Apoptosis Of Vascular Smooth Muscle Cells Research Articles

PTP1B Modulates Carotid Plaque Vulnerability in Atherosclerosis Through Rab5-PDGFRβ-Mediated Endocytosis Disruption and Apoptosis.

Protein tyrosine phosphatase 1B (PTP1B) is a protein tyrosine phosphatase and modulates platelet-derived growth factor (PDGF)/platelet-derived growth factor receptor (PDGFR) signaling in vascular smooth muscle cells (VSMCs) via endocytosis. However, the related molecular pathways that participated in the interaction of endo-lysosome and the trafficking of PDGFR are largely unknown. This study aims to determine the subcellular regulating mechanism of PTP1B to the endo-lysosome degradation of PDGFR in atherosclerotic carotid plaques, thereby offering a potential therapeutic target for the stabilization of carotid plaques. The immunohistochemical staining technique was employed to assess the expression levels of both PDGFR-β and Caspase 3 in stable and vulnerable carotid plaques. Tunnel staining was utilized to quantify the apoptosis of carotid plaques. Live-cell imaging was employed to observe endocytic motility, while cell apoptosis was evaluated through Propidium Iodide staining. In an invivo experiment, ApoE-/- mice were administered a PTP1B inhibitor to investigate the impact of PTP1B on atherosclerosis. The heightened expression of PDGFR-β correlates with apoptosis in patients with vulnerable carotid plaques. At the subcellular level of VSMCs, PDGFR-β plays a pivotal role in sustaining a balanced endocytosis system motility, regulated by the expression of Rab5, a key regulator of endocytic motility. And PTP1B modulates PDGFR-β signaling via Rab5-mediated endocytosis. Additionally, disrupted endocytic motility influences the interplay between endosomes and lysosomes, which is crucial for controlling PDGFR-β trafficking. Elevated PTP1B expression induces cellular apoptosis and impedes migration and proliferation of carotid VSMCs. Ultimately, mice with PTP1B deficiency exhibit a reduction in atherosclerosis. Our results illustrate that PTP1B induces disruption in endocytosis and apoptosis of VSMCs through the Rab5-PDGFRβ pathway, suggesting a potential association with the heightened vulnerability of carotid plaques.

Akkermansia muciniphila alleviates abdominal aortic aneurysms via restoring CITED2 activated by EPAS1.

Abdominal aortic aneurysm (AAA) is a life-threatening cardiovascular disease that has been linked to gut microbiome dysbiosis. Therefore, this study aims to investigate the effects of Akkermansia muciniphila (Am) on AAA mice and the biomolecules involved. AAA mice were generated using angiotensin II (Ang II), and 16sRNA sequencing was used to identify an altered abundance of microbiota in the feces of AAA mice. Vascular smooth muscle cell (VSMC) markers and apoptosis, and macrophage infiltration in mouse aortic tissues were examined. The abundance of Am was reduced in AAA mouse feces, and endothelial PAS domain-containing protein 1 (EPAS1) was downregulated in AAA mice and VSMC induced with Ang II. Am delayed AAA progression in mice, which was blunted by knockdown of EPAS1. EPAS1 was bound to the Cbp/p300-interacting transactivator 2 (CITED2) promoter and promoted CITED2 transcription. CITED2 reduced VSMC apoptosis and delayed AAA progression. Moreover, EPAS1 inhibited macrophage inflammatory response by promoting CITED2 transcription. In conclusion, gut microbiome dysbiosis in AAA induces EPAS1-mediated dysregulation of CITED2 to promote macrophage inflammatory response and VSMC apoptosis.

LTBP2 down-regulated FGF2 to repress vascular smooth muscle cell proliferation and vascular remodeling in a rat model of intracranial aneurysm

This work probed into the role of latent transforming growth factor beta binding protein 2 (LTBP2) in intracranial aneurysm (IA). The rats underwent IA modeling and then stereotactic injection of short hairpin RNA against LTBP2 (shLTBP2). Hematoxylin-eosin (HE) staining was employed to assess IA model and vascular remodeling. Rat vascular smooth muscle cells (VSMCs) were transfected with shLTBP2, LTBP2 overexpression plasmid and fibroblast growth factor 2 (FGF2) overexpression plasmid. The mRNA and protein expressions of LTBP2, FGF2 and mitochondrial apoptosis-related factors (Caspase-3, Cyt-c, Mcl-1) were tested through qRT-PCR and Western blot. Cell viability, proliferation and apoptosis were examined by cell counting kit-8, EdU assay and flow cytometry. The up-regulated LTBP2 and down-regulated FGF2 were detected in IA rats. LTBP2 knockdown promoted vascular remodeling and Mcl-1 level, and restrained cell apoptosis and expressions of Caspase-3 and Cyt-c in IA model rats. Moreover, LTBP2 knockdown potentiated cell viability, proliferation and FGF2 level, and repressed apoptosis in rat VSMCs, while overexpressed LTBP2 exerted opposite effects. FGF2 overexpression promoted proliferation and Mcl-1 level, and inhibited apoptosis and expressions of Caspase-3 and Cyt-c in rat VSMCs, which also reversed the effects of overexpressed LTBP2 on these aspects. Collectively, LTBP2 down-regulates FGF2 to repress VSMCs proliferation and vascular remodeling in an IA rat model.

