Abstract Body (Do not enter title and authors here): Background: Pulmonary arteriovenous malformations (PAVMs) are vascular complications that universally develop in patients with single ventricle congenital heart disease (CHD) after Glenn surgery. However, single ventricle PAVMs are poorly understood with limited cellular and molecular data. To improve our understanding of single ventricle PAVMs and comprehensively evaluate molecular changes post-Glenn, we performed single cell transcriptome analysis on unfiltered lung samples from rats after Glenn surgery.

Methods: Using adult Sprague Dawley rats, we performed a unilateral left-sided Glenn or sham surgery. To 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. To identify transcriptionally distinct cell clusters, we performed an unsupervised cell clustering with Seurat. We then identified differentially expressed genes (DEGs) (adjusted p<0.05 and Log2FC>|0.5|) between Glenn and sham rats within each cluster and used DEGs for subsequent pathway analysis to identify dysregulated biological processes (DAVID, IPA). To validate our scRNAseq data in endothelial cell (EC) clusters, we sorted CD31+ lung ECs from the left lung of additional post-surgery rats and isolated RNA for qRT-PCR.

Results: Using scRNAseq (n=4 Glenn, n=4 sham), we identified 13 distinct transcriptional clusters, including 3 EC clusters (general, capillary, lymphatic), with pronounced differences between Glenn and sham in the general EC cluster (>1300 DEGs, ~16.7% of expressed genes). Angiogenesis was the most significantly dysregulated biological pathway (p=3.0E-13); however, multiple other pathways were also dysregulated, including EC response to LPS, IL-1, TNF, and others. qRT-PCR validation using independent rat lung samples (n=5 Glenn, n=5 sham) confirmed scRNAseq findings that post-Glenn ECs have altered expression in angiogenesis (Angpt2, Acvrl1, Eng, Tmem100, Vegfa), vasodilation (Nos2, Edn1), inflammation (Glp1r, Serpina1), and others (Car4, Cyp26b1).

Conclusions: In this study, we report the novel application of scRNAseq to study single ventricle PAVMs in a surgical rat model. Multiple biological processes are dysregulated in rats post-Glenn, including angiogenesis, vasodilation, and inflammation. Our findings support further investigation of anti-angiogenic and anti-inflammatory therapies for single ventricle PAVMs.