Role of hypoxia and extracellular matrix-integrin binding in the modulation of angiogenic growth factors secretion by retinal pigmented epithelial cells.

The retinal pigmented epithelium (RPE) is a monolayer of polarized cells located between retinal photoreceptors and blood vessels of the choroid. The basal surface of RPE cells rests on Bruch's membrane, a complex extracellular matrix structure which becomes abnormal in several disease processes, including age-related macular degeneration (AMD). Ruptures or abnormalities in Bruch's membrane are frequently accompanied by choroidal neovascularization. Disturbed interaction of RPE cells with their extracellular matrix (ECM) could play a role in this process. The present study was undertaken to examine the complex interactions between hypoxia, integrin, and ECM in the regulation of RPE functions. Antibody blocking experiments demonstrated that RPE cell adhesion to vitronectin is mediated primarily through alphavbeta5 and adhesion to fibronectin occurs through alpha5beta1. RPE adhesion to immobilized laminin demonstrated highest level of non-RGD-mediated adhesion as compared to that with collagen IV or the RGD matrices such as vitronectin (alphavalpha5) , fibronectin (alpha5beta1), or thrombospondin (alpha5beta1 + alphavbeta5). Addition of soluble vitronectin, or fibrinogen to RPE cell cultures resulted in a small to moderate increase in VEGF and FGF2 in the media, while each of these growth factors was dramatically increased after addition of thrombospondin 1 (TSP1). In contrast, soluble fibronectin resulted in differential upregulation of VEGF but not FGF2. Similarly, immobilized TSP1 resulted in differential greater upregulation in VEGF but not FGF2 release from RPE as compared to other ECMs under either normoxic or hypoxic conditions. Additionally, hypoxia resulted in a time-dependent increase in VEGF, but not FGF2 release in the media. RPE cells grown on TSP1-coated plates showed increased VEGF and FGF2 in their media compared to cells grown on plates coated with type IV collagen, laminin, vitronectin, or fibronectin. The TSP1-induced increase in secretion of growth factors was partially blocked by anti-alpha5beta1, anti-alphavbeta3, and anti-alphavbeta5 antibodies indicating that it may be mediated in part by TSP1 binding to those integrins. These data suggest that alterations in oxygen levels (hypoxia/ischemia) and ECM of RPE cells, a prominent feature of AMD, can cause increased secretion of angiogenic growth factors that might contribute to the development of choroidal neovascularization. These data also suggest the potential modulatory role of VEGF release from RPE by ECM and alphavbeta5 and alpha5beta1 integrins.     

Vascular Permeability Factor/Vascular Endothelial Growth Factor Inhibits Anchorage-Disruption-Induced Apoptosis in Microvessel Endothelial Cells by Inducing Scaffold Formation

Survival and proliferation of endothelial cells requires both growth factors and an appropriate extracellular matrix to which cells can attach. In the absence of either, endothelial cells rapidly undergo apoptosis. Thus, when human microvascular endothelial cells (HDMEC) are plated on a hydrophobic surface such as untreated polystyrene, they rapidly undergo apoptosis and die. The present study demonstrates that vascular permeability factor/vascular endothelial growth factor (VPF/VEGF), an endothelial cell-selective cytokine, inhibits apoptosis of HDMEC cultured on untreated polystyrene and induces these cells to adhere, spread, and proliferate. VPF/VEGF-induced HDMEC adhesion was time-dependent, requiredde novoprotein synthesis, and was inhibited by a soluble RGD peptide but not by an inhibitor of collagen synthesis. Under the conditions of these experiments, VPF/VEGF downregulated expression of collagen IV and fibronectin but did not change collagen I mRNA levels. VPF/VEGF-induced HDMEC adhesion was inhibited by antibodies to αvβ5 and vitronectin but not by antibodies to αvβ3. Other endothelial growth factors and cytokines such as bFGF, HGF, and TGFβ did not reproduce the VPF/VEGF effect. We suggest that VPF/VEGF induces endothelial cells to deposit a scaffolding (likely involving vitronectin) that allows them to attach to and proliferate on an otherwise nonsupportive surface (hydrophobic polystyrene) and in this manner serves as both a survival factor and a growth factor.     

