Abstract: In the past decade, cell therapy has provided researchers with new inspirations in the treatment of secondary lymphedema. Adipose derived stem cell (ADSC) therapy and their derivatives are emerging as a promising treatment. This study aims at exploring the therapeutic effects of ADSC extracellular vesicles (EV) and its potential as an assistive therapy for vascularized lymph node transfer (VLNT) on a mouse lymphedema model. The mouse lymphedema model was established with three separate procedures, radiation 1 week before and 2 days after surgery (a dose of 20 Gy in two fractions), and surgery of resecting popliteal and inguinal lymph nodes. Mice are divided into 4 groups: therapeutic groups (PBS/EV injection on the 7th day postoperatively) and assistive therapy groups (VLNT immediately following the surgery and PBS/EV injection on the 7th day postoperatively). Circumferential measurement of hindlimb, weekly micro-CT imaging for volumetric measurement and the limb excessive volume % is used. The recovery of lymph flow is monitored monthly using near-infrared fluoroscopy. At 1 month after injection, histological staining, immunohistochemical staining, western blotting and qPCR analysis were conducted to observe any lymph vessel regeneration, local inflammation or fibrosis. Both EV group and EV+VLNT group indicated significant decrease in limb circumference and limb excessive volume % compared to PBS group and PBS+VLNT group respectively. ICG fluorescence results showed earlier lymph flow recovery in EV-treated groups. The numbers of lymphatic vessels by LYVE-1 immunohistochemistry, and VEGF-C expressing cells were significantly increased in EV group compared to PBS group (p < 0.05). The IHC results of type I collagen and pro-inflammatory factors indicated that the inflammatory level and fibrosis were significantly lowered by EV treatment. Lymphangiogenesis gene expression was upregulated in EV group compared to PBS group. The prescription of ADSC-EV alleviates the symptoms of lymphedema and increases the number of lymphatic vessels. It also helps accelerate the recovery of lymph flow after VLNT. This study offers a novel, promising approach in lymphedema management.
