RESUMO
Macrophage (MΦ) dysregulation is increasingly becoming recognized as a risk factor for a number of inflammatory complications including atherosclerosis, cancer, and the host response elicited by biomedical devices. It is still unclear what roles the pro-inflammatory (M1) MΦ and pro-healing (M2) MΦ phenotypes play during the healing process. However, it has been shown that a local overabundance of M1 MΦs can potentially lead to a chronically inflamed state of the tissue; while a local over-exuberant M2 MΦ response can lead to tissue fibrosis and even promote tumorigenesis. These notions strengthen the argument that the tight temporal regulation of this phenotype balance is necessary to promote inflammatory resolution that leads to tissue homeostasis. In this study, we have engineered pro-inflammatory MΦs, MΦ-cTLR4 cells, which can be activated to a M1-like MΦ phenotype with a small molecule, the chemical inducer of dimerization (CID) drug. The MΦ-cTLR4 cells when activated with the CID drug, express increased levels of TNFα, IL-6, and iNOS. Activated MΦ-cTLR4 cells stay stimulated for at least 48h; once the CID drug is withdrawn, the MΦ-cTLR4 cells return to baseline state within 18h. Further, in vitro CID-activated MΦ-cTLR4 cells induce upregulation of VCAM-1 and ICAM-1 on endothelial cells (EC) in a TNFα-dependent manner. With the ability to specifically modulate the MФ-cTLR4 cells with the presence or absence of a small molecule, we now have the tool necessary to observe a primarily M1 MФ response during inflammation. By isolating this phase of the wound healing response, it may be possible to determine conditions for ideal healing.
Assuntos
Engenharia Celular , Inflamação/metabolismo , Macrófagos/metabolismo , Neovascularização Patológica/metabolismo , Animais , Células Cultivadas , Macrófagos/efeitos dos fármacos , Macrófagos/patologia , Camundongos , Neovascularização Patológica/patologia , Tacrolimo/análogos & derivados , Tacrolimo/farmacologiaRESUMO
OBJECTIVES: Ectopic calcification is a common response to soft tissue injury and systemic mineral imbalance and can lead to devastating clinical consequences when present in joints, heart valves and blood vessels. We have hypothesized that mineralization of matrices in any tissue is normally controlled by a balance between procalcific and anticalcific regulatory proteins such that abnormal deposition of apatite is avoided. Alterations in this balance induced by injury, disease or genetic deficiency are postulated to induce ectopic mineral deposition. Over the past several years, we have developed in vitro and in vivo models of ectopic calcification to investigate potential inducers and inhibitors of this process. RESULTS: Osteopontin, a secreted phosphoprotein, has emerged as a major inhibitor of ectopic mineralization. Osteopontin is a potent inhibitor of vascular cell calcification in vitro and mice lacking osteopontin are highly susceptible to ectopic calcification. Furthermore, osteopontin treatment of biomaterials protected against ectopic mineralization. Our studies indicate that in addition to inhibiting apatite crystal initiation and growth, osteopontin stimulates resorption of ectopic calcification via peripheral macrophages and giant cells. In contrast, inorganic phosphate has emerged as a major inducer of mineralization in these systems. Elevated inorganic phosphate (Pi) was shown to induce smooth muscle cell matrix calcification with morphological properties similar to those observed in calcified human valves and atherosclerotic plaques. Furthermore, mineralization induced by inorganic phosphate was dependent on the activity of the sodium-dependent phosphate cotransporter, Pit-1. CONCLUSIONS: These studies implicate controlled, transcellular transport of Pi as a major requirement for matrix calcification.
