RESUMEN
BACKGROUND: Realistic reconstruction of the in vivo human atherosclerotic environment requires the coculture of different cell types arranged in atherosclerotic vessel-like structures with exposure to flow and circulating cells, presenting challenges for disease modeling. This study aimed to develop a 3-dimensional tubular microfluidic model with quadruple coculture of human aortic smooth muscle cells, human umbilical cord vein endothelial cells, and foam cells to recreate a complex human atherosclerotic vessel in vitro to study the effects of flow and circulating immune cells. METHODS: We developed a coculture protocol utilizing BFP (blue fluorescent protein)-labeled human aortic smooth muscle cells, GFP (green fluorescent protein)-labeled human umbilical cord vein endothelial cells, and THP-1 macrophage-derived, Dil-labeled oxidized LDL (low-density lipoprotein) foam cells within a fibrinogen/collagen I-based 3-dimensional ECM (extracellular matrix). Perfusion experiments were conducted for 24 hours on both atherosclerotic vessels and healthy vessels (BFP-labeled human aortic smooth muscle cells and GFP-labeled human umbilical cord vein endothelial cells without foam cells). Additionally, perfusion with circulating THP-1 monocytes was performed to observe cell extravasation and recruitment. RESULTS: The resulting vessels displayed early lesion morphology, with a layered composition including an endothelium and media, and foam cells accumulating in the subendothelial space. The layered wall composition of both atherosclerotic and healthy vessels remained stable under perfusion. Circulating THP-1 monocytes demonstrated cell extravasation into the atherosclerotic vessel wall and recruitment to the foam cell core. The qPCR analysis indicated increased expression of atherosclerosis markers in the atherosclerotic vessels and adaptation of vascular smooth muscle cell migration in response to flow and the plaque microenvironment, compared with control vessels. CONCLUSIONS: The human 3-dimensional atherosclerosis model demonstrated stability under perfusion and allowed for the observation of immune cell behavior, providing a valuable tool for the atherosclerosis research field.
RESUMEN
BACKGROUND: Arteriovenous fistulae (AVFs) are the preferred vascular access for hemodialysis in patients with end-stage kidney disease. Chronic kidney disease (CKD) is associated with endothelial injury, impaired AVF maturation, and reduced patency, as well as utilization. Because CKD is characterized by multiple pathophysiological processes that induce endothelial-to-mesenchymal transition (EndMT), we hypothesized that CKD promotes EndMT during venous remodeling and that disruption of endothelial TGF (transforming growth factor)-ß signaling inhibits EndMT to prevent AVF failure even in the end-stage kidney disease environment. METHODS: The mouse 5/6 nephrectomy and aortocaval fistula models were used. CKD was created via 5/6 nephrectomy, with controls of no (0/6) or partial (3/6) nephrectomy in C57BL/6J mice. AVFs were created in mice with knockdown of TGF-ßR1/R2 (TGF-ß receptors type 1/2) in either smooth muscle cells or endothelial cells. AVF diameters and patency were measured and confirmed by serial ultrasound examination. AVF, both murine and human, were examined using Western blot, histology, and immunofluorescence. Human and mouse endothelial cells were used for in vitro experiments. RESULTS: CKD accelerates TGF-ß activation and promotes EndMT that is associated with increased AVF wall thickness and reduced patency in mice. Inhibition of TGF-ß signaling in both endothelial cells and smooth muscle cells decreased smooth muscle cell proliferation in the AVF wall, attenuated EndMT, and was associated with reduced wall thickness, increased outward remodeling, and improved AVF patency. Human AVF also showed increased TGF-ß signaling and EndMT. CONCLUSIONS: CKD promotes EndMT and reduces AVF patency. Inhibition of TGF-ß signaling, especially disruption of endothelial cell-specific TGF-ß signaling, attenuates EndMT and improves AVF patency in mouse AVF. Inhibition of EndMT may be a therapeutic approach of translational significance to improve AVF patency in human patients with CKD.
RESUMEN
BACKGROUND: The long isoform of the Wnk1 (with-no-lysine [K] kinase 1) is a ubiquitous serine/threonine kinase, but its role in vascular smooth muscle cells (VSMCs) pathophysiology remains unknown. METHODS: AngII (angiotensin II) was infused in Apoe-/- to induce experimental aortic aneurysm. Mice carrying an Sm22-Cre allele were cross-bred with mice carrying a floxed Wnk1 allele to specifically investigate the functional role of Wnk1 in VSMCs. RESULTS: Single-cell RNA-sequencing of the aneurysmal abdominal aorta from AngII-infused Apoe-/- mice revealed that VSMCs that did not express Wnk1 showed lower expression of contractile phenotype markers and increased inflammatory activity. Interestingly, WNK1 gene expression in VSMCs was decreased in human abdominal aortic aneurysm. Wnk1-deficient VSMCs lost their contractile function and exhibited a proinflammatory phenotype, characterized by the production of matrix metalloproteases, as well as cytokines and chemokines, which contributed to local accumulation of inflammatory macrophages, Ly6Chi monocytes, and γδ T cells. Sm22Cre+Wnk1lox/lox mice spontaneously developed aortitis in the infrarenal abdominal aorta, which extended to the thoracic area over time without any negative effect on long-term survival. AngII infusion in Sm22Cre+Wnk1lox/lox mice aggravated the aortic disease, with the formation of lethal abdominal aortic aneurysms. Pharmacological blockade of γδ T-cell recruitment using neutralizing anti-CXCL9 (anti-CXC motif chemokine ligand 9) antibody treatment, or of monocyte/macrophage using Ki20227, a selective inhibitor of CSF1 receptor, attenuated aortitis. Wnk1 deletion in VSMCs led to aortic wall remodeling with destruction of elastin layers, increased collagen content, and enhanced local TGF-ß (transforming growth factor-beta) 1 expression. Finally, in vivo TGF-ß blockade using neutralizing anti-TGF-ß antibody promoted saccular aneurysm formation and aorta rupture in Sm22 Cre+ Wnk1lox/lox mice but not in control animals. CONCLUSION: Wnk1 is a key regulator of VSMC function. Wnk1 deletion promotes VSMC phenotype switch toward a pathogenic proinflammatory phenotype, orchestrating deleterious vascular remodeling and spontaneous severe aortitis in mice.
