Puerarin protects human umbilical vein endothelial cells against high glucose-induced apoptosis by upregulating heme oxygenase-1 and inhibiting calpain activation

The aim of this study was to investigate whether puerarin protects against high glucose (HG)-induced apoptosis by suppressing calpain activation in human umbilical vein endothelial cells (HUVECs). HUVECs were exposed to normal glucose (NG) (5.5 mm) or HG (33 mm) for 48 h; then, apoptosis and caspase-3 activity were determined. The expression of heme oxygenase-1 (HO-1) mRNA was evaluated by RT-PCR analysis. The activation of calpain and HO activity were also assessed. Compared with the NG group, exposure of HUVECs to HG for 48 h resulted in significant increases in calpain and caspase-3 activity as well as apoptosis, which were prevented by co-incubation with puerarin (1-100 μm) in a concentration-dependent manner. HO-1 mRNA expression and HO activity were decreased in HUVECs treated with HG for 48 h. Compared with the group exposed to HG alone, co-incubation of HUVECs with puerarin and HG induced increases in HO-1 mRNA expression and HO activity. The HO-1 inhibitor protoporphyrin IX zinc (II) abolished the inhibitory effect of puerarin on HG-induced calpain and caspase-3 activation, as well as apoptosis. The data show that puerarin protects against HG-induced endothelial cell apoptosis by a mechanism involving upregulation of HO-1 expression and inhibition of calpain activity.     

Oxidative stress causes enhanced endothelial cell injury in human heme oxygenase-1 deficiency

The first known human case of heme oxygenase-1 (HO-1) deficiency is presented in this report. The patient is a six-year-old boy with severe growth retardation. He has been suffering from persistent hemolytic anemia characterized by marked erythrocyte fragmentation and intravascular hemolysis, with paradoxical increase of serum haptoglobin and low bilirubin. An abnormal coagulation/fibrinolysis system, associated with elevated thrombomodulin and von Willebrand factor, indicated the presence of severe, persistent endothelial damage. Electron microscopy of renal glomeruli revealed detachment of endothelium, with subendothelial deposition of an unidentified material. Iron deposition was noted in renal and hepatic tissue. Immunohistochemistry of hepatic tissue and immunoblotting of a cadmium-stimulated Epstein-Barr virus–transformed lymphoblastoid cell line (LCL) revealed complete absence of HO-1 production. An LCL derived from the patient was extremely sensitive to hemin-induced cell injury. Sequence analysis of the patient's HO-1 gene revealed complete loss of exon-2 of the maternal allele and a two-nucleotide deletion within exon3 of the paternal allele. Growth retardation, anemia, iron deposition, and vulnerability to stressful injury are all characteristics observed in recently described HO–1 targeted mice. This study presents not only the first human case of HO-1 deficiency but may also provide clues to the key roles played by this important enzyme in vivo.     

The interaction of nitric oxide with distinct hemoglobins differentially amplifies endothelial heme uptake and heme oxygenase-1 expression

Heme is a strong inducer and substrate of the stress protein heme oxygenase-1 (HO-1), which produces carbon monoxide, iron, and bilirubin. We have reported recently that nitric oxide (NO) augments the incorporation of free hemin in endothelial cells, resulting in amplified HO-1 expression and production of bilirubin. Here, we extend our studies by showing that both NO+ and NO- donors interacted with reduced (HbA0) or oxidized (metHb) hemoglobin, as well as hemoglobin from sickle cell disease (HbS), to strongly magnify HO-1, with a pattern of induction dependent on the oxidation state of the hemoglobin used. A corresponding enhancement of endothelial heme uptake was observed following exposure of HbA0 or HbS to the NO donors, which also increased the uptake of free hemin. We postulated that this effect may be caused by formation of heme-nitrosyl (H-NO) complexes, and indeed endothelial cells exposed to preformed H-NO showed greater heme incorporation than free hemin. Furthermore, NO donors directly affected the permeability of membranes to free hemin. In conclusion, our data indicate a novel role for NO in the modulation of heme transport and HO-1 induction in endothelial cells, which may be relevant for hematological disorders characterized by disruption of the heme-NO equilibrium.     

