RESUMO
Wound healing is facilitated by neoangiogenesis, a complex process that is essential to tissue repair in response to injury. MicroRNAs are small, noncoding RNAs that can regulate the wound healing process including stimulation of impaired angiogenesis that is associated with type-2 diabetes (T2D). Expression of miR-409-3p was significantly increased in the nonhealing skin wounds of patients with T2D compared to the non-wounded normal skin, and in the skin of a murine model with T2D. In response to high glucose, neutralization of miR-409-3p markedly improved EC growth and migration in human umbilical vein endothelial cells (HUVECs), promoted wound closure and angiogenesis as measured by increased CD31 in human skin organoids, while overexpression attenuated EC angiogenic responses. Bulk mRNA-Seq transcriptomic profiling revealed BTG2 as a target of miR-409-3p, where overexpression of miR-409-3p significantly decreased BTG2 mRNA and protein expression. A 3' untranslated region (3'-UTR) luciferase assay of BTG2 revealed decreased luciferase activity with overexpression of miR-409-3p, while inhibition had opposite effects. Mechanistically, in response to high glucose, miR-409-3p deficiency in ECs resulted in increased mTOR phosphorylation, meanwhile BTG-anti-proliferation factor 2 (BTG2) silencing significantly decreased mTOR phosphorylation. Endothelial-specific and tamoxifen-inducible miR-409-3p knockout mice (MiR-409IndECKO ) with hyperglycemia that underwent dorsal skin wounding showed significant improvement of wound closure, increased blood flow, granulation tissue thickness (GTT), and CD31 that correlated with increased BTG2 expression. Taken together, our results show that miR-409-3p is a critical mediator of impaired angiogenesis in diabetic skin wound healing.
Assuntos
Diabetes Mellitus Tipo 2 , Proteínas Imediatamente Precoces , MicroRNAs , Proteínas Supressoras de Tumor , Animais , Humanos , Camundongos , Angiogênese , Proliferação de Células/fisiologia , Diabetes Mellitus Tipo 2/genética , Glucose , Células Endoteliais da Veia Umbilical Humana/metabolismo , Proteínas Imediatamente Precoces/genética , Luciferases , Camundongos Obesos , MicroRNAs/genética , MicroRNAs/metabolismo , RNA Mensageiro , Serina-Treonina Quinases TOR , Proteínas Supressoras de Tumor/genética , Cicatrização/genéticaRESUMO
BACKGROUND: Current paradigms suggest that nitric oxide (NO) produced by endothelial cells (ECs) through endothelial nitric oxide synthase (eNOS) in the vessel wall is the primary regulator of blood flow and blood pressure. However, red blood cells (RBCs) also carry a catalytically active eNOS, but its role is controversial and remains undefined. This study aimed to elucidate the functional significance of RBC eNOS compared with EC eNOS for vascular hemodynamics and nitric oxide metabolism. METHODS: We generated tissue-specific loss- and gain-of-function models for eNOS by using cell-specific Cre-induced gene inactivation or reactivation. We created 2 founder lines carrying a floxed eNOS (eNOSflox/flox) for Cre-inducible knockout (KO), and gene construct with an inactivated floxed/inverted exon (eNOSinv/inv) for a Cre-inducible knock-in (KI), which respectively allow targeted deletion or reactivation of eNOS in erythroid cells (RBC eNOS KO or RBC eNOS KI mice) or in ECs (EC eNOS KO or EC eNOS KI mice). Vascular function, hemodynamics, and nitric oxide metabolism were compared ex vivo and in vivo. RESULTS: The EC eNOS KOs exhibited significantly impaired aortic dilatory responses to acetylcholine, loss of flow-mediated dilation, and increased systolic and diastolic blood pressure. RBC eNOS KO mice showed no alterations in acetylcholine-mediated dilation or flow-mediated dilation but were hypertensive. Treatment with the nitric oxide synthase inhibitor Nγ-nitro-l-arginine methyl ester further increased blood pressure in RBC eNOS KOs, demonstrating that eNOS in both ECs and RBCs contributes to blood pressure regulation. Although both EC eNOS KOs and RBC eNOS KOs had lower plasma nitrite and nitrate concentrations, the levels of bound NO in RBCs were lower in RBC eNOS KOs than in EC eNOS KOs. Reactivation of eNOS in ECs or RBCs rescues the hypertensive phenotype of the eNOSinv/inv mice, whereas the levels of bound NO were restored only in RBC eNOS KI mice. CONCLUSIONS: These data reveal that eNOS in ECs and RBCs contribute independently to blood pressure homeostasis.
