scholarly journals Altered Expression of ORAI and STIM Isoforms in Activated Human Cardiac Fibroblasts

2021 ◽  
pp. S21-S30
Author(s):  
R. Čendula ◽  
N. Chomaničová ◽  
A. Adamičková ◽  
A. Gažová ◽  
J. Kyselovič ◽  
...  
Keyword(s):  
Cell Culture ◽  
Cardiac Fibroblasts ◽  
Collagen Content ◽  
Mrna Levels ◽  
Cellular Mechanisms ◽  
Altered Expression ◽  
Human Cardiac Fibroblasts ◽  
Collagen Genes ◽  
Molecular Components ◽  
Cell Medium

Cardiac fibrotization is a well-known process characteristic of many cardiac pathological conditions. The key element is excessive activation of cardiac fibroblasts, their transdifferentiation into myofibroblasts, increased production, and accumulation of extracellular matrix proteins, resulting in cardiac stiffness. The exact cellular mechanisms and molecular components involved in the process are not fully elucidated, but the SOCE mechanism could play an important role. Its key molecules are the molecular sensor of calcium in ER/SR – STIM and the highly selective calcium channels Orai located in the plasma membrane. This study aims to evaluate selected SOCE-associated genes in the activation of HCF cell culture by several known substances (phenylephrine, isoprenaline) that represent cardiovascular overload. After cell cultivation, cell medium was collected to measure the soluble collagen content. From the harvested cells, qRT-PCR was performed to determine the mRNA levels of the corresponding genes. The activation of cells was based on changes in the relative expression of collagen genes as well as the collagen content in the medium of the cell culture. We detected an increase in the expression of the Orai2 isoform, a change in the Orai1/Orai3 ratio and also an increase in the expression of the STIM2 isoform. These results suggest an increased activation of the SOCE mechanism under stress conditions of fibroblasts, which supports the hypothesis of fibroblast activation in pathological processes by altering calcium homeostasis through the SOCE mechanism.

IL-17 stimulates MMP-1 expression in primary human cardiac fibroblasts via p38 MAPK- and ERK1/2-dependent C/EBP-β, NF-κB, and AP-1 activation

2007 ◽  
Vol 293 (6) ◽  
pp. H3356-H3365 ◽  
Author(s):  
Dolores M. Cortez ◽  
Marc D. Feldman ◽  
Srinivas Mummidi ◽  
Anthony J. Valente ◽  
Bjorn Steffensen ◽  
...  
Keyword(s):  
Transcription Factor ◽  
P38 Mapk ◽  
Western Blotting ◽  
Cardiac Fibroblasts ◽  
Critical Role ◽  
Dominant Negative ◽  
Expression Vectors ◽  
Type I ◽  
Mrna Levels ◽  
Human Cardiac Fibroblasts

Matrix metalloproteinases (MMPs) degrade collagen and mediate tissue remodeling. The novel cytokine IL-17 is expressed during various inflammatory conditions and modulates MMP expression. We investigated the effect of IL-17 on MMP-1 expression in primary human cardiac fibroblasts (HCF) and delineated the signaling pathways involved. HCF were treated with recombinant human IL-17. MMP-1 expression was analyzed by Northern blotting, RT-quantitative PCR, Western blotting, and ELISA; transcriptional induction and transcription factor binding by EMSA, ELISA, and reporter assay; and p38 MAPK and ERK1/2 activation by protein kinase assays and Western blotting. Signal transduction pathways were investigated using pharmacological inhibitors, small interfering RNA (siRNA), and adenoviral dominant-negative expression vectors. IL-17 stimulated MMP-1 gene transcription, net mRNA levels, protein, and promoter-reporter activity in HCF. This response was blocked by IL-17 receptor-Fc chimera and IL-17 receptor antibodies, but not by IL-6, TNF-α, or IL-1β antibodies. IL-17-stimulated type I collagenase activity was inhibited by the MMP inhibitor GM-6001 and by siRNA-mediated MMP-1 knockdown. IL-17 stimulated activator protein-1 [AP-1 (c-Fos, c-Jun, and Fra-1)], NF-κB (p50 and p65), and CCAAT enhancer-binding protein (C/EBP)-β DNA binding and reporter gene activities, effects attenuated by antisense oligonucleotides, siRNA-mediated knockdown, or expression of dominant-negative signaling proteins. Inhibition of AP-1, NF-κB, or C/EBP activation attenuated IL-17-stimulated MMP-1 expression. IL-17 induced p38 MAPK and ERK1/2 activation, and inhibition by SB-203580 and PD-98059 blunted IL-17-mediated transcription factor activation and MMP-1 expression. Our data indicate that IL-17 induces MMP-1 in human cardiac fibroblasts directly via p38 MAPK- and ERK-dependent AP-1, NF-κB, and C/EBP-β activation and suggest that IL-17 may play a critical role in myocardial remodeling.

