CXCL5 participates in carotid plaque formation by inducing vascular calcification / 中国组织工程研究

BACKGROUND:CXC motif chemokine 5(CXCL5)is a neutrophil activating peptide derived from epithelial cells,which may be involved in arterial diseases.However,there is yet no report on the effect of CXCL5 in vascular calcification. OBJECTIVE:To explore the role of CXCL5 in the vascular calcification of carotid atherosclerosis(CAS). METHODS:(1)Cytological experiment:Mouse vascular smooth muscle cells(VSMCs)were divided into five groups:osteogenic medium group,Vector group(vector,blank plasmid transfected into VSMCs),CXCL5 group(CXCL5 plasmid transfected into VSMCs),si-NC group(CXCL5 negative control siRNA transfected into VSMCs),si-CXCL5 group(CXCL5 siRNA transfected into VSMCs),Vector+LY2157299 group and CXCL5+LY2157299 group(LY2157299 transferred into the cells 24 hours after cell transfection).Alizarin red staining,alkaline phosphatase staining,and calcium content determination were performed to evaluate the osteogenic differentiation level of VSMCs.(2)Animal experiment:Forty-eight ApoE-/-mice were randomly divided into four groups(n=12 per group):Con+si-NC group,Con+si-CXCL5 group,CAS+si-NC group and CAS+si-CXCL5 group.Animal models were not prepared in the first two groups,in which si-NC or si-CXCL5 lentivirus was injected into the tail vein;carotid atherosclerosis models were made in the latter two groups,in which si-NC or si-CXCL5 lentivirus was injected into the tail vein.Von Kossa staining and immunohistochemical staining were used to evaluate carotid vascular calcification and the expression of CXCL5 and transforming growth factor-β receptor 1(TGFBR1)in mice. RESULTS AND CONCLUSION:In the CXCL5 group,the protein level of runt-related transcription factor 2(RUNX2)was up-regulated and the level of α-smooth muscle actin was down-regulated,in contrary to the findings in the si-CXCL5 group.In addition,CXCL5 overexpression upregulated the level of TGFBR1,while CXCL5 knockdown inhibited the level of TGFBR1.Compared with the Vector group,the intensity of alizarin red staining,alkaline phosphatase activity and calcium content in the CXCL5 group increased significantly(P<0.05).Compared with the si-NC group,the intensity of alizarin red staining,alkaline phosphatase activity and calcium content in the si-CXCL5 group decreased significantly(P<0.05).When LY2157299 inhibited TGFBR1 expression,the osteogenic differentiation of VSMCs induced by CXCL5 was reduced.Compared with the Con+si-NC group,the expression of CXCL5 protein in the carotid artery and calcification area in the CAS+si-NC group increased significantly(P<0.05).Compared with the CAS+si-NC group,the expression of CXCL5 protein in the carotid artery and vascular calcification area in the CAS+si-CXCL5 group decreased significantly(P<0.05).In addition,compared with the Con+si-NC group,the expression of RUNX2 protein in the carotid artery in the CAS+si-NC group increased significantly(P<0.05),while the expression of α-smooth muscle actin protein decreased significantly(P<0.05).Compared with the CAS+si-NC group,the expression of RUNX2 protein in the carotid artery in CAS+si-CXCL5 group decreased significantly(P<0.05),while the expression of α-smooth muscle actin protein increased significantly(P<0.05).In conclusion,CXCL5 can induce osteogenic transformation of VSMCs by activating the TGFBR1 pathway,and inhibition of CXCL5 expression is effective in improving carotid arterial calcification in CAS mice.

Proliferation and metabolic patterns of smooth muscle cells during construction of tissue-engineered blood vessels / 中国组织工程研究

