Expression and spatial heterogeneity of dipeptidyl peptidases in endothelial cells of conduct vessels and capillaries

Dipeptidyl peptidase IV (DPPIV)/CD26 is by far the most extensively studied member of the prolyl oligopeptidase family of serine proteases. The discovery of the related enzymes DPP8 and DPP9 necessitates a (re-)evaluation of the DPPIV-like enzymatic activity in cells and organs. In this study, we aimed (1) to investigate the expression of the individual dipeptidyl peptidases in different types of endothelial cells (ECs) and (2) to reconsider published data in relation to our findings. Examination of DPP expression in rat primary ECs of aortic, endocardial and cardiac microvascular origin revealed the presence of DPPIV-like activity in all cell lysates. More than half of this activity could be attributed to DPP8/9. Western blot analysis revealed an abundance of the DPP8 protein as compared to DPP9. The expression of DPPIV and DPP8 was significantly higher in the cardiac microvascular endothelium than in the other ECs, suggesting a more pronounced role of these DPPs in the microvasculature. In situ, DPP activity in ventricular microvasculature was completely inhibited by sitagliptin, indicating that DPPIV is the predominant DPPIV-like enzyme in this organ. By contrast, immunohistochemical studies indicated DPP9 as the predominant DPP in human carotid artery ECs. In conclusion, our results support a highly regulated expression of individual DPPs in ECs, with a spatial heterogeneity in the cardiovascular tree.     

A polymorphism in the splice donor site of ZNF419 results in the novel renal cell carcinoma-associated minor histocompatibility antigen ZAPHIR

Nonmyeloablative allogeneic stem cell transplantation (SCT) can induce remission in patients with renal cell carcinoma (RCC), but this graft-versus-tumor (GVT) effect is often accompanied by graft-versus-host disease (GVHD). Here, we evaluated minor histocompatibility antigen (MiHA)-specific T cell responses in two patients with metastatic RCC who were treated with reduced-intensity conditioning SCT followed by donor lymphocyte infusion (DLI). One patient had stable disease and emergence of SMCY.A2-specific CD8+ T cells was observed after DLI with the potential of targeting SMCY-expressing RCC tumor cells. The second patient experienced partial regression of lung metastases from whom we isolated a MiHA-specific CTL clone with the capability of targeting RCC cell lines. Whole genome association scanning revealed that this CTL recognizes a novel HLA-B7-restricted MiHA, designated ZAPHIR, resulting from a polymorphism in the splice donor site of the ZNF419 gene. Tetramer analysis showed that emergence of ZAPHIR-specific CD8+ T cells in peripheral blood occurred in the absence of GVHD. Furthermore, the expression of ZAPHIR in solid tumor cell lines indicates the involvement of ZAPHIR-specific CD8+ T cell responses in selective GVT immunity. These findings illustrate that the ZNF419-encoded MiHA ZAPHIR is an attractive target for specific immunotherapy after allogeneic SCT.     

Intracellular serine protease inhibitor SERPINB4 inhibits granzyme M-induced cell death

Granzyme-mediated cell death is the major pathway for cytotoxic lymphocytes to kill virus-infected and tumor cells. In humans, five different granzymes (i.e. GrA, GrB, GrH, GrK, and GrM) are known that all induce cell death. Expression of intracellular serine protease inhibitors (serpins) is one of the mechanisms by which tumor cells evade cytotoxic lymphocyte-mediated killing. Intracellular expression of SERPINB9 by tumor cells renders them resistant to GrB-induced apoptosis. In contrast to GrB, however, no physiological intracellular inhibitors are known for the other four human granzymes. In the present study, we show that SERPINB4 formed a typical serpin-protease SDS-stable complex with both recombinant and native human GrM. Mutation of the P2-P1-P1' triplet in the SERPINB4 reactive center loop completely abolished complex formation with GrM and N-terminal sequencing revealed that GrM cleaves SERPINB4 after P1-Leu. SERPINB4 inhibited GrM activity with a stoichiometry of inhibition of 1.6 and an apparent second order rate constant of 1.3×10(4) M(-1) s(-1). SERPINB4 abolished cleavage of the macromolecular GrM substrates α-tubulin and nucleophosmin. Overexpression of SERPINB4 in tumor cells inhibited recombinant GrM-induced as well as NK cell-mediated cell death and this inhibition depended on the reactive center loop of the serpin. As SERPINB4 is highly expressed by squamous cell carcinomas, our results may represent a novel mechanism by which these tumor cells evade cytotoxic lymphocyte-induced GrM-mediated cell death.     

