Endotoxin-Induced Myeloid-Derived Suppressor Cells Inhibit Alloimmune Responses via Heme Oxygenase-1

Inflammation and cancer are associated with impairment of T-cell responses by a heterogeneous population of myeloid-derived suppressor cells (MDSCs) coexpressing CD11b and GR-1 antigens. MDSCs have been recently implicated in costimulation blockade-induced transplantation tolerance in rats, which was under the control of inducible NO synthase (iNOS). Herein, we describe CD11b+GR-1+MDSC-compatible cells appearing after repetitive injections of lipopolysaccharide (LPS) using a unique mechanism of suppression. These cells suppressed T-cell proliferation and Th1 and Th2 cytokine production in both mixed lymphocyte reaction and polyclonal stimulation assays. Transfer of CD11b+ cells from LPS-treated mice in untreated recipients significantly prolonged skin allograft survival. They produced large amounts of IL-10 and expressed heme oxygenase-1 (HO-1), a stress-responsive enzyme endowed with immunoregulatory and cytoprotective properties not previously associated with MDSC activity. HO-1 inhibition by the specific inhibitor, SnPP, completely abolished T-cell suppression and IL-10 production. In contrast, neither iNOS nor arginase 1 inhibition did affect suppression. Importantly, HO-1 inhibition before CD11b+ cell transfer prevented the delay of allograft rejection revealing a new MDSC-associated suppressor mechanism relevant for transplantation.     

Heme Oxygenase-1 Modulates Mesangial Cell Proliferation by P21Waf1 Upregulation

Mesangial cell (MC) proliferation is a hallmark of many progressive renal diseases. Heme oxygenase-1 (HO-1) has been shown to have an anti-proliferative effect on vascular smooth muscle cells. In the present study, we evaluated the role of HO-1 on MC proliferation and the involved molecular mechanism. Both epidermal growth factor (EGF) and hepatocyte growth factor (HGF) not only enhanced mesangial cell HO-1 expression but also stimulated proliferation of MCs. Interestingly, inhibition of HO-1 induction (by zinc protoporphyrin, ZnP) was associated with an accelerated mitogenic response to EGF and HGF in MCs. Induction of HO-1 was associated with enhanced mesangial cell p21 expression. On the other hand, hemoglobin and ZnP inhibited mesangial cell p21 expression. It appears that the effect of HO-1 on MC growth may be mediated through upregulation of p21 expression.     

血红素氧合酶－1与急性肺损伤

血红素加氧酶-1是一种最广泛存在的抗氧化防御酶．是热体克蛋白家族中的一个成员，可代谢血红素生成CO．胆红素和游离铁在人多数组织内呈低水平表达，可被多种伤害性刺激包括血红素、高氧．缺氧、热体克、内毒素．过氧化氢、细胞因子．紫外线．重金属、NO和阿司匹林等诱导产生高水平的表达HO-1基因表达的调控主要发生在转录水平HO-1具有抗炎．抗氧化、抗凋亡效应。HO-1的产生作为一种对伤害性刺激的适应性和保护性反应，与临床上一些相关疾病密切相关。有关此方面的研究现越来越多且取得了重大的进展。本文就HO—1的产生、基因调控、生物学特性、功能与急性肺损伤的研究现状作一综述。     

Heme Oxygenase-1 Regulates Ferrous Iron and Foxo1 in Control of Hepatic Gluconeogenesis

The liver is a key player for maintaining glucose homeostasis. Excessive hepatic glucose production is considered to be a key for the onset of type 2 diabetes. The primary function of heme oxygenase-1 (HO1) is to catalyze the degradation of heme into biliverdin, ferrous iron, and carbon monoxide. Previous studies have demonstrated that the degradation of heme by HO1 in the liver results in mitochondrial dysfunction and drives insulin resistance. In this study, by overexpressing HO1 in hepatocytes and mice, we showed that HO1 promotes gluconeogenesis in a Foxo1-dependent manner. Importantly, HO1 overexpression increased the generation of ferrous iron in the liver, which further activates nuclear factor-κB and phosphorylates Foxo1 at Ser273 to enhance gluconeogenesis. We further assessed the role of HO1 in insulin-resistant liver-specific knockout of IRS1 and IRS2 genes (L-DKO) mice, which exhibit upregulation of HO1 in the liver and hepatic ferrous iron overload. HO1 knockdown by shRNA or treatment of iron chelator rescued the aberrant gluconeogenesis in L-DKO mice. In addition, we found that systemic iron overload promotes gluconeogenesis by activating the hepatic protein kinase A→Foxo1 axis. Thus, our results demonstrate the role of HO1 in regulating hepatic iron status and Foxo1 to control gluconeogenesis and blood glucose.     

