Neuropeptide Y induces ischemic angiogenesis and restores function of ischemic skeletal muscles

Previously we showed that neuropeptide Y (NPY), a sympathetic vasoconstrictor neurotransmitter, stimulates endothelial cell migration, proliferation, and differentiation in vitro. Here, we report on NPY’s actions, receptors, and mediators in ischemic angiogenesis. In rats, hindlimb ischemia stimulates sympathetic NPY release (attenuated by lumbar sympathectomy) and upregulates NPY-Y2 (Y2) receptor and a peptidase forming Y2/Y5-selective agonist. Exogenous NPY at physiological concentrations also induces Y5 receptor, stimulates neovascularization, and restores ischemic muscle blood flow and performance. NPY-mediated ischemic angiogenesis is not prevented by a selective Y1 receptor antagonist but is reduced in Y2^–/– mice. Nonischemic muscle vascularity is also lower in Y2^–/– mice, whereas it is increased in NPY-overexpressing rats compared with their WT controls. Ex vivo, NPY-induced aortic sprouting is markedly reduced in Y2^–/– aortas and spontaneous sprouting is severely impaired in NPY^–/– mice. NPY-mediated aortic sprouting, but not cell migration/proliferation, is blocked by an antifetal liver kinase 1 antibody and abolished in mice null for eNOS. Thus, NPY mediates neurogenic ischemic angiogenesis at physiological concentrations by activating Y2/Y5 receptors and eNOS, in part due to release of VEGF. NPY’s effectiveness in revascularization and restoring function of ischemic tissue suggests its therapeutic potential in ischemic conditions.

Angiogenesis is a complex multistep process of formation of new vessels that is regulated by numerous growth factors (1). Most known angiogenic factors, such as bFGF (2) and VEGF (3), are derived from mesenchymal cells that are of either vascular or blood-borne origin. Angiogenesis begins with the activation of endothelial cells and proteases, which leads to the degradation of matrix and subsequent endothelial cell migration, proliferation, and differentiation into capillaries (4). Formation of arteries, or arteriogenesis, also requires migration and proliferation of VSMCs (4). While instrumental in tissue growth and development in the prenatal period, in the adult organism physiological angiogenesis is less active and is limited to certain organs such as the uterus during the menstrual cycle, pregnancy (5), or intensely exercising skeletal muscle (6).

In pathophysiological situations, however, angiogenesis may become reactivated, for example in tumors (7) and retinopathy (8) and in rheumatoid (9), ischemic heart, and peripheral vascular diseases (10, 11). Stimulating endogenous ischemia-driven angiogenesis recently became a promising treatment for salvaging ischemic cardiac and skeletal muscles (12, 13). Tissue hypoxia stimulates the release of bFGF (2) and VEGF (3) and upregulates the expression of their receptors (14). Consequently, many investigators have explored the therapeutic potential of revascularizing ischemic tissues through local administration of these and other exogenous angiogenic growth factors (11–13, 15–17). While often successful in partial therapeutic revascularization in animal models (2, 15–17), other factors may be active endogenously as permissive or driving mediators of ischemic angiogenesis. Sympathetic nerves are the major vascular regulatory system and have long been suspected to provide trophic influences on the innervated tissues (18), but their angiogenic potential has not been considered.

Recently, we found that neuropeptide Y (NPY1-36), also referred to as NPY, a sympathetic cotransmitter and vasoconstrictor (19), is a potent growth factor for VSMCs (20) and endothelial cells (21) at nonvasoconstrictive concentrations. In vitro, NPY stimulates human endothelial cell adhesion, migration, proliferation, and differentiation into capillaries (21). Human endothelium expresses receptors for NPY-Y1 (Y1) and Y2 as well as the NPY-converting enzyme DPPIV (21). The potency and efficacy of NPY in stimulating angiogenesis in vitro was found to be similar to those of bFGF and VEGF (21).

NPY exerts pleiotropic actions by activating multiple G_i/G_o-coupled receptors designated Y1–Y5 (22). Y1 is the predominant vascular receptor and the primary one mediating vasoconstriction (19) and VSMC proliferation (20). Conversely, NPY angiogenic activity in vitro is mediated not by Y1, but by either Y2 or Y5 or both receptors (20), and involves NPY’s processing to a Y2/Y5-selective agonist, NPY3-36 (23). This conversion of NPY1-36 from a vasoconstrictive Y1 agonist to a nonvasoconstrictive Y2/Y5 agonist occurs endogenously upon cleavage by endothelial DPPIV, which forms NPY3-36 (24).

Having determined in vitro the potent mitogenic and angiogenic activities of NPY on VSMCs and endothelial cells, we then sought to determine its physiological role and mechanisms in angiogenesis in vivo, using a variety of experimental models. Here, we demonstrate for the first time to our knowledge that neuronally released NPY is an endogenous mediator of ischemic angiogenesis via its Y2/Y5 receptors and NO- and VEGF-dependent pathways.

