αMß2 Is Antiatherogenic in Female but Not Male Mice.

Atherosclerosis is a complex inflammatory process characterized by monocyte recruitment into the arterial wall, their differentiation into macrophages, and lipid accumulation. Because integrin αMß2 (CD11b/CD18) mediates multiple diverse functions of leukocytes, we examined its role in atherogenesis. αM-/-/ApoE-/- and ApoE-/- mice were fed a control or high fat diet for 3 or 16 wk to induce atherogenesis. Unexpectedly, αM deficiency accelerated development of atherosclerosis in female but not in male mice. The size of aortic root lesions was 3-4.5-fold larger in female αM-/-/ApoE-/- than in ApoE-/- mice. Monocyte and macrophage content within the lesions was increased 2.5-fold in female αM-/-/ApoE-/- mice due to enhanced proliferation. αMß2 elimination promoted gender-dependent foam cell formation due to enhanced uptake of cholesterol by αM-/-/ApoE-/- macrophages. This difference was attributed to enhanced expression of lipid uptake receptors, CD36 and scavenger receptor A1 (SR-A1), in female mice. Macrophages from female αM-/-/ApoE-/- mice showed dramatically reduced expression of FoxM1 transcription factor and estrogen receptors (ER) α and ß. As their antagonists inhibited the effect of 17ß-estradiol (E2), E2 decreased CD36, SR-A1, and foam cell formation in ApoE-/- macrophages in an ERα- and ERß-dependent manner. However, female αM-/-/ApoE-/- macrophages failed to respond to E2 and maintained elevated CD36, SR-A1, and lipid accumulation. FoxM1 inhibition in ApoE-/- macrophages reduced ERs and enhanced CD36 and SR-A1 expression, whereas FoxM1 overexpression in αM-/-/ApoE-/- macrophages reversed their proatherogenic phenotype. We demonstrate a new, surprising atheroprotective role of αMß2 in female ApoE-/- mice. αMß2 maintains ER expression in macrophages and E2-dependent inhibition of foam cell formation.

Kindlin-2 interacts with endothelial adherens junctions to support vascular barrier integrity.

KEY POINTS: A reduction in Kindlin-2 levels in endothelial cells compromises vascular barrier function. Kindlin-2 is a previously unrecognized component of endothelial adherens junctions. By interacting directly and simultaneously with ß- or γ-catenin and cortical actin filaments, Kindlin-2 stabilizes adherens junctions. The Kindlin-2 binding sites for ß- and γ-catenin reside within its F1 and F3 subdomains. Although Kindlin-2 does not associate directly with tight junctions, its downregulation also destabilizes these junctions. Thus, impairment of both adherens and tight junctions may contribute to enhanced leakiness of vasculature in Kindlin-2+/- mice. ABSTRACT: Endothelial cells (EC) establish a physical barrier between the blood and surrounding tissue. Impairment of this barrier can occur during inflammation, ischaemia or sepsis and cause severe organ dysfunction. Kindlin-2, which is primarily recognized as a focal adhesion protein in EC, was not anticipated to have a role in vascular barrier. We tested the role of Kindlin-2 in regulating vascular integrity using several different approaches to decrease Kindlin-2 levels in EC. Reduced levels of Kindlin-2 in Kindlin-2+/- mice aortic endothelial cells (MAECs) from these mice, and human umbilical ECs (HUVEC) treated with Kindlin-2 siRNA showed enhanced basal and platelet-activating factor (PAF) or lipopolysaccharide-stimulated vascular leakage compared to wild-type (WT) counterparts. PAF preferentially disrupted the Kindlin-2+/- MAECs barrier to BSA and dextran and reduced transendothelial resistance compared to WT cells. Kindlin-2 co-localized and co-immunoprecipitated with vascular endothelial cadherin-based complexes, including ß- and γ-catenin and actin, components of adherens junctions (AJ). Direct interaction of Kindlin-2 with ß- and γ-catenin and actin was demonstrated in co-immunoprecipitation and surface plasmon resonance experiments. In thrombin-stimulated HUVECs, Kindlin-2 and cortical actin dissociated from stable AJs and redistributed to radial actin stress fibres of remodelling focal AJs. The ß- and γ-catenin binding site resides within the F1 and F3 subdomains of Kindlin-2 but not the integrin binding site in F3. These results establish a previously unrecognized and vital role of Kindlin-2 with respect to maintaining the vascular barrier by linking Vascuar endothelial cadherin-based complexes to cortical actin and thereby stabilizing AJ.

