Human Aortic Valve Research Articles

Preliminary Evidence for Aortopathy and an X-Linked Parent-of-Origin Effect on Aortic Valve Malformation in a Mouse Model of Turner Syndrome.

Turner syndrome (TS), most frequently caused by X-monosomy (45,X), is characterized in part by cardiovascular abnormalities, including aortopathy and bicuspid aortic valve (BAV). There is a need for animal models that recapitulate the cardiovascular manifestations of TS. Extracellular matrix (ECM) organization and morphometrics of the aortic valve and proximal aorta were examined in adult 39,XO mice (where the parental origin of the single X was paternal (39,XPO) or maternal (39,XMO)) and 40,XX controls. Aortic valve morphology was normal (tricuspid) in all of the 39,XPO and 40,XX mice studied, but abnormal (bicuspid or quadricuspid) in 15% of 39,XMO mice. Smooth muscle cell orientation in the ascending aorta was abnormal in all 39,XPO and 39,XMO mice examined, but smooth muscle actin was decreased in 39,XMO mice only. Aortic dilation was present with reduced penetrance in 39,XO mice. The 39,XO mouse demonstrates aortopathy and an X-linked parent-of-origin effect on aortic valve malformation, and the candidate gene FAM9B is polymorphically expressed in control and diseased human aortic valves. The 39,XO mouse model may be valuable for examining the mechanisms underlying the cardiovascular findings in TS, and suggest there are important genetic modifiers on the X chromosome that modulate risk for nonsyndromic BAV and aortopathy.

Quinazoline-4-piperidine sulfamides are specific inhibitors of human NPP1 and prevent pathological mineralization of valve interstitial cells.

Ectonucleotide pyrophosphatase/PDE1 (NPP1) is an ectoenzyme, which plays a role in several disorders including calcific aortic valve disease (CAVD). So far, compounds that have been developed as inhibitors of NPP1 lack potency and specificity. Quinazoline-4-piperidine sulfamides (QPS) have been described as potent inhibitors of NPP1. However, their mode of inhibition as well as their selectivity and capacity to modify biological processes have not been investigated. In the present series of experiments, we have evaluated the efficacy of two derivatives, QPS1-2, in inhibiting human NPP1, and we have evaluated the effect of the most potent derivative (QPS1) on other ectonucleotidases as well as on the ability of this compound to prevent phosphate-induced mineralization of human primary aortic valve interstitial cells (VICs). The QPS1 derivative is a potent (Ki 59.3 ± 5.4 nM) and selective non-competitive inhibitor of human NPP1. Moreover, QPS1 also significantly inhibited the K121Q NPP1 gene variant (Ki 59.2 ± 14.5 nM), which is prevalent in the general population. QPS1 did not significantly alter the activity of other nucleotide metabolizing ectoenzymes expressed at the cell surface, namely NPP3, NTPDases (1-3), ecto-5'-nucleotidase and ALP. Importantly, QPS1 in the low micromolar range (≤10 μM) prevented phosphate-induced mineralization of VICs and lowered the rise of osteogenic genes as expected for NPP1 inhibition. We have provided evidence that QPS1 is a potent and selective non-competitive inhibitor of NPP1 and that it prevented pathological mineralization in a cellular model.

MicroRNA-204 Targets Runx2 to Attenuate BMP-2-induced Osteoblast Differentiation of Human Aortic Valve Interstitial Cells.

