scholarly journals Substrate stiffness modulates integrin α5 expression and ECM-associated gene expression in fibroblasts

2021 ◽  
Author(s):  
Brijesh Kumar Verma ◽  
Aritra Chatterjee ◽  
Paturu Kondaiah ◽  
Namrata Gundiah
Keyword(s):  
Gene Expression ◽  
Gelatin Zymography ◽  
Confocal Imaging ◽  
Substrate Stiffness ◽  
Specific Substrate ◽  
Tgf Beta ◽  
Cell Nuclei ◽  
Compliant Substrates ◽  
Collagen Iii ◽  
Activated State

Biomaterials, like polydimethylsiloxane (PDMS), are soft, biocompatible, and tuneable, which makes them useful to delineate specific substrate factors that regulate the complex landscape of cell-substrate interactions. We used a commercial formulation of PDMS to fabricate substrates with moduli 40 kPa, 300 kPa, and 1.5 MPa, and cultured HMF3S fibroblasts on them. Gene expression analysis was performed by RT-PCR and Western blotting. Cellular and nuclear morphologies were analyzed using confocal imaging, and MMP-2 and MMP-9 activities were determined with gelatin zymography. Results, comparing mechanotransduction on PDMS substrates with control petridishes, show that substrate stiffness modulates cell morphologies and proliferations. Cell nuclei were rounded on compliant substrates and correlated with increased tubulin expression. Proliferations were higher on stiffer substrates with cell cycle arrest on softer substrates. Integrin alpha5 expression decreased on lower stiffness substrates, and correlated with inefficient TGF-beta; activation. Changes to the activated state of the fibroblast on higher stiffness substrates were linked to altered TGF-beta; secretion. Collagen I, collagen III, and MMP-2 expression levels were lower on compliant PDMS substrates as compared to stiffer ones; there was little MMP-9 activity on substrates. These results demonstrate critical feedback mechanisms between substrate stiffness and ECM regulation by fibroblasts which is highly relevant in diseases like tissue fibrosis.

scholarly journals Promoter-Targeted Histone Acetylation of Chromatinized Parvoviral Genome Is Essential for the Progress of Infection

2016 ◽  
Vol 90 (8) ◽  
pp. 4059-4066 ◽  
Author(s):  
Elina Mäntylä ◽  
Kari Salokas ◽  
Mikko Oittinen ◽  
Vesa Aho ◽  
Pekka Mäntysaari ◽  
...  
Keyword(s):  
Gene Expression ◽  
Histone Acetylation ◽  
High Throughput Sequencing ◽  
Critical Role ◽  
Confocal Imaging ◽  
Proximity Ligation Assay ◽  
Viral Gene ◽  
Cell Nuclei ◽  
Viral Dna ◽  
First Time

ABSTRACTThe association of host histones with parvoviral DNA is poorly understood. We analyzed the chromatinization and histone acetylation of canine parvovirus DNA during infection by confocal imaging andin situproximity ligation assay combined with chromatin immunoprecipitation and high-throughput sequencing. We found that during late infection, parvovirus replication bodies were rich in histones bearing modifications characteristic of transcriptionally active chromatin, i.e., histone H3 lysine 27 acetylation (H3K27ac). H3K27ac, in particular, was located in close proximity to the viral DNA-binding protein NS1. Importantly, our results show for the first time that in the chromatinized parvoviral genome, the two viral promoters in particular were rich in H3K27ac. Histone acetyltransferase (HAT) inhibitors efficiently interfered with the expression of viral proteins and infection progress. Altogether, our data suggest that the acetylation of histones on parvoviral DNA is essential for viral gene expression and the completion of the viral life cycle.IMPORTANCEViral DNA introduced into cell nuclei is exposed to cellular responses to foreign DNA, including chromatinization and epigenetic silencing, both of which determine the outcome of infection. How the incoming parvovirus resists cellular epigenetic downregulation of its genes is not understood. Here, the critical role of epigenetic modifications in the regulation of parvovirus infection was demonstrated. We showed for the first time that a successful parvovirus infection is characterized by the deposition of nucleosomes with active histone acetylation on the viral promoter areas. The results provide new insights into the regulation of parvoviral gene expression, which is an important aspect of the development of parvovirus-based virotherapy.

