Flow Chamber Assay Research Articles

Abstract 653: Endothelial ASC Modulates Early Atherosclerosis by Limiting Monocyte Recruitment

The inflammasome consists of an innate immune receptor, the adaptor molecule ASC, and the effector Caspase-1 that is responsible for processing signature cytokines such as IL-1beta. The exact role of the inflammasome in atherosclerosis is not clear since both pathogenic and dispensable roles have been reported. Notably, ASC has functions that may impact atherosclerosis but are unrelated to the inflammasome, for example the induction of apoptosis and the regulation of pro-inflammatory transcription factors. The various roles of ASC (i.e. IL-1beta processing, cell death, NF-kappaB and AP-1 regulation) may modulate the progression of atherosclerosis depending on lesion stage and explain the divergent findings highlighted above. To examine the role of ASC in lesion progression, we bred ASC -deficient mice onto the Ldlr -/- background and initiated a cholesterol-rich diet (CRD). In the early stages of atherogenesis (2 weeks of CRD), ASC -/- Ldlr -/- mice had a significantly increased lesion surface area, leukocyte content, and rate of Ly6C hi monocyte recruitment in the ascending aorta. Bone marrow transplant experiments suggested that ASC deficiency in the vessel wall was responsible for this phenotype. Knockdown of ASC by siRNA in cultured human umbilical vein endothelial cells lead to increased TNF-alpha-induced expression of VCAM1 and monocyte adhesion in parallel-plate flow chamber assays. Monocyte adhesion was not affected by silencing Caspase-1, suggesting that the effect of ASC was independent of the inflammasome. Preliminary findings implicate MAPK family members in ASC-mediated regulation of endothelial adhesion molecules. At 16 weeks of a CRD ASC -/- Ldlr -/- mice had a significantly decreased lesion size compared with controls. Lesions from ASC -/- Ldlr -/- mice had a smaller necrotic core area accompanied by decreased numbers of apoptotic leukocytes as determined by active Caspase-3 and TUNEL positivity. These data are in accordance with the pro-atherogenic role of ASC in later stages of atherosclerosis. However our findings demonstrate a novel protective role for ASC in the early stage, which represents a non-inflammasome function via modulation of endothelial-associated adhesion molecules.

Identification of sharpin as a molecule regulating leukocyte transmigration from siRNA screens (LB281)

Migration of leukocytes into tissues is a hallmark of an acute inflammation. The migration is regulated by molecular interactions between adhesion molecules on endothelium and their counterparts on leukocytes. Here we have used siRNA‐based screens to search for new molecules regulating leukocyte transmigration through endothelium. The functional significance of the hits was confirmed using time‐lapse microscopy in in vitro flow chamber assays, in which human leukocytes were perfused with a defined shear stress over a confluent monolayer of cultured endothelial cells. In peripheral blood mononuclear cells a cytosolic protein, SHARPIN, was localized to uropods of migrating leukocytes. Moreover, using siRNA silencing we found that after the transmigration, the leukocytes without SHARPIN trapped close to transmigration site while control leukocytes moved onwards. In endothelial cells the use of siRNA to silence one ephrin type‐B receptor, reduced granulocyte transmigration through endothelium while overexpressing of it increased transmigration. As a conclusion we have found new molecules in leukocytes and in endothelium that regulate leukocyte transmigration.

CD47 contributes to neutrophil recruitment (146.2)

Leukocyte recruitment plays a key role in an inflammatory response. CD47 associates “in cis” with β1, β2 and β3 integrins. CD47 also interacts “in trans” with Signal Regulatory Proteins (SIRP‐α and ‐γ) and thrombospondin (TSP). CD47 plays an important but incompletely understood role in the innate and adaptive immune responses. Neutrophil recruitment in CD47KO mice is reduced in models of peritonitis, lung injury and dermal air pouch inflammation, but the exact contribution(s) of CD47 to neutrophil (PMN) extravasation is unclear. Here we report that in the TNF‐α‐stimulated cremaster muscle microcirculation, PMN rolling velocity is elevated compared to WT (17.8±1.5μm/s vs. 14.3±1.0μm/s, respectively; p<0.05), whereas there was no difference between WT and CD47KO mice in CXCL1‐induced arrest. Also, PMN transendothelial migration (TEM) is reduced by 30% in CD47KO mice compared to WT mice (p<0.05). In vitro flow chamber assays demonstrate reduced adhesion (30% reduction vs. WT; p<0.05) and TEM (60% reduction vs. WT; p<0.05) of CD47KO PMN compared to WT PMN across WT TNF‐activated murine endothelium. We have previously reported that CD47 physically interacts with β2 integrins and regulates integrin affinity on T cells (Azcutia et al., Mol Biol Cell 2013). These previous observations and our current results suggest that CD47 plays a role in PMN recruitment through the regulation of β2 integrin adhesive function.Grant Funding Source: Supported by NIH HL‐36028 and AHA fellowship 11POST7730055.

