Modulation Of Vascular Smooth Muscle Cells Research Articles

Restoration of ARA metabolic disorders in vascular smooth muscle cells alleviates intimal hyperplasia

Intimal hyperplasia (IH) is an innegligible issue for patients undergoing interventional therapy. The proliferation and migration of vascular smooth muscle cells (VSMCs) induced by platelet-derived growth factor-BB (PDGF-BB) are critical events in the development of IH. While the exact mechanism and effective target for IH needs further investigation. Metabolic disorders of arachidonic acid (ARA) are involved in the occurrence and progression of various diseases. In this study, we found that the expressions of soluble epoxide hydrolase (sEH) and cyclooxygenase-2 (COX-2) were significantly increased in the VSMCs during balloon injury-induced IH. Then, we employed a COX-2/sEH dual inhibitor PTUPB to increase the concentration of epoxyeicosatrienoic acids (EETs) while prevent the release of pro-inflammatory prostaglandins. Results showed that PTUPB treatment significantly reduced neointimal thickening induced by balloon injury in rats in vivo and inhibited PDGF-BB-induced proliferation and migration of VSMCs in vitro. Our results showed that PTUPB may reverse the phenotypic transition of VSMCs by inhibiting Pttg1 expression. In conclusion, we found that the dysfunction of ARA metabolism in VSMCs contributes to IH, and the COX-2/sEH dual inhibitor PTUPB attenuates IH progression by reversing the phenotypic switch in VSMC through the Sirt1/Pttg1 pathway.

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 invitro and exvivo 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.

S100A4 Is a Key Facilitator of Thoracic Aortic Dissection.

Background: Thoracic aortic dissection (TAD) is one of the cardiovascular diseases with high incidence and fatality rates. Vascular smooth muscle cells (VSMCs) play a vital role in TAD formation. Recent studies have shown that extracellular S100A4 may participate in VSMCs regulation. However, the mechanism(s) underlying this association remains elusive. Consequently, this study investigated the role of S100A4 in VSMCs regulation and TAD formation. Methods: Hub genes were screened based on the transcriptome data of aortic dissection in the Gene Expression Synthesis database. Three-week-old male S100A4 overexpression (AAV9- S100A4 OE) and S100A4 knockdown (AAV9- S100A4 KD) mice were exposed to β-aminopropionitrile monofumarate through drinking water for 28 days to create the murine TAD model. Results: S100A4 was observed to be the hub gene in aortic dissection. Furthermore, overexpression of S100A4 was exacerbated, whereas inhibition of S100A4 significantly improved TAD progression. In the TAD model, the S100A4 was observed to aggravate the phenotypic transition of VSMCs. Additionally, lysyl oxidase (LOX) was an important target of S100A4 in TAD. S100A4 interacted with LOX in VSMCs, reduced mature LOX (m-LOX), and decreased elastic fiber deposition, thereby disrupting extracellular matrix homeostasis and promoting TAD development. Elastic fiber deposition in human aortic tissues was negatively correlated with the expression of S100A4, which in turn, was negatively correlated with LOX. Conclusions: Our data showed that S100A4 modulates TADprogression, induces lysosomal degradation of m-LOX, and reduces the deposition of elastic fibers by interacting with LOX, thus contributing to the disruption of extracellular matrix homeostasis in TAD. These findings suggest that S100A4 may be a new target for the prevention and treatment of TAD.

AT1R autoantibody promotes phenotypic transition of smooth muscle cells by activating AT1R-OAS2

