Ketogenic diet inhibits neointimal hyperplasia by suppressing oxidative stress and inflammation

ABSTRACT

Objective

Neointimal hyperplasia is the primary mechanism underlying atherosclerosis and restenosis after percutaneous coronary intervention. Ketogenic diet (KD) exerts beneficial effects in various diseases, but whether it could serve as non-drug therapy for neointimal hyperplasia remains unknown. This study aimed to investigate the effect of KD on neointimal hyperplasia and the potential mechanisms.

Methods and Results

Carotid artery balloon-injury model was employed in adult Sprague-Dawley rats to induce neointimal hyperplasia. Then, animals were subjected to either standard rodent chow or KD. For in-vitro experiment, impacts of β-hydroxybutyrate (β-HB), the main mediator of KD effects, on platelet-derived growth factor BB (PDGF-BB) induced vascular smooth muscle cell (VSMC) migration and proliferation were determined. Balloon injury induced event intimal hyperplasia and upregulation of protein expression of proliferating cell nuclear antigen (PCNA) and α-smooth muscle actin (α-SMA), and these changes were significantly ameliorated by KD. In addition, β-HB could markedly inhibit PDGF-BB induced VMSC migration and proliferation, as well as inhibiting expressions of PCNA and α-SMC. Furthermore, KD inhibited balloon-injury induced oxidative stress in carotid artery, indicated by reduced ROS level, malondialdehyde (MDA) and myeloperoxidase (MPO) activities, and increased superoxide dismutase (SOD) activity. We also found balloon-injury induced inflammation in carotid artery was suppressed by KD, indicated by decreased expressions of proinflammatory cytokines IL-1β and TNF-α, and increased expression of anti-inflammatory cytokine IL-10.

Conclusion

KD attenuates neointimal hyperplasia through suppressing oxidative stress and inflammation to inhibit VSMC proliferation and migration. KD may represent a promising non-drug therapy for neointimal hyperplasia associated diseases.

KEYWORDS:

• Neointimal hyperplasia
• ketogenic diet
• oxidative stress
• inflammation

Introduction

Neointimal hyperplasia is the primary mechanism underlying atherosclerosis and restenosis after percutaneous coronary intervention (1). The process is complex, involving multiple cell types and vascular wall components, and among them, vascular smooth muscle cells (VSMCs) typically play the most important roles (2,3). In response to vascular injury, inflammatory stimuli or growth factors, VSMCs switch from a quiescent contractile phenotype to an active synthetic phenotype. Synthetic VSMCs migrate and proliferate, and their accumulation directly contributes to neointima formation, and thereafter narrowing of vessels (4). The narrowing of vessels influences oxygen supply, thus resulting in the ischemia of organs including myocardium and brain (5,6). Although many researchers have devoted large amount of effort to the prevention and therapy of neointimal hyperplasia, few clinical strategies with satisfactory safety and efficacy have been developed. Therefore, it is urgent to explore new strategy for the treatment of neointimal hyperplasia.

Ketogenic diet (KD) is a high-fat and low-carbohydrate diet with moderate protein levels. KD increases hepatic ketogenic metabolism and markedly increases hematic β-hydroxybutyrate (β-HB) concentration and has been widely used as an effective non-pharmacological preventive strategy in children with drug-resistant epilepsy (7,8). In addition, recent studies have demonstrated that KD might also be beneficial in many other diseases. For example, it was reported that KD ameliorates oxidative stress and inflammation after spinal cord injury via activating Nrf2 and inhibiting the NF-κB signaling pathways (9). KD was also reported to help improve the spatial memory impairment caused by exposure to hypobaric hypoxia via increasing acetylation of histones in rats (10). Furthermore, KD could increase β-HB and alleviate urate crystal-induced gout without impairing immune defense against bacterial infection (11). These evidences suggest that KD may exert multiple beneficial effects in various pathological process. However, whether KD could inhibit neointimal hyperplasia remains unknown.

