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The plant cuticle is an important protective barrier on the plant surface, constructed mainly by polymerized cutin matrix and a complex wax mixture. Although the pathway of plant cuticle biosynthesis has been clarified, knowledge of the transcriptional regulation network underlying fruit cuticle formation remains limited. In the present work, we discovered that tomato fruits of the NAC transcription factor SlNOR-like1 knockout mutants (nor-like1) produced by CRISPR/Cas9 [clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9] displayed reduced cutin deposition and cuticle thickness, with a microcracking phenotype, while wax accumulation was promoted. Further research revealed that SlNOR-like1 promotes cutin deposition by binding to the promoters of glycerol-3-phosphate acyltransferase6 (SlGPAT6 ; a key gene for cutin monomer formation) and CUTIN DEFICIENT2 (SlCD2 ; a positive regulator of cutin production) to activate their expression. Meanwhile, SlNOR-like1 inhibits wax accumulation, acting as a transcriptional repressor by targeting wax biosynthesis, and transport-related genes 3-ketoacyl-CoA synthase1 (SlKCS1), ECERIFERUM 1-2 (SlCER1-2), SlWAX2 , and glycosylphosphatidylinositol-anchored lipid transfer protein 1-like (SlLTPG1-like). In conclusion, SlNOR-like1 executes a dual regulatory effect on tomato fruit cuticle development. Our results provide a new model for the transcriptional regulation of fruit cuticle formation. [ABSTRACT FROM AUTHOR]

A decrease in the bioavailability of endothelium-derived nitric oxide (NO) is linked to hypercholesterolemia. However, the mechanism by which low density lipoprotein (LDL) mediates endothelial NO synthase (eNOS) dysfunction remains controversial. We investigate the effect of LDL on eNOS regulation in human endothelial cells (ECs). In cultured ECs, a high level of LDL increased the abundance of eNOS and caveolin-1 (Cav-1) in the membrane caveolae and the association of eNOS with Cav-1. Furthermore, it decreased the basal level of NO and blocked NO production stimulated by the calcium ionophore A23187. LDL exposure also increased the formation of stress fibers and the membrane translocation of eNOS. These effects can be blocked by cytochalasin D, an actin cytoskeleton disruptor. In revealing the mechanism underlying the translocation of eNOS, we found that a high level of LDL increased the level of membrane-associated and GTP-formed RhoA and activated the RhoA downstream kinase ROCK-1 activity. Y-27632, a specific inhibitor of ROCK-1, blocked LDL-induced stress fiber formation, eNOS translocation and NO production. In conclusion, a high level of LDL increases the movement of eNOS to membrane caveolae via the increased stress fibers. The RhoA-mediated pathway may play a crucial role in this process in vascular ECs. [Copyright &y& Elsevier]

Objective: To generate systemic expression human cellular glutathione peroxidase-1 (GPx-1) (198Leu) transgenic mice model in order to investigate the functional variants in GPx-1 gene in oxidative stress-related diseases., Methods: After linearization with BamnH I and Acc I, the transgenic construct GPx-1 (198Leu) was microinjected into the zygotes of C57BL/6J mice to generate transgenic mice, whose genotype was detected by PCR with specific primers. The GPx-1 gene expression profile was determined by Western blotting., Results: 13 transgenic founder mice were successfully generated. Western blotting result showed that the protein expression level of 4 transgenic mice in hearts were higher than that of wild type mice., Conclusion: Human GPx-1PSL transgenic mice was successfully established. This kind of animal model is of significance for making further researches on oxidative stress-related diseases.

Objective: To identify the different serum proteins expressed in patients with Keshan disease (KD)., Methods: Two-dimensional gel electrophoresis (2-DE) was performed with serum samples from the patients with chronic KD and healthy controls to separate serum proteins. The gels were stained by sliver and scanned by Umax scanner. The data were analyzed by ImageMaster 2D software. KD related proteins were identified through searching the ExPASy SWISS-2DPAGE database., Results: Stable two-dimensional gel electrophoresis maps were established for serum samples of KD patients and healthy controls. A total of 808 and 814 protein spots were observed in KD patients and healthy controls, respectively. The two maps had 96.5475% identical protein spots and 44 differentially expressed protein spots. Eleven protein spots were expressed exclusively in KD patients and 12 protein spots only appeared in healthy controls. About 21 proteins were expressed in both groups but varied in quantities (14 proteins were over-expressed by more than 3 times and 7 proteins were under-expressed by more than 3 times in KD patients, P < 0.01). Among the 353 protein spots matched with the ExPASy-SWISS-2DPAGE databank, No. 1177 protein appeared in the KD patient was found to have the closest match with P02774 2-D0004T6 known as vitamin D binding protein (VDBP)., Conclusion: There is a significant difference in serum protein expression between KD patients and normal people. VDBP might play a role in cardiac muscle damage via inflammatory immune reactions.