Effects of ischemia on plasma NPY levels and expression of NPY receptors and DPPIV in rat hindlimb. Fourteen days after femoral artery occlusion in rats, femoral venous plasma NPY immunoreactivity levels in the occluded limb were 4.5-fold higher than in the nonoccluded limb (Figure 1a) or in the abdominal aorta (0.746 ± 0.200 pmol/ml, P < 0.01) (not shown). This response was decreased 35% by ipsilateral lumbar sympathectomy (Figure 1a). Administration of the NPY pellet doubled the ischemia-induced plasma peptide levels (Figure 1a). Femoral artery occlusion also decreased (by 60%) NPY immunoreactivity levels in the artery below the occlusion compared with the contralateral nonoccluded artery (Figure 1b). The NPY immunoreactivity level in the femoral artery below the occlusion was further reduced (to 6–10% of nonischemic levels) by ipsilateral sympathectomy (Figure 1b), indicating efficacy of denervation.

Figure 1

Rat hindlimb ischemia stimulates NPY release and upregulates its receptors and DPPIV. (a) Femoral venous NPY immunoreactivity levels in rat ischemic (isch) and contralateral nonischemic (non-isch) hindlimbs (n = 10 each) and in the ischemic limb with ipsilateral lumbar sympathectomy (n = 4). (b) Ischemia- and sympathectomy-induced depletion of the NPY immunoreactivity content in the femoral arteries below the occlusion. **P < 0.01. (c) Ischemia-induced upregulation of expression of Y2 receptor and DPPIV, and NPY-mediated induction of Y5 receptor mRNA in gastrocnemius muscles (semiquantitative RT-PCR).

Ischemia also altered NPY receptor expression (Figure 1c). In nonischemic gastrocnemius muscles of control unoperated and nonischemic contralateral muscles of the operated rats, no Y2 or Y5 receptor or DPPIV expression could be detected by RT-PCR; the only detectable mRNA was that of Y1 and NPY (Figure 1c). Conversely, ischemic muscles showed detectable expression of Y2 and DPPIV mRNA’s, and NPY administration also induced Y5 mRNA (Figure 1c).

Capillary angiogenesis and collateral formation in the rat ischemic hindlimb after administration of NPY. NPY was administered locally in a slow-release pellet at a dose of 1 μg per pellet delivered over 14 days, which was estimated to yield physiological levels. We also used an antagonist selective for the Y1 receptor, NPY’s main vasoconstrictive receptor (26), to block NPY-mediated vasoconstriction and to confirm whether Y1 receptors are angiogenic. Thus, NPY was given to three groups of rats: NPY combined with intra-arterial infusion of the Y1 antagonist H409/22, its inactive enantiomer H510/445, or vehicle alone.

Angiogenic effects of NPY in ischemic tissues were assessed by evaluating vascularization of gastrocnemius muscles and the footpad. Normal, nonischemic gastrocnemius muscles showed rich density of vessels evident by positive staining with two endothelial markers, vWF and cd31 (Figure 2, a and b). In contrast, ischemic muscles and the footpad were pale and had a tendency for reduced density of vessels stained positively for vWF and cd31 (Figure 2, a and b). The angiogenic index, indicating density of perfused vessels (latex-filled, vWF-positive) to total microvascular density (all vWF-positive vessels), was reduced in ischemic gastrocnemius muscle and the footpad (Figure 2a, P < 0.01 compared with nonischemic values).

Figure 2

NPY stimulates capillary angiogenesis (a and b) and collateral formation (c) in rat ischemic hindlimb. (a) Density of vWF- and cd31-positive (a measure of total microvascular density), and latex-filled (a measure of vessels perfused, red arrowheads) vessels in ischemic and nonischemic gastrocnemius muscles (×100). Angiogenic index is a ratio of latex-perfused to total microvascular density. (b) Density of vWF-positive vessels in ischemic and nonischemic tissues with and without NPY, Y1 receptor antagonist (active and inactive), and placebo (**P < 0.01 compared with ischemic and placebo group [white bars, n=10]. (c) Representative vessels in the area around the femoral artery occlusion in the adductor muscle (bars = 20 μm) following perfusion with latex (H&E staining) and graph showing number of these arteries/mm² (**P < 0.01, n = 10). Arrows indicate media thickness. Ant, antagonist.

NPY application markedly increased vWF- and cd31-positive vessels in ischemic tissues to the levels observed in nonischemic tissues (Figure 2, a and b). The angiogenic index of the ischemic tissues was also restored to normal by NPY in the muscle and the footpad (Figure 2a, P < 0.01, compared with ischemic values). The Y1 receptor antagonist H409/22 (0.02 μmol/kg/min for 14 days) had no effect of its own and also did not significantly alter the NPY effect in ischemic gastrocnemius muscles or the footpad compared with tissues treated with NPY and either placebo or Y1-inactive enantiomer H510/445 (Figure 2, a and b). NPY pellets placed in nonischemic tissues did not augment their vascular density significantly but there was a tendency for an increase (Figure 2b). Also, ipsilateral lumbar sympathectomy alone did not significantly change vascular density of ischemic gastrocnemius muscles (data not shown). This, together with incomplete blockade of ischemia-induced increase in plasma NPY suggests that ipsilateral lumbar sympathectomy may have been insufficient for complete limb denervation (due to crossover of fibers from the contralateral side) or that there are other, non-neurogenic sources of NPY (see Discussion).

