AZD-3965

Maintaining barrier integrity, along with cell adhesion to the extracellular matrix and the subsequent process of cell spreading, are essential functions of endothelial cells, including human retinal endothelial cells (HRECs). Disruptions in these processes can lead to vision-threatening conditions like diabetic retinopathy. However, the bioenergetic mechanisms that regulate HREC barrier function and cell spreading remain incompletely understood. This study investigates the role of lower glycolytic components in modulating these critical functions of HRECs. In vitro, Electric Cell-Substrate Impedance Sensing (ECIS) technology was used to measure real-time changes in HREC barrier integrity (electrical resistance) and cell spreading (capacitance). Pharmacological inhibitors targeting lower glycolytic components were tested: heptelidic acid for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), NG-52 for phosphoglycerate kinase (PGK), shikonin for pyruvate kinase M (PKM), galloflavin for lactate dehydrogenase (LDH), AZD3965 for lactate transporter (MCT1), and MSDC-0160 for the mitochondrial pyruvate carrier (MPC). GAPDH knockdown was performed using siRNA, and cell viability was assessed via LDH release assays. For in vivo studies, wild-type C57BL/6J mice received intravitreal injections of heptelidic acid, while control mice received the vehicle (dimethyl sulfoxide). Retinal vascular permeability was assessed by fluorescein angiography (FA) and retinal albumin leakage. The most significant decrease in electrical resistance and increase in capacitance of HRECs were observed following the dose-dependent inhibition of GAPDH and the resulting reduction in 1,3-bisphosphoglycerate (1,3-BPG) and NADH by heptelidic acid. LDH level analysis at 24 to 48 h post-treatment with heptelidic acid (1 and 10 μM) showed no significant difference compared to controls, indicating that the observed disruption of HREC functionality was not due to cell death. Supporting these findings, inhibition of downstream glycolytic steps that result in the accumulation of 1,3-BPG and NADH, such as treatment with NG-52 for PGK or shikonin for PKM, led to a significant increase in electrical resistance and a decrease in cell capacitance. Furthermore, GAPDH knockdown via siRNA also led to a significant decrease in cellular resistance in HRECs. In vivo, FA imaging demonstrated that intravitreal injection of heptelidic acid led to significant retinal vascular leakage, as further supported by increased albumin extravasation in treated eyes. Conversely, pharmacological inhibition of other lower glycolytic components, including LDH, MCT, and MPC, did not significantly alter HREC barrier function or spreading behavior. This study highlights the distinct roles of lower glycolytic components in regulating HREC functionality. GAPDH and its downstream products (1,3-BPG and NADH) are shown to play a pivotal role in maintaining barrier integrity and promoting HREC adhesion and spreading. These findings guide the development of targeted interventions that modulate HREC bioenergetics to treat endothelial dysfunction in various retinal disorders, while minimizing potential adverse effects on healthy endothelial cells.

During acute pancreatitis (AP), glycolysis is enhanced. The upregulation of glycolysis increases the level of metabolite lactate. Lactate has been shown to facilitate tissue repair across various pathological conditions. However, its role in the recovery following AP remains unclear. This study aims to explore the role of lactate in the regenerative processes following AP and to elucidate its underlying molecular mechanisms.

The caerulein-induced recovery AP model was established using wild type and 6-Phosphofructo-2-Kinase/Fructose-2,6-Biphosphatase 3 (Pfkfb3) heterozygous mice. Pancreatic repair was evaluated histologically, while lactate levels and inflammatory markers were measured serologically. Macrophages were isolated from pancreatic tissue using fluorescence-activated cell sorting for mRNA sequencing to identify phenotypes. In ex vivo, macrophages were indirectly co-cultured with inflammatory acinar, and the effect of lactate on macrophage phenotype were investigated through immunoprecipitation, fluorescence analysis, and western blotting.

We first found that exogenous lactate administration promoted pancreatic repair, while Pfkfb3 deficiency lowered lactate levels and ultimately delayed pancreatic repair. Mechanistically, lactate altered macrophage phenotype during recovery after AP, by reducing the proportion of pro-inflammatory macrophages and increasing the percentage of reparative macrophages. In the indirectly co-cultured macrophage, lactate increased lactylation levels and enhanced repair gene expression. Treatment with AZD3965, a chemical inhibitor of lactate transportation, blocked the effects on lactylation and gene expression. Besides, lactate repressed the JAK2-STAT1 pathway via GPR132 receptor, thereby suppressing the expression of pro-inflammatory genes.

Lactate facilitates pancreatic repair by promoting reparative macrophage polarization, achieved through promoting lactylation and inhibiting JAK2-STAT1 signaling. This phenotypic shift alleviates inflammation and facilitates tissue recovery, highlighting a potential therapeutic approach for AP.