RESUMEN
Multidirectional or disturbed flow promotes endothelial dysfunction and is associated with early atherogenesis. Here we investigated the role of Wnt signalling in flow-mediated endothelial dysfunction. The expression of Frizzled-4 was higher in cultured human aortic endothelial cells (ECs) exposed to disturbed flow compared to that seen for undisturbed flow, obtained using an orbital shaker. Increased expression was also detected in regions of the porcine aortic arch exposed to disturbed flow. The increased Frizzled-4 expression in cultured ECs was abrogated following knockdown of R-spondin-3. Disturbed flow also increased the nuclear localisation and activation of ß-catenin, an effect that was dependent on Frizzled-4 and R-spondin-3. Inhibition of ß-catenin using the small-molecule inhibitor iCRT5 or knockdown of Frizzled-4 or R-spondin-3 resulted in reduced expression of pro-inflammatory genes in ECs exposed to disturbed flow, as did inhibition of WNT5A signalling. Inhibition of the canonical Wnt pathway had no effect. Inhibition of ß-catenin also reduced endothelial paracellular permeability; this was associated with altered junctional and focal adhesion organisation and cytoskeletal remodelling. These data suggest the presence of an atypical Frizzled-4-ß-catenin pathway that promotes endothelial dysfunction in response to disturbed flow.
Asunto(s)
Células Endoteliales , beta Catenina , Animales , Humanos , beta Catenina/genética , beta Catenina/metabolismo , Células Endoteliales/metabolismo , Inflamación/metabolismo , Permeabilidad , Porcinos , Vía de Señalización Wnt , Receptores Frizzled/metabolismoRESUMEN
BACKGROUND: Late vein graft failure is caused by intimal thickening resulting from endothelial cell (EC) damage and inflammation which promotes vascular smooth muscle cell (VSMC) dedifferentiation, migration, and proliferation. Nonphosphorylatable PRH (proline-rich homeodomain) S163C:S177C offers enhanced stability and sustained antimitotic effect. Therefore, we investigated whether adenovirus-delivered PRH S163C:S177C protein attenuates intimal thickening via VSMC phenotype modification without detrimental effects on ECs. METHODS: PRH S163C:S177C was expressed in vitro (human saphenous vein-VSMCs and human saphenous vein-ECs) and in vivo (ligated mouse carotid arteries) by adenoviruses. Proliferation, migration, and apoptosis were quantified and phenotype was assessed using Western blotting for contractile filament proteins and collagen gel contraction. EC inflammation was quantified using VCAM (vascular cell adhesion protein)-1, ICAM (intercellular adhesion molecule)-1, interleukin-6, and monocyte chemotactic factor-1 measurement and monocyte adhesion. Next Generation Sequencing was utilized to identify novel downstream mediators of PRH action and these and intimal thickening were investigated in vivo. RESULTS: PRH S163C:S177C inhibited proliferation, migration, and apoptosis and promoted contractile phenotype (enhanced contractile filament proteins and collagen gel contraction) compared with virus control in human saphenous vein-VSMCs. PRH S163C:S177C expression in human saphenous vein-ECs significantly reduced apoptosis, without affecting cell proliferation and migration, while reducing TNF (tumor necrosis factor)-α-induced VCAM-1 and ICAM-1 and monocyte adhesion and suppressing interleukin-6 and monocyte chemotactic factor-1 protein levels. PRH S163C:S177C expression in ligated murine carotid arteries significantly impaired carotid artery ligation-induced neointimal proliferation and thickening without reducing endothelial coverage. Next Generation Sequencing revealed STAT-1 (signal transducer and activator of transcription 1) and HDAC-9 (histone deacetylase 9) as mediators of PRH action and was supported by in vitro and in vivo analyses. CONCLUSIONS: We observed PRH S163C:S177C attenuated VSMC proliferation, and migration and enhanced VSMC differentiation at least in part via STAT-1 and HDAC-9 signaling while promoting endothelial repair and anti-inflammatory properties. These findings highlight the potential for PRH S163C:S177C to preserve endothelial function whilst suppressing intimal thickening, and reducing late vein graft failure.
