Abstract 15843: A Novel Role for Telomerase in Calcific Aortic Valve Disease

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Rolando A Cuevas ◽  
Luis HORTELLS ◽  
Camille Boufford ◽  
Cailyn Regan ◽  
Claire Chu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Smooth Muscle ◽  
Aortic Valve ◽  
Smooth Muscle Cells ◽  
Knockout Mice ◽  
Aortic Valve Disease ◽  
Calcific Aortic Valve Disease ◽  
Calcification Process

Introduction: Calcific aortic valve disease (CAVD) is a disorder characterized by the slow, but a progressive thickening of the aortic valve leaflet that develops into severe calcification. The only current therapy is aortic valve replacement. Telomerase is an enzymatic complex best known for its telomere-extending activities on the ends of chromosomes yet, the catalytic subunit (TERT) has been implicated in multiple non-canonical transcriptional and epigenetic activities, including priming of mesenchymal stem cells (MSCs) to differentiate into osteoblasts, and transcriptional regulation of inflammatory genes. Hypothesis: We hypothesize that non-canonical TERT activity contributes to the progression of CAVD. Methods: We performed biochemical assays to study the role of TERT in the calcification process using primary tissues and valve interstitial cells (VICs) from control and CAVD patients, smooth muscle cells (SMCs) from WT and Tert knockout mice, and mesenchymal stem cells (MSCs). Results: We found that TERT protein is highly expressed in calcified aortic valves and VICs isolated from patients with calcified valves, compared to healthy valves. VICs can be induced to calcify under osteogenic differentiation conditions, and we found that TERT accumulated after fourteen days in this culture, with no effect on either telomere length, proliferation, or senescence. We expanded the scope of our approach by evaluating TERT's influence on calcification of mice aortic smooth muscle cells (mSMCs). We found WT mSMCs readily calcified in vitro, but mSMCs from Tert knockout mice did not, and Tert deletion also reduced valve calcification in a Ldlr / Tert double knockout mice model compared to Ldlr knockout alone. In VICs, shRNA mediated TERT downregulation reduced expression of RUNX2 . Finally, we found that inflammatory signals intensify in vitro calcification, induce TERT expression, and we show evidence that TERT interacts with STAT5. Conclusion: Our data suggest that TERT is required for valve calcification by stimulating transcriptional pathways promoting the osteogenic transition of quiescent VICs into calcifying VICs in the aortic valve. These results indicate that TERT is an active contributor to the calcification process of valve tissues.

212 PORCINE BONE MARROW-DERIVED MESENCHYMAL STEM CELLS DIFFERENTIATE IN VITRO INTO SMOOTH MUSCLE CELLS.

2012 ◽  
Vol 24 (1) ◽  
pp. 218
Author(s):  
B. Mohana Kumar ◽  
G. H. Maeng ◽  
Y. M. Lee ◽  
T. H. Kim ◽  
W. J. Lee ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Muscle Cells ◽  
Class Ii ◽  
Alizarin Red ◽  
Expression Levels

