RESUMEN
Lipid droplets (LDs) are distinct and dynamic organelles that affect the health of cells and organs. Much progress has been made in understanding how these structures are formed, how they interact with other cellular organelles, how they are used for storage of triacylglycerol in adipose tissue, and how they regulate lipolysis. Our understanding of the biology of LDs in the heart and vascular tissue is relatively primitive in comparison with LDs in adipose tissue and liver. The National Heart, Lung, and Blood Institute convened a working group to discuss how LDs affect cardiovascular diseases. The goal of the working group was to examine the current state of knowledge on the cell biology of LDs, including current methods to study them in cells and organs and reflect on how LDs influence the development and progression of cardiovascular diseases. This review summarizes the working group discussion and recommendations on research areas ripe for future investigation that will likely improve our understanding of atherosclerosis and heart function.
Asunto(s)
Enfermedades Cardiovasculares/metabolismo , Gotas Lipídicas/metabolismo , Miocardio/metabolismo , Animales , Enfermedades Cardiovasculares/genética , Consensus Development Conferences, NIH as Topic , Modelos Animales de Enfermedad , Interacción Gen-Ambiente , Humanos , Metabolismo de los Lípidos , National Heart, Lung, and Blood Institute (U.S.) , Estados UnidosRESUMEN
Pathophysiological, epidemiological, and genetic studies provide strong evidence that lipoprotein(a) [Lp(a)] is a causal mediator of cardiovascular disease (CVD) and calcific aortic valve disease (CAVD). Specific therapies to address Lp(a)-mediated CVD and CAVD are in clinical development. Due to knowledge gaps, the National Heart, Lung, and Blood Institute organized a working group that identified challenges in fully understanding the role of Lp(a) in CVD/CAVD. These included the lack of research funding, inadequate experimental models, lack of globally standardized Lp(a) assays, and inadequate understanding of the mechanisms underlying current drug therapies on Lp(a) levels. Specific recommendations were provided to facilitate basic, mechanistic, preclinical, and clinical research on Lp(a); foster collaborative research and resource sharing; leverage expertise of different groups and centers with complementary skills; and use existing National Heart, Lung, and Blood Institute resources. Concerted efforts to understand Lp(a) pathophysiology, together with diagnostic and therapeutic advances, are required to reduce Lp(a)-mediated risk of CVD and CAVD.
Asunto(s)
Estenosis de la Válvula Aórtica , Enfermedades Cardiovasculares , Lipoproteína(a)/sangre , Estenosis de la Válvula Aórtica/sangre , Estenosis de la Válvula Aórtica/prevención & control , Enfermedades Cardiovasculares/sangre , Enfermedades Cardiovasculares/prevención & control , Humanos , National Heart, Lung, and Blood Institute (U.S.) , Evaluación de Necesidades , Investigación , Factores de Riesgo , Estados UnidosAsunto(s)
National Heart, Lung, and Blood Institute (U.S.)/economía , Apoyo a la Investigación como Asunto , Investigación Biomédica Traslacional/economía , Difusión de Innovaciones , Predicción , Humanos , National Heart, Lung, and Blood Institute (U.S.)/tendencias , Desarrollo de Programa , Evaluación de Programas y Proyectos de Salud , Apoyo a la Investigación como Asunto/tendencias , Investigación Biomédica Traslacional/tendencias , Estados UnidosAsunto(s)
Investigación Biomédica/economía , Prioridades en Salud/economía , Necesidades y Demandas de Servicios de Salud/economía , National Institutes of Health (U.S.)/economía , Evaluación de Necesidades/economía , Apoyo a la Investigación como Asunto/economía , Impuestos/economía , Presupuestos , Relaciones Comunidad-Institución/economía , Costo de Enfermedad , Recesión Económica , Humanos , Relaciones Interinstitucionales , Opinión Pública , Estados UnidosRESUMEN
Closure of the ductus arteriosus (DA) at birth is essential for the transition from fetal to postnatal life. Before birth the DA bypasses the uninflated lungs by shunting blood from the pulmonary trunk into the systemic circulation. The molecular mechanism underlying DA closure and degeneration has not been fully elucidated, but is associated with apoptosis and cytolytic necrosis in the inner media and intima. We detected features of histology during DA degeneration that are comparable to Hutchinson Gilford Progeria syndrome and ageing. Immunohistochemistry on human fetal and neonatal DA, and aorta showed that lamin A/C was expressed in all layers of the vessel wall. As a novel finding we report that progerin, a splicing variant of lamin A/C was expressed almost selectively in the normal closing neonatal DA, from which we hypothesized that progerin is involved in DA closure. Progerin was detected in 16.2%±7.2 