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1.
Circ Res ; 112(1): 113-27, 2013 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-23048070

RESUMO

RATIONALE: Activin receptor-like kinase-1 (ALK1) is an endothelial transforming growth factor ß receptor involved in angiogenesis. ALK1 expression is high in the embryo vasculature, becoming less detectable in the quiescent endothelium of adult stages. However, ALK1 expression becomes rapidly increased after angiogenic stimuli such as vascular injury. OBJECTIVE: To characterize the molecular mechanisms underlying the regulation of ALK1 on vascular injury. METHODS AND RESULTS: Alk1 becomes strongly upregulated in endothelial (EC) and vascular smooth muscle cells of mouse femoral arteries after wire-induced endothelial denudation. In vitro denudation of monolayers of human umbilical vein ECs also leads to an increase in ALK1. Interestingly, a key factor in tissue remodeling, Krüppel-like factor 6 (KLF6) translocates to the cell nucleus during wound healing, concomitantly with an increase in the ALK1 gene transcriptional rate. KLF6 knock down in human umbilical vein ECs promotes ALK1 mRNA downregulation. Moreover, Klf6(+/-) mice have lower levels of Alk1 in their vasculature compared with their wild-type siblings. Chromatin immunoprecipitation assays show that KLF6 interacts with ALK1 promoter in ECs, and this interaction is enhanced during wound healing. We demonstrate that KLF6 is transactivating ALK1 gene, and this transactivation occurs by a synergistic cooperative mechanism with specificity protein 1. Finally, Alk1 levels in vascular smooth muscle cells are not directly upregulated in response to damage, but in response to soluble factors, such as interleukin 6, released from ECs after injury. CONCLUSIONS: ALK1 is upregulated in ECs during vascular injury by a synergistic cooperative mechanism between KLF6 and specificity protein 1, and in vascular smooth muscle cells by an EC-vascular smooth muscle cell paracrine communication during vascular remodeling.


Assuntos
Receptores de Activinas Tipo II/metabolismo , Receptores de Ativinas Tipo I/metabolismo , Células Endoteliais/metabolismo , Artéria Femoral/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Lesões do Sistema Vascular/metabolismo , Cicatrização , Receptores de Ativinas Tipo I/genética , Receptores de Activinas Tipo II/genética , Animais , Sítios de Ligação , Modelos Animais de Doenças , Células Endoteliais/patologia , Artéria Femoral/lesões , Artéria Femoral/patologia , Regulação da Expressão Gênica , Células HEK293 , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Interleucina-6/metabolismo , Fator 6 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/deficiência , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Comunicação Parácrina , Regiões Promotoras Genéticas , Transporte Proteico , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas/genética , Interferência de RNA , RNA Mensageiro/metabolismo , Fator de Transcrição Sp1/metabolismo , Fatores de Tempo , Transcrição Gênica , Ativação Transcricional , Transfecção , Lesões do Sistema Vascular/genética , Lesões do Sistema Vascular/patologia
2.
Eur J Clin Invest ; 44(1): 83-92, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24350923

RESUMO

BACKGROUND: Stem cell therapy offers a promising approach to reduce the long-term mortality rate associated with heart failure after acute myocardial infarction (AMI). To date, in vivo translational studies have not yet fully studied the immune response to allogeneic adipose tissue-derived mesenchymal stem cells (ATMSCs). We analysed the immune response and the histological and functional effects of allogeneic ATMSCs in a porcine model of reperfused AMI and determine the effect of administration timing. DESIGN: Pigs that survived AMI (24/26) received intracoronary administration of culture medium after reperfusion (n = 6), ATMSCs after reperfusion (n = 6), culture medium 7 days after AMI (n = 6) or ATMSCs 7 days after AMI (n = 6). At 3-week follow-up, cardiac function, alloantibodies and histological analysis were evaluated. RESULTS: Administration of ATMSCs after reperfusion and 7 days after AMI resulted in similar rates of cell engraftment; some of those cells expressed endothelial, smooth muscle and cardiomyogenic cell lineage markers. Delivery of ATMSCs after reperfusion compared with that performed at 7 days was more effective in increasing: vascular density (249 ± 64 vs. 161 ± 37 vessels/mm2; P < 0.01), T lymphocytes (1 ± 0.4 vs. 0.4 ± 0.3% of area CD3(+) ; P < 0.05) and expression of vascular endothelial growth factor (VEGF; 32 ± 7% vs. 20 ± 4% of area VEGF(+) ; P < 0.01). Allogeneic ATMSC-based therapy did not change ejection fraction but generated alloantibodies. CONCLUSIONS: The present study is the first to demonstrate that allogeneic ATMSCs elicit an immune response and, when administered immediately after reperfusion, are more effective in increasing VEGF expression and neovascularization.


