We investigated the expression of heme oxygenase-1 (HO-1) gene and production of endogenous carbon monoxide (CO) in the rat lung tissue at different time points of chronic hypoxic pulmonary hypertension and the effect of hemin on the expression of HO-1 gene and pulmonary hypertension. A rat model of hypoxic pulmonary hypertension was recreated by exposure to intermittent normobaric hypoxic environment (10% O2). Reverse transcriptase polymerase chain reaction (RT-PCR) was performed to determine the level of HO-1 mRNA in the rat lung tissue and double wave length spectrophotometry was used to evaluate the quantity of COHb in arterial blood. Cardiac catheterization was employed to measure the right ventricular systolic pressure (RVSP) and HE staining was performed in dissected lung tissue to observe the pathological changes of the intra-acinar pulmonary arteries (IAPA). It was found that (1) There was a low level of HO-1 mRNA in normal rat lung tissue, but the level of HO-1 mRNA increased by 2-4 times in the lung tissue of hypoxic rats (P<0.01). The quantity of COHb was 2-3 times those of control group (P<0.01 or P<0.05). These were accompanied by the increased of RVSP and the thickened IAPA; (2) Hemin could keep the HO-1 mRNA and COHb in the hypoxic rat lung tissue at a high level, and partially suppressed the increase of rat RVSP, thereby ameliorating the pathological changes of IAPA. In conclusion, the upregulation of the expression of HO-1 gene and production of CO in the rat lung of hypoxic pulmonary hypertension plays a role of inhibition in the development of hypoxic pulmonary hypertension. Hemin has a therapeutic effect on hypoxic pulmonary hypertension.
Animals ; Carbon Monoxide ; metabolism ; physiology ; Heme Oxygenase (Decyclizing) ; biosynthesis ; genetics ; Heme Oxygenase-1 ; Hypertension, Pulmonary ; etiology ; metabolism ; Hypoxia ; complications ; Lung ; metabolism ; Myocytes, Smooth Muscle ; drug effects ; pathology ; Pulmonary Artery ; metabolism ; pathology ; RNA, Messenger ; biosynthesis ; genetics ; Rats

The heme oxygenase-1 (HO-1), a rate-limiting enzyme in heme metabolism, has been recently defined as a novel stress-stimulated protein, since the intracellular expression of HO-1 in response to various stimuli as oxidation, ischemia and endotoxin injury has been proved to be able to protect the cells from damage. In this study, a retroviral vector containing human HO-1 gene was constructed and transfected to rat vascular smooth muscle cells (VSMCs). Using Southern and Northern blot analyses, the integration and mRNA expression of HO-1 gene in the transfected cells were confirmed. The profound protein expression of HO-1 as well as HO enzyme activity in the transfected cells increased by 1.8-fold and 2.0-fold respectively as compared with the non-transfected cells. It was found that the HO-1 transfected-VSMCs presented dominant resistance to toxicity produced by exposure to H2O2, as a significant protective effect of HO-1 marked by cell survival and LDH leakage was observed when 200, 400 and 600 micromol/L of H2O2 were used. The protection of HO-1 rapidly declined after the transfected-VSMCs were pretreated 24 h with an HO-1 specific inhibitor (ZnPP-IX). The results of this investigation suggest that the functional expression of HO-1 gene within VSMCs raises an alternative ability to protect the vascular cells against active oxygen injury.
Animals ; Cells, Cultured ; Gene Expression ; Genetic Vectors ; Heme Oxygenase (Decyclizing) ; biosynthesis ; genetics ; Heme Oxygenase-1 ; Hydrogen Peroxide ; toxicity ; Muscle, Smooth, Vascular ; enzymology ; pathology ; physiology ; Oxidants ; toxicity ; Rats ; Rats, Inbred WKY ; Retroviridae ; genetics ; Transfection

