Inhibition of the eukaryotic initiation factor-2-α kinase PERK decreases risk of autoimmune diabetes in mice.

Preventing the onset of autoimmune type 1 diabetes (T1D) is feasible through pharmacological interventions that target molecular stress-responsive mechanisms. Cellular stresses, such as nutrient deficiency, viral infection, or unfolded proteins, trigger the integrated stress response (ISR), which curtails protein synthesis by phosphorylating eIF2α. In T1D, maladaptive unfolded protein response (UPR) in insulin-producing beta cells renders these cells susceptible to autoimmunity. We found that inhibition of the eIF2α kinase PERK, a common component of the UPR and ISR, reversed the mRNA translation block in stressed human islets and delayed the onset of diabetes, reduced islet inflammation, and preserved ß cell mass in T1D-susceptible mice. Single-cell RNA sequencing of islets from PERK-inhibited mice showed reductions in the UPR and PERK signaling pathways and alterations in antigen processing and presentation pathways in ß cells. Spatial proteomics of islets from these mice showed an increase in the immune checkpoint protein PD-L1 in ß cells. Golgi membrane protein 1, whose levels increased following PERK inhibition in human islets and EndoC-ßH1 human ß cells, interacted with and stabilized PD-L1. Collectively, our studies show that PERK activity enhances ß cell immunogenicity, and inhibition of PERK may offer a strategy to prevent or delay the development of T1D.

Inhibition of the Eukaryotic Initiation Factor-2-α Kinase PERK Decreases Risk of Autoimmune Diabetes in Mice.

Preventing the onset of autoimmune type 1 diabetes (T1D) is feasible through pharmacological interventions that target molecular stress-responsive mechanisms. Cellular stresses, such as nutrient deficiency, viral infection, or unfolded proteins, trigger the integrated stress response (ISR), which curtails protein synthesis by phosphorylating eIF2α. In T1D, maladaptive unfolded protein response (UPR) in insulin-producing ß cells renders these cells susceptible to autoimmunity. We show that inhibition of the eIF2α kinase PERK, a common component of the UPR and ISR, reverses the mRNA translation block in stressed human islets and delays the onset of diabetes, reduces islet inflammation, and preserves ß cell mass in T1D-susceptible mice. Single-cell RNA sequencing of islets from PERK-inhibited mice shows reductions in the UPR and PERK signaling pathways and alterations in antigen processing and presentation pathways in ß cells. Spatial proteomics of islets from these mice shows an increase in the immune checkpoint protein PD-L1 in ß cells. Golgi membrane protein 1, whose levels increase following PERK inhibition in human islets and EndoC-ßH1 human ß cells, interacts with and stabilizes PD-L1. Collectively, our studies show that PERK activity enhances ß cell immunogenicity, and inhibition of PERK may offer a strategy to prevent or delay the development of T1D.

Plasma Exosomes Contribute to Microvascular Damage in Diabetic Retinopathy by Activating the Classical Complement Pathway.

Diabetic retinopathy (DR) is a microvascular complication of diabetes and is the leading cause of vision loss in working-age adults. Recent studies have implicated the complement system as a player in the development of vascular damage and progression of DR. However, the role and activation of the complement system in DR are not well understood. Exosomes, small vesicles that are secreted into the extracellular environment, have a cargo of complement proteins in plasma, suggesting that they can participate in causing the vascular damage associated with DR. We demonstrate that IgG-laden exosomes in plasma activate the classical complement pathway and that the quantity of these exosomes is increased in diabetes. Moreover, we show that a lack of IgG in exosomes in diabetic mice results in a reduction in retinal vascular damage. The results of this study demonstrate that complement activation by IgG-laden plasma exosomes could contribute to the development of DR.

ELOVL4-Mediated Production of Very Long-Chain Ceramides Stabilizes Tight Junctions and Prevents Diabetes-Induced Retinal Vascular Permeability.

