To generate PACAP knockout mice (PACAP^−/−), PACAP was deleted from the PACAP gene locus in C57BL/6 mice (30). Then, PACAP knockout mice (PACAP^−/−) were crossbred with ApoE^−/− (Charles River, Sulzfeld, Germany) to generate PACAP^−/−/ApoE^−/− mice (29). For this study, only male homozygous PACAP^−/−/ApoE^−/− and ApoE^−/− mice were used, which were fed either standard chow for 30 weeks (SC, LASQCdiet® Rod16 Rad; LASvendi, Soest, Germany) or were fed a cholesterol-enriched diet [CED; “western-type diet” (21% fat, 0.15% cholesterol, and 19.5% casein), Altromin GmbH, Lage, Germany] for an additional 20 weeks at 10 weeks of age. All animals had ad libitum access to water and feed in their cages which had a minimum area of 100 cm² and an adequate enrichment device. The procedures were approved by the Regierungspräsidium Gießen (V54–19 c 2015 h 01 MR 20/26 No. 21/2014) and complied with the regulations for animal experiments at the Philipps-University Marburg.

Mice 30 weeks of age were intraperitoneally analgized for tissue removal with a combination of ketamine (150 mg/kg) and xylazine (20 mg/kg) (29) and then weighed and measured. Local intercostal anesthesia was performed with lidocaine 2%. After opening the thorax, the tip of the left ventricle was opened and a cannula (8 G, B. Braun Melsungen AG, Melsungen, Germany) was inserted. The vasculature was perfused with a solution of phosphate-buffered saline (PBS) with 5 Ul/ml heparin (Liquemin® 25,000 Ul/5 ml, Roche, Grenzach, Germany) using an automatic syringe pump (Secura, B. Braun, Melsungen AG) at 30 ml (rate of 100 ml/h). The aortic arch was harvested using a binocular loupe, embedded in Tissue-Tek® (Sakura Finetek, Stauffen, Germany), and frozen in liquid nitrogen-cooled isopentane.

Genomic DNA was isolated from the mouse ear using a commercial kit (DNA Extraction Solution; PeqLab, VWR Company, Erlangen, Germany) according to the manufacturer's instructions (DirectPCR® lysis reagent ear; Peqlab, VWR International; Darmstadt, Germany). Homozygous transgenic mice were subsequently detected by polymerase chain reaction (PCR) using intron-spanning oligonucleotides (see Table 1) (29, 30).

Cryosection series (6 µm) of the aortic arch were prepared for morphometric and immunohistological studies. The extent of atherosclerotic plaques in the aortic arch was measured by computerized morphometry. These images were analyzed and quantified using Fiji software (31). For this purpose, standard hematoxylin-eosin (HE) staining was performed. Further immunohistochemical staining was performed with the antibodies listed in Table 2. The lumen stenosis was determined by recording the lumen and plaque areas along the internal elastic lamina (or luminal plaque circumference) and calculating [[plaque area (µm²)]/[lumen area (µm²)] × 100% = lumen stenosis (%)] (29). Media thickness was determined by calculating the area of the lumen along the internal elastic lamina and the area along the outer elastic lamina by calculating [lumen area to outer elastic lamina (µm²)]—[lumen area to internal elastic lamina (µm²)] = area of media (µm²)]. Quantification of immunoreactive plaque area was assessed as described previously (29, 32).

