Ji-Yun Huang

Regulation of the ischemia-induced autophagy-lysosome processes by nitrosative stress in endothelial cells.

Abstract:  The cellular mechanisms that underlie the diverse nitrosative stress‐mediated cellular events associated with ischemic complications in endothelial cells are not yet clear. To characterize whether autophagic elements are associated with the nitrosative stress that causes endothelial damage after ischemia injury, an in vitro sustained oxygen–glucose deprivation (OGD) and an in vivo microsphere embolism model were used in the present study. Consistent with OGD‐induced peroxynitrite formation, a rapid induction of microtubule‐associated protein 1 light chain 3 (LC3)‐I/II conversion and green fluorescent protein‐LC3 puncta accumulation were observed in endothelial cells. The Western blot analyses indicated that OGD induced elevations in lysosome‐associated membrane protein 2 and cathepsin B protein levels. Similar results were observed in the microvessel insult model, following occlusion of the microvessels using microsphere injections in rats. Furthermore, cultured endothelial cells treated with peroxynitrite (1–50 μm) exhibited a concentration‐dependent change in the pattern of autophagy–lysosome signaling. Intriguingly, OGD‐induced autophagy–lysosome processes were attenuated by PEP‐19 overexpression and by a small‐interfering RNA (siRNA)‐mediated knockdown of eNOS. The importance of nitrosative stress in ischemia‐induced autophagy–lysosome cascades is further supported by our finding that pharmacological inhibition of nitrosative stress by melatonin partially inhibits the ischemia‐induced autophagy–lysosome cascade and the degradation of the tight junction proteins. Taken together, the present results demonstrate that peroxynitrite‐mediated nitrosative stress at least partially potentiates autophagy–lysosome signaling during sustained ischemic insult‐induced endothelial cell damage.

Targeted therapy of brain ischaemia using Fas ligand antibody conjugated PEG-lipid nanoparticles.

The translation of experimental stroke research from the laboratory to successful clinical practice remains a formidable challenge. We previously reported that PEGylated-lipid nanoparticles (PLNs) effectively transport across the blood-brain barrier along with less inflammatory responses. In the present study, PLNs conjugated to Fas ligand antibody that selectively present on brain ischaemic region were used for therapeutic targeting. Fluorescent analysis of the mice brain show that encapsulated 3-n-Butylphthalide (dl-NBP) in PLNs conjugated with Fas ligand antibody effectively delivered to ipsilateral region of ischaemic brain. Furthermore, the confocal immunohistochemical study demonstrated that brain-targeted nanocontainers specifically accumulated on OX42 positive microglia cells in ischaemic region of mice model. Finally, dl-NBP encapsulated nano-drug delivery system is resulted in significant improvements in brain injury and in neurological deficit after ischaemia, with the significantly reduced dosages versus regular dl-NBP. Overall, these data suggests that PLNs conjugated to an antibody specific to the Fas ligand constituted an ideal brain targeting drug delivery system for brain ischaemia.

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 effect of lipid nanoparticle PEGylation on neuroinflammatory response in mouse brain

Nanocarrier-based drug delivery systems have attracted wide interest for the treatment of brain disease. However, neurotoxicity of nanoparticle has limited their therapeutic application. Here we demonstrated that lipid nanoparticles (LNs) accumulated in the brain parenchyma within 3 h of intravenous injection to mice and persisted for more than 24 weeks, coinciding with a dramatic activation of brain microglia. Morphological characteristic of microglial activation also observed in LNs-treated Cx3cr1GFP/+ mice. In vivo study with two-photon confocal microscopy revealed abnormal Ca²⁺ waves in microglia following LNs injection. The correlated activation of caspase-1, IL-1β and neurovascular damage following LNs injection was attenuated in P2X₇-/- mice. PEGylation of LNs reduced correlated nanoparticles aggregation. Moreover, PEGylation of LNs ameliorated the P2X₇/caspase-1/IL-1β signalling-dependent microglia activation and neurovascular damage. In conclusion, PEGylation of LNs is a promising biomaterial for brain-targeted therapy that inhibits P2X7₇-dependent neuroinflammatory response.

