Shakil Ahmad

Elevated Placental Soluble Vascular Endothelial Growth Factor Receptor-1 Inhibits Angiogenesis in Preeclampsia

Preeclampsia is an inflammatory disorder in which serum levels of vascular endothelial growth factor (VEGF) and its soluble receptor-1 (sVEGFR-1, also known as sFlt-1) are elevated. We hypothesize that VEGF and placenta growth factor (PlGF) are dysregulated in preeclampsia due to high levels of sVEGFR-1, which leads to impaired placental angiogenesis. Analysis of supernatants taken from preeclamptic placental villous explants showed a four-fold increase in sVEGFR-1 than normal pregnancies, suggesting that villous explants in vitro retain a hypoxia memory reflecting long-term fetal programming. The relative ratios of VEGF to sVEGFR-1 and PlGF to sVEGFR-1 released from explants decreased by 53% and 70%, respectively, in preeclampsia compared with normal pregnancies. Exposure of normal villous explants to hypoxia increased sVEGFR-1 release compared with tissue normoxia (P<0.001), as did stimulation with tumor necrosis factor-&agr; (P<0.01). Conditioned medium (CM) from normal villous explants induced endothelial cell migration and in vitro tube formation, which were both attenuated by pre-incubation with exogenous sVEGFR-1 (P<0.001). In contrast, endothelial cells treated with preeclamptic CM showed substantially reduced angiogenesis compared with normal CM (P<0.001), which was not further decreased by the addition of exogenous sVEGFR-1, indicating a saturation of the soluble receptor. Removal of sVEGFR-1 by immunoprecipitation from preeclamptic CM significantly restored migration (P<0.001) and tube formation (P<0.001) to levels comparable to that induced by normal CM, demonstrating that elevated levels of sVEGFR-1 in preeclampsia are responsible for inhibiting angiogenesis. Our finding demonstrates the dysregulation of the VEGF/PlGF axis in preeclampsia and offers an entirely new therapeutic approach to its treatment.

Negative Regulation of Soluble Flt-1 and Soluble Endoglin Release by Heme Oxygenase-1

Background— Preeclampsia is characterized clinically by hypertension and proteinuria. Soluble Flt-1 (sFlt-1; also known as soluble vascular endothelial growth factor receptor-1 [VEGFR-1]) and soluble endoglin (sEng) are elevated in preeclampsia, and their administration to pregnant rats elicits preeclampsia-like symptoms. Heme oxygenase-1 (HO-1) and its metabolite carbon monoxide (CO) exert protective effects against oxidative stimuli. Thus, we hypothesized that HO-1 upregulation may offer protection against preeclampsia by inhibiting sFlt-1 and sEng release. Methods and Results— Preeclamptic villous explants secreted high levels of sFlt-1 and sEng. Adenoviral overexpression of HO-1 in endothelial cells inhibited VEGF-mediated sFlt-1 release and interferon-&ggr;– and tumor necrosis factor-&agr;–induced sEng release, whereas HO-1 inhibition potentiated sFlt-1 and sEng production from endothelial cells and placental villous explants. Consistent with these findings, mice lacking HO-1 produced higher levels of sFlt-1 and sEng compared with wild-type mice. Using selective ligands (VEGF-E and placental growth factor) and a receptor-specific inhibitor (SU-1498), we demonstrated that VEGF-induced sFlt-1 release was VEGFR-2 dependent. Furthermore, CO-releasing molecule-2 (CORM-2) or CO decreased sFlt-1 release and inhibited VEGFR-2 phosphorylation. Treatment of endothelial cells with statins upregulated HO-1 and inhibited the release of sFlt-1, whereas vitamins C and E had no effect. Conclusions— The present study demonstrates that the HO-1/CO pathway inhibits sFlt-1 and sEng release, providing compelling evidence for a protective role of HO-1 in pregnancy, and identifies HO-1 as a novel target for the treatment of preeclampsia.

