RESUMO
BACKGROUND: Treatment of occluded vessels can involve angioplasty, stenting, and bypass grafting, which can be limited by restenosis and thrombosis. Drug-eluting stents attenuate restenosis, but the current drugs used are cytotoxic, causing smooth muscle cell (SMC) and endothelial cell (EC) death that may lead to late thrombosis. N-cadherin is a junctional protein expressed by SMCs, which promotes directional SMC migration contributing to restenosis. We propose that engaging N-cadherin with mimetic peptides can act as a cell type-selective therapeutic strategy to inhibit polarization and directional migration of SMCs without negatively impacting ECs. METHODS: We designed a novel N-cadherin-targeting chimeric peptide with a histidine-alanine-valine cadherin-binding motif, combined with a fibronectin-binding motif from Staphylococcus aureus. This peptide was tested in SMC and EC culture assays of migration, viability, and apoptosis. Rat carotid arteries were balloon injured and treated with the N-cadherin peptide. RESULTS: Treating scratch-wounded SMCs with the N-cadherin-targeting peptide inhibited migration and reduced polarization of wound-edge cells. The peptide colocalized with fibronectin. Importantly, EC junction, permeability, or migration was not impacted by peptide treatment in vitro. We also demonstrated that the chimeric peptide persisted for 24 hours after transient delivery in the balloon-injured rat carotid artery. Treatment with the N-cadherin-targeting chimeric peptide reduced intimal thickening in balloon-injured rat carotid arteries at 1 and 2 weeks after injury. Reendothelialization of injured vessels after 2 weeks was unimpaired by peptide treatment. CONCLUSIONS: These studies show that an N-cadherin-binding and fibronectin-binding chimeric peptide is effective in inhibiting SMC migration in vitro and in vivo and limiting neointimal hyperplasia after balloon angioplasty without affecting EC repair. These results establish the potential of an advantageous SMC-selective strategy for antirestenosis therapy.
Assuntos
Lesões das Artérias Carótidas , Trombose , Ratos , Animais , Fibronectinas/farmacologia , Lesões das Artérias Carótidas/patologia , Caderinas , Artérias Carótidas/patologia , Hiperplasia/patologia , Peptídeos/farmacologia , Trombose/patologiaRESUMO
Pulmonary hypertension (PH) is a multifaceted condition characterized by elevated pulmonary arterial pressure, which can result in right ventricular dysfunction and failure. Disorders of lung development can present with secondary PH, which is a leading cause of mortality in infants with bronchopulmonary dysplasia (BPD). DDR1 (discoidin domain receptor 1) is a collagen-binding receptor that regulates tissue fibrosis and inflammation and controls cellular growth and migration. However, the roles of DDR1 in lung development or the pathogenesis of PH are unknown. Studying mice with a DDR1 deletion (Ddr1-/-), we have noted 35% mortality between 1 and 4 months of age, and we demonstrate that DDR1 deficiency results in reduced right ventricular contractility and muscularization of distal pulmonary arteries, consistent with PH. Pathology analysis revealed enlarged alveolar spaces in Ddr1-/- mice by Postnatal Day 7, consistent with impaired alveolar development. Gene expression analysis showed that Ddr1-/- mice have reduced concentrations of alveologenesis factors and epithelial-to-mesenchymal transition markers. Mechanistic studies in vitro confirmed that DDR1 mediated epithelial-to-mesenchymal transition, migration, and growth of alveolar epithelial cells. Taken together, these data suggest that DDR1 plays important roles mediating alveolarization during lung development. Our studies also describe a new model of spontaneous PH and bronchopulmonary dysplasia in mice.
Assuntos
Displasia Broncopulmonar , Receptor com Domínio Discoidina 1 , Hipertensão Pulmonar , Animais , Humanos , Recém-Nascido , Camundongos , Receptor com Domínio Discoidina 1/genética , Receptor com Domínio Discoidina 1/metabolismo , Transição Epitelial-Mesenquimal/fisiologia , FibroseRESUMO
Arterial stiffening is a significant predictor of cardiovascular disease development and mortality. In elastic arteries, stiffening refers to the loss and fragmentation of elastic fibers, with a progressive increase in collagen fibers. Type VIII collagen (Col-8) is highly expressed developmentally, and then once again dramatically upregulated in aged and diseased vessels characterized by arterial stiffening. Yet its biophysical impact on the vessel wall remains unknown. The purpose of this study was to test the hypothesis that Col-8 functions as a matrix scaffold to maintain vessel integrity during extracellular matrix (ECM) development. These changes are predicted to persist into the adult vasculature, and we have tested this in our investigation. Through our in vivo and in vitro studies, we have determined a novel interaction between Col-8 and elastin. Mice deficient in Col-8 (Col8-/-) had reduced baseline blood pressure and increased arterial compliance, indicating an enhanced Windkessel effect in conducting arteries. Differences in both the ECM composition and VSMC activity resulted in Col8-/- carotid arteries that displayed increased crosslinked elastin and functional distensibility, but enhanced catecholamine-induced VSMC contractility. In vitro studies revealed that the absence of Col-8 dramatically increased tropoelastin mRNA and elastic fiber deposition in the ECM, which was decreased with exogenous Col-8 treatment. These findings suggest a causative role for Col-8 in reducing mRNA levels of tropoelastin and the presence of elastic fibers in the matrix. Moreover, we also found that Col-8 and elastin have opposing effects on VSMC phenotype, the former promoting a synthetic phenotype, whereas the latter confers quiescence. These studies further our understanding of Col-8 function and open a promising new area of investigation related to elastin biology.
