Fluoride impairs vascular smooth muscle A7R5 cell lines via disrupting amino acids metabolism.

Given the insidious and high-fatality nature of cardiovascular diseases (CVDs), the emergence of fluoride as a newly identified risk factor demands serious consideration alongside traditional risk factors. While vascular smooth muscle cells (VSMCs) play a pivotal role in the progression of CVDs, the toxicological impact of fluoride on VSMCs remains largely uncharted. In this study, we constructed fluorosis model in SD rats and A7R5 aortic smooth muscle cell lines to confirm fluoride impaired VSMCs. Fluoride aggravated the pathological damage of rat aorta in vivo. Then A7R5 were exposed to fluoride with concentration ranging from 0 to 1200 µmol/L over a 24-h period, revealing a dose-dependent inhibition of cell proliferation and migration. The further metabolomic analysis showed alterations in metabolite profiles induced by fluoride exposure, notably decreasing organic acids and lipid molecules level. Additionally, gene network analysis underscored the frequency of fluoride's interference with amino acids metabolism, potentially impacting the tricarboxylic acid (TCA) cycle. Our results also highlighted the ATP-binding cassette (ABC) transporters pathway as a central element in VSMC impairment. Moreover, we observed a dose-dependent increase in osteopontin (OPN) and α-smooth muscle actin (α-SMA) mRNA level and a dose-dependent decrease in ABC subfamily C member 1 (ABCC1) and bestrophin 1 (BEST1) mRNA level. These findings advance our understanding of fluoride as a CVD risk factor and its influence on VSMCs and metabolic pathways, warranting further investigation into this emerging risk factor.

Circ_0000006 and circ_0000160 regulate hsa-let-7e-5p/UBQLN4 axis in aortic dissection progression.

Aortic aneurysms (AA) and aorta dissection (AD) are life-threatening conditions with a rising incidence and high mortality rate. Recent research has linked non-coding RNAs to the regulation of AA and AD progression. In this study, we performed circRNA sequencing, microRNA (miRNA) sequencing, and messenger RNA (mRNA) sequencing on plasma samples from AA and AD patients to identify the key circRNA-miRNA-mRNA axis involved in the transition from AA to AD. Our results showed elevated levels of circ_0000006 and circ_0000160, along with decreased levels of hsa-let-7e-5p in AD samples compared to AA samples. Predictive analysis suggested that circ_0000006 and circ_0000160 potentially target hsa-let-7e-5p, which in turn may bind to the mRNA of Ubiquilin 4 (UBQLN4). In an AD cell model using vascular smooth muscle cells (VSMCs), silencing circ_0000006 and circ_0000160 attenuated the effects of platelet-derived growth factor (PDGF)-induced phenotypic changes, proliferation, and migration. This effect was partially reversed by inhibiting hsa-let-7e-5p. Furthermore, we found that overexpression of UBQLN4 counteracted the effects of hsa-let-7e-5p, suggesting UBQLN4 as a downstream mediator of hsa-let-7e-5p. In an animal model of AD, knockdown of circ_0000006 and circ_0000160 also showed protective effects against aortic septation. Overall, our findings indicate that the upregulation of circ_0000006 and circ_0000160 contributes to the progression from AA to AD by influencing abnormal phenotypic changes, migration, and proliferation of VSMCs. The Hsa-let-7e-5p/UBQLN4 axis may play a critical role in AD development. Targeting circ_0000006 and circ_0000160 could be a potential therapeutic strategy for preventing the progression of AD.

Overexpression of miR-204-5p Alleviates Osteogenic Differentiation and Calcification of Human Aortic Vascular Smooth Muscle Cells by Targeting Calcium/Calmodulin-dependent Protein Kinase 1.

