Contribution of increased VEGF receptors to hypoxic changes in fetal ovine carotid artery contractile proteins

Abstract

Recent studies suggest that vascular endothelial growth factor (VEGF) can modulate smooth muscle phenotype and, consequently, the composition and function of arteries upstream from the microcirculation, where angiogenesis occurs. Given that hypoxia potently induces VEGF, the present study explores the hypothesis that, in fetal arteries, VEGF contributes to hypoxic vascular remodeling through changes in abundance, organization, and function of contractile proteins. Pregnant ewes were acclimatized at sea level or at altitude (3,820 m) for the final 110 days of gestation. Endothelium-denuded carotid arteries from full-term fetuses were used fresh or after 24 h of organ culture in a physiological concentration (3 ng/ml) of VEGF. After 110 days, hypoxia had no effect on VEGF abundance but markedly increased abundance of the Flk-1 (171%) and Flt-1 (786%) VEGF receptors. Hypoxia had no effect on smooth muscle α-actin (SMαA), decreased myosin light chain (MLC) kinase (MLCK), and increased 20-kDa regulatory MLC (MLC(20)) abundances. Hypoxia also increased MLCK-SMαA, MLC(20)-SMαA, and MLCK-MLC(20) colocalization. Compared with hypoxia, organ culture with VEGF produced the same pattern of changes in contractile protein abundance and colocalization. Effects of VEGF on colocalization were blocked by the VEGF receptor antagonists vatalanib (240 nM) and dasatinib (6.3 nM). Thus, through increases in VEGF receptor density, hypoxia can recruit VEGF to help mediate remodeling of fetal arteries upstream from the microcirculation. The results support the hypothesis that VEGF contributes to hypoxic vascular remodeling through changes in abundance, organization, and function of contractile proteins.

Fig. 1.

Summary of our approach to test the hypothesis that VEGF contributes to hypoxic vascular remodeling through changes in abundance, organization, and function of contractile proteins in fetal arteries. First, we propose that hypoxia induces short-term increases in VEGF (arrow 1) through upregulation of the transcription factor hypoxia-inducible factor. We further propose that these increases in VEGF act on VEGF receptors (VEGF R1/R2) expressed by smooth muscle cells (arrow 2). In addition, we propose that chronic hypoxia increases expression of smooth muscle VEGF receptors (arrow 3). Finally, we propose that activation of smooth muscle VEGF receptors leads to changes in contractile protein abundance and organization that result in changes in arterial structure and function (arrow 4). In this manner, we propose that hypoxic increases in VEGF mediate not only microcirculatory angiogenesis, but also arterial remodeling. Separate experiments were performed to test each of the numbered arrows in fetal arteries.

Fig. 2.

Hypoxia remodels ovine carotid artery structure and function. Compared with normoxic arteries, arteries from chronically hypoxic animals exhibited an increased thickness (left) and stiffness (middle) of the medial layer. Determination of active stress-strain relations revealed that chronic hypoxia also significantly depressed myogenic tone but did not alter strain values at which contractile force was maximal (right). Values are means ± SE for arteries from normoxic (n = 17) and hypoxic (n = 12) fetuses. *P < 0.05.

Fig. 3.

Chronic hypoxia alters smooth muscle contractile protein abundances. Western blot quantification of smooth muscle α-actin (SMαA) abundance yielded similar values in normoxic and hypoxic fetal carotid arteries (left). Myosin light chain (MLC) kinase (MLCK) abundance was markedly less in hypoxic than normoxic arteries (middle). Abundance of 20-kDa regulatory MLC (MLC₂₀) was significantly greater in hypoxic than normoxic arteries (right). These results demonstrate that effects of chronic hypoxia on smooth muscle contractile protein abundances are highly protein-specific. Values are means ± SE; n ≥ 5 in all experimental groups. *P < 0.05 by ANOVA.

Fig. 4.