Assuntos
Pressão Sanguínea/fisiologia , Células Endoteliais/metabolismo , Eritrócitos/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Óxido Nítrico/metabolismo , Acetilcolina/farmacologia , Animais , Doenças da Aorta/tratamento farmacológico , Arginina/análogos & derivados , Arginina/farmacologia , Pressão Sanguínea/efeitos dos fármacos , Contagem de Eritrócitos/métodos , Hipertensão/metabolismo , Hipertensão/fisiopatologia , CamundongosRESUMO
The fat mass and obesity gene (FTO) is a N6-methyladenosine RNA demethylase that was initially linked by Genome-wide association studies to increased rates of obesity. Subsequent studies have revealed multiple mass-independent effects of the gene, including cardiac myocyte contractility. We created a mouse with a conditional and inducible smooth muscle cell deletion of Fto (Myh11 Cre+ Ftofl/fl) and did not observe any changes in mouse body mass or mitochondrial metabolism. However, the mice had significantly decreased blood pressure (hypotensive), despite increased heart rate and sodium, and significantly increased plasma renin. Remarkably, the third-order mesenteric arteries from these mice had almost no myogenic tone or capacity to constrict to smooth muscle depolarization or phenylephrine. Microarray analysis from Fto-/--isolated smooth muscle cells demonstrated a significant decrease in serum response factor (Srf) and the downstream effectors Acta2, Myocd, and Tagln; this was confirmed in cultured human coronary arteries with FTO siRNA. We conclude Fto is an important component to the contractility of smooth muscle cells.NEW & NOTEWORTHY We show a key role for the fat mass obesity (FTO) gene in regulating smooth muscle contractility, possibly by methylation of serum response factor (Srf).
Assuntos
Estudo de Associação Genômica Ampla , Fator de Resposta Sérica , Camundongos , Humanos , Animais , Fator de Resposta Sérica/genética , Miócitos de Músculo Liso/metabolismo , Obesidade/genética , Contração Muscular , Dioxigenase FTO Dependente de alfa-Cetoglutarato/genética , Dioxigenase FTO Dependente de alfa-Cetoglutarato/metabolismoRESUMO
RATIONALE: Increasing prevalence of obesity and its associated risk with cardiovascular diseases demands a better understanding of the contribution of different cell types within this complex disease for developing new treatment options. Previous studies could prove a fundamental role of FTO (fat mass and obesity-associated protein) within obesity; however, its functional role within different cell types is less understood. OBJECTIVES: We identify endothelial FTO as a previously unknown central regulator of both obesity-induced metabolic and vascular alterations. METHODS AND RESULTS: We generated endothelial Fto-deficient mice and analyzed the impact of obesity on those mice. While the loss of endothelial FTO did not influence the development of obesity and dyslipidemia, it protected mice from high-fat diet-induced glucose intolerance and insulin resistance by increasing AKT (protein kinase B) phosphorylation in endothelial cells and skeletal muscle. Furthermore, loss of endothelial FTO prevented the development of obesity-induced hypertension by preserving myogenic tone in resistance arteries. In Fto-deficient arteries, microarray analysis identified upregulation of L-Pgds with significant increases in prostaglandin D2 levels. Blockade of prostaglandin D2 synthesis inhibited the myogenic tone protection in resistance arteries of endothelial Fto-deficient mice on high-fat diet; conversely, direct addition of prostaglandin D2 rescued myogenic tone in high-fat diet-fed control mice. Myogenic tone was increased in obese human arteries with FTO inhibitors or prostaglandin D2 application. CONCLUSIONS: These data identify endothelial FTO as a previously unknown regulator in the development of obesity-induced metabolic and vascular changes, which is independent of its known function in regulation of obesity.
