Chronic hypoxia alters fetal cerebrovascular responses to endothelin-1.

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.

Chronic hypoxia attenuates the vasodilator efficacy of protein kinase G in fetal and adult ovine cerebral arteries.

Long-term hypoxia (LTH) attenuates nitric oxide-induced vasorelaxation in ovine middle cerebral arteries. Because cGMP-dependent protein kinase (PKG) is an important mediator of NO signaling in vascular smooth muscle, we tested the hypothesis that LTH diminishes the ability of PKG to interact with target proteins and cause vasorelaxation. Prominent among proteins that regulate vascular tone is the large-conductance Ca2+-sensitive K+ (BK) channel, which is a substrate for PKG and is responsive to phosphorylation on multiple serine/threonine residues. Given the influence of these proteins, we also examined whether LTH attenuates PKG and BK channel protein abundances and PKG activity. Middle cerebral arteries were harvested from normoxic and hypoxic (altitude of 3,820 m for 110 days) fetal and adult sheep. These arteries were denuded and equilibrated with 95% O2-5% CO2 in the presence of N-nitro-l-arginine methyl ester (l-NAME) to inhibit potential confounding influences of events upstream from PKG. Expression and activity of PKG-I were not significantly affected by chronic hypoxia in either fetal or adult arteries. Pretreatment with the BK inhibitor iberiotoxin attenuated vasorelaxation induced by 8-(4-chlorophenylthio)guanosine 3',5'-cyclic monophosphate in normoxic but not LTH arteries. The spatial proximities of PKG with BK channel α- and ß1-proteins were examined using confocal microscopy, which revealed a strong dissociation of PKG with these proteins after LTH. These results support our hypothesis that hypoxia reduces the ability of PKG to attenuate vasoconstriction in part through suppression of the ability of PKG to associate with and thereby activate BK channels in arterial smooth muscle.NEW & NOTEWORTHY Using measurements of contractility, protein abundance, kinase activity, and confocal colocalization in fetal and adult ovine cerebral arteries, the present study demonstrates that long-term hypoxia diminishes the ability of cGMP-dependent protein kinase (PKG) to cause vasorelaxation through suppression of its colocalization and interaction with large-conductance Ca2+-sensitive K+ (BK) channel proteins in cerebrovascular smooth muscle. These experiments are among the first to demonstrate hypoxic changes in BK subunit abundances in fetal cerebral arteries and also introduce the use of advanced methods of confocal colocalization to study interaction between PKG and its targets.

Role of the sympathetic autonomic nervous system in hypoxic remodeling of the fetal cerebral vasculature.

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. Although an extensive literature details effects of nonneuronal 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, which exert motor and trophic effects on cerebral arteries and influence dynamic transitions among SM phenotypes. Our data also suggest 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 SM 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.

Vasotrophic regulation of age-dependent hypoxic cerebrovascular remodeling.

Hypoxia can induce functional and structural vascular remodeling by changing the expression of trophic factors to promote homeostasis. While most experimental approaches have been focused on functional remodeling, structural remodeling can reflect changes in the abundance and organization of vascular proteins that determine functional remodeling. Better understanding of age-dependent hypoxic macrovascular remodeling processes of the cerebral vasculature and its clinical implications require knowledge of the vasotrophic factors that influence arterial structure and function. Hypoxia can affect the expression of transcription factors, classical receptor tyrosine kinase factors, non-classical G-protein coupled factors, catecholamines, and purines. Hypoxia's remodeling effects can be mediated by Hypoxia Inducible Factor (HIF) upregulation in most vascular beds, but alterations in the expression of growth factors can also be independent of HIF. PPARγ is another transcription factor involved in hypoxic remodeling. Expression of classical receptor tyrosine kinase ligands, including vascular endothelial growth factor, platelet derived growth factor, fibroblast growth factor and angiopoietins, can be altered by hypoxia which can act simultaneously to affect remodeling. Tyrosine kinase-independent factors, such as transforming growth factor, nitric oxide, endothelin, angiotensin II, catecholamines, and purines also participate in the remodeling process. This adaptation to hypoxic stress can fundamentally change with age, resulting in different responses between fetuses and adults. Overall, these mechanisms integrate to assure that blood flow and metabolic demand are closely matched in all vascular beds and emphasize the view that the vascular wall is a highly dynamic and heterogeneous tissue with multiple cell types undergoing regular phenotypic transformation.