclamptic serum, tube formation was restored (Figure 2). Recombinant human VEGF, human PIGF, and human Flt-lFc were used for these assays. These results suggested that the anti-angiogenic properties of pre-eclamptic serum were due to the antagonism of VEGF and PIGF by endogenous sFIt-1. These results also suggested that addition of purified VEGF and/or PIGF can reverse or mitigate the pre-eclamptic condition and can be used therapeutically. Example 3. sFlt-1 inhibits VEGF and PIGF induced vasodilation of renal microvessels. The causative role of sFh-1 in vasoconstriction was determined using an in vitj-o microvascular reactivity experiment. Microvascular reactivity experiments were done as described previously using rat renal microvessels (Sato et al., J. Surg. Res.. 90:135-143, 2000). ECidney artery microvessels (70-170 yxn internal diameter) were dissected from rat kidneys using a 1 Ox to 60x dissecting microscope (Olympus Optical, Tokyo, Japan). Microvessels were placed in an isolated microvessel chamber, cannulated with dual glass micropipettes measuriiig 30-60 ^un in diameter, and secured with a 10-0 nylon monofilament through the vessel chamber and a reservoir containing a total of 100 ml of the solution. The vessels were pressurized to 40 mmHg in a no-flow state using a burette manometer filled with a Krebs" buffer solution. With an inverted microscope (40x to 200x; Oiympus CK2_ 01>"mpus Optical) connected to video camera, the vessel image was projected onto a black-and-white television monitor. An electronic dimension analyzer (Living System Instrumentation, Burlington, VT) was used to measure the internal lumen diameter. Measurements were recorded with a strip-chart recorder (Graphtec, Irvine, CA). Vessels were allowed to batlie in the microvessel chamber for at least 30 minutes prior to any intervention. In all experimental groups, the relaxation responses of kidney microvessels were examined after pre-contraction of the microvessels with U46619 (tliromboxane agonist) to 40-60% of their baseline diameter at a distendmg pressure of 40 mmHg. Once the steady-state tone was reached, the responses to various reagents such as VEGF, PiGF, and sFlt-1 were examined. Recombinant rat VEGF, mouse PIGF, and mouse FIt-IFc were used for these assays. All drugs were applied extraiuminally. Measurements were made when the response had stabilized (usually 2-3 minutes after the drug was administered). One to four interventions were performed on each vessel. The vessels were washed with a Krebs" buffer solution and allowed to equilibrate in a drug-free Krebs" buffer solution for 20-30 minutes between interventions. We found that sFlt-1 alone did not cause significant vasoconstriction, however it blocked the dose responsive increase in vasodilation induced by VEGF or PlGF (Figure 3A). Furthermore, we found that VEGF and PIGF, at physiological levels seen in pregnancy, induced significant dose dependent irteriolar relaxation, and that this effect was blocked by the addition of 10 ng/ml ;Flt-l, a concentration observed in severely pre-eclamptic women (Figure 3B). This result suggested that circulating sFlt-1 in patients witli pre-eclampsia may tppose vasorelaxation, thus contributing to hypertension. These results support ie conclusion that sFlt-1 is responsible for many of the clinical and pathological symptoms ofpre-eclampsia, including hypertension. Inhibition ofsFIt-1, through the use of directed antibodies, for example, could reverse the effects of the protein in pre-eciamptic women and such sFlt-I inhibitors could potentially be used as a therapeutic agent. Example 4. Effects of sFlt-1 in an animal model ofpre-eclampsia. Based on the above results, we hypothesized that the addition of exogenous sFlt-1 would produce hypenension and proteinuria in an animal model Adenovirus expressing sFlt-1 has been shown to produce sustained systemic sFlt-1 levels associated with significant anti-tumor activity (Kuo et al., Proc. Natl Acad. Sci. USA, 98:4605-4610, 2O01). This recombinant adenovuTis encoding murine sFlt-1 was injected into the tail vein, of pregnant Sprs^ue-Dawley rats on day S-9 of pregnancy. Adenovirus encoding murine Fc and sFlkl-Fc (fusion protein of mouse VEGF receptor I Flkl ectodomain and Fc protein) in equivalent doses were used as controls. Flkl has been shown to bind to VEGF, but not PIGF. Hence, sFlk-lFc was chosen as a control to help discriminate between die and-VEGF and the anti-PlGF activity ofsFltl. Both pregnant and non-pregnant Sprague-Dawley rats were injected with I X lO^fuofAdFc, Ad sFlt-1,or Ad sFlk-lFc by tail vem injections. These adenoviruses have been described previously (Kuo et al., supra) and were generated at tlie Harvard Vector Core Laboratory. Pregnant rats were injected witla the adenoviruses at day S-9 of pregnancy (early second trimester) and blood pressure was measured at day 16-17 of pregnancy (early third trimester). In nonpregnant animals, BPs were measured at day 8 after injection of die adenoviruses. BPs were measured in the rats after anesthesia with pentobarbital sodium (60 nig/kg, i.p.). The carotid artery was isolated and cannulated with a 3-Fr high-fidelity microtip catheter coimecred to a pressure transducer (Millar Instruments, Houston, TX). The Millar Mikro-Tip catheter was advanced into the artery to record blood pressure. Blood pressure and heart rate were recorded in by chart-strip recorder (model 56-lX 40-006158, Gould Instrument Systems, Qeveland, OH) and averaged over a lO-minute period. Blood, tissue, and urine samples were men oDtained before euthanasia. Urinary albumin was measured by standard dipstick and quantitated by competitive enzyme-linked immimoassay (ELISA) as has been described elsewhere (Cohen et al.. Kidney Intl., 45: 1673-1679,1994). Urinary creatinine was measured by a picric acid colorimetric procedure kit (Sigma, St. Louis, MO). We measured intrarterial blood pressures in the eariy tliird trimester of the pregnancy to mimic lite natural pathology of pre-eclampsia. These experiments were also performed in non-pregnant female ■ Sprague-Dawley rats to determine if the effects of sFlt-1 is direct or indirect through its effects on the placenta. Systemic levels of sFlt-1 on the day of blood pressure measurement were confirmed by Western blot analysis to be in the range of 23-350 ng/niL in the various sFlt-1 treated animals on the day of BP measurements. Blood pressure and proteinuria in the different experimental groups is shown in Table I. Pregnant rats treated with sFIt-1 had significant hypertension and nephrotic range albuminuria compared with Fc controls. Nonpregnant rats administered sFItl also developed hypertension and proteinuria. Notably, the sFlk-Fc treated nonpregnant rats developed hypertension and proteinuria, whereas the sFlk-Fc treated pregnant rats did not In pregnancy, therefore, the antagonism of VEGF alone is insufficient to produce pre-eclampsia, possibly due to the presence of high levels of PIGF. In the nonpregnant state, where PIGF is virtually absent, antagonism of VEGF alone is sufficient to disrupt the pro/anti-angiogenic balance and produce renal pathologies similar to those associated with preeclampsia. Various stainmg techniques were used to examine the renal lesion diat was observed in all sFlt-1 treated rats (Figure 4). Harvested kidneys from tlie rats were fixed in Bouin"s solution, sectioned and stained with H&E and PAS stains. For electron microscopy, renal tissue was fixed in glutaraldehyde, embedded in araldite-epon mixture, and ultrathin kidney sections (1 fim) were cut, stained with Toluene blue and assessed using a Zeiss EM 10 at various magnifications. Immunofluorescence for fibrin deposits within tiie glomeruU was done using polyclonal anti-fibnn antibody (ICN, Switzerland). Global and diffuse glomerular endotheliosis was the renal lesion universally observed in the sFlt-1 treated rats. We detected glomerular enlargement with occlusion of the capillary loops by swelling and hypertrophy of endocapillary cells. Numerous apparent protein resorption droplets were seen in the glomerular epithelial cells. No segmental giomemlcsclerosis was observed. Isolated "double contours" and focal deposition of fibrin within the glomeruli were seen. This finding of fibrin deposition in the absence of significant mesangial interposition is similar to what has been described as typical of the pre-partum stage of the human disease (Kincaid-Simth, Am. J. KidjieyDis., 17:144-145, 1991). Immunofluorescence for fibrin showed foci of fibrin deposition within the glomeruli of sFIt-1 treated animals but not Fc treated animals. The sFlkl treated nonpregnant rats developed the same lesion. In fact, when sFlk! was used at the same levels as sFlt-1, the renal damage was more severe in the non-pregnant rats, as there are fewer circulating pro-angiogenic molecules for the sFh-1 to antagonize. These results suggested that elevated levels of sFlt-1 maybe responsible for the glomerular endotheliosis associated with pre-eclampsia, but that this effect was independent of the placenta since glomerular changes were detected in nonpregnant as well as pregnant rats. These results also suggested that antagonism of both VEGF and PIGF is important in the patliology of pre-eclampsia as hypertension and proteinuria occurred in sFlk-l treated non-pregnant mice but not in sFlk-1 treated pregnant mice where PIGF levels are high. " The animal model created herein can be used as an experimental model to test novel therapeutic compounds. Both the efficacy of potential therapeutic compounds and the pharmacology and toxicity can be studied using this animal model. Example 5. EfTects of sFlt-1 in an animal model of eclampsia. Pregnant rats in their early second trimester of pregnancy are injected with exogenous sFlt-1. The rats are then monitored and tested during their early third trimester for the development of eclampsia. Tests used for detection of eclampsia can include MRI of the rat brains for die development of edema, EEG of the rat brain for the development of seizures, and histology of the rat brains to determine if endothelial damage has occurred along the blood-brain barrier and clioroids-plexus using specific endothelial markers. The animal model created herein can be used as an experimental model to test novel therapeutic compounds. Both the efficacy of potential therapeutic compounds and the pharmacology and toxicity can be studied using this animal model. Example 6: PIGF/creatinine ratio in urine is diagnostic of pre-eclampsia. Urine samples were obtained from 10 women at 16 weeks gestation (five normals, four mild preeclamptics, and one severe pre-eclamptic). These samples were provided by Dr. Ravi Tbadhani at Massachusetts General Hospital. The average urinary free PIGF/creatinine ratios (pg PIGF pep mg of creatinine) for the - ■ lormal pregnant women were 78+/-10,7 and for the four mild pre-eclamptics were33i +/-5.0 and for the one severe preeclamptic patient was 17. Thus, an alteration in the ratio of PIGF to creatinine in urine is useful as a diagnostic indicator for pre-eclampsia in a patient. Example 7: sFIt-1 and PIGF protein levels as a diagnostic indicator of preeclampsia and eclampsia in women. For this study we used archived samples from the Calcium for Preeclampsia Prevention trial in order to analyze the gestational patterns of circulating sFIt-1, free PIGF. and free VBGF in normotensive and pre-eclamptic pregnancies. Calcium for Pre-eclampsia Prevention, or CPEP, was a randomized, double-blind clinical trial conducted during 1992-1995 to evaluate the effects of daily supplementation with 2 g elemental calcium or placebo on the incidence and severity of pre-eclampsia (Levine et al., N. £jjg/. /. Med. 377:69-76, i997;Levine et al., Control Clin. Trials 17:442-469, 1996). Healthy nuliiparous women with singleton pregnancies were enrolled between 13 and 21 weeks gestation al five participating U.S. medical centers and followed until 24 hours postpartum using a common protocol and identical data collection forms. At erurollment, all CPEP participants had blood pressure Participants We selected subjects having complete outcome information, serum ;araples obtained at without smoking history, 9 with hypertension not verified by chart review teams, and 32 others with stillbirths, leaving 4,257 women with adequate information and live births. Among these 2,156 had male infants. After excluding one woman whose infant had a chromosomal abnormality, 381 with gestational hypertension, and 43 without a baseline serum specimen, 1.731 women remained. Of these, 175 developed pre-eclampsia and 1,556 remained normotensive tliroughout pregnancy. Since calcium supplementation had no effect on the risk and severity of pTe-eclampsia and was unrelated to concentrations of pro- and anti-angiogenic molecules, cases and controls were chosen without regard to CPEP treatment For each pre-eclampsia case one normotensive control was selected, matched for enrollment site, gestational age at collection of the first serum specimen (within one week), and freezer storage time at -70° C (within 12 months). 