Kenichiro Yamashita

Heme oxygenase-1: unleashing the protective properties of heme

Heme oxygenase (HO)-1 catabolizes heme into three products: carbon monoxide (CO), biliverdin (which is rapidly converted to bilirubin) and free iron (which leads to the induction of ferritin, an iron-binding protein). HO-1 serves as a protective gene by virtue of the anti-inflammatory, anti-apoptotic and anti-proliferative actions of one or more of these three products. Administration of CO, biliverdin, bilirubin or iron-binding compounds is protective in rodent disease models of ischemia-reperfusion injury, allograft and xenograft survival, intimal hyperplasia following balloon injury or as seen in chronic graft rejection and others. We suggest that the products of HO-1 action could be valuable therapeutic agents and speculate that HO-1 functions as a therapeutic funnel, mediating the beneficial effects attributed to other molecules, such as interleukin-10 (IL-10), inducible nitric oxide synthase (NOS2; iNOS) and prostaglandins. This Review is the third in a series on the regulation of the immune system by metabolic pathways.

Heavy chain ferritin acts as an antiapoptotic gene that protects livers from ischemia reperfusion injury

Heme oxygenase‐1 (HO‐1) is induced under a variety of pro‐oxidant conditions such as those associated with ischemia‐reperfusion injury (IRI) of transplanted organs. HO‐1 cleaves the heme porphyrin ring releasing Fe2+, which induces the expression of the Fe2+ sequestering protein ferritin. By limiting the ability of Fe2+ to participate in the generation of free radicals through the Fenton reaction, ferritin acts as an anti‐oxidant. We have previously shown that HO‐1 protects transplanted organs from IRI. We have linked this protective effect with the anti‐apoptotic action of HO‐1. Whether the iron‐binding properties of ferritin contributed to the protective effect of HO‐1 was not clear. We now report that recombinant adenovirus mediated overexpression of the ferritin heavy chain (H‐ferritin) gene protects rat livers from IRI and prevents hepatocellular damage upon transplantation into syngeneic recipients. The protective effect of H‐ferritin is associated with the inhibition of endothelial cell and hepatocyte apoptosis in vivo. H‐ferritin protects cultured endothelial cells from apoptosis induced by a variety of stimuli. These findings unveil the anti‐apoptotic function of H‐ferritin and suggest that H‐ferritin can be used in a therapeutic manner to prevent liver IRI and thus maximize the organ donor pool used for transplantation.

Biliverdin, a natural product of heme catabolism, induces tolerance to cardiac allografts

Biliverdin, a product of heme oxygenase‐1 (HO‐1) enzymatic action, is converted into bilirubin, which has been considered a waste product in the past. We now show that administration of biliverdin has a salutary effect in organ transplantation. A brief course of treatment with biliverdin leads to long‐term survival of H‐2 incompatible heart allografts. Furthermore, those recipients harboring long‐surviving (>100 days) allografts were tolerant to donor antigens indicated by the acceptance of second donor strain hearts but not third‐party grafts. Treatment with biliverdin decreased intragraft leukocyte infiltration and inhibited T cell proliferation. Likely related to tolerance induction, biliverdin interferes with T cell signaling by inhibiting activation of nuclear factor of activated T cells (NFAT) and nuclear factor κB (NF‐κB), two transcription factors involved in interleukin‐2 (IL‐2) transcription and T cell proliferation, as well as suppressing Th1 interferon‐γ (IFN‐γ) production in vitro. These findings support the potential use of biliverdin, a natural product, in transplantation and other T cell mediated immune disorders.

