Dependent Adhesion Research Articles

Abstract 3928: N-cadherin(CDH2) expression in mesenchymal glioblastoma modulates radiation sensitivity

Abstract Background: Radiation is a mainstay of treatment in patients diagnosed with glioblastoma. Despite maximal surgical and chemo-radiotherapeutic treatment, the disease remains uniformly fatal, with local disease recurrence within the previously radiated field comprising the major pattern of progression. According to the molecular subtyping of GBM, there is a predominance of mesenchymal GBM cells within the recurrent tumor mass, with characteristic increased expression of CD44, N-cadherin, L1CAM, TWIST1 and phosphorylated STAT3. Moreover, these populations of cells are known to be resistant to additional salvage radiation. The current study aims to evaluate the role of N-cadherin in the relative radiation resistance of mesenchymal subtype GBM. Methods: Overall survival data from the TCGA was analyzed using Cox regression hazard ratios based on N-cadherin expression across multiple molecular subtypes of GBM along with age and MGMT covariates. Using the U3035 and U87MG cells, the analyses of N-cadherin expression or exogenous exposure in the setting of external beam radiation were performed via stable shRNA knockdowns or the use of recombinant human N-cadherin ectodomain-Fc fusion proteins in clonogenic survival assays. Orthotopic implantations were performed in NSG mice with the endpoints of overall survival and tumor size, as determined by luciferase imaging. Subcellular localization of RAD51 was accomplished by confocal microscopy. Statistical analyses were performed using SPSS and GraphPad Prism with significance at p<0.05. Results: N-cadherin expression above the median among the mesenchymal subtype of GBM in the TCGA dataset correlated with a significant decrease in overall survival (HR 1.924, p=0.029). The elimination of N-cadherin expression in normally high expressing U3035 GBM cells led to a significant increase in the ability of radiation to induce cell death in clonogenic assay (17.2% U3035 vs 2% U3035 Δ N-cadherin at 8Gy; 12.7% U3035 vs 1.75% U3035 Δ N-cadherin at 20Gy) and an increase in the presence of double stranded DNA breaks (DSBs) on TUNEL assay. N-cadherin ectodomain-Fc fusion protein exposure in U87 cells resulted in a decrease in NSG mouse mean OS (17.6±0.25 days) compared to control radiated mice (22.3±1.1 days). Conversely, N-cadherin knockdown in U3035 cells resulted in further decrease in tumor bioluminescence beyond that seen in 4Gy x 4 radiation treatment of native U3035 xenografts (21% vs 4% of baseline). Conclusions: The reduction of N-cadherin expression in mesenchymal U3035 GBM cells increases the efficacy of radiation-induced cell death. This finding is accompanied by a decrease in the nuclear localization of RAD51 following DSBs induced by ionizing radiation, indicating a potential crosstalk between DNA damage repair mechanisms and calcium dependent cell-cell adhesion. Citation Format: Christopher P. Cifarelli, J. Austin Vargo, Ian MacFawn, Steven M. Frisch. N-cadherin(CDH2) expression in mesenchymal glioblastoma modulates radiation sensitivity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3928.

Phosphorylated lipid-conjugated oligonucleotide selectively anchors on cell membranes with high alkaline phosphatase expression

Attachment of lipid tails to oligonucleotides has emerged as a powerful technology in constructing cell membrane-anchorable nucleic acid-based probes. In practice, however, conventional lipid-conjugated oligonucleotides fail to distinguish among different cell membranes. Herein, a phosphorylated lipid-conjugated oligonucleotide (DNA-lipid-P) is reported for alkaline phosphatase (ALP)-dependent cell membrane adhesion. In the absence of ALP, DNA-lipid-P with its poor hydrophobicity shows only weak interaction with cell membrane. However, in the presence of the highly expressed plasma membrane-associated ALP, DNA-lipid-P is converted to lipid-conjugated oligonucleotide (DNA-lipid) by enzymatic dephosphorylation. As a result of such conversion, the generated DNA-lipid has greater hydrophobicity than DNA-lipid-P and is thus able to insert into cell membranes in situ. Accordingly, DNA-lipid-P enables selective anchoring on cell membranes with elevated ALP level. Since elevated ALP level is a critical index of some diseases and even cancers, DNA-lipid-P holds promise for cell membrane engineering and disease diagnostics at the molecular level.