Endothelial integrin α3β1 stabilizes carbohydrate-mediated tumor/endothelial cell adhesion and induces macromolecular signaling complex formation at the endothelial cell membrane

Blood borne metastatic tumor cell adhesion to endothelial cells constitutes a critical rate-limiting step in hematogenous cancer metastasis. Interactions between cancer associated carbohydrate Thomsen-Friedenreich antigen (TF-Ag) and endothelium-expressed galectin-3 (Gal-3) have been identified as the leading molecular mechanism initiating tumor/endothelial cell adhesion in several types of cancer. However, it is unknown how these rather weak and transient carbohydrate/lectin mediated interactions are stabilized. Here, using Western blot and LC tandem mass spectrometry analyses of pull-downs utilizing TF-Ag loaded gold nanoparticles, we identified Gal-3, endothelial integrin α3β1, Src kinase, as well as 5 additional molecules mapping onto focal adhesion pathway as parts of the macromolecular complexes formed at the endothelial cell membranes downstream of TF-Ag/Gal-3 interactions. In a modified parallel flow chamber assay, inhibiting α3β1 integrin greatly reduced the strength of tumor/endothelial cell interactions without affecting the initial cancer cell adhesion. Further, the macromolecular complex induced by TF-Ag/Gal-3/α3β1 interactions activates Src kinase, p38, and ERK1/2, pathways in endothelial cells in a time- and α3β1-dependent manner. We conclude that, following the initial metastatic cell attachment to endothelial cells mediated by TF-Ag/Gal-3 interactions, endothelial integrin α3β1 stabilizes tumor/endothelial cell adhesion and induces the formation of macromolecular signaling complex activating several major signaling pathways in endothelial cells.

Neutrophils mediate edema formation but not mechanical allodynia during zymosan-induced inflammation

Inflammatory pain is based on stimulation and sensitization of peripheral endings of sensory neurons (nociceptors) by pronociceptive mediators. These mediators can be released by resident cells, as well as invading immune cells. Although neutrophils are known to release various mediators, which can stimulate or sensitize nociceptors, the extent of their contribution to nociceptive responses is unclear. Here, we studied the contribution of neutrophils to zymosan-induced inflammatory pain, which is characterized by an early recruitment of high numbers of neutrophils. Surprisingly, antibody-mediated neutrophil depletion caused a complete loss of edema formation but had no effect on mechanical pain thresholds. Blockage of the interaction between neutrophils and platelets or endothelial cells using antibodies directed against CD11b and CD162 reduced neutrophil recruitment to the site of inflammation. Again, the treatment decreased zymosan-induced edemas without altering mechanical pain thresholds. Also, HLB-219 mice, which have five to 10 times less platelets than WT mice, showed reduced neutrophil recruitment to the site of inflammation and decreased edema sizes, whereas, again, mechanical thresholds were unaltered. The effects observed in HLB-219 mice were relatively small and not reproduced in vWF-deficient mice or after antibody-mediated blockage of GPIbα. Flow chamber and transmigration assays showed that platelets were not necessary for neutrophil adhesion to endothelial cells but increased their transmigration. Taken together, zymosan-induced mechanical allodynia is, in contrast to edema formation, independent of neutrophils, and recruitment of neutrophils is only partly influenced by interactions with platelets.

