Abstract 139: Alterations to the Extracellular Matrix Composition Following Myocardial Infarction Impact Cardiac Progenitor Cell Fate In Vitro

Limitations associated with cardiac progenitor cell (CPC) therapy of myocardial infarction (MI) including poor engraftment, cell death and incomplete cardiac differentiation have hindered the efficacy of treatment in pre-clinical trials. Given that the extracellular environment plays an important role in regulating cell function and that it is significantly remodeled following MI, it is critical to understand how these changes impact the therapeutic potential of CPCs. In this study, we investigated how the alterations to the extracellular matrix (ECM) following MI impacted the regenerative potential of CPCs in vitro. Hearts were decellularized with 1% SDS prior to MI and 1 and 4 weeks post-MI (Fig A) and the composition of the left ventricle or scar was characterized through LC-MS/MS. While Periostin and Collagen I increased post-MI, Laminin decreased (Fig B). c-kit+ CPCs isolated from rat hearts 1 week post-MI were cultured on tissue culture plastic (TCP) coated with pepsin-solubilized ECM. Our results demonstrated that the healthy matrix promoted the expression of pro-angiogenic growth factors, while maintaining the cells in an undifferentiated state (Fig D,E). Alternatively, 1 week ECM promoted cell adherence (Fig C) and the expression of pro-survival growth factors (Fig D) and GATA-4 (Fig E). Cells cultured on 4 week ECM demonstrated significant differentiation towards vascular lineages through their expression of smooth muscle (TAGLN) and endothelial (VWF) markers. By characterizing how the changing ECM composition following MI impacts CPC fate, we may be able to develop therapeutic strategies that modulate cell fate/ function in vivo following implantation.

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Abstract 356: Krueppel-Like Factor 15 Regulates Wnt/β-Catenin Transcription and Controls Cardiac Progenitor Cell Fate in the Postnatal Heart

Circulation Research ◽

10.1161/res.111.suppl_1.a356 ◽

2012 ◽

Vol 111 (suppl_1) ◽

Author(s):

Claudia Noack ◽

Maria P Zafiriou ◽

Anke Renger ◽

Hans J Schaeffer ◽

Martin W Bergmann ◽

...

Keyword(s):

Cell Fate ◽

Transcriptional Activation ◽

Progenitor Cell ◽

Cellular Localization ◽

Target Genes ◽

Critical Role ◽

Isolated Cells ◽

Cardiac Progenitor Cell

Wnt/β-catenin signaling controls adult heart remodeling partly by regulating cardiac progenitor cell (CPC) differentiation. We now identified and characterized a novel cardiac interaction of the transcription factor Krueppel-like factor 15 (KLF15) with the Wnt/β-catenin signaling on adult CPCs. In vitro mutation, reporter gene assays and co-localization studies revealed that KLF15 requires two distinct domains for nuclear localization and for repression of β-catenin-mediated transcription. KLF15 had no effect on β-catenin stability or cellular localization, but interacted with its co-factor TCF4, which is required for activation of β-catenin target gene expression. Moreover, increased TCF4 ubiquitination was induced by KLF15. In line with this finding we found KLF15 to interact with the Nemo-like kinase, which was shown to phosphorylate and target TCF4 for degradation. In vivo analyses of adult Klf15 functional knock-out (KO) vs. wild-type (WT) mice showed a cardiac β-catenin-mediated transcriptional activation and reduced TCF4 degradation along with cardiac dysfunction assessed by echocardiography (n=10). FACS analysis of the CPC enriched-population of KO vs. WT mice revealed a significant reduction of cardiogenic-committed precursors identified as Sca1+/αMHC+ (0.8±0.2% vs. 1.8±0.1%) and Tbx5+ (3.5±0.3% vs. 5.2±0.5%). In contrast, endothelial Sca1+/CD31+ cells were significantly higher in KO mice (11.3±0.4% vs. 8.6±0.4%; n≥9). In addition, Sca1+ isolated cells of Klf15 KO showed increased RNA expression of endothelial markers von Willebrand Factor, CD105, and Flk1 along with upregulation of β-catenin target genes. CPCs co-cultured on adult fibroblasts resulted in increased endothelial Flk1 cells and reduction of αMHC and Hand1 cardiogenic cells in KO vs. WT CPCs (n=9). Treating these co-cultures with Quercetin, an inhibitor of nuclear β-catenin, resulted in partial rescue of the observed phenotype. This study uncovers a critical role of KLF15 for the maintenance of cardiac tissue homeostasis. Via inhibition of β-catenin transcription, KLF15 controls cardiomyogenic cell fate similar to embryonic cardiogenesis. This knowledge may provide a tool for activation of endogenous CPCs in the postnatal heart.

