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2. Angiogenic Markers In Pre Eclampsia Yukti 2012 3. Angiogenic Factors Endothelium+ VEGF + PIGF - sFlt1 4. Angiogenic Markers In Pre eclampsiaOut Line Of Presentation Endothelium+ VEGF + PIGF - sFlt1 Preeclampsia Disease of theories Need of the hour? Pathophysiology Role Of Placenta Angiogenic MarkersClinical significance Possibility of Prediction of PE in normal & High Risk.Pregnancies Gestational proteinuria, chronic glomerulonepritis with .super imposed preeclampsia. Endothelial cell Modulators The Future ? 5. Angiogenic Markers In Pre Eclampsia Introduction Preeclampsia is observed in 5-7% of pregnancies Etiology remains elusive Major cause of maternal & fetal morbidity & mortality world wide.* 50-75000 maternal deaths /year world wise . Confidential enquiry in to maternal deaths (UK) revealed PE eclampsia as second common cause of MM* Manifests clinically after 20 weeks of gestation. Phenotype varies from mild to severe as seizures,stroke,liver failure or rupture, HELLP,renal failure acute tubular necrosis death Fetal Prematurity ,low birth weight PND, NND. *WHO2004,2005*SLJOG2009;31:48-52 6. EndotheliumIntroduction+ VEGF + PIGFPE Future Risk- sFlt1 Recurrence with complications & Chronic hypertension Maternal endothelial dysfunction may persist years after theepisode Cardiovascular events. The risk factors of PE are similar as for underlying vasculardisease asinsulin resistantancehyperlipidemia,hypertention,thrombophilias & obesity. *Women with PE are at > risk for cardiac problems *Best Pract Res 2003;17(3):441-58,Lancet 2001;357(9273):2002 7. EndotheliumPathogenesis of PE + VEGF + PIGF Thought to involve 3 steps.- sFlt1 Defective placentation Placental ischemia Endothelial cell dysfunctiona key factor Placental dysarrangement may be reflected in the maternal circulation by alteration in the concentration of biological markers VEGF, PlGF ( pro angiogenic) & important regulators in human placenta 8. Endothelium + VEGF + PIGFPreeclampsia Basic Pathology- sFlt1 *Defective or abnormal cytotrophoblastinvasion of spiral arterioles >> Reduced uteroplacental perfusion . * Ischemic placenta secretes anti angiogenicsoluble factors into maternalvasculature, leading to endothelial damage.*Hypothesis Roberts et al *Maynard et al J .clin inves 2003 9. Placenta Extravillus invasive cyto trophoblast migrate to & invade theuterine spiral artereries & decidual arterioles . These initially express adhesion molecules characteristic ofepithelial cells as:integrine alpha6/beta1,alpha5/beta5,Ecadherin. As invasion proceeds instead of epithelial expression, endothelial cell adhesion molecule expressionoccurs.Integrine alpha 1/beta1,alpha 5 beta 3,vE cadherin isobserved.Pseudovascularisation. This results in replacement of endothelial layer of thesevessels and subsequent formation of high capacitance ,lowresistant system -- nutrition etc NORMAL. In PE pregnancies invasive cytotrophoblast fail to completelyinvade decidual arterioles, fail to show above vascularmimicry=placental hypoxia , & liberation of antiangiogenic factors. 10. Preeclampsia Disease of Theories Toxins Malnutrition Vitamin Deficiency Stress Inflammation Immunological Prostaglandin imbalance Angiogenic factors 11. PossibleMechanisms inPreeclampsia Friedman and Lindheimer,1999 12. Physiology ofVasculogenesis & Angiogenesis As the embryo develops ,mesodermal precursorsdifferentiate in to endothelial cells & assemble intoprimitive vascular network vasculogenesis. These net works under go extensive budding &branching ,associate with vascular smooth muscleelements termed Angiogenesis. This vasculature is capable of responding to systemic,local tissue needs ,nutrients ,o2,cellular product export ,hormones,vasoconstrictive & metabolic waste products And development of fetus. 