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BACKGROUND: Clearance of damaged mitochondria via mitophagy is crucial for cellular homeostasis. Apart from Parkin, little is known about additional Ub (ubiquitin) ligases that mediate mitochondrial ubiquitination and turnover, particularly in highly metabolically active organs such as the heart. METHODS: In this study, we have combined in silico analysis and biochemical assay to identify CRL (cullin-RING ligase) 5 as a mitochondrial Ub ligase. We generated cardiomyocytes and mice lacking RBX2 (RING-box protein 2; also known as SAG [sensitive to apoptosis gene]), a catalytic subunit of CRL5, to understand the effects of RBX2 depletion on mitochondrial ubiquitination, mitophagy, and cardiac function. We also performed proteomics analysis and RNA-sequencing analysis to define the impact of loss of RBX2 on the proteome and transcriptome. RESULTS: RBX2 and CUL (cullin) 5, 2 core components of CRL5, localize to mitochondria. Depletion of RBX2 inhibited mitochondrial ubiquitination and turnover, impaired mitochondrial membrane potential and respiration, increased cardiomyocyte cell death, and has a global impact on the mitochondrial proteome. In vivo, deletion of the Rbx2 gene in adult mouse hearts suppressed mitophagic activity, provoked accumulation of damaged mitochondria in the myocardium, and disrupted myocardial metabolism, leading to the rapid development of dilated cardiomyopathy and heart failure. Similarly, ablation of RBX2 in the developing heart resulted in dilated cardiomyopathy and heart failure. The action of RBX2 in mitochondria is not dependent on Parkin, and Parkin gene deletion had no impact on the onset and progression of cardiomyopathy in RBX2-deficient hearts. Furthermore, RBX2 controls the stability of PINK1 (PTEN-induced kinase 1) in mitochondria. CONCLUSIONS: These findings identify RBX2-CRL5 as a mitochondrial Ub ligase that regulates mitophagy and cardiac homeostasis in a Parkin-independent, PINK1-dependent manner.
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Ratones Noqueados , Mitocondrias Cardíacas , Mitofagia , Miocitos Cardíacos , Ubiquitinación , Animales , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Ratones , Mitocondrias Cardíacas/metabolismo , Mitocondrias Cardíacas/enzimología , Mitocondrias Cardíacas/genética , Mitocondrias Cardíacas/patología , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ratones Endogámicos C57BL , Humanos , Células Cultivadas , MasculinoRESUMEN
BACKGROUND: Pulmonary arterial hypertension (PAH) is high blood pressure in the lungs that originates from structural changes in small resistance arteries. A defining feature of PAH is the inappropriate remodeling of pulmonary arteries (PA) leading to right ventricle failure and death. Although treatment of PAH has improved, the long-term prognosis for patients remains poor, and more effective targets are needed. METHODS: Gene expression was analyzed by microarray, RNA sequencing, quantitative polymerase chain reaction, Western blotting, and immunostaining of lung and isolated PA in multiple mouse and rat models of pulmonary hypertension (PH) and human PAH. PH was assessed by digital ultrasound, hemodynamic measurements, and morphometry. RESULTS: Microarray analysis of the transcriptome of hypertensive rat PA identified a novel candidate, PBK (PDZ-binding kinase), that was upregulated in multiple models and species including humans. PBK is a serine/threonine kinase with important roles in cell proliferation that is minimally expressed in normal tissues but significantly increased in highly proliferative tissues. PBK was robustly upregulated in the medial layer of PA, where it overlaps with markers of smooth muscle cells. Gain-of-function approaches show that active forms of PBK increase PA smooth muscle cell proliferation, whereas silencing PBK, dominant negative PBK, and pharmacological inhibitors of PBK all reduce proliferation. Pharmacological inhibitors of PBK were effective in PH reversal strategies in both mouse and rat models, providing translational significance. In a complementary genetic approach, PBK was knocked out in rats using CRISPR/Cas9 editing, and loss of PBK prevented the development of PH. We found that PBK bound to PRC1 (protein regulator of cytokinesis 1) in PA smooth muscle cells and that multiple genes involved in cytokinesis were upregulated in experimental models of PH and human PAH. Active PBK increased PRC1 phosphorylation and supported cytokinesis in PA smooth muscle cells, whereas silencing or dominant negative PBK reduced cytokinesis and the number of cells in the G2/M phase of the cell cycle. CONCLUSIONS: PBK is a newly described target for PAH that is upregulated in proliferating PA smooth muscle cells, where it contributes to proliferation through changes in cytokinesis and cell cycle dynamics to promote medial thickening, fibrosis, increased PA resistance, elevated right ventricular systolic pressure, right ventricular remodeling, and PH.