cells of the DA. Progerin-expressing cells were predominantly located in intima and inner media where cytolytic necrosis accompanied by apoptosis will develop. Concomitantly we found loss of α-smooth muscle actin as well as reduced lamin A/C expression compared to the fetal and non-closing DA. In cells of the adjacent aorta, that remains patent, progerin expression was only sporadically detected in 2.5%±1.5 of the cells. Data were substantiated by the detection of mRNA of progerin in the neonatal DA but not in the aorta, by PCR and sequencing analysis. The fetal DA and the non-closing persistent DA did not present with progerin expressing cells. Our analysis revealed that the spatiotemporal expression of lamin A/C and progerin in the neonatal DA was mutually exclusive. We suggest that activation of LMNA alternative splicing is involved in vascular remodeling in the circulatory system during normal neonatal DA closure.
Asunto(s)
Conducto Arterial/metabolismo , Proteínas Nucleares/metabolismo , Precursores de Proteínas/metabolismo , Femenino , Humanos , Inmunohistoquímica , Técnicas In Vitro , Recién Nacido , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Proteínas Nucleares/genética , Embarazo , Progeria/metabolismo , Precursores de Proteínas/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Túnica Íntima/metabolismoRESUMEN
Hutchinson-Gilford progeria syndrome (HGPS), a devastating premature aging disease, is caused by a point mutation in the lamin A gene (LMNA). This mutation constitutively activates a cryptic splice donor site, resulting in a mutant lamin A protein known as progerin. Recent studies have demonstrated that progerin is also produced at low levels in normal human cells and tissues. However, the cause-and-effect relationship between normal aging and progerin production in normal individuals has not yet been determined. In this study, we have shown in normal human fibroblasts that progressive telomere damage during cellular senescence plays a causative role in activating progerin production. Progressive telomere damage was also found to lead to extensive changes in alternative splicing in multiple other genes. Interestingly, elevated progerin production was not seen during cellular senescence that does not entail telomere shortening. Taken together, our results suggest a synergistic relationship between telomere dysfunction and progerin production during the induction of cell senescence, providing mechanistic insight into how progerin may participate in the normal aging process.
Asunto(s)
Senescencia Celular/fisiología , Fibroblastos/fisiología , Proteínas Nucleares/metabolismo , Precursores de Proteínas/metabolismo , Telómero/metabolismo , Envejecimiento/fisiología , Animales , Células Cultivadas , Fibroblastos/citología , Humanos , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Proteínas Nucleares/genética , Progeria/genética , Progeria/fisiopatología , Precursores de Proteínas/genética , Telomerasa/genética , Proteína 2 de Unión a Repeticiones Teloméricas/genéticaRESUMEN
Asthma is etiologically and clinically heterogeneous, making the genomic basis of asthma difficult to identify. We exploited the strain-dependence of a murine model of allergic airway disease to identify different genomic responses in the lung. BALB/cJ and C57BL/6J mice were sensitized with the immunodominant allergen from the Dermatophagoides pteronyssinus species of house dust mite (Der p 1), without exogenous adjuvant, and the mice then underwent a single challenge with Der p 1. Allergic inflammation, serum antibody titers, mucous metaplasia, and airway hyperresponsiveness were evaluated 72 hours after airway challenge. Whole-lung gene expression analyses were conducted to identify genomic responses to allergen challenge. Der p 1-challenged BALB/cJ mice produced all the key features of allergic airway disease. In comparison, C57BL/6J mice produced exaggerated Th2-biased responses and inflammation, but exhibited an unexpected decrease in airway hyperresponsiveness compared with control mice. Lung gene expression analysis revealed genes that were shared by both strains and a set of down-regulated genes unique to C57BL/6J mice, including several G-protein-coupled receptors involved in airway smooth muscle contraction, most notably the M2 muscarinic receptor, which we show is expressed in airway smooth muscle and was decreased at the protein level after challenge with Der p 1. Murine strain-dependent genomic responses in the lung offer insights into the different biological pathways that develop after allergen challenge. This study of two different murine strains demonstrates that inflammation and airway hyperresponsiveness can be decoupled, and suggests that the down-modulation of expression of G-protein-coupled receptors involved in regulating airway smooth muscle contraction may contribute to this dissociation.
