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Cellular prion protein (PrPC) and superoxide dismutase (SOD) in vascular cells under oxidative stress
Experimental and Toxicologic Pathology 63 (2011) 229–236
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Cellular prion protein (PrPC) and superoxide dismutase (SOD) in vascular cells under oxidative stress He´len Zocche Soprana a, Liliete Canes Souza a,n, Victor Debbas b,1, Francisco Rafael Martins Laurindo b,I a b
Department of Clinical Analysis, Center of Health Sciences, Federal University of Santa Catarina, Brazil Vascular Biology Laboratory, Heart Institute, University of Sao Paulo School of Medicine, Brazil
a r t i c l e in fo
abstract
Article history: Received 1 May 2009 Accepted 14 December 2009
The PrPC is expressed in several cell types but its physiological function is unknown. Some studies associate the PrPC with copper metabolism and the antioxidant activity of SOD. Our hypothesis was that changes in PrPC expression lead to abnormal copper regulation and induce SOD downregulation in the vascular wall. Objectives: to study whether the PrPC expression undergoes induction by agents that trigger endoplasmic reticulum stress (ERS) and, in this context, to evaluate the SOD activity. Methods: To trigger ERS, in vitro, rabbit aortic smooth muscle cells were challenged for 4, 8 and 18 hours, with angiotensin-II, tunicamycin and 7-ketocholesterol. For in vivo studies rabbit aortic arteries were subjected to injury by balloon catheter. Results: In vitro baseline SOD activity, determined through inhibition of cytochrome-c reduction, was 13.9 7 1.2 U/mg protein, angiotensin-II exposed for 8 hours produced an increase in SOD activity, and cellular copper concentration was about 9 times greater only under these conditions. Western blotting analysis for SOD isoenzymes showed an expression profile that was not correlated with the enzymatic activity. PrPC expression decreased after exposure to all agents after different incubation periods. RT-PCR assay showed increased mRNA expression for PrPC only in cells stimulated for 8 hours with the different stressors. The PrPC mRNA expression in rabbit aortic artery fragments, subjected to balloon catheter injury, showed a pronounced increase immediately after overdistension. The results obtained indicated a PrPC protection factor during the early part of the ERS exposure period, but did not demonstrate a SOD-like profile for the PrPC. & 2009 Elsevier GmbH. All rights reserved.
Keywords: Superoxide dismutase Cellular prion protein Rabbit aortic smooth muscle cells Copper concentration Endoplasmic reticulum stress
1. Introduction The cellular prion protein (PrPC) is a glycoprotein of 33–35 kDa (Prusiner, 1998; Glockshuber et al., 1998). The physiological function of PrPC is still unclear; however, it is involved in neurodegenerative diseases, characterized by the accumulation of an abnormally folded isoform of the PrPC, denoted PrPSC, which represents the major component of infectious prion diseases (Prusiner, 1998). The superoxide dismutases (SOD) are the first and most important line of antioxidant enzyme defense system against reactive oxygen species (ROS) and particularly superoxide anion radicals (Zelko et al., 2002). The SOD expression and activity have an effect on cellular vascular responses to acute and chronic oxidative stress (Packer, 2002).
n Corresponding author at: Campus Universita´rio Trindade, Departamento de Ana´lises Clı´nicas, 88040-970, Floriano´polis, SC, Brazil. Tel.: +55 48 3721 9712x221; fax: + 55 48 3721 9542. E-mail address: [email protected] (L. Canes Souza). 1 Avenida Eneas de Carvalho Aguiar, 44 - 91 andar Bloco II, 05403-000, Sa~ o Paulo, SP, Brazil. Tel.: + 55 11 3069 5185; fax: + 55 11 3069 5920.
0940-2993/$ - see front matter & 2009 Elsevier GmbH. All rights reserved. doi:10.1016/j.etp.2009.12.004
SOD isoforms are cytosolic or copper/zinc SOD (CuZnSOD or SOD-1), manganese SOD (MnSOD or SOD-2), and an extracellular form of CuZnSOD (EcSOD or SOD-3) (Marklund, 1982; Fukai et al., 2002). EcSOD expression in vascular cells and within the vessel wall can be altered in response to a variety of stimuli including angiotensin II (Zelko et al., 2002; Fukai et al., 2002). The ROS in small quantities are essential in many processes, including intracellular signaling, protection against microorganisms and cellular function. In contrast, the excessive production and/or inadequate removal of ROS has been implicated in oxidation of biological macromolecules, such as DNA, proteins, carbohydrates and lipids, and this condition has commonly been referred to as oxidative stress (Fukai et al., 2002). Evidence suggests that oxidative stress is involved in the pathogenesis of many cardiovascular diseases, including hypercholesterolemia, atherosclerosis, hypertension, diabetes and heart failure (Fukai et al., 2002; Cai and Harrison, 2000). ROS production is prominently immediately after injury, it has been reported in neointima and adventitia during the later stages of vessel repair (Souza et al., 2000; Azevedo et al., 2000; Rey and Pagano, 2002). Despite the potential for ROS to induce vascular injury, they have been ascribed an important signaling role in
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physiologic and pathologic situations (Irani, 2000). The importance of redox-dependent signaling in vascular cell growth, apoptosis and senescense is increasingly apparent (Abe and Berk, 1998; Suzuki et al., 1997; Sen and Packer, 1996; Kunsch and Medford, 1999). Leite et al. examined in detail the pathophysiology of vascular repair after balloon injury and reported that a decrease in SOD activity plays a role in constrictive vascular remodeling. Although there was depletion in both cytoplasmic and extracellular SOD after injury, the authors showed that the administration of EcSOD was sufficient to normalize SOD activity and improve vascular caliber (Leite et al., 2003). The normal prion protein binds copper and the resulting complex apparently acts in the protection against oxidative stress (Brown, 2001; Brown et al., 1997a, b). Thus, PrP prevents copper from interacting with cells deleteriously (Brown et al., 1998; Rachidi et al., 2003). The loss of PrP function may result in an increased sensitivity to oxidative stress and copper toxicity. Therefore, the PrP physiological function may be involved not only in central nervous system diseases but also in other human illnesses, such as heart reperfusion injury, hepatic failure and sepsis (Martins et al., 2002). It has been reported that the enzymatic activity and copper loading of Cu/Zn superoxide dismutase (SOD1) are 50% of the normal level in brain and cultured cerebellar neurons from PrPnull mice and that SOD1 activity and copper loading are elevated in mice that overexpress PrP (Brown et al., 1997a, b). Thus, some authors have investigated whether PrP functions as an endocytic receptor for cellular uptake of copper ions, or facilitates some other aspect of copper trafficking such as efflux from the cell or intracellular sequestration of the metal (Brown et al., 1997a, b; Waggoner et al., 2000). The mechanism of the PrPC antioxidant effect is not clear and may be dependent on an increase in the endogenous SOD activity and its role in Cu2 + metabolism (Brown and Besinger, 1998). Our hypothesis is that PrPC levels contribute to a sustained decrease in SOD activity in the vascular wall stimulating negative remodeling after injury. In preliminary immuno-histochemical studies, we verified a significant PrPC expression in rabbit intact iliac arteries, with a significant decrease in this expression immediately after balloon injury (data not published). Moreover, Leite et al. observed that CuZnSOD and EcSOD expression after balloon injury has a similar pattern compared to its protein mRNA expression in the brain of knockout mice for PrPC (Leite et al., 2003; Brown et al., 2002). The aim of the present study was to evaluate the expression of PrPC under assay conditions typical of those used for studies on vascular redox signaling. Isolated rabbit aortic smooth muscle cells (RASMCs) were challenged with agonists of endoplasmic reticulum stress pathways and rabbit iliac arteries were submitted to overdistension injury to correlate SOD activity and its isoenzyme expression to PrPC expression, using both Western blotting and RT-PCR techniques.