scholarly journals Dye-Mediated Photo-Oxidation Biomaterial Fixation: Analysis of Bioinductivity and Mechanical Properties of Bovine Pericardium for Use in Cardiac Surgery

2021 ◽  
Vol 22 (19) ◽  
pp. 10768
Author(s):  
Simranjit S. Pattar ◽  
Vishnu Vasanthan ◽  
Guoqi Teng ◽  
Karl T. Wagner ◽  
Kristina Jeon ◽  
...  
Keyword(s):  
Cardiac Surgery ◽  
Cardiac Fibroblasts ◽  
Tensile Force ◽  
Biomechanical Testing ◽  
Biomechanical Properties ◽  
Cardiac Fibroblast ◽  
Bovine Pericardium ◽  
Cellular Mechanisms ◽  
Photo Oxidation ◽  
Human Cardiac Fibroblasts

Extracellular matrix bioscaffolds can influence the cardiac microenvironment and modulate endogenous cellular mechanisms. These materials can optimize cardiac surgery for repair and reconstruction. We investigated the biocompatibility and bioinductivity of bovine pericardium fixed via dye-mediated photo-oxidation on human cardiac fibroblast activity. We compared a dye-mediated photo-oxidation fixed bioscaffold to glutaraldehyde-fixed and non-fixed bioscaffolds reported in contemporary literature in cardiac surgery. Human cardiac fibroblasts from consenting patients were seeded on to bioscaffold materials to assess the biocompatibility and bioinductivity. Human cardiac fibroblast gene expression, secretome, morphology and viability were studied. Dye-mediated photo-oxidation fixed acellular bovine pericardium preserves human cardiac fibroblast phenotype and viability; and potentiates a pro-vasculogenic paracrine response. Material tensile properties were compared with biomechanical testing. Dye-mediated photo-oxidation fixed acellular bovine pericardium had higher compliance compared to glutaraldehyde-fixed bioscaffold in response to tensile force. The biocompatibility, bioinductivity, and biomechanical properties of dye-mediated photo-oxidation fixed bovine pericardium demonstrate its feasibility as a bioscaffold for use in cardiac surgery. As a fixed yet bioinductive solution, this bioscaffold demonstrates enhanced compliance and retains bioinductive properties that may leverage endogenous reparative pathways. Dye-mediated photo-oxidation fixed bioscaffold warrants further investigation as a viable tool for cardiac repair and reconstruction.

scholarly journals Diastolic dysfunction in a pre-clinical model of diabetes is associated with changes in the cardiac non-myocyte cellular composition

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Charles D. Cohen ◽  
Miles J. De Blasio ◽  
Man K. S. Lee ◽  
Gabriella E. Farrugia ◽  
Darnel Prakoso ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Diastolic Dysfunction ◽  
Systolic Function ◽  
Cardiac Fibroblasts ◽  
Detailed Examination ◽  
Diabetic Mice ◽  
Cellular Mechanisms ◽  
Clinical Model ◽  
Cardiac Ventricles

