RESUMO
BACKGROUND AND OBJECTIVE: Endothelin-1 (ET-1) plays a central role in the pathogenesis of several vascular diseases. Aminaftone is a drug used for the treatment of capillary disorders but which has a mechanism of action that is not fully understood. We investigated whether aminaftone may exert its effect by interfering with the production of ET-1. METHODS: Human ECV304 endothelial cells were incubated with interleukin-1beta (IL-1beta) 100 IU/mL with or without the addition of increasing concentrations of aminaftone (2, 4 or 6 microg/mL). ET-1 concentrations in surnatants were quantified by enzyme immunoassay kit at 3, 6 and 12 hours. Pre-pro-endothelin-1 (PPET-1) gene expressions were also analysed by real-time polymerase chain reaction (RT-PCR) at the same time points. Endothelin-converting enzyme (ECE) activity was also determined. RESULTS: Incubation with IL-1beta increased concentrations of ET-1 and PPET-1 relative gene expression. Incubation with aminaftone significantly reduced production of ET-1 in a concentration-dependent manner. A strong direct correlation was found between ET-1 concentrations and PPET-1 relative gene expression, but aminaftone did not influence ECE activity. CONCLUSION: Aminaftone inhibits ET-1 production in cell cultures by interfering with transcription of the PPET-1 gene. These findings may account for the clinical efficacy of aminaftone in the treatment of capillary disorders and may encourage conduct of further clinical trials.
Assuntos
Endotelina-1/biossíntese , Endotélio Vascular/metabolismo , Hemostáticos/farmacologia , para-Aminobenzoatos , Ácido 4-Aminobenzoico/farmacologia , Ácido Aspártico Endopeptidases/biossíntese , Ácido Aspártico Endopeptidases/genética , Linhagem Celular , Regulação para Baixo/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Enzimas Conversoras de Endotelina , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Ensaio de Imunoadsorção Enzimática , Humanos , Interleucina-1beta/sangue , Metaloendopeptidases/biossíntese , Metaloendopeptidases/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Prevention of graft dysfunction is a major objective in transplantation medicine. Previous research on experimental heart transplantation indicated that treatment with the immunomodulatory peptide alpha-melanocyte stimulating hormone (alpha-MSH) improves histopathology, prolongs allograft survival, and reduces expression of the main tissue injury mediators. Because calcium-handling is critical in heart graft function, we determined the effects of transplantation injury and influences of alpha-MSH treatment on representative calcium regulatory proteins in rat heart allografts. Hearts from Brown Norway rats were transplanted heterotopically into MHC incompatible Lewis rats. Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), protein kinase C epsilon (PKC epsilon), sarcoplasmic/endoplasmic reticulum calcium-ATPase 2 (SERCA2a), arrestin-beta1 (Arrb1), cholinergic receptor M2 (Chrm2), and inositol 1,4,5-triphosphate receptor 1 (InsP(3)R1) were examined in: (1) non-transplanted donor hearts; (2) allografts from saline-treated rats; and (3) allografts from rats treated with the synthetic alpha-MSH analog Nle4-DPhe7-alpha-MSH (NDP-alpha-MSH) (100 microg i.p. every 12h). Transplantation injury was associated with severe reduction in calcium regulatory protein transcription and expression level. NDP-alpha-MSH administration partly reversed inhibition of protein transcription and almost completely prevented protein loss. Finally, because certain effects of cyclic 3'-5'-adenosine monophosphate (cAMP) signaling on calcium handling in cardiac myocytes depend on activation of exchange protein directly activated by cAMP 1 (Epac1), we determined Epac1 mRNA and protein expression in heart allografts. Transplantation injury markedly reduced Epac1. NDP-alpha-MSH treatment significantly preserved both Epac1 protein and mRNA in the allografts. Administration of alpha-MSH or related melanocortins could reduce transplantation-induced dysfunction through protection of heart calcium regulatory proteins.