MiRNA-132/212 encapsulated by adipose tissue-derived exosomes worsen atherosclerosis progression

BackgroundVisceral adipose tissue in individuals with obesity is an independent cardiovascular risk indicator. However, it remains unclear whether adipose tissue influences common cardiovascular diseases, such as atherosclerosis, through its secreted exosomes.MethodsThe exosomes secreted by adipose tissue from diet-induced obesity mice were isolated to examine their impact on the progression of atherosclerosis and the associated mechanism. Endothelial apoptosis and the proliferation and migration of vascular smooth muscle cells (VSMCs) within the atherosclerotic plaque were evaluated. Statistical significance was analyzed using GraphPad Prism 9.0 with appropriate statistical tests.ResultsWe demonstrate that adipose tissue-derived exosomes (AT-EX) exacerbate atherosclerosis progression by promoting endothelial apoptosis, proliferation, and migration of VSMCs within the plaque in vivo. MicroRNA-132/212 (miR-132/212) was detected within AT-EX cargo. Mechanistically, miR-132/212-enriched AT-EX exacerbates palmitate acid-induced endothelial apoptosis via targeting G protein subunit alpha 12 and enhances platelet-derived growth factor type BB-induced VSMC proliferation and migration by targeting phosphatase and tensin homolog in vitro. Importantly, melatonin decreases exosomal miR-132/212 levels, thereby mitigating the pro-atherosclerotic impact of AT-EX.ConclusionThese data uncover the pathological mechanism by which adipose tissue-derived exosomes regulate the progression of atherosclerosis and identify miR-132/212 as potential diagnostic and therapeutic targets for atherosclerosis.Graphical

TRIM25 activates Wnt/β-catenin signalling by destabilising MAT2A mRNA to drive thoracic aortic aneurysm development.

This study explored the roles of methionine adenosyltransferase 2A (MAT2A) and tripartite motif containing 25 (TRIM25) in the progression of thoracic aortic aneurysm (TAA). The TAA model was established based on the β-aminopropionitrile method. The effects of MAT2A on thoracic aortic lesions and molecular levels were analyzed by several pathological staining assays (hematoxylin-eosin, Verhoeff-Van Gieson, TUNEL) and molecular biology experiments (qRT-PCR, Western blot). Angiotensin II (Ang-II) was used to induce injury in vascular smooth muscle cells (VSMCs) in vitro. The effects of MAT2A, shMAT2A, shTRIM25 and/or Wnt inhibitor (IWR-1) on the viability, apoptosis and protein expressions of VSMCs were examined by CCK-8, Annexin V-FITC/PI and Western blot assays. In TAA mice, overexpression of MAT2A alleviated thoracic aortic injury, inhibited the aberrant expressions of aortic contractile proteins and dedifferentiation markers, and blocked the activation of Wnt/β-catenin pathway. In Ang-II-induced VSMCs, up-regulation of MAT2A increased cellular activity and repressed the expression of β-catenin protein. TRIM25 knockdown promoted activity of VSMCs, inhibited apoptosis, and blocked the Wnt/β-catenin pathway activation by binding to MAT2A. IWR-1 partially counteracted the regulatory effects of shMAT2A. Collectively, TRIM25 destabilises the mRNA of MAT2A to activate Wnt/β-catenin signaling and ultimately exacerbate TAA injury.

Insulin receptors in vascular smooth muscle cells regulate plaque stability of atherosclerosis.

