Aberrant splicing of CaV1.2 calcium channel induced by decreased Rbfox1 enhances arterial constriction during diabetic hyperglycemia.

Diabetic hyperglycemia induces dysfunctions of arterial smooth muscle, leading to diabetic vascular complications. The CaV1.2 calcium channel is one primary pathway for Ca2+ influx, which initiates vasoconstriction. However, the long-term regulation mechanism(s) for vascular CaV1.2 functions under hyperglycemic condition remains unknown. Here, Sprague-Dawley rats fed with high-fat diet in combination with low dose streptozotocin and Goto-Kakizaki (GK) rats were used as diabetic models. Isolated mesenteric arteries (MAs) and vascular smooth muscle cells (VSMCs) from rat models were used to assess K+-induced arterial constriction and CaV1.2 channel functions using vascular myograph and whole-cell patch clamp, respectively. K+-induced vasoconstriction is persistently enhanced in the MAs from diabetic rats, and CaV1.2 alternative spliced exon 9* is increased, while exon 33 is decreased in rat diabetic arteries. Furthermore, CaV1.2 channels exhibit hyperpolarized current-voltage and activation curve in VSMCs from diabetic rats, which facilitates the channel function. Unexpectedly, the application of glycated serum (GS), mimicking advanced glycation end-products (AGEs), but not glucose, downregulates the expression of the splicing factor Rbfox1 in VSMCs. Moreover, GS application or Rbfox1 knockdown dynamically regulates alternative exons 9* and 33, leading to facilitated functions of CaV1.2 channels in VSMCs and MAs. Notably, GS increases K+-induced intracellular calcium concentration of VSMCs and the vasoconstriction of MAs. These results reveal that AGEs, not glucose, long-termly regulates CaV1.2 alternative splicing events by decreasing Rbfox1 expression, thereby enhancing channel functions and increasing vasoconstriction under diabetic hyperglycemia. This study identifies the specific molecular mechanism for enhanced vasoconstriction under hyperglycemia, providing a potential target for managing diabetic vascular complications.

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

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

Extracellular glucose and dysfunctional insulin receptor signaling independently upregulate arterial smooth muscle TMEM16A expression.

Diabetes alters the function of ion channels responsible for regulating arterial smooth muscle membrane potential, resulting in vasoconstriction. Our prior research demonstrated an elevation of TMEM16A in diabetic arteries. Here, we explored the mechanisms involved in Transmembrane protein 16A (TMEM16A) gene expression. Our data indicate that a Snail-mediated repressor complex regulates arterial TMEM16A gene transcription. Snail expression was reduced in diabetic arteries while TMEM16A expression was upregulated. The TMEM16A promoter contained three canonical E-box sites. Electrophoretic mobility and super shift assays revealed that the -154 nt E-box was the binding site of the Snail repressor complex and binding of the repressor complex decreased in diabetic arteries. High glucose induced a biphasic contractile response in pressurized nondiabetic mouse hindlimb arteries incubated ex vivo. Hindlimb arteries incubated in high glucose also showed decreased phospho-protein kinase D1 and TMEM16A expression. In hindlimb arteries from nondiabetic mice, administration of a bolus dose of glucose activated protein kinase D1 signaling to induce Snail degradation. In both in vivo and ex vivo conditions, Snail expression exhibited an inverse relationship with the expression of protein kinase D1 and TMEM16A. In diabetic mouse arteries, phospho-protein kinase D1 increased while Akt2 and pGSK3ß levels declined. These results indicate that in nondiabetic mice, high glucose triggers a transient deactivation of the Snail repressor complex to increase arterial TMEM16A expression independently of insulin signaling. Conversely, insulin resistance activates GSK3ß signaling and enhances arterial TMEM16A channel expression. These data have uncovered the Snail-mediated regulation of arterial TMEM16A expression and its dysfunction during diabetes.NEW & NOTEWORTHY The calcium-activated chloride channel, TMEM16A, is upregulated in the diabetic vasculature to cause increased vasoconstriction. In this paper, we have uncovered that the TMEM16A gene expression is controlled by a Snail-mediated repressor complex that uncouples with both insulin-dependent and -independent pathways to allow for upregulated arterial protein expression thereby causing vasoconstriction. The paper highlights the effect of short- and long-term glucose-induced dysfunction of an ion channel expression as a causative factor in diabetic vascular disease.

