Effects of Himatanthus lancifolius on human leukocyte chemotaxis and their adhesion to integrins

The aim of this work was to investigate the anti-inflammatory activities of the uleine-rich fraction extracted from the barks of Himatanthus lancifolius (Muell. Arg.) Woodson (Apocynaceae). To achieve this, we focused on its in vitro effects on some steps of the inflammatory response using peripheral human leukocytes. The results presented herein show that the uleine-rich fraction significantly inhibits the migration of casein-induced granulocytes and their adhesion to fibronectin and vitronectin, along with mononuclear cells, by down-regulating the expression of alpha 4beta1 and alpha5beta1 integrins. The data suggest that H. LANCIFOLIUS has the potential of interferring with leukocyte trafficking through its uleine-rich fraction, emphasizing its usefulness in inflammatory conditions. DEXA:dexamethasone disodium phosphate FN:fibronectin PMN:polymorphonuclear URF:uleine-rich fraction VN:vitronectin.     

Inhibition of angiogenesis by a tenascin-c peptide which is capable of activating beta1-integrins

In addition to humoral angiogenic factors, including vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), integrin-mediated adhesion of vascular endothelial cells to the extracellular matrix plays an important role in neovascularization. We recently found that TNIIIA2, a peptide derived from tenascin-C, induces functional activation of beta1 integrins. Here we investigated the effect of TNIIIA2 on vascular endothelial cell migration and proliferation, key processes for angiogenesis. TNIIIA2 was shown to activate beta1-integrins on human dermal microvascular endothelial cells (HDMEC). HDMEC adhered to fibronectin mainly via integrin alpha5beta1 and their haptotactic migration on that substrate was inhibited by TNIIIA2, in concomitant with a marked inhibition of Rac activation. TNIIIA2-treatment unaffected autophosphorylation of focal adhesion kinase (FAK), but induced its physical association with phospho-paxillin (Tyr118), suggesting the FAK/paxillin-dependent negative regulation of Rac activation. HDMEC proliferation on the fibronectin substrate was also inhibited by TNIIIA2-treatment, and this was accompanied either by an increase in the population of G 0/G1 cells and, conversely, a decrease in the population of S and G2/M cells or by dephosphorylation/inactivation of MAP-kinase (ERK1/2). Inhibited HDMEC migration and proliferation were both restored by pretreating the cells with a fibronectin peptide, FNIII14, which is capable of inactivating beta1-integrins. The chorioallantoic membrane assay demonstrated an antiangiogenic effect of TNIIIA2 in vivo. Thus, TNIIIA2 appears to negatively regulate angiogenesis by inhibiting migration and proliferation of endothelial cells. The ability to activate beta1-integrins may be responsible for the antiangiogenic effect of TNIIIA2, although it cannot be excluded the possibility that an additional mechanism(s) may play a role.     

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.     

Simvastatin disrupts cytoskeleton and decreases cardiac fibroblast adhesion, migration and viability

Statins reduce the isoprenoids farnesyl and geranylgeranyl pyrophosphate, essential intermediates, which control a diversity of cellular events such as cytoskeleton integrity, adhesion, migration and viability. Cardiac fibroblasts are the major non-myocyte cell constituent in the normal heart, and play a key role in the maintenance of extracellular matrix. The effects of simvastatin on cardiac fibroblast processes previously mentioned remain unknown. Our aims were to investigate the effects of simvastatin on cytoskeleton structure and focal adhesion complex assembly and their relationships with cell adhesion, migration and viability in cultured cardiac fibroblasts. To this end, cells were treated with simvastatin for 24 h and changes in actin cytoskeleton, levels of vimentin and paxillin as well as their subcellular localization were analyzed by Western blot and immunocytochemistry, respectively. Cell adhesion to plastic or collagen coated dishes, migration in Transwell chambers, and cell viability were analyzed after simvastatin treatment. Our results show that simvastatin disrupts actin cytoskeleton and focal adhesion complex evaluated by phalloidin stain and immunocytochemistry for paxillin and vinculin. All these effects occurred by a cholesterol synthesis-independent mechanism. Simvastatin decreased cell adhesion, migration and viability in a concentration-dependent manner. Finally, simvastatin decreased angiotensin II-induced phospho-paxillin levels and cell adhesion. We concluded that simvastatin disrupts cytoskeleton integrity and focal adhesion complex assembly in cultured cardiac fibroblasts by a cholesterol-independent mechanism and consequently decreases cell migration, adhesion and viability.     

