Mathison, A. J. | Search bioRxiv

bioRxiv2025

Decreased endothelial cell retinoic acid signaling accelerates progression of single ventricle pulmonary arteriovenous malformations

BackgroundPulmonary arteriovenous malformations (PAVMs) are vascular complications that universally develop in patients with single ventricle congenital heart disease after Glenn surgery. However, the pathophysiological mechanisms underlying single ventricle PAVMs are poorly understood. To comprehensively evaluate molecular changes post-Glenn, we performed single-cell RNA sequencing (scRNAseq) on rat lung samples after Glenn surgery. MethodsUsing adult Sprague Dawley rats, we performed scRNAseq on unfiltered lung samples 3 weeks after left-sided Glenn or sham surgery. We compared endothelial cell (EC) differentially expressed genes (DEGs) in our model to two mouse models of hereditary hemorrhagic telangiectasia (HHT), a hereditary condition characterized by visceral AVMs. Finally, we modified the vitamin A (Vit A) content of Glenn and sham rat diets and re-assessed PAVM shunting and EC transcriptional differences. ResultsUsing scRNAseq (n=4 Glenn, n=4 sham), we identified 13 transcriptionally distinct lung cell clusters, including 3 EC clusters (general, capillary, lymphatic), with pronounced differences between Glenn and sham in the general EC cluster ([~]17% of genes). Comparison to HHT mouse models confirmed overlap of [~]18% of DEGs, including identification of significantly downregulated genes involved in and regulated by all-trans retinoic acid (ATRA) signaling in all 3 models. Dietary deficiency of Vit A intake, a precursor of ATRA, caused increased PAVM shunting (p<0.01) that was mitigated with excess dietary Vit A intake. Lastly, EC-specific RNAseq identified Vit A diet-induced gene expression differences, including regulation of PI3K signaling. ConclusionsIn this study, we report the novel application of scRNAseq to study mechanisms underlying single ventricle PAVMs in a surgical rat model. We identified multiple dysregulated biological processes in rat lung ECs post-Glenn, including decreased ATRA signaling and conserved gene expression patterns with HHT. Dietary modification of Vit A intake altered post-Glenn shunting and represents a novel potential therapeutic strategy for single ventricle PAVMs and HHT AVMs.

physiology↗

bioRxiv2023

Beyond Structural Bioinformatics for Genomics with Dynamics Characterization of an Expanded KRAS Mutational Landscape

ABSTRACTCurrent capabilities in genomic sequencing outpace functional interpretations. Our previous work showed that 3D protein structure calculations enhance mechanistic understanding of genetic variation in sequenced tumors and patients with rare diseases. The KRAS GTPase is among the critical genetic factors driving cancer and germline conditions. Because KRAS-altered tumors frequently harbor one of three classic hotspot mutations, nearly all studies have focused on these mutations, leaving significant functional ambiguity across the broader KRAS genomic landscape observed in cancer and non-cancer diseases. Herein, we extend structural bioinformatics with molecular simulations to study an expanded landscape of 86 KRAS mutations. We identify multiple coordinated changes strongly associated with experimentally established KRAS biophysical and biochemical properties. The patterns we observe span hotspot and non-hotspot alterations, which can all dysregulate Switch regions, producing mutation-restricted conformations with different effector binding propensities. We experimentally measured mutation thermostability and identified shared and distinct patterns with simulations. Our results indicate mutation-specific conformations which show potential for future research into how these alterations reverberate into different molecular and cellular functions. The data we present is not predictable using current genomic tools, demonstrating the added functional information derived from molecular simulations for interpreting human genetic variation. Key PointsPlease provide 3 bullet points summarizing the manuscripts contribution to the field (100 characters max per point) 1) We functionally grouped 86 distinct KRAS mutations using MD scores, demonstrating scalability for genomics 2) MD-based groups explain experimental differences and mechanistic information about mutant proteins 3) Demonstrated added functional information from simulations for interpreting human genetic variation

genomics↗