RESUMO
Stress-induced p38 mitogen-activated protein kinase (MAPK) activity is implicated in pathological remodeling in the heart. For example, constitutive p38 MAPK activation in cardiomyocytes induces pathological features, including myocyte hypertrophy, apoptosis, contractile dysfunction, and fetal gene expression. However, the physiological function of cardiomyocyte p38 MAPK activity in beneficial compensatory vascular remodeling is unclear. This report investigated the functional role and the underlying mechanisms of cardiomyocyte p38 MAPK activity in cardiac remodeling induced by chronic stress. Using both in vitro and in vivo model systems, we found that p38 MAPK activity is required for hypoxia-induced pro-angiogenic activity from cardiomyocytes and that p38 MAPK activation in cardiomyocyte is sufficient to promote paracrine signaling-mediated, pro-angiogenic activity. We further demonstrate that VEGF is a paracrine factor responsible for the p38 MAPK-mediated pro-angiogenic activity from cardiomyocytes and that p38 MAPK pathway activation is sufficient for inducing VEGF secretion from cardiomyocytes in an Sp1-dependent manner. More significantly, cardiomyocyte-specific inactivation of p38α in mouse heart impaired compensatory angiogenesis after pressure overload and promoted early onset of heart failure. In summary, p38αMAPK has a critical role in the cross-talk between cardiomyocytes and vasculature by regulating stress-induced VEGF expression and secretion in cardiomyocytes. We conclude that as part of a stress-induced signaling pathway, p38 MAPK activity significantly contributes to both pathological and compensatory remodeling in the heart.
Assuntos
Endotélio Vascular/metabolismo , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Isquemia Miocárdica/metabolismo , Revascularização Miocárdica , Miócitos Cardíacos/metabolismo , Animais , Animais Recém-Nascidos , Hipóxia Celular , Células Cultivadas , Cruzamentos Genéticos , Endotélio Vascular/citologia , Endotélio Vascular/patologia , Ativação Enzimática , Regulação da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Masculino , Camundongos Knockout , Camundongos Transgênicos , Proteína Quinase 14 Ativada por Mitógeno/química , Proteína Quinase 14 Ativada por Mitógeno/genética , Isquemia Miocárdica/patologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/patologia , Interferência de RNA , Ratos Sprague-Dawley , Proteínas Recombinantes/metabolismo , Fator de Transcrição Sp1/antagonistas & inibidores , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp1/metabolismo , Sus scrofa , Fator A de Crescimento do Endotélio Vascular/agonistas , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/agonistas , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Bone morphogenetic protein (BMP) 10, a cardiac-restricted BMP family member, is essential in cardiomyogenesis, especially during trabeculation. Crossveinless-2 (CV2, also known as BMP endothelial cell precursor derived regulator [BMPER]) is a BMP-binding protein that modulates the activity of several BMPs. The objective of this study was to examine the combined effects of BMP10 and CV2 on cardiomyocyte differentiation using mouse dedifferentiated fat (mDFAT) cells, which spontaneously differentiate into cardiomyocyte-like cells, as a model. Our results revealed that CV2 binds directly to BMP10, as determined by co-immunoprecipitation, and inhibits BMP10 from initiating SMAD signaling, as determined by luciferase reporter gene assays. BMP10 treatment induced mDFAT cell proliferation, whereas CV2 modulated the BMP10-induced proliferation. Differentiation of cardiomyocyte-like cells proceeded in a reproducible fashion in mDFAT cells, starting with small round Nkx2.5-positive progenitor cells that progressively formed myotubes of increasing length that assembled into beating colonies and stained strongly for Troponin I and sarcomeric alpha-actinin. BMP10 enhanced proliferation of the small progenitor cells, thereby securing sufficient numbers to support formation of myotubes. CV2, on the other hand, enhanced formation and maturation of large myotubes and myotube-colonies and was expressed by endothelial-like cells in the mDFAT cultures. Thus BMP10 and CV2 have important roles in coordinating cardiomyogenesis in progenitor cells.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Proteínas de Transporte/metabolismo , Diferenciação Celular/fisiologia , Miócitos Cardíacos/citologia , Células-Tronco/citologia , Actinina/metabolismo , Adipócitos/citologia , Animais , Proliferação de Células , Células Cultivadas , Proteína Homeobox Nkx-2.5/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais/fisiologia , Proteínas Smad/metabolismo , Troponina I/metabolismoRESUMO