RESUMO
Objective- Vascular calcification is a common and severe complication in patients with atherosclerosis which is exacerbated by type 2 diabetes mellitus. Our laboratory recently reported that the collagen receptor discoidin domain receptor 1 (DDR1) mediates vascular calcification in atherosclerosis; however, the underlying mechanisms are unknown. During calcification, vascular smooth muscle cells transdifferentiate into osteoblast-like cells, in a process driven by the transcription factor RUNX2 (runt-related transcription factor 2). DDR1 signals via the phosphoinositide 3-kinase/Akt pathway, which is also central to insulin signaling, and upstream of RUNX2, and this led us to investigate whether DDR1 promotes vascular calcification in diabetes mellitus via this pathway. Approach and Results- Ddr1+/+ ; Ldlr-/- (single knock-out) and Ddr1-/- ; Ldlr-/- (double knock-out) mice were placed on high-fat diet for 12 weeks to induce atherosclerosis and type 2 diabetes mellitus. Von Kossa staining revealed reduced vascular calcification in the aortic arch of double knock-out compared with single knock-out mice. Immunofluorescent staining for RUNX2 was present in calcified plaques of single knock-out but not double knock-out mice. Primary vascular smooth muscle cells obtained from Ddr1+/+ and Ddr1-/- mice were cultured in calcifying media. DDR1 deletion resulted in reduced calcification, a 74% reduction in p-Akt levels, and an 88% reduction in RUNX2 activity. Subcellular fractionation revealed a 77% reduction in nuclear RUNX2 levels in Ddr1-/- vascular smooth muscle cells. DDR1 associated with phosphoinositide 3-kinase, and treatment with the inhibitor wortmannin attenuated calcification. Finally, we show that DDR1 is important to maintain the microtubule cytoskeleton which is required for the nuclear localization of RUNX2. Conclusions- These novel findings demonstrate that DDR1 promotes RUNX2 activity and atherosclerotic vascular calcification in diabetes mellitus via phosphoinositide 3-kinase/Akt signaling.
Assuntos
Aterosclerose/enzimologia , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Diabetes Mellitus Tipo 2/enzimologia , Angiopatias Diabéticas/enzimologia , Receptor com Domínio Discoidina 1/metabolismo , Músculo Liso Vascular/enzimologia , Miócitos de Músculo Liso/enzimologia , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Calcificação Vascular/enzimologia , Transporte Ativo do Núcleo Celular , Animais , Aterosclerose/genética , Aterosclerose/patologia , Células Cultivadas , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/patologia , Angiopatias Diabéticas/genética , Angiopatias Diabéticas/patologia , Dieta Hiperlipídica , Receptor com Domínio Discoidina 1/deficiência , Receptor com Domínio Discoidina 1/genética , Modelos Animais de Doenças , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologia , Fosforilação , Receptores de LDL/deficiência , Receptores de LDL/genética , Transdução de Sinais , Calcificação Vascular/genética , Calcificação Vascular/patologiaRESUMO
Collagens in the atherosclerotic plaque signal regulation of cell behavior and provide tensile strength to the fibrous cap. Type VIII collagen, a short-chain collagen, is up-regulated in atherosclerosis; however, little is known about its functions in vivo. We studied the response to arterial injury and the development of atherosclerosis in type VIII collagen knockout mice (Col8(-/-) mice). After wire injury of the femoral artery, Col8(-/-) mice had decreased vessel wall thickening and outward remodeling when compared with Col8(+/+) mice. We discovered that apolipoprotein E (ApoE) is an endogenous repressor of the Col8a1 chain, and, therefore, in ApoE knockout mice, type VIII collagen was up-regulated. Deficiency of type VIII collagen in ApoE(-/-) mice (Col8(-/-);ApoE(-/-)) resulted in development of plaques with thin fibrous caps because of decreased smooth muscle cell migration and proliferation and reduced accumulation of fibrillar type I collagen. In contrast, macrophage accumulation was not affected, and the plaques had large lipid-rich necrotic cores. We conclude that in atherosclerosis, type VIII collagen is up-regulated in the absence of ApoE and functions to increase smooth muscle cell proliferation and migration. This is an important mechanism for formation of a thick fibrous cap to protect the atherosclerotic plaque from rupture.
