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PubMed2005

Biological and molecular properties of a new alpha(v)beta3/alpha(v)beta5 integrin antagonist.

The aim of the present study was to identify specific alpha(v)beta3/alpha(v)beta5 integrin antagonists active on tumor-induced angiogenesis. To this purpose, in vitro integrin-binding assays were used to screen a library of conformationally constrained bicyclic lactam Arg-Gly-Asp-containing pseudopeptides. The results identified ST1646 as a high-affinity specific ligand for alpha(v)beta3 and alpha(v)beta5 integrins with negligible interacting with alpha5beta1 integrin. In all the assays, ST1646 was equipotent to or more potent than the well-characterized integrin antagonists c(RGDfV) and cyclo(Arg-Gly-Asp-d-Phe-[NMe]Val) (EMD121974). In the chorioallantoic membrane assay, topical administration of ST1646 was able to prevent the angiogenic responses elicited by recombinant fibroblast growth factor-2 or vascular endothelial growth factor. In addition, systemic administration of ST1646 in mice exerted a significant antiangiogenic activity on neovascularization triggered by mammary carcinoma MDA-MB435 cells implanted s.c. in a dorsal air sac via a (Millipore Filter Corporation, Bedford, MA) chamber. Moreover, ST1646 delivery via an osmotic pump inhibited the growth and vascularization of tumor xenografts originating from the injection of alpha(v)beta3/alpha(v)beta5-expressing human ovarian carcinoma cells in nude mice. In agreement with the biochemical and pharmacologic studies, Monte Carlo/Stochastic Dynamics simulation showed that the bicyclic scaffold in ST1646 forced the compound to assume a preferred conformation superimposable to the X-ray conformation of alpha(v)beta3-bound EMD121974. Accordingly, computer-docking studies indicated that the ST1646-alpha(v)beta3 integrin complex maintains the ligand-receptor distances and interactions observed in the crystalline EMD121974-alpha(v)beta3 integrin complex. Taken together, these observations indicate that ST1646 represents a dual alpha(v)beta3/alpha(v)beta5 integrin antagonist with interesting biochemical and biological features to be tested in cancer therapy.

Animals↗
PubMed2001

Cell surface tissue transglutaminase is involved in adhesion and migration of monocytic cells on fibronectin.

Expression of tissue transglutaminase (transglutaminase II, tTG) was shown to increase drastically during monocyte differentiation into macrophages; however, its role in monocytic cells remains largely unknown. This study describes a novel function of cell surface tTG as an adhesion and migration receptor for fibronectin (Fn). Two structurally related transglutaminases, tTG and the A subunit of factor XIII (FXIIIA), are expressed on the surface of monocytic cells, whereas only surface tTG is associated with multiple integrins of the beta1 and beta3 subfamilies. Both surface levels of tTG and the amounts of integrin-bound tTG are sharply up-regulated during the conversion of monocytes into macrophages. In contrast, a reduction in biosynthesis and surface expression of FXIIIA accompanies monocyte differentiation. Cell surface tTG is colocalized with beta1- and beta3-integrins in podosomelike adhesive structures of macrophages adherent on Fn. Down-regulation of surface tTG by expression of antisense tTG construct or its inhibition by function-blocking antibodies significantly decreases adhesion and spreading of monocytic cells on Fn and, in particular, on the gelatin-binding fragment of Fn consisting of modules I6II1,2I7-9. Likewise, interfering with the adhesive function of surface tTG markedly reduces migration of myeloid cells on Fn and its gelatin-binding fragment. These data demonstrate that cell surface tTG serves as an integrin-associated adhesion receptor that might be involved in extravasation and migration of monocytic cells into tissues containing Fn matrices during inflammation.

Antigens, CD↗
PubMed2001

Coxsackievirus-adenovirus receptor expression in ovarian cancer cell lines is associated with increased adenovirus transduction efficiency and transgene expression.