Tissue injury initiates a complex series of events that act to restore structure and physiological homeostasis. Infiltration of inflammatory cells and vascular remodeling are both keystones of this process. However, the role of inflammation and angiogenesis in general and, more specifically, the significance of inflammatory cell-derived VEGF in this context are unclear. To determine the role of inflammatory cell-derived VEGF in a clinically relevant and chronically inflamed injury, pulmonary fibrosis, we deleted the VEGF-A gene in myeloid cells. In a model of pulmonary fibrosis in mice, deletion of VEGF in myeloid cells resulted in significantly reduced formation of blood vessels; however, it causes aggravated fibrotic tissue damage. This was accompanied by a pronounced decrease in epithelial cell survival and a striking increase in myofibroblast invasion. The drastic increase in fibrosis following loss of myeloid VEGF in the damaged lungs was also marked by increased levels of hypoxia-inducible factor (HIF) expression and Wnt/beta-catenin signaling. This demonstrates that the process of angiogenesis, driven by myeloid cell-derived VEGF, is essential for the prevention of fibrotic damage.
Assuntos
Deleção de Genes , Fibrose Pulmonar/genética , Fator A de Crescimento do Endotélio Vascular/fisiologia , Animais , Bleomicina/toxicidade , Feminino , Hipóxia/patologia , Camundongos , Camundongos Mutantes , Fosforilação , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/patologia , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , beta Catenina/metabolismoRESUMO
Hypoxia-inducible factor-2alpha (HIF-2alpha) is highly expressed in embryonic vascular endothelial cells (ECs) and activates the expression of target genes whose products modulate vascular function and angiogenesis. In this report, we describe a genetic model designed to test the physiologic consequences of deleting HIF-2alpha in murine endothelial cells. Surprisingly, mice with HIF-2alpha-deficient ECs developed normally but displayed a variety of phenotypes, including increased vessel permeability, aberrant endothelial cell ultrastructure, and pulmonary hypertension. Moreover, these animals exhibited defective tumor angiogenesis associated with increased hypoxic stress and tumor cell apoptosis. Immortalized HIF-2alpha-deficient ECs displayed decreased adhesion to extracellular matrix proteins and expressed reduced levels of transcripts encoding fibronectin, integrins, endothelin B receptor, angiopoietin 2, and delta-like ligand 4 (Dll4). Together, these data identify unique cell-autonomous functions for HIF-2alpha in vascular endothelial cells.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Células Endoteliais/metabolismo , Neoplasias/irrigação sanguínea , Neoplasias/metabolismo , Neovascularização Patológica , Alelos , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/deficiência , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Linhagem Celular Tumoral , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Regulação Neoplásica da Expressão Gênica , Hipertensão Pulmonar/genética , Hipertensão Pulmonar/metabolismo , Hipertensão Pulmonar/fisiopatologia , Hipóxia/genética , Hipóxia/metabolismo , Hipóxia/patologia , Camundongos , Camundongos Knockout , Microscopia Eletrônica , Neoplasias/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Growth factor gradients regulate many developmental processes. VEGF-A is distributed in a graded fashion in growing tissues in order to direct sprouting of new vessels. Growth factor gradients can be formed by regulated production, retention, controlled release and degradation. VEGF-A production is controlled by hypoxia while its retention depends on the C-terminal heparin-binding motifs present in the longer splice-isoforms, VEGF164 and 188. This motif confers binding to the cell surface and the surrounding extracelluar matrix. The short isoform VEGF120 is diffusible and hence fails to direct endothelial tip cell migration. Conditional inactivation of heparan sulfate proteoglycans in the cells that produce VEGF results similarly in misguidance of the tip cells. Studying retinal developmental angiogenesis and pathological neovascularization side-by-side in the mouse retina, we find that endothelial tip cell guidance and stalk cell proliferation control are disrupted in neovascularization due to a loss of VEGF-A retention. The cause for this is proteolytic cleavage of VEGF-A by matrix metalloproteases (MMP) derived mostly from macrophages infiltrating the ischaemic retinal areas. Genetic or pharmacological inhibition of macrophage infiltration or MMP activity can rescue guided revascularization at the expense of pre-retinal neovascularization. Disruption of VEGF-A gradients provides a novel concept for the mechanism underlying pathological patterning in ocular disease.
