Enhancement of peri-implant bone osteogenic activity induced by a peptidomimetic functionalization of titanium.

Osteoblast cell adhesion to the extracellular matrix is established through two main pathways: one is mediated by the binding between integrin and a minimal adhesion sequence (RGD) on the extracellular protein, the other is based on the interactions between transmembrane proteoglycans and heparin-binding sequences found in many matrix proteins. The aim of this study is the evaluation in an in vivo endosseous implant model of the early osteogenic response of the peri-implant bone to a biomimetic titanium surface functionalized with the retro-inverso 2DHVP peptide, an analogue of Vitronectin heparin binding site. The experimental plan is based on a bilateral study design of Control and 2DHVP implants inserted respectively in the right and left femur distal metaphysis of adult male Wistar rats (n=16) weighing about 300grams and evaluated after 15days. Fluorochromic bone vital markers were given in a specific time frame, in order to monitor the dynamic of new bone deposition. The effect inducted by the peptidomimetic coating on the surrounding bone were qualitatively and quantitatively evaluated by means of static and dynamic histomorphometric analyses performed within three concentric and subsequent circular Regions of Interest (ROI) of equivalent thickness (220µm), ROI1 adjacent to the interface, ROI2, the middle, and ROI3 the farthest. The data indicated that these functionalized implants stimulated a higher bone apposition rate (p<0,01) and larger and rapid osteoblast activation in terms of mineralizing surface within ROI1 compared to the control (p<0,01). These higher osteoblast recruitment and activation leads to a greater bone-to-implant contact reached for DHVP samples (p<0,5). This represents an initial stimulus of the osteogenic activity that might results in a faster and better osteointegration process.

Blockade of alpha v beta3 and alpha v beta5 integrins by RGD mimetics induces anoikis and not integrin-mediated death in human endothelial cells.

Alpha v integrins are thought to play an important role in tumor angiogenesis. However, discrepancies between findings with Arg-Gly-Asp (RGD) mimetics, which block angiogenesis in animal models, and knockout mice, in which loss of some alpha v integrins enhances tumor angiogenesis, raise questions concerning the function of these integrins and the precise role of alpha v substrate mimetics in antiangiogenic therapies. We have examined the effects of a novel non-peptide RGD mimetic, S 36578-2, on human endothelial cells to elucidate its antagonist activity and to identify possible agonist functions. S 36578-2 is highly selective for alpha v beta3 and alpha v beta5 integrins and induces detachment, caspase-8 activation, and apoptosis in human umbilical endothelial cells (HUVECs) plated on vitronectin. Importantly, the compound has no effect on the morphology or survival of cells plated on interstitial matrix components such as fibronectin, and it does not potentiate the apoptotic process in suspended cells. Identical results were obtained with a cyclic RGD peptide with similar target specificity. In microvascular endothelial cells, S 36578-2-induced death was also linked to its antiadhesive effect, with established lines markedly more resistant than primary cultures to the antiadhesive and proapoptotic effects. Altogether, these findings have important implications for the development of this class of antiangiogenics.

Modulation in vitro of keratinocyte integrins by interferon-alpha and interferon-gamma.

BACKGROUND: Interferon-alpha and -gamma are glycoproteins with antiviral and immunoregulatory properties. In vitro studies have shown a role for these cytokines in the regulation of epidermal keratinocyte growth and differentiation. In the same way, integrins are adhesion molecules which regulate keratinocyte proliferation and differentiation. AIM: To determine whether the regulatory activity of interferons on keratinocyte proliferation and differentiation is related to a modulation of keratinocyte integrins. METHODS: Two different methods were used: monolayers and reconstituted skin, incubated either with 1,200 U/mL interferon-alpha or 500 U/mL interferon-gamma or control medium for 48 h. The integrin expression was assessed by flow cytometry and immunohistochemistry. RESULTS: In monolayers, only the alpha3 subunit was significantly inhibited by interferon-gamma. In reconstituted skin, where keratinocytes are differentiated, both interferons had an inductive effect on beta1 expression and interferon-alpha had an inhibitory effect on alpha6 expression. CONCLUSION: Interferon-alpha and -gamma induce a modulatory effect on alpha3, alpha6 and beta1 which appears to be related to the state of differentiation. Moreover, the decreased expression of alpha6 and alpha3 could be one of the mechanisms involved in the formation of bullous lesions during long-term interferon therapy.

