Rat Aortic Smooth Muscle Cells Research Articles

Huotan Jiedu Tongluo Decoction Inhibits Balloon-Injury-Induced Carotid Artery Intimal Hyperplasia in the Rat through the PERK-eIF2α-ATF4 Pathway and Autophagy Mediation.

In-stent restenosis (ISR) is the main factor affecting the outcome of percutaneous coronary intervention (PCI), and its main pathological feature is neointimal hyperplasia. Huotan Jiedu Tongluo decoction (HTJDTLD) is an effective traditional Chinese medicine (TCM) prescription for the treatment of vascular stenosis diseases. However, the precise anti-ISR mechanism of HTJDTLD remains unclear. Here, we investigated whether HTJDTLD can inhibit the excessive activation of endoplasmic reticulum stress (ERS) and reduce the level of autophagy factors through regulating the PERK-eIF2α-ATF4 pathway, thereby inhibiting the proliferation of the intima of blood vessels damaged by balloon injury (BI) and preventing the occurrence of ISR. In this study, a 2F Fogarty balloon was used to establish a common carotid artery (CCA) BI model in male Sprague-Dawley rats. Then, HTJDTLD (16.33 g/kg/d) or atorvastatin (1.19 mg/kg/d) was administered by gavage. Four weeks later, hematoxylin-eosin (HE) and Masson staining of the injured CCA were performed to observe the histological changes in the CCA. Immunohistochemistry (IHC) was used to assess the proliferation and dedifferentiation of vascular smooth muscle cells (VSMCs) in the CCA. Western blotting and RT-PCR were used to measure the expression of ERS- and autophagy-related proteins and mRNAs in the CCA. The results indicated that HTJDTLD significantly alleviated BI-induced carotid artery intimal hyperplasia and fibrosis and reduced the neointimal area (NIA) and NIA/medial area (MA) ratio. In addition, HTJDTLD inhibited the proliferation and dedifferentiation of VSMCs, reduced the expression of proliferating cell nuclear antigen (PCNA), and increased the smooth-muscle-α-actin- (SMα-actin-) positive area. HTJDTLD also significantly reduced the expression of the ERS-related factors: GRP78, p-PERK/PERK, p-eIF2α/eIF2α, ATF4, and CHOP. In addition, the expression of the autophagy-related factors, Beclin1, LC3B, and ATG12, was significantly decreased. In addition, in vitro experiments showed that HTJDTLD inhibited the above-mentioned ERS signal molecules in human umbilical vein endothelial cells (HUVEC) and rat aortic smooth muscle cells (A7R5) induced by tunicamycin (TM) and played a crucial role in protecting cells from damage. HTJDTLD may be a very promising drug for the treatment of ISR.

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.

5‐Fluorouracil Induces Vascular Calcification in Rat Aortic Smooth Muscle Cells

Background 5-Fluorouracil (5-FU) is a commonly prescribed chemotherapy used to treat malignancies of the gastrointestinal tract, breast, and head/neck. Despite its high clinical efficacy in treating cancer, it is associated with late occurring cardiotoxicity, specifically increases in arterial stiffness along with a heightened risk for developing heart failure and cardiovascular disease. Because increases in arterial stiffness and vessel calcification can occur concurrently and are associated with cardiovascular disease outcomes, we determined if 5-FU treated aortic smooth muscle cells adopted a calcified phenotype. We hypothesized 1) aortic smooth muscle cells resected from 5-FU treated rats would have a faster calcification onset when challenged with inorganic phosphate media and 2) a single in vitro dose of 5-FU in healthy cells would result in a calcified phenotype. Methods Primary aortic smooth muscle cell culture (ASMC) was performed via resection of rat thoracic aorta from 6-month-old Sprague Dawley rats (n= 8) that were treated with a clinically relevant continuous infusion of 5-FU (50mg/kg and 270mg/kg) or saline equivalent volume. After treatment and resection, ASMC were cultured using explant methods until cells were identifiable via smooth muscle actin staining. Cells derived from each group were cultured with normal media (NM) or media including a pathological concentration (3.2 mmol) of inorganic phosphate media (PI) for 5, 8, or 10 days to determine differences in calcification onset between conditions. In a second experiment, healthy rat aortic smooth muscle cells were incubated in NM or media containing 7x10-3 5-FU (NM + 5-FU) to determine if a single dose, in vitro, directly results in calcification. Calcification was quantified using an Alizarin Red staining assay and colorimetric calcium assay. Data are expressed as Alizarin Red Stain concentration (mM) and total calcium (mg/dL). Results In experiment 1, 5-FU+PI presented with significantly higher concentration of Alizarin Red at days 8 (.043 vs .031; p=.048) and 10 (.068 vs .017mM; p<0.0001) compared to 5-FU+NM whereas no differences were found between CON+PI and CON+NM at any timepoint (all p>0.05). These data were confirmed via Calcium assay in both 5-FU (.346 vs.235 mg/dl; p =0.0039) and CON (.293 vs .269 mg/dl; p=0.69) at the 8-day timepoint. Experiment 2 indicated that healthy rat aortic smooth muscle cells treated directly with 5-FU for 72 h expressed significantly greater calcium absorbance compared to CON (5-FU = .3347 mg/dl, CON = .2362 mg/dl; p=0.02). Conclusion After treatment with 5-FU, ASMCs from rat thoracic aorta explants are prone to a calcification phenotype compared to saline control treated cells. This predisposition with treatment with 5-FU is an ongoing investigation into the possible molecular mechanisms involved in oxidative stress signaling.

