Acta Diabetol DOI 10.1007/s00592-013-0541-3
ORIGINAL ARTICLE
DPP-4 inhibitors repress foam cell formation by inhibiting scavenger receptors through protein kinase C pathway Yao Dai • Xianwei Wang • Zufeng Ding Dongsheng Dai • Jawahar L. Mehta
•
Received: 24 October 2013 / Accepted: 2 December 2013 Ó Springer-Verlag Italia (outside the USA) 2013
Abstract Studies show that dipeptidyl peptidase-4 (DPP4) inhibitors may have an anti-atherosclerotic effect. Since foam cells are key components of atherosclerotic plaque, we studied the effect of DPP-4 inhibitors on foam cell formation. Foam cell formation was studied by treatment of THP-1 macrophages with oxidized low-density lipoprotein in the absence or presence of DPP-4 inhibitors (sitagliptin and NVPDPP728). The expression of scavenger receptors SRA, CD36 and LOX-1 was measured, and their role in foam cell formation in the presence of DPP-4 inhibitors was examined. In additional studies, role of protein kinase C and A in the effect of DPP-4 inhibitors was examined.
Foam cell formation was markedly reduced by both DPP-4 inhibitors, as was the expression of CD36 and LOX-1 (CD36 LOX-1), but not SRA. Simultaneously, there was a reduction in phosphorylated PKC, but not PKA, content. Recovery of phosphorylated PKC following treatment of cells negated the effect of DPP-4 inhibitors on foam cell formation. Further, overexpression of CD36 or LOX-1 blocked the effect of DPP-4 inhibitors on foam cell formation. DPP-4 inhibitors repress foam cell formation through the inhibition of SRs CD36 and LOX-1, most likely via the inhibition of PKC activity. This study provides novel insights into the mechanism of inhibition of atherosclerosis by DPP-4 inhibitors.
Communicated by Massimo Federici.
Keywords DPP-4 Foam cell formation LOX-1 CD36 PKC
Electronic supplementary material The online version of this article (doi:10.1007/s00592-013-0541-3) contains supplementary material, which is available to authorized users.
Introduction Y. Dai Department of Endocrinology, The First Affiliated Hospital of Anhui Medical University, Hefei, Anhui, People’s Republic of China Y. Dai X. Wang Z. Ding J. L. Mehta (&) Cardiovascular Division, Department of Cardiology, University of Arkansas for Medical Sciences and the Central Arkansas Veterans Healthcare System, Little Rock, AR 72212, USA e-mail: [email protected] X. Wang Department of Cell Biology, College of Life Science and Technology, Xinxiang Medical University, Xinxiang, Henan, People’s Republic of China D. Dai Department of Cardiology, The First Affiliated Hospital of Anhui Medical University, Hefei, Anhui, People’s Republic of China
Atherogenesis is a complex process involving interaction between a host of vascular cells and lipids and inflammatory mediators [1]. Oxidized low-density lipoprotein (oxLDL) is now thought to play a pivotal role in atherogenesis [2]. Binding of extracellular ox-LDL to scavenger receptors (SRs) on membrane of macrophages, such as SRA, CD36 and LOX-1 and its internalization leading to macrophage transformation into foam cells, is believed to be an early and significant process in atherogenesis [3]. LOX-1 abrogation has previously been shown to inhibit atherogenesis in LDLr-null mice [4]. Some studies suggest that abrogation of CD36 may also inhibit atherogenesis by preventing foam cell formation [5, 6]. Protein kinases (PK) are a large group of enzymes that modify other proteins by phosphorylating them, and
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resulting in their activation [7]. PKC and PKA regulate a wide range of biological activities in the development of atherosclerosis [8]. Studies suggest that activation of PKC and PKA plays an important role in foam cell formation [9, 10]. Dipeptidyl peptidase-4 (DPP-4) is an enzyme that degrades native glucagon-like peptide 1 (GLP-1), an incretin that stimulates insulin release, postpones gastric emptying and inhibits food intake [11]. DPP-4 inhibitors are widely used to lower glucose in the treatment of patients with type-2 diabetes. These agents are considered as a major milestone development in the treatment of type-2 diabetes, in part because these agents have the potential to modulate the evolution and progression of a variety of cardiovascular diseases [12]. A major complication of diabetes is atherosclerosis, and there are experimental studies that point to a salutary effect of DPP-4 inhibitors in atherogenesis [12, 13]. Most functions of DPP-4 inhibitors are dependent on GLP-1 receptor; however, DPP-4 inhibitors may also act in a GLP-1Rindependent manner [14]. Several investigators have shown the suppression of proliferation of vascular smooth muscle cells and monocyte inflammatory reaction [12], restoration of endothelial function [15] and inhibition of thrombosis [16] by DPP-4 inhibitors. While these agents modulate accumulation of monocytes/macrophages/foam cells in the atherosclerotic plaque, the effects of these agents on foam cell formation, the primary abnormality in atherogenesis, remain unclear [17, 18], especially in the in vitro setting. This study was designed to examine in detail whether DPP-4 inhibitors would inhibit the formation of foam cells.
