RESUMO
There is increasing evidence that radiation-induced damage to endothelial cells and loss of endothelial function may contribute to both acute radiation syndromes and long-term effects of whole-body nuclear irradiation. Therefore, several drugs are being developed to mitigate the effects of nuclear radiation, most of these drugs will target and protect or regenerate leukocytes and platelets. Our laboratory has demonstrated that TP508, a 23-amino acid thrombin peptide, activates endothelial cells and stem cells to revascularize and regenerate tissues. We now show that TP508 can mitigate radiation-induced damage to endothelial cells in vitro and in vivo. Our in vitro results demonstrate that human endothelial cells irradiation attenuates nitric oxide (NO) signaling, disrupts tube formation and induces DNA double-strand breaks (DSB). TP508 treatment reverses radiation effects on NO signaling, restores tube formation and accelerates the repair of radiation-induced DSB. The radiation-mitigating effects of TP508 on endothelial cells were also seen in CD-1 mice where systemic injection of TP508 stimulated endothelial cell sprouting from aortic explants after 8 Gy irradiation. Systemic doses of TP508 that mitigated radiation-induced endothelial cell damage, also significantly increased survival of CD-1 mice when injected 24 h after 8.5 Gy exposure. These data suggest that increased survival observed with TP508 treatment may be due to its effects on vascular and microvascular endothelial cells. Our study supports the usage of a regenerative drug such as TP508 to activate endothelial cells as a countermeasure for mitigating the effects of nuclear radiation.
Assuntos
Reparo do DNA/efeitos dos fármacos , Reparo do DNA/efeitos da radiação , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/efeitos da radiação , Fragmentos de Peptídeos/farmacologia , Trombina/farmacologia , Sequência de Aminoácidos , Animais , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Relação Dose-Resposta à Radiação , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Humanos , Masculino , Camundongos , Óxido Nítrico/biossíntese , Análise de SobrevidaRESUMO
Endothelial dysfunction (ED) is characterized by impaired nitric oxide (NO) signaling, decreased NO-dependent vasodilatation, increased vascular inflammation, and diminished response to angiogenic factors. TP508 (Chrysalin), an angiogenic tissue repair peptide, was tested for potential effects on myocardial revascularization and ED using a porcine model of chronic myocardial ischemia. TP508 increased perfusion in ischemic regions up to16-fold (P < .02) and doubled myocardial wall thickening (P < .02) relative to placebo controls. Ischemic arterioles exhibited impaired NO-mediated vasodilation and diminished NO production. TP508 reversed ischemic effects, increasing NO-mediated vasodilation (P < .05), endothelial nitric oxide synthase (eNOS) expression, and NO production. In human endothelial cells, TP508 stimulated eNOS activation (1.84 +/- 0.2-fold; P < .02), increased NO production (85 +/- 18%; P < .02), and prevented hypoxia-induced eNOS downregulation (P < .01). Thus, TP508 reverses ED both in porcine ischemic hearts and cultured human endothelial cells. These results suggest potential therapeutic benefit of TP508 in myocardial revascularization and treatment of ED-related diseases.
Assuntos
Indutores da Angiogênese/farmacologia , Circulação Coronária/efeitos dos fármacos , Vasos Coronários/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Isquemia Miocárdica/tratamento farmacológico , Revascularização Miocárdica/métodos , Fragmentos de Peptídeos/farmacologia , Animais , Hipóxia Celular , Células Cultivadas , Doença Crônica , Angiografia Coronária , Vasos Coronários/metabolismo , Vasos Coronários/fisiopatologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Ecocardiografia sob Estresse , Endotélio Vascular/metabolismo , Endotélio Vascular/fisiopatologia , Humanos , Isquemia Miocárdica/metabolismo , Isquemia Miocárdica/patologia , Isquemia Miocárdica/fisiopatologia , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Suínos , Porco Miniatura , Trombina , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologiaRESUMO
The precise biological role of Thy-1, a glycophosphatidyl-inositol (GPI)-linked cell surface glycoprotein in non-caveolar lipid raft microdomains, remains enigmatic. Evidence suggests that Thy-1 affects intracellular signaling through src-family protein kinases, and modulates adhesive and migratory events, such as thymocyte adhesion and neurite extension. Primary fibroblasts sorted based on presence or absence of cell surface Thy-1 display strikingly distinct morphologies and differ with respect to production of and response to cytokines and growth factors. It is unclear the extent to which Thy-1 mediates these differences. Findings reported here indicate a novel role for Thy-1 in regulating the activity of Rho GTPase, a critical regulator of cellular adhesion and cytoskeletal organization. Endogenous or heterologous Thy-1 expression promotes focal adhesion and stress fiber formation, characteristic of increased Rho GTPase activity, and inhibits migration. Immunoblotting following transfection of RFL6 fibroblasts with Thy-1 demonstrates that Thy-1 expression inhibits src-family protein tyrosine kinase (SFK) activation, resulting in decreased phosphorylation of p190 Rho GTPase-activating protein (GAP). This results in a net increase in active Rho, and increased stress fibers and focal adhesions. We therefore conclude that Thy-1 surface expression regulates fibroblast focal adhesions, cytoskeletal organization and migration by modulating the activity of p190 RhoGAP and Rho GTPase.