Differential activation of coronary and pulmonary endothelial cells by thermal injury.

Remote organ dysfunction during resuscitation of severe thermal injury is characterized by early, transient pulmonary insufficiency and cardiac contractile dysfunction. Thermal injury is typified by profound systemic alterations of endothelial immunological, vasoactive, and barrier functions. The unique location of this ubiquitous, fragile monolayer makes it vulnerable to circulating serum factors created at remote cutaneous wounds. We examined endothelial "activation" in 2 distinct cell types, human coronary and pulmonary endothelial cells (EC), after severe thermal injury. By using human serum isolated at specific times after thermal injury ("early" [2 h post-burn] or "late" [26 h post-burn]), the endothelial release of vasoactive mediators, ICAM-1 expression, and monolayer permeability were assessed in vitro. Early burn serum enhanced coronary EC vasoconstrictor (ET-1) release and ICAM expression, inhibited vasodilator (PGI2) release, but had no effect on permeability. Conversely, under similar conditions, pulmonary EC PGI2 release and permeability were enhanced, ET-1 release was diminished, but ICAM was unaffected. Late burn serum enhanced vasodilator (NO) release and permeability to albumin in both coronary and pulmonary EC, whereas ET-1 release was inhibited. Under these conditions, only pulmonary ICAM expression was significantly enhanced. These data suggest that human endothelium isolated from divergent vascular beds are activated by burn injury in a unique manner for time post-burn and vascular site of cell origin.     

Microglia regulate blood clearance in subarachnoid hemorrhage by heme oxygenase-1

Subarachnoid hemorrhage (SAH) carries a 50% mortality rate. The extravasated erythrocytes that surround the brain contain heme, which, when released from damaged red blood cells, functions as a potent danger molecule that induces sterile tissue injury and organ dysfunction. Free heme is metabolized by heme oxygenase (HO), resulting in the generation of carbon monoxide (CO), a bioactive gas with potent immunomodulatory capabilities. Here, using a murine model of SAH, we demonstrated that expression of the inducible HO isoform (HO-1, encoded by Hmox1) in microglia is necessary to attenuate neuronal cell death, vasospasm, impaired cognitive function, and clearance of cerebral blood burden. Initiation of CO inhalation after SAH rescued the absence of microglial HO-1 and reduced injury by enhancing erythrophagocytosis. Evaluation of correlative human data revealed that patients with SAH have markedly higher HO-1 activity in cerebrospinal fluid (CSF) compared with that in patients with unruptured cerebral aneurysms. Furthermore, cisternal hematoma volume correlated with HO-1 activity and cytokine expression in the CSF of these patients. Collectively, we found that microglial HO-1 and the generation of CO are essential for effective elimination of blood and heme after SAH that otherwise leads to neuronal injury and cognitive dysfunction. Administration of CO may have potential as a therapeutic modality in patients with ruptured cerebral aneurysms.     

Statin-mediated cytoprotection of human vascular endothelial cells: a role for Kruppel-like factor 2-dependent induction of heme oxygenase-1