BACKGROUND:Seed cells are seeded on three-dimensional scaffold materials,and three-dimensional culture in bioreactors is a common in vitro tissue engineering culture method,but the changes in cell proliferation and metabolic patterns in bioengineered blood vessel construction are still unclear. OBJECTIVE:To explore the metabolic changes of cells such as oxygen consumption and their causes during the whole process of biological vascular tissue construction by in vitro bioreactor. METHODS:The self-built vascular bioreactor system was used as the platform;bovine vascular smooth muscle cells were used as the seed cells,and a conventional CO2 incubator provided the external gas environment for the cultivation process.Seed cells were seeded on a tubular porous polyglycolic acid scaffold material for three-dimensional culture,and the whole process included a one-week resting period and a seven-week pulsating tensile stress stimulation loading period.A non-invasive monitoring system was built,and the optical dissolved oxygen patch method was used to monitor the changes of dissolved oxygen in the culture solution in the reactor,and the glucose consumption and lactic acid production were measured by regular sampling.CCK-8 assay was used to determine the proliferation of smooth muscle cells on polyglycolic acid three-dimensional scaffold materials.Nicotinamide adenine dinucleotide oxidation state and reduction state ratio(NAD+/NADH)was utilized to understand cell proliferation and metabolism in the early stage of culture.RT-qPCR and western blot assay were applied to detect the expression of proliferation-related genes(Ki67)and glycolysis-related genes(GLUT-1,LDHA). RESULTS AND CONCLUSION:(1)The dissolved oxygen level in the culture solution was(4.314±0.380)mg/L from the cell injection to the end of the resting period(the first week),and gradually stabilized at(1.960±0.866)mg/L after the tensile stress stimulation(the last seven weeks);the two had significant changes(P<0.05).(2)The ratio of glucose consumption to lactic acid production in the cell culture medium YL/G increased rapidly after the cells were injected,and the highest value was above 1 on the fifth day,and then slow down to 0.5(The mean value of YL/G in the resting period was 0.89 and the mean value in the pressurized period was 0.57,P<0.05).(3)CCK-8 assay results showed that A450 value gradually increased after the cells were injected,and reached the highest value on the fifth day,reaching 3.17,and then slowly decreased.At the same time,it was found that Ki67 mRNA was up-regulated on the third day of culture,and then declined.The expression level of Ki67 protein was higher from the third day to the fifth day.(4)The detection of NAD+/NADH showed that the increase was obvious from the fifth to the seventh day after the injection of cells,and the expression of glycolysis-related genes(GLUT-1 and LDHA)was up-regulated and changed synchronously,and the relative expression was higher in the first five days.(5)The results showed that the tissue-engineered blood vessels were constructed using the vascular bioreactor and the smooth muscle cells in the early stage mainly proliferated and exhibited a metabolic feature of low oxygen consumption.The metabolic characteristics of high oxygen consumption were observed during the pulsatile tensile stress stimulation stage.

Progression of research into the relationship between pyroptosis and atherosclerosis / 中国比较医学杂志

Pyroptosis is a programmed mode of cell death.Activated caspase-1 can induce the occurrence of pyroptosis,promote the release of inflammatory factors,and trigger a violent inflammatory response.Depending on the type of caspase involved,pyroptosis can be divided into a caspase-1-mediated typical inflammasome pathway and a human caspase-4/5(or mouse caspase-11)-mediated atypical inflammasome pathway.In recent years,studies have found that pyroptosis is closely related to the occurrence,development,and prognosis of atherosclerosis.This article reviews the roles and mechanisms of endothelial cells,vascular smooth muscle cells,and macrophage cells in the occurrence and development of atherosclerosis,with the aim of promoting new ideas for research into the pathogenesis,diagnosis,and treatment of atherosclerosis

Impact of the transforming growth factor-β pathway on vascular restenosis and its mechanism / 中南大学学报(医学版)

As a crucial regulatory molecule in the context of vascular stenosis, transforming growth factor-β (TGF-β), plays a pivotal role in its initiation and progression. TGF-β, a member of the TGF-β superfamily, can bind to the TGF-β receptor and transduce extracellular to intracellular signals through canonical Smad dependent or noncanonical signaling pathways to regulate cell growth, proliferation, differentiation, and apoptosis. Restenosis remains one of the most challenging problems in cardiac, cerebral, and peripheral vascular disease worldwide. The mechanisms for occurrence and development of restenosis are diverse and complex. The TGF-β pathway exhibits diversity across various cell types. Hence, clarifying the specific roles of TGF-β within different cell types and its precise impact on vascular stenosis provides strategies for future research in the field of stenosis.

A nonobstructive condition: Medial arterial calcification / 中南大学学报(医学版)

Vascular calcification, including intimal and medial calcification, is closely associated with a significant increase in cardiovascular diseases. Although increased understandings were achieved, people still know much more about intimal calcification than medial calcification because the latter doesn't obstruct the arterial lumen, commonly considered as a non-significant finding. We clarified the pathologic characteristic of medial calcification, its difference from intimal calcification, principally focused on its clinical relevance, such as diagnosis, nosogenesis, and hemodynamics. We underline the importance of identifying and distinguishing medial calcification, understanding its effect to local/systematic arterial compliance, and relationship to diabetic neuropathy. Recent studies emphasize do not ignore its predictive role in cardiovascular mortality. It is of great clinical significance to summarize the mechanisms of occurrence, lesion characteristics, diagnostic methods, pathogenic mechanisms, hemodynamic changes, and the distinction as well as association of intimal calcification with intimal calcification.

