Biology subjects

Papangeli, I.

Publications and source records attributed to Papangeli, I..

4 recordsLinked to original sources

bioRxiv2026

Sequence determinants of efficient exon 44 skipping in Duchenne muscular dystrophy define design principles for steric-blocking antisense oligonucleotides

Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene that disrupt the reading frame and abolish expression of functional dystrophin protein. Antisense oligonucleotides (ASO) can restore production of partially functional dystrophins by inducing exon skipping to restore the reading frame of dystrophin transcripts. While exon skipping is an FDA approved therapeutic strategy, there are currently no approved therapies for patients amenable to exon 44 skipping (8% of DMD patients). Here, we carried out a discovery campaign to identify phosphorothioate (PS) ASOs that efficiently induce exon 44 skipping and to define key sequence and chemistry features associated with activity. A tiling and micro-tiling approach with 18mer fully PS and 2-O-methoxyethyl (2MOE) modified ASOs in patient-derived myotubes identified five exonic target regions that promote skipping. ASO activity was strongly correlated across skeletal muscle and iPSC-derived cardiomyocytes, indicating similar exon 44 splicing regulation across cell types. Optimization studies showed that for 2MOE PS ASOs, 16-20mers were generally most active, while longer ASOs often had reduced activity due in part to impaired productive uptake into cells. Swapping out 2MOE modifications at both terminal positions for locked nucleic acids (LNAs) rarely improved activity and could also reduce it. Finally, promising candidates were tested in a humanized mouse model with an exon 44 skippable deletion, where one ASO outperformed others, inducing dose-dependent exon 44 skipping and dystrophin restoration in mouse models. These findings define practical design rules for exon 44-targeted ASOs and provide a foundation for therapeutic development.

molecular biology↗

bioRxiv2023

Transcriptional profile of the rat cardiovascular system at single cell resolution

BackgroundDespite the critical role of the cardiovascular system, our understanding of its cellular and transcriptional diversity remains limited. We therefore sought to characterize the cellular composition, phenotypes, molecular pathways, and communication networks between cell types at the tissue and sub-tissue level across the cardiovascular system of the healthy Wistar rat, an important model in preclinical cardiovascular research. We obtained high quality tissue samples under controlled conditions that reveal a level of cellular detail so far inaccessible in human studies. Methods and ResultsWe performed single nucleus RNA-sequencing in 78 samples in 10 distinct regions including the four chambers of the heart, ventricular septum, sinoatrial node, atrioventricular node, aorta, pulmonary artery, and pulmonary veins (PV), which produced an aggregate map of 505,835 nuclei. We identified 26 distinct cell types and additional subtypes, including a number of rare cell types such as PV cardiomyocytes and non-myelinating Schwann cells (NMSCs), and unique groups of vascular smooth muscle cells (VSMCs), endothelial cells (ECs) and fibroblasts (FBs), which gave rise to a detailed cell type distribution across tissues. We demonstrated differences in the cellular composition across different cardiac regions and tissue-specific differences in transcription for each cell type, highlighting the molecular diversity and complex tissue architecture of the cardiovascular system. Specifically, we observed great transcriptional heterogeneities among ECs and FBs. Importantly, several cell subtypes had a unique regional localization such as a subtype of VSMCs enriched in the large vasculature. We found the cellular makeup of PV tissue is closer to heart tissue than to the large arteries. We further explored the ligand-receptor repertoire across cell clusters and tissues, and observed tissue-enriched cellular communication networks, including heightened Nppa - Npr1/2/3 signaling in the sinoatrial node. ConclusionsThrough a large single nucleus sequencing effort encompassing over 500,000 nuclei, we broadened our understanding of cellular transcription in the healthy cardiovascular system. The existence of tissue-restricted cellular phenotypes suggests regional regulation of cardiovascular physiology. The overall conservation in gene expression and molecular pathways across rat and human cell types, together with our detailed transcriptional characterization of each cell type, offers the potential to identify novel therapeutic targets and improve preclinical models of cardiovascular disease.

cell biology↗

bioRxiv2022

Endothelial beta-arrestins Regulate Mechanotransduction by the Type II Bone Morphogenetic Protein Receptor in Primary Cilia

