3β‐Diol Attenuates COX‐2 Levels in a Neonatal In‐Vivo and In‐Vitro Ischemic Injury Rodent and Human Model

Abstract

Studies using experimental stroke models demonstrate that neuroprotective agents which provide protection from secondary injury, including inflammation, result in improved outcome. Estrogen receptor β (ESR2) has the potential to be a novel therapeutic target in the treatment of ischemic stroke. Previous studies have shown that ESR2 activation elicits beneficial effects in the cerebrovasculature by promoting vasorelaxation and by attenuating pro‐inflammatory mediator production. We previously observed that activation of the androgen, 5α‐androstane‐3β,17β‐diol (3β‐diol), reduced pro‐inflammatory mediator production in adult human vascular smooth muscle cells and rodent cerebral arteries, and this response was ESR2 dependent. The current study was designed to determine if ERS2 is expressed in both a human pediatric brain endothelial cell model and juvenile rat stroke model and whether 3β‐diol will attenuate cyclooxygenase‐2 (COX‐2) expression following ischemic injury. Male rat pups (P10–15) underwent 15 minutes of bilateral internal carotid artery occlusion (BCAO) or sham surgery and 24‐hour reperfusion. At the onset of reperfusion, pups were injected with 3β‐diol i.p. (2.5mg/kg) or vehicle (1.2% EtOH and 98.8% PEG). Next, animals were heavily sedated and whole brain removed at 24 hours. In an in‐vitro model of ischemic stroke, male primary pediatric human microvascular endothelial cells (HBMEC; Cell Systems) were exposed to hypoxia plus glucose deprivation (HGD; 1% O2) or normoxia (21% O2) plus or minus 3β‐diol (10nM) or vehicle (0.0001% ethanol) for 3h at passage 7, using a humidified chamber (BioSpherix®) housed within a 5% CO2 incubator. Total protein was extracted from rat whole brain and human endothelial cells, assayed for protein content, and COX‐2 levels assessed using western analysis. RT‐PCR analysis was used to explore whether 3β‐diol alters expression of its target receptor, ERS2. We observed that ESR2 was basally expressed in the prepubescent brain and HBMECs, and expression was not altered following ischemic injury. Proinflammatory COX‐2 protein levels were increased in both whole brain and HBMEC homogenates from BCAO and HGD groups compared to sham and vehicle controls, and the selective ERS2 agonist, 3β‐diol, attenuated this response. In conclusion, these studies suggest that 3β‐diol may have a role in modulating ischemia‐induced COX‐2 during stroke. Since ERS2 is expressed in the whole brain and brain endothelial cells, and ERS2 levels are not decreased during ischemic injury, ERS2 may be a viable target for future stroke therapeutics in the pediatric population by attenuating inflammatory modulators during ischemic injury in the developing brain.Support or Funding InformationUniversity of Arizona Valley Research Partnership Grant VRP04 P1 (RJG), American Heart Association 19AIREA34480018 (RJG).