Objective: To study the effect of peroxisome proliferator activated receptor γ (PPAR-γ) agonist on the angiotensin converting enzyme 2 (ACE2) mRNA expression in monocyte-derived macrophages of essential hypertensive patients., Methods: Totally 57 essential hypertensive patients were randomly divided into three groups: conventional treatment group (n=18), telmisartan group (n=19), and benazepril group (n=20); 20 patients with normal blood pressure were also selected as the control group. Monocyte-derived macrophages were isolated from blood samples of patients in all four groups. The expression of ACE2 mRNA in monocyte-derived macrophages was detected by RT-PCR before treatment and 4 and 12 weeks after treatment., Results: Four and 12 weeks after treatment, the systolic pressure and diastolic pressure of telmisartan group and benazepril group were significantly lower than that of the conventional treatment group (all P<0.01), and the systolic pressure and diastolic pressure of telmisartan group were significantly lower than that of the benazepril group(both P<0.01) .The expression of ACE2 mRNA in monocyte-derived macrophages were significantly lower in essential hypertensive patients than that in control group (P<0.01). After having been treated for 4 weeks and 12 weeks, the expression of ACE2 mRNA in monocyte-derived macrophages of hypertensive patients in telmisartan and benazepril groups were significantly higher than that in conventional treatment group (all P<0.01), and the expression of ACE2 mRNA in telmisartan group was significantly higher than that in benazepril group (both P<0.01)., Conclusion: PPAR-γ agonist could increase the ACE2 mRNA expression in monocyte-derived macrophages of essential hypertensive patients.

Objective: To investigate the effects of alpha-linolenic acid (ALA) on inflammation and oxidative stress in the diabetic rats., Methods: An experimental type 2 diabetes mellitus model was induced by feeding male SD rats with diet of high fat for 4 weeks and then injected them intraperitoneally with streptozocin (STZ) at 30 mg/kg. Then the animals were randomly divided into three groups (n = 10): control group, diabetic group and ALA group. Four weeks later, tumor necrosis factor (TNF)-a, soluble P-selectin (sP-selectin), soluble intercellular adhesion molecule-1 (sICAM-1), nitric oxide (NO) production, malondialdehyde (MDA), superoxide dismutase (SOD) and catalase (CAT) in the serum were determined., Results: Inflammatory agents including TNF-alpha, sP-selectin and sICAM-1 increased in diabetic rats to compare with control group. Treatment with ALA significantly decreased TNF-alpha, sP-selectin and slCAM-1 to compare with diabetic group. Furthermore, compared with control group, serum MDA production increased whereas NO production, SOD and CAT activities decreased in diabetic rats. Treatment with ALA reduced MDA production, increased NO production, promoted SOD and CAT activities compared with diabetic group., Conclusion: These results indicate that diet rich in ALA exerted the anti-inflammatory and anti-oxidative effects in diabetic rats, which may be beneficial to the prevention and treatment of diabetes.

