Erratum to: Gentamicin Reduces Calcific Nodule Formation by Aortic Valve Interstitial Cells In Vitro

Calcific Aortic Valve Disease (CAVD) is the third most common cause of cardiovascular disease, affecting nearly 5 million people in the United States alone. It is now the most common form of acquired valvular disease in industrialized countries and will likely affect more individuals in the coming years as the prevalence increases with life expectancy. It is known that the progression of CAVD is closely related to the behavior of aortic valve interstitial cells (AVICs); however the cellular mechanobiological mechanisms leading to dysfunction remain unclear. Generally, CAVD is characterized by the formation of calcified AVIC aggregates with an apoptotic core. These aggregates increase the leaflet stiffness and impede normal valve function. Multiple studies have investigated the effects of various biochemical cues on this process, such as transformation growth factor β1 (TGF-β1), on the regulation of nodule formation [1]. Additionally, Yip et al revealed that matrix stiffness controls nodule formation in vitro, with stiffer substrates promoting apoptotic nodule formation, while compliant substrates generated nodules containing cells with osteoblast markers [2]. This suggests that matrix stiffness is involved in the regulatory mechanisms of nodule formation and may initiate different types of nodule formation (i.e. osteogenic vs. dystrophic). In the current study, we examined the synergistic role of strain and TGF-β1 in the generation of calcified nodules AVICs.

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Gentamicin Reduces Calcific Nodule Formation by Aortic Valve Interstitial Cells In Vitro

Cardiovascular Engineering and Technology ◽

10.1007/s13239-012-0114-6 ◽

2012 ◽

Vol 4 (1) ◽

pp. 16-25 ◽

Cited By ~ 5

Author(s):

Aditya Kumar ◽

Dena C. Wiltz ◽

K. Jane Grande-Allen

Keyword(s):

Aortic Valve ◽

Interstitial Cells ◽

Nodule Formation ◽

Valve Interstitial Cells

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Cadherin-11 Regulates Calcific Nodule Formation by Aortic Valve Interstitial Cells

Volume 1B: Extremity; Fluid Mechanics; Gait; Growth, Remodeling, and Repair; Heart Valves; Injury Biomechanics; Mechanotransduction and Sub-Cellular Biophysics; MultiScale Biotransport; Muscle, Tendon and Ligament; Musculoskeletal Devices; Multiscale Mechanics; Thermal Medicine; Ocular Biomechanics; Pediatric Hemodynamics; Pericellular Phenomena; Tissue Mechanics; Biotransport Design and Devices; Spine; Stent Device Hemodynamics; Vascular Solid Mechanics; Student Paper and Design Competitions ◽

10.1115/sbc2013-14201 ◽

2013 ◽

Author(s):

Joseph Chen ◽

Joshua D. Hutcheson ◽

M. K. Sewell-Loftin ◽

Larisa M. Ryzhova ◽

Charles I. Fisher ◽

...

Keyword(s):

Aortic Valve ◽

Transforming Growth Factor ◽

Critical Role ◽

Transforming Growth Factor Β1 ◽

Interstitial Cells ◽

In Vitro Models ◽

Nodule Formation ◽

Calcific Aortic Valve Disease ◽

Valve Interstitial Cells

Calcific aortic valve disease (CAVD) is characterized by the stiffening and calcification of the aortic valve leaflets which result in impaired valve function and increased load on the myocardium. In vitro models of CAVD involve the formation the calcific nodules via aortic valve interstitial cells (AVICs). Transforming growth factor β1 (TGF-β1) induced myofibroblast differentiation of AVICs, which is evidenced by increased αSMA expression, has been shown to be a key mediator of dystrophic calcific nodule formation. Benton et al. demonstrated the critical role of αSMA in nodule formation in that when αSMA was suppressed, calcific nodules did not form [1]. Confoundingly, preventing phosphorylation of Erk1/2 with a MEK1/2 inhibitor leads to increased αSMA expression yet prevents calcific nodule formation [2], suggesting the requirement of another essential component of nodule formation that has yet to be revealed.