The inhibitory effects of endostatin on endothelial cells are modulated by extracellular matrix

We investigated the ability of extracellular matrix (ECM) proteins to modulate the response of endothelial cells to both promoters and inhibitors of angiogenesis. Using human dermal microvascular endothelial cells (HDMEC), we found that cells demonstrated different adhesive properties and proliferative responses to the growth factor VEGF depending upon which ECM protein with which they were in contact, with fibronectin having the most impact on VEGF-induced HDMEC proliferation and survival. More importantly, we observed that ECM could modulate the ability of the angiogenic inhibitor endostatin to prevent endothelial cell proliferation, survival and migration. We observed that growth on vitronectin or fibronectin impaired the ability of endostatin to inhibit VEGF-induced HDMEC proliferation to the greatest extent as determined by BrdU incorporation. We found that, following growth on collagen I or collagen IV, endostatin only inhibited VEGF-induced HDMEC proliferation at the highest dose tested (2500 ng/ml). In a similar manner, we observed that growth on ECM proteins modulated the ability of endostatin to induce endothelial cell apoptosis, with growth on collagen I, fibronectin and collagen IV impairing endostatin-induced apoptosis. Interestingly, endostatin inhibited VEGF-induced HDMEC migration following culture on collagen I, collagen IV and laminin, while migration was not inhibited by endostatin following HDMEC culture on other matrices including vitronectin, fibronectin and tenascin-C. These results suggest that different matrix proteins may affect different mechanisms of endostatin inhibition of angiogenesis. Taken together, our results suggest that the ECM may have a profound impact on the ability of angiostatic molecules such as endostatin to inhibit angiogenesis and thus may have impact on the clinical efficacy of such inhibitors.     

Insulin-like growth factor-I receptor mediates the prosurvival effect of fibronectin

We recently showed that extracellular matrix (ECM) proteins, which are abundant in desmoplastic pancreatic tumor, are as potent as growth factors in inhibiting apoptosis in pancreatic cancer (PaCa) cells. Here we show that fibronectin, a major ECM component, engages insulin-like growth factor-I receptor (IGF-IR) to inhibit PaCa cell death. We found that fibronectin-induced protection from apoptosis is fully mediated by IGF-IR and is independent of IGF-I. Pharmacologic and molecular inhibitions of IGF-IR stimulated apoptosis and prevented the prosurvival effect of fibronectin in PaCa cells. Our data indicate that fibronectin protects from apoptosis through trans-activation of IGF-IR. We showed that fibronectin stimulated complex formation between its receptor beta3 integrin and protein-tyrosine phosphatase SHP-2. This process of complex formation, in turn, prevents SHP-2 from dephosphorylating IGF-IR resulting in sustained phosphorylation of IGF-IR and leading to the downstream activation of Akt kinase, up-regulation of antiapoptotic Bcl(xL), and inhibition of apoptosis. Among ECM proteins tested only fibronectin and laminin but not vitronectin and collagen I stimulated trans-activation of IGF-IR. Interaction of fibronectin with beta3 but not beta1 integrin receptors mediates the survival pathway. In contrast, fibronectin-induced adhesion is mediated through beta1 integrin receptor and is IGF-IR-independent. Thus, our results indicate that the prosurvival effect of fibronectin in PaCa cells is mediated by trans-activation of IGF-IR induced by the beta3 integrin receptor. The data suggest IGF-IR as a key target for prevention of the prosurvival effects of ECM proteins and growth factors in pancreatic cancer.     

Neurite outgrowth,RGD-dependent,and RGD-independent adhesion of identified molluscan motoneurons on selected substrates

