WISP1 alleviates lipid deposition in macrophages via the PPARγ/CD36 pathway in the plaque formation of atherosclerosis

Abstract Lipid deposition in macrophages plays an important role in atherosclerosis. The WNT1‐inducible signalling pathway protein 1(WISP1) can promote proliferation and migration of smooth muscle cells. Its expression is up‐regulated in obesity, which is associated with atherosclerosis, but the effect of WISP1 on atherosclerosis remains unclear. Thus, the objective of our study was to elucidate the role of WISP and its mechanism of action in atherosclerosis via in vivo and in vitro experiments. In our experiment, ApoE‐/‐ mice were divided into 5 groups: control, high‐fat diet (HFD), null lentivirus (HFD + NC), lentivirus WISP1 (HFD + IvWISP1) and WISP1‐shRNA (HFD + shWISP1). Oil Red O staining, immunofluorescence and immunohistochemistry of the aortic sinuses were conducted. Macrophages (RAW264.7 cell lines and peritoneal macrophages) were stimulated with 50 μg/mL oxidized low‐density lipoprotein (ox‐LDL); then, the reactive oxygen species (ROS) level was measured. Oil Red O staining and Dil‐ox‐LDL (ox‐LDL with Dil dye) uptake measurements were used to test lipid deposition of peritoneal macrophages. WISP1, CD36, SR‐A and PPARγ expression levels were measured via Western blotting and ELISA. The results showed that HFD mice had increased WISP1, CD36 and SR‐A levels. The plaque lesion area increased when WISP1 was down‐regulated, and lipid uptake and foam cell formation were inhibited when WISP1 was up‐regulated. Treatment of RAW264.7 cell lines with ox‐LDL increased WISP1 expression via activation of the Wnt5a/β‐catenin pathway, whereas ROS inhibition reduced WISP1 expression. Moreover, WISP1 down‐regulated CD36 and SR‐A expression, and Oil Red O staining and Dil‐ox‐LDL uptake measurement showed that WISP1 down‐regulated lipid deposition in macrophages. These results clearly demonstrate that WISP1 is activated by ox‐LDL at high ROS levels and can alleviate lipid deposition in atherosclerosis through the PPARγ/CD36 pathway.


| INTRODUC TI ON
Atherosclerosis is the leading cause of cardiovascular disease. It is a complex process that includes endothelial dysfunction, lipid deposition, and proliferation and migration of smooth muscle cells. [1][2][3] Formation of macrophage foam cells is characteristic of early atherosclerotic lesions, 2 and oxidized low-density lipoprotein (ox-LDL) is absorbed by macrophages with the help of scavenger receptors (SRs). 4 Ox-LDL, lysophosphatidylcholine and oxidized fatty acids induce the expression of these scavenger receptors, such as CD36, SR class A (SR-A) and LOX-1. 5 Thus, peroxisome proliferator-activated receptor γ (PPARγ), a ligand-activated transcription factor known to regulate fatty acid metabolism, plays a role in lipid deposition. 6 After its activation, PPARγ gene transcription is regulated to modify lipid metabolism, and it has important effects on cell proliferation, differentiation and inflammatory responses. [7][8][9] Additionally, studies have found that PPARγ is the main factor regulating the uptake of ox-LDL by CD36, which affects the generation of foam cells by influencing lipid metabolism in macrophages. [10][11][12] CD36 belongs to the class B scavenger receptor family, which is localized in various cell types, such as monocytes/macrophages, adipocytes, endothelial and smooth muscle cells. [13][14][15][16] Ox-LDL, apoptotic cells and advanced glycation end products (AGEs) are the most noticeable substances involved in atherosclerosis development that interact with CD36. 17 Furthermore, CD36 and ox-LDL binding can activate signalling pathways, including those of protein kinases that lead to the development of atherosclerosis. [18][19][20] Additionally, it is reported that the knockout of SR-A, a kind of cell surface glycoprotein that belongs to the SR family, 21 can accelerate atherosclerosis. 22 The WNT signalling pathway is rather a conservative pathway in biological evolution that is closely related to embryo formation, development and cell differentiation. In recent years, studies on the role of the WNT pathway in cardiovascular diseases have attracted much attention, as it participates in the adhesion between endothelium and monocytes, regulates the function of smooth muscle cells, promotes vascular calcification 23,24 and plays an important role in the adipogenic differentiation of cells. 25 WNT-inducible signalling pathway protein 1 (WISP1) belongs to the CCN family of extracellular matrix proteins, and it is identified as a downstream target gene of the canonical WNT signalling pathway. 8 WISP1 plays an important role in the inflammatory process, and its expression is closely related to the severity of osteoarthritis due to its effect on the expression of chondrocytes, macrophage matrix metalloproteinases and proteoglycan enzymes. 26 Further, WISP1 expression was found to increase significantly in neurons under oxidative stress and fracture repair. 27 Studies have proven that WISP1 can accelerate the migration and proliferation of smooth muscle cells to thicken the intima of blood vessels. 28 Other studies have shown that obesity, which is associated with atherosclerosis, leads to the up-regulation of WISP1. 29 However, whether WISP1 plays a role in the lipid deposition in atherosclerosis remains unclear.
In this study, we investigated the relationship between WISP1 and atherosclerosis, the function of WISP1 during atherosclerotic plaque formation and progression, and the specific role(s) and molecular mechanism(s) of action of WISP1. The results of our in vitro and in vivo experiments point to a possible mechanism of WISP1 in the progression of atherosclerosis.

