Down-regulation of the Smad signaling by circZBTB46 via the Smad2-PDLIM5 axis to inhibit type I collagen expression / 老年心脏病学杂志（英文版）

BACKGROUND@#Abnormal type I collagen (COL1) expression is associated with the development of many cardiovascular diseases. The TGF-beta/Smad signaling pathway and circRNAs have been shown to regulate COL1 gene expression, but the underlying molecular mechanisms are still not fully understood.@*METHODS@#Gain- and loss-of-function experiments were prformed to study the effect of circZBTB46 on the expression of alpha 2 chain of type I collagen (COL1A2). Co-immunoprecipitation assay was performed to observe the interaction between two proteins. RNA immunoprecipitation assay and biotin pull-down assay were performed to observe the interaction of circZBTB46 with PDLIM5.@*RESULTS@#In this study, we investigated the role of circZBTB46 in regulating COL1A2 expression in human vascular smooth muscle cells (VSMCs). We found that circZBTB46 is expressed in VSMCs and that TGF-beta inhibits circZBTB46 formation by downregulating KLF4 expression through activation of the Smad signaling pathway. CircZBTB46 inhibits the expression of COL1A2 induced by TGF-beta. Mechanistically, circZBTB46 mediates the interaction between Smad2 and PDLIM5, resulting in the inhibition of Smad signaling and the subsequent downregulation of COL1A2 expression. Furthermore, we found that the expression of TGF-beta and COL1A2 is decreased, while circZBTB46 expression is increased in human abdominal aortic aneurysm tissues, indicating that circZBTB46-mediated regulation of TGF-beta/Smad signaling and COL1A2 synthesis in VSMCs plays a crucial role in vascular homeostasis and aneurysm development.@*CONCLUSIONS@#CircZBTB46 was identified as a novel inhibitor of COL1 synthesis in VSMCs, highlighting the importance of circZBTB46 and PDLIM5 in regulating TGF-beta/Smad signaling and COL1A2 expression.

Gene polymorphism of CYP450 2C9 and VKORC1 in Chinese population and their relationships to the maintaining dosage of warfarin / 中华流行病学杂志

Objective To investigate the distribution of gene polymorphism of CYP450 2C9 and VKORC1-1639A/G in the Chinese population as well as the difference of genetic polymorphism between Chinese Han population and other ethnic populations.Contribution of CYP2C9 and VKORC1 genotype to the maintenance doses on warfarin was also studied.Methods The genotype and allele frequencies were calculated and compared with those in other populations.One hundred and one patients with stable anticoagulation with warfarin under a target international normalized ratio(INR)of 2.0 to 3.0 were enrolled for studying the relationship between the CYP2C9 and VKORC1 gene polymorphism and the warfarin maintaining dosage.Results CYP450 2C9~*3 + 1075C/A allele frequencies were:AA in 449 cases(92.2%),AC in 36 cases(7.4%)and CC in 2 cases(0.4%),respectively.VKORC1-1639A/G allele frequencies were AA in 415 cases(85.2%),GA in 72 cases(14.8%),but GG in no case(0.0%),respectively.When linear stepwise regression analysis was used to identify factors contributing to warfarin stable dose,the final equation was:ln(D)=0.346 + 0.017(weight)-0.376(CYP450 2C9~*3 + 1075C/A)+ 0.148(VKORC1-1639A/G)-0.002(age)(r=0.827,P=0.02).Conclusion There existed significant gene polymorphism CYP450 2C9~*3 + 1075C/A and VKORC1-1639A/G in the Chinese Han population.Both Gene polymorphisms of CYP450 2C9*3 + 1075C/A and VKORC1-1639A/G were significantly affecting the maintaining dose of warfarin in the Chinese population.

