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BACKGROUND: Coronary atherosclerotic plaques susceptible to acute coronary syndrome have traditionally been characterized by their surrounding cellular architecture. However, with the advent of intravascular imaging, novel mechanisms of coronary thrombosis have emerged, challenging our contemporary understanding of acute coronary syndrome. These intriguing findings underscore the necessity for a precise molecular definition of plaque stability. Considering this, our study aimed to investigate the vascular microenvironment in patients with stable and unstable plaques using spatial transcriptomics. METHODS: Autopsy-derived coronary arteries were preserved and categorized by plaque stability (n=5 patients per group). We utilized the GeoMx spatial profiling platform and Whole Transcriptome Atlas to link crucial histological morphology markers in coronary lesions with differential gene expression in specific regions of interest, thereby mapping the vascular transcriptome. This innovative approach allowed us to conduct cell morphological and spatially resolved transcriptional profiling of atherosclerotic plaques while preserving crucial intercellular signaling. RESULTS: We observed intriguing spatial and cell-specific transcriptional patterns in stable and unstable atherosclerotic plaques, showcasing regional variations within the intima and media. These regions exhibited differential expression of proinflammatory molecules (eg, IFN-γ [interferon-γ], MHC class II, proinflammatory cytokines) and prothrombotic signaling pathways. By using lineage tracing through spatial deconvolution of intimal CD68+ (cluster of differentiation 68) cells, we characterized unique, intraplaque subpopulations originating from endothelial, smooth muscle, and myeloid lineages with distinct regional locations associated with plaque instability. In addition, unique transcriptional signatures were observed in vascular smooth muscle and CD68+ cells among plaques exhibiting coronary calcification. CONCLUSIONS: Our study illuminates distinct cell-specific and regional transcriptional alterations present in unstable plaques. Furthermore, we characterize the first spatially resolved, in situ evidence supporting cellular transdifferentiation and intraplaque plasticity as significant contributors to plaque instability in human coronary atherosclerosis. Our results provide a powerful resource for the identification of novel mediators of acute coronary syndrome, opening new avenues for preventative and therapeutic treatments.
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BACKGROUND: Calcium deficiency in women is strongly linked to an increased risk of developing preeclampsia. Mitochondrial calcium ([Ca2+]m) homeostasis is essential to regulate vascular smooth muscle cell (VSMC) function. However, the role of [Ca2+]m in preeclampsia development remains largely unknown. METHODS: To investigate this, human spiral arteries obtained from normotensive and preeclamptic women were collected for vascular function, RNA sequencing, and VSMC studies. N(ω)-nitro-L-arginine methyl ester-induced preeclampsia animal experiments were established to investigate the effects of intervening in [Ca2+]m to improve the outcome for preeclamptic mothers or their infants. RESULTS: Our initial findings revealed compromised vessel function in spiral arteries derived from patients with preeclampsia, as evidenced by diminished vasoconstriction and vasodilation responses to angiotensin II and sodium nitroprusside, respectively. Moreover, the spiral artery VSMCs from patients with preeclampsia exhibited phenotypic transformation and proliferation associated with the disrupted regulatory mechanisms of [Ca2+]m uptake. Subsequent in vitro experiments employing gain- and loss-of-function approaches demonstrated that the mitochondrial Na+/Ca2+ exchanger played a role in promoting phenotypic switching and impaired mitochondrial functions in VSMCs. Furthermore, mtNCLX (mitochondrial Na+/Ca2+ exchanger) inhibitor CGP37157 significantly improved VSMC phenotypic changes and restored mitochondrial function in both patients with preeclampsia-derived VSMCs and the preeclampsia rat model. CONCLUSIONS: This study provides comprehensive evidence supporting the disrupted regulatory mechanisms of [Ca2+]m uptake in VSMCs of spiral arteries of patients with preeclampsia and further elucidates its correlation with VSMC phenotypic switching and defective spiral artery remodeling. The findings suggest that targeting mtNCLX holds promise as a novel therapeutic approach for managing preeclampsia.