Assuntos
Dioxigenase FTO Dependente de alfa-Cetoglutarato/genética , Endotélio Vascular/metabolismo , Obesidade/metabolismo , Dioxigenase FTO Dependente de alfa-Cetoglutarato/metabolismo , Animais , Artérias/metabolismo , Artérias/patologia , Endotélio Vascular/patologia , Humanos , Oxirredutases Intramoleculares/metabolismo , Lipocalinas/metabolismo , Masculino , Camundongos , Tono Muscular , Músculo Esquelético/metabolismo , Obesidade/genética , Obesidade/patologia , Prostaglandina D2/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
RATIONALE: Resistance arteries and conduit arteries rely on different relative contributions of endothelial-derived hyperpolarization versus nitric oxide to achieve dilatory heterocellular signaling. Anatomically, resistance arteries use myoendothelial junctions (MEJs), endothelial cell projections that make contact with smooth muscle cells. Conduit arteries have very few to no MEJs. OBJECTIVE: Determine if the presence of MEJs in conduit arteries can alter heterocellular signaling. METHODS AND RESULTS: We previously demonstrated that PAI-1 (plasminogen activator inhibitor-1) can regulate formation of MEJs. Thus, we applied pluronic gel containing PAI-1 directly to conduit arteries (carotid arteries) to determine if this could induce formation of MEJs. We found a significant increase in endothelial cell projections resembling MEJs that correlated with increased biocytin dye transfer from endothelial cells to smooth muscle cells. Next, we used pressure myography to investigate whether these structural changes were accompanied by a functional change in vasodilatory signaling. Interestingly, PAI-1-treated carotids underwent a switch from a conduit to resistance artery vasodilatory profile via diminished nitric oxide signaling and increased endothelial-derived hyperpolarization signaling in response to the endothelium-dependent agonists acetylcholine and NS309. After PAI-1 application, we also found a significant increase in carotid expression of endothelial alpha globin, a protein predominantly expressed in resistance arteries. Carotids from mice with PAI-1, but lacking alpha globin (Hba1-/-), demonstrated that l-nitro-arginine methyl ester, an inhibitor of nitric oxide signaling, was able to prevent arterial relaxation. CONCLUSIONS: The presence or absence of MEJs is an important determinant for influencing heterocellular communication in the arterial wall. In particular, alpha globin expression, induced within newly formed endothelial cell projections, may influence the balance between endothelial-derived hyperpolarization and nitric oxide-mediated vasodilation.
Assuntos
Artérias Carótidas/efeitos dos fármacos , Comunicação Celular/fisiologia , Células Endoteliais/efeitos dos fármacos , Junções Intercelulares/fisiologia , Músculo Liso Vascular/citologia , Vasodilatação/fisiologia , Acetilcolina/farmacologia , Animais , Artérias Carótidas/fisiologia , Comunicação Celular/efeitos dos fármacos , Células Endoteliais/fisiologia , Endotélio Vascular/fisiologia , Inibidores Enzimáticos/farmacologia , Indóis/farmacologia , Junções Intercelulares/efeitos dos fármacos , Junções Intercelulares/metabolismo , Masculino , Camundongos , Miografia/métodos , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico/antagonistas & inibidores , Óxido Nítrico/metabolismo , Oximas/farmacologia , Inibidor 1 de Ativador de Plasminogênio/farmacologia , Inibidores de Serina Proteinase/farmacologia , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologia , alfa-Globinas/metabolismoRESUMO
Pannexin 1 (Panx1) is a ubiquitously expressed protein forming large conductance channels that are central to many distinct inflammation and injury responses. There is accumulating evidence showing ATP released from Panx1 channels, as well as metabolites, provide effective paracrine and autocrine signaling molecules that regulate different elements of the injury response. As channels with a broad range of permselectivity, Panx1 channels mediate the secretion and uptake of multiple solutes, ranging from calcium to bacterial derived molecules. In this review, we describe how Panx1 functions in response to different pro-inflammatory stimuli, focusing mainly on signaling coordinated by the vasculature. How Panx1 mediates ATP release by injured cells is also discussed. The ability of Panx1 to serve as a central component of many diverse physiologic responses has proven to be critically dependent on the context of expression, post-translational modification, interacting partners, and the mode of stimulation.
