RESUMEN
Vascular smooth muscle cell (VSMC) phenotypic conversion from a contractile to 'synthetic' state contributes to vascular pathologies including restenosis, atherosclerosis and vascular calcification. We have recently found that the secretion of exosomes is a feature of 'synthetic' VSMCs and that exosomes are novel players in vascular repair processes as well as pathological vascular thrombosis and calcification. Pro-inflammatory cytokines and growth factors as well as mineral imbalance stimulate exosome secretion by VSMCs, most likely by the activation of sphingomyelin phosphodiesterase 3 (SMPD3) and cytoskeletal remodelling. Calcium stress induces dramatic changes in VSMC exosome composition and accumulation of phosphatidylserine (PS), annexin A6 and matrix metalloproteinase-2, which converts exosomes into a nidus for calcification. In addition, by presenting PS, VSMC exosomes can also provide the catalytic surface for the activation of coagulation factors. Recent data showing that VSMC exosomes are loaded with proteins and miRNA regulating cell adhesion and migration highlight VSMC exosomes as potentially important communication messengers in vascular repair. Thus, the identification of signalling pathways regulating VSMC exosome secretion, including activation of SMPD3 and cytoskeletal rearrangements, opens up novel avenues for a deeper understanding of vascular remodelling processes.
Asunto(s)
Exosomas/metabolismo , Músculo Liso Vascular/metabolismo , Calcificación Vascular/metabolismo , Animales , Exosomas/patología , Humanos , Metaloproteinasa 2 de la Matriz/metabolismo , Músculo Liso Vascular/patología , Esfingomielina Fosfodiesterasa/metabolismo , Calcificación Vascular/patologíaRESUMEN
Emerging evidence from the last two decades has shown that vascular calcification (VC) is a regulated, cell-mediated process orchestrated by vascular smooth muscle cells (VSMCs) and that this process bears many similarities to bone mineralization. While many of the mechanisms driving VSMC calcification have been well established, it remains unclear what factors in specific disease states act to promote vascular calcification and in parallel, bone loss. Diabetes is a condition most commonly associated with VC and bone abnormalities. In this review, we describe how factors associated with the diabetic milieu impact on VSMCs, focusing on the role of oxidative stress, inflammation, impairment of the advanced glycation end product (AGE)/receptor for AGE system and, importantly, diabetic neuropathy. We also explore the link between bone and VC in diabetes with a specific emphasis on the receptor activator of nuclear factor κß ligand/osteoprotegerin system. Finally, we describe what insights can be gleaned from studying Charcot osteoarthropathy, a rare complication of diabetic neuropathy, in which the occurrence of VC is frequent and where bone lysis is extreme.
Asunto(s)
Artropatía Neurógena/etiología , Neuropatías Diabéticas/complicaciones , Calcificación Vascular/etiología , Artropatía Neurógena/fisiopatología , Neuropatías Diabéticas/fisiopatología , Humanos , Mediadores de Inflamación/metabolismo , Músculo Liso Vascular/patología , Estrés Oxidativo/fisiología , Ligando RANK/fisiología , Transducción de Señal/fisiología , Calcificación Vascular/patología , Calcificación Vascular/fisiopatologíaRESUMEN
Vascular calcification is associated with increased cardiovascular morbidity and mortality. A number of calcification inhibitors have been defined recently, including inorganic pyrophosphate (PP(i)), an important physicochemical inhibitor of hydroxyapatite crystal growth. Increased hydrolysis of PP(i) by tissue-nonspecific alkaline phosphatase (TNAP) may occur in renal failure and act to enhance mineralization of vessels.
Asunto(s)
Fosfatasa Alcalina/fisiología , Calcinosis/enzimología , Calcinosis/etiología , Insuficiencia Renal/complicaciones , Insuficiencia Renal/enzimología , Enfermedades Vasculares/enzimología , Enfermedades Vasculares/etiología , Animales , RatasRESUMEN
Chronic kidney disease (CKD) is associated with fatal cardiovascular consequences in part due to ectopic calcification of soft tissues particularly arteries, capillaries, and cardiac valves. An increasing body of evidence from experimental studies and in vivo data suggest that (I) a mineral imbalance with hyperphosphatemia and high-circulating calcium x phosphate product, (II) a deficiency of systemic or local calcification inhibitors, (III) death or 'damage' of vascular smooth muscle cells (VSMCs), and/or (IV) phenotypic transformation of VSMCs to osteo/chondrocytic cells may all act in concert to initiate and sustain vascular calcification. In CKD patients inhibitory systems are overwhelmed by a multitude of agents that induce VSMC damage and cell death resulting in the release of vesicles capable of nucleating basic calcium phosphate. Studies with genetically altered mice have identified both local and systemic calcification inhibitors that act to maintain VSMC differentiation or regulate vesicle properties. However, for many of these proteins the mechanisms and sites of action are still under investigation. In particular, it is unclear whether factors present in the circulation have an inhibitory role there and whether circulating levels of these proteins influence or are indicative of underlying disease processes in individual patients. A greater understanding of the origins and roles of potential circulating inhibitors may result in novel strategies aimed at the prevention or reversal of the life-limiting calcifying vasculopathies seen in CKD patients.
