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1.
Hum Immunol ; 72(11): 1064-7, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21925559

RESUMO

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.


Assuntos
Antígenos HLA-G/biossíntese , Insuficiência Cardíaca/diagnóstico , Insuficiência Cardíaca/imunologia , Adulto , Idoso , Biomarcadores/sangue , Citocinas/sangue , Progressão da Doença , Feminino , Antígenos HLA-G/sangue , Antígenos HLA-G/genética , Antígenos HLA-G/imunologia , Insuficiência Cardíaca/patologia , Insuficiência Cardíaca/fisiopatologia , Testes de Função Cardíaca , Humanos , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Sensibilidade e Especificidade , Regulação para Cima
2.
J Heart Lung Transplant ; 30(5): 583-8, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21273094

RESUMO

BACKGROUND: Post-transplant immunosuppression is associated with endothelial dysfunction that may lead to vasculopathy. We have previously demonstrated that cyclosporine causes vascular dysfunction. In this we study examined the effect of tacrolimus (Tac) in an identical model. METHODS: Lewis rats (n = 8 per group) were injected with Tac (low, medium or high dose) or saline (Con) daily for 2 weeks. Segments of thoracic aorta (TAo) were assessed for endothelium-dependent (Edep) and -independent (Eind) vasorelaxation (E(max)) and sensitivity to endothelin (ET)-induced vasoconstriction (C(max)). ET(A) and ET(B) receptor (Rc) expression levels were determined in TAo. Tumor necrosis factor-alpha (TNF-α) and transforming growth factor-beta (TGF-ß) expression were determined in myocardial (LV) tissue. Plasma ET levels and tissue oxidative injury were quantified by enzyme-linked immunoassay. RESULTS: Tac did not impair Edep relaxation when compared with Con (p = 0.69). Impairment of sodium nitroprusside-mediated Eind vasorelaxation was noted with Tac (E(max): Con 69 ± 2%, Tac high 54 ± 2%; p = 0.0001), whereas no such impairment was seen with diltiazem-mediated Eind vasorelaxation (p = 0.06). Tac decreased sensitivity to ET (C(max): Con 222 ± 19%, Tac high 162 ± 11%; p = 0.0002) and ET levels (Con 0.8 ± 0.1 fmol/ml, Tac 0.4 ± 0.1 fmol/ml; p = 0.02). Tac did not alter ET(A) Rc expression (p = 0.28), but increased ET(B) Rc levels (p = 0.02). Oxidative injury was similar in both LV (p = 0.43) and Ao (p = 0.73) tissue. Similarly, TNF-α expression (p = 0.16) was not different between groups, whereas expression of TGF-ß demonstrated a significant decrease with Tac treatment (p = 0.02). CONCLUSION: Our findings suggests that tacrolimus has beneficial effects with respect to endothelial function.


Assuntos
Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/fisiologia , Homeostase/efeitos dos fármacos , Imunossupressores/farmacologia , Tacrolimo/farmacologia , Sistema Vasomotor/efeitos dos fármacos , Sistema Vasomotor/fisiologia , Animais , Aorta Torácica/efeitos dos fármacos , Aorta Torácica/fisiologia , Homeostase/fisiologia , Masculino , Modelos Animais , Miocárdio/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Ratos , Ratos Endogâmicos Lew , Receptor de Endotelina A/metabolismo , Receptor de Endotelina B/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Vasodilatação/efeitos dos fármacos , Vasodilatação/fisiologia , Vasodilatadores/farmacologia
3.
Circulation ; 122(11 Suppl): S156-61, 2010 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-20837907

RESUMO

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% O(2)) followed by normoxia (21% O(2)) 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-κB 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-κB 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-κB nuclear localization and augmented inhibitor of nuclear factor-κB protein levels, suggesting that it inhibits nuclear factor-κB 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.