Assuntos
Conexinas/metabolismo , Inflamação/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Traumatismo por Reperfusão/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Humanos , Transdução de Sinais/fisiologiaRESUMO
Pannexin 1 (PANX1)-mediated ATP release in vascular smooth muscle coordinates α1-adrenergic receptor (α1-AR) vasoconstriction and blood pressure homeostasis. We recently identified amino acids 198-200 (YLK) on the PANX1 intracellular loop that are critical for α1-AR-mediated vasoconstriction and PANX1 channel function. We report herein that the YLK motif is contained within an SRC homology 2 domain and is directly phosphorylated by SRC proto-oncogene, nonreceptor tyrosine kinase (SRC) at Tyr198 We demonstrate that PANX1-mediated ATP release occurs independently of intracellular calcium but is sensitive to SRC family kinase (SFK) inhibition, suggestive of channel regulation by tyrosine phosphorylation. Using a PANX1 Tyr198-specific antibody, SFK inhibitors, SRC knockdown, temperature-dependent SRC cells, and kinase assays, we found that PANX1-mediated ATP release and vasoconstriction involves constitutive phosphorylation of PANX1 Tyr198 by SRC. We specifically detected SRC-mediated Tyr198 phosphorylation at the plasma membrane and observed that it is not enhanced or induced by α1-AR activation. Last, we show that PANX1 immunostaining is enriched in the smooth muscle layer of arteries from hypertensive humans and that Tyr198 phosphorylation is detectable in these samples, indicative of a role for membrane-associated PANX1 in small arteries of hypertensive humans. Our discovery adds insight into the regulation of PANX1 by post-translational modifications and connects a significant purinergic vasoconstriction pathway with a previously identified, yet unexplored, tyrosine kinase-based α1-AR constriction mechanism. This work implicates SRC-mediated PANX1 function in normal vascular hemodynamics and suggests that Tyr198-phosphorylated PANX1 is involved in hypertensive vascular pathology.
Assuntos
Tirosina/metabolismo , Quinases da Família src/metabolismo , Animais , Cálcio/metabolismo , Membrana Celular/metabolismo , Células Cultivadas , Conexinas/efeitos dos fármacos , Conexinas/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/efeitos dos fármacos , Proteínas do Tecido Nervoso/metabolismo , Fenilefrina/farmacologia , Fosforilação , Proto-Oncogene Mas , Quinases da Família src/químicaRESUMO
Discrete calcium signals within the vascular endothelium decrease with age and contribute to impaired endothelial-dependent vasodilation. Calreticulin (Calr), a multifunctional calcium binding protein and endoplasmic reticulum (ER) chaperone, can mediate calcium signals and vascular function within the endothelial cells (ECs) of small resistance arteries. We found Calr protein expression significantly decreases with age in mesenteric arteries and examined the functional role of EC Calr in vasodilation and calcium mobilization in the context of aging. Third-order mesenteric arteries from mice with or without EC Calr knockdown were examined for calcium signals and constriction to phenylephrine (PE) or vasodilation to carbachol (CCh) after 75 wk of age. PE constriction in aged mice with or without EC Calr was unchanged. However, calcium signals and vasodilation to endothelial-dependent agonist carbachol were significantly impaired in aged EC Calr knockdown mice. Ex vivo incubation of arteries with the ER stress inhibitor tauroursodeoxycholic acid (TUDCA) significantly improved vasodilation in mice lacking EC Calr. Our data suggests diminished vascular Calr expression with age can contribute to the detrimental effects of aging on endothelial calcium regulation and vasodilation.NEW & NOTEWORTHY Calreticulin (Calr) is responsible for key physiological processes in endoplasmic reticulum, especially in aging tissue. In particular, endothelial Calr is crucial to vascular function. In this study, we deleted Calr from the endothelium and aged the mice up to 75 wk to examine changes in vascular function. We found two key differences: 1) calcium events in endothelium were severely diminished after muscarinic stimulation, which 2) corresponded with a dramatic decrease in muscarinic vasodilation. Remarkably, we were able to rescue the effect of Calr deletion on endothelial-dependent vasodilatory function using tauroursodeoxycholic acid (TUDCA), an inhibitor of endoplasmic reticulum stress that is currently in clinical trials.
Assuntos
Envelhecimento/metabolismo , Calreticulina/metabolismo , Endotélio Vascular/metabolismo , Envelhecimento/fisiologia , Animais , Sinalização do Cálcio , Calreticulina/genética , Carbacol/farmacologia , Endotélio Vascular/fisiologia , Deleção de Genes , Masculino , Artérias Mesentéricas/efeitos dos fármacos , Artérias Mesentéricas/metabolismo , Artérias Mesentéricas/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Fenilefrina/farmacologia , Ácido Tauroquenodesoxicólico/farmacologia , Vasoconstritores/farmacologia , VasodilataçãoRESUMO
RATIONALE: Resistant hypertension is a major health concern with unknown cause. Spironolactone is an effective antihypertensive drug, especially for patients with resistant hypertension, and is considered by the World Health Organization as an essential medication. Although spironolactone can act at the mineralocorticoid receptor (MR; NR3C2), there is increasing evidence of MR-independent effects of spironolactone. OBJECTIVE: Here, we detail the unexpected discovery that Panx1 (pannexin 1) channels could be a relevant in vivo target of spironolactone. METHODS AND RESULTS: First, we identified spironolactone as a potent inhibitor of Panx1 in an unbiased small molecule screen, which was confirmed by electrophysiological analysis. Next, spironolactone inhibited α-adrenergic vasoconstriction in arterioles from mice and hypertensive humans, an effect dependent on smooth muscle Panx1, but independent of the MR NR3C2. Last, spironolactone acutely lowered blood pressure, which was dependent on smooth muscle cell expression of Panx1 and independent of NR3C2. This effect, however, was restricted to steroidal MR antagonists as a nonsteroidal MR antagonist failed to reduced blood pressure. CONCLUSIONS: These data suggest new therapeutic modalities for resistant hypertension based on Panx1 inhibition.