2. Materials and methods 2.1. Chemicals Angiotensin II, tunicamycin, xanthine and xanthine oxidase were purchased from Calbiochems (San Diego, CA, USA), 7ketocholesterol from Sigma-Aldrichs (St Louis, MO, USA), fetal calf serum (FCS) from Gibco-BRL Lifes (Grand Island, NY, USA) and lysis buffer plus protease inhibitors from Mercks (Darmstadt, Germany). The mouse anti-PrPC was kindly donated by Vilma Regina Martins Ph.D. (Ludwig Institute of Cancer Research, Sao Paulo, Brazil). Sheep anti-CuZnSOD antibody was purchased from
Oxis International Inc.s (Foster City, CA, USA); rabbit anti-MnSOD and anti-EcSOD from Stressgens (Victoria, Canada); primary monoclonal antibody anti-b-actin (mouse IgG) from SigmaAldrichs (St Louis, MO, USA); secondary antibody anti-IgG of mice with peroxidase conjugate from Amersham Pharmacia Biotechs (USA); antibody goat anti-rabbit IgG (MnSOD and EcSOD) from Stressgens (Victoria, Canada); secondary rabbit anti-sheep to CuZnSOD from Calbiochems (San Diego, CA, USA); and rabbit anti-mouse IgG to PrPC from Amersham Pharmacia Biotechs (USA), conjugate with peroxidase. The proteins were detected by a chemiluminescence method using an ECL reagent kit from Amersham Pharmacia Biotechs (USA). The reagents used for the RT-PCR reaction including Trizols were purchased from Invitrogens (Carlsbad, CA, USA). All other reagents were purchased from Sigma-Aldrichs (St Louis, MO, USA). All solutions were prepared with distilled water further purified in a Millipore Milli-Q system and treated with Chelex-100 before use. 2.2. Cell culture and homogenate Rabbit aortic smooth muscle cells (RASMCs) obtained from a previously established selection-immortalized line (Buonassisi and Venter, 1976) were cultured in Coon’s F12 medium supplemented with 10% FCS, 100 U/mL penicillin and 100 mg/mL streptomycin at 37 1C in a humidified atmosphere with 5% CO2 to 95% air. For the present study, cells between passages 2 and 7 were used. To induce oxidative stress, cells (approximately 90% confluence) were exposed to angiotensin II (Ang II – 100 nM), tunicamycin (Tn – 5 mg/mL) or 7-ketocholesterol (7KC – 5 mg/mL) for 4, 8 and 18 hours (n= 3 for each stimulus and time, incubated on different days). Viability of cells after stimuli exposure was determined by the trypan blue exclusion assay. Cell homogenates were prepared as described previously (Laurindo et al., 2002) and protein concentration was assessed using the Bradford method. 2.3. SOD activity assay Aliquots of the supernatant were assayed for total SOD activity as previously described (Azevedo et al., 2000). The inhibition rate of cytochrome-c reduction induced by xanthine oxidase-derived superoxide at pH 7.4 was monitored spectrophotometrically at 550 nm at 37 1C, and results were normalized for protein concentration (Bradford method). Statistical analysis was performed through the t-test, with a 0.05 significance level. 2.4. Cellular copper concentrations Levels of Cu in the homogenate samples were measured according to the method of Papageorgiou et al. (2002) with the use of an atomic absorption spectrophotometer (AAS model 2380, Perkin-Elmer, USA) with a graphite furnace heated graphite atomizer (HGA model 300) and analytical wavelengths of 324.8 nm. The measurements were repeated twice and two quality controls (homogenate and aqueous) were used: (1) cation-cal collaboration and (2) an individual solution of a fixed concentration using spectrosol BDH standards. Statistical analysis was performed using Student’s t-test. The mean values are expressed as micrograms per milliliter. 2.5. Western blot analysis Samples were subjected to SDS-PAGE in polyacrylamide gels according to the protein molecular weight (CuZnSOD: 17.5%, EcSOD and PrPC: 15%, MnSOD: 12%). After electrophoresis, proteins were electro-transferred to a nitrocellulose membrane
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(Amersham Biosciences, Piscataway, NJ). Equal loading of samples (30 mg for SOD isozymes and 80 mg for PrPC) and transfer efficiency were monitored with the use of 0.5% Ponceau S staining of the blot membrane. The blotted membrane was then blocked (5% nonfat dry milk, 10 mM Tris–HCl, pH 7.6, 150 mM NaCl, and 0.1% Tween 20) for 2 h at room temperature and incubated (same buffer plus 1% nonfat dry milk) with specific antibodies (1:200 for mouse anti-PrPC; 1:1000 for sheep anti-CuZnSOD; 1:800 for rabbit anti-MnSOD and rabbit anti-EcSOD; and 1:10,000 for mouse anti-b-actin) overnight at 4 1C. Binding of the primary antibody was detected with the use of peroxidase-conjugated secondary antibodies (goat anti-rabbit IgG diluted 1:2000 for MnSOD and EcSOD; rabbit anti-sheep IgG diluted 1:2000 for CuZnSOD; rabbit anti-mouse IgG diluted 1:600 for PrPC; mouse anti-IgG diluted 1:20,000 for b-actin) for 1:30 h at room temperature and developed by enhanced chemiluminescence (Amersham Pharmacia Biotech) detected by autoradiography. Quantification analysis of blots was performed with the use of Scion Image software (Scion based on NIH Image). 2.6. Reverse transcriptase-polymerase chain reaction (RT-PCR) Total RNA was extracted from RASM cells using Trizols according to the manufacturer’s instructions. RT-PCR was performed using the thermocycler (Eppendorfs Mastercycler Personal). The quality of the isolated RNA was checked in a spectrophotometer (Hitachi Spectrophotometers U-2001) by measuring the absorbance at 260 and 280 nm (OD260/OD280 ratio of 1.6–1.8, as described by Sambrook and Russel, 2001). The RNA integrity was evaluated by 0.8% agarose gel electrophoresis (Amrescos) followed by ethidium bromide-staining (1 mg/mL) for the visualization of the 18S and 28S bands in a transilluminator (Eagle Eye IIs, Alpha Innotech). Total RNA (2 mg) was reverse transcribed following the manufacturer’s instructions (Invitrogens). For cDNA amplification, a 1 mL aliquot of the reverse transcription reaction was amplified in a volume of 25 mL, composed of 2.5 mL of 10 PCR buffer, 0.5 mL of 10 mM dNTP, 0.75 mL of 50 mM MgCl2, and 1.0 mL of each oligonucleotide primer (10 mM): CAC CAA AGG GGA GAA CTT CA (sense) and ATC CCA CGA TCA GGA AGA TG (anti-sense). The PCR protocol was performed in a thermocycler (Eppendorfs Mastercycler Personal) with an initial cycle of 2 min at 94 1C, followed by 30 cycles of 1 min at 94 1C, 1 min at 55 1C and 1 min at 72 1C. PCR amplification products were visualized, after electrophoresis in a 2%
Fig. 1. SOD enzymatic activity (UI/mg Protein) in RASMCs homogenate (n= 3) stimulated for 4, 8 and 18 hours with 100 nM angiotensin II (Ang II), 5 mg/mL tunicamycin (Tn) or 5 mg/mL 7-ketocholesterol (7KC). nP o0.05 versus control (C).
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agarose gel, using ethidium bromide (1.0 mg/mL) staining. The obtained PCR amplification products gave DNA fragments of the expected size (185 bp). Primers specific to the GAPDH gene for rabbit (sense: TCA CCA TCT TCC AGG GA AGC, and anti-sense: CAC AAT GCC GAA GTG GTC GT) were used as the positive control. 2.7. Balloon injury of iliac artery Iliac artery overdistension injury was performed in pentobarbital-anesthetized normolipemic male New Zealand White rabbits, as described previously (Leite et al., 2003; Laurindo et al., 2002) using a coronary angioplasty-type balloon with diameter of 2.75 mm, inflated at 8.0 atm. After 7 and 14 days, the rabbits were euthanized with pentobarbital sodium; both the injured right and uninjured left iliac arteries were removed immediately. In addition to these two groups, there was a third group (‘‘zero time’’) in which the animal was killed immediately after angioplasty. The procedure was performed on two animals from each group with survival, at 7 and 14 days and ‘‘zero time’’. The two iliac arteries taken from a rabbit not submitted to any prior injury were used as negative controls. After surgical removal of the arteries, the fragments were quickly frozen in liquid nitrogen, macerated and later transferred to an RNAse-free tube. RNA extraction and other procedures to obtain the PCR product were performed in the same way as described for the cell cultures. This protocol was approved by a scientific/ethics committee of our institution (InCor/USP).
3. Results 3.1. SOD activity The baseline of SOD activity was 13.971.2 U/mg protein (Fig. 1). A significant decrease in SOD activity was observed after RASMCs incubation for 4 hours with Tn (approx. 29.0%) and 7KC (approx.12.0%), 8 hours with Tn (approx. 37.0%), and 18 hours with Ang II (approx. 42.0%) and 7KC (approx. 32.0%). A significant increase in SOD activity was observed after 8 hours of incubation with Ang II (approx. 41.0%). 3.2. Copper concentration The most interesting observation in relation to the copper concentration was for Ang II. RASMCs incubation with Ang II for 8
Fig. 2. Copper concentration (mg/mL) in RASMCs homogenate (n =3) at different incubation times (4, 8 and 18 hours) with 100 nM angiotensin II (Ang II), 5 mg/mL tunicamycin (Tn) or 5 mg/mL 7-ketocholesterol (7KC).