Materials and methods Cell culture and transfection and treatments THP-1 macrophages (ATCC, Manassas, VA, USA) were cultured in 1640 medium (ATCC) containing 10 % FBS (ATCC) in 75-mm2 flask. Macrophages were seeded onto multiwell plates and incubated with phorbol 12-myristate 13-acetate (PMA, 5 ng/mL; Sigma-Aldrich, St. Louis, MO, USA) for 24 h [19]. The cells were then washed with PBS, and cells (&1.5 9 106/mL per plate) that remained attached to the plates were reincubated with fresh medium and treated with ox-LDL (50 lg/mL; TBARS 50.00 ± 0.54 nmol/mg; Biomedical Technologies Inc, Stoughton, MA, USA) for another 24 h at 37 °C to obtain foam cells [20]. Additional studies were conducted in human primary monocytes that were isolated as described previously [21].
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To study the modulation of foam cell formation by DPP4 inhibitors, THP-1 cells were treated with sitagliptin (25 lM) [22] or NVPDPP728 (270 lM) [23] (both purchased from Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 24 h followed by incubation with ox-LDL for additional 24 h. To study the role of PKC in the effect of DPP-4 inhibitors, THP-1-induced macrophages were co-incubated with PMA (100 ng/mL, PKC activator) or diacylglycerol (DAG, Sigma-Aldrich, St. Louis, MO, USA) and sitagliptin or NVPDPP728 for 24 h followed by incubation with ox-LDL for additional 24 h [24]. To study the role of SRs, cells were seeded in six-well plates. When confluent (&70 %), cells were transfected with PCI-neo empty plasmid (vector) or CD36 cDNA (hCD36) (R&D Systems, Minneapolis, MN, USA) or human LOX-1 cDNA (hLOX-1) for 24 h followed by treatment with DPP-4 inhibitors and ox-LDL (50 lg/mL) for another 24 h. The transfection was confirmed by measurements of CD36 and LOX-1 Western blotting. Cells were also used to study Dil-ox-LDL uptake and oil red O staining. Ox-LDL uptake Ox-LDL uptake in macrophages was studied as uptake of Dil-ox-LDL (Biomedical Technologies Inc, Stoughton, MA, USA) as described previously [24]. The uptake was quantified by flow cytometry, and the data were analyzed by WinMDI 2.9 software. Determination for foam cell formation (oil red O staining) Foam cell formation was evaluated by oil red O staining. Oil red O powder (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in 99 % isopropanol and diluted to 60 % with ddH2O (working solution) 2 h before staining. Cells were fixed with 10 % formalin for 30 min after being washed with ice-cold PBS gently. After rinsing with 60 % isopropanol for 5 min, cells were stained with filtered oil red O working solution for 5 min followed by another rinse with distilled water to reduce the background and were imaged with LSM510 Zeiss Laser inverted microscope. The percentage area of red color per cell was used for statistical analysis. Western blotting SRA, CD36, LOX-1, CD68, interleukin-6 (IL-6), phospho-PKC (Ser) and PKA (Ser/Thr) were measured by Western blotting. Anti-human SRA, CD36, LOX-1,
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CD68, IL-6 and b-actin antibodies were obtained from Abcam (Cambridge, MA, USA); phospho-PKC and PKA antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Concentrations of primary antibodies were as follows: SRA (1:1,500), CD36 (1:800), LOX-1 (1:1,000), CD68 (1:1,000), IL-6 (1:1,500), phosphate-PKC (1:1,000) and phosphate-PKA (1:1,200). The protocol used in a previous study was followed [24]. bActin was used for normalizing relative expression of proteins. Statistical analysis All experiments were performed at least in triplicate. Values were analyzed by using two-tailed Newman–Keuls–Student’s t test. Data are presented as mean ± SD. A P value \0.05 was considered significant.
Fig. 1 The effects of DPP-4 inhibitors on foam cell formation, Dilox-LDL uptake and expression of scavenger receptors in THP-1 macrophages. a Foam cell formation was repressed following treatment of macrophages with DPP-4 inhibitors (oil red O staining; magnification 940). b Dil-ox-LDL uptake was also reduced by DPP-4 inhibitors (Dil-ox-LDL uptake fluorescence on the left, uptake measured by flow cytometry and its quantitation on the right). Scale
Results DPP-4 inhibitors and foam cell formation and Dil-oxLDL uptake As shown in Fig. 1a, a large number of macrophages following treatment with ox-LDL turned into foam cells (P \ 0.05). The formation of foam cells was blocked by both DPP-4 inhibitors, sitagliptin and NVPDPP728 (P \ 0.05 vs. ox-LDL alone treatment group). The most effective dose of sitagliptin in inhibiting foam cell formation was 25 lM, shown in Figure supplement 1A. This concentration of sitagliptin was used in all subsequent experiments. Dil-ox-LDL uptake increased rapidly and significantly in macrophages treated with ox-LDL, and both DPP-4 inhibitors reduced Dil-ox-LDL uptake markedly (Fig. 1b).
bars in panels a and b: 20 lm. c Expression of CD36 and LOX-1 was inhibited by DPP-4 inhibitors (Western blotting), quantitative data on the right. *P \ 0.05 versus control, P \ 0.05 versus ox-LDL alone treatment group. Ox-LDL oxidized low-density lipoprotein, LOX-1 oxidized low-density lipoprotein receptor 1, SRA scavenger receptor A
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DPP-4 inhibitors and expression of SRs
PKC stimulation and DPP-4 inhibitors
SRA, CD36 and LOX-1 are three major scavenger receptors on macrophage membrane, which bind and internalize ox-LDL into the cytoplasm [25]. Therefore, we checked the expression of these receptors on macrophages. As shown in Fig. 1c, ox-LDL caused a marked increase in the expression of all 3 receptors. Both DPP-4 inhibitors induced a decrease in the expression of CD36 and LOX-1 (&20 and 50 %, respectively; P \ 0.05 vs. ox-LDL alone treatment group). However, the expression of SRA remained unchanged following treatment with DPP-4 inhibitors. To confirm the effects of DPP-4 inhibitors on SRs in THP-1 cells, we conducted studies in primary human macrophages and found similar results. We also checked the expression of CD68, another scavenger receptor that binds modified LDL, and found that both DPP-4 inhibitors reduced its expression (Figure supplement 1B).
