Shivraj Tyagi

Carbon monoxide suppresses arteriosclerotic lesions associated with chronic graft rejection and with balloon injury

Carbon monoxide (CO), one of the products of heme oxygenase action on heme, prevents arteriosclerotic lesions that occur following aorta transplantation; pre-exposure to 250 parts per million of CO for 1 hour before injury suppresses stenosis after carotid balloon injury in rats as well as in mice. The protective effect of CO is associated with a profound inhibition of graft leukocyte infiltration/activation as well as with inhibition of smooth muscle cell proliferation. The anti-proliferative effect of CO in vitro requires the activation of guanylate cyclase, the generation of cGMP, the activation of p38 mitogen-activated protein kinases and the expression of the cell cycle inhibitor p21Cip1. These findings demonstrate a protective role for CO in vascular injury and support its use as a therapeutic agent.

Bilirubin A Natural Inhibitor of Vascular Smooth Muscle Cell Proliferation

Background—Bilirubin, a natural product of heme catabolism by heme oxygenases, was considered a toxic waste product until 1987, when its antioxidant potential was recognized. On the basis of observations that oxidative stress is a potent trigger in vascular proliferative responses, that heme oxygenase-1 is antiatherogenic, and that several studies now show that individuals with high-normal or supranormal levels of plasma bilirubin have a lesser incidence of atherosclerosis-related diseases, we hypothesized that bilirubin would have salutary effects on preventing intimal hyperplasia after balloon injury. Methods and Results—We found less balloon injury–induced neointima formation in hyperbilirubinemic Gunn rats and in wild-type rats treated with biliverdin, the precursor of bilirubin, than in controls. In vitro, bilirubin and biliverdin inhibited serum-driven smooth muscle cell cycle progression at the G1 phase via inhibition of the mitogen-activated protein kinase signal transduction pathways and inhibition of phosphorylation of the retinoblastoma tumor suppressor protein. Conclusions—Bilirubin and biliverdin might be potential therapeutics in vascular proliferative disorders.

Heme oxygenase-1-derived carbon monoxide protects hearts from transplant associated ischemia reperfusion injury

Heme oxygenase‐1 (HO‐1) degrades heme into iron, biliverdin, and carbon monoxide (CO). HO‐ 1 expression can be used therapeutically to ameliorate undesirable consequences of ischemia reperfusion injury (IRI), but the mechanism by which this occurs, remains to be established. Rat hearts, exposed to a prolonged period (24 h) of cold (4°C) ischemia, failed to function upon transplantation into syngeneic recipients. Induction of HO‐1 expression by administration of cobalt protoporphyrin IX (CoPPIX) to the graft donor restored graft function. Inhibition of HO‐1 enzymatic activity, by administration of zinc protoporphyrin (ZnPPIX) at the time of transplantation, reversed the protective effect of HO‐1. Exposure of the graft donor as well as the graft (during ischemia) to exogenous CO mimicked the protective effect of HO‐1. This was associated with a significant reduction in the number of cells undergoing apoptosis in the graft with no apparent decrease of intravascular fibrin polymerization, platelet aggregation, or P‐ selectin expression. In conclusion, HO‐1‐derived CO prevents IRI associated with cardiac transplantation based on its antiapoptotic action. The observation that exposure of the donor and the graft to CO is sufficient to afford this protective effect should have important clinical implications in terms of preventing IRI associated with heart transplantation in humans.

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.

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.

Expression and function of C1q receptors and C1q binding proteins at the cell surface

C1q is the recognition unit of the first component of complement that binds not only IgG and IgM containing immune complexes, but also recognizes foreign structures such as the lipid A of endotoxin, and molecules expressed at the surface of apoptotic cells. In this review, the plasma membrane receptors and binding proteins for C1q are discussed and new data are presented on calreticulin expression on human peripheral blood cells. Although much is known about C1q receptors and binding molecules there are still many questions regarding their role in vivo.