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Nitric oxide differentially affects ERK and Akt in type 1 and type 2 diabetic rats
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Available online at www.sciencedirect.com
journal homepage: www.JournalofSurgicalResearch.com
Nitric oxide differentially affects ERK and Akt in type 1 and type 2 diabetic rats Monica P. Rodriguez, MD,a,1 Zachary M. Emond, MD,a,1 Vinit N. Varu, MD,a Sadaf S. Ahanchi, MD,a Janet Martinez, AAS,a,b and Melina R. Kibbe, MDa,b,* a
Division of Vascular Surgery and Institute for BioNanotechnology in Medicine, Northwestern University Feinberg School of Medicine, Chicago, Illinois b Jesse Brown VA Medical Center, Chicago, Illinois
article info
abstract
Article history:
Background: We have shown that nitric oxide (NO) is more effective at inhibiting neointimal
Received 20 February 2013
hyperplasia in type 2 diabetic rats than in nondiabetic rats, but is not effective in type 1 diabetic
Received in revised form
rats. Insulin signaling is mediated by the ERK and Akt pathways, and thus we hypothesized
20 February 2013
that NO differentially affects ERK and Akt activity in type 1 versus type 2 diabetic rats.
Accepted 27 February 2013
Materials and methods: To investigate this hypothesis, we induced type 2 diabetes in Zucker
Available online 19 March 2013
diabetic fatty (ZDF) rats by feeding them Purina 5008 chow. To induce type 1 diabetes, lean Zucker (LZ) rats were injected with streptozotocin (STZ; 60 mg/kg). The carotid artery injury
Keywords:
model was performed. Groups included injury and injury þ PROLI/NO (20 mg/kg) (n ¼ 6/group).
Nitric oxide
Results: Three days following injury, all animal models exhibited an increase in pERK levels.
Akt
Whereas NO reduced pERK levels in LZ and STZ rats, NO had no effect on pERK levels in
ERK
ZDF rats. Following a similar pattern, NO reduced pAkt levels in LZ and STZ rats but
Neointimal hyperplasia
increased pAkt levels in ZDF rats. Fourteen days following injury, NO increased total pERK
Type 1 diabetes
levels throughout the arterial wall in both the STZ and ZDF rats. These changes were
Type 2 diabetes
greatest in the adventitia. Interestingly, whereas NO decreased total pAkt levels in LZ and
Metabolic environment
STZ rats, NO increased pAkt levels in ZDF rats. Evaluation of the pERK:pAkt ratio revealed that NO increased this ratio in LZ and STZ rats but decreased the ratio in ZDF rats. Conclusions: We report that NO differentially affects the expression of pERK and pAkt in type 1 versus type 2 diabetic rats. Given that NO is more effective at inhibiting neointimal hyperplasia in type 2 diabetic animals, the pERK:pAkt ratio may be the best surrogate to predict efficacy. Published by Elsevier Inc.
1.
Introduction
Patients with diabetes have a 4-fold greater risk of developing peripheral arterial disease, coronary arterial disease, and cerebrovascular disease [1e3]. This population also experiences higher failure rates following revascularization procedures due to aggressive rates of restenosis from neointimal
hyperplasia [4]. Nitric oxide (NO) is a naturally occurring molecule that has been shown to effectively inhibit neointimal hyperplasia in animal models of arterial injury [5e9]. We previously reported that NO was more effective at inhibiting neointimal hyperplasia after arterial injury in a rodent model of uncontrolled type 2 diabetes and less effective in a rodent model of uncontrolled type 1 diabetes [5,9]. These
* Corresponding author. Division of Vascular Surgery, Northwestern University Feinberg School of Medicine, 676 N. St. Clair Street, #650, Chicago, IL 60611. Tel.: þ1 312 503 6701; fax: þ1 312 503 1222. E-mail address: [email protected] (M.R. Kibbe). 1 These authors contributed equally to this manuscript. Drs. Rodriguez and Emond share first author status. 0022-4804/$ e see front matter Published by Elsevier Inc. http://dx.doi.org/10.1016/j.jss.2013.02.055
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data suggest a role for insulin in mediating the effects of NO, given that a main difference between these animal models is hyperinsulinemia versus hypoinsulinemia. The insulin signaling pathway has been well characterized. The two predominant signaling pathways downstream from the insulin receptor are the mitogen-activated protein kinase (MAPK) and PI3K/Akt pathways, which are known as the mitogenic and metabolic pathways, respectively. Hyperinsulinemia causes derangement of these pathways by stimulating the MAPK pathway, which leads to aggressive proliferation and neointimal hyperplasia, and by inhibiting the PI3 kinase/Akt pathway, which leads to lower NO production [10e14]. The effect of NO on the MAP kinase and AKT pathway in type 1 and type 2 diabetic environments is unknown. Thus, we hypothesized that NO differentially affects ERK and Akt in type 1 versus type 2 diabetic environments. Our goal was to evaluate the role of ERK and Akt in mediating the differential effects of NO in type 1 and type 2 diabetic rat models.
2.
Materials and methods
2.1.
Type 1 diabetic animal model
Eleven-week-old male lean Zucker (LZ) rats were obtained from Charles River Laboratories (Wilmington, MA). Type 1 diabetes was induced in LZ rats with a single intraperitoneal injection of streptozotocin (STZ, 60 mg/kg). Daily serum glucose levels were assessed with a glucometer via tail vein puncture and animals with glucose concentrations of 300 mg/dL or above were considered diabetic and included in the study. The nonfasting daily blood glucose concentration was recorded.
2.2.
Type 2 diabetic animal model
Zucker diabetic fatty (ZDF) rats were obtained from Charles River Laboratories. The ZDF strain has a homozygous leptin receptor mutation predisposing the rats to type 2 diabetes. Inbred ZDF male rats were fed the Purina 5008 diet, which is manufactured high in carbohydrates and fats. This induces a metabolic state of hyperinsulinemia, hyperglycemia, hypercholesterolemia, and hypertriglyceridemia, mimicking a type 2 diabetic state. Rats were kept in their respective group assignments for 3 wk prior to and 2 wk following surgery.
2.3.
Animal surgery
All animal procedures were performed in accordance with the Guide for 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. Rats were anesthetized with inhaled isoflurane (0.5%e3%). Atropine was administered subcutaneously (0.1 mg/kg) to decrease airway secretions. Weight was documented and blood glucose was measured daily following administration of STZ. The neck was shaved and prepped with betadine and alcohol (70%). Following a midline neck incision, the rat carotid artery balloon injury model was performed using a 2 French Fogarty catheter (provided by Edwards Lifesciences, Irvine, CA) as previously described [5e9].
