Interaction of inflammatorily activated retinal pigment epithelium with retinal microglia and neuronal cells.

In age-related macular degeneration, inflammatory events are presumed to contribute to disease development. A primary suspect of this contribution is the microglia, the innate immune cell of the retina. In addition, retinal pigment epithelium (RPE) cells can be inflammatorily activated. In this study, we investigate the effect of activated RPE cells on retinal microglia and on neuronal cells. RPE cells and microglia were harvested from porcine eyes. In addition, a neuronal cell line (SHSY-5Y) of human origin was used. For inflammatory activation, agonists of toll-like receptors in different concentrations were used: Pam2CSK4 (Pam; TLR-2), Polyinosinic:polycytidylic acid (Poly I:C; TLR-3) and lipopolysaccharid (LPS; TLR-4). Cell viability was investigated with an MTT assay. The secretion of cytokines was assessed in an ELISA and their expression in real-time PCR. There was no effect of the agonists on cell viability in RPE cells. All agonists induced the secretion of IL-6 and IL-8 in RPE cells with the strongest effect induced by LPS. In microglia, pro-inflammatory stimulation increased the metabolic activity. All agonists induced the secretion of IL-1ß, IL-8, and TNFα in microglia cells while in real-time PCR, LPS and Pam induced the expression of IL-6, IL-1ß and iNOS. Direct stimulation of SHSY-5Y with the agonists induced only minor alterations of viability. Stimulated RPE cell supernatant reduced the secretion of TNFα and IL-8 irrespective of the inducing agent in microglia cells. Additionally a slight induction of IL-1ß was found in microglia treated with supernatant of RPE cells treated with Pam. In real time PCR, the supernatant of RPE cells stimulated with LPS significantly reduced the expression of iNOS and IL-6, but not of IL-1ß. Of note, the expression of iNOS was also reduced by naive RPE cells. The treatment of the SHSY-5Y with supernatant of microglia previously treated with RPE conditioned medium significantly decreased SHSY-5Y viability with and without pro-inflammatory treatment. In conclusion, inflammatory activated RPE cells have a regulatory effect on the pro-inflammatory activation of microglia, stressing the importance of the interaction between these two retinal cell types. Microglia treated with RPE supernatant reduced viability of a neuronal cell line, indicating a neurotoxic effect.

The multifunctional growth factor midkine promotes proliferation and migration in pancreatic cancer.

UNLABELLED: Pancreatic ductal adenocarcinoma (PDAC) has a devastating prognosis among solid tumors and despite increased knowledge of the molecular mechanisms contributing to progression and metastasis, minimal progress has been done in establishing new targeted therapies for this deadly disease. The expression of the multifunctional growth/differentiation factor midkine (MK) promotes a variety of cellular functions leading to increased angiogenesis, proliferation, migration, and survival. Moreover, MK is intensively discussed as a potential new-therapy target and as biomarker for cancer progression and chemotherapeutic resistance in multiple cancers. Therefore, the present study investigated the molecular role of MK in pancreatic cancer. It was found that MK is elevated in PDAC and differentially expressed in other histologic subtypes of pancreatic cancer, whereas normal pancreatic cells did not express MK, thus making it an attractive candidate for targeted therapies. As a secreted growth/differentiation factor, MK was investigated as a biomarker in clinical serum specimens using ELISA. In addition, knockdown studies of MK revealed a link to proliferation and migration status in vitro. Finally, upstream signaling pathways were analyzed, with TNF-α and EGF being the main inductors of MK expression in PDAC. IMPLICATIONS: This study presents novel MK functions and new upstream signaling effectors that induce its expression to promote PDAC and therefore defines an attractive new therapeutic target in pancreatic cancer.