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Niacin Suppresses Progression of Atherosclerosis by Inhibiting Vascular Inflammation and Apoptosis of Vascular Smooth Muscle Cells

Background Niacin is a broad-spectrum lipid-regulating drug used for the clinical therapy of atherosclerosis; however, the mechanisms by which niacin ameliorates atherosclerosis are not clear. Material/Methods The effect of niacin on atherosclerosis was assessed by detection of atherosclerotic lesion area. Adhesion molecules in arterial endothelial cells were determined by using qRT-PCR and Western blot analysis. The levels of serum inflammatory cytokines in ApoE−/− mice were detected by using ELISA. We detected the expression levels of phosphorylated nuclear factors-κB (NF-κB) p65 in aortic endothelial cells of mice using Western blot analysis. Furthermore, we investigated the anti-inflammation effect and endothelium-protecting function of niacin and their regulatory mechanisms in vitro. Results Niacin inhibited the progress of atherosclerosis and decreased the levels of serum inflammatory cytokines and adhesion molecules in ApoE−/− mice. Niacin suppressed the activity of NF-κB and apoptosis of vascular smooth muscle cells (VSMCs). Furthermore, niacin induced phosphorylated focal adhesion kinase (FAK) and FAK inhibitor PF-573228 reduced the level of Bcl-2 and elevated the level of cleaved caspase-3 in VSMCs. Conclusions Niacin inhibits vascular inflammation and apoptosis of VSMCs via inhibiting the NF-κB signaling and the FAK signaling pathway, respectively, thus protecting ApoE−/− mice against atherosclerosis.


Background
Atherosclerosis, the main cause of death in developed and some developing countries, is a systemic inflammatory disease characterized by the formation of atherosclerotic plaques [1]. Hyperlipidemia and obesity may increase the risk of atherosclerosis by triggering chronic inflammation of blood vessels [2]. The cell adhesion molecules, including intercellular adhesion molecule-1 (ICAM-1), E-selectin and vascular cell adhesion molecule-1 (VCAM-1), and oxLDL are involved in the initiation and development of atherosclerosis, and oxidized low density lipoprotein (oxLDL) can enhances hypoxia-reoxygenation-mediated apoptosis of human coronary artery endothelial cells (HCAECs) [3,4]. During the initiation of atherosclerosis, expression levels of cellular adhesion molecules are up-regulated by pro-inflammatory cytokines [5]. Monocytes adhere to vascular endothelial cells and accelerate the formation of plaque in atherosclerosis [6]. Nuclear factors-kB (NF-kB) has been proved to regulate expression of the inflammatory mediators in atherosclerosis and numerous negative regulators of NF-kB have been identified [7,8]. Caspase-3 is a dominant executioner caspase, which plays a central role in the execution-phase of cell apoptosis. Previous studies have shown that caspase-9 and cleaved caspase-3, but not caspase-8, play a key function in the ox-LDLinduced apoptotic signaling pathway in HCAECs [4]. Elucidating the molecular mechanisms of inflammation in atherosclerosis will contribute to identifying better therapeutic strategies.
Niacin (nicotinic acid, vitamin B3) can lower blood fat and prevent atherosclerosis, which is confirmed in large-scale clinical trials [9,10]. Peroxisome proliferators-activated receptor g (PPARg), a transcription factor for several genes, is involved in lipid metabolism. Niacin stimulates the ATP-binding cassette transporter A1 in monocytes and macrophages, up-regulates PPARg, and ultimately results in reverse cholesterol transport [11]. Kamanna et al. provided direct evidence for anti-inflammatory properties of niacin in human aortic endothelial cells (HAECs) [9]. A study in guinea pig showed that niacin inhibited vascular inflammation by down-regulating the NF-kB signaling pathway [12]. However, how niacin participates in anti-inflammation and protecting endothelium in atherosclerosis remains largely unknown.
High concentrations of pro-inflammatory cytokines increase oxidative stress, down-regulate endothelial nitric oxide synthase (eNOS) bioactivity and induce endothelial cell apoptosis [13]. Vascular smooth muscle cells (VSMCs) are structural components of atherosclerotic plaque caps, and they play an important role in protection of plaque stability [14]. DNA damage has been identified in human VSMCs and alters plaque phenotype by inhibiting fibrous cap areas in advanced lesions [15,16]. Damaged smooth muscle cells will affect vascular tone and ultimately lead to plaque instability. Wang et al. found that VSMC apoptosis was an early trigger for hypothyroid atherosclerosis [17]. Oxidized LDL has been shown to promote VSMC apoptosis in culture media, in part by down-regulation of Bcl-2 and activation of caspase-3 [18]. Interleukin 1b (IL-1b) is a pro-inflammatory cytokine secreted by various cell types, including endothelial cells [19]. IL-1b acts on endothelial cells which line the arterial wall to up-regulate adhesion molecules during the initiation phase of atherosclerosis and drives smooth muscle cell expression and activation of proteases, such as matrix metalloproteinases, leading to plaque rupture and resulting occlusive thrombosis, in the progression and rupture of atherosclerotic plaques [20]. VSMC apoptosis promotes both thrombin generation and vascular calcification [21,22]. In this study, we used ApoE -/mice with high fat diet to explore the protective mechanisms of niacin in atherosclerosis. In human aortic endothelial cells (HAECs), regulatory effect of niacin on the NF-kB signaling pathway was deeply investigated. In addition, the protective effect of niacin against VSMCs apoptosis was investigated.

