Differentiation Of Valvular Interstitial Cells Research Articles

Enhancing aortic valve drug delivery with PAR2-targeting magnetic nano-cargoes for calcification alleviation

Calcific aortic valve disease is a prevalent cardiovascular disease with no available drugs capable of effectively preventing its progression. Hence, an efficient drug delivery system could serve as a valuable tool in drug screening and potentially enhance therapeutic efficacy. However, due to the rapid blood flow rate associated with aortic valve stenosis and the lack of specific markers, achieving targeted drug delivery for calcific aortic valve disease has proved to be challenging. Here we find that protease-activated-receptor 2 (PAR2) expression is up-regulated on the plasma membrane of osteogenically differentiated valvular interstitial cells. Accordingly, we develop a magnetic nanocarrier functionalized with PAR2-targeting hexapeptide for dual-active targeting drug delivery. We show that the nanocarriers effectively deliver XCT790—an anti-calcification drug—to the calcified aortic valve under extra magnetic field navigation. We demonstrate that the nano-cargoes consequently inhibit the osteogenic differentiation of valvular interstitial cells, and alleviate aortic valve calcification and stenosis in a high-fat diet-fed low-density lipoprotein receptor-deficient (Ldlr−/−) mouse model. This work combining PAR2- and magnetic-targeting presents an effective targeted drug delivery system for treating calcific aortic valve disease in a murine model, promising future clinical translation.

Upregulation of NOR-1 in calcified human vascular tissues: impact on osteogenic differentiation and calcification

Cardiovascular calcification is a significant public health issue whose pathophysiology is not fully understood. NOR-1 regulates critical processes in cardiovascular remodeling, but its contribution to ectopic calcification is unknown. NOR-1 was overexpressed in human calcific aortic valves and calcified atherosclerotic lesions colocalizing with RUNX2, a factor essential for osteochondrogenic differentiation and calcification. NOR-1 and osteogenic markers were upregulated in calcifying human valvular interstitial cells (VICs) and human vascular smooth muscle cells (VSMCs). Gain- and loss-of-function approaches demonstrated that NOR-1 negatively modulates the expression of osteogenic genes relevant for the osteogenic transdifferentiation (RUNX2, IL-6, BMP2, and ALPL) and calcification of VICs. VSMCs from transgenic mice overexpressing NOR-1 in these cells (TgNOR-1VSMC) expressed lower basal levels of osteogenic genes (IL-6, BMP2, ALPL, OPN) than cells from WT littermates, and their upregulation by a high-phosphate osteogenic medium (OM) was completely prevented by NOR-1 transgenesis. Consistently, this was associated with a dramatic reduction in the calcification of both transgenic VSMCs and aortic rings from TgNOR-1VSMC mice exposed to OM. Atherosclerosis and calcification were induce in mice by the administration of AAV-PCSK9D374Y and a high-fat/high-cholesterol diet. Challenged-TgNOR-1VSMC mice exhibited decreased vascular expression of osteogenic markers, and both less atherosclerotic burden (assessed in whole aorta and lesion size in aortic arch and brachiocephalic artery) and less vascular calcification (assessed either by near-infrared fluorescence imaging or histological analysis) than WT mice. Our data indicate that NOR-1 negatively modulates the expression of genes critically involved in the osteogenic differentiation of VICs and VSMCs, thereby restraining ectopic cardiovascular calcification.

Oxidized phospholipids facilitate calcific aortic valve disease by elevating ATF4 through the PERK/eIF2α axis.

In this study we sought to analyze the critical role of oxidized phospholipid (OxPL) in the progression of calcific aortic valve disease (CAVD) with the involvement of activating transcription factor 4 (ATF4). Differentially expressed genes related to CAVD were identified using bioinformatics analysis. Expression of ATF4 was examined in mouse models of aortic valve calcification (AVC) induced by the high cholesterol (HC) diet. Valvular interstitial cells (VICs) were then isolated from mouse non-calcified valve tissues, induced by osteogenic induction medium (OIM) and co-cultured with OxPAPC-stimulated macrophages. The effect of OxPLs regulating ATF4 on the macrophage polarization and osteogenic differentiation of VICs was examined with gain- and loss-of-function experiments in VICs and in vivo. In aortic valve tissues and OIM-induced VICs, ATF4 was highly expressed. ATF4 knockdown alleviated the osteogenic differentiation of VICs, as evidenced by reduced expression of bone morphogenetic protein-2 (BMP2), osteopontin (OPN), and osteocalcin. In addition, knockdown of ATF4 arrested the AVC in vivo. Meanwhile, OxPL promoted M1 polarization of macrophages and mediated osteogenic differentiation of VICs. Furthermore, OxPL up-regulated ATF4 expression through protein kinase R-like endoplasmic reticulum kinase (PERK)/eukaryotic translation initiation factor 2 subunit alpha (eIF2α) pathway. In conclusion, OxPL can potentially up-regulate the expression of ATF4, inducing macrophages polarized to M1 phenotype, osteogenic differentiation of VICs and AVC, thus accelerating the progression of CAVD.