Phenotypic transition of vascular smooth muscle cells (VSMCs) is an early event in the onset and progression of several cardiovascular diseases. As an important mediator of the renin-angiotensin system (RAS), activation of the angiotensin II type 1 receptor (AT1R) induces phenotypic transition of VSMCs. AT1R autoantibodies (AT1-AAs), which are agonistic autoantibodies of AT1R, have been detected in the sera of patients with a variety of cardiovascular diseases associated with phenotypic transition. However, the effect of AT1-AA on phenotypic transition is currently unknown. In this study, AT1-AA-positive rat model was established by active immunization to detect markers of VSMCs phenotypic transition. The results showed that AT1-AA-positive rats showed phenotypic transition of VSMCs, which was evidenced by the decrease of contractile markers, while the increase of synthetic markers in the thoracic aorta. However, in AT1-AA-positive AT1R knockout rats, the phenotypic transition-related proteins were not altered. In vitro, after stimulating human aortic smooth muscle cells with AT1-AA for 48 h, 2′-5′ oligoadenylate synthase 2 (OAS2) was identified as the key differentially expressed gene by RNA sequencing and bioinformatics analysis. Furthermore, high expression of OAS2 was found in aorta of AT1-AA-positive rats; knockdown of OAS2 by siRNA can reverse the phenotypic transition of VSMCs induced by AT1-AA. In summary, this study suggests that AT1-AA can promote phenotypic transition of VSMCs through AT1R-OAS2 pathway, and OAS2 might serve as a potential therapeutic target to prevent pathological phenotypic transition of smooth muscle cells.

Bone Morphogenetic Protein-4 Promotes Phenotypic Modulation via SMAD-4/MCT-4 Axis in Vascular Smooth Muscle Cells

Introduction: This study aimed to determine whether bone morphogenetic protein-4 (BMP-4), which increases in response to intimal hyperplasia, promotes phenotype transition in vascular smooth muscle cells (VSMCs). Methods: Balloon injury was used to induce intimal hyperplasia in rats. Hematoxylin-eosin staining was used to detect the alteration of vascular structure. Serum levels of BMP-4 and lactate were detected by ELISA. Human aortic smooth muscle cells (HA-SMCs) were cultured. Protein and mRNA expression levels were detected through Western blot and real-time PCR. Cell migration was measured by transwell assay. Results: Our data showed that serum concentration of BMP-4 was upregulated after balloon injury. Treatment with BMP-4 inhibitor DMH1 (4-(6-(4-isopropoxyphenyl)pyrazolo(1,5-a)pyrimidin-3-yl)quinoline) suppressed the abnormal expression of BMP-4 and inhibited the intimal hyperplasia induced by balloon injury. Compared to BMP-4-negative medium, BMP-4-positive medium was associated with higher synthetic VSMC marker expression levels and lower in contractile gene markers in cultured HA-SMCs. Transfection of monocarboxylic acid transporters-4 (MCT-4) siRNA inhibited the excretion of lactate induced by BMP-4. Conclusion: Our analyses provided evidence that BMP-4 and its regulator Smad-4 are key regulators in MCT-4-mediated lactate excretion. This indicates that BMP-4 stimulates the phenotypic transition of VSMCs via SMAD-4/MCT-4 signaling pathway.

The Long non-coding RNA MALAT1 functions as a competing endogenous RNA to regulate vascular remodeling by sponging miR-145-5p/HK2 in hypertension

ABSTRACT Long non-coding RNAs (LncRNAs) have been found to play a regulatory role in the pathophysiology of vascular remodeling-associated illnesses through the lncRNA-microRNA (miRNA) regulation axis. LncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is thought to be involved in proliferation, migration, apoptosis, and calcification of vascular smooth muscle cells (VSMCs). The purpose of this study was to investigate the regulatory role of MALAT1 on vascular remodeling in hypertension. Our data indicate that the expression of MALAT1 is significantly upregulated in hypertensive aortic smooth muscle. Knockdown of MALAT1 inhibited the proliferation, migration, and phenotypic transition of VSMCs induced by Ang II. Bioinformatics analysis was used to predict the complementary binding of miR-145-5p to the 3’-untranslated region of MALAT1. Besides, the expressions of MALAT1 and miR-145-5p were negatively correlated, while luciferase reporter assays and RNA immunoprecipitation assay validated the interaction between miR-145-5p and MALAT1. The proliferation, migration and phenotypic transformation of VSMCs induced by overexpression of MALAT1 were reversed in the presence of miR-145-5p. Furthermore, we verified that miR-145-5p could directly target and bind to hexokinase 2 (HK2) mRNA, and that HK2 expression was negatively correlated with miR-145-5p in VSMCs. Knockdown of HK2 significantly inhibited the effects of overexpression of MALAT1 on Ang II-induced VSMCs proliferation, migration and phenotypic transformation. Taken together, the MALAT1/miR-145-5p/HK2 axis may play a critical regulatory role in the vascular remodeling of VSMCs in hypertension.

