R Kuddus

Enhanced synthesis and reduced metabolism of endothelin-1 (ET-1) by hepatocytes - an important mechanism of increased endogenous levels of ET-1 in liver cirrhosis

BACKGROUND/AIMS Hepatic concentration of endothelin-1 (ET-1) is increased in human and experimental liver cirrhosis. Because of its potent actions in the liver, ET-1 has been suggested to play an important role in the pathophysiology of cirrhosis. Since hepatocytes are the major cell type to metabolize ET-1, we investigated whether their reduced capacity to degrade ET-1 is a mechanism of its elevated levels in cirrhosis. METHODS The expression of ET-1 receptors, ET-1 and endothelin converting enzyme (ECE), and metabolism of ET-1 and ECE activity were compared in hepatocytes isolated from control and carbon tetrachloride-induced cirrhotic rats. RESULTS ET-1 receptor density and receptor-mediated internalization of ET-1 were significantly increased in cirrhotic hepatocytes relative to the control cells. However, compared to control hepatocytes, metabolism of ET-1 by the cirrhotic cells was reduced significantly. Interestingly, hepatocytes were found to contain preproET-1 mRNA, ECE-1 mRNA and ET-1. PreproET-1 mRNA and ET-1 levels were increased in cirrhotic hepatocytes but their ECE mRNA and ECE activity were not altered. CONCLUSIONS These results provide the first evidence that hepatocytes have the ability to synthesize ET-1 and demonstrate that decreased metabolism and enhanced synthesis, of ET-1 in hepatocytes are an important mechanism of its elevated levels in cirrhosis.

Endothelin stimulates transforming growth factor-β1 and collagen synthesis in stellate cells from control but not cirrhotic rat liver

Interactions between hepatic stellate cells and endothelin-1 are implicated in liver fibrosis. We determined endothelin-1, its receptors and its effects on the synthesis of a fibrogenic agent transforming growth factor (TGF)-beta1 and collagen in stellate cells from control and CCl(4)-induced cirrhotic rats. The basal synthesis of endothelin-1, TGF-beta1 and collagen was much higher in cirrhotic stellate cells than in control cells. Endothelin-1 stimulated TGF-beta1 and collagen synthesis via endothelin ET(A) and endothelin ET(B) receptors, respectively, in control stellate cells, but did not elicit these effects in the cirrhotic cells despite increased density of the respective receptor subtypes in them. These results indicate that the actions of endothelin-1 on stellate cells may be an important physiological mechanism in maintenance of hepatic architecture. However, inability of endothelin-1 to stimulate TGF-beta1 and collagen synthesis in cirrhotic stellate cells suggests that it does not influence fibrogenic activity by direct action on them probably because the processes are already maximally activated.

Superoxide-induced changes in endothelin (ET) receptors in hepatic stellate cells

BACKGROUND/AIMS Reactive oxygen species are mediators of various pathophysiologic events, including postischemic reperfusion injury and inflammation. Generation of reactive oxygen species and consequent organ injury are associated with increased levels of a powerful vasoconstrictor peptide endothelin-1. Current evidence suggests that actions of endothelin-1 on the contractile and fibrogenic transdifferentiated stellate cells may play a critical role in hepatic pathophysiology. The aim of this investigation was to determine whether reactive oxygen species modulate the synthesis of endothelin-1 and its receptors in stellate cells. METHODS Primary cultures of transdifferentiated stellate cells were exposed to reactive oxygen species-generating system, hypoxanthine/xanthine oxidase, before determination of endothelin-1 and its receptors. RESULTS The treatment caused an initial decrease in ET-1 receptor density (about 30% at 30 min), followed by a significant increase over the basal level at 6 h. The increase in the receptors, which occurred specifically in the ET(B) subtype, progressed thereafter up to 24 h and was accompanied by an augmented functional response, as indicated by an enhanced endothelin-1-induced release of [3H]arachidonic acid from the prelabeled cells. Furthermore, treatment of cells for 24 h but not 30 min caused increased expression of ET(B) mRNA as determined by semi-quantitative polymerase chain reaction. The release of endothelin-1 in the culture medium was also enhanced by hypoxanthine/xanthine oxidase treatment. These effects of hypoxanthine/xanthine oxidase were inhibited by superoxide dismutase and dimethyl sulfoxide. ET-1-induced [3H]arachidonic acid release was also inhibited by the ET(B) receptor antagonist BQ788, but not by the ET(A) receptor antagonist BQ123. CONCLUSIONS These findings indicate that interactions between ET-1 and stellate cells during episodes of the generation of reactive oxygen species can be an important mechanism in the pathophysiology of hepatic disorders.