Assuntos
Tecido Adiposo/citologia , Transplante de Células-Tronco Mesenquimais , Infarto do Miocárdio/terapia , Animais , Modelos Animais de Doenças , Sobrevivência de Enxerto , Células-Tronco Mesenquimais/metabolismo , Microscopia Confocal , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Miofibroblastos/metabolismo , Miofibroblastos/patologia , Volume Sistólico , Suínos , Fatores de Tempo , Transplante Homólogo , Resultado do Tratamento , Fator A de Crescimento do Endotélio Vascular/metabolismo
3.
Arterioscler Thromb Vasc Biol ; 30(6): 1203-11, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20299686

RESUMO

OBJECTIVE: Vascular smooth muscle cells (VSMCs) contribute significantly to occlusive vascular diseases by virtue of their ability to switch to a noncontractile, migratory, and proliferating phenotype. Although the participation of ion channels in this phenotypic modulation (PM) has been described previously, changes in their expression are poorly defined because of their large molecular diversity. We obtained a global portrait of ion channel expression in contractile versus proliferating mouse femoral artery VSMCs, and explored the functional contribution to the PM of the most relevant changes that we observed. METHODS AND RESULTS: High-throughput real-time polymerase chain reaction of 87 ion channel genes was performed in 2 experimental paradigms: an in vivo model of endoluminal lesion and an in vitro model of cultured VSMCs obtained from explants. mRNA expression changes showed a good correlation between the 2 proliferative models, with only 2 genes, Kv1.3 and Kvbeta2, increasing their expression on proliferation. The functional characterization demonstrates that Kv1.3 currents increased in proliferating VSMC and that their selective blockade inhibits migration and proliferation. CONCLUSIONS: These findings establish the involvement of Kv1.3 channels in the PM of VSMCs, providing a new therapeutical target for the treatment of intimal hyperplasia.


Assuntos
Proliferação de Células , Canal de Potássio Kv1.3/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Animais , Movimento Celular , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Análise por Conglomerados , Modelos Animais de Doenças , Artéria Femoral/metabolismo , Artéria Femoral/patologia , Perfilação da Expressão Gênica , Genótipo , Hiperplasia , Canal de Potássio Kv1.3/antagonistas & inibidores , Canal de Potássio Kv1.3/genética , Potenciais da Membrana , Camundongos , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/lesões , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/patologia , Fenótipo , Bloqueadores dos Canais de Potássio/farmacologia , RNA Mensageiro/metabolismo , Superfamília Shaker de Canais de Potássio/genética , Superfamília Shaker de Canais de Potássio/metabolismo , Regulação para Cima , Vasoconstrição
4.
J Card Fail ; 16(4): 357-66, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20350704