Protective mechanism of salvianolic acid B on blood vessels / 中国中药杂志

Salvianolic acid B(Sal B) is the main water-soluble component of Salvia miltiorrhiza Bunge. Studies have found that Sal B has a good protective effect on blood vessels. Sal B can protect endothelial cells by anti-oxidative stress, inducing autophagy, inhibiting endoplasmic reticulum stress(ERS), inhibiting endothelial inflammation and adhesion molecule expression, inhibiting endothelial cell permeability, anti-thrombosis, and other ways. In addition, Sal B can alleviate endothelial cell damage caused by high glucose(HG). For vascular smooth muscle cell(VSMC), Sal B can reduce the synthesis and secretion of inflammatory factors by inhibiting cyclooxygenase. It can also play a vasodilatory role by inhibiting Ca~(2+) influx. In addition, Sal B can inhibit VSMC proliferation and migration, thereby alleviating vascular stenosis. Sal B also inhibits lipid deposition in the subendothelium, inhibits macrophage conversion to foam cells, and reduces macrophage apoptosis, thereby reducing the volume of subendothelial lipid plaques. For some atherosclerosis(AS) complications, such as peripheral artery disease(PAD), Sal B can promote angiogenesis, thereby improving ischemia. It should be pointed out that the conclusions obtained from different experiments are not completely consistent, which needs further research. In addition, previous pharmacokinetics showed that Sal B was poorly absorbed by oral administration, and it was unstable in the stomach, with a large first-pass effect in the liver. Sal B had fast distribution and metabolism in vivo and short drug action time. These affect the bioavailability and biological effects of Sal B, and the development of clinically valuable Sal B non-injectable delivery systems remains a great challenge.

Terpine-4-ol on inhibiting vascular smooth muscle cell calcification by improving mitochondrial dynamics / 药学学报

The aim of this study was to investigate the role and mechanism of terpinen-4-ol (T4O) on high glucose (HG) -induced calcification in vascular smooth muscle cell (VSMC). To investigate the role of T4O on HG-induced calcium deposition, osteogenic phenotypic transformation and mitochondrial dynamics in VSMC, Mdivi-1, a mitochondrial dynamin-related protein 1 (Drp-1) inhibitor, was used to analyze the correlation between mitochondrial dynamics and VSMC calcification and the role of T4O. Alizarin red S staining was used to observe calcium salt deposition and flow cytometry to detect intracellular Ca2+ content; Western blot and immunofluorescence were used to detect the expression of phenotypic switching-related markers α-smooth muscle actin (α-SMA), bone morphogenetic protein 2 (BMP2) and Runt related transcription factor 2 (Runx2), and mitochondrial dynamics-related markers mitofusin 1 (MFN1), mitofusin 2 (MFN2) and Drp-1. The results showed that low and high doses of T4O could inhibit HG-induced down-regulation of α-SMA, MFN1 and MFN2 expression levels, and up-regulation of BMP2, Runx2 and Drp-1 expression levels, reduce intracellular Ca2+ content and calcium salt deposition, and effectively inhibit HG-induced VSMC calcification and mitochondrial dynamics disorders. The T4O group, Mdivi-1 group and T4O+Mdivi-1 group were able to up-regulate the expression levels of HG-induced α-SMA, MFN1 and MFN2, down-regulate the protein expression levels of BMP2, Runx2 and Drp-1, and inhibit calcium salt deposition, and there was no significant difference between the above indexes in the T4O and T4O+Mdivi-1 groups. The above findings suggest that T4O can inhibit the expression level of Drp-1, regulate the disturbance of mitochondrial dynamics, and suppress HG-induced VSMC calcification.

Mechanism of Huoxue Jiedu Huayu Recipe Ameliorates Kidney Vascular Smooth Muscle Cell Pyroptosis in the Contralateral Kidneys of 6-Month UUO Rats via MR/NLRP3 Pathway / 世界科学技术-中医药现代化

