Wei-Feng Ye

Nitrosative stress induces peroxiredoxin 1 ubiquitination during ischemic insult via E6AP activation in endothelial cells both in vitro and in vivo.

AIMS Although there is accumulating evidence that increased formation of reactive nitrogen species in cerebral vasculature contributes to the progression of ischemic damage, but the underlying molecular mechanisms remain elusive. Peroxiredoxin 1 (Prx1) can initiate the antioxidant response by scavenging free radicals. Therefore, we tested the hypothesis that Prx1 regulates the susceptibility to nitrosative stress damage during cerebral ischemia in vitro and in vivo. RESULTS Proteomic analysis in endothelial cells revealed that Prx1 was upregulated after stress-related oxygen-glucose deprivation (OGD). Although peroxynitrite upregulated Prx1 rapidly, this was followed by its polyubiquitination within 6 h after OGD mediated by the E3 ubiquitin ligase E6-associated protein (E6AP). OGD colocalized E6AP with nitrotyrosine in endothelial cells. To assess translational relevance in vivo, mice were studied after middle cerebral artery occlusion (MCAO). This was accompanied by Prx1 ubiquitination and degradation by the activation of E6AP. Furthermore, brain delivery of a lentiviral vector encoding Prx1 in mice inhibited blood-brain barrier leakage and neuronal damage significantly following MCAO. INNOVATION AND CONCLUSIONS Nitrosative stress during ischemic insult activates E6AP E3 ubiquitin ligase that ubiquitinates Prx1 and subsequently worsens cerebral damage. Thus, targeting the Prx1 antioxidant defense pathway may represent a novel treatment strategy for neurovascular protection in stroke.

Ischemic injury promotes Keap1 nitration and disturbance of antioxidative responses in endothelial cells: a potential vasoprotective effect of melatonin.

Clinical epidemiology has indicated that the endothelial injury is a potential contributor to the pathogenesis of ischemic neurovascular damage. In this report, we assessed S‐nitrosylation and nitration of Keap1 to identify downstream nitric oxide redox signaling targets into endothelial cells during ischemia. Here, oxygen–glucose deprivation (OGD) exposure initiates the nuclear import of Keap1 in endothelial cells, which interacted with nuclear‐localized Nrf2, as demonstrated through co‐immunoprecipitation and immunocytochemical assay. Paralleling the ischemia‐induced nuclear import of Keap1, increased nitrotyrosine immunoreactivity in endothelial cells was also observed. Consistently, the addition of peroxynitrite provoked nuclear import of Keap1 and a concomitant Nrf2 nuclear import in the endothelial cells. Importantly, pharmacological inhibition of nitrosative stress by melatonin partially inhibited the OGD‐induced constitutive nuclear import of Keap1 and subsequently disturbance of Nrf2/Keap1 signaling. Moreover, the effect of melatonin on nitration and S‐nitrosylation of keap1 was examined in endothelial cells with 6 hr OGD exposure. Here, we demonstrated that OGD induced tyrosine nitration of Keap1, which was blocked by melatonin treatment, while there were no significant changes in S‐nitrosylation of Keap1. The specific amino acid residues of Keap1 involved in tyrosine nitration were identified as Y473 by mass spectrometry. Moreover, the protective role of melatonin against damage to endothelial tight junction integrity was addressed by ZO‐1 expression, paralleled with the restored heme oxygenase‐1 levels during OGD. Together, our results emphasize that upon nitrosative stress, the protective effect of melatonin on endothelial cells is likely mediated at least in part by inhibition of ischemia‐evoked protein nitration of Keap1, hence contributing to relieve the disturbance of Nrf2/Keap1 antioxidative signaling.

The γ-Secretase Blocker DAPT Reduces the Permeability of the Blood-Brain Barrier by Decreasing the Ubiquitination and Degradation of Occludin During Permanent Brain Ischemia.

Tight junction protein degradation is a principal characteristic of the blood–brain barrier (BBB) damage that occurs during brain ischemia.

The Disturbance of Hippocampal CaMKII/PKA/PKC Phosphorylation in Early Experimental Diabetes Mellitus

Defining the impact of diabetes and related risk factors on brain cognitive function is critically important for patients with diabetes.

