title: Nicotine increases the VEGF/PEDF ratio in retinal pigment epithelium: a possible mechanism for CNV in passive smokers with AMD.
abstract: OBJECTIVE
  Cigarette smoking is the strongest environmental risk factor for wet age-related macular degeneration (AMD). Inappropriate expression of proangiogenic vascular endothelial growth factor (VEGF) and antiangiogenic pigment epithelium derived factor (PEDF) may cause choroidal neovascularization (CNV), a key event in wet AMD, resulting in vision loss. Nicotine (NT), a potent angiogenic agent abundant in second-hand smoke, may play a major role in the pathogenesis of wet AMD. The purpose of this study was to evaluate the expression of nicotinic acetylcholine receptors (nAchR) in retinal pigment epithelium (RPE) and determine the effects of NT on RPE-derived VEGF and PEDF expression in the context of passive smoking.
  
  
  METHODS
  Human RPE cells were treated with NT (10(-8) M), with or without the nAchR-nonspecific antagonist hexamethonium (HXM) (10(-5) M) for 72 hours. RPE sheets were microdissected from rats exposed to NT in drinking water (100 μg/mL), with or without HXM (40 mg/kg/d, intraperitoneally), for 72 hours. Cell death was determined by cell count and proliferation by Western blot for proliferating cell nuclear antigen (PCNA). nAchR expression was examined by real-time PCR and Western blot. ERK activation was evaluated by Western blot analysis. VEGF and PEDF expression was assessed by ELISA, Western blot, and real-time PCR.
  
  
  RESULTS
  Cultured RPE cells constitutively expressed the nAchR α3, α10, and β1 subunits, with β1 being the most prevalent. The nAchR α4, α5, α7, and β2 subunits were detected in RPE sheets from rats, among which α4 is the predominant subtype. NT, which did not result in either cell death or proliferation, induced β1 nAchR, upregulated VEGF, and downregulated PEDF expression through nAChR in ARPE-19 cells. Transcriptional activation of the nAchR α4 subunit and nAChR-mediated upregulation of VEGF and PEDF were observed in RPE from rats exposed to NT.
  
  
  CONCLUSIONS
  NT increased the VEGF-to-PEDF ratio in the RPE through nAchR in vitro and in vivo. This alteration in the ratio may play a key role in the progression to wet AMD in passive smokers.
authors.affiliation: Bascom Palmer Eye Institute, Department of Ophthalmology, University of Miami Miller School of Medicine, Miami, Florida 33136, USA.
authors.forename: Marianne
authors.initials: M
authors.lastname: Pons
authors.forename: Maria E
authors.initials: ME
authors.lastname: Marin-Castaño
mesh_terms: D000818:Animals; D015153:Blotting, Western; D016923:Cell Death; D049109:Cell Proliferation; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay; D005136:Eye Proteins / Q000378:metabolism*; D018738:Hexamethonium / Q000494:pharmacology; D006801:Humans; D008297:Male; D009414:Nerve Growth Factors / Q000378:metabolism*; D009538:Nicotine / Q000494:pharmacology*; D018733:Nicotinic Antagonists / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D011978:Receptors, Nicotinic / Q000378:metabolism; D055213:Retinal Pigment Epithelium / Q000187:drug effects* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015843:Serpins / Q000378:metabolism*; D014028:Tobacco Smoke Pollution / Q000009:adverse effects*; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism*; D057135:Wet Macular Degeneration / Q000209:etiology* / Q000378:metabolism; D000099174:Pigment Epithelium-Derived Factor
chemical_list: D005136:Eye Proteins; D018738:Hexamethonium; D009414:Nerve Growth Factors; D009538:Nicotine; D018733:Nicotinic Antagonists; D011978:Receptors, Nicotinic; D015843:Serpins; D014028:Tobacco Smoke Pollution; D042461:Vascular Endothelial Growth Factor A; D000099174:Pigment Epithelium-Derived Factor; C467484:VEGFA protein, human