Asunto(s)
Alérgenos , Antígenos Dermatofagoides/inmunología , Asma/genética , Hiperreactividad Bronquial/genética , Broncoconstricción/genética , Pulmón/inmunología , Resistencia de las Vías Respiratorias/genética , Animales , Proteínas de Artrópodos , Asma/inmunología , Asma/fisiopatología , Hiperreactividad Bronquial/inmunología , Hiperreactividad Bronquial/fisiopatología , Pruebas de Provocación Bronquial , Broncoconstricción/efectos de los fármacos , Broncoconstrictores , Cisteína Endopeptidasas , Citocinas/metabolismo , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Predisposición Genética a la Enfermedad , Inmunoglobulina E/sangre , Inmunoglobulina G/sangre , Pulmón/fisiopatología , Masculino , Cloruro de Metacolina , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Mucinas/metabolismo , Fenotipo , Receptores Acoplados a Proteínas G/genética , Células Th2/inmunología , Factores de TiempoRESUMEN
Despite the success of protein farnesyltransferase inhibitors (FTIs) in the treatment of certain malignancies, their mode of action is incompletely understood. Dissecting the molecular pathways affected by FTIs is important, particularly because this group of drugs is now being tested for the treatment of Hutchinson-Gilford progeria syndrome. In the current study, we show that FTI treatment causes a centrosome separation defect, leading to the formation of donut-shaped nuclei in nontransformed cell lines, tumor cell lines, and tissues of FTI-treated mice. Donut-shaped nuclei arise during chromatin decondensation in late mitosis; subsequently, cells with donut-shaped nuclei exhibit defects in karyokinesis, develop aneuploidy, and are often binucleated. Binucleated cells proliferate slowly. We identified lamin B1 and proteasome-mediated degradation of pericentrin as critical components in FTI-induced "donut formation" and binucleation. Reducing pericentrin expression or ectopic expression of nonfarnesylated lamin B1 was sufficient to elicit donut formation and binucleated cells, whereas blocking proteasomal degradation eliminated FTI-induced donut formation. Our studies have uncovered an important role of FTIs on centrosome separation and define pericentrin as a (indirect) target of FTIs affecting centrosome position and bipolar spindle formation, likely explaining some of the anticancer effects of these drugs.
