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Introduction: Cardiovascular calcification is an important public health issue with an unmeet therapeutic need. We had previously shown that lysyl oxidase (LOX) activity critically influences vascular wall smooth muscle cells (VSMCs) and valvular interstitial cells (VICs) calcification by affecting extracellular matrix remodeling. We have delved into the participation of LOX in atherosclerosis and vascular calcification, as well as in the mineralization of the aortic valve., Methods: Immunohistochemical and expression studies were carried out in human atherosclerotic lesions and experimental models, valves from patients with aortic stenosis, VICs, and in a genetically modified mouse model that overexpresses LOX in CMLV (TgLOX CMLV ). Hyperlipemia and atherosclerosis was induced in mice through the administration of adeno-associated viruses encoding a PCSK9 mutated form (AAV-PCSK9 D374Y ) combined with an atherogenic diet., Results: LOX expression is increased in the neointimal layer of atherosclerotic lesions from human coronary arteries and in VSMC-rich regions of atheromas developed both in the brachiocephalic artery of control (C57BL/6J) animals transduced with PCSK9 D374Y and in the aortic root of ApoE -/- mice. In TgLOX CMLV mice, PCSK9 D374Y transduction did not significantly alter the enhanced aortic expression of genes involved in matrix remodeling, inflammation, oxidative stress and osteoblastic differentiation. Likewise, LOX transgenesis did not alter the size or lipid content of atherosclerotic lesions in the aortic arch, brachiocephalic artery and aortic root, but exacerbated calcification. Among lysyl oxidase isoenzymes, LOX is the most expressed member of this family in highly calcified human valves, colocalizing with RUNX2 in VICs. The lower calcium deposition and decreased RUNX2 levels triggered by the overexpression of the nuclear receptor NOR-1 in VICs was associated with a reduction in LOX., Conclusions: Our results show that LOX expression is increased in atherosclerotic lesions, and that overexpression of this enzyme in VSMC does not affect the size of the atheroma or its lipid content, but it does affect its degree of calcification. Further, these data suggest that the decrease in calcification driven by NOR-1 in VICs would involve a reduction in LOX. These evidences support the interest of LOX as a therapeutic target in cardiovascular calcification., (Copyright © 2024 Sociedad Española de Arteriosclerosis. Publicado por Elsevier España, S.L.U. All rights reserved.)

Background and Aims: Diabetes is one of the major causes of cardiovascular disease (CVD). As high as 29 % of patients with diabetes develop atherosclerosis. Vascular Smooth Muscle Cells (VSMCs) are a key mediator in the pathogenesis of atherosclerosis, generating pro-inflammatory and proliferative characteristics in atherosclerotic lesions., Methods: We used human atherosclerotic samples, developed diabetes-induced atherosclerotic mice, and generated loss of function and gain of function in Klotho human aortic smooth muscle cells to investigate the function of Klotho in atherosclerosis., Results: We found that Klotho expression is decreased in smooth muscle actin-positive cells in patients with diabetes and atherosclerosis. Consistent with human data, we found that Apoe knockout mice with streptozotocin-induced diabetes fed on a high-fat diet showed decreased expression of Klotho in SMCs. Additionally, these mice showed increased expression of TGF-β, MMP9, phosphorylation of ERK and Akt. Further, we utilized primary Human Aortic Smooth Muscle Cells (HASMCs) with d-glucose under dose-response and in time-dependent conditions to study the role of Klotho in these cells. Klotho gain of function and loss of function studies showed that Klotho inversely regulated the expression of atherosclerotic markers TGF-β, MMP2, MMP9, and Fractalkine. Further, High Glucose (HG) induced Akt, and ERK1/2 phosphorylation were enhanced or mitigated by endogenous Klotho deficiency or its overexpression respectively. PI3K/Akt and MAPK/ERK inhibition partially abolished the HG-induced upregulation of TGF-β, MMP2, MMP9, and Fractalkine. Additionally, Klotho knockdown increased the proliferation of HASMCs and enhanced α-SMA and TGF-β expression., Conclusions: Taken together, these results indicate that local vascular Klotho is involved in diabetes-induced atherosclerosis, which is via PI3K/Akt and ERK1/2-dependent signaling pathways., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Obstructive sleep apnea (OSA), characterized by episodes of intermittent hypoxia (IH), is highly prevalent in patients with abdominal aortic aneurysm (AAA). However, whether IH serves as an independent risk factor for AAA development remains to be investigated. Here, we determined the effects of chronic (6 mo) IH on angiotensin (Ang II)-induced AAA development in C57BL/6J male mice and investigated the underlying mechanisms of IH in cultured vascular smooth muscle cells (SMCs). IH increased the susceptibility of mice to develop AAA in response to Ang II infusion by facilitating the augmentation of the abdominal aorta's diameter as assessed by transabdominal ultrasound imaging. Importantly, IH with Ang II augmented aortic elastin degradation and the expression of matrix metalloproteinases (MMPs), mainly MMP8, MMP12, and a disintegrin and metalloproteinase-17 (ADAM17) as measured by histology and immunohistochemistry. Mechanistically, IH increased the activities of MMP2, MMP8, MMP9, MMP12, and ADAM17, while reducing the expression of the MMP regulator reversion-inducing cysteine-rich protein with Kazal motifs (RECK) in cultured SMCs. Aortic samples from human AAA were associated with decreased RECK and increased expression of ADAM17 and MMPs. These data suggest that IH facilitates AAA development when additional stressors are superimposed and that this occurs in association with an increased presence of aortic MMPs and ADAM17, potentially due to IH-induced modulation of RECK expression. These findings support a plausible synergistic link between OSA and AAA and provide a better understanding of the molecular mechanisms underlying the pathogenesis of AAA. NEW & NOTEWORTHY IH facilitates Ang II-induced abdominal aortic diameter expansion and AAA development in C57BL/6J male mice. IH upregulates the expression of specific MMPs such as MMP8, MMP12, and ADAM17. IH directly suppresses RECK expression and increases MMPs activity in SMCs. Human AAA tissues exhibit a downregulation of RECK and an upregulation of ADAM17 and MMPs.

Hypertension, a disease with known sexual dimorphism, accelerates aging-associated arterial stiffening, partly because of the activation of matrix remodeling caused by increased biomechanical load. In this study, we tested the effect of biological sex and the role of the matrix remodeling enzyme lysyl oxidase-like 2 (LOXL2) in hypertension-induced arterial stiffening. Hypertension was induced by angiotensin II (ANG II) infusion via osmotic minipumps in 12- to 14-wk-old male and female mice. Blood pressure and pulse wave velocity (PWV) were measured noninvasively. Wire myography and uniaxial tensile testing were used to test aortic vasoreactivity and mechanical properties. Aortic wall composition was examined by histology and Western blotting. Uniaxial stretch of cultured cells was used to evaluate the effect of biomechanical strain. LOXL2's catalytic function was examined using knockout and inhibition. ANG II infusion-induced hypertension in both genotypes and sexes. Wild-type (WT) males exhibited arterial stiffening in vivo and ex vivo. Aortic remodeling with increased wall thickness, intralamellar distance, higher LOXL2, and collagen I and IV content was noted in WT males. Female mice did not exhibit increased PWV despite the onset of hypertension. LOXL2 depletion improved vascular reactivity and mechanics in hypertensive males. LOXL2 depletion improved aortic mechanics but worsened hypercontractility in females. Hypertensive cyclic strain contributed to LOXL2 upregulation in the cell-derived matrix in vascular smooth muscle cells (VSMCs) but not endothelial cells. LOXL2's catalytic function facilitated VSMC alignment in response to biomechanical strain. In conclusion, in males, arterial stiffening in hypertension is driven both by VSMC response and matrix remodeling. Females are protected from PWV elevation in hypertension. LOXL2 depletion is protective in males with improved mechanical and functional aortic properties. VSMCs are the primary source of LOXL2 in the aorta, and hypertension increases LOXL2 processing and shifts to collagen I accumulation. Overall, LOXL2 depletion offers protection in young hypertensive males and females. NEW & NOTEWORTHY We examined the effect of sex on the evolution of angiotensin II (ANG II)-induced hypertension and the role of lysyl oxidase-like 2 (LOXL2), an enzyme that catalyzes matrix cross linking. While ANG II led to hypertension and worsening vascular reactivity in both sexes, aortic remodeling and stiffening occurred only in males. LOXL2 depletion improved outcomes in males but not females. Thus males and females exhibit a distinct etiology of hypertension and LOXL2 is an effective target in males.

An increased intracellular Ca 2+ concentration ([Ca 2+ ] i ) is a key trigger for pulmonary arterial smooth muscle cell (PASMC) proliferation and contributes greatly to pulmonary hypertension (PH). Extracellular Ca 2+ influx via a store-operated Ca 2+ channel, termed store-operated Ca 2+ entry (SOCE), is a crucial mechanism for [Ca 2+ ] i increase in PASMCs. Calcium release-activated calcium modulator (Orai) proteins, consisting of three members (Orai1-3), are the main components of the store-operated Ca 2+ channel. Sodium houttuyfonate (SH) is a product of the addition reaction of sodium bisulfite and houttuynin and has antibacterial, antiinflammatory, and other properties. In this study, we assessed the contributions of Orai proteins to monocrotaline (MCT)-enhanced SOCE, [Ca 2+ ] i , and cell proliferation in PASMCs and determined the effect of SH on MCT-PH and the underlying mechanism, focusing on Orai proteins, SOCE, and [Ca 2+ ] i in PASMCs. Our results showed that: 1 ) Orai1 and Orai2 were selectively upregulated in the distal pulmonary arteries and the PASMCs of MCT-PH rats; 2 ) knockdown of Orai1 or Orai2 reduced SOCE, [Ca 2+ ] i , and cell proliferation without affecting their expression in PASMCs in MCT-PH rats; 3 ) SH significantly normalized the characteristic parameters in a dose-dependent manner in the MCT-PH rat model; and 4 ) SH decreased MCT-enhanced SOCE, [Ca 2+ ] i , and PASMC proliferation via Orai1 or Orai2. These results indicate that SH likely exerts its protective role in MCT-PH by inhibiting the Orai1,2-SOCE-[Ca 2+ ] i signaling pathway.

