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PubMed1998

VLA-4 is involved in the engraftment of the human pre-B acute lymphoblastic leukaemia cell line NALM-6 in SCID mice.

Attachment of human pre-B leukaemic cells to human or murine bone marrow stromal cells in vitro is largely mediated by the beta1 integrin VLA-4 binding to VCAM-1. Cells subsequently migrate within the stroma, a process also involving VLA-4. A variant of the pre-B acute lymphoblastic leukaemia cell line NALM-6, designated 4A1, lacking expression of VLA-4, was generated by radiation-induced mutagenesis followed by several rounds of negative selection with immunomagnetic beads, fluorescence activated cell sorting and clonal expansion. In vitro assays using 4A1 cells showed reduced binding to, and migration under, the murine stromal line M2-10B4. Sublethally irradiated mice (n=19) with severe combined immunodeficiency were injected intravenously with NALM-6 cells. Animals developed signs of leukaemia with hind-limb paralysis at a median of 30 d (95% confidence interval 28-30). Although there were no gross abnormal findings at autopsy, histological analysis revealed extensive marrow replacement and focal liver infiltration with leukaemic blasts, which were confirmed to be of human origin by flow cytometry. 12 mice were injected with a similar number of cells from the VLA-4-negative variant cell line 4A1. Six mice developed signs of leukaemia after 43-74d, with the remaining six being free of signs of disease after > 100d (P<0.001). Mice in this group with leukaemia had a lower incidence of hind-limb paralysis and less leukaemic infiltration in the marrow, but in some cases had large tumour nodules elsewhere. After a single 500 microg intraperitoneal injection of anti-murine VCAM-1 monoclonal antibody (MK2.7), five additional mice were injected with an identical number of wild-type (VLA-4+) NALM-6. All animals developed signs of leukaemia after a similar period to those injected with wild-type NALM-6 only. These results demonstrate that the beta1 integrin VLA-4 is involved in the engraftment of the pre-B-cell leukaemic cell line NALM-6 in SCID mice, although the interaction with VCAM-1 is unlikely to be the sole explanation.

Animals↗
PubMed2002

H-Ras/mitogen-activated protein kinase pathway inhibits integrin-mediated adhesion and induces apoptosis in osteoblasts.

We have studied the relevance of H-Ras and its downstream effectors to osteoblast functions. 1) Purified human osteoblasts highly expressed integrins beta1, alpha4, alpha5, alpha6 and the activation epitope of beta1. However, these molecules were markedly down-regulated on osteoblasts transfected with expression vector encoding fully activated H-Ras(V12), H-Ras(V12)T35S, activating Raf-1/mitogen-activated protein kinase (MAPK), or an active Raf-1 but not on cells having H-Ras(V12)Y40C, a phosphoinositide 3-kinase (PI3K)-binding mutant. 2) Although osteoblasts spontaneously adhered to fibronectin and laminin in beta1-dependent manner, the expression of H-Ras(V12) or H-Ras(V12)T35S, but not H-Ras(V12)Y40C, in osteoblasts reduced their adhesion. 3) Osteoblasts bearing H-Ras(V12), H-Ras(V12)T35S, or Raf-1 failed to proliferate, whereas those with H-Ras(V12)Y40C proliferated well. (4) The up-regulation of Fas and down-regulation of Bcl-2 were observed in osteoblasts expressing H-Ras(V12), H-Ras(V12)T35S, or Raf-1. (5) Most of the cells having H-Ras(V12), H-Ras(V12)T35S, or Raf-1 became annexin-V(high)/propidium iodide (PI)(high or low) and terminal deoxynucleotidyl-transferase-mediated dUTP nick-end labeling (TUNEL)(high)/PI(low) after 24 and 72 h incubation, respectively. Thus, we propose that H-Ras signals followed by Raf-1/MAPK pathway but not PI3K not only reduces beta(1)-mediated adhesion of osteoblasts to matrix proteins but induces apoptosis presumably via the Fas up-regulation and Bcl-2 down-regulation.

