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1.
Aging (Albany NY) ; 13(23): 25241-25255, 2021 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-34887361

RESUMO

The abnormal proliferation of vascular smooth muscle cells (VSMCs) plays an important role in the development and progression of diabetic vascular complications. In high-glucose (HG) conditions, endothelial cells (ECs) act as the first barrier to damaging stimuli and trigger a multi-response, including EC and VSMC crosstalk. However, the crosstalk pathways between ECs and VSMCs under HG conditions remain unclear. This study aimed to explore the roles and underlying mechanism of exosomes derived from ECs in the crosstalk between ECs and VSMCs. Our results showed that mouse aortic endothelial cell (MAEC)-secreted exosomes could promote the proliferation and inhibit the apoptosis of VSMCs induced by HG. Furthermore, we isolated the exosomes secreted by MAECs and found that exosomes derived from MAECs that were exposed to HG could transfer circHIPK3, which is enriched in MAEC-derived exosomes, to VSMCs. Exosomal circHIPK3 promoted the proliferation and inhibited the apoptosis of VSMCs. circHIPK3 sponged miR-106a-5p to relieve its repression of forkhead box O1 (Foxo1) expression. The increased expression of Foxo1 acted as a transcription factor to promote Vcam1 expression, thus facilitating the uptake of MAEC-derived exosomes by VSMCs. The results of this study suggested that exosomal circHIPK3 derived from MAECs promotes the proliferation of VSMCs induced by HG via the miR-106a-5p/Foxo1/Vcam1 pathway.


Assuntos
Proliferação de Células , Proteína Forkhead Box O1/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , MicroRNAs/metabolismo , Músculo Liso Vascular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Molécula 1 de Adesão de Célula Vascular/metabolismo , Animais , Apoptose , Proliferação de Células/efeitos dos fármacos , Exossomos/metabolismo , Glucose/farmacologia , Células HEK293 , Humanos , Camundongos , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/crescimento & desenvolvimento , Transdução de Sinais/efeitos dos fármacos
2.
J Biomed Mater Res A ; 109(12): 2493-2505, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34096176

RESUMO

Long-term in vivo observation in large animal model is critical for evaluating the potential of small diameter tissue engineering vascular graft (SDTEVG) in clinical application, but is rarely reported. In this study, a SDTEVG is fabricated by the electrospinning of poly(ε-caprolactone) and subsequent heparin modification. SDTEVG is implanted into canine's abdominal aorta for 511 days in order to investigate its clinical feasibility. An active and robust remodeling process was characterized by a confluent endothelium, macrophage infiltrate, extracellular matrix deposition and remodeling on the explanted graft. The immunohistochemical and immunofluorescence analysis further exhibit the regeneration of endothelium and smooth muscle layer on tunica intima and tunica media, respectively. Thus, long-term follow-up reveals viable neovessel formation beyond graft degradation. Furthermore, the von Kossa staining exhibits no occurrence of calcification. However, although no TEVG failure or rupture happens during the follow-up, the aneurysm is found by both Doppler ultrasonic and gross observation. Consequently, as-prepared TEVG shows promising potential in vascular tissue engineering if it can be appropriately strengthened to prevent the occurrence of aneurysm.


Assuntos
Prótese Vascular , Vasos Sanguíneos/transplante , Heparina/química , Poliésteres/química , Aneurisma/prevenção & controle , Animais , Aorta Abdominal/transplante , Cães , Endotélio Vascular/crescimento & desenvolvimento , Matriz Extracelular/ultraestrutura , Humanos , Macrófagos , Músculo Liso Vascular/crescimento & desenvolvimento , Projetos Piloto , Alicerces Teciduais , Túnica Íntima , Túnica Média
3.
Mol Med Rep ; 24(1)2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33982767

RESUMO

Accumulating evidence suggests that pulmonary expression of a disintegrin and metalloproteinase­33 (ADAM33) serves a key role in the pathogenesis of airway remodeling­related diseases, including asthma. Airway vascular proliferation has been recognized as a key feature of airway remodeling. Our previous study showed that ADAM33 is constitutively expressed in airway vascular smooth muscle cells in patients with asthma, suggesting a potential role of ADAM33 in regulating airway vascular remodeling. Using in vitro human aortic smooth muscle cells (HASMCs) and lentiviral vector carrying short hairpin RNA for ADAM33, the present study aimed to evaluate the influence of ADAM33 silencing on the proliferation and apoptosis of HASMCs and the underlying molecular pathways. Cellular proliferation was observed using the Cell Counting Kit­8 method. Cellular apoptosis was evaluated with Annexin V­PE/7­AAD staining and flow cytometry. Reverse transcription­quantitative PCR and western blotting were used to evaluate the changes in mRNA and protein levels of involved signaling molecules. It was found that silencing of ADAM33 expression in HASMCs significantly inhibited proliferation, but induced the apoptosis of HASMCs. These changes were accompanied by inhibition of the PI3K/AKT/ERK pathway and Bcl­2, but an increase in Bax expression. These results suggested that constitutive expression of ADAM33 may be important to maintain a proliferative phenotype in HASMCs. The influences of ADAM33 on proliferation and apoptosis of HASMCs may involve regulation of PI3K/AKT/ERK and Bax/Bcl­2 pathways. These findings suggested an important role of ADAM33 in airway vascular remodeling and potential therapeutic significance of ADAM33 inhibition in airway remodeling­related diseases.


