ResearchPad - extracellular-space https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[An electrodiffusive, ion conserving Pinsky-Rinzel model with homeostatic mechanisms]]> https://www.researchpad.co/article/elastic_article_7780 Neurons generate their electrical signals by letting ions pass through their membranes. Despite this fact, most models of neurons apply the simplifying assumption that ion concentrations remain effectively constant during neural activity. This assumption is often quite good, as neurons contain a set of homeostatic mechanisms that make sure that ion concentrations vary quite little under normal circumstances. However, under some conditions, these mechanisms can fail, and ion concentrations can vary quite dramatically. Standard models are thus not able to simulate such conditions. Here, we present what to our knowledge is the first multicompartmental neuron model that accounts for ion concentration variations in a way that ensures complete and consistent ion concentration and charge conservation. In this work, we use the model to explore under which activity conditions the ion concentration variations become important for predicting the neurodynamics. We expect the model to be of great value for the field of neuroscience, as it can be used to simulate a range of pathological conditions, such as spreading depression or epilepsy, which are associated with large changes in extracellular ion concentrations.

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<![CDATA[Regulation of Feto-Maternal Barrier by Matriptase- and PAR-2-Mediated Signaling Is Required for Placental Morphogenesis and Mouse Embryonic Survival]]> https://www.researchpad.co/article/5989da53ab0ee8fa60b8e578

The development of eutherian mammalian embryos is critically dependent on the selective bi-directional transport of molecules across the placenta. Here, we uncover two independent and partially redundant protease signaling pathways that include the membrane-anchored serine proteases, matriptase and prostasin, and the G protein-coupled receptor PAR-2 that mediate the establishment of a functional feto-maternal barrier. Mice with a combined matriptase and PAR-2 deficiency do not survive to term and the survival of matriptase-deficient mice heterozygous for PAR-2 is severely diminished. Embryos with the combined loss of PAR-2 and matriptase or PAR-2 and the matriptase partner protease, prostasin, uniformly die on or before embryonic day 14.5. Despite the extensive co-localization of matriptase, prostasin, and PAR-2 in embryonic epithelia, the overall macroscopic and histological analysis of the double-deficient embryos did not reveal any obvious developmental abnormalities. In agreement with this, the conditional deletion of matriptase from the embryo proper did not affect the prenatal development or survival of PAR-2-deficient mice, indicating that the critical redundant functions of matriptase/prostasin and PAR-2 are limited to extraembryonic tissues. Indeed, placentas of the double-deficient animals showed decreased vascularization, and the ability of placental epithelium to establish a functional feto-maternal barrier was severely diminished. Interestingly, molecular analysis suggested that the barrier defect was associated with a selective deficiency in the expression of the tight junction protein, claudin-1. Our results reveal unexpected complementary roles of matriptase-prostasin- and PAR-2-dependent proteolytic signaling in the establishment of placental epithelial barrier function and overall embryonic survival.

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<![CDATA[Exposure to Ozone Modulates Human Airway Protease/Antiprotease Balance Contributing to Increased Influenza A Infection]]> https://www.researchpad.co/article/5989db10ab0ee8fa60bcc095

Exposure to oxidant air pollution is associated with increased respiratory morbidities and susceptibility to infections. Ozone is a commonly encountered oxidant air pollutant, yet its effects on influenza infections in humans are not known. The greater Mexico City area was the primary site for the spring 2009 influenza A H1N1 pandemic, which also coincided with high levels of environmental ozone. Proteolytic cleavage of the viral membrane protein hemagglutinin (HA) is essential for influenza virus infectivity. Recent studies suggest that HA cleavage might be cell-associated and facilitated by the type II transmembrane serine proteases (TTSPs) human airway trypsin-like protease (HAT) and transmembrane protease, serine 2 (TMPRSS2), whose activities are regulated by antiproteases, such as secretory leukocyte protease inhibitor (SLPI). Based on these observations, we sought to determine how acute exposure to ozone may modulate cellular protease/antiprotease expression and function, and to define their roles in a viral infection. We utilized our in vitro model of differentiated human nasal epithelial cells (NECs) to determine the effects of ozone on influenza cleavage, entry, and replication. We show that ozone exposure disrupts the protease/antiprotease balance within the airway liquid. We also determined that functional forms of HAT, TMPRSS2, and SLPI are secreted from human airway epithelium, and acute exposure to ozone inversely alters their expression levels. We also show that addition of antioxidants significantly reduces virus replication through the induction of SLPI. In addition, we determined that ozone-induced cleavage of the viral HA protein is not cell-associated and that secreted endogenous proteases are sufficient to activate HA leading to a significant increase in viral replication. Our data indicate that pre-exposure to ozone disrupts the protease/antiprotease balance found in the human airway, leading to increased influenza susceptibility.

