Resveratrol inhibits macrophage expression of EMMPRIN by activating PPARgamma.

The effect of resveratrol on macrophage EMMPRIN expression and its potential mechanism was investigated. Both EMMPRIN expression and MMP-9 activity, respectively assayed by Western blot and zymography, were greatly up-regulated during PMA-induced macrophage differentiation from THP-1 monocytes. Both resveratrol and a PPARgamma agonist, pioglitazone, significantly inhibited EMMPRIN expression and MMP-9 activity in a concentration-dependent manner. The effects of pioglitazone and resveratrol were reversed by pretreatment of THP-1 cells with a PPARgamma antagonist, GW9662, prior to PMA induction. Thus, data suggest that resveratrol may down-regulate EMMPRIN and MMP-9 through PPARgamma activation. This possibility was further examined in resveratrol-or pioglitazone-treated U937 cells, which had been co-transfected with a PPARgamma expression vector and a luciferase reporter vector containing three tandem repeats of PPRE in cis. Results of the agonist-activated luciferase assay showed that resveratrol activated PPARgamma in a concentration-dependent manner. Since EMMPRIN and MMP-9 up-regulation is associated with activation of the NF-kappaB pathway, we investigated the effect of pioglitazone and resveratrol on TNF-alpha-induced NF-kappaB activation. Western blot results indicated that both pioglitazone and resveratrol markedly inhibited the NF-kappaB pathway through suppressing IkappaB protein phosphorylation in macrophages, although this effect of resveratrol was not reversed by GW9662. In conclusion, resveratrol can down-regulate EMMPRIN expression by macrophages via activating PPARgamma. This may be a primary mechanism of its inhibitory effect on MMP-9.

[Effect of retinoid X receptor activation on oxidized low-density lipoprotein induced cell differentiation of murine macrophage cell line into dendritic like cells].

OBJECTIVE: To investigate the effect and related mechanism of retinoid X receptor (RXR) activation on oxidized low-density lipoprotein (ox-LDL) induced differentiation of macrophage into dendritic cell. METHODS: RAW264.7 murine macrophage cell line was cultured with ox-LDL for 48 h in the absence and presence of RXR activator 9-cisRA or SR11237. Cell morphology was observed by phase contrast microscope and cell surface markers involved in dendritic cell immune maturation and activation was analyzed by FACS. Cellular reactive oxygen species production was detected by CM-H2DCFDA fluorescent probe. RESULTS: ox-LDL-treated RAW264.7 murine macrophage cell line differentiated into dendritic like cells after 48 h and cell surface markers CD40, CD86, CD83, MHC Class II and CD1d were upregulated. These changes could be attenuated by cotreatment with 9-cisRA or SR11237. Upregulated cell surface markers CD40, CD86, CD83, MHC Class II and CD1d by ox-LDL were decreased about 47%, 43%, 48%, 32% and 17% respectively by 9-cisRA and 38%, 38%, 46%, 36% and 32% respectively by SR11237. The effect of 9-cisRA and SR11237 was dose dependent. Cellular reactive oxygen species were significantly increased in ox-LDL-treated RAW264.7 cells (MFI 38.24 +/- 4.20 vs. 4.46 +/- 0.39, P < 0.05) and which was significantly reduced by 9-cisRA (10(-7) mol/L) and SR11237 (10(-6) mol/L) to 12.60 +/- 1.52 and 17.89 +/- 1.91 respectively (all P < 0.05). CONCLUSION: RXR activation partly inhibits the differentiation of ox-LDL induced macrophage into dendritic cell by reducing oxidative stress injury.

[Resveratrol inhibits expression of EMMPRIN from macrophages].

AIM: To investigate the effect of resveratrol on EMMPRIN expression of macrophages. METHODS: Human monocytic cell line THP-1 cells were co-cultured with EMMPRIN-highly-expressed MCF-7 cells; MMP-9 production was assayed by zymography. THP-1 cells were induced by PMA, expression of EMMPRIN was assayed by Western blotting. Cells were treated with resveratrol or PPARgamma agonist--pioglitazone during differentiation, EMMPRIN expression and MMP-9 activity were assayed. U937 cells were co-transfected with PPARy expression and luciferase-coding reporter vector, then cultured with pioglitazone or resveratrol, the activating capability of resveratrol on PPARgamma was evaluated by measuring the luciferase activity. THP-1 cells were pretreated with PPARgamma antagonist--GW9662 before pioglitazone or resveratrol treatment, then assayed for EMMPRIN expression and MMP-9 production. RESULTS: EMMPRIN expression was greatly increased during the differentiation from monocytes to macrophages; co-culturing with MCF-7 cells significantly increased MMP-9 production by monocytes. Both resveratrol and pioglitazone markedly inhibited EMMPRIN expression during monocytes differentiation. Resveratrol significantly activated PPARgamma and GW9662 greatly decreased the effect of resveratrol on EMMPRIN and MMP-9. CONCLUSION: EMMPRIN expression is greatly up-regulated from monocytes to macrophages, which may play a role in inducing MMPs production by monocytes/macrophages. Resveratrol can significantly inhibit EMMPRIN expression via activating PPARgamma, which may be the underlying mechanism of its inhibitory effect on MMPs production by monocytes/macrophages.