Objective The intervention effect of Huoxue Jiedu Huayu Recipe on the pyroptosis of vascular smooth muscle cells(VSMC)on the contralateral renal vessel of 6-month unilateral ureteral obstruction(UUO)rat model was observed,and part of the treatment mechanism of Huoxue Jiedu Huayu Recipe on chronic kidney disease(CKD)was investigated.Methods Forty male wistar rats were randomly divided into 4 groups:the sham operation(Sham)group,the unilateral ureteral obstruction(UUO)group,the eplerenone treatment(EPL)group and the Huoxue Jiedu Huayu Recipe intervention(HJHR)group,10 rats in each group.The EPL group was given eplerenone and the HJHR group was given traditional Chinese medicine.The right kidney was collected after 6 months.Pathological changes were assessed by HE staining and Masson staining.The expressions of Cysteinyl aspartate specific proteinase1(Caspase-1),Interleukin-1β(IL-1β),Serum and glucocorticoid-induced kinase 1(SGK1)and nuclear factor κappa-B(NF-κB)were detected by immunohistochemistry.The DNA damage in renal VSMC was examined by TUNEL staining.Rat VSMCs were used for in vitro experiments.The cells were divided into 4 groups using the random number method:the control(CON)group,the aldosterone(ALD)group,the aldosterone plus eplerenone treatment(EPL)group and the aldosterone plus Huoxue Jiedu Huayu Recipe treatment(HJHR)group.The ALD group was given aldosterone stimulation for 24 h.The EPL group and the HJHR group were given eplerenone and rat serum containing 10%Huoxue Jiedu Huayu Recipe pretreatment before aldosterone stimulation,respectively.The apoptosis rate was detected by flow cytometry.Perforation of gasdermin D(GSDMD)and nuclear translocation of NR3C2(Encode MR)in cell membranes were examined by immunofluorescence.The protein expression of inflammation and pyroptosis-related signaling molecules were examined by western blotting.The mRNA expression of pyroptosis signaling pathway were detected by qRT-PCR.Results Compared with the Sham group,DNA damage of renal blood vessels and VSMCs was increased in UUO group,and expressions of Caspase-1,IL-1β,SGK1 and NF-κB were increased.Compared with the UUO group,the DNA damage of renal blood vessels and VSMCs in EPL and HJHR groups was reduced,and the expressions of Caspase-1,IL-1β,SGK1 and NF-κB were decreased.Compared with the CON group,the ALD group showed pyroptosis and DNA damage of rat VSMC.The expression of NR3C2,NLRP3,Caspase-1,GSDMD,IL-1β,SGK1 and NF-κB was up-regulated.Compared with the ALD group,the pyroptosis and DNA damage were alleviated,and the expression of NR3C2,NLRP3,Caspase-1,GSDMD,IL-1β,SGK1 and NF-κB was decreased in EPL and HJHR groups.Conclusion Huoxue Jiedu Huayu Recipe reduce the vascular damage of the contralateral kidneys of UUO by inhibiting the activation of the MR/NLRP3 inflammasome pathway and inhibiting the pyroptosis of VSMC.

Role of nicotinamide phosphoribosyltransferase in delaying smooth muscle cell senescence and protecting abdominal aortic aneurysm / 中华危重病急救医学

Objective:To investigate the protective effect of nicotinamide phosphoribosyltransferase (NAMPT) on abdominal aortic aneurysm by delaying the senescence of aortic vascular smooth muscle cells (VSMC).Methods:The primary VSMC cells from normal and patients with abdominal aortic aneurysm were cultured by tissue adherence method. Cells were divided into normal human-derived VSMC group (Ctrl-VSMC group), abdominal aortic aneurysm patient-derived VSMC group (AAA-VSMC group), and angiotensinⅡ(AngⅡ) in vitro abdominal aortic aneurysm model group (AngⅡ-VSMC group, 100 nmol/L AngⅡ treated normal human-derived VSMC for 48 hours), AngⅡ+P7C3 group and AAA+P7C3 group after NAMPT agonist P7C3 intervention (adding 5 μmol/L P7C3 on the basis of AngⅡ-VSMC group and AAA-VSMC group, respectively). Immunofluorescence staining was used to identify VSMC; cell proliferation-associated antigen Ki67 staining was used to detect cell proliferation; senescence associated β-galactosidase (SA-β-gal) staining was used to detect cell senescence in each group; Western blotting was used to detect the protein expression levels of senescence-related proteins p21, p16 and NAMPT in each group. Results:Compared with the Ctrl-VSMC group, the positive rate of SA-β-gal staining and the expression levels of senescence-related proteins p21 and p16 in the AAA-VSMC group and AngⅡ-VSMC group were significantly increased [SA-β-gal staining positive rate: (74.1±4.4)%, (68.6±5.5)% vs. (36.8±10.3)%, p21/GAPDH: 0.61±0.07, 0.51±0.03 vs. 0.31±0.03, p16/GAPDH: 0.77±0.03, 0.72±0.06 vs. 0.33±0.26, all P < 0.01]. However, the expression of NAMPT was significantly decreased (NAMPT/GAPDH: 0.88±0.07, 0.79±0.14 vs. 1.29±0.02, both P < 0.01). Compared with the AngⅡ-VSMC group, the positive rate of SA-β-gal staining and the expressions levels of senescence-related proteins p21 and p16 in the AngⅡ+P7C3 group were significantly lower [SA-β-gal staining positive rate: (49.1±3.2)% vs. (68.6±5.5)%, p21/GAPDH: 0.35±0.06 vs. 0.51±0.03, p16/GAPDH: 0.47±0.08 vs. 0.72±0.06, all P < 0.05], while the expression of NAMPT was significantly increased (NAMPT/GAPDH: 1.15±0.06 vs. 0.79±0.14, P < 0.01). Compared with the AAA-VSMC group, the positive rate of SA-β-gal staining and the expression levels of senescence-related proteins p21 and p16 in the AAA+P7C3 group were significantly lower [SA-β-gal staining positive rate: (54.1±6.0)% vs. (74.1±4.4)%, p21/GAPDH: 0.38±0.02 vs. 0.61±0.07, p16/GAPDH: 0.50±0.13 vs. 0.77±0.03, all P < 0.05], but the expression of NAMPT was significantly increased (NAMPT/GAPDH: 1.25±0.28 vs. 0.88±0.07, P < 0.01). Conclusion:NAMPT agonist P7C3 can delay the senescence of VSMC and play a protective role in abdominal aortic aneurysm.

