Rat Aortic Vascular Smooth Muscle Research Articles

Fenoldopam-mediated increase in autophagy associates with recruiting WIPI2 protein in early endosomes in vascular smooth muscle cells

Autophagy, a crucial cellular homeostatic process, has protective roles in the pathogenesis of hypertension. Dopamine, a monoamine neurotransmitter, regulates autophagy in the kidney and vascular system. However, the exact roles and mechanisms of dopaminergic system on the regulation of autophagy is not well known. In rat aortic vascular smooth muscle cells, fenoldopam (FEN, 25 nM/15 min), a D1-like receptor agonist, increased the ATG5 and LC3-II (a late autophagosome maker) protein expression. Consistently, FEN decreased p62/SQSTM1 protein expression. The FEN-mediated increase in ATG5 was attenuated by SCH39166 (1 μM/15 min), a D1- like receptor antagonist, indicating that FEN increased autophagy in the autophagosome formation stage through its D1-like receptors. FEN increased the protein expression of WD Repeat Domain, Phosphoinositide Interacting 2 (WIPI2) in a concentration- and time-dependent manner, which is important in the early steps of autophagosome formation. FEN (25 nM, 15 min) increased the WIPI2 puncta fluorescence intensity and the colocalization of WIPI2 with EEA1 (Veh: 7.12±1.1% vs FEN: 9.96±0.9%, N=5, P < 0.001, Student’s t test), a typical phosphatidylinositol-3-phosphate binding protein in early endosomes, indicating that WIPI2 recruitment in early endosomes is important in the early stage of FEN-mediated autophagic process. Taken together, FEN increases autophagy with WIPI2 recruitment in the early endosomes in vascular smooth muscle cells. This work was supported by US National Institutes of Health grants R01DK134574 and R01DK119652. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Exploring the Impact of Nitric Oxide and Epidermal Growth Factor Receptor on Vascular Remodeling Induced by Mechanical Stretch

Hypertension is a key risk factor in many cardiovascular diseases like heart failure, kidney failure and vascular hypertrophy. Mechanical stretch/hypertension, promotes vascular smooth muscle cells remodeling by inducing leptin expression and other hypertrophic proteins like ERK1/2, AKT, and cofilin. Also the Epidermal growth factor receptor (EGFR) transactivation has shown to mediate vascular remodeling by enhancing VSMC hypertrophy, migration, and proliferation. Nitric oxide (NO) has been shown to be a critical factor for many cellular physiological function, it has the ability to induce VSMC relaxation leading to reduce hypertension. Moreover NO has received extensive attention as an anti- hypertrophic agent. Several Studies have shown that nitric oxide does not only promote vasodilation but it also inhibits VSMC proliferation. The major aim of this study was to investigate the inhibitory effect of NO and the effect of blocking the EGFR in mechanical stretch induced vascular remodeling. Methods: Rat portal veins (RPVs) were isolated and pretreated with/without NO donor (SNAP) or EGFR kinase blocker (AG1478) and then stretched to study their effects on mechanical stretch induced leptin synthesis and VSMC remolding. Western blot analysis was done to detect protein expression for leptin, adiponectin, eNOS p-ERK1/2 and p-AKT. Immunocytochemistry was performed on rat aortic vascular smooth muscle (RASMC) to detect GATA-4 nuclear translocation. Moreover, we used laser confocal microscopy on frozen RPV sections to detect the level of ROS formation and leptin. Finally, we performed qPCR for adiponectin, adipoR1 and adipoR2 mRNA. Results: In this study we showed the effect of mechanical stretch at different time points (1hour and 24 hours). Mechanical stretch-induced leptin expression was mediated by ERK1/2, AKT activations and ROS formation. These effects were associated with eNOS and adiponectin downregulation. Mechanical stretch had no effect on the expression of adiponectin receptors (adipoR1 and adipoR2). RASMCs pretreated with 3.1 nmol/L for 3 hours had induced GATA-4 nuclear translocation. Pre-incubation the RPV with SNAP significantly decreased mechanical stretch induced leptin expression, ERK1/2 and AKT phosphorylation. Moreover, SNAP significantly inhibited the adiponectin downregulation by mechanical stretch. Leptin-induced GATA-4 nuclear translocation was inhibited by SNAP. The involvement of EGFR transactivation in mechanical stretch-induced leptin expression was investigated using AG1478. Pretreatment of RPVs with AG1478 had no effect on mechanical stretch-induced early leptin expression (1 hour) while it inhibited mechanical stretch-induced late leptin expression (24 hours), ROS formation, and adiponectin and eNOS downregulation. However, treatment with AG1478 showed no effect on leptin-induced GATA-4 nuclear translocation. Conclusion: This study showed that NO has a direct inhibitory effect on mechanical stretch-induced AKT and ERK1/2 pathways and inhibits ROS formation that leads to vascular remodeling. On the other hand, EGFR transactivation was able to mediate mechanical stretch-induced vascular hypertrophy via ROS formation and leptin production, thus making EGFR and NO a promising therapeutic target for hypertension induced hypertrophy. This work was funded by Qatar University grants award UREP27-050-3-020 from Qatar National Research Fund (a member of Qatar Foundation) to AZ. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Adverse cardiovascular effects of long-term exposure to diethyl phthalate in the rat aorta

