Role of myocardin-related transcription factors in myofibroblastic activation of valvular interstitial cells from aortic valves

Calcific aortic valve stenosis (CAVS) is a heart disease characterized by the progressive fibro-calcific remodeling of the aortic valves, an actively regulated process with the involvement of the reactive oxygen species-mediated differentiation of valvular interstitial cells (VICs) into osteoblast-like cells. Nuclear factor erythroid 2-related factor 2 (Nrf2) regulates the expression of a variety of antioxidant genes, and plays a protective role in valve calcification. Heme oxygenase-1 (HO-1), an Nrf2-target gene, is upregulated in human calcified aortic valves. Therefore, we investigated the effect of Nrf2/HO-1 axis in VIC calcification. We induced osteogenic differentiation of human VICs with elevated phosphate and calcium-containing osteogenic medium (OM) in the presence of heme. Heme inhibited Ca deposition and OM-induced increase in alkaline phosphatase and osteocalcin (OCN) expression. Heme induced Nrf2 and HO-1 expression in VICs. Heme lost its anti-calcification potential when we blocked transcriptional activity Nrf2 or enzyme activity of HO-1. The heme catabolism products bilirubin, carbon monoxide, and iron, and also ferritin inhibited OM-induced Ca deposition and OCN expression in VICs. This study suggests that heme-mediated activation of the Nrf2/HO-1 pathway inhibits the calcification of VICs. The anti-calcification effect of heme is attributed to the end products of HO-1-catalyzed heme degradation and ferritin.

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Upregulated Autophagy in Calcific Aortic Valve Stenosis Confers Protection of Valvular Interstitial Cells

International Journal of Molecular Sciences ◽

10.3390/ijms20061486 ◽

2019 ◽

Vol 20 (6) ◽

pp. 1486 ◽

Cited By ~ 6

Author(s):

Miguel Carracedo ◽

Oscar Persson ◽

Peter Saliba-Gustafsson ◽

Gonzalo Artiach ◽

Ewa Ehrenborg ◽

...

Keyword(s):

Aortic Valve ◽

Aortic Valve Stenosis ◽

Interstitial Cells ◽

Left Ventricular ◽

Autophagic Flux ◽

Valvular Interstitial Cells ◽

Survival Mechanism ◽

Calcified Tissue ◽

Left Ventricular Outflow

Autophagy serves as a cell survival mechanism which becomes dysregulated under pathological conditions and aging. Aortic valve thickening and calcification causing left ventricular outflow obstruction is known as calcific aortic valve stenosis (CAVS). CAVS is a chronic and progressive disease which increases in incidence and severity with age. Currently, no medical treatment exists for CAVS, and the role of autophagy in the disease remains largely unexplored. To further understand the role of autophagy in the progression of CAVS, we analyzed expression of key autophagy genes in healthy, thickened, and calcified valve tissue from 55 patients, and compared them with nine patients without significant CAVS, undergoing surgery for aortic regurgitation (AR). This revealed a upregulation in autophagy exclusively in the calcified tissue of CAVS patients. This difference in autophagy between CAVS and AR was explored by LC3 lipidation in valvular interstitial cells (VICs), revealing an upregulation in autophagic flux in CAVS patients. Inhibition of autophagy by bafilomycin-A1 led to a decrease in VIC survival. Finally, treatment of VICs with high phosphate led to an increase in autophagic activity. In conclusion, our data suggests that autophagy is upregulated in the calcified tissue of CAVS, serving as a compensatory and pro-survival mechanism.

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3D-Bioprinting of Valvular Interstitial Cells of Ovine Aortic Valves: Impact of Printing Parameters on Cell Viability

10.1055/s-0041-1725707 ◽

2021 ◽

Author(s):

M. B. Immohr ◽

M. Barth ◽

F. Santos ◽

Y. Sugimura ◽

A. Lichtenberg ◽

...