转染人血红素加氧酶-1基因抑制移植静脉血管内膜增生

目的　应用含人血红素加氧酶 - 1基因 (human Heme Oxygenase- 1,h HO- 1)的重组腺病毒 (Adeno- XTMh HO- 1,Ad- h HO- 1)转染静脉移植血管 ,观察 h HO- 1基因预防静脉移植血管内膜增生的作用。　方法　将 2 1只日本大耳白兔分为 3组 ,对照组 ,Ad- null组和 Ad- h HO- 1组 ,每组各 7只。在兔颈外静脉移植于颈总动脉的血管移植术前分别应用肝素生理盐水、Ad- null和 Ad- h HO- 1病毒液常温浸泡静脉移植血管 30 min。术后 2 8d病理切片观察移植血管内膜增生的情况 ,计算机图象分析仪计算新生内膜厚度、中膜厚度及二者比值 ;采用免疫组织化学染色方法 (S- P法 )观察术后 14 d、2 8d移植血管壁 h HO- 1蛋白表达情况。　结果　Ad- h HO- 1组内膜厚度、内膜厚度与中膜厚度比均显著低于Ad- null组和对照组 (P<0 .0 1) ,中膜厚度差别无统计学意义 (P>0 .0 5 )。 Ad- h HO- 1组静脉血管壁细胞 h HO- 1免疫组化染色阳性。　结论　 Ad- h HO- 1转染兔静脉旁路移植血管能够抑制内膜增生。     

Gene Transfer of Heme Oxygenase-1 and Carbon Monoxide Delivery Inhibit Chronic Rejection

The hallmark of chronic rejection is the occlusion of the artery lumen by intima hyperplasia as a consequence of leukocyte infiltration and vascular smooth muscle cell (VSMC) migration and proliferation. Heme oxygenase-1 (HO-1) is a tissue protective molecule which degrades heme into carbon monoxide (CO), free iron and biliverdin. We analyzed the effects of HO-1 gene transfer into the vessel wall using an adenoviral vector (AdHO-1) and of CO delivery in a model of chronic allogeneic aorta rejection in rats. Carbon monoxide treatment was achieved by a new pharmacological approach in transplantation using methylene chloride (MC), which releases CO after degradation. AdHO-1-mediated gene transfer into aorta endothelial cells (ECs) or CO delivery resulted in a significant reduction in intimal thickness compared to untreated or noncoding adenovirus-treated controls. Aortas transduced with AdHO-1 or treated with CO showed a reduction in the number of leukocytes as well as in the expression of adhesion molecules, costimulatory molecules and cytokines, with the gene transfer treatment displaying a more pronounced effect than the CO treatment. Conversely, CO inhibited VSMC accumulation in the intima more efficiently than AdHO-1 treatment. Gene transfer of HO-1 and pharmacological manipulation of CO are novel approaches to the analysis and treatment of chronic rejection.     

Heme Oxygenase-1 Overexpression Protects Rat Livers from Ischemia/Reperfusion Injury with Extended Cold Preservation

This study analyzes the effects and mechanisms of heme oxygenase-1 (HO-1)-mediated cytoprotection in rat livers exposed to cold preservation. In the first series, rats were pretreated with cobalt protoporphyrin (CoPP) or zinc protoporphyrin (ZnPP), HO-1 inducer and antagonist, respectively. Livers were stored at 4 degrees C for 24 h, and then perfused ex vivo for 2 h. Livers pretreated with CoPP had significantly higher portal venous blood flow and increased total bile production, as compared with the ZnPP group. This correlated with histologic (Banff) criteria of hepatocyte injury/liver function. In the second series, rat livers were stored at 4 degrees C for 24 h or 40 h, and then transplanted into syngeneic recipients. After 24 h of preservation, 80% of rats bearing CoPP-pretreated liver grafts survived 21 days (vs. 50% in controls). After 40h of cold preservation, liver transplant survival at day 1, 7 and 21 for the CoPP group was: 100%, 71% and 57%, respectively (vs. 50%, 50% and 33% in controls). This correlated with improved hepatic function/histologic (Suzuki) criteria of hepatocyte injury after HO-1 overexpression (immunohistology/Western blots) by infiltrating macrophages. This study documents the potential utility of HO-1-inducing agents in preventing ischemia/reperfusion injury resulting from prolonged storage of liver transplants.     