Asunto(s)
Calcinosis/etiología , Calcinosis/metabolismo , Enfermedades Renales/complicaciones , Enfermedades Vasculares/etiología , Enfermedades Vasculares/metabolismo , Animales , Calcinosis/sangre , Enfermedad Crónica , Humanos , Ratones , Ratones Mutantes , Enfermedades Vasculares/sangreRESUMEN
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.
Asunto(s)
Arteriosclerosis/metabolismo , Calcinosis/metabolismo , Proteínas de Unión al Calcio/genética , Proteínas de la Matriz Extracelular , Músculo Liso Vascular/metabolismo , Sialoglicoproteínas/genética , Proteínas de Unión al Calcio/fisiología , Diferenciación Celular , Humanos , Inmunohistoquímica , Proteínas de Microfilamentos , Músculo Liso Vascular/citología , Osteopontina , ARN Mensajero/análisis , Sialoglicoproteínas/fisiología , Calponinas , Proteína Gla de la MatrizRESUMEN
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.
Asunto(s)
Calcinosis/prevención & control , Proteínas de Unión al Calcio/metabolismo , Calcio/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Procesamiento Proteico-Postraduccional , Ácido 1-Carboxiglutámico/metabolismo , Adolescente , Adulto , Secuencia de Aminoácidos , Calcinosis/metabolismo , Proteínas de Unión al Calcio/química , Membrana Celular/metabolismo , Células Cultivadas , Proteínas de la Matriz Extracelular/química , Femenino , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Músculo Liso Vascular/efectos de los fármacos , Miocitos del Músculo Liso/efectos de los fármacos , Fragmentos de Péptidos/metabolismo , Fosforilación , Estructura Terciaria de Proteína , Serina/metabolismo , Vesículas Transportadoras/metabolismo , Vitamina K/antagonistas & inhibidores , Vitamina K/metabolismo , Vitamina K 1/farmacología , Warfarina/farmacología , Proteína Gla de la MatrizRESUMEN
Transgenic mice containing a sheep metallothionein 1a-sheep growth hormone fusion gene exhibited low, tissue-specific basal levels of transgene mRNA expression, resulting in slightly elevated levels of circulating growth hormone that did not lead to a detectable increase in growth. After zinc stimulation, high levels of transgene mRNA expression were induced in a number of tissues; these levels correlated with increased levels of circulating growth hormone, resulting in growth increases of up to 1.5 times the levels of controls and unstimulated transgenic mice. After removal of the zinc stimulus, transgene expression and circulating growth hormone concentrations returned to basal levels. Additional evidence from the pattern of developmental expression of the transgene suggests that zinc is the main regulator of this promoter in mice. The demonstrated regulation and low basal level of expression of the sheep metallothionein 1a promoter make it a candidate for use in other mouse transgenic studies and for use in transgenic livestock, in which regulation of expression is essential.
Asunto(s)
Regulación de la Expresión Génica , Genes , Hormona del Crecimiento/genética , Metalotioneína/genética , Ratones Transgénicos/crecimiento & desarrollo , Animales , Northern Blotting , Femenino , Genes/efectos de los fármacos , Hormona del Crecimiento/biosíntesis , Hormona del Crecimiento/sangre , Masculino , Metalotioneína/biosíntesis , Ratones , ARN/genética , ARN/aislamiento & purificación , ARN Mensajero/análisis , Proteínas Recombinantes de Fusión/biosíntesis , Mapeo Restrictivo , Ovinos , Transcripción Genética , Zinc/farmacologíaRESUMEN
Non-enzymatic glycation of extracellular matrix with (U-13C5)-d-ribose-5-phosphate (R5P), enables in situ 2D ssNMR identification of many deleterious protein modifications and crosslinks, including previously unreported oxalamido and hemiaminal (CH3-CH(OH)NHR) substructures. Changes in charged residue proportions and distribution may be as important as crosslinking in provoking and understanding harmful tissue changes.