In the current study, we induced neointimal hyperplasia in the carotid arteries via balloon-injury and tested whether KD exerts protective effect against neointimal hyperplasia and explored potential mechanisms. We observed that KD treatment could significantly attenuate neointimal hyperplasia in the carotid arteries of balloon-injury rats, as well as inhibit VSMC migration and proliferation. Furthermore, beneficial effect of KD on neointimal hyperplasia could be simulated by β-HB in vitro, as indicated by inhibited VSMC migration and proliferation. For mechanisms, KD might ameliorate neointimal hyperplasia through suppressing oxidative stress and inflammation.

Methods

Arterial injury model and KD treatment

The methods for establishing a rat carotid artery balloon-injury model were described as previously published (12). All animal experiments were performed in accordance with Institutional Animal Care and Use Committee (IACUC) of Henan Provincial Chest Hospital and all protocols complied with the US National Institutes of Health Guide for the Care and Use of Laboratory Animals. Briefly, Sprague-Dawley rats (weight, 240–260 g) were subjected to carotid artery balloon-injury. After anesthetized, a midline incision was performed on the skin of the anterior neck and the left common carotid, external and internal carotid arteries were exposed. Via the external carotid artery, a 2.0 F balloon catheter was introduced and advanced toward the common carotid artery. The balloon was inflated and the common carotid artery was injured by passing the inflated balloon back and forth slowly three times. After that, the catheter was removed and the external carotid arteries and incision were closed. The sham animals underwent the same surgery procedure except balloon-injury. After the surgery, animals were subjected to either standard rodent chow or ketogenic diet for 28 days. The standard diet consists of 18.6% protein, 6.2% fat, 59.8% carbohydrates and 4.5% fiber. The KD consists of 8.4% protein, 78.8% fat, 0.8% carbohydrates and 5% fiber.

Histological examination

Carotid arteries of animals were harvested 28 days after injury and fixed in 4% paraformaldehyde before being embedded in paraffin and sectioned at 5 μm thickness. Next, the sections were stained with hematoxylin and eosin (HE, Sigma) according to standard procedures and observed under a light microscope. Then the images were analyzed using Image J software and ratio of intima/tunica media and neointima area was calculated.

Primary VSMC culture

VSMCs were isolated from the thoracic aorta of adult Sprague-Dawley rats and cultured in Dulbecco’s modified Eagle’s medium (DMEM, Thermo Fisher Scientific) containing 10% fetal bovine serum (FBS, Thermo Fisher Scientific) as previously published (13). All VSMCs were used for experiments between the 3rd and 5th passages.

Cell proliferation and migration assay

For proliferation assay, VSMCs were seeded in 96-well plates at 10⁴ cells per well in 200 ml culture medium. After treated with PDGF (25 ng/mL) or combination of PDGF with various dose of β-HB (5 μM, 10 μM, 15 μM, 20 μM), 20 μl of CCK-8 (Dojindo) was added after 24 h and measured for absorbance at 450 nm.

To measure VSMCs migration activity, Modified Boyden chamber assays and wound scratch assays were performed. For Modified Boyden chamber assays, 10⁵ VSMCs were seeded into the upper chamber in the serum-free medium and treated with PDGF (25 ng/mL) or combination of PDGF with various doses of β-HB (5 μM, 10 μM, 15 μM, 20 μM) for 24 h. Then migrated cells on the bottom of the membrane were fixed in methanol and stained with 0.5% crystal violet. The number of migrated cells was manually counted under a microscope. For wound scratch assays, monolayer confluent VSMCs were growth arrested and scraped, and then treated with PDGF (25 ng/mL) or combination of PDGF with various dose of β-HB (5 μM, 10 μM, 15 μM, 20 μM). Images were captured by microscopy at 24 h after treatment. Wound area was measured to valuate cell migration activity.

Measurement of ROS production and oxidative stress

Reactive oxygen species (ROS) level in carotid artery was evaluated by Dihydroethidium (DHE, Sigma-Aldrich, USA) staining. Frozen carotid artery sections (transversely cut at 5 μm thick) were made two weeks after balloon-injury and incubated with DHE (10 μM) in a light-protected humidified chamber in the dark for 20 min at 37°C. Then the sections were washed with PBS to remove free DHE and images were captured with a fluorescence microscope (Olympus, Japan). To further evaluate oxidative stress, malondialdehyde (MDA), myeloperoxidase (MPO) and superoxide dismutase (SOD) activity in carotid artery two weeks after balloon-injury were examined with commercially available kits (Sigma, USA) according to the manufacturer’s protocols.