Tight junctions (TJs) involve close apposition of transmembrane proteins between cells. Although TJ proteins have been studied in detail, the role of lipids is largely unknown. We addressed the role of very long-chain (VLC ≥26) ceramides in TJs using diabetes-induced loss of the blood-retinal barrier as a model. VLC fatty acids that incorporate into VLC ceramides are produced by elongase elongation of very long-chain fatty acids protein 4 (ELOVL4). ELOVL4 is significantly reduced in the diabetic retina. Overexpression of ELOVL4 significantly decreased basal permeability, inhibited vascular endothelial growth factor (VEGF)- and interleukin-1ß-induced permeability, and prevented VEGF-induced decrease in occludin expression and border staining of TJ proteins ZO-1 and claudin-5. Intravitreal delivery of AAV2-hELOVL4 reduced diabetes-induced increase in vascular permeability. Ultrastructure and lipidomic analysis revealed that ω-linked acyl-VLC ceramides colocalize with TJ complexes. Overall, normalization of retinal ELOVL4 expression could prevent blood-retinal barrier dysregulation in diabetic retinopathy through an increase in VLC ceramides and stabilization of TJs.

Come to the Light Side: In Vivo Monitoring of Pseudomonas aeruginosa Biofilm Infections in Chronic Wounds in a Diabetic Hairless Murine Model.

The presence of bacteria as structured biofilms in chronic wounds, especially in diabetic patients, is thought to prevent wound healing and resolution. Chronic mouse wounds models have been used to understand the underlying interactions between the microorganisms and the host. The models developed to date rely on the use of haired animals and terminal collection of wound tissue for determination of viable bacteria. While significant insight has been gained with these models, this experimental procedure requires a large number of animals and sampling is time consuming. We have developed a novel murine model that incorporates several optimal innovations to evaluate biofilm progression in chronic wounds: a) it utilizes hairless mice, eliminating the need for hair removal; b) applies pre-formed biofilms to the wounds allowing for the immediate evaluation of persistence and effect of these communities on host; c) monitors biofilm progression by quantifying light production by a genetically engineered bioluminescent strain of Pseudomonas aeruginosa, allowing real-time monitoring of the infection thus reducing the number of animals required per study. In this model, a single full-depth wound is produced on the back of STZ-induced diabetic hairless mice and inoculated with biofilms of the P. aeruginosa bioluminescent strain Xen 41. Light output from the wounds is recorded daily in an in vivo imaging system, allowing for in vivo and in situ rapid biofilm visualization and localization of biofilm bacteria within the wounds. This novel method is flexible as it can be used to study other microorganisms, including genetically engineered species and multi-species biofilms, and may be of special value in testing anti-biofilm strategies including antimicrobial occlusive dressings.

Increase in acid sphingomyelinase level in human retinal endothelial cells and CD34+ circulating angiogenic cells isolated from diabetic individuals is associated with dysfunctional retinal vasculature and vascular repair process in diabetes.

BACKGROUND: Diabetic retinopathy is a microvascular disease that results from retinal vascular degeneration and defective repair due to diabetes-induced endothelial progenitor dysfunction. OBJECTIVE: Understanding key molecular factors involved in vascular degeneration and repair is paramount for developing effective diabetic retinopathy treatment strategies. We propose that diabetes-induced activation of acid sphingomyelinase (ASM) plays essential role in retinal endothelial and CD34+ circulating angiogenic cell (CAC) dysfunction in diabetes. METHODS: Human retinal endothelial cells (HRECs) isolated from control and diabetic donor tissue and human CD34+ CACs from control and diabetic patients were used in this study. ASM messenger RNA and protein expression were assessed by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. To evaluate the effect of diabetes-induced ASM on HRECs and CD34+ CACs function, tube formation, CAC incorporation into endothelial tubes, and diurnal release of CD34+ CACs in diabetic individuals were determined. RESULTS: ASM expression level was significantly increased in HRECs isolated from diabetic compared with control donor tissue, as well as CD34+ CACs and plasma of diabetic patients. A significant decrease in tube area was observed in HRECs from diabetic donors compared with control HRECs. The tube formation deficiency was associated with increased expression of ASM in diabetic HRECs. Moreover, diabetic CD34+ CACs with high ASM showed defective incorporation into endothelial tubes. Diurnal release of CD34+ CACs was disrupted with the rhythmicity lost in diabetic patients. CONCLUSION: Collectively, these findings support that diabetes-induced ASM upregulation has a marked detrimental effect on both retinal endothelial cells and CACs.

Dual Anti-Inflammatory and Anti-Angiogenic Action of miR-15a in Diabetic Retinopathy.