To confirm whether PKC regulates the inhibitory effect of DPP-4 inhibitors on foam cell formation, we used PMA, a PKC activator [9]. As shown in Fig. 3, PMA almost completely blocked the effect of sitagliptin and NVDPP728 on phospho-PKC, while phospho-PKA substrate remained unchanged (data not shown; P \ 0.05 vs. non-PMA treatment groups, respectively). As shown in Fig. 4a, the inhibitory effect of DPP-4 inhibitors on foam cell formation was reversed by PKC stimulation with PMA (P \ 0.05 vs. non-PMA treatment groups, respectively). Similar pattern was seen in experiments designed to study Dil-ox-LDL uptake (Fig. 4b). Lastly, the inhibitory effects of DPP-4 inhibitors on CD36 and LOX-1 protein expression were also reversed by PMA (P \ 0.05 vs. non-PMA treatment groups, respectively; Fig. 4c). The expression of SRA still remained unchanged. In other experiments, we used another PKC stimulus, DAG, to confirm our findings with PMA. As shown in Figure supplement 2A, the inhibitory effect of DPP-4 inhibitors was blocked by DAG-induced activation of PKC.
DPP-4 inhibitors and protein kinases As shown earlier, protein kinases play a role in foam cell formation [26, 27]. Accordingly, we measured phosphoPKA and phospho-PKC protein levels and their modulation by DPP-4 inhibitors. As shown in Fig. 2, the expression of phospho-PKC was stimulated by ox-LDL, and this effect was inhibited by both DPP-4 inhibitors (P \ 0.05 vs. oxLDL alone treatment group). The expression of phosphoPKA also increased with ox-LDL treatment; however, it was not affected by either DPP-4 inhibitor.
Fig. 2 DPP-4 inhibitors and expression of protein kinases (PK) C and A. Phospho-PKC was enhanced by ox-LDL and DPP-4 inhibitors suppressed the effect of ox-LDL (representative Western blotting, quantitative data on the right). *P \ 0.05 versus control, P \ 0.05 versus oxLDL alone treatment group
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Scavenger receptor overexpression and DPP-4 inhibitors To examine the role of LOX-1 and CD36 in the effects of DPP-4 inhibitors on foam cell formation, we transfected macrophages (pretreated with DPP-4 inhibitors) with human LOX-1 and CD36 cDNAs. The transfection efficiency was determined by CD36 and LOX-1 protein
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Fig. 3 PKC activation and neutralization of the effects of DPP-4 inhibitors. The reduction in phospho-PKC expression by DPP-4 inhibitors was neutralized by PMA (representative Western blotting and quantitative data below). *P \ 0.05 versus ox-LDL plus
sitagliptin treatment, P \ 0.05 versus ox-LDL plus NVPDPP728 treatment. Note that Phospho-PKA expression was not affected by DPP-4 inhibitors
Fig. 4 PKC activation by PMA and reversal of the effect of DPP-4 inhibitors. a PMA treatment reversed the inhibitory effects of DPP-4 inhibitors on foam cell formation (oil red O staining) (magnification 940). b PMA treatment also reversed the inhibitory effects of DPP-4 inhibitors on Dil-ox-LDL uptake (Dil-ox-LDL uptake measurement by fluorescence on the left, and uptake measured by flow cytometry
and its quantitation on the right). Scale bars in panels a and b: 20 lm. c PMA reversed the inhibitory effects of DPP-4 inhibitors on CD36 and LOX-1 expression. (representative Western blotting, quantitative data on the right). *P \ 0.05 versus ox-LDL plus sitagliptin treatment, P \ 0.05 versus ox-LDL plus NVPDPP728 treatment. Abbreviations same as in previous figures
measurement in respective groups (Figure supplement 3). As shown in Fig. 5, macrophages that expressed high levels of CD36 exhibited almost 2–3 times more foam cell formation and greater Dil-ox-LDL uptake compared with
non-transfected group (P \ 0.05). Macrophages with enhanced hLOX-1 exhibited almost 1.5–2 times more foam cell formation and greater Dil-ox-LDL uptake than vector alone transfected cells (P \ 0.05).
