TRAF5 Deficiency Accelerates Atherogenesis in Mice by Increasing Inflammatory Cell Recruitment and Foam Cell Formation.

Circ Res. 2010 Jul 22;

Authors: Missiou A, Rudolf P, Stachon P, Wolf D, Varo N, Aichele P, Colberg C, Hoppe N, Ernst S, Münkel C, Walter C, Sommer B, Hilgendorf I, Nakano H, Bode C, Zirlik A

Rationale: Tumor necrosis factor receptor-associated factors (TRAFs) are cytoplasmic adaptor proteins for the TNF/interleukin-1/Toll-like receptor superfamily. Ligands of this family comprise multiple important cytokines such as TNFalpha, CD40L, and interleukin-1beta that promote chronic inflammatory diseases such as atherosclerosis. We recently reported overexpression of TRAF5 in murine and human atheromata and that TRAF5 promotes inflammatory functions of cultured endothelial cells and macrophages. Objective: This study tested the hypothesis that TRAF5 modulates atherogenesis in vivo. Methods and Results: Surprisingly, TRAF5(-/-)/LDLR(-/-) mice consuming a high-cholesterol diet for 18 weeks developed significantly larger atherosclerotic lesions than did TRAF5(+/+)/LDLR(-/-) controls. Plaques of TRAF5-deficient animals contained more lipids and macrophages, whereas smooth muscle cells and collagen remained unchanged. Deficiency of TRAF5 in endothelial cells or in leukocytes enhanced adhesion of inflammatory cells to the endothelium in dynamic adhesion assays in vitro and in murine vessels imaged by intravital microscopy in vivo. TRAF5 deficiency also increased expression of adhesion molecules and chemokines and potentiated macrophage lipid uptake and foam cell formation. These findings coincided with increased activation of JNK and appeared to be independent of TRAF2. Finally, patients with stable or acute coronary heart disease had significantly lower amounts of TRAF5 mRNA in blood compared with healthy controls. Conclusions: Unexpectedly, TRAF5 deficiency accelerates atherogenesis in mice, an effect likely mediated by increased inflammatory cell recruitment to the vessel wall and enhanced foam cell formation.

PMID: 20651286 [PubMed - as supplied by publisher]

Imaging of protein translocation in situ in skeletal muscle of living mice.

Methods Mol Biol. 2010;637:231-44

Authors: Lauritzen HP

Skeletal muscle plays a key role in regulating whole body glucose homeostasis and severe dysfunction in insulin-mediated glucose uptake is the hallmark of insulin-resistant states and type II diabetes. Therefore it is highly pathophysiologically relevant to perform detailed studies of insulin signaling inside skeletal muscle cells in order to elucidate the specific molecular events during both normal and insulin-resistant conditions. So far, cell biology imaging techniques have been limited to in vitro cultured muscle originating from primary or cell line-based myoblasts. However, these types of cultured muscle lack many characteristics of fully differentiated muscle cells. By performing intravital protein translocation analysis directly in situ in living animals, we have been able to give a high-resolution account of the spatial and temporal details during insulin signaling in vivo in muscle that does not have the limitations of in vitro cultures. We have shown that after i.v. insulin injection, PI3-kinase activation and, in turn, GLUT4 translocation are initiated at the plasma membrane proper, the sarcolemma. Then insulin signaling progresses into the t-tubules with a velocity corresponding to the diffusion of sulforhodamine B-conjugated insulin molecules. By using intravital confocal time-lapse analysis we have revealed that the t-tubules are the membrane surface where the majority of the insulin signaling is located.

PMID: 20419438 [PubMed - indexed for MEDLINE]

Development of a liposomal delivery system for temperature-triggered release of a tumor targeting agent, Ln(III)-DOTA-phenylboronate.