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Hipertensión Arterial Pulmonar , Arteria Pulmonar , Remodelación Vascular , Animales , Humanos , Ratas , Ratones , Masculino , Hipertensión Arterial Pulmonar/metabolismo , Hipertensión Arterial Pulmonar/genética , Hipertensión Arterial Pulmonar/fisiopatología , Hipertensión Arterial Pulmonar/patología , Arteria Pulmonar/metabolismo , Arteria Pulmonar/patología , Arteria Pulmonar/fisiopatología , Modelos Animales de Enfermedad , Ratas Sprague-Dawley , Proliferación Celular , Ratones Endogámicos C57BL , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Quinasas de Proteína Quinasa Activadas por MitógenosRESUMEN
BACKGROUND: Obesity is associated with increased risk of cardiovascular disease, but underlying mechanisms remain elusive. Metabolic dysfunction, especially hyperglycemia, is thought to be a major contributor, but how glucose impacts vascular function is unclear. GAL3 (galectin-3) is a sugar-binding lectin upregulated by hyperglycemia, but its role as a causative mechanism of cardiovascular disease remains poorly understood. Therefore, the objective of this study was to determine the role of GAL3 in regulating microvascular endothelial vasodilation in obesity. METHODS: GAL3 was measured and found to be markedly increased in the plasma of overweight and obese patients, as well as in the microvascular endothelium of diabetic patients. To investigate causative mechanisms in cardiovascular disease, mice deficient in GAL3 were bred with obese db/db mice to generate lean, lean GAL3 knockout, obese, and obese GAL3 knockout genotypes. Endothelial cell-specific GAL3 knockout mice with novel AAV-induced obesity recapitulated whole-body knockout studies to confirm cell specificity. RESULTS: Deletion of GAL3 did not alter body mass, adiposity, or plasma indices of glycemia and lipidemia, but levels of plasma reactive oxygen species as assessed by plasma thiobarbituric acid reactive substances were normalized in obese GAL3 knockout mice. Obese mice exhibited profound endothelial dysfunction and hypertension, both of which were rescued by GAL3 deletion. Isolated microvascular endothelial cells from obese mice had increased expression of NOX1 (nicotinamide adenine dinucleotide phosphate oxidase 1), which we have previously shown to contribute to increased oxidative stress and endothelial dysfunction, which was normalized in microvascular endothelium from mice lacking GAL3. Cell-specific deletion confirmed that endothelial GAL3 regulates obesity-induced NOX1 overexpression and subsequent microvascular function. Furthermore, improvement of metabolic syndrome by increasing muscle mass, improving insulin signaling, or treating with metformin decreased microvascular GAL3, and thereby NOX1, expression levels. CONCLUSIONS: Deletion of GAL3 normalizes microvascular endothelial function in obese db/db mice, likely through a NOX1-mediated mechanism. Pathological levels of GAL3, and in turn NOX1, are amenable to improvements in metabolic status, presenting a potential therapeutic target to ameliorate pathological cardiovascular consequences of obesity.
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Enfermedades Cardiovasculares , Hiperglucemia , Hipertensión , Animales , Humanos , Ratones , Células Endoteliales/metabolismo , Endotelio Vascular/metabolismo , Galectina 3/genética , Galectina 3/metabolismo , Hiperglucemia/metabolismo , Ratones Noqueados , Ratones Obesos , NADPH Oxidasa 1/metabolismo , NADPH Oxidasas/metabolismo , Obesidad/complicaciones , Obesidad/genética , Obesidad/metabolismo , Estrés OxidativoRESUMEN
Sepsis is a major cause of mortality in intensive care units, which results from a severely dysregulated inflammatory response that ultimately leads to organ failure. While antibiotics can help in the early stages, effective strategies to curtail inflammation remain limited. The high mobility group (HMG) proteins are chromosomal proteins with important roles in regulating gene transcription. While HMGB1 has been shown to play a role in sepsis, the role of other family members including HMGXB4 remains unknown. We found that expression of HMGXB4 is strongly induced in response to lipopolysaccharide (LPS)-elicited inflammation in murine peritoneal macrophages. Genetic deletion of Hmgxb4 protected against LPS-induced lung injury and lethality and cecal ligation and puncture (CLP)-induced lethality in mice, and attenuated LPS-induced proinflammatory gene expression in cultured macrophages. By integrating genome-wide transcriptome profiling and a publicly available ChIP-seq dataset, we identified HMGXB4 as a transcriptional activator that regulates the expression of the proinflammatory gene, Nos2 (inducible nitric oxide synthase 2) by binding to its promoter region, leading to NOS2 induction and excessive NO production and tissue damage. Similar to Hmgxb4 ablation in mice, administration of a pharmacological inhibitor of NOS2 robustly decreased LPS-induced pulmonary vascular permeability and lethality in mice. Additionally, we identified the cell adhesion molecule, ICAM1, as a target of HMGXB4 in endothelial cells that facilitates inflammation by promoting monocyte attachment. In summary, our study reveals a critical role of HMGXB4 in exacerbating endotoxemia via transcriptional induction of Nos2 and Icam1 gene expression and thus targeting HMGXB4 may be an effective therapeutic strategy for the treatment of sepsis.
