Biomedical subjects

M A Horton

Publications and source records attributed to M A Horton.

At least 37 records · Page 2Linked to original sources

PubMed2000

Divergent effects of platelet-endothelial cell adhesion molecule-1 and beta 3 integrin blockade on leukocyte transmigration in vivo.

The final stage in the migration of leukocytes to sites of inflammation involves movement of leukocytes through the endothelial cell layer and the perivascular basement membrane. Both platelet-endothelial cell adhesion molecule-1 (PECAM-1/CD31) and the integrin alphavbeta3 have been implicated in this process, and in vitro studies have identified alphavbeta3 as a heterotypic ligand for PECAM-1. In the present study we have addressed the roles of these molecules by investigating and comparing the effects of PECAM-1 and alphavbeta3 blockade on leukocyte migration in vivo. For this purpose we have examined the effects of neutralizing Abs directed against PECAM-1 (domain 1-specific, mAb 37) and beta3 integrins (mAbs 7E3 and F11) on leukocyte responses in the mesenteric microcirculation of anesthetized rats using intravital microscopy. The anti-PECAM-1 mAb suppressed leukocyte extravasation, but not leukocyte rolling or firm adhesion, elicited by IL-1beta in a dose-dependent manner (e.g., 67% inhibition at 10 mg/kg 37 Fab), but had no effect on FMLP-induced leukocyte responses. Analysis by electron microscopy suggested that this suppression was due to an inhibition of neutrophil migration through the endothelial cell barrier. By contrast, both anti-beta3 integrin mAbs, 7E3 F(ab')2 (5 mg/kg) and F11 F(ab')2 (5 mg/kg), selectively reduced leukocyte extravasation induced by FMLP (38 and 46%, respectively), but neither mAb had an effect on IL-1beta-induced leukocyte responses. These findings indicate roles for both PECAM-1 and beta3 integrins in leukocyte extravasation, but do not support the concept that these molecules act as counter-receptors in mediating leukocyte transmigration.

Abciximab↗

PubMed2000

New technologies in scanning probe microscopy for studying molecular interactions in cells.

Atomic force microscopy (AFM) is a specialised form of scanning probe microscopy, which was invented by Binnig and colleagues in 1986. Since then, AFM has been increasingly used to study biomedical problems. Because of its high resolution, AFM has been used to examine the topography or shape of surfaces, such as during the molecular imaging of proteins. This, combined with the ability to operate under known force regimes, makes AFM technology particularly useful for measuring intermolecular bond forces and assessing the mechanical properties of biological materials. Many of the constraints (e.g. complex instrumentation, slow acquisition speeds and poor vertical range) that previously limited the use of AFM in cell biology are now beginning to be resolved. Technological advances will enable AFM to challenge both confocal laser scanning microscopy and scanning electron microscopy as a method for carrying out three-dimensional imaging. Its use as both a precise micro-manipulator and a measurement tool will probably result in many novel and exciting applications in the future. In this article, we have reviewed some of the current biological applications of AFM, and illustrated these applications using studies of the cell biology of bone and integrin-mediated adhesion.

Journal Article↗

PubMed2000

Adapting atomic force microscopy for cell biology.

We present details of our AFM modifications to produce an adaptable imaging system for the cell biologist. We have designed and validated a new inverted microscope interface and a scan head with increased Z-range, based upon the TopoMetrix Explorer AFM. We have utilised these changes, together with home-made glass ball cantilevers, to obtain topographical information over cells with large Z-dimension (over 15 microm high), and mapped calcitonin-calcitonin receptor binding forces in living bone cells. We conclude that modified AFM can be used to evaluate intermolecular events in living cells and that this approach will ensure general application to the study of receptor-ligand interactions under truly physiological conditions.

Animals↗

PubMed2000

An antisense oligonucleotide targeting the alphaV integrin gene inhibits adhesion and induces apoptosis in breast cancer cells.