Animals and induction of atherosclerosis
Twelve 8-week-old male ApoE -/mice with a C57BL/6J background (weighing 20-25 g) were purchased from Beijing Biocytogen (Beijing, China). Mice were kept in a temperature-controlled room with a 12-hour light/dark cycle and fed a high-fat diet composed of 20% fat, 20% sugar, and 1.25% cholesterol (Ssniff special diets, Soest, Germany) with free access to water for 10 weeks. At the same time, six of them were fed with niacin (100 mg/kg) (Sigma-Aldrich, St Louis, MO, USA) by oral gavage once daily for 10 weeks. The use of animals was in strict accordance with Guidance Suggestions for the Care of Laboratory Animals published by the Chinese Ministry of Science and Technology. The animal experimental protocol was approved by the Institutional Animal Care and Use Committee of the First Affiliated Hospital of Zhengzhou University. The number and suffering of animals was as minimized as much as possible.

Oil red O staining
The aortas were stored at -20°C for subsequent analysis. The aortic section was soak in 60% isopropanol (Aladdin, shanghai, China) for 25 s and then stained with oil red O (Sigma-Aldrich) for 8 min. Next, redundant dye was removed by soaking the slide in 60% isopropanol for10 s again. To measure the lesion area in the aortic root, sections of the aortic root were soaked in 60% isopropanol for 30 s and then in oil red O for 20 min. Rinsed sections were counterstained with hematoxylin (Sigma-Aldrich). The entire inner surface of aortic intima and sections of aortic roots were photographed, and positive lesions were analyzed using ImageJ software (NIH, Bethesda, MD, USA). Data were expressed as average lesion size in percentage of the total aortic surface or as the absolute lesion area in square micrometers.

Cell culture and treatment
HAECs (ATCC) were maintained in endothelial basal medium-2 and supplemented with 2% fetal bovine serum (FBS, Gibco, Carlsbad, CA, USA) in a 5% CO 2 /95% air incubator at 37°C. The medium was changed every two days until the cells became confluent. To study the Anti-inflammatory effect of niacin on HAECs, HAECs (1×10 5 /ml) were incubated with platelet (1.5×10 7 /ml) or 25 mM D-glucose in the absence or presence of niacin (1 mM) for 24 h. The expression levels of TNF-a, IL-1b, and IL-6 were measured by using Western blot analysis. HAECs (1×10 5 /ml) were incubated with high glucose (25 mM D-glucose) in the absence or presence of niacin (1 mM) for 24 h. The expression level of MCP-1 was determined using Western blot analysis.
Mouse aortic SMCs were isolated and maintained as described previously [23]. To study the effect of the FAK signaling on apoptosis of VSMCs, VSMCs were treated with 50 µM of focal adhesion kinase (FAK) inhibitor PF-573228 for 24 h. The levels of Bcl-2, pro-caspase-3, and cleaved caspase-3 were determined using Western blot analysis.

