Overexpression of HTRA1 Leads to Down-Regulation of Fibronectin and Functional Changes in RF/6A Cells and HUVECs Jingjing Jiang1,2,3., Lvzhen Huang1,2., Wenzhen Yu1,2, Xi Wu1,2, Peng Zhou1,2,4, Xiaoxin Li1,2* 1 Department of Ophthalmology, Peking University People’s Hospital, Beijing, China, 2 Key Laboratory of Vision Loss and Restoration, Ministry of Education, Beijing, China, 3 Department of Ophthalmology, China-Japan Friendship Hospital, Beijing, China, 4 Department of Ophthalmology, Eye and ENT Hospital of Fudan University, Shanghai, China Abstract Multiple genetic studies have suggested that high-temperature requirement serine protease (HTRA1) is associated with polypoidal choroidal vasculopathy (PCV). To date, no functional studies have investigated the biological effect of HTRA1 on vascular endothelial cells, essential vascular components involved in polypoidal vascular abnormalities and arteriosclerosis- like changes. In vitro studies were performed to investigate the effect of HTRA1 on the regulation of fibronectin, laminin, vascular endothelial growth factor (VEGF), platelet derived growth factor receptor (PDGFR) and matrix metalloparoteinases 2 (MMP-2) and the role of HTRA1 in choroid-retina endothelial (RF/6A) and human umbilical vein endothelial (HUVEC) cells. Lentivirus-mediated overexpression of HTRA1 was used to explore effects of the protease on RF/6A and HUVEC cells in vitro. HTRA1 overexpression inhibited the proliferation, cell cycle, migration and tube formation of RF/6A and HUVEC cells, effects that might contribute to the early stage of PCV pathological lesions. Fibronectin mRNA and protein levels were significantly down-regulated following the upregulation of HTRA1, whereas the expressions of laminin, VEGF and MMP-2 were unaffected by alterations in HTRA1 expression. The decreased biological function of vascular endothelial cells and the degradation of extracellular matrix proteins, such as fibronectin, may be involved in a contributory role for HTRA1 in PCV pathogenesis. Citation: Jiang J, Huang L, Yu W, Wu X, Zhou P, et al. (2012) Overexpression of HTRA1 Leads to Down-Regulation of Fibronectin and Functional Changes in RF/6A Cells and HUVECs. PLoS ONE 7(10): e46115. doi:10.1371/journal.pone.0046115 Editor: Gianfranco Pintus, University of Sassari, Italy Received January 3, 2012; Accepted August 28, 2012; Published October 8, 2012 Copyright: 2012 Jiang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This research was supported by the National Basic Research Program of China (973 Program (2011CB510200)). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: drlixiaoxin@163.com . These authors contributed equally to this work. Introduction Polypoidal choroidal vasculopathy (PCV) is a major cause of serosanguinous maculopathy, a condition associated with a reduction of vision in the elderly Asian population. PCV was first described by Yannuzzi and associates in 1990 as an idiopathic PCV presenting polypoidal and choroidal vascular lesions [1]. The incidences of PCV among Chinese and Japanese populations with fundus characteristics of neovascular age-related macular degen- eration (nAMD) are 24.5% and 54.7%, respectively, which are much higher than the incidence noted in Caucasians [2,3,4]. Distinct from the clinical features of AMD, PCV is characterized on indocyanine green (ICG) by a network of branching abnormal choroid vessels and polypoidal vascular dilations. The etiology and pathogenesis of PCV are largely unclear. HTRA1 (high-temperature requirement factor A-1), a member of the high-temperature requirement A family of serine proteases, is ubiquitously expressed in various normal adult human tissues, such as the epidermis, vascular endothelia and neuronal cells [5]. HTRA1 mutations have been associated with familial ischemic cerebral small-vessel disease (CARASIL), which is characterized by non-hypertensive cerebral small-vessel arteriopathy [6]. Previ- ous studies have reported raised levels of HTRA1 expression in drusen, abnormal retinal pigment epithelial (RPE) cells and choroidal neovascular membranes [7,8,9]. It has been well established that variation in HTRA1 has a strong genetic effect on AMD, a disease sharing certain common environmental risk factors and genetic determinants with PCV [10]. The functional single nucleotide polymorphism (SNP) rs11200638, located in the promoter region of the HTRA1 gene within the 10q26 locus, has been identified as one of the most closely associated AMD risk factors [7,11]. Recently, an increasing number of studies have investigated the possible association of PCV in Asian populations with rs11200638 in HTRA1 [12,13,14,15]. However, a summary of the genetic effects of this variant on the susceptibility to PCV has not been reviewed. The mechanism by which HTRA1 instigates the ocular tissue abnormalities of AMD has been discussed in functional studies. Many of these studies suggest a link between HTRA1, fibronectin and stabilization of the extracellular matrix in AMD pathogenesis [8,16,17]. As an important signal protein promoting angiogenesis, VEGF should also be studied to determine if there is any connection with HTRA1 in AMD [18,19]. Thus, we speculated that the expression of HTRA1 could be associated with fibronectin PLOS ONE | www.plosone.org 1 October 2012 | Volume 7 | Issue 10 | e46115