Differential susceptibility of osteosarcoma cells and primary osteoblasts to cell detachment caused by snake venom metalloproteinase protein.

The interaction of extracellular matrix with cells plays a key role in the regulation of cell adhesion, migration, proliferation as well as differentiation. Transformed cells express a different profile of adhesion molecules, which may mediate metastasis under specific matrix microenvironment. We here found that ROS 17/2.8 osteosarcoma cells and osteoblasts have different expression of alpha5 integrin, executing different fibronectin fibrillogenesis. As compared with ROS 17/2.8 cells, osteoblasts have higher expression of fibronectin, collagen, alpha5, beta1, alpha2 integrins and focal adhesion kinase as examined by immunostaining and flow cytometry. Crovidisin, a PIII snake venom metalloproteinase (SVMP) purified from venom of Crotalus viridis, exhibits collagen-binding activity and matrix metalloproteinase activity. Crovidisin selectively caused the detachment of ROS 17/2.8 osteosarcoma cells but not of primary cultured osteoblasts. On the other hand, triflavin, an RGD-dependent disintegrin purified from venom of Trimeresurus flavoviridis, did not cause the detachment of both osteoblasts and ROS 17/2.8 cells. Although ROS 17/2.8 cells detached from substratum after crovidisin treatment for 24 h, the loss of mitochondrial membrane potential was not observed unless a prolonged treatment for longer than 36 h. These results suggest that cultured primary rat osteoblasts and ROS 17/2.8 osteosarcoma cells possess different expression of integrins and matrix environment, and ROS 17/2.8 is much more susceptible to be detached by crovidisin. The matrix degradation by crovidisin may be responsible for the preferential detachment of ROS 17/2.8 osteosarcoma cells.     

Inhibition of human monocyte adhesion to endothelial cells by the coumarin derivative, cloricromene.

1. The ability of the coumarin derivative cloricromene (8-monochloro-3-beta-diethylaminoethyl-4-methyl-7-ethoxy- carbonylmethoxycoumarin) to inhibit monocyte adhesion to human cultured umbilical vein endothelial cells (HUVEC) was investigated. 2. Cloricromene (10-200 microM) inhibited, in a concentration-dependent manner, the adhesion of both resting and activated monocytes to HUVEC. Significant inhibition was reached with drug concentrations ranging between 15 to 30 microM. 3. The inhibitory activity was, at least in large part, directed to monocytes since no inhibition was observed after selective preincubation of HUVEC with cloricromene and the drug maintained its effect also on monocyte adhesion to paraformaldehyde-treated HUVEC. 4. Inhibition was maximal after 1 min of exposure of monocytes to cloricromene and persisted even in the absence of the drug. 5. Both basal and chemoattractant-mediated monocyte adhesion was inhibited by cloricromene as it was by TS1/18, a monoclonal antibody (mAb) directed to beta 2 integrins; however, cytofluorimetric analysis showed that cloricromene was unable to modulate the expression of beta 2 integrins on the monocyte surface. 6. When monocyte adhesion was mediated by a large set of adhesive receptors, as obtained after treatment of HUVEC with either interleukin 1 beta (IL-1; 50 ng ml-1) or tumour necrosis factor-alpha (TNF; 100 u ml-1), the inhibitory effect of cloricromene was considerably reduced. 7. The results of this study show that cloricromene may regulate monocyte adhesion to HUVEC, an event relevant in vivo in the pathogenesis of inflammatory and atherosclerotic processes.     

Differential modulation of integrin expression in chondrocytes during expansion for tissue engineering

