arteriogenesis. Moreover, as described above, the peptide also activates other angiogenic pathways, such as VEGF and FGF-2. Thus, by combining its direct effects on vascular cells and actions of other NPYinduced growth factors, the peptide initiates a complex, multi-step cascade of events leading to revascularization of the ischemic tissues and restoration of its functions.

6. NPY in Wound Healing

Wound repair is another example of angiogenesis which occurs throughout the adult life. It is a complicated process involving inflammation, changes in extracellular matrix, proliferation and migration of cells, as well as angiogenesis. After injury, formation of new blood vessels is necessary to restore oxygen and nutrient supply to the healing tissue.41 Thus, wound healing is associated with induction of various angiogenic pathways, including NPY.

It has been shown that after wounding of confluent human umbilical vein-derived endothelial cells, NPY stimulates their migration and wound closure. This effect is mimicked by Y2/Y5 agonist, NPY3−36, and accompanied by upregulation of DPPIV expression in migrating cells and its localization to the edge of wound.23 Additionally, DPPIV neutralizing antibody completely blocks endothelial cell proliferation induced by NPY1−36, whereas it has no effect on the same actions of NPY3−36.23 These findings once again support the crucial role of Y2 receptor and DPPIV in NPY-induced angiogenesis. The fact that DPPIV is necessary for NPY-mediated chemotaxis may be associated with high constitutive expression of Y1 receptors observed in endothelial cells.21 Since full length NPY can bind both Y1 and Y2 receptors, and the Y1 receptors are normally more abundant, the selectivity for the Y2 activation is provided by DPPIV-mediated cleavage to NPY3−36, which has no affinity for the Y1 and is selective for Y2/Y5 receptors Thus, DPPIV acts here as an “angiogenic switch.”

The role of NPY in physiological wound healing has been further confirmed in vivo. In mice, NPY accelerates the healing of excisional full skin wounds and this is associated with increased vascularization of the regenerating tissue. Consistently, wound repair is significantly

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delayed in Y−/− mice, which is accompanied by decreased vessel density of the regenerating skin.22 Exogenous NPY does not improve the healing process in Y2−/− mice, whereas other growth factors, such as FGF-2, maintain their wound healing abilities22 supporting the notion that NPY is upstream from them. Interactions with the immune cells are also possible but not supported by the initial data which indicate no differences in the number of immune cells in the wounds of Y2−/− and wild-type mice.22

7. NPY in Adipose Tissue Growth and Obesity

One tissue which undergoes a continued growth and remodeling during an adult life is the white adipose tissue (WAT). Since the seminal observation by Rupnick et al.,42 who reported that anti-angiogenic agents lead to reduction of adipose tissue mass and weight loss by blocking vessel development and inducing their apoptosis, the interplay of adipogenesis with angiogenesis has become a hot topic. WAT is also well innervated by autonomic nerves, primarily sympathetic, which, in addition to their vasoconstrictive properties, have long been known to influence lipolysis and cell proliferation.43,44 These effects were primarily ascribed to catecholamines and their β-adrenergic receptors on preadipocytes and adipocytes, whose activation leads to lipolysis and reduced WAT mass.43,44 Hence, sympathetic activity is considered the body’s major weight loss mechanism.

The role of NPY in this process was unknown until Turtzo et al.45,46 reported that this sympathetic co-transmitter actually opposes β- adrenergic activity in WAT and leads to lipogenesis. They showed that co-culture of 3T3-L1 preadipocytes with rat sympathetic neurons leads to upregulation of NPY in the neuronal cells, and released peptide almost completely prevents isoproterenol-induced lipolysis. Our recent studies extended these observations to NPY-mediated control of adipogenesis. In addition to direct stimulation of preadipocyte proliferation, NPY is potently angiogenic in normal and obese mice. As in other tissues where NPY stimulated angiogenesis, this effect is mediated primarily by Y2 receptors in WAT. Inhibition by either local administration of Y2 antagonist or by receptor knockout reduces adipose tissue mass