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A naturally derived cardiac extracellular matrix enhances cardiac progenitor cell behavior in vitro

Acta Biomaterialia ◽

10.1016/j.actbio.2012.07.033 ◽

2012 ◽

Vol 8 (12) ◽

pp. 4357-4364 ◽

Cited By ~ 91

Author(s):

Kristin M. French ◽

Archana V. Boopathy ◽

Jessica A. DeQuach ◽

Loice Chingozha ◽

Hang Lu ◽

...

Keyword(s):

Extracellular Matrix ◽

Progenitor Cell ◽

Cell Behavior ◽

Cardiac Progenitor Cell

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Abstract 340: Short Telomeres Induce Autophagy and Modulate Cardiac Progenitor Cell Fate

Circulation Research ◽

10.1161/res.121.suppl_1.340 ◽

2017 ◽

Vol 121 (suppl_1) ◽

Author(s):

Nirmala Hariharan ◽

Collin Matsumoto ◽

Jacqueline Emathinger ◽

Saba Daneshpooy ◽

Minyoung Shin ◽

...

Keyword(s):

Cell Fate ◽

Progenitor Cell ◽

Smooth Muscle Actin ◽

Wild Mouse ◽

Degradation Process ◽

Mouse Strains ◽

Therapeutic Effects ◽

Cardiac Progenitor Cell ◽

Altered Cell ◽

The Impact

Aging severely limits myocardial regeneration. Delineating the impact of age-associated factors such as short telomeres is critical to enhance the regenerative potential of cardiac progenitor cells (CPCs). We hypothesize that short telomeres induce autophagy and elicit the age-associated change in cardiac progenitor cell fate. We compared mouse strains with different telomere lengths (TL) for phenotypic characteristics of aging and also isolated CPCs from them. Naturally occurring wild mouse strain Mus musculus castaneus (CAST) possessing short telomeres (TL:18Kb) exhibits early cardiac aging with diastolic dysfunction, hypertrophy, fibrosis and increase in senescence markers p53 and p16, as compared to common lab strains FVB (TL:75Kb) and C57 (TL:50Kb). CAST CPCs with short TLs have altered cell fate as characterized by slower proliferation (p<0.01); increased senescence identified by beta-galactosidase activity (p<0.05); increased basal commitment as determined by expression of lineage markers smooth muscle actin, Tie2, and sarcomeric actinin (16.6, 1.7 and 1.75, p<0.05); as well as loss of quiescence marker expression. Consistent findings of altered cell fate are also evident in old CPCs isolated from aged mice with significantly shorter TLs. Cell fate changes occurring downstream from short TL are at least partially p53 dependent, as p53 inhibition rescues the irreversible cell cycle arrest observed in CAST CPCs. Mechanistically, short TLs induce autophagy, a catabolic protein degradation process. Autophagy flux is increased in CAST CPCs as evidenced by increased LC3 (p<0.05), reduced p62 expression (-52%, p<0.05) and increased accumulation of autophagic puncta. Pharmacological inhibition of autophagosome formation, but not autolysosome formation reverses the cell fate to a more youthful phenotype. Overall the data suggests that short TLs activate autophagy to accommodate cell fate changes that tip the equilibrium away from quiescence and proliferation into differentiation and senescence, leading to age-associated exhaustion of CPCs. The study provides the mechanistic basis underlying age-associated cell fate changes that will enable identification of molecular strategies to enhance the therapeutic effects of aged CPCs.