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BACKGROUND: STIM1 (stromal interaction molecule 1) regulates store-operated calcium entry and is involved in pulmonary artery vasoconstriction and pulmonary artery smooth muscle cell proliferation, leading to pulmonary arterial hypertension (PAH). METHODS: Bioinformatics analysis and a 2-stage matched case-control study were conducted to screen for noncoding variants that may potentially affect STIM1 transcriptional regulation in 242 patients with idiopathic PAH and 414 healthy controls. Luciferase reporter assay, real-time quantitative polymerase chain reaction, western blot, 5-ethynyl-2'-deoxyuridine (EdU) assay, and intracellular Ca2+ measurement were performed to study the mechanistic roles of those STIM1 noncoding variants in PAH. RESULTS: Five noncoding variants (rs3794050, rs7934581, rs3750996, rs1561876, and rs3750994) were identified and genotyped using Sanger sequencing. Rs3794050, rs7934581, and rs1561876 were associated with idiopathic PAH (recessive model, all P<0.05). Bioinformatics analysis showed that these 3 noncoding variants possibly affect the enhancer function of STIM1 or the microRNA (miRNA) binding to STIM1. Functional validation performed in HEK293 and pulmonary artery smooth muscle cells demonstrated that the noncoding variant rs1561876-G (STIM1 mutant) had significantly stronger transcriptional activity than the wild-type counterpart, rs1561876-A, by affecting the transcriptional regulatory function of both hsa-miRNA-3140-5p and hsa-miRNA-4766-5p. rs1561876-G enhanced intracellular Ca2+ signaling in human pulmonary artery smooth muscle cells secondary to calcium-sensing receptor activation and promoted proliferation of pulmonary artery smooth muscle cells under both normoxia and hypoxia conditions, suggesting a possible contribution to PAH development. CONCLUSIONS: The potential clinical implications of the 3 noncoding variants of STIM1, rs3794050, rs7934581, and rs1561876, are 2-fold, as they may help predict the risk and prognosis of idiopathic PAH and guide investigations on novel therapeutic pathway(s).
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Arteria Pulmonar , Molécula de Interacción Estromal 1 , Humanos , Molécula de Interacción Estromal 1/genética , Molécula de Interacción Estromal 1/metabolismo , Masculino , Femenino , Estudios de Casos y Controles , Persona de Mediana Edad , Adulto , Proteínas de Neoplasias/genética , Miocitos del Músculo Liso/metabolismo , Hipertensión Pulmonar Primaria Familiar/genética , Hipertensión Pulmonar Primaria Familiar/fisiopatología , Predisposición Genética a la Enfermedad , Músculo Liso Vascular/metabolismo , Regulación de la Expresión Génica , Proliferación Celular/genética , Polimorfismo de Nucleótido SimpleRESUMEN
BACKGROUND: Vascular smooth muscle cells (VSMCs) are highly plastic. Vessel injury induces a phenotypic transformation from differentiated to dedifferentiated VSMCs, which involves reduced expression of contractile proteins and increased production of extracellular matrix and inflammatory cytokines. This transition plays an important role in several cardiovascular diseases such as atherosclerosis, hypertension, and aortic aneurysm. TGF-ß (transforming growth factor-ß) is critical for VSMC differentiation and to counterbalance the effect of dedifferentiating factors. However, the mechanisms controlling TGF-ß activity and VSMC phenotypic regulation under in vivo conditions are poorly understood. The extracellular matrix protein TN-X (tenascin-X) has recently been shown to bind TGF-ß and to prevent it from activating its receptor. METHODS: We studied the role of TN-X in VSMCs in various murine disease models using tamoxifen-inducible SMC-specific knockout and adeno-associated virus-mediated knockdown. RESULTS: In hypertensive and high-fat diet-fed mice, after carotid artery ligation as well as in human aneurysmal aortae, expression of Tnxb, the gene encoding TN-X, was increased in VSMCs. Mice with smooth muscle cell-specific loss of TN-X (SMC-Tnxb-KO) showed increased TGF-ß signaling in VSMCs, as well as upregulated expression of VSMC differentiation marker genes during vascular remodeling compared with controls. SMC-specific TN-X deficiency decreased neointima formation after carotid artery ligation and reduced vessel wall thickening during Ang II (angiotensin II)-induced hypertension. SMC-Tnxb-KO mice lacking ApoE showed reduced atherosclerosis and Ang II-induced aneurysm formation under high-fat diet. Adeno-associated virus-mediated SMC-specific expression of short hairpin RNA against Tnxb showed similar beneficial effects. Treatment with an anti-TGF-ß antibody or additional SMC-specific loss of the TGF-ß receptor reverted the effects of SMC-specific TN-X deficiency. CONCLUSIONS: In summary, TN-X critically regulates VSMC plasticity during vascular injury by inhibiting TGF-ß signaling. Our data indicate that inhibition of vascular smooth muscle TN-X may represent a strategy to prevent and treat pathological vascular remodeling.
