Wide gene expression profiling of ischemia-reperfusion injury in human liver transplantation.

Ischemia-reperfusion injury (IRI) causes up to 10% of early liver failures in humans and can lead to a higher incidence of acute and chronic rejection. So far, very few studies have investigated wide gene expression profiles associated with the IRI process. The discovery of novel genes activated by IRI might lead to the identification of potential target genes for the prevention or treatment of the injury. In our study, we compared gene expression levels in reperfused livers (RL group) vs. the basal values before retrieval from the donor (basal liver [BL] group) using oligonucleotide array technology. We examined 10 biopsies from 5 livers, analyzing approximately 33,000 genes represented on the Affymetrix HG-U133APlus 2.0 oligonucleotide arrays (Affymetrix, Santa Clara, CA). About 13,000 individual genes were considered expressed in at least 1 condition. A total of 795 genes whose expression is significantly modified by ischemia-reperfusion in human liver transplantation were identified in this study. Some of them are likely to be completely activated by IRI, as they are not expressed in basal livers. The supervised gene expression analysis revealed that at least 12% of the genes involved in the apoptotic process, 12.5% of the genes involved in inflammatory processes, and 22.5% of the genes encoding for heat shock proteins are differentially expressed in RL samples vs. BL samples. Furthermore, IRI induces the upregulation of some genes' coding for adhesion molecules and integrins. In conclusion, we have identified a relevant amount of early genes regulated in the human liver after 7-9 hours of cold ischemia and 2 hours from reperfusion, many of them not having been described before in this process. Their analyses may help us to better understand the pathophysiology of IRI and to characterize potential target genes for the prevention or treatment of the liver injury in order to increase the number of patients that successfully undergo transplantation.

Flavoridin inhibits Yersinia enterocolitica uptake into fibronectin-adherent HeLa cells.

In this study, three structurally distinct disintegrins (flavoridin, echistatin, kistrin) were used as molecular probes to further characterize the molecular mechanisms underlying Yersinia enterocolitica infection of host cells. The activity of the three disintegrins on Y. enterocolitica uptake into fibronectin-adherent HeLa cells was evaluated at disintegrin doses which were non-cytotoxic and unable to induce cell detachment. Flavoridin resulted to be the most effective in inhibiting bacterial entry into host cells; echistatin was almost 50% less effective than flavoridin, whereas kistrin was definitely inactive. Our results suggest that alpha(5)beta(1) integrin receptor, which binds flavoridin with higher affinity than the other two disintegrins, plays a major role in Y. enterocolitica uptake into HeLa cells. Furthermore, flavoridin binding to this integrin prevented the disruption of the functional complex FAK-Cas, which occurs in the Y. enterocolitica uptake process.

Pro-apoptotic signaling pathway activated by echistatin in GD25 cells.

Disintegrins, low molecular weight RGD-containing polypeptides isolated from snake venoms, have seen use as integrin antagonists in the field of tumor biology and angiogenesis. In this study, we investigated the molecular mechanism by which the disintegrin echistatin affects cell adhesion and signaling resulting in an apoptotic response in the GD25 cell system. Wild-type GD25 cells, which lack expression of the beta(1) family of integrin, and stable transfectants expressing the A isoform of beta(1) integrin subunit were used. Nanomolar concentrations of echistatin detached fibronectin- and vitronectin-adherent GD25 cells from immobilized substratum. However, prior to inducing detachment of adherent cells, echistatin caused apoptosis as measured by caspase-3 activation. Either cell detachment or apoptotic response induced by echistatin were more pronounced on fibronectin-adherent GD25 cells than on vitronectin-adherent ones. GD25 cell exposure to echistatin caused a reduction of tyrosine phosphorylation levels of pp125(FAK), whereas it didn't affect either Shc tyrosine phosphorylation levels or Shc-Grb2 functional association. The down-regulation of pp125(FAK) preceded apoptosis and cell detachment induced by echistatin. Our results indicate that pp125(FAK) and not Shc pathway is involved in echistatin-induced apoptotic response in the GD25 cell system.

Ochratoxin A affects COS cell adhesion and signaling.

Ochratoxin A (OTA), a metabolite produced by strains of Aspergillus and Penicillium, has nephritogenic, carcinogenic, and teratogenic activity in animals and humans. Nanomolar concentrations of OTA promote apoptosis in a cell-type specific fashion. In this study, we have analyzed the molecular mechanism by which OTA affects COS cell adhesion and signaling resulting in an apoptotic response. OTA, at noncytotoxic doses, was able to detach collagen- and fibronectin-adherent cells from immobilized substratum. However, prior to inducing detachment of adherent cells, OTA caused apoptosis as measured by caspase-3 activation. The treatment of adherent cells by OTA caused a reduction of tyrosine phosphorylation levels of FAK and of the adapter protein paxillin. The down-regulation of FAK preceded apoptosis and cell detachment induced by OTA. The mycotoxin was also able to cause a decrease of the phosphorylation levels of the two Shc isoforms, P66 and P52, in adherent cells. Since these Shc isoforms have been implicated in the activation of protein kinase c-Src, which is required for FAK tyrosine phosphorylation, the observed dephosphorylation of FAK and of the FAK substrate paxillin by OTA could be ascribed to the early down-regulation of Shc isoforms. However, whether FAK and Shc phosphorylation contribute both to the same pathway leading to the induction of apoptosis by OTA or are involved in two parallel signaling pathways remains to be investigated.