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1.
Circ Res ; 108(4): 427-36, 2011 Feb 18.
Artículo en Inglés | MEDLINE | ID: mdl-21193738

RESUMEN

RATIONALE: Vascular smooth muscle cell (VSMC) proliferation causes intimal thickening in atherosclerosis and restenosis. Previously, we demonstrated that Wnt/ß-catenin signaling upregulates VSMC proliferation in vitro. OBJECTIVE: We examined this pathway in vivo and investigated the involvement of specific Wnt proteins in VSMC proliferation. METHODS AND RESULTS: Left carotid arteries of TOPgal (ß-catenin signaling reporter) transgenic mice were ligated to induce intimal thickening. ß-Catenin signaling was induced in the media and intima at 3 and 28 days after ligation, respectively, and was associated with VSMC proliferation and cyclin D1 expression. In vitro, a Wnt agonist promoted mouse VSMC proliferation, whereas Wnt inhibitory factor (WIF)-1 retarded platelet-derived growth factor-BB (PDGF-BB)-induced VSMC proliferation. Microarray analysis and quantitative PCR detected a significant induction of Wnt2 and Wnt4 mRNA in PDGF-BB-treated (proliferating) VSMCs compared to quiescent VSMCs. Western blotting revealed this increase was only translated into protein for Wnt4. Specific silencing RNA knockdown of Wnt4, but not Wnt2, significantly reduced VSMC proliferation. Recombinant Wnt4, but not Wnt2, significantly increased VSMC proliferation by ≈2-fold and silencing RNA knockdown revealed this is via Frizzled 1. Immunohistochemistry showed that increased Wnt4 protein correlated with VSMC proliferation and cyclin D1 expression (P<0.05 and P<0.001, respectively) during intimal thickening after rat carotid artery injury. Importantly, we also showed that intimal thickening and VSMC proliferation after carotid artery ligation was significantly retarded in Wnt4(+/-) compared to Wnt4(+/+) mice. CONCLUSIONS: This study demonstrates that Wnt/ß-catenin signaling occurs in proliferating VSMCs during intimal thickening and indicates that this is a result of Wnt4 upregulation.


Asunto(s)
Proliferación Celular , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/citología , Transducción de Señal/fisiología , Túnica Íntima/citología , Proteínas Wnt/fisiología , beta Catenina/fisiología , Animales , Ciclina D1/fisiología , Receptores Frizzled/fisiología , Ratones , Ratones Mutantes , Ratones Transgénicos , Modelos Animales , Músculo Liso Vascular/fisiología , Miocitos del Músculo Liso/fisiología , Túnica Íntima/fisiología , Regulación hacia Arriba/fisiología , Proteínas Wnt/genética , Proteína wnt2/genética , Proteína wnt2/fisiología , Proteína Wnt4 , beta Catenina/genética
2.
J Vasc Surg ; 52(5): 1301-9, 2010 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-20630685

RESUMEN

OBJECTIVES: Inhibition of vascular smooth muscle cell (VSMC) migration is a potential strategy for reducing intimal thickening during in-stent restenosis and vein graft failure. In this study, we examined the effect of disrupting the function of the VSMC adhesion molecule, N-cadherin, using antagonists, neutralizing antibodies, and a dominant negative, on VSMC migration and intimal thickening. Migration was assessed by the scratch-wound assay of human saphenous vein VSMCs and in a human saphenous vein ex vivo organ culture model of intimal thickening. RESULTS: Inhibition of cadherin function using a pan-cadherin antagonist, significantly reduced migration by 53%±8% compared with the control peptide (n=3; P<.05). Furthermore, inhibition of N-cadherin function with an N-cadherin antagonist, neutralizing antibodies, and adenoviral expression of dominant negative N-cadherin (RAd dn-N-cadherin), significantly reduced migration by 31%±2%, 23%±1% and 32%±7% compared with controls, respectively (n=3; P<.05). Inhibition of cadherin function significantly increased apoptosis by between 1.5- and 3.3-fold at the wound edge. In an ex vivo model of intimal thickening, inhibition of N-cadherin function by infection of human saphenous vein segments with RAd dn-N-cadherin significantly reduced VSMC migration by 55% and increased VSMC apoptosis by 2.7-fold. As a result, intimal thickening was significantly suppressed by 54%±14%. Importantly, there was no detrimental effect of dn-N-cadherin on endothelial coverage; in fact, it was significantly increased, as was survival of cultured human saphenous vein endothelial cells. CONCLUSIONS: Under the condition of this study, cell-cell adhesion mediated by N-cadherin regulates VSMC migration via modulation of viability. Interestingly, inhibition of N-cadherin function significantly retards intimal thickening via inhibition of VSMC migration and promotion of endothelial cell survival. We suggest that disruption of N-cadherin-mediated cell-cell contacts is a potential strategy for reducing VSMC migration and intimal thickening.