120 matched pairs ("cases" and "controls") were randomly chosen for analysis of all 657 serum specimens obtained before labor (Table 2, below). Mean gestational age at collection of the first serum specimen was 112.8 and 113.6 days in cases and controls, respectively; mean duration of freeze: storage was 9.35 and 9.39 years. For this study, hypertension, was defined as a diastolic biood pressure of at least 90 mm Hg on two occasions 4-168 hours apart. Severe hypertension was defined as a diastolic bloodpressureofatleast 110 mm Hg on two occasions 4-16S hours apart, or one occasion if the woman had received anti-hypertensive therapy. Proteinuria was defined as 300 mg or more protein in a 24-hour urine collection, two random urine specimens 4-16S hours apart containing at least 1+ protein by dipstick, a single urine sample with a protein / creatinine ratio at least 0-35, or a single random urine specimen containing at least 2+ protein by dipstick. Severe proteinuria was diagnosed by a 24-hour urine collection sample containing at least 3.5 g protein or by two random urine specimens with at least 3+ protein by dipstick. Pre-eclampsia was defmed as hypertension and proteinuria occurring within 7 days of each other; severe pre-eclampsia was defmed as pre-eclampsia with severe hypertension, severe proteinuria, HELLH syndrome (hemolysis, ;levated liver enzymes, low platelets), or eclampsia. The onset of pre-eclampsia vas thie time of detection of the first elevated blood pressure or proteinuria in the rine sample leading to the diagnosis of pre-eclampsia. Small for gestational age (SGA) was defined as birdi weight lower than the O"" percentile for gestational age according to US tables of birth weight for ?stational age by race, parity, and infant sex (Zhang and Bowes 1995, supra). "ocedures Assays were performed at the Beth Israel Deaconess Medical Center by ioratoty personnel who were blinded to patients" diagnoses and other relevant uical information. Specimens were randomly ordered for analysis. Enzyme-ked inimunosorfaent assays (ELISA) for human sFlt-1, free PIGF, and free ■,GF were performed according to the manufacturer"s instructions, using kits chased from R&D Systems (Minneapolis, MN). Aliquots of serum samples ich had been stored at -70°C, were thawed to room temperature, diluted with VTris-buffered saline, and incubated for 2 hours in a 96-well plate pre-coated 1 a capture antibody directed against sFU-1, plGF, or VEGF- The wells were then washed three times, incubated 20 minutes with a substrate solution containing hydrogen peroxide and tetramethylbenzidine, and the reaction quenched with 2N sulfuric acid. Optical density was determined at 450 nm (wavelength correction 550 mn). All assays were performed in duphcate. Protein concentrations were calculated using a standard curve derived from known concentrations of the respective recombinant proteins. If the difference between duplicates exceeded 25%, the assay was repeated and initial results discarded. The assays had sensitivities of 5, 7, and 5 pg/ml for sFIt 1, PIGF, and VEGF, respectively, with inter- and intra-assay coefficients of variation of 7.6% and 3.3% for sFlt I, 11.2% and 5.4% for PIGF, and 7.3% and 5.4% for VEGF. Statistical analysis Chi-square and t tests were used in analyses of maternal or infant characteristics to compare categorical or continuous variables, respectively. Although aritlimetic mean values of concentrations are given in text and figures, statistical testing was performed after logarithmic transformation unless noted otherwise. Adjustment was perfomied using logistic regression on logarithmically transformed concentrations. Results Of the 120 cases, 80 developed mild and 40 severe pre-eclampsia, ncluding 3 with HELLP syndrome and 3 with eclampsia. Case patients were horter than control patients, had a higher body mass index, and higher baseline lood pressure (Table 2). In addition, larger proponions of case patients had regnancies complicated by pre-term delivery or small-for-gestational age (SGA) ifants, Case patients contributed an average of 2.9 serum specimens to the udy; controls, 2.6 specimens. We first confirmed that sFlt-I, PIGF, and VEGF were altered in patients ith pre-eclampsia at the time of active disease as compared to gestationally atched controls from this CPEP study group. Specunens drawn at the time of established clinical pre-eclampsia (endpoint specimens) had dramatically increased sFlt-I levels, decreased P!GF levels, and decreased VEGF levels compared to controls with gestational ages (43S2 vs. 1643 pg/ml sFltl, p substantial differences were observed (Figure 5C). These data suggest that the higher sFlt-1 concentration in case patients prior to onset of pre-eclampsia is due to acute rises in sF!t-l within the 5 weeks before onset of clinical disease. We then plotted the gestational pattern of PIGF protein in the same patient group as shown in Figure 6A. Control PIGF protein concentrations rose during he first two trimesters, peaked at 29-32 weeks, and fell during late gestation. among case patients, prior to pre-eclampsia, PIGF protein concentrations bllowed a similar gestational pattern, but were significantly lower than controls from 13-16 weeks. Overall, differences in PlGF between cases patients and ontrols measured before the onset of clinical symptoms were 35% (p VEGF concentrations throughout pregnancy were very low and similar in controls and cases before pre-eclampsia, except for a significant decrease in case ients at 37-41 weeks. Mean VEGF concentrations at 23-32 weeks in cases ;luding specimens obtained 5 weeks before pre-eclampsia did noi differ nificantly from controls (11.6 vs. 12.S pg/ml), whereas concentrations in cases -[udingspecimens At 33-41 weeks case VEGF concentrations >5 or were higher and lower than controls, respectively (U.2pg/ml and 8.3 vs. 9.7 pg/ml), although these differences were not significant. Figure 7 depicts sFlt-1 and PIGF at 23-52 weekss (Figure 7A) and 33-41 weeks (Figure 7B) by pre-eclampsia status and severity. The graphs show that sFlt-1 increases and PIGF decreases before onset of pre-eclampsia were associated with disease severity, time of onset, and the presence of an SGA infant. At 23-32 weeks, sFlt-1 and PIGF in case patients with an SGA infant before onset of pre-eclampsia were significantly higher or lower, respectively, than corresponding concentrations in control patients with an SGA infant. Moreover, in comparison to control patients who delivered pre-term, case patients with pre-term delivery had higher sFlt-l and significantly lower PIGF. We then determined if we could use circulating concentrations of PtGF and/or sFlt-1 during the first trimester to identify women at risk for the development of pre-eclampsia. At S-20 weeks, after adjustment for gestational age, body mass index, and sFlt-I, case patients with PlGF in the lowest quartile of the distribution of control values had almost a I2-foId increased risk of preeclampsia at These results demonstrate that sFIt-I levels, begin to rise dramatically about 5 weeks before the onset of pre-eclampsia symptoms. Parallel with the rise in sFlt-1, free PIGF and free VEGF levels fall, suggesting that the decrease in FIGF and VEGF may be due at least partially to anmgonism by sFIt-1 and not due to a decrease in placental production of PIGF and VEGF. Three pre-eclampsia subgroups - severe pre-eclampsia, early onset of disease, and SGA infants - had higlier sFlt-1 and lower PIGF concentrations at 23-32 weeks and at 33-41 weeks than controls or women with mild pre-eclampsia. We have also demonstrated a small but significant decrease in free PlGF beginning early in the second trimester among women destined to develop pre-eclanipsia. These results demonstrate that a decrease in PIGF levels may be a useful predictor of early onset pre-eclampsia. We describe here for the first time the gestational pattern of sFlt-1 in normal pregnancy, observing relatively stable levels throughout gestation followed by a steady increase beginning at 33-36 weeks. This rise corresponds to the late gestational fall in PlGF observed in normal pregnancy by others (Tony et al„ J. 5oc. Gynecoilnvest. 10:178-188, 1998; Taylor etal.,v4jH./. Obstet. Gynecol- 1SS:177-IS2, 2003-) and in the results described herein. The temporal association, together with the knowledge that sFlt-1 mterferes with PIGF ELISA measurement (Maynard ei al., supra) suggests thai the fall in free PIGF levels during late gestation may be due to the rise in sFlt-1 levels. During first and second trimesters, when placental growth is needed to keep pace with increasing fetal demands, PIGF concentrations are high and sFlt-1 concentrations are low, creating a relatively pro-angiogenie state. Later in gestation, when placental vascular growth may need to be tempered and halted, there is a rise in the anti-angiogenic sFlt-I and resulting decrease in PIGF. In women with pre-eclampsia, the sFlt-1 rise begins earlier in gestation, approximately five weeks before symptom onset, at about 29-32 weeks gestation on average. Thus, in preeclampsia, the anti"angiogenic "brakes" may be applied too soon and too strongly, resulting in an exaggeration of a nonnal physiologic process which arrests placental growth. It seems clear that the pathologic placental changes that characterize pre-eclampsia occur early in gestation (10-14 weeks), well before the dramatic rise in sFlt-l. The resulting placental ischemia itself may enhance sFh-1 production, ultimately triggering a burst in sFlt-I. In addition to the large differences seen in the five weeks prior to the development of clinical symptoms, women destined to develop pre-eclampsia had small, but statistically significant, decreases in free PIGF as early as 13-16 weeks gestation. This fall in PIGF generally was not accompained by a reciprocal increase m sFlt-1 levels. However, there was a tendency towards slightly higher sFIt-1 levels in cases during the first trimester though it was not statistically significant (For example at the 17-20 week window, average sFlt-1 levels in cases were 365.77 pg/ml vs. 795.25 in controls). This decrease in PIGF levels early on in gestation might reflect a smaller placental production of PIGF in pregnancies compromised by conditions such as pre-eclampsia or SGA. Importantly, in patients with pre-eclampsia complicated by SGA, we found a statistically significant increase in both sFl:-l elevation and PIGF fall prior to the disease presentation. It is also possible that there is no change in placental production of PIGF in pre-eclamptics and that elevation of local sFlt-I levels in the placenta may contribute to the decrease in circulating free PIGF. This is supported by the finding that placental PIGF, measured by immunohistochemistry, is not altered in pre-eclampsia (Zhou et al.. Am. J. Pathol. 160:1405-1423, 2002). In summary, we have shown thal sFlt-i starts rising in pre-eclampsia at lest 5 weeks before the onset of clinical disease which is accompanied by decreases in circulating free PIGF and free VEGF. Decreased PIGF during the first trimester may serve as a predictor of pre-eclampsia and elevated sFlt-1 may serve as a predictor of proximity to clinical disease. This data in conjunction with the animal work described above demonstrating sFlt-1 alone induces preeclampsia like symptoms in rodents suggests aprobable etiological role for sFlt-1 in the padiogenesis of pre-eclarapsia. Our limited data on SGA infants and preterm delivery in controls, as compared to case patients, suggest that the increased alterations in protein levels observed in pre -eclamptic pregnancies with an SGA infant are more substantial than a difference due only to intrauterine growth restriction or pre-term delivery in the absence of pre-eclampsia. Diagnostics The present ivention features diagnostic assays for the detection of preeclampsia, eclampsia, or the propensity" to develop such conditions. Levels of VEGF, PIGF, or sFlt-1, either free or total levels, are measured in a subject sample and used as an indicator of pre-eclampsia, eclampsia, or the propensity to develop such conditions. In one embodiment, a metric is used to determine whether a relationship between levels of at least two of the proteins is indicative of pre-eclampsia or eclampsia. Standard methods may be used to measure levels of VEGF, PIGF, or sFlt-1 polypeptide in any bodily fluid, including, but not limited to, urine, serum, plasma, saliva, amniotic fluid, or cerebrospinal fluid. Such methods include immunoassay, ELISA, western blotting using antibodies duected to VEGF, PIGF or sFlt-1, and quantitative enzyme immunoassay techniques such as those described in Ong et al {Obslet. Oyyiecol. 