Mechanistic Roles of Matrilin-2 and Klotho in Modulating the Inflammatory Activity of Human Aortic Valve Cells

Background: Calcific aortic valve disease (CAVD) is a chronic inflammatory disease. Soluble extracellular matrix (ECM) proteins can act as damage-associated molecular patterns and may induce valvular inflammation. Matrilin-2 is an ECM protein and has been found to elevate the pro-osteogenic activity in human aortic valve interstitial cells (AVICs). Klotho, an anti-aging protein, appears to have anti-inflammatory properties. The effect of matrilin-2 and Klotho on AVIC inflammatory responses remains unclear. Methods and Results: Isolated human AVICs were exposed to matrilin-2. Soluble matrilin-2 induced the production of ICAM-1, MCP-1, and IL-6. It also induced protein kinase R (PKR) activation via Toll-like receptor (TLR) 2 and 4. Pretreatment with PKR inhibitors inhibited NF-κB activation and inflammatory mediator production induced by matrilin-2. Further, recombinant Klotho suppressed PKR and NF-κB activation and markedly reduced the production of inflammatory mediators in human AVICs exposed to matrilin-2. Conclusions: This study revealed that soluble matrilin-2 upregulates AVIC inflammatory activity via activation of the TLR-PKR-NF-κB pathway and that Klotho is potent to suppress AVIC inflammatory responses to a soluble ECM protein through inhibiting PKR. These novel findings indicate that soluble matrilin-2 may accelerate the progression of CAVD by inducing valvular inflammation and that Klotho has the potential to suppress valvular inflammation.

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

International Journal of Molecular Sciences ◽

10.3390/ijms21238917 ◽

2020 ◽

Vol 21 (23) ◽

pp. 8917

Author(s):

Francesco Vieceli Dalla Sega ◽

Francesca Fortini ◽

Paolo Cimaglia ◽

Luisa Marracino ◽

Elisabetta Tonet ◽

...

Keyword(s):

Aortic Valve ◽

Clinical Analysis ◽

Dystrophic Calcification ◽

Calcific Aortic Valve Disease ◽

Aortic Valves ◽

Inflammatory Drugs ◽

Cox 2 ◽

Anti Inflammatory ◽

Induced Apoptosis ◽

Human Aortic Valve

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-β-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.

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TLR4 Stimulation Promotes Human AVIC Fibrogenic Activity through Upregulation of Neurotrophin 3 Production

International Journal of Molecular Sciences ◽

10.3390/ijms21041276 ◽

2020 ◽

Vol 21 (4) ◽

pp. 1276

Author(s):

Qingzhou Yao ◽

Erlinda The ◽

Lihua Ao ◽

Yufeng Zhai ◽

Maren K. Osterholt ◽

...

Keyword(s):