TRPM4: New actor in osteogenic differentiation of human valvular interstitial cells

Transient receptor potential melastatin 4 (TRPM4) protein, a non-selective cation channel activated by calcium, is involved in cardiac fibroblasts remodeling. Its expression was reported in human valvular interstitial cells (VIC) with similar properties than fibroblasts, and its protein expression is increased on calcified aortic valves from patients suffering of aortic stenosis. VIC osteogenic differentiation is a key element in valvular calcification, which is, at term, responsible of aortic stenosis. The purpose of this study is to evaluate the implication of TRPM4 in human VIC osteogenic differentiation. VIC are isolated by enzymatic digestion from human aortic valves obtained from patients undergoing surgical aortic valve replacement. They are maintained 14 days in procalcifying media (2.7 mM calcium/2.5 mM phosphate), with or without 9-phenanthrol, a pharmacological TRPM4 inhibitor. Channel expression and functionality were followed respectively by PCR, Western Blot and electrophysiological recordings in the inside-out configuration of the patch clamp technique. Osteogenic differentiation of VIC was evaluated by measuring hydroxyapatite crystals by Alizarin red staining and by monitoring the protein expression of the osteogenic markers, BMP2 and RUNX2. A typical TRPM4 current was detected on VIC: its current-voltage relationship was linear with a conductance of 20.3 pS; the channel exhibited similar permeability for Na+ and K+; it was more activated in positive voltages and with increasing concentrations of internal Ca2+; the current was dose-dependently inhibited by 9-phenanthrol. TRPM4 mRNA and protein expression on VIC was confirmed by PCR and Western Blot. When maintained in procalcifying media, VIC developed hydroxyapatite crystals. Application of 9-phenanthrol (3.10-6 M) reduced mineralization by 30% without affecting cell viability. Moreover, it significantly reduced BMP2 and RUNX2 expression. Thereby, TRPM4 participates in osteogenic differentiation of human VIC in culture. Next studies of our unit are to evaluate whether TRPM4 repression by shRNA confirms those results.

Rho A/ROCK1 signaling-mediated metabolic reprogramming of valvular interstitial cells toward Warburg effect accelerates aortic valve calcification via AMPK/RUNX2 axis

The aberrant differentiation of valvular interstitial cells (VICs) to osteogenic lineages promotes calcified aortic valves disease (CAVD), partly activated by potentially destructive hemodynamic forces. These involve Rho A/ROCK1 signaling, a mechano-sensing pathway. However, how Rho A/ROCK1 signaling transduces mechanical signals into cellular responses and disrupts normal VIC homeostasis remain unclear. We examined Rho A/ROCK1 signaling in human aortic valves, and further detected how Rho A/ROCK1 signaling regulates mineralization in human VICs. Aortic valves (CAVD n = 22, normal control (NC) n = 12) from patients undergoing valve replacement were investigated. Immunostaining and western blotting analysis indicated that Rho A/ROCK1 signaling, as well as key transporters and enzymes involved in the Warburg effect, were markedly upregulated in human calcified aortic valves compared with those in the controls. In vitro, Rho A/ROCK1-induced calcification was confirmed as AMPK-dependent, via a mechanism involving metabolic reprogramming of human VICs to Warburg effect. Y-27632, a selective ROCK1 inhibitor, suppressed the Warburg effect, rescued AMPK activity and subsequently increased RUNX2 ubiquitin-proteasome degradation, leading to decreased RUNX2 protein accumulation in human VICs under pathological osteogenic stimulus. Rho A/ROCK1 signaling, which is elevated in human calcified aortic valves, plays a positive role in valvular calcification, partially through its ability to drive metabolic switching of VICs to the Warburg effect, leading to altered AMPK activity and RUNX2 protein accumulation. Thus, Rho A/ROCK1 signaling could be an important and unrecognized hub of destructive hemodynamics and cellular aerobic glycolysis that is essential to promote the CAVD process.

