scholarly journals Hypoxic Culture Maintains Cell Growth of the Primary Human Valve Interstitial Cells with Stemness

2021 ◽  
Vol 22 (19) ◽  
pp. 10534
Author(s):  
Kaho Kanno ◽  
Tomohisa Sakaue ◽  
Mika Hamaguchi ◽  
Kenji Namiguchi ◽  
Daisuke Nanba ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Cell Cycle ◽  
Aortic Valve ◽  
Molecular Mechanisms ◽  
Interstitial Cells ◽  
Oxidative Stress Marker ◽  
Marker Genes ◽  
Low Oxygen ◽  
Valve Interstitial Cells

The characterization of aortic valve interstitial cells (VICs) cultured under optimal conditions is essential for understanding the molecular mechanisms underlying aortic valve stenosis. Here, we propose 2% hypoxia as an optimum VIC culture condition. Leaflets harvested from patients with aortic valve regurgitation were digested using collagenase and VICs were cultured under the 2% hypoxic condition. A significant increase in VIC growth was observed in 2% hypoxia (hypo-VICs), compared to normoxia (normo-VICs). RNA-sequencing revealed that downregulation of oxidative stress-marker genes (such as superoxide dismutase) and upregulation of cell cycle accelerators (such as cyclins) occurred in hypo-VICs. Accumulation of reactive oxygen species was observed in normo-VICs, indicating that low oxygen tension can avoid oxidative stress with cell-cycle arrest. Further mRNA quantifications revealed significant upregulation of several mesenchymal and hematopoietic progenitor markers, including CD34, in hypo-VICs. The stemness of hypo-VICs was confirmed using osteoblast differentiation assays, indicating that hypoxic culture is beneficial for maintaining growth and stemness, as well as for avoiding senescence via oxidative stress. The availability of hypoxic culture was also demonstrated in the molecular screening using proteomics. Therefore, hypoxic culture can be helpful for the identification of therapeutic targets and the evaluation of VIC molecular functions in vitro.

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

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.

Calcific Nodule Morphogenesis by Aortic Valve Interstitial Cells: Synergism of Applied Strain and TGF-β1

2011 ◽  
Author(s):  
Joseph Chen ◽  
Charles I. Fisher ◽  
M. K. Sewell-Loftin ◽  
W. David Merryman
Keyword(s):  
Aortic Valve ◽  
The United States ◽  
Interstitial Cells ◽  
Nodule Formation ◽  
Matrix Stiffness ◽  
Calcific Aortic Valve Disease ◽  
Valve Interstitial Cells ◽  
Compliant Substrates ◽  
Tgf Β1

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.

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

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.

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

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.

scholarly journals Protection of Tong-Sai-Mai Decoction against Apoptosis Induced by H2O2in PC12 Cells: Mechanisms via Bcl-2-Mitochondria-ROS-INOS Pathway

2014 ◽  
Vol 2014 ◽  
pp. 1-18 ◽  
Author(s):  
Maxwell Kim Kit Lee ◽  
Yin Lu ◽  
Liu-qing Di ◽  
Hui-qin Xu
Keyword(s):  
Oxidative Stress ◽  
Pc12 Cells ◽  
Molecular Mechanisms ◽  
Cell Types ◽  
In Vivo Studies ◽  
Marker Genes ◽  
Mitochondria Membrane Potential ◽  
Stress Dependent

Tong-Sai-Mai decoction (TSM) is a Chinese materia medica polyherbal formulation that has been applied in treating brain ischemia for hundreds of years. Because it could repress the oxidative stress in in vivo studies, now we focus on the in vitro studies to investigate the mechanism by targeting the oxidative stress dependent signaling. The relation between the neurogenesis and the reactive oxygen species (ROS) production remains largely unexamined. PC12 cells are excitable cell types widely used as in vitro model for neuronal cells. Most marker genes that are related to neurotoxicity, apoptosis, and cell cycles are expressed at high levels in these cells. The aim of the present study is to explore the cytoprotection of TSM against hydrogen peroxide- (H2O2-) induced apoptosis and the molecular mechanisms underlying PC12 cells. Our findings revealed that TSM cotreatment with H2O2restores the expression of bcl-2, inducible nitric oxide synthase (INOS), and mitochondria membrane potential. Meanwhile, it reduces intracellular [Ca2+] concentration, lactate dehydrogenase (LDH) release, and the expression of caspase-3 and bax. The results of the present study suggested that the cytoprotective effects of the TSM might be mediated, at least in part, by the bcl-2-mitochondria-ROS-INOS pathway. Due to its nontoxic characteristics, TSM could be further developed to treat the neurodegenerative diseases which are closely associated with the oxidative stress.

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

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

scholarly journals Gene Profiling of Aortic Valve Interstitial Cells under Elevated Pressure Conditions: Modulation of Inflammatory Gene Networks

2011 ◽  
Vol 2011 ◽  
pp. 1-10 ◽  
Author(s):  
James N. Warnock ◽  
Bindu Nanduri ◽  
Carol A. Pregonero Gamez ◽  
Juliet Tang ◽  
Daniel Koback ◽  
...  
Keyword(s):  
Aortic Valve ◽  
Network Model ◽  
Gene Networks ◽  
Gene Network ◽  
Molecular Mechanisms ◽  
Elevated Pressure ◽  
Interstitial Cells ◽  
Gene Profiling ◽  
Differentially Expressed ◽  
Valve Interstitial Cells

The study aimed to identify mechanosensitive pathways and gene networks that are stimulated by elevated cyclic pressure in aortic valve interstitial cells (VICs) and lead to detrimental tissue remodeling and/or pathogenesis. Porcine aortic valve leaflets were exposed to cyclic pressures of 80 or 120 mmHg, corresponding to diastolic transvalvular pressure in normal and hypertensive conditions, respectively. Linear, two-cycle amplification of total RNA, followed by microarray was performed for transcriptome analysis (with qRT-PCR validation). A combination of systems biology modeling and pathway analysis identified novel genes and molecular mechanisms underlying the biological response of VICs to elevated pressure. 56 gene transcripts related to inflammatory response mechanisms were differentially expressed. TNF-α, IL-1α, and IL-1βwere key cytokines identified from the gene network model. Also of interest was the discovery that pentraxin 3 (PTX3) was significantly upregulated under elevated pressure conditions (41-fold change). In conclusion, a gene network model showing differentially expressed inflammatory genes and their interactions in VICs exposed to elevated pressure has been developed. This system overview has detected key molecules that could be targeted for pharmacotherapy of aortic stenosis in hypertensive patients.

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

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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