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ABSTRACT: 1. Diabetes mellitus predisposes to and female sex protects against arterial thrombosis. The aim of the present study was to determine whether advanced glycation end-products (AGE), which accumulate in diabetes, impair platelet function through effects on platelet nitric oxide (NO) generation and whether this can be prevented by 17beta-oestradiol. 2. Aggregation responses of human platelet-rich plasma to ADP were determined in the absence or presence of 200 mg/L AGE-modified albumin (AGE-albumin), 10(-5) mol/L 17beta-oestradiol and 10(-5) mol/L ICI 182 780 (the pure oestrogen receptor antagonist). 3. Intraplatelet cGMP, an index of bioactive NO, was measured by radioimmunoassay and expression of nitric oxide synthase (NOS)-3, phosphoserine-1177-NOS-3 and O-glycosylated NOS-3 was quantified by western blotting in response to these same treatments. 4. Advanced glycation end-products-albumin increased platelet aggregatory responses to ADP. This increase was largely prevented by 17beta-oestradiol. Advanced glycation end-products-albumin decreased and 17beta-oestradiol increased intraplatelet NO-attributable cGMP and 17beta-oestradiol attenuated the AGE-albumin-induced decrease in NO-attributable cGMP. Despite no effect on NOS-3 expression, AGE-albumin decreased and 17beta-oestradiol increased phosphoserine-1177-NOS-3 and 17beta-oestradiol largely prevented the decrease in phosphoserine-1177-NOS-3 induced by AGE-albumin. Alone, AGE-albumin increased O-glycosylation of NOS-3 by N-acetylglucosamine, an effect largely inhibited by 17beta-oestradiol. 5. In conclusion, AGE-albumin inhibits platelet NO biosynthesis through effects on serine phosphorylation and O-glycosylation of platelet NOS-3 and this may explain, at least in part, the increase in platelet aggregability induced by AGE-albumin. These effects of AGE-albumin are largely prevented by 17beta-oestradiol. These actions may contribute to the effects of diabetes and sex on arterial thrombosis in vivo.
Clinical and Experimental Pharmacology and Physiology 11/2007; 34(10):972-8. · 1.85 Impact Factor
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ABSTRACT: Low-density lipoprotein (LDL) inhibits endothelium-dependent vasorelaxation. The aim of this study was to determine whether pyridoxine supplementation improves indices of LDL-induced endothelial dysfunction. Human umbilical vein endothelial cells (HUVEC) were incubated with native LDL (nLDL) from healthy subjects, oxidized LDL (oxLDL, formed by nLDL oxidation) or nLDL from type II diabetic patients (dLDL), in the absence or presence of pyridoxine; nitric oxide synthase (NOS) activity, cyclic GMP and expression of NOS isoforms were measured, as well as thiobarbituric acid reactive substances (TBARS) in HUVEC supernatants and amino acid concentrations in HUVEC lysates. All LDL species inhibited total NOS activity, whilst increasing the much smaller Ca2+-independent component of NOS activity, the effects of oxLDL being greatest and those of nLDL smallest; in accordance with these findings, NOS type 3 expression decreased and NOS type 2 expression increased, with a resultant decrease in bioactive nitric oxide (NO), in HUVEC treated with each LDL species, with the same rank order of potency. LDL species also increased TBARS in HUVEC supernatants as well as homocysteine concentrations in HUVEC lysates, nLDL < dLDL < oxLDL. Pyridoxine largely prevented all LDL-induced changes in NOS activity and isoform expression, as well as in TBARS and homocysteine. The findings suggest that pyridoxine prevents LDL-induced dysfunction of endothelial cell NO generation, most likely through its antioxidant effects as well as through its effects on cellular homocysteine metabolism. This has important potential therapeutic implications for cardiovascular disease prevention.