Assuntos
Calcificação Fisiológica/fisiologia , Calcinose/metabolismo , Fosfatos/metabolismo , Sialoglicoproteínas/metabolismo , Animais , Aterosclerose/metabolismo , Humanos , Osteopontina , Proteínas Cotransportadoras de Sódio-Fosfato Tipo III/metabolismoRESUMO
Endothelial cell survival and antiapoptotic pathways, including those stimulated by extracellular matrix, are critical regulators of vasculogenesis, angiogenesis, endothelial repair, and shear-stress-induced endothelial activation. One of these pathways is mediated by alpha(v)beta(3) integrin ligation, downstream activation of nuclear factor-kappaB, and subsequent up-regulation of osteoprotegerin (OPG). In this study, the mechanism by which OPG protects endothelial cells from death was examined. Serum-starved human microvascular endothelial cells (HMECs) plated on the alpha(v)beta(3) ligand osteopontin were protected from cell death. Immunoprecipitation experiments indicated that OPG formed a complex with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in HMECs under these conditions. Furthermore, inhibitors of TRAIL, including recombinant soluble TRAIL receptors and a neutralizing antibody against TRAIL, blocked apoptosis of serum-starved HMECs plated on the nonintegrin attachment factor poly-d-lysine. Whereas TRAIL was unable to induce apoptosis in HMECs plated on osteopontin, the addition of recombinant TRAIL did increase the percentage of apoptotic HMECs plated on poly-d-lysine. This evidence indicates that OPG blocks endothelial cell apoptosis through binding TRAIL and preventing its interaction with death-inducing TRAIL-receptors
Assuntos
Células Endoteliais/citologia , Células Endoteliais/metabolismo , Glicoproteínas/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Anticorpos Monoclonais/imunologia , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose , Linhagem Celular , Membrana Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Meios de Cultura Livres de Soro/farmacologia , Células Endoteliais/efeitos dos fármacos , Proteínas Ligadas por GPI , Glicoproteínas/genética , Glicoproteínas/farmacologia , Humanos , Imunoprecipitação , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Glicoproteínas de Membrana/farmacologia , Osteopontina , Osteoprotegerina , Polilisina/metabolismo , Ligação Proteica , Receptores Citoplasmáticos e Nucleares/genética , Receptores do Ligante Indutor de Apoptose Relacionado a TNF , Receptores do Fator de Necrose Tumoral/metabolismo , Membro 10c de Receptores do Fator de Necrose Tumoral , Proteínas Recombinantes de Fusão/farmacologia , Sialoglicoproteínas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Ligante Indutor de Apoptose Relacionado a TNF , Receptores Chamariz do Fator de Necrose Tumoral , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Bovine aortic smooth muscle cell (BASMC) cultures undergo mineralization on addition of the organic phosphate donor, beta-glycerophosphate (betaGP). Mineralization is characterized by apatite deposition on collagen fibrils and the presence of matrix vesicles, as has been described in calcified vascular lesions in vivo as well as in bone and teeth. In the present study, we used this model to investigate the molecular mechanisms driving vascular calcification. We found that BASMCs lost their lineage markers, SM22alpha and smooth muscle alpha-actin, within 10 days of being placed under calcifying conditions. Conversely, the cells gained an osteogenic phenotype as indicated by an increase in expression and DNA-binding activity of the transcription factor, core binding factor alpha1 (Cbfa1). Moreover, genes containing the Cbfa1 binding site, OSE2, including osteopontin, osteocalcin, and alkaline phosphatase were elevated. The relevance of these in vitro findings to vascular calcification in vivo was further studied in matrix GLA protein null (MGP(-/-)) mice whose arteries spontaneously calcify. We found that arterial calcification was associated with a similar loss in smooth muscle markers and a gain of osteopontin and Cbfa1 expression. These data demonstrate a novel association of vascular calcification with smooth muscle cell phenotypic transition, in which several osteogenic proteins including osteopontin, osteocalcin, and the bone determining factor Cbfa1 are gained. The findings suggest a positive role for SMCs in promoting vascular calcification.
Assuntos
Calcinose/metabolismo , Proteínas da Matriz Extracelular , Músculo Liso Vascular/metabolismo , Proteínas de Neoplasias , Animais , Antígenos de Diferenciação/metabolismo , Aorta/metabolismo , Aorta/patologia , Calcinose/induzido quimicamente , Calcinose/patologia , Fosfatos de Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/deficiência , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Artérias Carótidas/metabolismo , Artérias Carótidas/patologia , Bovinos , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core , Fatores de Ligação ao Core , Glicerofosfatos , Humanos , Camundongos , Camundongos Knockout , Modelos Biológicos , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/patologia , Osteocalcina/metabolismo , Osteopontina , Fenótipo , RNA Mensageiro/metabolismo , Sialoglicoproteínas/genética , Sialoglicoproteínas/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína de Matriz GlaRESUMO
Vascular calcification is highly correlated with elevated serum phosphate levels in uremic patients. To shed light on this process, we examined the ability of extracellular inorganic phosphate (Pi) levels to regulate human aortic smooth muscle cell (HSMC) culture mineralization in vitro. When cultured in media containing normal physiological levels of Pi (1.4 mmol/L Pi), HSMC grew in monolayers and did not mineralize. In contrast, HSMC cultured in media containing Pi levels comparable to those seen in hyperphosphatemic individuals (>1.4 mmol/L), showed dose-dependent increases in cell culture calcium deposition. Mechanistic studies showed that elevated Pi treatment of HSMC also enhanced the expression of the osteogenic markers, osteocalcin and Cbfa-1. The effects of elevated Pi on HSMC were mediated by a sodium-dependent phosphate cotransporter (NPC), as indicated by the ability of the specific NPC inhibitor, phosphonoformic acid (PFA), to dose-dependently inhibit Pi-induced calcium deposition as well as osteocalcin and Cbfa-1 gene expression. Using polymerase chain reaction and Northern blot analyses, the NPC in HSMC was identified as Pit-1 (Glvr-1), a member of the type III NPCs. Interestingly, platelet-derived growth factor-BB (PDGF-BB), a potent atherogenic stimulus, increased the maximum velocity (Vmax) but not the affinity (Km) of phosphate uptake, enhanced the expression of Pit-1 mRNA, and induced HSMC culture calcification in a time- and dose-dependent manner. Importantly, in the presence of PDGF, HSMC culture calcification occurred under normophosphatemic conditions. These data suggest that elevated Pi may directly stimulate HSMC to undergo phenotypic changes that predispose to calcification and may help explain both the phenomena of human metastatic calcification under hyperphosphatemic conditions as well as increased calcification in PDGF-rich atherosclerotic lesions.
