Expression Of Focal Adhesion Proteins Research Articles

Functional profiling of resident cells during vascular aging

Introduction: Depending on surrounding cell types, substrate mechanics, and chemical agonists, vascular cell types – including endothelial (ECs) and smooth muscle cells (VSMC) — modulate their behavior to maintain homeostasis. Aging perturbs this homeostasis resulting in EC and SMC dysfunction, marked by impaired barrier integrity and pathological ECM remodeling. Endothelial-to-mesenchymal transition (EndMT) in ECs and “dedifferentiation” of VSMCs towards a synthetic phenotype are phenomenon under investigation as cellular drivers of the widespread dysregulation seen with aging. The overall goal of this project is to understand the effect of cross-talk between ECM and growth factor signaling on vascular cell plasticity during aging. Materials and Methods: Human aortic endothelial cells (HAECs) were seeded onto transwell inserts after seven days treatment with or without TGF-β. After growing to confluence on the insert, cells were subjected to a FITC-dextran transwell assay. A parallel group of cells was seeded onto fibronectin coated glass-bottom dishes and stained for the expression of focal adhesion proteins and cell-type markers. Mouse aortic smooth muscle cells (maSMCs) were isolated from C57Bl6 mice at 3 (young), 6 (early middle-aged; EMA), 9 (middle-age; MA), or greater than 18 months of age (old). Cells were seeded onto dye-quenched (DQ) collagen type I (Col-I) substrate before gain of fluorescence was measured in proximity to the cell as well as in the surrounding media. A parallel group of cells were allowed to grow in media containing FITC Col-I, and gain of fluorescence was measured in proximity to the cells. Each cell type (3M, 6M, 9M, and 18M) in both experiments (DQ and FITC) were further treated with MMP inhibitor, TG2 inhibitor and TGF-β1 to measure their impacts on Col-I turnover. Results: Barrier function was not impaired in ECs cultured with TGF-β1, relative to their untreated counterparts. Mouse aortic SMCs isolated from older mice had decreased rates of Col-I cleavage and were more susceptible to MMP inhibition than their younger counterparts. Conclusions: maSMCs from older mice have lower Col-I turnover than their younger counterparts. This is consistent with bulk tissue behavior, as it is well known that relative collagen levels increase in the elastic vasculature as we age. TGF-β1 treatment did not have an effect on permeability of 4kd FITC-dextran, it is feasible that there will be an impact on the larger 70kd fragments. Travis Brady is supported by the NASEM Ford Foundation Pre-doctoral Fellowship. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Pip5k1c expression in osteocytes regulates bone remodeling in mice

Research backgroundThe role of osteocytes in maintaining bone mass has been progressively emphasized. Pip5k1c is the most critical isoform among PIP5KIs, which can regulate cytoskeleton, biomembrane, and Ca2+ release of cells and participate in many processes, such as cell adhesion, differentiation, and apoptosis. However, its expression and function in osteocytes are still unclear. Materials and methodsTo determine the function of Pip5k1c in osteocytes, the expression of Pip5k1c in osteocytes was deleted by breeding the 10-kb mouse Dmp1-Cre transgenic mice with the Pip5k1cfl/fl mice. Bone histomorphometry, micro-computerized tomography analysis, immunofluorescence staining and western blotting were used to determine the effects of Pip5k1c loss on bone mass. In vitro, we explored the mechanism by siRNA knockdown of Pip5k1c in MLO-Y4 cells. ResultsPip5k1c expression was decreased in osteocytes in senescent and osteoporotic tissues both in humans and mice. Loss of Pip5k1c in osteocytes led to a low bone mass in long bones and spines and impaired biomechanical properties in femur, without changes in calvariae. The loss of Pip5k1c resulted in the reduction of the protein level of type 1 collagen in tibiae and MLO-Y4 cells. Osteocyte Pip5k1c loss reduced the osteoblast and bone formation rate with high expression of sclerostin, impacting the osteoclast activities at the same time. Moreover, Pip5k1c loss in osteocytes reduced expression of focal adhesion proteins and promoted apoptosis. ConclusionOur studies demonstrate the critical role and mechanism of Pip5k1c in osteocytes in regulating bone remodeling. The translational potential of this articleOsteocyte has been considered to a key role in regulating bone homeostasis. The present study has demonstrated that the significance of Pip5k1c in bone homeostasis by regulating the expression of collagen, sclerostin and focal adhesion expression, which provided a possible therapeutic target against human metabolic bone disease.