Abstract: Angiomotin-like 2 (AMOTL2) is related to numerous physiological and pathological conditions by affecting signal transduction. However, whether AMOTL2 is linked to pulmonary arterial hypertension (PAH) has not been addressed. This work aimed to investigate the potential role of AMOTL2 in PAH. A decrease in AMOTL2 abundance was observed in the lungs of PAH rats. The upregulation of AMOTL2 significantly decreased right ventricle systolic pressure and right ventricular hypertrophy in PAH rats. Overexpression of AMOTL2 also led to a noteworthy decrease in vascular wall thickness, pulmonary artery area, and collagen deposition in rats with PAH. AMOTL2 was downregulated in hypoxia-stimulated pulmonary arterial smooth muscle cells (PASMCs). Moreover, AMOTL2 overexpression impeded hypoxia-evoked proliferation, migration, and phenotypic transformation in rat PASMCs. Mechanistic investigation revealed that Yes-associated protein 1 (YAP1) activation in PAH rats or hypoxia-stimulated PASMCs was markedly inhibited by AMOTL2 overexpression, which was associated with increased large tumor suppressor 1/2 phosphorylation. The inhibition of large tumor suppressor 1/2 reversed the AMOTL2-mediated inactivation of YAP1. Restoring the activity of YAP1 reversed the inhibitory effect of AMOTL2 on hypoxia-evoked proliferation, migration, and phenotypic transformation of PASMCs. Collectively, these results suggest that AMOTL2 can ameliorate PAH in a rat model by interfering with pulmonary arterial remodeling via the inactivation of YAP1 signaling. Our work indicates that AMOTL2 may be a candidate target for novel drug development for the treatment of PAH., Competing Interests: The authors report no conflicts of interest., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Vascular calcification is a prevalent hallmark of cardiovascular risk in elderly and diabetic individuals. Senescent vascular smooth muscle cells (VSMCs) participate in calcification; however, the associated underlying mechanisms remain unknown. Aberrant activation of the cytosolic DNA sensing adaptor stimulator of interferon gene 1 (STING1) caused by cytosolic DNA, particularly that leaked from damaged mitochondria, is a catalyst for aging-related diseases. Although oleoylethanolamide (OEA) is an endogenous bioactive lipid mediator with lipid overload-associated vasoprotective effects, its benefit in diabetic vascular calcification remains uncharacterized. This study focused on the role of STING1 in mitochondrial dysfunction-mediated calcification and premature VMSC senescence in diabetes and the effects of OEA on these pathological processes. In diabetic in vivo rat/mouse aorta calcification models and an in vitro VSMC calcification model induced by N ε -carboxymethyl-lysine (CML), senescence levels, STING1 signaling activation, and mitochondrial damage markers were significantly augmented; however, these alterations were markedly alleviated by OEA, partially in a nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent manner, and similar anti-calcification and senescence effects were observed in STING1-knockout mice and STING1-knockdown VSMCs. Mechanistically, mitochondrial DNA (mtDNA) damage was aggravated by CML in a reactive oxygen species-dependent manner, followed by mtDNA leakage into the cytosol, contributing to VSMC senescence-associated calcification via STING1 pathway activation. OEA treatment significantly attenuated the aforementioned cytotoxic effects of CML by enhancing cellular antioxidant capacity through the maintenance of Nrf2 translocation to the nucleus. Collectively, targeting STING1, a newly defined VSMC senescence regulator, contributes to anti-vascular calcification effects., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Pulmonary arterial hypertension (PAH) is a progressive disease characterized by vasoconstriction and remodeling of small pulmonary arteries (PAs). Central to the remodeling process is a switch of pulmonary vascular cells to a proliferative, apoptosis-resistant phenotype. Plasminogen activator inhibitors-1 and -2 (PAI-1 and PAI-2) are the primary physiological inhibitors of urokinase-type and tissue-type plasminogen activators (uPA and tPA), but their roles in PAH are unsettled. Here, we report that: 1 ) PAI-1, but not PAI-2, is deficient in remodeled small PAs and in early-passage PA smooth muscle and endothelial cells (PASMCs and PAECs) from subjects with PAH compared with controls; 2 ) PAI-1 -/- mice spontaneously develop pulmonary vascular remodeling associated with upregulation of mTORC1 signaling, pulmonary hypertension (PH), and right ventricle (RV) hypertrophy; and 3 ) pharmacological inhibition of uPA in human PAH PASMCs suppresses proproliferative mTORC1 and SMAD3 signaling, restores PAI-1 levels, reduces proliferation, and induces apoptosis in vitro, and prevents the development of SU5416/hypoxia-induced PH and RV hypertrophy in vivo in mice. These data strongly suggest that downregulation of PAI-1 in small PAs promotes vascular remodeling and PH due to unopposed activation of uPA and consequent upregulation of mTOR and transforming growth factor-β (TGF-β) signaling in PASMCs, and call for further studies to determine the potential benefits of targeting the PAI-1/uPA imbalance to attenuate and/or reverse pulmonary vascular remodeling and PH. NEW & NOTEWORTHY This study identifies a novel role for the deficiency of plasminogen activator inhibitor (PAI)-1 and resultant unrestricted uPA activity in PASMC remodeling and PH in vitro and in vivo, provides novel mechanistic link from PAI-1 loss through uPA-induced Akt/mTOR and TGFβ-Smad3 upregulation to pulmonary vascular remodeling in PH, and suggests that inhibition of uPA to rebalance the uPA-PAI-1 tandem might provide a novel approach to complement current therapies used to mitigate this pulmonary vascular disease.

Increased proliferation and reduced apoptosis of pulmonary artery smooth muscle cells (PASMCs) is recognised as a universal hallmark of pulmonary arterial hypertension (PAH), in part related to the association with reduced pyruvate dehydrogenase (PDH) activity, resulting in decreased oxidative phosphorylation of glucose and increased aerobic glycolysis (Warburg effect). Perhexiline is a well-recognised carnitine palmitoyltransferase-1 (CPT1) inhibitor used in cardiac diseases, which reciprocally increases PDH activity, but is associated with variable pharmacokinetics related to polymorphic variation of the cytochrome P450-2D6 (CYP2D6) enzyme, resulting in the risk of neuro and hepatotoxicity in 'slow metabolisers' unless blood levels are monitored and dose adjusted. We have previously reported that a novel perhexiline fluorinated derivative (FPER-1) has the same therapeutic profile as perhexiline but is not metabolised by CYP2D6, resulting in more predictable pharmacokinetics than the parent drug. We sought to investigate the effects of perhexiline and FPER-1 on PDH flux in PASMCs from patients with PAH. We first confirmed that PAH PASMCs exhibited increased cell proliferation, enhanced phosphorylation of AKT Ser473 , ERK 1/2 Thr202/Tyr204 and PDH-E1α Ser293 , indicating a Warburg effect when compared to healthy PASMCs. Pre-treatment with perhexiline or FPER-1 significantly attenuated PAH PASMC proliferation in a concentration-dependent manner and suppressed the activation of the AKT Ser473 but had no effect on the ERK pathway. Perhexiline and FPER-1 markedly activated PDH (seen as dephosphorylation of PDH-E1α Ser293 ), reduced glycolysis, and upregulated mitochondrial respiration in these PAH PASMCs as detected by Seahorse analysis. However, both perhexiline and FPER-1 did not induce apoptosis as measured by caspase 3/7 activity. We show for the first time that both perhexiline and FPER-1 may represent therapeutic agents for reducing cell proliferation in human PAH PASMCs, by reversing Warburg physiology., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Aortic aneurysm and dissection (AAD) is a cardiovascular disease that poses a severe threat to life and has high morbidity and mortality rates. Clinical and animal-based studies have irrefutably shown that fluoroquinolones, a commonly prescribed antibiotic for treating infections, significantly increase the risk of AAD. Despite this, the precise mechanism by which fluoroquinolones cause AAD remains unclear. Therefore, this study aims to investigate the molecular mechanism and role of Ciprofloxacin definitively-a type of fluoroquinolone antibiotic-in the progression of AAD. Aortic transcriptome data were collected from GEO datasets to detect the genes and pathways expressed differently between healthy donors and AAD patients. Human primary Vascular Smooth Muscle Cells (VSMCs) were isolated from the aorta. After 72 h of exposure to 110ug/ml Ciprofloxacin or 100 nmol/L AngII, either or combined, the senescent cells were identified through SA-β-gal staining. MitoTracker staining was used to examine the morphology of mitochondria in each group. Cellular Reactive Oxygen Species (ROS) levels were measured using MitoSox and DCFH-DA staining. Western blot assay was performed to detect the protein expression level. We conducted an analysis of transcriptome data from both healthy donors and patients with AAD and found that there were significant changes in cellular senescence-related signaling pathways in the latter group. We then isolated and identified human primary VSMCs from healthy donors (control-VSMCs) and patients' (AAD-VSMCs) aortic tissue, respectively. We found that VSMCs from patients exhibited senescent phenotype as compared to control-VSMCs. The higher levels of p21 and p16 and elevated SA-β-gal activity demonstrated this. We also found that pretreatment with Ciprofloxacin promoted angiotensin-II-induced cellular senescence in control-VSMCs. This was evidenced by increased SA-β-gal activity, decreased cell proliferation, and elevation of p21 and p16 protein levels. Additionally, we found that Angiotensin-II (AngII) induced VSMC senescence by promoting ROS generation. We used DCFH-DA and mitoSOX staining to identify that Ciprofloxacin and AngII pretreatment further elevated ROS levels than the vehicle or alone group. Furthermore, JC-1 staining showed that mitochondrial membrane potential significantly declined in the Ciprofloxacin and AngII combination group compared to others. Compared to the other three groups, pretreatment of Ciprofloxacin plus AngII could further induce mitochondrial fission, demonstrated by mitoTracker staining and western blotting assay. Mechanistically, we found that Ciprofloxacin impaired the balance of mitochondrial fission and fusion dynamics in VSMCs by suppressing the phosphorylation of AMPK signaling. This caused mitochondrial dysfunction and ROS generation, thereby elevating AngII-induced cellular senescence. However, treatment with the AMPK activator partially alleviated those effects. Our data indicate that Ciprofloxacin may accelerate AngII-induced VSMC senescence through modulating AMPK/ROS signaling and, subsequently, hasten the progression of AAD., (© 2024. The Author(s).)