Myo1f, an Unconventional Long-Tailed Myosin, Is a New Partner for the Adaptor 3BP2 Involved in Mast Cell Migration.

Mast cell chemotaxis is essential for cell recruitment to target tissues, where these cells play an important role in adaptive and innate immunity. Stem cell factor (SCF) is a major chemoattractant for mast cells. SCF binds to the KIT receptor, thereby triggering tyrosine phosphorylation in the cytoplasmic domain and resulting in docking sites for SH2 domain-containing molecules, such as Lyn and Fyn, and the subsequent activation of the small GTPases Rac that are responsible for cytoskeletal reorganization and mast cell migration. In previous works we have reported the role of 3BP2, an adaptor molecule, in mast cells. 3BP2 silencing reduces FcεRI-dependent degranulation, by targeting Lyn and Syk phosphorylation, as well as SCF-dependent cell survival. This study examines its role in SCF-dependent migration and reveals that 3BP2 silencing in human mast cell line (LAD2) impairs cell migration due to SCF and IgE. In that context we found that 3BP2 silencing decreases Rac-2 and Cdc42 GTPase activity. Furthermore, we identified Myo1f, an unconventional type-I myosin, as a new partner for 3BP2. This protein, whose functions have been described as critical for neutrophil migration, remained elusive in mast cells. Myo1f is expressed in mast cells and colocalizes with cortical actin ring. Interestingly, Myo1f-3BP2 interaction is modulated by KIT signaling. Moreover, SCF dependent adhesion and migration through fibronectin is decreased after Myo1f silencing. Furthermore, Myo1f silencing leads to downregulation of β1 and β7 integrins on the mast cell membrane. Overall, Myo1f is a new 3BP2 ligand that connects the adaptor to actin cytoskeleton and both molecules are involved in SCF dependent mast cell migration.

Subcellular localization of mutated β-catenins with different incidences of cis-peptide bonds at the Xaa246-P247 site in HepG2 cells.

Mutations may ultimately change the local conformation of proteins; however, little attention has been paid to alterations in protein function caused by the incidence of cis-peptide bonds (ICPB) in mammalian cells. In this study, a statistical approach, coimmunoprecipitation, and immunofluorescence staining have been used to confirm that S246→Y and S246→W missense mutations, which help increase the ICPB in Xaa246-P247 (Xaa is any amino acid) in human β-catenin, can reduce the interactions between β-catenin and adenomatous polyposis coli (APC) and between β-catenin and Ca2+-dependent cell adhesion molecule family in epithelial tissue (E-cadherin), eventually leading to increased nuclear migration of β-catenin in the HepG2 cell line (an immortalized cell line consisting of human liver carcinoma cells). Conversely, S246→L and S246→M missense mutations, which reduce the ICPB in Xaa246-P247 in human β-catenin, can enhance interactions between β-catenin and APC and between β-catenin and E-cadherin, leading to decreased nuclear migration of β-catenin. These results not only indicate that a change in the ICPB may be an important cause of functional protein changes but also provide a new basis for the study of genetic disease prediction, gene diagnosis, individualized treatment, and protein modification at the gene level for clinicians and other professionals.—Yu, S., Zhang, Y., Wu, Y., Yang, H., Chen, Y., Yang, Y., Zhang, Z. Subcellular localization of mutated β-catenins with different incidences of cis-peptide bonds at the Xaa246-P247 site in HepG2 cells.

Correlation of in vitro cell adhesion, local shear flow and cell density.

Investigating cell adhesion behavior on biocompatible surfaces under dynamic flow conditions is not only of scientific interest but also a principal step towards development of new medical implant materials. Driven by the improvement of the measurement technique for microfluidic flow fields (scanning particle image velocimetry, sPIV), a semi-automatic correlation of the local shear velocity and the cell detachment probability became possible. The functionality of customized software entitled ‘PIVDAC’ (Particle Image Velocimetry De-Adhesion Correlation) is demonstrated on the basis of detachment measurements using standard sand-blasted titanium implant material. A thermodynamic rate model is applied to describe the process of cell adhesion and detachment. A comparison of the model and our experimental findings, especially in a mild regime, where the shear flow does not simply tear away all cells from the substrate, demonstrates, as predicted, an increase of detachment rate with increasing shear force. Finally, we apply the method to compare experimentally obtained detachment rates under identical flow conditions as a function of cell density and find excellent agreement with previously reported model simulations that consider pure geometrical effects. The demonstrated method opens a wide field of applications to study various cell lines on novel substrates or in time dependent flow fields.