[The clinical value of sixteen-detector row computed tomography angiography for the assessment of coronary artery bypass graft].

OBJECTIVE: The aim of this study was to evaluate the value of sixteen-detector row computed tomography angiography (CTA) for the assessment of coronary artery bypass graft (CABG). METHODS: Sixty-two consecutive patients undergoing coronary artery bypass grafting were recruited. Among them, 6 patients were excluded from the study due to unfavorable control of heart rate. A total of 56 patients with 152 coronary artery bypass grafts (internal mammary artery, n = 48; saphenous venous grafts, n = 104) were examined by computed tomography angiography (CTA) with sixteen-detector row CT and by conventional invasive coronary angiography (CAG). All CT procedures were performed with retrospective electrocardiogram gating method. The patients' mean heart rate was 58 +/- 6 beats/minute. 120 ml of Visipaque 320 were continuously injected with the rate of 4.0 ml/sec during the procedure. The patency and the stenosis of coronary artery bypass grafts were evaluated by two experienced readers. RESULTS: All the coronary artery bypass grafts were visualized by CTA, and all the proximal bypass anastomoses and 71% of the distal bypass anastomoses were also visualized by CTA. Furthermore, 29 occlusions and 13 significant stenoses of coronary artery bypass grafts were detected by CTA. The comparison of the results between CTA and CAG showed that among all the 42 occluded and stenosed coronary artery bypass grafts detected by CTA, 34 were confirmed by CAG; among all the 110 normal coronary artery bypass grafts detected by CTA, 108 were confirmed by CAG. There were 8 false positive and 2 false negative findings, resulting in a sensitivity of 94%, a specificity of 95%, a positive predictive value of 86%, and a negative predictive value of 99%. CONCLUSION: Sixteen-detector row CTA technology may provide a reliable visualization and higher diagnostic accuracy of coronary artery bypass grafts lesions. This technique can be used as a noninvasive procedure for the diagnosis of suspected coronary artery bypass grafts dysfunction.

[Effects of TIMP-2 gene transfer on atherosclerotic plaque in rabbits].

OBJECTIVE: To evaluate the effects of TIMP-2 local gene transfer on atherosclerotic plaque. METHODS: Atherosclerosis models were induced by denuding femoral artery endothelium plus high lipid diet in rabbits. TIMP-2 gene was transferred locally by balloons eluted with pcDNA3-TIMP-2. RT-PCR and Western blot were performed to verify exogenous genes transfer. MMPs activity in atherosclerotic plaque was evaluated by zymography. HE and VG staining and automatic image analysis system were used for pathological analysis of atherosclerotic femoral arteries. The lumen area of the vessel and the collagen contents in the atherosclerotic plaque were measured. RESULTS: The expression of TIMP-2 gene in pcDNA3-TIMP-2 transferred group was significantly higher than control-vector transferred group at the end of week 2 after operation and reached the peak at the end of week 4. Comparing with the control group, the expression of TIMP-2 protein in treated group was also higher at the end of week 2, 4, and 8 after operation. Correspondingly, the MMP-2 and MMP-9 activities were lower in treated group. The thickness of fibrous cap of atherosclerotic plaque and the amount of collagen of the lesion were increased significantly in treated group compared with the control group, but there were no significant differences in vessel lumen area. CONCLUSION: TIMP-2 gene transfer locally in atherosclerotic plaque could inhibit the activities of MMP-2 and MMP-9 in the lesion, increase the thickness of fibrous cap and the amount of collagen of the lesion, but may have no effect on the degree of the stenosis.

[Effect of danshensu, protocatechualdehyde and danshen injection on calcium ion concentration in cytoplasm of human erythrocytes].

OBJECTIVE: To study the predominant calcium-antagonist components of Danshen injection. METHOD: The effects of danshensu, protocatechualdehyde and Danshen injection on calcium concentration in cytoplasm of erythrocytes were examined in vitro by the fluorescent Ca+ -chelator fura-2. RESULT: Either DS182 or PCAD can decrease in dose-dependent cytosolic free calcium concentration in human erythrocytes. They had additive effect when mixed, which was similar to Danshen injection. CONCLUSION: DS182 and PCAD may be predominant calcium-antagonist components of Danshen injection.

Oxidized low-density lipoprotein induces differentiation of RAW264.7 murine macrophage cell line into dendritic-like cells.