Keyword(s):

Cell Viability ◽

Interstitial Cells ◽

3D Bioprinting ◽

Valvular Interstitial Cells ◽

Aortic Valves ◽

Printing Parameters

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Abstract 673: KLF10 is a Critical Mediator of Wnt Signaling in Aortic Valve Interstitial Cells

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.34.suppl_1.673 ◽

2014 ◽

Vol 34 (suppl_1) ◽

Author(s):

Nalini M Rajamannan ◽

Muzaffer Cicek ◽

John Hawse ◽

Thomas Spelsberg ◽

Malayannan Subramaniam

Keyword(s):

Wnt Signaling ◽

Wnt Signaling Pathway ◽

Fold Increase ◽

Interstitial Cells ◽

Aortic Valves ◽

Valve Interstitial Cells ◽

Over Expression ◽

Differentiated Cells ◽

Undifferentiated Cells

We have previously demonstrated that β-catenin plays important roles in valve calcification with a specific osteogenic phenotype defined by increased bone mineral content and overall valve thickening. Recent studies indicate that KLF10 may be involved in mediating the Wnt signaling pathway in bone, which is known to play critical roles in osteoblast differentiation and mineralization. Therefore, we sought to test the role of KLF10 in mediating Wnt signaling, as well as differentiation and mineralization, in valve interstitial cells (VICs) isolated from porcine valves. Exposure of VICs to differentiation media led to increased expression of Runx2, Sox9 and osteocalcin. Differentiated cells also stained positive with Von Kossa while undifferentiated cells stained negative confirming the induction of an osteogenic phenotype. As expected, over-expression of both Lef1 and β-catenin led to activation of the top-flash reporter when transfected into VICs. Interestingly, over-expression of KLF10 also significantly up-regulated the top-flash reporter alone and further enhanced the activity of both Lef1 and β-catenin when co-transfected. We further confirmed the role of TIEG in an atherosclerotic mouse model ApoE/LRP5 double KO and found a two-fold increase in KLF10, Lrp6, and Runx2 expression in the cholesterol treated aortic valves as compared to controls. These data suggested that KLF10, Lef1 and β-catenin interact with each other to form a transcriptionally active protein complex leading to enhanced Wnt signaling in VICs. This possibility was further confirmed by the observation that KLF10 and β-catenin co-localize with one another in the nucleus of VICs following stimulation with LiCl and/or TGF-β. Taken together, these data implicate an important role for KLF10 in mediating Wnt signaling and Lef1 transcriptional activity in VICs, and implicate a potential role for canonical Wnt signaling in the observed osteogenic bone phenotype of cardiac aortic valves.

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Abstract 303: CD44 Activation Protects Human Sclerotic-derived Valvular Interstitial Cells from Calcification

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.33.suppl_1.a303 ◽

2013 ◽

Vol 33 (suppl_1) ◽

Author(s):

Paolo Poggio ◽

Emanuela Branchetti ◽

Rachana Sainger ◽

Juan Grau ◽

Eric Lai ◽

...

Keyword(s):

Ex Vivo ◽

Early Stage ◽

Interstitial Cells ◽

Calcium Deposition ◽

Proximity Ligation Assay ◽

Valvular Interstitial Cells ◽

Direct Role ◽

Aortic Sclerosis

Introduction. The activation of Valvular Interstitial Cells (VIC) towards an osteoblast-like phenotype is a cellular hallmark of pathological progression towards Aortic Stenosis (AS). In recent years several clinical trials have failed to halt or revert the progression of this prevalent disease. The ability to prevent end-stage AS requires the understanding of the molecular events associated with the early phase of valve degeneration, a condition known as Aortic Sclerosis (ASc). In the last few years the transmembrane receptor CD44 has been studied as a putative molecule for cardiovascular drug therapy. We reported that the functional interaction between CD44 and one of its ligand, Osteopontin (OPN), protects vascular smooth muscle cells from calcification. More recently, we demonstrated that sclerotic tissues show increased expression of Bone Morphogenetic Protein 4 (BMP4) and BMP4 directly stimulates osteoblast-like transdifferentiation and calcification of VICs. Therefore we hypothesized a direct role of CD44 activation in protecting human Aortic Sclerosis-derived VICs from calcification. Methods. Human VICs from Control, ASc, and AS (n=5 each group) were isolated. Histological, cellular and molecular analysis, and in situ Proximity Ligation Assay were used to investigate the role of CD44 and OPN in VIC calcification. BMP4 treatments were used to promote VIC activation. Osteoblast-like transdifferentiation was analyzed using Alkaline Phosphatase (ALP) expression. Results. CD44 and OPN, as well as their functional binding, were increased in sclerotic and stenotic tissues compared to healthy controls in vitro and ex vivo. CD44-OPN binding prevented in vitro calcification induced by inorganic phosphate on human ASc-derived VICs. A neutralizing antibody against CD44, under BMP4 treatments, promoted calcium deposition along with increased expression of OPN and ALP. Conclusion. Our results generate an important insight into the molecular mechanism of VIC calcification. We proved that CD44-OPN direct interaction inhibits calcification of Aortic Sclerosis-derived VICs, suggesting that CD44 activation could have a protective role against VIC osteoblast-like transdifferentiation and calcification in the early stage of the disease.