The expression of coxsackievirus-adenovirus receptor (CAR) and the integrins alpha(v)beta3 and alpha(v)beta5 was analyzed quantitatively (flow cytometry) and qualitatively (immunocytochemistry) in five human ovarian cancer cell lines (PEO1, PEO4, PEO14, SKOV-3, and OVCAR-3) and three control cell lines (293, HeLa, and CHO-K1). The transduction efficiencies were evaluated by adv/rsv-beta-Gal transduction followed by X-gal staining. The effects of 17beta-estradiol on cell growth, CAR and integrins alpha(v)beta3/5 expression, adenovirus transduction efficiency, and cell-killing efficacy of adv/rsv-tk plus ganciclovir were determined. The levels of CAR, integrin alpha(v)beta3, and integrin alpha(v)beta5 showed great variation between the cell lines. Whereas the expression of CAR appeared to be essential for and positively correlated with adenovirus transduction efficiency, the integrins alpha(v)beta3 and alpha(v)beta5 were not absolutely necessary for adenovirus transduction even though their presence may facilitate transduction. In PEO4 and PEO1 cells, proliferation was stimulated by 17beta-estradiol in a dose-dependent manner. In PEO4 cells, and much less pronounced in PEO1 cells, this was accompanied by an increase in CAR expression. The stimulation of CAR expression was paralleled by an increased transduction efficiency resulting in an increased cell-killing efficacy. Our data suggest that the expression of CAR is one of the most important prerequisites for successful adenovirus-mediated gene therapy of ovarian cancer.

Adenoviridae↗
PubMed2003

Modulation of integrin-binding selectivity by mutation within the RGD-loop of snake venom proteins: a novel drug development approach.

Integrins are a family of heterodimeric class I transmembrane receptors, many of which bind to the RGD sequence in adhesive proteins and mediate the adhesive interactions of a variety of cells. The RGD motif has also been found in snake venom proteins that specifically inhibit integrin binding function and serve as potent integrin antagonists. The majority of these proteins interact with beta1 and beta3 associated integrins and their potency is at least 500-2000 times higher than short RGD peptides. Structural and functional studies suggest that the inhibitory potency of these proteins lies in subtle positional requirements of the tripeptide RGD that is harboured in a defined flexible loop. The integrin-binding specificity and selectivity of each of the proteins is controlled by amino acid residues in this loop in close vicinity to the RGD-motif. The review includes an overview of the structure and function of snake-venom integrin antagonists. The ability of these proteins to control platelet aggregation, cell adhesion and ligand binding is compared to that of short linear, cyclic RGD-peptides and RGD-containing proteins and the influence of modulation of amino acid residues flanking the RGD motif is also considered. The review is intended to provide insight into the development of novel inhibitors as drugs.

Amino Acid Sequence↗
PubMed2000

Disruption of integrin-dependent adhesion and survival of endothelial cells by recombinant adenovirus expressing isolated beta integrin cytoplasmic domains.

We explored the possibility of using a genetic approach to inhibit integrin-mediated endothelial cell adhesion and survival. We constructed recombinant adenoviruses (Ads) expressing chimeric proteins consisting of the cytoplasmic and transmembrane domains of integrin beta1 (CH1), beta3 (CH3) or the beta1 transmembrane domain alone (CH2) connected to the extracellular domain of L3T4 placed under the control of the CMV promoter (AdCMV) or the endothelial cell specific Tie-1 promoter (AdTie). All constructs were expressed in a dose- and time-dependent manner with over 90% of cells expressing the constructs within 24 h (AdCMVs) or 72 h (AdTies) after infection. Confluent monolayers of HUVEC infected with AdCMVCH1 or AdCMVCH3 detached from the substrate in a time- and dose-dependent manner with over 95% of the cells being detached 2 days (AdCMVs) or 3 to 4 days (AdTies) after infection. Cell detachment was preceded by the disruption of focal adhesions and reorganization of the actin cytoskeleton and was associated with a reduced ligand-binding activity of beta1, while cell surface density of beta1 integrins remained unchanged. Detached cells failed to re-adhere to different matrix proteins, without, however, any specificity toward beta1 or beta3 integrin-mediated adhesion. Upon detachment, HUVEC rapidly died by apoptosis. These results demonstrate that dominant negative inhibition of integrin function is an effective approach to disrupt endothelial cell adhesion and survival in vitro.

Adenoviridae↗
PubMed2005

Intracellular signaling in platelets.

