Search

Standardin vitrocell culture is one of the pillars of biomedical science. However, there is increasing evidence that 2D systems provide biological responses that are often in disagreement within vivoobservations, partially due to limitations in reproducing the native cellular microenvironment. 3D materials that are able to mimic the native cellular microenvironment to a greater extent tackle these limitations. Here, we report Porous yet Dense (PyD) type I collagen materials obtained by ice-templating followed by topotactic fibrillogenesis. These materials combine extensive macroporosity, favouring the cell migration and nutrients exchange, as well as dense collagen walls, which mimic locally the Extracellular Matrix. When seeded with Normal Human Dermal Fibroblasts (NHDFs), PyD matrices allow for a faster and more extensive colonisation when compared with equivalent Non-Porous matrices. The textural properties of the PyD materials also impact cytoskeletal and nuclear 3D morphometric parameters. Due to the effectiveness in creating a biomimetic 3D environment for NHDFs and the ability to promote cell culture for more than 28 days without subculture, we anticipate that PyD materials could configure an important step towardsin vitrosystems applicable to other cell types and with higher physiological relevance.

The effects of an increase in intracellular cAMP concentration on proteoglycan (PG) synthesis by peritubular (PT) cells from immature rat testis were investigated. In the presence of dBcAMP for 72 h, the [3H]-hexosamine incorporation in secreted PG and in cell-associated PG was reduced, whereas [35S]-sulfate radioactivity was enhanced in secreted PG and not affected in cell-associated PG. Cholera toxin and IBMX, known to generate high intracellular cAMP levels, induced similar changes. Cyclic AMP did not alter PG protein moiety synthesis but enhanced PG turnover. Cholera toxin and dBcAMP profoundly modified PG characteristics: (1) Apparent molecular weight of PG was increased. (2) This was due to an increase in glycosaminoglycans (heparan sulfate (HS) and chondroitin sulfate (CS)) length. (3) The number of glycosaminoglycan chains was presumably reduced. (4) Heparan sulfate and chondroitin sulfate chains of medium and cell layer-associated PG appeared oversulfated. (5) The pattern of cell layer associated PG was modified with a decrease in HSPG and a correlative increase in CSPG. Cholera toxin and dBcAMP also dramatically stimulated hyaluronan synthesis by possible phosphorylation induced activation of hyaluronan synthase(s).

The exposure of confluent peritubular (PT) cells from immature rat testis to insulin-like growth factor-1 (IGF-1) induced a time and dose-dependent increase of [35S]-sulfate and [3H]-d-glucosamine incorporations in newly synthesized proteoglycans (PG). This increased content of PG was the result of an enhancement of PG synthesis rather than a decreased rate of degradation. IGF-1 had no effect on the molecular weight of synthesized PG nor on the nature and distribution of the constitutive glycosaminoglycan chains, both in medium and in cell layer. The stimulation of PG synthesis by IGF-1 appeared to be due, at least partially, to an increase of glycosylation processes. IGF-1 effect was mediated by the classical tyrosine kinase signalling process, since IGF-1 action on PG synthesis was abolished by genistein and tyrphostin A9, two well known tyrosine kinase inhibitors. The increase of PG synthesis was accompanied with an undersulfation of constitutive glycosaminoglycan (GAG) chains (chondroitin sulfate and heparan sulfate chains) since the [35S]/[3H] ratio was reduced by about 20–25% in presence of IGF-1. Although the mechanism of hyaluronic acid synthesis was completely different from those of other GAG, IGF-1 also dramatically enhanced its production by PT cells.

Confluent testicular peritubular cells derived from immature rats were used to study membrane associated proteoglycans (PG). Peripheral material (heparin releasable), membrane and intracellular material (Triton X-100 releasable) were collected, purified by anion exchange chromatography then characterized by gel filtration and by hydrophobic interaction chromatography, followed by enzymatic digestion and chemical treatment. The peripheral material was constituted of two populations of PG (Kav = 0 and 0.10 on Superose 6 column), each containing both heparan sulfate proteoglycans (HSPG) and chondroitin proteoglycans (CSPG) and perhaps a hybrid PG (HSCSPG). These PG being not retained on an octyl Sepharose column, they were devoided of hydrophobic properties. The integral membrane proteoglycans isolated on the basis of their hydrophobic properties represented 20% of the Triton X-100 releasable material, and were exclusively constituted of proteoheparan sulfate. There were no relationships between this membrane HSPG and the peripheral HSPG as evidenced by pulse chase experiments. The mode of intercalation of the hydrophobic HSPG in the cell membrane was studied. The majority of these macromolecules (80%) were sensitive to trypsin and only a minor proportion (20%) were sensitive to phosphatidylinositol specific phospholipase C. Thus, about 80% of the hydrophobic HSPG were intercalated in the cell membrane by a hydrophobic segment of the core protein whereas about 20% were associated with the cell membrane via a phosphatidylinositol residue covalently bound to the core protein of the PG.