Asunto(s)
Angiotensina II , Aneurisma de la Aorta Abdominal , Aortitis , Músculo Liso Vascular , Miocitos del Músculo Liso , Proteína Quinasa Deficiente en Lisina WNK 1 , Animales , Aortitis/genética , Aortitis/metabolismo , Aortitis/patología , Ratones , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Aneurisma de la Aorta Abdominal/genética , Aneurisma de la Aorta Abdominal/metabolismo , Aneurisma de la Aorta Abdominal/patología , Humanos , Proteína Quinasa Deficiente en Lisina WNK 1/genética , Proteína Quinasa Deficiente en Lisina WNK 1/metabolismo , Ratones Endogámicos C57BL , Masculino , Células Cultivadas , Ratones Noqueados para ApoE , Modelos Animales de Enfermedad , Inflamación/metabolismo , Inflamación/genética , Inflamación/patología , Aorta Abdominal/metabolismo , Aorta Abdominal/patologíaRESUMEN
BACKGROUND: Atherosclerosis is driven by the infiltration of the arterial intima by diverse immune cells and smooth muscle cells (SMCs). CD8+ T cells promote lesion growth during atherosclerotic lesion development, but their role in advanced atherosclerosis is less clear. Here, we studied the role of CD8+ T cells and their effects on SMCs in established atherosclerosis. METHODS: CD8+ T cells were depleted in (SMC reporter) low-density lipoprotein receptor-deficient (Ldlr-/-) mice with established atherosclerotic lesions. Atherosclerotic lesion formation was examined, and single-cell RNA sequencing of aortic SMCs and their progeny was performed. Additionally, coculture experiments with primary aortic SMCs and CD8+ T cells were conducted. RESULTS: Although we could not detect differences in atherosclerotic lesion size, an increased plaque SMC content was noted in mice after CD8+ T-cell depletion. Single-cell RNA sequencing of aortic lineage-traced SMCs revealed contractile SMCs and a modulated SMC cluster, expressing macrophage- and osteoblast-related genes. CD8+ T-cell depletion was associated with an increased contractile but decreased macrophage and osteoblast-like gene signature in this modulated aortic SMC cluster. Conversely, exposure of isolated aortic SMCs to activated CD8+ T cells decreased the expression of genes indicative of a contractile SMC phenotype and induced a macrophage and osteoblast-like cell state. Notably, CD8+ T cells triggered calcium deposits in SMCs under osteogenic conditions. Mechanistically, we identified transcription factors highly expressed in modulated SMCs, including Runx1, to be induced by CD8+ T cells in cultured SMCs in an IFNγ (interferon-γ)-dependent manner. CONCLUSIONS: We here uncovered CD8+ T cells to control the SMC phenotype in atherosclerosis. CD8+ T cells promote SMC dedifferentiation and drive SMCs to adopt features of macrophage-like and osteoblast-like, procalcifying cell phenotypes. Given the critical role of SMCs in atherosclerotic plaque stability, CD8+ T cells could thus be explored as therapeutic target cells during lesion progression.
Asunto(s)
Aterosclerosis , Linfocitos T CD8-positivos , Desdiferenciación Celular , Modelos Animales de Enfermedad , Músculo Liso Vascular , Miocitos del Músculo Liso , Placa Aterosclerótica , Animales , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Miocitos del Músculo Liso/patología , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/inmunología , Ratones , Aterosclerosis/patología , Aterosclerosis/metabolismo , Aterosclerosis/genética , Aterosclerosis/inmunología , Músculo Liso Vascular/patología , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/inmunología , Ratones Endogámicos C57BL , Ratones Noqueados , Células Cultivadas , Masculino , Receptores de LDL/genética , Receptores de LDL/deficiencia , Fenotipo , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Aorta/patología , Aorta/inmunología , Aorta/metabolismo , Técnicas de Cocultivo , Enfermedades de la Aorta/patología , Enfermedades de la Aorta/genética , Enfermedades de la Aorta/inmunología , Enfermedades de la Aorta/metabolismoRESUMEN
BACKGROUND: The N6-methyladenosine (m6A) modification of RNA and its regulators have important roles in the pathogenesis of pulmonary hypertension (PH). Ythdf2 (YTH N6-methyladenosine RNA binding protein 2) is best known for its role in degrading m6A-modified mRNAs such as Hmox1 mRNA, which leads to alternative activation of macrophages in PH. Recent studies have also linked Ythdf2 to the proliferation of pulmonary artery smooth muscle cells (PASMCs). However, its specific roles in PASMCs and downstream targets during the development of PH remain unclear. METHODS: The expression and biological function of Ythdf2 in PASMCs were investigated in human and experimental models of PH. Smooth muscle cell-specific Ythdf2-deficient mice were used to assess the roles of Ythdf2 in PASMCs in vivo. Proteomic analysis, m6A sequencing, and RNA immunoprecipitation analysis were used to screen for potential downstream targets. RESULTS: Ythdf2 was significantly upregulated in human and rodent PH-PASMCs, and smooth muscle cell-specific Ythdf2 deficiency ameliorated PASMC proliferation, right ventricular hypertrophy, pulmonary vascular remodeling, and PH development. Higher expression of Ythdf2 promoted PASMC proliferation and PH by paradoxically stabilizing Myadm mRNA in an m6A-dependent manner. Loss of Ythdf2 decreased the expression of Myadm in PASMCs and pulmonary arteries, both in vitro and in vivo. Additionally, silencing Myadm inhibited the Ythdf2-dependent hyperproliferation of PASMCs by upregulating the cell cycle kinase inhibitor p21. CONCLUSIONS: We have identified a novel mechanism where the increased expression of Ythdf2 stimulates PH-PASMC proliferation through an m6A/Myadm/p21 pathway. Strategies targeting Ythdf2 in PASMCs might be useful additions to the therapeutic approach to PH.