Asunto(s)
Antineoplásicos/farmacología , Núcleo Celular , Centrosoma , Mitosis/efectos de los fármacos , Prenilación de Proteína/efectos de los fármacos , Huso Acromático , Animales , Antígenos/biosíntesis , Antígenos/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , Núcleo Celular/patología , Centrosoma/metabolismo , Centrosoma/patología , Células Hep G2 , Humanos , Lamina Tipo B/biosíntesis , Lamina Tipo B/genética , Ratones , Ratones Transgénicos , Mitosis/genética , Progeria/genética , Progeria/metabolismo , Progeria/patología , Prenilación de Proteína/genética , Huso Acromático/genética , Huso Acromático/metabolismo , Huso Acromático/patologíaRESUMEN
RATIONALE: Arginine methylation by protein N-arginine methyltransferases (PRMTs) is an important posttranslational modification in the regulation of protein signaling. PRMT2 contains a highly conserved catalytic Ado-Met binding domain, but the enzymatic function of PRMT2 with respect to methylation is unknown. The JAK-STAT pathway is proposed to be regulated through direct arginine methylation of STAT transcription factors, and STAT3 signaling is known to be required for leptin regulation of energy balance. OBJECTIVE: To identify the potential role of STAT3 arginine methylation by PRMT2 in the regulation of leptin signaling and energy homeostasis. METHODS AND RESULTS: We identified that PRMT2(-/-) mice are hypophagic, lean, and have significantly reduced serum leptin levels. This lean phenotype is accompanied by resistance to food-dependent obesity and an increased sensitivity to exogenous leptin administration. PRMT2 colocalizes with STAT3 in hypothalamic nuclei, where it binds and methylates STAT3 through its Ado-Met binding domain. In vitro studies further clarified that the Ado-Met binding domain of PRMT2 induces STAT3 methylation at the Arg31 residue. Absence of PRMT2 results in decreased methylation and prolonged tyrosine phosphorylation of hypothalamic STAT3, which was associated with increased expression of hypothalamic proopiomelanocortin following leptin stimulation. CONCLUSIONS: These data elucidate a molecular pathway that directly links arginine methylation of STAT3 by PRMT2 to the regulation of leptin signaling, suggesting a potential role for PRMT2 antagonism in the treatment of obesity and obesity-related syndromes.
Asunto(s)
Metabolismo Energético/fisiología , Leptina/metabolismo , Metiltransferasas/metabolismo , Obesidad/metabolismo , Proteína-Arginina N-Metiltransferasas/metabolismo , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/fisiología , Animales , Núcleo Arqueado del Hipotálamo/metabolismo , Peso Corporal/fisiología , Ingestión de Alimentos/fisiología , Glucógeno/metabolismo , Hígado/metabolismo , Metilación , Metiltransferasas/genética , Ratones , Ratones Mutantes , Obesidad/fisiopatología , Fosforilación/fisiología , Estructura Terciaria de Proteína , Proteína-Arginina N-Metiltransferasas/química , Proteína-Arginina N-Metiltransferasas/genéticaRESUMEN
OBJECTIVE: Children with Hutchinson-Gilford progeria syndrome (HGPS) exhibit dramatically accelerated cardiovascular disease (CVD), causing death from myocardial infarction or stroke between the ages of 7 and 20 years. We undertook the first histological comparative evaluation between genetically confirmed HGPS and the CVD of aging. METHODS AND RESULTS: We present structural and immunohistological analysis of cardiovascular tissues from 2 children with HGPS who died of myocardial infarction. Both had features classically associated with the atherosclerosis of aging, as well as arteriolosclerosis of small vessels. In addition, vessels exhibited prominent adventitial fibrosis, a previously undescribed feature of HGPS. Importantly, although progerin was detected at higher rates in the HGPS coronary arteries, it was also present in non-HGPS individuals. Between the ages of 1 month and 97 years, progerin staining increased an average of 3.34% per year (P<0.0001) in coronary arteries. CONCLUSIONS: We find concordance among many aspects of cardiovascular pathology in both HGPS and geriatric patients. HGPS generates a more prominent adventitial fibrosis than typical CVD. Vascular progerin generation in young non-HGPS individuals, which significantly increases throughout life, strongly suggests that progerin has a role in cardiovascular aging of the general population.
Asunto(s)
Aterosclerosis/patología , Enfermedad de la Arteria Coronaria/patología , Progeria/patología , Adolescente , Envejecimiento/patología , Enfermedades Cardiovasculares/patología , Niño , Femenino , Humanos , Lamina Tipo A/análisis , Masculino , Infarto del Miocardio/etiología , Progeria/complicacionesRESUMEN
Cyclin-dependent kinase inhibitors, p21(Cip1) and p27(Kip1), are upregulated during vascular cell proliferation and negatively regulate growth of vascular cells. We hypothesized that absence of either p21(Cip1) or p27(Kip1) in apolipoprotein E (apoE)-deficiency may increase atherosclerotic plaque formation. Compared to apoE(-/-) aortae, both apoE(-/-)/p21(-/-) and apoE(-/-)/p27(-/-) aortae exhibited significantly more atherosclerotic plaque following a high-cholesterol regimen. This increase was particularly observed in the abdominal aortic regions. Deficiency of p27(Kip1) accelerated plaque formation significantly more than p21(-/-) in apoE(-/-) mice. This increased plaque formation was in parallel with increased intima/media area ratios. Deficiency of p21(Cip1) and p27(Kip1) accelerates atherogenesis in apoE(-/-) mice. These findings have significant implications for our understanding of the molecular basis of atherosclerosis associated with excessive proliferation of vascular cells.
