Static Adhesion Research Articles

Construction of heparinylated multilayer films on Ti[sbnd]O via streptavidin/biotin interaction

Construction of heparinylated multilayer films on TiO via streptavidin/biotin interaction was conducted in the present study. An organic layer of 3-aminopropylphosphonic acid (APP) was first introduced on TiO by self-assembling, and then biotin was immobilized by photochemical methods. So streptavidin and biotinylated heparin were assembling through biorecognition, and a desired 6-layer heparinylated multilayer was obtained through layer-by-layer driven by streptavidin/biotin interaction. The in vitro platelet adhesion and activation were investigated by a static platelet adhesion test. The clotting time was examined by activated partial thromboplastin time (APTT). Results show that the heparinylated multilayer coated TiO can significantly decrease platelet adhesion and activation, and prolong clotting time of APTT compared to untreated TiO, which indicates the heparinylated multilayer coated TiO displays more excellent anticoagulation performance than that of the bare TiO.

The Role for Decorin in Delayed-Type Hypersensitivity

Decorin, a small leucine-rich proteoglycan, regulates extracellular matrix organization, growth factor-mediated signaling, and cell growth. Because decorin may directly modulate immune responses, we investigated its role in a mouse model of contact allergy (oxazolone-mediated delayed-type hypersensitivity [DTH]) in decorin-deficient (Dcn(-/-)) and wild-type mice. Dcn(-/-) mice showed a reduced ear swelling 24 h after oxazolone treatment with a concurrent attenuation of leukocyte infiltration. These findings were corroborated by reduced glucose metabolism, as determined by (18)fluordeoxyglucose uptake in positron emission tomography scans. Unexpectedly, polymorphonuclear leukocyte numbers in Dcn(-/-) blood vessels were significantly increased and accompanied by large numbers of flattened leukocytes adherent to the endothelium. Intravital microscopy and flow chamber and static adhesion assays confirmed increased adhesion and reduced transmigration of Dcn(-/-) leukocytes. Circulating blood neutrophil numbers were significantly increased in Dcn(-/-) mice 24 h after DTH elicitation, but they were only moderately increased in wild-type mice. Expression of the proinflammatory cytokine TNF-α was reduced, whereas syndecan-1 and ICAM-1 were overexpressed in inflamed ears of Dcn(-/-) mice, indicating that these adhesion molecules could be responsible for increased leukocyte adhesion. Decorin treatment of endothelial cells increased tyrosine phosphorylation and reduced syndecan-1 expression. Notably, absence of syndecan-1 in a genetic background lacking decorin rescued the attenuated DTH phenotype of Dcn(-/-) mice. Collectively, these results implicated a role for decorin in mediating DTH responses by influencing polymorphonuclear leukocyte attachment to the endothelium. This occurs via two nonmutually exclusive mechanisms that involve a direct antiadhesive effect on polymorphonuclear leukocytes and a negative regulation of ICAM-1 and syndecan-1 expression.

Increased Neutrophil Adhesive Properties In Patients with Venous Thromboembolism and Residual Vein Thrombosis and High D-Dimer Levels

Abstract 2301 Introduction:Venous Thromboembolism (VTE) is a multifactorial disease that affects 1:1000 individuals worldwide, with a recurrence rate of about 25% in 10 years. Although many risk factors for VTE are well defined, first presentation and recurrence depend, at least in part, on as yet unknown etiologic factors. Studies in animal models show a tight relation between inflammation and hemostasis, as well as the infiltration of neutrophils in the venous wall after the induction of venous thrombosis. Neutrophils also participate in different stages in the formation and resolution of venous thrombosis. Methods: In this study, we investigated the adhesive properties of neutrophils in VTE patients. We hypothesized that increased adhesive properties of these cells, either as an individual baseline characteristic or as an acquired alteration after a previous VTE episode, could be associated with the thrombotic process. The patient population consisted of 22 VTE patients (14F:8M; median age: 46.1 years) that had completed at least 6 months of oral anticoagulation. Twenty-two healthy volunteers matched to VTE patients by age, gender and ethnic background were used as controls. Neutrophil adhesion was measured by a static adhesion assay in triplicate. Peripheral blood was collected with heparin and neutrophils were separated on Histopaque® (Sigma-Aldrich, St. Louis, MO, USA). Isolated neutrophils (2.2×106 cells/ml) were allowed to adhere to fibronectin (FN)-coated 96-well plates (30 min, 37°C, 5%CO2). Non-adherent cells were then removed by washing and adherent cells calculated as the percentage of cells adhered, compared to a standard curve of known cell concentrations and using a colorimetric enzyme assay. Results are expressed as means ± standard error of mean (SEM) and were compared using the Mann-Whitney test. Results: Overall, adhesion of neutrophils from VTE patients (25.40% ±2.35) was not increased when compared to the control group (21.25%±1.20 p=0.2). However when only patients at a higher risk of recurrence (n=13) - here defined as the presence of elevated D-dimer (higher than 0.5mg/L) and residual vein thrombosis - were analyzed, a statistically significant increase in cell adhesion compared to matched controls was observed (26.70%±2.08 and 21.36%±1.26, respectively, p = 0.04). When these patients (higher recurrence risk; n=13) were compared to the remaining VTE patients (standard recurrence risk, n=9), a non significant increase in neutrophil adhesion was observed (26.70%±2.08 vs 23.51%±5.03 respectively, p=0.1). Conclusions: We demonstrate that neutrophil adhesion is increased in patients with VTE with characteristics associated with increased recurrence risk. In addition, we also observed a non-significant difference in neutrophil adhesion in these patients compared to other VTE patients. Our results suggest that the increased adhesive properties of neutrophils in VTE patients could play a role in the exacerbation of inflammation, and in the pathophysiology of VTE. Further studies are warranted to study whether neutrophil adhesiveness could be used as a biomarker of VTE recurrence. Disclosures:No relevant conflicts of interest to declare.