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After carotid artery injury and restoration of blood flow, 20 mg/kg of the diazeniumdiolate NO donor disodium 1-[(2carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate (PROLI/ NO) was applied evenly to the external surface of the injured common carotid artery of rats in the treatment group and the neck incision was closed. Treatment groups for STZ and ZDF group assignments included (1) injury and (2) injury þ PROLI/ NO (n ¼ 6e7/treatment group). Carotid arteries were harvested at 14 d after injury for morphometric analysis. Blood was collected to measure insulin and glucose levels. Insulin levels were determined using an ELISA-based insulin assay kit (SPIBio; Bertin Pharma, Montigny-le-Bretonneux, France). The diazeniumdiolate PROLI/NO was used for the in vivo experiments as our laboratory has established that this NO donor is highly effective at inhibiting neointimal hyperplasia [7].
2.4.
Carotid artery lysate preparation
For carotid artery lysates, vessels were harvested 3 d after arterial balloon injury (n ¼ 4e7/group). Rats were anesthetized and 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 immediately explanted en bloc. Arteries were washed in cold 1 phosphate-buffered saline (PBS) and opened en face, and the injured section of the common carotid artery was excised, snap-frozen, and stored in liquid nitrogen. Lysis was performed mechanically using a ceramic mortar and pestle by resuspending the artery in a lysis buffer (50 mM Hepes [pH 7.5], 150 mM NaCl, 10% glycerol [vol/vol], 10 mM sodium pyrophosphate [Na4P2O7], 1 mM EDTA, 1 mM ethylene glycol tetraacetic acid [EGTA], 1% Triton X-100, 10 mg/mL aprotinin, 10 mg/mL leupeptin, 50 mM sodium fluoride [NaF], 1 mM Na3VO4, and 1 mM PMSF). Protein concentration was determined via a bicinchonic assay, and samples were stored at 80 C until Western blot analysis was performed.
2.5.
Western blot analysis
Carotid artery lysate was subjected to sodium dodecyl sulfateepolyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Membranes were hybridized with antibodies against ERK, pERK (1:1000; Cell Signaling Technologies, Inc, Danvers, MA), Akt 1/2/3, and pAkt (1:500, Cell Signaling Technologies, Inc) in 1% bovine serum albumin followed by a horseradish peroxidaseelinked goat anti-rabbit or goat anti-mouse secondary antibody (1:10,000; Thermo Fisher Scientific Inc, Hanover Park, IL). Proteins were visualized by using chemiluminescence reagents according to the manufacturer’s instructions (SuperSignal West Pico; Thermo Fisher Scientific Inc). Beta-actin expression was used to confirm equal loading. Levels of pERK and pAkt 1/2/3 were quantified from Western blots using integrated density obtained from ImageJ (National Institutes of Health, Bethesda, MD) and normalized to ERK and Akt 1/2/3, respectively.
2.6.
Tissue processing
Carotid arteries were harvested 14 d after arterial balloon injury following in situ perfusionefixation with cold PBS
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Table e Baseline characteristics.
n Weight (post), g Weight (at harvest), g Glucose (pre-STZ), mg/dL Glucose (post-STZ), mg/dL Insulin, pmol/L Cholesterol, mg/dL Triglycerides, mg/dL
LZ
LZ þ PROLI/NO
STZ
STZ þ PROLI/NO
ZDF
ZDF þ PROLI/NO
10 321 8 335 7 136 2 108 7 41 10 72 4 77 8
14 321 7 348 8 122 5 111 3 30 0 68 3 65 4
9 374 9 297 11 123 6 554 28* 22 4* 91 3* 163 34
14 366 5 302 13 97 3 453 55* 16 5* 85 4* 125 34
10 356 7y 356 20y 459 30*,y 467 27*,y 158 4* 333 20* 114 8
14 359 11y 389 18y 416 24*,y 345 56*,y 148 12* 366 45* 119 10
Values are means SE for all rats used in the 3-d and 14-d experiments; n, number of rats. * P < 0.05 compared with LZ rats. y P < 0.05 compared with LZ rats and preoperative glucose.
(250 mL) and 2% paraformaldehyde (500 mL). Vessels were placed in paraformaldehyde at 4 C for 1 h, then cryoprotected in 30% sucrose at 4 C overnight. The tissue was quick-frozen in Optimum Cutting Temperature O.C.T.(TM) compound (Tissue Tek, Hatfield, PA) and 5-mm sections were cut throughout the entire injured segment of the common carotid artery for histologic analysis.
2.7.
Immunofluorescent staining
Carotid artery sections of uninjured, injured, and NO-treated arteries 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 pERK and pAkt 1/2/3 antibody (1:50) for 1 h at 4 C. Negative controls were incubated without primary antibody. After incubation with goat anti-rabbit AlexaFluor 555 antibody (1:100 in PBS; 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 Spot Advanced software (Diagnostic Instruments, Sterling Heights, MI) on a Nikon Eclipse 50i Microscope (Nikon Instruments, Inc, Melville, NY) with a 40 objective. To quantify the pERK or pAkt staining, the integrated density of the positive (red) fluorescent signal was quantified using the Spot Advanced software.
2.8.
Statistical analysis
Results are expressed as mean the standard error of the mean (SEM). Differences between multiple groups were analyzed using one-way analysis of variance with the Student-Newman-Keuls post hoc test for all pairwise comparisons (SigmaStat; SPSS, Chicago, IL). Statistical significance was assumed when P < 0.05.
3.
Results
3.1.
Metabolic characteristics
Insulin and glucose measurements of all rats confirmed the desired nondiabetic or diabetic state of all rats in this study. Lean Zucker rats exhibited a normoglycemic, normoinsulinemic
state weighing 321e348 g (Table). The rats injected with STZ required 3e5 d to become hyperglycemic (>300 mg/dL). At sacrifice, STZ rats were found to be appropriately hypoinsulinemic compared with their LZ counterparts (P < 0.05). ZDF rats weighed significantly more than LZ controls (P < 0.05) and exhibited hyperglycemia and hyperinsulinemia (Table).
3.2. Nitric oxide therapy results in differential activation of ERK and Akt pathways in type 1 and type 2 diabetic rats at 3 d following arterial injury Carotid arteries from control, injury, and injury plus NO donor treatment groups were harvested and homogenized 3 d postarterial injury. Western blot analysis was performed. Following arterial injury, all animal models exhibited an increase in pERK levels compared with control artery (Fig. 1). With NO therapy, we observed inhibition of pERK levels in control (LZ) and type 1 diabetic rats (STZ) but no effect on pERK levels in type 2 diabetic rats (ZDF). Evaluation of the Akt pathway revealed that in control LZ rats, injury resulted in a 1.4-fold increase in pAkt levels and exposure to NO decreased pAkt levels (Fig. 1). In type 1 diabetic rats, injury resulted in a small decrease in pAkt levels from control; NO therapy resulted in a further decrease from injury alone. On the contrary, injury in type 2 diabetic rats resulted in a 1.3-fold increase in pAkt levels versus control and the addition of NO therapy resulted in a further increase in pAkt levels from injury alone. Thus, at 3 d following arterial injury, although there was activation of the ERK pathway in all three metabolic environments, there was no activation of the Akt pathway in insulin-deficient environments. This finding is opposite to the insulin-resistant environment where both injury and NO treatment resulted in an increase in pAkt levels.