Abstract Background Diabetes is associated with a significantly elevated risk of cardiovascular disease and its specific pathophysiology remains unclear. Recent studies have changed our understanding of cardiac cellularity, with cellular changes accompanying diabetes yet to be examined in detail. This study aims to characterise the changes in the cardiac cellular landscape in murine diabetes to identify potential cellular protagonists in the diabetic heart. Methods Diabetes was induced in male FVB/N mice by low-dose streptozotocin and a high-fat diet for 26-weeks. Cardiac function was measured by echocardiography at endpoint. Flow cytometry was performed on cardiac ventricles as well as blood, spleen, and bone-marrow at endpoint from non-diabetic and diabetic mice. To validate flow cytometry results, immunofluorescence staining was conducted on left-ventricles of age-matched mice. Results Mice with diabetes exhibited hyperglycaemia and impaired glucose tolerance at endpoint. Echocardiography revealed reduced E:A and e’:a’ ratios in diabetic mice indicating diastolic dysfunction. Systolic function was not different between the experimental groups. Detailed examination of cardiac cellularity found resident mesenchymal cells (RMCs) were elevated as a result of diabetes, due to a marked increase in cardiac fibroblasts, while smooth muscle cells were reduced in proportion. Moreover, we found increased levels of Ly6Chi monocytes in both the heart and in the blood. Consistent with this, the proportion of bone-marrow haematopoietic stem cells were increased in diabetic mice. Conclusions Murine diabetes results in distinct changes in cardiac cellularity. These changes—in particular increased levels of fibroblasts—offer a framework for understanding how cardiac cellularity changes in diabetes. The results also point to new cellular mechanisms in this context, which may further aid in development of pharmacotherapies to allay the progression of cardiomyopathy associated with diabetes.

scholarly journals Age-related decline in the expression of GDF9 and BMP15 genes in follicle fluid and granulosa cells derived from poor ovarian responders

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Yan Gong ◽  
Jesse Li-Ling ◽  
Dongsheng Xiong ◽  
Jiajing Wei ◽  
Taiqing Zhong ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Enzyme Linked Immunosorbent Assay ◽  
Mrna Levels ◽  
Clinical Trial Registry ◽  
Altered Expression ◽  
Vitro Fertilization ◽  
Age Related ◽  
Tertiary Teaching ◽  
Poor Ovarian Responders

Abstract Background Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) genes play important roles in folliculogenesis. Altered expression of the two have been found among patients with poor ovarian response (POR). In this prospective cohort study, we have determined the expression of the GDF9 and BMP15 genes in follicle fluid (FF) and granulosa cells (GCs) derived from poor ovarian responders grouped by age, and explored its correlation with the outcome of in vitro fertilization and embryo transfer (IVF-ET) treatment. Methods A total of 196 patients with POR were enrolled from a tertiary teaching hospital. The patients were diagnosed by the Bologna criteria and sub-divided into group A (< 35 year old), group B (35–40 year old), and group C (> 40 year old). A GnRH antagonist protocol was conducted for all patients, and FF and GCs were collected after oocyte retrieval. Expression of the GDF9 and BMP15 genes in the FF and GCs was determined with enzyme-linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting. Results Compared with group C, groups A and B had significantly more two pronuclei (2PN) oocytes and transplantable embryos, in addition with higher rates of implantation and clinical pregnancy (P <  0.05). The expression level of GDF9 and BMP15 genes in the FF and GCs differed significantly among the three groups (P <  0.05), showing a trend of decline along with age. The ratio of GDF9/BMP15 mRNA levels were similar among the three groups (P > 0.05). The relative levels of GDF9 and BMP15 proteins in GCs have correlated with the relative mRNA levels in GCs and protein concentrations in FF (P <  0.05). Conclusions For poor ovarian responders, in particular those over 40, the expression of GDF9 and BMP15 is declined along with increased age and in accompany with poorer oocyte quality and IVF outcome, whilst the ratio of GDF9/BMP15 mRNA levels remained relatively constant. Trial registration Chinese Clinical Trial Registry Center (ChiCTR1800016107). Registered on 11 May 2018.