The aim of this study was to show the anti-adhesive potential of an antisense oligodeoxynucleotide (ODN) approach when designed to suppress the cellular function of the alphaV integrin subunit in breast cancer cells. The alphaV integrins play major roles in favouring breast cancer spreading. In this study, we inhibited alphaV subunit synthesis in the human breast carcinoma cell line, MDA-MB231, by a partially phosphorothioated antisense oligodeoxynucleotide (5543-ODN). The alphaV antisense 5543-ODN reduced alphaV, but not actin, mRNA transcription and protein expression by 55% and 65% respectively (1 microM, 72 h). Control sense and mismatch reagents were inactive. The antisense, but not the sense and mismatch, 5543-ODN induced dose- and time-dependent inhibition of MDA-MB231 adhesion to serum, vitronectin, fibrinogen and fibronectin substrates but was inactive on adhesion to laminin. Thus, the alphaV integrin was located in adhesion structures, which were disrupted by treatment with the alphaV antisense 5543-ODN. Antisense treated cells also showed evidence of programmed cell death with the appearance of apoptotic bodies. MDA-MB231 cells express a mutant form of the pro-apoptotic factor p53; however, no changes in the expression of p53 were observed by Western blotting. Immunofluorescence did reveal an increased nuclear translocation of p53 suggesting activation of the protein, but such a translocation did not lead to significant changes in either the expression of the cyclin dependent kinase inhibitor, p21(WAF1/CIP1) the cell survival factor Bcl-2 or the pro-apoptotic factor Bax.

Antigens, CD↗

PubMed2000

Vasoactive intestinal peptide regulates osteoclast activity via specific binding sites on both osteoclasts and osteoblasts.

Clinical and experimental observations, together with immunohistochemical findings, suggest that neuro-osteogenic interactions may occur in the skeleton. In this study, we have examined the effect of vasoactive intestinal peptide (VIP), one of the neuropeptides present in bone, on the activity of the bone-resorbing osteoclast. Effects on bone resorption were assessed by counting the number of pits formed by rat osteoclasts incubated on devitalized slices of bovine cortical bone. Under conditions with an initially sparse density of stromal cells/osteoblasts, VIP caused a rapid cytoplasmic contraction and decreased motility of osteoclasts. This was coupled with a decrease in the number of resorption lacunae and a decrease in the total area resorbed by the osteoclasts in 48-h cultures. Time-course experiments revealed that the inhibitory effects on contraction and motility were transient and that the cells gradually regained their activity, such that, when culture time was prolonged to 120 h, a stimulatory effect by VIP on bone resorption was observed. When osteoclasts were incubated on bone slices, in the presence of an initially large number of stromal cells/osteoblasts, VIP treatment increased the number of resorption pits and total bone area resorbed in 48-h cultures. Using atomic force microscopy, we provide direct evidence that both osteoclasts and stromal cells/osteoblasts bind VIP. Also, VIP was shown to cause a rapid rise of intracellular calcium in osteoclasts and in a proportion (20%) of stromal cells/osteoblasts. Taken together, these data suggest that differentiated osteoclasts are equipped with receptors for VIP that are linked to a transient inhibition of osteoclast activity and, in addition, that stromal cells/osteoblasts have VIP receptors coupled to a delayed stimulation of osteoclastic resorption.

Animals↗

PubMed2000

A new specialized cell-matrix interaction in actively resorbing osteoclasts.

We have identified a novel cell-matrix interaction in activated osteoclasts. Resorbing osteoclasts maintain a barrier adjacent to the bone surface that prevents the leakage of secreted protons and proteases from the resorption area. Using a series of fluorescent dyes of known molecular mass and different surface charge we established that negatively charged molecules with M(r )up to 10,000 rapidly accumulate underneath actively resorbing osteoclasts. Live cell imaging shows that staining could be detected underneath the osteoclasts as early as 30 seconds after the addition of the low molecular mass markers. We provide evidence that the actin cytoskeleton and the adhesion substrate in contact with the cells are critically involved in the maintenance of the sealing barrier. These data taken together suggest that the accumulation under resorbing osteoclasts is by diffusion rather than transcytotic delivery. Our results indicate that the net concentration of secreted and resorbed components is a balance between generation rate and limited diffusion rather than the presence of an impermeable barrier as previously suggested. This dynamic osteoclast sealing zone may, thus, provide the mechanism by which osteoclast migration and resorption can occur simultaneously.

