Shift of syndecan-1 expression from epithelial to stromal cells during progression of solid tumours.

Syndecan-1 (SDC-1), a protein found on cells and in the extracellular matrix, participates in cell proliferation, cell migration and cell-matrix interactions. SDC-1 expression correlates with the maintenance of epithelial morphology and inhibition of invasiveness. In the present study, a second SDC-1 mRNA isoform was identified and the expression of both transcripts was investigated in various normal and malignant tissues. Both transcripts were coexpressed at equal levels in all tissues and organs analysed. Cancer-profiling array (CPA) analysis of 241 non-enriched tumour and normal cDNAs revealed stronger upregulation of SDC-1 in tumour tissues as compared with oligonucleotide array-based expression analysis of SDC-1 in microdissected breast, prostate, lung, and colon carcinoma cells. With in situ hybridisation and immunohistochemistry it was demonstrated that this difference in SDC-1 expression originates from stromal cells present in tumour connective tissue. But only the cells in connective tissue surrounding breast, lung, colon and bladder carcinoma showed upregulation of SDC-1. These stromal cells were characterised as spindle cells with myofibroblastic differentiation and they may contribute to the dedifferentiation of tumour cells and the development of metastasis.

Human SPRY2 inhibits FGF2 signalling by a secreted factor.

Growth factor signalling pathways and their inhibitors coordinate the formation of three-dimensional patterns of vertebrates and invertebrates. Temporal and spatial restriction of the response to a few well-defined cells is crucial and needs the integration of positive and negative signals. Recently, Spry has been identified as an inhibitor of fibroblast growth factor (FGF) signalling during Drosophila trachea development. Spry has been described as an intracellular protein that can exert its function in a cell autonomous or a paracrine manner. Here we describe the role of SPRY2, a human homologue of Spry, in human FGF2 signalling. We show that in primary human dermal endothelial cells (MVEC) SPRY2 mRNA is transiently upregulated in response to FGF2. Overexpression of SPRY2 in A375 cells leads to the secretion of a soluble factor that inhibits FGF2- but not VEGF-stimulated proliferation of MVEC. Direct administration of recombinant SPRY2 protein has no effect on MVEC proliferation. However, SPRY2 protein binds the intracellular adaptor protein GRB2, indicating an intracellular localization. A SPRY2/GFP fusion protein remains in the cell, further supporting the intracellular localization of SPRY2. So the intracellular protein SPRY2 is involved in the non-cell autonomous inhibitory effect indirectly, via regulating the secretion of an inhibitor of FGF2 signalling in vertebrates, the evidence of which is presented here for the first time.

Differential gene expression by endothelial cells in distinct angiogenic states.

Angiogenesis is a complex process that can be regarded as a series of sequential events comprising a variety of tissue cells. The major problem when studying angiogenesis in vitro is the lack of a model system mimicking the various aspects of the process in vivo. In this study we have used two in vitro models, each representing different and distinct aspects of angiogenesis. Differentially expressed genes in the two culture forms were identified using the suppression subtractive hybridization technique to prepare subtracted cDNA libraries. This was followed by a differential hybridization screen to pick up overexpressed clones. Using comparative multiplex RT-PCR we confirmed the differential expression and showed differences up to 14-fold. We identified a broad range of genes already known to play an important role during angiogenesis like Flt1 or TIE2. Furthermore several known genes are put into the context of endothelial cell differentiation, which up to now have not been described as being relevant to angiogenesis, like NrCAM, Claudin14, BMP-6, PEA-15 and PINCH. With ADAMTS4 and hADAMTS1/METH-1 we further extended the set of matrix metalloproteases expressed and regulated by endothelial cells.

PTK787/ZK 222584, a novel and potent inhibitor of vascular endothelial growth factor receptor tyrosine kinases, impairs vascular endothelial growth factor-induced responses and tumor growth after oral administration.