The evaluation of DNA aptamer monolayers on silicon surface for the promotion of cell-surface interaction

DNA aptamers are very specific towards biological targets and are useful in many biosensor applications. However, there had been little reports that systematically examined the potential of DNA aptamers as surface thin film for promoting cell-surface adhesion. In this work, three aptamers (IDA, NC3S and SYL3C) were grafted on silicon surface and were only selective towards epithelial cell type. To better demonstrate this selectivity, click chemistry on underlying PEG layer was performed to attach these DNA aptamers on the surfaces so as to discourage non-specific cell attachment. The physicochemical properties of the surfaces were examined via XPS, AFM, FTIR and contact angle goniometry. A range of different cell types (MDA-MB 231, A549, MES-SA, N2A and HaCaT) were incubated to these surfaces and their viability, morphology and focal adhesion protein expression levels were quantified after a period of 24 h. From our observations, we had shown that NC3S and SYL3C can actively promote cell adhesion while integrin targeting IDA did not help to improve the outcomes of cell-surface interaction. Moreover, aptamer functionalized surfaces were observed to be only favorably towards epithelial cell types and our current findings here had help shed light on DNA aptamer as potential bioactive thin films that are cell type selective.

Exploiting the Biophysical Cues toward Dual Differentiation of hMSC's within Geometrical Patterns.

A wide variety of cues from the extracellular matrix (ECM) have been known to affect the differentiation of stem cells in vivo. In particular, the biophysical cues and cell shape have been known to affect the stem cell function, yet very little is known about the interplay between how these cues control differentiation. For the first time, by using photolithography to pattern poly(ethylene glycol) (PEG), patterns of square and triangular geometries were created, and the effect of these structures and the biophysical cues arising were utilized to differentiate cells into multiple lineages inside a same pattern without the use of any adhered protein or growth factors. The data from these studies showed that the cells present at the edges were well elongated, exhibit high aspect ratios, and differentiated into osteogenic lineage, whereas the cells present at the center exhibit lower aspect ratio and were primarily adipogenic lineage regardless of the geometry. This was correlated to the higher expression of focal adhesion proteins at the edges, the expression of which have been known to affect the osteogenic differentiation. By showing MSC lineage commitment relationships due to physical signals, this study highlights the importance of these cues and cell shape in further understanding stem cell behavior for tissue engineering applications.

Focal adhesion proteins in hepatocellular carcinoma: RSU1 a novel tumour suppressor with prognostic significance

AimHepatocellular carcinoma (HCC) is a common cause a cancer-related death. Focal adhesions (FAs) represent multiprotein complexes at integrin-mediated cell-extracellular matrix adhesion sites that orchestrate vital cellular functions. The heterotrimeric ILK-PINCH-PARVB (IPP) complex, RSU1, a PINCH binding protein and CTEN, a member of the tensin family of proteins exert a critical role in FAs, where they regulate important cancer related functions such as cell adhesion, migration, proliferation and survival. Previous studies implicate these FA proteins in liver pathophysiology but their detailed role in human HCC is not fully understood. Here in we investigated expression and function of IPP, RSU1 and CTEN in human HCC. MethodsThe expression of focal adhesion proteins was studied in human HCC by immunohistochemistry in relation to clinicopathological parameters, previous studied genomic instability markers and patient’s survival. Effects on cell proliferation and FA proteins expression upon ILK inhibition and RSU1 silencing were also investigated in HCC in vitro. ResultsIPP complex and CTEN proteins are overexpressed while RSU1 expression is decreased in human HCC. CTEN expression correlates with reduced patients’ survival while RSU1 represents an independent favorable prognostic indicator in human HCC. Nuclear ILK expression correlates with markers of genomic instability. Pharmacological targeting of ILK suppresses, while RSU1 silencing promotes cell growth of HCC cells in vitro, while in both experimental conditions expression and/or localization of focal adhesion proteins is deregulated. ConclusionOur results suggest that FA signaling is implicated in hepatocellular carcinogenesis with prognostic significance. RSU1 seems to exert tumor suppressive functions in HCC and represents a novel favorable prognostic indicator.