Bioorg Med Chem. 2010 Jun 18;

Authors: Djanashvili K, Ten Hagen TL, Blangé R, Schipper D, Peters JA, Koning GA

Liposomes, capable of temperature-triggered content release at the site of interest, can be of great importance for imaging and therapy of tumors. The delivery of imaging agents or therapeutics can be improved by application of liposomes with a gel-to-liquid phase-transition temperature suitable for mild hyperthermia (41-43 degrees C), and by prolonging their circulation time by incorporation of lipids containing polyethyleneglycol moieties. Still, the rapid wash out of the delivered material from the tumor tissue is a major obstacle for both imaging and therapy. In this study, we developed an optimized temperature sensitive liposomal system to be used with mild hyperthermia: highly stable at physiological temperature and with a sharp transition of the bilayer at 41.5 degrees C, with subsequent rapid release of entrapped compounds such as calcein or tumor cell-targeting contrast agents. Intravital microscopy on calcein/rhodamine containing liposomes was applied to demonstrate the applicability of this system in vivo. The calcein loaded liposomes were injected iv into nude mice with a human BLM melanoma tumor implanted in a dorsal skin-fold window chamber. Arrival of the liposomes at the tumor site and content release after temperature increase were monitored. The results demonstrated not only accumulation of the liposomes at the tumor site, but also a massive release of calcein after increase of the temperature to 41 degrees C. The versatility of the thermosensitive liposomes was further demonstrated by encapsulation of a tumor cell-targeting DOTA-phenylboronate conjugate and its release at elevated temperatures. The DOTA ligand in this system is able to chelate a variety of metals suitable for both diagnostic and therapeutic applications, whereas the phenylboronate function is able to target specifically to tumor cells through a covalent binding with sialic acid moieties over-expressed on their surface upon heat-triggered release from the liposomal carrier.

PMID: 20624680 [PubMed - as supplied by publisher]

Triggered content release from optimized stealth thermosensitive liposomes using mild hyperthermia.

J Control Release. 2010 Apr 19;143(2):274-9

Authors: Li L, ten Hagen TL, Schipper D, Wijnberg TM, van Rhoon GC, Eggermont AM, Lindner LH, Koning GA

Liposomes are potent nanocarriers to deliver chemotherapeutic drugs to tumors. However, the inefficient drug release hinders their application. Thermosensitive liposomes (TSL) can release drugs upon heat. This study aims to identify the optimum 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-PEG(2000) (DSPE-PEG(2000)) concentration in stealth TSL to improve content release efficiency under mild hyperthermia (HT). TSL were prepared with DSPE-PEG(2000) from 1 to 10 mol%, around 80 nm in size. Quenched carboxyfluorescein (CF) in aqueous phase represented encapsulated drugs. In vitro temperature/time-dependent CF release and TSL stability in serum were quantified by fluorometry. In vivo CF release in dorsal skin flap window chamber models implanted with human BLM melanoma was captured by confocal microscopy. In vitro heat triggered CF release increased with increasing DSPE-PEG(2000) density. However, 6 mol% and higher DSPE-PEG(2000) caused CF leakage at physiological temperature. TSL with 5 mol% DSPE-PEG(2000) were stable at 37 degrees C, while released 60% CF in 1 min and almost 100% CF in 1h at 42 degrees C. In vivo optical intravital imaging showed immediate massive CF release above 41 degrees C. In conclusion, incorporation of 5 mol% DSPE-PEG(2000) optimized stealth TSL content release triggered by HT.

PMID: 20074595 [PubMed - indexed for MEDLINE]

Near-Infrared Fluorescent Probe for Imaging of Pancreatic beta Cells.

Bioconjug Chem. 2010 Jun 29;

Authors: Reiner T, Kohler RH, Liew CW, Hill JA, Gaglia J, Kulkarni RN, Weissleder R

The ability to image and ultimately quantitate beta-cell mass in vivo will likely have far reaching implications in the study of diabetes biology, in the monitoring of disease progression or response to treatment, and for drug development. Here, using animal models, we report on the synthesis, characterization, and intravital microscopic imaging properties of a near-infrared fluorescent exendin-4 analogue with specificity for the GLP-1 receptor on beta cells (E4(K12)-Fl). The agent demonstrated subnanomolar EC(50) binding concentrations, with high specificity and binding that could be inhibited by GLP-1R agonists. Following intravenous administration to mice, pancreatic islets were readily distinguishable from exocrine pancreas, achieving target-to-background ratios within the pancreas of 6:1, as measured by intravital microscopy. Serial imaging revealed rapid accumulation kinetics (with initial signal within the islets detectable within 3 min and peak fluorescence within 20 min of injection), making this an ideal agent for in vivo imaging.