| Animals
All mouse studies were sanctioned by the Animal Ethics Committee of Shandong University; the care and use of animals followed the guidelines on animal ethics. Male ApoE-/-mice (8 weeks old, n = 100) were obtained from HFK Bioscience Company (Beijing, China). All HFD + shWISP1 (WISP1-shRNA, 5'-CCA CTA GAG GAA ACG ACT A-3') (GenePharma, China). Group 1 mice were fed with a diet of 5% fat without cholesterol, whereas the other groups were fed with an HFD (16% fat and 0.25% cholesterol). All mice were provided with food and water and subjected to a light-dark cycle in an environment at 20℃-22℃ and 50%-60% humidity. After 7 days of feeding to adapt to the environment, the mice were injected with null lentivirus (Group 3), shWISP1 (Group 5) or lentivirus WISP1 (Group 4) via the caudal vein with a total lentivector dose of 2 × 10 7 TU/mouse. After 12 weeks, the mice were anaesthetized intraperitoneally with 3% pentobarbital sodium (40 mg/kg) and killed. The hearts were gathered for histological staining, the aortas were gathered for Western blotting and Oil Red O staining, and the serum samples were collected for blood lipid detection and ELISA. The effectiveness of the lentivirus was confirmed by the expression of WISP1 in the serum and tissues of the mice injected with lentivirus.

| Cell culture
In this study, macrophages (peritoneal macrophages and RAW264.7

| Western blot analysis
Proteins from mice or cells were separated by 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a polyvinylidene fluoride (PVDF) membrane for 90 min. Skim milk (5%) was used to block the PVDF membranes, and then, the membranes were incubated with primary and secondary antibodies. Finally, enhanced chemiluminescence (Millipore) was used for exposure via Amersham Imager 600.

| ROS levels
The level of intracellular ROS was measured with the peroxidesensitive fluorescent probe 2', 7'-diacetate (Sigma-Aldrich, Shanghai, China). The experimental group was treated with NAC (5 mmol/L) for 2 h before ox-LDL stimulation for 24 h. RAW264.7 cell lines were exposed to DCFH-DA (10 μmol/L) for 30 min at 37°C, and the fluorescent signal was detected via fluorescence microscopy (488 nm filter, Olympus, Tokyo, Japan).

