Endothelial prostaglandin E 2 regulates neuronal injury after seizure via activation of astrocytes

Astrocytes interact closely with neurons via glutamate; this astrocyteneuron circuit may play a pivotal role in synaptic transmission. In addition, astrocytes contact vascular endothelial cells (ECs) with their end-feet; therefore, ECs may have some role in regulating neuronal activity via astrocytes in the brain. In our studies, we found that kainic acid (KA) microinjection induced the expression of microsomal prostaglandin E synthase-1 (mPGES-1) in venous ECs and the expression of the prostaglandin E2 (PGE2) receptor EP3 on astrocytes. Moreover, endothelial mPGES-1 exacerbated KA-induced neuronal injury in the mouse brain. In in vitro experiments, mPGES-1 produced PGE2, which increased astrocytic Ca2+ levels and Ca2+-dependent glutamate release, thus aggravating neuronal injury. We found ECs had a role under pathological conditions and brain ECs are not merely a physiological barrier between the blood and brain; instead, they may also act as a signal transducer or amplifier. Moreover, the endotheliumastrocyte-neuron signaling pathway may be crucial for driving neuronal injury elicited by repetitive seizures and may be a new therapeutic target for epilepsy.


Introduction
Prostaglandin E 2 (PGE 2 ) is an important modulator in inflammation.In the brain, PGE 2 is associated with neuroinflammation because PGE 2 is involved in pathological processes such as seizure and cerebral ischemia 1,2 .PGE 2 is sequentially synthesized from arachidonic acid by cyclooxygenase (COX) and PGE 2 synthase (PGES).Inducible COX-2 expression is known to be associated with acute neurotoxicity [1][2][3][4] and is also involved in delayed proinflammatory activities, which aggravate the neuronal damage found in neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD), multiple sclerosis (MS) and Alzheimer's disease (AD) 4,5 .We find that COX-2 is induced in non-neuronal cells late after seizure and facilitates neuronal injury in the hippocampus 6 .
In this review, we show the induction of microsomal prostaglandin E synthase-1 (mPGES-1) in brain endothelial cells (ECs) as well as the role of endothelial mPGES-1 in neuronal loss in the hippocampus after seizures.Furthermore, we present a novel mechanism for exacerbation of neuronal injury by PGE 2 derived from endothelial mPGES-1 and discuss the intercellular signaling pathway among endothelia, astrocytes and neurons in the process.

Induction of COX-2 and mPGES-1 in the brain
PGE 2 is synthesized by mPGES-1 coupling with COX-2 in brain ECs in lipopolysaccharide (LPS)-induced fever 7 , and mPGES-1 is also coinduced with COX-2 during fever or inflammation [8][9][10][11] .Moreover, we find that mPGES-1 is induced in the hippocampus after epileptic seizures caused by kainic acid (KA) microinjection 12 .KA is an analogue of the excitatory amino acid glutamate, and it is used in research to investigate the mechanisms of hippocampal neuronal loss after seizures because it induces generalized convulsion and causes neuronal loss in the hippocampus after seizures 13 .Unilateral KA microinjection induces COX-2 in bilateral neurons in the hippocampi, but in ipsilateral blood vessels both at 8 h and 24 h after KA injection 6 .Moreover, mPGES-1 is also localized in the blood vessels at 8 h, lasting until 24 h after KA microinjection 12 .Double immunostaining for both mPGES-1 and von Willebrand factor (an endothelial cell marker) shows that mPGES-1 is induced with COX-2 in the ECs for 48 h after the microinjection 12 .Finally, neuronal loss is caused in the KA microinjection side 6 , therefore we judge that PGE 2 synthesized by endothelial COX-2 and mPGES-1 facilitates neuronal injury in the hippocampus.Meanwhile, general injection of KA or pilocarpine causes general convulsion and induces COX-2 protein 18 h or 24 h after injection in hippocampal neurons.In addition COX-2 inhibitor blocks the neuronal injury 14,15 , suggesting that neuronal COX-2 has an effect to facilitate neuronal injury after strong seizure.

