Essential Role of Transglutaminase 2 in Vascular Endothelial Growth Factor–Induced Vascular Leakage in the Retina of Diabetic Mice

Abstract

Diabetic retinopathy is predominantly caused by vascular endothelial growth factor (VEGF)–induced vascular leakage; however, the underlying mechanism is unclear. Here we designed an in vivo transglutaminase (TGase) activity assay in mouse retina and demonstrated that hyperglycemia induced vascular leakage by activating TGase2 in diabetic retina. VEGF elevated TGase2 activity through sequential elevation of intracellular Ca2+ and reactive oxygen species (ROS) concentrations in endothelial cells. The TGase inhibitors cystamine and monodansylcadaverin or TGase2 small interfering RNA (siRNA) prevented VEGF-induced stress fiber formation and vascular endothelial (VE)–cadherin disruption, which play a critical role in modulating endothelial permeability. Intravitreal injection of two TGase inhibitors or TGase2 siRNA successfully inhibited hyperglycemia-induced TGase activation and microvascular leakage in the retinas of diabetic mice. C-peptide or ROS scavengers also inhibited TGase activation in diabetic mouse retinas. The role of TGase2 in VEGF-induced vascular leakage was further supported using diabetic TGase2−/− mice. Thus, our findings suggest that ROS-mediated activation of TGase2 plays a key role in VEGF-induced vascular leakage by stimulating stress fiber formation and VE-cadherin disruption.

The benzodiazepine anesthetic midazolam prevents hyperglycemia‐induced microvascular leakage in the retinas of diabetic mice

“…Because midazolam can cross the blood-brain barrier (36), some intravitreally injected midazolam is expected to pass through the blood-retina barrier into the blood stream. In the present study, the amount of intravitreally injected midazolam was ;0.072 mg/kg, estimated by the weight of the diabetic mice (12). Mice intravitreally injected with the dose of midazolam did not show any differences in behavior, including breathing and sleeping, food consumption, or water intake, from the behaviors of the control mice.…”

“…HRECs were fixed, permeabilized, and incubated overnight with a VE-cadherin mAb (1:200; Santa Cruz Biotechnology, Dallas, TX, USA). Cells were then probed with FITC-conjugated goat anti-mouse antibody (1:200; MilliporeSigma, Billerica, MA, USA) and visualized using confocal microscopy (Fluoview-300; Olympus) (12). VE-cadherin is represented by histograms and was quantitatively analyzed using peak fluorescence intensities of the histograms at the single-cell level for 10 cells/ experiment (randomly selected).…”

“…In vitro permeability assay was performed, as previously described by Mohler et al (12). Briefly, HRECs were grown on gelatin-coated inserts (0.4-mm polycarbonate membranes) of Transwell Permeable Supports (CoStar Group, Washington DC, USA) up to confluence.…”

“…In vivo TGase transamidating activity in mouse retina was determined by confocal microscopy (12). Briefly, mice were deeply anesthetized with 2.5% avertin, and 48 ml of 100 mM 5-(biotinamido)pentylamine was injected into the left ventricle and allowed to circulate for 10 min.…”

“…VEGF induces stress fiber formation and vascular endothelial (VE)-cadherin disruption and internalization, which result in adherens junction disassembly and vascular leakage in diabetic retinas (9)(10)(11). Recently, we demonstrated that reactive oxygen species (ROS)-mediated activation of transglutaminase (TGase)-2 has a key role in VEGF-induced retinal vascular leakage in diabetic mice (12). Although various drugs against VEGF, oxidative stress, and inflammation have been evaluated for the treatment of DR, there is strong demand for longer-acting and noninvasive therapies with minimal complications to prevent and treat diabetic retinal vascular disease (13,14).…”