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PubMed1995

Megakaryocytes and fibroblasts--interactions as determined in normal human bone marrow specimens.

An in vitro study was performed to investigate possible interactions between megakaryocytes and bone marrow fibroblasts, both obtained from healthy donors. We were able to demonstrate that the proliferation of fibroblasts increased significantly by co-culturing these cells with megakaryocytes for 6 days. Addition of neutralizing antibodies for PDGF and TGF beta 1, caused a significant reduction of fibroblast growth. Inhibition of cell to cell contacts via tissue culture inserts generated a conspicuous impairment of fibroblast proliferation compared with megakaryocyte-fibroblast co-cultures, where contact was allowed. Hence, our findings suggest that a close spatial relationship between megakaryocytes and fibroblasts is needed for the activation of growth in normal human bone marrow. Neighbouring of megakaryocytes and fibroblasts seems to be necessary in order to achieve a certain threshold of local growth factor concentration. Our results are in keeping with the assumption that PDGF and TGF beta 1, are secreted by normal human megakaryocytes in very low concentrations and promote significantly fibroblast proliferation.

Adult↗
PubMed1995

Repertoire cloning of a human IgG inhibitor of alpha IIB beta 3 function. The OG idiotype.

A patient (OG) with Glanzmann thrombasthenia became refractory to platelet transfusion following the production of IgG antibodies (Ab1) specific for the integrin subunit beta 3. We generated recombinant VH and VL cDNA libraries using IgG-specific mRNA isolated from OG peripheral blood B-lymphocytes that had been selected for binding to antigen (alpha IIb beta 3 adsorbed to plastic dishes). These antigen-specific B-lymphocytes contain rearranged VH DNA segments that belong exclusively to the VH4 gene family. Recombinant Fab were expressed on the surface of filamentous phage coinfected with VH and VL segments cloned into the phagemid pHEN1 or the phage fd-tet-DOG1. To facilitate selection of the desired recombinant Ab1 Fab, we developed a rabbit polyclonal antibody specific for affinity-purified OG anti-beta 3 Fab (Ab2). Ab2 reacts specifically with Ab1, and this interaction is inhibited by purified alpha IIb beta 3. Following three rounds of phage selection on Ab2 adsorbed to plastic dishes and random reassociation of heavy and light chains, we isolated Ab1 Fab and tested their binding to alpha IIb beta 3. Five Id-positive Fab were selected for further characterization. These Fab use one of two VH genes (H21 or H23) complexed with one of three V lambda genes. Subsequent sequence data demonstrated that all three lambda genes are the same clone L22 which uses a germline V lambda gene segment. Fab using H23 bind to alpha IIb beta 3, while those using H21 do not. Based on sequence homology, both H21 and H23 use VH gene segments belonging to the VH4 gene family. Thus, the idiotype OG is restricted to the VH4 gene family and is the first sequenced prototype of human antibodies that bind close to or at a functional epitope(s) of alpha IIb beta 3.

Amino Acid Sequence↗
PubMed1996

Platelet activation studies with anti-CD41/61 monoclonal antibodies.

The purpose of this study was to monitor platelet activation by following alpha IIb beta 3 integrin (GpIIb/IIIa complex or CD41/CD61) on the platelet surface by flow cytometry (FCM) analysis using workshop mAbs. The results obtained with the mAbs showed increased expression of the GpIIb/IIIa complex (about 40%) on the platelet membrane surface under thrombin stimulation.

Animals↗
PubMed1996

Biochemical characterization of antigens detected with anti-platelet monoclonal antibodies.

A panel of 18 monoclonal antibodies (mAbs) defined by the third workshop as specific for platelets, clustered in three preliminary groups: PC7, PC13 and PC27. These mAbs were further analysed by immunoprecipitation using extracts of iodinated and biotinylated peripheral blood mononucleated cells (PBMC) and platelets. We could confirm the existence of mAbs with specificities to WC9 (in PC7) and CD41/61 (in PC13). Two mAbs formed a new cluster, WC13, which may be homologous to human CD31 (in PC27). The influence of EDTA and thrombin on the expression of the different antigens on the platelet membrane was assessed by flow cytometry (FCM) analysis, as well as cross-reactivity with platelets from different species.

Animals↗
PubMed1991

Immunohistochemical localization of vitronectin, its receptor and beta-3 integrin in Alzheimer brain tissue.

