PACAP regulates VPAC1 expression, inflammatory processes and lipid homeostasis in M1- and M2-macrophages

Background Pituitary adenylate cyclase-activating polypeptide (PACAP) acts as an anti-atherogenic neuropeptide and plays an important role in cytoprotective, as well as inflammatory processes, and cardiovascular regulation. Therefore, the aim of this study is to investigate the regulatory effects of PACAP and its receptor VPAC1 in relation to inflammatory processes and lipid homeostasis in different macrophage (MΦ) subtypes. Methods To investigate the role of PACAP deficiency in the pathogenesis of atherosclerosis under standard chow (SC) or cholesterol-enriched diet (CED) in vivo, PACAP−/− mice were crossbred with ApoE−/− to generate PACAP−/−/ApoE−/− mice. Lumen stenosis in the aortic arch and different MΦ-subtypes were analyzed in atherosclerotic plaques by quantitative immunohistochemistry. Undifferentiated bone marrow-derived cells (BMDC) from 30-weeks-old ApoE−/− and PACAP−/−/ApoE−/− mice were isolated, differentiated into BMDM1- and BMDM2-MΦ, and incubated with oxidized low-density lipoprotein (oxLDL). In addition, PMA-differentiated human THP-1 MΦ were further differentiated into M1-/M2-MΦ and subsequently treated with PACAP38, the VPAC1 agonist [(Ala11,22,28)VIP], the antagonist (PG 97–269), and/or oxLDL. Uptake/accumulation of oxLDL was analyzed by oxLDL-DyLight™488 and Bodipy™ 493/503. The mRNA expression was analyzed by qRT-PCR, protein levels by Western blot, and cytokine release by ELISA. Results In vivo, after 30 weeks of SC, PACAP−/−/ApoE−/− mice showed increased lumen stenosis compared with ApoE−/− mice. In atherosclerotic plaques of PACAP−/−/ApoE−/− mice under CED, immunoreactive areas of VPAC1, CD86, and CD163 were increased compared with ApoE−/− mice. In vitro, VPAC1 protein levels were increased in PACAP−/−/ApoE−/− BMDM compared with ApoE−/− BMDM, resulting in increased TNF-α mRNA expression in BMDM1-MΦ and decreased TNF-α release in BMDM2-MΦ. Concerning lipid homeostasis, PACAP deficiency decreased the area of lipid droplets in BMDM1-/M2-MΦ with concomitant increasing adipose differentiation-related protein level. In THP-1 M1-/M2-MΦ, the VPAC1 antagonist increased the uptake of oxLDL, whereas the VPAC1 agonist decreased the oxLDL-induced intracellular triglyceride content. Conclusion Our data suggest that PACAP via VPAC1 signaling plays an important regulatory role in inflammatory processes in atherosclerotic plaques and in lipid homeostasis in different MΦ-subtypes, thereby affecting foam cell formation. Therefore, VPAC1 agonists or PACAP may represent a new class of anti-atherogenic therapeutics.


Introduction
Atherosclerosis is a chronic inflammatory disease (1) enhanced not only by lipid deposits in the arterial wall, but also by cellular waste products, calcium or fibrin.However, these factors play only a minor role in our study.At arterial branch points and bends, circulating lipoprotein particles can enter the arterial wall and accumulate in the intima, promoting the development of atherosclerotic lesions.
Low-density lipoprotein (LDL), especially in its modified forms, is the major contributor to lipid accumulation in atherosclerotic lesions (2).In this context, modifications of LDL, such as oxidation, trigger an immune response leading to the formation of circulating LDL-containing immune complexes that are exceedingly atherogenic (3,4).Moreover, the progression of atherosclerosis is associated with phenotypic diversity of MΦ.In general, MΦ are a fundamental immune system component and can be classified into two subtypes.The pro-inflammatory or immunosuppressive M1-MΦ are classically activated and produce inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α) (5)(6)(7)(8).M1-MΦ are a predominant phenotype in rupture-prone zones, plaque progression and advanced lesions, whereas they are abundant in unstable plaques (9,10).The antiinflammatory M2-MΦ are important for tissue and wound healing (6,8,11) and present in the early stages of atherosclerotic plaques (12-14).Thus, identifying the phenotype is also used as a marker of plaque stability (15).MΦ in atherosclerotic lesions are actively involved in the uptake and accumulation of lipoproteins, forming foam cells.The accumulation of foam cells contributes to further and increased lipid storage and, thus, to the progression of atherosclerotic plaque (16).Therefore, it is essential to understand and decipher the biomolecular and phenotypic diversity of MΦ to reveal their functions and roles in atherosclerotic plaques to develop specific therapies for atherosclerosis.In addition, the microenvironment of the atherosclerotic plaque (lipids, growth factors, cytokines, chemokines) regulates the MΦ subtypes in the plaque itself (31).By targeting the MΦ microenvironment of the atherosclerotic plaque, previous research shows potential strategies for selective treatment of atherosclerosis (3).
In this context, this study investigated the expression of PACAP and the PACAP receptor VPAC1 concerning lipid homeostasis and inflammatory processes in M1-/M2-MΦ.Our data show that PACAP regulates VPAC1 protein levels, which has implications for cellular inflammatory processes and affects lipid homeostasis in MΦ subtypes.Therefore, we consider it fundamental to further investigate the efficacy of VPAC1 or PACAP in regards to anti-atherogenic therapeutics.