Preparation and functional evaluation of RGD-modified proteins as alpha(v)beta(3) integrin directed therapeutics.

Tumor blood vessels can be selectively targeted by RGD-peptides that bind to alpha(v)beta(3) integrin on angiogenic endothelial cells. By inhibiting the binding of these integrins to its natural ligands, RGD-peptides can serve as antiangiogenic therapeutics. We have prepared multivalent derivatives of the cyclic RGD-peptide c(RGDfK) by covalent attachment of the peptide to side chain amino groups of a protein. These RGDpep-protein conjugates inhibited alpha(v)beta(3)-mediated endothelial cell adhesion in vitro, while conjugates prepared with a control RAD-peptide showed no activity. Radiobinding and displacement studies with endothelial cells demonstrated an increased affinity of the RGDpep-protein conjugates compared to the free peptide, with IC(50) values ranging from 23 to 0.6 nM, depending on the amount of coupled RGDpep per protein. Compared to the parental RGD-peptide and the related RGD-peptide ligand c(RGDfV), the RGDpep-protein conjugates showed a considerable increase in affinity (IC(50) parent RGDpep: 818 nM; IC(50) c(RGDfV): 158 nM). We conclude that the conjugation of RGD-peptides to a protein, resulting in products that can bind multivalently, is a powerful approach to increase the affinity of peptide ligands for alpha(v)beta(3)/alpha(v)beta(5) integrins.

A fibronectin fragment inhibits tumor growth, angiogenesis, and metastasis.

We have shown previously that a polymeric form of fibronectin is strongly antimetastatic when administered systemically to tumor-bearing mice. The polymeric fibronectin, sFN, is formed in vitro by treating soluble fibronectin with a 76-aa peptide, III1-C, which is derived from the first type III repeat in fibronectin. Here we show that the III1-C peptide and sFN also reduce tumor growth in mice, and that this effect correlates with a low density of blood vessels in the tumors of the treated mice. III1-C also polymerized fibrinogen, and the fibrinogen polymer, sFBG, had antitumor and antiangiogenic effects similar to those of sFN. Mice that had been injected s.c. with three different types of human tumor cells and treated with biweekly i.p. injections of III1-C, sFN, or sFBG over a 5-week period had tumors that were 50-90% smaller than those of control mice. Blood vessel density in the tumors of the treated mice was reduced by 60-80% at the end of the experiment. Xenograft tumors from a human breast carcinoma line (MDA-MB-435) were particularly susceptible to these treatments. Metastasis into the lungs from the primary s.c. tumors also was inhibited in the mice treated with III1-C and the two polymers. The III1-C peptide is an antiangiogenic and antimetastatic agent. Because of its ability to suppress tumor growth, angiogenesis, and metastasis, we have named the III1-C peptide anastellin [from anastello (Greek), inhibit, force a retreat].

Manganese induces neurite outgrowth in PC12 cells via upregulation of alpha(v) integrins.