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Comparison of transplantation effects of cardiac progenitor cell types on the mitochondrial energy-producing ability after myocardial infarction in rats

Proceedings for Annual Meeting of The Japanese Pharmacological Society ◽

10.1254/jpssuppl.93.0_2-p-208 ◽

2020 ◽

Vol 93 (0) ◽

pp. 2-P-208

Author(s):

Yuiki Saotome ◽

Tetsuro Marunouchi ◽

Emi Yano ◽

Kouichi Tanonaka

Keyword(s):

Myocardial Infarction ◽

Progenitor Cell ◽

Cell Types ◽

Cardiac Progenitor Cell

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Multiscale molecular profiling of pathological bone resolves sexually dimorphic control of extracellular matrix composition

Disease Models & Mechanisms ◽

10.1242/dmm.048116 ◽

2021 ◽

Vol 14 (3) ◽

pp. dmm048116 ◽

Cited By ~ 1

Author(s):

Aikta Sharma ◽

Alice Goring ◽

Peter B. Johnson ◽

Roger J. H. Emery ◽

Eric Hesse ◽

...

Keyword(s):

Extracellular Matrix ◽

Ex Vivo ◽

Second Harmonic ◽

Collagen Fibre ◽

Matrix Composition ◽

Label Free ◽

Fibre Number ◽

Backscattered Electron ◽

Matrix Mineralisation

ABSTRACTCollagen assembly during development is essential for successful matrix mineralisation, which determines bone quality and mechanocompetence. However, the biochemical and structural perturbations that drive pathological skeletal collagen configuration remain unclear. Deletion of vascular endothelial growth factor (VEGF; also known as VEGFA) in bone-forming osteoblasts (OBs) induces sex-specific alterations in extracellular matrix (ECM) conformation and mineralisation coupled to vascular changes, which are augmented in males. Whether this phenotypic dimorphism arises as a result of the divergent control of ECM composition and its subsequent arrangement is unknown and is the focus of this study. Herein, we used murine osteocalcin-specific Vegf knockout (OcnVEGFKO) and performed ex vivo multiscale analysis at the tibiofibular junction of both sexes. Label-free and non-destructive polarisation-resolved second-harmonic generation (p-SHG) microscopy revealed a reduction in collagen fibre number in males following the loss of VEGF, complemented by observable defects in matrix organisation by backscattered electron scanning electron microscopy. This was accompanied by localised divergence in collagen orientation, determined by p-SHG anisotropy measurements, as a result of OcnVEGFKO. Raman spectroscopy confirmed that the effect on collagen was linked to molecular dimorphic VEGF effects on collagen-specific proline and hydroxyproline, and collagen intra-strand stability, in addition to matrix carbonation and mineralisation. Vegf deletion in male and female murine OB cultures in vitro further highlighted divergence in genes regulating local ECM structure, including Adamts2, Spp1, Mmp9 and Lama1. Our results demonstrate the utility of macromolecular imaging and spectroscopic modalities for the detection of collagen arrangement and ECM composition in pathological bone. Linking the sex-specific genetic regulators to matrix signatures could be important for treatment of dimorphic bone disorders that clinically manifest in pathological nano- and macro-level disorganisation.This article has an associated First Person interview with the first author of the paper.

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Sca-1+ Cardiac Progenitor Cell Therapy With Cells Overexpressing Integrin-Linked Kinase Improves Cardiac Function After Myocardial Infarction

Transplantation Journal ◽

10.1097/tp.0b013e31828a9423 ◽

2013 ◽

Vol 95 (10) ◽

pp. 1187-1196 ◽

Cited By ~ 8

Author(s):

Lin Ling ◽

Jian Bai ◽

Rong Gu ◽

Chunying Jiang ◽

Ran Li ◽

...