BACKGROUND: Heme oxygenase-1 (HO-1), by exerting anti-inflammatory, antiproliferative, antiapoptotic and antioxidant effects in the vasculature, protects against atherosclerosis and post-transplant vasculopathy. We noted the overlap between the effects of HO-1 and those attributed to 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins). This led to an investigation of the role of HO-1 in statin-mediated cytoprotection in primary human endothelial cells (ECs), and the ability of Kruppel-like factor 2 (KLF2) to regulate HO-1 function. METHODS/RESULTS: Treatment of human umbilical vein and aortic ECs with atorvastatin significantly upregulated HO-1 promoter activity, mRNA expression and protein expression, increasing HO-1 enzymatic activity as shown by raised intracellular bilirubin IXalpha. This effect was indirect, dependent upon inhibition of HMG-CoA reductase and geranylgeranylation, and independent of nitric oxide or changes in mRNA stability. Atorvastatin protected ECs against the generation of reactive oxygen species and H(2)O(2)-induced injury. HO-1 inhibition, with small interfering RNA (siRNA) or zinc protoporphyrin IX, abrogated atorvastatin-mediated cytoprotection. Atorvastatin upregulated KLF2 expression, whereas KLF2 siRNA attenuated statin-induced HO-1 and its associated antioxidant cytoprotective effects. Iron chelation, adenoviral-mediated overexpression of ferritin or supplementation of culture media with biliverdin reversed the inhibitory effects of HO-1 and KLF2 siRNA, suggesting that bile pigments and ferritin mediate the antioxidant actions of statin-induced HO-1. CONCLUSIONS: We have identified a novel link between KLF2 and HO-1 in human vascular ECs, demonstrating that atorvastatin-mediated HO-1 upregulation, and its associated antioxidant effect, is KLF2-dependent. The relationship between KLF2 and HO-1 is likely to represent an important component of the vasculoprotective profile of statins.     

After vascular injury, heme oxygenase-1/carbon monoxide enhances re-endothelialization via promoting mobilization of circulating endothelial progenitor cells

Summary.  Backgound: Heme oxygenase-1 (HO-1), a heme degradation enzyme with multiple vasoprotective functions, is systemically induced in pathophysiological states associated with oxidative stress. Objectives: To evaluate the impact of systemic HO-1 expression on circulating endothelial progenitor cells (EPCs) and re-endothelialization after vascular injury in an animal model. Methods: Mice received an intravenous (i.v.) injection of the adenovirus-bearing HO-1 gene (Adv-HO-1). The serum levels of vascular endothelial growth factor (VEGF) and stromal cell-derived factor-1 (SDF-1) were determined by ELISA and gene expression examined by quantitative real-time PCR. Circulating EPCs were characterized by flow cytometry and in vitro culture. EPC recruitment and re-endothelialization in injured arteries were assessed in mice receiving GFP⁺-bone marrow transplantation and guide wire-induced carotid injury. The effect of carbon monoxide (CO), a byproduct from heme degradation by HO-1, was assessed by exposing mice to 250 p.p.m. CO for 2 h day⁻¹. Results: Systemic HO-1 induction led to elevated serum levels of VEGF and SDF-1 and an increase in circulating EPCs. The re-endothelialization of denuded vessels was accelerated in mice with systemic HO-1 overexpression. A further experiment demonstrated that both EPC mobilization and re-endothelialization were significantly attenuated in mice with HO-1 deficiency. The increase in EPC mobilization and enhanced re-endothelialization was also observed in mice exposed to CO prior to carotid injury. The CO-mediated effect was associated with an increase in circulating SDF-1 but not VEGF. Conclusion: These findings support a vital role of HO-1 and its reaction byproduct, CO, in vascular repair through enhancing EPC mobilization.     

Differential effects of heme oxygenase isoforms on heme mediation of endothelial intracellular adhesion molecule 1 expression.