13. Angiogenesis VEGF Angiogenisis is controlled by positive and negative influences. VEGF (VPF) comprised of six different proteins encoded bydistinct genes ,=VEGF ABC,or VEGF- E & PlGF These act by Stimulation of endothelial angiogenesis ,endothelial survival by tkT path way ,controls vascular permeability, stimulates expressionof tissue plasminogen activators,urokinase ,collagenase . Physiological regulators for erythropoiesis & synthesis oferythropoietin. VEGF- an attractive target for pro & anti angiogenic gene therapybecause of its association with normal & abnormal angiogenesis Lack of 1 or 2 allele in embryo leads to defective angiogenesis anomaly 14. Endothelium + VEGF + PIGF Pro angiogenic Factor- sFlt1 PlGF a member of VEGF made predominantly in placentaalso binds to VEGFR-1 *VEGFR-1 has 2 isomers:Trans membrane isoform &soluble isoform.(sFlt-1) *VEGF & PlGF which are pro angiogenic, reduce vasculartone thus blood pressure. *N Eng J Med 2004;350:672,683*J Clin inves 2003;1111:6491, AJ Path2002;160:1405, ObGyn2000;95:3 15. Anti Angiogenic FactorsFlt-1 sFlk-1 Soluble fm like tyrosine kinase -1(sFlt-1)also known assVEGFR-1,is a naturally occurring circulating antagonist Soluble form of new receptor for VEGF family is identifiedas sFlk-1 or VEGFR-2 Its action is mediated by inter action with high affinityreceptors as tyrosine kinase,VEGFR-1,Flt-1VEGFR-2,kinasedomain region (KDR)Flk-1 which are selectively expressedon vascular endothelial cell surface and targeted tissueslike kidney* *Mol Cancer Rea 2004;2:315-26 16. Endothelium + VEGF + PIGF Anti Angiogenic Factor sFlt -1 - sFlt1 sFlt-1 acts, by binding to placental growth factor & VEGF,preventing the interaction with endothelial receptors on the cellsurface & inducing endothelial dysfunction. Among the several isomers of sFlt-1 14 is expressed in humans &primates It differs by lacking the C-terminal 31 amino acids andcontaining intron 14 coded unique 28 amino acids sFlt-14 is identified as the primary isoform produced by placentain PE Placental *syncytial knots identified as the major source for sFlt-* induced by hypoxia Exogenous administration of sFlt-1 in pregnant rats induces HTN, proteinuria& glomerular endotheliosis. 17. sFlt-1 *Lowered oxygen tension in primary cytotrophoblastculture & villus explants causes increased sFltexpression, may be stimulated by hypoxia. *Other path ways inducing sFlt probably are Deficient hemooxygenase expression, placentalhypoxia, genetic factors ,oxidativestress, inflammation,auto antibodies against angiotensintype 1 receptor, altered NK cell signaling & deficientcatechol-o methyl transferase The exact role of these pathways in humans is still debated. *Endocrinology 2004;145(11):4838-45 18. Endothelium + VEGF + PIGF Path physiology- sFlt1 Current data indicates that placental ischemia &trophoblastic hyperperfusion with endothelialdysfunction as the most consistent change *Placental ischemia occur at an early stage & upregulates placental production of soluble antiangiogenesis protein soluble fms like tyrosine kinase1(sFlt-1)leading to endothelial dysfunction & clinicalmanifestations. 19. Endothelium + VEGFAngiogenic Factors sFlt-1 + PIGF - sFlt1 Increased placental expression of sFlt-1 appears to play a central role in pathogenesis of PE. sFlt1 Differential gene expression profiling in placentas extracted from women delivering in a normal or PE context was reported by the group of Ananth Karumanchi in Boston, in 2003 [3]. This signal study identified sFlt-1 as a major gene up-regulated in PE*J Clin inves 2003;1111:6491,AJ Path 2002;160:1405,Obs Gyn 2000;95:353 AJOG 2004;190:1541,AJOG 2007;197:1240 20. Pathogenesis of Systemic Endothelial Dysfunction Balance between pro-angiogenic - VEGF, PlGF & antiangiogenic sFlt-1& soluble endoglin gene factors ,isessential for normal function of placenta.