RESUMO
Deficient angiogenesis may contribute to worsen the prognosis of myocardial ischemia, peripheral arterial disease, ischemic stroke, etc. Dyslipidemic and inflammatory environments attenuate endothelial cell (EC) proliferation and angiogenesis, worsening the prognosis of ischemia. Under these dyslipidemic and inflammatory environments, EC-caspase-1 becomes activated and induces inflammatory cell death that is defined as pyroptosis. However, the underlying mechanism that correlates caspase-1 activation with angiogenic impairment and the prognosis of ischemia remains poorly defined. By using flow cytometric analysis, enzyme and receptor inhibitors, and hind limb ischemia model in caspase-1 knock-out (KO) mice, we examined our novel hypothesis, i.e. inhibition of caspase-1 in ECs under dyslipidemic and inflammatory environments attenuates EC pyroptosis, improves EC survival mediated by vascular endothelial growth factor receptor 2 (VEGFR-2), angiogenesis, and the prognosis of ischemia. We have made the following findings. Proatherogenic lipids induce higher caspase-1 activation in larger sizes of human aortic endothelial cells (HAECs) than in smaller sizes of HAECs. Proatherogenic lipids increase pyroptosis significantly more in smaller sizes of HAECs than in larger sizes of the cells. VEGFR-2 inhibition increases caspase-1 activation in HAECs induced by lysophosphatidylcholine treatment. Caspase-1 activation inhibits VEGFR-2 expression. Caspase-1 inhibition improves the tube formation of lysophosphatidylcholine-treated HAECs. Finally, caspase-1 depletion improves angiogenesis and blood flow in mouse hind limb ischemic tissues. Our results have demonstrated for the first time that inhibition of proatherogenic caspase-1 activation in ECs improves angiogenesis and the prognosis of ischemia.
Assuntos
Caspase 1/metabolismo , Inibidores de Caspase/farmacologia , Isquemia/tratamento farmacológico , Neovascularização Fisiológica/efeitos dos fármacos , Animais , Caspase 1/deficiência , Caspase 1/genética , Morte Celular/efeitos dos fármacos , Tamanho Celular , Células Cultivadas , Modelos Animais de Doenças , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/enzimologia , Técnicas de Silenciamento de Genes , Membro Posterior/irrigação sanguínea , Humanos , Isquemia/enzimologia , Isquemia/patologia , Lipídeos/química , Lipídeos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Hyperhomocysteinemia (HHcy) impairs re-endothelialization and accelerates vascular remodeling. The role of CD34(+)/VEGF receptor (VEGFR) 2(+) progenitor cells (PCs) in vascular repair in HHcy is unknown. We studied the effect of HHcy on PCs and its role in vascular repair in severe HHcy (â¼150 µM), which was induced in cystathionine-ß synthase heterozygous mice fed a high-methionine diet for 8 weeks. Vascular injury was introduced by carotid air-dry endothelium denudation. CD34(+)/VEGFR2(+) cells were examined by flow cytometry. HHcy reduced bone marrow (BM) CD34(+)/VEGFR2(+) cells and suppressed replenishment of postinjury CD34(+)/VEGFR2(+) cells in peripheral blood (PB). Donor green fluorescent protein-positive PC homing to the injured vessel was reduced in HHcy after CD34(+) PCs from enhanced green fluorescent protein mice were adoptively transferred following carotid injury. CD34(+) PC transfusion partially reversed HHcy-suppressed re-endothelialization and HHcy-induced neointimal formation. Furthermore, homocysteine (Hcy) inhibited proliferation, adhesion, and migration and suppressed ß1-integrin expression and activity in human CD34(+) endothelial colony-forming cells (ECFCs) isolated from PBs in a dose-dependent manner. A functional-activating ß1-integrin antibody rescued Hcy-suppressed adhesion and migration in CD34(+) ECFCs. In conclusion, HHcy reduces BM CD34(+)/VEGFR2(+) generation and suppresses CD34(+)/VEGFR2(+) cell mobilization and homing to the injured vessel via ß1-integrin inhibition, which partially contributes to impaired re-endothelialization and vascular remodeling.
