Differentiation Of Valvular Interstitial Cells Research Articles

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.

Heme-Mediated Activation of the Nrf2/HO-1 Axis Attenuates Calcification of Valve Interstitial Cells.

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

Biofabrication of poly(l-lactide-co-ε-caprolactone)/silk fibroin scaffold for the application as superb anti-calcification tissue engineered prosthetic valve

In this study, electrospun scaffolds were fabricated by blending poly(l-lactide-co-ε-caprolactone) (PLCL) and silk fibroin (SF) with different ratios, and further the feasibility of electrospun PLCL/SF scaffolds were evaluated for application of tissue engineered heart valve (TEHV). Scanning electron microscopy (SEM) results showed that the surface of PLCL/SF electrospun scaffolds was smooth and uniform while the mechanical properties were appropriate as valve prosthesis. In vitro cytocompatibility evaluation results demonstrated that all of the PLCL/SF electrospun scaffolds were cytocompatible and valvular interstitial cells (VICs) cultured on PLCL/SF scaffolds of 80/20 & 70/30 ratios exhibited the best cytocompatibility. The in vitro osteogenic differentiation of VICs including alkaline phosphatase (ALP) activity and quantitative polymerase chain reaction (qPCR) assays indicated that PLCL/SF scaffolds of 80/20 & 90/10 ratios behaved better anti-calcification ability. In the in vivo calcification evaluation model of rat subdermal implantation, PLCL/SF scaffolds of 80/20 & 90/10 ratios presented better anti-calcification ability, which was consistent with the in vitro results. Moreover, PLCL/SF scaffolds of 80/20 & 70/30 ratios showed significantly enhanced cell infiltration and M2 macrophage with higher CD206+/CD68+ ratio. Collectively, our data demonstrated that electrospun scaffolds with the PLCL/SF ratio of 80/20 hold great potential as TEHV materials.

LncRNA RMST Promotes Progression of Calcification of Aortic Valve Via the miR-195-5p/Smad7 Axis

Calcified aortic valve disease (CAVD) is a cardiovascular disease closely related to cardiovascular morbidity and mortality. Osteogenesis differentiation of valvular interstitial cells (VICs) is the key process of CAVD. In this study, we investigated whether the circulating exosomes participated in the pathogenesis and progression of CAVD and explored the underlying mechanism. We constructed the CAVD model in rats to perform in-vivo experiments, and isolated the VICs to perfume in-vitro studies. A series of methods were used including exosomes extraction and identification, RNA sequencing, ALP activity assay, alizarin red staining, qRT-PCR, western blot, and luciferase reporter assay. RNA sequencing showed that the circulating exosomes from CAVD patients and the normal people exhibited aberrant long non-coding RNA level, among which RMST was significantly upregulated in the CAVD-derived circulating exosomes and in the calcified aortic valve tissues. ALP activity assay and alizarin red staining showed that RMST promoted the VICs calcification. Luciferase reporter assay indicated that RMST acted as the miR-195-5p sponge, and miR-195-5p could target the 3’UTR of Smad7. Further functional experiments revealed that RMST was enriched in the circulating exosomes, leading to the upregulation of Smad7 in through sponging miR-195-5p to promote osteogenic differentiation in VICs. Moreover, adipose-derived exosomes showed the same effect as the CAVD-derived circulating exosomes. In conclusion, we found that circulating exosomes from CAVD patients promoted the VICs calcification through delivering the lncRNA RMST which acted as the miR-195-5p sponge to regulate the Smad7 expression. These results provided a new understanding of the molecular mechanism of the progression of CAVD. Funding: This work was supported by National Natural Science Foundation of China (grant no. 81974033). Declaration of Interest: None to declare. Ethical Approval: The present research program had been reviewed and approved by the Ethics Committee of The First Affiliated Hospital of Nanjing Medical University.

Acetylsalicylic acid prevents the pro-calcific differentiation of valvular interstitial cells through the production of anti-inflammatory lipoxins

Background and Aims: To date, no pharmacological therapy has proven to slow down the progression of calcific aortic valve disease (CAVD). Our specific aim is to study the effects of acetylsalicylic acid (ASA) and ASA-triggered lipoxins (ATL) on valvular interstitial cells (VIC) calcification.

