Rapid Adhesion Research Articles

Isolation of Human Umbilical Vein Endothelial Cells and Their Use in the Study of Neutrophil Transmigration Under Flow Conditions

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system. During acute inflammation, neutrophils are the first inflammatory cells to migrate to the site of injury. Recruitment of neutrophils to an injury site is a stepwise process that includes first, dilation of blood vessels to increase blood flow; second, microvascular structural changes and escape of plasma proteins from the bloodstream; third, rolling, adhesion and transmigration of the neutrophil across the endothelium; and fourth accumulation of neutrophils at the site of injury. A wide array of in vivo and in vitro methods has evolved to enable the study of these processes. This method focuses on neutrophil transmigration across human endothelial cells. One popular method for examining the molecular processes involved in neutrophil transmigration utilizes human neutrophils interacting with primary human umbilical vein endothelial cells (HUVEC). Neutrophil isolation has been described visually elsewhere; thus this article will show the method for isolation of HUVEC. Once isolated and grown to confluence, endothelial cells are activated resulting in the upregulation of adhesion and activation molecules. For example, activation of endothelial cells with cytokines like TNF-α results in increased E-selectin and IL-8 expression. E-selectin mediates capture and rolling of neutrophils and IL-8 mediates activation and firm adhesion of neutrophils. After adhesion neutrophils transmigrate. Transmigration can occur paracellularly (through endothelial cell junctions) or transcellularly (through the endothelial cell itself). In most cases, these interactions occur under flow conditions found in the vasculature. The parallel plate flow chamber is a widely used system that mimics the hydrodynamic shear stresses found in vivo and enables the study of neutrophil recruitment under flow condition in vitro. Several companies produce parallel plate flow chambers and each have advantages and disadvantages. If fluorescent imaging is needed, glass or an optically similar polymer needs to be used. Endothelial cells do not grow well on glass. Here we present an easy and rapid method for phase-contrast, DIC and fluorescent imaging of neutrophil transmigration using a low volume ibidi channel slide made of a polymer that supports the rapid adhesion and growth of human endothelial cells and has optical qualities that are comparable to glass. In this method, endothelial cells were grown and stimulated in an ibidi μslide. Neutrophils were introduced under flow conditions and transmigration was assessed. Fluorescent imaging of the junctions enabled real-time determination of the extent of paracellular versus transcellular transmigration.

The Integrin-Ligand Interaction Regulates Adhesion and Migration through a Molecular Clutch

Adhesive and migratory behavior can be cell type, integrin, and substrate dependent. We have compared integrin and substrate differences using three integrin receptors: α5β1, α6β1, and αLβ2 expressed in a common cell type, CHO.B2 cells, which lack integrin α subunits, as well as in different cell types that express one or more of these integrins. We find that CHO.B2 cells expressing either α6β1 or αLβ2 integrins migrate and protrude faster and are more directionally persistent on laminin or ICAM-1, respectively, than CHO.B2 cells expressing α5β1 on fibronectin. Despite rapid adhesion maturation and the presence of large adhesions in both the α6β1- and αLβ2-expressing cells, they display robust tyrosine phosphorylation. In addition, whereas myosin II regulates adhesion maturation and turnover, protrusion rates, and polarity in cells migrating on fibronectin, surprisingly, it does not have comparable effects in cells expressing α6β1 or αLβ2. This apparent difference in the integration of myosin II activity, adhesion, and migration arises from alterations in the ligand–integrin–actin linkage (molecular clutch). The elongated adhesions in the protrusions of the α6β1-expressing cells on laminin or the αLβ2-expressing cells on ICAM-1 display a novel, rapid retrograde flux of integrin; this was largely absent in the large adhesions in protrusions of α5β1-expressing cells on fibronectin. Furthermore, the force these adhesions exert on the substrate in protrusive regions is reduced compared to similar regions in α5-expressing cells, and the adhesion strength is reduced. This suggests that intracellular forces are not efficiently transferred from actomyosin to the substratum due to altered adhesion strength, that is, avidity, affinity, or the ligand-integrin-actin interaction. Finally, we show that the migration of fast migrating leukocytes on fibronectin or ICAM-1 is also largely independent of myosin II; however, their adhesions are small and do not show retrograde fluxing suggesting other intrinsic factors determine their migration differences.