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.

An in-silico Investigation Into the Role of Strain and Structure on Vascular Smooth Muscle Cell Growth.

The orientation of vascular cells can greatly influence the in vivo mechanical properties and functionality of soft vascular tissues. How cell orientation mediates the growth response of cells is of critical importance in understanding the response of soft tissues to mechanical stimuli or injury. To date, considerable evidence has shown that cells align with structural cues such as collagen fibers. However, in the presence of uniaxial cyclic strain on unstructured substrates, cells generally align themselves perpendicularly to the mechanical stimulus, such as strain, a phenomenon known as “strain avoidance.” The cellular response to this interplay between structural cues and a mechanical stimulus is poorly understood. A recent in vitro experimental study in our lab has investigated both the individual and collective response of rat aortic smooth muscle cells (RASMC) to structural (collagenous aligned constructs) and mechanical (cyclic strain) cues. In this study, a 2D agent-based model (ABM) is developed to simulate the collective response of RASMC to varying amplitudes of cyclic strain (0–10%, 2–8%, 4–6%) when seeded on unstructured (PDMS) and structured (decellularized collagenous tissue) constructs. An ABM is presented that is fit to the experimental outcomes in terms of cellular alignment and cell growth on PDMS substrates, under cyclic strain amplitudes of (4–6%, 2–8%, 0–10%) at 24 and 72 h timepoints. Furthermore, the ABM can predict RASMC alignment and change in cell number on a structured construct at a cyclic strain amplitude of 0–10% after 10 days. The ABM suggests that strain avoidance behavior observed in cells is dominated by selective cell proliferation and apoptosis at these early time points, as opposed to cell re-orientation, i.e., cells perpendicular to the strain increase their rate of proliferation, whilst the rate of apoptosis simultaneously increases in cells parallel to the strain direction. The development of in-silico modeling platforms, such as that presented here, allow for further understanding of the response of cells to changes in their mechanical environment. Such models offer an efficient and robust means to design and optimize the compliance and topological structure of implantable devices and could be used to aid the design of next-generation vascular grafts and stents.

Adropin inhibits the phenotypic modulation and proliferation of vascular smooth muscle cells during neointimal hyperplasia by activating the AMPK/ACC signaling pathway.

In-stent restenosis (ISR) remains an inevitable problem for some patients receiving drug-eluting stent (DES) implantation. Intimal hyperplasia is an important biological cause of ISR. It has been previously reported that adropin is a potentially protective factor in cardiovascular disease. Therefore, the present study investigated the function of adropin in inhibiting smooth muscle cell (SMC) phenotype modulation and proliferation, causing intimal hyperplasia. A total of 56 patients who visited the hospital consecutively (25 with ISR and 31 without ISR), who were followed up between April 2016 and March 2019, 1 year following DES, were analyzed to evaluate the association between in-stent neointimal volume and adropin serum levels. Rat aorta smooth muscle cells (RASMCs) were used to determine the effects of adropin on their phenotypic modulation and proliferation using western blot, MTT, PCR and immunofluorescence analyses. Adropin serum levels in the ISR group were significantly lower than those in the non-ISR group. Furthermore, linear regression analysis revealed that only adropin levels were negatively associated with neointimal volume in both groups. The overall adropin levels of the 56 patients and the percentages of neointimal volume revealed a strong negative association. In vitro, adropin suppressed angiotensin II (Ang II)-induced phenotypic modulation in RASMCs by restoring variations of osteopontin and α-smooth muscle actin. Furthermore, compared with the Ang II group, adropin markedly decreased the percentage of G2/M-phase cells. Finally, adropin negatively regulated the phenotypic modulation and proliferation of RASMCs via the AMP-activated protein kinase/acetyl-CoA carboxylase (AMPK/ACC) signaling pathway. In conclusion, an independent, negative association was revealed between adropin and intimal hyperplasia; specifically, adropin inhibited the phenotypic modulation and proliferation of RASMCs by activating the AMPK/ACC signaling pathway. Therefore, adropin may be used as a potential predictor and therapeutic target for intimal hyperplasia and ISR.