RESUMO

BACKGROUND: Cell-based therapies offer a promising approach to reducing the short-term mortality rate associated with heart failure after a myocardial infarction. The aim of the study was to analyze histological and functional effects of adipose tissue-derived stem cells (ADSCs) after myocardial infarction and compare 2 types of administration pathways. METHODS AND RESULTS: ADSCs from 28 pigs were labeled by transfection. Animals that survived myocardial infarction (n = 19) received: intracoronary culture media (n = 4); intracoronary ADSCs (n = 5); transendocardial culture media (n = 4); or transendocardial ADSCs (n = 6). At 3 weeks' follow-up, intracoronary and transendocardial administration of ADSCs resulted in similar rates of engrafted cells (0.85 [0.19-1.97] versus 2 [1-2] labeled cells/cm(2), respectively; P = NS) and some of those cells expressed smooth muscle cell markers. The intracoronary administration of ADSCs was more effective in increasing the number of small vessels than transendocardial administration (223 +/- 40 versus 168 +/- 35 vessels/mm(2); P < .05). Ejection fraction was not modified by stem cell therapy. CONCLUSIONS: This is the first study to compare intracoronary and transendocardial administration of autologous ADSCs in a porcine model of myocardial infarction. Both pathways of ADSCs delivery are feasible, producing a similar number of engrafted and differentiated cells, although intracoronary administration was more effective in increasing neovascularization.


Assuntos
Tecido Adiposo/transplante , Endocárdio/cirurgia , Infarto do Miocárdio/cirurgia , Transplante de Células-Tronco/métodos , Tecido Adiposo/citologia , Animais , Células Cultivadas , Endocárdio/patologia , Feminino , Seguimentos , Infarto do Miocárdio/patologia , Suínos , Fatores de Tempo
5.
Age (Dordr) ; 35(1): 117-28, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22102320

RESUMO

The time-course for aging-associated effects on vascular reactivity to U46619, a stable analogue of thromboxane A(2) (TXA(2)), was studied in aorta from female senescence-accelerated mice-prone (SAMP8), a murine model of accelerated senescence. SAMP8 and senescence-accelerated mice-resistant (SAMR1) were divided into three groups: 3-, 6- and 10-month-old. Contractile curves to U46619 (10(-9) to 10(-6) M) were performed in aortic rings in the absence or in the presence of nitric oxide synthase (NOS) inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME; 10(-4) M) and/or cyclooxygenase (COX) inhibitor indomethacin (10(-5) M). Protein and gene expression for COX-1 and COX-2 were determined by immunofluorescence and real-time PCR, respectively. Maximal contraction to U46619 was markedly higher in SAMP8 at all ages. In SAMR1, increases were seen at 10 months, while SAMP8 displays augmented contraction at 6 months, which was further increased at 10 months. L-NAME enhanced U46619 contractions in both 6-month-old groups, although the increase was higher on vessels from SAMR1 at this age. Indomethacin equally increased U46619 contractions in both 3-month-old groups, suggesting the production of vasodilator prostaglandin in young animals. In contrast, at 6 and 10 months indomethacin decreased U46619 contractions in both groups, indicating an aging-associated swap to a release of contractile prostanoids in aorta. In conclusion, aging enhances contractile responses to TXA(2) in aorta from female mice by a mechanism involving a decrease of NO production and increased action of contractile prostanoids. This process occurs earlier in SAMP8 mice, establishing these mice as good model to study cardiovascular aging in a convenient and standard time-course.


Assuntos
Envelhecimento/fisiologia , Aorta Torácica/fisiologia , Estresse Oxidativo/fisiologia , Tromboxano A2/farmacologia , Vasoconstrição/efeitos dos fármacos , Envelhecimento/genética , Animais , Aorta Torácica/efeitos dos fármacos , Ciclo-Oxigenase 1/biossíntese , Ciclo-Oxigenase 1/genética , Ciclo-Oxigenase 2/biossíntese , Ciclo-Oxigenase 2/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Camundongos , RNA/biossíntese , RNA/genética , Reação em Cadeia da Polimerase em Tempo Real
6.
PLoS One ; 6(9): e25335, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21966501

