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.

Zelluläre Kontrolle adulter Neurogenese - Expression und Funktion des VEGF-Rezeptor-1 (Flt-1) im adulten Säugerhirn

Neurale Stammzellen sind im adulten Säugerhirn in der Subventrikulären Zone (SVZ) der Lateralventrikel und dem Hippokampus lokalisiert. In der SVZ entstandene neurale Zellen migrieren entlang eines von Astrozyten umgebenen Pfades, dem Rostralmigratorischen Strom (RMS), zum Olfaktorischen Bulbus (OB), wo sie zu olfaktorischen Interneuronen differenzieren. Vaskuläre Wachstumsfaktoren, wie VEGF-A beeinflussen die adulte Neurogenese. Die vorliegende Arbeit beschreibt erstmalig detailliert die spezifische Expression des VEGF-Rezeptor-1 (VEGFR-1) in den Regionen olfaktorischer und hippokampaler Neurogenese des adulten ZNS. Die Ergebnisse zeigen, dass VEGFR-1 im adulten Hirn hauptsächlich in GFAP-positiven Zellen in der SVZ, dem RMS, dem OB, dem Corpus callosum und dem Hippokampus exprimiert ist. In vivo-Analysen transgener Mäuse (Flt-1TK-/-), denen die Signaltransduktionsdomäne des VEGFR-1 fehlt, demonstrieren hier erstmals eine Rolle des VEGFR-1 in adulter Neurogenese. Flt-1TK-/- weisen eine erhöhte Proliferation neuronaler Vorläuferzellen der SVZ auf. Im RMS ist jedoch 6 Tage nach BrdU-Administration die Anzahl markierter Zellen im Vergleich zum Wildtyp (wt) um 47,97% reduziert, ohne dass es zu einer Akkumulation in der SVZ kommt. Zusammen mit der in Kulturversuchen stark erhöhten Migrationsgeschwindigkeit von Neuroblasten der Flt-1TK-/- und einer verminderten Abwanderung von Zellen aus dem RMS ins Corpus callosum der Flt-1Tk-/-, weist dies auf eine gesteigerte Migration zum OB hin. Tatsächlich war der OB der Flt-1TK-/-, vor allem die Plexiform- und Periglomerulärzellschicht (PGL), signifikant vergrößert. Im OB der transgenen Tiere migrieren zudem signifikant mehr BrdU-markierte Zellen in die PGL. Dort differenzieren signifikant mehr Neurone als im wt. Subtypisierungen zeigen, zudem eine erhöhte Differenzierung in dopaminerge Interneurone in der PGL der Flt-1TK-/-. Im Gehirn Flt-1TK-/- war die Konzentration von VEGF-A erhöht. Intrazerebroventrikuläre Infusion von VEGF-A in wt-Tiere erbrachte den eindeutigen Nachweis, dass die Erhöhung der VEGF-A-Konzentration im Gehirn der Flt-1TK-/- ursächlich für die in diesen Tieren beobachtete Reduktion der BrdU-positiven Zellen im RMS ist. Dies ist gleichzeitig der erste Nachweis einer Wirkung von VEGF-A auf Neuroblasten im RMS in vivo unter physiologischen Bedingungen. Die erhöhte VEGF-A-Konzentration könnte auch den anderen hier dargelegten Effekten zugrunde liegen. VEGFR-1 ist somit ein regulatorischer Faktor für die adulte olfaktorische Neurogenese und spielt eine potentielle Rolle in der Differenzierung dopaminerger Interneurone.

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.

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.