Animals↗

PubMed2000

Study of the nonresorptive phenotype of osteoclast-like cells from patients with malignant osteopetrosis: a new approach to investigating pathogenesis.

Osteopetrosis manifests as failure of osteoclastic bone resorption. The cause of the disease lies either in the hematopoietic lineage or in the bone marrow stromal microenvironment. It has not been possible to define the cell type involved in the various forms of the human disease because of the inability to form human osteoclasts in vitro. Using the recently described method for generating human osteoclasts from peripheral blood in coculture with rat osteoblastic UMR 106 cells, we demonstrate that a defect lies in the mature osteoclast-like cells in four cases of this disease. Control and osteopetrotic cocultures generated large numbers of osteoclast-like cells (calcitonin and vitronectin receptor positive, and F-actin ring-positive cells) with similar morphology. Bone resorption did not occur in three of the four osteopetrotic cultures. In case 1, in which bone resorption was identified, the area of resorption was negligible compared with the number of osteoclast-like cells in the culture and was detected only by scanning electron microscopy. In contrast, up to 20% of the bone surface in controls was resorbed. The normal and osteopetrotic osteoclast-like cells had a similar phenotype except that two of the osteopetrotic cases did not express CD44 and two expressed CD44 weakly, whereas CD44 was strongly expressed in the controls. This study shows that it is possible to reproduce in vitro the pathological features of human osteopetrosis, and the assay provides a means of acquiring a greater understanding of the pathogenesis of human osteopetrosis.

Animals↗

PubMed1999

Single integrin molecule adhesion forces in intact cells measured by atomic force microscopy.

Cross-talk between cells and the extracellular matrix is critically influenced by the mechanical properties of cell surface receptor-ligand interactions; these interactions are especially well defined and regulated in cells capable of dynamically modifying their matrix environment. In this study, attention was focused on osteoclasts, which are absolutely dependent on integrin extracellular matrix receptors in order to degrade bone; other bone cells, osteoblasts, were used for comparison. Integrin binding forces were measured in intact cells by atomic force microscopy (AFM) for several RGD-containing (Arg-Gly-Asp) ligands and ranged from 32 to 97 picoNewtons (pN); they were found to be cell and amino acid sequence specific, saturatable and sensitive to the pH and divalent cation composition of the cellular culture medium. In contrast to short linear RGD hexapeptides, larger peptides and proteins containing the RGD sequence, such as osteopontin (a major non-collagenous bone protein) and echistatin (a high affinity RGD sequence containing antagonist snake venom protein), showed different binding affinities. This demonstrates that the context of the RGD sequence within a protein has considerable influence upon the final binding force for receptor interaction. These data also demonstrate that AFM, as a methodological approach, can be adapted to cell biology studies wherever cell-matrix interactions play a critical role, and, moreover, may have applicability to the analysis of receptor-ligand interactions in cell membranes in general.

Animals↗

PubMed1999

Beta1 integrin antisense oligodeoxynucleotides: utility in controlling osteoclast function.

The involvement of beta1 integrins in osteoclast function has been investigated by utilising an antisense oligodeoxynucleotide (ODN) approach. 18-mer antisense and control phosphorothioate ODNs were made to a conserved internal region of beta1 integrin sequence (nucleotide positions 1634-1651 of the human beta1 fibronectin receptor). These were tested on rabbit osteoclasts for anti-adhesive and resorptive effects mediated by alphaVbeta3 and alpha2beta1, the major integrins of osteoclasts. Antisense, but not control, beta1 ODNs inhibited osteoclast adhesion to collagen-coated glass (by up to 70%), but not to glass coated with vitronectin, fibronectin or fibrinogen. Adhesion to dentine and subsequent resorption were also inhibited (up to 60%) in a sequence-specific manner. The mechanism of action was verified using both a melanoma cell line, DX3, which expresses multiple integrins at high level including alphaVbeta3 and alpha2beta1, and in a rabbit osteoclast marrow culture (BMC) system. Exposure of DX3 cells to antisense ODN for up to 48 hours reduced adhesion to FCS- and collagen-coated glass, and concomitantly inhibited beta1 protein expression assessed by FACS and Western blot analysis; expression of other integrin subunits, alphaV and beta3, was unaffected. Similarly, the beta1 protein levels in the BMC were reduced by > 75% without any effect on actin expression. These data reveal the utility of antisense ODNs in exploring osteoclast biology and further define the functional role of osteoclastic beta1 integrin(s).