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Interleucina-6 , Túnica Íntima , Ratones , Animales , Humanos , Interleucina-6/metabolismo , Túnica Íntima/patología , Proliferación Celular , Neointima/patología , Factores Quimiotácticos/metabolismo , Factores Quimiotácticos/farmacología , Miocitos del Músculo Liso/metabolismo , Movimiento CelularRESUMEN
Human abdominal aortic aneurysms (AAAs) are characterized by increased activity of matrix metalloproteinases (MMP), including MMP-12, alongside macrophage accumulation and elastin degradation, in conjunction with superimposed atherosclerosis. Previous genetic ablation studies have proposed contradictory roles for MMP-12 in AAA development. In this study, we aimed to elucidate if pharmacological inhibition of MMP-12 activity with a phosphinic peptide inhibitor protects from AAA formation and progression in angiotensin (Ang) II-infused Apoe-/- mice. Complimentary studies were conducted in a human ex vivo model of early aneurysm development. Administration of an MMP-12 inhibitor (RXP470.1) protected hypercholesterolemia Apoe-/- mice from Ang II-induced AAA formation and rupture-related death, associated with diminished medial thinning and elastin fragmentation alongside increased collagen deposition. Proteomic analyses confirmed a beneficial effect of MMP-12 inhibition on extracellular matrix remodeling proteins combined with inflammatory pathways. Furthermore, RXP470.1 treatment of mice with pre-existing AAAs exerted beneficial effects as observed through suppressed aortic dilation and rupture, medial thinning, and elastin destruction. Our findings indicate that pharmacological inhibition of MMP-12 activity retards AAA progression and improves survival in mice providing proof-of-concept evidence to motivate translational work for MMP-12 inhibitor therapy in humans.
Asunto(s)
Angiotensina II , Aneurisma de la Aorta Abdominal , Apolipoproteínas E , Metaloproteinasa 12 de la Matriz , Inhibidores de la Metaloproteinasa de la Matriz , Animales , Aneurisma de la Aorta Abdominal/metabolismo , Aneurisma de la Aorta Abdominal/patología , Aneurisma de la Aorta Abdominal/inducido químicamente , Aneurisma de la Aorta Abdominal/prevención & control , Aneurisma de la Aorta Abdominal/etiología , Metaloproteinasa 12 de la Matriz/metabolismo , Ratones , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Humanos , Inhibidores de la Metaloproteinasa de la Matriz/farmacología , Inhibidores de la Metaloproteinasa de la Matriz/uso terapéutico , Masculino , Modelos Animales de Enfermedad , Ratones Noqueados , Ratones Endogámicos C57BL , Elastina/metabolismo , Proteómica/métodosRESUMEN
The sudden exposure of venous endothelial cells (vECs) to arterial fluid shear stress (FSS) is thought to be a major contributor to coronary artery bypass vein graft failure (VGF). However, the effects of arterial FSS on the vEC secretome are poorly characterised. We propose that analysis of the vEC secretome may reveal potential therapeutic approaches to suppress VGF. Human umbilical vein endothelial cells (HUVECs) pre-conditioned to venous FSS (18 h; 1.5 dynes/cm2) were exposed to venous or arterial FSS (15 dynes/cm2) for 24 h. Tandem Mass Tagging proteomic analysis of the vEC secretome identified significantly increased fibroleukin (FGL2) in conditioned media from HUVECs exposed to arterial FSS. This increase was validated by Western blotting. Application of the NFκB inhibitor BAY 11-7085 (1 µM) following pre-conditioning reduced FGL2 release from vECs exposed to arterial FSS. Exposure of vECs to arterial FSS increased apoptosis, measured by active cleaved caspase-3 (CC3) immunocytochemistry, which was likewise elevated in HUVECs treated with recombinant FGL2 (20 ng/mL) for 24 h under static conditions. To determine the mechanism of FGL2-induced apoptosis, HUVECs were pre-treated with a blocking antibody to FcγRIIB, a receptor FGL2 is proposed to interact with, which reduced CC3 levels. In conclusion, our findings indicate that the exposure of vECs to arterial FSS results in increased release of FGL2 via NFκB signalling, which promotes endothelial apoptosis via FcγRIIB signalling. Therefore, the inhibition of FGL2/FcγRIIB signalling may provide a novel approach to reduce arterial FSS-induced vEC apoptosis in vein grafts and suppress VGF.
Asunto(s)
Apoptosis , Puente de Arteria Coronaria , Células Endoteliales de la Vena Umbilical Humana , Receptores de IgG , Transducción de Señal , Estrés Mecánico , Humanos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Puente de Arteria Coronaria/efectos adversos , Puente de Arteria Coronaria/métodos , Receptores de IgG/metabolismo , FN-kappa B/metabolismo , Arterias/metabolismo , Células Endoteliales/metabolismoRESUMEN
Coronary artery bypass grafting remains the treatment of choice for a large cohort of patients with significant coronary disease. Despite the increased use of arterial grafts, the long saphenous vein remains the most commonly used conduit. Long-term graft patency continues to be the Achilles heel of saphenous vein grafts. This is due to the development of intimal hyperplasia, a chronic inflammatory disease that results in the narrowing and occlusion of a significant number of vein grafts. Research models for intimal hyperplasia are essential for a better understanding of pathophysiological processes of this condition. Large animal models resemble human anatomical structures and have been used as a surrogate to study disease development and prevention over the years. In this paper, we systematically review all published studies that utilized large animal models of vein graft disease with a focus on the type of model and any therapeutic intervention, specifically the use of external stents/mesh.