Osteoblast differentiation of valve interstitial cells (VICs) is a key step in valve calcification, but the molecular mechanisms involved are not fully understood. In this study, we aimed to investigate whether microRNA (miR)-204-regulated VICs differentiation through modulation of runt-related transcription factor 2 (Runx2), a key transcription factor for osteogenesis. Our data demonstrated that miR-204 was markedly downregulated in both human calcified aortic valves and bone morphogenetic protein (BMP)-2-stimulated aortic VICs. In vitro experiments showed that miR-204 acted as a negative regulator of osteogenic differentiation by repressing Runx2 and thereby inhibiting expression of osteoblast-related genes, including alkaline phosphatase and osteocalcin, which were all induced by BMP-2. Luciferase reporter assays validated Runx2 as the direct target of miR-204. Furthermore, increased alkaline phosphatase activity and osteocalcin expression after miR-204 inhibition were abolished by small interfering RNA-mediated silencing of Runx2. Overall, these data suggested miR-204 as a possible molecular switch inhibiting osteoblastic transdifferentiation of human aortic VICs and targeting miR-204 may have therapeutic potential for human aortic valve calcification.

Over-expression of neurotrophin 3 in human aortic valves affected by calcific disease induces the osteogenic responses via the Trk–Akt pathway

Calcific aortic valve disease (CAVD) is a leading cardiovascular disorder in the elderly. While aortic valve interstitial cells (AVICs) are the main cells that express osteogenic mediators, the molecular mechanism that mediates AVIC osteogenic responses is incompletely understood. This study aims to identify pro-osteogenic factors in human AVICs affected by CAVD. Methods and resultsMicroarray analysis identified 11 up-regulated genes in AVICs of diseased valves. Among these genes, mRNA levels of neurotrophin 3 (NT3) increased by 2 fold. Higher levels of NT3 protein in diseased aortic valves and diseased AVICs were confirmed by immunofluorescent staining and immunoblotting, respectively. An exposure of AVICs of normal valves to recombinant human NT3 (0.025–0.10μg/mL) up-regulated the production of Runx2, TGF-β1 and BMP-2 in a dose-dependent fashion. NT3 also promotes calcium deposit formation. The pro-osteogenic effect of NT3 was not affected by neutralization of Toll-like receptor 2 or 4. Interestingly, mRNA encoding neural growth factor receptors (TrkA, TrkB, TrkC and p75 NTR) was detectable in human AVICs. Inhibition of Trk receptors markedly reduced the effects of NT3 on Runx2, TGF-β1 and BMP-2 production, calcium deposit formation and Akt phosphorylation. Further, inhibition of Akt also reduced the pro-osteogenic effects of NT3. ConclusionsAVICs of diseased human aortic valves express higher levels of NT3. NT3 up-regulates the production of Runx2, TGF-β1 and BMP-2, and promotes calcium deposit formation in human AVICs via the Trk–Akt pathway. Thus, NT3 is a novel pro-osteogenic factor in aortic valves and may play a role in valvular calcification.

Activating transcription factor 3 (ATF3) and its downstream inflammatory targets are dysregulated in human calcific aortic valve disease

Activating transcription factor 3 (ATF3) and its downstream inflammatory targets are dysregulated in human calcific aortic valve disease

Abstract 165: Microrna-486 Regulates Fibroblast-to-myofibroblast Transition in Human Aortic Valve Interstitial Cells

