RESUMO
Elevated blood concentrations of lipoprotein(a) [Lp(a)] and its constituent, apolipoprotein(a) [apo(a)], constitute a major risk factor for atherosclerosis, but their physiological activities remain obscure. Lp(a) and purified apo(a) stimulated the growth of human smooth muscle cells in culture. This effect resulted from inhibition of plasminogen activation, and consequently the activation by plasmin of latent transforming growth factor-beta, which is an inhibitor of smooth muscle cell growth. Because smooth muscle proliferation is one of the hallmarks of atherosclerotic lesions, these results point to a plausible mechanism for the atherogenic activity of Lp(a).
Assuntos
Lipoproteína(a)/fisiologia , Músculo Liso Vascular/citologia , Animais , Apolipoproteínas/fisiologia , Apoproteína(a) , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Células Cultivadas , Fibrinolisina/fisiologia , Humanos , Músculo Liso Vascular/metabolismo , Ativadores de Plasminogênio/metabolismo , Ratos , Tamoxifeno/farmacologia , Fator de Crescimento Transformador beta/fisiologiaRESUMO
Calcification is common in atheromatous plaques and may contribute to plaque rupture and subsequent thrombosis. However, little is known about the mechanisms which regulate the calcification process. Using in situ hybridization and immunohistochemistry we show that two bone-associated proteins, osteopontin (OP) and matrix Gla protein (MGP), are highly expressed in human atheromatous plaques. High levels of OP mRNA and protein were found in association with necrotic lipid cores and areas of calcification. The predominant cell type in these areas was the macrophage-derived foam cell, although some smooth muscle cells could also be identified. MGP was expressed uniformly by smooth muscle cells in the normal media and at high levels in parts of the atheromatous intima. Highest levels of this matrix-associated protein were found in lipid-rich areas of the plaque. The pattern of expression of these two genes contrasted markedly with that of calponin and SM22 alpha, genes expressed predominantly by differentiated smooth muscle cells and whose expression was generally confined to the media of the vessel. The postulated function of OP and MGP as regulators of calcification in bone and the high levels and colocalization of both in atheromatous plaques suggest they have an important role in plaque pathogenesis and stability.
Assuntos
Arteriosclerose/metabolismo , Calcinose/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteínas da Matriz Extracelular , Músculo Liso Vascular/metabolismo , Sialoglicoproteínas/genética , Proteínas de Ligação ao Cálcio/fisiologia , Diferenciação Celular , Humanos , Imuno-Histoquímica , Proteínas dos Microfilamentos , Músculo Liso Vascular/citologia , Osteopontina , RNA Mensageiro/análise , Sialoglicoproteínas/fisiologia , Calponinas , Proteína de Matriz GlaRESUMO
BACKGROUND: Matrix Gla protein (MGP) is a small vitamin K-dependent protein containing five gamma-carboxyglutamic acid (Gla) residues that are believed to be important in binding Ca(2+), calcium crystals and bone morphogenetic protein. In addition, MGP contains phosphorylated serine residues that may further regulate its activity. In vivo, MGP has been shown to be a potent inhibitor of vascular calcification; however, the precise molecular mechanism underlying the function of MGP is not yet fully understood. METHODS AND RESULTS: We investigated the effects of MGP in human vascular smooth muscle cell (VSMC) monolayers that undergo calcification after exposure to an increase in Ca(2+) concentration. Increased calcium salt deposition was found in cells treated with the vitamin K antagonist warfarin as compared to controls, whereas cells treated with vitamin K(1) showed decreased calcification as compared to controls. With conformation-specific antibodies, it was confirmed that warfarin treatment of VSMCs resulted in uncarboxylated (Gla-deficient) MGP. To specifically test the effects of MGP on VSMC calcification, we used full-length synthetic MGP and MGP-derived peptides representing various domains in MGP. Full length MGP, the gamma-carboxylated motif (Gla) (amino acids 35-54) and the phosphorylated serine motif (amino acids 3-15) inhibited calcification. Furthermore, we showed that the peptides were not taken up by VSMCs but bound to the cell surface and to vesicle-like structures. CONCLUSIONS: These data demonstrate that both gamma-glutamyl carboxylation and serine phosphorylation of MGP contribute to its function as a calcification inhibitor and that MGP may inhibit calcification via binding to VSMC-derived vesicles.
