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1.
Nat Commun ; 11(1): 3953, 2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32769974

RESUMO

Many important cell types in adult vertebrates have a mesenchymal origin, including fibroblasts and vascular mural cells. Although their biological importance is undisputed, the level of mesenchymal cell heterogeneity within and between organs, while appreciated, has not been analyzed in detail. Here, we compare single-cell transcriptional profiles of fibroblasts and vascular mural cells across four murine muscular organs: heart, skeletal muscle, intestine and bladder. We reveal gene expression signatures that demarcate fibroblasts from mural cells and provide molecular signatures for cell subtype identification. We observe striking inter- and intra-organ heterogeneity amongst the fibroblasts, primarily reflecting differences in the expression of extracellular matrix components. Fibroblast subtypes localize to discrete anatomical positions offering novel predictions about physiological function(s) and regulatory signaling circuits. Our data shed new light on the diversity of poorly defined classes of cells and provide a foundation for improved understanding of their roles in physiological and pathological processes.


Assuntos
Diferenciação Celular , Fibroblastos/fisiologia , Células-Tronco Mesenquimais/fisiologia , Miócitos de Músculo Liso/fisiologia , Pericitos/fisiologia , Animais , Separação Celular , Vasos Coronários/citologia , Matriz Extracelular/metabolismo , Fibroblastos/citologia , Citometria de Fluxo , Intestinos/irrigação sanguínea , Intestinos/citologia , Masculino , Camundongos , Músculo Esquelético/irrigação sanguínea , Músculo Esquelético/citologia , Músculo Liso Vascular/citologia , Miocárdio/citologia , Miócitos de Músculo Liso/citologia , Pericitos/citologia , RNA-Seq , Análise de Célula Única , Bexiga Urinária/irrigação sanguínea , Bexiga Urinária/citologia
2.
Proc Natl Acad Sci U S A ; 117(28): 16127-16137, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32601214

RESUMO

Thrombogenic reaction, aggressive smooth muscle cell (SMC) proliferation, and sluggish endothelial cell (EC) migration onto bioinert metal vascular stents make poststenting reendothelialization a dilemma. Here, we report an easy to perform, biomimetic surface engineering strategy for multiple functionalization of metal vascular stents. We first design and graft a clickable mussel-inspired peptide onto the stent surface via mussel-inspired adhesion. Then, two vasoactive moieties [i.e., the nitric-oxide (NO)-generating organoselenium (SeCA) and the endothelial progenitor cell (EPC)-targeting peptide (TPS)] are clicked onto the grafted surfaces via bioorthogonal conjugation. We optimize the blood and vascular cell compatibilities of the grafted surfaces through changing the SeCA/TPS feeding ratios. At the optimal ratio of 2:2, the surface-engineered stents demonstrate superior inhibition of thrombosis and SMC migration and proliferation, promotion of EPC recruitment, adhesion, and proliferation, as well as prevention of in-stent restenosis (ISR). Overall, our biomimetic surface engineering strategy represents a promising solution to address clinical complications of cardiovascular stents and other blood-contacting metal materials.


Assuntos
Adesivos/química , Materiais Revestidos Biocompatíveis/química , Peptídeos/química , Stents , Adesivos/síntese química , Animais , Materiais Biomiméticos/síntese química , Materiais Biomiméticos/química , Adesão Celular , Movimento Celular , Proliferação de Células , Células Cultivadas , Química Click , Células Progenitoras Endoteliais/citologia , Endotélio Vascular/citologia , Endotélio Vascular/fisiologia , Humanos , Miócitos de Músculo Liso/citologia , Óxido Nítrico/química , Compostos Organosselênicos/química , Peptídeos/síntese química , Proteínas/química , Coelhos , Stents/efeitos adversos , Trombose/etiologia , Trombose/prevenção & controle
3.
Life Sci ; 256: 118009, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32603819

RESUMO

AIMS: Abnormal mitochondrial metabolism is an essential factor for excessive proliferation of pulmonary artery smooth muscle cells (PASMCs), which drives the pathological process of pulmonary arterial hypertension (PAH). 3-Bromopyruvate (3-BrPA) is an effective glycolytic inhibitor that improves mitochondrial metabolism, thereby repressing anomalous cell proliferation. MAIN METHODS: An experimental PAH model was established by injection of monocrotaline (MCT) in male Sprague Dawley rats, following which rats were assigned to three groups: control, MCT, and 3-BrPA groups. Three days post injection of MCT, rats were treated with 3-BrPA or vehicle for 4 weeks. At the end of the study, hemodynamic data were measured to confirm PAH condition. Indicators of pulmonary arterial and right ventricular (RV) remodeling as well as the proliferative ability of PASMCs were assayed. Additionally, mitochondrial morphology and function, and antiglycolytic and antiproliferative pathways and genes were analyzed. KEY FINDINGS: Treatment with 3-BrPA effectively improved pulmonary vascular remodeling and right ventricular function, inhibited PASMC proliferation, and preserved mitochondrial morphology and function. Besides, 3-BrPA treatment inhibited the PI3K/AKT/mTOR pathway and regulated the expression of antiproliferative genes in PASMCs. However, bloody ascites, bloating, and cirrhosis of organs were observed in some 3-BrPA treated rats. SIGNIFICANCE: 3-BrPA acts as an important glycolytic inhibitor to improve energy metabolism and reverse the course of PAH. However, 3-BrPA is associated with side effects in MCT-induced rats, indicating that it should be caution in drug delivery dosage, and further studies are needed to evaluate this toxicological mechanism.