Assuntos
Anti-Hipertensivos/farmacologia , Conexinas/antagonistas & inibidores , Diuréticos/farmacologia , Hipertensão/tratamento farmacológico , Antagonistas de Receptores de Mineralocorticoides/farmacologia , Proteínas do Tecido Nervoso/antagonistas & inibidores , Espironolactona/farmacologia , Animais , Anti-Hipertensivos/uso terapêutico , Arteríolas/efeitos dos fármacos , Conexinas/metabolismo , Diuréticos/uso terapêutico , Células HEK293 , Humanos , Células Jurkat , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Antagonistas de Receptores de Mineralocorticoides/uso terapêutico , Proteínas do Tecido Nervoso/metabolismo , Espironolactona/uso terapêuticoRESUMO
Cardiovascular disease is a leading cause of death worldwide and accounts for >17.3 million deaths per year, with an estimated increase in incidence to 23.6 million by 2030. 1 Cardiovascular death represents 31% of all global deaths 2 -with stroke, heart attack, and ruptured aneurysms predominantly contributing to these high mortality rates. A key risk factor for cardiovascular disease is hypertension. Although treatment or reduction in hypertension can prevent the onset of cardiovascular events, existing therapies are only partially effective. A key pathological hallmark of hypertension is increased peripheral vascular resistance because of structural and functional changes in large (conductive) and small (resistance) arteries. In this review, we discuss the clinical implications of vascular remodeling, compare the differences between vascular smooth muscle cell remodeling in conductive and resistance arteries, discuss the genetic factors associated with vascular smooth muscle cell function in hypertensive patients, and provide a prospective assessment of current and future research and pharmacological targets for the treatment of hypertension.
Assuntos
Artérias/fisiopatologia , Hipertensão/fisiopatologia , Músculo Liso Vascular/fisiopatologia , Remodelação Vascular , Animais , Anti-Hipertensivos/uso terapêutico , Artérias/patologia , Doenças Cardiovasculares/fisiopatologia , Humanos , Hipertensão/tratamento farmacológico , Hipertensão/genética , Hipertensão/patologia , Inflamação/patologia , Inflamação/fisiopatologia , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologia , Miócitos de Músculo Liso/fisiologia , Fatores de Risco , Transdução de Sinais , Transmissão Sináptica/fisiologia , Resistência VascularRESUMO
OBJECTIVE: In resistance arteries, endothelial cell (EC) extensions can make contact with smooth muscle cells, forming myoendothelial junction at holes in the internal elastic lamina (HIEL). At these HIEL, calcium signaling is tightly regulated. Because Calr (calreticulin) can buffer ≈50% of endoplasmic reticulum calcium and is expressed throughout IEL holes in small arteries, the only place where myoendothelial junctions form, we investigated the effect of EC-specific Calr deletion on calcium signaling and vascular function. APPROACH AND RESULTS: We found Calr expressed in nearly every IEL hole in third-order mesenteric arteries, but not other ER markers. Because of this, we generated an EC-specific, tamoxifen inducible, Calr knockout mouse (EC Calr Δ/Δ). Using this mouse, we tested third-order mesenteric arteries for changes in calcium events at HIEL and vascular reactivity after application of CCh (carbachol) or PE (phenylephrine). We found that arteries from EC Calr Δ/Δ mice stimulated with CCh had unchanged activity of calcium signals and vasodilation; however, the same arteries were unable to increase calcium events at HIEL in response to PE. This resulted in significantly increased vasoconstriction to PE, presumably because of inhibited negative feedback. In line with these observations, the EC Calr Δ/Δ had increased blood pressure. Comparison of ER calcium in arteries and use of an ER-specific GCaMP indicator in vitro revealed no observable difference in ER calcium with Calr knockout. Using selective detergent permeabilization of the artery and inhibition of Calr translocation, we found that the observed Calr at HIEL may not be within the ER. CONCLUSIONS: Our data suggest that Calr specifically at HIEL may act in a non-ER dependent manner to regulate arteriolar heterocellular communication and blood pressure.