Atherosclerosis 09/2006; 188(1):84-94. · 3.79 Impact Factor
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Yaoyu Chen,
Xiaohua Wang,
Jingjing Ben,
Shen Yue,
Hui Bai,
Xiaoxiang Guan,
Xiaoming Bai,
Li Jiang,
Yong Ji,
Leming Fan, Qi Chen
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ABSTRACT: The di-leucine motif exists in the intracellular domains of certain cell surface receptors, participating in the receptor-mediated endocytosis. The present study was aimed at determining the role of the di-leucine motif in class A scavenger receptor (SR-A)-mediated ligand endocytosis.
cDNA coding for a mutant (SR-A mutant N3132LM) with deletion of the di-leucine structure was transfected into Chinese hamster ovary (CHO) cells. Compared with wild-type SR-A-expressing cells, the cells expressing the SR-A mutant N3132LM showed a significant decrease in uptake but almost no change in binding of the SR-A ligand acetylated low-density lipoprotein (AcLDL). Western blot analysis revealed coimmunoprecipitation of SR-A mutant and clathrin from the lysates of the mutant but not wild-type CHO cells, suggesting that AcLDL-bound SR-A mutant N3132LM is associated with the clathrin-coated pit of cellular membrane. Removal of the first 27 amino acid residues from the SR-A N-terminus further reduced AcLDL uptake by the cells with the di-leucine motif mutation.
The di-leucine motif of SR-A intracellular domain contributes to the SR-A-mediated cellular internalization of AcLDL. Di-leucine pair exists in the cytoplasmic domain of class A scavenger receptor. The cells expressing di-leucine mutants showed decreased uptake and unchanged binding of AcLDL. The di-leucine pair was not associated to coated pits. It suggests that di-leucine motif acts as a signal sequence to mediate SR-A into cell.
Arteriosclerosis Thrombosis and Vascular Biology 07/2006; 26(6):1317-22. · 6.37 Impact Factor
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ABSTRACT: Ultrasound-targeted microbubble destruction had been employed in gene delivery and promised great potential. Liver has unique features that make it attractive for gene therapy. However, it poses formidable obstacles to hepatocyte-specific gene delivery. This study was designed to test the efficiency of therapeutic gene transfer and expression mediated by ultrasound/microbubble strategy in HepG2 cell line. Air-filled albumin microbubbles were prepared and mixed with plasmid DNA encoding low density lipoprotein receptor (LDLR) and green fluorescent protein. The mixture of the DNA and microbubbles was administer to cultured HepG2 cells under variable ultrasound conditions. Transfection rate of the transferred gene and cell viability were assessed by FACS analysis, confocal laser scanning microscopy, Western blot analysis and Trypan blue staining. The result demonstrated that microbubbles with ultrasound irradiation can significantly elevate exogenous LDLR gene expression and the expressed LDLRs were functional and active to uptake their ligands. We conclude that ultrasound-targeted microbubble destruction has the potential to promote safe and efficient LDLR gene transfer into hepatocytes. With further refinement, it may represent an effective nonviral avenue of gene therapy for liver-involved genetic diseases.
Biochemical and Biophysical Research Communications 06/2006; 343(2):470-4. · 2.48 Impact Factor
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ABSTRACT: Elevation in plasma triglycerides (TG) has been widely accepted as a coronary artery disease (CAD) risk predictor. Recently, a new apolipoprotein playing an important role in TG metabolism named apolipoprotein AV (apoAV) was discovered, which is encoded by the APOA5 gene. Several single nucleotide polymorphisms (SNPs) of APOA5 associated with increased TG concentrations have been identified. We here report that a recently identified genetic variant, c.553G>T in the APOA5 gene which causes a substitution of a cysteine for a glycine residue at amino acid residue 185(G185C) is also associated with increased TG levels. To investigate the association between this genetic variation and the risk of CAD, a case-control study comprising 232 patients with CAD and 302 controls from the same area of China was performed. The minor allele frequencies of c.553G > T for the CAD and control groups were 7.76 and 3.97%, respectively (P = 0.008). In both the CAD and control groups, the T allele carriers had higher serum TG levels than homozygous carriers of the major G allele (CAD group: 2.67 +/- 1.48 mmol/l versus 1.95 +/- 1.02 mmol/l, P = 0.021; controls: 2.31 +/- 1.20 mmol/l versus 1.68 +/- 0.95 mmol/l, P = 0.002). After adjustment for age, gender, body mass index, smoking status, glucose and presence of hypertension, the odds ratio (OR) for CAD in the T allele carriers was 2.089 (95% CI = 1.140-3.830, P = 0.017), in comparison to the individuals without the T allele. These results suggest that the APOA5 c.553G > T polymorphism is an important predictor for hypertriglyceridemia and CAD.
