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1.
Arterioscler Thromb Vasc Biol ; 38(2): 414-424, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29217510

RESUMO

OBJECTIVE: Pressure-induced myogenic tone is involved in autoregulation of local blood flow and confers protection against excessive pressure levels in small arteries and capillaries. Myogenic tone is dependent on smooth muscle microRNAs (miRNAs), but the identity of these miRNAs is unclear. Furthermore, the consequences of altered myogenic tone for hypertension-induced damage to small arteries are not well understood. APPROACH AND RESULTS: The importance of smooth muscle-enriched microRNAs, miR-143/145, for myogenic tone was evaluated in miR-143/145 knockout mice. Furthermore, hypertension-induced vascular injury was evaluated in mesenteric arteries in vivo after angiotensin II infusion. Myogenic tone was abolished in miR-143/145 knockout mesenteric arteries, whereas contraction in response to calyculin A and potassium chloride was reduced by ≈30%. Furthermore, myogenic responsiveness was potentiated by angiotensin II in wild-type but not in knockout mice. Angiotensin II administration in vivo elevated systemic blood pressure in both genotypes. Hypertensive knockout mice developed severe vascular lesions characterized by vascular inflammation, adventitial fibrosis, and neointimal hyperplasia in small mesenteric arteries. This was associated with depolymerization of actin filaments and fragmentation of the elastic laminae at the sites of vascular lesions. CONCLUSIONS: This study demonstrates that miR-143/145 expression is essential for myogenic responsiveness. During hypertension, loss of myogenic tone results in potentially damaging levels of mechanical stress and detrimental effects on small arteries. The results presented herein provide novel insights into the pathogenesis of vascular disease and emphasize the importance of controlling mechanical factors to maintain structural integrity of the vascular wall.


Assuntos
Pressão Arterial , Hipertensão/metabolismo , MicroRNAs/metabolismo , Músculo Liso Vascular/metabolismo , Remodelação Vascular , Vasoconstrição , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/patologia , Angiotensina II , Animais , Sinalização do Cálcio , Células Cultivadas , Modelos Animais de Doenças , Tecido Elástico/metabolismo , Tecido Elástico/patologia , Feminino , Fibrose , Técnicas de Inativação de Genes , Hiperplasia , Hipertensão/genética , Hipertensão/patologia , Hipertensão/fisiopatologia , Masculino , Artérias Mesentéricas/metabolismo , Artérias Mesentéricas/patologia , Artérias Mesentéricas/fisiopatologia , Camundongos Knockout , MicroRNAs/genética , Músculo Liso Vascular/patologia , Músculo Liso Vascular/fisiopatologia , Neointima , Resistência Vascular
3.
EMBO J ; 36(2): 183-201, 2017 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-27940654

RESUMO

Shear detection and mechanotransduction by arterial endothelium requires junctional complexes containing PECAM-1 and VE-cadherin, as well as firm anchorage to the underlying basement membrane. While considerable information is available for junctional complexes in these processes, gained largely from in vitro studies, little is known about the contribution of the endothelial basement membrane. Using resistance artery explants, we show that the integral endothelial basement membrane component, laminin 511 (laminin α5), is central to shear detection and mechanotransduction and its elimination at this site results in ablation of dilation in response to increased shear stress. Loss of endothelial laminin 511 correlates with reduced cortical stiffness of arterial endothelium in vivo, smaller integrin ß1-positive/vinculin-positive focal adhesions, and reduced junctional association of actin-myosin II In vitro assays reveal that ß1 integrin-mediated interaction with laminin 511 results in high strengths of adhesion, which promotes p120 catenin association with VE-cadherin, stabilizing it at cell junctions and increasing cell-cell adhesion strength. This highlights the importance of endothelial laminin 511 in shear response in the physiologically relevant context of resistance arteries.


