Increased serum 2-oxoglutarate associated with high myocardial energy expenditure and poor prognosis in chronic heart failure patients.

Myocardial energy expenditure (MEE) and 2-oxoglutarate are elevated in chronic heart failure (CHF) patients compared with healthy controls. To explore whether 2-oxoglutarate could reflect the levels of MEE and predict the prognosis of CHF, 219 CHF patients and 66 healthy controls were enrolled. 2-Oxoglutarate was assayed with Liquid Chromatography-Mass Spectrometry/Mass Spectrometry (LC/MS/MS). CHF patients were divided into 4 groups according to interquartile range of MEE and followed for death or recurrent hospital admission due to CHF for the mean follow-up time 6.64±0.16months. 2-Oxoglutarate was increased in CHF patients compared with controls (P<0.01) and correlated with estimated glomerular filtration rate (r=0.142, P=0.036), age (r=-0.269, P<0.01) and MEE levels (r=0.307, P<0.01) in a multiple linear correlation analysis in CHF patients. Furthermore, 2-oxoglutarate (OR=3.470, 95% CI=1.557 to 7.730, P=0.002), N-terminal pro-B-type natriuretic peptide (OR=4.013, 95% CI=1.553 to 10.365, P=0.004), age (OR=1.611, 95% CI=1.136 to 2.283, P=0.007) and left ventricular ejection fraction (OR=7.272, 95% CI=3.110 to 17.000, P<0.001) were independently associated with MEE on multiple logistic regression analysis. Kaplan-Meier event curves showed that high 2-oxoglutarate levels were associated with adverse outcomes (Log Rank, Chi(2)=4.026, P=0.045). This study showed that serum 2-oxoglutarate is associated with MEE levels, which can be used as potential biomarkers for MEE, and it can reflect the clinical severity and short-term outcome of CHF.

Probucol alleviates atherosclerosis and improves high density lipoprotein function.

BACKGROUND: Probucol is a unique hypolipidemic agent that decreases high density lipoprotein cholesterol (HDL-C). However, it is not definite that whether probucol hinders the progression of atherosclerosis by improving HDL function. METHODS: Eighteen New Zealand White rabbits were randomly divided into the control, atherosclerosis and probucol groups. Control group were fed a regular diet; the atherosclerosis group received a high fat diet, and the probucol group received the high fat diet plus probucol. Hepatocytes and peritoneal macrophages were isolated for [(3)H] labeled cholesterol efflux rates and expression of ABCA1 and SR-B1 at gene and protein levels; venous blood was collected for serum paraoxonase 1, myeloperoxidase activity and lipid analysis. Aorta were prepared for morphologic and immunohistochemical analysis after 12 weeks. RESULTS: Compared to the atherosclerosis group, the paraoxonase 1 activity, cholesterol efflux rates, expression of ABCA1 and SR-BI in hepatocytes and peritoneal macrophages, and the level of ABCA1 and SR-BI in aortic lesions were remarkably improved in the probucol group, But the serum HDL cholesterol concentration, myeloperoxidase activity, the IMT and the percentage plaque area of aorta were significantly decreased. CONCLUSION: Probucol alleviated atherosclerosis by improving HDL function. The mechanisms include accelerating the process of reverse cholesterol transport, improving the anti-inflammatory and anti-oxidant functions.

ISO-1, a macrophage migration inhibitory factor antagonist, inhibits airway remodeling in a murine model of chronic asthma.

Airway remodeling is the process of airway structural change that occurs in patients with asthma in response to persistent inflammation and leads to increasing disease severity. Drugs that decrease this persistent inflammation play a crucial role in managing asthma episodes. Mice sensitized (by intraperitoneal administration) and then challenged (by inhalation) with ovalbumin (OVA) develop an extensive eosinophilic inflammatory response, goblet cell hyperplasia, collagen deposition, airway smooth muscle thickening, and airway wall area increase, similar to pathologies observed in human asthma. We used OVA-sensitized/challenged mice as a murine model of chronic allergic airway inflammation with subepithelial fibrosis (i.e., asthma). In this OVA mouse model, mRNA and protein of macrophage migration inhibitory factor (MIF) are upregulated, a response similar to what has been observed in the pathogenesis of acute inflammation in human asthma. We hypothesized that MIF induces transforming growth factor-ß1 (TGF-ß1) synthesis, which has been shown to play an important role in asthma and airway remodeling. To explore the role of MIF in the development of airway remodeling, we evaluated the effects of an MIF small-molecule antagonist, (S,R)3-(4-hy-droxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester (ISO-1), on pathologies associated with the airway-remodeling process in the OVA mouse model. We found that administration of ISO-1 significantly mitigated all symptoms caused by OVA treatment. In addition, the treatment of OVA-sensitized mice with the MIF antagonist ISO-1 significantly reduced TGF-ß1 mRNA levels in pulmonary tissue and its protein level in bronchial alveolar lavage fluid supernatants. We believe the repression of MIF in the ISO-1 treatment group led to the significant suppression observed in the inflammatory responses associated with the allergen-induced lung inflammation and fibrosis in our murine asthma (OVA) model. Our results implicate a possible function of MIF in the pathogenesis of chronic asthma and suggest that MIF might be an important therapeutic target for airway remodeling.