Background: Hyponatremia, frequently observed in patients with chronic kidney disease, is associated with increased cardiovascular morbidity and mortality. Hyponatremia or low osmolality induces oxidative stress and cell death, both of which accelerate vascular calcification (VC), a critical phenotype in patients with chronic kidney disease. Whether hyponatremia or low osmolality plays a role in the pathogenesis of VC is unknown., Methods: Human vascular smooth muscle cells (VSMCs) and mouse aortic rings were cultured in various osmotic conditions and calcifying medium supplemented with high calcium and phosphate. The effects of low osmolality on phenotypic change and oxidative stress in the cultured VSMCs were examined. Microarray analysis was conducted to determine the main signaling pathway of osmolality-related VC. The transcellular sodium and calcium ions flux across the VSMCs were visualized by live imaging. Furthermore, the effect of osmolality on calciprotein particles (CPPs) was investigated. Associations between arterial intimal calcification and hyponatremia or low osmolality were examined by a cross-sectional study using human autopsy specimens obtained in the Hisayama Study., Results: Low osmolality exacerbated calcification of the ECM (extracellular matrix) of cultured VSMCs and mouse aortic rings. Oxidative stress and osteogenic differentiation of VSMCs were identified as the underlying mechanisms responsible for low osmolality-induced VC. Microarray analysis showed that low osmolality activated the Rac1 (Ras-related C3 botulinum toxin substrate 1)-Akt (protein kinase B) pathway and reduced NCX1 (Na-Ca exchanger 1) expression. Live imaging showed synchronic calcium ion efflux and sodium ion influx via NCX1 when extracellular sodium ion concentrations were increased. An NCX1 inhibitor promoted calcifying media-induced VC by reducing calcium ion efflux. Furthermore, low osmolality accelerated the generation and maturation steps of CPPs. The cross-sectional study of human autopsy specimens showed that hyponatremia and low osmolality were associated with a greater area of arterial intimal calcification., Conclusions: Hyponatremia and low osmolality promote VC through multiple cellular processes, including the Rac1-Akt pathway activation., Competing Interests: None.

The rupture of intracranial aneurysm (IA) is the primary reason contributing to the occurrence of life-threatening subarachnoid hemorrhages. The oxidative stress-induced phenotypic transformation from the contractile phenotype to the synthetic phenotype of vascular smooth muscle cells (VSMCs) plays a pivotal role in IA formation and rupture. Our study aimed to figure out the role of phoenixin-14 in VSMC phenotypic switching during the pathogenesis of IA by using both cellular and animal models. Primary rat VSMCs were isolated from the Willis circle of male Sprague-Dawley rats. VSMCs were stimulated by hydrogen peroxide (H 2 O 2 ) to establish a cell oxidative damage model. After pretreatment with phoenixin-14 and exposure to H 2 O 2 , VSMC viability, migration, and invasion were examined through cell counting kit-8 (CCK-8), wound healing, and Transwell assays. Intracellular reactive oxygen species (ROS) production in VSMCs was evaluated by using 2',7'-Dichlorofluorescin diacetate (DCFH-DA) fluorescence probes and flow cytometry. Rat IA models were established by ligation of the left common carotid arteries and posterior branches of both renal arteries. The histopathological changes of rat intracranial blood vessels were observed through hematoxylin and eosin staining. The levels of contractile phenotype markers (alpha-smooth muscle actin [α-SMA] and smooth muscle 22 alpha [SM22α]) in VSMCs and rat arterial rings were determined through real-time quantitative polymerase chain reaction (RT-qPCR) and western blot analysis. Our results showed that H 2 O 2 stimulated the production of intracellular ROS and induced oxidative stress in VSMCs, while phoenixin-14 pretreatment attenuated intracellular ROS levels in H 2 O 2 -exposed VSMCs. H 2 O 2 exposure promoted VSMC migration and invasion, which, however, was reversed by phoenixin-14 pretreatment. Besides, phoenixin-14 administration inhibited IA formation and rupture in rat models. The decrease in α-SMA and SM22α levels in H 2 O 2 -exposed VSMCs and IA rat models was antagonized by phoenixin-14. Collectively, phoenixin-14 ameliorates the progression of IA through preventing the loss of the contractile phenotype of VSMCs., (© 2024 Wiley Periodicals LLC.)

Introduction: Vascular smooth muscle cells are important in intimal hyperplasia. Thrombospondin-1 is a matricellular protein involved in the vascular injury response. Statins are cholesterol lowering drugs that have beneficial cardiovascular effects. Statis have been shown to inhibit smooth muscle migration through the mevalonate pathway. This effect is thought to be mediated by small G protein Ras and Rho turnover which requires many hours. While many patients undergoing treatment for vascular disease are on statins, many are not. Thus immediate pretreatment with statins before surgery may be beneficial. We hypothesized that statins have effects independent of the mevalonate pathway and thus have an immediate effect., Methods: Human vascular smooth muscle cells were pretreated for 20 h (long-term) or 20 min (short-term) with fluvastatin, or mevalonolactone plus fluvastatin. Thrombospondin-1-induced migration, activation of p42/p44 extracellular signal-regulated kinase, c-Src, focal adhesion kinase and PI3 kinase was determined. The effect of fluvastatin on thrombospondin-1-induced expression of THBS1 , FOS , HAS2 and TGFB2 was examined., Results: Both treatments inhibited thrombospondin-1-induced chemotaxis back to the control group. Mevalonolactone reversed the long-term statin effect by increasing migration but had no effect on the short-term statin response. p42/p44 extracellular signal-regulated kinase was activated by thrombospondin-1 and both treatments augmented activation. Neither treatment affected c-Src activity, but both inhibited focal adhesion kinase and PI3 kinase activity. Only long-term statin treatment inhibited THBS1 expression while both treatments inhibited FOS and TGFB2 expression. Neither treatment affected HAS2 . FOS knockdown inhibited thrombospondin-1-induced HAS2 but not TGFβ2 gene expression., Conclusion: Long-term fluvastatin inhibited thrombospondin-1-induced chemotaxis through the mevalonate pathway while short-term fluvastatin inhibited chemotaxis through an alternate mechanism. Short-term stains have immediate effects independent of the mevalonate pathway. Acute local treatment with statins followed by longer term therapy may limit the vascular response to injury., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Background: Abdominal aortic aneurysm (AAA) is a complex vascular disorder characterized by the progressive dilation of the abdominal aorta, with a high risk of rupture and mortality. Understanding the cellular interactions and molecular mechanisms underlying AAA development is critical for identifying potential therapeutic targets., Methods: This study utilized datasets GSE197748, GSE164678 and GSE183464 from the GEO database, encompassing bulk and single-cell RNA sequencing data from AAA and control samples. We performed principal component analysis, differential expression analysis, and functional enrichment analysis to identify key pathways involved in AAA. Cell-cell interactions were investigated using CellPhoneDB, focusing on fibroblasts, vascular smooth muscle cells (VSMCs), and macrophages. We further validated our findings using a mouse model of AAA induced by porcine pancreatic enzyme infusion, followed by gene expression analysis and co-immunoprecipitation experiments., Results: Our analysis revealed significant alterations in gene expression profiles between AAA and control samples, with a pronounced immune response and cell adhesion pathways being implicated. Single-cell RNA sequencing data highlighted an increased proportion of pro-inflammatory macrophages, along with changes in the composition of fibroblasts and VSMCs in AAA. CellPhoneDB analysis identified critical ligand-receptor interactions, notably collagen type I alpha 1 chain (COL1A1)/COL1A2-CD18 and thrombospondin 1 (THBS1)-CD3, suggesting complex communication networks between fibroblasts and VSMCs. In vivo experiments confirmed the upregulation of these genes in AAA mice and demonstrated the functional interaction between COL1A1/COL1A2 and CD18., Conclusion: The interaction between fibroblasts and VSMCs, mediated by specific ligand-receptor pairs such as COL1A1/COL1A2-CD18 and THBS1-CD3, plays a pivotal role in AAA pathogenesis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Ferroptosis of vascular smooth muscle cells (VSMCs) is related to the incidence of aortic dissection (AD). Long non-coding RNA (lncRNA) NORAD plays a crucial role in the progression of various diseases. The present study aimed to investigate the effects of NORAD on the ferroptosis of VSMCs and the molecular mechanisms. The expression of NORAD, HUR, and GPX4 was detected using quantitative real-time PCR (qPCR) or western blot. Ferroptosis was evaluated by detecting lactate dehydrogenase (LDH) activity, lipid reactive oxygen species (ROS), malonaldehyde (MDA) content, L-Glutathione (GSH) level, Fe 2+ content, and ferroptosis-related protein levels. The molecular mechanism was assessed using RNA pull-down, RNA-binding protein immunoprecipitation (RIP), and luciferase reporter assay. The histology of aortic tissues was assessed using H&E, elastic Verhoeff-Van Gieson (EVG), and Masson staining assays. The data indicated that NORAD was downregulated in patients with AD and AngII-treated VSMCs. Overexpression of NORAD promoted VSMC growth and inhibited the ferroptosis induced by AngII. Mechanistically, NORAD interacted with HUR, which promoted GPX4 mRNA stability and elevated GPX4 levels. Knockdown of GPX4 abrogated the effects of NORAD on cell growth and ferroptosis of AngII-treated VSMCs. Moreover, METTL3 promoted m6A methylation of NORAD in an YTHDF2-dependent manner. In addition, NORAD attenuated AAD symptoms, incidence, histopathology, inflammation, and ferroptosis in AAD mice. In conclusion, METTL3-mediated NORAD inhibited ferroptosis of VSMCs via the HUR/GPX4 axis and decelerated AAD progression, suggesting that NORAD may be an AD therapeutic target., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Atherosclerosis (AS) is a pivotal pathological basis of cardiovascular and cerebrovascular diseases, and circular RNAs (circRNAs) has been disclosed to exert a vital part in the progression of AS. However, the functions of circ_0004872 in the progression of AS is indistinct. In this context, we aimed to elucidate the role of circ_0004872 and the potential mechanism in AS. The level of circ_0004872, miR-424-5p and fibroblast growth factor receptor substrate 2 (FRS2) was detected using quantitative real-time polymerase chain reaction (qRT-PCR). Cell proliferation was monitored by Cell Counting Kit-8 and 5-ethynyl-2'-deoxyuridine (EDU) assays. The invasion and migration capabilities of VSMCs were tested by transwell assays and wound-healing assay, respectively. Western blot was adopted to check the protein levels of CyclinD1, Vimentin and FRS2. Dual-luciferase reporter and RNA immunoprecipitation assay were executed to manifest the interaction between miR-424-5p and circ_0004872 or FRS2. The level of circ_0004872 was increased in the serum samples of AS patients and ox-LDL-exposed VSMCs. Ox-LDL exposure triggered cell proliferation, invasion and migration ability of VSMCs. depletion of circ_0004872 partly weakened ox-LDL-mediated effects in VSMCs. Mechanistically, circ_0004872 functioned as a sponge of miR-424-5p, and miR-424-5p inhibition partly alleviated circ_0004872 deficiency-mediated influences in VSMCs. Additionally, miR-424-5p interacted with FRS2, and miR-424-5p constrained dysfunction in ox-LDL-stimulated VSMCs via reducing FRS2 level. Notably, circ_0004872 functioned as a sponge of miR-424-5p to elevate FRS2 expression. Circ_0004872 accelerated ox-LDL-induced damage via mediating miR-424-5p/FRS2 axis., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Cellular senescence, macrophages infiltration, and vascular smooth muscle cells (VSMCs) osteogenic transdifferentiation participate in the pathophysiology of vascular calcification in chronic kidney disease (CKD). Senescent macrophages are involved in the regulation of inflammation in pathological diseases. In addition, senescent cells spread senescence to neighboring cells via Interferon-induced transmembrane protein3 (IFITM3). However, the role of senescent macrophages and IFITM3 in VSMCs calcification remains unexplored., Aims: To explore the hypothesis that senescent macrophages contribute to the calcification and senescence of VSMCs via IFITM3., Methods: Here, the macrophage senescence model was established using Lipopolysaccharides (LPS). The VSMCs were subjected to supernatants from macrophages (MCFS) or LPS-induced macrophages (LPS-MCFS) in the presence or absence of calcifying media (CM). Senescence-associated β-galactosidase (SA-β-gal), Alizarin red (AR), immunofluorescent staining, and western blot were used to identify cell senescence and calcification., Results: The expression of IFITM3 was significantly increased in LPS-induced macrophages and the supernatants. The VSMCs transdifferentiated into osteogenic phenotype, expressing higher osteogenic differentiation markers (RUNX2) and lower VSMCs constructive makers (SM22α) when cultured with senescent macrophages supernatants. Also, senescence markers (p16 and p21) in VSMCs were significantly increased by senescent macrophages supernatants treated. However, IFITM3 knockdown inhibited this process., Conclusions: Our study showed that LPS-induced senescence of macrophages accelerated the calcification of VSMCs via IFITM3. These data provide a new perspective linking VC and aging, which may provide clues for diagnosing and treating accelerated vascular aging in patients with CKD.