Apoptosis↗
PubMed2005

Platelet glycoprotein I(b)alpha and integrin alpha2 beta1 polymorphisms: gene frequencies and linkage disequilibrium in a population diversity panel.

Genetic variants in the GP1BA and ITGA2 genes have been proposed as potential modifiers for arterial vascular disease and bleeding disorders. Since ancestry may play an important role in the prevalence of these variants, we sought to determine their allele frequency and linkage disequilibrium in a collection of 1064 DNA samples from 51 ethnic groups. We studied haplotypes of ITGA2 defined by single nucleotide substitutions at positions -52, 807, and 1648, and GP1BA variants defined by sequence changes in positions -5 (Kozak), 1018 (T145M, HPA-2) and 1285 (VNTR A, B, C and D). Frequency of haplotypes of ITGA2 showed considerable variation across the different groups, with a higher prevalence of the haplotype -52C or T/807C/1648A observed in African compared with caucasian and Asian populations. The haplotypes 52C/807T/1648A and -52T/807T/1648A were not observed in caucasians or South Americans. While relative frequencies of the GP1BA Kozak alleles were comparable across groups, the methionine allele (HPA-2b) showed a higher frequency in Africa (0.26) than in the other groups. We also observed a high prevalence of the VNTR B allele in the African and Israeli populations. Haplotype analysis revealed incomplete linkage disequilibrium between the HPA-2 and VNTR alleles. Incorporation of GP1BA variants into the set of SNPs already genotyped by the HapMap project disrupted the pre-existing haplotype block. These data provide a valuable resource for optimal selection of variants best tailored for association studies of vascular disease or bleeding disorders when examining individuals of different ancestral origins.

Alleles↗
PubMed2002

Porcine mononuclear cells adhere to human fibronectin independently of very late antigen-5: implications for donor-specific tolerance induction in xenotransplantation.

To combat the shortage of donor organs, transplantation across species barriers has been proposed. Induction of tolerance would overcome the substantial immunologic barriers to xenotransplantation and would avoid the chronic use of immunosuppressive agents. Successful transplantation of hematopoietic cells induces robust specific tolerance to donor antigens in allogeneic and xenogeneic models. The beta1 integrin class of adhesion molecules and their interactions with extracellular matrix components are thought to be integral to the engraftment and maturation of hematopoietic stem cells. We therefore examined the efficacy of porcine very late antigen-5 (VLA-5) and VLA-4 interactions with the human extracellular matrix (ECM) protein, fibronectin. Peripheral blood mononuclear cells (PBMCs) from humans and miniature swine were flourochrome labeled and adhesion to plates coated with whole human fibronectin (whFN) or its 120 KDa fragment containing the VLA-5 binding region was determined. Flow cytometry and immuno- precipitation were used to identify a monoclonal antibody that cross-reacted on porcine VLA-5. Human and pig PBMC adhesion to human fibronectin (hFN) or 120 kDa fragment-coated plates was assessed following incubation with control ab, anti-VLA-4, anti-VLA-5, or soluble fibronectin. Using rabbit complement, cells expressing VLA-5 were purged from PBMC preparations before performing the adhesion assay. Porcine and human PBMC both adhered to hFN in a divalent cation-dependent and activation-dependent manner. Adhesion to hFN of human but not pig PBMC was blocked by anti-VLA-5 monoclonal antibody SAM-1, although this mAb immunoprecipitated a heterodimeric cell surface molecule (155/135 kDa) resembling VLA-5 from pig PBMC. Complement-mediated depletion of VLA-5-expressing cells ablated specific binding of human but not porcine cells to hFN and its 120 kDa fragment. Addition of soluble fibronectin was capable of blocking adhesion of PBMC of both species to hFN. Anti-VLA-4 reduced the binding of PBMC from both species to hFN to a similar extent. Human and pig cells can specifically adhere to hFN and its 120 kDa fragment, suggesting that this critical cell-ECM interaction is preserved across species. While human cells exclusively use VLA-5 for binding to the 120 kDa fragment, porcine cells could not be shown to adhere to whFN or its 120 kDa fragment via VLA-5. However, porcine VLA-4 is capable of mediating adhesion to human FN. We conclude that disparities in the adhesive interactions of beta1 integrins may be a barrier to the use of porcine hematopoietic stem cell transplantation as a means of inducing donor-specific tolerance in the pig to human species combination.

