Redox-active antioxidant modulation of lipid signaling in vascular endothelial cells: vitamin C induces activation of phospholipase D through phospholipase A2, lipoxygenase, and cyclooxygenase

We have earlier reported that the redox-active antioxidant, vitamin C (ascorbic acid), activates the lipid signaling enzyme, phospholipase D (PLD), at pharmacological doses (mM) in the bovine lung microvascular endothelial cells (BLMVECs). However, the activation of phospholipase A(2) (PLA(2)), another signaling phospholipase, and the modulation of PLD activation by PLA(2) in the ECs treated with vitamin C at pharmacological doses have not been reported to date. Therefore, this study aimed at the regulation of PLD activation by PLA(2) in the cultured BLMVECs exposed to vitamin C at pharmacological concentrations. The results revealed that vitamin C (3-10 mM) significantly activated PLA(2) starting at 30 min; however, the activation of PLD resulted only at 120 min of treatment of cells under identical conditions. Further studies were conducted utilizing specific pharmacological agents to understand the mechanism(s) of activation of PLA(2) and PLD in BLMVECs treated with vitamin C (5 mM) for 120 min. Antioxidants, calcium chelators, iron chelators, and PLA(2) inhibitors offered attenuation of the vitamin C-induced activation of both PLA(2) and PLD in the cells. Vitamin C was also observed to significantly induce the formation and release of the cyclooxygenase (COX)- and lipoxygenase (LOX)-catalyzed arachidonic acid (AA) metabolites and to activate the AA LOX in BLMVECs. The inhibitors of PLA(2), COX, and LOX were observed to effectively and significantly attenuate the vitamin C-induced PLD activation in BLMVECs. For the first time, the results of the present study revealed that the vitamin C-induced activation of PLD in vascular ECs was regulated by the upstream activation of PLA(2), COX, and LOX through the formation of AA metabolites involving oxidative stress, calcium, and iron.     

Intermittent hypoxia reduces upper airway stability in lean but not obese Zucker rats

Obstructive sleep apnea involves intermittent periods of airway occlusions that lead to repetitive oxygen desaturations. Exposure to chronic intermittent hypoxia (IH) in rats increases diurnal blood pressure and alters skeletal muscle physiology. The impact of IH on upper airway muscle function is unknown. We hypothesize that IH exposure increases upper airway collapsibility in rats due to alterations of the muscles surrounding the upper airway. Lean and obese rats were exposed to cyclic alterations in O(2) levels (20.6%-5%) every 90 s, 8 h/day for 6 days/wk for 12 wk. Following the exposure period, arterial pressure was recorded via the tail artery in conscious unrestrained rats. Mean arterial pressure was increased in lean IH but not in obese IH-exposed Zucker rats (P < 0.05). The pharyngeal pressure associated with airway collapse (P(crit)) was measured under anesthesia during baseline conditions and then during supramaximal stimulation of the hypoglossal nerve (cnXII). Baseline P(crit) was more positive (more collapsible) in lean but not obese rats following 12 wk of IH (P < 0.05), while supramaximal stimulation of cnXII increased airway stability (decreased P(crit)) in both lean and obese Zucker rats following IH to levels that were similar to their respective room air controls. The in vitro peak tension and the expression of the individual myosin heavy chain isoforms from the upper airway muscles were unaltered following IH. We conclude that IH leads to increases in baseline collapsibility in lean Zucker rats exposed to IH by nonmyogenic mechanisms.     

Vitamin C-induced activation of phospholipase D in lung microvascular endothelial cells: regulation by MAP kinases

Our earlier studies have shown that vitamin C at pharmacological doses (mM) induces loss of redox-dependent viability in bovine lung microvascular endothelial cells (BLMVECs) that is mediated by oxidative stress. Therefore, here, we investigated the vitamin C-induced activation of the lipid signaling enzyme, phospholipase D (PLD) in BLMVECs. Monolayer cultures of BLMVECs were treated with vitamin C (0-10 mM) for different time periods (0-2 h) and the activity of PLD was determined. Vitamin C induced activation of PLD in BLMVECs in a time- and dose-dependent fashion that was significantly attenuated by antioxidants, p38 mitogen-activated protein kinase (p38 MAPK)-specific inhibitor (SB203580), extracellular signal-regulated protein kinase (ERK)-specific inhibitor (PD98059), and transient transfection of cells with dominant-negative (DN)-p38 MAPK and DN-ERK1/ERK2. Vitamin C also induced phosphorylation and enhanced the activities of p38 MAPK and ERK in BLMVECs in a time-dependent fashion. It was also evident that vitamin C induced translocation of PLD(1) and PLD(2), association of p38 MAPK and ERK with PLD(1) and PLD(2), threonine phosphorylation of PLD(1) and PLD(2) and SB203580- and PD98059-inhibitable threonine phosphorylation of PLD(1) in BLMVECs. Transient transfection of BLMVECs with DN-p38 MAPK and DN-ERK1/ERK2 resulted in marked attenuation of vitamin C-induced phosphorylation of threonine in PLD(1) and PLD(2). We, for the first time, showed that vitamin C at pharmacological doses, activated PLD in the lung microvascular ECs through oxidative stress and MAPK activation.     