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hours promoted a 9-fold increase in the copper concentration compared to the control. The same stimulus for 18 hours of incubation resulted in an approx. 3.7-fold increase in the copper concentration (Fig. 2). 3.3. Western blotting The RASMCs protein expression for SOD isoenzymes showed no differences for 4 and 18 hours of incubation (data not shown).
The CuZnSOD expression for RASMCs incubated for 8 hours is represented in Fig. 3. An increased protein expression was obtained for comparisons with the control, with percentages of 101.1% for Ang II, 124.2% for Tn and 123.1% for 7KC. The MnSOD expression for RASMCs incubated for 8 hours, with different stimuli, showed no change compared to the control. The EcSOD expression decreased in all groups of samples incubated for 8 hours, and the percentages of protein expression were 54.5% for Ang II, 77.3% for Tn and 66.7% for 7KC (Fig. 4). RASMCs incubated for 8 hours showed a decrease in the PrPC expression for all stimuli. There was a pronounced protein expression reduction in cells exposed to the 7-ketocholesterol. The percentages of protein expression for comparison with the control were as follows: 82.6% for Ang II, 74.8% for Tn and 45.2% for 7KC (Fig. 5). 3.4. RT-PCR The RT-PCR for the RASMCs incubated for 8 hours showed an increase in the PrPC mRNA expression with the three stimuli used. The sample exposed to Ang II showed the greatest increase. The percentages of mRNA expression for comparison with the control were 128.2% for Ang II, 114.1% for Tn and 115.5% for 7KC (Fig. 6). 3.5. RT-PCR for mRNA PrPC in rabbit arteries Fig. 7 shows the results obtained with the PrPC mRNA expression for the rabbit arteries that underwent angioplasty, compared to the control arteries (C). The PrPC mRNA expression for the arteries of ‘‘zero time’’ (L0) was approximately 3.6 times greater than that for the control arteries (without injury). For the L0 arteries the PrPC mRNA expression was around 3.3 times higher than the expression in the contra-lateral arteries (CL0).
Fig. 3. Western blotting analysis for CuZnSOD expression, normalized against bactin expression. RASMCs were incubated for 8 hours with 100 nM angiotensin II (Ang II), 5 mg/mL tunicamycin (Tn) or 5 mg/mL 7-ketocholesterol (7KC). The upper panels show blots of representative experiments. The bar graphs represent the means obtained from densitometric analysis of blots from three independent experiments. nP o0.05 versus control (C).
Fig. 4. Western blotting analysis for EcSOD expression, normalized against b-actin expression. RASMCs were incubated for 8 hours with 100 nM angiotensin II (Ang II), 5 mg/mL tunicamycin (Tn) or 5 mg/mL 7-ketocholesterol (7KC). The upper panels show blots of representative experiments. The bar graphs represent the means obtained from densitometric analysis of blots from three independent experiments. nP o0.05 versus control (C).
4. Discussion The SOD assay showed a significant increase in enzyme activity (approx 0.41 times) only in the samples stimulated with Ang II for 8 hours, compared with the enzyme activity in the control samples (not stimulated) (Fig. 1). Since Nox1 is expected
Fig. 5. Western blotting analysis for PrPC expression, normalized against b-actin expression. RASMCs were incubated for 8 hours with 100 nM angiotensin II (Ang II), 5 mg/mL tunicamycin (Tn) or 5 mg/mL 7-ketocholesterol (7KC). The upper panels show blots of representative experiments. The bar graphs represent the means obtained from densitometric analysis of blots from three independent experiments. nPo 0.05 versus control (C).
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Fig. 6. PrPC mRNA expression after RASMCs exposure for 8 hours to 100 nM angiotensin II (Ang II), 5 mg/mL tunicamycin (Tn) or 5 mg/Ml 7-ketocholesterol (7KC). The bar graphs represent the means obtained from densitometric analysis of blots from three independent experiments. Gene expression was corrected by the use of GAPHD as an internal control. nP o0.05 versus control (C).
Fig. 7. PrPC mRNA expression in the injured rabbit iliac arteries (I) and contralateral uninjured rabbit iliac arteries (CL) at zero, 7 and 14 days after balloon injury, compared with the mRNA expression in the control arteries (C). The PrPC mRNA expression was corrected by GAPDH gene expression in the same samples (n= 2 for each group).
to be present both in the plasma membrane and in ER, and Nox4 is predicted to be in ER only (Lasse gue et al., 2001), this increase may be due to the production of ROS through Nox4, as observed by O’Brien et al. (2006) in rabbit corneal epithelial cells. Recently, Zwirska-Korczala et al. (2007), using preadipocyte cell cultures, showed that incubation with Ang II (2.5 10 9 M, 5.0 10 6 M) also resulted in an increase in SOD activity. Our results are consistent with those reported by Griendling et al. (1994) which showed that after the treatment of vascular smooth muscle cells (VSMC) with Ang II (100 nmol/L) for 4–6 h there was a 2.7(70.4)-fold increase in superoxide anion formation. With respect to Ang II, for incubation periods of 4 and 18 hours, the SOD activity was lower or similar to the control. These
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findings are consistent with those obtained in previous experiments conducted in the Vascular Biology Laboratory of InCor/ FMUSP (not published), where the RASMCs were stimulated with Ang II (100 nM) for 30 mins, 1, 2, 4 and 14 hours. The superoxide production mediated by 7KC, reported by Pedruzzi et al. (2004), demonstrated that 7KC specifically induces the overexpression of Nox4 mRNA (approx. 3-fold increase) in SMCs. In contrast, O’Callaghan et al. (2001) showed that after 12 hours of incubation with 7KC (30 mmol/L) the SOD activity remained unchanged. Thus, we can infer that superoxide production is increased when the cells are exposed to 7KC and therefore an increase of SOD activity in these cells is to be expected. As our experimental model did not show this response to stress, it could be that the stimulus concentration and/or the incubation time applied were not sufficient to induce such a response. Recently, Suzuki et al. (2006), using human neuroblastoma cells, showed that the ROS level was significantly increased after 3 and 6 hours of incubation with Tn (0.3 mM=0.25 mg/mL). With similar concentrations, Shimazawa et al. (2007) showed that ERS induced by Tn (1–4 mg/mL) was associated with proteins modified by binding to ubiquitin in a culture of rat retinal ganglion cells, and observed that these protein levels started to increase 2–6 hours after the start of Tn treatment. In this context, PrPC may be one of the proteins linked to the ubiquitin–proteasome system, which would be removed from the cell when submitted to stress, since in the presence of Tn our data showed a reduced PrPC expression for all three incubation times. Some studies (Brown and Besinger, 1998; Brown et al., 1997a, b, 2002; Herms et al., 1999) have reported that the brains of Prnp0/0 mice have a reduced Cu2 + content and decreased enzymatic activity of several Cu2 + -dependent enzymes, such as CuZnSOD. Others (Brown et al., 1998, 2001, 2002; White et al., 1999; Wong et al., 2001a, b) have suggested that the absence of PrPC results in increased neuron sensitivity to various models of oxidative injury, a condition that may be dependent on the Cu2 + ions binding to PrPC. The copper concentration in cellular homogenates (Fig. 2) increased (approx 9-fold) for the samples stimulated with Ang II after 8 hours, compared to the control, and subsequently an increased SOD activity was observed. However, the CuZnSOD expression remained unchanged under this condition and only the EcSOD expression changed, with a decrease of approx 45% compared to the control. Although the increase in copper concentration is consistent with the increased SOD activity, the PrPC expression did not follow the same profile, suggesting that PrPC do not have an antioxidant activity similarly to the Cu2 + dependent enzymes. It was also observed that the PrPC mRNA expression increased (approx 28%) under the same conditions, probably in response to a greater availability of Cu2 + for incorporation into the PrPC, considering that the PrPC expression after 18 hours of exposure to Ang II was similar to that observed with the control cells (data not shown). The increase in PrPC mRNA expression may be linked to an increased availability of Cu2 + ions in the sample stimulated after 8 hours with Ang II and is consistent with the hypothesis of Brown and Besinger (1998) that PrPC functions as a sensor for Cu2 + ions. Although the PrPC mRNA expression is also increased in the presence of Tn and 7KC after 8 hours of exposure, this increase is less intense than that of Ang II, and also the Cu2 + concentration in these samples did not alter compared with the respective controls. The protein expression for MnSOD increased (approx 36%) only with Tn after 4 hours of incubation (data not shown). After 8 hours of incubation the MnSOD expression decreased with Tn, while Ang II and 7KC did not alter the MnSOD expression. In our model, the results relating to Ang II exposure were in agreement