Animals↗
PubMed2004

Enhanced podocalyxin expression alters the structure of podocyte basal surface.

Glomerular basement membrane (GBM) and podocalyxin are essential for podocyte morphology. We provide evidence of functional interconnections between basement membrane components (collagen IV and laminin), the expression of podocalyxin and the morphology of human glomerular epithelial cells (podocytes). We demonstrated that GBM and laminin, but not collagen IV, up-regulated the expression of podocalyxin. Scanning electron microscopy revealed that laminin induced a modified morphology of podocytes with process formation, which was more extensive in the presence of GBM. Under high magnification, podocytes appeared ruffled. Using transmission electron microscopy we observed that raised areas occurred in the basal cell surface. Furthermore, the presence of anti-podocalyxin antibody increased the extent of adhesion and spreading of podocytes to both collagen IV and laminin, thus podocalyxin apparently inhibits cell-matrix interactions. We also performed adhesion and spreading assays on podocytes grown under increased glucose concentration (25 mM). Under these conditions, the expression of podocalyxin was almost totally suppressed. The cells adhered and spread to basement membrane components but there was no increase in the extent of adhesion and spreading in the presence of anti-podocalyxin antibody, or ruffling of the cell edges. Additionally, in podocytes expressing podocalyxin, the presence of anti-podocalyxin antibody partially reversed the inhibition of adhesion to collagen IV provoked by anti-beta1 integrin antibody, thus podocalyxin should compete with beta1-related cell adhesion. We suggest that the observed podocalyxin-mediated inhibition of binding to the matrix could be in part responsible for the specialized conformation of the basal surface of podocytes.

Amino Acids, Diamino↗
PubMed1999

Phenotypical and functional properties of human bone marrow mesenchymal progenitor cells.

Bone marrow stroma provides the microenvironment for hematopoiesis and is also the source of mesenchymal progenitors (mesenchymal or marrow stromal cells [MSC]) that may serve as long-lasting precursors for bone, cartilage, lung, and muscle. While several studies have indicated the differentiation potential of MSC, few studies have been performed on the cells themselves. In an attempt to further expand our knowledge on these cells, we have performed studies on their cell cycle, immuno- and adhesive-phenotype, ex vivo expansion, and differentiation properties. MSC cultures have been initiated from human bone marrow low-density mononuclear cells and maintained in the absence of differentiation stimuli and hematopoietic cells. The homogenous layer of adherent cells thus formed exhibits a typical fibroblastlike morphology, a population doubling time of 33 h, a large expansive potential, and cell cycle characteristics including a subset (20%) of quiescent cells. The antigenic phenotype of MSC is not unique, borrowing features of mesenchymal, endothelial, and epithelial cells. Together, MSC express several adhesion-related antigens, like the integrin subunits alpha4, alpha5, beta1, integrins alphavbeta3 and alphavbeta5, ICAM-1, and CD44H. MSC produce and functionally adhere to extracellular matrix molecules. When incubated under proper stimuli, MSC differentiate into osteoblasts or adipocytes. Taken together, these results demonstrate that adherent marrow-derived cells cultured in the absence of hematopoietic cells and differentiation stimulus give rise to a population of cells with phenotypical and functional features of mesenchymal progenitors. The existence of a subset of quiescent cells in MSC cultures seems to be extremely significant, since their number and properties should be enough to sustain a steady supply of cells that upon proliferation and commitment may serve as precursors for a number of nonhematopoietic tissues.

Adipocytes↗
PubMed1999

Tenascin-C inhibits beta1 integrin-dependent T lymphocyte adhesion to fibronectin through the binding of its fnIII 1-5 repeats to fibronectin.