MiR-214 Attenuates Aortic Valve Calcification by Regulating Osteogenic Differentiation of Valvular Interstitial Cells

Calcific aortic valve disease (CAVD) is a common heart valve disease in aging populations, and aberrant osteogenic differentiation of valvular interstitial cells (VICs) plays a critical role in the pathogenesis of ectopic ossification of the aortic valve. miR-214 has been validated to be involved in the osteogenesis process. Here, we aim to investigate the role and mechanism of miR-214 in CAVD progression. miR-214 expression was significantly downregulated in CAVD aortic valve leaflets, accompanied by upregulation of osteogenic markers. Overexpression of miR-214 suppressed osteogenic differentiation of VICs, while silencing the expression of miR-214 promoted this function. miR-214 directly targeted ATF4 and Sp7 to modulate osteoblastic differentiation of VICs, which was proved by dual luciferase reporter assay and rescue experiment. miR-214 knockout rats exhibited higher mean transvalvular velocity and gradient. The expression of osteogenic markers in aortic valve leaflets of miR-214 knockout rats was upregulated compared to that of the wild-type group. Taken together, our study showed that miR-214 inhibited aortic valve calcification via regulating osteogenic differentiation of VICs by directly targeting ATF4 and Sp7, indicating that miR-214 may act as a profound candidate of targeting therapy for CAVD.

Transforming growth factor-β1 promotes fibrosis but attenuates calcification of valvular tissue applied as a three-dimensional calcific aortic valve disease model

Calcific aortic valve disease (CAVD) is characterized by valvular fibrosis and calcification and driven by differentiating valvular interstitial cells (VICs). Expression data from patient biopsies suggest that transforming growth factor (TGF)-β1 is implicated in CAVD pathogenesis. However, CAVD models using isolated VICs failed to deliver clear evidence on the role of TGF-β1. Thus, employing cultures of aortic valve leaflets, we investigated effects of TGF-β1 in a tissue-based three-dimensional (3-D) CAVD model. We found that TGF-β1 induced phosphorylation of Mothers against decapentaplegic homolog (SMAD) 3 and expression of SMAD7, indicating effective downstream signal transduction in valvular tissue. Thus, TGF-β1 increased VIC contents of rough endoplasmic reticulum, Golgi, and secretory vesicles as well as tissue levels of RNA and protein. In addition, TGF-β1 raised expression of proliferation marker cyclin D1, attenuated VIC apoptosis, and upregulated VIC density. Moreover, TGF-β1 intensified myofibroblastic VIC differentiation as evidenced by increased α-smooth muscle actin and collagen type I along with diminished vimentin expression. In contrast, TGF-β1 attenuated phosphorylation of SMAD1/5/8 and upregulation of β-catenin while inhibiting osteoblastic VIC differentiation as revealed by downregulation of osteocalcin expression, alkaline phosphatase activity, and extracellular matrix incorporation of hydroxyapatite. Collectively, these effects resulted in blocking of valvular tissue calcification and associated disintegration of collagen fibers. Instead, TGF-β1 induced development of fibrosis. Overall, in a tissue-based 3-D CAVD model, TGF-β1 intensifies expressional and proliferative activation along with myofibroblastic differentiation of VICs, thus triggering dominant fibrosis. Simultaneously, by inhibiting SMAD1/5/8 activation and canonical Wnt/β-catenin signaling, TGF-β1 attenuates osteoblastic VIC differentiation, thus blocking valvular tissue calcification. These findings question a general phase-independent CAVD-promoting role of TGF-β1.NEW & NOTEWORTHY Employing aortic valve leaflets as a tissue-based three-dimensional disease model, our study investigates the role of transforming growth factor (TGF)-β1 in calcific aortic valve disease pathogenesis. We find that, by activating Mothers against decapentaplegic homolog 3, TGF-β1 intensifies expressional and proliferative activation along with myofibroblastic differentiation of valvular interstitial cells, thus triggering dominant fibrosis. Simultaneously, by inhibiting activation of Mothers against decapentaplegic homolog 1/5/8 and canonical Wnt/β-catenin signaling, TGF-β1 attenuates apoptosis and osteoblastic differentiation of valvular interstitial cells, thus blocking valvular tissue calcification. These findings question a general phase-independent calcific aortic valve disease-promoting role of TGF-β1.

