scholarly journals Vascular Endothelial Growth Factor Receptors in Human Mesangiumin Vitroand in Glomerular Disease

2000 ◽  
Vol 11 (7) ◽  
pp. 1236-1243 ◽  
Author(s):  
STEPHEN THOMAS ◽  
JOHANN VANUYSTEL ◽  
GABRIELLA GRUDEN ◽  
VERÓNICA RODRÍGUEZ ◽  
DAVINA BURT ◽  
...  
Keyword(s):  
Vascular Endothelial Growth Factor ◽  
Growth Factor ◽  
Ex Vivo ◽  
Fold Increase ◽  
Vascular Endothelial ◽  
Vegf Receptors ◽  
Glomerular Diseases ◽  
Neuropilin 1 ◽  
Endothelial Growth Factor

Abstract. Mesangial cell proliferation and growth factor over-expression are characteristic features of several glomerular diseases. Vascular endothelial growth factor (VEGF), a potent mitogen, is expressed in podocytes in the glomerulus, and VEGF receptors (flt-1, KDR, and neuropilin-1) are present on endothelial cells and other cell types. This study examined whether human mesangial cells (HMC) express VEGF receptorsin vitroandex vivoand evaluated the effect of VEGF on HMC proliferation. All receptor types were detected in HMCin vitroby immunofluorescence and Western blotting. VEGF165induced a dose-responsive increase in3H-thymidine incorporation (25 ng/ml VEGF165: 2.3-fold increase ; 50 ng/ml : 3.8-fold ; 100 ng/ml : 4.8-fold ; 200 ng/ml : 3.4-fold ;P= 0.016) and in cell number (50 ng/ml VEGF165: 1.2-fold increase ; 100 ng/ml : 1.6-fold ; 200 ng/ml : 1.4-fold ;P= 0.005), effects prevented by an anti-VEGF165polyclonal neutralizing antibody (100 μg/ml). The proliferative effect was confirmed by a tetrazolium dye-based assay (100 ng/ml VEGF165: 1.4-fold increase). Inex vivoexperiments, VEGF receptors in biopsy material from normal and diseased kidneys were detected by immunohistochemistry. No mesangial flt-1 receptor staining was seen in normal renal cortical tissue samples, and only weak mesangial KDR staining was detected. In contrast, mesangial flt-1 and KDR receptor staining were both clearly seen in biopsy samples from proliferative renal diseases. In conclusion, flt-1, KDR, and neuropilin-1 are present on cultured HMC, and VEGF165induces HMC proliferation. In addition, the flt-1 and KDR receptors are expressed in the mesangium in mesangioproliferative disease.

Direct identification of a major autophosphorylation site on vascular endothelial growth factor receptor Flt-1 that mediates phosphatidylinositol 3′-kinase binding

2001 ◽  
Vol 358 (2) ◽  
pp. 465-472 ◽  
Author(s):  
Ying YU ◽  
Jeffrey D. HULMES ◽  
Mark T. HERLEY ◽  
Ronald G. WHITNEY ◽  
John W. CRABB ◽  
...  
Keyword(s):  
Vascular Endothelial Growth Factor ◽  
Growth Factor ◽  
Insect Cells ◽  
Pi3 Kinase ◽  
Vascular Endothelial ◽  
Vegf Receptors ◽  
Phosphatidylinositol 3 Kinase ◽  
Endothelial Growth Factor ◽  
Autophosphorylation Site

Progress has been made in our understanding of the mechanism by which the binding of vascular endothelial growth factor (VEGF) to cognate receptors induces a range of biological responses, but it is far from complete. Identification of receptor autophosphorylation sites will allow us to determine how activated VEGF receptors are coupled to specific downstream signalling proteins. In the present study, we have expressed human VEGF receptors in insect cells using the baculovirus expression system, identified a major autophosphorylation site on the VEGF receptor fms-like tyrosine kinase-1 (Flt-1) by HPLC–electrospray ionization (ESI)–MS, and characterized in vitro interactions between Flt-1 and phosphatidylinositol 3′-kinase (PI3-kinase). Infection of High 5 insect cells with Flt-1 recombinant virus resulted in the expression of a 170kDa glycoprotein, which bound VEGF with a Kd of 2×10−10M in intact insect cells. The overexpressed recombinant Flt-1 receptors exhibited tyrosine kinase activity and were constitutively phosphorylated. Analysis of Flt-1 tryptic peptides by HPLC–ESI–MS with selective phosphate ion monitoring identified a hexapeptide (YVNAFK; where single-letter amino-acid code has been used) containing a phosphotyrosine (pTyr) residue at position 1213. Using synthetic phosphopeptides, this pTyr residue was found to be directly involved in the binding of PI3-kinase in vitro even though it did not fall within a consensus pYM/VXM PI3-kinase binding motif. These results suggest that phosphorylated Flt-1 associates with PI3-kinase at pTyr1213 to mediate the activation of this pathway in VEGF signalling.