Rho-kinase inhibitor upregulates migration by altering focal adhesion formation via the Akt pathway in colon cancer cells

Although Rho-kinase is reportedly implicated in carcinogenesis and the progression of human cancers, its precise mechanism has not been fully elucidated. We recently reported that Rho-kinase negatively regulates epidermal growth factor (EGF)-stimulated cancer progression in SW480 colon cancer cells. In the present study, we investigated the effect of Rho-kinase on the migration of SW480 colon cancer cells and the mechanism underlying the involvement of Rho-kinase. Interestingly, (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide, 2HCl (Y27632), a specific inhibitor of Rho-kinase, dose-dependently enhanced cell migration. SW480 cells spontaneously release vascular endothelial growth factor (VEGF), however, Y27632 had little effect on its release. While Rho-kinase, which is generally phosphorylated in unstimulated cells, was clearly suppressed by Y27632, exogenous VEGF did not affect its phosphorylation. Immunofluorescence microscopy revealed that Y27632 caused a dramatic change in the localization of focal adhesion components, vinculin, phosphorylated caveolin-1 and tyrosine-phosphorylated proteins in SW480 cells. Furthermore, Akt inhibitor restored the loss of vinculin-stained focal adhesion formation induced by Y27632. We also observed similar effects for Y27632 on the migration and localization of focal adhesion components such as vinculin in another colon cancer cell line, HT29. Taken together, these results strongly suggest that Rho-kinase negatively regulates the migration of colon cancer cells by altering focal adhesion formation via the Akt pathway.     

Inhibition of adipogenesis by RGD-dependent disintegrin

Adipogenesis plays a central role in obesity development. The processes of adipogenesis include migration, adhesion, proliferation and survival of preadipocytes and differentiation to mature adipocytes. Many of these biological functions are related to integrins. Here, we found that snake venom-derived arginine-glycine-aspartic acid (RGD)-containing disintegrin inhibited adipogenesis. Rhodostomin but not rhodostomin RGD mutants (RGE-Rn and AKGDWN-Rn) caused the detachment of primary cultured preadipocyte. Furthermore, rhodostomin also inhibited focal adhesion of preadipocyte, including the inhibition of the expression of focal adhesion kinase (FAK) and FAK phosphorylation, assembly of vinculin and reorganization of actin cytoskeleton. Cell viability of preadipocytes was decreased after rhodostomin treatment in a concentration-dependent manner. The results of flow cytometric analysis showed that rhodostomin induced cell apoptosis. In addition, chromatin condensation was observed in DAPI staining. The increase of Bax expression and activation of capsase-3 was detected following rhodostomin treatment. Addition of dexamethasone, IBMX and insulin induced differentiation of preadipocytes into mature adipocytes and treatment of cells with rhodostomin during the initial 3 days showed less mature adipocytes following 9-10 days of differentiating period. The triglyceride content and gene expression of peroxisome proliferators-activated receptor gamma (PPARgamma) and leptin also decreased in response to the treatment of rhodostomin. These results suggest that disintegrin inhibits processes of adipogenesis and may be developed to treat obesity.     

Neutrophil inhibitory factor abrogates neutrophil adhesion by blockade of CD11a and CD11b beta(2) integrins.

