Abstract 218: Chemical Induced Reductive Stress Causes Cardiomyocyte Hypertrophy

2015 ◽  
Vol 117 (suppl_1) ◽  
Author(s):  
Sandeep B Shelar ◽  
Madhusudhanan Narasimhan ◽  
Gobinath Shanmugam ◽  
Neelu E Vargees ◽  
Ramasamy Sakthivel ◽  
...  
Keyword(s):  
Small Molecules ◽  
Cardiac Remodeling ◽  
Cell Types ◽  
Molecular Signature ◽  
Cardiomyocyte Hypertrophy ◽  
Reductive Stress ◽  
Vitro Model ◽  
C 50 ◽  
Sustained Activation

Background: Progressive accumulation of misfolded or unfolded proteins is a symbol of impaired proteostasis and proteotoxicity. Such a chronic proteotoxicity is amenable to cell types that are post mitotically matured with lack of further differentiation or proliferation. Our recent discovery using a mouse model of familial human cardiac disease displayed protuberant shift in the redox state towards reductive stress (RS) in association with accumulation of toxic protein aggregates. Further, sustained trans-activation of Nrf2/antioxidant signaling caused RS in the myopathy hearts. Accordingly, we hypothesized that whether profound activation of Nrf2/antioxidant signaling and subsequent RS may cause pathological remodeling in cardiomyocyte. The aim of this study was to investigate the effect of sustained pharmacological activation of Nrf2 on cardiac remodeling. Methods: HL1 cardiomyocytes were used as an in vitro model to study the RS-mediated cardiac remodeling. They were treated with 2-10 μM of potential Nrf2-inducers; sulforaphane (SF), di-methyl fumarate (DMF) and novel small molecules (C-38, C-50, C-63 and C-66) to establish RS by sustained activation of Nrf2/antioxidant signaling. Next, we investigated the implications of RS in cardiomyocyte remodeling by analyzing transcriptional and translational mechanisms using immunoblotting, qPCR, immunofluorescence, GSH and NADPH redox measurements in HL1 cells. Results: Dose dependent effects for individual small molecules including known Nrf2 inducers (SF and DMF) revealed distinct pro-reductive and reductive intracellular (i.e. reductive stress) environments. In fact, the obligatory activation of Nrf2 signaling was associated with significant upregulation of antioxidant enzymes and small molecular thiols including glutathione (GSH). Surprisingly, while pro-reductive condition in HL1 cells was subdued, the RS induced cardiomyocyte hypertrophy was evident from microscopic examination and molecular signature (increased expression of ANF and BNF) after 24-48 hrs of Nrf2 activation. Conclusion: In summary, the chemical induced sustained activation of Nrf2 leading to formation of reductive stress showed hypertrophic remodeling in HL1 cardiomyocytes.

SOX9 is required for kidney fibrosis and activates NAV3 to drive renal myofibroblast function

2021 ◽  
Vol 14 (672) ◽  
pp. eabb4282 ◽  
Author(s):  
Sayyid Raza ◽  
Elliot Jokl ◽  
James Pritchett ◽  
Katherine Martin ◽  
Kim Su ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Actin Polymerization ◽  
Kidney Diseases ◽  
Kidney Injury ◽  
Pharmacological Intervention ◽  
Kidney Fibrosis ◽  
Genome Wide ◽  
Vitro Model ◽  
Sustained Activation

Renal fibrosis is a common end point for kidney injury and many chronic kidney diseases. Fibrogenesis depends on the sustained activation of myofibroblasts, which deposit the extracellular matrix that causes progressive scarring and organ failure. Here, we showed that the transcription factor SOX9 was associated with kidney fibrosis in humans and required for experimentally induced kidney fibrosis in mice. From genome-wide analysis, we identified Neuron navigator 3 (NAV3) as acting downstream of SOX9 in kidney fibrosis. NAV3 increased in abundance and colocalized with SOX9 after renal injury in mice, and both SOX9 and NAV3 were present in diseased human kidneys. In an in vitro model of renal pericyte transdifferentiation into myofibroblasts, we demonstrated that NAV3 was required for multiple aspects of fibrogenesis, including actin polymerization linked to cell migration and sustained activation of the mechanosensitive transcription factor YAP1. In summary, our work identifies a SOX9-NAV3-YAP1 axis involved in the progression of kidney fibrosis and points to NAV3 as a potential target for pharmacological intervention.