98:608-611, 2001) and Su et al {Obstet. Gynecol., 97:898-904, 2001). ELISA assays are the preferred method for . . measuring levels of VEGF, PIGF, or sFlt-1. Serum levels of sFIt-1 greater than 2 ng/"ml are considered a positive indicator of pre-eclampsia. Additionally, any detectable alteration in levels of sFlt-l, VEGF, or PIGF relative to nonnal levels is indicative of eclampsia, pre-eclampsia, or the propensity to develop such conditians. Preferably sFlt-1 is measured, more preferably measurement of VEGF and PIGF are combined with this measurement, and most preferably all three proteins (or mRNA levels indicative of protein levels) are measured. In another embodiment, the PAAI (sFlt-1/ VEGF ^ PIGF) is used as an anti-angiogenic index that is diagnostic of pre-eclampsia, eclampsia, or the propensity to develop such conditions. If the PAAI is greater than 20 then the subject is considered to have pre-eclampsia or to be in imminent risk of developing the same. The PAAI (sFlt-l/ VEGF + PIGF) ratio is merely one example of a useful metric that may be used as a diagnostic indicator. It is not intended to limit the invention. Vutually any metric that detects an alteration in the anti-angiogenic index in a subject having eclampsia relative to a normal control may be used as a diagnostic indicator. Expression levels of particular nucleic acids or polypeptides may be correlated with a particular disease state (e.g., pre-eclampsia or eclampsia), and thus are useful in diagnosis. Oligonucleotides or longer fragments derived from a sFlt-1. PlGF, or VEGF nucleic acid sequence may be used as a probe not only to monitor expression, but also to identify subjects having a genetic variation, mutation, or polymorphism in an sFlt-1, PIGF, or VEGF nucleic acid molecule that are indicative of a predisposition to develop the conditions. Such 3olymorphisms are known to the skilled artisan and are described by Parry et al. "Eur. JImmunogenet. 26:321-3, 1999). Such genetic alterations may be present n the promoter sequence, an open reading frame, intronic sequence, or intranslated 3" region of an sFlt-1 gene. Information related to genetic alterations ;an be used to diagnose a subject as having pre-eclampsia, eclampsia, or a tropensity to develop such condidons. As noted tluoughoul, specific alterations 1 the levels of biological activity of sFlt-1, X-^GF, and/or PIGF can be correlated /ith the likelihood of pre-eclampsia or eclampsia, or the predisposition to die . -ame. As a result, one skilled in the art, having detected a given mutation, can then assay one or more metrics of the biological activity of the protein to determine if the mutation causes or increases the likelihood of pre-eclampsia at eclampsia. In one embodiment, a subject having pre-eclampsia, eclampsia, or a propensity to develop such conditions will show an increase in the expression of a nucleic acid encoding sFlt-1 or an alteration in PIGF or VEGF levels. Methods for detecting such alterations are standard in the art and are described in Ausubel et al., supra. In one example northern blotting or real-time PCR is used to detect sFlt-1, PIGF, or VEGF mRNA levels. In another embodiment, hybridization with PCR probes that are capable of detecting an sFlt-1 nucleic acid molecule, including genomic sequences, or closely related molecules, may be used to hybridize to a nucleic acid sequence derived from a subject having pre-eclampsia or eclampsia or at risk of developing such conditions. The specificity of the probe, whether it is made from a highly specific region, e.g., the 5" regulatory region, or from a less specific region, e.g., a conserved motif, and the sningency of the hybridization or amplification (maximal, high, intermediate, or low), determine whether the probe hybridizes to a naturally occurring sequence, allelic variants, or other related sequences. Hybridization tecliniques may be used to identify mutations indicative of a preeclampsia or eclampsia in an sFh-1 nucleic acid molecule, or may be used to monitor expression levels of a gene encoding an sFlt-l polypeptide (for example, by Northern analysis, Ausubel et al., supra). In yet another embodiment, humans may be diagnosed for a propensity to develop pre-eclampsia or eclampsia by direct analysis of the sequence of an sF!t-1, VEGF, or PIGF nucleic acid molecule. A subject having pre-eclampsia, eclampsia, or a propensity to develop such conditions will show an increase in tlie expression of an sFlt-1 polypeptide. An antibody that specifically binds an sFlt-1 polypeptide may be used for the diagnosis of pre-eclampsia or eclampsia or to identify a subject at risk of developing such conditions. A variety of protocols for measuring an alteration in ■he expression of such polypeptides are known, including immunological methods (such as ELISAs and RIAs), and provide a basis for diagnosing preeclampsia or eclampsia or a risk of developing such conditions. Again, an increase in the level of the polypeptide is diagnostic of a subject having pre-iclampsia, eclampsia, or a propensity to develop such conditions. In one embodiment, the level of sFlt-L VEGF, or PIGF polypeptide or lucleic acid, or any combination thereof, is measured at least two different times md an alteration in the levels as compared to normal reference levels over time is used as an indicator of pre-ec!ampsia, eclampsia, or the propensity to develop ;uch conditions. The level of sFlt-1, VEGF, or PIGF in the bodily fluids of a subject having 3re-eclampsia, eclampsia, or the propensity to develop such conditions may be iltered by as little as 10%, 20%, 30%, or 40%, or by as much as 50%, 60%, 70%, 80%, or 90% relative to the level of sFlt-l,VEGF, or PIGF in a normal control. fhe level of sFlt-1 presentin the bodily fluids of a subject having pre-eclampsia, xlampsia, or the propensity to develop such conditions may be increased by 1.5-bld, 2-fold, 3-fold, 4-fold or even by as much as 10-fold or more relative to evels in a normal control subject. In one embodiment, a subject sample of a bodily fluid (e.g., urine, plasma, ;erum, anrmiotic fluid") is collected early in pregnancy prior to the onset of pre-jclampsia symptoms. In another example, the sample can be a tissue or cell ;onected early in pregnancy prior to the onset of pre-eclampsia symptoms. Non-imiting examples include placenta! tissue, placental cells, endothelial cells, and eukocytes such as moncytes. In humans, for example, maternal blood serum amples are collected from the antecubital vein of pregnant women during the irst, second, or third trimesters of the pregnancy. Preferably, the assay is carried lut during the first trimester, for example, at 4, 6, 8, 10, or 12 weeks, or during he second trimester, for example at 14,16, IS, 20, 22, or 24 weeks. Such assays nay also be conducted at the end of the second trimester or beginning of the third rimester (around 28 weeks). It is preferable that levels of sFlt-1, VEGF, or PIGF le measured twice during rhis period of time. For tlie diagnosis of post-partum pre-eclampsia or eclampsia, assays for sFlt-1, VEGF, or PIGF may be carried out postpartum. In one particular example, serial blood samples can be collected during pregnancy and the levels of soluble sFlt-1 determined by ELISA. In one study using this technique, the alternatively spliced mRNA encoding sFlt-1 is highly expressed by trophoblast cells and the protein was readily detectable in the plasma of pregnant women. It was observed that the levels of sFlt-1 increased approximately 3-fold between 20 and 36 weeks gestation. Levels were observed to be significantly higher in high-risk women who subsequently went on to develop pre-eclampsia (Chamock-Jones et al, / Soc. Gyiiecol. Investig. 10(2):230, 2003). In veterinary practice, assays may be carried out at any time during the pregnancy, but are. preferably, carried out early in pregnancy, prior to the onset of pre-eclampsia symptoms. Given that the temi of pregnancies varies widely between species, the timing of ihe assay will be determined by a veterinarian, but will generally correspond to the timing of assays during a human pregnancy. The diagnostic methods described herein can be used individually or in combination with any other diagnostic method described herein for a more accurate diagnosis of the presence of, severity of, or estimated time of onset of pre-eclampsia or eclampsia. In addition, the diagnostic methods described herein can be used in combination with any other diagnostic methods determined to be useful for the accurate diagnosis of the presence of, severity of, or estimated time of onset of pre-eclampsia or eclampsia. The diagnostic methods described herein can also be used to monitor and manage pre-eclampsia or eclampsia in a subject. In one example, if a subject is determined to have a serum sFh-1 protein level of 10 ng/mL and a serum level of free PIGF of 100 pg/mL, then \"EGF can be administered until the serum PIGF level rises to approximately 400 pg/mL. In this embodiment, the levels of sFlt-1, PIGF, and \VEGF, or any and all of these, are measured repeatedly as a method of not only diagnosing disease but monitoring the treatment and management of the pre-eclampsia and eclampsia Diagnostic Kits The invention also provides for a diagnostic test kit. For example, a diagnostic test kit can include antibodies to sFIt-L VEGF, or PIGF, and means for detecting, and more preferably evaluating, binding between the antibodies and the sFlt-1, VEGV, or PIGF polypeptide. For detection, either the antibody or the sFlt-1, VEGF, or PIGF polypeptide is labeled, and either the antibody or the sFlt-1, VEGF, or PIGF polypeptide is substrate-bouad, such dut the sFlt-1, VEGF, or PIGF polypeptide-anybody interaction can be estabhshed by determining the amount of label attached to the substrate following binding between the antiTiody ind the sFIt-1, VEGF, or PIGF polypeptide. A conventional ELISA is a lommon, art-known method for detecting antibody-substrate interaction and can .e provided with the kit of the invention. sFlt-1, VEGF, or PIGF polypeptides an be detected in virtually any bodily fluid including, but not limited to urine, erum, plasma, saliva, amniotic fluid, or cerebrospinal fluid. A kit that etemiines an alteration in the level of sFlt-1, VEGF, or PIGF polypeptide relative to a reference, such as the level present in a normal control, is useful as a agnostic kit in the methods of the invention. ireeoing Assays As discussed above, the expression of an sFlt-1 nucleic acid or polypeptide increased in a subject having pre-eclampsia, eclampsia, or a propensity to velop such conditions. Based on these discoveries, compositions of the "ention are useful for the high-throughput low-cost screening of candidate ■npounds to identify those that modulate the expression of a sFlt-1, VEGF, or oF polypeptide or nucleic acid molecule whose expression is altered in a iject having a pre-eclampsia or eclampsia. Any number of methods are available for carrying out screening assays to ntify new candidate compounds that alter the expression of a sFlt-I, VEGF, or IF nucleic acid molecule. In one working example, candidate compounds aie ed at varying concennations to the culture medium of cultured cells cessing a sFIt-I, VEGF, or PIGF nucleic acid sequence. Gene expression is then measured, for example, by microarray analysis. Northern blot analysis (Ausubel et al., supra), or RT-POl, using any appropriate fragment prepared from the nucleic acid molecule as a hybridization probe. The level of gene expression in the presence of the candidate compound is compared to the level measured in a control culture medium lacking the candidate compound A compound that promotes an alteration such as an increase in the expression of a VEGF or PIGF gene, nucleic acid molecule, or polypeptide, or a decrease in the expression of an sFU-1 gene, nucleic acid molecule, or polypeptide, or a fuctional equivalent thereof, is considered useful in the invention; snch a nolecule may be used, for example, as a therapeutic to treat pre-eciampsia or clampsia in a subject. In another working example, the effect of candidate compounds may be leasured at the level of polypeptide production using the same general approach id standard immunological techniques, such as Western blotting or imunoprecipitation with an antibody specific for a sFlt-1, VEGF, or PIGF ilypeptide. For example, immunoassays may be used to detect or monitor the pression of at least one of the polypeptides of the invention in an organism, ■lyclonal