Myofibroblasts play an important role in aortic valve fibrosis. In normal aortic valves, the interstitial cells (VICs) are primarily fibroblasts while myofibroblasts are dominant among VICs in stenotic aortic valves. Suppression of VIC phenotypic change may prevent the progression of aortic stenosis. However, the molecular mechanism underlying the fibroblast-to-myofibroblast transition in human VICs is not well understood. We hypothesized that an epigenetic mechanism controls the phenotypic transition in VICs of human aortic valves. Methods and results: Immunoblotting and immunofluorescence staining confirmed that VICs of diseased human aortic valves had higher levels of α-SMA and α-SMA fibers. MicroRNA (miR) microarray and real-time qPCR analysis revealed higher levels of miR-486 in VICs of diseased aortic valves. Lentiviral expression of miR-486 antagomir in diseased VICs markedly suppressed α-SMA expression and α-SMA fiber formation. Conversely, lentiviral expression of miR-486 mimic in normal VICs increased α-SMA levels and enhanced α-SMA fiber formation. Further, expression of miR-486 mimic in normal VICs activated Akt and up-regulated cellular levels of myosin light chain (MLC) kinase. Inhibition of Akt reduced α-SMA expression induced by miR-486 mimic. Inhibition of MLC kinase attenuated α-SMA fiber formation without affecting α-SMA levels. Prolonged treatment of normal VICs with miR-486 increased the production of collagen I, MMP-2 and MMP-9. Stimulation of normal VICs with TGF-β1 or BMP-2 caused a marked increase in cellular miR-486 level that was accompanied by up-regulated α-SMA expression, α-SMA fiber formation and matrix protein production. MiR-486 antagomir suppressed these changes associated with myofibroblastic transition and activity in cells exposed to TGF-β1 or BMP-2. Conclusions: MiR-486 regulates fibroblast-to-myofibroblast transition in human VICs, and pro-fibrogenic factors up-regulate miR-486 expression in VICs. Elevated levels of miR-486 accounts, at least in part, for the myofibroblastic phenotype of VICs in stenotic aortic valves. These novel findings indicate that modulation of miR-486 expression and/or function has therapeutic potential for slowing down the progression of aortic stenosis.

Abstract 192: Neurotrophin 3 Mediates the Fibrogenic Response to Pro-inflammatory Stimulation in Human Aortic Valve Interstitial Cells

Background: Calcific aortic valve disease is characterized by inflammation, fibrosis and calci[[Unable to Display Character: fi]]cation. Aortic valve interstitial cell (AVIC) proliferation and over-production of extracellular matrix (ECM) proteins result in valvular fibrosis and thickening, and an inflammatory milieu in valvular tissue is believed to promote aortic valve fibrosis and calcification. However, the factors that mediate AVIC fibrogenic response remain to be identified. Recently, we observed that AVICs of aortic valves affected by calcific disease express higher levels of neurotrophin 3 (NT3). It is unknown whether NT3 induces the fibrogenic response in AVICs and whether this growth factor plays a role in valvular fibrogenic response to pro-inflammatory stimulation. Hypotheses: NT3 is a pro-fibrogenic factor in the aortic valve and mediates the fibrogenic response to pro-inflammatory stimulation in human AVICs. Methods and Results: Stimulation of human AVICs with lipopolysaccharide (LPS, 0.20 μg/ml) induced proliferation and up-regulated production of MMP-9 and collagen III. TLR4-dependent NT3 over-production was observed in cells exposed to LPS and was attenuated by inhibition of the NF-κB, ERK1/2 or p38 MAPK pathway. Neutralization of NT3 or blocking the Trk receptors (neurotrophin receptors) with K252a suppressed LPS-induced fibrogenic response and reduced collagen deposition. Treatment of human AVICs with recombinant NT3 (0.10 μg/ml) also induced proliferation accompanied by up-regulated production of MMP-9 and collagen III. NT3-induced fibrogenic response was preceded by Akt phosphorylation and cyclin D1 up-regulation mediated by the Trk receptors. Inhibition of the Trk receptors, Akt or cyclin abrogated NT3-induced fibrogenic response. Conclusions: This study provides the first evidence that NT3 is capable of inducing human AVIC proliferation and ECM protein production via the Trk-Akt-cyclin D1 axis and plays an important role in mediating the TLR4-evoked fibrogenic response in human AVICs. Thus, NT3 is a pro-fibrogenic mediator in aortic valves. Up-regulation of AVIC production of NT3 by pro-inflammatory stimuli may have a mechanistic role in aortic valve fibrosis associated with calcific aortic valve disease.