Huanglian Jiedu decoction inhibits vascular smooth muscle cell-derived foam cell formation by activating autophagy via suppressing P2RY12.

ETHNOPHARMACOLOGICAL RELEVANCE: Huanglian Jiedu Decoction (HLJDD) is a Chinese medicine with a long history of therapeutic application. It is widely used in treating atherosclerosis (AS) in Chinese medicine theory and clinical practice. However, the mechanism of HLJDD in treating AS remains unclear. AIM OF THE STUDY: To investigate the efficacy and mechanism of HLJDD in treating AS. MATERIALS AND METHODS: AS was induced on high-fat diet-fed ApoE-/- mice, with the aorta pathological changes evaluated with lipid content and plaque progression. In vitro, foam cells were induced by subjecting primary mouse aortic vascular smooth muscle cells (VSMCs) to oxLDL incubation. After HLJDD intervention, VSMCs were assessed with lipid stack, apoptosis, oxidative stress, and the expression of foam cell markers. The effects of P2RY12 were tested by adopting clopidogrel hydrogen sulfate (CDL) in vivo and transfecting P2RY12 over-expressive plasmid in vitro. Autophagy was inhibited by Chloroquine or transfecting siRNA targeting ATG7 (siATG7). The mechanism of HLJDD treating atherosclerosis was explored using network pharmacology and validated with molecular docking and co-immunoprecipitation. RESULTS: HLJDD exhibited a dose-dependent reduction in lipid deposition, collagen loss, and necrosis within plaques. It also reversed lipid accumulation and down-regulated the expression of foam cell markers. P2RY12 inhibition alleviated AS, while P2RY12 overexpression enhanced foam cell formation and blocked the therapeutic effects of HLJDD. Network pharmacological analysis suggested that HLJDD might mediate PI3K/AKT signaling pathway-induced autophagy. P2RY12 overexpression also impaired autophagy. Similarly, inhibiting autophagy counteracted the effect of CDL, exacerbated AS in vivo, and promoted foam cell formation in vitro. However, HLJDD treatment mitigated these detrimental effects by suppressing the PI3K/AKT signaling pathway. Immunofluorescence and molecular docking revealed a high affinity between P2RY12 and PIK3CB, while co-immunoprecipitation assays illustrated their interaction. CONCLUSIONS: HLJDD inhibited AS in vivo and foam cell formation in vitro by restoring P2RY12/PI3K/AKT signaling pathway-suppressed autophagy. This study is the first to reveal an interaction between P2RY12 and PI3K3CB.

The role of macrophage polarization in vascular calcification.

Vascular calcification is an important factor in the high morbidity and mortality of Cardiovascular and cerebrovascular diseases. Vascular damage caused by calcification of the intima or media impairs the physiological function of the vascular wall. Inflammation is a central factor in the development of vascular calcification. Macrophages are the main inflammatory cells. Dynamic changes of macrophages with different phenotypes play an important role in the occurrence, progression and stability of calcification. This review focuses on macrophage polarization and the relationship between macrophages of different phenotypes and calcification environment, as well as the mechanism of interaction, it is considered that macrophages can promote vascular calcification by releasing inflammatory mediators and promoting the osteogenic transdifferentiation of smooth muscle cells and so on. In addition, several therapeutic strategies aimed at macrophage polarization for vascular calcification are described, which are of great significance for targeted treatment of vascular calcification.

Oxidized phospholipid POVPC contributes to vascular calcification by triggering ferroptosis of vascular smooth muscle cells.

Vascular calcification is an actively regulated biological process resembling bone formation, and osteogenic differentiation of vascular smooth muscle cells (VSMCs) plays a crucial role in this process. 1-Palmitoyl-2-(5'-oxo-valeroyl)-sn-glycero-3-phosphocholine (POVPC), an oxidized phospholipid, is found in atherosclerotic plaques and has been shown to induce oxidative stress. However, the effects of POVPC on osteogenic differentiation and calcification of VSMCs have yet to be studied. In the present study, we investigated the role of POVPC in vascular calcification using in vitro and ex vivo models. POVPC increased mineralization of VSMCs and arterial rings, as shown by alizarin red staining. In addition, POVPC treatment increased expression of osteogenic markers Runx2 and BMP2, indicating that POVPC promotes osteogenic transition of VSMCs. Moreover, POVPC increased oxidative stress and impaired mitochondria function of VSMCs, as shown by increased ROS levels, impairment of mitochondrial membrane potential, and decreased ATP levels. Notably, ferroptosis triggered by POVPC was confirmed by increased levels of intracellular ROS, lipid ROS, and MDA, which were decreased by ferrostatin-1, a ferroptosis inhibitor. Furthermore, ferrostatin-1 attenuated POVPC-induced calcification of VSMCs. Taken together, our study for the first time demonstrates that POVPC promotes vascular calcification via activation of VSMC ferroptosis. Reducing the levels of POVPC or inhibiting ferroptosis might provide a novel strategy to treat vascular calcification.

