Role Of Extracellular Superoxide Dismutase

Published Date: 02 Nov 2017

Obstructive sleep apnoea (OSA) syndrome, is characterised by chronic intermittent hypoxia (CIH), 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, inflammation and sympathetic activation 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)[14]. For example, CBs from Epas1+/− mice, which lack HIF-2α partially, 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[15].

Superoxide dismutases(SODs) are antioxidative enzymes that catalyze the degradation of ROS. Three SOD isoforms are expressed in mammalian cells: copper/zinc SOD(CuZn-SOD, SOD1), which is located in the cytoplasm[16]; manganese SOD(Mn-SOD, SOD2), which is localized in the mitochondrial matrix[17]; and extracellular SOD (EC-SOD, SOD3)[18], which 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 respiratory and cardiovascular homeostasis[15,19,20]. In addition, reported increased levels of EC-SOD 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 secretion of an excitatory transmitter. The inhibition of K+ channels in glomus cells is a critical initial step in hypoxia chemosensation, which leads to a cascade event including facilitation of membrane depolarization, voltage-dependent Ca2+ entry through L-type Ca2+ channels, and neurosecretion, as in previous study[21]. It has been shown Ad CuZnSOD enhanced IK of glomus cell in CHF rabbits[22]. 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 pro-inflammatory cytokines, such as interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α, and cytokine receptors (IL-1r1, gp130 and TNFr1)[23]. In animal models with inflammatory disease, EC-SOD exhibited anti-inflammatory effects through down-regulation of the expression of pro-inflammatory mediators and up-regulation of the expression of anti-inflammatory cytokines[24]. EC-SOD is an important mediator of reduced CD68+ macrophage migration into the inflammatory area, which due to decreased cytokine and adhesion molecule expression[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[25]. Therefore, I hypothesize that EC-SOD overexpression might affect the expression of inflammatory mediates and macrophage migration through activation of NF-κB and HIF-1α.

Methodology:

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 either 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[26].

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 extracellular SOD. The adenoviral vectors are produced by the University of Iowa Gene Transfer Vector Core[27].

3. Gene Transfer to the CBs

The left and right sinus region is temporarily vascularly isolated (including the common carotid artery, internal carotid artery, and external carotid artery), and the tip of a PE-10 catheter is positioned at the level of the CB via the external maxillary artery. After these arteries are occluded with snares, 200μl of Adβ-gal (as control group) or AdEC-SOD[1×108 plaque-forming units(pfu) per mL, dissolved in 0.9% sodium chloride] is slowly injected into the CB via the catheter[22].

4. EC-SOD Activity Measurements

EC-SOD is separated from intracellular SODs (Cu,Zn SOD and MnSOD) using a concavalin A sepharose column. EC-SOD activity levels are measured using a commercially available SOD assay kit[28].

5. Immunohistochemistry assessments for EC-SOD

Unfixed frozen CBs are cut on a cryostat at 220°C. Sections are incubated with primary antibody(goat anti-human EC-SOD, 1:500) overnight. After they are washed with PBS, sections are incubated for 30 minutes with the secondary antibody, rinsed again with PBS, and developed with Vectastain ABC Kit alkaline phosphatase (Vector Laboratories Inc)[27].

Western blot analysis for EC-SOD in CBs, HIF-1α, HIF-2α and NF-κB in glomus cells

Proteins are extracted from tissues or cells, separated by electrophoresis, and transferred onto polyvinylidene difluoride 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[25].

7. Electrophysiological recording of carotid sinus nerve(CSN) activity

The carotid bifurcations are excised from rabbits and placed in a lucite chamber containing 100% O2-equilibrated modified Tyrode solution at 0–4°C. Each CB along with its attached nerve is carefully dissected from the artery and cleaned of surrounding connective tissue. Preparations are then placed in a conventional flow chamber where the CB is continuously superfused (up to 4 h) with modified Tyrode solution maintained at 37°C and equilibrated with a selected gas mixture. The CSN is drawn up into the tip (~100-μm inner diameter) of a glass suction electrode for monopolar recording in 3% sucrose) of chemoreceptor activity. Basal neural activity is established in superfusates maintained at PO2=450 Torr. The PO2 is lowered to 120 Torr in superfusates equilibrated with air to provide a moderately hypoxic stimulus. Neural activity is led to an alternating current (AC)-coupled preamplifier, filtered, and transferred to a window discriminator and a frequency-to-voltage converter. Signals are processed by an analog-to-digital converter for display of frequency histograms on a PC monitor[29].

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 CB.

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 two-step enzymatic digestion protocol and cultured at 37°C in a humidified atmosphere of 95% air-5% CO2, and studied within 24 h of dissociation. Patch pipettes had resistances of 4-6MV when filled with intracellular solution. Currents are measured in the whole-cell configuration of the patch-clamp technique using a Warner PC-501A patch-clamp amplifier (Warner Instrument Corp., Hamden, CT, USA) and pClamp 8.1 program (Axon Instruments, CA, USA)[21,22].

11. Measurement of intracellular calcium

To estimate the intracellular Ca2+ concentration ([Ca2+]i), cultured CB glomus cells are loaded with fura-2 AM (2μm) in standard external solution described above for 10 min at 37°C followed by several rinses with the external solution for 15 min. [Ca2+]i is measured using dual-wavelength ratio fluorometry. Relative changes in [Ca2+]i are calculated from the ratio F340 ⁄ F380[21].

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 followed by SNK post hoc tests when necessary. 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.