Deficiency in catechol-O-methyltransferase and 2-methoxyoestradiol is associated with pre-eclampsia

Despite intense investigation, mechanisms that facilitate the emergence of the pre-eclampsia phenotype in women are still unknown. Placental hypoxia, hypertension, proteinuria and oedema are the principal clinical features of this disease. It is speculated that hypoxia-driven disruption of the angiogenic balance involving vascular endothelial growth factor (VEGF)/placenta-derived growth factor (PLGF) and soluble Fms-like tyrosine kinase-1 (sFLT-1, the soluble form of VEGF receptor 1) might contribute to some of the maternal symptoms of pre-eclampsia. However, pre-eclampsia does not develop in all women with high sFLT-1 or low PLGF levels, and it also occurs in some women with low sFLT-1 and high PLGF levels. Moreover, recent experiments strongly suggest that several soluble factors affecting the vasculature are probably elevated because of placental hypoxia in the pre-eclamptic women, indicating that upstream molecular defect(s) may contribute to pre-eclampsia. Here we show that pregnant mice deficient in catechol-O-methyltransferase (COMT) show a pre-eclampsia-like phenotype resulting from an absence of 2-methoxyoestradiol (2-ME), a natural metabolite of oestradiol that is elevated during the third trimester of normal human pregnancy. 2-ME ameliorates all pre-eclampsia-like features without toxicity in the Comt-/- pregnant mice and suppresses placental hypoxia, hypoxia-inducible factor-1α expression and sFLT-1 elevation. The levels of COMT and 2-ME are significantly lower in women with severe pre-eclampsia. Our studies identify a genetic mouse model for pre-eclampsia and suggest that 2-ME may have utility as a plasma and urine diagnostic marker for this disease, and may also serve as a therapeutic supplement to prevent or treat this disorder.

Release and Complex Formation of Soluble VEGFR-1 from Endothelial Cells and Biological Fluids

One of the key molecules promoting angiogenesis is the endothelial cell-specific mitogen, vascular endothelial growth factor (VEGF or VEGF-A), which acts through two high-affinity receptor tyrosine kinases (VEGFR), VEGFR-1 (or Flt-1) and VEGFR-2 (or KDR/Flk-1). It was shown before that a soluble variant of VEGFR-1 (sVEGFR-1) can be generated by differential splicing of the flt-1 mRNA. This soluble receptor is an antagonist to VEGF action, reducing the level of free, active VEGF-A, and therefore, plays a pivotal role in the generation of vascular diseases like pre-eclampsia or intra-uterine growth retardation. Here we show that sVEGFR-1 is produced by cultured human microvascular and macrovascular endothelial cells and a human melanoma cell line. The soluble receptor is mainly complexed with ligands; only 5–10% remains detectable as free, uncomplexed receptor protein. Furthermore, we show the time course of total and free sVEGFR-1 release together with its putative ligands, VEGF-A and placenta growth factor (PlGF), from macrovascular endothelial cells. The release of sVEGFR-1 was quantitatively measured in two different ELISA types. The release of sVEGFR-1 was strongly enhanced by phorbol-ester (PMA); the cells produced up to 22 ng/ml of sVEGFR-1 after 48 hours. The expression of VEGF-A and PlGF was moderately influenced by PMA. We also show a hypoxia-induced increase of sVEGFR-1 expression in cells cultured from placenta, a tissue that has a high flt-1 gene expression. Moreover, we demonstrate that sVEGFR-1 in amniotic fluids acts as a sink for exogenous VEGF165 and PlGF-2. Here, for the first time, to what extent recombinant ligands have to be added to compensate for the sink function of amniotic fluids was analyzed. In conclusion, human endothelial cells produce high levels of sVEGFR-1, which influences the availability of VEGF-A or related ligands. Therefore, sVEGFR-1 may reduce the ligand binding to transmembrane receptors and interfere with their signal transduction.

Autoantibody From Women With Preeclampsia Induces Soluble Fms-Like Tyrosine Kinase-1 Production via Angiotensin Type 1 Receptor and Calcineurin/Nuclear Factor of Activated T-Cells Signaling

Preeclampsia is a pregnancy-specific hypertensive syndrome that causes substantial maternal and fetal morbidity and mortality. Recent evidence indicates that maternal endothelial dysfunction in preeclampsia results from increased soluble Fms-like tyrosine kinase-1 (sFlt-1), a circulating antiangiogenic protein. Factors responsible for excessive production of sFlt-1 in preeclampsia have not been identified. We tested the hypothesis that angiotensin II type 1 (AT1) receptor activating autoantibodies, which occur in women with preeclampsia, contribute to increased production of sFlt-1. IgG from women with preeclampsia stimulates the synthesis and secretion of sFlt-1 via AT1 receptor activation in pregnant mice, human placental villous explants, and human trophoblast cells. Using FK506 or short-interfering RNA targeted to the calcineurin catalytic subunit mRNA, we determined that calcineurin/nuclear factor of activated T-cells signaling functions downstream of the AT1 receptor to induce sFlt-1 synthesis and secretion by AT1-receptor activating autoantibodies. AT1-receptor activating autoantibody–induced sFlt-1 secretion resulted in inhibition of endothelial cell migration and capillary tube formation in vitro. Overall, our studies demonstrate that an autoantibody from women with preeclampsia induces sFlt-1 production via angiotensin receptor activation and downstream calcineurin/nuclear factor of activated T-cells signaling. These autoantibodies represent potentially important targets for diagnosis and therapeutic intervention.