Asunto(s)
Colágeno/química , Matriz Extracelular/química , Productos Finales de Glicación Avanzada/química , Modelos Biológicos , Resonancia Magnética Nuclear BiomolecularRESUMEN
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.
Asunto(s)
Apoptosis , Calcinosis/metabolismo , Músculo Liso Vascular/metabolismo , Clorometilcetonas de Aminoácidos/farmacología , Antraquinonas , Calcinosis/tratamiento farmacológico , Calcinosis/patología , Calcio/metabolismo , Radioisótopos de Calcio , División Celular/efectos de los fármacos , Células Cultivadas , Colorantes , Cicloheximida/farmacología , Microanálisis por Sonda Electrónica , Humanos , Inmunoglobulina M/farmacología , Etiquetado Corte-Fin in Situ , Antígeno Ki-67/metabolismo , Músculo Liso Vascular/citología , Músculo Liso Vascular/patología , Inhibidores de la Síntesis de la Proteína/farmacología , Receptor fas/inmunologíaRESUMEN
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.
Asunto(s)
Calcinosis/inducido químicamente , Diferenciación Celular/efectos de los fármacos , Lipoproteínas LDL/farmacología , Músculo Liso Vascular/efectos de los fármacos , Osteoblastos/patología , Adolescente , Adulto , Anciano , Antígenos de Diferenciación/biosíntesis , Apoptosis/efectos de los fármacos , Calcinosis/patología , Enfermedades de las Arterias Carótidas/metabolismo , Enfermedades de las Arterias Carótidas/patología , Células Cultivadas , Medios de Cultivo/farmacología , Humanos , Hibridación in Situ , Técnicas In Vitro , Metabolismo de los Lípidos , Lipoproteínas/deficiencia , Lipoproteínas/farmacología , Masculino , Persona de Mediana Edad , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Poli I/farmacología , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Receptores Inmunológicos/antagonistas & inhibidores , Receptores Depuradores , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
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.
Asunto(s)
Arterias/metabolismo , Calcinosis/metabolismo , Proteínas de Unión al Calcio/genética , Proteínas de la Matriz Extracelular , Músculo Liso Vascular/citología , Sialoglicoproteínas/genética , Adulto , Anciano , Anciano de 80 o más Años , Arterias/patología , Proteínas Morfogenéticas Óseas/análisis , Proteínas Morfogenéticas Óseas/genética , Calcio/metabolismo , Proteínas de Unión al Calcio/análisis , Células Cultivadas , Femenino , Humanos , Masculino , Persona de Mediana Edad , Músculo Liso Vascular/metabolismo , Osteopontina , Esclerosis , Sialoglicoproteínas/análisis , Proteína Gla de la MatrizRESUMEN
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.
Asunto(s)
Arteriosclerosis/metabolismo , Quimiocinas CC/genética , Quimiocinas CX3C/genética , Cromosomas Humanos Par 16 , Macrófagos/inmunología , Proteínas de la Membrana/genética , Adolescente , Adulto , Anciano , Arterias/metabolismo , Arteriosclerosis/patología , Biomarcadores/análisis , Técnicas de Cultivo de Célula , Quimiocina CCL17 , Quimiocina CCL22 , Quimiocina CX3CL1 , Quimiocinas CC/biosíntesis , Quimiocinas CC/fisiología , Quimiocinas CX3C/biosíntesis , Quimiocinas CX3C/fisiología , Quimiotaxis de Leucocito , Células Dendríticas/metabolismo , Femenino , Ligamiento Genético , Humanos , Interleucinas/farmacología , Masculino , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/fisiología , Persona de Mediana Edad , ARN Mensajero/biosíntesis , Células Th2/inmunología , Regulación hacia ArribaRESUMEN
Vascular calcification is commonplace in patients with end-stage renal disease where it develops rapidly and predicts a variety of adverse outcomes. The processes responsible for vascular calcification have been the focus of much research, aided in recent decades by molecular genetic techniques and in vitro models. Converging evidence now suggests that vascular calcification is an active, regulated process, with abundant similarities to the process of skeletal mineralization. Using an in vitro model of calcifying vascular smooth muscle cells (VSMCs), we have shown that a mineral imbalance induces VSMC apoptosis, and that VSMC apoptotic bodies and vesicles can nucleate basic calcium phosphate in the form of hydroxyapatite, the same mineral found in bone. Gene expression studies suggest that the normal vessel wall expresses proteins such as matrix Gla protein that inhibit calcification. In addition, circulating proteins such as fetuin-A are produced at remote sites and act to inhibit soft tissue calcification systemically. However, down-regulation or perturbation of these proteins may lead to a phenotypic transformation of VSMCs to osteo/chondrocytic-like cells while the calcified environment may stimulate macrophages to adopt osteoclastic properties. Both clinical and basic research findings indicate an inverse relationship between bone mineralization and vascular calcification. The mechanisms linking these two processes are a topic for further investigation, with current theories proposing a role for lipids, common regulatory molecules, and calcium and bone turnover. We have synthesized these findings into a theoretical model offering a putative pathway for the development of severe vascular calcification in end-stage renal disease.
