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Carolina Franco Nitta, Finny Monickaraj, Amy Lucero, Paul McGuire, Arup Das; Sphingosine-1-Phosphate Pathway: Role in Pericyte-Endothelial Interactions in Diabetic Retinopathy. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5824.
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Diabetic macular edema is the major cause of vision loss in diabetes. Sphingosine-1-Phosphate (S1P) is a bioactive lipid with multiple functions, including a prominent role in ocular angiogensis by maintaining vascular barrier integrity. It signals through a family of five G-protein coupled receptors (S1PR1-5), and is synthesized by two sphingosine kinases (Sphk1 and Sphk2). It increases the physical interactions of pericytes and endothelial cells by upregulating cadherins. Due to its role in the vasculature, we sought to investigate alterations in the S1P pathway in different models of diabetes and diabetic retinopathy.
Human Retinal Endothelial Cells (HRECs) or Pericytes (HRPs) were cultured in the presence of low (5mM), high (30mM), or alternating daily low and high glucose (glucose flux) for 7 days. Additionally, C57Bl/6 mice were made diabetic with 5 daily injections of streptozotocin (50mg/kg/day) and retinas harvested 4 or 8 weeks after the last injection. RNA was isolated from cells or retinas and the levels of S1PR1-5, Sphk1 and Sphk2 were quantified by real-time PCR. To determine the mechanistic effect of Sphk1 in murine retinas, NG2-Cre/SphK1-flox mice were treated with tamoxifen for temporal knock-down in pericytes. Retinal vascular permeability in these mice was measured by retinal albumin protein levels by western blot.
High and glucose flux treatment of HRECs increased the expression levels of S1PR1 and decreased Sphk1. Similar treatments of HRPs decreased S1PR3, Sphk1, and Sphk2. Diabetic animals showed diminished levels of S1PR1 and Sphk1 at 4 and 8 weeks. NG-2-Cre/SphK1-flox mice 2 and 4 weeks after tamoxifen treatment showed increased retinal albumin as compared to controls.
Since S1P is responsible for vascular integrity, the leakiness seen in diabetic retinopathy patients can be due to decreases in S1P receptor levels and/or decreases in S1P levels directly. Both HRECs and HRPs treated for 7 days in diabetic conditions and diabetic mice at 4 and 8 weeks reduced the levels of S1P receptors as well as the kinases responsible for production of S1P. The increase in albumin levels in NG-2-Cre/SphK1-flox mice indicate heightened retinal vascular leakiness. In conclusion, S1P and its related receptors seem to play an important role in diabetic retinopathy, and can be a potential novel target for this disease.
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