PURPOSE OF REVIEW: Over the past few years, a large portion of platelet research has focused on intracellular signaling events that contribute to stable platelet adhesion and aggregation. RECENT FINDINGS: Studies of knockout mice have suggested critical roles for several previously unappreciated signaling molecules including phosphatidylinositol 3-kinase, the exchange factor CalDAG-GEFI, and the small GTPase Rap1b. These proteins may function to remodel the platelet cytoskeleton and thereby regulate both adhesion and aggregation. The abundant cytoskeletal protein talin appears to be a key regulator of the platelet integrin alphaIIbbeta3. Recent evidence suggests that talin binding to the cytoplasmic tail of beta3 promotes integrin oligomerization, thereby increasing the binding avidity the alphaIIbbeta3 complex for fibrinogen. SUMMARY: The identification of platelet signaling pathways not only has clinical implications for diagnosis, but perhaps more importantly for rationale drug design. Aspirin, dipyridamole (Persantine), and thienopyridines (ticlopidine and clopidogrel) are all examples of agents that specifically target discrete platelet signaling pathways. These drugs have already been proven to be beneficial in the treatment of cardiovascular disease. Novel agents that target newly identified signaling pathways hold promise of greater specificity and efficacy.

Blood Platelets↗
PubMed2004

Interactions of foot-and-mouth disease virus with soluble bovine alphaVbeta3 and alphaVbeta6 integrins.

At least four members of the integrin family of receptors, alphaVbeta1, alphaVbeta3, alphaVbeta6, and alphaVbeta8, have been identified as receptors for foot-and-mouth disease virus (FMDV) in vitro. Our investigators have recently shown that the efficiency of receptor usage appears to be related to the viral serotype and may be influenced by structural differences on the viral surface (H. Duque and B. Baxt, J. Virol. 77:2500-2511, 2003). To further examine these differences, we generated soluble alphaVbeta3 and alphaVbeta6 integrins. cDNA plasmids encoding the individual complete integrin alphaV, beta3, and beta6 subunits were used to amplify sequences encoding the subunits' signal peptide and ectodomain, resulting in subunits lacking transmembrane and cytoplasmic domains. COS-1 cells were transfected with plasmids encoding the soluble alphaV subunit and either the soluble beta3 or beta6 subunit and labeled with [35S]methionine-cysteine. Complete subunit heterodimeric integrins were secreted into the medium, as determined by radioimmunoprecipitation with specific monoclonal and polyclonal antibodies. For the examination of the integrins' biological activities, stable cell lines producing the soluble integrins were generated in HEK 293A cells. In the presence of divalent cations, soluble alphaVbeta6 bound to representatives of type A or O viruses, immobilized on plastic dishes, and significantly inhibited viral replication, as determined by plaque reduction assays. In contrast, soluble alphaVbeta3 was unable to bind to immobilized virus of either serotype; however, virus bound to the immobilized integrin, suggesting that FMDV binding to alphaVbeta3 is a low-affinity interaction. In addition, soluble alphaVbeta3 did not neutralize virus infectivity. Incubation of soluble alphaVbeta6 with labeled type A12 or O1 resulted in a significant inhibition of virus adsorption to BHK cells, while soluble alphaVbeta3 caused a low (20 to 30%), but consistent, inhibition of virus adsorption. Virus incubated with soluble alphaVbeta6 had a lower sedimentation rate than native virus on sucrose density gradients, but the particles retained all of their structural proteins and still contained bound integrin and, therefore, were not exhibiting characteristics of a picornavirus A particle.

Animals↗
PubMed2006

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.

Anoikis↗
PubMed2006

Macrophage matrix metalloproteinase-2/-9 gene and protein expression following adhesion to ECM-derived multifunctional matrices via integrin complexation.