Asunto(s)
Apolipoproteínas E/genética , Aterosclerosis/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/deficiencia , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/deficiencia , Animales , Aorta/metabolismo , Aorta/patología , Aterosclerosis/genética , Aterosclerosis/patología , Proliferación Celular , Colesterol/administración & dosificación , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/biosíntesis , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/biosíntesis , Dieta Aterogénica , Ratones , Ratones MutantesRESUMEN
Vascular proliferative diseases are characterized by VSMC proliferation and migration. Kinase interacting with stathmin (KIS) targets 2 key regulators of cell proliferation and migration, the cyclin-dependent kinase inhibitor p27Kip1 and the microtubule-destabilizing protein stathmin. Phosphorylation of p27Kip1 by KIS leads to cell-cycle progression, whereas the target sequence and the physiological relevance of KIS-mediated stathmin phosphorylation in VSMCs are unknown. Here we demonstrated that vascular wound repair in KIS-/- mice resulted in accelerated formation of neointima, which is composed predominantly of VSMCs. Deletion of KIS increased VSMC migratory activity and cytoplasmic tubulin destabilizing activity, but abolished VSMC proliferation through the delayed nuclear export and degradation of p27Kip1. This promigratory phenotype resulted from increased stathmin protein levels, caused by a lack of KIS-mediated stathmin phosphorylation at serine 38 and diminished stathmin protein degradation. Downregulation of stathmin in KIS-/- VSMCs fully restored the phenotype, and stathmin-deficient mice demonstrated reduced lesion formation in response to vascular injury. These data suggest that KIS protects against excessive neointima formation by opposing stathmin-mediated VSMC migration and that VSMC migration represents a major mechanism of vascular wound repair, constituting a relevant target and mechanism for therapeutic interventions.
Asunto(s)
Movimiento Celular , Núcleo Celular/enzimología , Proliferación Celular , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Miocitos del Músculo Liso/enzimología , Proteínas Serina-Treonina Quinasas/metabolismo , Estatmina/metabolismo , Túnica Media/enzimología , Transporte Activo de Núcleo Celular/genética , Animales , Movimiento Celular/genética , Núcleo Celular/genética , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Ratones , Ratones Noqueados , Miocitos del Músculo Liso/patología , Fosforilación/genética , Proteínas Serina-Treonina Quinasas/genética , Estatmina/genética , Túnica Media/lesiones , Túnica Media/patología , Cicatrización de Heridas/genéticaRESUMEN
Hutchinson-Gilford progeria syndrome (HGPS) is the most dramatic form of human premature aging. Death occurs at a mean age of 13 years, usually from heart attack or stroke. Almost all cases of HGPS are caused by a de novo point mutation in the lamin A (LMNA) gene that results in production of a mutant lamin A protein termed progerin. This protein is permanently modified by a lipid farnesyl group, and acts as a dominant negative, disrupting nuclear structure. Treatment with farnesyltransferase inhibitors (FTIs) has been shown to prevent and even reverse this nuclear abnormality in cultured HGPS fibroblasts. We have previously created a mouse model of HGPS that shows progressive loss of vascular smooth muscle cells in the media of the large arteries, in a pattern that is strikingly similar to the cardiovascular disease seen in patients with HGPS. Here we show that the dose-dependent administration of the FTI tipifarnib (R115777, Zarnestra) to this HGPS mouse model can significantly prevent both the onset of the cardiovascular phenotype as well as the late progression of existing cardiovascular disease. These observations provide encouraging evidence for the current clinical trial of FTIs for this rare and devastating disease.
