The Effects Of Growth Arrest Specific Protein-6 On The Senescence Process Of VSMC

BackgroundAging is a phenomenon that the body’s functions, adaptability and resistance decreases with time. Nowadays, with the accelerating pace of aging of global population, aging is becoming one of the focuses of the world. Accordingly aging is an issue of vital importance that should be delayed for our human beings due to it relates to quality of life and life-span. The aging of multiple organs finally lead to diseases, the whole individual aging or even death, and the cardiovascular system is no exception; Furthermore, aging is also becoming the first risk factor for the cardiovascular system:Aging can influence the metabolism of the cardiovascular system and may result in metabolic disorder of extracellular matrix, abnormal apoptosis and inflammation, that further affects the structure and function of the cardiovascular system, and the final result calls cardiovascular remodeling.Gas6 is a member of vitamin-K dependent protein family which is encoded by growth arrest-specific gene 6, The receptor of Gas6, Axl was first discovered in primary human myeloid leukemia cells, and the Axl receptor was found to one of receptor tyrosine kinases, Gas6 and Axl have been proven that they participate in the pathological process of vascular calcification, vascular remodeling and atherosclerosis. So pathophysiological processes of Gas6 and Axl on vascular may be realized by VSMCs. Moreover, Gas6 gene is associated with stroke and acute coronary syndrome in humans through gene polymorphism analysis.Briefly, we can conclude that Gas6 and Axl are critical for the process of cardiovascular diseases, through the regulation of survival, proliferation and migration of vascular cells, and various functions of circulating blood cells, so that Gas6 and Axl are closely associated with vascular remodeling, especially for VSMCs.Objectives1. To explore the effect of Gas6 on cell senescence.2. To explore the downstream signaling pathway of Gas6/Axl what will shed light on the specific mechanism in this process.3. To explore the specific mechanism of Gas6 that delay the senescent process of VSMC.Methods and SubjectsWe build the cellular senescence models-the replicative senescence (RS) and the induced senescence (IS) and hypothesize that Gas6 delays the senescent process of cells. Results showed that cells with Gas6 treated their dyeing rate of SA-β-Gal, the percentage of G1 phase, the level of p16INK4a and p21Cipl decreased while the percentage of S phase and the level of proliferation increased. Both the IS and RS under Gas6 treatment circumstance, the level of PI3K, p-Akt, and p-FoxO3a decreased after inhibiting Axl by R428; the level of p-Akt, p-FoxO3a also decreased after inhibiting PI3K by LY294002.ResultsThe identification of cellular senescence modelsWe chose the 10th cells as the replicative senescence model (RS). And we found that the passage 4th cells could become senescent when they were given Ang Ⅱ for 48 h at the concentration of 1*10-6 mol/L, according to the expression of p21Cipl and p16INK4a. Its SA-β-gal dyeing rate also increased significantly at this concentration and time points.Gas6 was involved in the VSMCs senescence processWhen cells were given Gas6 with the dilution of 250 ng/mL for 36 h, and Gas6 was added every 12 h, their expression level of p21Cipl and p16INK4a decreased significantly than the others. In order to study between Gas6 and cellular senescence, we gave the senescence models (including both RS and IS models) 250 ng/mL Gas6 for 36 h.Axl played a key role in Gas6 mediated the anti-senescence effectThe results showed that in the cells with the treatment of R428, regardless of whether Gas6 treatment or not, the protein expression level of p16INK4a and p21Cipl increased significantly compared with the other two group of cells.Gas6 promoted cells transition from G1 phase to S phaseIn the IS, cells treated with Gas6 showed a higher S phase and a lower G1 phase compared with the control; and in the RS, cells with Gas6 intervened showed a higher S phase and a lower G1 phase compared with the control, while there was no significant difference in Young cells whether Gas6 treated or not. In addition, in the IS, compared with the control, the positive rate of EdU staining after Gas6 treating showed a higher proportion. Meanwhile, in the RS, cells treated with Gas6 also showed a higher proportion in contrast with the control. While, there was no significant difference in young cells whether Gas6 treated or not.Gas6/Axl involved in cellular senescent process by PI3K/Akt/Fox03a signaling pathwayCells with R428 treated showed a low expression level of p110α (PI3K), p-Akt, p-FoxO3a and cyclin E, while the protein expression level of p27Kipl increased significantly; And in the RS, we got the similar results: the level of p110α (PI3K), p-Akt, p-FoxO3a and cyclin E decreased but the protein expression level of p27Kipl increased significantly after being treatment with R248.Cells with ly294002-treated showed a low expression level of p-Akt, p-FoxO3a and cyclin E, while the protein expression level of p27Kipl increased significantly, And in the RS, the results were similar with IS:the level of p-Akt, p-FoxO3a and cyclin E decreased but the level of p27Kipl increased.FoxO played a key role in the processWe infer that FoxO is the key factor in this pathway, and it is the target that Gas6 exerts this effect in the senescent process of VSMCs.Conclusions1. Gas6 could delay the senescenct process of VSMC;2. Axl plays a key role in Gas6 mediated the anti-senescence effect;3. Gas6 promots cells transition from G1 phase to S phase and improves the proliferation level;4. PI3K/Akt/FoxO3a signaling pathway is involved in the process that Gas6 delay cellular senescence;5. FoxO is the target molecular that is regulated by Gas6.