Assuntos
Proteínas ADAM/genética , Músculo Liso Vascular/enzimologia , Músculo Liso Vascular/crescimento & desenvolvimento , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Remodelação Vascular/genética , Proteínas ADAM/biossíntese , Idoso , Apoptose/genética , Ciclo Celular/genética , Linhagem Celular , Proliferação de Células/genética , Feminino , Inativação Gênica , Humanos , Sistema de Sinalização das MAP Quinases/genética , Masculino , Pessoa de Meia-Idade , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Interferente Pequeno/farmacologia , Transdução de Sinais/genética , Proteína X Associada a bcl-2/metabolismo
4.
Development ; 148(7)2021 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-33789914

RESUMO

Smooth muscle cells (SMCs) represent a major structural and functional component of many organs during embryonic development and adulthood. These cells are a crucial component of vertebrate structure and physiology, and an updated overview of the developmental and functional process of smooth muscle during organogenesis is desirable. Here, we describe the developmental origin of SMCs within different tissues by comparing their specification and differentiation with other organs, including the cardiovascular, respiratory and intestinal systems. We then discuss the instructive roles of smooth muscle in the development of such organs through signaling and mechanical feedback mechanisms. By understanding SMC development, we hope to advance therapeutic approaches related to tissue regeneration and other smooth muscle-related diseases.


Assuntos
Desenvolvimento Embrionário , Músculo Liso/crescimento & desenvolvimento , Miócitos de Músculo Liso/fisiologia , Vertebrados/crescimento & desenvolvimento , Animais , Animais Geneticamente Modificados , Sistema Cardiovascular , Diferenciação Celular/fisiologia , Trato Gastrointestinal , Pulmão , Mesoderma , Músculo Liso/citologia , Músculo Liso/embriologia , Músculo Liso Vascular/embriologia , Músculo Liso Vascular/crescimento & desenvolvimento , Miócitos de Músculo Liso/citologia , Organogênese/fisiologia , Sistema Respiratório , Vertebrados/embriologia
6.
Int J Mol Sci ; 21(24)2020 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-33322781

RESUMO

Amine-coated biodegradable materials based on synthetic polymers have a great potential for tissue remodeling and regeneration because of their excellent processability and bioactivity. In the present study, we have investigated the influence of various chemical compositions of amine plasma polymer (PP) coatings and the influence of the substrate morphology, represented by polystyrene culture dishes and polycaprolactone nanofibers (PCL NFs), on the behavior of vascular smooth muscle cells (VSMCs). Although all amine-PP coatings improved the initial adhesion of VSMCs, 7-day long cultivation revealed a clear preference for the coating containing about 15 at.% of nitrogen (CPA-33). The CPA-33 coating demonstrated the ideal combination of good water stability, a sufficient amine group content, and favorable surface wettability and morphology. The nanostructured morphology of amine-PP-coated PCL NFs successfully slowed the proliferation rate of VSMCs, which is essential in preventing restenosis of vascular replacements in vivo. At the same time, CPA-33-coated PCL NFs supported the continuous proliferation of VSMCs during 7-day long cultivation, with no significant increase in cytokine secretion by RAW 264.7 macrophages. The CPA-33 coating deposited on biodegradable PCL NFs therefore seems to be a promising material for manufacturing small-diameter vascular grafts, which are still lacking on the current market.


Assuntos
Aminas/química , Materiais Revestidos Biocompatíveis/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Nanofibras/química , Plasma/química , Polímeros/química , Aminas/efeitos adversos , Aminas/imunologia , Aminas/farmacologia , Animais , Adesão Celular/efeitos dos fármacos , Adesão Celular/imunologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Materiais Revestidos Biocompatíveis/efeitos adversos , Materiais Revestidos Biocompatíveis/química , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Músculo Liso Vascular/citologia , Músculo Liso Vascular/crescimento & desenvolvimento , Miócitos de Músculo Liso/metabolismo , Nanofibras/efeitos adversos , Espectroscopia Fotoeletrônica , Plasma/imunologia , Poliésteres/química , Polímeros/efeitos adversos , Polímeros/farmacologia , Células RAW 264.7 , Ratos , Propriedades de Superfície/efeitos dos fármacos , Alicerces Teciduais/efeitos adversos , Alicerces Teciduais/química
7.
Sci Rep ; 10(1): 13858, 2020 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-32807822

RESUMO

The aim of this study is to investigate the therapeutic role of Tanshinone II A, a key integrant from salvia miltiorrhiza, against pathological vascular remodeling. Completed ligation of mouse left common carotid arteries animal model and rat smooth muscle cells used to investigate the role of Tanshinone II A in regulating pathological vascular remodeling through hematoxylin and eosin staining, immunohistochemistry staining, immunofluorescence staining, adenovirus infection, real time PCR and western blotting. Our data demonstrated that Tanshinone II A treatment suppresses vascular injury-induced neointima formation. In vitro studies on rat smooth muscle cell indicated that Tanshinone II A treatment attenuates PDGF-BB induced cell growth, and promotes smooth muscle cell differentiated marker genes expression that induced by rapamycin treatment. Tanshinone II A treatment significant inhibits rat smooth muscle cell proliferation and migration. Tanshinone II A promotes KLF4 expression during smooth muscle phenotypic switching. Overexpression of KLF4 exacerbates Tanshinone II A mediated smooth muscle cell growth inhibition. Tanshinone II A plays a pivotal role in regulating pathological vascular remodeling through KLF4 mediated smooth muscle cell phenotypic switching. This study demonstrated that Tanshinone II A is a potential therapeutic agent for vascular diseases.