Global gene expression profiles reveal an increase in mRNA levels of collagens, MMPs, and TIMPs in late radiation enteritis

2004 ◽  
Vol 287 (4) ◽  
pp. G875-G885 ◽  
Author(s):  
Carine Strup-Perrot ◽  
Denis Mathé ◽  
Christine Linard ◽  
Dominique Violot ◽  
Fabien Milliat ◽  
...  
Keyword(s):  
Gene Expression ◽  
Extracellular Matrix ◽  
Expression Profiles ◽  
Cdna Array ◽  
Muscularis Propria ◽  
Gelatin Zymography ◽  
Tissue Inhibitors Of Metalloproteinases ◽  
Radiation Enteritis ◽  
Mrna Levels ◽  
Fixed Tissue

Radiation enteritis, a common complication of radiation therapy for abdominal and pelvic cancers, is characterized by severe transmural fibrosis associated with mesenchymal cell activation, tissue disorganization, and deposition of fibrillar collagen. To investigate the mechanisms involved in this pathological accumulation of extracellular matrix, we studied gene expression of matrix components along with that of genes involved in matrix remodeling, matrix metalloproteinases (MMPs), and tissue inhibitors of metalloproteinases (TIMPs). Hybrid selection on high-density cDNA array, real-time RT-PCR, gelatin zymography and imunohistochemistry were used to characterize the mRNA expression profile, activity, and tissue location of extracellular matrix-related genes in radiation enteritis compared with healthy ileum. cDNA array analysis revealed a strong induction of genes coding for collagens I, III, IV, VI, and VIII, SPARC, and tenascin-C, extracellular-matrix degrading enzymes (MMP-1, -2, -3, -14, -18+19), and metalloproteinase inhibitors (TIMP-1, -2, plasminogen activator inhibitor-1) in radiation enteritis. This increase was correlated with the degree of infiltration of the mucosa by inflammatory cells, and the presence of differentiated mesenchymal cells in the submucosa and muscularis propria. Despite the fact that expression of collagens, MMPs, and TIMPs simultaneously increase, quantification of net collagen deposition shows an overall accumulation of collagen. Our results indicate that late radiation enteritis tissues are subjected to active process of fibrogenesis as well as fibrolysis, with a balance toward fibrogenesis. This demonstrates that established fibrotic tissue is not scarred fixed tissue but is subjected to a dynamic remodeling process.

Neutralisation of TGF-beta 1 and TGF-beta 2 or exogenous addition of TGF-beta 3 to cutaneous rat wounds reduces scarring

1995 ◽  
Vol 108 (3) ◽  
pp. 985-1002 ◽  
Author(s):  
M. Shah ◽  
D.M. Foreman ◽  
M.W. Ferguson
Keyword(s):  
Wound Healing ◽  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Collagen I ◽  
Tgf Beta ◽  
Early Stages ◽  
Collagen Iii ◽  
Exogenous Addition ◽  
Growth Factor Beta ◽  
Beta 2