The extracellular matrix (ECM) provides structural support to cells and tissues and is involved in the regulation of various essential physiological processes, including neurite outgrowth. Most of the adhesive interactions between cells and ECM proteins are mediated by integrins. Integrins typically recognize short linear amino acid sequences in ECM proteins, one of the most common being Arginine-Glycine-Aspartate (RGD). The present study investigated neurite outgrowth and adhesion of identified molluscan neurons on a selection of substrates in vitro. Involvement of RGD binding sites in adhesion to the different substrates was investigated using soluble synthetic RGD peptides. The cells adhered to native (i.e., nondenatured) laminin and type IV collagen, but not to native plasma fibronectin. Denaturation of fibronectin dramatically enhanced cell adhesion. Only the adhesion to denatured fibronectin was inhibited by RGD peptides, indicating that denaturation uncovers a RGD binding site in the protein. Laminin as well as denatured fibronectin, but not type IV collagen, induced neurite outgrowth from a percentage of the RPA neurons. These results demonstrate that molluscan neurons can attach to various substrates using both RGD-dependent and RGD-independent adhesion mechanisms. This suggests that at least two different cell adhesion receptors, possibly belonging to the integrin family, are expressed in these neurons. Moreover, the results show that vertebrate ECM proteins can induce outgrowth from these neurons, suggesting that the mechanisms involved in adhesion as well as outgrowth promoting are evolutionarily well conserved. © 1998 John Wiley & Sons, Inc. J Neurobiol 35: 37–52, 1998     

Inhibition of in vitro tumor cell invasion by Arg-Gly-Asp-containing synthetic peptides [published erratum appears in J Cell Biol 1989 Jun;108(6):following 2546]

The interaction of cells with extracellular matrix components such as fibronectin, vitronectin, and type I collagen has been shown to be mediated through a family of cell-surface receptors that specifically recognize an arginine-glycine-aspartic acid (RGD) amino acid sequence within each protein. Synthetic peptides containing the RGD sequence can inhibit these receptor-ligand interactions. Here, we use novel RGD-containing synthetic peptides with different inhibition properties to investigate the role of the various RGD receptors in tumor cell invasion. The RGD-containing peptides used include peptides that inhibit the attachment of cells to fibronectin and vitronectin, a peptide that inhibits attachment to fibronectin but not to vitronectin, a cyclic peptide with the opposite specificity, and a peptide, GRGDTP, that inhibits attachment to type I collagen in addition to inhibiting attachment to fibronectin and vitronectin. The penetration of two human melanoma cell lines and a glioblastoma cell line through the human amniotic basement membrane and its underlying stroma was inhibited by all of the RGD-containing peptides except for the one that inhibits only the vitronectin attachment. Various control peptides lacking RGD showed essentially no inhibition. This inhibitory effect on cell invasion was dose-dependent and nontoxic. A hexapeptide, GRGDTP, that inhibits the attachment of cells to type I collagen in addition to inhibiting fibronectin- and vitronectin-mediated attachment was more inhibitory than those RGD peptides that inhibit only fibronectin and vitronectin attachment. Analysis of the location of these cells that were prevented from invading indicated that they attached to the amniotic basement membrane but did not proceed further into the tissue. These results suggest that interactions between RGD-containing extracellular matrix adhesion proteins and cells are necessary for cell invasion through tissues and that fibronectin and type I collagen are important for this process.     

Extracellular matrix regulates apoptosis in mammary epithelium through a control on insulin signaling

Adherent epithelial cells require interactions with the extracellular matrix for their survival, though the mechanism is ill-defined. In long term cultures of primary mammary epithelial cells, a laminin-rich basement membrane (BM) but not collagen I suppresses apoptosis, indicating that adhesion survival signals are specific in their response (. J. Cell Sci. 109:631-642). We now demonstrate that the signal from BM is mediated by integrins and requires both the alpha6 and beta1 subunits. In addition, a hormonal signal from insulin or insulin-like growth factors, but not hydrocortisone or prolactin, is necessary to suppress mammary cell apoptosis, indicating that BM and soluble factors cooperate in survival signaling. Insulin induced autophosphorylation of its receptor whether mammary cells were cultured on collagen I or BM substrata. However, both the tyrosine phosphorylation of insulin receptor substrate-1 and its association with phosphatidylinositol 3-kinase were enhanced in cells cultured on BM, as was the phosphorylation of the phosphatidylinositol 3-kinase effector, protein kinase B. These results suggest a novel extracellular matrix-dependent restriction point in insulin signaling in mammary epithelial cells. The proximal signal transduction event of insulin receptor phosphorylation is not dependent on extracellular matrix, but the activation of downstream effectors requires adhesion to BM. Since phosphatidylinositol 3-kinase was required for mammary epithelial cell survival, we propose that a possible mechanism for BM-mediated suppression of apoptosis is through its facilitative effects on insulin signaling.     