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with those of Guo et al. (2003) for MnSOD expression, except for the samples stimulated for 18 hours (data not shown), in which we observed a protein expression decrease. The EcSOD expression in RASMCs showed a slight difference in comparison to MnSOD. After 8 hours of incubation (Fig. 4), we can see that the EcSOD expression declined in the case of all ER stressor agents used, with a greater reduction for the samples stimulated with Ang II. Because of the SOD activity increase in the cells incubated with Ang II for 8 hours, we analyzed the CuZnSOD expression by western blotting under the same conditions with the three ER stressors, in an attempt to correlate the results obtained with the enzyme activity and the Cu2 + concentrations. We found that the CuZnSOD protein maintained the same profile of expression observed in a previous study conducted at the Vascular Biology Laboratory of InCor (data not published), for sample incubation periods of 4 and 18 hours. For the cells stimulated for 8 hours in the presence of Ang II we observed that the enzyme expression remained unchanged when compared to the control. However, the samples exposed to Tn and 7KC showed a slight increase in protein expression. In relation to these results, considering the profile of CuZnSOD expression, we could not correlate the SOD activity with the increase in Cu2 + concentration for the samples stimulated with Ang II for 8 hours. Exposure to Ang II can lead to increased SOD activity without the need to increase the isoenzyme expression. The Cu2 + concentration increase may be related to the PrPC mRNA expression, as mentioned previously. The reduced PrPC expression observed in the cells subjected to stimuli exposure for 8 hours may be due to a destruction of the protein in response to the initial stress. After 18 hours of incubation (data not shown) a change was observed in the cells exposed to Ang II, which showed a PrPC expression similar to that observed for the control samples under the same conditions. Eighteen hours of exposure to Tn and 7KC also promoted a decrease in the protein expression, but with some attenuation, because the PrPC expression was not as reduced as in the first 4 hours of incubation (data not shown). Possibly, the ER stress caused by the initial exposure to Ang II favored the response of RASMCs to produce PrPC (after 18 hours) in relation to other stimuli. The PrPC mRNA expression of the RASMCs cultures was assessed by RT-PCR. The samples incubated for 8 hours with the three ER stressors showed increased PrPC mRNA expression, compared to the control. Samples stimulated with Ang II showed greater expression of those incubated with Tn and 7KC. The increased PrPC mRNA expression after 8 hours of incubation with Ang II may be due to a cellular response to stress in an attempt to enhance the protein production, so that it could serve as a Cu2 + sensor in the ER stress response, considering that the PrPC is a Cu2 + -dependent protein. In addition, observing the supposed protective function (neuroprotective function) previously reported by some authors, this increase in the mRNA transcription could also reflect a tissue cellular response for protection against reactive species. The mRNA transcription increase may explain the protein expression normalization observed by western blotting after Ang II incubation for 18 hours (data not shown). Thus, the increase in the PrPC mRNA transcription within 8 hours of exposure resulted in an increased PrPC production observed after 18 hours of incubation. The immuno-histochemical observation of different amounts of PrPC in affected and normal rabbit arteries that underwent angioplasty by balloon catheter (unpublished data) led to the search for PrPC mRNA expression under the same conditions. An increase in oxidative stress (O2 production) after injury has been observed by Souza et al. (2000) in a study with rabbits where the
arterial injury resulting from a balloon catheter caused an immediate and sharp increase in O2 . Subsequently, similar results were shown by Jacobson et al. (2003), using mice as the experimental model. In the present study, the PrPC mRNA expression showed no difference between animals with a survival of 7 and 14 days after injury. However, a pronounced increase in the PrPC mRNA expression was seen when iliac arteries were examined immediately after injury. This result is in agreement with the increase in O2 reported by the above-cited authors and with the immunohistochemical observation of the arteries as mentioned above. In 2003, Leite et al. (2003), using a model of the distension of rabbit iliac arteries, observed that the vascular SOD activity had decreased by 45% and 34% at 7 and 14 days after injury, respectively. The results of the western blotting analysis for CuZnSOD showed that, on the 7th day after injury, the reduction in the enzyme expression was greater than that on the 14th day after injury. In contrast with the CuZnSOD expression, the EcSOD expression was significantly higher at 7 and 14 days after injury, and the MnSOD expression remained unchanged in the same period. With these results, Leite et al. showed that the decrease in the SOD isozymes activity was not accompanied by parallel changes in their expressions. In our experiments with rabbit iliac arteries, injured and uninjured, we noted that the PrPC mRNA expression remained unchanged at 7 and 14 days after injury. Compared with the data presented by Leite et al. (2003), we suggest that the PrPC had no influence on the antioxidant activity in response to the oxidative stress to which the arteries were submitted. In addition, the increase in the protein expression immediately after injury, observed in the histological sections by immuno-histochemistry (unpublished data), was consistent with the increase in PrPC mRNA expression observed in the iliac arteries soon after angioplasty (group ‘‘zero time ‘‘) in our study. The increase in PrPC mRNA expression in the arteries soon after injury (zero time) may be due to a protective action of the protein, as reported for the neural pathways (Roucou et al., 2004). This could explain the initial increase in mRNA as an attempt to protect the injured tissue, signaling the stimulus for the protein production. Given the superoxide increase in the arteries after angioplasty verified by Jacobson et al., 2003, the unchanged mRNA expression (7 and 14 days after injury) found in our results makes unlikely the association of PrPC with antioxidant activity, at least in this model of injury; since the PrPC mRNA expression in the arteries without lesions was similar to the expression observed in the control arteries (of animals not subjected to angioplasty). It is possible that analysis after a shorter period (24 hours after injury, for example) would show a relationship of the PrPC with the antioxidant activity exerted by SOD, but this time period is not usually studied in experimental models with survival after arterial injury. In terms of the PrPC mRNA expression our data can be compared with another stress model used by Weise et al. (2004), which showed, in the initial hours after permanent focal ischemia in the brain, a significant upregulation of PrPC in the ischemic hemisphere. Our results show increased PrPC mRNA expression soon after injury, in agreement with the data provided by Weise et al., although the mRNA expression returned to the basal levels after 7 and 14 days of injury. There are many questions related to whether the PrPC has a SOD-like activity. Some authors argue in favor of this function, while others disregard the possibility of PrPC action in the cellular antioxidant system. Indeed, a deeper discussion regarding the physiological functions of the PrPC, mainly in the neuronal tissue, has been conducted for some time by several authors. While some attempt
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to verify a possible SOD-like activity for PrPC (Brown et al., 1997b; Rachidi et al., 2003; Klamt et al., 2001; Treiber et al., 2007), others are in disagreement with this hypothesis for PrPC (Waggoner et al., 2000; Brown et al., 1999; Hutter et al., 2003, Jones et al., 2005; Legleiter et al., 2007). We suggest that the PrPC involvement in antioxidant activity proposed by other authors must be cell- and/or response-specific to the type of stress caused, since we could not attribute an antioxidant behavior to PrPC. Most experiments that verify PrPC antioxidant activity were carried out with PrPC normal and knockout mice, in extracts from cells and brain tissue, where PrPC is more abundant. Another possible explanation for PrPC SOD-like activity would be the need for a high PrPC concentration in these cells. This was not evaluated in our model, since we chose to investigate only the cell behavior according to the stimuli used, and not the response dependence on the PrPC concentration.
5. Conclusions In RASMCs submitted to ERS with Ang II, Tn or 7KC no SOD-like activity for PrPC was detected. This does not negate the possibility of its involvement in other types of cellular response to oxidative stress, considering the intense PrPC mRNA expression observed in the iliac arteries soon after injury. Thus, this protein may represent an important tool in the treatment of cardiovascular diseases by angioplasty, where the oxidative stress to which the organ is submitted requires a greater antioxidant response generated by the organism.
Acknowledgments This work was supported by grants from Fundac- a~ o de Amparo a Pesquisa do Estado de Sa~ o Paulo (FAPESP) and fellowships from Coordenac- a~ o de Aperfeic- oamento de Pessoal de Nı´vel Superior (CAPES) and Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq) of Brazil. We are grateful to Prof. Vilma R. Martins, Ph.D. (Ludwig Institute for Cancer Research, Sa~ o Paulo, Brazil) for helpful discussions and for providing the PrPC antibody. We acknowledge Leonora Loppnow for technical support in performing the angioplasty. We also would like to thank Prof. Jamil Assreuy, Ph.D. and Bettina Tomio Heckert, MD (Department of Pharmacology, Federal University of Santa Catarina, Brazil) for technical support in the western blotting analysis.
