Abstract
Key points Combining nitric oxide (NO)‐mediated increased blood flow with angiopoietin‐1–Tie2 receptor signalling induces arteriolargenesis – the formation of arterioles from capillaries – in a model of physiological angiogenesis.This NO–Tie‐mediated arteriolargenesis requires endogenous vascular endothelial growth factor (VEGF) signalling.Inhibition of VEGF signalling increases pericyte coverage in microvessels.Together these findings indicate that generation of functional neovasculature requires close titration of NO–Tie2 signalling and localized VEGF induction, suggesting that the use of exogenous VEGF expression as a therapeutic for neovascularization may not be successful. Signalling through vascular endothelial growth factor (VEGF) receptors and the tyrosine kinase with IgG and EGF domains‐2 (Tie2) receptor by angiopoietins is required in combination with blood flow for the formation of a functional vascular network. We tested the hypothesis that VEGF and angiopoietin‐1 (Ang1) contribute differentially to neovascularization induced by nitric oxide (NO)‐mediated vasodilatation, by comparing the phenotype of new microvessels in the mesentery during induction of vascular remodelling by over‐expression of endothelial nitric oxide synthase in the fat pad of the adult rat mesentery during inhibition of angiopoietin signalling with soluble Tie2 (sTie2) and VEGF signalling with soluble Fms‐like tyrosine kinase receptor‐1 (sFlt1). We found that NO‐mediated angiogenesis was blocked by inhibition of VEGF with sFlt1 (from 881 ± 98% increase in functional vessel area to 279 ± 72%) and by inhibition of angiopoietin with sTie2 (to 337 ± 67%). Exogenous angiopoietin‐1 was required to induce arteriolargenesis (8.6 ± 1.3% of vessels with recruitment of vascular smooth muscle cells; VSMCs) in the presence of enhanced flow. sTie2 and sFlt1 both inhibited VSMC recruitment (both 0%), and VEGF inhibition increased pericyte recruitment to newly formed vessels (from 27 ± 2 to 54 ± 3% pericyte ensheathment). We demonstrate that a fine balance of VEGF and angiopoietin signalling is required for the formation of a functional vascular network. Endogenous VEGF signalling prevents excess neovessel pericyte coverage, and is required for VSMC recruitment during increased nitric oxide‐mediated vasodilatation and angiopoietin signalling (NO–Tie‐mediated arteriogenesis). Therapeutic vascular remodelling paradigms may therefore require treatments that modulate blood flow to utilize endogenous VEGF, in combination with exogenous Ang1, for effective neovascularization.
Highlights
Chronic occlusive vascular disorders represent a significant hurdle in global healthcare
Ad.soluble Fms-like tyrosine kinase receptor-1 (sFlt1) and Ad.soluble tyrosine kinase with IgG and EGF domains-2 (Tie2) (sTie2), both of which were expressed in adipocytes within 24 h after infection (Fig. 1B), blocked the increase in blood vessels that could be visualized by intravital microscopy 6 days after infection (Fig. 1C)
Staining of vessels for endothelial cells, proliferating endothelial cells and pericytes (Fig. 2A) demonstrated that sFlt1 and sTie2 inhibited, respectively, the vascular endothelial growth factor (VEGF)- and Ang1-mediated increase in both vessel density (Fig. 2B) and proliferating endothelial cell density (Fig. 2C). sFlt1 inhibited VEGF-induced sprouting (Fig. 2D) and branching (Fig. 2E), and sFlt1 and sTie2 reversed the effect of VEGF and Ang1, respectively, on both vessel length (Fig. 2F) and diameter (Fig. 2G). sTie2 reduced the increased pericyte coverage induced by Ang1 (Fig. 2H)
Summary
Chronic occlusive vascular disorders represent a significant hurdle in global healthcare. Advances in the field of interventional medicine have significantly improved clinical outcome, a considerable proportion of patients cannot be managed adequately by ‘traditional’ therapies. For these patients, therapeutic induction of blood vessel growth remains an attractive treatment option (Gupta et al 2009). Translation from pre-clinical studies to clinical practice has been limited (Laitinen et al 2000; Comerota et al 2002; Grines et al 2002), possibly reflecting the inability of any single factor to induce the growth of a complete, functional vascular network. Regulated perfusion requires the formation of capillary, arterial and venous networks. Defining the molecular and physical signals that regulate neovascularization from endothelial sprouting through to arteriogenesis could facilitate the future development of therapeutics
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