Atherosclerotic Plaques Were Increased in GARKO-Low-Density Lipoprotein Receptor–Deficient Mice But Decreased in Monocytes/Macrophage AR Knockout-Low-Density Lipoprotein Receptor–Deficient Mice Compared With the Wild-Type-Low-Density Lipoprotein Receptor–Deficient Littermate Control Mice

Using the Cre-loxP system, we either knocked out AR ubiquitously or selectively in 3 major cell types associated with atherosclerosis: monocytes/macrophages, ECs, and SMCs, and the resultant monocytes/macrophage ARKO (MARKO), ECs-ARKO (EARKO), and SMC-ARKO (SARKO) mice were then crossed with the low-density lipoprotein receptor (LDLR)–deficient (LDLR^−/−) mice to develop GARKO-LDLR^−/−, MARKO-LDLR^−/−, EARKO-LDLR^−/−, and SARKO-LDLR^−/− mice. Figure S1A in the online-only Data Supplement shows the genotyping results of 4 types of the generated ARKO mice and Figure S1B shows the depletion of AR in the bone marrow–derived macrophages obtained from MARKO-LDLR^−/− mice. No significant change in body weight was observed in the MARKO-LDLR^−/− mice, but we observed increases in body weights in the other 2 types of mice (EARKO-LDLR^−/− and SARKO-LDLR^−/−) (Figure S1C).

After atherosclerosis was developed by feeding mice with high-fat diet for 16 weeks, aortas were excised from each group of mice and stained with Oil red O to visualize atherosclerotic plaques. Compared with the wild-type (WT)-LDLR^−/− littlermate control mice, the GARKO-LDLR^−/− showed increased atherosclerotic plaques and reduced androgen levels, which are consistent with previous findings (Figure 1A–1D).¹³ However, the MARKO-LDLR^−/− mice showed significantly less atherosclerosis (25.9% reduction; P value<0.001; n=9) without an altered androgen level as compared with the WT-LDLR^−/− littermate control mice (Figure 2A, 2B, and 2E). In contrast, we found little differences in plaque areas in the EARKO-LDLR^−/− and SARKO-LDLR^−/− mice as compared with their littermate controls. The results indicate that there are no significant differences in androgen levels among EARKO-LDLR^−/−, SARKO-LDLR^−/−, and their littermate controls (Figure 2A, 2B, and 2F). We then performed hematoxylin and eosin staining of aortic tissues of these mice and detected reduced plaque area in the MARKO-LDLR^−/− mice (38% reduction; P value <0.001; n=6) compared with the littermate control mice (n=5; Figure 2C; quantification of relative plaque areas is shown in Figure 2D).

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Figure 1.

Plaque formation was accelerated in general androgen receptor knockout in low-density lipoprotein receptor–deficient (GARKO-LDLR^−/−) mice compared with the wild-type (WT)-LDLR^−/− littermate control mice. A, Plaque area was analyzed after staining with Oil red O stain. Graph on the right is quantification results of plaque area over total area. B, Masson Trichrome staining was used to analyze collagen deposition in WT-LDLR^−/− and GARKO-LDLR^−/− mice. Graph on right shows the quantification results of collagen positive area over total area. Arrows indicate collagen area (blue color). Magnification, ×400. Androgens, (C) testosterone, and (D) dihydrotestosterone (DHT) were measured using ELISA kit in WT-LDLR^−/− and GARKO-LDLR^−/− mice. *P<0.05, **P<0.01, and ***P<0.001.

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Figure 2.

Plaque area was reduced in the monocyte/macrophage androgen receptor knockout (ARKO) in low-density lipoprotein receptor–deficient (MARKO-LDLR^−/−) mice compared with the wild-type (WT)-LDLR^−/− control mice, but not reduced in the endothelial cell-ARKO (EARKO)-LDLR^−/− and smooth muscle cell-ARKO (SARKO)-LDLR^−/− mice. A, Oil red O staining of aortae obtained from the MARKO-LDLR^−/−, EARKO-LDLR^−/−, and SARKO-LDLR^−/− mice and their WT-LDLR^−/− littermate control mice. Aortae were stained with Oil red O and positive red stained area indicates plaques formed. B, Quantification results of positive plaque area over total area from A. C. Hematoxylin–eosin staining of aortic tissues of MARKO-LDLR^−/−, EARKO-LDLR^−/−, and SARKO-LDLR^−/− mice and their WT-LDLR^−/− littermate control mice. Magnification, ×100 (inset, ×400). Two arrowheads indicate the plaque area. D, Quantification of plaque area over total area from C. E, Testosterone levels were determined in MARKO-LDLR^−/− mice and their WT-LDLR^−/− littermate controls. F, Testosterone levels in WT-LDLR^−/−, SARKO-LDLR^−/−, and EARKO-LDLR^−/− mice. **P<0.01.

