Clinical and Experimental Pharmacology and Physiology (2015) 42, 648–652

doi: 10.1111/1440-1681.12398

ORIGINAL ARTICLE

Role of Peptide YY in blood vessel function and atherosclerosis in a rabbit model Renee M Smith,* Rudi Klein,* Peter Kruzliak† and Anthony Zulli* *Centre for Chronic Disease Prevention & Management (CCDPM), College of Health & Biomedicine, Victoria University, Melbourne, Vic., Australia and †International Clinical Research Center, St. Anne0 s University Hospital and Masaryk University, Brno, Czech Republic

SUMMARY Cardiovascular disease remains a burden for Westernized countries. Peptide YY (PYY) raises blood pressure, yet its role has not yet been determined in diseased arteries. This study aimed at identifying PYY and eNOS in diseased blood vessels and to determine which blood vessels respond to PYY. New Zealand White rabbits were fed an atherogenic diet (n = 6, 0.5% cholesterol + 1% methionine + 5% peanut oil) and control animals fed a normal diet (n = 6) for 4 weeks. Immunohistochemistry was used to determine the localization of PYY and eNOS in the aorta. The aorta, carotid, renal, iliac, inferior mesenteric, and renal interlobular arteries were removed, mounted in organ baths, and subjected to doses of PYY (109–107 mol/L) and then acetylcholine (106 mol/L). Immunohistochemistry of the aorta shows PYY staining in plaque macrophages, smooth muscle cells and endothelium, and these cells co-expressed eNOS. PYY caused a minor vasoconstrictive response in all blood vessels studied but was blunted in arteries from control animals. Acetylcholine caused relaxation of PYY constricted blood vessels. This data clearly shows that PYY is present in atherosclerotic plaque and is a minor constrictor of the vasculature tree. Further studies aimed at understanding the role of PYY in cardiovascular disease are warranted. Key words: atherosclerosis, cholesterol, eNOS, peptide YY.

INTRODUCTION Pancreatic polypeptide YY (PYY), a small peptide consisting of 36 amino acids, was originally isolated from porcine intestine1 and is secreted from the neuroendocrine cells (L cells) in

Correspondence: Dr Peter Kruzliak, International Clinical Research Center, St. Anne0 s University Hospital and Masaryk University, Pekarska 53, 656 91 Brno, Czech Republic. Email: [email protected] Dr Anthony Zulli, Centre for Chronic Disease Prevention & Management (CCDPM), College of Health & Biomedicine, Victoria University, Melbourne, Victoria, Australia. Email: [email protected] Received 5 February 2015; revision 13 March 2015; accepted 28 March 2015. © 2015 Wiley Publishing Asia Pty Ltd

the mucosa of the gastrointestinal tract, but it has been localized to other locations associated with the digestive system such as the islets of Langerhans in the pancreas, the oral cavity, as well as certain regions of the brain, including neurons located in the brain stem, hindbrain, and hypoglossal nucleus.2–6 Two different forms of this peptide have been identified and characterised, PYY1–36 and PYY3–36, which are produced by the enzymatic cleavage of PYY1–36 by dipeptidyl peptidase-IV.7 PYY1–36 binds to and activates all members of the human NPY family of receptors (Y1-Y5)8 with equal affinity, all of which are widely distributed throughout the central nervous system (CNS) and peripheral tissues.9 However, this is not the case for the main circulating form of PYY,10 PYY3–36, which is selective for and exhibits highest affinity binding only to the G-protein coupled Y2 receptor (Y2R).10–12 Maximum PYY levels can be observed 1–2 h after a meal, and remain elevated for up to 6 h postprandially in humans.13 While the initial rise in plasma PYY is due to an endocrine or neural pathway, postprandial PYY levels have been shown to be proportional to meal size14 and PYY release becomes greater as nutrients arrive at the neurendocrine L cells in the intestines giving rise to a more sustained release of PYY for up to 6 h postprandially. The strongest stimuli for the release of plasma PYY is through the macromolecules protein and fats.15 Interestingly, both forms of PYY can induce vasoconstriction via Y1 agonsim.16,17 Thus, PYY is clearly more than just an appetite regulator.18 As PYY causes systemic vasoconstriction, it is unclear which particular blood vessels constrict to PYY. In addition, it is unknown if the neurotransmitter acetylcholine, which induces blood vessel dilatation, can cause relaxation of PYY preconstricted arteries, suggestive of a counterbalancing physiological response. Moreover, it is unknown if PYY can be localized to atherosclerotic plaques, which would suggest a role for PYY in atherogenesis. To this end, this study was designed in rabbits to immunolocalize PYY to atherosclerotic blood vessels and to determine which blood vessels respond to the vasoconstrictive effects of PYY, and if the preconstricted blood vessels can dilate to acetylcholine. This data would provide evidence to suggest that this polypeptide has a potential functional role in the development of atherosclerosis.