PTK787/ZK 222584 (1-[4-chloroanilino]-4-[4-pyridylmethyl] phthalazine succinate) is a potent inhibitor of vascular endothelial growth factor (VEGF) receptor tyrosine kinases, active in the submicromolar range. It also inhibits other class III kinases, such as the platelet-derived growth factor (PDGF) receptor beta tyrosine kinase, c-Kit, and c-Fms, but at higher concentrations. It is not active against kinases from other receptor families, such as epidermal growth factor receptor, fibroblast growth factor receptor-1, c-Met, and Tie-2, or intracellular kinases such as c-Src, c-Abl, and protein kinase C-alpha. PTK787/ZK 222584 inhibits VEGF-induced autophosphorylation of kinase insert domain-containing receptor (KDR), endothelial cell proliferation, migration, and survival in the nanomolar range in cell-based assays. In concentrations up to 1 microM, PTK787/ZK 222584 does not have any cytotoxic or antiproliferative effect on cells that do not express VEGF receptors. After oral dosing (50 mg/kg) to mice, plasma concentrations of PTK787/ZK 222584 remain above 1 microM for more than 8 h. PTK787/ZK 222584 induces dose-dependent inhibition of VEGF and PDGF-induced angiogenesis in a growth factor implant model, as well as a tumor cell-driven angiogenesis model after once-daily oral dosing (25-100 mg/kg). In the same dose range, it also inhibits the growth of several human carcinomas, grown s.c. in nude mice, as well as a murine renal carcinoma and its metastases in a syngeneic, orthotopic model. Histological examination of tumors revealed inhibition of microvessel formation in the interior of the tumor. PTK787/ZK 222584 is very well tolerated and does not impair wound healing. It also does not have any significant effects on circulating blood cells or bone marrow leukocytes as a single agent or impair hematopoetic recovery after concomitant cytotoxic anti-cancer agent challenge. This novel compound has therapeutic potential for the treatment of solid tumors and other diseases where angiogenesis plays an important role.

Modulation of cytokine release from mononuclear cells by prostacyclin, IL-4 and IL-13.

In a previous study, we reported that cicaprost, a stable prostacyclin analogue can inhibit the release of granulocyte-macrophage colony-stimulating factor (GM-CSF) from activated human peripheral mononuclear blood cells (PBMCs). Since interleukin (IL-4) and IL-13 have been shown to inhibit the release of cytokines from PBMCs we tested the hypothesis that prostacyclin in combination with IL-4 or IL-13 can act synergistically to modulate the release of IL-10, generally associated with anti-inflammatory properties, and the pro-inflammatory cytokine tumour necrosis factor alpha (TNF-alpha). For this purpose, PBMCs were isolated over Ficoll, stimulated with lipopolysaccharide (LPS) and incubated in the presence of cicaprost, IL-4 or IL-13. There was a significant reduction in TNF-alpha as well as IL-10 secretion from LPS-stimulated PBMCs following incubation with IL-4 or IL-13. In contrast, cicaprost reduced the secretion of TNF-alpha but led to a slight enhancement of IL-10 release from PBMCs. When LPS-activated PBMCs were incubated in the presence of cicaprost and IL-4 or IL-13 there was a selective, synergistic inhibition of the TNF-alpha release which was not observed for IL-10. Thus, our data suggest that prostacyclin can synergize with cytokines to selectively inhibit the release of pro-inflammatory cytokines from PBMCs.

Vascular endothelial growth factor (VEGF) receptor II-derived peptides inhibit VEGF.

Vascular endothelial growth factor (VEGF) directly stimulates endothelial cell proliferation and migration via tyrosine kinase receptors of the split kinase domain family. It mediates vascular growth and angiogenesis in the embryo but also in the adult in a variety of physiological and pathological conditions. The potential binding site of VEGF with its receptor was identified using cellulose-bound overlapping peptides of the extracytosolic part of the human vascular endothelial growth factor receptor II (VEGFR II). Thus, a peptide originating from the third globular domain of the VEGFR II comprising residues 247RTELNVGIDFNWEYP261 was revealed as contiguous sequence stretch, which bound 125I-VEGF165. A systematic replacement with L-amino acids within the peptide representing the putative VEGF-binding site on VEGFR II indicates Asp255 as the hydrophilic key residue for binding. The dimerized peptide (RTELNVGIDFNWEYPAS)2K inhibits VEGF165 binding with an IC50 of 0.5 microM on extracellular VEGFR II fragments and 30 microM on human umbilical vein cells. VEGF165-stimulated autophosphorylation of VEGFR II as well as proliferation and migration of microvascular endothelial cells was inhibited by the monomeric peptide RTELNVGIDFNWEYPASK at a half-maximal concentration of 3-10, 0.1, and 0.1 microM, respectively. We conclude that transduction of the VEGF165 signal can be interrupted with a peptide derived from the third Ig-like domain of VEGFR II by blockade of VEGF165 binding to its receptor.

