This study explored the hypothesis that intracerebral hemorrhage (ICH) promotes release of diffusible factors that can significantly influence the structure and function of cerebral arteries remote from the site of injury, through action on platelet-derived growth factor (PDGF) receptors. Four groups of adult male Sprague-Dawley rats were studied (n = 8 each): 1) sham; 2) sham + 60 mg/kg ip imatinib; 3) ICH (collagenase method); and 4) ICH + 60 mg/kg ip imatinib given 60 min after injury. At 24 h after injury, sham artery passive diameters (+3 mM EGTA) averaged 244 ± 7 µm (at 60 mmHg). ICH significantly increased passive diameters up to 6.4% and decreased compliance up to 42.5%. For both pressure- and potassium-induced contractions, ICH decreased calcium mobilization up to 26.2% and increased myofilament calcium sensitivity up to 48.4%. ICH reduced confocal colocalization of smooth muscle α-actin (αActin) with nonmuscle myosin heavy chain (MHC) and increased its colocalization with smooth muscle MHC, suggesting that ICH promoted contractile differentiation. ICH also enhanced colocalization of myosin light chain kinase (MLCK) with both αActin and regulatory 20-kDa myosin light chain. All effects of ICH on passive diameter, compliance, contractility, and contractile protein colocalization were significantly reduced or absent in arteries from animals treated with imatinib. These findings support the hypothesis that ICH promotes release into the cerebrospinal fluid of vasoactive factors that can diffuse to and promote activation of cerebrovascular PDGF receptors, thereby altering the structure, contractile protein organization, contractility, and smooth muscle phenotype of cerebral arteries remote from the site of hemorrhage.

In utero hypoxia influences the structure and function of most fetal arteries, including those of the developing cerebral circulation. Whereas the signals that initiate this hypoxic remodeling remain uncertain, these appear to be distinct from the mechanisms that maintain the remodeled vascular state. The present study explores the hypothesis that chronic hypoxia elicits sustained changes in fetal cerebrovascular reactivity to endothelin-1 (ET-1), a potent vascular contractant and mitogen. In fetal lambs, chronic hypoxia (3,820-m altitude for the last 110 days of gestation) had no significant effect on plasma ET-1 levels or ETA receptor density in cerebral arteries but enhanced contractile responses to ET-1 in an ETA-dependent manner. In organ culture (24 h), 10 nM ET-1 increased medial thicknesses less in hypoxic than in normoxic arteries, and these increases were ablated by inhibition of PKC (chelerythrine) in both normoxic and hypoxic arteries but were attenuated by inhibition of CaMKII (KN93) and p38 (SB203580) in normoxic but not hypoxic arteries. As indicated by Ki-67 immunostaining, ET-1 increased medial thicknesses via hypertrophy. Measurements of colocalization between MLCK and SMαA revealed that organ culture with ET-1 also promoted contractile dedifferentiation in normoxic, but not hypoxic, arteries through mechanisms attenuated by inhibitors of PKC, CaMKII, and p38. These results support the hypothesis that chronic hypoxia elicits sustained changes in fetal cerebrovascular reactivity to ET-1 through pathways dependent upon PKC, CaMKII, and p38 that cause increased ET-1-mediated contractility, decreased ET-1-mediated smooth muscle hypertrophy, and a depressed ability of ET-1 to promote contractile dedifferentiation.