PMID: 20583828 [PubMed - as supplied by publisher]

Fucose-deficient hematopoietic stem cells have decreased self-renewal and aberrant marrow niche occupancy.

Transfusion. 2010 Jun 21;

Authors: Myers J, Huang Y, Wei L, Yan Q, Huang A, Zhou L

BACKGROUND: Modification of Notch receptors by O-linked fucose and its further elongation by the Fringe family of glycosyltransferase has been shown to be important for Notch signaling activation. Our recent studies disclose a myeloproliferative phenotype, hematopoietic stem cell (HSC) dysfunction, and abnormal Notch signaling in mice deficient in FX, which is required for fucosylation of a number of proteins including Notch. The purpose of this study was to assess the self-renewal and stem cell niche features of fucose-deficient HSCs. STUDY DESIGN AND METHODS: Homeostasis and maintenance of HSCs derived from FX(-/-) mice were studied by serial bone marrow transplantation, homing assay, and cell cycle analysis. Two-photon intravital microscopy was performed to visualize and compare the in vivo marrow niche occupancy by fucose-deficient and wild-type (WT) HSCs. RESULTS: Marrow progenitors from FX(-/-) mice had mild homing defects that could be partially prevented by exogenous fucose supplementation. Fucose-deficient HSCs from FX(-/-) mice displayed decreased self-renewal capability compared with the WT controls. This is accompanied with their increased cell cycling activity and suppressed Notch ligand binding. When tracked in vivo by two-photon intravital imaging, the fucose-deficient HSCs were found localized farther from the endosteum of the calvarium marrow than the WT HSCs. CONCLUSIONS: The current reported aberrant niche occupancy by HSCs from FX(-/-) mice, in the context of a faulty blood lineage homeostasis and HSC dysfunction in mice expressing Notch receptors deficient in O-fucosylation, suggests that fucosylation-modified Notch receptor may represent a novel extrinsic regulator for HSC engraftment and HSC niche maintenance.

PMID: 20573072 [PubMed - as supplied by publisher]

Inflammatory response against different carbon fiber-reinforced PEEK wear particles compared with UHMWPE in vivo.

Acta Biomater. 2010 Jun 8;

Authors: Utzschneider S, Becker F, Grupp TM, Sievers B, Paulus A, Gottschalk O, Jansson V

Poly(ether ether ketone) (PEEK) and its composites are recognized as alternative load-bearing materials for use in arthroplasty because of their mechanical properties. The objective of this project was to evaluate the biological response of two different kinds of carbon fiber-reinforced (CFR) PEEK compared with ultra-high molecular weight polyethylene (UHMWPE) in vivo as a standard load-bearing material. Wear particles of the particulate biomaterials were injected into the left knee joint of female BALB/c mice. Assessment of the synovial microcirculation using intravital fluorescence microscopy as well as histological evaluation of the synovial layer were performed 7days after particle injection. Enhanced leukocyte-endothelial cell interactions and an increase in functional capillary density as well as histological investigations revealed that all tested biomaterials caused significantly (P<0.05) increased inflammatory reactions compared with control animals (injected with sterile phosphate-buffered saline), without any difference between the tested biomaterials (P>0.05). These data suggest that wear debris of CFR-PEEK is comparable with UHMWPE in its biological activity. Therefore, CFR-PEEK represents an alternative load-bearing material because of its superior mechanical and chemical behavior without any increased biological activity of the wear particles, compared with a standard load-bearing material.