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Endotoxemia/metabolismo , Animales , Células Endoteliales/metabolismo , Endotoxemia/etiología , Endotoxemia/genética , Femenino , Molécula 1 de Adhesión Intercelular/genética , Molécula 1 de Adhesión Intercelular/metabolismo , Lipopolisacáridos/toxicidad , Pulmón/metabolismo , Pulmón/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/metabolismo , TranscriptomaRESUMEN
AIMS: Proliferation of vascular smooth muscle cells (VSMCs) is a hallmark of pulmonary hypertension (PH). Proliferative cells utilize purine bases from the de novo purine synthesis (DNPS) pathways for nucleotide synthesis; however, it is unclear whether DNPS plays a critical role in VSMC proliferation during development of PH. The last two steps of DNPS are catalysed by the enzyme 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/inosine monophosphate cyclohydrolase (ATIC). This study investigated whether ATIC-driven DNPS affects the proliferation of pulmonary artery smooth muscle cells (PASMCs) and the development of PH. METHODS AND RESULTS: Metabolites of DNPS in proliferative PASMCs were measured by liquid chromatography-tandem mass spectrometry. ATIC expression was assessed in platelet-derived growth factor-treated PASMCs and in the lungs of PH rodents and patients with pulmonary arterial hypertension. Mice with global and VSMC-specific knockout of Atic were utilized to investigate the role of ATIC in both hypoxia- and lung interleukin-6/hypoxia-induced murine PH. ATIC-mediated DNPS at the mRNA, protein, and enzymatic activity levels were increased in platelet-derived growth factor-treated PASMCs or PASMCs from PH rodents and patients with pulmonary arterial hypertension. In cultured PASMCs, ATIC knockdown decreased DNPS and nucleic acid DNA/RNA synthesis, and reduced cell proliferation. Global or VSMC-specific knockout of Atic attenuated vascular remodelling and inhibited the development and progression of both hypoxia- and lung IL-6/hypoxia-induced PH in mice. CONCLUSION: Targeting ATIC-mediated DNPS compromises the availability of purine nucleotides for incorporation into DNA/RNA, reducing PASMC proliferation and pulmonary vascular remodelling and ameliorating the development and progression of PH.
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Hipertensión Pulmonar , Hipertensión Arterial Pulmonar , Ratones , Animales , Roedores/metabolismo , Remodelación Vascular/fisiología , Arteria Pulmonar , Purinas/metabolismo , Células Cultivadas , Hipoxia/metabolismo , ARN Mensajero/metabolismo , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Proliferación Celular , Miocitos del Músculo Liso/metabolismoRESUMEN
Acute lung injury (ALI) is characterized by lung vascular endothelial cell (EC) barrier compromise resulting in increased endothelial permeability and pulmonary edema. The infection of gram-negative bacteria that produce toxins like LPS is one of the major causes of ALI. LPS activates Toll-like receptor 4, leading to cytoskeleton reorganization, resulting in lung endothelial barrier disruption and pulmonary edema in ALI. However, the signaling pathways that lead to the cytoskeleton reorganization and lung microvascular EC barrier disruption remain largely unexplored. Here we show that LPS induces calpain activation and talin cleavage into head and rod domains and that inhibition of calpain attenuates talin cleavage, RhoA activation, and pulmonary EC barrier disruption in LPS-treated human lung microvascular ECs in vitro and lung EC barrier disruption and pulmonary edema induced by LPS in ALI in vivo. Moreover, overexpression of calpain causes talin cleavage and RhoA activation, myosin light chain (MLC) phosphorylation, and increases in actin stress fiber formation. Furthermore, knockdown of talin attenuates LPS-induced RhoA activation and MLC phosphorylation and increased stress fiber formation and mitigates LPS-induced lung microvascular endothelial barrier disruption. Additionally, overexpression of talin head and rod domains increases RhoA activation, MLC phosphorylation, and stress fiber formation and enhances lung endothelial barrier disruption. Finally, overexpression of cleavage-resistant talin mutant reduces LPS-induced increases in MLC phosphorylation in human lung microvascular ECs and attenuates LPS-induced lung microvascular endothelial barrier disruption. These results provide the first evidence that calpain mediates LPS-induced lung microvascular endothelial barrier disruption in ALI via cleavage of talin.
