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Rosiglitazone Decreases Trafficking Of Bone Marrow-derived Mesenchymal Cells And Increases Presence Of Mature Adipocytes At Sites Of Lymphatic Injury
Ashley Emily Siegel, MD, Samerender Nagam Hanumantharao, PhD, Sheida Shokohyar, PhD, Zachary Williams, BS, Pratik Koirala, PhD, Shailesh Agarwal, MD.
Brigham and Women's Hospital, Boston, MA, USA.
PURPOSE: Lymphedema is an inflammatory condition characterized by dermal thickening and fibroadipose tissue deposition caused by the disruption of lymphatic vessels. This process is mediated by infiltration of inflammatory cells such as T-cells, and mesenchymal cells marked by PDGFRα and PDGFRβ which contribute to fibrosis and neo-angiogenesis, respectively. We have previously shown that rosiglitazone, an adipogenic PPAR-γ agonist, reduces lymphedema based on skin thickness, edema and fibrosis; however, the mechanism for this effect remains unclear. Here, we examine how rosiglitazone modifies the trafficking of bone marrow resident cells to the site of lymphatic injury after 7- and 21- days.
METHODS: 6-8 week-old C57BL/6 male mice (n=5 per group/timepoint) underwent 4 days of busulfan treatment (10mg/kg) via i.p. injection for bone marrow ablation, followed by bone marrow transplantation from same strain male fluorescent (tdTomato) mice on day 5 (2 x 10
6 cells in 100uL PBS) via retro-orbital injection. Mice then underwent hindlimb lymphadenectomy 48 hours after transplant, followed by twice-weekly systemic treatment of rosiglitazone (10 mg/kg) or control (corn oil) via i.p. injection. Animals were euthanized at 7- and 21-days following surgery and reconstitution with donor marrow was verified via flow cytometry. Next, hindlimb skin was excised, sectioned and imaged with confocal microscopy using immunofluorescence to identify specific cell populations (PDGFRα, PDGFRβ, CD45, CD4, CD8, perilipin-1).
RESULTS: Flow cytometry verified >90% of bone marrow reconstitution in all mice. We confirmed that bone-marrow derived cells trafficked to the site of lymphedema in the hindlimb skin of mice based on the presence of tdTomato+ cells. Lymphedema tissue obtained from control-treated mice at 1 and 3 weeks showed infiltration of PDGFRα+, PDGFRβ+, CD45+, CD4+, and CD8+ cells throughout the dermis. Treatment with rosiglitazone observationally decreased all cell populations at 1 and 3 weeks, most prominently at 1 week (Fig A). There were more adipocytes (perilipin+) in rosiglitazone-treated tissue at 1 and 3 weeks compared to control (p = 0.03) (Fig B).
CONCLUSION: Our results reveal that bone-marrow derived (tdTomato+) inflammatory and mesenchymal cells are prominent in the development of lymphedema after lymphadenectomy. Rosiglitazone was observed to have an overall anti-inflammatory effect with decreased recruitment of bone marrow-derived hematopoietic cells to lymphedema tissue, as evidenced by a decreased CD45+, CD4+ and CD8+ cell populations at 1 and 3 weeks. Pro-fibrotic and angiogenic cells marked by PDGFRα and PDGFRβ were similarly reduced after treatment with rosiglitazone at both timepoints. Increased presence of adipocytes (perilipin+) after treatment with rosiglitazone, a potent adipogenic compound, suggests that adipogenesis in injured lymphatic tissue may play a positive role in lymphedema treatment. Further studies are required to examine how rosiglitazone affects cell fate and transcriptional profile of cells involved in lymphedema.
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