Hydrogen sulfide as an anti-calcification stratagem in human aortic valve: Altered biogenesis and mitochondrial metabolism of H2S lead to H2S deficiency in calcific aortic valve disease

Hydrogen sulfide (H2S) was previously revealed to inhibit osteoblastic differentiation of valvular interstitial cells (VICs), a pathological feature in calcific aortic valve disease (CAVD). This study aimed to explore the metabolic control of H2S levels in human aortic valves.Lower levels of bioavailable H2S and higher levels of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) were detected in aortic valves of CAVD patients compared to healthy individuals, accompanied by higher expression of cystathionine γ-lyase (CSE) and same expression of cystathionine β-synthase (CBS). Increased biogenesis of H2S by CSE was found in the aortic valves of CAVD patients which is supported by increased production of lanthionine. In accordance, healthy human aortic VICs mimic human pathology under calcifying conditions, as elevated CSE expression is associated with low levels of H2S. The expression of mitochondrial enzymes involved in H2S catabolism including sulfide quinone oxidoreductase (SQR), the key enzyme in mitochondrial H2S oxidation, persulfide dioxygenase (ETHE1), sulfite oxidase (SO) and thiosulfate sulfurtransferase (TST) were up-regulated in calcific aortic valve tissues, and a similar expression pattern was observed in response to high phosphate levels in VICs. AP39, a mitochondria-targeting H2S donor, rescued VICs from an osteoblastic phenotype switch and reduced the expression of IL-1β and TNF-α in VICs. Both pro-inflammatory cytokines aggravated calcification and osteoblastic differentiation of VICs derived from the calcific aortic valves. In contrast, IL-1β and TNF-α provided an early and transient inhibition of VICs calcification and osteoblastic differentiation in healthy cells and that effect was lost as H2S levels decreased. The benefit was mediated via CSE induction and H2S generation.We conclude that decreased levels of bioavailable H2S in human calcific aortic valves result from an increased H2S metabolism that facilitates the development of CAVD. CSE/H2S represent a pathway that reverses the action of calcifying stimuli.

Unravelling the role of SOCS3 in aortic valve stenosis

Abstract Background Calcific aortic valve stenosis (CAVD) is the most prevalent heart valve disease worldwide and, in advanced stages, leads to clinical deterioration and poor prognosis. Until now, no medical treatment is available. Thought to be a merely degenerative disease, we now know that disease initiation and progression are actively regulated by immune cell infiltration, chronic inflammation, osteogenic differentiation of valvular interstitial cells (VIC) and endothelial-to-mesenchymal transition (EndMT) of valvular endothelial cells (VEC). Suppressor of Cytokine Signaling 3 (SOCS3) is known to be a key regulator of inflammation. It promotes the polarisation of macrophages to an inflammatory phenotype and thus, contributes to disease progression in atherosclerosis. In CAVD, not only inflammation but also heterotopic bone formation leads to the thickening and obstruction of the aortic valve cusps and as SOCS3 is one of the main regulators of physiological bone formation, it could play a critical role in the progression of CAVD. Purpose We hypothesize that SOCS3 plays a crucial role during calcification of the aortic valve through regulation of EndMT, calcification of valvular interstitial cells and macrophage polarization. Methods and results In initial screening experiments, we investigated SOCS3-protein expression in explanted human aortic valves from patients undergoing surgical aortic valve replacement. SOCS3 is expressed in both stenotic and non-stenotic valve tissues. Immunofluorescence-staining of human aortic valves shows the co-localisation of SOCS3 with the interstitial cell-marker Vimentin and SOCS1, which is known to regulate macrophage-polarisation together with SOCS3 (Fig. 1). Staining of the macrophage-marker CD68 revealed its co-localisation with calcified areas of the aortic valve cusp. We validated our findings in our in vitro model of VIC calcification using two different calcifying conditions. Upregulation of RUNX2 and BMP2 verified successful osteogenic differentiation of VICs. During osteogenic differentiation for 7 days, SOCS3 and SOCS1 are significantly upregulated (Fig. 2A). We were able to induce EndMT by stimulating VEC with either TNFα or TGFβ/IL1β in vitro. We show that EndMT leads to the loss of endothelial cell markers like eNOS and VWF and to an upregulation of interstitial cell markers (such as Vimentin and α-SMA) and markers of EndMT (SNAI2). During EndMT, we were also able to observe a significant upregulation of SOCS1 and SOCS3 after 7 days (Fig. 2B). Ongoing knockdown experiments will help to elucidate the role of SOCS1 and SOCS3 during initiation and progression of CAVD. Conclusion We aim towards a better understanding of SOCS3 and its role in inflammation and calcification as hallmarks of the pathogenesis of CAVD. Since Zoledronic acid has been shown to induce a decreased expression of SOCS3 in macrophages, this might present as a possible treatment strategy to target SOCS3 therapeutically. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): Deutsche Forschungsgemeinschaft