Adhesive and Inflammatory Properties Are Increased in Blood Outgrowth Endothelial Cells From Patients with Sickle Cell Anemia

Abstract 2133The endothelium plays an important role in the pathophysiology of sickle cell anemia (SCA) due to interactions of endothelial cells with other cell types in the vaso-occlusive process and endothelial dysfunction caused by chronic inflammation and decreased nitric oxide bioavailability. Inflammatory molecules, such as cytokine TNF-α, may alter the endothelial cell surface and, thus, modulate the adhesion molecules that bind red blood cells (RBC). Damaged or activated endothelium produces cytokines and chemokines that result in the high levels of circulating inflammatory molecules found in the SCA. Blood Outgrowth Endothelial Cells (BOEC) are circulating endothelial cells isolated from the peripheral blood of adults, they have a high proliferative capacity in vitro, with the characteristic phenotype of mature cells. BOEC cultures were established to serve as a model for an in vitro study of endothelial properties and function in SCA, to evaluate RBC adhesion, expression of adhesion molecules and production of inflammatory cytokines in BOEC from healthy controls (Con) and SCA patients. Three cultures of BOEC from Con and three cultures from SCA patients in steady state were established and identification confirmed by flow cytometry (CD31+; CD146+; VEGF R2+; CD45-; CD133-). BOEC were used under basal conditions or following treatment with 10ng/ml TNF-α (3 H). RBC were isolated from Con, SCA and SCA patients on hydroxyurea treatment (SCAHU; 20–30 mg/Kg/day) and their adhesion to confluent Con and SCA BOEC was evaluated (4×106 cell/ml in EGM-2), using static adhesion assays. BOEC expression of adhesion molecules VCAM-1, E-selectin, P-selectin, CD36, the Vitronectin receptor and BCAM/Lu was evaluated by flow cytometry. The production and release of GM-CSF and IL-8 to the supernatant of BOEC in the presence and absence of an antiinflammatory stimulus (1μM Simvastatin, 3.5H) was quantified by ELISA. Notably, Con RBC adhered significantly more to SCA BOEC (16.6 ± 4.6%; n=14; p<0.001; Wilcoxon Test) than to Con BOEC (9.4 ± 3.2%; n=14). Furthermore, TNF-α stimulation further increased the adhesion of Con RBC to both Con BOEC (16.6 ± 5.6%; n=14 p<0.001 comp. to non-stimulated Con BOEC) and SCA BOEC (20.6 ±7.0%; n=14; p<0.05 comp. to non-stimulated SCA BOEC). Similarly, SCA RBC adhered significantly more to SCA BOEC (18.5 ± 4.8%; n=10; p<0.001; Wilcoxon Test) than to Con BOEC (14.3 ± 4.2%; n=10) and TNF-α stimulation further increased SCA RBC adhesion to Con BOEC (18.3 ± 5.4%; n=10; p<0.01) and SCA BOEC (20.9 ± 6.4%; n=10; p<0.05; Wilcoxon). SCAHU RBC also adhered more to SCA BOEC (19.5 ± 7.4%; p<0.01; Wilcoxon) compared to Con BOEC (14.3 ± 4.2%; n=10) and to TNF-α stimulated Con BOEC (19.1 ± 6.0%; n=10; p<0.01) and SCA BOEC (23.8 ± 6.3%; n=10; p<0.01), compared to respective non-stimulated BOEC. Con and SCA BOEC presented low-level expression of the P-selectin and CD36 adhesion molecules, even following TNF-α inflammatory stimulus; VCAM-1 was increased on both Con and SCA BOEC under TNF-α conditions, but no difference in expression was observed between the Con and SCA BOEC (data not shown); however, after TNF-α stimulus the expression of E-selectin on SCA BOEC was significantly higher (6613 ± 729 MFI; n=3; p=0.03; Unpaired t Test) than on Con BOEC (3645 ± 547 MFI; n=3). Vitronectin receptor and BCAM/Lu expression was slightly higher on SCA BOEC (6248.3 ± 552.9MFI; 1152 ± 51.4MFI respect; n=3; p<0.05, Unpaired t Test) than on Con BOEC (5059.7 ± 426.2MFI; 904.7 ± 70.9MFI; respect; n=3). Following TNF-α stimulus, SCA BOEC released significantly more GM-CSF (271.5 ± 8.7pg/ml; n=3; p<0.01; Unpaired t Test) than Con BOEC (175.2 ± 18.4pg/ml; n=3); where co-incubation with Simvastatin reduced the release of GM-CSF from TNF-α stimulated SCA (189.0 ± 7.1pg/ml; n=3; p<0.05 comp. to TNF-α SCA BOEC; Paired t Test) and Con BOEC (118.9 ± 21.9pg/ml; p<0.01; Paired t Test). SCA BOEC released more IL-8 (10977.4 ± 379.7pg/ml) than BOEC Con (8913.7 ± 234.7pg/ml) under TNF-α inflammatory conditions; however Simvastatin reduced the production of IL-8 only from Con BOEC (data not shown). Results suggest that SCA BOEC present inherent endothelial abnormalities that promote increased adhesion of red cells and augmented inflammatory properties, which are maintained during in vitro culture and following inflammatory stimulus. These cells may serve as a model for in vitro study of aberrant endothelial function in SCA.Support by FAPESP and CNPq Disclosures:No relevant conflicts of interest to declare.