Assuntos
Calcinose/prevenção & controle , Proteínas de Ligação ao Cálcio/metabolismo , Cálcio/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Processamento de Proteína Pós-Traducional , Ácido 1-Carboxiglutâmico/metabolismo , Adolescente , Adulto , Sequência de Aminoácidos , Calcinose/metabolismo , Proteínas de Ligação ao Cálcio/química , Membrana Celular/metabolismo , Células Cultivadas , Proteínas da Matriz Extracelular/química , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Fragmentos de Peptídeos/metabolismo , Fosforilação , Estrutura Terciária de Proteína , Serina/metabolismo , Vesículas Transportadoras/metabolismo , Vitamina K/antagonistas & inibidores , Vitamina K/metabolismo , Vitamina K 1/farmacologia , Varfarina/farmacologia , Proteína de Matriz GlaRESUMO
The mechanisms involved in the initiation of vascular calcification are not known, but matrix vesicles, the nucleation sites for calcium crystal formation in bone, are likely candidates, because similar structures have been found in calcified arteries. The regulation of matrix vesicle production is poorly understood but is thought to be associated with apoptotic cell death. In the present study, we investigated the role of apoptosis in vascular calcification. We report that apoptosis occurs in a human vascular calcification model in which postconfluent vascular smooth muscle cell (VSMC) cultures form nodules spontaneously and calcify after approximately 28 days. Apoptosis occurred before the onset of calcification in VSMC nodules and was detected by several methods, including nuclear morphology, the TUNEL technique, and external display of phosphatidyl serine. Inhibition of apoptosis with the caspase inhibitor ZVAD.fmk reduced calcification in nodules by approximately 40%, as measured by the cresolphthalein method and alizarin red staining. In addition, when apoptosis was stimulated in nodular cultures with anti-Fas IgM, there was a 10-fold increase in calcification. Furthermore, incubation of VSMC-derived apoptotic bodies with (45)Ca demonstrated that, like matrix vesicles, they can concentrate calcium. These observations provide evidence that apoptosis precedes VSMC calcification and that apoptotic bodies derived from VSMCs may act as nucleating structures for calcium crystal formation.
Assuntos
Apoptose , Calcinose/metabolismo , Músculo Liso Vascular/metabolismo , Clorometilcetonas de Aminoácidos/farmacologia , Antraquinonas , Calcinose/tratamento farmacológico , Calcinose/patologia , Cálcio/metabolismo , Radioisótopos de Cálcio , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Corantes , Cicloeximida/farmacologia , Microanálise por Sonda Eletrônica , Humanos , Imunoglobulina M/farmacologia , Marcação In Situ das Extremidades Cortadas , Antígeno Ki-67/metabolismo , Músculo Liso Vascular/citologia , Músculo Liso Vascular/patologia , Inibidores da Síntese de Proteínas/farmacologia , Receptor fas/imunologiaRESUMO
Despite Fas expression, many cells resist Fas-induced apoptosis. Although differences in surface Fas expression can explain Fas resistance, multiple proteins below receptor level also inhibit Fas-induced apoptosis. To examine the mechanism of Fas resistance, we studied Fas-induced apoptosis in human medial vascular smooth muscle cells (VSMCs) from healthy coronary arteries. VSMCs showed marked heterogeneity to Fas-induced apoptosis, exhibiting both Fas-resistant (98.1+/-2.3% viable, n = 4, P = NS) and Fas-sensitive (31.3+/-2.6% viable, n = 3, P<0.01) cells. Fas-resistant VSMCs expressed surface Fas and could recruit RIP, indicating that functional receptor complexes were formed. However, Fas-resistant cells showed reduced expression of FADD, Fas ligand, and caspases 3, 7, and 8 and increased expression of FLIP and c-IAP-1. Fas-induced apoptosis was associated with cleavage of caspase 3 and blocked by inhibitors of caspase 3 or 8 but not caspase 1, 6, or 7. Selective inhibition of caspase 3 or 8 by antisense transfection inhibited Fas-induced apoptosis, but their reexpression could not rescue the Fas-resistant phenotype. In vivo, medial VSMCs showed marked heterogeneity of expression of caspase 3. We conclude that Fas sensitivity is determined not only by expression of surface Fas but by differential expression of Fas-signaling proteins below receptor level. Subpopulations of cells within the same tissue have different sensitivities to apoptosis, determined by expression of specific death-signaling proteins.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Apoptose/fisiologia , Músculo Liso Vascular/fisiologia , Receptor fas/fisiologia , Arteriosclerose/metabolismo , Proteínas de Transporte/metabolismo , Caspase 3 , Caspase 8 , Caspase 9 , Caspases/metabolismo , Células Cultivadas , Meios de Cultura Livres de Soro , Resistência a Medicamentos , Proteína de Domínio de Morte Associada a Fas , Humanos , Músculo Liso Vascular/citologia , Transdução de Sinais , Receptor fas/metabolismoRESUMO
Apoptosis of vascular smooth muscle cells (VSMCs) is increased in atherosclerosis compared with normal vessels, where it may contribute to plaque rupture. We have previously found that human plaque-derived VSMCs (pVSMCs) are intrinsically sensitive to apoptosis and not responsive to the protective effects of insulin-like growth factor-1 (IGF-1). We therefore examined the mechanism underlying this defect. Human pVSMCs showed <25% (125)I-IGF-1 surface binding, <20% IGF-1 receptor (IGF-1R) expression than that of normal medial VSMCs, and <40% Akt kinase activity in response to IGF-1. pVSMCs expressed and secreted high levels of IGF-1 binding proteins (IGFBPs), and the IGF-1 analogues, long R3 and Des 1,3 IGF-1, which do not bind to IGFBPs, were able to increase pVSMC survival to normal medial VSMC levels. The long R3 survival effect was phosphatidylinositol 3-kinase-mediated, but it was not dependent on Akt activity alone. Intimal pVSMCs in vivo showed reduced IGF-1R expression compared with medial VSMCs, in particular at the shoulder regions of plaques. We conclude that human pVSMCs show an intrinsic sensitivity to apoptosis caused in part by defective expression of IGF-1R, impaired IGF-1-mediated survival signaling and increased IGFBP secretion. This impaired IGF-1 protection against apoptosis may promote VSMC loss and plaque instability in atherosclerosis.
