Heme oxygenase (HO-1, encoded by Hmox1) is an inducible protein activated in systemic inflammatory conditions by oxidant stress. Vascular injury is characterized by a local reparative process with inflammatory components, indicating a potential protective role for HO-1 in arterial wound repair. Here we report that HO-1 directly reduces vasoconstriction and inhibits cell proliferation during vascular injury. Expression of HO-1 in arteries stimulated vascular relaxation, mediated by guanylate cyclase and cGMP, independent of nitric oxide. The unexpected effects of HO-1 on vascular smooth muscle cell growth were mediated by cell-cycle arrest involving p21Cip1. HO-1 reduced the proliferative response to vascular injury in vivo; expression of HO-1 in pig arteries inhibited lesion formation and Hmox1-/- mice produced hyperplastic arteries compared with controls. Induction of the HO-1 pathway moderates the severity of vascular injury by at least two adaptive mechanisms independent of nitric oxide, and is a potential therapeutic target for diseases of the vasculature.
Nitric oxide (NO), which accounts for the biological properties of endothelium-derived relaxing factor, is generated by NO synthase (NOS). The vascular endothelium contains two types of NOS: one is constitutively expressed (cNOS), and the other is inducible. Endothelium-mediated vasorelaxation is impaired in atherosclerotic vessels. To determine whether tumor necrosis factor (TNF)-alpha, which is commonly found in atherosclerotic lesions, has an effect on NOS message, we measured cNOS mRNA levels in TNF-treated human umbilical vein endothelial cells (HUVECs) by RNA blot analysis with a cNOS cDNA probe. TNF-alpha markedly reduced cNOS mRNA levels in HUVECs in a dose- and time-dependent manner. In response to 3 ng/mL TNF-alpha, cNOS mRNA levels began to decrease at 4 hours and diminished to only 5% of control levels at 24 hours. As little as 0.1 ng/mL TNF-alpha reduced cNOS mRNA levels by 50%. This reduction in cNOS message in response to TNF-alpha depended on protein synthesis as it was blocked by cycloheximide. In nuclear runoff experiments, TNF-alpha did not change the rate of cNOS gene transcription. cNOS mRNA is very stable under basal conditions, with a half-life of 48 hours; however, treatment with TNF-alpha shortened this half-life to 3 hours. TNF-alpha thus appears to decrease cNOS mRNA levels by increasing the rate of mRNA degradation. TNF-induced reductions in cNOS mRNA levels may have an important effect on impaired endothelium-mediated vasorelaxation in atherosclerosis.
Abstract-Heme oxygenase (HO)-1 degrades the pro-oxidant heme and generates carbon monoxide and antioxidant bilirubin. We have previously shown that in response to hypoxia, HO-1-null mice develop infarcts in the right ventricle of their hearts and that their cardiomyocytes are damaged by oxidative stress. To test whether HO-1 protects against oxidative injury in the heart, we generated cardiac-specific transgenic mice overexpressing different levels of HO-1. By use of a Langendorff preparation, hearts from transgenic mice showed improved recovery of contractile performance during reperfusion after ischemia in an HO-1 dose-dependent manner. In vivo, myocardial ischemia and reperfusion experiments showed that infarct size was only 14.7% of the area at risk in transgenic mice compared with 56.5% in wild-type mice. Hearts from these transgenic animals had reduced inflammatory cell infiltration and oxidative damage. Our data demonstrate that overexpression of HO-1 in the cardiomyocyte protects against ischemia and reperfusion injury, thus improving the recovery of cardiac function. Key Words: heart Ⅲ infarction Ⅲ Langendorff preparation Ⅲ cytoprotection Ⅲ inflammation O xidative stress in the heart caused by ischemia and reperfusion leads to cardiomyocyte death. 1-3 Several studies have shown that increased expression of myocardial stress proteins and/or antioxidant enzymes protects against postischemic injury. 4 -6 In response to stress, elevated expression of heat shock proteins may protect the myocardium. 7 These heat shock proteins are thought to mediate cardioprotection through their biological functions as molecular chaperones by preventing protein denaturation. 7 Heme oxygenase (HO)-1, a stress response and cytoprotective protein, also known as hsp32, protects cells from death due to pathophysiological stress. 8 -12 By degrading the pro-oxidant heme and generating the antioxidant bilirubin, 13,14 HO-1 may protect cells against oxidative injury. In addition, carbon monoxide (CO), another HO-1 reaction product, contributes to the regulation of vascular tone and is reported to have antiinflammatory properties, which may contribute to the cytoprotective action of HO-1. 15,16 HO-1 is upregulated in the heart and blood vessels in response to hemodynamic stress in rats 17,18 and ischemia/ reperfusion injury in pigs, 19,20 implicating an important role for HO-1 in cardiovascular homeostasis. We have recently shown that in response to hypoxia, HO-1-null mice develop right ventricular infarcts with organized mural thrombi. Furthermore, increased lipid peroxidation and oxidative damage occur in right ventricular cardiomyocytes from HO-1-null but not wild-type mice. 12 Thus, we hypothesized that HO-1 may play a central role in cardiac homeostasis by protecting cardiomyocytes from ischemia/reperfusioninduced injury and secondary oxidative damage. To gain insight into the cardioprotective role of HO-1 in vivo, we generated transgenic mice overexpressing HO-1 specifically in the heart. We measured cardiac performance during ...
Members of the erythroid Krü ppel-like factor (EKLF) multigene family contain three C-terminal zinc fingers, and they are typically expressed in a limited number of tissues. EKLF, the founding member, transactivates the -globin promoter by binding to the CACCC motif. EKLF is essential for expression of the -globin gene as demonstrated by gene deletion experiments in mice. Using a DNA probe from the zinc finger region of EKLF, we cloned a cDNA encoding a member of this family from a human vascular endothelial cell cDNA library. Sequence analysis indicated that our clone, hEZF, is the human homologue of the recently reported mouse EZF and GKLF. hEZF is a single-copy gene that maps to chromosome 9q31. By gel mobility shift analysis, purified recombinant hEZF protein bound specifically to a probe containing the CACCC core sequence. In co-transfection experiments, we found that sense but not antisense hEZF decreased the activity of a reporter plasmid containing the CACCC sequence upstream of the thymidine kinase promoter by 6-fold. In contrast, EKLF increased the activity of the reporter plasmid by 3-fold. By fusing hEZF to the DNA-binding domain of GAL4, we mapped a repression domain in hEZF to amino acids 181-388. We also found that amino acids 91-117 of hEZF confer an activation function on the GAL4 DNA-binding domain.
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