Santiago Miriuka

Elevated endothelin-1 levels impair nitric oxide homeostasis through a PKC-dependent pathway.

Background— Endothelin-1 (ET-1) plays an important role in the maintenance of vascular tone and pathological states such as ischemia/reperfusion (I/R) injury, coronary vasospasm, and cardiac allograft vasculopathy. We assessed the effects of elevated ET-1 levels as seen after I/R to determine if ET-1 modulates nitric oxide (NO) production via the translocation of specific protein kinase C (PKC) isoforms. Methods and Results— Human saphenous vein endothelial cells (HSVECs) (n=8) were incubated with ET-1 or phosphate-buffered saline (PBS) for 24 hours. NO production was determined in the supernatant by measuring nitrate/nitrite levels. Protein expression of endothelial nitric oxide synthase (eNOS), inducible NOS (iNOS), caveolin-1 and PKC were determined. Lastly, PKC translocation and activity were assessed after exposure to the drug of interest. HSVECs exposed to ET-1 displayed decreased NO production. PKC inhibition reduced NO production, whereas PKC activation increased production. NO production was maintained when HSVECs exposed to ET-1 were treated with the PKC agonist, PMA. eNOS protein expression was reduced after ET-1 treatment. PKC inhibition also downregulated eNOS protein expression, whereas PMA upregulated expression. ET-1 exposure led to a significant increase in PKCΔ and PKCϵ translocation compared with control, whereas translocation of PKC&lgr; was inhibited. ET-1 exposure significantly reduced overall PKC activity compared with control. Conclusions— Our study demonstrates that high levels of ET-1 impair endothelial NO production via an isoform-specific PKC-mediated inhibition of eNOS expression. ET-1 antagonism with bosentan stimulates translocation of PKC&lgr; and leads to increased PKC activity and NO production. ET-1 antagonism may provide a novel therapeutic strategy to improve vascular homeostasis.

mTOR inhibition induces endothelial progenitor cell death.

Immunosuppressants are necessary to prevent graft rejection after solid organ transplantation. However, they are also known to have significant side effects, including endothelial toxicity. Endothelial progenitor cells originate in the bone marrow and are recognized by their angiogenic and endothelial reparative properties. The effects of the immunosuppressants cyclosporine A (CyA), tacrolimus and rapamycin were analyzed on endothelial progenitor‐like cells. Rapamycin induced rapid cell death, even at concentrations much lower than those used clinically, in peripheral blood mononuclear cells (PBMC) cultured to favor outgrowth of endothelial progenitors. Cyclosporine A and tacrolimus had no significant effects at clinical concentrations. The effect of rapamycin was specific to endothelial progenitor cells, in particular to the early stages of differentiation, as a lesser effect was observed in late outgrowth endothelial progenitors, mature aortic endothelial cells, and macrophages derived from the same PBMCs. The mechanism of cell death appeared to be apoptosis; however, its induction was probably multifactorial and did not depend on caspase or cathepsin activation. In conclusion, rapamycin induces endothelial progenitor cell death, possibly because it blocks survival signals given by growth factors critically required by these cells.

Role Of Endothelin-1 and Nitric Oxide Bioavailability in Transplant-Related Vascular Injury Comparative Effects of Rapamycin and Cyclosporine

Background— Cyclosporine (CyA) is associated with many side effects, including endothelial dysfunction and transplant vasculopathy (TxV). We previously demonstrated that CyA results in impairment of nitric oxide bioavailability and enhanced sensitivity to endothelin-1 (ET-1). In this study, we evaluated rapamycin (SRL) for its effects on the endothelium. Methods and Results— Lewis rats (n =8) were injected with SRL (1.5 mg/kg), CyA (5 mg/Kg), or saline (Con) intraperitoneally daily for 2-weeks. Thoracic aortic segments were assessed for endothelial-dependent (Edep) and independent (Eind) relaxation after exposure to acetylcholine and sodium nitroprusside by deriving the percent maximum relaxation (Emax). ET-1 plasma levels were also measured. Thoracic aortic expression of endothelial nitric oxide synthase (eNOS), ETA and ETB receptors (Rc), were determined. Oxidative injury was assessed by changes in 8-isoprostane levels. CyA exposure resulted in lower Edep vasorelaxation compared with control and SRL (Emax: SRL, 58±4%; CyA, 24±7%; Con, 52±8%; P=0.001). No differences in Eind vasorelaxation were seen. CyA exposure also increased sensitivity to ET-1 (% maximum contraction [Cmax]: Con, 211±8%; SRL, 230±5%; CyA, 259±3%; P=0.04). Only SRL treatment reduced ET-1 plasma levels. CyA reduced eNOS expression by 30% and increased ETA Rc expression by 34% compared with both Con and SRL (P=0.02). CyA resulted in higher 8-isoprostane levels (CyA, 50±2%; SRL, 3±3%; Con, 2±5%; P=0.02). Conclusions— CyA results in vascular dysfunction characterized by impairment of Edep vasorelaxation and enhanced sensitivity to vasospasm. SRL did not impair Edep vasorelaxation or increase sensitivity to vasospasm while lowering ET-1 levels and preserving eNOS protein expression. We conclude that SRL is less deleterious to the vasculature than CyA and may prevent TxV by these mechanisms.