Asunto(s)
Anticuerpos/farmacología , Apoptosis/efectos de los fármacos , Cadherinas/antagonistas & inhibidores , Movimiento Celular/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , Miocitos del Músculo Liso/efectos de los fármacos , Péptidos Cíclicos/farmacología , Túnica Íntima/efectos de los fármacos , Antígenos CD/genética , Antígenos CD/metabolismo , Cadherinas/genética , Cadherinas/metabolismo , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Humanos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Técnicas de Cultivo de Órganos , Vena Safena/efectos de los fármacos , Vena Safena/metabolismo , Vena Safena/patología , Factores de Tiempo , Transfección , Túnica Íntima/metabolismo , Túnica Íntima/patología
3.
Arterioscler Thromb Vasc Biol ; 29(2): 195-201, 2009 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-19008530

RESUMEN

OBJECTIVE: Vascular smooth muscle cell (VSMC) apoptosis contributes to atherosclerotic plaque instability and myocardial infarction. Consequently, reducing VSMC apoptosis may be beneficial for reducing plaque instability and acute coronary events. We previously demonstrated that N-cadherin, a cell-cell adhesion molecule, reduces VSMC apoptosis in vitro. In this study, we examined whether a soluble form of N-cadherin (SNC) affected VSMC apoptosis and plaque stability. METHODS AND RESULTS: SNC significantly inhibited VSMC apoptosis in vitro by approximately 50% via activation of fibroblast growth factor receptor, phosphoinositide-3 kinase, and Akt signaling. SNC also significantly reduced macrophage and foam cell-macrophage apoptosis in vitro by >50%, without affecting monocyte invasion or macrophage proliferation. Elevation of plasma levels of SNC in male apolipoprotein E-deficient mice with existing atherosclerosis via adenoviral delivery significantly reduced VSMC and macrophage apoptosis in brachiocephalic artery plaques by approximately 60%. Additionally, SNC promoted plaques of a more stable phenotype by elevating VSMC:macrophage ratio and presence of VSMC-rich fibrous cap, as well as attenuating macrophage number and incidence of buried fibrous caps (a surrogate plaque rupture marker). CONCLUSIONS: In summary, this study demonstrates that SNC suppressed plaque instability by attenuation of apoptosis, suggesting that SNC may have a therapeutic potential for retarding plaque instability.


Asunto(s)
Apoptosis , Aterosclerosis/terapia , Tronco Braquiocefálico/patología , Cadherinas/biosíntesis , Terapia Genética/métodos , Adenoviridae/genética , Animales , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Aterosclerosis/genética , Aterosclerosis/metabolismo , Aterosclerosis/patología , Tronco Braquiocefálico/metabolismo , Células CHO , Cadherinas/genética , Adhesión Celular , Cricetinae , Cricetulus , Modelos Animales de Enfermedad , Células Espumosas/metabolismo , Células Espumosas/patología , Vectores Genéticos , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Monocitos/metabolismo , Monocitos/patología , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Fragmentos de Péptidos/biosíntesis , Transducción de Señal , Factores de Tiempo , Transducción Genética , Regulación hacia Arriba
4.
Sci Rep ; 7(1): 17342, 2017 12 11.
Artículo en Inglés | MEDLINE | ID: mdl-29229950

RESUMEN

There is an unmet need for treatments to reduce abdominal aortic aneurysm (AAA) progression. Vascular smooth muscle cell (VSMC) apoptosis precipitates AAA formation, whereas VSMC proliferation repairs the vessel wall. We previously demonstrated that over-expression of EC4-Fc (truncated N-cadherin), or deletion of matrix-metalloproteinase-7 (Mmp-7) reduced VSMC apoptosis in mouse atherosclerotic plaques. Additionally, MMP-7 promotes VSMC apoptosis by cleavage of N-cadherin. We investigated their combined effect on AAA formation. Increased apoptosis and proliferation were observed in human AAA (HAAA) sections compared to normal aortae (HA). This coincided with increased MMP-7 activity and reduced N-cadherin protein levels in HAAA sections compared to HA. Using a mouse model of aneurysm formation, we showed that the combination of Mmp-7 deletion and EC4-Fc overexpression significantly increased AAA severity. Medial apoptosis and proliferation were both significantly reduced in these mice compared to control mice. In vitro, MMP-7 inhibition and EC4-Fc administration significantly supressed human aortic VSMC apoptosis (via activation of PI-3 kinase/Akt signalling) and proliferation. In conclusion, combined Mmp-7 deletion and systemic over-expression of EC4-Fc reduced both proliferation and apoptosis. Reduced proliferation-mediated repair over-rides any benefit of reduced apoptosis, increasing aneurysm severity. Future studies should therefore focus on retarding VSMC apoptosis whilst promoting VSMC proliferation.