Keyword(s):

Myocardial Infarction ◽

Cell Therapy ◽

Cardiac Function ◽

Progenitor Cell ◽

Cardiac Progenitor Cell ◽

Integrin Linked Kinase

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Hypoxia – as a Possible Regulator of the Activity of Epicardial Mesothelial Cells After Myocardial Infarction

Kardiologiia ◽

10.18087/cardio.2021.6.n1476 ◽

2021 ◽

Vol 61 (6) ◽

pp. 59-68

Author(s):

K. V. Dergilev ◽

Z. I. Tsokolaeva ◽

Yu. D. Vasilets ◽

I. B. Beloglazova ◽

B. N. Kulbitsky ◽

...

Keyword(s):

Myocardial Infarction ◽

Extracellular Matrix ◽

Matrix Protein ◽

Mesothelial Cells ◽

Immunofluorescent Staining ◽

Extracellular Matrix Protein ◽

Hypoxic Exposure ◽

Mesenchymal Transition ◽

Epicardial Cells

Aim To study the effect of hypoxia on the activity of epithelial-mesenchymal transition (EMT) in epicardial cells, which provides formation of a specialized microenvironment.Material and methods This study used a model of experimental myocardial infarction created by ligation of the anterior descendent coronary artery. The activity of epicardial cells after a hypoxic exposure was studied with the hypoxia marker, pimonidazole, bromodeoxyuridine, immunofluorescent staining of heart cryosections, and in vitro mesothelial cell culture.Results The undamaged heart maintained the quiescent condition of mesothelial cells and low levels of their proliferation, extracellular matrix protein production, and of the EMT activity. Acute ischemic injury induced moderate hypoxia in the epicardial/subepicardial region. This caused a global rearrangement of this region due to the initiation of EMT in cells, changes in the cell composition, and accumulation of extracellular matrix proteins. We found that the initiation of EMT in mesothelial cells may result in the formation of smooth muscle cell precursors, fibroblasts, and a population of Sca-1+ cardiac progenitor cells, which may both participate in construction of new blood vessels and serve as a mesenchymal link for the paracrine support of microenvironmental cells. In in vitro experiments, we showed that 72‑h hypoxia facilitated activation of EMT regulatory genes, induced dissembling of intercellular contacts, cell uncoupling, and increased cell plasticity.Conclusion The epicardium of an adult heart serves as a “reparative reserve” that can be reactivated by a hypoxic exposure. This creates a basis for an approach to influence the epicardium to modulate its activity for regulating reparative processes.

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Regulation and Directing Stem Cell Fate by Tissue Engineering Functional Microenvironments: Scaffold Physical and Chemical Cues

Stem Cells International ◽

10.1155/2019/2180925 ◽

2019 ◽

Vol 2019 ◽

pp. 1-16 ◽

Cited By ~ 8

Author(s):

Fei Xing ◽

Lang Li ◽

Changchun Zhou ◽

Cheng Long ◽

Lina Wu ◽

...

Keyword(s):

Tissue Engineering ◽

Stem Cells ◽

Stem Cell ◽

Growth Factors ◽

Cell Fate ◽

Chemical Cues ◽

Physical Factors ◽

Stem Cell Fate ◽

Physical And Chemical

It is well known that stem cells reside within tissue engineering functional microenvironments that physically localize them and direct their stem cell fate. Recent efforts in the development of more complex and engineered scaffold technologies, together with new understanding of stem cell behavior in vitro, have provided a new impetus to study regulation and directing stem cell fate. A variety of tissue engineering technologies have been developed to regulate the fate of stem cells. Traditional methods to change the fate of stem cells are adding growth factors or some signaling pathways. In recent years, many studies have revealed that the geometrical microenvironment played an essential role in regulating the fate of stem cells, and the physical factors of scaffolds including mechanical properties, pore sizes, porosity, surface stiffness, three-dimensional structures, and mechanical stimulation may affect the fate of stem cells. Chemical factors such as cell-adhesive ligands and exogenous growth factors would also regulate the fate of stem cells. Understanding how these physical and chemical cues affect the fate of stem cells is essential for building more complex and controlled scaffolds for directing stem cell fate.

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