Assuntos
Vasos Sanguíneos/embriologia , Padronização Corporal , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Animais , Vasos Sanguíneos/citologia , Movimento Celular , Proliferação de Células , Células Endoteliais/citologia , Camundongos , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Angiogenesis is implicated in the pathogenesis of cancer, rheumatoid arthritis, and atherosclerosis and in the treatment of coronary artery and peripheral vascular disease. Here, cholesterol-lowering agents, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, are shown to interfere with angiogenesis. In vivo, the HMG-CoA reductase inhibitor simvastatin dose-dependently inhibited capillary growth in both vascular endothelial growth factor-stimulated chick chorioallantoic membranes and basic fibroblast growth factor-stimulated mouse corneas. In vitro, the development of tubelike structures by human microvascular endothelial cells cultured on 3D collagen gels was inhibited at simvastatin concentrations similar to those found in the serum of patients on therapeutic doses of this agent. HMG-CoA reductase inhibitors interfered with angiogenesis via inhibition of the geranylgeranylation and membrane localization of RhoA. Simvastatin inhibited membrane localization of RhoA with a concentration dependence similar to that for the inhibition of tube formation, whereas geranylgeranyl pyrophosphate, the substrate for the geranylgeranylation of Rho, reversed the effect of simvastatin on tube formation and on the membrane localization of RhoA. Furthermore, tube formation was inhibited by GGTI, a specific inhibitor of the geranylgeranylation of Rho; by C3 exotoxin, which inactivates Rho; and by the adenoviral expression of a dominant-negative RhoA mutant. The expression of a dominant-activating RhoA mutant reversed the effect of simvastatin on tube formation. Finally, HMG-CoA reductase inhibitors inhibited signaling by vascular endothelial growth factor, Akt, and focal adhesion kinase, three RhoA-dependent pathways known to be involved in angiogenesis. This study demonstrates a new relationship between lipid metabolism and angiogenesis and an antiangiogenic effect of HMG-CoA reductase inhibitors with possible important therapeutic implications.
Assuntos
Inibidores da Angiogênese/farmacologia , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Neovascularização Fisiológica/efeitos dos fármacos , Sinvastatina/farmacologia , Proteína rhoA de Ligação ao GTP/antagonistas & inibidores , Animais , Membrana Celular/química , Células Cultivadas , Colágeno/farmacologia , Córnea/irrigação sanguínea , Córnea/efeitos dos fármacos , Fatores de Crescimento Endotelial/antagonistas & inibidores , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/crescimento & desenvolvimento , Fator 2 de Crescimento de Fibroblastos/antagonistas & inibidores , Humanos , Linfocinas/antagonistas & inibidores , Camundongos , Mutação , Prenilação de Proteína/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio Vascular , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Endothelial-mesenchymal transition (EndMT) drives endothelium to contribute to normal development and disease processes. Here, we report that EndMTs occur in the diabetic endothelium of Ins2Akita/wt mouse, and show that induction of sex determining region Y-box 2 (Sox2) is a mediator of excess BMP signaling that results in activation of EndMTs and increased vascular calcification. We also find an induction of a complex of serine proteases in the diabetic endothelium, required for the up-regulation of Sox2. Our results suggest that EndMTs contribute to vascular calcification in diabetic arteries.