Assuntos
Aterosclerose/patologia , Colágeno Tipo VIII/fisiologia , Placa Aterosclerótica/patologia , Animais , Apolipoproteínas E/deficiência , Apolipoproteínas E/fisiologia , Aterosclerose/metabolismo , Movimento Celular/fisiologia , Proliferação de Células , Células Cultivadas , Colágeno/metabolismo , Colágeno Tipo VIII/deficiência , Colágeno Tipo VIII/genética , Elastina/metabolismo , Feminino , Artéria Femoral/lesões , Gelatinases/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso Vascular/patologia , Necrose , Placa Aterosclerótica/metabolismo , RNA Mensageiro/genética , Transdução de Sinais/fisiologia , Regulação para Cima/fisiologiaRESUMO
The extracellular matrix signals and regulates the behavior of vascular cells during the pathogenesis of atherosclerosis. Type VIII collagen, a short chain collagen, is scarcely present in normal arteries, but is dramatically upregulated in atherosclerosis and after other types of vascular injury. Cell culture studies have revealed that this protein supports smooth muscle cell (SMC) adhesion and stimulates migration, however little is known about the signaling or the mechanisms by which this occurs. SMCs isolated from wild-type C57BL/6 and type VIII collagen deficient mice were studied using assays to measure chemotactic and haptotactic migration, and remodeling and contraction of 3-dimensional type I collagen gels. Col8(-/-) SMCs exhibited impairments in migration, and a strongly adhesive phenotype with prominent stress fibers, stable microtubules and pronounced central basal focal adhesions. The addition of exogenous type VIII collagen to the Col8(-/-) SMCs rescued the impairments in migration, and restored cytoskeletal architecture so that it was similar to Col8(+/+) cells. We measured elevated levels of active GTP-RhoA in the Col8(-/-) cells, and this too was reversed by treatment with exogenous type VIII collagen. We showed that type VIII collagen normally suppresses RhoA activation through a beta-1 integrin dependent mechanism. MMP-2 levels were reduced in the Col8(-/-) SMCs, and knockdown of MMP-2 in Col8(+/+) SMCs partially recapitulated the decreases in migration and 3D gel contraction seen in Col8(-/-) cells, showing that type VIII collagen-stimulated migration was dependent on MMP-2. Inhibition of Rho restored MMP-2 activity in the Col8(-/-) cells, and partially rescued migration, demonstrating that the elevations in RhoA activity were responsible for the suppression of migration of these cells. In conclusion, we have shown that type VIII collagen signals through beta-1 integrin receptors to suppress RhoA, allowing optimal configuration of the cytoskeleton, and the stimulation of MMP-2-dependent cell migration.
Assuntos
Colágeno Tipo VIII/genética , Citoesqueleto/metabolismo , Matriz Extracelular/metabolismo , Integrina beta1/genética , Metaloproteinase 2 da Matriz/genética , Miócitos de Músculo Liso/metabolismo , Proteínas rho de Ligação ao GTP/genética , Animais , Adesão Celular , Técnicas de Cultura de Células , Movimento Celular , Colágeno Tipo VIII/deficiência , Colágeno Tipo VIII/farmacologia , Citoesqueleto/genética , Citoesqueleto/ultraestrutura , Matriz Extracelular/genética , Matriz Extracelular/ultraestrutura , Géis , Regulação da Expressão Gênica , Teste de Complementação Genética , Integrina beta1/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miócitos de Músculo Liso/patologia , Inibidores de Proteínas Quinases/farmacologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Proteínas rho de Ligação ao GTP/antagonistas & inibidores , Proteínas rho de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTPRESUMO
Collagen receptors expressed on vascular smooth muscle cells include the discoidin domain receptors (DDR1 and DDR2). DDR1 is known to play important roles in mediating smooth muscle cell responses to vascular injury, including neointimal hyperplasia, but much less is known about the function of DDR2. In this study, we harvested smooth muscle cells from DDR2 wild-type and knockout mice and studied the cells using in vitro models of migration and growth. There were no significant differences in the ability of Ddr2(+/+) or Ddr2(-/-) smooth muscle cells to attach to, migrate, or proliferate on type I collagen. Furthermore, neither matrix metalloproteinase (MMP) 2 nor MMP-9 activity nor type I collagen expression was different between the cell types. We conclude that in vitro, endogenous DDR2 is not required for smooth muscle cell hyperplastic responses to collagen.