Objective: To assess the association of blood selenium and polymorphism of glutathione peroxidase-1 (GPx-1) genes in patients with Keshan Disease (KD) and provide genetic evidence for KD susceptibility., Methods: The levels of whole blood selenium and the activity of GPx-1 were measured with spectrophotometric and enzymatic method among 71 KD patients and 290 controls (including 78 internal controls and 212 external controls). The genotype of GPx-1 at 198 site was analyzed by sequencing and PCR-RFLP. The functions of two GPx-1 variants were studied by rat neonatal cardiomyocytes transfection and expression plasmid., Results: Blood level of selenium in KD patients was (0.8 ± 0.2) µmol/L, the internal controls' was (0.9 ± 0.2) µmol/L, and the external controls' was (1.2 ± 0.2) µmol/L (F = 4.888, P < 0.001).GPx-1 activity of KD patients was (73.0 ± 12.6) × 10(-10)U/RBC, internal controls' was (80.9 ± 9.2) × 10(-10)U/RBC, and external controls' was (115.8 ± 21.1) × 10(-10)U/RBC (F = 5.324, P < 0.001). Those of KD patients were significantly lower than controls. The polymorphism (Pro198Leu) of GPx-1 were identified; the frequency of Pro198Leu of KD patients was 21.1%, the frequency of controls was 10.7% (χ(2) = 5.588, P = 0.018). The level of blood selenium in variant subgroup (Pro198Leu or Leu198Leu) was (0.9 ± 0.2) µmol/L, and its in non-variant subgroup was (1.1 ± 0.3) µmol/L (t = 3.183, P < 0.01); The GPx-1 activity in variant subgroup was (86.1 ± 23.0) × 10(-10)U/RBC, and its in non-variant subgroup was (101.8 ± 25.9) × 10(-10)U/RBC (t = 5.784, P < 0.01). Further analysis revealed a synergistic-multiplicative interaction between presence of GPx-1 codon198 alleles and low blood selenium level. Over-expression of GPx-1 (198Leu) in rat cardiomyocytes caused 30% lower enzyme activity and less response to increasing concentrations of selenium than with over-expression of GPx-1 (198Pro)., Conclusion: Low blood selenium in carriers with the 198Leu-susceptible genotype of GPx-1 is associated with low GPx-1 activity, synergistic-multiplicative interaction was found between these two factors. And these two factors may increase the risk of KD.

Objective: To explore the electrophysiologic characteristics of Ito in myocytes from cardiac Koch triangle., Methods: Patch clamp technique was employed to investigate the I-V and D-V relation, steady-state activation and inactivation kinetics of Ito from myocytes in Koch triangle of rabbit hearts., Results: (1) The maximum peak current (pA) and peak current density (pA/pF) at +20 mV in PC (pacing cell), TC (transitonal cell) alpha,beta, AC (atrial cell) and PL (purkinje-like cell) cells were different from each other (P < 0. 05); The cells also had different current density (P < 0.05) except between TCalpha, TCbeta and PL cells (P > 0.05). (2) The half activation potential (V(mIto1/2), mV) of steady state activation among PC, TCalpha, TCbeta, AC and PL were significantly different (P < 0.05); The paired comparison between TCalpha and TCbeta, AC and PL, and TCbeta and PC showed significant difference (P < 0.05); but the differences between TCa and PC, TCbeta and AC, and TCbeta and PL were not significant (P > 0.05). (3) The half inactivation potential (V5(hIto1/2), mV) of steady state inactivation between TCalpha and PC, TCa and PL, TCbeta and PC, and TCbeta and PL demonstrated significant differences(P < 0.05). The differences of K(hIto) between TCalpha and TCbeta, PC and PL, TCbeta and PC, and AC and PL were significant (P < 0.05)., Conclusions: Ito of cardiac cells from Koch triangle in rabbit hearts are heterogeneous, but relatively specified and distributed in different groups.

Objective: To construct prokaryotic expression system for expressing, purifying and identifying truncated fragment of extra-cellular segment of sodium pump alpha3 subunit with pGEX-6P-1 GST gene fusion system in Escherichia coli by in-fusion technology., Methods: According to the conservative sequence of M1-M2 and M3-M4 extra-cellular gene fragments of sodium pump a3 subunit, which published in GenBank, a serial of primers and gene fragments was designed, and directly synthesized to fuse the above two gene fragments. The fusion gene was fused with gene-specific primers by PCR, and then fusion gene fragment was fused into the single stranded homology regions of vector pGEX-6P-1 by in-fusion cloning to construct recombinant vector pGEX-Trf-alpha3 (Truncated fragment of extracellular segment of sodium pump alpha3 subunit, Trf-alpha3). After DH10bac was transferred with it, the pGEX-Trf-alpha3 plasmid was purified and identified by PCR and sequenced. Then the recombinant plasmid pGEX-Trf-alpha3 was expressed in E. coli BL21 cells, inducted by IPTG. GST-Trf-alpha3 fusion protein was purified with Glutathione Sepharose 4B purifying system and analyzed by SDS-PAGE., Results: The results of PCR and sequencing demonstrated that the M1-M2 and M3-M4 extra-cellular gene was inserted in plasmid pGEX-6P-1 vector successfully. And the sequence was correct. Protein sequence analysis showed that the GST-Trf-alpha3 fusion protein was consisted of 262 amino-acid residues. Relative molecular mass in theory was 33.22 X 10(3). The amount of recombinant protein was 10% of the total bacteria protein. The soluble fusion protein was about 80.8%. After affinity purification, the purity of GST-Trf-alpha3 fusion protein was over 95%. There was some extent binding activity between GST-Trf-alpha3 fusion protein and ouabain, but the activity was very low., Conclusion: Prokaryotic expression system for expressing truncated fragment of extra-cellular segment of sodium pump alpha3 subunit with pGEX-6P-1 GST gene fusion system in Escherichia coli by in-fusion technology had been constructed. The purified method had also established. High purified GST-Trf-alpha3 fusion protein was obtained. These have found the foundation of further study on its biological function and potential pharmacology function.

