Nitric oxide decreases activity and levels of the 11S proteasome activator PA28 in the vasculature

Nitric Oxide 27 (2012) 50–58

Contents lists available at SciVerse ScienceDirect

Nitric Oxide journal homepage: www.elsevier.com/locate/yniox

Nitric oxide decreases activity and levels of the 11S proteasome activator PA28 in the vasculature Nick D. Tsihlis a,1, Muneera R. Kapadia a,1, Ashley K. Vavra a, Qun Jiang a, Bo Fu a, Janet Martinez a, Melina R. Kibbe a,b,⇑ a b

Division of Vascular Surgery, Northwestern University, IL, USA The Jesse Brown VA Medical Center, 820 S. Damen Ave., Chicago, IL 60612, USA

a r t i c l e

i n f o

Article history: Received 12 September 2011 Revised 16 April 2012 Available online 24 April 2012 Keywords: Arterial injury Neointimal hyperplasia Nitric oxide Proteasome activator PA28 Vascular smooth muscle cells

a b s t r a c t The 11S proteasome activator (PA28) binds to the 20S proteasome and increases its ability to degrade small peptides. Expression of PA28 subunits (a, b, c) is induced by interferon-c stimulation. Inflammation plays a role in the development of neointimal hyperplasia, and we have previously shown that nitric oxide (NO) reduces neointimal hyperplasia in animal models and 26S proteasome activity in rat aortic smooth muscle cells (RASMC). Here, we show that PA28 increased 26S proteasome activity in RASMC, as measured by a fluorogenic assay, and the NO donor S-nitroso N-acetylpenicillamine significantly inhibits this activation. This effect was abrogated by the reducing agents dithiothreitol and HgCl2, suggesting that NO affects the activity of PA28 through S-nitrosylation. NO did not appear to affect PA28 levels or intracellular localization in RASMC in vitro. Three days following rat carotid artery balloon injury, levels of PA28a, b and c subunits were decreased compared to uninjured control arteries (n = 3/group) in vivo. The NO donor proline NONOate further decreased PA28a, b and c levels by 1.9-, 2.3- and 3.4-fold, respectively, compared to uninjured control arteries. Fourteen days following arterial injury, levels of PA28a, b and c subunits were increased throughout the arterial wall compared to uninjured control arteries, but were greatest for the a and b subunits. NO continued to decrease the levels of all three PA28 subunits throughout the arterial wall at this time point. Since the PA28 subunits are involved in the breakdown of peptides during inflammation, PA28 inhibition may be one mechanism by which NO inhibits neointimal hyperplasia. Ó 2012 Published by Elsevier Inc.

Introduction Oxidative stress and the immune response are increasingly being linked to the development of neointimal hyperplasia which occurs after interventions to treat cardiovascular disease, a leading cause of death and disability in the United States [1–4]. Oxidative stress is known to increase protein damage and accumulation, which has been implicated in a host of disorders, including Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis, heart disease, and atherosclerosis [5–8]. First described in 1983, the proteolytic complex known as the 20S proteasome contains the three catalytic activities (i.e., trypsin-, chymotrypsin-, and caspase-like) responsible for degrading the majority of proteins in eukaryotic cells, including those damaged by oxidation [9]. This ATP-independent complex degrades small ⇑ Corresponding author. Address: Division of Vascular Surgery, Northwestern University, 676 N. St. Clair St., Suite 650, Chicago, IL 60611, USA. Fax: +1 312 503 1222. E-mail address: [email protected] (M.R. Kibbe). 1 These authors contributed equally to this manuscript. 1089-8603/$ - see front matter Ó 2012 Published by Elsevier Inc. http://dx.doi.org/10.1016/j.niox.2012.04.006

proteins and can combine with one of two activators (19S or 11S) to form a 26S complex. Combination with the former leads to formation of the 26S proteasome that is responsible for degrading ubiquitinated proteins in an ATP-dependent manner [10]. Alternatively, expression of the immune protein interferon-c (IFNc) induces combination of the 20S proteasome with the 11S cap also known as proteasome activator 28 (PA28) [11]. There are three PA28 subunits – a, 31 kDa; b, 29 kDa; c, 32 kDa – which are homologous and evolutionarily conserved [11–15]. PA28a and b are mainly found in the cytoplasm, while PA28c is mainly found in the nucleus and on some components of the cytoskeleton [16,17]. These subunits are also distinctly distributed throughout the body, with PA28a/b found in immune tissues, and PA28c found primarily in the brain, where PA28a and b are noticeably absent [12]. While PA28a can readily form a homoheptamer in vitro, PA28b monomers act primarily to stabilize the PA28a/b complex [18]. Interestingly, PA28b monomers are potent inhibitors of 20S proteasome activity, but high concentrations of PA28b allow for oligomerization and proteasome activation [19]. In vivo, there are two versions of the PA28 cap, the a3b4 heteroheptamer and the c homoheptamer, both of which are responsible

