Trypanosoma cruzi–cardiomyocyte interaction: role of fibronectin in the recognition process and extracellular matrix expression in vitro and in vivo

Abstract Background Invadopodia are actin-based cell-membrane protrusions associated with the extracellular matrix degradation accompanying cancer invasion. The elucidation of the molecular mechanisms leading to invadopodia formation and activity is central for the prevention of tumor spreading and growth. Protein tyrosine kinases such as Src are known to regulate invadopodia assembly, little is however known on the role of protein tyrosine phosphatases in this process. Among these enzymes, we have selected the tyrosine phosphatase Shp1 to investigate its potential role in invadopodia assembly, due to its involvement in cancer development. Methods Co-immunoprecipitation and immunofluorescence studies were employed to identify novel substrate/s of Shp1AQ controlling invadopodia activity. The phosphorylation level of cortactin, the Shp1 substrate identified in this study, was assessed by immunoprecipitation, in vitro phosphatase and western blot assays. Short interference RNA and a catalytically-dead mutant of Shp1 expressed in A375MM melanoma cells were used to evaluate the role of the specific Shp1-mediated dephosphorylation of cortactin. The anti-invasive proprieties of glycerophosphoinositol, that directly binds and regulates Shp1, were investigated by extracellular matrix degradation assays and in vivo mouse model of metastasis. Results The data show that Shp1 was recruited to invadopodia and promoted the dephosphorylation of cortactin at tyrosine 421, leading to an attenuated capacity of melanoma cancer cells to degrade the extracellular matrix. Controls included the use of short interference RNA and catalytically-dead mutant that prevented the dephosphorylation of cortactin and hence the decrease the extracellular matrix degradation by melanoma cells. In addition, the phosphoinositide metabolite glycerophosphoinositol facilitated the localization of Shp1 at invadopodia hence promoting cortactin dephosphorylation. This impaired invadopodia function and tumor dissemination both in vitro and in an in vivo model of melanomas. Conclusion The main finding here reported is that cortactin is a specific substrate of the tyrosine phosphatase Shp1 and that its phosphorylation/dephosphorylation affects invadopodia formation and, as a consequence, the ability of melanoma cells to invade the extracellular matrix. Shp1 can thus be considered as a regulator of melanoma cell invasiveness and a potential target for antimetastatic drugs.

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The extracellular matrix of the developing cornea: diversity, deposition and function*

Development ◽

10.1242/dev.103.supplement.195 ◽

1988 ◽

Vol 103 (Supplement) ◽

pp. 195-205

Author(s):

J. B. L. Bard ◽

M. K. Bansal ◽

A. S. A. Ross

Keyword(s):

Extracellular Matrix ◽

Chondroitin Sulphate ◽

Corneal Stroma ◽

Experimental System ◽

Collagen I ◽

And Function ◽

20 Nm

This paper examines the role of the extracellular matrix (ECM) in the development of the cornea. After a brief summary of the corneal structure and ECM, we describe evidence suggesting that the differentiation of neural crest (NC) cells into endothelium and fibroblasts is under the control of ocular ECM. We then examine the role of collagen I in stromal morphogenesis by comparing normal corneas with those of homozygous Movl3 mice which do not make collagen I. We report that, in spite of this absence, the cellular morphology of the Movl3 eye is indistinguishable from that of the wild type. In the 16-day mutant stroma, however, the remaining collagens form small amounts of disorganized, thin fibrils rather than orthogonally organized 20 nm-diameter fibrils; a result implying that collagen I plays only a structural role and that its absence is not compensated for. It also suggests that, because these remaining collagens will not form the normal fibrils that they will in vitro, fibrillogenesis in the corneal stroma differs from that elsewhere. The latter part of the paper describes our current work on chick stromal deposition using corneal epithelia isolated with an intact basal lamina that lay down in vitro ∼3μm-thick stromas of organized fibrils similar to that seen in vivo. This experimental system has yielded two unexpected results. First, the amount of collagen and proteoglycans produced by such epithelia is not dependent on whether its substratum is collagenous and we therefore conclude that stromal production by the intact epithelium is more autonomous than hitherto thought. Second, chondroitin sulphate (CS), the predominant proteoglycan, appears to play no role in stromal morphogenesis: epithelia cultured in testicular hyaluronidase, which degrades CS, lay down stromas whose organization and fibrildiameter distribution are indistinguishable from controls. One possible role for CS, however, is as a lubricant which facilitates corneal growth: it could allow fibrils to move over one another without deforming their orthogonal organization. Finally, we have examined the processes of fibrillogenesis in the corneal stroma and conclude that they are different from those elsewhere in the embryo and in vitro, perhaps because there is in the primary stroma an unidentified, highly hydrated ECM macromolecule that embeds the fibrils and that may mediate their morphogenesis.