The vitronectin receptor (VNR) is an integrin which consists of an alpha-subunit which can associate with multiple beta-subunits. A polyclonal antibody to this integrin weakly stained resting microglia in white matter of control brain and strongly stained reactive microglia in both gray and white matter of Alzheimer brain. This antibody, as well as a monoclonal antibody to beta 3, stained some platelets in capillaries of both control and Alzheimer tissue. When the antiserum was immunoabsorbed with a preparation enriched in the alpha-chain of the vitronectin receptor, it failed to stain microglial cells, but continued to stain platelets. When it was immunoabsorbed with a peripheral blood platelet preparation, all immunostaining was abolished. These results indicate that the vitronectin receptor of microglia is associated with a beta-chain different from beta 3, but that beta 3 is expressed by some platelets in brain capillaries. An antibody to vitronectin itself stained senile plaques and neurofibrillary tangles in Alzheimer entorhinal cortex, but only residual plasma in control tissue. Senile plaques positive for vitronectin had microglial cores strongly positive for the vitronectin receptor. The high levels of vitronectin receptor on reactive microglia in areas containing extracellular vitronectin suggest the possibility that vitronectin is serving an opsonizing function for microglial phagocytosis.

Aged↗
PubMed1992

Binding of glycoprotein IIIa-derived peptide 217-231 to fibrinogen and von Willebrand factors and its inhibition by platelet glycoprotein IIb/IIIa complex.

Based on previous reports in the literature and the high homology between platelet glycoprotein (GP) IIIa 217-231 and similar portions of other beta subunits of integrin receptors, we hypothesized that this region may participate in ligand binding. Using a polyclonal antibody against GPIIIa 217-231(YC), we tested the interaction of a synthetic peptide representing this region with fibrinogen (Fg), in the enzyme-linked immunosorbent assay (ELISA) system. Results show a calcium-independent, dose-related, direct interaction between GPIIIa 217-231(Y) and immobilized Fg. This peptide also bound to von Willebrand Factor (vWF) and fibronectin (Fn), but did not attach to a 50 kDa Fn fragment which is deficient in the cell attachment site. In addition, purified GPIIb/IIIa displaced GPIIIa 217-231(Y) from Fg and vWF. Binding of 125I-GPIIIa 217-231(Y) to Fg coated tubes was inhibited by soluble Fg and by the GPIIb/IIIa complex. We synthesized this peptide with several alterations; similar peptides with Pro-219 replaced with an Ala showed significantly reduced binding to Fg and vWF. The decreased binding of the peptides with Pro-219 substitutes suggests that the confirmation of GPIIIa 217-230 is important for its ability to bind to adhesive ligands. In conclusion, the amino acid residues between 217 and 231 of GPIIIa appear to be involved in ligand binding and Pro-219 probably plays a significant role in this interaction.

Amino Acid Sequence↗
PubMed2005

RANK-L induces the expression of NFATc1, but not of NFkappaB subunits during osteoclast formation.

We report the program of gene expression during osteoclast formation from RAW264.7 cell precursors in response to RANK-ligand (RANK-L) using a combination of quantitative real time PCR and Affymetrix gene chip assays. We found that genes obligatory to osteoclast formation and function, namely tartrate-resistant acid phosphatase, cathepsin K, beta3 integrin, and calcitonin receptors, were up-regulated by RANK-L markedly by up to approximately 2000-fold. In contrast, we found a cluster of genes that were significantly down-regulated: these included interleukin-18, insulin-like growth factor-1, interleukin-6 receptor, and cathepsins B, C, and L. These results from real time PCR were broadly concordant with those obtained from Affymetrix. We also explored the expression of the transcription factors of the NFAT and NFkappaB family at days 3 and 5 of culture. Whereas NFATc1 expression was increased significantly at days 3 and 5 following RANK-L exposure, there were no significant increases in the expression of NFkappaB subunits, namely p65, p50, c-Rel, IkappaBalpha, and IkappaBbeta. There were also no significant differences in transcription modulator expression between days 3 and 5, except for c-Rel and NFATc4, which were both decreased significantly at day 5. The studies suggest RANK-L regulates the expression only of NFATc1, while it signals through both NFATc1 and NFkappaB.

Animals↗
PubMed2004

Inhibition of osteoclast differentiation and bone resorption by tanshinone IIA isolated from Salvia miltiorrhiza Bunge.