FIB Patterning of Stainless Steel for the Development of Nano-Structured Stent Surfaces for Cardiovascular Applications

Coronary artery disease is a major problem worldwide. Stent implantation is a percutaneous interventional procedure that mitigates vessel stenosis, providing mechanical support within the artery. However, stenting causes physical damage to the arterial wall. The research presented here develops novel nano-structured features on stent surfaces to promote rapid endothelial cell adhesion to reduce in-stent re-stenosis. Nano-structured features (concaves) ordered in rectangular arrays were patterned on 316L Stainless Steel (SS) surfaces using focused ion beam (FIB) milling after electropolishing using linear sweep voltammetry and chronoamperometry. Various dose test experiments were performed, aiming at an array of 120 nm diameter holes with pitch of 240 nm and depth of 50-100 nm on sample area of 400 μm × 400 μm. Studies on FIB milling rates were carried out to optimise the time and to create a uniform array of holes. Based on the SEM examination of the hole arrays (plane view and cross section) it can be concluded that a low ion beam current created well shaped uniform concave structures (Gaussian shape) with good depth profile, while a high current resulted in an array of holes with sine profile. Further, a higher current created larger diameter holes with less defined depth profile and deviation from Gaussian shape. We demonstrate that the ability to perform nano-structuring with FIB milling is greatly affected by the polycrystalline nature of SS.

Polymerized laminin-332 matrix supports rapid and tight adhesion of keratinocytes, suppressing cell migration.

Laminin-332 (α3ß3γ2) (Lm332) supports the stable anchoring of basal keratinocytes to the epidermal basement membrane, while it functions as a motility factor for wound healing and cancer invasion. To understand these contrasting activities of Lm332, we investigated Lm332 matrices deposited by normal human keratinocytes and other Lm332-expressing cell lines. All types of the cells efficiently deposited Lm332 on the culture plates in specific patterns. On the contrary, laminins containing laminin ß1 and/or γ1 chains, such as Lm511 and Lm311, were not deposited on the culture plates even if secreted into culture medium. The Lm332 deposition was not inhibited by function-blocking antibodies to the α3 and α6 integrins but was inhibited by sodium selenate, suggesting that sulfated glycosaminoglycans on cell surface, e.g. heparan sulfate proteoglycans, might be involved in the process. HEK293 cells overexpressing exogenous Lm332 (Lm332-HEK) almost exclusively deposited Lm332 on the plates. The deposited Lm332 matrix showed a mesh-like network structure as analyzed by electron microscopy, suggesting that Lm332 was highly polymerized. When biological activity was analyzed, the Lm332 matrix rather suppressed the migration of keratinocytes as compared with purified Lm332, which highly promoted the cell migration. The Lm332 matrix supported adhesion of keratinocytes much more strongly and stably than purified Lm332. Integrin α3ß1 bound to the Lm332 matrix at a three times higher level than purified Lm332. Normal keratinocytes prominently showed integrin α6ß4-containing, hemidesmosome-like structures on the Lm332 matrix but not on the purified one. These results indicate that the polymerized Lm332 matrix supports stable cell adhesion by interacting with both integrin α6ß4 and α3ß1, whereas unassembled soluble Lm332 supports cell migration.

AMP‐Activated Protein Kinase Reduces Vascular Smooth Muscle Growth by Inhibiting Actin Cytoskeleton Dynamics Necessary for Migration

Abluminal vascular smooth muscle cell (VSMC) migration is a functional underpinning of vasculoproliferative disorders. Upon activation, the dynamic nature of the actin cytoskeleton and rapid focal adhesion turnover provides the structural basis necessary for cell motility. Preliminary data show that activation of the metabolic sensor AMP‐activated protein kinase (AMPK) inhibits migration in rat primary VSMCs. In the current study we tested the hypothesis that AMPK enhances focal adhesion stability and inhibits vasodilator‐stimulated phosphoprotein (VASP) anti‐capping function on microfilament elongation. Treatment of VSMCs with the AMPK activator AICAR (1 mM) significantly (~50%; p<0.05) increased phosphorylated VASP at Thr278, a reported AMPK‐specific site, thus inhibiting microfilament anti‐capping by VASP. This inhibition of VASP was independent of the traditional VASP‐activating kinases PKA and PKG. In addition, AICAR inhibited PDGF‐stimulated autophosphorylation of focal adhesion kinase (FAK) at Ser397, a necessary event for kinase activity and focal adhesion turnover. Inhibition of phospho‐FAK at Ser397 induced focal adhesion stability necessary for cytostasis. These data lend strong support for the notion that AMPK has capacity to promote focal adhesion stability and to inhibit cytoskeletal rearrangement necessary for VSMC migration.