Assuntos
Abietanos/farmacologia , Diferenciação Celular/genética , Expressão Gênica/efeitos dos fármacos , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Músculo Liso Vascular/crescimento & desenvolvimento , Remodelação Vascular/efeitos dos fármacos , Abietanos/uso terapêutico , Animais , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Células Cultivadas , Fator 4 Semelhante a Kruppel , Camundongos , Fenótipo , Fitoterapia , Ratos Sprague-Dawley , Salvia miltiorrhiza , Doenças Vasculares/tratamento farmacológico
8.
Clin Sci (Lond) ; 134(15): 2019-2035, 2020 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-32706027

RESUMO

OBJECTIVE: Transient receptor potential (TRP) melastatin 7 (TRPM7) cation channel, a dual-function ion channel/protein kinase, regulates vascular smooth muscle cell (VSMC) Mg2+ homeostasis and mitogenic signaling. Mechanisms regulating vascular growth effects of TRPM7 are unclear, but epidermal growth factor (EGF) may be important because it is a magnesiotropic hormone involved in cellular Mg2+ regulation and VSMC proliferation. Here we sought to determine whether TRPM7 is a downstream target of EGF in VSMCs and if EGF receptor (EGFR) through TRPM7 influences VSMC function. Approach and results: Studies were performed in primary culture VSMCs from rats and humans and vascular tissue from mice deficient in TRPM7 (TRPM7+/Δkinase and TRPM7R/R). EGF increased expression and phosphorylation of TRPM7 and stimulated Mg2+ influx in VSMCs, responses that were attenuated by gefitinib (EGFR inhibitor) and NS8593 (TRPM7 inhibitor). Co-immunoprecipitation (IP) studies, proximity ligation assay (PLA) and live-cell imaging demonstrated interaction of EGFR and TRPM7, which was enhanced by EGF. PP2 (c-Src inhibitor) decreased EGF-induced TRPM7 activation and prevented EGFR-TRPM7 association. EGF-stimulated migration and proliferation of VSMCs were inhibited by gefitinib, PP2, NS8593 and PD98059 (ERK1/2 inhibitor). Phosphorylation of EGFR and ERK1/2 was reduced in VSMCs from TRPM7+/Δkinase mice, which exhibited reduced aortic wall thickness and decreased expression of PCNA and Notch 3, findings recapitulated in TRPM7R/R mice. CONCLUSIONS: We show that EGFR directly interacts with TRPM7 through c-Src-dependent processes. Functionally these phenomena regulate [Mg2+]i homeostasis, ERK1/2 signaling and VSMC function. Our findings define a novel signaling cascade linking EGF/EGFR and TRPM7, important in vascular homeostasis.


Assuntos
Fator de Crescimento Epidérmico/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Canais de Cátion TRPM/metabolismo , Animais , Proteína Tirosina Quinase CSK/metabolismo , Cálcio/metabolismo , Proteínas de Transporte de Cátions/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células HEK293 , Humanos , Magnésio/metabolismo , Camundongos Endogâmicos C57BL , Morfogênese , Músculo Liso Vascular/crescimento & desenvolvimento , Fosforilação , Cultura Primária de Células , Ratos Endogâmicos WKY
9.
Mol Med Rep ; 22(2): 886-894, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32467985

RESUMO

Increasing evidence suggests that T­cell immunoglobulin and mucin domain 3 (TIM­3) displays anti­atherosclerotic effects, but its role in vascular smooth muscle cells (VSMCs) has not been reported. The present study aimed to investigate the function of TIM­3 and its roles in human artery VSMCs (HASMCs). A protein array was used to investigate the TIM­3 protein expression profile, which indicated that TIM­3 expression was increased in the serum of patients with lower extremity arteriosclerosis obliterans disease (LEAOD) compared with healthy individuals. Immunohistochemistry and western blotting of arterial tissue further revealed that TIM­3 expression was increased in LEAOD artery tissue compared with normal artery tissue. Additionally, platelet­derived growth factor­BB (PDGF­BB) displayed a positive correlation with TIM­3 expression in HASMCs. TIM­3 decreased the migration and proliferation of PDGF­BB­induced HASMCs, and anti­TIM­3 blocked the effects of TIM­3. The effect of TIM­3 on the proliferation and migration of HASMCs was further investigated using LV­TIM­3­transduced cells. The results revealed that TIM­3 also inhibited PDGF­BB­induced expression of the inflammatory factors interleukin­6 and tumor necrosis factor­α by suppressing NF­κB activation. In summary, the present study revealed that TIM­3 displayed a regulatory role during the PDGF­BB­induced inflammatory reaction in HASMCs, which indicated that TIM­3 may display anti­atherosclerotic effects.