Native (n)LDL (Cell sciences, MA, USA) oxidation was performed as described by Galle and Wanner (38) and Steinbrecher (39). nLDL was suspended in endotoxin-free PBS without Ca²⁺, Mg²⁺ (LONZA, Ratingen, Germany) to a final concentration of 2 mg protein/ml, and dialyzed using Vivaspin™ 20—System (Thermo Fisher Scientific GmbH, Schwerte, Germany). The Vivaspin™ 20 centrifugal concentrator was sterilized with 70% EtOH for 10 min at 3,000 × g. Afterward, the Vivaspin™ 20 was washed with aqua dest (endotoxin-free). Then, nLDL suspended in PBS was transferred into the Vivaspin™ 20 and centrifuged for 20 min at 4,500 × g. Two washing steps with PBS were performed to remove ethylene diamine tetraacetic acid (EDTA) from the nLDL. CuSO₄ was added to the EDTA-free nLDL and incubated overnight in the dark by constantly rotating. After 24 h, oxidation was stopped by adding EDTA (50 μM) and set in the dark for 1 h by continually rotating. After that, the oxLDL was washed three times with PBS. Subsequently, the oxLDL/PBS mixture was transferred by filtration through a 0.2 μm syringe filter to an endotoxin-free tube. The protein concentration was measured by Pierce™ BCA (bicinchoninic acid) Protein Assay (Thermo Scientific, Rockford, USA). We used different methods to determine the degree of oxidation: (1) trinitrobenzene sulfonic acid (TNBSA, Thermo Fisher Scientific GmbH), which measures free amino groups (40), (2) relative electrophoretic mobility (REM) by agarose gel electrophoresis and visualization by staining with Coomassie Blue (41), and 3. by spectrophotometric analysis (absorbance spectrum between 400 and 700 nm) (38).

The qRT-PCR was described before (34). QuantiTect primer assays (QIAGEN GmbH, Hilden, Germany) were used for qRT-PCR. All primers were purchased from QIAGEN GmbH (Hilden, Germany) (Table 3). Absorbance measurements at 260 nm and 280 nm (A260/A280 = 1.9–2.1) using a NanoDrop 8,000 spectrophotometer (Thermo Fisher Scientific GmbH) were used to determine RNA concentration and purity. Total RNA integrity was confirmed by lab-on-a-chip technology, using an RNA 6,000 NanoChip kit on an Agilent 2,100 Bioanalyzer (Stratagene-Agilent Technologies, Waldbronn, Germany). RNA was only used with an RNA Integrity Number (RIN) of ≥8.0. 1.0 μg of template RNA was used for cDNA synthesis. RNA was reverse transcribed using Oligo(dT)_12–18 primer and 20 units of the Affinity-Script™ Multiple Temperature cDNA synthesis reverse transcriptase (Agilent Technologies) and 24 units of Ribo Lock™ RNAse inhibitor (Thermo Fisher Scientific) (1 h; 42°C). The cDNA (diluted 1:20) was amplified using the Brilliant III Ultra-Fast SYBR® Green qRT-PCR Master Mix (Stratagene-Agilent Technologies). Amplification and data analyses were performed using the Mx3005P™ qPCR System (Stratagene-Agilent Technologies). The data were analyzed using the relative standard curve method. For each sample, the relative quantity was calculated by linear regression analysis from the respective standard curves. The NormFinder software program was used to ascertain the most suitable reference gene (actin, beta, ACTB) to normalize the RNA input as described earlier (42).

The release of IL-6, IL-10, and TNF-α was quantified using an enzyme-linked immunosorbent assay (ELISA). According to the manufacturer's instructions, cytokines were determined in the culture medium using the assay DuoSet ELISA™ Development kit (R&D Systems Europe, Ltd., Abingdon, UK) (Table 4). The Capture Antibody was coated to a 96-well MaxiSorp™-ELISA Microplate (Nunc, San Diego, USA) and incubated overnight at room temperature. After the blocking, 100 µl samples or standards were added to the well. After the incubation with the detection antibody and streptavidin-horseradish peroxidase (HRP), the substrate solution [SigmaFast™ OPD (o-Phenylendiamin-dihydrochlorid), Sigma-Aldrich Chemie GmbH] was added to each well and incubated for 30 min in the dark. The reaction was stopped with 50 μl 3 M HCl and the optical density (OD) was measured at 490 nm and reference at 655 nm (OD 490/655) using a Sunrise microplate ELISA reader (Tecan Deutschland GmbH). The concentration of cytokines released into the medium was calculated by interpolation from the respective standard curves and normalized against the protein concentration.