N.D. Tsihlis et al. / Nitric Oxide 27 (2012) 50–58

for removing oxidatively damaged proteins from the cell, while not affecting intact proteins [20,21]. Indeed, previous work has shown that overexpression of PA28a protects against oxidative stress by clearing oxidized proteins out of cells [22]. In stark contrast, overexpression of PA28c is observed in colorectal and thyroid cancers [23,24]. The corollary to this is that PA28c knockout mice exhibit reduced body size, and embryonic cells lacking PA28c are prevented from transitioning from G1 to S-phase [25,26]. More clinically relevant, decreases in proteasome activity have also been shown to reverse cardiac hypertrophy [27]. Additionally, it is known that expression of IFNc peaks 7 days after arterial balloon injury [28]. This induction prevents apoptosis of vascular smooth muscle cells (VSMC), which contributes to the development of neointimal hyperplasia. Others have also shown, via transcriptome analysis of human atherectomy specimens, that IFNc plays an important role in neointima formation [29]. Indeed, inhibition of IFNc secretion by administration of a small molecule can prevent neointimal hyperplasia [28,30]. While inhibition of NO synthase has been shown to increase neointimal hyperplasia in a balloon injury model [31], our lab and others have shown that administration of exogenous nitric oxide (NO) inhibits neointima formation in a number of large and small animal arterial injury models [32–34]. We have also previously shown that NO inhibits 26S proteasome activity in rat aortic smooth muscle cells (RASMC) [35]. While it is known that arterial injury induces inflammation and production of IFNc, neither the role of PA28 nor the effect of NO on PA28 levels or activity in the arterial injury process has been described. Thus, the goals of this study were to evaluate the effect of arterial balloon injury, with and without administration of NO, on the levels of PA28a/b/ c in vivo, and to determine the effect of NO on PA28 levels and activity in RASMC in vitro. Since balloon injury increases IFNc, which increases PA28a/b expression and inhibits RASMC apoptosis to cause neointimal hyperplasia, and since administration of NO or inhibition of IFNc decreases neointimal hyperplasia, we hypothesize that NO prevents neointimal hyperplasia by reducing the level and/or activity of PA28. Materials and methods Cell culture Rat aortic smooth muscle cells (RASMC) were cultured from the abdominal aorta of Sprague Dawley rats (Harlan; Indianapolis, IN) using the collagenase method as described previously [36] and in accordance with protocols that conform to the principles outlined in the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication 85-23, 1996) and approved by the Northwestern University Animal Care and Use Committee. Cultured cells had the characteristic appearance of hills and valleys and were routinely more than 95% pure by smooth muscle cell a-actin staining. Cells were maintained in DMEM (low glucose)/Ham’s F12, 1:1 vol:vol (JRH; Lenexa, KS) supplemented with 10% fetal bovine serum (FBS, Invitrogen; Carlsbad, CA), 100 U/mL penicillin and 100 lg/mL streptomycin (Invitrogen) and maintained in an incubator at 37 °C, 95% air, 5% CO2. Whole cell lysate RASMC were collected after media was removed from dishes and cells were rinsed with phosphate buffered saline (PBS). Cells were scraped from the dish, collected, and centrifuged at 1200 rpm for 5 min at 4 °C. After the supernatant was discarded, the pellet was resuspended in lysis buffer (1 M NaCl, 0.1 M Tris [pH 7.5], 0.5 M EDTA, Triton X-100, 1 M MgCl2, 1 M sucrose, and

51

0.1 M ATP), incubated on ice for 15 min, and centrifuged at 20,800 rcf for 15 min at 4 °C. The supernatant was transferred to a microfuge tube and stored at 80 °C. Lysate concentration was measured by bicinchoninic acid (BCA) protein assay kit according to manufacturer’s instructions (Pierce; Rockford, IL). Proteasome activity assay and nitric oxide concentration determination Whole cell lysate was collected from RASMC. The specific conditions for each experiment can be found in the accompanying figure legends. Reaction buffer (5 mM MgCl2, 50 mM Tris [pH 7.8], 20 mM KCl, 5 mM MgOAc), ATP (5 mM), ± varying concentrations of recombinant PA28 (Boston Biochem; Cambridge, MA), and the NO-donor S-nitroso-N-acetylpenicillamine (SNAP), were added to 20 lg of lysate. This mixture was incubated at 37 °C for 10 min. To this reaction, one of three 26S proteasome-specific fluorogenic peptide substrates was added: Suc-LLVY-AMC (chymotrypsin-like), Bz-VGR-AMC (trypsin-like) or Z-LLE-AMC (caspase-like) (Boston Biochem). The final reaction volume was 200 lL. Proteasome activity was determined using a fluorogenic plate reader with excitation and emission wavelengths of 355 and 460 nm, respectively, at time points between 0 and 120 min. To determine the concentration of NO released by SNAP in our proteasome activity assay, an Apollo 4000 Free Radical Analyzer (World Precision Instruments; Sarasota, FL) and 100-lm probe (ISO NOPF 100) were employed. After polarizing the probe, a standard curve was created using SNAP, per the manufacturer’s instructions. To a vial containing 5 mL of proteasome activity assay reaction buffer, sufficient SNAP (dissolved in distilled, deionized water) was added to generate a final SNAP concentration of 10 or 50 lM. Each concentration was assessed twice. The amount of NO generated was determined by comparing the standard curve to the difference in picoamps (pA) from the baseline to the highest point of the peak. Western blot analysis RASMC whole cell suspension was collected following treatment ±SNAP for 24 h. Cells were rinsed with PBS, scraped, and centrifuged at 1200 rpm for 5 min. The supernatant was removed and the pellet was resuspended in buffer (20 mM Tris [pH7.4]) with 1 mM phenylmethylsulfonyl fluoride (PMSF; Sigma, St Louis, MO), 1 lg/mL leupeptin (Sigma) and 1 mM sodium orthovanadate (Na3VO4, Sigma). Protein concentration was measured via BCA assay. Samples were subjected to SDS–PAGE on 10–13% polyacrylamide gels, and transferred to nitrocellulose membranes (Schleich and Schuell; Keene, NH). Membranes were hybridized with antibodies to PA28a (1:1000), PA28b (1:1000), or PA28c (1:1000) (Cell Signaling Technologies; Danvers, MA), followed by incubation with goat antirabbit antibody (1:10,000, Pierce). Proteins were visualized using chemiluminescence reagents (SuperSignal, Pierce) and exposed to film according to the manufacturer’s instructions. Equal protein loading was verified via Western blotting for b-actin. Animal surgery All animal procedures performed were approved by the Northwestern University Animal Care and Use Committee, and according to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication 85-23, 1996). Male 11-week-old Sprague Dawley rats were anesthetized using inhaled isoflurane (0.5–3%). Atropine was administered subcutaneously (0.1 mg/kg) to decrease airway secretions. After a midline neck incision, the left common carotid artery (CCA), external carotid artery (ECA), and the internal carotid artery (ICA) were dissected and proximal and distal control obtained with microclips. A