Assuntos
Artérias/metabolismo , Aterosclerose/metabolismo , Becaplermina/antagonistas & inibidores , Receptor Celular 2 do Vírus da Hepatite A/biossíntese , Receptor Celular 2 do Vírus da Hepatite A/sangue , Músculo Liso Vascular/metabolismo , Idoso , Artérias/citologia , Artérias/crescimento & desenvolvimento , Arteriosclerose Obliterante/sangue , Aterosclerose/induzido quimicamente , Becaplermina/efeitos adversos , Linhagem Celular , Movimento Celular , Proliferação de Células , Feminino , Humanos , Interleucina-6/metabolismo , Extremidade Inferior/irrigação sanguínea , Masculino , Pessoa de Meia-Idade , Músculo Liso Vascular/citologia , Músculo Liso Vascular/crescimento & desenvolvimento , NF-kappa B/metabolismo , Análise Serial de Proteínas , Transcriptoma , Fator de Necrose Tumoral alfa/metabolismo
10.
Am J Physiol Heart Circ Physiol ; 318(4): H976-H984, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32142377

RESUMO

There is a sustained reduction in arterial blood pressure that occurs in aged adults following exposure to acute leg heating. We tested the hypothesis that acute leg heating would decrease arterial blood pressure in aged adults secondary to sympathoinhibition. We exposed 13 young and 10 aged adults to 45 min of leg heating. Muscle sympathetic nerve activity (radial nerve) was measured before leg heating (preheat) and 30 min after (recovery) and is expressed as burst frequency. Neurovascular transduction was examined by assessing the slope of the relation between muscle sympathetic nerve activity and leg vascular conductance measured at rest and during isometric handgrip exercise performed to fatigue. Arterial blood pressure was well maintained in young adults (preheat, 86 ± 6 mmHg vs. recovery, 88 ± 7 mmHg; P = 0.4) due to increased sympathetic nerve activity (preheat, 16 ± 7 bursts/min vs. recovery, 22 ± 10 bursts/min; P < 0.01). However, in aged adults, sympathetic nerve activity did not differ from preheat (37 ± 5 bursts/min) to recovery (33 ± 6 bursts/min, P = 0.1), despite a marked reduction in arterial blood pressure (preheat, 101 ± 7 mmHg vs. recovery, 94 ± 6 mmHg; P < 0.01). Neurovascular transduction did not differ from preheat to recovery for either age group (P ≥ 0.1). The reduction in arterial blood pressure that occurs in aged adults following exposure to acute leg heating is mediated, in part, by a sympathoinhibitory effect that alters the compensatory neural response to hypotension.NEW & NOTEWORTHY There is a sustained reduction in arterial blood pressure that occurs in aged adults following exposure to acute leg heating. However, the neurovascular mechanisms mediating this response remain unknown. Our findings demonstrate for the first time that this reduction in arterial blood pressure is mediated, in part, by a sympathoinhibitory effect that alters the compensatory neural response to hypotension in aged adults.


Assuntos
Envelhecimento/fisiologia , Pressão Sanguínea , Resposta ao Choque Térmico , Sistema Nervoso Simpático/fisiologia , Adulto , Idoso , Feminino , Força da Mão , Humanos , Perna (Membro)/crescimento & desenvolvimento , Perna (Membro)/fisiologia , Masculino , Pessoa de Meia-Idade , Músculo Liso Vascular/crescimento & desenvolvimento , Músculo Liso Vascular/fisiologia , Condução Nervosa , Sistema Nervoso Simpático/crescimento & desenvolvimento
11.
J Cell Mol Med ; 24(8): 4762-4772, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32155686

RESUMO

Vascular smooth muscle cell (VSMC) proliferation is the pathological base of vascular remodelling diseases. Circular RNAs (circRNAs) are important regulators involved in various biological processes. However, the function of circRNAs in VSMC proliferation regulation remains largely unknown. This study was conducted to identify the key differentially expressed circRNAs (DEcircRNAs) and predict their functions in human aortic smooth muscle cell (HASMC) proliferation. To achieve this, DEcircRNAs between proliferative and quiescent HASMCs were detected using a microarray, followed by quantitative real-time RT-PCR validation. A DEcircRNA-miRNA-DEmRNA network was constructed, and functional annotation was performed using Gene Ontology (GO) and KEGG pathway analysis. The function of hsa_circ_0002579 in HASMC proliferation was analysed by Western blot. The functional annotation of the DEcircRNA-miRNA-DEmRNA network indicated that the four DEcircRNAs might play roles in the TGF-ß receptor signalling pathway, Ras signalling pathway, AMPK signalling pathway and Wnt signalling pathway. Twenty-seven DEcircRNAs with coding potential were screened. Hsa_circ_0002579 might be a pro-proliferation factor of HASMC. Overall, our study identified the key DEcircRNAs between proliferative and quiescent HASMCs, which might provide new important clues for exploring the functions of circRNAs in vascular remodelling diseases.