Animals↗

PubMed1999

Oligodeoxynucleotide targeted to the alphav gene inhibits alphav integrin synthesis, impairs osteoclast function, and activates intracellular signals to apoptosis.

The alphav integrin subunit is highly expressed in osteoclasts where it dimerizes with beta1 and beta3 subunits to form receptors for vitronectin and bone sialoproteins. Inhibition of osteoclast adhesion and function has previously been achieved by alphavbeta3 antibodies or Arg-Gly-Asp-containing peptides which have the disadvantages of blocking a single receptor type, or of being rather nonspecific, respectively. Here we show that alphav integrin expression in rabbit osteoclasts can be inhibited by partially phosphorothioated antisense oligodeoxynucleotide (ODN) spanning the adenine-uracil-guanine (AUG) translational start site of the human/rabbit alphav gene, a procedure which offers the advantage of affecting all the alphav receptors with high efficiency. The alphav antisense ODN caused a dose-dependent, substrate-specific reduction of osteoclast adhesion and bone resorption. Control ODNs, such as sense, inverted, and mismatch, were without effect, providing evidence of specificity of the antisense reagent. It is likely as a consequence of loss of substrate interaction, the antisense ODN induced osteoclast retraction and apoptosis, increase of the cyclin/cyclin-dependent kinase complex inhibitor p21WAF1/CIP1, and inhibition of the cell survival gene, bcl-2. Although the expression of the cell death-promoting gene, bax, remained unchanged, a reduction of the bcl-2/bax ratio, known to underlie the intracellular signal to apoptosis, was observed. This finding led us to hypothesize that these changes could provide a link between reduction of alphav synthesis and osteoclast programmed death. In conclusion, this study provides novel insights into the use of alphav antisense ODN as an efficacious mechanism for blocking osteoclast function and underscores for the first time the involvement of integrins in bone cell apoptosis. In vivo studies may verify potential application of this ODN as alternative therapy for bone diseases.

Animals↗

PubMed1997

Interleukin-5 expression in the lung epithelium of transgenic mice leads to pulmonary changes pathognomonic of asthma.

We have generated transgenic mice that constitutively express murine interleukin (IL)-5 in the lung epithelium. Airway expression of this cytokine resulted in a dramatic accumulation of peribronchial eosinophils and striking pathologic changes including the expansion of bronchus-associated lymphoid tissue (BALT), goblet cell hyperplasia, epithelial hypertrophy, and focal collagen deposition. These changes were also accompanied by eosinophil infiltration of the airway lumen. In addition, transgenic animals displayed airway hyperresponsiveness to methacholine in the absence of aerosolized antigen challenge. These findings demonstrate that lung-specific IL-5 expression can induce pathologic changes characteristic of asthma and may provide useful models to evaluate the efficacy of potential respiratory disease therapies or pharmaceuticals.

Animals↗

PubMed1997

A peptidomimetic antagonist of the alpha(v)beta3 integrin inhibits bone resorption in vitro and prevents osteoporosis in vivo.

Osteoclastic bone degradation requires intimacy between the matrix and the resorptive cell. While the precise role the integrin alpha(v)beta3 plays in the process is not yet understood, occupancy of the heterodimer by soluble ligand or by blocking antibody effectively inhibits bone resorption in vitro and in vivo, suggesting that alpha(v)beta3 blockade may prevent postmenopausal osteoporosis. Thus, we identified a synthetic chemical peptide mimetic, beta-[2-[[5-[(aminoiminomethyl)amino]-1-oxopentyl]amino]-1-+ ++oxoethyl]amino-3-pyridinepropanoic acid, bistrifluoroacetate (SC56631) based upon the alpha(v)beta3 ligand, Arg-Gly-Asp (RGD), which recognizes the isolated integrin, and its relative, alpha(v)beta5, as effectively as does the natural peptide. The mimetic dampens osteoclastic bone resorption in vitro and in vivo. Most importantly, intravenous administration of the mimetic prevents the 55% loss of trabecular bone sustained by rats within 6 wk of oophorectomy. Histological examination of bones taken from SC56631-treated, oophorectomized animals also demonstrates the compound's bone sparing properties and its capacity to decrease osteoclast number. Thus, an RGD mimetic prevents the rapid bone loss that accompanies estrogen withdrawal.