Triflavin,an Arg-Gly-Asp-containing peptide,inhibits B16-F10 mouse melanoma cell adhesion to matrix proteins via direct binding to tumor cells

Triflavin, an Arg-Gly-Asp (RGD)-containing snake venom peptide, inhibits B16-F10 mouse melanoma cell adhesion to extracellular matrices, e.g., fibronectin, vitronectin, fibrinogen, and collagen type I. In this study, GRGDS inhibits B16-F10 mouse melanoma cell adhesion to immobilized triflavin in a dose-dependent manner. In addition, flow-cytometric analysis and the fluorescence staining method in which FITC-triflavin is utilized as a binding ligand were used. GRGDS inhibits the binding of FITC-triflavin to B16-F10 cells. Additionally, the above results suggest that triflavin directly binds to its receptors expressed on B16-F10 cell surface primarily via its RGD sequence, there-by inhibiting B16-F10 cell adhesion to extracellular matrices.     

Characterization of the integrin alpha v beta 6 as a fibronectin-binding protein.

Integrins are a complex family of divalent cation-dependent cell adhesion receptors composed of one alpha and one beta subunit noncovalently bound to one another. A subset of integrins contains the alpha v subunit in association with one of several beta subunits (e.g. beta 3, beta 5, beta 1). We have recently identified a novel integrin beta subunit, beta 6, that is present in a number of epithelial cell lines. Using a polyclonal antibody raised against the carboxyl-terminal peptide of beta 6, we have now identified the integrin heterodimer, alpha v beta 6, on the surface of two human carcinoma cell lines. Using affinity chromatography of lysates from the pancreatic carcinoma cell line, FG-2, we demonstrate that alpha v beta 6 binds to fibronectin, but not to vitronectin or collagen I. In contrast, the alpha v beta 5 integrin, which is also expressed on FG-2 cells, binds exclusively to vitronectin. Immobilized collagen I does not interact with alpha v integrins, but binds beta 1-containing integrins. Both alpha v beta 6 and alpha v beta 5 are eluted from their respective immobilized ligands by a hexa-peptide containing the sequence Arg-Gly-Asp (RGD). RGD is highly effective in the presence of Ca2+, somewhat less effective in Mg2+, and virtually inactive in Mn2+. These results suggest that alpha v beta 6 functions as an RGD-dependent fibronectin receptor in FG-2 carcinoma cells. In agreement with this notion, cell adhesion assays show that FG-2 cell attachment to fibronectin is only partially inhibited by anti-beta 1 integrin antibodies, implying that other fibronectin receptors may be involved. Taken together with recent reports on the vitronectin receptor function of alpha v beta 5, our results suggest that the previously described carcinoma cell integrin, alpha v beta x (Cheresh, D. A., Smith, J. W., Cooper, H. M., and Quaranta, V. (1989) Cell 57, 59-69), is a mixture of at least two different receptors: alpha v beta 5, mediating adhesion to vitronectin, and alpha v beta 6, mediating adhesion to fibronectin.     

Extracellular matrix protein mediated regulation of the osteoblast differentiation of bone marrow derived human mesenchymal stem cells

The biomimetic approach of tissue engineering exploits the favorable properties of the extracellular matrix (ECM), to achieve better scaffold performance and tissue regeneration. ECM proteins regulate cell adhesion and differentiation through integrin mediated signal transduction. In the present study, we have examined the role of ECM proteins such as collagen type I, fibronectin, laminin and vitronectin in regulating the proliferation and osteogenic differentiation of bone marrow derived human mesenchymal stem cells (hMSCs). hMSCs were grown on selected ECM protein treated tissue culture plates. The growth kinetics was assessed by calculating the doubling time of the cells on different ECM treated plates. The cells were directed to osteoblast lineage by growing them in osteogenic induction media for 21 day. Differentiation was evaluated at different time points by osteoblast differentiation associated gene expression, alkaline phosphatase (ALP) activity, histochemical staining for mineralized matrix and calcium quantification. The doubling time of hMSCs cultured on collagen type I was significantly low, which was followed by laminin and fibronectin treated plates. However, doubling time of hMSCs cultured on vitronectin treated plate was not significantly different than that of the untreated control. High ALP gene (ALPL) expression and associated enhancement of mineralization were observed on collagen type I, fibronectin and vitronectin treated plates. Collagen type I showed early onset of mineralization with high ALP activity and up-regulation of osteopontin, ALPL, bone sialoprotein and osteocalcin genes. Vitronectin also up-regulated these genes and showed the highest amount of calcium in the secreted mineral matrix. Therefore, we conclude that, ECM proteins indeed modified the growth patterns and induced the osteoblast differentiation of hMSCs. Our findings have significant implication for bone tissue engineering applications.     