Assuntos
Membrana Basal/fisiologia , Endotélio Vascular/fisiologia , Laminina/metabolismo , Estresse Mecânico , Estresse Fisiológico , Animais , Células Cultivadas , Humanos , Camundongos , Camundongos Knockout
4.
J Cell Physiol ; 232(11): 3088-3102, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28019664

RESUMO

Modulation from contractile to synthetic phenotype of vascular smooth muscle cells is a central process in disorders involving compromised integrity of the vascular wall. Phenotype modulation has been shown to include transition from voltage-dependent toward voltage-independent regulation of the intracellular calcium level, and inhibition of non-voltage dependent calcium influx contributes to maintenance of the contractile phenotype. One possible mediator of calcium-dependent signaling is the FAK-family non-receptor protein kinase Pyk2, which is activated by a number of stimuli in a calcium-dependent manner. We used the Pyk2 inhibitor PF-4594755 and Pyk2 siRNA to investigate the role of Pyk2 in phenotype modulation in rat carotid artery smooth muscle cells and in cultured intact arteries. Pyk2 inhibition promoted the expression of smooth muscle markers at the mRNA and protein levels under stimulation by FBS or PDGF-BB and counteracted phenotype shift in cultured intact carotid arteries and balloon injury ex vivo. During long-term (24-96 hr) treatment with PF-4594755, smooth muscle markers increased before cell proliferation was inhibited, correlating with decreased KLF4 expression and differing from effects of MEK inhibition. The Pyk2 inhibitor reduced Orai1 and preserved SERCA2a expression in carotid artery segments in organ culture, and eliminated the inhibitory effect of PDGF stimulation on L-type calcium channel and large-conductance calcium-activated potassium channel expression in carotid cells. Basal intracellular calcium level, calcium wave activity, and store-operated calcium influx were reduced after Pyk2 inhibition of growth-stimulated cells. Pyk2 inhibition may provide an interesting approach for preserving vascular smooth muscle differentiation under pathophysiological conditions.


Assuntos
Lesões das Artérias Carótidas/enzimologia , Diferenciação Celular/efeitos dos fármacos , Quinase 2 de Adesão Focal/antagonistas & inibidores , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Vasoconstrição/efeitos dos fármacos , Animais , Becaplermina , Canais de Cálcio Tipo L/genética , Canais de Cálcio Tipo L/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Lesões das Artérias Carótidas/genética , Lesões das Artérias Carótidas/fisiopatologia , Artéria Carótida Primitiva/efeitos dos fármacos , Artéria Carótida Primitiva/enzimologia , Artéria Carótida Primitiva/fisiopatologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Quinase 2 de Adesão Focal/genética , Quinase 2 de Adesão Focal/metabolismo , Regulação da Expressão Gênica , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Músculo Liso Vascular/enzimologia , Músculo Liso Vascular/fisiopatologia , Miócitos de Músculo Liso/enzimologia , Proteína ORAI1/genética , Proteína ORAI1/metabolismo , Técnicas de Cultura de Órgãos , Fenótipo , Proteínas Proto-Oncogênicas c-sis/farmacologia , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Sprague-Dawley , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Fatores de Tempo , Transfecção
5.
Physiol Rep ; 2(7)2014 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-25347863

RESUMO

Stretch of vascular smooth muscle stimulates growth and proliferation as well as contraction and expression of contractile/cytoskeletal proteins, all of which are also regulated by calcium-dependent signals. We studied the role of the calcium- and integrin-activated proline-rich tyrosine kinase 2 (PYK2) in stretch-induced responses of the rat portal vein loaded by a hanging weight ex vivo. PYK2 phosphorylation at Tyr-402 was increased both by a 10-min stretch and by organ culture with load over several days. Protein and DNA synthesis were reduced by the novel PYK2 inhibitor PF-4594755 (0.5-1 µmol/L), while still sensitive to stretch. In 3-day organ culture, PF-4594755 caused maintained myogenic spontaneous activity but did not affect contraction in response to high-K(+) (60 mmol/L) or to α1-adrenergic stimulation by cirazoline. Basal and stretch-induced PYK2 phosphorylation in culture were inhibited by PF-4594755, closely mimicking inhibition of non-voltage-dependent calcium influx by 2-APB (30 µmol/L). In contrast, the L-type calcium channel blocker, nifedipine (1 µmol/L) eliminated stretch-induced but not basal PYK2 phosphorylation. Stretch-induced Akt and ERK1/2 phosphorylation was eliminated by PF-4594755. PYK2 inhibition had no effect on mRNA expression of several smooth muscle markers, and stretch-sensitive SM22α synthesis was preserved. Culture of portal vein with the Ang II inhibitor losartan (1 µmol/L) eliminated stretch sensitivity of PYK2 and Akt phosphorylation, but did not affect mRNA expression of smooth muscle markers. The results suggest that PYK2 signaling functionally distinguishes effects of voltage- and non-voltage-dependent calcium influx. A small-molecule inhibitor of PYK2 reduces growth and DNA synthesis but does not affect contractile differentiation of vascular smooth muscle.

