血管細胞老化會造成血管老化，是血管疾病的主要危險因子。細胞老化意指許多因素，例如DNA損壞或氧化壓力(oxidative stress)，導致細胞無法進入細胞週期，進行分裂。當氧自由基(reactive oxygen species)產生過量時，可能會增加細胞的DNA損壞，最後導致細胞凋亡或細胞老化的現象。NADPH氧化酶(Nox)和粒線體為血管平滑肌細胞產生氧自由基的主要來源。此外，粒線體也是氧自由基傷害的主要目標，過多的氧自由基會造成粒線體的損傷。先前的研究指出，血管收縮素刺激氧化壓力，進而導致血管平滑肌細胞老化，但其詳細機制仍尚未釐清。本研究主要探討NADPH氧化酶以及粒線體在血管收縮素所誘導的氧自由基生成與人類主動脈血管平滑肌細胞老化的角色。將血管平滑肌細胞以血管收縮素處理24、48及72小時後，利用能夠標記細胞老化的senescence-associated β-galactosidase活性測量，發現老化細胞的比例顯著上升。有趣的是，以血管收縮素處理細胞短時間(5分鐘)與長時間(24 至72小時)都會增加氧自由基的產生。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氧自由基產生，並抑制細胞老化。此外，以干擾RNA(siRNA) 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細胞老化。在相同的條件下，Nox1的干擾RNA完全抑制短暫性(transient)氧自由基的產生，但對持續性(sustained)的氧自由基產生的影響較不顯著。相對地，使用粒線體氧自由基的專一性抑制劑，mito-Q10，能抑制血管收縮素所誘導的細胞老化，同時抑制持續性(sustained)的氧自由基產生，對短暫性的氧自由基產生的影響則不顯著。粒線體專一性染色結果顯示，血管收縮素處理72小時後，氧自由基的位置與粒線體高度吻合。此外，Nox1的干擾RNA與mito-Q10皆顯著抑制血管收縮素造成的粒線體膜電位去極化的現象。上述結果顯示，血管收縮素在前期透過Nox1產生短暫性的氧化壓力，在後期則透過粒線體，產生持續性的氧化壓力，導致粒線體功能失調，進而導致血管平滑肌細胞的老化。

Vascular aging manifested by vascular cell senescence is a major risk factor that facilitates vascular diseases. Cellular senescence, characterized by inability of cells to enter cell cycle in response to mitogens, can be caused by multiple factors including telomere dysfunction, DNA damage and oxidative stress. Excessive production of reactive oxygen species (ROS) increases DNA damage and ultimately results in apoptosis or cellular senescence. NADPH oxidases (Nox) and mitochondria are major sources for ROS production in vascular smooth muscle cells (VSMCs). Mitochondria are both major source of detrimental ROS production and oxidation targets, eventually leading to their dysfunction. Oxidative stress plays a role in angiotensin II (Ang II)-induced cellular senescence of VSMCs, but the mechanisms remain unclear. This study investigated the role of NADPH oxidases and mitochondria in Ang II-induced ROS production and cellular senescence of human aortic VSMC. Ang II teatment (10-7 M) for 24 h, 48 h or 72 h induced VSMC senescence assessed with senescence-associated β-galactosidase activity. Interestingly, Ang II stimulated a transient ROS production peaked at 5 min and a sustained ROS production between 24 h and 72 h. ROS scavenging with antioxidants N-acetyl-L-cysteine and apocynin or a membrane-permeable catalase abolished Ang II-induced ROS production and cellular senescence. Furthermore, small interfering RNA (siRNA) for NADPH oxidase catalytic subunit isoform Nox1 markedly inhibited Ang II-induced cellular senescence. Under this condition, Nox1 siRNA abolished Ang II-induced transient ROS production but was less effective in inhibiting the sustained ROS production. In contrast, a mitochondria-specific ROS inhibitor, mito-Q10, inhibited Ang II-induced sustained ROS production and cellular senescence of VSMCs. Mitochondria-specific staining showed that ROS and mitochondria were highly co-localized at 72 h Ang II treatment. Both Nox1 siRNA and mito-Q10 effectively inhibited Ang II-induced mitochondria membrane depolarization. These results suggested that oxidative stress involving Nox1-dependent NADPH oxidase in the transient phase and mitochondria in the sustained phase leads to mitochondrial dysfunction and mediates Ang II-induced cellular senescence of VSMCs.

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