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<![CDATA[Non-Dioxin-Like Polychlorinated Biphenyls Inhibit G-Protein Coupled Receptor-Mediated Ca2+ Signaling by Blocking Store-Operated Ca2+ Entry]]> https://www.researchpad.co/article/5989da7eab0ee8fa60b99866

Polychlorinated biphenyls (PCBs) are ubiquitous pollutants which accumulate in the food chain. Recently, several molecular mechanisms by which non-dioxin-like (NDL) PCBs mediate neurodevelopmental and neurobehavioral toxicity have been elucidated. However, although the G-protein coupled receptor (GPCR) is a significant target for neurobehavioral disturbance, our understanding of the effects of PCBs on GPCR signaling remains unclear. In this study, we investigated the effects of NDL-PCBs on GPCR-mediated Ca2+ signaling in PC12 cells. We found that ortho-substituted 2,2’,6-trichlorinated biphenyl (PCB19) caused a rapid decline in the Ca2+ signaling of bradykinin, a typical Gq- and phospholipase Cβ-coupled GPCR, without any effect on its inositol 1,4,5-trisphosphate production. PCB19 reduced thapsigargin-induced sustained cytosolic Ca2+ levels, suggesting that PCB19 inhibits SOCE. The abilities of other NDL-PCBs to inhibit store-operated Ca2+ entry (SOCE) were also examined and found to be of similar potencies to that of PCB19. PCB19 also showed a manner equivalent to that of known SOCE inhibitors. PCB19-mediated SOCE inhibition was confirmed by demonstrating the ability of PCB19 to inhibit the SOCE current and thapsigargin-induced Mn2+ influx. These results imply that one of the molecular mechanism by which NDL-PCBs cause neurobehavioral disturbances involves NDL-PCB-mediated inhibition of SOCE, thereby interfering with GPCR-mediated Ca2+ signaling.

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<![CDATA[Lactation-Related MicroRNA Expression Profiles of Porcine Breast Milk Exosomes]]> https://www.researchpad.co/article/5989db38ab0ee8fa60bd4024

Breast milk is the primary source of nutrition for newborns, and is rich in immunological components. MicroRNAs (miRNAs) are present in various body fluids and are selectively packaged inside the exosomes, a type of membrane vesicles, secreted by most cell types. These exosomal miRNAs could be actively delivered into recipient cells, and could regulate target gene expression and recipient cell function. Here, we analyzed the lactation-related miRNA expression profiles in porcine milk exosomes across the entire lactation period (newborn to 28 days after birth) by a deep sequencing. We found that immune-related miRNAs are present and enriched in breast milk exosomes (p<10−16, χ2 test) and are generally resistant to relatively harsh conditions. Notably, these exosomal miRNAs are present in higher numbers in the colostrums than in mature milk. It was higher in the serum of colostrum-only fed piglets compared with the mature milk-only fed piglets. These immune-related miRNA-loaded exosomes in breast milk may be transferred into the infant body via the digestive tract. These observations are a prelude to in-depth investigations of the essential roles of breast milk in the development of the infant’s immune system.