ABSTRACT: Vascular calcification (VC), a major complication in chronic kidney disease (CKD), is predominantly driven by osteoblastic differentiation. Recent studies have highlighted the crucial role of microRNAs in CKD's pathogenesis. Here, our research focused on the effects of miR-204-5p and its molecular mechanisms within VC. We initially found a notable decrease in miR-204-5p levels in human aortic vascular smooth muscle cells stimulated with inorganic phosphate, using this as a VC model in vitro. Following the overexpression of miR-204-5p, a decrease in VC was observed, as indicated by alizarin red S staining and measurements of calcium content. This decrease was accompanied by lower levels of the osteogenic marker, runt-related transcription factor 2, and higher levels of α-smooth muscle actin, a marker of contractility. Further investigation showed that calcium/calmodulin-dependent protein kinase 1 (CAMK1), which is a predicted target of miR-204-5p, promotes VC. Conversely, overexpressing miR-204-5p reduced VC by suppressing CAMK1 activity. Overexpressing miR-204-5p also effectively mitigated aortic calcification in an in vivo rat model. In summary, our research indicated that targeting the miR-204-5p/CAMK1 pathway could be a viable strategy for mitigating VC in CKD patients.

Deciphering smooth muscle cell heterogeneity in atherosclerotic plaques and constructing model: a multi-omics approach with focus on KLF15/IGFBP4 axis.

BACKGROUND: Ruptured atherosclerotic plaques often precipitate severe ischemic events, such as stroke and myocardial infarction. Unraveling the intricate molecular mechanisms governing vascular smooth muscle cell (VSMC) behavior in plaque stabilization remains a formidable challenge. METHODS: In this study, we leveraged single-cell and transcriptomic datasets from atherosclerotic plaques retrieved from the gene expression omnibus (GEO) database. Employing a combination of single-cell population differential analysis, weighted gene co-expression network analysis (WGCNA), and transcriptome differential analysis techniques, we identified specific genes steering the transformation of VSMCs in atherosclerotic plaques. Diagnostic models were developed and validated through gene intersection, utilizing the least absolute shrinkage and selection operator (LASSO) and random forest (RF) methods. Nomograms for plaque assessment were constructed. Tissue localization and expression validation were performed on specimens from animal models, utilizing immunofluorescence co-localization, western blot, and reverse-transcription quantitative-polymerase chain reaction (RT-qPCR). Various online databases were harnessed to predict transcription factors (TFs) and their interacting compounds, with determination of the cell-specific localization of TF expression using single-cell data. RESULTS: Following rigorous quality control procedures, we obtained a total of 40,953 cells, with 6,261 representing VSMCs. The VSMC population was subsequently clustered into 5 distinct subpopulations. Analyzing inter-subpopulation cellular communication, we focused on the SMC2 and SMC5 subpopulations. Single-cell subpopulation and WGCNA analyses revealed significant module enrichments, notably in collagen-containing extracellular matrix and cell-substrate junctions. Insulin-like growth factor binding protein 4 (IGFBP4), apolipoprotein E (APOE), and cathepsin C (CTSC) were identified as potential diagnostic markers for early and advanced plaques. Notably, gene expression pattern analysis suggested that IGFBP4 might serve as a protective gene, a hypothesis validated through tissue localization and expression analysis. Finally, we predicted TFs capable of binding to IGFBP4, with Krüppel-like family 15 (KLF15) emerging as a prominent candidate showing relative specificity within smooth muscle cells. Predictions about compounds associated with affecting KLF15 expression were also made. CONCLUSION: Our study established a plaque diagnostic and assessment model and analyzed the molecular interaction mechanisms of smooth muscle cells within plaques. Further analysis revealed that the transcription factor KLF15 may regulate the biological behaviors of smooth muscle cells through the KLF15/IGFBP4 axis, thereby influencing the stability of advanced plaques via modulation of the PI3K-AKT signaling pathway. This could potentially serve as a target for plaque stability assessment and therapy, thus driving advancements in the management and treatment of atherosclerotic plaques.

Divergent and Compensatory Effects of BMP2 and BMP4 on the VSMC Phenotype and BMP4's Role in Thoracic Aortic Aneurysm Development.

