Search

Introduction: Technetium 99m (99mTc)-NC100692 is being developed as a marker of vitronectin receptor expression. The purpose of this study was to confirm the binding affinity [dissociation constant (Kd)] of 99mTc-NC100692 for a range of integrin receptors including alphavbeta3 and alphavbeta5 as well as to establish the biodistribution and metabolic stability of 99mTc-NC100692 in Wistar rats., Methods: The Kd of 99mTc-NC100692 for a range of human integrin receptors was established in an in vitro saturation binding assay. The biodistribution and metabolic stability of 99mTc-NC100692 in normal Wistar rats was investigated., Results: The Kd of 99mTc-NC100692 to alphavbeta3 and alphavbeta5 was less than 1 nM. It was not possible to saturate the binding of 99mTc-NC10092 towards alphaIIbbeta3, alpha5beta1, alpha3beta1 or alpha1beta1, and as a result, accurate Kd values could not be determined. The biodistribution of 99mTc-NC100692 in male and female Wistar rats showed that radioactivity was rapidly excreted, predominantly into the urine, with very little background tissue retention apart from the liver and kidneys. Kidney and liver retention was reduced in the presence of excess NC100692 ligand. In vivo, there was little systemic metabolism of 99mTc-NC100692., Conclusions: 99mTc-NC100692 has a high affinity for the vitronectin receptors that are associated with angiogenesis. 99mTc-NC100692 is metabolically stable in the systemic circulation of rats with a biodistribution that is favourable for imaging purposes. This evidence suggests that 99mTc-NC100692 might be a useful marker of vitronectin receptor expression in vivo.

The vitronectin receptor (integrin alphavbeta3), a cell-surface adhesion receptor, has been shown to play a significant role in endothelial cell migration, apoptosis, atherosclerosis, and T-lymphocyte activation. This study was undertaken to test the hypothesis that cardiac allograft rejection is associated with increased expression of alphavbeta3. We also determined whether fibronectin receptor (alpha5beta1) and tissue factor are up-regulated in the presence of acute cellular rejection. We evaluated endomyocardial biopsy specimens with histologic evidence of different degrees of acute cellular rejection (grade 0, n = 10; grade 1A, n = 10; grade 2, n = 10; grade 3A, n = 10). Biopsies were obtained 2-4weeks after cardiac transplantation. Immunoperoxidase staining was performed for alphavbeta3, tissue factor, and alpha5beta1, and protein levels were further determined by Western blot analysis. Specimens with grade 2 and grade 3A rejection showed positive staining of alphavbeta3 in lymphocytic aggregates and vascular endothelial cells. By immunoblotting, we identified significantly increased expression of alphavbeta3 in the presence of acute rejection, grade 2 (3-fold, p = 0.01) and grade 3A (3.6-fold, p = 0.005) compared to grade 0 and 1 A specimens. There was no evidence of increased expression of alpha5beta1 or tissue factor. Acute cellular rejection, a process characterized by T-lymphocyte activation and release of inflammatory cytokines, is associated with increased expression of alphavbeta3.

Background: Signal transduction by mesangial cell (MC) integrins regulates cell growth and survival, extracellular matrix production, and organization. The aim of the study was to investigate human MC integrin modulation by differently glycosylated IgA and macromolecular IgA, which are thought to play a pathogenetic role in IgA nephropathy (IgAN)., Methods: MCs were incubated with purified human polymeric IgA, heat-aggregated IgA, IgA glycoforms generated by enzymatic hydrolysis of saccharide residues and serum fractions from IgAN patients, and controls isolated by lectin affinity and containing IgA with peculiar glycan patterns. Integrins were quantitated by flow cytometry., Results: Cultured MCs highly expressed alphavbeta3 and some alpha3beta1; alphavbeta3 was up-regulated by matrix components (P < 0.02). In vitro desialylated and degalactosylated polymeric human IgA enhanced alphavbeta3 expression on cultured MCs (P < 0.001). Serum IgA glycoforms isolated from IgAN patients with high exposure of internal sugars, GalNAc, Neu5Ac2,6GalNAc, and Man enhanced alphav expression on cultured MCs more than healthy controls. CONCLUSIONS.: These data support the hypothesis that IgA glycation plays a role in modulating the cell-matrix interaction, and that this mechanism can be operating in IgAN.

Macrophage colony-stimulating factor (M-CSF) is essential for murine osteoclast formation and its role in human hematopoiesis in vitro is not fully defined. Therefore, we have investigated the effect of M-CSF on the formation of human osteoclasts in vitro. M-CSF was found to induce substantial bone resorption and osteoclast formation in a dose-responsive and time-dependent manner above that induced by 1,25 dihydroxyvitamin D3 (1,25 vitamin D3) in cultures of human bone marrow (BM) stromal cells sedimented onto devitalized bone. By day 14 there was a mean of approximately 50% of the surfaces of the bone slices resorbed compared with only 6% in cultures treated with 1,25 vitamin D3 alone. Osteoclasts were identified as 23c6+ cells (an antibody that recognizes the vitronectin receptor), 87.5% of which coexpressed the calcitonin receptor. The number of 23c6+ cells correlated strongly with bone resorption spatially, and in a dose-responsive and time-dependent manner; the correlation coefficient in cultures treated with 1,25 vitamin D3 alone was 0.856 and those treated with both M-CSF and 1,25 vitamin D3 was 0.880. Granulocyte-macrophage colony-stimulating factor, IL-1 beta, IL-3, IL-6, tumor necrosis factor-alpha, transforming growth factor-beta, leukemia inhibitory factor, and IL-11 did not increase bone resorption above that in 1,25 vitamin D3-treated cultures. We also found that 1,25 vitamin D3 increased, to a minor but significant degree, both bone resorption and the concentration of M-CSF in the culture supernatants above that in vehicle-treated cultures, indicating that M-CSF is present in our BM cultures, but that there is insufficient to induce substantial osteoclast formation. These results define a critical role for M-CSF in the formation of human osteoclasts.

