Nicole Girard

Hyaluronan and hyaluronectin in the extracellular matrix of human brain tumour stroma

Hyaluronan (HA) and the hyaluronan-binding glycoprotein hyaluronectin (HN) were measured in 23 gliomas and 8 meningiomas and their location was revisited in 35 tumours. A clear-cut difference was found in the HN/HA ratio values of glioblastomas (below 0.5) and that of astrocytomas (above 0.5 P < 0.001). Besides their location in the intercellular part of gliomas, HA and HN displayed a perivascular location in 1/3 astrocytomas, 17/24 glioblastomas, and 3/7 meningiomas, suggesting they could be produced also by the vascular stroma of tumours and that they would characterise the neoangiogenesis. All cultivated glioma cells tested produced HA in vitro, whereas only 1/11 cell lines produced HN, at a low level. The results obtained suggest that glioma HA and HN are produced by both cancer cells and vascular stroma cells, which contribute to the edification of the extracellular matrix. In meningiomas only the stroma would be responsible for HA and HN production.

Increased hyaluronidase levels in breast tumor metastases

Hyaluronidase, a matrix‐degrading enzyme, was assayed in extracts from breast primary tumors and regional metastases using a pool of human sera as a standard. Optimal activities of tumor extracts and serum were found for concentrations of 0.15–0.20 M NaCl in pH 3.8–4.0 buffer. In evaluating contamination by serum due to vascular proliferation, we expressed our results as the ratio of the entire activity (mU/l extract) on serum albumin content of tumors (g/l). Median (interquartile range) activities were 9.02 (6.04–14.34) for primary tumors and 37.36 (24.06–99.63) mU/g albumin for metastases. The difference was significant. Zymographic analysis showed that 3 bands of activity were detected which corresponded to 68, 53 and 49 kDa for tumoral hyaluronidase. The same pattern was observed for cellular extracts of breast cancer cell line CAL51, demonstrating that hyaluronidase detected in tumor extracts had mainly a cellular origin. Our results suggest that hyaluronidase may be involved in the metastatic process. Int. J. Cancer 73:327–331, 1997.

Localization of Hyaluronectin in the nervous system

The localization of hyaluronectin was determined by immunofluorescence and immunoperoxidase methods, in the rat, the sheep and the human. The study of the peripheral nervous system revealed the localization of this protein at the node of Ranvier. It was also present at this site in the central nervous system where the appearance was less characteristic than in the peripheral nervous system. The protein was also observed around about 10% of neurones in all of the regions studied. The subcellular structures labelled could not be precisely defined with the optical microscope.

CD44 and Hyaluronan Binding by Human Myeloid Cells

The CD44 cell surface molecule has been shown to be the principal cell surface receptor for hyaluronan (or hyaluronic acid), a glycosaminoglycan component of marrow extracellular matrix. However, its affinity for hyaluronan is not constitutive, since it depends on the cell type, the stage of differentiation and on activation by external stimuli including certain anti-CD44 antibodies and phorbol esters. Except for a few lymphoid cell lines, hematopoietic cells do not spontaneously bind hyaluronan and initial studies reported that, contrary to lymphocytes, myeloid cells could not be activated to bind hyaluronan. Because CD44 plays an important role in the initial phases of hematopoiesis, as shown by experiments using blocking anti-CD44 monoclonal antibodies, its capacity to mediate adhesion of primitive myeloid cells has been investigated. It was found that CD44 could mediate spontaneous adhesion to hyaluronan of immature myeloid cell lines KG1, KG1a, and TF1, which serve as a model for hematopoietic progenitors. However, despite expressing high amounts of CD44, no more than 15% of bone marrow progenitors could adhere to hyaluronan. Recent experiments have shown that a very important feature of CD44 is its capacity to be rapidly activated by certain antibodies and cytokines (GM-CSF and KL) from a low affinity to a high affinity state for hyaluronan. These data shed light on striking similarities in the functional regulation of CD44 and of the two integrin receptors VLA-4 (a4b1), and VLA-5 (a5b1), which are also expressed on hematopoietic progenitors. The relevance of these data to the regulation of normal hematopoiesis and mobilization of CD34+ progenitors in the view of cell grafting is analyzed. In addition, we show that in idiopathic myelofibrosis, the amount of hyaluronan is markedly increased in the extracellular matrix from the myeloproliferative spleen. Considering that the production of cytokines is enhanced in this disease, we discuss whether CD44-hyaluronan interaction may have a role in the pathophysiology of this myeloproliferative syndrome.