Asunto(s)
Aorta/patología , Aneurisma de la Aorta Abdominal/patología , Cadherinas/metabolismo , Modelos Animales de Enfermedad , Metaloproteinasa 7 de la Matriz/fisiología , Angiotensina II/efectos adversos , Animales , Aorta/metabolismo , Aneurisma de la Aorta Abdominal/etiología , Aneurisma de la Aorta Abdominal/metabolismo , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Apoptosis , Cadherinas/genética , Proliferación Celular , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Índice de Severidad de la Enfermedad , Transducción de Señal
5.
Vascul Pharmacol ; 78: 53-62, 2016 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-26586312

RESUMEN

Reoccurrence of symptoms occurs in 30-50% of coronary artery disease patients receiving vein grafts or bare-metal stents due to intimal thickening (restenosis). Restenosis is caused by vascular smooth muscle cell (VSMC) migration and proliferation. New therapeutic approaches that reduce VSMC migration and proliferation while promoting endothelial cell (EC) coverage are required. We assessed the effect of a soluble form of N-cadherin (SNC-Fc, a fusion of the extracellular portion of N-Cadherin to a mutated Fc fragment of IgG), a cell-cell junction molecule, on human saphenous VSMC proliferation and migration in vitro. We also assessed its effect on intimal thickening in a validated human ex vivo organ culture model. We observed that SNC-Fc significantly inhibited VSMC proliferation and to a lesser extent migration. The anti-proliferative effect of SNC-Fc was mediated by the interaction of SNC-Fc with the FGFR, rather than through inhibition of ß-catenin signalling. SNC-Fc also significantly reduced intimal thickening by ~85% in the ex vivo organ culture model. SNC-Fc treatment inhibited proliferation of the intimal cells but did not affect migration. SNC-Fc reduced EC apoptosis, without detrimental effects on EC proliferation and migration in vitro. Importantly SNC-Fc increased EC coverage in the ex vivo model of intimal thickening. In conclusion, we suggest that SNC-Fc may have potential as an anti-proliferative therapeutic agent for reducing restenosis which has no detrimental effects on endothelial cells.


Asunto(s)
Cadherinas/farmacología , Proliferación Celular/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , Miocitos del Músculo Liso/efectos de los fármacos , Apoptosis/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Reestenosis Coronaria/prevención & control , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Humanos , Músculo Liso Vascular/citología , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Técnicas de Cultivo de Órganos , Vena Safena/citología , Vena Safena/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Túnica Íntima/efectos de los fármacos , beta Catenina/metabolismo
6.
Mol Ther Methods Clin Dev ; 1: 14004, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-26015951

RESUMEN

Atherosclerotic plaque instability is precipitated by vascular smooth muscle cell apoptosis in the fibrous cap, weakening it and leading to plaque rupture. We previously showed that reducing smooth muscle cell apoptosis with soluble N-cadherin (SNC) increased features of plaque stability. We have now identified the active site of SNC and examined whether a truncated form containing this site retains the antiapoptotic effect. SNC was mutated to prevent interaction with N-cadherin or fibroblast growth factor receptor (FGFR). Interaction with FGFR in the extracellular (EC) 4 domain of SNC was essential for the antiapoptotic effect. Therefore, we made a truncated form consisting of the EC4 domain. EC4 significantly reduced smooth muscle cell, macrophage, and endothelial cell apoptosis in vitro by ~70%, similar to SNC. Elevation of plasma levels of EC4 in male apolipoprotein E-deficient mice with existing atherosclerosis significantly reduced apoptosis in brachiocephalic artery plaques by ~50%. EC4 reduced plaque size and the incidence of buried fibrous layers and the macrophage:smooth muscle cell ratio (surrogate markers of plaque instability). Interaction of EC4 with FGFR induced potent antiapoptotic signaling in vitro and in vivo. EC4 modulates atherosclerosis in mice demonstrating its therapeutic potential for retarding plaque size and instability.

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