Effect Of Mfn-2 On Cell Cycle Of RVSMCs

Part One:Difference between Mfn-1and Mfn-2Expression in the Cell Cycle of Vascular Smooth Muscle Cells[Aims] To explore whether Mfn-2or Mfn-1could play some roles in the regulation of cell cycle, we investigated the endogenous expressional difference between Mfn-2and Mfn-1in the cell cycle of vascular smooth muscle cells.[Methods] Cell cycle arrest was achieved by24h of serum deprivation,18h of serum culture after48h of serum deprivation and treatment of nocodazole for12h after thimidine for12h. Cells induced to the same phase were divided into two groups, one for analysis by propidium iodide staining and the other for Western Blot.[Results] Flow cytometry analysis showed the cells were arrested to Go/Gi, S and G2/M phase of the cell cycle of the vascular smooth muscle cells. The untreated cells with70%confluence were used as controls, indicating the average distributions of G0/G1, S and G2/M phase51.36%,39.52%and9.12%respectively. Western Blotting demonstrated more pronounced expression of endogenous Mfn-2, p27and p21and decrease of pRb in the G0/G1phase of the vascular smooth muscle cells, while no change of the expression of endogenous Mfh-1in the G0/G1phase.[Conclusion] G0/G1cell cycle arrest induced the upregulation of Mfn-2not Mfn-1, suggesting a regulatory role of Mfn-2on the cell cycle progression of vascular smooth muscle cells. Part Two:Morphological and Metabolic Changes in the Cell Cycle of Vascular Smooth Muscle Cells[Aims] To study the relationship between the morphology and function of Mfn-2, we examined the morphological and metabolic changes in the cell cycle of vascular smooth muscle cells.[Methods] MitoTracker Red was stained in the vascular smooth muscle cells induced into the different phase of the cell cycle to show the mitochondrial morphology by confocal fluorescence microscopy. Using transmission electron microscopy, we observed the ultrastructure variation during the cell cycle of vascular smooth muscle cells. Mitochondrial membrane potential was detected by MitoProbeTM JC-1assay and the energization alteration by QuantiChrom Glucose Assay Kit, L-Amino Acid Quantitation Kit, ATP colorimetric assay and NAD(P)+/NAD(P)H measurements.[Results] Laser confocal fluorescence microscopy showed that the mitochondria were in a tubular network state in the Go/G1phase but in a fragmented solitary spheroid state in the S phase and in a separated group state in the G2/M phase. Using transmission electron microscopy, we confirmed that there were many more mitochondria present and the endoplasmic reticulum (ER) was tethered to the mitochondria in the G0/G1phase, swelling mitochondria with destructed cristae in the S phase and autophagosome accumulation in the G2/M phase. Although glucose measurement, L-Amino Acid Quantitation and ATP production remained unchanged during the cell cycle progression, NAD+levels were lower in the G0/G1phase and mitochondrial membrane potential decreased in the G2/M phase.[Conclusion] The energization has no change during the cell cycle of vascular smooth muscle cells. However, mitochondria were in a tubular network state and endoplasmic reticulum was tethered to mitochondria when Mfn-2was upregulated in the G0/G1phase. Furthermore, mitochondria were in a fragmented solitary spheroid state and autophagosomes were accumulated with decreased mitochondrial membrane potential when Mfn-2was downregulated in the S and G2/M phase. Part Three:Effect of Overexpression of Mfn-2On the Mitochondrial remodeling in the Vascular Smooth Muscle Cells[Aims] To investigate the effect of Mfn-2on mitochondrial remodeling, we applied Adenovirus vector of Mfn-2in the vascular smooth muscle cells.[Methods] Adv-Mfn-2-S442A was constructed as previously. Adenoviral infection was carried out by adding25pfu cell-1and50pfu cell-1Adv-Mfn-2-S442A into the vascular smooth muscle cells after synchronized for48h. Adv-LacZ was served as a control. Then the treated cells were stained with MitoTracker Red and observed by laser confocal fluorescence microscopy.[Results] Adenovirus vector Adv-Mfn-2-S442A was successfully constructed. Using laser confocal fluorescence microscopy, the vascular smooth muscle cells stained with MitoTracker Red after the treatment of adenoviral infection displayed a solitary group gathering distribution when infected with25pfu cell-1of Adv-Mfn-2-S442A and a reticulum state with perinuclear clustering with50pfu cell-1of Adv-Mfn-2-S442A. [Conclusion] Overexpression of Mfn-2promotes the mitochondria to be in a tubular network state in the vascular smooth muscle cells.