Physiologic levels of resistin induce a shift from proliferation to apoptosis in macrophage and VSMC co-culture.

BACKGROUND: Resistin, an adipokine with inflammatory properties, has been associated with plaque vulnerability. Vascular smooth muscle cells and macrophages are the major cellular components in advanced atherosclerotic plaques and interdependently affect plaque stability. The purpose of this study was to examine the effects of resistin on the interactions of vascular smooth muscle cells and macrophages using co-culture systems. METHODS: Human monocytes were differentiated into macrophages. Vascular smooth muscle cells were grown and starved prior to co-culture condition. Indirect co-culture was performed by treating macrophages with resistin at 10 ng/mL for 24 hours with/without εV1-2, a selective protein kinase C epsilon inhibitor. Macrophages supernatants were then used to treat vascular smooth muscle cells for 24 hours. Direct co-culture was performed by culturing macrophages and vascular smooth muscle cells together for 24 to 48 hours. Cultures were evaluated for changes in proliferation, apoptosis, and gene expression of apoptosis, proliferation, and inflammation-associated genes. RESULTS: Macrophages induced vascular smooth muscle cells proliferation, which was further exaggerated in resistin-treated macrophages in the indirect co-culture model. Resistin also upregulated cyclin D1 and proliferating cell nuclear antigen via protein kinase C epsilon in the indirect co-culture. Augmented proliferation was further confirmed in the direct co-culture model, particularly at increased macrophage ratios. However, resistin treatment induced apoptosis in the presence of direct cell to cell interactions. Along with the shift to apoptosis, expressions of caspase 3 and caspase 8 were upregulated. The expression of kappa-light-chain-enhancer of activated B cells 1 and 2 was similar in direct and indirect co-cultures. CONCLUSION: Resistin promotes a shift from proliferation to apoptosis in vascular smooth muscle cells and macrophage co-culture systems with cellular composition similar to that found in vulnerable regions of plaques. Protein kinase C epsilon mediates the effects of resistin, suggesting that protein kinase C epsilon may represent a therapeutic strategy in resistin-associated atherosclerotic complications.

PKC-epsilon and TLR4 synergistically regulate resistin-mediated inflammation in human macrophages.

BACKGROUND AND AIMS: Resistin has been associated with atherosclerotic inflammation and cardiovascular complications. We and others have previously shown that PKC-epsilon (PKCε) is involved in resistin-induced smooth muscle cell (VSMC) dysfunction at a high pathological concentration. This study aimed to evaluate the role and potential pathways of resistin at a physiological concentration, in atherosclerosis-related inflammation. METHODS: Plasma from patients with atherosclerosis was analyzed for resistin concentration. Patients were divided into tertiles based on resistin levels and cytokines were compared between tertiles. Macrophages were then treated with resistin in the presence or absence of PKCε inhibitor and/or TLR4 blocking-antibody, and their inflammatory state was evaluated with ELISA, RT-PCR, immunocytochemistry, and Western blot. RESULTS: We observed significant associations between plasma resistin levels and TNF-α, IL-6, IL-12, MIP-1α, MIP-1ß, and CD40L. Our in vitro analyses revealed that resistin activated PKCε via TLR4. This was followed by NF-kB activation and induction of a pro-inflammatory phenotype in macrophages, significantly upregulating CD40, downregulating CD206 and stimulating gene expression and secretion of the inflammatory cytokines, for which we found association in our plasma analysis. Resistin also induced persistent TRAM and CD40L upregulation up to 36 h after resistin treatment. PKCε and TLR4 inhibitors suppressed gene expression to levels similar to control, especially when used in combination. CONCLUSIONS: Resistin, at a physiological concentration, exacerbates the inflammatory response of macrophages. PKCε is a key upstream mediator in resistin-induced inflammation that may interact synergistically with TLR4 to promote NF-kB activation, while TRAM is an important signal. PKCε and TRAM may represent novel molecular targets for resistin-associated chronic atherosclerotic inflammation.