52

N.D. Tsihlis et al. / Nitric Oxide 27 (2012) 50–58

transverse arteriotomy was created on the ECA. A 2 French Fogarty catheter (generously provided by Edwards Lifesciences; Irvine, CA) was inserted into the CCA through the ECA, and the CCA injured by inflating the balloon to 5 atmospheres of pressure for 5 min. Following injury, the catheter was removed, the ECA ligated, and flow restored to the CCA and ICA, as previously described [32,33,37]. After injury and restoration of flow, 20 mg of proline NONOate (PROLI/NO) powder was applied evenly to the periadventitial surface of the injured CCA of rats in the treatment group as previously described [32,33,37]. The neck incision was closed. Carotid arteries were harvested at 3 or 14 days post-injury. Control groups included uninjured contralateral arteries and injury alone (n = 3/group). Tissue processing for histology For sectioning and staining, carotid arteries were harvested 14 days after balloon injury as follows. Rats were anesthetized Ò with isoflurane and euthanized with 0.5 mL EUTHASOL (Virbac Animal Health; Ft. Worth, TX) and bilateral thoracotomies. A long midline incision was made to expose both carotid arteries. Vessels were perfused and fixed in situ using cold 1X PBS (250 mL) and 2% paraformaldehyde (500 mL), and then explanted en bloc from the aortic arch to the carotid bifurcation. Vessels were frozen in TissueTek O.C.T. compound (Sakura Finetek USA; Torrance, CA) and cut into 5-lm sections throughout the entire injured segment, as previously described [34]. Tissue processing for lysates For carotid lysates, vessels were harvested 3 days after balloon injury (n = 3/group). Rats were anesthetized, euthanized, and the vessels exposed as described above; however, the animals did not undergo perfusion and fixation. Each carotid artery was ligated at the aortic arch, and then explanted en bloc as quickly as possible. Vessels were identified, washed in cold 1X PBS, opened en face, and the injured section of the CCA was excised, snap-frozen, and stored in liquid nitrogen until lysed via mechanical means (ceramic mortar and pestle) and resuspended in lysis buffer (50 mM Hepes [pH 7.5], 150 mM NaCl, 10% glycerol (v/v), 10 mM sodium pyrophosphate [Na4P2O7], 1 mM EDTA, 1 mM ethylene glycol tetraacetic acid [EGTA], 1% Triton X-100, 10 lg/mL aprotinin, 10 lg/mL leupeptin, 50 mM sodium fluoride [NaF], 1 mM Na3VO4, and 1 mM PMSF). Protein concentration was determined via BCA assay, and samples were stored at 80 °C until Western blot analysis was performed as described above. Levels of PA28 subunits were quantified from Western blots using integrated density obtained from ImageJ and normalized to b-actin. Immunofluorescent staining RASMC plated in 24-well plates (4  104 cells/well) with glass coverslips (VWR International; West Chester, PA) were exposed to growth medium ± SNAP for 24 h. Cells were fixed with 2% paraformaldehyde (Sigma), followed by permeabilization with 0.3% Triton X-100 in PBS (Fisher Scientific; Fair Lawn, NJ). Cells were then blocked with goat serum (Sigma) in 0.5% bovine serum albumin (BSA, Vector Laboratories; Burlingame, CA). Primary antibody (PA28a, b, or c, 1:25) in BSA was applied for 1 h, followed by application of secondary antibody (goat anti-rabbit Alexa Fluor 555, 1:3000; Invitrogen) in BSA for 1 h. Nuclei were stained with DAPI for 5 min (600 nM; Invitrogen). Coverslips were affixed to slides with ProLong Anti Fade Reagent (Invitrogen), and digital images were acquired using an Eclipse 50i microscope (Nikon Instruments, Inc.; Melville, NY) and a 40X objective. Carotid artery sections of uninjured, injured, and NO-treated rats were fixed in 2% paraformaldehyde, followed by permeabilization

with 0.3% Triton X-100 in PBS for 10 min. The sections were then blocked with goat serum (1:20 in BSA) for 30 min, followed by incubation with the appropriate PA28 antibody (1:50), or S-nitrosocysteine (S-NO-Cys; Abcam, Cambridge, MA) antibody (1;1000), overnight at 4 °C. Negative controls were incubated without primary antibody, or, for S-NO-Cys, soaked in 5 mM dithiothreitol (DTT) for 5 min prior to incubation with primary antibody. After incubation with goat anti-rabbit AlexaFluor 555 or goat anti-mouse AlexaFluor 555 (1:100 and 1:1000 in PBS, respectively; Invitrogen) for 30 min, sections were stained with DAPI (1:500 in PBS) for 30 s, then coverslipped with ProLong Anti Fade Reagent (Invitrogen) and allowed to dry overnight. Digital images were acquired using an Eclipse 50i microscope (Nikon Instruments, Inc.) and a 40X objective, or a Zeiss Imager.A2 (Hallbergmoos, Germany) and a 20X objective. Statistical analysis Results are expressed as the mean ± standard error of the mean. Differences between groups were analyzed by one-way analysis of variance (ANOVA) with the Student–Newmann–Keuls post hoc test for all pairwise comparisons (SigmaStat, SPSS; Chicago, IL). Statistical significance was assumed for P < 0.05. Results PA28 increases 26S proteasome activity in RASMC Proteasome activity was assessed in RASMC with or without PA28 (1 nM). The enzymatic activities of all three catalytically active sites of the proteasome were increased in the presence of PA28 (Fig. 1A). The chymotrypsin-like and trypsin-like activities were increased by approximately 60% each with the addition of PA28 (P < 0.05 vs. control). However, the largest increase compared to baseline was seen with the caspase-like activity; PA28 more than doubled the caspase-like activity (P < 0.05 vs. control). Next, the effect of various PA28 concentrations (0.1–5 nM) on the 26S proteasome was measured at time points between 0 and 120 min. PA28 increased all three catalytic activities of the 26S proteasome from RASMC in a time- and concentration-dependent manner (Fig. 1B–D). NO completely inhibits PA28-induced proteasome activity To evaluate the effect of NO on PA28-induced proteasome activity, proteasome activity was assessed in RASMC in the presence of PA28 ± SNAP (500 lM). Interestingly, at this SNAP concentration, there was complete inhibition of PA28-induced proteasome activity for all three catalytic sites (P < 0.05 vs. PA28, Fig. 2A). With the addition of SNAP, the enzymatic activity was suppressed below baseline 26S proteasome activity levels. Next, the inhibitory effect of SNAP was tested at increasing concentrations (10–500 lM) at time points from 0 to 120 min. The peak amount of NO generated by 10 lM SNAP during the experiment was 2 lM, and the peak amount generated by 50 lM SNAP was 4 lM. In RASMC, SNAP inhibited the PA28-induced activity of all three catalytic sites in a time- and concentration-dependent manner (Fig. 2B–D). The greatest inhibition occurred with the highest concentration of SNAP (500 lM). DTT reverses the NO-mediated inhibition of PA28 To investigate the mechanism by which NO inhibits PA28-induced 26S proteasome activity, DTT was added to RASMC lysate prior to the addition of SNAP. DTT is a disulfide reducing agent

