Danny Ramzy

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.

Dual immunosuppression enhances vasomotor injury: Interactive effect between endothelin-1 and nitric oxide bioavailability

OBJECTIVE Cyclosporine A and corticosteroids are associated with many side effects, such as endothelial dysfunction and transplant vasculopathy. We examined the effects of cyclosporine A and hydrocortisone exposure on endothelial function of the rat thoracic aorta. METHODS Lewis rats were injected with cyclosporine A, hydrocortisone, cyclosporine A + hydrocortisone, or intraperitoneal saline daily for 2 weeks. Endothelial-dependent and independent vascular relaxation were assessed in isolated segments of thoracic aorta, as well as endothelin-1-induced vasoreactivity. Protein expression of endothelial nitric oxide synthase, endothelin(A), and endothelin(B) receptors were also determined in the thoracic aorta. RESULTS Exposure to cyclosporine A and cyclosporine A + hydrocortisone resulted in a reduction in endothelial-dependent vasorelaxation compared with control and hydrocortisone (P = .001). Cyclosporine A and hydrocortisone-treated rats demonstrated increased vasoreactivity to endothelin-1 compared with control, whereas cyclosporine A + hydrocortisone treatment resulted in a synergistic increase (P = .04). All treatment groups displayed a significant reduction in endothelial nitric oxide synthase expression compared with control (P = .001). Endothelin(A) receptor expression was increased in all treatment groups with a synergistic effect seen after cyclosporine A + hydrocortisone treatment. No differences were seen in endothelin(B) receptor expression. CONCLUSION Cyclosporine A and hydrocortisone induce vasomotor dysfunction with a synergistic impairment observed after concomitant exposure. Our findings suggest that the resultant vasomotor dysfunction is the result of alterations in both nitric oxide and endothelin-1 regulation.

Orthotopic Cardiac Transplantion in a Patient With Pectus Excavatum: Approach Via Left Anterior Thoracotomy

Abstract  Severe pectus excavatum, although a rare condition, is likely to be encountered by large transplant centers. The main concerns for transplant centers are the associated cardiopulmonary abnormalities along with the technical details of the transplant. We describe our experience managing a patient with severe pectus excavatum with associated cardiorespiratory abnormalities. This case report demonstrates that orthotopic cardiac transplantation through a left anterior thoracotomy is a feasible method in patients with significant displacement of the heart into the left chest.