Autocrine activity of soluble Flt-1 controls endothelial cell function and angiogenesis
- Shakil Ahmad†1,
- Peter W Hewett†2,
- Bahjat Al-Ani2,
- Samir Sissaoui2,
- Takeshi Fujisawa1,
- Melissa J Cudmore1 and
- Asif Ahmed1, 2Email author
© Ahmad et al; licensee BioMed Central Ltd. 2011
Received: 4 April 2011
Accepted: 13 July 2011
Published: 13 July 2011
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.
These results show that endothelial sFlt-1 expression is regulated by VEGF and acts as an autocrine regulator of endothelial cell function.
Vascular endothelial growth factor-A (VEGF-A) is a multifunctional cytokine induced by hypoxic stress . It plays a pivotal role in many aspects of embryonic cardiovascular development, including formation of blood vessels, cardiac morphogenesis, and development of the nervous system [2–6]. Loss and gain of function studies in mice indicate that VEGF-A levels have to be maintained within a narrow range to ensure proper cardiovascular development and embryo survival [7–9]. It has been shown that the effects of VEGF-A can be deleterious if uncontrolled. Over-expression of VEGF in experimental animals increases the leakiness of blood vessels, which may lead to severe edema, loss of limb and death [10, 11]. Excess VEGF-A expression in skeletal muscle results in the induction of vascular tumors (hemangiomas) [12–14], whereas loss of VEGF-A activity due to increased production of its natural antagonist, sFlt-1 (soluble VEGF receptor-1/sVEGFR-1), as in preeclampsia, reduces angiogenesis . Thus, homeostasis requires mechanisms to regulate the functional activity of VEGF-A.
Soluble Flt-1 is generated by alternative splicing of the fms-like tyrosine kinase (flt-1) gene , and binds to all isoforms of VEGF-A and placenta growth factor (PlGF) with high affinity [16, 17]. It acts as a potent soluble inhibitor of both VEGF-A and PlGF-mediated biological activities  and can also form dominant-negative complexes with competent full-length VEGF receptors . In pregnancies complicated with preeclampsia, sFlt-1 levels are elevated [15, 19–21]. Maternal serum levels of sFlt-1 are elevated five weeks prior to the onset of preeclampsia , supporting the premise that sFlt-1 is a key factor responsible for the clinical manifestation of this disorder . The demonstration that sFlt-1 is fundamental to the clinical onset of preeclampsia  highlights the importance of understanding the intracellular mechanism underlying its regulation and release in endothelial cells. Recently it was shown that autocrine VEGF signaling is required for vascular homeostasis . Here we demonstrate that endothelial sFlt-1 expression is regulated by VEGF and sFlt-1 is an autocrine regulator of endothelial cell function.
Materials and methods
Recombinant growth factors were purchased from RELIATech (Brauschweig, Germany). Rabbit polyclonal antibodies against phospho- endothelial nitric oxide synthase (eNOS) at serine-1177 (p-eNOSSer1177), phospho-ERK-1/-2 MAPK and phospho-VEGF receptor-2 (VEGFR-2) tyrosine-951 antibodies were purchased from Calbiochem (Nottingham, UK). Small inhibitory RNAs (siRNA) and oligonucleotide primers were purchased from Eurogentec (Southampton, UK). Luciferase reporter assay and cDNA synthesis kits were from Promega (Southampton, UK). All other cell culture reagents and chemicals were obtained from Sigma Aldrich (Poole, UK).
Human placental tissue was obtained from normal pregnancies and gestationally-matched pregnancies complicated by preeclampsia. Preeclampsia was defined as blood pressure > 140/90 mm Hg on at least two consecutive measurements and proteinuria of at least 300 mg per 24 hours. Informed consent was obtained from the patients and the study had the approval of the South Birmingham Ethical Committee (Birmingham, UK).