Assuntos
Arteriosclerose/patologia , Sobrevivência Celular/efeitos dos fármacos , Fator de Crescimento Insulin-Like I/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Proteínas Serina-Treonina Quinases , Androstadienos/farmacologia , Apoptose/efeitos dos fármacos , Arteriosclerose/metabolismo , Arteriosclerose/prevenção & controle , Células Cultivadas , Relação Dose-Resposta a Droga , Humanos , Imuno-Histoquímica , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Ligação Proteica , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Ensaio Radioligante , Receptor IGF Tipo 1/metabolismo , Receptor IGF Tipo 2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , WortmaninaRESUMO
BACKGROUND: Vascular smooth muscle cells (VSMCs) in atherosclerotic lesions display an osteogenic phenotype, and calcification commonly occurs in association with lipid. We therefore tested the hypothesis that lipid components in atherosclerotic lesions influenced VSMC phenotype and calcification using an in vitro model of calcification. METHODS AND RESULTS: In situ hybridization of human atherosclerotic plaques (n=10) collected from patients undergoing carotid endarterectomy demonstrated that subsets of lipid-filled VSMCs adjacent to sites of calcification expressed alkaline phosphatase, bone Gla protein, and bone sialoprotein, suggesting an osteogenic phenotype. Treatment of VSMCs in culture with acetylated low-density lipoprotein (acLDL) or lipoprotein-deficient serum altered the time course of bone-associated protein gene expression and calcification. AcLDL increased nodule calcification 3-fold, whereas lipoprotein-deficient serum significantly inhibited it. Reverse transcriptase-polymerase chain reaction and Western analysis demonstrated the presence of the acLDL receptor, SRA1, exclusively in calcifying nodular VSMCs, and blockade of SRA with polyinosinic acid inhibited acLDL-induced calcification. Because apoptotic bodies can serve as nucleation sites for calcification, we investigated whether acLDL could stimulate apoptosis in nodules. Apoptosis of nodular VSMCs was unaltered, but the number of apoptotic bodies per nodule increased approximately 3-fold, implying a defect in phagocytosis. Consistent with these observations, binding of apoptotic bodies to VSMCs was decreased in the presence of acLDL. CONCLUSIONS: These studies suggest that modified lipoproteins stimulate calcification by enhancing osteogenic differentiation of VSMCs and by a novel mechanism whereby acLDL interacts with SRA on VSMCs and blocks phagocytic removal of apoptotic bodies.
Assuntos
Calcinose/induzido quimicamente , Diferenciação Celular/efeitos dos fármacos , Lipoproteínas LDL/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Osteoblastos/patologia , Adolescente , Adulto , Idoso , Antígenos de Diferenciação/biossíntese , Apoptose/efeitos dos fármacos , Calcinose/patologia , Doenças das Artérias Carótidas/metabolismo , Doenças das Artérias Carótidas/patologia , Células Cultivadas , Meios de Cultura/farmacologia , Humanos , Hibridização In Situ , Técnicas In Vitro , Metabolismo dos Lipídeos , Lipoproteínas/deficiência , Lipoproteínas/farmacologia , Masculino , Pessoa de Meia-Idade , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Poli I/farmacologia , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Receptores Imunológicos/antagonistas & inibidores , Receptores Depuradores , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: Calcification of the media of peripheral arteries is referred to as Mönckeberg's sclerosis (MS) and occurs commonly in aged and diabetic individuals. Its pathogenesis is unknown, but its presence predicts risk of cardiovascular events and leg amputation in diabetic patients. Several studies have documented expression of bone-associated genes in association with intimal atherosclerotic calcification, leading to the suggestion that vascular calcification may be a regulated process with similarities to developmental osteogenesis. Therefore, we examined gene expression in vessels with MS to determine whether there was evidence for a regulated calcification process in the vessel media. METHODS AND RESULTS: In situ hybridization, immunohistochemistry, and semiquantitative reverse-transcription polymerase chain reaction were used to examine the expression of mineralization-regulating proteins in human peripheral arteries with and without MS. MS occurred in direct apposition to medial vascular smooth muscle cells (VSMCs) in the absence of macrophages or lipid. These VSMCs expressed the smooth muscle-specific gene SM22alpha and high levels of matrix Gla protein but little osteopontin mRNA. Compared with normal vessels, vessels with MS globally expressed lower levels of matrix Gla protein and osteonectin, whereas alkaline phosphatase, bone sialoprotein, bone Gla protein, and collagen II, all indicators of osteogenesis/chondrogenesis, were upregulated. Furthermore, VSMCs derived from MS lesions exhibited osteoblastic properties and mineralized in vitro. CONCLUSIONS: These data indicate that medial calcification in MS lesions is an active process potentially orchestrated by phenotypically modified VSMCs.
