Overexpression of HTRA1 in RF/6A Cells and HUVECs Figure 6. Effect of HTRA1 on tube formation in RF/6A cells and HUVECs. After 24 h of incubation, untransfected control cells (A) and lentiviral vector control group cells (B) formed well-organized, capillary-like structures, while the capacity of HTRA1-transfected group cells to organize was severely compromised (C, p,0.05). doi:10.1371/journal.pone.0046115.g006 the expression of matrix metalloprotease 1 and matrix metallo- protease 3 [39]. Activities of MMP-2 is closely associated with the migratory capacity of cells. We did not find significant change of MMP-2 expression in different groups. Maybe the effects of HTRA1 on different types of matrix metalloprotease are different. Davide V et al showed that matrix metalloproteinase 2 regulate human aortic smooth muscle cell migration differently in young cells and aged cells [40]. Therefore we conclude that the over- expression of HTRA1 did not regulate the activation of MMP-2, and the migratory capacity changes in our study were not induced by MMP-2. Moreover, laminin is an essential basement mem- brane protein as fibronectin and plays a role in the stability or maturation of vessels and their barrier functions [41]. However, our results showed that HTRA1 has no effect on Laminin. The different results maybe reasoned form the property differences of fibronectin and Laminin [42]. The role of VEGF in the pathogenesis of PCV has been controversial in previous studies. Matsuoka et al. found that VEGF was strongly expressed in the tissues from an eye with PCV and in the vascular endothelial cells in human choroidal neovascular membranes [19]. In contrast, according to Nakashizuka et al., within PCV lesion histologic specimens, vascular endothelial cells were negative for VEGF [9]. In addition,, the interrelationship between VEGF and HTRA1 is also open to question. Ng et al. revealed that HTRA1 and VEGF were upregulated in fetal RPE cells during stress induction, but they do not mutually regulate their expression [18]. VEGF was up-regulated in transgenic mice overexpressing human HTRA1, as reported by Jones et al [32]. To explore if upregulation of HTRA1 has any effect on the expression levels of VEGF in RF/6A cells and HUVECs, we measured mRNA and secreted protein levels of HTRA1. There were no significant differences in VEGF expression levels between HTRA1-transfected groups and control groups of RF/6A cells and HUVECs, except for a slight down-regulation of protein level in cultured HUVEC supernatants collected at 48 h post-transfec- tion. The difference between the two cell types may be attributable to species differences: RF/6A cells are derived from rhesus monkeys, and HUVECs are derived from humans. The mild decrease in the amount of VEGF secreted by HUVECs at 48 h was possibly due to the significant decrease of cell numbers. Moreover, it is reported that VEGF binding to fibronectin has a function in angiogenesis [43]. As noted above the downregulation of fibronectin could lead to the degradation of the capacity of VEGF to act as a promoter of angiogenesis. However, the short time span used in collecting the cells and supernatants limited the experimental data to that which reflected early changes in cell factor levels. With this in mind, we cannot exclude the possibility that the VEGF level changes in later stages through a negative feedback induced by hypofunction of vascular endothelial cells and related signaling pathways. Thus, we can only conclude that VEGF is not influenced by upregulation of HTRA1 during the initial lesion formation stages. Platelet derived growth factor (PDGF) are also vital elements of neoangiogenesis. Inhibitors targeting both VEGFR and PDGFR can contribute to the regression of neovascular vessels in tumors [44]. However, neovascularization in DR and AMD may differ significantly from angiogenesis in other pathological processes such as tumor angiogenesis [45]. Jo et al. have demonstrated that a combination therapy with anti-VEGF and anti-PDGFR is more effective for CNV prevention and regression [46].Nevertheless, in a model of injury-induced choroidal neovascularization, a VEGF inhibitor decreases CNV but administration of a PDGF inhibitor does not decrease CNV [47]. Moreover, PDGFR do not act on vascular endothelial cells, but on pericytes. PDGF induces pericyte recruitment and supports vascular maturation. HTRA1 have no effect upon the expression and activity of PDGFR in RF/6A cells and the role of HTRA1 on neovascularization may need further study on other cells, such as pericytes. In summary, the results of our study indicate that HTRA1 rs11200638 variants are a risk factor for PCV. Upregulation of HTRA1 expression in RF/6A cell and HUVECs inhibited their proliferation, migration, tube formation, and fibronectin expres- sion, whereas laminin, VEGF and MMP-2 expressions were not demonstrably affected. This study highlights the important role that HTRA1 may play in the initial formation of vascular lesions, and provides a better understanding of the effect of HTRA1 on PCV pathogenesis. Supporting Information Table S1 General characteristics of case-control studies included in meta-analysis. (DOC) Analysis of the HTRA1 gene polymorphisms Table S2 and PCV. (DOC) Figure S1 Meta-analysis of HTRA1 rs11200638 poly- morphism and PCV. Forest plot for meta-analysis of association between HTRA1 rs11200638 G.A polymorphism and PCV risk. Each study is shown by the point estimate of the odds ratio (OR) and 95% confidence interval (CI) for the OR. (TIF) PLOS ONE | www.plosone.org 7 October 2012 | Volume 7 | Issue 10 | e46115