Connective Tissue Mediated Vascular Disease

National Heart, Lung, And Blood Institute

Investigators

Linked publications, trials & patents

Abstract

DArea 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. Using computational methods, we have 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 elastin genes were present in these individuals, transcription had been limited to a single allele. Long read genome sequencing currently underway will determine whether the silenced allele is in cis or trans with deletion region and additional techniques such as ATAC-seq and Hi-ChIP may be able to provide information on key genomic interactions that promote elastin transcription that can potentially be manipulated to re-initiate elastin gene expression outside of the typical developmental window. A review summarizing what is currently known about the control of elastin transcriptional control was published earlier this year. 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. We have modeled elastin-mediated vasculopathy in a dish using patient-derived iPSCs carrying different mutations in ELN (n=2) gene as well as CRISPRed iPSC lines targeting ELN (n=3). We confirmed that the cell genomes had been minimally altered by reprogramming. Likewise, minimal off target genetic effects were detected in the CRISPR lines. This year, we confirmed the loss of expression level of ELN in CRISPRed iPSC line with both copies of ELN gene altered. We also demonstrated 50% loss of ELN expression in patient-derived iPSCs carrying a stopgain mutation. We are now working to quantify the deposition of elastic fiber using immunostaining, respectively in these ELN-insufficient iPS lines. We are also piloting new methods using 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. In addition to elastin quantity and quality, we will evaluate for functional outcomes on the cell, such as changes to cell proliferation and mobility and their responses to stretch. Area 3: Identification of novel genes important for elastic fiber assembly As noted above, 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 up-regulated. To identify additional genes that may play a role in elastic fiber production, we have developed new computational methods using single cell data drawn from the literature to identify genes that are consistently co-expressed in the same cell 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 and a protein involved in cell cycle control. We have hired a new scientist to follow up the role of these potentially important proteins in the coming year. Incorporation of novel techniques like RNAscope will offer the opportunity to study genes thought to be important for elastic fiber assembly that are not made concurrently by the same cells. Area 4: Mutations in the copper binding domain of lysyl oxidase produce aberrant elastic fibers We recently published a manuscript outlining the mechanism by which variants in the copper binding domain in lysyl oxidase (LOX) produce aortic dilation and aneurysm. Taken together our work showed that while elastin was laid down by Loxb2b370.2Clo (c.G854T; p.C285F) mouse aortic smooth muscle cells in normal amounts, the fibers generated were poor quality and began to prematurely turn over with repetitive stretch-recoil cycles in the aorta. This phenotype shows both sex and genetic background effects, with males and animals in a congenic hypertensive background dilating faster. Transcriptomic analysis shows derangement of dexamethasone and TGF-responsive genes. In particular, upregulation of both elastase and proteoglycans such as aggrecan were noted in the vessel wall. Ultrastructural imaging using Fib-SEM shows in detail the structural derangements that occur in the Lox mutants. Likewise, aortas from Lox+/ c.G854T mice exposed to elastase dilate faster suggesting that the elastin and collagen produced in these mutants is structurally inferior, making it increasingly susceptible to proteolytic damage. Current investigations are focused on using population-based cohort data to understand the impact of missense variation on aneurysm risk in the general population.