Research advance of Nrf2 on atherosclerosis by regulating vascular smooth muscle cell / 浙江大学学报·医学版

Atherosclerosis is a common pathological change in cardiovascular disease. Vascular smooth muscle cell is the main source of plaque cell and extracellular matrix, and nuclear factor-erythroid 2-related factor 2 (Nrf2) is a key transcription factor regulating the function of vascular smooth muscle cell. In the process of atherosclerosis, Nrf2 signaling pathway has the following regulatory effects on vascular smooth muscle cell: regulating the phenotype of vascular smooth muscle cell to change to the direction conducive to the alleviation of disease progression; inhibiting the proliferation and migration of vascular smooth muscle cell; mitigating the level of blood lipid; alleviating vascular smooth muscle cell calcification, aging and apoptosis process. This article reviews the specific mechanisms of Nrf2 regulating atherosclerosis, such as phenotypic transformation, proliferation and migration, lipid metabolism, calcification, aging and apoptosis in atherosclerosis, in order to provide a basis for understanding the molecular mechanism of atherosclerosis development and finding therapeutic targets.

Functional role and underlying mechanisms of neuropilin-1 in proliferation and migration of rat vascular smooth muscle cells / 中国胸心血管外科临床杂志

@#Objective    To investigate the role and potential mechanisms of neuropilin-1 (NRP1) in the pathogenesis of vein graft failure. Methods    The rat vascular smooth muscle cells (VSMCs) were transfected with NRP1-shRNA adenovirus and negative control adenovirus respectively. Cell counting kit-8, flow cytometry, Transwell and Western blot were used to investigate the effects of inhibition of NRP1 on VSMCs proliferation viability, apoptosis, migration capacity and its downstream signaling pathway protein expression. Results    The proliferation and migration of rat VSMCs could be inhibited after down-regulation of NRP1, and the increase of apoptosis was also observed. Moreover, inhibition of NRP1 significantly reduced Akt and NF-κB phosphorylation in rat VSMCs, but had little effect on activation of ERK1/2. Conclusion    NRP1 may promote vein graft hyperplastic remodeling by regulating the proliferation and migration of VSMCs through PI3K/Akt and NF-κB pathways, but further animal study is required.

Quercetin-induced apoptosis ameliorates vascular smooth muscle cell senescence through AMP-activated protein kinase signaling pathway

Aging is one of the risk factors for the development of cardiovascular diseases. During the progression of cellular senescence, cells enter a state of irreversible growth arrest and display resistance to apoptosis. As a flavonoid, quercetin induces apoptosis in various cells. Accordingly, we investigated the relationship between quercetin-induced apoptosis and the inhibition of cellular senescence, and determined the mechanism of oxidative stress-induced vascular smooth muscle cell (VSMC) senescence. In cultured VSMCs, hydrogen peroxide (H₂O₂) dose-dependently induced senescence, which was associated with increased numbers of senescence-associated β-galactosidase-positive cells, decreased expression of SMP30, and activation of p53-p21 and p16 pathways. Along with senescence, expression of the anti-apoptotic protein Bcl-2 was observed to increase and the levels of proteins related to the apoptosis pathway were observed to decrease. Quercetin induced apoptosis through the activation of AMP-activated protein kinase. This action led to the alleviation of oxidative stress-induced VSMC senescence. Furthermore, the inhibition of AMPK activation with compound C and siRNA inhibited apoptosis and aggravated VSMC senescence by reversing p53-p21 and p16 pathways. These results suggest that senescent VSMCs are resistant to apoptosis and quercetin-induced apoptosis attenuated the oxidative stress-induced senescence through activation of AMPK. Therefore, induction of apoptosis by polyphenols such as quercetin may be worthy of attention for its anti-aging effects.

The effect of PKD1 gene on autophagy in aortic smooth muscle cells / 中国胸心血管外科临床杂志