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Músculo Liso Vascular , Miocitos del Músculo Liso , Transducción de Señal , Tenascina , Remodelación Vascular , Animales , Humanos , Masculino , Ratones , Angiotensina II , Aneurisma de la Aorta/metabolismo , Aneurisma de la Aorta/patología , Aneurisma de la Aorta/genética , Aneurisma de la Aorta/prevención & control , Traumatismos de las Arterias Carótidas/patología , Traumatismos de las Arterias Carótidas/metabolismo , Traumatismos de las Arterias Carótidas/genética , Células Cultivadas , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Hipertensión/metabolismo , Hipertensión/patología , Hipertensión/fisiopatología , Hipertensión/genética , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Noqueados para ApoE , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Neointima , Fenotipo , Tenascina/metabolismo , Tenascina/genética , Tenascina/deficiencia , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
BACKGROUND: GPCRs (G-protein-coupled receptors) play a central role in the regulation of smooth muscle cell (SMC) contractility, but the function of SMC-expressed orphan GPCR class C group 5 member C (GPRC5C) is unclear. The aim of this project is to define the role of GPRC5C in SMC in vitro and in vivo. METHODS: We studied the role of GPRC5C in the regulation of SMC contractility and differentiation in human and murine SMC in vitro, as well as in tamoxifen-inducible, SMC-specific GPRC5C knockout mice under basal conditions and in vascular disease in vivo. RESULTS: Mesenteric arteries from tamoxifen-inducible, SMC-specific GPRC5C knockout mice showed ex vivo significantly reduced angiotensin II (Ang II)-dependent calcium mobilization and contraction, whereas responses to other relaxant or contractile factors were normal. In vitro, the knockdown of GPRC5C in human aortic SMC resulted in diminished Ang II-dependent inositol phosphate production and lower myosin light chain phosphorylation. In line with this, tamoxifen-inducible, SMC-specific GPRC5C knockout mice showed reduced Ang II-induced arterial hypertension, and acute inactivation of GPRC5C was able to ameliorate established arterial hypertension. Mechanistically, we show that GPRC5C and the Ang II receptor AT1 dimerize, and knockdown of GPRC5C resulted in reduced binding of Ang II to AT1 receptors in HEK293 cells, human and murine SMC, and arteries from tamoxifen-inducible, SMC-specific GPRC5C knockout mice. CONCLUSIONS: Our data show that GPRC5C regulates Ang II-dependent vascular contraction by facilitating AT1 receptor-ligand binding and signaling.
