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).

     

Mir‐29b promotes human aortic valve interstitial cell calcification via inhibiting TGF‐β3 through activation of wnt3/β‐catenin/Smad3 signaling

Herein, we hypothesized that pro‐osteogenic MicroRNAs (miRs) could play functional roles in the calcification of the aortic valve and aimed to explore the functional role of miR‐29b in the osteoblastic differentiation of human aortic valve interstitial cells (hAVICs) and the underlying molecular mechanism. Osteoblastic differentiation of hAVICs isolated from human calcific aortic valve leaflets obtained intraoperatively was induced with an osteogenic medium. Alizarin red S staining was used to evaluate calcium deposition. The protein levels of osteogenic markers and other proteins were evaluated using western blotting and/or immunofluorescence while qRT‐PCR was applied for miR and mRNA determination. Bioinformatics and luciferase reporter assay were used to identify the possible interaction between miR‐29b and TGF‐β3. Calcium deposition and the number of calcification nodules were pointedly and progressively increased in hAVICs during osteogenic differentiation. The levels of osteogenic and calcification markers were equally increased, thus confirming the mineralization of hAVICs. The expression of miR‐29b was significantly increased during osteoblastic differentiation. Furthermore, the osteoblastic differentiation of hAVICs was significantly inhibited by the miR‐29b inhibition. TGF‐β3 was markedly downregulated while Smad3, Runx2, wnt3, and β‐catenin were significantly upregulated during osteogenic induction at both the mRNA and protein levels. These effects were systematically induced by miR‐29b overexpression while the inhibition of miR‐29b showed the inverse trends. Moreover, TGF‐β3 was a direct target of miR‐29b. Inhibition of miR‐29b hinders valvular calcification through the upregulation of the TGF‐β3 via inhibition of wnt/β‐catenin and RUNX2/Smad3 signaling pathways.     

Deficiency of CCAAT/enhancer‐binding protein homologous protein (CHOP) prevents diet‐induced aortic valve calcification in vivo

Aortic 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.     

Polymorphism in exon 6 of the human NT5E gene is associated with aortic valve calcification

ABSTRACT

NT5E encodes ecto-5′-nucleotidase (e5NT, CD73) which hydrolyses extracellular AMP to adenosine. Adenosine has been shown to play a protective role against aortic valve calcification (AVC). We identified two nonsynonymous missense single nucleotide polymorphisms (c.1126A > G, p.T376A and c.1136T > C, p.M379T) in exon 6 of the human NT5E gene. Since both substitutions might affect e5NT activity and consequently alter extracellular adenosine levels, we evaluated the association between NT5E alleles and calcific aortic valve disease in 119 patients (95 patients with AVC and 24 controls). In AVC patients, the frequency of the G allele at c.1126 and the frequency of the GG genotype as well as the frequency of the C allele at c.1136, and the frequencies of CC and TC genotypes tended to be higher as compared to controls. The allele and genotype frequencies in AVC patients and controls were also compared to those calculated from the 1000 Genomes Project data for control individuals of European ancestry (n = 503). We found that the frequency of the C allele at c.1136 is significantly higher in patients with AVC than in the European controls (0.111 vs. 0.054, P = 0.0052). Moreover, e5NT activity in aortic valves showed a trend toward lower levels in AVC patients with CC and TC genotypes than in those with the TT genotype. Our findings indicate that the genetic polymorphism of NT5E may contribute to the pathogenesis of calcific aortic valve disease and that the C allele of SNP c.1136 is associated with an increased risk of AVC.     

Back Cover: In‐vitro analysis of early calcification in aortic valvular interstitial cells using Laser‐Induced Breakdown Spectroscopy (LIBS) (J. Biophotonics 1/2018)

Quantitative laser‐induced breakdown spectroscopy (LIBS) is successfully used for in‐vitro analysis of early stage calcification in aortic valvular interstitial cells (VICs). LIBS results indicate 5‐fold improvement in the detection limit of calcium deposition in VICs over cell histology techniques involving staining and colorimetric calcium assays. These results can establish LIBS at the forefront of early detection of calcification in VICs for pathological studies on Calcific Aortic Valve Disease (CAVD). Further details can be found in the article by Seyyed Ali Davari et al. ( e201600288 ).