Expression profiling of Ca2+/calmodulin-dependent signaling molecules in the rat dorsal and ventral hippocampus after acute lead exposure

The septal and temporal poles of the hippocampus differ markedly in their anatomical organization, but whether these distinct regions exhibit differential neurochemical profiles underlying lead (Pb(2+)) neurotoxicity remains to be determined. In the present study, we examined changes in the expression of Ca(2+)/calmodulin-dependent enzymes, including calpain, calcineurin, phospho-CaMKII (Thr286) and neuronal nitric oxide synthase (nNOS), in the rat dorsal and ventral hippocampus (DH and VH) after acute Pb(2+) exposure. Five days after Pb(2+) exposure, we observed constitutively active forms of calcineurin (45 kDa and 48 kDa) in ventral portions of the hippocampus, a result consistent with the observed calpain activation that is indicated by the breakdown of spectrin in this region. Our data demonstrate that nNOS expression is significantly higher in the ventral region of the hippocampus when compared to the dorsal region, whereas phosphorylation of CaMKII (Thr286) is less pronounced in the ventral portion of the hippocampus and more pronounced in dorsal regions after acute Pb(2+) exposure. Thus, it appears likely that the ventral region of hippocampus is more vulnerable to the neurotoxic effects of Pb(2+) than the dorsal region. Taken together, the present data suggest that acute lead exposure leads to differential expression patterns of Ca(2+)/calmodulin-dependent enzymes along the dorsoventral axis of the hippocampus.

Peroxiredoxin 1 participates in ischemia-triggered endothelial polarization

The balance between endothelial cell survival and death plays a pivotal role in brain remodeling and repair after stroke [1]. Extensive changes in endothelial cell behavior have been implicated in angiogenesis and vasculogenesis [2]. Angiogenesis is a prominent feature of ischemic stroke recovery, and increasing endothelial polarity might stimulate early angiogenesis and reflect a beneficial mechanism of revascularization and neuroprotection after stroke [3]. Polarization is important for various aspects of the endothelial response to a variety of stressful stimuli, such as hypoxia and inflammation [4]. However, the precise molecular cues that regulate endothelial polarity and function remain elusive. In the current study, we set out to address the potential role of peroxiredoxin 1 in ischemia-induced endothelial polarization. It is unknown whether peroxiredoxin 1 plays a role in angiogenesis, a process that involves the generation of new blood vessels and relies largely on vascular endothelial cell polarization and proliferation. We previously reported that peroxiredoxin 1 is a pivotal molecule for the protection of endothelial cells and microvessels from ischemia-induced neurovascular damage both in vitro and in vivo [5]. Earlier studies have shown that peroxiredoxin 1 carries out a wide range of cellular functions, including exerting a proliferative effect and playing an antioxidant protective role [5]. It is also known that peroxiredoxin 1 is important for vascular endothelial growth factor (VEGF) expression [6], and it is therefore likely that peroxiredoxin 1 is involved in the induction of angiogenesis following a stroke. Here, we first investigated the involvement of peroxiredoxin 1 in oxygen-glucose deprivation (OGD)-induced endothelial cell polarization. EA.hy926 endothelial cells were exposed to OGD for 6 and 12 h, and the expression of peroxiredoxin 1 was assayed using confocal immunocytochemistry. We defined polarized cells according to the proportion of the longest axis of the cell to that of the shortest axis of the cell [7]. As shown in Figure 1A, peroxiredoxin 1-positive endothelial cells in the OGD 6 h group exhibited polarized characteristics. After 12 h had elapsed, the cells respread, and their elongation became even more pronounced. They exhibited a polarized cell shape with a morphologically defined leading process and a uropod, whereas the control cells displayed oval-shaped morphology. The results of quantitative analyses from three independent experiments are depicted in Figure 1B, indicating that cell elongation significantly increased after OGD, whereas cell ellipsoid decreased (Figure 1C). This elongation might involve directional spreading via a protrusion at the front of the cells mediated by various intracellular signaling mechanisms [7]. These data raise the possibility that peroxiredoxin 1 plays a crucial role in the establishment of endothelial cell polarity in response to ischemic stimuli, which might be related to its proliferative effect and its contributions toward angiogenesis and brain repair. To further ascertain the impact of the upregulation of peroxiredoxin 1 on endothelial polarization in vivo, a mouse transient middle cerebral artery occlusion (tMCAO) model was employed in the present study. Staining brain sections from tMCAO mice for peroxiredoxin 1 and the endothelial marker Tie2 revealed that peroxiredoxin 1 was localized to the endothelial cells of the brain (Figure 2). Peroxiredoxin 1 immunoreactivity was observed predominantly in ipsilateral brain microvessels 12 h after administering tMCAO (Figure 2A) and was accompanied by colocalization with Tie2 immunoreactivity (Figure 2B,C,D). Compared with sham-operated animals, peroxiredoxin 1 expression was increased

Endothelial ErbB4 deficit induces alterations in exploratory behavior and brain energy metabolism in mice

The receptor tyrosine kinase ErbB4 is present throughout the primate brain and has a distinct functional profile. In this study, we investigate the potential role of endothelial ErbB4 receptor signaling in the brain.