Dissection and tissue harvesting
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.

Morphometry and immunohistology
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 (µm 2 )]/[lumen area (µm 2 )] × 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 (µm 2 )]-[lumen area to internal elastic lamina (µm 2 )] = area of media (µm 2 )].Quantification of immunoreactive plaque area was assessed as described previously (29,32).

MΦ from bone marrow-derived cells
At the age of 30 weeks, mice were weighed, and anesthetized by inhalation of isoflurane.The body size was measured by measuring the length from nose-tip to tail-base before euthanasia by neck dislocation.The procedures were in accordance with the animal

LDL-oxidation
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 Ca 2+ , Mg 2+ (LONZA, Ratingen, Germany) to a final concentration of 2 mg protein/ml, and dialyzed using Vivaspin TM 20-System (Thermo Fisher Scientific GmbH, Schwerte, Germany).The Vivaspin TM 20 centrifugal concentrator was sterilized with 70% EtOH for 10 min at 3,000 × g.Afterward, the Vivaspin TM 20 was washed with aqua dest (endotoxin-free).Then, nLDL suspended in PBS was transferred into the Vivaspin TM 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.CuSO 4 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 TM 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).

Real-time quantitative (qRT) -PCR
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 TM Multiple Temperature cDNA synthesis reverse transcriptase (Agilent Technologies) and 24 units of Ribo Lock TM 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 Frontiers in Cardiovascular Medicine using the Mx3005P TM 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).

Measurements of cytokine releases
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 TM Development kit (R&D Systems Europe, Ltd., Abingdon, UK) (Table 4).The Capture Antibody was coated to a 96-well MaxiSorp TM -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 TM OPD (o-Phenylendiamindihydrochlorid), 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.

Total cholesterol assay of THP-1 Mф
Total cholesterol was determined using the Cholesterol/ Cholesteryl Ester Quantitation Assay (Abcam plc., Cambridge, UK) (Table 4).THP-1 M1-/M2-MΦ (3*10 5 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.

SDS page and western blot
THP-1 M1-/M2-MΦ (3*10 5 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 TM 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')2fragment; 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 TM 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
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.

Expression of VPAC1 receptors and Mф markers in atherosclerotic plaque
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, VPAC1immunoreactive 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).
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.

Effect of PACAP-deficiency on inflammation in BMDM1-and BMDM2-MΦ
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.