Western blot analysis

Western blot analysis was performed to measure protein expression in VSMCs and carotid artery. Artery tissues or cultured VSMCs were homogenized in lysis buffer and the lysates were centrifuged at 12 000 g/min for 30 min, then the supernatants were collected. Bradford protein assay kit was performed to measure protein concentration. 50 μg of protein was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred to polyvinylidine difluoride membranes. The membranes were blocked with 5% nonfat milk at room temperature for 2 h followed by incubation with primary antibodies over night at 4°C. After being washed with TBS, the membranes were treated with corresponding secondary antibodies at room temperature for 2 h. Visualization of bounds was performed using the Odyssey Infrared Imaging System (Li-Cor Biosciences, Lincoln, NE). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and histone H3 were used as internal control.

Quantitative real-time RT-PCR

Quantitative real-time RT-PCR was performed to measure mRNA expression in carotid artery. Total RNA was isolated from artery tissues using TRIzol (Thermo Fisher Scientific) following the manufacturers’ protocols. Reverse transcription was performed with reverse transcriptase enzyme. Then SYBR Select Master Mix (Thermo Fisher Scientific) was used with ABI 7500 Real-Time PCR System for qPCR. GAPDH was used as internal reference and double delta Cq method was performed to calculate the relative expression of mRNA.

Statistical analysis

Data are expressed as Means ± SD and analyzed using SPSS 18.0 statistical package. Comparison of means among more than two groups was performed using one way analysis of variance (ANOVA) followed by the Student-Newman-Keuls (SNK) post hoc test. Comparison of means between two groups was performed using two-tailed Student’s t-test. Statistical significance was defined as P < .05.

Results

KD inhibits neointimal hyperplasia in the carotid arteries of balloon-injured rats

Growing evidence has demonstrated that KD has multiple beneficial effects in many diseases, including colitis (14), Alzheimer’s disease (15), hypertension (16), as well as improves survival and memory in aging rat (17). As abnormal growth of the intimal layer of blood vessels in response to injury is a key event in the development of vascular occlusive diseases, such as atherosclerosis, we wondered whether KD exerts protective effect against neointimal hyperplasia. In the present study, after subjected to balloon-injury of the left common carotid artery or sham operation, rats were treated with standard chow or KD for 4 weeks to determine effect of KD on neointimal hyperplasia. As shown in Figure 1a, the neointima as indicated by the area between the lumen and internal elastic lamina was evident in control rat after injury, compared with that in sham group, indicating successful establishment of animal model of neointimal hyperplasia. In contrast, KD treatment significantly attenuated balloon-injury induced neointimal hyperplasia of carotid arteries, as indicated by decreased ratio of intima/tunica media and neointima area. Abnormal proliferation and migration of VSMCs play pivotal roles in neointimal hyperplasia and development of restenosis after angioplasty and atherosclerosis. We, therefore, further examined effect of KD on expression of PCNA and α-SMC using western blot analysis. The results showed that protein levels of both PCNA and α-SMC in control rat after injury were significantly higher than that in sham group, indicating remarkable proliferation and migration of VSMC (Figure 1b). Interestingly, KD treatment markedly blocked balloon-injury induced increased expressions of PCNA and α-SMC (Figure 1b), suggesting that VSMC proliferation and migration was inhibited by KD. Collectively, these results suggested that KD, through inhibiting VSMC proliferation and migration, exerts a protective effect against neointimal hyperplasia.

Figure 1. Ketogenic diet inhibits neointimal hyperplasia in the carotid arteries of balloon-injured rats. Adult Sprague-Dawley rats were subjected to balloon-injury of the left common carotid artery or sham operation and then treated with standard chow (control) or ketogenic diet (KD) for 4 weeks. (a) Carotid artery tissues were collected and subjected to hematoxylin and eosin staining. Representative histologic sections of carotid arteries were presented (A1, magnification, X100) and ratio of intima/tunica media (A2) and neointima area (A3) in each group were calculated. Scale bars = 200 μM. N = 5. (b) Protein expressions of α-SMA and PCNA in rat carotid arteries were examined by western blot analysis. Representative blots (B1) and quantification of α-SMA (B2) and PCNA (B3) protein expression were shown. N = 6. Data are expressed as means ± SD. *p < .05 vs. control.