scholarly journals A Central Role for the Armadillo Protein Plakoglobin in the Autoimmune Disease Pemphigus Vulgaris

2001 ◽  
Vol 153 (4) ◽  
pp. 823-834 ◽  
Author(s):  
Reto Caldelari ◽  
Alain de Bruin ◽  
Dominique Baumann ◽  
Maja M. Suter ◽  
Christiane Bierkamp ◽  
...  
Keyword(s):  
Steric Hindrance ◽  
In Vitro Model ◽  
Molecular Mechanisms ◽  
Pemphigus Vulgaris ◽  
Cell Types ◽  
Linear Distribution ◽  
Igg Binding ◽  
Vitro Model ◽  
First Time

In pemphigus vulgaris (PV), autoantibody binding to desmoglein (Dsg) 3 induces loss of intercellular adhesion in skin and mucous membranes. Two hypotheses are currently favored to explain the underlying molecular mechanisms: (a) disruption of adhesion through steric hindrance, and (b) interference of desmosomal cadherin-bound antibody with intracellular events, which we speculated to involve plakoglobin. To investigate the second hypothesis we established keratinocyte cultures from plakoglobin knockout (PG−/−) embryos and PG+/+ control mice. Although both cell types exhibited desmosomal cadherin-mediated adhesion during calcium-induced differentiation and bound PV immunoglobin (IgG) at their cell surface, only PG+/+ keratinocytes responded with keratin retraction and loss of adhesion. When full-length plakoglobin was reintroduced into PG−/− cells, responsiveness to PV IgG was restored. Moreover, in these cells like in PG+/+ keratinocytes, PV IgG binding severely affected the linear distribution of plakoglobin at the plasma membrane. Taken together, the establishment of an in vitro model using PG+/+ and PG−/− keratinocytes allowed us (a) to exclude the steric hindrance only hypothesis, and (b) to demonstrate for the first time that plakoglobin plays a central role in PV, a finding that will provide a novel direction for investigations of the molecular mechanisms leading to PV, and on the function of plakoglobin in differentiating keratinocytes.

scholarly journals Involvement of leukocyte function-associated antigen-1 (LFA-1) in the invasion of hepatocyte cultures by lymphoma and T-cell hybridoma cells.

1987 ◽  
Vol 105 (1) ◽  
pp. 553-559 ◽  
Author(s):  
E Roos ◽  
F F Roossien
Keyword(s):  
T Cell ◽  
Tumor Cells ◽  
Cell Types ◽  
Hybridoma Cells ◽  
Lymphoma Cells ◽  
Hepatocyte Cultures ◽  
Leukocyte Function ◽  
Vitro Model

We studied the interaction of MB6A lymphoma and TAM2D2 T cell hybridoma cells with hepatocyte cultures as an in vitro model for in vivo liver invasion by these tumor cells. A monoclonal antibody against leukocyte function-associated antigen-1 (LFA-1) inhibited adhesion of the tumor cells to the surface of hepatocytes and consequently strongly reduced invasion. This effect was specific since control antibodies, directed against Thy.1 and against T200, of the same isotype, similar affinity, and comparable binding to these cells, did not inhibit adhesion. This suggests that LFA-1 is involved in the formation of liver metastases by lymphoma cells. TAM2D2 T cell hybridoma cells were agglutinated by anti-LFA-1, but not by control antibodies. Reduction of adhesion was not due to this agglutination since monovalent Fab fragments inhibited adhesion as well, inhibition was also seen under conditions where agglutination was minimal, and anti-LFA-1 similarly affected adhesion of MB6A lymphoma cells that were not agglutinated. The two cell types differed in LFA-1 surface density. TAM2D2 cells exhibited 400,000 surface LFA-1 molecules, 10 times more than MB6A cells. Nevertheless, the level of adhesion and the extent of inhibition by the anti-LFA-1 antibody were only slightly larger for the TAM2D2 cells.

