Human placental tissue stimulates bovine capillary endothelial cell growth,migration and protease production

A crude extract of human placenta has been demonstrated to stimulate growth, motility and the production of the proteases plasminogen activator and collagenase in cultured bovine capillary endothelial cells. These data are in keeping with the presence of an angiogenic factor(s) in human placenta.     

Processing, secretion, and biological properties of a novel growth factor of the fibroblast growth factor family with oncogenic potential.

We recently reported that the protein encoded in a novel human oncogene isolated from Kaposi sarcoma DNA was a growth factor with significant homology to basic and acidic fibroblast growth factors (FGFs). To study the properties of this growth factor (referred to as K-FGF) and the mechanism by which the K-fgf oncogene transforms cells, we have studied the production and processing of K-FGF in COS-1 cells transfected with a plasmid encoding the K-fgf cDNA. The results show that, unlike basic and acidic FGFs, the K-FGF protein is cleaved after a signal peptide, glycosylated, and efficiently secreted as a mature protein of 176 or 175 amino acids. Inhibition of glycosylation impaired secretion, and the stability of the secreted K-FGF was greatly enhanced by the presence of heparin in the cultured medium. We have used the conditioned medium from transfected COS-1 cells to test K-FGF biological activity. Similar to basic FGF, the K-FGF protein was mitogenic for fibroblasts and endothelial cells and induced the growth of NIH 3T3 mouse cells in serum-free medium. Accordingly, K-fgf-transformed NIH 3T3 cells grew in serum-free medium, consistent with an autocrine mechanism of growth. We have also expressed the protein encoded in the K-fgf protooncogene in COS-1 cells, and it was indistinguishable in its molecular weight, glycosylation, secretion, and biological activity from K-FGF. Taken together, these results suggest that the mechanism of activation of this oncogene is due to overexpression rather than to mutations in the coding sequences.     

Multiple forms of an angiogenesis factor: basic fibroblast growth factor

An angiogenesis factor has been isolated from human placenta and human hepatoma cells on the basis of its ability to stimulate protease production in cultured capillary endothelial cells. The purified angiogenesis factor also stimulated DNA synthesis and motility in capillary endothelial cells and induced angiogenesis in vivo. Amino acid sequence data revealed that the angiogenesis factor was human basic fibroblast growth factor (bFGF). Other angiogenesis factors isolated on the basis of their ability to stimulate endothelial cell proliferation have also been identified as bFGFs. The bFGFs that have been sequenced show variability in their N-termini. These different forms of bFGF may be naturally occurring processed forms or may be generated by proteases released during the isolation procedure. Recently a bFGF with a large N-terminal extension has been identified. This Mr 25,000 bFGF has the same biological activity and the same affinity for the bFGF receptor as the typical Mr 18,000 bFGFs. The Mr 25,000 bFGF can be converted into an Mr 18,000 form by treatment with low concentrations of trypsin, suggesting that it may be a precursor to the Mr 18,000 bFGF.     

In vitro neurite extension by granule neurons is dependent upon astroglial-derived fibroblast growth factor

When grown in the absence of astroglial cells, purified mouse cerebellar granule neurons survive less than 36 hr and do not extend neurites. Here we report that low concentrations of basic fibroblast growth factor (bFGF, 1-25 ng/ml) maintained the viability and promoted the differentiation of purified granule neurons. The effect of bFGF on granule cell neurite outgrowth was dose dependent. Neurite outgrowth was stimulated markedly in the presence of 1-25 ng/ml bFGF, but effects were not seen below 1 ng/ml or above 50 ng/ml. When affinity-purified antibodies against bFGF (1-5 micrograms/ml) were added either to purified granule cells or to co-cultures of neurons and astroglial cells, process extension by granule neurons was severely impaired. The inhibition of neurite outgrowth in the presence of anti-bFGF antibodies was reversed by the addition of 25 ng/ml of exogenous bFGF. In addition to neuronotrophic effects, bFGF influenced the rate of growth of the astroglial cells. This result depended on whether the astroglia were grown in isolation from neurons, where low doses of bFGF (10-25 ng) stimulated glial growth, or in coculture with neurons, where much higher doses of bFGF (100-250 ng/ml) were needed for glial mitogenesis. Immunoprecipitation of lysates from 35S-labeled cerebellar astroglial cells with anti-bFGF antibodies revealed a single band after SDS-PAGE at 18,000 Da, the molecular weight of bFGF. These results indicate that glial cells synthesize bFGF and are possibly an endogenous source of bFGF in cerebellar cultures. Thus, astroglial cells synthesize soluble factors needed for neuronal differentiation.     