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Fig. 5 Overexpression of LOX-1 and CD36 blocks the effects of DPP-4 inhibitors on foam cell formation and Dil-ox-LDL uptake. a Transfection with hCD36 and hLOX-1 cDNAs reversed the inhibitory effects of DPP-4 inhibitors on foam cell formation (oil red O staining). b Transfection with hCD36 and hLOX-1 cDNAs reversed the inhibitory effects of DPP-4 inhibitors on Dil-ox-LDL
uptake in macrophages (Dil-ox-LDL uptake by fluorescence on the left, uptake measured by flow cytometry and its quantitation on the right). Scale bars in panels a and b: 20 lm. *P \ 0.05 versus ox-LDL plus sitagliptin treatment transfected with vector, P \ 0.05 versus ox-LDL plus NVPDPP728 treatment transfected with vector. Abbreviations same as in previous figures
DPP-4 inhibitor blocks pro-inflammatory cytokine IL-6
atherogenesis, related most likely to the formation of oxLDL and AGEs, both of which induce endothelial dysfunction, create a pro-inflammatory milieu and induce foam cell formation [29]. Therefore, it is logical to suggest that drugs that improve upon the diabetic state may attenuate many of the steps in atherogenesis. The incretinbased therapy carries much promise in this regard. Recently, DPP-4 inhibitors have been shown to reduce accumulation of monocytes and macrophages in the vascular walls and around the aortic valve and attenuate atherogenesis in Apo E-null mice without affecting glucose tolerance or body weight [12]. Shah et al. [13] showed that alogliptin reduced visceral adipose tissue macrophage content in LDLr-null mice given high-fat diet. Terasaki et al. [17] observed that foam cell formation was suppressed by 40 % in peritoneal macrophages obtained from DPP-4-treated Apo E-null mice. Same investigators, however, were not able to demonstrate foam cell formation inhibitory effect of DPP-4 inhibitors in vitro [18]. These
Inflammation is another significant process in atherogenesis. Thus, we chose pro-inflammatory cytokine, IL-6, to detect the effect of DPP-4 inhibitors on inflammatory process mediated by the activation of monocytes/macrophages. As shown in Figure supplement 3B, the expression of IL-6 was significantly inhibited by both DPP-4 inhibitors. This further suggests that DPP-4 inhibitors can reduce inflammatory process.
Discussion Foam cells play an important role in atherogenesis by release of a variety of growth factors and inflammatory mediators [28]. The therapeutic strategies designed to reduce atherogenesis accordingly involve inhibition of foam cell formation. Diabetes is a major risk factor for
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observations collectively suggest that DPP-4 inhibitors might potentially reduce atherogenesis primarily by inhibiting inflammatory response; however, their effect on foam cell formation remains unclear. In this study, we show that two different DPP-4 inhibitors in clinically achieved concentrations have the potential to reduce foam cell formation from human primary and THP-1 macrophages exposed to ox-LDL. The effects of DPP-4 inhibitors were readily evident. This effect appears to be related to a marked suppressive effect on the expression of three major SRs, CD36, CD68 and LOX-1. Ox-LDL binds to these receptors and is internalized in macrophages resulting in foam cell formation [3]. The reduction in Dil-ox-LDL uptake lends credence to the concept that internalization of ox-LDL was blunted by DPP-4 inhibitors. Notably, while SRA was upregulated by ox-LDL, it was not suppressed by DPP-4 inhibitors. The SRs CD36 and LOX-1 have been reported to regulate PKC pathway in atherosclerotic regions [30, 31]. Our study shows that DPP-4 inhibitors repressed PKC activation in concert with reduced expression of CD36 and LOX-1. However, PKA activity was not affected by DPP-4 inhibitors. To confirm the role of PKC activation in the effects of DPP-4 inhibitors, we incubated macrophages with PMA and DAG, two different specific activators of PKC. As shown in Fig. 4 and Figure supplement 2, PKC activation overcame the inhibitory effect of DPP-4 inhibitors on foam cell formation. PKC activation by PMA and DAG also overcame the suppressive effect of DPP-4 inhibitors on SRs CD36 and LOX-1. In keeping with the latter observation, suppressive effect of DPP-4 inhibitors on Dil-oxLDL uptake was also blunted. In order to confirm the major role for suppression of SRs (CD36 and LOX-1) by DPP-4 inhibition, we forcibly upregulated the expression of these SRs by human CD36 and LOX-1 cDNA transfection. As shown in Fig. 5, upregulation of these SRs overcame the inhibitory effect of DPP-4 inhibitors on Dil-ox-LDL uptake and subsequent foam cell formation. It should also be noted that SRA seems to be not affected by DPP-4 inhibitors. This lack of sensitivity of SRA to DPP-4 inhibitors needs further study. Inflammation is another key element for atherogenesis. Thus, we chose pro-inflammatory cytokine IL-6 to assess the effect of DPP-4 inhibitors on the pro-inflammatory function of monocytes/macrophages [32]. Our study suggests that besides inhibiting foam cell formation, DPP-4 inhibitors also inhibit inflammatory signals, which may further impact atherogenesis. These observations collectively suggest that DPP-4 inhibitor exerts a potent inhibitory effect on foam cell formation from human macrophage cell line in response to ox-LDL. This effect is primarily mediated by decrease in
the expression of two different SRs on monocytes/macrophages CD36 and LOX-1. This results in a decrease in oxLDL internalization. DPP-4 inhibitors also exert a potent inhibitory effect on PKC activation, perhaps mediated by membrane-bound DPP-4, which plays a critical role in foam cell formation and inflammation. Acknowledgments This study was supported by funds from the Department of Veterans Affairs, Veterans Health Administration, Office of Research and Development, Biomedical Laboratory Research and Development, Washington, DC. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. Conflict of interest Yao Dai, Xianwei Wang, Zufeng Ding, Dongsheng Dai, Jawahar L. Mehta declare that they have no conflict of interest.
