Shang Z. Guo

Cardiomyocyte NF-κB p65 promotes adverse remodelling, apoptosis, and endoplasmic reticulum stress in heart failure

AIMS the role of nuclear factor (NF)-κB in heart failure (HF) is not well defined. We sought to determine whether myocyte-localized NF-κB p65 activation in HF exacerbates post-infarction remodelling and promotes maladaptive endoplasmic reticulum (ER) stress. METHODS AND RESULTS non-transgenic (NTg) and transgenic (Tg) mice with myocyte-restricted overexpression of a phosphorylation-resistant inhibitor of κBα (IκBα(S32A,S36A)) underwent coronary ligation (to induce HF) or sham operation. Over 4 weeks, the remote myocardium of ligated hearts exhibited robust NF-κB activation that was almost exclusively p65 beyond 24 h. Compared with sham at 4 weeks, NTg HF hearts were dilated and dysfunctional, and exhibited hypertrophy, fibrosis, up-regulation of inflammatory cytokines, increased apoptosis, down-regulation of ER protein chaperones, and up-regulation of the ER stress-activated pro-apoptotic factor CHOP. Compared with NTg HF, Tg-IκBα(S32A,S36A) HF mice exhibited: (i) improved survival, chamber remodelling, systolic function, and pulmonary congestion, (ii) markedly diminished NF-κB p65 activation, cytokine expression, and fibrosis, and (iii) a three-fold reduction in apoptosis. Moreover, Tg-IκBα(S32A,S36A) HF hearts exhibited maintained expression of ER chaperones and CHOP when compared with sham. In cardiomyocytes, NF-κB activation was required for ER stress-mediated apoptosis, whereas abrogation of myocyte NF-κB shifted the ER stress response to one of adaptation and survival. CONCLUSION persistent myocyte NF-κB p65 activation in HF exacerbates cardiac remodelling by imparting pro-inflammatory, pro-fibrotic, and pro-apoptotic effects. p65 modulation of cell death in HF may occur in part from NF-κB-mediated transformation of the ER stress response from one of adaptation to one of apoptosis.

Nitric oxide synthase and intermittent hypoxia-induced spatial learning deficits in the rat.

Intermittent hypoxia (IH) during sleep induces significant neurobehavioral deficits in the rat. Since nitric oxide (NO) has been implicated in ischemia-reperfusion-related pathophysiological consequences, the temporal effects of IH (alternating 21% and 10% O(2) every 90 s) and sustained hypoxia (SH; 10% O(2)) during sleep for up to 14 days on the induction of nitric oxide synthase (NOS) isoforms in the brain were examined in the cortex of Sprague-Dawley rats. No significant changes of endothelial NOS (eNOS) and neuronal NOS (nNOS) occurred over time with either IH or SH. Similarly, inducible NOS (iNOS) was not affected by SH. However, increased expression and activity of iNOS were observed on days 1 and 3 of IH (P < 0.01 vs. control; n = 12/group) and were followed by a return to basal levels on days 7 and 14. Furthermore, IH-mediated neurobehavioral deficits in the water maze were significantly attenuated in iNOS knockout mice. We conclude that IH is associated with a time-dependent induction of iNOS and that the increased expression of iNOS may play a critical role in the early pathophysiological events leading to IH-mediated neurobehavioral deficits.

Increased susceptibility to intermittent hypoxia in aging rats: changes in proteasomal activity, neuronal apoptosis and spatial function

Obstructive sleep apnea (OSA) is a frequent medical condition characterized by intermittent hypoxia (IH) during sleep, and is associated with neurodegenerative changes in several brain regions along with learning deficits. We hypothesized that aging rats exposed to IH during sleep would be particularly susceptible. Young (3–4 months) and aging (20–22 months) Sprague–Dawley rats were therefore exposed to either room air or IH for 14 days. Learning and memory was assessed with a standard place‐training version of the Morris water maze. Aging rats exposed to room air (RA) or IH displayed significant spatial learning impairments compared with similarly exposed young rats; furthermore, the decrements in performance between RA and IH were markedly greater in aging compared with young rats (p < 0.01), and coincided with the magnitude of IH‐induced decreases in cyclic AMP response element binding (CREB) phosphorylation. Furthermore, decreases in proteasomal activity occurred in both young and aging rats exposed to IH, but were substantially greater in the latter (p < 0.001). Neuronal apoptosis, as shown by cleaved caspase 3 expression, was particularly increased in aging rats exposed to IH (p < 0.01 versus young rats exposed to IH). Collectively, these findings indicate unique vulnerability of the aging rodent brain to IH, which is reflected at least in part, by the more prominent decreases in CREB phosphorylation and a marked inability of the ubiquitin‐proteasomal pathway to adequately clear degraded proteins.