This study explores the hypothesis that chronic hypoxia modulates endothelial regulation of smooth muscle phenotype and function in fetal cerebral arteries. Endothelium denuded and intact middle cerebral arteries harvested from term fetuses from ewes kept at sea level (Normoxic) or 3, 820 m (Hypoxic) altitudes for the last 110 days of gestation were assessed for contractile function and smooth muscle phenotype as quantified by changes in the colocalization of Non‐Muscle (NM) and Smooth Muscle (SM) Myosin Heavy Chain (MHC) with Smooth Muscle alpha‐actin (SM‐αA). Chronic hypoxia reduced maximum myogenic stress, but increased maximum K+‐induced stress. Organ culture of arteries from normoxic animals with VEGF reproduced this increase in K+‐induced stress but only in the presence of an intact endothelium. For NM‐MHC/AA colocalization, an index of synthetic smooth muscle, organ culture for normoxic arteries with VEGF had little effect in the absence of endothelium but in the presence of endothelium enhanced colocalization. This effect of the endothelium on NM‐MHC/AA colocalization was enhanced in hypoxic arteries. For SM‐MHC/AA colocalization, a marker for contractile smooth muscle, organ culture of normoxic arteries with VEGF had little effect in the presence or the absence of endothelium, but in hypoxic arteries endothelium dramatically amplified VEGF‐induced increases in colocalization. These results demonstrate that chronic hypoxia modulates VEGF‐dependent endothelial regulation of smooth muscle phenotype in fetal cerebral arteries.This work was supported by National Institutes of Health Grants HL‐54120, HD‐31266, HL‐64867, and NS‐076945 and the Loma Linda University School of Medicine.

To better understand how hypoxia‐induced vascular remodeling alters intracellular responses to ET‐1, we hypothesized that chronic hypoxia alters ET‐1 induced phenotypic transformation in arterial smooth muscle cells within the fetal cerebrovasculature. Endothelium denuded middle cerebral arteries (MCA) harvested from term fetuses of pregnant adult sheep kept at sea level or 3820m for 110 days were serum starved for 24h, then cultured 24h with ET‐1 and various kinase inhibitors. Fetal normoxic (FN) MCAs incubated with ET‐1 exhibited decreased colocalization between MLCK and α‐actin compared to control (starved), suggesting a shift of smooth muscle cells (SMCs) from a contractile to a more synthetic phenotype. This appeared to be mediated by multiple intracellular pathways, with PKC playing the largest role in normoxic arteries. Chronic hypoxia attenuated the ET‐1 induced decrease in MLCK and α‐actin colocalization, decreased the effects of PKC inhibitors, but increased the apparent effects of the CaMK‐dependent pathway, on colocalization. Given that plasma levels of ET‐1 were unaltered by chronic hypoxia, these results suggest that chronic hypoxia shifts the coupling of ET‐1 from PKC to CaMK to alter gene expression and thereby influence vascular remodeling. Grant Funding Source: Supported by National Institutes of Health Grants HD‐31266, HL‐64867, and NS‐076945

Cerebral arteries adapt to chronic hypoxia by altering the phenotypes of the plastic and heterogeneous population of cells in the artery wall. Perivascular nerves also undergo differentiation during hypoxia and alter vasotrophic effects on arteries. Here we test the hypothesis that nerves from the superior cervical ganglia (SCG) mediate hypoxic remodeling of ovine cerebral arteries. Using a chronic hypoxia (CH) and unilateral sympathectomy (SANX) model, we tested effects of CH and SANX on tone, contractile proteins and cell proliferation. Contractile responses to electrical nerve stimulation were upregulated (794%) by CH. CH ↑ dopa beta hydroxylase by 360% and SANX ablated this. CH↓ colocalization of a synthetic isoform of myosin heavy chain (SMemb) with smooth muscle alpha actin (AA) by 69%. SANX ↑ the colocalization by 600% in hypoxic arteries. NE in culture ↓ the colocalization by 91% and co‐culture of NE with prazosin ↑ it by 1363%. Also CH ↓ area per cell by 9% (media) and 45% (adventitia) whereas SANX ↑ it by 42% and 89% respectively. Also, CH ↓ colocalization of Ki67 (marker for cell proliferation) with AA by 15% (media) and 69% (adventitia). Together, these results suggest that the sympathetic cerebrovascular innervation helps mediate hypoxic remodeling through potentiation of NE synthesis, increased stimulation of α‐1 adrenoceptors, and transformation toward a contractile smooth muscle phenotype. Grant Funding Source: Supported by NIH Grants HD‐31266 and NS‐076945