Assuntos
Colágeno Tipo I/farmacologia , Miócitos de Músculo Liso/efeitos dos fármacos , Receptores Proteína Tirosina Quinases/genética , Receptores Mitogênicos/genética , Animais , Adesão Celular/efeitos dos fármacos , Adesão Celular/genética , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Quimiotaxia/efeitos dos fármacos , Quimiotaxia/genética , Receptores com Domínio Discoidina , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/fisiologia , Deleção de Genes , Expressão Gênica/efeitos dos fármacos , Inativação Gênica , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Knockout , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/fisiologia , Receptores Proteína Tirosina Quinases/deficiência , Receptores Mitogênicos/deficiênciaRESUMO
Directed migration of smooth muscle cells (SMCs) from the media to the intima in arteries occurs during atherosclerotic plaque formation and during restenosis after angioplasty or stent application. The polarized orientation of the microtubule-organizing center (MTOC) is a key determinant of this process, and we therefore investigated factors that regulate MTOC polarity in vascular SMCs. SMCs migrating in vivo from the medial to the intimal layer of the rat carotid artery following balloon catheter injury were rear polarized, with the MTOC located posterior of the nucleus. In tissue culture, migrating neointimal cells maintained rear polarization, whereas medial cells were front polarized. Using phosphoproteomic screening and mass spectrometry, we identified ARPC5 and RHAMM as protein kinase C (PKC)-phosphorylated proteins associated with rear polarization of the MTOC in neointimal SMCs. RNA silencing of ARPC5 and RHAMM, PKC inhibition, and transfection with a mutated nonphosphorylatable ARPC5 showed that these proteins regulate rear polarization by organizing the actin and microtubule cytoskeletons in neointimal SMCs. Both ARPC5 and RHAMM, in addition to PKC, were required for migration of neointimal SMCs.
Assuntos
Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Polaridade Celular , Proteínas da Matriz Extracelular/metabolismo , Receptores de Hialuronatos/metabolismo , Centro Organizador dos Microtúbulos/metabolismo , Miócitos de Músculo Liso/patologia , Proteína Quinase C-alfa/metabolismo , Túnica Íntima/patologia , Complexo 2-3 de Proteínas Relacionadas à Actina/antagonistas & inibidores , Animais , Artérias/metabolismo , Artérias/patologia , Contagem de Células , Movimento Celular , Núcleo Celular/metabolismo , Proteínas da Matriz Extracelular/antagonistas & inibidores , Inativação Gênica , Masculino , Modelos Biológicos , Mutação/genética , Fosforilação , Proteína Quinase C-alfa/antagonistas & inibidores , Pseudópodes/metabolismo , Ratos , Ratos Sprague-Dawley , Túnica Íntima/metabolismo , Túnica Média/metabolismo , Túnica Média/patologiaRESUMO
RATIONALE: Discoidin domain receptor (DDR)1 is a collagen receptor expressed on both smooth muscle cells (SMCs) and macrophages, where it plays important roles regulating cell and matrix accumulation during atherogenesis. Systemic deletion of DDR1 resulted in attenuated plaque growth but accelerated matrix accumulation in LDLR-deficient mice. Deletion of DDR1 solely on bone marrow-derived cells resulted in decreased macrophage accumulation and plaque growth but no change in matrix accumulation. OBJECTIVE: These findings led us to hypothesize that accelerated matrix accumulation was attributable to the increased synthetic ability of Ddr1(-/-) resident vascular wall SMCs. METHODS AND RESULTS: We used bone marrow transplantation to generate chimeric mice and investigate the role of SMC DDR1 during atherogenesis. Mice with deficiency of DDR1 in vessel wall-derived cells (Ddr1(+/+-->-/-)) or control mice (Ddr1(+/+-->+/+)) were fed an atherogenic diet for 12 weeks. We observed a 3.8-fold increase in the size of aortic sinus plaques in Ddr1(+/+-->-/-) compared to Ddr1(+/+-->+/+) mice. This was attributed to pronounced accumulation of collagen, elastin, proteoglycans, and fibronectin and resulted in a thickened fibrous cap. The enhanced matrix accumulation decreased the proportion of plaque area occupied by cells but was associated with a shift in the cellular composition of the lesions toward increased numbers of vessel wall-derived SMCs compared to bone marrow-derived macrophages. In vitro studies confirmed that Ddr1(-/-) SMCs expressed more matrix, proliferated more, and migrated farther than Ddr1(+/+) SMCs. CONCLUSIONS: DDR1 expression on resident vessel wall SMCs limits proliferation, migration and matrix accumulation during atherogenesis.
