Overexpression of HTRA1 in RF/6A Cells and HUVECs Figure 1. Effect of pGC-FU-3FLAG-HTRA1 on HTRA1, fibronectin and VEGF expression in RF/6A cells and HUVECs as determined by real-time RT-PCR and western blotting or ELISA. Messenger RNA (A) and protein (B) expression of HTRA1 in RF/6A cells and HUVECs transfected with pGC-FU-3FLAG-HTRA1 were increased when compared with the control groups (**P,0.01). The fibronectin mRNA (C) and protein (B) levels were reduced in HTRA1-overexpressing cells (**P,0.01), while no significant differences were noted in the expression levels of VEGF mRNA (D) and secreted protein, except for a slight decrease in protein levels in cultured HUVEC supernatants at 48 hours (*P,0.05). doi:10.1371/journal.pone.0046115.g001 between HTRA1-transfected HUVECs and NC HUVECs in G2/ M phase was not significant (6.93% versus 4.74%, p.0.05). To explore the role of HTRA1 in the migration of RF/6A cells and HUVECs, we used a modified Boyden chamber in which these cells migrated through a porous membrane. The mean counts of migrating cells among the HTRA1 group of the RF/6A cells and HUVECs were significantly lower than in the NC group (p,0.05, Figure 5). In contrast, the mean counts of migrated cells in the NC and UT groups were not significantly different (p.0.05). HTRA1 has a slight effect on tube formation in RF/6A cells and HUVECs To investigate whether HTRA1 could affect the angiogenic properties of RF/6A and HUVEC cells, we performed a capillary- like tube formation assay in matrigel. The HTRA1-transfected RF/6A cells and HUVECs both demonstrated little impairment of their capacities to form regular networks, and the differences between HTRA1-transfected cells and NC cells were slight in RF/ 6A cells (p,0.05, Figure 6) and HUVECs (p,0.05). There was no significant difference between the NC and UT groups. Discussion A meta-analysis focused on the relationship between HTRA1 variants and PCV was performed as described previously [20] (Table S1). The meta-analysis indicated a 2.6-fold increase in susceptibility to PCV among persons with the A allele of the HTRA1 rs11200638 SNP. Compared to GG homozygotes, the AA homozygous and AG heterozygous variants carry a 6.4-fold and 1.8-fold increased risk for PCV, respectively (Table S2 and Figure S1). These data support the notion that the HTRA1 variant may be a risk factor for PCV. The functional SNP, located in the promoter region of the HTRA1 gene, has been reported to increase expression levels of HTRA1 [25,26]. Using HTRA1 overexpression strategies, our experimental results show that this risk may result partly from decreased vascular endothelial cell biological function and degraded extracellular matrix proteins, such as fibronectin. It has been well established that PCV lesions arise from the inner choroid vascular network and are characterized by polypoidal vascular abnormality, which implied that vascular endothelial cells may play an important role in the pathogenesis of PCV. HTRA1 has been reported in vascular endothelia, and some PLOS ONE | www.plosone.org 4 October 2012 | Volume 7 | Issue 10 | e46115