β-HB attenuates PDGF-induced VSMC migration and proliferation

Next, we further confirmed effect of KD on VSMC migration and proliferation. As β-HB, a major ketone body in mammals, is the key mediator of beneficial effect of KD (18), we used β-HB in-vitro to simulate in-vivo effect of KD on VSMC migration and proliferation. PDGF-BB was used to stimulated VSMC migration and proliferation and various concentrations of β-HB (5 μM, 10 μM, 15 μM, 20 μM) were administrated. As shown in Figure 2a, Modified Boyden chamber assays demonstrated that PDGF-BB significantly induced VSMC migration, which was consistent with previously study. β-HB could markedly block PDGF-BB induced VSMCs migration via a dose-dependent manner, with minimum effective dose of 10 μM and peaking optimum effect from dose of 15 μM. Furthermore, wound scratch assays of confluent monolayers of VSMCs in the presence of PDGF-BB also showed that β-HB significantly inhibited directional cell migration in response to wound injury with similar range of effective doses (Figure 2b). Then we further examined effect of β-HB on VSMC proliferation using CCK-8 assay and found PDGF-BB-induced proliferation of VMSC was significantly attenuated by β-HB via a dose-dependent manner (Figure 2c). To further confirm effect of β-HB on VMSC migration and proliferation, we also measured protein expression of PCNA and α-SMC using western blot analysis. We found PDGF-BB induced significant increase in protein expressions of PCNA and α-SMC, and it is notably that expressions of both PCNA and α-SMC were marked inhibited by β-HB, via a dose dependent manner (Figure 2d), which was consistent with in-vivo results. Taken together, these results suggested that protective effect of KD on neointimal hyperplasia can be simulated by β-HB in-vitro, further confirming the beneficial effect KD on neointimal hyperplasia.

Figure 2. β-hydroxybutyrate attenuates vascular smooth muscle cell migration and proliferation stimulated by platelet-derived growth factor. Rat vascular smooth muscle cells (VSMCs) were treated with platelet-derived growth factor (PDGF, 25 ng/mL) or combination of PDGF with various dose of β-hydroxybutyrate (β-HB; 5 μM, 10 μM, 15 μM, 20 μM) for 24 h. (a) Cell migration was measured by the modified Boyden chamber assay after stimulation with PDGF. Representative images of migrated cells (A1) were shown and relative cell invasion numbers were calculated (A2). Scale bars = 20 μM. N = 4. (b) Monolayer confluent cells were growth arrested and scraped in the presence of PDGF to stimulate VSMC migration toward the wound area. Representative images of cell migration were shown and relative wound areas were calculated (B2). Scale bars = 50 μM. N = 4. (c) The cell proliferation was measured using Cell Counting Kit-8 after stimulation with PDGF for indicated periods. N = 4. (d) Protein expressions of α-SMA and PCNA in VSMC were examined by western blot analysis after stimulation with PDGF. Representative blots (D1) and quantification of α-SMA (D2) and PCNA (D3) protein expression were shown. N = 4. Data are expressed as means ± SD. *p < .05 vs. control; ^#p <.05 vs. PDGF.

KD attenuates oxidative stress in the carotid arteries of balloon-injured rats

It is widely reported that the vascular injury-induced oxidative stress critically contributes to the VSMC phenotype switch and neointimal hyperplasia, therefore, oxidative stress is regarded as an important therapeutic target in neointimal hyperplasia. To determine whether oxidative stress involves beneficial effect of KD in neointimal hyperplasia, we examined effect of KD on oxidative stress in the carotid arteries of balloon-injured rats. Results of DHE staining showed that ROS level was significantly increased in balloon-injured rats, compared with that in sham group (Figure 3a), indicating event oxidative stress in neointimal hyperplasia, which is consistent with previous study (19). However, KD treatment significantly blunted the increase in ROS level (Figure 3a), indicating an inhibitive effect on oxidative stress in neointimal hyperplasia. In addition, we found MDA and MPO activity was increased in control rat after injury, compared with that in sham group, and KD treatment blocked their activity increases (Figures 3b,c). On the contrary, SOD activity was decreased in control rat after injury, compared with that in sham group, and KD treatment rescued its activity (Figure 3d). Therefore, these data indicate that KD inhibited ROS production and enhanced ROS scavenging, thus attenuating oxidative stress in the carotid arteries of balloon-injured rats.

