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Vitreous fibrovascular membranes (FVMs), the hallmark of proliferative diabetic retinopathy (PDR), cause retinal hemorrhage, detachment, and eventually blindness. However, little is known about the pathophysiology of FVM. In this study, we used single-cell RNA sequencing on surgically harvested PDR-FVMs and generated a comprehensive cell atlas of FVM. Eight cellular compositions were identified, with microglia as the major cell population. We identified a GPNMB+ subpopulation of microglia, which presented both profibrotic and fibrogenic properties. Pseudotime analysis further revealed the profibrotic microglia was uniquely differentiated from retina-resident microglia and expanded in the PDR setting. Ligand-receptor interactions between the profibrotic microglia and cytokines upregulated in PDR vitreous implicated the involvement of several pathways, including CCR5, IFNGR1, and CD44 signaling, in the microglial activation within the PDR microenvironment. Collectively, our description of the novel microglia phenotypes in PDR-FVM may offer new insight into the cellular and molecular mechanism underlying the pathogenesis of DR, as well as potential signaling pathways amenable to disease-specific intervention. [ABSTRACT FROM AUTHOR]

In the endoplasmic reticulum (ER), the translocation-associated protein complex (TRAP), also called signal sequence receptor (SSR), includes four integral membrane proteins TRAPα/SSR1, TRAPβ/SSR2, and TRAPδ/SSR4 with the bulk of their extramembranous portions primarily in the ER lumen, whereas the extramembranous portion of TRAPγ/SSR3 is primarily cytosolic. Individually diminished expression of either TRAPα/SSR1, TRAPβ/SSR2, or TRAPδ/SSR4 mRNA is known in each case to lower TRAPα/SSR1 protein levels, leading to impaired proinsulin biosynthesis, whereas forced expression of TRAPα/SSR1 at least partially suppresses the proinsulin biosynthetic defect. Here, we report that diminished TRAPγ/SSR3 expression in pancreatic β-cells leaves TRAPα/SSR1 levels unaffected while nevertheless inhibiting cotranslational and posttranslational translocation of preproinsulin into the ER. Crucially, acute exposure to high glucose leads to a rapid upregulation of both TRAPγ/SSR3 and proinsulin protein without change in the respective mRNA levels, as observed in cultured rodent β-cell lines and confirmed in human islets. Strikingly, pancreatic β-cells with suppressed TRAPγ/SSR3 expression are blocked in glucose-dependent upregulation of proinsulin (or insulin) biosynthesis. Most remarkably, overexpression of TRAPγ/SSR3 in control β-cells raises proinsulin levels, even without boosting extracellular glucose. The data suggest the possibility that TRAPγ/SSR3 may fulfill a rate-limiting function in preproinsulin translocation across the ER membrane for proinsulin biosynthesis. [ABSTRACT FROM AUTHOR]