Activation of pro-inflammatory and pro-angiogenic pathways in the retina and the bone marrow contributes to pathogenesis of diabetic retinopathy. We identified miR-15a as key regulator of both pro-inflammatory and pro-angiogenic pathways through direct binding and inhibition of the central enzyme in the sphingolipid metabolism, ASM, and the pro-angiogenic growth factor, VEGF-A. miR-15a was downregulated in diabetic retina and bone marrow cells. Over-expression of miR-15a downregulated, and inhibition of miR-15a upregulated ASM and VEGF-A expression in retinal cells. In addition to retinal effects, migration and retinal vascular repair function was impaired in miR-15a inhibitor-treated circulating angiogenic cells (CAC). Diabetic mice overexpressing miR-15a under Tie-2 promoter had normalized retinal permeability compared to wild type littermates. Importantly, miR-15a overexpression led to modulation toward nondiabetic levels, rather than complete inhibition of ASM and VEGF-A providing therapeutic effect without detrimental consequences of ASM and VEGF-A deficiencies.

Imbalances in Mobilization and Activation of Pro-Inflammatory and Vascular Reparative Bone Marrow-Derived Cells in Diabetic Retinopathy.

Diabetic retinopathy is a sight-threatening complication of diabetes, affecting 65% of patients after 10 years of the disease. Diabetic metabolic insult leads to chronic low-grade inflammation, retinal endothelial cell loss and inadequate vascular repair. This is partly due to bone marrow (BM) pathology leading to increased activity of BM-derived pro-inflammatory monocytes and impaired function of BM-derived reparative circulating angiogenic cells (CACs). We propose that diabetes has a significant long-term effect on the nature and proportion of BM-derived cells that circulate in the blood, localize to the retina and home back to their BM niche. Using a streptozotocin mouse model of diabetic retinopathy with GFP BM-transplantation, we have demonstrated that BM-derived circulating pro-inflammatory monocytes are increased in diabetes while reparative CACs are trapped in the BM and spleen, with impaired release into circulation. Diabetes also alters activation of splenocytes and BM-derived dendritic cells in response to LPS stimulation. A majority of the BM-derived GFP cells that migrate to the retina express microglial markers, while others express endothelial, pericyte and Müller cell markers. Diabetes significantly increases infiltration of BM-derived microglia in an activated state, while reducing infiltration of BM-derived endothelial progenitor cells in the retina. Further, control CACs injected into the vitreous are very efficient at migrating back to their BM niche, whereas diabetic CACs have lost this ability, indicating that the in vivo homing efficiency of diabetic CACs is dramatically decreased. Moreover, diabetes causes a significant reduction in expression of specific integrins regulating CAC migration. Collectively, these findings indicate that BM pathology in diabetes could play a role in both increased pro-inflammatory state and inadequate vascular repair contributing to diabetic retinopathy.

Role of Acid Sphingomyelinase in Shifting the Balance Between Proinflammatory and Reparative Bone Marrow Cells in Diabetic Retinopathy.

The metabolic insults associated with diabetes lead to low-grade chronic inflammation, retinal endothelial cell damage, and inadequate vascular repair. This is partly due to the increased activation of bone marrow (BM)-derived proinflammatory monocytes infiltrating the retina, and the compromised function of BM-derived reparative circulating angiogenic cells (CACs), which home to sites of endothelial injury and foster vascular repair. We now propose that a metabolic link leading to activated monocytes and dysfunctional CACs in diabetes involves upregulation of a central enzyme of sphingolipid signaling, acid sphingomyelinase (ASM). Selective inhibition of ASM in the BM prevented diabetes-induced activation of BM-derived microglia-like cells and normalized proinflammatory cytokine levels in the retina. ASM upregulation in diabetic CACs caused accumulation of ceramide on their cell membrane, thereby reducing membrane fluidity and impairing CAC migration. Replacing sphingomyelin with ceramide in synthetic membrane vesicles caused a similar decrease in membrane fluidity. Inhibition of ASM in diabetic CACs improved membrane fluidity and homing of these cells to damaged retinal vessels. Collectively, these findings indicate that selective modulation of sphingolipid metabolism in BM-derived cell populations in diabetes normalizes the reparative/proinflammatory cell balance and can be explored as a novel therapeutic strategy for treating diabetic retinopathy.