to determine the role of NPY receptor oligomerization in its growthpromoting activity and its possible physiological, clinical and therapeutic implications. However, the ability of NPY to exert potent angiogenic activity at sub-picomolar concentrations makes the peptide an attractive molecule for either angiogenic or anti-angiogenic therapies

3. DPPIV: A Molecular Switch of the NPY Angiogenic System

An important feature of NPY’s potent growth promoting activity in VSM and endothelial cells is that they are mediated by different receptors (Fig. 2). The mitogenic effect of the peptide in endothelial cells is mediated by Y2/Y5 receptors, while Y1/Y5 receptors are involved in VSMC proliferation.8,20,21,28,29 Additionally, Y1 receptors present on VSMCs are also responsible for NPY’s vasoconstrictive effects. These divergent receptor-specific actions of NPY in vascular cells implicate a “converting enzyme,” DPPIV, as an important receptor switch and a regulator of angiogenesis. This membrane protease is abundantly expressed in endothelium and often considered a marker of microvascular endothelial cells.23 As mentioned before, the enzyme cleaves the full length NPY1−36 to NPY3−36, a fragment which is not able to bind to the Y1 receptors but is a specific Y2/Y5 agonist.19 In this way, the protease acts as an endogenous antagonist of Y1 receptor-mediated vasoconstriction and VSMC proliferation and an enhancer/amplifier of the Y2/Y5 receptor-mediated growth-promoting effects of NPY in endothelial cells. The protease also controls the balance between peptide’s effects is VSMC and endothelial cells. Such a balance is an important factor regulating NPY actions at different stages of vascularization. The initial steps of vascularization involve mainly changes in endothelium and require Y2 receptor-dependent activity of the peptide. However, maturation of the vessels and formation of collateral vessels involves also Y1 receptor-mediated VSMC proliferation. Hence, changes in DPPIV proteolytic activity may be an important mechanism controlling NPY-induced angioand arteriogenesis.

A crucial role of DPPIV in NPY-driven angiogenesis has been confirmed using a variety of models. As described above, DPPIV is coexpressed with NPY and its receptors in endothelial cells and, along

98 J. B. Kitlinska & Z. Zukowska

with them, upregulated in tissues undergoing active angiogenesis, e.g. during ischemia and wound healing (see below).20,23 On the other hand, age-dependent impairment of spontaneous and NPY-driven angiogenesis is associated with downregulation of the Y2 as well as DPPIV expression in old animals.27

Taken together, DPPIV is an integral element of NPY’s angiogenic system, augmenting its growth-promoting effects in endothelial cells. The protease also regulates the balance between Y1 receptor-mediated effects of NPY on VSMCs and its Y2 receptor-dependent actions in endothelial cells.

4. Downstream Mediators of NPY Actions

NPY is not only a potent but also a multifunctional peptide and involved in several steps of angiogenesis: endothelial cell adhesion, migration, proliferation and differentiation, as well as growth of VSMCs.8,21,29 NPY treatment leads to the formation of what appears to be fully functional vessels (Fig. 2), unlike those formed by vascular endothelial growth factor (VEGF), which are often poorly matured and leaky.13,21,22 This phenomenon may be associated with the fact that NPY is positioned upstream from other angiogenic factors, and acts not only directly, but also indirectly, via stimulation of various angiogenic pathways. Several lines of evidence support this notion. For example, NPY-induced aortic sprouting is completely abolished in mice deficient in endothelial nitric oxide synthase (eNOS), indicating that nitric oxide is a critical mediator of the NPY angiogenic effect.20 Blockade of VEGF also inhibits NPY-driven formation of capillaries, however, the inhibition is incomplete and does not affect peptide-induced endothelial cell migration.20 Hence, VEGF is involved in some, but not all, steps of the NPY angiogenic activity. Recent unpublished data from our laboratory indicate that NPY-induced angiogenesis may be also partially mediated by fibroblast growth factor-2 (FGF-2). NPY upregulates expression of FGF-2, its receptors, as well as VEGF receptor-2 in endothelial cells. Consequently, NPY-induced endothelial cell proliferation is blocked by both anti-FGF-2 neutralizing antibody and the VEGF antagonist, VEGF R2/Fc chimera.