Distinct Effects of Integrins αXß2 and αMß2 on Leukocyte Subpopulations during Inflammation and Antimicrobial Responses.

Integrins αMß2 and αXß2 are homologous adhesive receptors that are expressed on many of the same leukocyte populations and bind many of the same ligands. Although αMß2 was extensively characterized and implicated in leukocyte inflammatory and immune functions, the roles of αXß2 remain largely obscure. Here, we tested the ability of mice deficient in integrin αMß2 or αXß2 to deal with opportunistic infections and the capacity of cells derived from these animals to execute inflammatory functions. The absence of αMß2 affected the recruitment of polymorphonuclear neutrophils (PMN) to bacterial and fungal pathogens as well as to model inflammatory stimuli, and αMß2-deficient PMN displayed defective inflammatory functions. In contrast, deficiency of αXß2 abrogated intraperitoneal recruitment and adhesive functions of monocytes and macrophages (Mϕ) and the ability of these cells to kill/phagocytose Candida albicans or Escherichia coli cells both ex vivo and in vivo During systemic candidiasis, the absence of αXß2 resulted in the loss of antifungal activity by tissue Mϕ and inhibited the production of tumor necrosis factor alpha (TNF-α)/interleukin-6 (IL-6) in infected kidneys. Deficiency of αMß2 suppressed Mϕ egress from the peritoneal cavity, decreased the production of anti-inflammatory IL-10, and stimulated the secretion of IL-6. The absence of αXß2, but not of αMß2, increased survival against a septic challenge with lipopolysaccharide (LPS) by 2-fold. Together, these results suggest that αMß2 plays a primary role in PMN inflammatory functions and regulates the anti-inflammatory functions of Mϕ, whereas αXß2 is central in the regulation of inflammatory functions of recruited and tissue-resident Mϕ.

Dual-modality gene reporter for in vivo imaging.

The ability to track cells and their patterns of gene expression in living organisms can increase our understanding of tissue development and disease. Gene reporters for bioluminescence, fluorescence, radionuclide, and magnetic resonance imaging (MRI) have been described but these suffer variously from limited depth penetration, spatial resolution, and sensitivity. We describe here a gene reporter, based on the organic anion transporting protein Oatp1a1, which mediates uptake of a clinically approved, Gd(3+)-based, hepatotrophic contrast agent (gadolinium-ethoxybenzyl-diethylenetriamine pentaacetic acid). Cells expressing the reporter showed readily reversible, intense, and positive contrast (up to 7.8-fold signal enhancement) in T1-weighted magnetic resonance images acquired in vivo. The maximum signal enhancement obtained so far is more than double that produced by MRI gene reporters described previously. Exchanging the Gd(3+) ion for the radionuclide, (111)In, also allowed detection by single-photon emission computed tomography, thus combining the spatial resolution of MRI with the sensitivity of radionuclide imaging.

Short fungal fractions of ß-1,3 glucans affect platelet activation.

Platelets are capable of binding, aggregating, and internalizing microorganisms, which enhances the elimination of pathogens from the blood. The yeast Candida albicans is a pathobiont causing life-threatening invasive infections. Its cell wall contains ß-1,3 glucans that are known to trigger a wide range of host cell activities and to circulate during infection. We studied the effect of ß-1,3 glucan fractions (BGFs) consisting of diglucosides (Glc2), tetraglucosides (Glc4), and pentaglucosides (Glc5) on human platelets, their mechanisms of action, and their possible impact on host defenses. The effect of BGFs on the coagulation process was determined by measuring thrombin generation. Platelets pretreated with BGFs were analyzed in terms of activation, receptor expression, aggregation, and adhesion to neutrophils and to C. albicans The results show that BGFs affected the endogenous thrombin potential in a concentration-dependent manner. For platelet activation, BGFs at a low concentration (2 µmol/l) reduced ATP release and prevented the phosphorylation of protein kinase C. BGFs diminished the expression of P-selectin and the activation of αIIbß3 BGFs decreased platelet aggregation and the interaction between thrombin-stimulated platelets and neutrophils, fibrinogen, and C. albicans GLc5 decreased ATP release and TGF-ß1 production in response to TLR4 upregulation in thrombin-stimulated platelets, but TLR4 blockage abolished the effect of BGFs on platelets. This study provides evidence that fungal pentaglucosides modulate platelet activity mediated via TLR4 stimulation and reduce platelet-neutrophil interaction.