The molecular mechanisms of SM22alpha in cytoskeleton remodeling of vascular smooth muscle cells / 中国应用生理学杂志

<p><b>AIM</b>To investigate the molecular mechanisms of smooth muscle 22 alpha (SM22alpha) whereby cytoskeleton remodeling of vascular smooth muscle cells (VSMCs) is regulated.</p><p><b>METHODS</b>Synthetic (dedifferentiated) VSMCs were converted to contractile (differentiated) VSMCs by serum deprivation. Cells were transfected with pEGFP-SM22alpha, and localization of SM22alpha and its relationship with F-actin were observed through fluorescence microscopy. Fractional extraction of proteins and Western blotting were used to detect polymerization of SM alpha-actin in antisense-pcD2-SM22alpha-transfected VSMCs.Furthermore, effect of SM22alpha on F-actin cross-linking was observed by F-actin polymerization experiment.</p><p><b>RESULTS</b>Fluorescence microscopy showed that SM22alpha co-localized with F-actin in contractile VSMCs. Western blotting of protein extracts from F-/G-actin fractions revealed that polymerization of SM alpha-actin was lower in antisense-pcD2-SM22alpha-transfected VSMCs, in which SM alpha-actin mostly existed as soluble G-actin. Moreover, F-actin polymerization in vitro also showed that GST-SM22alpha could promote cross-linking of F-actin to form thick and bundled stress fibres,while extracts from VSMCs transfected with antisense-pcD2-SM22alpha could not effectively induce the polymerization of F-actin.</p><p><b>CONCLUSION</b>SM22alpha acts as a modulator to participate in VSMC cytoskeleton remodeling. It can not only induce polymerization of G-actin to F-actin, but also promote cross-linking of F-actin to bundled stress fibres, indicating its vital role in cytoskeleton remodeling.</p>

Expression of osteopontin RGD peptide in E. coli and its biological activities / 中国应用生理学杂志

<p><b>AIM</b>The six copies of RGD sequences present in osteopontin were expressed in the E. coli, and the biological activity of the purified products was studied.</p><p><b>METHODS</b>cDNA fragments containing six copies of RGD sequences were subcloned into prokaryotic expression vector pGEX-3X including GST coding sequence to construct pGEX-3X-RGD plasmids. E. coli DH5alpha transformed by pGEX-3X-RGD(6) plasmid was induced by different IPTG concentrations for different times to identify the optimal induction condition. Induced GST-RGD fusion protein was purified via GST-Sepharose 4B affinity resin.</p><p><b>RESULTS</b>GST-RGD fusion proteins containing six copies of RGD sequences could be successfully induced and were mainly located in inclusion bodies. After being denatured and dialyzed, renatured fusion proteins were purified via GST-Sepharose 4B affinity resin. GST-RGD(6) fusion protein could specifically inhibit adhesion and migration of VSMC stimulated by osteopontin, which could be considered as a basis on producing small peptides containing RGD sequences for inhibition of VSMC adhesion and migration.</p><p><b>CONCLUSION</b>The six copies of osteopontin RGD peptide were successfully expressed in E. coli DH5alpha. The purified GST-RGD fusion protein could inhibit the adhesion and migration of VSMC stimulated by osteopontin.</p>

Inulicin inhibits activation and expression of vascular remodeling enzymes induced by de-endothelium / 中国应用生理学杂志

<p><b>AIM</b>To observe the effects of inulicin on the neointimal hyperplasia and expression of matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinase-2 (TIMP-2) after balloon injury in rat aorta.</p><p><b>METHODS</b>The intimal hyperplasia model was replicated by balloon injury. The histological changes in vascular wall were observed by HE staining. MMP-2 activation was tested by gelatin zymogram analysis; MMP-2 and TIMP-2 expression was detected by Western blot and immunohistochemistry analysis.</p><p><b>RESULTS</b>Neointimal hyperplasia induced by balloon injury was significantly inhibited by inulicin. The proteolytic activity and the expression of MMP-2 and ratio of MMP-2 and TIMP-2 were also returned to control level by inulicin after balloon injury.</p><p><b>CONCLUSION</b>Inulicin inhibits hyperplasia of neointima by modulating the balance of MMP-2 and TIMP-2.</p>