Mir-195-5p targets Smad7 regulation of the Wnt/ß-catenin pathway to promote osteogenic differentiation of vascular smooth muscle cells.

In this study, we sought to investigate the mechanisms of action of miR-195-5p in the osteogenic differentiation of vascular smooth muscle cells (VSMCs), and thereby provide novel insights and a reference for the targeted therapy of arterial media calcification. VSMC differentiation was induced using sodium ß-glycerophosphate, and we investigated the effects of transfecting cells with miR-195-5p mimics, vectors overexpressing Smad7, and the Wnt/ß-catenin pathway inhibitor (KYA1797K) on VSMC differentiation by determining cell viability and apoptosis, and the mRNA and protein expression of factors associated with osteogenic differentiation and the Wnt/ß-catenin pathway. The results revealed that miR-195-5p mimics enhanced the osteogenic differentiation of VSMCs induced by ß-glycerophosphate, whereas the overexpression of Smad7 reversed this phenomenon. In addition, KYA1797K was found to promote the effects of Smad7 overexpression. In conclusion, by targeting, Smad7, miR-195-5p promotes the Wnt/ß-catenin pathway. and thus the osteogenic differentiation of VSMCs. These findings will provide a reference for elucidating the mechanisms whereby miR-195-5p regulates osteogenic differentiation.

Toll-like receptor signaling pathway involved in pathogenesis of thromboangiitis obliterans through activating of NF-κB.

OBJECTIVES: The pathogenic mechanisms of Thromboangiitis Obliterans (TAO) are not entirely known and autoimmune inflammation plays a vital role in the initiation and continuance of TAO activity. The authors investigated in this study the role of the TLR signaling pathway in the pathogenesis of TAO. METHODS: First, the authors detected the expressions of MyD88, TRIF and NF-κB in vascular walls of 46 patients with TAO and 32 patients with trauma and osteosarcoma by western blot assay. Second, the authors detected the cellular localization of MyD88, TRIF and NF-κB in vascular walls of patients with TAO by immunofluorescent assay. RESULTS: The protein expressions of MyD88, TRIF and NF-κB were much higher in vascular walls of TAO patients (p < 0.05). Higher expressions of MyD88 and NF-κB were detected both on vascular endothelial and vascular smooth muscle cells of TAO patients. However, higher expression of TRIF was just detected on vascular smooth muscle cells of TAO patients. CONCLUSIONS: These dates suggest that the TLR signaling pathway might play an important role in the pathogenesis of TAO, it might induce vasospasm, vasculitis and thrombogenesis to lead to the pathogenesis and progression of TAO.

Colchicine Blocks Abdominal Aortic Aneurysm Development by Maintaining Vascular Smooth Muscle Cell Homeostasis.

Development of non-surgical treatment of human abdominal aortic aneurysm (AAA) has clinical significance. Colchicine emerges as an effective therapeutic regimen in cardiovascular diseases. Yet, whether colchicine slows AAA growth remain controversy. Here, we demonstrated that daily intragastric administration of low-dose colchicine blocked AAA formation, prevented vascular smooth muscle cell (SMC) phenotype switching and apoptosis, and vascular inflammation in both peri-aortic CaPO4 injury and subcutaneous angiotensin-II infusion induced experimental AAA mice models. Mechanistically, colchicine increased global mRNA stability by inhibiting the METTL14/YTHDC1-mediated m6A modification, resulting in increased sclerostin (SOST) expression and consequent inactivation of the WNT/ß-catenin signaling pathway in vascular SMCs from mouse AAA lesions and in cultured human aortic SMCs. Moreover, human and mouse AAA lesions all showed increased m6A methylation, decreased SOST expression, and skewed synthetic SMC de-differentiation phenotype, compared to those without AAA. This study uncovers a novel mechanism of colchicine in slowing AAA development by using the METTL14/SOST/WNT/ß-catenin axis to control vascular SMC homeostasis in mouse aortic vessels and in human aortic SMCs. Therefore, use of colchicine may benefit AAA patients in clinical practice.