We studied the basis of inhibition of polymorphonuclear leukocyte (PMN) adhesion induced by neutrophil inhibitory factor (NIF), a 41-kDa CD11/CD18 beta(2) integrin-binding protein isolated from the canine hookworm (Ancylostoma caninum). NIF blocked PMN adhesion in a concentration-dependent manner with complete blockade occurring at approximately 10 nM NIF. Because CD11a and CD11b beta(2) integrins are functionally active on stimulated PMNs, and yet NIF is postulated to inhibit only CD11b integrin by binding to its I domain, we evaluated the contributions of CD11a and CD11b beta(2) integrins in the mechanism of inhibition of PMN adhesion to endothelial cells. We observed an additive inhibitory effect (>90% inhibition) of PMN adhesion to endothelial cells when NIF was used in combination with anti-CD11b monoclonal antibodies, which alone at saturating concentrations reduced PMN adhesion by only 50%. NIF also prevented aggregation of phorbol ester-stimulated JY lymphoblastoid cells that expressed only the functionally active CD11a, suggesting that NIF also can inhibit CD11a-dependent response. We transduced the NIF cDNA into human dermal microvessel endothelial cells in which NIF synthesis and release prevented PMN adhesion to the transduced human dermal microvessel endothelial cells. These data indicated that the potent antiadhesive effect of NIF may be the result of inhibition of CD11a and CD11b beta(2) integrins on PMNs. Moreover, the strategy of NIF release from transduced endothelial cells suggests the feasibility of blocking the CD11a- and CD11b beta(2) integrin-dependent PMN adhesion and PMN migration responses specifically at sites of endothelial cell activation.     

Suppressive effect and mechanism of saxatilin, a disintegrin from Korean snake (Gloydius saxatilis), in vascular smooth muscle cells

RGD-peptides can inhibit the binding of ligands to certain β3 integrins, αIIbβ3 and αvβ3, both of which are involved in neointimal hyperplasia that contributes to atherosclerosis and restenosis of arterial walls. Saxatilin, a disintegrin from a Korean snake (Gloydius saxatilis), interacts with integrins αIIbβ3 and αvβ3. It suppressed the adhesion of human coronary artery smooth muscle cells (HCASMCs) to vitronectin with an IC₅₀ of 2.5 μM, and growth factor (PDGF-BB or bFGF)-induced proliferation was inhibited at an IC₅₀ of 25 μM. Saxatilin disassembled the actin cytoskeleton of focal adhesion and induced cell detachment. This disassembly of focal adhesion in saxatilin-treated HCASMCs involved caspase-induced paxillin degradation. Saxatilin temporally phosphorylated FAK and ERKs and affected the cell cycle of HCASMCs by increasing CDK inhibitors (p21 and p27) and reducing cyclins (D1/2 and E). These results may have significant implications for integrin antagonistic therapy used for the treatment of atherosclerosis and restenosis.     

Integrin Targeting and Toxicological Assessment of Peptide‐Conjugated Liposome Delivery Systems to Activated Endothelial Cells

Utilization of functionalized liposomes as the means of targeted delivery of therapeutics may enhance specific transport of biologically active drugs to target tissues, while avoiding or reducing undesired side effects. In the present investigation, peptide‐conjugated cationic liposomes were constructed with the aim of targeting integrins (i.e. vitronectin and/or fibronectin receptors) on activated endothelial cells. The peptide‐conjugated liposomes induced only cytotoxicity at the highest concentration in non‐activated or activated endothelial cells, as well as in co‐culture of endothelial cells and macrophages. There was unaltered secretion of cytokines after exposure of peptide‐conjugated liposomes to endothelial cells, indicating that the materials were not inflammogenic. Liposomes with a peptide targeting the fibronectin receptor (integrin α5β1) were more effective in targeting of activated endothelial cells, as compared to a liposome with a peptide that targeted both the fibronectin and vitronectin receptors, as well as liposomes with a control peptide. The liposome targeted to the fibronectin receptor also displayed uptake in endothelial cells in co‐culture with activated macrophages. Therefore, this study demonstrates the feasibility of constructing a peptide‐conjugated cationic liposome, which displays targeting to activated endothelial cells at concentrations that are not cytotoxic or inflammogenic to the cells.     