In contrast, NPY receptor antagonists do not reduce FGF-2- and VEGF-stimulated endothelial cell proliferation. Moreover, both bFGF and VEGF induce full sprouting response in Y2−/− aortic rings, where NPY’s angiogenesis is reduced. Therefore, NPY is an upstream angiogenic factor acting, at least partially, via bFGF and VEGF pathways. Interactions of NPY with other angiogenic factors also cannot be excluded. Such activation of multiple downstream pathways by NPY leads to a complete, tightly controlled process of angiogenesis and to formation of mature, functional blood vessels. As a result, in mouse corneas, for example, NPY induces formation of microvessels that are well separated and organized, as opposed to the leaky and hemorrhagic vasculature stimulated by VEGF.22 Similar effects can also be observed in aortic sprouts.21

5. NPY in Revascularization of Ischemic Tissues

Ischemia is one of the most important stimuli activating angiogenic processes. A number of angiogenic factors, such as VEGF, are known to be directly upregulated by hypoxia-inducible factor (HIF-1α).36 However, hypoxia is also a powerful activator of perivascular nerves. All blood vessels, except the thoracic aorta, are innervated by sensory and sympathetic nerves, which alert the body of vascular injury or tissue ischemia. In consequence, sympathetic activity is augmented in ischemic conditions, which is associated with increased release of norepinephrine and its co-transmitters — NPY and purines.5,37−39 For years, sympathetic activity was believed to be deleterious for ischemic tissues by impairing tissue blood flow, due to their vasoconstrictive effects. However, discovery of angiogenic and vascular growth-promoting activity of NPY has put this notion in doubt, as new trophic effects of sympathetic nerves have emerged.

The role of NPY in revascularization of ischemic tissues was established using the rodent hindlimb model, where femoral artery occlusion results in calf muscle ischemia and capillary angiogenesis (Fig. 2). At the same time, in the area of vessel occlusion, in the thigh, shear stress above the occluded artery activates arteriogenesis and formation of collaterals.20 Both processes are associated with upregulation of the NPY system. Venous plasma levels of the peptide are elevated

100 J. B. Kitlinska & Z. Zukowska

in ischemic leg, as compared to the contralateral non-ischemic limb.20 This increase is reduced by ipsilateral lumbar sympathectomy, indicating it is, at least in part, associated with hyperactivity of sympathetic nerves, while the remaining, sympathectomy-resistant NPY release may be due to non-neuronal sources, such as platelets, immune or endothelial cells.20

Hindlimb ischemia is also associated with changes in NPY receptor expression. In the gastrocnemius muscle, which is located below the occlusion and represents the “capillary angiogenesis zone,” ischemia upregulates expression of Y2 receptors and DPPIV, which converts NPY to the Y2/Y5 agonist.20 In contrast, in the adductor muscle, above the occlusion, Y2 receptor mRNA levels are only slightly increased, whereas the expression of Y1 receptors is markedly upregulated.40 Such differential regulation of NPY receptor expression may reflect divergent functions of the peptide in the particular areas of the injured limb. In the muscles below the occlusion, ischemia upregulates NPY angiogenic system, Y2 and DPPIV, mediating growth-promoting effects of the peptide in endothelial cells and leading to capillary angiogenesis. On the other hand, in the areas above the occlusion, which correspond to the “collateralization zone,” Y1 receptors responsible for NPY mitogenic effect in VSMCs are upregulated, supporting the role of these receptors in arteriogenesis.

Despite activation of various angiogenic mechanisms, spontaneous revascularization does not fully restore blood flow in the ischemic muscle. This effect, however, can be achieved by treatment with exogenous NPY. The peptide, administered to the ischemic leg as a slow release pellet induces additional expression of Y5 receptors, which are the amplifiers of angiogenesis, and this leads to stimulation of capillary formation in the gastrocnemius muscle.20 Such amplification may be due to dimerization of the Y2 and Y5 receptors. In addition to its angiogenic effect, the exogenous NPY also stimulates formation of collateral vessels above the occlusion, which, along with its angiogenic effect, leads to the complete restoration of the blood flow and improvement of contractile functions of the ischemic muscle.20

Taken together, due to its growth-promoting effects on both endothelial and VSMCs NPY stimulates both capillary angiogenesis and