Vascular smooth muscle cells (VSMCs) play a key role in aortic aneurysm formation. Bone morphogenetic proteins (BMPs) have been implicated as important regulators of VSMC phenotype, and dysregulation of the BMP pathway has been shown to be associated with vascular diseases. The aim of this study was to investigate for the first time the effects of BMP-4 on the VSMC phenotype and to understand its role in the development of thoracic aortic aneurysms (TAAs). Using the angiotensin II (AngII) osmotic pump model in mice, aortas from mice with VSMC-specific BMP-4 deficiency showed changes similar to AngII-infused aortas, characterised by a loss of contractile markers, increased fibrosis, and activation of matrix metalloproteinase 9. When BMP-4 deficiency was combined with AngII infusion, there was a significantly higher rate of apoptosis and aortic dilatation. In vitro, VSMCs with mRNA silencing of BMP-4 displayed a dedifferentiated phenotype with activated canonical BMP signalling. In contrast, BMP-2-deficient VSMCs exhibited the opposite phenotype. The compensatory regulation between BMP-2 and BMP-4, with BMP-4 promoting the contractile phenotype, appeared to be independent of the canonical signalling pathway. Taken together, these results demonstrate the impact of VSMC-specific BMP-4 deficiency on TAA development.

Prevotella copri promotes vascular calcification via lipopolysaccharide through activation of NF-κB signaling pathway.

Emerging evidence indicates that alteration of gut microbiota plays an important role in chronic kidney disease (CKD)-related vascular calcification (VC). We aimed to investigate the specific gut microbiota and the underlying mechanism involved in CKD-VC. We identified an increased abundance of Prevotella copri (P. copri) in the feces of CKD rats (induced by using 5/6 nephrectomy followed by a high calcium and phosphate diet) with aortic calcification via amplicon sequencing of 16S rRNA genes. In patients with CKD, we further confirmed a positive correlation between abundance of P. copri and aortic calcification scores. Moreover, oral administration of live P. copri aggravated CKD-related VC and osteogenic differentiation of vascular smooth muscle cells in vivo, accompanied by intestinal destruction, enhanced expression of Toll-like receptor-4 (TLR4), and elevated lipopolysaccharide (LPS) levels. In vitro and ex vivo experiments consistently demonstrated that P. copri-derived LPS (Pc-LPS) accelerated high phosphate-induced VC and VSMC osteogenic differentiation. Mechanistically, Pc-LPS bound to TLR4, then activated the nuclear factor κB (NF-κB) and nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 (NLRP3) inflammasome signals during VC. Inhibition of NF-κB reduced NLRP3 inflammasome and attenuated Pc-LPS-induced VSMC calcification. Our study clarifies a novel role of P. copri in CKD-related VC, by the mechanisms involving increased inflammation-regulating metabolites including Pc-LPS, and activation of the NF-κB/NLRP3 signaling pathway. These findings highlight P. copri and its-derived LPS as potential therapeutic targets for VC in CKD.

Glucagon-like peptide-1 receptor agonist exendin 4 ameliorates diabetes-associated vascular calcification by regulating mitophagy through the AMPK signaling pathway.