Assuntos
Mitocôndrias/efeitos dos fármacos , Hipertensão Arterial Pulmonar/tratamento farmacológico , Piruvatos/farmacologia , Animais , Modelos Animais de Doenças , Masculino , Mitocôndrias/metabolismo , Monocrotalina , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Hipertensão Arterial Pulmonar/fisiopatologia , Artéria Pulmonar/citologia , Artéria Pulmonar/efeitos dos fármacos , Piruvatos/toxicidade , Ratos , Ratos Sprague-Dawley , Serina-Treonina Quinases TOR/metabolismo
4.
Life Sci ; 257: 118053, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32634424

RESUMO

AIMS: Vascular smooth muscle cells (VSMCs) play a crucial role in the progression of atherosclerosis. Paired box 9 (Pax9) is a member of the Pax gene family which participates in the development of various tissues and organs. However, the effect of Pax9 on atherosclerosis and VSMCs and the underlying mechanisms remain unclear. MAIN METHODS: Western blotting was performed to assess Pax9 expression in atherosclerosis and VSMCs. Pax9 siRNA and overexpression plasmid were constructed to explore the biological function. Cell proliferation assay, phalloidin staining, and Transwell assay, accompanied by the sonic hedgehog (Shh) signaling pathway antagonist, cyclopamine (5 µM) and agonist, SAG (100 nM), were used to evaluate the VSMC phenotype, proliferation, and migration, as well as explore the associated mechanisms. KEY FINDINGS: We first discovered Pax9 to be significantly increased in atherosclerotic mice and platelet-derived growth factor-BB (PDGF-BB)-induced VSMCs. Pax9 knockdown inhibited the phenotypic transformation, proliferation, and migration of VSMCs, whereas the opposite effect was observed when Pax9 was overexpressed. Next, we established that Shh was activated in PDGF-BB-induced VSMCs. Moreover, Pax9 overexpression further activated Shh and exacerbated the phenotypic transformation, proliferation, and migration of PDGF-BB-induced VSMCs. These changes were effectively inhibited by treatment with the Shh signaling pathway antagonist. Consistently, Pax9 knockdown down-regulated Shh expression and inhibited the phenotypic transformation, proliferation, and migration of PDGF-BB-induced VSMCs. Treatment with the Shh signaling pathway agonist prevented these changes. SIGNIFICANCE: Pax9 regulated VSMC phenotypic transformation, proliferation, and migration via Shh, which may represent a novel target for the treatment of atherosclerosis.


Assuntos
Aterosclerose/genética , Proteínas Hedgehog/genética , Miócitos de Músculo Liso/citologia , Fator de Transcrição PAX9/genética , Animais , Aterosclerose/patologia , Becaplermina/metabolismo , Movimento Celular/genética , Proliferação de Células/genética , Cicloexilaminas/farmacologia , Técnicas de Silenciamento de Genes , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Transdução de Sinais/genética , Tiofenos/farmacologia , Alcaloides de Veratrum/farmacologia
5.
Life Sci ; 256: 117964, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32534036

RESUMO

AIMS: Vascular smooth muscle cells (VSMCs) are important regulators of vascular functions and their conversion to osteoblasts is a key to development of vascular calcification. This study aimed to characterize in vitro effect of hepatoma-derived growth factor (HDGF) on phenotypic conversion of cultured aortic VSMCs into osteoblast-like cells. MATERIALS AND METHODS: Cell proliferation and migration assays were used to examine cell behaviors. Western blotting, alkaline phosphatase activity and calcium staining were used to evaluate osteoblastic marker expression and function, respectively. KEY FINDINGS: Recombinant HDGF treatment enhanced VSMC growth and motility. Treatment of osteogenic medium (OM) increased expression of not only HDGF but also osteoblastic markers, including Runx2 and osteopontin (OPN), while VSMC marker α-smooth muscle actin (α-SMA) declined. Coincidentally, HDGF and OM treatment alone stimulated signaling activities in both PI3K/Akt and MAPK pathways. Conversely, inhibition of Akt and p38 significantly blocked the OM-upregulated HDGF, Runx2, and OPN expression and NF-κB phosphorylation, but did not reversed the α-SMA downregulation, implicating the involvement of Akt and p38 activities in the osteoblastic transformation of VSMCs. Small interfering RNA-mediated HDGF gene silencing effectively prevented the Runx2 and OPN upregulation, alkaline phosphatase activation, and calcium deposition, but did not affect the α-SMA levels in the transformed cells, supporting the involvement of HDGF in regulation of Runx2 and OPN expression. SIGNIFICANCE: In conclusion, in synergism with other osteogenic factor, HDGF may promote the progression of osteobastic transformation of VSMCs via Akt and p38 signaling pathways and contribute to vascular calcification in arteriosclerosis. CHEMICAL COMPOUNDS STUDIED IN THIS STUDY: HDGF (PubChem CID:); LY294002 (PubChem CID: 3973); PD98059 (PubChem CID: 4713); SB203580 (PubChem CID: 176155); SB431542 (PubChem CID: 4521392); SP600125 (PubChem CID: 8515); Wortmannin (PubChem CID: 312145).


Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/citologia , Osteoblastos/citologia , Animais , Biomarcadores/metabolismo , Linhagem Celular Transformada , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Inativação Gênica/efeitos dos fármacos , Cinética , Miócitos de Músculo Liso/efeitos dos fármacos , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteogênese/efeitos dos fármacos , Osteopontina/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos Sprague-Dawley , Regulação para Cima/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
6.
Proc Natl Acad Sci U S A ; 117(27): 15818-15826, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32541024

RESUMO

Atherosclerosis is the process underlying heart attack and stroke. Despite decades of research, its pathogenesis remains unclear. Dogma suggests that atherosclerotic plaques expand primarily via the accumulation of cholesterol and inflammatory cells. However, recent evidence suggests that a substantial portion of the plaque may arise from a subset of "dedifferentiated" vascular smooth muscle cells (SMCs) which proliferate in a clonal fashion. Herein we use multicolor lineage-tracing models to confirm that the mature SMC can give rise to a hyperproliferative cell which appears to promote inflammation via elaboration of complement-dependent anaphylatoxins. Despite being extensively opsonized with prophagocytic complement fragments, we find that this cell also escapes immune surveillance by neighboring macrophages, thereby exacerbating its relative survival advantage. Mechanistic studies indicate this phenomenon results from a generalized opsonin-sensing defect acquired by macrophages during polarization. This defect coincides with the noncanonical up-regulation of so-called don't eat me molecules on inflamed phagocytes, which reduces their capacity for programmed cell removal (PrCR). Knockdown or knockout of the key antiphagocytic molecule CD47 restores the ability of macrophages to sense and clear opsonized targets in vitro, allowing for potent and targeted suppression of clonal SMC expansion in the plaque in vivo. Because integrated clinical and genomic analyses indicate that similar pathways are active in humans with cardiovascular disease, these studies suggest that the clonally expanding SMC may represent a translational target for treating atherosclerosis.


Assuntos
Aterosclerose/metabolismo , Clonagem Molecular , Ativação do Complemento , Miócitos de Músculo Liso/metabolismo , Fagocitose/fisiologia , Animais , Antígeno CD47/metabolismo , Linhagem da Célula , Proliferação de Células , Complemento C3/genética , Complemento C3/metabolismo , Feminino , Humanos , Inflamação , Macrófagos/metabolismo , Masculino , Camundongos Knockout para ApoE , Miócitos de Músculo Liso/citologia , Placa Aterosclerótica/metabolismo , Análise de Sequência de RNA , Regulação para Cima
7.
Zhongguo Ying Yong Sheng Li Xue Za Zhi ; 36(1): 45-50, 2020 Jan 28.
Artigo em Chinês | MEDLINE | ID: mdl-32476372

RESUMO

OBJECTIVE: To investigate the probable roles of the novel C2H2 zinc finger transcription factor ZFP580 on all-transretinoic acid (ATRA)-regulated VSMCs migration and underlying mechanisms. METHODS: Rat aortic VSMCs were isolated, cultured and identified. VSMCs were treated with ATRA at the concentrations of 0, 5, 10 or 20 µmol/L for 24 hours. The migration ability of VSMCs was observed in each group and compared with control group which was treated by 0 µmol/L ATRA. The mRNA and protein expression levels of ZFP580 were detected by QPCR and Western blot. ZFP580 protein expression in VSMCs was detected under ATRA stimulation when ERK inhibitor PD98059 was used to inhibit the protein expression of ERK. Adenovirus transfection technology was used to obtain VSMCs with overexpression or low expression of ZFP580, and QPCR and Western blot were used to detect the mRNA and protein levels of MMP-2, MMP-9 and ZFP580. RESULTS: On the 10th day of VSMCs culture, immunofluorescence showed that SM22 alpha antibody, as a specific marker of smooth muscle cells, was positive. Compared to the control group, VSMCs migration was reduced by 32%, 43%, and 59% in the group of 5, 10, and 20 µmol/L ATRA pretreatment. Compared with the control group, VSMCs treated by 20 µmol/L ATRA reduced the cell migration by 49%, 36% and 22% at 24, 48 and 72 h. The mRNA and protein expression levels of ZFP580 were increased with the increase of ATRA stimulation solubility and the extension of stimulation time. ERK was increased significantly after 15 min of ATRA stimulation. Pretreatment with ERK inhibitor PD98059 (20 µmol/L) inhibited the expression of ERK protein and reduced the expression of ATRA-induced ZFP580 protein. Overexpression of ZFP580 inhibited the expressions of MMP-2 and MMP-9, whereas down-expression of ZFP580 promoted the expressions of MMP-2 and MMP-9. CONCLUSION: ATRA increased the expression of ZFP580 through the ERK signaling pathway, while ZFP580 was involved in ATRA's inhibition of VSMCs migration by affecting the expression of downstream MMP-2 and MMP-9.


Assuntos
Movimento Celular , Miócitos de Músculo Liso/citologia , Fatores de Transcrição/metabolismo , Tretinoína/farmacologia , Animais , Células Cultivadas , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Ratos , Transdução de Sinais
8.
Life Sci ; 255: 117822, 2020 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-32450174