The extracellular matrix consists of different proteins interacting to form a meshwork-like structure. T lymphocyte adhesion to individual matrix proteins is mainly regulated at the adhesion receptor level, but it is conceivable that the composition of the matrix itself may affect T lymphocyte adhesion to individual proteins. We have addressed the latter point by studying the effect of the matrix protein tenascin-C (TN-C) on T lymphocyte adhesion to fibronectin. Here we report that TN-C inhibits adhesion of T lymphocytes and MOLT-4 lymphoma cells to fibronectin. We demonstrate that a TN-C fragment consisting of fibronectin type III repeats 1-5 (TNfnIII 1-5) but not TNfnIII A-D and TNfnIII 6-8 inhibited alpha5beta1 and alpha4beta1 integrin-mediated T lymphocyte and MOLT-4 adhesion to fibronectin. At concentrations that did not inhibit adhesion, TNfnIII 1-5 still prevented MOLT-4 cells from spreading on fibronectin. Preincubation and co-immobilization of TNfnIII 1-5 with fibronectin was more effective in inhibiting MOLT-4 adhesion to fibronectin than soluble TNfnIII 1-5 present during the adhesion test. Using an enzyme-linked immunosorbent assay we could demonstrate binding of TNfnIII 1-5 to fibronectin and fibronectin fragments. Taken together, these data demonstrate that the TNfnIII 1-5 domain is implicated in the inhibition of T lymphocyte adhesion to fibronectin caused by TN-C, and indicate that this effect involves the binding of TN-C repeats TNfnIII 1-5 to fibronectin.

Binding Sites↗
PubMed2002

NG2 proteoglycan mediates beta1 integrin-independent cell adhesion and spreading on collagen VI.

Collagens V and VI have been previously identified as specific extracellular matrix (ECM) ligands for the NG2 proteoglycan. In order to study the functional consequences of NG2/collagen interactions, we have utilized the GD25 cell line, which does not express the major collagen-binding beta(1) integrin heterodimers. Use of these cells has allowed us to study beta(1) integrin-independent phenomena that are mediated by binding of NG2 to collagens V and VI. Heterologous expression of NG2 in the GD25 line endows these cells with the capability of attaching to surfaces coated with collagens V and VI. The specificity of this effect is emphasized by the failure of NG2-positive GD25 cells to attach to other collagens or to laminin-1. More importantly, NG2-positive GD25 cells spread extensively on collagen VI. beta(1) integrin-independent extension of ruffling lamellipodia demonstrates that engagement of NG2 by the collagen VI substratum triggers signaling events that lead to rearrangement of the actin cytoskeleton. In contrast, even though collagens V and VI each bind to the central segment of the NG2 ectodomain, collagen V engagement of NG2 does not trigger cell spreading. The distinct morphological consequences of NG2/collagen VI and NG2/collagen V interaction indicate that closely-related ECM ligands for NG2 differ in their ability to initiate transmembrane signaling via engagement of the proteoglycan.

Animals↗
PubMed2006

Regulation of phosphatidylinositol 3-kinase (PI3K)/Akt and nuclear factor-kappa B signaling pathways in dystrophin-deficient skeletal muscle in response to mechanical stretch.

Phosphatidylinositol 3-kinase (PI3K)/Akt and nuclear factor-kappa B (NF-kappaB) signaling pathways play a critical role in mediating survival signals. In this study we have investigated how loss of dystrophin (the primary cause of Duchenne muscular dystrophy) modulates the activation of PI3K/Akt and NF-kappaB signaling pathways in skeletal muscle in response to mechanical stimulation. Activation of Akt was significantly higher in diaphragm muscle from dystrophin-deficient mdx mice compared to normal mice at both prenecrotic and necrotic states. Higher activation of Akt was also observed in cultured dystrophin-deficient primary myotubes differentiated in vitro. Application of passive mechanical stretch ex vivo synergistically increased the activation of Akt in diaphragm of mdx mice. Stretch-induced activation of PDK-1 and PI3K were also higher in diaphragm of mdx mice compared to normal mice. Pretreatment of diaphragm with PI3K inhibitor LY294002 blocked the activation of Akt in normal and mdx mice. Higher activation of Akt was associated with increased phosphorylation of its downstream targets glycogen synthase kinase 3beta (GSK3beta), FKHR, and mammalian target of rapamycin (mTOR). Treatment of diaphragm muscle with LY294002 inhibited the stretch-induced activation of IkappaB (IkappaB) kinase (IKK) and NF-kappaB transcription factor in normal and mdx mice. Mechanical stretch also reduced the interaction of HDAC1 with RelA subunit of NF-kappaB in diaphragm muscle. Finally, cellular levels of Bcl-2, cIAP1, and integrin beta1 and activation of integrin linked kinase were higher in diaphragm muscle of mdx mice compared to normal mice. Taken together, our data suggest that loss of dystrophin and/or mechanical stretch results in the up-regulation of P13K/Akt and NF-kappaB signaling pathways in skeletal muscle.

