The α7-nicotinic acetylcholine receptor and MMP-2/-9 pathway mediate the proangiogenic effect of nicotine in human retinal endothelial cells.
OBJECTIVE
Nicotine, the active component of cigarette smoke, has been found to stimulate angiogenesis in several experimental systems. In this study, the Matrigel duplex assay (Matrigel; BD Biosciences, Franklin Lakes, NJ) and the rat retinal explant assay were used to explore the molecular mechanisms underlying the proangiogenic effects of nicotine in endothelial cells.


METHODS
Western blot analysis was performed to determine the nicotinic acetylcholine receptor (nAChR) subtypes expressed on primary human retinal microvascular endothelial cells (HRMECs). The angiogenic effect of nicotine in the retina was evaluated with the duplex assay. The results obtained from the assay were confirmed by the rat retinal explant angiogenesis assay. ELISAs were used to measure MMP-2, -9, and -13 levels in HRMEC culture supernatants. The role of α7-nAChRs in nicotine-induced angiogenesis was examined by siRNA techniques.


RESULTS
Nicotine-induced angiogenesis required nAChR function and was associated with the upregulation of MMP-2 and -9 in HRMECs. Specifically, α7-nAChRs mediated the stimulatory effects of nicotine on retinal angiogenesis and MMP levels. Treatment of HRMECs with α7-nAChR antagonists ablated nicotine-induced angiogenesis. The inhibitory actions of α7-nAChR antagonists correlated with the suppression of MMP-2 and -9 levels in HRMECs.


CONCLUSIONS
The α7-nAChR is vital for the proangiogenic activity of nicotine. The α7-nAChRs expressed on HRMECs upregulate levels of MMP-2 and -9, which stimulate retinal angiogenesis. The data also suggest that α7-nAChR antagonists could be useful agents for the therapy of angiogenesis-related retinal diseases.
Dom
Aaron M
AM
Department of Pharmacology, Physiology, and Toxicology, Joan C. Edwards School of Medicine, Marshall University, Huntington, West Virginia 25755, USA
Buckley
Adam W
AW
Brown
Kathleen C
KC
Egleton
Richard D
RD
Marcelo
Aileen J
AJ
Proper
Nancy A
NA
Weller
Donald E
DE
Shah
Yashoni H
YH
Lau
Jamie K
JK
Dasgupta
Piyali
P
D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D004195:Disease Models, Animal; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D008297:Male; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism*; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism*; D009538:Nicotine / Q000633:toxicity*; D051381:Rats; D011924:Rats, Zucker; D011978:Receptors, Nicotinic / Q000378:metabolism*; D012160:Retina / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D015861:Retinal Neovascularization / Q000139:chemically induced / Q000378:metabolism* / Q000473:pathology; D064569:alpha7 Nicotinic Acetylcholine Receptor
C579167:Chrna7 protein, human; C579165:Chrna7 protein, rat; D011978:Receptors, Nicotinic; D064569:alpha7 Nicotinic Acetylcholine Receptor; D009538:Nicotine; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9