Asunto(s)
Calcinosis/etiología , Calcinosis/fisiopatología , Uremia/complicaciones , Enfermedades Vasculares/etiología , Enfermedades Vasculares/fisiopatología , Animales , Apoptosis , Proteínas Sanguíneas/fisiología , Calcificación Fisiológica/fisiología , Calcinosis/metabolismo , Proteínas de Unión al Calcio/genética , Proteínas de la Matriz Extracelular/genética , Humanos , Ratones , Miocitos del Músculo Liso/fisiología , Polimorfismo Genético , Uremia/metabolismo , Uremia/fisiopatología , Enfermedades Vasculares/metabolismo , alfa-2-Glicoproteína-HS , Proteína Gla de la MatrizRESUMEN
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.
Asunto(s)
Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Músculo Liso Vascular/citología , Acetamidas/farmacología , Animales , División Celular/efectos de los fármacos , Células Cultivadas , Hematínicos/farmacología , Heparina/farmacología , Humanos , RatasRESUMEN
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.
Asunto(s)
Biopolímeros/genética , ADN/análisis , Músculo Liso Vascular/química , Ubiquitinas/genética , Secuencia de Aminoácidos , Animales , Aorta , Secuencia de Bases , Northern Blotting , Células Cultivadas , Marcadores Genéticos , Hibridación in Situ , Datos de Secuencia Molecular , Poliubiquitina , Ratas , Ratas Wistar , Análisis de Secuencia de ADNRESUMEN
Prelamin A accumulation and persistent DNA damage response (DDR) are hallmarks of vascular smooth muscle cell (VSMC) ageing and dysfunction. Although prelamin A is proposed to interfere with DNA repair, our understanding of the crosstalk between prelamin A and the repair process remains limited. The extracellular signal-regulated kinases 1 and 2 (ERK1/2) have emerged as key players in the DDR and are known to enhance ataxia telangiectasia-mutated protein (ATM) activity at DNA lesions, and in this study, we identified a novel relationship between prelamin A accumulation and ERK1/2 nuclear compartmentalisation during VSMC ageing. We show both prelamin A accumulation and increased DNA damage occur concomitantly, before VSMC replicative senescence, and induce the localisation of ERK1/2 to promyelocytic leukaemia protein nuclear bodies (PML NBs) at the sites of DNA damage via nesprin-2 and lamin A interactions. Importantly, VSMCs treated with DNA damaging agents also displayed prelamin A accumulation and ERK compartmentalisation at PML NBs, suggesting that prelamin A and nesprin-2 are novel components of the DDR. In support of this, disruption of ERK compartmentalisation at PML NBs, by either depletion of nesprin-2 or lamins A/C, resulted in the loss of ATM from DNA lesions. However, ATM signalling and DNA repair remained intact after lamins A/C depletion, whereas nesprin-2 disruption ablated downstream Chk2 activation and induced genomic instability. We conclude that lamins A/C and PML act as scaffolds to organise DNA-repair foci and compartmentalise nesprin-2/ERK signalling. However, nesprin-2/ERK signalling fidelity, but not their compartmentalisation at PML NBs, is essential for efficient DDR in VSMCs.
Asunto(s)
Daño del ADN , Proteínas de Microfilamentos/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Músculo Liso Vascular/citología , Músculo Liso Vascular/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/metabolismo , Adulto , Ciclo Celular/fisiología , Senescencia Celular/fisiología , Reparación del ADN , Femenino , Humanos , Cuerpos de Inclusión Intranucleares/genética , Cuerpos de Inclusión Intranucleares/metabolismo , Lamina Tipo A/metabolismo , Masculino , Proteínas de Microfilamentos/genética , Persona de Mediana Edad , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/genética , Músculo Liso Vascular/enzimología , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/genética , Transfección , Adulto JovenRESUMEN
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.