Aortic Valve ◽

Critical Role ◽

Chronic Inflammatory Disease ◽

Collagen Deposition ◽

Calcific Aortic Valve Disease ◽

Aortic Valves ◽

Neurotrophin 3 ◽

Collagen Iii ◽

Inflammatory Stimuli ◽

Stimulation Of

Background: Calcific aortic valve disease (CAVD) is a chronic inflammatory disease that manifests as progressive valvular fibrosis and calcification. An inflammatory milieu in valvular tissue promotes fibrosis and calcification. Aortic valve interstitial cell (AVIC) proliferation and the over-production of the extracellular matrix (ECM) proteins contribute to valvular thickening. However, the mechanism underlying elevated AVIC fibrogenic activity remains unclear. Recently, we observed that AVICs from diseased aortic valves express higher levels of neurotrophin 3 (NT3) and that NT3 exerts pro-osteogenic and pro-fibrogenic effects on human AVICs. Hypothesis: Pro-inflammatory stimuli upregulate NT3 production in AVICs to promote fibrogenic activity in human aortic valves. Methods and Results: AVICs were isolated from normal human aortic valves and were treated with lipopolysaccharide (LPS, 0.20 µg/mL). LPS induced TLR4-dependent NT3 production. This effect of LPS was abolished by inhibition of the Akt and extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) pathways. The stimulation of TLR4 in human AVICs with LPS resulted in a greater proliferation rate and an upregulated production of matrix metallopeptidases-9 (MMP-9) and collagen III, as well as augmented collagen deposition. Recombinant NT3 promoted AVIC proliferation in a tropomyosin receptor kinase (Trk)-dependent fashion. The neutralization of NT3 or the inhibition of Trk suppressed LPS-induced AVIC fibrogenic activity. Conclusions: The stimulation of TLR4 in human AVICs upregulates NT3 expression and promotes cell proliferation and collagen deposition. The NT3-Trk cascade plays a critical role in the TLR4-mediated elevation of fibrogenic activity in human AVICs. Upregulated NT3 production by endogenous TLR4 activators may contribute to aortic valve fibrosis associated with CAVD progression.

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Wound-induced Ca2+ wave propagates through a simple release and diffusion mechanism

Molecular Biology of the Cell ◽

10.1091/mbc.e16-10-0695 ◽

2017 ◽

Vol 28 (11) ◽

pp. 1457-1466 ◽

Cited By ~ 14

Author(s):

L. Naomi Handly ◽

Roy Wollman

Keyword(s):

Diffusion Model ◽

Tissue Damage ◽

Atp Hydrolysis ◽

Inflammatory Responses ◽

Diffusion Mechanism ◽

Fast Activation ◽

Molecular Patterns ◽

Damage Associated Molecular Patterns ◽

And Diffusion ◽

Source And Sink

Damage-associated molecular patterns (DAMPs) are critical mediators of information concerning tissue damage from damaged cells to neighboring healthy cells. ATP acts as an effective DAMP when released into extracellular space from damaged cells. Extracellular ATP receptors monitor tissue damage and activate a Ca2+ wave in the surrounding healthy cells. How the Ca2+ wave propagates through cells after a wound is unclear. Ca2+ wave activation can occur extracellularly via external receptors or intracellularly through GAP junctions. Three potential mechanisms to propagate the Ca2+ wave are source and sink, amplifying wave, and release and diffusion. Both source and sink and amplifying wave regulate ATP levels using hydrolysis or secretion, respectively, whereas release and diffusion relies on dilution. Here we systematically test these hypotheses using a microfluidics assay to mechanically wound an epithelial monolayer in combination with direct manipulation of ATP hydrolysis and release. We show that a release and diffusion model sufficiently explains Ca2+-wave propagation after an epithelial wound. A release and diffusion model combines the benefits of fast activation at short length scales with a self-limiting response to prevent unnecessary inflammatory responses harmful to the organism.

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miRNA Expression Profiling Uncovers a Role of miR-139-5p in Regulating the Calcification of Human Aortic Valve Interstitial Cells

Frontiers in Genetics ◽

10.3389/fgene.2021.722564 ◽

2021 ◽

Vol 12 ◽

Author(s):

Fan Zhang ◽

Naixuan Cheng ◽

Yingchun Han ◽

Congcong Zhang ◽

Haibo Zhang

Keyword(s):