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KPT-330 Prevents Aortic Valve Calcification via a Novel C/EBPβ Signaling Pathway

Circulation Research ◽

10.1161/circresaha.120.318503 ◽

2021 ◽

Vol 128 (9) ◽

pp. 1300-1316

Author(s):

Punashi Dutta ◽

Karthik M. Kodigepalli ◽

Stephanie LaHaye ◽

J. Will Thompson ◽

Sarah Rains ◽

...

Keyword(s):

Aortic Valve ◽

Nuclear Export ◽

The United States ◽

Interstitial Cells ◽

Nodule Formation ◽

Calcific Aortic Valve Disease ◽

Valve Interstitial Cells ◽

Subsequent Decrease ◽

Inhibitor Drug

Rationale: Calcific aortic valve disease (CAVD) affects >5.2 million people in the United States. The only effective treatment is surgery, and this comes with complications and no guarantee of long-term success. Objective: Outcomes from pharmacological initiatives remain unsubstantiated and, therefore, the aim of this study is to determine if repurposing a selective XPO1 (exportin-1) inhibitor drug (KPT-330) is beneficial in the treatment of CAVD. Methods and Results: We show that KPT-330 prevents, attenuates, and mitigates calcific nodule formation in heart valve interstitial cells in vitro and prevents CAVD in Klotho −/− mice. Using RNA-sequencing and mass spectrometry, we show that KPT-330’s beneficial effect is mediated by inhibiting nuclear export of the C/EBPβ (transcription factor CCAAT/enhancing-binding protein) in valve interstitial cells, leading to repression of canonical Wnt signaling, in part, through activation of the Wnt antagonist Axin1 , and a subsequent decrease in proosteogenic markers and cell viability. Conclusions: Our findings have met a critical need to discover alternative, pharmacological-based therapies in the treatment of CAVD.

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Metformin alleviates the calcification of aortic valve interstitial cells through activating the PI3K/AKT pathway in an AMPK dependent way

Molecular Medicine ◽

10.1186/s10020-021-00416-x ◽

2021 ◽

Vol 27 (1) ◽

Author(s):

Qiao En ◽

Huang Zeping ◽

Wang Yuetang ◽

Wang Xu ◽

Wang Wei

Keyword(s):

Aortic Valve ◽

Signaling Pathways ◽

Signaling Pathway ◽

Interstitial Cells ◽

Akt Signaling ◽

Akt Signaling Pathway ◽

Aortic Valves ◽

Valve Interstitial Cells ◽

Pathological Mechanism

Abstract Background Calcific aortic valve disease (CAVD) is the most prevalent valvular disease worldwide. However, no effective treatment could delay or prevent the progression of the disease due to the poor understanding of its pathological mechanism. Many studies showed that metformin exerted beneficial effects on multiple cardiovascular diseases by mediating multiple proteins such as AMPK, NF-κB, and AKT. This study aims to verify whether metformin can inhibit aortic calcification through the PI3K/AKT signaling pathway. Methods We first analyzed four microarray datasets to screen differentially expressed genes (DEGs) and signaling pathways related to CAVD. Then aortic valve samples were used to verify selected genes and pathways through immunohistochemistry (IHC) and western blot (WB) assays. Aortic valve interstitial cells (AVICs) were isolated from non-calcific aortic valves and then cultured with phosphate medium (PM) with or without metformin to verify whether metformin can inhibit the osteogenic differentiation and calcification of AVICs. Finally, we used inhibitors and siRNA targeting AMPK, NF-κB, and AKT to study the mechanism of metformin. Results We screened 227 DEGs; NF-κB and PI3K/AKT signaling pathways were implicated in the pathological mechanism of CAVD. IHC and WB experiments showed decreased AMPK and AKT and increased Bax in calcific aortic valves. PM treatment significantly reduced AMPK and PI3K/AKT signaling pathways, promoted Bax/Bcl2 ratio, and induced AVICs calcification. Metformin treatment ameliorated AVICs calcification and apoptosis by activating the PI3K/AKT signaling pathway. AMPK activation and NF-κB inhibition could inhibit AVICs calcification induced by PM treatment; however, AMPK and AKT inhibition reversed the protective effect of metformin. Conclusions This study, for the first time, demonstrates that metformin can inhibit AVICs in vitro calcification by activating the PI3K/AKT signaling pathway; this suggests that metformin may provide a potential target for the treatment of CAVD. And the PI3K/AKT signaling pathway emerges as an important regulatory axis in the pathological mechanism of CAVD.

