Connective Tissue Mediated Vascular Disease

National Heart, Lung, And Blood Institute

Investigators

Linked publications, trials & patents

Abstract

Area 1: Defining domains and modifying elastin gene expression: Through our work with patients with elastic fiber disease, we have identified several individuals with overlapping deletions upstream of ELN, who present with vascular features of elastin insufficiency, including supravalvar aortic stenosis. We subsequently identified a minimal region of overlap for the deletions and have identified underlying areas containing likely enhancers of elastin expression. By combining genomic and transcriptomic techniques, we were able to show that although two complete copies of ELN were present in each individual, transcription had been limited to a single allele. Long read genome sequencing showed the silenced allele to be cis with the deletion region. In the last year, we identified an additional patient with a smaller deletion overlying one of candidate enhancer regions who also exhibited the SVAS phenotype. Experiments to characterize the putative cis-regulatory element within the patient deletions is ongoing using CRISPR technology. Additional techniques such as ATAC-seq and Hi-C may be able to provide information on key genomic interactions that promote elastin transcription that may be manipulated to re-initiate elastin gene expression outside of the typical developmental window. Area 2: Using induced pluripotent stem cells to investigate elastic fiber assembly We have also continued to work with our newly optimized a platform to generate induced pluripotent stem cell (iPS)-derived vascular smooth muscle cells (iVSMC) using chemically defined and serum free media. Transcriptomic analysis of control and elastin insufficient lines revealed changes in genes associated with extracellular matrix organization which parallel findings identified in our human modifier analysis. We also identified new biological processes that may be associated with ELN haploinsufficiency, including differences in cell-cell adhesion and regulation of protein secretion and trafficking. Based on the analysis of all differentially expressed genes, potential upstream transcriptional regulators were also identified. We are now working to validate these findings and quantify the deposition of elastic fiber using immunostaining, respectively in these ELN-insufficient iPS lines. We continue to develop new machine learning approaches to assess elastic fiber quantity in non-sense and frameshift alleles and quality-based differences in elastic fibers secreted by iVSMCs produced by donor lines with missense rather than nonsense variants. Area 3: Identification of novel genes important for elastic fiber assembly As in our iPS lines, elastin mRNA is readily detectable on days 2 and 3 of differentiation, but then disappears by day 5. Elastin protein, on the other hand, is detectable in cells by day 5 and appears in the matrix in robust quantities by day 9. Interestingly, deposition of elastin in the extracellular matrix does not occur until other elastic fiber assembly genes such as EFEMP2, FBLN5, and LOX are also up-regulated. To identify additional genes that may play a role in elastic fiber production, we are using single cell data drawn from the literature to identify genes that are consistently co-expressed with elastin across tissue types and time periods. Current efforts have confirmed the co-expression of a set of previously implicated elastic fiber assembly genes but have also revealed new candidates likely to be key to this process. Of particular interest are a novel chaperone thought to be active in acidic compartments, a novel gene involved in vesicle formation, and a protein involved in cell cycle control. RNAscope techniques enabled spatial analysis of novel co-expressed genes and subsequent protein analysis will allow us to test how the newly identified gene products may contribute to the elastin-assembly process. Area 4: Identification of novel Elastin spicoforms We have recently conducted long read RNA sequencing on 6-month old and 14-day old mouse lung and aortas in order to investigate the variation in Elastin splicoforms across both age and tissue type. Through this effort, we showed that although the canonical full length elastin transcript the most common single transcript type, it makes up a minority of the mature elastin mRNA pool, with all tissues showing a large repertoire of alternatively spliced transcripts. Non-canonical splice forms were higher in aortas compared to lung and in younger as opposed to older tissue. Alternative transcripts included proximal and distal lncRNAs, transcripts missing multiple central exons termed missing middle transcripts, and those with retained introns. Current work is focused on creating cell line overexpressing the top missing middle transcript tagged with a fluorophor to investigate the impact of this transcript on elastin assembly or post-transcriptional regulation. Data from all four aims were presented at the Elastin and Elastic Fibers Gordon research conference in summer of 2023 while the enhancer project was presented at several genetics meetings.