Down-regulation of endothelin receptors by transforming growth factor β1 in hepatic stellate cells

BACKGROUND/AIMS Hepatic endothelin-1 (ET-1) receptor density as well as the levels of both ET-1 and transforming growth factor beta1 (TGF-beta1) increase in liver cirrhosis. Considering their potent contractile (ET-1) and fibrogenic (TGF-beta1) actions on myofibroblastic stellate cells found in the fibrotic/cirrhotic liver, we aimed to investigate the effects of TGF-beta1 on ET-1 receptors and ET-1 synthesis in these cells. METHODS Stellate cells isolated from rat liver by enzymatic digestion were cultured and subjected to TGF-beta1 treatment. Cellular ET-1 receptors and ET-1 released in the medium were determined. RESULTS TGF-beta1 treatment produced time- and dose-dependent decrease in ET-1 binding sites, but did not affect the affinity of the receptors for ET-1. TGF-beta1 also stimulated the release of ET-1 from stellate cells. The extent of TGF-beta1-induced inhibition of [125I]ET-1 binding was much greater for ETB subtype (73+/-18% inhibition), which comprised a major portion (78+/-12%) of the total ET-1 receptors, than for ETA subtype (35+/-11% inhibition). The mRNA expression of the ET-1 receptors also was reduced by TGF-beta1 treatment. TGF-beta1-induced reduction in ET-1 receptor density was coupled to the inhibition of ET-1-stimulated release of [3Hlarachidonic acid from the prelabeled cells. The effects of TGF-beta1 were inhibited by a TGF-beta1 neutralizing monoclonal antibody. CONCLUSIONS These results suggest that the TGF-beta1-induced decrease in ET-1 receptor density may be an important mechanism in limiting the pathologic actions of ET-1 on stellate cells in chronic liver disease.

Endotoxin causes up‐regulation of endothelin receptors in cultured hepatic stellate cells via nitric oxide‐dependent and ‐independent mechanisms

Hepatic stellate cells (HSC) and their transformed phenotype found in the chronically injured liver play important roles in hepatic physiology and pathology. HSC produce and react to a potent contractile peptide endothelin‐1 (ET‐1) and also synthesize a vasorelaxant nitric oxide (NO) upon stimulation with endotoxin. However, whether endotoxin affects ET‐1 system of HSC and if this is a mechanism of endotoxin‐induced hepatic injury is not known. We characterized synthesis of ET‐1 and NO and ET‐1 receptors in cultured quiescent and transformed HSC subjected to endotoxin treatment. Endotoxin (1–1000 ng ml−1) stimulated synthesis of ET‐1 and NO and up‐regulated ET‐1 receptors in both cell types. Inhibition of NO synthesis by NG‐monomethyl‐L‐homoarginine strongly inhibited endotoxin‐induced increase in ET‐1 receptors in transformed HSC but produced small additional increase in quiescent HSC. Inhibition of soluble guanylyl cyclase by 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one blocked the effect of endotoxin on ET‐1 receptors in both cell types. Moreover, ET‐1 receptors were increased in both cell types during earlier time points (1–4 h) of endotoxin treatment in the absence of the stimulation of NO synthesis. These results demonstrate that endotoxin up‐regulates ET‐1 receptors in HSC by NO‐dependent and ‐independent mechanisms. Such effects of endotoxin can be of importance in acute endotoxemia and during chronic injury of the liver.

Endotoxin treatment causes an upregulation of the endothelin system in the liver: Amelioration of increased portal resistance by endothelin receptor antagonism

Background: Mechanisms underlying hepatic microcirculatory failure during endotoxemia are incompletely understood. Because endothelin‐1 (ET‐1) has been implicated in endotoxin‐induced liver injury, we investigated the hepatic ET‐1 system in endotoxin‐treated rats.

