Extracellular Matrix Fibronectin Research Articles

Dehydrocostus lactone inhibits BLM-induced pulmonary fibrosis and inflammation in mice via the JNK and p38 MAPK-mediated NF-κB signaling pathways

Idiopathic pulmonary fibrosis (IPF) is a chronic and irreversible inflammatory disease with a high mortality rate and limited therapeutic options. This study explored the potential role and mechanisms of Dehydrocostus lactone (DHL) in the inflammatory and fibrotic responses in a bleomycin (BLM) induced model. Treatment with DHL significantly reduced pathological injury and fibrosis, the secretion of BLM-induced pro-fibrotic mediators TGF-β and α-SMA, and components of the extracellular matrix (fibronectin). Additionally, in the early stages of inflammation, DHL administration inhibited the infiltration of inflammatory cells and downregulated the expression of TGF-β, TNF-α, and IL-6, indicating that DHL treatment effectively alleviated BLM-induced pulmonary fibrosis and inflammation in a dose-dependent manner. Furthermore, BLM induced the production of IL-33 in vivo, which initiated and progressed pulmonary fibrosis by activating macrophages and enhancing the production of IL-13 and TGF-β. In contrast, a significant decrease in the expression of IL-33 after DHL treatment in vitro showed that DHL strongly reduced IL-13 and TGF-β. Regarding the mechanism, BLM-induced phosphorylation of JNK, p38 MAPK, and NF-κB were significantly reduced after DHL treatment, which further led to the down-regulation of IL-33 expression, thereby decreasing IL-13 and TGF-β. Collectively, our data suggested that DHL could exert its anti-fibrosis effect via inhibiting the early inflammatory response by downregulating the JNK/p38 MAPK-mediated NF-κB signaling pathway to suppress macrophage activation. Therefore, DHL has therapeutic potential for pulmonary fibrosis.

Extracellular Matrix: Emerging Roles and Potential Therapeutic Targets for Breast Cancer.

Increasing evidence shows that the extracellular matrix (ECM) is an important regulator of breast cancer (BC). The ECM comprises of highly variable and dynamic components. Compared with normal breast tissue under homeostasis, the ECM undergoes many changes in composition and organization during BC progression. Induced ECM proteins, including fibrinogen, fibronectin, hyaluronic acid, and matricellular proteins, have been identified as important components of BC metastatic cells in recent years. These proteins play major roles in BC progression, invasion, and metastasis. Importantly, several specific ECM molecules, receptors, and remodeling enzymes are involved in promoting resistance to therapeutic intervention. Additional analysis of these ECM proteins and their downstream signaling pathways may reveal promising therapeutic targets against BC. These potential drug targets may be combined with new nanoparticle technologies. This review summarizes recent advances in functional nanoparticles that target the ECM to treat BC. Accurate nanomaterials may offer a new approach to BC treatment.

Defining optimal enzyme and matrix combination for replating of human induced pluripotent stem cell-derived cardiomyocytes at different levels of maturity

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) create an unlimited cell source for basic and translational research. Depending on the maturity of cardiac cultures and the intended applications, obtaining hiPSC-CMs as a single-cell, monolayer or three-dimensional clusters can be challenging. Here, we defined strategies to replate hiPSC-CMs on early days (D15-30) or later more mature (D60-150) differentiation cultures. After generation of hiPSCs and derivation of cardiomyocytes, four dissociation reagents Collagenase A/B, Collagenase II, TrypLE, EDTA and five different extracellular matrix materials Laminin, iMatrix-511, Fibronectin, Matrigel, and Geltrex were comparatively evaluated by imaging, cell viability, and contraction analysis. For early cardiac differentiation cultures mimicking mostly the embryonic stage, the highest adhesion, cell viability, and beating frequencies were achieved by treatment with the TrypLE enzyme. Video-based contraction analysis demonstrated higher beating rates after replating compared to before treatment. For later differentiation days of more mature cardiac cultures, dissociation with EDTA and replating cells on Geltrex or Laminin-derivatives yielded better recovery. Cardiac clusters at various sizes were detected in several groups treated with collagenases. Collectively, our findings revealed the selection criteria of the dissociation approach and coating matrix for replating iPSC-CMs based on the maturity and the requirements of further downstream applications.