PMID: 20570640 [PubMed - as supplied by publisher]

Strategies for high-resolution imaging of epithelial ovarian cancer by laparoscopic nonlinear microscopy.

Transl Oncol. 2010;3(3):181-94

Authors: Williams RM, Flesken-Nikitin A, Ellenson LH, Connolly DC, Hamilton TC, Nikitin AY, Zipfel WR

Ovarian cancer remains the most frequently lethal of the gynecologic cancers owing to the late detection of this disease. Here, by using human specimens and three mouse models of ovarian cancer, we tested the feasibility of nonlinear imaging approaches, the multiphoton microscopy (MPM) and second harmonic generation (SHG) to serve as complementary tools for ovarian cancer diagnosis. We demonstrate that MPM/SHG of intrinsic tissue emissions allows visualization of unfixed, unsectioned, and unstained tissues at a resolution comparable to that of routinely processed histologic sections. In addition to permitting discrimination between normal and neoplastic tissues according to pathological criteria, the method facilitates morphometric assessment of specimens and detection of very early cellular changes in the ovarian surface epithelium. A red shift in cellular intrinsic fluorescence and collagen structural alterations have been identified as additional cancer-associated changes that are indiscernible by conventional pathologic techniques. Importantly, the feasibility of in vivo laparoscopic MPM/SHG is demonstrated by using a "stick" objective lens. Intravital detection of neoplastic lesions has been further facilitated by low-magnification identification of an indicator for cathepsin activity followed by MPM laparoscopic imaging. Taken together, these results demonstrate that MPM may be translatable to clinical settings as an endoscopic approach suitable for high-resolution optical biopsies as well as a pathology tool for rapid initial assessment of ovarian cancer samples.

PMID: 20563260 [PubMed - in process]

Intravital imaging of IL-1beta production in skin.

J Invest Dermatol. 2010 Jun;130(6):1571-80

Authors: Matsushima H, Ogawa Y, Miyazaki T, Tanaka H, Nishibu A, Takashima A

IL-1 is a prototypic inflammatory cytokine that has pathogenic roles in various skin disorders. Although Langerhans cells (LCs) have been reported to express IL-1beta mRNA upon application of contact sensitizers, it remains unclear whether other cell types produce IL-1beta in skin. Thus, we sought to directly identify IL-1beta-producing cells in living animals by construction of transgenic mice expressing DsRed fluorescence protein gene under the control of IL-1beta promoter. Little DsRed fluorescence signal was detected in skin under steady-state conditions. Striking increases in DsRed signal were observed after topical application of a contact sensitizer, oxazolone, which also induced markedly elevated IL-1beta mRNA and protein expression. DsRed signal was expressed primarily by CD45(+)/CD11b(+) myeloid leukocytes in both epidermal and dermal compartments and was detected only in small fractions of epidermal LCs. Interestingly, DsRed(+) cells emerged preferentially as clusters around hair follicles. Intravital confocal imaging experiments revealed highly motile potentials of DsRed(+) cells-they constantly crawled around hair follicles via amoeba-like movements with a mean velocity of 1.0+/-0.4 microm min(-1) (epidermis) or 2.7+/-1.4 microm min(-1) (dermis). The newly developed in vivo imaging system represents a useful tool for studying spatial regulation of IL-1beta production in skin.

PMID: 20147964 [PubMed - indexed for MEDLINE]

A chemotactic gradient sequestered on endothelial heparan sulfate induces directional intraluminal crawling of neutrophils.