Assuntos
Calcinose/etiologia , Calcinose/metabolismo , Doenças Cardiovasculares/etiologia , Doenças Cardiovasculares/metabolismo , Proteínas de Membrana , Músculo Liso Vascular/metabolismo , Proteínas de Neoplasias , Fosfatos/farmacocinética , Uremia/complicações , Actinas/efeitos dos fármacos , Actinas/metabolismo , Animais , Proteínas de Transporte/genética , Células Cultivadas , Relação Dose-Resposta a Droga , Regulação para Baixo , Foscarnet/farmacologia , Expressão Gênica , Glicerofosfatos/farmacologia , Humanos , Osteocalcina/metabolismo , Fosfatos/metabolismo , Proteínas de Transferência de Fosfolipídeos , Fator de Crescimento Derivado de Plaquetas/metabolismo , Fator de Crescimento Derivado de Plaquetas/farmacologia , RNA Mensageiro/metabolismo , Proteínas Cotransportadoras de Sódio-Fosfato , Proteínas Cotransportadoras de Sódio-Fosfato Tipo III , Simportadores/antagonistas & inibidores , Simportadores/metabolismo , Fatores de Transcrição/genéticaRESUMO
BACKGROUND: Osteopontin is a secreted phosphoprotein that has a number of diverse biological functions, including cell signaling, mediation of cell adhesion, migration, and chemoattraction of monocytes/macrophages. Up-regulation of osteopontin expression by proximal tubular epithelium has been demonstrated in both human and rodent models of renal injury in association with macrophage influx. METHODS: We studied the expression of osteopontin protein and mRNA in renal donor biopsies (N = 7) and renal transplant biopsies with cyclosporine A toxicity (N = 23) by immunohistochemistry and in situ hybridization. Serial tissue sections were immunostained with a monocyte/macrophage marker, CD68, to demonstrate the pattern of macrophage infiltration. RESULTS: Strong osteopontin expression was observed in the majority of pretransplant donor biopsies in the absence of any macrophage infiltration. In the biopsies with cyclosporine toxicity, osteopontin expression was widespread and demonstrated moderate immunohistochemical signal intensity that did not correlate with the number of interstitial macrophages present. CONCLUSIONS: Strong osteopontin protein and mRNA expression by tubular epithelium was observed in pretransplant donor biopsies and in biopsies with cyclosporine toxicity without an inflammatory cell infiltration. Therefore, osteopontin expression alone is insufficient to serve as the principal mediator of intrarenal monocyte/macrophage influx in the transplant setting.
Assuntos
Ciclosporina/toxicidade , Imunossupressores/toxicidade , Transplante de Rim , Sialoglicoproteínas/genética , Biópsia por Agulha , Expressão Gênica/efeitos dos fármacos , Humanos , Rim/química , Rim/imunologia , Rim/patologia , Macrófagos/imunologia , Osteopontina , RNA Mensageiro/análise , Sialoglicoproteínas/análise , Doadores de TecidosRESUMO
Ectopic calcification is a common response to soft tissue injury and systemic mineral imbalance, and can lead to devastating clinical consequences when present in heart valves or blood vessels. It is hypothesized that mesenchymal and inflammatory cells normally maintain the balance between procalcific and anti-calcific regulatory proteins in soft tissues such that ectopic deposition of apatite is avoided. Alterations in this balance induced by injury or disease is postulated to induce ectopic apatite deposition. We have developed in vitro and in vivo models of ectopic calcification and found that: 1) elevated extracellular phosphate levels induce smooth muscle culture mineralization morphologically similar to that observed in calcified human valves and atherosclerotic plaques, 2) sodium-dependent phosphate cotransporter function is required for smooth muscle cell culture mineralization, 3) smooth muscle cell culture mineralization is associated with a dramatic loss of smooth muscle-specific gene expression and gain of osteoblast-like properties, including expression of osteoblast differentiation factor, Cbfa-1, and 4) osteopontin, a secreted phosphoprotein abundant in macrophages found in human calcified atherosclerotic and valvular lesions, is a potent inhibitor of ectopic calcification in vitro and in vivo. These studies suggest that both constitutively expressed (surveillance) and inducible (damage control) mineralization inhibitory molecules are produced by mesenchymal and inflammatory cells to control ectopic calcification.
