Ligation of ICAM-1 on human aortic valve interstitial cells induces the osteogenic response: A critical role of the Notch1-NF-κB pathway in BMP-2 expression

Calcific aortic valve disease (CAVD) is a chronic inflammatory condition and affects a large number of elderly people. Aortic valve interstitial cells (AVICs) occupy an important role in valvular calcification and CAVD progression. While pro-inflammatory mechanisms are capable of inducing the osteogenic responses in AVICs, the molecular interaction between pro-inflammatory and pro-osteogenic mechanisms remains poorly understood. This study tested the hypothesis that intercellular adhesion molecule-1 (ICAM-1) plays a role in mediating pro-osteogenic factor expression in human AVICs. AVICs were isolated from normal human aortic valves and cultured in M199 medium. Treatment with leukocyte function-associated factor-1 (LFA-1, an ICAM-1 ligand) up-regulated the expression of bone morphogenetic protein-2 (BMP-2) and resulted in increased alkaline phosphatase activity and formation of calcification nodules. Pre-treatment with lipopolysaccharide (LPS, 0.05 μg/ml) increased ICAM-1 levels on cell surfaces and exaggerated the pro-osteogenic response to LFA-1, and neutralization of ICAM-1 suppressed this response. Further, ligation of ICAM-1 by antibody cross-linking also up-regulated BMP-2 expression. Interestingly, LFA-1 elicited Notch1 cleavage and NF-κB activation. Inhibition of NF-κB markedly reduced LFA-1-induced BMP-2 expression, and inhibition of Notch1 cleavage with a γ-secretase inhibitor suppressed LFA-1-induced NF-κB activation and BMP-2 expression. Ligation of ICAM-1 on human AVICs activates the Notch1 pathway. Notch1 up-regulates BMP-2 expression in human AVICs through activation of NF-κB. The results demonstrate a novel role of ICAM-1 in translating a pro-inflammatory signal into a pro-osteogenic response in human AVICs and suggest that ICAM-1 on the surfaces of AVICs contributes to the mechanism of aortic valve calcification.     

Deficient signaling via Alk2 (Acvr1) leads to bicuspid aortic valve development

Bicuspid aortic valve (BAV) is the most common congenital cardiac anomaly in humans. Despite recent advances, the molecular basis of BAV development is poorly understood. Previously it has been shown that mutations in the Notch1 gene lead to BAV and valve calcification both in human and mice, and mice deficient in Gata5 or its downstream target Nos3 have been shown to display BAVs. Here we show that tissue-specific deletion of the gene encoding Activin Receptor Type I (Alk2 or Acvr1) in the cushion mesenchyme results in formation of aortic valve defects including BAV. These defects are largely due to a failure of normal development of the embryonic aortic valve leaflet precursor cushions in the outflow tract resulting in either a fused right- and non-coronary leaflet, or the presence of only a very small, rudimentary non-coronary leaflet. The surviving adult mutant mice display aortic stenosis with high frequency and occasional aortic valve insufficiency. The thickened aortic valve leaflets in such animals do not show changes in Bmp signaling activity, while Map kinase pathways are activated. Although dysfunction correlated with some pro-osteogenic differences in gene expression, neither calcification nor inflammation were detected in aortic valves of Alk2 mutants with stenosis. We conclude that signaling via Alk2 is required for appropriate aortic valve development in utero, and that defects in this process lead to indirect secondary complications later in life.     

Expression of functional Toll-like receptors 2 and 4 in human aortic valve interstitial cells: potential roles in aortic valve inflammation and stenosis

Calcific aortic valve stenosis is the most common indication for surgical valve replacement. Inflammation appears to be one of the mechanisms involved in aortic valve calcification, and valve interstitial cells seem to contribute to that process. Although Toll-like receptors (TLRs) play an important role in the cellular inflammatory response, it is unknown whether human aortic valve interstitial cells (HAVICs) express functional TLRs. We examined the expression of TLR2 and TLR4 in human aortic valve leaflets and in isolated HAVICs and analyzed the response of cultured HAVICs to the TLR2 and TLR4 agonists peptidoglycan (PGN) and LPS. Abundant TLR2 and TLR4 proteins were found in human aortic valve leaflets and in isolated HAVICs, and both receptors were detected in the membrane and cytoplasm of cultured HAVICs. Stimulation by either PGN or LPS resulted in the activation of the NF-kappaB signaling pathway and the production of multiple proinflammatory mediators, including IL-6, IL-8, and ICAM-1. In addition, stimulation by either PGN or LPS upregulated the expression of bone morphogenetic protein-2 (BMP-2) and Runx2, factors associated with osteogenesis. This study demonstrates for the first time that HAVICs express TLR2 and TLR4 and that stimulation of HAVICs by PGN or LPS induces the expression of proinflammatory mediators and the upregulation of osteogenesis-associated factors. These results suggest that TLR2 and TLR4 may play a role in aortic valve inflammation and stenosis.     