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Abstract 18216: Epigenetic Up-regulation of α-smooth Muscle Actin Expression and Cell Aggregation in Human Aortic Valve Interstitial Cells Promotes Calcific Nodule Formation

Circulation ◽

10.1161/circ.130.suppl_2.18216 ◽

2014 ◽

Vol 130 (suppl_2) ◽

Author(s):

Rui Song ◽

David A. Fullerton ◽

Lihua Ao ◽

Kesen Zhao ◽

Xianzhong Meng

Keyword(s):

Smooth Muscle ◽

Aortic Valve ◽

Smooth Muscle Actin ◽

Cell Aggregation ◽

Interstitial Cells ◽

Nodule Formation ◽

Muscle Actin ◽

Aortic Valves ◽

Valve Interstitial Cells ◽

Tgf Β1

Older people are at risk of calcific aortic valve disease. Aortic valve interstitial cells (AVICs) play an important role in nodular calcification in aortic valve leaflets. AVICs in human aortic valves consist of fibroblasts and myofibroblasts that express α-smooth muscle actin (α-SMA). We have observed that AVICs of diseased aortic valves have greater osteogenic activities. However, molecular mechanism underlying AVIC formation of calcification nodules is not well understood. We hypothesized that an epigenetic mechanism promotes AVIC calcification nodule formation through induction of α-SMA expression and cell aggregation. Methods and Results: MiRNA profiles in AVICs from normal and diseased human aortic valves were analyzed by miRNA array and real-time qPCR. Diseased AVICs displayed higher levels of miR-486. Immunoblotting and immunofluorescence staining revealed that diseased AVICs had higher levels of α-SMA and α-SMA fibers. Inhibition of miR-486 by lentiviral-delivered miR-486 antagomir in diseased AVICs suppressed α-SMA expression and cell aggregation, resulting in reduced calcification nodule formation. Conversely, lentiviral-delivered miR-486 mimic in normal AVICs induced α-SMA expression and cell aggregation, leading to exacerbated calcification nodule formation. Stimulation of normal AVICs with pro-osteogenic mediators TGF-β1 and BMP-2 up-regulated miR-486 levels. MiR-486 antagomir reduced α-SMA expression, cell aggregation and calcification nodule formation in cells exposed to TGF-β1 or BMP-2. Further, miR-486 mimic induced AKT phosphorylation. Inhibition of AKT decreased α-SMA expression and cell aggregation induced by miR-486 mimic in normal AVICs. Knockdown of α-SMA suppressed cell aggregation and calcification nodule formation. Conclusions: The pro-osteogenic phenotype of AVICs of diseased aortic valves is associated with up-regulated levels of miR-486 and α-SMA. MiR-486 modulates the AKT pathway to up-regulate α-SMA expression and cell aggregation that are required for calcification nodule formation. These novel findings indicate that miR-486 contributes to the mechanism underlying aortic valve calcification and appears to be a therapeutic target for suppression of valvular osteogenic activity.

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Strain and Substrate Stiffness Affect Calcium Accumulation in Aortic Valve Interstitial Cells

ASME 2011 Summer Bioengineering Conference, Parts A and B ◽

10.1115/sbc2011-53611 ◽

2011 ◽

Author(s):

Joshua D. Hutcheson ◽

M. K. Sewell-Loftin ◽

W. David Merryman

Keyword(s):