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Puente de Arteria Coronaria , Oclusión de Injerto Vascular , Animales , Humanos , Grado de Desobstrucción Vascular/fisiología , Hiperplasia/patología , Puente de Arteria Coronaria/métodos , Vena Safena/cirugía , Modelos AnimalesRESUMEN
Vascular endothelial cell stimulation is associated with the activation of different signalling pathways and transcription factors. Acute shear stress is known to induce different pro-inflammatory mediators such as IL-8. Nrf2 is activated by prolonged high shear stress promoting an antiinflammatory and athero-protective environment. However, little is known about the impact of acute shear stress on Nrf2 and Keap1 function and its role in IL-8 regulation. We aimed to examine Nrf2-Keap1 complex activation in-vitro and its role in regulating IL-8 transcripts under acute arterial shear stress (12 dyn/cm2) in venous endothelial cells (ECs). We note that acute high shear stress caused a significant upregulation of Nrf2 target genes, HO-1 and GCLM and an increased IL-8 upregulation at 90 and 120 minutes. Mechanistically, acute high shear did not affect Nrf2 nuclear translocation but resulted in reduced nuclear Keap1, suggesting that the reduction in nuclear Keap1 may result in increased free nuclear nrf2 to induce transcription. Consistently, the suppression of Keap1 using shRNA (shKeap1) resulted in significant upregulation of IL-8 transcripts in response to acute shear stress. Interestingly; the over expression of Nrf2 using Nrf2-Ad-WT or Sulforaphane was also associated with significant upregulation of IL-8 compared to controls. This study highlights the role of Keap1 in Nrf2 activation under shear stress and indicates that activation of Nrf2 may be deleterious in ECs in the context of acute haemodynamic injury.
Asunto(s)
Células Endoteliales , Factor 2 Relacionado con NF-E2 , Células Endoteliales/metabolismo , Humanos , Interleucina-8/genética , Proteína 1 Asociada A ECH Tipo Kelch/genética , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Estrés Oxidativo/fisiología , Estrés MecánicoRESUMEN
RATIONALE: In response to blood vessel wall injury, aberrant proliferation of vascular smooth muscle cells (SMCs) causes pathological remodeling. However, the controlling mechanisms are not completely understood. OBJECTIVE: We recently showed that the human long noncoding RNA, SMILR, promotes vascular SMCs proliferation by a hitherto unknown mechanism. Here, we assess the therapeutic potential of SMILR inhibition and detail the molecular mechanism of action. METHODS AND RESULTS: We used deep RNA-sequencing of human saphenous vein SMCs stimulated with IL (interleukin)-1α and PDGF (platelet-derived growth factor)-BB with SMILR knockdown (siRNA) or overexpression (lentivirus), to identify SMILR-regulated genes. This revealed a SMILR-dependent network essential for cell cycle progression. In particular, we found using the fluorescent ubiquitination-based cell cycle indicator viral system that SMILR regulates the late mitotic phase of the cell cycle and cytokinesis with SMILR knockdown resulting in ≈10% increase in binucleated cells. SMILR pulldowns further revealed its potential molecular mechanism, which involves an interaction with the mRNA of the late mitotic protein CENPF (centromere protein F) and the regulatory Staufen1 RNA-binding protein. SMILR and this downstream axis were also found to be activated in the human ex vivo vein graft pathological model and in primary human coronary artery SMCs and atherosclerotic plaques obtained at carotid endarterectomy. Finally, to assess the therapeutic potential of SMILR, we used a novel siRNA approach in the ex vivo vein graft model (within the 30 minutes clinical time frame that would occur between harvest and implant) to assess the reduction of proliferation by EdU incorporation. SMILR knockdown led to a marked decrease in proliferation from ≈29% in controls to ≈5% with SMILR depletion. CONCLUSIONS: Collectively, we demonstrate that SMILR is a critical mediator of vascular SMC proliferation via direct regulation of mitotic progression. Our data further reveal a potential SMILR-targeting intervention to limit atherogenesis and adverse vascular remodeling.