Eotaxin Augments Calcification in Vascular Smooth Muscle Cells.

Calcification of atherosclerotic plaques in elderly patients represents a potent risk marker of cardiovascular events. Plasma analyses of patients with or without calcified plaques reveal significant differences in chemokines, particularly eotaxin, which escalates with increased calcification. We therefore, hypothesize that eotaxin in circulation augments calcification of vascular smooth muscle cells (VSMCs) possibly via oxidative stress in the vasculature. We observe that eotaxin increases the rate of calcification significantly in VSMCs as evidenced by increased alkaline phosphatase activity, calcium deposition, and osteogenic marker expression. In addition, eotaxin promotes proliferation in VSMCs and triggers oxidative stress in a NADPH oxidase dependent manner. These primary novel observations support our proposition that in the vasculature eotaxin augments mineralization. Our findings suggest that eotaxin may represent a potential therapeutic target for prevention of cardiovascular complications in the elderly. J. Cell. Biochem. 118: 647-654, 2017. © 2016 Wiley Periodicals, Inc.

PKCε mediates resistin-induced NADPH oxidase activation and inflammation leading to smooth muscle cell dysfunction and intimal hyperplasia.

BACKGROUND AND AIMS: Resistin has been implicated in cardiovascular disease and poor interventional cardiovascular outcomes. Previous studies by our group demonstrated resistin promoted vascular smooth muscle cell (VSMC) migration through protein kinase C epsilon (PKCε) pathways, while few others showed that resistin induced reactive oxygen species (ROS) generation in various cell types. In this study, we aim to systemically examine the functional role of resistin at the cellular and tissue levels as well as the potential mechanistic relationship between resistin-induced PKCε activation and ROS production. METHODS: Plasma collected from patients undergoing carotid interventions was analyzed for resistin level and ROS. VSMCs were treated with resistin in the presence or absence of PKCε and NADPH oxidase (Nox)-specific inhibitors. Intracellular ROS production was analyzed using confocal microscopy and Nox activity with chemiluminescence. In vivo studies were performed in apolipoprotein E knock out (ApoE-/-) mice to determine therapeutic effects of PKCε-specific inhibitor, using the guide-wire injury model. RESULTS: We observed significant correlation between plasma resistin and circulating levels of oxidative stress in patients with severe atherosclerotic disease. We also demonstrated that resistin induced ROS production via PKCε-mediated Nox activation. Resistin-induced ROS production was time-dependent, and Nox4 was the primary isoform involved. Inhibition of Nox completely abolished resistin-exaggerated VSMC proliferation, migration and dedifferentiation, as well as pro-inflammatory cytokine release. Upstream modulation of PKCε significantly reduced resistin-mediated cytosolic ROS, Nox activity and VSMC dysfunction. Moreover, PKCε-specific inhibitor mitigated resistin-induced Nox activation and intimal hyperplasia in ApoE-/- mice. CONCLUSIONS: Resistin-associated VSMC dysfunction and intimal hyperplasia are related to PKCε-dependent Nox activation and ROS generation. Targeting the PKCε-Nox pathway may represent a novel strategy in managing resistin-associated atherosclerotic complications.

A Prospective Evaluation of Systemic Biomarkers and Cognitive Function Associated with Carotid Revascularization.

