Neuropilin-1 is essential for enhanced VEGF(165)-mediated vasodilatation in collateral-dependent coronary arterioles of exercise-trained pigs

Abstract

Background/aims: Exercise training enhances vasodilatation to vascular endothelial growth factor (VEGF(165)) in collateral-dependent coronary arterioles. Interaction of VEGF receptor 2 (VEGFR-2) and the non-tyrosine-kinase receptor, neuropilin-1 has been reported to potentiate VEGF(165)-mediated signaling. In the current study, we tested the hypotheses that neuropilin-1 mediates the exercise-enhanced VEGF(165)-mediated vasodilatation in collateral-dependent arterioles and that neuropilin-1 and/or VEGFR-2 protein levels are increased in these arterioles.

Methods: Ameroid occluders were surgically placed around the proximal left circumflex coronary artery of miniature swine. Eight weeks after surgery, the animals were randomized into sedentary or exercise training (treadmill run; 5 days/week; 14 weeks) protocols. Coronary arterioles (approximately 100 microm diameter) were isolated from both collateral-dependent and control (left anterior descending) myocardial regions and studied by in vitro videomicroscopy or frozen for immunoblot analysis.

Results: Exercise-enhanced VEGF(165)-mediated vasodilatation in collateral-dependent arterioles was reversed by inhibition of the VEGF(165)-neuropilin-1 interaction. VEGF(121), which does not interact with neuropilin-1, induced similar vasodilatation in arterioles from all treatment groups. Immunoblot revealed significantly elevated VEGFR-1, VEGFR-2 and neuropilin-1 protein levels in collateral-dependent arterioles of exercise-trained pigs.

Conclusions: Neuropilin-1 plays a vital role in the exercise-enhanced VEGF(165)-mediated vasodilatation of collateral-dependent coronary arterioles and is associated with increased neuropilin-1 receptor protein levels.

Fig. 1.

VEGF-mediated vasodilatation responses of coronary arterioles isolated from collateral-dependent and nonoccluded myocardial regions of hearts subjected to chronic occlusion. A VEGF₁₆₅-induced vasodilatation was significantly enhanced in collateral-dependent arterioles of exercise-trained compared with other treatment groups. B VEGF₁₂₁-mediated vasodilatation was not altered by chronic occlusion and/or exercise training. C Enhanced VEGF₁₆₅-mediated vasodilatation in the collateral-dependent arterioles of exercise-trained pigs was abolished in the presence of a synthetic peptide inhibitor of the VEGF₁₆₅-neuropilin-1 interaction. SED = Sedentary; EX = exercise-trained; LAD = arterioles from nonoccluded control region; LCX = arterioles from collateral-dependent region. Values are means ± SEM of the number of animals in parentheses. * Significantly different from other treatment groups.

Fig. 2.

Effect of chronic occlusion and exercise training on neuropilin-1, VEGFR-2, pVEGFR-2 and VEGFR-1 receptor protein levels as determined by immunoblot. Proteins of interest were quantified by densitometry analysis, normalized to β-actin, and expressed relative to SED LAD density. SED = Sedentary; EX = exercise-trained; LAD = arterioles from nonoccluded control region; LCX = arterioles from collateral-dependent region. Values are means ± SEM from arterioles of sedentary (n = 6) and exercise-trained (n = 6) animals. * Significantly different from other treatment groups. A Representative immunoblot images for proteins of interest from arterioles of 1 sedentary and 1 exercise-trained animal. Lanes 1–4 are SED LAD, SED LCX, EX LAD, and EX LCX, respectively. Protein levels for neuropilin-1 (B), cell surface VEGFR-2 (C), the soluble, immature form of VEGFR-2 (D), pVEGFR-2 (pY1175; E), and VEGFR-1 (F).