LDs are ubiquitous, dynamic organelles and serve as storage depots for neutral lipids, including triglycerides and cholesterol esters (43). The fluorescent neutral lipid dye 4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY), which displays excitation (Ex)/emission (Em) maxima of 493/503 nm (Ex/Em = 493/503 nm) allows quantifying of fluorescence area containing neutral lipid. 5% PFA-fixed MΦ were stained with 4 µM BODIPY, (Thermo Fisher Scientific) for 15 min and visualized using a Nikon Eclipse Ti laser scanning microscope (Nikon GmbH, Düsseldorf, Germany). Cells were analyzed and fluorescence area was determined using ImageJ software (NIH). The mean BODIPY™ fluorescence area was normalized to nuclear DAPI counterstain for each sample.

THP-1 MΦ (3*10⁵ cells/ml) were differentiated as described above, treated with oxLDL-DyLight™ 488 (Cayman Chemical, Ann Arbor, MI, USA) 1:50 and incubated at 37°C for 24 h. After that, the culture medium was removed and Hoechst 33342 staining was performed (Cayman Chemical, Ann Arbor, MI, USA; 1:2,000) to determine the total nuclei fluorescence. Finally, we visualized cells on Laser-Scanning-Microscope Nikon Eclipse Ti (Nikon GmbH, Düsseldorf, Germany). According to the manufactureŕs instruction, the fluorometric measurement of treated MΦ was done at Ex/Em = 493/518 nm for and Ex/Em = 350/461 nm for Hoechst dye using the Cytation3 microplate reader (BioTek Instruments Inc., Winooski, VT, USA). The oxLDL-uptake was normalized against the total nuclei fluorescence stained with Hoechst 33342 (Cayman Chemical).

Total cholesterol was determined using the Cholesterol/Cholesteryl Ester Quantitation Assay (Abcam plc., Cambridge, UK) (Table 4). THP-1 M1- /M2-MΦ (3*10⁵ cells/ml) treated in 6-well plates, as described above, were washed with ice-cold PBS and scraped in 150 μl PBS on ice. The suspension was transferred into a tube and centrifuged (10 min/250 × g/4°C). Thereafter, the supernatant was removed and the pellet was resuspended with a solution consisting of chloroform, isopropanol and NP40 (7:11:0.1). An additional centrifugation step was performed (10 min/15,000 × g/4°C). Subsequently, all phases of the supernatant were transferred to a fresh tube and air-dried overnight under the hood to remove the chloroform. The sample was then dissolved in assay buffer. 25 μl of the sample or a standard serial dilution were pipetted into a 96-well plate, refilled to 50 μl with the total cholesterol reaction mix and incubated for 1 h at RT in the dark. The total cholesterol concentration was measured with a microplate reader (Tecan) at OD595/655 nm and determined using a standard curve.

Triglycerides were determined using Triglyceride Quantification Colorimetric Assay Kit (Abcam plc.) (Table 4) according to manufacturer's instructions. The M1-/M2-MΦ (3*10⁵ cells/ml) treated in 6-well plates, as described above, were homogenized in 5% NP-40/ddH₂O and heated at 92°C for 5 min. After that, the samples were centrifuged by 13,000 × g and the supernatant was diluted 1:10 with aqua dest. Additionally, lipase was added to the samples and incubated for 20 min RT. The master mix of Triglyceride Assay Buffer, Triglyceride Probe and Triglyceride Enzyme Mix was prepared according to the manufacturer's instructions, added to the samples and incubated in the dark for 60 min. The fluorometric measurements were done at Ex/Em = 535/585 nm using the Cytation3 microplate reader (BioTek Instruments Inc.). Triglyceride concentration was determined by using a standard curve.