Figure 3. KD attenuates oxidative stress in the carotid arteries of balloon-injured rats. (a) Reactive oxygen species (ROS) level in carotid arteries from animals 7 days after injury was measured incubation with dihydroethidine (DHE). Representative images of DHE staining were shown (A1) and quantification of fluorescence intensity was performed (A2). Scale bars = 50 μM. N = 4. (b-d) Quantitative analysis of MDA (B), MPO (C) and SOD (D) activity in carotid arteries from animals 7 days after injury was performed. N = 5. Data are expressed as means ± SD. *p < .05 vs. control.

KD attenuates inflammation in the carotid arteries of balloon-injured rats

Besides oxidative stress, inflammation is also a factor that critically contributes to the VSMC phenotype switch and neointimal hyperplasia and thus inflammation is regarded as another important therapeutic target in neointimal hyperplasia. Therefore, we also examined the effect of KD on inflammation in the carotid arteries of balloon-injured rats. In the present study, the mRNA expression levels of proinflammatory cytokines IL-1β and TNF-α were significantly increased in control rat after vascular injury (Figures 4a,b), while expression level of anti-inflammatory cytokine IL-10 was significantly decreased (Figures 4c), indicating event inflammation in the carotid arteries of balloon-injured rats. Interestingly, the alterations of cytokines IL-1β, TNF-α and IL-10 were all blunted by KD (Figures 4a,c), suggesting an anti-inflammatory effect of KD in carotid arteries of balloon-injured rats. In addition, results of western blot analysis confirmed that protein expression levels of proinflammatory cytokines IL-1β and TNF-α were significantly increased in control rat after vascular injury while expression level of anti-inflammatory cytokines IL-10 was significantly decreased (Figure 4d), which was consistent with mRNA expression levels. Meanwhile, the alterations of protein levels of cytokines IL-1β, TNF-α and IL-10 were all blunted by KD (Figure 4d), further confirming the anti-inflammatory effect of ketogenic diet in carotid arteries of balloon-injured rats. We also observed that balloon-injury induced nuclear upregulation of the vital proinflammatory transcription factor NF-κB, and the change was blocked by KD (Figure 4e). Taken together, these results suggested that KD attenuates inflammation in the carotid arteries of balloon-injured rats and this property may be important mechanism for beneficial effect of KD in neointimal hyperplasia.

Figure 4. Ketogenic diet attenuates inflammation in the carotid arteries of balloon-injured rats. (a-c) The mRNA expressions of inflammatory cytokines IL-1β (A), TNF-α (B) and IL-10 (C) in the carotid arteries tissues were measured using Qrt-PCR. N = 5. (d) Protein expressions of IL-1β, TNF-α and IL-10 in the carotid arteries tissues were examined by western blot analysis. Representative blots (D1) and quantification of IL-1β (D1), TNF-α (D2) and IL-10 (D3) protein expression were shown. N = 5. (e) Protein expressions of nuclear NF-κB in the carotid arteries tissues were examined by western blot analysis. Representative blots (E1) and quantification of nuclear NF-κB (E2) protein were shown. N = 5. Data are expressed as means ± SD. *p < .05 vs. control.