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<![CDATA[A New Human 3D-Liver Model Unravels the Role of Galectins in Liver Infection by the Parasite Entamoeba histolytica]]> https://www.researchpad.co/article/5989d9feab0ee8fa60b72e8e

Investigations of human parasitic diseases depend on the availability of appropriate in vivo animal models and ex vivo experimental systems, and are particularly difficult for pathogens whose exclusive natural hosts are humans, such as Entamoeba histolytica, the protozoan parasite responsible for amoebiasis. This common infectious human disease affects the intestine and liver. In the liver sinusoids E. histolytica crosses the endothelium and penetrates into the parenchyma, with the concomitant initiation of inflammatory foci and subsequent abscess formation. Studying factors responsible for human liver infection is hampered by the complexity of the hepatic environment and by the restrictions inherent to the use of human samples. Therefore, we built a human 3D-liver in vitro model composed of cultured liver sinusoidal endothelial cells and hepatocytes in a 3D collagen-I matrix sandwich. We determined the presence of important hepatic markers and demonstrated that the cell layers function as a biological barrier. E. histolytica invasion was assessed using wild-type strains and amoebae with altered virulence or different adhesive properties. We showed for the first time the dependence of endothelium crossing upon amoebic Gal/GalNAc lectin. The 3D-liver model enabled the molecular analysis of human cell responses, suggesting for the first time a crucial role of human galectins in parasite adhesion to the endothelial cells, which was confirmed by siRNA knockdown of galectin-1. Levels of several pro-inflammatory cytokines, including galectin-1 and -3, were highly increased upon contact of E. histolytica with the 3D-liver model. The presence of galectin-1 and -3 in the extracellular medium stimulated pro-inflammatory cytokine release, suggesting a further role for human galectins in the onset of the hepatic inflammatory response. These new findings are relevant for a better understanding of human liver infection by E. histolytica.

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<![CDATA[Dimerization Capacities of FGF2 Purified with or without Heparin-Affinity Chromatography]]> https://www.researchpad.co/article/5989dab4ab0ee8fa60bac4eb

Fibroblast growth factor-2 (FGF2) is a pleiotropic growth factor exhibiting a variety of biological activities. In this article, we studied the capacity of FGF2 purified with or without heparin affinity chromatography to self-associate. Analyzing the NMR HSQC spectra for different FGF2 concentrations, heparin-affinity purified FGF2 showed perturbations that indicate dimerization and are a higher-order oligomerization state. HSQC perturbation observed with different FGF2 concentrations revealed a heparin-binding site and two dimer interfaces. Thus, with increasing protein concentrations, FGF2 monomers make contacts with each other and form dimers or higher order oligomers. On the contrary, FGF2 purified with ion-exchange chromatography did not show similar perturbation indicating that self-association of FGF2 is eliminated if purification is done without heparin-affinity chromatography. The HSQC spectra of heparin-affinity purified FGF2 can be reproduced to some extent by adding heparin tetra-saccharide to ion exchange chromatography purified FGF2. Heparin-affinity purified FGF2 bound to acceptor and donor beads in a tagged form using His-tagged or GST-tagged proteins, also dimerized in the AlphaScreen assay. This assay was further validated using different experimental conditions and competitors. The assay constitutes an interesting tool to study dimerization of other FGF forms as well.

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<![CDATA[The Anti-Inflammatory Effects of Matrix Metalloproteinase-3 on Irreversible Pulpitis of Mature Erupted Teeth]]> https://www.researchpad.co/article/5989da38ab0ee8fa60b86d89

Matrix metalloproteinases (MMPs) are involved in extracellular matrix degradation and the modulation of cell behavior. These proteinases have also been implicated in tissue repair and regeneration. Our previous studies have demonstrated that MMP-3 elicits stimulatory effects on the proliferation and the migration of endothelial cells as well as anti-apoptotic effects on these cells in vitro. In addition, we found that MMP-3 enhanced the regeneration of lost pulp tissue in a rat incisor pulp injury model. However, continuously erupting rodent incisors exhibit significantly different pulp organization compared with mature erupted teeth. Therefore, we have further extended these studies using a canine irreversible pulpitis model to investigate the effects of MMP-3. In this study, the crowns of the canine mature premolars were removed and the pulp tissues were amputated. The amputated pulp tissues remained exposed for 24 or 72 hours to induce mild or severe irreversible pulpitis, respectively, followed by sealing of the cavities. In both models, the whole pulp tissues became necrotic by day 14. In this mild pulpitis model, the regeneration of pulp tissue with vasculature and nerves was observed until 14 days after sealing with MMP-3, followed by extracellular matrix formation in the regenerated pulp tissues until day 28. The treatment with MMP-3 resulted in a decrease in the number of macrophage and antigen-presenting cells and a significant inhibition of IL-6 expression on day 3. The inhibition of MMP-3 activity abolished these anti-inflammatory effects. Immunofluorescence staining demonstrated that MMP-3 was involved in the modification of serum-derived hyaluronan-associated proteins and hyaluronan (SHAP-HA) complexes possibly through the degradation of versican. These results demonstrate that MMP-3 can act as an anti-inflammatory agent and suggest that MMP-3 might represent a useful therapy for the treatment of mild irreversible pulpitis.