Lidocaine Reduces Neutrophil Recruitment by Abolishing Chemokine-Induced Arrest and Transendothelial Migration in Septic Patients

The inappropriate activation, positioning, and recruitment of leukocytes are implicated in the pathogenesis of multiple organ failure in sepsis. Although the local anesthetic lidocaine modulates inflammatory processes, the effects of lidocaine in sepsis are still unknown. This double-blinded, prospective, randomized clinical trial was conducted to investigate the effect of lidocaine on leukocyte recruitment in septic patients. Fourteen septic patients were randomized to receive either a placebo (n = 7) or a lidocaine (n = 7) bolus (1.5 mg/kg), followed by continuous infusion (100 mg/h for patients >70 kg or 70 mg/h for patients <70 kg) over a period of 48 h. Selectin-mediated slow rolling, chemokine-induced arrest, and transmigration were investigated by using flow chamber and transmigration assays. Lidocaine treatment abrogated chemokine-induced neutrophil arrest and significantly impaired neutrophil transmigration through endothelial cells by inhibition of the protein kinase C-θ while not affecting the selectin-mediated slow leukocyte rolling. The observed results were not attributable to changes in surface expression of adhesion molecules or selectin-mediated capturing capacity, indicating a direct effect of lidocaine on signal transduction in neutrophils. These data suggest that lidocaine selectively inhibits chemokine-induced arrest and transmigration of neutrophils by inhibition of protein kinase C-θ while not affecting selectin-mediated slow rolling. These findings may implicate a possible therapeutic role for lidocaine in decreasing the inappropriate activation, positioning, and recruitment of leukocytes during sepsis.

Analysis of L-selectin-mediated cellular interactions under flow conditions.

Lymphocyte homing is mediated by a specific interaction between L-selectin expressed on lymphocytes and its ligands expressed on high endothelial venules (HEVs) in lymph nodes under physiological flow conditions. In this chapter, two methods for detecting L-selectin-mediated cellular interactions under shear stress mimicking physiological flow conditions are described. First, a modified Stamper-Woodruff cell-binding assay using leukocytes labeled with a fluorescent orange dye, CMTMR, is introduced. In this method, leukocytes are allowed to bind to frozen lymph node sections under shear stress and their binding to HEVs can be clearly visualized by fluorescence microscopy. Second, a parallel flow chamber assay is described. In this assay, leukocytes are allowed to roll on L-selectin ligand-expressing cells under various levels of shear stress and their adhesive interactions are recorded by a video camera equipped with an inverted microscope. These methods can be applied to determine the effects of various agents that might affect L-selectin-mediated lymphocyte homing and recruitment.

Ultrasound Molecular Imaging of E-Selectin in Tumor Vessels Using Poly n-Butyl Cyanoacrylate Microbubbles Covalently Coupled to a Short Targeting Peptide

The purposes of this study were the development and preclinical evaluation of clinically translatable E-selectin-specific ultrasound contrast agents based on a peptide ligand with the recognition sequence IELLQAR. The E-selectin-specific peptide was synthesized through solid phase peptide synthesis and covalently attached to poly n-butylcyanoacrylate-stabilized microbubbles with an air core. Quantification of the microbubble surface coverage with peptides was performed through flow cytometry. Targeted adhesion of peptide-coated microbubbles was investigated in vitro using parallel plate flow chamber assays on tumor necrosis factor-α-stimulated human umbilical vein endothelial cells. In vivo imaging was performed in nude mice bearing human ovarian carcinoma xenografts (MLS), followed by ex vivo immunohistochemistry validation of E-selectin expression. Success of peptide synthesis was validated through preparative reverse phase high-pressure liquid chromatography and electronspray ionization-mass spectrometry. Results of the flow cytometry revealed approximately 4000 E-selectin-specific peptides/microbubble surface. Results of the in vitro experiments demonstrated the specificity of peptide-coated microbubbles to E-selectin (1.10 ± 0.48 vs 0.19 ± 0.09 bound microbubbles per cell, before and after competition respectively; P < 0.01). The in vivo imaging enabled specific assessment of E-selectin expression in MLS carcinoma xenografts (5.21 ± 3.41 vs 1.37 ± 0.67 contrast intensity before and after competition, respectively; P < 0.05). Clinically translatable microbubbles that were covalently coupled to the short E-selectin-specific peptide (IELLQAR) enabled specific imaging of the E-selectin expression in tumor vessels in vivo.