Blood. 2010 Jun 8;

Authors: Massena S, Christoffersson G, Hjertström E, Zcharia E, Vlodavsky I, Ausmees N, Rolny C, Li JP, Phillipson M

During infection, chemokines sequestered on endothelium induce recruitment of circulating leukocytes into the tissue where they chemotax along chemokine gradients towards the afflicted site. The aim of this in vivo study was to determine if a chemokine gradient was formed intravascularly and influenced intraluminal neutrophil crawling and transmigration. A chemokine gradient was induced by placing a MIP-2-containing (CXCL2) gel on the cremaster muscle of anesthetized wild-type mice or heparanase over-expressing (hpa-tg) mice with truncated heparan sulfate (HS) side chains. Neutrophil-endothelial interactions were visualized by intravital microscopy and chemokine gradients detected by confocal microscopy. Localized extravascular chemokine release (MIP-2 gel) induced directed neutrophil crawling along a chemotactic gradient immobilized on the endothelium and accelerated their recruitment into the target tissue compared to homogenous extravascular chemokine concentration (MIP-2 superfusion). Endothelial chemokine sequestration occurred exclusively in venules and was HS-dependent, and neutrophils in hpa-tg mice exhibited random crawling. Despite similar numbers of adherent neutrophils in hpa-tg and wild-type mice, the altered crawling in hpa-tg mice was translated into decreased number of emigrated neutrophils and ultimately decreased ability to clear bacterial infections. In conclusion, an intravascular chemokine gradient sequestered by endothelial HS effectively directs crawling leukocytes towards transmigration loci close to the infection site.

PMID: 20530797 [PubMed - as supplied by publisher]

Fluorescent nanoprobes as a biomarker for increased vascular permeability: implications in diagnosis and treatment of cancer and inflammation.

Bioconjug Chem. 2010 Jan;21(1):93-101

Authors: Sandanaraj BS, Gremlich HU, Kneuer R, Dawson J, Wacha S

This article describes the use of a fluorescent nanoprobe as a functional biomarker for the identification of increased vascular permeability in cancer/arthritis disease models. Synthesis of the fluorescent nanoprobe was achieved by passive loading of a fluorophore inside the nanoparticle using thin film hydration method. The outer layer of the nanoprobe was decorated with poly(ethylene glycol) arms to increase the bioavailability of the fluorophore. Stability studies of the nanoprobe showed that the particles were stable up to 70 days. The uptake and internalization of the fluorescent nanoprobe inside target cells was confirmed by fluorescence microscopy studies. Co-localization of the probe with the target tissue in vivo was unambiguously identified using intravital microscopy. Results from in vivo imaging studies showed that the particles had a long half-life in the circulation and passively targeted tumor or arthritic tissue. The increased and specific uptake of the fluorescent nanoprobe in tumor/arthritic tissue is attributed to an enhanced permeation and retention (EPR) effect. Use of an optical method to validate anti-inflammatory drugs in an arthritis disease model is demonstrated in this study. In general, this methodology could be used for detection of leaky vasculature in different pathological states.

PMID: 19958018 [PubMed - indexed for MEDLINE]

Agent-based simulation of T-cell activation and proliferation within a lymph node.

Immunol Cell Biol. 2010 Feb;88(2):172-9

Authors: Bogle G, Dunbar PR

Recent intravital microscopy experiments have revealed the complex behavior of T cells within lymph nodes. Modeling T-cell responses in lymph nodes now requires integration of cell trafficking and motility with the molecular processes involved in T-cell activation. We describe an agent-based model that allows such integration, in which T cells undertake a random walk through a three-dimensional representation of the lymph node paracortex, integrating signals from dendritic cells (DCs), and proliferating in response. The model accommodates simulation of a large number of T cells packed at realistic densities, and includes dynamic cell trafficking that allows the lymph nodes to swell and shrink as the immune response progresses. The results from the model, including the kinetics of cognate T-cell proliferation and release, and the changes in their avidity profile, are similar to those observed in vivo. We therefore propose that this modeling framework is capable of successfully simulating T-cell activation while also accounting for new spatiotemporal knowledge of how T cells and DCs interact. Although some of the parameters used to drive the model are not yet experimentally validated, the model is capable of testing the effects of alternative values for any parameter on the T-cell response. We intend to refine each aspect of the model in collaboration with both theoreticians and experimentalists.

PMID: 19884904 [PubMed - indexed for MEDLINE]

Increased interstitial pressure improves nucleic acid delivery to skin enabling a comparative analysis of constitutive promoters.