[Effects of a new houttuyfonate derivative on proliferation of NIH3T3 cell and expression of syndecan-4 induced by tumor necrosis factor alpha].

OBJECTIVE: To investigate the effect of a new houttuyfonate derivative (NHD) on proliferation of NIH3T3 cell and expression of Syndecan-4 induced by TNF-alpha in vitro. METHODS: NIH3T3 cells were cultured and exposed to TNF-alpha or NHD respectively, and then cotreated with TNF-alpha and NHD. All the groups were cultured for 24 hour in vitro, in addition to the untreated control group established for comparison. The ratio of proliferation of NIH3T3 cell was determined by non-radioactive MTS/PMS assay and the expression of Syndecan-4 was evaluated by western blot using anti-Syndecan-4 antibody. RESULTS: Statistical analysis showed that, compared with the control group, NHD had no effect on VSMCs growth, but significantly inhibited NIH3T3 cell proliferation while induced by TNF-alpha. It also showed that compared with control group, NHD had no effect on the expression of Syndecan-4, but significantly inhibited its expression while induced by TNF-alpha (P < 0.05). CONCLUSIONS: NHD can inhibit the proliferation of NIH3T3 cell and the expression of syndecan-4 protein induced by TNF-alpha in vitro.

Contraction-dependent TGF-ß1 activation is required for thrombin-induced remodeling in human airway smooth muscle cells.

AIMS: Thrombin is a serine proteinase that is not only involved in coagulation cascade, but also mediates a number of biological responses relevant to tissues repair, and induces bronchoconstriction. TGF-ß plays a pivotal role in airway remodeling due to its effects on airway smooth muscle proliferation and extracellular matrix (ECM) deposition. Recently, bronchoconstriction itself is found to constitute a form of strain and is highly relevant to asthmatic airway remodeling. However, the underlying mechanisms remain unknown. Here, we investigated the role of contraction- dependent TGF-ß activation in thrombin-induced remodeling in human airway smooth muscle (HASM) cells. MATERIALS AND METHODS: Primary HASM cells were treated with or without thrombin in the absence or presence of anti-TGF-ß antibody, cytochalasin D and formoterol. CFSE labeling index or CCK-8 assay were performed to test cell proliferation. RT-PCR and Western blotting were used to examined ECM mRNA level and collagen Iα1, α-actin protein expression, respectively. Immunofluorescence was also used to confirm contraction induced by thrombin in HASM cells. KEY FINDING: Thrombin stimulation enhanced HASM cells proliferation and activated TGF-ß signaling. Thrombin induced ECM mRNA and collagen Iα1 protein expression, and these effects are mediated by TGF-ß. Abrogation of TGF-ß activation by contraction inhibitors cytochalasin D and formoterol prevents the thrombin-induced effects. SIGNIFICANCE: These findings suggest that contraction-dependent TGF-ß activation could be a mechanism by which thrombin leads to the development of asthmatic airway remodeling. Blocking physical forces with bronchodilator would be an intriguing way in reducing airway remodeling in asthma.

[Association of polymorphism of Met764Thr locus allele in ADAM33 gene with bronchial asthma and lung function of asthmatic subjects].

