Rohit Sheshgiri

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.

Progesterone Induces Human Leukocyte Antigen-G Expression in Vascular Endothelial and Smooth Muscle Cells

Background— Human leukocyte antigen-G (HLA-G) expression in heart transplant patients has been negatively associated with acute cellular rejection and cardiac allograft vasculopathy. We assessed HLA-G expression in vascular human endothelial and smooth muscle cell cultures to determine if future therapeutic agents can be targeted toward inducing HLA-G expression to protect against allograft rejection and vasculopathy. Methods and Results— Human coronary artery endothelial, aortic endothelial, and coronary artery smooth muscle cell cultures were exposed to cytokines (interferon-γ or interleukin-10), hypoxia/reoxygenation stress, immunosuppressive agents (cyclosporine, sirolimus, or tacrolimus), or progesterone. HLA-G was not expressed by untreated, normoxic cells. Furthermore, maximal doses of interferon-γ, interleukin-10, cyclosporine, sirolimus, or tacrolimus, as well as exposure to hypoxia/reoxygenation, failed to induce HLA-G expression. HLA-G, which has previously not been detected in adult vascular endothelial and smooth muscle cells, was detected by enzyme-linked immunosorbent assay and flow cytometry in human coronary artery endothelial, human coronary aortic endothelial, and human coronary artery smooth muscle cultures after incubation with progesterone in a dose-dependent manner (P<0.001) with no change in cellular proliferation ability or viability. This effect was partially blocked in the presence of mifepristone, a progesterone receptor antagonist (human coronary artery endothelial: 48.8±15.6%; human coronary aortic endothelial: 59.5±9.5%; human coronary artery smooth muscle: 59.8±9.8% of control; P<0.05). Progesterone-induced HLA-G expression was not protective against hypoxia/reoxygenation injury. Conclusions— HLA-G is not expressed at baseline in vascular endothelial and smooth muscle cells but can be induced by exposure to progesterone. Although tightly regulated, induction of HLA-G expression in these cells may represent a promising and novel therapeutic strategy to protect against rejection and cardiac allograft vasculopathy after heart transplantation.

Epidermal Growth Factor-Like Domain 7 Suppresses Intercellular Adhesion Molecule 1 Expression in Response to Hypoxia/Reoxygenation Injury in Human Coronary Artery Endothelial Cells

Background— Epidermal growth factor-like domain 7 (Egfl7) is a chemoattractant for endothelial cells, and its expression is restricted to endothelial cells. Hypoxia/reoxygenation (H/R) induced endothelial injury that occurs during transplantation contributes to the subsequent development of allograft vasculopathy. We investigated the effect of Egfl7 on endothelial cell intercellular adhesion molecule 1 expression in response to H/R injury. Methods and Results— Human coronary artery endothelial cells were submitted to hypoxia (0.1% O2) followed by normoxia (21% O2) in the presence or absence of Egfl7 (100 ng/mL). Hypoxia alone increased the expression of Egfl7×140±8% of control at 3 hours (n=6; P<0.05) and 385±50% of control at 6 hours (n=6; P<0.001). Incubation with Egfl7 during the reoxygenation period prevented intercellular adhesion molecule 1 upregulation (mean fluorescence intensity: 5.37±0.92 versus 3.81±0.21; P<0.05; n=4 per group). Nuclear factor-&kgr;B nuclear localization on H/R injury was blocked by Egfl7 administration (cytosolic/nuclear ratio of 0.93±0.01 versus 1.44±0.24; P<0.05; n=4 per group). Inhibitor of nuclear factor-&kgr;B protein level was significantly reduced on H/R injury (26±4.6% of control expression; P<0.05; n=4 per group); however, concurrent incubation with Egfl7 attenuated this reduction (46±6.2% of control expression; P<0.05 when compared with H/R injury alone; n=4 per group). Conclusions— Our study reveals the novel observation that hypoxia upregulates human coronary artery endothelial cells expression of Egfl7 and that Egfl7 inhibits expression of intercellular adhesion molecule 1 subsequent to H/R injury. Mechanistically, Egfl7 prevented nuclear factor-&kgr;B nuclear localization and augmented inhibitor of nuclear factor-&kgr;B protein levels, suggesting that it inhibits nuclear factor-&kgr;B activation, a key step in the inflammatory activation of endothelial cells. Egfl7 may be protective against H/R injury incurred during transplantation and may modulate the events that lead to the development of graft vasculopathy.