BACKGROUND: Vascular calcification (VC) is a complication in diabetes mellitus (DM) patients. Osteogenic phenotype switching of vascular smooth muscle cells (VSMCs) plays a critical role in diabetes-related VC. Mitophagy can inhibit phenotype switching in VSMCs. This study aimed to investigate the role of the glucagon-like peptide-1 receptor (GLP-1R) agonist exendin 4 (EX4) in mitophagy-induced phenotype switching. MATERIALS AND METHODS: The status of VC in T2DM mice was monitored using Von Kossa and Alizarin Red S (ARS) staining in mouse aortic tissue. Human aortic smooth muscle cells were cultured in high glucose (HG) and ß-glycerophosphate (ß-GP) conditioned medium. Accumulation of LC3B and p62 was detected in the mitochondrial fraction. The effect of EX4 in vitro and in vivo was investigated by knocking down AMPKα1. RESULTS: In diabetic VC mice, EX4 decreased the percentage of von Kossa/ARS positive area. EX4 inhibited osteogenic differentiation of HG/ß-GP-induced VSMCs. In HG/ß-GP-induced VSMCs, the number of mitophagosomes was increased, whereas the addition of EX4 restored mitochondrial function, increased the number of mitophagosome-lysosome fusions, and reduced p62 in mitochondrial frictions. EX4 increased the phosphorylation of AMPKα (Thr172) and ULK1 (Ser555) in HG/ß-GP-induced VSMCs. After knockdown of AMPKα1, ULK1 could not be activated by EX4. The accumulation of LC3B and p62 could not be reduced after AMPKα1 knockdown. Knockdown of AMPKα1 negated the therapeutic effects of EX4 on VC of diabetic mice. CONCLUSION: EX4 could promote mitophagy by activating the AMPK signaling pathway, attenuate insufficient mitophagy, and thus inhibit the osteogenic phenotype switching of VSMCs.

Asiatic acid alleviates vascular remodeling in BAPN-induced aortic dissection through inhibiting NF-κB p65/CX3CL1 signaling.

Inflammation assumes a pivotal role in the aortic remodeling of aortic dissection (AD). Asiatic acid (AA), a triterpene compound, is recognized for its strong anti-inflammatory properties. Yet, its effects on ß-aminopropionitrile (BAPN)-triggered AD have not been clearly established. The objective is to determine whether AA attenuates adverse aortic remodeling in BAPN-induced AD and clarify potential molecular mechanisms. In vitro studies, RAW264.7 cells pretreated with AA were challenged with lipopolysaccharide (LPS), and then the vascular smooth muscle cells (VSMCs)-macrophage coculture system was established to explore intercellular interactions. To induce AD, male C57BL/6J mice at three weeks of age were administered BAPN at a dosage of 1 g/kg/d for four weeks. To decipher the mechanism underlying the effects of AA, RNA sequencing analysis was conducted, with subsequent validation of these pathways through cellular experiments. AA exhibited significant suppression of M1 macrophage polarization. In the cell coculture system, AA facilitated the transformation of VSMCs into a contractile phenotype. In the mouse model of AD, AA strikingly prevented the BAPN-induced increases in inflammation cell infiltration and extracellular matrix degradation. Mechanistically, RNA sequencing analysis revealed a substantial upregulation of CX3CL1 expression in BAPN group but downregulation in AA-treated group. Additionally, it was observed that the upregulation of CX3CL1 negated the beneficial impact of AA on the polarization of macrophages and the phenotypic transformation of VSMCs. Crucially, our findings revealed that AA is capable of downregulating CX3CL1 expression, accomplishing this by obstructing the nuclear translocation of NF-κB p65. The findings indicate that AA holds promise as a prospective treatment for adverse aortic remodeling by suppressing the activity of NF-κB p65/CX3CL1 signaling pathway.

Linarin Ameliorates Restenosis After Vascular Injury in Type 2 Diabetes Mellitus via Regulating ADAM10-Mediated Notch Signaling Pathway.