Understanding the cellular response of human tenon fibroblast on polycaprolactone-Aloe vera blend fiber.

The signalling response is determined by the cell's reaction to different biochemical and biophysical inputs such as stiffness, topological, and structural alignment. The surface patterns at the nano-scale can be an influential factor in cell signalling behaviour. It is important to understand the cellular response to the biophysical cues for biomedical applications. Biomaterials have an important role in regenerative tissue engineering. In this study, we have fabricated electrospun polycaprolactone (PCL) and PCL-Aloe vera (PCL-AV) nanofibrous matrix and studied its effect on the human tenon fibroblast (HTF) cellular and morphological changes. The electrospun fibers were characterized using Scanning Electron Microscope (SEM), Fourier Transform Infrared spectroscopy (FTIR), Atomic Force Microscopy (AFM) and Brunaur, Emette and Teller (BET) analysis for their morphology, composition, topography, surface area and porosity. The results revealed fiber size, roughness and porosity has been altered by addition of AV. The HTF cell viability, proliferation and expression of focal adhesion proteins, such as FAK, Ezrin, Vasp and Cofilin on the PCL-AV fiber matrix were examined. The results showed a change in cellular morphology and a significant change in the cofilin phosphorylation on PCL-AV nanofiber. The influence of Aloe vera composition on the nano-dimension of the PCL has made a significant impact on the cellular morphology at both gene and protein levels. This observation suggests that AV composition in the nanofiber can significantly influence the HTF cellular adhesions.

Mechanistic Insight in Surface Nanotopography Driven Cellular Migration.

Cellular migration plays a vital role in many physiological processes. To elucidate the role of surface nanotopography on the downstream signaling pathways underlying cell migration, model surfaces having well-defined hill-like surface nanotopography and uniform surface chemistry were designed and implemented using plasma polymerization and covalent attachment of nanoparticles of predetermined size. A scratch wound assay, immunostaining, and gene expression of focal adhesion (FA) proteins were performed to determine the influence of surface nanotopography on cell migration. The results of this study demonstrate that the gap closure between cell monolayers is faster on surfaces having greater nanoscale topography. The phenomenon is predominantly driven by cell migration and was independent from cell proliferation. Qualitative and quantitative assessment of proteins involved in the signaling pathways underlying cell migration showed significant modulation by surface nanotopography. Specifically, focal adhesion sites decreased with the increase in surface nanotopography scale while the expression of FA proteins increased. This implies that nanotopography mediated modulation of cell migration is directly governed by the recruitment of receptor and adapter proteins responsible for cell-surface interaction. The results of this study indicate that biomaterial devices and constructs having rationally designed surface nanotopography and chemistry could be utilized to regulate wound healing and tissue regeneration.

Enhancing Extracellular Adenosine Levels Restores Barrier Function in Acute Lung Injury Through Expression of Focal Adhesion Proteins.