Dual role of the leukocyte integrin αMß2 in angiogenesis.

Polymorphonuclear neutrophils (PMNs) and macrophages are crucial contributors to neovascularization, serving as a source of chemokines, growth factors, and proteases. α(M)ß(2)(CD11b/CD18) and α(L)ß(2)(CD11a/CD18) are expressed prominently and have been implicated in various responses of these cell types. Thus, we investigated the role of these ß2 integrins in angiogenesis. Angiogenesis was analyzed in wild-type (WT), α(M)-knockout (α(M)(-/-)), and α(L)-deficient (α(L)(-/-)) mice using B16F10 melanoma, RM1 prostate cancer, and Matrigel implants. In all models, vascular area was decreased by 50-70% in α(M)(-/-) mice, resulting in stunted tumor growth as compared with WT mice. In contrast, α(L) deficiency did not impair angiogenesis and tumor growth. The neovessels in α(M)(-/-) mice were leaky and immature because they lacked smooth muscle cell and pericytes. Defective angiogenesis in the α(M)(-/-) mice was associated with attenuated PMN and macrophage recruitment into tumors. In contrast to WT or the α(L)(-/-) leukocytes, the α(M)(-/-) myeloid cells showed impaired plasmin (Plm)-dependent extracellular matrix invasion, resulting from 50-75% decrease in plasminogen (Plg) binding and pericellular Plm activity. Surface plasmon resonance verified direct interaction of the α(M)I-domain, the major ligand binding site in the ß(2) integrins, with Plg. However, the α(L)I-domain failed to bind Plg. In addition, endothelial cells failed to form tubes in the presence of conditioned medium collected from TNF-α-stimulated PMNs derived from the α(M)(-/-) mice because of severely impaired degranulation and secretion of VEGF. Thus, α(M)ß(2) plays a dual role in angiogenesis, supporting not only Plm-dependent recruitment of myeloid cells to angiogenic niches, but also secretion of VEGF by these cells.

Kindlin-2 regulates hemostasis by controlling endothelial cell-surface expression of ADP/AMP catabolic enzymes via a clathrin-dependent mechanism.

Kindlin-2, a widely distributed cytoskeletal protein, has been implicated in integrin activation, and its absence is embryonically lethal in mice. In the present study, we tested whether hemostasis might be perturbed in kindlin-2(+/-) mice. Bleeding time and carotid artery occlusion time were significantly prolonged in kindlin-2(+/-) mice. Whereas plasma concentrations/activities of key coagulation/fibrinolytic proteins and platelet counts and aggregation were similar in wild-type and kindlin-2(+/-) mice, kindlin-2(+/-) endothelial cells (ECs) showed enhanced inhibition of platelet aggregation induced by adenosine 5'-diphosphate (ADP) or low concentrations of other agonists. Cell-surface expression of 2 enzymes involved in ADP/adenosine 5'-monophosphate (AMP) degradation, adenosine triphosphate (ATP) diphosphohydrolase (CD39) and ecto-5'-nucleotidase (CD73) were increased twofold to threefold on kindlin-2(+/-) ECs, leading to enhanced ATP/ADP catabolism and production of adenosine, an inhibitor of platelet aggregation. Trafficking of CD39 and CD73 at the EC surface was altered in kindlin-2(+/-) mice. Mechanistically, this was attributed to direct interaction of kindlin-2 with clathrin heavy chain, thereby controlling endocytosis and recycling of CD39 and CD73. The interaction of kindlin-2 with clathrin was independent of its integrin binding site but still dependent on a site within its F3 subdomain. Thus, kindlin-2 regulates trafficking of EC surface enzymes that control platelet responses and hemostasis.

Integrin αXß2 is a leukocyte receptor for Candida albicans and is essential for protection against fungal infections.

