The expression of growth factors and their receptors in retinal and endothelial cells cocultured in the rotating bioreactor.

Vascular endothelial growth factor (VEGF) has been strongly implicated in the development of choroidal neovascularization, which is seen in age-related macular degeneration. This study investigates whether retinal cells cultured individually or as cocultures with endothelial cells and maintained in the horizontally rotating bioreactor will express more VEGF and VEGF receptors. We measured the expression of VEGF isotypes and VEGF receptors for cells maintained in monolayer and horizontally rotating bioreactor culture at various times by using reverse transcription PCR and Western blot analysis. Retinal cells showed a twofold increase in VEGF-A mRNA expression after five days of culture in the bioreactor, compared with monolayer cultures (77 +/- 3 vs 42 +/- 2, P < .006). Further, we found that the expression of mRNA for VEGF-A growth factor was increased fivefold for retinal cells cocultured with endothelial cell (52 +/- 4 for one day vs 240 +/- 15, P < .001, cultured in the bioreactor for five days). Where the expression of VEGF receptors (FLK-1 and FLT-4) was low for monolayer retinal cultures, we found the expression of both VEGF receptors was higher after 5, 10, and 15 days of culture. Increased expression of these receptors was also found for cocultured retinal/endothelial cells. Further, we found that cultured retinal cells showed higher VEGF-C protein expression compared to monolayer cultures. Our protein analysis data showed that the expression of VEGF-A was increased by twofold (780 +/- 30 for one day vs 1520 +/- 36 for five days, P < .001) after five days in bioreactor cocultures. These findings suggest that retinal/endothelial cell coculture in the horizontally rotating bioreactor may be a very good model for investigating the role of growth factors in the neovascularization seen in human ocular disorders.

Inhibition of cyclooxygenase-2 decreases breast cancer cell motility, invasion and matrix metalloproteinase expression.

BACKGROUND: Cyclooxygenase (COX) is the rate-limiting enzyme that catalyzes the formation of prostaglandins. The inducible isoform of COX (COX-2) is highly expressed in aggressive metastatic breast cancers and may play a critical role in cancer progression (i.e. growth and metastasis). However, the exact mechanism(s) for COX-2-enhanced metastasis has yet to be clearly defined. It is well established that one of the direct results of COX-2 action is increased prostaglandin production, especially prostaglandin E2 (PGE2). Here, we correlate the inhibition of COX-2 activity with decreased breast cancer cell proliferation, migration, invasion and matrix metalloproteinase (MMP) expression. METHODS: Breast cancer cells (Hs578T, MDA-MB-231 and MCF-7) were treated with selective COX-2 inhibitors (NS-398 and Niflumic acid, NA). Cell proliferation was measured by staining with erythrosin B and counting the viable cells using a hemacytometer. Cell migration and invasion were measured using migration and invasion chamber systems. MMP expression was determined by enzyme immunoassay (secreted protein) and real-time quantitative polymerase chain reaction (mRNA). RESULTS: Our results show that there is a decline in proliferation, migration and invasion by the Hs578T and MDA-MB-231 breast cancer cell lines in the presence of either low concentrations (1 microM or lower) NA or NS-398. We also report that MMP mRNA and protein expression by Hs578T cells is inhibited by NS-398; there was a 50% decrease by 100 muM NS-398. PGE2 completely reversed the inhibitory effect of NS-398 on MMP mRNA expression. CONCLUSION: Our data suggests that COX-2-dependent activity is a necessary component for cellular and molecular mechanisms of breast cancer cell motility and invasion. COX-2 activity also modulates the expression of MMPs, which may be a part of the molecular mechanism by which COX-2 promotes cell invasion and migration. The studies suggest that COX-2 assists in determining and defining the metastatic signaling pathways that promote the breast cancer progression to metastasis.

Three-dimensional growth of endothelial cells in the microgravity-based rotating wall vessel bioreactor.

We characterized bovine aortic endothelial cells (BAEC) continuously cultured in the rotating wall vessel (RWV) bioreactor for up to 30 d. Cultures grew as large tissue-like aggregates (containing 20 or more beads) after 30 d. These cultures appeared to be growing in multilayers around the aggregates, where single beads were covered with confluent BAEC, which displayed the typical endothelial cell (EC) morphology. The 30-d multibead aggregate cultures have a different and smoother surface when viewed under a higher-magnification scanning electron microscope. Transmission electron microscopy of these large BAEC aggregates showed that the cells were viable and formed multilayered sheets that were separated by an extracellular space containing matrix-like material. These three-dimensional cultures also were found to have a basal production of nitric oxide (NO) that was 10-fold higher for the RWV than for the Spinner flask bioreactor (SFB). The BAEC in the RWV showed increased basal NO production, which was dependent on the RWV rotation rate: 73% increase at 8 rpm, 262% increase at 15 rpm, and 500% increase at 20 rpm as compared with control SFB cultures. The addition of l-arginine to the RWV cultures resulted in a fourfold increase in NO production over untreated RWV cultures, which was completely blocked by L-NAME [N(G)-nitro-L-arginine-methylester]. Cells in the SFB responded similarly. The RWV cultures showed an increase in barrier properties with an up-regulation of tight junction protein expression. We believe that this study is the first report of a unique growth pattern for ECs, resulting in enhanced NO production and barrier properties, and it suggests that RWV provides a unique model for investigating EC biology and differentiated function.