scholarly journals A protocol for transdifferentiation of human cardiac fibroblasts into endothelial cells via activation of innate immunity

2021 ◽  
Vol 2 (2) ◽  
pp. 100556
Author(s):  
Chun Liu ◽  
Pedro Medina ◽  
Dilip Thomas ◽  
Ian Y. Chen ◽  
Karim Sallam ◽  
...  
Keyword(s):  
Innate Immunity ◽  
Endothelial Cells ◽  
Cardiac Fibroblasts ◽  
Human Cardiac Fibroblasts

Effects of methylglyoxal on human cardiac fibroblast: roles of transient receptor potential ankyrin 1 (TRPA1) channels

2014 ◽  
Vol 307 (9) ◽  
pp. H1339-H1352 ◽  
Author(s):  
Gaku Oguri ◽  
Toshiaki Nakajima ◽  
Yumiko Yamamoto ◽  
Nami Takano ◽  
Tomofumi Tanaka ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Diabetic Cardiomyopathy ◽  
Cell Cycle Progression ◽  
Transient Receptor Potential ◽  
Cardiac Fibroblasts ◽  
Receptor Potential ◽  
Rt Pcr ◽  
Cycle Progression ◽  
Transient Receptor ◽  
Human Cardiac Fibroblasts

Cardiac fibroblasts contribute to the pathogenesis of cardiac remodeling. Methylglyoxal (MG) is an endogenous carbonyl compound produced under hyperglycemic conditions, which may play a role in the development of pathophysiological conditions including diabetic cardiomyopathy. However, the mechanism by which this occurs and the molecular targets of MG are unclear. We investigated the effects of MG on Ca2+ signals, its underlying mechanism, and cell cycle progression/cell differentiation in human cardiac fibroblasts. The conventional and quantitative real-time RT-PCR, Western blot, immunocytochemical analysis, and intracellular Ca2+ concentration [Ca2+]i measurement were applied. Cell cycle progression was assessed using the fluorescence activated cell sorting. MG induced Ca2+ entry concentration dependently. Ruthenium red (RR), a general cation channel blocker, and HC030031 , a selective transient receptor potential ankyrin 1 (TRPA1) antagonist, inhibited MG-induced Ca2+ entry. Treatment with aminoguanidine, a MG scavenger, also inhibited it. Allyl isothiocyanate, a selective TRPA1 agonist, increased Ca2+ entry. The use of small interfering RNA to knock down TRPA1 reduced the MG-induced Ca2+ entry as well as TRPA1 mRNA expression. The quantitative real-time RT-PCR analysis showed the prominent existence of TRPA1 mRNA. Expression of TRPA1 protein was confirmed by Western blotting and immunocytochemical analyses. MG promoted cell cycle progression from G0/G1 to S/G2/M, which was suppressed by HC030031 or RR. MG also enhanced α-smooth muscle actin expression. The present results suggest that methylglyoxal activates TRPA1 and promotes cell cycle progression and differentiation in human cardiac fibroblasts. MG might participate the development of pathophysiological conditions including diabetic cardiomyopathy via activation of TRPA1.

