Clopidogrel Protects Endothelium by Hindering TNFα-Induced VCAM-1 Expression through CaMKKβ/AMPK/Nrf2 Pathway

Clopidogrel (INN), an oral antiplatelet drug, has been revealed to have a number of biological properties, for instance, anti-inflammation and antioxidation. Oxidative stress plays an imperative role in inflammation, diabetes mellitus, atherosclerosis, and cancer. In the present study, human aortic endothelial cells (HAECs) were employed to explore the anti-inflammatory activity of INN. INN reduced TNFα-induced reactive oxygen species (ROS) generation and time-dependently prompted the expression and activity of heme oxygenase 1 (HO-1). Cellular glutathione (GSH) levels were augmented by INN. shHO-1 blocked the INN suppression of TNFα-induced HL-60 cell adhesion. The CaMKKβ/AMPK pathway and Nrf2 transcriptional factor were implicated in the induction of HO-1 by INN. Additionally, TNFα dramatically augmented VCAM-1 expression at protein and mRNA levels. INN treatment strikingly repressed TNFα-induced expression of VCAM-1 and HL-60 cell adhesion. Compound C, an AMPK inhibitor, and shNrf2 abolished TNFα-induced expression of VCAM-1 and HL-60 cell adhesion. Our data suggest that INN diminishes TNFα-stimulated VCAM-1 expression at least in part via HO-1 induction, which is CaMKKβ/AMPK pathway-dependent.


Introduction
Inflammation is common for patients with cardiovascular diseases and is considered to be a sign or atherogenic response. Reactive oxygen species (ROS) such as superoxide anions, hydrogen peroxide, peroxynitrite, and hydroxyl radicals plays an important role in inflammation, leading to endothelial oxidative damage and dysfunction of the cardiovascular system. Proinflammatory cytokines, for example, tumor necrosis factor-alpha (TNF ), are able to stimulate ROS liberation [1]. Superfluous ROS production not only causes endothelial dysfunction [2] but also stimulates signaling transduction pathways implicated in augmented gene expression of inflammation-related cytokine [3]. In cardiovascular cells, the normal intracellular ROS levels rely on the proper balance between ROS formation and antioxidant defense systems.
Sophisticated interactions between leukocytes and the endothelium are involved in inflammatory reaction. The endothelial cell (EC) surface is relatively smooth and nonadhesive. In cardiovascular diseases, the interactions between the ECs and components of the blood are altered by adhesion molecules, for example, ICAM-1 and VCAM-1 [4]. The endothelial cells are activated, leading to overexpression of adhesion molecules at inflammation sites. The proinflammatory molecule TNF-prominently elevates adhesion molecules on the endothelium [5][6][7]. Additionally, substantial evidence demonstrates an augmented expression of VCAM-1 in inflammatory animal models and human atherosclerotic plaques [8].
Clopidogrel (INN) is an oral antiplatelet drug hindering blood clots and has been discovered to possess several biological properties such as anti-inflammation, antioxidation, and antiatherosclerosis [19][20][21]. Anti-inflammation and antioxidation can be used as therapeutic strategies to prevent or treat cardiovascular diseases. The present study was carried out to elucidate the role of AMPK and HO-1 in the INN inhibition of TNF -stimulated expression of VCAM-1 and the underlying mechanisms implicated.

Western
Blotting. ECs were lysed in the lysis buffer. Equivalent amounts of protein samples were separated by SDS-PAGE and then transferred to nitrocellulose membrane followed by immunoblotting with the primary antibodies including phosphor-AMPK-Thr172, AMPK, HO-1, Nrf2, PARP, and -actin.

HO-1 Activity
Assay. HO-1 activity was determined as described previously [23]. The HO-1 activity in EC lysates was calculated as picomoles bilirubin produced per hour per milligram of total protein (pmol BR h-1 mg-1) and the data were expressed as fold HO-1 activity compared to control cells.

Subcellular
Fractionation. Subcellular fractionation was performed with a Subcellular Protein Fractionation Kit (Thermo Fisher Scientific Inc., Rockford, IL, USA) according to the manufacturer's protocol. Nrf2 protein levels in the nuclear fractions were measured by Western analysis. Expression of PARP was utilized as loading controls for the purity of the nuclear extracts.