Assuntos
Aorta/crescimento & desenvolvimento , Proliferação de Células/genética , Músculo Liso Vascular/crescimento & desenvolvimento , RNA Circular/genética , Aorta/metabolismo , Técnicas de Cultura de Células , Regulação da Expressão Gênica no Desenvolvimento/genética , Ontologia Genética , Redes Reguladoras de Genes/genética , Humanos , MicroRNAs/genética , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Transdução de Sinais/genética
12.
Life Sci ; 246: 117419, 2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-32045592

RESUMO

AIMS: Although resistin-like molecule ß (RELM-ß) is involved in the pathological processes of various lung diseases, such as pulmonary inflammation, asthma and fibrosis, its potential roles in hypoxic pulmonary arterial hypertension (PAH) remain largely unknown. The study aims to investigate whether RELM-ß contributes to hypoxia-induced excessive proliferation of human pulmonary artery smooth muscle cells (PASMCs) and to explore the potential mechanisms of this process. MAIN METHODS: Human PASMCs were exposed to normoxia or hypoxia (1% O2) for 24 h. siRNA targeting RELM-ß was transfected into cells. Protein levels of KCNK3, RELM-ß, pSTAT3 and STAT3 were determined by immunoblotting. The translocation of NFATc2 and expression of KCNK3 were visualized by immunofluorescence. 5-ethynyl-2'-deoxyuridine assays and cell counting kit-8 assays were performed to assess the proliferation of PASMCs. KEY FINDINGS: (1) Chronic hypoxia significantly decreased KCNK3 protein levels while upregulating RELM-ß protein levels in human PASMCs, which was accompanied by excessive proliferation of cells. (2) RELM-ß could promote human PASMCs proliferation and activate the STAT3/NFAT axis by downregulating KCNK3 protein under normoxia. (3) Inhibition of RELM-ß expression effectively prevented KCNK3-mediated cell proliferation under hypoxia. (4) Phospholipase C (PLC) inhibitor U-73122 could not only prevent the hypoxia/RELM-ß-induced decrease in KCNK3 protein, but also inhibit the enhanced cell viability caused by hypoxia/RELM-ß. (5) Both hypoxia and RELM-ß could downregulate membrane KCNK3 protein levels by enhancing endocytosis. SIGNIFICANCE: RELM-ß activation is responsible for hypoxia-induced excessive proliferation of human PASMCs. Interfering with RELM-ß may alleviate the progression of hypoxic PAH by upregulating PLC-dependent KCNK3 expression.


Assuntos
Hipóxia/complicações , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Músculo Liso Vascular/efeitos dos fármacos , Proteínas do Tecido Nervoso/metabolismo , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Artéria Pulmonar/efeitos dos fármacos , Fosfolipases Tipo C/metabolismo , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Imunofluorescência , Humanos , Hipóxia/tratamento farmacológico , Músculo Liso Vascular/citologia , Músculo Liso Vascular/crescimento & desenvolvimento , Artéria Pulmonar/fisiopatologia , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais
13.
Sci Rep ; 9(1): 18224, 2019 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-31796853

RESUMO

Vascular smooth muscle cell (VSMC) dysfunction is a hallmark of small vessel disease, a common cause of stroke and dementia. Two of the most frequently mutated genes in familial small vessel disease are HTRA1 and NOTCH3. The protease HTRA1 cleaves the NOTCH3 ligand JAG1 implying a mechanistic link between HTRA1 and Notch signaling. Here we report that HTRA1 is essential for VSMC differentiation into the contractile phenotype. Mechanistically, loss of HTRA1 increased JAG1 protein levels and NOTCH3 signaling activity in VSMC. In addition, the loss of HTRA1 enhanced TGFß-SMAD2/3 signaling activity. Activation of either NOTCH3 or TGFß signaling resulted in increased transcription of the HES and HEY transcriptional repressors and promoted the contractile VSMC phenotype. However, their combined over-activation led to an additive accumulation of HES and HEY proteins, which repressed the expression of contractile VSMC marker genes. As a result, VSMC adopted an immature phenotype with impaired arterial vasoconstriction in Htra1-deficient mice. These data demonstrate an essential role of HTRA1 in vascular maturation and homeostasis by controlling Notch and TGFß signaling.


Assuntos
Serina Peptidase 1 de Requerimento de Alta Temperatura A/metabolismo , Músculo Liso Vascular/crescimento & desenvolvimento , Animais , Western Blotting , Imunofluorescência , Serina Peptidase 1 de Requerimento de Alta Temperatura A/fisiologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Contração Muscular , Músculo Liso Vascular/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Receptor Notch3/metabolismo , Receptor Notch3/fisiologia , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta/fisiologia
14.
Circ Res ; 125(11): 1006-1018, 2019 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-31590613