scholarly journals CELLULAR IMMUNITY IN VITRO

1971 ◽  
Vol 133 (6) ◽  
pp. 1377-1389 ◽  
Author(s):  
Harvey B. Simon ◽  
John N. Sheagren
Keyword(s):  
Cellular Immunity ◽  
In Vitro Model ◽  
Specific Antigen ◽  
Cell Types ◽  
Intracellular Bacteria ◽  
Migration Inhibition ◽  
Vitro Model ◽  
And Control ◽  
Macrophage Migration Inhibition

An in vitro model of cellular immunity in the guinea pig was established. Animals were immunized with tubercle bacilli, bovine gamma globulin, or picrylated human serum albumin in complete Freund's adjuvant. Oil-induced peritoneal exudates from immune and control animals were cultured overnight with and without specific antigen. The cultures were washed and the macrophage monolayers were infected with Listeria monocytogenes. At intervals the monolayers were lysed and the numbers of viable intracellular bacteria were quantitated by pour plate cultures. Random monolayers were also evaluated in sequence by visually counting the intracellular bacteria on Gram-stained plates. Both methods demonstrated that the macrophages from immune animals had markedly enhanced listericidal activity when the peritoneal exudates were cultured with antigen before infection. Macrophage migration inhibition was also demonstrated under these conditions. The experiments reported here describe an in vitro model of cellular immunity which will allow separation and recombination of cell types and direct assay of cell products in efforts to elucidate further the mechanisms of the immunologically mediated enhancement of macrophage bactericidal capacity.

scholarly journals Mesenchymal Stem Cells as a Promising Cell Source for Integration in Novel In Vitro Models

Biomolecules ◽  
2020 ◽  
Vol 10 (9) ◽  
pp. 1306
Author(s):  
Ann-Kristin Afflerbach ◽  
Mark D. Kiri ◽  
Tahir Detinis ◽  
Ben M. Maoz
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Cell Types ◽  
In Vitro Models ◽  
Cell Source ◽  
Fundamental Research ◽  
Multiple Cell ◽  
Vitro Model

The human-relevance of an in vitro model is dependent on two main factors—(i) an appropriate human cell source and (ii) a modeling platform that recapitulates human in vivo conditions. Recent years have brought substantial advancements in both these aspects. In particular, mesenchymal stem cells (MSCs) have emerged as a promising cell source, as these cells can differentiate into multiple cell types, yet do not raise the ethical and practical concerns associated with other types of stem cells. In turn, advanced bioengineered in vitro models such as microfluidics, Organs-on-a-Chip, scaffolds, bioprinting and organoids are bringing researchers ever closer to mimicking complex in vivo environments, thereby overcoming some of the limitations of traditional 2D cell cultures. This review covers each of these advancements separately and discusses how the integration of MSCs into novel in vitro platforms may contribute enormously to clinical and fundamental research.

P5994Role of BGP-15 treatment in hypertensive heart failure progression and mitochondrial protection

2019 ◽  
Vol 40 (Supplement_1) ◽  
Author(s):  
O Horvath ◽  
L Deres ◽  
K Ordog ◽  
K Bruszt ◽  
B Sumegi ◽  
...  
Keyword(s):  
Heart Failure ◽  
Mitochondrial Function ◽  
Cardiac Remodeling ◽  
In Vitro Model ◽  
Mitochondrial Dynamics ◽  
Treated Group ◽  
Mitochondrial Structure ◽  
Vitro Model ◽  
And Function