Macrophages are commonly observed at the biomaterial-tissue interface and interact with the extracellular matrix (ECM) mainly by integrin receptors to play a critical role in ECM turnover by secreting matrix metalloproteinases (MMPs). To investigate beta1 and beta3 containing integrin-mediated adhesion and subsequent MMP-2/-9 protein and gene expression in human blood-derived monocytes, biofunctional peptides immobilized onto flexible polyethylene glycol (PEG) arms were grafted onto a gelatin-based interpenetrating network (IPN). Adherent monocyte density was dramatically greater in the presence of RGD immobilized onto flexible PEG arms of the gelatin-based IPN. Pretreatment of monocytes with either anti-integrin beta1 or beta3 led to a significant decrease in adherent cell density on RGD-PEG-grafted IPNs. MMP-2 and MMP-9 protein and MMP-9 mRNA expression increased in the presence of IPNs initially, independent of ligand identity. Anti-integrin beta1 or beta3 antibody pretreatment of monocytes led to a general decrease in MMP-2/-9 protein expression. These results demonstrate the importance of beta1 and beta3 containing integrins in mediating monocyte adhesion onto RGD immobilized onto flexible PEG arms of the IPN. The results also reveal that MMP-2/-9 protein and gene expression is influenced by the presence of gelatin and not the ligands immobilized on the PEG arms of the IPN.

Cell Adhesion↗
PubMed1997

A 2.7-kb portion of the 5' flanking region of the murine glycoprotein alphaIIb gene is transcriptionally active in primitive hematopoietic progenitor cells.

The continuous generation of mature blood cells from primitive multipotent progenitor cells requires a highly complex series of cellular events that are still largely unknown. To examine the molecular events associated with the commitment of these hematopoietic progenitor cells to the megakaryocytic lineage, the alpha subunit of the platelet integrin alphaIIb beta3 was used as marker. Despite an abundance of information regarding the role of this integrin in platelet adhesion and aggregation, the mechanisms that control the expression of the genes that code for these proteins are poorly understood and the earliest hematopoietic cell capable of expressing them has not been clearly identified. Thus, a strategy was developed to eradicate, using a conditional toxigene, all the hematopoietic cells capable of expressing the alphaIIb gene in mice. This was achieved by targeting the expression of the gene encoding the herpes simplex virus thymidine kinase (tk), specifically to these cell types, using a 2.7-kb fragment of the 5'-flanking region of the murine alphaIIb gene. Three transgenic lines having 1, 3, and 4 copies of the transgene, respectively were produced and analyzed. Administration of ganciclovir (GCV) to these mice induced a severe thrombocytopenia, which was due to the depletion of the entire megakaryocytic lineage, as shown by bone marrow (BM) culture and electron microscopy analysis. The time required to attain a severe thrombocytopenia was dependent on the level of the expression of the transgene and varied from 7 to 11 days. This condition was completely reversed when GCV treatment was discontinued. Progenitor cell assays showed that the alphaIIb promoter was active in primitive hematopoietic progenitor cells possessing myeloid, erythroid, and megakaryocytic potential and that the transcriptional activity of the promoter decreased progressively as differentiation proceeded towards the erythroid and myeloid lineages.

Animals↗
PubMed2002

A novel 196Leu to Pro substitution in the beta3 subunit of the alphaIIbbeta3 integrin in a patient with a variant form of Glanzmann thrombasthenia.

Glanzmann thrombasthenia (GT) is an inherited disorder where an absence of platelet aggregation is associated with quantitative or qualitative abnormalities of the alphaIIbbeta3 integrin. In rare patients, amino acid substitutions have provided information on the functional significance of specific domains within alphaIIb or beta3. We now report an elderly male GT patient (R.M.) from the south west of France whose platelets possess a small residual expression of alphaIIbbeta3. Furthermore, the integrin failed to undergo the necessary conformational changes following platelet activation to permit the binding of fibrinogen or activation-dependent monoclonal antibodies despite the presence of an RGD-binding site. Screening of the alphaIIb and beta3 genes by PCR-SSCP revealed a heterozygous mutation at position 685 in exon 5 of the beta3 gene leading to a 196Leu to Pro substitution. 196Leu is a highly conserved amino acid of beta3. The other beta3 allele appeared to be silent. This mutation, inherited from his mother and present in other family members with intermediate levels of alphaIIbbeta3, was close to the MIDAS-like domain of beta3, a fact that appears to explain its effect on alphaIIbbeta3 activation and fibrinogen binding.

Aged↗
PubMed2006

Tumor-specific expression of alphavbeta3 integrin promotes spontaneous metastasis of breast cancer to bone.