3.3. Nitric oxide differentially affects pERK levels in type 1 versus type 2 diabetic environments 14 d following arterial injury To examine the effect of NO on the ERK and Akt pathways at a later time point, and throughout the different layers of the arterial wall, we collected arteries for immunohistologic examination. At 14 d following injury, in control LZ rats NO was found to decrease pERK levels by half compared with injury alone (P ¼ 0.005) in the intimal layer (Fig. 2A). However, NO had no effect on pERK levels in the media or adventitia
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Fig. 1 e Western blot analysis of carotid artery lysate 3 d following arterial injury in LZ, STZ, and ZDF rats (n [ 4e7/group). The pERK:ERK ratio increased from control uninjured arteries in all animal models following injury but decreased with NO treatment in LZ and STZ rats. In ZDF rats, NO treatment had no effect on the pERK:ERK ratio compared with injury alone. The pAkt:Akt ratio decreased with NO treatment in LZ and STZ rats but increased in ZDF rats.
(Fig. 2B, C). In the insulin-deficient environment of type 1 diabetes (STZ), NO had no effect on pERK levels in the intima, reduced pERK levels in the media by half, yet increased pERK levels in the adventitia 1.7-fold compared with injury alone (P < 0.001, Fig. 2B, C). In the insulin-resistant environment of type 2 diabetes (ZDF), NO had no significant effect on pERK levels in the intima or media compared with injury alone. However, NO increased pERK levels in the adventitia 1.9-fold compared with injury alone (P < 0.04, Fig. 2C). Upon evaluation of pERK levels in all layers of the arterial wall (Fig. 2D), NO had no significant effect on pERK levels in control LZ rats but increased pERK levels in both the STZ and ZDF rats 1.23- and 1.4-fold, respectively (P < 0.05). Of note, although the latter two metabolic environments have different insulin levels, they are both profoundly hyperglycemic.
3.5. Ratio of pERK:pAkt correlates with neointimal hyperplasia in type 2 diabetic rats at 14 d after arterial injury Given the patterns we observed in pERK and pAkt levels following exposure to NO in the different metabolic environments, we assessed the ratio of pERK to pAkt levels (pERK:pAkt). Interestingly, NO induced an increase in the pERK:pAkt ratio in LZ and STZ rats compared with the uninjured and injured treatment groups (Fig. 4). Yet, in the ZDF rats, NO did not increase the pERK:pAkt ratio, but decreased it (0.83) compared with the uninjured (1.2) and injured (0.94) treatment groups. Interestingly, this ratio correlated with our prior findings on the effect of NO on neointimal hyperplasia, with NO inhibiting neointimal hyperplasia to the greatest extent in ZDF rats, compared with LZ and STZ rats [5,9].
3.4. Nitric oxide causes a paradoxical effect on pAkt levels in type 1 versus type 2 diabetic environments 14 d following arterial injury
4.
Evaluation of the effect of NO on pAkt levels revealed an interesting pattern. Whereas NO either decreased or had no effect on pAkt levels in the intima, media, and adventitia of LZ and STZ rats (Fig. 3A, C), NO dramatically increased pAkt levels in the adventitia of ZDF rats (2-fold increase, P < 0.001, Fig. 3C). An evaluation of total pAkt levels throughout all layers of the arterial wall mirrored this pattern (Fig. 3D).
In this study we have demonstrated that treatment with NO in type 1 and type 2 diabetic rat models causes differential activation of the ERK and Akt pathway. At 3 d following injury, NO inhibited activation of the ERK pathway in control and type 1 diabetic rats but had no effect in type 2 diabetic rats. By 14 d, NO caused inhibition of pERK in control animals but activation of pERK in both type 1 and 2 diabetic rat models. For Akt, NO
Discussion
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Fig. 2 e Immunofluorescent pERK staining of carotid artery cross sections at 14 d following arterial injury in LZ, STZ, and ZDF rats (n [ 6/treatment group). (Top) Composite of representative images of the immunofluorescent staining. (Bottom) Integrated density of the positive pERK staining (red in top images) was quantified in the (A) intima, (B) media, (C) adventitia, and (D) total artery. *P < 0.05. (Color version of figure is available online.)
inhibited Akt activation in control and type 1 diabetic rats at 3 and 14 d following injury but increased Akt activation in type 2 diabetic rats at both time points. We also demonstrated that the largest changes occur in the adventitia of all animal models. To further characterize the effect of NO on these two pathways, we assessed the pERK:pAkt ratio and found that NO increased this ratio in the control and type 1 diabetic rats but decreased this ratio in type 2 diabetic rats. Taking all of this into account, it appears that the ratio of activation of the ERK pathway to the Akt pathway may be the best surrogate to predict efficacy of NO at inhibiting neointimal hyperplasia. The insulin signaling pathway has been well characterized. Under normoglycemic and normoinsulinemic conditions, insulin binds to the insulin receptor (IR), which consists of alpha and beta subunits [10]. This binding results in the phosphorylation of the beta subunit (IRbeta), which then leads to
tyrosine phosphorylation of the insulin receptor substrate (IRS1) or its homolog IRS2. The C-terminal tail of IRS1 binds to phosphoinositide 3-kinase (PI3K), which then converts PI(4,5) P2 to PI(3,4,5)P3, which in turn recruits protein kinase B, also known as Akt, and 3-phosphoinositide-dependent kinase-1 to the plasma membrane, where PKD1 activates Akt. Akt phosphorylates many substrates important for cell survival and growth and, importantly, phosphorylates and activates endothelial NO synthase, thereby increasing NO production. This pathway is typically referred to as the metabolic or survival pathway. Insulin also triggers the activation of the MAPK pathway through tyrosine-phosphorylated IRS1 or SHC. Phosphorylated IRS1 or SHC lead to binding of the growth factor receptor binding protein 2, which is prebound to mammalian son of sevenless, a nucleotide exchange protein that catalyzes the
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Fig. 3 e Immunofluorescent pAkt staining of carotid artery cross sections at 14 d following arterial injury in LZ, STZ, and ZDF rats (n [ 6/treatment group). (Top) Composite of representative images of the immunofluorescent staining. (Bottom) Integrated density of the positive pAkt staining (red in top images) was quantified in the (A) intima, (B) media, (C) adventitia, and (D) total artery. *P < 0.05. (Color version of figure is available online.)