HIV-1 matrix protein p17 binds to the IL-8 receptor CXCR1 and shows IL-8–like chemokine activity on monocytes through Rho/ROCK activation

AbstractExogenous HIV-1 matrix protein p17 was found to deregulate biologic activities of many different immune cells that are directly or indirectly involved in AIDS pathogenesis after binding to unknown cellular receptor(s). In particular, p17 was found to induce a functional program in monocytes related to activation and inflammation. In the present study, we demonstrate that CXCR1 is the receptor molecule responsible for p17 chemokine–like activity on monocytes. After CXCR1 binding, p17 was capable of triggering rapid adhesion and chemotaxis of monocytes through a pathway that involved Rho/ROCK. Moreover, CXCR1-silenced primary monocytes lost responsiveness to p17 chemoattraction, whereas CXCR1-transfected Jurkat cells acquired responsiveness. Surface plasmon resonance studies confirmed the capacity of p17 to bind CXCR1 and showed that the p17/CXCR1 interaction occurred with a low affinity compared with that measured for IL-8, the physiologic CXCR1 ligand. In all of its activities, p17 mimicked IL-8, the natural high-affinity ligand of CXCR1. Recent studies have highlighted the role of IL-8 and CXCR1 in HIV-1 replication and AIDS pathogenesis. Our findings herein call for an exploration of the therapeutic potential of blocking the p17/IL-8/CXCR1 axis in HIV-1 infection.

Chemically programmed cell adhesion with membrane-anchored oligonucleotides.

Cell adhesion organizes the structures of tissues and mediates their mechanical, chemical, and electrical integration with their surroundings. Here, we describe a strategy for chemically controlling cell adhesion using membrane-anchored single-stranded DNA oligonucleotides. The reagents are pure chemical species prepared from phosphoramidites synthesized in a single chemical step from commercially available starting materials. The approach enables rapid, efficient, and tunable cell adhesion, independent of proteins or glycans, by facilitating interactions with complementary labeled surfaces or other cells. We demonstrate the utility of this approach by imaging drug-induced changes in the membrane dynamics of non-adherent human cells that are chemically immobilized on a passivated glass surface.

Enzymatically degradable mussel-inspired adhesive hydrogel.

Mussel-inspired adhesive hydrogels represent innovative candidate medical sealants or glues. In the present work, we describe an enzyme-degradable mussel-inspired adhesive hydrogel formulation, achieved by incorporating minimal elastase substrate peptide Ala-Ala into the branched poly(ethylene glycol) (PEG) macromonomer structure. The system takes advantage of neutrophil elastase expression upregulation and secretion from neutrophils upon recruitment to wounded or inflamed tissue. By integrating adhesive degradation behaviors that respond to cellular cues, we expand the functional range of our mussel-inspired adhesive hydrogel platforms. Rapid (<1 min) and simultaneous gelation and adhesion of the proteolytically active, catechol-terminated precursor macromonomer was achieved by addition of sodium periodate oxidant. Rheological analysis and equilibrium swelling studies demonstrated that the hydrogel is appropriate for soft tissue-contacting applications. Notably, hydrogel storage modulus (G′) achieved values on the order of 10 kPa, and strain at failure exceeded 200% strain. Lap shear testing confirmed the material’s adhesive behavior (shear strength: 30.4 ± 3.39 kPa). Although adhesive hydrogel degradation was not observed during short-term (27 h) in vitro treatment with neutrophil elastase, in vivo degradation proceeded over several months following dorsal subcutaneous implantation in mice. This work represents the first example of an enzymatically degradable mussel-inspired adhesive and expands the potential biomedical applications of this family of materials.