THP-1 M1-/M2-MΦ (3*10⁵ cells/ml), treated in 6-well plates as described above, were washed in ice-cold PBS and lysed with radioimmunoprecipitation buffer (RIPA) pH 7.5 (Cell Signaling Technology, Frankfurt, Germany) containing protease/phosphatase inhibitor cocktail (Cell Signaling Technology). Protein concentrations were determined spectrophotometrically using the Pierce™ BCA Protein Assay (Thermo Scientific). 30 µg of proteins were loaded onto a NuPAGE® Novex® 4%–12% Bis-Tris gel (Life Technologies GmbH, Darmstadt, Germany). By wet blot, proteins were transferred to a 0.45 μm nitrocellulose membrane (Millipore, Billerica, MA, USA). Primary antibodies (Table 2) were incubated overnight at 4°C in blocking buffer (5% nonfat milk in Tris-buffered saline with Tween20). Incubation of the 2nd antibody [donkey anti-rabbit IgG, HRP-linked F(ab’)2-fragment; Table 2] was performed at room temperature for 1 h at room temperature. The peroxidase reaction was visualized using AceGlow chemiluminescent substrate (PEQLAB GmbH, Erlangen, Germany) and documented using the Fusion-SL Advance™ Imaging System (PEQLAB GmbH) according to the instructions in the manual. The intensity of specific Western blot bands was quantified using ImageJ software from the National Institutes of Health (Bethesda, USA). Normalization was performed against α-tubulin.

Statistical analyses were performed using SigmaPlot 12 (Systat Software Inc., USA). After testing for normality (by Shapiro-Wilk), the unpaired Student's t-test or one-way analysis of variance (ANOVA) was performed. Data are reported as mean + standard deviation (SD), p ≤ 0.05 was considered statistically significant.

Given the increased lumen stenosis in PACAP^−/−/ApoE^−/− mice after 30 weeks of standard chow (SC), which we have demonstrated in this study (3.8-fold; p = 0.024) (Figures 1A,C–F) as well as in a recent study (29), we also investigated the effects on media thickness and body weight of PACAP deficiency in ApoE^−/− mice. After 10 weeks of SC following 20 weeks of CED, lumen stenosis increased 3.8-fold in ApoE^−/− mice only (p = 0.021), whereas no change in lumen stenosis was observed in PACAP^−/−/ApoE^−/− mice after this observation period (Figures 1A,E,F). Media thickness in ApoE^−/− and PACAP^−/−/ApoE^−/− at 30 weeks of SC or 20 weeks of CED remained unaffected (Figure 1B). Body weight in PACAP^−/−/ApoE^−/− mice compared to ApoE^−/− mice was decreased by 14% (p = 0.056) after 30 weeks of SC (Figure 1G). After 20 weeks of CED, body weight increased by 25% (p = 0.058) in PACAP^−/−/ApoE^−/− mice (Figure 1G).

We immunohistochemically determined the expression of VPAC1 in atherosclerotic plaques of the aortic arch of PACAP^−/−/ApoE^−/− and ApoE^−/− mice after 30 weeks of SC and 20 weeks of CED (Figures 2A,E–H), because it has been shown that the VPAC1 agonist (Ala11,22,28)-VIP aggravated early atherosclerosis in hypercholesterolemic ApoE^−/− mice (44). In PACAP^−/−/ApoE^−/− mice after 20 weeks of CED, VPAC1-immunoreactive plaque area was increased by 5.78% (p = 0.033) compared with ApoE^−/− mice (Figure 2A). VPAC1-positive cells were found to be located in the fibrotic cap and shoulder regions, as well as in the media (Figures 2E–H).