The opportunistic fungus Candida albicans is one of the leading causes of infections in immunocompromised patients, and innate immunity provides a principal mechanism for protection from the pathogen. In the present work, the role of integrin α(X)ß2 in the pathogenesis of fungal infection was assessed. Both purified α(X)ß2 and α(X)ß2-expressing human epithelial kidney 293 cells recognized and bound to the fungal hyphae of SC5314 strain of C. albicans but not to the yeast form or to hyphae of a strain deficient in the fungal mannoprotein, Pra1. The binding of the integrin to the fungus was inhibited by ß-glucans but not by mannans, implicating a lectin-like activity in recognition but distinct in specificity from that of α(M)ß2. Mice deficient in α(X)ß2 were more prone to systemic infection with the LD50 fungal inoculum decreasing 3-fold in α(X)ß2-deficient mice compared with wild-type mice. After challenging i.v. with 1.5 × 104 cell/g, 60% of control C57BL/6 mice died within 14 d compared with 100% mortality of α(X)ß2-deficient mice within 9 d. Organs taken from α(X)ß2-deficient mice 16 h postinfection revealed a 10-fold increase in fungal invasion into the brain and a 2-fold increase into the liver. These data indicate that α(X)ß2 is important for protection against systemic C. albicans infections and macrophage subsets in the liver, Kupffer cells, and in the brain, microglial cells use α(X)ß2 to control fungal invasion.

Multi-scale in vivo imaging of tumour development using a germline conditional triple-reporter system.

Imaging reporter genes are indispensable for visualising biological processes in living subjects, particularly in cancer research where they have been used to observe tumour development, cancer cell dissemination, and treatment response. Engineering reporter genes into the germline frequently involves single imaging modality reporters operating over limited spatial scales. To address these limitations, we developed an inducible triple-reporter mouse model (Rosa26LSL - NRL) that integrates reporters for complementary imaging modalities, flfluorescence, bioluminescence and positron emission tomography (PET), along with inducible Cre-lox functionality for precise spatiotemporal control of reporter expression. We demonstrated robust reporter inducibility across various tissues in the Rosa26LSL - NRL mouse, facilitating effective tracking and characterisation of tumours in liver and lung cancer mouse models. We precisely pinpointed tumour location using multimodal whole-body imaging which guided in situ lung microscopy to visualise cell-cell interactions within the tumour microenvironment. The triple-reporter system establishes a robust new platform technology for multi-scale investigation of biological processes within whole animals, enabling tissue-specific and sensitive cell tracking, spanning from the whole-body to cellular scales.

Noninvasive Stratification of Colon Cancer by Multiplex PET Imaging.

PURPOSE: The current approach for molecular subtyping of colon cancer relies on gene expression profiling, which is invasive and has limited ability to reveal dynamics and spatial heterogeneity. Molecular imaging techniques, such as PET, present a noninvasive alternative for visualizing biological information from tumors. However, the factors influencing PET imaging phenotype, the suitable PET radiotracers for differentiating tumor subtypes, and the relationship between PET phenotypes and tumor genotype or gene expression-based subtyping remain unknown. EXPERIMENTAL DESIGN: In this study, we conducted 126 PET scans using four different metabolic PET tracers, [18F]fluorodeoxy-D-glucose ([18F]FDG), O-(2-[18F]fluoroethyl)-l-tyrosine ([18F]FET), 3'-deoxy-3'-[18F]fluorothymidine ([18F]FLT), and [11C]acetate ([11C]ACE), using a spectrum of five preclinical colon cancer models with varying genetics (BMT, AKPN, AK, AKPT, KPN), at three sites (subcutaneous, orthograft, autochthonous) and at two tumor stages (primary vs. metastatic). RESULTS: The results demonstrate that imaging signatures are influenced by genotype, tumor environment, and stage. PET imaging signatures exhibited significant heterogeneity, with each cancer model displaying distinct radiotracer profiles. Oncogenic Kras and Apc loss showed the most distinctive imaging features, with [18F]FLT and [18F]FET being particularly effective, respectively. The tissue environment notably impacted [18F]FDG uptake, and in a metastatic model, [18F]FET demonstrated higher uptake. CONCLUSIONS: By examining factors contributing to PET-imaging phenotype, this study establishes the feasibility of noninvasive molecular stratification using multiplex radiotracer PET. It lays the foundation for further exploration of PET-based subtyping in human cancer, thereby facilitating noninvasive molecular diagnosis.

Binding of CD40L to Mac-1's I-domain involves the EQLKKSKTL motif and mediates leukocyte recruitment and atherosclerosis--but does not affect immunity and thrombosis in mice.