Smooth muscle cell (SMC) phenotypic modulation, primarily driven by PDGFRβ signaling, is implicated in occlusive cardiovascular diseases. However, the promotive and restrictive regulation mechanism of PDGFRβ and the role of protein tyrosine phosphatase non-receptor type 14 (PTPN14) in neointimal hyperplasia remain unclear. Our study observes a marked upregulation of PTPN14 in SMCs during neointimal hyperplasia. PTPN14 overexpression exacerbates neointimal hyperplasia in a phosphatase activity-dependent manner, while SMC-specific deficiency of PTPN14 mitigates this process in mice. RNA-seq indicates that PTPN14 deficiency inhibits PDGFRβ signaling-induced SMC phenotypic modulation. Moreover, PTPN14 interacts with intracellular region of PDGFRβ and mediates its dephosphorylation on Y692 site. Phosphorylation of PDGFRβ Y692 negatively regulates PDGFRβ signaling activation. The levels of both PTPN14 and phospho-PDGFRβ Y692 are correlated with the degree of stenosis in human coronary arteries. Our findings suggest that PTPN14 serves as a critical modulator of SMCs, promoting neointimal hyperplasia. PDGFRβ Y692 , dephosphorylated by PTPN14, acts as a self-inhibitory site for controlling PDGFRβ activation., (© 2024. The Author(s).)

Restenosis following percutaneous revascularization is a major challenge in patients with insulin resistance and diabetes. Currently, the vascular effects of insulin are not fully understood. In vitro, insulin's effects on endothelial cells (ECs) are beneficial, whereas on vascular smooth muscle cells (SMCs), they are mitogenic. We previously demonstrated a suppressive effect of insulin on neointimal growth under insulin-sensitive conditions that was abolished in insulin-resistant conditions. Here, we aimed to determine the cell-specific effects of insulin on neointimal growth in a model of restenosis under insulin-sensitive and insulin-resistant conditions. Vascular cell-specific insulin receptor (IR)-deficient mice were fed a low-fat diet (LFD) or a high-fat, high-sucrose diet (HFSD) and implanted with an insulin pellet or vehicle prior to femoral artery wire injury. In insulin-sensitive conditions, insulin decreased neointimal growth only in controls. However, under insulin-resistant conditions, insulin had no effect in either control, EC-specific or SMC-specific IR-deficient mice. These data demonstrate that EC and SMC IRs are required for the anti-restenotic effect of insulin in insulin-sensitive conditions and that, in insulin resistance, insulin has no adverse effect on vascular SMCs in vivo.

Background: Vascular smooth muscle cell (VSMC) intimal migration, proliferation, and phenotypic transformation from a contractile to a synthetic state are hallmarks of the progression of atherosclerotic plaques. This study aims to explore the effects of exosomes derived from M2 macrophages (Exo M2 ) on the pathological changes of VSMCs in atherosclerosis (AS)., Methods: Cell Counting Kit-8 (CCK8) and wound healing assays were used to examine the impact of Exo M2 on platelet-derived growth factor-BB (PDGF-BB)-induced VSMC proliferation and migration, respectively. Western blotting was employed to analyze changes in the expression levels of contractile markers (e.g., alpha-smooth muscle actin [α-SMA]) and synthetic ones (e.g., osteopontin [OPN]) in VSMCs with or without Exo M2 treatment. ApoE -⁣/- mice on a high fat diet were utilized to observe the effects of Exo M2 on plaque progression and stability. Serial histopathological analysis was performed to elucidate the cellular mechanisms underlying the atheroprotective effects of Exo M2 ., Results: Compared with controls, Exo M2 significantly inhibited PDGF-BB-induced VSMC proliferation, migration, and phenotypic transformation in vitro . In ApoE -⁣/- mice, Exo M2 treatment led to a marked reduction in plaque size, necrotic core area, the CD68/α-SMA ratio, and matrix metalloproteinase 9 (MMP9) and OPN levels, while enhancing plaque stability., Conclusions: Exo M2 inhibit AS progression by regulating VSMC proliferation, migration, and phenotypic transformation., Competing Interests: The authors declare no conflict of interest., (© 2024 The Author(s). Published by IMR Press.)

Atherosclerosis is a leading cause of cardiovascular diseases (CVDs), often resulting in major adverse cardiovascular events (MACEs), such as myocardial infarction and stroke due to the rupture or erosion of vulnerable plaques. Ferroptosis, an iron-dependent form of cell death, has been implicated in the development of atherosclerosis. Despite its involvement in CVDs, the specific role of ferroptosis in atherosclerotic plaque stability remains unclear. In this study, we confirmed the presence of ferroptosis in unstable atherosclerotic plaques and demonstrated that the ferroptosis inhibitor ferrostatin-1 (Fer-1) stabilizes atherosclerotic plaques in apolipoprotein E knockout (Apoe -/- ) mice. Using bioinformatic analysis combining RNA sequencing (RNA-seq) with single-cell RNA sequencing (scRNA-seq), we identified Yes-associated protein 1 (YAP1) as a potential key regulator of ferroptosis in vascular smooth muscle cells (VSMCs) of unstable plaques. In vitro, we found that YAP1 protects against oxidized low-density lipoprotein (oxLDL)-induced ferroptosis in VSMCs. Mechanistically, YAP1 exerts its anti-ferroptosis effects by regulating the expression of glutaminase 1 (GLS1) to promote the synthesis of glutamate (Glu) and glutathione (GSH). These findings establish a novel mechanism where the inhibition of ferroptosis promotes the stabilization of atherosclerotic plaques through the YAP1/GLS1 axis, attenuating VSMC ferroptosis. Thus, targeting the YAP1/GLS1 axis to suppress VSMC ferroptosis may represent a novel strategy for preventing and treating unstable atherosclerotic plaques., (© 2024 Federation of American Societies for Experimental Biology.)

Microplastics (MPs) have attracted widespread attention because they can lead to combined toxicity by adsorbing heavy metals from the environment. Exposure to lead (Pb), a frequently adsorbed heavy metal by MPs, is common. In the current study, the coexistence of MPs and Pb was assessed in human samples. Then, mice were used as models to examine how co-exposure to MPs and Pb promotes aortic medial degeneration. The results showed that MPs and Pb co-exposure were detected in patients with aortic disease. In mice, MPs and Pb co-exposure promoted the damage of elastic fibers, loss of vascular smooth muscle cells (VSMCs), and release of inflammatory factors. In vitro cell models revealed that co-exposure to MPs and Pb induced excessive reactive oxygen species generation, impaired mitochondrial function, and triggered PANoptosome assembly in VSMCs. These events led to PANoptosis and inflammation through the cAMP/PKA-ROS signaling pathway. However, the use of the PKA activator 8-Br-cAMP or mitochondrial ROS scavenger Mito-TEMPO improved, mitochondrial function in VSMCs, reduced cell death, and inhibited inflammatory factor release. Taken together, the present study provided novel insights into the combined toxicity of MPs and Pb co-exposure on the aorta., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The phenotypic switch of vascular smooth cells (VSMCs) from a contractile to a synthetic state is associated with the development and progression of aortic aneurysm (AA). However, the mechanism underlying this process remains unclear. In this issue of the JCI, Song et al. identified SLC44A2 as a regulator of the phenotypic switch in VSMCs. Inhibition of SLC44A2 facilitated the switch to the synthetic state, contributing to the development of AA. Mechanistically, SLC44A2 interacted with NRP1 and ITGB3 to activate the TGF-β/SMAD signaling pathway, resulting in VSMCs with a contractile phenotype. Furthermore, VSMC-specific SLC44A2 overexpression by genetic or pharmacological manipulation reduced AA in mouse models. These findings suggest the potential of targeting the SLC44A2 signaling pathway for AA prevention and treatment.

Background: Vascular smooth muscle cell (VSMC) proliferation is involved in many types of arterial diseases, including neointima hyperplasia, in which Ca 2+ has been recognized as a key player. However, the physiological role of Ca 2+ release via inositol 1,4,5-trisphosphate receptors (IP 3 Rs) from endoplasmic reticulum in regulating VSMC proliferation has not been well determined., Methods and Results: Both in vitro cell culture models and in vivo mouse models were generated to investigate the role of IP 3 Rs in regulating VSMC proliferation. Expression of all 3 IP 3 R subtypes was increased in cultured VSMCs upon platelet-derived growth factor-BB and FBS stimulation as well as in the left carotid artery undergoing intimal thickening after vascular occlusion. Genetic ablation of all 3 IP 3 R subtypes abolished endoplasmic reticulum Ca 2+ release in cultured VSMCs, significantly reduced cell proliferation induced by platelet-derived growth factor-BB and FBS stimulation, and also decreased cell migration of VSMCs. Furthermore, smooth muscle-specific deletion of all IP 3 R subtypes in adult mice dramatically attenuated neointima formation induced by left carotid artery ligation, accompanied by significant decreases in cell proliferation and matrix metalloproteinase-9 expression in injured vessels. Mechanistically, IP 3 R-mediated Ca 2+ release may activate cAMP response element-binding protein, a key player in controlling VSMC proliferation, via Ca 2+ /calmodulin-dependent protein kinase II and Akt. Loss of IP 3 Rs suppressed cAMP response element-binding protein phosphorylation at Ser133 in both cultured VSMCs and injured vessels, whereas application of Ca 2+ permeable ionophore, ionomycin, can reverse cAMP response element-binding protein phosphorylation in IP 3 R triple knockout VSMCs., Conclusions: Our results demonstrated an essential role of IP 3 R-mediated Ca 2+ release from endoplasmic reticulum in regulating cAMP response element-binding protein activation, VSMC proliferation, and neointima formation in mouse arteries.