Background: Acute respiratory distress syndrome (ARDS) is a clinical presentation of acute lung injury (ALI) with often fatal lung complication. Adenosine, a nucleoside generated following cellular stress provides protective effects in acute injury. The levels of extracellular adenosine can be depleted by equilibrative nucleoside transporters (ENTs). ENT inhibition by pharmaceutical agent dipyridamole promotes extracellular adenosine accumulation and is protective in ARDS. However, the therapeutic potential of dipyridamole in acute lung injury has not yet been evaluated. Methods: Adenosine acts on three adenosine receptors, the adenosine A1 (Adora1), A2a (Adora2a), the A2b (Adora2b) or the adenosine A3 (Adora 3) receptor. Accumulation of adenosine is usually required to stimulate the low-affinity Adora2b receptor. In order to investigate the effect of adenosine accumulation and the contribution of epithelial-specific ENT2 or adora2b expression in experimental ALI, dipyridamole, and epithelial specific ENT2 or Adora2b deficient mice were utilized. MLE12 cells were used to probe downstream Adora2b signaling. Adenosine receptors, transporters, and targets were determined in ARDS lungs. Results: ENT2 is mainly expressed in alveolar epithelial cells and is negatively regulated by hypoxia following tissue injury. Enhancing adenosine levels with ENT1/ENT2 inhibitor dipyridamole at a time when bleomycin-induced ALI was present, reduced further injury. Mice pretreated with the ADORA2B agonist BAY 60-6583 were protected from bleomycin-induced ALI by reducing vascular leakage (558.6 ± 50.4 vs. 379.9 ± 70.4, p < 0.05), total bronchoalveolar lavage fluid cell numbers (17.9 ± 1.8 to 13.4 ± 1.4 e4, p < 0.05), and neutrophil infiltration (6.42 ± 0.25 vs. 3.94 ± 0.29, p < 0.05). While mice lacking Adora2b in AECs were no longer protected by dipyridamole. We also identified occludin and focal adhesion kinase as downstream targets of ADORA2B, thus providing a novel mechanism for adenosine-mediated barrier protection. Similarly, we also observed similar enhanced ADORA2B (3.33 ± 0.67 to 16.12 ± 5.89, p < 0.05) and decreased occludin (81.2 ± 0.3 to 13.3 ± 0.4, p < 0.05) levels in human Acute respiratory distress syndrome lungs. Conclusion: We have highlighted a role of dipyridamole and adenosine signaling in preventing or treating ALI and identified Ent2 and Adora2b as key mediators in important for the resolution of ALI.

Multiscale Analysis of Extracellular Matrix Remodeling in the Failing Heart.

Cardiac ECM (extracellular matrix) comprises a dynamic molecular network providing structural support to heart tissue function. Understanding the impact of ECM remodeling on cardiac cells during heart failure (HF) is essential to prevent adverse ventricular remodeling and restore organ functionality in affected patients. We aimed to (1) identify consistent modifications to cardiac ECM structure and mechanics that contribute to HF and (2) determine the underlying molecular mechanisms. We first performed decellularization of human and murine ECM (decellularized ECM) and then analyzed the pathological changes occurring in decellularized ECM during HF by atomic force microscopy, 2-photon microscopy, high-resolution 3-dimensional image analysis, and computational fluid dynamics simulation. We then performed molecular and functional assays in patient-derived cardiac fibroblasts based on YAP (yes-associated protein)-transcriptional enhanced associate domain (TEAD) mechanosensing activity and collagen contraction assays. The analysis of HF decellularized ECM resulting from ischemic or dilated cardiomyopathy, as well as from mouse infarcted tissue, identified a common pattern of modifications in their 3-dimensional topography. As compared with healthy heart, HF ECM exhibited aligned, flat, and compact fiber bundles, with reduced elasticity and organizational complexity. At the molecular level, RNA sequencing of HF cardiac fibroblasts highlighted the overrepresentation of dysregulated genes involved in ECM organization, or being connected to TGFβ1 (transforming growth factor β1), interleukin-1, TNF-α, and BDNF signaling pathways. Functional tests performed on HF cardiac fibroblasts pointed at mechanosensor YAP as a key player in ECM remodeling in the diseased heart via transcriptional activation of focal adhesion assembly. Finally, in vitro experiments clarified pathological cardiac ECM prevents cell homing, thus providing further hints to identify a possible window of action for cell therapy in cardiac diseases. Our multiparametric approach has highlighted repercussions of ECM remodeling on cell homing, cardiac fibroblast activation, and focal adhesion protein expression via hyperactivated YAP signaling during HF.