We used different MФ markers (CD86, CD68, CD163) to measure the respective immunoreactive atherosclerotic plaque areas in the aortic arch of ApoE^−/− and PACAP^−/−/ApoE^−/− mice (Figures 2B–D,I–T). After 20 weeks of CED, PACAP^−/−/ApoE^−/− mice revealed an 8.45% (p < 0.001) higher CD86-immunoreactive area than ApoE^−/− mice (Figure 2B). Moreover, after 30 weeks SC, PACAP^−/−/ApoE^−/− mice showed a 3.82% (p = 0.053) increased CD68-immunoreactive area compared to ApoE^−/− (Figure 2C). Additionally, ApoE^−/− mice fed for 20 weeks with CED revealed 6.8% elevated CD68-immunoreactive area compared to ApoE^−/− after SC for 30 weeks (Figure 2C). After 20 weeks CED, PACAP^−/−/ApoE^−/− mice showed a significant (p = 0.017) 7.44% higher CD163-immunoreactive area than ApoE^−/− mice (Figure 2D). ApoE^−/− mice fed for 20 weeks with CED had a 3.76% (p = 0.07) lower CD163-immunoreactive area than ApoE^−/− mice after SC (Figure 2D). However, CD86-positive cells were ubiquitously found in atherosclerotic plaques predominantly in the fibrotic cap (Figures 2I–L). CD68-immunoreactive areas were localized around the necrotic core and shoulder regions (Figures 2M–P), whereas the CD163-positive cells were predominantly found in the media, and fibrotic cap regions of the atherosclerotic plaque (Figures 2Q–T).

Based on our findings that VPAC1 immunoreactivity was found in regions where CD163-positive cells were localized, we further aimed to investigate in vitro different MΦ subtypes and the role of VPAC1 and PACAP concerning inflammatory processes and lipid homeostasis.

To determine whether VPAC1 receptors are expressed in different MΦ subtypes, BMDM were isolated from the femur of ApoE^−/− and PACAP^−/−/ApoE^−/− mice and differentiated into BMDM1- and BMDM2-MΦ (Figure 3A). The mRNA and protein expressions of VPAC1 were measured after incubation with/without oxLDL. VPAC1 mRNA expression was increased 5-fold (p = 0.014) in ApoE^−/− BMDM2-MΦ and 6-fold (p = 0.044) in PACAP^−/−/ApoE^−/− BMDM2-MΦ compared to ApoE^−/− and PACAP^−/−/ApoE^−/− BMDM1-MΦ, respectively (Figure 3B). Interestingly, PACAP^−/−/ApoE^−/− BMDM1- and BMDM2-MΦ showed a 4-fold (p = 0.045) and 2-fold (p = 0.045) higher VPAC-1 protein level compared to ApoE^−/− BMDM (Figures 3C,D). In either BMDM-subtypes of ApoE^−/− or PACAP^−/−/ApoE^−/− mice incubation with oxLDL for 24 h did not affect VPAC1 mRNA expression or VPAC1 protein level (Figures 3B–D).

In the present study, we analyzed the anti-inflammatory properties of PACAP in classically activated (inflammatory) M1-MΦ and anti-inflammatory M2-MΦ in vitro.

IL-10 mRNA expression was significantly increased in ApoE^−/− BMDM2-MΦ and PACAP^−/−/ApoE^−/− BMDM2-MΦ compared with the corresponding BMDM1-MΦ (ApoE^−/− BMDM2-MΦ with medium: 330%; with oxLDL: 285%; PACAP^−/−/ApoE^−/− BMDM2-MΦ with medium: 205%; with oxLDL: 178%; p ≤ 0.05) (Figure 4A). PACAP deficiency or treatment with oxLDL did not affect IL-10 mRNA expression in either BMDM-subtype (Figure 4A). Additionally, the IL-10 release is independent of PACAP, oxLDL and BMDM-subtype (Figure 4B).

In BMDM2-MΦ, TNF-α mRNA expression was significantly lower than in BMDM1-MΦ (ApoE^−/− BMDM2-MΦ with medium: 41%; with oxLDL: 44%; PACAP^−/−/ApoE^−/− BMDM2-MΦ with medium: 312%; with oxLDL: 285%; p ≤ 0.008) (Figure 4C). Moreover, in BMDM1-MΦ of PACAP^−/−/ApoE^−/− mice TNF-α mRNA expression was increased by 269% (p = 0.005) without oxLDL addition and by 256% (p = 0.014) with oxLDL, respectively (Figure 4C). In general, the TNF-α release was significantly lower in BMDM2-MΦ than in BMDM1-MΦ (ApoE^−/− BMDM 2-MΦ with medium: 84%; with oxLDL: 84%; PACAP^−/−/ApoE^−/− BMDM2-MΦ with medium: 100%; with oxLDL: 98%; p ≤ 0.009) (Figure 4D). PACAP^−/−/ApoE^−/− BMDM2-MΦ showed significantly decreased TNF-α release compared to ApoE^−/− BMDM2-MΦ (PACAP^−/−/ApoE^−/− BMDM2-MΦ with medium: 100%; with oxLDL: 93%; p ≤ 0.002) independent of oxLDL-treatment (Figure 4D).