RESUMO

RATIONALE: Elastin is an important ECM (extracellular matrix) protein in large and small arteries. Vascular smooth muscle cells (SMCs) produce the layered elastic laminae found in elastic arteries but synthesize little elastin in muscular arteries. However, muscular arteries have a well-defined internal elastic lamina (IEL) that separates endothelial cells (ECs) from SMCs. The extent to which ECs contribute elastin to the IEL is unknown. OBJECTIVE: To use targeted elastin (Eln) deletion in mice to explore the relative contributions of SMCs and ECs to elastic laminae formation in different arteries. METHODS AND RESULTS: We used SMC- and EC-specific Cre recombinase transgenes with a novel floxed Eln allele to focus gene inactivation in mice. Inactivation of Eln in SMCs using Sm22aCre resulted in depletion of elastic laminae in the arterial wall with the exception of the IEL and SMC clusters in the outer media near the adventitia. Inactivation of elastin in ECs using Tie2Cre or Cdh5Cre resulted in normal medial elastin and a typical IEL in elastic arteries. In contrast, the IEL was absent or severely disrupted in muscular arteries. Interruptions in the IEL resulted in neointimal formation in the ascending aorta but not in muscular arteries. CONCLUSIONS: Combined with lineage-specific fate mapping systems, our knockout results document an unexpected heterogeneity in vascular cells that produce the elastic laminae. SMCs and ECs can independently form an IEL in most elastic arteries, whereas ECs are the major source of elastin for the IEL in muscular and resistance arteries. Neointimal formation at IEL disruptions in the ascending aorta confirms that the IEL is a critical physical barrier between SMCs and ECs in the large elastic arteries. Our studies provide new information about how SMCs and ECs contribute elastin to the arterial wall and how local elastic laminae defects may contribute to cardiovascular disease.


Assuntos
Tecido Elástico/metabolismo , Elastina/metabolismo , Células Endoteliais/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Animais , Artérias/crescimento & desenvolvimento , Artérias/metabolismo , Pressão Sanguínea , Linhagem da Célula , Proliferação de Células , Tecido Elástico/crescimento & desenvolvimento , Tecido Elástico/ultraestrutura , Elastina/deficiência , Elastina/genética , Células Endoteliais/ultraestrutura , Feminino , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso Vascular/crescimento & desenvolvimento , Músculo Liso Vascular/ultraestrutura , Miócitos de Músculo Liso/ultraestrutura , Neointima , Transdução de Sinais
15.
Cell Death Differ ; 26(12): 2790-2806, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31024075

RESUMO

TEAD1 (TEA domain transcription factor 1), a transcription factor known for the functional output of Hippo signaling, is important for tumorigenesis. However, the role of TEAD1 in the development of vascular smooth muscle cell (VSMC) is unknown. To investigate cell-specific role of Tead1, we generated cardiomyocyte (CMC) and VSMC-specific Tead1 knockout mice. We found CMC/VSMC-specific deletion of Tead1 led to embryonic lethality by E14.5 in mice due to hypoplastic cardiac and vascular walls, as a result of impaired CMC and VSMC proliferation. Whole transcriptome analysis revealed that deletion of Tead1 in CMCs/VSMCs downregulated expression of muscle contractile genes and key transcription factors including Pitx2c and myocardin. In vitro studies demonstrated that PITX2c and myocardin rescued TEAD1-dependent defects in VSMC differentiation. We further identified Pitx2c as a novel transcriptional target of TEAD1, and PITX2c exhibited functional synergy with myocardin by directly interacting with myocardin, leading to augment the differentiation of VSMC. In summary, our study reveals a critical role of Tead1 in cardiovascular development in mice, but also identifies a novel regulatory mechanism, whereby Tead1 functions upstream of the genetic regulatory hierarchy for establishing smooth muscle contractile phenotype.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Músculo Liso Vascular/metabolismo , Fatores de Transcrição/metabolismo , Animais , Diferenciação Celular/fisiologia , Proteínas de Ligação a DNA/genética , Feminino , Deleção de Genes , Masculino , Camundongos , Camundongos Knockout , Músculo Liso Vascular/citologia , Músculo Liso Vascular/crescimento & desenvolvimento , Fatores de Transcrição de Domínio TEA , Fatores de Transcrição/genética
16.
ACS Appl Mater Interfaces ; 11(18): 16402-16411, 2019 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-30998317

RESUMO

Fully integrated hydrogel channels were fabricated via interfacial bioorthogonal cross-linking, a diffusion-controlled method for the creation and patterning of synthetic matrices based on the rapid bioorthogonal reaction between s-tetrazines (Tz) and trans-cyclooctene (TCO) dienophiles. Injecting an aqueous solution of a bisTCO cross-linker into a reservoir of tetrazine-modified hyaluronic acid (HA-Tz), while simultaneously drawing the syringe needle through the reservoir, yielded a cross-linked hydrogel channel that was mechanically robust. Fluorescent tags and biochemical signals were spatially patterned into the channel wall through time-dependent perfusion of TCO-conjugated molecules into the lumen of the channel. Different cell populations were spatially encapsulated in the channel wall via temporal alteration of cells in the HA-Tz reservoir. The interfacial approach enabled the spatial patterning of vascular cells, including human abdominal aorta endothelial cells, aortic vascular smooth muscle cells, and aortic adventitial fibroblasts, into the hydrogel channels with high viability and proper morphology in the anatomical order found in human arteries. The bioorthogonal platform does not rely on external triggers and represents the first step toward the engineering of functional and implantable arteries.