| Plasma and supernatant analyses
Designated adipokines were quantitatively assessed in plasma and cell supernatant using the respective ELISA kits (R&D Systems GmbH) in accordance with the manufacturer's instructions. The TC (total cholesterol), LDL-C (low-density lipoprotein cholesterol) and HDL-C (high-density lipoprotein cholesterol) levels in plasma were measured by an enzyme-labelled instrument (Molecular Devices) following the manufacturer's instructions. All mice plasma samples were isolated from blood by eyeball extirpation.

| Immunofluorescence
The slides were rinsed with PBS thrice, blocked with 3% BSA for 30 min at 37℃ and then incubated with rabbit anti-CD36 antibody

| Statistical analysis
Data analyses and statistical predictions in this study were conducted through GraphPad prism 5.0 and SPSS 20.0. One-way ANOVA was used for analysing differences among groups, and an unpaired t test was used for analysing discrepancies between groups. All experiments were independently conducted in triplicates or greater, and data have been shown as means ± standard deviation. P < .05 was considered statistically significant.

| Basic characteristics of HFD ApoE-/-mouse model
After 12 weeks, the HFD (16% fat and 0.25% cholesterol) groups showed higher TC and LDL-C levels and weight, but lower HDL-C levels than the control group. However, WISP1 overexpression and down-regulation had no effect on the TC, LDL-C, HDL-C or weight of the mice (Figure 1). Compared to the control group, HFD was found to increase the WISP1 level in serum via activation of the Wnt5a/βcatenin pathway ( Figure 4D,E,F), whereas IvWISP1 and shWISP1 significantly increased and decreased WISP1 levels in serum, respectively, compared to the HFD + NC group.

| WISP1 affects plaque formation in ApoE-/-mice
We studied images of haematoxylin and eosin ( Figure 2A) and Oil Red O staining ( Figure 2B) of cross-sections from aortic sinuses. We found that IvWISP1 reduced the size of HFD-induced atherosclerotic lesions in the aortic tissue of the mice. On the contrary, atherosclerotic lesions in the shWISP1 group were larger. The same results were observed in Oil Red O staining images of whole aortas ( Figure 2C).

| WISP1 alleviates lipid deposition and recruitment of macrophages in ApoE-/-mice
CD36 and SR-A are important factors in lipid deposition. 30 The expression of CD36 and SR-A proteins increased significantly in HFD mice compared to those in the control group. Down-regulation of WISP1 contributed to higher CD36 and SR-A levels, while IvWISP1 mice showed decreased CD36 and SR-A levels compared to mice in the NC group ( Figure 3A,C, Figure 4A,B,C). Moreover, more macrophage recruitment in plaques was observed in HFD mice than in mice on a regular diet. Additionally, down-regulation of WISP1 resulted in macrophage recruitment, which decreased in IvWISP1treated mice compared to NC group mice (P < .05; Figure 3B).

| ROS mediates WISP1 expression with ox-LDL stimulation in macrophages via Wnt5a/βcatenin pathway
WISP1 protein expression apparently increased after stimulation with ox-LDL, thereby confirming that ox-LDL stimulation promoted WISP1 expression. As ROS production was promoted by ox-LDL, we inspected possible mechanisms of ox-LDL stimulation of WISP1 in macrophages. To investigate this, NAC (N-Acetyl-Lcysteine), an ROS inhibitor, was used to reduce the process of ROS generation. We found that WISP1 expression in the ox-LDL + NAC group was evidently down-regulated compared to that in the ox-LDL group through suppression of the Wnt5a/β-catenin pathway.

| WISP1 can alleviate lipid deposition in macrophages
We then determined whether WISP1 was involved in lipid depo-

| Down-regulation of WISP1 is involved in the activation of PPARγ and promotion of lipid deposition
To analyse the function of WISP1 in the activation of PPARγ, lentivirus WISP1 and WISP1-shRNA were used to increase and reduce WISP1 expression, respectively. Down-regulation of WISP1 led to higher levels of PPARγ, CD36 and SR-A compared to the NC group (P < .05), whereas IvWISP1 treatment had the opposite effect (P < .05) ( Figure 7A). To further understand the mechanism, we used T0070907, an inhibitor of PPARγ, and found that it inhibited the upregulation of lipid deposition and CD36 expression under the condition of down-regulated WISP1 ( Figure 7B).

