Contribution of Bone Morphogenetic Protein-2 to Aortic Valve Calcification in Aged Rat

Seya, Kazuhiko; Yu, Zaiqiang; Kanemaru, Kouta; Daitoku, Kazuyuki; Akemoto, Yui; Shibuya, Hiroyuki; Fukuda, Ikuo; Okumura, Ken; Motomura, Shigeru; Furukawa, Ken‐Ichi

doi:10.1254/jphs.10198fp

Cited by 25 publications

(9 citation statements)

References 20 publications

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“…Osteogenic differentiation of the VICs cultured in osteogenic condition advances over time and can be evaluated by change in the identified osteogenic markers. In a study on rat VICs cultured in osteogenic media for 7 and 14 days, the amounts of osteogenic markers increased significantly at day 14 compared to day 7, showing that longer duration of culture is associated with increased calcification . Our calcium assay results are consistent with previous studies showing that amount of calcium increases over the time range of 4 to 21 days.…”

Section: Resultssupporting

confidence: 90%

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

Davari

Masjedi

Ferdous

et al. 2017

Journal of Biophotonics

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Keywords: calcific aortic valve disease, valvular interstitial cells, calcium deposition, 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 AE 0.04 mg 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).

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Section: Resultssupporting

confidence: 90%

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

Davari

Masjedi

Ferdous

et al. 2017

Journal of Biophotonics

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“…1,4 In experimental models, activated endothelial cells have been shown to secrete BMP-2 and BMP-4 in response to changes in laminar flow patterns, and BMP-2 has been detected in VICs isolated from the aortic valve of aged rats. 5,6 BMPs are capable of driving VICs to an osteoblast-like phenotype where these cells express all of the markers of functional osteoblasts including the master osteoblast transcription factor runt-related transcription factor 2 (Runx2) and calcificationrelated protein osteocalcin, elaborate bone matrix proteins, and mineralize to form calcific nodules by activating Smad and Wnt/b-catenin signaling and upregulating expression of alkaline phosphatase (ALP). 4 MicroRNAs (miRNAs) are small noncoding RNAs (z22 nucleotides) that lead to silencing of genetic information by annealing inexactly to complementary sequences in the 3 0 -untranslated regions (3 0 -UTR) of the target mRNA causing mRNA destabilization and/or translational inhibition.…”

Section: Introductionmentioning

confidence: 99%

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

Wang

Chen

Deng

et al. 2015

Journal of Cardiovascular Pharmacology

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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.

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“…Calcified valves show BMP-2 staining around the diseased area suggesting its involvement in valvular diseases [Wirrig et , 2011]. Members of the TGF-β superfamily, such as BMP-2, BMP-4, and TGF-β 1 , have been reported to participate in calcification in various in vitro studies [Kizu et al, 2004;Balachandran et al, 2009;Balachandran et al, 2010;Seya et al, 2011]. Downstream signaling components of the BMP pathway such as SMADs, Runx2, and αSMA have therefore been commonly studied to understand their involvement in calcification.…”

Section: Discussionmentioning

confidence: 99%

Strain Magnitude-Dependent Calcific Marker Expression in Valvular and Vascular Cells

Ferdous

Jo²,

Nerem³

2013

Cells Tissues Organs

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Aortic valve disease and atherosclerosis tend to coexist in most patients with cardiovascular disease; however, the causes and mechanisms of disease development in heart valves are still not clearly understood. To understand the contributions of the magnitude of cyclic strain (5% hypotension, 10% physiological, and 15% hypertension) in calcification, we used a model system of tissue-engineered collagen gels containing human aortic smooth muscle cells and human aortic valvular interstitial cells, both isolated from noncalcific heart transplant tissue. The compacted collagen gels were cultured in osteogenic media for 3 weeks in a custom-designed bioreactor and all assessments were performed at the end of the culture period. The major finding of this study is that bone morphogenic protein (BMP)-2 and BMP-4 and transforming growth factor-β1 mRNA expression significantly changed in response to the magnitude of applied strain in valvular cells, while the lowest expression was observed for the representative physiological strain. On the other hand, mRNA expression in vascular cells did not vary in response to the magnitude of strain. Regarding BMP-2 and BMP-4 protein expression determined by immunostaining, trends were similar to mRNA expression in vascular and valvular cells, where only valvular cells showed a varied protein expression depending on the magnitude of the strain applied. Our results suggest that cellular differences exist between vascular and valvular cells in their response to altered levels of cyclic strain during calcification.

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Contribution of Bone Morphogenetic Protein-2 to Aortic Valve Calcification in Aged Rat

Cited by 25 publications

References 20 publications

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

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

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

Strain Magnitude-Dependent Calcific Marker Expression in Valvular and Vascular Cells

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