Objective: To assess the binding activity of polypeptide containing human Na+, K+-ATPase alpha1 subunit M1-M2 extracellular segment (HES1 derivative)., Methods: HES1 derivative was synthesized by Fmoc method and purified by high-performance liquid chromatography-mass spectrometry, and its binding activity was identified by radioligand binding assay., Results: 3H-ouabain and synthetic HES1 derivative showed some binding activity with the equilibrium dissociation constant (KD) of 24.58 nmol/L, with the the receptor density of 492.43 fmol x mg(-1) pro. and IC50 of 3.078 x 10(-7) mol/L., Conclusion: HES1 derivative can bind to ouabain and has the potential of becoming an effective therapeutic agent.

Arginine vasopressin (AVP), a neurohormone and hemodynamic factor implicated in the pathophysiology of hypertension and congestive heart failure, can also act as a growth-stimulating factor. Our previous work demonstrated that AVP is a mitogen for neonatal rat cardiac fibroblasts (CFs). In the present study, we extended our investigations to adult rat CFs to explore whether AVP could induce adult rat CF proliferation and, if so, to identify the mechanism involved. Adult rat CFs were isolated, cultured and subjected to AVP treatment. DNA synthesis and cell cycle distribution were analyzed by [(3)H]-thymidine incorporation and flow cytometry. Cellular extracellular signal-regulated kinase 1/2 (ERK1/2) activity was measured by in vitro kinase assay using myelin basic protein (MBP) as a substrate. Protein expressions of total- and phospho-ERK1/2, p27(Kip1), cyclins D1, A, E were assessed by Western blot. The results showed that AVP stimulated DNA synthesis in adult rat CFs, and the effect was abolished by a V1 receptor antagonist, d(CH(2))(5)[Tyr(2)(Me), Arg(8)]-vasopressin (0.1 μmol/L), but not by a V2 receptor antagonist, desglycinamide-[d(CH(2))(5), D-Ile(2), Ile(4), Arg8]-vasopressin (0.1 μmol/L). AVP induced an activation of ERK1/2, which could be mimicked by the protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA, 30 nmol/L, 5 min), but abolished by depletion of PKC via chronic PMA incubation (2.5 μmol/L, 24 h). In addition, AVP down-regulated protein expression of p27(Kip1), increased protein expressions of cyclins D1, A and E, and induced cell cycle progression from G(0)/G(1) into S stage. Inhibition of ERK1/2 activation by PD98059 (30 μmol/L) abolished the effect of AVP on DNA synthesis, protein expressions of p27(Kip1), cyclins D1, A and E as well as cell cycle progression. These results suggest that AVP is also a growth factor for adult rat CFs. The mitogenic effect of AVP is mediated via V1 receptors and PKC-ERK1/2 pathway. Moreover, AVP modulates the expressions of cell cycle regulatory proteins p27(Kip1) and cyclins D1, A and E, which lie downstream of ERK1/2 activation, and induces cell cycle progression in adult rat CFs.