This study explores the hypothesis that chronic hypoxia alters the expression and intracellular coupling of endothelin receptors to induce phenotypic transformations of smooth muscle cells within the arteries of fetal cerebrovasculature. Endothelium‐denuded middle cerebral arteries (MCAs) harvested from term fetuses of pregnant adult sheep kept at sea level or 3820m for 110 days were used in contractility experiments or cultured with ET‐1 and various kinase inhibitors. Chronic hypoxia had no effect on plasma levels of ET‐1 but altered contractile responses to ET‐1 and increased ETA receptor expression in fetal MCAs. ET‐1 treatment increased arterial thicknesses in both normoxic and hypoxic arteries. Organ culture of MCAs with ET‐1 decreased colocalization of MLCK and α‐actin and this effect was attenuated by chronic hypoxia. ET‐1 also decreased MLC20 and MLCK colocalization, but this effect was similar in normoxic and hypoxic arteries. The effects of ET‐1 on colocalization were reversed by inhibitors of either PKC (7 µM chelerythrine) or p38 (10 µM SB203580) in both normoxic and hypoxic arteries. From these results we conclude that chronic hypoxia modulates phenotypic responses of fetal cerebrovascular smooth muscle to ET‐1 through elevated expression of ET‐1 receptors, which act downstream through PKC and p38 dependent pathways on contractile protein expression.This work was supported by National Institutes of Health Grants HL‐54120, HD‐31266, HL‐64867, and NS‐076945 and the Loma Linda University School of Medicine.

The sympathetic perivascular innervation has long been suggested to exert trophic influences on cerebral arteries, particularly during hypoxic adaptation. Although most of this effect has been attributed to the actions of norepinephrine, sympathetic nerves also co‐release NPY together with norepinephrine. This study tests the hypothesis that NPY released from sympathetic nerves also contributes to hypoxic remodeling of fetal lamb cerebral arteries. Hypoxic acclimatization for 110 days increased NPY levels by 230% in nerve intact arteries. Whereas sympathectomy (SANX) had no significant effect in normoxic arteries, it decreased NPY levels in hypoxic arteries (25%). Hypoxia also increased Y1 receptor levels (70%) but only in nerve intact arteries. SANX decreased Y1 levels (30%) in hypoxic arteries. Confocal microscopy revealed that organ culture with 100 nM NPY potently decreased the colocalization of Non‐Muscle Myosin with Smooth Muscle Alpha‐Actin, indicating increased contractile differentiation. In nerve intact arteries, NPY decreased myogenic tone whereas in SANX arteries NPY increased myogenic tone. Co‐culture with the Y1 antagonist BIBP‐3226 (5 µM) reversed these effects of NPY. Taken together, these results suggest that NPY released from sympathetic nerves contributes to remodeling of the fetal cerebral vasculature during hypoxic acclimatization.

Prenatal, intra‐partum or post partum exposure of a fetus to hypoxia results in structural and functional changes in cerebral arteries, known as remodeling. Sympathetic nerves have been suggested to contribute to this process. Whereas NE is suggested as the major mediator of adrenergic effects, our preliminary experiments show that there is a guanethidine‐resistant (norepinephrine‐independent) component. This study explores the hypothesis that NPY contributes to hypoxic cerebrovascular remodeling in the fetal cerebral circulation. Chronic hypoxia (CH) produced a 254% ↑ in K‐induced tone and a 30% ↓ in stretch‐induced myogenic tone in fresh nerve‐intact fetal cerebral arteries. CH also increased the magnitude of nerve stimulation induced guanethidine‐resistant contractile tone. Immunohistochemistry revealed that CH significantly ↑ cerebrovascular NPY whereas sympathectomy (SANX) ablated this effect. Likewise, Western blot quantification of NPY‐Y1 receptors revealed that CH increased Y1 receptors 73%. Whereas SANX in normoxic tissues had no significant effect, SANX produced a 30% ↓ in Y1 receptors in hypoxic arteries. Together these results suggest that NPY released from sympathetic cerebrovascular nerves contributes to hypoxic remodeling through increased NPY content/release and Y1 receptor density. Grant Funding Source: Supported by NIH Grants HD‐31266 and NS‐076945