Environment‐Dependent Adhesion Energy of Mica Nanolayers Determined by a Nanomanipulation‐Based Bridging Method

AbstractMica nanolayers have great potential for applications in the future flexible electronics due to their excellent flexibility and dielectric properties. Characterization of the adhesion behaviors of mica nanolayers is thus essential, as they would substantially affect the reliability of the electronic devices. In this work, a new delamination method is developed based on the optical microscopic nanomanipulation for measuring the adhesion energy of mica nanolayers. In particular, the tests are performed under different environmental conditions with relative humidity varying from 10% to 90% and temperature ranging from 25 to 300 °C. The average surface energy of mica nanolayers is 221.5 ± 25.4 mJ m−2 measured at the room temperature of 25 °C and decreases with the increase in relative humidity or temperature. The finding of the environmentally dependent adhesion is of importance for the design of flexible electronics products based on mica nanolayers.

Propiedades Adhesivas Húmedas de la Proteína del Pie de Mejillones Verdes Asiático (Perna viridis) Pvfp-5: Una Base Adhesiva Subacuática

Los mejillones verdes asiáticos (Perna viridis) son bivalvos que se adhieren firmemente a las rocas en las costas intermareales golpeadas por las olas a través de una secreción proteica. Los mejillones de P. viridis siguen una secreción de proteínas adhesivas regulada en el tiempo, donde la proteína del pie-5 de P. viridis (Pvfp-5) fue identificada como la primera proteína que se secreta durante la formación de la placa adhesiva. El alto contenido de aminoácido catecólico 3,4-dihidroxi-L-fenilalanina (DOPA) (~11% en moles) y cisteína (CYS) (~15% en moles) en Pvfp-5 y su localización cerca de la interfaz placa-sustrato provocó la especulación de que la proteína de vanguardia Pvfp-5 desempeña un papel clave como imprimación adhesiva en la adhesión bajo el agua de los mejillones de P. viridis. El aparato de fuerza de superficie (SFA) se usó para probar las propiedades adhesivas de Pvfp-5 a escala nanométrica, donde se investigó la adhesión húmeda dependiente del pH y la actividad antioxidante de Pvfp-5 purificado y extraído. El estudio reveló que Pvfp-5 con su alto contenido de DOPA y CYS mantiene la adhesión incluso a un pH más alto al superar la oxidación espontánea de DOPA a quinona. Los resultados de SFA son consistentes con la función aparente de que Pvfp-5 actúa como imprimación adhesiva, superando las fuerzas de hidratación repulsivas al desplazar el agua unida a la superficie y generando una adhesión superficial fuerte y duradera. Nuestros hallazgos revelan conocimientos a escala molecular que deberían ser relevantes e impactar en las ciencias de los materiales al desarrollo de la nueva generación de adhesivos, recubrimientos y pegamentos resistentes a la humedad para aplicaciones biomédicas, terapéuticas y antiincrustantes.

Structural Immunology of Complement Receptors 3 and 4.

Complement receptors (CR) 3 and 4 belong to the family of beta-2 (CD18) integrins. CR3 and CR4 are often co-expressed in the myeloid subsets of leukocytes, but they are also found in NK cells and activated T and B lymphocytes. The heterodimeric ectodomain undergoes considerable conformational change in order to switch the receptor from a structurally bent, ligand-binding in-active state into an extended, ligand-binding active state. CR3 binds the C3d fragment of C3 in a way permitting CR2 also to bind concomitantly. This enables a hand-over of complement-opsonized antigens from the cell surface of CR3-expressing macrophages to the CR2-expressing B lymphocytes, in consequence acting as an antigen presentation mechanism. As a more enigmatic part of their functions, both CR3 and CR4 bind several structurally unrelated proteins, engineered peptides, and glycosaminoglycans. No consensus motif in the proteinaceous ligands has been established. Yet, the experimental evidence clearly suggest that the ligands are primarily, if not entirely, recognized by a single site within the receptors, namely the metal-ion dependent adhesion site (MIDAS). Comparison of some recent identified ligands points to CR3 as inclined to bind positively charged species, while CR4, by contrast, binds strongly negative-charged species, in both cases with the critical involvement of deprotonated, acidic groups as ligands for the Mg2+ ion in the MIDAS. These properties place CR3 and CR4 firmly within the realm of modern molecular medicine in several ways. The expression of CR3 and CR4 in NK cells was recently demonstrated to enable complement-dependent cell cytotoxicity toward antibody-coated cancer cells as part of biological therapy, constituting a significant part of the efficacy of such treatment. With the flexible principles of ligand recognition, it is also possible to propose a response of CR3 and CR4 to existing medicines thereby opening a possibility of drug repurposing to influence the function of these receptors. Here, from advances in the structural and cellular immunology of CR3 and CR4, we review insights on their biochemistry and functions in the immune system.