Novel antibodies directed against the extracellular domain of the human VEGF-receptor type II.

Vascular endothelial cell growth factor (VEGF) plays a pivotal role in the regulation of angiogenesis by binding to its cognate receptor molecule type II (VEGFr-II, KDR). VEGFr-II is an endothelial cell-specific transmembrane tyrosine kinase important for vascular endothelial cell development and differentiation during embryogenesis, angiogenic processes under physiological conditions, and various diseases. An increasing number of reports indicate that VEGF/VEGFr-II also play a fundamental role for tumor angiogenesis. We present the generation and in vitro characterization of the monoclonal antibodies 2-7-9 and 2-10-1. Both antibodies are highly specific for VEGFr-II as demonstrated by Western blotting and immunoprecipitation. MAbs 2-10-1 and 2-7-9 bind to a disulphide bridge-stabilized epitope within domains 6 and 7 of the human VEGFr-II with an affinity of 8 and 80 nM, respectively. Furthermore, the antibodies are suitable for immunohistochemistry and ELISA techniques. Because both antibodies recognize their epitope on living cells, they also have the potential for drug targeting and diagnostic purposes.

Prostacyclin modulates granulocyte/macrophage colony-stimulating factor release by human blood mononuclear cells.

Although production and immunological activity of granulocyte-macrophage colony stimulating factor (GM-CSF) have been implicated in the pathogenesis of various disorders, little has been reported concerning the factors involved in the regulation of GM-CSF release. Therefore, we examined the effect of the stable prostacyclin agonist, cicaprost, on the in vitro production of GM-CSF by peripheral blood mononuclear cells (PBMC) obtained from normal subjects by enzyme-linked immunosorbent assay (ELISA) and reverse transcriptase polymerase chain reaction (RT-PCR). Incubation of PBMC (10(6) cells/ml1) with the bacterial lipopolysaccharide (LPS; 0.1 microgram/ml) for 24 h caused a more than 10-fold concentration-dependent increase of GM-CSF release (401 +/- 58 pg/ml x 10(6) cells-1). Addition of cicaprost (0.01 ng/ml to 1 microgram/ml) resulted in a concentration- and time-dependent reduction of LPS-induced GM-CSF secretion by PBMC with a mean IC50 of 6.7 ng/ml (n = 9). Furthermore, cicaprost also inhibited the LPS-elicited expression of GM-CSF mRNA, as determined by RT-PCR. These results demonstrate that prostacyclin inhibits LPS-induced GM-CSF release and that its effects are related to the level of transcription. Hence, our data suggest that cicaprost or related PGI2 agonists may represent immunomodulators of mononuclear cell function and may offer a therapeutic approach to GM-CSF-mediated inflammatory disorders.

Splice variants of the human EP3 receptor for prostaglandin E2.

The EP3 receptor for prostaglandin E2 (PGE2) mediates various biological activities such as uterine contraction, inhibition of gastric acid secretion, presynaptic inhibition of neurotransmitter release and potentiation of platelet aggregation. In an attempt to understand the molecular basis of this diversity of biological function, we cloned full-length cDNAs encoding EP3 receptors for PGE2 from human uterus cDNA libraries. Seven cDNA variants were identified which code for six distinct EP3-receptor isoforms. Sequencing revealed that the receptor isoforms differ in their intracellular C-terminal domains. Southern blot experiments indicate that the isoforms are generated by alternative splicing. The EP3-receptor gene is expressed in various tissues with high expression in kidney and pancreas, as demonstrated by Northern blot analysis. All receptors, stably expressed in baby hamster kidney (BHK) cells, bind PGE2 specifically with similar Kd of 2.2-5.8 nM. The binding of [3H]PGE2 is competed with by unlabelled prostaglandins in the order sulprostone (a PGE2-like agonist) approximately PGE2 >> PGF2 alpha > Iloprost (a prostacyclin analogue) > PGD2, which is specific for EP3 receptors. Analysis of the signal-transduction pathways demonstrated that all receptors respond with inhibition of forskolin-induced cAMP accumulation with an IC50 of 0.1-3 nM PGE2. In addition, some isoforms induce an increase in intracellular free calcium ([Ca2+]i) at PGE2 concentrations greater than or equal to 10 nM. These results may offer an explanation for the different physiological responses observed in various tissues following activation of EP3 receptors.

