Elsevier

Peptides

Volume 28, Issue 2, February 2007, Pages 269-280
Peptides

The Y2 receptor mediates increases in collateral-dependent blood flow in a model of peripheral arterial insufficiency

https://doi.org/10.1016/j.peptides.2006.09.026Get rights and content

Abstract

We have utilized a rat model of peripheral artery disease (PAD) to examine whether the known angiogenic activity of the Y2 receptor would translate into a meaningful increase in collateral blood flow. The maximal increase in collateral blood flow capacity of ∼60% (p < 0.001) was obtained with a 10 μg/kg day (IA infusion, 14 days) of either PYY or PYY3–36 and did not differ from that obtained with a maximally angiogenic dose of VEGF165. Pharmacodynamic modeling based upon single dose pharmacokinetic plasma profiles of both agonists suggests that Emax is reached when the Y2 receptor is occupied by ≥50%. Furthermore, for PYY3–36, occupancy of the Y2 receptor is sufficient to promote a significant benefit in collateral blood flow.

Introduction

Peripheral arterial disease (PAD) is a condition that affects approximately 27 million adults in Europe and North America and 20% of people over the age of 55 [12]. It is a chronic obstructive disease in which blood flow to the lower extremities is compromised, usually as a result of atherosclerosis. PAD is characterized by symptoms of intermittent claudication (leg pain) that substantially limit mobility and quality of life, and in a small minority of patients the disease progresses to the point of critical limb ischemia for which there is no alternative other than amputation. Current management of PAD includes smoking cessation, exercise, and treatment of the underlying hypertension and atherosclerosis [12]. There are currently only two approved drugs in the United States for the treatment of intermittent claudication, pentoxifylline and cilostazol, with only the latter producing a modest benefit [16]. Surgical bypass is an effective means of revascularization of the affected tissue, but not all patients are viable surgical candidates [14]. For this reason, therapeutic angiogenesis, or the restoration of collateral-dependent blood flow by agents that are capable of promoting the formation of mature collateral vessels either de novo or from existing conduits, has been offered as an alternative treatment for PAD [29].

Neuropeptide Y (NPY) is a 36 amino acid peptide that is released by sympathetic neurons surrounding the heart and blood vessels. NPY and the related Peptide YY (PYY) interact with 4 known receptors in primates, all of which have been cloned (Y1, Y2, Y4, and Y5), and the potent vasoconstrictor action of these peptides is mediated by the Y1 receptor [36]. The angiogenic activity of NPY has been demonstrated in a variety of models (reviewed in [36]), and the nonselective Y receptor agonist NPY has been shown to increase collateral-dependent blood flow in a setting of rat hindlimb ischemia [21]. It has been demonstrated that both the angiogenic activity of NPY peptides in the mouse cornea micro-pocket assay and the wound-healing response to NPY are impaired in Y2−/− mice [10], demonstrating that the Y2 receptor is necessary for the angiogenic activity of NPY and PYY peptides.

Additional lines of evidence exist for a role of the Y2 receptor in pathological angiogenesis. Both NPY and PYY are converted to the 3–36 variant by the enzyme dipeptidyl peptidase IV (DPPIV). The action of DPPIV converts the nonselective receptor agonist to one that is incapable of activating the Y1 receptor except at high doses [36]. Both the Y2 receptor and DPPIV were up-regulated in the setting of hindlimb ischemia [21], implying that the pathology also skews the receptor specificity of NPY towards a preferential interaction with the Y2 receptor.

While it appears from these studies that the Y2 receptor is necessary for the angiogenic activity of NPY and PYY peptides, it is not known if activation of the Y2 receptor is sufficient for the angiogenic and arteriogenic effects of NPY. In the present study, we have utilized a rat model of intermittent claudication and the more potent PYY peptides to demonstrate that both PYY and the Y2-selective agonist PYY3–36 produce a measurable increase in collateral-dependent blood flow with efficacy comparable to that of the known angiogenic agent VEGF165. Furthermore, we have utilized a pharmacodynamic model that relates the plasma concentration of unchanged peptide at the administered dose to the receptor occupancy based upon our in vitro data for Gi-mediated inhibition of forskolin-stimulated cAMP production to demonstrate that this arteriogenic effect can be explained solely by occupancy of the Y2 receptor.

Section snippets

Materials

Human PYY and PYY3–36 were purchased from Bachem (King of Prussia, PA). Tween 20 was purchased from BioRad (Hercules, CA). Hydron polymer was obtained from Interferon Sciences (New Brunswick, NJ). Radiolabeled microspheres (141Ce and 85Sr, 15 ± 1.0 μm diameter) were purchased from NEN (Boston, MA). Alzet® minipumps were purchased from DURECT Corporation (Cupertino, CA). All TaqMan reagents and probe/primers were purchased from Applied Biosystems Inc. (Forest City, CA). RNeasy mini columns were

Determination of the potency of NPY and PYY peptides at the various NPY receptors in the cAMP assay

It has previously been demonstrated that PYY is more potent than is NPY at the Y2 receptor (17). Indeed, this is the case at the rhesus receptor as well, where PYY is approximately one order of magnitude more potent at the Y2 receptor than is NPY (PYY Y2 Log EC50 = −10.58 ± 0.08, n = 10, EC50 = 2.62 × 10−11 M, versus Y2 Log EC50 = −9.56 ± 0.64, n = 5, EC50 = 2.77 × 10−10 M, for NPY). We therefore chose to determine the angiogenic activity of these peptides using the PYY peptide backbone, and to use the in vitro

Discussion

Although it has previously been demonstrated that the nonselective agonist NPY produced angiogenic activity in a variety of in vitro models [20], [21], [37] and it has been demonstrated that the Y2 receptor is necessary for the angiogenic activity of NPY peptides in the mouse cornea as well as the wound healing effects of NPY [10], it is not known if activation of the Y2 receptor is sufficient for angiogenesis. Furthermore, the mouse cornea model is a model of angiogenic vessel growth

Acknowledgements

The authors thank Drs. Kenneth R. Wehmeyer, Kevin G. Peters, and Thue W. Schwartz for helpful comments and suggestions during the preparation of this manuscript. The authors gratefully acknowledge receipt of the rhesus NPY receptor cDNAs from Eli Lilly and Company.

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    Present address: Ikaria, Inc., 1616 Eastlake Avenue E Suite 340, Seattle, WA 98102, USA.

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    3

    Present address: Department of Integrative Pharmacology, Abbott Laboratories, D-46 AP9, 100 Abbott Park Rd., Abbott Park, IL 60064-6119, USA.

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