Losing control over adenosine 5′-triphosphate release: Implications for the red blood cell storage lesion*

Losing control over adenosine 5′-triphosphate release: Implications for the red blood cell storage lesion*

The effects of low frequency electromagnetic fields on the proliferation of epidermal stem cells cultured on two different scaffolds

Objective To observe the effects of low frequency electromagnetic fields (LFEMFs) on the proliferation of human epidermal stem cells (hESCs) cultured in a three dimensional environment so as to provide an experimental basis for applying LFEMF in skin tissue engineering.Methods hESCs from human prepuces were isolated and purified by the method of rapid adherence to collagen type ⅣV. They were grafted into a type-I collagen sponge or chitosan scaffold in vitro, and then stimulated with different frequencies of LFEMF ( 1 Hz, 10 Hz or 50 Hz) at a magnetic field intensity of 5 mT for 30 min/d. The cells' growth and proliferation were tracked using hematoxylin and eosin (HE) and diamine pheny1 indole (DAPI) staining and observed under the scanning electron microscope at different time points ( on 2nd, 7th, 10th and 14th days of LFEMF intervention). The amounts of cell proliferation at every time point were analyzed and compared.Results LFEMFs of different frequencies showed significantly different efficacy in promoting hESC proliferation. The two scaffolds also showed significantly different effects.By the 10th day, hESCs had grown significantly better on collagen sponge scaffolds than on the chitosan ones. All LFEMF frequencies could promote proliferation of hESCs, but the differences in their effects were statistically significant.Conclusion Collagen sponge may be a preferable scaffold for hESCs cultured in vitro. Rapid proliferation of ESCs in three-dimensional settings can be promoted by LFEMF intervention. LFEMF has relatively great potential in skin tissue engineering. Key words: Low frequency electromagnetic fields; Epidermal stem cells; Proliferation; Collagen sponge scaffolds; Chitosan scaffolds; Tissue engineering

The Nod2 Sensor Promotes Intestinal Pathogen Eradication via the Chemokine CCL2-Dependent Recruitment of Inflammatory Monocytes

The intracellular sensor Nod2 is activated in response to bacteria, and the impairment of this response is linked to Crohn's disease. However, the function of Nod2 in host defense remains poorly understood. We found that Nod2-/- mice exhibited impaired intestinal clearance of Citrobacter rodentium, an enteric bacterium that models human infection by pathogenic Escherichia coli. The increased bacterial burden was preceded by reduced CCL2 chemokine production, inflammatory monocyte recruitment, and Th1 cell responses in the intestine. Colonic stromal cells, but not epithelial cells or resident CD11b+ phagocytic cells, produced CCL2 in response to C. rodentium in a Nod2-dependent manner. Unlike resident phagocytic cells, inflammatory monocytes produced IL-12, a cytokine that induces adaptive immunity required for pathogen clearance. Adoptive transfer of Ly6C(hi) monocytes restored the clearance of the pathogen in infected Ccr2-/- mice. Thus, Nod2 mediates CCL2-CCR2-dependent recruitment of inflammatory monocytes, which is important in promoting bacterial eradication in the intestine.

Presence of Xenogenic Mouse RNA in RPE and IPE Cells Cultured on Mitotically Inhibited 3T3 Fibroblasts

Mitotically inhibited 3T3 fibroblasts are used as feeder layers to culture a variety of cells. However, transplantation of human cells cultured on mitotically arrested mouse cells poses potential risks, such as disease transfer and contamination with 3T3 cells. Bovine RPE and IPE cells were cultured on mitomycin-treated 3T3 fibroblasts, to examine cell characteristics and contamination by 3T3 products. IPE or RPE cells cultured on mitomycin-treated 3T3 fibroblasts were evaluated for adhesion, morphology, and tight junction formation by microscopy and immunohistochemistry. ROS phagocytosis was used to examine functional activity. Gene expression was evaluated by quantitative real-time PCR. In the presence of 3T3 fibroblasts, primary IPE and RPE cells adhere, spread and acquire a hexagonal shape within 12 hours. When cultured on 3T3 fibroblasts, IPE and RPE cells exhibited stable expression of pigment epithelial genes, but expression of mouse collagen type I was also observed. Culturing IPE and RPE cells on mitomycin-treated 3T3 fibroblasts resulted in rapid adhesion and growth of primary pigment cells. However, the presence of potentially hazardous xenogeneic mRNA of mouse origin in the cultures limits the use of these cells for transplantation.

