Extracellular vesicle-mimetic nanovesicles transport LncRNA-H19 as competing endogenous RNA for the treatment of diabetic wounds

Abstract

Diabetic wounds, one of the most enervating complications of diabetes mellitus, affect millions of people worldwide annually. Vascular insufficiency, caused by hyperglycemia, is one of the primary causes and categories of diabetic impaired wound healing. Recently, long noncoding RNA (LncRNA)-H19, which is significantly decreased in diabetes and may be crucial in triggering angiogenesis, has attracted increasing interest. The possible relationship between the decrease of LncRNA-H19 and the impairment of angiogenesis in diabetes could involve impairment of the insulin-phosphatidylinositol 3-kinase (PI3K)-Akt pathway via the interdiction of LncRNA-H19. Thus, a therapeutic strategy utilizing LncRNA-H19 delivery is feasible. In this study, we investigated the possibility of using high-yield extracellular vesicle-mimetic nanovesicles (EMNVs) as an effective nano-drug delivery system for LncRNA, and studied the function of EMNVs with a high content of LncRNA-H19 (^H19EMNVs). The results, which were exciting, showed that ^H19EMNVs had a strong ability to neutralize the regeneration-inhibiting effect of hyperglycemia, and could remarkably accelerate the healing processes of chronic wounds. Our results suggest that bioengineered EMNVs can serve as a powerful instrument to effectively deliver LncRNA and will be an extremely promising multifunctional drug delivery system in the immediate future.

Keywords: Long noncoding RNA; angiogenesis; competing endogenous RNA; diabetes; diabetic wound; nanovesicles.

Figure 1.

Preparation and characterization of EMNVs. (a) PAGE analysis immediately after RT-PCR in HEK-293 cells transfected with empty vector or H19-OE. (b) The size distribution of EMNVs directly tracked using a DLS system. (c) Representative TEM images of EMNVs; scale bar, 100 nm. (d) Western blot analysis of CD9, CD63 and CD81. (e) PAGE analysis immediately after RT-PCR (initial total RNA content of each sample set as 100 ng) in ²⁹³EMNVs and ^H19EMNVs treated with RNase (2 mg/mL) alone or combined with Triton X-100 (0.1%) for 15 min.

Figure 2.

^H19EMNVs as transporters of LncRNA-H19. (a) Uptake of DiL-labeled ^H19EMNVs (^H19EMNVs-DiL) by HMEC-1. Scale bar, 50 μm. (b) PAGE analysis immediately after RT-PCR in HMEC-1 cultured in normal medium or in HG medium with or without ^H19EMNVs or ²⁹³EMNVs, along with the results of real-time qPCR. *p < .05 compared with control. (c) Proliferation of HMEC-1 analyzed by flow cytometry (FCM) using an EdU kit after culture in normal medium or HG medium with or without ^H19EMNVs or ²⁹³EMNVs. (d) Proliferation of HMEC-1 detected using a CCK-8 kit on day 0, 1, 3 and 5 in normal medium or HG medium with or without ^H19EMNVs or ²⁹³EMNVs. *p < .05 compared with control.

Figure 3.

^H19EMNVs rescued the hyperglycemia-induced impairment of angiogenesis by sustaining the vitality of Akt activation through regulation of the LncRNA-H19. (a) PAGE analysis immediately after RT-PCR in HMEC-1 transfected with NC or H19-SS, along with the results of real-time qPCR. *p < .05 compared with NC. (b) Western blot analysis of the phosphorylation level of Akt. (c) Representative photomicrographs of transwell assays; scale bar, 50 μm, and representative photomicrographs of tubule formation; scale bar, 50 μm. (d) Statistical results of (c) in migrated cells, percentage of HMEC-1 tube numbers relative to control and percentage of HMEC-1 branch points relative to control. *p < .05 compared with control. (e) Western blot analysis of the phosphorylation level of Akt.

Figure 4.

^H19EMNVs promoted healing of diabetic wounds. (a) Representative images of full-thickness skin defects in a diabetic rat model, left untreated (control) or treated with SAH, SAH-²⁹³EMNVs, SAH-^H19EMNVs or SAH-^H19EMNVs together with RI (BMS-754807), at 0, 3, 7 and 14 days after operation. Scale bar: 10 mm. (b) Percentage wound closure of untreated defects and defects treated with SAH, SAH-²⁹³EMNVs, SAH-^H19EMNVs or SAH-^H19EMNVs together with RI (BMS-754807) at 3, 7 and 14 days after surgery. *p < .05 compared with control. #p < .05 compared between SAH-^H19EMNVs and SAH-²⁹³EMNVs. (c) Transmitted light images of HE-stained sections of the untreated defects (control) and the defects treated with SAH, SAH-²⁹³EMNVs, SAH-^H19EMNVs or SAH-^H19EMNVs together with RI (BMS-754807) at 14 days after operation (scale bar = 2 mm). The total width of the image represents the initial defect size (1.8 cm) while the white arrows indicate the length of full thickness wound healing. (d) Total length of full thickness wound healing (renascent skin) in the skin defects left untreated (control), treated with SAH, SAH-²⁹³EMNVs, SAH-^H19EMNVs or SAH-^H19EMNVs together with RI (BMS-754807) at 14 days after operation. *p < .05 compared with control. #p < .05 compared between SAH-^H19EMNVs and SAH-²⁹³EMNVs. (e) Immunofluorescence images of p-Akt counterstained with DAPI; scale bar, 100 μm. (f) Rate of positive cells (%) in (e). *p < .05 compared with control. #p < .05 compared between SAH-^H19EMNVs and SAH-²⁹³EMNVs.

Figure 5.

The deposition and remodeling of collagen, and the regeneration of epithelium. (a) Transmitted light images of Masson's trichrome-stained sections of the untreated defects (control) and the defects treated with SAH, SAH-²⁹³EMNVs, SAH-^H19EMNVs or SAH-^H19EMNVs together with RI (BMS-754807) at 7 and 14 days after operation, showing collagen deposition. Scale bar, 100 μm (100×), 25 μm (400×). (b) Immunofluorescence images of cytokeratin 14 (K14) counterstained with DAPI; scale bar, 2 mm.

Figure 6.

^H19EMNVs promoted angiogenesis in diabetic wounds. (a) Micro-CT evaluation of blood supply in full-thickness skin defects left untreated (control) or treated with SAH, SAH-²⁹³EMNVs, SAH-^H19EMNVs or SAH-^H19EMNVs together with RI (BMS-754807) at 14 days after surgery; scale bar, 2 mm. (b) Morphometric analysis of the new blood vessel area and the number of blood vessels. *p < .05 compared with control. (c) IF staining of CD31 and α-SMA. Endothelial cells (CD31), smooth muscle cells (α-SMA) and cell nuclei are stained. CD31 and ?-SMA co-staining indicates mature blood vessels. Scale bar: 100 μm. (d) Number of regenerated mature blood vessels in the untreated defects (control) and the defects treated with SAH, SAH-²⁹³EMNVs, SAH-^H19EMNVs or SAH-^H19EMNVs together with RI (BMS-754807) at 7 and 14 days after operation. *p < .05 compared with control.