| Background and ObjectiveAge-related macular degeneration (AMD) is a sight threatening eye disease that affects millions of people over the 50 years old around the world. The etiology of AMD is a complex with the combination factors including age, environmental, heredity and other factors, among which cigarette smoke is the most important and modifiable risk factor. The wet form is characterized by the growth of leaky new abnormal blood vessels that originate from the subjacent choroid through Bruch’s membrane into the sub-RPE and subretinal space. Although wetAMD occurs in only 10～15% of eyes with AMD, it is responsible for 85% of the severe vision loss in AMD. The choroidal neovascularization (CNV), a key event in wetAMD, can cause bleeding and fluid leakage, which along with RPE and photoreceptor destruction, lead to rapid vision loss.Nicotine (NT) and nornicotine, two potent angiogenic modulators in cigarette smoke, may play a major role in the pathogenesis of wetAMD. Overwhelming evidence showed that NT has been shown to be mitogenic for endothelial cells, to induce cell proliferation, migration and tube formation. Although previous studies have reported that NT stimulates VEGF expression in some kinds of cells, including RPE and endothelial cells, there are few reports about the role of nornicotine, the main metabolite of NT, in angiogenesis. The purpose of the current study was to evaluate the influences of nornicotine and NT on VEGF and PEDF expression in retinal pigment epithelium (RPE) cells and human umbilical vein endothelial cells (HUVECs). In addition, we assessed the changes in cellular behaviors of endothelial cells treated with nornicotine and NT. The identification of different facets on the development of AMD will provide further evidence in the prevention and treatment of AMD.Methods1. Cell culture:ARPE-19 cells, a human retinal pigment epithelial cell line, were grown in DMEM/F12 medium supplemented with 10% FBS. HUVECs, a human umbilical vein endothelial cell line, were cultured in sterile RPMI 1640 with 10% FBS culture media. The concentrations ranging from 0.01umol/L to 100umol/L of nornicotine and NT were prepared by serial dilutions of the reagents in the respective medium.2. Cellular proliferation assay:Cell proliferation was evaluated with the method of MTT. ARPE-19 cells and HUVECs were incubated with indicated concentrations (0.01～100umol/L) of nornicotine or NT for 24h or at the concentration of lOumol/L for 0～48h.3. Migration assay:HUVECs migratory function was examined by using a wound healing assay. HUVECs wounded monolayers were incubated in medium containing 0.01～10umol/L nornicotine or NT for 24h.4. Matrigel tube formation assay:Tube formation by endothelial cells was assessed using the Matrigel models. HUVECs were incubated in medium containing 0.01～10umol/L nornicotine or NT for 6-8h.5. Real time-PCR analysis:ARPE-19 cells and HUVECs were treated with 0.01～10umol/L nornicotine or NT for 24h or at the concentration of 10umol/L for 0～48h.6. Western blotting assay:ARPE-19 cells and HUVECs were treated with 0.01～10umol/L nornicotine or NT for 24h or at the concentration of 10umol/L for 0-48h. Results1. The effect of nornicotine and NT on cell proliferation:nornicotine and NT did not significantly alter the viability of ARPE-19 cells at any concentration and time points (P> 0.05). In HUVECs, nornicotine and NT can significantly caused cell proliferation in a dose-and time-dependent manner (P< 0.05). Nevertheless, two types of cells viabilities were compromised by 100umol/L nornicotine and NT incubation (P < 0.01).2. Nornicotine and NT enhanced HUVECs migration:When the cells were stimulated with nornicotine or NT on concentrations of 0.01～10umol/L, the migratory abilities of HUVECs were significantly enhanced in a dose-dependent manner compared to controls (P< 0.05).3. Nornicotine and NT induced HUVECs tubular formation:More tubes were formed in HUVECs treated with nornicotine or NT than that in control in a concentration-dependent manner (P< 0.05).4. The expression of VEGF mRNA was increased (P< 0.05) and the expression of PEDF mRNA was decreased (P< 0.05)with the incubation of nornicotine or NT in two types of cells. These led a significantly increase of the VEGF/PEDF mRNA ratios in a dose-and time-dependent manner (P< 0.05).5. Nornicotine and NT increased VEGF protein expression and decreased PEDF protein expression in two types of cells:The expression of VEGF protein was increased (P< 0.05) and the expression of PEDF protein was decrease (P< 0.05) in a dose-and time-dependent manner. Conclusion1. No cell proliferation was detected in ARPE-19 cells. Nornicotine and NT promoted endothelial cellular proliferation, migration and angiogenesis of HUVECs in vitro.2. Nornicotine and NT increased of the VEGF/PEDF mRNA ratios in ARPE-19 cells and HUVECs. The angiogenic behaviors of HUVECs with nornicotine and NT treatment might be partly through simultaneous modulation of VEGF/PEDF signaling in two types of cells. It was helpful to understand the mechanism, which might be the potential therapeutic target for wetAMD. |
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