Intermittent hypoxic exposure during light phase induces changes in cAMP response element binding protein activity in the rat CA1 hippocampal region: water maze performance correlates.

Intermittent hypoxia (IH) during sleep, a characteristic feature of sleep-disordered breathing (SDB) is associated with time-dependent apoptosis and spatial learning deficits in the adult rat. The mechanisms underlying such neurocognitive deficits remain unclear. Activation of the cAMP-response element binding protein (CREB) transcription factor mediates critical components of neuronal survival and memory consolidation in mammals. CREB phosphorylation and DNA binding, as well as the presence of apoptosis in the CA1 region of the hippocampus were examined in Sprague-Dawley male rats exposed to IH. Spatial reference task learning was assessed with the Morris water maze. IH induced significant decreases in Ser-133 phosphorylated CREB (pCREB) without changes in total CREB, starting as early as 1 h IH, peaking at 6 h-3 days, and returning toward normoxic levels by 14-30 days. Double-labeling immunohistochemistry for pCREB and Neu-N (a neuronal marker) confirmed these findings. The expression of cleaved caspase 3 (cC3) in the CA1, a marker of apoptosis, peaked at 3 days and returned to normoxic values at 14 days. Initial IH-induced impairments in spatial learning were followed by partial functional recovery starting at 14 days of IH exposure. We postulate that IH elicits time-dependent changes in CREB phosphorylation and nuclear binding that may account for decreased neuronal survival and spatial learning deficits in the adult rat. We suggest that CREB changes play an important role in the neurocognitive morbidity of SDB patients.

Temporal aspects of spatial task performance during intermittent hypoxia in the rat: evidence for neurogenesis

Intermittent hypoxia (IH) during sleep, such as occurs in obstructive sleep apnea, leads to degenerative changes in the hippocampus, and is associated with spatial learning deficits in the adult rat. We report that in Sprague–Dawley rats the initial IH‐induced impairments in spatial learning are followed by a partial functional recovery over time, despite continuing IH exposure. These functional changes coincide with initial decreases in basal neurogenesis as shown by the number of positively colabelled cells for BrdU and neurofilament in the dentate gyrus of the hippocampus, and are followed by increased expression of neuronal progenitors and mature neurons (nestin and BrdU‐neurofilament positively labelled cells, respectively). In contrast, no changes occurred during the course of IH exposures in the expression of the synaptic proteins synaptophysin, SNAP25, and drebrin. Collectively, these findings indicate that the occurrence of IH during the lights on period results in a biphasic pattern of neurogenesis in the hippocampus of adult rats, and may account for the observed partial recovery of spatial function.

Platelet-activating factor receptor-deficient mice are protected from experimental sleep apnea-induced learning deficits

Intermittent hypoxia (IH) during sleep, a hallmark of sleep apnea, is associated with neurobehavioral impairments, regional neurodegeneration and increased oxidative stress and inflammation in rodents. Platelet‐activating factor (PAF) is an important mediator of both normal neural plasticity and brain injury. We report that mice deficient in the cell surface receptor for PAF (PAFR–/–), a bioactive mediator of oxidative stress and inflammation, are protected from the spatial reference learning deficits associated with IH. Furthermore, PAFR–/– exhibit attenuated elevations in inflammatory signaling (cyclo‐oxygenase‐2 and inducible nitric oxide synthase activities), degradation of the ubiquitin–proteasome pathway and apoptosis observed in wild‐type littermates (PAFR+/+) exposed to IH. Collectively, these findings indicate that inflammatory signaling and neurobehavioral impairments induced by IH are mediated through PAF receptors.

γ-Amino Butyric Acid Type B Receptors Stimulate Neutrophil Chemotaxis during Ischemia-Reperfusion