Discussion
The main novel findings of our studies were that PACAP deficiency in ApoE −/− mice promoted the development of atherosclerotic plaques in the aortic arch under SC (30 weeks) and led to an increase of VPAC1-immunoreactive area after 20 weeks of CED.These data confirm, previously published findings that PACAP deficiency aggravates atherosclerosis in the brachiocephalic trunk in ApoE −/− mice after 30 weeks of SC (29).To VPAC1, it is known that VIP and PACAP can bind to the receptor with equal affinity (Kd = 1.0 nM) (45).VIP is known to regulate cholesterol efflux in MΦ and to reduce foam cell formation (46).Further studies showed that systemic VIP treatment reduced the number and size of atherosclerotic plaques in the carotid artery, aorta, and sinus in hypercholesterolemic mice (46).Therefore, the presence of VPAC1 in atherosclerotic plaques suggests the possibility that PACAP/VIP signaling plays a critical role in atherogenesis.
In addition to increased lumen stenosis, weight loss in PACAP −/− /ApoE −/− mice after 30 weeks of SC was also verified within this study.This is consistent with the previous research by Rasbach et al. (29).PACAP neurons of the paraventricular neuron are thought to promote appetite through synaptic connections of NPY/AgRP neurons (47).Loss of this signaling through PACAP deficiency may explain the reports of hypoinsulinemia, decreased adiposity, lower body weight, and increased insulin sensitivity in PACAP-null transgenic mice (48).
In view of inflammatory processes, VPAC1 mediates the immunomodulatory properties of PACAP and VIP (49).After demonstrating that PACAP deficiency in ApoE −/− mice leads to an increase in VPAC1-immunoreactive area in the plaque after 20 weeks of CED feeding, we examined the differences in MΦ subtypes in plaque between PACAP −/− /ApoE −/− and ApoE −/− mice.In this context, we were able to show, that after 20 weeks of CED PACAP −/− /ApoE −/− mice led to an increased CD86-and CD163-immunoreactive areas compared to ApoE −/− mice.CD86 located on the surface of antigen-presenting cells, including classical M1-and M2b-MΦ provides costimulatory signals necessary for T cell activation and survival (50).Activated T cells proliferate, release effector molecules (IFN-γ, IL-2), and enhance cytotoxic activity (50, 51).CD163 is a monocyte/macrophagespecific marker for M2-MΦ expressed primarily on cells with strong anti-inflammatory potential (52).Our data show that CD163-positive cells were most frequently localized on the cap of the plaque and in the media.Interestingly, VPAC1-positive cells were also predominantly located on the cap of atherosclerotic plaques and the subendothelial space.Additionally, after 30 weeks of SC, the CD68-immunoreactive area was more pronounced in PACAP −/− /ApoE −/− mice compared with ApoE −/ − mice and was predominantly located in the necrotic core and shoulder region.CD68 is generally considered to be a selective marker and a lysosomal protein that is highly expressed by human monocytes and tissue MΦ (50).However, several immunohistochemical studies showed that CD68 antibodies can also react with other hematopoietic and nonhematopoietic cell types (53).Accordingly, it is generally known that pro-inflammatory M1-MΦ are pro-atherogenic, whereas the antiinflammatory M2-MΦ subtype appears to be anti-atherogenic (15, 16).The recruited MΦ in the subendothelial space represent several different polarized phenotypes, which have multiple implications for lesion development and progression (54).M1-MΦ exist in symptomatic plaques and are predominantly localized in shoulder regions, whereas M2-MΦ are present in stable, asymptomatic plaques, predominantly localized in the adventitia and fibrotic cap of plaques (9,10).M1-MΦ characterize progressive lesions, whilst regressing plaques are enriched in M2-MΦ (55).However, M1-and M2-MΦ contribute to diverse stages of plaque development and are localized in distinct morphological areas within atherosclerotic lesions.Our data show that the proportion of anti-inflammatory markers, like CD163 is increased in PACAP-deficient ApoE −/− mice, suggesting a stabilization of the atherosclerotic plaque.Therefore, we further investigated M1-and M2-MΦ in the context of PACAP and the receptor VPAC1.