N.D. Tsihlis et al. / Nitric Oxide 27 (2012) 50–58

53

Fig. 1. PA28 increases all three catalytic activities of the 26S proteasome in rat aortic smooth muscle cells (RASMC). (A) Proteasome activity assay was performed in RASMC with PA28 (1 nM) at a time point of 120 min (⁄P < 0.05 vs. control). (B) Chymotrypsin-, (C) trypsin- and (D) caspase-like activities were assessed with various PA28 concentrations (0.1–5 nM) over 0–120 min. Data are representative of three separate experiments.

which keeps monothiols in the reduced state, and stabilizes enzymes which possess free sulfhydryl groups. DTT prevented the NO-mediated inhibition of PA28-induced proteasome activity for all three substrates (Fig. 3A; P < 0.05), and not only returned PA28-induced proteasome activity to baseline, but also more than doubled the caspase-like activity. Mercuric chloride inhibits the PA28 activator in RASMC DTT prevented the effect of NO on PA28-induced proteasome activity. Since the mechanism by which NO inhibits the 26S proteasome may be through reversible S-nitrosylation, mercuric chloride (HgCl2) was employed to more specifically elucidate this mechanism. HgCl2 binds to sulfide groups in proteins and disrupts the proper functioning of sulfhydryl enzymes. For all three substrate groups, when HgCl2 (50 lM) was added to RASMC lysate, it inhibited PA28-augmented 26S proteasome activity (Fig. 3B; P < 0.05 vs. control). The addition of DTT (5 mM) reversed the effects of HgCl2. These data suggest that the activity of the 26S proteasome is susceptible to regulation through modification of active thiol groups. This provides further support for the hypothesis that NO inhibits the PA28-induced effect on the 26S proteasome through reversible S-nitrosylation.

NO does not affect PA28 subunit protein levels or localization in RASMC In order to examine whether NO affected the protein levels of the PA28 subunits, Western blot analysis was conducted. RASMC were exposed to medium containing increasing concentrations of SNAP (125–1000 lM) or the proteasome inhibitor MG132 (1 lM) for 24 h, and then collected. Neither SNAP nor MG132 altered the levels of the PA28a, b, and c subunits in RASMC, as determined by Western blotting (Fig. 4). To examine whether NO affected subcellular localization of the PA28 subunits, RASMC plated on glass coverslips were exposed to SNAP (1000 lM) for 24 h and stained for PA28 subunits by immunofluorescence. As seen in Fig. 5, SNAP had no effect on subcellular localization or overall levels of any of the PA28 subunits in RASMC. NO decreases PA28 subunit levels in the carotid artery early following arterial injury To ascertain the effects of NO on PA28 subunit levels early following arterial injury, balloon-injured carotid arteries harvested after 3 days were homogenized and subjected to Western blot analysis. Surprisingly, balloon injury actually decreased levels of

54

N.D. Tsihlis et al. / Nitric Oxide 27 (2012) 50–58

Fig. 2. Nitric oxide inhibits PA28-induced proteasome activity in RASMC. (A) Proteasome activity assay with PA28 (1 nM) was performed in RASMC with SNAP (500 lM) at a time point of 120 min. PA28-induced (B) chymotrypsin-, (C) trypsin-, and (D) caspase-like activities were assessed with increasing concentrations of SNAP (10–500 lM) over 0–120 min. Data points were read at 120 min and are representative of three separate experiments.

Fig. 3. NO may prevent PA28-induced proteasome activity by S-nitrosolyation. (A) The inhibitory effects of NO on PA28-induced proteasome activity are prevented by pretreatment with the reducing agent DTT (5 mM) in RASMC at a time point of 120 min. (B) PA28-induced proteasome activity is inhibited by mercuric chloride (HgCl2, 50 lM), a known sulfhydryl reacting agent, in RASMC at 120 min, suggesting that the activity of the 26S proteasome is modified by a cysteine residue. PA28 = 1 nM. Data are representative of three separate experiments.

N.D. Tsihlis et al. / Nitric Oxide 27 (2012) 50–58

55

Fig. 4. NO does not affect PA28 subunit protein levels RASMC. Western blot analysis was conducted on RASMC collected following exposure to SNAP (0–1000 lM) or the proteasome inhibitor MG132 (1 lM) for 24 h. Beta smooth muscle cell actin served as a loading control. Data are representative of three separate experiments.

all PA28 subunits (Fig. 6). NO exposure further decreased the levels of the PA28a, b, and c subunits by 1.9-, 2.3- and 3.4-fold compared to control arteries, respectively, with the most profound effect observed in PA28c levels.

NO decreases injury-induced PA28 subunit levels in the carotid artery late following arterial injury To investigate the effects of NO on the levels of PA28 subunits at a later time point in vivo, balloon-injured rat carotid arteries harvested at 14 days were subjected to immunofluorescent staining. While baseline levels of all three subunits were consistently low, balloon injury qualitatively increased levels of all three subunits at this time point, particularly in the neointima and media (Fig. 7). PA28c staining showed no injury-induced increase in the adventitia, unlike PA28a and b. Injured arteries treated with PROLI/NO qualitatively showed marked decreases in PA28b and c levels in all three arterial layers, while PA28a levels decreased only in the neointima and media. Of note, similar to the data at 3 days, NO had the greatest effect on PA28c levels in carotid arteries.

Fig. 6. NO decreases PA28 subunit levels in the carotid artery early following arterial injury. Balloon-injured carotid arteries harvested after 3 days (n = 3/group) were homogenized and subjected to Western blot analysis (top panel). Balloon injury decreased levels of all PA28 subunits. Treatment with NO (PROLI/NO) further decreased PA28 subunit levels, with the most profound effect observed in PA28c levels. (Bottom panel) Quantitation of the PA28 subunit Western blots in the top panel was performed using ImageJ and normalized to b-actin.