Basic science: Beta cells, islets and complications

Introduction: Inflammation has been linked to hemichannel-mediated release of adenosine triphosphate (ATP) and downstream fibrosis in various tissue type-specific complications of diabetes. In the current study we examined how inflammatory cytokines alter markers of renal tubular injury ahead of evaluating the efficacy of the connexin-43 (Cx43) hemichannel blocker Tonabersat in negating these effects. Methods: Human Kidney (HK-2) proximal tubule epithelial cells were cultured in low (5mM) or high (25mM) glucose ± interleukin (IL)-1β (10ng) and tumour necrosis factor alpha (TNFα; 10ng) for 48 hours. Immunoblotting determined expression of key proteins, whilst carboxyfluorescein (200µM) dye uptake examined hemichannel activity in treated cells +/- Tonabersat (10µM). Results: Under high glucose conditions, IL-1β and TNFα significantly increased hemichannel activity to 161±16%, (P<0.05, n=5) an effect which was negated when co-incubated with hemichannel blocker Tonabersat (123±8.5%, n=5). Moreover, stimulation with IL-1β and TNFα increased expression of the inflammatory protein IL-6 (206±42%, P<0.05, n=5) and the extracellular matrix proteins, fibronectin (365±81%, P<0.05, n=4), collagen-I (516±36%, P<0.05, n=4) and collagen-IV (830±217%, P<0.01, n=5) as compared to low glucose control. The effect was augmented at high (25mM) glucose for IL-6 (491±164%, P<0.05, n=5), fibronectin (455±112%, n=6), collagen-I (725±85%, P<0.05, n=4) and colIagen-IV (868±77%, P<0.001, n=6). Conclusion: We demonstrate that IL1β and TNFα increase expression of inflammatory and profibrotic proteins in glucose treated HK-2 cells. Paralleled by a Tonabersat-sensitive increase in Cx43 hemichannel activity, we believe that blocking hemichannels has therapeutic potential and protects against changes associated with tubular injury in the diabetic kidney.

POS-437 CHARACTERIZATION OF MATRICELLULAR PROTEIN EXPRESSION SIGNATURES IN MECHANISTICALLY DIVERSE MOUSE MODELS OF KIDNEY INJURY

The development of an assortment of chronic kidney diseases (CKDs) is associated with fibrosis, a maladaptive repair response characterized by excessive deposition and processing of extracellular matrix (ECM) materials, like collagen and fibronectin. Embedded within the ECM are a family of proteins called Matricellular Proteins (MCPs), which have emerged over the last 20 years as essential regulators to ECM processing and cell-ECM interactions. They also exhibit contextual expression, typically induced transiently during a normal repair process.

In vitro and in vivo evaluation of a biomimetic scaffold embedding silver nanoparticles for improved treatment of oral lesions

BackgroundNew materials are currently designed for efficient treatment of oral tissue lesions by guided tissue regeneration. The aim of this study was to develop a multifunctional 3D hybrid biomaterial consisting of extracellular matrix components, collagen, chondroitin 4-sulfate and fibronectin, functionalised with silver nanoparticles, intended to improve periodontitis treatment protocols. MethodsStructural observations were performed by autometallography, scanning and transmission electron microscopy. In vitro tests of 3D constructs of embedded gingival fibroblasts within hybrid biomaterial were performed by MTS and Live/Dead assays. Genotoxicity was assessed by comet assay. In vivo experiments using chick embryo chorioallantoic membrane (CAM) assay analysed the degradation and nanoparticles release, but also angiogenesis, new tissue formation in 3D constructs and the regenerative potential of the hybrid material. Biological activity was investigated in experimental models of inflamed THP-1 macrophages and oral specific bacterial cultures. ResultsLight micrographs showed distribution of silver nanoparticles on collagen fibrils. Scanning electron micrographs revealed a microstructure with interconnected pores, which favoured cell adhesion and infiltration. Cell viability and proliferation were significantly higher within the 3D hybrid biomaterial than in 2D culture conditions, while absence of the hybrid material's genotoxic effect was found. In vivo experiments showed that the hybrid material was colonised by cells and blood vessels, initiating synthesis of new extracellular matrix. Besides the known effect of chondroitin sulfate, incorporated silver nanoparticles increased the anti-inflammatory activity of the hybrid biomaterial. The silver nanoparticles maintained their antibacterial activity even after embedding in the polymeric scaffold and inhibited the growth of F. nucleatum and P. gingivalis. ConclusionThe novel biomimetic scaffold functionalised with silver nanoparticles presented regenerative, anti-inflammatory and antimicrobial potential for oral cavity lesions repair.