Asunto(s)
Proliferación Celular/fisiología , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Microfilamentos/metabolismo , Mitosis/fisiología , Músculo Liso Vascular/metabolismo , ARN Largo no Codificante/biosíntesis , Remodelación Vascular/fisiología , Ciclo Celular/fisiología , Células Cultivadas , Proteínas Cromosómicas no Histona/genética , Humanos , Proteínas de Microfilamentos/genética , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/metabolismo , Técnicas de Cultivo de Órganos , ARN Largo no Codificante/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Vena Safena/citología , Vena Safena/metabolismoRESUMEN
OBJECTIVE: Galectin-3 (formerly known as Mac-2), encoded by the LGALS3 gene, is proposed to regulate macrophage adhesion, chemotaxis, and apoptosis. We investigated the role of galectin-3 in determining the inflammatory profile of macrophages and composition of atherosclerotic plaques. Approach and Results: We observed increased accumulation of galectin-3-negative macrophages within advanced human, rabbit, and mouse plaques compared with early lesions. Interestingly, statin treatment reduced galectin-3-negative macrophage accrual in advanced plaques within hypercholesterolemic (apolipoprotein E deficient) Apoe-/- mice. Accordingly, compared with Lgals3+/+:Apoe-/- mice, Lgals3-/-:Apoe-/- mice displayed altered plaque composition through increased macrophage:smooth muscle cell ratio, reduced collagen content, and increased necrotic core area, characteristics of advanced plaques in humans. Additionally, macrophages from Lgals3-/- mice exhibited increased invasive capacity in vitro and in vivo. Furthermore, loss of galectin-3 in vitro and in vivo was associated with increased expression of proinflammatory genes including MMP (matrix metalloproteinase)-12, CCL2 (chemokine [C-C motif] ligand 2), PTGS2 (prostaglandin-endoperoxide synthase 2), and IL (interleukin)-6, alongside reduced TGF (transforming growth factor)-ß1 expression and consequent SMAD signaling. Moreover, we found that MMP12 cleaves macrophage cell-surface galectin-3 resulting in the appearance of a 22-kDa fragment, whereas plasma levels of galectin-3 were reduced in Mmp12-/-:Apoe-/- mice, highlighting a novel mechanism where MMP12-dependent cleavage of galectin-3 promotes proinflammatory macrophage polarization. Moreover, galectin-3-positive macrophages were more abundant within plaques of Mmp12-/-:Apoe-/- mice compared with Mmp12+/+:Apoe-/- animals. CONCLUSIONS: This study reveals a prominent protective role for galectin-3 in regulating macrophage polarization and invasive capacity and, therefore, delaying plaque progression.
Asunto(s)
Aterosclerosis/patología , Galectina 3/fisiología , Macrófagos/fisiología , Animales , Cruzamientos Genéticos , Femenino , Galectina 3/análisis , Galectina 3/deficiencia , Humanos , Inflamación/patología , Macrófagos/química , Macrófagos/patología , Masculino , Metaloproteinasa 12 de la Matriz/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Noqueados para ApoE , Persona de Mediana Edad , Placa Aterosclerótica/patología , Transducción de Señal/fisiología , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
BACKGROUND: The saphenous vein remains the most frequently used conduit for coronary artery bypass grafting, despite reported unsatisfactory long-term patency rates. Understanding the pathophysiology of vein graft failure and attempting to improve its longevity has been a significant area of research for more than three decades. This article aims to review the current understanding of the pathophysiology and potential new intervention strategies. METHODS: A search of three databases: MEDLINE, Web of Science, and Cochrane Library, was undertaken for the terms "pathophysiology," "prevention," and "treatment" plus the term "vein graft failure." RESULTS: Saphenous graft failure is commonly the consequence of four different pathophysiological mechanisms, early acute thrombosis, vascular inflammation, intimal hyperplasia, and late accelerated atherosclerosis. Different methods have been proposed to inhibit or attenuate these pathological processes including modified surgical technique, topical pretreatment, external graft support, and postoperative pharmacological interventions. Once graft failure occurs, the available treatments are either surgical reintervention, angioplasty, or conservative medical management reserved for patients not eligible for either procedure. CONCLUSION: Despite the extensive amount of research performed, the pathophysiology of saphenous vein graft is still not completely understood. Surgical and pharmacological interventions have improved early patency and different strategies for prevention seem to offer some hope in improving long-term patency.
