Photodynamic therapy induces caspase-dependent apoptosis in rat CNV model
Photodynamic therapy induces caspase-dependent apoptosis in rat CNV model
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DOI：
10.1167/iovs.06-1534
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发表时间：
2007-10-01
影响因子：
4.4
通讯作者：
Miller, Joan W.
中科院分区：
文献类型：
--
作者：
Matsubara, Akihisa;Nakazawa, Toru;Miller, Joan W.
目的.探讨光动力学疗法（PDT）诱导脉络膜新生血管（CNV）的细胞死亡机制。在Brown-Norway大鼠静脉注射剂量为3、6和12 mg/m2的维替泊芬后，使用波长为689 nm、辐照度为600 mW/cm（2）、能量密度为25 J/cm（2）的激光进行PDT。TUNEL法检测PDT后1、3、6、15、24、48 h CNV细胞凋亡情况。PDT后1、3、6、15和24小时的胱天蛋白酶活化通过免疫组织化学（IHC）用裂解的胱天蛋白酶-3或-9抗体测定。通过蛋白质印迹和用磷酸化Akt（pAkt）抗体的IHC测定Akt活性。为探讨Akt在PDT诱导的细胞凋亡中的作用，在PDT前玻璃体内注射Akt激活剂胰岛素样生长因子（IGF）-1和PI 3 K-Akt通路抑制剂渥曼青霉素（wortmannin）。PDT后3 h CNV中TUNEL阳性细胞数增加，6 h达高峰，呈剂量依赖性。TUNEL阳性细胞中检测到Caspase活化。CNV中Akt的去磷酸化在1小时内发生。IGF-1可显著激活CNV中Akt表达，抑制CNV中TUNEL阳性细胞数，且其作用可被wortmannin减弱。PDT诱导CNV中的半胱天冬酶依赖性凋亡。这些结果表明，PDT导致Akt的去磷酸化和随后的半胱天冬酶依赖性途径的激活。了解光动力疗法中细胞凋亡的细胞内信号传导机制可能会导致对继发于年龄相关性黄斑变性的CNV的更有选择性和更有效的治疗。
PURPOSE. To investigate the mechanism of cell death in laser-induced choroidal neovascularization (CNV) after photodynamic therapy (PDT).METHODS. 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.RESULTS. 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.CONCLUSIONS. 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.