Improvement of thermal stability, adhesion strength and corrosion performance of diamond-like carbon films with titanium doping

Ti-doped diamond-like carbon (Ti-doped DLC) films were deposited on 304 stainless steel through a pulsed filtered cathodic vacuum arc deposition with an individual cathodic arc source. Structural dependent thermal stability, mechanical properties, adhesion, and corrosion performance were thoroughly investigated as a function of the Ti content. Only 0.8 at.% Ti content in the DLC films offers the relatively high hardness (28.8 GPa), high corrosion resistance, enhanced adhesion strength as well as improved thermal stability compared to the undoped DLC films. The reduction in the internal stress and the sp3 content associated with a slight decrease in the mechanical properties is from Ti doping. Higher thermal stability is due to the TiC phase in the Ti-doped DLC structure. Enhancement of the adhesion strength is owing to the relief of the internal stress and the occurrence of the strong atomic intermixing bond at a Ti-doped DLC/Ti intermediate layer interface. Interestingly, the formation of the TiO2 film on the Ti-doped DLC surface due to a small amount of Ti doping significantly exhibited a better corrosion performance. Ti-doped DLC films, therefore, are a promising coating for tribological applications.

Thioglycosides Are Efficient Metabolic Decoys of Glycosylation that Reduce Selectin Dependent Leukocyte Adhesion

Metabolic decoys are synthetic analogs of naturally occurring biosynthetic acceptors. These compounds divert cellular biosynthetic pathways by acting as artificial substrates that usurp the activity of natural enzymes. While O-linked glycosides are common, they are only partially effective even at millimolar concentrations. In contrast, we report that N-acetylglucosamine (GlcNAc) incorporated into various thioglycosides robustly truncate cell surface N- and O-linked glycan biosynthesis at 10-100μM concentrations. The >10-fold greater inhibition is in part due to the resistance of thioglycosides to hydrolysis by intracellular hexosaminidases. The thioglycosides reduce β-galactose incorporation into lactosamine chains, cell surface sialyl Lewis-X expression, and leukocyterolling on selectin substrates including inflamed endothelial cells under fluid shear. Treatment of granulocytes with thioglycosides prior to infusion into mouse inhibited neutrophil homing to sites of acute inflammation and bone marrow by ∼80%-90%. Overall, thioglycosides represent an easy to synthesize class of efficient metabolic inhibitors or decoys. They reduce N-/O-linked glycan biosynthesis and inflammatory leukocyte accumulation.

Interface interactions between βγ-crystallin domain and Ig-like domain render Ca2+ -binding site inoperative in abundant perithecial protein of Neurospora crassa.

We describe a set of proteins in which a βγ-crystallin domain pairs with an Ig-like domain, and which are confined to microbes, like bacteria, slime molds and fungi. DdCAD-1 (Ca2+ -dependent cell adhesion molecule-1) and abundant perithecial protein (APP) represent this class of molecules. Using the crystal structure of APP-NTD (N-terminal domain of APP), we describe its mode of Ca2+ binding and provide a generalized theme for correct identification of the Ca2+ -binding site within this class of molecules. As a common feature, one of the two Ca2+ -binding sites is non-functional in the βγ-crystallin domains of these proteins. While APP-NTD binds Ca2+ with a micromolar affinity which is comparable to DdCAD-1, APP surprisingly does not bind Ca2+ . Crystal structures of APP and Ca2+ -bound APP-NTD reveal that the interface interactions in APP render its Ca2+ -binding site inoperative. Thus, heterodomain association provides a novel mode of Ca2+ -binding regulation in APP. Breaking the interface interactions (mutating Asp30Ala, Leu132Ala and Ile135Ala) or separation from the Ig-like domain removes the constraints upon the required conformational transition and enables the βγ-crystallin domain to bind Ca2+ . In mechanistic detail, our work demonstrates an interdomain interface adapted to distinct functional niches in APP and its homolog DdCAD-1.