RATIONALE: CD40L figures prominently in chronic inflammatory diseases such as atherosclerosis. However, since CD40L potently regulates immune function and hemostasis by interaction with CD40 receptor and the platelet integrin GPIIb/IIIa, its global inhibition compromises host defense and generated thromboembolic complications in clinical trials. We recently reported that CD40L mediates atherogenesis independently of CD40 and proposed Mac-1 as an alternate receptor. OBJECTIVE: Here, we molecularly characterized the CD40L-Mac-1 interaction and tested whether its selective inhibition by a small peptide modulates inflammation and atherogenesis in vivo. METHODS AND RESULTS: CD40L concentration-dependently bound to Mac-1 I-domain in solid phase binding assays, and a high-affinity interaction was revealed by surface-plasmon-resonance analysis. We identified the motif EQLKKSKTL, an exposed loop between the α1 helix and the ß-sheet B, on Mac-1 as binding site for CD40L. A linear peptide mimicking this sequence, M7, specifically inhibited the interaction of CD40L and Mac-1. A cyclisized version optimized for in vivo use, cM7, decreased peritoneal inflammation and inflammatory cell recruitment in vivo. Finally, LDLr(-/-) mice treated with intraperitoneal injections of cM7 developed smaller, less inflamed atherosclerotic lesions featuring characteristics of stability. However, cM7 did not interfere with CD40L-CD40 binding in vitro and CD40L-GPIIb/IIIa-mediated thrombus formation in vivo. CONCLUSIONS: We present the novel finding that CD40L binds to the EQLKKSKTL motif on Mac-1 mediating leukocyte recruitment and atherogenesis. Specific inhibition of CD40L-Mac-1 binding may represent an attractive anti-inflammatory treatment strategy for atherosclerosis and other inflammatory conditions, potentially avoiding the unwanted immunologic and thrombotic effects of global inhibition of CD40L.

Radiosynthesis and Analysis of (S)-4-(3-[18F]Fluoropropyl)-L-Glutamic Acid.

PURPOSE: (S)-4-(3-[18F]Fluoropropyl)-L-glutamic acid ([18F]FSPG) is an L-glutamate derivative used as a PET biomarker to assess intracellular redox status in vivo through targeting of the cystine/glutamate antiporter protein, xc- transporter. In this report, we describe a radiosynthesis of [18F]FSPG for use in PET studies that address specific challenges in relation to the radiotracer purity, molar activity, and quality control testing methods. PROCEDURES: The radiosynthesis of [18F]FSPG was performed using a customised RNPlus Research automated radiosynthesis system (Synthra GmbH, Hamburg, Germany). [18F]FSPG was labelled in the 3-fluoropropylmoiety at the 4-position of the glutamic acid backbone with fluorine-18 via substitution of nucleophilic [18F]fluoride with a protected naphthylsulfonyloxy-propyl-L-glutamate derivative. Radiochemical purity of the final product was determined by radio HPLC using a new method of direct analysis using a Hypercarb C18 column. RESULTS: The average radioactivity yield of [18F]FSPG was 4.2 GBq (range, 3.4-4.8 GBq) at the end of synthesis, starting from 16 GBq of [18F]fluoride at the end of bombardment (n = 10) in a synthesis time of 50 min. The average molar activity and radioactivity volumetric concentration at the end of synthesis were 66 GBq µmol-1 (range, 48-73 GBq µmol-1) and 343-400 MBq mL-1, respectively. CONCLUSION: Stability tests using a 4.6 GBq dose with a radioactivity volumetric concentration of 369 MBq mL-1 at the end of synthesis showed no observable radiolysis 3 h after production. The formulated product is of high radiochemical purity (> 95%) and higher molar activity compared to previous methods and is safe to inject into mice up to 3 h after production.

Positron emission tomography imaging of the sodium iodide symporter senses real-time energy stress in vivo.