OBJECTIVE: To determine factors affecting cognition and identify predictors of long-term cognitive impairment following carotid revascularization procedures. BACKGROUND: Cognitive impairment is common in older patients with carotid occlusive diseases. METHODS: Patients undergoing carotid intervention for severe occlusive diseases were prospectively recruited. Patients received neurocognitive testing before, 1, and 6 months after carotid interventions. Plasma samples were also collected within 24 hours after carotid intervention and inflammatory cytokines were analyzed. Univariate and multivariate logistic regressions were performed to identify risk factors associated with significant cognitive deterioration (>10% decline). RESULTS: A total of 98 patients (48% symptomatic) were recruited, including 55 patients receiving carotid stenting and 43 receiving endarterectomy. Mean age was 69 (range 54-91 years). Patients had overall improvement in cognitive measures 1 month after revascularization. When compared with carotid stenting, endarterectomy patients demonstrated postoperative improvement in cognition at 1 and 6 months compared with baseline. Carotid stenting (odds ratio 6.49, P = 0.020) and age greater than 80 years (odds ratio 12.6, P = 0.023) were associated with a significant long-term cognitive impairment. Multiple inflammatory cytokines also showed significant changes after revascularization. On multivariate analysis, after controlling for procedure and age, IL-12p40 (P = 0.041) was associated with a higher risk of significant cognitive impairment at 1 month; SDF1-α (P = 0.004) and tumor necrosis factor alpha (P = 0.006) were independent predictors of cognitive impairment, whereas interleukin-6 (P = 0.019) demonstrated cognitive protective effects at 6 months after revascularization. CONCLUSIONS: Carotid interventions affect cognitive function. Systemic biomarkers can be used to identify patients at risk of significant cognitive decline postprocedures that benefit from targeted cognitive training.

Protein kinase G-regulated production of H2S governs oxygen sensing.

Reflexes initiated by the carotid body, the principal O2-sensing organ, are critical for maintaining cardiorespiratory homeostasis during hypoxia. O2 sensing by the carotid body requires carbon monoxide (CO) generation by heme oxygenase-2 (HO-2) and hydrogen sulfide (H2S) synthesis by cystathionine-γ-lyase (CSE). We report that O2 stimulated the generation of CO, but not that of H2S, and required two cysteine residues in the heme regulatory motif (Cys(265) and Cys(282)) of HO-2. CO stimulated protein kinase G (PKG)-dependent phosphorylation of Ser(377) of CSE, inhibiting the production of H2S. Hypoxia decreased the inhibition of CSE by reducing CO generation resulting in increased H2S, which stimulated carotid body neural activity. In carotid bodies from mice lacking HO-2, compensatory increased abundance of nNOS (neuronal nitric oxide synthase) mediated O2 sensing through PKG-dependent regulation of H2S by nitric oxide. These results provide a mechanism for how three gases work in concert in the carotid body to regulate breathing.

Inherent variations in CO-H2S-mediated carotid body O2 sensing mediate hypertension and pulmonary edema.

Oxygen (O2) sensing by the carotid body and its chemosensory reflex is critical for homeostatic regulation of breathing and blood pressure. Humans and animals exhibit substantial interindividual variation in this chemosensory reflex response, with profound effects on cardiorespiratory functions. However, the underlying mechanisms are not known. Here, we report that inherent variations in carotid body O2 sensing by carbon monoxide (CO)-sensitive hydrogen sulfide (H2S) signaling contribute to reflex variation in three genetically distinct rat strains. Compared with Sprague-Dawley (SD) rats, Brown-Norway (BN) rats exhibit impaired carotid body O2 sensing and develop pulmonary edema as a consequence of poor ventilatory adaptation to hypobaric hypoxia. Spontaneous Hypertensive (SH) rat carotid bodies display inherent hypersensitivity to hypoxia and develop hypertension. BN rat carotid bodies have naturally higher CO and lower H2S levels than SD rat, whereas SH carotid bodies have reduced CO and greater H2S generation. Higher CO levels in BN rats were associated with higher substrate affinity of the enzyme heme oxygenase 2, whereas SH rats present lower substrate affinity and, thus, reduced CO generation. Reducing CO levels in BN rat carotid bodies increased H2S generation, restoring O2 sensing and preventing hypoxia-induced pulmonary edema. Increasing CO levels in SH carotid bodies reduced H2S generation, preventing hypersensitivity to hypoxia and controlling hypertension in SH rats.

Role of oxidative stress-induced endothelin-converting enzyme activity in the alteration of carotid body function by chronic intermittent hypoxia.