OBJECTIVE: To analyze the association of the polymorphism of Met764Thr with bronchial asthma and lung function of asthmatic subjects of Han nationality in Southern China. METHODS: In 164 unrelated patients with asthma and 112 unrelated healthy controls, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and DNA sequencing were used to determine polymorphism of Met764Thr locus allele in ADAM33 gene. The clinical indexes associated with lung function (FVC%, FEV(1)%) were compared among the three genotypes (Met764/Met764, Met764/Thr764, Thr764/Thr764) in asthmatic subjects. RESULTS: No significant difference was found in the allele (Met764, Thr 764) frequency among populations in UK, US, German, Korean, and Southern China (chi(2) = 6.77, P > 0.05). The frequencies of the genotypes (Met764/Met764, Met764/Thr764, Thr764/Thr764) were respectively 78.7% (129), 18.3% (30), 3.0% (5) in 164 asthmatic subjects and respectively 91.1% (102), 6.3% (7), 2.7% (3) in the 112 controls. There was a significant difference in the distributions of the genotypes (Met764/Met764, Met764/Thr764, Thr764/Thr764) between asthmatic subjects and controls (chi(2) = 8.46, P < 0.05). The frequencies of alleles (Thr764) were respectively 12.2% in the asthmatic subjects and 5.8% in the controls. Significant difference was observed in the allele (Met764, Thr 764) frequency between the two groups (chi(2) = 6.27, P < 0.05). The presence of Thr764 allele of ADAM33 gene was found to be a greater risk factor in asthmatic subjects than in controls. The odds ratio (OR) of Met764/Thr764 and Met764/Thr764 + Thr764/Thr764 were 3.389 (1.430 - 8.030), 2.767 (1.308 - 5.854), respectively. When compared with Met764/Met764 genotype, all P < 0.05. There was a significant decrease in the FVC% and FEV(1)% levels of Met764/Thr764, Thr764/Thr764 and Met764/Met764 genotype. CONCLUSIONS: These results suggest that Met764Thr locus genetic polymorphism is associated with susceptibility of asthma and clinical indexes of lung function of asthmatic subjects of Han nationality in Southern China.

[Effects of flavone from leaves of Diospyros kaki on expression of apoptosis signal-regulating kinase 1 and rat vascular smooth muscle cells proliferation by tumor necrosis factor alpha in vitro].

OBJECTIVE: To observe the effects of flavone from leaves of Diospyros kaki on expression of apoptosis signal-regulating kinase 1 (ASK1) and rat vascular smooth muscle cells (VSMCs) proliferation by tumor necrosis factor alpha in vitro. METHODS: Rat aortic VSMCs were cultured in vitro and treated with tumor necrosis factor alpha (TNF-alpha) and flavone from leaves of Diospyros kaki, respectively, and were observed in comparison with the control group. The ratio of cell proliferation was determined by non-radioactive MTS/PES as-say. The expression of ASK1 protein was evaluated by the immunoblotting technique using anti-ASKL antibody. RESULTS: The ratio of cell proliferation was 0.817 +/- 0.074 in the control group, and was 1.865 +/- 0.093 in TNF-alpha20 ng/ml group. It was shown that TNF-alpha significantly induced rat VSMCs proliferation (P < 0.05). The ratio of cell proliferatioh was 0.905 +/- 0.044 in flavone from leaves of Diospyros kaki group corresponding to concentration of 50 microg/ml. It was shown that flavone from leaves of Diospyros kaki alone had no effect on rat VSMCs proliferation (p > 0.05). With TNF-alpha stimulation, flavone from leaves of Diospyros kaki significantly inhibited rat expression of ASK1 protein enhanced by TNF-alpha was significantly inhibited rat-VSMNCs proliferation (1.247 +/- 0.061 vs. 1.865 +/- 0.093, p < 0.05). The expression of ASK1 protein enhanced by TNF-alpha was significantly inhibited by flavone from leaves of Diospyros kaki in VSMCs. CONCLUSION: Flavone from leaves of Diospyros kaki can significantly inhibit expression of ASK1 protein stimulated by TNF-alpha of rat VSMcs in vitro.

[A novel polymorphism in the ATP-binding cassette transporter A1 gene (M233V) in Chinese patients with coronary artery disease].

OBJECTIVE: To identify ATP-binding cassette transporter A1 (ABCA1) gene polymorphism in Chinese patients with coronary artery disease (CAD). METHODS: Single Nucleotide Polymorphisms in the ABCA1 gene were detected by polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP)-DNA sequence and restriction-fragment length polymorphism (RFLP) method in 112 patients with CAD. RESULTS: A novel polymorphism in the ABCA1 gene was found in two patients: M233V which exists in exon7 of ABCA1 gene and it's cDNA location is A1092G and converse 233 amino acid from Methionine to Valeric. We further collected the blood samples from 16 family members of one proband and M233V polymorphism was found in 5 out 16 family members. CONCLUSION: M233V is a novel polymorphism in the ATP-binding cassette transporter A1 gene and this AG genotype had family proneness.

