Effects of SGLT2 Inhibition on Diabetic Retinopathy

Background and Hypothesis: Diabetic retinopathy (DR), a microvascular complication of diabetes, is the leading cause of blindness in the working-age population, and its prevalence is increasing. New treatment modalities must be developed to slow the progression of DR. SGLT2 inhibition has shown promise in treating other diabetic complications; however, its effect on DR remains unknown, therefore, for this study, we hypothesize that SGLT2 inhibition will reduce the harmful effects of DR. Methods: Diabetic (db/db) mice were fed 10 mg/kg of the SGLT2 inhibitor dapagliflozin in their diet for 6 months, non-diabetic (db/m) mice on a regular diet served as controls. In parallel, human retinal endothelial cells (HREC) were used as in-vitro models and treated with dapagliflozin to assess glucose uptake via a 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose (2-NBDG) assay. Results: Our studies show that db/db mice with dapagliflozin had significantly fewer acellular capillaries compared to untreated db/db mice. Furthermore, Dapagliflozin treatment at 1 and 10 µM concentrations of dapagliflozin yielded a significant decrease in glucose uptake compared to respective vehicle controls. Conclusion: Our study shows that SGLT2 inhibition has a promise in treating DR by reducing acellular capillaries and retinal glucose transport suggesting the potential of dapagliflozin treatment in DR.

Reduced Levels of Drp1 Protect against Development of Retinal Vascular Lesions in Diabetic Retinopathy

High glucose (HG)-induced Drp1 overexpression contributes to mitochondrial dysfunction and promotes apoptosis in retinal endothelial cells. However, it is unknown whether inhibiting Drp1 overexpression protects against the development of retinal vascular cell loss in diabetes. To investigate whether reduced Drp1 level is protective against diabetes-induced retinal vascular lesions, four groups of mice: wild type (WT) control mice, streptozotocin (STZ)-induced diabetic mice, Drp1+/− mice, and STZ-induced diabetic Drp1+/− mice were examined after 16 weeks of diabetes. Western Blot analysis indicated a significant increase in Drp1 expression in the diabetic retinas compared to those of WT mice; retinas of diabetic Drp1+/− mice showed reduced Drp1 level compared to those of diabetic mice. A significant increase in the number of acellular capillaries (AC) and pericyte loss (PL) was observed in the retinas of diabetic mice compared to those of the WT control mice. Importantly, a significant decrease in the number of AC and PL was observed in retinas of diabetic Drp1+/− mice compared to those of diabetic mice concomitant with increased expression of pro-apoptotic genes, Bax, cleaved PARP, and increased cleaved caspase-3 activity. Preventing diabetes-induced Drp1 overexpression may have protective effects against the development of vascular lesions, characteristic of diabetic retinopathy.

Opa1 Deficiency Promotes Development of Retinal Vascular Lesions in Diabetic Retinopathy

This study investigates whether reduced optic atrophy 1 (Opa1) level promotes apoptosis and retinal vascular lesions associated with diabetic retinopathy (DR). Four groups of mice: wild type (WT) control mice, streptozotocin (STZ)-induced diabetic mice, Opa1+/− mice, and diabetic Opa1+/− mice were used in this study. 16 weeks after diabetes onset, retinas were assessed for Opa1 and Bax levels by Western blot analysis, and retinal networks were examined for acellular capillaries (AC) and pericyte loss (PL). Apoptotic cells were detected in retinal capillaries using TUNEL assay, and caspase-3 activity was assessed using fluorometric analysis. Opa1 expression was significantly downregulated in retinas of diabetic and Opa1+/− mice compared with those of WT mice. Inducing diabetes further decreased Opa1 expression in retinas of Opa1+/− mice. Increased cytochrome c release concomitant with increased level of pro-apoptotic Bax and elevated caspase-3 activity were observed in retinas of diabetic and Opa1+/− mice; the number of TUNEL-positive cells and AC/PL was also significantly increased. An additional decrease in the Opa1 level in retinas of diabetic Opa1+/− mice exacerbated the development of apoptotic cells and AC/PL compared with those of diabetic mice. Diabetes-induced Opa1 downregulation contributes, at least in part, to the development of retinal vascular lesions characteristic of DR.