Asunto(s)
Proliferación Celular , Hipertensión Pulmonar , Músculo Liso Vascular , Miocitos del Músculo Liso , Arteria Pulmonar , Proteínas de Unión al ARN , Remodelación Vascular , Animales , Humanos , Masculino , Ratones , Células Cultivadas , Modelos Animales de Enfermedad , Hipertensión Pulmonar/metabolismo , Hipertensión Pulmonar/genética , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/metabolismo , Arteria Pulmonar/metabolismo , Estabilidad del ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Remodelación Vascular/fisiología , Remodelación Vascular/genéticaAsunto(s)
Aterosclerosis , Miocitos del Músculo Liso , Humanos , Aterosclerosis/patología , Aterosclerosis/metabolismo , Miocitos del Músculo Liso/patología , Miocitos del Músculo Liso/metabolismo , Animales , Neoplasias/patología , Músculo Liso Vascular/patología , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/citologíaRESUMEN
BACKGROUND: Thromboembolic events secondary to rupture or erosion of advanced atherosclerotic lesions is the global leading cause of death. The most common and effective means to reduce these major adverse cardiovascular events, including myocardial infarction and stroke, is aggressive lipid lowering via a combination of drugs and dietary modifications. However, we know little regarding the effects of reducing dietary lipids on the composition and stability of advanced atherosclerotic lesions, the mechanisms that regulate these processes, and what therapeutic approaches might augment the benefits of lipid lowering. METHODS: Smooth muscle cell lineage-tracing Apoe-/- mice were fed a high-cholesterol Western diet for 18 weeks and then a zero-cholesterol standard laboratory diet for 12 weeks before treating them with an IL (interleukin)-1ß or control antibody for 8 weeks. We assessed lesion size and remodeling indices, as well as the cellular composition of aortic and brachiocephalic artery lesions, indices of plaque stability, overall plaque burden, and phenotypic transitions of smooth muscle cell and other lesion cells by smooth muscle cell lineage tracing combined with single-cell RNA sequencing, cytometry by time-of-flight, and immunostaining plus high-resolution confocal microscopic z-stack analysis. RESULTS: Lipid lowering by switching Apoe-/- mice from a Western diet to a standard laboratory diet reduced LDL cholesterol levels by 70% and resulted in multiple beneficial effects including reduced overall aortic plaque burden, as well as reduced intraplaque hemorrhage and necrotic core area. However, contrary to expectations, IL-1ß antibody treatment after diet-induced reductions in lipids resulted in multiple detrimental changes including increased plaque burden and brachiocephalic artery lesion size, as well as increasedintraplaque hemorrhage, necrotic core area, and senescence as compared with IgG control antibody-treated mice. Furthermore, IL-1ß antibody treatment upregulated neutrophil degranulation pathways but downregulated smooth muscle cell extracellular matrix pathways likely important for the protective fibrous cap. CONCLUSIONS: Taken together, IL-1ß appears to be required for the maintenance of standard laboratory diet-induced reductions in plaque burden and increases in multiple indices of plaque stability.
Asunto(s)
Aterosclerosis , Modelos Animales de Enfermedad , Interleucina-1beta , Ratones Noqueados para ApoE , Miocitos del Músculo Liso , Placa Aterosclerótica , Animales , Interleucina-1beta/metabolismo , Aterosclerosis/patología , Aterosclerosis/prevención & control , Aterosclerosis/metabolismo , Aterosclerosis/genética , Ratones , Miocitos del Músculo Liso/patología , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/efectos de los fármacos , Masculino , Dieta Occidental , Ratones Endogámicos C57BL , Aorta/patología , Aorta/metabolismo , Aorta/efectos de los fármacos , Enfermedades de la Aorta/patología , Enfermedades de la Aorta/prevención & control , Enfermedades de la Aorta/genética , Enfermedades de la Aorta/metabolismo , Dieta Alta en Grasa , Músculo Liso Vascular/patología , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/efectos de los fármacos , Tronco Braquiocefálico/patología , Tronco Braquiocefálico/metabolismo , Tronco Braquiocefálico/efectos de los fármacosRESUMEN
The implementation of human induced pluripotent stem cell (hiPSC) models has introduced an additional tool for identifying molecular mechanisms of disease that complement animal models. Patient-derived or CRISPR/Cas9-edited induced pluripotent stem cells differentiated into smooth muscle cells (SMCs) have been leveraged to discover novel mechanisms, screen potential therapeutic strategies, and model in vivo development. The field has evolved over almost 15 years of research using hiPSC-SMCs and has made significant strides toward overcoming initial challenges such as the lineage specificity of SMC phenotypes. However, challenges both specific (eg, the lack of specific markers to thoroughly validate hiPSC-SMCs) and general (eg, a lack of transparency and consensus around methodology in the field) remain. In this review, we highlight the recent successes and remaining challenges of the hiPSC-SMC model.