Dexamethasone and olmesartan as potential antiremodelling agents of valvular interstitial cell into myofibroblast

Rheumatic heart disease is a late complication of valvular inflammation caused by rheumatic fever. Studies have shown that the differentiation of valvular interstitial cells (VIC) into fibroblasts plays an important role in valvular remodeling and fibrosis. Various strategies to minimize valvular fibrosis has increased recently. This study aims to analyze the effect of dexamethasone, olmesartan, and its combination in inhibiting TGF-β1-induced VIC differentiation into myofibroblast. In vitro laboratory experimental-posttest only control group design was conducted. Isolated VIC of Oryctolagus cuniculus was pretreated using 2,5 ng/mL of TGF-β1 and divided into groups of dexamethasone (0.1 uM/L), olmesartan (100 nmol/L), and its combination. Inhibition of myofibroblast differentiation was quantified by the expression of α-SMA levels detected by immunofluorescence. Dexamethasone, olmesartan, and its combination administration were significantly reduced TGF-β1-induced VIC differentiation into myofibroblast expressed by α-SMA levels (dexamethasone 6823 ± 1735.3, olmesartan 6683.7 ± 2795.05). Combination of dexamethasone and olmesartan exhibit the most potent inhibition compared to control (5051.87 ± 1612.210 vs 22286.73 ± 2780.2; p < 0.000). In conclusion, dexamethasone, olmesartan, and the combination can significantly reduce the differentiation of VIC into myofibroblasts. The greatest potential is the combined effect of dexamethasone and olmesartan, while dexamethasone and olmesartan have the same potential.

Implication of TRPM4 ionic channel in osteogenic differentiation of human valvular interstitial cells

The Ca2 + -activated non selective cationic channel, TRPM4, is involved in cardiac fibroblasts remodelling. In valvular interstitial cells (VIC), with similar properties than fibroblasts, its expression at the mRNA level was reported to be higher in conditions of valvular calcification leading to aortic stenosis. Endothelial dysfunction, followed by inflammation and fibrosis, promote the differentiation of VIC into osteoblastic phenotype cells which is, at term, responsible of valve ossification. The purpose of this study is to search for a potential implication of TRPM4 into osteogenic differentiation of human VIC. Human aortic valves are obtained from surgery for valve replacement. VIC are isolated by enzymatic digestion then maintained in culture. VIC are incubated during 14 days in a pro-calcific mixture in the presence or not of 9-phenanthrol, a pharmacological inhibitor of TRPM4. Expression and functionality of this channel were followed respectively by Western Blot and electrophysiological recordings in the inside-out configuration of the patch clamp technique. The implication of TRPM4 in VIC mineralization process was observed by measuring calcific crystals using alizarin red staining. A typical TRPM4 single channel current was detected in VIC in culture with a single channel conductance of 20 pS, a monovalent cationic permeability as well as voltage and calcium sensitivity. Protein expression of TRPM4 was confirmed by Western Blot. Pharmacological inhibition of the channel by 9-phenanthrol (3.10–6 M), during culture, reduced the mineralization by 30% without affecting cell viability. Thereby, TRPM4 would participate in osteogenic differentiation of human VIC in culture. Further studies are needed to understand which mecanisms link TRPM4 and ostegenic differentiation.

Iron promotes Slc7a11-deficient valvular interstitial cell osteogenic differentiation: A possible mechanism by which ferroptosis participates in intraleaflet hemorrhage-induced calcification

Calcific aortic valve disease (CAVD) is the most frequent pathogeny of aortic valve replacement in developed countries. Iron deposits are found in the intraleaflet hemorrhage (IH) areas of calcific aortic valves. Ferroptosis is a form of regulated cell death that involves metabolic dysfunction resulting from iron overload-dependent excessive lipid peroxidation. In this study, histological analysis showed that ferroptosis occurs in the IH areas of calcific aortic valves. We also demonstrated that Slc7a11 is expressed at low levels in OM-treated valvular interstitial cells (VICs) and IH areas and that low Slc7a11 expression is associated with calcification in CAVD. However, iron overload treatment did not promote VIC calcification under osteogenic conditions in vitro. Using lentiviral transfection to knockdown Slc7a11 in VICs, we found that the degree of iron overload-induced ferroptosis was positively increased in vitro. Finally, we also found that Slc7a11 knockdown promoted the osteogenic differentiation of VICs in vitro. In summary, this study reports a novel mechanism linking ferroptosis and CAVD development in which iron may promote Slc7a11-deficient VIC osteogenic differentiation by aggravating ferroptosis in vitro, thereby accelerating the progression of aortic valve calcification.

