Cursiefen Claus, Masli Sharmila, Ng Tat Fong, Dana M Reza, Bornstein Paul, Lawler Jack, Streilein J Wayne
Schepens Eye Research Institute, Department of Ophthalmology, Harvard Medical School, Boston Massachusetts 02114, USA.
Invest Ophthalmol Vis Sci. 2004 Apr;45(4):1117-24. doi: 10.1167/iovs.03-0940.
Thrombospondin (TSP)-1 and -2 are important antiangiogenic factors thought to be involved in maintaining corneal avascularity (angiogenic privilege). This study was undertaken to investigate whether deficiencies of these factors altered developmental and inflammation-induced angiogenesis in the cornea and developmental angiogenesis of the iris of mice.
Expression of TSP-1 and -2 mRNA and protein was assayed in cornea and iris stroma by RT-PCR and Western blot. Corneas and irides of TSP-1(-/-), TSP-2(-/-), and TSP-1,2(-/-) mice aged 2, 3, and 6 months, and wild-type control mice, were analyzed for spontaneous angiogenesis biomicroscopically, histologically, and with CD31 immunohistochemistry. The mouse model of suture-induced, inflammatory corneal neovascularization was used to evaluate the lack of TSP-1,2 and both TSPs on induced-corneal angiogenesis. Seven days after intrastromal placement of three 11-0 sutures, vascularized areas were analyzed morphometrically on CD31-stained corneal flatmounts.
Corneas and irises from normal mouse eyes constitutively expressed TSP-1 and -2 mRNAs and proteins. Corneas of TSP-1(-/-), -2(-/-), and -1,2(-/-) mice displayed no evidence of spontaneous developmental-postnatal angiogenesis, although irises of these mice contained significantly increased iris vessel density compared with wild-type animals (P < 0.01). One week after suturing, corneas of all TSP(-/-) mice had significantly greater corneal angiogenesis than those of control mice (P < 0.05). TSP-1(-/-) had a significantly greater effect on induced corneal neovascularization than did TSP-2(-/-), with the opposite being the case in developmental iris angiogenesis (P < 0.01).
Corneal avascularity during development is redundantly regulated, shown by the fact that lack of the antiangiogenic factors TSP-1 and/or -2 resulted in no spontaneous corneal angiogenesis. By contrast, TSP-1, more than TSP-2, helps to suppress inflammation-induced corneal angiogenesis postnatally, implying that angiogenic privilege in the cornea is actively maintained.
血小板反应蛋白（TSP）-1和-2是重要的抗血管生成因子，被认为参与维持角膜无血管状态（血管生成特权）。本研究旨在调查这些因子的缺乏是否会改变角膜的发育性和炎症诱导性血管生成以及小鼠虹膜的发育性血管生成。
通过逆转录聚合酶链反应（RT-PCR）和蛋白质印迹法检测角膜和虹膜基质中TSP-1和-2的信使核糖核酸（mRNA）及蛋白表达。对2、3和6月龄的TSP-1基因敲除（-/-）、TSP-2基因敲除（-/-）、TSP-1,2双基因敲除（-/-）小鼠以及野生型对照小鼠的角膜和虹膜进行生物显微镜检查、组织学分析及CD31免疫组织化学分析，以检测自发血管生成情况。采用缝线诱导的炎症性角膜新生血管小鼠模型，评估TSP-1,2缺乏以及两种TSP均缺乏对诱导性角膜血管生成的影响。在角膜基质内植入三根11-0缝线7天后，对CD31染色的角膜平铺片上的血管化区域进行形态计量分析。
正常小鼠眼睛的角膜和虹膜持续表达TSP-1和-2的mRNA及蛋白。TSP-1基因敲除（-/-）、-2基因敲除（-/-）和-1,2双基因敲除（-/-）小鼠的角膜未显示出出生后自发的发育性血管生成迹象，尽管与野生型动物相比，这些小鼠的虹膜血管密度显著增加（P < 0.01）。缝合1周后，所有TSP基因敲除（-/-）小鼠角膜的血管生成均显著多于对照小鼠（P < 0.05）。TSP-1基因敲除（-/-）对诱导性角膜新生血管的影响显著大于TSP-2基因敲除（-/-），而在虹膜发育性血管生成中情况则相反（P < 0.01）。
发育过程中角膜的无血管状态受到冗余调节，这表现为抗血管生成因子TSP-1和/或-2的缺乏未导致角膜自发血管生成。相比之下，TSP-1比TSP-2更有助于抑制出生后炎症诱导的角膜血管生成，这意味着角膜的血管生成特权是被积极维持的。