The integrin LFA-1 signals through ZAP-70 to regulate expression of high-affinity LFA-1 on T lymphocytes

AbstractThe integrin lymphocyte function-associated antigen 1 (LFA-1) controls many functions of T lymphocytes and is particularly essential during lymphocyte migration from blood into tissues. LFA-1 is considered to initiate “outside-in” signaling when bound to ligand intercellular adhesion molecule 1 (ICAM-1), but little is known about the proteins involved or where in the cell such LFA-1–mediated signaling might be operating. Here we show that LFA-1 is constitutively associated with the protein tyrosine kinases Lck and zeta chain–associated protein of 70 kDa (ZAP-70). When LFA-1 binds ICAM-1, both kinases become phosphorylated and the consequence of kinase activation is the conversion of intermediate- to high-affinity LFA-1 and an increase in close contact with ICAM-1. In the polarized T lymphocyte, phospho-ZAP-70 is concentrated within a region of high-affinity LFA-1 that includes talin and encompasses the lamella/lamellipodial interface as well as further back in the cell. Deficiency of ZAP-70 through inhibition or knockdown in T lymphocytes decreases the speed of migration on ICAM-1, as well as reducing firm adhesion under shear-flow conditions. Through its control of high-affinity LFA-1, the LFA-1/Lck/ZAP-70 complex is in position to initiate the rapid adhesion strengthening and migration necessary for T-lymphocyte responses when stimulated vasculature is encountered at sites of infection or injury.

Isolation, culture and phenotypic investigation of dedifferentiation-derived epidermal stem cells

Objective To isolate the dedifferentiation-derived epidermal stem cells (DDESCs)to further investigate their phenotypic characteristics. Methods The sheets of human foreskin were digested overnight after removal of adipose tissues and then the epidermis was separated from the dermis.The epidermis sheets which eliminated basal stem cells by repeated adhesion to type Ⅳ collagen and flushing were transplanted onto the full-thickness skin wounds on the back of BALB/c nude mice. After five days, the sheets were collected and digested into single cells, after which the percentages of positive cells of CK10, CK19 and β1 integrin were detected by flow cytometric analysis. DDESCs were isolated by rapid adhesion to type Ⅳ collagen. The expressions of CK19, β1 integrin, Oct4 and Nanog in the cells were examined using immunofluorescence and quantitative real-time polymerase chain reaction (RTPCR). Results The percentages of positive cells of CK19 and 31 integrin were increased (P ＜0.01 )and those of CK10 in the transplanted sheets decreased ( P ＜0.01 ) five days after transplantation. Isolation of DDESCs by repeated adhesion to type Ⅳ collagen showed 4.56% adhering cells in the transplantation group within 10 minutes. The in vitro phenotypic assays showed that the expressions of CK19, β1 integrin, Oct4 and Nanog in DDESCs were similar to those of original epidermal stem cells ( P ＞0.05 ) but remarkably higher than those in the control group ( P ＜ 0.01 ). Conclusion The phenotypic characteristics of DDESCs cultured in vitro are similar to those of epidermal stem cells, indicating a new approach for wound repair and regeneration. Key words: Epidermis; Stem cells; Dedifferentiation; Cell separation; Phenotype

Ex Vivo Generated Donor Central Memory CD8 T Cells, Previously Shown to Enhance Engraftment of Allogeneic Bone Marrow, Also Exhibit Significant GVL Activity without Causing Gvhd In An In Vivo b Cell Lymphoma Model