Phthalates are classified as priority environmental pollutants, since they are ubiquitous in the environment, have endocrine disrupting properties and can contribute to impaired health. Used primarily in personal care products and excipients for pharmaceuticals, diethyl phthalate (DEP) is a short-chain alkyl phthalate that has been linked to decreased blood pressure, glucose tolerance, and increased gestational weight gain in humans, while in animals it has been associated with atherosclerosis and metabolic syndrome. Although all these findings are related to risk factors or cardiovascular diseases, DEP's vascular impacts still need to be clarified. Thus, performing ex vivo and in vitro experiments, we aimed to understand the vascular DEP effects in rat. To evaluate the vascular contractility of rat aorta exposed to different doses of DEP (0.001–1000 μM), an organs bath was used; and resorting to a cell line of the rat aorta vascular smooth muscle, electrophysiology experiments were performed to analyse the effects of a rapid (within minutes with no genomic effects) and a long-term (24 h with genomic effects) exposure of DEP on the L-type Ca2+ current (ICa,L), and the expression of several genes related with the vascular function. For the first time, vascular electrophysiological properties of an EDC were analysed after a long-term genomic exposure. The results show a hormetic response of DEP, inducing a Ca2+ current inhibition of the rat aorta, which may be responsible for impaired cardiovascular electrical health. Thus, these findings contribute to a greater scientific knowledge about DEP's effects in the cardiovascular system, specifically its implications in the development of electrical disturbances like arrhythmias and its possible mechanisms.

Apelin Regulation of K-Cl Cotransport in Vascular Smooth Muscle Cells as a Potential Target for Cardiovascular Disease

Background/Aims: Apelin and its signaling through the G-protein coupled receptor (APJ, gene symbol APLNR) regulate cardiovascular function via two mechanisms: 1) By promoting nitric oxide (NO)-mediated vasodilation, impaired by oxidized low-density lipoproteins (oxLDL); and 2) By inducing cell proliferation via the phosphatidylinositol-3-kinase (PI3K)/protein kinase B/Akt pathway (PI3K/Akt) and mitogen activated protein kinase (MAPK) pathways. The potassium chloride cotransporter (KCC1,3,4; SLC12A4,6,7) controls cell volume, and regulates cardiovascular function through proliferation, migration, and blood pressure control. Importantly, KCC regulatory mechanisms and apelin/APJ signaling pathways overlap placing KCC as a potential target for apelin/APJ to elicit its cardioprotective effects. Therefore, apelin’s action on KCC activity was examined in contractile and synthetic rat aortic vascular smooth muscle cells (VSMCs). Methods: KCC activity was measured by atomic absorption spectrophotometry in chloride (Cl-) and Cl--free medium with sulfamate (Sf-) as Cl- replacement, and with rubidium (Rb+) as a potassium (K+) congener. The calculated difference between Rb+ transport in the presence of chloride (Cl-) and sulfamate (Sf-) is the Cl--dependent Rb+ influx (i.e., K-Cl cotransport activity). Apelin-13 (1 µM) was added either during flux (acute effect) and/or in the growth media (chronic effect) based on the experimental goals. KCC activity was characterized with respect to the VSMC phenotypes, in the presence or absence of apelin and corresponding inhibitors of the signaling pathways, oxLDL, and as a function of various physiological factors described below. Results: The APJ receptor was expressed in both contractile and synthetic VSMC phenotypes, the former also possessing the soluble guanylyl cyclase-coupled protein kinase G (PKG) receptor, critical for NO-mediated signaling. In general, KCC activity was higher in synthetic vs. contractile VSMCs, consistent with enhanced migration and proliferation in the former. In addition, apelin-mediated activation of KCC was modulated by extracellular sodium [Na+]o, osmolality, length of apelin treatment (acute or chronic) and VSMC phenotype (contractile vs synthetic). Based on selective inhibitors, apelin activated KCC through the (NO)/soluble guanylate cyclase (sGC)/protein kinase G (PKG) (NO/sGC/PKG)-, PI3K/Akt- and MAPK-dependent pathway(s). Furthermore, apelin rescued the inhibition of KCC by oxLDL. Altogether, results suggest apelin/APJ as an important modulator of KCC activity to sustain cell volume regulation and cardiovascular function. More recently, the apelinergic system has been proposed as a novel target for the treatment of Corona virus disease 2019 (COVID-19) and, given the significant overlap between the regulatory mechanisms of this system and KCC, and their role in cardiovascular disease (CVD), this study opens new avenues to identify potential targets for diverse implementation strategies. Conclusion: A better understanding of apelin effects on KCC will help design a novel therapeutic approach to treat atherosclerosis-linked cardiovascular diseases, including COVID-19-associated mortality.