Abstract Introduction The deterioration of mitochondrial quality control greatly contributes to the hypertension induced cardiac remodeling and progression of heart failure. Our previous in vitro results demonstrated the mitochondrial protective effect of antioxidant BGP-15 compound in the presence of cellular stress. Purpose In our recent study we investigated the effect of BGP-15 on cardiac remodeling in spontaneously hypertensive rats (SHR) with manifested heart failure and on mitochondrial dynamics and function in cell culture model. Methods 15-month-old male SHR received 25 mg/kg/day BGP-15 (SHR-B) or placebo (SHR-C) for 18 weeks. Age matched Wistar rats (WKY) were used as normotensive control. The heart function was monitored by echocardiography. Histological preparations were made from cardiac tissue. Neonatal rat cardiomyocytes (NRCMs) were used as in vitro model. 150 μM H2O2 stress and 50 μM BGP-15 treatment was applied. Mitochondrial network was stained with MitoTracker Red. Mitochondrial membrane potential was detected using JC-1 dye, while mitochondrial function was monitored by the Agilent Seahorse XFp, Cell Mito Stress Test. In both model the cellular levels of mitochondrial dynamics proteins were measured in Western blot. To study the ultrastructure we used electron microscopy in our in vivo and in vitro model. Results Left ventricular (LV) mass and LV wall thickness were increased significantly in SHR-C group compared to the initial values (p<0.05). These parameters were decreased considerably in the SHR-B group. Ejection fraction (EF%) decreased in both SHR group although this downturn was minimal because of the treatment. Chronic high blood pressure caused higher collagen deposition in SHR-C rats that was significantly diminished in the SHR-B group. Regarding the mitochondrial function decrease in the levels of fusion proteins OPA1 and MFN2 was observed in the SHR-C group. These differences were significantly reduced by BGP-15 treatment (p<0.05). Mitigation of the level of fission protein DRP1 was however reduced by BGP-15 (p<0.05). In our cellular model, we observed that the H2O2-induced mitochondrial fragmentation was decreased by BGP-15 treatment (p<0.05). BGP-15 treatment prevented mitochondrial membrane potential fall in H2O2 stress (p<0.05). There was no significant difference in basal respiration among groups by monitoring the mitochondrial function. The maximal respiration capacity and ATP production were significantly higher in the BGP-15 treated group in comparison to the stressed group (p<0.05). Conclusion BGP-15 treatment has beneficial effects on mitochondrial dynamics and structure by promoting fusion processes. It also supports the maintenance of mitochondrial function through the preservation of the mitochondrial structure. The mitigation of remodeling processes and the preserved EF in the treated group are results at least partly of the comprehensible effects of BGP-15 on mitochondrial structure and function. Acknowledgement/Funding GINOP-2.3.2-15-2016-00049; GINOP-2.3.2-15-2016-00048; GINOP-2.3.3-15-2016-00025

scholarly journals Rho family GTPase Rnd2 interacts and co-localizes with MgcRacGAP in male germ cells

2003 ◽  
Vol 372 (1) ◽  
pp. 105-112 ◽  
Author(s):  
Nathalie NAUD ◽  
Aminata TOURÉ ◽  
Jianfeng LIU ◽  
Charles PINEAU ◽  
Laurence MORIN ◽  
...  
Keyword(s):  
Germ Cells ◽  
In Vitro Model ◽  
Cell Types ◽  
Glutathione S Transferase ◽  
Rac Gtpase ◽  
Male Germ Cells ◽  
Rho Family ◽  
Vitro Model ◽  
Null Mutant Mice

The male-germ-cell Rac GTPase-activating protein gene (MgcRacGAP) was initially described as a human RhoGAP gene highly expressed in male germ cells at spermatocyte stage, but exhibits significant levels of expression in most cell types. In somatic cells, MgcRacGAP protein was found to both concentrate in the midzone/midbody and be required for cytokinesis. As a RhoGAP, MgcRacGAP has been proposed to down-regulate RhoA, which is localized to the cleavage furrow and midbody during cytokinesis. Due to embryonic lethality in MgcRacGAP-null mutant mice and to the lack of an in vitro model of spermatogenesis, nothing is known regarding the role and mode of action of MgcRacGAP in male germ cells. We have analysed the expression, subcellular localization and molecular interactions of MgcRacGAP in male germ cells. Whereas MgcRacGAP was found only in spermatocytes and early spermatids, the widespread RhoGTPases RhoA, Rac1 and Cdc42 (which are, to various extents, in vitro substrates for MgcRacGAP activity) were, surprisingly, not detected at these stages. In contrast, Rnd2, a Rho family GTPase-deficient G-protein was found to be co-expressed with MgcRacGAP in spermatocytes and spermatids. MgcRacGAP was detected in the midzone of meiotic cells, but also, unexpectedly, in the Golgi-derived pro-acrosomal vesicle, co-localizing with Rnd2. In addition, a stable Rnd2–MgcRacGAP molecular complex could be evidenced by glutathione S-transferase pull-down and co-immunoprecipitation experiments. We conclude that Rnd2 is a probable physiological partner of MgcRacGAP in male germ cells and we propose that MgcRacGAP, and, quite possibly, other RhoGAPs, may participate in signalling pathways involving Rnd family proteins.