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Lesión Pulmonar Aguda , Edema Pulmonar , Humanos , Lipopolisacáridos/farmacología , Calpaína/metabolismo , Talina/metabolismo , Pulmón/metabolismo , Lesión Pulmonar Aguda/inducido químicamente , Lesión Pulmonar Aguda/metabolismo , Cadenas Ligeras de Miosina/metabolismo , Permeabilidad CapilarRESUMEN
BACKGROUND: Vascular homeostasis is maintained by the differentiated phenotype of vascular smooth muscle cells (VSMCs). The landscape of protein coding genes comprising the transcriptome of differentiated VSMCs has been intensively investigated but many gaps remain including the emerging roles of noncoding genes. METHODS: We reanalyzed large-scale, publicly available bulk and single-cell RNA sequencing datasets from multiple tissues and cell types to identify VSMC-enriched long noncoding RNAs. The in vivo expression pattern of a novel smooth muscle cell (SMC)-expressed long noncoding RNA, Carmn (cardiac mesoderm enhancer-associated noncoding RNA), was investigated using a novel Carmn green fluorescent protein knock-in reporter mouse model. Bioinformatics and quantitative real-time polymerase chain reaction analysis were used to assess CARMN expression changes during VSMC phenotypic modulation in human and murine vascular disease models. In vitro, functional assays were performed by knocking down CARMN with antisense oligonucleotides and overexpressing Carmn by adenovirus in human coronary artery SMCs. Carotid artery injury was performed in SMC-specific Carmn knockout mice to assess neointima formation and the therapeutic potential of reversing CARMN loss was tested in a rat carotid artery balloon injury model. The molecular mechanisms underlying CARMN function were investigated using RNA pull-down, RNA immunoprecipitation, and luciferase reporter assays. RESULTS: We identified CARMN, which was initially annotated as the host gene of the MIR143/145 cluster and recently reported to play a role in cardiac differentiation, as a highly abundant and conserved, SMC-specific long noncoding RNA. Analysis of the Carmn GFP knock-in mouse model confirmed that Carmn is transiently expressed in embryonic cardiomyocytes and thereafter becomes restricted to SMCs. We also found that Carmn is transcribed independently of Mir143/145. CARMN expression is dramatically decreased by vascular disease in humans and murine models and regulates the contractile phenotype of VSMCs in vitro. In vivo, SMC-specific deletion of Carmn significantly exacerbated, whereas overexpression of Carmn markedly attenuated, injury-induced neointima formation in mouse and rat, respectively. Mechanistically, we found that Carmn physically binds to the key transcriptional cofactor myocardin, facilitating its activity and thereby maintaining the contractile phenotype of VSMCs. CONCLUSIONS: CARMN is an evolutionarily conserved SMC-specific long noncoding RNA with a previously unappreciated role in maintaining the contractile phenotype of VSMCs and is the first noncoding RNA discovered to interact with myocardin.
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Contracción Muscular , Músculo Liso Vascular/metabolismo , Músculo Liso/metabolismo , Proteínas Nucleares/metabolismo , ARN Largo no Codificante/metabolismo , Transactivadores/metabolismo , Animales , Humanos , Ratones , Proteínas Nucleares/genética , ARN Largo no Codificante/genética , Ratas , Transactivadores/genéticaRESUMEN
Rationale: Posttranscriptional modifications are implicated in vascular remodeling of pulmonary hypertension (PH). m6A (N6-methyladenosine) is an abundant RNA modification that is involved in various biological processes. Whether m6A RNA modification and m6A effector proteins play a role in pulmonary vascular remodeling and PH has not been demonstrated.Objectives: To determine whether m6A modification and m6A effectors contribute to the pathogenesis of PH.Methods: m6A modification and YTHDF1 expression were measured in human and experimental PH samples. RNA immunoprecipitation analysis and m6A sequencing were employed to screen m6A-marked transcripts. Genetic approaches were employed to assess the respective roles of YTHDF1 and MAGED1 in PH. Primary cell isolation and cultivation were used for function analysis of pulmonary artery smooth muscle cells (PASMCs).Measurements and Main Results: Elevated m6A levels and increased YTHDF1 protein expression were found in human and rodent PH samples as well as in hypoxic PASMCs. The deletion of YTHDF1 ameliorated PASMC proliferation, phenotype switch, and PH development both in vivo and in vitro. m6A RNA immunoprecipitation analysis identified MAGED1 as an m6A-regulated gene in PH, and genetic ablation of MAGED1 improved vascular remodeling and hemodynamic parameters in SU5416/hypoxia mice. YTHDF1 recognized and promoted translation of MAGED1 in an m6A-dependent manner that was absent in METTL3-deficient PASMCs. In addition, MAGED1 silencing inhibited hypoxia-induced proliferation of PASMCs through downregulating PCNA.Conclusions: YTHDF1 promotes PASMC proliferation and PH by enhancing MAGED1 translation. This study identifies the m6A RNA modification as a novel mediator of pathological changes in PASMCs and PH.
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Hipertensión Pulmonar/etiología , Hipertensión Pulmonar/patología , Proteínas de Neoplasias/metabolismo , Proteínas de Unión al ARN/metabolismo , Remodelación Vascular/fisiología , Adenosina/análogos & derivados , Adenosina/metabolismo , Animales , Técnicas de Cultivo de Célula , Proliferación Celular , Modelos Animales de Enfermedad , Humanos , Hipertensión Pulmonar/metabolismo , Ratones , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Miocitos del Músculo Liso , Arteria Pulmonar/metabolismo , Arteria Pulmonar/patologíaRESUMEN
Pulmonary Arterial Hypertension (PAH) is a progressive vascular disease arising from the narrowing of pulmonary arteries (PA) resulting in high pulmonary arterial blood pressure and ultimately right ventricular (RV) failure. A defining characteristic of PAH is the excessive remodeling of PA that includes increased proliferation, inflammation, and fibrosis. There is no cure for PAH nor interventions that effectively impede or reverse PA remodeling, and research over the past several decades has sought to identify novel molecular mechanisms of therapeutic benefit. Galectin-3 (Gal-3; Mac-2) is a carbohydrate-binding lectin that is remarkable for its chimeric structure, comprised of an N-terminal oligomerization domain and a C-terminal carbohydrate-recognition domain. Gal-3 is a regulator of changes in cell behavior that contribute to aberrant PA remodeling including cell proliferation, inflammation, and fibrosis, but its role in PAH is poorly understood. Herein, we summarize the recent literature on the role of Gal-3 in the development of PAH and provide experimental evidence supporting the ability of Gal-3 to influence reactive oxygen species (ROS) production, NOX enzyme expression, inflammation, and fibrosis, which contributes to PA remodeling. Finally, we address the clinical significance of Gal-3 as a target in the development of therapeutic agents as a treatment for PAH.