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Hyaluronic acid-functionalized gelatin hydrogels reveal extracellular matrix signals temper the efficacy of erlotinib against patient-derived glioblastoma specimens

10.1101/556324 ◽

2019 ◽

Author(s):

Sara Pedron ◽

Gabrielle L. Wolter ◽

Jee-Wei E. Chen ◽

Sarah E. Laken ◽

Jann N. Sarkaria ◽

...

Keyword(s):

Extracellular Matrix ◽

Hyaluronic Acid ◽

Tumor Microenvironment ◽

Kinase Inhibitor ◽

Egfr Mutations ◽

Primary Glioblastoma ◽

Stat3 Phosphorylation

AbstractTherapeutic options to treat primary glioblastoma (GBM) tumors are scarce. GBM tumors with epidermal growth factor receptor (EGFR) mutations, in particular a constitutively active EGFRvIII mutant, have extremely poor clinical outcomes. GBM tumors with concurrent EGFR amplification and active phosphatase and tensin homolog (PTEN) are sensitive to the tyrosine kinase inhibitor erlotinib, but the effect is not durable. A persistent challenge to improved treatment is the poorly understood role of cellular, metabolic, and biophysical signals from the GBM tumor microenvironment on therapeutic efficacy and acquired resistance. The intractable nature of studying GBM cell in vivo motivates tissue engineering approaches to replicate aspects of the complex GBM tumor microenvironment. Here, we profile the effect of erlotinib on two patient-derived GBM specimens: EGFR+ GBM12 and EGFRvIII GBM6. We use a three-dimensional gelatin hydrogel to present brain-mimetic hyaluronic acid (HA) and evaluate the coordinated influence of extracellular matrix signals and EGFR mutation status on GBM cell migration, survival and proliferation, as well as signaling pathway activation in response to cyclic erlotinib exposure. Comparable to results observed in vivo for xenograft tumors, erlotinib exposure is not cytotoxic for GBM6 EGFRvIII specimens. We also identify a role of extracellular HA (via CD44) in altering the effect of erlotinib in GBM EGFR+ cells by modifying STAT3 phosphorylation status. Taken together, we report an in vitro tissue engineered platform to monitor signaling associated with poor response to targeted inhibitors in GBM.

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Extracellular Matrix and Integrins in Embryonic Stem Cell Differentiation

Biochemistry Insights ◽

10.4137/bci.s30377 ◽

2015 ◽

Vol 8s2 ◽

pp. BCI.S30377 ◽

Cited By ~ 18

Author(s):

Han Wang ◽

Xie Luo ◽

Jake Leighton

Keyword(s):

Extracellular Matrix ◽

Pancreatic Beta Cells ◽

Embryonic Stem ◽

Stem Cell Differentiation ◽

Embryonic Stem Cell Differentiation ◽

In Vitro Differentiation ◽

Integrin Receptors

Embryonic stem cells (ESCs) are pluripotent cells with great therapeutic potentials. The in vitro differentiation of ESC was designed by recapitulating embryogenesis. Significant progress has been made to improve the in vitro differentiation protocols by toning soluble maintenance factors. However, more robust methods for lineage-specific differentiation and maturation are still under development. Considering the complexity of in vivo embryogenesis environment, extracellular matrix (ECM) cues should be considered besides growth factor cues. ECM proteins bind to cells and act as ligands of integrin receptors on cell surfaces. Here, we summarize the role of the ECM and integrins in the formation of three germ layer progenies. Various ECM–integrin interactions were found, facilitating differentiation toward definitive endoderm, hepatocyte-like cells, pancreatic beta cells, early mesodermal progenitors, cardiomyocytes, neuroectoderm lineages, and epidermal cells, such as keratinocytes and melanocytes. In the future, ECM combinations for the optimal ESC differentiation environment will require substantial study.

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Enhanced β-Catenin/Foxo1 Activity Alleviates Pulmonary Fibrosis by Inhibiting β-Catenin/T Cell Factor Signaling

Journal of Biomaterials and Tissue Engineering ◽

10.1166/jbt.2020.2241 ◽

2020 ◽

Vol 10 (2) ◽

pp. 182-188

Author(s):

Kun Gui ◽

Yu Huang ◽

Meijin Wang ◽

Jun Yang

Keyword(s):