Interaction of Multimerin 1 with Collagens: Role in Platelet Adhesion

Abstract 2207Multimerin 1 (MMRN1) is a large, homopolymeric adhesive protein stored in platelets and endothelium that binds to activated platelets, endothelial cells and the extracellular matrix after agonist stimulation. MMRN1 supports platelet adhesion by von Willebrand factor (VWF) dependent and independent mechanisms, and also increases platelet adhesion to Horm collagen. Mice deficient in Mmrn1/Snca (α-synuclein) showed defective platelet adhesion to collagen in vitro and in vivo which was corrected by MMRN1. The ability of MMRN1 to support platelet adhesion in vivo and enhance platelet adhesion to collagen ex vivo, led us to explore the molecular basis of MMRN1 interactions with collagen. Solid phase binding assays were used to test MMRN1 binding to human fibril forming collagen (types I, II, III and V) and to types IV and VI collagen. Binding assays were also used to map the MMRN1 binding sites on collagen using collagen peptide toolkits III and II (which has significant similarity to type I collagen). Static adhesion assays were used to test selected collagen peptides for platelet adhesion. Platelet adhesion assays at high shear rates 1500s−1, were used to test the adhesion of washed platelets from normal and VWF-deficient subjects to type I collagen pre-treated or in fluid phase with: BSA (negative control), MMRN1, VWF or their combination. Human collagens types I, II, III and VI (p-values <0.001) but not types IV or V (p-values = 0.84 and 0.09, respectively) supported MMRN1 binding. Peptide toolkits binding studies indicated that MMRN1 bound to a single site on collagen III (peptide III-38) and to two sites on collagen II, with peptide II-9 showing much stronger binding than peptide II-44. Like the VWF binding peptide III-23 (which did not overlap the MMRN1 binding site), peptide III-38 supported platelet adhesion in combination with GFOGER, the peptide with high affinity for platelet α2β1. The possibility that MMRN1 binds to collagen at sites distinct from VWF was supported by the observations that pre-treatment of collagen I matrices, with the combination of MMRN1 and VWF, increased platelet adhesion more than MMRN1 or VWF alone (p-values< 0.001). Moreover, adhesion deficit of VWF-deficient platelets on collagen type I matrix pre-treated with a combination of MMRN1 and VWF was corrected by adding fluid phase VWF but not MMRN1 (p-values < 0.0001). Taken together, our data indicates that MMRN1 binds to different forms of human collagen that support platelet adhesion. As MMRN1 binds to sites on collagen distinct from VWF or integrin α2β1, it may be important for maximizing platelet adhesion at sites of vascular injury. Disclosures:No relevant conflicts of interest to declare.