Non-adsorbing macromolecules promote endothelial adhesion of erythrocytes with reduced sialic acids

BackgroundAbnormal adhesion of red blood cells (RBCs) to vascular endothelium is often associated with reduced levels of sialic acids on RBC membranes and with elevated levels of pro-adhesive plasma proteins. However, the synergistic effects of these two factors on the adhesion are not clear. In this work, we tested the hypothesis that macromolecular depletion interaction originating from non-adsorbing macromolecules can promote the adhesion of RBCs with reduced sialic acid content to the endothelium. MethodsRBCs are treated with neuraminidase to specifically remove sialic acids from their surface followed by the evaluation of their deformability, zeta potential and membrane proteins. The adhesion of these enzyme-treated RBCs to cultured human umbilical vein endothelial cells (ECs) is studied in the presence of 70 or 500kDa dextran with a flow chamber assay. ResultsOur results demonstrate that removal of sialic acids from RBC surface can induce erythrocyte adhesion to endothelial cells and that such adhesion is significantly enhanced in the presence of high-molecular weight dextran. The adhesion-promoting effect of dextran exhibits a strong dependence on dextran concentration and molecular mass, and it is concluded to originate from macromolecular depletion interaction. ConclusionThese results suggest that elevated levels of non-adsorbing macromolecules in plasma might play a significant role in promoting endothelial adhesion of erythrocytes with reduced sialic acids. General significanceOur findings should therefore be of great value in understanding abnormal RBC–EC interactions in pathophysiological conditions (e.g., sickle cell disease and diabetes) and after blood transfusions.

Simultaneously Capturing Real-time Images in Two Emission Channels Using a Dual Camera Emission Splitting System: Applications to Cell Adhesion

Multi-color immunofluorescence microscopy to detect specific molecules in the cell membrane can be coupled with parallel plate flow chamber assays to investigate mechanisms governing cell adhesion under dynamic flow conditions. For instance, cancer cells labeled with multiple fluorophores can be perfused over a potentially reactive substrate to model mechanisms of cancer metastasis. However, multi-channel single camera systems and color cameras exhibit shortcomings in image acquisition for real-time live cell analysis. To overcome these limitations, we used a dual camera emission splitting system to simultaneously capture real-time image sequences of fluorescently labeled cells in the flow chamber. Dual camera emission splitting systems filter defined wavelength ranges into two monochrome CCD cameras, thereby simultaneously capturing two spatially identical but fluorophore-specific images. Subsequently, psuedocolored one-channel images are combined into a single real-time merged sequence that can reveal multiple target molecules on cells moving rapidly across a region of interest.

Simultaneously Capturing Real-time Images in Two Emission Channels Using a Dual Camera Emission Splitting System: Applications to Cell Adhesion

Multi-color immunofluorescence microscopy to detect specific molecules in the cell membrane can be coupled with parallel plate flow chamber assays to investigate mechanisms governing cell adhesion under dynamic flow conditions. For instance, cancer cells labeled with multiple fluorophores can be perfused over a potentially reactive substrate to model mechanisms of cancer metastasis. However, multi-channel single camera systems and color cameras exhibit shortcomings in image acquisition for real-time live cell analysis. To overcome these limitations, we used a dual camera emission splitting system to simultaneously capture real-time image sequences of fluorescently labeled cells in the flow chamber. Dual camera emission splitting systems filter defined wavelength ranges into two monochrome CCD cameras, thereby simultaneously capturing two spatially identical but fluorophore-specific images. Subsequently, psuedocolored one-channel images are combined into a single real-time merged sequence that can reveal multiple target molecules on cells moving rapidly across a region of interest.

CD36 Recruits α5β1 Integrin to Promote Cytoadherence of P. falciparum-Infected Erythrocytes

The adhesion of Plasmodium falciparum-infected erythrocytes (IRBC) to receptors on different host cells plays a divergent yet critical role in determining the progression and outcome of the infection. Based on our ex vivo studies with clinical parasite isolates from adult Thai patients, we have previously proposed a paradigm for IRBC cytoadherence under physiological shear stress that consists of a recruitment cascade mediated largely by P-selectin, ICAM-1 and CD36 on primary human dermal microvascular endothelium (HDMEC). In addition, we detected post-adhesion signaling events involving Src family kinases and the adaptor protein p130CAS in endothelial cells that lead to CD36 clustering and cytoskeletal rearrangement which enhance the magnitude of the adhesive strength, allowing adherent IRBC to withstand shear stress of up to 20 dynes/cm2. In this study, we addressed whether CD36 supports IRBC adhesion as part of an assembly of membrane receptors. Using a combination of flow chamber assay, atomic force and confocal microscopy, we showed for the first time by loss- and gain-of function assays that in the resting state, the integrin α5β1 does not support adhesive interactions between IRBC and HDMEC. Upon IRBC adhesion to CD36, the integrin is recruited either passively as part of a molecular complex with CD36, or actively to the site of IRBC attachment through phosphorylation of Src family kinases, a process that is Ca2+-dependent. Clustering of β1 integrin is associated with an increase in IRBC recruitment as well as in adhesive strength after attachment (∼40% in both cases). The adhesion of IRBC to a multimolecular complex on the surface of endothelial cells could be of critical importance in enabling adherent IRBC to withstand the high shear stress in the microcirculations. Targeting integrins may provide a novel approach to decrease IRBC cytoadherence to microvascular endothelium.