Increased prevalence of acute myocardial infarction related to diabetes and insulin resistance is associated with elevated risk for unstable atherosclerotic plaques, which are characterized by reduced vascular smooth muscle cells (VSMC) and extracellular matrix (ECM) and increased inflammation. Thus, insulin resistance may reduce plaque stability as deleting insulin receptors (IR) in VSMC decreased their proliferation and enhanced apoptosis. Direct effects of insulin on VSMC to alter plaque composition were studied using mice with double knockout of ApoE and IR genes in VSMC with SMIRKO/ApoE-/- and Myh11-CreERT2EYFP+/ApoE-/- and Myh11-CreERT2EYFP+IRKO/ApoE-/- mice, which were also used for lineage tracing studies. Compared to ApoE-/- mice, SMIRKO/ApoE-/-- mice exhibited more atherosclerotic plaques, which contained less VSMC and collagen, but increased levels of VSMC apoptosis and necrotic areas. Lineage tracing studies showed that Icam1+ Vcam1+ VSMC was inflammatory VSMC, which increased in aortas of Myh11-CreERT2EYFP+IRKO/ApoE-/- mice compared to control mice. Isolated VSMC lacking IR expressed higher inflammatory cytokines than cells with IR. Cell based studies indicated that insulin's anti-apoptotic and pro-proliferative effects in VSMC were mediated via activation of IR/Akt pathway, which were decreased in VSMC from SMIRKO or high fat diet (HFD) mice. Analysis of IR targets that regulated inflammatory cytokines in VSMC showed that thrombospondin 1 (Thbs1) and Mmp2 were consistently increased with loss of IR. Insulin inhibited Thbs1 expression, but not Mmp2, by p-Akt/p-FoxO1 pathways in VSMC from ApoE-/- mice, which was impaired in cells from SMIRKO/ApoE-/-- mice. Thbs1 further induced Icam1 and Mmp2 expressions in VSMC. Insulin via IR has significant actions in VSMC to decrease inflammation, apoptosis and ECM turnover via the activation of Akt and FoxO1 pathways. Inhibition of insulin actions and related pathways related to insulin resistance and diabetes may contribute to the formation of unstable atherosclerotic plaques.

Reversal of heavy arterial calcification in a rat model of chronic kidney disease using targeted ethylene diamine tetraacetic acid-loaded albumin nanoparticles.

Elastin degradation and severe calcification in the medial layer of the vessel wall, known as medial arterial calcification (MAC), is typical in the aging population and patients with metabolic disorders, such as diabetes and chronic kidney disease (CKD). We have previously reported that ethylene diamine tetraacetic acid (EDTA) delivery to the site of calcification can be achieved by tagging nanoparticles with an elastin antibody that recognizes explicitly damaged elastin, and such systemic therapy can remove focal calcium deposits from the calcified arteries in CKD rodent model. The current study aims to test whether heavy calcification seen throughout arterial tree and kidneys in CKD can be reversed with nanoparticle therapy. Thirty healthy male Sprague-Dawley rats weighing approximately 300 g, were placed on an adenine diet for 21 non-consecutive days to induce kidney failure, followed by daily vitamin D3 (VitD3) injections for 4 sequential days to cause severe calcification throughout the cardiovascular system and kidneys. DiR-dye loaded and elastin antibody conjugated albumin nanoparticles were used to confirm the targeting of nanoparticles to the calcification area. The rats were divided into two groups for targeted removal of calcification starting at day 7 of the last doses of VitD3. The experimental group received biweekly IV injections of anti-elastin antibody conjugated EDTA loaded human serum albumin nanoparticles (EDTA-HSA-El-Ab NPs), while the sham controls received blank nanoparticles (Blank-HSA-El-Ab NPs) (5 injections in total). Micro-computed tomography (microCT) was used to analyze the extent of calcification. Reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry studies were performed for osteogenic markers, including bone morphogenic protein 2 (BMP2), runt-related transcription factor 2 (RUNX2), and tissue non-specific alkaline phosphatase (TNAP). For comparison, aortic ring organ cultures from healthy rats were treated with high phosphate to induce calcification in vitro, and then they were treated with EDTA. Human calcified femoral arteries were also treated ex vivo with EDTA-HSA-EL-Ab NPs to test if nanoparticles remove heavy calcification. EDTA-loaded nanoparticles that specifically target degraded elastin reversed existing heavy mineral deposits in arteries, as per elemental calcium analysis (124.161±34.410 µg Ca per mg of the dry aorta in Blank-HSA-El-Ab NPs vs. 100.520±19.131 µg in EDTA-HSA-El-Ab NPs group, P=0.04) and microCT (object volume, 129.001±37.785 vs. 29.815±24.169 mm3, P=0.0005). The reversal of aortic calcification was accompanied by a significant reduction of bone-associated mRNA expression of BMP2 and RUNX2 (P=0.01). Immunohistochemistry studies corroborated RT-PCR results, showing a reduction of BMP2 and RUNX2 stains in the vessel wall. The rat aortic ring culture study also showed similar results, where osteogenic genes (BMP2, RUNX2) and proteins (BMP2, RUNX2, TNAP) were suppressed upon reversal of calcification with EDTA (P=0.001). We also show ex vivo reversal of human femoral artery calcification by microCT (calcium intensity: untreated, 57.721±28.551 vs. day 6 of treatment, 5.441±3.615, P=0.01) by EDTA nanoparticle therapy. This is the first study showing the removal of calcium from heavily calcified arteries by using intravenous targeted EDTA therapy. Such therapy also reversed vascular smooth muscle cell osteoblastic transition and apoptosis in the arterial tissue, thereby potentially creating an environment for suitable tissue repair.