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Hipertensión Pulmonar , Hipertensión Arterial Pulmonar , Animales , Modelos Animales de Enfermedad , Fibrosis , Galectina 3/genética , Inflamación/patología , Arteria Pulmonar/patología , Especies Reactivas de Oxígeno , Remodelación VascularRESUMEN
During development, ventricular chamber maturation is a crucial step in the formation of a functionally competent postnatal heart. Defects in this process can lead to left ventricular noncompaction cardiomyopathy and heart failure. However, molecular mechanisms underlying ventricular chamber development remain incompletely understood. Neddylation is a posttranslational modification that attaches ubiquitin-like protein NEDD8 to protein targets via NEDD8-specific E1-E2-E3 enzymes. Here, we report that neddylation is temporally regulated in the heart and plays a key role in cardiac development. Cardiomyocyte-specific knockout of NAE1, a subunit of the E1 neddylation activating enzyme, significantly decreased neddylated proteins in the heart. Mice lacking NAE1 developed myocardial hypoplasia, ventricular noncompaction, and heart failure at late gestation, which led to perinatal lethality. NAE1 deletion resulted in dysregulation of cell cycle-regulatory genes and blockade of cardiomyocyte proliferation in vivo and in vitro, which was accompanied by the accumulation of the Hippo kinases Mst1 and LATS1/2 and the inactivation of the YAP pathway. Furthermore, reactivation of YAP signaling in NAE1-inactivated cardiomyocytes restored cell proliferation, and YAP-deficient hearts displayed a noncompaction phenotype, supporting an important role of Hippo-YAP signaling in NAE1-depleted hearts. Mechanistically, we found that neddylation regulates Mst1 and LATS2 degradation and that Cullin 7, a NEDD8 substrate, acts as the ubiquitin ligase of Mst1 to enable YAP signaling and cardiomyocyte proliferation. Together, these findings demonstrate a role for neddylation in heart development and, more specifically, in the maturation of ventricular chambers and also identify the NEDD8 substrate Cullin 7 as a regulator of Hippo-YAP signaling.
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Ventrículos Cardíacos/metabolismo , Miocardio/metabolismo , Miocitos Cardíacos/metabolismo , Proteína NEDD8/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Proteínas de Ciclo Celular , Proteínas Cullin/genética , Proteínas Cullin/metabolismo , Ventrículos Cardíacos/patología , Vía de Señalización Hippo , Ratones , Ratones Noqueados , Miocardio/patología , Miocitos Cardíacos/patología , Proteína NEDD8/genética , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Proteínas Señalizadoras YAPRESUMEN
Maintenance of the endothelial cell (EC) barrier is critical to vascular homeostasis and a loss of barrier integrity results in increased vascular permeability. While the mechanisms that govern increased EC permeability have been under intense investigation over the past several decades, the processes regulating the preservation/restoration of the EC barrier remain poorly understood. Herein we show that the extracellular purines, adenosine (Ado) and adenosine 5'-[γ-thio]-triphosphate (ATPγS) can strengthen the barrier function of human lung microvascular EC (HLMVEC). This ability involves protein kinase A (PKA) activation and decreases in myosin light chain 20 (MLC20) phosphorylation secondary to the involvement of MLC phosphatase (MLCP). In contrast to Ado, ATPγS-induced PKA activation is accompanied by a modest, but significant decrease in cyclic adenosine monophosphate (cAMP) levels supporting the existence of an unconventional cAMP-independent pathway of PKA activation. Furthermore, ATPγS-induced EC barrier strengthening does not involve the Rap guanine nucleotide exchange factor 3 (EPAC1) which is directly activated by cAMP but is instead dependent upon PKA-anchor protein 2 (AKAP2) expression. We also found that AKAP2 can directly interact with the myosin phosphatase-targeting protein MYPT1 and that depletion of AKAP2 abolished ATPγS-induced increases in transendothelial electrical resistance. Ado-induced strengthening of the HLMVEC barrier required the coordinated activation of PKA and EPAC1 in a cAMP-dependent manner. In summary, ATPγS-induced enhancement of the EC barrier is EPAC1-independent and is instead mediated by activation of PKA which is then guided by AKAP2, in a cAMP-independent mechanism, to activate MLCP which dephosphorylates MLC20 resulting in reduced EC contraction and preservation.