exchange of GDP for GTP on Ras, as a small GTP-binding protein. This results in activation of Ras and, subsequently, Raf-1. Raf-1 then activates MEK1, which phosphorylates ERK1 and ERK2. Activation of the MEK1-ERK1/2 complex results in the formation of ERK monomers and dimers, which translocate to the nucleus and activate a host of growth-promoting genes. This pathway is commonly referred to as the mitogenic or proliferative pathway. Thus, under normal conditions, insulin promotes both the PI3K/Akt and MAPK pathways, supporting cell growth and survival. The role of MAPK and P13K/Akt pathways in the development of neointimal hyperplasia has been examined. Indolfi et al. evaluated the role of Ras in rats with type 1 diabetes with and without insulin therapy [15]. Rat carotid arteries were transfected with DNA vectors for Ras-dominant negative mutants at the time of vascular injury in type 1 diabetic rats
with insulin therapy compared with control rats not receiving the DNA vectors or insulin therapy. These investigators found markedly less neointimal hyperplasia in the transfected rats, comparable to insulin-deficient rats. Thus, neointimal hyperplasia is mediated through the ras-MAPK pathway and inhibition of MAPK genes results in inhibition of neointimal hyperplasia [16]. In our study, we found that arterial injury resulted in increased levels of pERK in type 1 diabetic rats at 3 d and 14 d. Furthermore, NO therapy resulted in an even further increase in pERK levels at 3 and 14 d. Previous studies have shown that type 1 diabetic rats form less neointimal hyperplasia following balloon arterial injury due to the insulin-deficient state, and that NO is not effective at inhibiting neointimal hyperplasia in this rat model [9]. In our investigation, we observed an increase in pERK levels overall in an insulin-deficient state, where little neointimal
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Fig. 4 e Evaluation of the pERK:pAkt immunofluorescent staining in LZ, STZ, and ZDF rats 14 d following arterial injury (n [ 6 for all treatment groups). Integrated density was measured using ImageJ software and the ratio of pERK:pAkt was determined.
hyperplasia is formed and NO is not at all effective. This leads us to the hypothesis that dysregulation in ERK metabolism also occurs in insulin-deficient environments. Jonas et al. examined the vascular response to stent injury in animal models of type 1 and type 2 diabetic rats [17]. Insulin-resistant rats exhibited more neointimal hyperplasia, as in our previous studies, and 98% greater pERK levels, but 54% less pAkt levels. Furthermore, the ratio of pERK to pAkt statistically correlated with the degree of neointimal hyperplasia. We found comparable activation of pERK in type 2 diabetic rats following arterial injury, but a significantly greater level of activation in rats treated with NO. Similar to the findings of Jonas et al., we found decreased activation of Akt pathway following arterial injury in type 2 diabetic rats. However, NO treatment resulted in a significant increase in pAkt levels following arterial injury. The ratio of pERK to pAkt was therefore decreased in type 2 diabetic rats, and NO caused further reduction in this ratio. As we have shown, NO causes a substantial decrease in neointimal hyperplasia in type 2 diabetic rats following arterial injury, and thus correlates with the pERK:pAkt ratio [5]. Koyama et al. evaluated activation of ERK activity at several time points up to 14 d following balloon arterial injury and found that ERK activation occurred early, within 1e2 d, but returned to baseline by 7 d [18]. Similarly, we observed activation of pERK with injury in all three rat models at 3 d. However, we found that pERK levels remained elevated above the levels of uninjured control arteries at 14 d, with the highest levels observed in the type 1 and type 2 diabetic rats. These findings differ from those of Koyama et al., and this is likely due to the difference in metabolic environments. These data provide further evidence that the metabolic environment influences the activation of the pERK pathway. Our study has revealed limitations. First, different methodologies were used to evaluate two different time points for Akt and MAPK pathway activation. Second, whole-vessel lysate risks the potential of including uninjured control
portions of the artery into treatment groups, thus diluting results from the injured segment. To avoid this possibility, we used only portions of the carotid artery that underwent balloon injury and increased the number of arteries included per rat to compensate for the decreased amount of tissue obtained. Third, lysate does not allow making observations regarding particular arterial layers. Fourth, we did not measure changes in downstream proteins to assess the direct effect of pAkt and pERK level differences. Thus, while the data presented here provide important insights, the potential for future studies on the effects of NO on the insulin signaling pathway in the vasculature of type 1 and 2 diabetic animals is very encouraging. Further studies are necessary to determine how the addition of insulin therapy influences the activation of the Akt and ERK pathways by NO in type 1 and type 2 diabetic rats. Also, obtaining carotid artery lysate at 7 and 14 d following arterial injury would be helpful for making comparisons to lysate at earlier time points; however, there are high costs involved with maintaining the animals necessary to complete this endeavor. In conclusion, the development of neointimal hyperplasia after arterial injury in diabetic populations is of increasing importance as the incidence of diabetes continues to rise. We have shown the efficacy of nitric oxide to be substantially different in type 1 and type 2 diabetic rats, and this may be due to differential activation of Akt and MAPK pathways in insulin-deficient versus insulin-resistant states. Understanding the mechanism by which NO affects the ERK and Akt pathway may help to advance the knowledge of diabetic vascular disease and may provide a mechanism to explore as a potential therapeutic intervention in the future.
Acknowledgment The authors would like to express their thanks to Lynnette Dangerfield for her administrative support, and to Edwards Lifesciences for providing the Fogarty balloon catheters. Funding: This work was supported in part by funding from the Department of Veterans Affairs, VA Merit Review Grant (I01 BX000409, MRK), the Eleanor B. Pillsbury Grant-University of Illinois, the American Medical Association Foundation Seed Grant program (MPR, SSA, VNV), and the EthiconeSociety of University Surgeons Scholarship Award (MPR), and by the generosity of Mrs Hilda Rosenbloom and Mrs Eleanor Baldwin.
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[1] Kornowski R, Mintz GS, Popma JJ, et al. Increased restenosis in diabetes mellitus following transcatheter coronary interventions is due to exaggerated intimal hyperplasia. J Am Coll Cardiol 1997;29:7335. [2] Capek P, Mclean GK, Berkowitz HD. Femoropopliteal angioplasty - factors influencing long-term success. Circulation 1991;83:70. [3] Kip KE, Faxon DP, Detre KM, Yeh WL, Kelsey SF, Currier JW. Coronary angioplasty in diabetic patients - the National Heart, Lung, and Blood Institute percutaneous transluminal corollary angioplasty registry. Circulation 1996;94:1818.
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[4] Kornowski R, Mintz GS, Kent KM, et al. Increased restenosis in diabetes mellitus after coronary interventions is due to exaggerated intimal hyperplasia - a serial intravascular ultrasound study. Circulation 1997;95:1366. [5] Ahanchi SS, Varu VN, Tsihlis ND, et al. Heightened efficacy of nitric oxide-based therapies in type II diabetes mellitus and metabolic syndrome. Am J Physiol Heart Circ Physiol 2008; 295:H2388. [6] Kapadia MR, Chow LW, Tsihlis ND, et al. Nitric oxide and nanotechnology: a novel approach to inhibit neointimal hyperplasia. J Vasc Surg 2008;47:173. [7] Pearce CG, Najjar SF, Kapadia MR, et al. Beneficial effect of a short-acting NO donor for the prevention of neointimal hyperplasia. Free Radic Biol Med 2008;44:73. [8] Shears LL, Kibbe MR, Murdock AD, et al. Efficient inhibition of intimal hyperplasia by adenovirus-mediated inducible nitric oxide synthase gene transfer to rats and pigs in vivo. J Am Coll Surg 1998;187:295. [9] Varu VN, Ahanchi SS, Hogg ME, et al. Insulin enhances the effect of nitric oxide at inhibiting neointimal hyperplasia in a rat model of type 1 diabetes. Am J Physiol Heart Circ Physiol 2010;299:H772. [10] Avogaro A, de Kreutzenberg SV, Fadini GP. Oxidative stress and vascular disease in diabetes: is the dichotomization of insulin signaling still valid? Free Radic Biol Med 2008;44:1209.
