Hubbell MC, Semotiuk AJ, Thorpe RB, Adeoye OO, Butler SM, Williams JM, Khorram O, Pearce WJ. Chronic hypoxia and VEGF differentially modulate abundance and organization of myosin heavy chain isoforms in fetal and adult ovine arteries. Am J Physiol Cell Physiol 303: C1090 -C1103, 2012. First published September 19, 2012 doi:10.1152/ajpcell.00408.2011.-Chronic hypoxia increases vascular endothelial growth factor (VEGF) and thereby promotes angiogenesis. The present study explores the hypothesis that hypoxic increases in VEGF also remodel artery wall structure and contractility through phenotypic transformation of smooth muscle. Pregnant and nonpregnant ewes were maintained at sea level (normoxia) or 3,820 m (hypoxia) for the final 110 days of gestation. Common carotid arteries harvested from term fetal lambs and nonpregnant adults were denuded of endothelium and studied in vitro. Stretch-dependent contractile stresses were 32 and 77% of normoxic values in hypoxic fetal and adult arteries. Hypoxic hypocontractility was coupled with increased abundance of nonmuscle myosin heavy chain (NM-MHC) in fetal (ϩ37%) and adult (ϩ119%) arteries. Conversely, hypoxia decreased smooth muscle MHC (SM-MHC) abundance by 40% in fetal arteries but increased it 123% in adult arteries. Hypoxia decreased colocalization of NM-MHC with smooth muscle ␣-actin (SM-␣A) in fetal arteries and decreased colocalization of SM-MHC with SM-␣A in adult arteries. Organ culture with physiological concentrations (3 ng/ml) of VEGF-A165 similarly depressed stretch-dependent stresses to 37 and 49% of control fetal and adult values. The VEGF receptor antagonist vatalanib ablated VEGF's effects in adult but not fetal arteries, suggesting age-dependent VEGF receptor signaling. VEGF replicated hypoxic decreases in colocalization of NM-MHC with SM-␣A in fetal arteries and decreases in colocalization of SM-MHC with SM-␣A in adult arteries. These results suggest that hypoxic increases in VEGF not only promote angiogenesis but may also help mediate hypoxic arterial remodeling through age-dependent changes in smooth muscle phenotype and contractility.

The present study explores the hypothesis that arterial smooth muscle cells are organized into layers with similar phenotypic characteristics that vary with the relative position between the lumen and the adventitia due to transmural gradients in vasotrophic factors. A corollary hypothesis is that vascular endothelial growth factor (VEGF) is a factor that helps establish transmural variations in smooth muscle phenotype. Organ culture of endothelium-denuded ovine carotid arteries with 3 ng/ml VEGF-A(165) for 24 h differentially and significantly influenced potassium-induced (55% increase) and stretch-induced (36% decrease) stress-strain relations in adult (n = 18) but not term fetal (n = 21) arteries, suggesting that smooth muscle reactivity to VEGF is acquired during postnatal maturation. Because inclusion of fetal bovine serum significantly inhibited all contractile effects of VEGF (adult: n = 11; fetus: n = 11), it was excluded in all cultures. When assessed in relation to the distance between the lumen and the adventitia in immunohistochemically stained coronal artery sections, expression of smooth muscle α-actin (SMαA), myosin light chain kinase (MLCK), and 20-kDa regulatory myosin light chain exhibited distinct protein-dependent and age-dependent gradients across the artery wall. VEGF depressed regional SMαA abundance up to 15% in adult (n = 6) but not in fetal (n = 6) arteries, increased regional MLCK abundance up to 140% in fetal (n = 8) but not in adult (n = 10) arteries, and increased regional MLC(20) abundance up to 28% in fetal arteries (n = 7) but decreased it by 17% in adult arteries (n = 9). Measurements of mRNA levels verified that VEGF receptor transcripts for both Flt-1 and kinase insert domain receptor (KDR) were expressed in both fetal and adult arteries. Overall, the present data support the unique hypothesis that smooth muscle cells are organized into lamina of similar phenotype with characteristics that depend on the relative position between the lumen and the adventitia and involve the direct effects of growth factors such as VEGF, which acts independently of the vascular endothelium in an age-dependent manner.

More than 40% of patients with luminal breast cancer treated with endocrine therapy agent tamoxifen demonstrate resistance. Emerging evidence suggests tumor initiating cells (TICs) and aberrant activation of Src and Akt signaling drive tamoxifen resistance and relapse. We previously demonstrated that aryl hydrocarbon receptor ligand aminoflavone (AF) inhibits the expression of TIC gene α6-integrin and disrupts mammospheres derived from tamoxifen-sensitive breast cancer cells. In the current study, we hypothesize that tamoxifen-resistant (TamR) cells exhibit higher levels of α6-integrin than tamoxifen-sensitive cells and that AF inhibits the growth of TamR cells by suppressing α6-integrin-Src-Akt signaling. In support of our hypothesis, TamR cells and associated mammospheres were found to exhibit elevated α6-integrin expression compared with their tamoxifen-sensitive counterparts. Furthermore, tumor sections from patients who relapsed on tamoxifen showed enhanced α6-integrin expression. Gene expression profiling from the TCGA database further revealed that basal-like breast cancer samples, known to be largely unresponsive to tamoxifen, demonstrated higher α6-integrin levels than luminal breast cancer samples. Importantly, AF reduced TamR cell viability and disrupted TamR mammospheres while concomitantly reducing α6-integrin messenger RNA and protein levels. In addition, AF and small interfering RNA against α6-integrin blocked tamoxifen-stimulated proliferation of TamR MCF-7 cells and further sensitized these cells to tamoxifen. Moreover, AF reduced Src and Akt signaling activation in TamR MCF-7 cells. Our findings suggest elevated α6-integrin expression is associated with tamoxifen resistance and AF suppresses α6-integrin-Src-Akt signaling activation to confer activity against TamR breast cancer.

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.

Fetal hypoxia triggers compensatory angiogenesis and remodeling through mechanisms not fully elucidated. In response to hypoxia, hypoxia inducible factor drives expression of cytokines that exert multiple effects on cerebral structures. Among these, the artery wall is composed of a heterogeneous cell mix, and exhibits distinct patterns of cellular differentiation and reactivity. Governing these patterns are the vascular endothelium, smooth muscle (SM), adventitia, sympathetic perivascular nerves (SPN) and the parenchyma. Whereas an extensive literature details effects of non-neuronal factors on cerebral arteries, the trophic role of perivascular nerves remains unclear. Hypoxia increases sympathetic innervation with subsequent release of norepinephrine (NE), neuropeptide-y (NPY) and adenosine triphosphate (ATP), which exert motor and trophic effects on cerebral arteries and influence dynamic transitions among smooth muscle phenotypes. Our data also suggests that the cerebrovasculature reacts very differently to hypoxia in fetuses and adults, and we hypothesize that these differences arise from age-related differences in arterial smooth muscle phenotype reactivity and proximity to trophic factors, particularly of neural origin. We provide an integration of recent literature focused on mechanisms by which SPN mediate hypoxic remodeling. Our recent findings suggest that trophic effects of SPN on cerebral arteries accelerate functional maturation through shifts in SM phenotype in an age-dependent manner.