Pharmacologic manipulation of sphingosine kinase in retinal endothelial cells: implications for angiogenic ocular diseases.
OBJECTIVE
The increased vascular permeability and pathogenic angiogenesis observed in diabetic retinopathy are induced, at least in part, by local inflammation and vascular endothelial growth factor (VEGF). Therefore, inhibition of signaling from VEGF and tumor necrosis factor-alpha (TNFalpha) is a promising approach to the treatment of this disease, as well as ocular diseases with similar etiologies, including age-related macular degeneration. A growing body of evidence demonstrates that sphingosine kinase (SK) plays an important role in cellular proliferation and angiogenesis. This study was undertaken to examine the effects of SK inhibitors on the responses of retinal endothelial cells (RECs) to VEGF and TNFalpha and their therapeutic efficacy in a diabetic retinopathy model.


METHODS
The expression and function of SK in bovine and human RECs were examined by immunoblot analysis. The involvement of SK in mediating responses to VEGF and TNFalpha was examined by using pharmacologic inhibitors of SK in cellular and in vivo assays, including a 3-month streptozotocin-induced diabetic retinopathy model in rats.


RESULTS
SK was present and active in human and bovine RECs, and SK activity in these cells was stimulated by VEGF. Inhibitors of SK blocked VEGF-induced production of sphingosine 1-phosphate and markedly attenuated VEGF-induced proliferation and migration of RECs. In addition, SK inhibitors were shown to block TNFalpha-induced expression of adhesion proteins, suppress VEGF-induced vascular leakage in an in vivo mouse model, and reduce retinal vascular leakage in the rat diabetic retinopathy model.


CONCLUSIONS
Overall, these studies demonstrate that inhibitors of SK attenuate the effects of proliferative and inflammatory stimuli on RECs both in vitro and in vivo, and so could be significant therapeutics in the treatment of diabetic retinopathy.
Maines
Lynn W
LW
Apogee Biotechnology Corporation, Hershey, Pennsylvania, USA.
French
Kevin J
KJ
Wolpert
Ellen B
EB
Antonetti
David A
DA
Smith
Charles D
CD
D000818:Animals; D015153:Blotting, Western; D002199:Capillary Permeability; D002417:Cattle; D018929:Cell Culture Techniques; D049109:Cell Proliferation; D003921:Diabetes Mellitus, Experimental / Q000201:enzymology / Q000473:pathology; D003930:Diabetic Retinopathy / Q000201:enzymology* / Q000473:pathology; D004195:Disease Models, Animal; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000201:enzymology / Q000473:pathology; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D008297:Male; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015861:Retinal Neovascularization / Q000517:prevention & control*; D012171:Retinal Vessels / Q000166:cytology*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D042461:Vascular Endothelial Growth Factor A / Q000494:pharmacology; D000099157:Sphingosine Kinase
D004791:Enzyme Inhibitors; D017853:Phosphotransferases (Alcohol Group Acceptor); D014409:Tumor Necrosis Factor-alpha; D042461:Vascular Endothelial Growth Factor A; D000099157:Sphingosine Kinase