Assuntos
Artérias/metabolismo , Calcinose/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteínas da Matriz Extracelular , Músculo Liso Vascular/citologia , Sialoglicoproteínas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Artérias/patologia , Proteínas Morfogenéticas Ósseas/análise , Proteínas Morfogenéticas Ósseas/genética , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/análise , Células Cultivadas , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Músculo Liso Vascular/metabolismo , Osteopontina , Esclerose , Sialoglicoproteínas/análise , Proteína de Matriz GlaRESUMO
BACKGROUND: Atherosclerotic plaque rupture is usually a consequence of inflammatory cell activity within the plaque. Current imaging techniques provide anatomic data but no indication of plaque inflammation. The glucose analogue [18F]-fluorodeoxyglucose (18FDG) can be used to image inflammatory cell activity non-invasively by PET. In this study we tested whether 18FDG-PET imaging can identify inflammation within carotid artery atherosclerotic plaques. METHODS AND RESULTS: Eight patients with symptomatic carotid atherosclerosis were imaged using 18FDG-PET and co-registered CT. Symptomatic carotid plaques were visible in 18FDG-PET images acquired 3 hours post-18FDG injection. The estimated net 18FDG accumulation rate (plaque/integral plasma) in symptomatic lesions was 27% higher than in contralateral asymptomatic lesions. There was no measurable 18FDG uptake into normal carotid arteries. Autoradiography of excised plaques confirmed accumulation of deoxyglucose in macrophage-rich areas of the plaque. CONCLUSIONS: This study demonstrates that atherosclerotic plaque inflammation can be imaged with 18FDG-PET, and that symptomatic, unstable plaques accumulate more 18FDG than asymptomatic lesions.
Assuntos
Arteriosclerose/diagnóstico por imagem , Doenças das Artérias Carótidas/diagnóstico por imagem , Fluordesoxiglucose F18 , Compostos Radiofarmacêuticos , Tomografia Computadorizada de Emissão/métodos , Idoso , Arteriosclerose/metabolismo , Arteriosclerose/patologia , Doenças das Artérias Carótidas/metabolismo , Doenças das Artérias Carótidas/patologia , Feminino , Fluordesoxiglucose F18/farmacocinética , Humanos , Inflamação/diagnóstico por imagem , Inflamação/metabolismo , Masculino , Pessoa de Meia-Idade , Compostos Radiofarmacêuticos/farmacocinética , Tomografia Computadorizada por Raios XRESUMO
Human atherosclerotic plaques that rupture are characterized by relatively low vascular smooth muscle cell (VSMC) and high inflammatory cell contents. Ruptured plaques also contain higher numbers of apoptotic VSMCs than do stable lesions, suggesting that VSMC apoptosis may promote plaque rupture. We examined the ability of human monocytes/macrophages to induce apoptosis of VSMCs derived from human carotid plaque, aortic media, and coronary media. Macrophages, but not T lymphocytes, induced a dose-dependent apoptosis of VSMCs, which required monocyte maturation to macrophages and direct cell-cell contact/proximity. VSMC apoptosis was inhibited by neutralizing antibodies to Fas-ligand (Fas-L) or an Fas-Fc fusion protein, indicating the requirement for membrane-bound Fas and Fas-L. Monocyte maturation was associated with increased surface expression of Fas-L, coincident with the onset of cytotoxicity. VSMCs expressed surface Fas, which was increased in plaque VSMCs, and plaque VSMCs also underwent Fas-induced apoptosis. We conclude that human macrophages potently induce human VSMC apoptosis, which requires direct cell-cell interactions and is in part dependent on Fas/Fas-L interactions. Macrophage-induced VSMC apoptosis may therefore directly promote plaque rupture.