Gene Ther. 2010 May 13;

Authors: González-González E, Ra H, Spitler R, Hickerson RP, Contag CH, Kaspar RL

Nucleic acid-based therapies hold great promise for treatment of skin disorders if delivery challenges can be overcome. To investigate one mechanism of nucleic acid delivery to keratinocytes, a fixed mass of expression plasmid was intradermally injected into mouse footpads in different volumes, and reporter expression was monitored by intravital imaging or skin sectioning. Reporter gene expression increased with higher delivery volumes, suggesting that pressure drives nucleic acid uptake into cells after intradermal injections similar to previously published studies for muscle and liver. For spatiotemporal analysis of reporter gene expression, a dual-axis confocal (DAC) fluorescence microscope was used for intravital imaging following intradermal injections. Individual keratinocytes expressing hMGFP were readily visualized in vivo and initially appeared to preferentially express in the stratum granulosum and subsequently migrate to the stratum corneum over time. Fluorescence microscopy of frozen skin sections confirmed the patterns observed by intravital imaging. Intravital imaging with the DAC microscope is a noninvasive method for probing spatiotemporal control of gene expression and should facilitate development and testing of new nucleic acid delivery technologies.Gene Therapy advance online publication, 13 May 2010; doi:10.1038/gt.2010.74.

PMID: 20463756 [PubMed - as supplied by publisher]

Effect of vibration on skin blood flow in an in vivo microcirculatory model.

Biosci Trends. 2007 Dec;1(3):161-6

Authors: Nakagami G, Sanada H, Matsui N, Kitagawa A, Yokogawa H, Sekiya N, Ichioka S, Sugama J, Shibata M

The effect of vibration on skin microcirculation was studied to investigate the possibility of clinical use of vibration to prevent and treat pressure ulcers. Vibrations at a vibrational intensity of 600, 800, or 1,000 mVpp with a fixed frequency of 47 Hz were applied horizontally to the ear of male hairless mice (n = 6 for each group) under inhalation anesthesia. The control group (n = 6) received no vibrations. Venular blood flow was measured by an intravital videomicroscope at the baseline and at 0, 5, and 15 min after the application of vibrations. A significant increase was observed in the 600 mVpp group 5 and 15 min after vibration in comparison to the control group (P = 0.002 and P = 0.046, respectively). We also detected increased blood flow in the 800 mVpp group (P = 0.028) and the 1,000 mVpp group (P = 0.012) 5 min after vibration; however, these increases attenuated after 15 min. These results indicate that direct skin vibration at a frequency of 47 Hz improves skin blood flow. The present study gives further support to the role of vibration on a short-term increase in skin blood flow.

PMID: 20103887 [PubMed - indexed for MEDLINE]

Impact of glycoprotein VI and platelet adhesion on atherosclerosis- a possible role of fibronectin.

J Mol Cell Cardiol. 2010 Apr 26;

Authors: Bültmann A, Li Z, Wagner S, Peluso M, Schönberger T, Weis C, Konrad I, Stellos K, Massberg S, Gawaz M, Ungerer M, Münch G

Glycoprotein VI (GPVI) mediates binding of platelets to subendothelial collagen during acute arterial thrombosis. GPVI interactions with the activated atherosclerotic vascular endothelium during early atherosclerosis, however, are not well understood. In ApoE-/- mice, platelet adhesion to atherosclerotic arteries was increased, as measured by intravital microscopy. This platelet adhesion was significantly inhibited by IV injection of GPVI-Fc (1mg/kg body weight). Atherosclerosis in ApoE-/- mice was attenuated both after 7 and 10weeks of treatment with the anti-GPVI antibody JAQ1 (2mg/kg body weight i.p. twice weekly). Binding of GPVI-Fc (1mg/kg IV) occurred to deeper layers, but also to the luminal site of plaques in atherosclerotic rabbits, but not to the vessel wall of healthy littermates. Gene transfer of GPVI-Fc to the carotid vascular wall significantly attenuated athero-progression and endothelial dysfunction in atherosclerotic rabbits in vivo. Specific binding of the soluble GPVI receptor (GPVI-Fc) to fibronectin was found in vitro to coated ELISA plates. Platelet adhesion to fibronectin was significantly inhibited both by GPVI-Fc and by the anti-GPVI antibody 5C4 ex vivo in flow chamber experiments. CONCLUSION: GPVI plays a role in platelet adhesion to atherosclerotic endothelium in the absence of plaque rupture. Inhibition of GPVI both via GPVI-Fc and anti-GPVI-antibodies results in protection against atherosclerosis in both cholesterol-fed rabbits and ApoE-/- mice. This novel mechanism of GPVI-mediated platelet adhesion - possibly via fibronectin - could relevantly contribute to platelet-triggered atheroprogression.