Asunto(s)
Puente de Arteria Coronaria/métodos , Enfermedad de la Arteria Coronaria/cirugía , Oclusión de Injerto Vascular/prevención & control , Oclusión de Injerto Vascular/terapia , Disfunción Primaria del Injerto/prevención & control , Disfunción Primaria del Injerto/terapia , Vena Safena/trasplante , Injerto Vascular/métodos , Oclusión de Injerto Vascular/etiología , Humanos , Disfunción Primaria del Injerto/etiología , Resultado del Tratamiento , Grado de Desobstrucción VascularRESUMEN
RATIONALE: Atherosclerosis and aneurysms are leading causes of mortality worldwide. MicroRNAs (miRs) are key determinants of gene and protein expression, and atypical miR expression has been associated with many cardiovascular diseases; although their contributory role to atherosclerotic plaque and abdominal aortic aneurysm stability are poorly understood. OBJECTIVE: To investigate whether miR-181b regulates tissue inhibitor of metalloproteinase-3 expression and affects atherosclerosis and aneurysms. METHODS AND RESULTS: Here, we demonstrate that miR-181b was overexpressed in symptomatic human atherosclerotic plaques and abdominal aortic aneurysms and correlated with decreased expression of predicted miR-181b targets, tissue inhibitor of metalloproteinase-3, and elastin. Using the well-characterized mouse atherosclerosis models of Apoe-/- and Ldlr-/-, we observed that in vivo administration of locked nucleic acid anti-miR-181b retarded both the development and the progression of atherosclerotic plaques. Systemic delivery of anti-miR-181b in angiotensin II-infused Apoe-/- and Ldlr-/- mice attenuated aneurysm formation and progression within the ascending, thoracic, and abdominal aorta. Moreover, miR-181b inhibition greatly increased elastin and collagen expression, promoting a fibrotic response and subsequent stabilization of existing plaques and aneurysms. We determined that miR-181b negatively regulates macrophage tissue inhibitor of metalloproteinase-3 expression and vascular smooth muscle cell elastin production, both important factors in maintaining atherosclerotic plaque and aneurysm stability. Validation studies in Timp3-/- mice confirmed that the beneficial effects afforded by miR-181b inhibition are largely tissue inhibitor of metalloproteinase-3 dependent, while also revealing an additional protective effect through elevating elastin synthesis. CONCLUSIONS: Our findings suggest that the management of miR-181b and its target genes provides therapeutic potential for limiting the progression of atherosclerosis and aneurysms and protecting them from rupture.
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Aneurisma de la Aorta Abdominal/metabolismo , Aterosclerosis/metabolismo , Elastina/fisiología , MicroARNs/biosíntesis , Inhibidor Tisular de Metaloproteinasa-3/fisiología , Animales , Aneurisma de la Aorta Abdominal/patología , Aneurisma de la Aorta Abdominal/prevención & control , Aterosclerosis/patología , Aterosclerosis/prevención & control , Dieta Alta en Grasa/efectos adversos , Elastina/antagonistas & inhibidores , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , MicroARNs/antagonistas & inhibidores , Persona de Mediana Edad , Inhibidor Tisular de Metaloproteinasa-3/antagonistas & inhibidoresRESUMEN
OBJECTIVE: Increased vascular smooth muscle cell (VSMC) migration leads to intimal thickening which acts as a soil for atherosclersosis, as well as causing coronary artery restenosis after stenting and vein graft failure. Investigating factors involved in VSMC migration may enable us to reduce intimal thickening and improve patient outcomes. In this study, we determined whether Wnt proteins regulate VSMC migration and thereby intimal thickening. APPROACH AND RESULTS: Wnt2 mRNA and protein expression were specifically increased in migrating mouse aortic VSMCs. Moreover, VSMC migration was induced by recombinant Wnt2 in vitro. Addition of recombinant Wnt2 protein increased Wnt1-inducible signaling pathway protein-1 (WISP-1) mRNA by ≈1.7-fold, via ß-catenin/T-cell factor signaling, whereas silencing RNA knockdown of Wnt-2 reduced WISP-1 mRNA by ≈65%. Treatment with rWISP-1 significantly increased VSMC migration by ≈1.5-fold, whereas WISP-1 silencing RNA knockdown reduced migration by ≈40%. Wnt2 and WISP-1 effects were integrin-dependent and not additive, indicating that Wnt2 promoted VSMC migration via WISP-1. Additionally, Wnt2 and WISP-1 were significantly increased and colocated in human coronary arteries with intimal thickening. Reduced Wnt2 and WISP-1 levels in mouse carotid arteries from Wnt2(+/-) and WISP-1(-/-) mice, respectively, significantly suppressed intimal thickening in response to carotid artery ligation. In contrast, elevation of plasma WISP-1 via an adenovirus encoding WISP-1 significantly increased intimal thickening by ≈1.5-fold compared with mice receiving control virus. CONCLUSIONS: Upregulation of Wnt2 expression enhanced WISP-1 and promoted VSMC migration and thereby intimal thickening. As novel regulators of VSMC migration and intimal thickening, Wnt2 or WISP-1 may provide a potential therapy for restenosis and vein graft failure.