Exogenous addition of neutralising antibody to transforming growth factor-beta 1,2 to cutaneous wounds in adult rodents reduces scarring. Three isoforms of transforming growth factor-beta (1, 2 and 3) have been identified in mammals. We investigated the isoform/isoforms of TGF-beta responsible for cutaneous scarring by: (i) reducing specific endogenous TGF-beta isoforms by exogenous injection of isoform specific neutralising antibodies; and (ii) increasing the level of specific TGF-beta isoforms by exogenous infiltration into the wound margins. Exogenous addition of neutralising antibody to TGF-beta 1 plus neutralising antibody to TGF-beta 2 reduced the monocyte and macrophage profile, neovascularisation, fibronectin, collagen III and collagen I deposition in the early stages of wound healing compared to control wounds. Treatment with neutralising antibodies to TGF-betas 1 and 2 markedly improved the architecture of the neodermis to resemble that of normal dermis and reduced scarring while the control wounds healed with scar formation. Exogenous addition of neutralising antibody to TGF-beta 1 alone also reduced the monocyte and macrophage profile, fibronectin, collagen III and collagen I deposition compared to control wounds. However, treatment with neutralising antibody to TGF-beta 1 alone only marginally reduced scarring. By contrast, wounds treated with neutralising antibody to TGF-beta 2 alone did not differ from control wounds. Interestingly, exogenous addition of the TGF-beta 3 peptide also reduced the monocyte and macrophage profile, fibronectin, collagen I and collagen III deposition in the early stages of wound healing and markedly improved the architecture of the neodermis and reduced scarring. By contrast, wounds treated with either TGF-beta 1 or with TGF-beta 2 had more extracellular matrix deposition in the early stages of wound healing but did not differ from control wounds in the final quality of scarring. This study clearly demonstrates isoform specific differences in the role of TGF-betas in wound healing and cutaneous scarring. TGF-beta 1 and TGF-beta 2 are implicated in cutaneous scarring. This study also suggests a novel therapeutic use of exogenous recombinant, TGF-beta 3 as an anti-scarring agent.

scholarly journals Effects of anti-inflammatory (NSAID) treatment on human tendinopathic tissue

2017 ◽  
Vol 123 (5) ◽  
pp. 1397-1405 ◽  
Author(s):  
Katja Maria Heinemeier ◽  
Tommy F. Øhlenschlæger ◽  
Ulla Ramer Mikkelsen ◽  
Freja Sønder ◽  
Peter Schjerling ◽  
...  
Keyword(s):  
Gene Expression ◽  
Color Doppler ◽  
Surrounding Tissue ◽  
Tendon Tissue ◽  
Nsaid Treatment ◽  
Collagen Iii ◽  
Tendon Thickness ◽  
Inflammatory Drugs ◽  
Anti Inflammatory ◽  
Pain At Rest

Nonsteroidal anti-inflammatory drugs (NSAIDs) are commonly used to treat tendinopathy, but evidence for this treatment is lacking, and little is known regarding effects of NSAIDs on human tendinopathic tendon. This study investigated the effects of NSAID treatment (ibuprofen) on human tendinopathic tendon, with changes in gene expression as the primary outcome, and tendon pain, function, and blood flow as secondary outcomes. Twenty-six adults (16 men, 10 women), diagnosed with chronic Achilles tendinopathy, were randomized to 1-wk treatment with ibuprofen (600 mg ×3/day) ( n = 13) or placebo ( n = 13) (double-blinded). Ibuprofen content in blood, visual analog scale score for tendon pain at rest and activity, Victorian Institute of Sports Assessment-Achilles (VISA-A) scores for tendon function, tendon thickness (with ultrasonography), and color Doppler were measured before and 1 h after treatment. After the last posttreatment test, a full-width tendon biopsy was taken from the affected area. Real-time-RT-PCR was used to assess expression of collagen I, collagen III, transforming growth factor (TGF-β) isoforms, cyclooxygenase-2 (COX-2), angiopoietin-like 4 (ANGPTL4), and cyclic AMP-dependent transcription factor (ATF3) in tendon tissue. Expression of collagens and TGF-β isoforms showed relatively low variation and was unaffected by ibuprofen treatment. Further, no changes were seen in tendon thickness or VISA-A score. The placebo treatment reduced the color Doppler (in tendon plus surrounding tissue) compared with the ibuprofen group and also increased the perception of pain at rest. In conclusion, there was no indication that short-term ibuprofen treatment affects gene expression in human chronic tendinopathic tendon or leads to any clear changes in tendon pain or function. NEW & NOTEWORTHY Nonsteroidal anti-inflammatory drugs are widely used in the treatment of tendinopathy, but little is known of the effects of these drugs on tendon tissue. We find that 1 wk of ibuprofen treatment has no effect on gene expression of collagen and related growth factors in adult human tendinopathic tendon in vivo (in spite of relatively low levels of variation in gene expression), suggesting that tendinopathic cells are not responsive to ibuprofen.