Soluble Integrin Ligands and Growth Factors Independently Rescue Neuroblastoma Cells from Apoptosis under Nonadherent Conditions

We have investigated the role of extracellular matrix (ECM) and growth factors in the survival of nonadherent human neuroblastoma cells (line SK-N-BE). Cells cultured in serum-free medium under nonadherent conditions died with apoptotic-like features (cromatin condensation and nuclear fragmentation). SK-N-BE cells underwent neuronal differentiation in response to retinoic acid (RA). While RA itself did not induce apoptosis, differentiation increased the susceptibility of SK-N-BE cells to detachment-induced apoptosis. The appearance of the apoptotic-like phenotype required the maintenance in suspension of SK-N-BE cells for at least 16 h (12.43 ± 1.40% of cells undergoing apoptosis) and the percentage increased up to 46.84 ± 3.15% after 24 h. Suspension-induced apoptosis did not depend on increased intracellular Ca²⁺levels nor onde novoprotein synthesis and was not associated with extensive DNA degradation. Stimulation by soluble collagen I rescued suspended cells from apoptosis, even in the absence of cell adhesion and spreading. The survival promoting effect of ECM was mediated by the integrin receptors, since ([1]) the protective effect of soluble collagen I was blocked by anti-integrin antibodies to β₁and α₁subunits and ([2]) the antibody-induced clustering of α₁, α₃, α_v, β₁, and β₃integrins rescued SK-N-BE cells cultured in suspension from apoptosis. As expected, adhesion on immobilized ECM proteins, collagen I, or laminin (0.1 to 10 μg/ml) also rescued SK-N-BE cells from apoptosis in a dose-dependent manner. Thede novoprotein synthesis was required to promote the survival effect of ECM, since cycloheximide completely abolished the protective effect of collagen I and protection from apoptosis by ECM or by anti-β₁antibody was associated with the increased expression ofbcl-2.In addition to integrin stimulation, serum, insulin, and nerve growth factor inhibited suspension-induced apoptosis of SK-N-BE cells. The survival effect of serum and growth factors did not require the synthesis of new proteins, unlike the ECM effect. These data show that matrix proteins can promote cell survival in neuronal cells via integrin receptors. This effect does not require cell adhesion and the subsequent changes in cell shape as it can be mediated by soluble integrin ligands in suspended cells and involves a signaling pathway different from that triggered by growth factors.     

Effect of hypoxia on cellular adhesion to vitronectin and fibronectin

Cellular invasion of extracellular matrix (ECM) occurs during normal and pathological settings. For cells to invade, they must adhere to the underlying substratum, break down barrier molecules, and detach from the substratum prior to migrating through the ECM. We previously demonstrated that incubation under reduced oxygen levels increases the in vitro invasiveness of trophoblast and breast carcinoma cells, an effect linked to elevated expression of the cell surface receptor for urokinase-type plasminogen activator (uPAR). This study examined the role of oxygen, integrins and the urokinase-type plasminogen activator (uPA) system on the adhesion of trophoblast and breast carcinoma cells to the ECM molecules vitronectin and fibronectin. Compared to exposure to 20 and 5% oxygen, exposure to 1% oxygen decreased adhesion of these cells to vitronectin and fibronectin, an effect that was reversible by re-exposure to 20% oxygen. Incubation in 1% oxygen also resulted in reduced expression of surface alpha(5) integrin. Furthermore, adhesion to vitronectin and fibronectin was reduced by compounds that interfere with integrin function, such as EDTA, anti-integrin antibodies, or by antibodies that interfere with the binding of pro-uPA to uPAR, soluble uPAR, soluble vitronectin, phosphatidylinositol-specific phospholipase C, as well as plasminogen activator inhibitor-1. These findings suggest an important role for oxygen in the regulation of cellular invasion, possibly in part through its effects on integrin and uPAR-mediated mechanisms of adhesion.     