Preparation of oxLDL
Native LDL was obtained from Sigma. Native LDL (200 µg protein/ml) was oxidized by exposure to CuSO4 (5 µmol/l free Cu 2+ ) in phosphate-buffered saline at 37°C for 20 h. Control incubations were done in the presence of 200 µmol/l EDTA without CuSO4. Oxidation was terminated by refrigeration. Oxidation of LDL was identified using thiobarbituric acid-reactive substances (TBARS) with malonaldehyde bis (dimethyl acetal) as the standard. The levels of malondialdehyde (MDA) were detected by a spectrophotometric measurement of TBARS, depending on kit (Nanjing Jiancheng Bioengineering Institute, China). Protein concentration was measured by bicinchoninic acid (BCA) kit (Pierce Chemical, Rockford, IL, USA).

Measurement of inflammatory factors in vivo
According to the instructions of ELISA kit (R&D systems, Minneapolis, MN, USA), the serum levels of IL-6, TNF-a, MCP-1 and IL-1b which can be secreted during the process of inflammation were measured following the manufacturer's instructions. Results were calculated as pg/L.

Luciferase reporter assay
HAECs seeded in 24-well plates (2×10 5 per well) were cotransfected with NF-kB promoter-luciferase vector (Promega, Madison, WI, USA) for 14 h. Then, the transfected cells were incubated in presence of 2 ng/ml of TNF-a with or without 1 mM niacin for an additional 12 h. HAECs were washed with PBS and then lysed in a reporter lysis reagent (Promega). The luciferase activity in cell lysate was determined by the dualluciferase reporter assay system (Promega).

Data analysis
Unpaired Student's t-test was used to analyze differences between two groups. ANOVA was used to compare the means of three or more groups. All the data are presented as the means ±SD. A value of P<0.05 was considered to indicate a statistically significant difference. All data analyses were performed using the SPSS version 16.0 software (SPSS Inc., Chicago, IL, USA).

Niacin suppressed atherosclerosis in ApoE -/mice
To test whether niacin alleviates atherosclerosis, we measured the plaque area of aortic arches and staining area of aortic roots. Using oil red O staining, we found that aortic arches plaque area (%) ( Figure 1A, 1B) and aortic root slice staining area ( Figure 1C, 1D) of ApoE -/mice fed niacin significantly decreased compared with the control group. The data suggest that niacin has an inhibitory effect on the initiation and progression of atherosclerosis in ApoE -/mice.

Niacin restrained the expression of adhesion molecules in ApoE -/mice
To explore the inhibition mechanism of niacin on the progression of atherosclerosis in mice, we determined the expression of adhesion molecules in ApoE -/mice after treatment with niacin by using qRT-PCR and Western blot assay. Significantly decreased mRNA (Figure 2A) expression levels of ICAM-1, Eselectin and VCAM-1 were observed in niacin group compared with control group and the protein ( Figure 2B) expression levels were obviously lower than that in control group.

Niacin decreased the levels of inflammatory cytokines in vivo
Previous studies have strongly suggested that inflammatory factors are involved in the initiation and progression of coronary atherosclerotic diseases. In this study, the concentrations of four major inflammatory cytokines (IL-1b, IL-6, TNF-a, and MCP-1) were determined by ELISA. As shown in Figure 3A-3D, the serum levels of TNF-a, IL-1b, IL-6 and MCP-1 in ApoE -/mice treated with niacin decreased significantly versus the control group.

Inflammatory responses in vascular endothelial cells were inhibited by niacin
The activation of the NF-kB signaling pathway is known to result in the production of inflammatory cytokines and chemokines, which is constitutively activated in atherosclerosis [24]. To assess the role played by niacin in the NF-kB signaling, we  This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License evaluated the phosphorylation of NF-kB subunit p65 by Western blot analysis. In endothelial cells of ApoE -/mice treated with niacin, we found that p65 protein phosphorylation was significantly decreased in contrast to the control group. However, there were no changes in p65protein expression ( Figure 4A).
To further affirm the anti-inflammation effect of niacin, experiments in HAECs were conducted. In the platelet-stimulated HAECs, we found that the inflammatory cytokines including IL-1b, IL-6 and TNF-a increased; In contrast, HAECs co-cultured with niacin showed dramatically lower levels of inflammatory cytokines ( Figure 4B). High glucose accelerates MCP-1 production [25][26][27][28]; In the presence of high glucose and niacin, the MCP-1 level of HAECs was lower than high glucose group ( Figure 4C). Western blot results demonstrated that niacin significantly decreased p-p65 overexpression induced by platelet or high glucose ( Figure 4D, 4E). To further verify the inhibitory effect of niacin on NF-kB, we examined the effect of niacin on the TNF-a-induced NF-kB activation in HAECs by luciferase reporter assay. As shown in Figure 4F, TNF-a-induced NF-kB transcriptional activity in HAECs was significantly inhibited by niacin. In summary, the above results indicated that niacin reduces the degree of inflammation in vascular endothelial cells via inhibiting NF-kB signaling pathway.