Uncoupling the Vicious Cycle of Mechanical Stress and Inflammation in Calcific Aortic Valve Disease.

Calcific aortic valve disease (CAVD) is a common acquired valvulopathy, which carries a high burden of mortality. Chronic inflammation has been postulated as the predominant pathophysiological process underlying CAVD. So far, no effective medical therapies exist to halt the progression of CAVD. This review aims to outline the known pathways of inflammation and calcification in CAVD, focussing on the critical roles of mechanical stress and mechanosensing in the perpetuation of valvular inflammation. Following initiation of valvular inflammation, dysregulation of proinflammatory and osteoregulatory signalling pathways stimulates endothelial-mesenchymal transition of valvular endothelial cells (VECs) and differentiation of valvular interstitial cells (VICs) into active myofibroblastic and osteoblastic phenotypes, which in turn mediate valvular extracellular matrix remodelling and calcification. Mechanosensitive signalling pathways convert mechanical forces experienced by valve leaflets and circulating cells into biochemical signals and may provide the positive feedback loop that promotes acceleration of disease progression in the advanced stages of CAVD. Mechanosensing is implicated in multiple aspects of CAVD pathophysiology. The mechanosensitive RhoA/ROCK and YAP/TAZ systems are implicated in aortic valve leaflet mineralisation in response to increased substrate stiffness. Exposure of aortic valve leaflets, endothelial cells and platelets to high shear stress results in increased expression of mediators of VIC differentiation. Upregulation of the Piezo1 mechanoreceptor has been demonstrated to promote inflammation in CAVD, which normalises following transcatheter valve replacement. Genetic variants and inhibition of Notch signalling accentuate VIC responses to altered mechanical stresses. The study of mechanosensing pathways has revealed promising insights into the mechanisms that perpetuate inflammation and calcification in CAVD. Mechanotransduction of altered mechanical stresses may provide the sought-after coupling link that drives a vicious cycle of chronic inflammation in CAVD. Mechanosensing pathways may yield promising targets for therapeutic interventions and prognostic biomarkers with the potential to improve the management of CAVD.

MicroRNA-22 promoted osteogenic differentiation of valvular interstitial cells by inhibiting CAB39 expression during aortic valve calcification.

Calcific aortic valve disease (CAVD) is a common valve disease characterized by the fibro-calcific remodeling of the aortic valves, which is an actively regulated process involving osteogenic differentiation of valvular interstitial cells (VICs). MicroRNA (miRNA) is an essential regulator in diverse biological processes in cells. The present study aimed to explore the role and mechanism of miR-22 in the osteogenic differentiation of VICs. The expression profile of osteogenesis-related miRNAs was first detected in aortic valve tissue from CAVD patients (n = 33) and healthy controls (n = 12). miR-22 was highly expressed in calcified valve tissues (P < 0.01), and the expression was positively correlated with the expression of OPN (rs = 0.820, P < 0.01) and Runx2 (rs = 0.563, P < 0.01) in VICs isolated from mild or moderately calcified valves. The sustained high expression of miR-22 was also validated in an in-vitro VICs osteogenic model. Adenovirus-mediated gain-of-function and loss-of-function experiments were then performed. Overexpression of miR-22 significantly accelerated the calcification process of VICs, manifested by significant increases in calcium deposition, alkaline phosphate activity, and expression of osteoblastic differentiation markers. Conversely, inhibition of miR-22 significantly negated the calcification process. Subsequently, calcium-binding protein 39 (CAB39) was identified as a target of miR-22. Overexpression of miR-22 significantly reduced the expression of CAB39 in VICs, leading to decreased catalytic activity of the CAB39-LKB1-STRAD complex, which, in turn, exacerbated changes in the AMPK-mTOR signaling pathway, and ultimately accelerated the calcification process. In addition, ROS generation and autophagic activity during VIC calcification were also regulated by miR-22/CAB39 pathway. These results indicate that miR-22 is an important accelerator of the osteogenic differentiation of VICs, and a potential therapeutic target in CAVD.

Endothelial cell-derived tetrahydrobiopterin prevents aortic valve calcification.