Angiotensin II Induces Soluble fms-Like Tyrosine Kinase-1 Release via Calcineurin Signaling Pathway in Pregnancy

Maternal endothelial dysfunction in preeclampsia is associated with increased soluble fms-like tyrosine kinase-1 (sFlt-1), a circulating antagonist of vascular endothelial growth factor and placental growth factor. Angiotensin II (Ang II) is a potent vasoconstrictor that increases concomitant with sFlt-1 during pregnancy. Therefore, we speculated that Ang II may promote the expression of sFlt-1 in pregnancy. Here we report that infusion of Ang II significantly increases circulating levels of sFlt-1 in pregnant mice, thereby demonstrating that Ang II is a regulator of sFlt-1 secretion in vivo. Furthermore, Ang II stimulated sFlt-1 production in a dose- and time-dependent manner from human villous explants and cultured trophoblasts but not from endothelial cells, suggesting that trophoblasts are the primary source of sFlt-1 during pregnancy. As expected, Ang II–induced sFlt-1 secretion resulted in the inhibition of endothelial cell migration and in vitro tube formation. In vitro and in vivo studies with losartan, small interfering RNA specific for calcineurin and FK506 demonstrated that Ang II–mediated sFlt-1 release was via Ang II type 1 receptor activation and calcineurin signaling, respectively. These findings reveal a previously unrecognized regulatory role for Ang II on sFlt-1 expression in murine and human pregnancy and suggest that elevated sFlt-1 levels in preeclampsia may be caused by a dysregulation of the local renin/angiotensin system.

Reduction of circulating soluble Flt-1 alleviates preeclampsia-like symptoms in a mouse model

Preeclampsia (PE) is characterized by widespread endothelial damage with hypertension, proteinuria, glomeruloendotheliosis and elevated soluble Flt‐1 (sFlt‐1), a natural occurring antagonist of vascular endothelial growth factor (VEGF). Cancer patients receiving anti‐VEGF therapy exhibit similar symptoms. We suggested that a decrease in circulating sFlt‐1 would alleviate the symptoms associated with PE. Adenoviral (Adv) overexpression of sFlt‐1 induced proteinuria, caused glomerular damage and increase in blood pressure in female Balb/c mice. Circulating level of sFlt‐1 above 50 ng/ml plasma induced severe vascular damage and glomerular endotheliosis. Albumin concentration in urine was elevated up to 30‐fold, compared to control AdvGFP‐treated animals. The threshold of kidney damage was in the range of 20–30 ng/ml sFlt‐1 in plasma (8–15 ng/ml in urine). Co‐administration of AdvsFlt‐1 with AdvVEGF to neutralize circulating sFlt‐1 resulted in more than a 70% reduction in free sFlt‐1 in plasma, more than 80% reduction in urine and rescued the damaging effect of sFlt‐1 on the kidneys. This demonstrates that below a critical threshold sFlt‐1 fails to elicit damage to the fenestrated endothelium and that co‐expression of VEGF is able to rescue effects mediated by sFlt‐1 overexpression.

Direct evidence for endothelial vascular endothelial growth factor receptor-1 function in nitric oxide-mediated angiogenesis

Vascular endothelial growth factor-A (VEGF) is critical for angiogenesis but fails to induce neovascularization in ischemic tissue lesions in mice lacking endothelial nitric oxide synthase (eNOS). VEGF receptor-2 (VEGFR-2) is critical for angiogenesis, although little is known about the precise role of endothelial VEGFR-1 and its downstream effectors in this process. Here we have used a chimeric receptor approach in which the extracellular domain of the epidermal growth factor receptor was substituted for that of VEGFR-1 (EGLT) or VEGFR-2 (EGDR) and transduced into primary cultures of human umbilical vein endothelial cells (HUVECs) using a retroviral system. Activation of HUVECs expressing EGLT or EGDR induced rapid phosphorylation of eNOS at Ser1177, release of NO, and formation of capillary networks, similar to VEGF. Activation of eNOS by VEGFR-1 was dependent on Tyr794 and was mediated via phosphatidylinositol 3-kinase, whereas VEGFR-2 Tyr951 was involved in eNOS activation via phospholipase C&ggr;1. Consistent with these findings, the VEGFR-1–specific ligand placenta growth factor-1 activated phosphatidylinositol 3-kinase and VEGF-E, which is selective for VEGFR-2–activated phospholipase C&ggr;1. Both VEGFR-1 and VEGFR-2 signal pathways converged on Akt, as dominant-negative Akt inhibited the NO release and in vitro tube formation induced following activation of EGLT and EGDR. The identification Tyr794 of VEGFR-1 as a key residue in this process provides direct evidence of endothelial VEGFR-1 in NO-driven in vitro angiogenesis. These studies provide new sites of modulation in VEGF-mediated vascular morphogenesis and highlight new therapeutic targets for management of vascular diseases.