BACKGROUND: Tissue environment is critical in determining tumour metabolic vulnerability. However, in vivo drug testing is slow and waiting for tumour growth delay may not be the most appropriate endpoint for metabolic treatments. An in vivo method for measuring energy stress would rapidly determine tumour targeting in a physiologically relevant environment. The sodium-iodide symporter (NIS) is an imaging reporter gene whose protein product co-transports sodium and iodide, and positron emission tomography (PET) radiolabelled anions into the cell. Here, we show that PET imaging of NIS-mediated radiotracer uptake can rapidly visualise tumour energy stress within minutes following in vivo treatment. METHODS: We modified HEK293T human embryonic kidney cells, and A549 and H358 lung cancer cells to express transgenic NIS. Next, we subjected these cells and implanted tumours to drugs known to induce metabolic stress to observe the impact on NIS activity and energy charge. We used [18F]tetrafluoroborate positron emission tomography (PET) imaging to non-invasively image NIS activity in vivo. RESULTS: NIS activity was ablated by treating HEK293T cells in vitro, with the Na+/K+ ATPase inhibitor digoxin, confirming that radiotracer uptake was dependent on the sodium-potassium concentration gradient. NIS-mediated radiotracer uptake was significantly reduced (- 58.2%) following disruptions to ATP re-synthesis by combined glycolysis and oxidative phosphorylation inhibition in HEK293T cells and by oxidative phosphorylation inhibition (- 16.6%) in A549 cells in vitro. PET signal was significantly decreased (- 56.5%) within 90 min from the onset of treatment with IACS-010759, an oxidative phosphorylation inhibitor, in subcutaneous transgenic A549 tumours in vivo, showing that NIS could rapidly and sensitively detect energy stress non-invasively, before more widespread changes to phosphorylated AMP-activated protein kinase, phosphorylated pyruvate dehydrogenase, and GLUT1 were detectable. CONCLUSIONS: NIS acts as a rapid metabolic sensor for drugs that lead to ATP depletion. PET imaging of NIS could facilitate in vivo testing of treatments targeting energetic pathways, determine drug potency, and expedite metabolic drug development.

High molar activity [18F]tetrafluoroborate synthesis for sodium iodide symporter imaging by PET.

BACKGROUND: Sodium iodide symporter (NIS) imaging by positron emission tomography (PET) is gaining traction in nuclear medicine, with an increasing number of human studies being published using fluorine-18 radiolabelled tetrafluoroborate ([18F]TFB). Clinical success of any radiotracer relies heavily on its accessibility, which in turn depends on the availability of robust radiolabelling procedures providing a radiotracer in large quantities and of high radiopharmaceutical quality. RESULTS: Here we publish an improved radiolabelling method and quality control procedures for high molar activity [18F]TFB. The use of ammonium hydroxide for [18F]fluoride elution, commercially available boron trifluoride-methanol complex dissolved in acetonitrile as precursor and removal of unreacted [18F]fluoride on Florisil solid-phase extraction cartridges resulted in the reliable production of [18F]TFB on SYNTHRA and TRACERLAB FXFN automated synthesizers with radiochemical yields in excess of 30%, radiochemical purities in excess of 98% and molar activities in the range of 34-217 GBq/µmol at the end of synthesis. PET scanning of a mouse lung tumour model carrying a NIS reporter gene rendered images of high quality and improved sensitivity. CONCLUSIONS: A novel automated radiosynthesis procedure for [18F]tetrafluoroborate has been developed that provides the radiotracer with high molar activity, suitable for preclinical imaging of NIS reporter gene.

Regulation of innate immune response to Candida albicans infections by αMß2-Pra1p interaction.

Candida albicans is a common opportunistic fungal pathogen and is the leading cause of invasive fungal diseases in immunocompromised individuals. The induction of cell-mediated immunity to C. albicans is one of the main tasks of cells of the innate immune system, and in vitro evidence suggests that integrin α(M)ß2 (CR3, Mac-1, and CD11b/CD18) is the principal leukocyte receptor involved in recognition of the fungus. Using α(M)ß2-KO mice and mutated strains of C. albicans in two models of murine candidiasis, we demonstrate that neutrophils derived from mice deficient in α(M)ß2 have a reduced ability to kill C. albicans and that the deficient mice themselves exhibit increased susceptibility to fungal infection. Disruption of the PRA1 gene of C. albicans, the primary ligand for α(M)ß2, protects the fungus against leukocyte killing in vitro and in vivo, impedes the innate immune response to the infection, and increases fungal virulence and organ invasion in vivo. Thus, recognition of pH-regulated antigen 1 protein (Pra1p) by α(M)ß2 plays a pivotal role in determining fungal virulence and host response and protection against C. albicans infection.