Together, the results of these studies using the LDLR^−/− derivatives of the 4 different ARKO mice demonstrated that the AR in monocytes/macrophages, but not in ECs and SMCs, played an important role in the promotion of atherosclerosis.

Depletion of AR in Monocytes/Macrophages Reduces Monocytes Infiltration into the Aorta Plaque Area

A crucial step in atherosclerosis is an infiltration of monocytes into the subendothelium of the arteries where they differentiate into macrophages and become functionally active.¹⁴ We therefore investigated monocytes infiltration in aortic tissues of the MARKO-LDLR^−/− and WT-LDLR^−/− littermate control mice by staining tissues with the monocyte-specific antibody, Mac-3. We found that the Mac-3 positively stained cell numbers (represented as positively stained cell percentage) were significantly decreased in the MARKO-LDLR^−/− mice (5.28±2.11%; n=7) compared with their littermate control mice (20.57±4.42%; n=6; Figure 3A).

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Figure 3.

Macrophage infiltration and collagen deposition were reduced, but smooth muscle cells (SMCs) invasion was increased in the monocyte/macrophage androgen receptor knockout in low-density lipoprotein receptor–deficient (MARKO-LDLR^−/−) mice compared with the wild-type (WT)-LDLR^−/− mice. A, Aortic tissues of the MARKO-LDLR^−/− and WT-LDLR^−/− mice were stained with Mac-3 antibody. Arrows indicate stained Mac-3 positively stained cells. Magnification, ×100. Quantification is shown below images **P<0.01. B, Aortic tissues of the MARKO-LDLR^−/− and WT-LDLR^−/− mice were used in the Masson Trichrome staining. Arrows indicate positively stained cells. Magnification, ×100. Quantification is shown below images. *P<0.05. C, Immunohistochemical staining of SMCs invasion. Aortic tissues of the MARKO-LDLR^−/− and WT-LDLR^−/− mice stained with α-smooth muscle actin (SMA). Arrows indicate positively stained cells. Magnification, ×100. Quantification is shown below images. **P<0.01.

We also examined collagen deposition in aortas of MARKO-LDLR^−/− and WT-LDLR^−/− mice via Masson Trichrome staining, and results demonstrated significantly lower collagen deposition in the MARKO-LDLR^−/− mice (10.14±1.08%; n=5) compared with the littermate control mice (14.48±1.28%; n=5; Figure 3B).

We further investigated whether invaded SMC numbers were modulated by the ARKO in monocytes/macrophages and found that the α-smooth muscle actin positively stained SMC numbers were increased in the MARKO-LDLR^−/− mice (28.70±1.94%; n=5) compared with the control littermate mice (12.37±1.71%; n=6; Figure 3C). This suggested that higher numbers of the migrated SMCs may stabilize the plaques leaving them less susceptible to rupture.^15,16 Therefore, we speculate that the ARKO in monocytes/macrophages might contribute to the stability of plaques as shown by the increased numbers of the invaded SMCs in MARKO-LDLR^−/− mice, thereby protecting mice from developing into the acute coronary syndrome.

Together, results of Figure 3A to 3C demonstrate that the loss of AR in monocytes/macrophages resulted in decreased infiltrating monocytes/macrophages in the subendothelium of arteries with decreased collagen deposition and increased SMCs, and all these combined effects led to the suppression of atherosclerosis.