Extracellular Matrix ◽

Pulmonary Fibrosis ◽

Inflammatory Cytokines ◽

Underlying Mechanism ◽

Control Group ◽

Kidney Fibrosis ◽

Mrc5 Cell

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive fibrosing interstitial pneumonia, resulting in chronic respiratoryfailure and eventually death. β-catenin/Foxo1 showed a protective property in kidney fibrosis, but the role of β-catenin/Foxo1 in IPF was unclear. Our study aimed to investigate the role of β-catenin/Foxo1 in IPF and explore its underlying mechanism. The IPF model was established by injection of bleomycin (BLM) in vivo and stimulation by TGF-β1 in MRC5 cell in vitro. Haematoxylin-eosin staining and Masson’s trichrome staining were performed to examine histopathological injury in lung. Protein expression of corresponding genes was detected using western blot. Immunofluorescence staining assay was carried out to detect the expression of β-catenin, Foxo1, TCF and α-SMA. The expression levels of inflammatory cytokines were determined using ELISA kit assay. The results showed that BLM induced a serious pulmonary injury and proliferated fibroblasts. A higher interaction of β-catenin with TCF and a lower interaction of β-catenin with Foxo1 was found in BLM group compared to the control group. TGF-β1 promoted β-catenin/TCF, whereas ICG-001 inhibited β-catenin/TCF, and promoted β-catenin/Foxo1. Furthermore, ICG-001 reversed TGF-β1 induced largely production of inflammatory cytokines and accumulation of extracellular matrix, as well as high expression of α-SMA. However, AS1842856, a FOXO1 antagonist, strengthened the effects induced by TGF-β1. In summary, our study revealed that β-catenin/Foxo1 protected against IPF through inhibiting inflammatory response and extracellular matrix accumulation, providing an alternative approach to explain the potential mechanism of IPF and seek for more effective therapeutic drugs.

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Nanostructures Control the Hepatocellular Responses to a Cytotoxic Agent “Cisplatin”

BioMed Research International ◽

10.1155/2015/925319 ◽

2015 ◽

Vol 2015 ◽

pp. 1-10 ◽

Cited By ~ 2

Author(s):

Shimaa A. Abdellatef ◽

Riho Tange ◽

Takeshi Sato ◽

Akihiko Ohi ◽

Toshihide Nabatame ◽

...

Keyword(s):

Extracellular Matrix ◽

Chemotherapeutic Agent ◽

Chromatin Condensation ◽

Cellular Responses ◽

Flat Surfaces ◽

Flat Substrate ◽

Cells Cultured

In drug discovery programs, the alteration betweenin vivoandin vitrocellular responses to drug represents one of the main challenges. Since the variation in the native extracellular matrix (ECM) betweenin vivoand 2Din vitroconditions is one of the key reasons for such discrepancies, thus the utilization of substrate that likely mimics ECM characteristics (topography, stiffness, and chemical composition) is needed to overcome such problem. Here, we investigated the role of substrate nanotopography as one of the major determinants of hepatic cellular responses to a chemotherapeutic agent “cisplatin.” We studied the substratum induced variations in cisplatin cytotoxicity; a higher cytotoxic response to cisplatin was observed for cells cultured on the nanopattern relative to a flat substrate. Moreover, the nanofeatures with grating shapes that mimic the topography of major ECM protein constituents (collagen) induced alterations in the cellular orientation and chromatin condensation compared to flat surfaces. Accordingly, the developments of biomimetic substrates with a particular topography could have potentials in drug development analyses to reflect more physiological mimicry conditionsin vitro.

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Interleukin-9 in Immunopathology of Trypanosoma cruzi Experimental Infection

Frontiers in Cellular and Infection Microbiology ◽

10.3389/fcimb.2021.756521 ◽

2021 ◽

Vol 11 ◽

Author(s):

Nadjania Saraiva de Lira Silva ◽

Cristina Mary Orikaza ◽

Fabiana Rodrigues de Santana ◽

Luana Aguiar dos Santos ◽

Bruno Ramos Salu ◽

...

Keyword(s):