We investigated the expression of heme oxygenase-1 (HO-1) gene and production of endogenous carbon monoxide (CO) in the rat lung tissue at different time points of chronic hypoxic pulmonary hypertension and the effect of hemin on the expression of HO-1 gene and pulmonary hypertension. A rat model of hypoxic pulmonary hypertension was recreated by exposure to intermittent normobaric hypoxic environment (10% O2). Reverse transcriptase polymerase chain reaction (RT-PCR) was performed to determine the level of HO-1 mRNA in the rat lung tissue and double wave length spectrophotometry was used to evaluate the quantity of COHb in arterial blood. Cardiac catheterization was employed to measure the right ventricular systolic pressure (RVSP) and HE staining was performed in dissected lung tissue to observe the pathological changes of the intra-acinar pulmonary arteries (IAPA). It was found that (1) There was a low level of HO-1 mRNA in normal rat lung tissue, but the level of HO-1 mRNA increased by 2-4 times in the lung tissue of hypoxic rats (P<0.01). The quantity of COHb was 2-3 times those of control group (P<0.01 or P<0.05). These were accompanied by the increased of RVSP and the thickened IAPA; (2) Hemin could keep the HO-1 mRNA and COHb in the hypoxic rat lung tissue at a high level, and partially suppressed the increase of rat RVSP, thereby ameliorating the pathological changes of IAPA. In conclusion, the upregulation of the expression of HO-1 gene and production of CO in the rat lung of hypoxic pulmonary hypertension plays a role of inhibition in the development of hypoxic pulmonary hypertension. Hemin has a therapeutic effect on hypoxic pulmonary hypertension.
Anoxia/complications ; Carbon Monoxide/*metabolism ; Carbon Monoxide/physiology ; Heme Oxygenase (Decyclizing)/*biosynthesis ; Heme Oxygenase (Decyclizing)/genetics ; Heme Oxygenase-1 ; Hypertension, Pulmonary/etiology ; Hypertension, Pulmonary/*metabolism ; Lung/metabolism ; Myocytes, Smooth Muscle/drug effects ; Myocytes, Smooth Muscle/pathology ; Pulmonary Artery/metabolism ; Pulmonary Artery/*pathology ; RNA, Messenger/biosynthesis ; RNA, Messenger/genetics

BACKGROUNDUnder an insulin resistance (IR) state, overproduction of reactive oxygen species (ROS) may be playing a major role in the pathogenesis of endothelial dysfunction, hypertension and atherosclerosis. Recently, increasing attention has been drawn to the beneficial effects of heme oxygenase-1 (HO-1) in the cardiovascular system. This study aimed to investigate the effects of HO-1 on vascular function of thoracic aorta in IR rats and demonstrate the probable mechanisms of HO-1 against endothelial dysfunction in IR states.

METHODSSprague-Dawley (SD) rats fed with high-fat diet for 6 weeks and the IR models were validated with hyperinsulinemic-euglycemic clamp test. Then the IR rat models (n = 44) were further randomized into 3 subgroups, namely, the IR control group (n = 26, in which 12 were sacrificed immediately and evaluated for all study measures), a hemin treated IR group (n = 10) and a zinc protoporphyrin-IX (ZnPP-IX) treated IR group (n = 8) that were fed with a high-fat diet. Rats with standardized chow diet were used as the normal control group (n = 12). The rats in IR control group, hemin treated IR group and ZnPP-IX treated IR group were subsequently treated every other day with an intraperitoneal injection of normal saline, hemin (inducer of HO-1, 30 micromol/kg) or ZnPP-IX (inhibitor of HO-1, 10 micromol/kg) for 4 weeks. Rats in the normal control group remained on a standardized chow diet and were treated with intraperitoneal injections of normal saline every other day for 4 weeks. Systolic arterial blood pressure (SABP) was measured by tail-cuffed microphotoelectric plethysmography. The blood carbon monoxide (CO) was measured by blood gas analysis. The levels of nitric oxide (NO), inducible nitric oxide synthase (iNOS), endothelial nitric oxide synthase (eNOS), blood glucose (BG), insulin, total cholesterol (TC) and triglyceride (TG) in serum, and the levels of total antioxidant capacity (TAOC), malondialdehyde (MDA) and superoxide dismutase (SOD) in the aorta were measured. The expression of HO-1 mRNA and HO-1 protein in aortal tissue were detected by semi-quantitative RT-PCR and Western blot. The vasoreactive tensometry was performed with thoracic aortic rings (TARs).