Assuntos
Doenças da Aorta/enzimologia , Aterosclerose/enzimologia , Proteínas da Matriz Extracelular/metabolismo , Músculo Liso Vascular/enzimologia , Miócitos de Músculo Liso/enzimologia , Receptores Proteína Tirosina Quinases/deficiência , Animais , Doenças da Aorta/genética , Doenças da Aorta/patologia , Aterosclerose/genética , Aterosclerose/patologia , Transplante de Medula Óssea , Movimento Celular , Proliferação de Células , Colágeno/metabolismo , Receptor com Domínio Discoidina 1 , Modelos Animais de Doenças , Elastina/metabolismo , Proteínas da Matriz Extracelular/genética , Feminino , Fibronectinas/metabolismo , Fibrose , Macrófagos/metabolismo , Masculino , Metaloproteinases da Matriz/metabolismo , Camundongos , Camundongos Knockout , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologia , Proteoglicanas/metabolismo , RNA Mensageiro/metabolismo , Receptores Proteína Tirosina Quinases/genética , Fatores de Tempo , Quimeras de Transplante , Regulação para CimaRESUMO
RATIONALE: We described a critical role for the discoidin domain receptor (DDR)1 collagen receptor tyrosine kinase during atherosclerotic plaque development. Systemic deletion of Ddr1 in Ldlr(-/-) mice accelerated matrix accumulation and reduced plaque size and macrophage content. However, whether these effects reflected an independent role for macrophage DDR1 during atherogenesis remained unresolved. METHODS: In the present study, we performed sex-mismatched bone marrow transplantation using Ddr1(+/+);Ldlr(-/-) and Ddr1(-/-);Ldlr(-/-) mice to investigate the role of macrophage DDR1 during atherogenesis. Chimeric mice with deficiency of DDR1 in bone marrow-derived cells (Ddr1(-/--->+/+)) or control chimeric mice that received Ddr1(+/+);Ldlr(-/-) marrow (Ddr1(+/+-->+/+)) were fed an atherogenic diet for 12 weeks. RESULTS: We observed a 66% reduction in atherosclerosis in the descending aorta and a 44% reduction in plaque area in the aortic sinus in Ddr1(-/--->+/+) mice compared to Ddr1(+/+-->+/+) mice. Furthermore, we observed a specific reduction in the number of donor-derived macrophages in Ddr1(-/--->+/+) plaques, suggesting that bone marrow deficiency of DDR1 attenuated atherogenesis by limiting macrophage accumulation in the plaque. We have also demonstrated that the effects of DDR1 on macrophage infiltration and accumulation can occur at the earliest stage of atherogenesis, the formation of the fatty streak. Deficiency of DDR1 limited the appearance of 5-bromodeoxyuridine-labeled monocytes/macrophages in the fatty streak and resulted in reduced lesion size in Ldlr(-/-) mice fed a high fat diet for 2 weeks. In vitro studies to investigate the mechanisms involved revealed that macrophages from Ddr1(-/-) mice had decreased adhesion to type IV collagen and decreased chemotactic invasion of type IV collagen in response to monocyte chemoattractant protein-1. CONCLUSIONS: Taken together, our data support an independent and critical role for DDR1 in macrophage accumulation at early and late stages of atherogenesis.
Assuntos
Aterosclerose/patologia , Células da Medula Óssea/patologia , Macrófagos/patologia , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/imunologia , Receptores Mitogênicos/genética , Receptores Mitogênicos/imunologia , Animais , Antimetabólitos , Aterosclerose/imunologia , Aterosclerose/metabolismo , Transplante de Medula Óssea , Bromodesoxiuridina , Colágeno Tipo IV/metabolismo , Receptores com Domínio Discoidina , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Feminino , Lipídeos/sangue , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Mutantes , Monócitos/patologia , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de LDL/genética , Receptores Mitogênicos/metabolismo , Proteína da Região Y Determinante do Sexo/genética , Quimeras de Transplante , Vasculite/imunologia , Vasculite/metabolismo , Vasculite/patologiaRESUMO
Vascular smooth muscle cells (VSMCs) maintain the ability to modulate their phenotype in response to changing environmental stimuli. This phenotype modulation plays a critical role in the development of most vascular disease states. In these studies, stimulation of cultured vascular smooth muscle cells with platelet-derived growth factor resulted in marked induction of c-jun expression, which was attenuated by protein kinase Cdelta and calcium/calmodulin-dependent protein kinase inhibition. Given that these signaling pathways have been shown to relieve the repressive effects of class II histone deacetylases (HDACs) on myocyte enhancer factor (MEF) 2 proteins, we ectopically expressed HDAC4 and observed repression of c-jun expression. Congruently, suppression of HDAC4 by RNA interference resulted in enhanced c-jun expression. Consistent with these findings, mutation of the MEF2 cis-element in the c-jun promoter resulted in promoter activation during quiescent conditions, suggesting that the MEF2 cis-element functions as a repressor in this context. Furthermore, we demonstrate that protein kinase A attenuates c-Jun expression by promoting the formation of a MEF2.HDAC4 repressor complex by inhibiting salt-inducible kinase 1. Finally, we document a physical interaction between c-Jun and myocardin, and we document that forced expression of c-Jun represses the ability of myocardin to activate smooth muscle gene expression. Thus, MEF2 and HDAC4 act to repress c-Jun expression in quiescent VSMCs, protein kinase A enhances this repression, and platelet-derived growth factor derepresses c-Jun expression through calcium/calmodulin-dependent protein kinases and novel protein kinase Cs. Regulation of this molecular "switch" on the c-jun promoter may thus prove critical for toggling between the activated and quiescent VSMC phenotypes.