Purification, structural characteristics and anti-atherosclerosis activity of a novel green tea polysaccharide

A new homogeneous polysaccharide (TPS3A) was isolated and purified from Tianzhu Xianyue fried green tea by DEAE-52 cellulose and Sephacryl S-500 column chromatography. Structural characterization indicated that TPS3A mainly consisted of arabinose, galactose, galacturonic acid and rhamnose in a molar ratio of 5.84: 4.15: 2.06: 1, with an average molecular weight of 1.596 × 104 kDa. The structure of TPS3A was characterized as a repeating unit consisting of 1,3-Galp, 1,4-Galp, 1,3,6-Galp, 1,3-Araf, 1,5-Araf, 1,2,4-Rhap and 1-GalpA, with two branches on the C6 of 1,3,6-Galp and C2 of 1,2,4-Rhap, respectively. To investigate the preventive effects of TPS3A on atherosclerosis, TPS3A was administered orally to ApoE-deficient (ApoE−/−) mice. Results revealed that TPS3A intervention could effectively delay the atherosclerotic plaque progression, modulate dyslipidemia, and reduce the transformation of vascular smooth muscle cells (VSMCs) from contractile phenotype to synthetic phenotype by activating the expression of contractile marker alpha-smooth muscle actin (α-SMA) and inhibiting the expression of synthetic marker osteopontin (OPN) in high-fat diet-induced ApoE−/− mice. Our findings suggested that TPS3A markedly alleviated atherosclerosis by regulating dyslipidemia and phenotypic transition of VSMCs, and might be used as a novel functional ingredient to promote cardiovascular health.

LKB1 delays atherosclerosis by inhibiting phenotypic transformation of vascular smooth muscle cells

Background and objectiveAlthough liver kinase B1 (LKB1) is a well-known tumor suppressor gene, and its encoded protein has important biological functions, it is not clear whether LKB1 can inhibit atherosclerosis by regulating vascular smooth muscle cells (VSMCs). The purpose of this study is to explore the relationship among LKB1, VSMCs and atherosclerosis. Methods and resultsApoE−/− mice with VSMCs-specific overexpression of LKB1 were constructed by adeno-associated virus transfection technique, and then fed with high-fat diet for eight weeks. The effect of LKB1 overexpression on atherosclerosis in mice was investigated by oil red O staining, HE staining, immunofluorescence and Western Blot. The results showed that the expression of LKB1 mRNA and protein in arterial tissue of mice increased significantly after overexpression of LKB1. The degree of atherosclerosis, smooth muscle fiber proliferation and lipid accumulation were significantly alleviated in the overexpression group. The results of Western Blot showed that the expression of α-SMA was increased, while the expression of OPN and CD68 was significantly decreased in the overexpression group (P < 0.05). The Immunofluorescence results of Image Pro Plus software analysis showed that the co-localization relationship between α-SMA and CD68 was more obvious in the control group (P < 0.01). ConclusionOur results suggested that LKB1 can delay the progression of atherosclerosis by inhibiting the phenotypic transition of VSMCs.

KAP1 modulates osteogenic differentiation via the ERK/Runx2 cascade in vascular smooth muscle cells.

Osteoblast phenotypic transition in vascular smooth muscle cells (VSMCs) has been unveiled as a common cause of vascular calcification (VC). Krüppel-Associated Box (KRAB)-Associated Protein 1(KAP1) is a transcriptional corepressor that modulates various intracellular pathological processes from gene expression to DNA repair to signal transduction. However, the function and mechanism of KAP1 on the osteoblastic differentiation of VSMCs have not been evaluated yet. We demonstrate that the expression of KAP1 in VSMCs is significantly enhanced in vivo and in vitro calcification models. Downregulating the expression of KAP1 suppresses the osteoblast phenotypic transition of VSMCs, which is indicated by a decrease in the expression of osteoblast marker collagenase type I (COL I) and an increase in the expression of VSMC marker α-smooth muscle actin (α-SMA). Conversely, exogenous overexpression of KAP1 could promote osteoblast phenotypic transition of VSMCs. Moreover, KAP1 upregulated the expression of RUNX family transcription factor 2 (Runx2), an inducer of osteoblast that positively regulates many osteoblast-related genes, such as COL I. Evaluation of the potential mechanism demonstrated that KAP1 promoted osteoblast phenotypic transition of VSMCs by activating the extracellular regulated protein kinases (ERK) signaling pathway, which could activate Runx2. In support of this finding, KAP1-induced cell osteoblast phenotypic transition is abolished by treatment with PD0325901, a specific ERK inhibitor. The present study suggested that KAP1 participated in the osteoblast differentiation of VSMCs via the ERK/Runx2 cascade and served as a potential diagnostics and therapeutics target for vascular calcification.

