Differential gene expression in human valve interstitial cells of normal aortic valves and calcified aortic valves
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ABSTRACT:
INSTRUMENT(S): [HuGene-1_0-st] Affymetrix Human Gene 1.0 ST Array [transcript (gene) version]
ORGANISM(S): Homo Sapiens
SUBMITTER: 
fei li
PROVIDER: GSE88803 | GEO | 2016-10-23
SECONDARY ACCESSION(S): PRJNA348688
REPOSITORIES: GEO
Dataset's files
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Project description:Aortic valve stenosis (AVS), a consequence of increased fibrosis and calcification of the aortic valve leaflets, causes progressive narrowing of the aortic valve. Proteoglycans, structural components of the aortic valve, accumulate in regions with fibrosis and moderate calcification. Particularly, proteoglycan 4 (PRG4) has been identified in fibrotic parts of aortic valves. However, the role of PRG4 in the context of AVS and aortic valve calcification has not yet been determined. Here, transcriptomics, histology, and immunohistochemistry were performed in human aortic valves from patients undergoing aortic valve replacement. Human valve interstitial cells (VICs) were used for calcification experiments and RNA expression analysis. PRG4 was significantly upregulated in thickened and calcified regions of aortic valves compared with healthy regions. In addition, mRNA levels of PRG4 positively associated with mRNA for proteins involved in cardiovascular calcification. Treatment of VICs with recombinant human PRG4 enhanced phosphate-induced calcification and increased the mRNA expression of bone morphogenetic protein 2 and the runt-related transcription factor 2. In summary, PRG4 was upregulated in the development of AVS and promoted VIC osteogenic differentiation and calcification. These results suggest that an altered valve leaflet proteoglycan composition may play a role in the progression of AVS.
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Project description:Valve interstitial cells (VICs) are crucial in the development of calcific aortic valve disease. The purpose of the present investigation was to compare the phenotype, differentiation potential and stem cell-like properties of cells from calcified and healthy aortic valves. VICs were isolated from human healthy and calcified aortic valves. Calcification was induced with osteogenic medium. Unlike VICs from healthy valves, VICs from calcified valves cultured without osteogenic medium stained positively for calcium deposits with Alizarin Red confirming their calcific phenotype. Stimulation of VICs from calcified valves with osteogenic medium increased calcification (p = 0.02), but not significantly different from healthy VICs. When stimulated with myofibroblastic medium, VICs from calcified valves had lower expression of myofibroblastic markers, measured by flow cytometry and RT-qPCR, compared to healthy VICs. Contraction of collagen gel (a measure of myofibroblastic activity) was attenuated in cells from calcified valves (p = 0.04). Moreover, VICs from calcified valves, unlike cells from healthy valves had lower potential to differentiate into adipogenic pathway and lower expression of stem cell-associated markers CD106 (p = 0.04) and aldehyde dehydrogenase (p = 0.04). In conclusion, VICs from calcified aortic have reduced multipotency compared to cells from healthy valves, which should be considered when investigating possible medical treatments of aortic valve calcification.
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Project description:OBJECTIVE:Extracellular matrix proteinases are implicated in the pathogenesis of calcific aortic valve disease. The ADAMTS5 (a disintegrin and metalloproteinase with thrombospondin motifs 5) enzyme is secreted, matrix-associated metalloendopeptidase, capable of degrading extracellular matrix proteins, particularly matrilin 2. We sought to determine the role of the ADAMTS5/matrilin 2 axis in mediating the phenotype transition of valvular interstitial cells (VICs) associated with calcific aortic valve disease. APPROACH AND RESULTS:Levels of ADAMTS5, matrilin 2, and ?-SMA (?-smooth muscle actin) were evaluated in calcified and normal human aortic valve tissues and VICs. Calcified aortic valves have reduced levels of ADAMTS5 and higher levels of matrilin 2 and ?-SMA. Treatment of normal VICs with soluble matrilin 2 caused an increase in ?-SMA level through Toll-like receptors 2 and 4, which was accompanied by upregulation of runt-related transcription factor 2 and alkaline phosphatase. In addition, ADAMTS5 knockdown in normal VICs enhanced the effect of matrilin 2. Matrilin 2 activated nuclear factor (NF) ?B and NF of activated T cells complex 1 and induced the interaction of these 2 NFs. Inhibition of either NF-?B or NF of activated T cells complex 1 suppressed matrilin 2's effect on VIC phenotype change. Knockdown of ?-SMA reduced and overexpression of ?-SMA enhanced the expression of pro-osteogenic factors and calcium deposit formation in human VICs. CONCLUSIONS:Matrilin 2 induces myofibroblastic transition and elevates pro-osteogenic activity in human VICs via activation of NF-?B and NF of activated T cells complex 1. Myofibroblastic transition in human VICs is an important mechanism of elevating the pro-osteogenic activity. Matrilin 2 accumulation associated with relative ADAMTS5 deficiency may contribute to the mechanism underlying calcific aortic valve disease progression.