PMID: 20430036 [PubMed - as supplied by publisher]

Crucial importance of the endothelial K+ channel SK3 and connexin40 in arteriolar dilations during skeletal muscle contraction.

FASEB J. 2010 Apr 28;

Authors: Milkau M, Köhler R, de Wit C

Skeletal muscle activity requires substantial increases in blood flow, and the underlying vasodilation involves endothelial activity, but the contribution of the endothelium-dependent hyperpolarizing factor (EDHF) is only poorly defined. In EDHF signaling, endothelial hyperpolarization mediated by the Ca(2+)-activated K(+) channels SK3 and IK1 is a key step and also initiates gap junction-dependent conducted dilations. We assessed the role of SK3, IK1, and connexin40 (Cx40) in muscular contraction-induced dilations in the microcirculation in vivo. Hitherto, arterioles were observed in the electrically stimulated cremaster skeletal muscle of anesthetized mice lacking SK3, IK1, or Cx40 using intravital microscopy. Genetic deficiency of SK3, but not of IK1, strongly attenuated dilations to muscular contraction. Similarly, pharmacologic blockade of SK3 by the specific blocker UCL1684 impaired such dilations in wild-type and IK1-deficient mice. In contrast, IK1 was required for acetylcholine-induced dilations. Genetic deficiency of Cx40 also attenuated dilations induced by muscular contraction but not by acetylcholine. These data support the concept that endothelial hyperpolarization through activation of SK3 is contributing to exercise hyperemia and the hyperpolarization ascends the vascular tree through gap junctions formed by Cx40 to orchestrate dilation. The differential impact of SK3- and IK1-deficiency on dilations to distinct stimuli suggests stimulus-dependent activation of these endothelial channels.-Milkau, M., Köhler, R., de Wit, C. Crucial importance of the endothelial K(+) channel SK3 and connexin40 in arteriolar dilations during skeletal muscle contraction.

PMID: 20427707 [PubMed - as supplied by publisher]

Stabilizing role of platelet P2Y(12) receptors in shear-dependent thrombus formation on ruptured plaques.

PLoS One. 2010;5(4):e10130

Authors: Nergiz-Unal R, Cosemans JM, Feijge MA, van der Meijden PE, Storey RF, van Giezen JJ, oude Egbrink MG, Heemskerk JW, Kuijpers MJ

BACKGROUND: In most models of experimental thrombosis, healthy blood vessels are damaged. This results in the formation of a platelet thrombus that is stabilized by ADP signaling via P2Y(12) receptors. However, such models do not predict involvement of P2Y(12) in the clinically relevant situation of thrombosis upon rupture of atherosclerotic plaques. We investigated the role of P2Y(12) in thrombus formation on (collagen-containing) atherosclerotic plaques in vitro and in vivo, by using a novel mouse model of atherothrombosis. METHODOLOGY: Plaques in the carotid arteries from Apoe(-/-) mice were acutely ruptured by ultrasound treatment, and the thrombotic process was monitored via intravital fluorescence microscopy. Thrombus formation in vitro was assessed in mouse and human blood perfused over collagen or plaque material under variable conditions of shear rate and coagulation. Effects of two reversible P2Y(12) blockers, ticagrelor (AZD6140) and cangrelor (AR-C69931MX), were investigated. PRINCIPAL FINDINGS: Acute plaque rupture by ultrasound treatment provoked rapid formation of non-occlusive thrombi, which were smaller in size and unstable in the presence of P2Y(12) blockers. In vitro, when mouse or human blood was perfused over collagen or atherosclerotic plaque material, blockage or deficiency of P2Y(12) reduced the thrombi and increased embolization events. These P2Y(12) effects were present at shear rates >500 s(-1), and they persisted in the presence of coagulation. P2Y(12)-dependent thrombus stabilization was accompanied by increased fibrin(ogen) binding. CONCLUSIONS/SIGNIFICANCE: Platelet P2Y(12) receptors play a crucial role in the stabilization of thrombi formed on atherosclerotic plaques. This P2Y(12) function is restricted to high shear flow conditions, and is preserved in the presence of coagulation.