Atherosclerosis 04/2006; 185(2):433-7. · 3.79 Impact Factor
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ABSTRACT: It is known that glycine protects mammalian cells against ischaemic cell injury by preventing cellular membrane leakage. However, the molecular mechanisms have not yet been clearly elucidated. The purpose of the present study was to clarify whether GlyR (glycine receptor) acts as a key mediator in cytoprotection of glycine. cDNA encoding human GlyRa1 (a1-subunit of glycine receptor) was transfected into HEK-293 cells. The membrane integrity of the cells with or without GlyRa1 was examined by the uptake of marker compounds, the release of LDH (lactate dehydrogenase) and the exclusion of Trypan Blue. Glycine prevented the permeability of 70 kDa dextrans and 140 kDa LDH in the cells in which GlyR was expressed under conditions of ATP depletion. The inhibition of endogenous GlyR expression by RNA interference attenuated the cytoprotection by glycine. Furthermore, the mutation of Tyr202 to phenylalanine in GlyRa1 blocked the glycine-mediated cytoprotection, while the mutation of Tyr202 to leucine abolished the cytoprotection by strychnine. Our results suggested that the cytoprotection of glycine against ATP-depletion-induced injury might be mediated by GlyR.
Biochemical Journal 10/2005; 390(Pt 2):447-53. · 4.90 Impact Factor
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ABSTRACT: To investigate the association of R219K and M883I polymorphisms of ATP binding cassette transporter 1 gene with lipid metabolism and the susceptibility to coronary atherosclerotic heart disease in Chinese population.
Genotypes were determined by PCR-restriction fragment length polymorphism and Primer introduced restriction analysis-PCR techniques, respectively, in 248 unrelated CHD-free controls and 224 CHD cases.
Smoking, high blood pressure and high serum glucose were independent risk factors for CHD. Multivariate logistic regression analysis revealed that individuals carrying at least one 219K variant allele (RK + KK genotypes) had a significantly decreased risk for CHD (adjusted OR = 0.41; 95% CI = 0.27-0.61) compared with the wild-type genotype (219RR) and only 883II homozygotes displayed a decreased risk for CHD (adjusted OR = 0.54; 95% CI = 0.26-1.11) compared with 883MM and 883MI genotypes. Furthermore, compared with individuals with both wild genotypes (219 RR and 883 MM or 883 MI) other individuals with all other assembly genotypes had a significantly decreased risk (adjusted OR = 0.39, 95% CI = 0.26-0.60). Plasma HDL-C in 219K allele carriers were markedly higher than those in 219 K non-carriers in controls (P = 0.037).
The ABCA1 R219K polymorphism may be involved in the variability of serum HDL-C and the susceptibility to coronary artery disease.
Zhonghua xin xue guan bing za zhi [Chinese journal of cardiovascular diseases] 08/2005; 33(7):627-30.
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ABSTRACT: We report here testosterone deficiency in diet-induced hypercholesterolemic mice. We attempted to compare the liver protein profiles between hypercholesterolemic and normal mice by using the technology of proteomics. Protein extractions from mice livers were separated by two-dimensional electrophoresis and the gels were analysed with image analysis software 2D Elite 4.0. The differentially expressed proteins were identified primarily by comparing with reference gel images in the SWISS 2DPAGE database and then confirmed by peptide mass fingerprinting. Sixteen differentially expressed protein spots (>2-fold) were detected and 8 of which were identified as major urinary proteins, carbonic anhydrase III, and glutathione S-transferase P2, which are known to be regulated by androgens. The expression of these three proteins was statistically lower in the livers of hypercholesterolemic mice. Meanwhile, the testosterone levels in serum, testis, and liver were lower in hypercholesterolemic mice than those in normal mice, when human chorionic gonadotrophin was injected intramuscularly. These results suggest a testosterone deficiency in diet-induced hypercholesterolemic mice.