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Experimental and Toxicologic Pathology journal homepage: www.elsevier.de/etp
Cellular prion protein (PrPC) and superoxide dismutase (SOD) in vascular cells under oxidative stress He´len Zocche Soprana a, Liliete Canes Souza a,n, Victor Debbas b,1, Francisco Rafael Martins Laurindo b,I a b
Department of Clinical Analysis, Center of Health Sciences, Federal University of Santa Catarina, Brazil Vascular Biology Laboratory, Heart Institute, University of Sao Paulo School of Medicine, Brazil
a r t i c l e in fo
abstract
Article history: Received 1 May 2009 Accepted 14 December 2009
The PrPC is expressed in several cell types but its physiological function is unknown. Some studies associate the PrPC with copper metabolism and the antioxidant activity of SOD. Our hypothesis was that changes in PrPC expression lead to abnormal copper regulation and induce SOD downregulation in the vascular wall. Objectives: to study whether the PrPC expression undergoes induction by agents that trigger endoplasmic reticulum stress (ERS) and, in this context, to evaluate the SOD activity. Methods: To trigger ERS, in vitro, rabbit aortic smooth muscle cells were challenged for 4, 8 and 18 hours, with angiotensin-II, tunicamycin and 7-ketocholesterol. For in vivo studies rabbit aortic arteries were subjected to injury by balloon catheter. Results: In vitro baseline SOD activity, determined through inhibition of cytochrome-c reduction, was 13.9 7 1.2 U/mg protein, angiotensin-II exposed for 8 hours produced an increase in SOD activity, and cellular copper concentration was about 9 times greater only under these conditions. Western blotting analysis for SOD isoenzymes showed an expression profile that was not correlated with the enzymatic activity. PrPC expression decreased after exposure to all agents after different incubation periods. RT-PCR assay showed increased mRNA expression for PrPC only in cells stimulated for 8 hours with the different stressors. The PrPC mRNA expression in rabbit aortic artery fragments, subjected to balloon catheter injury, showed a pronounced increase immediately after overdistension. The results obtained indicated a PrPC protection factor during the early part of the ERS exposure period, but did not demonstrate a SOD-like profile for the PrPC. & 2009 Elsevier GmbH. All rights reserved.
Keywords: Superoxide dismutase Cellular prion protein Rabbit aortic smooth muscle cells Copper concentration Endoplasmic reticulum stress
1. Introduction The cellular prion protein (PrPC) is a glycoprotein of 33–35 kDa (Prusiner, 1998; Glockshuber et al., 1998). The physiological function of PrPC is still unclear; however, it is involved in neurodegenerative diseases, characterized by the accumulation of an abnormally folded isoform of the PrPC, denoted PrPSC, which represents the major component of infectious prion diseases (Prusiner, 1998). The superoxide dismutases (SOD) are the first and most important line of antioxidant enzyme defense system against reactive oxygen species (ROS) and particularly superoxide anion radicals (Zelko et al., 2002). The SOD expression and activity have an effect on cellular vascular responses to acute and chronic oxidative stress (Packer, 2002).
n Corresponding author at: Campus Universita´rio Trindade, Departamento de Ana´lises Clı´nicas, 88040-970, Floriano´polis, SC, Brazil. Tel.: +55 48 3721 9712x221; fax: + 55 48 3721 9542. E-mail address: [email protected] (L. Canes Souza). 1 Avenida Eneas de Carvalho Aguiar, 44 - 91 andar Bloco II, 05403-000, Sa~ o Paulo, SP, Brazil. Tel.: + 55 11 3069 5185; fax: + 55 11 3069 5920.
0940-2993/$ - see front matter & 2009 Elsevier GmbH. All rights reserved. doi:10.1016/j.etp.2009.12.004
SOD isoforms are cytosolic or copper/zinc SOD (CuZnSOD or SOD-1), manganese SOD (MnSOD or SOD-2), and an extracellular form of CuZnSOD (EcSOD or SOD-3) (Marklund, 1982; Fukai et al., 2002). EcSOD expression in vascular cells and within the vessel wall can be altered in response to a variety of stimuli including angiotensin II (Zelko et al., 2002; Fukai et al., 2002). The ROS in small quantities are essential in many processes, including intracellular signaling, protection against microorganisms and cellular function. In contrast, the excessive production and/or inadequate removal of ROS has been implicated in oxidation of biological macromolecules, such as DNA, proteins, carbohydrates and lipids, and this condition has commonly been referred to as oxidative stress (Fukai et al., 2002). Evidence suggests that oxidative stress is involved in the pathogenesis of many cardiovascular diseases, including hypercholesterolemia, atherosclerosis, hypertension, diabetes and heart failure (Fukai et al., 2002; Cai and Harrison, 2000). ROS production is prominently immediately after injury, it has been reported in neointima and adventitia during the later stages of vessel repair (Souza et al., 2000; Azevedo et al., 2000; Rey and Pagano, 2002). Despite the potential for ROS to induce vascular injury, they have been ascribed an important signaling role in
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physiologic and pathologic situations (Irani, 2000). The importance of redox-dependent signaling in vascular cell growth, apoptosis and senescense is increasingly apparent (Abe and Berk, 1998; Suzuki et al., 1997; Sen and Packer, 1996; Kunsch and Medford, 1999). Leite et al. examined in detail the pathophysiology of vascular repair after balloon injury and reported that a decrease in SOD activity plays a role in constrictive vascular remodeling. Although there was depletion in both cytoplasmic and extracellular SOD after injury, the authors showed that the administration of EcSOD was sufficient to normalize SOD activity and improve vascular caliber (Leite et al., 2003). The normal prion protein binds copper and the resulting complex apparently acts in the protection against oxidative stress (Brown, 2001; Brown et al., 1997a, b). Thus, PrP prevents copper from interacting with cells deleteriously (Brown et al., 1998; Rachidi et al., 2003). The loss of PrP function may result in an increased sensitivity to oxidative stress and copper toxicity. Therefore, the PrP physiological function may be involved not only in central nervous system diseases but also in other human illnesses, such as heart reperfusion injury, hepatic failure and sepsis (Martins et al., 2002). It has been reported that the enzymatic activity and copper loading of Cu/Zn superoxide dismutase (SOD1) are 50% of the normal level in brain and cultured cerebellar neurons from PrPnull mice and that SOD1 activity and copper loading are elevated in mice that overexpress PrP (Brown et al., 1997a, b). Thus, some authors have investigated whether PrP functions as an endocytic receptor for cellular uptake of copper ions, or facilitates some other aspect of copper trafficking such as efflux from the cell or intracellular sequestration of the metal (Brown et al., 1997a, b; Waggoner et al., 2000). The mechanism of the PrPC antioxidant effect is not clear and may be dependent on an increase in the endogenous SOD activity and its role in Cu2 + metabolism (Brown and Besinger, 1998). Our hypothesis is that PrPC levels contribute to a sustained decrease in SOD activity in the vascular wall stimulating negative remodeling after injury. In preliminary immuno-histochemical studies, we verified a significant PrPC expression in rabbit intact iliac arteries, with a significant decrease in this expression immediately after balloon injury (data not published). Moreover, Leite et al. observed that CuZnSOD and EcSOD expression after balloon injury has a similar pattern compared to its protein mRNA expression in the brain of knockout mice for PrPC (Leite et al., 2003; Brown et al., 2002). The aim of the present study was to evaluate the expression of PrPC under assay conditions typical of those used for studies on vascular redox signaling. Isolated rabbit aortic smooth muscle cells (RASMCs) were challenged with agonists of endoplasmic reticulum stress pathways and rabbit iliac arteries were submitted to overdistension injury to correlate SOD activity and its isoenzyme expression to PrPC expression, using both Western blotting and RT-PCR techniques.