scholarly journals Triazolopeptides Inhibiting the Interaction between Neuropilin-1 and Vascular Endothelial Growth Factor-165

Molecules ◽  
2019 ◽  
Vol 24 (9) ◽  
pp. 1756 ◽  
Author(s):  
Bartlomiej Fedorczyk ◽  
Piotr F. J. Lipiński ◽  
Anna K. Puszko ◽  
Dagmara Tymecka ◽  
Beata Wilenska ◽  
...  
Keyword(s):  
Vascular Endothelial Growth Factor ◽  
Growth Factor ◽  
Inhibitory Activity ◽  
General Structure ◽  
Peptide Bond ◽  
Vascular Endothelial ◽  
Present Contribution ◽  
Neuropilin 1 ◽  
Endothelial Growth Factor

Inhibiting the interaction of neuropilin-1 (NRP-1) with vascular endothelial growth factor (VEGF) has become an interesting mechanism for potential anticancer therapies. In our previous works, we have obtained several submicromolar inhibitors of this interaction, including branched pentapeptides of general structure Lys(Har)-Xxx-Xxx-Arg. With the intent to improve the proteolytic stability of our inhibitors, we turned our attention to 1,4-disubstituted 1,2,3-triazoles as peptide bond isosteres. In the present contribution, we report the synthesis of 23 novel triazolopeptides along with their inhibitory activity. The compounds were synthesized using typical peptide chemistry methods, but with a conversion of amine into azide completely on solid support. The inhibitory activity of the synthesized derivatives spans from 9.2% to 58.1% at 10 μM concentration (the best compound Lys(Har)-GlyΨ[Trl]GlyΨ[Trl]Arg, 3, IC50 = 8.39 μM). Synthesized peptidotriazoles were tested for stability in human plasma and showed remarkable resistance toward proteolysis, with half-life times far exceeding 48 h. In vitro cell survival test resulted in no significant impact on bone marrow derived murine cells 32D viability. By means of molecular dynamics, we were able to propose a binding mode for compound 3 and discuss the observed structure–activity relationships.

scholarly journals Activating CD137 Signaling Promotes Sprouting Angiogenesis via Increased VEGFA Secretion and the VEGFR2/Akt/eNOS Pathway

2020 ◽  
Vol 2020 ◽  
pp. 1-19
Author(s):  
Bo Li ◽  
Yue Zhang ◽  
Runting Yin ◽  
Wei Zhong ◽  
Rui Chen ◽  
...  
Keyword(s):  
Vascular Endothelial Growth Factor ◽  
Growth Factor ◽  
Ex Vivo ◽  
Growth Factor Receptor ◽  
Aortic Ring ◽  
Vascular Endothelial ◽  
Knock Out ◽  
Sprouting Angiogenesis ◽  
Endothelial Growth Factor

Combination of antiangiogenesis and immunotherapy may be an effective strategy for treatment of solid tumors. Our previous work reported that activation of CD137 signaling promotes intraplaque angiogenesis. A number of studies have demonstrated that vascular endothelial growth factor receptor 2 (VEGFR2) is a key target for angiogenesis. However, it is unknown whether CD137-mediated angiogenesis is related to VEGFR2. In this study, we investigated the effect of CD137 on the VEGFR2 expression and explored the underlying mechanisms of CD137-mediated angiogenesis. Knock-out of CD137 in ApoE-/- mice significantly decreased neovessel density in atherosclerotic plaques. CD137 silencing or inhibition attenuated endothelial cell (ECs) proliferation, migration, and tube formation. We found activation of CD137 signaling for increased VEGFR2 transcription and translation steadily. Moreover, CD137 signaling activated phosphorylated VEGFR2 (Tyr1175) and the downstream Akt/eNOS pathway, whereas neutralizing CD137 signaling weakened the activation of VEGFR2 and the downstream Akt/eNOS pathway. The aortic ring assay further demonstrated that CD137 signaling promoted ECc sprouting. Inhibition of VEGFR2 by siRNA or XL184 (cabozantinib) and inhibition of downstream signaling by LY294002 (inhibits AKT activation) and L-NAME (eNOS inhibitor) remarkably abolished proangiogenic effects of CD137 signaling both in vitro and ex vivo. In addition, the condition medium from CD137-activated ECs and vascular endothelial growth factor A (VEGFA) had similar effects on ECs that expressed high VEGFR2. Additionally, activating CD137 signaling promoted endothelial secretion of VEGFA, while blocking CD137 signaling attenuated VEGFA secretion. In conclusion, activation of CD137 signaling promoted sprouting angiogenesis by increased VEGFA secretion and the VEGFR2/Akt/eNOS pathway. These findings provide a basis for stabilizing intraplaque angiogenesis through VEGFR2 intervatioin, as well as cancer treatment via combination of CD137 agonists and specific VEGFR2 inhibitors.

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