BMP4 is increased in the aortas of diabetic ApoE knockout mice and enhances uptake of oxidized low density lipoprotein into peritoneal macrophages

C57BL/6J ApoE KO mice were purchased from Jackson Laboratory (Bar Harbor, ME, USA) and were housed under standard conditions, including humidity, room temperature, and dark–light cycles. Mice were given free access to food and water throughout the study. The study protocol was approved by the Laboratory Animal Care and Use Committee of Fukuoka University. ApoE KO mice (6 weeks old) were intraperitoneally injected with 55 mg kg^–1 day^–1 STZ or vehicle (citrate buffer, control) over 5 consecutive days[22, 23]. Blood glucose levels were measured 2 weeks after STZ administration to assess the induction of diabetes; only diabetic mice (defined as non-fasting glucose > 250 mg/dL) were used in this study. Both groups of ApoE KO mice were fed a high-fat diet (1.25% cholesterol, 15% cacao butter, and 0.5% sodium cholate, F2HFD1, Oriental Yeast CO, Tokyo, Japan) for 4 weeks, starting from 8 weeks old. At 12 weeks old, the animals were killed, and the aortas removed for comparisons between STZ-induced diabetes and control mice.

To quantify the extent of atherosclerotic lesions, immediately after the mice were killed, the whole length of the aorta (n = 9 mice in each group) was excised for quantification of the en face plaque area, as previously described[24, 25]. Briefly, after carefully removing adventitial tissue, the aortic arch and the thoracic to abdominal aorta were opened longitudinally, pinned on a black wax surface, and stained with Oil red O (Sigma, St. Louis, MO, USA). En face images were obtained by a stereomicroscope and analyzed using a public domain software Image J (NIH Image, Bethesda, MD, USA) (n = 9 mice/group). The percentage of the luminal surface area stained by Oil red-O was determined[24, 25].

After the mouse was sacrificed and perfused with ice-cold phosphate-buffered saline (PBS), the heart and the ascending aorta were removed en bloc and snap-freezed in O.C.T. compound (Sakura FineTech, Tokyo, Japan) for histological and immunohistochemical analyses. Serial cryostat sections (6 μm thick) of the aortic root were prepared as previously described[24, 25]. Briefly, atherosclerotic plaques were examined in five independent sets of sections taken 60 μm apart. Oil red O staining was performed to identify the lipid-rich core. The Oil red O-stained areas, as a marker of lipid accumulation, were analyzed using Image J software. In each mouse, the mean for five independent sections was used for the analysis.

For staining the frozen sections, fresh mouse aortas were excised from ApoE KO mice, placed in Tissue-Tek O.C.T. compound, snap-freezed in lipid nitrogen, and stored at -80°C until use. After removing the O.C.T. compound and blocking, the samples were incubated with antibodies against BMP4 (1:100; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) and MOMA2 (1:500; BMA Biomedicals, Augst, Switzerland) overnight at 4°C. For double-immunofluorescence staining, the samples were incubated with FITC (Nacalai Tesque, Kyoto, Japan) and AlexaFluor 594®-conjugated secondary antibodies (Molecular Probes, Eugene, OR, USA), respectively, for 1 h at room temperature. Nuclei were counterstained with DAPI (Vector Laboratories, Burlingame, CA, USA). MOMA2-stained areas, as a marker of macrophage accumulation, were analyzed using Image J software.

The aorta was immediately snap-freezed in liquid nitrogen. Aortic proteins were isolated using lysis buffer (50 mM HEPES, 50 mM NaCl, 5 mM EDTA, 1% Triton X-100, 1 mM Na₃VO₄, 50 mM NaF, 10 mM sodium pyrophosphate decahydrate and 1 mM phenylmethylsulfonyl fluoride), containing 1% phosphatase inhibitor cocktail 1 (Sigma), 1% phosphatase inhibitor cocktail 2 (Sigma), and 1% protease inhibitor cocktail (Sigma). After tissue homogenization, particulate material was removed by centrifugation, and protein concentrations measured using the Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA, USA). Equal amounts of total protein (30 μg/sample) were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (10%) and electrophoretically transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). Non-specific antibody binding was blocked by incubating the membranes with Blocking One (Nacalai Tesque) for 60 min at room temperature. The primary antibodies used were mouse monoclonal anti-BMP4 (1:100, Santa Cruz Biotechnology Inc.) and anti-SMAD1 (1:100, Santa Cruz Biotechnology Inc.), and rabbit polyclonal anti-phospho-SMAD1/5/8 (1:1000, Cell Signaling, Danvers, MA) and anti-β-actin (1:2000, Abcam, Cambridge, UK). Blots were incubated overnight at 4°C with primary antibodies, and then incubated with horseradish peroxidase-conjugated secondary antibodies (1:5000 dilution) for 60 min at room temperature. The blot was developed using an ECL detection kit (Amersham International, Little Chalfont, UK). Signal intensities were normalized using beta-actin. Band images were digitally captured with a FluorChem SP imaging system (Alpha Innotech, San Leandro, CA, USA) and band intensities quantified using Image J software.