Endothelial mPGES-1 exacerbates neuronal injury
PGE 2 produced in ECs could have a direct effect on the adjacent astrocytes because brain ECs are surrounded by astrocytic end-feet 16 .In addition, several lines of evidence indicate that prostaglandin E receptors (EPs) are present on astrocytes, and exogenous PGE 2 immediately evokes Ca 2+ -dependent glutamate release from astrocytes 17 ; therefore, astrocytes may be directly activated by endogenous PGE 2 to elevate the intracellular Ca 2+ levels ([Ca 2+ ] i ) through the EP receptors.Furthermore, astrocytes can modulate synaptic transmission through the release of glutamate [18][19][20] , which may stimulate delayed neuronal injury after seizures 21 .Therefore, we hypothesized that PGE 2 produced by endothelial mPGES-1 directly activated EP receptors on astrocytes, elevating the astrocytic [Ca 2+ ] i , subsequently evoking sustained glutamate release and ultimately facilitating neuronal injury.
We found that the PGE 2 concentration was significantly elevated by KA treatment in cultured hippocampal slices from wild type (wt) mice, but that increase was not observed in slices from mPGES-1 knockout mice (mPGES-1 −/− ) 22 .The astrocytic [Ca 2+ ] i was significantly higher in the hippocampal CA3 region in the wt slice cultures than in the mPGES-1 −/− slice cultures 22 .These results suggest that the PGE 2 derived from mPGES-1 upregulates the astrocytic [Ca 2+ ] i in the hippocampal CA3 region.
Next, we examined the effects of each EP receptor antagonist and agonist on the [Ca 2+ ] i in astrocytes in the KA-treated wt and mPGES-1 −/− slice cultures.An EP3 receptor antagonist 23 decreased the [Ca 2+ ] i in astrocytes in the KA-treated wt slices 22 , conversely, an EP3 receptor agonist 23 increased the [Ca 2+ ] i in astrocytes in the KA-treated mPGES-1 −/− slices, suggesting that the EP3 receptor has a crucial role in astrocytic Ca 2+ elevation 22 .EP3 immunoreactivity was rarely detected in the astrocytes in naive control mice; in contrast, it was enhanced in the astrocytic end-feet with swelling after KA microinjection 22 .In the mPGES-1 −/− mice, the end-feet also showed swelling, but the EP3 immunoreactivity was not increased as much as in the wt mice 22 , indicating that the EP3 receptor was locally induced by KA in hippocampal astrocytes, which might receive PGE 2 from ECs.Previous publications have reported that EP3 mRNA is expressed in cultured astrocytes 24 and that EP3 protein expression is induced in astrocytomas by interleukin-1β 25 .These findings indicate that astrocytic EP3 receptors may be upregulated under pathological conditions, and endothelial PGE 2 may directly activate EP3 receptors on astrocytic end-feet in neurotoxic brain diseases, such as epileptic seizures.PGE 2 also acts on other three receptors, namely EP1, EP2 and EP4, and activation of their receptors has been found to contribute to PGE 2mediated neurotoxicity 26 .Block of EP1 receptor reduces proinflammatory responses and neuronal damage in the hippocampus after KA injection in mice 27 .Moreover, selective EP2 antagonism by small molecules prevents up-regulation of COX-2 in microglia, leading to reduce neuronal injury induced by pilocarpine 28 .In addition, EP4 receptor-associated protein promotes proinflammatory activation of microglia which modulates neuronal damage 29 .Activation of these receptors is concerned with regulation of neuronal injury by PGE 2 .Furthermore, we observed that treatment with KA for 17 h dramatically increased the level of glutamate release in the wt slices but not in the mPGES-1 −/− slices 22 .To verify whether mPGES-1 regulates hippocampal neuronal death, we stained the cells with propidium iodide (PI).The results showed greater PI uptake in the CA3 region of the wt slices than in that of the mPGES-1 −/− slices 22 .This significant increase in PI uptake in the wt slices suggests that neuronal injury may be enhanced by mPGES-1, which regulates the Ca 2+ -dependent glutamate release from astrocytes.
We next added exogenous PGE 2 to the mPGES-1 −/− slices to validate the above findings on the endogenous PGE 2 .PGE 2 enhanced the astrocytic [Ca 2+ ] i in the CA3 region 22 .Moreover, PGE 2 caused an increase in the glutamate concentration and exacerbated the PI uptake in the CA3 region 22 .These results indicate that the PGE 2 derived from mPGES-1 is an important mediator that regulates neuronal injury.Exogenous PGE 2 also increased neuronal [Ca 2+ ] i in co-cultures with astrocytes, but the increase was not found without astrocytes (unpublished data).In addition, the [Ca 2+ ] i increase in neurons was observed to follow the [Ca 2+ ] i increase in astrocytes (unpublished data).These results suggest that PGE 2 indirectly increases the neuronal [Ca 2+ ] i via the astrocytic [Ca 2+ ] i increase and subsequent glutamate release.Finally, we investigated whether this PGE 2 -evoked glutamate release occurred in a Ca 2+ -dependent manner.A membrane-permeable Ca 2+ chelator, BAPTA-AM, diminished the increase in the [Ca 2+ ] i in the astrocytes in the wt slices and abolished the increase in glutamate concentration 22 .Moreover, BAPTA-AM partially ameliorated the neuronal injury in the CA3 region, suggesting that CA3 neuronal injury is locally regulated by Ca 2+ -dependent glutamate release from neighboring astrocytes 22 .Together, these results suggest that the PGE 2 produced by endothelial mPGES-1 activates the astrocytic EP3 receptors to elevate the [Ca 2+ ] i in astrocytes, causing Ca 2+ -dependent glutamate release and facilitating neuronal injury 22 .

