Dataset Information

Collagen fiber regulation in human pediatric aortic valve development and disease.

ABSTRACT: Congenital aortic valve stenosis (CAVS) affects up to 10% of the world population without medical therapies to treat the disease. New molecular targets are continually being sought that can halt CAVS progression. Collagen deregulation is a hallmark of CAVS yet remains mostly undefined. Here, histological studies were paired with high resolution accurate mass (HRAM) collagen-targeting proteomics to investigate collagen fiber production with collagen regulation associated with human AV development and pediatric end-stage CAVS (pCAVS). Histological studies identified collagen fiber realignment and unique regions of high-density collagen in pCAVS. Proteomic analysis reported specific collagen peptides are modified by hydroxylated prolines (HYP), a post-translational modification critical to stabilizing the collagen triple helix. Quantitative data analysis reported significant regulation of collagen HYP sites across patient categories. Non-collagen type ECM proteins identified (26 of the 44 total proteins) have direct interactions in collagen synthesis, regulation, or modification. Network analysis identified BAMBI (BMP and Activin Membrane Bound Inhibitor) as a potential upstream regulator of the collagen interactome. This is the first study to detail the collagen types and HYP modifications associated with human AV development and pCAVS. We anticipate that this study will inform new therapeutic avenues that inhibit valvular degradation in pCAVS and engineered options for valve replacement.

SUBMITTER: Clift CL

PROVIDER: S-EPMC8105334 | biostudies-literature | 2021 May

REPOSITORIES: biostudies-literature

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Collagen fiber regulation in human pediatric aortic valve development and disease.

Clift Cassandra L CL Su Yan Ru YR Bichell David D Jensen Smith Heather C HC Bethard Jennifer R JR Norris-Caneda Kim K Comte-Walters Susana S Ball Lauren E LE Hollingsworth M A MA Mehta Anand S AS Drake Richard R RR Angel Peggi M PM

Scientific reports 20210507 1

Congenital aortic valve stenosis (CAVS) affects up to 10% of the world population without medical therapies to treat the disease. New molecular targets are continually being sought that can halt CAVS progression. Collagen deregulation is a hallmark of CAVS yet remains mostly undefined. Here, histological studies were paired with high resolution accurate mass (HRAM) collagen-targeting proteomics to investigate collagen fiber production with collagen regulation associated with human AV development ...[more]

PMID: 33963260

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Project description:Congenital aortic valve stenosis (AS) progresses as an obstructive narrowing of the aortic orifice due to deregulated extracellular matrix (ECM) production by aortic valve (AV) leaflets and leads to heart failure with no effective therapies. Changes in glycoprotein and proteoglycan distribution are a hallmark of AS, yet valvular carbohydrate content remains virtually uncharacterized at the molecular level. While almost all glycoproteins clinically linked to stenotic valvular modeling contain multiple sites for N-glycosylation, there are very few reports aimed at understanding how N-glycosylation contributes to the valve structure in disease. Here, we tested for spatial localization of N-glycan structures within pediatric congenital aortic valve stenosis. The study was done on valvular tissues 0-17 years of age with de-identified clinical data reporting pre-operative valve function spanning normal development, aortic valve insufficiency (AVI), and pediatric endstage AS. High mass accuracy imaging mass spectrometry (IMS) was used to localize N-glycan profiles in the AV structure. RNA-Seq was used to identify regulation of N-glycan related enzymes. The N-glycome was found to be spatially localized in the normal aortic valve, aligning with fibrosa, spongiosa or ventricularis. In AVI diagnosed tissue, N-glycans localized to hypertrophic commissures with increases in pauci-mannose structures. In all valve types, sialic acid (N-acetylneuraminic acid) N-glycans were the most abundant N-glycan group. Three sialylated N-glycans showed common elevation in AS independent of age. On-tissue chemical methods optimized for valvular tissue determined that aortic valve tissue sialylation shows both α2,6 and α2,3 linkages. Specialized enzymatic strategies demonstrated that core fucosylation is the primary fucose configuration and localizes to the normal fibrosa with disparate patterning in AS. This study identifies that the human aortic valve structure is spatially defined by N-glycomic signaling and may generate new research directions for the treatment of human aortic valve disease.

Project description:Recent studies indicated that small calcified particles observable by scanning electron microscopy (SEM) may initiate calcification in cardiovascular tissues. We hypothesized that if the calcified particles precede gross calcification observed in calcific aortic valve disease (CAVD), they would exhibit a regional asymmetric distribution associated with CAVD development, which always initiates at the base of aortic valve leaflets adjacent to the aortic outflow in a region known as the fibrosa. Testing this hypothesis required counting the calcified particles in histological sections of aortic valve leaflets. SEM images, however, do not provide high contrast between components within images, making the identification and quantification of particles buried within tissue extracellular matrix difficult. We designed a new unique pattern-matching based technique to allow for flexibility in recognizing particles by creating a gap zone in the detection criteria that decreased the influence of non-particle image clutter in determining whether a particle was identified. We developed this flexible pattern particle-labeling (FpPL) technique using synthetic test images and human carotid artery tissue sections. A conventional image particle counting method (preinstalled in ImageJ) did not properly recognize small calcified particles located in noisy images that include complex extracellular matrix structures and other commonly used pattern-matching methods failed to detect the wide variation in size, shape, and brightness exhibited by the particles. Comparative experiments with the ImageJ particle counting method demonstrated that our method detected significantly more (p < 2 × 10-7) particles than the conventional method with significantly fewer (p < 0.0003) false positives and false negatives (p < 0.0003). We then applied the FpPL technique to CAVD leaflets and showed a significant increase in detected particles in the fibrosa at the base of the leaflets (p < 0.0001), supporting our hypothesis. The outcomes of this study are twofold: (1) development of a new image analysis technique that can be adapted to a wide range of applications and (2) acquisition of new insight on potential early mediators of calcification in CAVD.

