BACKGROUND AND OBJECTIVES: Adenosine diphosphate (ADP), which is usually secreted from activated platelets, may activate integrins on vascular smooth muscle cells, resulting in adhesion and proliferation. Integrins, mediating the ADP-stimulated adhesion and proliferation of vascular smooth muscle cells, was investigated in this study. MATERIALS AND METHODS: Prothrombin (PT) and bone sialoprotein (BSP) were used as activation-dependent ligands in an adhesion assay. The adhesion of human aortic smooth muscle cells (HASMC) were measured after ADP stimulation, using ligand-coated 24-well plates. The 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay was used to evaluate the ADP-stimulated proliferation of the HASMC.RESULTS: ADP activated the HASMC to increase their adhesion to the PT or BSP, and their proliferation in a dose-dependent manner. The adhesion of the ADP-stimulated HASMC to the PT was completely blocked by P5H9, a blocking monoclonal Ab (mAb) to integrin alphavbeta5 (92% inhibition), but was only slightly inhibited by LM609, a blocking mAb to integrin alphavbeta3 (30% inhibition). The adhesion of the ADP-stimulated HASMC to the BSP was partially inhibited by both P5H9 (46% inhibition) and JBS5, a blocking mAb to integrin alpha5beta1 (75% inhibition), but was not affected by c7E3, a blocking mAb to integrin beta3. The ADP-stimulated proliferation of the HASMC was inhibited by both c7E3 and LM609 (98% and 93% inhibition, respectively), but not by either P1F5, a blocking mAb to integrin alphavbeta5 or JBS5. CONCLUSION: These results indicate the different roles of integrins on vascular smooth muscle cells after ADP stimulation; the integrins alphavbeta5 and alpha5beta1 for adhesion, and the integrin alphavbeta3 for proliferation.
Adenosine Diphosphate ; Humans ; Integrin alpha5beta1 ; Integrin alphaVbeta3 ; Integrin beta3 ; Integrin-Binding Sialoprotein ; Integrins* ; Ligands ; Muscle, Smooth, Vascular* ; Myocytes, Smooth Muscle ; Negotiating* ; Prothrombin

In order to generate a pharmacophore model of integrin alphavbeta3 receptor antagonists and design lead compounds which have potent and selective activity against alphavbeta3 receptor with the help of this model. Thirty compounds (four categories) with highly inhibitory activity against the integrin alphavbeta3 receptor (IC50 < 110 nmol x L(-1)), amide, piperazine, piperidine, gamma-valerolactam as the intermediate junction, separately, were selected as a training set to construct a three-dimensional pharmacophore models of integrin alphavbeta3 receptor antagonists with the Catalyst software. The best pharmacophore model of integrin alphavbeta3 receptor antagonists with RMS = 0.73, Correl = 0.90, Weight = 1.17, Config = 14.00 is found out, which consisting of four features: a neg ionizable core (NI), two aliphatic hydrophobic core (HP) and an aromatic ring center (RA). Some new and easily obtained compounds with fine ADME properties and highly potent activity against alphavbeta3 receptor were designed with the new pharmacophore models.
Computer-Aided Design ; Drug Design ; Integrin alphaVbeta3 ; antagonists & inhibitors ; chemistry ; Models, Molecular ; Molecular Structure

Animals ; Humans ; Integrin alphaVbeta3 ; physiology ; Receptors, Virus ; physiology ; Signal Transduction ; Virus Diseases ; etiology