Calpain inhibition decreases myocardial fibrosis in chronically ischemic hypercholesterolemic swine

ObjectivesCalpain activation during ischemia is known to play critical roles in myocardial remodeling. We hypothesize that calpain inhibition (CI) may serve to reverse and/or prevent fibrosis in chronically ischemic myocardium. MethodsYorkshire swine were fed a high-cholesterol diet for 4 weeks followed by placement of an ameroid constrictor on the left circumflex artery to induce myocardial ischemia. 3 weeks later, animals received either: no drug; high-cholesterol control group (CON; n = 8); low-dose CI (0.12 mg/kg; LCI, n = 9); or high-dose CI (0.25 mg/kg; HCI, n = 8). The high-cholesterol diet and CI were continued for 5 weeks, after which myocardial tissue was harvested. Tissue samples were analyzed by western blot for changes in protein content. ResultsIn the setting of hypercholesterolemia and chronic myocardial ischemia, CI decreased the expression of collagen in ischemic and nonischemic myocardial tissue. This reduced collagen content was associated with a corresponding decrease in Jak/STAT/MCP-1 signaling pathway, suggesting a role for Jak 2 signaling in calpain activity. CI also decreases the expression of focal adhesion proteins (vinculin) and stabilizes the expression of cytoskeletal and structural proteins (N-cadherin, α-fodrin, desmin, vimentin, filamin, troponin-I). CI had no significant effect on metabolic and hemodynamic parameters. ConclusionsCalpain inhibition may be a beneficial medical therapy to decrease collagen formation in patients with coronary artery disease and associated comorbidities.

Tuning of Titanium Microfiber Scaffold with UV-Photofunctionalization for Enhanced Osteoblast Affinity and Function.

Titanium (Ti) is an osteoconductive material that is routinely used as a bulk implant to fix and restore bones and teeth. This study explored the effective use of Ti as a bone engineering scaffold. Challenges to overcome were: (1) difficult liquid/cell infiltration into Ti microfiber scaffolds due to the hydrophobic nature of Ti; and (2) difficult cell attachment on thin and curved Ti microfibers. A recent discovery of UV-photofunctionalization of Ti prompted us to examine its effect on Ti microfiber scaffolds. Scaffolds in disk form were made by weaving grade 4 pure Ti microfibers (125 µm diameter) and half of them were acid-etched to roughen the surface. Some of the scaffolds with original or acid-etched surfaces were further treated by UV light before cell culture. Ti microfiber scaffolds, regardless of the surface type, were hydrophobic and did not allow glycerol/water liquid to infiltrate, whereas, after UV treatment, the scaffolds became hydrophilic and immediately absorbed the liquid. Osteogenic cells from two different origins, derived from the femoral and mandibular bone marrow of rats, were cultured on the scaffolds. The number of cells attached to scaffolds during the early stage of culture within 24 h was 3–10 times greater when the scaffolds were treated with UV. The development of cytoplasmic projections and cytoskeletal, as well as the expression of focal adhesion protein, were exclusively observed on UV-treated scaffolds. Osteoblastic functional phenotypes, such as alkaline phosphatase activity and calcium mineralization, were 2–15 times greater on UV-treated scaffolds, with more pronounced enhancement on acid-etched scaffolds compared to that on the original scaffolds. These effects of UV treatment were associated with a significant reduction in atomic carbon on the Ti microfiber surfaces. In conclusion, UV treatment of Ti microfiber scaffolds tunes their physicochemical properties and effectively enhances the attachment and function of osteoblasts, proposing a new strategy for bone engineering.

Controlling the Multiscale Network Structure of Fibers To Stimulate Wound Matrix Rebuilding by Fibroblast Differentiation.

The extracellular matrix (ECM) plays the role of a double-edged sword for controlling the differentiation of fibroblasts toward contractile myofibroblasts in the wound healing process. However, the exact structure-function relationship between ECM morphology and fibroblast behaviors still remains unclear. To better understand this relationship, herein, we designed and prepared a series of biocompatible polycaprolactone (PCL)-based fibers with different fiber diameters (nano vs micro) and different alignments (random vs aligned) using a simple electrospinning process, with a particular attention to the morphological effect of PCL fiber scaffolds on guiding fibroblast behaviors. Microfibers with the larger fiber diameters induce less cell spreading, adhesion, differentiation, and migration because of their lower surface tension. In contrast, nanofibers will retain fibroblast cells with typical spindle shapes and promote the expression of focal adhesion proteins through the integrin pathway. Furthermore, nanofibers upregulate the expression of α-smooth muscle actin (α-SMA), transforming growth factor, and vimentin filaments, indicating that the size change of the PCL fiber matrix from micrometers to nanometers indeed alters fibroblast differentiation to activate more α-SMA-expressed contractile myofibroblasts. Such a fiber size-dependent fibroblast behavior is largely attributed to the enhanced surface tension from the dressing matrix, which helps to promote the conversion of fibroblasts to myofibroblasts via either tissue regeneration or fibrosis. Therefore, this work further indicated that the rearrangement of collagen from nano-tropocollagen to micro-collagen bundles during the wound healing process can reverse fibroblasts to myofibroblasts from motivated to demise. This finding allows us to achieve the structural-based design of a new fibrous matrix for promoting wound healing and tissue regeneration.