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Cálcio/metabolismo , Transplante de Coração/métodos , alfa-MSH/farmacologia , Animais , Arrestinas/genética , Arrestinas/metabolismo , Western Blotting , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , AMP Cíclico/genética , AMP Cíclico/metabolismo , Expressão Gênica/efeitos dos fármacos , Perfilação da Expressão Gênica , Rejeição de Enxerto/tratamento farmacológico , Sobrevivência de Enxerto/efeitos dos fármacos , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Receptores de Inositol 1,4,5-Trifosfato , Masculino , Miocárdio/metabolismo , Miocárdio/patologia , Proteína Quinase C-épsilon/efeitos dos fármacos , Proteína Quinase C-épsilon/genética , Proteína Quinase C-épsilon/metabolismo , Ratos , Ratos Endogâmicos BN , Ratos Endogâmicos Lew , Receptores Colinérgicos/genética , Receptores Colinérgicos/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/efeitos dos fármacos , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , beta-Arrestina 1 , beta-ArrestinasRESUMO
Natural melanocortin peptides exert broad effects on the host and they have remarkable therapeutic potential. However, successful use of melanocortins as therapeutic agents depends on the design of molecules that have more stable pharmacological profiles. The synthetic peptide (CKPV)(2), based on the C-terminal sequence of alpha-melanocyte stimulating hormone (alpha-MSH), has anti-tumor necrosis factor-alpha (TNF-alpha) effects in vitro and in vivo and is a promising candidate to treat inflammation. Because neutrophil activity is a major target for anti-inflammatory therapies, we determined whether (CKPV)(2) modulates human neutrophil functions in vitro. Incubation of freshly-separated human neutrophils with 10(-12)-10(-6)M (CKPV)(2) significantly inhibited activities relevant to the inflammatory reaction. Neutrophil migration toward the two chemoattractants interleukin 8 (IL-8) and N-formyl-methionyl-leucyl-phenylalanine (fMLP) was significantly inhibited by (CKPV)(2). (CKPV)(2) also inhibited reactive oxygen intermediate (ROI) production induced by phorbol 12-myristate 13-acetate (PMA), but not that induced by fMLP. Because these effects of (CKPV)(2) were abolished by the adenylyl cyclase inhibitor 2',5'-dideoxyadenosine (ddAdo), they appear to be cAMP-dependent. Finally, the peptide reduced lipopolysaccharide (LPS)-stimulated expression of TNF-alpha, interleukin-1beta (IL-1beta), interleukin-8 (IL-8), and intercellular adhesion molecule 1 (ICAM-1), as well as TNF-alpha protein release in cell supernatants. The data indicate that (CKPV)(2) modulates broad cAMP-dependent, anti-inflammatory pathways in human neutrophils.
Assuntos
Anti-Inflamatórios/farmacologia , Melanocortinas/farmacologia , Neutrófilos/efeitos dos fármacos , Células Cultivadas , Humanos , Luminescência , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Alpha-melanocyte-stimulating hormone (alpha-MSH) is a peptide with broad anti-inflammatory effects. The present research was designed to determine production and effects of alpha-MSH in acute bleomycin-induced lung injury in rats. Intratracheal bleomycin instillation induced alpha-MSH expression in lung infiltrating cells and a marked peptide increase in the circulation. In experiments on the therapeutic potential of alpha-MSH on lung injury, we determined influences of the synthetic alpha-MSH analogue [Nle4-dPhe7]-alpha-MSH (NDP-alpha-MSH) on pulmonary edema, circulating nitric oxide, and gene expression profile in lungs 8 and 24 h after bleomycin instillation. Three main gene categories, known to be involved in the development of acute lung injury, were explored: stress response, inflammation, and fluid homeostasis. Peptide treatment was associated with a significant reduction in interstitial edema, with a virtually normal wet/dry weight ratio. Several stress-related genes, which were either upregulated or reduced by bleomycin, were only marginally altered during NDP-alpha-MSH treatment. NDP-alpha-MSH prevented bleomycin-related transcriptional alterations in genes involved in lung fluid homeostasis, including upregulation of Na/K-transporting ATPase and epithelial sodium channels and downregulation of cystic fibrosis transmembrane conductance regulator. Bleomycin-induced expression of proinflammatory and profibrotic factors (interleukin 6, tumor necrosis factor-alpha, transforming growth factor-beta1, and inducible nitric oxide synthase) and chemokines (chemokine [C-C motif] ligand 2 and chemokine [C-C motif] ligand 5) was likewise significantly reduced by NDP-alpha-MSH. In conclusion, treatment with the alpha-MSH analogue NDP-alpha-MSH greatly improved the clinical and molecular picture of bleomycin-induced lung injury. Treatment with alpha-MSH-related agents can exert beneficial effects in acute lung injury.