RESUMO

AIMS: Aging is among the major causes for the lack of cardiovascular protection by estrogen (E2) during postmenopause. Our study aims to determine the mechanisms whereby aging changes E2 effects on nitric oxide (NO) production in a mouse model of accelerated senescence (SAM). METHODS AND RESULTS: Although we found no differences on NO production in females SAM prone (SAMP, aged) compared to SAM resistant (SAMR, young), by either DAF-2 fluorescence or plasmatic nitrite/nitrate (NO2/NO3), in both cases, E2 treatment increased NO production in SAMR but had no effect in SAMP. Those results are in agreement with changes of eNOS protein and gene expression. E2 up-regulated eNOS expression in SAMR but not in SAMP. E2 is also known to increase NO by decreasing its catabolism by superoxide anion (O(2)(-)). Interestingly, E2 treatment decreased O(2)(-) production in young females, while increased O(2)(-) in aged ones. Furthermore, we observed that aging changed expression ratio of estrogen receptors (ERß/ERα) and levels of DNA methylation. Increased ratio ERß/ERα in aged females is associated to a lack of estrogen modulation of NO production and with a reversal in its antioxidant effect to a pro-oxidant profile. CONCLUSIONS: Together, our data suggest that aging has detrimental effects on E2-mediated benefits on NO bioavailability, partially by affecting the ability of E2 to induce up regulation of eNOS and decrease of O(2)(-). These modifications may be associated to aging-mediated modifications on global DNA methylation status, but not to a specific methylation at 5'flanking region of ERα gene.


Assuntos
Envelhecimento/fisiologia , Estrogênios/farmacologia , Óxido Nítrico/metabolismo , Receptores de Estrogênio/metabolismo , Animais , Western Blotting , Metilação de DNA/efeitos dos fármacos , Feminino , Imunofluorescência , Camundongos , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Estrogênio/genética
7.
J Exp Med ; 208(10): 2125-39, 2011 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-21930771

RESUMO

Artery wall remodeling, a major feature of diseases such as hypertension, restenosis, atherosclerosis, and aneurysm, involves changes in the tunica media mass that reduce or increase the vessel lumen. The identification of molecules involved in vessel remodeling could aid the development of improved treatments for these pathologies. Angiotensin II (AngII) is a key effector of aortic wall remodeling that contributes to aneurysm formation and restenosis through incompletely defined signaling pathways. We show that AngII induces vascular smooth muscle cell (VSMC) migration and vessel remodeling in mouse models of restenosis and aneurysm. These effects were prevented by pharmacological inhibition of calcineurin (CN) or lentiviral delivery of CN-inhibitory peptides. Whole-genome analysis revealed >1,500 AngII-regulated genes in VSMCs, with just 11 of them requiring CN activation. Of these, the most sensitive to CN activation was regulator of CN 1 (Rcan1). Rcan1 was strongly activated by AngII in vitro and in vivo and was required for AngII-induced VSMC migration. Remarkably, Rcan1(-/-) mice were resistant to AngII-induced aneurysm and restenosis. Our results indicate that aneurysm formation and restenosis share mechanistic elements and identify Rcan1 as a potential therapeutic target for prevention of aneurysm and restenosis progression.


Assuntos
Aneurisma/patologia , Calcineurina/metabolismo , Reestenose Coronária/patologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Musculares/metabolismo , Músculo Liso Vascular/patologia , Músculo Liso Vascular/fisiologia , Aneurisma/fisiopatologia , Angiotensina II/metabolismo , Animais , Aorta/patologia , Calcineurina/genética , Proteínas de Ligação ao Cálcio , Movimento Celular , Reestenose Coronária/fisiopatologia , Ciclosporina/metabolismo , Regulação da Expressão Gênica , Imunossupressores/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Musculares/genética , Músculo Liso Vascular/citologia , Fatores de Transcrição NFATC/genética , Fatores de Transcrição NFATC/metabolismo , Ratos , Ratos Wistar , Transdução de Sinais/fisiologia
8.
Exp Gerontol ; 45(11): 868-74, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20708673