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<![CDATA[Fibronectin Affects Transient MMP2 Gene Expression through DNA Demethylation Changes in Non-Invasive Breast Cancer Cell Lines]]> https://www.researchpad.co/article/5989dab2ab0ee8fa60babda6

Metastasis accounts for more than 90% of cancer deaths. Cells from primary solid tumors may invade adjacent tissues and migrate to distant sites where they establish new colonies. The tumor microenvironment is now recognized as an important participant in the signaling that induces cancer cell migration. An essential process for metastasis is extracellular matrix (ECM) degradation by metalloproteases (MMPs), which allows tumor cells to invade local tissues and to reach blood vessels. The members of this protein family include gelatinase A, or MMP-2, which is responsible for the degradation of type IV collagen, the most abundant component of the basal membrane, that separates epithelial cells in the stroma. It is known that fibronectin is capable of promoting the expression of MMP-2 in MCF7 breast cancer cells in culture. In addition, it was already shown that the MMP2 gene expression is regulated by epigenetic mechanisms. In this work, we showed that fibronectin was able to induce MMP2 expression by 30% decrease in its promoter methylation. In addition, a histone marker for an open chromatin conformation was significantly increased. These results indicate a new role for fibronectin in the communication between cancer cells and the ECM, promoting epigenetic modifications.

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<![CDATA[Tumour Microenvironments Induce Expression of Urokinase Plasminogen Activator Receptor (uPAR) and Concomitant Activation of Gelatinolytic Enzymes]]> https://www.researchpad.co/article/5989db01ab0ee8fa60bc6bd1

Background

The urokinase plasminogen activator receptor (uPAR) is associated with poor prognosis in oral squamous cell carcinoma (OSCC), and increased expression of uPAR is often found at the invasive tumour front. The aim of the current study was to elucidate the role of uPAR in invasion and metastasis of OSCC, and the effects of various tumour microenvironments in these processes. Furthermore, we wanted to study whether the cells’ expression level of uPAR affected the activity of gelatinolytic enzymes.

Methods

The Plaur gene was both overexpressed and knocked-down in the murine OSCC cell line AT84. Tongue and skin tumours were established in syngeneic mice, and cells were also studied in an ex vivo leiomyoma invasion model. Soluble factors derived from leiomyoma tissue, as well as purified extracellular matrix (ECM) proteins, were assessed for their ability to affect uPAR expression, glycosylation and cleavage. Activity of gelatinolytic enzymes in the tissues were assessed by in situ zymography.

Results

We found that increased levels of uPAR did not induce tumour invasion or metastasis. However, cells expressing low endogenous levels of uPAR in vitro up-regulated uPAR expression both in tongue, skin and leiomyoma tissue. Various ECM proteins had no effect on uPAR expression, while soluble factors originating from the leiomyoma tissue increased both the expression and glycosylation of uPAR, and possibly also affected the proteolytic processing of uPAR. Tumours with high levels of uPAR, as well as cells invading leiomyoma tissue with up-regulated uPAR expression, all displayed enhanced activity of gelatinolytic enzymes.

Conclusions

Although high levels of uPAR are not sufficient to induce invasion and metastasis, the activity of gelatinolytic enzymes was increased. Furthermore, several tumour microenvironments have the capacity to induce up-regulation of uPAR expression, and soluble factors in the tumour microenvironment may have an important role in the regulation of posttranslational modification of uPAR.