Association Between HLA-G Expression and C4d Staining in Cardiac Transplantation

After heart transplantation, allograft survival is often limited by rejection and vasculopathy. Human leukocyte antigen-G (HLA-G) is a nonclassical major histocompatibility complex-I protein expressed primarily by fetal cytotrophoblasts during early pregnancy, and it plays an essential role in maintaining maternal-fetal immune tolerance (1). Previous studies have demonstrated that HLA-G expression postheart transplant seems to confer protection against acute cellular rejection and cardiac allograft vasculopathy (2,3), possibly through inhibition of immune responses directed against the allograft. However, the relationship between HLA-G expression and antibody-mediated rejection remains unknown. Indeed, humoral rejection posttransplant has been linked to poor clinical outcomes (4). Therefore, this preliminary investigation was designed to assess the association between soluble HLA-G (sHLA-G) expression and C4d staining in heart transplant patients, because this is a routinely used technique in characterizing antibodymediated rejection (5). Patients who had undergone heart transplantation at our center were enrolled in this study, which was approved by the research ethics board. Blood samples were collected in EDTA-containing tubes during routine endomyocardial biopsy procedures, stored at 4°C, and centrifuged for 20 min. Plasma was isolated, flash frozen, and stored at 80°C within 4 hr of blood collection until further analysis. Soluble HLA-G levels were detected by an ELISA specific for all HLA-G isoforms and quantified according to a purified protein standard curve, as previously described (6). All patients were followed up after approximately 6 months to monitor changes in HLA-G levels. Clinically significant acute cellular rejection episodes (International Society for Heart and Lung Transplantation grade 2R) and C4d expression within 1 year of HLA-G measurement were assessed by a cardiac pathologist blinded to the clinical information and HLA-G status of study patients. This investigation included 50 patients (40 men and 10 women) with a mean age of 48 12 years (range 23– 64 years), who received a heart transplant between 1986 and 2008. Among study patients, 34 were HLA-G ( 100 ng/ mL), whereas 16 were HLA-G ( 100 ng/mL). Six-month follow-up revealed no changes in HLA-G status among all patients. All patients had normal left ventricular function by echocardiogram at the time of HLA-G determination. There were no significant differences in demographic characteristics, pretransplant conditions, time of HLA-G measurement posttransplant, or immunosuppressive therapy between groups (Table 1). However, only 13% of HLA-G patients suffered clinically significant acute cellular rejection (International Society for Heart and Lung Transplantation grade 2R) compared with 63% HLA-G patients (P 0.01), 1 year before and after HLA-G determination. Interestingly, the prevalence of patients who had biopsies with C4d deposition was also significantly lower in the HLA-G (13%) versus the HLA-G (38%) group (P 0.03), within 1 year of the HLA-G measurement.

Human leukocyte antigen–G is upregulated in heart failure patients: A potential novel biomarker

Immune activation and inflammation play critical roles in the development of heart failure (HF). Human leukocyte antigen-G (HLA-G) is a nonclassical, major histocompatibility complex class I (MHC-I) protein, upregulated in the context of transplantation, malignancy, and inflammation, and has been correlated with various clinical outcomes. We sought to evaluate the utility of plasma HLA-G in identifying patients with HF. We conducted a single-center, cross-sectional pilot study involving 82 patients diagnosed with HF and 10 healthy controls. Concentrations of circulating HLA-G and inflammatory markers were detected with specific enzyme-linked immunosorbent assay kits and quantified according to purified protein standards. The mean age of the patients was 49.1 ± 12.0 years and 62.2% were male. The median and interquartile range of HLA-G levels (U/ml) were significantly higher (p < 0.001) in HF patients (63, 36-98) compared with controls (28, 22-40). Moreover, HLA-G levels that were similarly (p = 0.766) upregulated across all New York Heart Association functional classes. There was no significant correlation between serum HLA-G and other biomarkers. In conclusion, HLA-G is upregulated in patients diagnosed with HF. Its marked elevation even in New York Heart Association class I patients might indicate that serum HLA-G is a more sensitive marker than other classical HF biomarkers.

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.