Inhibition of PP2A enhances the osteogenic differentiation of human aortic valvular interstitial cells via ERK and p38 MAPK pathways

AimsTo investigate the role of PP2A in calcified aortic valve disease (CAVD). Materials and methodsThe expressions of PP2A subunits were detected by real-time polymerase chain reaction (RT-PCR) and western blot in aortic valves from patients with CAVD and normal controls, the activities of PP2A were analyzed by commercial assay kit at the same time. Aortic valve calcification of mice was evaluated through histological and echocardiographic analysis. ApoE−/− mice and ApoE−/− mice injected intraperitoneally with PP2A inhibitor LB100 were fed a high-cholesterol diet for 24 weeks. Immunofluorescent staining was used to locate the cell-type in which PP2A activity was decreased, the PP2A activity of valvular interstitial cells (VICs) treated with osteogenic induction medium was assessed by western blot and commercial assay kit. After changing the activity of VICs through pharmacologic and genetic intervention, the osteoblast differentiation and mineralization were assessed by western blot and Alizarin Red staining. Finally, the mechanism was clarified by using several specific inhibitors. Key findingsPP2A activity was decreased both in calcified aortic valves and human VICs under osteogenic induction. The PP2A inhibitor LB100 aggravated the aortic valve calcification of mice. Furthermore, PPP2CA overexpression inhibited osteogenic differentiation of VICs, whereas PPP2CA knockdown promoted the process. Further study revealed that the ERK/p38 MAPKs signaling pathways mediated the osteogenic differentiation of VICs induced by PP2A inactivation. SignificanceThis study demonstrated that PP2A plays an important role in CAVD pathophysiology, PP2A activation may provide a novel strategy for the pharmacological treatment of CAVD.

Calcification in human vessels and valves: from pathological point of view

Vascular and valvular calcification are commonly encountered in clinical medicine and a greater understanding of their significance and pathophysiology remain a subject of immense importance. In the coronary arteries, vascular calcification burden correlates with the severity of luminal stenosis and atherosclerotic plaque burden. While in progressive lesions, the presence of coronary calcification is not binary but rather depends on the type of calcification. Racial and gender differences, and comorbidities like diabetes mellitus and chronic kidney disease, all affect the presence and severity of calcification. The peripheral arteries of the lower extremities are affected by both medial calcification and intimal calcification, and the former barely contributes to luminal stenosis. The character of atherosclerosis differs between above-knee and below-knee lesions. Valvular calcification generally occurs on the aortic valve leaflets, and pathologic findings range from minimal fibrocalcific changes in early disease to end-stage lesions characterized by fibrotic thickening and nodular calcification. Valvular calcification is similar to atherosclerotic changes, in terms of lipid deposition, inflammation, osteogenic differentiation of valvular interstitial cells, and oxidative stress. However, the mechanisms of vascular and valvular calcification are still not well understood. A deeper understanding of vascular and valvular calcification is needed in order to develop effective anti-calcification therapies and to improve outcomes in these patients.

Molecular aspects of the pathological activation and differentiation of valvular interstitial cells during the development of calcific aortic stenosis

Calcific aortic stenosis is the most common valvular heart disease. The pathogenesis of this disease is complex and resembles the atherosclerotic process in the blood vessels. It is known that valvular interstitial cell activation and subsequent differentiation into osteoblast- and myofibroblast-like cells is the main driving force of fibrous and calcified aortic valve tissue. However, the molecular mechanisms behind these processes are still not fully understood. Current information on this issue is collected and analyzed in this article. The main molecular pathways mediating the pathological differentiation of the valvular interstitial cells and the reasons for their activation are considered.

IL-33 promotes the progression of nonrheumatic aortic valve stenosis via inducing differential phenotypic transition in valvular interstitial cells