Expression of extracellular matrix components and cytokine receptors in human fibrocytes during rheumatoid arthritis

ABSTRACT Purpose: Fibroblast-like synoviocytes (FLS) represent one of the principal effectors of joint damage in rheumatoid arthritis (RA). Recent discovery of the circulating fibrocyte, a potential precursor of FLS, has raised issues regarding the characterization of fibrocytes with respect to their morphology and their biological role. In this study, we evaluated the morphology of fibrocytes in vitro and their ability to produce different extracellular matrix (ECM) components in comparison with two populations of RA FLS: synovial fluid FLS (fd-FLS) and intimal lining FLS (td-FLS). We also studied the expression of ECM regulators and a set of cytokine receptors involved in the pathogenesis of RA. Materials and Methods: Fibrocytes were cultured from peripheral blood of patients with RA. FLS were cultured from synovial fluids and tissues. ECM proteins (collagen I (col I) and fibronectin), Matrix metalloproteinases (MMP) (MMP3, and MMP9), ECM regulators (β catenin, TCF4, and c-fos), and cytokine receptors (CXCR1, CXCR2, CXCR3, IL1RI, IL1RII, and IL6Rα) were analyzed using qRT-PCR and/or western blot. Results: Our results demonstrated that fibronectin and MMP3 levels were higher in FLS compared to fibrocytes. Although MMP9 was expressed in the three cell types, its level was greater in fibrocytes than in td/fd FLS. The three cell types expressed CXCR3, IL1RI, IL1RII, and IL6Rα, while the expression of CXCR1 and CXCR2 was restricted to fibrocytes. Conclusion: Our results demonstrated that fibrocytes express ECM molecules and cytokines receptors. The observed differences between fibrocytes and FLS may be due to their distinct functions or differentiation state during RA. Abbreviations: RA: Rheumatoid ArthritisFLS: fibroblast-like synoviocytestd: tissue derivedfd: fluid derivedSF: Synovial FluidWnt: WinglessMMP: Matrix MetalloproteinaseCIA: murine collagen induced arthritisECM: Extracellular matrixcol I: Collagen ITCF/LEF: T-cell factor/lymphoid enhancer-binding factorAP1: Activator Protein 1

Quantitative proteomics reveal lineage-specific protein profiles in iPSC-derived Marfan syndrome smooth muscle cells

Marfan syndrome (MFS) is a connective tissue disorder caused by mutations in the FBN1 gene that produces wide disease phenotypic variability. The lack of ample genotype–phenotype correlation hinders translational study development aimed at improving disease prognosis. In response to this need, an induced pluripotent stem cell (iPSC) disease model has been used to test patient-specific cells by a proteomic approach. This model has the potential to risk stratify patients to make clinical decisions, including timing for surgical treatment. The regional propensity for aneurysm formation in MFS may be related to distinct smooth muscle cell (SMC) embryologic lineages. Thus, peripheral blood mononuclear cell (PBMC)-derived induced pluripotent stem cells (iPSC) were differentiated into lateral mesoderm (LM, aortic root) and neural crest (NC, ascending aorta/transverse arch) SMC lineages to model MFS aortic pathology. Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) proteomic analysis by tandem mass spectrometry was applied to profile LM and NC iPSC SMCs from four MFS patients and two healthy controls. Analysis revealed 45 proteins with lineage-dependent expression in MFS patients, many of which were specific to diseased samples. Single protein-level data from both iPSC SMCs and primary MFS aortic root aneurysm tissue confirmed elevated integrin αV and reduced MRC2 in clinical disease specimens, validating the iPSC iTRAQ findings. Functionally, iPSC SMCs exhibited defective adhesion to a variety of extracellular matrix proteins, especially laminin-1 and fibronectin, suggesting altered cytoskeleton dynamics. This study defines the aortic embryologic origin-specific proteome in a validated iPSC SMC model to identify novel protein markers associated with MFS aneurysm phenotype. Translating iPSC findings into clinical aortic aneurysm tissue samples highlights the potential for iPSC-based methods to model MFS disease for mechanistic studies and therapeutic discovery in vitro.

Influence of Human Platelet Lysate on Extracellular Matrix Deposition and Cellular Characteristics in Adipose-Derived Stem Cell Sheets.