Regulatory factor X7 Represses Ox-LDL-Induced Proliferation and Migration of VSMCs via SIRT4-Mediated Inactivation of JAK2/STAT3 Pathway.

The regulatory factor X7 (RFX7) is a vital mediator in atherosclerosis. This study aims to discuss the effect and underlying mechanism of RFX7 on the regulation of oxidized low-density lipoprotein (ox-LDL) -induced proliferation and migration of vascular smooth muscle cells (VSMCs).Ox-LDL was used to construct atherosclerosis in vitro model. The mRNA and protein levels of RFX7 and Sirtuin 4 (SIRT4) were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR) or western blot assays. The cellular functions were measured via 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT), EdU, flow cytometry, and wound healing assay assays. The interaction between RFX7 and SIRT4 promoter was validated using chromatin immunoprecipitation and dual-luciferase reporter assays.The stimulation with ox-LDL elevated the viability of VSMCs and decreased the mRNA and protein levels of RFX7 and SIRT4 in VSMCs in a dose-dependent manner. Functionally, RFX7 overexpression restrained the VSMC viability, proliferation, and migration induced by ox-LDL, but facilitated VSMC apoptosis. RFX7 elevated SIRT4 expression via binding to its promoter. Furthermore, overexpressing either SIRT4 or RFX7 inactivated JAK2/STAT3 signaling, causing a decrease in VSMC proliferation and migration and an increase in VSMC apoptosis when exposed to ox-LDL. The impact of RFX7 overexpression on JAK2/STAT3 signaling and cellular function following ox-LDL exposure was abrogated by SIRT4 silencing.The heightened RFX7 expression restrained the proliferation and migration of ox-LDL-stimulated VSMCs via SIRT4-mediated inactivation of JAK2/STAT3 pathway.

ADAR1 Is Essential for Smooth Muscle Homeostasis and Vascular Integrity.

Vascular smooth muscle cells (VSMCs) play a critical role in maintaining vascular integrity. VSMC dysfunction leads to numerous vascular diseases. Adenosine deaminases acting on RNA 1 (ADAR1), an RNA editing enzyme, has shown both RNA editing and non-editing functions. Global deletion of ADAR1 causes embryonic lethality, but the phenotype of homozygous ADAR1 deletion specifically in SMCs (ADAR1sm-/-) remains to be determined. By crossing ADAR1fl/fl mice with Myh11-CreERT2 mice followed by Tamoxifen induction, we found that ADAR1sm-/- leads to lethality in adult mice 14 days after the induction. Gross examination revealed extensive hemorrhage and detrimental vascular damage in different organs. Histological analyses revealed destruction of artery structural integrity with detachment of elastin laminae from VSMCs in ADAR1sm-/- aortas. Furthermore, ADAR1sm-/- resulted in severe VSMC apoptosis and mitochondrial dysfunction. RNA sequencing analyses of ADAR1sm-/- aorta segments demonstrated profound transcriptional alteration of genes impacting vascular health including a decrease in fibrillin-1 expression. More importantly, ADAR1sm-/- disrupts the elastin and fibrillin-1 interaction, a molecular event essential for artery structure. Our results indicate that ADAR1 plays a critical role in maintaining SMC survival and vascular stability and resilience.

MiR-140-3p Normalizes Vascular Smooth Muscle Cell Behavior by Inhibiting the RhoA/ROCK Signaling Pathway in Lower-extremity Arteriosclerosis Obliterans.

Excessive vascular smooth muscle cell (VSMC) proliferation and migration are the main contributors to the symptoms of lower-extremity arteriosclerosis obliterans (ASO). Previous studies suggested that microRNAs (miRNAs) regulate VSMC activity. Nevertheless, the molecular mechanisms by which they do so are unclear. The present study aimed to identify the biological processes accounting for the effects of miR-140-3p on VSMCs in ASO. The expression levels of miR-140-3p in clinical samples were analyzed by real-time polymerase chain reaction. An ASO cell model was established to investigate the expression of miR-140-3p on VSMCs. The transwell® assays and MTT assays were used to assess migration and proliferation. The interaction between RhoA and miR-140-3p was verified using the Dualluciferase reporter assay. Western blot technique was used to identify RhoA, RhoA-associated protein kinase 1 (ROCK1), and ROCK2. We discovered that miR-140-3p inhibited the proliferation, migration, and invasion but promoted the apoptosis of VSMCs, and RhoA was its downstream target gene. RhoA, ROCK1, and ROCK2 were upregulated in vascular tissues damaged by ASO compared to normal, healthy arteries. MiR-140-3p also decreased RhoA, ROCK1, and ROCK2 mRNA and protein expression. Overall, the present work partially elucidated the mechanism by which miR-140-3p regulates VSMC function and offered novel insights into potential therapeutic approaches for patients with lower-extremity arteriosclerosis obliterans.