In the context of multipotent stem cells, mesenchymal stem cells (MSC) derived from bone marrow have been identified as most promising cell types for the treatment of smooth muscle related injured tissues and organs. In the present study, the ability of porcine bone marrow derived MSC to differentiate in vitro into smooth muscle cells (SMC) was examined. MSC were isolated from domestic pig bone marrow by their readily adherent property to tissue culture plastic with fibroblast-like morphology. Cells were analysed for the expression of MSC specific markers by flow cytometer and mesenchymal lineage differentiation by following previously published protocols. Differences in values were analysed by one-way ANOVA using SPSS and data are presented as mean ± SD. Flow cytometry analysis of MSC showed the positive expression of markers, such as CD29 (97.33 ± 2.08%), CD44 (97.67 ± 1.15%), CD73 (62.33 ± 2.89%), CD90 (96.67 ± 2.08%) and vimentin (59.33 ± 2.52%). In contrast, the expression levels were significantly lower for CD34 (3.33 ± 1.53%), CD45 (3.67 ± 1.53%), major histocompatibility complex class II (MHC class II, 10.33 ± 2.52%) and swine leukocyte antigen-DR (SLA-DR, 9.67 ± 2.08%). The MSC were further confirmed by their ability to differentiate in vitro along the distinct lineages of adipocytes (Oil red O), osteocytes (von Kossa and Alizarin red) and chondrocytes (Alcian blue). Induction of SMC differentiation was performed with supplementation of porcine transforming growth factor-β (TGF-β) and recombinant human bone morphogenic protein 4 (BMP4) as described earlier (Wang et al. 2010 Tissue Eng. A 1201–1213) with minor modifications. Upon induction, porcine MSC acquired myoblast-like morphology with intracellular thin filaments. Immunofluorescence staining showed the presence of early and late markers of smooth muscle differentiation, such as α-smooth muscle actin (α-SMA), calponin, smooth muscle 22 α (SM22α) and smooth muscle-myosin heavy chain (SM-MHC) and their expression levels varied from 22.65% to 56.75%. Later, the expression of selected markers was demonstrated by Western blotting analysis. Consistent with this phenotypic characterisation, reverse transcription-polymerase chain reaction (RT-PCR) and quantitative PCR (RT-qPCR) further showed the expression and a sequential up-regulation of transcripts for α-SMA, calponin, SM22α and SM-MHC. However, no expression of SMC-specific markers was observed in untreated MSC. In conclusion, these findings suggest the ability of porcine MSC from bone marrow to differentiate in vitro into SMC in the presence of growth factors. Further understanding of SMC differentiation with functional properties would be essential for employing porcine MSC as a useful model for cell-based tissue engineering and regeneration strategies. This work was supported by Basic Science Research Program through the National Research Foundation (NRF) funded by the Ministry of Education, Science and Technology (2010-0010528) and BioGreen 21 (20070301034040), Republic of Korea.

scholarly journals Creation of disease-inspired biomaterial environments to mimic pathological events in early calcific aortic valve disease

2017 ◽  
Vol 115 (3) ◽  
pp. E363-E371 ◽  
Author(s):  
Ana M. Porras ◽  
Jennifer A. Westlund ◽  
Austin D. Evans ◽  
Kristyn S. Masters
Keyword(s):  
Aortic Valve ◽  
Disease Progression ◽  
Aortic Valve Disease ◽  
Low Density Lipoprotein ◽  
Treatment Strategies ◽  
Density Lipoprotein ◽  
Valve Disease ◽  
Calcific Aortic Valve Disease ◽  
Valve Lesion

An insufficient understanding of calcific aortic valve disease (CAVD) pathogenesis remains a major obstacle in developing treatment strategies for this disease. The aim of the present study was to create engineered environments that mimic the earliest known features of CAVD and apply this in vitro platform to decipher relationships relevant to early valve lesion pathobiology. Glycosaminoglycan (GAG) enrichment is a dominant hallmark of early CAVD, but culture of valvular interstitial cells (VICs) in biomaterial environments containing pathological amounts of hyaluronic acid (HA) or chondroitin sulfate (CS) did not directly increase indicators of disease progression such as VIC activation or inflammatory cytokine production. However, HA-enriched matrices increased production of vascular endothelial growth factor (VEGF), while matrices displaying pathological levels of CS were effective at retaining lipoproteins, whose deposition is also found in early CAVD. Retained oxidized low-density lipoprotein (oxLDL), in turn, stimulated myofibroblastic VIC differentiation and secretion of numerous inflammatory cytokines. OxLDL also increased VIC deposition of GAGs, thereby creating a positive feedback loop to further enrich GAG content and promote disease progression. Using this disease-inspired in vitro platform, we were able to model a complex, multistep pathological sequence, with our findings suggesting distinct roles for individual GAGs in outcomes related to valve lesion progression, as well as key differences in cell–lipoprotein interactions compared with atherosclerosis. We propose a pathogenesis cascade that may be relevant to understanding early CAVD and envision the extension of such models to investigate other tissue pathologies or test pharmacological treatments.

scholarly journals Simulating early calcific aortic valve disease within novel in vitro 3D tissue platform