CaMKIIδB Mediates Aberrant NCX1 Expression and the Imbalance of NCX1/SERCA in Transverse Aortic Constriction-Induced Failing Heart

Ca2+/calmodulin-dependent protein kinase II δB (CaMKIIδB) is one of the predominant isoforms of CaMKII in the heart. The precise role of CaMKIIδB in the transcriptional cross-talk of Ca2+-handling proteins during heart failure remains unclear. In this work, we aim to determine the mechanism of CaMKIIδB in modulating the expression of sarcolemmal Na+–Ca2+ exchange (NCX1). We also aim to address the potential effects of calmodulin antagonism on the imbalance of NCX1 and sarcoendoplasmic reticulum Ca2+ ATPase (SERCA) during heart failure. Eight weeks after transverse aortic constriction (TAC)-induced heart failure in mice, we found that the heart weight/tibia length (HW/TL) ratio and the lung weight/body weight (LW/BW) ratio increased by 59% and 133%, respectively. We further found that the left ventricle-shortening fraction decreased by 40% compared with the sham-operated controls. Immunoblotting revealed that the phosphorylation of CaMKIIδB significantly increased 8 weeks after TAC-induced heart failure. NCX1 protein levels were also elevated, whereas SERCA2 protein levels decreased in the same animal model. Moreover, transfection of active CaMKIIδB significantly increased NCX1 protein levels in adult mouse cardiomyocytes via class IIa histone deacetylase (HDAC)/myocyte enhancer factor-2 (MEF2)-dependent signaling. In addition, pharmacological inhibition of calmodulin/CaMKIIδB activity improved cardiac function in TAC mice, which partially normalized the imbalance between NCX1 and SERCA2. These data identify NCX1 as a cellular target for CaMKIIδB. We also suggest that the CaMKIIδB-induced imbalance between NCX1 and SERCA2 is partially responsible for the disturbance of intracellular Ca2+ homeostasis and the pathological process of heart failure.

P2RX7 sensitizes Mac-1/ICAM-1-dependent leukocyte-endothelial adhesion and promotes neurovascular injury during septic encephalopathy.

Septic encephalopathy (SE) is a critical factor determining sepsis mortality. Vascular inflammation is known to be involved in SE, but the molecular events that lead to the development of encephalopathy remain unclear. Using time-lapse in vivo two-photon laser scanning microscopy, we provide the first direct evidence that cecal ligation and puncture in septic mice induces microglial trafficking to sites adjacent to leukocyte adhesion on inflamed cerebral microvessels. Our data further demonstrate that septic injury increased the chemokine CXCL1 level in brain endothelial cells by activating endothelial P2RX7 and eventually enhanced the binding of Mac-1 (CD11b/CD18)-expressing leukocytes to endothelial ICAM-1. In turn, leukocyte adhesion upregulated endothelial CX3CL1, thereby triggering microglia trafficking to the injured site. The sepsis-induced increase in endothelial CX3CL1 was abolished in CD18 hypomorphic mutant mice. Inhibition of the P2RX7 pathway not only decreased endothelial ICAM-1 expression and leukocyte adhesion but also prevented microglia overactivation, reduced brain injury, and consequently doubled the early survival of septic mice. These results demonstrate the role of the P2RX7 pathway in linking neurovascular inflammation to brain damage in vivo and provide a rationale for targeting endothelial P2RX7 for neurovascular protection during SE.

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.

Melatonin reverses the decreases in hippocampal protein serine/threonine kinases observed in an animal model of autism.

Lower global cognitive function scores are a common symptom of autism spectrum disorders (ASDs). This study investigates the effects of melatonin on hippocampal serine/threonine kinase signaling in an experimental ASD model. We found that chronic melatonin (1.0 or 5.0 mg/kg/day, 28 days) treatment significantly rescued valproic acid (VPA, 600 mg/kg)‐induced decreases in CaMKII (Thr286), NMDAR1 (Ser896), and PKA (Thr197) phosphorylation in the hippocampus without affecting total protein levels. Compared with control rats, the immunostaining of pyramidal neurons in the hippocampus revealed a decrease in immunolabeling intensity for phospho‐CaMKII (Thr286) in the hippocampus of VPA‐treated rats, which was ameliorated by chronic melatonin treatment. Consistent with the elevation of CaMKII/PKA/PKC phosphorylation observed in melatonin‐treated rat, long‐term potentiation (LTP) was enhanced after chronic melatonin (5.0 mg/kg) treatment, as reflected by extracellular field potential slopes that increased from 56 to 60 min (133.4 ± 3.9% of the baseline, P < 0.01 versus VPA‐treated rats) following high‐frequency stimulation (HFS) in hippocampal slices. Accordingly, melatonin treatment also significantly improved social behavioral deficits at postnatal day 50 in VPA‐treated rats. Taken together, the increased phosphorylation of CaMKII/PKA/PKC signaling might contribute to the beneficial effects of melatonin on autism symptoms.

In Vivo Two-photon Fluorescence Microscopy Reveals Disturbed Cerebral Capillary Blood Flow and Increased Susceptibility to Ischemic Insults in Diabetic Mice

Diabetes mellitus increases the risk of stroke, but the mechanisms are unclear. The present study tested the hypothesis that diabetes mellitus disturbs the brain microcirculation and increases the susceptibility to cerebral damage in a middle cerebral artery occlusion (MCAO) model of ischemia.