Assuntos
Vasos Coronários/metabolismo , Células Endoteliais/metabolismo , Fatores de Crescimento Endotelial/metabolismo , Regulação da Expressão Gênica , Molécula 1 de Adesão Intercelular/biossíntese , Transporte Ativo do Núcleo Celular , Proteínas de Ligação ao Cálcio , Hipóxia Celular , Núcleo Celular/metabolismo , Células Cultivadas , Vasos Coronários/patologia , Família de Proteínas EGF , Células Endoteliais/patologia , Humanos , Proteínas I-kappa B/metabolismo , NF-kappa B/metabolismo , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia
5.
Transpl Int ; 23(6): 641-8, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20102553

RESUMO

Endothelial progenitor cells (EPCs) may contribute to rejection and cardiac allograft vasculopathy (CAV) by being intrinsically involved in the rejection process and causing neointimal hyperplasia. The mammalian target of rapamycin inhibitors (mTORi), sirolimus and everolimus, have been demonstrated to attenuate the progression of CAV and are cytotoxic to EPC. Thus, one mechanism by which mTORi may protect against CAV is by altering EPC function. Our study measured circulating EPC function and correlated this assessment with rejection episodes in heart transplant (HT) recipients. In addition, we examined the effect of mTORi on EPCs. Patients who received HT at our institution between 1995 and 2007 were included and stratified by International Society for Heart and Lung Transplantation (ISHLT) rejection grade. Group A (n = 13) consisted of patients with at least one moderate/severe rejection episode (grade > or = 2). Group B (n = 28) patients had no moderate/severe episodes (grade < 2). Patients were also independently stratified based on exposure as mTORi (n = 21) vs. non mTORi (n = 20). To assess EPC functional capacity, we counted the number of colony-forming units (CFU) of EPCs in peripheral blood samples from HT recipients. There were no significant differences in baseline characteristics between groups. The mean EPC-CFU counts/plate for group A (rejecting) were 30 +/- 6 vs.16 +/- 3 for group B (nonrejecting) (P = 0.03). The EPC-CFU counts/plate in the mTORi group (15 +/- 3) were lower compared to the non mTORi (27 +/- 5) group (P = 0.04). We found that EPC colony-forming capacity was higher in HT patients who experienced moderate/severe rejection episodes. Patients on mTORi showed a reduced EPC colony count consistent with our previous findings of EPC cytotoxicity. Detection of circulating EPC function post-transplant may reliably identify patient risk level for subsequent allograft rejection and allow for appropriate adjustments to immunosuppression. Converting to mTORi therapy may reduce EPC function and provide a novel mechanism to prevent rejection and possibly attenuate the development of CAV.


Assuntos
Células Endoteliais/fisiologia , Rejeição de Enxerto/imunologia , Transplante de Coração , Células-Tronco/fisiologia , Adulto , Idoso , Ensaio de Unidades Formadoras de Colônias , Everolimo , Feminino , Rejeição de Enxerto/prevenção & controle , Humanos , Imunossupressores/uso terapêutico , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Masculino , Pessoa de Meia-Idade , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Estudos Retrospectivos , Risco , Sirolimo/análogos & derivados , Sirolimo/uso terapêutico , Serina-Treonina Quinases TOR
7.
J Heart Lung Transplant ; 28(11): 1193-7, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19783164

RESUMO

BACKGROUND: Human leukocyte antigen-G (HLA-G), a protein primarily expressed during pregnancy, helps maintain maternal-fetal immune tolerance. Myocardial and/or soluble HLA-G (sHLA-G) expression confers protection against rejection and vasculopathy after heart transplantation. Although the precise mechanisms remain unclear, immunosuppressive therapy has been reported to influence this expression. METHODS: We compared sHLA-G expression in heart transplant recipients receiving two different anti-proliferative agents: mycophenolate mofetil (MMF) and everolimus (RAD). Twelve-hour pharmacokinetic (PK) studies were conducted in patients after cyclosporine (CsA) administration in conjunction with RAD or MMF, during which plasma HLA-G concentrations were measured by enzyme-linked immunoassay (ELISA). RESULTS: Among patients receiving RAD, 78% expressed detectable levels of plasma HLA-G (1,002 +/- 511 ng/ml) compared with 25% of patients receiving MMF (612 +/- 438 ng/ml, p = 0.03). In all sHLA-G(+) patients, expression remained constant, with no significant changes in HLA-G levels throughout the 12-hour PK study period. CsA did not appear to influence sHLA-G expression, as there was no correlation between HLA-G levels and CsA exposure (R(2) = 0.43, p = 0.08). CONCLUSIONS: These preliminary findings suggest a disproportionate expression of HLA-G in patients under two distinct immunosuppression strategies after heart transplantation. Although CsA administration does not influence sHLA-G levels, RAD but not MMF is associated with sHLA-G expression. Larger prospective clinical investigations are required to confirm whether RAD is independently associated with increased HLA-G expression.