(Arg-Gly-Asp)n-albumin conjugates as a model substratum for integrin-mediated cell adhesion

We have prepared protein-peptide conjugates composed of bovine serum albumin (BSA) derivatized with short peptides containing the Arg-Gly-Asp (RGD) sequence derived from the adhesion site of fibronectin. The RGD-BSA conjugates were used to coat tissue culture plastic surfaces which then served as substrata in cell adhesion experiments. Our results indicate that the efficiency of adhesion to RGD-BSA-coated surfaces is highly dependent on the valency of the (RGD)n-BSA conjugates. For example, on surfaces with approximately equal amounts of RGD ligand, CHO cells adhered virtually 100% to the (RGD)n-BSA (n = 20.8) conjugate and not at all to the (RGD)n-BSA (n = 3.5) conjugate. Adhesion on (RGD)n-BSA-coated substrata and on fibronectin- or vitronectin-coated substrata was also examined in terms of the relationship between cell adhesion and the intermolecular distances of adsorbed proteins. It was observed that for substrata coated with relatively compact, symmetric molecules, such as RGD-BSA or vitronectin, adhesion dropped off sharply as intermolecular distances increased; by contrast, for fibronectin, a large asymmetric molecule, adhesion declined more gradually as intermolecular distances increased. Finally, we have examined the role of different cell-surface receptors in the process of adhesion to RGD-BSA substrata. Interestingly, competition and blocking experiments with antibodies and with soluble competing proteins suggest that it is the vitronectin receptor rather than the fibronectin receptor which mediates adhesion to RGD-BSA.     

Collagen modulates gene activation of plasminogen activator system molecules

Skin extracellular matrix (ECM) molecules regulate a variety of cellular activities, including cell movement, which are central to wound healing and metastasis. Regulated cell movement is modulated by proteases and their associated molecules, including the serine proteases urinary-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA) and their inhibitors (PAIs). As a result of wounding and loss of basement membrane structure, epidermal keratinocytes can become exposed to collagen. To test the hypothesis that during wounding, exposed collagen, the most abundant ECM molecule in the skin, regulates keratinocyte PA and PAI gene expression, we utilized an in vitro model in which activated keratinocytes were cultured in dishes coated with collagen or other ECM substrates. tPA, uPA, and PAI-1 mRNA and enzymatic activity were detected when activated keratinocytes attached to fibronectin, vitronectin, collagen IV, and RGD peptide. In contrast, adhesion to collagen I and collagen III completely suppressed expression of PAI-1 mRNA and protein and further increased tPA expression and activity. Similarly, keratinocyte adhesion to laminin-1 suppressed PAI-1 mRNA and protein expression and increased tPA activity. The suppressive effect of collagen I on PAI-1 gene induction was dependent on the maintenance of its native fibrillar structure. Thus, it would appear that collagen- and laminin-regulated gene expression of molecules associated with plasminogen activation provides an additional dimension in the regulation of cell movement and matrix remodeling in skin wound healing.     

Vitronectin production by human yolk sac carcinoma cells resembling parietal endoderm

Normal mesenchymal cells, normal epithelial cells and many transformed epithelial cells require serum attachment factors and extracellular matrix proteins for growth and differentiation in vitro, and recent evidence strongly supports a role for extracellular matrix molecules in the regulation of cell movement in vivo during early embryogenesis. We previously described the isolation and characterization of cell lines representative of three types of stem cells most commonly found in human adult testicular teratomas, namely embryonal carcinoma cells, yolk sac carcinoma cells resembling visceral endoderm and yolk sac carcinoma cells resembling parietal endoderm (endodermal sinus tumour cells). Of these three cell types, only endodermal sinus tumour cells, which show particularly malignant behaviour in vivo, have no serum requirement for attachment and growth in vitro. Supernatants from endodermal sinus tumour cells support the attachment of embryonal carcinoma cells in serum-free medium. We demonstrate here that endodermal sinus tumour cells, but not other cell types isolated from testicular teratomas, secrete the serum attachment protein, vitronectin (also known as serum-spreading factor, S-protein or epibolin), as well as fibronectin, laminin and type IV collagen, into serum-free medium. Purified vitronectin from medium conditioned by endodermal sinus tumour cells supported both attachment and spreading of embryonal carcinoma cells in vitro, whereas cells attached but did not spread properly on surfaces coated with fibronectin or laminin. Peptides containing the RGD cell recognition sequence common to many attachment proteins blocked attachment of endodermal sinus tumour cells to untreated tissue-culture plastic in serum-free medium. The results suggest a possible role for vitronectin in regulating cell motility and growth in early development, and in the invasion and spread of teratomas in vivo.     

