Sympathetic nerves have long been suspected of trophic activity, but the nature of their angiogenic factor has not been determined. Neuropeptide Y (NPY), a sympathetic cotransmitter, is the most abundant peptide in the heart and the brain. It is released during nerve activation and ischemia and causes vasoconstriction and smooth muscle cell proliferation. Here we report the first evidence that NPY is angiogenic. At low physiological concentrations, in vitro, it promotes vessel sprouting and adhesion, migration, proliferation, and capillary tube formation by human endothelial cells. In vivo, in a murine angiogenic assay, NPY is angiogenic and is as potent as a basic fibroblast growth factor. The NPY action is specific and is mediated by Y1 and Y2 receptors. The expression of both receptors is upregulated during cell growth; however, Y2 appears to be the main NPY angiogenic receptor. Its upregulation parallels the NPY-induced capillary tube formation on reconstituted basement membrane (Matrigel); the Y2 agonist mimics the tube-forming activity of NPY, whereas the Y2 antagonist blocks it. Endothelium contains not only NPY receptors but also peptide itself, its mRNA, and the “NPY-converting enzyme” dipeptidyl peptidase IV (both protein and mRNA), which terminates the Y1 activity of NPY and cleaves the Tyr 1-Pro 2 from NPY to form an angiogenic Y2 agonist, NPY 3–36. Endothelium is thus not only the site of action of NPY but also the origin of the autocrine NPY system, which, together with the sympathetic nerves, may be important in angiogenesis during tissue development and repair.
Angiogenesis, the process of new vessel formation or neovascularization, has aroused increasing interest over the last 25 years. Normal angiogenic activity is low in the adult organism but increases during injury and in diseases such as cancer, retinopathies, or arthritis, where it contributes to pathological changes. Conversely, in states of inadequate tissue perfusion such as myocardial or limb ischemia, enhanced angiogenesis is essential and beneficial. Inhibition or enhancement of angiogenesis may thus prove an attractive strategy for the treatment of several disorders. Although numerous growth factors stimulating vessel development are known (eg, bFGF or VEGF), the exact mechanisms controlling angiogenesis are poorly understood. The sympathetic nerves have long been known to have in vivo trophic effects on blood vessel growth. This process was believed to be mediated by catecholamines; at physiological plasma concentrations, however, catecholamines have weak growth-promoting effects on vascular cells. Moreover, the sympathetic nerves release not only norepinephrine but also other nonadrenergic neurotransmitters, such as NPY and purines.
NPY is present in all sympathetic nerves innervating the cardiovascular system and is the most abundant peptide in the brain and the heart. Additionally, in rats but not in humans, NPY is also found extraneuronally in platelets and endothelium. Originally, NPY was known as a potent vasoconstrictor and a neuromodulator. Released by stress, exercise, and myocardial ischemia, NPY has been implicated in coronary heart disease, congestive heart failure, and hypertension. More recently, because of the potent ability of NPY to stimulate food intake, it is suspected to play a role in obesity and diabetes. Our latest findings indicate that NPY is also a mitogen for rat aortic vascular smooth muscle cells. These diverse functions, together with its highly evolutionarily preserved peptide structure and its high degree of identity among mammalian species, suggest an important physiological role for NPY. The hypothesis tested here is that NPY functions as a trophic/angiogenic factor.
The NPY system encompasses several NPY-related peptides, receptors, and processing enzymes. NPY activates six receptors, designated as Y1 to Y6, of which Y1 and, more recently, Y2, Y4, and Y5 have been cloned. NPY-induced vasoconstriction is mediated primarily by Y1 (the predominant vascular receptor), with little or no contribution of the Y2 receptor, the functions of which in blood vessels are less clear. In addition to NPY, PYY, a gastrointestinal peptide with 75% homology to NPY, activates all NPY-Y receptors except Y3. The endogenous products of NPY and PYY metabolism, such as NPY/PYY 3–36, are selective Y2 agonists. Interestingly, NPY 3–36 is produced from NPY by an endothelial serine protease, DPPIV, which itself has been implicated in endothelial-matrix interactions in cancer.
In the present study we demonstrate for the first time that NPY is an angiogenic factor. We investigated the effect of NPY and related compounds in vitro, on capillary tube formation by HUVECs and on rat aortic ring capillary sprouting, as well as in vivo using the murine reconstituted basement membrane (Matrigel) assay. Additionally, we examined the receptor specificity of the angiogenic activity and the endothelial expression of NPY, its receptors, and the NPY-processing enzyme DPPIV.