Objective: To investigate the mechanism of rosiglitazone (RSG, the activator of peroxisome proliferators activated receptor lambda) for inhibiting endothelin-1 (ET-1)-induced neonatal rat cardiac myocyte hypertrophy and the role of protein kinase C (PKC) and c-fos., Methods: In vitro cultured neonatal rat cardiac myocytes were treated with ET-1, phorbol ester (PMA, the PKC activator), ET-1+RSG, ET-1+chelerythrine (che, the PKC inhibitor), PMA+RSG, or without treatment (control), respectively. The effects of RSG on the protein content, (3)H-leucine incorporation, PKC activity and C-fos protein expression were observed in the cardiac myocytes stimulated with ET-1 or PMA., Results: After two days of culture, the intracellular protein content in ET-1 group and PMA group were increased by 15% (339-/+15 microg/ml) and 13% (329-/+14 microg/ml) as compared with the control cells (290-/+13 microg/ml), respectively (P<0.01). Compared with the ET-1 group, cells treated with ET-1+10(-8) mol/L RSG, ET-1+10(-7) mol/L RSG, and ET-1+che showed decreased intracellular protein content by 10% (303-/+14 microg/ml, P<0.05), 12% (292-/+11 microg/ml, P<0.05), and 13% (291-/+12 microg/ml, P<0.01), respectively. The intracellular protein content in PMA+10(-7) mol/LRSG group was decreased by 10% (P<0.05) in comparison with the PMA group. RSG inhibited protein synthesis enhancement and increased (3)H-leucine incorporation induced by ET-1 and PMA, and antagonized the effects of ET-1 and PMA in promoting PKC activity and c-fos protein expression in the myocytes., Conclusion: The inhibitory effect of RSG on ET-1- or PMA-induced myocyte hypertrophy is associated with PKC-c-fos pathway.

In order to explore the activity of a peptide containing rat sodium pump α2 subunit M1-M2 extramembrane fragment (RES2 derivative) in vitro, the peptide (Leu-Ala-Ala-Met-Glu-Asp-Glu-Pro-Ser-Asn-Asp-Asn-Gly-Gly-Gly-Ser) was synthesized by peptide synthesizer with Fmoc method and purified by high performance liquid chromatography (HPLC). Its binding activity was identified by radioligand-receptor binding assay (RRA) and its bioactivity was measured by erythrocyte (86)Rb uptake. The results of saturation binding experiment and competitive binding experiment showed that the synthesized RES2 derivative had the capability to bind to (3)H-ouabain. The dissociation constant (K(d)) was 38.46 nmol/L and IC(50) was 6.353 nmol/L. Erythrocyte (86)Rb uptake experiment showed that the RES2 derivative blocked the inhibitory effect of ouabain on the sodium pump on erythrocyte membrane in a dose-dependent manner. The results showed that the RES2 derivative is capable of binding to ouabain and improving the activity of sodium pump on erythrocyte membrane, suggesting that the RES2 derivative might become an effective antihypertensive drug in the future.

Objective: To investigate the effects of peroxisome proliferator-activated receptor-gamma (PPAR-gamma) ligand on angiotensin II (AngII)-induced endothelin-1 (ET-1) and NO secretion by endothelial cells in comparison with AngII type I receptor (AT1R) antagonist losartan, so as to reveal the relationship between PPAR gamma and essential hypertension., Methods: Cultured human umbilical vein endothelial cells (HUVECs) were treated with AngII, PPAR gamma ligand troglitazone, AngII plus troglitazone, and AngII plus AT1R antagonist losartan, respectively, and the concentrations of NO and ET-1 in the cell culture supernatant were measured to evaluate the effects of troglitazone and losartan on AngII-induced NO and ET-1 production by human endothelial cells., Results: Treatment of the HUVECs with troglitazone at 10 micromol/L and 50 micromol/L did not produce significant changes in ET-1 concentration in the cell culture supernatants, but significantly increased NO concentration as compared with the control group (P<0.05). Triglitazone at the concentration of 50 micromol/L significantly inhibited AngII (1x10(-6) mol/L)-induced ET-1 production (P<0.05), and at both 10 and 50 micromol/L, troglitazone inhibited the NO release-lowering effect of AngII in the endothelial cells (P<0.05). Both troglitazone and losartan inhibited AngII-induced ET-1 production by the endothelial cells, but losartan showed more potent effect (P<0.05). Similarly, both troglitazone and losartan inhibited decreased NO production in response to AngII treatment, and again losartan showed stronger effect (P<0.05)., Conclusion: PPAR gamma ligand troglitazone can inhibit AngII-induced ET-1 production enhancement and decreased NO release by the endothelial cells, but its effect is not so strong as losartan, suggesting that troglitazone modulates blood pressure not solely through AT1R pathway.

