scholarly journals Calpastatin-Mediated Inhibition of Calpain Ameliorates Skin Scar Formation after Burn Injury

2021 ◽  
Vol 22 (11) ◽  
pp. 5771
Author(s):  
Cheong Hoon Seo ◽  
Hui Song Cui ◽  
June-Bum Kim
Keyword(s):  
Extracellular Matrix ◽  
Transforming Growth Factor ◽  
Human Fibroblasts ◽  
Scar Formation ◽  
Type I ◽  
Mrna Levels ◽  
Hypertrophic Scars ◽  
Burn Wound ◽  
Antifibrotic Effect ◽  
Calpain Inhibition

Hypertrophic scars, the most common complication of burn injuries, are characterized by excessive deposition of fibroblast-derived extracellular matrix proteins. Calpain, a calcium-dependent protease, is involved in the fibroblast proliferation and extracellular matrix production observed in certain fibrotic diseases. However, its role in the formation of post-burn hypertrophic skin scars remains largely unknown. Here, calpain expression and activity were assessed in skin fibroblasts obtained directly from patients with third-degree burns, who consequently developed post-burn hypertrophic scars. Furthermore, the antifibrotic effect of calpastatin, an endogenous calpain inhibitor, was evaluated in human fibroblasts and a murine burn model. The activity, mRNA levels, and protein levels of calpain were markedly higher in fibroblasts from the burn wounds of patients than in normal cells. Selective calpain inhibition by calpastatin markedly reduced not only the proliferation of burn-wound fibroblasts but also the mRNA and protein expression of calpain, transforming growth factor-beta 1, α-smooth muscle actin, type I and type III collagens, fibronectin, and vimentin in burn-wound fibroblasts. The anti-scarring effects of calpastatin were validated using a murine burn model by molecular, histological, and visual analyses. This study demonstrates the pathological role of calpain and the antifibrotic effect of calpastatin via calpain inhibition in post-burn hypertrophic scar formation.

scholarly journals Platelet-Released Growth Factors Induce Genes Involved in Extracellular Matrix Formation in Human Fibroblasts

2021 ◽  
Vol 22 (19) ◽  
pp. 10536
Author(s):  
Andreas Bayer ◽  
Bernard Wijaya ◽  
Franziska Rademacher ◽  
Lena Möbus ◽  
Mark Preuß ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Growth Factors ◽  
Growth Factor ◽  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Lysyl Oxidase ◽  
Human Fibroblasts ◽  
Dermal Fibroblasts ◽  
Type I ◽  
Cellular Mechanisms

Platelet concentrate products are increasingly used in many medical disciplines due to their regenerative properties. As they contain a variety of chemokines, cytokines, and growth factors, they are used to support the healing of chronic or complicated wounds. To date, underlying cellular mechanisms have been insufficiently investigated. Therefore, we analyzed the influence of Platelet-Released Growth Factors (PRGF) on human dermal fibroblasts. Whole transcriptome sequencing and gene ontology (GO) enrichment analysis of PRGF-treated fibroblasts revealed an induction of several genes involved in the formation of the extracellular matrix (ECM). Real-time PCR analyses of PRGF-treated fibroblasts and skin explants confirmed the induction of ECM-related genes, in particular transforming growth factor beta-induced protein (TGFBI), fibronectin 1 (FN1), matrix metalloproteinase-9 (MMP-9), transglutaminase 2 (TGM2), fermitin family member 1 (FERMT1), collagen type I alpha 1 (COL1A1), a disintegrin and metalloproteinase 19 (ADAM19), serpin family E member 1 (SERPINE1) and lysyl oxidase-like 3 (LOXL3). The induction of these genes was time-dependent and in part influenced by the epidermal growth factor receptor (EGFR). Moreover, PRGF induced migration and proliferation of the fibroblasts. Taken together, the observed effects of PRGF on human fibroblasts may contribute to the underlying mechanisms that support the beneficial wound-healing effects of thrombocyte concentrate products.