To test whether NPY stimulates formation of collateral vessels, we analyzed arterial densities in the adductor muscle around the site of femoral artery occlusion. Since arteriogenesis requires proliferation of VSMCs and leads to formation of corkscrew-like, thick-walled vessels (30), we used the latter feature as a marker for collateral formation. Femoral artery occlusion alone (in placebo-treated limbs) increased density of thick-walled muscular arteries in the adductor muscle compared with the contralateral side. NPY stimulated this process by an additional 42% (Figure 2c).

Effects of NPY on blood flow and contractile function of ischemic muscles. In gastrocnemius muscle, femoral artery occlusion alone induced a 78% reduction in blood flow compared with contralateral nonischemic muscles (Figure 3a). In the more distal tissues of the footpad, blood flow was even more reduced, reaching 9.6% of that in contralateral nonischemic tissues (Figure 3b). NPY (1 μg per pellet delivered over 14 days) completely restored blood flow in the ischemic gastrocnemius muscle (Figure 3a) and increased footpad flow up to 50% (Figure 3b). The NPY responses were resistant to Y1 receptor antagonist (H409/22, 0.02 μmol/kg/min for 14 days). The Y1 receptor antagonist also had no effect of its own on blood flow in ischemic tissues (data not shown).

Figure 3

NPY normalizes blood flow of ischemic gastrocnemius muscle (a) and improves it in the footpad (b), and restores function of ischemic gastrocnemius muscle. (c, d, and e) Individual recordings of 112 tetanic contractions, superimposed on each other, in nonischemic, ischemic, and NPY-treated ischemic muscles. Note the peak force of the first (uppermost) and last (lowest curve) contractions, indicating declining force due to prominent fatigue. (f) Isometric speed (time to attain the peak slope of the first tetanic contraction) and the fatigue index (the percent ratio of the peak force of the first to the 52nd contraction) in nonischemic, ischemic, and NPY-treated ischemic muscles (*P < 0.001, **P < 0.01, n = 6–8).

To determine whether NPY improves not only the structure but also ischemic tissue function, we studied the contractile performance and fatigability of the rat ischemic gastrocnemius muscle in response to repeated stimulations of the gastrocnemius nerves with six series of an increasing number of trains (total of 112 stimulations) under isometric conditions. The maximal peak tetanic force of gastrocnemius muscle was significantly reduced in ischemic (3.6 ± 0.4 N) compared with nonischemic conditions (4.8 ± 0.3 N, P < 0.05) (Figure 3), but its value increased toward nonischemic levels with administration of the NPY pellet (4.4 ± 0.4 N, Figure 3e). Ischemia also slowed the rate of development (isometric speed) of the first tetanic contraction (Figure 3f). Over the course of stimulations, there was a progressive decrease in the peak value of contractions (the lowest being the last, 112th contraction) in all groups (Figure 3, c–e), indicating the development of fatigue. The fatigue index (determined as the percent ratio of the peak force of the 52nd contraction to the first tetanic contraction) was increased in ischemic compared with nonischemic muscle (Figure 3f), indicating decreased fatigability of ischemic muscle. Both parameters — isometric speed and fatigue index — were fully normalized by NPY (Figure 3f).

Ischemic and nonischemic angiogenesis in NPY-Tg rats and NPY^–/– and Y2^–/– mice. To test the angiogenic effects of long-term elevation of endogenous levels of NPY, rats overexpressing the NPY gene (NPY-Tg mice) under the control of the natural promoter (31) were examined. NPY-Tg rats had markedly increased vascularization of gastrocnemius muscles (Figure 4, a and b). At 22 months of age, skeletal muscles of the NPY-Tg rats possessed a threefold greater number of capillaries per muscle fiber (Figure 4e, P < 0.01) and 30% greater muscle fiber area (data not shown) than muscles from WT rats.

Figure 4

Vascularization is increased in nonischemic muscles of NPY-Tg rats (a, b, and e) and reduced in ischemic and nonischemic muscles of Y2^–/– mice (c–e). (a, b, and e) Vascularization of nonischemic gastrocnemius muscles in 22-month-old NPY-overexpressing rats (NPY-Tg, **P < 0.01, n = 6–8). (c–e) Reduced vascularization (measured by latex-filled capillaries, blue arrowheads) of nonischemic and ischemic gastrocnemius muscles of Y2^+/+ and Y2^–/– mice (**P < 0.01 and ^‡P < 0.01 compared with corresponding WT and Y2^–/– mice in ischemic group, n = 6). (f) Inhibition of spontaneous and/or NPY-induced aortic sprouting in Y2^–/– and NPY^–/– mice compared with WT mice (**P < 0.01 and ^‡P < 0.01 compared with corresponding basal group, n = 4–8).