Human breast-cancer metastasis formation in a nude-mouse model: Studies of hyaluronidase, hyaluronan and hyaluronan-binding sites in metastatic cells

Few animal models are available to study metastasis formation. The purpose of the present study was to obtain a useful model of metastasis formation in nude mice in an attempt to analyze the stroma reaction and in particular the production and the expression of hyaluronan (HA), hyaluronidase, and HA‐binding sites by cultivated cells, and HA and hyaluronectin (HN) in the invasive areas of tumors. Nude mice were subjected to i.p. injections of several human cancer cell lines (PLC/PRF/5, HepG2, CB 191, CB 193, PC3, CAL 51, SA 87 and SA 98), and formation of metastases was analyzed in different organs (lung, liver, kidney, spleen and axillary nodes) by immunohistochemical techniques. CAL 51, a breast‐cancer‐metastasis‐derived cell line with a normal karyotype, produced i.p. tumors in 75% animals and metastases in 90% animals (detected in the liver and axillary nodes). Two modes of invasion by CAL 51 cells were observed in the liver: one, direct, from the surface of the liver and the other, indirect, via the bloodstream. HA and HN were strongly expressed at the invasion areas. A cell line derived from hepatic metastasis of CAL 51 (HMD CAL 51) presented an abnormal karyotype. HMD CAL 51 produced more hyaluronidase (12‐fold) and HA (10‐fold) and expressed more CD44 (1.6‐fold) and other HA‐binding sites (9.5‐fold) than the established cell line CAL 51. Our results show that i.p. injection of the CAL 51 cell line into nude mice provides a useful model of metastasis formation. The passage of the CAL 51 cells from the primary state to the metastatic state was characterized by a dramatic increase of HA and hyaluronidase production, and expression of HA, HN and HA‐binding sites. Int. J. Cancer 82:77–83, 1999.

Expression and Effects of Hyaluronan and of the Hyaluronan‐Binding Protein Hyaluronectin in Newborn Rat Brain Glial Cell Cultures

Abstract: Hyaluronan (HA) is a polymerized nonsulfated extracellular matrix glycosaminoglycan that may be involved in brain development. We have tested the expression of HA and the HA‐binding protein hyaluronectin (HN) in glial cell cultures from newborn rat brain. HA was secreted into the culture medium by type 1 astrocytes in the first stages of the primary cultures. The secretion was high during cell proliferation, reached a maximum when they were confluent, and then decreased. HA was not secreted at a detectable level by total O‐2A lineage cell‐ enriched cultures. HA labeled small O‐2A progenitor cells (GFA‐, A2B5+, HA+), small O‐2A progenitorlike (GFA−, A2B5−, HA+) cells, and type 2 astrocytes (GFA+, A2B5+, HA+), but not mature oligodendrocytes (Galc+, HA−). In contrast to HA, hyaluronectin labeled oligodendrocyte membranes (i.e., more mature cells) from day 8. A2B5+ GFA− cells were found to be either HA+ or HN+ at days 7–9, suggesting intermediary stages. The addition of HA to primary cultures and to O‐2A progenitor‐enriched cultures decreased significantly the increase in the number of O‐2A progenitors, of mature (Galc+) oligodendrocytes proportionally to the decrease of the O‐2A progenitor number, and of BrdU+ cells, suggesting that HA acts (directly or indirectly) on O‐2A cell proliferation. This effect, which was seen for concentrations as low as 0.1 μg/ml, was HA specific and was not observed with other glycosaminogly‐ cans. When primary cultures were performed in the presence of hyaluronidase‐digested or HA‐depleted (by passage on a HN column) fetal calf serum, the total number of O‐2A lineage cells was dramatically increased (100%, p<10–4) in comparison with control cultures in standard fetal calf serum. Platelet‐derived growth factor increased the total number of O‐2A lineage cells and of (Galc+) oligodendrocytes. This effect was opposed by HA dose dependently. The effect of HA was significantly inhibited by HN (30%, p<10–4). HN had, however, no effect when it was added to culture in the presence of hyaluronidase in fetal calf serum, suggesting its effect was only due to its binding to HA. During cell maturation, HA disappears as HN appears. This and the fact that HA and PDGF have opposite effects suggest an effect of these factors, or of their balance, on myelination.

Characterization of hyaluronic acid on tissue sections with hyaluronectin.

An affinity immunological procedure for hyaluronic acid detection on tissue sections is described. This new, sensitive, and specific technique is based on the high affinity of hyaluronectin for hyaluronic acid, utilizing anti-hyaluronectin-hyaluronectin immune complexes. Elimination of binding when the reagent was supplemented with hyaluronic acid or when Streptomyces hyaluronidase-digested tissue sections were used emphasizes the specificity of the assay. This technique made possible accurate HA localization in embryonic mesenchyme, in neural tissue, in kidney medulla, and in tumors.