[Red blood cell distribution width combined with lipoprotein-associated phospholipase A2 detection for improving diagnostic accuracy of coronary artery stenosis in patients with coronary artery disease].

OBJECTIVE: To study the association of red blood cell distribution width (RDW) and lipoprotein-associated phospholipase A2 (LP-PLA2) with the degree of coronary artery stenosis in patients with coronary artery disease (CAD) and the value of RDW combined with LP-PLA2 detection in accurate evaluation of coronary artery stenosis. METHODS: A total of 224 patients including 119 non-CAD cases and 105 CAD cases admitted in our hospital between June, 2013 and June, 2014 were enrolled in this study. The patients' baseline clinical data were collected and venous blood samples were obtained for detecting WBC, RDW-CV and LP-PLA2. The Gensini score of the CAD patients was calculated based on coronary angiographic findings. RESULTS: Compared with the non-CAD patients, CAD patients had significantly higher RDW-CV (P=0.009) and LP-PLA2 (P=0.004) levels. The CAD patients with high Gensini scores had also significantly higher RDW-CV (P=0.001) and LP-PLA2 (P<0.001) levels than those with low scores; RDW-CV and LP-PLA2 were significantly correlated with the Gensini score, and the area under curve of their combined detection was 0.931. CONCLUSION: Combination of RDW and LP-PLA2 can improve the diagnostic accuracy of the degree of coronary artery stenosis in patients with CAD.

[Effects of dexamethasone on the expression of muscarinic receptor mRNA in asthmatic guinea pig airway smooth muscle and eosinophil infiltration in bronchoalveolar lavage fluid].

OBJECTIVE: To investigate the effect of dexamethasone on the expression of muscarinic receptor (MR) mRNA in smooth muscle and infiltration of eosinophils (Eos) in the airway of asthmatic guinea pigs. METHODS: Thirty healthy guinea pigs were randomized into 3 equal groups, the control group, asthmatic group and dexamethasone therapy group. Asthma was induced in the latter 2 groups with the asthma-inducing agents and received treatments as indicated. Bronchial alveolar lavage fluid(BALF) were collected subsequently from the guinea pigs for examining the total cell number and cell classification, and histopathologic examination of the lung tissue was performed. Semi-quantitative analysis with reverse transcriptional- polymerase chain reaction (RT-PCR) was performed for M(2) and M(3) receptor mRNA in airway smooth muscle. RESULTS: Compared with the control and the asthmatic group, the number of Eos in the BALF of dexamethasone therapy group was significantly lower (P<0.01). In spite of the presence of hyperemia and edema in the lung tissues of the dexamethasone therapy group, Eos infiltration was less severe than that in the asthmatic group. As found by RT-PCR, the quantity of M(2) receptor mRNA in the airway smooth muscle of the dexamethasone therapy group was significantly higher than those in both the control and asthmatic groups (P<0.01), and the quantity of M(3) receptor mRNA in the airway smooth muscle of dexamethasone therapy group was significantly higher than that in the asthmatic group, but did not significantly differ from that in the control group. The quantities of M(2) and M(3) receptor mRNAs in the control group were both significantly higher than that in asthmatic group (P<0.01). CONCLUSION: The expression of M(2) receptor is increased in antigen- challenged guinea pigs, and that of M(3) receptor decreased. Dexamethasone can treat asthma by inhibiting inflammatory action involving Eos infiltration, regulating the expressions of M(2) and M(3) receptors and restoring the function of M(2) receptor.

[Transient cytosolic free calcium changes in cultured neonatal rat cardiac myocytes in response to advanced glycation end-product treatment].

OBJECTIVE: To investigate the effects of advanced glycation end-products (AGEs) on transient cytosolic free calcium in neonatal rat cardiac myocytes (CMs) cultured in vitro. METHODS: CMs cultured for 3 to 5 days in vitro were incubated with Ca(2+)-sensitive fluorescent indicator Fluo-3AM with light screening at 37 degrees celsius; with 5% CO(2) for 60 min. Changes of the fluorescence signal of free calcium caused by AGEs were measured under laser scanning confocal microscope (LSCM). RESULTS: Compared with the control cells, AGEs caused an increase in the concentration of cytosolic free calcium in a dose-dependent manner. CONCLUSION: AGEs may impair neonatal rat CMs by altering cytosolic free calcium concentration.