Journal of Nanoscience and Nanotechnology 08/2005; 5(8):1273-6. · 1.56 Impact Factor
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ABSTRACT: To investigate the relationship between apolipoprotein A5(apoA5) - 1131T > C polymorphism and the susceptibility of coronary artery disease (CAD) in Chinese.
The restriction fragment length polymorphism of apoA5 gene - 1131T > C was studied using PCR in a case-control study which enrolled 235 patients with CAD diagnosed by angiography and 262 healthy controls from Jiangsu province.
The frequencies of T, C allele were 59.57%ì40.43% and 65.65%, 34.35% in CAD group and control group respectively. There was statistically significant difference in allele frequencies between CAD group and control group (P < 0.05). The susceptibility to CAD for the CC genotype was much higher than that for wild type TT (OR = 1.872, 95% CI = 1.039 - 3.376, P = 0.037), even after the use of Logistic regression models (OR = 2.285, 95% CI = 1.222 - 4.274, P = 0.012). In control group, there was significant difference in TG levels among different genotypes, the C allele carriers had higher serum TG concentration (P = 0.007).
apoA5 - 1131T > C polymorphism is associated with an increased risk of CAD and is also in strong association with serum TG levels.
Zhonghua yi xue yi chuan xue za zhi = Zhonghua yixue yichuanxue zazhi = Chinese journal of medical genetics 06/2005; 22(3):281-3.
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ABSTRACT: To investigate the role of MyD88-dependent nuclear factor-kappaB (NF-(B) activation signaling pathway in the development of cardiac hypertrophy in vivo.
Dominant negative myeloid differentiation protein (dn-MyD) 88 fragment was inserted into pShuttle plasmid and then fused into adenovirus so as to construct Ad5-dn-MyD88. Some Sprague-Dawley (SD) rats underwent banding of aorta (aorta binding group) and some SD rats underwent sham operation (sham operation group). Part of the rats in the aorta banding group were transfected with Ad5-dn-MyD88 into the myocardium tissue (Ad5-dn-MyD88 transfection group) or adenovirus expressing dnMyD88 (Ad5-GFP) (control group) so as to determine the effect of blocking down stream of MyD88 signaling on the development of cardiac hypertrophy. Three days after the hearts of some rats from the 4 groups were collected and Western blotting was used to detect the expression of dn-MyD88 protein and fluorescent microscopy was used to detect the expression of GFP. Three weeks after the beginning of experiment the hearts were collected to calculate the heart weight/body weight (HW/BW) ratio and extract the plasma protein and nuclear protein. Electrophoretic mobility shift assay (EMSA) was used to determine the NFkappaB binding activity. Western blotting was used to examine the phosphorylation of IkappaBalpha and IKKalpha/beta with appropriate specific anti-phospho antibodies.
Flag and dn-MyD88 were effectively expressed 3 days after the transfection of Ad5-dn-MyD88 into the myocardium. Three weeks after the HW/BW ratio was 0.47 +/- 0.01 in the aorta banding group, significantly higher, by 37.8%, than that of the sham operation group (0.34 +/- 0.01, P < 0.01), and was 0.41 +/- 0.02 in the Ad5-dn-MyD88 transfection group, significantly lower, by 11.58%, than that of the aorta banding group (P < 0.01); the myocardial ANP protein expression level of the aorta binding group was significantly higher, by 43.5%, than that of the sham operation group (P < 0.01) and 36.2% higher than that of the Ad5-dnMyD88 transfection group (P < 0.01); the ANP/GAPDH in the aorta binding group was significantly higher than that of the sham operation group (P < 0.01) and that of the Ad5-dn-MyD88 transfection group (P < 0.01); the NF-kappaB binding activity in the myocardium of the aorta banding group was 9.94 +/- 1.58, significantly higher, by 144.8%, than that of the sham operation group (4.06 +/- 0.52, P < 0.01) and significantly lower, by 41.8%, than that of the Ad5-dn-MyD88 transfection group (5.79 +/- 0.52, P < 0.05); the phospho-(p-) IkappaBalpha level and p-IkappaBalpha/IkappaBalpha of the aorta binding group were significantly higher than those of the sham operation group (P < 0.01) and significantly higher, by 26.7%, than that of the Ad5-dn-MyD88 transfection group (P < 0.05); the p-IKKalphabeta/IKKalphabeta in the myocardium of the aorta binding group was significantly higher, by 318.0%, than that of the sham operation group (P < 0.01), and significantly higher, by 77.4%, than that of the Ad5-dn-MyD88 transfection group (P < 0.01).