Serine/threonine kinase Akt, or protein kinase B, has been shown to regulate a number of neutrophil functions. We sought to identify Akt binding proteins in neutrophils to provide further insights into understanding the mechanism by which Akt regulates various neutrophil functions. Proteomic and immunoprecipitation studies identified γ-amino butyric acid (GABA) type B receptor 2 (GABABR2) as an Akt binding protein in human neutrophils. Neutrophil lysates subjected to Akt immunoprecipitation followed by immunoblotting with anti-GABABR2 demonstrated Akt association with the intact GABABR. Similar results were obtained when reciprocal immunoprecipitations were performed with anti-GABABR2 Ab. Additionally, GABABR2 and Akt colocalization was demonstrated by confocal microscopy. A GABABR agonist, baclofen, activated Akt and stimulated neutrophil-directed migration in a PI3K-dependent manner, whereas CGP52432, a GABABR antagonist blocked such effects. Baclofen, stimulated neutrophil chemotaxis and tubulin reorganization in a PI3K-dependent manner. Additionally, a GABABR agonist failed to stimulate neutrophil superoxide burst. We are unaware of the association of GABABR with Akt in any cell type. The present study shows for the first time that a brain-specific receptor, GABABR2 is present in human neutrophils and that it is functionally associated with Akt. Intraventricular baclofen pretreatment in rats subjected to a stroke model showed increased migration of neutrophils to the ischemic lesion. Thus, the GABABR is functionally expressed in neutrophils, and acts as a chemoattractant receptor via an Akt-dependent pathway. The GABABR potentially plays a significant role in the inflammatory response and neutrophil-dependent ischemia-reperfusion injury such as stroke.

Exogenous growth hormone attenuates cognitive deficits induced by intermittent hypoxia in rats

Sleep disordered breathing (SDB), which is characterized by intermittent hypoxia (IH) during sleep, causes substantial cardiovascular and neurocognitive complications and has become a growing public health problem. SDB is associated with suppression of growth hormone (GH) secretion, the latter being integrally involved in the growth, development, and function of the CNS. Since GH treatment is able to attenuate neurocognitive deficits in a hypoxic-ischemic stroke model, GH, GH receptor (GHR) mRNA expression, and GH protein expression were assessed in rat hippocampus after exposures to chronic sustained hypoxia (CH, 10% O(2)) or IH (10% O(2) alternating with 21% O(2) every 90 s). In addition, the effect of GH treatment (50 μg/kg daily s.c. injection) on erythropoietin (EPO), vascular endothelial growth factor (VEGF), heme oxygenase-1 (HO-1), and GLUT-1 mRNA expression and neurobehavioral function was assessed. CH significantly increased GH mRNA and protein expression, as well as insulin-like growth factor-1 (IGF-1). In contrast, IH only induced a moderate increase in GH mRNA and a slight elevation in GH protein at day 1, but no increases in IGF-1. CH, but not IH, up-regulated GHR mRNA in the hippocampus. IH induced marked neurocognitive deficits compared with CH or room air (RA). Furthermore, exogenous GH administration increased hippocampal mRNA expression of IGF-1, EPO, and VEGF, and not only reduced IH-induced hippocampal injury, but also attenuated IH-induced cognitive deficits. Thus, exogenous GH may provide a viable therapeutic intervention to protect IH-vulnerable brain regions from SDB-associated neuronal loss and associated neurocognitive dysfunction.

Monocarboxylate Transporter 2 and Stroke Severity in a Rodent Model of Sleep Apnea

Stroke is not only more prevalent but is also associated with more severe adverse functional outcomes among patients with sleep apnea. Monocarboxylate transporters (MCT) are important regulators of cellular bioenergetics, have been implicated in brain susceptibility to acute severe hypoxia (ASH), and could underlie the unfavorable prognosis of cerebrovascular accidents in sleep apnea patients. Rodents were exposed to either intermittent hypoxia (IH) during sleep, a characteristic feature of sleep apnea, or to sustained hypoxia (SH), and expression of MCT1 and MCT2 was assessed. In addition, the functional recovery to middle cerebral artery occlusion (MCAO) in rats and hMCT2 transgenic mice and of hippocampal slices subjected to ASH was assessed, as well as the effects of MCT blocker and MCT2 antisense oligonucleotides and siRNAs. IH, but not SH, induced significant reductions in MCT2 expression over time at both the mRNA and protein levels and in the functional recovery of hippocampal slices subjected to ASH. Similarly, MCAO-induced infarcts were significantly greater in IH-exposed rats and mice, and overexpression of hMCT2 in mice markedly attenuated the adverse effects of IH. Exogenous pyruvate treatment reduced infarct volumes in normoxic rats but not in IH-exposed rats. Administration of the MCT2 blocker 4CN, but not the MCT1 antagonist p-chloromercuribenzene sulfonate, increased infarct size. Thus, prolonged exposures to IH mimicking sleep apnea are associated with increased CNS vulnerability to ischemia that is mediated, at least in part, by concomitant decreases in the expression and function of MCT2. Efforts to develop agonists of MCT2 should provide opportunities to ameliorate the overall outcome of stroke.