Long Noncoding RNA MIAT Modulates the Extracellular Matrix Deposition in Leiomyomas by Sponging MiR-29 Family

Endocrinology ◽  
2021 ◽  
Vol 162 (11) ◽  
Author(s):  
Tsai-Der Chuang ◽  
Derek Quintanilla ◽  
Drake Boos ◽  
Omid Khorram
Keyword(s):  
Extracellular Matrix ◽  
Long Noncoding Rna ◽  
Noncoding Rna ◽  
Collagen Type ◽  
Transforming Growth Factor ◽  
Transforming Growth Factor Β ◽  
Luciferase Reporter ◽  
Cycle Phase ◽  
Type I ◽  
Matrix Deposition

Abstract The objective of this study was to determine the expression and functional role of a long noncoding RNA (lncRNA) MIAT (myocardial infarction–associated transcript) in leiomyoma pathogenesis. Leiomyoma compared with myometrium (n = 66) expressed significantly more MIAT that was independent of race/ethnicity and menstrual cycle phase but dependent on MED12 (mediator complex subunit 12) mutation status. Leiomyomas bearing the MED12 mutation expressed higher levels of MIAT and lower levels of microRNA 29 family (miR-29a, -b, and -c) compared with MED12 wild-type leiomyomas. Using luciferase reporter activity and RNA immunoprecipitation analysis, MIAT was shown to sponge the miR-29 family. In a 3-dimensional spheroid culture system, transient transfection of MIAT siRNA in leiomyoma smooth muscle cell (LSMC) spheroids resulted in upregulation of miR-29 family and downregulation of miR-29 targets, collagen type I (COL1A1), collagen type III (COL3A1), and TGF-β3 (transforming growth factor β-3). Treatment of LSMC spheroids with TGF-β3 induced COL1A1, COL3A1, and MIAT levels, but repressed miR-29 family expression. Knockdown of MIAT in LSMC spheroids blocked the effects of TGF-β3 on the induction of COL1A1 and COL3A1 expression. Collectively, these results underscore the physiological significance of MIAT in extracellular matrix accumulation in leiomyoma.

Abstract 14664: Reduced Activin Like Kinase 1 Activity Promotes Cardiac Fibrosis in Heart Failure

Circulation ◽  
2015 ◽  
Vol 132 (suppl_3) ◽  
Author(s):  
Kevin Morine ◽  
Vikram Paruchuri ◽  
Xiaoying Qiao ◽  
Emily Mackey ◽  
Mark Aronovitz ◽  
...  
Keyword(s):  
Heart Failure ◽  
Cardiac Remodeling ◽  
Cardiac Fibrosis ◽  
Type I Collagen ◽  
Transforming Growth Factor ◽  
Left Ventricular ◽  
Lv Function ◽  
Type I ◽  
Mrna Levels ◽  
Protein Levels

Introduction: Activin receptor like kinase 1 (ALK1) mediates signaling via transforming growth factor beta-1 (TGFb1), a pro-fibrogenic cytokine. No studies have defined a role for ALK1 in heart failure. We tested the hypothesis that reduced ALK1 expression promotes maladaptive cardiac remodeling in heart failure. Methods and Results: ALK1 mRNA expression was quantified by RT-PCR in left ventricular (LV) tissue from patients with end-stage heart failure and compared to control LV tissue obtained from the National Disease Research Interchange (n=8/group). Compared to controls, LV ALK1 mRNA levels were reduced by 85% in patients with heart failure. Next, using an siRNA approach, we tested whether reduced ALK1 levels promote TGFb1-mediated collagen production in human cardiac fibroblasts. Treatment with an ALK1 siRNA reduced ALK1 mRNA levels by 75%. Compared to control, TGFb1-mediated Type I collagen and pSmad-3 protein levels were 2.5-fold and 1.7-fold higher, respectively, after ALK1 depletion. To explore a role for ALK1 in heart failure, ALK1 haploinsufficient (ALK1) and wild-type mice (WT; n=8/group) were studied 2 weeks after thoracic aortic constriction (TAC). Compared to WT, baseline LV ALK1 mRNA levels were 50% lower in ALK1 mice. Both LV and lung weights were higher in ALK1 mice after TAC. Cardiomyocyte area and LV mRNA levels of BNP, RCAN, and b-MHC were increased similarly, while SERCa levels were reduced in both ALK1 and WT mice after TAC. Compared to WT, LV fibrosis (Figure) and Type 1 Collagen mRNA and protein levels were higher among ALK1 mice. Compared to WT, LV fractional shortening (48±12 vs 26±10%, p=0.01) and survival (Figure) were lower in ALK1 mice after TAC. Conclusions: Reduced LV expression of ALK1 is associated with advanced heart failure in humans and promotes early mortality, impaired LV function, and cardiac fibrosis in a murine model of heart failure. Further studies examining the role of ALK1 and ALK1 inhibitors on cardiac remodeling are required.

scholarly journals Stimulation of the chemotactic migration of human fibroblasts by transforming growth factor beta.