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Regulação da Expressão Gênica , Histona Desacetilases/metabolismo , Fatores de Regulação Miogênica/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Animais , Núcleo Celular/metabolismo , Fatores de Transcrição MEF2 , Camundongos , Modelos Biológicos , Músculo Liso Vascular/metabolismo , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Ratos , Transativadores/metabolismoRESUMO
The migration and proliferation of vascular smooth muscle cells (vSMCs) are critical events in neointima formation during atherosclerosis and restenosis. The transcription factor nuclear factor of activated T-cells-isoform c1 (NFATc1) is regulated by atherogenic cytokines, and has been implicated in the migratory and proliferative responses of vSMCs through the regulation of gene expression. In T-cells, calcineurin de-phosphorylates NFATc1, leading to its nuclear import, while glycogen synthase kinase 3 beta (GSK3beta) phosphorylates NFATc1 and promotes its nuclear export. However, the relationship between NFATc1 and GSK3beta has not been studied during SMC migration and proliferation. We investigated this by scrape wounding vSMCs in vitro, and studying wound repair. NFATc1 protein was transiently increased, reaching a peak at 8 h after wounding. Cell fractionation and immunocytochemistry revealed that NFATc1 accumulation in the nucleus was maximal at 4 h after injury, and this was coincident with a significant 9 fold increase in transcriptional activity. Silencing NFATc1 expression with siRNA or inhibition of NFAT with cyclosporin A (CsA) attenuated wound closure by vSMCs. Phospho-GSK3beta (inactive) increased to a peak at 30 min after injury, preceding the nuclear accumulation of NFATc1. Overexpression of a constitutively active mutant of GSK3beta delayed the nuclear accumulation of NFATc1, caused a 50% decrease in NFAT transcriptional activity, and attenuated vSMC wound repair. We conclude that NFATc1 promotes the vSMC response to injury, and that inhibition of GSK3beta is required for the activation of NFAT during wound repair.
Assuntos
Quinase 3 da Glicogênio Sintase/metabolismo , Músculo Liso Vascular , Miócitos de Músculo Liso/metabolismo , Fatores de Transcrição NFATC/metabolismo , Isoformas de Proteínas/metabolismo , Animais , Calcineurina/genética , Calcineurina/metabolismo , Fracionamento Celular , Movimento Celular/fisiologia , Núcleo Celular/metabolismo , Células Cultivadas , Ciclosporina/metabolismo , Citoplasma/metabolismo , Inibidores Enzimáticos/metabolismo , Quinase 3 da Glicogênio Sintase/genética , Glicogênio Sintase Quinase 3 beta , Masculino , Músculo Liso Vascular/citologia , Músculo Liso Vascular/lesões , Miócitos de Músculo Liso/citologia , Fatores de Transcrição NFATC/genética , Isoformas de Proteínas/genética , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Sprague-Dawley , Transcrição GênicaRESUMO
Integrin-mediated interactions between smooth muscle cells (SMCs) and the extracellular matrix regulate cell migration and proliferation during neointimal hyperplasia. Integrin-linked kinase (ILK) is a serine-threonine kinase and scaffolding molecule that acts downstream of integrin receptors to modulate cell adhesion; therefore, we examined ILK function in SMCs during wound repair. Silencing of ILK expression with siRNA in vitro decreased cell adhesion to fibronectin and accelerated both cell proliferation and wound closure in the cell monolayer; it also resulted in the rearrangement of focal adhesions and diminished central actin stress fibers. Akt and GSK3beta are ILK substrates that are important in cell motility; however, ILK siRNA silencing did not attenuate injury-induced increases in Akt and GSK3beta phosphorylation. Following balloon catheter injury of the rat carotid artery in vivo, a dramatic decrease in ILK levels coincided with both the proliferation and migration of SMCs, which leads to the formation of a thickened neointima. Immunostaining revealed decreased ILK levels in the media and deep layers of the neointima, but increased ILK levels in the subluminal layers of the intima. Taken together, these results suggest that ILK functions to maintain SMC quiescence in the normal artery. A decrease in ILK levels after injury may permit SMC migration, proliferation, and neointimal thickening, and its re-expression at the luminal surface may attenuate this process during later stages of the injury response.
