Research Of AMPK In Rat Vascular Smooth Muscle Cell Proliferation Through The MTOR Pathway

BackgroundCoronary stent implantation has been the main method for the coronary heart disease, which significantly reduced acute and chronic coronary incidence and mortality, but the stent is still facing the problem of restenosis. Although drug-eluting stents can decline the restenosis to 5% within 6 months, but patients with coronary risk factors and complex lesions also have high restenosis rate from 10% to 50% [1].PCI and CABG in patients with multi-vessel coronary artery disease on long-term safety and efficacy: ARTS, ERACI-II, MASS-II, and SoS trial meta-analysis of 5 years, PCI and CABG is similar to the long-term security, but the patients needed to re-PCI are relatively low in CABG group, so in 5 years CABG group's major adverse cardiac events and cardiovascular events were significantly lower than the PCI group[2], this also explains the importance to once again PCI or CABG. Study confirmed: thrombosis,inflammation and the proliferation of vascular smooth muscle cells (VSMCs) are the main result of the restenosis and vascular reconstruction, of which the VSMCs'proliferation is the trigger factor to the initiating intimal hyperplasia[3]. How to prevent and stop the proliferation of VSMCs, is important to the prevent the restenosis of angioplasty.AMP-activated protein kinase (AMPK) is a heterogeneous trimer. The absence of glucose, hypoxia, ischemia, and thermal shock which caused the rising of AMP/ATP ratio can lead to AMPK activation[4]. In addition to regulate energy metabolism, the activation of AMPK plays an important role in gene transcription and cell division cycle[5], caused the cycle arrest and the inhibition of proliferation[6].Mammalian target of rapamycin (mTOR) is a conserved serine/threonine protein kinase, mTOR plays an important role in cell growth, proliferation and differentiation [7].mTOR induced translation of initiation codon[8], affect the changes of cell division cycle G1/S/G2, DNA replication and subsequent cell division. Kimura[9] show that the activation of AMPK can inhibit the mTOR signaling pathway caused by amino acid stimulated, some investigators found the activation of AMPK can reduce protein synthesis by inhibiting translation elongation factor (EF2)[10].The role of AMPK provides a new direction to the prevention and treatment of diseases which caused by the proliferationof VSMCs. This study observed on the expression and activity of mTOR and explored the ways which affect VSMCS proliferation, when the AMPK activated.PurposeCultured and identified SD rat aortic VSMCs in vitro. Observe the proliferation of VSMCs in serum culture conditions and under factor PDGF-bb when AMPK activated. Detection the expression and activity of mTOR activation under the activation of AMPK to provide a new clues or theories to prevention and treatment of the abnormal proliferation of VSMCs.Methods1. Culture SD rat aortic VSMCsMale SD rats were purchased from Experimental Animal Center of Guangdong (Permit No. 2008A002), quality 200～300g, pentobarbital anesthesia, sterile remove the thoracic aorta, using tissue adherent culture, usingα-actin to identify the cells. 0.25% trypsin for passage. using 4～6th generation cell in the following experiment. 2. Mapping the VSMCs growth curveUsed 4～6th generation cell, observe and count the growth of VSMCs, draw the cell growth curve, determine the time of drug intervention.3. Western blot to detect the activation of AMPKTake 4th generation VSMCs, after AICAR intervention, extract the total cellular protein in the time at 30min, 1h, 3h, 6h, 12h, 24h, Western blot to detect the activation of AMPK.4. Different concentrations of AICAR at different time detect the proliferation of VSMCsMTT detect the cell proliferation in different concentrations with AICAR intervention in 24h, 48h, 72h. Using microplate Reader(570nm) to determine the value, each of 6 holes.5. Semi-RT-PCR detect the mTOR expression levels of mRNA with AICAR interventionUsing semi-RT-PCR method to detect the changes of mTOR's mRNA level in the 4th generation VSMCs, intervented by different concentration of AICAR.6. Western blot to detect the change of mTOR phosphorylation level after AICAR interventionUsing Western blotting detect the changes of p-mTOR in different time, after intervention by the same concentration of AICAR in the 4th generation VSMCs.7. Statistical AnalysisMeasurement data were presented as mean±standard deviation (x±s). The data were analyzed with SPSS 13.0(SPSS Inc,Chicago, IL,USA). Multiple comparisons between groups using analysis of variance. P <0.05 as statistically significant difference.Results1. VSMCS Culture and identificationWith adherent tissue culture, cells began to climb out from the tissue edge in 3 days(72h). From the 4th day the cells climbing out of the tissue gradually increased, to 14th day the cells covered with tissue surrounding the microscope view. Subcultured to 4th generagtion, cells showed the typical vascular smooth muscle cells"peak-valley","gully-like"growth form. After immunohistochemical staining, showing positive staining in the cytoplasm of filamentousα-actin distribution, identified as VSMCS and the purity> 99%.2. VSMCs Growth CurveMapping VSMCs growth curve in 4th generation of VSMCs: from the inoculated cells to 5.5 days, curve "S" shape, from 2th to 4.5th day in the logarithmic phase which can be excluded from cell itself and other factors on cell proliferation, drug intervention in this period has practical significance.3. Detect the activation of p-AMPK at different time after AICAR interventionUsing Quantity One image analysis software collate data, AICAR(0.5mmol/L) activates AMPK from 30min, reaching the maximum in 6h, then gradually decrease. Indicating that with AICAR, the activation of AMPK increases gradually over time, then gradually reduced.4. The proliferation of VSMCS with different concentrations of AICAR at different timeThe cells'proliferation can be inhibited in dose-dependent with different concentrations of AICAR intervention in 24h, 48h, 72h under the two conditions (serum culture and PDGF-bb factor)( P<0.01) using MTT method.5. The expression of mTOR's mRNA under the AICARAnalysis the images using Quantity One software. The mTOR's mRNA expression decreased with the increasing concentration of AICAR in 24h (P<0.01).6. The expression of p-mTOR with AICAR interventionAnalysis the western blot result show: AICAR(0.5mmol/L) can inhibit the expression of p-mTOR with time-dependent. With AICAR intervention, the expression of p-mTOR was inhibited starting from 10min and almost completely inhibited in 12h(P<0.01). Conclusion1. The experiments show that AICAR can activate AMPK in smooth muscle cells, AICAR intervention can significantly inhibit the proliferation of vascular smooth muscle cells both in serum and PDGF-bb conditions.2. The experiments show: the activation of AMPK by AICAR can inhibit the expression of mTOR's mRNA and p- mTOR significantly in the SD rat vascular smooth muscle cells.3. The activation of AMPK inhibit the proliferation of vascular smooth muscle cells may through the mTOR pathway.