Membrane surface roughness characterization and its influence on ultrafine particle adhesion

Knowledge of the surface properties of ceramic membranes is useful for understanding and improving their filtration performance. In this study surface roughness was determined using a surface roughness tester (SRT). Roughness values were found to increase with increasing scan-length up to a scan-length of 4mm after which it remained relatively constant. Scanning electron microscopy (SEM), vertical scanning interferometry (VSI) and atomic force microscopy (AFM) were used to characterize the membrane morphology. Compared to AFM, VSI and SEM allow for rapid and accurate characterization of membrane roughness at substantially larger scan-sizes. Static adhesion tests showed that rougher surfaces have more adhesion to nanosized particles than smooth ones. In crossflow filtration, adhesion of nanosized particles smaller than or similar in size to the asperities depended mainly on the membrane roughness. At a certain roughness, a highest flux was obtained. But for microsized particles larger than the asperities, roughness had no evident effect on the flux. According to experimental results and the estimation of hydrodynamic forces acting on particles, fouling could be controlled by modification of surface roughness of ceramic membranes.

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

Imaging Leukocyte Adhesion to the Vascular Endothelium at High Intraluminal Pressure

Worldwide, hypertension is reported to be in approximately a quarter of the population and is the leading biomedical risk factor for mortality worldwide. In the vasculature hypertension is associated with endothelial dysfunction and increased inflammation leading to atherosclerosis and various disease states such as chronic kidney disease2, stroke3 and heart failure4. An initial step in vascular inflammation leading to atherogenesis is the adhesion cascade which involves the rolling, tethering, adherence and subsequent transmigration of leukocytes through the endothelium. Recruitment and accumulation of leukocytes to the endothelium is mediated by an upregulation of adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1), intracellular cell adhesion molecule-1 (ICAM-1) and E-selectin as well as increases in cytokine and chemokine release and an upregulation of reactive oxygen species5. In vitro methods such as static adhesion assays help to determine mechanisms involved in cell-to-cell adhesion as well as the analysis of cell adhesion molecules. Methods employed in previous in vitro studies have demonstrated that acute increases in pressure on the endothelium can lead to monocyte adhesion, an upregulation of adhesion molecules and inflammatory markers6 however, similar to many in vitro assays, these findings have not been performed in real time under physiological flow conditions, nor with whole blood. Therefore, in vivo assays are increasingly utilised in animal models to demonstrate vascular inflammation and plaque development. Intravital microscopy is now widely used to assess leukocyte adhesion, rolling, migration and transmigration7-9. When combining the effects of pressure on leukocyte to endothelial adhesion the in vivo studies are less extensive. One such study examines the real time effects of flow and shear on arterial growth and remodelling but inflammatory markers were only assessed via immunohistochemistry10. Here we present a model for recording leukocyte adhesion in real time in intact pressurised blood vessels using whole blood perfusion. The methodology is a modification of an ex vivo vessel chamber perfusion model9 which enables real-time analysis of leukocyte -endothelial adhesive interactions in intact vessels. Our modification enables the manipulation of the intraluminal pressure up to 200 mmHg allowing for study not only under physiological flow conditions but also pressure conditions. While pressure myography systems have been previously demonstrated to observe vessel wall and lumen diameter11 as well as vessel contraction this is the first time demonstrating leukocyte-endothelial interactions in real time. Here we demonstrate the technique using carotid arteries harvested from rats and cannulated to a custom-made flow chamber coupled to a fluorescent microscope. The vessel chamber is equipped with a large bottom coverglass allowing a large diameter objective lens with short working distance to image the vessel. Furthermore, selected agonist and/or antagonists can be utilized to further investigate the mechanisms controlling cell adhesion. Advantages of this method over intravital microscopy include no involvement of invasive surgery and therefore a higher throughput can be obtained. This method also enables the use of localised inhibitor treatment to the desired vessel whereas intravital only enables systemic inhibitor treatment.

Imaging Leukocyte Adhesion to the Vascular Endothelium at High Intraluminal Pressure