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Angiotensina II , Músculo Liso Vascular , Receptores Acoplados a Proteínas G , Animales , Humanos , Masculino , Ratones , Angiotensina II/farmacología , Células Cultivadas , Hipertensión/metabolismo , Hipertensión/fisiopatología , Hipertensión/inducido químicamente , Hipertensión/genética , Arterias Mesentéricas/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Contracción Muscular , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/genética , VasoconstricciónRESUMEN
BACKGROUND: Epigenetic age estimators (clocks) are predictive of human mortality risk. However, it is not yet known whether the epigenetic age of atherosclerotic plaques is predictive for the risk of cardiovascular events. METHODS: Whole-genome DNA methylation of human carotid atherosclerotic plaques (n=485) and of blood (n=93) from the Athero-Express endarterectomy cohort was used to calculate epigenetic age acceleration (EAA). EAA was linked to clinical characteristics, plaque histology, and future cardiovascular events (n=136). We studied whole-genome DNA methylation and bulk and single-cell transcriptomics to uncover molecular mechanisms of plaque EAA. We experimentally confirmed our in silico findings using in vitro experiments in primary human coronary endothelial cells. RESULTS: Male and female patients with severe atherosclerosis had a median chronological age of 69 years. The median epigenetic age was 65 years in females (median EAA, -2.2 [interquartile range, -4.3 to 2.2] years) and 68 years in males (median EAA, -0.3 [interquartile range, -2.9 to 3.8] years). Patients with diabetes and a high body mass index had higher plaque EAA. Increased EAA of plaque predicted future events in a 3-year follow-up in a Cox regression model (univariate hazard ratio, 1.7; P=0.0034) and adjusted multivariate model (hazard ratio, 1.56; P=0.02). Plaque EAA predicted outcome independent of blood EAA (hazard ratio, 1.3; P=0.018) and of plaque hemorrhage (hazard ratio, 1.7; P=0.02). Single-cell RNA sequencing in plaque samples from 46 patients in the same cohort revealed smooth muscle and endothelial cells as important cell types in plaque EAA. Endothelial-to-mesenchymal transition was associated with EAA, which was experimentally confirmed by TGFß-triggered endothelial-to-mesenchymal transition inducing rapid epigenetic aging in coronary endothelial cells. CONCLUSIONS: Plaque EAA is a strong and independent marker of poor outcome in patients with severe atherosclerosis. Plaque EAA was linked to mesenchymal endothelial and smooth muscle cells. Endothelial-to-mesenchymal transition was associated with EAA, which was experimentally validated. Epigenetic aging mechanisms may provide new targets for treatments that reduce atherosclerosis complications.
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Metilación de ADN , Células Endoteliales , Epigénesis Genética , Placa Aterosclerótica , Humanos , Masculino , Femenino , Anciano , Pronóstico , Persona de Mediana Edad , Células Endoteliales/patología , Células Endoteliales/metabolismo , Factores de Edad , Enfermedades de las Arterias Carótidas/genética , Enfermedades de las Arterias Carótidas/patología , Enfermedades de las Arterias Carótidas/cirugía , Células Cultivadas , Factores de Riesgo , Medición de RiesgoRESUMEN
BACKGRUOUND: Glucagon-like peptide-1 receptor agonist (GLP-1RA), which is a therapeutic agent for the treatment of type 2 diabetes mellitus, has a beneficial effect on the cardiovascular system. METHODS: To examine the protective effects of GLP-1RAs on proliferation and migration of vascular smooth muscle cells (VSMCs), A-10 cells exposed to angiotensin II (Ang II) were treated with either exendin-4, liraglutide, or dulaglutide. To examine the effects of GLP-1RAs on vascular calcification, cells exposed to high concentration of inorganic phosphate (Pi) were treated with exendin-4, liraglutide, or dulaglutide. RESULTS: Ang II increased proliferation and migration of VSMCs, gene expression levels of Ang II receptors AT1 and AT2, proliferation marker of proliferation Ki-67 (Mki-67), proliferating cell nuclear antigen (Pcna), and cyclin D1 (Ccnd1), and the protein expression levels of phospho-extracellular signal-regulated kinase (p-Erk), phospho-c-JUN N-terminal kinase (p-JNK), and phospho-phosphatidylinositol 3-kinase (p-Pi3k). Exendin-4, liraglutide, and dulaglutide significantly decreased the proliferation and migration of VSMCs, the gene expression levels of Pcna, and the protein expression levels of p-Erk and p-JNK in the Ang II-treated VSMCs. Erk inhibitor PD98059 and JNK inhibitor SP600125 decreased the protein expression levels of Pcna and Ccnd1 and proliferation of VSMCs. Inhibition of GLP-1R by siRNA reversed the reduction of the protein expression levels of p-Erk and p-JNK by exendin-4, liraglutide, and dulaglutide in the Ang II-treated VSMCs. Moreover, GLP-1 (9-36) amide also decreased the proliferation and migration of the Ang II-treated VSMCs. In addition, these GLP-1RAs decreased calcium deposition by inhibiting activating transcription factor 4 (Atf4) in Pi-treated VSMCs. CONCLUSION: These data show that GLP-1RAs ameliorate aberrant proliferation and migration in VSMCs through both GLP-1Rdependent and independent pathways and inhibit Pi-induced vascular calcification.
