br Results br Discussion Impaired BBB functions evoke vasoge

Results
Discussion Impaired BBB functions evoke vasogenic edema, which elevates intracranial pressure and leads to a life-threatening condition (Sperk, 1994, Unterberg et al., 2004, Simard et al., 2007). In addition, vasogenic edema formation results in the worsen consequence contributing to pharmacoresistant epilepsy in patients and in chronic epilepsy rats, since vasogenic edema increases p-glycoprotein (a drug efflux transporter) activity in the am580 synthesis (Rigau et al., 2007, Ko and Kang, 2015). During SE-induced vasogenic edema formation, various vasoactive effectors influence on the vascular permeability (Rodgers et al., 2009, Deli et al., 2005). One of them, TRPC3 is highly up-regulated in endothelial cells during pathophysiological processes including SE, although it is expressed at low levels under physiological condition (Balzer et al., 1999, Yu, 2004, Poteser, 2006, Ryu et al., 2013, Kim et al., 2016). Recently, we have suggested that TRPC3 may regulate NFκB activity in a positive feedback manner, which increases vascular permeability via enhancing ETB receptor-mediated eNOS activation (Min and Kang, 2016). eNOS is a critical regulator of vascular tone and plays an especially prominent role in blood flow and vascular permeability. eNOS activity is activated by vasoactive signals such as ET-1 (Kim et al., 2013). In addition, TRPC3 activates eNOS via sustained Ca2+ bursts (Yi et al., 2010), and increases vascular permeability (Ahmmed and Malik, 2005, Huang et al., 2011). In the present study, we found that Pyr-3 (a TRPC3 inhibitor) effectively abolished SE-induced PI3K/AKT phosphorylations accompanied by reduction in eNOS expression. Furthermore, both wortmannin (a PI3K inhibitor) and 3CAI (an AKT inhibitor) inhibited SE-induced eNOS expression and mitigated vasogenic edema lesion. With respect to TRPC3-mediated PI3K/AKT activation (Tano et al., 2011), our findings indicate that TRPC3 may be involved in vasogenic edema formation via PI3K/AKT-mediated eNOS activation in endothelial cells. Following SE, up-regulated ETB receptor activates eNOS activity (Kim et al., 2013), which disrupts tight junction integrity in endothelial cells and leads to vasogenic edema (Kim et al., 2015). In addition, ETB receptor-mediated PI3K/AKT phosphorylations stimulate eNOS (Liu et al., 2014). Similar to this report, the present study shows that BQ788 (an ETB receptor antagonist) alleviated PI3K/AKT phosphorylations with reduced eNOS expression. Consistent with our previous study (Min and Kang, 2016), however, inhibition of ETB receptor by BQ788 could not affect SE-induced up-regulations of TRPC3 and VEGF expression. Therefore, our findings indicate that ETB receptor may activate PI3K/AKT independent of TRPC3 or VEGF activity. Indeed, NFκB inhibition by SN50 could abolish PI3K/AKT phosphorylation accompanied by abrogation of up-regulated TRPC3 and ETB receptor expressions induced by SE. These findings indicate that PI3K/AKT may be one of the common down-stream signaling pathways of both TRPC3 and ETB receptor. VEGF is a growth factor regulating neovascularization and provokes vasogenic edema by impaired BBB integrity (Pavlicek et al., 2000, Walter et al., 2001, Argaw et al., 2009, Bates, 2010). In a previous study (Kim et al., 2016), we have reported that p38 MAPK-mediated VEGF over-expression impair BBB integrity leading to increased vascular permeability, which potentially causes vasogenic edema formation following SE. However, we could not find the downstream effectors/signaling pathways concerning VEGF-mediated vasogenic edema formation. In the present study, we found that LMB attenuated SE-induced vasogenic edema by reducing VEGF expression and PI3K/AKT activation. Since VEGF activates PI3K followed by AKT phosphorylation and increases eNOS activity (Guo et al., 1995, Suzuma et al., 2000, Tanimoto et al., 2002), our findings suggest that VEGF-mediated PI3K/AKT phosphorylation may be one of the important signaling pathways in vasogenic edema formation. On the other hand, the present data reveal that reduced VEGF expression by LMB inhibited SE-induced PI3K/AKT-mediated eNOS activation without altered TRPC3 expression. Thus, it is not excluded the possibility that VEGF may directly regulate PI3K/AKT phosphorylation independent of TRPC3. However, VEGF-induced increase in vascular permeability is mediated by stimulating endothelial Ca2+ influx via TRPC3 (Pocock et al., 2004, Cheng et al., 2006, Poteser, 2006). With respect to these previous reports, it is presumable that TRPC3 would be required for VEGF-mediated vasogenic edema formation. Therefore, our findings indicate that TRPC3 may be one of the important downstream targets of VEGF signaling or a synergist for VEGF effects, while VEGF could not affect TRPC3 expression. Furthermore, it is important to note that TRPC3 may link VEGF signaling to TNF-α/NFκB/ETB receptor-mediated eNOS activation during vasogenic edema formation, since NFκB activation induced TRPC3 expression in endothelial cells following SE (Min and Kang, 2016). In addition, it is likely that VEGF may be involved in a TRPC3-mediated positive feedback loop of NFκB activity, which may integrate ETB receptor/eNOS activation (Fig. 6).