Assuntos
Apoptose , Arteriosclerose/patologia , Macrófagos/imunologia , Glicoproteínas de Membrana/metabolismo , Músculo Liso Vascular/patologia , Receptor fas/metabolismo , Aorta/citologia , Arteriosclerose/imunologia , Células Sanguíneas/imunologia , Sobrevivência Celular , Células Cultivadas , Proteína Ligante Fas , Humanos , Inflamação/imunologia , Inflamação/patologia , Monócitos/imunologia , Músculo Liso Vascular/metabolismo , Linfócitos T/imunologiaRESUMO
Chemokines are important mediators of macrophage and T-cell recruitment in a number of inflammatory pathologies, and chemokines expressed in atherosclerotic lesions may play an important role in mononuclear cell recruitment and macrophage differentiation. We have analyzed the expression of the linked chromosome 16q13 genes that encode macrophage-derived chemokine (MDC/CCL22), thymus- and activation-regulated chemokine (TARC/CCL17), and the CX(3)C chemokine fractalkine (CX(3)CL1) in primary macrophages and human atherosclerotic lesions by reverse transcription-polymerase chain reaction and immunohistochemistry. We show that macrophage expression of the chemokines MDC, fractalkine, and TARC is upregulated by treatment with the Th2-type cytokines interleukin-4 and interleukin-13. High levels of MDC, TARC, and fractalkine mRNA expression are seen in some, but not all, human arteries with advanced atherosclerotic lesions. Immunohistochemistry shows that MDC, fractalkine, and TARC are expressed by a subset of macrophages within regions of plaques that contain plaque microvessels. We conclude that MDC, fractalkine, and TARC, which are chromosome 16q13 chemokines, could play a role in mononuclear cell recruitment into atherosclerotic lesions and influence the subsequent inflammatory response. Macrophage-expressed chemokines upregulated by interleukin-4 may be useful surrogate markers for the presence of Th2-type immune responses in human atherosclerotic lesions.
Assuntos
Arteriosclerose/metabolismo , Quimiocinas CC/genética , Quimiocinas CX3C/genética , Cromossomos Humanos Par 16 , Macrófagos/imunologia , Proteínas de Membrana/genética , Adolescente , Adulto , Idoso , Artérias/metabolismo , Arteriosclerose/patologia , Biomarcadores/análise , Técnicas de Cultura de Células , Quimiocina CCL17 , Quimiocina CCL22 , Quimiocina CX3CL1 , Quimiocinas CC/biossíntese , Quimiocinas CC/fisiologia , Quimiocinas CX3C/biossíntese , Quimiocinas CX3C/fisiologia , Quimiotaxia de Leucócito , Células Dendríticas/metabolismo , Feminino , Ligação Genética , Humanos , Interleucinas/farmacologia , Masculino , Proteínas de Membrana/biossíntese , Proteínas de Membrana/fisiologia , Pessoa de Meia-Idade , RNA Mensageiro/biossíntese , Células Th2/imunologia , Regulação para CimaRESUMO
The effect of reducing systemic arterial pressure with an arteriovenous fistula on left and right ventricular myocardial blood flow was studied in 17 anaesthetised, open chest, autonomically blocked dogs. Global and regional myocardial blood flows were measured with radioactive microspheres. As mean arterial pressure was reduced from 133 mmHg to 78 mmHg left ventricular myocardial blood flow and the left ventricular inner to outer flow ratio decreased progressively. By contrast, right ventricular myocardial blood flow remained constant (range 78-81 ml.min-1.100 g-1) whereas right ventricular vascular resistance fell linearly (from 235 to 130 kPa.litre-1.min.100 g-1). The inner to outer right ventricular free wall flow ratio (range 1.04-1.10) and blood flow to the right side of the interventricular septum also did not change significantly. It is concluded that the right ventricular myocardium shows effective autoregulation of total and regional tissue blood flow during changes in coronary perfusion pressure.
Assuntos
Velocidade do Fluxo Sanguíneo , Pressão Sanguínea , Circulação Coronária , Ventrículos do Coração/fisiopatologia , Animais , Vasos Coronários/fisiopatologia , Cães , Hemodinâmica , Resistência Vascular , Obstrução do Fluxo Ventricular ExternoRESUMO
OBJECTIVES: Various heparins have been reported to inhibit the proliferation of vascular smooth muscle cells (VSMCs). The effects of eight chemically distinct heparins on the cell cycle and differentiation of primary and passaged cultures of rat aortic VSMCs have been characterised and the mechanism of heparin action investigated. METHODS: VSMCs from adult rat aorta were prepared by enzyme dispersion and stimulated to enter the cell cycle with 10% serum in the presence or absence of heparin. Progressions through S phase and M phase were measured by [3H]-thymidine incorporation and cell counting respectively. Flow cytometry was used to confirm the effects of heparin on VSMC cell cycle progression. The effect of heparin on VSMC differentiation was investigated by analysing smooth muscle specific myosin heavy chain content of the cells after heparin treatment. RESULTS: Eight heparins at concentrations between 5 micrograms.ml-1 and 100 micrograms.ml-1 partially inhibited VSMC proliferation (27% to 76% 96 hours after addition of heparin), but did not affect the entry of the cells into S phase. Flow cytometry confirmed that VSMC populations in the presence of heparin contained significantly (p < 0.005) more cells in the G2/M phase of the cell cycle than control populations. Heparin also blocked the dedifferentiation of primary cultures of VSMCs stimulated by serum. These effects of heparin were completely reversed by the presence of a neutralising antiserum to transforming growth factor beta (TGF beta) and heparin attached to agarose beads was as effective as free heparin as a growth inhibitor of VSMCs. CONCLUSIONS: Heparins of varying molecular weight and anticoagulant properties all partially inhibited VSMC proliferation predominantly by extending the G2/M phase of the cell cycle. Heparin also inhibited dedifferentiation of primary cultures of VSMCs. Heparin (< 100 micrograms.ml-1) acted extracellularly to release TGF beta from serum, which accounted for the effects of heparin on proliferation and differentiation.