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Diabetes Mellitus Tipo 2 , Calcificación Vascular , Humanos , Angiotensina II/farmacología , Angiotensina II/metabolismo , Exenatida/farmacología , Liraglutida/farmacología , Músculo Liso Vascular/metabolismo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Antígeno Nuclear de Célula en Proliferación/farmacología , Receptores de Péptidos Similares al Glucagón , Diabetes Mellitus Tipo 2/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatidilinositol 3-Quinasas/farmacología , Fosfatos/metabolismo , Fosfatos/farmacología , Proliferación Celular , Calcificación Vascular/metabolismoRESUMEN
BACKGROUND: Extracellular vesicles (EVs) contain bioactive cargo including miRNAs and proteins that are released by cells during cell-cell communication. Endothelial cells (ECs) form the innermost lining of all blood vessels, interfacing with cells in the circulation and vascular wall. It is unknown whether ECs release EVs capable of governing recipient cells within these 2 separate compartments. Given their boundary location, we propose ECs use bidirectional release of distinct EV cargo in quiescent (healthy) and activated (atheroprone) states to communicate with cells within the circulation and blood vessel wall. METHODS: EVs were isolated from primary human aortic ECs (plate and transwell grown; ±IL [interleukin]-1ß activation), quantified, visualized, and analyzed by miRNA transcriptomics and proteomics. Apical and basolateral EC-EV release was determined by miRNA transfer, total internal reflection fluorescence and electron microscopy. Vascular reprogramming (RNA sequencing) and functional assays were performed on primary human monocytes or smooth muscle cells±EC-EVs. RESULTS: Activated ECs increased EV release, with miRNA and protein cargo related to atherosclerosis. EV-treated monocytes and smooth muscle cells revealed activated EC-EV altered pathways that were proinflammatory and atherogenic. ECs released more EVs apically, which increased with activation. Apical and basolateral EV cargo contained distinct transcriptomes and proteomes that were altered by EC activation. Notably, activated basolateral EC-EVs displayed greater changes in the EV secretome, with pathways specific to atherosclerosis. In silico analysis determined compartment-specific cargo released by the apical and basolateral surfaces of ECs can reprogram monocytes and smooth muscle cells, respectively, with functional assays and in vivo imaging supporting this concept. CONCLUSIONS: Demonstrating that ECs are capable of polarized EV cargo loading and directional EV secretion reveals a novel paradigm for endothelial communication, which may ultimately enhance the design of endothelial-based therapeutics for cardiovascular diseases such as atherosclerosis where ECs are persistently activated.