In contrast with NPY-Tg rats, Y2^–/– mice had a 30% lower vascular density in nonischemic gastrocnemius muscles than WT (Y2^+/+) mice had (Figure 4e, P < 0.01). Like in WT rats, in Y2^+/+ mice, limb ischemia markedly (73%) decreased vascular density of the latex-filled vessels in gastrocnemius muscles (Figure 4, c–e, P < 0.01). In Y2^–/– mice, this decrease became even more pronounced, and vascular density of ischemic muscle in these mice remained 58% lower than that of Y2^+/+ mice (Figure 4e, P < 0.01).

The Y2^–/– mice also showed markedly diminished (by 76%) NPY-induced aortic sprouting ex vivo compared with that of Y2^+/+ aortas (Figure 4f, P < 0.01). Aortic sprouting was even more impaired in NPY^–/– mice compared with WT 129SV mice (Figure 4f). While WT murine aortas began to sprout within 3–5 days, those from NPY^–/– mice took 7–10 days to sprout and their spontaneous sprouting was markedly diminished compared with WT mice (Figure 4f, P < 0.01). NPY administration restored aortic sprouting of NPY^–/– aortic rings to the same level as that of the WT mice (Figure 4f).

VEGF and NO in NPY-mediated angiogenesis. To test whether NPY’s angiogenic activity is mediated by release of VEGF and NO, and if so, whether these factors alter NPY receptor expression, we first used NO donors or inhibitors of NOS in a human endothelial cell line, ECV, which has undetectable NPY receptor expression when quiescent (Figure 5a, control). In ECV cells, NPY induced/upregulated expression of both Y1 and Y2 receptor mRNA as shown by RT-PCR. These effects were mimicked by sodium nitroprusside, an NO donor, and inhibited by 1 μM L-NAME (Figure 5a). Moreover, NPY-induced aortic sprouting was completely eliminated in eNOS^–/– mice (Figure 5, b–d), similar to that of VEGF-treated mice (Figure 5d). This dramatic effect was not assay-specific since deficiency in eNOS also blocked in vivo NPY-mediated capillary tube formation on Matrigel (data not shown).

Figure 5

NPY-induced angiogenesis is mediated by eNOS and VEGF. (a) Semiquantitative RT-PCR showing NPY- and sodium nitroprusside–induced upregulation of NPY receptors and inhibition of receptors by L-NAME in human endothelial cell line ECV. (b–d) Inhibition of basal-, NPY-, and VEGF-induced sprouting in eNOS^–/– aortas (**P < 0.001, n = 6–8). (e–j) Inhibition of NPY-induced aortic sprout formation but not cell migration/proliferation (mean cell density) by anti-flk1 antibody (**P < 0.001 and *P < 0.01 compared with control [white bars] n = 8). SNP, sodium nitroprusside. ECGS, endothelial cell growth supplement (Sigma-Aldrich).

NPY-induced aortic sprouting in WT mice was also markedly inhibited by a neutralizing anti-flk1 antibody (20 ng/ml) (32) (Figure 5, e–j, P < 0.01). Interestingly, anti-flk1 antibody did not completely block, but reduced by 20%, NPY-induced increase in cellular density in the area of the aortic rings, indicating that cell proliferation and migration was affected much less (Figure 5j).

The hypothesis that NPY is a physiological mediator of angiogenesis in vivo was tested in pathophysiological conditions where both neovascularization and sympathetic nerve activity are stimulated, i.e., in ischemia. Both angiogenesis and NPY levels are known to be the highest early in development (33), coinciding with organogenesis and growth, and they decline with age (34). Also, both become activated during myocardial ischemia (35), exercise, and hypoxia (36). Here we demonstrate for the first time to our knowledge, using a variety of models, that NPY is an endogenously active neurogenic factor stimulating ischemic angiogenesis via Y2/Y5 receptor- and VEGF/NO-dependent pathways.

Ischemia stimulates NPY release and upregulates NPY receptor expression. In rats, femoral artery occlusion–induced limb ischemia increases local venous outflow and lowers femoral arterial content of NPY, indicating its release from vascular stores. This release is at least in part neurogenic since it is decreased by ipsilateral lumbar sympathectomy. The remaining sympathectomy-resistant NPY release may be due to the incompleteness of one-sided denervation, as well as NPY release from the endothelium, which was previously shown to synthesize its own NPY and to internalize sympathetically derived NPY (21).

A major shift occurs in the Y2/Y5 receptor system during ischemia. While nonischemic muscles express only Y1 receptor (and NPY) mRNA’s, ischemic muscles show an induction/upregulation of Y2 receptors and DPPIV, an enzyme forming Y2/Y5-selective agonist (24). These effects are further augmented by the NPY pellet, which also induces Y5 mRNA. Similar NPY-dependent positive regulation of its own receptors was previously found in VSMCs in vitro (20, 21), and in the present study is seen in endothelial cells. Although the exact mechanisms of NPY’s upregulation of its own receptors remain unclear, NO appears to play a role. We now show that an NO donor increases and an NOS inhibitor reduces both NPY-mediated upregulation of receptors and endothelial cell proliferation (E.W. Lee et al., unpublished data). Moreover, in eNOS^–/– mice, NPY-induced aortic sprouting is completely abolished, as is that of VEGF (37). Thus, endothelial NO appears to be essential for NPY’s angiogenic signaling, in part due to its upregulation of NPY receptor expression.