Assuntos
Pressão Sanguínea , Calbindina 2/metabolismo , Sinalização do Cálcio , Células Endoteliais/metabolismo , Junções Intercelulares/metabolismo , Artérias Mesentéricas/metabolismo , Miócitos de Músculo Liso/metabolismo , Comunicação Parácrina , Vasoconstrição , Animais , Pressão Sanguínea/efeitos dos fármacos , Calbindina 2/deficiência , Calbindina 2/genética , Sinalização do Cálcio/efeitos dos fármacos , Células Cultivadas , Células Endoteliais/efeitos dos fármacos , Humanos , Junções Intercelulares/efeitos dos fármacos , Masculino , Artérias Mesentéricas/efeitos dos fármacos , Camundongos Endogâmicos DBA , Camundongos Knockout , Miócitos de Músculo Liso/efeitos dos fármacos , Comunicação Parácrina/efeitos dos fármacos , Fenilefrina/farmacologia , Vasoconstrição/efeitos dos fármacos , Vasoconstritores/farmacologia , VasodilataçãoRESUMO
Objective- Sympathetic nerve innervation of vascular smooth muscle cells (VSMCs) is a major regulator of arteriolar vasoconstriction, vascular resistance, and blood pressure. Importantly, α-adrenergic receptor stimulation, which uniquely couples with Panx1 (pannexin 1) channel-mediated ATP release in resistance arteries, also requires localization to membrane caveolae. Here, we test whether localization of Panx1 to Cav1 (caveolin-1) promotes channel function (stimulus-dependent ATP release and adrenergic vasoconstriction) and is important for blood pressure homeostasis. Approach and Results- We use in vitro VSMC culture models, ex vivo resistance arteries, and a novel inducible VSMC-specific Cav1 knockout mouse to probe interactions between Panx1 and Cav1. We report that Panx1 and Cav1 colocalized on the VSMC plasma membrane of resistance arteries near sympathetic nerves in an adrenergic stimulus-dependent manner. Genetic deletion of Cav1 significantly blunts adrenergic-stimulated ATP release and vasoconstriction, with no direct influence on endothelium-dependent vasodilation or cardiac function. A significant reduction in mean arterial pressure (total=4 mm Hg; night=7 mm Hg) occurred in mice deficient for VSMC Cav1. These animals were resistant to further blood pressure lowering using a Panx1 peptide inhibitor Px1IL2P, which targets an intracellular loop region necessary for channel function. Conclusions- Translocalization of Panx1 to Cav1-enriched caveolae in VSMCs augments the release of purinergic stimuli necessary for proper adrenergic-mediated vasoconstriction and blood pressure homeostasis.
Assuntos
Pressão Sanguínea/fisiologia , Caveolina 1/metabolismo , Conexinas/metabolismo , Homeostase , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Trifosfato de Adenosina/metabolismo , Agonistas de Receptores Adrenérgicos alfa 1/farmacologia , Animais , Membrana Celular/metabolismo , Células Cultivadas , Humanos , Masculino , Camundongos Knockout , Músculo Liso Vascular/citologia , Músculo Liso Vascular/inervação , Fenilefrina/farmacologia , Sistema Nervoso Simpático/fisiologia , Vasoconstrição/fisiologiaRESUMO
Ischemia-reperfusion (I/R) injury (IRI), which involves inflammation, vascular permeability, and edema, remains a major challenge after lung transplantation. Pannexin-1 (Panx1) channels modulate cellular ATP release during inflammation. This study tests the hypothesis that endothelial Panx1 is a key mediator of vascular inflammation and edema after I/R and that IRI can be blocked by Panx1 antagonism. A murine hilar ligation model of IRI was used whereby left lungs underwent 1 h of ischemia and 2 h of reperfusion. Treatment of wild-type mice with Panx1 inhibitors (carbenoxolone or probenecid) significantly attenuated I/R-induced pulmonary dysfunction, edema, cytokine production, and neutrophil infiltration versus vehicle-treated mice. In addition, VE-Cad-CreERT2+/Panx1fl/fl mice (tamoxifen-inducible deletion of Panx1 in vascular endothelium) treated with tamoxifen were significantly protected from IRI (reduced dysfunction, endothelial permeability, edema, proinflammatory cytokines, and neutrophil infiltration) versus vehicle-treated mice. Furthermore, extracellular ATP levels in bronchoalveolar lavage fluid is Panx1-mediated after I/R as it was markedly attenuated by Panx1 antagonism in wild-type mice and by endothelial-specific Panx1 deficiency. Panx1 gene expression in lungs after I/R was also significantly elevated compared with sham. In vitro experiments demonstrated that TNF-α and/or hypoxia-reoxygenation induced ATP release from lung microvascular endothelial cells, which was attenuated by Panx1 inhibitors. This study is the first, to our knowledge, to demonstrate that endothelial Panx1 plays a key role in mediating vascular permeability, inflammation, edema, leukocyte infiltration, and lung dysfunction after I/R. Pharmacological antagonism of Panx1 activity may be a novel therapeutic strategy to prevent IRI and primary graft dysfunction after lung transplantation.