INTRODUCTION: Studies in xenograft models and experimental models of metastasis have implicated several beta3 integrin-expressing cell populations, including endothelium, platelets and osteoclasts, in breast tumor progression. Since orthotopic human xenograft models of breast cancer are poorly metastatic to bone and experimental models bypass the formation of a primary tumor, however, the precise contribution of tumor-specific alphavbeta3 to the spontaneous metastasis of breast tumors from the mammary gland to bone remains unclear. METHODS: We used a syngeneic orthotopic model of spontaneous breast cancer metastasis to test whether exogenous expression of alphavbeta3 in a mammary carcinoma line (66cl4) that metastasizes to the lung, but not to bone, was sufficient to promote its spontaneous metastasis to bone from the mammary gland. The tumor burden in the spine and the lung following inoculation of alphavbeta3-expressing 66cl4 (66cl4beta3) tumor cells or control 66cl4pBabe into the mammary gland was analyzed by real-time quantitative PCR. The ability of these cells to grow and form osteolytic lesions in bone was determined by histology and tartrate-resistant acid phosphatase staining of bone sections following intratibial injection of tumor cells. The adhesive, migratory and invasive properties of 66cl4pBabe and 66cl4beta3 cells were evaluated in standard in vitro assays. RESULTS: The 66cl4beta3 tumors showed a 20-fold increase in metastatic burden in the spine compared with 66cl4pBabe. A similar trend in lung metastasis was observed. alphavbeta3 did not increase the proliferation of 66cl4 cells in vitro or in the mammary gland in vivo. Similarly, alphavbeta3 is not required for the proliferation of 66cl4 cells in bone as both 66cl4pBabe and 66cl4beta3 proliferated to the same extent when injected directly into the tibia. 66cl4beta3 tumor growth in the tibia, however, increased osteoclast recruitment and bone resorption compared with 66cl4 tumors. Moreover, alphavbeta3 increased 66cl4 tumor cell adhesion and alphavbeta3-dependent haptotactic migration towards bone matrix proteins, as well as their chemotactic response to bone-derived soluble factors in vitro. CONCLUSION: These results demonstrate for the first time that tumor-specific alphavbeta3 contributes to spontaneous metastasis of breast tumors to bone and suggest a critical role for this receptor in mediating chemotactic and haptotactic migration towards bone factors.

Animals↗
PubMed2006

Bacitracin reveals a role for multiple thiol isomerases in platelet function.

The platelet-specific integrin alphaIIb beta3 has endogenous thiol isomerase activity associated with the CXXC motifs within the beta subunit. Using a highly purified form of bacitracin, a thiol isomerase inhibitor, we now provide further evidence of the functional significance of this enzymatic activity in integrin activation. In addition, we demonstrate a role for multiple thiol isomerases in platelet function. This bacitracin prevented platelet aggregation to thrombin and collagen, and directly inhibited alphaIIb beta3 activation, as detected by PAC-1 binding. In parallel, bacitracin inhibited the endogenous thiol isomerase activity of purified alphaIIb beta3 with a 50% inhibitory concentration of 15.5 micromol/l. In order to determine whether the effects of bacitracin are solely mediated by inhibition of integrin enzymatic activity, we examined integrin-independent indices of platelet activation. We found bacitracin inhibited both platelet secretion (CD62P and CD63) and thromboxane (TxA2) production, with complete inhibition at different concentrations. Thus, we demonstrated a role for multiple thiol isomerases in platelet function. Taken together, these studies support a role for the endogenous integrin thiol isomerase activity in activation of alphaIIb beta3 and highlight the novel regulation of platelet function by other, as yet undefined thiol isomerases.

Bacitracin↗
PubMed2000

Snake venom modulators of platelet adhesion receptors and their ligands.

