Agarwal Mahesh, Chandrakumar Sathishkumar, Tierno Irene Santiago, Lessieur Emma M, Bollinger Zak R, Kern Timothy S, Ghosh Kaustabh
Department of Ophthalmology, University of California, Los Angeles, CA, USA 90095.
Doheny Eye Institute, Pasadena, CA, USA 91103.
bioRxiv. 2025 May 7:2025.05.02.651525. doi: 10.1101/2025.05.02.651525.
Activated neutrophils contribute to retinal endothelial cell (EC) death and capillary degeneration associated with early diabetic retinopathy (DR). However, the factors and mechanisms driving neutrophil activation and cytotoxicity in diabetes remain insufficiently understood. Here we show that lysyl oxidase (LOX), a collagen crosslinking and matrix stiffening enzyme that increases retinal EC susceptibility to activated neutrophils, simultaneously activates neutrophils in its alternate soluble form. Specifically, treatment of diabetic mice with LOX inhibitor β-aminopropionitrile (BAPN) prevented the diabetes-induced increase in neutrophil activation (extracellular release of neutrophil elastase and superoxide) and cytotoxicity towards co-cultured mouse retinal ECs. Mouse neutrophils and differentiated (neutrophil-like) human HL-60 cells treated with recombinant LOX alone exhibited similar activation and cytotoxicity. Mechanistically, this LOX-induced neutrophil activation was associated with biphasic F-actin remodeling, with the initial and rapid (<15 min) F-actin depolymerization followed by a significant increase in F-actin polymerization and polarization. Preventing the initial F-actin depolymerization blocked LOX-induced neutrophil activation and cytotoxicity towards co-cultured retinal ECs. Finally, we show that this biphasic F-actin remodeling is essential for LOX-induced membrane clustering of azurophilic granule marker CD63 and NADPH organizer p47 that are associated with extracellular release of neutrophil elastase and superoxide, respectively. By revealing a causal and previously unrecognized link between LOX and actin-dependent neutrophil activation in diabetes, these findings provide fresh mechanistic insights into the proinflammatory role of LOX in early DR that goes beyond its canonical matrix-stiffening effects.
活化的中性粒细胞会导致与早期糖尿病视网膜病变（DR）相关的视网膜内皮细胞（EC）死亡和毛细血管变性。然而，糖尿病中驱动中性粒细胞活化和细胞毒性的因素及机制仍未得到充分了解。在此我们表明，赖氨酰氧化酶（LOX）是一种参与胶原蛋白交联和基质硬化的酶，它会增加视网膜EC对活化中性粒细胞的易感性，同时以其交替的可溶性形式激活中性粒细胞。具体而言，用LOX抑制剂β-氨基丙腈（BAPN）治疗糖尿病小鼠可预防糖尿病诱导的中性粒细胞活化增加（中性粒细胞弹性蛋白酶和超氧化物的细胞外释放）以及对共培养的小鼠视网膜EC的细胞毒性。单独用重组LOX处理的小鼠中性粒细胞和分化的（类中性粒细胞）人HL-60细胞表现出相似的活化和细胞毒性。从机制上讲，这种由LOX诱导的中性粒细胞活化与双相F-肌动蛋白重塑有关，最初是快速的（<15分钟）F-肌动蛋白解聚，随后是F-肌动蛋白聚合和极化的显著增加。阻止最初的F-肌动蛋白解聚可阻断LOX诱导的中性粒细胞活化以及对共培养的视网膜EC的细胞毒性。最后，我们表明这种双相F-肌动蛋白重塑对于LOX诱导的嗜天青颗粒标志物CD63和NADPH组织者p47的膜聚集至关重要，它们分别与中性粒细胞弹性蛋白酶和超氧化物的细胞外释放有关。通过揭示糖尿病中LOX与肌动蛋白依赖性中性粒细胞活化之间因果关系及此前未被认识的联系，这些发现为LOX在早期DR中的促炎作用提供了新的机制见解，这一作用超出了其经典的基质硬化效应。