Previous studies have demonstrated that the divalent cation manganese (Mn) causes PC12 cells to form neurites in the absence of NGF. Since divalent cations modulate the binding affinity and specificity of integrins, and integrin function affects neurite outgrowth, we tested the hypothesis that Mn induces neurite outgrowth through an integrin-dependent signaling pathway. Our studies support this hypothesis. Function-blocking antisera specific for beta(1) integrins block the neurite-promoting activity of Mn by 90-95%. Bioassays and biochemical studies with antisera specific for the alpha(v), alpha(5), or alpha(8) integrin subunit suggest that the alpha(v)beta(1) heterodimer is one of the principal beta(1) integrins mediating the response of PC12 cells to Mn. This is corroborated by studies in which Mn failed to induce neurite outgrowth in a clone of PC12 cells that does not express alpha(v) at levels detectable by immunoprecipitation or immunocytochemistry. SDS-PAGE analysis of biotinylated surface proteins immunoprecipitated from Mn-responsive PC12 cells, as well as confocal laser microscopy of PC12 immunostained for surface alpha(v) indicate that Mn increases the surface expression of alpha(v) integrins. This increase appears to be due in part to synthesis of alpha(v) since specific inhibitors of RNA and protein synthesis block the neurite-promoting activity of Mn. These data indicate that Mn induces neurite outgrowth in PC12 cells by upregulating alpha(v) integrins, suggesting that Mn potentially represents an additional mechanism for regulating the rate and direction of neurite outgrowth during development and regeneration.

Blockade of the alpha(v)beta(3) integrin adversely affects implantation in the mouse.

The role of endometrial and embryonic integrins during implantation remains unresolved although work in animal models and in humans supports their involvement in this process. Temporal and spatial distribution of the alpha(v)beta(3) integrin on both embryo and endometrium in women and mice coincides with the time of initial attachment during implantation. In mice, the endometrial and embryonic alpha(v)beta(3) integrin is present at the time of implantation, as shown by reverse transcription-polymerase chain reaction and immunohistochemistry. In situ hybridization demonstrates the presence of the alpha(v)beta(3) integrin on the subluminal stromal cells of the uterus. Functional blockade of this integrin on the day of implantation by intrauterine injection of neutralizing monoclonal antibodies against alpha(v) or beta(3) integrin subunits, arg-gly-asp (RGD)-containing peptides, or of the disintegrin echistatin, reduced the number of implantation sites compared to controls receiving BSA. These studies demonstrate that, like the human, the murine alpha(v)beta(3) integrin is expressed at the time of implantation in the endometrium and on the blastocyst, and may play a critical role in the cascade of events leading to successful implantation.

Identification of CD47/integrin-associated protein and alpha(v)beta3 as two receptors for the alpha3(IV) chain of type IV collagen on tumor cells.

Previous studies from our laboratories demonstrated that a peptide from the noncollagenous domain of the alpha3 chain of basement membrane collagen (COL IV), comprising residues 185-203, inhibits polymorphonuclear leukocyte activation and melanoma cell proliferation independently of its ability to promote cell adhesion; these properties require the presence of the triplet -SNS- at residues 189-191 (J. C. Monboisse et al., J. Biol. Chem., 269: 25475-25482, 1994; J. Han et al., J. Biol. Chem., 272: 20395-20401, 1997). More recently, we demonstrated that native COL IV and -SNS-containing synthetic peptides (10 microg/ml) added to culture medium inhibit the proliferation of not only melanoma cells but also breast, pancreas, and stomach tumor cells up to 82% and prostate tumor cells by 15%. This inhibition was shown to be dependent on a COL IV- or peptide-induced increase in intracellular cAMP (T. A. Shahan et al., Connect. Tissue Res., 40: 221-232, 1999). Attempts to identify the putative receptor(s) on tumor cells led to the isolation of five proteins (Mr 33,000, 52,000, 72,000, 95,000, and 250,000) from melanoma and prostate cells by affinity purification with the alpha3(IV)179-208 peptide. The Mr 52,000, 95,000, and 250,000 proteins were shown to be CD47/integrin-associated protein(IAP), the integrin beta3 subunit, and the alpha(v)beta3 integrin complex, respectively. The Mr 33,000 and 72,000 proteins have not yet been identified. To confirm the specificity of ligand binding to the receptors, cell membranes from either melanoma or prostate tumor cells were pretreated with the unlabeled ligand alpha3(IV)187-191 (-YYSNS-); alternatively, the peptide was pretreated with a peptide-reactive monoclonal antibody (A5D7) before receptor isolation. These treatments inhibited the purification of CD47/IAP, the integrin beta3 subunit, and the alpha(v)beta3 integrin complex from tumor cells. Furthermore, cells treated with CD47/IAP- or the alpha(v)beta3 integrin-reactive antibodies prevented the alpha3(IV)185-203 peptide from inhibiting cell proliferation and the subsequent rise in intracellular cAMP. Pretreating cells with the alpha3(IV)187-191 (-YYSNS-) peptide also inhibited their adhesion to the alpha3(IV)185-203 peptide substrate, whereas the inactive alpha1(IV)185-203 peptide, from the same region of the alpha1 chain as the alpha3(IV)185-203 peptide, had no effect. Incubation of cells with either CD47/IAP and/or alpha(v)beta3 integrin-reactive antibodies inhibited their adhesion to the alpha3(IV)185-203 peptide, whereas antibodies to the beta1 and beta2 integrin subunits were without effect. These data suggest that ALC-COL IV, through its alpha3(IV) chain, inhibits tumor cell proliferation using the receptors CD47/IAP and the alpha(v)beta3 integrin.

