Overexpression of HTRA1 in RF/6A Cells and HUVECs Figure 4. Effect of HTRA1 on the cell cycles of RF/6A cells and HUVECs. Flow cytometric analysis demonstrated that the fraction of G1-phase cells increased and the proportion of S-phase cells decreased in the RF/6A cells (A and B) and HUVECs (C and D) after increased expression of HTRA1. The proportions of G0/G1, G2, and S phase cells decreased in HTRA1-transfected RF/6A cells compared to control RF/6A cells transduced with the lentiviral vector (E, *p,0.05, **p,0.01). The proportions of G0/G1-and S-phase cells were decreased in HTRA1-transfected HUVECs compared with the control HUVECs transduced with the lentiviral vector (F, **p,0.01). doi:10.1371/journal.pone.0046115.g004 HTRA1 could have a detrimental effect on physiologic functions and homeostasis of vascular endothelial cells, which might lead to the destruction or dysfunction of vascular endothelium and might contribute to the early stage of PCV pathological lesions. Fibronectin, as a major component of the vascular basement membrane and a mediator of extracellular matrix assembly, was found to be strongly associated with HTRA1 in ocular fundus lesions. Bajenaru et al. revealed that the loss of astrocytic fibronectin results in a mild vascular defect characterized by delayed vessel extension [37]. More abundant expression of fibronectin in the RPE of AMD donors was reported by An et al. [38]. Vierkotten et al. demonstrated that HTRA1 degrades fibronectin in RPE cells, and a higher level of expression of active fibronectin fragments was detected in HtrA1 transgenic mice [17]. In this study, we observed that fibronectin was expressed in both RF/6A cells and HUVECs and was significantly decreased by overexpressing HTRA1. The down-regulated fibronectin may impact the PCV lesion process by involving aneurysm or dilation of the abnormal vessels. Fibronectin has been reported to regulate the complement pathway [8]. Meanwhile, fibronectin fragments, produced by increased fibronectin digestion related to HTRA1, could act as a downstream protagonist of intraocular inflammation and catabolism [16]. The fibronectin fragments also have the capacity to degrade the stabilization of the extracellular matrix and to stimulate the release of cytokines and matrix metallopro- teinases (MMPs) from RPE cells, which might further play a contributory role in PCV pathogenesis. HtrA1 or HtrA1- generated fibronectin fragments was reported be able to induce Figure 5. Effect of HTRA1 on the migration of RF/6A cells and HUVECs. The migratory activities of both cell lines were estimated based on the numbers of cells that had migrated through the filter of the chamber. The numbers of migrating cells in the HTRA1-transfected group were less than the number observed in the untransfected control group and the lentiviral vector control group (G, *p,0.01). doi:10.1371/journal.pone.0046115.g005 PLOS ONE | www.plosone.org 6 October 2012 | Volume 7 | Issue 10 | e46115