Animals↗
PubMed1996

Tumor gangliosides enhance alpha2 beta1 integrin-dependent platelet activation.

Gangliosides, sialic acid-containing glycosphingolipids, enhance platelet adhesion to collagen and consequent platelet activation. For example, gangliosides shed by neuroblastoma tumor cells (NBTG) added to a subthreshold (non-activating) concentration (1 microgram/ml) of collagen, cause platelet aggregation (59 +/- 10%) and ATP release (2.3 +/- 0.2 nmol) equivalent to that caused by 10 micrograms/ml collagen alone. Here we report further studies to characterize this effect. Platelet aggregation and ATP release were not induced by NBTG in combination with subthreshold concentrations of adenosine diphosphate, epinephrine, thrombin or arachidonic acid, suggesting that NBTG specifically influences collagen-mediated platelet activation. Maximal platelet aggregation and ATP release required extracellular magnesium and only a short (1 min) preincubation with NBTG, suggesting a collagen receptor-mediated mechanism of this ganglioside activity. Since gangliosides interact with several integrin receptors, we determined whether NBTG influences alpha 2 beta 1, a major integrin collagen receptor on platelets. Incubation of platelets with a monoclonal antibody directed against the alpha 2 chain (5E8) blocked the increase in platelet aggregation (9 +/- 3% vs. 80 +/- 2%) and ATP release ( < 0.2 vs 2.5 +/- 0.1 nmol) induced by NBTG and 1 microgram/ml collagen. Incubation with an antibody to the non-integrin collagen receptor, CD36, or with an isotype control antibody did not abrogate the effect of NBTG. Finally, NBTG and its major component, GD2, enhanced alpha 2 beta 1-mediated platelet adhesion to immobilized collagen in an antibody 5E8-inhibitable manner. These findings implicate the alpha 2 beta 1-collagen interaction as a target of the effect of tumor-derived gangliosides.

Cations, Divalent↗
PubMed1999

Functional cloning of ARM-1, an adhesion-regulating molecule upregulated in metastatic tumor cells.

Interactions of tumor cells with the endothelium and tissue stroma are considered to be critical steps in metastasis formation and progression of cancer. To identify cellular receptors that mediate the binding of tumor cells to endothelium, a murine T cell lymphoma-derived expression library was screened for adhesion-inducing cDNA clones. We identified a novel cell adhesion-promoting molecule, termed ARM-1 (adhesion regulating molecule-1), which is homologous to a human Mr 110,000 tumor-associated antigen. The ARM-1 cDNA codes for a type I transmembrane protein of 407 amino acids with potential O- and N-glycosylation sites that does not belong to any of the known families of cell adhesion molecules. Overexpression of ARM-1 in 293T human embryonic kidney cells significantly increased adhesion to different endothelial cells. ARM-1 expression in 293T cells did not alter integrin expression or beta1-integrin-mediated cell adhesion. Northern blot analysis of human breast cancer cell lines revealed 3- to 5-fold elevated ARM-1 mRNA levels in metastatic as compared to non-metastatic cells. In conclusion, we have identified ARM-1 as a novel cell adhesion-promoting receptor that is upregulated in metastatic cancer cells.

Amino Acid Sequence↗
PubMed2006

Plasma-membrane-associated sialidase (NEU3) differentially regulates integrin-mediated cell proliferation through laminin- and fibronectin-derived signalling.