Intercellular signaling pathway among endothelia, astrocytes and neurons
Neuron-to-astrocyte signaling controls arterial blood flow in the brain [30][31][32] .Conversely, there is also mounting evidence for dynamic astrocyte-to-neuron interactions; for example, astrocytes modulate synaptic transmission [18][19][20] .The interactions are also involved in neuronal synchrony 33 and epileptic discharges 14,34 , which contribute to a delayed neuronal injury after seizures 21 .Neurons are vulnerable to glutamate in the hippocampus, and it is thought to be mediated by N-methyl-d-aspartate (NMDA) receptors (NMDARs) 35 .In particular, glutamate release from astrocytes activates the extrasynaptic NMDAR subunit NR2B, which induces neuronal currents 21 or triggers neuronal loss 21,36,37 .This suggests that extrasynaptic NR2B receptors play crucial roles in the neurotoxicity caused by the glutamate released from astrocytes.Conversely, neuronal glutamate activates astrocytic mGluR5 to cause an increase in [Ca 2+ ] i in astrocytes, which may in turn release glutamate and generate feedback to extrasynaptic NR2B 21 .Thus, the neuron-astrocyte circuit may amplify the glutamate signaling, which aggravates neuronal excitotoxicity following seizures.
In this review, we propose an advanced mechanism for excitotoxicity via ECs and astrocytes.We demonstrated that endothelial mPGES-1 regulated Ca 2+ signaling in astrocytes and Ca 2+ -dependent glutamate release, consistent with the findings that application of exogenous PGE 2 propagated astrocytic [Ca 2+ ] i and evoked Ca 2+dependent glutamate release 17 .However, PGE 2 alone did not increase astrocytic [Ca 2+ ] i (unpublished data); therefore, PGE 2 may require another factor, such as a concomitant activation of astrocytic EP3, to elevate [Ca 2+ ] i in astrocytes.Brain ECs are not merely a physiological barrier between the blood and brain; instead, they may also act as a signal transducer or amplifier.In particular, we found ECs had a role under pathological conditions, such as in epileptic seizure.The interaction among neurons, astrocytes and ECs may be key to understanding the processes of seizureinduced neuronal injury in epilepsy.

Conclusions
PGE 2 is synthesized by inducible mPGES-1 and COX-2 in vascular ECs in response to KA microinjection.In addition, endothelial PGE 2 activates astrocytic EP3 receptor to elevate [Ca 2+ ] i levels in astrocytes, causing Ca 2+ -dependent glutamate release which stimulates neuronal injury.This is a new mechanism underlying neuronal injury regulated by ECs; therefore, this review emphasizes that brain ECs act as a signal transducer or amplifier, especially, under pathological conditions, such as epileptic seizure.The analysis of the interactions among neurons, astrocytes and ECs provides a better understanding of the processes of seizure induced neuronal injury and will facilitate the development of new treatments.