Paracrine action of sFLT-1 secreted by stably-transfected Ehrlich ascites tumor cells and therapy using sFLT-1 inhibits ascites tumor growth in vivo

Background Vascular endothelial growth factor (VEGF) is known to play a major role in angiogenesis. A soluble form of Flt-1, a VEGF receptor, is potentially useful as an antagonist of VEGF, and accumulating evidence suggests the applicability of sFlt-1 in tumor suppression. In the present study, we have developed and tested strategies targeted specifically to VEGF for the treatment of ascites formation.Methods As an initial strategy, we produced recombinant sFLT-1 in the baculovirus expression system and used it as a trap to sequester VEGF in the murine ascites carcinoma model. The effect of the treatment on the weight of the animal, cell number, ascites volume and proliferating endothelial cells was studied. The second strategy involved, producing Ehrlich ascites tumor (EAT) cells stably transfected with vectors carrying cDNA encoding truncated form of Flt-1 and using these cells to inhibit ascites tumors in a nude mouse model. Results The sFLT-1 produced by the baculovirus system showed potent antiangiogenic activity as assessed by rat cornea and tube formation assay. sFLT-1 treatment resulted in reduced peritoneal angiogenesis with a concomitant decrease in tumor cell number, volume of ascites, amount of free VEGF and the number of invasive tumor cells as assayed by CD31 staining. EAT cells stably transfected with truncated form of Flt-1 also effectively reduced the tumor burden in nude mice transplanted with these cells, and demonstrated a reduction in ascites formation and peritoneal angiogenesis. Conclusions The inhibition of peritoneal angiogenesis and tumor growth by sequestering VEGF with either sFlt-1 gene expression by recombinant EAT cells or by direct sFLT-1 protein therapy is shown to comprise a potential therapy. Copyright (C) 2009 John Wiley & Sons, Ltd.

SFlt-1 and IP-10 in women with early-onset preeclampsia

Intense inflammatory response and an anti-angiogenic state have been implicated in the pathogenesis of preeclampsia. Here, we investigated this hypothesis evaluating the serum concentrations of CXCL10/IP-10, sFlt-1, and PlGF in women with early-onset preeclampsia. CXCL10/IP-10 was measured by Cytometric Bead Array. sFlt-1 and PlGF were measured by automated electrochemiluminescence immunoassay. The median serum concentration of CXCL10/IP-10 was 109.5 pg/mL in preeclamptic women, as compared with 52.28 pg/mL in the controls (P = 0.0028). The mean serum level of sFlt-1 was 13,636 pg/mL in preeclamptic women, as compared with 2020 pg/mL in the controls (P < 0.0001). PlGF levels were significantly lower in women with preeclampsia. © 2011 International Society for the Study of Hypertension in Pregnancy. Published by Elsevier B.V. All rights reserved.

SFlt-1/PlGF ratio as a prognostic marker of adverse outcomes in women with early-onset preeclampsia

Soluble fms-like tyrosine kinase 1 (sFlt-1) is an anti-angiogenic factor released in higher amounts by preeclamptic placentas and it has been implicated in the endothelial dysfunction observed in the disease. In this study we evaluated if circulating sFlt-1/PlGF ratio is useful to predict adverse outcomes in women with early-onset preeclampsia. This is a cohort study of 88 preeclamptic women with singleton pregnancies at ≤35 weeks of gestation. According to definitions used, adverse outcomes occurred in 46.5% (N = 43) of the patients. The median sFlt1/PlGF ratio (25th-75th centile) for all patients evaluated was of 42.26 (13.1-226.1). The median sFlt-1/PlGF ratio among women who had any adverse outcome (N = 43) versus no adverse outcomes (N = 45) was of 227.6 (80.3-346.1) versus 14.4 (3.35-30.0), (P < 0.0001). According to our analyses a sFlt-1/PlGF ratio cut-point of ≥85 gave a sensitivity of 74.0% and specificity of 97.0%. The positive predictive value and the negative predictive value were 96.0% and 80.0%, respectively. The median sFlt-1/PlGF ratio (25th-75th centile) for patients who delivered within <7 days was 260.0 (127.7-404.7) as compared to 14.4 (3.35-34.97) for those patients who delivered within two weeks or more (P < 0.0001). Our results suggest that sFlt-1/PlGF ratio is a promising marker for adverse outcomes in women with early-onset preeclampsia. © 2013 International Society for the Study of Hypertension in Pregnancy. Published by Elsevier B.V. All rights reserved.