Objective: To observe the relationship between protein sythesis and cardiomyocyte viability in neonatal rats., Methods: The protein sythesis in neonatal rat cardiomyocytes was measured according to Brandford's method, the absorbance at 490 nm (A(490 nm)) of the cells was measured with MTT assay and the cell viability evaluated by the ratio of A(490 nm) to the total cell number., Results: ET-1 increased cardiomyocyte protein synthesis dose-dependently, and this effect was attenuated by the application of lacidipine and tetramethylpyrazines Higher doses of ET-1 resulted in lower A(490 nm)/total cell number ratio, which was further lowered by larcidipine and tetramethylpyrazine., Conclusion: The status of protein synthesis is not associated with the viability of neonatal rat cardiomyocytes.

Objective: To quantitatively detect the expression of connexins (Cx) mRNA in the posterior nodal extension (PNE) of adult rat heart and understand the relationship between Cx expression and atrial ventricular nodal reentrant tachycardia (AVNRT)., Methods: PNE was separated from adult rat heart by means of laser microdissection (LCM), and the cells were also isolated from the atrioventricular node (AVN), sinoatrial node (SAN), Purkinje fiber (PF), right atrium (RA) and right ventricle (RV), to serve as the controls. The Cx mRNA level was detected in these cells with quantitative real-time PCR (QRT-PCR)., Results: The cells were successfully isolated from the PNE and other regions of adult rat heart, where heterogeneous expression of the 3 Cx isoforms (Cx43, Cx45, and Cx40) were observed. Cx45 mRNA showed higher expression in the PNE than in the working myocardium, whereas Cx43 mRNA level was about 25 times higher (P<0.05) in the working myocardium and 18 times higher (P<0.05) in the PF than in the PNE. In the PF, Cx40 mRNA level was proximately 6.8 times (P<0.01) as much as that in the PNE. Cx expression in the PNE was, however, similar to that in the SAN and AVN., Conclusion: Cx mRNAs exhibit heterogeneous expression in the PNE to allow the formation of the slow pathway. In addition, Cx expression in the PNE is very different from that in the adjacent myocardium, resulting in conduction discontinuity at the cellular junction, where, on certain occasion, unidirectional block may occur to cause AVNRT.

Objective: To investigate HLA-DRB1 gene polymorphism in patients with Keshan disease (KD) in the north of China, and its relation to KD in the core families., Methods: Polymerase chain reaction-sequence specific oligonucleotide probe (PCR-SSOP) method was used to determine HLA-DRB1 genotypes in 118 KD patients, including 63 with latent KD and 55 with chronic KD. Sixty-five normal from the same area were selected as controls. The haplotype based haplotype relative risk (HHRR) and transmission disequilibrium test (TDT) methods were used to analyze the genetic association and linkage of HLA-DRB1 with KD in 18 KD core families., Results: (1) Thirteen kinds of alleles of HLA-DRB1 gene were found in all patients and the controls. (2) The distributive frequency of DR7 allele was significantly lower in chronic KD patients than that in controls (P< 0.01, OR is 0.1695). (3) The distributive frequency of DR7 allele was statistically lower in chronic KD (P< 0.01, OR is 0.091) and showed no differences in latent KD patients as compared with the controls. (4) DR15 allele of HLA-DRB1 gene showed significant association (chi square is 9.32, P< 0.01) and linkage (chi square is 7.40, P< 0.01) with KD patients in the core families., Conclusion: The results show that there might be the genetic susceptibility in the pathogenesis of KD. DR7 allele of HLA-DRB1 gene might be the protective gene of KD. Patients with DR7 allele might be more difficult to become to chronic KD. DR15 allele of HLA-DRB1 gene might be linked to the susceptive site of KD.

Angiotensin (Ang) II plays a pivotal role in vascular fibrosis, which leads to serious complications in hypertension and diabetes. Connective tissue growth factor (CTGF) is a potent profibrotic factor implicated in the Ang II-induced pathologic fibrosis process. PPAR-gamma activators thiazolidinediones have been recently reported to have beneficial vascular effects. However, their effects and related molecular mechanisms on extracellular matrix (ECM) turnover in vascular smooth muscle cells (VSMCs) are unknown. The present study evaluated the regulation of Ang II-induced CTGF, ECM production and cell growth by rosiglitazone in VSMCs. In aorta of Ang II-infused rats, CTGF expression was markedly increased, and type III collagen and fibronectin overexpression was observed. Cotreatment with rosiglitazone diminished these changes, whereas increased nuclear PPAR-gamma expression in VSMCs. In growth-arrested VSMCs, rosiglitazone attenuated the proliferation and apoptosis, increased PPAR-gamma production and activation, and reduced CTGF and ECM production in response to Ang II in a dose-dependent fashion. These inhibitory effects were attenuated by the pretreatment of cells with PPAR-gamma antagonist GW9662 or bisphenol A diglycidyl ether (BADGE). Furthermore, rosiglitazone inhibited Ang II-induced Smad2 production and phosphorylation but had no effect on transforming growth factor-beta(1) (TGF-beta(1)) expression. These results suggest that in Ang II-stimulated VSMCs, rosiglitazone caused an antiproliferative, antiapototic effect and reduces ECM production through mechanisms that include reducing CTGF expression, and a crosstalk between PPAR-gamma and Smad may be involved in the inhibitory effects of rosiglitazone. This novel finding suggests a role of PPAR-gamma activators in preventing Ang II-induced vascular fibrosis.