Cartilage tissue engineering has an important role to play in the generation of graft material for reconstructive surgery. In cultured chondrocytes, the dedifferentiation of cells seems unavoidable for multiplication. Dedifferentiated cells produce matrix of less quality. Normal cartilage is composed of chondrocytes, which are embedded within an extracellular matrix (ECM). The ECM plays a key role in controlling cellular characteristics and contains the integrins as a large family of heterodimeric cell adhesion receptors involved in cell-cell and cell-matrix interactions. In this study, the characteristic changes of integrin expression and expression of matrix proteins during the course of dedifferentiation of chondrocytes in cell culture for 1, 6 and 21 days, analyzed at the mRNA level by microarray analysis and at the protein level by immunohistochemistry, are described. The components of the fibronectin receptor, integrin beta1,alpha5, in conjunction with the ligand fibronectin, were up-regulated during dedifferentiation. Integrin beta3 was expressed in the grey area. The components of the vitronectin-receptor, integrin alpha2b, alpha v, as well as integrin beta5, were activated on day 21, but neither vitronectin nor osteopontin were expressed by the cells. With ongoing dedifferentiation, activation of the GPIIb/GPIIIa receptor was found. The integrins beta2, beta4, beta6, beta8 and alpha2, alpha4, alpha6, alpha7 and alpha11 were never expressed. ILK, CD47 and ICAP1, as components of the intracellular signalling cascade of several integrins, were activated with ongoing dedifferentiation. In conclusion, a candidate for signal transmission during dedifferentiation is the fibronectin receptor (integrin alpha5beta1) in conjunction with its ligand fibronectin. Other receptors, e.g. for vitronectin and osteopontin (alphaVbeta3) or laminin (alpha6beta1) or their ligands, do not seem to be involved in signal transmission for dedifferentiation. In addition, the GPIIb/IIIa-receptor seems to assist the process of dedifferentiation. Intracellularly, ILK, ICAP1 and CD47 might assist the transduction of the integrin-dependent signals.     

The lipoxygenase inhibitor, baicalein, modulates cell adhesion and migration by up-regulation of integrins and vinculin in rat heart endothelial cells

BACKGROUND AND PURPOSE: Endothelial cell proliferation, migration and adhesion are necessary for the formation of new blood vessels. We reported previously that baicalein strongly inhibited proliferation of rat heart endothelial cells and here we assess effects on migration and adhesion of these cells. EXPERIMENTAL APPROACH: Effects of baicalein on endothelial migration and adhesion were determined by in vitro wound assays and in modified Boyden chambers. Protein expression and subcellular distribution in rat heart endothelial cells were analysed by immunoblots and immunofluorescence staining. RESULTS: Pretreatment with baicalein for 48 h resulted in a concentration-dependent inhibition of endothelial migration, with an IC(50) of approximately 20 microM. Adhesion assays revealed that baicalein stimulated endothelial cell adhesion to fibronectin and vitronectin, effects blocked by the synthetic peptide Arg-Gly-Asp (RGD). Moreover, treatment with a blocking antibody against integrin alpha5beta1 drastically attenuated baicalein-mediated endothelial adhesion to fibronectin, but not to vitronectin. Furthermore, baicalein-mediated anti-migration effect and adhesion promotion could be partially reversed by the addition of 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE). Western blot analysis indicated that baicalein increased expression levels of integrin-alpha5beta1, -alphavbeta3 and vinculin proteins. Immunofluorescence staining showed that baicalein induced a marked reorganization of actin stress fibres and the recruitment of vinculin and integrins to focal adhesion plaques, with consequently increased formation of focal adhesion contacts. CONCLUSIONS AND IMPLICATIONS: Baicalein markedly inhibited the migration and enhanced the adhesion of rat heart endothelial cells, possibly by up-regulation of the integrins (alpha5beta1 and alphavbeta3) and vinculin and by promotion of actin reorganization and focal adhesion contact formation.     

Antiadhesive sites present in the fibronectin type III-like repeats of human plasma fibronectin