Kahweol, a Diterpenoid Molecule, Inhibits CTGF-Dependent Synthetic Phenotype Switching and Migration in Vascular Smooth Muscle Cells.

Vascular smooth muscle cell (VSMC) phenotype switching from contractile to synthetic is essential for proliferation and migration in vascular pathophysiology. Connective tissue growth factor (CTGF) is a matricellular protein involved in cell adhesion, migration, and proliferation. Kahweol, a diterpene molecule in arabica coffee beans, has been reported to have anti-inflammatory, antiproliferative, and apoptotic effects in many cells. However, in VSMCs, the effects of kahweol on CTGF activities have not been investigated. Thus, in this study, the effects and associated mechanisms of kahweol in CTGF-dependent phenotype switching and migration in VSMCs were examined. Experiments were performed on primary rat aortic smooth muscle cells and a rat VSMC line, A7r5. Western blot analysis was used to determine the protein levels. The mRNA levels of synthetic markers were measured by qRT-PCR. Migration of VSMCs was evaluated by wound healing and transwell assays. Kahweol reduced the angiotensin II (Ang II)-induced CTGF expression. Further, kahweol inhibited expressions of synthetic phenotype markers of VSMC. The kahweol-reduced synthetic marker protein levels were reversed by the administration of rCTGF. However, expressions of contractile phenotype markers of VSMC were not affected. Kahweol suppressed Ang II-stimulated VSMC migration. Moreover, kahweol downregulated Ang II-induced p-FAK, p-Erk, and Yes-associated protein (YAP) protein expressions. Taken together, in Ang II-stimulated VSMCs, kahweol inhibited CTGF-dependent synthetic phenotype switching and migration, with focal adhesion kinase (FAK), Erk, and YAP involved in the underlying mechanisms of the kahweol effects. These results suggest that kahweol has a potential as a therapeutic agent to inhibit CTGF, which is a molecular target in sclerogenic vascular disease.

Involvement of Mitofusin2 on functional interaction between SR and mitochondria in rat aortic smooth muscle cells

Involvement of Mitofusin2 on functional interaction between SR and mitochondria in rat aortic smooth muscle cells

In Vivo Targeted Metabolomic Profiling of Prostanit, a Novel Anti-PAD NO-Donating Alprostadil-Based Drug.

Prostanit is a novel drug developed for the treatment of peripheral arterial diseases. It consists of a prostaglandin E1 (PGE1) moiety with two nitric oxide (NO) donor fragments, which provide a combined vasodilation effect on smooth muscles and vascular spastic reaction. Prostanit pharmacokinetics, however, remains poorly investigated. Thus, the object of this study was to investigate the pharmacokinetics of Prostanit-related and -affected metabolites in rabbit plasma using the liquid chromatography-mass spectrometry (LC-MS) approach. Besides, NO generation from Prostanit in isolated rat aorta and human smooth muscle cells was studied using the Griess method. In plasma, Prostanit was rapidly metabolized to 1,3-dinitroglycerol (1,3-DNG), PGE1, and 13,14-dihydro-15-keto-PGE1. Simultaneously, the constant growth of amino acid (proline, 4-hydroxyproline, alanine, phenylalanine, etc.), steroid (androsterone and corticosterone), and purine (adenosine, adenosine-5 monophosphate, and guanosine) levels was observed. Glycine, aspartate, cortisol, and testosterone levels were decreased. Ex vivo Prostanit induced both NO synthase-dependent and -independent NO generation. The observed pharmacokinetic properties suggested some novel beneficial activities (i.e., effect prolongation and anti-inflammation). These properties may provide a basis for future research of the effectiveness and safety of Prostanit, as well as for its characterization from a clinical perspective.

Vascular Cells Proteome Associated with Bradykinin and Leptin Inflammation and Oxidative Stress Signals.