Potential non-glycoprotein IIb/IIIa effects of abciximab.

The antithrombotic effect of abciximab is believed to be primarily due to its blockade of platelet glycoprotein IIb/IIIa receptors, leading to the inhibition of platelet aggregation. Studies have, however, identified that antibody 7E3, the parent molecule of abciximab, and/or abciximab itself, binds to both "activated" alphaMbeta2 receptors and alphaVbeta3 receptors. Because alphaMbeta2 receptors are present on granulocytes and monocytes, cells that have been implicated in contributing to atherosclerosis, intimal hyperplasia after vascular injury, reperfusion injury, and thrombin generation, it is possible that some of abciximab's effects relate to this reactivity. Similarly, because alphaVbeta3 has been implicated in platelet adhesion to osteopontin, intimal hyperplasia after vascular injury, and platelet-mediated thrombin generation, it is possible that some of abciximab's beneficial effects relate to this reactivity. Blockade of alphaVbeta3 receptors may also be beneficial in other disease states because, in animal models, such blockade inhibits tumor angiogenesis and sickle cell adhesion to blood vessel endothelium. Despite these intriguing observations, there are no direct data to support any beneficial roles or any unwanted side effects related to the reactivities of abciximab with "activated" alphaMbeta2 or alphaVbeta3 receptors.

Mechanisms and consequences of affinity modulation of integrin alpha(V)beta(3) detected with a novel patch-engineered monovalent ligand.

Integrin alpha(V)beta(3) mediates diverse responses in vascular cells, ranging from cell adhesion, migration, and proliferation to uptake of adenoviruses. However, the extent to which alpha(V)beta(3) is regulated by changes in receptor conformation (affinity), receptor diffusion/clustering (avidity), or post-receptor events is unknown. Affinity regulation of the related integrin, alpha(IIb)beta(3), has been established using a monovalent ligand-mimetic antibody, PAC1 Fab. To determine the role of affinity modulation of alpha(V)beta(3), a novel monovalent ligand-mimetic antibody (WOW-1) was created by replacing the heavy chain hypervariable region 3 of PAC1 Fab with a single alpha(V) integrin-binding domain from multivalent adenovirus penton base. Both WOW-1 Fab and penton base bound selectively to activated alpha(V)beta(3), but not to alpha(IIb)beta(3), in receptor and cell binding assays. alpha(V)beta(3) affinity varied with the cell type. Unstimulated B-lymphoblastoid cells bound WOW-1 Fab poorly (apparent K(d) = 2.4 microM), but acute stimulation with phorbol 12-myristate 13-acetate increased receptor affinity >30-fold (K(d) = 80 nM), with no change in receptor number. In contrast, alpha(V)beta(3) in melanoma cells was constitutively active, but ligand binding could be suppressed by overexpression of beta(3) cytoplasmic tails. Up-regulation of alpha(V)beta(3) affinity had functional consequences in that it increased cell adhesion and spreading and promoted adenovirus-mediated gene transfer. These studies establish that alpha(V)beta(3) is subject to rapid regulated changes in affinity that influence the biological functions of this integrin.

