The relationship between hypoxia and regulation of nitric oxide synthase (NOS) in myocardial tissue is not well understood. We investigated the role of hypoxia inducible factor-1 (HIF-1) on expression of the inducible NOS (iNOS) in myocardial cells in vivo and in vitro. In situ hybridization in myocardial tissue from rats exposed to hypoxia for 3 weeks demonstrated increased iNOS mRNA expression. Northern analysis of RNA from hearts of those animals and from cells exposed to hypoxia for 12 hours in vitro demonstrated an increase of HIF-1 RNA expression. Electrophoretic mobility shift assays using oligonucleotides containing the iNOS HIF-1 DNA binding site and nuclear extracts from cardiac myocytes showed induction of specific DNA binding in cells subjected to hypoxia. Transient transfection of cardiac myocytes using the murine iNOS promoter resulted in a 3.43-fold increase in promoter activity under hypoxia compared with normoxia. Mutation or deletion of the HIF-1 site eliminated the hypoxic response. As cytokines have been shown to regulate iNOS expression in myocardial cells, cultured neonatal cardiac myocytes were stimulated with interleukin-1beta causing a dramatic induction of iNOS protein expression under normoxia, with further augmentation under hypoxia. Transient transfection of cells stimulated with interleukin-1beta showed an increased iNOS promoter activity under normoxic conditions compared with unstimulated cells, with a further increase in response to hypoxia, which was dependent on HIF-1. These results demonstrate that hypoxia causes an increase in iNOS expression in cardiac myocytes and that HIF-1 is essential for the hypoxic regulation of iNOS gene expression.
Pulmonary hypertension is characterized by structural and morphological changes to the lung vasculature. To determine the potential role of nitric oxide in the vascular remodeling induced by hypoxia, we exposed wild-type [WT(+/+)] and endothelial nitric oxide synthase (eNOS)-deficient [(-/-)] mice to normoxia or hypoxia (10% O(2)) for 2, 4, and 6 days or for 3 wk. Smooth muscle alpha-actin and von Willebrand factor immunohistochemistry revealed significantly less muscularization of small vessels in hypoxic eNOS(-/-) mouse lungs than in WT(+/+) mouse lungs at early time points, a finding that correlated with decreases in proliferating vascular cells (5-bromo-2'-deoxyuridine positive) at 4 and 6 days of hypoxia in the eNOS(-/-) mice. After 3 wk of hypoxia, both mouse types exhibited similar percentages of muscularized small vessels; however, only the WT(+/+) mice exhibited an increase in the percentage of fully muscularized vessels and increased vessel wall thickness. eNOS protein expression was increased in hypoxic WT(+/+) mouse lung homogenates at all time points examined, with significantly increased percentages of small vessels expressing eNOS protein after 3 wk. These results indicate that eNOS deficiency causes decreased muscularization of small pulmonary vessels in hypoxia, likely attributable to the decrease in vascular cell proliferation observed in these mice.
The nitric oxide (NO)-cGMP signal transduction pathway plays an important role in the regulation of pulmonary vascular tone and resistance in pulmonary hypertension. A number of studies have demonstrated that endothelial (e) and inducible nitric oxide synthases (NOS) are upregulated in hypoxia-exposed rat lung. These changes in NOS expression have been found to correlate with the process of pulmonary vascular remodeling in hypoxia-induced pulmonary hypertension, and remodeling is increased in the absence of eNOS. In this study, we examined the expression and localization of soluble guanylate cyclase (sGC), the primary receptor for NO, in hypoxia- and normoxia-treated rat lungs. Male Sprague-Dawley rats were exposed to hypoxia (10% O(2), normobaric) or normoxia for 1, 3, 5, and 21 days. The lungs were used for Western analysis of sGC protein, sGC enzyme activity, immunohistochemistry using antiserum against sGC alpha(1)- and beta(1)-subunits, and nonradioactive in situ hybridization (NRISH) using a digoxigenin-labeled sGC alpha(1)-subunit cRNA probe. Western blot analysis revealed a more than twofold increase of sGC protein alpha(1)-subunit in rat lungs exposed to 3, 5, and 21 days of hypoxia, correlating well with sGC enzyme activity. Immunohistochemistry and NRISH demonstrated increased expression of sGC in the smooth muscle cells of the pulmonary arteries and arterioles in the hypoxic rat lungs when compared with normoxic controls. Based on our results, the upregulation of sGC may play an important role in the regulation of smooth muscle tone and pressure in the pulmonary circulation during chronic hypoxia.
Highlights d CNTF induces neuronal production of IL-6 upon peripheral nerve injury d CNTF-STAT3-IL-6 mediates neuroinflammation from peripheral to central nervous systems d Schwann cells-neurons-microglia transmit neuroinflammation to the central nervous system d CNTF acts as a danger signal for nerve injury response and pain enhancement
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