To isolate peritoneal macrophages, we intraperitoneally injected wild-type mice with 2 mL of 4% thioglycollate. Cells were collected from the peritoneal cavity 3 days after injection and were incubated in 12-well plates in complete medium (RPMI1640 media containing 10% fetal bovine serum and 100 U/mL penicillin/streptomycin). After 2 h, the cells were washed three times with PBS and cultured in media. The adherent cells, considered to be peritoneal macrophages, were used in the experiments[26]. For a single experiment, peritoneal macrophages were collected from one mouse.

OxLDL was purchased from HARBOR BIO-PRODUCTS (Norwood, MA, USA). Recombinant BMP4 and Noggin (a BMP4 antagonist) were purchased from R&D systems (Minneapolis, MN, USA). Peritoneal macrophages were serum starved for 3 h and then pretreated with or without 150 ng/ml Noggin for 3 h. OxLDL (15 μg/ml) and BMP4 (100ng/ml) were added to Noggin-treated or untreated peritoneal macrophages for 20 h. After washing three times with PBS, cells were fixed in 4% paraformaldehyde, and stained with Oil red-O and hematoxylin to evaluate uptake of oxLDL. The average percentage of Oil red O-labeled macrophages for each well was obtained by counting the number of labeled and unlabeled cells in 15 microscopic fields selected from each of three separate wells/group. The average value was calculated for each group. The experiment was repeated four times and the average percentage of four separate experiments was shown in Figure 1C.

At 12 weeks of age, blood was collected to measure blood glucose, plasma total cholesterol and triglyceride levels. Glucose was measured directly from the tail tip with a glucometer (Glutest sensor, Sanwa Kagaku Kenkyusho, Mie, Japan). Plasma total cholesterol and triglyceride levels were determined using commercial available kits (Wako Chemical Co., Osaka, Japan).

ApoE KO mice were rendered diabetic with an intraperitoneal administration of STZ, and were then fed a high-fat diet for 4 weeks. Approximately 80% of the mice were diabetic at 2 weeks after STZ administration, defined as non-fasting glucose > 250 mg/dL. Six weeks after STZ, glucose levels in the control and diabetic ApoE KO mice were 102 ± 12 and 454 ± 123 mg/dL, respectively (Table 1). After being fed the high-fat diet for 4 weeks, fasting plasma cholesterol levels in the diabetic ApoE KO mice were 1.4-fold higher than those in the control mice (2529 ± 513 vs. 1815 ± 302 mg/dL). Plasma triglyceride levels were not significantly different between the two groups of mice (n = 18, control mice; n = 16, diabetic ApoE KO mice).

At 12 weeks of age, the control and diabetic ApoE KO mice were killed to measure plaque area. The non-diabetic control ApoE KO mice had Oil red O-stained atherosclerotic plaques extending from the ascending aorta to the aortic arch. By contrast, the diabetic mice had more severe aortic plaque formation, with an increase in en face plaque area of 15% (Figure 2A–D). In addition, STZ-induced diabetes markedly accelerated the formation of atherosclerotic plaques in the aortic sinus (Figure 2E,F).

BMP4 protein expression levels in the aortas of control and diabetic ApoE KO mice were evaluated by western blot. Aortic BMP4 protein expression was significantly increased, by approximately 60%, in diabetic ApoE KO mice compared with control ApoE KO mice (n = 9 mice/group) (Figure 3B). Cross-sections taken at the aortic sinus were evaluated to determine the structural features of the atherosclerotic lesions. At 12 weeks of age, the plaques in the aortic root consisted of a lipid-rich core and massive macrophage infiltration into the intima in control ApoE KO mice. By contrast, the formation of lipid- and macrophage-rich plaques was remarkably increased at the aortic root in the diabetic ApoE KO mice.

Next, to test whether BMP4 was expressed in atherosclerotic lesions in the aortic sinus of ApoE KO mice, we stained several sections of the aortic root with anti-BMP4 antibody. The low magnification images (Figure 4A) revealed BMP4 protein in the internal layer. The localization of BMP4 in the atherosclerotic lesion was confirmed by double-immunofluorescence staining with anti-MOMA2 antibody (specific for monocytes/macrophages) and anti-BMP4 antibody. The BMP4-positive area appeared to correspond to the macrophage-rich area of the atherosclerotic lesion in the aortic root (Figure 4A). Furthermore, MOMA2-stained areas were larger at the aortic root in diabetic ApoE KO mice, than in controls (Figure 4B). BMP4 expression in monocytes/macrophages in the atherosclerotic lesions was much greater in diabetic mice than in control ApoE KO mice.

Western blot analysis showed SMAD1/5/8 phosphorylation was clearly induced in the whole aorta in diabetic ApoE KO mice, compared with controls (Figure 5A). Summarized data of SMAD1/5/8 phosphorylation is shown (Figure 5B; n = 6/group).

OxLDL incorporation in peritoneal macrophages obtained from wild-type mice was markedly increased by BMP4 treatment compared with untreated peritoneal macrophages (Figure 1). Noggin, a BMP4 antagonist, inhibited BMP4-induced oxLDL uptake in peritoneal macrophages. In the absence of oxLDL, few Oil red-O-positive peritoneal macrophages were observed in each group. On the other hand, we observed few Oil red-O-positive peritoneal macrophages in the absence of oxLDL. BMP4 alone did not increase the number of Oil red-O positive peritoneal macrophages (Figure 1).