Heme oxygenase (HO), by catabolizing heme to bile pigments, down-regulates cellular hemoprotein, hemoglobin, and heme; the latter generates pro-oxidant products, including free radicals. Two HO isozymes, the products of distinct genes, have been described; HO-1 is the inducible isoform, whereas HO-2 is suggested to be constitutively expressed. We studied the inducing effect of several metal compounds (CoCl(2), stannic mesoporphyrin, and heme) on HO activity. Additionally, we studied HO-1 expression in experimental models of adhesion molecule expression produced by heme in endothelial cells, and the relationship of HO-1 expression to the induced adhesion molecules. Flow cytometry analysis showed that heme induces intracellular adhesion molecule 1 (ICAM-1) expression in a concentration (10-100 microM)- and time (1-24 h)-dependent fashion in human umbilical vein endothelial cells. Pretreatment with stannic mesoporphyrin, an inhibitor of HO activity, caused a 2-fold increase in heme-induced ICAM-1 expression. In contrast, HO induction by CoCl(2) decreased heme-induced ICAM-1 expression by 33%. To examine the contribution of HO-1 and HO-2 to endothelial HO activity, specific antisense oligonucleotides (ODNs) of each isoform were tested for their specificity to inhibit HO activity in cells exposed to heme. Endothelial cells exposed to heme elicited increased HO activity, which was prevented (70%) by HO-1 antisense ODNs. HO-2 antisense ODN inhibited heme-induced HO activity by 21%. Addition of HO-1 antisense ODNs prevented heme degradation and resulted in elevation of microsomal heme. Western blot analysis showed that HO-1 antisense ODNs selectively inhibited HO-1 protein and failed to inhibit HO-2 protein. Incubation of endothelial cells with HO-1 antisense enhanced heme-dependent increase of ICAM-1. In contrast, addition of HO-2 antisense to endothelial cells failed to increase adhesion molecules. The role of glutathione, an important antioxidant, was examined on heme-induced ICAM-1 expression. Endothelial cells pretreated with a glutathione precursor, N-acetylcysteine, or glutathione ester, showed a decrease in heme-induced ICAM-1 expression of 37 and 44%, respectively, suggesting that the mechanism of ICAM-1 induction by heme may be partly dependent on the levels of antioxidant. It is possible that amelioration of the heme-induced oxidative stress and expression of ICAM-I is due, in part, to the induction of HO-1 activity. Regulation of HO activity in this manner may have clinical applications.     

Induction of long-term cardiac allograft survival by heme oxygenase-1 gene transfer

Elevated expression of heme oxygenase-1 (HO-1), an intracellular enzyme that degrades heme into carbon monoxide (CO), biliverdine and free iron, has anti-inflammatory and antiapoptotic effects in diverse models. Here, we analyzed the effects of specific overexpression of HO-1 following adenovirus-mediated (AdHO-1) gene transfer in an acute cardiac allograft rejection model. The intragraft (i.g.) injection of AdHO-1 into cardiac allografts, as well as intramuscular (i.m.) or intravenous (i.v.) administration, prolonged allograft survival with, respectively, 13.3, 62.5 and 80% of the grafts surviving long term (>100 days), whereas control grafts were rejected with acute kinetics. HO-1 overexpression was associated with inhibited allogeneic responses in MLRs using graft-infiltrating leukocytes and splenocytes, but not with lymph node cells. The inhibition of splenocyte proliferation was mediated by soluble factors and was dependent on the presence of APCs, since purified T cells proliferated normally. i.v. but not i.g. AdHO-1 administration decreased the number of graft-infiltrating leukocytes, cytokine mRNA accumulation and apoptosis in transplanted hearts, whereas i.v. and i.g. AdHO-1 did not modify normal immune responses against cognate antigens, indicating that there was no general immunosuppression. These results indicate that HO-1 overexpression prolongs the survival of vascularized allografts by promoting tolerogenic mechanisms acting on allogeneic cellular immune responses.     

Leydig cell-derived heme oxygenase-1 regulates apoptosis of premeiotic germ cells in response to stress