RESUMO

The aim of this work was to characterize a mouse model of experimental menopause and cardiovascular aging that closely reflects menopause in women. Senescence accelerated mouse (SAM)-Resistant type 1 (SAMR1, n=30) and SAM-Prone type 8 (SAMP8, n=30) were separated at 5months of age into three groups: 1) sham-operated (Sham); 2) ovariectomized (Ovx); and 3) ovariectomized chronically-treated with estrogen (Ovx+E2). Contractile responses to KCl (60mM) and thromboxane A(2) were greater in aorta from SAMP8 mice compared with SAMR1 in all groups. Neither ovariectomy nor estrogen replacement modified the contractile responses from SAMR1 mice. Conversely, in Ovx SAMP8 the increased maximal contractions were reversed by estrogen treatment. Rings with endothelium from all SAMR1 groups showed a greater relaxation to acetylcholine than SAMP8 groups. In SAMR1, endothelium-dependent relaxation was not altered in Ovx or Ovx+E2 groups. Rings from Ovx SAMP8 showed a decreased maximal response to acetylcholine compared to Sham SAMP8. Estrogen replacement restored the response to acetylcholine altered by ovariectomy. Nitric oxide inhibition by L-NAME markedly reduced acetylcholine responses in all groups, but this effect was less pronounced in SAMP8 and Ovx groups (determined by area under the curve reduction). These results indicate that SAMP8 exhibit a significant decreased endothelium-dependent and NO-mediated relaxation and increased vasoconstrictor responses that are potentiated by the lack of estrogen. Because these responses are closely in agreement with vascular dysfunction observed in menopausal women, we propose SAMP8 Ovx as a new model to concomitantly study the effects of aging and menopause in female mice.


Assuntos
Senilidade Prematura/fisiopatologia , Vasos Sanguíneos/fisiopatologia , Estrogênios/metabolismo , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacologia , Acetilcolina/farmacologia , Animais , Aorta/metabolismo , Regulação para Baixo , Inibidores Enzimáticos/farmacologia , Estradiol/farmacologia , Terapia de Reposição de Estrogênios , Estrogênios/farmacologia , Feminino , Técnicas In Vitro , Camundongos , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico/antagonistas & inibidores , Ovariectomia , Receptores de Tromboxano A2 e Prostaglandina H2/metabolismo , Vasoconstrição/efeitos dos fármacos , Vasoconstritores/farmacologia , Vasodilatação , Vasodilatadores/farmacologia
9.
Hypertension ; 56(3): 405-11, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20606108

RESUMO

Conjugated equine estrogen therapy is the most common hormone replacement strategy used to treat postmenopausal women. However, the ability of an individual conjugated equine estrogen to modulate NO production and, therefore, to induce cardiovascular protection is largely unknown. The effects of equine and naturally occurring estrogens on NO generation were evaluated in human aortic endothelial cells by measuring in vivo NO production, as well as NO synthase (eNOS) activity and expression. The transcriptional activity on the eNOS gene was determined by the ability of estrogen receptors (alpha and beta) to activate the eNOS promoter and induce transcription. Docking and molecular dynamics simulations were used to study structural features of the interaction between estrogenic compounds and estrogen receptor-alpha. After 24 hours of incubation, we found that estrone upregulated NO production almost as effectively as estradiol by increasing eNOS activity and expression. However, the effect of equine estrogens (equilin, equilenin, and their metabolites) were marked decreased. eNOS promoter activity by equine estrogens was 30% to 50% lower than the naturally occurring estrogens. Computational analysis of estrogen molecules revealed that position 17 and the saturation of estrogenic compounds in ring B are important determinants for estrogen receptor-alpha transcriptional activity. Equine estrogens increase NO production less effectively than naturally occurring estrogens, partially because of their lesser ability to activate the eNOS promoter and induce transcription. Differences in NO production by different estrogens may account for the differences in cardiovascular benefits achieved by the distinct estrogen replacement therapies.


Assuntos
Células Endoteliais/efeitos dos fármacos , Estradiol/farmacologia , Estrogênios Conjugados (USP)/farmacologia , Óxido Nítrico Sintase Tipo III/metabolismo , Óxido Nítrico/biossíntese , Células Cultivadas , Células Endoteliais/metabolismo , Humanos , Modelos Biológicos , Simulação de Dinâmica Molecular , Óxido Nítrico Sintase Tipo III/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Gênica/efeitos dos fármacos
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