Biliverdin therapy protects rat livers from ischemia and reperfusion injury

Heme oxygenase (HO‐1) provides a cellular defense mechanism during oxidative stress and catalyzes the rate‐limiting step in heme metabolism that produces biliverdin (BV). The role of BV and its potential use in preventing ischemia/reperfusion injury (IRI) had never been studied. This study was designed to explore putative cytoprotective functions of BV during hepatic IRI in rat liver models of ex vivo perfusion and orthotopic liver transplantation (OLT) after prolonged periods of cold ischemia. In an ex vivo hepatic IRI model, adjunctive BV improved portal venous blood flow, increased bile production, and decreased hepatocellular damage. These findings were correlated with amelioration of histological features of IRI, as assessed by Suzukis criteria. Following cold ischemia and syngeneic OLT, BV therapy extended animal survival from 50% in untreated controls to 90% to 100%. This effect correlated with improved liver function and preserved hepatic architecture. Additionally, BV adjuvant after OLT decreased endothelial expression of cellular adhesion molecules (P‐selectin and intracellular adhesion molecule 1), and decreased the extent of infiltration by neutrophils and inflammatory macrophages. BV also inhibited expression of inducible nitric oxide synthase and proinflammatory cytokines (interleukin 1β, tumor necrosis factor α, and interleukin 6) in OLTs. Finally, BV therapy promoted an increased expression of antiapoptotic molecules independently of HO‐1 expression, consistent with BV being an important mediator through which HO‐1 prevents cell death. In conclusion, this study documents and dissects potent cytoprotective effects of BV in well‐established rat models of hepatic IRI. Our results provide the rationale for a novel therapeutic approach using BV to maximize the function and thus the availability of donor organs. (HEPATOLOGY 2004;40:1333–1341.)

Heme oxygenase-1 is essential for and promotes tolerance to transplanted organs

This investigation focused on obtaining a further understanding of the role of heme oxygenase‐1 (HO‐1) in tolerance induction. Hearts from C57BL/6 (H‐2b) mice survived long‐term when transplanted into BALB/c (H‐2d) recipients treated with the tolerance‐inducing regimen of anti‐CD40L antibody (MR‐1) plus donor‐specific transfusion (DST). Grafts did not, however, survive long‐term in (HO‐1−/−) recipients given the same treatment. Similarly, long‐term survival induced by DST was ablated when HO‐1 activity was blocked by zinc protoporphyrin IX (ZnPPIX). We further asked whether modulation of HO‐1 expression/activity could be used to promote the induction of graft tolerance. DST alone (day 0) failed to promote any prolongation of survival of DBA/2 (H‐2d) hearts transplanted into B6AF1 (H‐2b,k/d) recipients. However, long‐term survival and (dominant peripheral) tolerance were readily induced when DST was combined with induction of HO‐1 expression by cobalt protoporphyrin IX (CoPPIX). HO‐1 induction plus DST led to a significant up‐regulation of Foxp3, TGF‐β, IL‐10, and CTLA4, which suggests a prominent role for CD4+CD25+ regulatory T cells (Tregs). In fact, the tolerogenic effect of HO‐1 plus DST was dependent on CD4+CD25+ Tregs as suggested by adoptively transferring these cells into irradiated recipients under various regimens. Taken together, these findings show that expression of HO‐1 in a graft recipient can be essential for long‐term graft survival and for induction of tolerance and that modulation of HO‐1 expression/activity can be used therapeutically to synergize in the generation of graft tolerance.

Heme oxygenase-1 modulates the allo-immune response by promoting activation-induced cell death of T cells

Heme oxygenase‐1 (HO‐1), which degrades heme into three products (carbon monoxide, free iron, and biliverdin), plays a protective role in many models of disease via its anti‐inflammatory, anti‐apoptotic, and anti‐proliferative actions. Overexpression of HO‐1 has been shown to suppress immune responses and prolong the survival of allografts; however, the underlying mechanism is not clear. We demonstrate two “new” properties of HO‐1 that mediate activation induced cell death (AICD) of allo‐antigen‐responsive murine CD4+ T cells, resulting in immunomodulation. First, it functions in vivo and in vitro to “boost” the proliferative response of CD4+ T cells to allo‐antigens in the early phase of allo‐antigen‐driven immune responses. This “boosting” effect is accompanied with a significant increase of activation markers and IL‐2 production. Second, it exerts a pro‐apoptotic effect in those activated T cells after the initial burst of proliferation. We further show that the AICD effect is mediated through the Fas/CD95‐FasL signal transduction pathway. Correlating with the above‐mentioned findings is the observed prolongation of mouse heart graft survival when HO‐1 is expressed in vivo in both donor and recipient. In conclusion, induction of HO‐1 expression accelerates clonal deletion of peripheral alloreactive CD4+ T cells by promoting AICD, which is presumably a key mechanism for its immunomodulatory effects such as in prolonging the survival of transplanted organs.

