A decrease in integrin α5β1/FAK is associated with increased apoptosis of aortic smooth muscle cells in acute type a aortic dissection
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SUBMITTER: Xue M
PROVIDER: S-EPMC10964683 | biostudies-literature | 2024 Jan
REPOSITORIES: biostudies-literature
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Project description:TNFα activates NADPH oxidase 1 (Nox1) in vascular smooth muscle cells (VSMCs). The extracellular superoxide anion (O2•-) produced is essential for the pro-inflammatory effects of the cytokine but the specific contributions of O2•- to signal transduction remain obscure. Extracellular superoxide dismutase (ecSOD, SOD3 gene) is a secreted protein that binds to cell surface heparin sulfate proteoglycans or to Fibulin-5 (Fib-5, FBLN5 gene), an extracellular matrix protein that also associates with elastin and integrins. ecSOD converts O2•- to hydrogen peroxide (H2O2) which prevents NO• inactivation, limits generation of hydroxyl radical (OH•), and creates high local concentrations of H2O2. We hypothesized that ecSOD modifies TNFα signaling in VSMCs. Knockdown of ecSOD (siSOD3) suppressed downstream TNFα signals including MAPK (JNK and ERK phosphorylation) and NF-κB activation (luciferase reporter and IκB phosphorylation), interleukin-6 (IL-6) secretion, iNOS and VCAM expression, and proliferation (Sulforhodamine B assay, PCNA western blot). These effects were associated with significant reductions in the expression of both Type1 and 2 TNFα receptors. Reduced Fib-5 expression (siFBLN5) similarly impaired NF-κB activation by TNFα, but potentiated FAK phosphorylation at Y925. siSOD3 also increased both resting and TNFα-induced phosphorylation of FAK and of glycogen synthase kinase-3β (GSK3β), a downstream target of integrin linked kinase (ILK). These effects were dependent upon α5β1 integrins and siSOD3 increased resting sulfenylation (oxidation) of both integrin subunits, while preventing TNFα-induced increases in sulfenylation. To determine how ecSOD modified TNFα-induced inflammation in intact blood vessels, mesenteric arteries from VSMC-specific ecSOD knockout (KO) mice were exposed to TNFα (10 ng/ml) in culture for 48 h. Relaxation to acetylcholine and sodium nitroprusside was impaired in WT but not ecSOD KO vessels. Thus, ecSOD association with Fib-5 supports pro-inflammatory TNFα signaling while tonically inhibiting α5β1 integrin activation.
| S-EPMC10841345 | biostudies-literature
Project description:Aortic dissection (AD) is a catastrophic disease with high mortality and morbidity, characterized with fragmentation of elastin and loss of smooth muscle cells. Although AD has been largely attributable to polymorphisms defect in the elastin-coding gene, tropoelastin (TE), other undermined factors also appear to play roles in AD onset. Here, we investigated the effects of post-transcriptional control of TE by microRNAs (miRNAs) on elastin levels in aortic smooth muscle cells (ASMC). We found that miR-144-3p is a miRNA that targets TE mRNA in both human and mouse. Bioinformatics analyses and dual luciferase reporter assay showed that miR-144-3p inhibited protein translation of TE, through binding to the 3'-UTR of the TE mRNA. Interestingly, higher miR-144-3p levels and lower TE were detected in the ASMC obtained from AD patients, compared to those from non-AD controls. In a mouse model for human AD, infusion of adeno-associated viruses (serotype 6) carrying antisense for miR-144-3p (as-miR-144-3p) under CAG promoter significantly reduced the incidence and severity of AD, seemingly through enhancement of TE levels in ASMC. Thus, our data suggest an essential role of miR-144-3p on the pathogenesis of AD.
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Project description:Aortic aneurysms (AA) are characterized by structural deterioration leading to progressive dilation. During the development of AA, two key structural changes are pronounced, one being degradation of extracellular matrix and the other loss of smooth muscle cells (SMCs) through apoptosis. Reactive oxygen species (ROS) are produced above physiological levels in dilated (aneurismal) part of the aorta compared to the nondilated part and they are known to be associated with both the extracellular matrix degradation and the loss of SMCs. In this study, we hypothesized that aneurismal SMCs are more prone to apoptosis and that at least some cells undergo apoptosis due to elevated ROS in the aortic wall. To test this hypothesis, we first isolated SMCs from thoracic aneurismal tissue and compared their apoptotic tendency with normal SMCs in response to H(2)O(2), oxidized sterol, or UV treatment. Exposed cells exhibited morphological changes characteristic of apoptosis, such as cell shrinkage, membrane blebbing, chromatin condensation, and DNA fragmentation. Terminal deoxynucleotidyl transferased UTP nick end labeling (TUNEL) further confirmed the fragmentation of nuclear DNA in these cells. Vascular SMCs were analyzed for their micronuclei (MN) and binucleate (BN) frequency as indicators of genomic abnormality. These data were then compared to patient parameters, including age, gender, hypertension, or aortic diameter for existing correlations. While the tendency for apoptosis was not significantly different compared to normal cells, both the %MN and %BN were higher in aneurismal SMCs. The data suggest that there is increased DNA damage in TAA samples, which might play a pivotal role in disease development.