scholarly journals Gene Expression Profiling of Human Hepatocytes Grown on Differing Substrate Stiffness

2019 ◽  
Vol 16 (S2) ◽  
pp. 131-131
Author(s):  
Fan Feng ◽  
Tingting Xia ◽  
Runze Zhao ◽  
Mengyue Wang ◽  
Li Yang
Keyword(s):  
Gene Expression ◽  
Gene Expression Profiling ◽  
Expression Profiling ◽  
Human Hepatocytes ◽  
Substrate Stiffness

Transcriptional modulation of transin gene expression by epidermal growth factor and transforming growth factor beta

1988 ◽  
Vol 8 (6) ◽  
pp. 2479-2483
Author(s):  
C M Machida ◽  
L L Muldoon ◽  
K D Rodland ◽  
B E Magun
Keyword(s):  
Gene Expression ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Prolonged Treatment ◽  
Transcriptional Level ◽  
Tgf Beta ◽  
Epidermal Growth ◽  
Growth Factor Beta

Transin is a transformation-associated gene which is expressed constitutively in rat fibroblasts transformed by a variety of oncogenes and in malignant mouse skin carcinomas but not benign papillomas or normal skin. It has been demonstrated that, in nontransformed Rat-1 cells, transin RNA expression is modulated positively by epidermal growth factor (EGF) and negatively by transforming growth factor beta (TGF-beta); other peptide growth factors were found to have no effect on transin expression. Results presented here indicate that both protein synthesis and continuous occupancy of the EGF receptor by EGF were required for sustained induction of transin RNA. Treatment with TGF-beta inhibited the ability of EGF to induce transin, whether assayed at the transcriptional level by nuclear run-on analysis or at the level of transin RNA accumulation by Northern (RNA) blot analysis of cellular RNA. TGF-beta both blocked initial induction of transin transcription by EGF and halted established production of transin transcripts during prolonged treatment. These results suggest that TGF-beta acts at the transcriptional level to antagonize EGF-mediated induction of transin gene expression.

Abstract 312: Regulation of Fibrotic Signaling Pathways by Desmosomal Armadillo Proteins in Cardiac Tissue

2014 ◽  
Vol 115 (suppl_1) ◽  
Author(s):  
Adi D Dubash ◽  
Kathleen J Green
Keyword(s):  
Gene Expression ◽  
Signaling Pathways ◽  
Cardiac Disease ◽  
Cardiac Tissue ◽  
Cardiac Fibroblasts ◽  
Fibrous Tissue ◽  
Tgf Beta ◽  
Extracellular Matrix Components ◽  
Different Types ◽  
P38mapk Signaling

