Age dependent endothelial responses to pneumonia-induced Acute Lung Injury

Abstract

Objective: Children with Pediatric Acute Respiratory Distress Syndrome (PARDS) have better outcomes than adults with ARDS. Animal studies suggest that the reasons for improved outcomes are physiologic. Yes-associated protein (YAP) is necessary for lung growth and post-pneumonia regeneration. We sought to determine YAP responses to pneumonia-induced Acute Lung Injury (ALI). Hypothesis: Endothelial YAP responses to pneumonia determine ALI. Methods: We induced ALI by intranasal instillation of Pseudomonas aeruginosa strain K (PAK, 2.5 x 10 5 colonyforming units (CFUs)) in anesthetized mature (10-week-old) and juvenile (3-week-old) mice. The bacteria were in exponential growth phase. Control mice were instilled with sterile saline. 24 hours later, we assessed ALI by quantification of protein and leukocytes in the bronchoalveolar lavage (BAL), assessed lung YAP expression by immunoblot, and isolated primary endothelial cells (PECs) for RNA sequencing (RNA-seq). To knockdown YAP expression, we injected mature mice with YAP-targeted siRNA by tail vein and control mice with scrambled siRNA. We isolated lung PECs by negative selection with magnetic beads attached to CD45 and CD41 antibodies followed by positive selection with anti-CD31 beads. We extracted total RNA. Bulk RNA-seq was performed by Azenta. We mined our RNA-seq database for proteins known to interact with YAP, setting the threshold for significantly different gene expression at >1.5-fold, p<0.05. Results: We confirmed PAK was instilled in equal doses in juvenile and adult mice through quantification of BAL CFUs. In mature mice, PAK instillation induced an increase in BAL protein and leukocytes by 30- and 6-fold, respectively, compared with saline-treated controls. In juvenile mice, the corresponding increases were 9- and 3-fold (p<0.05 vs mature). Following PAK instillation, YAP expression increased by >5-fold in adult mice. In contrast, YAP expression decreased by 70% in juvenile mice. Following vascular knockdown of YAP in mature mice, PAK-induced BAL protein and leukocyte count increases were inhibited (p<0.05). We confirmed 95% purity of isolated PECs by immunostaining for VE-cadherin and CD45. PAK instillation significantly decreased RNA expression of TEAD2—a YAP-related transcription factor—in juvenile mice, whereas PAK instillation did not affect TEAD2 RNA expression in mature mice. Conclusions: Pneumonia-induced ALI was worse in mature mice than in juveniles. Surprisingly, YAP expression increased in response to ALI in mature mice but decreased in juveniles. Indeed, we noted protection from ALI in mature mice with YAP knockdown. RNA-seq revealed a PAK-induced decrease in a YAP-related transcription factor in juvenile PECs that was absent in mature PECs. Together, these data suggest that YAP expression contributes to ALI and that it underlies age-dependent responses ALI. K08 HL148403 Parker B. Francis Fellowship Program Louis V. Gerstner Scholars Program Department of Pediatrics and Innovation Nucleation Fund This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.