AbstractAbstract 424Generation of T cells endowed with graft versus leukemia (GVL) and depleted of graph versus host (GVH) activity represents a highly desirable goal in BM transplantation (BMT). Donor CTLs directed against 3rd party stimulators were shown to effectively enhance engraftment of BM allografts without causing GVHD. Recently, we demonstrated that activated anti 3rd party CD8 cells with central memory phenotype (Tcm), can further support and improve BM engraftment. This is likely due to the improved lymph node homing of the Tcms, their proliferative capacity and prolonged persistence in BMT recipients. Surprisingly, when tested in vitro, anti 3rd party human CTLs were found to eliminate B CLL and other lymphoma cell types. Investigating the mechanism of the cytotoxicity revealed a unique TCR independent effect mediated against both autologous and allogeneic targets. Inhibition studies using specific blocking Ab revealed initial binding of CTLs to their targets via a rapid ICAM1-LFA1 adhesion, followed by slow induction of apoptosis following interaction of the CD8 on the CTL with MHCI on the tumor cell. The killing did not involve classical CTL death molecules. To establish an appropriate mouse model, we initially verified that anti 3rd party Tcm derived from (B6xBALB/c)F1 exhibit TCR independent killing of A20 lymphoma cells of BALB/c origin (34.8±12.1% in 4 experiments, 5:1 Tcm/lymphoma cell ratio, in comparison to A20 cells incubated without Tcm, p<0.05). Moreover, after 16 hours of incubation with Tcm, AnnexinV staining of A20 cells was enhanced compared to basal staining (14.8±4.5% and 5.2±2.2% respectively, in three experiments, p<0.05), suggesting an apoptosis based mechanism as previously described for the killing of human lymphoma. Using luciferase expressing A20 cells, we were able to follow the fate of the malignant cells in vivo, and study the effect of added donor anti 3rd party Tcm to either syngeneic or allogeneic BMT in a model simulating minimal residual disease. In the syngeneic model, 3×106 Nude BALB/c BM cells were transplanted into lethally irradiated BALB/c mice together with 5000 A20 cells. On the next day, syngeneic Tcm were infused. As can be seen in Fig. 1, none of the untreated mice survived (0/7) 100 days post BMT (23 days median survival). Administration of 1×107 or 2×107 syngeneic Tcms led to significantly diminished tumor burden (Fig. 1A) and improved overall survival of 28% (2/7, P<0.0001) and 40% (2/5, P<0.002) with median survival of 49 and 80 days, respectively (Fig. 1B). As expected, further improvement of tumor eradication was found in the allogeneic setting, in which an additional GVL effect, mediated by residual alloreactivity in the absence of GVHD, occurred in addition to the TCR independent killing. In the allogeneic model, 3×106 allogeneic Nude B6 BM cells were transplanted together with 5000 A20 cells into lethally irradiated BALB/c mice, and mice were treated with donor type Tcm. Similar to the results in the syngeneic model, none of the untreated mice survived 100 days (0/8) (23 days median survival), while administration of 5×106 donor Tcms, led to remarkable overall survival of 100% (7/7) at 100 days post BMT (Fig. 2). Although the allogeneic Tcm displayed enhanced GVL activity compared to syngeneic Tcm, this effect was not associated with any manifestation of GVHD. Thus, as previously described, the weight and overall appearance of mice receiving allogeneic anti 3rd party Tcms were the same as that of mice in the control group, radioprotected with a transplant of Nude BM alone. Collectively, we demonstrate for the first time, by in vivo imaging the GVL reactivity of murine anti 3rd party Tcm. Considering our previous finding that anti 3rd party Tcm markedly enhance BM allografting, these results suggest that anti 3rd party Tcm can provide a ‘double supportive effect’ by promoting BM engraftment, and concurrently inducing GVL reactivity without causing GVHD. Such cell therapy is highly attractive, in particular for elderly patients with B CLL and other B cell malignancies who might not tolerate aggressive conditioning. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

The F-BAR domain of SRGP-1 facilitates cell-cell adhesion during C. elegans morphogenesis.

Robust cell-cell adhesion is critical for tissue integrity and morphogenesis, yet little is known about the molecular mechanisms controlling cell-cell junction architecture and strength. We discovered that SRGP-1 is a novel component of cell-cell junctions in Caenorhabditis elegans, localizing via its F-BAR (Bin1, Amphiphysin, and RVS167) domain and a flanking 200-amino acid sequence. SRGP-1 activity promotes an increase in membrane dynamics at nascent cell-cell contacts and the rapid formation of new junctions; in addition, srgp-1 loss of function is lethal in embryos with compromised cadherin-catenin complexes. Conversely, excess SRGP-1 activity leads to outward bending and projections of junctions. The C-terminal half of SRGP-1 interacts with the N-terminal F-BAR domain and negatively regulates its activity. Significantly, in vivo structure-function analysis establishes a role for the F-BAR domain in promoting rapid and robust cell adhesion during embryonic closure events, independent of the Rho guanosine triphosphatase-activating protein domain. These studies establish a new role for this conserved protein family in modulating cell-cell adhesion.

Upregulation of coronary endothelial P-selectin in a monkey heart ischemia reperfusion model

The design of targeted ultrasound contrast agents for molecular imaging of myocardial ischemia-reperfusion (IR) requires the availability of an adequate in vivo model in a species in which cross reactivity with the target occurs. P-selectin (Psel) is an activation-dependent endothelial receptor that supports rapid and reversible cell adhesion in a flowing system. Together with E- and L-selectins it constitutes the selectin family of adhesion molecules. We investigated the myocardial expression of selectins in a closed chest minimally invasive monkey myocardial IR model. Catheter-based occlusion (30-50 min) followed by reperfusion (3 or 5 h) of left anterior descending artery (LAD) was performed in anesthetised monkeys. At the end of each procedure animals were killed, and their hearts were excised. The tissues were analyzed immunohistochemically using an anti-human Psel antibody (AK-6 clone) that cross reacts with rhesus monkey. Histopathological features confirm the presence of IR injuries in myocardial tissues. There was significant increase in the Psel expression in vessels from the IR areas. However, significantly higher Psel immunoreactivity was also seen in areas which are distant from IR injuries.