Worldwide, hypertension is reported to be in approximately a quarter of the population and is the leading biomedical risk factor for mortality worldwide. In the vasculature hypertension is associated with endothelial dysfunction and increased inflammation leading to atherosclerosis and various disease states such as chronic kidney disease2, stroke3 and heart failure4. An initial step in vascular inflammation leading to atherogenesis is the adhesion cascade which involves the rolling, tethering, adherence and subsequent transmigration of leukocytes through the endothelium. Recruitment and accumulation of leukocytes to the endothelium is mediated by an upregulation of adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1), intracellular cell adhesion molecule-1 (ICAM-1) and E-selectin as well as increases in cytokine and chemokine release and an upregulation of reactive oxygen species5. In vitro methods such as static adhesion assays help to determine mechanisms involved in cell-to-cell adhesion as well as the analysis of cell adhesion molecules. Methods employed in previous in vitro studies have demonstrated that acute increases in pressure on the endothelium can lead to monocyte adhesion, an upregulation of adhesion molecules and inflammatory markers6 however, similar to many in vitro assays, these findings have not been performed in real time under physiological flow conditions, nor with whole blood. Therefore, in vivo assays are increasingly utilised in animal models to demonstrate vascular inflammation and plaque development. Intravital microscopy is now widely used to assess leukocyte adhesion, rolling, migration and transmigration7-9. When combining the effects of pressure on leukocyte to endothelial adhesion the in vivo studies are less extensive. One such study examines the real time effects of flow and shear on arterial growth and remodelling but inflammatory markers were only assessed via immunohistochemistry10. Here we present a model for recording leukocyte adhesion in real time in intact pressurised blood vessels using whole blood perfusion. The methodology is a modification of an ex vivo vessel chamber perfusion model9 which enables real-time analysis of leukocyte -endothelial adhesive interactions in intact vessels. Our modification enables the manipulation of the intraluminal pressure up to 200 mmHg allowing for study not only under physiological flow conditions but also pressure conditions. While pressure myography systems have been previously demonstrated to observe vessel wall and lumen diameter11 as well as vessel contraction this is the first time demonstrating leukocyte-endothelial interactions in real time. Here we demonstrate the technique using carotid arteries harvested from rats and cannulated to a custom-made flow chamber coupled to a fluorescent microscope. The vessel chamber is equipped with a large bottom coverglass allowing a large diameter objective lens with short working distance to image the vessel. Furthermore, selected agonist and/or antagonists can be utilized to further investigate the mechanisms controlling cell adhesion. Advantages of this method over intravital microscopy include no involvement of invasive surgery and therefore a higher throughput can be obtained. This method also enables the use of localised inhibitor treatment to the desired vessel whereas intravital only enables systemic inhibitor treatment.

Enzymatic Single-Chain Antibody Tagging

Rationale: Antibody-targeted delivery of imaging agents can enhance the sensitivity and accuracy of current imaging techniques. Similarly, homing of effector cells to disease sites increases the efficacy of regenerative cell therapy while reducing the number of cells required. Currently, targeting can be achieved via chemical conjugation to specific antibodies, which typically results in the loss of antibody functionality and in severe cell damage. An ideal conjugation technique should ensure retention of antigen-binding activity and functionality of the targeted biological component. Objective: To develop a biochemically robust, highly reproducible, and site-specific coupling method using the Staphylococcus aureus sortase A enzyme for the conjugation of a single-chain antibody (scFv) to nanoparticles and cells for molecular imaging and cell homing in cardiovascular diseases. This scFv specifically binds to activated platelets, which play a pivotal role in thrombosis, atherosclerosis, and inflammation. Methods and Results: The conjugation procedure involves chemical and enzyme-mediated coupling steps. The scFv was successfully conjugated to iron oxide particles (contrast agents for magnetic resonance imaging) and to model cells. Conjugation efficiency ranged between 50% and 70%, and bioactivity of the scFv after coupling was preserved. The targeting of scFv-coupled cells and nanoparticles to activated platelets was strong and specific as demonstrated in in vitro static adhesion assays, in a flow chamber system, in mouse intravital microscopy, and in in vivo magnetic resonance imaging of mouse carotid arteries. Conclusions: This unique biotechnological approach provides a versatile and broadly applicable tool for procuring targeted regenerative cell therapy and targeted molecular imaging in cardiovascular and inflammatory diseases and beyond.

Structural characterization and in vitro inhibitory activities in P-selectin-mediated leukocyte adhesion of polysaccharide fractions isolated from the roots of Physalis alkekengi

Selectin-mediated leukocyte initial attachment and rolling over vessel endothelial surface are crucial steps for inflammatory responses. As P-selectin is a promising target for anti-inflammation therapeutic strategy, recent works have focused on searching for more potent and non-toxic P-selectin antagonists among various natural carbohydrate products. Here, we isolated three water-soluble polysaccharide fractions (PPS-1, PPS-2 and PPS-3) from the roots of Physalis alkekengi by DEAE-cellulose and Sephacryl S-200 chromatography. Their physicochemical and structural characterizations were determined by chemical methods, GC (gas chromatography), HPLC (high performance liquid chromatography), FT-IR (Fourier transform infrared spectrometry), partial acid hydrolysis, methylation and GC–MS (gas chromatography–mass spectrometry) analyses. The inhibitory capacity of the polysaccharide fractions in P-selectin-mediated leukocyte adhesion was evaluated by flow cytometric, static adhesion and laminar flow assays. Results showed that different polysaccharide fractions possess distinct physicochemical and structural properties, including carbohydrate, protein and uronic acid contents, molecular weight, monosaccharide composition and glycosidic linkage type. Among the polysaccharide fractions, PPS-2 could effectively block the interaction between P-selectin and its native ligand.

Foxc2 overexpression enhances benefit of endothelial progenitor cells for inhibiting neointimal formation by promoting CXCR4-dependent homing

Endothelial progenitor cells (EPCs) are capable of enhancing re-endothelialization and attenuating neointimal formation. However, inefficient homing limits the therapeutic efficacy of EPCs transplantation. CXCR4 plays a critical role in regulating EPCs homing. Here, we studied the effect of Foxc2 overexpression on CXCR4 expression and the homing capacity of EPCs as well as the EPCs-mediated therapeutic benefit after artery injury. Bone marrow-derived EPCs were transfected with Foxc2 expression vector (Foxc2-EPCs) or empty control vector (Ctrl-EPCs) and examined 48 hours later. CXCR4 expression of EPCs was detected by flow cytometry and quantitative reverse transcriptase-polymerase chain reaction. The migration of EPCs toward SDF-1α was evaluated in a transwell migration assay, and the adhesion to fibronectin was determined using a static adhesion assay. For in vivo studies, EPCs were injected intravenously into the mice subjected to carotid injury. At 3 days after green fluorescent protein (GFP)/EPCs delivery, the recruited cells to the injury sites were detected by fluorescent microscopy. Re-endothelialization and neointimal formation were, respectively, assessed by Evans blue dye at 7 days and by the morphometric analysis for neointima and media area ratio (N/M) at 28 days after EPCs transfusion. Foxc2 overexpression significantly increased the surface expression of CXCR4 on EPCs (about 1.9-fold of Ctrl-EPCs, P < .05). Foxc2-EPCs showed an increased migration toward SDF-1α (P < .05); Foxc2 overexpression increased also the adhesion capacity of EPCs (P < .05). In vivo, the number of recruited GFP cells was significantly higher in the mice transfused with Foxc2-GFP/EPCs compared with Ctrl-GFP/EPCs (about 2-fold of Ctrl-GFP/EPCs). The degree of re-endothelialization was higher in mice transfused with Foxc2-EPCs compared with Ctrl-EPCs (90.3% ± 1.6% vs 57.2% ± 1.3%; P < .05). Foxc2-EPCs delivery resulted in a greater inhibition of neointimal hyperplasia than Ctrl-EPCs administration (N/M: 0.38 ± 0.03 vs 0.67 ± 0.05, P < .05). Preincubation with CXCR4-Ab, AMD3100, or LY294002 significantly attenuated the enhanced in vitro and in vivo effects of Foxc2-EPCs. Our findings indicate that Foxc2 overexpression increases CXCR4 expression of EPCs and efficiently enhances the homing potential of EPCs, thereby improving EPCs-mediated therapeutic benefit after endothelial injury. Foxc2 may be a novel molecular target for improving the therapeutic efficacy of EPCs transplantation.

Activation of β1Integrins on Blood Eosinophils by P-Selectin

Activation of β(1) integrins of blood eosinophils, assessed by mAb N29, correlates inversely with FEV(1) in two paradigms for studying control of human asthma. We asked whether P-selectin causes eosinophil β(1) integrin activation and results in increased adhesivity. By dual-label flow cytometry, eosinophils with high levels of surface-associated P-selectin had higher reactivity with the activation-sensitive anti-β(1) mAbs N29, 8E3, and 9EG7 than eosinophils with no or with a low-level of surface-associated P-selectin. Among patients with nonsevere asthma, surface P-selectin correlated with N29, 8E3, and 9EG7 signals. By immunofluorescence microscopy, surface-associated P-selectin was present in patches on eosinophils, some of which stained for the platelet marker thrombospondin-1. Activated β(1) and P-selectin partially colocalized on eosinophils. Soluble P-selectin added to whole blood enhanced activation of eosinophil β(1), but not β(2), integrins. In contrast, IL-5 activated eosinophil β(2), but not β(1), integrins. Eosinophils that did not attach to vascular cell adhesion molecule-1 (VCAM-1) in a static adhesion assay had a lower N29 signal than the original population. Soluble P-selectin added to whole blood enhanced eosinophil adhesion to VCAM-1. These findings are compatible with a scenario whereby P-selectin, on eosinophil-associated activated platelets or acquired from plasma or from prior interactions with endothelial cells or platelets, activates eosinophil α(4)β(1) integrin and stimulates eosinophils to adhere to VCAM-1 and move to the airway in asthma.

Surface Modification of COC Microfluidic Devices: A Comparative Study of Nitrogen Plasma Treatment and its Advantages Over Argon and Oxygen Plasma Treatments

AbstractControl of surface properties is very important in high performance microfluidic devices because appropriate functionalization of the surface of the microchannel helps to minimize adsorption of certain analytes thus improving its performance. In this regard, both the argon and oxygen plasma techniques have been used to improve the adaption of polymer surfaces to biological environments. However, the less common nitrogen plasma technique has not been used for surface modification of cyclic olefin copolymer (COC) in microfluidic applications. This paper presents a comparative study between the argon, oxygen, and nitrogen plasma treatments with the aim to identify the most suitable process for the development of a smart, disposable chip for the Bio‐MEMS application. The chemical and morphological changes of the plasma modified COC surfaces were characterized using X‐ray photoelectron spectroscopy, atomic force microscopy, and water contact angle measurements. The effect of plasma treatment on the strength of thermally bonded lap‐shear specimens was studied. The influence of plasma treatment on the integrity of thermally sealed microdevices was assessed using burst pressure tests. The plasma treatments had a significant impact on the electroosmosis flow mobility in the microchannels. The hemocompatibility of the various plasma‐modified COC surfaces was determined using the static platelets adhesion experiment. It was shown that the nitrogen plasma treatment was more effective than the argon and oxygen treatments for the modification of COC based microfluidic devices. magnified image

Immunoglobulin subclass determines ability of immunoglobulin (Ig)G to capture and activate neutrophils presented as normal human IgG or disease-associated anti-neutrophil cytoplasm antibody (ANCA)-IgG

Immunoglobulin G (IgG) is a potent neutrophil stimulus, particularly when presented as anti-neutrophil cytoplasm antibody (ANCA) in ANCA-associated vasculitis. We assessed whether IgG subclasses had differential effects on neutrophil activation and whether differences were dependent on specific Fc-receptor engagement. Using a physiologically relevant flow model, we compared adhesion of neutrophils to different subclasses of normal IgG coated onto solid surfaces, with adhesion of neutrophils treated with different subclasses of soluble ANCA IgG to P-selectin surfaces or endothelial cells (EC). Normal IgG captured flowing neutrophils efficiently in the order IgG3 > IgG1 > IgG2 > IgG4. Fc-receptor blockade reduced capture, IgG3 being more dependent on CD16 and IgG1/2 on CD32. Blockade of the integrin CD18 reduced neutrophil spreading, while inhibition of calcium-dependent signalling reduced both capture and spreading, suggesting that both were active processes. Neutrophils treated with ANCA IgG subclasses 1, 3 and 4 showed stabilization of adhesion to P-selectin surfaces and EC. ANCA changed neutrophil behaviour from rolling to static adhesion and the potency of the subclasses followed the same pattern as above: IgG3 > IgG1 > IgG4. Blockade of Fc receptors resulted in neutrophils continuing to roll, i.e. they were not ANCA-activated; differential utilization of Fc receptor by particular IgG subclasses was not as apparent as during neutrophil capture by normal IgG. IgG3 is the most effective subclass for inducing neutrophil adhesion and altered behaviour, irrespective of whether the IgG is surface bound or docks onto neutrophil surface antigens prior to engaging Fc receptors. Engagement of Fc receptors underpins these responses; the dominant Fc receptor depends on IgG subclass.

A time course study on prothrombotic parameters and their modulation by anti-platelet drugs in hyperlipidemic hamsters

The present study was undertaken to assess the chronology of major pathological events associated with high cholesterol (HC) diet and their modulation by anti-platelet drugs. Male Golden Syrian hamsters were fed HC diet up to 90 days. Plasma lipid, glucose and coagulation parameters (commercial kits), platelet activation (whole blood aggregation and static adhesion), endothelial dysfunction (aortic ring vasoreactivity), splenocyte TNF-α, IFN-γ and iNOS mRNA transcripts (RT-PCR), and ferric chloride (time to occlusion) induced thrombosis were monitored at 15, 30, 60, and 90 days after HC feeding and compared with normolipidemic hamsters. A significant increase in plasma lipid levels was observed at 15 days of HC feeding, but other parameters remain unaltered. Enhanced ADP, collagen, and thrombin-induced platelet aggregation, splenocyte TNF-α expression along with endothelial dysfunction were observed from 30 to 90 days of HC feeding. Platelet adhesion on collagen-/fibrinogen-coated surface and IFN-γ expression were augmented only after 60 days, while enhanced iNOS expression, reduction in thrombin time, and potentiation of ferric chloride-induced thrombosis was observed only at 90 days of HC feeding. Thus, pathological changes induced by HC diet depend on the duration and extent of hyperlipidemia. Moreover, hamsters treated with anti-platelet drugs aspirin (5 mg/kg) or clopidogrel (10 mg/kg) along with HC feeding exhibited reduction in platelet activation as well as subsequent changes observed in the abovementioned parameters following HC feeding. Since reduction in TNF-α was associated with reversion in endothelial dysfunction and prothrombotic state, the role of platelets is implicated in the pathological changes associated with HC feeding.

Achieving Ultralow Fouling Performance for Poly(Hydroxy-Functional Methacrylates) Grafted Surfaces via Atom Transfer Radical Polymerization

The development of nonfouling biomaterials to prevent nonspecific protein adsorption and cell/bacterial adhesion is critical for many biomedical applications, such as antithrombogenic implants and biosensors. In this work, we synthesize and characterize two polymer brushes on the gold substrate by using surface-initiated atom transfer radical polymerization (SI-ATRP) of Hydroxy-Functional Methacrylates monomers: HEMA and HPMA. We investigate the effect of film thickness on protein adsorption from single protein solution to complex human blood media, as well as bacterial adhesion. Surface plasmon resonance (SPR) results show a correlation between antifouling properties and film thickness, that is, optimal film thickness of 25-45 nm for polyHPMA and 20-45 nm for polyHEMA is obtained to achieve almost zero protein adsorption (< 0.3 ng/cm2) from single protein solution, 10% human blood plasma, and 10% human blood serum. Furthermore, polyHEMA brushes remain its excellent resistance to 100% human serum and plasma with < 5 ng/cm2 adsorption amount, while polyHPMA brushes adsorb more proteins of 13.5 ng/cm2 and 50.0 ng/cm2 from 100% human serum and plasma, respectively. More strikingly, static bacteria adhesion assay shows that there are almost no bacteria adhered to polyHEMA and polyHPMA surfaces as compared to full coverage of bacteria on the bare gold surface. In addition, stability tests show a very stable polyHPMA and polyHEMA surfaces without polymer degradation within 20 days. PolyHEMA and PolyHPMA provide effective nonfouling biomaterials, alternative to poly (ethylene glycol), to highly resist nonspecific protein adsorption and cell/bacteria adhesion.

Platelet Adhesion to Keratin Biomaterial Hydrogels under Physiologic Flow Conditions

Keratin biomaterial-based hydrogels have demonstrated hemostatic characteristics in animal models. However, little is known about the mechanisms contributing to these properties. Platelet adhesion to keratin appears to be mediated by β1 and β3 integrins in static adhesion assays. However, these assays did not replicate physiological flow conditions where other factors are known to play a role.In this study we investigated how keratin biomaterial hydrogels influence platelet adhesion under physiologic flow conditions. The goal of these experiments was to quantify the adhesion of platelets to the surface of keratin hydrogels and determine which integrins are involved under physiologically relevant shear stress. Whole blood was taken from healthy volunteers and the platelets fluorescently dyed with mepacrine. The blood was pumped through a flow chamber situated on an inverted fluorescence microscope. Digital images were recorded every 15 seconds for the duration of the experiment and the number of adhered platelets counted. Results show that keratin biomaterial hydrogels promote platelet adhesion in numbers similar to collagen controls.

A novel family of RGD-containing disintegrins (Tablysin-15) from the salivary gland of the horsefly Tabanus yao targets αIIbβ3 or αVβ3 and inhibits platelet aggregation and angiogenesis.

A novel family of RGD-containing molecules (Tablysin-15) has been molecularly characterised from the salivary gland of the haematophagous horsefly Tabanus yao. Tablysin-15 does not share primary sequence homology to any disintegrin discovered so far, and displays an RGD motif in the N-terminus of the molecule. It is also distinct from disintegrins from Viperidae since its mature form is not released from a metalloproteinase precursor. Tablysin-15 exhibits high affinity binding for platelet αIIbβ3 and endothelial cell αVβ3 integrins, but not for α5β1 or α2β1. Accordingly, it blocks endothelial cell adhesion to vitronectin (IC50 ~1 nM) and marginally to fibronectin (IC50 ~1 μM), but not to collagen. It also inhibits fibroblast growth factor (FGF)-induced endothelial cell proliferation, and attenuates tube formation in vitro. In platelets, Tablysin-15 inhibits aggregation induced by collagen, ADP and convulxin, and prevents static platelet adhesion to immobilised fibrinogen. In addition, solid-phase assays and flow cytometry demonstrates that αIIbβ3 binds to Tablysin-15. Moreover, immobilised Tablysin-15 supports platelet adhesion by a mechanism which was blocked by anti-integrin αIIbβ3 monoclonal antibody (e.g. abciximab) or by EDTA. Furthermore, Tablysin-15 dose-dependently attenuates thrombus formation to collagen under flow. Consistent with these findings, Tablysin-15 displays antithrombotic properties in vivo suggesting that it is a useful tool to block αIIbβ3, or as a prototype to develop antithrombotics. The RGD motif in the unique sequence of Tablysin-15 represents a novel template for studying the structure-function relationship of the disintegrin family of inhibitors.