Studies in experimental mice have indicated that treating cerebral ischemia using a selective EP4 agonist can significantly reduce the level of brain damage and benefit long-term behavioral deficits. In vitro and in vivo studies by Stanford University School of Medicine researchers found that neuronally and endothelially expressed EP4 exert separate cell-specific effects that act to reduce infarct size and increase reperfusion, respectively, and that both actions can be boosted through administration of an EP4 agonist. Conversely, conditional genetic inactivation of neuronal EP4 worsened stroke outcome, while endothelial deletion of EP4 also had adverse effects with respect to stroke injury and decreased cerebral reperfusion.
Katrin Andreasson, M.D., and colleagues, reported their findings in the Journal of Clinical Investigation, in a paper titled “Signaling via the prostaglandin E2 receptor EP4 exerts neuronal and vascular protection in a mouse model of cerebral ischemia.”
The cyclooxygenases COX-1 and COX-2 catalyze the initial set of reactions that lead to prostaglandin formation and signaling, the researchers explain. Studies have shown that prostaglandin receptor signaling exacerbates injury in models of cerebral ischemia and related models of CNS trauma, and contributes to neurodegeneration in models of Parkinson disease, amyotrophic lateral sclerosis, and Alzheimer disease. Recent research has, however, also indicated that chronic blockade of cyclooxygenase through treatment with COX-2 inhibitors can lead to cerebrovascular and cardiovascular complications, suggesting that some prostaglandin signaling pathways may be protective. “Selective targeting of prostaglandin GPCRs, both toxic and beneficial, therefore represents a promising approach in the treatment of brain disorders,” the Stanford team remarks.
PGE2 is a major product downstream of COX-2 enzymatic activity, and activates the four distinct GPCRs, EP1–EP4, which trigger divergent downstream signaling cascades, cellular expression patterns, and functional effects, depending on the physiological or pathological context. To investigate the functions of the PGE2 receptor EP4 in stroke more closely, the team used both drug-based and genetic strategies to target cell-specific EP4 signaling in the brain, using a middle cerebral artery occlusion–reperfusion (MCAo-RP) model of transient focal cerebral ischemia.
They first treated three-month-old male C57B6 mice using the selective EP4 agonist AE1-329, at two hours after middle cerebral artery occlusion (MCAo) and again after eight hours. This double dose reduced the hemispheric infarct size by over 50% at 24 hours. A single dose of either a high- or low-dose AE1-329 administered at three hours after MCAo led to 67.3% and 51.6% reductions in infarct size, respectively, at 24 hours.
The team then investigated whether there were any longer-term benefits of treatment with AE1-329, this time in three-month-old F1 hybrid B6D2F1/J male mice. Animals were treated with a single low dose of AE1-329 after MCAo, and their performance on the rotorod test was evaluated at 48 hours and 7 days post-treatment. The results showed that even the lower dose of agonist therapy resulted in improved rotorod performance at both time points, despite the fact that in comparison with control mice, the treated mice showed no differences in infarct volume measured by cresyl violet staining.
To test the function of neuronal EP4 in vitro, the GPCR was expressed basally in cultured cortical neurons in a perinuclear distribution and in processes. Adding AE1-329 to the cultured cells led to significant induction of phospho-CREB, and reduced injury induced by administration of glutamate. AE1-329 treatment also led to dose-dependent rescue of cortical neurons subjected to oxygen-glucose deprivation, an effect that was blocked by administration of the PKA inhibitor H89 at doses low enough not to impact on cell survival. The protective effect of EP4 signaling was separately confirmed in hippocampal organotypic slices subjected to NMDA excitotoxicity.
The researchers then went on to evaluate whether neuronal EP4 was protective in the MCAo-RP model: their previous research had demonstrated increased expression of EP4 in neurons, four hours after MCAo-RP in the peri-infarct area region. They subjected both Thy-1Cre;EP4lox/lox conditional neuronal EP4 knockout mice and Thy-1Cre;EP4+/+ control mice to transient focal ischemia using the MCAo-RP model. Compared with the control animals, the conditional EP4 knockouts demonstrated a 115% larger hemispheric infarct size, a 331% increase in cortical infarct size, and a 33% increase in caudate-putamen infarct size.
Separate cohorts of Thy-1Cre;EP4lox/lox and Thy-1Cre;EP4+/+ mice were then subjected to MCAo, followed by 23 hours of reperfusion and treatment with AE1 329 three hours after injury. Interestingly AE1-329 therapy still reduced the infarct size in Thy-1 Cre;EP4lox/lox mice by nearly 35%, and significantly increased the numbers of mice who demonstrated no cortical infarction.
The ability of AE1-329 to reverse ischemic injury in neuronal EP4 cKO mice suggested that although neuronal EP4 exerts endogenous neuroprotection, there may be an additional non-neuronal EP4 signaling pathway that contributes to AE1-329 cerebroprotection, the researchers note. They were therefore prompted to look more closely at the role of endothelial EP4, specifically, in MCAo-RP-injured mice. They generated inducible, conditional endothelial EP4 knockout animals, and first confirmed that at four and 24 hours after MCAo, endotheial EP4 was induced in the microvasculature of control mice, but not the endothelial EP4 knockout animals. Compared with control animals, the endothelial EP4 knockouts demonstrated significantly larger hemispheric and striatal infarct sizes, but this increase could be ameliorated by AE1-329 treatment, similar to the effect of the drug in neuronal EP4 cKO mice.
Because PGE2 elicits vasodilation in systemic vasculature through EP2 and EP4 signaling, the team evaluated whether deletion of endothelial EP4 would alter cerebral perfusion during and after ischemia. Cerebral blood flow measurements in mice subjected to 60 minutes of ischemia followed by 60 minutes of reperfusion showed that deleting endothelial EP4 resulted in significant reductions in reperfusion blood flow, without any associated changes in systemic mean arterial pressure during or after MCAo-RP. Evaluation of microvasculature following administration of AE1-329 in both endothelial EP4 knockouts and control animals suggested that endothelial EP4 signaling in vivo may enhance cerebral perfusion through increased eNOS expression and activation.
The overall data indicate that EP4 signaling protects against damage following stroke via the two independent cell-specific mechanisms of neuroprotection and enhanced vascular perfusion, the authors conclude. Neuronal EP4 signaling elicited protection through a PKA-dependent pathway, while systemic administration of AE1-329 increased protein levels of eNOS and activated phospho-Ser1177 eNOS in cerebral microvessels, to increase reperfusion.
“Our findings broaden the concept of beneficial prostaglandin receptors in cerebrovascular disease,” they state. “The dual neuronal and vascular mechanisms of cerebroprotection triggered by activation of EP4 support the concept of protective prostaglandin receptors that could be targeted therapeutically after stroke. These data also support the concept of targeting non-neuronal as well as neuronal mechanisms, which could recruit more robust and less time-sensitive pathways than neuroprotective strategies alone...As human MRI studies now suggest that rescue of cerebral perfusion may be possible at later time points than previously realized, reperfusion-based strategies may extend the effective time window for therapeutic interventions in stroke."