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Adenosina Trifosfato/análogos & derivados , Permeabilidad Capilar/efectos de los fármacos , Microvasos/efectos de los fármacos , Agonistas del Receptor Purinérgico P1/farmacología , Receptores Purinérgicos P1/efectos de los fármacos , Proteínas de Anclaje a la Quinasa A/genética , Proteínas de Anclaje a la Quinasa A/metabolismo , Adenosina Trifosfato/farmacología , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Impedancia Eléctrica , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Células HEK293 , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Microvasos/metabolismo , Cadenas Ligeras de Miosina/metabolismo , Fosfatasa de Miosina de Cadena Ligera/genética , Fosfatasa de Miosina de Cadena Ligera/metabolismo , Fosforilación , Receptores Purinérgicos P1/genética , Receptores Purinérgicos P1/metabolismo , Transducción de SeñalRESUMEN
A defining characteristic of pulmonary hypertension (PH) is the extensive remodeling of pulmonary arteries (PAs), which results in progressive increases in vascular resistance and stiffness and eventual failure of the right ventricle. There is no cure for PH and identification of novel molecular mechanisms that underlie increased proliferation, reduced apoptosis, and excessive extracellular matrix production in pulmonary artery smooth muscle cells (PASMCs) is a vital objective. Galectin-3 (Gal-3) is a chimeric lectin and potent driver of many aspects of fibrosis, but its role in regulating PASMC behavior in PH remains poorly understood. Herein, we evaluated the importance of increased Gal-3 expression and signaling on PA vascular remodeling and cardiopulmonary function in experimental models of PH. Gal-3 expression was quantified by qRT-PCR, immunoblotting, and immunofluorescence imaging, and its functional role was assessed by specific Gal-3 inhibitors and CRISPR/Cas9-mediated knockout of Gal-3 in the rat. In rat models of PH, we observed increased Gal-3 expression in PASMCs, which stimulated migration and resistance to apoptosis, whereas silencing or genetic deletion reduced cellular migration and PA fibrosis and increased apoptosis. Gal-3 inhibitors attenuated and reversed PA remodeling and fibrosis, as well as hemodynamic indices in monocrotaline (MCT)-treated rats in vivo. These results were supported by genetic deletion of Gal-3 in both MCT and Sugen Hypoxia rat models. In conclusion, our results suggest that elevated Gal-3 levels contribute to inappropriate PA remodeling in PH by enhancing multiple profibrotic mechanisms. Therapeutic strategies targeting Gal-3 may be of benefit in the treatment of PH.
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Apoptosis , Proliferación Celular , Galectina 3/biosíntesis , Regulación de la Expresión Génica , Hipertensión Pulmonar/metabolismo , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Fibrosis Pulmonar/metabolismo , Animales , Proteínas Sanguíneas , Modelos Animales de Enfermedad , Galectina 3/genética , Galectinas , Humanos , Hipertensión Pulmonar/genética , Hipertensión Pulmonar/patología , Masculino , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/patología , Fibrosis Pulmonar/genética , Fibrosis Pulmonar/patología , Ratas , Ratas Sprague-DawleyAsunto(s)
Dependovirus , Enfermedades Vasculares , Ratones , Animales , Dependovirus/genética , Obesidad/genética , Hiperfagia/genética , EncéfaloRESUMEN
The circadian clock is rhythmically expressed in blood vessels, but the interaction between the circadian clock and disturbed blood flow remains unclear. We examined the relationships between BMAL1 and CLOCK and 2 regulators of endothelial function, AKT1 and endothelial nitric oxide synthase (eNOS), in vascular regions of altered blood flow. We found that the aortic arch from WT mice exhibited reduced sensitivity to acetylcholine (Ach)-mediated relaxation relative to the thoracic aorta. In Clock-mutant (mut) mice the aorta exhibited a reduced sensitivity to Ach. In WT mice, the phosphorylated forms of eNOS and AKT were decreased in the aortic arch, while BMAL1 and CLOCK expression followed a similar pattern of reduction in the arch. In conditions of surgically induced flow reduction, phosphorylated-eNOS (serine 1177) increased, as did p-AKT in the ipsilateral left common carotid artery (LC) of WT mice. Similarly, BMAL1 and CLOCK exhibited increased expression after 5 days in the remodeled LC. eNOS expression was increased at 8 p.m. versus 8 a.m. in WT mice, and this pattern was abolished in mut and Bmal1-KO mice. These data suggest that the circadian clock may be a biomechanical and temporal sensor that acts to coordinate timing, flow dynamics, and endothelial function.