Assuntos
Vasos Sanguíneos/patologia , Calcinose/patologia , Endotélio Vascular/patologia , Insulina/genética , Mesoderma/patologia , Animais , Diabetes Mellitus Experimental/genética , Endotélio Vascular/enzimologia , Camundongos , Camundongos Transgênicos , Fatores de Transcrição SOXB1/fisiologia , Serina Proteases/metabolismo , Regulação para CimaRESUMO
BACKGROUND AND PURPOSE: Acceleration of healing mechanisms is a promising approach to improve current limitations of endovascular aneurysm therapy with the use of platinum coils. We evaluated a new endovascular therapeutic, bioabsorbable polymeric material (BPM), which may promote cellular reaction in the aneurysms. METHODS: Four different concentrations of lactide/glycolic acid copolymer [poly(D-L-lactic-co-glycolic acid)] (PLGA), 85/15, 75/25, 65/35, and 50/50, were used as BPMs. Sixteen experimental aneurysms were created in 8 swine. Eight-millimeter-long spiral-shaped BPMs were surgically implanted in the aneurysms without tight packing (n=3 for each BPM). Guglielmi detachable coils (GDCs) were used as control (n=4). The animals were killed 14 days after embolization, and angiographic, histological, and immunohistochemical analyses were performed. RESULTS: Despite loose packing of aneurysms with BPMs, faster BPMs such as 50/50 or 65/35 PLGA demonstrated more mature collagen formation and fibrosis in the sac and neck of the aneurysm. One aneurysm treated with 65/35 PLGA, 1 treated with 75/25 PLGA, and all 3 treated with 85/15 PLGA showed a neck remnant on angiography. There was a linear relationship between collagen levels and polymer degradation properties (r=-0.9513). CONCLUSIONS: This preliminary animal study indicates that acceleration of aneurysm healing with the use of BPM is feasible. This concept can be applied to decrease and perhaps prevent aneurysmal recanalization after endovascular treatment of cerebral aneurysms.
Assuntos
Implantes Absorvíveis , Aneurisma/cirurgia , Implante de Prótese Vascular , Prótese Vascular , Doenças das Artérias Carótidas/cirurgia , Ácido Láctico , Ácido Poliglicólico , Polímeros , Implantes Absorvíveis/efeitos adversos , Aneurisma/complicações , Aneurisma/diagnóstico por imagem , Angiografia , Animais , Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacologia , Prótese Vascular/efeitos adversos , Implante de Prótese Vascular/métodos , Doenças das Artérias Carótidas/complicações , Doenças das Artérias Carótidas/patologia , Artéria Carótida Primitiva/metabolismo , Artéria Carótida Primitiva/patologia , Artéria Carótida Primitiva/cirurgia , Colágeno Tipo I/metabolismo , Modelos Animais de Doenças , Embolização Terapêutica/instrumentação , Embolização Terapêutica/métodos , Estudos de Viabilidade , Feminino , Fibrose/etiologia , Reação a Corpo Estranho/etiologia , Imuno-Histoquímica , Implantes Experimentais/efeitos adversos , Ácido Láctico/química , Ácido Láctico/farmacologia , Masculino , Ácido Poliglicólico/química , Ácido Poliglicólico/farmacologia , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Polímeros/química , Polímeros/farmacologia , Suínos , Trombose/etiologia , Cicatrização/efeitos dos fármacosRESUMO
HIV-1 infection is associated with serious cardiovascular complications, but the roles of HIV-1, viral proteins, and highly active antiretroviral therapy (HAART) drugs are not understood. HAART decreases the overall risk of heart disease but leads to metabolic disturbances and possibly coronary artery disease. We investigated toxicities of HIV-1, HIV-1 glycoprotein 120 (gp120), and HAART drugs for human coronary artery endothelial cells (CAECs), brain microvascular endothelial cells, and neonatal rat ventricular myocytes (NRVMs). HIV-1 and gp120, but not azidothymidine (AZT), induced apoptosis of NRVMs and CAECs. Ethylisothiourea, an inhibitor of nitric oxide synthase, inhibited apoptosis induction by gp120. AZT, HIV-1, and gp120 all damaged mitochondria of cardiomyocytes. HAART drugs, AZT, and indinavir, but not HIV-1, produced intercellular gaps between confluent endothelial cells and decreased transendothelial electrical resistance. In conclusion, HIV-1 and gp120 induce toxicity through induction of cardiomyocyte and endothelial cell apoptosis. HAART drugs disrupt endothelial cell junctions and mitochondria and could cause vascular damage.