Asunto(s)
Diferenciación Celular , Células Madre Pluripotentes Inducidas , Miocitos del Músculo Liso , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/patología , Miocitos del Músculo Liso/patología , Miocitos del Músculo Liso/metabolismo , Animales , Fenotipo , Músculo Liso Vascular/patología , Músculo Liso Vascular/metabolismo , Linaje de la CélulaAsunto(s)
Aterosclerosis , Modelos Animales de Enfermedad , Interleucina-1beta , Aterosclerosis/inmunología , Aterosclerosis/metabolismo , Aterosclerosis/patología , Aterosclerosis/prevención & control , Interleucina-1beta/metabolismo , Animales , Humanos , Transducción de Señal , Antiinflamatorios/uso terapéutico , Placa Aterosclerótica , Ratones , Proteína Antagonista del Receptor de Interleucina 1/uso terapéuticoRESUMEN
BACKGROUND: Pulmonary hypertension (PH) is a progressive and life-threatening disease characterized by pulmonary vascular remodeling, which involves aberrant proliferation and apoptosis resistance of the pulmonary arterial smooth muscle cells (PASMCs), resembling the hallmark characteristics of cancer. In cancer, the HMGB2 (high-mobility group box 2) protein promotes the pro-proliferative/antiapoptotic phenotype. However, the function of HMGB2 in PH remains uninvestigated. METHODS: Smooth muscle cell (SMC)-specific HMGB2 knockout or HMGB2-OE (HMGB2 overexpression) mice and HMGB2 silenced rats were used to establish hypoxia+Su5416 (HySu)-induced PH mouse and monocrotaline-induced PH rat models, respectively. The effects of HMGB2 and its underlying mechanisms were subsequently elucidated using RNA-sequencing and cellular and molecular biology analyses. Serum HMGB2 levels were measured in the controls and patients with pulmonary arterial (PA) hypertension. RESULTS: HMGB2 expression was markedly increased in the PAs of patients with PA hypertension and PH rodent models and was predominantly localized in PASMCs. SMC-specific HMGB2 deficiency or silencing attenuated PH development and pulmonary vascular remodeling in hypoxia+Su5416-induced mice and monocrotaline-treated rats. SMC-specific HMGB2 overexpression aggravated hypoxia+Su5416-induced PH. HMGB2 knockdown inhibited PASMC proliferation in vitro in response to PDGF-BB (platelet-derived growth factor-BB). In contrast, HMGB2 protein stimulation caused the hyperproliferation of PASMCs. In addition, HMGB2 promoted PASMC proliferation and the development of PH by RAGE (receptor for advanced glycation end products)/FAK (focal adhesion kinase)-mediated Hippo/YAP (yes-associated protein) signaling suppression. Serum HMGB2 levels were significantly increased in patients with PA hypertension, and they correlated with disease severity, predicting worse survival. CONCLUSIONS: Our findings indicate that targeting HMGB2 might be a novel therapeutic strategy for treating PH. Serum HMGB2 levels could serve as a novel biomarker for diagnosing PA hypertension and determining its prognosis.
Asunto(s)
Modelos Animales de Enfermedad , Proteína HMGB2 , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Liso Vascular , Miocitos del Músculo Liso , Arteria Pulmonar , Remodelación Vascular , Animales , Proteína HMGB2/genética , Proteína HMGB2/metabolismo , Humanos , Masculino , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Arteria Pulmonar/metabolismo , Arteria Pulmonar/fisiopatología , Arteria Pulmonar/patología , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Músculo Liso Vascular/fisiopatología , Ratas , Ratones , Proliferación Celular , Índice de Severidad de la Enfermedad , Transducción de Señal , Hipertensión Arterial Pulmonar/metabolismo , Hipertensión Arterial Pulmonar/fisiopatología , Ratas Sprague-Dawley , Femenino , Células Cultivadas , Persona de Mediana Edad , Hipertensión Pulmonar/metabolismo , Hipertensión Pulmonar/fisiopatologíaRESUMEN
BACKGROUND: Abdominal aortic aneurysm (AAA) is a severe aortic disease without effective pharmacological approaches. The nuclear hormone receptor LXRα (liver X receptor α), encoded by the NR1H3 gene, serves as a critical transcriptional mediator linked to several vascular pathologies, but its role in AAA remains elusive. METHODS: Through integrated analyses of human and murine AAA gene expression microarray data sets, we identified NR1H3 as a candidate gene regulating AAA formation. To investigate the role of LXRα in AAA formation, we used global Nr1h3-knockout and vascular smooth muscle cell-specific Nr1h3-knockout mice in 2 AAA mouse models induced with angiotensin II (1000 ng·kg·min; 28 days) or calcium chloride (CaCl2; 0.5 mol/L; 42 days). RESULTS: Upregulated LXRα was observed in the aortas of patients with AAA and in angiotensin II- or CaCl2-treated mice. Global or vascular smooth muscle cell-specific Nr1h3 knockout inhibited AAA formation in 2 mouse models. Loss of LXRα function prevented extracellular matrix degeneration, inflammation, and vascular smooth muscle cell phenotypic switching. Uhrf1, an epigenetic master regulator, was identified as a direct target gene of LXRα by integrated analysis of transcriptome sequencing and chromatin immunoprecipitation sequencing. Susceptibility to AAA development was consistently enhanced by UHRF1 (ubiquitin-like containing PHD and RING finger domains 1) in both angiotensin II- and CaCl2-induced mouse models. We then determined the CpG methylation status and promoter accessibility of UHRF1-mediated genes using CUT&Tag (cleavage under targets and tagmentation), RRBS (reduced representation bisulfite sequencing), and ATAC-seq (assay for transposase-accessible chromatin with sequencing) in vascular smooth muscle cells, which revealed that the recruitment of UHRF1 to the promoter of miR-26b led to DNA hypermethylation accompanied by relatively closed chromatin states, and caused downregulation of miR-26b expression in AAA. Regarding clinical significance, we found that underexpression of miR-26b-3p correlated with high risk in patients with AAA. Maintaining miR-26b-3p expression prevented AAA progression and alleviated the overall pathological process. CONCLUSIONS: Our study reveals a pivotal role of the LXRα/UHRF1/miR-26b-3p axis in AAA and provides potential biomarkers and therapeutic targets for AAA.