Tetrahydrobiopterin (BH4) is a critical determinant of the biological function of endothelial nitric oxide synthase. The present study was to investigate the role of valvular endothelial cell (VEC)-derived BH4 in aortic valve calcification. Plasma and aortic valve BH4 concentrations and the BH4:BH2 ratio were significantly lower in calcific aortic valve disease patients than in controls. There was a significant decrease of the two key enzymes of BH4 biosynthesis, guanosine 5'-triphosphate cyclohydrolase I (GCH1) and dihydrofolate reductase (DHFR), in calcified aortic valves compared with the normal ones. Endothelial cell-specific deficiency of Gch1 in Apoe-/- (Apoe-/-Gch1fl/flTie2Cre) mice showed a marked increase in transvalvular peak jet velocity, calcium deposition, runt-related transcription factor 2 (Runx2), dihydroethidium (DHE), and 3-nitrotyrosine (3-NT) levels in aortic valve leaflets compared with Apoe-/-Gch1fl/fl mice after a 24-week western diet (WD) challenge. Oxidized LDL (ox-LDL) induced osteoblastic differentiation of valvular interstitial cells (VICs) co-cultured with either si-GCH1- or si-DHFR-transfected VECs, while the effects could be abolished by BH4 supplementation. Deficiency of BH4 in VECs caused peroxynitrite formation increase and 3-NT protein increase under ox-LDL stimulation in VICs. SIN-1, the peroxynitrite generator, significantly up-regulated alkaline phosphatase (ALP) and Runx2 expression in VICs via tyrosine nitration of dynamin-related protein 1 (DRP1) at Y628. Finally, folic acid (FA) significantly attenuated aortic valve calcification in WD-fed Apoe-/- mice through increasing DHFR and salvaging BH4 biosynthesis. The reduction in endothelial-dependent BH4 levels promoted peroxynitrite formation, which subsequently resulted in DRP1 tyrosine nitration and osteoblastic differentiation of VICs, thereby leading to aortic valve calcification. Supplementation of FA in diet attenuated hypercholesterolaemia-induced aortic valve calcification by salvaging BH4 bioavailability.

Basic fibroblast growth factor inhibits aortic valvular interstitial cells calcification via Notch1 pathway

Calcific aortic valve disease (CAVD) is an active pathological process mediated by abnormal activation and transdifferentiation of valvular interstitial cells (VICs). The present study aims to investigate the function and...

Calcified aortic valve disease complicated with and without diabetes mellitus: the underlying pathogenesis.

As the most prevalent valvular heart disease, calcific aortic valve disease (CAVD) is a major health problem with risk of severe morbidity and mortality in the absence of effective medical treatment beyond surgical or interventional aortic valve replacement. The pathology involved in CAVD is multifactorial, including valvular endothelial cells damage, valvular interstitial cells differentiation, extracellular matrix remodeling, inflammation, fibrosis and calcification. Various risk factors for CAVD have been reported, such as age, gender, smoking, hyperlipidemia, hypertension, obesity and bicuspid aortic valves. Recently, diabetes mellitus has also been shown to accelerate the progression of CAVD. CAVD patients complicated with diabetes mellitus may benefit from early aortic valve replacement when compared with those without diabetes mellitus. Hence, diabetes mellitus is considered as an independent risk factor for CAVD. Therefore, in-depth understanding of the pathogenesis of these two diseases and their relationship may help us find appropriate prevention and therapeutic strategies for CAVD patients complicated with diabetes mellitus.

Transcription factor Sp2 promotes TGFB-mediated interstitial cell osteogenic differentiation in bicuspid aortic valves through a SMAD-dependent pathway

Calcification of the bicuspid aortic valve (BAV) involves differential expression of various RNA genes, which is achieved through complex regulatory networks that are controlled in part by transcription factors and microRNAs. We previously found that miR-195–5p regulates the osteogenic differentiation of valvular interstitial cells (VICs) by targeting the TGF-β pathway. However, the transcriptional regulation of miR-195–5p in calcified BAV patients is not yet clear. In this study, stenotic aortic valve tissues from patients with BAVs and tricuspid aortic valves (TAVs) were collected. Candidate transcription factors of miR-195–5p were predicted by bioinformatics analysis and tested in diseased valves and in male porcine VICs. SP2 gene expression and the corresponding protein levels in BAV were significantly lower than those in TAV, and a low SP2 expression level environment in VICs resulted in remarkable increases in RNA expression levels of RUNX2, BMP2, collagen 1, MMP2, and MMP9 and the corresponding proteins. ChIP assays revealed that SP2 directly bound to the transcription promoter region of miR-195–5p. Cotransfection of SP2 shRNA and a miR-195–5p mimic in porcine VICs demonstrated that SP2 repressed SMAD7 expression via miR-195–5p, while knockdown of SP2 increased the mRNA expression of SMAD7 and the corresponding protein and attenuated Smad 2/3 expression. Immunofluorescence staining of diseased valves confirmed that the functional proteins of osteogenesis differentiation, including RUNX2, BMP2, collagen 1, and osteocalcin, were overexpressed in BAVs. In Conclusion, the transcription factor Sp2 is expressed at low levels in VICs from BAV patients, which has a negative impact on miR-195–5p expression by binding its promoter region and partially promotes calcification through a SMAD-dependent pathway.