Length Polymorphism in Heme Oxygenase-1 and Risk of CKD among Patients with Coronary Artery Disease

The length polymorphism of guanosine thymidine dinucleotide repeats in the heme oxygenase-1 gene promoter is associated with cardiovascular events and mortality in high-risk populations. Experimental data suggest that heme oxygenase-1 protects against kidney disease. However, the association between this polymorphism and long-term risk of CKD in high-risk patients is unknown. We analyzed the allelic frequencies of guanosine thymidine dinucleotide repeats in the heme oxygenase-1 gene promoter in 386 patients with coronary artery disease recruited from January 1999 to July 2001 and followed until August 31, 2012. The S allele represents short repeats (<27), and the L allele represents long repeats (≥27). The primary renal end points consisted of sustained serum creatinine doubling and/or ESRD requiring long-term RRT. The secondary end points were major adverse cardiovascular events and mortality. At the end of study, the adjusted hazard ratios (95% confidence intervals) for each L allele in the additive model were 1.99 (1.27 to 3.14; P=0.003) for the renal end points, 1.70 (1.27 to 2.27; P<0.001) for major adverse cardiovascular events, and 1.36 (1.04 to 1.79; P=0.03) for mortality. With cardiac events as time-dependent covariates, the adjusted hazard ratio for each L allele in the additive model was 1.91 (1.20 to 3.06; P=0.01) for the renal end points. In conclusion, a greater number of guanosine thymidine dinucleotide repeats in the heme oxygenase-1 gene promoter is associated with higher risk for CKD, cardiovascular events, and mortality among patients with coronary artery disease.     

Cytoprotective and Antiapoptotic Effects of IL-13 in Hepatic Cold Ischemia/Reperfusion Injury Are Heme Oxygenase-1 Dependent

Liver injury caused by ischemia/reperfusion (I/R) insult represents the major problem following orthotopic liver transplantation (OLT). I/R damage has been linked to Th1-like cytokine producers. This study evaluates putative cytoprotective effects/mechanisms of Th2-type IL-13 gene transfer. IL-13 overexpression prevented hepatic insult in a rat model of 24 h cold ischemia followed by OLT, as assessed: (i) profoundly decreased hepatocellular damage (sGOT levels), and ameliorated histological signs of I/R injury (Suzuki criteria), consistent with long-term OLT survival; (ii) prevented hepatic apoptosis (TUNEL stains) and up-regulated expression of antiapoptotic (A20, Bcl-2/Bcl-xl)/antioxidant (HO-1) genes. However, inhibition of HO-1 with tin protoporphyrin reversed cytoprotective/antiapoptotic effects of IL-13. In conclusion, cytoprotection rendered by virally induced IL-13 against hepatic I/R injury in this clinically relevant rat hepatic cold I/R injury model was accomplished via decreased apoptosis and induction of antiapoptotic/antioxidant molecules. HO-1 neutralization studies suggest that HO-1 represents one of putative IL-13 downstream effectors. This study provides the rationale for novel approaches to maximize organ donor pool through the safer use of OLTs despite prolonged periods of cold ischemia.     

Minimization of Immunosuppressive Therapy After Islet Transplantation: Combined Action of Heme Oxygenase-1 and PEGylation to Islet

We previously established a type of PEGylated islets to attenuate cellular immune reactions by immobilizing polyethylene glycol (PEG) molecules on islet surfaces, thereby synergistically reducing the dose of immunosuppressant cyclosporine A (CsA; 3 mg/kg/day) to protect transplanted islets. However, higher doses of immunosuppressants should be administered after islet transplantation due to nonspecific inflammation. This study documents that PEGylated islets can be cooperatively protected by the systemic overexpression of heme oxygenase-1 (HO-1), which has a potent cytoprotective function in preventing nonspecific inflammation during an early stage following islet transplantation. Under this scheme, the viability of PEGylated islets was improved; that is, PEG molecules could block cellular immunity and HO-1 could exert its cytoprotective property against inflammation. Interestingly, when employed with a low dose of CsA (1 mg/kg/day), a cooperative action of PEG molecules and HO-1 in immune reactions could result in the complete survival of transplanted islets for 100 days without islet function impairment. However, unmodified islets (control) were completely rejected within 2 weeks despite cotreatment with HO-1 expression and CsA. These results demonstrated that the combinatorial protocol of initial induction of HO-1 expression, followed by the daily administration of a low dose CsA after transplantation of PEGylated islets can be employed as a successful cell therapy in clinical islet transplantation.     