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Project description:One key risk factor of aortic valve stenosis in clinical practice is bicuspid aortic valve (BAV). Increasing evidence indicates that numerous microRNAs (miRs/miRNAs) are involved in BAV calcification via their target genes. miR?330?3p was found to be involved in the deterioration of BAV calcification by miR profiling in human calcified BAV and tricuspid aortic valve (TAV) tissues in the present study and the underlying mechanism was investigated. RNA sequencing was performed on four BAV and four TAV tissues from patients with aortic stenosis before these leaflets were examined for the expression levels of miR?330?3p and CREB?binding protein (CREBBP) by reverse transcription?PCR. The alteration of functional factors associated with calcification was also assessed by Western blotting and immunohistochemistry in human aortic tissue samples. The putative target of miR?330?3p was detected by dual?luciferase assay in 293 cells. Furthermore, the influence of miR?330?3p expression on osteogenic progression was explored in cultured porcine valve interstitial cells (VICs). Rescue experiments of CRBBP were performed to confirm the influence of the miR?330?3p?CREBBP pathway in the calcification progress in porcine VICs. RNA sequencing indicated distinct expression of miR?330?3p in human BAV tissues compared with TAV, which was then confirmed by PCR. CREBBP expression levels in human BAV and TAV leaflets also demonstrated the opposite alterations. This negative correlation was then confirmed in cultured porcine VICs. Under an osteogenic environment, cellular calcification was promoted in miR?330?3p?overexpressed porcine VICs expressing higher bone morphogenetic protein 2, Runt?related transcription factor 2, matrix metalloproteinase (MMP)?2, MMP?9 and collagen I compared with controls. Rescue experiments further confirmed that miR?330?3p played its role via targeting CREBBP in porcine VICs. Collectively, miR?330?3p was upregulated in calcified BAV compared with TAV. The upregulation of miR?330?3p promotes the calcification progress partially via targeting CREBBP.
| S-EPMC7411470 | BioStudies
Project description:<b>Background:</b> Aortic valve calcification is an active proliferative process, where interstitial cells of the valve transform into either myofibroblasts or osteoblast-like cells causing valve deformation, thickening of cusps and finally stenosis. This process may be triggered by several factors including inflammation, mechanical stress or interaction of cells with certain components of extracellular matrix. The matrix is different on the two sides of the valve leaflets. We hypothesize that inflammation and mechanical stress stimulate osteogenic differentiation of human aortic valve interstitial cells (VICs) and this may depend on the side of the leaflet. <b>Methods:</b> Interstitial cells isolated from healthy and calcified human aortic valves were cultured on collagen or elastin coated plates with flexible bottoms, simulating the matrix on the aortic and ventricular side of the valve leaflets, respectively. The cells were subjected to 10% stretch at 1 Hz (FlexCell bioreactor) or treated with 0.1 ?g/ml lipopolysaccharide, or both during 24 h. Gene expression of myofibroblast- and osteoblast-specific genes was analyzed by qPCR. VICs cultured in presence of osteogenic medium together with lipopolysaccharide, 10% stretch or both for 14 days were stained for calcification using Alizarin Red. <b>Results:</b> Treatment with lipopolysaccharide increased expression of osteogenic gene bone morphogenetic protein 2 <i>(BMP2)</i> (5-fold increase from control; <i>p</i> = 0.02) and decreased expression of mRNA of myofibroblastic markers: ?-smooth muscle actin <i>(ACTA2)</i> (50% reduction from control; <i>p</i> = 0.0006) and calponin <i>(CNN1)</i> (80% reduction from control; <i>p</i> = 0.0001) when cells from calcified valves were cultured on collagen, but not on elastin. Mechanical stretch of VICs cultured on collagen augmented the effect of lipopolysaccharide. Expression of periostin <i>(POSTN)</i> was inhibited in cells from calcified donors after treatment with lipopolysaccharide on collagen (70% reduction from control, <i>p</i> = 0.001), but not on elastin. Lipopolysaccharide and stretch both enhanced the pro-calcific effect of osteogenic medium, further increasing the effect when combined for cells cultured on collagen, but not on elastin. <b>Conclusion:</b> Inflammation and mechanical stress trigger expression of osteogenic genes in VICs in a side-specific manner, while inhibiting the myofibroblastic pathway. Stretch and lipopolysaccharide synergistically increase calcification.