Asunto(s)
Proteínas CCN de Señalización Intercelular/metabolismo , Traumatismos de las Arterias Carótidas/metabolismo , Movimiento Celular , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Neointima , Proteínas Proto-Oncogénicas/metabolismo , Proteína wnt2/metabolismo , Animales , Proteínas CCN de Señalización Intercelular/deficiencia , Proteínas CCN de Señalización Intercelular/genética , Proteínas CCN de Señalización Intercelular/farmacología , Enfermedades de las Arterias Carótidas/metabolismo , Enfermedades de las Arterias Carótidas/patología , Traumatismos de las Arterias Carótidas/genética , Traumatismos de las Arterias Carótidas/patología , Movimiento Celular/efectos de los fármacos , Proliferación Celular , Células Cultivadas , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Genotipo , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/patología , Fenotipo , Proteínas Proto-Oncogénicas/deficiencia , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/farmacología , Interferencia de ARN , Proteínas Recombinantes/farmacología , Factores de Transcripción TCF/metabolismo , Transfección , Vía de Señalización Wnt , Proteína wnt2/deficiencia , Proteína wnt2/genética , Proteína wnt2/farmacología , beta Catenina/metabolismoRESUMEN
AIMS: The long-term failure of autologous saphenous vein bypass grafts due to neointimal thickening is a major clinical burden. Identifying novel strategies to prevent neointimal thickening is important. Thus, this study aimed to identify microRNAs (miRNAs) that are dysregulated during neointimal formation and determine their pathophysiological relevance following miRNA manipulation. METHODS AND RESULTS: We undertook a microarray approach to identify dysregulated miRNAs following engraftment in an interpositional porcine graft model. These profiling experiments identified a number of miRNAs which were dysregulated following engraftment. miR-21 levels were substantially elevated following engraftment and these results were confirmed by quantitative real-time PCR in mouse, pig, and human models of vein graft neointimal formation. Genetic ablation of miR-21 in mice or grafted veins dramatically reduced neointimal formation in a mouse model of vein grafting. Furthermore, pharmacological knockdown of miR-21 in human veins resulted in target gene de-repression and a significant reduction in neointimal formation. CONCLUSION: This is the first report demonstrating that miR-21 plays a pathological role in vein graft failure. Furthermore, we also provided evidence that knockdown of miR-21 has therapeutic potential for the prevention of pathological vein graft remodelling.
Asunto(s)
MicroARNs/genética , Neointima/genética , Vena Safena/metabolismo , Injerto Vascular , Animales , Arteria Carótida Común/metabolismo , Células Cultivadas , Técnicas de Silenciamiento del Gen , Rechazo de Injerto/genética , Rechazo de Injerto/metabolismo , Humanos , Ratones , Ratones Noqueados , MicroARNs/metabolismo , Análisis por Micromatrices , Vena Safena/trasplante , Porcinos , Venas Cavas/metabolismoRESUMEN
Hypertension induces cardiac fibrotic remodelling characterised by the phenotypic switching of cardiac fibroblasts (CFs) and collagen deposition. We tested the hypothesis that Wnt1-inducible signalling pathway protein-1 (WISP-1) promotes CFs' phenotypic switch, type I collagen synthesis, and in vivo fibrotic remodelling. The treatment of human CFs (HCFs, n = 16) with WISP-1 (500 ng/mL) induced a phenotypic switch (α-smooth muscle actin-positive) and type I procollagen cleavage to an intermediate form of collagen (pC-collagen) in conditioned media after 24h, facilitating collagen maturation. WISP-1-induced collagen processing was mediated by Akt phosphorylation via integrin ß1, and disintegrin and metalloproteinase with thrombospondin motifs 2 (ADAMTS-2). WISP-1 wild-type (WISP-1+/+) mice and WISP-1 knockout (WISP-1-/-) mice (n = 5-7) were subcutaneously infused with angiotensin II (AngII, 1000 ng/kg/min) for 28 days. Immunohistochemistry revealed the deletion of WISP-1 attenuated type I collagen deposition in the coronary artery perivascular area compared to WISP-1+/+ mice after a 28-day AngII infusion, and therefore, the deletion of WISP-1 attenuated AngII-induced cardiac fibrosis in vivo. Collectively, our findings demonstrated WISP-1 is a critical mediator in cardiac fibrotic remodelling, by promoting CFs' activation via the integrin ß1-Akt signalling pathway, and induced collagen processing and maturation via ADAMTS-2. Thereby, the modulation of WISP-1 levels could provide potential therapeutic targets in clinical treatment.