Assuntos
Células da Medula Óssea/patologia , Células da Medula Óssea/fisiologia , Hiper-Homocisteinemia/patologia , Hiper-Homocisteinemia/fisiopatologia , Integrina beta1/fisiologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Antígenos CD34/metabolismo , Lesões das Artérias Carótidas/patologia , Lesões das Artérias Carótidas/fisiopatologia , Adesão Celular , Movimento Celular , Ensaio de Unidades Formadoras de Colônias , Cistationina beta-Sintase/deficiência , Cistationina beta-Sintase/genética , Cistationina beta-Sintase/metabolismo , Modelos Animais de Doenças , Proteínas de Fluorescência Verde/genética , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/patologia , Células-Tronco Hematopoéticas/fisiologia , Humanos , Hiper-Homocisteinemia/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neointima/patologia , Neointima/fisiopatologia , Neointima/prevenção & controle , Remodelação VascularRESUMO
RATIONALE: Hyperhomocysteinemia (HHcy) accelerates atherosclerosis and increases inflammatory monocytes (MC) in peripheral tissues. However, its causative role in atherosclerosis is not well established and its effect on vascular inflammation has not been studied. The underlying mechanism is unknown. OBJECTIVE: This study examined the causative role of HHcy in atherogenesis and its effect on inflammatory MC differentiation. METHODS AND RESULTS: We generated a novel HHcy and hyperlipidemia mouse model, in which cystathionine ß-synthase (CBS) and low-density lipoprotein receptor (LDLr) genes were deficient (Ldlr(-/-) Cbs(-/+)). Severe HHcy (plasma homocysteine (Hcy)=275 µmol/L) was induced by a high methionine diet containing sufficient basal levels of B vitamins. Plasma Hcy levels were lowered to 46 µmol/L from 244 µmol/L by vitamin supplementation, which elevated plasma folate levels. Bone marrow (BM)-derived cells were traced by the transplantation of BM cells from enhanced green fluorescent protein (EGFP) transgenic mice after sublethal irradiation of the recipient. HHcy accelerated atherosclerosis and promoted Ly6C(high) inflammatory MC differentiation of both BM and tissue origins in the aortas and peripheral tissues. It also elevated plasma levels of TNF-α, IL-6, and MCP-1; increased vessel wall MC accumulation; and increased macrophage maturation. Hcy-lowering therapy reversed HHcy-induced lesion formation, plasma cytokine increase, and blood and vessel inflammatory MC (Ly6C(high+middle)) accumulation. Plasma Hcy levels were positively correlated with plasma levels of proinflammatory cytokines. In primary mouse splenocytes, L-Hcy promoted rIFNγ-induced inflammatory MC differentiation, as well as increased TNF-α, IL-6, and superoxide anion production in inflammatory MC subsets. Antioxidants and folic acid reversed L-Hcy-induced inflammatory MC differentiation and oxidative stress in inflammatory MC subsets. CONCLUSIONS: HHcy causes vessel wall inflammatory MC differentiation and macrophage maturation of both BM and tissue origins, leading to atherosclerosis via an oxidative stress-related mechanism.