Thoracic aortic aneurysm (TAA) is mainly sporadic and with higher incidence in the presence of a bicuspid aortic valve (BAV) for unknown reasons. The lack of drug therapy to delay TAA progression lies in the limited knowledge of pathophysiology. We aimed to identify the molecular hallmarks that differentiate the aortic dilatation associated with BAV and tricuspid aortic valve (TAV). Aortic vascular smooth muscle cells (VSMCs) isolated from sporadic TAA patients with BAV or TAV were analyzed by mass spectrometry. DNA oxidative damage assay and cell cycle profiling were performed in three independent cohorts supporting proteomics data. The alteration of secreted proteins was confirmed in plasma. Stress phenotype, oxidative stress, and enhanced DNA damage response (increased S-phase arrest and apoptosis) were found in BAV-TAA patients. The increased levels of plasma C1QTNF5, LAMA2, THSB3, and FAP confirm the enhanced stress in BAV-TAA. Plasma FAP and BGN point to an increased inflammatory condition in TAV. The arterial wall of BAV patients shows a limited capacity to counteract drivers of sporadic TAA. The molecular pathways identified support the need of differential molecular diagnosis and therapeutic approaches for BAV and TAV patients, showing specific markers in plasma which may serve to monitor therapy efficacy.

Background: The long isoform of the Wnk1 (with-no-lysine [K] kinase 1) is a ubiquitous serine/threonine kinase, but its role in vascular smooth muscle cells (VSMCs) pathophysiology remains unknown., Methods: AngII (angiotensin II) was infused in Apoe -/- to induce experimental aortic aneurysm. Mice carrying an Sm22-Cre allele were cross-bred with mice carrying a floxed Wnk1 allele to specifically investigate the functional role of Wnk1 in VSMCs., Results: Single-cell RNA-sequencing of the aneurysmal abdominal aorta from AngII-infused Apoe -/- mice revealed that VSMCs that did not express Wnk1 showed lower expression of contractile phenotype markers and increased inflammatory activity. Interestingly, WNK1 gene expression in VSMCs was decreased in human abdominal aortic aneurysm. Wnk1 -deficient VSMCs lost their contractile function and exhibited a proinflammatory phenotype, characterized by the production of matrix metalloproteases, as well as cytokines and chemokines, which contributed to local accumulation of inflammatory macrophages, Ly6C hi monocytes, and γδ T cells. Sm22Cre+Wnk1 lox/lox mice spontaneously developed aortitis in the infrarenal abdominal aorta, which extended to the thoracic area over time without any negative effect on long-term survival. AngII infusion in Sm22Cre+Wnk1 lox/lox mice aggravated the aortic disease, with the formation of lethal abdominal aortic aneurysms. Pharmacological blockade of γδ T-cell recruitment using neutralizing anti-CXCL9 (anti-CXC motif chemokine ligand 9) antibody treatment, or of monocyte/macrophage using Ki20227, a selective inhibitor of CSF1 receptor, attenuated aortitis. Wnk1 deletion in VSMCs led to aortic wall remodeling with destruction of elastin layers, increased collagen content, and enhanced local TGF-β (transforming growth factor-beta) 1 expression. Finally, in vivo TGF-β blockade using neutralizing anti-TGF-β antibody promoted saccular aneurysm formation and aorta rupture in Sm22 Cre+ Wnk1 lox/lox mice but not in control animals., Conclusion: Wnk1 is a key regulator of VSMC function. Wnk1 deletion promotes VSMC phenotype switch toward a pathogenic proinflammatory phenotype, orchestrating deleterious vascular remodeling and spontaneous severe aortitis in mice., Competing Interests: None.

Fluoride exposure has been implicated as a potential risk factor for hypertension, but the underlying mechanisms remain unclear. This study investigated the role of the RhoA/ROCK signaling pathway in fluoride-induced hypertension. Male Wistar rats were divided into different groups and exposed to varying concentrations of sodium fluoride (NaF) or sodium chloride (NaCl) via drinking water. The rats' blood pressure was measured, and their aortic tissue was utilized for high-throughput sequencing analysis. Additionally, rat and A7r5 cell models were established using NaF and/or Fasudil. The study evaluated the effects of fluoride exposure on blood pressure, pathological changes in the aorta, as well as the protein/mRNA expression levels of phenotypic transformation indicators (a-SMA, calp, OPN) in vascular smooth muscle cells (VSMCs), along with the RhoA/ROCK signaling pathway (RhoA, ROCK1, ROCK2, MLC/p-MLC). The results demonstrated that fluoride exposure in rats led to increased blood pressure. High-throughput sequencing analysis revealed differential gene expression associated with vascular smooth muscle contraction, with the RhoA/ROCK signaling pathway emerging as a key regulator. Pathological changes in the rat aorta, such as elastic membrane rupture and collagen fiber deposition, were observed following NaF exposure. However, fasudil, a ROCK inhibitor, mitigated these pathological changes. Both in vitro and in vivo models confirmed the activation of the RhoA/ROCK signaling pathway and the phenotypic transformation of VSMCs from a contractile to a synthetic state upon fluoride exposure. Fasudil effectively inhibited the activities of ROCK1 and ROCK2 and attenuated the phenotypic transformation of VSMCs. In conclusion, fluoride has the potential to induce hypertension through the activation of the RhoA/ROCK signaling pathway and phenotypic changes in vascular smooth muscle cells. These results provide new insights into the mechanism of fluoride-induced hypertension., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Cellular communication among different types of vascular cells is indispensable for maintaining vascular homeostasis and preventing atherosclerosis. However, the biological mechanism involved in cellular communication among these cells and whether this biological mechanism can be used to treat atherosclerosis remain unknown. We hypothesized that endothelial autophagy mediates the cellular communication in vascular tissue through exosome-mediated delivery of atherosclerosis-related genes., Methods: Rapamycin and adeno-associated virus carrying Atg7 short hairpin RNA under the Tie (TEK receptor tyrosine kinase) promoter were used to activate and inhibit vascular endothelial autophagy in high-fat diet-fed ApoE -/- mice, respectively. miRNA microarray, in vivo and in vitro experiments, and human vascular tissue were used to explore the effects of endothelial autophagy on endothelial function and atherosclerosis and its molecular mechanisms. Quantitative polymerase chain reaction and miRNA sequencing were performed to determine changes in miRNA expression in exosomes. Immunofluorescence and exosome coculture experiments were conducted to examine the role of endothelial autophagy in regulating the communication between endothelial cells and smooth muscle cells (SMCs) via exosomal miRNA., Results: Endothelial autophagy was inhibited in thoracic aortas of high-fat diet-fed ApoE -/ - mice. Furthermore, rapamycin alleviated high-fat diet-induced atherosclerotic burden and endothelial dysfunction, while endothelial-specific Atg7 depletion aggravated the atherosclerotic burden. miRNA microarray, in vivo and in vitro experiments, and human vascular tissue analysis revealed that miR-204-5p was significantly increased in endothelial cells after high-fat diet exposure, which directly targeted Bcl2 to regulate endothelial cell apoptosis. Importantly, endothelial autophagy activation decreased excess miR-204-5p by loading miR-204-5p into multivesicular bodies and secreting it through exosomes. Moreover, exosomal miR-204-5p can effectively transport to SMCs, alleviating SMC calcification by regulating target proteins such as RUNX2 (runt-related transcription factor 2)., Conclusions: Our study revealed the exosomal pathway by which endothelial autophagy protects atherosclerosis: endothelial autophagy activation transfers miR-204-5p from endothelial cells to SMCs via exosomes, both preventing endothelial apoptosis and alleviating SMC calcification., Registration: URL: https://www.chictr.org.cn/; Unique identifier: ChiCTR2200064155., Competing Interests: None.