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ORIGINAL ARTICLE
DPP-4 inhibitors repress foam cell formation by inhibiting scavenger receptors through protein kinase C pathway Yao Dai • Xianwei Wang • Zufeng Ding Dongsheng Dai • Jawahar L. Mehta
•
Received: 24 October 2013 / Accepted: 2 December 2013 Ó Springer-Verlag Italia (outside the USA) 2013
Abstract Studies show that dipeptidyl peptidase-4 (DPP4) inhibitors may have an anti-atherosclerotic effect. Since foam cells are key components of atherosclerotic plaque, we studied the effect of DPP-4 inhibitors on foam cell formation. Foam cell formation was studied by treatment of THP-1 macrophages with oxidized low-density lipoprotein in the absence or presence of DPP-4 inhibitors (sitagliptin and NVPDPP728). The expression of scavenger receptors SRA, CD36 and LOX-1 was measured, and their role in foam cell formation in the presence of DPP-4 inhibitors was examined. In additional studies, role of protein kinase C and A in the effect of DPP-4 inhibitors was examined.
Foam cell formation was markedly reduced by both DPP-4 inhibitors, as was the expression of CD36 and LOX-1 (CD36 LOX-1), but not SRA. Simultaneously, there was a reduction in phosphorylated PKC, but not PKA, content. Recovery of phosphorylated PKC following treatment of cells negated the effect of DPP-4 inhibitors on foam cell formation. Further, overexpression of CD36 or LOX-1 blocked the effect of DPP-4 inhibitors on foam cell formation. DPP-4 inhibitors repress foam cell formation through the inhibition of SRs CD36 and LOX-1, most likely via the inhibition of PKC activity. This study provides novel insights into the mechanism of inhibition of atherosclerosis by DPP-4 inhibitors.
Communicated by Massimo Federici.
Keywords DPP-4 Foam cell formation LOX-1 CD36 PKC
Electronic supplementary material The online version of this article (doi:10.1007/s00592-013-0541-3) contains supplementary material, which is available to authorized users.
Introduction Y. Dai Department of Endocrinology, The First Affiliated Hospital of Anhui Medical University, Hefei, Anhui, People’s Republic of China Y. Dai X. Wang Z. Ding J. L. Mehta (&) Cardiovascular Division, Department of Cardiology, University of Arkansas for Medical Sciences and the Central Arkansas Veterans Healthcare System, Little Rock, AR 72212, USA e-mail: [email protected] X. Wang Department of Cell Biology, College of Life Science and Technology, Xinxiang Medical University, Xinxiang, Henan, People’s Republic of China D. Dai Department of Cardiology, The First Affiliated Hospital of Anhui Medical University, Hefei, Anhui, People’s Republic of China
Atherogenesis is a complex process involving interaction between a host of vascular cells and lipids and inflammatory mediators [1]. Oxidized low-density lipoprotein (oxLDL) is now thought to play a pivotal role in atherogenesis [2]. Binding of extracellular ox-LDL to scavenger receptors (SRs) on membrane of macrophages, such as SRA, CD36 and LOX-1 and its internalization leading to macrophage transformation into foam cells, is believed to be an early and significant process in atherogenesis [3]. LOX-1 abrogation has previously been shown to inhibit atherogenesis in LDLr-null mice [4]. Some studies suggest that abrogation of CD36 may also inhibit atherogenesis by preventing foam cell formation [5, 6]. Protein kinases (PK) are a large group of enzymes that modify other proteins by phosphorylating them, and
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resulting in their activation [7]. PKC and PKA regulate a wide range of biological activities in the development of atherosclerosis [8]. Studies suggest that activation of PKC and PKA plays an important role in foam cell formation [9, 10]. Dipeptidyl peptidase-4 (DPP-4) is an enzyme that degrades native glucagon-like peptide 1 (GLP-1), an incretin that stimulates insulin release, postpones gastric emptying and inhibits food intake [11]. DPP-4 inhibitors are widely used to lower glucose in the treatment of patients with type-2 diabetes. These agents are considered as a major milestone development in the treatment of type-2 diabetes, in part because these agents have the potential to modulate the evolution and progression of a variety of cardiovascular diseases [12]. A major complication of diabetes is atherosclerosis, and there are experimental studies that point to a salutary effect of DPP-4 inhibitors in atherogenesis [12, 13]. Most functions of DPP-4 inhibitors are dependent on GLP-1 receptor; however, DPP-4 inhibitors may also act in a GLP-1Rindependent manner [14]. Several investigators have shown the suppression of proliferation of vascular smooth muscle cells and monocyte inflammatory reaction [12], restoration of endothelial function [15] and inhibition of thrombosis [16] by DPP-4 inhibitors. While these agents modulate accumulation of monocytes/macrophages/foam cells in the atherosclerotic plaque, the effects of these agents on foam cell formation, the primary abnormality in atherogenesis, remain unclear [17, 18], especially in the in vitro setting. This study was designed to examine in detail whether DPP-4 inhibitors would inhibit the formation of foam cells.