Adipose-derived stem cell (ASC) is a valuable source of cell therapy. By stimulating extracellular matrix (ECM) secretion, ASC sheets can be fabricated with enhanced regenerative capabilities. In recent years, human platelet lysate (HPL) provides an attractive alternative to fetal bovine serum (FBS) for the ex vivo expansion of ASCs for clinical use. However, the effect of HPL on ASC sheet formation has not been previously determined. In this study, we compared ECM composition and cellular characteristics of ASC sheets cultured in growth medium supplemented with either FBS or HPL. HPL supplement significantly enhanced ASC proliferation without obvious change in the expression pattern of cell surface markers. We found that culturing ASCs with HPL rendered thicker cell sheets with significantly more ECM deposition, including collagen and fibronectin. Proteomic analysis of the FBS or HPL-cultured cell sheets showed diversity in ECM composition. HPL-cultured ASC sheets exhibited up-regulation of interleukin-6 and an anti-inflammatory cytokine, C1q/tumor necrosis factor-related protein-3. Conditioned medium of HPL-cultured ASC sheets significantly enhanced fibroblast migration and tube formation of endothelial cells in vitro, while it inhibited the migration of macrophages toward stimulated macrophages in vitro. TGF-β1-stimulated fibroblasts cultured in ASC sheet-conditioned medium showed down-regulation of α-SMA and TGF-β1. By adding an anti-hepatocyte growth factor (HGF) neutralizing antibody in conditioned medium, we indicated that an anti-fibrosis effect of HPL-cultured ASC sheets is partially mediated through the increased secretion of HGF. Moreover, chick embryo chorioallantoic membrane (CAM) assay showed comparable capillary density after applying either FBS or HPL-cultured ASC sheets, both of which were significantly higher than the control. In conclusion, robust ECM formation with altered ECM composition was noted in ASC sheets cultured in HPL-supplemented medium. Their immunomodulatory and pro-angiogenesis capabilities were largely maintained. Our findings paved the way to elucidate the potential of HPL-cultured ASC sheets for clinical application in tissue regeneration.

Extracellular Control of Radial Glia Proliferation and Scaffolding During Cortical Development and Pathology.

During the development of the cortex, newly generated neurons migrate long-distances in the expanding tissue to reach their final positions. Pyramidal neurons are produced from dorsal progenitors, e.g., radial glia (RGs) in the ventricular zone, and then migrate along RG processes basally toward the cortex. These neurons are hence dependent upon RG extensions to support their migration from apical to basal regions. Several studies have investigated how intracellular determinants are required for RG polarity and subsequent formation and maintenance of their processes. Fewer studies have identified the influence of the extracellular environment on this architecture. This review will focus on extracellular factors which influence RG morphology and pyramidal neuronal migration during normal development and their perturbations in pathology. During cortical development, RGs are present in different strategic positions: apical RGs (aRGs) have their cell bodies located in the ventricular zone with an apical process contacting the ventricle, while they also have a basal process extending radially to reach the pial surface of the cortex. This particular conformation allows aRGs to be exposed to long range and short range signaling cues, whereas basal RGs (bRGs, also known as outer RGs, oRGs) have their cell bodies located throughout the cortical wall, limiting their access to ventricular factors. Long range signals impacting aRGs include secreted molecules present in the embryonic cerebrospinal fluid (e.g., Neuregulin, EGF, FGF, Wnt, BMP). Secreted molecules also contribute to the extracellular matrix (fibronectin, laminin, reelin). Classical short range factors include cell to cell signaling, adhesion molecules and mechano-transduction mechanisms (e.g., TAG1, Notch, cadherins, mechanical tension). Changes in one or several of these components influencing the RG extracellular environment can disrupt the development or maintenance of RG architecture on which neuronal migration relies, leading to a range of cortical malformations. First, we will detail the known long range signaling cues impacting RG. Then, we will review how short range cell contacts are also important to instruct the RG framework. Understanding how RG processes are structured by their environment to maintain and support radial migration is a critical part of the investigation of neurodevelopmental disorders.

Marein ameliorates diabetic nephropathy by inhibiting renal sodium glucose transporter 2 and activating the AMPK signaling pathway in db/db mice and high glucose–treated HK-2 cells