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<![CDATA[Transglutaminases (TGs) in Ocular and Periocular Tissues: Effect of Muscarinic Agents on TGs in Scleral Fibroblasts]]> https://www.researchpad.co/article/5989dad3ab0ee8fa60bb6f25

Objective

To investigate the expression of transglutaminases (TGs) in the ocular surface, the eyelid margin and associated glands and to determine effect of muscarinic agents on TGs in scleral fibroblasts (SF).

Materials and Methods

Primary SFs cultured from mouse and human sclera were treated with atropine and carbachol for 5 days. Lysed cell RNA was used for real-time PCR, protein was used for Western blot analysis and TG-2 transamidase activity was measured by ELISA. Immunohistochemistry was done to determine the expression of TGases.

Results

Immunohistochemistry and western blot confirmed the expression of TGs-1, 2, 3 and 5 proteins in cultured SFs and eye tissues. Real time PCR showed TG-1, 2, 5 transcript levels to be down regulated 3 fold (p<0.05) in cultured human and mouse SFs after incubation with atropine and this was reversed by carbachol. However, TG-3 expression was increased with atropine and decreased with carbachol at all concentrations. Atropine abrogated the carbachol-induced activation of SF in a dose-dependent manner. TGs-1, 3, 5 were localized in the entire mouse corneal epithelium, stroma and endothelium but TG-2 was present only in the corneal subepithelium and stroma. All TGs were localized in mouse Meibomian glands however TG-2 had a weak expression.

Conclusions

Our results confirm that TGs-1, 2, 3 and 5 are expressed in human SF and murine ocular tissues, eyelid and associated Meibomian glands. Real-time PCR and Western blot results showed that muscarinic antagonist down-regulates TGs-1, 2 and 5 in both cultured human and mouse SFs and upregulates TG-3. Atropine abrogated the carbachol-induced activation of SF in a dose-dependent manner. These results suggest that manipulation of TGs by way of muscarinic receptor acting drugs may be a plausible method of intervention in wound healing and scleral remodeling.

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<![CDATA[Identification of Histological Patterns in Clinically Affected and Unaffected Palm Regions in Dupuytren's Disease]]> https://www.researchpad.co/article/5989da09ab0ee8fa60b76dfd

Dupuytren's disease is a fibro-proliferative disease characterized by a disorder of the extracellular matrix (ECM) and high myofibroblast proliferation. However, studies failed to determine if the whole palm fascia is affected by the disease. The objective of this study was to analyze several components of the extracellular matrix of three types of tissues—Dupuytren's diseased contracture cords (DDC), palmar fascia clinically unaffected by Dupuytren's disease contracture (NPF), and normal forehand fascia (NFF). Histological analysis, quantification of cells recultured from each type of tissue, mRNA microarrays and immunohistochemistry for smooth muscle actin (SMA), fibrillar ECM components and non-fibrillar ECM components were carried out. The results showed that DDC samples had abundant fibrosis with reticular fibers and few elastic fibers, high cell proliferation and myofibroblasts, laminin and glycoproteins, whereas NFF did not show any of these findings. Interestingly, NPF tissues had more cells showing myofibroblasts differentiation and more collagen and reticular fibers, laminin and glycoproteins than NFF, although at lower level than DDC, with similar elastic fibers than DDC. Immunohistochemical expression of decorin was high in DDC, whereas versican was highly expressed NFF, with no differences for aggrecan. Cluster analysis revealed that the global expression profile of NPF was very similar to DDC, and reculturing methods showed that cells corresponding to DDC tissues proliferated more actively than NPF, and NPF more actively than NFF. All these results suggest that NPF tissues may be affected, and that a modification of the therapeutic approach used for the treatment of Dupuytren's disease should be considered.