We have found that fibronectin (FN) has a functional cryptic site opposing cell adhesion to extracellular matrix (ECM): a synthetic FN peptide derived from the 14th FN type III-like (FN-III) repeat, termed peptide FNIII14, inhibits cell adhesion to the FN without binding to beta1 integrins. This antiadhesive activity of peptide FNIII14 depends on its C-terminal amino acid sequence YTIYVIAL. A 50-kDa membrane protein (p50) has been detected as a specific binding protein of peptide FNIII14. Here we showed that antiadhesive activity of peptide FNIII14 was depedent upon the presence of p50 on cell surfaces. Furthermore, we found that there exists a sequence, analogous to the YTIYVIAL, in the 10th FN-III repeat of the FN molecule and that a FN peptide containing this analogous sequence, termed peptide FNIII10, inhibited cell adhesion to the FN. Peptide FNIII10 appeared to share p50 with peptide FNIII14 in expressing the antiadhesive activity. As a physiological consequence of decreased adhesion, peptides FNIII10 and FNIII14 accelerated the anoikis-like apoptosis of normal fibroblasts by down-regulating Bcl-2 expression through blocking the FAK/PI3K/Akt signaling pathway. Thus, the YTIYVIAL-related sequences of the FN molecule may be involved in cell regulation by modulating negatively cell adhesion to the ECM, in which p50 probably serves as a membrane receptor.     

C3a and C5a enhance granulocyte adhesion to endothelial and epithelial cell monolayers: epithelial and endothelial priming is required for C3a-induced eosinophil adhesion

Effects of the anaphylatoxins C3a and C5a on eosinophil and neutrophil adhesion to HUVEC and to primary culture human bronchial epithelial cells (HBEC) were investigated. Activities on both leukocytes and on structural cells were examined. C3a upregulated beta2 integrin expression and caused shedding of L-selectin on eosinophils, but had no effect on neutrophil adhesion molecule expression. C5a upregulated beta2 integrins and caused shedding of L-selectin on both eosinophils and neutrophils. The potency of C5a was equivalent on both cell types; however, the magnitude of the changes in each of these adhesion molecules was significantly greater in neutrophils than eosinophils. Neither C3a nor C5a altered expression of ICAM-1, VCAM-1, E-selectin or P-selectin on either HUVEC or HBEC. C5a induced adhesion of both neutrophils and eosinophils to unstimulated HUVEC or HBEC, and adhesion was further enhanced when HUVEC and HBEC were "primed" with TNF-alpha and IFN-gamma, respectively. C3a failed to enhance adhesion of either eosinophils or neutrophils to unprimed HUVEC or HBEC, and enhanced only eosinophil adhesion to cytokine-primed HUVEC or HBEC. Similar to C3a, C3a(desArg) and a C3a-analog peptide E7 also enhanced eosinophil adhesion only to cytokine-primed HUVEC and HBEC. These results support the traditional view of anaphylatoxins as leukocyte-specific mediators. The specificity of C3a for eosinophils implicates this molecule as a potential participant in allergic inflammation. The pro-adhesive effects of C3a(desArg) suggest that this molecule, previously characterized as a spasmogenically inactive derivative of C3a, may also alter leukocyte dynamics and migration. Finally, activation of endothelium may represent an important control mechanism for C3a-mediated adhesion preventing unchecked eosinophil adhesion to uninflamed systemic vasculature.     

DNA/Lipid complex incorporated with fibronectin to cell adhesion enhances transfection efficiency in prostate cancer cells and xenografts

Previously we have described the development and applications of lipid-based nanoparticles for gene delivery vector. In an attempt to improve transfection efficiency using the cell adhesion of extracellular matrix (ECM) to DNA/lipid complex (nanoplex), the mRNA expression of integrin alpha2beta1 and CD44 in prostate cancer cells was detected as adhesion molecules for fibronectin (Fn), collagen I (Col) and laminin (Lam) using a commercially available cDNA array (GEArray) system. These ECM proteins could enhance DNA transfection activity in cells when coated on the nanoplex. Among the ECM proteins, Fn-coating nanoplexes significantly increased transfection activity 2-fold in prostate cancer PC-3 cells, and exhibited higher DNA transfection activities to PC-3 xenografts, compared with commercially available cationic polymer in vivo jetPEI. These results indicated that Fn-coating nanoplexes could facilitate efficient transfection of prostate tumor cells.     

alpha(4) integrin-dependent eosinophil recruitment in allergic but not non-allergic inflammation