[Significance of -191G/C single nucleotide polymorphisms in the promoter region of ATP-binding cassette transporter gene in coronary artery disease].

OBJECTIVE: To investigate the effect of -191G/C single nucleotide polymorphisms (SNP) in the promoter region of ATP-binding cassette transporter A1(ABCA1) gene on plasma lipids and its significance in coronary artery disease (CAD). METHODS: By polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), -191G/C SNP in the promoter region of ABCA1 gene was analyzed in 204 patients with CAD and 114 control subjects and the distribution of the -191G/C genotypes compared between the two groups and also between different clinical phenotypes of CAD. The clinical indexes associated with CAD were also compared between the patients with the three genotypes of CAD. RESULTS: The frequency distribution of GG, GC, and CC genotypes significantly differed between CAD group and the control group, and the former group had obvious higher CC genotype frequency and the C allele frequency (P<0.05 and P<0.01, respectively). In CAD patients, the frequency distribution of GG, GC, and CC genotypes varied significantly between those with acute coronary syndrome (ACS) and those with stable angina pectoris (SAP). The CC genotype showed obviously higher frequency in ACS group than in SAP group and the C allele was more frequent in the former group (P<0.05 and P<0.01, respectively). However, no significant difference was noted in the body mass index, total cholesterol, triglyceride, high-density lipoprotein, low-density lipoprotein, or very low-density lipoprotein cholesterols between the three genotypes. CONCLUSIONS: The -191G/C SNP in the promoter region of ABCA1 is associated with increased CAD and the C allele may relate to the stability of CAD without detectable changes in plasma lipids.

[Effects of captopril and losartan on expression of kidney aquaporin-2 mRNA and urine aquaporin-2 excretion in rats].

OBJECTIVE: To investigate effects of captopril and losartan on the expression of kidney aquaporin-2 (AQP2) mRNA and the excretion of urine AQP2 in rats. METHODS: Thirty healthy rats were randomized into 3 groups, namely the control group, captopril group and losartan group, respectively. Blood and urine samples were collected from the rats for detecting serum Na(+), urine volume and urine osmolality in the course of medication. Urine AQP2 concentration was measured by enzyme-linked immunosorbent assay (ELISA). Semi-quantitative RT-PCR was performed for measurement of kidney inner medullary AQP2 and vasopressin V(2) receptor mRNA. RESULTS: Urine volume was increased in rats of captopril and losartan groups as compared with that of the control group. However, urine osmolality was lower in captopril group than in the other two groups (P<0.05). RT-PCR revealed decreased quantity of the inner medullary AQP2 mRNA of the captopril group than that of the other two groups, but the quantity of V(2) receptor mRNA did not differ significantly between the 3 groups. Urine AQP2 concentration was significantly higher in captopril group than in the control (P<0.05) and losartan groups (P0<0.01). CONCLUSION: Captopril can reduce the expression of the kidney inner medullary AQP2 mRNA and accelerate the excretion of the urine AQP2 in normal rats.

Rosiglitazone attenuates atherosclerosis and increases high-density lipoprotein function in atherosclerotic rabbits.

Rosiglitazone has been found to have anti-atherogenic effects and to increase serum high-density lipoprotein (HDL) cholesterol (HDL-C) levels. However, in vivo studies investigating the regulation of adenosine triphosphate-binding cassette transporter A1 (ABCA1) and scavenger receptor class B type I (SR-BI) by rosiglitazone are limited. Moreover, the effects of rosiglitazone on the function and levels of HDL are unclear. In the present study, we investigated the effects of rosiglitazone on HDL function and its mechanisms of action in atherosclerotic rabbits. Our results revealed that rosiglitazone induced a significant increase in serum HDL-C levels, paraoxonase 1 (PON1) activity, [(3)H]cholesterol efflux rates, and the expression of ABCA1 and SR-BI in hepatocytes and peritoneal macrophages. The expression of ABCA1 was also increased in aortic lesions. Rosiglitazone markedly reduced serum myeloperoxidase (MPO) activity, aortic intima-media thickness (IMT) and the percentage of plaque area in the aorta. It can thus be concluded that in atherosclerotic rabbits, rosigitazone increases the levels of HDL-C and hinders atherosclerosis. Thus, it improves HDL quality and function, as well as the HDL-induced cholesterol efflux, exerting anti-inflammatory and antioxidant effects.

