Critical role of endoplasmic reticulum stress in chronic endothelial activation-induced visual deficits in tie2-tumor necrosis factor mice.

Diabetic retinopathy (DR) is the leading cause of vision loss among working-age adults. The interplay between hyperglycemia and endothelial activation in inducing endoplasmic reticulum (ER) stress pathways and visual deficits in DR is not fully understood. To address this, we used a mouse model of chronic vascular activation using endothelial-specific tumor necrosis factor-α (TNF-α)-expressing (tie2-TNF) mice to induce diabetes with streptozotocin. At 4 weeks post streptozotocin, a significant 2-fold to 10-fold increase in retinal neurovascular inflammatory gene transcript response in tie2-TNF mice was further increased in diabetic tie2-TNF mice. A decrease in visual acuity and scotopic b-wave amplitude in tie2-TNF mice was further accentuated in diabetic tie2-TNF mice and these changes correlated with a multi-fold increase in retinal ER stress markers and a reduction in adherens junctions. Cultured retinal endothelial cells showed a significant decrease in trans-endothelial resistance as well as VE-cadherin expression under TNF-α and high glucose stress. These changes were partly rescued by tauroursodeoxycholic acid, a potent ER stress inhibitor. Taken together, constant endothelial activation induced by TNF-α further exacerbated by hyperglycemia results in activation of ER stress and chronic proinflammation in a feed forward loop ultimately resulting in endothelial junction protein alterations leading to visual deficits in the retina. Inhibition of ER stress and endothelial activation may prove to be a novel therapeutic target in DR.

GRP78 translocation to the cell surface and O-GlcNAcylation of VE-Cadherin contribute to ER stress-mediated endothelial permeability.

Increased O-GlcNAcylation, a well-known post-translational modification of proteins causally linked to various detrimental cellular functions in pathological conditions including diabetic retinopathy (DR). Previously we have shown that endothelial activation induced by inflammation and hyperglycemia results in the endoplasmic reticulum (ER) stress-mediated intercellular junction alterations accompanied by visual deficits in a tie2-TNF-α transgenic mouse model. In this study, we tested the hypothesis that increased ER stress via O-GlcNAcylation of VE-Cadherin likely contribute to endothelial permeability. We show that ER stress leads to GRP78 translocation to the plasma membrane, increased O-GlcNAcylation of proteins, particularly VE-Cadherin resulting in a defective complex partnering leading to the loss of retinal endothelial barrier integrity and increased transendothelial migration of monocytes. We further show an association of GRP78 with the VE-Cadherin under these conditions. Interestingly, cells exposed to ER stress inhibitor, tauroursodeoxycholic acid partially mitigated all these effects. Our findings suggest an essential role for ER stress and O-GlcNAcylation in altering the endothelial barrier function and reveal a potential therapeutic target in the treatment of DR.

Featured Article: Deterioration of visual function mediated by senescence-associated endoplasmic reticulum stress in inflammatory tie2-TNF mice.

Stress-associated premature senescence plays a major role in retinal diseases. In this study, we investigated the relationship between endothelial dysfunction, endoplasmic reticulum (ER) stress, and cellular senescence in the development of retinal dysfunction. We tested the hypothesis that constant endothelial activation by transmembrane tumor necrosis factor-α (tmTNF-α) exacerbates age-induced visual deficits via senescence-mediated ER stress in this model. To address this, we employed a mouse model of chronic vascular activation using endothelial-specific TNF-α-expressing (tie2-TNF) mice at 5 and 10 months of age. Visual deficits were exhibited by tie2-TNF mice at both 5 months and 10 months of age, with the older mice showing statistically significant loss of visual acuity compared with tie2-TNF mice at age 5 months. The neural defects, as measured by electroretinogram (ERG), also followed a similar trend in an age-dependent fashion, with 10-month-old tie2-TNF mice showing the greatest decrease in "b" wave amplitude at 25 cd.s.m2 compared with age-matched wildtype (WT) mice and five-month-old tie2-TNF mice. While gene and protein expression from the whole retinal extracts demonstrated increased inflammatory (Icam1, Ccl2), stress-associated premature senescence (p16, p21, p53), and ER stress (Grp78, p-Ire1α, Chop) markers in five-month-old tie2-TNF mice compared with five-month-old WT mice, a further increase was seen in 10-month-old tie2-TNF mice. Our data demonstrate that tie2-TNF mice exhibit age-associated increases in visual deficits, and these data suggest that inflammatory endothelial activation is at least partly at play. Given the correlation of increased premature senescence and ER stress in an age-dependent fashion, with the loss of visual functions and increased endothelial activation, our data suggest a possible self-enhanced loop of unfolded protein response pathways and senescence in propagating neurovascular defects in this model. Impact statement Vision loss in most retinal diseases affects the quality of life of working age adults. Using a novel animal model that displays constant endothelial activation by tmTNF-α, our results demonstrate exacerbated age-induced visual deficits via premature senescence-mediated ER stress. We have compared mice of 5 and 10 months of age, with highly relevant human equivalencies of approximately 35- and 50-year-old patients, representing mature adult and middle-aged subjects, respectively. Our studies suggest a possible role for a self-enhanced loop of ER stress pathways and senescence in the propagation of retinal neurovascular defects, under conditions of constant endothelial activation induced by tmTNF-α signaling.