1. Although anti-alpha(4) integrin mAbs reduce eosinophil accumulation in several models of allergic inflammation, it is not clear whether this occurs via a direct action to block eosinophil alpha(4) integrins or indirectly on another cell type. The role of alpha(4) integrins on the accumulation of (111)In-labelled eosinophils in allergic and non-allergic inflammation in guinea-pig skin was therefore investigated. 2. Intradermal injection of antigen in sensitized skin sites induced accumulation of (111)In-eosinophils that was reduced up to 70% by two anti-alpha(4) integrin mAbs. In contrast, accumulation of (111)In-eosinophils to intradermal chemoattractants was unaffected by the same mAbs. 3. Accumulation of (111)In-eosinophils in allergic and non-allergic conditions was partly inhibited by a low dose of an anti-beta(2) integrin mAb. In combination with anti-alpha(4) integrin mAb, responses were not further reduced suggesting that these adhesion pathways are not additive or synergic. 4. Pretreating skin sites with antiserum or contaminating LPS did not reveal an alpha(4) integrin dependent pathway for chemoattractant-induced (111)In-eosinophil accumulation. These data suggest that alpha(4) integrins are involved in the response to antigen in sensitized skin sites. 5. Pretreating (111)In-eosinophil with alpha(4) integrin mAb blocked their adhesion to fibronectin in vitro but did not inhibit their accumulation in allergic inflammation suggesting that the blocking effect in vivo was eosinophil independent. 6. These data support the concept that targeting alpha(4) integrins on cells other than eosinophils could control eosinophil accumulation and have therapeutic potential in allergic diseases such as asthma and atopic dermatitis.     

A novel RGD antagonist that targets both alphavbeta3 and alpha5beta1 induces apoptosis of angiogenic endothelial cells on type I collagen

Integrin-mediated cell adhesion is necessary for endothelial cell proliferation and apoptosis, which is a major determinant in tumor-induced angiogenesis. In this study, we compared two novel, structurally similar, Arg-Gly-Asp (RGD) peptidomimetic compounds having different integrin selectivities, for their inhibition of endothelial cell proliferation and induction of apoptosis on functionally relevant extracellular matrices (ECM) for angiogenesis. BCH-14661 was specific for integrin alphavbeta3, whereas BCH-15046 nonselectively antagonized integrins alphavbeta3, alphavbeta5, and alpha5beta1. Both compounds were potent inducers of endothelial cell apoptosis when plated on RGD-dependent ECM (vitronectin, VN), which was dependent on the ability to induce cell detachment. However, with endothelial cells plated on RGD-independent ECM (type I collagen, COL), only BCH-15046 was able to significantly prevent growth and induce apoptosis. This effect was not dependent on the induction of detachment. Experiments using the matrix metalloproteinase (MMP) inhibitor GM 6001 revealed that cleavage of COL was not required for the ability of BCH-15046 to induce apoptosis. However, the inhibition of growth factor-stimulated endothelial cell proliferation, required MMPs, and correlated with BCH-15046s' potent inhibition of endothelial cell attachment to denatured collagen. Antibody inhibition experiments showed that adhesion to denatured collagen required integrins alphavbeta3 and beta1, but not alphavbeta5. In addition, BCH-15046 exerted a significant inhibition of VEGF-stimulated angiogenesis in the chick chorioallontoic membrane in vivo. These results suggest that integrin antagonism of both alphavbeta3 and alpha5beta1 are important for MMP-independent induction of apoptosis on COL and MMP-dependent inhibition of endothelial cell-denatured collagen interactions required for proliferation.     

Jararhagin, a snake venom metalloproteinase-disintegrin, stimulates epithelial cell migration in an in vitro restitution model

The snake venom metalloproteinase-disintegrin jararhagin (JG) has no chemotactic activity but stimulates the migration of neutrophils in vivo through a mechanism still unclear. In this study we investigated the effects of jararhagin on epithelial cell adhesion and migration in vitro. F-actin arrangement and the distribution of laminin, fibronectin, several integrins and phosphorylated Focal Adhesion Kinase (FAK) were studied using rhodamine–phalloidin and immunofluorescence. Maximum stimulation of migration (about 100%) was obtained with 5 μg/ml JG, with about 38% inhibition of cellular adhesion. In migratory cells the toxin stimulated the formation of filopodia, lamellipodia and stress fibers. The pericellular fibronectin matrix was lost in migrating cells, while laminin was less affected. The toxin stimulated FAK phosphorylation and the recruitment of v-containing integrins to focal contacts, whereas integrins containing the 2 subunit were reduced in these junctions. Inactivation of the toxin with 1,10 phenanthroline showed that the catalytic activity is important for the effect of jararhagin on cell migration, FAK phosphorylation and for the recruitment of v, but not as much for the anti-adhesive effect. In conclusion, jararhagin stimulates the migration of epithelial cells in vitro through a mechanism that involves its proteolytic activity, qualitative changes in cellular adhesion and the formation of actin-rich cellular processes.     