Aortic Valve ◽

Osteogenic Differentiation ◽

Mirna Expression ◽

Structural Heart Disease ◽

Interstitial Cells ◽

Calcific Aortic Valve Disease ◽

Valve Interstitial Cells ◽

Functional Studies ◽

Human Aortic Valve ◽

Osteogenic Gene

Calcific aortic valve disease (CAVD) is the most common structural heart disease, and the morbidity is increased with elderly population. Several microRNAs (miRNAs) have been identified to play crucial roles in CAVD, and numerous miRNAs are still waiting to be explored. In this study, the miRNA expression signature in CAVD was analyzed unbiasedly by miRNA-sequencing, and we found that, compared with the normal control valves, 152 miRNAs were upregulated and 186 miRNAs were downregulated in calcified aortic valves. The functions of these differentially expressed miRNAs were associated with cell differentiation, apoptosis, adhesion and immune response processes. Among downregulated miRNAs, the expression level of miR-139-5p was negatively correlated with the osteogenic gene RUNX2, and miR-139-5p was also downregulated during the osteogenic differentiation of primary human aortic valve interstitial cells (VICs). Subsequent functional studies revealed that miR-139-5p overexpression inhibited the osteogenic differentiation of VICs by negatively modulating the expression of pro-osteogenic gene FZD4 and CTNNB1. In conclusion, these results suggest that miR-139-5p plays an important role in osteogenic differentiation of VICs via the Wnt/β-Catenin pathway, which may further provide a new therapeutic target for CAVD.

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Untangling Local Pro-Inflammatory, Reparative, and Regulatory Damage-Associated Molecular-Patterns (DAMPs) Pathways to Improve Transplant Outcomes

Frontiers in Immunology ◽

10.3389/fimmu.2021.611910 ◽

2021 ◽

Vol 12 ◽

Author(s):

Gaelen K. Dwyer ◽

Hēth R. Turnquist

Keyword(s):

Organ Transplantation ◽

Immune Cells ◽

Inflammatory Responses ◽

Ischemia Reperfusion ◽

Solid Organ Transplantation ◽

Experimental Studies ◽

Surgical Trauma ◽

Solid Organ ◽

Molecular Patterns ◽

Damage Associated Molecular Patterns

Detrimental inflammatory responses after solid organ transplantation are initiated when immune cells sense pathogen-associated molecular patterns (PAMPs) and certain damage-associated molecular patterns (DAMPs) released or exposed during transplant-associated processes, such as ischemia/reperfusion injury (IRI), surgical trauma, and recipient conditioning. These inflammatory responses initiate and propagate anti-alloantigen (AlloAg) responses and targeting DAMPs and PAMPs, or the signaling cascades they activate, reduce alloimmunity, and contribute to improved outcomes after allogeneic solid organ transplantation in experimental studies. However, DAMPs have also been implicated in initiating essential anti-inflammatory and reparative functions of specific immune cells, particularly Treg and macrophages. Interestingly, DAMP signaling is also involved in local and systemic homeostasis. Herein, we describe the emerging literature defining how poor outcomes after transplantation may result, not from just an over-abundance of DAMP-driven inflammation, but instead an inadequate presence of a subset of DAMPs or related molecules needed to repair tissue successfully or re-establish tissue homeostasis. Adverse outcomes may also arise when these homeostatic or reparative signals become dysregulated or hijacked by alloreactive immune cells in transplant niches. A complete understanding of the critical pathways controlling tissue repair and homeostasis, and how alloimmune responses or transplant-related processes disrupt these will lead to new immunotherapeutics that can prevent or reverse the tissue pathology leading to lost grafts due to chronic rejection.

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Cell-Type Transcriptome Atlas of Human Aortic Valves Reveal Cell Heterogeneity and Endothelial to Mesenchymal Transition Involved in Calcific Aortic Valve Disease

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvbaha.120.314789 ◽

2020 ◽

Vol 40 (12) ◽

pp. 2910-2921

Author(s):

Kang Xu ◽

Shangbo Xie ◽

Yuming Huang ◽

Tingwen Zhou ◽

Ming Liu ◽

...