We have found previously that human plasma-membrane-associated sialidase (NEU3), a key glycosidase for ganglioside degradation, was markedly up-regulated in human colon cancers, with an involvement in suppression of apoptosis. To elucidate the molecular mechanisms underlying increased NEU3 expression, in the present study we investigated its role in cell adhesion of human colon cancer cells. DLD-1 cells transfected with NEU3 exhibited increased adhesion to laminins and consequent cell proliferation, but decreased cell adhesion to fibronectin and collagens I and IV, compared with control cells. When triggered by laminins, NEU3 clearly stimulated phosphorylation of FAK (focal adhesion kinase) and ERK (extracellular-signal-regulated kinase), whereas there was no activation on fibronectin. NEU3 markedly enhanced tyrosine phosphorylation of integrin beta4 with recruitment of Shc and Grb-2 only on laminin-5, and NEU3 was co-immunoprecipitated by an anti-(integrin beta4) antibody, suggesting that association of NEU3 with integrin beta4 might facilitate promotion of the integrin-derived signalling on laminin-5. In addition, the promotion of phosphorylation of integrin beta1 and ILK (integrin-linked kinase) was also observed on laminins. G(M3) depletion as the result of NEU3 overexpression, assessed by TLC, appeared to be one of the causes of the increased adhesion on laminins and, in contrast, of the decreased adhesion on fibronectin - NEU3 probably having bimodal effects. These results indicate that NEU3 differentially regulates cell proliferation through integrin-mediated signalling depending on the extracellular matrix and, on laminins, NEU3 did indeed activate molecules often up-regulated in carcinogenesis, which may cause an acceleration of the malignant phenotype in cancer cells.

Cell Adhesion↗
PubMed1998

Interactions of dendritic cells with fibronectin and endothelial cells.

We studied the phenotypic characteristics of spontaneously migrated skin dendritic cells (sDC) and monocyte-derived dendritic cells (moDC), generated under different culture conditions, and their interactions with fibronectin (FN) and endothelial cells. Monocyte-derived dendritic cells were obtained after culturing monocytes with granulocyte-macrophage colony-stimulating factor (GM-CSF) (800 U/ml) and interleukin-4 (IL-4) (500 U/ml) with either 10% fetal bovine serum (FBS) or 10% allogeneic human serum (HS). Regardless of the type of serum used, the majority of moDC expressed human leucocyte antigen-DR (HLA-DR) and CD86. On day 5 of incubation, 20-67% of moDC cultured in the presence of HS (HS-moDC) expressed CD1a, b and c versus 94-97% when cultured in the presence of FBS (FBS-moDC). DC showed a differential gradient of adhesion to FN: FBS-moDC>HS-moDC>sDC approximately monocytes. Both FBS-moDC and HS-moDC were strongly positive for CD49e (alpha5-integrin) and CD29 (beta1-integrin) but negative for CD49d (alpha4-integrin). A monoclonal antibody (mAb) against CD49e blocked the adhesion of both types of moDC to FN. Although both FBS-moDC and HS-moDC attached to endothelium (a 76% and 63% increase, respectively), only HS-moDC were able to migrate through non-activated endothelium. Overall, these results suggest that spontaneously migrated sDC are less adherent to FN than moDC, that HS and FBS induce differences in CD1 expression, that HS-moDC are less adhesive to FN and endothelial cells but more motile than FBS-moDC, and that alpha5beta1-integrin is the molecule involved in moDC adhesion to FN.

Animals↗
PubMed2004

AlphaPIX associates with calpain 4, the small subunit of calpain, and has a dual role in integrin-mediated cell spreading.