Administration of NPY revascularizes and restores blood flow to ischemic tissues. When administered locally to the rat ischemic hindlimb, NPY stimulates angiogenesis and restores vascularization of ischemic tissues to nonischemic levels. This occurs at physiological plasma NPY concentrations (<10 nM), which may be vasoconstrictive, similar to those induced by stress or exercise in rats (19, 26). There is no indication, however, that NPY-mediated vasoconstriction occurs in these conditions. First, NPY-induced angiogenesis is accompanied by the restoration of blood flow to the ischemic gastrocnemius muscles and a 50% improvement of flow to the footpad. Second, both structural and functional NPY-mediated improvements are resistant to the Y1-selective receptor antagonist (H402/22), indicating that Y1-mediated vasoconstriction does not play a role and that Y1 receptors are not angiogenic in the ischemic hindlimb.

The possibility, however, that NPY may exert an angiogenic effect in nonischemic tissues by activating Y1 receptors is suggested by the tendency for NPY to increase vascularization of the nonischemic muscle, which expresses only this receptor type (as shown by RT-PCR). Also our previous findings of the ability of Y1 receptor antagonist to reduce already markedly impaired aortic sprouting in Y2^–/– mice (38) suggest that Y1 receptors play a compensatory role in the absence of the major angiogenic Y2 receptor.

NPY also stimulates formation of collateral vessels in the adductor muscle at the level of the occluded femoral artery. This process, called arteriogenesis, differs from capillary angiogenesis by being hypoxia-independent but shear stress–dependent (30), and also requires proliferation of VSMCs (39). The newly formed collateral arteries often assume corkscrew shapes (39, 40), possibly due to the nonuniformity of their VSMC proliferation and formation of hypertrophied media. NPY, due to its potent growth-promoting activities for VSMCs (20) and endothelial cells (21), stimulates both collateralization and capillary angiogenesis, leading to the full revascularization of ischemic tissues. Although stimulation of VSMC proliferation is necessary for collateral formation and salvaging of ischemic tissue, it may also carry the risk of promoting neointima formation and atherosclerosis. Such concerns have been raised for several angiogenic factors, such as VEGF and bFGF (41), and may also apply to NPY. However, although NPY promotes angioplasty-induced neointima formation in rat carotid artery, its effects appear to be mediated by Y1, and not Y2, receptors (L. Li et al., unpublished data).

The loss of the angiogenic influence of NPY may explain the long-term ineffectiveness of regional sympathectomy in patients with severe peripheral vascular insufficiency (42) when it is used to alleviate ischemic pain. Although in our studies sympathectomy did not significantly reduce ischemic revascularization, this could be due to the short observation time (2 weeks) and incomplete removal of NPY by unilateral sympathectomy (endothelial NPY and/or crossover of contralateral fibers).

Administration of NPY restores ischemic muscle contractile function. Along with blood flow, NPY restores the contractile performance of the ischemic gastrocnemius muscle. Ischemia led to several impairments of muscle function, such as a decrease in maximal peak force of tetanic contraction and slower development of peak contraction (isometric speed). In contrast, the fatigability of the ischemic muscles in response to long series of tetanic stimulations decreased compared with nonischemic muscles. Such changes have been reported previously (43) and are believed to be due to the muscle’s selective loss of ischemia-sensitive fast glycolytic fibers and its switch to fast-oxidative ones, which are less fatigued (44). NPY increased the maximal peak force of tetanic contraction of the ischemic gastrocnemius muscle toward normal and fully restored its isometric speed of contraction and fatigability. These data demonstrate the peptide’s ability to restore not only ischemic tissue vascularization and blood flow but also its contractile function.

Endogenous NPY and Y2 receptors are angiogenic. To test whether the angiogenic activity of NPY plays a long-term role during tissue growth and development, we used rats overexpressing the NPY gene under the control of the natural promoter (45, 46). These rats, which do not have elevated plasma NPY at rest but have slightly elevated tissue NPY levels (31), have a remarkable hypervascularity of gastrocnemius muscles at 5, 12 (data not shown), and 22 months of age, suggesting enhanced angiogenesis during their lifelong skeletal muscle growth. This hypervascularization appears to be functional as it is associated with skeletal muscle hypertrophy (data not shown), lower blood pressure, and increased longevity of these rats (45). Chronically elevated NPY levels may also protect against the age-dependent decline in angiogenesis. Previously we noted that aging impairs NPY angiogenic activity in vitro and also inhibits expression of Y2 receptors and DPPIV (47). In NPY-Tg rats, NPY-induced protection against declining angiogenesis could be due to the peptide’s ability to upregulate its own angiogenic receptors (20).

