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. 2010 Aug 15;49(4):548-58.
doi: 10.1016/j.freeradbiomed.2010.04.039. Epub 2010 May 20.

Pluronic-modified superoxide dismutase 1 attenuates angiotensin II-induced increase in intracellular superoxide in neurons

Xiang Yi  1 , Matthew C Zimmerman, Ruifang Yang, Jing Tong, Serguei Vinogradov, Alexander V Kabanov
Affiliations

Pluronic-modified superoxide dismutase 1 attenuates angiotensin II-induced increase in intracellular superoxide in neurons

Xiang Yi et al. Free Radic Biol Med. .

Abstract

Overexpressing superoxide dismutase 1 (SOD1; also called Cu/ZnSOD), an intracellular superoxide (O(2)(*-))-scavenging enzyme, in central neurons inhibits angiotensin II (AngII) intraneuronal signaling and normalizes cardiovascular dysfunction in diseases associated with enhanced AngII signaling in the brain, including hypertension and heart failure. However, the blood-brain barrier and neuronal cell membranes impose a tremendous impediment for the delivery of SOD1 to central neurons, which hinders the potential therapeutic impact of SOD1 treatment on these diseases. To address this, we developed conjugates of SOD1 with poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) block copolymer (Pluronic) (SOD1-P85 and SOD1-L81), which retained significant SOD1 enzymatic activity. The modified SOD1 effectively scavenged xanthine oxidase/hypoxanthine-derived O(2)(*-), as determined by HPLC and the measurement of 2-hydroxyethidium. Using catecholaminergic neurons, we observed an increase in neuronal uptake of SOD1-Pluronic after 1, 6, or 24h, compared to neurons treated with pure SOD1 or PEG-SOD1. Importantly, without inducing neuronal toxicity, SOD1-Pluronic conjugates significantly inhibited AngII-induced increases in intraneuronal O(2)(*-) levels. These data indicate that SOD1-Pluronic conjugates penetrate neuronal cell membranes, which results in elevated intracellular levels of functional SOD1. Pluronic conjugation may be a new delivery system for SOD1 into central neurons and therapeutically beneficial for AngII-related cardiovascular diseases.

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Figures

Fig. 1
Fig. 1. Representative SDS-PAGE gel electrophoresis of SOD1 conjugates
All conjugates were prepared in 20% aqueous EtOH supplemented by borate buffer (0.1M, pH8.0). The reaction molar ratio of Pluronic to SOD1 in SOD1-P85 (DSS), SOD1-L81 (DSS), SOD1-P85 (EDC) and SOD1-P85 (DSP) were 10, 5, 18 and 60 respectively. The mixture (i.e. no covalent modification) of SOD1 and P85 (4:1 by weight) or SOD1 and L81 (16:3 by weight) is prepared based on the weight percentage of SOD1 attached by one Pluronic chain.
Fig. 2
Fig. 2. Representative mass spectra of SOD1-Pluronic conjugates obtained by MALDI-TOF
The conjugates were obtained using the reaction conditions described in Fig. 1. (m)SOD1 and (d)SOD1 represent monomer (16 kDa) and dimer (32 kDa) respectively. The average molecular weight at the centroid of the peak was labeled for (m)SOD1 (16 kDa) and its conjugate form: (m)SOD1-P85(1:1), 21 kDa; (m)SOD1-P85(1:2), 26 kDa; (m)SOD1-L81(1:1), 19 kDa; (m)SOD1-L81(1:2), 22 kDa.
Fig. 3
Fig. 3. SOD1-Pluronic conjugates retain SOD1 activity
(A) Representative in-gel SOD activity assay. The conjugates and mixtures of SOD1 and Pluronic were prepared as described in Fig. 1. SOD activity is indicated by the appearance of achromatic bands. (B) HPLC profile of authentic DHE, pure 2-hydroxyethidum (2-OH-E+) and DHE incubated with xanthine oxidase (XO) and hypoxanthine (HX) in the presence of PEG-SOD1, purified SOD1-Pluronic conjugates, or Pluronic alone. The amount of Pluronic used was calculated based on the weight percentage of Pluronic in the SOD1-Pluronic conjugates.
Fig. 4
Fig. 4. Cellular uptake of SOD1-Pluronic conjugates in CATH.a neurons
(A) Cellular internalization of SOD1 and SOD1-P85 labeled by Alex Fluor 680 within CATH.a neuronal cells at various time intervals. (B) Representative SOD1 activity assay showing SOD1 activity in CATH.a neurons exposed to SOD1-P85, SOD1-L81, or native SOD1 at 37°C for 1, 6, and 24 hrs.
Fig. 5
Fig. 5
Viability of CATH.a neurons treated with SOD1-P85 or SOD1-L81 conjugates. CATH.a neurons were exposed to SOD1-P85, SOD1-L81, or native SOD1 (80 μg/mL) at 37°C for 1, 3, 6, 18 and 24 hrs. Data were obtained in triplicate and present as means ± SEM. There is no statistical significance between groups.
Fig. 6
Fig. 6. SOD1-P85 and SOD1-L81 conjugates Inhibit Ang-II induced O2•− level in CATH.a neurons
(A) Representative confocal images of DHE-loaded CATH.a neurons, showing the effects of AngII (100 nM) treatment after 10 min in cells pre-exposed to SOD1-P85, SOD1-L81, SOD1 alone or media for 24 hrs. (B) Summary data of the fluorescence in DHE-loaded CATH.a neurons recorded for 20 min after AngII treatment. Images were analyzed for fluorescence intensity per cell in equal numbers of cells (10 cells from each field, 5 fields total). Data are means ± SEM and expressed relative to the cell fluorescence before AngII treatment. * P<0.01 vs control cells; # P<0.01 vs cells treated with AngII alone at 10, 12, 14, 16 and 18 min; NS not significant between SOD1 treated cells and cells treated with AngII alone. (C) Representative HPLC profile of the cell extract from CATH.a neurons pre-treated with SOD1 samples for 24 hrs followed by AngII stimulation (100 nM) for 1 hrs and DHE (25 μM) incubation for 20 min. (D) Quantification of actual concentrations of 2-OH-E+ generated under the conditions described in (C) using 2-OH-E+ as standard. Data are present as the amount of 2-OH-E+ normalized by cell protein content obtained by MicroBCA assay (n=3). Data are mean ± SEM. * P < 0.05 vs. Control. # P < 0.05 vs. AngII.
Sch. 1
Sch. 1
Conjugation of SOD1 with mono-amine P85 through biodegradable linker, DSP and non-degradable linkers, DSS and EDC.
See this image and copyright information in PMC

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