In thrombosis, platelet aggregation is initiated by a specific membrane glycoprotein (GP) Ib-IX-V complex binding to its adhesive ligand, von Willebrand factor, in the matrix of ruptured atherosclerotic plaques or in plasma exposed to high hydrodynamic shear stress. This process closely resembles normal haemostasis at high shear, where GP Ib-IX-V-dependent platelet adhesion to von Willebrand factor in the injured blood vessel wall initiates platelet activation and integrin alphaIIb beta3 (GP IIb-IIIa)-dependent platelet aggregation. At low shear, other receptors such as those that bind collagen, the integrin alpha2beta1 (GP Ia-IIa) or GP VI, mediate platelet adhesion. Recently, snake venom proteins have been identified that selectively modulate platelet function, either promoting or inhibiting platelet aggregation by targeting GP Ib-IX-V, alpha2beta1, GP VI, alphaIIb beta3, or their respective ligands. Interestingly, these venom proteins typically belong to one of two major protein families, the C-type lectin family or the metalloproteinase-disintegrins. This review focuses on recent insights into structure-activity relationships of snake venom proteins that regulate platelet function, and the ways in which these novel probes have contributed in unexpected ways to our understanding of the molecular mechanisms underlying thrombosis.

Amino Acid Sequence↗
PubMed1996

Domain-specific interactions between entactin and neutrophil integrins. G2 domain ligation of integrin alpha3beta1 and E domain ligation of the leukocyte response integrin signal for different responses.

Extracellular matrix proteins activate neutrophils to up-regulate many physiologic functions that are necessary at sites of tissue injury. To elucidate the ligand-receptor interactions that mediate these functions, we examined neutrophil activation by the basement membrane protein, entactin. Entactin is structurally and functionally organized into distinct domains; therefore, we utilized glutathione S-transferase -fusion proteins encompassing its four major domains, G1, G2, E, and G3, to assess interactions between entactin and neutrophil integrin receptors. We show that the E domain, which contains the single RGD sequence of entactin, is sufficient for ligation of the beta3-like integrin, leukocyte response integrin, and signaling for chemotaxis. Moreover, the G2 domain signals for stimulation of Fc receptor-mediated phagocytosis via ligation of alpha3beta1. This receptor-ligand interaction was revealed only after stimulation of neutrophil by immune complexes or phorbol esters. Interestingly, the E domain does not enhance phagocytosis, and the G2 domain is not chemotactic. Furthermore, cleavage of entactin with the matrix metalloproteinase, matrilysin, liberates peptides that retain E domain-mediated chemotaxis and G2 domain-mediated enhancement of phagocytosis. These studies indicate that multiple domains of entactin have the ability to ligate individual integrins expressed by neutrophils and to activate distinct functions.

Antigen-Antibody Complex↗
PubMed1998

Molecular identification of the cross-reacting epitope on alphaM beta2 integrin I domain recognized by anti-alphaIIb beta3 monoclonal antibody 7E3 and its involvement in leukocyte adherence.

The monoclonal antibody (mAb) 7E3 directed to the platelet integrin alphaIIb beta3 was tested for its cross-reactivity with the homologous leukocyte integrin alphaM beta2. Nested recombinant fragments of alphaM I domain were expressed as glutathione S-transferase fusion proteins and analyzed for antibody recognition. In enzyme-linked immunosorbent assay, mAb 7E3 bound alphaM I domain fragments containing the amino-terminal sequence Cys128-Ser172, whereas the carboxyl-terminal region Leu173-Pro291 was ineffective. A synthetic peptide designated R1.1 and duplicating the alphaM sequence G127CPQEDSDIAFLIDGSGSIIPHDF150 bound mAb 7E3. In contrast, the adjacent alphaM region F150RRMKEFVSTVMEQLKKSKTLFS172 or a control peptide with a scrambled R1.1 sequence was not recognized by mAb 7E3. Binding of mAb 7E3 to alphaM I domain blocked monocyte and neutrophil adhesion to immobilized fibrinogen and fibrinogen-dependent leukocyte-endothelium bridging, indistinguishably from bona fide anti-beta2 mAb IB4. In contrast, leukocyte binding to stable transfectants expressing intercellular adhesion molecule-1 was not affected by mAb 7E3. Balloon-mediated injury of iliofemoral arteries in rabbits resulted in prominent deposition of fibrinogen and increased monocyte adhesion to the injured vessel, in a reaction inhibited by mAb 7E3, but unaffected by control mAb 14E11. Through its cross-reactivity between alphaIIb beta3 and alphaM beta2, mAb 7E3 may initiate a new class of integrin antagonists, capable of simultaneously targeting platelet and leukocyte adhesion mechanisms in vascular injury.