Materials and methods Cell culture and transfection and treatments THP-1 macrophages (ATCC, Manassas, VA, USA) were cultured in 1640 medium (ATCC) containing 10 % FBS (ATCC) in 75-mm2 flask. Macrophages were seeded onto multiwell plates and incubated with phorbol 12-myristate 13-acetate (PMA, 5 ng/mL; Sigma-Aldrich, St. Louis, MO, USA) for 24 h [19]. The cells were then washed with PBS, and cells (&1.5 9 106/mL per plate) that remained attached to the plates were reincubated with fresh medium and treated with ox-LDL (50 lg/mL; TBARS 50.00 ± 0.54 nmol/mg; Biomedical Technologies Inc, Stoughton, MA, USA) for another 24 h at 37 °C to obtain foam cells [20]. Additional studies were conducted in human primary monocytes that were isolated as described previously [21].
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To study the modulation of foam cell formation by DPP4 inhibitors, THP-1 cells were treated with sitagliptin (25 lM) [22] or NVPDPP728 (270 lM) [23] (both purchased from Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 24 h followed by incubation with ox-LDL for additional 24 h. To study the role of PKC in the effect of DPP-4 inhibitors, THP-1-induced macrophages were co-incubated with PMA (100 ng/mL, PKC activator) or diacylglycerol (DAG, Sigma-Aldrich, St. Louis, MO, USA) and sitagliptin or NVPDPP728 for 24 h followed by incubation with ox-LDL for additional 24 h [24]. To study the role of SRs, cells were seeded in six-well plates. When confluent (&70 %), cells were transfected with PCI-neo empty plasmid (vector) or CD36 cDNA (hCD36) (R&D Systems, Minneapolis, MN, USA) or human LOX-1 cDNA (hLOX-1) for 24 h followed by treatment with DPP-4 inhibitors and ox-LDL (50 lg/mL) for another 24 h. The transfection was confirmed by measurements of CD36 and LOX-1 Western blotting. Cells were also used to study Dil-ox-LDL uptake and oil red O staining. Ox-LDL uptake Ox-LDL uptake in macrophages was studied as uptake of Dil-ox-LDL (Biomedical Technologies Inc, Stoughton, MA, USA) as described previously [24]. The uptake was quantified by flow cytometry, and the data were analyzed by WinMDI 2.9 software. Determination for foam cell formation (oil red O staining) Foam cell formation was evaluated by oil red O staining. Oil red O powder (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in 99 % isopropanol and diluted to 60 % with ddH2O (working solution) 2 h before staining. Cells were fixed with 10 % formalin for 30 min after being washed with ice-cold PBS gently. After rinsing with 60 % isopropanol for 5 min, cells were stained with filtered oil red O working solution for 5 min followed by another rinse with distilled water to reduce the background and were imaged with LSM510 Zeiss Laser inverted microscope. The percentage area of red color per cell was used for statistical analysis. Western blotting SRA, CD36, LOX-1, CD68, interleukin-6 (IL-6), phospho-PKC (Ser) and PKA (Ser/Thr) were measured by Western blotting. Anti-human SRA, CD36, LOX-1,
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CD68, IL-6 and b-actin antibodies were obtained from Abcam (Cambridge, MA, USA); phospho-PKC and PKA antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Concentrations of primary antibodies were as follows: SRA (1:1,500), CD36 (1:800), LOX-1 (1:1,000), CD68 (1:1,000), IL-6 (1:1,500), phosphate-PKC (1:1,000) and phosphate-PKA (1:1,200). The protocol used in a previous study was followed [24]. bActin was used for normalizing relative expression of proteins. Statistical analysis All experiments were performed at least in triplicate. Values were analyzed by using two-tailed Newman–Keuls–Student’s t test. Data are presented as mean ± SD. A P value \0.05 was considered significant.
Fig. 1 The effects of DPP-4 inhibitors on foam cell formation, Dilox-LDL uptake and expression of scavenger receptors in THP-1 macrophages. a Foam cell formation was repressed following treatment of macrophages with DPP-4 inhibitors (oil red O staining; magnification 940). b Dil-ox-LDL uptake was also reduced by DPP-4 inhibitors (Dil-ox-LDL uptake fluorescence on the left, uptake measured by flow cytometry and its quantitation on the right). Scale
Results DPP-4 inhibitors and foam cell formation and Dil-oxLDL uptake As shown in Fig. 1a, a large number of macrophages following treatment with ox-LDL turned into foam cells (P \ 0.05). The formation of foam cells was blocked by both DPP-4 inhibitors, sitagliptin and NVPDPP728 (P \ 0.05 vs. ox-LDL alone treatment group). The most effective dose of sitagliptin in inhibiting foam cell formation was 25 lM, shown in Figure supplement 1A. This concentration of sitagliptin was used in all subsequent experiments. Dil-ox-LDL uptake increased rapidly and significantly in macrophages treated with ox-LDL, and both DPP-4 inhibitors reduced Dil-ox-LDL uptake markedly (Fig. 1b).
bars in panels a and b: 20 lm. c Expression of CD36 and LOX-1 was inhibited by DPP-4 inhibitors (Western blotting), quantitative data on the right. *P \ 0.05 versus control, P \ 0.05 versus ox-LDL alone treatment group. Ox-LDL oxidized low-density lipoprotein, LOX-1 oxidized low-density lipoprotein receptor 1, SRA scavenger receptor A
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DPP-4 inhibitors and expression of SRs
PKC stimulation and DPP-4 inhibitors
SRA, CD36 and LOX-1 are three major scavenger receptors on macrophage membrane, which bind and internalize ox-LDL into the cytoplasm [25]. Therefore, we checked the expression of these receptors on macrophages. As shown in Fig. 1c, ox-LDL caused a marked increase in the expression of all 3 receptors. Both DPP-4 inhibitors induced a decrease in the expression of CD36 and LOX-1 (&20 and 50 %, respectively; P \ 0.05 vs. ox-LDL alone treatment group). However, the expression of SRA remained unchanged following treatment with DPP-4 inhibitors. To confirm the effects of DPP-4 inhibitors on SRs in THP-1 cells, we conducted studies in primary human macrophages and found similar results. We also checked the expression of CD68, another scavenger receptor that binds modified LDL, and found that both DPP-4 inhibitors reduced its expression (Figure supplement 1B).