Asunto(s)
Enfermedades Cardiovasculares/prevención & control , Inhibidores Enzimáticos/farmacología , Farnesiltransferasa/antagonistas & inhibidores , Progeria/complicaciones , Animales , Enfermedades Cardiovasculares/etiología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/uso terapéutico , Ratones , Progeria/tratamiento farmacológico , Quinolonas/farmacología , Quinolonas/uso terapéuticoRESUMEN
Cyclin-dependent kinase inhibitors, including p21Cip1, are implicated in cell turnover and are active players in cardiovascular wound repair. Here, we show that p21Cip1 orchestrates the complex interactions between local vascular and circulating immune cells during vascular wound repair. In response to femoral artery mechanical injury, mice with homozygous deletion of p21Cip1 displayed accelerated proliferation of VSMCs and increased immune cell infiltration. BM transplantation experiments indicated that local p21Cip1 plays a pivotal role in restraining excessive proliferation during vascular wound repair. Increased local vascular stromal cell-derived factor-1 (SDF-1) levels were observed after femoral artery injury in p21+/+ and p21-/- mice, although this was significantly greater in p21-/- animals. In addition, disruption of SDF-1/CXCR4 signaling inhibited the proliferative response during vascular remodeling in both p21+/+ and p21-/- mice. We provide evidence that the JAK/STAT signaling pathway is an important regulator of vascular SDF-1 levels and that p21Cip1 inhibits STAT3 binding to the STAT-binding site within the murine SDF-1 promoter. Collectively, these results suggest that p21Cip1 activity is essential for the regulation of cell proliferation and inflammation after arterial injury in local vascular cells and that the SDF-1/CXCR4 signaling system is a key mediator of vascular proliferation in response to injury.
Asunto(s)
Apoptosis , Quimiocina CXCL12/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/biosíntesis , Receptores CXCR4/metabolismo , Cicatrización de Heridas , Animales , Proliferación Celular , Arteria Femoral/patología , Homocigoto , Ratones , Ratones Transgénicos , Modelos Biológicos , Linfocitos T/metabolismo , Factores de Tiempo , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
The cyclin-dependent kinase inhibitor p27(Kip1) arrests cell cycle progression through G1/S phases and is regulated by phosphorylation of serine/threonine residues. Recently, we identified the serine/threonine kinase, KIS, which phosphorylates p27(Kip1) on serine 10 leading to nuclear export of p27(Kip1) and protein degradation. However, the molecular mechanisms of transcriptional activation of the human KIS gene and its biological activity are not known. We mapped the transcription initiation site approximately 116 bp 5' to the translation start site, and sequences extending to -141 were sufficient for maximal promoter activity. Mutation in either of two Ets-binding sites in this region resulted in an approximately 75-80% decrease in promoter activity. These sites form at least 3 specific complexes, which contained GA-binding protein (GABP). Knocking down GABPalpha by siRNA in vascular smooth muscle cells (VSMCs) diminished KIS gene expression and reduced cell migration. Correspondingly, in serum stimulated GABPalpha-deficient mouse embryonic fibroblasts (MEFs), KIS gene expression was also significantly reduced, which was associated with an increase in p27(Kip1) protein levels and a decreased percentage of cells in S-phase. Consistent with these findings, following vascular injury in vivo, GABPalpha-heterozygous mice demonstrated reduced KIS gene expression within arterial lesions and these lesions were significantly smaller compared to GABP+/+ mice. In summary, serum-responsive GABP binding to Ets-binding sites activates the KIS promoter, leading to KIS gene expression, cell migration, and cell cycle progression.