Determination of oxidation state of iron in normal and pathologically altered human aortic valves

In order to investigate changes in chemical state of iron in normal and pathologically altered human aortic valves X-ray absorption spectroscopy was applied. Since Fe is suspected to play detrimental role in aortic valve stenosis pathogenesis the oxidation state of this element has been determined. The experimental material consisted of 10μm sections of valves excised during routine surgery and from autopsies. The experiment was performed at the MicroXAS beamline of the SLS synchrotron facility in Villigen (Switzerland). The Fe K-edge XANES spectra obtained from tissue samples were carefully analyzed and compared with the spectra of reference compounds containing iron in various chemical structures. The analysis of absorption edge position and shape of the spectra revealed that both chemical forms of iron are presented in valve tissue but Fe3+ is the predominant form. Small shift of the absorption edge toward higher energy in the spectra from stenotic valve samples indicates higher content of the Fe3+ form in pathological tissue. Such a phenomenon suggests the role of Fenton reaction and reactive oxygen species in the etiology of aortic valve stenosis. The comparison of pre-edge regions of XANES spectra for control and stenotic valve tissue confirmed no differences in local symmetry or spin state of iron in analyzed samples.

NOTCH1 regulates matrix gla protein and calcification gene networks in human valve endothelium

Valvular and vascular calcification are common causes of cardiovascular morbidity and mortality. Developing effective treatments requires understanding the molecular underpinnings of these processes. Shear stress is thought to play a role in inhibiting calcification. Furthermore, NOTCH1 regulates vascular and valvular endothelium, and human mutations in NOTCH1 can cause calcific aortic valve disease. Here, we determined the genome-wide impact of altering shear stress and NOTCH signaling on human aortic valve endothelium. mRNA-sequencing of primary human aortic valve endothelial cells (HAVECs) with or without knockdown of NOTCH1, in the presence or absence of shear stress, revealed NOTCH1-dependency of the atherosclerosis-related gene connexin 40 (GJA5), and numerous repressors of endochondral ossification. Among these, matrix gla protein (MGP) is highly expressed in aortic valve and vasculature, and inhibits soft tissue calcification by sequestering bone morphogenetic proteins (BMPs). Altering NOTCH1 levels affected MGP mRNA and protein in HAVECs. Furthermore, shear stress activated NOTCH signaling and MGP in a NOTCH1-dependent manner. NOTCH1 positively regulated endothelial MGP in vivo through specific binding motifs upstream of MGP. Our studies suggest that shear stress activates NOTCH1 in primary human aortic valve endothelial cells leading to downregulation of osteoblast-like gene networks that play a role in tissue calcification.

0171 : Contribution of progenitor cells sharing some serotonergic proteins in the pathophysiology of human aortic and mitral valves degeneration

Many compounds (pergolide, fenfluramine, ectasy) were described as inducers of fibrotic valvular lesions. All these compounds share in common to activate the 5HT2B serotonergic receptor. This observation leads to the hypothesis that cardiac valves express a « serotonergic system » that could be activated by serotonin (5-HT) or 5-HT receptor (5-HTR) agonists. In this work, we characterized the expression pattern of 5-HT2A, 2B, 4 R and the 5- HT transporter (SERT) in whole and cell subpopulations of 30 human mitral and aortic valves collected at the time of surgical valve replacement (Aortic: 11 calcified, 5 sclerotic, 4 bicuspid; Mitral: 12 dystrophic). All samples express 5HT2A, 2B, 4 R and SERT, the amount of 5HT2B R mRNA being higher than the 5HT2A R whatever the valve and etiology. 5HT2BR expression is found in endothelial cells (CD31+) at the valve surface, but also inside valve lesions, expressed by interstitial cells (smooth muscle α-actin and vimentin positive cells) located in an abundant glycosaminoglycan matrix. In fact, fibromyxoid lesions and calcified aortic valves express a high amount of CD34+ cells. These cells are endothelial progenitors because they express VEGFR2 and eNOS together with 5-HT2R. After collagenase treatment, valve samples were labeled with CD31 and CD34 antibodies: 60.1 (± 11) % of all mitral valvular cells are CD34+ compared to 36.1 (±8) % of aortic valvular cells (FACS analysis and sorting). To summarize, 5HT2A, 2B, 4 receptors and SERT are expressed in aortic and mitral diseased valves. The amounts of 5HT2A, 2B R mRNA are equivalent between mitral and aortic valves. High amount of CD34+ endothelial progenitors, expressing 5HT2AR and 5HT2BR, are found in degenerated valves. The contribution of the two 5-HT2 receptors and endothelial progenitors in valve degeneration is now under investigation.