Angiostatin is derived from enzymatic degradation of plasminogen and it has endogenous anti-angiogenic properties. Although tumor cells, macrophages, platelets, and neutrophils generate high amount of angiostatin, its expression is increased in inflammatory conditions. Moreover, angiostatin binds to integrin alpha(v)beta(3), ATP synthase, and angiomotin, which expressed on neutrophils. Activated neutrophils are essential to innate immune response, but also cause tissue damage through production of reactive oxygen species (ROS) and increase lifespan. In this article, it suggests several mechanism of angiostatin as immune regulator for neutrophils in inflammatory conditions; complex with integrin alpha(v)beta(3) and F(1)F(0) ATP synthase on lipid raft, attenuate polarization, and ROS production. These data provide possible exploit of double-edged role of neutrophils in acute inflammatory pathologies to preserve beneficial effect and minimize tissue damage.
Adenosine Triphosphate ; Angiostatins* ; Apoptosis ; Immunity, Innate ; Integrin alphaVbeta3 ; Macrophages ; Neutrophil Activation* ; Neutrophils* ; Pathology ; Plasminogen ; Reactive Oxygen Species

OBJECTIVETo assess the binding ability of microbubbles targeted to alphavbeta3-integrin (MBp) for thrombus-targeted contrast-enhanced ultrasound.

METHODSTargeted microbubbles were prepared by conjugating the monoclonal antibody against alphavbeta3-integrin to lipid shell of the microbubble via the avidin-biotin bridges. Equivalent isotype control microbubbles (MB) or targeted ultrasound microbubbles (MBp) were randomly added into the flow chamber. After a 30-min incubation with the thrombus fixed in an agarose flow chamber model, the thrombus was washed with a continuous flow of PBS solution (15 cm/s) for 2, 4, 6, 8 and 10 min, followed by thrombus imaging using contrast-enhanced ultrasound and measurement of the video intensity (VI) values of the images.

RESULTSThe VI of the thrombus in MBp group was reduced by 28%-66%, while that in control MB group was decreased by 87%-94%, and the VI values of the thrombus group were significantly greater in former group at each of the time points (P<0.05).

CONCLUSIONMBP has good targeting ability to the thrombus with resistance to the shear stress after adhesion to the thrombus. In vitro evaluation of the thrombus-binding capability of the targeted microbubble (MBp) by simulating the shear stress in vivo can be helpful for predicting the in vivo effects of ultrasonic molecular imaging using MBp.

Antibodies, Monoclonal ; chemistry ; immunology ; Contrast Media ; chemistry ; Humans ; Integrin alphaVbeta3 ; immunology ; metabolism ; Microbubbles ; Sepharose ; Thrombosis ; diagnostic imaging ; Ultrasonography

This study investigated a nano drug delivery system built by one sort of modified trimethyl chitosan (TMC). The TMC was modified by cRGDyk, ligand of integrin receptor avβ3. Single factor screening was used to optimize the prescription in which the particle sizes of TMC nanoparticle (TMC NPs) and cRGDyk modified TMC nanoparticle (C-TMC NPs) were (240.3 ± 4.2) nm and (259.5 ± 3.3) nm. Electric potential of those two nanoparticles were (33.5 ± 0.8) mV and (25.7 ± 1.6) mV. Encapsulation efficiencies were (76.0 ± 2.2) % and (74.4 ± 2.0) %. Drug loading efficacies were (50.1 ± 2.1) % and (26.1 ± 1.0) %. Then the cellular uptake, uptake mechanism and transport efficacy of TMC NPs and C-TMC NPs were investigated using Caco-2 cell line. The uptake rate and accumulating drug transit dose of C-TMC NPs were 1.98 and 2.84 times higher than TMC NPs, separately. Mechanism investigations revealed that caveolae-mediated endocytosis, clathrin-mediated endocytosis and macropinocytosis were involved in the intercellular uptake of both TMC NPs and C-TMC NPs. What is more, free cRGDyk could remarkably inhibit the uptake of C-TMC NPs.
Biological Transport ; Caco-2 Cells ; Caveolae ; Chitosan ; chemistry ; Clathrin ; Endocytosis ; Humans ; Integrin alphaVbeta3 ; chemistry ; Nanoparticles ; Particle Size ; Pinocytosis

OBJECTIVETo investigate the regulatory effect of Jingang Jiangu pill (see text, JGJG) on expression of integrin in ovariectomized rats.