Cultured rat aortic vascular smooth muscle cells do not express a functional TRPV1.

We showed previously that capsaicin, an active compound of chili peppers, can inhibit platelet-derived growth factor-induced proliferation in primary rat vascular smooth muscle cells (VSMCs). The inhibition of BrdU incorporation by capsaicin in these cells was revoked by BCTC, which might be explained by a role of TRPV1 in VSMCs proliferation. To further pursue the hypothesis of a TRPV1-dependent effect of capsaicin, we investigated TRPV1 expression and function. Commercially available antibodies against two different TRPV1 epitopes (N-terminus and C-terminus) were rendered invalid in detecting TRPV1, as shown: i) in western blot experiments using control lysates of TRPV1-expressing (PC-12 and hTRPV1 transfected HEK293T) and TRPV1-downregulated (CRISPR/Cas gene edited A10) cells, and ii) by substantial differences in staining patterns between the applied antibodies using fluorescence confocal microscopy. The TRPV1 agonists capsaicin, resiniferatoxin, piperine and evodiamine did not increase intracellular calcium levels in primary VSMCs and in A10 cells. Using RT qPCR, we could detect a rather low TRPV1 expression in VSMCs at the mRNA level (Cp value around 30), after validating the primer pair in NGF-stimulated PC-12 cells. We conclude that rat vascular smooth muscle cells do not possess canonical TRPV1 channel activity, which could explain the observed antiproliferative effect of capsaicin.

Trimethylamine-N-oxide (TMAO) promotes balloon injury-induced neointimal hyperplasia via upregulating Beclin1 and impairing autophagic flux

Background and aimsTMAO is a microbiota-dependent metabolite associated with increased risk of various cardiovascular diseases. However, the relationship between TMAO and vascular injury-related neointimal hyperplasia is unclear. This study aimed to explore whether TMAO promotes neointimal hyperplasia after balloon injury and elucidate the underlying mechanism. Methods and ResultsThrough hematoxylin and eosin staining and immunohistochemistry staining, we found that supplementary TMAO promoted balloon injury-induced neointimal hyperplasia, while reducing TMAO by antibiotic administration produced the opposite result. TMAO showed limited effect on rat aortic vascular smooth muscle cells (RAOSMCs) proliferation and migration. However, TMAO notably induced dysfunction of rat aortic vascular endothelial cells (RAOECs) in vitro and attenuated reendothelialization of carotid arteries after balloon injury in vivo. Autophagic flux was measured by fluorescent mRFP-GFP-LC3, transmission electron microscopy, and western blot. TMAO impaired autophagic flux, as evidenced by the accumulation of p62 and LC3II and high autophagosome to autolysosome ratios. Furthermore, we confirmed that Beclin1 level increased in TMAO-treated RAOECs and carotid arteries. Knocking down Beclin1 alleviated TMAO-induced autophagic flux impairment and neointimal hyperplasia. ConclusionsTMAO promoted neointimal hyperplasia through Beclin1-induced autophagic flux blockage, suggesting that TMAO is a potential target for improvement of vascular remodeling after injury.

Sclerostin is involved in osteogenic transdifferentiation of vascular smooth muscle cells in chronic kidney disease-associated vascular calcification with non-canonical Wnt signaling

Vascular calcification is prominent in patients with chronic kidney disease (CKD) and is a strong predictor of cardiovascular mortality in the CKD population. However, the mechanism underlying CKD-associated vascular calcification remains unclear. To identify potential therapeutic targets, a 5/6 nephrectomy rat model was established by feeding of a high-phosphorous diet as the CKD group and compared with sham group rats at 4 and 16 weeks. Sequencing analyses of the rat aorta revealed 643 upregulated and 1023 downregulated genes at 4 weeks, as well as 899 upregulated and 1185 downregulated genes at 16 weeks in the CKD group compared to the sham group. Bioinformatics analyses suggested that SOST (which encodes sclerostin) and Wnt signaling are involved in CKD-associated vascular calcification. Furthermore, protein-protein interactions analysis revealed interactions between SOST, WNT5A, and WNT5B, that involved runt-related transcription factor 2 (RUNX2) and transgelin (TAGLN). SOST was increased in CKD-associated vascular calcification following reduction of the Wnt signaling, including WNT5A and WNT5B, both in vivo and in vitro. TargetScan was used to predict the microRNAs (miRNAs) targeting WNT5A and WNT5B. The expression levels of miR-542-3p, miR-298-3p, miR-376b-5p, and miR-3568 were significantly reduced, whereas that of miR-742-3p was significantly increased in calcified rat aortic vascular smooth muscle cells (VSMCs). In CKD rat aortas, the expression of miR-542-3p, miR-298-3p, miR-376b-5p, miR-3568, miR-742-3p, and miR-22-5p were significantly reduced at both 4 and 16 weeks. Altogether, owing to several assessments, potentially diagnostic and prognostic biomarkers for improving common CKD diagnostic tools were identified in this study. Abbreviations: BUN: blood urea nitrogen; CKD: chronic kidney disease; CKD-MBD: chronic kidney disease-mineral bone disorder; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GO: the Gene Ontology; HE: hematoxylin-eosin; HRP: horseradish peroxidase; KEGG: Kyoto Encyclopedia of Genes and Genomes; MiRNAs: microRNAs; PAS: periodic acid-Schiff; RUNX2: runt-related transcription factor 2; SCr: serum creatinine; STRING: the Search Tool for the Retrieval of Interacting Genes/Proteins; TAGLN: transgelin; VSMC: vascular smooth muscle cell.