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.     

Augmented Osteogenic Responses in Human Aortic Valve Cells Exposed to oxLDL and TLR4 Agonist: A Mechanistic Role of Notch1 and NF-κB Interaction

Aortic valve calcification causes the progression of calcific aortic valve disease (CAVD). Stimulation of aortic valve interstitial cells (AVICs) with lipopolysaccharide (LPS) up-regulates the expression of osteogenic mediators, and NF-κB plays a central role in mediating AVIC osteogenic responses to Toll-like receptor 4 (TLR4) stimulation. Diseased aortic valves exhibit greater levels of oxidized low-density lipoprotein (oxLDL). This study tested the hypothesis that oxLDL augments the osteogenic responses in human AVICs through modulation of NF-κB and Notch1 activation. AVICs isolated from normal human aortic valves were treated with LPS (0.1 µg/ml), oxLDL (20 µg/ml) or LPS plus oxLDL for 48 h. OxLDL alone increased cellular bone morphogenetic protein-2 (BMP-2) levels while it had no effect on alkaline phosphatase (ALP) levels. Cells exposed to LPS plus oxLDL produced higher levels of BMP-2 and ALP than cells exposed to LPS alone. Further, LPS plus oxLDL induced greater NF-κB activation, and inhibition of NF-κB markedly reduced the expression of BMP-2 and ALP in cells treated with LPS plus oxLDL. OxLDL also induced Notch1 activation and resulted in augmented Notch1 activation when it was combined with LPS. Inhibition of Notch1 cleavage attenuated NF-κB activation induced by LPS plus oxLDL, and inhibition of NF-κB suppressed the expression of BMP-2 and ALP induced by the synergistic effect of Jagged1 and LPS. These findings demonstrate that oxLDL up-regulates BMP-2 expression in human AVICs and synergizes with LPS to elicit augmented AVIC osteogenic responses. OxLDL exerts its effect through modulation of the Notch1-NF-κB signaling cascade. Thus, oxLDL may play a role in the mechanism underlying CAVD progression.     

趋化因子受体CX3CR1调控人主动脉瓣膜间质细胞成骨分化的作用研究

目的:探究趋化因子受体CX3CR1调控人主动脉瓣膜间质细胞成骨分化的作用和机制,为钙化性主动脉瓣膜疾病的早期干预和治疗提供新思路。方法:取非钙化主动脉瓣(3例)和钙化主动脉瓣(5例),免疫组织化学染色检测成骨相关转录因子Runx2、骨桥蛋白OPN和骨钙蛋白OCN的表达;取3例非钙化的主动脉瓣,采用胶原酶连续消化法分离人主动脉瓣膜间质细胞,观察细胞形态及生长状态,并采用细胞免疫荧光进行表型鉴定。对成骨诱导培养的人主动脉瓣膜间质细胞分别过表达和干扰趋化因子受体CX3CR1,平行设置CM组、OM组和negative control+OM组,采用qPCR和Western blot检测Runx2、OPN和OCN的表达,Western blot检测AKT和p-AKT的表达。茜素红S染色评价晚期钙结节形成情况。结果:临床标本显示钙化的主动脉瓣较非钙化的主动脉瓣高表达CX3CR1(P 0. 05);成功分离人主动脉瓣膜间质细胞,α-SMA和Vimentin阳性,vWF阴性。与CM、OM、negative control组比较,CX3CR1+OM组Runx2、OPN和p-AKT表达上调(P 0. 05),且茜素红S染色可见明显钙结节;与CM、OM、negative siRNA control+OM组比较,si CX3CR1+OM组Runx2、OPN和p-AKT表达下调(P 0. 05),且茜素红S染色可见钙结节减少。结论:趋化因子受体CX3CR1可能通过AKT信号通路促进人主动脉瓣膜间质细胞成骨分化。     

Lysophosphatidic acid-induced Ca2+ mobilization requires intracellular sphingosine 1-phosphate production. Potential involvement of endogenous EDG-4 receptors