or monoclonal antibodies (produced as described above) that are sable of binding to such a polypeptide may be used in any standard munoassay fomiat (e.g., ELISA, western blot, or RIA assay) to measure the e! of the polypeptide. In some embodiments, a cxjmpound that promotes an tration such as an increase in the expression or biological activity of a VEGF FIGF polypeptide or a decrease in the expression or biological activity of an 1 polypeptide is considered particularly useful. Again, such a molecule may ised, for example, as a therapeutic to delay, ameliorate, or treat a pre-impsia or eclampsia, or the symptoms of a pre-eclampsia or eclampsia, m a iect. In yet another working example, candidate compounds may be screened hose that specifically bind to an sFlt-1, VEGF, or PIGF polypeptide. The acy of such a candidate compound is dependent upon its -ability to interact such a polypeptide or a tuncuonal equivalent thereof. Such an interaction functional assays (e.g., those described in Ausubel et al., supra). In one embodiment, a candidate compound may be tested in intro for its ability to specifically bind apolypeptide of the invention. In another embodiment, a candidate compound is tested for its ability to decrease the biological activity of an sFlt-I polypeptide by decreasing binding of an sFlt-1 polypeptide and a growth factor, such as VEGF or P!GF. In another working example, an sFlt-i, VEGF. orPlGF nucleic acid is expressed as a transcriptional or tanslational fusion with a detectable reporter, and expressed in an isolated cell (e.g., mammalian or insect cell) under the control of a heterologous promoter, such as an inducible promoter. The cell expressing the fusion protein is then contacted with a candidate compound, and the expression of the detectable reporter in that cell is compared to the expression of the detectable reporter in an untreated control cell. A candidate compound that decreases the expression of an sFIt-1 detectable reporter, or that increases the expression of a VEGF or PIGF detectable reporter is a compound that is useful for the treatment of pre-eclampsia or eclampsia. In preferred embodiments, the candidate compound alters the expression of a reporter gene fused to a nucleic acid or nucleic acid. In one particular working example, a candidate compound that binds to an Flt-1 polypeptide may be identified using a chromatography-baed technique, "or example, a recombinant polypeptide of the invention may be purified by tandard techniques from cells engineered to express the polypeptide (e.g., those escribed above) and may be immobilized on a column. A solution of candidate impounds is then passed through the column, and a compound specific for the ■lt-1 polypeptide is identified on the basis of its ability to bind to the polypeptide id be immobilized on the column. To isolate the compound, the column is ished to remove non-specifically bound molecules, and the compound of :erest is then released from the column and collected. Similar methods may be ed to isolate a compound bound to a polypeptide microarray". Compounds lated by this method (or any other appropriate method) may, if desired, be further purified (e.g., by high performance liquid chromatography). In addition, these candidate compoiuds may be tested for their ability to decrease the activity of ail sFlt-1 polypeptide or to increase the activity of a VEGF signaling pathway {e.g., as described herein). Compounds isolated by this approach may also be used, for example, as therapeutics to treat pre-eclampsia or eclampsia in a human subject. Compounds that are identified as binding to a polypeptide of die invention with an affinity constant less than or equal to 10 mM are considered particularly usefull in the invention. Alternatively, any in vivo protein interaction detection system, for example, any two-hybrid assay may be utilized to identify compounds or proteins that bind to a polypeptide of the invention. Potential antagonists include organic molecules, peptides, peptide mimetics, polypeptides, nucleic acids, and antibodies that bind to an sFlt-1 nucleic acid sequence or sFlt-1 polypeptide. sFlt-1 DNA. sequences may also be used in the discovery and development of a therapeutic compound for the treatment of pre-eclampsia or eclampsia. The encoded protein, upon expression, can be used as a target for the screening of drugs. Additionally, the DNA sequences encoding the amino terminal regions of he encoded protein or Shine-Delgamo or other translation facilitating sequences of the respective mRNA can be used to construct sequences that decrease the xpression of an sFlt-I coding sequence. Such sequences may be isolated by tandard techniques (Ausubel et al., supra). Optionally, compounds identified in any of the above-described assays lay be confirmed as useful in an assay for compounds that decrease the iological activity of sFlt-1 or that increase the activity of a VEGF signahng ithway. Small molecules of the invention preferably have a molecular weight below 2,000 daltons, more preferably between 300 and 1,000 daltons, and most preferably between 400 and 700 daltons. It is preferred that these small molecules are organic molecules. Therapeutics targeting the VEGF signaling pathway VEGF is a potent etidothelial cell-specific mitogen that stimulates angiogenesis, vascular hypeipenneability, and vasodilation. Three tyrosine-kinase signaling receptors for VEGF have been identified. VEGF-receptor binding triggers a signaling cascade that results in tyrosine phosphorylation of phospholipase Cyl, leading to increases in intracellular levels of inositol 1,4,5-triphosphate and increases in intracellular calcium that activates nitric oxide synthase to produce nitric oxide (NO). NO formation activates guanylale cyclase within vascular smooth muscle ceils and endothelial cells, causing cGMP production. This NO/cGMP cascade is thought to mediate the vasoactive effects of VEGF. Another pathway that appears to be involved in mediating the vasoactive effects of VEGF is the prostacyclin release pathway. VEGF induces PGI2 production via activation of phospholipase A2 as a consequence of initiation of the MAPK cascade. Increased VEGF levels are useful for the treatment of pre-eclampsia and eclampsia. Therapeuric compounds that target VEGF signaling pathways, or components of a VEGF signaling pathway, and enhance the activity of a VEGF signaling pathway are also useful in for thetreament of pre-eclampsia and eclampsia. Such compounds include sildenafil, prostacyclin analogs, such as Flolan, Remodulin, and Tracleer. Pest compounds and extracts In general, compounds capable of decreasing the activity of a sFit-1 polypeptide or increasing the activity of VEGF or PIGF are identified from large libraries of both natural product or synthetic (or semi-synthetic) extracts or chemical libraries or from polypeptide or nucleic acid libraries, according to methods known in the art, Those skilled in the field of drug discovery and development will understand that the precise source of test extracts or compounds is not critical to the screening procedure(s) of the invention. Compounds used in screens may include known compounds (for example, known therapeutics used for other diseases or disorders). Alternatively, virtually any number of unknown t!ie methods described herein. Examples of such extracts or compounds include, but are not limited to, plant-, ftingal-, prokar/otic- or animal-based extracts, fenrientation broths, and synthetic compounds, as well as modification of existing compounds. Numerous methods are also available for generating random or directed synthesis (e.g., semi-synthesis or total synthesis) of any number of chemical compounds, including, but not limited to, saccharide-, lipid-, peptide-, and nucleic acid-based compounds. Syiithetic compound libraries are commercially available from Brandon Associates (Merrimack, NH) and Aldrich Chemical (Milwaukee, WI). Alternatively, libraries of natural compounds in the fomi of bacterial, ftingal, plant, and animal extracts are commercially available from a number of sources, including Biotics (Sussex, UK), Xenova (Slough, UK), Harbor Branch Oceangraphics Institute (Ft, Pierce, FL), and PhaxmaMar, U.S.A. (Cambridge, MA). In addition, natural and synthetically produced libraries are produced, if desired, according to methods known in the art, e.g., by standard extraction and fractionation methods. Furthermore, if desired, any library or compound is ■eadily modified using standard chemical, physical, or biochemical meUiods-In addition, those skilled in the art of drug discovery and development eadily understand that methods for dereplication (e.g., taxonomic dereplication, "iological dereplicatior,, and chemical dereplication, or any combination thereof) r the elimination of replicates or repeats of materials already known for their lolt-dismpling activity should be employed whenever possible. When a crude extract is found to decrease the activity of ai\ sFlt" I Dlypeptide, or to binding an sFlt-1 polypeptide, further fractionation of the jsitive lead extract is necessary- to isolate chemical constituent responsible for e observed effect. Thus, the goal of the extraction, fractionation, and irification process is the carefull characterization and identification of a chemical tiny within the crude extract that decrease the activity of ansFlt-1 polypeptide. ;thods of fractionation and purificarion of such heterogeneous extracts are own in the art. If desired, compounds shown to be usefiil as therapeutics for chemically modified according to methods known in the art. Therapeutics The present invention features methods for treating or preventing preeclampsia or eclampsia in a subject. Preferably the therapeutic is administered during pregnancy for the treatment or prevention of pre-eclampsia or eclampsia or after pregnancy to treat post-partum pre-eclampsia or eclampsia. Techniques and dosages for administration vary depending on the type of compound (antibody, antisense, nucleic acid vector, etc.) and are well known to those skilled in the art or are readily determined. Therapeutic compounds of the present invention may be administered with a pharmaceutically acceptable diluent, carrier, or excipient, in unit dosage form. Administration may be parenteral, intravenous, subcutaneous, oral or local by direct injection into the amniotic fluid. Intravenous delivery by continuous infusion is the preferred method for administering the therapeutic compounds of the present invention. The composition can be in the form of a pill, tablet, capsule, liquid, or sustained release tablet for oral administration; or a liquid for intravenous, subcutaneous or parenteral administration; or a polymer or other sustained release vehicle for local administration. Methods well known in the art for making formulations are found, for example, in "Remington: The Science and Practice of Pharmacy" (20th ed., ed. A..R. Gennaro AR., 2000, Lippincott Williams & Wilkins, Philadelphia, PA), Forraulations for parenteral adminisnation may, for example, contain excipients, ;terile water, saline, polyalkylene glycols such as polyethylene glycol, oils of -egetable origin, or hydrogenated napthalenes. Biocompatible, biodegradable actide polymer, lactide/glycolide copolymer, or polyoxyethylene-olyoxypropyiene copolymers may be used to control the release of the impounds. Nanopaniculate formulations (e.g., biodegradable nanoparlicles, solid lipid nanoparticles, liposomes) may be used to control the biodistribution of the compounds. Other potentially useful parenteral delivery systems include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. The concentration of the compound in the formulation varies depending upon a number of factors, including the dosage of the drug to be administered, and theroute of administration. The compound may be optionally administered as a pharmaceutically acceptable salts, such as non-toxic acid addition salts or metal complexes that are commonly used in the pharmaceutical industry. Examples of acid addition salts include organic acids such as acetic, lactic, pamoic, maleic, citric, malic, ascorbic, succinic, benzoic, palmitic, suberic, salicylic, tartaric, methanesulfonic, toluenesulfonic. or trifluoroacetic acids or the like; polymeric acids such as tannic acid, carboxymethyl cellulose, or the like; and inorganic acid such as hydrochloric acid, hydrobromic acid, suifuric acid phosphoric acid, or the like. Metal complexes include zinc, iron, and the like. Formulations for oral use include tablets containing the active ingredient(s) in a mixture with non-toxic pharmaceutically acceptable excipients. These excipients may be, for example, inert diluents or fillers (e.g., sucrose and sorbitol), lubricating