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RESULTS Pancreatic polypeptide YY immunoreactivity was abundantly present in atherosclerotic aorta as depicted by positive brown immunoreactivity (Figs 1–3). PYY was identified in the endothelial layer, within atherosclerotic plaques, and adventitia. As well, cells within plaques showed positive immunoreactivity for PYY/ RAM-11 macrophage/HHF-35 smooth muscle cells as shown in Fig. 1, and this is exemplified in Fig. 2 (dashed circles). Negative control sections showed no staining (Neg ()). Also, as presented in Fig. 3, positive PYY immunoreactivity was observed in the same cells that also show positive eNOS immunoreactivity (the enzyme that produces nitric oxide in endothelial cells) within atherosclerotic plaques (dashed black and pink circles). Pharmacological data shows that PYY caused vasoconstriction in all blood vessels tested, including abdominal aorta, iliac, mesenteric, renal interlobar, carotid and renal arteries. Interestingly, in control arteries, only the highest dose of PYY showed constriction (range from 0 to 68 mg), but in blood vessels from the atherogenic dietary group, constriction was observed at lower doses (range from 6 to 605 mg, Table 1, P < 0.05. The addition of a bolus dose of acetylcholine (1 lmol/L) was able to cause relaxation of all blood vessels which constricted to PYY (Fig. 4).

Fig. 2 Magnified section of plaque showing cap formation. Peptide YY (PYY), HHF-35 and RAM-11are clearly visible in the same cells (dashed circles) as identified by brown precipitation.

DISCUSSION The main findings of this study are: (i) PYY is localised to blood vessels and atherosclerotic plaques; (ii) PYY causes vasoconstriction of the vascular tree; and (iii) acetylcholine can cause relaxation of preconstricted blood vessels. Being a gut hormone, studies on PYY have focussed mainly on obesity and satiety,19 as well as mood manipulation.20 Interestingly, few studies have also linked PYY to the prevention of pancreatic cancer,21 and early studies have shown that PYY

Fig. 3 Magnified section of plaque showing early stage atherosclerosis including foam cells. Peptide YY (PYY) and eNOS are clearly visible in the same cells (dashed black and pink circles) as identified by brown precipitation.

Fig. 1 Immunohistochemical localisation of peptide YY (PYY), HHF-35 (alpha smooth muscle cell actin) and RAM-11 (macrophage) in rabbit atherosclerotic aorta as identified by brown precipitation (Diaminobenzidine). Negative control shows no staining.

receptors are located mainly in pancreatic smooth muscle cells.22 In the study presented here, we show clearly that PYY immunoreactivity is present throughout the blood vessel wall, including cells within atherosclerotic plaques and strong immunoreactivity in the adventitia. The cells within atherosclerotic plaques have been identified as smooth muscle cells (using HHF35 marker) and macrophages (using RAM11), indicating a role for PYY in the regulation of smooth muscle cell and macrophage in atherogenesis. Indeed, early in vitro studies using PYY showed that PYY activated the cell signalling cascade which promoted smooth muscle proliferation,23 and recently this was evidenced in preglomerular vascular smooth muscle cells.24 Additionally, early in vitro studies using PYY revealed anti-inflammatory properties, whereby a reduction in macrophage adhesion to inflammatory site was observed,25 yet another study showed that PYY stimulates macrophage function, such as adherence, chemotaxis, phagocytosis and production of superoxide anion.26 Taken together, these studies indicate that

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RM Smith et al. 6  4 mg 17  9 mg 92  31 mg* ND ND 8  6 mg 22  12 mg 66  19 mg 128  27 mg* ND ND ND

Fig. 4 Real time recordings of force generated by peptide YY (PYY) in arteries. Force data enlarged on Y axis for ease of reference.

ND ND 18  8 mg *p < 0.05. **p < 0.01. MC, Methionine + Cholesterol; ND, not detected.

ND 75  8 mg 115  15 mg* 0.001 0.01 0.1

ND ND 68  22 mg

ND ND 8  3 mg

229  25 mg 348  45 mg 605  115 mg**

ND ND 98  12 mg*

ND ND 37  16 mg

ND ND 62  6 mg*

MC Control MC Control MC Control MC Control MC Control MC Control

Abdominal aorta PYY dose (lmol/L)