Binding of integrins to the extracellular matrix results in actin cytoskeletal rearrangements, e.g. during cell spreading, by regulating the activity of Rho GTP-ases. We have shown previously that alphaPIX (Cool-2 or ARHGEF6), a Rac1/Cdc42-specific guanine nucleotide exchange factor (GEF), binds to beta-parvin/affixin and colocalizes with integrin-linked kinase in actively spreading cells, suggesting that alphaPIX is involved in integrin-induced signaling leading to activation of Rac1/Cdc42. Here we report calpain 4, the small subunit of the proteases mu-calpain and m-calpain, as a novel binding partner of alphaPIX. This association was identified by the CytoTrap system and confirmed by coimmunoprecipitation and glutathione S-transferase pull-down assays. The alphaPIX triple domain SH3-DH-PH was found to be required for calpain 4 binding. During integrin-dependent spreading of CHO-K1 cells, alphaPIX colocalized with mu- and m-calpain, integrin-linked kinase, and beta1 integrin in early integrin-containing clusters. Overexpression of alphaPIX wild type but not the GEF-deficient mutant (L386R/L387S) resulted in enhanced formation of characteristic cellular protrusions during cell spreading, suggesting that alphaPIX GEF activity is necessary for this specific actin cytoskeletal reorganization. The calpain inhibitors calpeptin and calpain inhibitor IV significantly inhibited integrin-dependent cell spreading. However, concomitant overexpression of alphaPIX wild type or the L386R/L387S mutant restored cell spreading. Together, these data suggest that alphaPIX is a component of early integrin clusters and plays a dual role in integrin-dependent cell spreading. Whereas alphaPIX GEF activity contributes to enhanced formation of cellular protrusions, the GEF-independent association with calpain 4 leads to induction of a yet unknown signaling cascade resulting in cell spreading.

Amino Acid Substitution↗
PubMed1998

Integrin alphavbeta3 requirement for sustained mitogen-activated protein kinase activity during angiogenesis.

Angiogenesis depends on growth factors and vascular cell adhesion events. Integrins and growth factors are capable of activating the ras/MAP kinase pathway in vitro, yet how these signals influence endothelial cells during angiogenesis is unknown. Upon initiation of angiogenesis with basic fibroblast growth factor (bFGF) on the chick chorioallantoic membrane (CAM), endothelial cell mitogen-activated protein (MAP) kinase (ERK) activity was detected as early as 5 min yet was sustained for at least 20 h. The initial wave of ERK activity (5-120 min) was refractory to integrin antagonists, whereas the sustained activity (4-20 h) depended on integrin alphavbeta3, but not beta1 integrins. Inhibition of MAP kinase kinase (MEK) during this sustained alphavbeta3-dependent ERK signal blocked the formation of new blood vessels while not influencing preexisting blood vessels on the CAM. Inhibition of MEK also blocked growth factor induced migration but not adhesion of endothelial cells in vitro. Therefore, angiogenesis depends on sustained ERK activity regulated by the ligation state of both a growth factor receptor and integrin alphavbeta3.

Animals↗
PubMed2001

[Phenotyping of chondrocytes from human osteoarthritic cartilage: chondrocyte expression of beta integrins and correlation with anatomic injury]

Chondrocyte-ECM (extracellular matrix) interactions are believed to play a pivotal role in the development and metabolic homeostasis of articular cartilage. Cell surface adhesion molecules have been reported to modulate chondrocyte binding to ECM (collagen, fibronectin, laminin) and they also act as transducers of critical signals in many biological processes such as growth, differentiation, migration and matrix synthesis. Recently, it has been shown that normal human articular chondrocytes strongly express beta1 integrins, which are constituted by a common chain (beta1) and a variable alphachain, but the behaviour of these molecules in human osteoarthritic cartilage has not been extensively investigated. We studied the expression of beta integrins (beta1-5, alpha1-6, av chains), LFA-1, ICAM-1 and CD44, on freshly isolated chondrocytes obtained from 10 osteoarthritic patients undergoing surgical knee replacement. Chondrocytes were isolated by enzymatic digestion from three zones of each articular cartilage with a differing degree of macroscopic and microscopic damage. Integrin expression and cell cycle analysis were carried out by flowcytometry. Chondrocytes from costal cartilages of 5 human foetuses were also studied. Chondrocytes from osteoarthritic cartilage expressed high levels of beta1 integrin and, at different percentages, all the alphachains. The alphachain most frequently expressed was alpha1, foilowed by alpha3, alpha5, alpha2, alphav. Integrin expression decreased from the least to the most damaged zone of articular cartilage and cell cycle analysis showed that proliferating chondrocytes (S phase) were prevalent on the latter zone. beta2, beta3, beta2, beta5, CD44, LFA-1/ICAM-1 complex were very low expressed. Fetal chondrocytes strongly expressed beta1 and beta5 chains. These data provide evidence to show that integrin expression on human chondrocytes changes in osteoarthritis and suggest that perturbations of chondrocyte-ECM signalling occur in the development of the disease. The different pattern of expression of beta1 and beta5 chains on adult and fetal chondrocytes leads to speculate that integrins play a key role in control of cartilage morphogenesis and differentiation.