* Increased production of anti angiogenic factors disturbsthe balance . Endothelial dysfunction & PE. (AnanthKarumanchi 12 January 2012) sFlt-1 a split variant of VEGF receptor Flt-1,which lackstransmembrane and cytoplasmic domain ,is made inlarge amounts by PE placenta,& released in to maternalcirculation. 21. EndotheliumsEng AntiAngiogenic+ VEGF + PIGF- sFlt1 Soluble Endoglin(sEng) truncated form of endoglin(CD105), a cell receptor for transforming growthfactor-beta (TGF-), has been localized to bothplacental syncytiotrophoblasts and endothelial cells. It binds and antagonizes TGF-b in extra cellularmilieu. sEng is up regulated in PE , acts with sFlt-1& causesendothelial dysfunction. Circulating soluble endoglin levels were shown to increase 2-3 months before the onset of PE. Peak at onset of clinical symptoms. (Levineetal) 22. Endothelium+ PIGFAngiogenic Markers In Healthy+ VEGFPregnancy- sFlt1sFlt-1 levels are stable during early & mid gestation ,thenincrease significantly during late stages. N Eng J.Med.2004;350:672-83 23. sFlt-1 By ELISA The group of Karumanchi recently publishedencouraging data about the positive and negativepredictive values of the sFlt1 concentration inmaternal serum. Using a cut-off value of 14,000 pg/ml in women followed prospectively, and from whom blood was drawn around 30 weeks of gestation, the area under the receiving operating characteristic (ROC) curve was 0.98, and a sensitivity and specificity of 96 and 96%, respectively, for the diagnosis of PE . 24. Changes in circulating angiogenic factors fromfirst to second trimester as predictors of PE Low or no increase in serum concentration of free PlGF, VEGF & high concentration of sFlt-1 a strong predictor ofearly PE.* Low increase in PlGF & low increase in sFlt are associatedwith 10 fold higher risk of pre term PE. Low increase in PlGF in early pregnancy ,independent ofchange in sFlt-1 is associated with high risk PE. sFlt-1 increase observed approximately 5 weeks beforeonset of PE. J Clin Investigation 2003;111:649-58 ,AJOG March 2007 21 25. Mean sFlt-1 concentration and gestational age 26. Endothelium+ VEGF + PIGF Placental Growth Factor Clinical Significance- sFlt1 PlGF concentration increased in 1,2 trimester in controls,peaked at 29-32 weeks, decreased thereafter. In PE women the increase was significantly lower from13-16 weeks onwards. Similar to sFlt-1 PGF began to decrease 11-9 weeks beforethe onset of PE, with substantial reduction 5 weeks before the onset of hypertension or proteinuria. Sri Lancan J.Ob,Gy 203w2 27. Mean PIGF concentration and gestational age 28. Endothelium+ VEGF + PIGF Soluble Endoglin- sFlt1 Soluble endoglin as a second trimester marker for preeclampsiaSoluble endoglin elevated in patients destined to, developsevere early-onset preeclampsia Circulating sFlt-1 and sEng may synergize and contribute to PE,via different but additive mechanism, probably by inhibition ofNO production. Robinson Johnson D. AJOG 2007:197 29. Endothelium Angiogenic Factors Prediction of PE+ VEGF+ PIGFIn High Risk Women- sFlt1 High risk women Previous H/O PE,Multiple gestation,pre gestational DM,Chronic hypertension, chronic kidney disease, obesity & adolescent age. Study revealed that Rapid rise in the sFlt-1 to PlGFratio with advancing gestation may be predictive of PE. Mean serum sFlt-1 ,sFlt-1/PlGF were higher with early onset PE (< 34 weeks) Above ratio at 22-26 weeks was highly predictive of early onset PE. A 2 tiered screening approach may be a useful tool for prediction. *AJOG September 2007-244-248,BMJ2005;330:565,J of Med 2006;355:992-1005 30. Endothelium Angiogenic factors in + VEGF + PIGF high risk women- sFlt1 Twin pregnancies are associated with 2-3 fold increased riskfor PE. Circulating levels of sFlt-1 PlGF ratio were twice as high as insingleton . But not accompanied by any change in levels of sFlt-1 mRNA &HIF alpha protein, in the twine placentas, but werecorrelated with placental weight. Trisomy 13 a risk factor as gene for sFlt-1 is located on thatchromosome. Smokers have low sFlt-1levels & less Incidence of PE. N Eng J Med 2006;335(10):992-1005.AJOG2005 :193(1):185-91. 