Dataset Information

COX-2 Is Downregulated in Human Stenotic Aortic Valves and Its Inhibition Promotes Dystrophic Calcification.

ABSTRACT: Calcific aortic valve disease (CAVD) is the result of maladaptive fibrocalcific processes leading to a progressive thickening and stiffening of aortic valve (AV) leaflets. CAVD is the most common cause of aortic stenosis (AS). At present, there is no effective pharmacotherapy in reducing CAVD progression; when CAVD becomes symptomatic it can only be treated with valve replacement. Inflammation has a key role in AV pathological remodeling; hence, anti-inflammatory therapy has been proposed as a strategy to prevent CAVD. Cyclooxygenase 2 (COX-2) is a key mediator of the inflammation and it is the target of widely used anti-inflammatory drugs. COX-2-inhibitor celecoxib was initially shown to reduce AV calcification in a murine model. However, in contrast to these findings, a recent retrospective clinical analysis found an association between AS and celecoxib use. In the present study, we investigated whether variations in COX-2 expression levels in human AVs may be linked to CAVD. We extracted total RNA from surgically explanted AVs from patients without CAVD or with CAVD. We found that COX-2 mRNA was higher in non-calcific AVs compared to calcific AVs (0.013 ± 0.002 vs. 0.006 ± 0.0004; p < 0.0001). Moreover, we isolated human aortic valve interstitial cells (AVICs) from AVs and found that COX-2 expression is decreased in AVICs from calcific valves compared to AVICs from non-calcific AVs. Furthermore, we observed that COX-2 inhibition with celecoxib induces AVICs trans-differentiation towards a myofibroblast phenotype, and increases the levels of TGF-?-induced apoptosis, both processes able to promote the formation of calcific nodules. We conclude that reduced COX-2 expression is a characteristic of human AVICs prone to calcification and that COX-2 inhibition may promote aortic valve calcification. Our findings support the notion that celecoxib may facilitate CAVD progression.

SUBMITTER: Vieceli Dalla Sega F

PROVIDER: S-EPMC7727817 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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COX-2 Is Downregulated in Human Stenotic Aortic Valves and Its Inhibition Promotes Dystrophic Calcification.

Vieceli Dalla Sega Francesco F Fortini Francesca F Cimaglia Paolo P Marracino Luisa L Tonet Elisabetta E Antonucci Antonio A Moscarelli Marco M Campo Gianluca G Rizzo Paola P Ferrari Roberto R

International journal of molecular sciences 20201124 23

Calcific aortic valve disease (CAVD) is the result of maladaptive fibrocalcific processes leading to a progressive thickening and stiffening of aortic valve (AV) leaflets. CAVD is the most common cause of aortic stenosis (AS). At present, there is no effective pharmacotherapy in reducing CAVD progression; when CAVD becomes symptomatic it can only be treated with valve replacement. Inflammation has a key role in AV pathological remodeling; hence, anti-inflammatory therapy has been proposed as a s ...[more]

PMID: 33255450

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Project description:Purpose: The goal of this study is to characterize the gene expression profiles and identify genes of interest (GOI) in stenotic (AS) and regurgitant (AI) human aortic valves using RNA sequencing technology. Methods: Aortic valve leaflets were collected from non-matched transplant donor hearts (NC) and from aortic valve replacement operations (AS or AI). Leaflets were washed in cold PBS, snap frozen, and stored at -80°C until RNA extraction. For each sample, total RNA was extracted, a cDNA library was generated, and an Illumina HiSeq 2500 was used to sequence each sample. 150-bp paired-ends reads were generated with approximately 30 million reads per sample. Sequencing was performed in duplicate for each sample. Paired-end clean reads were aligned to the reference genome using STAR. HTSeq v0.6.1 was used to count the read numbers mapped to each gene. Fragments per kilobases per million (FPKM) were calculated to estimate the level of gene expression. Differential gene expression analysis was performed using the DESeq2 R package. A model based on the negative binomial distribution was used to determine differential gene expression (log 2 [fold change]). P-values were adjusted using the Benjamini-Hochberg approach for controlling false discovery rate. Genes with adjusted P-values <0.05 found by DESeq2 were assigned as differentially-expressed genes (DEGs). Results: RNA sequencing analysis identified 8,621 DEGs among the AS, AI, and NC valves. Specifically, 6,438 genes were found to be differentially expressed between the AS and NC groups; 4,994 genes were found to be differentially expressed between the AI and NC groups; and 2,771 genes were found to be differentially expressed between the AS and AI groups. Of these, 1,979 (31%) genes were expressed at statistically different levels between the AS and NC groups, and 1,428 (29%) genes were expressed at statistically different levels between the AI and NC groups Conclusions: Overall, we have sequenced aortic leaflets from non-matched transplant donor hearts , thereby establishing a new standard for normal aortic valve transcriptional profiling. We have also sequenced human aortic valves with AI and demonstrated that valves with AI and AS possess unique gene expression patterns. The plethora of DEGs identified and our novel PPI networks for AS and AI have revealed new pathways missed by previous aortic valve studies. Our data lays the foundation for future gene editing studies and may guide researchers in developing novel therapeutic interventions for AS and AI.

Project description:Calcific aortic valve disease (CAVD) is an inexorably degenerative pathology characterized by progressive calcific lesion formation on the valve leaflets. The interaction of valvular cells in advanced lesion environments is not well understood yet highly relevant as clinically detectable CAVD exhibits calcifications composed of non-stoichiometric hydroxyapatite (HA). In this study, Fourier transform infrared spectroscopic imaging was used to spatially analyze mineral properties as a function of disease progression. Crystallinity (size and perfection) increased with increased valve calcification. To study the relationship between crystallinity and cellular behavior in CAVD, valve cells were seeded into 3D mineral-rich collagen gels containing synthetic HA particles, which had varying crystallinities. Lower crystallinity HA drove myofibroblastic activation in both valve interstitial and endothelial cells, as well as osteoblastic differentiation in interstitial cells. Additionally, calcium accumulation within gels depended on crystallinity, and apoptosis was insufficient to explain differences in HA-driven cellular activity. The protective nature of endothelial cells against interstitial cell activation and calcium accumulation was completely inhibited in the presence of less crystalline HA particles. Elucidating valve cellular behavior post-calcification is of vital importance to better predict and treat clinical pathogenesis, and mineral-containing hydrogel models provide a unique 3D platform to evaluate valve cell responses to a later stage of valve disease.Statement of significanceWe implement a 3D in vitro platform with embedded hydroxyapatite (HA) nanoparticles to investigate the interaction between valve interstitial cells, valve endothelial cells, and a mineral-rich extracellular environment. HA nanoparticles were synthesized based on analysis of the mineral properties of calcific regions of diseased human aortic valves. Our findings indicate that crystallinity of HA drives activation and differentiation in interstitial and endothelial cells. We also show that a mineralized environment blocks endothelial protection against interstitial cell calcification. Our HA-containing hydrogel model provides a unique 3D platform to evaluate valve cell responses to a mineralized ECM. This study additionally lays the groundwork to capture the diversity of mineral properties in calcified valves, and link these properties to progression of the disease.

Dataset Information

COX-2 Is Downregulated in Human Stenotic Aortic Valves and Its Inhibition Promotes Dystrophic Calcification.

Publications

COX-2 Is Downregulated in Human Stenotic Aortic Valves and Its Inhibition Promotes Dystrophic Calcification.

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