Asunto(s)
Ciclo Celular/fisiología , Movimiento Celular/fisiología , Factor de Transcripción de la Proteína de Unión a GA/fisiología , Regulación Enzimológica de la Expresión Génica/fisiología , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas Serina-Treonina Quinasas/genética , Secuencia de Bases , Células Cultivadas , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Cartilla de ADN , Ensayo de Cambio de Movilidad Electroforética , Factor de Transcripción de la Proteína de Unión a GA/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Fosforilación , Reacción en Cadena de la Polimerasa , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Interferente PequeñoRESUMEN
Heme oxygenase (HO)-1 (encoded by Hmox1) catalyzes the oxidative degradation of heme to biliverdin and carbon monoxide. HO-1 is induced during inflammation and oxidative stress to protect tissues from oxidative damage. Because intravascular thrombosis forms at sites of tissue inflammation, we hypothesized that HO-1 protects against arterial thrombosis during oxidant stress. To investigate the direct function of HO-1 on thrombosis, we used photochemical-induced vascular injury in Hmox1-/- and Hmox1+/+ mice. Hmox1-/- mice developed accelerated, occlusive arterial thrombus compared with Hmox1+/+ mice, and we detected several mechanisms accounting for this antithrombotic effect. First, endothelial cells in Hmox1-/- arteries were more susceptible to apoptosis and denudation, leading to platelet-rich microthrombi in the subendothelium. Second, tissue factor, von Willebrand Factor, and reactive oxygen species were significantly elevated in Hmox1-/- mice, consistent with endothelial cell damage and loss. Third, following transplantation of Hmox1-/- donor bone marrow into Hmox1+/+ recipients and subsequent vascular injury, we observed rapid arterial thrombosis compared with Hmox1+/+ mice receiving Hmox1+/+ bone marrow. Fourth, inhaled carbon monoxide and biliverdin administration rescued the prothrombotic phenotype in Hmox1-/- mice. Fifth, using a transcriptional analysis of arterial tissue, we found that HO-1 determined a transcriptional response to injury, with specific effects on cell cycle regulation, coagulation, thrombosis, and redox homeostasis. These data provide direct genetic evidence for a protective role of HO-1 against thrombosis and reactive oxygen species during vascular damage. Induction of HO-1 may be beneficial in the prevention of thrombosis associated with vascular oxidant stress and inflammation.
Asunto(s)
Monóxido de Carbono/metabolismo , Endotelio Vascular/enzimología , Hemo-Oxigenasa 1/genética , Trombosis/metabolismo , Trombosis/fisiopatología , Administración por Inhalación , Animales , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Biliverdina/farmacología , Monóxido de Carbono/farmacología , Endotelio Vascular/patología , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Regulación Enzimológica de la Expresión Génica , Células Madre Hematopoyéticas/enzimología , Hemo-Oxigenasa 1/deficiencia , Hemostasis/fisiología , Ratones , Ratones Mutantes , Estrés Oxidativo/fisiología , Fenotipo , Tromboplastina/metabolismo , Trombosis/patología , Factor de von Willebrand/metabolismoRESUMEN
The retinoblastoma gene product (RB) is an important regulator of E2F activity. RB recruits a number of proteins, including HDACs, SWI/SNF complex, lysine methyl transferase (SUV39H1) and DNA methyltransferase (DNMT1), all of which negatively regulate E2F activity with RB. Here, we show that RB interacts with PRMT2, a member of the protein arginine methyltransferase family, to regulate E2F activity. PRMT2 directly bound and interacted with RB through its AdoMet binding domain, in contrast to other PRMT proteins, including PRMT1, PRMT3 and PRMT4. In reporter assays, PRMT2 repressed E2F1 transcriptional activity in an RB-dependent manner. PRMT2 formed a ternary complex with E2F1 in the presence of RB. To further explore the role of endogenous PRMT2 in the regulation of E2F activity, the PRMT2 gene was ablated in mice by gene targeting. Compared with PRMT2(+/+) mouse embryonic fibroblasts (MEFs), PRMT2(-/-) MEFs demonstrated increased E2F activity and early S phase entry following release of serum starvation. Vascular injury to PRMT2(-/-) arteries results in a hyperplastic response, consistent with increased G1-S phase progression. Taken together, these findings demonstrate a novel mechanism for the regulation of E2F activity by a member of the protein arginine methyltransferase family.