Chronic hypoxia increases colocalization among smooth muscle contractile proteins. As revealed by confocal microscopy, MLCK-SMαA colocalization was 42% greater in hypoxic than normoxic fetal arteries. Similarly, MLC₂₀-SMαA colocalization was 42% greater and MLCK-MLC₂₀ colocalization was 123% greater in hypoxic than normoxic fetal arteries. All these differences were statistically significant and suggest that the contractile proteins were becoming more compact and highly organized in response to chronic hypoxia. UR%, percentage of pixels in upper right quadrant. Values are means ± SE; n ≥ 5 in all experimental groups. *P < 0.05 by ANOVA.

Fig. 5.

Effects of long-term hypoxia on VEGF abundance. Endogenous VEGF levels quantified via Western blot analysis exhibited similar abundances in normoxic and hypoxic fetal sheep. These results suggest that the well-documented increases in VEGF induced on exposure to hypoxia are transient and disappear after 110 days of hypoxic acclimatization. Values are means ± SE for normoxic (n = 7) and hypoxic (n = 5) fetal arteries.

Fig. 6.

Effects of long-term hypoxia on VEGF receptor expression. In homogenized endothelium-denuded fetal arteries, abundances of VEGF receptor 1 (Flt-1) and VEGF receptor 2 (Flk-1) were significantly increased by chronic hypoxia. Multiple bands on Western blots indicate different glycosylation states of the receptors. Values represent total of all glycosylation states for Flk-1. Because of the absence of the 230-kDa form of Flt-1 in hypoxic arteries, abundances of the 250-kDa form are compared. Standards on Western blots were prepared from mixed samples of normoxic adult arteries. Values are means ± SE; n = 6 in all experimental groups. *P < 0.05 by ANOVA.

Fig. 7.

VEGF activation of its tyrosine kinase receptors mediates increased contractile protein colocalization. Organ culture of endothelium-denuded normoxic fetal arteries with VEGF (3 ng/ml) significantly increased MLC₂₀-SMαA colocalization. Addition of the VEGF receptor antagonists vatalanib (240 nM) and dasatinib (6.3 nM) completely blocked this effect of VEGF. Values are means ± SE; n = 5 in all experimental groups. *P < 0.05 by ANOVA.

Fig. 8.

Effects of VEGF on contractile protein abundances are highly protein-specific. As revealed by Western blot quantification, organ culture with 3 ng/ml VEGF had no significant effect on SMαA abundance in endothelium-denuded arteries from normoxic or hypoxic fetuses. In contrast, organ culture with 3 ng/ml VEGF significantly increased MLCK abundance in endothelium-denuded arteries from normoxic fetuses but decreased it in arteries from hypoxic fetuses. Conversely, organ culture with 3 ng/ml VEGF had no significant effect on MLC₂₀ abundance in arteries from normoxic fetuses but significantly increased MLC₂₀ in arteries from hypoxic fetuses. This pattern of effects emphasizes that effects of VEGF on contractile protein abundance are highly protein-specific, markedly influenced by hypoxic acclimatization, and closely similar to effects of chronic hypoxia (Fig. 3). Values are means ± SE; n = 5 in all experimental groups. *P < 0.05 by ANOVA.

Fig. 9.

VEGF increases colocalization among smooth muscle contractile proteins. In endothelium-denuded arteries from normoxic fetuses, organ culture with 3 ng/ml VEGF increased MLCK-SMαA colocalization by 55%, MLC₂₀-SMαA colocalization by 237%, and MLCK-MLC₂₀ colocalization by 75% compared with corresponding untreated controls. These results demonstrate that VEGF can act directly on arterial smooth muscle to enhance contractile protein colocalization. This pattern of effects was also closely similar to effects of chronic hypoxia on contractile protein colocalization (Fig. 4). Values are means ± SE; n ≥ 5 in all experimental groups. *P < 0.05 by ANOVA.