Amino Acids↗

PubMed1997

Trafficking of matrix collagens through bone-resorbing osteoclasts.

An intracellular pathway for proteins liberated from mineralized matrix during resorption was identified in osteoclasts. Analysis by confocal microscopy of sites of active bone resorption showed that released matrix proteins, including degraded type I collagen, were endocytosed along the ruffled border within the resorption compartment and transcytosed through the osteoclast to the basolateral membrane. Intracellular trafficking of degraded collagen, as typified by the resorbing osteoclast, may provide the cell with a regulatory mechanism for the control of tissue degradation.

Bone Matrix↗

PubMed1997

Expression of IL-5 in thymocytes/T cells leads to the development of a massive eosinophilia, extramedullary eosinophilopoiesis, and unique histopathologies.

Transgenic mice were generated using regulatory elements from the CD3delta gene to drive T cell expression of IL-5. Expression of this cytokine resulted in white blood cell counts that expand virtually unabated (approximately 400,000 cells/mm3). This expansion is characterized by a profound eosinophilia (>60%) and commensurate increases in the absolute numbers of all other white blood cell types. In particular, circulating B220+ B lymphocyte populations increased >30-fold over wild-type (+/+) levels. Cell differentials and expression studies using a marker for eosinophil precursor cells (major basic protein gene expression) suggest that the peripheral eosinophilia is induced primarily through the establishment of extramedullary sites of eosinophilopoiesis. These mice display a massive peritoneal cavity cell exudate (1-2 x 10(8) cells) dominated by eosinophils (approximately 50%) and the infiltration of eosinophils in nearly all organ systems. Sudden unexplained death occurs in 70% of all transgenic animals by 12 mo of age. Surviving transgenic animals display severe inflammatory pathologies that include ulcerating skin lesions as well as lower bowel inflammation. These pathologies parallel clinical observations of patients with a profound eosinophilia and imply that IL-5 effector functions during some inflammatory responses may be contingent upon peripheral lymphohemopoietic expression.

Animals↗

PubMed1997

The alpha v beta 3 integrin "vitronectin receptor".

The alpha v beta 3 "vitronectin receptor" is a member of the integrin superfamily of adhesion molecules. As such, this 160/85 kDa heterodimeric protein exhibits many of the typical structural and functional features of integrins. It mediates cell adhesion to extracellular matrix by recognizing the conserved arg-gly-asp (RGD) sequence of several plasma and matrix proteins. Recently, it has also been shown that alpha v beta 3 is involved in signal transduction and cell to cell interactions. alpha v beta 3 is highly expressed in bone resorbing cells, osteoclasts, and upregulated in response to vascular damage, during angiogenesis and in certain types of malignancy. Antagonists of alpha v beta 3 are being developed for use in a variety of diseases associated with altered receptor function or level.

Animals↗

PubMed1996

Cloning of the murine eosinophil peroxidase gene (mEPO): characterization of a conserved subgroup of mammalian hematopoietic peroxidases.

The mouse eosinophil peroxidase (mEPO) gene was cloned by screening a random-primed bone marrow cDNA library at reduced criteria using a hEPO cDNA. An mEPO cDNA was subsequently used to isolate the mEPO gene from a lambda-genomic library. The mEPO gene displays a high degree of conservation with its human homologue: the transcription units are approximately the same size, conserve the relative size and position of the 12 exons associated with each gene, and at a nucleotide level the mouse and human EPO genes are 86% identical in the protein coding regions and 66% identical in the 3'-untranslated trailer regions. This strong conservation extends to the encoded proteins which show approximately 90% amino acid identity. Expression of the mEPO gene is restricted to tissues containing eosinophil progenitor cells (e.g., bone marrow and spleen), a pattern similar to the expression of another murine eosinophil granule protein, major basic protein.

