F.H Bach

Expression of heme oxygenase-1 can determine cardiac xenograft survival

The rejection of concordant xenografts, such as mouse-to-rat cardiac xenografts, is very similar to the delayed rejection of porcine-to-primate discordant xenografts. In concordant models, this type of rejection is prevented by brief complement inhibition by cobra venom factor (CVF) and sustained T-cell immunosuppression by cyclosporin A (CyA) (refs. 7, 8, 9, 10). Mouse hearts that survive indefinitely in rats treated with CVF plus CyA express the anti-inflammatory gene heme oxygenase-1 (HO-1) in their endothelial cells and smooth muscle cells. The anti-inflammatory properties of HO-1 are thought to rely on the ability of this enzyme to degrade heme and generate bilirubin, free iron and carbon monoxide. Bilirubin is a potent anti-oxidant, free iron upregulates the transcription of the cytoprotective gene, ferritin, and carbon monoxide is thought to be essential in regulating vascular relaxation in a manner similar to nitric oxide. We show here that the expression of the HO-1 gene is functionally associated with xenograft survival, and that rapid expression of HO-1 in cardiac xenografts can be essential to ensure long-term xenograft survival.

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.

Carbon monoxide pretreatment prevents respiratory derangement and ameliorates hyperacute endotoxic shock in pigs

Endotoxic shock, one of the most prominent causes of mortality in intensive care units, is characterized by pulmonary hypertension, systemic hypotension, heart failure, widespread endothelial activation/injury, and clotting culminating in disseminated intravascular coagulation and multi‐organ system failure. In the last few years, studies in rodents have shown that administration of low concentrations of carbon monoxide (CO) exerts potent therapeutic effects in a variety of diseases/disorders. In this study, we have administered CO (one our pretreatment at 250 ppm) in a clinically relevant, well‐characterized model of LPS‐induced acute lung injury in pigs. Pretreatment only with inhaled CO significantly ameliorated several of the acute pathological changes induced by endotoxic shock. In terms of lung physiology, CO pretreatment corrected the LPS‐induced changes in resistance and compliance and improved the derangement in pulmonary gas exchange. In terms of coagulation and inflammation, CO reduced the development of disseminated intravascular coagulation and completely suppressed serum levels of the proinflammatory IL‐1β in response to LPS, while augmenting the anti‐inflammatory cytokine IL‐10. Moreover, the effects of CO blunted the deterioration of kidney and liver function, suggesting a beneficial effect in terms of end organ damage associated with endotoxic shock. Lastly, CO pretreatment prevents LPS‐induced ICAM expression on lung endothelium and inhibits leukocyte marginalization on lung parenchyma.

Heme oxygenase-1 in organ transplantation.

Cells have a plethora of defense mechanisms that are activated upon exposure to oxidative stress. These aim at limiting the deleterious effects of oxidative stress and re-establishing homeostasis. In the particular context of organ transplantation, these defense mechanisms contribute to sustain graft survival via at least two interrelated mechanisms. First, cytoprotection per se should support survival and function of cells within a transplanted organ. Second, cytoprotection could reduce immunogenicity of the graft and modulate the activation of the recipients immune system to promote regulatory (suppressive) responses that sustain graft survival. Others and we have gathered evidence, to suggest that the stress-responsive enzyme Heme Oxygenase-1 (HO-1 encoded by the gene Hmox1) acts in such a manner. Upon organ transplantation, HO-1 is ubiquitously expressed in a transplanted organ, becoming the rate-limiting enzyme in the catabolism of heme into carbon monoxide (CO), iron (Fe) and biliverdin (1). There is accumulating evidence to support the notion that HO-1 expression in a graft and in the recipient can prevent rejection and promote immune tolerance. We will argue that these effects are mediated to a large extent by limiting the deleterious effects of free heme as well as by the inherent cytoprotective and/or anti-inflammatory effects of the end-products generated via heme catabolism.

Biliverdin protects rat livers from ischemia/reperfusion injury.

OBJECTIVE To explore putative cytoprotective functions of biliverdin during hepatic ischemia/reperfusion (I/R) injury in rat models. MATERIAL AND METHODS Male Sprague Dawley (SD) rat livers were harvested and stored for 24 hours at 4 degrees C in University of Wisconsin (UW) solution (n=18), and then perfused with blood for 2 hours on an isolated rat liver perfusion apparatus equipped for temperature (37 degrees C), pressure (13 cm H2O), and pH (7.3) maintenance. Biliverdin was added to the blood at concentrations of 10 and 50 micromol in two groups of six animals. Portal vein blood flow, bile production, and GOT/GPT levels were assessed serially. At the conclusion of the experiment, liver samples were collected for histologic evaluation using Suzuki criteria. RESULTS BV exerted protective effects against liver I/R injury. Adjunctive biliverdin improved portal venous blood flow (mL/min/g) from the beginning of reperfusion (1.33+/-0.17 versus 0.98+/-0.15; P<.001) and increased bile production (mL/g) as compared with the control group (3.40 versus 1.88; P<.003). I/R-induced hepatocellular damage as measured by GOT/GPT release (IU/L) was diminished in the biliverdin group (91 versus 171 and 46 versus 144, respectively; P<.0001). Improved liver function by biliverdin was accompanied by preservation of the histologic structure as assessed by Suzuki criteria (3.7+/-1.4 versus 6.8+/-0.8 in untreated controls; P<.005). CONCLUSIONS Biliverdin attenuates the ischemia/early reperfusion injury of rat liver grafts as assessed by hemodynamics, function, enzyme analysis, and histology. This study provides the rationale for novel therapeutic approaches using biliverdin to maximize the organ donor pool through the safer use of liver transplants despite prolonged periods of cold ischemia.