Chronic intermittent hypoxia (CIH) leads to remodelling of the carotid body function, manifested by an augmented sensory response to hypoxia and induction of sensory long-term facilitation (LTF). It was proposed that endothelin-1 (ET-1) contributes to CIH-induced hypoxic hypersensitivity of the carotid body. The objectives of the present study were as follows: (i) to delineate the mechanisms by which CIH upregulates ET-1 expression in the carotid body; and (ii) to assess whether ET-1 also contributes to sensory LTF. Experiments were performed on adult, male rats exposed to alternating cycles of 5% O2 (15 s) and room air (5 min), nine episodes per hour and 8 h per day for 10 days. Chronic intermittent hypoxia increased ET-1 levels in glomus cells without significantly altering prepro-endothelin-1 mRNA levels. The activity of endothelin-converting enzyme increased with concomitant elevation of ET-1 levels in CIH-exposed carotid bodies, and MnTMPyP, a membrane-permeable antioxidant, prevented these effects. Hypoxia facilitated ET-1 release from CIH-treated carotid bodies, which is a prerequisite for activation of ET receptors; however, hypoxia had no effect on ET-1 release from control carotid bodies. In CIH-exposed carotid bodies, mRNAs encoding ETA receptor were upregulated, and an ETA receptor-specific antagonist abolished CIH-induced hypersensitivity of the hypoxic response, whereas it had no effect on the sensory LTF. These results suggest that ECE-dependent increased production of ET-1 coupled with hypoxia-evoked ET-1 release and the ensuing ETA receptor activation mediate the CIH-induced carotid body hypersensitivity to hypoxia, but the ETA signalling pathway is not associated with sensory LTF elicited by CIH.

Differential regulation of tyrosine hydroxylase by continuous and intermittent hypoxia.

Although continuous hypoxia (CH) and intermittent hypoxia (IH) cause reduction in oxygen availability, organisms adapt to the effects of chronic CH whereas IH adversely impacts autonomic functions. Catecholamines are expressed both in the central and peripheral nervous systems and they play important roles in the regulation of cardio-respiratory functions during hypoxia. Tyrosine hydroxylase (TH) is the rate-limiting enzyme for catecholamine synthesis. Several studies have examined the effects of hypoxia on catecholamines by focusing on the regulation of TH. In this article, we present a brief overview of the impact of chronic CH and IH on TH expression, activity and the associated cellular mechanism(s).

Endogenous H2S is required for hypoxic sensing by carotid body glomus cells.

H(2)S generated by the enzyme cystathionine-γ-lyase (CSE) has been implicated in O(2) sensing by the carotid body. The objectives of the present study were to determine whether glomus cells, the primary site of hypoxic sensing in the carotid body, generate H(2)S in an O(2)-sensitive manner and whether endogenous H(2)S is required for O(2) sensing by glomus cells. Experiments were performed on glomus cells harvested from anesthetized adult rats as well as age and sex-matched CSE(+/+) and CSE(-/-) mice. Physiological levels of hypoxia (Po(2) ∼30 mmHg) increased H(2)S levels in glomus cells, and dl-propargylglycine (PAG), a CSE inhibitor, prevented this response in a dose-dependent manner. Catecholamine (CA) secretion from glomus cells was monitored by carbon-fiber amperometry. Hypoxia increased CA secretion from rat and mouse glomus cells, and this response was markedly attenuated by PAG and in cells from CSE(-/-) mice. CA secretion evoked by 40 mM KCl, however, was unaffected by PAG or CSE deletion. Exogenous application of a H(2)S donor (50 µM NaHS) increased cytosolic Ca(2+) concentration ([Ca(2+)](i)) in glomus cells, with a time course and magnitude that are similar to that produced by hypoxia. [Ca(2+)](i) responses to NaHS and hypoxia were markedly attenuated in the presence of Ca(2+)-free medium or cadmium chloride, a pan voltage-gated Ca(2+) channel blocker, or nifedipine, an L-type Ca(2+) channel inhibitor, suggesting that both hypoxia and H(2)S share common Ca(2+)-activating mechanisms. These results demonstrate that H(2)S generated by CSE is a physiologic mediator of the glomus cell's response to hypoxia.

