231 - Platelets are Activated and Increased in the Lungs of Neonatal Mice with Experimental Bronchopulmonary Dysplasia and Pulmonary Hypertension

Publication Number: 231.6458

Jamie Archambault, University of Colorado School of Medicine, Denver, CO, United States; Janelle N. Posey, Anschutz Medical Campus, Aurora, CO, United States; Mariah S. Jordan, University of Colorado School of Medicine, Aurora, CO, United States; Eva Nozik, University of Colorado School of Medicine, Aurora, CO, United States; Cassidy Delaney, University of Colorado, Aurora, CO, United States

Background: Pulmonary hypertension (PH) is a life-threatening condition characterized by inflammation, vascular remodeling, and vasoconstriction. 25-60% of former-preterm infants with severe bronchopulmonary dysplasia (BPD) demonstrate co-existing PH. Current therapies target vasoconstriction and are often ineffective in severe disease; thus, identification of novel mechanisms driving PH is of important clinical relevance. Accumulating data from adult studies demonstrate platelets are mediators of vascular inflammation and play a role in the pathogenesis of PH, but whether platelets contribute to pulmonary vascular remodeling in neonatal PH is unknown.

Objective: Utilizing a translational science approach, we seek to test the hypothesis that platelets are activated and recruited to the lungs of hypoxic neonatal mice, a clinically relevant model of neonatal PH.

Design/Methods: C57BL/6 neonatal mice remained in normoxia or were placed in hypobaric hypoxia at day of life (DOL 1). Lung injury was established at DOL 15 by mean linear intercept (MLI) and PH was assessed by right ventricular systolic pressure (RVSP) and right ventricular hypertrophy (RVH). Whole blood was obtained via right ventricle cardiac puncture and cell surface markers of platelet activation were quantified by flow cytometry. Lungs were inflated for immunohistochemistry or homogenized for ELISA. Data were analyzed using unpaired t test and reported as mean+/-SE with significance defined as p< 0.05.

Results: Hypoxia resulted in increased MLI (51.4+/-1.0 vs. 40.6+/-1.9 [um], p< 0.001), increased RVSP (29.7+/-1.8 vs. 21.5+/-0.3 [mmHg], p< 0.0001), and RVH (0.4+/-0.0 vs. 0.3+/-0.0 [RV/LV+S], p< 0.01). Platelets from hypoxic mice were activated with increased expression of platelet P-selectin (11.7+/-1.9 vs. 3.3+/-1.0 [%], p< 0.01) and activation of the transmembrane integrin α2βB3 (14.0+/-2.6 vs. 4.2+/-0.7 [%], p< 0.01). Platelets were increased in the lungs of hypoxic mice, with more CD41+ cells (2.26+/-0.7 vs. 0.93+/-0.1 [%], p< 0.05) on immunostaining and increased platelet factor 4 (PF4), in lung homogenates (3331+/-474 vs. 1256+/-248 [ng/mL], p< 0.01).

Conclusion(s): Neonatal platelets are activated in the circulation and increased in the lungs of mice with BPD and PH. Our parallel clinical study will assess platelet activation in infants with BPD-associated PH, and future translational investigation will determine the contribution of platelets to PH using the thrombocytopenic mouse. These findings have the potential to lead to a paradigm shift in our understanding of BPD-associated PH, laying the foundation for the development of targeted therapies.

Effect of 14-day hypoxia on lung injury, pulmonary hypertension, and platelet activation in neonatal mice.
Hypoxic mice have increased (a) mean linear intercept, (b) right ventricular systolic pressure, and (c) right ventricular hypertrophy. Hypoxic mice have increased platelet expression of (d) the cell adhesion molecule P-selectin and (e) integrin αIIbβ3. (f) Representative CD41 stained sections from distal lungs of D14 neonatal mice at 20X magnification. HPX mice have increased (g) platelets in the distal sections of inflated lungs and (h) platelet factor 4 from lung homogenates. *p <.05; **p <.01; ***p <.001; ****p < 0.0001