Regulation of angiogenesis by the kallikrein-kinin system

High molecular weight kininogen (HK) is a plasma protein that is cleaved by plasma kallikrein in the clinical settings of sepsis and chronic inflammatory diseases such as rheumatoid arthritis and Crohn's disease. This proteolytic event results in a nonapeptide, bradykinin (BK), and a kinin-free derivative of HK, namely HKa. BK promotes angiogenesis by upregulation of bFGF through the B1 receptor or by stimulation of VEGF formation via the B2 receptor. Kininogen-deficient rats show diminished angiogenesis when neovascularization is stimulated. The formation of HKa results in exposure of domain 5 (D5). HKa or D5 inhibit endothelial cell migration and proliferation, both of which are needed for angiogenesis. In the chicken chorioallantoic membrane assay when neovascularization is stimulated by bFGF or VEGF, HKa or D5 inhibit angiogenesis. Monoclonal antibody C11C1, which prevents binding of HK to endothelial cells, also limits its conversion to BK thus downregulating angiogenesis. In vivo, mAb C11C1 inhibits tumor angiogenesis in mice as well as in experimental inflammatory arthritis and inflammatory bowel disease in Lewis rats. In vitro HKa or D5 inhibits endothelial cell adhesion to vitronectin and fibrinogen, resulting in anokis and apoptosis. The HKa receptor, uPAR, forms a signaling complex containing the integrin alphavbeta3 or alpha5beta1, caveolin, Src kinase Yes, focal adhesion kinase and paxcillin. HKa physically disrupts the complex by interfering with the binding of vitronectin to uPAR. Both mAb C11C1 and D5 have potential applications for controlling unwanted angiogenesis in inflammation and cancer.     

Effects of monocrotaline pyrrole and thrombin on pulmonary endothelial cell junction and matrix adhesion proteins

Previous work in our laboratory has shown that monocrotaline pyrrole (MCTP) interacts with actin and potentiates thrombin-mediated endothelial barrier permeability through increasing the overall surface area of intercellular gaps. To better characterize endothelial barrier leak in this model, we examined the effects of MCTP and thrombin on the localization and structure of three adhesion associated proteins that directly or indirectly interact with actin in regulating barrier function: cell-cell occludens junction molecule (ZO-1), the cell-cell adherens junction linker, ss-catenin, and the cell-matrix intermediary signaling protein, focal adhesion kinase (FAK). Immunohistochemistry demonstrated that thrombin treatment resulted in radial reorganization of focal adhesions and broader distribution of adherens and occludins junctions at the cell border suggestive of membrane stretching in contracture. MCTP pretreatment resulted in fewer and more disorganized focal adhesions and marked thinning of occludins and adherens junctions. MCTP pretreatment also interfered with thrombin stimulated junctional reorganization. Western blot analysis showed thrombin stimulated catalysis of ZO-1 and FAK while MCTP pretreatment resulted in FAK fragmentation similar to previous reports for apoptosis. We conclude that both MCTP and thrombin alter critical endothelial cell adhesion molecules and this may be an underlying mechanism for the potentiating effect MCTP has on thrombin induced vascular permeability in vitro.     

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.     

Sodium arsenite exposure alters cell migration, focal adhesion localization and decreases tyrosine phosphorylation of focal adhesion kinase in H9C2 myoblasts.

Exposure to the environmental toxicant arsenic is reported to produce a variety of effects including disruption of signal transduction pathways, cell proliferation, and apoptosis. This suggests that arsenite may not have specific targets but rather extremely broad effects. The present study was designed to test the hypothesis that arsenite alters signaling involved in focal adhesion structure and function in cultured myoblasts. H9C2 cells were exposed to 1, 2.5, 5, or 10 microM sodium arsenite for 48 h. MTT metabolism and staining by neutral red, trypan blue, and propidium iodide showed that sodium arsenite treatments of 5 microM or less were not overtly cytotoxic. At these doses, sodium arsenite did not affect the amount of polymerized actin in cells, rate of protein synthesis, or amounts of vinculin, talin, paxillin, and focal adhesion kinase (FAK) in cells. However, sodium arsenite-treated cells contained fewer focal adhesions with an altered distribution pattern. Sodium arsenite exposure caused a dose-dependent reduction in cell migration and cell attachment rates. The average area of substrate covered by a cell was also reduced, although the average volume of cells was not significantly affected. Sodium arsenite exposure resulted in reduced tyrosine phosphorylation of FAK, its substrate paxillin and the FAK auto- phosphorylation site, Tyr397. Our results indicate that sodium arsenite can alter focal adhesion structure and function, thus affecting cell attachment and migration and possibly other aspects of focal adhesion function such as integrin signaling. These diverse consequences may be mediated by a relatively specific inhibition of FAK tyrosine phosphorylation, modifying scaffolding proteins.     