agents, glidants, and anti-adhesives (e.g., magnesium stearate, zinc stearate. stearic acid, silicas, hydrogenated vegetable oils, or talc). Formulations for oral use may also be provided as chewable tablets, or as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium. The dosage and the timing of administering the compound depends on various clinical factors inciuding the overall health of the subject and the severity" of the symptoms of pre-eclampsia. In general, once pre-eclampsta or a propensitv" lo develop pre-eclampisa is detected, continuous infusion of the purified protein is used to treat or prevent further progression of the condition. Treatment can be continued for a period of time ranging from I to 100 days, more preferably 1 to 60 days, and most preferably 1 to 20 days, or until the completion of pregnancy. Dosages vary depending on each compound and the severity of the condition and are titrated to achieve a steady-state blood serum concentration ranging from 1 to 500 ng/mL VEGF or PIGF, or both, preferably 1 to 100 ng/mL, more preferably 5 to 50 ng/mL and most preferably 5 to lOng/mL VEGF or PIGF, or both. Methods to increase VEGF or PIGF protein expression The present invention features methods for increasing the levels of VEGF and PIGF in a subject diagnosed with pre-elampsia or eclampsia. The increased levels of VEGF or PIGF can be achieved using several different methodologies that are described below, among others. Purified proteins In a preferred embodiment of the present invention, purified forms of VEGF or PIGF or both are administered to the subject in order to treat or prevent pre-eclampsia or eclampsia. Purified VEGF or VEGF-like proteins include any protein with an amino acid sequence that is homologous, more desirably, substantially identical to the amino acid sequence of VEGF, or any VEGF family member, that can induce ingiogenesis or that is capable of promoting selective growth of vascular endotelial cells or umbilical vein endothelial cells. An example of a purified, /EGF compound is human recombinant VEGF from Geaentech, Inc. (San Fancisco,CA). Purified PIGF or PlGF-like proteins include any protein with an amino cid sequence that is homologous, more desirably, substantially identical to the mino acid sequence of PIGF, or any PIGF family member, that can induce igiogenesis or that is capable of promoting selective growth of vascular idothelial cells or umbilical vein endothelial cells. An example of commercially available purified PIGF is human recombinant PIGF from R&D Systems (catalog 264-PG, R&D Systems, Minneapolis, MN). ThromboGenics Ltd is also ;veIoping a purified form of PIGF for the treatment of ischemic stroke; esumably this fom of PIGF would be effective for the applications described in ; present invention. Therapeutic compounds that increase VEGF or PIGF activity The present invention provides for the use of any compound known to stimulate or increase biood serum levels of VEGF or PlGF, or the biological activity of these poly"peptides, for the treatment or prevention of pre-eclampsia m a subject. These compounds can be used alone or in combination with the purified proteins described above or any of the other methods used to increase VEGF or PIGF protein levels described herein. One example of a compound shown lo stimulate VEGF production is nicotine. Although smoking poses many risks for the overall health of a pregnant woman and her developing fetus, nicotine by itself is believed lo be safer than cigarettes and can be used for short-term therapy on high-risk subjects. Examples include Nicorette (nicotine polacrilex). which is an over-the-counter nicotine gum product made by SmithKline Beecham and NicoDerm CQ, which is an over-the counter nicotme patch made by Hoechst Marion Roussel Inc. {formerly Marion Merrell Dow). Nicotine delivered via tobacco is specifically excluded fom the methods of the invention where the patient has not also been diagnosed using the methods of the invention. Nicotine is admmistered after the diagnosis of pre-eclampsia or eclampsia using either the patch or gum. Dosages vary depending on the severity of the condition and the overall health of the subject. In general, the manufacturer"s instructions are followed to achieve a serum level of nicotine ranging from 5 to 500 ng/mL, more preferably 5 to 100 ng/mL, and most preferably 50 to 100 ng/mL. Theophylline is another example of an additional compound that can be ised to treat or prevent pre-eclampsia or eclampsia. Theophylline is a )ronchodilator which is often used for the treatment of asthma and is available mder many brand names (e.g., Aerolate Sr, .Asmelix, Elxophyllin, etc.) as well as he generic. Methods of administration and dosages vary with each manufacturer and are chosen based on the overall health of the subject and the severity of the ondition. In general dailvdosage range from1to 500 mg, more preferably 100 to 400 mg, and most preferably 250 to 350 mg given twice a day to achieve a serum level of theophylline of 5 to 50 ng/raL. Adenosine Is another example of an addirional compound that can be used to treat or prevent pre-ec!ampsia or eclampsia. Adenosine (Fujisawa Pharmaceutical Co.) is commonly used as an ami-hypertensive drug. Methods of administration and dosages vary with each manufacturer and are chosen based on the overail health of the subject and the severity of the condition. In general, a daily dosage of 50 mg/kg given twice a day is typicai for adenosine. Nifedipine is another example of an additional compound that can be used to treat or prevent pre-eclampsia or eclampsia. Nifedipine (Bayer Pharmaceuticals) is commonly used as an anti-hypertensive drug. Methods of administration and dosages vary with each manufacturer and are chosen based on the overall health of the subject and the severity of the condition. In general, a daily dosage of 1-2 mg/kg given twice a day orally or subcutaneously is t^ical for nifedipine-Minoxidil is another example of an additional compound that can be used to treat or prevent pre-eclampsia or eclampsia. Minoxidil (Pfizer, Inc.) is commonly used as an anti-hypertensive drug. Methods o"f administration and dosages vary with each manufacturer aid are chosen based on the overall health of the subject and the severity of the condition. In general, a daily dosage of 0.25 to 1.0 mg/kg given twice a day orally or subcutaneously is typical for mmoxidil. Magnesitmi sulfate is another example of an additional compound that can be used to treat or prevent pre-eclarapsia or eclampsia. Magnesium sulfate is a generic drug which is typically used as an anti-hypertensive drug. Methods of administration and dosages vary with each manufacturer and are chosen based on the overall health of the subject and the severity of the condition. In general, a daily dosage of 1-2 gm given intravenously ever four hours is a typical dosage for magnesium sulfate. In addition to the use of compounds that can increase serum levels of VEGF or PIGF, the invention provides for the use of any chronic hypertension medications used in combination with any of the VEGF or PIGF directed compounds. Medications used for the treatment of hypertension during pregnancy include methyldopa, hydralazine hydrochloride, or labetalol. For each of these medications, modes of administration and dosages are determined by the physician and by the manufacturer"s instructions. Therapeutic nucleic acids Recent work has shown that the delivenry of nucleic acid (DNA or RNA) capable of expressing an endothelial cell mitogen such as VEGF to the site of a blood vessel injury will induce proliferation and reendothelialization of the injured vessel. While the present invention does not relate to blood vessel injury, the techniques for the deliver." of nucleic acid encoding endothelial cell mitogens !uch as VEGF and PIGF used in these studies can also be employed in the present nvention. These techniques are described in U.S. Patent Nos. 5,830,879 and ;,25S,7S7 and are incorporated herein by reference. In the present invention the nucleic acid may be any nucleic acid (DNA or NA) including genomic DKA, cDNA. and mKSA, encoding VEGF or PIGF or ly VBGF or PIGF family members, The nucleic acid may also include any icleic acid which encodes a protein shown to bind to the sFlt-1 receptor. The icleic acids encoding the desired protein may be obtained using routine ocedures in the an, e.g. recombinant DNA, PCR amplification. lerapeutic nucleic acids that inhibit sFIt-1 expression The present invention also features the use of antisense nucleobase ^omers to downregulate expression of sFlt-1 mRNA directly. By binding to complementary nucleic acid sequence (the sense or coding strand), antisense leobase oligomers are able to inhibit protein expression presumably through ensyniatic cleavage of the RNA strand by RNAse H. Preferably the antisense leobase oligomer is capable of reducing sFlt-1 protein expression in a cell that ■esses excess levels of sFIt-1, Preferably the decrease in sFlt-1 protein ession is at least 10% relative to cells treated with a control oligonucleotide, : preferably 25%, and most preferably 50% or .greater. Method for;— example of the use of antisense nucleobase oligomers to downregulate VEGF expression seeU.S, Patent No. 6,4I0,3~, incorporated herein by reference. Methods for assaying levels of protein expression are also well known in the art and include western blotting, immunoprecipiution, and ELISA. The present invention also features the use of RNA interference (RNAi) to inhibit expression of sFlt-1. RNA interference (RNAi) is a recently discovered mechanism of post-transcriptional gene silencing (PTGS) in .which double-stranded RMA (dsRNA) corresponding to a gene or niRNA of interest is introduced into an organism resulting in the degradation of the corresponding mRNA. In the RNAi reaction, both the sense and anti-sense strands of a dsRNA molecule are processed into small RNA fragments or segments ranging in length from 21 to 23 nucleotides (nt) and having 2-nuc!eotide 3" tails. Alternatively, synthetic dsRNAs, which are 21 to 23 nl in length and have 2-nucleotide 3" tails, can be synthesized, purified and used in the reaction. These 21 to 23 nt dsRNAs are known as "guide RNAs" or "short interfering RNAs" (siRNAs). The siRNA duplexes then bind to a nuclease complex composed of proteins that target and destroy endogenous mRNAs having homology to the RNA within the complex. Although the identity of the proteins within the :omplex remains unclear., the function of the complex is to target the homologous nRNA molecule through base pairing interactions between one of the siRNA trands and the endogenous mRNA. The mRN"A is then cleaved approximately 2 nt from the 3" terminus of the siRNA and degraded. In this marmer, specific lnes can be targeted and degraded, thereby resulting in a toss of protein cpression from the targeted gene. The specific requirements and modifications of dsRNA are described in TT Pubhcation No. W00l/75164 (incorporated herein by reference). While RNA molecules can vary in length, it is most preferable to use siRNA .lecules which are 21- to 23- nucleotide dsRNAs with characteristic 2- to 3-rleotide 3" overhanging ends typically either (2 "-deaxy)thymidine,or. uracil. ; siRNAs typically comprise a 3" hydroxyl group. Single stranded siRNA as well as blunt ended forms of dsRNA can also be used. In order to further enhance the stability of the RNA, the 3" overhangs can be stabilized against degradation. In one such embodiment, the RNA is stabilized by including purine nucleotides, such as adenosine or guanosine. Alternatively, substitution of pryimidine nucleotides by modified analogs, e.g.,substutition of uridme 2-nucleotide overhangs by {2"-deQKy)chyTnide is tolerated and does not affect the efficiency of RNAi. Vest absence of a2" hydroxyl group significantly enhances the nuclease resistance of the overhang in tissue culture medium. Alternatively siRNA can be prepared using any of the methods set forth in PCX Publication No. WOO 1 "5164 (incorporated herein by reference) or usmg standard procedures for in vitro transcription of RNA and dsRNA annealing procedures as described in Elbashir et ai. (Genes & Dev., 15:lSS-200, 2001). siRNAs are also obtained as described in Elbashir et al. by incubation of dsRNA that corresponds to a sequence of the targe: gene in a cell-free Drosophila lysate from syncytial blastoderm Drosophila embryos under conditions in which the isRNA is processed lo generate siRNAs of about 31 to about 23 nucleotides, which are then isolated using techniques known to those of skill in the art. For example, gel electrophoresis can be used to separate"the 21-23 nt RNAs and the WAs can then be eluted from the gel slices,In addition, chromatography (e.g.. ize exclusion chromatography), glycerol gradient centrifugation, and affinity lurification with antibody can be used to isolate tlie 21 to 23 nt RNAs. In the present invention, the dsRNA. or siRNA, is complementary to the ■\RNA sequence of an sFU-1 mRNA and can reduce or inhibit expression of sFlt-. Preferably, the decrease m sFlt-1 protein expression is at least 10% relative lo ells treated with a control dsPNA or siRNA, more preferably 25%, and most referably at least 50%. Methods for assaying levels of protem expression are so well known in the art and include westem blotting, immunoprecipitation, and LISA. luveniion, ine nucleic acids used include any modification that enhances the stabihty or function of the nucleic acid in any way. Examples include modifications to the phosphate backbone, the intemucleotide linkage, or to the sugar moiety. To simplify the manipulation and handling of the nucleic acid encoding the sFlt-1 bmding protein, the nucleic acid is preferably inserted into a cassette where it is operably linked to a promoter. The promoter must be capable of driving expression of the sFlt-1 binding protein in the desired target host cell. The selection of appropriate promoters can readily be accomplished. Preferably, one would use a high expression promoter. A,n example of a suitable promoter is the 763-base-paiT cytoroegaloviru-us (CMV) promoter. The Rous sarcoma virus (RSV) (Davis, et al., Hum. Gene Tlxer. 