Assuntos
Arteriosclerose/patologia , Calcinose/patologia , Proteínas da Matriz Extracelular , Doenças das Valvas Cardíacas/patologia , Apatitas/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Valvas Cardíacas/patologia , Humanos , Músculo Liso Vascular/patologia , Osteoblastos/patologia , Osteopontina , Fosfatos/metabolismo , Sialoglicoproteínas/metabolismo , Proteína de Matriz GlaRESUMO
Osteopontin (OPN) is a glycosylated phosphoprotein found in all body fluids and in the proteinaceous matrix of mineralized tissues. It can function both as a cell attachment protein and as a cytokine, delivering signals to cells via a number of receptors including several integrins and CD44. Expression of OPN is enhanced by a variety of toxicants, especially those that activate protein kinase C. In its capacity as a signaling molecule, OPN can modify gene expression and promote the migration of monocytes/macrophages up an OPN gradient. It has both inflammatory and anti-inflammatory actions. Some experiments suggest that it may inhibit apoptosis, possibly contributing to the survival of cells in response to toxicant injury. Elevated OPN expression often correlates with malignancy and has been shown to enhance the tumorigenic and/or metastatic phenotype of the cancer cell. Recent studies have revealed that OPN plays critical roles in bone remodeling and cell-mediated immunity.
Assuntos
Sialoglicoproteínas/fisiologia , Transdução de Sinais/fisiologia , Toxinas Biológicas/toxicidade , Animais , Doenças Ósseas/fisiopatologia , Humanos , Neoplasias/fisiopatologia , Osteopontina , Receptores de Citocinas/fisiologiaRESUMO
Osteopontin is a secreted glycoprotein with a multidomain structure and functions characteristic of a matricellular protein. Osteopontin interacts with cell surface receptors via arginine-glycine-aspartate (RGD)- and non-RGD containing adhesive domains, in addition to binding to components of the structural extracellular matrix. While normally expressed in bone and kidney, osteopontin levels are elevated during wound healing and inflammation in most tissues studied to date. Since 1986, over one thousand studies have been published on osteopontin, including recent experiments in osteopontin-deficient mice. These studies reveal osteopontin as a cell adhesive, signaling, migratory, and survival stimulus for various mesenchymal, epithelial, and inflammatory cells, in addition to being a potent regulator of osseous and ectopic calcification. Based on these reports, a general picture of osteopontin as an important regulator of inflammation and biomineralization is emerging. A common denominator in osteopontin function in these situations is its ability to regulate the function of macrophage and macrophage-derived cells (i.e. osteoclasts). While we have learned much about osteopontin and the processes it appears to regulate over the past decade, many questions regarding this important multifunctional protein remain unanswered and provide important directions for future studies.
Assuntos
Calcificação Fisiológica/fisiologia , Sialoglicoproteínas/fisiologia , Animais , Humanos , Camundongos , Osteopontina , Sialoglicoproteínas/imunologia , Sialoglicoproteínas/metabolismoRESUMO
High plasma levels of plasminogen activator inhibitor-1 (PAI-1) are associated with an increased risk of cardiovascular disease. There is also a close relation between high plasma levels of PAI-1 and hypertriglyceridemia. Cell culture studies have shown that very low density lipoprotein (VLDL) increases the production and secretion of PAI-1 in endothelial cells and hepatocytes, suggesting a possible mechanism for this association. To determine whether VLDL stimulates PAI-1 production in vascular cells also in vivo, Sprague-Dawley rats were injected intravenously with 6 mg/kg of VLDL (derived from human subjects with type IV hyperlipidemia). Previous studies have demonstrated that this results in an accumulation of human VLDL in the aorta and other arteries followed by increased nuclear factor-kappa B (NF-kappaB) activation. Endothelial, but not smooth muscle cells, showed a basal PAI-1 mRNA and protein expression as assessed by in situ hybridization and immunohistochemistry, respectively. Six to twenty-four hours after the VLDL injection, lipoprotein particle accumulation was seen in the aortic wall, which was accompanied by increasing PAI-1 mRNA and protein expression in endothelial and smooth muscle cells. Within the rat PAI-1 promoter we identified a sequence located at -589 to -571 with 74% homology with the recently described VLDL responsive element in the human PAI-1 promoter and located adjacent to a 4-guanosine motif presumably corresponding to the human 4G/5G polymorphism. Transient transfection studies showed that VLDL exerts its stimulatory effects on rat PAI-1 gene expression in vascular cells by interaction with promoter sequences located within bp -656 and -505. Electrophoretic mobility shift assays showed that VLDL increases the binding of as yet incompletely characterized factors to this response element. Taken together these observations support a direct influence of VLDL on vascular PAI-1 gene expression ill vivo. This stimulation is exerted on the level of PAI-1 gene transcription, and involves transcription factor binding to a VLDL responsive element adjacent to a 4G motif within the PAI-1 promoter.