Cooperative effect of biliverdin and carbon monoxide on survival of mice in immune-mediated liver injury

Induction of the heme‐degrading enzyme heme oxygenase‐1 (HO‐1) has been shown to be beneficial in terms of improvement of liver allograft survival and prevention of CD95‐mediated apoptosis in the liver. In the present study, we investigated the effects of HO‐1, and its products carbon monoxide (CO), biliverdin (BV), and iron/ferritin, in a mouse model of inflammatory liver damage inducible by lipopolysaccharide (LPS) in mice sensitized with the hepatocyte‐specific transcription inhibitor D‐galactosamine (GalN). Our results show that HO‐1 induction by cobalt‐protoporphyrin‐IX (CoPP) reduced cytokine expression, protected mice from liver injury, and prolonged survival. While in contrast to ferritin overexpression, single administration of the CO donor methylene chloride (MC) or of BV also protected mice from liver damage, only coadministration of both HO products prolonged survival and reduced the expression of cytokines, e.g., tumor necrosis factor (TNF) and interferon γ (IFN‐γ). In conclusion, HO‐1–induced prolongation of survival, but not the protection from liver damage, seems to be dependent on down‐regulation of cytokine synthesis. (HEPATOLOGY 2004;40:1128–1135.)

Das Gallepigment Bilirubin: ein natürlicher Hemmer der Arteriosklerose?

Introduction: Induction of heme oxygenase 1 (HO-1) ameliorates the development of neointimal hyperplasia, which normally involves oxidative stress and vascular smooth muscle cell (VSMC) proliferation [1]. Bilirubin and biliverdin, generated during heme catabolism by HO-1, are potent anti-oxidants that play a role in maintenance of homeostasis [2]. Based on the clinical observation that healthy individuals with higher levels of bilirubin develop less arteriosclerosis3, we hypothesized that the bile pigments would inhibit VSMC proliferation in vitro and neointima formation in vivo. Methods: Balloon injury (BI) was performed in the left common carotid artery of congenitally hyperbilirubinemic Gunn rats. The artery was harvested two weeks after BI, and neointima formation was assessed by quantifying intima/media ratio and luminal cross-sectional area narrowing (LCAN). In vitro, proliferation of primary vascular smooth muscle cells (VSMC) was measured by [3H]-thymi-dine incorporation in the presence or absence of bilirubin/biliverdin. Cell cycle progression was monitored by DNA content analysis; apoptosis by the Annexin V binding assay. Protein levels were detected by western blot. Immunocytochemistry was performed using primary antibodies directed against cdk2 and phospho-p38 in growth stimulated primary VSMCs, in the presence or absence of bilirubin/biliverdin. To further delineate the pathways of bilirubin/biliverdin action on VSMC proliferation, we used VSMCs derived from p53 KO, p21 KO and HO-1 KO mice. Results: BI mediated neointima formation was significantly reduced in hyperbilirubinemic Gunn rats (serum bilirubin levels: 12.0 ± 2.5 mg/dl) as compared to control Wistar rats (LCAN 0.18 vs. 0.44; p<0.05). Systemic administration of biliverdin, the precursor of bilirubin, to normal rats also suppressed neointima proliferation (LCAN 0.29 vs. 0.43; p<0.05). In vitro, bilirubin inhibited VSMC proliferation by arresting VSMC cell cycle progression at the G0/G1 phase, while it did not increase apoptosis (p=0.2816). Inhibition of cell cycle progression was mediated by reduced phosphorylation of p38 MAPK and c-Jun NH2-terminal kinase 1/2 (JNK 1/2), down-regulation of cdk2, cyclins D1, E and A resulting in hypophosphorylation of pRb as demonstrated in nuclear extracts of cultured cells. Further the effect of bilirubin on VSMC proliferation was dependent on p53, but independent of p21 and HO-1. Conclusions: Our studies implicate bilirubin as the molecule to explain the observed associations between higher levels of plasma bilirubin in humans and a lesser incidence of atherosclerosis-related diseases and suggest the future use of bilirubin/biliverdin as a therapeutic to prevent restenosis, chronic rejection and atherosclerosis.

Heme Oxygenase-1 (HO-1) generiertes Biliverdin schützt vor Kolitis

Background nThe cytoprotective role of HO-1 has been observed in a wide variety of inflammatory diseases e.g. sepsis, arteriosclerosis, acute renal injury and transplant rejection. It is believed that the anti-inflammatory effects of HO-1 are mediated by the enzymatic products biliverdin/bilirubin, carbon monoxide (CO) and iron/ferritin. HO-1 induction in cultured enterocytes leads to inhibition of nitric oxide synthase activity; a pathway believed to play a crucial role in the mucosal injury associated with inflammatory bowel disease. We therefore tested whether induction of HO-1 can influence experimental colitis.