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[11] Feener EP, King GL. Endothelial dysfunction in diabetes mellitus: role in cardiovascular disease. Heart Fail Monit 2001;1:74. [12] Folli F, Saad MJ, Velloso L, et al. Crosstalk between insulin and angiotensin II signalling systems. Exp Clin Endocrinol Diabetes 1999;107:133. [13] Spinetti G, Kraenkel N, Emanueli C, Madeddu P. Diabetes and vessel wall remodelling: from mechanistic insights to regenerative therapies. Cardiovasc Res 2008;78:265. [14] Rask-Madsen C, King GL. Proatherosclerotic mechanisms involving protein kinase C in diabetes and insulin resistance. Arterioscler Thromb Vasc Biol 2005;25:487. [15] Indolfi C, Torella D, Cavuto L, et al. Effects of balloon injury on neointimal hyperplasia in streptozotocin-induced diabetes and in hyperinsulinemic nondiabetic pancreatic islet-transplanted rats. Circulation 2001;103:2980. [16] Indolfi C, Avvedimento EV, Rapacciuolo A, et al. Inhibition of cellular ras prevents smooth muscle cell proliferation after vascular injury in vivo. Nat Med 1995;1:541. [17] Jonas M, Edelman ER, Groothuis A, Baker AB, Seifert P, Rogers C. Vascular neointimal formation and signaling pathway activation in response to stent injury in insulinresistant and diabetic animals. Circ Res 2005;97:725. [18] Koyama H, Olson NE, Dastvan FF, Reidy MA. Cell replication in the arterial wall: activation of signaling pathway following in vivo injury. Circ Res 1998;82:713.
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Nitric oxide differentially affects ERK and Akt in type 1 and type 2 diabetic rats Monica P. Rodriguez, MD,a,1 Zachary M. Emond, MD,a,1 Vinit N. Varu, MD,a Sadaf S. Ahanchi, MD,a Janet Martinez, AAS,a,b and Melina R. Kibbe, MDa,b,* a
Division of Vascular Surgery and Institute for BioNanotechnology in Medicine, Northwestern University Feinberg School of Medicine, Chicago, Illinois b Jesse Brown VA Medical Center, Chicago, Illinois
article info
abstract
Article history:
Background: We have shown that nitric oxide (NO) is more effective at inhibiting neointimal
Received 20 February 2013
hyperplasia in type 2 diabetic rats than in nondiabetic rats, but is not effective in type 1 diabetic
Received in revised form
rats. Insulin signaling is mediated by the ERK and Akt pathways, and thus we hypothesized
20 February 2013
that NO differentially affects ERK and Akt activity in type 1 versus type 2 diabetic rats.
Accepted 27 February 2013
Materials and methods: To investigate this hypothesis, we induced type 2 diabetes in Zucker
Available online 19 March 2013
diabetic fatty (ZDF) rats by feeding them Purina 5008 chow. To induce type 1 diabetes, lean Zucker (LZ) rats were injected with streptozotocin (STZ; 60 mg/kg). The carotid artery injury
Keywords:
model was performed. Groups included injury and injury þ PROLI/NO (20 mg/kg) (n ¼ 6/group).
Nitric oxide
Results: Three days following injury, all animal models exhibited an increase in pERK levels.
Akt
Whereas NO reduced pERK levels in LZ and STZ rats, NO had no effect on pERK levels in
ERK
ZDF rats. Following a similar pattern, NO reduced pAkt levels in LZ and STZ rats but
Neointimal hyperplasia
increased pAkt levels in ZDF rats. Fourteen days following injury, NO increased total pERK
Type 1 diabetes
levels throughout the arterial wall in both the STZ and ZDF rats. These changes were
Type 2 diabetes
greatest in the adventitia. Interestingly, whereas NO decreased total pAkt levels in LZ and
Metabolic environment
STZ rats, NO increased pAkt levels in ZDF rats. Evaluation of the pERK:pAkt ratio revealed that NO increased this ratio in LZ and STZ rats but decreased the ratio in ZDF rats. Conclusions: We report that NO differentially affects the expression of pERK and pAkt in type 1 versus type 2 diabetic rats. Given that NO is more effective at inhibiting neointimal hyperplasia in type 2 diabetic animals, the pERK:pAkt ratio may be the best surrogate to predict efficacy. Published by Elsevier Inc.
1.
Introduction
Patients with diabetes have a 4-fold greater risk of developing peripheral arterial disease, coronary arterial disease, and cerebrovascular disease [1e3]. This population also experiences higher failure rates following revascularization procedures due to aggressive rates of restenosis from neointimal
hyperplasia [4]. Nitric oxide (NO) is a naturally occurring molecule that has been shown to effectively inhibit neointimal hyperplasia in animal models of arterial injury [5e9]. We previously reported that NO was more effective at inhibiting neointimal hyperplasia after arterial injury in a rodent model of uncontrolled type 2 diabetes and less effective in a rodent model of uncontrolled type 1 diabetes [5,9]. These
* Corresponding author. Division of Vascular Surgery, Northwestern University Feinberg School of Medicine, 676 N. St. Clair Street, #650, Chicago, IL 60611. Tel.: þ1 312 503 6701; fax: þ1 312 503 1222. E-mail address: [email protected] (M.R. Kibbe). 1 These authors contributed equally to this manuscript. Drs. Rodriguez and Emond share first author status. 0022-4804/$ e see front matter Published by Elsevier Inc. http://dx.doi.org/10.1016/j.jss.2013.02.055
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data suggest a role for insulin in mediating the effects of NO, given that a main difference between these animal models is hyperinsulinemia versus hypoinsulinemia. The insulin signaling pathway has been well characterized. The two predominant signaling pathways downstream from the insulin receptor are the mitogen-activated protein kinase (MAPK) and PI3K/Akt pathways, which are known as the mitogenic and metabolic pathways, respectively. Hyperinsulinemia causes derangement of these pathways by stimulating the MAPK pathway, which leads to aggressive proliferation and neointimal hyperplasia, and by inhibiting the PI3 kinase/Akt pathway, which leads to lower NO production [10e14]. The effect of NO on the MAP kinase and AKT pathway in type 1 and type 2 diabetic environments is unknown. Thus, we hypothesized that NO differentially affects ERK and Akt in type 1 versus type 2 diabetic environments. Our goal was to evaluate the role of ERK and Akt in mediating the differential effects of NO in type 1 and type 2 diabetic rat models.
2.
Materials and methods
2.1.
