Defective placentation is due to abnormal cytotrophoblast invasion
sFlt-1 administered to pregnant rats induces albuminuria. ,removal of sFlt-1 from PE tissue culture restores endothelial function Exogenous administration of VEGF PlGF reverses anti angiogenic state
Angiogenic markers in pre eclampsia
Dr. Indira Devi Ponugoti MD,DGO,FICOG,FIAMS,FCGP.• MBBS,MD.DGO, Osmania University, Hyderabad - 1965;1969; 70• FICOG 2009• FAMS 2010•Professor of Obstetrics and Gyneaecology - Osmania Medical College 1991--1999•Kamineni institute of Medical Sciences HOD 2001 - 06•Under and post graduate Examiner 1970 - 2010•Chair person womens wing IMA - Hyderabad 2007 - 08•Vice President OGSH 2007•President OGSH 2008 – 09•Coordinator APCOG 2008 - 09•Vice President IMA –HYD 2010 - 11•Presented papers at national conferences•Chaired the sessions at national and international conferences•Contributed to FOGSI focus on Maternal Nutrition•Life member IMA ISOPARB OGA AIAORAO•Presently•State council member IMA Hyderabad•Organizing Co- Chair person & Chair Person Scientific Committee - AICOG 2011•Dean Of Faculty CGP-IMA 2011
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 Markers Clinical significance Possibility of Prediction of PE in normal & High Risk . Pregnancies Gestational proteinuria, chronic glomerulonepritis with . super imposed preeclampsia. Endothelial cell Modulators The Future ?
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
Endothelium Introduction + VEGF + PIGF PE Future Risk - sFlt1 Recurrence with complications & Chronic hypertension Maternal endothelial dysfunction may persist years after the episode –Cardiovascular events. The risk factors of PE are similar as for underlying vascular disease –as insulin resistantance hyperlipidemia,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
EndotheliumPathogenesis of PE + VEGF + PIGF Thought to involve 3 steps. - sFlt1 Defective placentation Placental ischemia Endothelial cell dysfunction a 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
Endothelium + VEGF + PIGFPreeclampsia Basic Pathology - sFlt1 *Defective or abnormal cytotrophoblast invasion of spiral arterioles >> Reduced uteroplacental perfusion . * Ischemic placenta secretes anti angiogenic soluble factors into maternal vasculature, leading to endothelial damage. *Hypothesis Roberts et al *Maynard et al J .clin inves 2003
Placenta Extravillus invasive cyto trophoblast migrate to & invade the uterine spiral artereries & decidual arterioles . These initially express adhesion molecules characteristic of epithelial cells as:integrine alpha6/beta1,alpha5/beta5,E cadherin. As invasion proceeds instead of epithelial expression , endothelial cell adhesion molecule expression occurs.Integrine alpha 1/beta1,alpha 5 beta 3,vE cadherin is observed.Pseudovascularisation. This results in replacement of endothelial layer of these vessels and subsequent formation of high capacitance ,low resistant system -- nutrition etc NORMAL. In PE pregnancies invasive cytotrophoblast fail to completely invade decidual arterioles, fail to show above vascular mimicry=placental hypoxia , & liberation of anti angiogenic factors.
PossibleMechanisms inPreeclampsia Friedman and Lindheimer,1999
Physiology of Vasculogenesis & Angiogenesis As the embryo develops ,mesodermal precursors differentiate in to endothelial cells & assemble into primitive vascular network –vasculogenesis. These net works under go extensive budding & branching ,associate with vascular smooth muscle elements –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.
Angiogenesis VEGF Angiogenisis is controlled by positive and negative influences. VEGF (VPF) comprised of six different proteins encoded by distinct genes ,=VEGF ABC,or VEGF- E & PlGF These act by Stimulation of endothelial angiogenesis ,endothelial survival by tk T path way ,controls vascular permeability, stimulates expression of tissue plasminogen activators,urokinase ,collagenase . Physiological regulators for erythropoiesis & synthesis of erythropoietin. VEGF- an attractive target for pro & anti angiogenic gene therapy because of its association with normal & abnormal angiogenesis Lack of 1 or 2 allele in embryo leads to defective angiogenesis – anomaly
Endothelium + VEGF + PIGF Pro angiogenic Factor - sFlt1 PlGF a member of VEGF made predominantly in placenta also binds to VEGFR-1 *VEGFR-1 has 2 isomers:Trans membrane isoform & soluble isoform.(sFlt-1) *VEGF & PlGF which are pro angiogenic, reduce vascular tone thus blood pressure. *N Eng J Med 2004;350:672,683*J Clin inves 2003;1111:6491, AJ Path2002;160:1405, ObGyn2000;95:3
Anti Angiogenic Factor sFlt-1 sFlk-1 Soluble fm like tyrosine kinase -1(sFlt-1)also known as sVEGFR-1,is a naturally occurring circulating antagonist Soluble form of new receptor for VEGF family is identified as sFlk-1 or VEGFR-2 Its action is mediated by inter action with high affinity receptors –as tyrosine kinase,VEGFR-1,Flt-1VEGFR-2,kinase domain region (KDR)Flk-1 which are selectively expressed on vascular endothelial cell surface and targeted tissues like kidney* *Mol Cancer Rea 2004;2:315-26
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 cell surface & 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 and containing intron 14 coded unique 28 amino acids sFlt-14 is identified as the primary isoform produced by placenta in 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.
