Abstract
Purpose:
Diabetic retinopathy is characterized by increased retinal levels of vascular endothelial growth factor (VEGF) at an early disease stage. VEGF induces expression of leukocyte adhesion proteins and proinflammatory cytokines that trigger leukostasis, one of the complications of diabetic retinopathy. The nuclear factor of activated T-cells (NFAT) family of transcription factors is also known to upregulate inflammatory proteins. NFAT is dephosphorylated by calcium-activated calcineurin (CN), resulting in its translocation to the nucleus where it can affect gene transcription. We hypothesized that VEGF stimulates production of genes related to leukostasis through a mechanism involving intracellular calcium and NFAT.
Methods:
Primary human retinal microvascular endothelial cells (HRMEC) were plated in black wall 96-well plates. HRMEC were incubated with 1 µM Fura-2 for 1 hour before being stimulated with 50 or 100 ng/ml of VEGF or 50, 100, or 1000 nM thapsigargin (TG, calcium reuptake inhibitor). Intracellular calcium flux was measured over the following 600 seconds on a FLEXstation as a ratio of excitation at 340 and 380 nm. HRMEC were serum starved for 12 hours, pretreated with 1 µM FK-506 (CN inhibitor) for two hours, and then treated with 50 or 100 ng/ml VEGF or 100 nM TG. Cells were fixed and immunolabeled for NFAT. HRMEC were treated with 25 ng/ml VEGF and 1 µM FK-506 or 100 nM TG for 8 hours. Cells were lysed and collected for RT-PCR of CXCL2 and VCAM-1.
Results:
Both VEGF and TG stimulated intracellular calcium flux in HRMEC in a dose-dependent manner (p<0.0015). VEGF and TG also induced translocation of NFATc1 to the nucleus. Inhibiting calcineurin with FK-506 prevented VEGF-induced translocation of NFATc1. Finally, both VEGF and TG stimulated transcription of CXCL2 (p<0.005) and VCAM-1 (p<0.025) in HRMEC. FK-506 inhibited the VEGF-induced upregulation of CXCL2 (p<0.005) and VCAM-1 (p<0.025).
Conclusions:
VEGF induces an elevated intracellular calcium concentration in RMEC. Subsequently, increased calcium activates NFAT, resulting in the translocation of one NFAT isoform to the nucleus and stimulation of its transcription activity. Inhibition of CN/NFAT reduces the VEGF-induced production of proteins and cytokines related to leukostasis. CN and NFAT may be valuable therapeutic targets for treatment of early events in the pathology of diabetic retinopathy.
Keywords: 499 diabetic retinopathy •
739 transcription factors •
439 calcium