LncRNA CARMN inhibits abdominal aortic aneurysm formation and vascular smooth muscle cell phenotypic transformation by interacting with SRF.

Phenotypic transformation of vascular smooth muscle cells (VSMCs) plays a crucial role in abdominal aortic aneurysm (AAA) formation. CARMN, a highly conserved, VSMC-enriched long noncoding RNA (lncRNA), is integral in orchestrating various vascular pathologies by modulating the phenotypic dynamics of VSMCs. The influence of CARMN on AAA formation, particularly its mechanisms, remains enigmatic. Our research, employing single-cell and bulk RNA sequencing, has uncovered a significant suppression of CARMN in AAA specimens, which correlates strongly with the contractile function of VSMCs. This reduced expression of CARMN was consistent in both 7- and 14-day porcine pancreatic elastase (PPE)-induced mouse models of AAA and in human clinical cases. Functional analyses disclosed that the diminution of CARMN exacerbated PPE-precipitated AAA formation, whereas its augmentation conferred protection against such formation. Mechanistically, we found CARMN's capacity to bind with SRF, thereby amplifying its role in driving the transcription of VSMC marker genes. In addition, our findings indicate an enhancement in CAMRN transcription, facilitated by the binding of NRF2 to its promoter region. Our study indicated that CARMN plays a protective role in preventing AAA formation and restrains the phenotypic transformation of VSMC through its interaction with SRF. Additionally, we observed that the expression of CARMN is augmented by NRF2 binding to its promoter region. These findings suggest the potential of CARMN as a viable therapeutic target in the treatment of AAA.

Creation of an Atherosclerosis Model Using Palmitic Acid and Oleic Acid in the Vascular Smooth Muscle Cells of Rats.

BACKGROUND: Atherosclerosis (AS) is a chronic vascular inflammatory disease resulting from vascular endothelial injury and lipid deposition, closely linked to abnormal lipid metabolism within the body. The critical processes involved in atherosclerosis encompass lipid deposition, oxidation, metabolic disruptions, and inflammatory stimulation within the inner vessel wall. Lipid deposition emerges as a pivotal factor triggering these pathological changes, with vascular smooth muscle cells (VSMCs) playing a significant role in the development of AS. Therefore, the goal was to employ lipids, specifically palmitic acid (PA) and oleic acid (OA) solutions, to stimulate VSMCs and create an in vitro atherosclerosis model. This approach allows for the establishment of a rapid and efficient cell model for simulating atherosclerosis in vitro. METHODS: Primary vascular smooth muscle cells (VSMCs) were isolated and cultured from the thoracic aorta of healthy rats using the tissue-block method. VSMCs were identified through cell climbing slices and immunofluorescence. The growth of VSMCs was observed using light microscopy. The logarithmic growth phase of VSMCs was induced and stimulated by various concentrations of palmitic acid (PA) and oleic acid (OA) ranging from 0 to 650 µmol/L, with a gradient dilution of 50 µmol/L. VSMC activity was assessed using the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Intracellular lipid deposition was visualized through Oil Red O staining. The levels of total cholesterol (TC), triglyceride (TG), high-density lipoprotein-cholesterol (HDL-C), and low-density lipoprotein-cholesterol (LDL-C) within VSMCs were quantified using commercially available kits. RESULTS: The optimal conditions for VSMC proliferation were determined to be an OA concentration of 500 µmol/L, a PA concentration of 300 µmol/L, and a culture duration of 48 hours. In comparison to the control group, the presence of lipid droplets within VSMCs became significantly evident following treatment with OA or PA. Furthermore, the levels of TC, TG, and LDL-C increased, while the HDL-C content decreased after treatment with OA or PA. CONCLUSIONS: A research model for atherosclerosis (AS) and the early stages of cardiovascular events, specifically lipid deposition, was successfully established through the use of OA and PA solutions. This model has the potential to open up new research avenues for gaining a deeper understanding of the pathogenesis and progression of AS.

Knockdown of LINC01128 Downregulates FUT8 to Inhibit OxLDL-Induced Vascular Smooth Muscle Cell Apoptosis in Atherosclerosis.