NO, but not injury alone, increases levels of S-nitrosocysteine in balloon-injured carotid arteries Lastly, to ascertain whether balloon injury affected S-nitrosylated proteins, balloon-injured rat carotid arteries harvested at 14 days were subjected to immunofluorescent staining for S-nitrosocysteine (S-NO-Cys). As seen in Fig. 8, uninjured arteries and injured arteries not treated with NO showed no S-NO-Cys staining, but injured arteries treated with NO showed marked staining in the adventitia at the interface with the internal elastic lamina.

Fig. 5. NO does not affect PA28 subunit intracellular localization in RASMC. Immunofluorescent staining performed on RASMC plated on glass coverslips and treated with SNAP (1000 lM) showed that NO had no significant effect on PA28 subunit intracellular localization (red staining). Nuclei are stained blue with DAPI. Data are representative of two experiments. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

56

N.D. Tsihlis et al. / Nitric Oxide 27 (2012) 50–58

Fig. 7. NO decreases PA28 subunit levels in the carotid artery later following arterial injury. Carotid arteries harvested 14 days after balloon injury (n = 3/group) were sectioned and subjected to immunofluorescent staining for PA28 subunits (red). Uninjured vessels show little baseline staining, but injury caused a noticeable increase in PA28 subunit levels, especially PA28a and b. Administration of PROLI/NO (20 mg) caused a reversion to baseline levels throughout all three arterial layers for PA28b and c, while PA28a remained elevated in the adventitia, but was reduced in the media and neointima. Green staining shows the internal elastic lamina and nuclei are stained blue with DAPI. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 8. NO, but not injury alone, increases levels of S-nitrosocysteine in balloon-injured carotid arteries. Carotid arteries harvested 14 days after balloon injury (n = 3/group) were sectioned and subjected to immunofluorescent staining for S-nitrosocysteine (S-NO-Cys, red). Uninjured vessels show no baseline staining, and injury caused no noticeable increase in S-NO-Cys levels. Administration of PROLI/NO (20 mg) caused an increase in S-NO-Cys staining in the adventitia, especially at the interface with the internal elastic lamina. Green staining shows the internal elastic lamina and nuclei are stained blue with DAPI. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Discussion We show here that PA28 stimulates the activity of the 26S proteasome isolated from RASMC in a time- and concentration-dependent manner (Figs. 1 and 2). We also show, for the first time, that NO abrogates this stimulatory effect of PA28 in a time- and concentration-dependent manner (Figs. 1 and 2). The concentration of NO

generated by SNAP at 10 lM peaked at 2 lM. While this is higher than the reported values of 400 nM NO in the arteriolar wall [38,39], it is only slightly so, indicating that lower, near-physiological, concentrations of NO are beneficial. This effect of NO appears to be mediated by S-nitrosylation of active-site cysteines, as reducing agents reverse the inhibitory effects of NO on PA28-stimulated 26S proteasome activity (Fig. 3). Also, cross sections of NO-treated

N.D. Tsihlis et al. / Nitric Oxide 27 (2012) 50–58

carotid arteries show a marked increase in S-NO-Cys levels (Fig. 8). Western blotting of RASMC treated with NO indicated no effect of NO on PA28 subunit levels in vitro (Fig. 4). Immunofluorescent staining of RASMC treated with NO showed no effect of NO on PA28 subunit intracellular localization (Fig. 5). In vivo, at an early time point before neointimal hyperplasia has had time to develop, we observed a decrease in PA28 subunits with injury and a further decrease with NO treatment (Fig. 6). At a later time point, when neointimal hyperplasia has fully developed, we observed qualitative increases in PA28a, b and c staining throughout the carotid artery wall with injury, but to a lesser extent with PA28c (Fig. 7). Consistent with the 3-day time point, NO decreased the levels of all three PA28 subunits 14 days following arterial injury (Fig. 7). However, while NO decreased levels of PA28b and c subunits throughout all three layers of the arterial wall, NO decreased the PA28a subunit mostly in the neointima and media (Fig. 7). While much is known about the effects of NO on neointimal hyperplasia, and some is known about the effect of NO on the proteasome, little is known about the effect of vascular injury, with or without NO, on the PA28 subunits. There is evidence that local inhibition of proteasome activity via MG132 causes a decrease in neointimal hyperplasia and inflammation [40]. It has also recently been shown that PA28c breaks down the cyclin-dependent kinase inhibitor p21 without requiring ATP or ubiquitin [41,42]. It is well established that NO increases levels of p21 in association with inhibition of neointimal hyperplasia [43]. Thus, it stands to reason that NO-mediated inhibition of PA28, and, in particular, PA28c is responsible for the NO-mediated increases in p21 levels and associated inhibition of neointimal hyperplasia. Along these lines, we have shown that PA28c staining in balloon-injured carotid arteries treated with NO is markedly decreased (Fig. 7). Interestingly, though we saw little nuclear staining of PA28c in either RASMC or carotid artery sections, we did observe staining in the cytoplasm (Figs. 5 and 7). This is not unexpected, as PA28c is known to localize to parts of the cytoskeleton [12]. It is also known that PA28c is not as strongly induced by IFNc as PA28a and b are; indeed, some groups have reported a complete loss of PA28c after IFNc treatment [44]. What is interesting is that such low levels of PA28c can have such a dramatic effect on cellular proliferation. We have also shown here that injury causes a significant increase in PA28a and b levels in balloon-injured carotid artery sections, more than PA28c (Fig. 7). Since injury causes inflammation, which causes IFNc expression, and IFNc is known to increase PA28a/b expression, this result is consistent with published literature [11]. At an early time point, our in vivo data show that injury decreases levels of the PA28 subunits (Fig. 6), but by 14 days, injury increases levels of the PA28 subunits (Fig. 7). This is consistent with the development of neointimal hyperplasia. At the early time point of 3 days following injury, we and others have shown that neointimal hyperplasia has not had enough time to form [45]. At 3 days, proliferation of VSMC, adventitial fibroblasts, and macrophages is beginning [45–47]. By 14 days, neointimal hyperplasia has fully developed [45]. Thus, we believe our data are consistent with the arterial injury response. The fact that we saw no changes in levels of the PA28 subunits in RASMC in vitro after 24 h of exposure to NO simply means that: (1) NO is not effecting a change in PA28 subunit levels in RASMC; (2) RASMC in culture represents an artificial and isolated system, and NO requires the presence of other cells and/or molecules to initiate a change in PA28 levels; and/or, (3) longer exposure to the NO donor is required to effect a change in the levels of the PA28 subunits. We believe it is a combination of all three of these factors. The arterial wall has many cell types that contribute to the formation of neointimal hyperplasia, including inflammatory cells, adventitial fibroblasts, resident progenitor cells, and circulating stem cells [46–52]. Thus, NO may be