[Effects of Huobahuagen tablet on the expression of interleukin-3 receptor mRNA in asthmatic guinea pig bronchoalveolar lavage fluid and eosinophil infiltration].

OBJECTIVE: To investigate the effects of Huobahuagen tablet on the expression of interleukin-3 (IL-3) receptor mRNA in bronchoalveolar lavage fluid (BALF) and infiltration of eosinophil (Eos) in the airway of asthmatic guinea pigs. METHODS: Thirty-two healthy guinea pigs were randomized into 4 equal groups, the control group, asthmatic group, dexamethasone therapy group and Huobahuagen tablet therapy group. Asthma was induced in the latter 3 groups which were challenged with the asthma-inducing agents and at the same time received treatments as indicated. BALF were collected subsequently from the guinea pigs for examining the total cell number and cell classification, and histopathologic examination of the lung tissue was performed. Semi-quantitative analysis with reverse transcriptional-polymerase chain reaction (RT-PCR) of IL-3 receptor mRNA in the BALF was performed. RESULTS: Compared with the control and the asthmatic group, the number of Eos in the BALF of Huobahuagen tablet therapy group was significantly lower (P<0.05 and P<0.01, respectively). In spite of the presence of hyperemia and edema in the lung tissues of the Huobahuagen tablet therapy group, Eos infiltration was less severe than that in the asthmatic group. As found by RT-PCR, the quantity of IL-3 receptor mRNA in the BALF of the Huobahuagen tablet therapy group did not significantly differ from that in the dexamethasone therapy group, but was significantly higher than that in both the control and asthmatic group (P<0.01). CONCLUSION: Huobahuagen tablet can significantly lower the number of Eos in the airway of asthmatic guinea pigs, a finding that may potentially serve as the elementary theoretical basis for clinical therapy of asthma.

[Comparative study of T cell proliferative response in different types of coronary heart diseases].

OBJECTIVE: To investigate the association between proliferative response of T cells and plaque stability in coronary heart disease. METHODS: A total of 66 subjects were recruited in the study, including 13 patients with acute myocardial infarction (AMI), 22 with unstable angina pectoris (UAP), 15 with stable angina pectoris (SAP) and 16 control subjects without coronary heart disease. The proliferative response of T cells to phytohemagglutinin (PHA), oxidized low-density lipoprotein (oxLDL) and low-density lipoprotein (LDL) was examined by MTS/PMS colorimetric assay. RESULTS: Significantly stronger proliferative response of T cells to PHA was noted in AMI and UAP groups than in SAP and control groups (P<0.05), as with the response to oxLDL (at doses of 5 and 1 microg/ml, P<0.05). T cell proliferative response to oxLDL (at the doses of 10, 5 and 1 microg/ml) appeared significantly stronger than that to LDL (at the same dose as oxLDL) in AMI group and UAP group (P<0.05). CONCLUSION: Cellular immunity mediated by T cells, especially the immune response to oxLDL, may play an important role in the instability of plaque and the occurrence of acute coronary syndrome (ACS).

[Green tea polyphenols inhibit low-density lipoprotein-induced proliferation of rat vascular smooth muscle cells].

The proliferation and migration of vascular smooth muscle cells (VSMCs) is one of the major mechanisms of intimal thickening in atherosclerosis and post-angioplasty restenosis. Elevated plasma levels of low-density lipoprotein (LDL) have been implicated in the pathogenesis of atherosclerotic vascular diseases. The purpose of this study was to determine the effects of green tea polyphenols on the proliferation and p44/42 mitogen-activated protein kinase (MAPK) activity in rat VSMCs simulated by native LDL. Rat aortic VSMCs were cultured and treated with LDL (100 microg/ml) in the absence or presence of green tea polyphenols, and the cell proliferation was subsequently quantified by non-radioactive MTS/PES assay and the cell cycle analyzed by flow cytometry. The p44/42 MAPK activity was evaluated by immunoblotting using anti-p44/42 phospho-MAPK antibody. Compared with the cells without polyphenol treatment, the proliferation of the VSMCs induced by LDL was dose-dependently inhibited by green tea polyphenols (P<0.05), with more numerous cells in G(0)G(1) phase (P<0.05) as shown by flow cytometry analysis. LDL significantly enhanced the p44/42 MAPK activity, an effect obviously inhibited by green tea polyphenols (at 100 microg/ml). These results suggest that green tea polyphenols can inhibit high levels of LDL-induced proliferation of phosphorylated p44/42 MAPK expression in rat VSMCs. Green tea polyphenols may, therefore, offer vascular protection by inhibiting VSMC growth in response to hypercholesterolemia.