Discussion

The KD is a high-fat, low-carbohydrate diet with adequate protein and calories originally developed in the 1920s and it has been applied for over 100 years as a non-drug treatment for intractable epilepsy (20). KD is intended to increase ketone bodies (KB) synthesis and utilization. Ketogenesis, mostly occurring in the liver, leads to the synthesis of β-HB and acetoacetate (ACA), two main KB, from mitochondrial acetyl-CoA pool (21). Ketone metabolism has been shown to result in cellular changes that could potentially contribute to its neuroprotective properties (22). For example, enhanced ketone metabolism could improve mitochondrial respiratory complex activity and decrease oxidative stress (23). In recent years, more and more beneficial effects have been discovered for KD in many diseases. For example, KD is reported to alleviate bleomycin-induced pulmonary fibrosis in murine models by regulating autophagy and PI3K/AKT/mTOR signaling pathway, providing a novel therapeutic strategy for pulmonary fibrosis (24). Large evidences have also demonstrated KD as a promising opportunity to modulate the cellular metabolism of cancer cells and thus serve as an effective non-drug therapy for various types of cancer, such as nuroblastoma, melanoma and colon adenocarcinoma (25). However, whether KD also exerts beneficial effects against neointimal hyperplasia remains largely unknown. Our present study found KD could effectively attenuates neointimal hyperplasia in the carotid arteries of balloon-injured rats, and this effect can be simulated by β-HB in vitro, thus we speculate that KD may represent a promising non-drug therapy for neointimal hyperplasia associated diseases.

It is widely recognized that abnormal proliferation and migration of VSMCs are the underlying mechanism of neointimal hyperplasia (26). Therefore, VSMC proliferation and migration are the main targets for developing new strategies for prevention and therapy of neointimal hyperplasia. For example, semaphorin-3A protects against neointimal hyperplasia after vascular injury via suppressing VSMC proliferation and migration, as well as increasing differentiated gene expression (27). Overexpression of Cbx3 also attenuates injury-induced neointima formation via inhibiting Notch3 signaling to reduce VSMC proliferation and migration (28). Consistent with these previous studies, we found KD significantly blocked balloon-injury induced increase in expressions of PCNA and α-SMC in vivo. In addition, we also observed that β-HB, the main mediator of KD effects, could markedly inhibit PDGF-BB induced VMSC migration and proliferation, as well as inhibiting expressions of PCNA and α-SMC.

Although mechanisms involve in VSMC activation and phenotype switch from a quiescent contractile to active synthetic are complex, arterial oxidative stress and inflammation paly essential roles. Therefore, arterial oxidative stress and inflammation are important targets for inhibiting VSMC activation and neointimal hyperplasia. For example, cholinergic anti-inflammatory pathway could ameliorate neointimal hyperplasia by inhibiting inflammation and oxidative stress (29). Activation of the redox-sensitive transcription factor, nuclear factor erythroid 2-related factor 2 (Nrf2), could inhibiting oxidative stress via transcriptional upregulation of antioxidant proteins, thus attenuating VSMC migration and neointimal hyperplasia (30). Metformin could inhibit VSMC proliferation and migration via attenuating insulin resistance as well as inflammation (31). PPARγ inhibits VSMC proliferation and migration by suppressing TLR4-mediated inflammation and ultimately attenuates intimal hyperplasia after carotid injury (32). We also observed that KD treatment could reduce ROS level, MAD and MPO activity and increase SOD activity, thus we speculate that KD may exerts its anti-neointimal hyperplasia effect, at least to some extent, via inhibiting oxidative stress. In addition, KD reduced expressions of proinflammatory cytokines TNF-α while enhanced expression anti-inflammatory cytokine IL-10 in balloon-injured carotid arteries, suggesting that anti-inflammatory effect of KD may also involve in its anti-neointimal hyperplasia effect. These findings are consistent with previous studies, which demonstrated anti-oxidative stress and anti-inflammatory effect of KD (9,21,23,33).

In summary, KD treatment could significantly attenuate neointimal hyperplasia in the carotid arteries of balloon-injury rats, as well as inhibit VSMC migration and proliferation. For mechanisms, KD might ameliorate neointimal hyperplasia through suppressing oxidative stress and inflammation. Thus, our study suggests KD as a promising non-drug therapy for neointimal hyperplasia. Additional experimental studies are needed to further reveal underlying mechanism of beneficial effects of KD on neointimal hyperplasia and improve the efficacy of KD in attenuating neointimal hyperplasia.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

The author(s) reported there is no funding associated with the work featured in this article.