Abstract 40: Neutrophil Serine Protease PR3 Promotes Platelet Adhesion and Activation on von Willebrand Factor (vWF) Expressing Endothelial Cells

Introduction Neutrophils play important roles in inflammation, regulating vascular permeability and promoting thrombus formation. Upon activation, neutrophils release serine proteases that can activate coagulation factors and protease activated receptors (PARs). Proteinase 3 (PR3) is the most abundant serine protease in neutrophils; however, the role of PR3 in regulating vascular function and thrombosis is not clear. In this study we have used flow chamber assays and confocal microscopy to investigate PR3 activation of endothelial cells through PAR-2 and the recruitment and activation of platelets and leukocytes under shear conditions. Results After endothelial cells were incubated with PR3 or a PAR-2 activating peptide, we observed significant release and surface presentation of von Willebrand (vWF) compared to untreated cells. In addition, we also found that PR3 increased endothelial cell adhesion molecule expression and ERK phosphorylation. Using flow chamber assays, we observed that PR3-treated endothelial cells expressing vWF were able to capture platelets from whole blood under venous shear conditions; however, blocking antibodies against either platelet GpIb or GpIIb/IIIa significantly reduced platelet adhesion. Using whole blood, we also found that significant numbers of leukocytes were recruited to PR3-treated endothelial cells. However, in platelet-depleted whole blood, or blood treated with antibodies against GpIb or GpIIb/IIIa, we observed a dramatic reduction in leukocyte adhesion. Therefore, leukocyte recruitment to PR3-treated endothelial cells required platelet activation and adhesion and this was mediated in part by vWF. Conclusions Neutrophil PR3 can promote platelet adhesion to the vascular endothelium through a process requiring endothelial cell vWF, platelet GpIb and GpIIb/IIIa. PR3 also promotes leukocyte recruitment to the endothelium through a platelet-dependant process. This data demonstrates that PR3 can be a significant contributor to thrombus formation and leukocyte recruitment to the vascular endothelium and PR3 may be an important target for disrupting thrombus formation in vivo .

α4β7 integrin-mediated lymphocyte adhesion to VCAM-1 under physiologic shear requires L-selectin function (P5116)

Abstract Lymphocyte migration is facilitated by a cascade of adhesion molecule interactions. Specifically, lymphocyte rolling is mediated by selectins, while slow rolling and adhesion are attributed to integrin and Ig family members. α4β7 integrin interactions with its primary ligand, MAdCAM-1, synergize with L-selectin function to regulate the recirculation of naïve lymphocytes to the gut. However, subsets of regulatory T cells also express high levels of L-selectin and α4β7 integrin, and may instead bind to VCAM-1, a secondary ligand for α4β7 integrin. Therefore, potential synergism between L-selectin function and α4β7 integrin/VCAM-1 interactions were examined. To determine the ability of α4β7 integrin to interact with VCAM-1 under conditions of shear, transfected TK-1 T cell interactions with EA.hy926 endothelial cell lines were assessed using an in vitro flow chamber assay. While TK-1 cells, expressing α4β7 but not α4β1 integrin, showed little interaction with VCAM-1-transfected endothelial cells, the presence of L-selectin function enabled α4β7 integrin/VCAM-1 interactions to support slow rolling and adhesion. Importantly, α4β7 integrin interactions with VCAM-1 supported slow rolling and adhesion of murine regulatory T cells under shear stress in the presence of L-selectin function. Thus, functional synergy between L-selectin and α4β7 integrin/VCAM-1 interactions may promote regulatory T cell recruitment to sites of inflammation.

