Platelet Lysate-Derived Neuropeptide y Influences Migration and Angiogenesis of Human Adipose Tissue-Derived Stromal Cells

Abstract

Neuropeptide Y (NPY), a powerful neurotransmitter of the central nervous system, is a key regulator of angiogenesis and biology of adipose depots. Intriguingly, its peripheral vascular and angiogenic powerful activity is strictly associated to platelets, which are source of clinical hemoderivates, such as platelet lysate (PL), routinely employed in several clinical applications as wound healing, and to preserve ex vivo the progenitor properties of the adipose stromal cells pool. So far, the presence of NPY in PL and its biological effects on the adipose stromal cell fraction (ASCs) have never been investigated. Here, we aimed to identify endogenous sources of NPY such as PL-based preparations and to investigate which biological properties PL-derived NPY is able to exert on ASCs. The results show that PL contains a high amount of NPY, which is in part also excreted by ASCs when stimulated with PL. The protein levels of the three main NPY subtype receptors (Y1, Y2, Y5) are unaltered by stimulation of ASCs with PL, but their inhibition through selective pharmacological antagonists, considerably enhances migration, and a parallel reduction of angiogenic features of ASCs including decrease in VEGF mRNA and intracellular calcium levels, both downstream targets of NPY. The expression of VEGF and NPY is enhanced within the sites of neovascularisation of difficult wounds in patients after treatment with leuco-platelet concentrates. Our data highlight the presence of NPY in PL preparations and its peripheral effects on adipose progenitors.

Figure 1

(a–c) ELISA assay for the detection of soluble human NPY levels in PL-based preparations and expression of Y1, Y2 and Y5 receptors on ASC cultures. (a) The graph shows the amount of NPY contained in undiluted platelet lysate preparations as well as in 10% PL. DMEM has been used as negative control. N = 5). PL, platelet lysate. ^#p < 0.0001. (b) Western blot analysis shows that the stimulation with 10% PL for 48 hours does not alter the protein levels of each of the three main receptors for NPY. GAPDH was used as loading control. N = 3. Cropped images are from the same samples split on three different gel runs (Y1, Y2, Y5). Full-length blots are shown in Supplementary Fig. 1d. (c) Immunofluorescence for Y1, Y2 and Y5 on ASCs cultures confirms the expression of the three main receptors for NPY. Magnification 20×. PL, platelet lysate. DAPI (blue, nuclei); Y1, Y2, Y5 receptors (green).

Figure 2

(a,b) NPY amount from ASCs and ASC-conditioned media (a) ELISA assay for soluble human NPY. The levels of excreted NPY in ASC-conditioned media after 48 hours of stimulation with 10% PL are significantly higher compared to starvation. PL, platelet lysate; starv, starvation. *p < 0.05. N = 5. (b) Western blot analysis for NPY in ASCs. After 48 hours of stimulation with 10% PL, ASC cultures exhibit enhanced protein levels of NPY. Tubulin is the loading control. Cropped images are from samples ran on the same gel. Full-length blots are shown in Supplementary Fig. 2c. N = 4. PL, platelet lysate; starv, starvation. *p < 0.05.

Figure 3

(a–c) ASCs clonogenic activity, adipogenic differentiation and chemotactic activity. (a) Bar graph shows that the clonogenic capacity of ASC cultures is not influenced by the stimulation with 10% PL and the selective blocking for 48 hours of NPY receptors antagonists. %CFE (Colony forming efficiency) has been expressed as N. secondary colonies/N. plated cells x 100. N = 5. (b) Similarly, the rate of adipogenic differentiation of ASCs remains unaltered. The rate (%) of adipogenic differentiation has been calculated as: (N. adipogenic cells/N. Total cells) x 100. N = 5. (c) The chemotactic ability of ASCs is significantly enhanced by selective blocking of Y1, Y2 and Y5 receptors after stimulation with 10% PL. *p < 0.05, **p < 0.01, ^#p < 0.0001. N = 4. Representative images of migrated ASCs stained with Giemsa after stimulation with DMEM, 10% PL alone or in presence of NPY receptor antagonists. Magnification 10×. PL, platelet lysate. NPYRAnts, NPY receptor antagonists.

Figure 4

(a–c) Angiogenesis assay, VEGF expression and detection of nitric oxide soluble NO levels (a) Bar graph shows the decrease in vitro number of loops and network length found in Matrigel assays of ASCs cultured in 10% PL combined with or without NPY receptor antagonists, **p < 0.01. N = 5–6. Representative optical images of in vitro angiogenesis of ASCs. Magnification 4×. (b) Bar graph shows mRNA levels of VEGF detected by qPCR after stimulation with 10% PL and parallel blocking of NPY receptor in ASC cultures, compared to unstimulated control. N = 3. *p < 0.05. (c) Bar graph shows no significant alterations in the release of NO after blocking of NPY receptors with selective antagonists. However, 10% PL is able to significantly enhance the levels of NO respect to both unstimulated controls (DMEM) and recombinant NPY (10⁻⁹ M). N = 6. **p < 0.01. (rec NPY, recombinant NPY; PL, platelet lysate; NPYRAnts, NPY receptor antagonists.

Figure 5

(a,b) Flow cytometry Analysis of ASC cultures stained with Fluo4AM and Western Blot Analysis of ERK signalling. (a) Boxplot displays the mean green fluorescence intensity of ASCs stimulated for 48 hours with 10% PL in presence of the selective antagonists for NPY receptors is significantly decreased compared to the treatment with 10% PL. The stimulation with recombinant NPY and Ionomycin have been used as biological and positive control, respectively. N = 3 biological replicates. *p < 0.05 (two-way ANOVA and Fisher LSD post-test). MFI, mean fluorescent intensity; PL, Platelet lysate; NPYRAnts, NPY receptor antagonists; rec NPY, recombinant NPY. (b) Bar graph shows that no difference in ERK phosphorylation is found in ASCs cultures after 48 hour of treatment with 10% PL combined to NPY receptor antagonists compared to the 10% PL. Nevertheless, the stimulation with recombinant NPY (10⁻⁹ M) is able to induce a significant increase of phospho ERK with respect to both treatments. N = 6. ^#p < 0.001. Densitometry is presented as the ratio (pERK 1/2/Total ERK) normalized to control (10% PL). Cropped images are from samples ran on the same gel. Full-length blots are shown in Supplementary Fig. 5c. PL, platelet lysate; NPYRAnts, NPY receptor antagonists.

Figure 6

(a–c) Representative light microscopic appearance and staining for NPY, VEGF and CD31 of difficult wound biopsies from patients treated with leuco-platelet concentrates. (a) Bar graph showing the quantification of the expression of NPY and VEGF, which is significantly and concurrently enhanced after 48 hours of treatment. *p < 0.05. Results are expressed as the following ratio = No. Positive foci of angiogenesis/No. total foci of angiogenesis and normalized vs time 0. N = 3. (b) Staining for VEGF and NPY in patients difficult wound microscopic preparations. NPY is mainly distributed at the sites of angiogenesis including areas where it colocalizes with CD31 (c) a marker of mature endothelial cells. A similar localization of VEGF was also observed. Cell nuclei are counterstained with haematoxylin/eosin. In (c) CD31 stains blue and NPY red. Magnification 10X and 40X in b and c, respectively. The insert shows a higher magnification (20X and 60X in b and c, respectively).