Among the primary contributors to cardiovascular diseases are inflammation and oxidative imbalance within the vessel walls as well as the fibrosis of rat aortic smooth muscle cell (RASMC). Bradykinin (BK) and leptin are inflammatory modulators that are linked to vascular injury. In this study, we employed tandem LC-MS/MS to identify protein signatures that encompass protein abundance in RASMC treated with BK or leptin followed by systems biology analyses to gain insight into the biological pathways and processes linked to vascular remodeling. In the study, 1837 proteins were identified in control untreated RASMC. BK altered the expression of 72 (4%) and 120 (6.5%) proteins, whereas leptin altered the expression of 189 (10.2%) and 127 (6.5%) proteins after 24 and 48 h, respectively, compared to control RASMC. BK increased the protein abundance of leptin receptor, transforming growth factor-β. On the other hand, leptin increased the protein abundance of plasminogen activator inhibitor 1 but decreased the protein abundance of cofilin. BK and leptin induced the expression of inflammatory cytokines such as tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β) and pathway analysis revealed the activation of mitogen-activated protein kinases (MAPKs) and AKT pathways. The proteome profile in response to BK and leptin revealed mechanistic interplay of multiple processes that modulate inflammation and oxidative stress signals in the vasculature.

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.

Probucol decreases homocysteine-stimulated CRP production in rat aortic smooth muscle cells via regulating HO-1/NADPH oxidase/ROS/p38 pathway.

The elevated homocysteine level is an independent risk factor for atherosclerosis, which is characterized as a chronic inflammatory disease associated with oxidative stress. We have confirmed that homocysteine can stimulate the production of C-reactive protein (CRP) in rat aortic smooth muscle cells (RASMCs). In the present study, we investigated the role of probucol in homocysteine-induced CRP expression in cultured RASMCs and high-methionine-diet-induced hyperhomocysteinemic rats. The results showed that probucol decreased homocysteine-induced CRP mRNA and protein expression in RASMCs in a concentration-dependent manner. In addition, the animal experiment showed that probucol not only inhibited CRP expression in the vessel wall but also reduced the circulating CRP level in hyperhomocysteinemic rats. Further investigations revealed that probucol markedly increased heme oxygenase-1 activity, suppressed nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity, diminished superoxide anion generation, and decreased p38 phosphorylation in RASMCs and hyperhomocysteinemic rat aorta. These data demonstrate that probucol can inhibit homocysteine-induced CRP generation by interfering with the NADPH oxidase/p38 signal pathway in RASMCs, which will provide new evidence for the anti-inflammatory and anti-atherosclerotic effects of probucol.

Smooth Muscle Cell Responses to Poly(ε-Caprolactone) Triacrylate Networks with Different Crosslinking Time.

Poly(ε-caprolactone) triacrylate (PCLTA) is attractive in tissue engineering because of its good biocompatibility and processability. The crosslinking time strongly influences PCLTAs cellular behaviors. To investigate these influences, PCLTAs with different molecular weights were crosslinked under UV light for times ranging from 1 to 20 min. The crosslinking efficiency of PCLTA increased with decreasing the molecular weight and increasing crosslinking time which could increase the gel fraction and network stiffness and decrease the swelling ratio. Then, the PCLTA networks crosslinked for different time were used as substrates for culturing rat aortic smooth muscle cells (SMCs). SMC attachment and proliferation all increased when the PCLTA molecular weight increased from 8k to 10k and then to 20k at the same crosslinking time. For the same PCLTA, SMC attachment, proliferation, and focal adhesions increased with increasing the crosslinking time, in particular, between the substrates crosslinked for less than 3 min and longer than 5 min. This work will provide a good experimental basis for the application of PCLTA.

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.

Osteoglycin knockdown promotes vascular smooth muscle cell proliferation and migration in aortic dissection via the VEGF/VEGFR2 axis.

Extracellular matrix (ECM) proteins serve a major role in the pathogenesis of aortic dissection (AD). The aim of the present study was to investigate the effect of osteoglycin (OGN), an ECM proteoglycan, on aortic dissection (AD), as well as the underlying mechanism. Thoracic aortic tissues from 20patients with AD and healthy thoracic aortic tissue from 5patients undergoing coronary artery bypass grafting were collected to detect OGN expression levels. Following OGN knockdown in rat aortic smooth muscle cells, cell proliferation was detected by performing Cell Counting Kit‑8 and BrdU assays, cell migration was assessed by performing the wound healing assay, cell invasion was detected by performing the Transwell assay, and VEGFR/AKT signaling pathway‑related protein expression levels were measured via western blotting. The results demonstrated that OGN expression was significantly downregulated in patients with AD compared with healthy controls. Compared with the si‑negative control(NC) group, OGN knockdown promoted RASMC proliferation and migration. Compared with the si‑NC group, OGN knockdown also significantly enhanced the phosphorylation of the downstream signaling molecules of VEGFR, including AKT and ERK1/2, in VEGF‑stimulated RASMCs. Collectively, the present study indicated that OGN knockdown facilitated RASMC proliferation and migration by activating AKT and ERK1/2 signaling. Therefore, OGN may serve as a novel therapeutic target for AD.