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.     

Litebamine, a phenanthrene alkaloid from the wood of Litsea cubeba, inhibits rat smooth muscle cell adhesion and migration on collagen

Smooth muscle cells (SMCs) play an important role in the development of atherosclerosis and restenosis after angioplasty and coronary bypass grafting. The pathogenesis of these vascular diseases includes the abnormal production of extracellular matrix (ECM) proteins by SMCs and their interactions with this newly synthesized and preexisting ECM. Litebamine, a natural phenanthrene alkaloid from the wood of Litsea cubeba, has been shown to inhibit platelet aggregation and thromboxane B(2) formation, suggesting its antithrombotic activity. In the present study we examined litebamine effects on vascular SMC adhesion and migration. Our results indicated that litebamine inhibited rat aortic SMCs (RASMCs) and A10 thoracic SMCs adhesion to collagen but not to other matrix proteins, suggesting its specificity on collagen. This inhibition was possibly resulted from that litebamine attenuated immobilized collagen-induced focal adhesion kinase (FAK) phosphorylation and actin cytoskeleton reorganization in RASMCs, as determined by Western blotting and immunofluorescence microscopy. In a functional study, litebamine also inhibited platelet-derived growth factor (PDGF)-induced RASMC migration but did not affect PDGF-induced matrix metalloproteinases (MMPs) secretion. Strikingly, among the tested kinases involved in PDGF-induced migration, only PDGF-induced phosphatidylinositol-3 kinase (PI-3K) activation was inhibited by litebamine. Taken together, we demonstrated here that litebamine can functionally inhibit vascular SMC adhesion and migration and elucidated its possible mechanisms of action. As SMC adhesion and migration are critical events in disease-related vascular remodeling, this compound may have beneficial effects in preventing cardiovascular diseases.     

The effect of brown spider venom on endothelial cell morphology and adhesive structures

Spiders of the Loxosceles genus have been responsible for severe clinical cases of envenomation worldwide. Accidents involving brown spiders can cause dermonecrotic injury, hemorrhage, hemolysis, platelet aggregation and renal failure. Histological findings of animals treated by venom have shown subendothelial blebs, vacuoles and endothelial cell membrane degeneration of blood vessel walls, as well as fibrin and thrombus formation. The mechanisms by which the venom causes these disorders are poorly understood. In this work, with an endothelial cell line derived from rabbit aorta, we were able to demonstrate that venom binds to the cell surface and the extracellular matrix. Moreover, we observed that the venom also induced morphological alterations, such as cell retraction, homophilic disadhesion and an increasing in filopodia projections. We also demonstrated that toxins present in the venom disorganized focal adhesion points and actin microfilaments of endothelial cells. Nevertheless, endothelial cell viability showed no alterations compared to controls. Additionally, venom treatment changed the fibronectin matrix profile synthesized by these cells as well as cell adhesion to fibronectin. These results suggest that the deleterious effects of venom on blood vessel walls could be a consequence of the direct effect on the endothelial cell surface and adhesive structures involved in blood vessel stability. These effects indirectly lead to leukocyte and platelet activation, disseminated intravascular coagulation and an increase in vessel permeability.     

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.     