ObjectiveInterleukin (IL)-33 is a mediator in the pathogenesis of several inflammatory diseases. Its receptor, ST2, is overexpressed in nonrheumatic aortic valve stenosis (NR-AS). This study compared smooth muscle α-actin (α-SMA), osteopontin (OPN), and suppression of tumorigenicity 2 (ST2) expression between specimens from fibrotic and calcific stages of NR-AS and observed the effects and mechanisms of phenotypic transition of porcine valvular interstitial cells (VICs) in the presence of IL-33. MethodsPeripheral blood IL-1 family mRNA and protein levels in NR-AS patients and healthy adults were quantified by real-time quantitative polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay. Immunohistochemistry and immunofluorescence were used to detect the expression and coexpression of α-SMA, OPN, and ST2 in NR-AS specimens. Porcine VICs were stimulated with IL-33, IL-33+SB203580, or IL-33+SC75741. mRNA and protein expression levels of porcine VICs were detected by RT-qPCR and western blot. ResultsThe mRNA and protein levels of IL-33 and sST2 in peripheral blood of NR-AS patients were higher than those in healthy adults. Immunohistochemistry and immunofluorescence showed higher expression of α-SMA, OPN, and ST2 in the calcific stage of NR-AS than in the fibrotic stage. Coexpression of ST2/α-SMA or ST2/OPN was found only in the calcific stage. Nuclear factor (NF)-κB and p38 mitogen-activated protein kinase (MAPK) phosphorylation levels were associated with IL-33-induced porcine VIC differentiation into myofibroblasts and osteoblasts, respectively. IL-33 stimulation also promoted the coexpression of ST2/OPN or α-SMA/OPN/ST2. ConclusionIL-33 might be a potential biomarker for NR-AS. IL-33-induced porcine VIC differential phenotypic transition and differentiation into myofibroblasts and osteoblasts were dependent on the NF-κB and p38 MAPK signaling pathways, respectively.

Novel mechanisms for osteogenic differentiation of human aortic valve interstitial cells

ObjectiveAortic valve calcification is common in aging populations without effective pharmacologic interventions. Our previous in vitro data revealed a critical role for long noncoding RNA metastasis-associated lung adenocarcinoma transcript 1 as a positive regulator of osteogenic differentiation in aortic valve calcification pathogenesis. The current study sought to determine the mechanism by which metastasis-associated lung adenocarcinoma transcript 1 is regulated in aortic valve calcification. MethodsThe stability assay was used to examine the effect of human antigen R on metastasis-associated lung adenocarcinoma transcript 1 expression. Aortic valves from patients with aortic stenosis and normal controls were subjected to determination of RNA-binding protein human antigen R expression. Mineralized bone matrix formation was assessed by Alizarin Red staining. The interaction between metastasis-associated lung adenocarcinoma transcript 1 and miR-191-3p was confirmed via RNA pull-down, luciferase reporter, and RNA-binding protein immunoprecipitation assays. ResultsIn cultured human aortic valvular interstitial cells, we found human antigen R enhanced metastasis-associated lung adenocarcinoma transcript 1 stability and thus increased its concentration. Moreover, human antigen R was significantly upregulated in human calcific aortic valves and valvular interstitial cells after osteogenic induction. Human antigen R partly relied on metastasis-associated lung adenocarcinoma transcript 1 to positively regulate osteogenic differentiation of valvular interstitial cells. Luciferase reporter assays validated human antigen R as the direct target of miR-191-3p. Metastasis-associated lung adenocarcinoma transcript 1 positively regulated the expression of human antigen R through sponging miR-191-3p. ConclusionsThis study demonstrates the existence of a regulatory loop between metastasis-associated lung adenocarcinoma transcript 1 and human antigen R during osteogenic differentiation of valvular interstitial cells. Our findings provide novel mechanistic insights into a critical role of human antigen R in the aortic valve calcification progression and shed new light on RNA-binding protein-directed diagnostics and therapeutics in aortic valve calcification.

Lipoprotein(a) and Oxidized Phospholipids Promote Valve Calcification in Patients With Aortic Stenosis

BackgroundLipoprotein(a) [Lp(a)], a major carrier of oxidized phospholipids (OxPL), is associated with an increased incidence of aortic stenosis (AS). However, it remains unclear whether elevated Lp(a) and OxPL drive disease progression and are therefore targets for therapeutic intervention. ObjectivesThis study investigated whether Lp(a) and OxPL on apolipoprotein B-100 (OxPL-apoB) levels are associated with disease activity, disease progression, and clinical events in AS patients, along with the mechanisms underlying any associations. MethodsThis study combined 2 prospective cohorts and measured Lp(a) and OxPL-apoB levels in patients with AS (Vmax >2.0 m/s), who underwent baseline 18F-sodium fluoride (18F-NaF) positron emission tomography (PET), repeat computed tomography calcium scoring, and repeat echocardiography. In vitro studies investigated the effects of Lp(a) and OxPL on valvular interstitial cells. ResultsOverall, 145 patients were studied (68% men; age 70.3 ± 9.9 years). On baseline positron emission tomography, patients in the top Lp(a) tertile had increased valve calcification activity compared with those in lower tertiles (n = 79; 18F-NaF tissue-to-background ratio of the most diseased segment: 2.16 vs. 1.97; p = 0.043). During follow-up, patients in the top Lp(a) tertile had increased progression of valvular computed tomography calcium score (n = 51; 309 AU/year [interquartile range: 142 to 483 AU/year] vs. 93 AU/year [interquartile range: 56 to 296 AU/year; p = 0.015), faster hemodynamic progression on echocardiography (n = 129; 0.23 ± 0.20 m/s/year vs. 0.14 ± 0.20 m/s/year] p = 0.019), and increased risk for aortic valve replacement and death (n = 145; hazard ratio: 1.87; 95% CI: 1.13 to 3.08; p = 0.014), compared with lower tertiles. Similar results were noted with OxPL-apoB. In vitro, Lp(a) induced osteogenic differentiation of valvular interstitial cells, mediated by OxPL and inhibited with the E06 monoclonal antibody against OxPL. ConclusionsIn patients with AS, Lp(a) and OxPL drive valve calcification and disease progression. These findings suggest lowering Lp(a) or inactivating OxPL may slow AS progression and provide a rationale for clinical trials to test this hypothesis.