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Aterosclerosis , Vesículas Extracelulares , MicroARNs , Humanos , Células Endoteliales/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Vesículas Extracelulares/metabolismo , Comunicación Celular , Aterosclerosis/metabolismoRESUMEN
While arterial tone is generally determined by the phosphorylation of Ser19 in myosin light chain (p-MLC2), Thr18/Ser19 diphosphorylation of MLC2 (pp-MLC2) has been suggested to hinder the relaxation of smooth muscle. In a dual-wire myography of rodent pulmonary artery (PA) and mesenteric artery (MA), we noticed significantly slower relaxation in PA than in MA after 80 mM KCl-induced condition (80K-contraction). Thus, we investigated the MLC2 phosphorylation and the expression levels of its regulatory enzymes; soluble guanylate cyclase (sGC), Rho-A dependent kinase (ROCK) and myosin light chain phosphatase target regulatory subunit (MYPT1). Immunoblotting showed higher sGC-α and ROCK2 in PA than MA, while sGC-ß and MYPT1 levels were higher in MA than in PA. Interestingly, the level of pp-MLC2 was higher in PA than in MA without stimulation. In the 80K-contraction state, the levels of p-MLC2 and pp-MLC2 were commonly increased. Treatment with the ROCK inhibitor (Y27632, 10 µM) reversed the higher pp-MLC2 in PA. In the myography study, pharmacological inhibition of sGC (ODQ, 10 µM) slowed relaxation during washout, which was more pronounced in PA than in MA. The simultaneous treatment of Y27632 and ODQ reversed the impaired relaxation in PA and MA. Although treatment of PA with Y27632 alone could increase the rate of relaxation, it was still slower than that of MA without Y27632 treatment. Taken together, we suggest that the higher ROCK and lower MYPT in PA would have induced the higher level of MLC2 phosphorylation, which is responsible for the characteristic slow relaxation in PA.
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BACKGROUND: Smooth muscle cell (SMC) phenotypic switching has been increasingly detected in aortic aneurysm and dissection (AAD) tissues. However, the diverse SMC phenotypes in AAD tissues and the mechanisms driving SMC phenotypic alterations remain to be identified. METHODS: We examined the transcriptomic and epigenomic dynamics of aortic SMC phenotypic changes in mice with angiotensin II-induced AAD by using single-cell RNA sequencing and single-cell sequencing assay for transposase-accessible chromatin. SMC phenotypic alteration in aortas from patients with ascending thoracic AAD was examined by using single-cell RNA sequencing analysis. RESULTS: Single-cell RNA sequencing analysis revealed that aortic stress induced the transition of SMCs from a primary contractile phenotype to proliferative, extracellular matrix-producing, and inflammatory phenotypes. Lineage tracing showed the complete transformation of SMCs to fibroblasts and macrophages. Single-cell sequencing assay for transposase-accessible chromatin analysis indicated that these phenotypic alterations were controlled by chromatin remodeling marked by the reduced chromatin accessibility of contractile genes and the induced chromatin accessibility of genes involved in proliferation, extracellular matrix, and inflammation. IRF3 (interferon regulatory factor 3), a proinflammatory transcription factor activated by cytosolic DNA, was identified as a key driver of the transition of aortic SMCs from a contractile phenotype to an inflammatory phenotype. In cultured SMCs, cytosolic DNA signaled through its sensor STING (stimulator of interferon genes)-TBK1 (tank-binding kinase 1) to activate IRF3, which bound and recruited EZH2 (enhancer of zeste homolog 2) to contractile genes to induce repressive H3K27me3 modification and gene suppression. In contrast, double-stranded DNA-STING-IRF3 signaling induced inflammatory gene expression in SMCs. In Sting-/- mice, the aortic stress-induced transition of SMCs into an inflammatory phenotype was prevented, and SMC populations were preserved. Finally, profound SMC phenotypic alterations toward diverse directions were detected in human ascending thoracic AAD tissues. CONCLUSIONS: Our study reveals the dynamic epigenetic induction of SMC phenotypic alterations in AAD. DNA damage and cytosolic leakage drive SMCs from a contractile phenotype to an inflammatory phenotype.