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Factores de Transcripción ARNTL/metabolismo , Aorta Torácica/metabolismo , Proteínas CLOCK/metabolismo , Enfermedades de las Arterias Carótidas/metabolismo , Arteria Carótida Externa/metabolismo , Ritmo Circadiano , Mecanotransducción Celular , Factores de Transcripción ARNTL/deficiencia , Factores de Transcripción ARNTL/genética , Animales , Aorta Torácica/efectos de los fármacos , Proteínas CLOCK/genética , Enfermedades de las Arterias Carótidas/genética , Enfermedades de las Arterias Carótidas/fisiopatología , Arteria Carótida Externa/fisiopatología , Arteria Carótida Externa/cirugía , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Regulación de la Expresión Génica , Genotipo , Ligadura , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Mutantes , Mutación , Óxido Nítrico Sintasa de Tipo III/metabolismo , Fenotipo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Flujo Sanguíneo Regional , Estrés Mecánico , Factores de Tiempo , Vasodilatación , Vasodilatadores/farmacologíaRESUMEN
The type 1 angiotensin II (ANG II) receptor (AT1R) undergoes internalization following stimulation by ANG II. Internalization reduces cell surface AT1Rs, and it is required for AT1R resensitization. In this process AT1R may interact with caveolin-1 (Cav1), the main scaffolding protein of caveolae. We hypothesized that the interaction between Cav1 and AT1R delays AT1R resensitization and thereby prevents sustained ANG II-induced resistance artery (RA) constriction under normal conditions and in experimental obesity. In rat and mouse skeletal muscle RA (diameter: â¼90-120 µm) ANG II-induced constrictions were reduced upon repeated (30-min apart) administrations. Upon disruption of caveolae with methyl-ß-cyclodextrin or in RA of Cav1 knockout mice, repeated ANG II applications resulted in essentially maintained constrictions. In vascular smooth muscle cells, AT1R interacted with Cav1, and the degree of cell surface interactions was reduced by long-term (15-min), but not short-term (2-min), exposure to ANG II. When Cav1 was silenced, the amount of membrane-associated AT1R was significantly reduced by a short-term ANG II exposure. Moreover, Cav1 knockout mice fed a high-fat diet exhibited augmented and sustained RA constriction to ANG II and had elevated systemic blood pressure, when compared with normal or high-fat fed wild-type mice. Thus, Cav1, through a direct interaction, delays internalization and subsequent resensitization of AT1R. We suggest that this mechanism prevents sustained ANG II-induced RA constriction and elevated systemic blood pressure in diet-induced obesity.
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Arterias/fisiopatología , Caveolina 2/metabolismo , Hipertensión/metabolismo , Obesidad/metabolismo , Receptor de Angiotensina Tipo 1/metabolismo , Resistencia Vascular , Vasoconstricción , Angiotensina II/farmacología , Animales , Arterias/efectos de los fármacos , Arterias/metabolismo , Caveolas/metabolismo , Caveolina 2/genética , Membrana Celular/metabolismo , Hipertensión/etiología , Hipertensión/fisiopatología , Masculino , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/fisiología , Obesidad/complicaciones , Unión Proteica , Ratas , Ratas Wistar , Receptor de Angiotensina Tipo 1/agonistas , Vasoconstrictores/farmacologíaRESUMEN
OBJECTIVE: Pulmonary hypertension (PH) is a progressive disease arising from remodeling and narrowing of pulmonary arteries (PAs) resulting in high pulmonary blood pressure and ultimately right ventricular failure. Elevated production of reactive oxygen species by NADPH oxidase 4 (Nox4) is associated with increased pressure in PH. However, the cellular location of Nox4 and its contribution to aberrant vascular remodeling in PH remains poorly understood. Therefore, we sought to identify the vascular cells expressing Nox4 in PAs and determine the functional relevance of Nox4 in PH. APPROACH AND RESULTS: Elevated expression of Nox4 was detected in hypertensive PAs from 3 rat PH models and human PH using qualititative real-time reverse transcription polymerase chain reaction, Western blot, and immunofluorescence. In the vascular wall, Nox4 was detected in both endothelium and adventitia, and perivascular staining was prominently increased in hypertensive lung sections, colocalizing with cells expressing fibroblast and monocyte markers and matching the adventitial location of reactive oxygen species production. Small-molecule inhibitors of Nox4 reduced adventitial reactive oxygen species generation and vascular remodeling as well as ameliorating right ventricular hypertrophy and noninvasive indices of PA stiffness in monocrotaline-treated rats as determined by morphometric analysis and high-resolution digital ultrasound. Nox4 inhibitors improved PH in both prevention and reversal protocols and reduced the expression of fibroblast markers in isolated PAs. In fibroblasts, Nox4 overexpression stimulated migration and proliferation and was necessary for matrix gene expression. CONCLUSION: These findings indicate that Nox4 is prominently expressed in the adventitia and contributes to altered fibroblast behavior, hypertensive vascular remodeling, and development of PH.