6.
Cardiovasc Res ; 101(2): 288-96, 2014 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-24233972

RESUMO

AIMS: The myogenic response is the intrinsic ability of small arteries to constrict in response to increased intraluminal pressure. Although microRNAs have been shown to play a role in vascular smooth muscle function, their importance in the regulation of the myogenic response is not known. In this study, we investigate the role of microRNAs in the regulation of myogenic tone by using smooth muscle-specific and tamoxifen-inducible deletion of the endonuclease Dicer in mice. METHODS AND RESULTS: In order to avoid effects of Dicer deletion on smooth muscle differentiation and growth, we used an early time point (5 weeks) after the tamoxifen-induction of Dicer knockout (KO). At this time point, we found that myogenic tone was completely absent in the mesenteric arteries of Dicer KO mice. This was associated with a reduced pressure-induced Akt-phosphorylation, possibly via increased phosphatase and tensin homologue (PTEN) expression, which was found to be a target of miR-26a. Furthermore, loss of myogenic tone was associated with a decreased depolarization-induced calcium influx, and was restored by the L-type channel agonist Bay K 8644 or by transient stimulation with angiotensin II (Ang II). The effect of Ang II was dependent on AT1-receptors and activation of the PI3-kinase/Akt pathway. CONCLUSION: In this study we have identified novel mechanisms that regulate myogenic tone in resistance arteries, which involves microRNA-dependent control of PI3-kinase/Akt signalling and L-type calcium influx. Furthermore, we have demonstrated that transient stimulation by Ang II can have long-lasting effects by potentiating myogenic tone.


Assuntos
Pressão Arterial , Mecanotransdução Celular , MicroRNAs/metabolismo , Músculo Liso Vascular/enzimologia , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Vasoconstrição , Vasodilatação , Animais , Agonistas dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo L/metabolismo , Sinalização do Cálcio , Células Cultivadas , RNA Helicases DEAD-box/deficiência , RNA Helicases DEAD-box/genética , Ativação Enzimática , Genótipo , Mecanotransdução Celular/efeitos dos fármacos , Artérias Mesentéricas/enzimologia , Camundongos , Camundongos Knockout , MicroRNAs/genética , Músculo Liso Vascular/efeitos dos fármacos , PTEN Fosfo-Hidrolase/metabolismo , Fenótipo , Fosforilação , Sistema Renina-Angiotensina , Ribonuclease III/deficiência , Ribonuclease III/genética , Fatores de Tempo , Transfecção , Vasoconstrição/efeitos dos fármacos , Vasodilatação/efeitos dos fármacos
7.
Microcirculation ; 21(3): 230-8, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24238368

RESUMO

The mechanical forces acting on SMC in the vascular wall are known to regulate processes such as vascular remodeling and contractile differentiation. However, investigations to elucidate the underlying mechanisms of mechanotransduction in smooth muscle have been hampered by technical limitations associated with mechanical studies on pressurized small arteries, due primarily to the small amount of available tissue. The murine portal vein is a relatively large vessel showing myogenic tone that in many respects recapitulates the properties of small resistance vessels. Studies on stretched portal veins to elucidate mechanisms of mechanotransduction in the vascular wall have shown that stretch-sensitive regulation of contractile differentiation is mediated via Rho-activation and actin polymerization, while stretch-induced growth is regulated by the MAPK pathway. In this review, we have summarized findings on mechanotransduction in the portal vein with focus on stretch-induced contractile differentiation and the role of calcium, actin polymerization and miRNAs in this response.


Assuntos
Actinas/metabolismo , Músculo Liso Vascular/citologia , Músculo Liso Vascular/fisiologia , Veia Porta/citologia , Veia Porta/fisiologia , Animais , Fenômenos Biomecânicos , Sinalização do Cálcio , Diferenciação Celular , Humanos , Mecanotransdução Celular , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Modelos Cardiovasculares , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/fisiologia , Polimerização
8.
PLoS One ; 8(12): e82308, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24340017