Journal Article↗
PubMed1998

Influence of adhesion molecule expression by human brain microvessel endothelium on cancer cell adhesion.

Cultures of endothelial (En) cells derived from human brain microvessels were established in order to characterize adhesion molecule expression and to assay the adhesion properties of neoplastic cell lines to monolayers of En cells. Low constitutive expression of beta1 integrin (CD29), and ICAM-2 (CD102) was detected on human brain microvessel En cells. The beta1 chain of the VLA integrin family, ICAM-1, E-selectin (CD62E) and VCAM-1 (CD106) but not ICAM-2 and PECAM-1 (CD31) expression was upregulated by IL1-alpha, and TNF-alpha proinflammatory cytokines. High expression of PECAM-1 was found on non-activated human brain EN cells. In order to study the potential role of adhesion molecules in neoplastic cell adhesion two tumor cell lines were chosen. Adhesion of a cell line (DU145) derived from a cerebral metastasis of prostate carcinoma to human brain microvessel En cell monolayers was less pronounced compared to adhesion of a primary prostate carcinoma cell line (ND1). Adhesion of cerebral metastatic neoplastic cell line (DU145) was not significantly influenced by incubation of endothelial cells with different proinflammatory cytokines. The adhesion capability of primary prostate carcinoma line (NDI) was significantly upregulated by TNF-alpha proinflammatory cytokine. Furthermore, the adhesion of ND1 was partly inhibited using anti-E-selectin and VCAM-1 monoclonal antibodies. There was no significant effect of anti-adhesion antibodies on the adhesion characteristics of the cerebral metastatic (DU145) cell line. Our data demonstrate that different mechanisms are involved in the adhesion of neoplastic cells to cerebral En cells and turn our attention to the importance of adhesion molecule expression in the formation of metastases.

Antibodies, Monoclonal↗
PubMed2000

Mobilization of CD34+ haematopoietic stem cells is associated with a functional inactivation of the integrin very late antigen 4.

The beta1 integrin very late antigen 4 (VLA-4) plays a central role in mobilization and homing of CD34+ cells. In this study, we examined the activation state of VLA-4 on CD34+ cells from bone marrow (BM) and peripheral blood (PB) by flow cytometry using a vascular cell adhesion molecule I-immunoglobulin (VCAM-I/IgG) fusion protein as soluble ligand. In an intraindividual analysis, we found a significantly reduced affinity and avidity of the VLA-4 receptor on CD34+ cells from PB during granulocyte colony-stimulating factor (G-CSF)-enhanced marrow recovery in comparison with steady-state BM. Moreover, the amount of circulating CD34+ cells during marrow recovery was inversely related to the activation state but not to the expression level of VLA-4, suggesting that a modulation of the functional state of VLA-4 is involved in the mobilization of CD34+ cells. Moreover, VLA-4 function on CD34+ cells from BM was associated with the maturation state of CD34+ cells as high-affinity VLA-4 receptors were observed on the vast majority of more primitive CD34+ cells. In addition, we found that Mg2+ ions as well as co-incubation of CD34+ cells with endothelial cells resulted in an activation of the VLA-4 receptor. In conclusion, modulation of the functional state of VLA-4 appears to be of relevance for the mobilization and homing of CD34+ haematopoietic stem cells.

Antigens, CD34↗