Human mesenchymal stem cell-conditioned medium improves cardiac function following myocardial infarction

Recent studies suggest that the therapeutic effects of stem cell transplantation following myocardial infarction (MI) are mediated by paracrine factors. One of the main goals in the treatment of ischemic heart disease is to stimulate vascular repair mechanisms. Here, we sought to explore the therapeutic angiogenic potential of mesenchymal stem cell (MSC) secretions. Human MSC secretions were collected as conditioned medium (MSC-CM) using a clinically compliant protocol. Based on proteomic and pathway analysis of MSC-CM, an in vitro assay of HUVEC spheroids was performed identifying the angiogenic properties of MSC-CM. Subsequently, pigs were subjected to surgical left circumflex coronary artery ligation and randomized to intravenous MSC-CM treatment or non-CM (NCM) treatment for 7 days. Three weeks after MI, myocardial capillary density was higher in pigs treated with MSC-CM (645 ± 114 vs 981 ± 55 capillaries/mm(2); P = 0.021), which was accompanied by reduced myocardial infarct size and preserved systolic and diastolic performance. Intravenous MSC-CM treatment after myocardial infarction increases capillary density and preserves cardiac function, probably by increasing myocardial perfusion.     

A Dynamic Integrated Analysis of Truck Tires in Western Europe

By evaluating tires from a perspective of industrial metabolism, potential novel and practical ways to reduce their environmental impact can be found. This may be achieved by focusing on technological issues such as choosing materials, designing products, and recovering materials, or by looking at institutional and social barriers and incentives such as opening waste markets or changing consumer behavior. A model is presented for the life cycle of truck tires in Western Europe that is dynamic in nature and values both environmental and economic consequences. Various scenarios are simulated including longer tire lifetimes, better maintenance of tire pressure, increased use of less-expensive Asian tires, and increased use of fuel efficiency-enhancing tires ("eco-tires"). Tentative results indicate that, among other things, more than 95% of the overall environmental impact during the life of a tire occurs during the use of the tire, due to the impact of tires on automotive fuel efficiency. Better maintenance of tire pressure and use of eco-tires produce greater environmental and economics benefits than more-durable and/or less-expensive (Asian) tires. These results imply that the emphasis in environmental policies related to tires should shift from the production and the waste stages to the consumption stage. It also suggests that the focus on materials throughput and associated improvements through factor 4 or factor 10 advances in reduction in mass are less important than the quality of the tires and their management.     

Exogenous NO suppresses flow-induced endothelium-derived NO production because of depletion of tetrahydrobiopterin