Assuntos
Movimento Celular , Citoesqueleto/metabolismo , Fibroblastos/metabolismo , Adesões Focais/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas Nucleares/metabolismo , Antígenos Thy-1/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Células Cultivadas , Proteínas de Ligação a DNA , Ativação Enzimática , Fibroblastos/enzimologia , Proteínas Ativadoras de GTPase , Pulmão/citologia , Camundongos , Camundongos Knockout , Modelos Biológicos , Fosforilação , Proteínas Tirosina Quinases/metabolismo , Ratos , Ratos Endogâmicos Lew , Proteínas Repressoras , Quinases da Família src/antagonistas & inibidoresRESUMO
Recently, we identified that regulation of leukocyte recruitment by IL-6 requires shedding of the IL-6R from infiltrating neutrophils. In this study, experiments have examined whether other IL-6-related cytokines possess similar properties. Levels of oncostatin M (OSM) and leukemia inhibitory factor were analyzed in patients with overt bacterial peritonitis during the first 5 days of infection. Although no change in leukemia inhibitory factor was observed throughout the duration of infection, OSM was significantly elevated on day 1 and rapidly returned to baseline by days 2-3. The source of OSM was identified as the infiltrating neutrophils, and OSM levels correlated both with leukocyte numbers and i.p. soluble IL-6R (sIL-6R) levels. FACS analysis revealed that OSM receptor beta expression was restricted to human peritoneal mesothelial cells. Stimulation of human peritoneal mesothelial cells with OSM induced phosphorylation of gp130 and OSM receptor beta, which was accompanied by activation of STAT3 and secretion of CC chemokine ligand 2/monocyte chemoattractant protein-1 and IL-6. Although OSM itself did not modulate CXC chemokine ligand 8/IL-8 release, it effectively suppressed IL-1beta-mediated expression of this neutrophil-activating CXC chemokine. Moreover, OSM synergistically blocked IL-1beta-induced CXC chemokine ligand 8 secretion in combination with the IL-6/sIL-6R complex. Thus suggesting that OSM and sIL-6R release from infiltrating neutrophils may contribute to the temporal switch between neutrophil influx and mononuclear cell recruitment seen during acute inflammation.
Assuntos
Quimiocinas/biossíntese , Mediadores da Inflamação/metabolismo , Interleucina-6/biossíntese , Infiltração de Neutrófilos/imunologia , Neutrófilos/imunologia , Neutrófilos/metabolismo , Omento/imunologia , Omento/metabolismo , Peptídeos/metabolismo , Antígenos CD/fisiologia , Infecções Bacterianas/imunologia , Células Cultivadas , Receptor gp130 de Citocina , Citocinas/biossíntese , Inibidores do Crescimento/biossíntese , Humanos , Mediadores da Inflamação/fisiologia , Interleucina-1/farmacologia , Fator Inibidor de Leucemia , Linfocinas/biossíntese , Glicoproteínas de Membrana/fisiologia , Ativação de Neutrófilo/imunologia , Omento/citologia , Oncostatina M , Peptídeos/fisiologia , Diálise Peritoneal Ambulatorial Contínua/efeitos adversos , Peritonite/imunologia , Receptores de Citocinas/fisiologia , Receptores de Interleucina-6/fisiologia , Receptores de Oncostatina M , Transdução de Sinais/imunologia , SolubilidadeRESUMO
Following lung injury or inflammation, fibroblasts mediate either restorative repair or disordered remodeling. Interleukin (IL)-1beta is a key mediator in the transition from injury/inflammation to tissue remodeling, in part through its regulation of platelet-derived growth factor alpha receptor (PDGFalphaR). Based on prior demonstration of differential PDGFalphaR expression, we hypothesized that subpopulations of fibroblasts would have heterogeneous responses to IL-1. We report that IL-1beta significantly increases expression of PDGFalphaR in Thy-1-, but not Thy-1+ fibroblasts. Higher baseline expression of PDGFalphaR in Thy-1- fibroblasts is partially abrogated by IL-1 receptor antagonist. There are no differences in IL-1beta binding, as determined by flow cytometry, or in the presence of the type I IL-1 receptor (IL-1RtI) or its associated protein (IL-1RacP) by immunoblotting. IL-1beta induces DNA binding of both nuclear factor kappaB (NF-kappaB) and CAATT-enhancer binding protein (C/EBP), and activation of p38 mitogen-activated protein kinase in both subpopulations. However, IL-1beta-induced proliferation and expression of IL-6 are significantly higher in Thy-1- fibroblasts. Heterogeneous responses to IL-1beta despite equivalent presence of both proximal and distal signaling components indicates that parallel signaling pathways are activated selectively in Thy-1- cells, suggesting a prominent role for this subset in the transition from inflammation to lung remodeling.