Osteoclasts, multinuclear cells specialized for bone resorption, differentiate from the monocyte/macrophage lineage of hematopoietic cells. Intervention in osteoclast differentiation is considered an effective therapeutic approach to the treatment of bone diseases involving osteoclasts. In this study, we found that tanshinone IIA, originating from Salvia miltiorrhiza Bunge, inhibited the differentiation of osteoclasts. Addition of tanshinone IIA to the osteoclast precursor culture caused a significant decrease in the level of calcitonin receptor, c-Src, and integrin beta3 mRNA, which are normally upregulated during the osteoclast differentiation dependent on RANKL (receptor activator of nuclear factor kappa B ligand). RANKL activated the ERK, Akt, and NF-kappaB signal transduction pathways in osteoclast precursor cells, and tanshinone IIA suppressed this activation. Tanshinone IIA also inhibited the bone resorptive activity of differentiated osteoclasts, which was accompanied with the disruption of the actin ring. Thus, tanshinone IIA has the potential to ameliorate bone-resorption diseases in vivo by reducing both the number and activity of osteoclasts.

Abietanes↗
PubMed2006

Endometrium in PCOS: Implantation and predisposition to endocrine CA.

Polycystic ovarian syndrome (PCOS) is a common endocrinopathy characterized by oligo/anovulatiaon and elevated circulating androgens or evidence of hyperandrogenism after all known potential causes have been excluded. In addition, insulin resistance and accompanying hyperinsulinemia commonly occur in women with PCOS. There is increasing evidence that the endocrinologic and metabolic abnormalities in PCOS may have complex effects on the endometrium, contributing to the infertility and endometrial disorders observed in women with this syndrome. Androgen receptors and steroid receptor co-activators are over-expressed in the endometrium of women with PCOS. Also, biomarkers of endometrial receptivity to embryonic implantation-such as alpha(v)beta3-integrin and glycodelin-are decreased, and epithelial expression of estrogen receptor alpha (ERalpha) abnormally persists in the window of implantation in endometrium in women with PCOS. In addition to being responsive to the steroid hormones estradiol, progesterone, and androgens, the endometrium is also a target for insulin, the receptor for which is cyclically regulated in normo-ovulatory women. In vitro, insulin inhibits the normal process of endometrial stromal differentiation (decidualization). In addition, insulin-like growth factors (IGFs) and their binding proteins are regulated in and act on endometrial cellular constituents, and hyperinsulinemia down-regulates hepatic IGFBP-1, resulting in elevated free IGF-I in the circulation. Thus, elevated estrogen (without the opposing effects of progesterone in the absence of ovulation), hyperinsulinemia, elevated free IGF-I and androgens, and obesity all likely contribute to endometrial dysfunction, infertility, increased miscarriage rate, endometrial hyperplasia, and endometrial cancer common in women with PCOS. The potential mechanisms underlying these disorders, specifically in women with PCOS, are complex and await additional transdisciplinary research for their complete elucidation.

Abortion, Spontaneous↗
PubMed2006

Hepatocyte growth factor-regulated genes in differentiated RAW 264.7 osteoclast and undifferentiated cells.

Hepatocyte Growth Factor (HGF) and its protooncogene receptor c-Met regulate osteoclast function by activating pp60(c-Src) kinase and alpha(v)beta3 integrin. HGF causes transcription yet in osteoclast cells, this gene regulation is currently unknown. To begin characterization of HGF-regulated gene expression in osteoclast cells, we used a well characterized model of osteoclast cells. Using microarray, relative RT-PCR, and Western blot analyses, we have identified and confirmed differentially expressed genes in RAW 264.7 osteoclast cells in response to HGF. HGF regulation of transcription of these genes was concordant with microarray results. We report that HGF downregulates transcription factors, Distal-less 5 (Dlx-5), Distal-less 6 (Dlx-6) and Aristaless 4 (Alx-4), in RAW 264.7 osteoclast cells but has an inverse effect in undifferentiated RAW 264.7 cells.

Animals↗
PubMed2005

Clinicopathologic study of 24 cases of cherubism.

The authors reviewed 24 cases of familial or nonfamilial cherubism. The age at onset was between 6 and 10 years. It was characterized by bilateral painless swelling of jaws and eyes-to-heaven appearance was visible when the maxillae were affected as well. Radiographs showed well-defined multilocular radiolucencies and with age, thick sclerotic borders were visible. A malocclusive and abnormal dentition, worse in the mandible can be seen. Histopathologically, numerous randomly distributed multinucleated giant cells and vascular spaces within a fibrous connective tissue stroma with or without eosinophilic collagen perivascular cuffing were apparent. Multinucleated giant cells were positive for osteoclastic specific markers, tartrate-resistant acid phosphatase and human alphaV beta3 integrin, 23C6. Results after follow-up were available for 14 cases. Of these, no treatment was carried out in five cases, cherubism resolved (three cases) or grew slowly (two cases); curettage or surgical contouring was performed in seven cases, during the rapid growth of the lesions. This not only gave good immediate results, but also arrested active growth of remnant cherubic lesions and even stimulated bone regeneration. Segmental mandibulectomy followed by reconstruction was performed in two cases with extensive lesion and the risk of pathologic fracture of the mandible, and excellent results were obtained.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Application to immunoassays of the fusion protein between protein ZZ and enhanced green fluorescent protein.

