Disturbance Of Peroxiredoxin-1 Signaling Participates In Nitrosative Stress-mediated Neurovascular Injury During Brain Ischemia

Background:Brain micro vascular endothelial cells provide a barrier between the bloodstream and the brain that is critical in brain development, maturation, and homeostasis. The balance between endothelial cell survival and death is pivotal for brain remodeling and repair. Increased cell death of cerebrovascular endothelial cells exacerbates inflammatory, ischemic and degenerative brain diseases. Before a new strategy can be developed to counter these adverse effects of ischemia-induced endothelial dysfunction and neurovascular damage, it is necessary to define the factors responsible for ischemia-induced blood-brain barrier damage.Under conditions of intense nitrosative stress such as in stroke, neurodegenerative disorders., endothelial cells are the primary targets of nitrosative stress in neurovascular damage. Peroxiredoxins (Prxs) are thiol-specific antioxidant enzymes that maintain redox balance under both normal conditions and oxidative stress. Although Prx1 is the most abundant and widely distributed member of the mammalian Prxs and is a recognized peroxide-detoxifying enzyme, its pathophysiologic role during brain ischemia remains unclear.Objective:Although Prxl is a recognized antioxidant enzyme that maintains redox balance by scavenging free radicals, its pathophysiologic role during neurovascular damage remains unclear. Here, we tested the hypothesis that Prxl regulates the susceptibility to nitrosative stress damage in endothelial cells, thereby exacerbating cerebral ischemia injury.Methods and Results:Proteomic analysis in endothelial cells revealed that Prxl was upregulated after stress-related oxygen-glucose deprivation accompanied by the heat shock protein 27 (HSP27) increase. A time-dependent upregulation of Prxl was observed within 6 h after OGD, then followed by a decline after 12 h. The inbalance of Prx1 is is closely associated with nitrosative stress-induced Prxl ubiquitination and degradation, as well as expression inhibition. Nitrosative stress activated E6AP with the tyrosin nitration modification, which selectively targeted Prxl for ubiquitination and degradation. Moreover, peroxynitrite provoked nuclear import of Keapl and a concomitant Nrf2 nuclear import in the endothelial cells.The Y473 nitration modification in Keapl is closely associated with the disturbance of Keapl-Nrf2 signaling, which is the key modulator of Prxl antioxidative response in endothelial cell.The protective effects of Prxl against ischemia-induced neurological and functional deficit were evaluated by the rotarod test and neurological score measurements. The neurological scores were decreased significantly and rotarod time was increased in LV-Prxl-treated mice 24 h after tMCAO and the infarct area was reduced accordingly. The breakdown products of spectrin and calcineurin were increased after tMCAO, coincident with blood-brain barrier (BBB) leakage. LV-Prx1 treatment blocked degradation of tight junction proteins Zonula occludens-1 (ZO-1) and claudin5 and significantly reduced BBB leakage. LV-Prxl transduction reduced the superoxide level assessed from dihydroethidium staining in the penumbra region of mice 24 h after tMCAO.Innovation and Conclusion:our studies identify an intracellular link between nitrosative stress and Prx1 signaling in endothelial cells following ischemia-like injury. Our description of an early ischemia-induced activation of beneficial Prx1 generation followed by subsequent inactivation in ischemia represents a previously undescribed, nitrosative stress-dependent process mediated by E6AP-dependent Prx1 ubiquitination and Keap1-Nrf2-dependent Prx1 antioxidative response inhibition. Since Prx1 protected against oxidative stress in endothelial cells to reduce injury after ischemia both in vitro and in vivo. Specific inducers of the Prx1 pathway, or mechanisms to prevent its degradation and inhibition, may be targeted for therapeutic benefit in neurovascular disorders.