Asunto(s)
Aneurisma de la Aorta Abdominal , Proteínas Potenciadoras de Unión a CCAAT , Epigénesis Genética , Receptores X del Hígado , Ratones Noqueados , MicroARNs , Ubiquitina-Proteína Ligasas , Aneurisma de la Aorta Abdominal/genética , Aneurisma de la Aorta Abdominal/metabolismo , Aneurisma de la Aorta Abdominal/patología , Aneurisma de la Aorta Abdominal/inducido químicamente , Animales , Receptores X del Hígado/metabolismo , Receptores X del Hígado/genética , MicroARNs/genética , MicroARNs/metabolismo , Humanos , Proteínas Potenciadoras de Unión a CCAAT/genética , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Ratones , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Masculino , Modelos Animales de Enfermedad , Ratones Endogámicos C57BL , Metilación de ADN , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Angiotensina II/farmacologíaRESUMEN
In-stent restenosis (ISR) develops primarily due to neointimal hyperplasia. Gallic acid (GA) has anti-inflammatory, antioxidant, and cardioprotective effects. This study sought to investigate the effects of GA on neointimal hyperplasia and proliferation and migration of vascular smooth muscle cells (VSMCs) in a pig ISR model. In vitro proliferation and migration experiments were confirmed, after VSMCs were treated with platelet-derived growth factor (PDGF-BB) and GA (100 µM) using a 3-(4,5-dimethylthiazol)-2,5-diphenyltetrazolium bromide (MTT) assay and a scratch wound assay for 24 hours and 48 hours. A bare metal stent (BMS) was implanted in the pig coronary artery to induce ISR with overdilation (1.1-1.2:1), and GA (10 mg/kg/day) was administered for 4 weeks. At the 4-week follow-up, optical coherence tomography (OCT) and histopathological analyses were performed. GA decreased the proliferation of VSMCs by PDGF-BB for 24 hours (89.24±24.56% vs. 170.04±19.98%, p<0.001) and 48 hours (124.87±7.35% vs. 187.64±4.83%, p<0.001). GA inhibited the migration of VSMCs induced by PDGF-BB for 24 hours (26.73±2.38% vs. 65.38±9.73%, p<0.001) and 48 hours (32.96±3.04% vs. 77.04±10.07%, p<0.001). Using OCT, % neointimal hyperplasia was shown to have significantly decreased in the GA group compared with control vehicle group (28.25±10.07% vs. 37.60±10.84%, p<0.001). GA effectively reduced neointimal hyperplasia by inhibiting the proliferation and migration of VSMCs in a pig ISR model. GA could be a potential treatment strategy for reducing ISR after stent implantation.
RESUMEN
BACKGROUND: Abdominal aortic aneurysm (AAA) is a potentially life-threatening vascular condition, but approved medical therapies to prevent AAA progression and rupture are currently lacking. Sphingolipid metabolism disorders are associated with the occurrence and development of AAA. It has been discovered that ganglioside GM3, a sialic acid-containing type of glycosphingolipid, plays a protective role in atherosclerosis, which is an important risk factor for AAA; however, the potential contribution of GM3 to AAA development has not been investigated. METHODS: We performed a metabolomics study to evaluated GM3 level in plasma of human patients with AAA. We profiled GM3 synthase (ST3GAL5) expression in the mouse model of aneurysm and human AAA tissues through Western blotting and immunofluorescence staining. RNA sequencing, affinity purification and mass spectrometry, proteomic analysis, surface plasmon resonance analysis, and functional studies were used to dissect the molecular mechanism of GM3-regulating ferroptosis. We conditionally deleted and overexpressed St3gal5 in smooth muscle cells (SMCs) in vivo to investigate its role in AAA. RESULTS: We found significantly reduced plasma levels of GM3 in human patients with AAA. GM3 content and ST3GAL5 expression were decreased in abdominal aortic vascular SMCs in patients with AAA and an AAA mouse model. RNA sequencing analysis showed that ST3GAL5 silencing in human aortic SMCs induced ferroptosis. We showed that GM3 interacted directly with the extracellular domain of TFR1 (transferrin receptor 1), a cell membrane protein critical for cellular iron uptake, and disrupted its interaction with holo-transferrin. SMC-specific St3gal5 knockout exacerbated iron accumulation at lesion sites and significantly promoted AAA development in mice, whereas GM3 supplementation suppressed lipid peroxidation, reduced iron deposition in aortic vascular SMCs, and markedly decreased AAA incidence. CONCLUSIONS: Together, these results suggest that GM3 dysregulation promotes ferroptosis of vascular SMCs in AAA. Furthermore, GM3 may constitute a new therapeutic target for AAA.