Type 1 diabetic animal model
Eleven-week-old male lean Zucker (LZ) rats were obtained from Charles River Laboratories (Wilmington, MA). Type 1 diabetes was induced in LZ rats with a single intraperitoneal injection of streptozotocin (STZ, 60 mg/kg). Daily serum glucose levels were assessed with a glucometer via tail vein puncture and animals with glucose concentrations of 300 mg/dL or above were considered diabetic and included in the study. The nonfasting daily blood glucose concentration was recorded.
2.2.
Type 2 diabetic animal model
Zucker diabetic fatty (ZDF) rats were obtained from Charles River Laboratories. The ZDF strain has a homozygous leptin receptor mutation predisposing the rats to type 2 diabetes. Inbred ZDF male rats were fed the Purina 5008 diet, which is manufactured high in carbohydrates and fats. This induces a metabolic state of hyperinsulinemia, hyperglycemia, hypercholesterolemia, and hypertriglyceridemia, mimicking a type 2 diabetic state. Rats were kept in their respective group assignments for 3 wk prior to and 2 wk following surgery.
2.3.
Animal surgery
All animal procedures were performed in accordance with the Guide for 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. Rats were anesthetized with inhaled isoflurane (0.5%e3%). Atropine was administered subcutaneously (0.1 mg/kg) to decrease airway secretions. Weight was documented and blood glucose was measured daily following administration of STZ. The neck was shaved and prepped with betadine and alcohol (70%). Following a midline neck incision, the rat carotid artery balloon injury model was performed using a 2 French Fogarty catheter (provided by Edwards Lifesciences, Irvine, CA) as previously described [5e9].
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After carotid artery injury and restoration of blood flow, 20 mg/kg of the diazeniumdiolate NO donor disodium 1-[(2carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate (PROLI/ NO) was applied evenly to the external surface of the injured common carotid artery of rats in the treatment group and the neck incision was closed. Treatment groups for STZ and ZDF group assignments included (1) injury and (2) injury þ PROLI/ NO (n ¼ 6e7/treatment group). Carotid arteries were harvested at 14 d after injury for morphometric analysis. Blood was collected to measure insulin and glucose levels. Insulin levels were determined using an ELISA-based insulin assay kit (SPIBio; Bertin Pharma, Montigny-le-Bretonneux, France). The diazeniumdiolate PROLI/NO was used for the in vivo experiments as our laboratory has established that this NO donor is highly effective at inhibiting neointimal hyperplasia [7].
2.4.
Carotid artery lysate preparation
For carotid artery lysates, vessels were harvested 3 d after arterial balloon injury (n ¼ 4e7/group). Rats were anesthetized and 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 immediately explanted en bloc. Arteries were washed in cold 1 phosphate-buffered saline (PBS) and opened en face, and the injured section of the common carotid artery was excised, snap-frozen, and stored in liquid nitrogen. Lysis was performed mechanically using a ceramic mortar and pestle by resuspending the artery in a lysis buffer (50 mM Hepes [pH 7.5], 150 mM NaCl, 10% glycerol [vol/vol], 10 mM sodium pyrophosphate [Na4P2O7], 1 mM EDTA, 1 mM ethylene glycol tetraacetic acid [EGTA], 1% Triton X-100, 10 mg/mL aprotinin, 10 mg/mL leupeptin, 50 mM sodium fluoride [NaF], 1 mM Na3VO4, and 1 mM PMSF). Protein concentration was determined via a bicinchonic assay, and samples were stored at 80 C until Western blot analysis was performed.
2.5.
Western blot analysis
Carotid artery lysate was subjected to sodium dodecyl sulfateepolyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Membranes were hybridized with antibodies against ERK, pERK (1:1000; Cell Signaling Technologies, Inc, Danvers, MA), Akt 1/2/3, and pAkt (1:500, Cell Signaling Technologies, Inc) in 1% bovine serum albumin followed by a horseradish peroxidaseelinked goat anti-rabbit or goat anti-mouse secondary antibody (1:10,000; Thermo Fisher Scientific Inc, Hanover Park, IL). Proteins were visualized by using chemiluminescence reagents according to the manufacturer’s instructions (SuperSignal West Pico; Thermo Fisher Scientific Inc). Beta-actin expression was used to confirm equal loading. Levels of pERK and pAkt 1/2/3 were quantified from Western blots using integrated density obtained from ImageJ (National Institutes of Health, Bethesda, MD) and normalized to ERK and Akt 1/2/3, respectively.
2.6.
Tissue processing
Carotid arteries were harvested 14 d after arterial balloon injury following in situ perfusionefixation with cold PBS
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Table e Baseline characteristics.
n Weight (post), g Weight (at harvest), g Glucose (pre-STZ), mg/dL Glucose (post-STZ), mg/dL Insulin, pmol/L Cholesterol, mg/dL Triglycerides, mg/dL
LZ
LZ þ PROLI/NO
STZ
STZ þ PROLI/NO
ZDF
ZDF þ PROLI/NO
10 321 8 335 7 136 2 108 7 41 10 72 4 77 8
14 321 7 348 8 122 5 111 3 30 0 68 3 65 4
9 374 9 297 11 123 6 554 28* 22 4* 91 3* 163 34
14 366 5 302 13 97 3 453 55* 16 5* 85 4* 125 34
10 356 7y 356 20y 459 30*,y 467 27*,y 158 4* 333 20* 114 8
14 359 11y 389 18y 416 24*,y 345 56*,y 148 12* 366 45* 119 10
Values are means SE for all rats used in the 3-d and 14-d experiments; n, number of rats. * P < 0.05 compared with LZ rats. y P < 0.05 compared with LZ rats and preoperative glucose.
(250 mL) and 2% paraformaldehyde (500 mL). Vessels were placed in paraformaldehyde at 4 C for 1 h, then cryoprotected in 30% sucrose at 4 C overnight. The tissue was quick-frozen in Optimum Cutting Temperature O.C.T.(TM) compound (Tissue Tek, Hatfield, PA) and 5-mm sections were cut throughout the entire injured segment of the common carotid artery for histologic analysis.
2.7.
Immunofluorescent staining
Carotid artery sections of uninjured, injured, and NO-treated arteries 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 pERK and pAkt 1/2/3 antibody (1:50) for 1 h at 4 C. Negative controls were incubated without primary antibody. After incubation with goat anti-rabbit AlexaFluor 555 antibody (1:100 in PBS; 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 Spot Advanced software (Diagnostic Instruments, Sterling Heights, MI) on a Nikon Eclipse 50i Microscope (Nikon Instruments, Inc, Melville, NY) with a 40 objective. To quantify the pERK or pAkt staining, the integrated density of the positive (red) fluorescent signal was quantified using the Spot Advanced software.
2.8.
Statistical analysis
Results are expressed as mean the standard error of the mean (SEM). Differences between multiple groups were analyzed using one-way analysis of variance with the Student-Newman-Keuls post hoc test for all pairwise comparisons (SigmaStat; SPSS, Chicago, IL). Statistical significance was assumed when P < 0.05.
3.
Results
3.1.