Table 1 Small changes in tension observed in vascular tree

Iliac artery

Mesenteric artery

Renal interlobular artery

Carotid artery

Renal artery

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PYY would regulate atherogenesis by stimulating smooth muscle cell proliferation and regulating macrophage function. Further studies investigating the role of PYY in atherogenesis are warranted. Nitric oxide (NO) is a potent anti-atherogenic factor involved in diverse physiological functions, including inhibition of smooth muscle cell proliferation, and macrophage activation.27 The enzyme that produces nitric oxide in endothelial cells is endothelial nitric oxide synthase (eNOS). We have shown immunohistochemical evidence that cells within atherosclerotic plaque that show positive PYY reactivity are also positive to eNOS. This indicates a possible counter-regulatory role for NO, whereby stimulation of eNOS could reduce the effects of PYY. Indeed, this is confirmed in our investigation, showing that eNOS stimulation via acetylcholine was able to dilate all arteries which constricted to PYY. Therefore, in disease were NO bioavailability is impaired, such as CVD, diabetes, obesity and hypertension,28 PYY could aggravate disease progression. The PYY administration in vivo was first shown to elevate blood pressure in 1982,29 yet there is no data pertaining to the role of PYY in healthy and diseased blood vessels. In this investigation, it is shown that PYY only at the highest dose used (0.1 lmol/L) caused a minor constriction of blood vessels, yet in arteries excised from the group fed high dietary cholesterol, methionine and fat, vasoconstriction initiated from the lowest dose used (1 nmol/L). This led to an increase in maximal constriction in this group. This effect could be due to reduced basal NO bioavailability, as this diet has been previously shown in this laboratory to induce severe reduction in NO bioavailability.30,31 In conclusion, we report that PYY immunoreactivity is present in atherosclerotic plaques, and that aorta, carotid, iliac, mesenteric, renal interlobar and renal arteries constrict to PYY, which can be dilated using acetylcholine. This study confirms a role for PYY in atherogenesis and vasoconstriction, and studies aimed at further deciphering this role are now warranted.

MATERIALS AND METHODS Male New Zealand White rabbits at 3 months of age were either fed a control diet (n = 6) or an atherogenic diet (n = 6, MC

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Role of Peptide YY group) consisting of normal rabbit chow diet supplemented with 0.5% cholesterol plus 1% methionine plus 5% peanut oil for 4 weeks.30,32,33 The animals were housed in individual cages and maintained at a constant temperature of approximately 21°C. Food and water were supplied ad libitum. The experiments were carried out according to the National Health and Medical Research Council Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (6th edn, 1997). The animals were killed with a bolus dose of xylazine (6 mg/kg) and ketamine (20 mg/kg) as previously described in our laboratory.32 The abdominal aorta, the iliac, renal, carotid, mesenteric and renal interlobar arteries were excised, cleaned of connective tissue and fat, cut into 3 mm rings, and mounted in organ baths. Sections of abdominal aorta were also fixed in 4% paraformaldehyde in 1 x PBS overnight, then processed for paraffin, mounted on a single paraffin block to keep uniform cutting thickness and immunohistochemistry procedures consistent. Immunohistochemistry Tissue sections were randomly selected, de-waxed, rehydrated and placed in 10 mm TrisCl (pH 7.4). Tissue sections were preincubated with 1% goat serum in 10 mm TrisCl (pH 7.4) for 20 min before incubating with the primary antibody diluted in 1% goat serum in 10 mm TrisCl (pH 7.4). Mouse monoclonal IgG against PYY (Catalogue # ABS 029-01-02, diluted 1 : 150, Thermo Scientific, Scoresby, Australia), mouse monoclonal IgG against eNOS (Catalogue # 610297, diluted 1 : 150, Transduction Laboratories, Becton Dickinson, North Ryde, NSW, Australia), mouse monoclonal IgG against RAM11 and HHF35 (Cat#M0633 and M0635 respectively, Dakocytomation, North Sydney, Australia) were incubated overnight. As a negative control, a monoclonal antibody to Aspergillus niger glucose oxidase (Dakocytomation) was diluted 1 : 20 and also incubated overnight. Immunohistochemistry was performed using the ‘Envision’ commercially available kit,32,34–36 following the manufacturers’ directions (Catalogue # K4001, DAKO, Carpentaria, CA, USA) Antigenic sites were developed with diaminobenzidine, counterstained with Haematoxylin, dehydrated and mounted with DPX mounting media (BDH, Poole, UK). Isometric tension studies Blood vessels were mounted between two metal hooks in organ baths attached to force displacement transducers37(OB8, Zultek Engineering, Melbourne, Australia). The baths were filled with Krebs’ solution, kept at a constant temperature of 37°C and continuously bubbled with 95% O2 ⁄5% CO2. Vessel rings were precontracted with 0.001 lmol/L, 0.01 lmol/L and then 0.1 lmol/L PYY. After the contraction reached plateau, a bolus dose of acetylcholine (1 lmol/L) was added to induce vasodilatation via nitric oxide release. Statistical analysis Data (Emax) was analysed using Students’ t test with GRAPHPAD La Jolla, CA, USA. Results are presented as mean  SEM and significance was taken at P < 0.05 in all cases.

PRISM

651 ACKNOWLEDGEMENTS

The authors thank Renee Smith for her technical expertise, Rudi Klein for original concept, Peter Kruzliak and Anthony Zulli for laboratory management and manuscript preparation.

DISCLOSURE The authors declare no conflict of interest.

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Role of Peptide YY in blood vessel function and atherosclerosis in a rabbit model.

Cardiovascular disease remains a burden for Westernized countries. Peptide YY (PYY) raises blood pressure, yet its role has not yet been determined in...
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