HUVECs were isolated from freshly delivered umbilical cords after incubation at 37°C for 20 minutes with collagenase type I enzyme solution and plated on gelatin-coated T75 flasks. After the first passage, cells were grown on noncoated Nunc flasks. The HUVEC media consisted of medium 199 (GIBCO-BRL) supplemented with 20% FCS (HyClone), 1000 U/dL penicillin/streptomycin, 5 mg/dL gentamicin, 2 mmol/L glutamine, 500 U/dL sodium heparin, 2.5 mg/dL amphotericin B (Biofluids), and 2 mg/dL ECGS (Collaborative Research Inc). Aliquots of cells were preserved frozen between passages 2 and 4. Biological experiments were performed with cells between passages 3 and 7.
Subconfluent HUVECs preincubated with or without NPY (18 hours) were resuspended in the serum-free medium and plated (2×10 4 cells per well) on 96-well laminin-coated plates (5 mg per well; experiments were performed on two different cultures in triplicates). After 0 to 40 minutes, the adherent cells were fixed, stained (0.2% crystal violet/80% methanol), and quantified spectrophotometrically (A=560 nm). The method has been validated and extensively used in the laboratory of Dr H. Kleinman, as described in Reference 26 .
HUVECs in medium 199 were added to the upper wells (48-multiwell chemotaxis Boyden microchamber) at 3 to 4×10 4 cells per well; the lower wells contained NPY or analogues diluted in medium 199 (n=4 in triplicates). After 2 hours at 37°C in 5% CO 2, the membranes were fixed and stained, and the number of cells that migrated through to the lower surface of each membrane was counted. A negative control consisted of medium 199, and a positive control contained medium 199 supplemented with 20% FBS, ECGS, and heparin.
HUVECs plated onto 96-well dishes (10 4 cells per well) were growth-arrested in serum-free media supplemented with insulin, transferrin, and selenite for 24 hours and then treated for 24 hours with or without NPY or agonists in 10% FBS-DMEM (n=6); 0.5 μCi [3 H]thymidine per well was added for the last 6 hours. Cells were harvested in a 96-well harvester (Tomtec) and counted in a Betaplate liquid scintillation counter (model 1205; Wallac Inc).
Cells were incubated (18 hours, 37°C) on Matrigel-coated 24-well plates at 4×10 4 cells per well in the 10% FBS–containing medium with NPY, its analogues, or the vehicle (n=4 in duplicates). Cells were fixed and stained (DiffQuick Fixative and Solution II), and the area of the tube network was quantified at ×40 magnification with a Nikon microscope connected to an NIH image system.
Eight-week-old female C57BL mice were injected subcutaneously either with Matrigel alone or with Matrigel mixed with bFGF or NPY (n=4 in duplicates). After 14 days, Matrigel plugs were excised, fixed in 10% formaldehyde, and embedded in paraffin. Sections of the paraffin-embedded plugs were stained with Masson’s trichrome and photographed. Vessel ingrowth was quantified with the use of a Nikon microscope connected to an NIH image system. Results were expressed as mean area of tubes per square millimeter.
Rat aortic rings were prepared as previously described, with modifications. The thoracic and abdominal aorta was obtained from 100- to 150-g male Sprague-Dawley rats (Taconic, Germantown, NY). Excess perivascular tissue was removed, transverse sections (1 to 2 mm) were made, and the resulting aortic rings were then extensively washed in medium 199 (Mediatech Inc). The rings were then embedded in rat tail collagen 200 mg/dL in Nunc eight-well chamber slides (Nalgene Nunc International) so that the lumen was parallel to the base of the slide. After the collagen I gelled (by adjustment of pH to neutral with NaOH), serum-free medium (endothelial basal medium supplemented with antibiotics and ε-aminocaproic acid, 30 mg/dL) was added to each well, and the slides were incubated at 37°C for 3 days. Once sprouts began to appear, NPY was added at concentrations of 0.002 to 2.2 nmol/L (n=6 per dose). VEGF (1 nmol/L) was used as a positive control. The rings were incubated for 3 days, photographed, and fixed and stained for image analysis and quantification with the use of an NIH image system. The ring assay was repeated three times.
Subconfluent HUVECs were gently trypsinized (2 to 3 minutes of incubation with 0.025% versene-trypsin applied onto the cells and immediately removed), plated in their full growth medium on plastic- or Matrigel-coated T75 flasks, and allowed to adhere for 1 hour, at which point they were harvested for RNA (time zero); cells grown on plastic were used as a control for cells grown on Matrigel. Other cells were allowed to grow on plastic and on Matrigel for additional 1, 6, and 20 hours before being harvested for RNA for Northern blot analysis (n=3 to 4 per time point). Total RNA was purified from cells with the use of standard guanidinium-isothiocyanate and cesium chloride centrifugation. Ten micrograms of total RNA was electrophoresed through a 1% agarose denaturing gel and transferred to Nytran membranes.
