| Objective:Vascular diseases such as atherosclerosis are characterized by abnormal accumulation of vascular smooth muscle cells(VSMCs) within the intimal lining. The intimal VSMCs exihibit an increased expression of peroxisome proliferator-activated receptor-gamma (PPAR-γ). However, the factors that regulate PPAR-γexpression in the vasculature are pooly defined. Here we hoped to explore the effect of homocysteine on the expression of peroxisome proliferator-activated receptor-gamma and the possible mechanism in the vascular smooth muscle cells.Methods:1. Cell CultureVascular smooth muscle cells isolated separately from healthy donors'umbilical artery and rat aortic artery were cultured in DMEM/F12 medium containing 10% fetal calf serum in vitro. HUASMCs of passage 3 were characterized by immunocytochemistry analysis with anti-SM-α-actin antibody. More than 90% of cells revealed immunoreactivity. For all experiments, early-passage (passage 3 to 4) HUASMCs after primary culture were grown to 80% to 90% confluence and made quiescent by serum starvation for at least 24 hours.2. MTT AnalysisCultured vascular mooth muscle cells were pretreated for 0-24 h with or without 0-1 mM NAC before addition of 0-1 mM homocysteine for 0-48 h. The proliferation characteristics of the HUASMCs were measured by a rapid colorimetric assay (MTT assay).3. immunocytochemistry AnalysisHUASMCs on slides were fixed in cold methanol for 10 min and kept at 4℃until use. Cells were washed and blocked for 10 minutes with blocking solution containing goat serum. After incubation with specific antibodies for PPAR-γ(Santa Cruz Biotechnologies) at a dilution of 1:100 in PBS at 37℃for 1 hour, slides were washed and incubated with the secondary antibodies for 10 minutes at room temperature. The PPAR-γproteine were stained with diaminobenzidine and visualized with microscope.4. RT-PCR AnalysisSubconfluent HUASMCs cells in 25ml culture flasks were deprived of serum for at least 24 h. Cells were pretreated with and without 0-1 mM NAC, then homocysteine was added in the serum-free medium for a specified time period (0-48h). The maximum effect was attained and used as the basis of following experiments. Total RNA isolated from 2×106 cells was quantitated at 260 nm absorbance. The purity of total RNA was assessed by an absorbance ratio (260/280 nm) of 1.7-2.0. For semi-quantitative polymerase chaine reaction determinations, GAPDH cDNA was used as an internal control, and the linear amplification was determined for each experiment. The primers were synthesized according to published seqences. PCR products (lOul) were electrophoresed using 1% agarose gel.Results:1. MTT assay suggested that after the treatment of homocysteine for 24h, VSMCs showed marked proliferation as concentration and time increased. The preincubation of N-acetylcysteine had partially inhibitory effect on the growth of VSMCs as a well-known antioxidant, also in a dose- and time- dependent manner.2. RT-PCR showed that the expression of PPAR-γmRNA was upregulated by homocysteine after 2h treatment, gained maxium effect at 24h which was followed by slight downregulation at 48h. Immunocytochemistry Analysis showed that PPAR-γproteine was also upregulated'by homocysteine in a dose-dependent manner. Preincubation of antioxidant N- acetylcysteine indicated that N-acetylcysteine had partially inhibitory effect on the expression of PPAR-γ, also in a dose- and time- dependent manner. Conclution:Our results demonstrated that homocysteine had dual effect on VSMCs. In primary period the upregulated expression of PPAR-γwas disadvantageous to the proliferation of VSMCs by homocysteine. However, as a later effect, homocysteine enhanced the proliferation of vascular smooth muscle cells though PPAR-γwas lightly activated. Because we investigated that the preincubation of N-acetylcysteine could diminish the expression of PPAR-γor the proliferation of VSMCs induced by homocysteine, so we concluded that oxidation may be one of the possible mechanism involved in the process.
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