Abstract 10200: Enhanced Human VIC Calcification and Altered RNA Expression in Dynamic Culture with Induction Media in Microphysiological Systems

Introduction: Aortic valve (AV) calcification is the main characteristic in AV stenosis affected by fibrosis, endothelial cell layer damage and altered shear stress. Osteogenic differentiation of valvular interstitial cells (VIC) is research focus. VIC culturing in micro-physiological systems (MPS) that allows the adjustment of flow rate/shear force and oxygen concentration are new approaches to evaluate AV disease processes. Culture media inducing VIC calcification were applied in dynamic MPS VIC culture to investigate the impact on calcification and gene expression. Methods: A MPS able to modulate fluidic conditions (pulsatile flow of 2.7 μl/s resulting in average shear forces of 0.04 dyn/cm 2 ) was applied for human VICs. ADGMedium (50 μM ascorbic acid phosphate, 100 nM dexamethasone,10 mM β-glycerophosphate) was compared with PMedium (2 mmol/L NaH 2 PO 4 , 0.3 mM ascorbic acid) and standard DMEM (72 h). VIC viability was proven using a lactate dehydrogenase assay. Calcium accumulation was quantified in dynamic vs. static culture and mRNA expression of ACTA2, FN1, COL1A1, COL3A1, RUNX2 and SOX9 was quantified via qRT-PCR. Results: Viability of MPS VIC cultures was confirmed. ADGM induced calcium accumulation was significantly enhanced to 5.1 ± 1.4 Ca 2+ mol/kg protein in dynamic but not static culture compared to static DMEM control (0.1 ± 0.2 Ca 2+ mol/kg protein; n=4). PM resulted in a significantly higher calcium level compared to static DMEM control in static and dynamic chip culture (7.8 ± 5.2 and 9.2 ± 1.6 Ca 2+ mol/kg protein). Myofibroblastic ACTA2, Fibronectin and osteogenic factor RUNX2 were significantly induced but SOX9 (prevents AV calcification) was downregulated in ADGM but not in PM and independent from culture dynamics. Collagen COL1A1 mRNA level was significantly enhanced two-fold in ADGM and PM but exclusively in dynamic culture. An induction of COL3A1 was detected in ADGM dynamic but not static culture vs. DMEM counterparts. Conclusion: Dynamic culture significantly enhances calcium content in ADGM induced VIC cultures accompanied with enhanced collagen expression. MPS culture allows the investigation of VIC pathogenesis at defined shear forces and seeking the combined adjustment of oxygen supply. SMT and FSo contributed equally

Radiation Induces Valvular Interstitial Cell Calcific Response in an in vitro Model of Calcific Aortic Valve Disease

Background: Mediastinal ionizing radiotherapy is associated with an increased risk of valvular disease, which demonstrates pathological hallmarks similar to calcific aortic valve disease (CAVD). Despite advances in radiotherapy techniques, the prevalence of comorbidities such as radiation-associated valvular disease is still increasing due to improved survival of patients receiving radiotherapy. However, the mechanisms of radiation-associated valvular disease are largely unknown. CAVD is considered to be an actively regulated disease process, mainly controlled by valvular interstitial cells (VICs). We hypothesize that radiation exposure catalyzes the calcific response of VICs and, therefore, contributes to the development of radiation-associated valvular disease.Methods and Results: To delineate the relationship between radiation and VIC behavior (morphology, calcification, and matrix turnover), two different in vitro models were established: (1) VICs were cultured two-dimensional (2D) on coverslips in control medium (CM) or osteogenic medium (OM) and irradiated with 0, 2, 4, 8, or 16 Gray (Gy); and (2) three-dimensional (3D) hydrogel system was designed, loaded with VICs and exposed to 0, 4, or 16 Gy of radiation. In both models, a dose-dependent decrease in cell viability and proliferation was observed in CM and OM. Radiation exposure caused myofibroblast-like morphological changes and differentiation of VICs, as characterized by decreased αSMA expression. Calcification, as defined by increased alkaline phosphatase activity, was mostly present in the 2D irradiated VICs exposed to 4 Gy, while after exposure to higher doses VICs acquired a unique giant fibroblast-like cell morphology. Finally, matrix turnover was significantly affected by radiation exposure in the 3D irradiated VICs, as shown by decreased collagen staining and increased MMP-2 and MMP-9 activity.Conclusions: The presented work demonstrates that radiation exposure enhances the calcific response in VICs, a hallmark of CAVD. In addition, high radiation exposure induces differentiation of VICs into a terminally differentiated giant-cell fibroblast. Further studies are essential to elucidate the underlying mechanisms of these radiation-induced valvular changes.