Assuntos
Conexinas/metabolismo , Células Endoteliais/metabolismo , Pulmão/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Edema Pulmonar/metabolismo , Traumatismo por Reperfusão/metabolismo , Vasculite/metabolismo , Animais , Permeabilidade Capilar/efeitos dos fármacos , Permeabilidade Capilar/genética , Carbenoxolona/farmacologia , Conexinas/genética , Modelos Animais de Doenças , Células Endoteliais/patologia , Inflamação/tratamento farmacológico , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Pulmão/irrigação sanguínea , Pulmão/patologia , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Probenecid/farmacologia , Edema Pulmonar/dietoterapia , Edema Pulmonar/genética , Edema Pulmonar/patologia , Traumatismo por Reperfusão/tratamento farmacológico , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/patologia , Vasculite/tratamento farmacológico , Vasculite/genética , Vasculite/patologiaRESUMO
Recent smooth muscle cell (SMC) lineage-tracing studies have revealed that SMCs undergo remarkable changes in phenotype during development of atherosclerosis. Of major interest, we demonstrated that Kruppel-like factor 4 (KLF4) in SMCs is detrimental for overall lesion pathogenesis, in that SMC-specific conditional knockout of the KLF4 gene ( Klf4) resulted in smaller, more-stable lesions that exhibited marked reductions in the numbers of SMC-derived macrophage- and mesenchymal stem cell-like cells. However, since the clinical consequences of atherosclerosis typically occur well after our reproductive years, we sought to identify beneficial KLF4-dependent SMC functions that were likely to be evolutionarily conserved. We tested the hypothesis that KLF4-dependent SMC transitions play an important role in the tissue injury-repair process. Using SMC-specific lineage-tracing mice positive and negative for simultaneous SMC-specific conditional knockout of Klf4, we demonstrate that SMCs in the remodeling heart after ischemia-reperfusion injury (IRI) express KLF4 and transition to a KLF4-dependent macrophage-like state and a KLF4-independent myofibroblast-like state. Moreover, heart failure after IRI was exacerbated in SMC Klf4 knockout mice. Surprisingly, we observed a significant cardiac dilation in SMC Klf4 knockout mice before IRI as well as a reduction in peripheral resistance. KLF4 chromatin immunoprecipitation-sequencing analysis on mesenteric vascular beds identified potential baseline SMC KLF4 target genes in numerous pathways, including PDGF and FGF. Moreover, microvascular tissue beds in SMC Klf4 knockout mice had gaps in lineage-traced SMC coverage along the resistance arteries and exhibited increased permeability. Together, these results provide novel evidence that Klf4 has a critical maintenance role within microvascular SMCs: it is required for normal SMC function and coverage of resistance arteries. NEW & NOTEWORTHY We report novel evidence that the Kruppel-like factor 4 gene ( Klf4) has a critical maintenance role within microvascular smooth muscle cells (SMCs). SMC-specific Klf4 knockout at baseline resulted in a loss of lineage-traced SMC coverage of resistance arteries, dilation of resistance arteries, increased blood flow, and cardiac dilation.
