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 P13K 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. (c) 2006 Elsevier Inc. All rights reserved.

Interleukin- 6 ( IL- 6) may play multiple roles in angiogenesis and vascular remodeling. Our previous study showed that a promoter polymorphism ( 174G > C) in IL- 6 is associated with brain arteriovenous malformation hemorrhage; tissue expression is related to genotype. In this study, we investigated the effects of IL- 6 on human cerebral smooth muscle cells ( HCSMCs) and smooth muscle cells isolated from brain arteriovenous malformation surgical specimens ( AVM SMCs) and surgical controls ( control HCSMCs - from structurally normal temporal lobe taken during surgical treatment of epilepsy patients). We found that IL- 6 ( 1.16 +/- 0.27 versus 0.376 +/- 0.04 pg/ mL, n= 5, P < 0.05) and endogenous vascular endothelial growth factor ( VEGF) receptor II ( kinase domain- containing receptor ( KDR), 15 +/- 3 versus 1.5 +/- 3 pg/ mL, n= 5, P < 0 .05) were increased in brain AVM SMCs compared with control HCSMCs. Further research revealed that IL- 6 could stimulate SMC proliferation, VEGF release, and KDR activation in control HCSMCs. It could also stimulate KDR phosphorylation in control HCSMCs, further confirming a unique role of IL- 6 in the triggering of KDR. Interleukin- 6 could increase matrix metalloproteinase- 9 ( MMP- 9) secretion through activating KDR in control HCSMCs ( P < 0.05 versus control). Inhibiting IL- 6- induced KDR could reduce MMP- 9 activity at least 50% compared with the control group ( P < 0.05). Increased MMP- 9 activity was accompanied by increased control HCSMC proliferation, and blocking MMP- 9 activity significantly reduced IL- 6- induced control HCSMC proliferation ( P < 0.05). Collectively, our results show that IL- 6 could activate, amplify, and maintain the angiogenic cascade in HCSMCs. A novel role of IL- 6 during HCSMC proliferation is upregulating KDR expression and phosphorylation. The results may contribute to the angiogenic phenotype of human brain vascular diseases, such as brain AVM.

Abnormal vascular remodeling mediated by inflammatory cells has been identified as a key pathologic component of various vascular diseases, including abdominal aortic aneurysms, brain arteriovenous malformations and atherosclerosis. Based on findings from observational studies that analysed human intracranial aneurysms and experimental studies that utilized animal models, an emerging concept suggests that a key component of the pathophysiology of intracranial aneurysms is sustained abnormal vascular remodeling coupled with inflammation. This concept may provide a new treatment strategy to utilize agents to inhibit inflammation or cytokines produced by inflammatory cells such as matrix metalloproteinases. Such an approach would aim to stabilize these vascular lesions and prevent future expansion or rupture.

Brain arteriovenous malformations (BAVMs) are a potentially life-threatening disorder. Matrix metalloproteinase (MMP)-9 activity was greatly increased in BAVM tissue specimens. Doxycycline was shown to decrease cerebral MMP-9 activities and angiogenesis induced by vascular endothelial growth factor ( VEGF). In the present study, we determined the dose-response effects of doxycycline and minocycline on cerebral MMP-9 using our mouse model with VEGF focal hyperstimulation delivered with adenoviral vector (AdVEGF) in the brain. Mice were treated with doxycycline or minocycline, respectively, at 1, 5, 10, 30, 50, or 100 mg/kg/day through drinking water for 1 week. Our results have shown that MMP-9 messenger ribonucleic acid (mRNA) expression was inhibited by doxycycline starting at 10 mg/kg/day (P < 0.02). Minocycline showed more potent inhibition on MMP-9 mRNA expression, starting at 1 (P < 0.005) and further at more than 30 (P < 0.001) mg/kg/day. At the enzymatic activity level, doxycycline started to suppress MMP-9 activity at 5 mg/kg/day (P < 0.001), while minocycline had an effect at a lower dose, 1mg/kg/day (P < 0.02). The inhibition of cerebral MMP-9 mRNA and activity were highly correlated with drug levels in the brain tissue. We also assessed the potential relevant signaling pathway in vitro to elucidate the mechanisms underlying the MMP-9 inhibition by tetracyclines. In vitro, minocycline, but not doxycycline, inhibits MMP- 9, at least in part, via the extracellular signaling-related kinase 1/2 (ERK1/2)-mediated pathway. This study provided the evidence that the tetracyclines inhibit stimulated cerebral MMP- 9 at multiple levels and are effective at very low doses, offering great potential for therapeutic use.