Effect of Denosumab or Alendronic Acid on the Progression of Aortic Stenosis: A Double-Blind Randomized Controlled Trial.

Valvular calcification is central to the pathogenesis and progression of aortic stenosis, with preclinical and observational studies suggesting that bone turnover and osteoblastic differentiation of valvular interstitial cells are important contributory mechanisms. We aimed to establish whether inhibition of these pathways with denosumab or alendronic acid could reduce disease progression in aortic stenosis. In a single-center, parallel group, double-blind randomized controlled trial, patients >50 years of age with calcific aortic stenosis (peak aortic jet velocity >2.5 m/s) were randomized 2:1:2:1 to denosumab (60 mg every 6 months), placebo injection, alendronic acid (70 mg once weekly), or placebo capsule. Participants underwent serial assessments with Doppler echocardiography, computed tomography aortic valve calcium scoring, and 18F-sodium fluoride positron emission tomography and computed tomography. The primary end point was the calculated 24-month change in aortic valve calcium score. A total of 150 patients (mean age, 72±8 years; 21% women) with calcific aortic stenosis (peak aortic jet velocity, 3.36 m/s [2.93-3.82 m/s]; aortic valve calcium score, 1152 AU [655-2065 AU]) were randomized and received the allocated trial intervention: denosumab (n=49), alendronic acid (n=51), and placebo (injection n=25, capsule n=25; pooled for analysis). Serum C-terminal telopeptide, a measure of bone turnover, halved from baseline to 6 months with denosumab (0.23 [0.18-0.33 µg/L] to 0.11 µg/L [0.08-0.17 µg/L]) and alendronic acid (0.20 [0.14-0.28 µg/L] to 0.09 µg/L [0.08-0.13 µg/L]) but was unchanged with placebo (0.23 [0.17-0.30 µg/L] to 0.26 µg/L [0.16-0.31 µg/L]). There were no differences in 24-month change in aortic valve calcium score between denosumab and placebo (343 [198-804 AU] versus 354 AU [76-675 AU]; P=0.41) or alendronic acid and placebo (326 [138-813 AU] versus 354 AU [76-675 AU]; P=0.49). Similarly, there were no differences in change in peak aortic jet velocity or 18F-sodium fluoride aortic valve uptake. Neither denosumab nor alendronic acid affected progression of aortic valve calcification in patients with calcific aortic stenosis. Alternative pathways and mechanisms need to be explored to identify disease-modifying therapies for the growing population of patients with this potentially fatal condition. Registration: URL: https://www.clinicaltrials.gov; Unique identifier: NCT02132026.

Reproducible In Vitro Tissue Culture Model to Study Basic Mechanisms of Calcific Aortic Valve Disease: Comparative Analysis to Valvular Interstitials Cells.

The hallmarks of calcific aortic valve disease (CAVD), an active and regulated process involving the creation of calcium nodules, lipoprotein accumulation, and chronic inflammation, are the significant changes that occur in the composition, organization, and mechanical properties of the extracellular matrix (ECM) of the aortic valve (AV). Most research regarding CAVD is based on experiments using two-dimensional (2D) cell culture or artificially created three-dimensional (3D) environments of valvular interstitial cells (VICs). Because the valvular ECM has a powerful influence in regulating pathological events, we developed an in vitro AV tissue culture model, which is more closely able to mimic natural conditions to study cellular responses underlying CAVD. AV leaflets, isolated from the hearts of 6–8-month-old sheep, were fixed with needles on silicon rubber rings to achieve passive tension and treated in vitro under pro-degenerative and pro-calcifying conditions. The degeneration of AV leaflets progressed over time, commencing with the first visible calcified domains after 14 d and winding up with the distinct formation of calcium nodules, heightened stiffness, and clear disruption of the ECM after 56 d. Both the expression of pro-degenerative genes and the myofibroblastic differentiation of VICs were altered in AV leaflets compared to that in VIC cultures. In this study, we have established an easily applicable, reproducible, and cost-effective in vitro AV tissue culture model to study pathological mechanisms underlying CAVD. The valvular ECM and realistic VIC–VEC interactions mimic natural conditions more closely than VIC cultures or 3D environments. The application of various culture conditions enables the examination of different pathological mechanisms underlying CAVD and could lead to a better understanding of the molecular mechanisms that lead to VIC degeneration and AS. Our model provides a valuable tool to study the complex pathobiology of CAVD and can be used to identify potential therapeutic targets for slowing disease progression.