Assuntos
Fatores de Transcrição Kruppel-Like/metabolismo , Microvasos/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Miócitos de Músculo Liso/metabolismo , Animais , Fatores de Crescimento de Fibroblastos/metabolismo , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Macrófagos/metabolismo , Camundongos , Microvasos/citologia , Miofibroblastos/metabolismo , Fator de Crescimento Derivado de Plaquetas/metabolismo , RegeneraçãoAssuntos
Conexinas/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Conexinas/antagonistas & inibidores , Conexinas/genética , Deleção de Genes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , Traumatismo por Reperfusão Miocárdica/genética , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/genética , Probenecid/farmacologia , Probenecid/uso terapêuticoRESUMO
Control of blood flow distribution and tissue homeostasis depend on the tight regulation of and coordination between the microvascular network and circulating blood cells. Channels formed by connexins or pannexins that connect the intra- and extracellular compartments allow the release of paracrine signals, such as ATP and prostaglandins, and thus play a central role in achieving fine regulation and coordination of vascular function. This review focuses on vascular connexin hemichannels and pannexin channels. We review their expression pattern within the arterial and venous system with a special emphasis on how post-translational modifications by phosphorylation and S-nitrosylation of these channels modulate their function and contribute to vascular homeostasis. Furthermore, we highlight the contribution of these channels in smooth muscle cells and endothelial cells in the regulation of vasomotor tone as well as how these channels in endothelial cells regulate inflammatory responses such as during ischemic and hypoxic conditions. In addition, this review will touch on recent evidence implicating a role for these proteins in regulating red blood cell and platelet function.
Assuntos
Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/fisiologia , Conexinas/metabolismo , Inflamação/metabolismo , Inflamação/patologia , Proteínas do Tecido Nervoso/metabolismo , Animais , Conexinas/química , Conexinas/genética , Células Endoteliais/metabolismo , Humanos , Miócitos de Músculo Liso/metabolismo , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genéticaRESUMO
Connexin (Cx) 37 suppresses vascular and cancer cell proliferation. The C terminus and a channel able to function are necessary, and neither by itself is sufficient, for Cx37 to mediate growth suppression. Cx37 supports transmembrane and intercellular signaling by forming functional hemichannels (HCs) and gap junction channels (GJCs), respectively. Here we determined whether Cx37 with HC, but not GJC, functionality would suppress proliferation of rat insulinoma (Rin) cells comparably to wild-type Cx37 (Cx37-WT). We mutated extracellular loop residues hypothesized to compromise HC docking but not HC function (six cysteines mutated to alanine, C54A,C61A,C65A, C187A,C192A,C198A (designated as C6A); N55I; and Q58L). All three mutants trafficked to the plasma membrane and formed protein plaques comparably to Cx37-WT. None of the mutants formed functional GJCs, and Cx37-C6A did not form functional HCs. Cx37-N55I and -Q58L formed HCs with behavior and permeation properties similar to Cx37-WT (especially Q58L), but none of the mutants suppressed Rin cell proliferation. The data indicate that determinants of Cx37 HC function differ from other Cxs and that HC functions with associated HC-supported protein-protein interactions are not sufficient for Cx37 to suppress Rin cell proliferation. Together with previously published data, these results suggest that Cx37 suppresses Rin cell proliferation only when in a specific conformation achieved by interaction of the C terminus with a Cx37 pore-forming domain able to open as a GJC.
Assuntos
Proliferação de Células , Conexinas/fisiologia , Animais , Linhagem Celular , Linhagem Celular Tumoral , Junções Comunicantes/metabolismo , Insulinoma , Ativação do Canal Iônico , Potenciais da Membrana , Mutação de Sentido Incorreto , Transporte Proteico , Ratos , Transdução de Sinais , Proteína alfa-4 de Junções ComunicantesAssuntos
Betacoronavirus/patogenicidade , Conexinas/efeitos dos fármacos , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Proteínas do Tecido Nervoso/efeitos dos fármacos , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Betacoronavirus/efeitos dos fármacos , COVID-19 , Infecções por Coronavirus/tratamento farmacológico , Humanos , Pandemias , Pneumonia Viral/diagnóstico , Pneumonia Viral/tratamento farmacológico , SARS-CoV-2RESUMO
Pannexin channels are newly discovered ATP release channels expressed throughout the body. Pannexin 1 (Panx1) channels have become of great interest as they appear to participate in a multitude of signalling cascades, including regulation of vascular function. Although numerous Panx1 pharmacological inhibitors have been discovered, these inhibitors are not specific for Panx1 and have additional effects on other proteins. Therefore, molecular tools, such as RNA interference and knockout animals, are needed to demonstrate the role of pannexins in various cellular functions. This review focuses on the known roles of Panx1 related to purinergic signalling in the vasculature focusing on post-translational modifications and channel gating mechanisms that may participate in the regulated release of ATP.