CXCR4- and BCR-triggered integrin activation in B-cell chronic lymphocytic leukemia cells depends on JAK2-activated Bruton's tyrosine kinase.

Bruton's tyrosine kinase (BTK) regulates the B-cell receptor (BCR) signaling pathway, which, in turn, plays a critical role in B-cell chronic lymphocytic leukemia (B-CLL) pathogenesis. The BTK-specific inhibitor Ibrutinib blocks BCR signaling and is now approved as effective B-CLL therapy. Chemokines, such as the homeostatic chemokine CXCL12, play a central role in B-CLL pathogenesis and progression, by regulating CLL cell interaction with the stromal microenvironment, leading to cells survival and proliferation. In this study, we investigated, in normal versus CLL B-lymphocytes, the role of BTK in signal transduction activated by the CXCL12-CXCR4 signaling axis and its involvement in rapid integrin activation. We show that BTK is rapidly activated by CXCL12 in healthy as well as CLL B-lymphocytes, with a kinetic of tyr-phosphorylation coherent with rapid adhesion triggering. BTK inhibition prevents CXCL12-induced triggering of lymphocyte function-associated antigen-1 (LFA-1) and very late antigen-4 (VLA-4) integrins. Furthermore, BTK inhibition blocks the activation of the small GTP-binding protein RhoA, controlling integrin affinity. Very importantly, we show that BTK tyr-phosphorylation and activation by CXCL12 depends on upstream activation of JAK2 tyrosine kinase. A comparative analysis of 36 B-CLL patients demonstrates that JAK2-dependent BTK regulatory role on integrin activation by CXCL12 is fully conserved in CLL cells. Finally, we show that the JAK2-BTK axis also regulates signaling to integrin activation by BCR. Thus, BTK and JAK protein tyrosine kinases (PTKs) manifest a hierarchical activity both in chemokine- as well as BCR-mediated integrin activation and dependent adhesion, potentially suggesting the possibility of combined therapeutic approaches to B-CLL treatment.

Space dependent adhesion forces mediated by transient elastic linkages: New convergence and global existence results

In the first part of this work we show the convergence with respect to an asymptotic parameter ε of a delayed heat equation. It represents a mathematical extension of works considered previously by the authors [14–16]. Namely, this is the first result involving delay operators approximating protein linkages coupled with a spatial elliptic second order operator. For the sake of simplicity we choose the Laplace operator, although more general results could be derived. The main arguments are (i) new energy estimates and (ii) a stability result extended from the previous work to this more involved context. They allow to prove convergence of the delay operator to a friction term together with the Laplace operator in the same asymptotic regime considered without the space dependence in [14]. In a second part we extend fixed-point results for the fully non-linear model introduced in [16] and prove global existence in time. This shows that the blow-up scenario observed previously does not occur. Since the latter result was interpreted as a rupture of adhesion forces, we discuss the possibility of bond breaking both from the analytic and numerical point of view.

3D micro-environment regulates NF-\u03ba\u03b2 dependent adhesion to induce monocyte differentiation

Differentiation of monocytes entails their relocation from blood to the tissue, hence accompanied by an altered physicochemical micro-environment. While the mechanism by which the biochemical make-up of the micro-environment induces differentiation is known, the fluid-like to gel-like transition in the physical micro-environment is not well understood. Monocytes maintain non-adherent state to prevent differentiation. We establish that irrespective of the chemical makeup, a 3D gel-like micro-environment induces a positive-feedback loop of adhesion-MAPK-NF-κβ activation to facilitate differentiation. In 2D fluid-like micro-environment, adhesion alone is capable of inducing differentiation via the same positive-feedback signaling. Chemical inducer treatment in fluid-like micro-environment, increases the propensity of monocyte adhesion via a brief pulse of p-MAPK. The adhesion subsequently elicit differentiation, establishing that adhesion is both necessary and sufficient to induce differentiation in 2D/3D micro-environment. MAPK, and NF-κβ being key molecules of multiple signaling pathways, we hypothesize that biochemically inert 3D gel-like micro-environment would also influence other cellular functions.