Lysophosphatidic acid (LPA)-mediated Ca(2+) mobilization in human SH-SY5Y neuroblastoma cells does not involve either inositol 1,4, 5-trisphosphate (Ins(1,4,5)P(3))- or ryanodine-receptor pathways, but is sensitive to inhibitors of sphingosine kinase. This present study identifies Edg-4 as the receptor subtype involved and investigates the presence of a Ca(2+) signaling cascade based upon the lipid second messenger molecule, sphingosine 1-phosphate. Both LPA and direct G-protein activation increase [(3)H]sphingosine 1-phosphate levels in SH-SY5Y cells. Measurements of (45)Ca(2+) release in premeabilized SH-SY5Y cells indicates that sphingosine 1-phosphate, sphingosine, and sphingosylphosphorylcholine, but not N-acetylsphingosine are capable of mobilizing intracellular Ca(2+). Furthermore, the effect of sphingosine was attenuated by the sphingosine kinase inhibitor dimethylsphingosine, or removal of ATP. Confocal microscopy demonstrated that LPA stimulated intracellular Ca(2+) "puffs," which resulted from an interaction between the sphingolipid Ca(2+) release pathway and Ins(1,4,5)P(3) receptors. Down-regulation of Ins(1,4,5)P(3) receptors uncovered a Ca(2+) response to LPA, which was manifest as a progressive increase in global cellular Ca(2+) with no discernible foci. We suggest that activation of an LPA-sensitive Edg-4 receptor solely utilizes the production of intracellular sphingosine 1-phosphate to stimulate Ca(2+) mobilization in SH-SY5Y cells. Unlike traditional Ca(2+) release processes, this novel pathway does not require the progressive recruitment of elementary Ca(2+) events.     

Cell surface receptors in lysophospholipid signaling

The lysophospholipids, lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P), regulate various signaling pathways within cells by binding to multiple G protein-coupled receptors. Receptor-mediated LPA and S1P signaling induces diverse cellular responses including proliferation, adhesion, migration, morphogenesis, differentiation and survival. This review will focus on major components of lysophospholipid signaling: metabolism, identification and expression of LPA and S1P receptors, general signaling pathways and specific signaling mechanisms in mouse embryonic fibroblasts. Finally, in vivo effects of LP receptor gene deletion in mice will be discussed.     

Angiogenic activation of valvular endothelial cells in aortic valve stenosis

Here, we demonstrate the angiogenic response of valvular endothelial cells to aortic valve (AV) stenosis using a new ex vivo model of aortic leaflets. Histological analysis revealed neovascularization within the cusps of stenotic but not of non-stenotic aortic valves. Correspondingly, the number of capillary-like outgrowth in 3D collagen gel was significantly higher in stenotic than in non-stenotic valves. Capillary-like sprouting was developed significantly faster in stenotic than in non-stenotic valves. New capillary sprouts from stenotic aortic valves exhibited the endothelial cell markers CD31, CD34 and von-Willebrand factor (vWF) as well as carcinoembryonic antigen cell adhesion molecule-1 (CEACAM1), Tie-2 and angiogenesis inhibitor endostatin. Western blot analyses revealed a significant increase of CEACAM1 and endostatin in stenotic aortic valve tissue. Electron microscopic examinations demonstrate that these capillary-like tubes are formed by endothelial cells containing Weibel-Palade bodies. Remarkably, inter-endothelial junctions are established and basement membrane material is partially deposited on the basal side of the endothelial tubes. Our data demonstrate the capillary-like sprout formation from aortic valves and suggest a role of angiogenesis in the pathogenesis of aortic valve stenosis. These data provide new insights into the mechanisms of valvular disorders and open new perspectives for prevention and early treatment of calcified aortic stenosis.     

Activation of sphingosine kinase by the bradykinin B2 receptor and its implication in regulation of the ERK/MAP kinase pathway

Sphingosine kinase phosphorylates sphingosine to generate sphingosine 1-phosphate, a phospholipid that has been implicated in signaling by a number of transmembrane receptors and was recently shown to act as a ligand for a specific class of G protein-coupled receptors. Here we show that the G protein-coupled bradykinin B2 receptor activates sphingosine kinase leading to a time- and dose-dependent elevation of cellular sphingosine 1-phosphate levels that was blocked by the sphingosine kinase inhibitor dihydrosphingosine. Furthermore, increasing doses of this inhibitor partially affected the bradykinin-mediated ERK/MAP kinase activation and fully blocked the protein kinase C-independent component of the signaling pathway from the B2 receptor to the ERK/MAP kinase cascade. Overexpression of sphingosine kinase did not additionally increase the bradykinin-induced ERK/MAP kinase activity, indicating a permissive rather than activating role of sphingosine 1-phosphate in B2 receptor-mediated mitogenic signaling.     

