KD025 Shifts Pulmonary Endothelial Cell Bioenergetics and Decreases Baseline Lung Permeability

KD025 is a ROCK2 inhibitor currently under clinical investigation for treating fibrotic lung diseases. Its therapeutic benefits are thought to stem from inhibiting profibrotic pathways and influencing fat metabolism. However, the impact of KD025 on pulmonary microvascular endothelial cell (PMVEC) function remains unclear, despite the fact that disruption of the alveolar-capillary membrane is a major cause of mortality in fibrotic lung diseases. We hypothesized that KD025 regulates PMVEC metabolism, pH, migration, and survival—key functional characteristics that maintain pulmonary barrier integrity.

In our experiments, we utilized PMVECs isolated from Sprague Dawley rats. We found that KD025 reduced lactate production and glucose consumption in a dose-dependent manner. Its inhibitory effect was more pronounced compared to other metabolic modifiers, such as 2-deoxy-glucose, extracellular acidosis, dichloroacetate, and remogliflozin. Notably, KD025 increased oxidative phosphorylation, which was not observed with 2-deoxy-glucose. Additionally, KD025 lowered intracellular pH, leading to a compensatory rise in anion exchanger 2 activity. While KD025 inhibited PMVEC migration, the nonspecific ROCK inhibitor fasudil did not.

To assess endothelial permeability in vivo, we used Evans Blue dye in a bleomycin pulmonary fibrosis model. We observed that baseline permeability was reduced in KD025-treated animals, regardless of bleomycin treatment. Under hypoxic conditions, KD025 increased PMVEC necrosis, as indicated by elevated lactate dehydrogenase release and propidium iodide uptake, along with decreased ATP levels, although it did not affect Annexin V binding. Interestingly, ROCK2 knockdown did not alter PMVEC metabolism, pH, or migration, but it did enhance non-apoptotic caspase-3 activity.

In summary, our findings indicate that KD025 promotes oxidative phosphorylation, reduces glycolysis and intracellular pH, inhibits migration, and enhances pulmonary barrier integrity in a manner that is independent of ROCK2.