Recombinant C-terminal fragment of SM22 induces cytoskeleton reorganization / 中国应用生理学杂志

<p><b>AIM</b>To investigate the interaction between C-terminal domains of SM22alpha and cytoskeleton F-actin.</p><p><b>METHODS</b>Prokaryotic expression vector containing SM22alpha cDNA and GST sequence was constructed. The induction conditions were optimized to increase the product of soluble GST-SM22alpha fusion protein in E coli. Expression products were purified and rabbit anti-GST-SM22alpha polyclonal antibody was produced by the purified fusion protein. In order to explore the effect of SM22alpha on cytoskeleton reorganization, VSMCs were treated with serum withdrawal and then serum stimulation to induce contractile/synthetic phenotypic modulation. SM22alpha protein distribution in F-actin/G-actin fractions was detected by Western blotting. The interaction between SM22alpha and actin was examined by GST pull down assay and coimmunoprecipitation. Colocalization of endogenous SM22alpha with F-actin was observed by immunofluorescence.</p><p><b>RESULTS</b>The results showed that the expression of soluble GST-SM22alpha protein was the highest under condition induced by 30 degrees C, 0.5 mmol/L IPTG for 6 h. Immunofluorescence and Western blotting of protein extracts from F-actin/G-actin fractions revealed that SM22alpha colocalized with F-actin during VSMC redifferentiation. GST pull down assay and coimmunoprecipitation showed that SM22alpha interacted with F-actin by C-terminal domains to participate in cytoskeleton reorganization.</p><p><b>CONCLUSION</b>The recombinant SM22alpha C-terminal domains have the ability to bind F-actin, by which SM22alpha interacts with actin and participates in cytoskeleton reorganization.</p>

Experimental study on inhibition of restenosis by osteopontin oligopeptide antagonist after de-endothelium / 中国应用生理学杂志

<p><b>AIM</b>Osteopontin 13-peptide(Gly158-Lys170), containing multi-function domains was used to inhibit the VSMC adhesion, migration. The mechanism of 13-peptide inhibiting neointima formation was investigated.</p><p><b>METHODS</b>The effect of 13-peptide on VSMC adhesion was tested by adhesion assay. The restenosis model was prepared balloon injury after administration of 13-peptide for 1 h, and then the 13-peptide was given by an intravenous drip for 7 days. The expression changes of OPN, FAK, ILK in vessel wall were detected by immunohistochemistry and Western blot.</p><p><b>RESULTS</b>The 13-peptide dose-dependently reduced adhesion of VSMC in OPN matrix, and the infiltration of macrophage in vessel wall also was reduced in the treatment group after balloon injury. The expression of OPN, FAK, ILK was down-regulated following with the inhibition of neointima thickening.</p><p><b>CONCLUSION</b>The OPN 13-peptide can inhibit inflammation and neointima formation by blocking the binding of OPN to it's receptors.</p>

Expression, purification and antibody preparation of recombinant human SM22alpha / 中国应用生理学杂志

<p><b>AIM</b>The recombinant human smooth muscle 22 alpha (SM22alpha) was expressed by using Pichia pastoris.</p><p><b>METHODS</b>Using pGEM3z-SM22alpha as the template, SM22alpha coding region was amplified by PCR, and was inserted the expression vector pPIC9. Then the recombinant plasmid pPIC9-SM22alpha was transfected into Pichia pastoris. The products induced by methanol were precipitated by ammonium sulfate, then CM-cellulose chromatography was performed for SM22alpha. Polyclonal antibody against SM22alpha was produced by immunizing a rabbit with purified recombinant SM22alpha.</p><p><b>RESULTS</b>The positive clone with SM22alpha got high output at 84 hours after induction by methanol. The SM22alpha prepared by ammonium sulfate fractionation and chromatographic separation showed a single band whose apparent molecular weight was 22 kD on SDS-PAGE. Polyclonal antibody against SM22alpha could detect the SM22alpha expression in human or rat vascular walls.</p><p><b>CONCLUSION</b>High-level expression of SM22alpha is successfully achieved in Pichia pastoris. Antibody against SM22alpha can be used to explore the function of SM22alpha.</p>