Objective: To use the laser capture microdissection (LCM) to isolate the pure atrial cells from heart. To extract tiny amount RNA and amplify the genes of beta-actin and GAPDH., Methods: First, the prepared sample RNA was extracted and verified. Second, the frozen sections were scraped after haematoxylin-eosin staining, and then the RNA of scraped tissues was extracted and verified. Last, the tiny amount RNA was extracted from atrial cells captured by LCM, and the genes of beta-actin and GAPDH were amplified by reverse transcriptase polymerase chain reaction (RT-PCR)., Results: The atrial cells isolated with LCM had clear morphology after quick staining. The high quality RNA was extracted from LCM sample and scraped tissues, of which A260/A280 was 1.9-2.0. And 18S rRNA and 28S rRNA were seen distinctly on the gel of RNA formaldehyde denaturing gel electrophoresis, which indicated that RNA didn't degrade before cells captured. Too small amount of RNA extracted from captured cells was below the detectable limit of gel electrophoresis or ultraviolet spectrophotometer. Bands of 300 bp and 357 bp could be amplified by RT-PCR from tiny amount RNA., Conclusion: Highly pure atrial cells could be obtained by LCM. Tiny amount RNA of captured atrial cells could be extracted and amplified with genes of beta-actin and GAPDH.

Objective: In order to elucidate the relationship between PPAR-gamma and the development of hypertension, we detected the expression of peroxisome proliferator-activated receptors gamma (PPAR-gamma) in the vascular smooth muscle cells (VSMCs) and the VSMC proliferation of spontaneously hypertensive rats (SHR) at different ages., Methods: The expression of PPAR-gamma in the intact vascular tissues of rats at different ages were detected by immunohistochemistry. Aortic VSMCs were cultured. PPAR-gamma mRNA in primary and low-passage cultured VSMCs of various ages were determined by RT-PCR, and proteins were evaluated by immunohistochemistry and age matched Western Blot. Age matched Wistar-Kyoto rats (WKY) were used as control., Results: This experiment demonstrated that the expression of PPAR-gamma increased with age in the intact aorta tissues of SHR. The expression of PPAR-gamma did not continuously increase in 24w-old SHR when compared with that in 16w-old SHR. No difference between 4w-, 24w-old SHR and age matched WKY was observed, but the expression of PPAR-gamma was greater in 8w- and 16w old SHR than in age matched WKY. In primary and low-passage cultured VSMCs, the expression of PPAR-gamma mRNA and protein increased with age both in SHR VSMCs and WKY VSMCs of 4w, 8w and 16w old, and no difference between 4w- and 24w-old SHR and WKY was noted, but the expression of PPAR-gamma was higher in 8w- and 16w-old SHR than in age matched WKY. PPAR-gamma expression in 24w-old SHR did not increase and it was equal to 16w-old SHR and 24w-old WKY., Conclusion: These datas on SHR suggest that the expression of PPAR-gamma changes with age and the development of high blood pressure. PPAR-gamma expression may play a compensatory role in hypertension, but this compensatory action is limited.

Objective: To investigate the expression and distribution of L-type calcium channel alpha1C subunits in adult rat heart., Methods: HE staining was applied on the frozen sections of adult rat heart to identify the sinoatrial node (SAN), atrioventricular node (AVN), and posterior nodal extension (PNE). The protein expression of L-type calcium channel alpha1C in adult rat heart and its cellular localization were examined by Western blotting and immunohistochemistry, respectively., Results: L-type calcium channel alpha1C subunit was immunolocalized on the membrane of the myocardial cells, and its expression increased gradually in the SAN, AVN, PNE, right atrium and right ventricle. The protein level of L-type calcium channel alpha1C in the AVN was similar to that in the PNE (P>0.05), and its level in the right atrium and ventricle were significantly higher than those in the SAN and AVN (P<0.01). The results of Western blotting were well consistent with the results of immunohistochemistry., Conclusion: The expression of L-type calcium channel alpha1C subunit may play a role in the electrophysiological functions of the heart.