AR in Monocyte THP-1 Cells Promoted Their Migration and Adhesion to Human Umbilical Vein ECs and Foam Cell Formation

To further confirm the above in vivo findings, we applied in vitro assays to show the AR role in monocytes/macrophages in promoting atherosclerosis. We first investigated whether the AR expression in monocytes influences their migration. Monocyte THP-1 cells were infected with lentivirus carrying either scramble control sequence or AR-siRNA and Figure 4A-a (upper) shows successful knockdown of AR in these cells. In the migration assay, the THP-1 cells, either the AR knocked down (THP-1siAR) or the scramble control (THP-1sc) cells, were placed in the upper chamber, whereas the conditioned media of human umbilical vein ECs (HUVECs) were placed in the lower chamber of transwell plates as shown in Figure 4A-a (lower) cartoon. We found that the migration of the THP-1siAR cells was suppressed, when compared with the THP-1sc cells (Figure 4A-b). However, when we manipulated AR expression in HUVECs and used the conditioned media of the AR expressing and depleted HUVECs in the tests, migration of THP-1 cells was not influenced significantly (data not shown), suggesting that AR expression in monocytes, and not in ECs, is critical in mediating this migration process.

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Figure 4.

A, Androgen receptor (AR) in THP-1 cells play a stimulatory role to promote the migration and adhesion to human umbilical vein endothelial cells (HUVECs) and foam cell formation. a, THP-1 cells with AR knocked down (THP-1siAR) and scramble control (THP-1sc), and HUVECs conditioned media were placed in transwell plates as described in the cartoon (lower). b, The migrated cells were stained and the positively stained cells numbers were measured. **P<0.01. c, THP-1siAR and THP-1sc were tagged with green fluorescence dye and then incubated with HUVECs. The adhered THP-1 cells with green fluorescence were counted. Quantification is shown on the right. Magnification, ×100. **P<0.01. d, The THP-1siAR and THP-1sc cells were induced to differentiate into macrophages by mouse-derived colony-stimulating factor (M-CSF). The cells were then incubated with oxidized low-density lipoprotein (oxLDL) to evaluate the ability of cells to uptake oxLDL (cartoon at left). At the end of reaction, cells were stained with filtered Oil red O (middle). Magnification, ×100. Quantification is shown on the right. **P<0.01. B, The AR in THP-1 cells promotes migration to HUVECs. a, Quantitative polymerase chain reaction (qPCR) analysis testing mRNA expression levels of candidate molecules involved in migration process. **P<0.01. b, ELISA test showing tumor necrosis factor (TNF)-α secretion by the THP-1siAR and THP-1sc cells. ***P<0.001. c, Chromatin immunoprecipitation (ChIP) assay. AR antibody was used to pull down TNF-α promoter region, which contains androgen response elements (AREs; upper). d, TNF-α promoter transactivation was measured using luciferase constructs containing AR binding sites of the promoter region of TNF-α molecule in the presence of pBabe vector only or pBabe-AR. Human embryonic kidney (HEK)-293 cells were used in this assay. e, Blocking effect of migration of THP-1 cells to HUVECs on incubation with the TNF-α antibody. f, Immunohistochemical (IHC) staining of TNF-α in aortic tissues obtained from the monocyte/macrophage AR knockout in low-density lipoprotein receptor–deficient (MARKO-LDLR^−/−) and wild-type (WT)-LDLR^−/− mice. Arrowheads indicate positive stained areas. Magnification, ×100. Quantification is shown on the right. *P<0.05, **P<0.01, and ***P<0.001. C, The AR in THP-1 cells promotes adhesion to HUVECs. a, qPCR analysis testing mRNA expression levels of candidate molecules involved in adhesion process. b, Western blot analysis showing AR and integrin (ITG) β2 in the THP-1siAR and THP-1sc cells. GAPDH was used as control. c, ChiP assay. AR antibody was used to pull down ITGβ2 promoter region, which contains AREs. d, ITGβ2 promoter transactivation was measured using luciferase construct containing ITGβ2 promoter region in the presence or absence of AR with or without dihydrotestosterone (DHT). HEK-293 cells were used in this assay. e, Blocking effect of adhesion of THP-1siAR and THP-1sc cells onto the HUVECs on incubation with the ITGβ2 antibody. f, IHC staining of ITGβ2 in aortic tissues obtained from the MARKO-LDLR^−/− and WT-LDLR^−/− mice. Magnification, ×400. Arrowheads indicate positive stained area. Quantification is shown on the right. *P<0.05, **P<0.01, and ***P<0.001. D, The AR in macrophages promotes foam cell formation. a, qPCR analysis testing mRNA expression levels of candidate molecules involved in foam cell formation process. **P<0.01. b, AR and lectin-type oxidized LDL receptor 1 (LOX-1) expression levels were determined using Western blot in THP-1sc and THP-1siAR cells. GAPDH served as loading control. c, LOX-1 promoter region containing ARE (upper) was pulled down with AR antibody and amplified with primers specific targeting ARE region (lower). d, LOX-1 promoter region was constructed to pGL3 luciferase vector. The LOX-1 promoter transactivation was measured in the presence or absence of AR with or without DHT. e, Blocking effect on foam cell formation on incubation with the LOX-1 antibody. Quantification is shown on the right. f, IHC staining of LOX-1 in aortic tissues obtained from the MARKO-LDLR^−/− and WT-LDLR^−/− mice. Magnification, ×100. Quantification is shown on the right. *P<0.05, **P<0.01, and ***P<0.001.