2013 ◽  
Vol 34 (suppl 1) ◽  
pp. P3908-P3908 ◽  
Author(s):  
J. Hjortnaes ◽  
G. Gamci-Unal ◽  
C. Goettsch ◽  
K. Scherer ◽  
L. Lax ◽  
...  
Keyword(s):  
Aortic Valve ◽  
Aortic Valve Disease ◽  
Valve Disease ◽  
Calcific Aortic Valve Disease

scholarly journals Mesenchymal Stem Cells Attenuate Lipopolysaccharide-Induced Inflammatory Response in Human Uterine Smooth Muscle Cells

2020 ◽  
Vol 10 (03) ◽  
pp. e335-e341
Author(s):  
Arunmani Mani ◽  
John W. Hotra ◽  
Sean C. Blackwell ◽  
Laura Goetzl ◽  
Jerrie S. Refuerzo
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Smooth Muscle ◽  
Preterm Birth ◽  
Inflammatory Response ◽  
Smooth Muscle Cells ◽  
Muscle Cells ◽  
Uterine Smooth Muscle ◽  
Cell Based Therapy ◽  
Increased Risk

Abstract Objective The aim of this study was to determine if mesenchymal stem cells (MSCs) would suppress the inflammatory response in human uterine cells in an in vitro lipopolysaccharide (LPS)-based preterm birth (PTB) model. Study Design Cocultures of human uterine smooth muscle cells (HUtSMCs) and MSCs were exposed to 5 μg/mL LPS for 4 hours and further challenged with 1 μg/mL LPS for a subsequent 24 hours. Key elements of the parturition cascade regulated by toll-like receptors (TLRs) through activation of mitogen-activated protein kinases (MAPKs) were quantified in culture supernatant as biomarkers of MSC modulation. Results Coculture with MSCs significantly attenuated TLR-4, p-JNK, and p- extracellular signal-regulated kinase 1/2 (ERK1/2) protein levels compared with HUtSMCs monoculture (p = 0.05). In addition, coculture was associated with significant inhibition of proinflammatory cytokines interleukin (IL)-6 and IL-8 (p = 0.0001) and increased production of anti-inflammatory cytokines IL-10 and transforming growth factor (TGF)-β1 (p = 0.0001). Conclusion MSCs appear to play a role in significantly attenuating LPS-mediated inflammation via alteration of down-stream MAPKs. MSCs may represent a novel, cell-based therapy in women with increased risk of inflammatory-mediated preterm birth.

scholarly journals Phenotypic Change of Mesenchymal Stem Cells Into Smooth Muscle Cells Regulated By Dynamic Cell-Surface Interactions On Patterned Arrays of Ultrathin Graphene Oxide Substrates

2021 ◽  
Author(s):  
Rowoon Park ◽  
Jung Won Yoon ◽  
Jin-Ho Lee ◽  
Suck Won Hong ◽  
Jae Ho Kim
Keyword(s):  
Stem Cells ◽  
Cell Culture ◽  
Mesenchymal Stem Cells ◽  
Smooth Muscle ◽  
Graphene Oxide ◽  
Smooth Muscle Cells ◽  
Self Assembly ◽  
Muscle Cells ◽  
Phenotypic Change ◽  
Cell Functions

Abstract The topographical interface of the extracellular environment has been appreciated as a principal biophysical regulator for modulating cell functions, such as adhesion, migration, proliferation, and differentiation. Despite the existed approaches that use two-dimensional nanomaterials to provide beneficial effects, opportunities evaluating their impact on stem cells remain open to elicit unprecedented cellular responses. Herein, we report an ultrathin cell-culture platform with potential-responsive nanoscale biointerfaces for monitoring mesenchymal stem cells (MSCs). We designed an intriguing nanostructured array through self-assembly of graphene oxide sheets and subsequent lithographical patterning method to produce chemophysically defined regions. MSCs cultured on anisotropic micro/nanoscale patterned substrate were spontaneously organized in a highly ordered configuration mainly due to the cell-repellent interactions. Moreover, the spatially aligned MSCs were spontaneously differentiated into smooth muscle cells upon the specific crosstalk between cells. This work provides a robust strategy for directing stem cells and differentiation, which can be utilized as a potential cell culture platform to understand cell-substrate or cell-cell interactions, further developing tissue repair and stem cell-based therapies.

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