Asunto(s)
Proteínas CCN de Señalización Intercelular , Fibroblastos , Fibrosis , Miocardio , Proteínas Proto-Oncogénicas , Animales , Proteínas CCN de Señalización Intercelular/metabolismo , Proteínas CCN de Señalización Intercelular/genética , Fibroblastos/metabolismo , Fibroblastos/patología , Fibroblastos/efectos de los fármacos , Humanos , Ratones , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas/genética , Miocardio/patología , Miocardio/metabolismo , Colágeno/metabolismo , Angiotensina II/farmacología , Ratones Noqueados , Colágeno Tipo I/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Masculino , Transducción de Señal/efectos de los fármacos , Ratones Endogámicos C57BLRESUMEN
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/genéticaRESUMEN
The long saphenous vein is the most used conduit in cardiac surgery, but its long-term patency is limited by vein graft disease (VGD). Endothelial dysfunction is a key driver of VGD; its aetiology is multi-factorial. However emerging evidence identifies vein conduit harvest technique and preservation fluids as causal in their onset and propagation. This study aims to comprehensively review published data on the relationship between preservation solutions, endothelial cell integrity and function, and VGD in human saphenous veins harvested for CABG. The review was registered with PROSPERO (CRD42022358828). Electronic searches of Cochrane Central Register of Controlled Trials, MEDLINE, and EMBASE databases were undertaken from inception until August 2022. Papers were evaluated in line with registered inclusion and exclusion criteria. Searches identified 13 prospective, controlled studies for inclusion in the analysis. All studies used saline as a control solution. Intervention solutions included heparinised whole blood and saline, DuraGraft, TiProtec, EuroCollins, University of Wisconsin (UoW), buffered, cardioplegic and Pyruvate solutions. Most studies demonstrated that normal saline appears to have negative effects on venous endothelium and the most effective preservation solutions identified in this review were TiProtec and DuraGraft. The most used preservation solutions in the UK are heparinised saline or autologous whole blood. There is substantial heterogeneity both in practice and reporting of trials evaluating vein graft preservation solutions, and the quality of existing evidence is low. There is an unmet need for high quality trials evaluating the potential for these interventions to improve long-term patency in venous bypass grafts.
Asunto(s)
Soluciones Preservantes de Órganos , Enfermedades Vasculares , Humanos , Vena Safena/trasplante , Estudios Prospectivos , Endotelio Vascular , Reino UnidoRESUMEN
BACKGROUND: Osteopontin has been implicated in vascular calcification formation and vein graft intimal hyperplasia, and its expression can be triggered by pro-inflammatory activation of cells. The role of osteopontin and the temporal formation of microcalcification in vein grafts is poorly understood with a lack of understanding of the interaction between haemodynamic changes and the activation of osteopontin. METHODS: We used a porcine model of vein interposition grafts, and human long saphenous veins exposed to ex vivo perfusion, to study the activation of osteopontin using polymerase chain reaction, immunostaining, and 18F-sodium fluoride autoradiography. RESULTS: The porcine model showed that osteopontin is active in grafts within 1 week following surgery and demonstrated the presence of microcalcification. A brief pretreatment of long saphenous veins with dexamethasone can suppress osteopontin activation. Prolonged culture of veins after exposure to acute arterial haemodynamics resulted in the formation of microcalcification but this was suppressed by pretreatment with dexamethasone. 18F-sodium fluoride uptake was significantly increased as early as 1 week in both models, and the pretreatment of long saphenous veins with dexamethasone was able to abolish its uptake. CONCLUSIONS: Osteopontin is activated in vein grafts and is associated with microcalcification formation. A brief pretreatment of veins ex vivo with dexamethasone can suppress its activation and associated microcalcification.