Dendtritic cells (DCs) are potent antigen-presenting cells and have an important role in the pathogenesis of atherosclerosis. Recent data suggests oxidized low-density lipoprotein (oxLDL) promotes the transition of a differentiating monocyte to a mature dendritic cell. In this study, we examined whether oxLDL could induce the differentiation of mature macrophages into DCs. After 48 h treatment with oxLDL, RAW264.7 cells increased in cell size and exhibited dendritic morphology. At the optimal oxLDL dose (10 microg/ml), approximately 74% of RAW264.7 cells differentiated into dendritic-like cells. Flow cytometric analysis detected dendritic cell surface markers (CD83, CD40, CD86, MHC Class II, and CD1d), and their expression increased in a dose- and time-dependent manner. Moreover, oxLDL-treated RAW264.7 cells showed functional changes including reduced endocytic activity, increased allostimulatory activity, and IL-12 production. The findings of the present work demonstrate that RAW264.7 cells, incubated with oxLDL, acquire some dendritic cell features.

[Overexpression of hypoxia inducible factor-1alpha (HIF-1alpha) promotes the differentiation of endothelial progenitor cell ex vivo].

To investigate the influence of HIF-1alpha overexpression on the differentiation of endothelial progenitor cells (EPCs) ex vivo, EPCs were isolated from human peripheral blood by density gradient centrifugation, overexpressed HIF-1alpha was transfected to EPCs by electroporation; HIF1alpha, HIF1beta, vascular endothelial growth factor (VEGF) mRNA level were measured with RT-PCR; HIF-1alpha protein was detected with immunohistochemistry in a time course. CD31(+) cells were measured with flow cytometry. Cell morphology was observed after transfection. The results showed that the transfection efficiency of HIF-1alpha to EPCs was about 20%. HIF-1alpha and its controlled target gene VEGF were markedly induced by HIF-1alpha vector (P < 0.05). HIF1beta had its same level as it before interference (P > 0.05). HIF-1alpha protein was induced by HIF-1alpha transfection after 12 hours but was undetectable at 24 hours. After 7 - 14 days cultured in 21% oxygen pressure, fluorescence-trace experiments revealed that CD31 + EPCs/EC could be generated more efficiently from overexpressed HIF-1alpha than that from pEGFP transfected group (P > 0.05). EPC morphology was observed by light microscopy. HIF-1alpha-transfected cells under normoxia sprouted more rapidly from the EPC colonies than the untransfected cells or cells transfected with an GFP vector, which essentially maintained the original colony formation. HIF-1alpha transfected cells took on an array-like arrangement rather than random dispersal, suggesting that they were in an advanced state of differentiation. It is concluded that the utility of overexpression of HIF-1alpha can induce target genes which have influence on cell differentiation. HIF-1alpha transfection was found to give a prospected way to do the insight research on ischemic treatment in vivo.

[Inhibitory effect of siRNA targeting HIF-1alpha on differentiation of peripheral blood endothelial progenitor cells].

BACKGROUND & OBJECTIVE: Transcription factor hypoxia inducible factor-1 alpha (HIF-1alpha) is a key determinant of oxygen-dependent gene regulation in angiogenesis. Suppression of HIF-1alpha is important for exploring HIF-1-dependent processes and hypoxia-induced pathophysiologic events. This study applied RNA interference targeting HIF-1alpha to human peripheral blood endothelial progenitor cells (EPCs) for therapeutic antineovascularization in vitro. METHODS: Small interference RNA (siRNA) targeting HIF-1alpha was constructed and transfected into human EPCs. Transfection efficiency of siRNA-HIF-1alpha was measured by reverse transcription-polymerase chain reaction (RT-PCR), immunocytochemistry, and flow cytometry. The content of endothelial nitric oxide synthase (eNOS) was measured by ELISA. The morphology of EPCs/endothelial cells (ECs) cultured in normoxia or hypoxia was observed after siRNA-HIF-1alpha transfection. RESULTS: SiRNA-HIF-1alpha was successfully constructed with the transfection efficiency of about 20%. After transfected for 6 h, HIF-1alpha mRNA was detected in EPCs cultured in normoxia or hypoxia with suppression; the expression of vascular endothelial growth factor (VEGF) was significantly attenuated (P < 0.05); the expression of HIF-1beta had no obvious change (P > 0.05). Stable expression of HIF-1alpha protein was induced by hypoxia, and was inhibited by siRNA-HIF-1alpha (P < 0.05). When cultured in 1% or 21% oxygen pressure for 3 days, CD31+ EPCs were inhibited more efficiently by siRNA-HIF-1alpha than by pEGFP (P < 0.05). The content of eNOS was significantly attenuated by siRNA-HIF-1alpha in time- and VEGF concentration-dependent manners (P < 0.01). EPCs differentiation and proliferation were induced by hypoxic culture, and suppressed by siRNA-HIF-1alpha. CONCLUSIONS: The constitutive or hypoxia-induced expression of HIF-1alpha in EPCs is sufficient to affect the expression of downstream target genes. SiRNA targeting HIF-1alpha could inhibit the expression of the genes which promote neovascularization, and suppress the differentiation of EPC to EC.