Modulation of a new pathway prevent valvular interstitial cells calcification, a potential innovative therapeutic target in aortic valve stenosis

Aortic valve stenosis (AVS) is the narrowing of the aortic valve (AV) opening caused by fibrosis and calcification. Despite this paradigm, no targeted medical therapy has proven to prevent, slow down or treat AVS. Using transcriptomic approach on human valvular interstitial cells (VIC) isolated from fibrocalcific and normal valves, our team has identified an enzyme X downregulated in fibro-calcific VIC. This enzyme X and its product Y have documented anti-calcification effects in other cell types than VIC. Our goal is to decipher the implication of this pathway on an in vitro model of human primary cultures of VIC in ostegenic conditions. Normal AV unsuitable for grafting and fibrocalcific AV from valve replacement (ATHERAO protocol) were collected. Levels of enzyme X in AV was assessing by histology and by Western Blot on tissue homogenate. VIC isolation was realized by enzymatic digestion of normal AV. Primary cultures were exposed to ostegenic media (OM) or control media with or without the product Y of the enzyme (1–1000 nM) during 7 days. Evaluation of calcification was realized by alizarin red staining, mRNA levels of calcification markers. We confirmed a differential expression of enzyme X on tissue by histology and by western blot (− 5.2 fold, P < 0.05) from fibro-calcific AV compared to those from control AV. In OM, enzyme X is decreased at the mRNA (− 5.2 fold, P < 0.05) and protein level (− 5.2 fold, P < 0.05) compared to control media. VIC treatments with product Y in presence of OM decrease the formation of calcium nodules in a dose dependent manner (− 5.2 fold, P < 0.05, vs OM) and mRNA levels for RUNX2 (− 2.4 fold, P < 0.05 vs OM), attenuating osteoblastic VIC differentiation. Our results demonstrate the involvement of this new pathway in an in vitro model of AVS by decreasing human VIC calcification and associated markers. Enzyme X is a potential innovative pharmacological therapeutic target in AVS.

Histone deacetylase 6 reduction promotes aortic valve calcification via an endoplasmic reticulum stress-mediated osteogenic pathway

ObjectiveAortic valve (AoV) calcification occurs via a pathophysiologic process that includes osteoblastic differentiation of valvular interstitial cells (VICs). Histone deacetylases (HDACs) have been shown to be involved in the pathogenesis of vascular diseases. Here, we investigated the role of HDAC6 in AoV calcification. MethodsAoV cusps from patients with aortic stenosis (n = 7) and normal controls (n = 7) were subjected to determination of calcified nodules and HDAC6 expression. Human VICs were cultured in osteogenic media and treated with 10 uM tubacin or HDAC6 small interfering RNA silencing to inhibit HDAC6. Treatment with 100 uM tauroursodeoxycholic acid was used to suppress endoplasmic reticulum stress. Activating transcription factor 4 (ATF4) small interfering RNA was used to knock down ATF4. Alizarin red staining was used to evaluate calcified nodules formation of VICs cultured with osteogenic media for 14 days. ResultsHDAC6 expression was significantly reduced in AoV tissue of patients with aortic stenosis compared with controls. Tubacin treatment or HDAC6 silencing markedly promoted osteoblastic differentiation accompanied by endoplasmic reticulum stress activation in VICs. The HDAC6 inhibition-induced osteogenic pathway was mediated by endoplasmic reticulum stress/ATF4 pathway as indicated by tauroursodeoxycholic acid pretreatment or ATF4 silencing. Finally, alizarin red staining showed that HDAC6 inhibition promoted osteoblastic differentiation of VICs, which could be suppressed by tauroursodeoxycholic acid. ConclusionsHDAC6 inhibition promotes AoV calcification via an endoplasmic reticulum stress/ATF4-mediated osteogenic pathway. HDAC6 may be a novel target for AoV calcification prevention and treatment.