BACKGROUND: The apoptosis of vascular smooth muscle cells (VSMCs) contributes to the progression of atherosclerosis (AS). Long intergenic non-protein coding RNA 1128 (LINC01128) has been implicated in AS, and this study aims to uncover the role and mechanism of LINC01128 in regulating oxidized low-density lipoprotein (oxLDL)-induced VSMCs. METHODS: The position of LINC01128 in cells and its target genes were predicted using bioinformatics. The localization of LINC01128 in human VSMCs was determined through fluorescence in situ hybridization. VSMCs were transfected, and the interaction between LINC01128 and fucosyltransferase 8 (FUT8) was validated by chromatin immunoprecipitation assay. The apoptotic VSMC model was established using oxLDL. LINC01128 expression in VSMCs was analyzed by quantitative real-time polymerase chain reaction (qRT-PCR), and FUT8 expression was detected by qRT-PCR and western blot. VSMC viability, migration, invasion abilities, and apoptosis were assessed using cell counting kit-8, transwell assay, and flow cytometry, respectively. RESULTS: OxLDL (200 µg/mL) upregulated the expression of LINC01128 and FUT8 mRNA, as well as FUT8 protein, in VSMCs. LINC01128 was expressed in the nucleus of VSMCs and bound to FUT8. Knockdown of LINC01128 alleviated the inhibitory effects of oxLDL (200 µg/mL) on viability, migration, and invasion, and mitigated the promotion of apoptosis and FUT8 expression in VSMCs. On the other hand, FUT8 overexpression enhanced the suppressive effects of oxLDL (200 µg/mL) on viability, migration, and invasion activities, and amplified the facilitating effect of oxLDL on apoptosis in VSMCs. Moreover, FUT8 overexpression reversed the impact of LINC01128 silencing on viability, migration, invasion, and apoptosis in oxLDL-stimulated VSMCs. CONCLUSION: The knockdown of LINC01128 downregulates FUT8, inhibiting the progression of VSMCs in AS.

CircTLK1: A novel regulator involved in VSMC phenotypic switching and the developmental process of atherosclerosis.

Phenotypic switching of vascular smooth muscle cells (VSMCs) is essential for atherosclerosis development. Circular RNA (circRNA) is a specific non-coding RNA that is produced as a closed-loop structure in mammals, and its specific expression pattern is closely related to its cell type and tissue. To clarify the roles of circTLK1 in VSMC phenotypic switching, we performed qRT-PCR, immunoblotting, and immunostaining. qRT-PCR revealed that circTLK1 was upregulated in both mouse models of atherosclerosis in vivo and PDGF (platelet-derived growth factor)-BB-induced VSMCs in vitro. Furthermore, the overexpression of circTLK1 promoted PDGF-BB-induced VSMC phenotypic switching. Conversely, experiments performed in vivo demonstrate that the knockdown of SMC-specific circTLK1 led to a reduction in the development of atherosclerosis. The relationship between circTLK1 and miR-513a-3p and Krüppel-like factor 4 (KLF4) was detected by RNA immunoprecipitation (RIP), luciferase reporter assay, RNA pull-down, and RNA fluorescence in situ hybridization (RNA FISH). Mechanistically, circTLK1 acted as a sponge for miR-513a-3p, leading to the upregulation of KLF4, a key transcription factor for phenotypic switching. Targeting the circTLK1/miR-513a-3p/KLF4 axis may provide a potential therapeutic strategy for atherosclerosis.

Hypomethylation of the LncRNA H19 promoter accelerates osteogenic differentiation of vascular smooth muscle cells by activating the Erk1/2 pathways.

OBJECTIVE: Vascular calcification is a common chronic kidney disease complication. This study aimed to investigate the function of long non-coding RNA (LncRNA) H19 in vascular calcification to explore new therapeutic strategies. METHODS: We induced osteogenic differentiation and calcification of vascular smooth muscle cells (VSMCs) using ß-glycerophosphate. Then, we detected the LncRNA H19 promoter methylation status and Erk1/2 pathways using methylation-specific polymerase chain reaction and western blotting, respectively. RESULTS: Compared with the control group, high phosphorus levels induced VSMC calcification, accompanied by increases in LncRNA H19 and the osteogenic marker Runx2 and reduction of the contractile phenotype marker SM22a. LncRNA H19 knockdown inhibited osteogenic differentiation and calcification of VSMCs. However, the suppressed role of VSMC calcification caused by shRNA H19 was partially reversed by simultaneous activation of the Erk1/2 pathways. Mechanically, we found that the methylation rate of CpG islands in the LncRNA H19 promoter region was significantly lower in the high-phosphorus group, and the hypomethylation state elevated LncRNA H19 levels, which in turn regulated phosphorylated Erk1/2 expression. CONCLUSIONS: LncRNA H19 promoted osteogenic differentiation and calcification of VSMCs by regulating the Erk1/2 pathways. Additionally, hypomethylation of LncRNA H19 promoter CpG islands upregulated LncRNA H19 levels and subsequently activated Erk1/2 phosphorylation.

