Matsubara Akihisa, Nakazawa Toru, Noda Kosuke, She Haicheng, Connolly Edward, Young Tara A, Ogura Yuichiro, Gragoudas Evangelos S, Miller Joan W
Angiogenesis and Laser Laboratories, Retina Service, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, Massachusetts, USA.
Invest Ophthalmol Vis Sci. 2007 Oct;48(10):4741-7. doi: 10.1167/iovs.06-1534.
To investigate the mechanism of cell death in laser-induced choroidal neovascularization (CNV) after photodynamic therapy (PDT).
PDT was performed in Brown-Norway rats using laser light at a wavelength of 689 nm, irradiance of 600 mW/cm(2), and fluence of 25 J/cm(2) after intravenous injection of verteporfin at the doses of 3, 6, and 12 mg/m(2). Apoptotic cells in CNV were detected by TUNEL assay at 1, 3, 6, 15, 24, and 48 hours after PDT. Caspase activation at 1, 3, 6, 15, and 24 hours after PDT was determined by immunohistochemistry (IHC) with a cleaved caspase-3 or -9 antibody. Akt activity was determined by Western blot and IHC with a phosphorylated-Akt (pAkt) antibody. To investigate the roles of Akt in PDT-induced apoptosis, insulin-like growth factor (IGF)-1, an Akt activator, with or without wortmannin, an inhibitor of PI3K-Akt pathway, was injected into the vitreous before PDT.
The number of TUNEL-positive cells in CNV increased at 3 hours after PDT and peaked at 6 hours, showing a dose dependence of verteporfin. Caspase activation was detected in TUNEL-positive cells. Dephosphorylation of Akt in CNV occurred within 1 hour. IGF-1 significantly activated Akt and suppressed the number of TUNEL-positive cells in CNV, and the effects of IGF-1 were diminished by wortmannin.
PDT induced caspase-dependent apoptosis in CNV. These results suggest that PDT leads to dephosphorylation of Akt and subsequent activation of the caspase-dependent pathway. Understanding the intracellular signaling mechanisms of apoptosis in PDT may lead to more selective and effective treatment of CNV secondary to age-related macular degeneration.
研究光动力疗法（PDT）后激光诱导脉络膜新生血管（CNV）中细胞死亡的机制。
给棕色挪威大鼠静脉注射剂量分别为3、6和12mg/m²的维替泊芬后，使用波长689nm、辐照度600mW/cm²、能量密度25J/cm²的激光进行PDT。在PDT后1、3、6、15、24和48小时，通过TUNEL法检测CNV中的凋亡细胞。使用裂解的caspase-3或-9抗体，通过免疫组织化学（IHC）测定PDT后1、3、6、15和24小时的caspase激活情况。通过蛋白质免疫印迹法和使用磷酸化Akt（pAkt）抗体的IHC测定Akt活性。为了研究Akt在PDT诱导的凋亡中的作用，在PDT前将胰岛素样生长因子（IGF）-1（一种Akt激活剂）与或不与PI3K-Akt途径抑制剂渥曼青霉素注入玻璃体。
PDT后3小时CNV中TUNEL阳性细胞数量增加，并在6小时达到峰值，显示出维替泊芬的剂量依赖性。在TUNEL阳性细胞中检测到caspase激活。CNV中的Akt在1小时内发生去磷酸化。IGF-1显著激活Akt并抑制CNV中TUNEL阳性细胞的数量，渥曼青霉素减弱了IGF-1的作用。
PDT诱导CNV中caspase依赖性凋亡。这些结果表明，PDT导致Akt去磷酸化并随后激活caspase依赖性途径。了解PDT中凋亡的细胞内信号传导机制可能会导致对年龄相关性黄斑变性继发的CNV进行更具选择性和有效性的治疗。
