Interleukin (IL) 33, a member of the IL-1 superfamily, is an "alarmin" protein and is secreted in its active form from damaged cells undergoing necrotic cell death. Mast cells are one of the main effector cell types in allergic disorders. They secrete a variety of mediators, including T helper 2 cytokines. As mast cells have high-affinity IgE receptors (FcepsilonRI) on their surface, they can capture circulating IgE. IgE-bound mast cells degranulate large amounts of histamine, heparin, and proteases when they encounter antigens. As IL-33 is an important mediator of innate immunity and mast cells play an important role in adaptive immune responses, interactions between the two could link innate and adaptive immunity. IL-33 promotes the adhesion of mast cells to laminin, fibronectin, and vitronectin. IL-33 increases the expression of adhesion molecules, such as intracellular adhesion molecule-1 and vascular cell adhesion molecule-1, in endothelial cells, thus enhancing mast cell adhesion to blood vessel walls. IL-33 stimulates mast cell proliferation by activating the ST2/Myd88 pathway; increases mast cell survival by the activation of survival proteins such as Bcl-XL; and promotes the growth, development, and maturation of mast cell progenitors. IL-33 is also involved in the activation of mature mast cells and production of different proinflammatory cytokines. The interaction of IL-33 and mast cells could have important clinical implications in the field of clinical urology. Epithelial dysfunction and mast cells could play an important role in the pathogenesis of interstitial cystitis. Urinary levels of IL-33 significantly increase in patients with interstitial cystitis. In addition, the number of mast cells significantly increase in the urinary bladders of patients with interstitial cystitis. Therefore, inhibition of mast cell activation and degranulation in response to increase in IL-33 is a potential therapeutic target in the treatment of interstitial cystitis.
Adaptive Immunity* ; Allergy and Immunology ; Blood Vessels ; Cell Death ; Cystitis, Interstitial ; Cytokines ; Endothelial Cells ; Fibronectins ; Heparin ; Histamine ; Humans ; Immunity, Innate ; Immunoglobulin E ; Interleukin-1 ; Interleukins ; Laminin ; Mast Cells* ; Peptide Hydrolases ; Receptors, IgE ; Urinary Bladder ; Urology ; Vascular Cell Adhesion Molecule-1 ; Vitronectin

Integrin alphavbeta3 plays a major role in various signaling pathways, cell apoptosis, and tumor angiogenesis. To examine the functions and roles of alphavbeta3 integrin, a stable CHO-677 cell line expressing the murine alphavbeta3 heterodimer (designated as "CHO-677-malphavbeta3" cells) was established using a highly efficient lentiviral-mediated gene transfer technique. Integrin subunits alphav and beta3 were detected at the gene and protein levels by polymerase chain reaction (PCR) and indirect immunofluorescent assay (IFA), respectively, in the CHO-677-malphavbeta3 cell line at the 20th passage, implying that these genes were successfully introduced into the CHO-677 cells and expressed stably. A plaque-forming assay, 50% tissue culture infective dose (TCID50), real-time quantitative reverse transcription-PCR, and IFA were used to detect the replication levels of Foot-and-mouth disease virus (FMDV) in the CHO-677-malphavbeta3 cell line. After infection with FMDV/O/ZK/93, the cell line showed a significant increase in viral RNA and protein compared with CHO-677 cells. These findings suggest that we successfully established a stable alphavbeta3-receptor-expressing cell line with increased susceptibility to FMDV. This cell line will be very useful for further investigation of alphavbeta3 integrin, and as a cell model for FMDV research.
Animals ; Animals, Suckling ; CHO Cells ; Cloning, Molecular ; Cricetulus ; DNA, Complementary/genetics/metabolism ; Disease Susceptibility/virology ; Foot-and-Mouth Disease/*genetics/virology ; Foot-and-Mouth Disease Virus/*physiology ; Integrin alphaVbeta3/*genetics/metabolism ; Mice