Regulation of U-937 monocyte adhesion to cultured human mesangial cells by cytokines and vasoactive agents.

Leukocyte adhesion to kidney cells is an early event in renal inflammation, such as glomerulonephritis. We developed an experimental model of monocyte adhesion to cultured human mesangial cells. U-937 myelomonocytic leukaemia cells, similar to peripheral blood human monocytes, irreversibly bound to mesangial cell monolayers upon 30-180 min coincubations (to a max. of 13,600 +/- 1100/cm2 monolayer), as assessed by cell counting, U-937 labelling with 3H-thymidine, and colorimetry of nuclear staining with crystal violet. Adhesion was enhanced in mesangial cells proliferating in response to 17% fetal bovine serum, indicating expression of a proinflammatory phenotype. E. coli lipopolysaccharide (LPS), tumour necrosis factor-alpha (TNF-alpha) and protein kinase C activation with phorbol myristate acetate (PMA) potentiated monocyte binding during either coincubation or 24-h pretreatment (0.1 microM PMA, +200 +/- 21%). Binding was also promoted by pretreatment with vasoconstrictors, such as the thromboxane A2 mimetic, U-46619 (10 nM-1 microM, max. +35 +/- 3%), or 1 microM angiotensin II (+64 +/- 4%). To elucidate the mechanisms of monocyte adhesion, we analysed the adhesion molecules expressed by human mesangial cells, employing reverse transcription/polymerase chain reaction to detect ICAM-1, VCAM-1 and E-selectin gene expression. Proliferating cells express VCAM-1 and ICAM-1, confirmed by immunocytochemical staining and 79 +/- 3% inhibition of stimulated adhesion by pretreatment of mesangial cells with an anti-ICAM-1 monoclonal Ab. E-selectin transcription was not detectable.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of human platelets and polymorphonuclear neutrophils by the potent and metabolically stable prostaglandin D2 analog ZK 118.182.

The actions of the novel metabolically stable and selective prostaglandin D2 receptor agonist ZK 118.182 ((5Z,13E)-(9R,11R,15S)-9-chloro-15-cyclohexyl-15- hydroxy-16,17,18,19,20-pentanor-3-oxa-5,13-prostadienoic acid) were studied in human platelets and polymorphonuclear neutrophils in vitro and compared to the naturally occurring agonist prostaglandin D2. ZK 118.182 inhibited collagen and ADP induced platelet aggregation more potently than prostaglandin D2 (IC50: 15 nM versus 60 nM) but was less effective than the stable prostacyclin mimetic iloprost (IC50: 3 nM). The same rank order of potencies was observed for the inhibition of collagen-induced platelet ATP secretion. A dose-dependent activation of adenylate cyclase could be demonstrated by ZK 118.182 which was comparable to that of prostaglandin D2 with respect to the concentration needed for half maximal stimulation (ED50) maximal cAMP level achievable. ZK 118.182 also dose dependently reduced the formyl-methionyl-leucyl-phenylalanine (FMLP) or platelet-activating factor (PAF) induced activation of polymorphonuclear neutrophils. Both, the oxygen burst resulting in the generation of superoxide anions and the degranulation of polymorphonuclear neutrophils accompanied by release of the lysosomal enzyme beta-glucuronidase, were significantly and dose dependently inhibited. ZK 118.182 was more potent than prostaglandin D2 in inhibiting polymorphonuclear neutrophil activation in all tests performed. In summary, ZK 118.182 is a prostaglandin D2 mimetic exerting potent inhibitory effects on human platelets and polymorphonuclear neutrophils.