The primary receptor involved in NPY’s angiogenic actions in ischemia and during developmental growth appears to be Y2. Removal of Y2 receptors by gene knockout reduces vascularization by 30–60% in both nonischemic and ischemic gastrocnemius muscles compared with corresponding muscles of Y2^+/+ mice. Similarly, Y2 receptor knockout and the Y2-antagonist treatment of Y2^+/+ aortas (38) markedly inhibit NPY-mediated aortic sprouting ex vivo. The role of Y5 receptor cannot be similarly assessed due to the lack of data on Y5^–/– mice (investigation in process). In the in vitro aortic sprouting assay, however, NPY-mediated angiogenic effect in the WT aortas is blocked by Y5 receptors, while in the Y2^–/– mice, Y1 receptors may also play a compensatory angiogenic role (38). The latter notion is also supported by near-normal spontaneous aortic sprouting in Y2^–/– mice. However, knockout of the NPY gene itself severely impairs spontaneous sprouting of murine aortas, which administration of NPY fully restores. These data indicate that endogenous NPY is a required growth factor for stimulation of angiogenesis, at least in the model of murine aorta.

NPY-mediated angiogenesis is VEGF- and NO-dependent. Having determined potent mitogenic activities of NPY on VSMCs and endothelial cells in vitro and its angiogenic activities in vivo, we also sought to establish mechanisms of NPY’s angiogenesis. There are many mediators and signaling pathways that lead to angiogenesis (4). Some growth factors such as VEGF stimulate neovascularization partly by releasing NO, which by itself may be angiogenic (37). NO also appears to be involved in NPY’s actions: in cerebral vessels, it mediates NPY-induced vasodilation, which is Y2 receptor-dependent (48). In support of the role of VEGF, NPY-mediated aortic sprouting was blocked by an antibody neutralizing a VEGF receptor, flk1 (32). However, in spite of this dramatic effect, anti-flk1 antibody only slightly reduced the density of endothelial cells migrating out of the rings. This indicates that VEGF is not involved in NPY’s effect on cell migration and/or proliferation but mediates the later stage of angiogenesis, endothelial cell differentiation into capillaries. In contrast, deficiency of eNOS completely prevented all components of NPY-induced angiogenic response in aortic sprouting assays, implying that NO is required for full elaboration of NPY-mediated angiogenesis.

In conclusion, NPY is an endogenously active neurogenic (and possibly endothelial) ischemia-induced angiogenic factor that stimulates ischemic and growth-dependent angiogenesis by activating Y2/Y5 receptors and NO-dependent pathways, in part mediated by release of VEGF. Exogenous NPY, at physiological concentrations, restores not only vascularization but also the function of the ischemic tissues. Thus, NPY-derived drugs (Y2/Y5 agonists) might be therapeutic for revascularization of ischemic tissues in peripheral vascular and heart diseases.

This study was supported in part by NIH grants HL-67357 (to Z. Zukowska) and HL-57921 (to M. Michalkiewicz).

Conflict of interest: The authors have declared that no conflict of interest exists.

Nonstandard abbreviations used: neuropeptide Y (NPY); NPY-Y1 (Y1); NPY transgenic (NPY-Tg); anti–fetal liver kinase 1 antibody (anti-flk1).