Assuntos
Aorta/metabolismo , Lipoproteínas VLDL/metabolismo , Lipoproteínas VLDL/farmacologia , Inibidor 1 de Ativador de Plasminogênio/biossíntese , Animais , Aorta/patologia , Humanos , Hibridização In Situ , Ratos , Ratos Sprague-Dawley , Fatores de Transcrição/metabolismoRESUMO
Vascular calcification is a common finding in atherosclerosis and a serious problem in diabetic and uremic patients. Because of the correlation of hyperphosphatemia and vascular calcification, the ability of extracellular inorganic phosphate levels to regulate human aortic smooth muscle cell (HSMC) culture mineralization in vitro was examined. HSMCs cultured in media containing normal physiological levels of inorganic phosphate (1.4 mmol/L) did not mineralize. In contrast, HSMCs cultured in media containing phosphate levels comparable to those seen in hyperphosphatemic individuals (>1.4 mmol/L) showed dose-dependent increases in mineral deposition. Mechanistic studies revealed that elevated phosphate treatment of HSMCs also enhanced the expression of the osteoblastic differentiation markers osteocalcin and Cbfa-1. The effects of elevated phosphate on HSMCs were mediated by a sodium-dependent phosphate cotransporter (NPC), as indicated by the ability of the specific NPC inhibitor phosphonoformic acid, to dose dependently inhibit phosphate-induced calcium deposition as well as osteocalcin and Cbfa-1 gene expression. With the use of polymerase chain reaction and Northern blot analyses, the NPC in HSMCs was identified as Pit-1 (Glvr-1), a member of the novel type III NPCs. These data suggest that elevated phosphate may directly stimulate HSMCs to undergo phenotypic changes that predispose to calcification and offer a novel explanation of the phenomenon of vascular calcification under hyperphosphatemic conditions. The full text of this article is available at http://www.circresaha.org.
Assuntos
Calcificação Fisiológica , Cálcio/metabolismo , Proteínas de Transporte/fisiologia , Músculo Liso Vascular/fisiologia , Fosfatos/fisiologia , Simportadores , Artérias/metabolismo , Células Cultivadas , Expressão Gênica , Humanos , Receptores Virais/metabolismo , Proteínas Cotransportadoras de Sódio-Fosfato , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIIRESUMO
This study tests the hypothesis that alpha(v)beta(3) integrin receptors play a critical role in smooth muscle cell (SMC) migration after arterial injury and facilitate migration through the upregulation of matrix metalloproteinase (MMP) activity. We showed that beta(3) integrin mRNA was upregulated by SMCs in the balloon-injured rat carotid artery in coincidence with MMP-1 expression and early SMC migration. Treatment with the beta(3) integrin-blocking antibody F11 significantly decreased SMC migration into the intima at 4 days after injury, from 110.8+/-30.8 cells/mm(2) in control rats to 10.29+/-7.03 cells/mm(2) in F11-treated rats (P=0.008). By contrast, there was no effect on medial SMC proliferation or on medial SMC number in the carotid artery at 4 days. In vitro, we found that human newborn SMCs produced MMP-1 but that adult SMCs did not. This was possibly due to the fact that newborn SMCs expressed alpha(v)beta(3) integrin receptors, whereas adult SMCs did not. Stimulation of newborn (alpha(v)beta(3)+) SMCs with osteopontin, a matrix ligand for alpha(v)beta(3), increased MMP-1 production from 114.4+/-35.8 ng/mL at 0 nmol/L osteopontin to 232.5+/-57.5 ng/mL at 100 nmol/L osteopontin. Finally, we showed that stimulation of newborn SMCs with platelet-derived growth factor-BB and osteopontin together increased the SMC production of MMP-9. Thus, our results support the hypothesis that SMC alpha(v)beta(3) integrin receptors play an important role in regulating migration by stimulating SMC MMP production.
