Ren Sheng-Wei, Qi Xia, Jia Chang-Kai, Wang Yi-Qiang
Qingdao University-SEI Joint Ophthalmology Program, Qingdao University Medical College, Qingdao 266071, Shandong Province, China ; Shandong Provincial Key Lab of Ophthalmology, Shandong Eye Institute, Shandong Academy of Medical Sciences, Qingdao 266071, Shandong Province, China.
Shandong Provincial Key Lab of Ophthalmology, Shandong Eye Institute, Shandong Academy of Medical Sciences, Qingdao 266071, Shandong Province, China.
Int J Ophthalmol. 2014 Apr 18;7(2):187-93. doi: 10.3980/j.issn.2222-3959.2014.02.01. eCollection 2014.
To solidify the involvement of Saa-related pathway in corneal neovascularization (CorNV). The pathogenesis of inflammatory CorNV is not fully understood yet, and our previous study implicated that serum amyloid A (Saa) 1 (Saa1) and Saa3 were among the genes up-regulated upon CorNV induction in mice.
Microarray data obtained during our profiling project on CorNV were analyzed for the genes encoding the four SAA family members (Saa1-4), six reported SAA receptors (formyl peptide receptor 2, Tlr2, Tlr4, Cd36, Scarb1, P2rx7) and seven matrix metallopeptidases (Mmp) 1a, 1b, 2, 3, 9, 10, 13 reportedly to be expressed upon SAA pathway activation. The baseline expression or changes of interested genes were further confirmed in animals with CorNV using molecular or histological methods. CorNV was induced in Balb/c and C57BL/6 mice by placing either three interrupted 10-0 sutures or a 2 mm filter paper soaked with sodium hydroxide in the central area of the cornea. At desired time points, the corneas were harvested for histology examination or for extraction of mRNA and protein. The mRNA levels of Saa1, Saa3, Fpr2, Mmp2 and Mmp3 in corneas were detected using quantitative reverse transcription-PCR, and SAA3 protein in tissues detected using immunohistochemistry or western blotting.
Microarray data analysis revealed that Saa1, Saa3, Fpr2, Mmp2, Mmp3 messengers were readily detected in normal corneas and significantly up-regulated upon CorNV induction. The changes of these five genes were confirmed with real-time PCR assay. On the contrary, other SAA members (Saa2, Saa4), other SAA receptors (Tlr2, Tlr4, Cd36, P2rx7, etc), or other Mmps (Mmp1a, Mmp1b, Mmp9, Mmp10, Mmp13) did not show consistent changes. Immunohistochemistry study and western blotting further confirmed the expression of SAA3 products in normal corneas as well as their up-regulation in corneas with CorNV.
SAA-FPR2 pathway composing genes were expressed in normal murine corneas and, upon inflammatory stimuli challenge to the corneas, their expressions were up-regulated, suggesting their roles in pathogenesis of CorNV. The potential usefulness of SAA-FPR2 targets in future management of CorNV-related diseases deserves investigation.
确定与血清淀粉样蛋白A（Saa）相关的信号通路在角膜新生血管形成（CorNV）中的作用。炎症性角膜新生血管形成的发病机制尚未完全明确，我们之前的研究表明，血清淀粉样蛋白A（Saa）1（Saa1）和Saa3是小鼠角膜新生血管形成诱导后上调的基因之一。
对我们在角膜新生血管形成分析项目中获得的微阵列数据进行分析，以检测编码四个SAA家族成员（Saa1 - 4）、六个已报道的SAA受体（甲酰肽受体2、Tlr2、Tlr4、Cd36、Scarb1、P2rx7）以及七个据报道在SAA信号通路激活时表达的基质金属蛋白酶（Mmp）1a、1b、2、3、9、10、13的基因。使用分子或组织学方法在角膜新生血管形成的动物中进一步确认感兴趣基因的基线表达或变化。通过在Balb/c和C57BL/6小鼠角膜中央区域放置三根间断的10 - 0缝线或浸泡有氢氧化钠的2 mm滤纸来诱导角膜新生血管形成。在所需时间点，收获角膜用于组织学检查或提取mRNA和蛋白质。使用定量逆转录 - PCR检测角膜中Saa1、Saa3、Fpr2、Mmp2和Mmp3的mRNA水平，使用免疫组织化学或蛋白质印迹法检测组织中的SAA3蛋白。
微阵列数据分析显示，Saa1、Saa3、Fpr2、Mmp2、Mmp3信使RNA在正常角膜中易于检测到，并且在角膜新生血管形成诱导后显著上调。通过实时PCR分析证实了这五个基因的变化。相反，其他SAA成员（Saa2、Saa4）、其他SAA受体（Tlr2、Tlr4、Cd36、P2rx7等）或其他Mmp（Mmp1a、Mmp1b、Mmp9、Mmp10、Mmp13）未显示出一致的变化。免疫组织化学研究和蛋白质印迹法进一步证实了SAA3产物在正常角膜中的表达以及它们在角膜新生血管形成的角膜中的上调。
SAA - FPR2信号通路相关基因在正常小鼠角膜中表达，并且在角膜受到炎症刺激时，它们的表达上调，表明它们在角膜新生血管形成的发病机制中起作用。SAA - FPR2靶点在未来角膜新生血管形成相关疾病治疗中的潜在用途值得研究。