NADPH oxidase-derived H(2)O(2) contributes to angiotensin II-induced aldosterone synthesis in human and rat adrenal cortical cells.

BACKGROUND: The Renin-Angiotensin-Aldosterone-System plays a pivotal role in hypertension. Angiotensin II (Ang II) is a major regulator of aldosterone synthesis and secretion, and it is known to facilitate reactive oxygen species (ROS) generation in many cell types. AIMS: Here, we assessed the role of ROS signaling in Ang II-induced aldosterone synthesis by focusing on the regulation of aldosterone synthase (CYP11B2), a cytochrome P450 oxidase that catalyzes the final step in aldosterone biosynthetic pathway. RESULTS: Ang II increased CYP11B2 activity, mRNA and protein with a concomitant elevation of 6-Carboxy- 2',7'-dichlorodihydrofluorescein diacetate fluorescence, malondialdehyde and protein carbonyl levels (indices of ROS), NADPH oxidase (Nox) activity, and H(2)O(2) levels in human and rat adrenal cortical cells. The expression of nuclear receptor related 1 protein, a transcription factor known to regulate CYP11B2 expression, was also augmented by Ang II. These Ang II-evoked effects were either abolished or attenuated by pretreatment of cells with either Ang II type I receptor (AT(1)R) antagonist, or antioxidants or Nox inhibitor or siRNA silencing of Nox1, 2 and 4, or inhibitors of phospholipase C and protein kinase C. Exogenous H(2)O(2) mimicked the facilitatory effects of Ang II on CYP11B2 activity, mRNA, and protein expression, and these changes were significantly reduced by PEG-catalase. INNOVATION: ROS, particularly H(2)O(2), is identified as a key regulator of aldosterone production. CONCLUSION: Our results suggest that Ang II facilitates CYP11B2 activity and the ensuing aldosterone production via activation of AT(1)R-Nox-H(2)O(2) signaling pathway.

Endothelin-1 mediates attenuated carotid baroreceptor activity by intermittent hypoxia.

The objectives of the present study were to examine the effects of intermittent hypoxia (IH) on arterial baroreflex function and assess the underlying mechanism(s). Experiments were performed on adult male rats treated with 14 days of IH (15 s of hypoxia, 5 min of normoxia; 8 h/day) or normoxia (control). Arterial blood pressures were elevated in IH-treated rats, and this effect was associated with attenuated heart rate and splanchnic sympathetic nerve responses to arterial baroreflex activation. In IH-treated rats, carotid baroreceptor responses to elevated sinus pressures were attenuated. Endothelin-1 (ET-1) levels were elevated in the carotid sinus region of IH-treated rats, and this effect was associated with increased endothelin converting enzyme (ECE) activity, which generates biologically active ET-1. ET(A) receptor antagonist prevented the effects of IH on carotid baroreceptor activity. In IH-treated rats, reactive oxygen species (ROS) levels were elevated in the carotid sinus region, and antioxidant treatment prevented the effects of IH on ET-1 levels, ECE activity, carotid baroreceptor activity, and baroreflex function. These results demonstrate that 1) IH attenuates arterial baroreflex function, which is in part due to reduced carotid baroreceptor responses to elevated carotid sinus pressure, and 2) IH-induced carotid baroreceptor dysfunction involves reactive oxygen species-dependent upregulation of ET-1 signaling in the carotid sinus region.

Angiotensin II evokes sensory long-term facilitation of the carotid body via NADPH oxidase.