Microglial Activation Is Associated With Vasoprotection in a Rat Model of Inflammatory Retinal Vasoregression

Vascular dysfunction and vasoregression are hallmarks of a variety of inflammatory central nervous system disorders and inflammation-related retinal diseases like diabetic retinopathy. Activation of microglia and the humoral innate immune system are contributing factors. Anti-inflammatory approaches have been proposed as therapies for neurovascular diseases, which include the modulation of microglial activation. The present study aimed at investigating the effects of microglial activation by clodronate-coated liposomes on vasoregression in a model of retinal degeneration. Clodronate treatment over 5 weeks led to an increase in activated CD74+ microglia and completely prevented acellular capillaries and pericyte loss. Gene expression analyses indicated that vasoprotection was due to the induction of vasoprotective factors such as Egr1, Stat3, and Ahr while expression of pro-inflammatory genes remained unchanged. We concluded that activated microglia led to a shift toward induction of pleiotropic protective pathways supporting vasoprotection in neurovascular retinal diseases.

Modulation of p75NTR on Mesenchymal Stem Cells Increases Their Vascular Protection in Retinal Ischemia-Reperfusion Mouse Model

Mesenchymal stem cells (MSCs) are a promising therapy to improve vascular repair, yet their role in ischemic retinopathy is not fully understood. The aim of this study is to investigate the impact of modulating the neurotrophin receptor; p75NTR on the vascular protection of MSCs in an acute model of retinal ischemia/reperfusion (I/R). Wild type (WT) and p75NTR-/- mice were subjected to I/R injury by increasing intra-ocular pressure to 120 mmHg for 45 min, followed by perfusion. Murine GFP-labeled MSCs (100,000 cells/eye) were injected intravitreally 2 days post-I/R and vascular homing was assessed 1 week later. Acellular capillaries were counted using trypsin digest 10-days post-I/R. In vitro, MSC-p75NTR was modulated either genetically using siRNA or pharmacologically using the p75NTR modulator; LM11A-31, and conditioned media were co-cultured with human retinal endothelial cells (HREs) to examine the angiogenic response. Finally, visual function in mice undergoing retinal I/R and receiving LM11A-31 was assessed by visual-clue water-maze test. I/R significantly increased the number of acellular capillaries (3.2-Fold) in WT retinas, which was partially ameliorated in p75NTR-/- retinas. GFP-MSCs were successfully incorporated and engrafted into retinal vasculature 1 week post injection and normalized the number of acellular capillaries in p75NTR-/- retinas, yet ischemic WT retinas maintained a 2-Fold increase. Silencing p75NTR on GFP-MSCs coincided with a higher number of cells homing to the ischemic WT retinal vasculature and normalized the number of acellular capillaries when compared to ischemic WT retinas receiving scrambled-GFP-MSCs. In vitro, silencing p75NTR-MSCs enhanced their secretome, as evidenced by significant increases in SDF-1, VEGF and NGF release in MSCs conditioned medium; improved paracrine angiogenic response in HREs, where HREs showed enhanced migration (1.4-Fold) and tube formation (2-Fold) compared to controls. In parallel, modulating MSCs-p75NTR using LM11A-31 resulted in a similar improvement in MSCs secretome and the enhanced paracrine angiogenic potential of HREs. Further, intervention with LM11A-31 significantly mitigated the decline in visual acuity post retinal I/R injury. In conclusion, p75NTR modulation can potentiate the therapeutic potential of MSCs to harness vascular repair in ischemic retinopathy diseases.

Effects of Diabetes on Microcirculation and Leukostasis in Retinal and Non-Ocular Tissues: Implications for Diabetic Retinopathy

Changes in retinal microcirculation are associated with the development of diabetic retinopathy (DR). However, it is unclear whether such changes also develop in capillary beds of other non-retinal tissues. Here, we investigated microcirculatory changes involving velocity of rolling neutrophils, adherence of neutrophils, and leukostasis during development of retinal vascular lesions in diabetes in other non-retinal tissues. Intravital microscopy was performed on post-capillary venules of cremaster muscle and ear lobe of mice with severe or moderate diabetes and compared to those of non-diabetic mice. Additionally, number and velocity of rolling leukocytes, number of adherent leukocytes, and areas of leukostasis were quantified, and retinal capillary networks were examined for acellular capillaries (AC) and pericyte loss (PL), two prominent vascular lesions characteristic of DR. The number of adherent neutrophils and areas of leukostasis in the cremaster and ear lobe post-capillary venules of diabetic mice was increased compared to those of non-diabetic mice. Similarly, a significant increase in the number of rolling neutrophils and decrease in their rolling velocities compared to those of non-diabetic control mice were observed and severity of diabetes exacerbated these changes. Understanding diabetes-induced microcirculatory changes in cremaster and ear lobe may provide insight into retinal vascular lesion development in DR.