Assuntos
Lesão Pulmonar , alfa-MSH/metabolismo , Animais , Anti-Inflamatórios/farmacologia , Bleomicina/farmacologia , Perfilação da Expressão Gênica , Imuno-Histoquímica , Inflamação , Pulmão/metabolismo , Pulmão/patologia , Masculino , Modelos Biológicos , Óxido Nítrico/química , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Ratos , Ratos WistarRESUMO
BACKGROUND: There is evidence that brain death causes changes in peripheral organs. Marked inflammation is found in organs collected during experimental brain death and clinical studies indicate that, despite genetic mismatch, organs obtained from living donors show improved survival over those from brain-dead donors. The aim of the present clinical research was to explore changes in the transcriptional profile of livers from brain-dead organ donors. METHODS: Using the cDNA macroarray technique, we compared gene expression in liver biopsies from 21 brain-dead organ donors and in normal liver tissue obtained during resection of benign focal lesions. RESULTS: Analysis of gene expression showed significant differences in the mRNA levels of 117 genes. There was reduced expression of 93 genes whereas expression of 24 genes was enhanced. Downregulated pathways included transcripts related to morphogenesis, blood coagulation, complement cascade, amine metabolism, lipid metabolism, nucleic acid metabolism, biodegradation of xenobiotics, signal transduction, and transcription. Conversely, there was induction of genes related to acute phase response, damage-related response, electron transport, and energy metabolism. CONCLUSIONS: The present research demonstrates major changes in the transcriptional profile of livers from brain-dead organ donors. The presence of both down- and upregulated gene families suggests that the alteration in transcriptional profile is not a consequence of death-associated organ failure, but rather, an active change in regulatory mechanisms.
Assuntos
Morte Encefálica/metabolismo , Perfilação da Expressão Gênica , Transplante de Fígado , Fígado/metabolismo , Doadores Vivos , Adulto , Idoso , Humanos , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Transcrição GênicaRESUMO
OBJECTIVE: There is evidence that brain death has detrimental effects on peripheral organs. Clinical and experimental studies on organ donors showed marked inflammation in tissue samples of livers and kidneys collected during brain death. The inflammatory reaction is characterized by release of cytokines and inflammatory cell infiltration. Because melanocortins and their receptors are significant modulators of inflammation, we hypothesized that downregulation of melanocortin receptors during brain death could contribute to enhance inflammation. METHODS: Using real-time polymerase chain reaction (PCR) analysis, we determined expression of melanocortin receptors in liver biopsies obtained from brain-dead organ donors before cold ischemia and in normal liver tissue during resection of benign focal lesions of the liver. Tissue biopsies were also analyzed for expression of intercellular adhesion molecule-1 (ICAM-1), which has a central function in inflammatory cell migration. RESULTS: Expression of melanocortin-1 receptor (MC1R) mRNA was markedly reduced in liver samples obtained from brain-dead organ donors compared to hepatic tissue collected during resection of benign focal lesions of the liver. Conversely, expression of the adhesion molecule ICAM-1 was significantly increased in livers of brain-dead organ donors. CONCLUSIONS: Disruption of the endogenous anti-inflammatory circuit based on MC1R could contribute to tissue damage during brain death.