Like the majority of tumor cells, ovarian cancer cell growth is critically dependent on their neovascularization. Adhesion molecules and cellular events that lead to ovarian tumor cell interactions with endothelial extracellular matrix surrounding the vasculature are poorly identified. To understand the role of alphavbeta3 integrin and its ligand fibronectin in this process, we used in vitro coculture models with IGROV1 human ovarian adenocarcinoma cell line and human umbilical vein endothelial cells (HUVEC). Adhesion assays revealed a strong ability of IGROV1 cells to adhere to HUVEC-ECM. alphavbeta3 is mainly implicated and seems to cooperate with alpha5beta1 integrin in this event. Immunofluorescence staining revealed the presence of alphavbeta3 and alpha5beta1 in IGROV1 cells adhering on HUVEC-ECM at regions of cell sub-stratum contacts. Furthermore, our data showed the absence of fibronectin staining in IGROV1 cells and the disruption of the HUVEC-ECM fibrillar fibronectin network under IGROV1 cell influence. In situ experiments in ovarian neoplastic tissue corroborated the absence of fibronectin in the tumor and its strong detection in vasculature. These findings suggest the active participation of alphavbeta3 and alpha5beta1 integrins and the reorganization of endothelial fibronectin during the adhesion of IGROV1 cells to HUVEC-ECM whereas IGROV1 cells seem to be unable to synthesize fibronectin. Thus, fibronectin integrin receptors expressed by ovarian tumor cells and endothelial fibronectin may be of importance in ovarian carcinoma neovascularization and during tumor-vasculature interactions.

The influence of phorbol myristate acetate (PMA), dibutyryl cAMP and insulin-like growth factor (IGF-1) as well as cytoskeletal disrupting drugs on morphological changes has been studied in peritubular cells isolated from immature rat testis. Morphological studies were combined with immunofluorescence investigations of cytoskeletal elements and their rearrangements by various agents. The results were correlated with modulation of proteoglycan synthesis. Peritubular cells exposed to dibutyryl cAMP or cytochalasin D were transformed from flattened, fibroblast-like into neuronal-like morphology. In such cells, destruction of actin filaments was accompanied with a 50% decrease in cell-associated proteoglycan synthesis as well as with oversulfation of total proteoglycans. On the contrary, peritubular cell shape has been slightly altered after addition of PMA, IGF-1, vinblastine or colchicine. After these treatments, destruction or rearrangement of cytoskeletal elements was observed; cell-layer proteoglycan synthesis remained either unchanged or increased while total proteoglycans were always undersulfated. IGF-1, PMA and dibutyryl cAMP modified the peritubular cell morphology, cytoskeletal organization and proteoglycan production; the cytoskeleton disrupting drugs such as vinblastine, colchicine and cytochalasin D mimicked some of these effects. These observations suggest that alterations in proteoglycan biosynthesis, after activation of tyrosine kinase, protein kinase C and protein kinase A pathways might be mediated, at least in part, by the disorganization of the cytoskeleton structure.

In cultured peritubular cells (PT) from rat testis, protein kinase C (PKC) was activated by phorbol 12-myristate 13-acetate (PMA). PMA enhanced the synthesis of proteoglycans (PG) and to a lesser extent their catabolism; the stimulation of the synthesis appeared to be due to an increase in PG protein moiety production and, at the same time, to an increase in the glycanation process as revealed by the use of an exogenous acceptor, p-nitrophenyl-beta-d-xyloside. In the presence of PMA, the molecular weight of neosynthesized PG and the length of their constitutive glycosaminoglycan chains were not modified. Moreover, the distribution of proteochondroitin sulfate and proteoheparan sulfate in medium and in cell layer remained unchanged. However, PMA reduced the sulfation level of chondroitin sulfate and heparan sulfate chains, suggesting that PKC activation resulted in an independent modulation of the sugar chain formation and of the sulfate residue transfer. PMA effect on the synthesis of hyaluronan was also determined: PMA dramatically enhanced its production by PT cells.