We previously reported that reactive oxygen species generated by NADPH oxidase 2 (Nox2) induces sensory plasticity of the carotid body, manifested as a progressive increase in baseline sensory activity or sensory long-term facilitation (sLTF). ANG II, a peptide generated within the carotid body, is a potent activator of Nox2. In the present study, we tested the hypothesis that ANG II evokes sLTF of the carotid body via Nox2 activation. Experiments were performed on carotid bodies ex vivo from adult rats and mice. Sensory activity was recorded from the carotid sinus nerve. Repetitive (5 times for 30 s each at 5-min intervals), but not continuous (for 150 s), application of 60 pM ANG II evoked robust sLTF of the carotid body. ACh, ATP, substance P, and KCl, when applied repetitively, stimulated the carotid body but did not evoke sLTF. Reactive oxygen species levels increased in response to repetitive applications of ANG II, and this effect was blocked by apocynin, an inhibitor of Nox2, as well as losartan, an angiotensin type 1 (AT(1)) receptor antagonist. Losartan, apocynin, and 4-(2-aminoethyl)benzenesulfonyl fluoride prevented ANG II-induced sLTF, which was absent in mice deficient in gp91(phox), the catalytic subunit of the Nox2 complex. These results demonstrate that repetitive application of ANG II induces sLTF of the carotid body via activation of Nox2 by AT(1) receptors.

Enhanced neuropeptide Y synthesis during intermittent hypoxia in the rat adrenal medulla: role of reactive oxygen species-dependent alterations in precursor peptide processing.

Intermittent hypoxia (IH) associated with recurrent apneas often leads to cardiovascular abnormalities. Previously, we showed that IH treatment elevates blood pressure and increases plasma catecholamines (CAs) in rats via reactive oxygen species (ROS)-dependent enhanced synthesis and secretion from the adrenal medulla (AM). Neuropeptide Y (NPY), a sympathetic neurotransmitter that colocalizes with CA in the AM, has been implicated in blood pressure regulation during persistent stress. Here, we investigated whether IH facilitates NPY synthesis in the rat AM and assessed the role of ROS signaling. IH increased NPY-like immunoreactivity in many dopamine-ß-hydroxylase-expressing chromaffin cells with a parallel increase in preproNPY mRNA and protein. IH increased the activities of proNPY-processing enzymes, which were due, in part, to elevated protein expression and increased proteolytic processing. IH increased ROS generation, and antioxidants reversed IH-induced increases in ROS, preproNPY, and its processing to bioactive NPY in the AM. IH treatment increased blood pressure and antioxidants and inhibition of NPY amidation prevented this response. These findings suggest that IH-induced elevation in NPY expression in the rat AM is mediated by ROS-dependent augmentation of preproNPY mRNA expression and proNPY-processing enzyme activities and contributes to IH-induced elevation of blood pressure.

Post-translational modification of glutamic acid decarboxylase 67 by intermittent hypoxia: evidence for the involvement of dopamine D1 receptor signaling.

Intermittent hypoxia (IH) associated with sleep apnea leads to cardio-respiratory morbidities. Previous studies have shown that IH alters the synthesis of neurotransmitters including catecholamines and neuropeptides in brainstem regions associated with regulation of cardio-respiratory functions. GABA, a major inhibitory neurotransmitter in the CNS, has been implicated in cardio-respiratory control. GABA synthesis is primarily catalyzed by glutamic acid decarboxylase (GAD). In this study, we tested the hypothesis that IH like its effect on other transmitters also alters GABA synthesis. The impact of IH on GABA synthesis was investigated in pheochromocytoma 12 cells, a neuronal cell line which is known to express active form of GAD67 in the cytosolic fraction and also assessed the underlying mechanisms contributing to IH-evoked response. Exposure of cell cultures to IH decreased GAD67 activity and GABA level. IH-evoked decrease in GAD67 activity was caused by increased cAMP - protein kinase A (PKA) - dependent phosphorylation of GAD67, but not as a result of changes in either GAD67 mRNA or protein expression. PKA inhibitor restored GAD67 activity and GABA levels in IH treated cells. Pheochromocytoma 12 cells express dopamine 1 receptor (D1R), a G-protein coupled receptor whose activation increased adenylyl cyclase activity. Treatment with either D1R antagonist or adenylyl cyclase inhibitor reversed IH-evoked GAD67 inhibition. Silencing D1R expression with siRNA reversed cAMP elevation and GAD67 inhibition by IH. These results provide evidence for the role of D1R-cAMP-PKA signaling in IH-mediated inhibition of GAD67 via protein phosphorylation resulting in down-regulation of GABA synthesis.