Long-term evaluation of AAV-mediated sFlt-1 gene therapy for ocular neovascularization in mice and monkeys

Vascular endothelial growth factor (VEGF) is one of the major mediators of retinal ischemia-associated neovascularization. We have shown here that adeno-associated virus (AAV)-mediated expression of sFIt-1, a soluble form of the Flt-1 VEGF receptor, was maintained for up to 8 and 17 months postinjection in mice and in monkeys, respectively. The expression of sFIt-1 was associated with the long-term (8 months) regression of neovascular vessels in 85% of trVEGF029 eyes. In addition, it resulted in the maintenance of retinal morphology, as the majority of the treated trVEGF029 eyes (75%) retained high numbers of photoreceptors, and in retinal function as measured by electroretinography. AAV-mediated expression of sFIt-1 prevented the development of laser photocoagulation-incluced choroidal neovascularization in all treated monkey eyes. There were no clinically or histologically detectable signs of toxicity present in either animal model following AAV.sFlt injection. These results suggest that AAV-mediated secretion gene therapy could be considered for treatment of retinal and choroidal neovascularizations.

Resveratrol inhibits the release of soluble fms-like tyrosine kinase (sFlt-1) from human placenta

Objective - Soluble vascular endothelial growth factor receptor–1 (also know as soluble fms-like tyrosine kinase [sFlt]-1) is a key causative factor of preeclampsia. Resveratrol, a plant phytoalexin, has antiinflammatory and cardioprotective properties. We sought to determine the effect of resveratrol on sFlt-1 release. Study Design - Human umbilical vein endothelial cells, transformed human trophoblast-8 (HTR/SVneo)-8/SVneo trophoblast cells, or placental explants were incubated with cytokines and/or resveratrol. Conditioned media were assayed for sFlt-1 by enzyme-linked immunosorbent assay and cell proteins used for Western blotting. Results - Resveratrol inhibited cytokine-induced release of sFlt-1 from normal placental explants and from preeclamptic placental explants. Preincubation of human umbilical vein endothelial cells or HTR-8/SVneo cells with resveratrol abrogated sFlt-1 release. Resveratrol prevented the up-regulation of early growth response protein-1 (Egr-1), a transcription factor necessary for induction of the vascular endothelial growth factor receptor–1 gene and caused up-regulation of heme oxygenase–1, a cytoprotective enzyme found to be dysfunctional in preeclampsia. Conclusion - In summary, resveratrol can inhibit sFlt-1 release and up-regulate heme oxygenase–1; thus, may offer therapeutic potential in preeclampsia.

Regulation of neovascularization by S-glutathionylation via the Wnt5a/sFlt-1 pathway

S-glutathionylation occurs when reactive oxygen or nitrogen species react with protein-cysteine thiols. Glutaredoxin-1 (Glrx) is a cytosolic enzyme which enzymatically catalyses the reduction in S-glutathionylation, conferring reversible signalling function to proteins with redox-sensitive thiols. Glrx can regulate vascular hypertrophy and inflammation by regulating the activity of nuclear factor κB (NF-κB) and actin polymerization. Vascular endothelial growth factor (VEGF)-induced endothelial cell (EC) migration is inhibited by Glrx overexpression. In mice overexpressing Glrx, blood flow recovery, exercise function and capillary density were significantly attenuated after hindlimb ischaemia (HLI). Wnt5a and soluble Fms-like tyrosine kinase-1 (sFlt-1) were enhanced in the ischaemic-limb muscle and plasma respectively from Glrx transgenic (TG) mice. A Wnt5a/sFlt-1 pathway had been described in myeloid cells controlling retinal blood vessel development. Interestingly, a Wnt5a/sFlt-1 pathway was found also to play a role in EC to inhibit network formation. S-glutathionylation of NF-κB components inhibits its activation. Up-regulated Glrx stimulated the Wnt5a/sFlt-1 pathway through enhancing NF-κB signalling. These studies show a novel role for Glrx in post-ischaemic neovascularization, which could define a potential target for therapy of impaired angiogenesis in pathological conditions including diabetes.