MyD88-dependent NFkappaB signaling is a novel pathway for inducing the development of cardiac hypertrophy in vivo and blocking MyD88 mediated signaling pathway attenuates the development of cardiac hypertrophy.
Zhonghua yi xue za zhi 02/2005; 85(4):267-72.
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ABSTRACT: Although viral vectors are efficient systems to transfer foreign genes into cells or target tissues, safety issues remain in relation to human gene therapy. Microbubbles currently used as ultrasound contrast agents have been applied in transfection of genes. This study was designed to test the transfection efficiency and the expression of exogenous gene mediated by ultrasound irradiation enhanced air filled albumin microbubbles in ECV304 cell line in vitro and the heart of the mouse in vivo. Air filled microbubbles (2.0-4.0 microm in diameter) were created by sonicating the mixture of human albumin, glucose, mannitol and special additive that was designed for stabilization. Plasmid DNA loading the reporter genes was gently mixed with microbubbles. The mixture of plasmid DNA and microbubbles was administrated to cultured ECV304 cells and BALB/c mice (tail vein injection) under different ultrasound/microbubble conditions, and then the transfection and expression efficiency were examined. The results both in vivo and in vitro demonstrated that microbubble with ultrasound irradiation could significantly elevate the exogenous gene expression as compared with microbubble or ultrasound only. Overall, the present study showed that the ultrasound-target microbubble destruction method enhanced the exogenous gene expression in vivo and in vitro, and provided a gene therapy way not only efficient but also easy to be manipulated and carried out in clinical.
Acta Biochimica et Biophysica Sinica 01/2005; 36(12):824-31. · 1.38 Impact Factor
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ABSTRACT: We evaluated, in endothelial cells, the relative effectiveness of L-arginine and L-ascorbate in preventing the decrease in nitric oxide (NO) production in response to native low-density lipoprotein (LDL) from healthy subjects (nLDL), oxidized LDL (oxLDL, formed by nLDL oxidation) or native LDL from type 2 diabetic patients (dLDL). Human umbilical vein endothelial cells (HUVEC) were exposed to nLDL, dLDL or oxLDL (100 mg protein/L), in the absence or presence of L-arginine 10(-4)mol/L and/or L-ascorbate 10(-4)mol/L; NO synthase (NOS) activity and cyclic guanosine-3',5'-monophosphate (cGMP) were measured by the conversion of L-[3H]-arginine to L-[3H]citrulline and by radioimmunoassay, respectively. Both L-arginine and L-ascorbate increased cGMP in HUVEC co-incubated with any LDL species, although to lower levels than found in the absence of LDL. L-ascorbate did not affect NOS activity, whereas L-arginine increased it, both in the absence and presence of all LDL species. The effects of combined L-arginine and L-ascorbate on NOS activity and cGMP were no greater than those of L-arginine alone. Our results suggest that L-arginine or L-ascorbate can ameliorate, but not normalize, NO production in this situation, and that combining L-arginine with L-ascorbate is unlikely to produce additional benefit as compared with L-arginine alone.
Atherosclerosis 11/2004; 176(2):345-53. · 3.79 Impact Factor
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ABSTRACT: Renal cell hypertrophy is an important compensatory mechanism of chronic renal diseases, which has been shown closely correlated with the activation of intrarenal renin angiotensin system. However, the exact mechanism is still uncertain. The present study was to investigate the role of connective tissue growth factor (CTGF) in mediating the effect of angiotensin (AngII) induced human proximal tubular cell (HK-2) hypertrophy.