@#Objective    To explore the effects of PKD1 gene on mouse aortic smooth muscle (MOVAS) cells autophagy. Methods    The shRNA and over-expression lentiviral vectors for the target gene of PKD1 were constructed. MOVAS cells were infected by a number of successful packaging shRNA (PKD1 knockdown) or ETS-1 (PKD1 over-expressing) lentiviral vectors, and qPCR was used to test interference and over-expressing effects. Then qPCR and Western blotting were used to detect the expression levels of autophagy markers including Atg5, Beclin1 and LC3 in control group, shPKD1 group and ETS-1 group. Results    Compared with the control group, PKD1 mRNA level was decreased in the shPKD1 group (P<0.05); ETS-1 and PKD1 mRNA levels were increased in the ETS-1 group (P<0.05). In contrast with the control group, the mRNA levels of autophagy markers including Atg5 (P<0.05) and Beclin1 (P<0.01) were obviously decreased in the shPKD1 group, but they were obviously increased in the ETS-1 group (P<0.001). Protein levels of Atg5, Beclin1 and LC3 were significantly decreased in the shPKD1 group (P<0.05), but they were increased obviously in the ETS-1 group (P<0.05) in contrast with the control group. Conclusion    PKD1 gene is involved in MOVAS cells autophagy, low expression of PKD1 gene can inhibit autophagy and high expression of PKD1 promotes autophagy in vascular smooth muscle cells.

Berberine Inhibits Mechanical Stretch Stress induced MAPK Phosphorylation，Proliferation and Migration of Vascular Smooth Muscle Cells / 中山大学学报(医学科学版)

@#【Objective】To observe whether berberine can inhibit vascular smooth muscle cells（VSMC）proliferation induced by mechanical strength stress and to investigate the role of MAPK pathway in it.【Methods】The cultured VSMC were divided into 4 groups：negative control group（NC group），stretch stress group（SS group），berberine pretreated and stretch stress stimulation group（BBR+SS group），and berberine group. In NC group，phosphate buffer saline was used as a negative control；in SS group，stretch stress was given to VSMC；in BBR+SS group，VSMC were pretreated with berberine for 1 hour and then exposed to stretch stress；in BBR group，VSMC were treated only with berberine for 1 hour and cultured in serum- free DMEM afterwards. We collected VSMC in each group ，detected and analyzed their MAPK phosphorylation，proliferation and migration by using Western blotting，immunofluorescence and wound-healing assay respectively. 【Results】 Compare with NC group，stretch stress markedly induced VSMC proliferation and migration ，which could be inhibited significantly by berberine. Stretch stress obviously increased phosphorylation of MAPK （ERK，JNK，p38），which could be inhibited by berberine in a concentration dependent manner. 【Conclusion】 Berberine inhibits hypertension-induced proliferation and migration of VSMC through MAPK pathway. The results revealed the new use and mechanism of berberine，and provided important data for further study on the prevention and treatment of vascular remodeling caused by abnormal increase of mechanical stress in hypertension.

Therapeutic effects of albendazole on mice with artery stenosis and its mechanism / 上海交通大学学报（医学版）

Objective • To investigate the effect of albendazole (ABZ) on mouse femoral arteries restenosis and explore its possible mechanism. Methods • Vascular smooth muscle cells (VSMCs) were cultured in vitro with 0.5 and 1 μmol/L ABZ, and evaluated for cell proliferation, migration, and apoptosis by MTT, Transwell assay, and Annexin V-PI staining flow cytometry, respectively; and Western blotting was also used for the analysis of phosphorylation mechanism of cytoskeleton proteins cofilin (CFL) and myosin light chain (MLC). Stenosis was established in the unilateral femoral artery of 10-week-old wildtype male C57BL/6 mice by perivascular cuff placement and high fat chow breeding for 4 weeks. After successful modeling, mice were randomly divided into control group (equal volume of solvent) and ABZ group (1.5 mg/d) for gavage, and femoral arteries were collected 4 weeks later for H-E histological analysis of intimal area, medial area, and intima/media (I/M) ratio to clarify the severity of restenosis. Results • Both 0.5 and 1 μmol/L ABZ could significantly inhibit the proliferation and migration of VSMCs (both P<0.05), while 0.5 μmol/L had no significant effect on the apoptosis of VSMCs. ABZ gavage could significantly reduce the neointimal area and I/M ratio in the restenosis mice, while there were no effects on the median area. Both 0.5 and 1 μmol/L ABZ treatment could significantly inhibit CFL dephosphorylation and MLC phosphorylation, which showed a concentration-dependent trend (both P<0.05). Conclusion • ABZ inhibits VSMCs migration and intimal hyperplasia, via affecting the phosphorylation of cytoskeleton protein CFL and MLC, thereby resulting in therapeutic effects on restenosis of mice.