IL-6 mRNA expression was significantly reduced in BMDM2-MΦ compared with BMDM1-MΦ (ApoE^−/− BMDM2-MΦ with medium: 98%; with oxLDL: 123%; PACAP^−/−/ApoE^−/− BMDM2-MΦ with medium: 135%; with oxLDL: 140%) (Figure 4E). PACAP deficiency or incubation with oxLDL had no effect on IL-6 mRNA expression in either BMDM-subtype (Figure 4E). IL-6 release was significantly decreased in BMDM2-MΦ compared with BMDM1-MΦ (ApoE^−/− BMDM2-MΦ with medium: 99%; with oxLDL: 96%; PACAP^−/−/ApoE^−/− BMDM2-MΦ with medium: 100%; with oxLDL: 94%; p ≤ 0.02) independent from PACAP or oxLDL-treatment (Figure 4F).

MΦ actively participate in lipoprotein-uptake via scavenger receptors and thereby develop into foam cells. Due to its nonpolar structure, long-wavelength absorption, and fluorescence, BODIPY™ (Ex/Em = 493/503 nm) was used as a dye to detect intracellular triglycerides (TAGs). Thereby, 24 h oxLDL incubation significantly increased BODIPY™ fluorescence area [normalized to DAPI fluorescence (Ex/Em = 359/457 nm)] by 6-fold (p ≤ 0.001) in ApoE^−/− BMDM1- and by 88-fold (p ≤ 0.001) in ApoE^−/− BMDM2-MΦ compared to BMDM incubated with medium alone (∼ medium control) (Figures 5A,B). Likewise, oxLDL treatment of PACAP^−/−/ApoE^−/− BMDM1- and BMDM2-MΦ showed 3-fold (p ≤ 0.05) increased BODIPY™ fluorescence area compared to corresponding BMDM incubated in medium alone (∼control) (Figures 5A,B). However, interestingly, PACAP^−/−/ApoE^−/− BMDM revealed a significantly lower oxLDL-induced BODIPY™ fluorescence area by 2-fold (p = 0.026) in BMDM1- and by 3-fold (p = 0.021) in BMDM2-MΦ compared to the corresponding ApoE^−/− BMDM (Figures 5A,B). Additionally, investigations of the cell morphology by fluorescence images revealed that PACAP^−/−/ApoE^−/− BMDM1-MΦ incubated with oxLDL exhibited a foam cell-like morphology compared with the oxLDL-treated ApoE^−/− BMDM1-MΦ (Figure 5B): (1) the cells were larger and (2) the lipid droplets (LDs) were more concentrated at the cell edge (Figure 5B). The LDs in oxLDL-treated ApoE^−/− BMDM1-MΦ were predominantly localized in the periphery of the nucleus (Figure 5B).

The scavenger receptors CD36 and oxLDL receptor-1 (LOX-1) mRNA expression were determined in BMDM1-/M2-MΦ (Figures 5C,D). CD36 mRNA expression was increased by 84% (p ≤ 0.001) in ApoE^−/− BMDM2-MΦ and by 63% (p = 0.044) in PACAP^−/−/ApoE^−/− BMDM2-MФ after oxLDL-treatment (Figure 5C). LOX-1 mRNA expression was reduced in ApoE^−/− BMDM2-MΦ (with medium: 95%; with oxLDL: 84%; p ≤ 0.032) and PACAP^−/−/ApoE^−/− BMDM2-MΦ (with medium: 52%; with oxLDL: 111%; p ≤ 0. 032) compared to the corresponding BMDM1-MΦ (Figure 5D).