Interleukin-11 enhancement of VLA-5 mediated adhesion of CD34+ cells from cord blood to fibronectin is associated with the PI-3 kinase pathway

Adhesion is required for cell growth, differentiation, survival, and function. Cell adhesion is mediated by a structurally diverse group of plasma membrane receptors, each exhibiting specialized ligand-binding properties that are needed for specific tasks. Integrin-mediated adhesion is important for hematopoietic stem (HSC)/progenitor (HPC) cell survival and may prevent programmed cell death. Interleukin (IL)-11, a multi-functional cytokine secreted by the bone marrow environment, plays an important role in regulating growth and differentiation of HSCs/HPCs. In this report, we demonstrate that IL-11 enhanced adhesion of freshly isolated and 3 day-expanded CD34+ cells to immobilized fibronectin. the expression of very late antigen (VLA)-4 and VLA-5 integrins was detected on CD34+ cells. CD34+ cells also expressed a-chain and gp130 subunits of the IL-11 receptor (R). Enhanced adhesion by IL-11 was mediated via activation of VLA-5 integrins, since this action could be blocked by monoclonal antibodies against beta 1 and alpha 5, but not alpha 4, integrins. Addition of phosphatidylinositol (PI)-3 kinase inhibitors blocked IL-11 enhanced adhesion of CD34+ cells to fibronectin. The results suggest that this enhanced adhesion is associated with the PI-3 kinase pathway, an inside-out signaling pathway.     

Evidence for the involvement of carbohydrate moieties in the adhesion of U937 cells to tumor necrosis factor-alpha (TNFalpha)-stimulated vascular endothelium in vitro.

Treatment of U937 cells with fructose 1-phosphate (P) and fucoidan dose-dependently inhibited the adhesion of these monocytic cells to TNF alpha-stimulated human umbilical vein endothelial cells (HUVEC) (IC50 = 1 mM and 10 micrograms/ml respectively). These carbohydrates (CHO) failed to inhibit U937 adhesion to unstimulated (basal) HUVEC or phorbol 12, 13 dibutyrate (PdBu)-stimulated HUVEC. At 10 mM concentration, both fucose 1-P and lactose 1-P inhibited TNF alpha-stimulated adhesion while the latter also inhibited basal adhesion. Fructose 6-P, fucose, galactose 1-P, glucose 1-P, glucose 6-P, glucuronic acid, beta-glycerol 1-P, mannose 1-P, mannose 6-P, ribose 1-P and ribose 5-P tested at 10 mM did not inhibit U937 cells adhesion to basal or TNF alpha-stimulated HUVEC. These data suggest that CHO may play an important role in modulating monocytes adhesion to cytokine-induced adhesion molecule(s) on the surface of HUVEC.     

Genipin Selectively Inhibits TNF-��-activated VCAM-1 But Not ICAM-1 Expression by Upregulation of PPAR-�� in Human Endothelial Cells