Hydrogen sulphide rescues preeclampsia-like phenotype aggravated by high sFlt-1 in placenta growth factor (PlGF) deficiency

Placenta growth factor (PlGF) deficient mice are fertile at a Mendelian ratio. Interestingly, low maternal plasma levels of PlGF are strongly associated with early onset of preeclampsia, a pregnancy hypertensive disorder characterised by high blood pressure, proteinuria and fetal growth restriction. PlGF is increasingly being recognised as an early diagnostic biomarker, but the physiological importance of PlGF in the pathogenesis of preeclampsia is unknown. We investigated whether the decreased levels of PlGF in pregnancy exacerbate the fetal growth restriction associated with preeclampsia in the presence of high sFlt-1 and the potential of hydrogen sulphide to ameliorate these effects. Pregnant PlGF−/− mice were injected with adenovirus encoding sFlt-1 (Ad-sFlt-1) at 1 × 109 pfu/ml at E10.5 and mean arterial blood pressure (MAP), biochemical and histological analysis of maternal kidney, placenta and embryos were assessed at the end of pregnancy. Ad-sFlt-1 significantly increased MAP and induced severe glomerular endotheliosis in PlGF−/− mice compared to wild-type animals. Soluble Flt-1 also significantly elevated albumin–creatinine ratio and increased levels of urinary kidney injury molecule-1, a marker for proximal tubule injury. Furthermore, sFlt-1 over expression increased fetal resorption rate in the PlGF−/− mice and promoted abnormal placental vascularisation. To determine whether placental PlGF is critical for preventing fetal growth restriction associated with preeclampsia, we generated haploinsufficient PlGF+/− placentas and embryos in dams and exposed to high sFlt-1 environment. These mothers showed reduced fetal resorption, gestational hypertension and proteinuria when compared to pregnant PlGF−/− mice. Furthermore, treatment with hydrogen sulphide-releasing agent, GYY4137, significantly reduced resorption, hypertension and proteinuria observed in Ad-sFlt-1 treated pregnant PlGF−/− mice. Our study shows that placental PlGF is a critical protective factor against the damaging effects of high sFlt-1 associated with preeclampsia and activation of the hydrogen sulphide pathway may rescue preeclampsia phenotypes even under low PlGF environment.

Hydrogen sulphide rescues preeclampsia-like phenotype aggravated by high sFlt-1 in Placenta Growth Factor (PlGF) deficiency

INTRODUCTION: Low circulating levels of placenta growth factor (PlGF) is strongly associated with the onset of preeclampsia, a maternal hypertensive disorder characterized by high blood pressure and proteinuria after 20 weeks of gestation. Although, PlGF-deficient mice are born healthy and fertile at a Mendelian ratio, the physiological importance of PlGF in the pathogenesis of preeclampsia is unclear. We hypothesised that decreased levels of PlGF in pregnancy exacerbates the fetal growth restriction associated with preeclampsia in the presence of high sFlt-1. METHODS: Pregnant PlGF-/- mice were injected with adenovirus encoding sFlt-1 (Ad-sFlt-1) at high (i) 1.5x109 pfu/ml and low (ii) 0.5x109 pfu/ml doses. Mean arterial blood pressure (MBP), biochemical and histological assessments of maternal kidney, placenta and embryos were performed. RESULTS: Ad-sFlt-1 significantly increased MBP and induced severe glomerular endotheliosis in PlGF-/- mice at E10.5 gestation compared to wild-type animals. High sFlt-1 also significantly elevated albumincreatinine ratio and increased levels of urinary kidney injury molecule-1, a marker for proximal tubule injury.At a high dose of sFlt-1, there was complete fetal resorption in the pregnant PlGF-/- mice, and even the lower dose of sFlt-1 induced severe fetal resorption and abnormal placental vascularization. Hydrogen sulphide-releasing agent, GYY4137, significantly reduced resorption, hypertension and proteinuria in Ad-sFlt-1 treated pregnant PlGF-/- mice. To determine if placental PlGF is critical for preventing fetal growth restriction associated with preeclampsia, we generated haploinsufficient PlGF+/- placentas and embryos were generated in wild-time dams and exposed to high sFlt-1 environment. This resulted in reduced fetal resorption, gestational hypertension and proteinuria when compared to pregnant PlGF-/- mice. CONCLUSIONS: Placental PlGF is a critical protective factor against the damaging effects of high sFlt-1 in preeclampsia and the hydrogen sulphide pathway may rescue preeclampsia phenotypes.

The role of angiogenic and antiangiogenic factors in the second trimester in the prediction of preeclampsia in pregnant women with type 1 diabetes

OBJECTIVE To assess the association between circulating angiogenic and antiangiogenic factors in the second trimester and risk of preeclampsia in women with type 1 diabetes.

RESEARCH DESIGN AND METHODS Maternal plasma concentrations of placental growth factor (PlGF), soluble fms-like tyrosine kinase 1 (sFlt-1), and soluble endoglin (sEng) were available at 26 weeks of gestation in 540 women with type 1 diabetes enrolled in the Diabetes and Preeclampsia Intervention Trial.