Thromboembolism in heart transplantation: role of prothrombin G20210A and factor V Leiden.

Background. Thromboembolism has been reported as a frequent complication after cardiac transplantation. Many risk factors for thrombosis may explain this, such as metabolic alterations and the use of cyclosporine. In the general population, two single nucleotide polymorphisms (SNPs), factor V Leiden and prothrombin G20210A (PT G20210A), have been associated with a significant increase in the risk of thrombosis. However, these mutations have not been analyzed in cardiac transplant patients. We describe the protracted history of recurrent thromboembolism in a rare case of homozygosity for the PT G20210A variant. This prompted us to analyze the entire cardiac transplant cohort for the incidence of thromboembolic events and their association with these genetic polymorphisms. Methods. We report the study of 84 cardiac transplant recipients. We retrospectively analyzed the frequency of thromboembolic episodes. The genotypes for FVL and PT G20210A were determined and correlated with those episodes. Results. Our results confirm a very high incidence of thromboembolism in this population. We also found a significant increase in the likelihood of thromboembolism in subjects with the PTB G20210A variant (odds ratio 3.08; 95% confidence interval: 1.7–5.5). Conclusions. The incidence of thromboembolic complications after heart transplantation is increased and may be related in part to genetic predisposition.

Genetic polymorphisms predisposing to hyperhomocysteinemia in cardiac transplant patients

Genetic determinants for high homocysteine (Hcy) levels are now well known. We studied several single nucleotide polymorphisms (SNP) in Hcy‐regulating genes [methylenetetrahydrofolate reductase (MTHFR) C677T and A1298C; methionine synthase (MS) A2756G; methionine synthase reductase (MTRR) A66G] in relation to total plasma Hcy levels, transplant coronary artery disease and thromboembolic episodes in 84 heart transplant patients, and we compared the incidence of these polymorphisms with those in a healthy adult controls. At least one copy of the G allele of the MTRR A66G SNP was found in a significantly greater proportion of cardiac transplant (CTX) recipients compared with controls (94.0% vs. 79.9% respectively). None of the SNP analyzed were correlated with total Hcy plasma levels or the presence of transplant coronary artery disease. However, MS A2756G was significantly associated with cobalamin levels (AA genotype: 290 ± 122 pmol/l; AG: 381 ± 151 pmol/l and GG: 415 ± 100 pmol/l), as was MTRR A66G (AA: 478 ± 219 pmol/l, AG: 306 ± 124 pmol/l and GG: 306 ± 123 pmol/l). MTRR A66G was also correlated with serum folate. No association was found with thromboembolic events. In conclusion, there was a significant difference in the frequency of the G allele genotype of the MTRR A66G in CTX patients versus controls. Differences in cobalamin and folate levels with the MTRR A66G and MS A2756G polymorphisms were noted. Thus, SNP in Hcy‐regulating genes may be important determinants of vitamin metabolism in CTX, raising the question of increased vitamin requirements to minimize increased plasma Hcy in this high‐risk group.

Hyperhomocysteinemia in heart transplantation: from bench to bedside

Orthotopic heart transplantation (OHT) is a life-saving treatment for end-stage cardiac disease. Short-term results are improving, but long-term survival is limited by an aggressive form of atherosclerotic disease termed transplant coronary artery disease (TxCAD). In patients surviving more than 1 year after transplantation, TxCAD remains the primary cause of death. The disease tends to be a diffuse, panarterial process, with progressive, concentric obliteration of both small and large coronary vessels. The cause is multifactorial, and several risk factors have been implicated. Current evidence shows that histocompatibility mismatch and acute cellular rejection have a role in the development of TxCAD. Also non-immunologic factors such as donor-related factors (age, sex, pre-existing coronary disease, ischemic time), recipient-related factors (age, sex, and obesity), cytomegalovirus infection, hypertension, metabolic derangements (diabetes and increased cholesterol and triglyceride levels), and tobacco use have been associated with TxCAD. Homocysteine (Hcy) has been implicated in the development of TxCAD; however, the evidence to date is largely retrospective (Table I). Hyperhomocysteinemia (HHcy) is almost universal after OHT, and strong evidence links HHcy to vascular dysfunction. This article summarizes the current information available regarding the pathogenesis of the HHcy-induced vascular dysfunction and how these alterations can further explain the association between HHcy and TxCAD.