The stretch responsive microRNA miR-148a-3p is a novel repressor of IKBKB, NF-κB signaling, and inflammatory gene expression in human aortic valve cells.

Bicuspid aortic valves calcify at a significantly higher rate than normal aortic valves, a process that involves increased inflammation. Because we have previously found that bicuspid aortic valve experience greater stretch, we investigated the potential connection between stretch and inflammation in human aortic valve interstitial cells (AVICs). Microarray, quantitative PCR (qPCR), and protein assays performed on AVICs exposed to cyclic stretch showed that stretch was sufficient to increase expression of interleukin and metalloproteinase family members by more than 1.5-fold. Conditioned medium from stretched AVICs was sufficient to activate leukocytes. microRNA sequencing and qPCR experiments demonstrated that miR-148a-3p was repressed in both stretched AVICs (43% repression) and, as a clinical correlate, human bicuspid aortic valves (63% reduction). miR-148a-3p was found to be a novel repressor of IKBKB based on data from qPCR, luciferase, and Western blot experiments. Furthermore, increasing miR-148a-3p levels in AVICs was sufficient to decrease NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) signaling and NF-κB target gene expression. Our data demonstrate that stretch-mediated activation of inflammatory pathways is at least partly the result of stretch-repression of miR-148a-3p and a consequent failure to repress IKBKB. To our knowledge, we are the first to report that cyclic stretch of human AVICs activates inflammatory genes in a tissue-autonomous manner via a microRNA that regulates a central inflammatory pathway.

Morphological and chemical study of pathological deposits in human aortic and mitral valve stenosis: a biomineralogical contribution.

Aim of this study was to investigate heart valve calcification process by different biomineralogical techniques to provide morphological and chemical features of the ectopic deposit extracted from patients with severe mitral and aortic valve stenosis, to better evaluate this pathological process. Polarized light microscopy and scanning electron microscopy analyses brought to light the presence of nodular and massive mineralization forms characterized by different levels of calcification, as well as the presence of submicrometric calcified globular cluster, micrometric cavities containing disorganized tissue structures, and submillimeter pockets formed by organic fibers very similar to amyloid formations. Electron microprobe analyses showed variable concentrations of Ca and P within each deposit and the highest content of Ca and P within calcified tricuspid aortic valves, while powder X-ray diffraction analyses indicated in the nanometer range the dimension of the pathological bioapatite crystals. These findings indicated the presence of highly heterogeneous deposits within heart valve tissues and suggested a progressive maturation process with continuous changes in the composition of the valvular tissue, similar to the multistep formation process of bone tissue. Moreover the micrometric cavities represent structural stages of the valve tissue that immediately precedes the formation of heavily mineralized deposits such as bone-like nodules.

HIF-1α and VEGF: Immunohistochemical Profile and Possible Function in Human Aortic Valve Stenosis