Assuntos
Antígenos HLA/genética , Transplante de Coração/imunologia , Antígenos de Histocompatibilidade Classe I/genética , Imunossupressores/uso terapêutico , Ácido Micofenólico/análogos & derivados , Sirolimo/análogos & derivados , Adulto , Idoso , Área Sob a Curva , Ciclosporina/sangue , Ciclosporina/uso terapêutico , Quimioterapia Combinada , Everolimo , Feminino , Seguimentos , Antígenos HLA/sangue , Antígenos HLA-G , Antígenos de Histocompatibilidade Classe I/sangue , Humanos , Masculino , Troca Materno-Fetal/genética , Troca Materno-Fetal/imunologia , Pessoa de Meia-Idade , Ácido Micofenólico/uso terapêutico , Prednisona/uso terapêutico , Gravidez , Proteínas da Gravidez/genética , Sirolimo/uso terapêutico
8.
Circulation ; 120(11 Suppl): S206-14, 2009 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-19752369

RESUMO

BACKGROUND: Hypertonic saline (HTS) has been previously demonstrated to have immune modulatory and vascular protective effects. We assessed the effect of donor pretreatment with HTS on allograft preservation in a porcine model of orthotopic heart transplantation. METHODS AND RESULTS: Orthotopic transplants were performed after 6 hours of cold static allograft storage. Donor pigs were randomly assigned to pretreatment with (n=7) or without (n=6) HTS (4.5 mL/kg of 7.5% NaCl) administered 1 hour before donor heart arrest. Administration of HTS increased serum sodium level from 138+/-2 mmol/L to 154+/-4 mmol/L, which normalized to 144+/-3 mmol/L 1 hour after infusion. Successful weaning from cardiopulmonary bypass was significantly greater in HTS-treated hearts (6/7 vs 1/6; P=0.029). Preload recruitable stroke work after transplantation was improved compared to control (88+/-21% vs 35+/-8% of baseline; P=0.0001). Similarly, end-systolic elastance was improved compared to control (85+/-17% vs 42+/-12% of baseline; P=0.0002). Posttransplantation systolic blood pressure was significantly higher in the donor HTS group (60+/-9 mm Hg vs 35+/-6 mm Hg; P=0.04). Donor HTS treatment improved coronary artery endothelial-dependent vasorelaxation compared with control (Emax: HTS, 59+/-4%; control, 47+/-3%; P=0.04). HTS also resulted in improved endothelial-independent vasorelaxation compared with control (Emax: HTS, 71+/-3%; control, 59+/-4%; P=0.03; ED-50: HTS, 0.56x10 to 6+/-0.23 mol/L; control, 2.5x10 to 6+/-1.0 mol/L; P=0.04). Sensitivity to endothelin-1-induced vasospasm was reduced with HTS pretreatment (% maximum contraction [Cmax]: HTS, 338+/-15%; control, 419+/-40%; P=0.01). CONCLUSIONS: Donor HTS pretreatment attenuates posttransplantation cardiac allograft myocardial dysfunction, improves posttransplantation systemic hemodynamic function, and preserves posttransplantation cardiac allograft vascular function. HTS may be a novel organ donor intervention to prevent primary graft dysfunction.