Marein, an active component of the Coreopsis tinctoria Nutt. plant, is known to improve diabetic nephropathy (DN). However, its anti-diabetic functions in DN and potential mechanisms remain unclear. The aim of this study was to elucidate the effects and mechanisms of Marein in diabetic db/db mice with DN, and in high glucose–treated HK-2 cells. In vivo, treating diabetic db/db mice with Marein for 12 consecutive weeks restored diabetes-induced hyperglycemia and dyslipidemia, and ameliorated renal function deterioration, glomerulosclerosis, and renal ectopic lipid deposition. Marein exerted renoprotective effects by directly inhibiting renal tubule sodium glucose transporter 2 (SGLT2) expression, and then activating the AMP-activated protein kinase (AMPK)/acetyl CoA carboxylase (ACC)/peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) pathway in db/db mice. Meanwhile, Marein ameliorated fibrosis and inflammation by suppressing the pro-inflammatory factors interleukin-6 (IL-6) and monocyte chemotactic protein-1 (MCP-1), and expression of the extracellular matrix proteins, fibronectin (FN) and collagen 1 (COL1) in diabetic mice. In vitro, MDCK monolayer cells were established to explore the characteristics of Marein transmembrane transport. Marein was found to be absorbed across the membrane at a medium level that involved active transport and this was mediated by SGLTs. In HK-2 cells, Marein decreased uptake of the fluorescent glucose analog, 2-NBDG, by 22 % by inhibiting SGLT2 expression. In high glucose–treated HK-2 cells, Marein decreased SGLT2 expression and increased phosphorylated (p)-AMPK/p-ACC to improve high glucose–induced cellular dysfunction. Furthermore, Marein treatment decreased SGLT2 expression in SGLT2-overexpressing HK-2 cells. In addition, molecular docking and dynamics analysis revealed that SGLT2 was a direct target of Marein. Collectively, our results demonstrated that Marein ameliorates DN by inhibiting renal SGLT2 and activating p-AMPK, suggesting Marein can potentially prevent DN by suppressing renal SGLT2 expression directly.

Development of an In Vitro 3D Model for Investigating Ligamentum Flavum Hypertrophy

BackgroundLigamentum flavum hypertrophy (LFH) is among the most crucial factors in degenerative lumbar spinal stenosis, which can cause back pain, lower extremity pain, cauda equina syndrome and neurogenic claudication. The exact pathogenesis of LFH remains elusive despite extensive research. Most in vitro studies investigating LFH have been carried out using conventional two-dimensional (2D) cell cultures, which do not resemble in vivo conditions, as they lack crucial pathophysiological factors found in three-dimensional (3D) LFH tissue, such as enhanced cell proliferation and cell cluster formation. In this study, we generated ligamentum flavum (LF) clusters using spheroid cultures derived from primary LFH tissue.ResultsThe cultured LF spheroids exhibited good viability and growth on an ultra-low attachment 96-well plate (ULA 96-plate) platform according to live/dead staining. Our results showed that the 100-cell culture continued to grow in size, while the 1000-cell culture maintained its size, and the 5000-cell culture exhibited a decreasing trend in size as the culture time increased; long-term culture was validated for at least 28 days. The LF spheroids also maintained the extracellular matrix (ECM) phenotype, i.e., fibronectin, elastin, and collagen I and III. The 2D culture and 3D culture were further compared by cell cycle and Western blot analyses. Finally, we utilized hematoxylin and eosin (H&E) staining to demonstrate that the 3D spheroids resembled part of the cell arrangement in LF hypertrophic tissue.ConclusionsThe developed LF spheroid model has great potential, as it provides a stable culture platform in a 3D model that can further improve our understanding of the pathogenesis of LFH and has applications in future studies.

CD4+ T Cell Interstitial Migration Controlled by Fibronectin in the Inflamed Skin.

The extracellular matrix (ECM) is extensively remodeled during inflammation providing essential guidance cues for immune cell migration and signals for cell activation and survival. There is increasing interest in the therapeutic targeting of ECM to mitigate chronic inflammatory diseases and enhance access to the tumor microenvironment. T cells utilize the ECM as a scaffold for interstitial migration, dependent on T cell expression of matrix-binding integrins αVβ1/αVβ3 and tissue display of the respective RGD-containing ligands. The specific ECM components that control T cell migration are unclear. Fibronectin (FN), a canonical RGD-containing matrix component, is heavily upregulated in inflamed tissues and in vitro can serve as a substrate for leukocyte migration. However, limited by lack of tools to intravitally visualize and manipulate FN, the specific role of FN in effector T cell migration in vivo is unknown. Here, we utilize fluorescently-tagged FN to probe for FN deposition, and intravital multiphoton microscopy to visualize T cell migration relative to FN in the inflamed ear dermis. Th1 cells were found to migrate along FN fibers, with T cells appearing to actively push or pull against flexible FN fibers. To determine the importance of T cell interactions with FN, we used a specific inhibitor of FN polymerization, pUR4. Intradermal delivery of pUR4 (but not the control peptide) to the inflamed skin resulted in a local reduction in FN deposition. We also saw a striking attenuation of Th1 effector T cell movement at the pUR4 injection site, suggesting FN plays a key role in T cell interstitial migration. In mechanistic studies, pUR4 incubation with FN in vitro resulted in enhanced tethering of T cells to FN matrix, limiting productive migration. In vivo, such tethering led to increased Th1 accumulation in the inflamed dermis. Enhanced Th1 accumulation exacerbated inflammation with increased Th1 activation and IFNγ cytokine production. Thus, our studies highlight the importance of ECM FN fibrils for T cell migration in inflamed tissues and suggest that manipulating local levels of ECM FN may prove beneficial in promoting T cell accumulation in tissues and enhancing local immunity to infection or cancer.