Nesprin-2G knockout fibroblasts exhibit reduced migration, changes in focal adhesion composition, and reduced ability to generate traction forces.

The nuclear envelope protein nesprin-2G is a component of the linker of nucleoskeleton and cytoskeleton (LINC) complex and is responsible for mechanical and signaling crosstalk between the nucleus and cytoskeleton. A prior study has demonstrated that nesprin-2G knockout mice show delayed wound healing. The goal was to elucidate the mechanism underlying the delayed wound closure in this mouse model. Primary fibroblasts from wild-type and knockout neonatal mice were isolated. Knockout cells exhibited decreased focal adhesion (FA) size, number, and intensity. Consistent with this result, FA protein expression levels were decreased in knockout cells. Additionally, knockout fibroblasts displayed an abnormal actin cytoskeleton, as evidenced by loss of TAN line formation and both cytoplasmic and peri-nuclear actin staining. Using collective and single cell motility assays, it was found that knockout cells exhibited a reduction in both speed and directed migration. Traction force microscopy revealed that knockout fibroblasts generated fewer traction forces compared with WT fibroblasts. In summary, the data indicated that changes in actin organization and defects in FAs result in a reduced ability of knockout fibroblasts to generate traction forces needed for efficient motility.

Mussel-Inspired Polydopamine Coating: A General Strategy To Enhance Osteogenic Differentiation and Osseointegration for Diverse Implants.

Surface modifications play an important role in endowing implant surface with excellent biocompatibility and bioactivity. Among the bioinspired surface modifications, the mussel-inspired polydopamine (PDA) has aroused great interest of researchers. Herein, we fabricated PDA on diverse implant surfaces, including biopolymer, biometal, and bioceramic. Then the effects of PDA coating on cell responsive behaviors in vitro and bone formation capacity in vivo were evaluated in detail. The results showed that PDA coating was fabricated on diverse samples surface successfully, which could significantly improve the hydrophilicity of different material surfaces. Furthermore, the results indicated that PDA coating exerted direct enhancing on the adhesion, proliferation and osteogenic differentiation of bone marrow derived mesenchymal stromal cells (BMSCs) through FAK and p38 signaling pathways. During the process, the focal adhesion protein expression and osteogenic-related genes expression level (e.g., ALP, BMP2, BSP, and OPN) were considerably upregulated. Most importantly, the in vivo study confirmed that PDA coating remarkably accelerated new bone formation and enhanced osseointegration performance. Our study uncovered the biological responses stimulated by PDA coating to make a better understanding of cell/tissue-PDA interactions and affirmed that PDA, a bioinspired polymer, has great potential as a candidate and functional bioactive coating medium in bone regeneration and orthopedic application.

Claudin-7 modulates cell-matrix adhesion that controls cell migration, invasion and attachment of human HCC827 lung cancer cells.