sFlt-1 *Lowered oxygen tension in primary cytotrophoblast culture & villus explants causes increased sFlt expression, may be stimulated by hypoxia. *Other path ways inducing sFlt probably are Deficient hemooxygenase expression, placental hypoxia, genetic factors ,oxidative stress, inflammation,auto antibodies against angiotensin type 1 receptor, altered NK cell signaling & deficient catechol-o methyl transferase The exact role of these pathways in humans is still debated. *Endocrinology 2004;145(11):4838-45
Endothelium + VEGF + PIGF Path physiology - sFlt1 Current data indicates that placental ischemia & trophoblastic hyperperfusion with endothelial dysfunction as the most consistent change *Placental ischemia occur at an early stage & up regulates placental production of soluble anti angiogenesis protein –soluble fms like tyrosine kinase 1(sFlt-1)leading to endothelial dysfunction & clinical manifestations.
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 . 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
Pathogenesis of Systemic Endothelial Dysfunction Balance between pro-angiogenic - VEGF, PlGF & anti angiogenic –sFlt-1& soluble endoglin gene factors ,is essential for normal function of placenta.* Increased production of anti angiogenic factors disturbs the balance . Endothelial dysfunction & PE. (Ananth Karumanchi 12 January 2012) sFlt-1 a split variant of VEGF receptor Flt-1,which lacks transmembrane and cytoplasmic domain ,is made in large amounts by PE placenta,& released in to maternal circulation.
Endothelium sEng AntiAngiogenic + VEGF + PIGF - sFlt1 Soluble Endoglin(sEng) truncated form of endoglin (CD105), a cell receptor for transforming growth factor-beta (TGF-β), has been localized to both placental syncytiotrophoblasts and endothelial cells. It binds and antagonizes TGF-b in extra cellular milieu. sEng is up regulated in PE , acts with sFlt-1& causes endothelial dysfunction. Circulating soluble endoglin levels were shown to increase 2-3 months before the onset of PE. Peak at onset of clinical symptoms. (Levineetal)
Endothelium + PIGFAngiogenic Markers In Healthy + VEGFPregnancy - sFlt1 sFlt-1 levels are stable during early & mid gestation ,thenincrease significantly during late stages. N Eng J.Med.2004;350:672-83
sFlt-1 By ELISA The group of Karumanchi recently published encouraging data about the positive and negative predictive values of the sFlt1 concentration in maternal 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 .
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 of early PE.* Low increase in PlGF & low increase in sFlt are associated with 10 fold higher risk of pre term PE. Low increase in PlGF in early pregnancy ,independent of change in sFlt-1 is associated with high risk –PE. sFlt-1 increase observed approximately 5 weeks before onset of PE. J Clin Investigation 2003;111:649-58 ,AJOG March 2007 21
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 from 13-16 weeks onwards. Similar to sFlt-1 PGF began to decrease 11-9 weeks before the onset of PE, with substantial reduction 5 weeks before the onset of hypertension or proteinuria. Sri Lancan J.Ob,Gy 203w2
Endothelium + VEGF + PIGF Soluble Endoglin - sFlt1 Soluble endoglin as a second trimester marker for preeclampsia Soluble endoglin elevated in patients destined to, develop severe early-onset preeclampsia Circulating sFlt-1 and sEng may synergize and contribute to PE ,via different but additive mechanism, probably by inhibition of NO production. Robinson Johnson D. AJOG 2007:197
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
Endothelium Angiogenic factors in + VEGF + PIGF high risk women - sFlt1 Twin pregnancies are associated with 2-3 fold increased risk for PE. Circulating levels of sFlt-1 PlGF ratio were twice as high as in singleton . But not accompanied by any change in levels of sFlt-1 mRNA & HIF – alpha protein, in the twine placentas, but were correlated with placental weight. Trisomy 13 a risk factor as gene for sFlt-1 is located on that chromosome. Smokers have low sFlt-1levels & less Incidence of PE. N Eng J Med 2006;335(10):992-1005.AJOG2005 :193(1):185-91.