Assuntos
Heparina/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Fator de Crescimento Transformador beta/sangue , Animais , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Meios de Cultura , Citometria de Fluxo , Músculo Liso Vascular/citologia , RatosRESUMO
Abnormal proliferation of VSMC is a feature of atheromatous plaques and is responsible for obstructive neointimal lesions at the sites of mechanical or immunological intimal damage. We have discussed approaches to the development of specific inhibitors of VSMC proliferation based firstly on studies of the mechanism of action of known inhibitors and secondly on the identification of genes which are unique to and necessary for VSMC proliferation. The effects of angiotensin converting enzyme inhibitors, heparin and hexamethylene bisacetamide on VSMC proliferation have been discussed. Each of these types of agent has been shown to inhibit VSMC proliferation in vitro and/or in vivo. Examination of the mechanisms by which these agents inhibit VSMC proliferation reveals that transforming growth factor beta is an important mediator of their action and that the processes of de-differentiation and proliferation are independently regulated. Studies aimed at identifying genes involved in VSMC proliferation are at an early stage but have already provided strong evidence to support the hypothesis that VSMC in the vessel wall are heterogeneous and contain a subpopulation of cells with an enhanced proliferative capacity. Identification of the genes expressed by these cells may allow specific inhibitors of VSMC proliferation to be developed and may shed light on the pathogenesis of neointimal lesions.
Assuntos
Inibidores da Enzima Conversora de Angiotensina/farmacologia , Músculo Liso Vascular/citologia , Acetamidas/farmacologia , Animais , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Hematínicos/farmacologia , Heparina/farmacologia , Humanos , RatosRESUMO
OBJECTIVE: When vascular smooth muscle cells are dispersed into culture in the presence of serum they modulate their phenotype. The aim of this study was to determine the range of growth factors likely to stimulate replication of medial vascular smooth muscle cells in vivo by examining their responses when cultured in the absence of serum. METHODS: Freshly dispersed vascular smooth muscle cells from the healthy aortic media of adult rats were prepared and plated out in cell culture in the absence of fetal calf serum. DNA synthesis by these cells when plated onto fibronectin in response to various growth factors and vasoconstrictors was analysed by [3H]-thymidine incorporation. RESULTS: Less than 5% of cells plated on culture plastic spread, but 15-25% of cells plated onto fibronectin spread and survived for at least 8 d in culture. These cells responded to platelet derived growth factor BB homodimer (PDGF-BB), basic fibroblast growth factor, and epidermal growth factor by stimulating DNA synthesis at least 10-fold compared with cells in the absence of growth factors. Maximum rate of DNA synthesis occurred 36-42 h after addition of 10% fetal calf serum, whereas maximum rate of DNA synthesis occurred 80-88 h after stimulation with PDGF-BB or basic fibroblast growth factor. By contrast, PDGF-AA homodimer, transforming growth factor type beta, insulin-like growth factor I, angiotensin II, and endothelin I did not stimulate DNA synthesis by more than threefold. CONCLUSIONS: Freshly dispersed vascular smooth muscle cells plated onto fibronectin in the absence of serum proliferate in response to PDGF-BB, basic fibroblast growth factor, and epidermal growth factor by stimulating DNA synthesis. The range of mitogens and the time course of entry into DNA synthesis under these culture conditions suggest that serum-free culture provides a good model for the responses of medial vascular smooth muscle cells in vivo.
Assuntos
Substâncias de Crescimento/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Animais , Aorta/citologia , Aorta/metabolismo , Becaplermina , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Meios de Cultura Livres de Soro , DNA/biossíntese , Fator de Crescimento Epidérmico/farmacologia , Fibronectinas , Masculino , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Fator de Crescimento Derivado de Plaquetas/farmacologia , Proteínas Proto-Oncogênicas c-sis , Ratos , Ratos WistarRESUMO
The effect of one hour (40 mmHg) lower body positive pressure on blood pressure, plasma atrial natriuretic factor concentration, and urinary sodium and water excretion was studied in 10 healthy volunteers and seven cardiac transplant recipients. Both groups showed a sustained rise in blood pressure and plasma atrial natriuretic factor concentration. The healthy volunteers had a diuresis during the period of lower body positive pressure and a small natriuresis in the subsequent hour. In contrast, lower body positive pressure had no significant effect on urinary sodium and water excretion in the cardiac transplant group. The data suggest that cardiac innervation is not important as a mediator of the haemodynamic response to lower body positive pressure but is necessary for the renal response. Furthermore, small physiological rises in plasma atrial natriuretic factor concentrations do not cause a brisk natriuresis as has been reported with pharmacological plasma concentrations.