Claudins are a family of tight junction proteins, and serve important roles in epithelial barrier, selective ion transports and cancer metastasis. Although the exact role of claudin-7 in human lung cancer has not been completely elucidated, recent clinical studies have demonstrated that claudin-7 is associated with the survival of patients with lung cancer. Our previous studies have demonstrated that claudin-7 forms a protein complex with integrin β1 in human lung cancer cells. The knockdown (KD) of claudin-7 by short hairpin RNA (shRNA) reduced integrin β1 expression and increased the cell proliferative rate, whereas claudin-7 re-expression in the KD cells decreased the cell proliferation. It is unknown as to whether claudin-7 and integrin β1 regulate cell proliferation and invasion synergistically or independently. In the present study, it was observed that ectopic expression of integrin β1 in claudin-7 KD lung cancer cells did not reduce the cell proliferation. However, integrin β1-transfected cells migrated more effectively in wound healing and cell invasion assays and were more adhesive in a cell attachment assay when compared with those of claudin-7 KD cells. This indicates that claudin-7 controls cell proliferation, while cell attachment and motility were regulated partially through integrin β1. Additionally, claudin-7 overexpression in claudin-7 KD cells resulted in an improved ability to attach to the surface of cell culture plates and a higher expression of focal adhesion proteins when compared with claudin-7 non-KD control cells, which supports the role of claudin-7 in cell adhesion and motility. Taken together, these data suggest that claudin-7 regulates cell motility through integrin β1, providing additional insight into the roles of claudins in carcinogenesis and cancer cell metastasis.

1461 Nesprin-2G regulates focal adhesion protein expression and motility of fibroblasts

1461 Nesprin-2G regulates focal adhesion protein expression and motility of fibroblasts

Investigation of neuronal pathfinding and construction of artificial neuronal networks on 3D-arranged porous fibrillar scaffolds with controlled geometry

Herein, we investigated the neurite pathfinding on electrospun microfibers with various fiber densities, diameters, and microbead islands, and demonstrated the development of 3D connected artificial neuronal network within a nanofiber-microbead-based porous scaffold. The primary culture of rat hippocampal embryonic neurons was deposited on geometry-controlled polystyrene (PS) fiber scaffolds while growth cone morphology, neurite outgrowth patterns, and focal adhesion protein expression were cautiously examined by microscopic imaging of immunostained and live neuronal cells derived from actin-GFP transgenic mice. It was demonstrated that the neurite outgrowth was guided by the overall microfiber orientation, but the increase in fiber density induced the neurite path alteration, thus, the reduction in neurite linearity. Indeed, we experimentally confirmed that growth cone could migrate to a neighboring, but, spatially disconnected microfiber by spontaneous filopodium extrusion, which is possibly responsible for the observed neurite steering. Furthermore, thinner microfiber scaffolds showed more pronounced expression of focal adhesion proteins than thicker ones, suggesting that the neuron-microfiber interaction can be delicately modulated by the underlying microfiber geometry. Finally, 3D connected functional neuronal networks were successfully constructed using PS nanofiber-microbead scaffolds where enhanced porosity and vertical fiber orientation permitted cell body inclusion within the scaffold and substantial neurite outgrowth in a vertical direction, respectively.

Talin Modulation by a Synthetic N-Acylurea Derivative Reduces Angiogenesis in Human Endothelial Cells.

Talin is a focal adhesion protein that activates integrins and recruits other focal adhesion proteins. Talin regulates the interactions between integrins and the extracellular matrix, which are critical for endothelial cells during angiogenesis. In this study, we successfully synthesized a novel talin modulator, N-((2-(1H-indol-3-yl)ethyl)carbamoyl)-2-(benzo[d][1,3]dioxol-5-yloxy)acetamide, referred to as KCH-1521. KCH-1521 was determined to bind talin and modulate downstream signaling molecules of talin. After 24 h of treatment, KCH-1521 changed the cell morphology of human umbilical vein endothelial cells (HUVECs) and reduced focal adhesion protein expression including vinculin and paxillin. Talin downstream signaling is regulated via focal adhesion kinase (FAK), kinase B (AKT), and extracellular signal-regulated kinase (ERK) pathways, however, treatment with KCH-1521 decreased phosphorylation of FAK, AKT, and ERK, leading to reduction of cell proliferation, survival, and angiogenesis. Interestingly, the expression of various angiogenic genes was significantly decreased after treatment with KCH-1521. Also, in vitro tube forming assay revealed that KCH-1521 reduced angiogenic networks in a time-dependent manner. To investigate the reversibility of its effects, KCH-1521 was removed after treatment. HUVECs recovered their morphology through rearrangement of the cytoskeleton and the expression of angiogenic genes was also recovered. By further optimization and in vivo studies of KCH-1521, a novel drug of talin modulation could be used to achieve therapeutic anti-angiogenesis for vascular diseases and cancers.