M1 Macrophage-Derived Exosome LncRNA PVT1 Promotes Inflammation and Pyroptosis of Vascular Smooth Muscle Cells in Abdominal Aortic Aneurysm by Inhibiting miR-186-5p and Regulating HMGB1.

Abdominal aortic aneurysm (AAA) is a chronic vascular degenerative disease. Vascular smooth muscle cells (VSMCs) are essential for maintaining the integrity of healthy blood vessels. Macrophages play an important role in the inflammatory process of AAA. However, the effect of macrophage-derived exosome LncRNA PVT1 on VSMCs is unclear. Exosomes from M1 macrophages (M1φ-exos) were isolated and identified. The expression of LncRNA PVT1 in M1φ-exos was determined. AAA cell model was constructed by treating VSMCs with Ang-II. AAA cell model was treated with M1φ exosomes transfected with si-LncRNA PVT1 (M1φsi-LncRNA PVT1-exo). VSMCs were transfected with miR-186-5p mimic and oe-HMGB1. Cell viability was detected by CCK-8. The accumulation of LDH was detected by ELISA. Western blot was used to detect the expression of HMGB1, inflammatory factors (IL-6, TNF-α and IL-1ß) and pyroptosis-related proteins (GSDMD, N-GSDMD, ASC, NLRP3, Caspase-1 and Cleaved-Capase-1). Cell pyroptosis rate was detected by flow cytometry. At the same time, the targeting relationship between miR-186-5p and LncRNA PVT1 and HMGB1 was verified by double fluorescein experiment. Exosomes from M1φ were successfully extracted. The expression of LncRNA PVT1 in M1φ-exos was significantly increased. M1φ-exo promotes inflammation and pyroptosis of VSMCs. M1φsi-LncRNA PVT1-exos inhibited the inflammation and pyroptosis of VSMCs. LncRNA PVT1 can sponge miR-186-5p mimic to regulate HMGB1 expression. MiR-186-5p mimic further inhibited inflammation and pyroptosis induced by M1φsi-LncRNA PVT1-exos. However, oe-HMGB1 could inhibit the reversal effect of miR-186-5p mimic. LncRNA PVT1 in exosomes secreted by M1φ can regulate HMGB1 by acting as ceRNA on sponge miR-186-5p, thereby promoting cell inflammatory and pyroptosis and accelerating AAA progression.

Contractions Induced in Human Pulmonary Arteries by a H2S Donor, GYY 4137, Are Inhibited by Low-Frequency (20 kHz) Ultrasound.

The present study aimed to investigate the effect of a H2S donor, GYY 4137, on human pulmonary arteries and whether low-frequency ultrasound (20 kHz, 4 W/cm2) inhibits GYY 4137 contractions. Functional studies were conducted on human and rat pulmonary arteries mounted on microvascular myographs. We placed an ultrasonic gadget in the tissue organ bath to insonate the arteries with low-frequency ultrasound. To measure the effect of the low-frequency ultrasound on the entrance of extracellular Ca2+, the preparations were placed in a Ca2+-free solution, and the thromboxane agonist, U46619, and extracellular calcium were added in the presence of insonation. In isolated human pulmonary arteries, GYY 4137 induced contractions, which were most pronounced in the arteries contracted with the thromboxane analogue, U46619. The transient GYY4137 contractions were reversed by low-frequency ultrasound, a blocker of KV7 channels, XE-991 (10 µM), and glibenclamide (1 µM), a blocker of ATP-sensitive channels. Low-frequency ultrasound also inhibited the contractions induced by the smooth muscle entrance of increasing extracellular calcium concentrations. The present findings show that GYY 4137 can cause a transient contraction of pulmonary arteries in human arteries. GYY 4137 alone does not cause significant vascular contraction in rat lung arteries, but it contracts rat lung arteries precontracted with U46619. The transient contractions induced by GYY 4137 can be inhibited by low-frequency ultrasound, probably by counteracting the influx of external Ca2+. The effect of low-frequency ultrasound counteracts contraction in pulmonary arteries; therefore, a possibility could be to develop a larger device allowing treatment of patients with pulmonary hypertension.

