Fei Ping, Palenski Tammy L, Wang Shoujian, Gurel Zafer, Hankenson Kurt D, Sorenson Christine M, Sheibani Nader
1 Department of Ophthalmology and Visual Sciences, University of Wisconsin School of Medicine and Public Health , Madison, Wisconsin.
2 Department of Ophthalmology, Xinhua Hospital, School of Medicine, Shanghai Jiao Tong University , Shanghai, China .
J Ocul Pharmacol Ther. 2015 Sep;31(7):429-44. doi: 10.1089/jop.2014.0151. Epub 2015 May 7.
To determine thrombospondin-2 (TSP2) expression and its impact on postnatal retinal vascular development and retinal neovascularization.
The TSP2-deficient (TSP2(-/-)) mice and a line of TSP2 reporter mice were used to assess the expression of TSP2 during postnatal retinal vascular development and neovascularization. The postnatal retinal vascularization was evaluated using immunostaining of wholemount retinas prepared at different postnatal days by collagen IV staining and/or TSP2 promoter driven green fluorescent protein (GFP) expression. The organization of astrocytes was evaluated by glial fibrillary acidic protein (GFAP) staining. Retinal vascular densities were determined using trypsin digestion preparation of wholemount retinas at 3- and 6-weeks of age. Retinal neovascularization was assessed during the oxygen-induced ischemic retinopathy (OIR). Choroidal neovascularization (CNV) was assessed using laser-induced CNV.
Using the TSP2-GFP reporter mice, we observed significant expression of TSP2 mRNA in retinas of postnatal day 5 (P5) mice, which increased by P7 and remained high up to P42. Similar results were observed in retinal wholemount preparations, and western blotting for GFP with the highest level of GFP was observed at P21. In contrast to high level of mRNA at P42, the GFP fluorescence or protein level was dramatically downregulated. The primary retinal vasculature developed at a faster rate in TSP2(-/-) mice compared with TSP2(+/+) mice up to P5. However, the developing retinal vasculature in TSP2(+/+) mice caught up with that of TSP2(-/-) mice after P7. No significant differences in retinal vascular density were observed at 3- or 6-weeks of age. TSP2(-/-) mice also exhibited a similar sensitivity to the hyperoxia-mediated vessel obliteration and similar level of neovascularization during OIR as TSP2(+/+) mice. Lack of TSP2 expression minimally affected laser-induced CNV compared with TSP2(+/+) mice.
Lack of TSP2 expression was associated with enhanced retinal vascularization during early postnatal days but not at late postnatal times, and minimally affected retinal and CNV. However, the utility of TSP2 as a potential therapeutic target for inhibition of ocular neovascularization awaits further investigation.
确定血小板反应蛋白-2（TSP2）的表达及其对出生后视网膜血管发育和视网膜新生血管形成的影响。
利用TSP2基因缺陷（TSP2(-/-)）小鼠和TSP2报告基因小鼠品系，评估出生后视网膜血管发育和新生血管形成过程中TSP2的表达。通过对不同出生后天数制备的视网膜全层进行免疫染色，采用IV型胶原染色和/或TSP2启动子驱动的绿色荧光蛋白（GFP）表达，评估出生后视网膜血管生成情况。通过胶质纤维酸性蛋白（GFAP）染色评估星形胶质细胞的组织情况。在3周龄和6周龄时，使用胰蛋白酶消化制备的视网膜全层测定视网膜血管密度。在氧诱导的缺血性视网膜病变（OIR）过程中评估视网膜新生血管形成情况。使用激光诱导脉络膜新生血管形成（CNV）评估脉络膜新生血管形成情况。
利用TSP2-GFP报告基因小鼠，我们观察到出生后第5天（P5）小鼠视网膜中TSP2 mRNA有显著表达，到P7时增加，并在P42时一直保持高水平。在视网膜全层制备物中观察到类似结果，在P21时观察到GFP水平最高的western印迹法检测GFP。与P42时mRNA的高水平相反，GFP荧光或蛋白水平显著下调。在P5之前，TSP2(-/-)小鼠的初级视网膜血管系统发育速度比TSP2(+/+)小鼠快。然而，P7之后，TSP2(+/+)小鼠发育中的视网膜血管系统赶上了TSP2(-/-)小鼠。在3周龄或6周龄时未观察到视网膜血管密度有显著差异。在OIR期间，TSP2(-/-)小鼠对高氧介导的血管闭塞也表现出类似的敏感性，新生血管形成水平与TSP2(+/+)小鼠相似。与TSP2(+/+)小鼠相比，TSP2表达缺失对激光诱导的CNV影响最小。
TSP2表达缺失与出生后早期而非晚期视网膜血管生成增强有关，对视网膜和CNV影响最小。然而，TSP2作为抑制眼部新生血管形成的潜在治疗靶点的效用有待进一步研究。