Amino Acid Sequence↗

PubMed1996

Adhesive properties of isolated chick osteocytes in vitro.

Different functions have been proposed for osteocytes over time, but it is now generally accepted that their most important task lies in the sensing of strain caused by mechanical loading on bone. The fact that mechanical strain can be sensed as deformation of the extracellular matrix or as fluid shear stress along the cell, in the space between cell membrane and extracellular matrix, requires that osteocytes have close (specialized) contact with the bone matrix. We studied to which extracellular matrix proteins isolated chicken osteocytes adhere and whether this adhesion is mediated by specific cell adhesion receptors called integrins. The adhesive properties of the osteocytes were compared with that of osteoblasts. Osteocytes (and osteoblasts) adhere to the same substrates (i.e., collagen types I and II, collagen fibers, osteopontin, osteonectin, fibronectin, fibrinogen, thrombospondin, and laminin). Cell spreading varied between substrates, from all cells rounded on thrombospondin to all cells fully spread out on osteopontin, osteonectin, vitronectin, fibronectin, fibrinogen, and laminin. The percentage of osteocytes adhered was equivalent to that of osteoblasts adhered on all substrates except osteopontin and vitronectin, where osteocytes adhered less. The adhesion of osteocytes and osteoblasts to osteopontin, osteonectin, vitronectin, and fibrinogen was strongly inhibited, and to fibronectin and laminin moderately, by an RGD peptide. No RGD inhibition was found on collagen. An antibody against chicken integrin alpha v beta 3, the monoclonal antibody (MAb) 23C6, did not interfere with the adhesion of osteocytes and osteoblasts to matrix proteins, whereas an MAb against chicken integrin subunit beta 1 (CSAT) strongly inhibited adhesion to all substrates. Labeling with osteocyte-specific MAbs (OB7.3, OB37.4, and OB37.11) also did not hinder the adhesion of osteocytes to collagen type I, vitronectin, and osteopontin. Adhesion sites on osteocytes were small compared with the large adhesion plaques of osteoblasts, as demonstrated by interference reflection microscopy and immunocytochemically by staining for vinculin. Osteocyte adhesion is analogous to osteoblast adhesion with regard to the range of extracellular matrix proteins to which they adhere. The adhesion is mediated by the integrin subunit beta 1, but other integrins or nonintegrin adhesion receptors are also involved. Osteocytes make contact with the extracellular matrix via small attachment points which colocalize with vinculin. This connection between the bone matrix and the cytoskeleton may be important for osteocytic sensing of mechanical strain, as it supplies a transduction route of extracellular (mechanical) signals into intracellular messages.

Animals↗

PubMed1996

Beta 1 integrins and osteoclast function: involvement in collagen recognition and bone resorption.

The extracellular matrix of bone is composed mainly of type I collagen. In this report we studied the role and collagen-binding properties of osteoclast integrins (alpha v, alpha 2, beta 1, and beta 3). Cell adhesion assays with rat osteoclasts and affinity chromatography/SDS-PAGE analysis with purified human osteoclast membranes demonstrated adhesion of osteoclasts to native type I collagen in a divalent cation and Arg-Gly-Asp (RGD)-dependent way via alpha 2 beta 1 integrin, whereas osteoclast adhesion to denatured collagen predominantly involved alpha v beta 3. In receptor-binding assays, the involvement of human recombinant alpha v beta 3 in adhesion to denatured collagen was confirmed. Additionally, osteoclasts adhered to type I collagen fibers and to monomeric types II-V collagen with characteristics similar to those on native monomeric type I collagen. Osteoclastic bone resorption in vitro was inhibited (> 40%) in the presence of alpha 2 and beta 1 antibodies. Using scanning laser confocal microscopy, alpha v beta 3, alpha 2, and beta 1 integrin were detected within podosomes in nonresorbing osteoclasts and in the ruffled border area and basolateral membrane in resorbing osteoclasts, but not in the sealing zone of resorbing osteoclasts. These results demonstrate that alpha 2 beta 1, in addition to alpha v beta 3, has an important role in osteoclast function and acts as a receptor for native, but not denatured, collagen.

Amino Acid Sequence↗