Lysophosphatidic acid induces a migratory phenotype through a crosstalk between RhoA-Rock and Src-FAK signalling in colon cancer cells

Lysophosphatidic acid (LPA) acts as a potent stimulator of tumorigenesis. Cell-cell adhesion disassembly, actin cytoskeletal alterations, and increased migratory potential are initial steps of colorectal cancer progression. However, the role that LPA plays in these events in this cancer type is still unknown. We explored this question by using Caco-2 cells, as colon cancer model, and treatment with LPA or pretreatment with different cell signalling inhibitors. Changes in the location of adherent junction proteins were examined by immunofluorescence and immunoblotting. The actin cytoskeleton organisation and focal adhesion were analysed by confocal microscopy. Rho-GTPase activation was analysed by the pull-down assay, FAK and Src activation by immunoblotting, and cell migration by the wound healing technique. We show that LPA induced adherent junction disassembly, perijunctional actin cytoskeletal reorganisation, and increased cell migration. These events were dependent on Src, Rho and Rock because their chemical inhibitors PP2, toxin A and Y27632, respectively, abrogated the effects of LPA. Moreover, we showed that Src acts upstream of RhoA in this signalling cascade and that LPA induces focal adhesion formation and FAK redistribution and activation in confluent monolayers. Focal adhesion formation was also observed in the front of migrating cells in response to LPA, and Rock inhibitor abolished this effect. In conclusion, our findings show that LPA modulates adherent junction disassembly, actin cytoskeletal disorganisation, and focal adhesion formation, conferring a migratory phenotype in colon tumour cells. We suggest a functional regulatory cascade that integrates RhoA-Rock and Src-FAK signalling to control these events during colorectal cancer progression.     

Targeting vascular cell migration as a strategy for blocking angiogenesis: the central role of focal adhesion protein tyrosine kinase family

The formation of capillary-like structures during angiogenesis requires a series of well-orchestrated cellular events allowing endothelial cells and pericytes to migrate into the perivascular space. The proper activation of the migratory machinery in these cells is fine controlled by the presence of angiogenic challenges and by the interactions with extracellular matrix. The two members of the focal adhesion protein tyrosine kinases (FA-PTKs), FAK and PYK2, play a central role in modulating endothelial and vascular smooth muscle cells migration confirming the well consolidated observations in other migrating cell types. However accumulating data reveal that FAK and PYK2 are involved in several cell processes including cell proliferation and survival. FAK, once localized to focal adhesions, is thought to be one of the principal effectors in linking signals initiated by integrins and growth factor receptors to cytoskeleton, thus controlling migration. Although more obscure, and differently regulated, the function of PYK2 seems to be similar to that of FAK, but restricted to few cell types, including vasculature forming cells. FAK and PYK2 exert a primary role as adaptor proteins able to recruit, with high turnover, several proteins which in turn, through their docking domains and tyrosine kinase activity, determine both the turnover in focal adhesion assembly and the specificity of downstream signaling. The characterization of functional interactions of FA-PTKs may provide new potential therapeutic targets in order to control vascular pathological processes including angiogenesis.     

Involvement of Rho signaling in PAR2-mediated regulation of neutrophil adhesion to lung epithelial cells

Protease-activated receptor 2 (PAR2) has been implicated in the pathogenesis of airway inflammation. We report that epithelial PAR2 stimulation with trypsin (0.05-1 U/ml) or an agonist peptide (SLIGKV-NH2, 1-100 microM) for 0.5-3 h dose- and time-dependently enhanced neutrophil adhesion to alveolar type II epithelial cells (A549 cells) and that this stimulation also induced the formation of epithelial actin filaments. Both responses in neutrophil adhesion and epithelial actin reorganization were reduced by a Rho inhibitor, mevastatin and by a Rho-associated kinase (ROCK) inhibitor, Y-27632 ((R)-(+)-trans-N-(4-Pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide). Neutrophil adherence was also inhibited by an inhibitor of actin polymerization, cytochalasin D and a tyrosine kinase inhibitor, genistein. Further, the PAR2-mediated tyrosine phosphorylation of focal adhesion kinase (FAK), a major cytoskeleton protein, was detected, and this response was inhibited by mevastatin or Y-27632. These results suggest that PAR2 stimulation of alveolar epithelial cells enhances neutrophil adhesion presumably at least in part through Rho/ROCK signal-mediated actin cytoskeleton reorganization associated with the tyrosine phosphorylation of FAK.