D108-11 Mechanism of Ensifentrine Protection Against Endothelial Injury in COPD-associated Pulmonary Hypertension

Abstract Rationale Pulmonary hypertension is a severe complication of COPD characterized by pulmonary vascular remodeling and right ventricular failure with no specific therapies. Cigarette smoke (CS) plays a central role in COPD-associated pulmonary hypertension (COPD-PH) pathogenesis and directly injures endothelial cells (ECs) causing barrier dysfunction and apoptosis. A potential mechanism of EC injury is through inhibition of cAMP signaling, a critical EC homeostatic pathway reduced by CS. However, the role of cAMP or its downstream effectors protein kinase A (PKA) and exchange proteins directly activated by cAMP (EPAC) in COPD-PH is unknown. We hypothesized that reduced cAMP-PKA/EPAC signaling drives pulmonary EC injury in COPD-PH, and can be restored by phosphodiesterase (PDE) 3/4 inhibition with ensifentrine, which increases cAMP levels. Methods Spatial transcriptomics of lung tissue from patients with COPD-PH (n = 3) and COPD without PH (n = 3), confirmed by right heart catheterization prior to lung transplantation, was performed using the Visium HD platform and cell populations were annotated with the Human Lung Cell Atlas. Transformed human lung microvascular endothelial cells (HuLEC-5a) were exposed to cigarette smoke extract (CSE, 3-5%) for 6-20 hrs. cAMP signaling was activated by PDE3/4 inhibitor ensifentrine (5-10 μM) for 6-20 hrs, and downstream effectors inhibited by PKI (3 μM) or ESI-09 (0.3 μM). Barrier function was assessed by electric cell-substrate impedance sensing, junctional integrity by VE-cadherin and ZO-1 immunocytochemistry, and apoptosis and cell injury by western blotting and lactate dehydrogenase (LDH) release. Results Spatial transcriptomics identified several microvascular EC populations and revealed significant downregulation of key cAMP signaling components as well as upregulation of PDE4D in capillary ECs in COPD-PH lungs. KEGG pathway analysis revealed dysregulation of the cAMP pathway (p = 0.04), which included PDE3A upregulation. Ensifentrine preserved VE-cadherin and ZO-1 localization disrupted by CSE and maintained barrier function during CSE exposure (1.2-fold vs CSE, p 0.01). PKI and ESI-09 abrogated ensifentrine’s protective effects and worsened CSE-induced barrier disruption (0.85- and 0.74-fold vs CSE alone; p = 0.30 and p = 0.0024). Ensifentrine reduced cleaved PARP levels (0.6-fold vs vehicle, p 0.01) and LDH release (0.5-fold vs vehicle, p = 0.017) during CSE exposure, while PKI and ESI-09 abolished these protective effects. PKI or ESI-09 alone increased cleaved PARP under baseline and CSE exposure. Conclusion Suppression of cAMP-PKA/EPAC signaling may play a key role in endothelial dysfunction in COPD-PH. Restoration of this pathway via PDE3/4 inhibition may protect against CS-induced endothelial injury, supporting cAMP-PKA/EPAC signaling as a potential novel therapeutic target for COPD-PH. This abstract is funded by: •NIH Stimulating Access to Research in Residency (StARR) Award (RO) •Carson Family Foundation (IP) •Wollowick Endowed Chair in COPD Research (IP) •Post-doctoral Medical Science Concepts Incubator Funds