Mulberry Leaf and Neochlorogenic Acid Alleviates Glucolipotoxicity-Induced Oxidative Stress and Inhibits Proliferation/Migration via Downregulating Ras and FAK Signaling Pathway in Vascular Smooth Muscle Cell.

Mulberry leaf (Morus alba L.) has been used as a health food and in traditional medicine to treat several metabolic diseases, including diabetes, hypertension, and hyperlipidemia. However, the mechanism by which mulberry leaf and its functional components mediate atherosclerosis remains unclear. This study aimed to determine the effect of mulberry leaf extract (MLE) and its major component, neochlorogenic acid (nCGA), on the proliferation and migration of rat aortic vascular smooth muscle cells (VSMCs, A7r5 cell line) under diabetic cultured conditions (oleic acid and high glucose, OH). Our findings showed that MLE and nCGA significantly inhibited cell proliferation and migration in A7r5 cells as determined by a scratch wound assay and a Transwell assay. Furthermore, we observed MLE and nCGA inhibited cell proliferation and migration, such as reducing the phosphoinositide 3-kinases (PI3K)/protein kinase B (Akt), focal adhesion kinase (FAK), and small GTPase proteins using Western blot analysis. In conclusion, we confirmed the anti-atherosclerotic effects of MLE and nCGA in reducing vascular smooth muscle cell (VSMC) migration and proliferation under diabetic cultured conditions via inhibition of FAK/small GTPase proteins, PI3K/Akt, and Ras-related signaling.

RuBPY decreases intracellular calcium by decreasing influx and increasing storage.

cis-[Ru(bpy)2(py)NO2](PF6) (RuBPY) is a ruthenium complex nitric oxide (NO) donor that presents a nitrite in its moiety and has been shown to induce vasodilation in various arteries, as well as arterial pressure reduction with no changes in heart rate. Because vascular tone is highly dependent on the cytosolic calcium concentration ([Ca2+ ]c), the current study aimed to investigate the effects of RuBPY on the intracellular mobilization of calcium stores of rat aortic vascular smooth muscle cells. Vascular reactivity experiments were performed in isolated aortic rings that were contracted with a high concentration of KCl or phenylephrine (Phe). Moreover, primary cultured vascular smooth muscle cells were used to measure [Ca2+ ]c by confocal microscopy. The NO donor RuBPY decreased the [Ca2+ ]c and reduced KCl and Phe-induced contractile responses. The selective inhibitor of sarco-endoplasmic Ca-ATPase (SERCA) with thapsigargin impaired the effect of RuBPY on Phe-induced contractile response. RuBPY also reduced caffeine-induced contraction, and the contraction dependent on the capacitive Ca2+ influx. Therefore, our results suggest that NO released from RuBPY decreased [Ca2+ ]c by calcium influx blockade and activation of guanylyl-cyclase-cGMP-GK pathway. These results indicate that RuBPY increases Ca2+ storage in the sarcoplasmic reticulum by SERCA activation and also by capacitive Ca2+ influx inhibition, which is dependent on the intracellular release of nitric oxide from this compound.

Retraction of "Omarigliptin Prevents TNF-α-Induced Cellular Senescence in Rat Aorta Vascular Smooth Muscle Cells".

Retraction of "Omarigliptin Prevents TNF-α-Induced Cellular Senescence in Rat Aorta Vascular Smooth Muscle Cells".

Potential Actions of Baicalein for Preventing Vascular Calcification of Smooth Muscle Cells In Vitro and In Vivo.

