(654) Mechanism of Pulmonary Vascular Dysfunction in Cystic Fibrosis.

Background: Cystic Fibrosis (CF) is a life-shortening autosomal recessive genetic disorder caused by a deleterious mutation in the CFTR gene. CF affects the lungs and digestive systems of about 30,000 people in the U.S. Studies have reported loss of pulmonary blood vessels and perfusion abnormalities in 85% of 1-year old CF infants [1], which suggests a role of the vasculature in CF lung disease. Mucus plugging and bronchial wall abnormalities only explain 20% of the observed perfusion deficit [1]. Although CFTR has been shown to be expressed and functional on endothelial cells (ECs), poor effort has been made to investigate the vascular origin of CF lung disease. As a result, the mechanism of loss of pulmonary blood vessels in CF remains unknown.

Methods: This study investigates the effects of laminar and disturbed shear flows on ECs when CFTR is dysfunctional using microfluidic devices. Laminar shear stress is the primary trigger for EC nitric oxide production and provides important physiological cues for endothelial homeostasis. On the other hand, disturbed flow induces EC senescence and apoptosis. This study will determine how CFTR modulates EC migration, flow alignment, angiogenesis, wound healing, and apoptosis. These processes, in particular angiogenesis and wound repair, play an important role in maintaining the vasculature, especially when chronic inflammation and injuries are present. Therefore, their disruptions could explain the loss of pulmonary blood vessels in CF. We hypothesize that dysfunctional CFTR disrupts 1) the cellular response of ECs to chronic high shear stress and disturbed flow; 2) impairs cell alignment and migration; 3) disrupts angiogenesis and wound healing processes; and ultimately leads to premature death of ECs.

Results: Our results show that inhibition of CFTR causes ECs death in high shear stress conditions but not in static conditions. Similarly, inhibition of CFTR under disturbed flow causes significant cell death in ECs. We also show that CFTR inhibition prevents wound closure and creates gaps in the endothelial monolayer, while activation of CFTR (via forskolin-induced cAMP increase) leads to faster wound closure. Besides, tube formation assay data show that inhibition of CFTR leads to faster breakdown of tube network. Similar results were obtained by knocking down CFTR using siRNA. Furthermore, our results suggest that CFTR, but no other chloride channel, specifically increases stress-induced calcium entry. Finally, we identified a correlation between intracellular calcium overload and the effects caused by CFTR inhibition or knockdown. These effects were mitigated by preventing rise in intracellular calcium.

Conclusions: Loss of pulmonary blood vessels in CF could be due to a disruption of the angiogenic and wound repair processes, or a disruption of normal flow-induced calcium entry into the cells, which leads to cell death. Our results also show evidence of a modulation of cationic channels by CTFR.

Acknowledgements: This work is supported by the American Heart Association [grant number 903099].

References: [1] [1] Wielpütz MO, Puderbach M, Kopp-Schneider A, Stahl M, Fritzsching E, Sommerburg O, et al. Magnetic Resonance Imaging Detects Changes in Structure and Perfusion, and Response to Therapy in Early Cystic Fibrosis Lung Disease. American Journal of Respiratory and Critical Care Medicine. 2014;189(8):956-65. https://doi.org/10.1164/rccm.201309-1659OC