Reactive Oxygen Species (ROS) Measurement.
Intracellular ROS state was determined with the cell-permeant 2 ,7 -dichlorodihydrofluorescein diacetate (H2DCFDA) as previously described [25]. Concisely, HAECs were grown to 50% confluence followed by serum starvation in medium 199 supplemented with 0.5% (v/v) FBS for another 24 h. The ECs were maintained in serum-free medium without phenol red for 15 min preceding being exposed to TNF . ECs were incubated with H2DCFDA (10 M) for 10 min and instantly observed under a confocal microscope (Leica TCS SP2).

Cellular GSH Assay.
Cellular GSH assay was performed as described previously [26]. Briefly, ECs were washed two times with ice cold PBS. The homogenate was prepared in the potassium phosphate buffer (20 mM, pH 7.0) and centrifuged at 10,000 ×g for 20 min at 4 ∘ C. The supernatant was kept as the cell lysate. The protein was measured with a BCA Protein Assay Kit. Cell lysates (100 L) were incubated with 5% TCA (150 L) and centrifuged at 5000 ×g for 10 min at 4 ∘ C. The cell lysate was incubated with 0.4 M Tris buffer and 0.01 M DTNB. After incubation at room temperature for 5 min, the intracellular GSH production was measured with a microplate reader at 412 nm (Model 680, Bio-Rad).
2.9. Monocyte Adhesion Assay. The monocyte adhesion assay was executed as described by Chen et al. [27].

Statistical Analysis.
The results were expressed as mean ± SD. Data were analyzed with one-way analysis of variance and Fisher's protected least significant difference test. < 0.05 was considered as the level of significance.

Effect of Clopidogrel (INN) on TNF -Induced ROS Formation in HAECs.
To determine whether INN decreases TNF -stimulated ROS formation, we pretreated ECs with 10 mol/L INN for 24 h and then treated ECs with 1 ng/mL TNF for another 20 min. As illustrated in Figure 1, TNF promoted ROS liberation at 20 min, and pretreatment with 10 mol/L INN dramatically repressed this ROS liberation. N-Acetylcysteine (NAC) is a potent antioxidant and used as a positive control.

The CaMKK /AMPK Pathway Mediates INN-Induced
HO-1 Expression. Previous studies [30] suggest that AMPK modulates the antioxidant status of cardiovascular ECs by upregulating expression of genes implicated in antioxidant defense, for example, manganese superoxide dismutase, catalase, and thioredoxin. To determine the signaling pathways implicated in the INN-mediated HO-1 induction, we examined the phosphorylation of AMPK. ECs were incubated with INN (10 M) for different time periods. As illustrated in

AMPK and Nrf2 Are Responsible for INN Inhibition of TNF -Stimulated VCAM-1 Expression and Monocyte Adhesion in HAEC.
Next, we sought to determine whether INN hinders TNF -induced VCAM-1 activation. As indicated in Figure 5, TNF significantly increased expression of VCAM-1 and HL-60 cell adhesion. INN treatment dramatically suppressed TNF -induced VCAM-1 expression at mRNA and protein levels as well as HL-60 cell adhesion. To further identify the role of AMPK and Nrf2 in the TNF -stimulated VCAM-1 expression, we then used Compound C to inhibit AMPK and shNrf2 to knock down Nrf2 expression. As illustrated in Figure 5, Compound C and shNrf2 abolished TNFtriggered VCAM-1 expression and HL-60 cell adhesion. Together, these results suggest that AMPK and Nrf2 mediate INN inhibition of TNF -stimulated VCAM-1 expression and monocyte adhesion in HAEC.

