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.

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

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.

scholarly journals Computational Analysis of Fluid Flow Within a Device for Applying Biaxial Strain to Cultured Cells

2015 ◽  
Vol 137 (5) ◽  
Author(s):  
Jason Lee ◽  
Aaron B. Baker
Keyword(s):  
Shear Stress ◽  
Cultured Cells ◽  
Fluid Velocity ◽  
Mechanical Strain ◽  
Cell Types ◽  
Linear Increase ◽  
Shear Stresses ◽  
Biaxial Strain ◽  
Maximal Strain

In vitro systems for applying mechanical strain to cultured cells are commonly used to investigate cellular mechanotransduction pathways in a variety of cell types. These systems often apply mechanical forces to a flexible membrane on which cells are cultured. A consequence of the motion of the membrane in these systems is the generation of flow and the unintended application of shear stress to the cells. We recently described a flexible system for applying mechanical strain to cultured cells, which uses a linear motor to drive a piston array to create biaxial strain within multiwell culture plates. To better understand the fluidic stresses generated by this system and other systems of this type, we created a computational fluid dynamics model to simulate the flow during the mechanical loading cycle. Alterations in the frequency or maximal strain magnitude led to a linear increase in the average fluid velocity within the well and a nonlinear increase in the shear stress at the culture surface over the ranges tested (0.5–2.0 Hz and 1–10% maximal strain). For all cases, the applied shear stresses were relatively low and on the order of millipascal with a dynamic waveform having a primary and secondary peak in the shear stress over a single mechanical strain cycle. These findings should be considered when interpreting experimental results using these devices, particularly in the case when the cell type used is sensitive to low magnitude, oscillatory shear stresses.

Microdevice for Delivering Homogeneous Equi-Biaxial Strain to Live Cells Without the Use of Platen Structures

2013 ◽  
Author(s):  
Qian Wang ◽  
Yi Zhao
Keyword(s):  
Cultured Cells ◽  
Flexible Substrate ◽  
Mechanical Strain ◽  
Live Cells ◽  
Biaxial Strain ◽  
Thin Membrane ◽  
Strain Homogeneity ◽  
Homogeneous Strain

Live cells from most of the membranous tissues such as alveoli are subjected to equi-biaxial strain originated from their extracellular environments. To understand the role of equi-biaxial strain in live cells, a number of engineered methods have been developed for applying such mechanical strain to in vitro cultured cells. Among these methods, deforming a flexible substrate on a circular platen has been widely used [1], and has been miniaturized into millimeter scale for parallel stretching assay (Figure 1a). Nonetheless, the strain homogeneity becomes increasingly challenging at smaller scale, since it requires an ultra-thin membrane, an indentation platen with well controlled dimension, and the highly precise alignment. This obviously increases the fabrication and operation complexities. Devices that deliver homogeneous strain with minimal fabrication and assembling complexities are needed.

scholarly journals Regulation of TRH neurons and energy homeostasis-related signals under stress

2015 ◽  
Vol 224 (3) ◽  
pp. R139-R159 ◽  
Author(s):  
Patricia Joseph-Bravo ◽  
Lorraine Jaimes-Hoy ◽  
Jean-Louis Charli
Keyword(s):  
Energy Homeostasis ◽  
Molecular Mechanisms ◽  
Cell Types ◽  
Cellular Phenotype ◽  
Energy Demands ◽  
Rapid Responses ◽  
Hpt Axis

Energy homeostasis relies on a concerted response of the nervous and endocrine systems to signals evoked by intake, storage, and expenditure of fuels. Glucocorticoids (GCs) and thyroid hormones are involved in meeting immediate energy demands, thus placing the hypothalamo–pituitary–thyroid (HPT) and hypothalamo–pituitary–adrenal axes at a central interface. This review describes the mode of regulation of hypophysiotropic TRHergic neurons and the evidence supporting the concept that they act as metabolic integrators. Emphasis has been be placed on i) the effects of GCs on the modulation of transcription ofTrhin vivoandin vitro, ii) the physiological and molecular mechanisms by which acute or chronic situations of stress and energy demands affect the activity of TRHergic neurons and the HPT axis, and iii) the less explored role of non-hypophysiotropic hypothalamic TRH neurons. The partial evidence gathered so far is indicative of a contrasting involvement of distinct TRH cell types, manifested through variability in cellular phenotype and physiology, including rapid responses to energy demands for thermogenesis or physical activity and nutritional status that may be modified according to stress history.

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.

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.

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.

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