Deviation of eyes and head in acute cerebral stroke

Background  

It is a well-known phenomenon that some patients with acute left or right hemisphere stroke show a deviation of the eyes (Prévost's sign) and head to one side. Here we investigated whether both right- and left-sided brain lesions may cause this deviation. Moreover, we studied the relationship between this phenomenon and spatial neglect. In contrast to previous studies, we determined not only the discrete presence or absence of eye deviation with the naked eye through clinical inspection, but actually measured the extent of horizontal eye-in-head and head-on-trunk deviation. In further contrast, measurements were performed early after stroke onset (1.5 days on average).     

miR-155 deletion modulates lipopolysaccharide-induced sleep in female mice

Immune signaling is known to regulate sleep. miR-155 is a microRNA that regulates immune responses. We hypothesized that miR-155 would alter sleep regulation. Thus, we investigated the potential effects of miR-155 deletion on sleep-wake behavior in adult female homozygous miR-155 knockout (miR-155^KO) mice and littermate controls (WT). Mice were implanted with biotelemetry units and EEG/EMG biopotentials were recorded continuously for three baseline days. miR-155^KO mice had decreased bouts of NREM and REM sleep compared with WT mice, but no differences were observed in the length of sleep bouts or total time spent in sleep-wake states. Locomotor activity and subcutaneous temperature did not differ between WT and miR-155^KO mice. Following baseline recordings, mice were sleep-deprived during the first six hours of the rest phase (light phase; ZT 0–6) followed by an 18 h recovery period. There were no differences between groups in sleep rebound (% sleep and NREM δ power) after sleep deprivation. Following recovery from sleep deprivation, mice were challenged with a somnogen (viz., lipopolysaccharide (LPS)) one hour prior to the initiation of the dark (active) phase. Biopotentials were continuously recorded for the following 24 h, and miR-155^KO mice displayed increased wakefulness and decreased NREM sleep during the dark phase following LPS injection. Additionally, miR-155^KO mice had reduced EEG slow-wave responses (0.5–4 Hz) compared to WT mice. Together, our findings indicate that miR-155 deletion attenuates the somnogenic and EEG delta-enhancing effects of LPS.

Abbreviations: ANOVA: analysis of variance; EEG: electroencephalogram; EMG: electromyogram; h: hour; IL-1: interleukin-1; IL-6: interleukin-6; IP: intra-peritoneal; LPS: lipopolysaccharide; miR/miRNA: microRNA; miR-155^KO: miR-155 knockout; NREM: non-rapid eye movement; REM: rapid eye movement; TNF: tumor necrosis factor; SWS: slow-wave sleep; WT: wild-type.     

Adiponectin Protects Against Hyperoxic Lung Injury and Vascular Leak

Adiponectin (Ad), an adipokine exclusively secreted by the adipose tissue, has emerged as a paracrine metabolic regulator as well as a protectant against oxidative stress. Pharmacological approaches of protecting against clinical hyperoxic lung injury during oxygen therapy/treatment are limited. We have previously reported that Ad inhibits the NADPH oxidase-catalyzed formation of superoxide from molecular oxygen in human neutrophils. Based on this premise, we conducted studies to determine whether (i) exogenous Ad would protect against the hyperoxia-induced barrier dysfunction in the lung endothelial cells (ECs) in vitro, and (ii) endogenously synthesized Ad would protect against hyperoxic lung injury in wild-type (WT) and Ad-overexpressing transgenic (AdTg) mice in vivo. The results demonstrated that exogenous Ad protected against the hyperoxia-induced oxidative stress, loss of glutathione (GSH), cytoskeletal reorganization, barrier dysfunction, and leak in the lung ECs in vitro. Furthermore, the hyperoxia-induced lung injury, vascular leak, and lipid peroxidation were significantly attenuated in AdTg mice in vivo. Also, AdTg mice exhibited elevated levels of total thiols and GSH in the lungs as compared with WT mice. For the first time, our studies demonstrated that Ad protected against the hyperoxia-induced lung damage apparently through attenuation of oxidative stress and modulation of thiol-redox status.     

