Abstract 5370: Neuropeptide-Y (NPY)/NPY Y5R Ligand/Receptor Interaction Can Restore Aging Impaired Growth Potential Of Bone Marrow Stromal Cells
Background: Physiological aging impairs the proliferative potential of bone marrow derived stromal cells (BMSCs). We report here the pro-proliferative response and improved growth characteristics of the aging BMSCs subsequent to NPY/NPY Y5R ligand/receptor interaction.
Methods and Results: BMSCs were isolated from neonatal (2–3 weeks), young (8–12 weeks) and old (24–28 months) rats on the basis of their preferential adherence to plastic surface. After culturing the cells at initial seeding density of 1x104 cells/cm2, we estimated the doubling time (Dt) at 3–6 days from their respective growth curves which showed that OldBMSCs grew slower (302.6± 35.3 hours) than NeoBMSCs (135.5± 9.9 hours) and YngBMSCs (195.1± 26.3 hours). Saturation density (cells/cm2) declined with age (NeoBMSCs 5.5± 0.07x104, YngBMSCs 3.5± 0.1x104, OldBMSCs 2± 0.1x104). Real-time PCR and western blot showed that BMSCs in all different age groups constitutively expressed NPY and NPY receptor subtypes (Y1R, Y2R and Y5R). However, NPY and Y5R expression increased by more than 130 fold and decreased by 28 fold respectively in OldBMSCs as compared with YngBMSCs. NPY (10 nM) stimulated the proliferation of all BMSCs age groups and their proliferation was blocked Y5R antagonist. However, the pro-proliferative effect of NPY on OldBMSCs was weaker than other cell groups due to lower Y5R expression. Y5R gene transfection of OldBMSCs with subsequent NPY3–36 (10 nM) treatment significantly increased OldBMSCs proliferation (>56%) as compared with GFP transfected control OldBMSCs. On the other hand, NPY also inhibited forskolin-stimulated cAMP formation. Basal level of cAMP were higher (32.1± 2.3 pmol/mg protein) in OldBMSCs as compared with NeoBMSCs (p<0.007) however having insignificant difference with YngBMSCs (p>0.05), forskolin-stimulated cAMP production increased in OldBMSCs (8.5 fold) as compared with NeoBMSCs (3 fold) and YngBMSCs (6.8 fold). This effect of forskolin was inhibited by prior treatment with 10 nM NPY.
Conclusion: NPY treatment subsequent to Y5R overexpression in OldBMSCs can rejuvenate their growth characteristics. Moreover, physiological aging of BMSCs is associated with cAMP signal pathway.