1. Risau, W. Mechanisms of angiogenesis. Nature. 1997. 386:671-674.
   View this article via: PubMed CrossRef Google Scholar
2. Baffour, R, et al. Enhanced angiogenesis and growth of collaterals by in vivo administration of recombinant basic fibroblast growth factor in a rabbit model of acute lower limb ischemia: dose-response effect of basic fibroblast growth factor. J. Vasc. Surg. 1992. 16:181-191.
   View this article via: PubMed CrossRef Google Scholar
3. Shweiki, D, Itin, A, Soffer, D, Keshet, E. Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis. Nature. 1992. 359:843-845.
   View this article via: PubMed CrossRef Google Scholar
4. Carmeliet, P. Mechanisms of angiogenesis and arteriogenesis. Nat. Med. 2000. 6:389-395.
   View this article via: PubMed CrossRef Google Scholar
5. Gargett, CE, Rogers, PA. Human endometrial angiogenesis. Reproduction. 2001. 121:181-186.
   View this article via: PubMed CrossRef Google Scholar
6. Egginton, S, et al. Capillary growth in relation to blood flow and performance in overloaded rat skeletal muscle. J. Appl. Physiol. 1998. 85:2025-2032.
   View this article via: PubMed Google Scholar
7. Kerbel, RS. Tumor angiogenesis: past, present and the near future. Carcinogenesis. 2000. 21:505-515.
   View this article via: PubMed CrossRef Google Scholar
8. Breier, G, Damert, A, Plate, KH, Risau, W. Angiogenesis in embryos and ischemic diseases. Thromb. Haemost. 1997. 78:678-683.
   View this article via: PubMed Google Scholar
9. Folkman, J. Angiogenesis in cancer, vascular, rheumatoid and other disease. Nat. Med. 1995. 1:27-31.
   View this article via: PubMed CrossRef Google Scholar
10. Ware, JA, Simons, M. Angiogenesis in ischemic heart disease. Nat. Med. 1997. 3:158-164.
    View this article via: PubMed CrossRef Google Scholar
11. Isner, JM. Manipulating angiogenesis against vascular disease. Hosp. Pract. (Off. Ed.) 1999. 34:69-74, 76, 79–80 passim.
    View this article via: PubMed Google Scholar
12. Rivard, A, Isner, JM. Angiogenesis and vasculogenesis in treatment of cardiovascular disease. Mol. Med. 1998. 4:429-440.
    View this article via: PubMed Google Scholar
13. Folkman, J. Therapeutic angiogenesis in ischemic limbs. Circulation. 1998. 97:1108-1110.
    View this article via: PubMed Google Scholar
14. Ikeda, E, Achen, MG, Breier, G, Risau, W. Hypoxia-induced transcriptional activation and increased mRNA stability of vascular endothelial growth factor in C6 glioma cells. J. Biol. Chem. 1995. 270:19761-19766.
    View this article via: PubMed CrossRef Google Scholar
15. Isner, JM, Asahara, T. Angiogenesis and vasculogenesis as therapeutic strategies for postnatal neovascularization. J. Clin. Invest. 1999. 103:1231-1236.
    View this article via: JCI PubMed CrossRef Google Scholar
16. Mack, CA, et al. Biologic bypass with the use of adenovirus-mediated gene transfer of the complementary deoxyribonucleic acid for vascular endothelial growth factor 121 improves myocardial perfusion and function in the ischemic porcine heart. J. Thorac. Cardiovasc. Surg. 1998. 115:168-176; discussion 176–177.
    View this article via: PubMed CrossRef Google Scholar
17. Sellke, FW, Laham, RJ, Edelman, ER, Pearlman, JD, Simons, M. Therapeutic angiogenesis with basic fibroblast growth factor: technique and early results. Ann. Thorac. Surg. 1998. 65:1540-1544.
    View this article via: PubMed CrossRef Google Scholar
18. Bevan, RD. Trophic effects of peripheral adrenergic nerves on vascular structure. Hypertension. 1984. 6:III19-III26.
    View this article via: PubMed Google Scholar
19. Zukowska-Grojec, Z., and Wahlestedt, C. 1993. Origin and actions of neuropeptide Y in the cardiovascular system. In The biology of neuropeptide Y and related peptides. W.F. Colmers and C. Wahlestedt, editors. Humana Press. Totowa, New Jersey, USA. 315–388.
20. Zukowska-Grojec, Z, Karwatowska-Prokopczuk, E, Fisher, TA, Ji, H. Mechanisms of vascular growth-promoting effects of neuropeptide Y: role of its inducible receptors. Regul. Pept. 1998. 75–76:231-238.
21. Zukowska-Grojec, Z, et al. Neuropeptide Y: a novel angiogenic factor from the sympathetic nerves and endothelium. Circ. Res. 1998. 83:187-195.
    View this article via: PubMed Google Scholar
22. Ingenhoven, N, Beck-Sickinger, AG. Molecular characterization of the ligand-receptor interaction of neuropeptide Y. Curr. Med. Chem. 1999. 6:1055-1066.
    View this article via: PubMed Google Scholar
23. Ghersi, G, Chen, W, Lee, EW, Zukowska, Z. Critical role of dipeptidyl peptidase IV in neuropeptide Y-mediated endothelial cell migration in response to wounding. Peptides. 2001. 22:453-458.
    View this article via: PubMed CrossRef Google Scholar
24. Mentlein, R, Dahms, P, Grandt, D, Kruger, R. Proteolytic processing of neuropeptide Y and peptide YY by dipeptidyl peptidase IV. Regul. Pept. 1993. 49:133-144.
    View this article via: PubMed CrossRef Google Scholar
25. Doods, HN, et al. Pharmacological characterization of the selective nonpeptide neuropeptide Y Y1 receptor antagonist BIBP 3226. J. Pharmacol. Exp. Ther. 1995. 275:136-142.