Keyword(s):

Aortic Valve ◽

Single Cell ◽

Aortic Valve Disease ◽

Valve Disease ◽

Cell Heterogeneity ◽

Calcific Aortic Valve Disease ◽

Mesenchymal Transition ◽

Endothelial To Mesenchymal Transition ◽

Human Aortic Valve ◽

Insight Into

Objective: Leaflet thickening, fibrosis, and hardening are early pathological features of calcific aortic valve disease (CAVD). An inadequate understanding of the resident aortic valve cells involved in the pathological process may compromise the development of therapeutic strategies. We aim to construct a pattern of the human aortic valve cell atlas in healthy and CAVD clinical specimens, providing insight into the cellular origins of CAVD and the complex cytopathological differentiation process. Approach and Results: We used unbiased single-cell RNA sequencing for the high-throughput evaluation of cell heterogeneity in 34 632 cells isolated from 6 different human aortic valve leaflets. Cellular experiments, in situ localization, and bulk sequencing were performed to verify the differences between normal, healthy valves and those with CAVD. By comparing healthy and CAVD specimens, we identified 14 cell subtypes, including 3 heterogeneous subpopulations of resident valve interstitial cells, 3 types of immune-derived cells, 2 types of valve endothelial cells, and 6 novel valve-derived stromal cells found particularly in CAVD leaflets. Combining additional verification experiments with single-cell transcriptome profiling provided evidence of endothelial to mesenchymal transition involved in lesion thickening of the aortic valve leaflet. Conclusions: Our findings deconstructed the aortic valve cell atlas and suggested novel functional interactions among resident cell subpopulations. Our findings may provide insight into future targeted therapies to prevent CAVD.

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Cranberry Proanthocyanidins Neutralize the Effects of Aggregatibacter actinomycetemcomitans Leukotoxin

Toxins ◽

10.3390/toxins11110662 ◽

2019 ◽

Vol 11 (11) ◽

pp. 662 ◽

Cited By ~ 1

Author(s):

Amel Ben Lagha ◽

Amy Howell ◽

Daniel Grenier

Keyword(s):

Gene Expression ◽

Inflammatory Responses ◽

Disease Process ◽

Aggregatibacter Actinomycetemcomitans ◽

Aggressive Periodontitis ◽

Cytotoxic Activities ◽

Expression Of Genes ◽

Genes Encoding ◽

Molecular Patterns ◽

Damage Associated Molecular Patterns

Aggregatibacter actinomycetemcomitans is a Gram-negative bacterium that has been strongly associated with localized aggressive periodontitis. The capacity of A. actinomycetemcomitans to produce a leukotoxin (LtxA) that activates pyroptosis in macrophages and induces the release of endogenous danger signals is thought to play a key role in the disease process. The aim of the present study was to investigate the effects of cranberry proanthocyanidins (PACs) on gene expression and cytotoxic activities of LtxA. We showed that cranberry PACs dose-dependently attenuate the expression of genes making up the leukotoxin operon, including ltxB and ltxC, in the two strains of A. actinomycetemcomitans tested. Cranberry PACs (≥62.5 µg/mL) protected macrophages against the cytotoxic effect of purified LtxA. Moreover, cranberry PACs reduced caspase-1 activation in LtxA-treated macrophages and consequently decreased the release of both IL-1β and IL-18, which are known as damage-associated molecular patterns (DAMPs) and contribute to the progression of periodontitis by increasing cell migration and osteoclastogenesis. In addition, cranberry PACs reduced the expression of genes encoding the P2X7 receptor and NALP3 (NACHT, LRR and PYD domains-containing protein 3), which play key roles in pore formation and cell death. Lastly, cranberry PACs blocked the binding of LtxA to macrophages and consequently reduced the LtxA-mediated cytotoxicity. In summary, the present study showed that cranberry PACs reduced LtxA gene expression in A. actinomycetemcomitans and neutralized the cytolytic and pro-inflammatory responses of human macrophages treated with LtxA. Given these properties, cranberry PACs may represent promising molecules for prevention and treatment of the aggressive form of periodontitis caused by A. actinomycetemcomitans.

