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Vascular endothelial growth factor receptor-2-mediated mitogenesis is negatively regulated by vascular endothelial
1Department of Reproductive and Vascular Biology, Division of Reproductive and Child Health, The Medical School, University of Birmingham, Edgbaston, Birmingham, United Kingdom.
Abstract:
Vascular endothelial growth factor (VEGF) receptors are present on nonendothelial cells suggesting that VEGF may mediate nonendothelial effects during organogenesis and tumorigenesis. Here we show that VEGF receptor-1 (VEGFR-1) negatively regulates VEGFR-2-mediated proliferation via nitric oxide (NO) in an epithelial cancer cell line ECV304. Cell proliferation was assessed by [(3)H]thymidine incorporation, fluorescent-activated cell-sorting analysis, and cell number using a Coulter Counter. Total NO generated by the action of nitric oxide synthase was measured by Seivers NOA 280 Nitric Oxide Chemiluminescence Analyser. VEGF (1 ng/ml) stimulated DNA synthesis and increased ECV304 cell number in a manner that was inhibited by a neutralizing anti-VEGFR-2 antibody. In contrast, VEGF (50 ng/ml) stimulated NO release in a manner that was inhibited by functionally neutralizing anti-VEGFR-1 antibody. Blockage of the VEGFR-1 receptor signal with anti-VEGFR-1 stimulated DNA synthesis and increased cell number. Cell-cycle analysis showed that inhibition of VEGFR-1 increased the transition from G(1) to S phase whereas inhibition of VEGFR-2 blocked the VEGF-mediated transition from G(1) to S phase. Finally, the addition of NO donors suppressed both VEGF-mediated proliferation and the increase in growth after blockade of VEGFR-1. Conversely, inhibition of VEGF mediated NO release by nitric oxide synthase inhibitor, L-monomethyl-L-arginine, restored the mitogenic effect of VEGF. These findings identify a dose-dependent reciprocal regulatory mechanism for VEGF via its two receptors. It shows that VEGFR-1 induces cell cytostasis via NO and as such is a suitable target for molecular strategies suppressing tumorigenesis.
Insights
Vascular endothelial growth factor (VEGF) receptors regulate cancer cell growth. VEGFR-1 inhibits proliferation via nitric oxide (NO), while VEGFR-2 promotes it, offering therapeutic targets for tumorigenesis.
Area of Science:
- Cell biology
- Molecular biology
- Cancer research
Background:
- Vascular endothelial growth factor (VEGF) receptors are found on nonendothelial cells, suggesting roles beyond blood vessel formation.
- VEGF signaling is implicated in both organogenesis and tumorigenesis.
- The specific roles of VEGFR-1 and VEGFR-2 in nonendothelial cell proliferation require further elucidation.
Purpose of the Study:
- To investigate the distinct roles of VEGF receptor-1 (VEGFR-1) and VEGF receptor-2 (VEGFR-2) in regulating epithelial cancer cell proliferation.
- To determine the involvement of nitric oxide (NO) in VEGF-mediated nonendothelial cell responses.
- To explore the potential of targeting VEGF receptors for cancer therapy.
Main Methods:
- Cell proliferation was measured using [(3)H]thymidine incorporation, flow cytometry, and cell counting.
- Nitric oxide (NO) generation was quantified using a chemiluminescence analyzer.
- Functional blocking antibodies against VEGFR-1 and VEGFR-2, NO donors, and nitric oxide synthase inhibitors were employed.
Main Results:
- Low VEGF concentrations (1 ng/ml) stimulated proliferation via VEGFR-2, while high concentrations (50 ng/ml) induced NO release via VEGFR-1.
- Blocking VEGFR-1 enhanced cell proliferation and G(1) to S phase transition, whereas blocking VEGFR-2 inhibited it.
- Nitric oxide donors suppressed proliferation, and NO inhibition restored VEGF-mediated proliferation, confirming NO's inhibitory role via VEGFR-1.
Conclusions:
- VEGF exhibits a dose-dependent, reciprocal regulation of epithelial cancer cell proliferation through VEGFR-1 and VEGFR-2.
- VEGFR-1 acts as a negative regulator of proliferation by inducing cytostasis via nitric oxide.
- VEGFR-1 represents a potential molecular target for developing anti-tumorigenic strategies.