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<![CDATA[Integrating Macrophages into Organotypic Co-Cultures: A 3D In Vitro Model to Study Tumor-Associated Macrophages]]> https://www.researchpad.co/article/5989d9f8ab0ee8fa60b70dba

Tumor progression is controlled by signals from cellular and extra-cellular microenvironment including stromal cells and the extracellular matrix. Consequently, three-dimensional in vitro tumor models are essential to study the interaction of tumor cells with their microenvironment appropriately in a biologically relevant manner. We have previously used organotypic co-cultures to analyze the malignant growth of human squamous cell carcinoma (SCC) cell lines on a stromal equivalent in vitro. In this model, SCC cell lines are grown on a collagen-I gel containing fibroblasts. Since macrophages play a critical role in the progression of many tumor types, we now have expanded this model by integrating macrophages into the collagen gel of these organotypic tumor co-cultures. This model was established as a murine and a human system of skin SCCs. The effect of macrophages on tumor progression depends on their polarization. We demonstrate that macrophage polarization in organotypic co-cultures can be modulated towards and M1 or an M2 phenotype by adding recombinant IFN-γ and LPS or IL-4 respectively to the growth medium. IL-4 stimulation of macrophage-containing cultures resulted in enhanced tumor cell invasion evidenced by degradation of the basement membrane, enhanced collagenolytic activity and increased MMP-2 and MMP-9. Interestingly, extended co-culture with tumor cells for three weeks resulted in spontaneous M2 polarization of macrophages without IL-4 treatment. Thus, we demonstrate that macrophages can be successfully integrated into organotypic co-cultures of murine or human skin SCCs and that this model can be exploited to analyze macrophage activation towards a tumor supporting phenotype.

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<![CDATA[A Fractal Nature for Polymerized Laminin]]> https://www.researchpad.co/article/5989dae1ab0ee8fa60bbbfd7

Polylaminin (polyLM) is a non-covalent acid-induced nano- and micro-structured polymer of the protein laminin displaying distinguished biological properties. Polylaminin stimulates neuritogenesis beyond the levels achieved by ordinary laminin and has been shown to promote axonal regeneration in animal models of spinal cord injury. Here we used confocal fluorescence microscopy (CFM), scanning electron microscopy (SEM) and atomic force microscopy (AFM) to characterize its three-dimensional structure. Renderization of confocal optical slices of immunostained polyLM revealed the aspect of a loose flocculated meshwork, which was homogeneously stained by the antibody. On the other hand, an ordinary matrix obtained upon adsorption of laminin in neutral pH (LM) was constituted of bulky protein aggregates whose interior was not accessible to the same anti-laminin antibody. SEM and AFM analyses revealed that the seed unit of polyLM was a flat polygon formed in solution whereas the seed structure of LM was highly heterogeneous, intercalating rod-like, spherical and thin spread lamellar deposits. As polyLM was visualized at progressively increasing magnifications, we observed that the morphology of the polymer was alike independently of the magnification used for the observation. A search for the Hausdorff dimension in images of the two matrices showed that polyLM, but not LM, presented fractal dimensions of 1.55, 1.62 and 1.70 after 1, 8 and 12 hours of adsorption, respectively. Data in the present work suggest that the intrinsic fractal nature of polymerized laminin can be the structural basis for the fractal-like organization of basement membranes in the neurogenic niches of the central nervous system.

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<![CDATA[Col6a1 Null Mice as a Model to Study Skin Phenotypes in Patients with Collagen VI Related Myopathies: Expression of Classical and Novel Collagen VI Variants during Wound Healing]]> https://www.researchpad.co/article/5989da0fab0ee8fa60b79043

Patients suffering from collagen VI related myopathies caused by mutations in COL6A1, COL6A2 and COL6A3 often also display skin abnormalities, like formation of keloids or “cigarette paper” scars, dry skin, striae rubrae and keratosis pilaris (follicular keratosis). Here we evaluated if Col6a1 null mice, an established animal model for the muscle changes in collagen VI related myopathies, are also suitable for the study of mechanisms leading to the skin pathology. We performed a comprehensive study of the expression of all six collagen VI chains in unwounded and challenged skin of wild type and Col6a1 null mice. Expression of collagen VI chains is regulated in both skin wounds and bleomycin-induced fibrosis and the collagen VI α3 chain is proteolytically processed in both wild type and Col6a1 null mice. Interestingly, we detected a decreased tensile strength of the skin and an altered collagen fibril and basement membrane architecture in Col6a1 null mice, the latter being features that are also found in collagen VI myopathy patients. Although Col6a1 null mice do not display an overt wound healing defect, these mice are a relevant animal model to study the skin pathology in collagen VI related disease.