METHODSFifty ovariectomized 10 months old female rats were randomly divided into 5 groups: Fushanmei group (FSM), Jingang Jiangu pill (see text) group (JGJG), Gusongbao granule group (GSB), Model group (OVX), Sham group. After ovariectomized,the rats were raised in the same environment for 13 weeks. The rats in JGJG group took 0.13 g JGJG pill orally each day for each rat; the rats in GSB group took 0.86 g GSB granule orally each day for each rat; the rats in FSM group took 0.28 mg FSM orally each day for each rat; and the rats in OVX and sham groups took sodium. The treatment duration of rats in above 5 groups was 13 weeks. Bone mineral density (BMD) and the expression of integrin beta1 and alphavbeta3 were detected in each group after the treatment. RESYKTS: The BMD and the expression of integrin beta1 in FSM group, JGJG group and GSB group improved obviously than that of OVX group. There were statistical difference between these groups (P<0.05). The expression of integrin alphavbeta3 of the three treating groups significantly depressed.

CONCLUSIONThe JGJG pill improves BMD and express of integrin beta1, in ovariectomized rats and reduces express of integrin alphavbeta3 through the regulation of the coupling of osteoblasts and osteoclasts.

Animals ; Bone Density ; Disease Models, Animal ; Female ; Integrin alphaVbeta3 ; analysis ; Integrin beta1 ; analysis ; Medicine, Chinese Traditional ; Osteoporosis ; drug therapy ; metabolism ; Ovariectomy ; Rats ; Rats, Wistar

Netrins are secreted molecules and involved in axon guidance, cell migration and tumor development. Recent studies revealed that netrins perform novel functions in such processes as epithelial development and angiogenesis without operating through the classical netrin receptors, DCC (Deleted in Colorectal Cancer) and Unc5h. In the present study, we investigated the roles of netrin-1 and its receptors in cell spreading of human glioblastoma cells, and found that netrin-1 haptotactically enhanced fibronectin-induced cell spreading and focal adhesion formation in U373 glioblastoma cells. Netrin-1 binding to the U373 cell membrane was blocked by an antibody against alpha v integrin subunit, but not by an anti-DCC or anti-Unc5h antibody. In addition, enhancement of the fibronectin response by netrin-1 was abrogated by a function blocking antibody against integrin alpha v beta 3. Since the alpha v subunit of the integrin family plays an important role in the pathophysiological aspects of cell migration, including tumor angiogenesis and metastasis, our data provide important insight into the molecular mechanism of netrin function.
Axons ; Cell Membrane ; Cell Movement ; Fibronectins ; Focal Adhesions ; Glioblastoma ; Humans ; Integrin alphaV ; Integrin alphaVbeta3 ; Neoplasm Metastasis ; Nerve Growth Factors ; Receptors, Cell Surface ; Tumor Suppressor Proteins