208 CYTOLYTIC ANALYSIS AND NUCLEAR TRANSFER OF hCD46-TRANSGENIC PORCINE EMBRYONIC GERM CELLS TO DEVELOP AND IN VITRO MODEL OF XENOTRANSPLANTATION

2006 ◽  
Vol 18 (2) ◽  
pp. 212
Author(s):  
J. Y. Won ◽  
K. S. Ahn ◽  
S. Y. Heo ◽  
J. H. Kang ◽  
H. Shim
Keyword(s):  
Human Serum ◽  
Germ Cells ◽  
Nuclear Transfer ◽  
In Vitro Model ◽  
Regulatory Protein ◽  
Cell Types ◽  
Human Complement ◽  
Transgenic Pigs ◽  
Vitro Model

Pigs are considered the most likely source of organs for xenotransplantation due to their anatomical and physiological similarities to humans. Production of transgenic pigs including addition of human complement-regulatory protein genes and deletion of alpha-1,3-galactosyl transferase gene may overcome hyperacute rejection (HAR), the first and currently the most critical immunological hurdle in the development of xenogeneic organs for human transplantation. However, even after resolving HAR in pig-to-human xenotransplantation, a series of other transgenic pigs may be required to alleviate subsequent acute and chronic rejection and incompatibility of porcine proteins to human counterparts. The production of transgenic pigs is not only labor-intensive, time-consuming, and costly, but also the usefulness of such pigs in transplantation to humans is unpredictable. For these reasons, development of a reliable in vitro procedure to pre-evaluate effectiveness of the transgenic approach would be beneficial. This study was preformed to establish an in vitro model of xenotransplantation using porcine embryonic germ (EG) cells, undifferentiated stem cells derived from culture of primordial germ cells. Porcine EG cells were maintained in feeder-free state in DMEM containing 15% (v/v) fetal bovine serum and 1000 units/mL leukemia inhibitory factor. Human complement down-regulator hCD46 (also known as MCP, membrane cofactor protein) gene under the regulation of cytomegalovirus promoter was introduced into porcine EG cells. Transfected cells were selected by antibiotic treatment and confirmed by PCR. To test the resistance of hCD46-transgenic EG cells to human xenoreactive natural antibody and complement, EG cells were cultured for 1.5 days in DMEM containing 15% (v/v) normal human serum. The treatment with human serum did not affect the survival of hCD46-transgenic EG cells, whereas with the same treatment approximately one half of non-transfected EG cells failed to survive (P < 0.01). Transgenic EG cells presumably capable of overcoming HAR were used as nuclear donors for subsequent transfer of nuclei into enucleated oocytes. Among 110 reconstituted oocytes, 19 (17.3%) developed to the blastocyst stage. Analysis of individual nuclear transfer embryos by PCR indicated that 89.5% (17/19) of embryos contained transgene hCD46. The PCR-negative embryos might be due to an incomplete antibiotic selection of cells after transfection. Overall, the results of the present study demonstrate that the cell culture-based model of xenotransplantation may validate the usefulness of particular transgenic pigs prior to actual production. Further experiments on differentiation of transgenic EG cells into various cell types, cytolytic analysis of such cells to assess efficiency of xenotransplantation, and subsequent production and transfer of transgenic clone embryos to recipients may provide a useful new procedure to accelerate xenotransplantation research.