Vascular inflammation process has been suggested to be an important risk factor in the development of atherosclerosis. Recently we reported that induction of peroxisome proliferator-activated receptor-γ (PPAR-γ) selectively inhibits vascular cell adhesion molecule-1 (VCAM-1) but not intercellular cell adhesion molecule-1 (ICAM-1) in tumor necrosis factor (TNF)-α-activated human umbilical vein endothelial cells (HUVEC). In this study, we investigated whether genipin inhibits expression of cellular adhesion molecules, which is relevant to inflammation. Pretreatment with genipin reduced reactive oxygen species (ROS) production and expression of VCAM-1, but not ICAM-1 in TNF-α-activated HUVEC. Genipin dose- and time-dependently increased PPAR-γ expression and inhibited TNF-α-induced phosphorylation of Akt and PKC with different degrees. Finally, genipin prevented TNF-α-induced adhesion of U937 monocytic cells to HUVEC. Taken together, these results indicate that upregualtion of PPAR-γ by genipin selectively inhibits TNF-α-induced expression of VCAM-1, in which regulation of Akt and/or PKC play a key role. We concluded that genipin can be used for the treatment of cardiovascular disorders such as atherosclerosis.     

Anti-inflammatory activity of c(ILDV-NH(CH2)5CO), a novel, selective, cyclic peptide inhibitor of VLA-4-mediated cell adhesion.

1. Small, N- to C-terminal cyclized peptides containing the leucyl-aspartyl-valine (LDV) motif from fibronectin connecting segment-1 (CS-1) have been investigated for their effects on the adhesion of human T-lymphoblastic leukaemia cells (MOLT-4) to human plasma fibronectin in vitro mediated by the integrin Very Late Antigen (VLA)-4 (alpha4beta1, CD49d/CD29). 2. Cyclo(-isoleucyl-leucyl-aspartyl-valyl-aminohexanoyl-) (c(ILDV-NH(CH2)5CO)) was approximately 5 fold more potent (IC50 3.6+/-0.44 microM) than the 25-amino acid linear CS-1 peptide. Cyclic peptides containing two more or one less methylene groups had similar potency to c(ILDV-NH(CH2)5CO) while a compound containing three less methylene groups, c(ILDV-NH(CH2)2CO), was inactive at 100 microM. 3. c(ILDV-NH(CH2)5CO) had little effect on cell adhesion mediated by two other integrins, VLA-5 (alpha5,beta1, CD49e/CD29) (K562 cell adhesion to fibronectin) or Leukocyte Function Associated molecule-1 (LFA-1, alphabeta2, CD11a/CD18) (U937 cell adhesion to Chinese hamster ovary cells transfected with intercellular adhesion molecule-1) at concentrations up to 300 microM. 4. c(ILDV-NH(CH2)5CO) inhibited ovalbumin delayed-type hypersensitivity or oxazolone contact hypersensitivity in Balb/c mice when dosed continuously from subcutaneous osmotic mini-pumps (0.1-10 mg kg(-1) day(-1)). Maximum inhibition (approximately 40%) was similar to that caused by the monoclonal antibody PS/2 (7.5 mg kg(-1) i.v.) directed against the alpha4 integrin subunit. 5. c(ILDV-NH(CH2)5CO) also inhibited oxazolone contact hypersensitivity when dosed intravenously 20 h after oxazolone challenge (1-10 mg kg(-1)). Ear swelling was reduced at 3 h and 4 h but not at 1 h and 2 h post-dose (10 mg kg(-1)). 6. Small molecule VLA-4 inhibitors derived from c(ILDV-NH(CH2)5CO) may be useful as anti-inflammatory agents.     

Inhibitory effect of the water-soluble extract of Salvia miltiorrhiza on neutrophil-endothelial adhesion