Angiogenesis inhibitors. New anticancer strategy.

Neoangiogenesis has been proved to be crucial in neoplasmatic tumor growth and metastases. Over the last few years, the factors that have both a positive (angiogenic) and negative (antiangiogenic) influence on tumor growth have been identified. The potential use of natural and synthetic factors that suppress vasculature formation as anticancer drugs is currently under intense investigation. Recently, several antiangiogenic compounds, including TNP-470 or matrix metalloproteinase inhibitors, have entered clinical trials. This review will describe the main groups of angiogenesis inhibitors, their mechanisms of action and some data from clinical studies.

Vitronectin regulates smooth muscle contractility via alphav and beta1 integrin.

Previous work from this laboratory has established a method for maintaining physiological contractility of dissociated avian smooth muscle in a defined medium at low density. The present report emphasizes the dramatic potency of serum to alter smooth muscle phenotype and induce a loss of contractility. Vitronectin, a molecule purified from plasma, mimicked these effects of serum via an integrin that is RGD-sensitive. Studies utilizing blocking antibodies against vitronectin demonstrated that the presence of this specific adhesion molecule was necessary for the serum-induced loss of contractility. Based on the actions of function-blocking antibodies and RGD-containing peptides, the integrin alphavbeta1 appears to be the primary receptor involved in vitronectin's ability to induce phenotypic transformation in amniotic smooth muscle. The influence of vitronectin on smooth muscle contractility is particularly relevant, because this molecule is abundant in whole blood and plasma (approx. 400 microg/ml). The results suggest that smooth muscle needs to be continually protected from normal blood constituents in vivo. The implications of these results for smooth muscle-related diseases like atherosclerosis, restenosis and Kaposi's sarcoma are discussed.

Evidence for the involvement of endothelial cell integrin alphaVbeta3 in the disruption of the tumor vasculature induced by TNF and IFN-gamma.

Administration of tumor necrosis factor (TNF) and gamma interferon (IFN-gamma) to melanoma patients causes selective disruption of the tumor vasculature but the mechanism of this disruption is unknown. Here we report that exposure of human endothelial cells to TNF and IFN-gamma results in a reduced activation of integrin alphaVbeta3, an adhesion receptor that plays a key role in tumor angiogenesis, leading to a decreased alphaVbeta3-dependent endothelial cell adhesion and survival. Detachment and apoptosis of angiogenic endothelial cells was demonstrated in vivo in melanoma metastases of patients treated with TNF and IFN-gamma. These results implicate integrin alphaVbeta3 in the anti-vascular activity of TNF and IFN-gamma and demonstrate a new mechanism by which cytokines control cell adhesion.

Colony stimulating factor-1-induced osteoclast spreading depends on substrate and requires the vitronectin receptor and the c-src proto-oncogene.