BACKGROUND: We investigate the influence of cell surface adhesion receptor integrin alphavbeta3, alpha5beta1 contributes to proliferation and migration of tumor cell in osteosarcoma for carves out a new treatment model by regulation of integrin roles in human osteosarcoma. MATERIALS AND METHODS: We performed proliferation assay, total 11 cell lines including 7 osteosarcoma cell lines established from patients and 4 osteosarcoma standard cell lines. Murine monoclonal anti-alpha5beta1 and anti-alphavbeta3 (Chemicon International Inc. Temecula, CA) were used for growth inhibition assays. We also performed cell motility assay by using the Boyden chamber to evaluate the effect of integrin mediated cell migration. We used the HOS standard osteosarcoma cell lines and each separates contained serum free media with mouse IgG1 negative control antibody, anti-alpha5beta1 antibody and anti-alphavbeta3 antibody. RESULTS: Proliferation of cells decreased significantly in 10 out of 11 cell lines when blocking with alphavbeta3 or alpha5beta1 respectively. Blocking with anti-alphavbeta3 antibody decreased significantly tumor cell proliferation in 10 cell lines. Among the 10 cell lines, 7 cell lines showed significantly more decrease of proliferation with anti-alphavbeta3 antibody than with anti-alpha5beta1antibody. Blocking with anti-alpha5beta1 antibody decreased significantly tumor cell proliferation in 10 cell lines. Among the 10 cell lines, 3 cell lines showed significantly more decrease of proliferation with anti-alpha5beta1 antibody than with anti-alphavbeta3 antibody. Including statistically not significant 2 cell lines the growth inhibition of osteosarcoma cell lines was more obvious (10 out of 11) in blocking with anti-alphavbeta3 antibody. The migration of cells was significantly decreased when blocked with anti-alpha5beta1 antibody and anti-alphavbeta3 antibody. CONCLUSION: Under the based on the integrin alphavbeta3, alpha5 beta1 are central role on proliferation and migration of osteosarcoma cells, we could be more approach to new therapeutic endeavors with antibody to integrin alphavbeta3, alpha5beta1 molecular target of osteosarcoma.
Animals ; Cell Line ; Cell Migration Assays ; Cell Movement ; Cell Proliferation* ; Culture Media, Serum-Free ; Humans* ; Immunoglobulin G ; Integrin alphaVbeta3* ; Mice ; Osteosarcoma*

The selective targeting of an integrin alphavbeta3 receptor using radioligands may enable the assessment of angiogenesis and integrin alphavbeta3 receptor status in tumors. The aim of this research was to label a peptidomimetic integrin alphavbeta3 antagonist (PIA) with 99mTc(CO)3 and to test its receptor targeting properties in nude mice bearing receptor-positive tumors. PIA was reacted with tris-succinimidyl aminotriacetate (TSAT) (20 mM) as a PIA per TSAT. The product, PIA-aminodiacetic acid (ADA), was radiolabeled with [99mTc(CO)3(H2O)3](+1), and purified sequentially on a Sep-Pak C-18 cartridge followed by a Sep-Pak QMA anion exchange cartridge. Using gradient C-18 reverse-phase HPLC, the radiochemical purity of 99mTc(CO)3-ADA-PIA (retention time, 10.5 min) was confirmed to be > 95%. Biodistribution analysis was performed in nude mice (n = 5 per time point) bearing receptor-positive M21 human melanoma xenografts. The mice were administered 99mTc(CO)3-ADA-PIA intravenously. The animals were euthanized at 0.33, 1, and 2 hr after injection for the biodistribution study. A separate group of mice were also co-injected with 200 microg of PIA and euthanized at 1 hr to quantify tumor uptake. 99mTc(CO)3-ADA-PIA was stable in phosphate buffer for 21 hr, but at 3 and 6 hr, 7.9 and 11.5% of the radioactivity was lost as histidine, respectively. In tumor bearing mice, 99mTc(CO)3-ADA-PIA accumulated rapidly in a receptor-positive tumor with a peak uptake at 20 min, and rapid clearance from blood occurring primarily through the hepatobiliary system. At 20 min, the tumor-to-blood ratio was 1.8. At 1 hr, the tumor uptake was 0.47% injected dose (ID)/g, but decreased to 0.12% ID/g when co-injected with an excess amount of PIA, indicating that accumulation was receptor mediated. These results demonstrate successful 99mTc labeling of a peptidomimetic integrin antagonist that accumulated in a tumor via receptor-specific binding. However, tumor uptake was very low because of low blood concentrations that likely resulted from rapid uptake of the agent into the hepatobiliary system. This study suggests that for 99mTc(CO)3-ADA-PIA to be useful as a tumor detection agent, it will be necessary to improve receptor binding affinity and increase the hydrophilicity of the product to minimize rapid hepatobiliary uptake.
Animals ; Chromatography, High Pressure Liquid ; Histidine ; Humans ; Hydrophobic and Hydrophilic Interactions ; Integrin alphaVbeta3 ; Melanoma ; Mice ; Mice, Nude ; Radioactivity ; Succinimides ; Transplantation, Heterologous ; Ursidae