2. Materials and methods 2.1. Chemicals Angiotensin II, tunicamycin, xanthine and xanthine oxidase were purchased from Calbiochems (San Diego, CA, USA), 7ketocholesterol from Sigma-Aldrichs (St Louis, MO, USA), fetal calf serum (FCS) from Gibco-BRL Lifes (Grand Island, NY, USA) and lysis buffer plus protease inhibitors from Mercks (Darmstadt, Germany). The mouse anti-PrPC was kindly donated by Vilma Regina Martins Ph.D. (Ludwig Institute of Cancer Research, Sao Paulo, Brazil). Sheep anti-CuZnSOD antibody was purchased from
Oxis International Inc.s (Foster City, CA, USA); rabbit anti-MnSOD and anti-EcSOD from Stressgens (Victoria, Canada); primary monoclonal antibody anti-b-actin (mouse IgG) from SigmaAldrichs (St Louis, MO, USA); secondary antibody anti-IgG of mice with peroxidase conjugate from Amersham Pharmacia Biotechs (USA); antibody goat anti-rabbit IgG (MnSOD and EcSOD) from Stressgens (Victoria, Canada); secondary rabbit anti-sheep to CuZnSOD from Calbiochems (San Diego, CA, USA); and rabbit anti-mouse IgG to PrPC from Amersham Pharmacia Biotechs (USA), conjugate with peroxidase. The proteins were detected by a chemiluminescence method using an ECL reagent kit from Amersham Pharmacia Biotechs (USA). The reagents used for the RT-PCR reaction including Trizols were purchased from Invitrogens (Carlsbad, CA, USA). All other reagents were purchased from Sigma-Aldrichs (St Louis, MO, USA). All solutions were prepared with distilled water further purified in a Millipore Milli-Q system and treated with Chelex-100 before use. 2.2. Cell culture and homogenate Rabbit aortic smooth muscle cells (RASMCs) obtained from a previously established selection-immortalized line (Buonassisi and Venter, 1976) were cultured in Coon’s F12 medium supplemented with 10% FCS, 100 U/mL penicillin and 100 mg/mL streptomycin at 37 1C in a humidified atmosphere with 5% CO2 to 95% air. For the present study, cells between passages 2 and 7 were used. To induce oxidative stress, cells (approximately 90% confluence) were exposed to angiotensin II (Ang II – 100 nM), tunicamycin (Tn – 5 mg/mL) or 7-ketocholesterol (7KC – 5 mg/mL) for 4, 8 and 18 hours (n= 3 for each stimulus and time, incubated on different days). Viability of cells after stimuli exposure was determined by the trypan blue exclusion assay. Cell homogenates were prepared as described previously (Laurindo et al., 2002) and protein concentration was assessed using the Bradford method. 2.3. SOD activity assay Aliquots of the supernatant were assayed for total SOD activity as previously described (Azevedo et al., 2000). The inhibition rate of cytochrome-c reduction induced by xanthine oxidase-derived superoxide at pH 7.4 was monitored spectrophotometrically at 550 nm at 37 1C, and results were normalized for protein concentration (Bradford method). Statistical analysis was performed through the t-test, with a 0.05 significance level. 2.4. Cellular copper concentrations Levels of Cu in the homogenate samples were measured according to the method of Papageorgiou et al. (2002) with the use of an atomic absorption spectrophotometer (AAS model 2380, Perkin-Elmer, USA) with a graphite furnace heated graphite atomizer (HGA model 300) and analytical wavelengths of 324.8 nm. The measurements were repeated twice and two quality controls (homogenate and aqueous) were used: (1) cation-cal collaboration and (2) an individual solution of a fixed concentration using spectrosol BDH standards. Statistical analysis was performed using Student’s t-test. The mean values are expressed as micrograms per milliliter. 2.5. Western blot analysis Samples were subjected to SDS-PAGE in polyacrylamide gels according to the protein molecular weight (CuZnSOD: 17.5%, EcSOD and PrPC: 15%, MnSOD: 12%). After electrophoresis, proteins were electro-transferred to a nitrocellulose membrane
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(Amersham Biosciences, Piscataway, NJ). Equal loading of samples (30 mg for SOD isozymes and 80 mg for PrPC) and transfer efficiency were monitored with the use of 0.5% Ponceau S staining of the blot membrane. The blotted membrane was then blocked (5% nonfat dry milk, 10 mM Tris–HCl, pH 7.6, 150 mM NaCl, and 0.1% Tween 20) for 2 h at room temperature and incubated (same buffer plus 1% nonfat dry milk) with specific antibodies (1:200 for mouse anti-PrPC; 1:1000 for sheep anti-CuZnSOD; 1:800 for rabbit anti-MnSOD and rabbit anti-EcSOD; and 1:10,000 for mouse anti-b-actin) overnight at 4 1C. Binding of the primary antibody was detected with the use of peroxidase-conjugated secondary antibodies (goat anti-rabbit IgG diluted 1:2000 for MnSOD and EcSOD; rabbit anti-sheep IgG diluted 1:2000 for CuZnSOD; rabbit anti-mouse IgG diluted 1:600 for PrPC; mouse anti-IgG diluted 1:20,000 for b-actin) for 1:30 h at room temperature and developed by enhanced chemiluminescence (Amersham Pharmacia Biotech) detected by autoradiography. Quantification analysis of blots was performed with the use of Scion Image software (Scion based on NIH Image). 2.6. Reverse transcriptase-polymerase chain reaction (RT-PCR) Total RNA was extracted from RASM cells using Trizols according to the manufacturer’s instructions. RT-PCR was performed using the thermocycler (Eppendorfs Mastercycler Personal). The quality of the isolated RNA was checked in a spectrophotometer (Hitachi Spectrophotometers U-2001) by measuring the absorbance at 260 and 280 nm (OD260/OD280 ratio of 1.6–1.8, as described by Sambrook and Russel, 2001). The RNA integrity was evaluated by 0.8% agarose gel electrophoresis (Amrescos) followed by ethidium bromide-staining (1 mg/mL) for the visualization of the 18S and 28S bands in a transilluminator (Eagle Eye IIs, Alpha Innotech). Total RNA (2 mg) was reverse transcribed following the manufacturer’s instructions (Invitrogens). For cDNA amplification, a 1 mL aliquot of the reverse transcription reaction was amplified in a volume of 25 mL, composed of 2.5 mL of 10 PCR buffer, 0.5 mL of 10 mM dNTP, 0.75 mL of 50 mM MgCl2, and 1.0 mL of each oligonucleotide primer (10 mM): CAC CAA AGG GGA GAA CTT CA (sense) and ATC CCA CGA TCA GGA AGA TG (anti-sense). The PCR protocol was performed in a thermocycler (Eppendorfs Mastercycler Personal) with an initial cycle of 2 min at 94 1C, followed by 30 cycles of 1 min at 94 1C, 1 min at 55 1C and 1 min at 72 1C. PCR amplification products were visualized, after electrophoresis in a 2%
Fig. 1. SOD enzymatic activity (UI/mg Protein) in RASMCs homogenate (n= 3) stimulated for 4, 8 and 18 hours with 100 nM angiotensin II (Ang II), 5 mg/mL tunicamycin (Tn) or 5 mg/mL 7-ketocholesterol (7KC). nP o0.05 versus control (C).
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agarose gel, using ethidium bromide (1.0 mg/mL) staining. The obtained PCR amplification products gave DNA fragments of the expected size (185 bp). Primers specific to the GAPDH gene for rabbit (sense: TCA CCA TCT TCC AGG GA AGC, and anti-sense: CAC AAT GCC GAA GTG GTC GT) were used as the positive control. 2.7. Balloon injury of iliac artery Iliac artery overdistension injury was performed in pentobarbital-anesthetized normolipemic male New Zealand White rabbits, as described previously (Leite et al., 2003; Laurindo et al., 2002) using a coronary angioplasty-type balloon with diameter of 2.75 mm, inflated at 8.0 atm. After 7 and 14 days, the rabbits were euthanized with pentobarbital sodium; both the injured right and uninjured left iliac arteries were removed immediately. In addition to these two groups, there was a third group (‘‘zero time’’) in which the animal was killed immediately after angioplasty. The procedure was performed on two animals from each group with survival, at 7 and 14 days and ‘‘zero time’’. The two iliac arteries taken from a rabbit not submitted to any prior injury were used as negative controls. After surgical removal of the arteries, the fragments were quickly frozen in liquid nitrogen, macerated and later transferred to an RNAse-free tube. RNA extraction and other procedures to obtain the PCR product were performed in the same way as described for the cell cultures. This protocol was approved by a scientific/ethics committee of our institution (InCor/USP).
3. Results 3.1. SOD activity The baseline of SOD activity was 13.971.2 U/mg protein (Fig. 1). A significant decrease in SOD activity was observed after RASMCs incubation for 4 hours with Tn (approx. 29.0%) and 7KC (approx.12.0%), 8 hours with Tn (approx. 37.0%), and 18 hours with Ang II (approx. 42.0%) and 7KC (approx. 32.0%). A significant increase in SOD activity was observed after 8 hours of incubation with Ang II (approx. 41.0%). 3.2. Copper concentration The most interesting observation in relation to the copper concentration was for Ang II. RASMCs incubation with Ang II for 8
Fig. 2. Copper concentration (mg/mL) in RASMCs homogenate (n =3) at different incubation times (4, 8 and 18 hours) with 100 nM angiotensin II (Ang II), 5 mg/mL tunicamycin (Tn) or 5 mg/mL 7-ketocholesterol (7KC).