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DAMPening Inflammation by Modulating TLR Signalling

Mediators of Inflammation ◽

10.1155/2010/672395 ◽

2010 ◽

Vol 2010 ◽

pp. 1-21 ◽

Cited By ~ 452

Author(s):

A. M. Piccinini ◽

K. S. Midwood

Keyword(s):

Immune System ◽

Tissue Damage ◽

Tissue Repair ◽

Inflammatory Responses ◽

Current Knowledge ◽

Feedback Loops ◽

Inflammatory Gene Expression ◽

Molecular Patterns ◽

Damage Associated Molecular Patterns ◽

Tlr Signalling

Damage-associated molecular patterns (DAMPs) include endogenous intracellular molecules released by activated or necrotic cells and extracellular matrix (ECM) molecules that are upregulated upon injury or degraded following tissue damage. DAMPs are vital danger signals that alert our immune system to tissue damage upon both infectious and sterile insult. DAMP activation of Toll-like receptors (TLRs) induces inflammatory gene expression to mediate tissue repair. However, DAMPs have also been implicated in diseases where excessive inflammation plays a key role in pathogenesis, including rheumatoid arthritis (RA), cancer, and atherosclerosis. TLR activation by DAMPs may initiate positive feedback loops where increasing tissue damage perpetuates pro-inflammatory responses leading to chronic inflammation. Here we explore the current knowledge about distinct signalling cascades resulting from self TLR activation. We also discuss the involvement of endogenous TLR activators in disease and highlight how specifically targeting DAMPs may yield therapies that do not globally suppress the immune system.

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Double-stranded RNA upregulates the expression of inflammatory mediators in human aortic valve cells through the TLR3-TRIF-noncanonical NF-κB pathway

AJP Cell Physiology ◽

10.1152/ajpcell.00230.2016 ◽

2017 ◽

Vol 312 (4) ◽

pp. C407-C417 ◽

Cited By ~ 9

Author(s):

Qiong Zhan ◽

Rui Song ◽

Fei Li ◽

Lihua Ao ◽

Qingchun Zeng ◽

...

Keyword(s):

Aortic Valve ◽

Inflammatory Mediators ◽

Inflammatory Mediator ◽

Inflammatory Responses ◽

Poly I:C ◽

Aortic Valves ◽

Double Stranded Rna ◽

Inflammatory Mediator Production ◽

Polycytidylic Acid ◽

Chronic Inflammatory Condition

Calcific aortic valve disease is a chronic inflammatory condition, and the inflammatory responses of aortic valve interstitial cells (AVICs) play a critical role in the disease progression. Double-stranded RNA (dsRNA) released from damaged or stressed cells is proinflammatory and may contribute to the mechanism of chronic inflammation observed in diseased aortic valves. The objective of this study is to determine the effect of dsRNA on AVIC inflammatory responses and the underlying mechanism. AVICs from normal human aortic valves were stimulated with polyinosinic-polycytidylic acid [poly(I:C)], a mimic of dsRNA. Poly(I:C) increased the production of IL-6, IL-8, monocyte chemoattractant protein-1, and ICAM-1. Poly(I:C) also induced robust activation of ERK1/2 and NF-κB. Knockdown of Toll-like receptor 3 (TLR3) or Toll-IL-1 receptor domain-containing adapter-inducing IFN-β (TRIF) suppressed ERK1/2 and NF-κB p65 phosphorylation and reduced inflammatory mediator production induced by poly(I:C). Inhibition of NF-κB, not ERK1/2, reduced inflammatory mediator production in AVICs exposed to poly(I:C). Interestingly, inhibition of NF-κB by prevention of p50 migration failed to suppress inflammatory mediator production. NF-κB p65 intranuclear translocation induced by the TLR4 agonist was reduced by inhibition of p50 migration; however, poly(I:C)-induced p65 translocation was not, although the p65/p50 heterodimer is present in AVICs. Poly(I:C) upregulates the production of multiple inflammatory mediators through the TLR3-TRIF-NF-κB pathway in human AVICs. The NF-κB activated by dsRNA appears not to be the canonical p65/p50 heterodimers.

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