Hyaluronan as a modulator of VEGF expression in immune cells

SPINELLI FIORELLA; VITALE DAINA; DEMARCHI G; CRISTINA C; ALANIZ L

Florencia

Conferencia; 10th International Conference Hyaluronan 2015; 2015

IHAS

Hyaluronan (HA) is able to regulate several biological processes that include angiogenesis, not only in normal but also in cancer tissues (1). Besides, increasing evidence suggests hyaluronan as a modulator of immune responses (2,3). Biological functions of HA are mediated by its interaction with different cell surface receptors, being CD44 the main one (2). Moreover, Toll-like receptors (TLR) 2 and 4 have been reported to bind HA fragments, having an important role in innate immunity and dendritic cells function (2).Vascular endothelial growth factor (VEGF) is the key pro-angiogenic factor. The main mechanism of tumor angiogenesis is endothelial cell (EC) sprouting which crucially depends on VEGF and the interaction between ECs, pericytes, stroma cells as well as their association with the ECM(4). Recent studies shown that immune cells can secrete angiogenic factors, such as VEGF, that might modulate tumor angiogenesis(4) and in consequence affect efficacy of antiangiogenic therapy. Objective: To evaluate the action of HA as a modulator of VEGF expression in spleen cells and to investigate the mechanisms by which CD44-HA and TLR4-HA are involved in this angiogenic modulation in immune cells. Methods: Spleen cells were isolated from CD44-/-, TLR4-/-and WT mice and were then treated with different concentrations of low molecular weight (LMW) HA. VEGF mRNA expression levels were analyzed by real-time PCR and VEGF protein levels in supernatants were measured by ELISA. Angiogenic function was evaluated by tube formation assays in EC using Matrigel. Results: In basal conditions, VEGF expression detected by real-time PCR in WT spleen cells was significant higher than those of CD44-/-and TLR4-/-. When WT spleen cells were stimulated with LMW HA the expression of VEGF was diminished. However, CD44-/-spleen cells incubated with LMW HA the VEGF expression was higher than basal condition, while in TLR4-/-spleen cells its expression was down-regulate. VEGF protein was not detected in the supernatants of WT and CD44-/-spleen cells, but it was detected in TLR4-/-cells, and LMW HA treatment diminish it expression. Supernatants from spleen cells treated with HA could modulate EC tube formation in vitro. Conclusion: We suggest that HA could modulate angiogenic action in immune cells, but its regulation depends on HA receptors. Because CD44 absence allows increased expression and function of VEGF, however the lack of TLR4 expression was associated with a decrease in mRNA and protein level of this factor. Indicating that TLR4 receptor could mediate the pro-angiogenic effects of HA in immune cells. Our results provide the first evidence about the rol of HA in the regulation of tumor angiogenesis by immune cells. 1.Toole BP (2004) Nature Reviews Cancer 4:528-39.2.Jiang D et al. (2011) Physiological Reviews 91:221-64. 3.Alaniz L et al (2009) Mini reviews in medicinal chemistry 9:1538-46. 4. Stockmann C et al (2014). Frontiers in oncology 4:69.Financial support: Fundación Fiorini 2014, CONICET 3892-13.