RESULTS Preeclampsia developed in 17% of pregnancies (n = 94). At 26 weeks of gestation, women in whom preeclampsia developed later had significantly lower PlGF (median [interquartile range]: 231 pg/mL [120–423] vs. 365 pg/mL [237–582]; P < 0.001), higher sFlt-1 (1,522 pg/mL [1,108–3,393] vs. 1,193 pg/mL [844–1,630] P < 0.001), and higher sEng (6.2 ng/mL [4.9–7.9] vs. 5.1 ng/mL[(4.3–6.2]; P < 0.001) compared with women who did not have preeclampsia. In addition, the ratio of PlGF to sEng was significantly lower (40 [17–71] vs. 71 [44–114]; P < 0.001) and the ratio of sFlt-1 to PlGF was significantly higher (6.3 [3.4–15.7] vs. 3.1 [1.8–5.8]; P < 0.001) in women who later developed preeclampsia. The addition of the ratio of PlGF to sEng or the ratio of sFlt-1 to PlGF to a logistic model containing established risk factors (area under the curve [AUC], 0.813) significantly improved the predictive value (AUC, 0.850 and 0.846, respectively; P < 0.01) and significantly improved reclassification according to the integrated discrimination improvement index (IDI) (IDI scores 0.086 and 0.065, respectively; P < 0.001).

CONCLUSIONS These data suggest that angiogenic and antiangiogenic factors measured during the second trimester are predictive of preeclampsia in women with type 1 diabetes. The addition of the ratio of PlGF to sEng or the ratio of sFlt-1 to PlGF to established clinical risk factors significantly improves the prediction of preeclampsia in women with type 1 diabetes.

Preeclampsia is characterized by the development of hypertension and new-onset proteinuria during the second half of pregnancy (1,2), leading to increased maternal morbidity and mortality (3). Women with type 1 diabetes are at increased risk for development of preeclampsia during pregnancy, with rates being two-times to four-times higher than that of the background maternity population (4,5). Small advances have come from preventive measures, such as low-dose aspirin in women at high risk (6); however, delivery remains the only effective intervention, and preeclampsia is responsible for up to 15% of preterm births and a consequent increase in infant mortality and morbidity (7).

Although the etiology of preeclampsia remains unclear, abnormal placental vascular remodeling and placental ischemia, together with maternal endothelial dysfunction, hemodynamic changes, and renal pathology, contribute to its pathogenesis (8). In addition, over the past decade accumulating evidence has suggested that an imbalance between angiogenic factors, such as placental growth factor (PlGF), and antiangiogenic factors, such as soluble fms-like tyrosine kinase 1 (sFlt-1) and soluble endoglin (sEng), plays a key role in the pathogenesis of preeclampsia (8,9). In women at low risk (10–13) and women at high risk (14,15), concentrations of angiogenic and antiangiogenic factors are significantly different between women who later develop preeclampsia (lower PlGF, higher sFlt-1, and higher sEng levels) compared with women who do not.

Few studies have specifically focused on circulating angiogenic factors and risk of preeclampsia in women with diabetes, and the results have been conflicting. In a small study, higher sFlt-1 and lower PlGF were reported at the time of delivery in women with diabetes who developed preeclampsia (16). In a longitudinal prospective cohort of pregnant women with diabetes, Yu et al. (17) reported increased sFlt-1 and reduced PlGF in the early third trimester as potential predictors of preeclampsia in women with type 1 diabetes, but they did not show any difference in sEng levels in women with preeclampsia compared with women without preeclampsia. By contrast, Powers et al. (18) reported only increased sEng in the second trimester in women with pregestational diabetes who developed preeclampsia.

The aim of this study, which was significantly larger than the previous studies highlighted, was to assess the association between circulating angiogenic (PlGF) and antiangiogenic (sFlt-1 and sEng) factors and the risk of preeclampsia in women with type 1 diabetes. A further aim was to evaluate the added predictive ability and clinical usefulness of angiogenic factors and established risk factors for preeclampsia risk prediction in women with type 1 diabetes.

The reduction of serum soluble Flt-1 in patients with neovascular age-related macular degeneration

PURPOSE: To evaluate serum soluble Flt-1 (sFlt-1) in age-related degeneration (AMD) patients.

DESIGN: Case control study.

METHODS: Fifty-six non-AMD participants, fifty-three early AMD patients and ninety-seven neovascular AMD patients from Belfast in Northern Ireland. Serum samples were collected from each patient. Serum sFlt-1 was measured by human sVEGFR1/sFlt-1 ELISA kit. The results were analyzed by Excel and SPSS.