A Feedforward Regulatory Loop between HuR and Long Noncoding RNA MALAT1 Promotes Osteogenic Differentiation of Human Aortic Valve Interstitial Cells

Objective: Aortic valve calcification (AVC) is common in aging populations without effective pharmacological interventions. Our previous in vitro data revealed a critical role for long non-coding RNA (lncRNA) MALAT1 as a positive regulator of osteogenic differentiation in AVC pathogenesis. The current study sought to determine whether MALAT1 deficiency could prevent AVC progression in vivo and the mechanism by which MALAT1 is regulated in AVC. Approach and Results: AVC was evaluated in Apoe-/- mice (n = 10) or in mice with dual deficiencies of Apoe and MALAT1 (Apoe-/-MALAT1-/-, n = 10) fed a Western diet for 24 weeks. Genetic ablation of MALAT1 attenuated pro-calcification signaling activation, and valvular calcification in the leaflets of Apoe-/- mice. In cultured human aortic valvular interstitial cells (VICs), we found RNA-binding protein (RBP) human antigen R (HuR) enhanced the mRNA stability of MALAT1 and thus promoted MALAT1 expression. Moreover, HuR was significantly upregulated in mouse and human calcific aortic valves and VICs following osteogenic induction. HuR partly relied on MALAT1 to positively regulate osteogenic differentiation of VICs. Luciferase reporter assays validated HuR as the direct target of miR-191-3p. Importantly, MALAT1 positively regulated the expression of HuR through sponging miR-191-3p. Conclusions: This study demonstrates the existence of a regulatory loop in which the expression of MALAT1 and HuR is reciprocally regulated during osteogenic differentiation of VICs. Our findings provide novel mechanistic insights into a critical role of HuR in the AVC progression, and shed new light on RBPs-directed diagnostics and therapeutics in AVC. Funding: This work was supported by the National Natural Science Foundation of China (No. 81500300) and the National Key Research and Development Program of China (No. 2016YFA0101100). Declaration of Interest: None. Ethical Approval: All animal experimental protocols complied with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). All animal protocols were approved by the Institutional Animal Research Committee of Tongji Medical College. The protocol complied with the Declaration of Helsinki, was approved by the Ethic Board of Tongji Medical College of Huazhong University of Science and Technology. Written informed consents were obtained before surgeries from all patients.

Creation of disease-inspired biomaterial environments to mimic pathological events in early calcific aortic valve disease

An insufficient understanding of calcific aortic valve disease (CAVD) pathogenesis remains a major obstacle in developing treatment strategies for this disease. The aim of the present study was to create engineered environments that mimic the earliest known features of CAVD and apply this in vitro platform to decipher relationships relevant to early valve lesion pathobiology. Glycosaminoglycan (GAG) enrichment is a dominant hallmark of early CAVD, but culture of valvular interstitial cells (VICs) in biomaterial environments containing pathological amounts of hyaluronic acid (HA) or chondroitin sulfate (CS) did not directly increase indicators of disease progression such as VIC activation or inflammatory cytokine production. However, HA-enriched matrices increased production of vascular endothelial growth factor (VEGF), while matrices displaying pathological levels of CS were effective at retaining lipoproteins, whose deposition is also found in early CAVD. Retained oxidized low-density lipoprotein (oxLDL), in turn, stimulated myofibroblastic VIC differentiation and secretion of numerous inflammatory cytokines. OxLDL also increased VIC deposition of GAGs, thereby creating a positive feedback loop to further enrich GAG content and promote disease progression. Using this disease-inspired in vitro platform, we were able to model a complex, multistep pathological sequence, with our findings suggesting distinct roles for individual GAGs in outcomes related to valve lesion progression, as well as key differences in cell-lipoprotein interactions compared with atherosclerosis. We propose a pathogenesis cascade that may be relevant to understanding early CAVD and envision the extension of such models to investigate other tissue pathologies or test pharmacological treatments.

Deficiency of CCAAT/enhancer-binding protein homologous protein (CHOP) prevents diet-induced aortic valve calcification invivo.