GDF‐15 prevents platelet integrin activation and thrombus formation

Integrin-mediated platelet function plays an important role in primary hemostasis. Growth-differentiation factor 15 (GDF-15) has been shown to inhibit β(2) -integrin activation in leukocytes. We investigated the effect of GDF-15 on platelet integrin activation in vitro and in different in vivo models of thrombus formation. GDF-15(-/-) mice showed an accelerated thrombus formation and a reduced survival rate after collagen-induced pulmonary thromboembolism. In reconstitution experiments, recombinant GDF-15 decelerated thrombus formation and prolonged the bleeding time. In vitro experiments demonstrated that GDF-15 pretreated, agonist-stimulated platelets showed decreased binding to fibrinogen in flow chamber assays and reduced activation of β(1) - and β(3) -integrins in flow cytometry experiments. Pretreating human and mouse platelets with GDF-15 reduced platelet aggregation. Mechanistically, GDF-15 prevents agonist-induced Rap1- dependent α(II) (b) β(3) activation by activating PKA. Platelet P-selectin expression and dense granule secretion after stimulation were unaffected by GDF-15, indicating a specific effect of GDF-15 on integrin activation. GDF-15 specifically inhibits platelet integrin activation. These findings may have profound clinical implications for the treatment of hemostatic conditions involving platelets.

Immobilized immune complexes effectively promote the recruitment of FcyRIII (CD16)- expressing human blood dendritic cells

Fcy receptor (FCyR)-mediated interaction of soluble and tissue bond immune complexes (ICs) with dendritic cells (DCs) contributes to IC-triggered antimicrobial and autoimmune inflammatory responses. Among human blood CD1c- CD11c + DCs we previously identified the distinct subset of 6-sulfo LacNAc(slan)DCs, characterized by a pronounced expression of FcyRIII (CD16) and potent proinflammatory capacities. We show that CD16 expression equips slanDCs with an exceptional capacity to capture small soluble ICs. Moreover, by applying a flow chamber assay and time-lapse video microscopy, we show that surface-immobilized ICs alone are highly effective in mediating the firm arrest of slanDCs under physiological shear stress conditions. Contrarily, neither immobilized chemokine (ie. CX3CL1) nor complement (i.e. C5a) promote shear-resistant adhesion of slanDC, despite marked expression of the respective surface receptors. Notably, ICs fail to mediate adhesion of other human DC subsets (i.e. plasmocytoid DC and CD1c + DC) or isolated T cells. By blocking selective FcyRs on slanDCs, we demonstrate that their shear-resistant adhesion to immobilized ICs critically depends on CD16, while CD32 (FcyRII) expression is entirely dispensable. Additional studies confirmed enhanced CD16/IC-mediated arrest of slanDCs on monolayers of human dermal microvascular endothelial cells that were pre-incubated with anti-endothelial cell IgG antibodies. Collectively, we identified CD16 as a critical structure for 1) the capture of soluble ICs and 2) the effective, shear-resistant adhesion of circulating proinflammatory slanDCs to immobilized ICs at the endothelial interface. Our data provide first evidence for a novel conduit of rapid FcR-coordinated recruitment of DC in IC-mediated tissue inflammation. JSID AbstractsJournal of Dermatological ScienceVol. 69Issue 2Preview Full-Text PDF

Molecular Imaging of Inflammation in Inflammatory Bowel Disease with a Clinically Translatable Dual-Selectin–targeted US Contrast Agent: Comparison with FDG PET/CT in a Mouse Model

To develop and test a molecular imaging approach that uses ultrasonography (US) and a clinically translatable dual-targeted (P- and E-selectin) contrast agent (MBSelectin) in the quantification of inflammation at the molecular level and to quantitatively correlate selectin-targeted US with fluorodeoxyglucose (FDG) combined positron emission tomography (PET) and computed tomography (CT) in terms of visualization and quantification of different levels of inflammation in a murine acute colitis model. Animal studies were approved by the Institutional Administrative Panel on Laboratory Animal Care at Stanford University. MBSelectin was developed by covalently binding an analog of the naturally occurring binding ligand P-selectin glycoprotein ligand 1 fused to a human fragment crystallizable(or Fc) domain onto the lipid shell of perfluorobutane and nitrogen-containing MBs. Binding specificity of MBSelectin was assessed in vitro with a flow chamber assay and in vivo with a chemically induced acute colitis murine model. US signal was quantitatively correlated with FDG uptake at PET/CT and histologic grade. Statistical analysis was performed with the Student t test, analysis of variance, and Pearson correlation analysis. MBSelectin showed strong attachment to both human and mouse P- and E-selectin compared with MBControl in vitro (P ≤ .002). In vivo, US signal was significantly increased (P < .001) in mice with acute colitis (173.8 arbitrary units [au] ± 134.8 [standard deviation]) compared with control mice (5.0 au ± 4.5). US imaging signal strongly correlated with FDG uptake on PET/CT images (ρ = 0.89, P < .001). Ex vivo analysis enabled confirmation of inflammation in mice with acute colitis and high expression levels of P- and E-selectin in mucosal capillaries (P = .014). US with MBSelectin specifically enables detection and quantification of inflammation in a murine acute colitis model, leveraging the natural pathway of leukocyte recruitment in inflammatory tissue. US imaging with MBSelectin correlates well with FDG uptake at PET/CT imaging.