| D ISCUSS I ON
Atherosclerosis is known as one of the most widespread diseases, and its formation is complex, including lipid deposition, endothelial dysfunction and the propagation of these reactions. 1 Lipid deposition is considered as a chronic inflammatory disorder. 31 33 Some studies have demonstrated that WISP1 accelerates angiogenesis in oral squamous carcinoma via VEGF-A up-regulation. 34 In this study, we found that WISP1 expression was Interestingly, when we down-regulated WISP1 expression, plaque formation worsened, suggesting that WISP1 played an important role in the progression of atherosclerosis. The down-regulation of WISP1 can accelerate macrophage recruitment in plaques. Additionally, a highfat diet increased the weight and the levels of TC and LDL-C in mice, but decreased HDL-C levels. However, WISP1 inhibition and overexpression had no effect on the TC, LDL-C and HDL-C. Therefore, we concluded that WISP1 levels could be raised by HFD, and WISP1 can decelerate the progression of atherosclerosis.
Uncontrolled uptake of oxidized, low-density lipoprotein (ox-LDL) leads to the accumulation of cholesterol ester (CE), which is stored as cytoplasmic lipid droplets and subsequently triggers the formation of foam cells. The generation of foam cells is associated with the lipid homeostasis between cholesterol influx and efflux, and esterification. 35,36 The scavenger receptors (SRs) CD36 and SR class A (SR-A) are the major receptors responsible for the uptake of ox-LDL in macrophages. 19 In contrast, the efflux of intracellular cholesterol to high-density lipoprotein is mediated by reverse cholesterol transporters, including class B scavenger receptor type I (SR-BI) and ATP-binding cassette transporter A1 (ABCA1). [37][38][39] Additionally, CD36 is highly expressed in macrophages, and its expression is regulated by multiple factors. For example, CD36 levels in peritoneal macrophages are stimulated by peroxisome proliferator-activated receptor-γ (PPARγ).
PPARγ is a member of a nuclear hormone superfamily that heterodimerizes with RXR. They are transcriptional regulators of genes that can encode proteins involved in adipogenesis and lipid metabolism. 11 Interestingly, interaction of WISP1 with PPARγ leads to proteasome-dependent degradation of the latter, and therefore, results   43,44 Our study clearly demonstrated that WISP1 overexpression was triggered by ox-LDL in macrophages, by activation of the Wnt5a/β-catenin pathway (RAW264.7 cell line) ( Figure 8A). Additionally, ox-LDL treatment resulted in excessive synthesis of ROS in macrophages, but with ROS inhibition, Notably, we propose that the mechanism by which WISP1 alleviates lipid deposition of atherosclerosis is by reduction of SR-A and CD36 expression, which is crucial to lipid deposition. Also, WISP1 can inhibit the recruitment of macrophages in atherosclerosis.
When macrophages were stimulated by ox-LDL, the expression of WISP1 increased because of ROS mediation. However, up-regulation of WISP1 could suppress foam cell formation by suppressing the PPARγ/CD36 pathway and SR-A.

| CON CLUS ION
In conclusion, we demonstrated that WISP1 could alleviate lipid deposition and plaque formation in ApoE-/-mice. We also found that WISP1 was activated by ox-LDL, with high levels of ROS, which played a significant role in mediating WISP1 expression. Nonetheless, down-regulation of WISP1 can promote SR-A and CD36 expression through the PPARγ signalling pathway. Hence, this study successfully clarified the role of WISP1 in atherosclerotic plaques, thereby providing a new therapeutic goal for atherosclerosis.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest.