Fluorescein Isothiocyanate-bovine Serum Albumin Research Articles

Biphenyl-Induced Superhelix in l -Phenylalanine-Based Supramolecular Self-Assembly with Dynamic Morphology Transitions

Biphenyl-Induced Superhelix in <scp>l</scp> -Phenylalanine-Based Supramolecular Self-Assembly with Dynamic Morphology Transitions

MicroRNA-93-5p participates in type 2 diabetic retinopathy through targeting Sirt1.

To investigate the role of miR-93-5p in rats with type 2 diabetic retinopathy (DR) through targeting Sirt1. The targeting correlation between miR-93-5p and Sirt1 was validated by dual-luciferase reporter gene assay. Type 2 diabetes mellitus (T2DM) rat models were received intravitreal injection of antagomir NC (negative control), miR-93-5p antagomir, miR-93-5p agomir and/or recombinant Sirt1, followed by observation of pathological changes in retina via HE staining. Besides, retinal vascular permeability was determined by fluorescein isothiocyanate-bovine serum albumin (FITC-BSA), while the retinal vasculature was observed through retinal trypsin digestion. Expression of miR-93-5p and Sirt1 was measured by qRT-PCR and Western blotting, while the levels of VEGF, proinflammatory cytokines and anti-oxidative indicators were determined using corresponding kits. MiR-93-5p could target Sirt1 as analyzed by the luciferase reporter gene assay. Rats in the T2DM group presented the up-regulation of miR-93-5p and down-regulation of Sirt1 in the retina, and miR-93-5p inhibition could up-regulate Sirt1 expression in the T2DM rats. Recombinant Sirt1 decreased retinal vascular permeability and acellular capillaries with improved pathological changes in retina from T2DM rats, which was abolished by miR-93-5p agomir. Moreover, miR-93-5p inhibition or Sirt1 overexpression decreased the levels of VEGF and proinflammatory cytokines while enhancing the activity of anti-oxidative indicators. However, indicators above had no significant differences between T2DM group and T2DM + agomir + Sirt1 group. MiR-93-5p, via targeting Sirt1, could affect the vascular permeability and acellular capillaries and mitigate the inflammation and oxidative stress in the retinas, which may play a critical role in DR.

Locally Controlled Diffusive Release of Bone Morphogenetic Protein-2 Using Micropatterned Gelatin Methacrylate Hydrogel Carriers.

In this work, a novel and simple bone morphogenetic protein (BMP)-2 carrier is developed, which enables localized and controlled release of BMP-2 and facilitates bone regeneration. BMP-2 is localized in the gelatin methacrylate (GelMA) micropatterns on hydrophilic semi-permeable membrane (SNM), and its controlled release is regulated by the concentration of GelMA hydrogel and BMP-2. The controlled release of BMP-2 is verified using computational analysis and quantified using fluorescein isothiocyanate-bovine serum albumin (FITC-BSA) diffusion model. The osteogenic differentiation of osteosarcoma MG-63 cells is manipulated by localized and controlled BMP-2 release. The calcium deposits are significantly higher and the actin skeletal networks are denser in MG-63 cells cultured in the BMP-2-immobilized GelMA micropattern than in the absence of BMP-2. The proposed BMP-2 carrier is expected to not only act as a barrier membrane that can prevent invasion of connective tissue during bone regeneration, but also as a carrier capable of localizing and controlling the release of BMP-2 due to GelMA micropatterning on SNM. This approach can be extensively applied to tissue engineering, including the localization and encapsulation of cells or drugs.

Controlled Exposure of Bioactive Growth Factor in 3D Amyloid Hydrogel for Stem Cells Differentiation.

Amyloid based hydrogels can mimic the extracellular matrix and serve as matrices for tissue engineering both in vitro and in vivo. A pH responsive self-assembled amyloid hydrogel system is used to encapsulate various growth factors for driving stem cell differentiation toward neuronal lineage. Diffusion studies with fluorescence recovery after photobleaching and bulk release with the model protein fluorescein isothiocyanate-bovine serum albumin show that encapsulated protein molecules can be released in a sustained fashion from the hydrogel over a considerable period of time (30 d). Moreover, by modulating the porosity of the hydrogel by the simple addition of salt, the encapsulated protein molecules can be retained for a longer period of time within the hydrogel. Mesenchymal stem cells, when cultured in 3D amyloid hydrogels with growth factors fibroblast growth factor 8 and sonic hedgehog, show more neuron specific differentiation as compared to hydrogel alone. This higher differentiation potential of growth factor encapsulated amyloid hydrogels can be due to concomitant exposure of cells to biomechanical as well as biochemical cues during the course of differentiation. The present study suggests that amyloid based hydrogel can be exploited for controlled growth factor delivery as well as directed stem cell differentiation to neuron.

Dendritic Mesoporous Silica Nanospheres Synthesized by a Novel Dual-Templating Micelle System for the Preparation of Functional Nanomaterials.

Highly monodisperse, dendritic, and functionalized mesoporous silica nanospheres (MSNs) with sub-200 nm size were synthesized in a one-pot sol-gel reaction, by a dual-templating micelle system consisting of a partially fluorinated short-chain anionic fluorocarbon surfactant and cetyltrimethylammonium bromide. This kind of anionic fluorocarbon surfactant works simultaneously as a swelling agent to enlarge the pore of the MSNs, an ion-pair agent to the structure-directing silane in the preparation of amine-functionalized MSNs, and a surface tension reducing agent to make the system thermodynamically more stable for producing more uniform MSNs. The particle size and the morphology of the resultant MSNs can be fine-tuned by changing the amount of the fluorocarbon surfactant added and the ratio of the functional group containing organosilane to tetraethoxysilane. Subsequently, the as-prepared MSNs were used as base materials for the preparation of drug delivery nanomaterials through the surface grafting of a pH-sensitive drug-conjugated polymer and fluorescent nanomaterials through the embedding of europium(III) complex or the immobilization of large molecule fluorescein isothiocyanate-bovine serum albumin.

A novel study on the mechanisms of drug release in PLGA-mPEG microspheres with fluorescent drug

The purpose of this research was to proof the microspheres release mechanism by a novel method-detecting and comparing the drugs fluorescent changes on the microspheres surface. Fluorescein sodium (FS, 0.4 kDa) and fluorescein isothiocyanate-bovine serum albumin (FITC-BSA, 66.8 kDa) were employed as model drugs. FS and FITC-BSA were encapsulated into PLGA-mPEG microspheres through double emulsion evaporation method, and the drug-loaded microspheres in vitro degradation and release behaviors were evaluated by scanning electron microscope, gel permeation chromatography, confocal laser scanning microscopy (CLSM), BCA assay kit, and UV–vis spectrophotometry. FS-loaded microspheres revealed a severe initial burst release, followed by a sustained release, and we could observe a bright fluorescent on the microspheres surface during the early release period under the CLSM. The bright fluorescent gradually faded out in the later period as only 1~2% FS was remained after 14 days release. FITC-BSA-loaded microspheres revealed a typical tri-phase release profile, and we observed a weak fluorescent on the microspheres surface after the initial burst release, and the fluorescent came bright again after an obvious erosion appeared on the microspheres surface. In the later release stage, the fluorescent gradually faded out as the fast release of FITC-BSA.

Enhancement of Enzymatic Activity Using Microfabricated Poly(ε-caprolactone)/Silica Hybrid Microspheres with Hierarchically Porous Architecture

In this paper, we present a novel and facile microfluidic method to fabricate hierarchically porous poly(e-caprolactone)/silica hybrid microspheres and further investigate in detail their performance as enzyme carriers by three famous proteins and enzymes. Because of the synergy effect between sol–gel process and solvent extraction in microdroplets, hierarchically porous architecture can be formed in situ without the use of porogens and templates. More importantly, the surface porosity or the specific surface area of such microspheres can be precisely tuned via adjusting the hydrolysis/condensation rate by ammonia catalyst and thus the competition between the two above-mentioned processes. Fluorescein isothiocyanate-bovine serum albumin, alcohol dehydrogenase, and superoxide dismutase are immobilized via either physical adsorption or covalent binding to evaluate the performance of hierarchically porous microspheres as enzyme carriers. All the qualitative and quantitative data including fluorescence images...

Biocompatibility and Stability of Polysaccharide Polyelectrolyte Complexes Aimed at Respiratory Delivery.

Chitosan (CS) and chondroitin sulfate (CHS) are natural polymers with demonstrated applicability in drug delivery, while nanoparticles are one of the most explored carriers for transmucosal delivery of biopharmaceuticals. In this work we have prepared CS/CHS nanoparticles and associated for the first time the therapeutic protein insulin. Fluorescein isothiocyanate bovine serum albumin (FITC-BSA) was also used to enable comparison of behaviors regarding differences in molecular weight (5.7 kDa versus 67 kDa). Nanoparticles of approximately 200 nm and positive zeta potential around +20 mV were obtained. These parameters remained stable for up to 1 month at 4 °C. Proteins were associated with efficiencies of more than 50%. The release of FITC-BSA in PBS pH 7.4 was more sustained (50% in 24 h) than that of insulin (85% in 24 h). The biocompatibility of nanoparticles was tested in Calu-3 and A549 cells by means of three different assays. The metabolic assay MTT, the determination of lactate dehydrogenase release, and the quantification of the inflammatory response generated by cell exposure to nanoparticles have indicated an absence of overt toxicity. Overall, the results suggest good indications on the application of CS/CHS nanoparticles in respiratory transmucosal protein delivery, but the set of assays should be widened to clarify obtained results.

Electrokinetic concentrating with a nanofluidic device for magnetic beads-based antigen–antibody immunoassay

In this paper, a method of electrokinetic concentrating and a glass nanofluidic device for sensitivity-enhancing of magnetic beads-based antigen---antibody immunoassay (MBAAI) are presented. In the nanofluidic device, a polyacrylamide gel plug is deployed as the nanopores via photopolymerization reaction. To verify the capability of electrokinetic concentrating, the fluorescein isothiocyanate-bovine serum albumin (FITC-BSA) with initial concentration of 0.002 ng/ml is enriched and the concentration factor of a hundred million fold is achieved. And then the technique is implemented to improve the MBAAI sensitivity. In this experiment, magnetic beads coupled with sheep anti-rabbit IgG (immunoglobulin G) are firstly trapped into the immunoassay region via magnetic force, then the FITC-labeled rabbit IgG is electrokinetically concentrated and spontaneously bonded with the previous fixed magnetic beads. After the incubation, the total fluorescence intensity in immunoassay region is calculated. The result shows that the fluorescence intensity is improved by 58 % and the antigen-concentrating with the nanofluidic device can effectively enhance the sensitivity of MBAAI.

Curcumin-primed exosomes mitigate endothelial cell dysfunction during hyperhomocysteinemia

AimExosomes, the nano-units (<200nm), released from diverse cell types in the extracellular body fluid, possess non-immunogenic property and ability to cross the blood–brain barrier (BBB). Since exosomes carry biological information from their cells of origin, we hypothesize that priming cells with potential therapeutic agents release improved cellular contents through exosomes. Curcumin possesses anti-oxidative and anti-inflammatory properties and provides a promising treatment for cerebral diseases and therefore, the aim of the study is to establish that mouse brain endothelial cells (MBECs) when primed with curcumin (7.5μM), release an alleviated exosome population that can help recover the endothelial cell (EC) layer permeability. Main methodsHomocysteine is a well-known causative factor of BBB disruption; therefore, homocysteine-treated ECs were used as a model of BBB disruption and curcumin-primed exosomes were utilized to check their potential for mitigating EC disruption. MBECs were treated with curcumin and exosomes were isolated by using ultracentrifugation and immunoprecipitation. Expression levels of junction proteins were detected by Western blot and immunocytochemistry assays. Endothelial cell permeability was analyzed with Fluorescein isothiocyanate-Bovine serum albumin (FITC-BSA) leakage assay using transwell permeable supports. Key findingsExosomes derived from curcumin-treated (primed) cells (CUR-EXO) alleviated oxidative stress, tight junctions (ZO-1, claudin-5, occludin), adherent junction (VE-cadherin) proteins and EC layer permeability induced during EC damage due to high homocysteine levels (hyperhomocysteinemia). SignificanceIn conclusion, the study potentiates the use of CUR-EXO for cerebral diseases where drug delivery is still a challenge. The results also pave the way to novel translational therapies for cerebral diseases by maintaining and establishing therapeutic conservatories via primed exosomes.

PH-sensitive degradable nanoparticles for highly efficient intracellular delivery of exogenous protein.

BackgroundEncapsulating exogenous proteins into a nanosized particulate system for delivery into cells is a great challenge. To address this issue, we developed a novel nanoparticle delivery method that differs from the nanoparticles reported to date because its core was composed of cross-linked dextran glassy nanoparticles which had pH in endosome-responsive environment and the protein was loaded in the core of cross-linked dextran glassy nanoparticles.MethodsIn this study, dextran in a poly(ethylene glycol) aqueous two-phase system created a different chemical environment in which proteins were encapsulated very efficiently (84.3% and 89.6% for enhanced green fluorescent protein and bovine serum albumin, respectively) by thermodynamically favored partition. The structures of the nanoparticles were confirmed by confocal laser scanning microscopy and scanning electron microscopy.ResultsThe nanoparticles had a normal size distribution and a mean diameter of 186 nm. MTT assays showed that the nanoparticles were nontoxic up to a concentration of 2000 μg/mL in human hepatocarcinoma cell line SMMC-7721, HeLa, and BRL-3A cells. Of note, confocal laser scanning microscopy studies showed that nanoparticles loaded with fluorescein isothiocyanate-bovine serum albumin were efficiently delivered and released proteins into the cytoplasm of HeLa cells. Flow cytometry and terminal deoxynucleotidyl transferase dUTP nick end labeling assays showed that nanoparticles with a functional protein (apoptin) efficiently induced significant tumor cell apoptosis, which was confirmed by DAPI staining.ConclusionOur findings indicate that these nanoparticles meet the high demands for delivering protein medicines and have great potential in protein therapy.

Preparation of Calcium Phosphate – Peptide Composites with Highly pH-Sensitive Drug Release

Calcium phosphate (CaP) material has been used successfully for protein delivery. In the present work, our aim is the preparation of CaP combined with peptide and protein, and is the evaluation of pH-sensitive drug release ability. Protein used as the model drug was fluorescein isothiocyanate-bovine serum albumin (FITC-BSA). All compounds synthesized were low crystalline hydroxyapatite (HAp). The result of zeta potential indicated that incorporation of peptide in HAp could be changed the surface charge of particle. The CaP-BSA and CaP-BSA-pLys [Poly-L-lysine (pLys) was added to CaP composite.] were-23.1 mV and-12.0 mV, respectively. The ratio of protein release and dissolved Ca ion were measured by soaking CaP-peptide composites under various pH (pH 7.4, pH 6 and pH 5) conditions. BSA was released from CaP-BSA-pLys at only pH 5, not at pH 7.4 and pH 6.

Preliminary characteristic of alginate, heparin-chitosan-alginate, heparin microencapsulated hepatocytes system

Alginate, low mole mass heparin-chitosan-alginate, low mole mass heparin microcapsules (ALCAL) with good mechanical stability were made from the high voltage pulsing microcapsule shaping device. ALCAL was made of low-mole-mass heparin (LMWH), alginate (ALG) and chitosan (CS) by supramolecular layer upon layer self-assembled technique. It was found from the microscopic observation that the microcapsules had smooth surface and a porous structure with interconnected pores. The results of the permeability experiment of microcapsules using fluorescein isothiocyanate-bovine serum albumin (FITC-BSA) and fluorescein isothiocyanate-immunoglobulin G (FITC-IgG) showed that the ALCAL membrane could provide cell immuno-isolation; meanwhile, the ALCAL membrane had good biocompatibility. The potential of ALCAL microcapsules for the encapsulation of liver cells had been investigated and showed that the ALCAL membrane supports the survival, proliferation and protein secretion on encapsulating hepatocytes. The ALCAL microcapsule had several advantages compared to more widely used alginate-chitosan-alginate (ACA) microcapsules for the application of cell therapy.

Methylglyoxal induces hyperpermeability of the blood–retinal barrier via the loss of tight junction proteins and the activation of matrix metalloproteinases

One of the early signs of diabetic retinopathy is the alteration of the blood-retinal barrier (BRB), which may involve the breakdown of endothelial cell tight junctions. Methylglyoxal (MGO) is a cytotoxic metabolite that is produced from glycolysis in vivo. Elevated levels of MGO are observed in a number of pathological conditions, including neurodegenerative disorders and diabetic complications. Herein, we hypothesize that increased levels of MGO disrupt the tight junction protein known as occludin protein by matrix metalloproteinases (MMPs), leading to breakage of the BRB. MGO was intravitreally injected into eyes of rats. BRB leakage, MMPs activity, and occludin were investigated in intravitreally MGO-injected eyes. When normoglycemic rats were intravitreally injected with 400μM MGO, there was widespread leakage of fluorescein isothiocyanate-bovine serum albumin (FITC-BSA) from the retinal vasculature when compared to control retinas. In addition, MGO-injected retinas demonstrated increases of both activity and expression of MMP-2 and MMP-9, and the degradation of occludin was found in the MGO-injected retinas. The results suggest that the activation of MMPs by elevated levels of MGO in the retina may facilitate an increase in vascular permeability by a mechanism involving proteolytic degradation of occludin. These findings may have implications for the role of MGO in the pathogenesis of diabetic retinopathy.

Fibrinogen-Induced Increased Pial Venular Permeability in Mice

Elevated blood level of Fibrinogen (Fg) is commonly associated with vascular dysfunction. We tested the hypothesis that at pathologically high levels, Fg increases cerebrovascular permeability by activating matrix metalloproteinases (MMPs). Fibrinogen (4 mg/mL blood concentration) or equal volume of phosphate-buffered saline (PBS) was infused into male wild-type (WT; C57BL/6J) or MMP-9 gene knockout (MMP9-/-) mice. Pial venular leakage of fluorescein isothiocyanate-bovine serum albumin to Fg or PBS alone and to topically applied histamine (10(-5) mol/L) were assessed. Intravital fluorescence microscopy and image analysis were used to assess cerebrovascular protein leakage. Pial venular macromolecular leakage increased more after Fg infusion than after infusion of PBS in both (WT and MMP9-/-) mice but was more pronounced in WT compared with MMP9-/- mice. Expression of vascular endothelial cadherin (VE-cadherin) was less and plasmalemmal vesicle-associated protein-1 (PV-1) was greater in Fg-infused than in PBS-infused both mice groups. However, in MMP9-/- mice, VE-cadherin expression was greater and PV-1 expression was less than in WT mice. These data indicate that at higher levels, Fg compromises microvascular integrity through activation of MMP-9 and downregulation of VE-cadherin and upregulation of PV-1. Our results suggest that elevated blood level of Fg could have a significant role in cerebrovascular dysfunction and remodeling.

Mast cells are critically involved in serum-mediated vascular leakage in chronic urticaria beyond high-affinity IgE receptor stimulation

Chronic urticaria (CU) is one of the most common skin disorders whose pathogenic mechanisms are not fully clarified. Autoimmune aetiology can be ascribed to 45% of patients with CU, and basophil histamine release is positive in 40% of cases. Our aim was to use a novel approach to evaluate the serum permeabilizing effect to identify the mediators of endothelial cell (EC) leakage and to define the role of mast cells (MCs) in the process. Permeabilizing activity of sera from 19 patients with CU and 11 healthy blood donors was evaluated by measuring serum-induced degranulation of two MC lines, expressing (LAD2) or lacking (HMC-1) the IgE receptor. Mast cell supernatant (SN) was then incubated with an EC monolayer, and endothelial permeability was evaluated by Fluorescein isothiocyanate-bovine serum albumin leakage in a transwell system. All 19 patient sera failed to induce direct EC leakage, but 15/19 and 17/19 promoted degranulation of HMC-1 and LAD2, respectively. Interestingly, 85% of autologous serum skin test-negative sera were able to cause MC degranulation. Also, 17/19 SNs from HMC-1 and all SNs from LAD2 incubated with CU sera increased endothelial permeability. Endothelial cell leakage remained unchanged after Ig depletion and was prevented by antihistamine, platelet-activating factor or leukotriene antagonist. Our study shows that CU sera are able to degranulate MCs through an IgE- and IgG-independent mechanism. The nature of histamine-releasing factors involved is still unclear, but our finding opens new ways to the understanding of the pathogenesis of CU, particularly in patients not showing circulating autoantibodies to FcεRI or IgE.

Hydration Effects on Skin Microstructure as Probed by High-Resolution Cryo-Scanning Electron Microscopy and Mechanistic Implications to Enhanced Transcutaneous Delivery of Biomacromolecules

Although hydration is long known to improve the permeability of skin, penetration of macromolecules such as proteins is limited and the understanding of enhanced transport is based on empirical observations. This study uses high-resolution cryo-scanning electron microscopy to visualize microstructural changes in the stratum corneum (SC) and enable a mechanistic interpretation of biomacromolecule penetration through highly hydrated porcine skin. Swollen corneocytes, separation of lipid bilayers in the SC intercellular space to form cisternae, and networks of spherical particulates are observed in porcine skin tissue hydrated for a period of 4–10 h. This is explained through compaction of skin lipids when hydrated, a reversal in the conformational transition from unilamellar liposomes in lamellar granules to lamellae between keratinocytes when the SC skin barrier is initially established. Confocal microscopy studies show distinct enhancement in penetration of fluorescein isothiocyanate-bovine serum albumin (FITC-BSA) through skin hydrated for 4–10 h, and limited penetration of FITC-BSA once skin is restored to its natively hydrated structure when exposed to the environment for 2–3 h. These results demonstrate the effectiveness of a 4–10 h hydration period to enhance transcutaneous penetration of large biomacromolecules without permanently damaging the skin. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99:730–740, 2010

Development and in vitro cytotoxicity of microparticle drug delivery system for proteins using l-tyrosine polyphosphate

l-Tyrosine polyphosphate (LTP), a “pseudo” poly (amino acid) polymer is characterized by a rapid degradation rate. Subsequently, formulation of a drug delivery system has been investigated by encapsulating fluorescein isothiocyanate–bovine serum albumin (FITC-BSA) within LTP microparticles. Characterization of surface morphology shows a mixture of spherical and discoid particles with a slightly rough surface morphology for all microparticle formulations. Dynamic laser light scattering (DLS) shows a decrease in particle diameters and size distribution upon FITC-BSA encapsulation. LTP microparticles are found to degrade over a period of 7 days, and complete release of FITC-BSA is observed over a period of 6 days. Cytotoxicity evaluation of LTP microparticles indicates that these microparticles do not cause severe cell death in cultured primary human dermal fibroblasts over a period of 10 days. Therefore, the LTP microparticles are promising candidates for short-term protein delivery applications.

Effect of Moxonidine on the Uveoscleral Outflow: Role of α2-Adrenoceptors or I1 Imidazoline Receptors

Purpose: The purpose of this study is to assess the change of uveoscleral outflow induced by moxonidine and to investigate whether the increase of uveoscleral outflow induced by moxonidine is mediated by α1, α2, or I1 receptors. Methods: 0.05% moxonidine was topically and unilaterally administered in rabbit eyes with or without pretreatment of prazosin, yohimbine, efaroxan, or AGN 192403, as indicated. We injected fluorescein isothiocyanate-bovine serum albumin (FITC-BSA) into the anterior chamber and observed the fluorescence intensity of the uveoscleral outflow. Finally, the volume of uveoscleral outflow was calculated based on the fluorescence intensities captured. Results: A bilateral increase of fluorescence intensity was observed along the uveoscleral outflow pathway following moxonidine administration, especially in the ciliary body and supraciliochoroidal space. Pretreatment with prazosin further enhanced the bilateral increase of fluorescence intensity at between 2 and 4 hours after moxonidine administration. The response of moxonidine was antagonized by either yohimbine, an α2 receptor antagonist, or efaroxan, an I1/α2 receptor antagonist. The antagonizing effect of yohimbine was more potent than that of efaroxan. The moxonidne-induced response was not antagonized by AGN 192403, an I1 receptor antagonist. The bilateral volumes of aqueous humor within the uveoscleral pathway increased significantly induced by moxonidine (p < 0.01 versus control). The increased bilateral volumes of uveoscleral outflow were 0.381 ± 0.073 and 0.376 ± 0.095 μ l/min, respectively. Conclusion: These results suggest that topical, unilateral administration of moxonidine causes a bilateral increase of aqueous humor via the uveoscleral outflow pathway. The moxonidine-induced increase of uveoscleral outflow is mediated by α2 adrenergic receptors, not by I1 imidazoline receptors.

Binding of ADAM28 to P-selectin Glycoprotein Ligand-1 Enhances P-selectin-mediated Leukocyte Adhesion to Endothelial Cells

ADAMs (a disintegrin and metalloproteinases) are a recently discovered gene family of multifunctional proteins with the disintegrin-like and metalloproteinase domains. To analyze the biological functions of ADAM28, we screened binding molecules to secreted-type ADAM28 (ADAM28s) by the yeast two-hybrid system and identified P-selectin glycoprotein ligand-1 (PSGL-1). Binding between the disintegrin-like domain of ADAM28s and the extracellular portion of PSGL-1 was determined by yeast two-hybrid assays, binding assays of the domain-specific recombinant ADAM28s species using PSGL-1 stable transfectants and leukocyte cell lines expressing native PSGL-1 (HL-60 cells and Jurkat cells), and co-immunolocalization and co-immunoprecipitation of the molecules in these cells. Incubation of HL-60 cells with recombinant ADAM28s enhanced the binding to P-selectin-coated wells and P-selectin-expressing endothelial cells. In addition, intravenous injection of ADAM28s-treated HL-60 cells increased their accumulation in the pulmonary microcirculation and alveolar spaces in a mouse model of endotoxin-induced inflammation. These data suggest a novel function that ADAM28s promotes PSGL-1/P-selectin-mediated leukocyte rolling adhesion to endothelial cells and subsequent infiltration into tissue spaces through interaction with PSGL-1 on leukocytes under inflammatory conditions.