The effect of the water-soluble extract (WSE) of Salvia miltiorrhiza on neutrophil-endothelial cell adhesion was investigated. Cell adhesion was evaluated by testing neutrophil myeloperoxidase activity: expression of adhesion molecules in human umbilical vein endothelial cells (HUVEC) was measured by ELISA: the neutrophil activation rate induced by N-formyl-methionyl-leucyl-phenylalanine (fMLP) was tested by the method of nitroblue tetrazolium (NBT) reduction. The results showed that the adhesion rate of neutrophils to unstimulated HUVEC was very low. TNFalpha (50 - 800 U/ml) increased the adhesion of neutrophils to TNFalpha-stimulated HUVEC in a concentration- and time-dependent manner. The WSE of Salvia miltiorrhiza (0.01 - 1 mg/ml) dose-dependently inhibited the adhesion of neutrophils. The inhibitory rate of the WSE of Salvia miltiorrhiza at 0.01, 0.1 and 1 mg/ml was 6.2%, 17.0% and 28.0%, respectively. fMLP (10(-9) - 10(-5) M) increased the activation rate of neutrophils concentration-dependently. The WSE of Salvia miltiorrhiza also concentration-dependently inhibited the adhesion of fMLP-activated neutrophils to HUVEC. The inhibitory rate of the WSE of Salvia miltiorrhiza at 0.001, 0.01 and 0.1 mg/ml was 5.3%, 26.3% and 28.9%, respectively. Moreover, TNFalpha upregulated expression of adhesion molecule E-selectin, ICAM-1 and VCAM-1. The WSE of Salvia miltiorrhiza had an inhibitory effect on TNF alpha-induced expression of these molecules. These results indicated that the WSE of Salvia miltiorrhiza inhibited neutrophil-endothelial adhesion. The action mechanism of the WSE of Salvia miltiorrhiza was partly related to suppressing the expression of adhesion molecules.     

Evaluation of in-vitro anti-inflammatory activity of some 2-alkyl-4,6-dimethoxy-1,3,5-triazines

The ability of some 2-alkyl(aryl)-4,6-dimethoxy-1,3,5-triazine derivatives to interfere with production of reactive oxygen species (ROS) by human phagocytes was evaluated in an in-vitro cell model. Superoxide anion (O(2)(-*)) production by human polymorphonuclear cells (PMNs), challenged by the chemotactic agent N-formylmethionyl-leucyl-phenylalanine (FMLP), was inhibited in a dose-dependent manner by all the compounds tested, compounds 3, 4 and 5 being statistically the most active. Adhesion of PMNs to vascular endothelial cells (ECs) is a critical step in recruitment and infiltration of leucocytes into tissues during inflammation, and the effects of 1,3,5-triazine derivatives on PMN adhesion to ECs from the human umbilical vein (HUVEC) were also investigated. Triazines were incubated with PMNs and HUVEC; adhesion was quantitated by computerized micro-imaging fluorescence analysis. The 1,3,5-triazines tested inhibited the adhesion evoked by pro-inflammatory stimuli, such as platelet activating factor (PAF), FMLP, phorbol myristate acetate (PMA), tumour necrosis factor-alpha (TNF-alpha) and interleukin-1beta(IL-1beta) in a dose-response manner over the concentration range 10(-9) to 10(-4)M, compounds 5 and 6 being the most active. Both of these compounds inhibited PMN adhesion to HUVEC, even when endothelial or PMN stimuli were used. Indeed, when both cell populations were activated contemporarily, the anti-adhesive effect was enhanced. The study suggests that 2-aryl-4,6-dimethoxy-1,3,5-triazines deserve further evaluation as anti-inflammatory agents.     

槲皮素抑制血小板-内皮细胞粘附及粘附分子表达槲皮素抑制血小板-内皮细胞粘附及粘附分子表达

目的研究槲皮素(Que)对人脐静脉内皮细胞(HUVEC)和人血小板的粘附作用,及其粘附分子表达。方法用流式细胞仪测定肿瘤坏死因子(TNF-α)诱导HUVEC表达细胞间粘附分子(ICAM-1)及凝血酶诱导的人血小板P-选择素的表达，用［³H］-Adenine标记人血小板,检测其与HUVEC的粘附反应。结果Que与人血小板作用后，可抑制凝血酶诱导的人血小板表达P-选择素。HUVEC经TNF-α处理后，明显增加细胞表面ICAM-1的表达,加强其与血小板的粘附反应，Que在一定剂量范围内可抑制HUVEC表达ICAM-1,并可抑制其与血小板的粘附作用。结论Que可抑制内皮细胞和血小板粘附及粘附分子表达。     