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<![CDATA[Functionalized Positive Nanoparticles Reduce Mucin Swelling and Dispersion]]> https://www.researchpad.co/article/5989d9ecab0ee8fa60b6cd8f

Multi-functionalized nanoparticles (NPs) have been extensively investigated for their potential in household and commercial products, and biomedical applications. Previous reports have confirmed the cellular nanotoxicity and adverse inflammatory effects on pulmonary systems induced by NPs. However, possible health hazards resulting from mucus rheological disturbances induced by NPs are underexplored. Accumulation of viscous, poorly dispersed, and less transportable mucus leading to improper mucus rheology and dysfunctional mucociliary clearance are typically found to associate with many respiratory diseases such as asthma, cystic fibrosis (CF), and COPD (Chronic Obstructive Pulmonary Disease). Whether functionalized NPs can alter mucus rheology and its operational mechanisms have not been resolved. Herein, we report that positively charged functionalized NPs can hinder mucin gel hydration and effectively induce mucin aggregation. The positively charged NPs can significantly reduce the rate of mucin matrix swelling by a maximum of 7.5 folds. These NPs significantly increase the size of aggregated mucin by approximately 30 times within 24 hrs. EGTA chelation of indigenous mucin crosslinkers (Ca2+ ions) was unable to effectively disperse NP-induced aggregated mucins. Our results have demonstrated that positively charged functionalized NPs can impede mucin gel swelling by crosslinking the matrix. This report also highlights the unexpected health risk of NP-induced change in mucus rheological properties resulting in possible mucociliary transport impairment on epithelial mucosa and related health problems. In addition, our data can serve as a prospective guideline for designing nanocarriers for airway drug delivery applications.

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<![CDATA[HDAC6 Deacetylase Activity Is Critical for Lipopolysaccharide-Induced Activation of Macrophages]]> https://www.researchpad.co/article/5989da8bab0ee8fa60b9dd43

Activated macrophages play an important role in both innate and adaptive immune responses, and aberrant activation of macrophages often leads to inflammatory and immune disorders. However, the molecular mechanisms of how macrophages are activated are not fully understood. In this study, we identify a novel role for histone deacetylse 6 (HDAC6) in lipopolysaccharide (LPS)-induced macrophage activation. Our data show that suppression of HDAC6 activity significantly restrains LPS-induced activation of macrophages and production of pro-inflammatory cytokines. Further study reveals that the regulation of macrophage activation by HDAC6 is independent of F-actin polymerization and filopodium formation; instead, it is mediated by the effects of HDAC6 on cell adhesion and microtubule acetylation. These data thus suggest that HDAC6 is an important regulator of LPS-induced macrophage activation and might be a potential target for the management of inflammatory disorders.

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<![CDATA[Elevated Expression of Stromal Palladin Predicts Poor Clinical Outcome in Renal Cell Carcinoma]]> https://www.researchpad.co/article/5989daaaab0ee8fa60ba9265

The role that stromal renal cell carcinoma (RCC) plays in support of tumor progression is unclear. Here we sought to determine the predictive value on patient survival of several markers of stromal activation and the feasibility of a fibroblast-derived extracellular matrix (ECM) based three-dimensional (3D) culture stemming from clinical specimens to recapitulate stromal behavior in vitro. The clinical relevance of selected stromal markers was assessed using a well annotated tumor microarray where stromal-marker levels of expression were evaluated and compared to patient outcomes. Also, an in vitro 3D system derived from fibroblasts harvested from patient matched normal kidney, primary RCC and metastatic tumors was employed to evaluate levels and localizations of known stromal markers such as the actin binding proteins palladin, alpha-smooth muscle actin (α-SMA), fibronectin and its spliced form EDA. Results suggested that RCCs exhibiting high levels of stromal palladin correlate with a poor prognosis, as demonstrated by overall survival time. Conversely, cases of RCCs where stroma presents low levels of palladin expression indicate increased survival times and, hence, better outcomes. Fibroblast-derived 3D cultures, which facilitate the categorization of stromal RCCs into discrete progressive stromal stages, also show increased levels of expression and stress fiber localization of α-SMA and palladin, as well as topographical organization of fibronectin and its splice variant EDA. These observations are concordant with expression levels of these markers in vivo. The study proposes that palladin constitutes a useful marker of poor prognosis in non-metastatic RCCs, while in vitro 3D cultures accurately represent the specific patient's tumor-associated stromal compartment. Our observations support the belief that stromal palladin assessments have clinical relevance thus validating the use of these 3D cultures to study both progressive RCC-associated stroma and stroma-dependent mechanisms affecting tumorigenesis. The clinical value of assessing RCC stromal activation merits further study.