Primary human umbilical vein endothelial cells (HUVEC) were isolated and cultured as described . Cells were used at passage two or three for experiments and serum-starved in endothelial cell serum-free medium (Gibco-BRL, UK) supplemented with 0.2% bovine serum albumin for 24 hours prior to stimulation.
Adenoviral gene transfer
Quantitative Real-Time PCR
Sample preparation and real-time PCR was performed as described previously . Briefly, mRNA was prepared using TRIzol and DNase-1 digestion/purification on RNAeasy columns (Qiagen), and reverse transcribed with the cDNA Synthesis Kit (Promega). Triplicate cDNA samples and standards were amplified in SensiMix containing SYBR green (Quantace) with primers specific for sFlt-1 . The mean threshold cycle (CT) was normalized to β-actin and expressed relative to control.
siRNA knock-down of sFlt-1
Two siRNA sequences to the unique 3' sequence of sFlt-1 (sFlt-1 A sense: 5'-TAACAGUUGUCUCAUAUCAtt-3' and antisense: 5'-UGAUAUGAGACAACUGUUAtt-3'; sFlt-1 B sense: 5'-UCUCGGAUCUCCAAAUUUAtt-3' and antisense 5'-UAAAUUUGGAGAUCCGAGAtt-3') were designed using the Dharmacon siDESIGN tool . HUVEC (~ 1 × 106 cells) were electroporated with ~ 3 μg of sFlt-1, or a universal control siRNA (Dharmacon) using the HUVEC kit II and Amaxa nucleofector (Amaxa GmbH, Cologne, Germany) as described .
Transduction of chimeric VEGF Receptors in HUVEC
A chimeric VEGF/epidermal growth factor (EGF) receptor comprising the intracellular and transmembrane domains of VEGFR-2 fused to the extracellular domain of the human EGF receptor . EGF does not bind to VEGF receptors, therefore, it does not activate the endogenous VEGF receptors. EGDR and its tyrosine-to-phenylalanine mutants (EGDR-Y951F) were generated and cloned into the pMMP retroviral vector, and retrovirus-containing cell supernatant was harvested and used immediately to infect HUVEC . Following 16 hours of incubation, the medium was replaced with fresh growth medium and the HUVEC were used 48 hours after infection.
Nitric oxide (NO) Release
Total NO in conditioned media was assayed as nitrite, the stable breakdown product of NO, using a Sievers NO chemiluminescence analyzer (Analytix, Sunderland, UK) as described previously .
Tube Formation Assay
The formation of capillary-like structures was examined on growth factor-reduced Matrigel in 24-well plates as described previously . Tube formation was quantified by measuring the total tube length in five random x200 power fields per well using a Nikon phase-contrast inverted microscope with Image ProPlus image analysis software (Media Cybernetics, Silver Spring, USA). Mean total tube length was calculated from three independent experiments performed in duplicate.
flt-1gene promoter activity assay
A 1.3 Kb fragment of the human flt-1 gene corresponding to -1214 to +155 bp relative to the first exon in the pGL2 luciferase vector (Promega) was used to determine flt-1 promoter activity . Briefly, porcine aortic endothelial cells (PAEC) were transfected with the flt-1 promoter-reporter construct using Exgen 500 (Fermentas, UK) and the cell lysates assayed as described previously .
Cells lysates were immunoblotted as described previously . Membranes were probed with rabbit polyclonal antibodies against phospho-eNOS-Ser1177, anti-ERK-1/-2 or anti-VEGFR-2 phosphotyrosine-951 at 4°C overnight. Proteins were visualised using the ECL detection kit (Amersham-Pharmacia, UK).
Soluble Flt-1 (sFlt-1) levels in culture supernatants were measured as previously described .
Formalin-fixed, paraffin-embedded tissues were used for immunohistochemistry as previously described .
All data are expressed as mean ± SEM. Statistical comparisons were performed using one-way ANOVA followed by the Student-Newman-Keuls test as appropriate. Statistical significance was set at a value of p < 0.05.