Asunto(s)
Calcinosis , Osteopontina , Humanos , Porcinos , Animales , Osteopontina/metabolismo , Fluoruro de Sodio , Vena Safena/trasplante , Dexametasona/farmacología , Calcinosis/metabolismoAsunto(s)
Puente de Arteria Coronaria/estadística & datos numéricos , Enfermedad de la Arteria Coronaria/diagnóstico , Enfermedad de la Arteria Coronaria/cirugía , Animales , Enfermedad Crónica , Puente de Arteria Coronaria/tendencias , Humanos , Revascularización Miocárdica/estadística & datos numéricos , Revascularización Miocárdica/tendencias , Resultado del TratamientoRESUMEN
BACKGROUND: Coronary artery vein graft failure, resulting from thrombosis, intimal thickening, and atherosclerosis, is a significant clinical problem, with approximately 50% of vein grafts failing within 10 years. Intimal thickening is caused by migration of vascular smooth muscle cells from the media to the intima, where they proliferate. Interventions using gene transfer to inhibit vascular smooth muscle cells proliferation and migration are attractive because ex vivo access to the graft is possible. The involvement of matrix-degrading metalloproteinases in intimal thickening is well established, and we previously showed that adenoviral-delivered overexpression of an endogenous inhibitor, the tissue inhibitor of metalloproteinases-3 (TIMP-3), significantly retarded intimal thickening in short-term autologous porcine arteriovenous interposition grafts (28 days). However, it is essential to determine whether this approach will provide longer-term benefits. METHODS AND RESULTS: We assessed whether a recombinant adenovirus that overexpresses TIMP-3 (RAdTIMP-3) affects vein graft intimal thickening in the longer term (at 3 months). Porcine saphenous veins were subjected to luminal infection with 2.5×10(10) pfu/mL RAdTIMP-3 or RAd60 (control virus) or vehicle control, for 30 minutes before implantation into the carotid artery. Analysis of grafts harvested 3 months after delivery revealed that RAdTIMP-3-infected grafts had significantly reduced intimal areas compared with both controls (3.2 ± 0.4 mm(2) versus 5.6 ± 0.7 mm(2) and 5.9 ± 0.5 mm(2), RAdTIMP-3, RAd60, and vehicle, respectively). Medial areas were also significantly decreased by TIMP-3 (3.8 ± 0.3 mm(2) versus 6.7 ± 1.0 mm(2) and 5.2 ± 0.4 mm(2), RAdTIMP-3, RAd60, and vehicle, respectively). CONCLUSIONS: Overexpression of TIMP-3 provides a sustained retardation of vein graft intimal thickening and highlights the translational potential for ex vivo TIMP-3 gene therapy.
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Arterias Carótidas/cirugía , Terapia Genética/métodos , Neointima/prevención & control , Neointima/terapia , Vena Safena/trasplante , Inhibidor Tisular de Metaloproteinasa-3/genética , Injerto Vascular/métodos , Adenoviridae/genética , Animales , Arterias Carótidas/metabolismo , Arterias Carótidas/patología , Movimiento Celular/fisiología , Endotelio Vascular/metabolismo , Endotelio Vascular/patología , Modelos Animales , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Vena Safena/metabolismo , Vena Safena/patología , Porcinos , Factores de Tiempo , Inhibidor Tisular de Metaloproteinasa-3/metabolismo , Resultado del TratamientoRESUMEN
OBJECTIVE: Several matrix metalloproteinases (MMPs) have been implicated in extracellular matrix destruction and other actions that lead to plaque rupture and myocardial infarction. Conversely, other MMPs have been shown to promote vascular smooth muscle cell (VSMC)-driven neointima formation, which contributes to restenosis, fibrous cap formation, and plaque stability. MMP-3 knockout reduced VSMC accumulation in mouse atherosclerotic plaques, implicating MMP-3 in neointima formation. We therefore investigated the effect of MMP-3 knockout on neointima formation after carotid ligation in vivo and VSMC migration in vitro. METHODS AND RESULTS: Twenty-eight days after left carotid ligation, MMP-3 knockout significantly reduced neointima formation (75%, P<0.01) compared with wild-type (WT) littermates, and also reduced remodeling of ligated and contralateral carotid arteries. Gelatin zymography illustrated that MMP-3 knockout abolished MMP-9 activation in ligated carotids and scratch-wounded VSMC cultures. MMP-3 knockout also attenuated VSMC migration into a scratch wound by 59% compared with WT cells. Addition of exogenous MMP-3 or activated MMP-9 restored migration of MMP-3 knockouts to that of WT VSMCs, but exogenous MMP-3 had no effect on migration in MMP-9 knockout VSMCs. MMP-9 knockout or knockdown with small interfering RNA significantly retarded VSMC migration to the same extent as MMP-3 knockout. CONCLUSIONS: These results indicate for the first time that MMP-3 mediated activation of MMP-9 is required for efficient neointima formation after carotid ligation in vivo and for VSMC migration in vitro, whereas MMP-12 plays a redundant role. These findings add to the understanding of MMP action in plaque stability and restenosis.