Purpose: Previously, we demonstrated that ACE2 deficiency increases the severity of DR in Akita mice. In this study, we sought to determine the mechanism where by the gut microbiome, which is typically altered in T1D, contributes to the pathogenesis of DR in ACE2-/-Akita mice.
Methods: Wild type (WT), Akita and ACE2-/-Akita mice were studied at 9 months of diabetes and the gut microbiota was examined using 16S rRNA sequencing and metatranscriptomic analysis. Microbial load in the circulation was assessed by measuring circulating PGN levels along with markers of the gut vascular barrier (GVB). Next, human retinal endothelial cells (HREC) were treated with PGN, a known Toll-like receptor (TLR)-2 agonist, to determine whether PGN enhances permeability.
Results: 16S rRNA sequencing revealed a shift in beta diversity of the gut microbiota with an increase in Firmicutes and Bacteroidetes within the gut of ACE2-/-Akita mice. Metatranscriptomic analysis showed increases in PGN synthesis. Loss of ACE2 promoted gut barrier permeability through decreasing the turnover of VE-cadherin expression (41%, p<0.0001) in the intestine of ACE2-/-Akita mice. Increased vascular permeability was associated with barrier dysfunction, microbial translocation and increase PGN in the circulation (2.5 fold, p<0.007) in ACE2-/-Akita mice compared with Akita mice. Acellular capillaries (2 fold, p<0.05) were significantly higher in ACE2-/-Akita mice compared with Akita mice. PGN treatment caused dissociation of VE-cadherin junctional complexes in HRECs through a MyD88/ARNO/ARF6-mediated signaling pathway. This occurred by depleting p120-catenin (51%. p<0.004) and internalization of VE-cadherin.
Conclusions: These data suggest that microbiota-derived PGN has deleterious effects on retinal endothelial cells to increasing permeability. Strategies aimed at resorting gut health may serve to prevent development of DR.
R. Prasad: None. Y. Duan: None. J.L. Floyd: None. M.B. Grant: None.
National Eye Institute (EY028858)