Trypanosoma Cruzi ◽

Chagas Disease ◽

Experimental Infection ◽

Cardiac Fibrosis ◽

Tissue Injury ◽

Chronic Phase ◽

Protective Role

Chagas’ disease is a parasitosis caused by Trypanosoma cruzi, which affects approximately 8 million people worldwide. The balance between pro- and anti-inflammatory cytokines produced during immunological responses contributes to disease prognosis and progression. Parasite tissue persistence can induce chronic inflammatory stimuli, which can cause long-term tissue injury and fibrosis. Chronic Chagas’ patients exhibit increased levels of interleukin (IL)-9, an important cytokine in the regulation of inflammatory and fibrogenic processes. Data on the role of IL-9 in other pathologies are sometimes contradictory, and few studies have explored this cytokine’s influence in Chagas’ disease pathology. Hence, the aim of this study was to evaluate the role of IL-9 in the progression of T. cruzi infection in vivo and in vitro. In vitro infection demonstrated that IL-9 reduced the number of infected cells and decreased the multiplication of intracellular amastigotes in both C2C12 myoblasts and bone marrow-derived macrophages. In myoblasts, the increased production of nitric oxide (NO) was essential for reduced parasite multiplication, whereas macrophage responses resulted in increased IL-6 and reduced TGF-β levels, indicating that parasite growth restriction mechanisms induced by IL-9 were cell-type specific. Experimental infection of BALB/c mice with T. cruzi trypomastigotes of the Y strain implicated a major role of IL-9 during the chronic phase, as increased Th9 and Tc9 cells were detected among splenocytes; higher levels of IL-9 in these cell populations and increased cardiac IL-9 levels were detected compared to those of uninfected mice. Moreover, rIL9 treatment decreased serum IL-12, IL-6, and IL-10 levels and cardiac TNF-α levels, possibly attempting to control the inflammatory response. IL-9 neutralization increased cardiac fibrosis, synthesis of collagens I and III, and mastocyte recruitment in BALB/c heart tissue during the chronic phase. In conclusion, our data showed that IL-9 reduced the invasion and multiplication of T. cruzi in vitro, in both myoblasts and macrophages, favoring disease control through cell-specific mechanisms. In vivo, IL-9 was elevated during experimental chronic infection in BALB/c mice, and this cytokine played a protective role in the immunopathological response during this phase by controlling cardiac fibrosis and proinflammatory cytokine production.

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Arcanobacterium pyogenes: Virulence factors, importance in mastitis etiology and therapeutic (im)possibilities

Veterinarski glasnik ◽

10.2298/vetgl1104235m ◽

2011 ◽

Vol 65 (3-4) ◽

pp. 235-246

Author(s):

Dubravka Milanov ◽

Jelena Petrovic ◽

Milos Kapetanov ◽

Ljiljana Suvajdzic

Keyword(s):

Extracellular Matrix ◽

Virulence Factors ◽

Opportunistic Pathogen ◽

Clinical Mastitis ◽

Livestock Species ◽

Arcanobacterium Pyogenes ◽

Possible Cause

Arcanobacterium pyogenes is an opportunistic pathogen, a causative agent of suppurative infections of organs and tissues in economically important livestock species. Most frequently this bacteria is isolated from inflamed lung lesions in pigs and cattle, in samples of uterine mucus of cows with endometritis and milk from cows with clinical mastitis. A. pyogenes possesses a number of virulence factors: cholesterol-dependent cytolysin (pyolysin), two neuraminidases, several proteases, extracellular matrix-binding proteins, DNases, fimbriae. The virulence factors are well studied in laboratory conditions, but the role of these factors in the pathogenesis of A. pyogenes infections remains to be elucidated. Lately, the ability of A. pyogenes to form biofilm in vivo has also been implicated as a virulence factor and a possible cause of therapeutic failure. Despite the fact that A. pyogenes milk isolates in cows with mastitis in vitro are very sensitive to ?-lactam drugs and tetracycline, experience has shown that therapy is usually ineffective, prognosis is poor and the affected quarter is lost for milk production.

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The Role of Cardiac Fibroblasts in Extracellular Matrix-Mediated Signaling During Normal and Pathological Cardiac Development

Journal of Biomechanical Engineering ◽

10.1115/1.4024349 ◽

2013 ◽

Vol 135 (7) ◽

Cited By ~ 29

Author(s):

Kelly Elizabeth Sullivan ◽

Lauren Deems Black

Keyword(s):

Extracellular Matrix ◽

Cardiac Development ◽

Cardiac Fibroblasts ◽

Heart Defects ◽

Critical Role ◽

Heart Tissue ◽

Physical And Chemical

The extracellular matrix is no longer considered a static support structure for cells but a dynamic signaling network with the power to influence cell, tissue, and whole organ physiology. In the myocardium, cardiac fibroblasts are the primary cell type responsible for the synthesis, deposition, and degradation of matrix proteins, and they therefore play a critical role in the development and maintenance of functional heart tissue. This review will summarize the extensive research conducted in vivo and in vitro, demonstrating the influence of both physical and chemical stimuli on cardiac fibroblasts and how these interactions impact both the extracellular matrix and, by extension, cardiomyocytes. This work is of considerable significance, given that cardiovascular diseases are marked by extensive remodeling of the extracellular matrix, which ultimately impairs the functional capacity of the heart. We seek to summarize the unique role of cardiac fibroblasts in normal cardiac development and the most prevalent cardiac pathologies, including congenital heart defects, hypertension, hypertrophy, and the remodeled heart following myocardial infarction. We will conclude by identifying existing holes in the research that, if answered, have the potential to dramatically improve current therapeutic strategies for the repair and regeneration of damaged myocardium via mechanotransductive signaling.

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