Abstract 4368824: Decreased retinoic acid signaling in lung endothelial cells accelerates progression of single ventricle pulmonary arteriovenous malformations

Background: Pulmonary arteriovenous malformations (PAVMs) are vascular complications that universally develop in patients with single ventricle congenital heart disease after Glenn surgery. However, single ventricle PAVMs are poorly understood with no known medical treatments. Methods: We performed a unilateral left-sided Glenn or sham surgery on 6-8 week old Sprague Dawley rats. To comprehensively study the molecular changes early after surgery, we collected the lower half of the left lung and performed single cell RNA sequencing (scRNAseq) on unfiltered lung samples 3 weeks after surgery. After performing unsupervised cell clustering, we identified differentially expressed genes (DEGs) and dysregulated canonical pathways (Ingenuity Pathway Analysis; IPA) between Glenn and sham rats within each cluster. We then validated a DEGs with qPCR and immunohistochemistry and confirmed lung retinoid levels via HPLC. Based on these data, we modified Vitamin A (Vit A) content of Glenn and sham rat diets and assessed PAVM shunting with microspheres 3 weeks after surgery. Results: Using scRNAseq (n=4 Glenn, n=4 sham), we identified 13 distinct transcriptional clusters, including 3 endothelial cell (EC) clusters (general, capillary, lymphatic), with pronounced differences between Glenn and sham in cluster 1 general ECs (>1300 DEGs). Among DEGs within the general EC cluster, multiple DEGs are responsible for uptake and metabolism of Vit A metabolites (Lpl, Nceh1, Rdh12, Dhrs3, Cyp26b1) and are direct transcription factors for retinoic acid (Rarγ). IPA identified decreased canonical activity of retinoic acid signaling (Z-score -2.00) in cluster 1 ECs, and we confirmed decreased lung retinoid levels in the left lung of Glenn rats 3 weeks after surgery (retinol and retinyl esters both p<0.001). After varying dietary Vit A intake, Glenn rats on a Vit A deficient diet had increased shunting compared to diet-matched sham rats (p<0.0001) and compared to Glenn rats on Vit A control diet (p=0.002) and excess diet (p<0.001). In contrast, Glenn rats on Vit A excess diet did not have increased shunting compared to diet-matched sham rats (p=0.82). Conclusions: We report the novel application of scRNAseq to study single ventricle PAVMs. Additionally, we identified that deficient Vit A intake increases early post-Glenn PAVM shunting and excess Vit A intake may mitigate shunting. Our findings support Vit A as a novel potential therapy for single ventricle PAVMs.