Influence of DNA Methylation on Vascular Smooth Muscle Cell Phenotypic Switching.

Vascular smooth muscle cells (VSMCs) are crucial components of the arterial wall, controlling blood flow and pressure by contracting and relaxing the artery walls. VSMCs can switch from a contractile to a synthetic state, leading to increased proliferation and migratory potential. Epigenetic pathways, including DNA methylation, play a crucial role in regulating VSMC differentiation and phenotypic flexibility. DNA methylation involves attaching a methyl group to the 5' carbon of a cytosine base, which regulates gene expression by interacting with transcription factors. Understanding the key factors influencing VSMC plasticity may help to identify new target molecules for the development of innovative drugs to treat various vascular diseases. This review focuses on DNA methylation pathways in VSMCs, summarizing mechanisms involved in controlling vascular remodeling, which can significantly enhance our understanding of related mechanisms and provide promising therapeutic approaches for complex and multifactorial diseases.

Activation of the IKK2/NF-κB pathway in VSMCs inhibits calcified vascular stiffness in CKD.

IKK2/NF-κB pathway-mediated inflammation in vascular smooth muscle cells (VSMCs) has been proposed to be an etiologic factor in medial calcification and stiffness. However, the role of the IKK2/NF-κB pathway in medial calcification remains to be elucidated. In this study, we found that chronic kidney disease (CKD) induces inflammatory pathways through the local activation of the IKK2/NF-κB pathway in VMSCs associated with calcified vascular stiffness. Despite reducing the expression of inflammatory mediators, complete inhibition of the IKK2/NF-κB pathway in vitro and in vivo unexpectedly exacerbated vascular mineralization and stiffness. In contrast, activation of NF-κB by SMC-specific IκBα deficiency attenuated calcified vascular stiffness in CKD. Inhibition of the IKK2/NF-κB pathway induced cell death of VSMCs by reducing anti-cell death gene expression, whereas activation of NF-κB reduced CKD-dependent vascular cell death. In addition, increased calcification of extracellular vesicles through the inhibition of the IKK2/NF-κB pathway induced mineralization of VSMCs, which was significantly reduced by blocking cell death in vitro and in vivo. This study reveals that activation of the IKK2/NF-κB pathway in VSMCs plays a protective role in CKD-dependent calcified vascular stiffness by reducing the release of apoptotic calcifying extracellular vesicles.

Circ-ABCA1 promotes oxidized low-density lipoprotein-induced inflammation and phenotypic switch in vascular smooth muscle cells.

OBJECTIVE: Atherosclerosis (AS) is a chronic inflammatory disease of the arterial wall, in which Human Vascular Smooth Muscle Cells (HVSMCs) are involved. Nevertheless, the functions and mechanisms of circRNAs in oxidized Low-Density Lipoprotein (ox-LDL)-induced vascular smooth muscle cells remain unclear. METHODS: Circ-ABCA1 expression was measured in the models of AS. Then, in the vitro model, oligonucleotide transfection was performed, followed by an analysis of VSMC proliferation, migration, inflammation, and phenotypic switch. Also, in the in vivo model, mice were injected with shRNA lentivirus, followed by histological examination of aortic tissues. Finally, the interaction of circ-ABCA1, miR-885-5p, and ROCK2 was identified. RESULTS: Circ-ABCA1, was confirmed to be overexpressed in ox-LDL-induced VSMCs and mouse models of AS. Functionally, silencing circ-ABCA1 via oligonucleotide transfection suppressed VSMC proliferation, migration, inflammation, and phenotypic switch in vitro and prevented AS development in mice in vivo. Mechanistically, circ-ABCA1 absorbed miR-885-5p, which targeted ROCK2. CONCLUSION: Taken together, the data from this study suggest that circ-ABCA1 mediates cellular inflammation and phenotype switching through the miR-885-5p/ROCK2 axis in ox-LDL-induced VSMCs, and the circ-ABCA1/miR-885-5p/ROCK2 axis is a new potential biomarker for the treatment of AS.