Vascular calcification (VC) is associated with cardiovascular disease. Baicalein, a natural flavonoid extract of Scutellaria baicalensis rhizome has several biological properties which may inhibit VC. We investigated whether baicalein suppresses Runt-related transcription factor 2 (Runx2) and bone morphogenetic protein 2 (BMP-2) and upregulates smooth muscle 22-alpha (SM22-α) and alpha-smooth muscle actin (α-SMA). In an in vitro experiment, primary rat aortic vascular smooth muscle cells (VSMCs) were pretreated with 0.1, 1, and 5 μM baicalein, followed by β-glycerophosphate (β-GP) to induce calcification. In an in vivo experiment, VC was generated by vitamin D3 plus nicotine (VDN) administration to male Sprague Dawley (SD) rats randomly assigned into a control group, a VC group, a VC group pretreated with baicalein, and a baicalein alone group. Each group comprised 10 rats. Left ventricular (LV) morphology, function and performance were assessed by echocardiography. Calcium content was measured by Alizarin red S staining and alkaline phosphatase (ALP) activity assays. Apoptotic VSMCs were detected by flow cytometry. Protein levels and superoxide changes were evaluated using Western blotting and immunofluorescence assays respectively. Plasma malondialdehyde (MDA) was assayed. Baicalein pretreatment significantly reduced calcium content in calcified VSMCs (p < 0.001) as well as in VC rat aortic smooth muscle (p < 0.001). Additionally, ALP activity was decreased in calcified VSMCs and VC rat aortic smooth muscle (p < 0.001). Apoptosis was significantly attenuated by 1 μM baicalein pretreatment in calcified VSMCs. Runx2 and BMP-2 expressions were downregulated by the baicalein in calcified VSMCs. Baicalein pretreatment increased typical VSMCs markers SM22-α and α-SMA in calcified VSMCs. Baicalein pretreatment was associated with adverse changes in LV morphometry. Markers of oxidative stress declined, and endogenous antioxidants increased in VC rats pretreated with baicalein. Baicalein mitigates VC through the inhibition of Runx2/BMP-2 signaling pathways, enhancement of vascular contractile phenotype and oxidative stress reduction. However, our study is of basic experimental design; more advanced investigations to identify other molecular regulators of VC and their mechanisms of action is required.

Ubiquitin-specific protease 47 is associated with vascular calcification in chronic kidney disease by regulating osteogenic transdifferentiation of vascular smooth muscle cells

Chronic kidney disease (CKD) has recently become a serious health and social concern. Vascular calcification, a common complication of CKD, is a risk factor that increases the incidence and mortality of cardiovascular events in patients with CKD. However, there are currently no effective therapeutic targets that can facilitate treatment with fewer side effects for vascular calcification in CKD. To identify potential therapeutic targets, we performed label-free quantification (LFQ) analyses of protein samples from rat aortic vascular smooth muscle cells (RASMCs) after high-phosphorus treatment by nano-UPLC–MS/MS. We determined that ubiquitin-specific protease 47 (USP47) may be associated with CKD vascular calcification by regulating the osteogenic transdifferentiation of the vascular smooth muscle cell (VSMC) phenotype, thus suggesting a novel and potentially effective therapeutic target for CKD vascular calcification. USP47 knockdown significantly reduced the expression of β-transducin repeat-containing protein (BTRC), serine/threonine-protein kinase akt-1 (AKT1), Klotho, fibroblast growth factor (FGF23), and matrix Gla protein (MGP) in RASMCs after high-phosphorus treatment. Consistent with the results of protein–protein interaction (PPI) analyses, USP47 may be involved in regulating osteogenic transdifferentiation markers, such as runt-related transcription factor 2 (RUNX2), Klotho, FGF23, and MGP through the BTRC/AKT1 pathway upon CKD vascular calcification. These data indicate that USP47 may be associated with vascular calcification in CKD by regulating osteogenic differentiation of VSMCs. USP47 may regulate osteogenic transdifferentiation in VSMCs upon CKD vascular calcification through a process involving the BTRC/AKT1 pathway. This study identified a novel potential therapeutic target for the treatment of vascular calcification in CKD.

Profilin-1 is involved in macroangiopathy induced by advanced glycation end products via vascular remodeling and inflammation.