Assuntos
Aorta Abdominal/crescimento & desenvolvimento , Células Endoteliais/efeitos dos fármacos , Hidrogéis/farmacologia , Músculo Liso Vascular/crescimento & desenvolvimento , Aorta/efeitos dos fármacos , Aorta/crescimento & desenvolvimento , Aorta Abdominal/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Reagentes de Ligações Cruzadas/química , Ciclo-Octanos/química , Células Endoteliais/patologia , Fibroblastos/efeitos dos fármacos , Humanos , Hidrogéis/síntese química , Hidrogéis/química , Músculo Liso Vascular/efeitos dos fármacos , Tetrazóis/química , Engenharia Tecidual/tendências
17.
Am J Physiol Lung Cell Mol Physiol ; 316(6): L999-L1012, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-30908936

RESUMO

Lung diseases with hypoxia are complicated by pulmonary hypertension, leading to heart failure and death. No pharmacological treatment exists. Increased proinflammatory cytokines are found in hypoxic patients, suggesting an inflammatory pathogenesis. Caspase-1, the effector of the inflammasome, mediates inflammation through activation of the proinflammatory cytokines interleukin (IL)-18 and IL-1ß. Here, we investigate inflammasome-related mechanisms that can trigger hypoxia-induced pulmonary hypertension. Our aim was to examine whether caspase-1 induces development of hypoxia-related pulmonary hypertension and is a suitable target for therapy. Wild-type (WT) and caspase-1-/- mice were exposed to 10% oxygen for 14 days. Hypoxic caspase-1-/- mice showed lower pressure and reduced muscularization in pulmonary arteries, as well as reduced right ventricular remodeling compared with WT. Smooth muscle cell (SMC) proliferation was reduced in caspase-1-deficient pulmonary arteries and in WT arteries treated with a caspase-1 inhibitor. Impaired inflammation was shown in hypoxic caspase-1-/- mice by abolished pulmonary influx of immune cells and lower levels of IL-18, IL-1ß, and IL-6, which were also reduced in the medium surrounding caspase-1 abrogated pulmonary arteries. By adding IL-18 or IL-1ß to caspase-1-deficient pulmonary arteries, SMC proliferation was retained. Furthermore, inhibition of both IL-6 and phosphorylated STAT3 reduced proliferation of SMC in vitro, indicating IL-18, IL-6, and STAT3 as downstream mediators of caspase-1-induced SMC proliferation in pulmonary arteries. Caspase-1 induces SMC proliferation in pulmonary arteries through the caspase-1/IL-18/IL-6/STAT3 pathway, leading to pulmonary hypertension in mice exposed to hypoxia. We propose that caspase-1 inhibition is a potential target for treatment of pulmonary hypertension.


Assuntos
Caspase 1/genética , Hipóxia Celular/fisiologia , Hipertensão Pulmonar/patologia , Miócitos de Músculo Liso/fisiologia , Função Ventricular Direita/fisiologia , Animais , Linhagem Celular , Proliferação de Células/genética , Humanos , Inflamassomos/metabolismo , Interleucina-18/metabolismo , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso Vascular/citologia , Músculo Liso Vascular/crescimento & desenvolvimento , Artéria Pulmonar/citologia , Artéria Pulmonar/patologia , Fator de Transcrição STAT3/metabolismo
18.
Am J Respir Crit Care Med ; 200(5): 617-627, 2019 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-30817168

RESUMO

Rationale: Glycolytic shift is implicated in the pathogenesis of pulmonary arterial hypertension (PAH). It remains unknown how glycolysis is increased and how increased glycolysis contributes to pulmonary vascular remodeling in PAH.Objectives: To determine whether increased glycolysis is caused by 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) and how PFKFB3-driven glycolysis induces vascular remodeling in PAH.Methods: PFKFB3 levels were measured in pulmonary arteries of patients and animals with PAH. Lactate levels were assessed in lungs of animals with PAH and in pulmonary artery smooth muscle cells (PASMCs). Genetic and pharmacologic approaches were used to investigate the role of PFKFB3 in PAH.Measurements and Main Results: Lactate production was elevated in lungs of PAH rodents and in platelet-derived growth factor-treated PASMCs. PFKFB3 protein was higher in pulmonary arteries of patients and rodents with PAH, in PASMCs of patients with PAH, and in platelet-derived growth factor-treated PASMCs. PFKFB3 inhibition by genetic disruption and chemical inhibitor attenuated phosphorylation/activation of extracellular signal-regulated kinase (ERK1/2) and calpain-2, and vascular remodeling in PAH rodent models, and reduced platelet-derived growth factor-induced phosphorylation/activation of ERK1/2 and calpain-2, collagen synthesis and proliferation of PASMCs. ERK1/2 inhibition attenuated phosphorylation/activation of calpain-2, and vascular remodeling in Sugen/hypoxia PAH rats, and reduced lactate-induced phosphorylation/activation of calpain-2, collagen synthesis, and proliferation of PASMCs. Calpain-2 inhibition reduced lactate-induced collagen synthesis and proliferation of PASMCs.Conclusions: Upregulated PFKFB3 mediates collagen synthesis and proliferation of PASMCs, contributing to vascular remodeling in PAH. The mechanism is through the elevation of glycolysis and lactate that results in the activation of calpain by ERK1/2-dependent phosphorylation of calpain-2.