RAM, an RGDS analog, exerts potent anti-melanoma effects in vitro and in vivo

Peptides containing the RGD sequence are under continuous investigation given their ability to control cell adhesion and apoptosis. Since small peptides are quickly metabolized and degraded in vivo, developing analogs resistant to serum-induced degradation is a challenging task. RGD analogs developed so far are known as molecules mostly inhibiting cell adhesion; this feature may reduce cell proliferation and tumor development but may not induce regression of tumors or metastases already formed. In the current study, carried out in melanoma in vitro and in vivo models, we show that RAM, an RGD-non-peptide Analog-Molecule, strongly inhibits cells adhesion onto plastic, vitronectin, fibronectin, laminin and von Willebrand Factor while it does not inhibit cell adhesion onto collagen IV, similarly to the RGDS template peptide. It also strongly inhibits in vitro cell proliferation, migration and DNA-synthesis, increases melanoma cells apoptosis and reduces survivin expression. All such effects were observed in collagen IV seeded cells, therefore are most likely independent from the anti adhesive properties. Further, RAM is more stable than the template RGDS; in fact it maintains its anti-proliferation and anti-adhesion effects after long serum exposure while RGDS almost completely loses its effects upon serum exposure. In a mouse metastatic melanoma in vivo model, increasing doses of RAM significantly reduce up to about 80% lung metastases development, while comparable doses of RGDS are less potent. In conclusion these data show that RAM is a potent inhibitor of melanoma growth in vitro, strongly reduces melanoma metastases development in vivo and represents a novel candidate for further in vivo investigations in the cancer treatment field.     

Triflavin, an Arg-Gly-Asp-containing peptide, inhibits human cervical carcinoma (HeLa) cell-substratum adhesion through an RGD-dependent mechanism

Triflavin, a 7.5-kDa cysteine-rich polypeptide purified from Trimeresurus flavoviridis snake venom, belongs to a family of RGD-containing peptides, termed disintegrins, that have been isolated from the venoms of various vipers and shown to be potent inhibitors of platelet aggregation. The interaction of tumor cells with extracellular matrices such as fibronectin, vitronectin, and collagen has been shown to be mediated through a family of cell surface receptors that specifically recognize an arginine-glycine-aspartic acid (RGD) sequence within each adhesive protein. In this study, we show that triflavin dose-dependently inhibited adhesion of human cervical carcinoma (HeLa) cells to extracellular matrices (ECMs; i.e., fibronectin, fibrinogen, and vitronectin). On the other hand, triflavin exerted a limited inhibitory effect on cell adhesion to laminin and collagen (type I and IV). On a molar basis, triflavin is approximately 800 times more potent than Gly-Arg-Gly-Asp-Ser (GRGDS) at inhibiting cell adhesion. When immobilized on plate, triflavin significantly promoted HeLa cell adhesion, and this attachment was inhibited by GRGDS. Furthermore, FITC-conjugated triflavin bound to cells in a saturable manner and its binding was inhibited by GRGDS. In addition, triflavin did not affect [³H]thymidine uptake of HeLa cells during a 3-day incubation. These results suggest that triflavin probably binds to integrin receptors expressed on HeLa cell surface via its RGD sequence within its molecule, thereby inhibiting the adhesion of extracellular matrices to HeLa cells.     

Synthetic RGD-containing alpha-helical coiled coil peptides promote integrin-dependent cell adhesion.