Aortic Valve ◽

Molecular Mechanisms ◽

Cultured Cells ◽

Mechanical Strain ◽

Cell Types ◽

Interstitial Cells ◽

Nodule Formation ◽

Native Tissue

The progression of aortic valve (AV) disease is often characterized by the formation of calcific nodules on thickened AV leaflets, limiting the biomechanical function of the valve. Calcification is a major problem that often leads to the failure of bioprosthetic replacement valves [1]. In these cases, the association of extracellular Ca2+ with phosphates remaining in cellular debris within the decellularized scaffolds has been proposed to lead to the nucleation and growth of Ca3(PO4)2 nodules. In native tissue, calcification is thought to be a more active process involving AV interstitial cells (AVICs). The exact molecular mechanisms that lead to the formation of these calcific nodules in native tissue remain unclear; however, AVICs have been shown to form nodule-like structures in vitro through differentiation to a phenotype with osteogenic character [2]. Additionally, in vitro nodules are characterized by activated smooth muscle α-actin positive AVICs and high levels of apoptosis [2–3]. Mechanical strain has also been shown to influence nodule formation in excised AV leaflets [4]. Intracellular Ca2+ exhibits mechanodependency in cultured cells [5], and heightened levels of intracellular Ca2+ have been shown to be associated with apoptosis in many cell types [6] In this study, we assess the role of mechanically-induced changes in intracellular calcium and its function in modulating AVIC behavior. We hypothesized that intracellular Ca2+ will increase in strained AVICs and that over time, this will lead to apoptosis. We believe that the results from this study will help illustrate the mechanotransductive role of Ca2+ in AVICs and may elucidate early cellular changes that lead to AV calcification.

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Primary culture and in vitro calcification model establishment of human aortic valve interstitial cells

Academic Journal of Second Military Medical University ◽

10.3724/sp.j.1008.2013.00488 ◽

2013 ◽

Vol 33 (5) ◽

pp. 488-492

Author(s):

Mi ZHANG ◽

Xiao-hong LIU ◽

Bo-yao ZHANG ◽

Lin HAN ◽

Fang-lin LU ◽

...

Keyword(s):

Aortic Valve ◽

Primary Culture ◽

Interstitial Cells ◽

Valve Interstitial Cells ◽

Human Aortic Valve

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5-HT2B Antagonism Inhibits Strain- and Cytokine-Dependent Formation of Calcific Nodules by Aortic Valve Interstitial Cells

ASME 2012 Summer Bioengineering Conference, Parts A and B ◽

10.1115/sbc2012-80496 ◽

2012 ◽

Author(s):

Joshua D. Hutcheson ◽

Joseph Chen ◽

Larisa M. Ryzhova ◽

W. David Merryman

Keyword(s):

Aortic Valve ◽

Mechanical Strain ◽

Cyclic Strain ◽

Cell Types ◽

Epidemiological Studies ◽

Interstitial Cells ◽

Nodule Formation ◽

Type I ◽

Tgf Β1

The progression of aortic valve (AV) disease is often characterized by the formation of calcific nodules on thickened AV leaflets, limiting the biomechanical function of the valve. In these cases, the association of extracellular Ca2+ with phosphates remaining in cellular debris within the decellularized scaffolds has been proposed to lead to the nucleation and growth of calcific nodules. In native tissue, calcification is thought to be a more active process involving AV interstitial cells (AVICs). AVICs have been shown to form nodule-like structures in vitro through differentiation to a phenotype with osteogenic character. Additionally, in vitro nodules are characterized by activated smooth muscle α-actin (αSMA) positive AVICs and high levels of apoptosis [1–2]. Mechanical strain has also been shown to influence nodule formation in excised AV leaflets [3]. Our lab has recently developed a model system that recapitulates the formation of calcific nodules in vitro [4]. AVICs treated with TGF-β1 for 24 h prior to the addition of 15% cyclic strain in a Flexcell strain system form nodules that appear to be dependent upon the initiation of AVIC activation. These observations are consistent with previous studies that have shown that αSMA expression is required for nodule formation by AVICs in static culture, with statins shown to inhibit in vitro nodule formation [1]. However, retrospective epidemiological studies have shown that these drugs may not be as effective in preventing calcific valve disease in patients [5]. Additionally, the molecular target and relevant pathways for statins in AVICs remain largely unknown. Therefore, a therapeutically relevant target to prevent AVIC activation and subsequent nodule formation is greatly needed. In this study we investigated the ability of antagonists to 5-HT2B, a receptor known to be upstream of TGF-β1, to oppose strain- and TGF-β1-induced AVIC activation and nodule formation. We also assessed the efficacy of an antagonist to a receptor, the angiotensin II type I receptor (AT1R), known to crosstalk with both 5-HT2B and TGF-β1 signaling in other cell types in inhibiting AVIC nodule formation. Our results indicate that 5-HT2B antagonism inhibits AVIC activation and nodule formation by blocking non-canonical TGF-β1 signaling, whereas AT1R antagonism does not inhibit these outcomes. We believe that the results of this study may indicate novel therapeutic targets to prevent the progression of AV calcification.

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