The cell line, HK-2, was grown in Dulbeccos's Modified Eagle's Medium (DMEM) containing 10% heat inactivated fetal calf serum (FCS). After rested in serum-free medium for 24 hours, The influence of anti-CTGF antibody on AngII induced cell protein de novo synthesis and total protein content were determined by [(3)H]-leucine incorporation and Coomassie brilliant blue G250 technique respectively. Fluorescence-activated cell sorter (FACS) flow cytometer was used to analyze the effect of anti-CTGF antibody on cell cycle distribution. The change of cellular size was determined by scanning electronic microscope (SEM).
AngII significantly induced the increase of [(3)H] leucine incorporation in dose [AngII (mol/L): 0: 6926 +/- 1034; 10(-9): 8455 +/- 2137; 10(-7): 10 741 +/- 802; 10(-5): 12 945 +/- 1377] and time [AngII (10(-7)mol/L): 0 h: 5584 +/- 1016; 24 h: 7379 +/- 957; 48 h: 10 741 +/- 802; 72 h: 16 606 +/- 1177] dependent manner. Meanwhile, the influence of AngII on cell total protein content showed the similar manner. Anti-CTGF antibody significantly inhibited the AngII induced above effects dose and time dependently. 48 h after the stimulation by AngII (10(-7)mol/L), the percentage of cells in G0-G1 phase (76.09% +/- 1.82%) and the average cell diameter (20.6 microm +/- 3.8 microm) was significantly increased compared to the control (62.1% +/- 2.5%, 11.9 microm +/- 1.6 microm, P < 0.01 respectively), which could markedly reversed by treatment with anti-CTGF antibody (71.68% +/- 1.78%; 16.4 microm +/- 3.2 microm, P < 0.05, 0.01 respectively vs AngII group).
AngII could induce the development of tubular cell hypertrophy, which might be mediated by CTGF.
Zhonghua yi xue za zhi 10/2004; 84(17):1470-4.
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ABSTRACT: Intrarenal activation of the renin angiotensin system (RAS) plays an important role in mediating renal fibrosis. Both angiotensin converting enzyme inhibitors (ACEIs) and angiotensin II (AngII) receptor antagonists have been shown to exert a protective role against diabetic and non-diabetic nephropathy. However, the exact mechanism of how blocking local RAS prevents renal fibrosis is unclear. The present study was to investigate the influence of a new AngII receptor antagonist, irbesartan (Irb), on AngII-induced hypertrophy in human proximal tubular cell line (HK-2).
The cell line, HK-2, was grown in Dulbeccos's Modified Eagle's Medium containing 10% heat-inactivated fetal calf serum. After rested in serum-free medium for 24 hours, the effects of Irb on AngII (10(-7) mol/L)-induced [(3)H]-leucine incorporation, total protein content (measured by the Coomassie brilliant blue G250 method), and change in cell size (determined by scanning electron microscopy) were observed. The influence of Irb on the cell cycle was analyzed by fluorescence activated cell sorter (FACS) flow cytometry.
AngII induced cell hypertrophy in a time and dose dependent manner. Stimulation of cells with AngII for 48 hours resulted in a increase in [(3)H]-leucine incorporation [0 hour: (5584 +/- 1016) cpm/10(5) cells vs 48 hours: (10741 +/- 802) cpm/10(5) cells, P < 0.05], which was significantly attenuated by treatment with Irb. AngII significantly increased the total protein content in HK-2 cells [control: (0.169 +/- 0.011) mg/10(5) cells vs AngII group: (0.202 +/- 0.010) mg/10(5) cells, P < 0.05], which was also markedly inhibited by cotreatment with Irb (P < 0.01). Scanning electron microscopy showed that AngII induced an increase in average physical cell size, which was significantly inhibited by Irb [control: (11.92 +/- 1.62) microm; AngII group: (20.63 +/- 3.83) micro m; AngII + Irb group: (13.59 +/- 3.15) micro m; P < 0.01 vs control, respectively]. Furthermore, flow cytometry revealed that AngII arrested cells in the G(0)-G(1) phase, which was significantly reversed by treatment with Irb [G(0)-G(1) cells in AngII group: (76.09 +/- 1.82)%, in AngII + Irb group: (67.00 +/- 2.52)%, P < 0.05].
Irb can inhibit AngII-induced hypertrophy in HK-2 cells.