Quantitative assessment of ground squirrel mRNA levels in multiple stages of hibernation

2002 ◽  
Vol 10 (2) ◽  
pp. 93-102 ◽  
Author(s):  
L. Elaine Epperson ◽  
Sandra L. Martin
Keyword(s):  
Steady State ◽  
Core Body Temperature ◽  
Ground Squirrels ◽  
Apolipoprotein A1 ◽  
Mrna Levels ◽  
Cdna Subtraction ◽  
Global Issues ◽  
Α2 Macroglobulin ◽  
Steady State Levels ◽  
Molecular Components

Hibernators in torpor dramatically reduce their metabolic, respiratory, and heart rates and core body temperature. These extreme physiological conditions are frequently and rapidly reversed during the winter hibernation season via endogenous mechanisms. This phenotype must derive from regulated expression of the hibernator’s genome; to identify its molecular components, a cDNA subtraction was used to enrich for seasonally upregulated mRNAs in liver of golden-mantled ground squirrels. The relative steady-state levels for seven mRNAs identified by this screen, plus five others, were measured and analyzed for seasonal and stage-specific differences using kinetic RT-PCR. Four mRNAs show seasonal upregulation in which all five winter stages differ significantly from and are higher than summer (α2-macroglobulin, apolipoprotein A1, cathepsin H, and thyroxine-binding globulin). One of these mRNAs, α2-macroglobulin, varies during the winter stages with significantly lower levels at late torpor. None of the 12 mRNAs increased during torpor. The implications for these newly recognized upregulated mRNAs for hibernation as well as more global issues of maintaining steady-state levels of mRNA during torpor are discussed.

TGF-β1 RNA Interference in Mouse Primary Dura Cell Culture: Downstream Effects on TGF Receptors, FGF-2, and FGF-R1 mRNA Levels

2009 ◽  
Vol 124 (5) ◽  
pp. 1466-1473 ◽  
Author(s):  
Arun K. Gosain ◽  
Jacques A. Machol ◽  
Christy Gliniak ◽  
Nadine L. N. Halligan
Keyword(s):  
Cell Culture ◽  
Rna Interference ◽  
Mrna Levels ◽  
Downstream Effects ◽  
Tgf Β1

Abstract 107: Mir-431 as a Potential Master Regulator in Angiotensin Ii-induced Vascular Injury

Hypertension ◽  
2016 ◽  
Vol 68 (suppl_1) ◽  
Author(s):  
Kugeng Huo ◽  
Tlili Barhoumi ◽  
Julio C Fraulob-Aquino ◽  
Chantal Richer ◽  
Mathieu Lajoie ◽  
...  
Keyword(s):  
Vascular Injury ◽  
Molecular Networks ◽  
Type I ◽  
Mirna Cluster ◽  
Mrna Levels ◽  
Ang Ii ◽  
Master Regulator ◽  
Developmental Processes ◽  
Altered Expression ◽  
Total Rna

Objective: Vascular injury is an early manifestation and a cause of end-organ damage in hypertension. microRNAs (miRNAs) play an important role in cardiovascular disease, but their implication in vascular injury is remains unclear. We aim to use RNA sequencing (seq) and a systems biology approach to identify master regulators that mediate global gene expression changes in the course of vascular injury. Methods and Results: Ten week-old male C57BL/6 mice were infused or not with angiotensin (Ang) II (1 μg/kg/min, SC) for 14 days. Blood pressure (BP) was measured by telemetry. Total RNA was extracted from the mesenteric vasculature for total RNA and small RNA-seq. Differentially expressed (DE) miRNAs (23 up and 12 down) and mRNAs (550 up and 256 down) were identified (1.5-fold, q <0.05). Molecular networks were constructed to integrate predicted interactions between DE miRNAs and inversely expressed DE mRNAs and between DE transcription factors (TF) and DE genes. Gene enrichment analysis revealed DE mRNAs involved in extracellular matrix (ECM) and developmental processes regulated by DE miRNAs ( q <1.5E-11). Seventeen upregulated miRNAs are located in the miRNA cluster of the Dlk1-Dio3 region that is highly conserved in humans, 9 of which had expression levels correlated with BP ( P <0.05). Among those 9, miR-431 that ranked first as DE miRNA ( q <0.0005) and is 100% conserved in humans, and a conserved putative DE target, a BP-correlated ( P <0.05) TF ETS homologous factor ( Ehf ), which regulates numerous ECM genes including collagen type I α1 ( Col1a1 ), were selected for functional studies. Transfection of a miR-431 mimic in human aortic smooth muscle cells (HASMCs) decreased Ehf (0.1±0.1-fold, P <0.001) and increased Ehf -suppressing target Col1a1 (1.7±0.5-fold, P <0.001) mRNA levels. Transfection of a miR-431 inhibitor caused reciprocal effects ( P <0.05). Ehf siRNA knockdown increased Col1a1 (1.2±0.1-fold, P <0.001) mRNA levels. Conclusions: Ang II infusion altered expression of miRNAs in the Dlk1-Dio3 cluster and genes involved in ECM and developmental processes. miR-431 targets TF Ehf , which leads to increased Col1a1 in HASMCs. miR-431 may act as a master regulator for vascular injury and could be a potential therapeutic target.