    View this article via: PubMed Google Scholar
26. Zukowska-Grojec, Z, Dayao, EK, Karwatowska-Prokopczuk, E, Hauser, GJ, Doods, HN. Stress-induced mesenteric vasoconstriction in rats is mediated by neuropeptide Y Y1 receptors. Am. J. Physiol. 1996. 270:H796-H800.
    View this article via: PubMed Google Scholar
27. van Dielen, FM, et al. Effects of surgical sympathectomy on skin blood flow in a rat model of chronic limb ischemia. World J. Surg. 1998. 22:807-811.
    View this article via: PubMed CrossRef Google Scholar
28. Pettorossi, VE, Della Torre, G, Bortolami, R, Brunetti, O. The role of capsaicin-sensitive muscle afferents in fatigue-induced modulation of the monosynaptic reflex in the rat. J. Physiol. 1999. 515:599-607.
    View this article via: PubMed CrossRef Google Scholar
29. Nicosia, RF, Ottinetti, A. Growth of microvessels in serum-free matrix culture of rat aorta. A quantitative assay of angiogenesis in vitro. Lab. Invest. 1990. 63:115-122.
    View this article via: PubMed Google Scholar
30. Hershey, JC, et al. Revascularization in the rabbit hindlimb: dissociation between capillary sprouting and arteriogenesis. Cardiovasc. Res. 2001. 49:618-625.
    View this article via: PubMed CrossRef Google Scholar
31. Michalkiewicz, M., and Michalkiewicz, T. 2000. Developing transgenic neuropeptide Y rats. In Neuropeptide Y protocols. A. Balasubramaniam, editor. Humana Press. Totowa, New Jersey, USA. 73–89.
32. Conn, G, et al. Amino acid and cDNA sequences of a vascular endothelial cell mitogen that is homologous to platelet-derived growth factor. Proc. Natl. Acad. Sci. U. S. A. 1990. 87:2628-2632.
    View this article via: PubMed CrossRef Google Scholar
33. Gordon, L, et al. Development of the peptidergic innervation of human heart. J. Anat. 1993. 183:131-140.
    View this article via: PubMed Google Scholar
34. Swift, ME, Kleinman, HK, DiPietro, LA. Impaired wound repair and delayed angiogenesis in aged mice. Lab. Invest. 1999. 79:1479-1487.
    View this article via: PubMed Google Scholar
35. Morris, MJ, et al. Plasma neuropeptide Y levels rise in patients undergoing exercise tests for the investigation of chest pain. Clin. Exp. Pharmacol. Physiol. 1986. 13:437-440.
    View this article via: PubMed CrossRef Google Scholar
36. Kaijser, L, Pernow, J, Berglund, B, Grubbstrom, J, Lundberg, JM. Neuropeptide Y release from human heart is enhanced during prolonged exercise in hypoxia. J. Appl. Physiol. 1994. 76:1346-1349.
    View this article via: PubMed CrossRef Google Scholar
37. Ziche, M, Morbidelli, L. Nitric oxide and angiogenesis. J. Neurooncol. 2000. 50:139-148.
    View this article via: PubMed CrossRef Google Scholar
38. Lee, EW, Grant, DS, Movafagh, S, Zukowska, Z. Impaired angiogenesis in neuropeptide Y (NPY)-Y2 receptor knockout mice. Peptides. 2003. 24:99-106.
    View this article via: PubMed CrossRef Google Scholar
39. Heeschen, C, et al. Nicotine stimulates angiogenesis and promotes tumor growth and atherosclerosis. Nat. Med. 2001. 7:833-839.
    View this article via: PubMed CrossRef Google Scholar
40. Deindl, E, et al. Role of ischemia and of hypoxia-inducible genes in arteriogenesis after femoral artery occlusion in the rabbit. Circ. Res. 2001. 89:779-786.
    View this article via: PubMed CrossRef Google Scholar
41. Carmeliet, P, Collen, D. Molecular analysis of blood vessel formation and disease. Am. J. Physiol. 1997. 273:H2091-H2104.
    View this article via: PubMed Google Scholar
42. Alexander, JP. Chemical lumbar sympathectomy in patients with severe lower limb ischaemia. Ulster Med. J. 1994. 63:137-143.
    View this article via: PubMed Google Scholar
43. Lee, SL, Pevec, WC, Carlsen, RC. Functional outcome of new blood vessel growth into ischemic skeletal muscle. J. Vasc. Surg. 2001. 34:1096-1102.
    View this article via: PubMed CrossRef Google Scholar
44. Lieber, RL, Pedowitz, RA, Friden, J, Gershuni, DH. Decreased muscle speed, strength and fatigability following two hours of tourniquet-induced ischaemia. Scand. J. Plast. Reconstr. Surg. Hand Surg. 1992. 26:127-132.
    View this article via: PubMed CrossRef Google Scholar
45. Michalkiewicz, M, Knestaut, KM, Bytchkova, EY, Michalkiewicz, T. Hypotension and reduced catecholamines in neuropeptide Y transgenic rats. Hypertension. 2003. 41:1056-1062.
    View this article via: PubMed CrossRef Google Scholar
46. Michalkiewicz, M, Michalkiewicz, T, Kreulen, DL, McDougall, SJ. Increased blood pressure responses in neuropeptide Y transgenic rats. Am. J. Physiol. Regul. Integr. Comp. Physiol. 2001. 281:R417-R426.
    View this article via: PubMed Google Scholar
47. Kitlinska, J, Lee, EW, Movafagh, S, Pons, J, Zukowska, Z. Neuropeptide Y-induced angiogenesis in aging. Peptides. 2002. 23:71-77.
    View this article via: PubMed CrossRef Google Scholar
48. You, J, Edvinsson, L, Bryan (Jr), RM. Neuropeptide Y-mediated constriction and dilation in rat middle cerebral arteries. J. Cereb. Blood Flow Metab. 2001. 21:77-84.
    View this article via: PubMed Google Scholar