The origin of hyaluronectin in human tumors.

The origin of tumor stroma hyaluronectin (HN), a glycoprotein that binds to hyaluronan (HA), has long remained unknown. Histological observations of human tumors suggest that tumor HN could originate from stroma fibroblasts, and in some cases from inflammatory cells. The fibroblast origin was confirmed by the discovery of HN‐like antigen along with hyaluronan in culture medium of tumor‐derived fibroblasts. An HA‐binding protein was characterized in the culture medium of peripheral blood mononuclear cells (PBMC) in both normal subjects and tumor‐bearing patients and was found to be human HN. Cultivated monocytes did not produce HA. HN was not related to the HA‐binding site CD44. Sequencing of brain HN‐derived peptides demonstrated that each determined peptide sequence was similar to a sequence of the proteoglycan PG‐M/versican, suggesting that HN is the HA‐binding moiety of the proteoglycan. One probe was synthesized from human PBMC by polymerase chain reaction with primers derived from HN sequences also found in versican. Northern blots were positive only with HN‐producing cells. The main RNAs were in the 6–8 kb range, and there was a limited proportion of smaller RNA, which was compatible with the size expected from the HN molecular mass. Southern blotting of monocytes and tumor cells demonstrated that the gene was limited to a unique band. We conclude that HN, an extracellular component of brain, connective embryonic, inflammatory and tumoral tissues, is a PG‐M/versican‐derived molecule. Our results suggest that tumor HN, which originates from fibroblasts and monocytes of tumor stroma, is a molecular component of the host‐tumor relationship and could play a role in the regulation of HA activity in oncogenesis. Int. J. Cancer 72:942–948, 1997.

Characterization of a hyaluronic acid-binding protein from sheep brain comparison with human brain hyaluronectin.

1. A hyaluronic acid (HA)-binding glycoprotein from sheep brain was characterized. 2. The specific affinity for HA was shown in vitro by high performance liquid chromatography, polyacrylamide gel electrophoresis and ELISA methods. 3. The KD for high molecular weight HA was 5.4 10(-9) M at 37 degrees C and lower than 10(-10) M at 4 degrees C. 4. No link protein was found and HA molecules could bind up to 10 times their weight of the glycoprotein. 5. The specific site for interaction was the HA-derived decasaccharide HA10. 6. The protein is composed of one polypeptidic chain. Tryptophan and lysine play a prominent role in the conformation of the binding site to HA. 7. Enzyme analysis indicated that the protein different forms are due to differences in glycosylation and that N- and O-linkages coexist in the molecules. 8. Immunohistochemistry localized the glycoprotein at the nodes of Ranvier and at the periphery of neurons. The perineuronal labeling was seen around all neurons studied in the cerebellum whereas it was almost undetectable in the cerebral hemispheres. 9. HA is not saturated by hyaluronectin (HN) in the sheep nervous system. 10. The glycoprotein is largely similar to human brain HN, and different from the hyaluronate-binding protein characterized in the cartilage.

Caffeine decreases glial cell number and increases hyaluronan secretion in newborn rat brain cultures.

ABSTRACT: Newborn rat brain astrocytes (type 1 astrocytes, O-2A progenitor cells, and O-2A progenitor-derived cells, i.e. oligodendrocytes and type 2 astrocytes) were cultivated to investigate the effect of addition of caffeine to the culture medium on glial cell development and secretion of hyaluronan (hyaluronic acid, HA). HA is a glycosaminoglycan, secreted by type 1 astrocytes especially, which is a major component of the extracellular matrix of immature brain involved in morphogenesis and differentiation. Caffeine was added to the culture medium of primary glial cell cultures at concentrations of 102 μM (20 mg/L) or 255 μM (50 mg/L), considered therapeutic and toxic levels, respectively, in human newborns. HA was measured in the culture medium by immunoenzyme assay using sheep brain hyaluronectin, a glycoprotein that exhibits a strong affinity for HA, as probe. In primary glial cell cultures, 102 μM (20 mg/L) caffeine had no visible effect on cell number or on HA secretion. At 255 μM (50 mg/L), there was a significant reduction of cell number (i.e. type 1 astrocytes, O-2A progenitor cells, and progenitor-derived cells) and a significant increase of HA secretion per cell. These results suggest that caffeine at a high concentration in brain could have a prejudicial effect on the number of proliferating glial cells (astrocytes and oligodendrocytes) and on the composition of the extracellular matrix, which could affect myelination onset.