TXNIP Suppresses the Osteochondrogenic Switch of Vascular Smooth Muscle Cells in Atherosclerosis.

The osteochondrogenic switch of vascular smooth muscle cells (VSMCs) is a pivotal cellular process in atherosclerotic calcification. However, the exact molecular mechanism of the osteochondrogenic transition of VSMCs remains to be elucidated. Here, we explore the regulatory role of TXNIP (thioredoxin-interacting protein) in the phenotypical transitioning of VSMCs toward osteochondrogenic cells responsible for atherosclerotic calcification. The atherosclerotic phenotypes of Txnip-/- mice were analyzed in combination with single-cell RNA-sequencing. The atherosclerotic phenotypes of Tagln-Cre; Txnipflox/flox mice (smooth muscle cell-specific Txnip ablation model), and the mice transplanted with the bone marrow of Txnip-/- mice were analyzed. Public single-cell RNA-sequencing dataset (GSE159677) was reanalyzed to define the gene expression of TXNIP in human calcified atherosclerotic plaques. The effect of TXNIP suppression on the osteochondrogenic phenotypic changes in primary aortic VSMCs was analyzed. Atherosclerotic lesions of Txnip-/- mice presented significantly increased calcification and deposition of collagen content. Subsequent single-cell RNA-sequencing analysis identified the modulated VSMC and osteochondrogenic clusters, which were VSMC-derived populations. The osteochondrogenic cluster was markedly expanded in Txnip-/- mice. The pathway analysis of the VSMC-derived cells revealed enrichment of bone- and cartilage-formation-related pathways and bone morphogenetic protein signaling in Txnip-/- mice. Reanalyzing public single-cell RNA-sequencing dataset revealed that TXNIP was downregulated in the modulated VSMC and osteochondrogenic clusters of human calcified atherosclerotic lesions. Tagln-Cre; Txnipflox/flox mice recapitulated the calcification and collagen-rich atherosclerotic phenotypes of Txnip-/- mice, whereas the hematopoietic deficiency of TXNIP did not affect the lesion phenotype. Suppression of TXNIP in cultured VSMCs accelerates osteodifferentiation and upregulates bone morphogenetic protein signaling. Treatment with the bone morphogenetic protein signaling inhibitor K02288 abrogated the effect of TXNIP suppression on osteodifferentiation. Our results suggest that TXNIP is a novel regulator of atherosclerotic calcification by suppressing bone morphogenetic protein signaling to inhibit the transition of VSMCs toward an osteochondrogenic phenotype.

Phenotypic plasticity of vascular smooth muscle cells in vascular calcification: Role of mitochondria.

Vascular calcification (VC) is an important hallmark of cardiovascular disease, the osteo-/chondrocyte phenotype differentiation of vascular smooth muscle cells (VSMCs) is the main cause of vascular calcification. Accumulating evidence shows that mitochondrial dysfunction may ultimately be more detrimental in the VSMCs calcification. Mitochondrial participate in essential cellular functions, including energy production, metabolism, redox homeostasis regulation, intracellular calcium homeostasis, apoptosis, and signal transduction. Mitochondrial dysfunction under pathological conditions results in mitochondrial reactive oxygen species (ROS) generation and metabolic disorders, which further lead to abnormal phenotypic differentiation of VSMCs. In this review, we summarize existing studies targeting mitochondria as a treatment for VC, and focus on VSMCs, highlighting recent progress in determining the roles of mitochondrial processes in regulating the phenotype transition of VSMCs, including mitochondrial biogenesis, mitochondrial dynamics, mitophagy, mitochondrial energy metabolism, and mitochondria/ER interactions. Along these lines, the impact of mitochondrial homeostasis on VC is discussed.

