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Obstructive sleep apnoea syndrome, is characterised by chronic intermittent hypoxia, which is considered the primary risk factor for developing cardiovascular diseases[1-3]. Reported responses to CIH include increased blood pressure, augmented sympathetic nerve activity, elevated circulating catecholamines, enhanced long-term facilitation(LTF) of the respiratory motor activity and augmented ventilatory response to hypoxia[4-9]. It has been proposed that oxidative stress, sympathetic activation and inflammation are involved in the CIH-induced carotid body(CB) chemosensory potentiation[10-12].
Recent studies suggest that oxidative stress plays a significant role in respiratory changes caused by CIH[9,13], which may arise because of increased generation of ROS by pro-oxidants, such as Nox2, or because of decreased activity of antioxidant enzymes, such as superoxide dismutase(SOD). For example, CBs from Epas1+/− mice, which have a partial deficiency of HIF-2α, show augmented responses to hypoxia. HIF-2α is required for the production of anti-oxidant enzymes and the expression of SOD2 is significantly decreased in the CBs of Epas1+/− mice.
Superoxide dismutases(SODs) as antioxidative enzymes, are able to catalyze the degradation of ROS. There are three SOD isoforms expressed in mammalian cells, including copper/zinc SOD(CuZn-SOD, SOD1) located in the cytoplasm; manganese SOD(Mn-SOD, SOD2) localized in the mitochondrial matrix; and extracellular SOD (EC-SOD, SOD3) located in the extracellular matrix. It has been shown that SOD can attenuate tissue damage and inflammation, which is required for redox homeostasis in the CB, which in turn is required for the maintenance of cardiovascular and respiratory homeostasis[15,19,20]. In addition, reported increased EC-SOD levels are able to reduce oxidative tissue damage by decreasing superoxide and diminishing the direct and indirect reaction of ROS. Systemic administration of membrane permeable SOD mimetic prevented CIH-induced changes in the CB activity[19,20]. Thus, I investigate the expression of EC-SOD in CIH-induced CB as well as the effect of adenoviral EC-SOD (Ad EC-SOD) gene transfer to the CB on the enhanced CB chemoreceptor activity in CIH rabbits.
CIH-induced CB changes is due to inhibition of IK in CB glomus cells and release of excitatory transmitters. The inhibition of K+ channels in glomus cells serve as a critical step in hypoxia chemosensation, which brings about a cascade event including facilitation of membrane depolarization, voltage-dependent Ca2+ access via L-type Ca2+ channels, and neurosecretion, as in previous study. It has been shown Ad CuZnSOD enhanced IK of glomus cells in CHF rabbits. Thus, I hypothesize that restoring EC-SOD expression by Ad EC-SOD gene transfer to the CB is able to attenuate IK of glomus cells and decrease [Ca2+]i of CB glomus cells.
CIH induces macrophages infiltration and a local inflammation of the CB with functionally up-regulated various pro-inflammatory cytokines, such as interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α, and related cytokine receptors (IL-1r1, gp130 and TNFr1). In animal models with inflammatory disease, EC-SOD exhibited anti-inflammatory effects through down-regulating the expression of pro-inflammatory mediators and up-regulating the expression of anti-inflammatory mediators. EC-SOD can serve as an important mediator to reduce CD68+ macrophage migration into the inflammatory area, which due to decreased the expression of cytokines and adhesion molecule[24,25]. It has been shown EC-SOD has anti-inflammatory effects through regulation of HIF-1α, PKC, and NF-κB pathways after exposure to hypoxia. Therefore, I hypothesize that EC-SOD overexpression might affect the expression of inflammatory mediates and macrophage infiltration through activation of NF-κB and HIF-1α.
1. Experimental Animals and CIH
All experiments are carried out on 120 male New Zealand White rabbits weighing 2.5-3.5 kg. Rabbits are randomly divided into four group: (1)normoxic (Nx) groups; (2)CIH group; (3) CIH+Adβ-gal; (4)CIH+AdEC-SOD. The rabbits cages are kept in acrylic chambers under normobaric conditions and the rabbits had free access to water and chow. The Nx controls are kept in room air with maintenance matching other groups. For the CIH group, rabbits are exposed to hypoxic cycles of 5% inspired O2 for 20 s, followed by room air for 280 s, applied 12 times per hour, 8 h•day-1 for 21 days.
2. Recombinant Adenoviral Vectors
Recombinant adenoviruses are used for gene transfer: (1) AdCMVβ-gal carried the reporter gene of β-galactosidase and is used as a control virus; (2) AdCMVEC-SOD carried cDNA for human EC-SOD. The adenoviral vectors are obtained from the University of Iowa Gene Transfer Vector Core.
3. Gene Transfer to the CBs
Either sides of sinus region is temporarily vascularly isolated (including the common, internal and external carotid artery), and the tip of a PE-10 catheter is located at the level of the CB through the external maxillary artery. After the occlusion of these arteries by snares, Adβ-gal (as control group) or AdEC-SOD is slowly injected into the CBs via the catheter.