RESUMO

Recent work has uncovered a role of the microRNA (miRNA) miR-29 in remodeling of the extracellular matrix. Partial bladder outlet obstruction is a prevalent condition in older men with prostate enlargement that leads to matrix synthesis in the lower urinary tract and increases bladder stiffness. Here we tested the hypothesis that miR-29 is repressed in the bladder in outlet obstruction and that this has an impact on protein synthesis and matrix remodeling leading to increased bladder stiffness. c-Myc, NF-κB and SMAD3, all of which repress miR-29, were activated in the rat detrusor following partial bladder outlet obstruction but at different times. c-Myc and NF-κB activation occurred early after obstruction, and SMAD3 phosphorylation increased later, with a significant elevation at 6 weeks. c-Myc, NF-κB and SMAD3 activation, respectively, correlated with repression of miR-29b and miR-29c at 10 days of obstruction and with repression of miR-29c at 6 weeks. An mRNA microarray analysis showed that the reduction of miR-29 following outlet obstruction was associated with increased levels of miR-29 target mRNAs, including mRNAs for tropoelastin, the matricellular protein Sparc and collagen IV. Outlet obstruction increased protein levels of eight out of eight examined miR-29 targets, including tropoelastin and Sparc. Transfection of human bladder smooth muscle cells with antimiR-29c and miR-29c mimic caused reciprocal changes in target protein levels in vitro. Tamoxifen inducible and smooth muscle-specific deletion of Dicer in mice reduced miR-29 expression and increased tropoelastin and the thickness of the basal lamina surrounding smooth muscle cells in the bladder. It also increased detrusor stiffness independent of outlet obstruction. Taken together, our study supports a model where the combined repressive influences of c-Myc, NF-κB and SMAD3 reduce miR-29 in bladder outlet obstruction, and where the resulting drop in miR-29 contributes to matrix remodeling and altered passive mechanical properties of the detrusor.


Assuntos
Matriz Extracelular/metabolismo , MicroRNAs/biossíntese , Obstrução do Colo da Bexiga Urinária/metabolismo , Animais , Matriz Extracelular/genética , Matriz Extracelular/patologia , Humanos , Masculino , Camundongos , MicroRNAs/genética , NF-kappa B/genética , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Ratos , Proteína Smad3/genética , Proteína Smad3/metabolismo , Obstrução do Colo da Bexiga Urinária/genética , Obstrução do Colo da Bexiga Urinária/patologia
9.
PLoS One ; 8(5): e65135, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23705032

RESUMO

Vascular smooth muscle cells are constantly exposed to mechanical force by the blood pressure, which is thought to regulate smooth muscle growth, differentiation and contractile function. We have previously shown that the expression of microRNAs (miRNAs), small non-coding RNAs, is essential for regulation of smooth muscle phenotype including stretch-dependent contractile differentiation. In this study, we have investigated the effect of mechanical stretch on miRNA expression and the role of stretch-sensitive miRNAs for intracellular signaling in smooth muscle. MiRNA array analysis, comparing miRNA levels in stretched versus non-stretched portal veins, revealed a dramatic decrease in the miR-144/451 cluster level. Because this miRNA cluster is predicted to target AMPK pathway components, we next examined activation of this pathway. Diminished miR-144/451 expression was inversely correlated with increased phosphorylation of AMPKα at Thr172 in stretched portal vein. Similar to the effect of stretch, contractile differentiation could be induced in non-stretched portal veins by the AMPK activator, AICAR. Transfection with miR-144/451 mimics reduced the protein expression level of mediators in the AMPK pathway including MO25α, AMPK and ACC. This effect also decreased AICAR-induced activation of the AMPK signaling pathway. In conclusion, our results suggest that stretch-induced activation of AMPK in vascular smooth muscle is in part regulated by reduced levels of miR-144/451 and that this effect may play a role in promoting contractile differentiation of smooth muscle cells.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Regulação para Baixo/genética , MicroRNAs/genética , Músculo Liso Vascular/enzimologia , Transdução de Sinais , Estresse Mecânico , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Animais , Aorta/efeitos dos fármacos , Aorta/metabolismo , Sequência de Bases , Artérias Carótidas/efeitos dos fármacos , Artérias Carótidas/metabolismo , Regulação para Baixo/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/metabolismo , Dados de Sequência Molecular , Contração Muscular/efeitos dos fármacos , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/enzimologia , Fosforilação/efeitos dos fármacos , Veia Porta/efeitos dos fármacos , Veia Porta/metabolismo , Pressão , Ribonucleotídeos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transfecção
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