4:151-159, 1993) and mouse mammary tumor virus (MMTV) promoters may also be used. Certain proteins can be expressed using their- native promoter. Other elements that can enhance expression can also be included (e.g., enhancers or a system that results in high levels of expression such as a tat gene and tar element"). The recombinant vector :an be a plasmid vector such as pUCl IS. pBR522, or other known plasmid "ectors, that includes, for example, an E. coli origin of replication (see, lambrook, et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor -aboratory press, 19S9). The plasmid vector may also include a selectable marker such as the p lactamase gene for ampicillin resistance, provided that the marker polypeptide does not adversely affect the metabolism of the organism eing ti-eated. Thecassette can also be bound to a nucleic acid binding moiety in synthetic delivery system, such as the system disclosed in POT Publication No. ^095/22618. The nucleic acid can be introduced into the cells by any means appropriate r the vector employed. Many such methods are well known in the art ambrook et al., supra, and Watson et al., "Recombinant DNA", Chapter 12, 2d ition. Scientific American Books, 1993). Recombinant vectors can be usfetred by methods such as calcium phosphate precipitation, electroporation, osome-mediated transfection, gene gun,micro injection, viral caps id-mediated transfer, polybrene-medialed transfer, or protoplast fusion. For a review ot the procedures for liposome preparation, targeting and delivery of contents, see Mannino and Gouid-Fogerite, (Bio Techniques, 6:682-690, 1988), Feigner and Holni, {Bethesda Res. lab. Focus. 11:2 L 19S9) and Maurer (Betliesda Res. Lab. Focus. 11:25.1989). Transfer of the recombinant vector (either plasmid vector or viral vectors) can be accomplished through direct injection into the amniotic fluid or intravenous delivery. Gene delivery using adenoviral vectors or adeno-associated vectors (AAV) can also be used. Adenoviruses are present in a large number of animal species, are not very pathogenic, and can replicate equally well in dividing and quiescent cells. As a general rule, adenoviruses used for gene delivery are lacking one or more genes required for viral replication. Replication-defective recombinant adenoviral vectors used for the dehvery of VEGF, PIGF or any sFlt-1 bmding protein, can be produced in accordance with art-known techniques (see Quantin et ai., Proc. Nail. Acad. Sci. US.-i. S9:25S 1-25S4, 1992; So-atford-Perricadet et al, / Clm. Invest., 90:626-630, 1992; and Rosenfeld et al.. Ceil, 68:143-155, 1992). For an example of the use of gene therapy in uiero see U.S. Patent No. 6,399,585. A variety of methods are available for transfection, or introduction, of dsRNA or oligonucleotides into mammalian cells. For example, there are several commercially available transfection reagents including but not limited to; TransIT-TKQTM (Minis, Cat. = MIR 2150), Transmessengerf"" (Qiagen, Cat. # 301525), and Oligofeciamine™ (Invitrogen, Cat. # MIR 12252-011). Protocols for each transfection reagent are available from the manufacturer. Once transferred, the nucleic acid is expressed by tire cells at the site of injury for a period of time sufficient to mcrease blood serum levels of VEGF, PIGF, or any other sFIt-I binding protein. Because the vectors containing the nuckic acid are not normally incorporated into the genome of the cells, expression of the protein of interest takes place for only a limited time. Typically, the protein is expressed at therapeuic"leve!s for about two days to several weeks, preferably for about one to two weeks. Re-application of the ui\j\ can oe utilized to provide additional periods of expression of the therapeutic protein. Recent examples of gene therapy using VEGF for the treatment of vascular disease in mammals can be found in Deodato et al. (Gene Tlier., 9:111-785,2002); Isner et al. {Human Gem Tlier.. 12:1593-1594,2001); Lai et al. (Gene Ther., 9:804-813, 2002); and reviewed in Freedman and Isner {Ann. Intern. Med.. 136:54-71,2002) and Isner JM {Namra. 415:234-239,2002). Assays for gene and protein expression The following methods can be used to evaluate protein or gene expression and determine efficacy for any of the above-mentioned methods for increasing VEGF, PIGF or any other sFlt-1 binding protem levels, or for decreasing sFlt-1 protein levels. Blood serum from the subject is measured for levels of VEGF, PIGF, or any protein ligand known to bind to sFlt-I. Methods used to measure serum levels of proteins include ELISA, western blotting, or immunoassays using specific antibodies. In addition, in vitro angiogenesis assays can be perfonned to ietermine if the subject"s blood has converted from an anti-angiogenic state to a iro-angiogenic state. Such assays are described above in Example 2. A positive esult is considered an increase of at least 20%, preferably 30%, more preferably t least 50%, and most preferably at least 60% in the scrum levels of VEGF, IGF, or any protein ligand known to bind to sFlt-1. A positive result can also be Dnsidered conversion from an anti-angiogenic state to a pro-angiogenic state 5ing the in vitro angiogenesis assay. There are several art-known methods to assay for gene expression. Some lamples include the preparation of RNA from the blood samples of the subject id the use of the RNA for northern blotting, PCR based amplification, or RNAse oteclion assays. ;e of antibodies for therapeutic treatment The elevated levels of sFlt-i found in the serum samples taken from ;gnanitwomen suffering from pre-eclampsia suggests that sFlt-1 is acting as a "physiologic sink" to bind to and deplete the trophoblast cells and maternal endothelial cells of funcrional VEGF and PIGF. The use of compounds, such as antibodies, to bind to sFlt-1 and block VEGF or PIGF binding, may help prevent or treat pre-eclampsia or eclampsia, by producing an increase in free VEGF or PIGF. Such an mcrease would allow for an increase in trophoblast proliferation, migration and angiogenesis required for placenual development and feta! nourishment, and for systemic maternal endothelial cell health. The present invention provides antibodies that bind specifically to the ligand-bindingdomainof sFU-1. The antibodies are used to inlubit Flt-1 and the most effective mechanism is believed to be through direct blocking of the binding sites for VEGF or PIGF. however, other mechanisms cannot be ruled out. Methods for the preparation and use of antibodies for therapeutic purposes are described in several patents including U.S. Patent Numbers 6,054,297; 5,821,337; 6,365,157; and 6,165,464 and are incorporated herein by reference. Antibodies :an be polyclonal or monoclonal; monoclonal antibodies are preferred. Monoclonal antibodies, paniculariy those derived from rodents including nice, have been used for the treatment of various diseases; however, there are imitations to tlieir use including the Induction of a human anti-mouse mmunoglobulin response that causes rapid clearance and a reduction in the ■.fficacy of the treatment. For example, a major limitation in the clinical use of odent monoclonal antibodies is an anti-globulm response during therapy (Miller ;t a!., Blood, 62:988-995 1953; Schroff et al., Cancer Res.. 45:579-885, 1985). The art has attempted to overcome this problem by constructing chimeric" antibodies in which an animal antigen-binding variable domain is oupled to a human constant domain (U.S. Pat. No. 4,816,567; Morrison et al., •roc. Natl. Acad. Sci. USA, 51:6551-6555, 19S4; Boulianne etal., Nature. 12:643-646, 1984;Neuberger et al.,Nature. 314:268-370, 1985). The roduction and use of such chimenc antibodies are described below. Competitive inhibition of ligand binding to sFlt-1 is useful for the revention or treatment of pre-eclampsia or eclampsia. Antibodies directed to -It-l can block binding of VEGF or PIGF to sFlt-I resulting in increased levels ofVEGForPlGF. Such an increase can result in a rescue of endothelial dysfunction and a shiit in the balance of pro-angiogenie /anti-angiogenic factors towards angiogenesis. A coclctail of the monoclonal antibodies of the present invention can be used as an effective treatment for pre-eclampsia or eclampsia. The cocktail may include as few as two, three, or four different antibodies or as many as six, eight or ten different antibodies. In addition, the antibodies of the present invention can be combined with an anti-hypertensive drug (e.g., methyldopa, hydralazine hydrochloride, or labetalol or any other medication used to treat pre-eclampsia, eclampsia, or the symptoms associated with pre-eclampsia or eclampsia. Preparation of Antibodies Monoclonal antibodies that specifically bind to the sFlt-1 receptor may be produced by methods known in the art. These methods include the immunological method described by Kohler and Milstem (Nature. 256: 495-497, 1975) and Campbell ("Monoclonal Antibody Technology, The Production and Characterization of Rodent and Human Hybndomas" in Burdon et al., Eds., Laboratory Tecimiques in Biochemistry and Molecular Biology, Volume 13, Elsevier Science Publishers, .Amsterdam, 19S5), as well as by tlie recombinant DNA method described by Huse et al, {ScieJice, 246, 1275-12S1, 19S9). Monoclonal antibodies may be prepared from supematants of cultured hybridoma cells or from ascites induced by intra-peritoneal inoculation of hybridoma cells into mice. The hybridoma technique described originally by Kohler and Milstein (Eur J. Immunol, 6, 511-519,1976) has been widely apphed -.0 produce hybrid cell lines that secrete high levels of monoclonal antibodies igainst many specific antigens. The route and schedule of immunization of the host animal or cultured intibody-producing cells therefrom are generally in keeping with established and :onventional techniques for andbody stimulation and production. Typically, nice are used as the test model, however, any mammalian subject including - ■ uman subjects or antibody producing cells therefrom can be manipulated according to the processes of this invention to serve as the basis for production of mammalian, including human, hybrid cell lines. After immunization, immune lymphoid cells are fused with myeloma cells to generate a hybrid ceil line that can be cultivated and subcultivated indefinitely, to produce large quantities of monoclonal antibodies. For purposes of this invention, the immufle lymphoid ceils selected for fiision are lymphocytes and their normal differentiated progeny, taken either from lymph node tissue or spleen tissue from immunized animals. The use of spleen cells is preferred, since they offer a more concentrated and convenient source of antibody producing cells with respect to the mouse system. The myeloma cells provide the basis for continuous propagation of the fused hybnd. Myeloma cells are tumor cells derived from plasma cells. Mlurine myeloma cell lines can be obtained, for example, from the American Type Culture Collection (ATCC; Manassas, VA). Human myeloma and mouse-human heteromyeloma cell lines have also been described (Kozbor et a!., J. Immunol.. 