4. EC-SOD Activity Measurements
EC-SOD is separated from intracellular SODs (including Cu,Zn SOD and MnSOD) through a concavalin A sepharose column. The levels of EC-SOD activity are measured by an available SOD assay kit.
5. Immunohistochemistry assessments for EC-SOD
Unfixed frozen CBs are prepared for immunohistochemistry analysis. Sections are incubated with primary antibody overnight. After washing with PBS, sections are incubated for 30 minutes with the secondary antibody, washed again with PBS, and developed with Vectastain ABC Kit alkaline phosphatase.
Western blot analysis for EC-SOD in rabbit CBs, HIF-1α, HIF-2α and NF-κB in glomus cells
Proteins are extracted from tissues or cells, separated by electrophoresis, and then transferred onto polyvinylidene difluoride(PVDF) membranes. The specific primary antibodies include phospho-NF-κB p65, NF-κB p65, anti-HIF-1α and HIF-2α antibodies, anti-EC-SOD antibody and GAPDH.
7. Electrophysiological recording of carotid sinus nerve(CSN) activity
The carotid bifurcations are removed from rabbits and placed in a lucite chamber with 100% O2-equilibrated modified Tyrode's solution at 0-4°C. Each CB together with its attached nerve is dissected from the artery and cleaned of surrounding tissue. Then, the preparations placed in a flow chamber, is continuously superfused with modified Tyrode's solution at 37°C and equilibrated with a selected gas mixture. The CSN is drawn up into the tip of a glass suction electrode for electrophysiological recording of chemoreceptor activity. Basal neural activity is established in superfusates maintained at PO2=450 Torr. The PO2 is lowered in superfusates equilibrated with air for providing a modestly hypoxic stimulus. Signals are processed by an analog-to-digital converter for display of frequency histograms on a PC monitor.
Reverse transcription polymerase chain reaction(RT-PCR) detection of pro-inflammatory cytokine(IL-1β, IL-6 and TNF-α) and chemokines (MCP-1, CCR2, MIP-1α, MIP-1β and ICAM-1) in the CBs.
Immunofluorescence detection of CD68+ macrophage infiltration in CBs
Immunofluorescent double-labeling technique is used to determine the immunohistochemical localization of macrophage infiltration.
10. Recording of outward K+ currents (IK) in the CB glomus cells
CB glomus cells are isolated by a enzymatic digestion protocol and cultured at 37°C in a humidified condition with 95% air and 5% CO2, and studied within 24 h of dissociation. Patch pipettes had resistances of 4-6MV when containging intracellular solution. Currents are measured in the whole-cell configuration of the patch-clamp technique[21,22].
11. Measurement of intracellular calcium
To estimate the intracellular Ca2+ concentration ([Ca2+]i), cultured CB glomus cells are loaded in standard external solution for 10 min at 37°C followed by several washes with the external solution for 15 min. [Ca2+]i is detected by dual-wavelength ratio fluorometry.
12. Data analysis
Data are expressed as means ± SEM. Statistical significance is defined as p<0.05 (two-tailed), paired Student's t-tests or one-way ANOVA. Data are analyzed with SPSS10.0.
Outcomes and Value:
Immunohistochemistry assessments and Western blot analysis reveal the expression of EC-SOD is suppressed in the CBs from CIH rabbits. CIH induces an increased hypoxia-evoked nerve discharge, which indicates enhanced CB chemoreceptor activity during exposure to CIH. AdEC-SOD localized to the CB enhanced the expression of EC-SOD in the CB and reversed the enhanced CB chemoreflex activity in CIH.
Ad EC-SOD gene transfer to the CB attenuates IK of glomus cells and decreases [Ca2+]i of CB glomus cells. The effect of AdEC-SOD in modulating CSN chemoreflex activity induced by CIH through augmentation of IK in CB glomus cells and secretion of an excitatory transmitter.
RT-PCR and Western blot analysis indicate that CIH up-regulated the expression of various pro-inflammatory mediators (IL-1β, IL-6, TNF-α, and iNOS), chemokines (MCP-1, MIP-1α, MIP-1β and ICAM-1), activation of HIF-α and NF-κB subunits (p50 and p65), whereas EC-SOD might inhibit their expression. EC-SOD might inhibit CIH-induced expression of inflammatory mediators in glomus cells and rabbit CBs via down-regulation of HIF-1α and NF-κB pathways.
Immunofluorescence assessments indicate that increased numbers of invasive CD68+ macrophages are present in the CBs following CIH, whereas the increased macrophages infiltration in the CB might be inhibited by AdEC-SOD due to down-regulation of MCP-1 and ICAM. Reduced expression of adhesion molecules and inflammatory mediators increased by macrophages in CIH-induced CB highlights the anti-inflammatory and anti-migratory role of EC-SOD.
The study reveals the role of EC-SOD on the rabbit CB exposed to CIH. These results might also provide a foundation for future investigations to test new therapeutic interventions with novel antioxidant and anti-inflammatory strategies that target extracellular O2 and local inflammation in the CB. The relative contribution of the other isoforms of SOD to the oxidant and inflammatory effects in the CB is not known and deserves further study.