57

affecting PA28 subunit levels in any one or more of these other cell types. Of note, we believe the inhibition of PA28 activity in vitro is observed at short time points given the presumed mechanism of action, namely through S-nitrosylation. Indeed, we show that NO increases levels of S-NO-Cys in carotid arteries (Fig. 8), which correlates well with data previously published by our lab showing that NO S-nitrosylates the proteasome [35]. However, this is vastly different from how NO may be effecting a change in protein levels, which could occur through regulation of transcription, translation, or degradation, most of which require more time. This work is not without limitations. First, we did not stain sections for IFNc, nor did we assess carotid or RASMC lysates for IFNc via Western blot. Since it is well established that injury causes inflammation, that inflammation causes IFNc induction, and that NO reduces injury and inflammation, we did not believe staining for IFNc was necessary. We also did not assess PA28 subunit activity in vivo, as the balloon-injured area of the carotid artery is relatively small, and obtaining enough protein for activity assays would require sacrificing many animals. Additionally, due to the heterogeneous nature of the cells in the vessel wall, the results of an in vivo activity assay would be difficult to interpret. In conclusion, we report that NO decreases PA28 subunit levels at both 3 and 14 days after balloon injury (Figs. 6 and 7), and decreases PA28-stimulated 26S proteasome activity in RASMC in vitro (Figs. 1 and 2). The inhibition of PA28-induced activation by NO was reversible via reducing agents (Fig. 3), indicating that S-nitrosylation of active-site cysteines may be involved. As we have shown that NO decreases neointimal hyperplasia and PA28 subunit levels and activity, inhibition of PA28 may represent one mechanism by which NO inhibits neointimal hyperplasia, and may prove to be an effective therapy for preventing restenosis and improving patient outcomes following vascular interventions. Acknowledgments The authors would like to express their thanks to the Northwestern University Institute for BioNanotechnology in Medicine, the Northwestern University Feinberg Cardiovascular Research Institute, to Lynnette Dangerfield for her administrative support, and to Edwards Lifesciences for providing the Fogarty balloon catheters. Special thanks to Dr. Edward Moreira for his assistance with the free radical analyzer experiments and insightful discussions. This work was supported in part by funding from the National Institutes of Health (1K08HL0842-03 to Kibbe), the Society for Vascular Surgery Foundation (Mentored Clinical Scientist Development Award to Kibbe), the American Heart Association (0725766Z and 09POST2230028 to NDT), the Department of Veterans Affairs (VA Merit Review Grant to Kibbe), and by the generosity of Mrs. Hilda Rosenbloom and Mrs. Eleanor Baldwin. References [1] V.L. Roger, A.S. Go, D.M. Lloyd-Jones, R.J. Adams, J.D. Berry, T.M. Brown, M.R. Carnethon, S. Dai, S.G. de, E.S. Ford, C.S. Fox, H.J. Fullerton, C. Gillespie, K.J. Greenlund, S.M. Hailpern, J.A. Heit, P.M. Ho, V.J. Howard, B.M. Kissela, S.J. Kittner, D.T. Lackland, J.H. Lichtman, L.D. Lisabeth, D.M. Makuc, G.M. Marcus, A. Marelli, D.B. Matchar, M.M McDermott, J.B. Meigs, C.S. Moy, D. Mozaffarian, M.E. Mussolino, G. Nichol, N.P. Paynter, W.D. Rosamond, P.D. Sorlie, R.S. Stafford, T.N. Turan, M.B. Turner, N.D. Wong, J. Wylie-Rosett, Heart disease and stroke statistics – 2011 update: a report from the American heart association, Circulation 123 (2011) e18–e209. [2] E. Afergan, D.M. Ben, H. Epstein, N. Koroukhov, D. Gilhar, K. Rohekar, H.D. Danenberg, G. Golomb, Liposomal simvastatin attenuates neointimal hyperplasia in rats, AAPS J. 12 (2010) 181–187. [3] K. Toutouzas, A. Colombo, C. Stefanadis, Inflammation and restenosis after percutaneous coronary interventions, Eur. Heart J. 25 (2004) 1679–1687. [4] C. Rogers, E.R. Edelman, D.I. Simon, A mAb to the beta2-leukocyte integrin Mac-1 (CD11b/CD18) reduces intimal thickening after angioplasty or stent implantation in rabbits, Proc. Natl. Acad. Sci. USA 95 (1998) 10134–10139.