Assuntos
Morte Encefálica/imunologia , Morte Encefálica/fisiopatologia , Hepatite/imunologia , Fígado/imunologia , Receptor Tipo 1 de Melanocortina/genética , alfa-MSH/imunologia , Adulto , Idoso , Biópsia , Adesão Celular/imunologia , Morte Celular/imunologia , Regulação para Baixo/imunologia , Feminino , Hepatite/metabolismo , Hepatite/fisiopatologia , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Fígado/metabolismo , Fígado/fisiopatologia , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/metabolismo , Regulação para Cima/imunologia , alfa-MSH/metabolismoRESUMO
Novel therapies are sought to increase efficiency and survival of transplanted organs. Previous research on experimental heart transplantation showed that treatment with the anti-inflammatory peptide alpha-melanocyte-stimulating hormone (alpha-MSH) prolongs allograft survival. The aim of the present research was to determine the molecular mechanism of this protective activity. Gene expression profile was examined in heart grafts removed on postoperative days 1 and 4 from rats treated with saline or the synthetic alpha-MSH analog Nle4DPhe7 (NDP)-alpha-MSH. On postoperative day 1, the peptide induced expression of cytoskeleton proteins, intracellular kinases, transcription regulators, metallopeptidases, and protease inhibitors. Conversely, NDP-alpha-MSH repressed immune, inflammatory, cell cycle, and protein turnover mediators. Later effects of alpha-MSH treatment included down-regulation of oxidative stress response and up-regulation of ion channels, calcium regulation proteins, phosphatidylinositol signaling system, and glycolipidic metabolism. NDP-alpha-MSH exerted its effects on both Ag-dependent and -independent injury. The results indicate that NDP-alpha-MSH preserves heart function through a broad effect on multiple pathways and suggest that the peptide could improve the outcome of organ transplantation in combination with immunosuppressive treatments.
Assuntos
Perfilação da Expressão Gênica , Transplante de Coração , Miocárdio/metabolismo , alfa-MSH/análogos & derivados , Animais , Cálcio/metabolismo , Masculino , Ratos , Ratos Endogâmicos BN , Ratos Endogâmicos Lew , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transplante Homólogo , alfa-MSH/uso terapêuticoRESUMO
BACKGROUND: With the increasing need for organ transplantation and the use of "marginal" organs, novel approaches are sought to increase the efficiency and survival of transplanted tissue. We tested the idea that treatment with the anti-inflammatory peptide, alpha-melanocyte-stimulating hormone (alpha-MSH), an endogenous hormone that does not cause marked immunosuppression but does reduce reperfusion injury, may protect allografts and prolong their survival. METHODS: Donor cardiac grafts (Brown Norway) were transplanted heterotopically into the abdomen of recipient (Lewis) rats. Treatments consisted of intraperitoneal injections of Nle DPhe -alpha-MSH (NDP-alpha-MSH) or saline from the time of transplantation until sacrifice or spontaneous rejection. Allografts were removed on day 1, day 4, or at the time of rejection and examined for histopathology and expression of molecules prominent in reperfusion injury, transplant rejection, and apoptosis. RESULTS: NDP-alpha-MSH treatment caused a significant increase in allograft survival and a marked decrease in leukocyte infiltration. Expression of molecules such as endothelin 1, chemokines, and adhesion molecules, which are involved in allograft rejection, was significantly inhibited in NDP-alpha-MSH-treated rats. CONCLUSIONS: The results indicate that protection of the allograft from early injury with alpha-MSH can postpone rejection. Addition of this early protection with the peptide to usual treatment with immunosuppressive agents may, therefore, improve success of organ transplants.