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hours promoted a 9-fold increase in the copper concentration compared to the control. The same stimulus for 18 hours of incubation resulted in an approx. 3.7-fold increase in the copper concentration (Fig. 2). 3.3. Western blotting The RASMCs protein expression for SOD isoenzymes showed no differences for 4 and 18 hours of incubation (data not shown).
The CuZnSOD expression for RASMCs incubated for 8 hours is represented in Fig. 3. An increased protein expression was obtained for comparisons with the control, with percentages of 101.1% for Ang II, 124.2% for Tn and 123.1% for 7KC. The MnSOD expression for RASMCs incubated for 8 hours, with different stimuli, showed no change compared to the control. The EcSOD expression decreased in all groups of samples incubated for 8 hours, and the percentages of protein expression were 54.5% for Ang II, 77.3% for Tn and 66.7% for 7KC (Fig. 4). RASMCs incubated for 8 hours showed a decrease in the PrPC expression for all stimuli. There was a pronounced protein expression reduction in cells exposed to the 7-ketocholesterol. The percentages of protein expression for comparison with the control were as follows: 82.6% for Ang II, 74.8% for Tn and 45.2% for 7KC (Fig. 5). 3.4. RT-PCR The RT-PCR for the RASMCs incubated for 8 hours showed an increase in the PrPC mRNA expression with the three stimuli used. The sample exposed to Ang II showed the greatest increase. The percentages of mRNA expression for comparison with the control were 128.2% for Ang II, 114.1% for Tn and 115.5% for 7KC (Fig. 6). 3.5. RT-PCR for mRNA PrPC in rabbit arteries Fig. 7 shows the results obtained with the PrPC mRNA expression for the rabbit arteries that underwent angioplasty, compared to the control arteries (C). The PrPC mRNA expression for the arteries of ‘‘zero time’’ (L0) was approximately 3.6 times greater than that for the control arteries (without injury). For the L0 arteries the PrPC mRNA expression was around 3.3 times higher than the expression in the contra-lateral arteries (CL0).
Fig. 3. Western blotting analysis for CuZnSOD expression, normalized against bactin expression. RASMCs were incubated for 8 hours with 100 nM angiotensin II (Ang II), 5 mg/mL tunicamycin (Tn) or 5 mg/mL 7-ketocholesterol (7KC). The upper panels show blots of representative experiments. The bar graphs represent the means obtained from densitometric analysis of blots from three independent experiments. nP o0.05 versus control (C).
Fig. 4. Western blotting analysis for EcSOD expression, normalized against b-actin expression. RASMCs were incubated for 8 hours with 100 nM angiotensin II (Ang II), 5 mg/mL tunicamycin (Tn) or 5 mg/mL 7-ketocholesterol (7KC). The upper panels show blots of representative experiments. The bar graphs represent the means obtained from densitometric analysis of blots from three independent experiments. nP o0.05 versus control (C).
4. Discussion The SOD assay showed a significant increase in enzyme activity (approx 0.41 times) only in the samples stimulated with Ang II for 8 hours, compared with the enzyme activity in the control samples (not stimulated) (Fig. 1). Since Nox1 is expected
Fig. 5. Western blotting analysis for PrPC expression, normalized against b-actin expression. RASMCs were incubated for 8 hours with 100 nM angiotensin II (Ang II), 5 mg/mL tunicamycin (Tn) or 5 mg/mL 7-ketocholesterol (7KC). The upper panels show blots of representative experiments. The bar graphs represent the means obtained from densitometric analysis of blots from three independent experiments. nPo 0.05 versus control (C).
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Fig. 6. PrPC mRNA expression after RASMCs exposure for 8 hours to 100 nM angiotensin II (Ang II), 5 mg/mL tunicamycin (Tn) or 5 mg/Ml 7-ketocholesterol (7KC). The bar graphs represent the means obtained from densitometric analysis of blots from three independent experiments. Gene expression was corrected by the use of GAPHD as an internal control. nP o0.05 versus control (C).
Fig. 7. PrPC mRNA expression in the injured rabbit iliac arteries (I) and contralateral uninjured rabbit iliac arteries (CL) at zero, 7 and 14 days after balloon injury, compared with the mRNA expression in the control arteries (C). The PrPC mRNA expression was corrected by GAPDH gene expression in the same samples (n= 2 for each group).
to be present both in the plasma membrane and in ER, and Nox4 is predicted to be in ER only (Lasse gue et al., 2001), this increase may be due to the production of ROS through Nox4, as observed by O’Brien et al. (2006) in rabbit corneal epithelial cells. Recently, Zwirska-Korczala et al. (2007), using preadipocyte cell cultures, showed that incubation with Ang II (2.5 10 9 M, 5.0 10 6 M) also resulted in an increase in SOD activity. Our results are consistent with those reported by Griendling et al. (1994) which showed that after the treatment of vascular smooth muscle cells (VSMC) with Ang II (100 nmol/L) for 4–6 h there was a 2.7(70.4)-fold increase in superoxide anion formation. With respect to Ang II, for incubation periods of 4 and 18 hours, the SOD activity was lower or similar to the control. These
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findings are consistent with those obtained in previous experiments conducted in the Vascular Biology Laboratory of InCor/ FMUSP (not published), where the RASMCs were stimulated with Ang II (100 nM) for 30 mins, 1, 2, 4 and 14 hours. The superoxide production mediated by 7KC, reported by Pedruzzi et al. (2004), demonstrated that 7KC specifically induces the overexpression of Nox4 mRNA (approx. 3-fold increase) in SMCs. In contrast, O’Callaghan et al. (2001) showed that after 12 hours of incubation with 7KC (30 mmol/L) the SOD activity remained unchanged. Thus, we can infer that superoxide production is increased when the cells are exposed to 7KC and therefore an increase of SOD activity in these cells is to be expected. As our experimental model did not show this response to stress, it could be that the stimulus concentration and/or the incubation time applied were not sufficient to induce such a response. Recently, Suzuki et al. (2006), using human neuroblastoma cells, showed that the ROS level was significantly increased after 3 and 6 hours of incubation with Tn (0.3 mM=0.25 mg/mL). With similar concentrations, Shimazawa et al. (2007) showed that ERS induced by Tn (1–4 mg/mL) was associated with proteins modified by binding to ubiquitin in a culture of rat retinal ganglion cells, and observed that these protein levels started to increase 2–6 hours after the start of Tn treatment. In this context, PrPC may be one of the proteins linked to the ubiquitin–proteasome system, which would be removed from the cell when submitted to stress, since in the presence of Tn our data showed a reduced PrPC expression for all three incubation times. Some studies (Brown and Besinger, 1998; Brown et al., 1997a, b, 2002; Herms et al., 1999) have reported that the brains of Prnp0/0 mice have a reduced Cu2 + content and decreased enzymatic activity of several Cu2 + -dependent enzymes, such as CuZnSOD. Others (Brown et al., 1998, 2001, 2002; White et al., 1999; Wong et al., 2001a, b) have suggested that the absence of PrPC results in increased neuron sensitivity to various models of oxidative injury, a condition that may be dependent on the Cu2 + ions binding to PrPC. The copper concentration in cellular homogenates (Fig. 2) increased (approx 9-fold) for the samples stimulated with Ang II after 8 hours, compared to the control, and subsequently an increased SOD activity was observed. However, the CuZnSOD expression remained unchanged under this condition and only the EcSOD expression changed, with a decrease of approx 45% compared to the control. Although the increase in copper concentration is consistent with the increased SOD activity, the PrPC expression did not follow the same profile, suggesting that PrPC do not have an antioxidant activity similarly to the Cu2 + dependent enzymes. It was also observed that the PrPC mRNA expression increased (approx 28%) under the same conditions, probably in response to a greater availability of Cu2 + for incorporation into the PrPC, considering that the PrPC expression after 18 hours of exposure to Ang II was similar to that observed with the control cells (data not shown). The increase in PrPC mRNA expression may be linked to an increased availability of Cu2 + ions in the sample stimulated after 8 hours with Ang II and is consistent with the hypothesis of Brown and Besinger (1998) that PrPC functions as a sensor for Cu2 + ions. Although the PrPC mRNA expression is also increased in the presence of Tn and 7KC after 8 hours of exposure, this increase is less intense than that of Ang II, and also the Cu2 + concentration in these samples did not alter compared with the respective controls. The protein expression for MnSOD increased (approx 36%) only with Tn after 4 hours of incubation (data not shown). After 8 hours of incubation the MnSOD expression decreased with Tn, while Ang II and 7KC did not alter the MnSOD expression. In our model, the results relating to Ang II exposure were in agreement