Enhanced green fluorescent protein (EGFP) from Aequorea victoria was fused to the C terminal region of protein ZZ, an artificial synthetic IgG Fc fragment binding protein derived from tandem repeats of the B domain of protein A. The ZZ-EGFP fusion protein was expressed in Escherichia coli with a His(6) tag and purified in high yield by one-step Ni(2+) chelating affinity chromatography. It was then used in the immunoblot analysis of GST and TNFalpha as well as in immunofluorescent assays of 293T cells transfected with IRF3, an interferon regulatory factor which localized in cytoplasm without virus infection. The fusion protein also performed effectively in FACS analysis of surface integrin beta3 subunit on 293 T cells. The chimeric protein bound various antibodies from different animal sources, directed against a variety of proteins. Thus, ZZ-EGFP showed broad promise in potential immunological applications.

Amino Acid Sequence↗
PubMed2005

Decursin and PDBu: two PKC activators distinctively acting in the megakaryocytic differentiation of K562 human erythroleukemia cells.

Protein kinase C (PKC) plays an important role in the proliferation and differentiation of various cell types including normal and leukemic hematopoietic cells. Phorbol 12,13-dibutyrate (PDBu) induces the megakaryocytic differentiation of K562 human erythroleukemia cells through PKC activation. Decursin, a pyranocoumarin from Angelica gigas, exhibits the cytotoxic effects on various human cancer cell lines and in vitro PKC activation. We report here the differences between two PKC activators, tumor-suppressing decursin and tumor-promoting PDBu, in their actions on the megakaryocytic differentiation of K562 cells. First of all, decursin inhibited PDBu-induced bleb formation in K562 cells. Decursin also inhibited the PDBu-induced megakaryocytic differentiation of K562 cells that is characterized by an increase in substrate adhesion, the secretion of granulocyte/macrophage colony stimulating factor (GM-CSF) and interleukin-6 (IL-6), and the surface expression of integrin beta3. The binding of PDBu to PKC was competitively inhibited by decursin. Decursin induced the more rapid down-regulation of PKC alpha and betaII isozymes than that induced by PDBu in K562 cells. Unlike PDBu, decursin promoted the translocation of PKC alpha and betaII to the nuclear membrane. Decursin-induced faster down-regulation and nuclear translocation of PKC alpha and betaII were not affected by the presence of PDBu. All these results indicate that decursin and phorbol ester are PKC activators distinctively acting in megakaryocytic differentiation and PKC modulation in K562 leukemia cells.

Active Transport, Cell Nucleus↗
PubMed2005

Analysis of long-term gene expression in neurons of the hippocampal subfields following traumatic brain injury in rats.

After experimental traumatic brain injury (TBI), widespread neuronal loss is progressive and continues in selectively vulnerable brain regions, such as the hippocampus, for months to years after the initial insult. To clarify the molecular mechanisms underlying secondary or delayed cell death in hippocampal neurons after TBI, we compared long-term changes in gene expression in the CA1, CA3 and dentate gyrus (DG) subfields of the rat hippocampus at 24 h and 3, 6, and 12 months after TBI with changes in gene expression in sham-operated rats. We used laser capture microdissection to collect several hundred hippocampal neurons from the CA1, CA3, and DG subfields and linearly amplified the nanogram samples of neuronal RNA with T7 RNA polymerase. Subsequent quantitative analysis of gene expression using ribonuclease protection assay revealed that mRNA expression of the anti-apoptotic gene, Bcl-2, and the chaperone heat shock protein 70 was significantly downregulated at 3, 6 (Bcl-2 only), and 12 months after TBI. Interestingly, the expression of the pro-apoptotic genes caspase-3 and caspase-9 was also significantly decreased at 3, 6 (caspase-9 only), and 12 months after TBI, suggesting that long-term neuronal loss after TBI is not mediated by increased expression of pro-apoptotic genes. The expression of two aging-related genes, p21 and integrin beta3 (ITbeta3), transiently increased 24 h after TBI, returned to baseline levels at 3 months and significantly decreased below sham levels at 12 months (ITbeta3 only). Expression of the gene for the antioxidant glutathione peroxidase-1 also significantly increased 6 months after TBI. These results suggest that decreased levels of neuroprotective genes may contribute to long-term neurodegeneration in animals and human patients after TBI. Conversely, long-term increases in antioxidant gene expression after TBI may be an endogenous neuroprotective response that compensates for the decrease in expression of other neuroprotective genes.