Results and Discussion
VEGF-A stimulates sFlt-1 release
Activation of VEGFR-2, mediates the release of sFlt-1
VEGF stimulated sFlt-1 production, is mediated via PI3K
To investigate the role of the PI3K pathway in VEGF-A-induced sFlt-1 release, PI3K activity was inhibited through overexpression of PTEN (Phosphatase and Tensin homolog deleted on chromosome Ten), which dephosphorylates phosphatidylinositol 3,4,5-triphosphate and has been shown to reduce VEGF-mediated signaling and cellular function [28, 35]. HUVEC were infected overnight with an adenovirus encoding PTEN (PTEN(wt)) or a control adenovirus (CMV) and stimulated with VEGF-A for 24 hours. Inhibition of PI3K activity by PTEN overexpression led to a significant decrease in sFlt-1 release (Figure 2c). Furthermore, pre-incubation of HUVEC with LY294002, a pharmacological PI3K inhibitor, also attenuated the VEGF mediated release of sFlt-1 (Figure 2d) and of flt-1 gene promoter activity (Figure 2e).
Loss of sFlt-1 promotes angiogenesis
Excess sFlt-1 inhibits VEGFR-2 Y951 phosphorylation
Endothelial cell sFlt-1 expression is regulated by VEGF and sFlt-1 is an autocrine regulator of endothelial cell function.
- EGF :
epidermal growth factor
- eNOS :
endothelial nitric oxide synthase
- ERK-1/-2 :
extracellular signal regulated kinase -1/2
- HUVEC :
human umbilical vein endothelial cells
- MAPK :
mitogen activated kinase
- NO :
- PI3K :
- PlGF :
placenta growth factor
- PTEN :
Phosphatase and Tensin homolog deleted on chromosome Ten
- sFlt-1 :
soluble vascular endothelial growth factor receptor-1
- VEGF-A :
vascular endothelial growth factor-A
- VEGFR-1 :
vascular endothelial growth factor receptor-1
- VEGFR-2 :
vascular endothelial growth factor receptor-2.
Acknowledgements and funding
This work was supported by grants from the Medical Research Council (G0600270, G0601295 and G0700288) and British Heart Foundation (RG/09/001/25940 and PG/06/114).
- Shweiki D, Itin A, Soffer D, Keshet E: Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis. Nature. 1992, 359: 843-845. 10.1038/359843a0.View ArticlePubMedGoogle Scholar
- Dor Y, Camenisch TD, Itin A, Fishman GI, McDonald JA, Carmeliet P, Keshet E: A novel role for VEGF in endocardial cushion formation and its potential contribution to congenital heart defects. Development. 2001, 128: 1531-1538.PubMedGoogle Scholar
- Ferrara N, Gerber HP, LeCouter J: The biology of VEGF and its receptors. Nat Med. 2003, 9: 669-676. 10.1038/nm0603-669.View ArticlePubMedGoogle Scholar
- Giordano FJ, Gerber HP, Williams SP, VanBruggen N, Bunting S, Ruiz-Lozano P, Gu Y, Nath AK, Huang Y, Hickey R, et al: A cardiac myocyte vascular endothelial growth factor paracrine pathway is required to maintain cardiac function. Proc Natl Acad Sci USA. 2001, 98: 5780-5785. 10.1073/pnas.091415198.PubMed CentralView ArticlePubMedGoogle Scholar
- Oosthuyse B, Moons L, Storkebaum E, Beck H, Nuyens D, Brusselmans K, Van Dorpe J, Hellings P, Gorselink M, Heymans S, et al: Deletion of the hypoxia-response element in the vascular endothelial growth factor promoter causes motor neuron degeneration. Nat Genet. 2001, 28: 131-138. 10.1038/88842.View ArticlePubMedGoogle Scholar