| S-EPMC6256176 | BioStudies
Project description:<h4>Aims</h4>Aortic valve sclerosis (AVSc) is a hallmark of several cardiovascular conditions ranging from chronic heart failure and myocardial infarction to calcific aortic valve stenosis (AVS). AVSc, present in 25-30% of patients over 65 years of age, is characterized by thickening of the leaflets with marginal effects on the mechanical proprieties of the valve making its presentation asymptomatic. Despite its clinical prevalence, few studies have investigated the pathogenesis of this disease using human AVSc specimens. Here, we investigate in vitro and ex vivo BMP4-mediated transdifferentiation of human valve interstitial cells (VICs) towards an osteogenic-like phenotype in AVSc.<h4>Methods and results</h4>Human specimens from 60 patients were collected at the time of aortic valve replacement (AVS) or through the heart transplant programme (Controls and AVSc). We show that non-calcified leaflets from AVSc patients can be induced to express markers of osteogenic transdifferentiation and biomineralization through the combinatory effect of BMP4 and mechanical stimulation. We show that BMP4 antagonist Noggin attenuates VIC activation and biomineralization. Additionally, patient-derived VICs were induced to transdifferentiate using either cell culture or a Tissue Engineering (TE) Aortic Valve model. We determine that while BMP4 alone is not sufficient to induce osteogenic transdifferentiation of AVSc-derived cells, the combinatory effect of BMP4 and mechanical stretch induces VIC activation towards a phenotype typical of late calcified stage of the disease.<h4>Conclusion</h4>This work demonstrates, for the first time using AVSc specimens, that human sclerotic aortic valves can be induced to express marker of osteogenic-like phenotype typical of advanced severe aortic stenosis.
| S-EPMC3656614 | BioStudies
Project description:<b>Objective:</b> Aortic valve (AV) leaflets rely on a precise extracellular matrix (ECM) microarchitecture for appropriate biomechanical performance. The ECM structure is maintained by valvular interstitial cells (VICs), which reside within the leaflets. The presence of pigment produced by a melanocytic population of VICs in mice with dark coats has been generally regarded as a nuisance, as it interferes with histological analysis of the AV leaflets. However, our previous studies have shown that the presence of pigment correlates with increased mechanical stiffness within the leaflets as measured by nanoindentation analyses. In the current study, we seek to better characterize the phenotype of understudied melanocytic VICs, explore the role of these VICs in ECM patterning, and assess the presence of these VICs in human aortic valve tissues. <b>Approach and Results:</b> Immunofluorescence and immunohistochemistry revealed that melanocytes within murine AV leaflets express phenotypic markers of either neuronal or glial cells. These VIC subpopulations exhibited regional patterns that corresponded to the distribution of elastin and glycosaminoglycan ECM proteins, respectively. VICs with neuronal and glial phenotypes were also found in human AV leaflets and showed ECM associations similar to those observed in murine leaflets. A subset of VICs within human AV leaflets also expressed dopachrome tautomerase, a common melanocyte marker. A spontaneous mouse mutant with no aortic valve pigmentation lacked elastic fibers and had reduced elastin gene expression within AV leaflets. A hyperpigmented transgenic mouse exhibited increased AV leaflet elastic fibers and elastin gene expression. <b>Conclusions:</b> Melanocytic VIC subpopulations appear critical for appropriate elastogenesis in mouse AVs, providing new insight into the regulation of AV ECM homeostasis. The identification of a similar VIC population in human AVs suggests conservation across species.
| S-EPMC8257952 | BioStudies
Project description:Aortic valves are collected from patients with severe aortic valve stenosis undergoing aortic valve replacement at the Institut universitaire de cardiologie et de pneumologie de Québec (IUCPQ), Quebec City, Canada. From this biobank collection, transcriptomic analyses from 240 aortic valves were performed. All valves were tricuspid and had a fibro-calcific remodeling score of 3 or 4. RNA was extracted from valve leaflets and gene expression evaluated using the Illumina HumanHT-12 v4 Expression BeadChip. The main objective of this study was to perform a large-scale expression quantitative trait loci (eQTL) mapping study on human aortic valves. Overall design: Whole-genome gene expression was performed in 240 human aortic valves.
2018-03-14 | GSE102249 | GEO
Project description: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.
| S-EPMC7679242 | BioStudies
Project description:Calcific aortic valve disease (CAVD) is the most prevalent type of heart valve disease, affecting ?2% of the US population. CAVD is characterized by the presence of calcific nodules, resulting in aortic valve (AoV) stenosis; however, the underlying mechanisms driving disease remain unknown. Studies of human diseased AoV provide initial evidence that bone morphogenetic protein (BMP) signaling, essential for normal bone formation, is activated during CAVD. Mice deficient in Klotho, an FGF23 transmembrane coreceptor, exhibit premature aging and develop AoV calcific nodules as occurs in human CAVD. The role of BMP signaling in the development of CAVD was examined in porcine aortic valve interstitial cells (VICs) and Klotho(-/-) mice.We show that activation of BMP signaling, as indicated by pSmad1/5/8 expression, precedes and later localizes with AoV calcification in Klotho(-/-) mice. In addition, cellular and extracellular matrix changes resembling features of normal bone formation are accompanied by increased osteochondrogenic gene induction in calcified Klotho(-/-) AoV. Likewise, osteogenic media treatment of porcine VICs results in BMP pathway activation, increased osteochondrogenic gene induction, and formation of calcific nodules in vitro. We demonstrate that genetic inactivation of the BMP type IA receptor in Klotho(-/-) aortic VICs, as well as BMP pathway inhibition of osteogenic media-treated aortic VICs in vitro, results in the inhibition of AoV calcification.BMP signaling and osteochondrogenic gene induction are active in calcified Klotho(-/-) AoV in vivo and calcified porcine aortic VICs in vitro. Importantly, BMP signaling is required for the development of AoV calcification in vitro and in vivo.
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