BACKGROUNDThe accumulation of advanced glycation end products (AGEs) have been implicated in the development and progression of diabetic vasculopathy. However, the role of profilin-1 as a multifunctional actin-binding protein in AGEs-induced atherosclerosis (AS) is largely unknown.AIMTo explore the potential role of profilin-1 in the pathogenesis of AS induced by AGEs, particularly in relation to the Janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3) signaling pathway.METHODSEighty-nine individuals undergoing coronary angiography were enrolled in the study. Plasma cytokine levels were detected using ELISA kits. Rat aortic vascular smooth muscle cells (RASMCs) were incubated with different compounds for different times. Cell proliferation was determined by performing the MTT assay and EdU staining. An AGEs-induced vascular remodeling model was established in rats and histological and immunohistochemical analyses were performed. The mRNA and protein levels were detected using real-time PCR and Western blot analysis, respectively. In vivo, shRNA transfection was performed to verify the role of profilin-1 in AGEs-induced proatherogenic mediator release and aortic remodeling. Statistical analyses were performed using SPSS 22.0 software.RESULTSCompared with the control group, plasma levels of profilin-1 and receptor for AGEs (RAGE) were significantly increased in patients with coronary artery disease, especially in those complicated with diabetes mellitus (P < 0.01). The levels of profilin-1 were positively correlated with the levels of RAGE (P < 0.01); additionally, the levels of both molecules were positively associated with the degree of coronary artery stenosis (P < 0.01). In vivo, tail vein injections of AGEs induced the release of proatherogenic mediators, such as asymmetric dimethylarginine, intercellular adhesion molecule-1, and the N-terminus of procollagen III peptide, concomitant with apparent aortic morphological changes and significantly upregulated expression of the profilin-1 mRNA and protein in the thoracic aorta (P < 0.05 or P < 0.01). Downregulation of profilin-1 expression with an shRNA significantly attenuated AGEs-induced proatherogenic mediator release (P < 0.05) and aortic remodeling. In vitro, incubation of vascular smooth muscle cells (VSMCs) with AGEs significantly promoted cell proliferation and upregulated the expression of the profilin-1 mRNA and protein (P < 0.05). AGEs (200 μg/mL, 24 h) significantly upregulated the expression of the STAT3 mRNA and protein and JAK2 protein, which was blocked by a JAK2 inhibitor (T3042-1) and/or STAT3 inhibitor (T6308-1) (P < 0.05). In addition, pretreatment with T3042-1 or T6308-1 significantly inhibited AGEs-induced RASMC proliferation (P < 0.05). CONCLUSIONAGEs induce proatherogenic events such as VSMC proliferation, proatherogenic mediator release, and vascular remodeling, changes that can be attenuated by silencing profilin-1 expression. These results suggest a crucial role for profilin-1 in AGEs-induced vasculopathy.

Omarigliptin Prevents TNF-α-Induced Cellular Senescence in Rat Aorta Vascular Smooth Muscle Cells.

Cellular senescence is one of the most significant factors involved in aging and age-related diseases. Senescence of vascular smooth muscle cells (VSMCs) adversely affects the function of the cardiovascular system and contributes to the development of atherosclerosis, hypertension, and other cardiovascular diseases. Glucagon-like peptide-1 (GLP-1) is an important incretin hormone involved in insulin release and vascular tone. GLP-1 is quickly degraded by the enzyme dipeptidyl peptidase-4 (DPP-4). Omarigliptin is a new DPP-4 inhibitor that has demonstrated anti-inflammatory and antioxidative stress properties. In the present study, we investigated the effects of the selective DPP-4 inhibitor omarigliptin (OMG) on VSMCs exposed to insult from tumor necrosis factor-α (TNF-α), one of the main inflammatory signaling molecules involved in cellular senescence. We found that OMG could suppress TNF-α-induced expression of pro-inflammatory cytokines (interleukin-1β (IL-1β), IL-6, and IL-8) and inhibit oxidative stress by reducing the production of H2O2 and protein carbonyl. OMG ameliorated the increase in senescence-associated β-galactosidase (SA-β-gal) and telomerase activity induced by TNF-α. The plasminogen activator inhibitor-1 (PAI-1)/p53/p21 pathway is a key inducer of cellular senescence. OMG ameliorated the acetylation of p53 at lysine 382 (K382) and subsequent activation of p21 via inhibition of PAI-1. Importantly, our experiments revealed that blockage of silent information-regulator 1 (SIRT1) abolished the inhibitory effects of OMG on p53 acetylation, SA-β-gal activity, and telomerase activity in VSMCs. These results suggest that OMG may have the potential to delay or prevent the progression of age-related cardiovascular diseases by modulating the activity of SIRT1.

Angiotensin-II activates vascular inflammasome and induces vascular damage

Angiotensin-II (Ang-II), a major target for treatment of cardiovascular disease, promotes cardiovascular dysfunction by directly modulating structure and function of vascular cells. Inflammasome components are expressed in the vasculature and are activated by specific stimuli. However, whether Ang-II activates the inflammasome in vascular cells or inflammasome activation contributes to Ang-II-induced vascular damage is still not fully elucidated. We tested the hypothesis that Ang-II induces endothelial dysfunction, vascular remodeling, and high blood pressure via inflammasome activation. C57BL6/J wild type (WT) and Caspase-1 knockout (Casp1−/−) mice were infused with vehicle or Ang-II for two weeks (490 ng/Kg/day) to determine whether the inflammasome contributes to vascular damage induced by Ang-II. Rat Aortic Vascular Smooth Muscle cells (RASMC) were used to determine if the interaction between Ang-II and inflammasomes causes migration and proliferation of vascular smooth muscle cells. Ex vivo studies revealed that Ang-II infusion induced vascular oxidative stress, endothelial dysfunction and vascular remodeling in WT mice. Casp1−/− mice were protected against Ang-II-induced vascular injury. In vitro experiments, Ang-II activated the NLRP3 inflammasome in RASMC, i.e. Ang-II increased Caspase-1 (Casp1) activity and cleavage of pro-interleukin (IL)-1β. MCC950 (NLRP3 receptor antagonist) prevented Ang-II-induced vascular migration and proliferation, but failed to reduce reactive oxygen species production. In conclusion, Ang-II leads to inflammasome activation in the vasculature contributing to endothelial dysfunction and vascular remodeling. Taken together, we place inflammasomes as a possible therapeutic target in conditions associated with increased Ang-II levels.