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0312: Characterization of human valvular interstitial cells isolated from normal and fibrocalcified aortic valves

Archives of Cardiovascular Diseases Supplements ◽

10.1016/s1878-6480(14)71463-6 ◽

2014 ◽

Vol 6 ◽

pp. 73

Author(s):

Anais Arbesu Y Miar ◽

Anais Toursel ◽

Alexandre Ung ◽

Rodrigo Lorenzi ◽

Ahmed Elkalioubie ◽

...

Keyword(s):

Interstitial Cells ◽

Valvular Interstitial Cells ◽

Aortic Valves

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Abstract 15192: Leptin Effects on Osteoblast Differentiation of Human Valvular Interstitial Cells: New Insight Into Calcific Aortic Valve Disease

Circulation ◽

10.1161/circ.132.suppl_3.15192 ◽

2015 ◽

Vol 132 (suppl_3) ◽

Author(s):

Mickael Rosa ◽

Rodrigo Lorenzi ◽

Madjid Tagzirt ◽

Francis Juthier ◽

Antoine Rauch ◽

...

Keyword(s):

Aortic Valve ◽

Aortic Valve Disease ◽

Osteoblast Differentiation ◽

Interstitial Cells ◽

Valve Disease ◽

Calcium Deposition ◽

Valvular Interstitial Cells ◽

Calcific Aortic Valve Disease ◽

Aortic Valves ◽

Alp Activity

Introduction: Calcific aortic valve disease (CAVD) affects 2% to 6% of the population over 65 years and results from dysregulated processes such as calcification, supported in part by the osteoblast differentiation of valvular interstitial cells (VIC), the most prevalent cell type in the human aortic valves. Leptin has recently been linked to aortic valve calcification in ApoE-/- mice. Hypothesis: Our hypothesis is that leptin could play a role in the calcifying processes implicated in CAVD via direct effects on human VIC. Methods: Patients who underwent aortic valve replacement for severe CAVD (n=43) or with coronary artery disease (CAD) but without CAVD (n=129) were included in this study. Presence of leptin was analyzed in human explanted calcified aortic valves and blood samples. Leptin receptors expression was analyzed in aortic valves and VIC isolated from aortic valves. Leptin effects on osteoblast differentiation of VIC in presence or not of Akt and ERK inhibitors were investigated by alizarin red staining, alkaline phosphatase (ALP) activity, and RT-qPCR analysis for osteopontin, ALP, bone morphogenetic protein BMP-2, and RUNX2. Results: Patients with CAVD have significant higher serum leptin concentration than CAD patients (p=0.002). The presence of leptin was observed by immunochemistry in human calcified aortic valves, with higher concentrations in calcified vs non-calcified zones (p=0.01). Both short and long leptin receptor isoforms were expressed in VIC. Chronic leptin stimulation of VIC enhanced ALP, BMP-2 and RUNX2 expression and decreased osteopontin expression. This treatment led to a higher, dose dependent, ALP activity and calcium deposition in VIC. Inhibiting Akt or ERK during leptin stimulation led to a reduced calcification by bringing the expression of calcification genes to the control levels. Conclusions: Together, these novel findings depict the potential role of leptin in the process of CAVD by triggering calcification processes in human VIC.

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