The colony stimulating factor 1 (CSF-1) regulates osteoclastogenesis and bone resorption. Mutations in the CSF-1 gene cause an osteopetrosis characterized by the absence of osteoclasts. Mature osteoclasts respond to CSF-1 with inhibition of bone resorption and an increment of cell spreading. Herein we demonstrate that CSF-1-induced osteoclast spreading depends on the substrate the osteoclast interacts with and requires integrity of the vitronectin receptor and of the c-src proto-oncogene. Rabbit osteoclasts were allowed to attach to glass, serum, osteopontin, and bone substrates, and were treated with 10 ng/ml human recombinant CSF-1 for 4 h. In osteoclasts plated on glass, the cytokine induced 70% inhibition of bone resorption and 1.8-fold stimulation of cell spreading, without changes in podosome expression and microfilament array. In contrast, CSF-1 induced a 2.5-fold increase of osteoclasts showing filopodia, and a 9.5-fold increase of osteoclasts presenting lamellipodia, indicating that membrane motility was required for cell spreading. Osteoclasts plated on serum substrates showed a 50% reduction of spontaneous spreading. However, in this circumstance, CSF-1 still stimulated an increase of osteoclast area. In osteoclasts cultured on osteopontin substrate or on bone slices, an inhibition of CSF-1-induced osteoclast spreading was observed. To establish involvement of the vitronectin receptor and c-src proto-oncogene, cells were treated with the alpha vbeta3 integrin neutralizing antibody, LM609, or c-src antisense oligonucleotides, which reduced CSF-1-induced osteoclast spreading by 57% and 60%, respectively. The results demonstrate that CSF-1-induced osteoclast spreading requires both the vitronectin receptor and the c-src proto-oncogene and that this action is modulated by the adhesion substrata.

An interaction between penton base and alpha v integrins plays a minimal role in adenovirus-mediated gene transfer to hepatocytes in vitro and in vivo.

Studies in cultured cell lines have shown that adenovirus infection involves binding of adenovirus fiber to its cell surface receptor and binding of penton base to alpha v integrins. However, much less is known about the role of these interactions in cells that are targets for adenovirus-mediated gene transfer. Earlier work showed that hepatocytes are readily infected by adenovirus, making them an attractive target for gene therapy in several diseases. We found that addition of fiber protein blocked adenovirus infection of primary cultures of hepatocytes. This suggests an important role for fiber and its receptor. However, mutation of the integrin-binding motif in penton base did not inhibit infection of hepatocytes, even though the mutation impaired infection of HeLa cells. Hepatocytes had undetectable amounts of alpha v integrins on their cell surface and showed no specific adherence to vitronectin, the natural substrate of alpha v integrins. Adenovirus with an intact penton base enhanced infection of liver following intravenous injection, but only by three-fold as compared with virus in which the integrin-binding motif was disrupted. These studies suggest that interactions between cell surface integrins and penton base are not required for adenovirus infection of hepatocytes in vitro, but the interaction enhances infection to a small degree in vivo.

Melanoma cell migration on vitronectin: regulation by components of the plasminogen activation system.

Tumor cell migration and invasion require complex interactions between tumor cells and the surrounding extracellular matrix. These interactions are modified by cell adhesion receptors, as well as by proteolytic enzymes and their receptors. Here, we study the influence of the protease urokinasetype plasminogen activator (uPA) and its receptor (uPAR) on melanoma cell adhesion to, and migration on, the extracellular matrix protein vitronectin (VN). Cell adhesion to VN, but not to type I collagen, is significantly enhanced in the presence of either uPA or its amino-terminal fragment (ATF). Soluble uPAR can inhibit this effect, indicating that uPA/uPAR on melanoma cells can function as a VN receptor. In the absence of bivalent cations, uPA/uPAR can promote cell attachment on VN, but not cell spreading, suggesting that the glycosylphosphatidylinositol (GPI)-anchored uPAR alone is unable to organize the cytoskeleton. Chemotactic melanoma cell migration on a uniform VN matrix is inhibited by uPA and ATF, implying that cell motility decreases when uPA/uPAR acts as a VN receptor. In contrast, plasminogen activator inhibitor I (PAI-I) can stimulate melanoma cell migration on VN, presumably by inhibiting uPA/uPAR-mediated cell adhesion to VN and thereby releasing the inhibition of cell migration induced by uPA. Together, our data implicate components of the plasminogen activation system in the direct regulation of cell adhesion and migration, thereby modulating the behavior of malignant tumor cells.