31. Endothelium+ VEGF + PIGF - sFlt1Maternal serum levelsof sFlt1 (A) and the sFlt1to PlGF ratio (B) bygestational age in high-risk pregnant women 32. Pregnancy with chronic Glomerulo nephritis sFlt-1levels of super imposed pre eclampsia were significantly higher and PlGF were significantly lower ,than in women with sever protenuria with out hypertension, and those with normal course. conclusion--- it is the imbalance of circulating angiogenic factors which is important in CGN ,for the onset of PE. 33. Angiogenic Factors in gestational protenuria and PEsuper imposed on Chronic Glomerulo NephritisCirculating sFlt-1 and PlGF levels in pregnancy with CGN. Blood samples were collectedfrom CGN patients soon after onset of the disease. The samples from CGN patients withnormal clinical course were obtained at 36 to37 weeks of gestational ages. Values are shownas mean GSEM, asterisks indicate P ! .01 and double asterisks indicateP ! .05. 34. Circulating angiogenicfactors Endothelium+ VEGF + PIGFIncreasesFlt-1 - sFlt1Increase Endoglinin patients that will develop clinical preeclampsia Levine et al, NEJM; 2004 Robinson CJ, Johnson DD. AJOG 2007 35. Latest results :sFLT-1 free VEGF not usefulin the 1st trimester screeningCONCLUSION: Measurement of free-VEGF andsFlt-1 in maternal blood at 11 to 13 weeks ofgestation is not useful in the prediction ofpregnancies destined to develop PE. 32 36. Endothelium + VEGF + PIGFsFlt-1 to PlGF Ratio Predictor for PE - sFlt1 It is an index of antiangiogenic activity, that reflects changes in the balance between sFlt-1 & PlGF obviously seen in PE Women with PE have reduced uteroplacental blood flow . The transcription factor hypoxia inducible factor 1 (HIF 1 alpha) regulates VEGF& Flt-1gene transcription HIF-1 ,2 are elevated in pre eclamptic placenta. 37. Endothelium+ VEGF + PIGFBio Markers- sFlt1 In 2008, it was reported that mice with catechol-o-methyltransferase(COMT, the enzyme responsible for the degradation of catecholamines)deficiency displayed a mild but spontaneous PE-like phenotype duringpregnancy . The site of action of 2-ME could be upstream from the uncontrolledproduction of sFlt1 and sEng by the ischemic placenta, sinceCOMT/mice also exhibit defective placental vascularization. At firstsight, this is a very paradoxical finding: 2-ME was initially defined ashaving antiangiogenic property. placental insufficiency (the concentration of progestagens andandrogens was comparable in the different groups). 17-E2 hadpreviously been tested as a biomarker in PE, 38. Placental protein 13 (PP 13) PP13 is a 32-kDa homodimer found in the brush borderof syncytiotrophoblasts, at the maternalfetal interface(the placenta is the only organ that produces PP13). Itbelongs to the galectin family, and binds tocarbohydrates. The biological function of PP13 isthought to favor the implantation of the embryo andthe occurrence of vascular remodeling. However, three studies have reported that at an earlygestational age (before 14 weeks), the level of PP13 wasrelatively low in women with subsequent preeclamspiacompared to controls . 