The short-chain fatty acid butyrate accelerates vascular calcification via regulation of histone deacetylases and NF-κB signaling

Recent studies have shown that short-chain fatty acids (SCFAs), primarily acetate, propionate and butyrate, play a crucial role in the pathogenesis of cardiovascular disease. Whether SCFAs regulate vascular calcification, a common pathological change in cardiovascular tissues, remains unclear. This study aimed to investigate the potential role of SCFAs in vascular calcification. Using cellular and animal models of vascular calcification, we showed that butyrate significantly enhanced high phosphate (Pi)-induced calcification and osteogenic transition of vascular smooth muscle cells (VSMC) in vitro, whereas acetate and propionate had no effects. Subsequent studies confirmed that butyrate significantly promoted high Pi-induced aortic ring calcification ex vivo and high dose vitamin D3 (vD3)-induced mouse vascular calcification in vivo. Mechanistically, butyrate significantly inhibited histone deacetylase (HDAC) expression in VSMCs, and a pan HDAC inhibitor Trichostatin A showed similar inductive effects on calcification and osteogenic transition of VSMCs to butyrate. In addition, the SCFA sensing receptors Gpr41 and Gpr109a were primarily expressed by VSMCs, and butyrate induced the rapid activation of NF-κB, Wnt and Akt signaling in VSMCs. Intriguingly, the NF-κB inhibitor SC75741 significantly attenuated butyrate-induced calcification and the osteogenic gene Msx2 expression in VSMCs. We showed that knockdown of Gpr41 but not Gpr109a attenuated butyrate-induced VSMC calcification. This study reveals that butyrate accelerates vascular calcification via its dual effects on HDAC inhibition and NF-κB activation. Our data provide novel insights into the role of microbe-host interaction in vascular calcification, and may have implications for the development of potential therapy for vascular calcification.

Mechanisms of miR-29a-5p involvement in osteogenic phenotype transformation and cellular regulation of vascular smooth muscle and thus influencing calcification in VSMCs in chronic kidney disease

Vascular calcification is one of the major complications of chronic kidney disease (CKD), which could be further accelerated by the osteogenic transition and apoptosis of smooth muscle cells, thereby advancing the progression of renal diseases and increasing the mortality rate of cardiovascular events. MicroRNA is a kind of key regulator in the phenotypic transition of vascular smooth muscle cells (VSMCs), but its role remains unclear in VSMCs. In this study, VSMCs were stimulated by platelet-derived growth factors - BB (PDGF-BB) in varying concentrations to establish the VSMC dysfunction models. The relative expression of miR-29a-5p was quantified via the quantitative real-time polymerase chain reaction (qRT-PCR). The proliferation of VSMCs was determined via the BrdU method, analysis of cell cycle via flow cytometry, and the migration of VSMCs via Transwell assay. Expression of γ-secretase activating protein (GSAP) and markers of VSMC differentiation, including α-SMA, SM-22α, SMMHC and Calponin, was quantified via the Western blot. The targeting relationship between the 3'-UTR of miR-29a-5p and GSAP was validated through the dual-luciferase reporter gene assay. As a result, we found that PDGF-BB could trigger a decrease of miR-29a-5p in a time- and dose-dependent manner (P < 0.05). Overexpression of miR-29a-5p could curb the effect of PDGF-BB on the proliferation and migration of VSMCs while upregulating the expression of markers of differentiation (P < 0.05). In addition, the expression of GSAP was also affected by the negative regulation of miR-29a-5p, while the restoration of GSAP eliminated the effect of miR-29a-5p on the VSMCs partially (P < 0.05). Moreover, vascular calcification models were also established in the CKD rats, suggesting that the inhibition of GSAP could prevent PTH-induced vascular calcification in CKD rats. In conclusion, miR-29a-5p could inhibit the PDGF-BB-induced proliferation, migration and phenotypic transition of VSMCs via targeting GSAP. Thus, miR-29a-5p/GSAP might be a potential target for the treatment of vascular calcification.