Heme oxygenase enzyme activity in human seminal plasma of fertile and infertile males

This work aimed to assess heme oxygenase (HO) enzyme activity relationship with different human semen parameters. One hundred and twenty men were divided according to their sperm count and clinical examination into: obstructive azoospermia (n = 20), nonobstructive azoospermia (NOA) (n = 25), oligozoospermia (n = 35) and normozoospermia (n = 40). Semen analysis, western blot for HO-1 and HO-2, and estimation of seminal plasma HO enzyme activity chemically in the form of bilirubin concentration were carried out. Seminal plasma HO enzyme activity was very low in OA specimens, low in NOA, moderate in oligozoospermia while higher in normozoospermia (mean +/- SD; 6.26 +/- 2.2, 81.4 +/- 35.5, 283.8 +/- 90.1, 657.4 +/- 227.6 pmol ml(-1) min(-1)) with significant differences. Western blot analysis demonstrated HO-2 expression in all studied groups whereas HO-1 was highly expressed in fertile normozoospermic group compared with other groups. There was positive correlation between seminal plasma HO enzyme activity and sperm concentration, sperm motility percentage, motile spermatozoa ml(-1) and sperm normal morphology per cent. It is concluded that HO enzyme activity in the human seminal plasma is related to spermatogenesis and sperm-motility processes.     

血红素氧合酶2在去势大鼠阴茎海绵体内的表达

目的:研究去势大鼠阴茎海绵体血红素氧合酶2(HO-2)和内皮型一氧化氮合酶(eNOS)的表达,探讨雄激素与HO-2、eNOS在ED中的作用及相关性。方法:10周龄雄性SD大鼠40只,分为4、8、12周组和正常对照组各10只,实验组采取手术切除双侧睾丸,对照组采取假手术。分别于术后4、8、12周测定大鼠血清睾酮(T)、阴茎海绵体内压(ICP)、平均颈动脉压(MAP),取阴茎标本,采用Western印迹分析阴茎海绵体HO-2含量,免疫组化分析HO-2和eNOS的表达。结果:去势各组血清T水平较正常对照组显著下降(P<0.05)。经3V、5V电压刺激后去势各组ICP/MAP值明显下降(P<0.05)。HO-2在正常和去势大鼠阴茎海绵体组织均有表达,去势4周组HO-2光密度分布曲线下面积(341.50±99.70)较正常组(876±443.36)和去势8周组(705.00±152.74)明显下降(P<0.05),去势8周与正常组之间无显著变化(P>0.05),去势12周没有检测到HO-2的表达。eNOS主要表达于阴茎海绵体血管内皮细胞,去势组eNOS(123.94±30.23)较正常组(421.21±125.12)差异有显著性(P<0.05)。T与eNOS和HO-2表达呈高度正相关(r=0.976、0.946,P均<0.05)。结论:雄激素可能通过影响大鼠阴茎海绵体HO-2、eNOS的表达参与阴茎勃起功能调控。     

血红素氧合酶在去势大鼠阴茎海绵体的表达

目的:研究血红素氧合酶(HO)在去势大鼠阴茎海绵体的表达,探讨其在雄激素缺乏的勃起功能障碍发生中的机制。方法:40只10周龄雄性SD大鼠随机分成:假手术2周组(A组),假手术4周组(B组),去势2周组(C组),去势4周组(D组)。术后2、4周检测血清睾酮水平,免疫组化及RT-PCR技术检测HO及nNOS在大鼠阴茎海绵体的表达。结果:去势组大鼠较假手术组血清睾酮水平显著下降,[AvsC:(283.222±117.171)ng/dlvs(7.117±3.700)ng/dl;BvsD:(289.280±87.413)ng/dlvs(48.826±19.477)ng/dl](P<0.01)、HO-1、HO-2蛋白表达明显降低(P<0.01),去势组HO-1、HO-2及nNOSmRNA表达较假手术组显著降低(P<0.01)。结论:雄激素可能通过HO-CO系统部分调控阴茎勃起功能。