RESUMO

AIM: Proliferation and migration of pulmonary artery smooth muscle cells (PASMCs) are regarded as the primary factors resulting in pulmonary arterial remodeling in pulmonary hypertension (PH). Myeloid ecotropic viral integration site 1 (MEIS1) has been positioned as a negative cardiomyocyte cell cycle regulator and regulates proliferation of multiple kinds of cancer cells. Whether MESI1 is involved in the proliferation and migration of PASMCs deserves to be identified. MAIN METHODS: Sprague Dawley rats were exposed to hypoxia condition (10% O2) for 4 weeks to induce PH and primary rat PASMCs were cultured in hypoxia condition (3% O2) for 48 h to induce proliferation and migration. Immunohistochemistry, immunofluorescence, reverse transcription PCR and Western blot analysis were performed to detect the expressions of target mRNAs and proteins. EDU, CCK8 and wound healing assays were conducted to measure the proliferation and migration of PASMCs. KEY FINDINGS: Hypoxia down-regulated the expression of MEIS1 (both mRNA and protein) in pulmonary arteries and PASMCs. Over-expression of MEIS1 inhibited the proliferation and migration of PASMCs afforded by hypoxia. In contrast, knockdown of MEIS1 under normoxia condition like hypoxia induced the proliferation and migration of PASMCs. MEIS1 mediated hypoxia-induced the proliferation and migration of PASMCs via METTL14/MEIS1/p21 signaling. SIGNIFICANCE: The present study revealed that MEIS1 regulated the proliferation and migration of PASMCs during hypoxia-induced PH. Thus, MEIS1 may be a potential target for PH therapy.


Assuntos
Hipertensão Pulmonar/fisiopatologia , Proteína Meis1/genética , Miócitos de Músculo Liso/citologia , Artéria Pulmonar/citologia , Animais , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Células Cultivadas , Hipertensão Pulmonar/genética , Hipóxia , Masculino , Músculo Liso Vascular/citologia , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Remodelação Vascular/fisiologia
9.
Life Sci ; 255: 117835, 2020 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-32450169

RESUMO

AIMS: Emerging findings demonstrate the critical roles of noncoding RNA (ncRNA) in asthma development. Nevertheless, the biological roles of circular RNA (circRNA) in airway remodeling are still elusive. Here, the present research focuses on the regulation of circRNA circHIPK3 in airway smooth muscle cells (ASMCs) proliferation and migration. MATERIALS AND METHODS: The sequence of circRNA was detected using Sanger sequencing. Cellular phenotypes were detected using CCK-8 assay, transwell and flow cytometer assay. The potential binding of miRNA and downstream and upstream targets was detected using dual-luciferase reporter assay. KEY FINDINGS: Results showed that circHIPK3 was significantly upregulated in platelet-derived growth factor (PDGF) induced ASMCs. Functional analysis using CCK-8, transwell migration assays and flow cytometry analysis showed that circHIPK3 knockdown repressed proliferation, migration and up-regulated the apoptosis in ASMCs. Mechanistic assays showed that circHIPK3 sponged miR-326 in the cytoplasm, thereby targeting stromal interaction molecule 1 (STIM1) to regulate ASMCs' proliferation, migration and apoptosis. SIGNIFICANCE: Collectively, the data elucidates that circHIPK3 functions as a regulator in the airway remodeling during the asthma development through miR-326/STIM1 axis, providing a novel insight for the therapeutic target.


Assuntos
Asma/fisiopatologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , MicroRNAs/genética , Miócitos de Músculo Liso/citologia , Proteínas de Neoplasias/genética , Proteínas Serina-Treonina Quinases/genética , Molécula 1 de Interação Estromal/genética , Remodelação das Vias Aéreas/genética , Apoptose/efeitos dos fármacos , Asma/genética , Proliferação de Células/genética , Técnicas de Silenciamento de Genes , Humanos , Fator de Crescimento Derivado de Plaquetas/metabolismo , RNA Circular/genética , Sistema Respiratório/citologia
10.
Life Sci ; 255: 117758, 2020 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-32407845

RESUMO

AIMS: NLR family pyrin domain containing 3 (NLRP3) inflammasome activation contributes to the development of diabetic cardiovascular complications. CD38 regulates vascular inflammation through cyclic ADP-ribose (cADPR)-mediated Ca2+ signaling in vascular smooth muscle cells (VSMCs). Ca2+ mobilization may modulate inflammasome activation by impacting mitochondrial function. However, it remains unclear whether CD38 regulates NLRP3 inflammasome activation in VSMCs through cADPR-dependent Ca2+ release under diabetic condition. Main methods and key findings: In VSMCs, we observed that high glucose (HG, 30 mM) enhanced CD38 protein expression and ADP ribosyl cyclase activity. Moreover, along with less abundance of NLRP3, apoptosis-associated speck-like protein containing CARD (ASC) and their colocalization, the expression of active caspase-1(p20) and IL-1ß were significantly inhibited by CD38 gene deficiency with siRNA transfection in VSMCs. Further, CD38 regulated the release of intracellular cADPR-mediated Ca2+ and mitochondrial DNA (mtDNA) to the cytosol, which was associated with NLRP3 inflammasome activation and VSMCs proliferation and collagen I synthesis. Finally, we found that CD38 inhibitors, nicotinamide and telmisartan significantly improved the endothelium-independent contraction and vascular remodeling, which was also associated with the inhibition of NLRP3 inflammasome in the aorta media in the diabetic mice. SIGNIFICANCE: Our data suggested that CD38/cADPR-mediated Ca2+ signaling contributed to the mitochondrial damage, consequently released mtDNA to the cytosol, which was related with NLRP3 inflammasome activation and VSMCs remodeling in diabetic mice.