Rosmarinic Acid Inhibits Platelet Aggregation and Neointimal Hyperplasia In Vivo and Vascular Smooth Muscle Cell Dedifferentiation, Proliferation, and Migration In Vitro via Activation of the Keap1-Nrf2-ARE Antioxidant System.

The activation of platelets and proliferation of vascular smooth muscle cells (VSMCs) in the vascular intima play an essential role in the pathological mechanism of vascular restenosis (RS). Rosmarinic acid (RA) is a natural phenolic acid compound. However, its mechanism of action on platelets and VSMCs is still unclear. This study investigated the effects of RA on platelet function, VSMCs phenotypic conversion, proliferation, and migration in vascular remodeling with a specific focus on the Keap1-Nrf2-ARE signaling pathway. RA inhibited platelet aggregation and Ca2+ release and significantly reduced the release of platelet microvesicles. In addition, RA inhibited the phenotypic transition of VSMCs in vitro and in vivo. In vitro experiments showed that RA could effectively inhibit the proliferation and migration of VSMCs induced by the platelet-derived growth factor (PDGF)-BB. PDGF-BB triggered ROS generation and a decrease in mitochondrial membrane potential, which were inhibited by RA. Mechanistically, after artery injury or treatment with PDGF-BB, VSMCs presented with inhibition of the Nrf2/antioxidant response element (ARE) signaling pathway. RA treatment reversed this profile by activating the Nrf2/ARE signaling pathway; stabilizing Keap1 protein; upregulating HO-1, NQO1, GCLM, and GST protein levels; promoting typical Nrf2 nuclear translocation; and preventing VSMCs from oxidative stress damage. On the other hand, RA also inhibited the NF-κB pathway to reduce inflammation. In summary, these results indicate that RA inhibits platelet function and attenuates the proliferation, migration, and phenotypic transition of VSMCs induced by PDGF-BB in vitro and vascular remodeling in vivo. Therefore, RA treatment may be a potential therapy for preventing or treating RS.

IL-37 Expression in Patients with Abdominal Aortic Aneurysm and Its Role in the Necroptosis of Vascular Smooth Muscle Cells.

Background Our previous studies have shown that interleukin- (IL-) 37 plays a protective role in patients and animal models with coronary artery disease. However, the role of IL-37 in patients with abdominal aortic aneurysm (AAA), another artery disease, is yet to be elucidated. Methods and Results AAA tissues and plasma samples were obtained from patients with or without surgical intervention. Normal renal aortic tissues were collected from kidney transplant donors. Our findings established that in AAA, IL-37 was distributed in endothelial cells, macrophages, and vascular smooth muscle cells (VSMCs) and that it was chiefly concentrated in VSMCs. Furthermore, the expression was found to be downregulated compared with that in normal artery tissues. Immunofluorescence showed that, unlike normal arteries, IL-37 was translocated to the nucleus of VSMCs in AAA. Moreover, in patients with AAA, the expressions of IL-37, IL-6, and tumor necrosis factor- (TNF-) α were increased in the plasma in comparison with the healthy controls. Correlation analysis revealed that IL-37 was positively correlated with IL-6, TNF-α, age, aneurysm diameter, and blood pressure. Furthermore, human aortic vascular smooth muscle cells (HASMCs) were stimulated with angiotensin II (AngII) in vitro to simulate smooth muscle cell (SMC) damage in AAA. A decrease in IL-37 expression and an increase in receptor-interacting serine/threonine-protein kinase 3 (RIPK3) expression were observed in HASMCs stimulated with AngII. On this basis, inhibition of RIPK3 with GSK'872 significantly attenuated necroptosis. Moreover, the necroptosis rates were significantly lowered in HASMCs treated with recombinant IL-37, whereas the rates were enhanced when the cells were depleted of the interleukin. Immunoblotting results showed that both exogenous and endogenous IL-37 could affect the expressions of RIPK3, NLRP3, and IL-1β. Also, the phosphorylation of RIPK3 and p65 was affected. Meanwhile, IL-37 promoted the transition of SMC from proliferative type to contractile type. Conclusions The expression of IL-37 in VSMCs decreases in patients with AAA, whereas IL-37 supplementation suppresses RIPK3-mediated necroptosis and promotes the transition of VSMCs from proliferative to contractile type.