PMID: 20405028 [PubMed - in process]

Single-cell analysis of cytotoxic T cell function by intravital multiphoton microscopy.

Methods Mol Biol. 2010;616:181-92

Authors: Mempel TR

Novel nonlinear optical imaging modalities, most prominently multiphoton microscopy, allow for the direct in vivo visualization of dynamic biological processes in experimental animals at high spatial and temporal resolution. This has led to important new insights into the orchestration of adaptive immune responses. Here we describe in detail a technique that allows for the monitoring of the cytotoxic function of CD8(+) effector T cells in tumor-draining lymph nodes of mice at the single-cell level in vivo using multiphoton intravital microscopy.

PMID: 20379876 [PubMed - in process]

Junctional Adhesion Molecule A Expressed on Human CD34+ Cells Promotes Adhesion on Vascular Wall and Differentiation Into Endothelial Progenitor Cells.

Arterioscler Thromb Vasc Biol. 2010 Apr 8;

Authors: Stellos K, Langer H, Gnerlich S, Panagiota V, Paul A, Schönberger T, Ninci E, Menzel D, Mueller I, Bigalke B, Geisler T, Bültmann A, Lindemann S, Gawaz M

OBJECTIVE: To investigate the role of junctional adhesion molecule A (JAM-A) on adhesion and differentiation of human CD34(+) cells into endothelial progenitor cells. METHODS AND RESULTS: Tissue healing and vascular regeneration is a multistep process requiring firm adhesion of circulating progenitor cells to the vascular wall and their further differentiation into endothelial cells. The role of JAM-A in platelet-mediated adhesion of progenitor cells was investigated by adhesion assays in vitro and with the help of intravital fluorescence microscopy in mice. Preincubation of human CD34(+) progenitor cells with sJAM-A-Fc resulted in significantly decreased adhesion over immobilized platelets or inflammatory endothelium under high shear stress in vitro and after carotid ligation in vivo or ischemia/reperfusion injury in the microcirculation of mice. Human CD34(+) cells express JAM-A, as defined by flow cytometry and Western blot analysis. JAM-A mediates differentiation of CD34(+) cells to endothelial progenitor cells and facilitates CD34(+) cell-induced reendothelialization in vitro. Pretreatment of human CD34(+) cells with sJAM-A-Fc resulted in increased neointima formation 3 weeks after endothelial denudation in the carotid arteries of nonobese diabetic/severe combined immunodeficient mice. CONCLUSIONS: These results indicate that the expression of JAM-A on CD34(+) cells mediates adhesion to the vascular wall after injury and differentiation into endothelial progenitor cells, a mechanism potentially involved in vascular regeneration. Human CD34(+) cells express JAM-A, mediating their interaction with platelets and endothelial cells. Specifically, JAM-A expressed on human CD34(+) progenitor cells regulates their adhesion over immobilized platelets or inflammatory endothelium under high shear stress in vitro and after carotid ligation in vivo or ischemia/reperfusion injury in the microcirculation of mice. Moreover, it mediates differentiation of CD34(+) cells to endothelial progenitor cells and facilitates reendothelialization.

PMID: 20378847 [PubMed - as supplied by publisher]