Genetic influence on vascular smooth muscle cell apoptosis

Vascular smooth muscle cell (VSMC) proliferation, migration, and apoptosis play important roles in many physiological processes and pathological conditions. To identify genetic influences on VSMC behavior, we measured these traits and undertook genome-wide association studies in primary umbilical artery-derived VSMCs from >2000 individuals. Although there were no genome-wide significant associations for VSMC proliferation or migration, genetic variants at two genomic loci (7p15.3 and 7q32.3) showed highly significant associations with VSMC apoptosis (P = 1.95 × 10−13 and P = 7.47 × 10−9, respectively). The lead variant at the 7p51.3 locus was associated with increased expression of the GSDME and PALS2 genes in VSMCs. Knockdown of GSDME or PALS2 in VSMCs attenuated apoptotic cell death. A protein co-immunoprecipitation assay indicated that GSDME complexed with PALS2. PALS2 knockdown attenuated activated caspase-3 and GSDME fragmentation, whilst GSDME knockdown also reduced activated caspase-3. These findings provide new insights into the genetic regulation of VSMC apoptosis, with potential utility for therapeutic development.

The role of FGL2 in vascular smooth muscle cell dysfunction in coronary artery bypass vein graft failure

Abstract Funding Acknowledgements Type of funding sources: Other. Main funding source(s): British Heart Foundation Rationale The sudden exposure of venous endothelial cells (vECs) to arterial fluid shear stress (FSS) is thought to be a major contributor to coronary artery bypass vein graft failure (VGF). However, the acute effects of arterial FSS on the vEC secretome and the corresponding effects on underlying vascular smooth muscle cells (VSMCs) remains poorly characterised. Methods & Results Human umbilical vein endothelial cells (HUVECs; n=6) pre-conditioned to venous FSS (18h; 1.5 dynes/cm2) were further exposed to venous or arterial FSS (24h; 15 dynes/cm2) using an Ibidi pump system. Analysis of the EC secretome using Tandem Mass Tagging proteomics detected significantly increased fibroleukin (FGL2) in conditioned media from ECs exposed to arterial FSS compared to venous FSS (fold-change 1.97±0.72; P=0.0078). In addition, RT-qPCR analysis of FGL2 production showed a significant increase in vECs exposed to arterial FSS (2.11±0.27 vs. 0.05±0.04 log(copy number/mg); P=0.0312). Subsequently, pooled human saphenous vein VSMCs (3 donors per pool) were treated with conditioned media from ECs exposed to arterial FSS (n=6) which showed a significant increase in apoptosis, measured by active cleaved caspase-3 (CC3) immunocytochemistry (27±1% vs 22±1%; P=0.0391), and a significant decrease in the contractile markers smoothelin and desmin quantified by Western blotting (fold-change 0.76±0.06 and 0.44±0.11; P=0.004 and P=0.0421, respectively), in comparison to VSMCs treated with conditioned media from ECs exposed to venous FSS. Treatment of VSMCs (n=6) with recombinant FGL2 (20 ng/mL) for 72h likewise resulted in increased apoptosis (27±4% vs. 16±3%; P=0.0169) and decreased desmin and smoothelin (fold-change 0.68±0.03 and 0.61±0.07; P=0.0322 and P=0.0479, respectively). Conclusions The exposure of vECs to arterial FSS results in increased production and release of FGL2 which induced VSMC apoptosis and de-differentiation and therefore potentially contributes to VGF. Consequently, targeting FGL2 production or release may provide a therapeutic target to improve vein graft patency.

Pentamethylquercetin attenuates angiotensin II-induced abdominal aortic aneurysm formation by blocking nuclear translocation of C/EBPβ at Lys253