Assuntos
ADP-Ribosil Ciclase 1/metabolismo , Cálcio/metabolismo , Diabetes Mellitus Experimental/fisiopatologia , Inflamassomos/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Animais , Sinalização do Cálcio/fisiologia , ADP-Ribose Cíclica/metabolismo , DNA Mitocondrial/metabolismo , Diabetes Mellitus Experimental/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Mitocôndrias/patologia , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/citologia
11.
Life Sci ; 253: 117659, 2020 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-32283055

RESUMO

Abdominal aortic aneurysm (AAA) is a chronic vascular degenerative disease featured by progressive dilation and remodeling of the vascular wall, which may lead to aortic rupture and high mortality. The occurrence and development of AAA involve multiple mechanisms, including extracellular matrix degradation, chronic inflammation, oxidative stress, apoptosis of vascular smooth muscle cells and innate immunity. Extracellular matrix degradation is considered as the most important mechanism causing AAA. Matrix metalloproteinases (MMPs) are key factors in this process, contributing greatly to the occurrence and development of AAA. But whether the zinc-dependent endopeptidases (ADAM/ADAMTS) are involved in this process is very little known. This study is a review about the role of MMPs and ADAM/ADAMT as well as the existing MMP inhibitors in abdominal aortic aneurysm, with the purpose of providing reference for the clinical treatment of abdominal aortic aneurysm.


Assuntos
Proteínas ADAM/metabolismo , Aneurisma da Aorta Abdominal/tratamento farmacológico , Inibidores de Metaloproteinases de Matriz/farmacologia , Metaloproteinases da Matriz/metabolismo , Animais , Endopeptidases/metabolismo , Humanos , Miócitos de Músculo Liso/citologia , Proteólise , Transdução de Sinais
12.
Artigo em Inglês | MEDLINE | ID: mdl-32130030

RESUMO

Mechanical tension and humoral stimuli can induce transitions in airway smooth muscle phenotype between a synthetic inflammatory state that promotes cytokine secretion and a differentiated state that promotes the expression of smooth muscle phenotype-specific proteins. When tissues are maintained under high tension, Akt activation and eotaxin secretion are suppressed, but expression of the differentiation marker protein, smooth muscle myosin heavy chain (SmMHC), is promoted. When tissues are maintained under low tension, Akt activation and eotaxin secretion are stimulated, and the differentiated phenotype is suppressed. We hypothesized that mechanical stimuli are differentially transduced to Akt-mediated signaling pathways that regulate phenotype expression by α-parvin and ß-parvin integrin-linked kinase/PINCH/parvin (IPP) signaling complexes within integrin adhesomes. High tension or ACh triggered paxillin phosphorylation and the binding of phospho-paxillin to ß-parvin IPP complexes. This inhibited Akt activation and promoted SmMHC expression. Low tension or IL-4 did not elicit paxillin phosphorylation and triggered the binding of unphosphorylated paxillin to α-parvin IPP complexes, which promoted Akt activation and eotaxin secretion and suppressed SmMHC expression. Expression of a nonphosphorylatable paxillin mutant or ß-parvin depletion by siRNA promoted the inflammatory phenotype, whereas the depletion of α-parvin promoted the differentiated phenotype. Results demonstrate that phenotype expression is regulated by the differential interaction of phosphorylated and unphosphorylated paxillin with α-parvin and ß-parvin IPP complexes and that these complexes have opposite effects on the activation of Akt. Our results describe a novel molecular mechanism for transduction of mechanical and humoral stimuli within integrin signaling complexes to regulate phenotype expression in airway smooth muscle.


Assuntos
Actinina/genética , Mecanotransdução Celular , Músculo Liso/metabolismo , Paxilina/genética , Proteínas Proto-Oncogênicas c-akt/genética , Traqueia/metabolismo , Acetilcolina/farmacologia , Actinina/metabolismo , Animais , Quimiocina CCL11/genética , Quimiocina CCL11/metabolismo , Cães , Feminino , Regulação da Expressão Gênica , Interleucina-4/genética , Interleucina-4/metabolismo , Proteínas com Domínio LIM/genética , Proteínas com Domínio LIM/metabolismo , Masculino , Contração Muscular/efeitos dos fármacos , Contração Muscular/genética , Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Paxilina/metabolismo , Fenótipo , Fosforilação/efeitos dos fármacos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Miosinas de Músculo Liso/genética , Miosinas de Músculo Liso/metabolismo , Traqueia/efeitos dos fármacos
13.
Life Sci ; 250: 117548, 2020 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-32173312

RESUMO

BACKGROUND: Pulmonary arterial hypertension (PAH) is a severe pulmonary vascular disease characterized by unbalanced proliferation and apoptosis of pulmonary arterial smooth muscle cells (PASMCs). Prohibitin 1 (PHB1) is known for its significant anti-proliferative activity. However, the role of PHB1 in PASMCs and PAH have not been elucidated. METHODS: Monocrotaline (MCT 60 mg/kg) was used to build a PAH model in SD rats. Right ventricular systolic pressure (RVSP) and right ventricle (RV) hypertrophy were measured. Morphology of pulmonary vessels was observed by Hematoxylin-Eosin (HE) staining. Expression of PHB1 in pulmonary arteries and PASMCs was determinated by immunoblot and immunofluorescence. Cell proliferation was detected by CCK8 and EDU when PASMCs were stimulated by PDGF-BB (20 ng/mL). Furthermore, siRNA for PHB1 and Akt inhibitor were conducted to investigate the mechanism behind the role of PHB1 and AKT signaling pathway in PASMCs proliferation and apoptosis. RESULTS: The protein expression of PHB1 in PAH rats lung tissue was significantly up-regulated accompanied by elevated RVSP and enhanced RV hypertrophy. Immunohistochemistry showed that PHB1 was mainly localized in the pulmonary vascular smooth muscle layer. PDGF-BB significantly up-regulated the expression of PHB1 in rat primary PASMCs in a time- and dose-dependent manner. After PHB1 knock down, PASMCs proliferation was significantly suppressed while apoptosis was significantly recovered. Meanwhile the level of proliferating cell nuclear antigen (PCNA) and P-Akt were significantly down-regulated. Perifosine (Akt inhibitor) also significantly inhibit proliferation of PASMCs. CONCLUSION: PHB1 contributes to pulmonary vascular remodeling by accelerating proliferation of PASMCs which involves AKT phosphorylation.