Extracellular matrix, integrins, and mesenchymal cell function in the airways

Subepithelial fibrosis is one of the characteristic features of asthmatic airways. The fibrotic response includes an increase in volume occupied by extracellular matrix (ECM) tissue, and a change in the ECM composition favouring wound type collagens, fibronectin and a number of glycoproteins and proteoglycans normally associated with development. The altered ECM is likely to be deposited by the mesenchymal cells (including (myo) fibroblasts and smooth muscle) that are increased in number in asthmatic airways. In turn, the altered asthmatic ECM is likely to influence the function of the resident airway cells, and may be directly responsible for increasing proliferation, migration, ECM synthesis, inflammatory mediator release, and survival of resident mesenchymal cells. Therefore, the deposited ECM may perpetuate the disease phenotype. The different components of the ECM bi-directionally communicate with cells through a family of transmembrane receptors called integrins. Current research has begun to characterize: 1) the particular ECM components altered in airways disease; 2) the breadth of activity of different ECM components on airway cell function; and 3) the particular integrins responsible for mediating these effects. Further understanding of the role of integrins in transmitting responses of ECM in healthy or diseased airways may lead to novel targets for anti-asthma therapy.     

Nanomolar small molecule inhibitors for alphav(beta)6, alphav(beta)5, and alphav(beta)3 integrins

Integrin adhesion receptors frequently recognize a core amino acid sequence, Arg-Gly-Asp, in their target ligands. Inhibitors with the ability to inhibit one or a small subset of such RGD-dependent integrins have been invaluable in defining their biological function. Here, we have characterized low molecular weight inhibitors for their ability to specifically inhibit alphav(beta)6 integrin, a fibronectin/tenascin receptor. As of yet, no nonpeptidic inhibitor of alphav(beta)6 was known. New peptidomimetic and nonpeptidic compounds were examined in isolated integrin binding assays and in cell adhesion assays for their ability to block alphav(beta)6, alphav(beta)3, alphav(beta)5, and alphalIb(beta)3 integrins. The compounds are based on an aromatically substituted beta amino acid or glutaric acid derivative as an acidic center and an aminopyridyl or guanidyl residue as a basic mimetic. We found several classes of inhibitors with different selectivities, especially mono- or biselectivity on the alpha(v)-integrins alphav(beta)6 and alphav(beta)3, and nanomolar activity. Furthermore, nearly all compounds are inactive on alphaIIb(beta)3. Compound 11 is the first specific, peptidomimetic inhibitor of the alphav(beta)6 integrin receptor.     

Alphavbeta3 integrin binding affinity and specificity of SM256 in various species

This study was undertaken to define the alphavbeta3 binding affinity and specificity of the low-molecular-weight nonpeptide integrin antagonist, SM256. SM256 demonstrated high potency (IC50, 0.057+/-0.030 nM) in inhibiting vitronectin binding to purified human alphavbeta3 receptors. Additionally, SM256 inhibited alphavbeta3-mediated human umbilical vein endothelial cell (HUVEC) or 293/beta3 (beta3-transfected cell line) adhesion to fibrinogen with IC50 values of 0.0054+/-0.0058 and 0.0023+/-0.0012 microM, respectively. SM256 demonstrated a relatively high degree of specificity for human alphavbeta3-mediated functions as compared with other human integrins including alphavbeta5 (IC50, 0.92+/-0.69 microM), alphaIIbbeta3 (IC50, 0.72+/-0.07 microM), alpha4/beta1 (IC50, >100 microM) and alpha5/beta1 (IC50, 2.3+/-2.1 microM). SM256 demonstrated different degree of species specificity in blocking alphavbeta3-mediated cellular adhesion with relatively higher affinity to dog (IC50, 0.005+/-0.002 microM), rabbit (IC50, 0.021+/-0.01 microM), mouse (IC50, 0.035+/-0.01 microM), and pig (IC50, 0.41+/-0.24 microM) endothelial or smooth-muscle cell alphavbeta3-mediated adhesion. Additionally, SM256 demonstrated high degree of alphavbeta3 specificity as compared with alphavbeta5, alpha5beta1, or alphaIIbbeta3-mediated binding in these species. SM256 is a potent alphavbeta3, antagonist with high affinity and specificity for alphavbeta3-mediated functions. Additionally, a comparable alphavbeta3 affinity for SM256 was demonstrated with endothelial cells obtained from various species (dog, mouse, rabbit, and pig) as compared with that from human.