H3K4 Methyltransferase Smyd3 Mediates Vascular Smooth Muscle Cell Proliferation, Migration, and Neointima Formation.

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Parathyroid Hormone Induces Transition of Myofibroblasts in Arteriovenous Fistula and Increases Maturation Failure.

Arteriovenous fistula (AVF) maturation failure remains a clinical dilemma, and its pathobiology is largely unclear. Secondary hyperparathyroidism is a complication of chronic renal failure that is associated with cardiovascular disease. While parathyroid hormone (PTH) has a prosclerotic effect on vascular smooth muscle cells (VSMCs), its role in AVF maturation failure remained unknown. This work aimed to investigate the association between plasma PTH and AVF maturation. Patients receiving AVF creation were enrolled retrospectively. A mouse model of secondary hyperparathyroidism and aortocaval AVF was used to investigate the effect of PTH on an AVF lesion. A cell model of VSMCs treated with PTH in a pressurized culture system was used to disclose the signaling pathway underlying the effect of PTH on an AVF lesion. In patients receiving AVF creation, higher PTH was associated with an increased risk for maturation failure. In a mouse model, vascular wall thickness and myofibroblasts of AVF significantly increased with higher PTH. When the same mice were treated with cinacalcet, AVF lesions were attenuated by suppression of PTH. A cell model showed that PTH increased the marker of myofibroblasts, integrin β6 subunit (ITGB6), via the phosphorylated protein kinase B pathway. Finally, in the same model of mice AVF, higher PTH also increased the expression of ITGB6 in the smooth muscle layer of AVF, suggesting the transition to myofibroblast. Overall, our results suggest that higher PTH increased the risk of AVF maturation failure through increasing the transition of VSMCs to myofibroblasts. Lowering PTH may be a strategy to enhance AVF maturation.

Angiotensin-converting enzyme 2 augments the effects of endothelial progenitor cells-exosomes on vascular smooth muscle cell phenotype transition.

Phenotype transition of vascular smooth muscle cells (VSMCs) is implicated in vascular diseases. Angiotensin-converting enzyme 2 (ACE2) is a perspective cardiovascular target due to its ability of converting angiotensin (Ang II) to Ang (1-7). Our group recently showed that ACE2 can regulate the function of endothelial progenitor cell-derived exosomes (EPC-EXs). Here, we investigate whether ACE2 could affect the role of EPC-EXs on phenotype transition of VSMCs. After co-incubation with EXs released from EPC overexpressed ACE2 (EPC-EXsACE2), the ACE2 level and Ang II/Ang (1-7), proliferation/migration, phenotype gene, cytokine and NF-κB level on VSMCs were assessed. To determine the EX uptake route, VSMCs were pretreated with inhibitors. We found that (1) EPC-EXs and EPC-EXsACE2 were uptaken by VSMCs dominantly through caveolin-dependent endocytosis. (2) EPC-EXsACE2 remarkably increased the ACE2 level and decreased Ang II/Ang (1-7) in VSMCs activated by Ang II, whereas EPC-EXsACE2 pretreated by proteinase A blocked this effect. (3) EPC-EXsACE2 had better effects than EPC-EXs on reducing proliferation/migration activities and cytokine (MCP-1, TNF-α) secretion of Ang II-activated VSMCs. (4) EPC-EXs attenuated Ang II-induced VSMC synthetic phenotype change as evidenced by upregulated expressions of calponin and a-SMA and downregulated expressions of CRBP-1 and MYH10, associated with a decreased NF-κB level. EPC-EXsACE2 augmented these effects, which were attenuated by ACE2 inhibitor (DX600). In conclusion, EPC-EXsACE2 reduced Ang II-induced VSMC phenotype change by conveying functional ACE2 to downregulate the activated NF-κB pathway.