and vascularization in obese mice, and to a lesser degree, also in lean mice. This suggests that NPY is physiologically active during adipose tissue development and is an important nerve-derived regulator of tissue remodeling, in large part due to its angiogenic activity. While NPY has long been known for its pro-obesity actions, those actions were solely attributed to its centrally mediated stimulation of food intake and thermogenesis. Our study is the first to demonstrate that sympathetic nerves, via NPY, have powerful local effects on tissue growth, via angiogenesis and adipogenesis. Blockade of NPY-Y2 receptors directly in the adipose tissue presents a new avenue for treatment of obesity and site-specific reduction of adiposity.

8. NPY in Retinopathy

Angiogenesis is also a basis of several pathological conditions. One of them is retinopathy, a common and severe complication of diabetes mellitus and one of the leading causes of blindness. It is also a major complication of oxygen therapy of premature babies. In both forms, retinopathy occurs as a result of pathological neovascularization of the retina with abnormal leaky vessels, and is triggered by tissue ischemia.47 In retinopathy of prematurity, relative retinal hyperoxia caused by exposure of the preterm newborns to hyperbaric oxygen results in excessive vasoconstriction and subsequent decrease of retinal blood flow.25 On the other hand, in diabetic retinopathy damage of the vessels and ischemia are believed to be triggered by hyperglycemia.48,49 However, in both cases the initial vascular insult is of the ischemic nature and, consequently, leads to upregulation of angiogenic factors and induction of retinal neovascularization. Although, like in tumor angiogenesis, VEGF has been considered the main molecule implicated in this process, involvement of other factors has not been excluded.

NPY is also present in fetal and adult retinas, and its angiogenic activity implicated in the pathogenesis if retinopathy.50,51 In the mouse model of oxygen-induced retinopathy, both NPY and its Y2 receptors are significantly upregulated in the retinas of newborn mice subjected to hyperoxia In contrast, the retinal Y1 receptors, which are

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constitutively expressed, are unchanged in spite of increased oxygen level.25 Oxygen-induced retinopathy is also significantly reduced in Y2−/− mice, as well as in newborn rats treated systemically with Y2 receptor antisense (where scrambled or sense oligonucleotides are ineffective).24 The above data strongly suggest that NPY, acting via its Y2 receptors, is an important factor involved in pathological vascularization of the retina. Interestingly, in the mouse model of oxygeninduced retinopathy, NPY mRNA levels are elevated starting with the initial vasoconstrictive phase-induced oxygen exposure25 suggesting that vasoconstrictive activities of the peptide may also play a role, in spite of unchanged Y1 receptor expression. Thus, receptor-specific activities of NPY may contribute to retinopathy at both stages of its development.

The role of NPY in retinopathy is further supported by the clinical data. A functional polymorphism in NPY signal peptide, Leu7 Pro7, has been positively correlated with the occurrence of retinopathy in patients with type II diabetes, with 20% of patients who developed severe retinopathy having that mutation as compared to its frequency of 6–8% in the healthy population.24 This Leucine to Proline substitution appears to result in increased release of NPY in response to stress, and has been already linked to impaired metabolism and elevated levels of blood lipids, decreased levels of insulin and accelerated atherosclerosis.24,52−55 The exact mechanism by which this polymorphism may contribute to diabetic retinopathy has not been elucidated. However, the enhanced angiogenic activity of NPY associated with its increased secretion may be at least partially responsible for this phenomenon. Interestingly, no correlation between NPY polymorphism and retinopathy has been found in patients with type I diabetes.24 Thus, although in both types of diabetes retinopathy is believed to be triggered by hyperglycemia, the mechanisms underlying this disorder and, consequently, role of NPY in its development, are different.

Taken together, NPY angiogenic activity mediated by its Y2 receptors plays an important role in development of certain types of retinopathy. Hence, blocking NPY-induced angiogenesis may be an effective therapy in treatment of this disease.