Vascular lesions frequently arise as complication in patients diagnosed with diabetes mellitus (DM). Presently, percutaneous coronary intervention (PCI) and antithrombotic therapy serve as primary treatments. However, in-stent restenosis persists as a challenging clinical issue following PCI, lacking sustained and effective treatment. Linarin (LN) exhibits diverse pharmacological activities and is regarded as a potential drug for treating various diseases, including DM. But its specific role in restenosis after vascular injury in DM patients remains unclear. A rat model of diabetes-related restenosis was established to evaluate the role of LN on neointimal hyperplasia. Vascular smooth muscle cells (VSMCs) stimulated by high glucose (HG, 30 mM) underwent LN treatment. Additionally, an overexpression plasmid of A disintegrin and metalloproteinases (ADAM10) was constructed to transfect VSMCs. We employed CCK-8, Brdu, wound-healing scratch, and transwell migration assays to evaluate the proliferation and migration of VSMCs. Furthermore, western blot and immunofluorescence assays were utilized to investigate the expressions of ADAM10 and the downstream Notch signaling pathway in vivo and in vitro models. LN notably alleviated intimal hyperplasia after vascular injury in DM rats and reduced the protein expression of ADAM10, alongside its downstream Notch1 signaling pathway-related proteins (Notch1, NICD and Hes1) in rat carotid artery tissues. LN effectively suppressed the proliferation and migration of VSMCs induced by HG, downregulating the protein expression of ADAM10, Notch1, NICD and Hes1. Moreover, our findings indicated that ADAM10 overexpression significantly reversed LN's effects on proliferation, migration, and the expression of Notch1 signaling pathway-related proteins in HG-treated VSMCs. LN demonstrates potential therapeutic efficacy in addressing restenosis after diabetic-related vascular injury, with the ADAM10 mediated Notch signaling pathway playing a pivotal role.

USP18 Curbs the Progression of Metabolic Hypertension by Suppressing JAK/STAT Pathway.

Hypertension is a pathological state of the metabolic syndrome that increases the risk of cardiovascular disease. Managing hypertension is challenging, and we aimed to identify the pathogenic factors and discern therapeutic targets for metabolic hypertension (MHR). An MHR rat model was established with the combined treatment of a high-sugar, high-fat diet and ethanol. Histopathological observations were performed using hematoxylin-eosin and Sirius Red staining. Transcriptome sequencing was performed to screen differentially expressed genes. The role of ubiquitin-specific protease 18 (USP18) in the proliferation, apoptosis, and oxidative stress of HUVECs was explored using Cell Counting Kit-8, flow cytometry, and enzyme-linked immunosorbent assays. Moreover, USP18 downstream signaling pathways in MHR were screened, and the effects of USP18 on these signaling pathways were investigated by western blotting. In the MHR model, total cholesterol and low-density lipoprotein levels increased, while high-density lipoprotein levels decreased. Moreover, high vessel thickness and percentage of collagen were noted along with increased malondialdehyde, decreased superoxide dismutase and catalase levels. The staining results showed that the MHR model exhibited an irregular aortic intima and disordered smooth muscle cells. There were 78 differentially expressed genes in the MHR model, and seven hub genes, including USP18, were identified. USP18 overexpression facilitated proliferation and reduced apoptosis and oxidative stress in HUVECs treated with Ang in vitro. In addition, the JAK/STAT pathway was identified as a USP18 downstream signaling pathway, and USP18 overexpression inhibited the expression of JAK/STAT pathway-related proteins. Conclusively, USP18 restrained MHR progression by promoting cell proliferation, reversing apoptosis and oxidative stress, and suppressing the JAK/STAT pathway.

Inhibition of Slc39a14/Slc39a8 reduce vascular calcification via alleviating iron overload induced ferroptosis in vascular smooth muscle cells.