Asunto(s)
Factores de Transcripción E2F/metabolismo , Proteína-Arginina N-Metiltransferasas/metabolismo , Proteína de Retinoblastoma/metabolismo , Animales , Línea Celular Tumoral , Células Cultivadas , Regulación de la Expresión Génica , Células HeLa , Humanos , Metiltransferasas/metabolismo , Ratones , Ratones Noqueados , Unión Proteica , Proteína-Arginina N-Metiltransferasas/genética , Activación TranscripcionalRESUMEN
Children with Hutchinson-Gilford progeria syndrome (HGPS) suffer from dramatic acceleration of some symptoms associated with normal aging, most notably cardiovascular disease that eventually leads to death from myocardial infarction and/or stroke usually in their second decade of life. For the vast majority of cases, a de novo point mutation in the lamin A (LMNA) gene is the cause of HGPS. This missense mutation creates a cryptic splice donor site that produces a mutant lamin A protein, termed "progerin," which carries a 50-aa deletion near its C terminus. We have created a mouse model for progeria by generating transgenics carrying a human bacterial artificial chromosome that harbors the common HGPS mutation. These mice develop progressive loss of vascular smooth muscle cells in the medial layer of large arteries, in a pattern very similar to that seen in children with HGPS. This mouse model should prove valuable for testing experimental therapies for this devastating disorder and for exploring cardiovascular disease in general.
Asunto(s)
Modelos Animales de Enfermedad , Músculo Liso Vascular/anomalías , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/patología , Progeria/patología , Animales , Presión Sanguínea , Cromosomas Artificiales Bacterianos/genética , Progresión de la Enfermedad , Humanos , Lamina Tipo A/metabolismo , Ratones , Microscopía Electrónica de Transmisión , Músculo Liso Vascular/irrigación sanguínea , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/ultraestructura , Progeria/genética , Progeria/metabolismo , Progeria/ultraestructura , Transgenes/genéticaRESUMEN
The G1/S phase restriction point is a critical checkpoint that interfaces between the cell cycle regulatory machinery and DNA replicator proteins. Here, we report a novel function for the cyclin-dependent kinase inhibitor p27Kip1 in inhibiting DNA replication through its interaction with MCM7, a DNA replication protein that is essential for initiation of DNA replication and maintenance of genomic integrity. We find that p27Kip1 binds the conserved minichromosome maintenance (MCM) domain of MCM7. The proteins interact endogenously in vivo in a growth factor-dependent manner, such that the carboxyl terminal domain of p27Kip1 inhibits DNA replication independent of its function as a cyclin-dependent kinase inhibitor. This novel function of p27Kip1 may prevent inappropriate initiation of DNA replication prior to S phase.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Replicación del ADN , Proteínas de Unión al ADN/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Nucleares/metabolismo , Sitios de Unión , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/fisiología , Fase G1 , Sustancias de Crecimiento/farmacología , Humanos , Péptidos y Proteínas de Señalización Intracelular/fisiología , Componente 7 del Complejo de Mantenimiento de Minicromosoma , Unión Proteica , Técnicas del Sistema de Dos HíbridosRESUMEN
The cyclin-dependent kinase inhibitors are key regulators of cell cycle progression. Although implicated in carcinogenesis, they inhibit the proliferation of a variety of normal cell types, and their role in diverse human diseases is not fully understood. Here, we report that p27(Kip1) plays a major role in cardiovascular disease through its effects on the proliferation of bone marrow-derived (BM-derived) immune cells that migrate into vascular lesions. Lesion formation after mechanical arterial injury was markedly increased in mice with homozygous deletion of p27(Kip1), characterized by prominent vascular infiltration by immune and inflammatory cells. Vascular occlusion was substantially increased when BM-derived cells from p27(-/-) mice repopulated vascular lesions induced by mechanical injury in p27(+/+) recipients, in contrast to p27(+/+) BM donors. To determine the contribution of immune cells to vascular injury, transplantation was performed with BM derived from RAG(-/-) and RAG(+/+) mice. RAG(+/+) BM markedly exacerbated vascular proliferative lesions compared with what was found in RAG(-/-) donors. Taken together, these findings suggest that vascular repair and regeneration is regulated by the proliferation of BM-derived hematopoietic and nonhematopoietic cells through a p27(Kip1)-dependent mechanism and that immune cells largely mediate these effects.