This review focuses on the vital function that SIRT1 and other sirtuins play in promoting cellular senescence in vascular smooth muscle cells, which is a key element in the pathogenesis of vascular aging and associated cardiovascular diseases. Vascular aging is a gradual process caused by the accumulation of senescent cells, which results in increased vascular remodeling, stiffness, and diminished angiogenic ability. Such physiological alterations are characterized by a complex interplay of environmental and genetic variables, including oxidative stress and telomere attrition, which affect gene expression patterns and trigger cell growth arrest. SIRT1 has been highlighted for its potential to reduce cellular senescence through modulation of multiple signaling cascades, particularly the endothelial nitric oxide (eNOS)/NO signaling pathway. It also modulates cell cycle through p53 inactivation and suppresses NF-κB mediated expression of adhesive molecules at the vascular level. The study also examines the therapeutic potential of sirtuin modulation in vascular health, identifying SIRT1 and its sirtuin counterparts as potential targets for reducing vascular aging. This study sheds light on the molecular basis of vascular aging and the beneficial effects of sirtuins, paving the way for the development of tailored therapies aimed at enhancing vascular health and prolonging life., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Thoracic aortic aneurysm (TAA) is a serious vascular disease causing the death of elder people. Accumulating studies have reported that circular RNAs (circRNAs) are implicated in the regulation of aortic aneurysms. However, the role of circ_0000595 in the progression of TAA is still unclear., Methods: Quantitative real-time PCR (qRT-PCR) and western blotting were implemented to assess circ_0000595, microRNA (miR)-582-3p, guanine nucleotide-binding protein alpha subunit (ADAM10), PCNA, Bax, and Bcl-2 expression. The proliferation of vascular smooth muscle cells was determined using cell counting kit 8 (CCK-8) and 5-ethynyl-2-deoxyuridine (EdU). Cell apoptosis was measured using flow cytometry, and caspase-3 activity was analyzed using a commercial kit. After bioinformatics analysis, the interaction between miR-582-3p and circ_0000595 or ADAM10 was validated using a dual-luciferase reporter and RNA immunoprecipitation., Results: As compared with controls, TAA tissues and CoCl 2 -induced VSMCs displayed high expression of circ_0000595 and ADAM10, and low expression of miR-582-3p. CoCl 2 treatment evidently suppressed VSMC proliferation and promoted VSMCs apoptosis, and these impacts were reverted by circ_0000595 knockdown. Circ_0000595 acted as a molecular sponge for miR-582-3p, and circ_0000595 silencing-mediated influences in CoCl 2 -induced VSMCs were overturned by miR-582-3p inhibitor. ADAM10 was confirmed as a target gene of miR-582-3p, and miR-582-3p overexpression-induced influence was almost restored by overexpressed ADAM10 in CoCl 2 -induced VSMCs. Besides, circ_0000595 contributed to ADAM10 protein expression by sponging miR-582-3p., Conclusion: Our data verified that circ_0000595 silencing might attenuate CoCl2-mediated impacts in VSMCs by regulating the miR-582-3p/ADAM10 axis, providing new potential roads for treating TAA., Competing Interests: Declaration of conflicting interestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Objective: Proliferation and migration of vascular smooth muscle cells (VSMCs) contribute to vascular remodeling. Asprosin, a newly discovered protein hormone, is involved in metabolic diseases. Little is known about the roles of asprosin in cardiovascular diseases. This study focused on the role and mechanism of asprosin on VSMC proliferation and migration, and vascular remodeling in a rat model of hypertension., Methods and Results: VSMCs were obtained from the aortic media of 8-week-old male Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). Asprosin was upregulated in the VSMCs of SHR. For in vitro studies, asprosin promoted VSMC proliferation and migration of WKY and SHR, and increased Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) activity, NOX1/2/4 protein expressions and superoxide production. Knockdown of asprosin inhibited the proliferation, migration, NOX activity, NOX1/2 expressions and superoxide production in the VSMCs of SHR. The roles of asprosin in promoting VSMC proliferation and migration were not affected by hydrogen peroxide scavenger, but attenuated by superoxide scavenger, selective NOX1 or NOX2 inhibitor. Toll-like receptor 4 (TLR4) was upregulated in SHR, TLR4 knockdown inhibited asprosin overexpression-induced proliferation, migration and oxidative stress in VSMCs of WKY and SHR. Asprosin was upregulated in arteries of SHR, and knockdown of asprosin in vivo not only attenuated oxidative stress and vascular remodeling in aorta and mesentery artery, but also caused a subsequent persistent antihypertensive effect in SHR., Conclusions: Asprosin promotes VSMC proliferation and migration via NOX-mediated superoxide production. Inhibition of endogenous asprosin expression attenuates VSMC proliferation and migration, and vascular remodeling of SHR., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Background and Aims: Activation of vascular smooth muscle cell inflammation is recognised as an important early driver of vascular disease. We have previously identified the let-7 miRNA family as important regulators of inflammation in in vitro and in vivo models of atherosclerosis. Here we investigated a dual statin/let-7d-5p miRNA combination therapy approach to target human aortic SMC (HAoSMC) activation and inflammation., Methods: In vitro studies using primary HAoSMCs were performed to investigate the effects of let-7d-5p miRNA overexpression and inhibition. HAoSMCs were treated with combinations of the inflammatory cytokine tumor necrosis factor-α (TNF-α), and atorvastatin or lovastatin. HAoSMC Bulk RNA-seq transcriptomics of HAoSMCs revealed downstream regulatory networks modulated by let-7d-5p miRNA overexpression and statins. Proteome profiler cytokine array, Western blotting and quantitative PCR analyses were performed on HAoSMCs to validate key findings., Results: Let-7d-5p overexpression significantly attenuated TNF-α-induced upregulation of IL-6, ICAM1, VCAM1, CCL2, CD68, MYOCD gene expression in HAoSMCs (p<0.05). Statins (atorvastatin, lovastatin) significantly attenuated inflammatory gene expression and upregulated Let-7d levels in HAoSMCs (p<0.05). Bulk RNA-seq analysis of a dual Let-7d-5p overexpression/statin therapy in HAoSMCs revealed that let-7d-5p activation and statins converge on key inflammatory pathways (IL-6, IL-1β, TNF-α, IFN-γ). Let-7d-5p overexpression led to reduced expression of the ox-LDL receptor OLR1, and this was associated with lower ox-LDL uptake in HAoSMCs. In silico analysis of smooth muscle cell phenotypic switching shows that overexpression of let-7d-5p in HAoSMCs maintains a contractile phenotype., Conclusions: Targeting the Let-7 network alongside statins can modulate HAoSMC activation and attenuate key inflammatory pathway signals., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Abstract: Aneurysms are localized dilations of blood vessels, which can expand to 50% of the original diameter. They are more common in cardiovascular and cerebrovascular vessels. Rupture is one of the most dangerous complications. The pathophysiology of aneurysms is complex and diverse, often associated with progressive vessel wall dysfunction resulting from vascular smooth muscle cell death and abnormal extracellular matrix synthesis and degradation. Multiple studies have shown that long noncoding RNAs (lncRNAs) play a significant role in the progression of cardiovascular and cerebrovascular diseases. Therefore, it is necessary to find and summarize them. LncRNAs control gene expression and disease progression by regulating target mRNA or miRNA and are biomarkers for the diagnosis and prognosis of aneurysmal cardiovascular and cerebrovascular diseases. This review explores the role, mechanism, and clinical value of lncRNAs in aneurysms, providing new insights for a deeper understanding of the pathogenesis of cardiovascular and cerebrovascular aneurysms., Competing Interests: The authors report no conflicts of interest., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Changes in the extracellular matrix of pulmonary arteries (PAs) are a key aspect of vascular remodeling in pulmonary hypertension (PH). Yet, our understanding of the alterations affecting the proteoglycan (PG) family remains limited. We sought to investigate the expression and spatial distribution of major vascular PGs in PAs from healthy individuals and various PH groups (chronic obstructive pulmonary disease: PH-COPD, pulmonary fibrosis: PH-PF, idiopathic: IPAH). PG regulation, deposition, and synthesis were notably heightened in IPAH, followed by PH-PF, with minor alterations in PH-COPD. Single-cell analysis unveiled cell-type and disease-specific PG regulation. Agrin expression, a basement membrane PG, was increased in IPAH, with PA endothelial cells (PAECs) identified as a major source. PA smooth muscle cells (PASMCs) mainly produced large-PGs, aggrecan and versican, and small-leucine-like proteoglycan (SLRP) biglycan, whereas the major PGs produced by adventitial fibroblasts were SLRP decorin and lumican. In IPAH and PF-PH, the neointima-forming PASMC population increased the expression of all investigated large-PGs and SLRPs, except fibroblast-predominant decorin (DCN). Expression of lumican, versican, and biglycan also positively correlated with collagen 1α1/1α2 expression in PASMCs in patients with IPAH and PH-PF. We demonstrated that transforming growth factor-beta (TGF-β) regulates versican and biglycan expression, indicating their contribution to vessel fibrosis in IPAH and PF-PH. We furthermore show that certain circulating PG levels display a disease-dependent pattern, with increased decorin and lumican across all patient groups, while versican was elevated in PH-COPD and IPAH and biglycan reduced in IPAH. These findings suggest unique compartment-specific PG regulation in different forms of PH, indicating distinct pathological processes. NEW & NOTEWORTHY Idiopathic pulmonary arterial hypertension (IPAH) pulmonary arteries (PAs) displayed the greatest proteoglycan (PG) changes, with PH associated with pulmonary fibrosis (PH-PF) and PH associated with chronic obstructive pulmonary disease (PH-COPD) following. Agrin, an endothelial cell-specific PG, was solely upregulated in IPAH. Among all cells, neo-intima-forming smooth muscle cells (SMCs) displayed the most significant PG increase. Increased levels of circulating decorin, lumican, and versican, mainly derived from SMCs, and adventitial fibroblasts, may serve as systemic indicators of pulmonary remodeling, reflecting perivascular fibrosis and neointima formation.

Background: Exposure to ionizing radiation has been linked to cardiovascular diseases. However, the impact of moderate doses of radiation on abdominal aortic aneurysm (AAA) remains unknown., Methods: Angiotensin II-infused Apoe-/- mice were irradiated (acute, 1 Gray) either 3 days before (Day-3) or 1 day after (Day+1) pomp implantation. Isolated primary aortic vascular smooth muscle cells (VSMCs) were irradiated (acute 1 Gray) for mechanistic studies and functional testing in vitro., Results: Day-3 and Day+1 irradiation resulted in a significant reduction in aorta dilation (Control: 1.39+/-0.12; Day-3: 1.12+/-0.11; Day+1: 1.15+/-0.08 mm, P<0.001) and AAA incidence (Control: 81.0%; Day-3: 33.3%, Day+1: 53.3%) compared to the non-irradiated group. Day-3 and Day+1 irradiation led to an increase in collagen content in the adventitia (Thickness control: 23.64+/-2.9; Day-3: 54.39+/-15.5; Day+1 37.55+/-10.8 mm, P = 0.006). However, the underlying protective mechanisms were different between Day-3 and Day+1 groups. Irradiation before Angiotensin II (AngII) infusion mainly modulated vascular smooth muscle cell (VSMC) phenotype with a decrease in contractile profile and enhanced proliferative and migratory activity. Irradiation after AngII infusion led to an increase in macrophage content with a local anti-inflammatory phenotype characterized by the upregulation of M2-like gene and IL-10 expression., Conclusion: Moderate doses of ionizing radiation mitigate AAA either through VSCM phenotype or inflammation modulation, depending on the time of irradiation., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Riazi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Neoatherosclerosis (NA) within stents has become an important clinical problem after coronary artery stent implantation. In-stent restenosis and in-stent thrombosis are the two major complications following coronary stent placement and seriously affect patient prognosis. As the common pathological basis of these two complications, NA plaques, unlike native atherosclerotic plaques, often grow around residual oxidized lipids and stent struts. The main components are foam cells formed by vascular smooth muscle cells (VSMCs) engulfing oxidized lipids at lipid residue sites. Current research mainly focuses on optical coherence tomography (OCT) and intravascular ultrasound (IVUS), but the specific pathogenesis of NA is still unclear. A thorough understanding of the pathogenesis and pathological features of NA provides a theoretical basis for clinical treatment. This article reviews the previous research of our research group and the current situation of domestic and foreign research., (© 2024. Huazhong University of Science and Technology.)