BACKGROUND: Vascular calcification (VC) is an independent risk factor for cardiovascular diseases. Recently, ferroptosis has been recognised as a novel therapeutic target for cardiovascular diseases. Although an association between ferroptosis and vascular calcification has been reported, the role and mechanism of iron overload in vascular calcification are still poorly understood. Specifically, further in-depth research is required on whether metalloproteins SLC39a14 and SLC39a8 are involved in ferroptosis induced by iron overload. METHODS: R language was employed for the differential analysis of the dataset, revealing the correlation between ferroptosis and calcification. The experimental approaches encompassed both in vitro and in vivo studies, incorporating the use of iron chelators and models of iron overload. Additionally, gain- and loss-of-function experiments were conducted to investigate iron's effects on vascular calcification comprehensively. Electron microscopy, immunofluorescence, western blotting, and real-time polymerase chain reaction were used to elucidate how Slc39a14 and Slc39a8 mediate iron overload and promote calcification. RESULTS: Ferroptosis was observed in conjunction with vascular calcification (VC); the association was consistently confirmed by in vitro and in vivo studies. Our results showed a positive correlation between iron overload in VSMCs and calcification. Iron chelators are effective in reversing VC and iron overload exacerbates this process. The expression levels of the metal transport proteins Slc39a14 and Slc39a8 were significantly upregulated during calcification; the inhibition of their expression alleviated VC. Conversely, Slc39a14 overexpression exacerbates calcification and promotes intracellular iron accumulation in VSMCs. CONCLUSIONS: Our research demonstrates that iron overload occurs during VC, and that inhibition of Slc39a14 and Slc39a8 significantly relieves VC by intercepting iron overload-induced ferroptosis in VSMCs, providing new insights into the VC treatment.

Single-Cell Transcriptome Analysis Reveals Dynamic Populations of Vascular Cells in Neointimal Hyperplasia.

BACKGROUND: Neointimal hyperplasia (NIH) is the pathological basis of vascular injury disease. Vascular cells are the dominant cells in the process of NIH, but the extent of heterogeneity amongst them is still unclear. METHODS: A mouse model of NIH was constructed by inducing carotid artery ligation. Single-cell sequencing was then used to analyze the transcriptional profile of vascular cells. Cluster features were determined by functional enrichment analysis, gene set scoring, pseudo-time analysis, and cell-cell communication analysis. Additionally, immunofluorescence staining was conducted on vascular tissues from fibroblast lineage-traced (PdgfraDreER-tdTomato) mice to validate the presence of Pecam1+Pdgfra+tdTomato+ cells. RESULTS: The left carotid arteries (ligation) were compared to right carotid arteries (sham) from ligation-induced NIH C57BL/6 mice. Integrative analyses revealed a high level of heterogeneity amongst vascular cells, including fourteen clusters and seven cell types. We focused on three dominant cell types: endothelial cells (ECs), vascular smooth muscle cells (vSMCs), and fibroblasts. The major findings were: (1) four subpopulations of ECs, including ECs4, mesenchymal-like ECs (ECs1 and ECs2), and fibro-like ECs (ECs3); (2) four subpopulations of fibroblasts, including pro-inflammatory Fibs-1, Sca1+ Fibs-2, collagen-producing Fibs-3, and mesenchymal-like Fibs-4; (3) four subpopulations of vSMCs, including vSMCs-1, vSMCs-2, vSMCs-3, and vSMCs-3-derived vSMCs; (4) ECs3 express genes related to extracellular matrix (ECM) remodeling and cell migration, and fibro-like vSMCs showed strong chemokine secretion and relatively high levels of proteases; (5) fibro-like vSMCs that secrete Vegfa interact with ECs mainly through vascular endothelial growth factor receptor 2 (Vegfr2). CONCLUSIONS: This study presents the dynamic cellular landscape within NIH arteries and reveals potential relationships between several clusters, with a specific focus on ECs3 and fibro-like vSMCs. These two subpopulations may represent potential target cells for the treatment of NIH.

Unveiling cellular and molecular aspects of ascending thoracic aortic aneurysms and dissections.

Ascending thoracic aortic aneurysm (ATAA) remains a significant medical concern, with its asymptomatic nature posing diagnostic and monitoring challenges, thereby increasing the risk of aortic wall dissection and rupture. Current management of aortic repair relies on an aortic diameter threshold. However, this approach underestimates the complexity of aortic wall disease due to important knowledge gaps in understanding its underlying pathologic mechanisms.Since traditional risk factors cannot explain the initiation and progression of ATAA leading to dissection, local vascular factors such as extracellular matrix (ECM) and vascular smooth muscle cells (VSMCs) might harbor targets for early diagnosis and intervention. Derived from diverse embryonic lineages, VSMCs exhibit varied responses to genetic abnormalities that regulate their contractility. The transition of VSMCs into different phenotypes is an adaptive response to stress stimuli such as hemodynamic changes resulting from cardiovascular disease, aging, lifestyle, and genetic predisposition. Upon longer exposure to stress stimuli, VSMC phenotypic switching can instigate pathologic remodeling that contributes to the pathogenesis of ATAA.This review aims to illuminate the current understanding of cellular and molecular characteristics associated with ATAA and dissection, emphasizing the need for a more nuanced comprehension of the impaired ECM-VSMC network.