Distribution of microtubules during the growth of tobacco pollen tubes

Summary— The distribution of microtubules was investigated in Nicotiana tabacum pollen tubes at different stages of tube growth by immunofluorescence microscopy. Using specific antibodies, the presence of microtubules consisting of different tubulin isoforms was tested. α-, β- and tyrosinated α-tubulin were present within the tube, whereas the acetylated form was lacking. The presence of tubulin subunits in pollen tube extracts was also investigated by immunoblotting analyses. The use of a confocal laser scanning microscope integrated with computer-assisted imaging, allowed a detailed visualization of the microtubule distribution and organization. Cytoplasmic microtubules organized as short bundles with various orientations were detected at the apex of long tubes.     

Lipopolysaccharide inhibits activation of latent transforming growth factor-β in bovine endothelial cells

The activation of latent transforming growth factor-β (TGF-β) by vascular endothelial cells (ECs) is regulated by cellular plasminogen activator (PA)/plasmin, transglutaminase (TGase), and latent TGF-β levels. Because lipopolysaccharide (LPS) has been reported to reduce EC surface plasmin levels by increasing the production of the inhibitor of PA, PA inhibitor-1 (PAI-1), we have tested whether LPS might suppress latent TGF-β activation in ECs using two different systems, namely, bovine aortic ECs (BAECs) cocultured with smooth muscle cells (SMCs) and BAECs treated with retinol. BAECs were either cocultured with SMCs after treatment with 15 ng/ml LPS or were treated with 2 μM retinol and/or 10 ng/ml LPS, and the expression of PA, surface plasmin, TGase, and the amounts of active and latent TGF-β secreted into the culture modium were measured. The downregulation of surface PA/plasmin levels with LPS was accompanied by a profound decline of both TGase and latent TGF-β expression as well as the suppression of surface activation of latent TGF-β. The effect was dependent on the concentration of LPS and on treatment time. The formation of TGF-β did not occur in cells maintained in LPS-contaminated culture medium. © 1995 Wiley-Liss, Inc.     

Tetradecanoyl phorbol acetate stimulates latent collagenase production by cultured human endothelial cells

Angiogenesis is associated with the fragmentation of blood vessel basement membranes. Since collagen is a major constituent of basement membranes, cultured human endothelial cells derived from umbilical cord veins were assayed for their ability to produce collagenase. Unstimulated cultured human endothelial cells did not secrete detectable levels of active collagenase into the culture medium. However, if the post-culture medium was treated with trypsin or plasmin, low levels of collagenolytic activity were detected, indicating that endothelial cells secrete small amounts of latent collagenase. Addition of the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA) to the culture medium stimulated the secretion of collagenase by endothelial cells 5–30 fold. More than 90% of the collagenase was secreted in the latent form. Stimulation of collagenase production was detected at 10^?9 M TPA and was maximal at 10^?8 M TPA. An increase in the rate of collagenase production could be detected within 3 hr after the addition of TPA, and full induction occurred by 12 hr. Cycloheximide (3 μg/ml) or actinomycin D (0.1 μg/ml) inhibited both basal levels of collagenase production and the stimulation of collagenase production by TPA. Phorbol-12,13-didecanoate (PDD), a tumor-promoting analog of TPA, also stimulated collagenase production when administered at the same concentrations that were effective for TPA. However, 4-O-methyl TPA and 4-αPDD, two analogs of TPA which are not tumor promoters, did not stimulate collagenase production at concentrations up to 10^?7 M. The collagenase produced by endothelial cells was a typical vertebrate collagenase as judged by the following criteria: it cleaved collagen into only two fragments which were three quarters and one quarter of the length of the intact molecule; it was inhibited by EDTA and human serum; it was not inhibited by inhibitors of serine, thiol or aspartate proteases. Thus TPA causes an increase in the production of latent collagenase by cultured human endothelial cells.     