58

N.D. Tsihlis et al. / Nitric Oxide 27 (2012) 50–58

[5] B.M. Riederer, G. Leuba, A. Vernay, I.M. Riederer, The role of the ubiquitin proteasome system in Alzheimer’s disease, Exp. Biol. Med. (Maywood.) 236 (2011) 268–276. [6] Q. Huang, M.E. Figueiredo-Pereira, Ubiquitin/proteasome pathway impairment in neurodegeneration: therapeutic implications, Apoptosis 15 (2010) 1292– 1311. [7] Y.F. Li, X. Wang, The role of the proteasome in heart disease, Biochim. Biophys. Acta 2011 (1809) 141–149. [8] P. Libby, Inflammation in atherosclerosis, Nature 420 (2002) 868–874. [9] N. Orlowski, S. Wilk, A multicatalytic protease complex from pituitary that forms enkephalin and enkephalin containing peptides, Biochem. Biophys. Res. Commun. 101 (1981) 814–822. [10] A. Hershko, A. Ciechanover, The ubiquitin system, Annu. Rev. Biochem. 67 (1998) 425–479. [11] C. Realini, W. Dubiel, G. Pratt, K. Ferrell, M. Rechsteiner, Molecular cloning and expression of a gamma-interferon-inducible activator of the multicatalytic protease, J. Biol. Chem. 269 (1994) 20727–20732. [12] M. Rechsteiner, C.P. Hill, Mobilizing the proteolytic machine: cell biological roles of proteasome activators and inhibitors, Trends Cell Biol. 15 (2005) 27–33. [13] S. Wilk, W.E. Chen, R.P. Magnusson, Properties of the nuclear proteasome activator PA28gamma (REGgamma), Arch. Biochem. Biophys. 383 (2000) 265– 271. [14] C. Realini, C.C. Jensen, Z. Zhang, S.C. Johnston, J.R. Knowlton, C.P. Hill, M. Rechsteiner, Characterization of recombinant REGalpha, REGbeta, and REGgamma proteasome activators, J. Biol. Chem. 272 (1997) 25483–25492. [15] J.D. Mott, B.C. Pramanik, C.R. Moomaw, S.J. Afendis, G.N. DeMartino, C.A. Slaughter, PA28, an activator of the 20 S proteasome, is composed of two nonidentical but homologous subunits, J. Biol. Chem. 269 (1994) 31466– 31471. [16] P. Brooks, G. Fuertes, R.Z. Murray, S. Bose, E. Knecht, M.C. Rechsteiner, K.B. Hendil, K. Tanaka, J. Dyson, J. Rivett, Subcellular localization of proteasomes and their regulatory complexes in mammalian cells, Biochem. J. 346 (Pt 1) (2000) 155–161. [17] C. Wojcik, K. Tanaka, N. Paweletz, U. Naab, S. Wilk, Proteasome activator (PA28) subunits, alpha, beta and gamma (Ki antigen) in NT2 neuronal precursor cells and HeLa S3 cells, Eur. J. Cell Biol. 77 (1998) 151–160. [18] S.C. Johnston, F.G. Whitby, C. Realini, M. Rechsteiner, C.P. Hill, The proteasome 11S regulator subunit REG alpha (PA28 alpha) is a heptamer, Prot. Sci. 6 (1997) 2469–2473. [19] S. Wilk, W.E. Chen, R.P. Magnusson, Properties of the beta subunit of the proteasome activator PA28 (11S REG), Arch. Biochem. Biophys. 384 (2000) 174–180. [20] Z. Zhang, A. Krutchinsky, S. Endicott, C. Realini, M. Rechsteiner, K.G. Standing, Proteasome activator 11S REG or PA28: recombinant REG alpha/REG beta hetero-oligomers are heptamers, Biochemistry 38 (1999) 5651–5658. [21] C.P. Ma, C.A. Slaughter, G.N. DeMartino, Identification, purification, and characterization of a protein activator (PA28) of the 20t S proteasome (macropain), J. Biol. Chem. 267 (1992) 10515–10523. [22] J. Li, S.R. Powell, X. Wang, Enhancement of proteasome function by PA28α overexpression protects against oxidative stress, FASEB J. 25 (2011) 883–893. [23] T. Okamura, S. Taniguchi, T. Ohkura, A. Yoshida, H. Shimizu, M. Sakai, H. Maeta, H. Fukui, Y. Ueta, I. Hisatome, C. Shigemasa, Abnormally high expression of proteasome activator-gamma in thyroid neoplasm, J. Clin. Endocrinol. Metab 88 (2003) 1374–1383. [24] M. Roessler, W. Rollinger, L. Mantovani-Endl, M.L. Hagmann, S. Palme, P. Berndt, A.M. Engel, M. Pfeffer, J. Karl, H. Bodenmuller, J. Ruschoff, T. Henkel, G. Rohr, S. Rossol, W. Rosch, H. Langen, W. Zolg, M. Tacke, Identification of PSME3 as a novel serum tumor marker for colorectal cancer by combining twodimensional polyacrylamide gel electrophoresis with a strictly mass spectrometry-based approach for data analysis, Mol. Cell Proteomic. 5 (2006) 2092–2101. [25] S. Murata, H. Kawahara, S. Tohma, K. Yamamoto, M. Kasahara, Y. Nabeshima, K. Tanaka, T. Chiba, Growth retardation in mice lacking the proteasome activator PA28gamma, J. Biol. Chem. 274 (1999) 38211–38215. [26] L.F. Barton, H.A. Runnels, T.D. Schell, Y. Cho, R. Gibbons, S.S. Tevethia, G.S. Deepe Jr., J.J. Monaco, Immune defects in 28-kDa proteasome activator gamma-deficient mice, J. Immunol. 172 (2004) 3948–3954. [27] N. Hedhli, P. Lizano, C. Hong, L.F. Fritzky, S.K. Dhar, H. Liu, Y. Tian, S. Gao, K. Madura, S.F. Vatner, C. Depre, Proteasome inhibition decreases cardiac remodeling after initiation of pressure overload, Am. J. Physiol Heart Circ. Physiol 295 (2008) H1385–H1393. [28] K. Kusaba, H. Kai, M. Koga, N. Takayama, A. Ikeda, H. Yasukawa, Y. Seki, K. Egashira, T. Imaizumi, Inhibition of intrinsic interferon-gamma function prevents neointima formation after balloon injury, Hypertension 49 (2007) 909–915. [29] D. Zohlnhofer, T. Richter, F. Neumann, T. Nuhrenberg, R. Wessely, R. Brandl, A. Murr, C.A. Klein, P.A. Baeuerle, Transcriptome analysis reveals a role of

[30]

[31]

[32]

[33]

[34]

[35]

[36]

[37]

[38] [39]

[40]

[41]

[42]

[43]

[44]

[45]

[46]

[47]

[48] [49]

[50]

[51]

