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Phosphodiesterase Type 5 Inhibition Enhances The Angiogenic Profile Of Adipose-derived Stem Cells
Marc Soares, MD, Piul S. Rabbani, PhD, Clarence Ojo, MD, April Duckworth, MD, Hersh Patel, BS, Lukas Ramcharran, BS, Camille Kim, BS, Amanda Hua, BS, Jessica Chang, BS, Karan Mehta, BS, Nakul Rao, MD, Pierre B. Saadeh, MD, Daniel J. Ceradini, MD.
New York University Department of Plastic Surgery, New York, NY, USA.
Purpose: Autologous fat grafting is a powerful clinical technique limited by unpredictable long-term retention of transplanted fat. We have previously reported that the use of phosphodiesterase type 5 inhibitors (PDE5i) markedly increases long term fat graft survival and improves predictability of the technique. Here, we investigate the underlying cellular and molecular mechanisms that impact adipose-derived stem cells (ASCs) in hypoxic engraftment conditions, and potentially contribute to graft durability. We hypothesize that targeted inhibition of PDE5 using FDA approved compounds will enhance ASC function in ischemic conditions, which may contribute to graft survival.
METHODS: We harvested ASCs from wild type adult mice and maintained them in vitro as previously described. We treated ASCs with PDE5i and incubated the cells in either normoxia or hypoxia, along with untreated controls. We monitored the cells over several timepoints spanning 14 days. At each time point, we assayed cell numbers using MTT assays. We also performed RT-PCR to examine changes in expression of the angiogenic marker, vascular endothelial growth factor (VEGF), and inducible nitric oxide synthase (iNOS). Using a well described preclinical model of autologous fat grafting, we analyzed ASC number and survival in transplanted grafts using flow cytometry (CD45- CD31- CD36+ CD105+).
RESULTS: ASCs demonstrated at least 20% increase in cell number and viability when cultured in hypoxic conditions compared to normoxia. Specifically, at 10 days in culture hypoxic ASCs demonstrate a 100% increase in cell viability. Treatment with PDE5i further emphasized this difference as early as 1 day after induction and resulted in at least 25% increase in hypoxic ASCs. VEGF expression in hypoxic ASC (1.78 x 10-3 ± 3.64 x 10-04 units) is significantly increased, as compared to normoxic ASCs (5.60x 10-04 ± 3.69 x 10-04 units, p<0.01). Similar to the pattern seen with cell numbers, PDE5i treatment increases VEGF expression 2x in hypoxic ASCs (p<0.01). iNOS expression follows a similar trend where hypoxia alone provides an advantage, and PDE5i-treated hypoxic ASCs express 3 times more iNOS than that in hypoxia alone (p<0.01). In vivo, PDE5i treated grafts demonstrated statistically significant increase in survival of donor derived CD45- CD31- CD36+ CD105+ ASCs, and a nearly two-fold increase in volume retention at three months.
CONCLUSIONS: Here we demonstrate that targeted PDE5 inhibition significantly increases ASC survival in transplanted grafts, while promoting a more angiogenic and vasoprotective phenotype. The strategy of targeted protection of both the donor vasculature and the function of ASCs in preserving this vasculature is a novel approach to improve reliability of autologous fat grafting. Use of FDA approved PDE5i is a rapidly translatable approach to increase predictability of clinical fat graft survival and modulate ASC function.
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