We also tested adhesion of THP-1 cells onto HUVECs (a cartoon in Figure 4A-c, upper). Similarly, we used THP-1siAR and THP-1sc cells in the test and tagged THP-1 cells with green fluorescence dye before the reaction so that the adhered green fluorescence dye–labeled cells can easily be detected (middle). After incubation of 2 types of cells for 2 hours at 37°C, numbers of the adhered fluorescent THP-1 cells were counted. Similar to the migration test, we found that adhesion of the THP-1siAR onto HUVECs was significantly reduced (60%) compared with the THP-1sc (Figure 4A-c, lower; quantification, right; representative images in Figure S2A). However, the manipulation of AR expression in HUVECs did not influence adhesion of THP-1 cells onto HUVECs significantly (data not shown). These results also suggest that AR expression in monocytes, and not in ECs, is critical in mediating this migration process and in promoting the adhesion process.

In addition to migration and adhesion, foam cell formation is the third key step of atherosclerosis development.¹⁷ Therefore, we tested whether the AR expression in THP-1 cells can also affect this process. THP-1siAR and THP-1sc cells were treated with mouse-derived colony-stimulating factor to stimulate differentiation of monocytes into macrophages and these resultant macrophages were subsequently treated with oxidized LDL to induce foam cell formation. After staining of foam cells with Oil red O, the positively stained cells numbers were compared. As shown in Figure 4A-d, the THP-1siAR cells have significantly reduced foam cell formation (11.89±2.18%) as compared with the THP-1sc control cells (17.65±1.72%), indicating that the AR in THP-1 cells also plays a stimulatory role in this process.

Taken together, Figure 4A results suggest that the AR in monocytes/macrophages plays positive roles in promoting the 3 major inflammation-associated processes in atherosclerosis: (1) migration, (2) adhesion of monocytes to ECs, and (3) subsequent foam cell formation.

Key AR-Modulated Molecules That Affect Migration of Monocytes to ECs in THP-1 Cells

We next investigated the molecular mechanisms by which the AR in monocytes/macrophages promote the 3 inflammation-associated processes via modulation of expressions of the target molecules of these processes.

Among the migration-related molecules, including tumor necrosis factor (TNF)-α, interleukin-6, chemokine (C-C motif) ligand 2 and C-C chemokine receptor type 2 we assayed, we found that the mRNA expressions of TNF-α and interleukin-6 were the most significantly inhibited in THP-1siAR cells compared with those in THP-1sc cells (Figure 4B-a). We then selected TNF-α for further investigation. As shown in Figure 4B-b, we found that the secreted TNF-α protein was decreased dramatically in THP-1siAR cells compared with that in the THP-1sc cells, confirming the positive regulation of AR in TNF-α expression at both protein and mRNA levels.

We then asked whether AR could modulate TNF-α at transcriptional levels via chromatin immunoprecipitation assay. The results showed that the AR could bind to the androgen response element (ARE; TNF promoter ARE1: +36≈+41; ARE2: +50≈+55) on the 5′ promoter region of TNF-α (Figure 4B-c), and the luciferase assay also confirmed that AR could induce TNF-α expression at the transcriptional level (Figure 4B-d).

We further applied functional assays to test whether blocking TNF-α via a neutralizing antibody of TNF-α could suppress the AR effect in promoting migration of THP-1 cells into ECs. As shown in Figure 4B-e, addition of TNF-α neutralizing antibody reduces THP-1sc cells migration to the level of the THP-1siAR cells, indicating that TNF-α is indeed critical in mediating the promoter role of AR in this process.