Assuntos
Músculo Liso Vascular/enzimologia , Miócitos de Músculo Liso/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Cicatrização/fisiologia , Actinas/metabolismo , Animais , Western Blotting , Lesões das Artérias Carótidas/enzimologia , Movimento Celular/fisiologia , Proliferação de Células , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Imuno-Histoquímica , Masculino , Miócitos de Músculo Liso/patologia , Paxilina/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno , Ratos , Ratos Sprague-Dawley , Túnica Íntima/enzimologia , Túnica Íntima/lesõesRESUMO
Collagens are abundant within the atherosclerotic plaque, where they contribute to lesion volume and mechanical stability and influence cell signaling. The discoidin domain receptor 1 (DDR1), a receptor tyrosine kinase that binds to collagen, is expressed in blood vessels, but evidence for a functional role during atherogenesis is incomplete. In the present study, we generated Ddr1(+/+);Ldlr(-/-) and Ddr1(-/-);Ldlr(-/-) mice and fed them an atherogenic diet for 12 or 24 weeks. Targeted deletion of Ddr1 resulted in a 50% to 60% reduction in atherosclerotic lesion area in the descending aorta at both 12 and 24 weeks. Ddr1(-/-);Ldlr(-/-) plaques exhibited accelerated deposition of fibrillar collagen and elastin at 12 weeks compared with Ddr1(+/+);Ldlr(-/-) plaques. Expression analysis of laser microdissected lesions in vivo, and of Ddr1(-/-) smooth muscle cells in vitro, revealed increased mRNA levels for procollagen alpha1(I) and alpha1(III) and tropoelastin, suggesting an enhancement of matrix synthesis in the absence of DDR1. Furthermore, whereas plaque smooth muscle cell content was unchanged, Ddr1(-/-);Ldlr(-/-) plaques had a 49% decrease in macrophage content at 12 weeks, with a concomitant reduction of in situ gelatinolytic activity. Moreover, mRNA expression of both monocyte chemoattractant protein-1 and vascular cell adhesion molecule-1 was reduced in vivo, and Ddr1(-/-);Ldlr(-/-) macrophages demonstrated impaired matrix metalloproteinase expression in vitro. These data suggest novel roles for DDR1 in macrophage recruitment and invasion during atherogenesis. In conclusion, our data support a role for DDR1 in the regulation of both inflammation and fibrosis early in plaque development. Deletion of DDR1 attenuated atherogenesis and resulted in the formation of matrix-rich plaques.
Assuntos
Aterosclerose/imunologia , Aterosclerose/fisiopatologia , Receptores Proteína Tirosina Quinases/genética , Receptores de LDL/genética , Receptores Mitogênicos/genética , Vasculite/imunologia , Vasculite/fisiopatologia , Animais , Aorta Torácica/imunologia , Aorta Torácica/metabolismo , Aorta Torácica/patologia , Aterosclerose/patologia , Quimiocina CCL2/metabolismo , Colágeno/metabolismo , Dieta Aterogênica , Receptores com Domínio Discoidina , Modelos Animais de Doenças , Elastina/metabolismo , Matriz Extracelular/metabolismo , Feminino , Fibrose , Lipídeos/sangue , Macrófagos/patologia , Masculino , Camundongos , Camundongos Mutantes , Músculo Liso Vascular/imunologia , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Mutagênese/fisiologia , Receptores Proteína Tirosina Quinases/metabolismo , Receptores Mitogênicos/metabolismo , Molécula 1 de Adesão de Célula Vascular/metabolismo , Vasculite/patologiaRESUMO
Remodeling of injured blood vessels is dependent on smooth muscle cells and matrix metalloproteinase activity. Doxycycline is a broad spectrum matrix metalloproteinase inhibitor that is under investigation for the treatment of acute coronary syndromes and aneurysms. In the present study, we examine the mechanisms by which doxycycline inhibits smooth muscle cell responses using a series of in vitro assays that mimic critical steps in pathological vascular remodeling. Doxycycline treatment dramatically increased smooth muscle cell adhesion to the substrate, as evidenced by interference reflection microscopy and immunostaining for paxillin and phosphotyrosine. Cell aggregation was also potentiated after treatment with doxycycline. Treatment with 104 mumol/L doxycycline reduced thymidine uptake by 58% compared with untreated cells (P < 0.05) and inhibited closure of a scrape wound made in a smooth muscle cell monolayer by 20% (P < 0.05). Contraction of a three-dimensional collagen gel was used as an in vitro model for constrictive vessel remodeling, demonstrating that treatment with 416 mumol/L doxycycline for 12 hours inhibited collagen gel remodeling by 37% relative to control (P < 0.05). In conclusion, we have shown that doxycycline treatment leads to dramatically increased smooth muscle cell adhesion, which in turn might limit responses in pathological vascular remodeling.