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<![CDATA[Autotaxin-Lysophosphatidic Acid Axis Is a Novel Molecular Target for Lowering Intraocular Pressure]]> https://www.researchpad.co/article/5989da1fab0ee8fa60b7e481

Primary open-angle glaucoma is the second leading cause of blindness in the United States and is commonly associated with elevated intraocular pressure (IOP) resulting from diminished aqueous humor (AH) drainage through the trabecular pathway. Developing effective therapies for increased IOP in glaucoma patients requires identification and characterization of molecular mechanisms that regulate IOP and AH outflow. This study describes the identification and role of autotaxin (ATX), a secretory protein and a major source for extracellular lysophosphatidic acid (LPA), in regulation of IOP in a rabbit model. Quantitative proteomics analysis identified ATX as an abundant protein in both human AH derived from non-glaucoma subjects and in AH from different animal species. The lysophospholipase D (LysoPLD) activity of ATX was found to be significantly elevated (by ∼1.8 fold; n = 20) in AH derived from human primary open angle glaucoma patients as compared to AH derived from age-matched cataract control patients. Immunoblotting analysis of conditioned media derived from primary cultures of human trabecular meshwork (HTM) cells has confirmed secretion of ATX and the ability of cyclic mechanical stretch of TM cells to increase the levels of secreted ATX. Topical application of a small molecular chemical inhibitor of ATX (S32826), which inhibited AH LysoPLD activity in vitro (by >90%), led to a dose-dependent and significant decrease of IOP in Dutch-Belted rabbits. Single intracameral injection of S32826 (∼2 µM) led to significant reduction of IOP in rabbits, with the ocular hypotensive response lasting for more than 48 hrs. Suppression of ATX expression in HTM cells using small-interfering RNA (siRNA) caused a decrease in actin stress fibers and myosin light chain phosphorylation. Collectively, these observations indicate that the ATX-LPA axis represents a potential therapeutic target for lowering IOP in glaucoma patients.

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<![CDATA[Identification of Molecular Pathways Facilitating Glioma Cell Invasion In Situ]]> https://www.researchpad.co/article/5989d9f1ab0ee8fa60b6e73b

Gliomas are mostly incurable secondary to their diffuse infiltrative nature. Thus, specific therapeutic targeting of invasive glioma cells is an attractive concept. As cells exit the tumor mass and infiltrate brain parenchyma, they closely interact with a changing micro-environmental landscape that sustains tumor cell invasion.

In this study, we used a unique microarray profiling approach on a human glioma stem cell (GSC) xenograft model to explore gene expression changes in situ in Invading Glioma Cells (IGCs) compared to tumor core, as well as changes in host cells residing within the infiltrated microenvironment relative to the unaffected cortex. IGCs were found to have reduced expression of genes within the extracellular matrix compartment, and genes involved in cell adhesion, cell polarity and epithelial to mesenchymal transition (EMT) processes. The infiltrated microenvironment showed activation of wound repair and tissue remodeling networks. We confirmed by protein analysis the downregulation of EMT and polarity related genes such as CD44 and PARD3 in IGCs, and EFNB3, a tissue-remodeling agent enriched at the infiltrated microenvironment. OLIG2, a proliferation regulator and glioma progenitor cell marker upregulated in IGCs was found to function in enhancing migration and stemness of GSCs. Overall, our results unveiled a more comprehensive picture of the complex and dynamic cell autonomous and tumor-host interactive pathways of glioma invasion than has been previously demonstrated. This suggests targeting of multiple pathways at the junction of invading tumor and microenvironment as a viable option for glioma therapy.

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