To confirm whether PKC regulates the inhibitory effect of DPP-4 inhibitors on foam cell formation, we used PMA, a PKC activator [9]. As shown in Fig. 3, PMA almost completely blocked the effect of sitagliptin and NVDPP728 on phospho-PKC, while phospho-PKA substrate remained unchanged (data not shown; P \ 0.05 vs. non-PMA treatment groups, respectively). As shown in Fig. 4a, the inhibitory effect of DPP-4 inhibitors on foam cell formation was reversed by PKC stimulation with PMA (P \ 0.05 vs. non-PMA treatment groups, respectively). Similar pattern was seen in experiments designed to study Dil-ox-LDL uptake (Fig. 4b). Lastly, the inhibitory effects of DPP-4 inhibitors on CD36 and LOX-1 protein expression were also reversed by PMA (P \ 0.05 vs. non-PMA treatment groups, respectively; Fig. 4c). The expression of SRA still remained unchanged. In other experiments, we used another PKC stimulus, DAG, to confirm our findings with PMA. As shown in Figure supplement 2A, the inhibitory effect of DPP-4 inhibitors was blocked by DAG-induced activation of PKC.
DPP-4 inhibitors and protein kinases As shown earlier, protein kinases play a role in foam cell formation [26, 27]. Accordingly, we measured phosphoPKA and phospho-PKC protein levels and their modulation by DPP-4 inhibitors. As shown in Fig. 2, the expression of phospho-PKC was stimulated by ox-LDL, and this effect was inhibited by both DPP-4 inhibitors (P \ 0.05 vs. oxLDL alone treatment group). The expression of phosphoPKA also increased with ox-LDL treatment; however, it was not affected by either DPP-4 inhibitor.
Fig. 2 DPP-4 inhibitors and expression of protein kinases (PK) C and A. Phospho-PKC was enhanced by ox-LDL and DPP-4 inhibitors suppressed the effect of ox-LDL (representative Western blotting, quantitative data on the right). *P \ 0.05 versus control, P \ 0.05 versus oxLDL alone treatment group
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Scavenger receptor overexpression and DPP-4 inhibitors To examine the role of LOX-1 and CD36 in the effects of DPP-4 inhibitors on foam cell formation, we transfected macrophages (pretreated with DPP-4 inhibitors) with human LOX-1 and CD36 cDNAs. The transfection efficiency was determined by CD36 and LOX-1 protein
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Fig. 3 PKC activation and neutralization of the effects of DPP-4 inhibitors. The reduction in phospho-PKC expression by DPP-4 inhibitors was neutralized by PMA (representative Western blotting and quantitative data below). *P \ 0.05 versus ox-LDL plus
sitagliptin treatment, P \ 0.05 versus ox-LDL plus NVPDPP728 treatment. Note that Phospho-PKA expression was not affected by DPP-4 inhibitors
Fig. 4 PKC activation by PMA and reversal of the effect of DPP-4 inhibitors. a PMA treatment reversed the inhibitory effects of DPP-4 inhibitors on foam cell formation (oil red O staining) (magnification 940). b PMA treatment also reversed the inhibitory effects of DPP-4 inhibitors on Dil-ox-LDL uptake (Dil-ox-LDL uptake measurement by fluorescence on the left, and uptake measured by flow cytometry
and its quantitation on the right). Scale bars in panels a and b: 20 lm. c PMA reversed the inhibitory effects of DPP-4 inhibitors on CD36 and LOX-1 expression. (representative Western blotting, quantitative data on the right). *P \ 0.05 versus ox-LDL plus sitagliptin treatment, P \ 0.05 versus ox-LDL plus NVPDPP728 treatment. Abbreviations same as in previous figures
measurement in respective groups (Figure supplement 3). As shown in Fig. 5, macrophages that expressed high levels of CD36 exhibited almost 2–3 times more foam cell formation and greater Dil-ox-LDL uptake compared with
non-transfected group (P \ 0.05). Macrophages with enhanced hLOX-1 exhibited almost 1.5–2 times more foam cell formation and greater Dil-ox-LDL uptake than vector alone transfected cells (P \ 0.05).