SummaryAortic valve (AoV) calcification is common in aged populations. Its subsequent aortic stenosis has been linked with increased morbidity, but still has no effective pharmacological intervention. Our previous data show endoplasmic reticulum (ER) stress is involved in AoV calcification. Here, we investigated whether deficiency of ER stress downstream effector CCAAT/enhancer‐binding protein homology protein (CHOP) may prevent development of AoV calcification. AoV calcification was evaluated in Apoe−/− mice (n = 10) or in mice with dual deficiencies of ApoE and CHOP (Apoe−/− CHOP −/−, n = 10) fed with Western diet for 24 weeks. Histological and echocardiographic analysis showed that genetic ablation of CHOP attenuated AoV calcification, pro‐calcification signaling activation, and apoptosis in the leaflets of Apoe−/− mice. In cultured human aortic valvular interstitial cells (VIC), we found oxidized low‐density lipoprotein (oxLDL) promoted apoptosis and osteoblastic differentiation of VIC via CHOP activation. Using conditioned media (CM) from oxLDL‐treated VIC, we further identified that oxLDL triggered osteoblastic differentiation of VIC via paracrine pathway, while depletion of apoptotic bodies (ABs) in CM suppressed the effect. CM from oxLDL‐exposed CHOP‐silenced cells prevented osteoblastic differentiation of VIC, while depletion of ABs did not further enhance this protective effect. Overall, our study indicates that CHOP deficiency protects against Western diet‐induced AoV calcification in Apoe−/− mice. CHOP deficiency prevents oxLDL‐induced VIC osteoblastic differentiation via preventing VIC‐derived ABs releasing.

Multiscale Systems Biology Model of Calcific Aortic Valve Disease Progression.

Calcific aortic valve disease is a common cause of aortic stenosis, a life threatening condition. In this study, a mathematical model is developed to simulate the cascade of mechanosensitive biochemical events that occur upon damage to the endothelial layer, leading to calcification. The model contains two phases. In the initiation phase, the model accounts for low-density lipoprotein (LDL) penetration into the subendothelial space, oxidation of LDL, and monocyte penetration and differentiation to activated macrophages. In the calcification phase, transforming growth factor beta is secreted from macrophages, inducing differentiation of valvular interstitial cells into activated myofibroblasts that can enable calcium deposition. Wall shear stress and mechanical strain are taken into account with simplified models updated based on calcification progression. The model parameters are estimated based on experimental data. Next, a statin therapy simulation is performed to evaluate the effect of lipid lowering therapy on calcification progression, demonstrating an age-dependent effectiveness in statin therapy. A new potential therapy targeting transforming growth factor-β activation is proposed and simulated. The long-term evolution of calcification is compared to two sets of published longitudinal clinical data, showing promising agreement. The proposed model can provide clinically valuable data, potentially guiding surgeons in valve replacement decision makings.

In-vitro analysis of early calcification in aortic valvular interstitial cells using Laser-Induced Breakdown Spectroscopy (LIBS).

Calcific aortic valve disease (CAVD) is a major cardiovascular disorder caused by osteogenic differentiation of valvular interstitial cells (VICs) within aortic valves. Conventional methods like colorimetric assays and histology fail to detect small calcium depositions during in-vitro VIC cultures. Laser-induced breakdown spectroscopy (LIBS) is a robust analytical tool used for inorganic materials characterizations, but relatively new to biomedical applications. We employ LIBS, for the first time, for quantitative in-vitro detection of calcium depositions in VICs at various osteogenic differentiation stages. VICs isolated from porcine aortic valves were cultured in osteogenic media over various days. Colorimetric calcium assays based on arsenazo dye and Von Kossa staining measured the calcium depositions within VICs. Simultaneously, LIBS signatures for Ca I (422.67 nm) atomic emission lines were collected for estimating calcium depositions in lyophilized VIC samples. Our results indicate excellent linear correlation between the calcium assay and our LIBS measurements. Furthermore, unlike the assay results, the LIBS results could resolve calcium signals from cell samples with as early as 2 days of osteogenic culture. Quantitatively, the LIBS measurements establish the limit of detection for calcium content in VICs to be ∼0.17±0.04 μg which indicates a 5-fold improvement over calcium assay. Picture: Quantitative LIBS enables in-vitro analysis for early stage detection of calcium deposition within aortic valvular interstitial cells (VICs).