Niacin attenuated oxLDL-induced apoptosis of vascular smooth muscle cells
We determined the MDA levels in nLDL and oxLDL in order to guarantee the experimental rigor. Compared with that of the nLDL group, the level of MDA was significantly increased in ox-LDL group ( Figure 5A). When VSMCs were exposed to oxLDL, we observed a significant increase in the number of apoptotic cells and this increase was significantly reduced in the presence of niacin ( Figure 5B). The expression level of Bcl-2, pro-caspase-3 and cleaved caspase-3 in each treatment group were detected. The result showed that oxLDL notably decreased the expression of Bcl-2 and elevated the expression level of cleaved caspase-3, but niacin reversed the effects of oxLDL. The expression level of pro-caspase-3 was not affected by nLDL, oxLDL and niacin ( Figure 5C). To examined whether the FAK signaling participates in oxLDL-induced apoptosis of VSMCs and whether niacin inhibits apoptosis of VSMCs via blocking the FAK signaling, VSMCs were incubated with nLDL, oxLDL, oxLDL+0.25/0.50/1.00 mM niacin and the expression levels of p-FAK and FAK protein were detected. We observed a significant decrease of p-FAK after treatment of oxLDL compared with the control or nLDL group. Moreover, niacin increased the level of p-FAK in a concentrationdependent manner. However, no effects were observed on FAK expression levels in VSMCs treated under different conditions ( Figure 5D). To further confirm the effect of the FAK signaling on apoptosis of VSMCs, we determined the expression of Bcl-2 and caspase-3 in VSMCs after treatment with FAK inhibitor PF-573228. The results exhibited that PF-573228 inhibited the expression of Bcl-2 and enhanced the expression of cleaved caspase-3 ( Figure 5E (F) Luciferase reporter assay were used to detect the activity of NF-kB in HAECs. The relative luciferase activity was increased after treatment with TNF-a. However, niacin decreased the relative luciferase activity, showing that niacin inhibits the NF-kB signaling pathway. Data presented as mean ±SD (n=4

