Overexpression of HTRA1 in RF/6A Cells and HUVECs Figure 2. Effect of pGC-FU-3FLAG-HTRA1 on laminin, PDGFR and MMP-2 expression in RF/6A cells as determined by real-time RT- PCR and western blotting or ELISA. Messenger RNA expression of laminin, PDGFR and MMP-2 in RF/6A cells transfected with pGC-FU-3FLAG- HTRA1 showed no significant changes compared with the control groups (A, B, C). No significant differences were noted in the protein expression levels of laminin and MMP-2. doi:10.1371/journal.pone.0046115.g002 PLOS ONE | www.plosone.org 5 October 2012 | Volume 7 | Issue 10 | e46115 Figure 3. Effect of HTRA1 on the proliferation of RF/6A cells and HUVECs. Proliferation of RF/6A cells (A) and HUVECs (B) was measured with an MTT assay at 24, 48, 72 and 96 h. The values are listed as the means6SD of at least three independent experiments. Compared with the control groups, RF/6A cell proliferation was reduced by 7% at 48 h and this reduction peaked at 17% on the fourth day (**p,0.01); HUVEC proliferation was reduced by 10% (*P,0.05) at 48 h, and this reduction peaked at 23% (**P,0.01). doi:10.1371/journal.pone.0046115.g003 expression is found in the peripheral retinas [5,27]. Moreover, proteins of the transforming growth factor b (TGF-b) family, which have been associated with the most-documented function of HTRA1, have multifaceted roles in vascular endothelial cells, depending on the type of cell and extracellular matrix [6,28]. Dysregulation of TGF-b-family signaling results in hereditary vascular disorders [29]. However, previous studies investigating the functional effects of HTRA1 on PCV or AMD mainly focused on RPE cells or Bruch’s membrane [8,17,18,30], and potential effects on vascular endothelial cells have been only rarely observed. In this study, we demonstrated that overexpression of HTRA1 induces varying degrees of reduction in the proliferation, cell cycle, migration and tube formation of RF/6A cells and HUVECs. The inhibition of proliferation was apparent in the HTRA1-transfected cells after 48 h. The cell growth of both RF/6A cells and HUVECs was also slowed due to a decreased progression past the G1/S checkpoint of the cell cycle. These impacts on vascular endothelial cells may be partly explained by the contributions of HtrA serine proteases to programmed cell death [31,32]. Enhanced expression of HTRA1 markedly attenuates cell migration, whereas downregulation of HTRA1 promotes cell motility [33]. In addition, a crucial functional indicator of vascular endothelial cells, the tube formation capability of RF/6A cells and HUVECs, was degraded, as observed on matrigel. Thus, HTRA1 might play a role in maintaining the normal physiologic functions and homeostatic functions of RF/6A cells and HUVECs. Furthermore, normal vascular endothelial cells are thought to have atheroprotective functions [34]. Repeated or chronic endothelial cell loss and dysfunction might be the first event leading to atherosclerosis [35]. A key finding in the histopatho- logical features of PCV is vessel hyalinization, which suggests an association with arteriosclerotic changes [10] In addition, HTRA1 has been suggested to play a critical role in the regulation of angiogenesis via TGF-b signaling [36]. Therefore, an excess of