1987 ◽  
Vol 165 (1) ◽  
pp. 251-256 ◽  
Author(s):  
A E Postlethwaite ◽  
J Keski-Oja ◽  
H L Moses ◽  
A H Kang
Keyword(s):  
Growth Factor ◽  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Human Fibroblasts ◽  
Critical Role ◽  
Dermal Fibroblasts ◽  
Type I ◽  
Tgf Beta ◽  
Chemotactic Migration ◽  
Growth Factor Beta

Transforming growth factor beta (TGF-beta) is a potent chemoattractant in vitro for human dermal fibroblasts. Intact disulfide and perhaps the dimeric structure of TGF-beta is essential for its ability to stimulate chemotactic migration of fibroblasts, since reduction with 2-ME results in a marked loss of its potency as a chemoattractant. Although epidermal growth factor (EGF) appears to be capable of modulating some effects of TGF-beta, it does not alter the chemotactic response of fibroblasts to TGF-beta. Specific polyvalent rabbit antibodies to homogeneously pure TGF-beta block its chemotactic activity but has no effect on the other chemoattractants tested (platelet-derived growth factor, fibronectin, and denatured type I collagen). Since TGF-beta is secreted by a variety of neoplastic and normal cells including platelets, monocytes/macrophages, and lymphocytes, it may play a critical role in vivo in embryogenesis, host response to tumors, and the repair response that follows damage to tissues by immune and nonimmune reactions.

Transforming growth factor beta increases cell surface binding and assembly of exogenous (plasma) fibronectin by normal human fibroblasts

1988 ◽  
Vol 8 (10) ◽  
pp. 4234-4242
Author(s):  
B L Allen-Hoffmann ◽  
C L Crankshaw ◽  
D F Mosher
Keyword(s):  
Extracellular Matrix ◽  
Growth Factor ◽  
Cell Surface ◽  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Human Fibroblasts ◽  
Tgf Beta ◽  
Surface Binding ◽  
Amino Terminal ◽  
Cell Surface Binding

Transforming growth factor beta (TGF-beta) enhances the cell surface binding of 125I-fibronectin by cultured human fibroblasts. The effect of TGF-beta on cell surface binding was maximal after 2 h of exposure to TFG-beta and did not require epidermal growth factor or protein synthesis. The enhancement was dose dependent and was found with the 125I-labeled 70-kilodalton amino-terminal fragment of fibronectin as well as with 125I-fibronectin. Treatment of cultures with TGF-beta for 6 h resulted in a threefold increase in the estimated number of fibronectin binding sites. The increase in number of binding sites was accompanied by an increased accumulation of labeled fibronectin in detergent-insoluble extracellular matrix. The effect of TGF-beta was biphasic; after 6 h of exposure, less labeled fibronectin bound to treated cultures than to control cultures. Exposure of cells to TGF-beta for greater than 6 h caused a two- to threefold increase in the accumulation of cellular fibronectin in culture medium as detected by a quantitative enzyme-linked immunosorbent assay. The second phase of the biphasic effect and the increase in soluble cellular fibronectin were blocked by cycloheximide. Immunofluorescence staining of fibroblast cultures with antifibronectin revealed that TGF-beta caused a striking increase in fibronectin fibrils. The 70-kilodalton amino-terminal fragment of fibronectin, which blocks incorporation of fibronectin into extracellular matrix, blocked anchorage-independent growth of NRK-49F cells in the presence of epidermal growth factor. Our results show that an increase in the binding and rate of assembly of exogenous fibronectin is an early event preceding the increase in expression of extracellular matrix proteins. Such an early increase in cell surface binding of exogenous fibronectin may be a mechanism whereby TGF-beta can modify extracellular matrix characteristics rapidly after tissue injury or during embryonic morphogenesis.

scholarly journals Interleukin-10 inhibits the osteogenic activity of mouse bone marrow

Blood ◽  
1993 ◽  
Vol 82 (8) ◽  
pp. 2361-2370 ◽  
Author(s):  
P Van Vlasselaer ◽  
B Borremans ◽  
R Van Den Heuvel ◽  
U Van Gorp ◽  
R de Waal Malefyt
Keyword(s):  
Bone Marrow ◽  
Extracellular Matrix ◽  
Osteogenic Differentiation ◽  
Interleukin 10 ◽  
Mouse Bone Marrow ◽  
Type I ◽  
Mrna Levels ◽  
Differentiation Potential ◽  
Alp Activity ◽  
Bone Proteins