Metabolic characteristics
Insulin and glucose measurements of all rats confirmed the desired nondiabetic or diabetic state of all rats in this study. Lean Zucker rats exhibited a normoglycemic, normoinsulinemic
state weighing 321e348 g (Table). The rats injected with STZ required 3e5 d to become hyperglycemic (>300 mg/dL). At sacrifice, STZ rats were found to be appropriately hypoinsulinemic compared with their LZ counterparts (P < 0.05). ZDF rats weighed significantly more than LZ controls (P < 0.05) and exhibited hyperglycemia and hyperinsulinemia (Table).
3.2. Nitric oxide therapy results in differential activation of ERK and Akt pathways in type 1 and type 2 diabetic rats at 3 d following arterial injury Carotid arteries from control, injury, and injury plus NO donor treatment groups were harvested and homogenized 3 d postarterial injury. Western blot analysis was performed. Following arterial injury, all animal models exhibited an increase in pERK levels compared with control artery (Fig. 1). With NO therapy, we observed inhibition of pERK levels in control (LZ) and type 1 diabetic rats (STZ) but no effect on pERK levels in type 2 diabetic rats (ZDF). Evaluation of the Akt pathway revealed that in control LZ rats, injury resulted in a 1.4-fold increase in pAkt levels and exposure to NO decreased pAkt levels (Fig. 1). In type 1 diabetic rats, injury resulted in a small decrease in pAkt levels from control; NO therapy resulted in a further decrease from injury alone. On the contrary, injury in type 2 diabetic rats resulted in a 1.3-fold increase in pAkt levels versus control and the addition of NO therapy resulted in a further increase in pAkt levels from injury alone. Thus, at 3 d following arterial injury, although there was activation of the ERK pathway in all three metabolic environments, there was no activation of the Akt pathway in insulin-deficient environments. This finding is opposite to the insulin-resistant environment where both injury and NO treatment resulted in an increase in pAkt levels.
3.3. Nitric oxide differentially affects pERK levels in type 1 versus type 2 diabetic environments 14 d following arterial injury To examine the effect of NO on the ERK and Akt pathways at a later time point, and throughout the different layers of the arterial wall, we collected arteries for immunohistologic examination. At 14 d following injury, in control LZ rats NO was found to decrease pERK levels by half compared with injury alone (P ¼ 0.005) in the intimal layer (Fig. 2A). However, NO had no effect on pERK levels in the media or adventitia
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Fig. 1 e Western blot analysis of carotid artery lysate 3 d following arterial injury in LZ, STZ, and ZDF rats (n [ 4e7/group). The pERK:ERK ratio increased from control uninjured arteries in all animal models following injury but decreased with NO treatment in LZ and STZ rats. In ZDF rats, NO treatment had no effect on the pERK:ERK ratio compared with injury alone. The pAkt:Akt ratio decreased with NO treatment in LZ and STZ rats but increased in ZDF rats.
(Fig. 2B, C). In the insulin-deficient environment of type 1 diabetes (STZ), NO had no effect on pERK levels in the intima, reduced pERK levels in the media by half, yet increased pERK levels in the adventitia 1.7-fold compared with injury alone (P < 0.001, Fig. 2B, C). In the insulin-resistant environment of type 2 diabetes (ZDF), NO had no significant effect on pERK levels in the intima or media compared with injury alone. However, NO increased pERK levels in the adventitia 1.9-fold compared with injury alone (P < 0.04, Fig. 2C). Upon evaluation of pERK levels in all layers of the arterial wall (Fig. 2D), NO had no significant effect on pERK levels in control LZ rats but increased pERK levels in both the STZ and ZDF rats 1.23- and 1.4-fold, respectively (P < 0.05). Of note, although the latter two metabolic environments have different insulin levels, they are both profoundly hyperglycemic.
3.5. Ratio of pERK:pAkt correlates with neointimal hyperplasia in type 2 diabetic rats at 14 d after arterial injury Given the patterns we observed in pERK and pAkt levels following exposure to NO in the different metabolic environments, we assessed the ratio of pERK to pAkt levels (pERK:pAkt). Interestingly, NO induced an increase in the pERK:pAkt ratio in LZ and STZ rats compared with the uninjured and injured treatment groups (Fig. 4). Yet, in the ZDF rats, NO did not increase the pERK:pAkt ratio, but decreased it (0.83) compared with the uninjured (1.2) and injured (0.94) treatment groups. Interestingly, this ratio correlated with our prior findings on the effect of NO on neointimal hyperplasia, with NO inhibiting neointimal hyperplasia to the greatest extent in ZDF rats, compared with LZ and STZ rats [5,9].
3.4. Nitric oxide causes a paradoxical effect on pAkt levels in type 1 versus type 2 diabetic environments 14 d following arterial injury
4.
Evaluation of the effect of NO on pAkt levels revealed an interesting pattern. Whereas NO either decreased or had no effect on pAkt levels in the intima, media, and adventitia of LZ and STZ rats (Fig. 3A, C), NO dramatically increased pAkt levels in the adventitia of ZDF rats (2-fold increase, P < 0.001, Fig. 3C). An evaluation of total pAkt levels throughout all layers of the arterial wall mirrored this pattern (Fig. 3D).
In this study we have demonstrated that treatment with NO in type 1 and type 2 diabetic rat models causes differential activation of the ERK and Akt pathway. At 3 d following injury, NO inhibited activation of the ERK pathway in control and type 1 diabetic rats but had no effect in type 2 diabetic rats. By 14 d, NO caused inhibition of pERK in control animals but activation of pERK in both type 1 and 2 diabetic rat models. For Akt, NO
Discussion
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Fig. 2 e Immunofluorescent pERK staining of carotid artery cross sections at 14 d following arterial injury in LZ, STZ, and ZDF rats (n [ 6/treatment group). (Top) Composite of representative images of the immunofluorescent staining. (Bottom) Integrated density of the positive pERK staining (red in top images) was quantified in the (A) intima, (B) media, (C) adventitia, and (D) total artery. *P < 0.05. (Color version of figure is available online.)