Semicarbazide-sensitive amine oxidase inhibition ameliorates albuminuria and glomerulosclerosis but does not improve tubulointerstitial fibrosis in diabetic nephropathy.

Semicarbazide-sensitive amine oxidase (SSAO) is an enzyme with a unique dual function in controlling inflammation as well as reactive oxygen species (ROS) generation. We have demonstrated benefit of SSAO inhibition in acute kidney fibrosis. However the function of SSAO in chronic kidney disease (CKD) and diabetic kidney disease (DKD) is yet to be determined. We aimed to assess the effectiveness of a SSAO inhibitor (SSAOi; PXS-4728A) as an antifibrotic agent using a diabetic model of CKD. Diabetic mice were treated with SSAOi for 24 weeks and outcomes compared with untreated diabetic mice and telmisartan treated animals as a standard of care comparator. Extracellular matrix markers, fibronectin and oxidative stress, were downregulated in diabetic mice treated with SSAOi compared with untreated diabetic mice. Expression of the pan-leukocyte marker CD45 was also supressed by SSAOi. SSAO inhibition in diabetic mice resulted in a significant reduction in glomerulosclerosis and associated albuminuria compared to untreated diabetic mice. However, the effect of SSAO inhibition was less obvious in the tubulointerstitial compartment than in the glomeruli. Therefore, SSAO may be a potential target for diabetic glomerulosclerosis.

Inhibition of RNA-binding protein HuR reduces glomerulosclerosis in experimental nephritis.

Recent identification of an RNA-binding protein (HuR) that regulates mRNA turnover and translation of numerous transcripts via binding to an ARE in their 3′-UTR involved in inflammation and is abnormally elevated in varied kidney diseases offers a novel target for the treatment of renal inflammation and subsequent fibrosis. Thus, we hypothesized that treatment with a selective inhibition of HuR function with a small molecule, KH-3, would down-regulate HuR-targeted proinflammatory transcripts thereby improving glomerulosclerosis in experimental nephritis, where glomerular cellular HuR is elevated. Three experimental groups included normal and diseased rats treated with or without KH-3. Disease was induced by the monoclonal anti-Thy 1.1 antibody. KH-3 was given via daily intraperitoneal injection from day 1 after disease induction to day 5 at the dose of 50 mg/kg BW/day. At day 6, diseased animals treated with KH-3 showed significant reduction in glomerular HuR levels, proteinuria, podocyte injury determined by ameliorated podocyte loss and podocin expression, glomerular staining for periodic acid-Schiff positive extracellular matrix proteins, fibronectin and collagen IV and mRNA and protein levels of profibrotic markers, compared with untreated disease rats. KH-3 treatment also reduced disease-induced increases in renal TGFβ1 and PAI-1 transcripts. Additionally, a marked increase in renal NF-κB-p65, Nox4, and glomerular macrophage cell infiltration observed in disease control group was largely reversed by KH-3 treatment. These results strongly support our hypothesis that down-regulation of HuR function with KH-3 has therapeutic potential for reversing glomerulosclerosis by reducing abundance of pro-inflammatory transcripts and related inflammation.

Autophagy Inhibition Sensitizes Renal Tubular Epithelial Cell to G1 Arrest Induced by Transforming Growth Factor beta (TGF-β).

