Heme oxygenase (HO) catalyzes the oxidation of heme to generate carbon monoxide (CO) and bilirubin. CO increases cellular levels of cGMP, which regulates vascular tone and smooth muscle development. Bilirubin is a potent antioxidant. Hypoxia increases expression of the inducible HO isoform (HO-1) but not the constitutive isoform (HO-2). To determine whether HO-1 affects cellular adaptation to chronic hypoxia in vivo, we generated HO-1 null (HO-1^–/–) mice and subjected them to hypoxia (10% oxygen) for five to seven weeks. Hypoxia caused similar increases in right ventricular systolic pressure in wild-type and HO-1^–/– mice. Although ventricular weight increased in wild-type mice, the increase was greater in HO-1^–/– mice. Similarly, the right ventricles were more dilated in HO-1^–/– mice. After seven weeks of hypoxia, only HO-1^–/– mice developed right ventricular infarcts with organized mural thrombi. No left ventricular infarcts were observed. Lipid peroxidation and oxidative damage occurred in right ventricular cardiomyocytes in HO-1^–/–, but not wild-type, mice. We also detected apoptotic cardiomyocytes surrounding areas of infarcted myocardium by terminal deoxynucleotide transferase–mediated dUTP nick end-labeling (TUNEL) assays. Our data suggest that in the absence of HO-1, cardiomyocytes have a maladaptive response to hypoxia and subsequent pulmonary hypertension. J.Clin. Invest.103:R23–R29 (1999).

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.

Inactivation of glycogen synthase kinase 3β (GSK3β) and the resulting stabilization of free β-catenin are critical steps in the activation of Wnt target genes. While Akt regulates GSK3α/β in the phosphatidylinositide 3-OH kinase signaling pathway, its role in Wnt signaling is unknown. Here we report that expression of Wnt or Dishevelled (Dvl) increased Akt activity. Activated Akt bound to the Axin-GSK3β complex in the presence of Dvl, phosphorylated GSK3β and increased free β-catenin levels. Furthermore, in Wnt-overexpressing PC12 cells, dominant-negative Akt decreased free β-catenin and derepressed nerve growth factor-induced differentiation. Therefore, Akt acts in association with Dvl as an important regulator of the Wnt signaling pathway.

Background — Ischemia and oxidative stress are the leading mechanisms for tissue injury. An ideal strategy for preventive/protective therapy would be to develop an approach that could confer long-term transgene expression and, consequently, tissue protection from repeated ischemia/reperfusion injury with a single administration of a therapeutic gene. In the present study, we used recombinant adeno-associated virus (rAAV) as a vector for direct delivery of the cytoprotective gene heme oxygenase-1 (HO-1) into the rat myocardium, with the purpose of evaluating this strategy as a therapeutic approach for long-term protection from ischemia-induced myocardial injury. Methods and Results — Human HO-1 gene (hHO-1) was delivered to normal rat hearts by intramyocardial injection. AAV-mediated transfer of the hHO-1 gene 8 weeks before acute coronary artery ligation and release led to a dramatic reduction (>75%) in left ventricular myocardial infarction. The reduction in infarct size was accompanied by decreases in myocardial lipid peroxidation and in proapoptotic Bax and proinflammatory interleukin-1β protein abundance, concomitant with an increase in antiapoptotic Bcl-2 protein level. This suggested that the transgene exerts its cardioprotective effects in part by reducing oxidative stress and associated inflammation and apoptotic cell death. Conclusions — This study documents the beneficial therapeutic effect of rAAV-mediated transfer, before myocardial injury, of a cytoprotective gene that confers long-term myocardial protection from ischemia/reperfusion injury. Our data suggest that this novel “pre-event” gene transfer approach may provide sustained tissue protection from future repeated episodes of injury and may be beneficial as preventive therapy for patients with or at risk of developing coronary ischemic events.

To examine the role of heme oxygenase (HO)‐1 in the pathophysiology of vascular diseases, we generated mice deficient in both HO‐1 and apolipoprotein E (HO‐1 ^−/− apoE ^−/− ). Despite similar total plasma cholesterol levels in response to hypercholesterolemia, HO‐1 ^−/− apoE ^−/− mice, in comparison with HO‐1 ^+/+ apoE ^−/− mice, had an accelerated and more advanced atherosclerotic lesion formation. In addition to greater lipid accumulation, these advanced lesions from HO‐1 ^−/− apoE ^−/− mice contained macrophages and smooth muscle α‐actin‐positive cells. We further tested the role of HO‐1 on neointimal formation in a mouse model of vein graft stenosis. Autologous vein grafts in HO‐1 ^−/− mice showed robust neointima consisting of α‐actin‐positive vascular smooth muscle cells (VSMC) 10 days after surgery in comparison to the smaller neointima formed in autologous vein grafts in HO‐1 ^+/+ mice. However, at 14 days after surgery, the neointima from composite vessels of HO‐1 ^−/− mice was composed mainly of acellular material, indicative of substantial VSMC death. VSMC isolated from HO‐1 ^−/− mice were susceptible to oxidant stress, leading to cell death. Our data demonstrate that HO‐1 plays an essential protective role in the pathophysiology of atherosclerosis and vein graft stenosis.

In the postnatal vasculature, fully differentiated and quiescent vascular smooth muscle cells (VSMCs) in a "contractile" phenotype are required for the normal regulation of vascular tone. The transforming growth factor-β (TGF-β) superfamily of growth factors (TGF-βs and bone morphogenetic proteins (BMPs)) are potent inducers of contractile phenotype and mediate (i) induction of contractile genes, and (ii) inhibition of VSMC growth and migration. Transcription of contractile genes is positively regulated by a regulatory DNA element called a CArG box. The CArG box is activated by the binding of serum response factor and its coactivators, myocardin (Myocd) or Myocd-related transcription factors (MRTFs). Krüppel-like factor-4 (KLF4) is known to inhibit activation of the CArG box. However, the potential role of KLF4 in the contractile activities of TGF-β or BMP has not been explored. Here, we demonstrate that TGF-β and BMP4 rapidly down-regulate KLF4 through induction of microRNA-143 (miR-143) and miR-145, which leads to a reduction of KLF4 transcripts and decreased KLF4 protein expression. Inhibition of miR-145 prevents down-regulation of KLF4 and activation of contractile genes by TGF-β or BMP4, suggesting that modulation of KLF4 is a prerequisite for induction of contractile genes by TGF-β and BMP4. Interestingly, both TGF-β and BMP4 activate transcription of the miR-143/145 gene cluster through the CArG box, however, TGF-β mediates this effect through induction of Myocd expression, whereas BMP4 utilizes nuclear translocation of MRTF-A. Thus, this study sheds light on both the similarities and the differences of TGF-β and BMP4 signaling in the regulation of KLF4 and contractile genes.