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Synthetic chemically modified mRNAs (modRNA) encoding vascular endothelial growth factor (VEGF) represents an alternative to gene therapy for the treatment of ischemic cardiovascular injuries. However, novel delivery approaches of modRNA are needed to improve therapeutic efficacy in the diseased setting. We hypothesized that cell-mediated modRNA delivery may enhance the in vivo expression kinetics of VEGF protein thus promoting more potent angiogenic effects. Here, we employed skin fibroblasts as a "proof of concept" to probe the therapeutic potential of a cell-mediated mRNA delivery system in a murine model of critical limb ischemia (CLI). We show that fibroblasts pre-treated with VEGF modRNA have the potential to fully salvage ischemic limbs. Using detailed molecular analysis we reveal that a fibroblast-VEGF modRNA combinatorial treatment significantly reduced tissue necrosis and dramatically improved vascular densities in CLI-injured limbs when compared to control and vehicle groups. Furthermore, fibroblast-delivered VEGF modRNA treatment increased the presence of Pax7 + satellite cells, indicating a possible correlation between VEGF and satellite cell activity. Our study is the first to demonstrate that a cell-mediated modRNA therapy could be an alternative advanced strategy for cardiovascular diseases., (Copyright © 2019. Published by Elsevier B.V.)

Background: The proangiogenic capacity of adipose tissue and its derivatives has been demonstrated in a variety of studies. The paracrine mechanism of the cellular component is considered to play a critical role in the regenerative properties of these tissues. However, cell-based therapy for clinical application has been hindered by limitations such as safety, immunogenicity issues, and difficulties in cell preservation, transportation, and phenotype control. In the current study, we aimed to produce a cell-free extract directly from human fat tissue and evaluate its potential therapeutic efficacy., Methods: We developed a novel physical approach to produce a cell-free aqueous extract from human fat tissue (fat extract (FE)). The therapeutic potential of FE was investigated in the ischemic hindlimb model of nude mice. After establishment of hindlimb ischemia with ligation of the left femoral artery and intramuscular injection of FE, blood perfusion was monitored at days 0, 7, 14, 21, and 28. Tissue necrosis and capillary density were evaluated. Enzyme-linked immunosorbent assay was used to analyze the growth factors contained in FE. Moreover, the proliferation, migration, and tube formation ability were tested on human umbilical vein endothelial cells (HUVECs) in vitro when treated with FE. The proangiogenic ability of FE was further assessed in an in-vivo Matrigel plug assay., Results: FE was prepared and characterized. The intramuscular injection of FE into the ischemic hindlimb of mice attenuated severe limb loss and increased blood flow and capillary density of the ischemic tissue. Enzyme-linked immunosorbent assay showed that FE contained high levels of various growth factors. When added as a cell culture supplement, FE promoted HUVEC proliferation, migration, and tube formation ability in a dose-dependent manner. The subcutaneous injection of Matrigel infused with FE enhanced vascular formation., Conclusions: We developed a novel cell-free therapeutic agent, FE, produced from human adipose tissue. FE was able to attenuate ischemic injury and stimulate angiogenesis in ischemic tissues. This study indicates that FE may represent a novel cell-free therapeutic agent in the treatment of ischemic disorders.

Background: Adipose tissue and its derivatives, including adipose-derived stem cells, stromal vascular fraction (SVF), and SVF-gel, have been utilized in the treatment of many ischemic disorders. However, the utilization of these products is limited in clinical applications by concerns related to the presence of cells in these derivatives.Objectives: This study aimed to isolate a cell-free fat extract (FE) from fat tissue and to evaluate its proangiogenic ability in vitro as well as its protective effects on skin flap survival in vivo.Methods: FE was isolated from human fat via a mechanical approach. The concentrations of several growth factors in the FE were determined by enzyme-linked immunosorbent assay. The proangiogenic ability of FE was evaluated utilizing assays of the proliferation, migration, and tube formation in human umbilical vein endothelial cells in vitro. The protective effects of FE on the survival of random pattern skin flaps were investigated by subcutaneous injection into rats.Results: Enzyme-linked immunosorbent assay results revealed that FE contained proangiogenic growth factors that promoted proliferation, migration, and tube formation in human umbilical vein endothelial cells in vitro. In addition, FE reduced skin flap necrosis and increased survival, as demonstrated by macroscopic measurements and blood flow analysis. Histological analysis revealed that FE treatment increased the capillary density.Conclusions: FE is a cell-free, easy-to-prepare, and growth-factor-enriched liquid derived from human adipose tissue that possesses proangiogenic activity and improves skin flap survival by accelerating blood vessel formation. FE may be potentially used for treating ischemic disorders. [ABSTRACT FROM AUTHOR]