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

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

Integrins such as αvβ3, α5β1 play a key role in angiogenesis regulation, invasion and metastasis, inflammation, wound healing, etc. The up-regulation of integrin αvβ3 after cerebral ischemic stroke can promote angiogenesis, which in turn improves functional recovery. In addition, the integrin αvβ3 inhibitor can block the blood-brain barrier (BBB) leakage induced by vascular endothelial growth factor (VEGF) and also can reduce inflammatory reaction, decrease the deposition of fibrinogen. Other studies showed that integrin αvβ3 is not essential in revascularization. Therefore, the effect of integrin αvβ3 in the whole process of brain function recovery merits further study.
Animals ; Blood Vessels ; drug effects ; physiopathology ; Blood-Brain Barrier ; drug effects ; metabolism ; physiopathology ; Humans ; Integrin alphaVbeta3 ; antagonists & inhibitors ; metabolism ; Integrins ; metabolism ; Models, Biological ; Peptides, Cyclic ; pharmacology ; Stroke ; metabolism ; physiopathology ; Vascular Endothelial Growth Factor A ; metabolism

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

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

OBJECTIVETo observe the effect of Yiqixue Buganshen recipe(, YBR) on the expression of integrin ανβ3 in the endometrium of controlled ovarian hyperstimulation mice.

METHODSA total of 180 mice were divided into three groups: model group, treatment group and control group. The treatment and model groups were intraperitoneally injected with gonadotropin-releasing hormone analogue for 7 days; pregnant mare serum gonadotropin was also injected on the 7th day. After 48 h, human chorionic gonadotropin was injected. The control group was injected with an equal volume of saline at the same time. From the start of the experiment, the treatment group was intragastrically administered Jinghouzengzhi Recipe() and Cuhuangti Recipe(). The model group and the control group were intragastrically administered an equal volume of saline. Real-time reverse transcription polymerase chain reaction and Western blotting were used to detect the mRNA and protein expression of integrin ανβ3 in mouse endometrium.

RESULTSIntegrin ανβ3 was expressed in mouse endometrium in all groups. Integrin αββ3 expression increased gradually along with pregnancy, progressing from pregnant day (Pd) 1. Integrin ανβ3 expression significantly increased on Pd 4, then began to decrease on Pd 6. Integrin ανβ3 expression in the treatment group was higher than in the model group, and the difference was statistically significant (P <0.05). The difference between the treatment group and the control group was not statistically significant (P >0.05).

CONCLUSIONYBR improves endometrial receptivity, and may play an important role in embryonic implantation.

Animals ; Blotting, Western ; Drugs, Chinese Herbal ; pharmacology ; Embryo Implantation ; drug effects ; Endometrium ; drug effects ; metabolism ; Female ; Gene Expression Regulation ; drug effects ; Horses ; Humans ; Integrin alphaVbeta3 ; genetics ; metabolism ; Mice ; Ovulation Induction ; Pregnancy ; RNA, Messenger ; genetics ; metabolism

OBJECTIVETo construct angiogenesis-specific RGD10-NGR9 dual-targeting superparamagnetic iron oxide nanoparticles, and to evaluate its magnetic resonamce imaging (MRI) features in nude mice and potential diagnostic value in tumor MRI.

METHODSDual-targeting peptides RGD10-NGR9 were designed and synthesized. Ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles were synthesized by chemical co-precipitation method and the surface was modified to be hydrophilic by coating with dextran. The dual-targeting peptides RGD10-NGR9 were conjugated to USPIO. Cell binding affinity and up-taking ability of the dual-targeting USPIO nanoparticles to integrin ανβ3-APN positive cells were subsequently tested by Prussian blue staining and phenanthroline colorimetry in vitro. The RGD10-NGR9 conjugated with USPIO was injected intravenously into xenograft mice, which were scanned by MRI at predetermined time points. The MRI and contrast-to-noise ratio (CNR) values were calculated to evaluate the ability of dual-targeting USPIO as a potential contrast agent in nude mice.