The Subventricular Zone: A Key Player in Human Neocortical Development

2017 ◽  
Vol 24 (2) ◽  
pp. 156-170 ◽  
Author(s):  
J. Alberto Ortega ◽  
Fani Memi ◽  
Nevena Radonjic ◽  
Radmila Filipovic ◽  
Inseyah Bagasrawala ◽  
...  
Keyword(s):  
Cerebral Cortex ◽  
Subventricular Zone ◽  
Ventricular Zone ◽  
Cell Types ◽  
Projection Neurons ◽  
Cortical Projection ◽  
Intrauterine Development ◽  
Vitro Model ◽  
Primate Cerebral Cortex

One of the main characteristics of the developing brain is that all neurons and the majority of macroglia originate first in the ventricular zone (VZ), next to the lumen of the cerebral ventricles, and later on in a secondary germinal area above the VZ, the subventricular zone (SVZ). The SVZ is a transient compartment mitotically active in humans for several gestational months. It serves as a major source of cortical projection neurons as well as an additional source of glial cells and potentially some interneuron subpopulations. The SVZ is subdivided into the smaller inner (iSVZ) and the expanded outer SVZ (oSVZ). The enlargement of the SVZ and, in particular, the emergence of the oSVZ are evolutionary adaptations that were critical to the expansion and unique cellular composition of the primate cerebral cortex. In this review, we discuss the cell types and organization of the human SVZ during the first half of the 40 weeks of gestation that comprise intrauterine development. We focus on this period as it is when the bulk of neurogenesis in the human cerebral cortex takes place. We consider how the survival and fate of SVZ cells depend on environmental influences, by analyzing the results from in vitro experiments with human cortical progenitor cells. This in vitro model is a powerful tool to better understand human neocortex formation and the etiology of neurodevelopmental disorders, which in turn will facilitate the design of targeted preventive and/or therapeutic strategies.

scholarly journals Dual Function of iPSC-Derived Pericyte-Like Cells in Vascularization and Fibrosis-Related Cardiac Tissue Remodeling In Vitro

2020 ◽  
Vol 21 (23) ◽  
pp. 8947
Author(s):  
Monika Szepes ◽  
Anna Melchert ◽  
Julia Dahlmann ◽  
Jan Hegermann ◽  
Christopher Werlein ◽  
...  
Keyword(s):  
Cardiac Tissue ◽  
De Novo ◽  
Interstitial Fibrosis ◽  
Tissue Remodeling ◽  
Cell Types ◽  
Dual Function ◽  
Small Vessels ◽  
Mediated Effects ◽  
Vitro Model

Myocardial interstitial fibrosis (MIF) is characterized by excessive extracellular matrix (ECM) deposition, increased myocardial stiffness, functional weakening, and compensatory cardiomyocyte (CM) hypertrophy. Fibroblasts (Fbs) are considered the principal source of ECM, but the contribution of perivascular cells, including pericytes (PCs), has gained attention, since MIF develops primarily around small vessels. The pathogenesis of MIF is difficult to study in humans because of the pleiotropy of mutually influencing pathomechanisms, unpredictable side effects, and the lack of available patient samples. Human pluripotent stem cells (hPSCs) offer the unique opportunity for the de novo formation of bioartificial cardiac tissue (BCT) using a variety of different cardiovascular cell types to model aspects of MIF pathogenesis in vitro. Here, we have optimized a protocol for the derivation of hPSC-derived PC-like cells (iPSC-PCs) and present a BCT in vitro model of MIF that shows their central influence on interstitial collagen deposition and myocardial tissue stiffening. This model was used to study the interplay of different cell types—i.e., hPSC-derived CMs, endothelial cells (ECs), and iPSC-PCs or primary Fbs, respectively. While iPSC-PCs improved the sarcomere structure and supported vascularization in a PC-like fashion, the functional and histological parameters of BCTs revealed EC- and PC-mediated effects on fibrosis-related cardiac tissue remodeling.

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