P0888FIBROBLAST GROWTH FACTOR 23 PRODUCES ARTERIAL STIFFNESS THROUGH CHANGES IN VASCULAR SMOOTH MUSCLE CELL PHENOTYPE

Abstract Background and Aims In patients with chronic kidney disease (CKD), high levels of c-terminal fibroblast growth factor 23 (FGF23) are associated with cardiovascular disease and mortality. Vascular smooth muscle cells (VSMC) may present two clearly differentiated functional phenotypes, contractile and synthetic. The abundance of synthetic phenotype is associated with vascular dysfunction and it is unknown whether in FGF23 may promote transition from a contractile to a synthetic phenotype in VSMC causing vascular stiffness. The present study was conducted to evaluate whether FGF23 affects VSMC phenotype and arterial stiffness. Method and Results High levels of FGF23 promoted VSMC transition from a contractile to a synthetic phenotype. These effects were mediated through FGFR1 and Ras/MAPK signaling activation. Inhibition of both pathways enhanced contractile phenotype of VSMC. The pro-contractile microRNAs, miR-221 and miR-222 were reduced by FGF23 and miR-221 transfection recovered the contractile phenotype of VSMC decreased by FGF23. In experimental rats, exogenous infusion of FGF23 produced an increase in vascular wall thickness with VSMC exhibiting synthetic phenotype and reduction of plasma levels of miR-221. Functional studies performed on aortic arterial rings revealed that passive and active forces were altered in rats treated with FGF23. In a group of CKD stage 2-3 patients with rather high levels of FGF23 it was observed an increased in pulse pressure reflecting vascular stiffness together with low plasma levels of miR-221 and miR-222. Conclusion FGF23 favors the transition of VSMC from contractile to synthetic phenotype causing vascular dysfunction and arterial stiffness; this may be a mechanism by which FGF23 contribute directly to the development of vascular disease in CKD patients.

Interleukin-1β-Induced Senescence Promotes Osteoblastic Transition of Vascular Smooth Muscle Cells

Introduction: Interleukin (IL)-1β, as a key biomarker and mediator of vascular calcification in patients with end-stage renal disease (ESRD), may be involved in the process of premature senescence of vascular smooth muscle cells (VSMCs). This work sought to investigate whether IL-1β-induced premature senescence contributes to the process of osteoblastic transition and vascular calcification in VSMCs. Methods: Eighty-eight patients with ESRD (aged 25–81 years), 11 healthy individuals, and 15 cases of lesion-free distal radial arteries from dialysis ESRD patients with angiostomy were collected in this study. Immunohistochemical analysis was performed to detect expression of IL-1β, p21, and bone morphogenetic protein-2 (BMP2) in the distal radial arteries. Primary human VSMCs from healthy neonatal umbilical cords were incubated with test agents for 1–3 days. Intracellular levels of reactive oxygen species (ROS) and senescence-associated-β-galactosidase (SA-β-gal) staining were used to detect senescent cells. Alizarin red staining and the calcium content of the cell layer were used to detect mineral deposition in VSMCs. Results: Coincident with positive staining of IL-1β, p21, and BMP2 in the lesion-free distal radial arteries, 66.67% patients showed mineral deposition. Serum IL-1β was 0.24 ± 0.57, 1.20 ± 2.95, and 9.41 ± 40.52 pg/mL in 11 healthy individuals, 20 patients without calcification, and 53 patients with calcification, respectively. Analysis of the cross-table chi-square test showed cardiovascular calcification is not correlated with levels of serum IL-1β in patients with ESRD (p = 0.533). In response to IL-1β, VSMCs showed a senescence-like phenotype, such as flat and enlarged morphology, increased expression of p21, an increased activity of SA-β-gal, and increased levels of ROS. IL-1β-induced senescence of VSMCs was required for the activation of IL-1β/NF-κB/p53/p21 signaling pathway. IL-1β-induced senescent VSMCs underwent calcification due to osteoblastic transition mainly depending upon the upregulation of BMP2. Resveratrol, an activator of sirtuin-1, postponed the IL-1β-induced senescence through blocking the NF-κB/p53/p21 pathway and attenuated the osteoblastic transition and calcification in VSMCs. Conclusions: High levels of IL-1β in medial smooth muscles of arteries may play roles in inducing senescence-associated calcification. IL-1β-induced senescence depending on the activation of the NF-κB/p53/p21 signaling pathway and contributing to osteoblastic transition of VSMCs.