Calcific aortic stenosis (CAS) is the most common valvular disease in Western countries. Histological findings in patients with CAS extremely resemble those of atherosclerosis and include accumulation and modification of lipoproteins, inflammation, extracellular matrix remodeling, and calcification. Angiogenesis is another prominent feature of CAS; however, there is only a limited amount of data available regarding the mechanisms behind the pathological neovascularization of a structure that is originally avascular. The present study aims to identify the molecular basis that regulates blood vessel growth in stenotic aortic valves, focusing on the role of HIF-1α and VEGF pathway. A total of 19 native degenerating aortic valves obtained at valve replacement surgery have been processed for Western blot, immunohistochemical, morphometric, and ultrastructural analyses. First, we have demonstrated the adverse ECM remodeling and the significant thickening of the leaflet also showing that HIF-1α and VEGF are significantly upregulated in the stenotic valves, are locally produced and colocalize with angiogenesis and areas of calcification. Next, we have characterized, for the first time to the best of our knowledge, the morphological features of the neovasculature evidencing the presence of intact blood vessels in close proximity to the mineralized zones. These results suggest that the complex structural remodeling of the matrix might reduce oxygen availability in the valve cusp contributing to the stabilization of HIF-1α that in turn induces a metabolic adaptation through the upregulation of VEGF and the formation of new blood vessels not only to overcome the hypoxic state but also to sustain the calcification process.

Raman microspectroscopy of human aortic valves: investigation of the local and global biochemical changes associated with calcification in aortic stenosis.

Raman microimaging was applied to study the biochemical composition in the aortic valves obtained from patients with calcific aortic stenosis. This progressive disease affects an increasing number of elderly patients with hyperlipidemia and hypercholesterolemia. Lipid accumulation in the tissue is associated with pathogenesis and progression of cardiac valve calcification. This is in line with our finding that lipid deposits, predominantly composed of cholesterol and its esters, are frequently co-localized with calcium salt deposits, even at an early stage of their development. Overall changes in the biochemical composition of the tissue upon pathology progression are less obvious. Globally, although the cholesterol level rises, the relative lipid-to-protein content decreases. The results broaden the knowledge of biochemical alterations in dysfunctional human aortic valves and may be helpful in designing lipid lowering therapies.

Biological Niches within Human Calcified Aortic Valves: Towards Understanding of the Pathological Biomineralization Process.

Despite recent advances, mineralization site, its microarchitecture, and composition in calcific heart valve remain poorly understood. A multiscale investigation, using scanning electron microscopy (SEM), transmission electron microscopy (TEM), and energy dispersive X-ray spectrometry (EDS), from micrometre up to nanometre, was conducted on human severely calcified aortic and mitral valves, to provide new insights into calcification process. Our aim was to evaluate the spatial relationship existing between bioapatite crystals, their local growing microenvironment, and the presence of a hierarchical architecture. Here we detected the presence of bioapatite crystals in two different mineralization sites that suggest the action of two different growth processes: a pathological crystallization process that occurs in biological niches and is ascribed to a purely physicochemical process and a matrix-mediated mineralized process in which the extracellular matrix acts as the template for a site-directed nanocrystals nucleation. Different shapes of bioapatite crystallization were observed at micrometer scale in each microenvironment but at the nanoscale level crystals appear to be made up by the same subunits.

A multi-scale investigation of biological niches within human calcified aortic valves helps to understand the pathological biomineralization process

Calcific aortic valve stenosis (CAVS) is the most common form of heart valve disease in the industrialized countries, being an important public health problem [1]. Ectopic calcifications within aortic valve leaflets are strictly associated with CAVS, interfering with cusps opening, they lead to ventricular outflow obstruction [2]. Up to date no proven medical therapy stops CAVS course progression, so valve replacement is the only possible treatment of severe CAVS. Unfortunately, the degenerative valve calcification process, affects also bioprosthetic implants [3]. Being the molecular mechanisms leading to valve calcification still not understood, our aim was to carry on a multi-scale investigation using Scanning Electron Microscopy, Transmission Electron Microscopy and Energy Dispersive X-ray Spectrometry, to provide new insights into calcification process. Severely calcified aortic (tricuspid type, n = 29; bicuspid type, n = 3) and mitral valves (n = 4) were obtained from patients of both sexes (males=25) and different ages (mean age 72±10, range 41-90 years old) undergoing valve replacement due to severe aortic and mitral valve stenosis. We detected bioapatite crystals in two different mineralization sites: niches and extracellular matrix. This suggests the action of two different growth processes: the first occurs in biological niches and it is ascribed to a purely physico-chemical process; the second has the extracellular matrix acting ass the template for a site-directed nanocrystals nucleation. Different shapes of bioapatite crystallization were observed at micrometer scale in each microenvironment but at the nanoscale level crystals appear made up by the same subunits. We suggest that bioapatite nanocrystals in heart valve may activate a strong inflammatory process leading to irreversible pathological condition that, once activated,may aggravate the inflammatory response against bioapatite nanocrystals leading to a severe calcification process.

