Einfluss von PACAP bzw. des PACAP-Rezeptors PAC1 auf Morphologie und Expression Inflammations-/ Apoptose-/ Atheroskleroserelevanter Proteine/ Zytokine im Myokard bei ApoE defizienten Mäusen unter cholesterinreicher „Western Type Diet“

Atherosclerotic changes in our western society are a very common pathogenesis which can cause cardiovascular diseases like myocardial infarction or [chronic] heart failure. It seems that the PACAP-System [„Pituitary Adenylate Cyclase-Activating Polypeptide“] with its receptor PAC1 plays a very important role in the development of such pathologic processes. PACAP belongs to the “vasoactive intestinal Peptide [VIP]- sekretin- somatoliberin Glucagon superfamily and is the only ligand that is able to bind at the PAC1-receptor. The PAC1-receptor is expressed mainly in the myocardium and might be able to influence the occurrence of inflammatory and anti-inflammatory cytokines that could act cardioprotective. Furthermore, the PACAP-system seems like to play an important role in influencing the outcome and development of the ischemic heart disease. In this Study we examined the outcome of the deficiency of PACAP and PAC1 in the myocardium of mice based on inflammatory, atherosclerotic and apoptosis relevant cytokines and proteins. We also examined the rate of infiltration of macrophages in the myocardium. The mice we examined had the following genotypes: 1. PACAP+/+ / PAC1+/+ / ApoE+/+ [short: WT]; 2. PACAP+/+ / PAC1+/+ / ApoE-/- [short: ApoE-/-]; 3. PACAP-/- / PAC1+/+ / ApoE-/-; 4. PACAP+/+ / PAC1-/- / ApoE-/-. All mice have been fed with a “Western Type Diet” over the period of 20 weeks, starting with the age of 10 weeks. Blood samples were taken to measure the levels of plasma-cholesterol and plasma-triglycerides. The ratio of the heart weight and length of the tibia were calculated. We examined samples of the myocardium covering COX2+-, MuRF-1+-, Moma-2+-, LOX-1+-, IL1-ß+-, TNF-α+- and Lectin+-cells. Furthermore, the composition of every myocardium in regard of the amino acids were also studied. The plasma triglyceride levels of ApoE-/- -mice in comparison to WT-mice and of PAC1-/- / ApoE-/--mice in comparison to ApoE-/--mice didn’t differ significantly. However, PACAP-/- / ApoE-/--mice by comparison to ApoE-/--mice showed an 60,6% increase of this level. Plasma cholesterol only showed an increase [tenfold] with ApoE-/--mice in comparison to WT-mice, whereas the levels in PACAP-/- / ApoE-/--, and PAC1-/- / ApoE-/--mice in comparison to ApoE-/--mice were similar. The relative heart weight was in the PAC1-/- / ApoE-/--population 18,1% lower in comparison to the ApoE-/--population. ApoE-/--, in comparison to WT-mice and PACAP-/- / ApoE-/-- mice in comparison to ApoE-/--mice didn’t show a significant change in the relative heart weights. The COX2+-cells showed a higher occurrence of 22% in the ApoE-/--, versus WT-mice and of 55,7% in the PAC1-/- / ApoE-/--mice versus the ApoE-/--mice. MuRF-1+-cells had in ApoE-/--mice compared to the WT-mice a 116,7% higher density, furthermore PAC1-/- / ApoE-/--, and PAC1-/- / ApoE-/-- mice showed an 51,2% and 66,7% increase in comparison with ApoE-/--mice. In addition, the density of macrophages was measured with the Moma-2-antibody which showed a 25,9% decline in the PACAP-/- / ApoE-/--mice, and a 44,2% decrease in the PAC1-/- / ApoE-/--mice compared to the ApoE-/--mice. The capillary supply of the myocardium [density of Lectin+-cells] showed only at the PAC1-/- / ApoE-/--mice in comparison with the WT-mice a 24,5% lower density. Furthermore, LOX-1+-cells were counted [marker for the ongoing atherosclerotic occurrence] and showed only in PAC1-/- / ApoE-/--mice a 44,47% higher activity compared to ApoE-/--mice. Also, the concentration of the amino acids the myocardium contains showed in PACAP-/- / ApoE-/--, and PAC1-/- / ApoE-/--mice a 23,6% and 20,9% lower concentration in comparison to ApoE-/--mice. Furthermore, the aminoacids Phosphatidyl-serine which is part of the apoptose-signalpathway and the gamma-amino-butter-acid which has anti-inflammatory characteristics, showed a 22% and 36,7% lower concentration in PACAP-/- / ApoE-/-- mice and a 21,8% and 18,2% lower concentration in PAC1-/- / ApoE-/--mice, all compared to ApoE-/--mice. The findings of our study show by the higher density of COX2+-positive- cells an inflammatory environment in the myocardium under the deficiency of PACAP and PAC1. Only PAC1-/- / ApoE-/--mice showed in combination with an ongoing atherosclerotic process [higher density with LOX1+-cells] a higher density of MuRF-1+-cells which indicates atrophy of the myocardium. In addition, we found a positive correlation between the density of MuRF-1+-cells and COX2+-cells, which underlines a connection between the atrophy and inflammation. Interestingly, PACAP-/- / ApoE-/--mice didn’t show a significant change in heart weight, higher density in COX2+-cells and LOX1+-cells but a higher density in MuRF-1+-cells. Furthermore, the deficiency of PACAP and PAC1 didn’t change the expression of cytokines like TNF-α, IL1-ß and the infiltration of macrophages was also unchanged. The capillary supply measured with the density of Lectin+-cells also didn’t show any significant difference. The lower concentration of the amino acids seemed only to influence the heart weight of the PAC1-/- / ApoE-/--mice, while amino acids, like Phosphatidyl-serine and GABA, only influenced the environment in PAC1-/- / ApoE-/--mice as well. In comparison to another study of our researchgroup (Dissertation Gohlke 2017), mice with the same genotype were fed “Normal diet” over the time of 30 weeks before being examined. The findings showing lighter heart weights and lower triglyceride, and cholesterol levels in the plasma [effect of cholesterol-enriched-diet]. Furthermore, comparing it to the raw data of our study the density of COX1+-cells, MuRF-1+-, LOX-1+-, Moma-2+-, and Lectin+-cells was higher. The data of the amino acids also showed a lower concentration. In summary, we conclude that PAC1 is able to influence the environment in the myocardium, has anti-inflammatory and anti-atherogenic features and influences local atrophic processes. Comparing all the collected data, with all the collected references gives PAC1 the ability to effect inflammatory processes [COX2], the expression of pro-atherogenic mediators [LOX-1] positively and subdues cardiac atrophy [MuRF-1] under a “Western Type Diet”. PACAP doesn’t seem to have these abilities under the feeding with a “Western Type Diet”, so other mechanisms must play an additional role, like the VIP-System or the persisting hypertriglyceridemia over a longer period of time. For future studies, the period of feeding [longer vs. shorter period] and the impact of the deficiency of the VIP-system would represent a good possibility in regard of a possible effect on the inflammatory and pro-atherogenic environment in the myocardium.