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Aneurisma de la Aorta Torácica , Aneurisma de la Aorta , Disección Aórtica , Humanos , Ratones , Animales , Epigenómica , Fenotipo , Aneurisma de la Aorta Torácica/genética , Aneurisma de la Aorta Torácica/metabolismo , Disección Aórtica/genética , Miocitos del Músculo Liso/metabolismo , ADN/metabolismo , Cromatina/metabolismo , Epigénesis Genética , Células CultivadasAsunto(s)
Músculo Liso , Miocitos del Músculo Liso , Animales , Ratones , Ratones Transgénicos , IntegrasasRESUMEN
Objective:To investigate the impact of cleavage factor Im25(CFIm25)on VSMCs-specific knockdown in the context of hyperlipidemia.Methods:Mice models were constructed with specific knockout of CFIm25 in VSMCs(CFIm25f/+ TaglnCre)and control mice(TaglnCre).The mice were fed a normal diet or high-fat diet(HFD)for 18 weeks and their body weight changes were monitored.ELISA was used to measure serum total cholesterol(TC), triacylglycerol(TG), high-density lipoprotein(HDL-C)and low-density lipoprotein(LDL-C)levels.The extent of aortic lipid deposition in mice was assessed by oil red O staining.Results:During the feeding of a high-fat diet, CFIm25f/+ TaglnCre mice showed a significant increase in body weight compared to the control group[Male(1.01±0.06)g and(0.87±0.31)g, t=7.53, P<0.05; Female: (0.64±0.02)g and(0.35±0.04)g, t=9.68, P<0.05].After 18 weeks of high-fat diet feeding, CFIm25f/+ TaglnCre mice had significantly higher levels of TC[(6.80±0.35)mmol/L and(3.76±0.87)mmol/L, t=5.63, P=0.004], TG[(0.97±0.21)mmol/L and(0.42±0.10)mmol/L, t=4.08, P=0.015], and LDL-C[(5.20±0.30)mmol/L and(2.00±0.98)mmol/L, t=5.40, P=0.006]compared to the TaglnCre group.Specifically, TC levels increased by 80.72%, TG increased by 132.79%, and LDL-C increased by 160.32%.There was a significant increase in aorta lipid deposition and atherosclerotic plaque area in CFIm25f/+ TaglnCre mice( P<0.05). Conclusions:The research indicated that VSMCs-specific CFIm25 knockdown in mice further worsened hyperlipidemia and atherosclerotic lesions.
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BACKGRUOUND: Excessive proliferation and migration of vascular smooth muscle cells (VSMCs), which contributes to the development of occlusive vascular diseases, requires elevated mitochondrial oxidative phosphorylation to meet the increased requirements for energy and anabolic precursors. Therefore, therapeutic strategies based on blockade of mitochondrial oxidative phosphorylation are considered promising for treatment of occlusive vascular diseases. Here, we investigated whether DN200434, an orally available estrogen receptor-related gamma inverse agonist, inhibits proliferation and migration of VSMCs and neointima formation by suppressing mitochondrial oxidative phosphorylation. METHODS: VSMCs were isolated from the thoracic aortas of 4-week-old Sprague-Dawley rats. Oxidative phosphorylation and the cell cycle were analyzed in fetal bovine serum (FBS)- or platelet-derived growth factor (PDGF)-stimulated VSMCs using a Seahorse XF-24 analyzer and flow cytometry, respectively. A model of neointimal hyperplasia was generated by ligating the left common carotid artery in male C57BL/6J mice. RESULTS: DN200434 inhibited mitochondrial respiration and mammalian target of rapamycin complex 1 activity and consequently suppressed FBS- or PDGF-stimulated proliferation and migration of VSMCs and cell cycle progression. Furthermore, DN200434 reduced carotid artery ligation-induced neointima formation in mice. CONCLUSION: Our data suggest that DN200434 is a therapeutic option to prevent the progression of atherosclerosis.