RESULTS: Serum sFlt-1 concentration of non-AMD, early AMD, and neovascular AMD were 90.8±2.9 pg/mL (±SEM), 88.2±2.6 pg/mL and 79.9±2.2 pg/mL. sFlt-1 from neovascular AMD patients was significantly decreased compared to non-AMD and early AMD patients (ANOVA, p<0.01). For each 10 point increase in sFlt-1, the odds for having neovascular AMD compared with non-AMD and neovascular AMD decreases by 27.8% OR=0.722 (95% CI: 0.588-0.888, p=0.002) and 27.0% OR=0.730 (95% CI: 0.594-0.898, p=0.003), respectively. In patients over 73 years of age, serum sFlt-1 <80 pg/mL was associated with a >6-fold higher risk of neovascular AMD.

CONCLUSIONS: Reduced serum sFlt-1 differentiates those patients with neovascular AMD from both early AMD and non-AMD participants. In those aged over 73, serum sFlt <80 pg/mL seems to indicate a particularly high risk of neovascular AMD. Our results indicate serum sFlt-1 could be a biomarker for development of neovascular AMD.

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.

Autocrine activity of soluble Flt-1 controls endothelial cell function and angiogenesis

Background - The negative feedback system is an important physiological regulatory mechanism controlling angiogenesis. Soluble vascular endothelial growth factor (VEGF) receptor-1 (sFlt-1), acts as a potent endogenous soluble inhibitor of VEGF- and placenta growth factor (PlGF)-mediated biological function and can also form dominant-negative complexes with competent full-length VEGF receptors. Methods and results - Systemic overexpression of VEGF-A in mice resulted in significantly elevated circulating sFlt-1. In addition, stimulation of human umbilical vein endothelial cells (HUVEC) with VEGF-A, induced a five-fold increase in sFlt-1 mRNA, a time-dependent significant increase in the release of sFlt-1 into the culture medium and activation of the flt-1 gene promoter. This response was dependent on VEGF receptor-2 (VEGFR-2) and phosphoinositide-3'-kinase signalling. siRNA-mediated knockdown of sFlt-1 in HUVEC stimulated the activation of endothelial nitric oxide synthase, increased basal and VEGF-induced cell migration and enhanced endothelial tube formation on growth factor reduced Matrigel. In contrast, adenoviral overexpression of sFlt-1 suppressed phosphorylation of VEGFR-2 at tyrosine 951 and ERK-1/-2 MAPK and reduced HUVEC proliferation. Preeclampsia is associated with elevated placental and systemic sFlt-1. Phosphorylation of VEGFR-2 tyrosine 951 was greatly reduced in placenta from preeclamptic patients compared to gestationally-matched normal placenta. Conclusion - These results show that endothelial sFlt-1 expression is regulated by VEGF and acts as an autocrine regulator of endothelial cell function.

Therapeutic potential of statins and the induction of heme oxygenase-1 in preeclampsia

Heme oxygenase (Hmox) is an endogenous system that offers protection against placental cytotoxic damage associated with preeclampsia. The Hmox1/carbon monoxide (CO) pathway inhibits soluble Flt-1 (sFlt-1) and soluble Endoglin (sEng). More importantly, statins induce Hmox1 and suppress the release of sFlt-1 and sEng; thus, statins and Hmox1 activators are potential novel therapeutic agents for treating preeclampsia. The contribution of the Hmox system to the pathogenesis of preeclampsia has been further indicated by the incidence of preeclampsia being reduced by a third in smokers, who had reduced levels of circulating sFlt-1. Interestingly, preeclamptic women exhale less CO compared with women with healthy pregnancies. Hmox1 is reduced prior to the increase in sFlt-1 as Hmox1 mRNA expression in the trophoblast is decreased in the first trimester in women who go on to develop preeclampsia. Induction of Hmox1 or exposure to CO or bilirubin has been shown to inhibit the release of sFlt-1 and sEng in animal models of preeclampsia. The functional benefit of statins and Hmox1 induction in women with preeclampsia is valid not only because they inhibit sFlt-1 release, but also because statins and Hmox1 are associated with anti-apoptotic, anti-inflammatory, and anti-oxidant properties. The StAmP trial is the first randomized control trial (RCT) evaluating the use of pravastatin to ameliorate severe preeclampsia. This proof-of-concept study will pave the way for future global RCT, the success of which will greatly contribute to achieving the United Nations Millennium Development Goals (MDG4 and MDG5) and offering an affordable and easily accessible therapy for preeclampsia. © 2014 The Authors.

Negative regulation of soluble Flt-1 and soluble endoglin release by heme oxygenase-1

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.