Expression, activation, and function of integrin alphaMbeta2 (Mac-1) on neutrophil-derived microparticles.

Leukocyte-derived microparticles (MPs) are markers of cardiovascular diseases and contribute to pathogenesis by their interaction with various cell types. The presence and activation state of a multifunctional leukocyte receptor, integrin alpha(M)beta(2) (CD11b/18), on MPs derived from human neutrophils (PMNs) were examined. alpha(M)beta(2) expression was significantly enhanced on MPs derived from stimulated compared with resting PMNs. Furthermore, alpha(M)beta(2) on MPs from stimulated but not resting PMNs was in an activated conformation because it was capable of binding activation-specific monoclonal antibodies (CBRM1/5 and mAb24) and soluble fibrinogen. MPs expressing active alpha(M)beta(2) interacted with and were potent activators of resting platelets as assessed by induction of P-selectin expression and activation of alpha(IIb)beta(3). With the use of function-blocking antibodies and MPs obtained from alpha(M)(-/-)-deficient mice, we found that engagement of GPIbalpha on platelets by alpha(M)beta(2) on MPs plays a pivotal role in MP binding. Platelet activation by MPs occurs by a pathway dependent on Akt phosphorylation. PSGL-1/P-selectin interaction also is involved in the conjugation of MPs to platelets, and the combination of blocking reagents to both alpha(M)beta(2)/GPIbalpha and to PSGL-1/P-selectin completely abrogates MP-induced platelet activation. Thus, cooperation of these 2 receptor/counterreceptor systems regulates the prothrombotic properties of PMN-derived MPs.

Neutrophil apoptosis: selective regulation by different ligands of integrin alphaMbeta2.

Neutrophils undergo spontaneous apoptosis, but their survival can be extended during inflammatory responses. alpha(M)beta(2) is reported either to delay or accelerate neutrophil apoptosis, but the mechanisms by which this integrin can support such diametrically opposed responses are poorly understood. The abilities of closely related alpha(M)beta(2) ligands, plasminogen and angiostatin, derived from plasminogen, as well as fibrinogen and its two derivative alpha(M)beta(2) recognition peptides, P1 and P2-C, differed markedly in their effects on neutrophil apoptosis. Plasminogen, fibrinogen, and P2-C suppressed apoptosis via activation of Akt and ERK1/2 kinases, while angiostatin and P1 failed to activate these prosurvival pathways and did not prevent neutrophil apoptosis. Using cells transfected with alpha(M)beta(2) or its individual alpha(M) or beta(2) subunits, and purified receptors and its constituent chains, we show that engagement of both subunits with prosurvival ligands is essential for induction of the prosurvival response. Hence, engagement of a single integrin by closely related ligands can induce distinct signaling pathways, which can elicit distinct cellular responses.

18F-C2Am: a targeted imaging agent for detecting tumor cell death in vivo using positron emission tomography.

INTRODUCTION: Trialing novel cancer therapies in the clinic would benefit from imaging agents that can detect early evidence of treatment response. The timing, extent and distribution of cell death in tumors following treatment can give an indication of outcome. We describe here an 18F-labeled derivative of a phosphatidylserine-binding protein, the C2A domain of Synaptotagmin-I (C2Am), for imaging tumor cell death in vivo using PET. METHODS: A one-pot, two-step automated synthesis of N-(5-[18F]fluoropentyl)maleimide (60 min synthesis time, > 98% radiochemical purity) has been developed, which was used to label the single cysteine residue in C2Am within 30 min at room temperature. Binding of 18F-C2Am to apoptotic and necrotic tumor cells was assessed in vitro, and also in vivo, by dynamic PET and biodistribution measurements in mice bearing human tumor xenografts treated with a TRAILR2 agonist or with conventional chemotherapy. C2Am detection of tumor cell death was validated by correlation of probe binding with histological markers of cell death in tumor sections obtained immediately after imaging. RESULTS: 18F-C2Am showed a favorable biodistribution profile, with predominantly renal clearance and minimal retention in spleen, liver, small intestine, bone and kidney, at 2 h following probe administration. 18F-C2Am generated tumor-to-muscle (T/m) ratios of 6.1 ± 2.1 and 10.7 ± 2.4 within 2 h of probe administration in colorectal and breast tumor models, respectively, following treatment with the TRAILR2 agonist. The levels of cell death (CC3 positivity) following treatment were 12.9-58.8% and 11.3-79.7% in the breast and colorectal xenografts, respectively. Overall, a 20% increase in CC3 positivity generated a one unit increase in the post/pre-treatment tumor contrast. Significant correlations were found between tracer uptake post-treatment, at 2 h post-probe administration, and histological markers of cell death (CC3: Pearson R = 0.733, P = 0.0005; TUNEL: Pearson R = 0.532, P = 0.023). CONCLUSION: The rapid clearance of 18F-C2Am from the blood pool and low kidney retention allowed the spatial distribution of cell death in a tumor to be imaged during the course of therapy, providing a rapid assessment of tumor treatment response. 18F-C2Am has the potential to be used in the clinic to assess early treatment response in tumors.