We also examined whether the TNF-α expression is reduced in the aortic tissues of the MARKO-LDLR^−/− mice compared with their WT-LDLR^−/− littermate control mice and found significantly reduced expressions of TNF-α in aortic tissues of MARKO-LDLR^−/− mice compared with the WT-LDLR^−/− littermate control mice (Figure 4B-f), confirming the in vitro effect of AR modulation of TNF-α for promoting monocytes/macrophages migration to ECs.

Key AR-Modulated Molecules That Affect Adhesion of Monocytes to ECs in THP-1 Cells

Next we surveyed molecules that have been documented to play important roles in the adhesion process,^15,16 and found that the expression of integrin family members (integrin [ITG] αM, αV, β1, β2) and P-selectin glycoprotein ligand was suppressed in THP-1siAR cells compared with that in the THP-1sc control cells (Figure 4C-a). We further dissected the molecular mechanism how AR could modulate ITGβ2 in the adhesion process, and confirmed AR could modulate ITGβ2 expression at both mRNA and protein levels (Figure 4C-a and b).

We further examined AR modulation in ITGβ2 expression at the transcriptional level and found that AR could bind to ARE (AGACCAnnnTGATCA, location −4462 to −4447) on the 5′ promoter region of ITGβ2 via chromatin immunoprecipitation assay (Figure 4C-c). The luciferase assay further confirmed that AR could induce ITGβ2 expression at the transcriptional level in the 293T cells (Figure 4C-d).

When we introduced the neutralizing antibody of ITGβ2 into the culture, we were able to inhibit adhesion of THP-1sc cells, but not the THP-1siAR cells (Figure 4C-e; representative images in Figure S2B), indicating that the ITGβ2 molecule is critical in this AR-mediated adhesion process.

Finally, we examined ITGβ2 expression in the aortic tissues of the MARKO-LDLR^−/− mice and their WT-LDLR^−/− littermate control mice and found significantly reduced ITGβ2 expression in aortic tissues of MARKO-LDLR^−/− mice compared with their littermate control mice (Figure 4C-f), which also confirmed the AR-mediated reduction of ITGβ2 in vivo.

Key AR-Modulated Molecules That Affect Foam Cell Formation in THP-1 Cells

We then investigated the target molecules in the foam cell formation process, such as lectin-type oxidized LDL receptor 1 (LOX-1), lysosomal acid lipase, and acyl–coenzyme A: cholesterol acyltransferase, in THP-1siAR and THP-1sc cells and found that expressions of these 3 molecules were suppressed in the THP-1siAR cells (Figure 4D-a). Further analysis of LOX-1 expression in THP-1siAR and THP-1sc cells showed lower mRNA expression of LOX-1 in THP-1siAR cells compared with the THP-1sc cells (Figure 4D-b).

We also used the chromatin immunoprecipitation assay to determine whether AR could modulate LOX-1 expression at the transcriptional level, and the results showed that AR could bind to ARE (ATAAAAnnnTGTTTT, location −4209 to −4194) on the 5′ promoter region of LOX-1 (Figure 4D-c). As expected, the luciferase assay also confirmed AR could induce LOX-1 expression at the transcriptional level (Figure 4D-d).

We further tested whether the blocking of LOX-1 by treating the THP-1siAR and THP-1sc cells with the LOX-1 neutralizing antibody could inhibit foam cell formation. As shown in Figure 4D-e, the foam cell formation was significantly inhibited on antibody treatment in THP-1sc cells, but not in THP-1siAR cells.

We then examined LOX-1 expression in the aortic tissues of the MARKO-LDLR^−/− and their WT-LDLR^−/− littermate control mice and found significantly reduced expressions of LOX-1 in aortic tissues of MARKO-LDLR^−/− mice compared with their littermate control mice (Figure 4D-f), suggesting that AR could go through modulation of LOX-1 expression to influence form cell formation in vivo.

Together, results from Figure 4 show that the AR in monocytes/macrophages might play a stimulatory role to promote atherosclerosis via multiple modulations of various key molecules, which might explain why we could detect decreased plaque formation in the MARKO-LDLR^−/− mice (see Figure 2).