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Fig. 5 Overexpression of LOX-1 and CD36 blocks the effects of DPP-4 inhibitors on foam cell formation and Dil-ox-LDL uptake. a Transfection with hCD36 and hLOX-1 cDNAs reversed the inhibitory effects of DPP-4 inhibitors on foam cell formation (oil red O staining). b Transfection with hCD36 and hLOX-1 cDNAs reversed the inhibitory effects of DPP-4 inhibitors on Dil-ox-LDL
uptake in macrophages (Dil-ox-LDL uptake by fluorescence on the left, uptake measured by flow cytometry and its quantitation on the right). Scale bars in panels a and b: 20 lm. *P \ 0.05 versus ox-LDL plus sitagliptin treatment transfected with vector, P \ 0.05 versus ox-LDL plus NVPDPP728 treatment transfected with vector. Abbreviations same as in previous figures
DPP-4 inhibitor blocks pro-inflammatory cytokine IL-6
atherogenesis, related most likely to the formation of oxLDL and AGEs, both of which induce endothelial dysfunction, create a pro-inflammatory milieu and induce foam cell formation [29]. Therefore, it is logical to suggest that drugs that improve upon the diabetic state may attenuate many of the steps in atherogenesis. The incretinbased therapy carries much promise in this regard. Recently, DPP-4 inhibitors have been shown to reduce accumulation of monocytes and macrophages in the vascular walls and around the aortic valve and attenuate atherogenesis in Apo E-null mice without affecting glucose tolerance or body weight [12]. Shah et al. [13] showed that alogliptin reduced visceral adipose tissue macrophage content in LDLr-null mice given high-fat diet. Terasaki et al. [17] observed that foam cell formation was suppressed by 40 % in peritoneal macrophages obtained from DPP-4-treated Apo E-null mice. Same investigators, however, were not able to demonstrate foam cell formation inhibitory effect of DPP-4 inhibitors in vitro [18]. These
Inflammation is another significant process in atherogenesis. Thus, we chose pro-inflammatory cytokine, IL-6, to detect the effect of DPP-4 inhibitors on inflammatory process mediated by the activation of monocytes/macrophages. As shown in Figure supplement 3B, the expression of IL-6 was significantly inhibited by both DPP-4 inhibitors. This further suggests that DPP-4 inhibitors can reduce inflammatory process.
Discussion Foam cells play an important role in atherogenesis by release of a variety of growth factors and inflammatory mediators [28]. The therapeutic strategies designed to reduce atherogenesis accordingly involve inhibition of foam cell formation. Diabetes is a major risk factor for
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observations collectively suggest that DPP-4 inhibitors might potentially reduce atherogenesis primarily by inhibiting inflammatory response; however, their effect on foam cell formation remains unclear. In this study, we show that two different DPP-4 inhibitors in clinically achieved concentrations have the potential to reduce foam cell formation from human primary and THP-1 macrophages exposed to ox-LDL. The effects of DPP-4 inhibitors were readily evident. This effect appears to be related to a marked suppressive effect on the expression of three major SRs, CD36, CD68 and LOX-1. Ox-LDL binds to these receptors and is internalized in macrophages resulting in foam cell formation [3]. The reduction in Dil-ox-LDL uptake lends credence to the concept that internalization of ox-LDL was blunted by DPP-4 inhibitors. Notably, while SRA was upregulated by ox-LDL, it was not suppressed by DPP-4 inhibitors. The SRs CD36 and LOX-1 have been reported to regulate PKC pathway in atherosclerotic regions [30, 31]. Our study shows that DPP-4 inhibitors repressed PKC activation in concert with reduced expression of CD36 and LOX-1. However, PKA activity was not affected by DPP-4 inhibitors. To confirm the role of PKC activation in the effects of DPP-4 inhibitors, we incubated macrophages with PMA and DAG, two different specific activators of PKC. As shown in Fig. 4 and Figure supplement 2, PKC activation overcame the inhibitory effect of DPP-4 inhibitors on foam cell formation. PKC activation by PMA and DAG also overcame the suppressive effect of DPP-4 inhibitors on SRs CD36 and LOX-1. In keeping with the latter observation, suppressive effect of DPP-4 inhibitors on Dil-oxLDL uptake was also blunted. In order to confirm the major role for suppression of SRs (CD36 and LOX-1) by DPP-4 inhibition, we forcibly upregulated the expression of these SRs by human CD36 and LOX-1 cDNA transfection. As shown in Fig. 5, upregulation of these SRs overcame the inhibitory effect of DPP-4 inhibitors on Dil-ox-LDL uptake and subsequent foam cell formation. It should also be noted that SRA seems to be not affected by DPP-4 inhibitors. This lack of sensitivity of SRA to DPP-4 inhibitors needs further study. Inflammation is another key element for atherogenesis. Thus, we chose pro-inflammatory cytokine IL-6 to assess the effect of DPP-4 inhibitors on the pro-inflammatory function of monocytes/macrophages [32]. Our study suggests that besides inhibiting foam cell formation, DPP-4 inhibitors also inhibit inflammatory signals, which may further impact atherogenesis. These observations collectively suggest that DPP-4 inhibitor exerts a potent inhibitory effect on foam cell formation from human macrophage cell line in response to ox-LDL. This effect is primarily mediated by decrease in
the expression of two different SRs on monocytes/macrophages CD36 and LOX-1. This results in a decrease in oxLDL internalization. DPP-4 inhibitors also exert a potent inhibitory effect on PKC activation, perhaps mediated by membrane-bound DPP-4, which plays a critical role in foam cell formation and inflammation. Acknowledgments This study was supported by funds from the Department of Veterans Affairs, Veterans Health Administration, Office of Research and Development, Biomedical Laboratory Research and Development, Washington, DC. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. Conflict of interest Yao Dai, Xianwei Wang, Zufeng Ding, Dongsheng Dai, Jawahar L. Mehta declare that they have no conflict of interest.
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