Cell-layer dependent adhesion differences in wood bonds

The performance of wood adhesives depends on its internal cohesion and the adhesion to the wood surface which is structurally and chemically heterogeneous as it may be formed by different micro-surface, i.e. the compound middle lamella, the secondary cell wall layer, or the lumen surface. This study introduces an experimental protocol to directly evaluate adhesion to each of these micro-surfaces individually. Two wood fiber types presumed to differ in their surface chemistry are used as substrate. The results demonstrate that melamine-urea-formaldehyde adhesive shows excellent adhesion irrespective of the fiber type or micro-surface. While the same is the case for adhesion between untreated wood fiber surfaces and one component polyurethane, higher relative work of indentation between the lignin-rich compound middle lamella surface of medium density fiberboard fiber and one component polyurethane was observed. This observation is corroborated by a reduced polar character of this micro-surface as shown by X-ray photoelectron spectroscopy.

Non-Desmoglein Antibodies in Patients With Pemphigus Vulgaris.

Pemphigus vulgaris (PV) is a potentially life-threatening mucocutaneous autoimmune blistering disease. Patients develop non-healing erosions and blisters due to cell–cell detachment of keratinocytes (acantholysis), with subsequent suprabasal intraepidermal splitting. Identified almost 30 years ago, desmoglein-3 (Dsg3), a Ca2+-dependent cell adhesion molecule belonging to the cadherin family, has been considered the “primary” autoantigen in PV. Proteomic studies have identified numerous autoantibodies in patients with PV that have known roles in the physiology and cell adhesion of keratinocytes. Antibodies to these autoantibodies include desmocollins 1 and 3, several muscarinic and nicotinic acetylcholine receptor subtypes, mitochondrial proteins, human leukocyte antigen molecules, thyroid peroxidase, and hSPCA1—the Ca2+/Mn2+-ATPase encoded by ATP2C1, which is mutated in Hailey–Hailey disease. Several studies have identified direct pathogenic roles of these proteins, or synergistic roles when combined with Dsg3. We review the role of these direct and indirect mechanisms of non-desmoglein autoantibodies in the pathogenesis of PV.

Specific ion effects on thermoresponsive polymer brushes: Comparison to other architectures

Thermoresponsive polymers have received significant research attention as smart materials with particular interest in biomedical applications. The composition and architecture are known to strongly influence the thermoresponsive properties of the materials. For example, the strong overlap of end-grafted polymer chains in polymer brushes leads to a broader collapse transition relative to linear ungrafted chains as well as temperature dependent adhesion. The temperature response of free polymer has been widely reported to depend on the concentration and identity of ions in solution and is further modified by the composition of the solvent and presence of cosolutes. However, the influence of polymer architecture on these specific ion effects is relatively unknown. Herein, we compare the current understanding of specific ion effects on free polymer chains and gels with recent studies of polymer brushes. Further studies on mixed salt systems are found to be the next step to predicting the behaviour of these materials in biological systems.

CXCR3 Regulates CD4+ T Cell Cardiotropism and Maladaptive Cardiac Remodeling Through Mechanisms Involving ICAM1‐Mediated Adhesion