Regulated and endothelial cell-specific expression of Fas ligand : An in vitro model for a strategy aiming at inhibiting xenograft rejection

BACKGROUND Immunologically privileged sites have been shown to express Fas ligand (FasL) and may protect themselves by inducing apoptosis of infiltrating inflammatory cells. We asked whether the Fas/FasL interaction could be used to protect a xenograft from rejection. We proposed that endothelial cells that are resistant to Fas-mediated killing could be considered as a vehicle for expression of recombinant FasL. METHODS Based on the tetracycline-regulated expression system, constructs were designed that allow endothelial cell-specific and regulated expression of FasL by placing the tetracycline-dependent transactivator under control of the murine intercellular adhesion molecule-2 promoter. RESULTS Primary bovine endothelial cells transfected with FasL efficiently killed Fas-expressing cells in a regulated manner. Not only Fas-positive cell lines but also human peripheral blood lymphocytes underwent apoptosis upon exposure to FasL-transfected endothelial cells. CONCLUSION This in vitro model may provide tools for the generation of transgenic animals to be used as donors for vascularized xenograft transplantation.

Expression of heme oxygenase-1 by endothelial cells: a protective response to injury in transplantation

Endothelial cells (EC) play a pivotal role in the regulation of inflammation by expressing a series of pro- and anti-inflammatory genes that are associated with the activation of these cells. The nature of these genes and the regulation of their expression may be particularly important for the outcome of immediately vascularised transplants. We refer to the set of anti-inflammatory genes that are expressed during EC activation as protective genes because they can block the expression of pro-inflammatory genes associated with EC activation and prevent EC apoptosis. In this review we discuss data that supports the hypothesis that expression of these protective genes in a transplanted organ can promote its survival. We will focus on the description of one such protective gene, heme oxygenase-1 (HO-1). The first part of the review discusses the potential role of EC activation in regulating inflammatory responses such as those associated with the rejection of transplanted organs. The second part discusses the molecular mechanisms that regulate the expression of HO-1 in EC as well as the molecular mechanism by which the expression of this gene can regulate EC activation. The third part discusses potential mechanisms by which HO-1 may contribute to suppress different phases of the rejection of transplanted organs, e.g., ischaemia reperfusion injury, acute rejection and chronic failure. In the last part we discuss the role of HO-1 in establishing long-term survival of organs that are transplanted across different species, an approach referred to as xenotransplantation.

A 50-year retrospective: cell-mediated immunity and the major histocompatibility complex☆

HE AREA in which I was most involved in transplantation is best summarized by focusing on the cellmediated immune responses to antigens of the major histocompatibility complex (MHC). Many of these reminiscences are discussed in the text of my Medawar Lecture, which is also included in this volume. In the period around 1960, one could appreciate from the work of Gowans, Brent, Medawar and others that lymphocytes were both the cells that expressed the transplantation antigens and that responded to those antigens. Thus, in an attempt to develop an in vitro model of the then-called homograft (now, allograft) reaction, I mixed lymphocytes of two individuals in the hope that the antigens on the cells of each would stimulate the lymphocytes of the other to form blasts and divide. This was the result obtained and was the initiation of the mixed leukocyte culture (MLC) test, 1 from which we then developed a one-way MLC test, 2 in which the stimulating cells of the potential donor were treated with either mitomycin-C or x-irradiation. 3 The one-way MLC test has been used as a histocompatibility test to match donor and recipient and has become the basis for in vitro studies of alloimmunity since. I found that one of four sibling pairs failed to stimulate each other in MLC, and there was a single polymorphic locus that controlled reactivity in MLC. 4 With Bernard Amos, we were able to show that the MLC test was controlled by the same locus that controlled the serologically defined antigens being studied by Amos, Payne, van Rood, Dausset, and others. We suggested that this was the major histocompatibility complex in humans based on skin graft experiments that each of us had performed and on the observations that in other species the MHC controlled

Protective Responses of Endothelial Cells

Endothelial cells (EC) as they normally exist in their quiescent state perform critical functions in maintaining blood flow and avoiding thrombosis. Various proinflammatory stimuli can induce EC to be activated, which results in recruitment, trans-endothelial migration and activation of circulating leukocytes, procoagulation, platelet aggregation, and other responses associated with inflammation. In the case of an organ that is transplanted, these reactions associated with EC activation accompany the rejection of such organ. We suggested, several years ago, that EC activation is the underlying basis of rejection of organ xenografts, i.e., grafts such as a heart or kidney transplanted across different species. While antibodies and complement in the recipient are clearly implicated in EC activation and xenograft rejection, investigators in the 1980s showed that, under certain circumstances, grafts can survive indefinitely despite the presence of antigraft antibodies and complement. We referred to the survival of an organ in the presence of anti-organ antibodies and complement as “accommodation.” One possible mechanism that we proposed to explain accommodation of these grafts was that, under certain circumstances the EC in the graft up-regulate the expression of “protective genes” that would prevent those reactions associated with EC activation that presumably lead to rejection. We have since found that such protective genes do exist and that they can play such a role.