Dysregulation of Hydrogen Sulfide Producing Enzyme Cystathionine γ-lyase Contributes to Maternal Hypertension and Placental Abnormalities in Preeclampsia

Background— The exact etiology of preeclampsia is unknown, but there is growing evidence of an imbalance in angiogenic growth factors and abnormal placentation. Hydrogen sulfide (H2S), a gaseous messenger produced mainly by cystathionine &ggr;-lyase (CSE), is a proangiogenic vasodilator. We hypothesized that a reduction in CSE activity may alter the angiogenic balance in pregnancy and induce abnormal placentation and maternal hypertension. Methods and Results— Plasma levels of H2S were significantly decreased in women with preeclampsia (P<0.01), which was associated with reduced placental CSE expression as determined by real-time polymerase chain reaction and immunohistochemistry. Inhibition of CSE activity by DL-propargylglycine reduced placental growth factorproduction from first-trimester (8–12 weeks gestation) human placental explants and inhibited trophoblast invasion in vitro. Knockdown of CSE in human umbilical vein endothelial cells by small-interfering RNA increased the release of soluble fms-like tyrosine kinase-1 and soluble endoglin, as assessed by enzyme-linked immunosorbent assay, whereas adenoviral-mediated CSE overexpression in human umbilical vein endothelial cells inhibited their release. Administration of DL-propargylglycine to pregnant mice induced hypertension and liver damage, promoted abnormal labyrinth vascularization in the placenta, and decreased fetal growth. Finally, a slow-releasing H2S-generating compound, GYY4137, inhibited circulating soluble fms-like tyrosine kinase-1 and soluble endoglin levels and restored fetal growth in mice that was compromised by DL-propargylglycine treatment, demonstrating that the effect of CSE inhibitor was attributable to inhibition of H2S production. Conclusions— These results imply that endogenous H2S is required for healthy placental vasculature and that a decrease in CSE/H2S activity may contribute to the pathogenesis of preeclampsia. (Circulation. 2013;127:2514-2522.)

Hypoxia induces dilated cardiomyopathy in the chick embryo: mechanism; intervention; and long-term consequences.

Background Intrauterine growth restriction is associated with an increased future risk for developing cardiovascular diseases. Hypoxia in utero is a common clinical cause of fetal growth restriction. We have previously shown that chronic hypoxia alters cardiovascular development in chick embryos. The aim of this study was to further characterize cardiac disease in hypoxic chick embryos. Methods Chick embryos were exposed to hypoxia and cardiac structure was examined by histological methods one day prior to hatching (E20) and at adulthood. Cardiac function was assessed in vivo by echocardiography and ex vivo by contractility measurements in isolated heart muscle bundles and isolated cardiomyocytes. Chick embryos were exposed to vascular endothelial growth factor (VEGF) and its scavenger soluble VEGF receptor-1 (sFlt-1) to investigate the potential role of this hypoxia-regulated cytokine. Principal Findings Growth restricted hypoxic chick embryos showed cardiomyopathy as evidenced by left ventricular (LV) dilatation, reduced ventricular wall mass and increased apoptosis. Hypoxic hearts displayed pump dysfunction with decreased LV ejection fractions, accompanied by signs of diastolic dysfunction. Cardiomyopathy caused by hypoxia persisted into adulthood. Hypoxic embryonic hearts showed increases in VEGF expression. Systemic administration of rhVEGF165 to normoxic chick embryos resulted in LV dilatation and a dose-dependent loss of LV wall mass. Lowering VEGF levels in hypoxic embryonic chick hearts by systemic administration of sFlt-1 yielded an almost complete normalization of the phenotype. Conclusions/Significance Our data show that hypoxia causes a decreased cardiac performance and cardiomyopathy in chick embryos, involving a significant VEGF-mediated component. This cardiomyopathy persists into adulthood.