Human peritoneal mesothelial cells synthesize interleukin-6: induction by IL-1 beta and TNF alpha.

Recent studies have demonstrated increased levels of IL-6 in the peritoneal cavity during CAPD peritonitis. The current investigation was initiated (i) to examine the human peritoneal mesothelial cell (HPMC) as a possible source of this secreted IL-6 and (ii) to characterize the released product and examine its regulation by other cytokines. Unstimulated HPMC under growth arrested conditions released IL-6 in a time dependent manner. After 24-hour HPMC IL-6 release (mean +/- SEM, N = 13) (expressed as pg/micrograms cell protein) was 1.67 +/- 0.33. Stimulation of HPMC with IL-1 beta or TNF alpha resulted in a time (increasing up to 48 hr) and dose dependent IL-6 generation. After 24 hours the levels induced by IL-1 beta and TNF alpha (both at 1000 pg/ml) were (mean +/- SEM, N = 13) 19.08 +/- 2.98 and 6.62 +/- 1.72, respectively. Stimulation with combinations of IL-1 beta and TNF alpha resulted in additive increases in IL-6 release. This release could be inhibited by co-incubation with anti-IL-1 beta and/or anti-TNF alpha antibodies. The level of released HPMC IL-6 measured by immunometric assay (ELISA) correlated directly with that detected in the 7TD1 IL-6 bioassay (r = 0.63; P < 0.001). Western blot analysis of concentrated HPMC supernatants using specific anti-IL-6 antibody demonstrated immunoreactive bands at 23 and 28 Kd following IL-1 beta or TNF alpha treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Ureotelism as the prevailing mode of nitrogen excretion in larvae of the marsupial frog Gastrotheca riobambae (Fowler) (Anura, Hylidae).

1. Embryos from the pouch of the marsupial hylid frog Gastrotheca riobambae excrete urea. 2. Free-living tadpoles of G. riobambae excrete mainly urea, in comparison with tadpoles of other hylid frogs. 3. The activity of arginase is high in embryos from the pouch, and in tadpoles of G. riobambae. 4. The ureotelism of G. riobambae larvae is an adaptation for prolonged incubation in the pouch of the mother, and for development in limited amounts of water.

Stable 9 beta- or 11 alpha-halogen-15-cyclohexyl-prostaglandins with high affinity to the PGD2-receptor.

Various chemically stable prostaglandin analogues were studied for their affinity towards the PGD2-receptor in human platelet membranes in order to define the requirements for specific ligand binding to this receptor. On replacing the 11- or 9-hydroxyl groups of PGF2 alpha by an 11 alpha- or 9 beta-chloro- or fluoro atom, stable prostaglandin analogues were obtained, which showed high affinity towards the PGD2-receptor. The lower side chain consisted of a 15-cyclohexyl group or of the natural 15-n-pentyl group, other substitutents decreased the affinity substantially. The highest PGD2-mimetic activity with a relative affinity of 0.5 to the PGD2-receptor was found in 9-deoxy-9 beta-chloro-16,17,18,19,20-pentanor-15-cyclohexyl-PGF2 alpha (ZK 110 841, compound 16 in Table 1). ZK 110 841 is a chemically stable crystalline substance, which is orally active and which might thus turn out to be an interesting tool for the study of PGD2-receptor interactions. Some other prostaglandin as well as prostacyclin analogues with a 15-cyclohexyl or 15-n-pentyl group exhibited in addition to their known high affinity to the PGE2-receptor of human uterine membranes or the PGI2-receptor of human platelets also affinities to the PGD2-receptor. Generally, the receptor affinities correlate with the activities as stimulators of adenylate cyclase and inhibitors of thrombin induced elevation of cytoplasmic free calcium as well as their ability to inhibit ADP-induced platelet aggregation. The PGI2-character regarding the effector systems prevails in compounds with affinity to both the PGI2- and PGD2-receptor. Compounds which bind to the PGE2- and PGD2-receptor show a flat dose response curve regarding platelet activation suggesting a mixture of pro- and antiaggregatory properties within these molecules.