HOTAIR‐Loaded Mesenchymal Stem/Stromal Cell Extracellular Vesicles Enhance Angiogenesis and Wound Healing

Abstract

Chronic wounds remain a substantial source of morbidity worldwide. An emergent approach that may be well-suited to induce the complex, multicellular processes such as angiogenesis that are required for wound repair is the use of extracellular vesicles (EVs). EVs contain a wide variety of proteins and nucleic acids that enable multifactorial signaling. Here, the capability of EVs is leveraged to be engineered via producer cell modification to investigate the therapeutic potential of EVs from mesenchymal stem/stromal cells (MSCs) transfected to overexpress long non-coding RNA HOX transcript antisense RNA (HOTAIR). HOTAIR is previously shown by the authors' group to be critical in mediating angiogenic effects of endothelial cell EVs, and MSCs are chosen as EV producer cells for this study due to their widely reported intrinsic angiogenic properties. The results indicate that MSCs overexpressing HOTAIR (HOTAIR-MSCs) produce EVs with increased HOTAIR content that promote angiogenesis and wound healing in diabetic (db/db) mice. Further, endothelial cells exposed to HOTAIR-MSC EVs exhibit increased HOTAIR content correlated with upregulation of the angiogenic protein vascular endothelial growth factor. Thus, this study supports EV-mediated HOTAIR delivery as a strategy for further exploration toward healing of chronic wounds.

The Impact of Storage Condition and Duration on Function of Native and Cargo-Loaded Mesenchymal Stromal Cell Extracellular Vesicles

“…As lyophilization adequately preserved the pro-angiogenic and anti-inflammatory capabilities of BDMSC EVs in vitro , we assessed whether these effects translated to a clinically relevant animal model. Previously, we demonstrated that BDMSC EVs loaded with the lncRNA HOTAIR enhanced wound closure after inducing an 8 mm punch biopsy on the dorsum of db/db mice, whereas unaltered BDMSC EVs did not [26]. Thus, we lyophilized HOTAIR-loaded BDMSC EVs and stored them at RT for 4 weeks, before reconstitution and usage in the same db/db mouse model.…”

“…BDMSCs were grown in EV-depleted media for 2 days before the conditioned medium was collected and subjected to differential ultracentrifugation with a 100,000 x g final centrifugation step as previously described [26]. Pelleted EVs were resuspended in 1x PBS and subsequently washed using Nanosep 300 kDa MWCO spin columns (Pall Laboratories; OD300C35).…”

“…P4 BDMSCs were seeded at 500,000 cells/flask (T175) before transfection as previously described with a pCMV-HOTAIR plasmid [26]. Specifically, pCMV-HOTAIR was transfected into BDMSCs via Lipofectamine 3000 (ThermoFisher Scientific, L30000015) for one hour, before washing and growth in EV-depleted media.…”

“…8 mice were used for each treatment group. Tracing of wounds and wound closure were determined as previously described [26]. All animal experiments were performed in compliance with the National Institutes of Health guide for care and use of laboratory animals.…”

The Role of MSCs and Cell Fusion in Tissue Regeneration

“…Therefore, miRNA, siRNA or lncRNA can be loaded as cargo into the EVs. For example, EVs isolated from MSCs, which overexpressed lncRNA HOX transcript antisense RNA (HOTAIR), promoted angiogenesis and wound healing in diabetic mice [44]. In a study by Liu et al, it could be shown that the release of apoptotic bodies (a subgroup of EVs) by transplanted MSCs, led to the shift of macrophages towards an anti-inflammatory phenotype and promoted the healing of cutaneous wounds [45].…”