Assuntos
Metaloproteinase 1 da Matriz/biossíntese , Músculo Liso Vascular/enzimologia , Receptores de Vitronectina/fisiologia , Animais , Becaplermina , Plaquetas/fisiologia , Lesões das Artérias Carótidas/metabolismo , Cateterismo , Movimento Celular , Células Cultivadas , Regulação da Expressão Gênica , Humanos , Recém-Nascido , Masculino , Camundongos , Músculo Liso Vascular/citologia , Osteopontina , Fator de Crescimento Derivado de Plaquetas/farmacologia , Proteínas Proto-Oncogênicas c-sis , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de Vitronectina/genética , Sialoglicoproteínas/farmacologiaRESUMO
Osteopontin protects endothelial cells from apoptosis induced by growth factor withdrawal. This interaction is mediated by the alpha(v)beta(3) integrin and is NF-kappaB-dependent (Scatena, M., Almeida, M., Chaisson, M. L., Fausto, N., Nicosia, R. F., and Giachelli, C. M. (1998) J. Cell Biol. 141, 1083-1093). In the present study we used differential cloning to identify osteopontin-induced, NF-kappaB-dependent genes in endothelial cells. One of the genes identified in this screen was osteoprotegerin, a member of the tumor necrosis factor receptor superfamily. By Northern and Western blot analysis, osteoprotegerin mRNA and protein levels were very low in endothelial cells plated on the non-integrin cell attachment factor, poly-d-lysine. In contrast, osteoprotegerin mRNA and protein levels were induced 5-7-fold following alpha(v)beta(3) ligation by osteopontin. Osteoprotegerin induction by osteopontin was time-dependent and observed as early as 3 h following treatment. NF-kappaB inactivation achieved by over expression of an IkappaB super repressor in endothelial cells completely inhibited osteoprotegerin induction by osteopontin. Finally, purified osteoprotegerin protected endothelial cells with inactive NF-kappaB from apoptosis induced by growth factor deprivation. These data suggest that alpha(v)beta(3)-mediated endothelial survival depends on osteoprotegerin induction by NF-kappaB and indicate a new function for osteoprotegerin in endothelial cells.
Assuntos
Sobrevivência Celular/fisiologia , Endotélio Vascular/citologia , Endotélio Vascular/fisiologia , Regulação da Expressão Gênica , Glicoproteínas/genética , NF-kappa B/metabolismo , Receptores Citoplasmáticos e Nucleares , Receptores de Vitronectina/fisiologia , Animais , Aorta , Apoptose/efeitos dos fármacos , Células Cultivadas , Glicoproteínas/farmacologia , Glicoproteínas/fisiologia , Humanos , Cinética , Osteoprotegerina , RNA Mensageiro/genética , Ratos , Receptores do Fator de Necrose Tumoral/genética , Proteínas Recombinantes/farmacologia , Transcrição GênicaRESUMO
Osteopontin (OPN) is a non-collagenous, glycosylated phosphoprotein associated with biomineralization in osseous tissues, as well as ectopic calcification. We previously reported that osteopontin was co-localized with calcified deposits in atherosclerotic lesions, and that osteopontin potently inhibits calcium deposition in a human smooth muscle cell (HSMC) culture model of vascular calcification. In this report, the role of phosphorylation in osteopontin's mineralization inhibitory function was examined. The ability of OPN to inhibit calcification completely depended on post-translational modifications, since bacteria-derived recombinant OPN did not inhibit HSMC mineralization. Following casein kinase II treatment, phosphorylated OPN (P-OPN) dose-dependently inhibited calcification of HSMC cultured in vitro about as effectively as native OPN. The inhibitory effect of osteopontin depended on the extent of phosphorylation. To determine the specific structural domains of OPN important for inhibition of calcification, we compared OPN fragments (N-terminal, C-terminal, and full-length), and compared the inhibitory effect of both phosphorylated and non-phosphorylated fragments. While none of the non-phosphorylated OPN fragments effected calcification, P-OPN caused dose dependent inhibition of HSMC calcification. P-OPN was treated with alkaline phosphatase to create dephosphorylated OPN. Dephosphorylated OPN did not have an inhibitory effect on calcification. The expression of OPN mRNA and P-OPN secretion by HSMC were decreased in a time-dependent manner during culture calcification. These results indicate that phosphorylation is required for the inhibitory effect of OPN on HSMC calcification, and that regulation of OPN phosphorylation represents one way in which mineralization may be controlled by cells.
Assuntos
Cálcio/metabolismo , Músculo Liso Vascular/metabolismo , Sialoglicoproteínas/metabolismo , Sialoglicoproteínas/fisiologia , Fosfatase Alcalina/farmacologia , Animais , Animais Recém-Nascidos , Northern Blotting , Caseína Quinase II , Células Cultivadas , Relação Dose-Resposta a Droga , Humanos , Osteopontina , Fosforilação , Plasmídeos/metabolismo , Testes de Precipitina , Proteínas Serina-Treonina Quinases/farmacologia , RNA Mensageiro/metabolismo , Ratos , Proteínas Recombinantes/metabolismo , Sialoglicoproteínas/farmacologia , Fatores de TempoRESUMO
Extracellular matrix proteins play key roles in controlling the activities of osteoblasts and osteoclasts in bone remodeling. These bone-specific extracellular matrix proteins contain amino acid sequences that mediate cell adhesion, and many of the bone-specific matrix proteins also contain acidic domains that interact with the mineral surface and may orient the signaling domains. Here we report a fusion peptide design that is based on this natural approach for the display of signaling peptide sequences at biomineral surfaces. Salivary statherin contains a 15-amino acid hydroxyapatite binding domain (N15) that is loosely helical in solution. To test whether N15 can serve to orient active peptide sequences on hydroxyapatite, the RGD and flanking residues from osteopontin were fused to the C terminus. The fusion peptides bound tightly to hydroxyapatite, and the N15-PGRGDS peptide mediated the dose-dependent adhesion of Moalpha(v) melanoma cells when immobilized on the hydroxyapatite surface. Experiments with an integrin-sorted Moalpha(v) subpopulation demonstrated that the alpha(v)beta(3) integrin was the primary receptor target for the fusion peptide. Solid state NMR experiments showed that the RGD portion of the hydrated fusion peptide is highly dynamic on the hydroxyapatite surface. This fusion peptide framework may thus provide a straightforward design for immobilizing bioactive sequences on hydroxyapatite for biomaterials, tissue engineering, and vaccine applications.