RESULTSCompared with the normal control group, the levels of SABP, BG, insulin, TC, TG, NO, iNOS and MDA were higher, while the levels of CO, TAOC, SOD and eNOS were lower in IR control rats. After treatment of IR rats for 4 weeks a more intensive expression of HO-1 mRNA and HO-1 protein were observed in hemin treated IR group compared with the normal control group. And compared with 4-week IR control rats, the levels of CO, TAOC, SOD and eNOS were increased, while the levels of SABP and iNOS activity were lower in the hemin treated IR group. Administration of hemin in IR rats appeared to improve the disordered vasorelaxation of TARs to acetylcholine (ACh). Alternatively, the reverse results of SABP, CO, TAOC, SOD, iNOS and vasorelaxation responses to ACh were observed in IR rats with administration of ZnPP-IX.

CONCLUSIONSThe endothelial dysfunction in the aorta is present in the IR state. The protective effects of HO-1 against aortic endothelial dysfunction may be due to its antioxidation and regulative effect of vasoactive substances. It is proposed that hemin, inducer of HO-1, could be a potential therapeutic option for vascular dysfunction in IR states.

Animals ; Aorta ; drug effects ; physiology ; Carbon Monoxide ; blood ; Endothelium, Vascular ; drug effects ; physiology ; Enzyme Induction ; drug effects ; Heme Oxygenase-1 ; analysis ; biosynthesis ; genetics ; Hemin ; pharmacology ; Insulin Resistance ; Male ; Nitric Oxide ; blood ; Oxidative Stress ; Rats ; Rats, Sprague-Dawley ; Systole ; drug effects

In vascular smooth muscle cells (VSMCs), induction of the heme oxygenase-1 (HO-1) confers vascular protection against cellular proliferation mainly via its up-regulation of the cyclin-dependent kinase inhibitor p21(WAF1/CIP1) that is involved in negative regulation of cellular proliferation. In the present study, we investigated whether the phytochemical curcumin and its metabolite tetrahydrocurcumin could induce HO-1 expression and growth inhibition in rat VSMCs and, if so, whether their antiproliferative effect could be mediated via HO-1 expression. At non-toxic concentrations, curcumin possessing two Michael-reaction acceptors induced HO-1 expression by activating antioxidant response element (ARE) through translocation of the nuclear transcription factor E2-related factor-2 (Nrf2) into the nucleus and also inhibited VSMC growth triggered by 5% FBS in a dose-dependent manner. In contrast, tetrahydrocurcumin lacking Michael-reaction acceptor showed no effect on HO-1 expression, ARE activation and VSMC growth inhibition. The antiproliferative effect of curcumin in VSMCs was accompanied by the increased expression of p21(WAF1/CIP1). Inhibition of VSMC growth and expression of p21(WAF1/CIP1) by curcumin were partially, but not completely, abolished when the cells were co- incubated with the HO inhibitor tin protoporphyrin. In human aortic smooth muscle cells (HASMCs), curcumin also inhibited growth triggered by TNF-alpha and increased p21(WAF1/CIP1) expression via HO-1-dependent manner. Our findings suggest that curcumin has an ability to induce HO-1 expression, presumably through Nrf2-dependent ARE activation, in rat VSMCs and HASMCs, and provide evidence that the antiproliferative effect of curcumin is considerably linked to its ability to induce HO-1 expression.
Active Transport, Cell Nucleus ; Animals ; Aorta/cytology ; Cell Nucleus/metabolism ; Cell Proliferation/*drug effects ; Cells, Cultured ; Curcumin/analogs & derivatives/*pharmacology ; Cyclin-Dependent Kinase Inhibitor p21/biosynthesis/metabolism ; Gene Expression Regulation ; Heme Oxygenase (Decyclizing)/biosynthesis/genetics/*physiology ; Heme Oxygenase-1/biosynthesis/genetics/*physiology ; Humans ; Metalloporphyrins/pharmacology ; Muscle, Smooth, Vascular/drug effects/*physiology ; Myocytes, Smooth Muscle/drug effects/*physiology ; NF-E2-Related Factor 2/metabolism ; Protoporphyrins/pharmacology ; Rats ; Regulatory Sequences, Nucleic Acid ; Response Elements ; Tumor Necrosis Factor-alpha/pharmacology