Plasminogen activator and collagenase production by cultured capillary endothelial cells

Cultured bovine capillary endothelial (BCE) cells produce low levels of collagenolytic activity and significant amounts of the serine protease plasminogen activator (PA). When grown in the presence of nanomolar quantities of the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA), BCE cells produced 5-15 times more collagenolytic activity and 2-10 times more PA than untreated cells. The enhanced production of these enzymes was dependent on the dose of TPA used, with maximal response at 10(-7) to 10(-8) M. Phorbol didecanoate (PDD), an analog of TPA which is an active tumor promoter, also increased protease production. 4-O-methyl-TPA and 4α-PDD, two analogs of TPA which are inactive as tumor promoters, had no effect on protease production. Increased PA and collagenase activities were detected within 7.5 and 19 h, respectively, after the addition of TPA. The TPA-stimulated BCE cells synthesized a urokinase-type PA and a typical vertebrate collagenase. BCE cells were compared with bovine aortic endothelial (BAE) cells and bovine embryonic skin (BES) fibroblasts with respect to their production of protease in response to TPA. Under normal growth conditions, low levels of collagenolyic activity were detected in the culture fluids from BCE, BAE, and BES cells. BCE cells produced 5-13 times the basal levels of collagenolytic activity in response to TPA, whereas BAE cells and BES fibroblasts showed a minimal response to TPA. Both BCE and BAE cells exhibited relatively high basal levels of PA, the production of which was stimulated approximately threefold by the addition of TPA. The observation that BCE cells and not BAE cells produced high levels of both PA and collagenase activities in response to TPA demonstrates a significant difference between these two types of endothelial cells and suggests that the enhanced detectable activities are a property unique to bovine capillary and microvessel and endothelial cells.     

Opposite Roles of NMDA Receptors in Relapsing and Primary Progressive Multiple Sclerosis

Synaptic transmission and plasticity mediated by NMDA receptors (NMDARs) could modulate the severity of multiple sclerosis (MS). Here the role of NMDARs in MS was first explored in 691 subjects carrying specific allelic variants of the NR1 subunit gene or of the NR2B subunit gene of this glutamate receptor. The analysis was replicated for significant SNPs in an independent sample of 1548 MS subjects. The C allele of rs4880213 was found to be associated with reduced NMDAR-mediated cortical excitability, and with increased probability of having more disability than the CT/TT MS subjects. MS severity was higher in the CC group among relapsing-remitting MS (RR-MS) patients, while primary progressive MS (PP-MS) subjects homozygous for the T allele had more pronounced clinical worsening. Mean time to first relapse, but not to an active MRI scan, was lower in the CC group of RR-MS patients, and the number of subjects with two or more clinical relapses in the first two years of the disease was higher in CC compared to CT/TT group. Furthermore, the percentage of relapses associated with residual disability was lower in subjects carrying the T allele. Lesion load at the MRI was conversely unaffected by the C or T allele of this SNP in RR-MS patients. Axonal and neuronal degeneration at the optical coherence tomography was more severe in the TT group of PP-MS patients, while reduced retinal nerve fiber thickness had less consequences on visual acuity in RR-MS patients bearing the T allele. Finally, the T allele was associated with preserved cognitive abilities at the Rao’s brief repeatable neuropsychological battery in RR-MS. Signaling through glutamate NMDARs enhances both compensatory synaptic plasticity and excitotoxic neurodegeneration, impacting in opposite ways on RR-MS and PP-MS pathophysiological mechanisms.