Chinese medical journal 04/2004; 117(4):547-51. · 0.86 Impact Factor
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ABSTRACT: 1. We investigated the relative effectiveness of L-arginine, L-ascorbate and pyridoxine in preventing the impairment of endothelium-mediated vasorelaxation induced by native low-density lipoprotein (nLDL) from healthy subjects, oxidised LDL (oxLDL, formed by oxidation of nLDL) or nLDL from type II diabetic patients (dLDL). 2. Rabbit aortic rings were exposed to nLDL, dLDL or oxLDL (50-200 mg protein l-1), or corresponding vehicle, following which they were constricted with noradrenaline 10(-6) M; concentration-relaxation curves were determined to acetylcholine (ACh), A23187, or sodium nitroprusside (NP), in the absence or presence of L-arginine (10(-5)-10(-3) M), L-ascorbate (10(-5)-10(-3) M) and pyridoxine (0.5-2.0 mM). 3. nLDL, dLDL and oxLDL all inhibited relaxant responses to ACh and A23187, but not to NP, in a concentration-dependent manner (oxLDL>dLDL>nLDL). 4. In the presence of all LDL preparations, L-arginine, L-ascorbate or pyridoxine each improved ACh and A23187 responses, although none completely normalised endothelium-dependent relaxations. The maximal effect of L-arginine occurred at 10(-4) M. The combination of L-arginine 10(-4) M, L-ascorbate 10(-5) M and pyridoxine 2.0 mM was equally effective as L-arginine 10(-4) M alone. 5. Our results confirm that nLDL, dLDL and oxLDL exert inhibitory effects on endothelium dependent, but not endothelium independent, relaxation of rabbit aorta. ACh and A23187 responses in the presence of any LDL species can be ameliorated by supplementation with L-arginine, L-ascorbate or pyridoxine, either singly or in combination, with no agent or combination proving superior to L-arginine alone. Nevertheless, ACh and A23187 responses are not completely normalised with such supplements, suggesting that there also exists a component of LDL-induced inhibition of endothelium-mediated vasorelaxation that is independent of the nitric oxide system.
British Journal of Pharmacology 12/2003; 140(7):1272-82. · 4.41 Impact Factor
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ABSTRACT: To investigate low density lipoprotein receptor (LDLR) function and gene mutation in Chinese patients with familial hypercholesterolemia(FH).
Lymphocytes were isolated from 10 ml anticoagulated peripheral blood of the patients, then a flow-cytometric method (FCM) with 1,1'-dioctadecyl-3,3,3', 3-tetramethylindocarbocyanine perchlorate labelled low density lipoproetin (DiI-LDL) was used to identify the function of LDLR on the surface of lymphocytes. Genomic DNA was isolated from whole blood of FH patients and analyzed by PCR-single strand conformation polymorphism (SSCP) and nucleotide sequencing methods.
Defects of binding and uptaking of LDLR were identified by FCM in 2 FH patients in one family, and their parents were examined in the present study. Then they were analyzed genetically. The detected mutation was a deletion of A, which caused a frame shift in codon 297 of exon 6 and introduced a beforehand stop codon in codon 369.
A novel mutation of LDL receptor gene was detected by the combination of FCM and PCR-SSCP methods.
Zhonghua yi xue yi chuan xue za zhi = Zhonghua yixue yichuanxue zazhi = Chinese journal of medical genetics 05/2003; 20(2):138-42.
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ABSTRACT: To investigate the mechanisms of angiotensin II receptor antagonist irbesartan (Irb) in prevention of renal lesion in streptozotocin (STZ)-induced diabetic rats.
Sprague-Dawley (SD) rats were randomly divided into three groups: normal control (group N), diabetic nephropathy (group DN), and diabetic nephropathy treated with Irb (group DNI). Diabetes was induced by injection of STZ ip after rats had received uninephrectomy. Blood glucose (BG), body weight (BW), urinary albumin excretion (Ualb), and 24-h proteinuria (24hUpro) were observed in the rats at week 4, 8, and 12, respectively. Creatinine clearance (Ccr), the kidney weight (KW), profile of kidney hypertrophy (KW/BW), renal tissue protein contents (RTP), glomerular area (AG), glomerular volume (VG), and width of glomerular basement membrane (GBM) were determined after the rats were sacrificed at week 12. Renal expression of connective tissue growth factor (CTGF) and transforming growth factor-beta1 (TGF-beta1) were determined by immunohistochemistry.