Quantification of volume, mass, and density of thrombus formation using brightfield and differential interference contrast microscopy

Flow chamber assays, in which blood is perfused over surfaces of immobilized extracellular matrix proteins, are used to investigate the formation of platelet thrombi and aggregates under shear flow conditions. Elucidating the dynamic response of thrombi/aggregate formation to different coagulation pathway perturbations in vitro has been used to develop an understanding of normal and pathological cardiovascular states. Current microscopy techniques, such as differential interference contrast (DIC) or fluorescent confocal imaging, respectively, do not provide a simple, quantitative understanding of the basic physical features (volume, mass, and density) of platelet thrombi/aggregate structures. The use of two label-free imaging techniques applied, for the first time, to platelet aggregate and thrombus formation are introduced: noninterferometric quantitative phase microscopy, to determine mass, and Hilbert transform DIC microscopy, to perform volume measurements. Together these techniques enable a quantitative biophysical characterization of platelet aggregates and thrombi formed on three surfaces: fibrillar collagen, fibrillar collagen +0.1 nM tissue factor (TF), and fibrillar collagen +1 nM TF. It is demonstrated that label-free imaging techniques provide quantitative insight into the mechanisms by which thrombi and aggregates are formed in response to exposure to different combinations of procoagulant agonists under shear flow.

Neutrophil-Derived Cathelicidin Promotes Adhesion of Classical Monocytes

The leukocyte response in acute inflammation is characterized by an initial recruitment of neutrophils preceding a second wave of monocytes. Neutrophil-derived granule proteins were suggested to hold an important role in this cellular switch. The exact mechanisms by which neutrophils mediate these processes are only partially understood. To investigate the role of neutrophils and their granule contents in the adhesion of monocyte subpopulations in acute inflammation. Here, we show that neutrophil-derived cathelicidins (human: LL37, mouse: CRAMP) induce adhesion of classical monocytes but not of nonclassical monocytes in the mouse cremaster muscle and in in vitro flow chamber assays. CRAMP is released from emigrated neutrophils and then transported across the endothelium, where it is presented to rolling leukocytes. Endothelial-bound cathelicidin activates formyl-peptide receptor 2 on classical monocytes, resulting in monocytic β1- and β2-integrin conformational change toward an extended, active conformation that allows for adhesion to their respective ligands, vascular cell adhesion molecule 1 and intercellular adhesion molecule 1. These data elucidate a novel mechanism of neutrophil-mediated monocyte recruitment, which could be targeted in conditions where recruitment of classical monocytes plays an unfavorable role.

Transient adhesion mediated by ligand-receptor interaction on surfaces of variable nanotopography

Surface microtopography and nanotopography have been shown to influence cell adhesion and function, including proliferation and differentiation, leading both to fundamental questions and practical applications in the field of biomaterials and nanomedicine. However, the mechanisms of how cells sense topography remain obscure. In this study, we measured directly the effect of nanotopography on the kinetics of association and dissociation of ligand–receptor bonds, which are critically involved in the first steps of cell adhesion. We designed models of biological functionalised surfaces with controlled roughness varying from 2 to 400 nm of root mean square, and controlled ligand density. Tests of transient adhesion of receptor–coated microspheres on these surfaces were performed, using a laminar flow chamber assay. We probed Intercellular Adhesion Molecule ICAM–1–anti–ICAM–1 bond adhesion kinetics in the single molecule limit on smooth and rough substrates. Frequency of adhesion did not exhibit any noticeable dependence on roughness parameter, except at high bead velocity. Detachment rate was also independent of roughness. Finally, leucocyte transient adhesion tests were performed on similar substrates, using variable activating incubating media. Here also, no strong effect of roughness was observed in these conditions. Results are rationalised in terms of the role of local geometry on the access of ligands to receptors.