S1P metabolism in cancer and other pathological conditions

Nearly two decades ago, the sphingolipid metabolite sphingosine 1-phosphate was discovered to function as a lipid mediator and regulator of cell proliferation. Since that time, sphingosine 1-phosphate has been shown to mediate a diverse array of fundamental biological processes including cell proliferation, migration, invasion, angiogenesis, vascular maturation and lymphocyte trafficking. Sphingosine 1-phosphate acts primarily via signaling through five ubiquitously expressed G protein-coupled receptors. Intracellular sphingosine 1-phosphate molecules are transported extracellularly and gain access to cognate receptors for autocrine and paracrine signaling and for signaling at distant sites reached through blood and lymphatic circulation systems. Intracellular pools of sphingosine 1-phosphate available for signaling are tightly regulated primarily by three enzymes: sphinosine kinase, S1P lyase and S1P phosphatase. Alterations in sphingosine 1-phosphate as well as the enzymes involved in its synthesis and catabolism have been observed in many types of malignancy. These enzymes are being evaluated for their role in mediating cancer formation and progression, as well as their potential to serve as targets of anti-cancer therapeutics. In this review, the impact of sphingosine 1-phosphate, its cognate receptors, and the enzymes of sphingosine 1-phosphate metabolism on cell survival, apoptosis, autophagy, cellular transformation, invasion, angiogenesis and hypoxia in relation to cancer biology and treatment are discussed.     

Molecules mediating cell-ECM and cell-cell communication in human heart valves

The specific phenotype of different tissues depends on the interactions of cells with neighboring cells and the surrounding extracellular matrix, which is mediated by cell adhesion receptors including integrins, immunoglobulin family members, syndecans, and selectins. The aim of this study was to investigate the adhesion profile of native human valve interstitial cells (ICs) in situ and in vitro by analyzing these adhesion receptors. Flow cytometry and immunocytochemistry was used to quantify the expression of the specific receptors on ICs cultured from all human cardiac valves, and immunohistochemistry were used to profile their distribution pattern in valve tissue sections. The valve leaflets and cultured ICs from all valves expressed alpha1, alpha2, alpha3, alpha4, and alpha5 integrins to varying degrees and percentages with very little expression of alpha6 and alphaV. Valve leaflet ICs from all valves, expressed predominantly beta1 integrin but no beta3 or beta4 integrin. Syndecan-1 and Syndecan-4 were not detected. Intercellular adhesion molecule-1 was weakly detected, whereas vascular adhesion molecule-1 was barely detectable and E-selectin was not detected. This study has delineated the identity of some of the integrins synthesized and expressed by human valve ICs and the specificity of adhesion molecules with which the valve ICs interact with the extracellular matrix and mediate intercellular interactions. This pattern of expression of cell surface adhesion molecules may be considered as a basis for a fingerprint on which to base future cell alternatives and would provide useful information for valve tissue engineering.     

Characterization of the human and mouse sphingosine 1-phosphate receptor, S1P5 (Edg-8): structure-activity relationship of sphingosine1-phosphate receptors.

Five G protein-coupled receptors (S1P(1)/Edg-1, S1P(3)/Edg-3, S1P(2)/Edg-5, S1P(4)/Edg-6, and S1P(5)/Edg-8) for the intercellular lipid mediator sphingosine 1-phosphate have been cloned and characterized. We found human and mouse sequences closely related to rat S1P(5) (97% identical amino acids) and report now the characterization of the human and mouse S1P(5) gene products as encoding sphingosine 1-phosphate receptors. When HEK293T cells were cotransfected with S1P(5) and G protein DNAs, prepared membranes showed sphingosine 1-phosphate concentration-dependent increases in [gamma-(35)S]GTP binding (EC(50) = 12.7 nM). The lipid mediator inhibited forskolin-driven rises in cAMP by greater than 80% after introduction of the mouse or human S1P(5) DNAs into rat hepatoma RH7777 cells (IC(50) = 0.22 nM). This response is blocked fully by prior treatment of cultures with pertussis toxin, thus implicating signaling through G(i/o)alpha proteins. Northern blot analysis showed high expression of human S1P(5) mRNA in spleen, corpus collosum, peripheral blood leukocytes, placenta, lung, aorta, and fetal tissues. Mouse S1P(5) mRNA is also expressed in spleen and brain. Finally, we found that one enantiomer of a sphingosine 1-phosphate analogue wherein the 3-hydroxyl and 4,5-olefin are replaced by an amide functionality shows some selectivity as an agonist S1P(1) and S1P(3) vs S1P(2) and S1P(5).     