Assuntos
Transplante de Coração/efeitos adversos , Disfunção Primária do Enxerto/prevenção & controle , Solução Salina Hipertônica/farmacologia , Doadores de Tecidos , Animais , Ponte Cardiopulmonar , Citocinas/sangue , Endotélio Vascular/fisiologia , Feminino , Transplante de Coração/mortalidade , Modelos Animais , Sódio/sangue , Suínos , Transplante Homólogo , Função Ventricular Esquerda
9.
Circulation ; 118(14 Suppl): S58-64, 2008 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-18824770

RESUMO

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-gamma 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-gamma, 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.


Assuntos
Células Endoteliais/efeitos dos fármacos , Células Endoteliais/imunologia , Antígenos HLA/biossíntese , Antígenos de Histocompatibilidade Classe I/biossíntese , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/imunologia , Progesterona/farmacologia , Aorta/citologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Vasos Coronários/citologia , Citocinas/farmacologia , Relação Dose-Resposta a Droga , Células Endoteliais/citologia , Células Endoteliais/fisiologia , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Antígenos HLA-G , Antagonistas de Hormônios/farmacologia , Humanos , Imunossupressores/farmacologia , Mifepristona/farmacologia , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/fisiologia , Progesterona/administração & dosagem , Receptores de Progesterona/antagonistas & inibidores
10.
J Thorac Cardiovasc Surg ; 135(4): 938-44, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18374784

RESUMO

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.


Assuntos
Ciclosporina/farmacologia , Endotélio Vascular/efeitos dos fármacos , Hidrocortisona/farmacologia , Imunossupressores/farmacologia , Animais , Aorta/efeitos dos fármacos , Aorta/fisiologia , Endotelina-1/fisiologia , Endotélio Vascular/fisiologia , Glucocorticoides/farmacologia , Masculino , Óxido Nítrico/fisiologia , Ratos , Ratos Endogâmicos Lew , Vasodilatação/efeitos dos fármacos , Vasodilatação/fisiologia
11.
J Heart Lung Transplant ; 27(5): 522-7, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18442718

RESUMO

BACKGROUND: Human leukocyte antigen-G (HLA-G), a non-classical MHC I protein with restricted tissue expression, plays an essential role in immune tolerance and has been negatively associated with acute and chronic rejection after heart transplantation. We assessed myocardial HLA-G expression in adult heart transplant patients in an attempt to determine the value of this protein in identifying patients with a low risk of acute cellular rejection. METHODS: Two groups of patients were included in this study. Group A (non-rejecting) included 29 patients who had no moderate or severe rejection episodes (ISHLT Grade <2R) post-transplant. Group B (rejecting) included 38 patients with at least two moderate or severe rejection episodes (Grade >or=2R) within a 1-year period. Expression of HLA-G in three myocardial biopsies post-transplant from each patient was determined through immunohistochemical staining. RESULTS: In Group A, 86% of patients had HLA-G(+) biopsies compared with 11% of patients in Group B (p < 0.001; sensitivity 86%, specificity 87%). Whereas 60% of non-rejecting HLA-G(+) patients had at least two positive biopsies, all rejecting HLA-G(+) patients had only one positive biopsy. CONCLUSIONS: There is a significant negative association between myocardial HLA-G expression and acute cellular rejection after cardiac transplantation. Detection of HLA-G appears to reliably indicate a low risk of developing moderate or severe allograft rejection and may, subsequently, allow for a reduced immunosuppressive regimen.


Assuntos
Rejeição de Enxerto/genética , Rejeição de Enxerto/imunologia , Antígenos HLA/imunologia , Transplante de Coração/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Adolescente , Adulto , Idoso , Feminino , Expressão Gênica , Antígenos HLA-G , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Fatores de Risco
12.
J Thorac Cardiovasc Surg ; 133(5): 1137-46, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-17467420