Left ventricular (LV) dysfunction and heart failure (HF) progression are associated in humans and mice with increased levels of circulating chemokines CXCL9 and CXCL10, and increased frequency of T cells expressing their receptor CXCR3. CXCR3 signaling upon chemokine recognition in T cells results in integrin activation and T cell adhesion to endothelial cells. We recently reported that the integrin ligand ICAM1 is significantly upregulated in the LV endothelium of mice with HF, and that Icam1−/− mice have decreased LV T cell recruitment and HF. Additionally, we reported that Th1 cells, which highly express CXCR3, are major drivers of maladaptive cardiac remodeling. Thus, we hypothesized that chemokine signaling through CXCR3 regulates integrin dependent adhesion to ICAM1 and contributes to Th1 cell recruitment to the LV and HF. We used the mouse model of Transverse Aortic Constriction (TAC) to induce LV remodeling and HF in WT and CXCR3−/− mice, and in vitro adhesion studies to evaluate WT and CXCR3−/− Th1 cell chemokine‐mediated integrin‐dependent adhesion to ICAM1. WT mice expressed increased levels of CXCL9 and CXCL10 in the LV during the course of TAC. Activation of CD4+ T cells occurred in the mediastinal lymph nodes (mLN), which drain the heart, of both WT and CXCR3−/− mice; however T cell infiltration in the LV only occurred in WT but not CXCR3−/− mice, and heart infltrated T cells expressed high levels of CXCR3 and the ligand of ICAM‐1, LFA1. Moreover, CXCR3−/− TAC mice did not develop cardiac fibrosis, hypertrophy or inflammation and had preserved systolic and diastolic function as compared to WT TAC mice. Mechanistically, both CXCL9 and CXCL10 induced Th1 cell adhesion to ICAM1 under shear flow conditions in vitro in a CXCR3‐dependent manner. Our data support a CXCL9/CXCL10‐CXCR3‐ICAM1 axis regulating CD4+ T cell LV recruitment, and may represent a novel pathway potentially targetable in non‐ischemic HF.Support or Funding InformationNIH R01 HL123658‐01, T32AI007077‐34This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Nanoscale Tuning of VCAM-1 Determines VLA-4-Dependent Melanoma Cell Plasticity on RGD Motifs.

The biophysical fine-tuning of cancer cell plasticity is crucial for tumor progression but remains largely enigmatic. Although vascular cell adhesion molecule-1 (VCAM-1/CD106) has been implicated in melanoma progression, here its presentation on endothelial cells was associated with diminished melanoma cell spreading. Using a specific nanoscale modulation of VCAM-1 (tunable from 70 to 670 ligands/μm²) next to integrin ligands (RGD motifs) in a bifunctional system, reciprocal regulation of integrin α4 (ITGA4/VLA-4/CD49d)-dependent adhesion and spreading of melanoma cells was found. As the VCAM-1/VLA-4 receptor pair facilitated adhesion, while at the same time antagonizing RGD-mediated spreading, melanoma cell morphogenesis on these bifunctional matrices was directly regulated by VCAM-1 in a dichotomic and density-dependent fashion. This was accompanied by concordant regulation of F-actin cytoskeleton remodeling, Rac1-expression, and paxillin-related adhesion formation. The novel function of VCAM-1 was corroborated in vivo using two murine models of pulmonary metastasis. The regulation of melanoma cell plasticity by VCAM-1 highlights the complex regulation of tumor-matrix interactions.Implications: Nanotechnology has revealed a novel dichotomic function of the VCAM-1/VLA-4 interaction on melanoma cell plasticity, as nanoscale tuning of this interaction reciprocally determines adhesion and spreading in a ligand density-dependent manner. Mol Cancer Res; 16(3); 528-42. ©2017 AACR.

Salt-bridge modulates differential calcium-mediated ligand binding to integrin \u03b11- and \u03b12-I domains

Integrins are transmembrane cell-extracellular matrix adhesion receptors that impact many cellular functions. A subgroup of integrins contain an inserted (I) domain within the α–subunits (αI) that mediate ligand recognition where function is contingent on binding a divalent cation at the metal ion dependent adhesion site (MIDAS). Ca2+ is reported to promote α1I but inhibit α2I ligand binding. We co-crystallized individual I-domains with MIDAS-bound Ca2+ and report structures at 1.4 and 2.15 Å resolution, respectively. Both structures are in the “closed” ligand binding conformation where Ca2+ induces minimal global structural changes. Comparisons with Mg2+-bound structures reveal Mg2+ and Ca2+ bind α1I in a manner sufficient to promote ligand binding. In contrast, Ca2+ is displaced in the α2I domain MIDAS by 1.4 Å relative to Mg2+ and unable to directly coordinate all MIDAS residues. We identified an E152-R192 salt bridge hypothesized to limit the flexibility of the α2I MIDAS, thus, reducing Ca2+ binding. A α2I E152A construct resulted in a 10,000-fold increase in Mg2+ and Ca2+ binding affinity while increasing binding to collagen ligands 20%. These data indicate the E152-R192 salt bridge is a key distinction in the molecular mechanism of differential ion binding of these two I domains.