[C-reactive protein inhibits the expression of hypoxia-inducible factor-1alpha in human umbilical vein endothelial cells in hypoxic condition].

OBJECTIVE: To investigate the effect of C-reactive protein (CRP) on expression of hypoxia-inducible factor-1alpha (HIF-1alpha). METHODS: Using CoCl(2) (100 micromol/L) to simulate hypoxic condition for culturing human umbilical vein endothelial cells (HUVECs), we examined the effects of CRP on expression of HIF-1alpha. The proteins were extracted from the cells after a 24-hour exposure of the cells to CRP of varied concentrations in the presence of CoCl(2), and Western blotting was performed for quantification of HIF-1alpha expression, the results were analyzed statistically with SPSS software. RESULTS: CRP at the concentration of 5 microg/ml decreased the expression of HIF-1alpha (P<0.001), producing the maximum inhibitory effect at the concentration of 100 microg/ml, an effect exhibiting dose-dependence. CONCLUSION: CRP inhibits the expression of HIF-1alpha in HUVECs subjected to hypoxic condition, which serves as an important evidence for the inhibitory effect of CRP on angiogenesis in hypoxic condition.

Effects of recombinant Sagartia rosea cytolysin and Lapemis hardwickii phospholipase A2 on adventitial fibroblasts proliferation.

OBJECTIVE: To observe the effects of recombinant Sagartia rosea cytolysin (rSrc) and Lapemis hardwickii phospholipase A2(rPLA2) on adventitial fibroblasts proliferation. METHODS: NIH-3T3 cells were cultured and treated with rSrc and rPLA2 at different concentrations for observation of cell proliferation using non-radioactive MTS/PES assay in comparison with the control group. RESULTS: The ratio of cell proliferation was 0.840+/-0.061 in the control group, and was 0.263+/-0.044, 0.418+/-0.054, 0.605+/-0.063, 0.772+/-0.054 and 0.906+/-0.072 in rSrc groups corresponding to rSrc concentrations of 100, 10, 1 microg/ml and 100, 10 ng/ml respectively. rSrc was found to significantly inhibit fibroblast proliferation in a dose-dependent manner when the concentration used was above 1 microg/ml (P<0.05), as compared with the control group (P<0.05). The ratio of cell proliferation was 0.498+/-0.076, 0.937+/-0.112 and 0.978+/-0.145 in rPLA2 groups corresponding to rPLA2 concentrations of 100, 10, 1 microg/ml respectively, indicating that rPLA2 also significantly inhibited fibroblast proliferation at the concentration of 100 microg/ml (P<0.05). CONCLUSION: rSrc and rPLA2 can both significantly inhibit adventitial fibroblast proliferation.

[Regulation of cyclin-dependent kinase inhibitors by mitogen-activated protein kinase in angiotensin II-stimulated vascular smooth muscle cell hypertrophy].

OBJECTIVE: To investigate the role of mitogen-activated protein kinase (MAPK) in the regulation of cyclin-dependent kinase inhibitors (CDKI) in the process of vascular smooth muscle cell (VSMC) hypertrophy induced by angiotensin II stimulation. METHODS: The medial layer of male SD rat aorta was isolated for VSMC culture. After cultured in serum-free medium to arrest the cell growth, VSMCs were stimulated with angiotensin II (1x10(-6) mol/L) or/and phorbol myristate acetate (PMA, 20 nmol/L), with the cells cultured in serum-free medium serving as control. MAPK activity of the cells was assayed 90 min after stimulation with immunoprecipitation test, and the expression levels of CDKI p27, p57 and p21 were determined by Western blotting 6 and 24 h after stimulation respectively. RESULTS: Compared with the control level, MAPK activity of the VSMCs was up-regulated by 238% by treatment with angiotensin II alone which, however, did not inhibit p27 expression or induce VSMC proliferation. Costimulation with angiotensin II and PMA slightly inhibited p27 expression but VSMC proliferation was still not observed. CONCLUSION: MAPK pathway is an important channel for extracellular proliferative and hypertrophic signal transduction into the nucleus of VSMCs.