Endothelium + VEGF + PIGF - sFlt1Maternal serum levelsof sFlt1 (A) and the sFlt1to PlGF ratio (B) bygestational age in high-risk pregnant women
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.
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.
Circulating angiogenicfactors Endothelium + VEGF + PIGF Increase sFlt-1 - sFlt1 Increase Endoglin in patients that will develop clinical preeclampsia Levine et al, NEJM; 2004 Robinson CJ, Johnson DD. AJOG 2007
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
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.
Endothelium + VEGF + PIGF Bio 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 during pregnancy . The site of action of 2-ME could be upstream from the uncontrolled production of sFlt1 and sEng by the ischemic placenta, since COMT−/−mice also exhibit defective placental vascularization. At first sight, this is a very paradoxical finding: 2-ME was initially defined as having antiangiogenic property. placental insufficiency (the concentration of progestagens and androgens was comparable in the different groups). 17β-E2 had previously been tested as a biomarker in PE,
Placental protein 13 (PP 13) PP13 is a 32-kDa homodimer found in the brush border of syncytiotrophoblasts, at the maternal–fetal interface (the placenta is the only organ that produces PP13). It belongs to the galectin family, and binds to carbohydrates. The biological function of PP13 is thought to favor the implantation of the embryo and the occurrence of vascular remodeling. However, three studies have reported that at an early gestational age (before 14 weeks), the level of PP13 was relatively low in women with subsequent preeclamspia compared to controls .
+ VEGF + PIGF AT1-AA) - sFlt1 In 1999, auto-antibodies directed against the angiotensin-II type-1 receptor (AT1-AA) were detected in the blood from women with PE The authors reporting this discovery provocatively suggested that plasma exchange could be a way of prevention Undoubtedly, these questions will have to be addressed before the clearance of AT1-AA can be considered as a therapeutic option. As a marker, AT1- AA appears to be less efficient than sFlt-1 .
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.
Genes for pre-eclampsia discovered The US researchers from the Washington University School of Medicine in St. Louis analyzed DNA from over 300 pregnant women. 40 normal & remaining 250 were women who were being monitored for other health complications. Forty of these also went on to develop pre-eclampsia. DNA analysis revealed a few genetic errors shared by five of the 60 otherwise healthy women and seven of the 40 "higher-risk pregnancy" women who developed pre-eclampsia. The genes on which the errors were identified (MCP factor I and factor H) play a role in regulating immune response and the researchers believe this could explain their possible link to pre-eclampsia. Scientists have suspected that problems with the immune system provoke many cases of pre-eclampsia because women with lupus and certain other autoimmune diseases - like 250 of the women in the study - have an increased risk of the disorder. At best genes like these might identify 10-15% of pre-eclampsia, so its relative importance may not be sensational. But it may allow us to study new treatments to prevent or delay the onset of pre-eclampsia and to know which women need closer surveillance."
Endothelium + VEGF + PIGF Summary Proangiogenic markers ,PGF & VEGF decrease with - sFlt1 preeclampsia compared to normotensive women. PlGF increase was significantly lower in PE between 13-16 weeks onwards. The anti angiogenic marker sFlt-1increases comparatively in preeclampsia.. The decrease in PGF ,increase in sFlt-1 are evident 5 weeks before 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 second trimester in women destined for early onset PE.
Summary Current evidence suggests that clinical S/S of PE may be mediated by excess circulating anti angiogenic factors of placental origin . Above is due to abnormal placental remodelling,resulting in ischemia. Numerous hypothesis as genetic ,immunological,sugested to be the cause for placental vascular defect. Recent data suggests that abnormal NK cell signaling at the feto maternal inter phase may be the culprit.
Conclusion From the much better insights into the pathophysiology of Preeclampsia and the improved prediction possibilities with angiogenic markers and Doppler studies with close surveillance probably lead to better outcome. Restoration of angiogenesis balance in maternal circulation such as VEGF A 21 or neutralizing antibodies against sFlt-1 or sEnd may be the future therapy. Endothelin receptor modulators The future
Endothelium + VEGF + PIGF Conclusion - 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