BackgroundPentamethylquercetin (PMQ) is a natural polymethyl flavonoid that possesses anti-apoptotic and other biological properties. Abdominal aortic aneurysm (AAA), a fatal vascular disease with a high risk of rupture, is associated with phenotypic switching and apoptosis of medial vascular smooth muscle cells (VSMCs). This study aimed to investigate the protective effects of PMQ on the development of AAA and the underlying mechanism. MethodsApoE−/− mice were continuously infused with angiotensin II (Ang II) for 4 weeks to develop the AAA model. Intragastric administration of PMQ was initiated 5 days before Ang II infusion and continued for 4 weeks. In vitro, VSMCs were cultured and pretreated with PMQ, stimulated with Ang II. Real-time PCR, western blotting, and immunofluorescence staining were used to examine the roles and mechanisms of PMQ on the phenotypic switching and apoptosis of VSMCs. ResultsPMQ dose-dependently reduced the incidence of Ang II-induced AAA, aneurysm diameter enlargement, elastin degradation, VSMCs phenotypic switching and apoptosis. Furthermore, PMQ also inhibited phenotypic switching and apoptosis in Ang II-stimulated VSMCs. PMQ exerted protective effects by regulating the C/EBPβ/PTEN/AKT/GSK-3β axis. AAV-mediated overexpression of PTEN reduced the therapeutic effects of PMQ in the AAA model mice, suggesting that the effects of PMQ on Ang II-mediated AAA formation were related to the PTEN/AKT/GSK-3β axis. PMQ inhibited VSMCs phenotypic switching and apoptosis by bounding to C/EBPβ at Lys253 with hydrogen bond to regulate C/EBPβ nuclear translocation and PTEN/AKT/GSK-3β axis, thereby inhibiting Ang II-induced AAA formation. ConclusionsPentamethylquercetin inhibits angiotensin II-induced abdominal aortic aneurysm formation by bounding to C/EBPβ at Lys253. Therefore, PMQ prevents the formation of AAA and reduces the incidence of AAA.

Abstract 1006: Pathological PERK/ATF4 Activation In Vascular Smooth Muscle Cells Drives Abdominal Aortic Aneurysm Development

Introduction: Abdominal aortic aneurysms (AAA) are characterized by vascular smooth muscle cell (VSMC) apoptosis leading to pathological aortic remodeling. Activation of the endoplasmic reticulum (ER) stress response has been linked to VSMC dysfunction but the driving factors behind ER stress activation and its role on AAA development remains poorly defined. The epigenetic enzyme, MLL1, plays a critical role in establishing cellular phenotype and regulating gene expression. We examine the MLL-mediated epigenetic regulation of the ER stress response in VSMCs during AAA development and establish the ER stress pathways as a novel target in the treatment AAAs. Methods: Single-cell sequencing was conducted on human AAA and control tissue samples. For our murine model, C57BL/6 mice were injected with an AAV encoding a PCSK9 gain-of-function mutation and then fed a saturated fat-enriched diet and infused with AngII (1 μg/kg/min) with or without 4-PBA (20mg/kg/day). Mice with vascular smooth muscle cell-specific knockout of PERK (PERK SMKO ) underwent tamoxifen injection. AAA maximum diameters were quantified and VSMCs were isolated. Eif2a, Atf4 , and Chop gene expression was examined. Apoptosis was quantified by Annexin V staining. Results: Using single-cell RNA sequencing of human aortic tissue, we identified EIF2A, ATF4 , and CHOP were profoundly upregulated in aneurysmal VSMCs as well as two murine AAA models. Mechanistically, TNFα signaling in VSMCs leads to activation of MLL1 which trimethylates histone 3 lysine 4 (H3K4me3) at NF-κB binding sites on the EIF2A and ATF4 gene promoters thereby upregulating the ER stress response and driving VSMC apoptosis. Both In vivo pharmacological inhibition of the PERK/eIF2A/ATF4 ER stress response via 4-PBA injection and vascular smooth muscle depletion of PERK (PERK SMKO ) inhibited AAA formation resulting with in significant reduction in VSMC apoptosis, elastin fragmentation, and inflammatory cytokine infiltration. Conclusions: Taken together, our results identify MLL1-mediated activation of the PERK/eIF2A/ATF4 ER stress response as a critical regulator of VSMC apoptosis in AAA formation and suggest inhibition of ER stress response as a mechanistic target for the management of AAAs.

Spatiotemporal ATF3 Expression Determines VSMC Fate in Abdominal Aortic Aneurysm.