RESUMO

OBJECTIVES: The proinflammatory marker C-reactive protein has been demonstrated to play a role in the development of atherosclerosis. Endothelin-1 and nitric oxide homeostasis is crucial for normal vasomotor function, limiting inflammatory activation and maintaining a nonthrombogenic endothelial surface. In addition to its vasoactive properties, endothelin-1 is also an inflammatory cytokine. We have previously demonstrated that C-reactive protein impairs endothelial cell nitric oxide production. Protein kinase C, an important signal transducer within the cell, is involved in several cellular responses to external stimuli. We therefore sought to determine whether endothelin-1 exposure modulates C-reactive protein's effects on nitric oxide production via protein kinase C. METHODS: Endothelial cells were incubated with C-reactive protein (200 microg), endothelin-1 (100 nM), C-reactive protein + endothelin-1, or phosphate-buffered saline solution (control) for 24 hours. After exposure, endothelial nitric oxide synthase expression was determined in addition to total nitric oxide production and protein kinase C translocation and activity. RESULTS: Endothelial nitric oxide synthase protein expression was reduced following incubation with C-reactive protein and endothelin-1 treatment compared with baseline by 40% and 45%, respectively (P = .04); however, no additive effects were seen with coincubation. C-reactive protein produced a 47% decrease in nitric oxide production compared with control. Coincubation with endothelin-1 resulted in a synergistic 70% reduction in nitric oxide production (P = .001). C-reactive protein exposure inhibited translocation of protein kinase C lambda compared with control (P = .01). Furthermore, coincubation of C-reactive protein with endothelin-1 led to a synergistic inhibition of protein kinase C lambda translocation (P = .01). C-reactive protein exposure reduced protein kinase C activity by 40% compared with control (P = .02), although coincubation with endothelin-1 had a synergistic reduction in activity (P = .02). CONCLUSIONS: Our results indicate that endothelin-1 exposure accentuated C-reactive protein's impairment of endothelial nitric oxide production via synergistic inhibition of protein kinase C lambda translocation and activity. Our investigations suggest that endothelin-1 inhibition and protein kinase C stimulation may provide a novel therapeutic strategy to improve vascular nitric oxide homeostasis and mitigate the proatherosclerotic effects of C-reactive protein.


Assuntos
Aterosclerose/metabolismo , Proteína C-Reativa/farmacologia , Endotelina-1/farmacologia , Endotélio Vascular/metabolismo , Alcaloides/farmacologia , Benzofenantridinas/farmacologia , Bosentana , Células Cultivadas , Citocinas/farmacologia , Endotelina-1/antagonistas & inibidores , Endotélio Vascular/fisiopatologia , Homeostase , Humanos , Naftalenos/farmacologia , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Proteína Quinase C/metabolismo , Veia Safena , Sulfonamidas/farmacologia
13.
Circulation ; 114(1 Suppl): I319-26, 2006 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-16820593

RESUMO

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 PKCdelta and PKCalpha translocation compared with control, whereas translocation of PKClambda 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 PKClambda and leads to increased PKC activity and NO production. ET-1 antagonism may provide a novel therapeutic strategy to improve vascular homeostasis.


Assuntos
Células Endoteliais/enzimologia , Endotelina-1/farmacologia , Isoenzimas/fisiologia , Óxido Nítrico Sintase Tipo III/biossíntese , Óxido Nítrico/biossíntese , Proteína Quinase C/fisiologia , Alcaloides , Benzofenantridinas , Bosentana , Caveolina 1/biossíntese , Caveolina 1/genética , Membrana Celular/enzimologia , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/enzimologia , Células Endoteliais/efeitos dos fármacos , Endotelina-1/antagonistas & inibidores , Endotélio Vascular/citologia , Ativação Enzimática/efeitos dos fármacos , Indução Enzimática/efeitos dos fármacos , Homeostase/efeitos dos fármacos , Isoenzimas/antagonistas & inibidores , Naftalenos/farmacologia , Óxido Nítrico Sintase Tipo II/biossíntese , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo III/genética , Fenantridinas/farmacologia , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C-alfa/metabolismo , Proteína Quinase C-delta/metabolismo , Transporte Proteico/efeitos dos fármacos , Veia Safena/citologia , Sulfonamidas/farmacologia , Acetato de Tetradecanoilforbol/farmacologia
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