Upregulation of iNOS Protects Cyclic Mechanical Stretch-Induced Cell Death in Rat Aorta Smooth Muscle Cells.

Aortic dissection and aneurysm are associated with abnormal hemodynamic loads originating from hypertension. Our previous study demonstrated that cyclic mechanical stretch (CMS, mimicked hypertension) caused the death of rat aortic smooth muscle cells (RASMCs) in a mitogen activated-protein kinases (MAPKs)-dependent manner. The current study investigated the effects of inducible nitric oxide synthase (iNOS) on CMS-induced RASMC death. cDNA microarrays for CMS-treated RASMCs showed that iNOS expression levels were increased in response to CMS. Real-time polymerase chain reaction (PCR) analysis demonstrated that this increase was p38 MAPK (p38)-dependent. NO production was also increased. This increase could be inhibited by p38 and iNOS inhibitors. Thus, CMS-induced iNOS synthesized NO. CMS-induced cell death in RASMCs was increased by the iNOS inhibitor but abrogated by the long-acting NO donor DETA-NONOate. Increased iNOS expression was confirmed in the abdominal aortic constriction mouse model. Signal transducers and activators of transcription 1 (STAT1) was activated in stretched RASMCs, and iNOS expression and NO production were inhibited by the STAT1 inhibitor nifuroxazide. Our findings suggest that RASMCs were protected by iNOS from CMS-stimulated cell death through the STAT1 and p38 signal pathways independently.

On the biocompatibility of graphene oxide towards vascular smooth muscle cells

Graphene and its derivatives have shown fascinating potential in biomedical applications. However, the biocompatibility of graphene with vascular smooth muscle cells (VSMCs) and applications to vascular engineering have not been explored extensively. Using a rat aortic smooth muscle cell line, A7r5, as a VSMC model, we have explored the effects of graphene oxide (GO) on the growth and behaviours of VSMCs. Results demonstrated that GO had no obvious toxicity to VSMCs. Cells cultured on GO retained the expression of smooth muscle cell-specific markers CNN1, ACTA2 and SMTN, on both mRNA and protein levels. A wound healing assay demonstrated no effect of GO on cell migration. We also found that small-flaked GO favoured the proliferation of VSMCs, suggesting a potential of using surface chemistry or physical properties of GO to influence cell growth behaviour. These results provide insight into the suitability of GO as a scaffold for vascular tissue engineering.

Circulating Ligands of the Receptor for Advanced Glycation End Products and the Soluble Form of the Receptor Modulate Cardiovascular Cell Apoptosis in Diabetes.

We determined whether plasma concentrations of the receptor for advanced glycation end products (RAGE) and the soluble (s) form of RAGE (sRAGE) in healthy individuals and patients with type 2 diabetes (T2D) modulate vascular remodeling. Healthy individuals and patients with T2D were divided into two age groups: young = <35 years old or middle-aged (36–64 years old) and stratified based on normal glucose tolerance (NGT), impaired (IGT), and T2D. Plasma titers of sRAGE, the RAGE ligands, AGEs, S100B, S100A1, S100A6, and the apoptotic marker Fas ligand Fas(L) were measured by enzyme-linked immunosorbent assay (ELISA). The apoptotic potential of the above RAGE ligands and sRAGE were assessed in cultured adult rat aortic smooth muscle cells (ASMC). In NGT individuals, aging increased the circulating levels of AGEs and S100B and decreased sRAGE, S100A1 and S100A6. Middle-aged patients with T2D presented higher levels of circulating S100B, AGEs and FasL, but lower levels of sRAGE, S100A1 and S100A6 than individuals with NGT or IGT. Treatment of ASMC with either AGEs or S100B at concentrations detected in T2D patients increased markers of inflammation and apoptosis. Responses attenuated by concomitant administration of sRAGE. In middle-aged patients with T2D, lower circulating plasma levels of sRAGE may limit decoy and exogenous trapping of deleterious pro-apoptotic/pro-inflammatory RAGE ligands AGEs and S100B, increasing the risk for diabetic complications.

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.