Assuntos
Fator Natriurético Atrial/sangue , Pressão Sanguínea , Trajes Gravitacionais , Transplante de Coração , Sódio/urina , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Pressão , MicçãoRESUMO
OBJECTIVE: Medial vascular smooth muscle cells (VSMCs) in healthy vessels are phenotypically distinct from their intimal counterparts in vascular disease. To compare the genes expressed in these phenotypes we have previously performed a differential cDNA library screen on cultured rat VSMCs. The aim of this study was to identify and characterise a 2.8 kb transcript, 2E10, which was highly expressed in freshly dispersed rat aortic VSMCs and downregulated in multiply passaged cultured VSMCs. METHODS: Sequence analysis was used to identify the 2.8 kb rat cDNA. After trypsinisation of proliferating cultured rat and human VSMCs, or enzymatic digestion of aortic tunica media, total cytoplasmic RNA was isolated from VSMCs by lysis in Nonidet P-40 and extraction in phenol; 15 micrograms of total cytoplasmic RNA was used in Northern blot analysis with a 32P-[dCTP]-labelled 2E10 cDNA probe. 35S-[dATP]-labelled 2E10 riboprobe was hybridised in situ to frozen sections of normal and diseased human coronary arteries. RESULTS: DNA sequencing identified 2E10 as a rat polyubiquitin which is homologous to the human polyubiquitin, UbC. Northern blot analysis showed that this polyubiquitin was more highly expressed in differentiated, freshly dispersed rat and human aortic VSMCs compared with their dedifferentiated proliferating counterparts. This also identified a 3.2 kb transcript cross-reacting with the polyubiquitin probe which is specific to differentiated rat VSMCs only. However, expression in growth arrested and proliferating VSMCs was identical, suggesting that UbC does not have a role in VSMC growth arrest. In situ hybridisation of the polyubiquitin riboprobe to sections of diseased human coronary arteries indicated much higher expression in medial than in intimal VSMCs. Northern blot analysis of RNA from the developing rat aorta showed that polyubiquitin expression increased substantially after week 2 of neonatal life, coincident with expression of VSMC-specific contractile proteins. CONCLUSIONS: The greater expression of a UbC polyubiquitin transcript in contractile, differentiated VSMCs compared with proliferating, synthetic VSMCs provides a new gene marker for the phenotypic characterisation of VSMCs in vivo. This, and the finding that the developmental induction of expression of polyubiquitin (UbC) mirrors that of VSMC contractile proteins, suggests that ubiquitin, a protein known to associate with and degrade contractile proteins in skeletal muscle, is involved in the function or maintenance of the contractile phenotype of VSMCs.
Assuntos
Biopolímeros/genética , DNA/análise , Músculo Liso Vascular/química , Ubiquitinas/genética , Sequência de Aminoácidos , Animais , Aorta , Sequência de Bases , Northern Blotting , Células Cultivadas , Marcadores Genéticos , Hibridização In Situ , Dados de Sequência Molecular , Poliubiquitina , Ratos , Ratos Wistar , Análise de Sequência de DNARESUMO
OBJECTIVE: Human vascular smooth muscle cells (VSMC) prepared by enzyme dispersal have been reported to proliferate more slowly and remain more differentiated than VSMC derived by explant culture. The aim of this study was to investigate whether these differences are attributable to the production of transforming growth factor beta (TGF beta), a major modulator of VSMC properties in culture, by enzyme dispersal, but not explant, cultures. METHODS: Human aortic VSMC cultures were prepared by enzymatic dissociation of aortic media or by outgrowth of cells from explants derived from the vessel media. Properties, including rate of proliferation and differentiation, of the two different types of culture obtained from 10 donors (age 3-54 years, of either sex) were compared. The role of TGF beta as mediator of these differences was investigated. RESULTS: VSMC from enzyme dispersal cultures proliferated with a long doubling time in 20% fetal calf serum of 68(SEM 2) h, reaching a low saturation density with no "hills and valleys", and the cells were predominantly of stellate morphology. VSMC from explant cultures proliferated with a shorter doubling time of 35(2) h, reached a higher saturation density with "hills and valleys", and showed a spindle shaped morphology. The enzyme dispersed cells, but not the explant cells, released TGF beta into the medium. Addition of exogenous TGF beta to the explant cells extended the doubling time, while addition of a neutralising antibody to the enzyme dispersed cells decreased the doubling time. Tamoxifen, an anti-oestrogenic drug, decreased the rate of proliferation of the explant cells (ED50 = 50 nM; n = 3) but not enzyme dispersed cells by stimulating production of TGF beta. The explant and enzyme dispersal cultures also differed in response to growth factors. The explant cultures stimulated DNA synthesis in response to platelet derived growth factor (PDGF) AA and BB. Enzyme dispersal cultures stimulated DNA synthesis in response to PDGF-BB but response to AA was weaker and also variable. CONCLUSIONS: Human explant derived VSMC systematically differ from enzyme dispersed VSMC obtained from the same donor tissue. Wherever the potential role of TGF beta was investigated, the differences between enzyme dispersal and explant cultures were due to autocrine production of TGF beta by the enzyme dispersal, but not explant cultures.