- Storkebaum E, Lambrechts D, Dewerchin M, Moreno-Murciano MP, Appelmans S, Oh H, Van Damme P, Rutten B, Man WY, De Mol M, et al: Treatment of motoneuron degeneration by intracerebroventricular delivery of VEGF in a rat model of ALS. Nat Neurosci. 2005, 8: 85-92. 10.1038/nn1360.View ArticlePubMedGoogle Scholar
- Carmeliet P, Ferreira V, Breier G, Pollefeyt S, Kieckens L, Gertsenstein M, Fahrig M, Vandenhoeck A, Harpal K, Eberhardt C, et al: Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele. Nature. 1996, 380: 435-439. 10.1038/380435a0.View ArticlePubMedGoogle Scholar
- Ferrara N, Carver-Moore K, Chen H, Dowd M, Lu L, O'Shea KS, Powell-Braxton L, Hillan KJ, Moore MW: Heterozygous embryonic lethality induced by targeted inactivation of the VEGF gene. Nature. 1996, 380: 439-442. 10.1038/380439a0.View ArticlePubMedGoogle Scholar
- Miquerol L, Langille BL, Nagy A: Embryonic development is disrupted by modest increases in vascular endothelial growth factor gene expression. Development. 2000, 127: 3941-3946.PubMedGoogle Scholar
- Masaki I, Yonemitsu Y, Yamashita A, Sata S, Tanii M, Komori K, Nakagawa K, Hou X, Nagai Y, Hasegawa M, et al: Angiogenic gene therapy for experimental critical limb ischemia: acceleration of limb loss by overexpression of vascular endothelial growth factor 165 but not of fibroblast growth factor-2. Circ Res. 2002, 90: 966-973. 10.1161/01.RES.0000019540.41697.60.View ArticlePubMedGoogle Scholar
- Vajanto I, Rissanen TT, Rutanen J, Hiltunen MO, Tuomisto TT, Arve K, Narvanen O, Manninen H, Rasanen H, Hippelainen M, et al: Evaluation of angiogenesis and side effects in ischemic rabbit hindlimbs after intramuscular injection of adenoviral vectors encoding VEGF and LacZ. J Gene Med. 2002, 4: 371-380. 10.1002/jgm.287.View ArticlePubMedGoogle Scholar
- Carmeliet P: VEGF gene therapy: stimulating angiogenesis or angioma-genesis?. Nat Med. 2000, 6: 1102-1103. 10.1038/80430.View ArticlePubMedGoogle Scholar
- Lee RJ, Springer ML, Blanco-Bose WE, Shaw R, Ursell PC, Blau HM: VEGF gene delivery to myocardium: deleterious effects of unregulated expression. Circulation. 2000, 102: 898-901.View ArticlePubMedGoogle Scholar
- Springer ML, Chen AS, Kraft PE, Bednarski M, Blau HM: VEGF gene delivery to muscle: potential role for vasculogenesis in adults. Mol Cell. 1998, 2: 549-558. 10.1016/S1097-2765(00)80154-9.View ArticlePubMedGoogle Scholar
- Ahmad S, Ahmed A: Elevated placental soluble vascular endothelial growth factor receptor-1 inhibits angiogenesis in preeclampsia. Circ Res. 2004, 95: 884-891. 10.1161/01.RES.0000147365.86159.f5.View ArticlePubMedGoogle Scholar
- Kendall RL, Wang G, Thomas KA: Identification of a natural soluble form of the vascular endothelial growth factor receptor, FLT-1, and its heterodimerization with KDR. Biochem Biophys Res Commun. 1996, 226: 324-328. 10.1006/bbrc.1996.1355.View ArticlePubMedGoogle Scholar
- Park JE, Chen HH, Winer J, Houck KA, Ferrara N: Placenta growth factor. Potentiation of vascular endothelial growth factor bioactivity, in vitro and in vivo, and high affinity binding to Flt-1 but not to Flk-1/KDR. J Biol Chem. 1994, 269: 25646-25654.PubMedGoogle Scholar