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Aterosclerosis , Neointima , Ratas , Ratones , Masculino , Animales , Neointima/prevención & control , Neointima/tratamiento farmacológico , Neointima/metabolismo , Músculo Liso Vascular/metabolismo , Ratones Endogámicos C57BL , Proliferación Celular , Ratas Sprague-Dawley , Células Cultivadas , Arteria Carótida Común/metabolismo , Arterias Carótidas/cirugía , Arterias Carótidas/metabolismo , MamíferosRESUMEN
BACKGROUND: Pericytes and vascular smooth muscle cells, collectively known as mural cells, are recruited through PDGFB (platelet-derived growth factor B)-PDGFRB (platelet-derived growth factor receptor beta) signaling. MCs are essential for vascular integrity, and their loss has been associated with numerous diseases. Most of this knowledge is based on studies in which MCs are insufficiently recruited or fully absent upon inducible ablation. In contrast, little is known about the physiological consequences that result from impairment of specific MC functions. Here, we characterize the role of the transcription factor SRF (serum response factor) in MCs and study its function in developmental and pathological contexts. METHODS: We generated a mouse model of MC-specific inducible Srf gene deletion and studied its consequences during retinal angiogenesis using RNA-sequencing, immunohistology, in vivo live imaging, and in vitro techniques. RESULTS: By postnatal day 6, pericytes lacking SRF were morphologically abnormal and failed to properly comigrate with angiogenic sprouts. As a consequence, pericyte-deficient vessels at the retinal sprouting front became dilated and leaky. By postnatal day 12, also the vascular smooth muscle cells had lost SRF, which coincided with the formation of pathological arteriovenous shunts. Mechanistically, we show that PDGFB-dependent SRF activation is mediated via MRTF (myocardin-related transcription factor) cofactors. We further show that MRTF-SRF signaling promotes pathological pericyte activation during ischemic retinopathy. RNA-sequencing, immunohistology, in vivo live imaging, and in vitro experiments demonstrated that SRF regulates expression of contractile SMC proteins essential to maintain the vascular tone. CONCLUSIONS: SRF is crucial for distinct functions in pericytes and vascular smooth muscle cells. SRF directs pericyte migration downstream of PDGFRB signaling and mediates pathological pericyte activation during ischemic retinopathy. In vascular smooth muscle cells, SRF is essential for expression of the contractile machinery, and its deletion triggers formation of arteriovenous shunts. These essential roles in physiological and pathological contexts provide a rationale for novel therapeutic approaches through targeting SRF activity in MCs.
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Pericitos , Enfermedades de la Retina , Animales , Ratones , Pericitos/metabolismo , Proteínas Proto-Oncogénicas c-sis/metabolismo , ARN/metabolismo , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/genética , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Enfermedades de la Retina/metabolismo , Factor de Respuesta Sérica/genética , Factor de Respuesta Sérica/metabolismoRESUMEN
BACKGROUND: Vascular smooth muscle cells (SMCs) undergo complex phenotypic modulation with atherosclerotic plaque formation in hyperlipidemic mice, which is characterized by de-differentiation and heterogeneous increases in the expression of macrophage, fibroblast, osteogenic, and stem cell markers. An increase of cellular cholesterol in SMCs triggers similar phenotypic changes in vitro with exposure to free cholesterol due to cholesterol entering the endoplasmic reticulum, triggering endoplasmic reticulum stress and activating Perk (protein kinase RNA-like endoplasmic reticulum kinase) signaling. METHODS: We generated an SMC-specific Perk knockout mouse model, induced hyperlipidemia in the mice by AAV-PCSK9DY injection, and subjected them to a high-fat diet. We then assessed atherosclerotic plaque formation and performed single-cell transcriptomic studies using aortic tissue from these mice. RESULTS: SMC-specific deletion of Perk reduces atherosclerotic plaque formation in male hyperlipidemic mice by 80%. Single-cell transcriptomic data identify 2 clusters of modulated SMCs in hyperlipidemic mice, one of which is absent when Perk is deleted in SMCs. The 2 modulated SMC clusters have significant overlap of transcriptional changes, but the Perk-dependent cluster uniquely shows a global decrease in the number of transcripts. SMC-specific Perk deletion also prevents migration of both contractile and modulated SMCs from the medial layer of the aorta. CONCLUSIONS: Our results indicate that hypercholesterolemia drives both Perk-dependent and Perk-independent SMC modulation and that deficiency of Perk significantly blocks atherosclerotic plaque formation.