Differential transactivation of sphingosine-1-phosphate receptors modulates NGF-induced neurite extension

The process of neurite extension after activation of the TrkA tyrosine kinase receptor by nerve growth factor (NGF) involves complex signaling pathways. Stimulation of sphingosine kinase 1 (SphK1), the enzyme that phosphorylates sphingosine to form sphingosine-1-phosphate (S1P), is part of the functional TrkA signaling repertoire. In this paper, we report that in PC12 cells and dorsal root ganglion neurons, NGF translocates SphK1 to the plasma membrane and differentially activates the S1P receptors S1P1 and S1P2 in a SphK1-dependent manner, as determined with specific inhibitors and small interfering RNA targeted to SphK1. NGF-induced neurite extension was suppressed by down-regulation of S1P1 expression with antisense RNA. Conversely, when overexpressed in PC12 cells, transactivation of S1P1 by NGF markedly enhanced neurite extension and stimulation of the small GTPase Rac, important for the cytoskeletal changes required for neurite extension. Concomitantly, differentiation down-regulated expression of S1P2 whose activation would stimulate Rho and inhibit neurite extension. Thus, differential transactivation of S1P receptors by NGF regulates antagonistic signaling pathways that modulate neurite extension.     

BMP7基因沉默抑制钙盐诱导猪主动脉瓣膜间质细胞成骨分化

目的:探究小干扰RNA(small interference RNA,siRNA)介导的骨形态发生蛋白7(bone morphogenetic protein7,BMP7)基因沉默对钙盐诱导猪主动脉瓣膜间质细胞成骨分化的影响及机制,为钙化性主动脉瓣膜病(calcific aortic valve disease,CAVD)的干预及治疗提供理论依据。方法:非CAVD瓣膜组织(non-CAVD组)取自手术治疗的主动脉夹层患者,CAVD瓣膜组织(CAVD组)取自因钙化性主动脉瓣狭窄而进行主动脉瓣膜置换术的患者,采用免疫组化和Western blot法检测non-CAVD组和CAVD组中BMP7、Runt相关转录因子2(Runx2)的蛋白质表达水平。选取健康家猪处死后即刻于无菌条件下取主动脉瓣叶,采用胶原酶连续消化法分离主动脉瓣膜间质细胞,观察其形态特征,并用免疫荧光染色行表型鉴定。采用脂质体转染法将BMP7-siRNA转染猪主动脉瓣膜间质细胞,采用qPCR和Western blot法验证BMP7表达的变化;利用钙盐培养基诱导细胞成骨分化,建立体外主动脉瓣膜间质细胞钙化模型后,采用ALP染色和茜素红S染色实验分别检测细胞早期及晚期成骨分化能力;采用qPCR和Western blot法分别检测细胞成骨相关基因及蛋白质Runx2、OCN和OPN的表达情况。并用Western blot法检测BMP7下游信号通路中Smad1/5/8的磷酸化水平。结果:BMP7和Runx2蛋白在CAVD组中表达明显高于non-CAVD组。成功分离出原代猪主动脉瓣膜间质细胞,α-平滑肌肌动蛋白(α-SMA)及波形蛋白(vimentin)染色阳性,血管性血友病因子(von willebrand factor,vWF)染色阴性。转染BMP7-siRNA后猪主动脉瓣膜间质细胞中BMP7的mRNA和蛋白质水平均明显下调,早期及晚期成骨分化能力均明显降低。沉默BMP7基因的表达,可下调Runx2、OCN和OPN的基因及蛋白质表达,且磷酸化的Smad1/5/8(p-Smad1/5/8)蛋白水平明显降低。结论:BMP7基因沉默抑制钙盐诱导的主动脉瓣膜间质细胞的成骨分化能力,BMP7/Smads信号通路可能在该过程中发挥重要作用。     

Suppression of homodimerization of toll-like receptor 4 by isoliquiritigenin

Toll-like receptors (TLRs) play important inductive roles in innate immune responses for host defense against invading microbial pathogens. Activation of TLR4 by lipopolysaccharide (LPS) induces dimerization of TLR4 and, subsequently, activation of downstream signaling pathways including nuclear factor-kappa B and interferon regulatory factor 3. TLR4 dimerization may be an early regulatory event in activating signaling pathways induced by LPS. Here, biochemical evidence is reported that isoliquiritigenin, one of the major ingredients derived from licorice root, inhibits LPS-induced TLR4 dimerization resulting in inhibition of nuclear factor-kappa B and interferon regulatory factor 3 activation, and cyclooxygenase-2 and inducible nitric oxide synthase expression. These results suggest that isoliquiritigenin modulates TLR-mediated signaling pathways at the receptor level. Furthermore, these results suggest that TLRs themselves may be important targets for the prevention of chronic inflammatory diseases.     

