Oxidative stress-induced endothelial cells-derived exosomes accelerate skin flap survival through Lnc NEAT1-mediated promotion of endothelial progenitor cell function

Abstract

Background Flap transplantation is commonly used in reconstructive surgery. A prerequisite for skin flap survival is sufficient blood supply. However, such approaches remain unclear. This study aimed to explore the underlying mechanisms of exosomes derived from human umbilical vascular endothelial cells (HUVECs) exposed to oxidative stress on endothelial progenitor cells (EPCs) and their subsequent influence on the survival of skin flaps. Methods HUVECs were treated with various concentrations of H2O2 to establish an oxidative stress model. To investigate the effects of H2O2-HUVEC-Exos and HUVEC-Exos, Cell Counting Kit-8, tube formation, invasion assays, and quantitative real-time polymerase chain reaction (qRT-PCR) were performed in EPCs. Microarray analysis was used to reveal the differentially expressed long non-coding RNAs (lncRNAs) in the H2O2-HUVEC-Exos and HUVEC-Exos. In addition, gene silencing and western blotting were employed to determine the mechanism behind lncRNA nuclear enrichment enriched transcript 1 (Lnc NEAT1) in EPCs. Further, a rat skin flap model was used to determine the role of the exosomes in skin flap survival in vivo. Results HUVECs were stimulated with 100 μmol/L H2O2 for 12 h to establish an oxidative stress model. H2O2-HUVEC-Exos promoted the proliferation, tube formation, and invasion of EPCs and remarkably increased skin flap survival compared to the HUVEC-Exos and control groups. Sequencing of exosome RNAs revealed that the Lnc NEAT1 level was dramatically increased in the H2O2-HUVEC-Exos, leading to activation of the Wnt/β-catenin signaling pathway. Comparatively, knockdown of Lnc NEAT1 in HUVEC-Exos and H2O2-HUVEC-Exos significantly inhibits the angiogenic capacity of EPCs, reduced the survival area of skin flap and downregulated the expression levels of Wnt/β-catenin signaling pathway proteins, whereas Wnt agonist partly reversed the negative effect of NEAT1 downregulation on EPCs through the Wnt/β-catenin signaling pathway. Conclusions Exosomes derived from HUVECs stimulated by oxidative stress significantly promoted the pro-angiogenic ability of EPCs through the Wnt/β-catenin signaling pathway mediated by Lnc NEAT1 and hence enhanced random flap survival in vivo. Therefore, the application of H2O2-HUVEC-Exos may serve as an alternative therapy for improving random skin flap survival.

Exosomal Lnc NEAT1 from endothelial cells promote bone regeneration by regulating macrophage polarization via DDX3X/NLRP3 axis

“…Furthermore, the histological and IHC evaluation revealed significant higher levels of angiogenic and osteogenic markers, increased local infiltration of M2 polarized macrophages in the Exos group, leading to a better repair of rat cranial defect, which corresponded to their strongest ability in vitro. Our results are in parallel with the former conclusion that exosomal NEAT1 could promote angiogenesis [ 15 ]. Moreover, bone regeneration highly depends on angiogenesis, which is a vital step to restore blood flow providing nutrients, further exerting a positive feedback to bone healing [ 53 ].…”

“…Numerous studies in the past have found that exosomes can effectively participate in the immune response to facilitate tissue regeneration [37,38]. Exosomes from HUVECs were also reported as offering a positive effect in protecting nerve cells from ischemia/reperfusion injury, improving fibroblast photoaging, inducing endothelial progenitor cell homing and inhibiting osteoclast formation to reduce bone resorption [15,[39][40][41]. Particularly, we demonstrated that HUVEC-derived exosomes could directly promote osteogenic differentiation and increase the migratory capacity of BMSCs, and the aforementioned phenomenon was slightly inhibited with the knockdown of NEAT1 (Additional file 3: Fig.…”

“…Human umbilical vein endothelial cells (HUVECs) derived exosomes are effective for promoting diabetic wound healing, reducing reperfusion damage, and protecting nerve cells against ischemia/reperfusion injury [ 17 – 19 ]. Inspired by these studies, our previous study has confirmed that the exosomes derived from HUVECs significantly improved the angiogenesis ability of endothelial progenitor cells and increased the survival area of the flap in vivo through nuclear enrichment enriched transcript 1 (NEAT1)/Wnt/β-catenin signal pathway [ 15 ]. Such suggestions indicate that HUVECs-derived exosomes may serve as a novel therapeutic tool for tissue repair, such as bone regeneration.…”

Exosomal ncRNAs: The pivotal players in diabetic wound healing

“…Exosomes that are secreted by the above-mentioned cells also have similar functions to cells. Furthermore, lncRNA NEAT1 in exosomes shows overexpression stimulated by oxidative stress on EPC cells, which is the effective therapeutic ingredient of exosomes ( Figure 3C ) ( 51 ). Evidence suggested that ROS concentration in macrophages could be modulated by lncRNA Lethe and lncRNA NTF3-5.…”

“…(B) HOTAIR-MSC EVs improve wound healing in db/db mice via increasing angiogenesis [Adapted from Born et al, doi: 10.1002/adhm.202002070, ( 50 )]. (C) Schematic illustration of the function and mechanisms of HUVEC-Exos on EPCs [Adapted from Guo et al, doi: 10.1186/s13287-022-03013-9, ( 51 )]. *p < 0.05.…”