Assuntos
Proliferação de Células/efeitos dos fármacos , Músculo Liso Vascular/crescimento & desenvolvimento , Fosfofrutoquinase-2/sangue , Fosfofrutoquinase-2/metabolismo , Hipertensão Arterial Pulmonar/sangue , Hipertensão Arterial Pulmonar/fisiopatologia , Remodelação Vascular/fisiologia , Animais , Modelos Animais de Doenças , Humanos , Masculino , Camundongos , Ratos
19.
J Anat ; 234(4): 465-479, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30793310

RESUMO

Teeth are richly supported by blood vessels and peripheral nerves. The aim of this study was to describe in detail the developmental time-course and localization of blood vessels during early tooth formation and to compare that to innervation, as well as to address the putative role of vascular endothelial growth factor (VEGF), which is an essential regulator of vasculature development, in this process. The localization of blood vessels and neurites was compared using double immunofluorescence staining on sections at consecutive stages of the embryonic (E) and postnatal (PN) mandibular first molar tooth germ (E11-PN7). Cellular mRNA expression domains of VEGF and its signaling receptor VEGFR2 were studied using sectional radioactive in situ hybridization. Expression of VEGF mRNA and the encoded protein were studied by RT-PCR and western blot analysis, respectively, in the cap and early bell stage tooth germs, respectively. VEGFR2 was immunolocalized on tooth tissue sections. Smooth muscle cells were investigated by anti-alpha smooth muscle actin (αSMA) antibodies. VEGF showed developmentally regulated epithelial and mesenchymal mRNA expression domains including the enamel knot signaling centers that correlated with the growth and navigation of the blood vessels expressing Vegfr2 and VEGFR2 to the dental papilla and enamel organ. Developing blood vessels were present in the jaw mesenchyme including the presumptive dental mesenchyme before the appearance of the epithelial dental placode and dental neurites. Similarly, formation of a blood vessel plexus around the bud stage tooth germ and ingrowth of vessels into dental papilla at E14 preceded ingrowth of neurites. Subsequently, pioneer blood vessels in the dental papilla started to receive smooth muscle coverage at the early embryonic bell stage. Establishment and patterning of the blood vessels and nerves during tooth formation are developmentally regulated, stepwise processes that likely involve differential patterning mechanisms. Development of tooth vascular supply is proposed to be regulated by local, tooth-specific regulation by epithelial-mesenchymal tissue interactions and involving tooth target expressed VEGF signaling. Further investigations on tooth vascular development by local VEGF signaling, as well as how tooth innervation and development of blood vessels are integrated with advancing tooth organ formation by local signaling mechanisms, are warranted.


Assuntos
Vasos Sanguíneos/crescimento & desenvolvimento , Dente , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Animais , Padronização Corporal , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Músculo Liso Vascular/crescimento & desenvolvimento , Odontogênese , Transdução de Sinais , Dente/embriologia , Dente/inervação , Dente/metabolismo , Germe de Dente/embriologia , Germe de Dente/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo
20.
Mech Dev ; 156: 8-19, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30796970

RESUMO

Blood vessel maturation, which is characterized by the investment of vascular smooth muscle cells (vSMCs) around developing blood vessels, begins when vessels remodel into a hierarchy of proximal arteries and proximal veins that branch into smaller distal capillaries. The ultimate result of maturation is formation of the tunica media-the middlemost layer of a vessel that is composed of vSMCs and acts to control vessel integrity and vascular tone. Though many studies have implicated the role of various signaling molecules in regulating maturation, no studies have determined a role for hemodynamic force in the regulation of maturation in the mouse. In the current study, we provide evidence that a hemodynamic force-dependent mechanism occurs in the mouse because reduced blood flow mouse embryos exhibited a diminished or absent coverage of vSMCs around vessels, and in normal-flow embryos, extent of coverage correlated to the amount of blood flow that vessels were exposed to. We also determine that the cellular mechanism of force-induced maturation was not by promoting vSMC differentiation/proliferation, but instead involved the recruitment of vSMCs away from neighboring low-flow distal capillaries towards high-flow vessels. Finally, we hypothesize that hemodynamic force may regulate expression of specific signaling molecules to control vSMC recruitment to high-flow vessels, as reduction of flow results in the misexpression of Semaphorin 3A, 3F, 3G, and the Notch target gene Hey1, all of which are implicated in controlling vessel maturation. This study reveals another role for hemodynamic force in regulating blood vessel development of the mouse, and opens up a new model to begin elucidating mechanotransduction pathways regulating vascular maturation.


Assuntos
Vasos Sanguíneos/crescimento & desenvolvimento , Diferenciação Celular/genética , Desenvolvimento Embrionário/genética , Músculo Liso Vascular/crescimento & desenvolvimento , Animais , Artérias/crescimento & desenvolvimento , Artérias/metabolismo , Vasos Sanguíneos/metabolismo , Proliferação de Células/genética , Hemodinâmica , Mecanotransdução Celular/genética , Camundongos , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo
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