Stress-induced downregulation of spermatogenesis remains poorly understood. This study examined the induction of heme oxygenase-1 (HO-1), a carbon monoxide-generating inducible enzyme, in modulation of spermatogenesis. Rats were exposed to cadmium chloride (CdCl(2)), a stressor causing oligozoospermia, and HO-1-induction was monitored by following HO isozyme expression. CdCl(2)-treated testes increased HO-1 activity and suppressed microsomal cytochromes P450, which are required for steroidogenesis. CdCl(2)-elicited HO-1 occurred mostly in Leydig cells and coincided with CO generation, as judged by bilirubin-IXalpha immunoreactivity. Under these circumstances, germ cells in peripheral regions of seminiferous tubules exhibited apoptosis; laser flow cytometry revealed that these apoptotic cells involve diploid and tetraploid germ cells, suggesting involvement of spermatogonia and primary spermatocytes in CdCl(2)-elicited apoptosis. Pretreatment with zinc protoporphyrin-IX, an HO inhibitor, but not copper protoporphyrin-IX, which does not block the enzyme, attenuated the CdCl(2)-induced apoptosis. Such antiapoptotic effects of zinc protoporphyrin-IX were repressed by supplementation of dichloromethane, a CO donor. Upon CdCl(2)-treatment, both Sertoli cells and the germ cells upregulated Fas ligand; this event was also suppressed by zinc protoporphyrin-IX and restored by dichloromethane. Thus, Leydig cells appear to use HO-1-derived CO to trigger apoptosis of premeiotic germ cells and thereby modulate spermatogenesis under conditions of stress.     

Salvianolic acid B exerts vasoprotective effects through the modulation of heme oxygenase-1 and arginase activities

Salvia miltiorrhiza (Danshen), a traditional Chinese herbal medicine, is commonly used for the prevention and treatment of cardiovascular disorders including atherosclerosis. However, the mechanisms responsible for the vasoprotective effects of Danshen remain largely unknown. Salvianolic acid B (Sal B) represents one of the most bioactive compounds that can be extracted from the water-soluble fraction of Danshen. We investigated the effects of Danshen and Sal B on the inflammatory response in murine macrophages. Danshen and Sal B both induced the expression of heme oxygenase-1 (HO-1) and inhibited nitric oxide (NO) production and inducible NO synthase (iNOS) expression in lipopolysaccharide (LPS)-activated RAW 264.7 cells. Inhibition of HO activity using Sn-protoporphyrin-IX (SnPP) abolished the inhibitory effect of Sal B on NO production and iNOS expression. Sal B increased macrophage arginase activity in a dose-dependent manner and diminished LPS-inducible tumor necrosis factor-α production. These effects were also reversed by SnPP. These data suggest that HO-1 expression plays an intermediary role in the anti-inflammatory effects of Sal B. In contrast to the observations in macrophages, Sal B dose-dependently inhibited arginase activity in murine liver, kidney, and vascular tissue. Furthermore, Sal B increased NO production in isolated mouse aortas through the inhibition of arginase activity and reduction of reactive oxygen species production. We conclude that Sal B improves vascular function by inhibiting inflammatory responses and promoting endothelium-dependent vasodilation. Taken together, we suggest that Sal B may represent a potent candidate therapeutic for the treatment of cardiovascular diseases associated with endothelial dysfunction.     

Role of heme oxygenase-1 in polymyxin B-induced nephrotoxicity in rats

Polymyxin B (PMB) is a cationic polypeptide antibiotic with activity against multidrug-resistant Gram-negative bacteria. PMB-induced nephrotoxicity consists of direct toxicity to the renal tubules and the release of reactive oxygen species (ROS) with oxidative damage. This study evaluated the nephroprotective effect of heme oxygenase-1 (HO-1) against PMB-induced nephrotoxicity in rats. Adult male Wistar rats, weighing 286 ± 12 g, were treated intraperitoneally once a day for 5 days with saline, hemin (HO-1 inducer; 10 mg/kg), zinc protoporphyrin (ZnPP) (HO-1 inhibitor; 50 μmol/kg, administered before PMB on day 5), PMB (4 mg/kg), PMB plus hemin, and PMB plus ZnPP. Renal function (creatinine clearance, Jaffe method), urinary peroxides (ferrous oxidation of xylenol orange version 2 [FOX-2]), urinary thiobarbituric acid-reactive substances (TBARS), renal tissue thiols, catalase activity, and renal tissue histology were analyzed. The results showed that PMB reduced creatinine clearance (P < 0.05), with an increase in urinary peroxides and TBARS. The PMB toxicity caused a reduction in catalase activity and thiols (P < 0.05). Hemin attenuated PMB nephrotoxicity by increasing the catalase antioxidant activity (P < 0.05). The combination of PMB and ZnPP incremented the fractional interstitial area of renal tissue (P < 0.05), and acute tubular necrosis in the cortex area was also observed. This is the first study demonstrating the protective effect of HO-1 against PMB-induced nephrotoxicity.     