Inhibition Effect of Ginkgo biloba Extract(EGb761) on the Phenotypic Switch of Vascular Smooth Muscle Cells through Regulation of APMK/KLF4 Signaling Pathway / 中国药学杂志

OBJECTIVE: To explore the effects of Ginkgo biloba extract (EGb761) on platelet derived growth factor (PDGF)-induced phenotypic switch of vascular smooth muscle cells (VSMCs) and its potential mechanisms. METHODS: VSMCs were cultured in vitro, and 20 ng•mL-1 PDGF was used to induce the phenotypic switch of VSMCs. MTT assay and wound healing assay were performed to determine the effects of various concentration (1, 10, 100 μg•mL-1) of EGb761 on cell proliferation and migration, respectively; immunofluorescence and Western blot assay were used to detect the arrangement of myofilament, the expression of phenotypic proteins including α-SMA, calopnin and OPN, as well as the protein expression of AMPK/KLF4 signaling pathway. RESULTS: Compared with the control group, PDGF significantly promoted the proliferation and migration of VSMCs. However, EGb761 treatment inhibited PDGF-induced cell proliferation and migration in a concentration-dependent manner. Compared with the control group, PDGF treatment induced disordered arrangement of myofilament and reduced the fluorescence intensity of F-actin. In addition, PDGF significantly decreased the expression of α-SMA and calponin, whrease increased the expression of OPN in VSMCs, when compared with the control group. VSMCs in PDGF+EGb761 group showed well-aligned myofilament and enhanced the fluorescence intensity of F-actin; the expressions of α-SMA and calponin were increased and OPN was decreased in the PDGF+EGb761 group when compared with the PDGF group. Meanwhile, as compared with the control group, PDGF increased the level of phosphorylated AMPK and the expression levels of KLF4, which was inhibited by the addition of EGb761 in a concentration dependent manner. After inhibition of AMPK/KLF4 signaling pathway with the use of specific AMPK pathway inhibitor compound C, the inhibitory effect of EGb761 on PDGF-mediated phenotypic switch of VSMCs was enhanced; vice versa, the activation of AMPK/KLF4 signaling pathway with AMPK pathway activator AICAR and the inhibitory effect of EGb761 on PDGF-mediated phenotypic switch of VSMCs were reversed. CONCLUSION: EGb761 inhibits PDGF-mediated phenotypic switch of VSMCs by targeting APMK/KLF4 signaling pathway.

Effect and mechanism of rosuvastatin preconditioning on inflammatory cytokines release in vascular smooth muscle cells of middle cerebral artery of rats after focal cerebral ischemia-reperfusion / 解剖学报

Objective To explore the effect and probable mechanism of rosuvastatin preconditioning on inflammatory cytokines interleukin (IL) -1β, IL-6 and tumor necrosis factor a (TNF-α) release in vascular smooth muscle cells (VSMCs) of middle cerebral artery after ischemia-reperfusion. Methods Thirty-six healthy SD rats were randomly assigned into three groups; sham operation group, focal cerebral ischemia-reperfusion group and rosuvastatin preconditioning group. There were 12 rats in each group. At the 24th hour of reperfusion after middle cerebral artery occlusion (MCAO) for 2 hours, the mRNA and protein expression of IL-1β, IL-6 and TNF-α release in VSMCs of middle cerebral artery were detected by Real-time PCR and Western blotting, respectively. Also, the mRNA and protein expression of nuclear factor κB (NF-κB) were measured by Real-time PCR and Western blotting. Results At the 24th hour of reperfusion after MCAO for 2 hours, the mRNA and protein expression of IL-Iβ, IL-6 and TNF-ot were markedly up-regulated in rats of model group; rosuvastatin preconditioning can significantly inhibited overexpression of IL-1β, IL-6 and TNF-ot at the mRNA and protein levels. And the decreasing of mRNA and protein expression of NF-κB was also found in this study. Conclusion Rosuvastatin preconditioning can decrease the release of inflammatory cytokines IL-1β, IL-6 and TNF-α in VSMCs of MCA. It can relieve the inflammatory injury after ischemia-reperfusion in brain. The effect of rovastatin on IL-1β,IL-6 and TNF-ot may be related to the reduction of the expression of NF-κB in VSMCs.

rBMSCs/ITGA5B1 Promotes Human Vascular Smooth Muscle Cell Differentiation via Enhancing Nitric Oxide Production

BACKGROUND AND OBJECTIVES: Previous studies have shown that integrins alpha5beta1 (ITGA5B1) gene-modified rat bone marrow mesenchymal stem cells (rBMSCs) could prevent cell anoikis and increase the nitric oxide (NO) production. Here we examined the capability of rBMSCs/ITGA5B1 on the phenotype modulation of Human Pulmonary Artery Smooth Muscle Cell (HPASMC) in vitro. METHODS AND RESULTS: The synthetic (dedifferentiated) phenotype of HPASMC was induced by monocrotaline (MCT, 1μM) for 24 h and then co-cultured with rBMSCs/ITGA5B1 in a transwell culture system. The activation of NO/cGMP (nitric oxide/Guanosine-3′, 5′-cyclic monophosphate) signaling was investigated in HPASMC. The changes of pro-inflammatory factors, oxidative stress, vasodilator, vasoconstrictor, contractile and synthetic genes, and the morphological changes of HPASMC were investigated. The results of this study showed that the NO/cGMP signal, endothelial nitric oxide synthase (eNOS) expression, the expression of the vasoprotective genes heme oxygenase-1 (HMOX1) and prostaglandin-endoperoxide synthase 2 (PTGS2) were increased, but the expression of transforming growth factor-β1 (TGF-β1), CCAAT/enhancer-binding proteins delta (Cebpd), Krüppel-like factor 4 (KLF4), and activating transcription factor 4 (ATF4) were reduced in MCT treated HPASMC co-cultured with rBMSCs/ITGA5B1. The synthetic smooth muscle cells (SMCs) phenotype markers thrombospondin-1, epiregulin and the vasoconstrictor endothelin (ET)-1, thromboxane A2 receptor (TbxA2R) were down-regulated, whereas the contractile SMCs phenotype marker transgelin expression was up-regulated by rBMSCs/ITGA5B1. Furthermore, rBMSCs/ITGA5B1 promoted the morphological restoration from synthetic (dedifferentiation) to contractile (differentiation) phenotype in MCT treated HPASMC. CONCLUSIONS: rBMSCs/ITGA5B1 could inhibit inflammation and oxidative stress related genes to promote the HPASMC cell differentiation by activation NO/cGMP signal.