Abstract Murine bone marrow cells synthesize bone proteins, including alkaline phosphatase (ALP), collagen type I, and osteocalcin, and form a mineralized extracellular matrix when cultured in the presence of beta- glycerophosphate and vitamin C. Interleukin-10 (IL-10) suppressed the synthesis of these bone proteins and mineralization without affecting cell proliferation. In addition, mRNA levels for the latter proteins were reduced in IL-10-treated cultures. This inhibitory effect was most outspoken when IL-10 was added before ALP activity peaked, eg, day 15 of culture. No significant effect was observed when IL-10 was added at later time points. This finding suggests that IL-10 acts at osteogenic differentiation stages that precede ALP expression but is ineffective on cells that progressed beyond this maturation stage. Likewise, IL-10 appeared to be unable to block both ALP activity and collagen synthesis in the preosteosteoblastic cell lines MN7 and MC3T3 that constitutively synthesize these proteins. Whereas IL-10 did not alter the number of fibroblast colony-forming cells of the marrow, it significantly reduced their osteogenic differentiation potential. In contrast to control cultures, IL-10-treated stroma was unable to either synthesize osteocalcin or to mineralize when subcultured over a 25-day period in the absence of IL-10. The inhibitory activity of IL-10 coincided with significant changes in stroma morphology. Whereas control cultures contained mainly flat adherent polygonal cells, significant numbers of rounded semiadherent to nonadherent cells were observed in the presence of IL-10. Scanning and transmission electron microscopy showed that, in contrast to control cultures, IL-10-treated stromas completely lacked a mineralized extracellular matrix. Collectively, these data suggest that IL-10 may have important regulatory effects on bone biology because of its capacity to downregulate early steps of osteogenic differentiation.

scholarly journals TIMP-1 Induces α-Smooth Muscle Actin in Fibroblasts to Promote Urethral Scar Formation

2015 ◽  
Vol 35 (6) ◽  
pp. 2233-2243 ◽  
Author(s):  
Yinglong Sa ◽  
Chao Li ◽  
Hongbin Li ◽  
Hailin Guo
Keyword(s):  
Smooth Muscle ◽  
Molecular Mechanisms ◽  
Transforming Growth Factor ◽  
Smooth Muscle Actin ◽  
Human Fibroblasts ◽  
Mapk Signaling ◽  
Scar Formation ◽  
Dependent Manner ◽  
Muscle Actin ◽  
Mesenchymal Transition

Background/Aims: Tissue inhibitor of metalloproteinases-1 (TIMP-1) has been reported to upregulate in urethral scar. However, the underlying molecular mechanisms remain undefined. Methods: Here, we studied levels of TIMP-1 and α-smooth muscle actin (α-SMA) in the fibroblasts isolated from urethral scar tissues, compared to the fibroblasts isolated from normal urethra. Then we either overexpressed TIMP-1, or inhibited TIMP-1 by lentiviruses carrying a transgene or a short hairpin small interfering RNA for TIMP-1 in human fibroblasts. We examined the effects of modulation of TIMP-1 on α-SMA, and on epithelial-mesenchymal transition (EMT)-related genes. We also studied the underlying mechanisms. Results: We detected significantly higher levels of TIMP-1 and α-smooth muscle actin (α-SMA) in the fibroblasts isolated from urethral scar tissues, compared to the fibroblasts isolated from normal urethra. Moreover, the levels of TIMP-1 and α-SMA strongly correlated. Moreover, we found that TIMP-1 significantly increased levels of α-SMA, transforming growth factor β 1 (TGFβ1), Collagen I and some other key factors related to an enhanced EMT, suggesting that TIMP-1 may induce transformation of fibroblasts into myofibroblasts to promote tissue EMT to enhance the formation of urethral scar. Moreover, increases in TIMP-1 also induced an increase in fibroblast cell growth and cell invasion, in an ERK/MAPK-signaling-dependent manner. Conclusion: Our study thus highlights a pivotal role of TIMP-1 in urethral scar formation.