Osteogenesis inducers promote distinct biological responses in aortic and mitral valve interstitial cells

Both aortic and mitral valves calcify in pathological conditions; however, the prevalence of aortic valve calcification is high whereas mitral valve leaflet calcification is somewhat rare. Patterns of valvular calcification may differ due to valvular architecture, but little is known to that effect. In this study, we investigated the intrinsic osteogenic differentiation potential of aortic versus mitral valve interstitial cells provided minimal differentiation conditions. For the assessment of calcification at the cellular level, we used classic inducers of osteogenesis in stem cells: β-glycerophosphate (β-Gly), dexamethasone (Dex), and ascorbate (Asc). In addition to proteomic analyses, osteogenic markers and calcium precipitates were evaluated across treatments of aortic and mitral valve cells. The combination of β-Gly, Asc, and Dex induced aortic valve interstitial cells to synthesize extracellular matrix, overexpress osteoblastic markers, and deposit calcium. However, no strong evidence showed the calcification of mitral valve interstitial cells. Mitral cells mainly responded to Asc and Dex by cell activation. These findings provide a deeper understanding of the physiological properties of aortic and mitral valves and tendencies for calcific changes within each valve type, contributing to the development of future therapeutics for heart valve diseases.     

Aortic valve stenosis: an active atheroinflammatory process

PURPOSE OF REVIEW: To summarize the current understanding of the pathobiology of aortic valve stenosis and portray the major advances in this field. RECENT FINDINGS: Stenotic aortic valves are characterized by atherosclerosis-like lesions, consisting of activated inflammatory cells, including T lymphocytes, macrophages, and mast cells, and of lipid deposits, calcific nodules, and bone tissue. Active mediators of calcification and cells with osteoblast-like activity are present in diseased valves. Extracellular matrix remodeling, including collagen synthesis and elastin degradation by matrix metalloproteinases and cathepsins, contributes to leaflet stiffening. In experimental animals, hypercholesterolemia induces calcification and bone formation in aortic valves, which can be inhibited by statin treatment. The potential of statins to retard progression of aortic valve stenosis has also been recognized in clinical studies; however, further prospective trials are needed. Angiotensin II-forming enzymes are upregulated in stenotic valves. Angiotensin II may participate in profibrotic progression of aortic valve stenosis and may serve as a possible therapeutic target. SUMMARY: Recent findings regarding the interaction of inflammatory cells, lipids, mediators of calcification, and renin-angiotensin system in stenotic valves support the current opinion of aortic valve stenosis being an actively regulated disease, potentially amenable to targeted molecular therapy. Evidence from prospective clinical studies is eagerly awaited.     

The Long Non-Coding HOTAIR Is Modulated by Cyclic Stretch and WNT/β-CATENIN in Human Aortic Valve Cells and Is a Novel Repressor of Calcification Genes

Aortic valve calcification is a significant and serious clinical problem for which there are no effective medical treatments. Individuals born with bicuspid aortic valves, 1–2% of the population, are at the highest risk of developing aortic valve calcification. Aortic valve calcification involves increased expression of calcification and inflammatory genes. Bicuspid aortic valve leaflets experience increased biomechanical strain as compared to normal tricuspid aortic valves. The molecular pathogenesis involved in the calcification of BAVs are not well understood, especially the molecular response to mechanical stretch. HOTAIR is a long non-coding RNA (lncRNA) that has been implicated with cancer but has not been studied in cardiac disease. We have found that HOTAIR levels are decreased in BAVs and in human aortic interstitial cells (AVICs) exposed to cyclic stretch. Reducing HOTAIR levels via siRNA in AVICs results in increased expression of calcification genes. Our data suggest that β-CATENIN is a stretch responsive signaling pathway that represses HOTAIR. This is the first report demonstrating that HOTAIR is mechanoresponsive and repressed by WNT β-CATENIN signaling. These findings provide novel evidence that HOTAIR is involved in aortic valve calcification.