Asunto(s)
Aneurisma de la Aorta Abdominal , Ferroptosis , Humanos , Ratones , Animales , Gangliósido G(M3)/metabolismo , Proteómica , Músculo Liso Vascular/metabolismo , Aneurisma de la Aorta Abdominal/genética , Aneurisma de la Aorta Abdominal/prevención & control , Aneurisma de la Aorta Abdominal/metabolismo , Hierro , Miocitos del Músculo Liso/metabolismo , Modelos Animales de EnfermedadRESUMEN
BACKGROUND: Senescence is a series of degenerative changes in the structure and physiological function of an organism. Whether JPX (just proximal to XIST)-a newly identified age-related noncoding RNA by us-is associated with atherosclerosis is still unknown. Our study was to investigate the role of JPX and provide insights into potential therapies targeting atherosclerosis. METHODS: We analyzed clinical data from multiple tissues including meniscus tissue, leukemia cells, and peripheral blood monocytes to identify age-related noncoding RNAs in senescent vascular smooth muscle cells (VSMCs). The molecular mechanism of JPX was investigated by capture hybridization analysis of RNA targets and chromatin immunoprecipitation. IGVTools and real-time quantitative polymerase chain reaction were used to evaluate the JPX expression during phenotype regulation in age-related disease models. The therapeutic potential of JPX was evaluated after establishing an atherosclerosis model in smooth muscle-specific Jpx knockout mice. RESULTS: JPX expression was upregulated in activated ras allele (H-rasV12)-induced senescent VSMCs and atherosclerotic arteries. JPX knockdown substantially reduced the elevation of senescence-associated secretory phenotype (SASP) genes in senescent VSMCs. Cytoplasmic DNA leaked from mitochondria via mitochondrial permeability transition pore formed by VDAC1 (voltage-dependent anion channel 1) oligomer activates the STING (stimulator of interferon gene) pathway. JPX could act as an enhancer for the SASP genes and functions as a scaffold molecule through interacting with phosphorylated p65/RelA and BRD4 (bromodomain-containing protein 4) in chromatin remodeling complex, promoting the transcription of SASP genes via epigenetic regulation. Smooth muscle knockout of Jpx in ApoeKO mice resulted in a decrease in plaque area, a reduction in SASP gene expression, and a decrease in senescence compared with controls. CONCLUSIONS: As an enhancer RNA, JPX can integrate p65 and BRD4 to form a chromatin remodeling complex, activating SASP gene transcription and promoting cellular senescence. These findings suggest that JPX is a potential therapeutic target for the treatment of age-related atherosclerosis.
Asunto(s)
Aterosclerosis , ARN Largo no Codificante , Ratones , Animales , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Músculo Liso Vascular/metabolismo , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Cromatina , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Epigénesis Genética , Aterosclerosis/genética , Aterosclerosis/metabolismo , Senescencia Celular/genética , Ratones Noqueados , Miocitos del Músculo Liso/metabolismoRESUMEN
Objective To explore the mechanism by which CD137 signal regulates the aging of vas-cular smooth muscle cells(VSMCs).Methods Thirty 8-week-old male C57BL/6J mice were ran-domly divided into a young group(8 weeks old)and an aged group(80 weeks old),with 30 mice in each group.After corresponding periods of feeding,the mice were euthanized,and the plasma and aortic blood vessels were isolated.In the cell experiments,normal VSMCs were divided into a control group,bleomycin(BLM)group,combined agonist group,and combined inhibitor group.The cellular senescence level of VSMCs was assessed using a cellular senescence β-galactosidase staining kit.Western blotting and PCR were employed to examine the expression of senescence-related proteins in tissues and cells,while ELISA was utilized to measure the expression of senes-cence-related inflammatory factors.Results The expression of CD137 and γ-H2AX in the aorta was significantly higher,while that of PCNA was obviously lower in the aged group than the young group(P＜0.05).The plasma level of CD137 was notably higher in the aged group than the young group(154.0±4.1 pg/ml vs 98.0±2.3 pg/ml,P＜0.05).Compared with the normal control group,there were significantly more aged VSMCs in the BLM group(P＜0.05).While,treatment of combined agonist resulted in larger amount of aged VSMCs when compared with the BLM group(P＜0.05),which was reversed by combined inhibitor treatment(P＜0.05).The levels of TNF-α,IL-6 and IL-1β were significantly elevated in the BLM group than the normal control group(P＜0.05).The combined agonist group had even higher levels of TNF-α,IL-6,and IL-1βthan the BLM group(P＜0.05),but the levels were decreased in the combined inhibitor group(P＜0.05).Compared with the normal control group,the expression of Bcl-2,γ-H2AX,P53,and P21 were significantly increased in the BLM group,combined agonist group,and combined inhibi-tor group,while that of PCNA was significantly decreased(P＜0.05).Compared with the BLM group,the expression of P53 and P21 in the combined agonist group showed an increase(P＜0.05),and the expression of P53 was significantly decreased in the combined inhibitor group(P＜0.05).Conclusion CD137 signal regulates the P53/P21 pathway to promote VSMC aging.