Assuntos
Apoptose , Hipertensão Pulmonar/patologia , Monocrotalina/farmacologia , Miócitos de Músculo Liso/citologia , Proteínas Repressoras/metabolismo , Animais , Proliferação de Células , Modelos Animais de Doenças , Inativação Gênica , Ventrículos do Coração/efeitos dos fármacos , Hemodinâmica , Hipertensão Pulmonar/induzido quimicamente , Pulmão/metabolismo , Masculino , Fosfatidilinositol 3-Quinases/metabolismo , Artéria Pulmonar/citologia , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Sprague-Dawley
14.
Arterioscler Thromb Vasc Biol ; 40(4): e105-e113, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32075417

RESUMO

OBJECTIVE: Vascular progenitor cells (VPCs), which are able to differentiate into both endothelial cells and smooth muscle cells, have the potential for treatment of ischemic diseases. Generated by pluripotent stem cells, VPCs carry the risk of tumorigenicity in clinical application. This issue could be resolved by direct lineage conversion, the induction of functional cells from another lineage by using only lineage-restricted transcription factors. Here, we show that induced VPCs (iVPCs) can be generated from fibroblasts by ETS (E-twenty six) transcription factors, Etv2 and Fli1. Approach and Results: Mouse fibroblasts were infected with lentivirus encoding Etv2 and Fli1. Cell colonies appeared in Fli1- and Etv2/Fli1-infected groups and were mechanically picked. The identity of cell colonies was confirmed by proliferation assay and reverse-transcription polymerase chain reaction with vascular markers. Etv2/Fli1- infected cell colonies were sorted by CD144 (also known as CDH5, VE-cadherin). We defined that CD144-positive iVPCs maintained its own population and expanded stably at multiple passages. iVPCs could differentiate into functional endothelial cells and smooth muscle cells by a defined medium. The functionalities of iVPC-derived endothelial cells and smooth muscle cells were confirmed by analyzing LDL (low-density lipoprotein) uptake, carbachol-induced contraction, and tube formation in vitro. Transplantation of iVPCs into the ischemic hindlimb model enhanced blood flow without tumor formation in vivo. Human iVPCs were generated by human ETS transcription factors ETV2 and FLI1. CONCLUSIONS: We demonstrate that ischemic disease curable iVPCs, which have self-renewal and bipotency, can be generated from mouse fibroblasts by enforced ETS family transcription factors, Etv2 and Fli1 expression. Our simple strategy opens insights into stem cell-based ischemic disease therapy.


Assuntos
Fibroblastos/citologia , Isquemia/fisiopatologia , Proteína Proto-Oncogênica c-fli-1/fisiologia , Células-Tronco/fisiologia , Fatores de Transcrição/fisiologia , Animais , Antígenos CD , Caderinas , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Modelos Animais de Doenças , Células Endoteliais/citologia , Membro Posterior/irrigação sanguínea , Isquemia/terapia , Miócitos de Músculo Liso/citologia , Transplante de Células-Tronco , Células-Tronco/imunologia
15.
Biochem Biophys Res Commun ; 524(4): 916-922, 2020 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-32057367

RESUMO

Macrophage-mediated inflammation is a key pathophysiological component of cardiovascular diseases, but the underlying mechanisms by which the macrophage regulates inflammation have been unclear. In our study, we, for the first time, showed an endogenous sulfur dioxide (SO2) production in RAW267.4 macrophages by using HPLC and SO2-specific fluorescent probe assays. Moreover, the endogenous SO2 generating enzyme aspartate aminotransferase (AAT) was found to be expressed by the macrophages. Furthermore, we showed that AAT2 knockdown triggered spontaneous macrophage-mediated inflammation, as represented by the increased TNF-α and IL-6 levels and the enhanced macrophage chemotaxis; these effects could be reversed by the treatment with a SO2 donor. Mechanistically, AAT2 knockdown activated the NF-κB signaling pathway in macrophages, while SO2 successfully rescued NF-κB activation. In contrast, forced AAT2 expression reversed AngII-induced NF-κB activation and subsequent macrophage inflammation. Moreover, treatment with a SO2 donor also alleviated macrophage infiltration in AngII-treated mouse hearts. Collectively, our data suggest that macrophage-derived SO2 is an important regulator of macrophage activation and it acts as an endogenous "on-off switch" in the control of macrophage activation. This knowledge might enable a new therapeutic strategy for cardiovascular diseases.