PDE8 Regulates Rapid Teff Cell Adhesion and Proliferation Independent of ICER

BackgroundAbolishing the inhibitory signal of intracellular cAMP by phosphodiesterases (PDEs) is a prerequisite for effector T (Teff) cell function. While PDE4 plays a prominent role, its control of cAMP levels in Teff cells is not exclusive. T cell activation has been shown to induce PDE8, a PDE isoform with 40- to 100-fold greater affinity for cAMP than PDE4. Thus, we postulated that PDE8 is an important regulator of Teff cell functions.Methodology/Principal FindingsWe found that Teff cells express PDE8 in vivo. Inhibition of PDE8 by the PDE inhibitor dipyridamole (DP) activates cAMP signaling and suppresses two major integrins involved in Teff cell adhesion. Accordingly, DP as well as the novel PDE8-selective inhibitor PF-4957325-00 suppress firm attachment of Teff cells to endothelial cells. Analysis of downstream signaling shows that DP suppresses proliferation and cytokine expression of Teff cells from Crem −/− mice lacking the inducible cAMP early repressor (ICER). Importantly, endothelial cells also express PDE8. DP treatment decreases vascular adhesion molecule and chemokine expression, while upregulating the tight junction molecule claudin-5. In vivo, DP reduces CXCL12 gene expression as determined by in situ probing of the mouse microvasculature by cell-selective laser-capture microdissection.Conclusion/SignificanceCollectively, our data identify PDE8 as a novel target for suppression of Teff cell functions, including adhesion to endothelial cells.

484 Rapid adherence to collagen IV enriches for tumour initiating cells

484 Rapid adherence to collagen IV enriches for tumour initiating cells

Expression of tumour‐suppressing chemokine BRAK/CXCL14 reduces cell migration rate of HSC‐3 tongue carcinoma cells and stimulates attachment to collagen and formation of elongated focal adhesions in vitro

BRAK/CXCL14 (breast- and kidney-expressed chemokine/CXC chemokine ligand 14) is a chemokine that is expressed in many normal cells and tissues but is absent from or expressed at very low levels in transformed cells and cancerous tissues, including HNSCC (head and neck squamous cell carcinoma). We reported previously that the forced expression of BRAK/CXCL14 in HNSCC (HSC-3 BRAK) cells decreased the rate of tumour formation and size of tumour xenografts compared with mock-vector-introduced (HSC-3 Mock) cells in athymic nude mice, even though the growth rates of these cells were the same under in vitro culture conditions, suggesting that high-level expression of the gene is important for the suppression of tumour establishment in vivo. For the first step to study the mechanisms of BRAK-dependent tumour suppression, we compared characteristics between HSC-3 BRAK and HSC-3 Mock cells under in vitro culture conditions. The cell migration rate was lower in HSC-3 BRAK cells than in HSC-3 Mock cells. Also, HSC-3 BRAK cells showed more rapid adhesion than HSC-3 Mock cells when cultured on type I collagen-coated dishes but not on fibronectin or laminin 1-coated ones. This adhesion was mediated by alpha2beta1 integrin. Immunofluorescent analysis of the cells cultured on type I collagen showed that HSC-3 BRAK cells formed much more elongated focal adhesions co-localized with paxillin and actin stress fibres than did HSC-3 Mock cells. Treatment of parental HSC-3 cells with recombinant BRAK stimulated the activation of Rap1, which is a ras family small GTPase, and formation of elongated focal adhesions, indicating that the difference in cell character observed between HSC-3 Mock and HSC-3 BRAK was not due to selection of clones of different character but due to expression of BRAK in the cells. The characteristic morphology of focal adhesions in HSC-3 BRAK cells was perturbed by the introduction of an expression vector of the Rap-binding domain of the Ral guanine nucleotide dissociation stimulator, a target of Rap1, into HSC-3 BRAK cells, suggesting that Rap1 regulated the formation of the morphology of the focal adhesions. These data indicate that the expression of BRAK stimulated the formation of elongated focal adhesions of the HSC-3 cells in an autocrine or paracrine fashion, in which stimulation may be responsible for the reduced migration of the cells.