Assuntos
Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Doxiciclina/farmacologia , Colágenos Fibrilares/metabolismo , Miócitos de Músculo Liso/fisiologia , Actinas/metabolismo , Animais , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Masculino , Músculo Liso Vascular , Ratos , Ratos Sprague-Dawley , Fatores de TempoRESUMO
OBJECTIVE: Type VIII collagen is upregulated after vascular injury and in atherosclerosis. However, the role of type VIII collagen endogenously expressed by smooth muscle cells (SMCs) and in the context of the vascular matrix microenvironment, which is rich in type I collagen, is not known. To address this, we have compared aortic SMCs from wild-type (WT) mice to SMCs from type VIII collagen-deficient (KO) mice when plated on type I collagen. METHODS AND RESULTS: Type VIII collagen was upregulated after wounding of WT SMCs. KO SMCs exhibited greater adhesion to type I collagen than WT SMCs (optical density [OD595]=0.458+/-0.044 versus 0.193+/-0.071). By contrast, the WT SMCs spread more (389+/-75% versus 108+/-14% increase in cell area), migrated further (total distance 80.6+/-6.2 microm versus 64.2+/-4.4 microm), and exhibited increased [3H]-thymidine uptake (160,000+/-22,300 versus 63,100+/-12,100 counts per minute) when compared with KO SMCs. Gelatin zymograms showed that WT SMCs expressed latent matrix metalloproteinase 2, whereas KO SMCs did not. Addition of exogenous type VIII collagen returned levels of KO SMC adhesion (OD595=0.316+/-0.038), migration (79.5+/-5.8 microm), and latent matrix metalloproteinase 2 expression to levels comparable to WT SMCs. CONCLUSIONS: This study suggests that SMCs can modify the matrix microenvironment by producing type VIII collagen, using it to overlay type I collagen, and generating a substrate favorable for migration.
Assuntos
Movimento Celular/fisiologia , Colágeno Tipo VIII/genética , Colágeno Tipo VIII/metabolismo , Músculo Liso Vascular/citologia , Músculo Liso Vascular/fisiologia , Alelos , Animais , Aorta/citologia , Divisão Celular/fisiologia , Células Cultivadas , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Matriz Extracelular/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Knockout , Microscopia de Vídeo , Regulação para Cima/fisiologiaRESUMO
Rhoptries are apical organelles which play an important role in erythrocytic invasion. A Plasmodium falciparum cDNA clone, coding for a novel rhoptry protein PfRhop148, was obtained by antibody screening of a library. The deduced amino acid sequence consists of 1262 amino acids and is highly rich in Asn (22%). The Asn residues are distributed in clusters and in multiple units of repeats. Analysis of specific RNA transcript and protein showed that PfRhop148 was synthesized at around 21 h post-invasion. IFA and immunoelectron microscopic analyses revealed a rhoptry localization for the protein. The role of this protein in invasion and its relationship to the RhopH complex is now under further investigation.
Assuntos
Plasmodium falciparum/patogenicidade , Proteínas de Protozoários/análise , Proteínas de Protozoários/genética , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/metabolismo , Clonagem Molecular , Imunofluorescência , Humanos , Estágios do Ciclo de Vida , Proteínas de Membrana/química , Camundongos , Modelos Genéticos , Dados de Sequência Molecular , Plasmodium falciparum/genética , Plasmodium falciparum/imunologia , Plasmodium falciparum/ultraestrutura , Proteínas de Protozoários/metabolismoRESUMO
Smooth muscle cell (SMC) interactions with collagen mediate cell migration during the pathogenesis of atherosclerosis and restenosis. Discoidin domain receptors (DDRs) have been identified as novel collagen receptors. We used aortic SMCs from wild-type and DDR1(-/-) mice to evaluate the function of the DDR1 in regulating migration. DDR1(-/-) SMCs exhibited impaired attachment to and migration toward a type I collagen substrate. Matrix metalloproteinase-2 (MMP-2) and MMP-9 activities were concomitantly reduced in these cells. Transfection of a full-length cDNA for DDR1b rescued these deficits, whereas kinase-dead mutants of DDR1 restored attachment but not migration and MMP production. These results suggest that active DDR1 kinase is a central mediator of SMC migration.
Assuntos
Movimento Celular/fisiologia , Metaloproteinases da Matriz/biossíntese , Músculo Liso Vascular/metabolismo , Proteínas Tirosina Quinases/metabolismo , Receptores Proteína Tirosina Quinases , Receptores Mitogênicos/metabolismo , Animais , Adesão Celular/fisiologia , Receptores com Domínio Discoidina , Genótipo , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos , Camundongos Knockout , Microscopia Confocal , Músculo Liso Vascular/citologia , Receptores Mitogênicos/genética , TransfecçãoRESUMO
The collagen matrix constitutes a major portion of the vascular extracellular matrix and imparts blood vessels with tensile strength and, even more important, modulates smooth muscle cell (SMC) responses via specific receptors and signaling pathways. This review is focused on the interactions of SMCs with the collagen matrix, how these interactions are involved in sensing the local environment, and the receptors that mediate these processes. Better understanding of the pathways involved in cell matrix interactions promises to provide novel therapeutic targets and treatment strategies for the prevention of arterial occlusive diseases such as atherosclerosis and restenosis.