Assuntos
Aspartato Aminotransferases/genética , Miócitos Cardíacos/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , NF-kappa B/genética , Dióxido de Enxofre/farmacologia , Angiotensina II/farmacologia , Animais , Aspartato Aminotransferases/antagonistas & inibidores , Aspartato Aminotransferases/imunologia , Linhagem Celular , Quimiotaxia/efeitos dos fármacos , Regulação da Expressão Gênica , Inflamação , Interleucina-6/genética , Interleucina-6/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/citologia , Miócitos Cardíacos/imunologia , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/imunologia , NF-kappa B/imunologia , Células RAW 264.7 , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Transdução de Sinais , Sulfitos/química , Sulfitos/farmacologia , Dióxido de Enxofre/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia
16.
Biochem Biophys Res Commun ; 524(4): 853-860, 2020 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-32046856

RESUMO

Telmisartan, an angiotensin II type 1 receptor blocker (ARB), is widely used to treat hypertension. Dysfunction of vascular smooth muscle cells (VSMCs) is well-established to contribute to the pathogenesis of various vascular diseases. A growing body of evidence indicates that increased VSMC contractility plays a primary role in the development of pathological artery spasms. Nevertheless, effect of telmisartan on VSMC contractility, and its mechanism of action remain unknown. Here, we investigated the mechanism by which telmisartan inhibits VSMC contractility and vessel contraction in rat VSMCs and endothelium-deprived aortas. Telmisartan inhibited phenylephrine-induced vessel contraction in endothelium-deprived aortas, and decreased myosin light chain kinase (MLCK) levels (without altering corresponding mRNA levels) and myosin light chain (MLC) phosphorylation at Ser19 (p-MLC-Ser19) in VSMCs. MG-132 but not doxycycline significantly restored telmisartan-inhibited MLCK expression and p-MLC-Ser19. Telmisartan induced AMP-activated protein kinase (AMPK) phosphorylation at Thr172 (p-AMPK-Thr172), and compound C or ectopic expression of the dominant negative (dn)-AMPKα1 gene significantly reversed telmisartan-inhibited MLCK expression and p-MLC-Ser19. Of the ARBs tested (including losartan and fimasartan), only telmisartan increased p-AMPK-Thr172, and inhibited MLCK expression and p-MLC-Ser19. GW9662 had no effects on telmisartan-induced changes. Similar to the in vitro results, telmisartan enhanced p-AMPK-Thr172, and inhibited MLCK expression and p-MLC-Ser19 in endothelium-deprived aortas. Furthermore, the telmisartan-inhibited vessel contraction in the aortas was significantly reversed by MG-132 or compound C. In conclusion, we demonstrated that telmisartan inhibits VSMC contractility and vessel contraction by activating AMPK/proteasome/MLCK degradation signaling axis. These results suggest that telmisartan can be used to treat pathological vasospasms.


Assuntos
Proteínas Quinases Ativadas por AMP/genética , Anti-Hipertensivos/farmacologia , Contração Muscular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Quinase de Cadeia Leve de Miosina/genética , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Telmisartan/farmacologia , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Aorta/citologia , Aorta/efeitos dos fármacos , Aorta/metabolismo , Doxiciclina/farmacologia , Regulação da Expressão Gênica , Leupeptinas/farmacologia , Masculino , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/metabolismo , Quinase de Cadeia Leve de Miosina/metabolismo , Fenilefrina/antagonistas & inibidores , Fenilefrina/farmacologia , Fosforilação/efeitos dos fármacos , Cultura Primária de Células , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Vasoconstritores/antagonistas & inibidores , Vasoconstritores/farmacologia
17.
Cell Prolif ; 53(3): e12774, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32034930

RESUMO

OBJECTIVES: Postflight orthostatic intolerance has been regarded as a major adverse effect after microgravity exposure, in which cerebrovascular adaptation plays a critical role. Our previous finding suggested that dedifferentiation of vascular smooth muscle cells (VSMCs) might be one of the key contributors to cerebrovascular adaptation under simulated microgravity. This study was aimed to confirm this concept and elucidate the underlying mechanisms. MATERIALS AND METHODS: Sprague Dawley rats were subjected to 28-day hindlimb-unloading to simulate microgravity exposure. VSMC dedifferentiation was evaluated by ultrastructural analysis and contractile/synthetic maker detection. The role of T-type CaV 3.1 channel was revealed by assessing its blocking effects. MiR-137 was identified as the upstream of CaV 3.1 channel by luciferase assay and investigated by gain/loss-of-function approaches. Calcineurin/nuclear factor of activated T lymphocytes (NFAT) pathway, the downstream of CaV 3.1 channel, was investigated by detecting calcineurin activity and NFAT nuclear translocation. RESULTS: Simulated microgravity induced the dedifferentiation and proliferation in rat cerebral VSMCs. T-type CaV 3.1 channel promoted the dedifferentiation and proliferation of VSMC. MiR-137 and calcineurin/NFATc3 pathway were the upstream and downstream signalling of T-type CaV 3.1 channel in modulating the dedifferentiation and proliferation of VSMCs, respectively. CONCLUSIONS: The present work demonstrated that miR-137 and its target T-type CaV 3.1 channel modulate the dedifferentiation and proliferation of rat cerebral VSMCs under simulated microgravity by regulating calcineurin/NFATc3 pathway.


Assuntos
Calcineurina/metabolismo , Canais de Cálcio Tipo T/metabolismo , Artérias Cerebrais/citologia , MicroRNAs/metabolismo , Miócitos de Músculo Liso/citologia , Fatores de Transcrição NFATC/metabolismo , Animais , Encéfalo/irrigação sanguínea , Canais de Cálcio Tipo T/genética , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Artérias Cerebrais/metabolismo , Regulação da Expressão Gênica , MicroRNAs/genética , Miócitos de Músculo Liso/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Simulação de Ausência de Peso
18.
Clin Sci (Lond) ; 134(5): 513-527, 2020 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-32104886