Assuntos
Aorta/enzimologia , Aterosclerose/etiologia , Células da Medula Óssea/enzimologia , Diferenciação Celular , Hiper-Homocisteinemia/complicações , Inflamação/etiologia , Liases/deficiência , Macrófagos/enzimologia , Receptores de LDL/deficiência , Animais , Antioxidantes/farmacologia , Aorta/efeitos dos fármacos , Aorta/imunologia , Aorta/patologia , Aterosclerose/sangue , Aterosclerose/enzimologia , Aterosclerose/genética , Aterosclerose/patologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/imunologia , Transplante de Medula Óssea , Células Cultivadas , Quimiocina CCL2/sangue , Modelos Animais de Doenças , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Hiper-Homocisteinemia/sangue , Hiper-Homocisteinemia/enzimologia , Hiper-Homocisteinemia/genética , Hiper-Homocisteinemia/imunologia , Hiperlipidemias/complicações , Hiperlipidemias/enzimologia , Hiperlipidemias/imunologia , Inflamação/sangue , Inflamação/enzimologia , Inflamação/imunologia , Mediadores da Inflamação/sangue , Interleucina-6/sangue , Lipídeos/sangue , Liases/genética , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Estresse Oxidativo , Receptores de LDL/genética , Índice de Gravidade de Doença , Superóxidos/metabolismo , Fator de Necrose Tumoral alfa/sangue , Complexo Vitamínico B/farmacologiaRESUMO
BACKGROUND: Current angiogenic therapies for cancers and cardiovascular diseases have not yet achieved expected benefits, which reflects the need for improved understanding of angiogenesis. In this study, we focused on solving the problem of whether tissues have different angiogenic potentials (APs) in physiological conditions and how angiogenesis is regulated in various disease conditions. METHODS: In healthy and diseased human and mouse tissues, we profiled the expression of 163 angiogenic genes, including transcription regulators (TRs), growth factors and receptors (GF/Rs), cytokines and chemokines (C/Cs), and proteases and inhibitors (P/Is). TRs were categorized as inflammatory, homeostatic, and endothelial cell-specific TRs, and C/Cs were categorized as pro-angiogenic, anti-angiogenic, and bi-functional C/Cs. RESULTS: We made the following findings: (1) the human heart, muscle, eye, pancreas, and lymph node are among the tissues with the highest APs; (2) tissues with high APs have more active angiogenic pathways and angiogenic C/C responses; (3) inflammatory TRs dominate regulation of all angiogenic C/Cs; homeostatic TRs regulate all to a lower extent, while endothelial cell-specific TRs mainly regulate pro-angiogenic and bi-functional C/Cs; (4) tissue AP is positively correlated with the expression of oxygen sensors PHD2 and HIF1B, VEGF pathway gene VEGFB, and stem cell gene SOX2; (5) cancers of the digestive system tend to have increased angiogenesis dominated by endothelial cell-specific pro-angiogenic pathways, while lung cancer and prostate cancer have significantly decreased angiogenesis; and (6) endothelial cell-specific pro-angiogenic pathways are significantly increased in thrombus-derived leukocytes in patients with acute coronary artery disease. CONCLUSIONS: Our results demonstrate that thrombus-derived leukocytes express more endothelial cell-specific angiogenic markers to directly promote angiogenesis after myocardial infarction and that certain solid tumors may be more sensitive to anti-angiogenic therapies than others.
Assuntos
Síndrome Coronariana Aguda/fisiopatologia , Transdiferenciação Celular , Células Endoteliais , Leucócitos/patologia , Neovascularização Fisiológica , Trombose/patologia , Proteínas Angiogênicas/genética , Animais , Biomarcadores , Mineração de Dados , Humanos , Camundongos , Infarto do Miocárdio/fisiopatologia , Neoplasias/irrigação sanguínea , Neoplasias/fisiopatologia , Neovascularização Patológica/fisiopatologia , TranscriptomaRESUMO
Caspase-1 activation senses metabolic danger-associated molecular patterns (DAMPs) and mediates the initiation of inflammation in endothelial cells. Here, we examined whether the caspase-1 pathway is responsible for sensing hyperlipidemia as a DAMP in bone marrow (BM)-derived Stem cell antigen-1 positive (Sca-(1+)) stem/progenitor cells and weakening their angiogenic ability. Using biochemical methods, gene knockout, cell therapy and myocardial infarction (MI) models, we had the following findings: 1) Hyperlipidemia induces caspase-1 activity in mouse Sca-(1+) progenitor cells in vivo; 2) Caspase-1 contributes to hyperlipidemia-induced modulation of vascular cell death-related gene expression in vivo; 3) Injection of Sca-1+ progenitor cells from caspase-1(-/-) mice improves endothelial capillary density in heart and decreases cardiomyocyte death in a mouse model of MI; and 4) Caspase-1(-/-) Sca-(1+) progenitor cell therapy improves mouse cardiac function after MI. Our results provide insight on how hyperlipidemia activates caspase-1 in Sca-(1+) progenitor cells, which subsequently weakens Sca-(1+) progenitor cell repair of vasculature injury. These results demonstrate the therapeutic potential of caspase-1 inhibition in improving progenitor cell therapy for MI.