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with those of Guo et al. (2003) for MnSOD expression, except for the samples stimulated for 18 hours (data not shown), in which we observed a protein expression decrease. The EcSOD expression in RASMCs showed a slight difference in comparison to MnSOD. After 8 hours of incubation (Fig. 4), we can see that the EcSOD expression declined in the case of all ER stressor agents used, with a greater reduction for the samples stimulated with Ang II. Because of the SOD activity increase in the cells incubated with Ang II for 8 hours, we analyzed the CuZnSOD expression by western blotting under the same conditions with the three ER stressors, in an attempt to correlate the results obtained with the enzyme activity and the Cu2 + concentrations. We found that the CuZnSOD protein maintained the same profile of expression observed in a previous study conducted at the Vascular Biology Laboratory of InCor (data not published), for sample incubation periods of 4 and 18 hours. For the cells stimulated for 8 hours in the presence of Ang II we observed that the enzyme expression remained unchanged when compared to the control. However, the samples exposed to Tn and 7KC showed a slight increase in protein expression. In relation to these results, considering the profile of CuZnSOD expression, we could not correlate the SOD activity with the increase in Cu2 + concentration for the samples stimulated with Ang II for 8 hours. Exposure to Ang II can lead to increased SOD activity without the need to increase the isoenzyme expression. The Cu2 + concentration increase may be related to the PrPC mRNA expression, as mentioned previously. The reduced PrPC expression observed in the cells subjected to stimuli exposure for 8 hours may be due to a destruction of the protein in response to the initial stress. After 18 hours of incubation (data not shown) a change was observed in the cells exposed to Ang II, which showed a PrPC expression similar to that observed for the control samples under the same conditions. Eighteen hours of exposure to Tn and 7KC also promoted a decrease in the protein expression, but with some attenuation, because the PrPC expression was not as reduced as in the first 4 hours of incubation (data not shown). Possibly, the ER stress caused by the initial exposure to Ang II favored the response of RASMCs to produce PrPC (after 18 hours) in relation to other stimuli. The PrPC mRNA expression of the RASMCs cultures was assessed by RT-PCR. The samples incubated for 8 hours with the three ER stressors showed increased PrPC mRNA expression, compared to the control. Samples stimulated with Ang II showed greater expression of those incubated with Tn and 7KC. The increased PrPC mRNA expression after 8 hours of incubation with Ang II may be due to a cellular response to stress in an attempt to enhance the protein production, so that it could serve as a Cu2 + sensor in the ER stress response, considering that the PrPC is a Cu2 + -dependent protein. In addition, observing the supposed protective function (neuroprotective function) previously reported by some authors, this increase in the mRNA transcription could also reflect a tissue cellular response for protection against reactive species. The mRNA transcription increase may explain the protein expression normalization observed by western blotting after Ang II incubation for 18 hours (data not shown). Thus, the increase in the PrPC mRNA transcription within 8 hours of exposure resulted in an increased PrPC production observed after 18 hours of incubation. The immuno-histochemical observation of different amounts of PrPC in affected and normal rabbit arteries that underwent angioplasty by balloon catheter (unpublished data) led to the search for PrPC mRNA expression under the same conditions. An increase in oxidative stress (O2 production) after injury has been observed by Souza et al. (2000) in a study with rabbits where the
arterial injury resulting from a balloon catheter caused an immediate and sharp increase in O2 . Subsequently, similar results were shown by Jacobson et al. (2003), using mice as the experimental model. In the present study, the PrPC mRNA expression showed no difference between animals with a survival of 7 and 14 days after injury. However, a pronounced increase in the PrPC mRNA expression was seen when iliac arteries were examined immediately after injury. This result is in agreement with the increase in O2 reported by the above-cited authors and with the immunohistochemical observation of the arteries as mentioned above. In 2003, Leite et al. (2003), using a model of the distension of rabbit iliac arteries, observed that the vascular SOD activity had decreased by 45% and 34% at 7 and 14 days after injury, respectively. The results of the western blotting analysis for CuZnSOD showed that, on the 7th day after injury, the reduction in the enzyme expression was greater than that on the 14th day after injury. In contrast with the CuZnSOD expression, the EcSOD expression was significantly higher at 7 and 14 days after injury, and the MnSOD expression remained unchanged in the same period. With these results, Leite et al. showed that the decrease in the SOD isozymes activity was not accompanied by parallel changes in their expressions. In our experiments with rabbit iliac arteries, injured and uninjured, we noted that the PrPC mRNA expression remained unchanged at 7 and 14 days after injury. Compared with the data presented by Leite et al. (2003), we suggest that the PrPC had no influence on the antioxidant activity in response to the oxidative stress to which the arteries were submitted. In addition, the increase in the protein expression immediately after injury, observed in the histological sections by immuno-histochemistry (unpublished data), was consistent with the increase in PrPC mRNA expression observed in the iliac arteries soon after angioplasty (group ‘‘zero time ‘‘) in our study. The increase in PrPC mRNA expression in the arteries soon after injury (zero time) may be due to a protective action of the protein, as reported for the neural pathways (Roucou et al., 2004). This could explain the initial increase in mRNA as an attempt to protect the injured tissue, signaling the stimulus for the protein production. Given the superoxide increase in the arteries after angioplasty verified by Jacobson et al., 2003, the unchanged mRNA expression (7 and 14 days after injury) found in our results makes unlikely the association of PrPC with antioxidant activity, at least in this model of injury; since the PrPC mRNA expression in the arteries without lesions was similar to the expression observed in the control arteries (of animals not subjected to angioplasty). It is possible that analysis after a shorter period (24 hours after injury, for example) would show a relationship of the PrPC with the antioxidant activity exerted by SOD, but this time period is not usually studied in experimental models with survival after arterial injury. In terms of the PrPC mRNA expression our data can be compared with another stress model used by Weise et al. (2004), which showed, in the initial hours after permanent focal ischemia in the brain, a significant upregulation of PrPC in the ischemic hemisphere. Our results show increased PrPC mRNA expression soon after injury, in agreement with the data provided by Weise et al., although the mRNA expression returned to the basal levels after 7 and 14 days of injury. There are many questions related to whether the PrPC has a SOD-like activity. Some authors argue in favor of this function, while others disregard the possibility of PrPC action in the cellular antioxidant system. Indeed, a deeper discussion regarding the physiological functions of the PrPC, mainly in the neuronal tissue, has been conducted for some time by several authors. While some attempt
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to verify a possible SOD-like activity for PrPC (Brown et al., 1997b; Rachidi et al., 2003; Klamt et al., 2001; Treiber et al., 2007), others are in disagreement with this hypothesis for PrPC (Waggoner et al., 2000; Brown et al., 1999; Hutter et al., 2003, Jones et al., 2005; Legleiter et al., 2007). We suggest that the PrPC involvement in antioxidant activity proposed by other authors must be cell- and/or response-specific to the type of stress caused, since we could not attribute an antioxidant behavior to PrPC. Most experiments that verify PrPC antioxidant activity were carried out with PrPC normal and knockout mice, in extracts from cells and brain tissue, where PrPC is more abundant. Another possible explanation for PrPC SOD-like activity would be the need for a high PrPC concentration in these cells. This was not evaluated in our model, since we chose to investigate only the cell behavior according to the stimuli used, and not the response dependence on the PrPC concentration.
5. Conclusions In RASMCs submitted to ERS with Ang II, Tn or 7KC no SOD-like activity for PrPC was detected. This does not negate the possibility of its involvement in other types of cellular response to oxidative stress, considering the intense PrPC mRNA expression observed in the iliac arteries soon after injury. Thus, this protein may represent an important tool in the treatment of cardiovascular diseases by angioplasty, where the oxidative stress to which the organ is submitted requires a greater antioxidant response generated by the organism.
Acknowledgments This work was supported by grants from Fundac- a~ o de Amparo a Pesquisa do Estado de Sa~ o Paulo (FAPESP) and fellowships from Coordenac- a~ o de Aperfeic- oamento de Pessoal de Nı´vel Superior (CAPES) and Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq) of Brazil. We are grateful to Prof. Vilma R. Martins, Ph.D. (Ludwig Institute for Cancer Research, Sa~ o Paulo, Brazil) for helpful discussions and for providing the PrPC antibody. We acknowledge Leonora Loppnow for technical support in performing the angioplasty. We also would like to thank Prof. Jamil Assreuy, Ph.D. and Bettina Tomio Heckert, MD (Department of Pharmacology, Federal University of Santa Catarina, Brazil) for technical support in the western blotting analysis.
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