H2S mediates O2 sensing in the carotid body.

Gaseousmessengers, nitric oxide and carbon monoxide, have been implicated in O2 sensing by the carotid body, a sensory organ that monitors arterial blood O2 levels and stimulates breathing in response to hypoxia. We now show that hydrogen sulfide (H2S) is a physiologic gasotransmitter of the carotid body, enhancing its sensory response to hypoxia. Glomus cells, the site of O2 sensing in the carotid body, express cystathionine gamma-lyase (CSE), an H2S-generating enzyme, with hypoxia increasing H2S generation in a stimulus-dependent manner. Mice with genetic deletion of CSE display severely impaired carotid body response and ventilatory stimulation to hypoxia, as well as a loss of hypoxia-evoked H2S generation. Pharmacologic inhibition of CSE elicits a similar phenotype in mice and rats. Hypoxia-evoked H2S generation in the carotid body seems to require interaction of CSE with hemeoxygenase-2, which generates carbon monoxide. CSE is also expressed in neonatal adrenal medullary chromaffin cells of rats and mice whose hypoxia-evoked catecholamine secretion is greatly attenuated by CSE inhibitors and in CSE knockout mice.

Pattern-specific sustained activation of tyrosine hydroxylase by intermittent hypoxia: role of reactive oxygen species-dependent downregulation of protein phosphatase 2A and upregulation of protein kinases.

We investigated the role of protein phosphatases (PP) and protein kinases in tyrosine hydroxylase (TH) activation by two patterns of intermittent hypoxia (IH) in rat brainstem. Rats exposed to either IH(15s) (15 s, 5% O(2); 5 min, 21%O(2)) or IH(90s) (90 s each of 10% O(2) & 21%O(2)) for 10 days were used. IH(15s) but not IH(90s) caused a robust increase in TH activity, dopamine (DA) level, and TH phosphorylation at Ser-31 and Ser-40 in the medulla but not in the pons. Likewise, IH(15s) but not IH(90s) decreased activity and expression of protein phosphatase 2A (PP2A) and increased activity of multiple protein kinases. In vitro dephosphorylation with PP2A nearly abolished IH(15s)-induced increase in TH activity. IH(15s) increased generation of reactive oxygen species (ROS) in brainstem medullary regions which was nearly threefold higher than that evoked by IH(90s). Antioxidants prevented IH(15s)-induced downregulation of PP2A and increases in multiple protein kinase activity with subsequent reversal of serine phosphorylation of TH, TH activity, and DA to control levels. These findings demonstrate that IH in a pattern-specific manner activates TH involving ROS-mediated sustained increase in TH phosphorylation via downregulation of PP2A and upregulation of protein kinases.

Intermittent hypoxia activates peptidylglycine alpha-amidating monooxygenase in rat brain stem via reactive oxygen species-mediated proteolytic processing.