39. + VEGF + PIGFAT1-AA) - sFlt1 In 1999, auto-antibodies directed against theangiotensin-II type-1 receptor (AT1-AA) were detectedin the blood from women with PE The authorsreporting this discovery provocatively suggested thatplasma exchange could be a way of prevention Undoubtedly, these questions will have to beaddressed before the clearance of AT1-AA can beconsidered as a therapeutic option. As a marker, AT1-AA appears to be less efficient than sFlt-1 . 40. Angiotensin receptor activating autoantibodies (AT1-AAs) may underlie manyfeatures of preeclampsia.AT1-AAs from preeclamptic patients activate angiotensinreceptors (AT1R) on the surface of many cell types and may be responsible for manyfeatures of this serious pregnancy disorder. We have shown that antibody-induced receptoractivation results in the mobilization of intracellular calcium and the activation of manygenes. We propose that AT1-AAs activate AT1 receptors by promoting receptorhomodimerization. ROS, reactive oxygen species. SMC, smooth muscle cells; EC,endothelial cells. 41. Genes for pre-eclampsia discovered The US researchers from the Washington University School of Medicine in St. Louisanalyzed DNA from over 300 pregnant women. 40 normal & remaining 250 werewomen who were being monitored for other health complications. Forty of thesealso went on to develop pre-eclampsia. DNA analysis revealed a few genetic errors shared by five of the 60 otherwisehealthy women and seven of the 40 "higher-risk pregnancy" women who developedpre-eclampsia. The genes on which the errors were identified (MCP factor I and factor H) play arole in regulating immune response and the researchers believe this could explaintheir possible link to pre-eclampsia. Scientists have suspected that problems with the immune system provoke manycases of pre-eclampsia because women with lupus and certain other autoimmunediseases - like 250 of the women in the study - have an increased risk of thedisorder. At best genes like these might identify 10-15% of pre-eclampsia, so its relativeimportance may not be sensational. But it may allow us to study new treatments toprevent or delay the onset of pre-eclampsia and to know which women need closersurveillance." 42. Endothelium+ VEGF + PIGFSummary Proangiogenic markers ,PGF & VEGF decrease with - sFlt1preeclampsia compared to normotensive women. PlGF increase was significantly lower in PE between 13-16 weeksonwards. The anti angiogenic marker sFlt-1increases comparatively inpreeclampsia.. The decrease in PGF ,increase in sFlt-1 are evident 5 weeksbefore the onset of clinical preeclampia. sFlt-1 /PlGF ratio is crucial in predicting early or late onset PE,superimposed PE on CGN sEng which synchronize with sFlt-1 is increased in secondtrimester in women destined for early onset PE. 43. Summary Current evidence suggests that clinical S/S of PE may bemediated by excess circulating anti angiogenic factors ofplacental origin . Above is due to abnormal placental remodelling,resultingin ischemia. Numerous hypothesis as genetic ,immunological,sugestedto be the cause for placental vascular defect. Recent data suggests that abnormal NK cell signaling at thefeto maternal inter phase may be the culprit. 44. Conclusion From the much better insights into thepathophysiology of Preeclampsia and the improved prediction possibilities withangiogenic markers and Doppler studies with closesurveillance probably lead to better outcome. Restoration of angiogenesis balance in maternalcirculation such as VEGF A 21 or neutralizingantibodies against sFlt-1 or sEnd may be the futuretherapy. Endothelin receptor modulators The future 45. Endothelium+ VEGF + PIGFConclusion- sFlt1 Preeclampsia may never be totally predictable, But better prediction would help to focus on Antenatal care more effectively Chris Redman, Reykjavik, May 26, 2007 46. + PIGF+ VEGF - sFlt1