Bioengineered vascular grafts with a pathogenic TGFBR1 variant model aneurysm formation in vivo and reveal underlying collagen defects.

Thoracic aortic aneurysm (TAA) is a life-threatening vascular disease frequently associated with underlying genetic causes. An inadequate understanding of human TAA pathogenesis highlights the need for better disease models. Here, we established a functional human TAA model in an animal host by combining human induced pluripotent stem cells (hiPSCs), bioengineered vascular grafts (BVGs), and gene editing. We generated BVGs from isogenic control hiPSC-derived vascular smooth muscle cells (SMCs) and mutant SMCs gene-edited to carry a Loeys-Dietz syndrome (LDS)-associated pathogenic variant (TGFBR1A230T). We also generated hiPSC-derived BVGs using cells from a patient with LDS (PatientA230T/+) and using genetically corrected cells (Patient+/+). Control and experimental BVGs were then implanted into the common carotid arteries of nude rats. The TGFBR1A230T variant led to impaired mechanical properties of BVGs, resulting in lower burst pressure and suture retention strength. BVGs carrying the variant dilated over time in vivo, resembling human TAA formation. Spatial transcriptomics profiling revealed defective expression of extracellular matrix (ECM) formation genes in PatientA230T/+ BVGs compared with Patient+/+ BVGs. Histological analysis and protein assays validated quantitative and qualitative ECM defects in PatientA230T/+ BVGs and patient tissue, including decreased collagen hydroxylation. SMC organization was also impaired in PatientA230T/+ BVGs as confirmed by vascular contraction testing. Silencing of collagen-modifying enzymes with small interfering RNAs reduced collagen proline hydroxylation in SMC-derived tissue constructs. These studies demonstrated the utility of BVGs to model human TAA formation in an animal host and highlighted the role of reduced collagen modifying enzyme activity in human TAA formation.

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

BACKGROUND: Pentamethylquercetin (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. METHODS: ApoE-/- 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. RESULTS: PMQ 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/EBPß/PTEN/AKT/GSK-3ß 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-3ß axis. PMQ inhibited VSMCs phenotypic switching and apoptosis by bounding to C/EBPß at Lys253 with hydrogen bond to regulate C/EBPß nuclear translocation and PTEN/AKT/GSK-3ß axis, thereby inhibiting Ang II-induced AAA formation. CONCLUSIONS: Pentamethylquercetin inhibits angiotensin II-induced abdominal aortic aneurysm formation by bounding to C/EBPß at Lys253. Therefore, PMQ prevents the formation of AAA and reduces the incidence of AAA.

IL-1ß Inhibition Partially Negates the Beneficial Effects of Diet-Induced Atherosclerosis Regression in Mice.

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.

12,13-diHOME attenuates high glucose-induced calcification of vascular smooth muscle cells through repressing CPT1A-mediated HMGB1 succinylation.