Assuntos
Músculo Liso Vascular/citologia , Fator de Crescimento Transformador beta/farmacologia , Adolescente , Adulto , Aorta , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Criança , Pré-Escolar , DNA/biossíntese , Feminino , Heparina/farmacologia , Humanos , Masculino , Microscopia de Contraste de Fase , Pessoa de Meia-Idade , Músculo Liso Vascular/metabolismo , Fator de Crescimento Derivado de Plaquetas/farmacologia , Tamoxifeno/farmacologiaRESUMO
OBJECTIVES: The aim was to determine the mechanisms, particularly bicarbonate dependent mechanisms, of intracellular pH (pHi) recovery from various acidoses in vascular smooth muscle and to explore the ATP dependency of the respective mechanisms. METHODS: Experiments were conducted in rat aortic smooth muscle cells grown in primary culture and synchronised in a non-growing state by serum deprivation. pHi was measured in cells loaded with the pH sensitive fluorescent dye, 2',7'-bis-(2-carboxyethyl)-5-(and 6)-carboxyfluorescein (BCECF). Chloride efflux was studied by determination of the rate of efflux of 36Cl over 5 min. Cells were ATP depleted by substitution of glucose in the medium by 2-deoxyglucose. Acidoses were induced by CO2 influx and NH3 efflux techniques. RESULTS: In the absence of HCO3-, the 5-(N-ethyl-N-isopropyl) amiloride (EIPA) sensitive Na+/H+ exchange accounted for the recovery from intracellular acidosis. In the presence of HCO3- ions the response to respiratory acidosis (CO2 influx) was predominantly via activation of Na+/H+ exchange and an EIPA sensitive Na+ and HCO3- dependent mechanism. A 4-acetamido-4'-isothiocyanostilbene-2',2'-sulphonic acids (SITS) sensitive Na+ dependent Cl-/HCO3- mechanism which is also sensitive to EIPA makes a small contribution during severe intracellular acidosis. Under such conditions HCO3- dependent mechanisms contributed about 40% to the overall pHi regulating capacity of vascular smooth muscle cells. However, under conditions which deplete cellular ATP these pHi regulating mechanisms account for virtually all of theses cells' ability to regulate pHi. The inability of Na+/H+ exchange to participate in pHi recovery under these circumstances, reduces the ability of vascular smooth muscle cells to recover pHi by approximately 50-60%. Chloride efflux was approximately linear over 5 min and was increased by 36% in the presence of extracellular HCO3-. Efflux in the presence of HCO3- was inhibited similarly by both SITS and EIPA. CONCLUSIONS: At least three transporters contribute to recovery from acidosis in vascular smooth muscle: Na+/H+ exchange, an Na(+)-HCO3- cotransporter which is sensitive to EIPA, and an Na+ dependent HCO3-/Cl- exchange sensitive to both SITS and EIPA. The Na(+)-HCO3- cotransporter appears to be similar to that described in human vascular smooth muscle. When the Na+/H+ exchanger is attenuated by cellular ATP depletion, the alternative pathways, particularly the Na(+)-HCO3- cotransporter, ensure that substantial pHi regulatory capacity is maintained.
Assuntos
Bicarbonatos/metabolismo , Proteínas de Transporte/metabolismo , Músculo Liso Vascular/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-dissulfônico/farmacologia , Trifosfato de Adenosina/metabolismo , Amilorida/análogos & derivados , Amilorida/farmacologia , Animais , Antiporters/efeitos dos fármacos , Antiporters/metabolismo , Proteínas de Transporte/efeitos dos fármacos , Células Cultivadas , Antiportadores de Cloreto-Bicarbonato , Concentração de Íons de Hidrogênio , Músculo Liso Vascular/citologia , Ratos , Ratos Sprague-Dawley , Simportadores de Sódio-Bicarbonato , Trocadores de Sódio-Hidrogênio/efeitos dos fármacosRESUMO
The recent discovery of aquaporins, a family of highly conserved water channel proteins, which are expressed in both eukaryotes and prokaryotes, has provoked a re-evaluation of the physiology of water transport in all organisms. So far, seven distinct aquaporins have been characterised in mammals, but highly homologous family members have also been found in amphibians, insects, plants and bacteria. These transmembrane proteins serve to facilitate water transport down osmotic gradients with low activation energy. Alterations in channel expression, cellular targeting and perhaps channel permeability regulate membrane water transport. Naturally occurring and experimentally produced mutations in aquaporins cause a variety of perturbations of water homeostasis. Manipulation of aquaporin expression may have a therapeutic role in several disease processes including cardiac failure and the ascites associated with liver disease.