Transforming growth factor-β1 decreases cardiac muscle L-type Ca2+ current and charge movement by acting on the Cav1.2 mRNA

2007 ◽  
Vol 292 (1) ◽  
pp. H622-H631 ◽  
Author(s):  
Guillermo Avila ◽  
Irma M. Medina ◽  
Esperanza Jiménez ◽  
Guillermo Elizondo ◽  
Citlalli I. Aguilar
Keyword(s):  
Transforming Growth Factor ◽  
Ventricular Myocytes ◽  
Neonatal Rat ◽  
Charge Movement ◽  
Type I ◽  
Mrna Levels ◽  
Patch Clamp Technique ◽  
Rat Cardiomyocytes ◽  
Electrophysiological Effects ◽  
Tgf Β1

Transforming growth factors-β (TGF-βs) are essential to the structural remodeling seen in cardiac disease and development; however, little is known about potential electrophysiological effects. We hypothesized that chronic exposure (6–48 h) of primary cultured neonatal rat cardiomyocytes to the type 1 TGF-β (TGF-β1, 5 ng/ml) may affect voltage-dependent Ca2+ channels. Thus we investigated T- ( ICaT) and L-type ( ICaL) Ca2+ currents, as well as dihydropyridine-sensitive charge movement using the whole cell patch-clamp technique and quantified CaV1.2 mRNA levels by real-time PCR assay. In ventricular myocytes, TGF-β1 did not exert significant electrophysiological effects. However, in atrial myocytes, TGF-β1 reduced both ICaL and charge movement (55% at 24–48 h) without significantly altering ICaT, cell membrane capacitance, or channel kinetics (voltage dependence of activation and inactivation, as well as the activation and inactivation rates). Reductions of ICaL and charge movement were explained by concomitant effects on the maximal values of L-channels conductance ( Gmax) and charge movement (Qmax). Thus TGF-β1 selectively reduces the number of functional L-channels on the surface of the plasma membrane in atrial but not ventricular myocytes. The TGF-β1-induced ICaL reduction was unaffected by supplementing intracellular recording solutions with okadaic acid (2 μM) or cAMP (100 μM), two compounds that promote L-channel phosphorylation. This suggests that the decreased number of functional L-channels cannot be explained by a possible regulation in the L-channels phosphorylation state. Instead, we found that TGF-β1 decreases the expression levels of atrial CaV1.2 mRNA (70%). Thus TGF-β1 downregulates atrial L-channel expression and may be therefore contributing to the in vivo cardiac electrical remodeling.

scholarly journals Integrin β1Signaling Is Necessary for Transforming Growth Factor-β Activation of p38MAPK and Epithelial Plasticity

2001 ◽  
Vol 276 (50) ◽  
pp. 46707-46713 ◽  
Author(s):  
Neil A. Bhowmick ◽  
Roy Zent ◽  
Mayshan Ghiassi ◽  
Maureen McDonnell ◽  
Harold L. Moses
Keyword(s):  
Extracellular Matrix ◽  
Growth Factor ◽  
Transcriptional Activation ◽  
Intracellular Signaling ◽  
Transforming Growth Factor ◽  
Transforming Growth Factor Β ◽  
Dominant Negative ◽  
Receptor Expression ◽  
Type I ◽  
Type Ii

Transforming growth factor-β (TGF-β) can induce epithelial to mesenchymal transdifferentiation (EMT) in mammary epithelial cells. TGF-β-meditated EMT involves the stimulation of a number of signaling pathways by the sequential binding of the type II and type I serine/threonine kinase receptors, respectively. Integrins comprise a family of heterodimeric extracellular matrix receptors that mediate cell adhesion and intracellular signaling, hence making them crucial for EMT progression. In light of substantial evidence indicating TGF-β regulation of various β1integrins and their extracellular matrix ligands, we examined the cross-talk between the TGF-β and integrin signal transduction pathways. Using an inducible system for the expression of a cytoplasmically truncated dominant negative TGF-β type II receptor, we blocked TGF-β-mediated growth inhibition, transcriptional activation, and EMT progression. Dominant negative TGF-β type II receptor expression inhibited TGF-β signaling to the SMAD and AKT pathways, but did not block TGF-β-mediated p38MAPK activation. Interestingly, blocking integrin β1function inhibited TGF-β-mediated p38MAPK activation and EMT progression. Limiting p38MAPK activity through the expression of a dominant negative-p38MAPK also blocked TGF-β-mediated EMT. In summary, TGF-β-mediated p38MAPK activation is dependent on functional integrin β1, and p38MAPK activity is required but is not sufficient to induce EMT.

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