Animals↗
PubMed2005

Biological and molecular characterization of a canine hemangiosarcoma-derived cell line.

Canine hemangiosarcoma (HSA) is a devastating disease. Investigation of novel therapies has been limited by the limited availability of canine HSA-derived cell lines. We report the development of a canine HSA-derived cell line, DEN-HSA, which recapitulates features of angiogenic endothelium. DEN-HSA cells were derived from a spontaneous HSA arising in the kidney of a dog. DEN-HSA displayed surface molecules distinctive of endothelial histogenesis, including factor VIII-related antigen, ICAM-1 and alpha(v)beta3 integrin. In vitro, DEN-HSA formed microvascular tube-like structures on Matrigel, and proliferated in response to a variety of angiogenic growth factors. The cells expressed mRNA and protein specific for bFGF and its receptors, and VEGF and its receptors, among others. DEN-HSA conditioned medium evoked a marked angiogenic response in a murine corneal pocket assay, indicating potent proangiogenic activity of substances secreted by this cell line. This research confirms the DEN-HSA cell line as endothelial in origin, suggests the presence of angiogenic growth factor autocrine loops, and offers the potential to utilize DEN-HSA cells for the study of novel therapies that modulate endothelial proliferation.

Angiogenic Proteins↗
PubMed2005

Sonic activation of molecularly-targeted nanoparticles accelerates transmembrane lipid delivery to cancer cells through contact-mediated mechanisms: implications for enhanced local drug delivery.

Liquid perfluorocarbon nanoparticles serve as sensitive and specific targeted contrast and drug delivery vehicles by binding to specific cell surface markers. We hypothesized that application of acoustic energy at diagnostic power levels could promote nanoparticle-associated drug delivery by stimulating increased interaction between the nanoparticle's lipid layer and the targeted cell's plasma membrane. Ultrasound (mechanical index = 1.9) applied with a conventional ultrasound imaging system to nanoparticles targeted to alpha(v)beta3-integrins on C32 melanoma cancer cells in vitro produced no untoward effects. Within 5 min, lipid delivery from nanoparticles into cell cytoplasm was dramatically augmented. We also demonstrate the operation of a potential physical mechanism for this effect, the acoustic radiation force on the nanoparticles, which may contribute to the enhanced lipid delivery. Accordingly, we propose that local delivery of lipophilic substances (e.g., drugs) from targeted nanoparticles directly into cell cytoplasm can be augmented rapidly and safely with conventional ultrasound imaging devices through nondestructive mechanisms.

Cell Line, Tumor↗
PubMed2005

Proteolysis of cell-surface tissue transglutaminase by matrix metalloproteinase-2 contributes to the adhesive defect and matrix abnormalities in thrombospondin-2-null fibroblasts and mice.

Thrombospondin (TSP)-2-null dermal fibroblasts display an attachment defect that results from increased matrix metalloproteinase (MMP)-2 levels in their conditioned media. To investigate the molecular mechanisms responsible for this defect, we analyzed the activity of tissue transglutaminase (tTG) in TSP-2-null dermal fibroblasts and in tissues of TSP-2-null mice. tTG functions as a co-receptor for beta1 and beta3 integrins and stabilizes extracellular matrix proteins by introduction of isopeptide cross-links. Cell-surface tTG activity was reduced in TSP-2-null cells (0.50 +/- 0.05 arbitrary units versus 0.84 +/- 0.07 for wild type; P < or = 0.05), and addition of MMP-2 to the culture medium of wild-type cells caused a 35% reduction in cell-surface tTG activity. tTG was susceptible to proteolysis by MMP-2 in vitro, and addition of the MMP inhibitor TIMP-2 to TSP-2-null cells restored tTG activity (0.3 +/- 0.08 for untreated cells; 0.71 +/- 0.09 with TIMP-2). TSP-2-null mice had reduced tTG activity in skin, as measured by incorporation of fluorescein isothiocyanate-labeled cadaverine, and a threefold increase in acetic acid-extracted dermal collagen. Furthermore, isopeptide cross-links were reduced in both uninjured skin and in excisional wounds of TSP-2-null mice, as determined by morphometric immunohistochemical analysis, indicating that isopeptide cross-links are important for the stabilization of the collagenous matrix in dermis. These findings provide a mechanism for the reduced adhesion of TSP-2-null fibroblasts and an explanation for the increased collagen solubility and fragility of TSP-2-null skin.

Animals↗