Discussion
Improvement of intracellular antioxidant ability is thought to diminish the risk of oxidative stress-induced diseases. Clopidogrel (INN) is an oral antiplatelet drug used to impede blood clots in coronary artery disease (CAD). Previous studies have shown that INN has anti-inflammation and antioxidation activities [19][20][21]. Recent work has demonstrated that INN augments nitric oxide (NO) and prostacyclin production in endothelial cells [31]. In addition, INN impedes CD40 ligand both in vitro and in vivo [32], stimulating HO-1 expression [33]. These findings suggest that INN can preserve endothelial function via a mechanism independent of its antiplatelet activity. in type-2 diabetes patients [34]. The effect of INN was to improve vascular function, protect against oxidative stress, and inhibit apoptosis in patients with type-2 diabetes. This involved an increase in the expression of both phosphorylated Akt and AMPK. In the present study, we revealed that INN repressed TNF -stimulated ROS liberation, VCAM-1 expression, and cell adhesion and that this effect was related to the increase in HO-1 expression and GSH content via the CaMKK /AMPK/Nrf2 pathway.
TNF is a powerful proinflammatory cytokine secreted by various innate immune cells, predominantly activated macrophages, as well as neutrophils, mast cells, and eosinophils cells [35][36][37][38][39]. In macrophages, inflammatory stimuli result in TNF synthesis and release by constitutive exocytosis [38]. The plasma levels of TNF are augmented in some pathologies, such as cancer, atherosclerosis, rheumatoid arthritis, and preeclampsia [40]. Several studies have revealed that TNF , via activation of NF B, induces expression of proinflammatory cytokines and adhesion molecules, for example, IL-6 and VCAM-1 [41,42]. In the current study, we revealed that TNF increased ROS production, VCAM-1 expression, and cell adhesion. Intriguingly, these effects of TNF were abolished by INN. Heme oxygenase or haem oxygenase (HO) is a stressinducible enzyme. Increasing evidence supports that it protects against many chronic diseases such as cardiovascular 8 Journal of Diabetes Research diseases, hypertension, diabetes mellitus, and neurological disorders [43]. Using systemic manipulation of either HO-1 expression or activity, some studies suggested that HO-1 plays a vital role in atherosclerosis initiation and development. HO-1-null mice show a noteworthy increase in plasma lipid hydroperoxides [44]. Similarly, rabbits treated with SnPP, an HO-1 inhibitor, exhibit a significant lipid deposits in abdominal aortic plaques [45]. Contrarily, HO-1 induction alleviated the oxLDL-induced formation of foam cells [46]. Taken together, these studies substantiated the anti-inflammatory or antiatherosclerotic activity of HO-1.
The early step in atherogenesis is endothelial dysfunction resulting in several compensatory responses that change the vascular homeostasis [47]. Proinflammatory stimuli such as a diet rich in saturated fat, obesity, hypercholesterolemia, and hyperglycemia cause the expression of adhesion molecules, for example, vascular cell adhesion molecule-1 (VCAM-1) and P-selectin in endothelium, and these molecules facilitate the attachment of monocytes and lymphocyte [48,49]. In addition, turbulent flow resulting from an unfavorable serum lipid profile probably leads to overexpression of adhesion molecules in endothelial cells triggering atherosclerosis. Animals fed a proatherogenic diet quickly overexpress VCAM-1 [50]. Overexpression of VCAM-1 enhances recruitment of monocytes to endothelial injury locations; succeeding liberation of monocyte chemoattractant protein-1 (MCP-1) by leukocytes amplifies the inflammatory cascade through recruiting other leukocytes, stimulating leukocytes in the media, and initiating recruitment and proliferation of smooth muscle cells [51]. Here, we demonstrate that suppression of VCAM-1 expression by INN reduced HL-60 cell adhesion to TNF -stimulated HAECs. Nevertheless, this inhibition was eliminated by AMPK inhibitor Compound C, shHO-1, and shNrf2 (Figures 2(d), 5(c), and 5(f)). These data demonstrate the contribution of AMPK, Nrf2, and HO-1 to the suppression of HL-60 cell adhesion by INN.
Nrf2 is an imperative transcriptional factor implicated in cellular anti-inflammatory action and related to the induction of HO-1 and glutathione S-transferase [52,53]. Nrf2 interacts with its cytosolic inhibitor Keap1 under basal circumstances. In response to stress, Nrf2 is released from Keap1 and successively translocates to the nucleus, activating its target gene transcription via ARE [54]. In the current study, INN augmented Nrf2 nuclear translocation, and silencing Nrf2 abolished the INN induction of HO-1. These results indicate that this transcriptional factor is indispensable for INNinduced HO-1 expression. Nrf2 activation is controlled by several kinases, including JNK, p38, ERK, and PI3K/Akt [55]. In the current study, we demonstrated that INN activates CaMKK and AMPK. By using specific inhibitors of CaMKK and AMPK, we revealed that the CaMKK /AMPK pathway is implicated in the INN-triggered HO-1 induction.
In summary, we have revealed that INN hinders TNFinduced ROS formation, expression of VCAM-1, and HL-60 cell adhesion by upregulating HO-1 gene expression and elevating GSH levels through the CaMKK /AMPK/Nrf2 pathway ( Figure 6). The antioxidant and anti-inflammatory characteristic of INN is thought to protect against oxidative stress-induced diseases including arthrosclerosis.