RationaleModulation of endothelial cell behavior and phenotype by hemodynamic forces involves many signaling components, including cell surface receptors, intracellular signaling intermediaries, transcription factors, and epigenetic elements. Many of the signaling mechanisms that underlie mechanotransduction by endothelial cells are inadequately defined. ObjectiveWe sought to better understand how {beta}-arrestins, intracellular proteins that regulate agonist-mediated desensitization and integration of signaling by transmembrane receptors, may be involved in the endothelial cell response to shear stress. Methods and ResultsIn vitro studies with primary endothelial cells subjected to {beta}-arrestin knockdown, and in vivo studies using mice with endothelial specific deletion of {beta}-arrestin 1 and {beta}-arrestin 2 were conducted. We found that {beta}-arrestins are localized to primary cilia in endothelial cells, which are present in subpopulations of endothelial cells in relatively low shear states. Recruitment of {beta}-arrestins to cilia involved its interaction with IFT81, a component of the flagellar transport protein complex in the cilia. {beta}-arrestin knockdown led to marked reduction in shear stress response, including induction of NOS3 expression. Within the cilia, {beta}-arrestins were found to associate with the type II bone morphogenetic protein receptor (BMPR-II), whose disruption similarly led to an impaired endothelial shear response. {beta}-arrestins also regulated Smad transcription factor phosphorylation by BMPR-II. Mice with endothelial specific deletion of {beta}-arrestin 1 and {beta}-arrestin 2 were found to have impaired retinal angiogenesis. ConclusionWe have identified a novel role for endothelial {beta}-arrestins as key transducers of ciliary mechanotransduction that play a central role in shear signaling by BMPR-II and contribute to vascular development. NOVELTY AND SIGNIFICANCE What Is Known?O_LIEndothelial cells respond to flow-induced shear stress with biochemical changes, such as phosphorylation of endothelial nitric oxide synthase, that promote morphological changes, such as cell alignment. C_LIO_LIThe endothelial response to shear stress can involve primary cilia, microtubule-based sensory organelles that detect extracellular stimuli and generates intracellular signals. C_LIO_LIThe specific ciliary signaling pathways that regulate endothelial mechanotransduction have not been fully elucidated. C_LI What New Information Does This Article Contribute?O_LI{beta}-arrestins directly interact with the ciliary protein intraflagellar transport protein 81 (IFT81), which is present in the primary cilia of endothelial cells, and are required for the morphological response to flow-induced shear stress. C_LIO_LI{beta}-arrestins regulates type II bone morphogenetic protein receptor signaling, which is required for the endothelial response to shear stress, and is required for the phosphorylation of Smad transcription factors. C_LIO_LI{beta}-arrestins are required for endothelial nitric oxide synthase-mediated flow-induced shear stress response in endothelial cells. C_LIO_LIEndothelial cell-specific knockout of {beta}-arrestins results in abnormal vascular development, with a loss of vessel length and branchpoints. C_LI

molecular biology↗

bioRxiv2020

Transcriptional and Cellular Diversity of the Human Heart

IntroductionThe human heart requires a complex ensemble of specialized cell types to perform its essential function. A greater knowledge of the intricate cellular milieu of the heart is critical to increase our understanding of cardiac homeostasis and pathology. As recent advances in low input RNA-sequencing have allowed definitions of cellular transcriptomes at single cell resolution at scale, here we have applied these approaches to assess the cellular and transcriptional diversity of the non-failing human heart. MethodsMicrofluidic encapsulation and barcoding was used to perform single nuclear RNA sequencing with samples from seven human donors, selected for their absence of overt cardiac disease. Individual nuclear transcriptomes were then clustered based upon transcriptional profiles of highly variable genes. These clusters were used as the basis for between-chamber and between-sex differential gene expression analyses and intersection with genetic and pharmacologic data ResultsWe sequenced the transcriptomes of 287,269 single cardiac nuclei, revealing a total of 9 major cell types and 20 subclusters of cell types within the human heart. Cellular subclasses include two distinct groups of resident macrophages, four endothelial subtypes, and two fibroblasts subsets. Comparisons of cellular transcriptomes by cardiac chamber or sex reveal diversity not only in cardiomyocyte transcriptional programs, but also in subtypes involved in extracellular matrix remodeling and vascularization. Using genetic association data, we identified strong enrichment for the role of cell subtypes in cardiac traits and diseases. Finally, intersection of our dataset with genes on cardiac clinical testing panels and the druggable genome reveals striking patterns of cellular specificity. ConclusionsUsing large-scale single nuclei RNA sequencing, we have defined the transcriptional and cellular diversity in the normal human heart. Our identification of discrete cell subtypes and differentially expressed genes within the heart will ultimately facilitate the development of new therapeutics for cardiovascular diseases.

genomics↗