Project description:Calcific Aortic Valve Disease (CAVD) is the most frequent valvular heart disease in developed countries. It is characterized by a progressive calcification of aortic valve leaflets mediated by myeloid cells, such as monocytes and macrophages. However, the molecular mechanisms involved in their recruitment and activity are still poorly understood and limit the development of a pharmacological treatments. Previous studies have already demonstrated the role of macrophages in CAVD development. However, little is known about the implication of their precursors, monocytes. Thus, we hypothesized that circulating monocytes from CAVD patients have higher pro-calcific and pro-inflammatory capacities. We aimed first to assess the pro-calcifying potential of circulating monocytes from patients with CAVD and second to better understand the molecular mechanisms of this pro-calcific effect through a whole transcriptomic study. Methods : We first characterized monocytes sub-population, from PBMCs isolated from peripheral blood of healthy volunteers (Vol) and patients with CAVD or without CAVD (NCAVD), with flow cytometry. Then we analyzed the whole transcriptome of these monocytes by RNA sequencing (RNAseq) to identify dysregulated genes expression, further confirmed by RT-qPCR. Finally, after evaluation of monocytes secretome with multiplex analysis, we evaluated CAVD monocytes capacities to induce myofibroblastic transdifferenciation and osteoblastic differencitation of human valvular interstitial cells (hVIC) with immunocytochemistry (ICC) and O-cresolphtalein assay. Results : In CAVD (calcific aortic valve disease) monocytes, we observe a shift in their sub-populations, with an increased frequency of classical monocytes (CD14+CD16-) and a decreased frequency of non-classical monocytes (CD14+CD16++). Interestingly, we found specific upregulation of genes linked to both inflammation (PDK4) and calcification (ATP2B1), alongside the downregulation of immunomodulatory genes (DDR1, IKBKE) in monocytes from CAVD patients. Additionally, CAVD monocytes produce lower levels of immunomodulatory and anti-osteoblastogenic cytokines (IL3, CCL3), which promote myofibroblastic transdifferentiation (TIMP1, TNC) and osteoblastic differentiation (ALPL, OPG). This correlates with an increase in αsma+ and opn+ cells and a doubling of hVIC calcifications. Conclusions : We demonstrated for the first time that circulating monocytes from CAVD patients have higher pro-inflammatory and pro-calcific capacities that might promote CAVD development. Likewise, we identify dysregulated genes, which could be used as new therapeutic targets.

Project description:Calcific aortic valve disease (CAVD) is characterized by progressive stiffening of aortic valve (AV) tissues, inducing stenosis and insufficiency. Bicuspid aortic valve (BAV) is a common congenital defect in which the AV has two leaflets rather than three, with BAV patients developing CAVD decades years earlier than in the general population. Current treatment for CAVD remains surgical replacement with its continued durability problems, as there are no pharmaceutical therapies or other alternative treatments available. Before such therapeutic approaches can be developed, a deeper understanding of CAVD disease mechanisms is clearly required. It is known that AV interstitial cells (AVICs) maintain the AV extracellular matrix and are typically quiescent in the normal state, transitioning into an activated, myofibroblast-like state during periods of growth or disease. One proposed mechanism of CAVD is the subsequent transition of AVICs into an osteoblast-like phenotype. A sensitive indicator of AVIC phenotypic state is enhanced basal contractility (tonus), so that AVICs from diseased AV will exhibit a higher basal tonus level. The goals of the present study were thus to assess the hypothesis that different human CAVD states lead to different biophysical AVIC states. To accomplish this, we characterized AVIC basal tonus behaviors from diseased human AV tissues embedded in 3D hydrogels. Established methods were utilized to track AVIC-induced gel displacements and shape changes after the application of Cytochalasin D (an actin polymerization inhibitor) to depolymerize the AVIC stress fibers. Results indicated that human diseased AVICs from the non-calcified region of TAVs were significantly more activated than AVICs from the corresponding calcified region. In addition, AVICs from the raphe region of BAVs were more activated than from the non-raphe region. Interestingly, we observed significantly greater basal tonus levels in females compared to males. Furthermore, the overall AVIC shape changes after Cytochalasin suggested that AVICs from TAVs and BAVs develop different stress fiber architectures. These findings are the first evidence of sex-specific differences in basal tonus state in human AVICs in varying disease states. Future studies are underway to quantify stress fiber mechanical behaviors to further elucidate CAVD disease mechanisms.

Dataset Information

Collagen fiber regulation in human pediatric aortic valve development and disease.

Publications

Collagen fiber regulation in human pediatric aortic valve development and disease.

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