Abstract 58: AT2 Receptor Agonism Regulates TIMP1/MMP9 Axis in the Heart Preventing Cardiac Fibrosis and Improving Heart Function After Experimental Myocardial Infarction

Hypertension ◽  
2013 ◽  
Vol 62 (suppl_1) ◽  
Author(s):  
Dilyara Lauer ◽  
Svetlana Slavic ◽  
Manuela Sommerfeld ◽  
Christa Thöne-Reineke ◽  
Yuliya Sharkovska ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Left Ventricle ◽  
Late Stage ◽  
Heart Function ◽  
Cardiac Fibroblasts ◽  
Collagen Content ◽  
Vehicle Group ◽  
At2 Receptor ◽  
Down Regulation ◽  
Tgf Β1

Aims: A selective nonpeptide agonist for the angiotensin AT2 receptor compound 21 (C21) improved cardiac functions 7 days after myocardial infarction (MI). Here, we aimed to investigate what are the cellular mechanisms underlying cardiac protection in the late stage after MI. Methods and Results: MI was induced in Wistar rats by permanent ligation of the left coronary artery. Treatment with C21 (0.03mg/kg i.p. daily) started 6h after MI and continued for 6 weeks. Hemodynamic parameters were measured via transthoracic Doppler echocardiography and intracardiac Samba catheter. The expression of MMP9, TIMP1, TGF-β1 and collagen content were determined in left ventricle. Anti-proteolytic effects were additionally studied in primary cardiac fibroblasts. C21 significantly improved systolic and diastolic function 6 weeks after MI in comparison with the vehicle group as shown by ejection fraction (71.2±4.7 % vs. 53.4±7.0%; p<0.001), fractional shortening (40.8±2.3% vs. 30.9±3.1%; p<0.05), LVIDs (4.4±0.5mm vs. 5.2±0.8mm; p<0.05), LV EDP (16.9±1.2mmHg vs. 22.1±1.4mmHg; p<0.05), E/A ratio, dP/dt max and dP/dt min (p<0.05). Moreover, C21 improved arterial stiffness parameter (AIx) (18±1.2% vs. 25%±1.8, p<0.05) and reduced collagen content (15%; p<0.05) in postinfarcted myocardium. TIMP1 protein expression in the left ventricle was strongly up-regulated (17.7-fold; p<0.05) whereas MMP9 and TGF-β1 were significantly down-regulated (1.5-fold, p<0.05; 3.4-fold p<0.001, respectively) in the treated group. In cardiac fibroblasts, C21 primarily induced TIMP1 expression followed by attenuated MMP9 secretion and TGF-β1 down-regulation. Conclusion: C21 improves heart function in the late stage after MI and prevents cardiac remodeling. Activation of TIMP1 and subsequent inhibition of MMP9-mediated proteolysis as well as down-regulation of TGF-β1 followed by decreased collagen accumulation may attenuate disintegration of the extracellular matrix and reduce fibrosis.

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