Activation of TLR3 induces osteogenic responses in human aortic valve interstitial cells through the NF-κB and ERK1/2 pathways.

Calcific aortic valve disease (CAVD) is characterized by chronic inflammation and progressive calcification in valve leaflets. Aortic valve interstitial cells (AVICs) play a critical role in the pathogenesis of CAVD. Previous studies show that stimulation of Toll-like receptor (TLR) 2 or TLR4 in AVICs in vitro up-regulates the expression of osteogenic mediators. Double-stranded RNA (dsRNA) can activate pro-inflammatory signaling through TLR3, the NLRP3 inflammasome and RIG-I-like receptors. The objective of this study is to determine the effect of dsRNA on AVIC osteogenic activities and the mechanism of its action. Methods and results: AVICs isolated from normal human valves were exposed to polyinosinic-polycytidylic acid [poly(I:C)], a mimic of dsRNA. Treatment with poly(I:C) increased the production of bone morphogenetic protein-2 (BMP-2), transforming growth factor beta-1 (TGF-β1) and alkaline phosphatase (ALP), and resulted in calcium deposit formation. Poly(I:C) induced the phosphorylation of NF-κB and ERK1/2. Knockdown of TLR3 essentially abrogated NF-κB and ERK1/2 phosphorylation, and markedly reduced the effect of poly(I:C) on the production of BMP-2, TGF-β1 and ALP. Further, inhibition of either NF-κB or ERK1/2 markedly reduced the levels of BMP-2, TGF-β1 and ALP in cells exposed to poly(I:C). Conclusion: Poly(I:C) up-regulates the production of BMP-2, TGF-β1 and ALP, and promotes calcium deposit formation in human AVICs. The pro-osteogenic effect of poly(I:C) is mediated primarily by TLR3 and the NF-κB and ERK1/2 pathways. These findings suggest that dsRNA, when present in aortic valve tissue, may promote CAVD progression through up-regulation of AVIC osteogenic activities.

Lipopolysaccharide stimulation improves the odontoblastic differentiation of human dental pulp cells.

Lipopolysaccharide (LPS) is one of the causative agents of pulpitis and previous studies have demonstrated that the LPS stimulation of human aortic valve interstitial cells induces inflammatory mediators and the gene expression of osteogenic factors. Therefore, in the present study, it was hypothesized that LPS affects the odontoblastic differentiation of human dental pulp cells (hDPCs). In order to investigate this, an in vitro study using hDPCs was performed. Increased alkaline phosphatase (ALP) activity was observed in the hDPCs treated with LPS, which was more marked when the cells were costimulated with odontogenic induction medium (OM). LPS also appeared to increase the gene expression levels of dentin sialophosphoprotein and dentin matrix protein‑1 and the protein expression level of dental sialoprotein in the hDPCs, particularly in combination with OM. In addition, the size and the number of nodules formed in the hDPCs exposed to OM and LPS were increased compared to those stimulated by OM alone. To determine the role of nuclear factor κB (NF‑κB) during the LPS‑induced odontoblastic differentiation of hDPCs, immunofluorescence was performed. The nuclear translocation of NF‑κB, induced by LPS was confirmed, suggesting its involvement in the LPS‑induced increase in odontoblastic differentiation of hDPCs. In conclusion, there may be an association between LPS stimulation, with or without OM, and odontoblastic differentiation.

