Effect of HTRA1 on the migration of RF/6A cells and HUVECs.

<p>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).</p

Overexpression of HTRA1 Leads to Down-Regulation of Fibronectin and Functional Changes in RF/6A Cells and HUVECs

<div><p>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.</p> </div

Effect of HTRA1 on tube formation in RF/6A cells and HUVECs.

<p>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).</p

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.

<p>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).</p

Effect of HTRA1 on the cell cycles of RF/6A cells and HUVECs.

<p>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).</p

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.

<p>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.</p

RT-PCR analysis showed the mRNA expression of Slit2, Robo1, VEGF, and ß-actin in the FVMs of different patients.

<p>A: RT-PCR amplification of Slit2, Robo1, VEGF, and ß-actin in the FVMs of six patients with PDR (panels 1–6) and in the membranes of six patients with idiopathic epiretinal membranes (panels 7–12).B: A representative photograph of the mRNA analysis of Slit2 expression. C: A representative photograph of the mRNA analysis analysis of Robo1 expression. D: A representative photograph of the mRNA analysis analysis of VEGF expression. Asterisks denote values significantly different to the control group (<i>P</i><0.05).</p

Expression of Robo1 and VEGF in the FVMs of eyes with PDR and the preretinal membranes of control patients without DR.

<p>(A-D) Immunostaining for Robo1 (A), VEGF (B), and DAPI (C) inFVMs from eyes with PDR. Staining intensities of Robo1 were strong (A) and co-localized with VEGF (D). (E-H) Staining of Robo1 (E), VEGF (F), and DAPI (G) in the preretinal membranes of control patients without DR. Weak staining of Robo1 (E) and insignificant staining of VEGF (F) were observed. Scale bar 50 μm.</p

A–D: Robo1 antibody staining of rat retina sections.

<p>A: control group; B: 1 month after diabetes induction; C: 3 months after diabetes induction; D: 6 months after diabetes induction. Scale bar 100 μm. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; ONL, outer nuclear layer.</p

The effects of ES, M-ES, and PEG-M-ES on the migration of HRMEC s.

<p>The migratory activities of the control, the VEGF-treated group (20 ng/ml), the ES group, the M-ES group, and the PEG-M-ES group are presented. The upper left panel shows the statistical analysis results, and the other figures are representative images (10⁻⁶mg/ml agents) of various treatment groups. The cell nucleus was stained with DAPI, which is indicated in blue. Each experiment was repeated at least three times. Data are presented as the mean ±SD. One-way ANOVA followed by a post-hoc Dunnett's t-test was used to analyze the data. *P<0.05; **P<0.01; ***P<0.0001.</p