Abdominal aortic aneurysm (AAA) is a catastrophic disease with little effective therapy, likely due to the limited understanding of the mechanisms underlying AAA development and progression. ATF3 (activating transcription factor 3) has been increasingly recognized as a key regulator of cardiovascular diseases. However, the role of ATF3 in AAA development and progression remains elusive. Genome-wide RNA sequencing analysis was performed on the aorta isolated from saline or Ang II (angiotensin II)-induced AAA mice, and ATF3 was identified as the potential key gene for AAA development. To examine the role of ATF3 in AAA development, vascular smooth muscle cell-specific ATF3 knockdown or overexpressed mice by recombinant adeno-associated virus serotype 9 vectors carrying ATF3, or shRNA-ATF3 with SM22α (smooth muscle protein 22-α) promoter were used in Ang II-induced AAA mice. In human and murine vascular smooth muscle cells, gain or loss of function experiments were performed to investigate the role of ATF3 in vascular smooth muscle cell proliferation and apoptosis. In both Ang II-induced AAA mice and patients with AAA, the expression of ATF3 was reduced in aneurysm tissues but increased in aortic lesion tissues. The deficiency of ATF3 in vascular smooth muscle cell promoted AAA formation in Ang II-induced AAA mice. PDGFRB (platelet-derived growth factor receptor β) was identified as the target of ATF3, which mediated vascular smooth muscle cell proliferation in response to TNF-alpha (tumor necrosis factor-α) at the early stage of AAA. ATF3 suppressed the mitochondria-dependent apoptosis at the advanced stage by upregulating its direct target BCL2. Our chromatin immunoprecipitation results also demonstrated that the recruitment of NFκB1 and P300/BAF/H3K27ac complex to the ATF3 promoter induces ATF3 transcription via enhancer activation. NFKB1 inhibitor (andrographolide) inhibits the expression of ATF3 by blocking the recruiters NFKB1 and ATF3-enhancer to the ATF3-promoter region, ultimately leading to AAA development. Our results demonstrate a previously unrecognized role of ATF3 in AAA development and progression, and ATF3 may serve as a novel therapeutic and prognostic marker for AAA.

The role of transcription factors in the pathogenesis and therapeutic targeting of vascular diseases.

Transcription factors (TFs) constitute an essential component of epigenetic regulation. They contribute to the progression of vascular diseases by regulating epigenetic gene expression in several vascular diseases. Recently, numerous regulatory mechanisms related to vascular pathology, ranging from general TFs that are continuously activated to histiocyte-specific TFs that are activated under specific circumstances, have been studied. TFs participate in the progression of vascular-related diseases by epigenetically regulating vascular endothelial cells (VECs) and vascular smooth muscle cells (VSMCs). The Krüppel-like family (KLF) TF family is widely recognized as the foremost regulator of vascular diseases. KLF11 prevents aneurysm progression by inhibiting the apoptosis of VSMCs and enhancing their contractile function. The presence of KLF4, another crucial member, suppresses the progression of atherosclerosis (AS) and pulmonary hypertension by attenuating the formation of VSMCs-derived foam cells, ameliorating endothelial dysfunction, and inducing vasodilatory effects. However, the mechanism underlying the regulation of the progression of vascular-related diseases by TFs has remained elusive. The present study categorized the TFs involved in vascular diseases and their regulatory mechanisms to shed light on the potential pathogenesis of vascular diseases, and provide novel insights into their diagnosis and treatment.

G6PD maintains the VSMC synthetic phenotype and accelerates vascular neointimal hyperplasia by inhibiting the VDAC1-Bax-mediated mitochondrial apoptosis pathway.

Glucose-6-phosphate dehydrogenase (G6PD) plays an important role in vascular smooth muscle cell (VSMC) phenotypic switching, which is an early pathogenic event in various vascular remodeling diseases (VRDs). However, the underlying mechanism is not fully understood. An IP‒LC‒MS/MS assay was conducted to identify new binding partners of G6PD involved in the regulation of VSMC phenotypic switching under platelet-derived growth factor-BB (PDGF-BB) stimulation. Co-IP, GST pull-down, and immunofluorescence colocalization were employed to clarify the interaction between G6PD and voltage-dependent anion-selective channel protein 1 (VDAC1). The molecular mechanisms involved were elucidated by examining the interaction between VDAC1 and apoptosis-related biomarkers, as well as the oligomerization state of VDAC1. The G6PD level was significantly elevated and positively correlated with the synthetic characteristics of VSMCs induced by PDGF-BB. We identified VDAC1 as a novel G6PD-interacting molecule essential for apoptosis. Specifically, the G6PD-NTD region was found to predominantly contribute to this interaction. G6PD promotes VSMC survival and accelerates vascular neointimal hyperplasia by inhibiting VSMC apoptosis. Mechanistically, G6PD interacts with VDAC1 upon stimulation with PDGF-BB. By competing with Bax for VDAC1 binding, G6PD reduces VDAC1 oligomerization and counteracts VDAC1-Bax-mediated apoptosis, thereby accelerating neointimal hyperplasia. Our study showed that the G6PD-VDAC1-Bax axis is a vital switch in VSMC apoptosis and is essential for VSMC phenotypic switching and neointimal hyperplasia, providing mechanistic insight into early VRDs.