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Project description:MicroRNA 22 (miR-22) was found in diverse cardiovascular diseases to have a role in regulating multiple cellular processes. However, the regulatory role of miR-22 in aortic dissection (AD) was still unclear. The miR-22 expression in human aorta was explored. A series of mimic, inhibitor, or small interfering RNA (siRNA) plasmids were delivered into vascular smooth muscle cells (VSMCs) to explore the effects of miR-22 and p38 mitogen-activated protein kinase α (p38MAPKα) in controlling VSMC apoptosis in vitro. In addition, a mouse AD model was established, and histopathologic analyses were performed to evaluate the regulatory effects of miR-22. Reduced miR-22 and increased apoptosis of VSMCs was seen in human AD aorta. Downregulation of miR-22 increased the apoptosis of VSMCs in vitro. Bioinformatics analyses revealed that p38MAPKα was a target of miR-22. Inhibiting p38MAPKα expression could reverse the apoptosis of VSMCs induced by miR-22 downregulation. Knockdown of miR-22 in the AD mouse model significantly promoted the development of AD. Our data underscore the importance of vascular remodeling and VSMC function in AD. miR-22 may represent a new therapeutic approach for AD by regulating the apoptosis of VSMCs through the MAPK signaling pathway.
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Project description:BackgroundmiR-145 is closely related to vascular smooth muscle cells (VSMC) phenotype transformation; however, the regulatory mechanisms through which miR-145 regulates the VSMC phenotype transformation under mechanical stretching are unclear. In this study, we evaluated the roles of miR-145 in VSMCs subjected to mechanical stretching in aortic dissection (AD).MethodsThe expression of miR-145 in the aortic vessel wall of model animals and patients with AD was analyzed by quantitative polymerase chain reaction. miR-145-related protein-protein interaction networks and Wikipathways were used to analyze VSMC phenotypic transformation pathways regulated by miR-145. We used gain- and loss-of-function studies to evaluate the effects of miR-145 on VSMC differentiation under mechanical stretch induction and assessed whether Krüppel-like factor 4 (KLF4) was regulated by miR-145 in the aorta under mechanical stretch conditions.ResultsmiR-145 was abundantly expressed in the walls of the normal human aorta, but was significantly downregulated in animal models and the walls of patients with dissection. We found that contractile phenotype-related proteins were downregulated in VSMCs subjected to mechanical stretching, whereas the expression of secreted phenotype-related proteins increased. miR-145 overexpression also downregulated contractile phenotype-related proteins in VSMCs and suppressed upregulation of phenotype-related proteins. Finally, under mechanical stretching, KLF4 expression was significantly increased in VSMCs, and overexpression of miR-145 blocked this effect.ConclusionOur results confirmed that mechanical stretch-induced phenotypic transformation of VSMCs to promote AD via upregulation of KLF4; this mechanism was regulated by miR-145, which directly modulated KLF4 expression and VSMC differentiation.
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Project description:Enhancer of zeste homolog 2 (EZH2), a methyltransferase that di- and tri-methylates lysine-27 of histone H3, largely functions as a transcriptional repressor, and plays a critical role in various kinds of cancers. Here we report a novel function of EZH2 in regulating autophagic cell death (ACD) of vascular smooth muscle cells (VSMCs) that affect aortic dissection (AD). Inhibition of EZH2 activity by UNC1999 or knockdown EZH2 resulted in VSMC loss, while overexpression of EZH2 facilitated VSMC growth, and these effects of EZH2 on VSMCs were independent of proliferation and apoptosis. Interestingly, more autophagic vacuoles and increased LC3II protein levels were identified in VSMCs with EZH2 inhibition or deficiency. Moreover, when compared with counterparts, chloroquine alone, or chloroquine with rapamycin treatment led to more LC3II accumulation in EZH2 inhibited or knockdown VSMCs, which indicated that EZH2 negatively regulated autophagosome formation. In conjunction to this, ATG5 and ATG7 protein levels were remarkably increased in EZH2 inhibited or deficient VSMCs, and ATG5 or ATG7 knockdown virtually rescued VSMC loss induced by EZH2 inhibition or knockdown. In addition, we found that the MEK-ERK1/2 signaling pathway, but not AMPKα, mTOR, or AKT pathway, is responsible for the impact of EZH2 on ACD of VSMCs. Additionally, the adverse effects of EZH2 inhibition or knockdown on VSMCs were largely reversed by PD98059, an inhibitor of MEK1. More importantly, decreased EZH2 expression levels in the aortic wall of patients with AD indicated its contribution to VSMC loss and AD occurrence. Overall, these findings revealed that EZH2 affects ACD of VSMCs and the pathologic process of AD via regulating ATG5 and ATG7 expression and MEK-ERK1/2 signaling. Our hitherto unrecognized findings indicate that EZH2 activation has therapeutic or preventive potential for AD.