For this purpose, BMDM from ApoE −/− and PACAP −/ − /ApoE −/− mice were differentiated into M1-MФ using LPS/ IFNγ or into M2-MФ using IL-4/IL-13.Stein et al. (56) were the first to describe an alternative MΦ subtype induced by IL-4 and characterized by high mannose receptor expression (56).Moreover, the proinflammatory M1-MΦ phenotype is known to release proinflammatory cytokines (IL-1β, TNF-α), whereas the M2-MΦ phenotype releases factors such as transforming growth factor (TGF) β or IL-10, which limit the inflammatory process (54).In this study, we successfully differentiated ApoE −/− and PACAP −/− /ApoE −/− BMDM into BMDM1-MФ and BMDM2-MФ, with M1-MФ characterized by increased mRNA-expression and release of IL-6 and TNF-α and M2-MФ by increased IL-10 mRNA expression.Next, we analyzed the expression of VPAC1 in the different BMDM-subtypes.Independent of the mouse genotype (ApoE −/− , PACAP −/− /ApoE −/− ), higher VPAC1 mRNA expression was detected in BMDM2-MФ compared with BMDM1-MФ.Interestingly, analysis of VPAC1 protein level showed that PACAP −/− /ApoE −/− BMDM had increased VPAC1 protein level compared with ApoE −/− .VPAC1 is a member of the GPCR family, whose specific features include the presence of many introns in its gene organization (57,58).This suggests that alternative splicing events may occur within the VPAC1 receptor, resulting in different VPAC1 splice variants whose functional significance, among others, is not well understood (59).Additionally, Harikumar et al. have shown that VPAC1 can form homodimers (60).In BMDM cells expressing VPAC1, anti-VPAC1 recognized two broad bands with apparent molecular weights of 70 kDa, but not 140 kDa (Additional File Figure S2).Therefore, in this study, only the bands of 70 kDa were evaluated semiquantitative.Previous studies on CHO cell lines showed that bands with molecular weight between 45 and 100 kDa indicate non-glycosylated (ng)-VPAC1 receptor (61).It is also known that the VPAC1 receptor, with the help of its ligand VIP, can be internalized from the plasma membrane via endosomes and transported to the nuclear membrane or cytoplasm, where it is functional or proteasomal degraded (62-66).Thus, the regulation of the VPAC1 receptor is complex and needs to be further investigated concerning the influence of PACAP to understand the differences between mRNA expression and protein analysis.PACAP, along with VIP, is an important factor in the balance between proinflammatory and anti-inflammatory mediators by inhibiting LPS-induced production of IL-6, IL-12, TNF-α, and NO in vitro and in vivo and can protect mice from endotoxic shock (67,68).In our study, PACAP-deficiency in ApoE −/− mice resulted in an increased TNF-α mRNA expression in BMDM1-MΦ and a decreased TNF-α release in BMDM2-MΦ.However, it could not be shown, that PACAP deficiency in ApoE −/− BMDM affect the expression or release of IL-6 or the antiinflammatory cytokine IL-10.Likewise, this study did not show any effect of oxLDL on the expression of VPAC1 and mRNA expression or release of IL-10, TNF-α, or IL-6 in ApoE −/− or PACAP −/− /ApoE −/− BMDM1-and M2-MΦ.It is known, that PACAP controls inflammatory processes by inhibiting NF-κB transcriptional activity in mouse MΦ and LPS-induced THP-1 monocytes (67,(69)(70)(71).More precisely, the inhibition of the p65 nuclear translocation and subsequent DNA binding is mediated through the VPAC1 receptor and a non-cAMP transduction pathway (71).Previous publications demonstrate that PACAP inhibits IL-6 production in LPS-induced peritoneal MΦ of mice (72) but, on the other hand, enhances IL-6 release in resting peritoneal MΦ of mice (73).Martinez et al. (73) postulated that the dual effect of PACAP on IL-6 release would be important for immune homeostasis (72,73).Moreover, previous studies have described the inhibition of LPS-induced IL-6 release by the PACAP-specific receptor PAC1 and that IL-6 stimulation by PACAP is dependent on VPAC1 (72,73).