BackgroundCell cycle arrest and autophagy have been demonstrated to be involved in various transforming growth factor (TGF)-β-mediated phenotype alterations of tubular epithelial cells (TECs) and tubulointerstitial fibrosis. But the relationship between cell cycle arrest and the autophagy induced by TGF-β has not been explored well.Material/MethodsThe effects of autophagy inhibition on TGF-β-induced cell cycle arrest in TECs were explored in vitro. Human kidney-2 (HK-2) cells were stimulated by TGF-β with or without a combined treatment of autophagy inhibitor chloroquine (CQ) or bafilomycin A1 (Baf).ResultsAutophagy inhibition by CQ or Baf promotes the suppression of growth in TGF-β-treated HK-2 cells, as detected by the Cell Counting Kit-8 (CCK-8) method. In addition, CQ or Baf stimulation enhances G1 arrest in TGF-β treated HK-2 cells, as investigated using propidium iodide (PI) staining and flow cytometry, which was further confirmed by a decrease in the expression of phosphorylated retinoblastoma protein (p-RB) and cyclin-dependent kinase 4 (CDK4). The upregulation of p21 induced by CQ or Baf may mediate an enhanced G1 arrest in TGF-β treated HK-2 cells. Western blot analysis showed that TGF-β-induced expression of extracellular matrix fibronectin was notably upregulated in the presence of autophagy inhibitors.ConclusionsInhibition of autophagy sensitizes the TECs to G1 arrest and proliferation suppression induced by TGF-β that contributes to the induction of tubulointerstitial fibrosis.

P0675LEUCOCYTE PODOSOME FORMATION AND ADHESION TO ENDOTHELIUM IS MEDIATED BY INTEGRIN LINKED KINASE IN CHRONIC KIDNEY DISEASE (CKD)

Abstract Background and Aims Patients with CKD have a higher risk of developing cardiovascular diseases. Protein-bound uremic toxins, such as p-cresol (pc) and indoxyl-sulfate (IS), are retention solutes, poorly removed during hemodialysis. They lead to endothelial dysfunction inducing the expression of adhesion molecules and leucocyte adhesion to endothelium. Monocyte extravasation can be carried out by dynamic adhesion structures called podosomes. Integrin-linked kinase (ILK), a kinase and an intracellular scaffold protein, links the cell-adhesion receptors, integrins and growth factors to the actin cytoskeleton and to a range of signalling pathways. We demonstrate the role of ILK in the accumulation of integrin-associated proteins inside the podosomes. The aim of this study was to investigate if ILK is involved in uremic toxin-induced leucocyte podosome formation and adhesion under uremic conditions that simulate CKD. Method In vitro experiments were carried out in human cell line of leukemic monocytes, THP-1. We tested the effect of uremic toxins on cell viability and ILK expression levels or activation by performing dose and time-response experiments. Cells were exposed to IS (25-100 µg ml-1) plus pc (10-100 µg ml-1) both combined, for different times. ILK expression levels were determined by Western Blot and RT-qPCR and its kinase activity was tested by its downstream effector GSK-3β phosphorylated levels. Cell adhesion of THP-1 cells stained with cell tracker to a monolayer of human endothelial cells (EA.hy926) and podosome formation and cell adhesion of THP-1 cells stained with phalloidin to a fibronectin extracellular matrix, was determined by fluorescence confocal microscopy. ILK co-localization with podosome specific protein cortactin was assessed by fluorescence confocal microscopy. For ex vivo experiments in ILK conditional-knockdown mice (cKD-ILK), male CRE-LOX mice were injected with tamoxifen (cKD-ILK) or vehicle (wild-type, WT), to induce ILK deletion. Peripheral blood mononuclear cells (PBMCs) were obtained and treated with uremic toxins. Cell adhesion to a fibronectin extracellular matrix was determined by cell count of the percentage of attached cells against the total cells collected. Results Our data suggests that uremic toxins did not induce toxicity in THP-1 cells. Furthermore, ILK was activated by uremic toxins both at low and high concentrations, without changes in the protein expression. In the cell adhesion assay to the endothelium, uremic toxins induces an increase of THP-1 cells adhesion, which is completely abolished when ILK is knocked down by ILK siRNA, both at low and high toxins concentrations. Similar results were obtained from the cell adhesion assay to the fibronectin extracellular matrix. Moreover, uremic toxins induced podosome formation in THP-1 cells, even at low concentrations, compared to the control, while ILK knockdown abrogated almost completely fibronectin adhesion and podosome formation in uremic toxin-treated cells. Interestingly, we tested that ILK co-localize with cortactin in podosomes, which confirmed the implication of ILK in podosome structures formation induced by uremic toxins. In cKD-ILK PBMC mice the transgenic ILK depletion significantly decrease non-excised ILK mRNA levels. These cKD-ILK PBMC mice exhibited a lower adhesion to the fibronectin extracellular matrix compared to WT. By last, uremic toxins induced a significant increase of mice PBMCs adhesion in WT animals compared to the control that was significantly lower in PBMCs of cKD-ILK animals. Conclusion These data suggest that ILK plays a critical role in the required processes for leukocyte extravasation. ILK deletion may prevent podosome formation and adhesion of leucocytes, decreasing the endothelial and vascular damage caused by the accumulation of uremic toxins in CKD. Therefore, ILK could be a potential therapeutic target for the treatment of vascular damage associated with CKD.