The endothelial and smooth muscle integrin alphaVbeta3, a receptor for vitronectin and fibrinogen, participates in angiogenesis associated with wound healing and tumorigenicity. The microvascular expression of alphavbeta3 and fibrin during experimental middle cerebral artery occlusion and reperfusion in a nonhuman primate model was examined by computer-assisted video imaging microscopy. No microvascular expression of alphavbeta3 was seen in the control subjects (n = 3) or the non-ischemic basal ganglia of subjects undergoing 2-hour MCA:O (middle cerebral artery occlusion) or 3-hour occlusion with 1-hour (n = 3), 4-hour (n = 3), and 24-hour (n = 3) reperfusion. In the ischemic territory, alphavbeta3 appeared initially at 2 hours of middle cerebral artery occlusion. Up-regulation of alphavbeta3 was confined to the media of 30.0- to 50.0-micron-diameter arterioles in the ischemic core and correlated significantly with fibrin deposition in those vessels (P < 0.0005). Integrin alphavbeta3 and its ligand fibrinogen appear in a subpopulation of microvessels after focal cerebral ischemia.

During early granulation tissue formation of wound repair, new capillaries invade the fibrin clot, a process that undoubtedly requires an interaction of vascular cells with the wound provisional matrix composed mainly of fibrin, fibronectin, and vitronectin. Integrin alphaVbeta3 is the vascular cell receptor for these wound-associated adhesive proteins. Therefore, we investigated the expression of this receptor on new capillaries of healing full-thickness cutaneous porcine wounds. During granulation tissue formation, alphaVbeta3 was expressed specifically on capillary sprouts invading the central fibrin clot whereas the closely related integrin alphaVbeta5 failed to localize to these cells. Cyclic peptides or antibody antagonists of alphaVbeta3 specifically inhibited granulation tissue formation in a transient manner during the period of invasive angiogenesis. Immunolocalization studies revealed that alphaVbeta3 became aggregated and lost from sprouting vessels after treatment with a peptide antagonist. In contrast, beta 1 integrins were not modulated by this treatment. Once granulation tissue filled the wound and invasive angiogenesis terminated, the alphaVbeta3 showed little or no expression in the granulation tissue microvasculature. These data demonstrate that integrin alphaVbeta3 plays a fundamental, but transient, role during invasive angiogenesis and granulation tissue formation in a healing wound.

The ligand specificity of the integrin cell adhesion receptors probably determines the ability of specific integrins to promote tumor cell proliferation and metastasis. Therefore, we compared the expression of integrin alphaVbeta3, a promiscuous receptor that binds with high affinity to numerous cell matrix proteins, with the expression of integrin alphaVbeta5 and the integrin beta 1 subunit (which pairs with multiple alpha subunits) in neuroblastic tumors at various stages of differentiation. Undifferentiated neuroblastoma tumors rapidly invade and metastasize, whereas ganglioneuroblastomas rarely metastasize. Differentiating neuroblastomas are associated with an intermediate prognosis. Paraffin sections of neuroblastic tumors at various stages of differentiation obtained at biopsy from 17 patients were hybridized with antisense integrin subunit-specific alphaV, beta3, beta1, and beta5 riboprobes. All neuroblastic tumors and seven adrenal glands obtained at autopsy were analyzed immunohistochemically with antibodies directed toward the alphaV, beta3, beta1, and beta5 subunits. The alphaV subunit was expressed in neuroblastic tumors independent of the stage of differentiation, although mRNA and protein expression were generally weak in ganglioneuroblastomas, and was also detected in adrenal gland medullae. The beta1 subunit was detected in most neuroblastic tumors independent of the stage of differentiation as well as in adrenal gland medullae. In contrast, the beta3 subunit, which was not expressed in adrenal gland medullae, was expressed at the protein and mRNA levels in undifferentiated neuroblastomas (six of seven and seven of seven, respectively) but was not expressed in neuroblasts or ganglion cells in ganglioneuroblastomas (one case weakly positive out of five). The beta 5 subunit was expressed at the protein (five of five) and mRNA (four of five) levels in the ganglion cells of ganglioneuroblastomas and, although mRNA for this subunit was detectable in undifferentiated tumors, the protein was not detectable. The expression of integrin alphaVbeta3 in undifferentiated neuroblastomas may contribute to the rapid growth of these tumors and their tendency to metastasize.