Patrolling monocytes are recruited and activated by diabetes to protect retinal microvessels

In diabetes there is a long latency between onset of hyperglycemia and appearance of structural microangiopathy. Because Ly6C^low patrolling monocytes (PMo) behave as housekeepers of the vasculature, we tested whether PMo protect microvessels against diabetes. <p>We found that, in wild-type mice,<b> </b>diabetes reduced PMo in the general circulation but increased by 4-fold the absolute number of PMo adherent to retinal vessels (leukostasis). Conversely, in diabetic NR4A1^-/- mice ─ a model of absence of PMo ─ there was no increase in leukostasis at all; and at 6 months of diabetes the number of retinal acellular capillaries almost doubled when compared to diabetic wild-type mice. Circulating PMo showed gene expression changes indicative of enhanced migratory, vasculo-protective, and housekeeping activities; as well as profound suppression of genes related to inflammation and apoptosis. Pro-migratory CXCR4 was no longer upregulated at longer duration, when retinal acellular capillaries begin to increase.</p> <p>Thus, after short diabetes duration, PMo are the cells preferentially recruited to the retinal vessels and protect vessels from diabetic damage. These observations support the need for reinterpretation of the functional meaning of leukostasis in diabetes, and document within the natural history of diabetic retinopathy processes of protection-repair that can provide novel paradigms for prevention. </p>

Patrolling monocytes are recruited and activated by diabetes to protect retinal microvessels

In diabetes there is a long latency between onset of hyperglycemia and appearance of structural microangiopathy. Because Ly6C^low patrolling monocytes (PMo) behave as housekeepers of the vasculature, we tested whether PMo protect microvessels against diabetes. <p>We found that, in wild-type mice,<b> </b>diabetes reduced PMo in the general circulation but increased by 4-fold the absolute number of PMo adherent to retinal vessels (leukostasis). Conversely, in diabetic NR4A1^-/- mice ─ a model of absence of PMo ─ there was no increase in leukostasis at all; and at 6 months of diabetes the number of retinal acellular capillaries almost doubled when compared to diabetic wild-type mice. Circulating PMo showed gene expression changes indicative of enhanced migratory, vasculo-protective, and housekeeping activities; as well as profound suppression of genes related to inflammation and apoptosis. Pro-migratory CXCR4 was no longer upregulated at longer duration, when retinal acellular capillaries begin to increase.</p> <p>Thus, after short diabetes duration, PMo are the cells preferentially recruited to the retinal vessels and protect vessels from diabetic damage. These observations support the need for reinterpretation of the functional meaning of leukostasis in diabetes, and document within the natural history of diabetic retinopathy processes of protection-repair that can provide novel paradigms for prevention. </p>

Characterizing the Retinal Phenotype in the High-Fat Diet and Western Diet Mouse Models of Prediabetes

We sought to delineate the retinal features associated with the high-fat diet (HFD) mouse, a widely used model of obesity. C57BL/6 mice were fed either a high-fat (60% fat; HFD) or low-fat (10% fat; LFD) diet for up to 12 months. The effect of HFD on body weight and insulin resistance were measured. The retina was assessed by electroretinogram (ERG), fundus photography, permeability studies, and trypsin digests for enumeration of acellular capillaries. The HFD cohort experienced hypercholesterolemia when compared to the LFD cohort, but not hyperglycemia. HFD mice developed a higher body weight (60.33 g vs. 30.17g, p < 0.0001) as well as a reduced insulin sensitivity index (9.418 vs. 62.01, p = 0.0002) compared to LFD controls. At 6 months, retinal functional testing demonstrated a reduction in a-wave and b-wave amplitudes. At 12 months, mice on HFD showed evidence of increased retinal nerve infarcts and vascular leakage, reduced vascular density, but no increase in number of acellular capillaries compared to LFD mice. In conclusion, the HFD mouse is a useful model for examining the effect of prediabetes and hypercholesterolemia on the retina. The HFD-induced changes appear to occur slower than those observed in type 2 diabetes (T2D) models but are consistent with other retinopathy models, showing neural damage prior to vascular changes.