Targeting Monocyte/Macrophage AR With ASC-J9 to Battle Atherosclerosis

We then asked how we can target monocytes/macrophages AR in the body to mimic the MARKO-LDLR^−/− mice effect and use that strategy as a potential new therapeutic approach to battle atherosclerosis. ASC-J9 is an AR degradation enhancer, which functions through the proteasome machinery to selectively degrade AR. ASC-J9 could increase Akt-modulated AR and Mdm2 interaction and recruit the proteasome complex to enhance AR’s degradation.¹⁸ The earlier studies on wound healing demonstrated that ASC-J9 was able to selectively degrade AR in monocytes/macrophages and mimicked the MARKO mice effect with improved wound healing.¹¹

We first tested the in vitro ASC-J9 effects in inhibiting 3 processes: migration and adhesion of monocytes onto ECs and subsequent foam cell formation. Our results showed that ASC-J9 could enhance AR protein degradation in THP-1 cells starting at 2.5 μmol/L concentration (Figure 5A). When we used 5-μmol/L ASC-J9 to test its effect on THP-1 cell migration into HUVECs, we found significant reduction in THP-1 cells migration (Figure 5B). We then tested ASC-J9 effect on the adhesion of THP-1 cells onto HUVECs. As shown in Figure 5C, THP-1 cell adhesion was also significantly reduced on ASC-J9 treatment (representative images in Figure S2C). We further examined ASC-J9 effects on foam cell formation and obtained similar results showing suppressive effects of ASC-J9 (2.5–10 μmol/L; Figure 5d).

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Figure 5.

ASC-J9 treatment blocked THP-1 cell migration and adhesion into human umbilical vein endothelial cells (HUVECs) and foam cell formation. A, Western blot analysis showing androgen receptor (AR) protein levels in THP-1 cells on ASC-J9 treatment. B, In vitro test of ASC-J9 effect on migration of THP-1 cells into HUVECs. THP-1 cells were treated with ASC-J9 (10 μmol/L) for 2 days and used for the migration experiment. *P<0.05. C, In vitro test of ASC-J9 effect on adhesion of THP-1 cells onto HUVECs. **P<0.01. D, In vitro test of ASC-J9 effect on foam cell formation. THP-1 cells were treated with various concentrations of ASC-J9 for 2 days and used for the experiment. Quantification is shown on the right. **P<0.01.

We then extended our studies of ASC-J9 effects into in vivo mice. ASC-J9 was injected intraperitoneally every other day (75 mg/kg per day) into the 16-week-old WT-LDLR^−/− mice, 8 weeks after starting high-fat diet feeding and continued for 8 weeks. In parallel, control littermates received vehicle only. After a total of 16 weeks of high-fat diet feeding, mice were euthanized and atherosclerosis parameters were analyzed. Interestingly, the results showed that the WT-LDLR^−/− mice treated with ASC-J9 had significantly decreased plaque area (25.1±4.9%; n=11) compared with the vehicle control mice (19.2±5.7%; n=5; Figure 6A). We then examined whether the treatment with ASC-J9 leads to decreased AR expression in macrophages. The immunohistochemical staining revealed that expression of the monocyte/macrophage AR (Figure 6B) and macrophages infiltration (Figure 6C) were significantly decreased in these ASC-J9–treated mice. Importantly, we found that mice treated with ASC-J9 still had normal body weights with near normal serum testosterone and normal fertility, which is consistent with the previous results showing few side effects.^11,19

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Figure 6.

Therapeutic effect of ASC-J9 in reducing atherosclerosis by inhibiting monocytes infiltration. A, ASC-J9 effects on plaque formation in mice. Wild type low-density lipoprotein receptor–deficient (WT-LDLR^−/−) mice fed with high-fat diet (HFD) for 8 weeks were injected with ASC-J9 (75 mg/kg body weight) every other day for another 8 weeks with continued HFD treatment. Quantification is shown below images, *P<0.05. B, Immunohistochemical (IHC) staining results of androgen receptor (AR) in aortic tissues of vehicle and ASC-J9–treated mice. C, Mac-3 IHC staining results in aortic tissues of vehicle and ASC-J9–treated mice. For both B and C, magnification, ×100; insets, ×400. Arrowheads indicate positive stained cells. Quantification is shown below images, **P<0.01.

Together, the results from in vitro cell lines (Figure 5) and in vivo mice studies (Figure 6) suggest that ASC-J9 may have a therapeutic potential to battle the atherosclerosis in the future.