Glutaredoxin-1 Is up-regulated in preeclampsia and increases soluble Flt-1 expression

INTRODUCTION: Preeclampsia is a vascular disorder in pregnancyand is biochemical characterization by high soluble Flt-1 and lowplacenta growth factor as well as an imbalance in redox homeostasis.During conditions of high oxidative stress, cysteine residues on keyproteins are reversibly altered by S-glutathionylation, modifying theirfunction. Glutaredoxin-1 (Glrx) enzymatically catalyzes the removal of S-glutathione adducts, conferring reversible signaling dynamics toproteins with redox-sensitive cysteines. The role of Glrx in preeclampsiais unknown.METHODS: Immunohistochemistry and Western blot analysis for Glrx orglutathione were conducted on human placenta samples collected pre-termfrom early onset preeclamptic patients (n=10) or non-preeclamptic induceddeliveries (n=9). Human endothelial cells were infected with adenovirusencoding Glrx or LacZ prior to the cells being exposed to hypoxia (0.1%O2, 24h) to measure changes in soluble Flt-1 (sFlt-1). Quantitative PCRand ELISA were used to measure sFlt-1 at mRNA and protein level.RESULTS: Immunohistochemical staining for GSH revealed lowerS-glutathionylation adducts in preeclampsia placenta in comparison tocontrols. Glrx expression, which catalyses de-glutathionylation wasenhanced in early onset preeclampsia compared to pre-term controlsamples. In contrast, no change was observed in preeclamptic and IUGRplacentas at full term. In endothelial cells overexpressing Glrx, sFlt-1expression was dramatically enhanced at mRNA (3-fold P<0.05) andprotein level (5 fold P>0.01, n=4) after hypoxia andoverexpressing Glrxin mice enhanced levels of circulating sFlt-1 during in vivo ischemia.CONCLUSIONS: Enhanced Glrx expression in preeclamptic placentain line with an apparent decrease in S-glutathionylation may leavekey proteins susceptible to irreversible oxidation in conditions of highoxidative stress.

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.

Down-Regulation of Platelet Endothelial Cell Adhesion Molecule-1 Results in Thrombospondin-1 Expression and Concerted Regulation of Endothelial Cell Phenotype

bEND.3 cells are polyoma middle T-transformed mouse brain endothelial cells that express very little or no thrombospondin-1, a natural inhibitor of angiogenesis, but express high levels of platelet endothelial cell adhesion molecule-1 (PECAM-1) that localizes to sites of cell–cell contact. Here, we have examined the role of PECAM-1 in regulation of bEND.3 cell proliferation, migration, morphogenesis, and hemangioma formation. We show that down-regulating PECAM-1 expression by antisense transfection of bEND.3 cells has a dramatic effect on their morphology, proliferation, and morphogenesis on Matrigel. There is an optimal level for PECAM-1 expression such that high levels of PECAM-1 inhibit, whereas moderate levels of PECAM-1 stimulate, endothelial cell morphogenesis. The down-regulation of PECAM-1 in bEND.3 cells resulted in reexpression of endogenous thrombospondin-1 and its antiangiogenic receptor CD36. The expression of the vascular endothelial growth factor receptors flk-1 and flt-1, as well as integrins and metalloproteinases (which are involved in angiogenesis), were also affected. These observations are consistent with the changes observed in proliferation, migration, and adhesion characteristics of the antisense-transfected bEND.3 cells as well as with their lack of ability to form hemangiomas in mice. Thus, a reciprocal relationship exists between thrombospondin-1 and PECAM-1 expression, such that these two molecules appear to be constituents of a “switch” that regulates in concert many components of the angiogenic and differentiated phenotypes of endothelial cells.

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.

The role of heterodimerization between VEGFR-1 and VEGFR-2 in the regulation of endothelial cell homeostasis

VEGF-A activity is tightly regulated by ligand and receptor availability. Here we investigate the physiological function of heterodimers between VEGF receptor-1 (VEGFR-1; Flt-1) and VEGFR-2 (KDR; Flk-1) (VEGFR(1-2)) in endothelial cells with a synthetic ligand that binds specifically to VEGFR(1-2). The dimeric ligand comprises one VEGFR-2-specific monomer (VEGF-E) and a VEGFR-1-specific monomer (PlGF-1). Here we show that VEGFR(1-2) activation mediates VEGFR phosphorylation, endothelial cell migration, sustained in vitro tube formation and vasorelaxation via the nitric oxide pathway. VEGFR(1-2) activation does not mediate proliferation or elicit endothelial tissue factor production, confirming that these functions are controlled by VEGFR-2 homodimers. We further demonstrate that activation of VEGFR(1-2) inhibits VEGF-A-induced prostacyclin release, phosphorylation of ERK1/2 MAP kinase and mobilization of intracellular calcium from primary endothelial cells. These findings indicate that VEGFR-1 subunits modulate VEGF activity predominantly by forming heterodimer receptors with VEGFR-2 subunits and such heterodimers regulate endothelial cell homeostasis.