133:3001-3005, 19S4; Brodeur et al, Monoclonal Antibody Production Tecliniques and Applications, Marcel Dekker, Inc., New York, pp. 51-63, 19S7). This hybrid cell lines can be maintained in viiro in cell culture media. Once the hybridon:ia cell line is established, it can be maintained on a variety of nutritionally adequate media such as hypoxanthine-aminoptenn-thymidine (HAT) medium. Moreover, the hybrid cell lines can be stored and preserved in any number of conventional ways, including freezing and storage under liquid nitrogen. Frozen cell lines can be revived and cultured indefinitely with resumed syntliesis and secretion of monoclonal antibody. The secreted antibody is recovered from tissue culture supernatant by conventional methods such as precipitation, ion exchange chromotography, affinity chromatography, or the like. The antibody may be prepared in an;" mammal, including mice, rats, rabbits, goats, and humans. The antibody may be a member of one of the following immunoglobulin classes: IgG, IgM, Ig.A, IgD, or IgE, and the subclasses thereof, and preferably is an IgG antibody. wnue me preferred animal for producing monoclonal antibodies is mouse, the invention is not so limited; in fact, human antibodies may be used and may prove to be preferable. Such antibodies can be obtained by using human hybridomas (Cole et al., "Monoclonal Antibodies and Cancer Therapy", Alan R. Liss Inc., p. 77-96, 19S5). In the present invention, techniques developed for the production of chimeric antibodies by splicing the genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule can be used (Morrison et al., Proc. Noul. Acad. Sci. S1,6S51-6S55, 19S4;Neuberger et al.,Natw-s3l2, 604-608, 1984; Takeda et al.,Naiwe 314, 452-454, 19S5); such antibodies are within the scope of this invention and are described below. As another alternative to the cell fusion technique, Epstien-Bart virus (EBV) immortalized B cells are used to produce the monoclonal antibodies of the present invention (Crawford D. etal.,7: of Gen. Virol. 64:697-700, 1983;Ko2bor and Roder, J. Immunol., 4:1275-1280, 19S1; Kozbor et al., Methods in Enzymology, 121:120-140, 1986). In general, the procedure consists of isolating Epstein-Bart virus from a suitable source, generally an infected cell time and exposing the target antibody secreimg cells to supematants containing the virus. The cells are washed, and cultured in an appropriate cell culture medium. Subsequently, virally transformed cells present in the cell culmre can be identified by the presence of the Epstein-Bair viral nuclear antigen, and transformed antibody secreting cells can be identified using standard methods known in the art. Other methods for producing monoclonal antibodies, such as recombinant DNA, are also included within the scope of the invention. Preparation ofsFlt-l Immimogens sFlt-1 may be used by itself as an immunogen, or may be attached to a ;arrier protein or to other objects, such as sepharose beads, sFlt-i may be injurified from cells known, to express the endogenous protein such, as human imbiiical vein endothehal cells (HUVEC; Kendall et al., Biochem. Biophys. Rss. Zomvi; 226:324-325,1996). AddirJooally, nucleic acid molecules that encode sFIt-l, or portions thereof, can be inserted into known vectcrffor expression i host cells using standard recombinant DNA techniques. Suitable host cells for sFIT-1 expression include baculovirus cells (e.g., SB cells), bacterial cells (e.g., E. coli), and mammalian cells (e.g., \IK3T3 cells). In addidon. peptides can be synthesized and used as immunogens. The methods for making anribody to pepndes are well known in the art and generally require coupling the peptide to a suitable carrier molecule, such as serum albumin. Peptides include any ammo acid sequence that is substantially identical to any part of the sFlt-1 amino acid sequence corresponding to GenBank accession number U0n34. Peptides can be any length, preferably 10 amino acids or greater, more preferably 25 amino acids or greater, and most preferably 40, 50, 60, 70, 80, or 100 amino acids or greater. Preferably, the amino acid sequences are at least 60%, more preferably 85%, and, most preferably 95% identical to the sequence of U0U34. Tlie peptides can be commercially obtained or made using techniques well known in the art, such as. for example, the Merrifield solid-phase method (Science, 232:341-347, 19S5), The procedure may use commercially available sytithesizers such as a Biosearth 9500 automated peptide machine, with cleavage of the blocked amino acids being achieved with hydrogen fluoride, and the peptides purified by preparative EIPLC using a Waters Delta Prep 3000 instrument on a 15-20 μm Vydac C4 PrepPAK column. Functional equivalents of antibodies Tlie invention also includes functional equivalents of the antibodies described in this specification. functional equivalents include polypeptides with amino acid sequences substantially identical to the amino acid sequence of the variable or hypervariable regions of the antibodies of the invention. Functional equivalents have binding charactenstics comparable to those of the antibodies, and include, for example, chimerizsd. humanized and single chain antibodies as well as fragments thereof Method; of producmg such functional equivalents are disclosed ui PCT Publication No. V."093/21319; European Patent Application No, 239,400; PCT Publication No. WO.S9/09622; European Patent Application No. 338,745; European Patent Application No. 332424; and U.S. Patent No. 4,816,567; each of which is herein incorporated by reference. Chimerized anlfDodies preferably have constant regions derived substantially or exclusively from human antibody constant regions and variable regions derived substantially or exclusively from the sequence of the variable region from a mammal other than a human. Such humanized antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab", F{ab")2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Methods for humanizing non-human antibodies are well known in the art (for reviews see Vasv/axii and EamiltoiL Ann Allergy Asthma Immunol.. 81:105-119, 1998 and Cartsr,NalureReT,"iews Cancer, 1:118-129, 2001). Generally, ahumanized antibody has one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as import residues, which are typically taken from an import variable domain. Humanization can be essentially performed following the methods known in the art (Jones et al.. Nature, 321 ;522-525, 1986; Riechmann et al.. Nature. 332:323-329, 1988; and Verhoeyen et al, Science, 239:1534-1536 198S). by substituting " rodent CDRs or other CDR sequences for the corresponding sequences of a human antibody. Accordingly, such humanized antibodies are chimeric antibodies wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species (see For example, U.S. Patent No. 4,816,567). In practice, humanized antibodies are ypically human antibodies in which some CDR residues and possibly some FR ■esidues are substituted by residues from analogous sites in rodent antibodies Presta, Cun: Op. Struct. Biol. 2:593-596,1992). Additional methods for the preparation of humanized antibodies can be bund inU.S. PatentNos. 5,821,337, and 6,054,297, and Carter, {supra) which re all incorporated herein hy reference. The humanized antibody is selected -om any class of immunogiobulins.-including IgM, IgG, IgD, IgA and IgE, and ny isotype, including IgG,, IgGi, IgGj, and IgGa. Where cytotoxic activity is not needed, such as in the present invention, the constant domain is preferably of the IgGa class. The humanized antibody may comprise sequences from more than one class or isotype, and selecting particular constant domains to optimize desired effector functions is within the ordinarv" skill in the art. Human antibodies can also be produced using various techniques known in the art, including phage display libraries (Marks cta.l,J. MoL Biol.. 222:581-597, 1991 and Winter et ai. Aiinu. Rev. ImimwoL, 12:433-455, 1994). The tecliniques of Cole et al. andBoemer et a.1. are also useful for the preparation of human monoclonal antibodies (Cole et al., supra; Boemer et al., J. Immunol.. 147: 86-95, 1991). Suitable mammals other than a human include any mammal from which monoclonal antibodies may be made. Examples of mammals other than a human include, for example a rabbit rat, mouse, horse, goat, or primate; a mouse is preferred. Functional equivalents of antibodies also include single-chain antibody fragments, also known as single-chain antibodies (scFvs). Single-chain antibody fragments are recombinant polypeptides which typically bind antigens or receptors; these fragments contain at least one fragment of an antibody variable heavy-chain amino acid sequence (VH) tethered to at least one fragment of an antibody variable lighr-chain sequence (VL) with or without one or more interconnecting linkers. Such a linker may be a short, flexible peptide selected to assure that the proper three-dimensional folding of the VL and VH domains occurs once they are linked so as to maintain the target molecule bindmg-specificity of the whole antibody from which the single-chain antibody fragment is derived. Generally, the carboxyl teminus of the VL or VH sequence is covalently linked by such a peptide linker to the amino acid temiinus of a complementary VL and Vg sequence. Single-chain antibody fragments can be generated by molecular cloning, antibody phage display library or similar techniques. These proteins can be produced either in eukaryotic cells or prokaryotic ceils, including bacteria. Single-chain andbody fragments contain amino acid sequences havmg at least one of the variable regions or CDRs of the whole antibodies described in this specification, but are lacking some or all of the constant domains of those antibodies. These constant domains are not necessary for antigen binding, but constitute a major portion of the structure of whole antibodies. Single-chain antibody fragments may therefore overcome some of the problems associated with the use of antibodies containing part or all of a constant domain. For example, single-chain antibody fragments tend to be free of undesired interactions between biological molecules and the heavy-chain constant region, or other unwanted biological activity. Additionally, single-chain antibody fragments are considerably smaller than whole antibodies and may therefore have greater capillary permeability than whole antibodies, allowing smgle-chain antibody fragments to localize and bind to target antigen-binding sites more efficiently. Also, antibody fragnients can be produced on a relatively large scale in prokaryotic cells, thus facilitating their production. Furtheraiore, the relatively small size of single-chain antibody fragments makes them less likely than whole antibodies to provoke an immune response in a recipient. Functional equivalents further include fragments of antibodies that have he same or comparable binding characteristics to those of the whole antibody. Such fragments may contain one or body Fab fragments or die F{ab")2 fragment. Preferably the antibody fragments contain all six CDRs of the whole antibody, Ithough fragments containing fewer than all of such regions, such as diree, four T five CDRs, are also functional. Further, the functional equivalents may be or may combine members of ly one of the following immunoglobulin classes; IgG, IgM, IgA, IgD, or IgE, id the subclasses thereof Preparation of Functional Equivalents of Antibodies Equivalents of antibodies are prepared by methods known in the art. For ample, fragments of antibodies may be prepared enzymatically from whole tibodies. Preferably, equivalents of antibodies are prepared from DNA encoding such equivalems. DNA encoding fragments of antibod.may be-prepared by deleting all but the desired portion of die DNA that encodes the full-length antibody. DNA encoding chimerized antibodies may be prepared by recombining DNA substantially or exclusively encoding human constant regions and DNA encoding variable regions derived substantially or exclusivtiy from the sequence of the variable region of a mammal other than a human. DNA encoding humanized antibodies may be prepared by recombining DNA encoding constant regions and variable regions other than the CDRs derived substantially or exclusively from the corresponding human antibody regions and DNA encoding CDRs derived substantially or exclusively from a mammal odier than a human. Suitable sources of DNA molecules that encode fragments of antibodies include cells, such as hybridomas, that express the full-length antibody. The fragments may be used by themselves as antibody equivalents, or may be recombined into equivalents, as described above. The DNA deletions and recombinations described in this section may be carried out by known methods, such as those described in the published patent applications listed above. Antibody Screening and Selection Monoclonal antibodies are isolated and purified using standard art-known methods. For example, antibodies