[18F]fluoroethyltyrosine-induced Cerenkov Luminescence Improves Image-Guided Surgical Resection of Glioma.

The extent of surgical resection is significantly correlated with outcome in glioma; however, current intraoperative navigational tools are useful only in a subset of patients. We show here that a new optical intraoperative technique, Cerenkov luminescence imaging (CLI) following intravenous injection of O­(2-[18F]fluoroethyl)-L-tyrosine (FET), can be used to accurately delineate glioma margins, performing better than the current standard of fluorescence imaging with 5-aminolevulinic acid (5-ALA). Methods: Rats implanted orthotopically with U87, F98 and C6 glioblastoma cells were injected with FET and 5-aminolevulinic acid (5-ALA). Positive and negative tumor regions on histopathology were compared with CL and fluorescence images. The capability of FET CLI and 5-ALA fluorescence imaging to detect tumor was assessed using receptor operator characteristic curves and optimal thresholds (CLIOptROC and 5-ALAOptROC) separating tumor from healthy brain tissue were determined. These thresholds were used to guide prospective tumor resections, where the presence of tumor cells in the resected material and in the remaining brain were assessed by Ki-67 staining. Results: FET CLI signal was correlated with signal in preoperative PET images (y = 1.06x - 0.01; p < 0.0001) and with expression of the amino acid transporter SLC7A5 (LAT1). FET CLI (AUC = 97%) discriminated between glioblastoma and normal brain in human and rat orthografts more accurately than 5-ALA fluorescence (AUC = 91%), with a sensitivity >92% and specificity >91%, and resulted in a more complete tumor resection. Conclusion: FET CLI can be used to accurately delineate glioblastoma tumor margins, performing better than the current standard of fluorescence imaging following 5-ALA administration, and is therefore a promising technique for clinical translation.

Leukocyte engagement of fibrin(ogen) via the integrin receptor alphaMbeta2/Mac-1 is critical for host inflammatory response in vivo.

The leukocyte integrin alpha(M)beta(2)/Mac-1 appears to support the inflammatory response through multiple ligands, but local engagement of fibrin(ogen) may be particularly important for leukocyte function. To define the biological significance of fibrin(ogen)-alpha(M)beta(2) interaction in vivo, gene-targeted mice were generated in which the alpha(M)beta(2)-binding motif within the fibrinogen gamma chain (N(390)RLSIGE(396)) was converted to a series of alanine residues. Mice carrying the Fibgamma(390-396A) allele maintained normal levels of fibrinogen, retained normal clotting function, supported platelet aggregation, and never developed spontaneous hemorrhagic events. However, the mutant fibrinogen failed to support alpha(M)beta(2)-mediated adhesion of primary neutrophils, macrophages, and alpha(M)beta(2)-expressing cell lines. The elimination of the alpha(M)beta(2)-binding motif on fibrin(ogen) severely compromised the inflammatory response in vivo as evidenced by a dramatic impediment in leukocyte clearance of Staphylococcus aureus inoculated into the peritoneal cavity. This defect in bacterial clearance was due not to diminished leukocyte trafficking but rather to a failure to fully implement antimicrobial functions. These studies definitively demonstrate that fibrin(ogen) is a physiologically relevant ligand for alpha(M)beta(2), integrin engagement of fibrin(ogen) is critical to leukocyte function and innate immunity in vivo, and the biological importance of fibrinogen in regulating the inflammatory response can be appreciated outside of any alteration in clotting function.