Ischemia/reperfusion injury induces chronic changes in the small bowel.

Ischemia/Reperfusion (I/R) injury is a common corollary of organ preservation, transplantation, and acute cellular rejection of intestinal allografts.1–3 Long-term studies have been precluded by the prevailing notion that in the small bowel (SB), the changes induced by I/R injury resolve within a few days postoccurrence. However, having recently documented that a protracted deleterious effect of a single episode of I/R injury of kidneys,4,5 we proceeded to ascertain if a similar outcome is also witnessed in the SB.

A semiquantitative PCR technique for detecting chimerism in hamster-to-rat bone marrow xenotransplantation

Although bone marrow transplantation has been used to induce donor-specific tolerance in many allogeneic models, similar effort in xenogeneic transplantation is met with obstacles like more severe graft versus host disease (GVHD). We are currently engaged in developing a GVHD-free hamster-to-rat xenotransplantation model using splenectomy, total body irradiation, and donor bone marrow transplantation. To test donor cell chimerism, particularly in the solid tissues, we developed a semiquantitative polymerase chain reaction (PCR) method using primers specific for hamster beta-actin and mitochondrial cytochrome C oxidase I and II (MCO I and II) genes and rat sex determination region on the Y chromosome (SRY) gene. Using this method, we estimated the level of hamster cells chimerism in rats subjected to splenectomy, total body irradiation (10 Gy), and hamster bone marrow transplantation (3 x 10(8) cell/recipient) and observed high levels of donor cells in all recipient tissues tested.

Use of recombinase activation gene-2 deficient mice to ascertain the role of cellular and humoral immune responses in the development of chronic rejection

INTRODUCTION Given its multifactorial etiology, the relative contribution of anti-donor cellular and humoral immune responses in the pathogenesis of chronic rejection is as yet ambiguous. We hypothesized that alloreactive T and B cells play a seminal role in the development of this lesion. METHODS To address this hypothesis, RAG-2(-/-) mice were used as donors and recipients in a well-established murine model of aortic transplantation. Grafts were transplanted across the following groups: Group I: C3H --> C3H; Group II: Wild-type [WT] 129Sv (H-2(b)) --> C3H (H-2(k)); Group III: C3H --> WT 129Sv; Group IV: 129SvEv RAG-2(-/-) --> C3H; and Group V: C3H --> 129SvEv RAG-2(-/-). Grafts were harvested at d40 to 146 post-transplantation for morphologic and immunohistochemical analyses and semi-quantitative RT-PCR was employed to evaluate the intragraft mRNA expression of various immune mediators. Mixed lymphocyte reaction and complement-mediated alloantibody cytotoxicity assays were performed to determine anti-donor proliferative and humoral responses, respectively. RESULTS Unlike that across the syngeneic combination (Group I), marked intimal thickening with corresponding luminal narrowing was observed in the majority of the aortic allografts (Groups II-IV). On the contrary, the morphology of C3H aortic allografts harvested from the majority of the RAG-2(-/-) was remarkably preserved. Correspondingly, anti-donor proliferative and humoral immune responses were undetectable in C3H --> RAG-2(-/-) recipients as was the intragraft mRNA expression of the Th(1) and the Th(2)-type cytokines. CONCLUSIONS Taken together, these data suggest that in this murine model of aortic allotransplantation, donor-specific cellular and humoral responses play a dominant role in the initiation and perpetuation of chronic rejection.

Xenogeneic Humoral Graft-Vs-Host Disease Following Hamster-to-Rat Bone Marrow Transplantation

Bone marrow transplantation (BMT) is an effective strategy to induce tolerance across xenogeneic barriers.1 This approach has been used in the hamster-to-rat BMT model.2 However, all animals suffered from graft-vs-host disease (GVHD). Although xenogeneic GVHD has been observed in other studies, it is not yet well characterized.3 Because xenograft rejection is primarily mediated by humoral mechanisms, we tested the hypothesis that, unlike allogeneic GVHD in which cellular mechanisms predominate, xenogeneic GVHD could also be humorally mediated.