Oxidized low-density lipoprotein induced hepatoma-derived growth factor upregulation mediates foam cell formation of cultured rat aortic vascular smooth muscle cells

Vascular smooth muscle cells (SMCs) are important vascular components that are essential for the regulation of vascular functions during vascular atherosclerogenesis and vascular injury. Oxidized low-density lipoprotein (oxLDL) is known to induce SMC activation and foam cell transformation. This study characterized the role of hepatoma-derived growth factor (HDGF) in oxLDL-induced foam cell formation in cultured primary rat aortic SMCs. OxLDL exposure significantly increased HDGF expression and extracellular release. It also upregulated atherogenic regulators in SMCs, including TLR4, MyD88, LOX-1, and CD36. Exogenous HDGF stimulation not only increased the expression of cognate receptor nucleolin, but also the innate immunity regulators TLR4/MyD88 and lipid metabolism regulators, including LOX-1 and CD36. Oil red O staining showed that HDGF did not initiate, but enhanced oxLDL-driven foam cell formation in SMCs. Further signaling characterization demonstrated that oxLDL evoked activation of PI3K/Akt and p38 MAPK signaling pathways, both of which were involved in the upregulation of HDGF, LOX-1, and CD36 induced by oxLDL. Gene knockdown experiments using LOX-1 targeted siRNA demonstrated that LOX-1 expression was critical for oxLDL-induced HDGF upregulation, while HDGF gene depletion completely abolished oxLDL-triggered TLR4, LOX-1, and CD36 overexpression and foam cell formation in SMCs. These findings strongly suggest that oxLDL-induced HDGF upregulation participates in subsequent LOX-1 and CD36 expression in aortic SMCs and mechanistically contributes to the formation of SMC-derived foam cells. The oxLDL/LOX-1/HDGF axis may serve as a target for anti-atherogenesis therapy.

Fetuin‐A Induces Endothelial and Vascular Smooth Muscle Cell Dysfunction Through Nox1 and TLR4 Activation

Introduction Although studies demonstrate an important role for fetuin-A (FetA) in the inhibition of vascular calcification, compiling evidence suggests that FetA is also an endogenous ligand to toll-like receptor 4 (TLR4) and is involved in insulin resistance, inflammation and cardiovascular damage. The present study seeks to unravel the vascular effects and associated molecular mechanisms of FetA, focusing on oxidative stress and TLR4. Methods Rat aortic endothelial cells (RAEC), mesenteric arteries and vascular smooth muscle cells (VSMC) from Wistar-Kyoto (WKY) rats and wild-type (WT) or Nox1 KO mice were studied. Vascular function was analysed by wire myography and in some experiments in the presence or absence of FetA (50 ng/mL) and/or CLI095 (CLI – 1 μM – TLR4 inhibitor) and Tempol (10 μM – SOD mimetic). Levels of reactive oxygen species (ROS) were measured by chemiluminescence, Amplex Red and ELISA in the presence or absence of FetA (50 ng/mL) and/or CLI095 (CLI – 1 μM – TLR4 inhibitor) and a Nox1 inhibitor NoxA1ds (10 μM). Activation of eNOS inhibitory site (Thr495) was assessed by immunoblotting. Results FetA decreased acetylcholine (LogEC50: 7.32±0.06 vs control: 8.02±0.08) and sodium nitroprusside (LogEC50: 6.48±0.19 vs control: 7.38±0.12) induced relaxation (p<0.05), all effects reversed by Tempol and CLI095. Similar findings were observed in arteries from WT but not Nox1 KO mice. In RAECs, FetA increased eNOS inactivation (C=100% vs. FetA=194%±32.4 vs. FetA+CLI095=88.39±3.87) and ROS production (C=100% vs. FetA=231%±49.2 vs. FetA+CLI095=85.62±15.4) via TLR4 (p<0.05). ROS production (66±12.2%), as well as, H2O2 (45±8%) and ONOO- (105±31.6%) levels, were increased by FetA in VSMCs (vs. control, p<0.05); effects blocked by NoxA1ds and CLI095. Conclusion FetA influences vascular function through Nox1-ROS dependent mechanisms. FetA-induced endothelial and VSMC dysfunction involve TLR4. Our findings identify a novel system whereby FetA differentially influences vascular function through Nox1-ROS and TLR4, which in turn may influence vascular dysfunction during cardiovascular diseases independent of vascular calcification.

Dihydroartemisinin ameliorates balloon injury-induced neointimal formation through suppressing autophagy in vascular smooth muscle cells.