- Roeckl W, Hecht D, Sztajer H, Waltenberger J, Yayon A, Weich HA: Differential binding characteristics and cellular inhibition by soluble VEGF receptors 1 and 2. Exp Cell Res. 1998, 241: 161-170. 10.1006/excr.1998.4039.View ArticlePubMedGoogle Scholar
- Ahmad S AA: Regulation of soluble VEGFR-1 by VEGF and oxygen and its elevation in pre-eclampsia and fetal growth restriction. Placenta . 2001, 22 (A7): (Editor ed.^eds.), CityGoogle Scholar
- Zhou Y, McMaster M, Woo K, Janatpour M, Perry J, Karpanen T, Alitalo K, Damsky C, Fisher SJ: Vascular endothelial growth factor ligands and receptors that regulate human cytotrophoblast survival are dysregulated in severe preeclampsia and hemolysis, elevated liver enzymes, and low platelets syndrome. Am J Pathol. 2002, 160: 1405-1423. 10.1016/S0002-9440(10)62567-9.PubMed CentralView ArticlePubMedGoogle Scholar
- Vuorela P, Helske S, Hornig C, Alitalo K, Weich H, Halmesmaki E: Amniotic fluid--soluble vascular endothelial growth factor receptor-1 in preeclampsia. Obstet Gynecol. 2000, 95: 353-357. 10.1016/S0029-7844(99)00565-7.View ArticlePubMedGoogle Scholar
- Levine RJ, Maynard SE, Qian C, Lim KH, England LJ, Yu KF, Schisterman EF, Thadhani R, Sachs BP, Epstein FH, et al: Circulating angiogenic factors and the risk of preeclampsia. N Engl J Med. 2004, 350: 672-683. 10.1056/NEJMoa031884.View ArticlePubMedGoogle Scholar
- Ahmed A, Dunk C, Kniss D, Wilkes M: Role of VEGF receptor-1 (Flt-1) in mediating calcium-dependent nitric oxide release and limiting DNA synthesis in human trophoblast cells. Lab Invest. 1997, 76: 779-791.PubMedGoogle Scholar
- Maynard SE, Min JY, Merchan J, Lim KH, Li J, Mondal S, Libermann TA, Morgan JP, Sellke FW, Stillman IE, et al: Excess placental soluble fms-like tyrosine kinase 1 (sFlt1) may contribute to endothelial dysfunction, hypertension, and proteinuria in preeclampsia. J Clin Invest. 2003, 111: 649-658.PubMed CentralView ArticlePubMedGoogle Scholar
- Lee S, Chen TT, Barber CL, Jordan MC, Murdock J, Desai S, Ferrara N, Nagy A, Roos KP, Iruela-Arispe ML: Autocrine VEGF signaling is required for vascular homeostasis. Cell. 2007, 130: 691-703. 10.1016/j.cell.2007.06.054.PubMed CentralView ArticlePubMedGoogle Scholar
- Bussolati B, Dunk C, Grohman M, Kontos CD, Mason J, Ahmed A: Vascular endothelial growth factor receptor-1 modulates vascular endothelial growth factor-mediated angiogenesis via nitric oxide. Am J Pathol. 2001, 159: 993-1008. 10.1016/S0002-9440(10)61775-0.PubMed CentralView ArticlePubMedGoogle Scholar
- Tseng JF, Farnebo FA, Kisker O, Becker CM, Kuo CJ, Folkman J, Mulligan RC: Adenovirus-mediated delivery of a soluble form of the VEGF receptor Flk1 delays the growth of murine and human pancreatic adenocarcinoma in mice. Surgery. 2002, 132: 857-865. 10.1067/msy.2002.127680.View ArticlePubMedGoogle Scholar