There was no significant difference in BG between group DN and DNI (P >0.05). Irb prevented the increasing of Ualb excretion, 24 hUpro, and Ccr in diabetic rats ( P < 0.01). Furthermore, Irb markedly inhibited the increasing of KW, KW/BW, RTP, AG, and VG shown in diabetic rats (P <0.05, P <0.01, respectively). Irb prevented the thickening of GBM and immunostaining of CTGF (P <0.01). The extent of CTGF expression was positively correlated with the glomerular immunostaining for TGF-beta1 and size of VG (P <0.01).
Irb exerts an early renal protective role to diabetic nephropathy, possibly through inhibition of renal hypertrophy and expression of CTGF.
Acta Pharmacologica Sinica 01/2003; 24(1):67-73. · 1.95 Impact Factor
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ABSTRACT: To analyse the LDL receptor (LDLR) function and gene mutation in a familial hypercholesterolemia (FH) patient and illustrate the effects of gene mutation type on LDL receptor function.
The pedigree of a FH proband was set up according to the serum lipid analysis and clinical presentations. The LDLR functions of cultured fibroblasts were investigated by radiolabelled ligand method. PCR-SSCP and DNA sequencing were performed on the genomic DNA isolated from whole blood
11 heterozygotes and 1 homozygote of FH were confirmed by pedigree analysis. The binding of LDL by LDLR of the proband was nearly normal while the uptake and degradation of LDL were only 3.6% and 1.7% as compared with controls. A frameshift mutation resulted from a G insert in codon 599 and a null mutation caused by CCA-->CCG base shift in codon 842 were found in exon 17.
A novel mutation of LDLR gene was reported. This mutation may severely affect the function of LDLR.
Zhonghua nei ke za zhi [Chinese journal of internal medicine] 10/2002; 41(10):667-70.
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ABSTRACT: To study the effects of praeruptorin C (pra-C) on proliferation of cattle aortic smooth muscle cells (SMC).
The DNA synthesis of SMC was measured using the incorporation of [3H]thymidine([3H]TdR). Cell cycle phase was evaluated by flow cytometry and cytotoxicity was evaluated by measuring lactic dehydrogenase (LDH) activity.
Whether or not treated with angiotensin II (Ang-II), SMC proliferation was suppressed by pra-C in a concentration-dependent manner at range from 0.001 micromol/L to 10 micromol/L. The inhibitory effects appeared to be related to G1-S block in cell cycle traverse while the LDH activities did not change dramatically.
Pra-C can completely inhibit SMC proliferation induced by Ang II and partly inhibit the growth of SMC- induced by bovine serum, which is important in prevention and treatment of vascular hyperplastic disease.
Acta Pharmacologica Sinica 03/2002; 23(2):129-32. · 1.95 Impact Factor
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ABSTRACT: To investigate the effects of ginkgolide B (GB) on proliferation of bovine aortic smooth muscle cells (SMC) and its related mechanisms.
After pretreating with GB or the mixture of ginkgolide A and B (GA:GB) at 37 degrees C for 0.5 h, the VSMC were treated with or without angiotensin II (Ang II) for 24 h. The proliferation of SMC was evaluated by 3H-thymidine incorporation and the cell cycle phase was measured by flow cytometry. Cytotoxicity was reflected by MTT and lactate dehydrogenase (LDH) activity of the supernatant.
Whether or not treated with Ang II, GB and GA:GB were shown to suppress SMC proliferation in concentration-dependent fashion at concentrations ranging from 10(-9) mol.L-1 to 10(-5) mol.L-1. The inhibitory effects appeared to be related to a G1-->S block in cell cycle traverse.
The suppression of SMC proliferation by GB might not only be contributed by blockage of the PAF receptor activity.
Yao xue xue bao = Acta pharmaceutica Sinica 02/2002; 37(2):90-3.