血红素氧合酶1提高骨髓间充质干细胞在梗死后心脏的存活

背景:移植骨髓间充质干细胞(bone mesenchymal stem cells,BMSCs)修复梗死后心功能受到围移植期移植细胞大量死亡的限制.因此寻找一种保护因子对移植细胞提供保护至关重要.目的:观察血红素氧合酶1(heme oxygenase,HO-1)对BMSCs在梗死后心脏生存的影响.方法:体外分离扩增培养大鼠BMSCs,Adv-hHO-1、Adv-GFP分别转染,移植前DAPI标记.结扎左前降支1 h后,分别将DAPI-hHO-1-BMSCs、DAPI-GFP-BMSCs多点注射到大鼠心脏梗死区周边,对照组注射等量PBS.结果与结论:Adv-hHO-1转染BMSCs后获稳定表达.仅hHO-1-BMSCs组稳定表达hHO-1 mRNA;hHO-1-BMSCs组血管内皮生长因子、碱性成纤维细胞生长因子、肝细胞生长因子表达均高于其他两组(P < 0.01);存活BMSCs数量在第3,7天均明显高于GFP-BMSCs组(P < 0.05);大鼠心功能各项参数明显优于其他两组(P < 0.01).移植4周后,HO-1-BMSCs组梗死区周边毛细血管密度明显高于GFP-BMSCs组和对照组(P < 0.01),且胶原蛋白沉积减少,心室壁变厚,梗死面积较其他两组明显缩小(P < 0.01).提示HO-1提高移植到梗死心脏BMSCs的存活,HO-1协同BMSCs抑制心室重构,改善心功能.     

血红素氧合酶1提高骨髓间充质干细胞在梗死后心脏的存活

背景：移植骨髓间充质干细胞（bone mesenchymal stem cells,BMSCs）修复梗死后心功能受到围移植期移植细胞大量死亡的限制。因此寻找一种保护因子对移植细胞提供保护至关重要。目的：观察血红素氧合酶1（heme oxygenase,HO-1）对BMSCs在梗死后心脏生存的影响。方法：体外分离扩增培养大鼠BMSCs,Adv-hHO-1、Adv-GFP分别转染,移植前DAPI标记。结扎左前降支1h后,分别将DAPI-hHO-1-BMSCs、DAPI-GFP-BMSCs多点注射到大鼠心脏梗死区周边,对照组注射等量PBS。结果与结论：Adv-hHO-1转染BMSCs后获稳定表达。仅hHO-1-BMSCs组稳定表达hHO-1 mRNA;hHO-1-BMSCs组血管内皮生长因子、碱性成纤维细胞生长因子、肝细胞生长因子表达均高于其他两组（P〈0.01）;存活BMSCs数量在第3,7天均明显高于GFP-BMSCs组（P〈0.05）;大鼠心功能各项参数明显优于其他两组（P〈0.01）。移植4周后,HO-1-BMSCs组梗死区周边毛细血管密度明显高于GFP-BMSCs组和对照组（P〈0.01）,且胶原蛋白沉积减少,心室壁变厚,梗死面积较其他两组明显缩小（P〈0.01）。提示HO-1提高移植到梗死心脏BMSCs的存活,HO-1协同BMSCs抑制心室重构,改善心功能。