Asunto(s)
Acuaporinas , Membrana Celular/metabolismo , Canales Iónicos/metabolismo , Proteínas de la Membrana , Agua/metabolismo , Animales , Acuaporina 1 , Acuaporina 2 , Acuaporina 3 , Acuaporina 4 , Acuaporina 5 , Acuaporina 6 , Bacterias , Transporte Biológico , Homeostasis , Canales Iónicos/química , PlantasRESUMEN
SM22 alpha is a 22-kDa protein of unknown function, the mRNA of which is highly and specifically expressed in smooth muscle cells (SMC). The 5' untranslated leader sequence of the rat SM22 alpha gene was found to contain two introns of 3.6 and 2.9 kb. Two transcripts of SM22 alpha exist in all SMC types examined, and genomic mapping of the gene suggests these transcripts result from different 5' transcription start points, split by the 2.9-kb intron. A small intron (102 bp), which contains an E-box consensus sequence, was found within the coding region 178 bp from the ATG start codon. The 3.6-kb intron contains 82 bp which show 98% homology at the RNA level with the rat identifier sequence (ID). Transient reporter gene assays demonstrate that a 576-bp fragment, including the ID, contains a regulatory element which may contribute to the SMC-specific expression of SM22 alpha.
Asunto(s)
Proteínas de Microfilamentos , Proteínas Musculares/genética , Músculo Liso Vascular/química , Secuencias Reguladoras de Ácidos Nucleicos , Animales , Secuencia de Bases , Cloranfenicol O-Acetiltransferasa/biosíntesis , Clonación Molecular , Secuencia de Consenso , Elementos de Facilitación Genéticos , Exones , Genes Reporteros , Intrones , Datos de Secuencia Molecular , Músculo Liso Vascular/enzimología , Regiones Promotoras Genéticas , Ratas , Secuencias Repetitivas de Ácidos Nucleicos , Análisis de Secuencia de ADNRESUMEN
Arterial calcification occurs with increasing age and in association with a diverse range of diseases, including atherosclerosis, diabetes, and uremia. It occurs at two sites in the vessel wall--in the media where it is known as Monckeberg's sclerosis and in the intima where it is invariably associated with atherosclerosis. Although there are similarities between them, the molecular mechanisms underlying these two forms of calcification may be distinct. Evidence is accumulating that vascular calcification is an active process that has many similarities with ossification, including local expression of bone-associated collagenous and noncollagenous proteins. The recent generation of a matrix gamma-carboxyglutamic acid (Gla) protein (MGP) knockout mouse, which exhibits extensive and lethal calcification and cartilaginous metaplasia of the media of all elastic arteries, has refocused attention on the role of Gla-containing proteins in vascular calcification. Gla-containing proteins have glutamic acid residues that must by gamma-carboxylated by vitamin-K-dependent gamma-carboxylase to enable them to bind calcium and function normally. Therefore, there is considerable scope for both transcriptional and posttranslational modifications of Gla protein function. Recent studies in humans have shown that although MGP mRNA is constitutively expressed by normal vascular smooth muscle cells (VSMCs), it is substantially upregulated in cells adjacent to both medial and intimal calcification. Studies in rats and on cultured human VSMCs showing that inhibition of MGP function by warfarin can accelerate spontaneous calcification have emphasized the potential importance of posttranslational processing in determining MGP function. It is therefore plausible that environmental influences such as diet and medication may have significant effects on vascular calcification. Furthermore, recent studies have shown that several other Gla-containing proteins with the potential to regulate or perhaps contribute to vascular calcification are present in the human vasculature. Future studies on the role of Gla-containing proteins combined with advances in noninvasive imaging techniques to quantify vascular calcification may lead to identification of individuals at particular risk of vascular calcification and the evaluation of novel therapies aimed at regulating its development or progression.
Asunto(s)
Arterias/fisiopatología , Calcinosis/fisiopatología , Proteínas de Unión al Calcio/fisiología , Proteínas de la Matriz Extracelular , Ácido 1-Carboxiglutámico , Animales , Proteínas de Unión al Calcio/genética , Regulación de la Expresión Génica , Humanos , Ratones , Procesamiento Proteico-Postraduccional , Ratas , Transcripción Genética , Proteína Gla de la MatrizRESUMEN
The structures of the M110 and M21 regulatory subunits of protein phosphatase-1M, the major enzyme which dephosphorylates myosin in smooth muscle, have been deduced from cloned cDNAs. The N-terminus of the M110 subunit from rat aorta contains seven ankyrin repeats, while the C-terminus of the M21 subunit from chicken gizzard contains a leucine zipper motif. The M110 subunit is expressed in two different forms which differ in their C-terminal sequences. One of these is highly homologous to the whole of the M21 subunit.