- Cai J, Ahmad S, Jiang WG, Huang J, Kontos CD, Boulton M, Ahmed A: Activation of vascular endothelial growth factor receptor-1 sustains angiogenesis and Bcl-2 expression via the phosphatidylinositol 3-kinase pathway in endothelial cells. Diabetes. 2003, 52: 2959-2968. 10.2337/diabetes.52.12.2959.View ArticlePubMedGoogle Scholar
- Bergmann A, Ahmad S, Cudmore M, Gruber AD, Wittschen P, Lindenmaier W, Christofori G, Gross V, Gonzalves A, Grone HJ, et al: Reduction of circulating soluble Flt-1 alleviates preeclampsia-like symptoms in a mouse model. J Cell Mol Med. 2010, 14: 1857-1867.PubMed CentralView ArticlePubMedGoogle Scholar
- Cudmore M, Ahmad S, Al-Ani B, Fujisawa T, Coxall H, Chudasama K, Devey LR, Wigmore SJ, Abbas A, Hewett PW, Ahmed A: Negative regulation of soluble Flt-1 and soluble endoglin release by heme oxygenase-1. Circulation. 2007, 115: 1789-1797. 10.1161/CIRCULATIONAHA.106.660134.View ArticlePubMedGoogle Scholar
- Zhou CC, Ahmad S, Mi T, Xia L, Abbasi S, Hewett PW, Sun C, Ahmed A, Kellems RE, Xia Y: Angiotensin II induces soluble fms-Like tyrosine kinase-1 release via calcineurin signaling pathway in pregnancy. Circ Res. 2007, 100: 88-95. 10.1161/01.RES.0000254703.11154.18.PubMed CentralView ArticlePubMedGoogle Scholar
- Reynolds A, Leake D, Boese Q, Scaringe S, Marshall WS, Khvorova A: Rational siRNA design for RNA interference. Nat Biotechnol. 2004, 22: 326-330. 10.1038/nbt936.View ArticlePubMedGoogle Scholar
- Ahmad S, Hewett PW, Wang P, Al-Ani B, Cudmore M, Fujisawa T, Haigh JJ, le Noble F, Wang L, Mukhopadhyay D, Ahmed A: Direct evidence for endothelial vascular endothelial growth factor receptor-1 function in nitric oxide-mediated angiogenesis. Circ Res. 2006, 99: 715-722. 10.1161/01.RES.0000243989.46006.b9.View ArticlePubMedGoogle Scholar
- Al-Ani B, Hewett PW, Cudmore MJ, Fujisawa T, Saifeddine M, Williams H, Ramma W, Sissaoui S, Jayaraman PS, Ohba M, et al: Activation of proteinase-activated receptor 2 stimulates soluble vascular endothelial growth factor receptor 1 release via epidermal growth factor receptor transactivation in endothelial cells. Hypertension. 2010, 55: 689-697. 10.1161/HYPERTENSIONAHA.109.136333.View ArticlePubMedGoogle Scholar
- Huang J, Kontos CD: PTEN modulates vascular endothelial growth factor-mediated signaling and angiogenic effects. J Biol Chem. 2002, 277: 10760-10766. 10.1074/jbc.M110219200.View ArticlePubMedGoogle Scholar
- Papapetropoulos A, Garcia-Cardena G, Madri JA, Sessa WC: Nitric oxide production contributes to the angiogenic properties of vascular endothelial growth factor in human endothelial cells. J Clin Invest. 1997, 100: 3131-3139. 10.1172/JCI119868.PubMed CentralView ArticlePubMedGoogle Scholar
- Murohara T, Asahara T, Silver M, Bauters C, Masuda H, Kalka C, Kearney M, Chen D, Symes JF, Fishman MC, et al: Nitric oxide synthase modulates angiogenesis in response to tissue ischemia. J Clin Invest. 1998, 101: 2567-2578. 10.1172/JCI1560.PubMed CentralView ArticlePubMedGoogle Scholar
- Kroll J, Waltenberger J: VEGF-A induces expression of eNOS and iNOS in endothelial cells via VEGF receptor-2 (KDR). Biochem Biophys Res Commun. 1998, 252: 743-746. 10.1006/bbrc.1998.9719.View ArticlePubMedGoogle Scholar
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