The present study was designed to investigate the therapeutic effects of injection of dihydroartemisinin (DHA) into the balloon-injured carotid arteries on balloon injury-induced neointimal formation and to explore whether autophagy is involved in the action of DHA. Percutaneous transluminal balloon angioplasty was performed in Sprague-Dawley rats to induce neointimal formation, immediately after which DHA (100μmol/l×1ml) and/or Rapamycin (1mg/100μl), were injected into the balloon-injured carotid arteries. After 14days, the serum samples and carotid artery tissues were harvested for analysis. Rat aortic vascular smooth muscle cells (VSMCs) were pretreated with DMSO (vehicle), DHA (1, 10, and 100μmol/l), or 3-methyladenine (3-MA; 10mM) for 1h and then stimulated with platelet-derived growth factor-BB (PDGF-BB; 10ng/ml) for another 24h. Animal experiments showed that DHA attenuated the balloon injury-induced neointimal formation, inflammation and VSMC phenotypic transition by inhibiting the balloon injury-induced autophagy activation. In vitro results showed that DHA attenuated the PDGF-BB-induced VSMC phenotypic transition, proliferation, and migration by inhibiting the PDGF-BB-induced autophagy activation. Taken together, DHA ameliorates balloon injury-induced neointimal formation through suppressing autophagy. This study provides insights into the development of a drug-eluting stent using DHA.

Heat Shock Protein 70 Is Associated With Cardioversion Outcome and Recurrence of Symptomatic Recent Onset Atrial Fibrillation in Hypertensive Patients.

Accumulating evidence indicates that heat shock proteins (HSPs) may represent a suitable biomarker to predict atrial fibrillation (AF). We investigated the relation of circulating serum HSP70 (sHSP70) with inflammatory cytokines and recurrence of symptomatic recent onset AF (ROAF). We enrolled 90 patients with ROAF (the duration from onset of symptoms ≤24 hours) and 30 controls. Patients received amiodarone for cardioversion and rhythm control. The association of serum HSP70, serum interleukin-2 (sIL-2), and serum interleukin-4 (sIL-4) with the presence of cardioversion and AF recurrence within a year was investigated. Toll-like receptor 4 (TLR4) signaling dependence for IL-2 and IL-4 induction in response to stimulation with HSP70 was tested in rat aortic vascular smooth muscle cell cultures. Patients had higher sHSP70 and sIL-2 and lower sIL-4 compared with controls. Serum HSP70 was independently associated with ROAF (P = 0.005) and correlated with sIL-2 (r = 0.494, P < 0.001) and sIL-4 (r = -0.550, P < 0.001). By 48 hours, 71 of the 90 patients were cardioverted, with noncardioverted patients having higher sHSP70 and sIL-2 and lower sIL-4, which were the only independent factors associated with cardioversion. AF recurred in 38 of the 71 cardioverted patients in 1 year. A cutoff value of sHSP70 ≥0.65 ng/mL and sIL-2 ≥0.21 pg/mL was the only independent factor associated with AF recurrence (hazard ratio: 3.311, 95% confidence interval: 1.503-7.293, P = 0.003 and hazard ratio: 3.144, 95% confidence interval: 1.341-7.374, P = 0.008, respectively). The exposure of smooth muscle cell to HSP70 in vitro increased the expression of IL-2 (5×) and IL-4 (1.5×) through TLR4-dependent and receptor-independent mechanisms. In conclusion, sHSP70 and sIL-2 might constitute a prognostic tool for determining the cardioversion and recurrence likelihood in ROAF.

CDKN2B-AS1 Aggravates the Pathogenesis of Human Thoracic Aortic Dissection by Sponge to miR-320d.

In the present study, the role and molecular mechanism of long noncoding RNA CDKN2B-AS1 in human thoracic aortic dissection (TAD), a highly lethal cardiovascular disease, was investigated. The expression of CDKN2B-AS1 in human TAD and normal aortic tissues of donors were examined by quantitative real-time polymerase chain reaction. RNA pull-down assay and a series of luciferase reporter assays were performed to predict the relationships between CDKN2B-AS1, miR-320d, and STAT3. Cell counting kit 8 (CCK-8), TUNEL, and western blot assays were applied to validate the biological functions of CDKN2B-AS1 in rat aortic vascular smooth muscle cells. Results showed that CDKN2B-AS1 was expressed at a higher level in human TAD than in normal aortic tissues. CDKN2B-AS1 overexpression significantly promoted apoptosis and suppressed the proliferation of vascular smooth muscle cells. CDKN2B-AS1 silence exhibited the opposite effects. Mechanistically, CDKN2B-AS1 was identified as a molecular sponge for miR-320d and positively modulated STAT3 expression via repressing miR-320d. In conclusion, our study revealed that CDKN2B-AS1 was dysregulated and displayed multiple potential functions in human TAD. These findings suggested that CDKN2B-AS1 was a novel potential therapeutic target for human TAD.