Abstract
Purpose :
Diabetic Retinopathy (DR) is characterized by inflammation and microvascular abnormalities leading to vision loss. Muller cells (MC) are a major source of cytokines whose vitreous elevation correlates with DR progression. We previously showed that, after direct stimulation of human retinal microvascular endothelial cells (hRMEC) with IL1β, TNFα, IL8, and IL6, only TNFα and IL1β significantly affected cytokine auto-amplification and cell adhesion molecule expression in hRMEC. Now, we explore paracrine effects of inflammatory cytokine-stimulated human MC (hMC) on NF-kB translocation, and expression of inflammatory cytokines and cell adhesion molecules in hRMEC.
Methods :
Primary hMC were stimulated with IL1β, TNFα, IL8, and IL6 vs vehicle for 2 hours and the media replaced. 6 hrs after replacement, hMC-conditioned media was collected. hRMEC were stimulated directly with 1ng/mL of IL1β, TNFα, IL8, and IL6 vs vehicle, or with conditioned media from stimulated hMC. hRMEC were fixed after 30 min stimulation and assayed by immunocytochemistry for NF-KB P65 nuclear translocation. hRMEC were lysed 4hr after stimulation with hMC-conditioned media, mRNA isolated, and cDNA generated and subjected to qRT-PCR with IL1β, TNFα, CXCL8/IL8, IL6, ICAM, VCAM, SELE versus TBP (housekeeping control) TaqMan probes.
Results :
NFkB nuclear translocation was observed after direct hRMEC stimulation with either TNFα or IL1β (P<0.0001) but only after hRMEC treatment with conditioned media from IL1β- (and not TNFα, IL8 or IL6-) stimulated hMC (P<0.0001). Likewise, only conditioned media from IL1β-stimulated hMC significantly increased hRMEC expression of IL1β, TNFα, CXCL8/IL8, and IL6 by 31.2, 17.9, 10.4 and 8.8 fold, respectively (P<0.0001) and of ICAM, VCAM, SELE by 8, 200, and 70 fold, respectively (P< 0.001). IL6 and IL8 stimulation did not significantly affect any outcomes measured.
Conclusions :
We observed NFkB nuclear translocation and increased expression of IL1β, TNFα, IL8/CXCL8, IL6, ICAM, VCAM, and E-SELECTIN in hRMEC only after direct TNFα and IL1β stimulation or after stimulation with conditioned media from IL1β-stimulated hMC. This data suggests that NFkB activation may be involved in hRMEC pro-inflammatory transcriptome alterations and that IL1β may be a key regulator of inflammatory amplification and a valuable target for early DR therapies.
This is a 2021 ARVO Annual Meeting abstract.