Cordycepin inhibits migration of human glioblastoma cells by affecting lysosomal degradation and protein phosphatase activation

Cordycepin, a nucleoside-derivative-isolated form Cordyceps militaris, has been reported to suppress tumor cell proliferation and cause apoptosis. This study investigates the effect of cordycepin on the migration of human glioblastoma cells. Cordycepin suppressed the migration of the human glioblastoma cell lines U87MG and LN229 in transwell and wound healing assays. Cordycepin decreased protein expression of integrin α1, focal adhesion kinase (FAK), p-FAK, paxillin and p-paxillin. The lysosomal inhibitor NH4Cl blocked the ability of cordycepin to inhibit focal adhesion protein expression and glioma cell migration. In addition, the protein phosphatase inhibitors calyculin A and okadaic acid blocked the cordycepin-mediated reduction in p-Akt, p-FAK and migration. Hematoxylin and eosin staining of mouse xenografts demonstrated that cordycepin reduced brain tumor size in vivo. In conclusion, cordycepin inhibited migration of human glioblastoma cells by affecting lysosomal degradation and protein phosphatase activation. This pathway may be a useful target for clinical therapy in the future.

Nanotopographical cell stimulation by the electrodeposited carbon nanotubes-based surfaces

Event Abstract Back to Event Nanotopographical cell stimulation by the electrodeposited carbon nanotubes-based surfaces Chinmaya Mahapatra1, 2, Kapil Dev Patel1, 2, Banani D. Kundu1, 2, Rajendra D. Kumar Singh1, 2, Roman A. Perez1, 2 and Hae D. Won- Kim1, 2, 3 1 Dankook University, Nanobiomedical Science, Korea 2 Dankook University, Institute of Tissue Regeneration Engineering (ITREN), Korea 3 Dankook University, Department of Biomaterials Science, College of Dentistry, Korea Introduction: Nano-topological bio-interfaces of metallic implants have been a critical feature in modulating cellular responses and achieving bio-functional success. The electrophoretic deposition (EPD) is a simple process where a wide range of coating materials can be usedwith tunable composition and thickness in a time and cost effective manner. In present study, we produced carbon nanotube (CNT) /chitosan (Chi) hybrid surfaces on titanium plate using electrophoretic deposition (EPD) technique. Materials and Methods: Acid treated CNTs were dispersed in chitosan(0.125-0.5mg/mL). The CNT-chitosansolutions were electrodeposited on titanium substrate under DC field. The coatings were characterized by TGA, ATR-FTIR and XRD. Stability and bioactivity of the coatings were performed by incubation in PBS and simulated body fluid, respectively. The potential of the coatings to load and deliver biomolecules was studied usinga model protein BSA.Murine pre-osteoblastic cells were cultured on the surface to investigate the effects of nano-topographic surfaces on the cell adhesion events. Results and Discussion: Depending on the electrophoretic deposition parameters, a wide range of CNT-chitosan compositions with varying thickness were successfully produced. The coatings presented good stability in a saline solution and a fast deposition of an apatitemineral phase when immersed in a simulated body fluid. Due to the increased surface area of the nanostructured surfaces as well as the higher electrostatic charges of the composition, the adsorption of proteins was substantially enhanced on the CNT-chitosan surfaces. The CNT-chitosan surfaces allowed for a sustained release of BSA molecules over two weeks through a diffusion controlled mechanism. The nanostructuredsurfaces up-regulated a series of cell adhesion events, includingcell anchorage, spreading, and the expression of focal adhesion proteins. Conclusion: The CNT-basednanostructured hybrid surfaces stimulated the initial cellular activity through the nano-topological features, andenhancedthe loadingand delivery capacity of biomolecules. The nanostructures can be considered as a possible interfacethat mediates the interactions of metallic implants with biomolecules and cells. This work was supported by the grants from the National Research Foundation, Republic of Korea; Priority Research Centers Program (No. 2009-0093829) and Global Research Lab Program (2015032163).