KD025 is a ROCK2 inhibitor currently being tested in clinical trials for the treatment of fibrotic lung diseases. The therapeutic effects of KD025 are partly due to its inhibition of profibrotic pathways and fat metabolism. However, whether KD025 affects pulmonary microvascular endothelial cell (PMVEC) function is unknown, despite evidence that alveolar–capillary membrane disruption constitutes major causes of death in fibrotic lung diseases. We hypothesized that KD025 regulates PMVEC metabolism, pH, migration, and survival, a series of interrelated functional characteristics that determine pulmonary barrier integrity. We used PMVECs isolated from Sprague Dawley rats. KD025 dose-dependently decreased lactate production and glucose consumption. The inhibitory effect of KD025 was more potent compared with other metabolic modifiers, including 2-deoxy-glucose, extracellular acidosis, dichloroacetate, and remogliflozin. Interestingly, KD025 increased oxidative phosphorylation, whereas 2-deoxy-glucose did not. KD025 also decreased intracellular pH and induced a compensatory increase in anion exchanger 2. KD025 inhibited PMVEC migration, but fasudil (nonspecific ROCK inhibitor) did not. We tested endothelial permeability in vivo using Evans Blue dye in the bleomycin pulmonary fibrosis model. Baseline permeability was decreased in KD025-treated animals independent of bleomycin treatment. Under hypoxia, KD025 increased PMVEC necrosis as indicated by increased lactate dehydrogenase release and propidium iodide uptake and decreased ATP; it did not affect Annexin V binding. ROCK2 knockdown had no effect on PMVEC metabolism, pH, and migration, but it increased nonapoptotic caspase-3 activity. Together, we report that KD025 promotes oxidative phosphorylation; decreases glycolysis, intracellular pH, and migration; and strengthens pulmonary barrier integrity in a ROCK2-independent manner.

KD025 是一种 ROCK2 抑制剂，目前正在进行治疗纤维化肺部疾病的临床试验。 KD025 的治疗作用部分归因于其对促纤维化途径和脂肪代谢的抑制。然而，尽管有证据表明肺泡毛细血管膜破坏是纤维化肺疾病死亡的主要原因，但 KD025 是否影响肺微血管内皮细胞 (PMVEC) 功能尚不清楚。我们假设 KD025 调节 PMVEC 代谢、pH、迁移和存活，这是决定肺屏障完整性的一系列相互关联的功能特征。我们使用从 Sprague Dawley 大鼠中分离出的 PMVEC。 KD025 剂量依赖性地降低乳酸产生和葡萄糖消耗。与其他代谢调节剂（包括 2-脱氧葡萄糖、细胞外酸中毒、二氯乙酸和雷莫格列净）相比，KD025 的抑制作用更有效。有趣的是，KD025 增加了氧化磷酸化，而 2-脱氧葡萄糖则没有。 KD025 还降低细胞内 pH 值并诱导阴离子交换剂 2 代偿性增加。KD025 抑制 PMVEC 迁移，但法舒地尔（非特异性 ROCK 抑制剂）则不然。我们在博莱霉素肺纤维化模型中使用伊文思蓝染料测试了体内内皮通透性。 KD025 治疗动物的基线通透性降低，与博莱霉素治疗无关。在缺氧条件下，KD025 增加了 PMVEC 坏死，表现为乳酸脱氢酶释放增加、碘化丙啶摄取增加以及 ATP 减少；它不影响膜联蛋白 V 结合。 ROCK2 敲低对 PMVEC 代谢、pH 和迁移没有影响，但增加了非凋亡 caspase-3 活性。 我们一起报告 KD025 促进氧化磷酸化；降低糖酵解、细胞内 pH 值和迁移；并以不依赖于 ROCK2 的方式增强肺屏障的完整性。