Correction: deviation of eyes and head in acute cerebral stroke

This is a correction article     

Photoperiod alters duration and intensity of non-rapid eye movement sleep following immune challenge in Siberian hamsters (Phodopus sungorus)

Sleep is regulated by circadian and homeostatic processes, but can be altered by infectious disease. During infection or exposure to inflammatory stimuli, such as bacterial lipopolysaccharide (LPS), the duration and intensity of non-rapid eye movement sleep (NREMS), as measured by electoencephalogram (EEG) delta waves (.5-4 Hz), increase. These sleep alterations are hypothesized to conserve or redirect energy for immune system activation. Many vertebrates exhibit seasonal changes in immune function and sleep-wake cycle, and photoperiod (day length) serves as a reliable environmental cue. For example, winter is energetically demanding for most animals, and Siberian hamsters (Phodopus sungorus) adapted to short winter day lengths display reduced fever after LPS administration to presumably conserve energy. We hypothesized that short days increase the duration and intensity of NREMS after LPS challenge to create additional energy savings, despite evidence to the contrary that high fever is associated with increased NREMS. Male hamsters were housed under long (16 h light (L):8 h dark (D)) or short (8L:16D) day lengths, and chronically implanted with transmitters that recorded EEG and electromyogram (EMG) biopotentials simultaneously or core body temperature. After >10 wks in photoperiod conditions, hamsters received an i.p. injection of LPS or saline (control), and vigilance states (duration and distribution of NREMS, rapid eye movement sleep (REMS), and wakefulness) and EEG delta power spectra (NREMS intensity) were assessed. As expected, LPS treatment increased the duration and intensity of NREMS compared to controls. Hamsters adapted to short photoperiods exhibited cumulatively larger increases in NREMS duration and EEG delta wave amplitude 0-8 h after LPS injection compared to long-day LPS-treated hamsters despite short-day attenuation of fever. These results suggest a seasonal decoupling of LPS-induced fever with sleep to promote energy conservation during predictable energy shortages. Ultimately, the combination of increased sleep and reduced fever could represent a suite of physiological adaptations that increase the probability of surviving winter.     

Eicosanoid Signaling and Vascular Dysfunction: Methylmercury-Induced Phospholipase D Activation in Vascular Endothelial Cells

Mercury, especially methylmercury (MeHg), is implicated in the etiology of cardiovascular diseases. Earlier, we have reported that MeHg induces phospholipase D (PLD) activation through oxidative stress and thiol-redox alteration. Hence, we investigated the mechanism of the MeHg-induced PLD activation through the upstream regulation by phospholipase A₂ (PLA₂) and lipid oxygenases such as cyclooxygenase (COX) and lipoxygenase (LOX) in the bovine pulmonary artery endothelial cells (BPAECs). Our results showed that MeHg significantly activated both PLA₂ (release of [³H]arachidonic acid, AA) and PLD (formation of [³²P]phosphatidylbutanol) in BPAECs in dose- (0–10 μM) and time-dependent (0–60 min) fashion. The cPLA₂-specific inhibitor, arachidonyl trifluoromethyl ketone (AACOCF₃), significantly attenuated the MeHg-induced [³H]AA release in ECs. MeHg-induced PLD activation was also inhibited by AACOCF₃ and the COX- and LOX-specific inhibitors. MeHg also induced the formation of COX- and LOX-catalyzed eicosanoids in ECs. MeHg-induced cytotoxicity (based on lactate dehydrogenase release) was protected by PLA₂-, COX-, and LOX-specific inhibitors and 1-butanol, the PLD-generated PA quencher. For the first time, our studies showed that MeHg activated PLD in vascular ECs through the upstream action of cPLA₂ and the COX- and LOX-generated eicosanoids. These results offered insights into the mechanism(s) of the MeHg-mediated vascular endothelial cell lipid signaling as an underlying cause of mercury-induced cardiovascular diseases.