RESULTSP-CLN-Dextran-USPIO nanoparticles with stable physical properties were successfully constructed. The average diameter of Fe3O4 nanoparticles was 8-10 nm, that of Dextran-USPIO was about 20 nm and P-CLN-Dextran-USPIO had an average diameter about 30 nm. The in vitro studies showed a better specificity of dual-targeting USPIO nanoparticles on proliferating human umbilical vein endothelia cells (HUVEC). In vivo, RGD10-NGR9-USPIO showed a significantly reduced contrast in signal intensity and 2.83-times increased the CNR in the tumor MRI in xenograft mice.

CONCLUSIONThis novel synthesized RGD10-NGR9 dual-targeting USPIO is with better specific affinity in vitro and in vivo, and might be used as a molecular contrast agent for tumor angiogenesis MRI.

Adenocarcinoma ; diagnosis ; metabolism ; pathology ; Aminopeptidases ; analysis ; Animals ; Cell Line, Tumor ; Cells, Cultured ; Contrast Media ; chemistry ; Dextrans ; chemistry ; Ferrosoferric Oxide ; metabolism ; Human Umbilical Vein Endothelial Cells ; cytology ; metabolism ; Humans ; Integrin alphaVbeta3 ; analysis ; Lung Neoplasms ; diagnosis ; metabolism ; pathology ; Magnetic Resonance Imaging ; Magnetite Nanoparticles ; chemistry ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Transplantation ; Oligopeptides ; chemistry ; Particle Size ; Signal-To-Noise Ratio

BACKGROUNDAngiogenesis is an essential step for tumor development and metastasis. The cell adhesion molecule avβ3 integrin plays an important role in angiogenesis and is a specific marker of tumor angiogenesis. A novel avβ3 integrin- targeted magnetic resonance (MR) imaging contrast agent utilizing Arg-Gly-Asp (RGD) and ultrasmall superparamagnetic iron oxide particles (USPIO) (referred to as RGD-USPIO) was designed and its uptake by endothelial cells was assessed both in vitro and in vivo to evaluate the angiogenic profile of lung cancer.

METHODSUSPIO were coated with -NH3+ and conjugated with RGD peptides. Prussian blue staining was performed to evaluate the specific uptake of RGD-USPIO by human umbilical vein endothelial cells (HUVECs). Targeted uptake and subcellular localization of RGD-USPIO in HUVECs were confirmed by transmission electron microscopy (TEM). The ability of RGD-USPIO to noninvasively assess avβ3 integrin positive vessels in lung adenocarcinoma A549 tumor xenografts was evaluated with a 4.7T MR scanner. Immunohistochemistry was used to detect avβ3 integrin expression and vessel distribution in A549 tumor xenografts.

RESULTSHUVECs internalized RGD-USPIO significantly more than plain USPIO. The uptake of RGD-USPIO by HUVECs could be competitively inhibited by addition of free RGD. A significant decrease in T2 signal intensity (SI) was observed at the periphery of A549 tumor xenografts at 30 minutes (P < 0.05) and 2 hours (P < 0.01) after RGD-USPIO was injected via the tail vein. Angiogenic blood vessels were mainly distributed in the periphery of tumor xenografts with positive avβ3 integrin expression.

CONCLUSIONSRGD-USPIO could specifically label avβ3 integrin and be taken up by HUVECs. This molecular MR imaging contrast agent can specifically evaluate the angiogenic profile of lung cancer using a 4.7T MR scanner.

Animals ; Cells, Cultured ; Dextrans ; therapeutic use ; Humans ; Integrin alphaVbeta3 ; analysis ; Lung Neoplasms ; blood supply ; drug therapy ; Magnetic Resonance Imaging ; Magnetite Nanoparticles ; therapeutic use ; Mice ; Mice, Inbred BALB C ; Neovascularization, Pathologic ; prevention & control ; Oligopeptides ; therapeutic use