RESUMO

An important link exists between hypertension and inflammation. Hypertensive patients present elevated circulating levels of proinflammatory cytokines, including interleukin-17A (IL-17A). This cytokine participates in host defense, autoimmune and chronic inflammatory pathologies, and cardiovascular diseases, mainly through the regulation of proinflammatory factors. Emerging evidence also suggests that IL-17A could play a role in regulating blood pressure and end-organ damage. Here, our preclinical studies in a murine model of systemic IL-17A administration showed that increased levels of circulating IL-17A raised blood pressure induced inward remodeling of small mesenteric arteries (SMAs) and arterial stiffness. In IL-17A-infused mice, treatment with hydralazine and hydrochlorothiazide diminished blood pressure elevation, without modifying mechanical and structural properties of SMA, suggesting a direct vascular effect of IL-17A. The mechanisms of IL-17A seem to involve an induction of vascular smooth muscle cell (VSMC) hypertrophy and phenotype changes, in the absence of extracellular matrix (ECM) proteins accumulation. Accordingly, treatment with an IL-17A neutralizing antibody diminished SMA remodeling in a model of angiotensin II (Ang II) infusion. Moreover, in vitro studies in VSMCs reported here, provide further evidence of the direct effects of IL-17A on cell growth responses. Our experimental data suggest that IL-17A is a key mediator of vascular remodeling of the small arteries, which might contribute, at least in part, to blood pressure elevation.


Assuntos
Pressão Sanguínea/efeitos dos fármacos , Interleucina-17/farmacologia , Artérias Mesentéricas/efeitos dos fármacos , Remodelação Vascular/efeitos dos fármacos , Angiotensina II/administração & dosagem , Angiotensina II/farmacologia , Animais , Forma Celular/efeitos dos fármacos , Humanos , Hipertensão/fisiopatologia , Interleucina-17/administração & dosagem , Masculino , Artérias Mesentéricas/fisiologia , Camundongos Endogâmicos C57BL , Músculo Liso Vascular , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Vasoconstritores/administração & dosagem , Vasoconstritores/farmacologia
19.
Mol Med Rep ; 21(2): 589-596, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31974617

RESUMO

Pulmonary arterial hypertension (PAH) is a progressive syndrome. When PAH occurs, the circulatory resistance of the pulmonary vasculature will gradually increase, which may lead to right heart failure and death. Pathological features of PAH include abnormal proliferation of pulmonary vascular smooth muscle cells and pulmonary vascular remodeling. Hypoxia is the main cause of PAH, which directly induces the contraction and proliferation of pulmonary artery smooth muscle cells (PASMCs), and eventually leads to pulmonary vascular remodeling. Recent studies have shown that long non­coding RNAs (lncRNAs) play key roles in numerous biological processes, including cell proliferation and the occurrence and development of cardiovascular diseases. Studies have also shown that lncRNA antisense noncoding RNA in the INK4 locus (ANRIL) can promote the proliferation of vascular smooth muscle cells. Therefore, the hypothesis of the present study was that ANRIL may be expressed in PASMCs and play a regulatory role. In this study, the expression of ANRIL was analyzed by quantitative PCR. The effects of ANRIL on human pulmonary artery smooth muscle cells (HPASMCs) were assessed by MTT assay, flow cytometry, bromodeoxyuridine incorporation assay, Transwell assay, scratch­wound assay, immunofluorescence assay and western blotting. These experiments revealed that the expression of ANRIL was significantly downregulated in HPASMCs induced by hypoxia. The downregulation of ANRIL affected the cell cycle, making more HPASMCs move from the G0/G1 phase to the G2/M+S phase and strengthening the cell proliferation. Moreover, downregulated ANRIL increased the migration of HPASMCs under hypoxia. This study identified ANRIL as a critical regulator in HPASMCs induced by hypoxia and demonstrated the potential of gene therapy and drug development for treating PAH.


Assuntos
Movimento Celular/genética , Regulação para Baixo/genética , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/metabolismo , Artéria Pulmonar/citologia , RNA Longo não Codificante/genética , Ciclo Celular/genética , Hipóxia Celular/genética , Proliferação de Células/genética , Sobrevivência Celular/genética , Ciclinas/metabolismo , Humanos , RNA Longo não Codificante/metabolismo
20.
Bull Exp Biol Med ; 168(3): 334-340, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31940128

RESUMO

The changes in endothelial progenitor cells and progenitor cells of angiogenesis, pericytes and smooth muscle cells, were studied in female C57BL/6 mice with a combination of metabolic impairments induced by injections of sodium glutamate and lung emphysema modeled by the administration of cigarette smoke extract. It was observed that sodium glutamate significantly enhances pathological changes in the lungs (inflammation and lung emphysema) induced by the administration of cigarette smoke extract. Recruiting of endothelial progenitor cells (CD45-CD31+CD34+ and CD31+CD34+CD146-) and progenitor cells of angiogenesis (CD45-CD117+CD309+) was registered in the injured lungs. Angiogenesis impairment induced by combined exposure is related to altered migration of pericytes (CD31-CD34-CD146+) and smooth muscle cells (CD31-CD34+CD146+) in emphysema-like enlarged lung tissue.


Assuntos
Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/metabolismo , Pericitos/citologia , Pericitos/metabolismo , Enfisema Pulmonar/metabolismo , Enfisema Pulmonar/patologia , Animais , Antígenos CD34/metabolismo , Antígeno CD146/metabolismo , Fumar Cigarros/efeitos adversos , Células Progenitoras Endoteliais/citologia , Células Progenitoras Endoteliais/efeitos dos fármacos , Células Progenitoras Endoteliais/metabolismo , Feminino , Antígenos Comuns de Leucócito/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Miócitos de Músculo Liso/efeitos dos fármacos , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Proteínas Proto-Oncogênicas c-kit/metabolismo
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