[52]

interferon-gamma in human neointima formation, Mol. Cell 7 (2001) 1059– 1069. J. George, J. Sack, I. Barshack, P. Keren, I. Goldberg, R. Haklai, G. Elad-Sfadia, Y. Kloog, G. Keren, Inhibition of intimal thickening in the rat carotid artery injury model by a nontoxic Ras inhibitor, Arterioscler. Thromb. Vasc. Biol. 24 (2004) 363–368. J.W. Fischer, S. Hawkins, A.W. Clowes, Pharmacologic inhibition of nitric oxide synthases and cyclooxygenases enhances intimal hyperplasia in ballooninjured rat carotid arteries, J. Vasc. Surg. 40 (2004) 115–122. M.R. Kapadia, L.W. Chow, N.D. Tsihlis, S.S. Ahanchi, J.W. Eng, J. Murar, J. Martinez, D.A. Popowich, Q. Jiang, J.A. Hrabie, J.E. Saavedra, L.K. Keefer, J.F. Hulvat, S.I. Stupp, M.R. Kibbe, Nitric oxide and nanotechnology: a novel approach to inhibit neointimal hyperplasia, J. Vasc. Surg. 47 (2008) 173– 182. C.G. Pearce, S.F. Najjar, M.R. Kapadia, J. Murar, J. Eng, B. Lyle, O.O. Aalami, Q. Jiang, J.A. Hrabie, J.E. Saavedra, L.K. Keefer, M.R. Kibbe, Beneficial effect of a short-acting NO donor for the prevention of neointimal hyperplasia, Free Radic. Biol. Med. 44 (2008) 73–81. L.L. Shears, M.R. Kibbe, A.D. Murdock, T.R. Billiar, A. Lizonova, I. Kovesdi, S.C. Watkins, E. Tzeng, Efficient inhibition of intimal hyperplasia by adenovirusmediated inducible nitric oxide synthase gene transfer to rats and pigs in vivo, J. Am. Coll. Surg. 187 (1998) 295–306. M.R. Kapadia, J.W. Eng, Q. Jiang, D.A. Stoyanovsky, M.R. Kibbe, Nitric oxide regulates the 26S proteasome in vascular smooth muscle cells, Nitric Oxide 20 (2009) 279–288. A.N. Orekhov, E.R. Andreeva, A.V. Krushinsky, V.N. Smirnov, Primary cultures of enzyme-isolated cells from normal and atherosclerotic human aorta, Med. Biol. 62 (1984) 255–259. S.S. Ahanchi, V.N. Varu, N.D. Tsihlis, J. Martinez, C.G. Pearce, M.R. Kapadia, Q. Jiang, J.E. Saavedra, L.K. Keefer, J.A. Hrabie, M.R. Kibbe, Heightened efficacy of nitric oxide-based therapies in type II diabetes mellitus and metabolic syndrome, Am. J. Physiol Heart Circ. Physiol 295 (2008) H2388–H2398. J.M. Lash, G.P. Nase, H.G. Bohlen, Acute hyperglycemia depresses arteriolar NO formation in skeletal muscle, Am. J. Physiol 277 (1999) H1513–H1520. H.G. Bohlen, G.P. Nase, Dependence of intestinal arteriolar regulation on flowmediated nitric oxide formation, Am. J. Physiol Heart Circ. Physiol 279 (2000) H2249–H2258. S. Meiners, M. Laule, W. Rother, C. Guenther, I. Prauka, P. Muschick, G. Baumann, P.M. Kloetzel, K. Stangl, Ubiquitin-proteasome pathway as a new target for the prevention of restenosis, Circulation 105 (2002) 483–489. X. Li, L. Amazit, W. Long, D.M. Lonard, J.J. Monaco, B.W. O’Malley, Ubiquitinand ATP-independent proteolytic turnover of p21 by the REGgammaproteasome pathway, Mol. Cell 26 (2007) 831–842. X. Chen, L.F. Barton, Y. Chi, B.E. Clurman, J.M. Roberts, Ubiquitin-independent degradation of cell-cycle inhibitors by the REGgamma proteasome, Mol. Cell 26 (2007) 843–852. M.R. Kibbe, J. Li, S. Nie, S.C. Watkins, A. Lizonova, I. Kovesdi, R.L. Simmons, T.R. Billiar, E. Tzeng, Inducible nitric oxide synthase (iNOS) expression upregulates p21 and inhibits vascular smooth muscle cell proliferation through p42/44 mitogen-activated protein kinase activation and independent of p53 and cyclic guanosine monophosphate, J. Vasc. Surg. 31 (2000) 1214–1228. N. Tanahashi, K. Yokota, J.Y. Ahn, C.H. Chung, T. Fujiwara, E. Takahashi, G.N. DeMartino, C.A. Slaughter, T. Toyonaga, K. Yamamura, N. Shimbara, K. Tanaka, Molecular properties of the proteasome activator PA28 family proteins and gamma-interferon regulation, Genes Cells 2 (1997) 195–211. A.W. Clowes, M.A. Reidy, M.M. Clowes, Kinetics of cellular proliferation after arterial injury 1. smooth-muscle growth in the absence of endothelium, Lab. Invest. 49 (1983) 327–333. E. Okamoto, T. Couse, L.H. De, J. Vinten-Johansen, R.B. Goodman, N.A. Scott, J.N. Wilcox, Perivascular inflammation after balloon angioplasty of porcine coronary arteries, Circulation 104 (2001) 2228–2235. J.N. Wilcox, R. Waksman, S.B. King, N.A. Scott, The role of the adventitia in the arterial response to angioplasty: the effect of intravascular radiation, Int. J. Radiat. Oncol. Biol. Phys. 36 (1996) 789–796. N.M. Tigerstedt, H. Savolainen-Peltonen, S. Lehti, P. Hayry, Vascular cell kinetics in response to intimal injury ex vivo, J. Vasc. Res. 47 (2010) 35–44. D. Kong, L.G. Melo, M. Gnecchi, L. Zhang, G. Mostoslavsky, C.C. Liew, R.E. Pratt, V.J. Dzau, Cytokine-induced mobilization of circulating endothelial progenitor cells enhances repair of injured arteries, Circulation 110 (2004) 2039–2046. Y. Shi, J.E. O’Brien, A. Fard, J.D. Mannion, D. Wang, A. Zalewski, Adventitial myofibroblasts contribute to neointimal formation in injured porcine coronary arteries, Circulation 94 (1996) 1655–1664. A.C. van der Wal, P.K. Das, v.d.B. Bentz, C.M. van der Loos, A.E. Becker, Atherosclerotic lesions in humans. In situ immunophenotypic analysis suggesting an immune mediated response, Lab. Invest. 61 (1989) 166–170. L. Jonasson, J. Holm, O. Skalli, G. Bondjers, G.K. Hansson, Regional accumulations of T cells, macrophages, and smooth muscle cells in the human atherosclerotic plaque, Arteriosclerosis 6 (1986) 131–138.