Vascular calcification in diabetes patients is a major independent risk factor for developing diabetic cardiovascular complications. However, the mechanisms by which diabetes leads to vascular calcification are complex and not yet fully understood. Our previous study revealed that miR-32-5p is a potential new diagnostic marker for coronary artery calcification. In this study, we found that miR-32-5p levels were significantly greater in the plasma of type 2 diabetes patients with coronary artery calcification and were positively correlated with the coronary artery calcification score. In type 2 diabetic mice, miR-32-5p levels were also elevated in the aorta, and knockout of miR-32-5p inhibited the osteogenic differentiation of vascular smooth muscle cells in vivo. Furthermore, overexpression of miR-32-5p promoted vascular smooth muscle cell calcification, while antagonism of miR-32-5p inhibited vascular smooth muscle cell calcification under high-glucose conditions. GATA binding protein 6 (GATA6) was identified as the key target gene through which miR-32-5p promotes vascular smooth muscle cell calcification. Overexpression of GATA6 antagonized the effects of miR-32-5p on vascular calcification. Additionally, high glucose levels were shown to induce the upregulation of miR-32-5p by activating CCAAT/enhancer binding protein beta (CEBPB). These results suggest that miR-32-5p is an important procalcification factor in vascular calcification associated with type 2 diabetes and identify the CEBPB/miR-32-5p/GATA6 axis as a potential biomarker and therapeutic target for preventing and treating vascular calcification in type 2 diabetes., Competing Interests: Declaration of Competing Interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background: Remodeling of vascular smooth muscle cells (VSMCs), as a pathological hallmark of cardiovascular diseases, is related to the molecular rewiring of Calcium signaling, which induces upregulation of stromal interaction molecule (STIM) proteins. This study analyzed the influence of STIM1 proteins on the remodeling of VSMCs in atherosclerosis (AS)., Methods: After oxidized low-density lipoprotein (ox-LDL) treatment and transfection, VSMC viability, migration, and invasion were separately measured using Cell Counting Kit-8, Scratch assay, and Transwell assay. An animal AS model was constructed, and histological analysis via hematoxylin-eosin staining was conducted on the aorta., Results: Ox-LDL promoted expression of STIM1 and Orai calcium release-activated calcium modulator 1 (Orai1). STIM1 or Orai1 downregulation suppressed viability, migration, invasion, and phenotypic switching of ox-LDL-treated VSMCs, whereas STIM1 or Orai1 upregulation had opposite effects. Orai1 level was upregulated by STIM1 overexpression. Orai1 silencing reversed the effects of STIM1 overexpression in VSMCs. STIM1 deficiency alleviated AS and regulated expression of Orai1 and phenotypic switch-related factors in vivo ., Conclusion: STIM1 deficiency suppresses viability, migration, invasion, and phenotypic switching of ox-LDL-induced VSMCs and alleviates AS by inhibiting Orai1.

Purpose of Review: Our work is to establish more distinct association between specific stress and vascular smooth muscle cells (VSMCs) phenotypes to alleviate atherosclerotic plaque burden and delay atherosclerosis (AS) progression., Recent Finding: In recent years, VSMCs phenotypic transition has received significant interests. Different stresses were found to be associated with VSMCs phenotypic transition. However, the explicit correlation between VSMCs phenotype and specific stress has not been elucidated clearly yet. We discover that VSMCs phenotypic transition, which is widely involved in the progression of AS, is associated with specific stress. We discuss approaches targeting stresses to intervene VSMCs phenotypic transition, which may contribute to develop innovative therapies for AS., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Vascular calcification (VC) is a characteristic feature in patients with diabetes mellitus (DM) and is closely associated with the osteogenic differentiation of vascular smooth muscle cells (VSMCs). Ubiquitin-Specific Protease 10 (USP10) has been shown to regulate multiple cellular processes; however, its relationship with diabetic VC remains unclear. This study aims to elucidate the role of USP10 in VC development and the underlying regulatory mechanisms. Nε-carboxymethyl lysine (CML) was significantly increased in calcified ateries from diabetic atherosclerosis ApoE -/- mice fed with high-fat diets. CML downregulated USP10 expression in VSMCs and calcified mice coronary arteries, as assessd by Western blotting, RT-qPCR,immunofluorescence and immunohistochemistry. Loss-and gain-of-function experiments were conducted both in vitro and in vivo to verify the biological functions of USP10. Ectopic expression of USP10 mitigated the severity of VC. With regard to the mechanism, the interaction between USP10 and AMPKα was investigated through double-label immunofluorescence and Co-immunoprecipitation. In vitro ubiquitination assay revealed that USP10 was capable of mediating AMPKα ubiquitination and caused increased AMPKα phosphorylation level at Thr172. Moreover, the anticalcification effect of USP10 was reversed by pharmacological inhibition of AMPK signaling pathway. The current fundings suggest an important role of USP10 in diabetic VC progression, at least in part, via mediating the ubiquitination and activation of AMPKα. USP10 may serve as a novel therapeutic target for the treatment of diabetic VC., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: Vascular smooth muscle cells (VSMCs) are highly plastic. Vessel injury induces a phenotypic transformation from differentiated to dedifferentiated VSMCs, which involves reduced expression of contractile proteins and increased production of extracellular matrix and inflammatory cytokines. This transition plays an important role in several cardiovascular diseases such as atherosclerosis, hypertension, and aortic aneurysm. TGF-β (transforming growth factor-β) is critical for VSMC differentiation and to counterbalance the effect of dedifferentiating factors. However, the mechanisms controlling TGF-β activity and VSMC phenotypic regulation under in vivo conditions are poorly understood. The extracellular matrix protein TN-X (tenascin-X) has recently been shown to bind TGF-β and to prevent it from activating its receptor., Methods: We studied the role of TN-X in VSMCs in various murine disease models using tamoxifen-inducible SMC-specific knockout and adeno-associated virus-mediated knockdown., Results: In hypertensive and high-fat diet-fed mice, after carotid artery ligation as well as in human aneurysmal aortae, expression of Tnxb , the gene encoding TN-X, was increased in VSMCs. Mice with smooth muscle cell-specific loss of TN-X (SMC-Tnxb-KO) showed increased TGF-β signaling in VSMCs, as well as upregulated expression of VSMC differentiation marker genes during vascular remodeling compared with controls. SMC-specific TN-X deficiency decreased neointima formation after carotid artery ligation and reduced vessel wall thickening during Ang II (angiotensin II)-induced hypertension. SMC-Tnxb-KO mice lacking ApoE showed reduced atherosclerosis and Ang II-induced aneurysm formation under high-fat diet. Adeno-associated virus-mediated SMC-specific expression of short hairpin RNA against Tnxb showed similar beneficial effects. Treatment with an anti-TGF-β antibody or additional SMC-specific loss of the TGF-β receptor reverted the effects of SMC-specific TN-X deficiency., Conclusions: In summary, TN-X critically regulates VSMC plasticity during vascular injury by inhibiting TGF-β signaling. Our data indicate that inhibition of vascular smooth muscle TN-X may represent a strategy to prevent and treat pathological vascular remodeling., Competing Interests: None.

Background: Intimal hyperplasia is the response to endothelial injury. Platelet-derived growth factor is released early and favors the formation of intimal hyperplasia. Although multiple treatments, from open surgery to endovascular techniques, have been used they remain controversial. There is currently interest in developing pharmacological strategies to address this pathology. Local vascular inflammation induced by vessel barotrauma generates intimal hyperplasia due to mechanical stress over the venous endothelium. Cilostazol is a selective phosphodiesterase type 3 (PDE3) selective inhibitor with a regulatory effect over intimal hyperplasia. The objective was to investigate cilostazol's role in inhibiting smooth muscle cell proliferation due to changes in the expression and release of PDGF-BB isoform and the effect on developing IH using an experimental model of vascular barotrauma (balloon-induced injury model)., Methods: We included 12 New Zealand rabbits. The balloon-induced injury model (BIIM) and experimental group cilostazol (20 mg/kg/day) included 6 rabbits each. Contralateral veins from 6 rabbits used in BIIM model has been taken as control group. We measured and compared the expression of PDGF-BB and the development of IH. A pathologist board chooses a PDGFRα antibody to localized its expression by immunohistochemistry analysis. Subsequently, using an automated immunohistochemical staining machine, the PDGFR expression was evaluated using a Zeiss Primo Star 4 light microscope., Results: The measurement obtained in the intimal layer was: 126.12 μm2 in the CG, 232 μm2 in the BIIM group, and 178 μm2 in the EG. A statistically significant difference was observed. Baseline serum concentrations of PDGF-BB in the BIIM group were 0.22 pg/mL. At 12 h 0.42 pg/mL, and 0.17 pg/mL at seven days. In the experimental group, the basal levels were 0.33 pg/mL. With the use of cilostazol, a lower peak was obtained at 12 h (0.08 pg/mL). This difference was statistically significant., Conclusions: Cilostazol induced a significant reduction of IH caused by barotrauma in the venous endothelium, which correlates with decrease in the PDGF-BB in serum. This could be attributed to the pharmacologic effect on PDGFR expression., Competing Interests: Declaration of conflicting interestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Pulmonary hypertension (PH) arises from increased pulmonary vascular resistance due to contraction and remodeling of the pulmonary arteries. The structural changes include thickening of the smooth muscle layer from increased proliferation and resistance to apoptosis. The mechanisms underlying apoptosis resistance in PH are not fully understood. In cancer cells, high expression of aquaporin 1 (AQP1), a water channel, is associated with apoptosis resistance. We showed AQP1 protein was expressed in pulmonary arterial smooth muscle cells (PASMCs) and upregulated in preclinical PH models. In this study, we used PASMCs isolated from control male rats and the SU5416 plus hypoxia (SuHx) model to test the role of AQP1 in modulating susceptibility to apoptosis. We found the elevated level of AQP1 in PASMCs from SuHx rats was necessary for resistance to apoptosis and that apoptosis resistance could be conferred by increasing AQP1 in control PASMCs. In exploring the downstream pathways involved, we found AQP1 levels influence the expression of Bcl-2, with enhanced AQP1 levels corresponding to increased Bcl-2 expression, reducing the ratio of BAX to Bcl-2, consistent with apoptosis resistance. These results provide a mechanism by which AQP1 can regulate PASMC fate., (© 2024 The Author(s). Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

