Interleukin-6 triggers human cerebral endothelial cells proliferation and migration: The role for KDR and MMP-9

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Abstract

Interleukin-6 (IL-6) is involved in angiogenesis. However, the underlying mechanisms are unknown. Using human cerebral endothelial cell (HCEC), we report for the first time that IL-6 triggers HCEC proliferation and migration in a dose-dependent manner, specifically associated with enhancement of VEGF expression, up-regulated and phosphorylated VEGF receptor-2 (KDR), and stimulated MMP-9 secretion. We investigated the signal pathway of IL-6/IL-6R responsible for KDR’s regulation. Pharmacological inhibitor of PI3K failed to inhibit IL-6-mediated VEGF overexpression, while blocking ERK1/2 with PD98059 could abolish IL-6-induced KDR overexpression. Further, neutralizing endogenous VEGF attenuated KDR expression and phosphorylation, suggesting that IL-6-induced KDR activation is independent of VEGF stimulation. MMP-9 inhibitor GM6001 significantly decreases HCEC proliferation and migration (p < 0.05), indicating the crucial function of MMP-9 in promoting angiogenic changes in HCECs. We conclude that IL-6 triggers VEGF-induced angiogenic activity through increasing VEGF release, up-regulates KDR expression and phosphorylation through activating ERK1/2 signaling, and stimulates MMP-9 overexpression.

Section snippets

Materials and methods

Human cerebral endothelial cell culture. HCECs were purchased from Cell systems (St. Katharinen, Germany) and their homogeneous nature was confirmed by immunostaining with CD-31 antibody. Cultured cells were maintained at 37 °C in 5% CO2/95% ambient mixed air and the culture media were changed every two days. All experiments were performed on HCEC 2–4 passages. In IL-6 treatment experiments, HCECs were grown in the EC growth medium (Clonetics, Walkersville, MD) in 35 mm polystyrene plates to 90%

Effect of IL-6 on VEGF expression in HCECs

Involvement of IL-6 in angiogenesis is related to VEGF production [16]. Because mitogenic activity of HCECs could be stimulated by VEGF or IL-6, we therefore investigated the interaction between IL-6 and VEGF expression. The data showed that IL-6 stimulates VEGF expression by two-fold in both mRNA and protein levels as compared to the control group (Figs. 1A and B). Dose–response results indicated that this effect was maximal at 100 ng/ml IL-6. The maximal increase in the mRNA expression was at

Discussion

Our results provide evidence that IL-6 augments VEGF-induced HCEC proliferation and migration, up-regulates KDR phosphorylation, and enhances MMP-9 activity. Our findings reveal a physiologically important feedback mechanism for the amplification of the angiogenic response in vitro, which may be relevant in many pathological situations.

IL-6 can be produced by HCECs and IL-6 receptors are expressed throughout CNS, including endothelial cells, astrocytes, microglia, and neurons [3], [4]. The

Acknowledgments

The authors thank Voltaire Gungab for editorial assistance and the collaborative support of the staff of the Center for Cerebrovascular Research <http://avm.ucsf.edu/>.

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    The study was supported by a Grant from the National Institutes of Health P01 NS44144 (GYY, WLY), R01 NS27713 (WLY), and R21 NS45123 (GYY).

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