RESUMEN
Objective:To evaluate the effect of Salvianolic acid B (Sal B) on the inflammatory responses of vascular smooth muscle cells (VSMCs) in septic mice and the role of circACTA2.Methods:In vivo experiment Eighty-one healthy male C57BL/6 mice, aged 6-8 weeks, were divided into 3 groups ( n=27 each) by a random number table method: sham operation group, sepsis group and Sal B group. Sepsis model was developed by cecal ligation and puncture. After sucessful preparation of the model, Sal B 7 mg/kg/d was intraperitoneally injected once a day for 2 consecutive days in Sal B group. Twenty mice in each group were randomly selected to measure systolic blood pressure (SBP), diastolic blood pressure (DBP), mean arterial pressure (MAP) and whole blood lactic acid (Lac) and to record the survival within 7 days after developing the model. Seven mice in each group were randomly selected at 48 h after developing the model, and the arterial vascular tissues were collected for determination of the expression of interleukin-1beta (IL-1β) (by immunofluorescence staining), expression of IL-1β, tumor necrosis factor-alpha (TNF-α) and IL-6 protein and mRNA (by Western blot and quantitative real-time polymerase chain reaction, respectively), and expression of circACTA2 (by quantitative real-time polymerase chain reaction). Cell experiment Mouse VSMCs were cultured and divided into 6 groups ( n=3 each) by a random number table method: control group (C group), lipopolysaccharide (LPS) group, Sal B group, si-circACTA2+ C group, si-circACTA2+ LPS group, and si-circACTA2+ Sal B group. The cells were incubated for 24 h with LPS (final concentration 1 μg/ml) in LPS group and with LPS (final concentration 1 μg/ml) and Sal B (final concentration 5 μmol/L) in Sal B group. VSMCs were transfected with si-circACTA2 only in si-circACTA2+ C group. At 24 h after transfection of si-circACTA2 into VSMCs, the cells were incubated with LPS (final concentration 1 μg/ml) in si-circACTA2+ LPS group and with LPS (final concentration 1 μg/ml) and Sal B (final concentration 5 μmol/L) for 24 h in si-circACTA2+ Sal B group. The expression of IL-1β, TNF-α and IL-6 protein and mRNA was detected using Western blot and quantitative real-time polymerase chain reaction, and the expression of circACTA2 was determined by the quantitative real-time polymerase chain reaction. Results:In vivo experiment Compared with sham operation group, SBP, DBP and MAP were significantly decreased, the concentrations of whole blood Lac were increased, 7-day survival rate was decreased, the expression of IL-1β, TNF-α and IL-6 protein and mRNA in arterial vascular tissues was up-regulated, circACTA2 expression was down-regulated ( P<0.05), and the fluorescence of IL-1β was enhanced in sepsis group. Compared with sepsis group, SBP, DBP and MAP were significantly increased, whole blood Lac concentrations were decreased, 7-day survival rate was increased, the expression of IL-1β, TNF-α and IL-6 protein and mRNA in arterial vascular tissues was down-regulated, the expression of circACTA2 was up-regulated ( P<0.05), and the fluorescence of IL-1β was weakened in Sal B group. Cell experiment Compared with group C, the expression of IL-1β, TNF-α and IL-6 protein and mRNA was significantly up-regulated, and the expression of circACTA2 was down-regulated in LPS group ( P<0.05). Compared with LPS group, the expression of IL-1β, TNF-α and IL-6 protein and mRNA was significantly down-regulated, and the expression of circACTA2 was up-regulated in Sal B group ( P<0.05). Compared with si-circACTA2+ C group, the expression of IL-1β, TNF-α and IL-6 protein and mRNA was significantly up-regulated in si-circACTA2+ LPS group ( P<0.05). There were no significant differences in the expression of IL-1β, TNF-α and IL-6 protein and mRNA between si-circACTA2+ LPS group and si-circACTA2+ Sal B group ( P>0.05). Conclusions:Sal B can reduce the inflammatory responses of VSMCs, and the mechanism may be related to promoting the expression of circACTA2 in septic mice.
RESUMEN
BACKGROUND: Pulmonary hypertension (PH) is a chronic vascular disease characterized, among other abnormalities, by hyperproliferative smooth muscle cells and a perturbed cellular redox and metabolic balance. Oxidants induce cell cycle arrest to halt proliferation; however, little is known about the redox-regulated effector proteins that mediate these processes. Here, we report a novel kinase-inhibitory disulfide bond in cyclin D-CDK4 (cyclin-dependent kinase 4) and investigate its role in cell proliferation and PH. METHODS: Oxidative modifications of cyclin D-CDK4 were detected in human pulmonary arterial smooth muscle cells and human pulmonary arterial endothelial cells. Site-directed mutagenesis, tandem mass-spectrometry, cell-based experiments, in vitro kinase activity assays, in silico structural modeling, and a novel redox-dead constitutive knock-in mouse were utilized to investigate the nature and definitively establish the importance of CDK4 cysteine modification in pulmonary vascular cell proliferation. Furthermore, the cyclin D-CDK4 oxidation was assessed in vivo in the pulmonary arteries and isolated human pulmonary arterial smooth muscle cells of patients with pulmonary arterial hypertension and in 3 preclinical models of PH. RESULTS: Cyclin D-CDK4 forms a reversible oxidant-induced heterodimeric disulfide dimer between C7/8 and C135, respectively, in cells in vitro and in pulmonary arteries in vivo to inhibit cyclin D-CDK4 kinase activity, decrease Rb (retinoblastoma) protein phosphorylation, and induce cell cycle arrest. Mutation of CDK4 C135 causes a kinase-impaired phenotype, which decreases cell proliferation rate and alleviates disease phenotype in an experimental mouse PH model, suggesting this cysteine is indispensable for cyclin D-CDK4 kinase activity. Pulmonary arteries and human pulmonary arterial smooth muscle cells from patients with pulmonary arterial hypertension display a decreased level of CDK4 disulfide, consistent with CDK4 being hyperactive in human pulmonary arterial hypertension. Furthermore, auranofin treatment, which induces the cyclin D-CDK4 disulfide, attenuates disease severity in experimental PH models by mitigating pulmonary vascular remodeling. CONCLUSIONS: A novel disulfide bond in cyclin D-CDK4 acts as a rapid switch to inhibit kinase activity and halt cell proliferation. This oxidative modification forms at a critical cysteine residue, which is unique to CDK4, offering the potential for the design of a selective covalent inhibitor predicted to be beneficial in PH.