Objective: To investigate the effect of endothelin-1 (ET-1) on the expression of peroxisome proliferators-activated receptor-gamma in vascular smooth muscle cells., Methods: The first to the third passages of aortic vascular smooth muscle cells (VSMCs) isolated from 16-week-old Wistar rats were cultured in vitro and stimulated by ET-1 for 12, 24, 48, and 72 h, respectively, and at different concentrations of ET-1 for 48 h. PPAR-gamma mRNA expression of the cells was detected by reverse transcription (RT)-PCR and PPAR-gamma protein by Western blotting after the stimulations, and the proliferation of VSMCs was evaluated by MTT assay., Results: No changes in PPAR-gamma mRNA and protein expressions were observed in the VSMCs after ET-1 stimulation for 12 h (P>0.05), and when the stimulation was prolonged to 24 h, slight decrease in the expressions occurred but the difference was not statistically significant in comparison with the control group (P<0.05). After 48 to 72 hour's ET-1 stimulation, the expressions of both PPAR-gamma mRNA and protein were markedly decreased as compares with the control group (P<0.01). The expressions of PPAR-gamma in mRNA and protein were both decreased with the increase of ET-1 concentration after stimulation for 48 h. VSMC proliferation occurred at 12 h during ET-1 stimulation and increased with time and ET-1 concentration., Conclusion: ET-1 stimulation can induce VSMC proliferation and decrease the expressions of both PPAR-gamma mRNA and protein.

Objective: To investigate the effect of Shenmai Injection (SMI) on immunologic function in patients with dilated cardiomyopathy (DCM)., Methods: Fifty-six patients were divided into two groups, the control group treated with conventional western medicine, and the SMI group treated with conventional western medicine plus SMI. The indices including red blood cell (RBC) C3b receptor rosette (RBC-C3bRR) and immune complex rosette (RBC-ICR), T-lymphocyte subsets (CD3, CD4, CD8, CD4/CD8) were determined before and after treatment., Results: The level of RBC-C3bRR, CD4, CD8 and CD3 in patients with DCM were significantly decreased (P <0 .01, P < 0.05), RBC-ICR and CD4/CD8 were significantly increased than those in the normal control group (P < 0.01); While the level of RBC-C3bRR, CD4, CD8 and CD3 in the SMI group after treatment were significantly higher, and the level of RBC-ICR and CD4/CD8 were significantly lower (P < 0.01, P < 0.05) than those in the control group., Conclusion: The RBC immune adherence and cellular immune function are lower in patients with DCM, and SMI has the effect in regulating immune function in patients with DCM.

Objective: To evaluate the relationship between hypertension and the expression level of PPAR-gamma in endothelial cells (ECs) of spontaneously hypertensive rats (SHR)., Methods: PPAR-gamma protein expression in ECs of 4-week and 16-week SHR was detected by immunohistochemistral technique and analyzed by image acquiring and analysis system, and age matched Wistar-Kyoto rats (WKY) were used as controls. The aortic ECs of different-aged rats were primarily cultured, and the protein and mRNA level of PPAR-gamma in cultured ECs (< or =3 passages) were detected by Western blotting and reverse transcription-polymerase chain reaction (RT-PCR)., Results: The blood pressure of 4-week SHR was slightly higher than those of 4-week WKY (P < 0.05). The protein and mRNA levels of PPAR-gamma in 4-week SHR were also higher than that of 4-week WKY, but the differences had no statistical significance (P > 0.05). Compared with the 16-week WKY, the blood pressure of 16-week SHR greatly increased (P < 0.01), and the protein and mRNA levels of PPAR-gamma in ECs from 16w SHR were approximately 1.5 times higher than those of the age matched WKY (P < 0.01). The protein and mRNA levels of PPAR-gamma in ECs from 16-week SHR were 2.5 times higher than those of 4-week SHR (P < 0.01), while in 16-week WKY it was only 1.5 times higher than those of 4-week WKY (P < 0.01). Conclusion In accordance with the hypertension progress, the protein and mRNA levels of PPAR-gamma in SHR can significantly increase.