Assuntos
Adesão Celular/efeitos dos fármacos , Durapatita/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas e Peptídeos Salivares/metabolismo , Sialoglicoproteínas/metabolismo , Adsorção , Sequência de Aminoácidos , Animais , Sítios de Ligação , Ligação Competitiva , Dicroísmo Circular , Humanos , Espectroscopia de Ressonância Magnética , Melanoma/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Oligopeptídeos/metabolismo , Osteopontina , Fragmentos de Peptídeos/farmacologia , Ligação Proteica , Estrutura Secundária de Proteína , Receptores de Vitronectina/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Proteínas e Peptídeos Salivares/química , Sialoglicoproteínas/química , Células Tumorais CultivadasRESUMO
Synthetic biomaterials are widely used in medical implants with success in improving and extending quality of life. However, these materials were not originally designed to interact with cells through specific signaling pathways. As a result, the interaction with the body is mediated through passive adsorption of a disorganized protein monolayer. Next generation biomaterials have been proposed to be active in modifying the biological response of the host through the incorporation of specific biorecognition moieties. An important tool in the development of these novel active biomaterials is the scanning force microscope (SFM). The SFM allows for interrogation of bioactive biomaterials in mapping or spectroscopic modes. In this work, micropatterned protein surfaces were prepared using biomolecules implicated in wound healing. The surfaces were imaged via SFM and the specific binding forces between surface associated biomolecules and antibody functionalized tips were quantified.
Assuntos
Materiais Biocompatíveis , Sialoglicoproteínas/química , Anticorpos/química , Microscopia de Força Atômica , Osteopontina , Sialoglicoproteínas/ultraestruturaRESUMO
UNLABELLED: Osteopontin expression in human crescentic glomerulonephritis. BACKGROUND: Osteopontin is a molecule with diverse biological functions, including cell adhesion, migration, and signaling. The expression of osteopontin has been demonstrated in a number of models of renal injury in association with accumulations of monocyte/macrophages, including recent reports of osteopontin expression in glomerular crescents in a rat model of anti-glomerular basement membrane glomerulonephritis. METHODS: Glomerular expression of osteopontin in biopsies of human crescentic glomerulonephritis (N = 25), IgA nephropathy with crescents (N = 2), and diffuse proliferative lupus glomerulonephropathy with crescents (N = 1) was studied by immunohistochemistry, in situ hybridization, and combined immunohistochemistry/in situ hybridization. Additionally, antibodies to cell-specific phenotypic markers were used to identify cellular components of the glomerular crescent, which express osteopontin protein and mRNA. RESULTS: All of the crescents present in the biopsies studied contained a significant number of cells that expressed osteopontin protein and mRNA, demonstrated by immunohistochemistry and in situ hybridization, respectively. Using replicate tissue sections and combined immunohistochemistry/in situ hybridization, we showed that the majority of the strongly osteopontin-positive cells are monocyte/macrophages. In addition to the very strong and cell-associated localization, a weaker and more diffuse pattern of osteopontin protein and mRNA expression could be seen in a number of crescents. None of the osteopontin mRNA-expressing cells could be identified as parietal epithelial cells, CD3-positive T cells, or alpha-smooth muscle actin-positive myofibroblasts. Interstitial monocyte/macrophages did not express osteopontin, except when located in a periglomerular inflammatory infiltrate. CONCLUSIONS: Macrophages present in the human glomerular crescent express osteopontin protein and mRNA at a high level. This expression supports a role for osteopontin in the formation and progression of the crescentic lesion via chemotactic and signaling properties of the molecule.
Assuntos
Glomerulonefrite/metabolismo , Sialoglicoproteínas/metabolismo , Humanos , Imuno-Histoquímica , Hibridização In Situ , Osteopontina , Fenótipo , RNA Mensageiro/genética , Sialoglicoproteínas/genéticaRESUMO
The transcription factor NF-kappa B is an important regulator of gene expression during immune and inflammatory responses, and can also protect against apoptosis. Here we show that endothelial cells undergo apoptosis when deprived of growth factors. Surviving viable cells exhibit increased activity of NF-kappa B, whereas apoptotic cells show caspase-mediated cleavage of the NF-kappa B p65/ReIA subunit. This cleavage leads to loss of carboxy-terminal transactivation domains and a transcriptionally inactive p65 molecule. The truncated p65 acts as a dominant-negative inhibitor of NF-kappa B, promoting apoptosis, whereas an uncleavable, caspase-resistant p65 protects the cells from apoptosis. The generation of a dominant-negative fragment of p65 during apoptosis may be an efficient pro-apoptotic feedback mechanism between caspase activation and NF-kappa B inactivation.