Diabetes is closely associated with vascular calcification (VC). Exorbitant glucose concentration activates pro-calcific effects in vascular smooth muscle cells (VSMCs). This study enrolled 159 elderly patients with type 2 diabetes and divided them into three groups, T1, T2 and T3, according to brachial-ankle pulse wave velocity(BaPWV). There were statistically significant differences in the waist circumference, waist hip ratio, systolic blood pressure, 12,13-diHOME (a lipokin) concentration among T1, T2 and T3. 12,13-diHOME levels were positively correlated to high density lipoprotein cholesterol and total cholesterol, but negatively correlated to with waist circumference, waist hip ratio, systolic blood pressure and baPWV. Studies in vitro showed that 12,13-diHOME effectively inhibits calcification in VSMCs under high glucose conditions. Notably, 12,13-diHOME suppressed the up-regulation of carnitine O-palmitoyltransferase 1 (CPT1A) and CPT1A-induced succinylation of HMGB1. The succinylation of HMGB1 at the K90 promoted the protein stability and induced the enrichment of HMGB1 in cytoplasm, which induced the calcification in VSMCs. Together, 12,13-diHOME attenuates high glucose-induced calcification in VSMCs through repressing CPT1A-mediated HMGB1 succinylation.

Urotensin II receptor deficiency ameliorates ligation-induced carotid intimal hyperplasia partially through the RhoA-YAP1 pathway.

Intimal hyperplasia (IH) is a common pathological feature of vascular proliferative diseases, such as atherosclerosis and restenosis after angioplasty. Urotensin II (UII) and its receptor (UTR) are widely expressed in cardiovascular tissues. However, it remains unclear whether the UII/UTR system is involved in IH. Right unilateral common carotid artery ligation was performed and maintained for 21 days to induce IH in UTR knockout (UTR-/-) and wild-type (WT) mice. Histological analysis revealed that compared with WT mice, UTR-deficient mice exhibited a decreased neointimal area, angiostenosis and intima-media ratio. Immunostaining revealed fewer smooth muscle cells (SMCs), endothelial cells and macrophages in the lesions of UTR-/- mice than in those of WT mice. Protein interaction analysis suggested that the UTR may affect cell proliferation by regulating YAP and its downstream target genes. In vitro experiments revealed that UII can promote the proliferation and migration of SMCs, and western blotting also revealed that UII increased the protein expression of RhoA, CTGF, Cyclin D1 and PCNA and downregulated p-YAP protein expression, while these effects could be partly reversed by urantide. To evaluate the translational value of UTRs in IH management, WT mice were also treated with two doses of urantide, a UTR antagonist, to confirm the benefit of UTR blockade in IH progression. A high dose of urantide (600 µg/kg/day), rather than a low dose (60 µg/kg/day), successfully improved ligation-induced IH compared with that in mice receiving vehicle. The results of the present study suggested that the UII/UTR system may regulate IH partly through the RhoA-YAP signaling pathway.

Spatiotemporal ATF3 Expression Determines VSMC Fate in Abdominal Aortic Aneurysm.

BACKGROUND: 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. METHODS: 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. RESULTS: 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 ß) 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. CONCLUSIONS: 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.

KLF13 promotes VSMCs phenotypic dedifferentiation by directly binding to the SM22α promoter.

Krüppel-like factor 13 (KLF13), a zinc finger transcription factor, is considered as a potential regulator of cardiomyocyte differentiation and proliferation during heart morphogenesis. However, its precise role in the dedifferentiation of vascular smooth muscle cells (VSMCs) during atherosclerosis and neointimal formation after injury remains poorly understood. In this study, we investigated the relationship between KLF13 and SM22α expression in normal and atherosclerotic plaques by bioanalysis, and observed a significant increase in KLF13 levels in the atherosclerotic plaques of both human patients and ApoE-/- mice. Knockdown of KLF13 was found to ameliorate intimal hyperplasia following carotid artery injury. Furthermore, we discovered that KLF13 directly binds to the SM22α promoter, leading to the phenotypic dedifferentiation of VSMCs. Remarkably, we observed a significant inhibition of platelet-derived growth factor BB-induced VSMCs dedifferentiation, proliferation, and migration when knocked down KLF13 in VSMCs. This inhibitory effect of KLF13 knockdown on VCMC function was, at least in part, mediated by the inactivation of p-AKT signaling in VSMCs. Overall, our findings shed light on a potential therapeutic target for treating atherosclerotic lesions and restenosis after vascular injury.