Intermittent hypoxia (IH) associated with sleep apneas leads to cardiorespiratory abnormalities that may involve altered neuropeptide signaling. The effects of IH on neuropeptide synthesis have not been investigated. Peptidylglycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3) catalyzes the alpha-amidation of neuropeptides, which confers biological activity to a large number of neuropeptides. PAM consists of O(2)-sensitive peptidylglycine alpha-hydroxylating monooxygenase (PHM) and peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL) activities. Here, we examined whether IH alters neuropeptide synthesis by affecting PAM activity and, if so, by what mechanisms. Experiments were performed on the brain stem of adult male rats exposed to IH (5% O(2) for 15 s followed by 21% O(2) for 5 min; 8 h/day for up to 10 days) or continuous hypoxia (0.4 atm for 10 days). Analysis of brain stem extracts showed that IH, but not continuous hypoxia, increased PHM, but not PAL, activity of PAM and that the increase of PHM activity was associated with a concomitant elevation in the levels of alpha-amidated forms of substance P and neuropeptide Y. IH increased the relative abundance of 42- and 35-kDa forms of PHM ( approximately 1.6- and 2.7-fold, respectively), suggesting enhanced proteolytic processing of PHM, which appears to be mediated by an IH-induced increase of endoprotease activity. Kinetic analysis showed that IH increases V(max) but has no effect on K(m). IH increased generation of reactive oxygen species in the brain stem, and systemic administration of antioxidant prevented IH-evoked increases of PHM activity, proteolytic processing of PHM, endoprotease activity, and elevations in substance P and neuropeptide Y amide levels. Taken together, these results demonstrate that IH activates PHM in rat brain stem via reactive oxygen species-dependent posttranslational proteolytic processing and further suggest that PAM activation may contribute to IH-mediated peptidergic neurotransmission in rat brain stem.

IFN-beta-mediated up-regulation of CD1d in bacteria-infected APCs.

The expression of CD1d molecules is essential for the selection and activation of a unique subset of T cells, invariant NKT cells, which express limited TCR diversity and have been demonstrated to function in both regulatory and antimicrobial immune responses. Although it has been reported that the levels of CD1d expression can be modulated during infection, the mechanisms that mediate this effect are poorly defined. In this study, we show that infection of dendritic cells and macrophages both in vitro and in vivo with the intracellular pathogen Listeria monocytogenes leads to up-regulation of CD1d. IFN-beta is required to mediate this up-regulation in L. monocytogenes infection, as well as being sufficient to up-regulate CD1d expression in vitro. Unlike MHC class I molecules, the increased surface expression of CD1d by IFN-beta is not regulated at the transcriptional level. Confocal microscopy and metabolic labeling experiments show that the total pool of CD1d protein is increased in IFN-beta-treated cells and that increased surface expression of CD1d is not due to the redistribution of the intracellular pool of CD1d. IFN-beta treatment increases the de novo synthesis of CD1d. This change in surface CD1d expression was functionally relevant, as IFN-beta-treated dendritic cells are more efficient in stimulating invariant NKT cells than untreated controls. Taken together, these data support a role for early IFN-beta-mediated up-regulation of CD1d in NKT cell activation during infection.

HLA-B44 polymorphisms at position 116 of the heavy chain influence TAP complex binding via an effect on peptide occupancy.

A single residue polymorphism distinguishes HLA-B*4402(D116) from HLA-B*4405(Y116), which was suggested to allow HLA-B*4405 to acquire peptides without binding to tapasin-TAP complexes. We show that HLA-B*4405 is not inherently unable to associate with tapasin-TAP complexes. Under conditions of peptide deficiency, both allotypes bound efficiently to TAP and tapasin, and furthermore, random nonamer peptides conferred higher thermostability to HLA-B*4405 than to HLA-B*4402. Correspondingly, under conditions of peptide sufficiency, more rapid peptide-loading, dissociation from TAP complexes, and endoplasmic reticulum exit were observed for HLA-B*4405, whereas HLA-B*4402 showed greater endoplasmic reticulum retention and enhanced tapasin-TAP binding. Together, these studies suggest that position 116 HLA polymorphisms influence peptide occupancy, which in turn determines binding to tapasin and TAP. Relative to HLA-B*4405, inefficient peptide loading of HLA-B*4402 is likely to underlie its stronger tapasin dependence for cell surface expression and thermostability, and its enhanced susceptibility to pathogen interference strategies.