Creation of a contractile biomaterial from a decellularized spinach leaf without ECM protein coating: An in vitro study.

Myocardial infarction (MI) results in the death of cardiac tissue, decreases regional contraction, and can lead to heart failure. Tissue engineered cardiac patches containing human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) can restore contractile function. However, cells within thick patches require vasculature for blood flow. Recently, we demonstrated fibronectin coated decellularized leaves provide a suitable scaffold for hiPS-CMs. Yet, the necessity of this additional coating step is unclear. Therefore, we compared hiPS-CM behavior on decellularized leaves coated with collagen IV or fibronectin extracellular matrix (ECM) proteins to noncoated leaves for up to 21 days. Successful coating was verified by immunofluorescence. Similar numbers of hiPS-CMs adhered to coated and noncoated decellularized leaves for 21 days. At Day 14, collagen IV coated leaves contracted more than noncoated leaves (3.25 ± 0.39% vs. 1.54 ± 0.60%; p < .05). However, no differences in contraction were found between coated leaves, coated tissue culture plastic (TCP), noncoated leaves, or noncoated TCP at other time points. No significant differences were observed in hiPS-CM spreading or sarcomere lengths on leaves with or without coating. This study demonstrates that cardiac scaffolds can be created from decellularized leaves without ECM coatings. Noncoated decellularized leaf surfaces facilitate robust cell attachment for an engineered tissue patch.

Induction of ligand promiscuity of αVβ3 integrin by mechanical force.

αVβ3 integrin can bind to multiple extracellular matrix proteins, including vitronectin (Vn) and fibronectin (Fn), which are often presented to cells in culture as homogenous substrates. However, in tissues, cells experience highly complex and changing environments. To better understand integrin ligand selection in such complex environments, we employed binary-choice substrates of Fn and Vn to dissect αVβ3 integrin-mediated binding to different ligands on the subcellular scale. Super-resolution imaging revealed that αVβ3 integrin preferred binding to Vn under various conditions. In contrast, binding to Fn required higher mechanical load on αVβ3 integrin. Integrin mutations, structural analysis and chemical inhibition experiments indicated that the degree of hybrid domain swing-out is relevant for the selection between Fn and Vn; only a force-mediated, full hybrid domain swing-out facilitated αVβ3-Fn binding. Thus, force-dependent conformational changes in αVβ3 integrin increased the diversity of available ligands for binding and therefore enhanced the ligand promiscuity of this integrin.This article has an associated First Person interview with the first author of the paper.

High Glucose Increases Binding of Lysyl Oxidase to Extracellular Matrix Proteins: Implications for Diabetic Retinopathy.

PurposeTo determine whether high glucose (HG) compromises internalization of lysyl oxidase (LOX) through excess binding of LOX with extracellular matrix (ECM) proteins.MethodsTo determine whether HG promotes binding of LOX with ECM proteins, fibronectin (FN) and collagen IV (Coll IV), total or ECM-only proteins from rat retinal endothelial cells grown in normal (N; 5 mM) or HG (30 mM) medium were analyzed by coimmunoprecipitation and Western blot (WB). In parallel, coimmunostaining was performed to determine changes in LOX binding to FN or Coll IV. To determine the effect of HG on extracellular LOX levels, medium in which cells were grown for 1, 3, 5, and 7 days were assessed for LOX levels.ResultsWB analysis using total protein showed LOX overexpression and elevated levels of LOX bound to Coll IV or FN in HG condition. Similarly, a significant increase in LOX bound to FN or Coll IV was observed in ECM-only protein. These data were supported by Z-stack confocal microscopy images from coimmunostaining. Furthermore, immunostaining performed on ECM layer revealed increased presence of LOX bound to Coll IV or FN. Additionally, when media from cells grown in HG was monitored, a maximal increase in LOX level was observed by day 3, which declined by day 7.ConclusionsFindings indicate that HG promotes binding of LOX to FN and Coll IV extracellularly that results in reduced LOX internalization, attenuation of negative feedback, and upregulation of LOX expression associated with diabetic retinopathy.