Effects of Astragalus and saponins of Panax notoginseng on MMP-9 in patients with type 2 diabetic macroangiopathy / 中国中药杂志

<p><b>OBJECTIVE</b>To investigate the role and mechanism of Astragalus (AS) and saponins of Panax notoginseng (PNS) in treating type 2 diabetic macroangiopathy.</p><p><b>METHOD</b>94 patients with type 2 diabetic macroangiopathy were divided into two groups randomly: group treated with Simvastatin and group treated with AS and PNS, compared with 40 healthy control subjects. Serum level of MMP-9 and lipid in patients and healthy subjects were measured before and after treatment.</p><p><b>RESULT</b>The serum levels of MMP-9, TG, TC, LDL-C, VLDL-C in patients with type 2 diabetic macroangiopathy were improved, while the levels of HDL-C were decreased. Like Simvastatin AS and PNS had the function of reducing MMP-9 and accommodating lipid metabolism.</p><p><b>CONCLUSION</b>Besides accommodating lipid metabolism, AS and PNS can also reduce the level of serum MMP-9 soas to treat type 2 diabetic macroangiopathy.</p>

Effect of yiqi huoxue huayu recipe on vascular collagen turnover and relevant gene expression / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To explore the effect of Yiqi Huoxue recipe (YHR), a Chinese herbal medicine for supplementing Qi, activating blood circulation to remove stasis, on vascular extracellular matrix (ECM) remodeling and its molecular mechanism.</p><p><b>METHODS</b>Vascular smooth muscle cell (VSMC) proliferation activity and collagen turnover rate were detected by 3H-TdR test and hydroxyproline amount determined by 3H-Pro incorporation. Expression activity of MMP-2 and osteopontin genes was detected by Northern blotting and MMP-2 zymography analysis.</p><p><b>RESULTS</b>YHR could markedly inhibit VSMC collagen synthesis stimulated by blasic fibroblast growth factor (bFGF) and lower the collagen turnover rate induced by vascular de-endothelialization. The expression level of MMP-2 and osteopontin genes was down-regulated by YHR in cultured VSMC and vascular wall with endothelial injury, and VSMC proliferation was inhibited by the serum obtained from YHR treated rats. Removing protein from the drug serum made no change on the effect of YHR to VSMC.</p><p><b>CONCLUSION</b>YHR could inhibit and/or retard ECM remodeling through regulating the expression of MMP-2 and osteopontin genes and lowering the collagen turnover rate.</p>

Change of enzyme activity correlation with nucleic acid metabolism during the vascular intimal hyperplasia / 中国应用生理学杂志

<p><b>AIM AND METHODS</b>To determine the relationship between the nuclear envelope nucleoside triphosphatase (EC 3. 6. 1. 15, NTPase) activity and the phenotypic modulation of vascular smooth muscle cell (VSMC), the NTPase activity was detected during restenosis after de-endothelialization in vascular wall. The activities of three enzymes involved in carbohydrate and nucleic acid metabolism were also investigated by spectrophotometry.</p><p><b>RESULTS</b>The activity of NTPase increased continuously and associated with the process of intimal thickening. Western blotting showed that expression of SMalpha-actin, as the marker of contractile phenotype of VSMC, decreased continuously. Osteopontin (OPN), the marker of synthetic phenotype of VSMC, was up-regulated during the process. These suggested that intimal injury induced phenotypic modulation of VSMC. The activities of 5'-nucleotidase, adenosine deaminase and succinate dehydrogenase increased and reached their peaks on 7 days after de-endothelialization. The changes of three enzymes were associated with proliferation in VSMC.</p><p><b>CONCLUSION</b>The efflux of mRNA and the changes of enzyme activity involved in carbohydrate or nucleic acid metabolism may be the biochemical basis in the development and progression of restenosis.</p>