Exogenous nitric oxide (NO) suppresses endothelium-derived NO production. We were interested in determining whether this is also the case in flow-induced endothelium-derived NO production. If so, then is the mechanism because of intracellular depletion of tetrahydrobiopterin [BH4; a cofactor of NO synthase (NOS)], which results in superoxide production by uncoupled NOS? Isolated canine femoral arteries were perfused with 100 microM S-nitroso-N-acetylpenicillamine (SNAP; an NO donor) and/or 64 microM BH4. Perfusion of SNAP suppressed flow-induced NO production, which was evaluated as a change in the slope of the linear relationship between perfusion rate and NO production rate (P < 0.02 vs. control; n = 7). Subsequent BH4 perfusion returned the slope to the control level. Concomitant perfusion of SNAP and BH4 retained the control-level NO production (n = 7). Concomitant perfusion of SNAP and 4,5-dihydroxy-1,3-benzene disulfonic acid (Tiron; 1 mM; a membrane-permeable superoxide scavenger) also retained the control-level NO production (n = 7), whereas perfusion of Tiron after SNAP could not return the NO production to the control level (P < 0.02 vs. control; n = 7). We also found a significant decrease in BH4 concentration in the endothelial cells after SNAP perfusion. In conclusion, these results indicate that exogenous NO suppresses the flow-induced, endothelium-derived NO production by superoxide released from uncoupled NOS because of intracellular BH4 depletion.     

Protein kinase C epsilon induces systolic cardiac failure marked by exhausted inotropic reserve and intact Frank-Starling mechanism

Myofilament dysfunction is a common point of convergence for many forms of heart failure. Recently, we showed that cardiac overexpression of PKC epsilon initially depresses myofilament activity and then leads to a progression of changes characteristic of human heart failure. Here, we examined the effects of PKC epsilon on contractile reserve, Starling mechanism, and myofilament activation in this model of end-stage dilated cardiomyopathy. Pressure-volume loop analysis and echocardiography showed that the PKC epsilon mice have markedly compromised systolic function and increased end-diastolic volumes. Dobutamine challenge resulted in a small increase in contractility in PKC epsilon mice but failed to enhance cardiac output. The PKC epsilon mice showed a normal length-dependent tension development in skinned cardiac muscle preparations, although Frank-Starling mechanism appeared to be compromised in the intact animal. Simultaneous measurement of tension and ATPase demonstrated that the maximum tension and ATPase were markedly lower in the PKC epsilon mice at any length or Ca2+ concentration. However, the tension cost was also lower indicating less energy expenditure. We conclude 1) that prolonged overexpression of PKC epsilon ultimately leads to a dilated cardiomyopathy marked by exhausted contractile reserve, 2) that PKC epsilon does not compromise the Frank-Starling mechanism at the myofilament level, and 3) that the Starling curve excursion is limited by the inotropic state of the heart. These results reflect the significance of the primary myofilament contractilopathy induced by phosphorylation and imply a role for PKC epsilon-mediated phosphorylation in myofilament physiology and the pathophysiology of decompensated cardiac failure.     

Validation of lactose[15N,15N]ureide as a tool to study colonic nitrogen metabolism

In vitro experiments have shown that fermentation of carbohydrates prevents accumulation of nitrogen in the colon. Variable results have been obtained on modulation of dietary intakes in vivo. Lactose[15N,15N]-labeled ureide has been proposed as a tool to study colonic nitrogen metabolism. However, on oral administration of the marker, different urinary excretion patterns of the 15N label have been found. In this study, 50 mg lactose[15N,15N]ureide was directly instilled in the colon through an orocecal tube to investigate the colonic handling of this molecule in a direct way. In basal conditions, 42% (range, 37-48%) of labeled nitrogen administered as lactose[15N,15N]ureide was retrieved in urine after 72 h. A substantial variability in total urinary excretion of the label was found, but the urinary excretion pattern of the label was similar in all volunteers. When inulin, a fermentable carbohydrate, was administered together with the labeled marker, a significant decrease in urinary excretion of 15N after 72 h was found, to 29% (range, 23-34%). The effect of a smaller dose of inulin (250 mg) on colonic handling of lactose[15N,15N]ureide (50 mg), was investigated in another group of volunteers, and this time, fecal excretion of the marker was also evaluated. The results seem to indicate that fermentation of inulin causes an increased fecal excretion of the marker, thereby reducing urinary excretion but not retention in the human nitrogen pool. This instillation study shows that lactose[15N,15N]ureide is a tool with good properties to investigate the effect of different types of carbohydrates on nitrogen metabolism in the proximal colon in vivo.