inhibited Akt activation in control and type 1 diabetic rats at 3 and 14 d following injury but increased Akt activation in type 2 diabetic rats at both time points. We also demonstrated that the largest changes occur in the adventitia of all animal models. To further characterize the effect of NO on these two pathways, we assessed the pERK:pAkt ratio and found that NO increased this ratio in the control and type 1 diabetic rats but decreased this ratio in type 2 diabetic rats. Taking all of this into account, it appears that the ratio of activation of the ERK pathway to the Akt pathway may be the best surrogate to predict efficacy of NO at inhibiting neointimal hyperplasia. The insulin signaling pathway has been well characterized. Under normoglycemic and normoinsulinemic conditions, insulin binds to the insulin receptor (IR), which consists of alpha and beta subunits [10]. This binding results in the phosphorylation of the beta subunit (IRbeta), which then leads to
tyrosine phosphorylation of the insulin receptor substrate (IRS1) or its homolog IRS2. The C-terminal tail of IRS1 binds to phosphoinositide 3-kinase (PI3K), which then converts PI(4,5) P2 to PI(3,4,5)P3, which in turn recruits protein kinase B, also known as Akt, and 3-phosphoinositide-dependent kinase-1 to the plasma membrane, where PKD1 activates Akt. Akt phosphorylates many substrates important for cell survival and growth and, importantly, phosphorylates and activates endothelial NO synthase, thereby increasing NO production. This pathway is typically referred to as the metabolic or survival pathway. Insulin also triggers the activation of the MAPK pathway through tyrosine-phosphorylated IRS1 or SHC. Phosphorylated IRS1 or SHC lead to binding of the growth factor receptor binding protein 2, which is prebound to mammalian son of sevenless, a nucleotide exchange protein that catalyzes the
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Fig. 3 e Immunofluorescent pAkt staining of carotid artery cross sections at 14 d following arterial injury in LZ, STZ, and ZDF rats (n [ 6/treatment group). (Top) Composite of representative images of the immunofluorescent staining. (Bottom) Integrated density of the positive pAkt staining (red in top images) was quantified in the (A) intima, (B) media, (C) adventitia, and (D) total artery. *P < 0.05. (Color version of figure is available online.)
exchange of GDP for GTP on Ras, as a small GTP-binding protein. This results in activation of Ras and, subsequently, Raf-1. Raf-1 then activates MEK1, which phosphorylates ERK1 and ERK2. Activation of the MEK1-ERK1/2 complex results in the formation of ERK monomers and dimers, which translocate to the nucleus and activate a host of growth-promoting genes. This pathway is commonly referred to as the mitogenic or proliferative pathway. Thus, under normal conditions, insulin promotes both the PI3K/Akt and MAPK pathways, supporting cell growth and survival. The role of MAPK and P13K/Akt pathways in the development of neointimal hyperplasia has been examined. Indolfi et al. evaluated the role of Ras in rats with type 1 diabetes with and without insulin therapy [15]. Rat carotid arteries were transfected with DNA vectors for Ras-dominant negative mutants at the time of vascular injury in type 1 diabetic rats
with insulin therapy compared with control rats not receiving the DNA vectors or insulin therapy. These investigators found markedly less neointimal hyperplasia in the transfected rats, comparable to insulin-deficient rats. Thus, neointimal hyperplasia is mediated through the ras-MAPK pathway and inhibition of MAPK genes results in inhibition of neointimal hyperplasia [16]. In our study, we found that arterial injury resulted in increased levels of pERK in type 1 diabetic rats at 3 d and 14 d. Furthermore, NO therapy resulted in an even further increase in pERK levels at 3 and 14 d. Previous studies have shown that type 1 diabetic rats form less neointimal hyperplasia following balloon arterial injury due to the insulin-deficient state, and that NO is not effective at inhibiting neointimal hyperplasia in this rat model [9]. In our investigation, we observed an increase in pERK levels overall in an insulin-deficient state, where little neointimal
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Fig. 4 e Evaluation of the pERK:pAkt immunofluorescent staining in LZ, STZ, and ZDF rats 14 d following arterial injury (n [ 6 for all treatment groups). Integrated density was measured using ImageJ software and the ratio of pERK:pAkt was determined.
hyperplasia is formed and NO is not at all effective. This leads us to the hypothesis that dysregulation in ERK metabolism also occurs in insulin-deficient environments. Jonas et al. examined the vascular response to stent injury in animal models of type 1 and type 2 diabetic rats [17]. Insulin-resistant rats exhibited more neointimal hyperplasia, as in our previous studies, and 98% greater pERK levels, but 54% less pAkt levels. Furthermore, the ratio of pERK to pAkt statistically correlated with the degree of neointimal hyperplasia. We found comparable activation of pERK in type 2 diabetic rats following arterial injury, but a significantly greater level of activation in rats treated with NO. Similar to the findings of Jonas et al., we found decreased activation of Akt pathway following arterial injury in type 2 diabetic rats. However, NO treatment resulted in a significant increase in pAkt levels following arterial injury. The ratio of pERK to pAkt was therefore decreased in type 2 diabetic rats, and NO caused further reduction in this ratio. As we have shown, NO causes a substantial decrease in neointimal hyperplasia in type 2 diabetic rats following arterial injury, and thus correlates with the pERK:pAkt ratio [5]. Koyama et al. evaluated activation of ERK activity at several time points up to 14 d following balloon arterial injury and found that ERK activation occurred early, within 1e2 d, but returned to baseline by 7 d [18]. Similarly, we observed activation of pERK with injury in all three rat models at 3 d. However, we found that pERK levels remained elevated above the levels of uninjured control arteries at 14 d, with the highest levels observed in the type 1 and type 2 diabetic rats. These findings differ from those of Koyama et al., and this is likely due to the difference in metabolic environments. These data provide further evidence that the metabolic environment influences the activation of the pERK pathway. Our study has revealed limitations. First, different methodologies were used to evaluate two different time points for Akt and MAPK pathway activation. Second, whole-vessel lysate risks the potential of including uninjured control
portions of the artery into treatment groups, thus diluting results from the injured segment. To avoid this possibility, we used only portions of the carotid artery that underwent balloon injury and increased the number of arteries included per rat to compensate for the decreased amount of tissue obtained. Third, lysate does not allow making observations regarding particular arterial layers. Fourth, we did not measure changes in downstream proteins to assess the direct effect of pAkt and pERK level differences. Thus, while the data presented here provide important insights, the potential for future studies on the effects of NO on the insulin signaling pathway in the vasculature of type 1 and 2 diabetic animals is very encouraging. Further studies are necessary to determine how the addition of insulin therapy influences the activation of the Akt and ERK pathways by NO in type 1 and type 2 diabetic rats. Also, obtaining carotid artery lysate at 7 and 14 d following arterial injury would be helpful for making comparisons to lysate at earlier time points; however, there are high costs involved with maintaining the animals necessary to complete this endeavor. In conclusion, the development of neointimal hyperplasia after arterial injury in diabetic populations is of increasing importance as the incidence of diabetes continues to rise. We have shown the efficacy of nitric oxide to be substantially different in type 1 and type 2 diabetic rats, and this may be due to differential activation of Akt and MAPK pathways in insulin-deficient versus insulin-resistant states. Understanding the mechanism by which NO affects the ERK and Akt pathway may help to advance the knowledge of diabetic vascular disease and may provide a mechanism to explore as a potential therapeutic intervention in the future.
Acknowledgment The authors would like to express their thanks to Lynnette Dangerfield for her administrative support, and to Edwards Lifesciences for providing the Fogarty balloon catheters. Funding: This work was supported in part by funding from the Department of Veterans Affairs, VA Merit Review Grant (I01 BX000409, MRK), the Eleanor B. Pillsbury Grant-University of Illinois, the American Medical Association Foundation Seed Grant program (MPR, SSA, VNV), and the EthiconeSociety of University Surgeons Scholarship Award (MPR), and by the generosity of Mrs Hilda Rosenbloom and Mrs Eleanor Baldwin.
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