Effects of rosuvastatin in homocysteine induced mouse vascular smooth muscle cell dedifferentiation and endoplasmic reticulum stress and its mechanisms / 中国应用生理学杂志

OBJECTIVE@#To investigate the effect of rosuvastatin on homocysteine (Hcy) induced mousevascular smooth muscle cells(VSMCs) dedifferentiation and endoplasmic reticulum stress(ERS).@*METHODS@#VSMCs were co-cultured with Hcy and different concentration of rosuvastatin (0.1, 1.0 and 10 μmol/L). Cytoskeleton remodeling, VSMCs phenotype markers (smooth muscle actin-α, calponin and osteopontin) and ERS marker mRNAs (Herpud1, XBP1s and GRP78) were detected at predicted time. Tunicamycin was used to induce, respectively 4-phenylbutyrate(4-PBA) inhibition, ERS in VSMCs and cellular migration, proliferation and expression of phenotype proteins were analyzed. Mammalian target of rapamycin(mTOR)-P70S6 kinase (P70S6K) signaling agonist phosphatidic acid and inhibitor rapamycin were used in Rsv treated VSMCs. And then mTOR signaling and ERS associated mRNAs were detected.@*RESULTS@#Compared with Hcy group, Hcy+ Rsv group (1.0 and 10 μmol/L) showed enhanced α-SMA and calponin expression (<0.01), suppressed ERS mRNA levels (<0.01) and promoted polarity of cytoskeleton. Compared with Hcy group, Hcy+Rsv group and Hcy+4-PBA group showed suppressed proliferation, migration and enhanced contractile protein expression (<0.01); while tunicamycin could reverse the effect of Rsv on Hcy treated cells. Furthermore, alleviated mTOR-P70S6K phosphorylation and ERS (<0.01)were observed in Hcy+Rsv group and Hcy+rapamycin group, compared with Hcy group; while phosphatidic acid inhibited the effect of Rsv on mTOR signaling activation and ERS mRNA levels (<0.01).@*CONCLUSIONS@#Rosuvastatin could inhibit Hcy induced VSMCs dedifferentiation suppressing ERS, which might be regulated by mTOR-P70S6K signaling.

Correlation and modulation of smooth muscle cells phenotypic switching induced by cytomegalovirus / 中华实验和临床病毒学杂志

Objective@#To explore the influence of murine cytomegalovirus on phenotypic modulation of vascular smooth muscle cell and modulation of PI3K/Akt pathway.@*Methods@#Male apoE knockout mice were injected abdominally with 2×105 PFU MCMV, followed by 16 weeks feeding. Then aortas were sectioned for HE staining and immunohistochemical staining of smooth muscle 22 alpha (SM22α) and osteopontin (OPN). Mouse aortic smooth muscle cells(MOVAS)were incubated with MCMV, then proliferation of MOVAS and expression of SM22a and OPN were tested. Western blotting test was applied to reveal MCMV’s modulation of PI3K/Akt pathway.@*Results@#The degree of atherosclerosis of apoE-/- mice in MCMV infection group was severe than that in control group, and OPN stain positive signals predominated in the arterial wall. After 24 hours of incubation with MCMV by 3×104 PFU, the expression of SM22a decreased (P=0.023), while OPN increased (P=0.034) in MOVAS. MCMV increased expression of Akt phosphorylation compared with the control group (P=0.035). The inhibitor of PI3K pathway LY294002 not only inhibited the phenotypic modulation of smooth muscle by MCMV, but also blocked the Akt phosphorylation after MCMV infection (P=0.031), however no significant influence was observed in control group.@*Conclusions@#MCMV induces phenotypic modulation of vascular smooth muscle, PI3K/Akt signaling pathway may be involved in the process of MCMV promoting the phenotype transformation of smooth muscle cells.