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.

Antiangiogenic effect of soluble vascular endothelial growth factor receptor-1 in placental angiogenesis

Differential splicing of the flt-1 mRNA generates soluble variant of vascular endothelial growth factor (VEGF) receptor-1 (sVEGFR-1, also known as sFlt-1). The action of VEGF is antagonized by sVEGFR-1. Soluble VEGFR-1 binds to VEGF with a high affinity and therefore works to modulate VEGF and VEGF signaling pathway. In this study, the authors tested the hypothesis that VEGF-mediated endothelial cell angiogenesis is tightly modulated by the release of sVEGFR-1 and placental expression of sVEGFR-1 is upregulated by hypoxia. Immunolocalization studies showed progressively intense staining for sVEGFR-1 and VEGF in the trophoblast of placental villous explants throughout gestation. Endothelial cell migration studies using a modified Boyden's chamber showed a significant increase in cell migration in response to VEGF that was significantly attenuated in the presence of exogenous sVEGFR-1. Furthermore, stimulation of endothelial cells with VEGF led to a dose-dependent increase in the release of sVEGFR-1 as determined by enzyme-linked immunosorbent assay (ELISA). Exposure of normal placental villous explants to hypoxia (1% pO2) increased trophoblast expression of sVEGFR-1 when compared with tissue normoxia (5% pO2). In addition, conditioned media from hypoxia treated placental villous explants induced a significant increase in endothelial cell migration that was significantly reduced in presence of sVEGFR-1. Our study demonstrates that hypoxia positively regulates sVEGFR-1 protein expression in ex vivo trophoblasts, which control VEGF-driven angiogenesis.

Heparin elevates circulating soluble fms-like tyrosine kinase-1 immunoreactivity in pregnant women receiving anticoagulation therapy

Background—Alterations in circulating levels of pro- and antiangiogenic factors have been associated with adverse pregnancy outcomes. Heparin is routinely administered to pregnant women, but without clear knowledge of its impact on these factors. Methods and Results—We conducted a longitudinal study of 42 pregnant women. Twenty-one women received prophylactic heparin anticoagulation, and 21 healthy pregnant women served as controls. Compared with gestational age-matched controls, heparin treatment was associated with increased circulating levels of soluble fms-like tyrosine kinase-1 (sFlt-1) in the third trimester (P<0.05), in the absence of preeclampsia, placental abruption, or fetal growth restriction. Heparin had no effect on circulating levels of vascular endothelial growth factor, placenta growth factor, or soluble endoglin as assessed by ELISA. In vitro, low-molecular weight and unfractionated heparins stimulated sFlt-1 release from placental villous explants, in a dose- and time-dependent manner. This effect was not due to placental apoptosis, necrosis, alteration in protein secretion, or increased transcription. Western blot analysis demonstrated that heparin induced shedding of the N-terminus of Flt-1 both in vivo and in vitro as indicated by a predominant band of 100–112 kDa. By using an in vitro angiogenesis assay, we demonstrated that serum of heparin-treated cases inhibited both basal and vascular endothelial growth factor-induced capillary-like tube formation. Conclusions—Heparin likely increases the maternal sFlt-1 through shedding of the extracellular domain of Flt-1 receptor. Our results imply that upregulation of circulating sFlt-1 immunoreactivity in pregnancy is not always associated with adverse outcomes, and that heparin's protective effects, if any, cannot be explained by promotion of angiogenesis.

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.