Vulnerable atherosclerotic plaque rupture, the leading cause of fatal atherothrombotic events, is associated with an increased risk of mortality worldwide. Peroxisome proliferator-activated receptor delta (PPARδ) has been shown to modulate vascular smooth muscle cell (SMC) phenotypic switching, and, hence, atherosclerotic plaque stability. Melatonin reportedly plays a beneficial role in cardiovascular diseases; however, the mechanisms underlying improvements in atherosclerotic plaque vulnerability remain unknown. In this study, we assessed the role of melatonin in regulating SMC phenotypic switching and its consequential contribution to the amelioration of atherosclerotic plaque vulnerability and explored the mechanisms underlying this process. We analyzed features of atherosclerotic plaque vulnerability and markers of SMC phenotypic transition in high-cholesterol diet (HCD)-fed apolipoprotein E knockout (ApoE -/- ) mice and human aortic SMCs (HASMCs). Melatonin reduced atherosclerotic plaque size and necrotic core area while enhancing collagen content, fibrous cap thickness, and smooth muscle alpha-actin positive cell coverage on the plaque cap, which are all known phenotypic characteristics of vulnerable plaques. In atherosclerotic lesions, melatonin significantly decreased the synthetic SMC phenotype and KLF4 expression and increased the expression of PPARδ, but not PPARα and PPARγ, in HCD-fed ApoE -/- mice. These results were subsequently confirmed in the melatonin-treated HASMCs. Further analysis using PPARδ silencing and immunoprecipitation assays revealed that PPARδ plays a role in the melatonin-induced SMC phenotype switching from synthetic to contractile. Collectively, we provided the first evidence that melatonin mediates its protective effect against plaque destabilization by enhancing PPARδ-mediated SMC phenotypic switching, thereby indicating the potential of melatonin in treating atherosclerosis., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Atherosclerosis (AS) is a dominant pathological basis of cardiovascular disease. Circular RNAs (circRNAs) have been proposed to have crucial functions in regulating pathological progressions of AS. Hence, the aim of this study was to investigate the potential function of circ&#95;0090231 in AS progression. Oxidized low densitylipoprotein (ox-LDL)-challenged vascular smooth muscle cells (VSMCs) were used for in vitro functional analysis. Levels of genes and proteins were measured by qRT-PCR and Western blot. The proliferation, migration and invasion were assessed using cell counting kit-8, 5-ethynyl-2'-deoxyuridine, and transwell assays. The interaction between miR-942-5p and circ&#95;0090231 or PPM1B (Protein Phosphatase, Mg 2+ /Mn 2+  Dependent 1B) was evaluated by dual-luciferase reporter and pull-down assays. Circ&#95;0090231 is a stable circRNA, and was increased in the serum of AS patients and ox-LDL-challenged VSMCs. Functionally, silencing of circ&#95;0090231 could reverse ox-LDL-induced proliferation, migration and invasion in VSMCs. Mechanistically, circ&#95;0090231 directly targeted miR-942-5p, and PPM1B was a target of miR-942-5p. Besides, circ&#95;0090231 sequestered miR-942-5p to release PPM1B expression, suggesting the circ&#95;0090231/miR-942-5p/PPM1B axis. Further rescue experiments showed that miR-942-5p inhibition or ectopic overexpression of PPM1B dramatically attenuated the suppressing influences of circ&#95;0090231 knockdown on VSMC proliferative, migratory and invasive abilities under ox-LDL treatment. Silencing of circ&#95;0090231 could reverse ox-LDL-induced proliferation, migration and invasion in VSMCs via miR-942-5p/PPM1B axis, providing a theoretical basis for elucidating the mechanism of AS process., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background and Aims: Circular RNA (circRNA) is closely related to atherosclerosis (AS) incidence and progression, but its regulatory mechanism in AS needs further elucidation. AS development is significantly influenced by abnormal vascular smooth muscle cells (VSMCs) growth and migration. This study explored the potential protein role of circLARP1B in VSMC proliferation and migration., Methods: We performed whole-transcriptome sequencing in human normal arterial intima and advanced atherosclerotic plaques to screen for differentially expressed circRNAs. The sequencing results were combined with database analysis to screen for circRNAs with coding ability. Real-time quantitative polymerase chain reaction was utilized to assess circLARP1B expression levels in atherosclerotic plaque tissues and cells. circLARP1B-243aa function and pathway in VSMCs growth and migration were studied by scratch, transwell, 5-ethynyl-2'-deoxyuridine, cell counting kit-8, and Western blot experiments., Results: We found that circLARP1B was downregulated in atherosclerotic plaque tissue and promoted the proliferation and migration of VSMCs. circLARP1B encodes a novel protein with a length of 243 amino acids. Through functional experiments, we confirmed the role of circLARP1B-243aa in enhancing VSMCs migration and proliferation. Mechanistically, circLARP1B-243aa promotes VSMCs migration and growth by upregulating phosphodiesterase 4C to inhibit the cyclic adenosine monophosphate signaling pathway., Conclusions: Our results suggested that circLARP1B could promote VSMCs growth and migration through the encoded protein circLARP1B-243aa. Therefore, it could be a treatment target and biomarker for AS., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: The dysfunction of human vascular smooth cells (hVSMCs) is significantly connected to the development of intracranial aneurysms (IAs). By suppressing the activity of microRNAs (miRNAs), circular RNAs (circRNAs) participate in IA pathogenesis. Nevertheless, the role of hsa&#95;circ&#95;0008571 in IAs remains unclear., Methods: circRNA sequencing was used to identify circRNAs from human IA tissues. To determine the function of circ&#95;0008571, Transwell, wound healing, and cell proliferation assays were conducted. To identify the target of circ&#95;0008571, the analyses of CircInteractome and TargetScan, as well as the luciferase assay were carried out. Furthermore, circ&#95;0008571 knockdown and over-expression were performed to investigate its functions in IA development and the underlying molecular mechanisms., Results: Both hsa&#95;circ&#95;0008571 and Integrin beta 8 (ITGB8) were downregulated, while miR-145-5p transcription was elevated in the aneurysm wall of IAs patients compared to superficial temporal artery tissues. In vitro, cell migration and growth were dramatically suppressed after hsa&#95;circ&#95;0008571 overexpression. Mechanistically, has&#95;circ&#95;0008571 could suppress miR-145-5p activity by direct sponging. Moreover, we found that ITGB8 expression and the activation of the TGF-β-mediated signaling pathway were significantly enhanced., Conclusion: The hsa&#95;circ&#95;0008571-miR-145-5p-ITGB8 axis plays an essential role in IA progression., Competing Interests: Declaration of competing interest The authors have no relevant financial or non-financial interests to disclose., (Copyright © 2024 Elsevier B.V. All rights reserved.)

In this study, we investigated the protective role of Mzb1 in atherosclerotic plaque vulnerability. To explore the impact of Mzb1, we analyzed Mzb1 expression, assessed apoptosis, and evaluated mitochondrial function in atherosclerosis (AS) mouse models and human vascular smooth muscle cells (HVSMCs). We observed a significant decrease in Mzb1 expression in AS mouse models and ox-LDL-treated HVSMCs. Downregulation of Mzb1 increased ox-LDL-induced apoptosis and cholesterol levels of HVSMCs, while Mzb1 overexpression alleviated these effect. Mzb1 was found to enhance mitochondrial function, as evidenced by restored ATP synthesis, mitochondrial membrane potential, and reduced mtROS production. Moreover, Mzb1 overexpression attenuated atherosclerotic plaque vulnerability in ApoE -/- mice. Our findings suggest that Mzb1 overexpression regulates the AMPK/SIRT1 signaling pathway, leading to the attenuation of atherosclerotic plaque vulnerability. This study provides compelling evidence for the protective effect of Mzb1 on atherosclerotic plaques by alleviating apoptosis and modulating mitochondrial function in ApoE -/- mice., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Atherosclerosis is driven by the infiltration of the arterial intima by diverse immune cells and smooth muscle cells (SMCs). CD8 + T cells promote lesion growth during atherosclerotic lesion development, but their role in advanced atherosclerosis is less clear. Here, we studied the role of CD8 + T cells and their effects on SMCs in established atherosclerosis., Methods: CD8 + T cells were depleted in (SMC reporter) low-density lipoprotein receptor-deficient ( Ldlr -/- ) mice with established atherosclerotic lesions. Atherosclerotic lesion formation was examined, and single-cell RNA sequencing of aortic SMCs and their progeny was performed. Additionally, coculture experiments with primary aortic SMCs and CD8 + T cells were conducted., Results: Although we could not detect differences in atherosclerotic lesion size, an increased plaque SMC content was noted in mice after CD8 + T-cell depletion. Single-cell RNA sequencing of aortic lineage-traced SMCs revealed contractile SMCs and a modulated SMC cluster, expressing macrophage- and osteoblast-related genes. CD8 + T-cell depletion was associated with an increased contractile but decreased macrophage and osteoblast-like gene signature in this modulated aortic SMC cluster. Conversely, exposure of isolated aortic SMCs to activated CD8 + T cells decreased the expression of genes indicative of a contractile SMC phenotype and induced a macrophage and osteoblast-like cell state. Notably, CD8 + T cells triggered calcium deposits in SMCs under osteogenic conditions. Mechanistically, we identified transcription factors highly expressed in modulated SMCs, including Runx1 , to be induced by CD8 + T cells in cultured SMCs in an IFNγ (interferon-γ)-dependent manner., Conclusions: We here uncovered CD8 + T cells to control the SMC phenotype in atherosclerosis. CD8 + T cells promote SMC dedifferentiation and drive SMCs to adopt features of macrophage-like and osteoblast-like, procalcifying cell phenotypes. Given the critical role of SMCs in atherosclerotic plaque stability, CD8 + T cells could thus be explored as therapeutic target cells during lesion progression., Competing Interests: None.

Background: The N6-methyladenosine (m 6 A) modification of RNA and its regulators have important roles in the pathogenesis of pulmonary hypertension (PH). Ythdf2 (YTH N6-methyladenosine RNA binding protein 2) is best known for its role in degrading m 6 A-modified mRNAs such as Hmox1 mRNA, which leads to alternative activation of macrophages in PH. Recent studies have also linked Ythdf2 to the proliferation of pulmonary artery smooth muscle cells (PASMCs). However, its specific roles in PASMCs and downstream targets during the development of PH remain unclear., Methods: The expression and biological function of Ythdf2 in PASMCs were investigated in human and experimental models of PH. Smooth muscle cell-specific Ythdf2 -deficient mice were used to assess the roles of Ythdf2 in PASMCs in vivo. Proteomic analysis, m 6 A sequencing, and RNA immunoprecipitation analysis were used to screen for potential downstream targets., Results: Ythdf2 was significantly upregulated in human and rodent PH-PASMCs, and smooth muscle cell-specific Ythdf2 deficiency ameliorated PASMC proliferation, right ventricular hypertrophy, pulmonary vascular remodeling, and PH development. Higher expression of Ythdf2 promoted PASMC proliferation and PH by paradoxically stabilizing Myadm mRNA in an m 6 A-dependent manner. Loss of Ythdf2 decreased the expression of Myadm in PASMCs and pulmonary arteries, both in vitro and in vivo. Additionally, silencing Myadm inhibited the Ythdf2 -dependent hyperproliferation of PASMCs by upregulating the cell cycle kinase inhibitor p21., Conclusions: We have identified a novel mechanism where the increased expression of Ythdf2 stimulates PH-PASMC proliferation through an m 6 A/Myadm/p21 pathway. Strategies targeting Ythdf2 in PASMCs might be useful additions to the therapeutic approach to PH., Competing Interests: None.