老化是造成心血管疾病的主要危險因子。然而，目前對於血管老化的作用機制仍所知甚少。在先前的研究中發現，Akt 是調控細胞的代謝與生存的一個重要蛋白激酶，但在最近的研究中也發現，若使Akt不正常的長期活化，則會導致老化細胞的增加；反之，在人類內皮細胞的實驗中，若抑制Akt不正常的活化，則可以延長細胞壽命。細胞內已知能調控Akt活化的因子主要有phosphatidylinositol 3-kinase (PI3K) 以及 小G蛋白Ras。目前已知由血管收縮素(Angiotensin II)所誘導的血管平滑肌細胞老化，主要與細胞內NADPH oxidase所產生的氧自由基有關。在血管平滑肌細胞中，血管收縮素透過增加細胞內的氧自由基，進一步去刺激Akt的活化。然而，在血管收縮素所誘導的細胞老化中，Akt扮演的角色仍不清楚。因此，在此研究中我主要探討的是Akt在血管收縮素所誘導的人類主動脈平滑肌細胞老化中所扮演的角色。我藉由分析三個已知的細胞老化標誌，senescence-associated β-galactosidase (SA-β-gal)的活性，以及兩個細胞週期抑制蛋白；p21和p16的表現量來鑑別細胞老化。以血管收縮素刺激人類主動脈平滑肌細胞24,48或72小時之後會增加SA-β-gal的活性與p21及p16的表現量。此外，血管收縮素亦會刺激反映DNA複製的BrdU攝取量並會增加細胞數。有趣的是，在血管收縮素的刺激下，Akt只在48及72小時明顯地被活化，在24小時卻沒有。若在血管收縮素刺激時；共同處理PI3K的抑制劑LY294002或mTOR的抑制劑rapamycin，或是在細胞內過量表現不活化的Akt突變蛋白AAA-Akt，皆可有效抑制血管收縮素所誘導的Akt活化，並抑制細胞老化。反之，在無血管收縮素的情況下，在細胞內過量表現活化的Akt突變蛋白myr-Akt，則會增加細胞老化的現象。若使用氧自由基清除劑N-acetyl-L-cysteine(NAC) 與apocynin，或可通透細胞膜之過氧化氫酶(membrane-permeable catalase)，均會抑制血管收縮素所誘導的Akt活化，並抑制細胞老化。此外，使用干擾RNA抑制NADPH氧化酶的分子異構物Nox1，也顯著抑制血管收縮素所誘導的Akt活化與細胞老化。上述結果顯示，血管收縮素所誘導的Akt活化與細胞內Nox1產生的氧化壓力有關，且Akt持續的活化，在血管收縮素所誘導的細胞老化過程中扮演重要的角色。

Aging is the major risk factor for cardiovascular diseases. Mechanisms accounting for vascular aging remain poorly understood. Previous studies showed that protein kinase Akt is a nutrient sensor involved in regulating cellular metabolism and cell survival. Long-term activation of Akt increases cellular senescence and inhibition of Akt extends the lifespan of primary cultured human endothelial cells. Upstream signaling pathways leading to Akt activation include phosphatidylinositol 3-kinase (PI3K) and small G protein Ras. Ang II induces cellular senescence of vascular smooth muscle cells (VSMCs) involving NADPH oxidase-derived reactive oxygen species (ROS). In VSMCs, Ang II stimulates Akt activation through ROS, but the role of Akt activation in Ang II-induced cellular senescence remains unclear. This study investigated the role of Akt in Ang II-induced cellular senescence of human aortic VSMCs. Cellular senescence was assessed with senescence-associated β-galactosidase (SA-β-gal) activity and the expression of cell cycle inhibitors p21 and p16. Ang II (10-7M) treatment for 24 h, 48 h or 72 h induced cellular senescence of VSMCs. Under this condition, Ang II treatment increased BrdU-positive cells and cell number in a time-dependent manner. These results suggested that Ang II induced premature senescence of VSMCs. Interestingly, Ang II treatment for 48 h or 72 h, but not 24 h, induced Akt activation. The co-treatment of Ang II with LY294002, a selective inhibitor of PI3K, or rapamycin, an inhibitor of mTOR, blocked Ang II-induced Akt activation, SA-β-gal activity, and expression of p21 and p16. Ang II-induced senescence was eliminated by overexpressing dominant-negative Akt while overexpressing constitutively active Akt alone increased VSMCs senescence. In addition, co-treating VSMCs with Ang II and antioxidants, N-acetyl-cysteine and apocynin, or a membrane-permeable catalase inhibited Ang II-induced Akt activation, SA-β-gal activity, and expression of p21 and p16. Moreover, small interfering RNA for NADPH oxidase catalytic subunit isoform NOX1 markedly inhibited Ang II-induced Akt activation and cellular senescence. These results suggest that NOX1-derived ROS-dependent, sustained Akt activation plays an important role in promoting Ang II-induced cellular senescence of VSMCs.

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