Discussion
Although many factors, such as aging, gender, smoking, diabetes, dyslipidemia, hypertension, physical and psychological stressors, and genetic factors are reported to have connection with atherosclerotic disease, the origination of atherosclerosis remains obscure [29]. A large number of researches have shown that atherosclerosis is a chronic inflammatory disease characterized by the presence of plaques and apoptosis of VSMCs [30]. Niacin is a broad-spectrum lipid drug, which has beneficial effects on plasma lipoproteins and has demonstrated clinical benefits in reducing cardiovascular events and   [31]. Niacin has been studied in 6 major clinical trials with cardiovascular endpoints and these clinical trials provide strong and consistent evidence that the use of niacin prevents atherosclerotic cardiovascular disease [32]. Numerous research studies have provided evidence that niacin has protective effects on cardiovascular disease through its vascular anti-inflammatory and antioxidant properties in addition to its function on regulating lipid metabolism. The ability of niacin to lower plasma levels of cholesterol was discovered 60 years ago by Rudolf Altschul [33]. The treatment with niacin, alone or in combination with other lipid-lowering agents, significantly reduces total mortality and coronary events and slows down the progression of coronary atherosclerosis [34]. Moreover, niacin attenuates obesity-induced adipose tissue inflammation and reduced pro-inflammatory cytokine expression in high fat diet-fed mice [35]. Besides its effects on lipid regulation, more and more researchers pay attention to the pharmacological potential of niacin. In the present study, the first part of results showed that niacin decreased the plaque area of atherosclerotic mice. In order to further explain the protection mechanism of niacin in the occurrence and development of atherosclerosis, we observed the effects of niacin on the expression of adhesion molecules and inflammatory cytokines in ApoE -/mice. We provided evidence that niacin inhibited inflammatory responses and apoptosis of VSMCs through regulating the NF-kB and FAK signaling pathway, respectively. NF-kB is a transcription factor that activates inflammation through regulating the gene expression of cytokines and adhesion molecules [36][37][38]. NF-kB causes early phenomenon in AS and the disruption of NF-kB activation has been shown to delay or prevent atherogenesis [39]. Oh et al. have shown that lipopolysaccharide-induced pro-inflammatory responses by Buddleja officinalis could be inhibited via negative regulation of the NF-kB and ERK1/2 signaling pathway. On the contrary, NF-kB activation promoted inflammation by TNF-a and IL-6 upregulation in serum [40]. So, we speculate that niacin suppresses inflammation via inhibiting the NF-kB signaling pathway. In human aortic endothelial cells in vitro, Ganji's team found that niacin inhibited reactive oxygen species (ROS) production, low density lipoprotein (LDL) oxidation, TNF-a-induced NF-kB activation, VCAM-1, MCP-1 secretion, and TNF-a-induced monocyte adhesion to HAECs in varying degrees [41], but it was unknown whether niacin had the same effects or not in vivo. Phosphorylation of several serine residues in p65 has been proved to be crucial for full activation of NF-kB [42]. To verify its function in vivo, ApoE -/mice treated with niacin or not were studied. Phosphorylation level of p65 in aortic endothelial cells of mice was detected by immunoblot. We found that, as expected, niacin reduced the degree of inflammation via inhibiting the NF-kB signaling pathway.
A main function of VSMCs is to regulate the caliber of the blood vessels in the body. VSMCs compose the majority of atherosclerotic plaque caps, and they maintain the stability of plaque [14]. Apoptosis of VSMCs and macrophages were found in unstable plaques. To further confirm the protection of niacin on VSMCs, its effect on oxLDL-induced apoptosis of VSMCs was studied. OxLDL plays an important role in the development of atherosclerosis and under condition of high levels of ox-LDL, monocytes are converted to activated macrophages. As a nonreceptor tyrosine kinase, FAK plays crucial roles in intracellular regulatory events, such as cell adhesion, proliferation, survival, angiogenesis and migration [43]. Peng et al. highlighted the in vivo functions of FAK in vascular endothelial cells [44]. Through complicated molecular mechanisms, FAK influences the cytoskeleton, structures of cell adhesion sites and membrane protrusions to regulate cell movement [45]. Blocking the FAK pathway is a key event in the inhibition of apoptosis induced by oxLDL. Our Western blot results indicated that niacin up-regulated the level of p-FAK in VSMCs. Si et al. found that niacin was able to inhibit vascular inflammation via down-regulating NF-kB signaling pathway in guinea pigs and human umbilical vein endothelial cells (HUVECs). Moreover, niacin attenuated oxLDL-induced apoptosis of HUVECs as well [12]. However, the protective mechanism of niacin against apoptosis of VSMCs remains unclear. In this paper, the regulation of niacin on FAK signaling pathway in apoptosis was studied. The data revealed that niacin induced phosphorylation of FAK in a concentration-dependent manner.
In summary, we verified that niacin alleviates atherosclerosis through restraining the expression of adhesion molecules and inflammatory cytokines secretion in serum. Niacin inhibits overexpression of TNF-a, IL-1b, IL-6, MCP-1 and p-p65 in HAECs treated with platelet or high glucose. The anti-inflammatory property of niacin is realized by down-regulating the NF-kB signaling pathway. Additionally, our data suggest that niacin attenuates oxLDL-induced apoptosis of VSMCs, accompanied by expression changes of Bcl-2, cleaved caspase-3 and p-FAK. However, the limitations of our study should be considered. The sample size of animals, a group of 6 mice only, is small. The molecular mechanisms by which niacin alleviates atherosclerosis should further explore. Together, these results indicated that niacin suppresses progression of atherosclerosis by inhibiting vascular inflammation and apoptosis of vascular smooth muscle cells.

Conclusions
Niacin inhibits vascular inflammation and apoptosis of VSMCs via inhibiting the NF-kB signaling and the FAK signaling pathway, respectively, thus protecting ApoE -/mice against atherosclerosis.