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* Department of Physiology and Biophysics, Georgetown University Medical Center, Washington, D.C., USA;
Department of Biochemistry, Virginia Commonwealth University School of Medicine, Richmond, Virginia, USA; and
National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland, USA
1Correspondence: Department of Physiology and Biophysics, Georgetown University Medical Center, Washington, DC, 20057, USA. E-mail: zzukow01{at}georgetown.edu
SPECIFIC AIMS
We have discovered previously that NPY is a potent angiogenic factor and exerts strong stimulatory effects on endothelial cell migration, proliferation, and differentiation into capillary tubes in vitro. Using a limited number of receptor agonist and antagonists in in vitro assays, we also found a bimodal pattern of chemotaxis and the important role of the Y2 receptor in NPY-induced angiogenesis. However, full characterization of NPY’s dose-response curves in in vitro angiogenic assays and the underlying receptor subtype(s) remained to be investigated. This project was designed to examine NPY’s angiogenic effect on two different (umbilical vein, HUVEC; microdermal, SVEC4–10) human endothelial cell types at a wide concentration range of NPY (10–14 to 10–7 M). To determine NPY’s angiogenic receptor subtype(s), migration, proliferation, and differentiation of these cells in the presence of NPY, with and without specific and selective NPY receptor agonists and antagonists, were tested in a variety of in vitro assays [Boyden chemotaxis chamber, [3H]-thymidine uptake, Matrigel tube formation, and RT-polymerase chain reaction (RT-PCR)].
PRINCIPAL FINDINGS
1. NPY potently and bimodally enhances endothelial cell migration, proliferation, and differentiation in vitro by 2-fold
NPY (at a concentration range of 10–14 to 10–7 M) reproducibly stimulated migration, proliferation, and differentiation of endothelial cells (HUVEC and SVEC4–10) in a bimodal fashion. A high-affinity (pM) peak occurred at 10–13-10–11M, and a low-affinity peak occurred at 10–8M, with an average of a 2-fold increase. Both cell types responded similarly to NPY when all three Y1, Y2, and Y5 receptor subtypes were expressed (RT-polymerase chain reaction).
2. Both the high- and low-affinity peaks of NPY activity are mediated via activation of multiple NPY receptor subtypes: Y1, Y2, and Y5
NPY1–36 is an agonist of all of its receptor subtypes: Y1-Y5. Our data revealed that agonists of multiple NPY receptors (Y1, Y2, Y3, and Y5) such as [Leu31-Pro34]-NPY (Y1/Y3/Y5), NPY3–36 (Y2/Y3/Y5), and human pancreatic polypeptide (hPP, Y4/Y5) could mimic NPY in stimulating migration, proliferation, and differentiation of endothelial cells in vitro. However, peptide YY (PYY, all receptors except Y3), [Ala31,Aib32]NPY (specific Y5 agonist), and [Ahx 5–24]NPY (specific Y2 agonist) did not influence endothelial cell migration. Our antagonist data illustrated that migration of endothelial cells in vitro was inhibited by any single NPY receptor antagonist (Y1, Y2, or Y5), proliferation by a combination of at least two receptor antagonists (Y1+Y2, Y1+Y5, or Y2+Y5), and differentiation only by the combination of all three receptor antagonists (Y1+Y2+Y5). The results were similar for both the high- and low-affinity peaks.
3. Y5 receptor acts as an enhancer of NPY’s angiogenic activity
The expression of the Y5 receptor, unlike those of the Y1 and the Y2 receptors, was not detected constitutively in SVEC4–10 cells (RT-PCR). However, 24 preincubation of these cells in low serum, with or without NPY, resulted in up-regulation of the Y5 receptor and enhanced endothelial cell migration toward pM concentrations of the peptide. NPY also dose-dependently, with higher levels at pM and 10 nM concentrations, increased its own expression and the expression of the Y5 receptor, correlating exactly with the two peaks of endothelial cell proliferation. Moreover, pretreatment with NPY 24 h prior to the experiment resulted in a further increase in the Y5 receptor expression and several fold increase in endothelial cell proliferation.
CONCLUSIONS AND SIGNIFICANCE
The angiogenesis cascade is multistep and complex because of the large number of participating proteins that function to promote cell migration, proliferation, and differentiation. Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are two ubiquitous, potent angiogenic peptides that participate in many angiogenic cascades. NPY, by virtue of being present in the brain, endothelium, and sympathetic nerves, which are tonically active at the angiogenic sites, is a potential participant of many angiogenic cascades. We discovered earlier that NPY is an angiogenic factor and exerts strong stimulatory effects on endothelial cell migration, proliferation, and differentiation into capillary tubes in vitro. NPY enhances angiogenesis in various ex vivo and in vivo models of angiogenesis, such as aortic sprouting, Matrigel plug assay, and ischemic hind limb revascularization. In vitro and in vivo studies revealed that NPY’s angiogenic potential is comparable to that of bFGF and VEGF.
In this study we demonstrate that NPY’s angiogenic activity in vitro is bimodal. The presence of two maxima, one at very low NPY concentrations (pM) and one at very high concentrations (10 nM), not only indicates that NPY is one of the most potent angiogenic peptides known, but that it can induce blood vessel formation over a wide range of concentrations. For instance, NPY-induced angiogenesis can occur in tissues that contain low NPY concentrations, such as the nonsympathetically innervated aorta or growing organs lacking a mature NPY system. However, it can also occur in tissues containing high NPY concentrations, such as the heart, mature vessels, or muscles during high sympathetic activity induced by stress, ischemia, or injury. Our study also shows that both the high- and the low-affinity peaks of NPY activity are mediated via activation of multiple NPY receptor subtypes: Y1, Y2, and Y5. Activation of these three receptor subtypes is necessary for endothelial cell migration, any two for their proliferation, and only one for their differentiation. We found an important role for the Y5 receptor as an enhancer of NPY’s angiogenic activity. Up-regulated by starvation, injury, or NPY, the Y5 receptor expression has correlated with an increased potency and efficacy of NPY in stimulating migration and proliferation of endothelial cells in vitro.
Therefore, we propose that NPY’s bimodal angiogenic effect on the endothelium in vitro is through oligomerization of NPY receptor subtypes Y1, Y2, and Y5: heterotrimers for migration, heterodimers for proliferation, and monomers for differentiation (Fig. 1
). Based on the inactivity of PYY, we suggest that the heterotrimer of Y1/Y2/Y5 is the uncloned Y3 receptor. We further hypothesize that the peaks of NPY-stimulated activity is the heteromeric state of the receptors, while the valleys result from either desensitization/dissociation of the NPY receptor oligomers or a transitional state. Finally, we speculate that NPY receptor oligomers function as a part of a larger receptor complex that includes other groups of receptors, enzymes, and membrane molecules, such as VEGF or bFGF receptors and dipeptidyl peptidase IV (DPPIV) and endothelial nitric oxide synthase enzymes.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4770fje
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