Cross Talk between NOTCH Signaling and Biomechanics in Human Aortic Valve Disease Pathogenesis.

Aortic valve disease is a burgeoning public health problem associated with significant mortality. Loss of function mutations in NOTCH1 cause bicuspid aortic valve (BAV) and calcific aortic valve disease. Because calcific nodules manifest on the fibrosa side of the cusp in low fluidic oscillatory shear stress (OSS), elucidating pathogenesis requires approaches that consider both molecular and mechanical factors. Therefore, we examined the relationship between NOTCH loss of function (LOF) and biomechanical indices in healthy and diseased human aortic valve interstitial cells (AVICs). An orbital shaker system was used to apply cyclic OSS, which mimics the cardiac cycle and hemodynamics experienced by AVICs in vivo. NOTCH LOF blocked OSS-induced cell alignment in human umbilical vein endothelial cells (HUVECs), whereas AVICs did not align when subjected to OSS under any conditions. In healthy AVICs, OSS resulted in decreased elastin (ELN) and α-SMA (ACTA2). NOTCH LOF was associated with similar changes, but in diseased AVICs, NOTCH LOF combined with OSS was associated with increased α-SMA expression. Interestingly, AVICs showed relatively higher expression of NOTCH2 compared to NOTCH1. Biomechanical interactions between endothelial and interstitial cells involve complex NOTCH signaling that contributes to matrix homeostasis in health and disorganization in disease.

Abstract 18216: Epigenetic Up-regulation of α-smooth Muscle Actin Expression and Cell Aggregation in Human Aortic Valve Interstitial Cells Promotes Calcific Nodule Formation

Older people are at risk of calcific aortic valve disease. Aortic valve interstitial cells (AVICs) play an important role in nodular calcification in aortic valve leaflets. AVICs in human aortic valves consist of fibroblasts and myofibroblasts that express α-smooth muscle actin (α-SMA). We have observed that AVICs of diseased aortic valves have greater osteogenic activities. However, molecular mechanism underlying AVIC formation of calcification nodules is not well understood. We hypothesized that an epigenetic mechanism promotes AVIC calcification nodule formation through induction of α-SMA expression and cell aggregation. Methods and Results: MiRNA profiles in AVICs from normal and diseased human aortic valves were analyzed by miRNA array and real-time qPCR. Diseased AVICs displayed higher levels of miR-486. Immunoblotting and immunofluorescence staining revealed that diseased AVICs had higher levels of α-SMA and α-SMA fibers. Inhibition of miR-486 by lentiviral-delivered miR-486 antagomir in diseased AVICs suppressed α-SMA expression and cell aggregation, resulting in reduced calcification nodule formation. Conversely, lentiviral-delivered miR-486 mimic in normal AVICs induced α-SMA expression and cell aggregation, leading to exacerbated calcification nodule formation. Stimulation of normal AVICs with pro-osteogenic mediators TGF-β1 and BMP-2 up-regulated miR-486 levels. MiR-486 antagomir reduced α-SMA expression, cell aggregation and calcification nodule formation in cells exposed to TGF-β1 or BMP-2. Further, miR-486 mimic induced AKT phosphorylation. Inhibition of AKT decreased α-SMA expression and cell aggregation induced by miR-486 mimic in normal AVICs. Knockdown of α-SMA suppressed cell aggregation and calcification nodule formation. Conclusions: The pro-osteogenic phenotype of AVICs of diseased aortic valves is associated with up-regulated levels of miR-486 and α-SMA. MiR-486 modulates the AKT pathway to up-regulate α-SMA expression and cell aggregation that are required for calcification nodule formation. These novel findings indicate that miR-486 contributes to the mechanism underlying aortic valve calcification and appears to be a therapeutic target for suppression of valvular osteogenic activity.