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Project description:Rationale: Aortic dissection (AD) is caused by functional disorder of cells in the aortic wall, which is largely attributed to vascular remodeling. Therapeutic strategies for AD remain limited due to our incomplete understanding of the role of endothelial cells (ECs) in AD pathogenesis. This study aimed to identify the regulatory role of miR-27a in AD and provide a mechanistic basis for a non-invasive treatment of AD. Methods: We harvested aortas from normal and AD patients to explore the expression of miR-27a. In vitro and in vivo assays were preformed to explore the biological effects of differential expression of miR-27a in ECs and its regulatory effect on AD. Results: MiR-27a was lower in intima of AD samples than in healthy individuals. Downregulation of miR-27a in EC was due to up-regulated expression of fas-associated protein with death domain (FADD) and the activation of apoptosis pathway, which led to apoptosis of ECs. Migration of vascular smooth muscle cells was promoted by EC after downregulation of miR-27a due to enhancement of growth/differentiation factor 8 (GDF8) and repression of matrix metalloproteinase-20 (MMP20) in the co-culture system supernatants. Increase in FADD and apoptosis of ECs to induce AD was shown using mouse models of AD in which miR-27a was stably knocked-down by antagomir. Up-regulation of miR-27a by agomir led to a protective effect on AD. Conclusion: Treatment with miR-27a activator that targets apoptosis of ECs strongly diminished occurrence of AD, providing a new strategy for this disease.
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Project description:BackgroundThis study aimed to investigate whether miR-146a-5p was involved in the pathogenesis of thoracic aortic dissection (AD) via regulating the biological function of vascular smooth muscle cells (VSMCs).MethodsCirculating miR-146a-5p level was measured by quantitative polymerase chain reaction (qPCR) in AD patients and healthy controls. Human dissected aortic samples were obtained from patients with thoracic AD Stanford type A undergoing surgical repair, and normal control samples were from organ donors who died from nonvascular diseases. The expression level of miR-146a-5p was detected using qPCR in each sample. The expression of SMAD4, which is involved in the TGF-β pathway and indicated as the target gene of miR-146a-5p, was measured by qPCR and Western blot analysis at the mRNA level and protein level, respectively. Subsequently, VSMCs were transfected with miR-146a-5p mimics or inhibitors in vitro. VSMC proliferation and migration were detected using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and Transwell assay, respectively. Flow cytometry was used to identify apoptosis. The expression of SMAD4 in VSMCs was determined using qPCR and Western blot analysis.ResultsPlasma level of miR-146a-5p is significantly higher in the AD group as compared with the control group. The expression of miR-146a-5p was significantly upregulated in dissected aorta compared with controls (P < 0.05). The overexpression of miR-146a-5p significantly induced VSMC proliferation and migration in vitro.ConclusionsThe expression of SMAD4 was modulated by miR-146a-5p. miR-146a-5p induced VSMC proliferation and migration through targeting SMAD4 and hence might be potentially involved in the development of AD.
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Project description:Purpose: This study aims to illustrate the cellular landscape in the aorta of experimental aortic dissection (AD) and elaborate on the smooth muscle cells (SMCs) heterogeneity and functions among various cell types. Methods: Male Apolipoprotein deficient (ApoE-/-) mice at 28 weeks of age were infused with Ang II (2,500 ng/kg/min) to induce AD. Aortas from euthanized mice were harvested after 7 days for 10×Genomics single-cell RNA sequencing (scRNA-seq), followed by the identification of cell types and differentially expressed genes (DEGs). Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was conducted. Results: AD was successfully induced in ApoE-/- mice. scRNA-seq identified 15 cell clusters and nine cell types, including non-immune cells (endothelials, fibroblasts, and SMCs) and immune cells (B cells, natural killer T cell, macrophages, dendritic cells, neutrophils, and mast cells). The relative numbers of SMCs were remarkably changed, and seven core DEGs (ACTA2,IL6,CTGF,BGN,ITGA8,THBS1, and CDH5) were identified in SMCs. Moreover, we found SMCs can differentiate into 8 different subtypes through single-cell trajectory analysis. Conclusion: scRNA-seq technology can successfully identify unique cell composition in experimental AD. To our knowledge, this is the first study that provided the complete cellular landscape in AD tissues from mice, seven core DEGs and eight subtypes of SMCs were identified, and the SMCs have evolution from matrix type to inflammatory type.
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