Paraspeckle protein NONO attenuates vascular calcification by inhibiting bone morphogenetic protein 2 transcription

Vascular calcification is a pathological process commonly associated with atherosclerosis, chronic kidney disease, and diabetes. Paraspeckle protein NONO is a multifunctional RNA/DNA binding protein involved in many nuclear biological processes but its role in vascular calcification remains unclear. Here, we observed that NONO expression was decreased in calcified arteries of mice and patients with CKD. We generated smooth muscle-specific NONO-knockout mice and established three different mouse models of vascular calcification by means of 5/6 nephrectomy, adenine diet to induce chronic kidney failure, or vitamin D injection. The knockout mice were more susceptible to the development of vascular calcification relative to control mice, as verified by an increased calcification severity and calcium deposition. Likewise, aortic rings from knockout mice showed more significant vascular calcification than those from control mice ex vivo. In vitro, NONO deficiency aggravated high phosphate-induced vascular smooth muscle cell osteogenic differentiation and apoptosis, whereas NONO overexpression had a protective effect. Mechanistically, we demonstrated that the regulation of vascular calcification by NONO was mediated by bone morphogenetic protein 2 (BMP2). NONO directly bound to the BMP2 promoter using its C-terminal region, exerting an inhibitory effect on the transcription of BMP2. Thus, our study reveals that NONO is a novel negative regulator of vascular calcification, which inhibits osteogenic differentiation of vascular smooth muscle cell and vascular calcification via negatively regulating BMP2 transcription. Hence, NONO may provide a promising target for the prevention and treatment of vascular calcification.

CircCDYL Contributes to Apoptosis, Ferroptosis, and Oxidative Stress of Ang II-Induced Vascular Smooth Muscle Cells in Thoracic Aortic Aneurysm.

Circular RNAs (circRNAs) have important regulation in thoracic aortic aneurysm (TAA). The function and mechanism of circCDYL (circ_0008285) was explored in TAA here. Angiotensin II (Ang II) was used to construct a TAA model. Real-time quantitative polymerase chain reaction (RT-qPCR) was performed for the detection of circCDYL, miR-1270, and a disintegrin and metalloproteinase 10 (ADAM10). Cell viability was examined via cell counting kit-8 (CCK-8) assay and proliferation was analyzed using Ethynyl-2'-deoxyuridine (EdU) assay. Apoptosis rate was assessed via flow cytometry. Western blot was used for protein detection. Oxidative stress was evaluated by commercial kits. CircCDYL was upregulated in TAA tissues and Ang II-induced circCDYL upregulation in vascular smooth muscle cells (VSMCs). Knockdown of circCDYL weakened Ang II-aroused inhibition of viability, proliferation, and promotion of apoptosis, ferroptosis, and oxidative stress in VSMCs. CircCDYL served as a miR-1270 sponge. The mitigated regulation of circCDYL knockdown for Ang II-induced injury was restored after miR-1270 downregulation. CircCDYL positively regulated ADAM10 through interacting with miR-1270. Overexpression of miR-1270 abated Ang II-induced injury by downregulating ADAM10. In conclusion, circCDYL was involved in the Ang II-induced VSMC injury in TAA via the miR-1270/ADAM10 axis.

Cannabidiol protects against acute aortic dissection by inhibiting macrophage infiltration and PMAIP1-induced vascular smooth muscle cell apoptosis

Acute aortic dissection (AAD) progresses rapidly and is associated with high mortality; therefore, there remains an urgent need for pharmacological agents that can protect against AAD. Herein, we examined the therapeutic effects of cannabidiol (CBD) in AAD by establishing a suitable mouse model. In addition, we performed human AAD single-cell RNA sequencing and mouse AAD bulk RNA sequencing to elucidate the potential underlying mechanism of CBD. Pathological assays and in vitro studies were performed to verify the results of the bioinformatic analysis and explore the pharmacological function of CBD. In a β-aminopropionitrile (BAPN)-induced AAD mouse model, CBD reduced AAD-associated morbidity and mortality, alleviated abnormal enlargement of the ascending aorta and aortic arch, and suppressed macrophage infiltration and vascular smooth muscle cell (VSMC) apoptosis. Bioinformatic analysis revealed that the pro-apoptotic gene PMAIP1 was highly expressed in human and mouse AAD samples, and CBD could inhibit Pmaip1 expression in AAD mice. Using human aortic VSMCs (HAVSMCs) co-cultured with M1 macrophages, we revealed that CBD alleviated HAVSMCs mitochondrial-dependent apoptosis by suppressing the BAPN-induced overexpression of PMAIP1 in M1 macrophages. PMAIP1 potentially mediates HAVSMCs apoptosis by regulating Bax and Bcl2 expression. Accordingly, CBD reduced AAD-associated morbidity and mortality and mitigated the progression of AAD in a mouse model. The CBD-induced effects were potentially mediated by suppressing macrophage infiltration and PMAIP1 (primarily expressed in macrophages)-induced VSMC apoptosis. Our findings offer novel insights into M1 macrophages and HAVSMCs interaction during AAD progression, highlighting the potential of CBD as a therapeutic candidate for AAD treatment.