Asunto(s)
Ciclinas , Hipertensión Arterial Pulmonar , Humanos , Ratones , Animales , Ciclinas/metabolismo , Hipertensión Arterial Pulmonar/metabolismo , Cisteína/metabolismo , Células Endoteliales/metabolismo , Proliferación Celular , Arteria Pulmonar/metabolismo , Fosforilación , Puntos de Control del Ciclo Celular , Ciclina D/metabolismo , Células Cultivadas , Quinasa 4 Dependiente de la Ciclina/genética , Quinasa 4 Dependiente de la Ciclina/metabolismoRESUMEN
BACKGROUND: Although single-cell RNA-sequencing is commonly applied to dissect the heterogeneity in human tissues, it involves the preparation of single-cell suspensions via cell dissociation, causing loss of spatial information. In this study, we employed high-resolution single-cell transcriptome imaging to reveal rare smooth muscle cell (SMC) types in human thoracic aortic aneurysm (TAA) tissue samples. METHODS: Single-molecule spatial distribution of transcripts from 140 genes was analyzed in fresh-frozen human TAA samples with region and sex-matched controls. In vitro studies and tissue staining were performed to examine human CART prepropeptide (CARTPT) regulation and function. RESULTS: We captured thousands of cells per sample including a spatially distinct CARTPT-expressing SMC subtype enriched in male TAA samples. Immunoassays confirmed human CART (cocaine- and amphetamine-regulated transcript) protein enrichment in male TAA tissue and truncated CARTPT secretion into cell culture medium. Oxidized low-density lipoprotein, a cardiovascular risk factor, induced CARTPT expression, whereas CARTPT overexpression in human aortic SMCs increased the expression of key osteochondrogenic transcription factors and reduced contractile gene expression. Recombinant human CART treatment of human SMCs further confirmed this phenotype. Alizarin red staining revealed calcium deposition in male TAA samples showing similar localization with human CART staining. CONCLUSIONS: Here, we demonstrate the feasibility of single-molecule imaging in uncovering rare SMC subtypes in the diseased human aorta, a difficult tissue to dissociate. We identified a spatially distinct CARTPT-expressing SMC subtype enriched in male human TAA samples. Our functional studies suggest that human CART promotes osteochondrogenic switch of aortic SMCs, potentially leading to medial calcification of the thoracic aorta.
Asunto(s)
Aneurisma de la Aorta Torácica , Calcinosis , Humanos , Masculino , Transcriptoma , Aneurisma de la Aorta Torácica/metabolismo , Aorta Torácica/metabolismo , Perfilación de la Expresión Génica/métodos , Calcinosis/metabolismo , Miocitos del Músculo Liso/metabolismoRESUMEN
BACKGROUND: Activation of vascular smooth muscle cell (VSMC) inflammation is vital to initiate vascular disease. The role of human-specific long noncoding RNAs in VSMC inflammation is poorly understood. METHODS: Bulk RNA sequencing in differentiated human VSMCs revealed a novel human-specific long noncoding RNA called inflammatory MKL1 (megakaryoblastic leukemia 1) interacting long noncoding RNA (INKILN). INKILN expression was assessed in multiple in vitro and ex vivo models of VSMC phenotypic modulation as well as human atherosclerosis and abdominal aortic aneurysm. The transcriptional regulation of INKILN was verified through luciferase reporter and chromatin immunoprecipitation assays. Loss-of-function and gain-of-function studies and multiple RNA-protein and protein-protein interaction assays were used to uncover a mechanistic role of INKILN in the VSMC proinflammatory gene program. Bacterial artificial chromosome transgenic mice were used to study INKILN expression and function in ligation injury-induced neointimal formation. RESULTS: INKILN expression is downregulated in contractile VSMCs and induced in human atherosclerosis and abdominal aortic aneurysm. INKILN is transcriptionally activated by the p65 pathway, partially through a predicted NF-κB (nuclear factor kappa B) site within its proximal promoter. INKILN activates proinflammatory gene expression in cultured human VSMCs and ex vivo cultured vessels. INKILN physically interacts with and stabilizes MKL1, a key activator of VSMC inflammation through the p65/NF-κB pathway. INKILN depletion blocks interleukin-1ß-induced nuclear localization of both p65 and MKL1. Knockdown of INKILN abolishes the physical interaction between p65 and MKL1 and the luciferase activity of an NF-κB reporter. Furthermore, INKILN knockdown enhances MKL1 ubiquitination through reduced physical interaction with the deubiquitinating enzyme USP10 (ubiquitin-specific peptidase 10). INKILN is induced in injured carotid arteries and exacerbates ligation injury-induced neointimal formation in bacterial artificial chromosome transgenic mice. CONCLUSIONS: These findings elucidate an important pathway of VSMC inflammation involving an INKILN/MKL1/USP10 regulatory axis. Human bacterial artificial chromosome transgenic mice offer a novel and physiologically relevant approach for investigating human-specific long noncoding RNAs under vascular disease conditions.