Amino Acid Sequence↗
PubMed2000

Distribution of alphavbeta3, alphavbeta5 integrins and the integrin associated protein--IAP (CD47) in human glomerular diseases.

The alphav integrins present on the membrane of numerous cells, mediate attachment to matrix proteins, cell proliferation, migration and survival. We studied the expression of alphav integrinis and CD47 (a beta3 chain integrin associated protein) in various forms of glomerulonephritis (GN) characterized by mesangial proliferation and/or increased mesangial matrix. In normal glomeruli, epithelial cells expressed alphavbeta3, alphavbeta5 and CD47; endothelial cells expressed alpha5beta1 and CD47; mesangial cells expressed alphavbeta5, CD47, and to a less extent alphavbeta3. In acute post infectious GN (APIGN), membrano-proliferative GN (MPGN) and diabetic nephropathy(DN), we observed that the beta3 chain, normally expressed by mesangial cells, was not detectable in the mesangium while its expression by epithelial cells was not modified. Parallel to the disappearance of alphavbeta3, the CD47 expression was decreased on the mesangial cells in MPGN, APIGN and DN. The expression of alphavbeta5 was clearly increased on podocytes and on proliferating mesangial cells in APIGN. By contrast, the mesangial expression of alphavbeta was normal or decreased in DN. The alpha5 chain of integrin, absent on normal mesangial cell, was expressed on proliferating mesangial cells in MPGN and APIGN. Thus, we observed modifications of alphavbeta3 and alphavbeta5 expression during human GN. The modulations of alphavbeta3 and alphavbeta5 expression differed according to the different glomerular cell types and were not parallel in glomerular cells: alphavbeta3 was decreased (and alphavbeta5 unchanged) on proliferating mesangial cells and alphavbeta5 was increased (and alphavbeta3 unchanged) in podocytes. This may reflect the existence of two distinct regulatory pathways.

Antibodies, Monoclonal↗
PubMed2006

Altered expression of disintegrin metalloproteinases and their inhibitor in human dilated cardiomyopathy.

BACKGROUND: Disintegrin metalloproteinases (ADAMs) may contribute to structural cardiac remodeling by altering cell-surface matrix receptors (integrins) and activating potent biomolecules. We compared expression of ADAMs, their endogenous inhibitor tissue inhibitor of metalloproteinases (TIMP)-3, and integrins in human heart tissue with varied patterns of structural remodeling. METHODS AND RESULTS: Myocardium was obtained from patients with dilated cardiomyopathy (n=20), hypertrophic obstructive cardiomyopathy (n=5), and nonfailing donor hearts (n=7). Paired samples (n=10) were obtained before left ventricular assist device insertion and at transplantation. The expressions of ADAM10, ADAM12, ADAM15, and ADAM17, TIMP-3, and integrin receptors beta1D and beta3 were determined by quantitative immunoblotting. Integrin shedding was assessed by the ratio of integrin cleavage products to intact protein abundance. Confocal microscopy was performed. Dilated cardiomyopathy was characterized by increased ADAM10 and ADAM15 expression and reduced TIMP-3 expression. The integrin beta1D cleavage ratio was elevated, indicating receptor shedding. ADAM10 and ADAM15 expressions correlated with the cleavage ratio. ADAM10 colocalized with integrin beta1D by confocal microscopy. ADAM10 expression correlated with clinical indices of chamber dilatation and systolic dysfunction. Hemodynamic unloading reduced ADAM10 and ADAM12 expressions and increased integrin beta1D expression. ADAM12 and integrin beta1D expressions were increased in HOCM. ADAM17 was increased in both dilated cardiomyopathy and hypertrophic obstructive cardiomyopathy. CONCLUSIONS: Disintegrin metalloproteinases are differentially expressed in human myocardium, reflecting the underlying pattern of structural remodeling. ADAM10 and ADAM15 may contribute to cardiac dilatation by reducing cell-matrix interactions via integrin shedding. Targeting disintegrin metalloproteinases, perhaps by restoring deficient TIMP-3 levels with gene or cell-based therapies, may prevent progressive chamber dilatation in human dilated cardiomyopathy.

ADAM Proteins↗