Integrin receptors are the main mediators of cell adhesion to the extracellular matrix. They bind to their ligands by interacting with short amino acid sequences, such as the RGD sequence. Soluble, small RGD-based peptides have been used to block integrin-binding to ligands, thereby interfering with cell adhesion, migration and survival, while substrate-immobilized RGD sequences have been used to enhance cell binding to artificial surfaces. This approach has several important medical applications, e.g. in suppression of tumor angiogenesis or stimulation of bone formation around implants. However, the relatively weak affinity of short RGD-containing peptides often results in incomplete integrin inhibition or ineffective ligation. In this work, we designed and synthesized several new multivalent RGD-containing molecules and tested their ability to inhibit or to promote integrin-dependent cell adhesion when used in solution or immobilized on substrates, respectively. These molecules consist of an oligomeric structure formed by alpha-helical coiled coil peptides fused at their amino-terminal ends with an RGD-containing fragment. When immobilized on a substrate, these peptides specifically promoted integrin alphaVbeta3-dependent cell adhesion, but when used in solution, they blocked alphaVbeta3-dependent cell adhesion to the natural substrates fibronectin and vitronectin. One of the peptides was nearly 10-fold more efficient than fibronectin or vitronectin in promoting cell adhesion, and almost 100-fold more efficient than a linear RGD tripeptide in blocking adhesion. These results indicate that alpha-helical coiled coil peptides carrying an amino-terminal RGD motif can be used as soluble antagonists or surface-immobilized agonists to efficiently inhibit or promote integrin alphaVbeta3-mediated cell adhesion, respectively.     

Integrin activation and focal complex formation in cardiac hypertrophy

Cardiac hypertrophy is characterized by both remodeling of the extracellular matrix (ECM) and hypertrophic growth of the cardiocytes. Here we show increased expression and cytoskeletal association of the ECM proteins fibronectin and vitronectin in pressure-overloaded feline myocardium. These changes are accompanied by cytoskeletal binding and phosphorylation of focal adhesion kinase (FAK) at Tyr-397 and Tyr-925, c-Src at Tyr-416, recruitment of the adapter proteins p130(Cas), Shc, and Nck, and activation of the extracellular-regulated kinases ERK1/2. A synthetic peptide containing the Arg-Gly-Asp (RGD) motif of fibronectin and vitronectin was used to stimulate adult feline cardiomyocytes cultured on laminin or within a type-I collagen matrix. Whereas cardiocytes under both conditions showed RGD-stimulated ERK1/2 activation, only collagen-embedded cells exhibited cytoskeletal assembly of FAK, c-Src, Nck, and Shc. In RGD-stimulated collagen-embedded cells, FAK was phosphorylated only at Tyr-397 and c-Src association occurred without Tyr-416 phosphorylation and p130(Cas) association. Therefore, c-Src activation is not required for its cytoskeletal binding but may be important for additional phosphorylation of FAK. Overall, our study suggests that multiple signaling pathways originate in pressure-overloaded heart following integrin engagement with ECM proteins, including focal complex formation and ERK1/2 activation, and many of these pathways can be activated in cardiomyocytes via RGD-stimulated integrin activation.     

Epigallocatechin gallate protects BEAS-2B cells from lipopolysaccharide-induced apoptosis through upregulation of gastrin-releasing peptide

Gastrin-releasing peptide (GRP) plays a major role in the development and maintenance of lung epithelial cells by promoting cell division, whereas its suppression causes growth arrest and apoptosis. The present study shows that human bronchial epithelial BEAS-2B cells challenged with lipopolysaccharide (LPS), an endotoxin from gram-negative bacteria, downregulated GRP expression and induced apoptosis via upregulation of p53 and active caspase-3, signifying the importance of GRP in lung epithelial cell survival. However, in the presence of epigallocatechin-3-gallate (EGCG), a polyphenol in green tea, BEAS-2B cells resisted LPS-induced apoptosis and restored the expression of GRP and its downstream effectors such as epidermal growth factor receptor and NF-κB, as analysed by immunoblotting and qPCR. Based on our findings, we objectify that cytoprotective functions of EGCG, via upregulation of GRP in cells challenged with LPS, are novel and can be further explored in a therapeutic point of view for diseases such as septic shock.