Assuntos
Terapia Antirretroviral de Alta Atividade/efeitos adversos , Apoptose/efeitos dos fármacos , Junções Comunicantes/efeitos dos fármacos , Proteína gp120 do Envelope de HIV/toxicidade , Indinavir/toxicidade , Mitocôndrias Cardíacas/efeitos dos fármacos , Zidovudina/toxicidade , Animais , Animais Recém-Nascidos , Fármacos Anti-HIV/toxicidade , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/patologia , Células Cultivadas/virologia , Vasos Coronários/efeitos dos fármacos , Vasos Coronários/patologia , Relação Dose-Resposta a Droga , Combinação de Medicamentos , Impedância Elétrica , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/patologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/patologia , Junções Comunicantes/patologia , Infecções por HIV/sangue , HIV-1/fisiologia , Humanos , Mitocôndrias Cardíacas/patologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Tioureia/farmacologiaAssuntos
Inibidores da Angiogênese/uso terapêutico , Neovascularização Patológica/tratamento farmacológico , Neovascularização Fisiológica/fisiologia , Animais , Resistência a Medicamentos , Oftalmopatias/tratamento farmacológico , Oftalmopatias/patologia , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia , Neovascularização Patológica/patologia , Neovascularização Fisiológica/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Receptores de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fatores de Transcrição/fisiologia , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/fisiologiaRESUMO
PURPOSE: To use proteomic analysis to identify upregulated and downregulated proteins in thrombosed hemodialysis graft specimens. One of these significantly upregulated proteins was a disintegrin and metalloproteinase thrombospondin-1 (ADAMTS-1), and its expression and activity were determined in thrombosed hemodialysis grafts. MATERIALS AND METHODS: Hemodialysis vascular access samples (thrombosed veins, n = 8; control veins, n = 6) were obtained from patients who required surgical revision. Proteomic analysis was performed with isotope-coded affinity tag labeling with multidimensional liquid chromatography followed by tandem mass spectrometry on four thrombosed hemodialysis graft specimens with control veins. Expression of ADAMTS-1 was confirmed by performing immunoprecipitation followed by Western blot analysis. Finally, immunohistochemistry was used to localize expression in a separate group of patients with thrombosed grafts. RESULTS: Thirty-nine unique proteins were common to all four patients. ADAMTS-1 was one of the only significantly upregulated protein (>38 fold). ADAMTS-1 expression was confirmed by performing immunoprecipitation and Western blot analysis and was significantly increased. ADAMTS-1 expression was localized to adventitial macrophages and neutrophils of thrombosed grafts. CONCLUSIONS: ADAMTS-1 was significantly upregulated in thrombosed hemodialysis grafts by mass spectrometric analysis and Western blot analysis. Expression was localized to adventitial macrophages and leukocytes. It is hypothesized that ADAMTS-1 may be related to intimal hyperplasia in hemodialysis vascular access grafts. Future work is planned on inhibiting ADAMTS-1 expression and determining the effect on intimal hyperplasia in hemodialysis grafts.