The process of fibrosis, described as accumulation of myofibroblasts and excessive deposition of extracellular matrix components, is a key development in the progression of multiple different types of cardiac disease. Nevertheless, little is known about the molecular mechanisms which cause the onset of fibrosis in cardiac disease. Fibrosis is a significant component of arrhythmogenic cardiomyopathy (AC), a genetic disorder characterized by replacement of healthy cardiomyocytes (CMs) with fibrous tissue, leading to arrhythmia and in certain cases, sudden death. AC is often characterized as a “disease of the desmosome”, as mutations for all obligate desmosome proteins have been found in cases of AC, including the desmosome armadillo proteins Plakophilin-2 (PKP2) and Plakoglobin (PG). PKP2 and PG are multi-functional proteins involved in both mechanical stabilization of the cardiac area composita, as well as mediation of desmosome-related signaling pathways. We have determined that loss of PKP2 or PG in neonatal CMs causes an aberrant increase in gene expression of pro-fibrotic stimuli such as transforming growth factor beta 1 (TGF-beta1) and Interleukin-6 (IL-6). In addition, p38 MAPK, a known mediator of inflammatory fibrosis, is activated upon loss of PKP2/PG. We hypothesize that mutation or loss of PKP2 or PG cause the recruitment and activation of cardiac fibroblasts via pro-fibrotic TGF-beta and p38MAPK signaling, resulting in pathological fibrosis characteristic of AC. Indeed, conditioned media from PKP2-silenced CMs causes an increase in fibronectin gene expression by freshly isolated cardiac fibroblasts. Our future experiments will investigate whether inhibition of TGF-beta or p38MAPK signaling can alleviate fibrotic gene production. By highlighting a novel link between desmosome armadillo proteins and pro-fibrotic signaling in cardiac tissue, this study provides mechanistic insights into the pathogenesis of AC, as well as advances our knowledge of potential therapeutic targets for combating fibrosis in multiple different types of heart disease or injury.

Regulated expression and growth inhibitory effects of transforming growth factor-beta isoforms in mouse mammary gland development

Development ◽  
1991 ◽  
Vol 113 (3) ◽  
pp. 867-878 ◽  
Author(s):  
S.D. Robinson ◽  
G.B. Silberstein ◽  
A.B. Roberts ◽  
K.C. Flanders ◽  
C.W. Daniel
Keyword(s):  
Gene Expression ◽  
Mammary Gland ◽  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Slow Release ◽  
Mouse Mammary Gland ◽  
Tgf Beta ◽  
Growth Factor Beta ◽  
Beta 2

Transforming Growth Factor-beta 1 (TGF-beta 1) was previously shown to inhibit reversibly the growth of mouse mammary ducts when administered in vivo by miniature slow-release plastic implants. We now report a comparative analysis of three TGF-beta isoforms with respect to gene expression and localization of protein products within the mouse mammary gland. Our studies revealed overlapping expression patterns of TGF-beta 1, TGF-beta 2 and TGF-beta 3 within the epithelium of the actively-growing mammary end buds during branching morphogenesis, as well as within the epithelium of growth-quiescent ducts. However, TGF-beta 3 was the only isoform detected in myoepithelial progenitor cells (cap cells) of the growing end buds and myoepithelial cells of the mature ducts. During pregnancy, TGF-beta 2 and TGF-beta 3 transcripts increased to high levels, in contrast to TGF-beta 1 transcripts which were moderately abundant; TGF-beta 2 was significantly transcribed only during pregnancy. Molecular hybridization in situ revealed overlapping patterns of expression for the three TGF-beta isoforms during alveolar morphogenesis, but showed that, in contrast to the patterns of TGF-beta 1 and TGF-beta 2 expression, TGF-beta 3 is expressed more heavily in ducts than in alveoli during pregnancy. Developing alveolar tissue and its associated ducts displayed striking TGF-beta 3 immunoreactivity which was greatly reduced during lactation. All three isoforms showed dramatically reduced expression in lactating tissue. The biological effects of active, exogenous TGF-beta 2 and TGF-beta 3 were tested with slow-release plastic implants. These isoforms, like TGF-beta 1, inhibited mammary ductal elongation in situ by causing the disappearance of the proliferating stem cell layer (cap cells) and rapid involution of ductal end buds. None of the isoforms were active in inhibiting alveolar morphogenesis. We conclude that under the limited conditions of these tests, the three mammalian isoforms are functionally equivalent. However, striking differences in patterns of gene expression and in the distribution of immunoreactive peptides suggest that TGF-beta isoforms may have distinct roles in mammary growth regulation, morphogenesis and functional differentiation.

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