PACAP is thought to play a role in lipid metabolism, as previous studies have demonstrated an accumulation of lipids in heart tissue, skeletal muscle, and liver tissue (74).With regard to atherosclerosis, lipid uptake and intracellular accumulation are early events in the development of atherosclerosis, including atherogenic oxLDL causing lipid accumulation that contributes to the formation of foam cells in the intima (75).This modified LDL can be uninhibitedly taken up by MΦ based on scavenger receptors or pinocytosis (75, 76), by which oxLDL uptake was primarily mediated via CD36 (77).In our study, CD36 and LOX-1 mRNA expressions were detected in both BMDM subtypes.Moreover, this study showed that BMDM2-MФ had significantly increased CD36 mRNA expression after oxLDL incubation independent of the genotype of mice.The fact is that endocytosed oxLDL triggers important and complex transcriptional changes in MΦ, including upregulation of CD36 expression (78).Interestingly, in this regard, anti-inflammatory M2-MΦ are more prone to foam cell formation than pro-inflammatory M1-MΦ (79) possibly due to oxLDLinduced upregulation of scavenger receptor CD36 in M2-MΦ.LOX-1 was predominantly expressed in BMDM1-MФ independent of PACAP-deficiency or oxLDL.LOX-1 is a type II integral membrane glycoprotein oxLDL-binding receptor in endothelial cells, which, however, can be up-regulated in MΦ during atherosclerosis (80, 81).Moreover, the lipid accumulation and foam cell formation in the different MΦ-subtypes are diversely discussed, because oxLDL can also be internalized via alternative pathways like micropinocytosis or other scavenger receptors (75, 82).Additionally, Endemann et al. (83) showed that blocking the CD36 functional site with the mouse monoclonal antibody OKM5 in PMA-treated THP-1 resulted only in a 52% reduction of oxLDL-binding (83).In hyperlipidemic CD36 −/ − /ApoE −/− mice the aortic sinus lesions were characterized by electron microscopy and immunohistochemistry and showed an abundance of MΦ foam cells, indicating that lipid uptake by intimal MΦ may occur in the absence of CD36 or SR-A, too (76).In addition, it is entirely unclear what role the intracellular VPAC1 receptor plays in foam cell formation or lipid uptake and accumulation.It is also completely unclear how the relationship between oxLDL, PACAP and the VPAC1 receptor is.A previous study described PACAP as a critical regulator of lipid and/or carbohydrate metabolism (74).Using the detection method of BODIPY TM 493/503, a lower accumulation of neutral lipids was detected in PACAP deficient ApoE −/− BMDM.On the other hand, analyses of fluorescence images (BODIPY TM 493/503) showed that LDs were more concentrated at the cell edge and had lager shape in PACAP −/− /ApoE −/− BMDM1-MΦ than in ApoE −/− BMDM1-MΦ, where LDs were located in the periphery of the nucleus and smaller shape.Therefore, PACAP −/− /ApoE −/− BMDM1-MΦ showed a more foam cell-like morphology than ApoE −/− BMDM1-MΦ.Considering the data, this possibly indicates an impaired lipid buffer function via LDs and thus an increased risk of lipotoxicity in PACAP −/− /ApoE −/− BMDM1-MФ (43).Previous studies showed that PACAP38 reduced lipid accumulation in human THP-1 M0-MФ (29) and that VIP impairs the formation of foam cells by increasing cholesterol efflux in mice MΦ (46).The expression of adipose differentiation-related protein (ADFP) showed that PACAP deficiency in ApoE −/− BMDM increased the ADFP protein level.So far described that the downregulation of ADFP inhibits lipid droplet accumulation and thus reduces the probability of conversion of MФ into foam cells (84,85).With this in mind, we investigated the VIP/PACAP receptor VPAC1 with regard to intracellular cholesterol and triglyceride concentrations after oxLDL treatment of human THP-1 M1-and M2-MΦ.In our study, intracellular triglyceride concentrations were significantly increased in both THP-1 MΦ subtypes after incubation with oxLDL or oxLDL in combination with the VPAC1 antagonist PG 97-269.Conversely, the VPAC1 agonist [Ala11,22,28]VIP decreased the oxLDL-induced increase in triglyceride concentration in M2-MΦ.Rasbach et al. (29) showed increased plasma triglyceride levels after CED feeding to PACAP −/ − / ApoE −/− mice compared with ApoE −/− mice (29).From these data, we conclude that PACAP and especially VPAC1 play important roles in regulating plasmatic and intracellular lipid homeostasis and foam cell formation.Further studies are needed to analyze the interplay of the individual components better.

Conclusions
The present data provide the background for further research on VPAC1 and PACAP38 as anti-atherogenic therapeutics, because PACAP deficiency impairs luminal stenosis in the aortic arch of ApoE −/− mice as well as VPAC1 receptor expression, inflammatory processes, and lipid homeostasis in M1/M2-MФ.

TABLE 1
Oligonucleotides used for genotyping.

TABLE 3
List of used primer for qRT-PCR.All primers were purchase by Qiagen GmbH, Hilden, Germany.

TABLE 4
List of used kits.