can be screened using standard art-known metliods such as ELISA against the sFlt-1 peptide antigen or western blot analysis. Examples of such techniques are described in Examples II and III of U.S. Patent No. 6,365,157, herein incorporated by reference. T/ierapeutic Uses of Amibodies When used in vivo for the treatment or prevention of pre-eclampsia or eclampsia, the antibodies of the subject invenlion are administered to the subject in therapeutically effective amounts- Preferably, the antibodies are administered larenterally or intravenously by continuous infrision.. The dose and dosage regimen depends upon the severity of the disease, and the overall health of the subject. The amount of antibody administered is typically in the range of about 0.01 to about 10 mg/kg of subject weight For parenteral adrainistration, the antibodies are formulated in a unit dosage injectable form (solution, suspension, emulsion) in association with a pharmaceutically acceptable parenteral vehicle. Such vehicles are inherently nontoxic, and non-therapeutic. Examples of such vehicles are water, saline, Ringer"s solution, dextrose solution, and 5% human serum albumin. Nonaqueous vehicles such as fixed oils and ethyl oleate may also be used. Liposomes may be used as carriers. The vehicle may contain minor amounts of additives such as substances that enhance isotoniciry- and chemical stability, e.g., buffers and preservatives. The antibodies typically are formulated in such vehicles at concentrations of about 1 mg/nij to 10 mg/ml. Therapeutic compounds that inhibit sFlt-1 Given that levels of sFlt-1 are increased in subjects having pre-eclampsia, eclampsia, or having a propensity to develop such conditions, any agent that decreases the expression of an sFIt-1 poTypeptide or nucleic acid molecule is useful in the methods of the invention. Such agents include small molecules that can disrupt sFlt-1 binding to VEGF or PIGF, antisense nucleobase oligomers, and dsRNAs used to mediate RNA interference. Combination therapies Optionally, a pre-eclampsia or eclampsia therapeutic may be administered in combination with any other standard pre-eclampsia or eclampsia therapy; such methods are known to the skilled artisan and described herein. A pre-eclampsia or eclampsia therapeutic of the invention may be administered in combination with any compound that increases the activity of a VEGF pathway. Non-limiting examples of agents which also induce endoeenous VEGF production include nicotine, Minoxidil, Ntfidepine, Adenosine, Magnesium sulfate, and theophyllme. in one embodiment, PIGF protein can be used in combination with any of the agents which induce endogenous VEGF produciion listed above. Subject monitoring The disease store or trearment of a subjecr having pre-eclampsia, eclampsia, or apropensity to develop such a condition can be monitored using the methods and compositions of the invention. In one embodiment, the expression of an sFlt-1, VEGF, or PIGF polypeptide present in a bodily fluid, such as urine, plasma, amniotic fluid, or CSF, is monitored. Such monitoring may be useful for example, in assessing the efficacy of a particular drug in a subject or in assessing disease progression. Therapeutics that decrease the expression of an sFh-1 nucleic acid molecule orpolypeptide or that increase the expression of a VEGF or PIGF nucleic acid molecule or polypeptide are taken as particularly useftil in the invention. Other Embodiments From the foregoing description. it is apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and condtions. Such embodiments are also within the scope of the following claims. Ml publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent publication or patent appUcation was specifically and individually indicated to be incorporated by reference. In addition. U.S. Provisional AppUcation Kos. 60/451796, filed March 3, 2003, 60/397,481, filed July 19, 2002, and 60/467,390 filed iMay 2. 2003, are hereby incorporated fay reference in their entirery what is claimed is: WE CLAIM: 1. A method of identifying a compound that ameliorates pre-eclampsia or eclampsia, said method comprising contacting a cell that expresses a sFlt-1, VEGF, or PIGF nucleic acid molecule with a candidate compound, and comparing the level of expression of said nucleic acid molecule in said cell contacted by said candidate compound with the level of expression in a control cell not contacted by said candidate compound, wherein an alteration in expression of said sFlt-l, VEGF, or PIGF nucleic acid molecule identifies said candidate compound as a compound that ameliorates pre-eclampsia or eclampsia. 2. The method as claimed in claim 1, wherein said alteration is a decrease in the level of sFlt-1. 3. The method as claimed in claim 1, wherein said alteration is an increase in the level ofVEGF or PIGF. 4. The method as claimed in claim 1, wherein said alteration in expression is an alteration in transcription. 5. The method as claimed in claim 1, wherein said alteration in expression is an alteration in translation. 6. A method of identifying a compound that ameliorates pre-eclampsia or eclampsia, said method comprising contacting a cell that expresses an sFlt-1, VEGF, or PIGF polypeptide with a candidate compound, and comparing the level of expression of said polypeptide in said cell contacted by said candidate compound with the level of polypeptide expression in a control cell not contacted by said candidate compound, wherein an alteration in the expression of said sFIt- 1, VEGF, or PIGF polypeptide identifies said candidate compoimd as a compound that ameliorates preeclampsia or eclampsia. 7. The method as claimed in claim 6, wherein said alteration in expression is assayed using an immunological assay, an enzymatic assay, or a immunoassay. 8. The method as claimed in claim 6, wherein said alteration in expression is a decrease in the level of sFlt-1. 9. The method as claimed in claim 6, wherem said alteration in expression is an increase in the level of VEGF or P1GF. 10. A method of identifying a compound that ameliorates pre-eclampsia or eclampsia, the method comprising contacting a cell that expresses an sFlt-1, VEGF, or PIGF polypeptide with a candidate compound, and comparing the biological activity of said polypeptide in said cell contacted by said candidate compound with the level of biological activity in a control cell not contacted by said candidate compound, wherein an alteration in the biological activity of said sFlt-1, VEGF, or PIGF polypeptide identifies said candidate compound as a compound that ameliorates preeclampsia or eclampsia. 11. The method as claimed in claim 10, wherein said alteration in biological activity is a decrease in the activity of sFlt-1. 12. The method as claimed in claim 10, wherein said alteration in biological activity is an increase in the activity of VEGF or PIGF. 13. The method as claimed in claim 10, wherein said alteration in biological activity is assayed using an immunological assay, an enzymatic assay, or an immunoassay. !4. A method of identifying a compound that ameliorates pre-eclampsia or eclampsia, said method comprising detecting binding between an sFIt-1 polypeptide and a growth factor in the presence of a candidate compound, wherein a decrease in said binding, relative to binding between said sFlt-1 polypeptide and said growth factor in the absence of said candidate compound identifies said candidate compound as a compound that ameliorates pre-eclampsia or eclampsia. 15. A method of identifying a polypeptide, or fragment thereof, that prevents binding between an sFlt-1 polypeptide and a growth factor, said method comprising detecting binding between an sFlt-1 polypeptide and a growth factor in the presence of said candidate polypeptide, wherein a decrease in said binding, relative to binding between said sFlt-1 polypeptide and said growth factor in the absence of said candidate polypeptide identifies said candidate polypeptide as a polypeptide that prevents binding between an sFlt-l polypeptide and a growth factor. 16. The method as claimed in claim 14 or 15, wherein said growth factor is VEGF. 17. The method as claimed in claim 14 or 15, wherein said growth factor is PIGF. 18. A method of identifying a compound that ameliorates pre-eclampsia or eclampsia, comprising detecting binding between an sFlt-1 polypeptide and a candidate compound, wherein a compound that binds said sFlt-1 polypeptide ameliorates pre-eclampsia or eclampsia. 19. A compound identified according to the method of claim 18, wherein said compound comprises a polypeptide that specifically binds an sFlt-1 polypeptide and prevents said sFlt-1 polypeptide from binding VEGF or PIGF. 20. The compound as claimed in claim 19, wherein said polypeptide is an antibody. 21. The compound as claimed in claim 19, wherein said polypeptide is a fragment ofsFlt-l,VEGF, orPlGF. 22. A pharmaceutical composition for treating or preventing pre-eclampsia or eclampsia comprising (a) a compound that increases the biological activity or level of unbound forms growth factors such as VEGF and PIGF capable of binding to the soluble Flt-1 receptor (sFlt-1) and (b) an known anti-hypertensive compound, formulated in a pharmaceutically acceptable carrier. 23. The pharmaceutical composition as claimed in claim 22, wherein the compound (a) is a VEGF or PIGF polypeptide, or portion thereof, formulated in a pharmaceutically acceptable carrier suitable for administration to a pregnant mammal. 24. The pharmaceutical composition as claimed in claim 22, wherein the compound (a) is VEGF or PIGF nucleic acid molecule, or portion thereof, formulated in a pharmaceutically acceptable carrier suitable for administration to a pregnant mammal. 25. The pharmaceutical composition as claimed in claim 22, wherein the antihypertensive compound of (b) is selected from the group consisting of adenosine, nifedipine, minoxidil, and magnesium sulfate. 26. The composition as claimed in claim 22 wherein the compound (a) is purified antibody or antigen-binding fragment thereof that specifically binds sFlt-1. 27. The composition as claimed in claim 26, wherein said antibody or antigen binding fragment prevents binding of a growth factor to sFlt-1. 28. The composition as claimed in claim 26, wherein said antibody is a monoclonal antibody. 29. The composition as claimed in claim 26, wherein said antibody or antigen binding fragment thereof is a human or humanized antibody. 30. The composition as claimed in claim 26, wherein said antibody lacks an Fc portion. 31. The composition as claimed in claim 26, wherein said antibody is an F(ab") 2, an Fab, or an Fv structure. 32. The composition as claimed in claim 26, wherein said antibody or antigen binding fragment thereof is present in a pharmaceutical ly acceptable carrier. 33. The composition as claimed in claim 22 wherein the compound (a) is an antisense nuleobase oligomer complementary to atleast a portion of an sFlt-1 nucleic acid sequence. 34. The composition as claimed in claim 33 wherein said antisense nucleobase oligomer is 8 to 30 nucleotides in length. 35. The composition as claimed in claim 22 wherein the compound (a) is a double stranded RNA that comprises at least a portion of an sFlt-1 nucleic acid sequence. 36. The composition as claimed in claim 35 wherein said double stranded RNA is processed into small interfering RNAs (siRNAs) 19 to 25 nucleotides in length. 37. The composition as claimed in claim 22 wherein the compound (a) is selected irom the group consisting of nicotine, theophylline, adenosine, nifedipine and minoxidil. |
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218-chenp-2005 abstract-duplicate.pdf
218-chenp-2005 claims-duplicate.pdf
218-chenp-2005 correspondence-others.pdf
218-chenp-2005 correspondence-po.pdf
218-chenp-2005 description (complete).pdf
218-chenp-2005 drawings-duplicate.pdf
218-chenp-2005 pct search report.pdf
218-chenp-2005 description (complete)-duplicate.pdf
Patent Number | 218780 | ||||||||||||||||
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Indian Patent Application Number | 218/CHENP/2005 | ||||||||||||||||
PG Journal Number | 21/2008 | ||||||||||||||||
Publication Date | 23-May-2008 | ||||||||||||||||
Grant Date | 16-Apr-2008 | ||||||||||||||||
Date of Filing | 18-Feb-2005 | ||||||||||||||||
Name of Patentee | BETH ISRAEL DEACONESS MEDICAL CENTER | ||||||||||||||||
Applicant Address | 330 Brookline Avenue, Boston, MA 02215, | ||||||||||||||||
Inventors:
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PCT International Classification Number | A61K 31/522 | ||||||||||||||||
PCT International Application Number | PCT/US03/22892 | ||||||||||||||||
PCT International Filing date | 2003-07-21 | ||||||||||||||||
PCT Conventions:
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