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Adventicia/enzimología , Hipertensión Pulmonar/enzimología , NADPH Oxidasas/metabolismo , Arteria Pulmonar/enzimología , Adventicia/efectos de los fármacos , Adventicia/patología , Animales , Antihipertensivos/farmacología , Movimiento Celular , Proliferación Celular , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Hipertensión Pulmonar Primaria Familiar , Fibroblastos/enzimología , Fibroblastos/patología , Células HEK293 , Humanos , Hipertensión Pulmonar/tratamiento farmacológico , Hipertensión Pulmonar/etiología , Hipertensión Pulmonar/genética , Hipertensión Pulmonar/patología , Hipertrofia Ventricular Derecha/enzimología , Hipertrofia Ventricular Derecha/patología , Hipertrofia Ventricular Derecha/prevención & control , Hipoxia/complicaciones , Indoles , Masculino , Ratones , Ratones Endogámicos C57BL , Monocrotalina , NADPH Oxidasa 4 , NADPH Oxidasas/antagonistas & inhibidores , NADPH Oxidasas/genética , Arteria Pulmonar/efectos de los fármacos , Arteria Pulmonar/patología , Pirroles , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Factores de Tiempo , Transfección , Regulación hacia ArribaRESUMEN
RATIONALE: Disruption of the circadian clock in mice produces vascular dysfunction as evidenced by impairments in endothelium-dependent signaling, vasomotion, and blood vessel remodeling. Although the altered function of endothelial NO synthase and the overproduction of reactive oxygen species are central to dysfunction of the endothelium, to date, the impact of the circadian clock on endothelial NO synthase coupling and vascular reactive oxygen species production is not known. OBJECTIVE: The goals of the present study were to determine whether deletion of a critical component of the circadian clock, Bmal1, can influence endothelial NO synthase coupling and reactive oxygen species levels in arteries from Bmal1-knockout (KO) mice. METHODS AND RESULTS: Endothelial function was reduced in aortae from Bmal1-KO mice and improved by scavenging reactive oxygen species with polyethylene glycol-superoxide dismutase and nonselectively inhibiting cyclooxygenase isoforms with indomethacin. Aortae from Bmal1-KO mice exhibited enhanced superoxide levels as determined by electron paramagnetic resonance spectroscopy and dihydroethidium fluorescence, an elevation that was abrogated by administration of nitro-l-arginine methyl ester. High-performance liquid chromatography analysis revealed a reduction in tetrahydrobiopterin and an increase in dihydrobiopterin levels in the lung and aorta of Bmal1-KO mice, whereas supplementation with tetrahydrobiopterin improved endothelial function in the circadian clock KO mice. Furthermore, levels of tetrahydrobiopterin, dihydrobiopterin, and the key enzymes that regulate biopterin bioavailability, GTP cyclohydrolase and dihydrofolate reductase exhibited a circadian expression pattern. CONCLUSIONS: Having an established influence in the metabolic control of glucose and lipids, herein, we describe a novel role for the circadian clock in metabolism of biopterins, with a significant impact in the vasculature, to regulate coupling of endothelial NO synthase, production of superoxide, and maintenance of endothelial function.
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Factores de Transcripción ARNTL/deficiencia , Aorta/metabolismo , Arterias/metabolismo , Relojes Circadianos/fisiología , Óxido Nítrico Sintasa de Tipo III/metabolismo , Superóxidos/metabolismo , Factores de Transcripción ARNTL/genética , Factores de Transcripción ARNTL/metabolismo , Animales , Aorta/citología , Arterias/citología , Biopterinas/análogos & derivados , Biopterinas/metabolismo , Células Cultivadas , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , GTP Ciclohidrolasa/metabolismo , Masculino , Ratones , Ratones Noqueados , Modelos Animales , Especies Reactivas de Oxígeno/metabolismo , Tetrahidrofolato Deshidrogenasa/metabolismoAsunto(s)
Anticoagulantes/uso terapéutico , Galectina 3/efectos adversos , Galectina 3/uso terapéutico , Hipertensión Pulmonar/etiología , Hipertensión Pulmonar/fisiopatología , Infarto del Miocardio/tratamiento farmacológico , Remodelación Vascular/efectos de los fármacos , Adulto , Anciano , Anciano de 80 o más Años , Animales , Femenino , Humanos , Masculino , Persona de Mediana Edad , Modelos Animales , RatasRESUMEN
Endothelial cells (ECs) form a semi-permeable barrier between the interior space of blood vessels and the underlying tissues. Pulmonary endothelial barrier integrity is maintained through coordinated cellular processes involving receptors, signaling molecules, junctional complexes, and protein-regulated cytoskeletal reorganization. In acute lung injury (ALI) or its more severe form acute respiratory distress syndrome (ARDS), the loss of endothelial barrier integrity secondary to endothelial dysfunction caused by severe pulmonary inflammation and/or infection leads to pulmonary edema and hypoxemia. Pro-inflammatory agonists such as histamine, thrombin, bradykinin, interleukin 1ß, tumor necrosis factor α, vascular endothelial growth factor, angiopoietin-2, and platelet-activating factor, as well as bacterial toxins and reactive oxygen species, cause dynamic changes in cytoskeletal structure, adherens junction disorganization, and detachment of vascular endothelial cadherin (VE-cadherin) from the actin cytoskeleton, leading to an increase in endothelial permeability. Endothelial interactions with leukocytes, platelets, and coagulation enhance the inflammatory response. Moreover, inflammatory infiltration and the associated generation of pro-inflammatory cytokines during infection cause EC death, resulting in further compromise of the structural integrity of lung endothelial barrier. Despite the use of potent antibiotics and aggressive intensive care support, the mortality of ALI is still high, because the mechanisms of pulmonary EC barrier disruption are not fully understood. In this review, we summarized recent advances in the studies of endothelial cytoskeletal reorganization, inter-endothelial junctions, endothelial inflammation, EC death, and endothelial repair in ALI and ARDS, intending to shed some light on the potential diagnostic and therapeutic targets in the clinical management of the disease.