Assuntos
Apoptose/fisiologia , Caspases/metabolismo , NF-kappa B/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação/genética , Sobrevivência Celular , Células Cultivadas , Primers do DNA/genética , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Ativação Enzimática , Retroalimentação , Humanos , Dados de Sequência Molecular , Mutação , NF-kappa B/química , NF-kappa B/genética , Transdução de Sinais , Especificidade por Substrato , Fator de Transcrição RelA , Transcrição GênicaRESUMO
BACKGROUND: Osteopontin is a macrophage adhesive protein that is expressed by renal tubules in tubulointerstitial disease. METHODS: To investigate the function of OPN, we induced tubulointerstitial disease in OPN null mutant (OPN-/-) and wild-type (OPN+/+) mice by unilateral ureteral ligation. Tissue was analyzed for macrophages (ED-1), types I and IV collagen deposition, TGF-beta expression, and for tubular and interstitial cell apoptosis. RESULTS: Obstructed kidneys from both OPN-/- and OPN+/+ mice developed hydronephrosis, tubular atrophy, interstitial inflammation and fibrosis. OPN was absent in OPN-/- kidneys but was increased in obstructed OPN+/+ kidneys. Macrophage influx, measured by computer-assisted quantitative immunostaining, was less in OPN-/- mice compared to OPN+/+ mice at day 4 (threefold, P < 0.02), day 7 (fivefold, P < 0.02), but not at day 14. Interstitial deposition of types I and IV collagen were also two- to fourfold less in obstructed OPN-/- kidneys (P < 0.02). There was also a reduction of TGF-beta mRNA expression in the interstitium at day 7 (by in situ hybridization) and a near significant 34% reduction in cortical TGF-beta activity (P = 0.06) compared to obstructed OPN+/+ kidneys at day 14. Obstructed kidneys from OPN-/- mice also had more interstitial and tubular apoptotic cells (TUNEL assay) compared to obstructed OPN+/+ mice at all time points. The ability of OPN to act as a cell survival factor was also documented by showing that the apoptosis of serum-starved NRK52E renal epithelial cells was markedly enhanced in the presence of neutralizing anti-OPN antibody. CONCLUSION: OPN mediates early interstitial macrophage influx and interstitial fibrosis in unilateral ureteral obstruction. OPN may also function as a survival factor for renal tubulointerstitial cells.
Assuntos
Apoptose/imunologia , Sialoglicoproteínas/genética , Obstrução Ureteral/genética , Obstrução Ureteral/patologia , Animais , Sobrevivência Celular/fisiologia , Colágeno/análise , Colágeno/metabolismo , Modelos Animais de Doenças , Células Epiteliais/química , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Feminino , Fibrose , Hibridização In Situ , Marcação In Situ das Extremidades Cortadas , Túbulos Renais/química , Túbulos Renais/metabolismo , Túbulos Renais/fisiologia , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Knockout , Osteopontina , Fenótipo , RNA Mensageiro/análise , Sialoglicoproteínas/imunologia , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/imunologia , Fator de Crescimento Transformador beta/metabolismo , Obstrução Ureteral/imunologiaRESUMO
Vascular smooth muscle cells (SMCs) undergo a dramatic phenotypic transition in response to injury and ex vivo culture that includes enhanced proliferation, migration, matrix deposition, and alterations in gene expression. Osteopontin is a good marker for the injury-induced SMC phenotypic state in vivo and in vitro. To identify transcription factors that might control the regulation of osteopontin expression, we investigated cultured vascular SMCs that express high and low levels of osteopontin. Using nuclear run-on assays, mRNA stability studies, and deletion analysis, we demonstrate that regulation of osteopontin steady-state mRNA levels in SMCs occurs at the transcriptional level. Transient transfection and gel-shift analyses of osteopontin promoter indicated that a region between -123 and +66 was involved in the expression of osteopontin. Supershift EMSAs identified the bHLH-leucine zipper transcription factor upstream stimulatory factor-1 (USF1) as the protein binding to this sequence. Finally, we show that USF1 protein is induced in vivo within 24 h of balloon angioplasty of rat carotids coordinately with osteopontin induction. These data suggest that USF1 governs expression of osteopontin in cultured vascular SMCs and might contribute to initial osteopontin expression observed post carotid injury and in vascular pathologies in vivo.