PACAP^−/−/ApoE^−/− BMDM1-MΦ showed higher ADFP protein levels compared to ApoE^−/− BMDM1-MΦ (with medium: 3-fold; with oxLDL: 3-fold; p = 0.002) (Figures 5E,F). Additionally, PACAP^−/−/ApoE^−/− BMDM1-MΦ showed a 7-fold (p = 0.017) increase in ADFP levels compared to PACAP^−/−/ApoE^−/− BMDM2-MΦ (Figures 5E,G). Treatment with oxLDL resulted in a 4-fold (p = 0.002) increase in the amount of ADFP in PACAP^−/−/ApoE^−/− BMDM1- and a 10-fold increase in the amount of ADFP in PACAP^−/−/ApoE^−/− BMDM2-MΦ (p = 0.004) compared to the medium control (Figures 5E,F).

Concerning the data from BMDM, we analyzed the percentage of oxLDL-uptake per cell in human M1- and M2-MΦ. For this purpose, we used the PMA-differentiated human THP-1, a cell line routinely used in atherosclerosis research. The uptake of oxLDL could be detected in all THP-1 M1-and M2-MΦ based on the fluorescence intensity of oxLDL-DyLight™488(Ex/Em = 493/518) compared with MΦ incubated in medium alone (∼control) (M1-MΦ: with oxLDL: 4-fold; with PACAP38/oxLDL: 4-fold; with [Ala11,22,28]VIP/oxLDL: 6-fold; with PG 97–269/PACAP38/oxLDL: 6-fold; M2-MΦ: with oxLDL: 3-fold; with PACAP38/oxLDL: 3-fold; with [Ala11,22,28]VIP/oxLDL: 3-fold; with PG 97–269/PACAP38/oxLDL: 5-fold; p ≤ 0.001) (Figures 6A–D). To investigate the function of VPAC1, THP-1 M1- and M2-MΦ were additionally treated with the VPAC1-antagonist PG 97–269 or the VPAC1 agonist [Ala11,22,28]VIP. Our data show that incubation with the VPAC1-antagonist PG 97–269 in combination with PACAP38 significantly increased the uptake of oxLDL-DyLight™488 by 2-fold (p ≤ 0.004) in THP-1 M1- and M2-MΦ compared to PACAP38/oxLDL-DyLight™488 (Figures 6A–D).

Analyses of total cholesterol and triglyceride levels in human THP-1 MΦ should provide information, whether PACAP38 or VPAC1 plays a role in lipid homeostasis in THP-1 M1-/M2-MΦ. However, neither oxLDL with or without PACAP38 nor [Ala11,22,28]VIP altered intracellular total cholesterol concentrations in either MΦ subtype (Figures 6E,F). Analyses of intracellular triglyceride content showed that incubation of both THP-1 MΦ subtypes with oxLDL resulted in an increase in intracellular triglyceride (M1-MΦ: 39%; M2-MΦ: 42%; p ≤ 0.03) compared to cells incubated in medium alone (∼control) (Figures 6G,H). Moreover, co-incubation of THP-1 MΦ with oxLDL as well as with the VPAC1 antagonist PG 97–269/PACAP38 significantly increased triglyceride concentration in both MΦ subtypes (M1-MΦ: 29%; M2-MΦ: 37%; p ≤ 0.034) compared to THP-1 MΦ incubated with PG 97–269/PACAP38 without oxLDL (Figures 6G,H). On the other hand, co-incubation of THP-1 MΦ with oxLDL as well as with PACAP38 or with [Ala11,22,28]VIP in both MΦ subtypes did not result in an increase in triglyceride concentration compared with PACAP38 or [Ala11,22,28]VIP alone (Figures 6G,H). In THP-1 M2-MΦ, even co-incubation of oxLDL with the VPAC1 agonist [Ala11,22,28]VIP significantly (p = 0.026) lowered intracellular triglyceride concentration by 20% compared to oxLDL alone (Figure 6H).