Small extracellular vesicles derived from miRNA-486 overexpressed dental pulp stem cells mitigate high altitude pulmonary edema through PTEN/PI3K/AKT/eNOS pathway

Abstract

High altitude pulmonary edema (HAPE) is a life-threatening, non-cardiogenic pulmonary edema characterized by rapid onset and high mortality. Extracellular vesicles of mesenchymal stem cells are used in the treatment of a variety of lung diseases, but their use in HAPE remains underreported. This study explores the therapeutic potential of miRNA-486 modified extracellular vesicles from dental pulp stem cells (sEV^miR-486) against HAPE, aiming to decipher the associated molecular mechanisms. The rat HAPE model was established by exposing subjects to a simulated high-altitude, low-oxygen environment within a specialized chamber. The HAPE-afflicted rats received sEV^Null and sEV^miR-486 intravenously, and the therapeutic effect was assessed through histopathological analysis, pulmonary artery pressure, lung water content, as well as markers of oxidative stress and inflammation. To supplement in vivo findings, pulmonary microvascular endothelial cells (PMVEC) were stressed with cobalt chloride to emulate hypoxic damage, and then treated with sEV^Null and sEV^miR-486 to unravel the mechanism of action. The sEV^Null mitigated pathological changes in the lungs, reduced pulmonary artery pressure and lung water content, and alleviated oxidative stress and inflammatory responses in cases of HAPE. Moreover, sEV^Null enhanced vascular reactivity and restored pulmonary permeability and tight junction integrity, these effects were intensified by miRNA-486 overexpression. Notably, sEV^miR-486 attenuated oxidative damage in hypoxic PMVEC cells by modulating the PTEN/PI3K/Akt/eNOS signaling pathway. miRNA-486 fortified DPSC-sEVs intervention as a novel and potent treatment strategy for HAPE.

Keywords: Extracellular vesicles; High-altitude pulmonary edema; Oxidative stress; miR-486.

Fig. 1

Isolation and identification of sEVs. (A) Depicts nanoparticle tracking analysis employed to ascertain the particle size distribution of sEVs. (B) Shows the application of transmission electron microscopy in visualizing the structure and size of sEVs with a scale bar denoting 100 nm. (C) Illustrates the utilization of Western blotting to detect the hallmark proteins of sEVs. (D) Demonstrates the uptake of sEVs by PMVEC, as captured by a confocal fluorescence microscope with a scale bar of 10 μm. (E) Presents in vivo imaging techniques to monitor the sEVs penetration into mouse lung tissue.

Fig. 2

sEV^miR−486 repair the damaged lung tissue in HAPE rat model (A) Expression of miR-486 in DPSCs following infection with Ad.Null or Ad.miR-486. (B) A volcano plot illustrating differentially expressed small RNA between sEV^Null and sEV^miR−486 treatments. (C) Cardiac ultrasound images accompanied by (D) a statistical histogram quantifying the results. (E) Hematoxylin and eosin (HE) stained sections of lung tissue; alveolar walls are marked by red arrows, smooth muscle by black arrows, and alveolar septa by yellow arrows. (F) Measurement of water content in rat lung tissue. (G) Quantification of protein levels in lung tissue homogenates and bronchoalveolar lavage fluid. Images are annotated with a scale bar representing 50 μm. Data represent means ± SD. Statistical significance is indicated as follows: ^#p < 0.05, ^###p < 0.001, versus the control group; ∗∗p < 0.01, ∗∗∗p < 0.001, versus the HAPE group; ^@@p < 0.01, versus the sEV^Null group.

Fig. 3

sEV^miR−486 mitigates lung injury by modulating oxidative stress and inflammation (A) Presents the concentrations of MDA and the activity of SOD in lung tissue homogenates. (B) Details the gene expression of SOD2 and CAT in PMVEC, as measured by quantitative PCR. (C)The protein expression levels of Nrf2, keap1, and HO-1 in PMVEC were revealed through Western blot analysis. (D) ELISA was used to detect the expression levels of IL-1β and IL-6 in the lung tissue homogenates. (E) Depicts the serum concentrations of a spectrum of cytokines—IL-1β, IL-6, IL-12, IL-17A, TNF-α, IL-4, and IL-10 quantified via ELISA. Data are expressed as means ± SD. Statistical significance is denoted by ^###p < 0.001 compared with the control group; ∗∗p < 0.01, ∗∗∗p < 0.001 compared with the HAPE group or the hypoxia group; ^@@p < 0.01, ^@@@p < 0.001, compared with sEV^Null group.

Fig. 4

sEV^miR−486 ameliorates lung injury by restoring intercellular tight junctions and permeability (A) Exhibits representative immunofluorescence images of Occludin protein levels in rat lung tissue. (B) Assesses the expression of tight junction proteins ZO-1 and Occludin in PMVEC using Western blot analysis. (C) Employs ELISA to measure plasma concentrations of TXA2, ET-1, NO, and PGI2 in rats. Moreover. (D) Quantifies mRNA levels of permeability-associated genes AQP-1 and AQP-5 in rat lung tissues via quantitative PCR. (E) Reports the corresponding mRNA levels in PMVEC. (F) Presents the protein expression levels of TRPV4, AQP-1, and AQP-5 in PMVEC. Data are expressed as means ± SD. Statistical significance is denoted as follows: ^###p < 0.001 versus the control group; ∗∗p < 0.01, ∗∗∗p < 0.001 versus the HAPE or hypoxia groups; ^@p < 0.05, ^@@p < 0.01, ^@@@p < 0.001 versus the sEV^Null group.

Fig. 5

miR-486 reduces oxidative stress by regulating the PTEN/PI3K/Akt/eNOS signaling pathway (A) A KEGG analysis identifies differentially expressed genes between sEV^Null and sEV^miR−486. (B) The mRNA levels of PTEN and Akt1 in PMVEC cells were quantified by qPCR. (C) NOS3 mRNA expression in PMVEC cells was assessed using qPCR. (D) The protein level of eNOS in PMVEC cells was evaluated by Western blot. Statistically significant differences are denoted by the following: ^#p < 0.05 and ^##p < 0.01 against the control group; ∗∗p < 0.01 and ∗∗∗p < 0.001 versus the hypoxia group; ^@@p < 0.01 and ^@@@p < 0.001 in comparison to the sEV^Null group. (E) qPCR was employed to measure the expression of miR-486 in PMVEC cells following transfection with either a miR-486 mimic or inhibitor. (F) Expressions of PTEN, Akt1, and PDK1 genes in PMVEC cells post-transfection were quantified by qPCR. (G) A phosphorylated protein Western blot analysis determined the levels of Akt1, p-Akt1, PI3K, and eNOS in PMVEC cells transfected with miR-486 mimic or inhibitor. (H) Flow cytometry was utilized to examine the ROS production in PMVEC cells following transfection. ^###p < 0.001, versus the Mimic-NC group; ∗∗∗p < 0.001, versus the Inhibitor-NC group. (I)Flow cytometry was used to detect the ROS production in PMVEC cells after Ly294002 was used to inhibit the PI3K signaling. ∗p < 0.05, versus the Mimic-NC group. ^#p < 0.05, versus the Mimic group. Data are expressed as means ± SD.

Fig. 6

Schematic diagram showing the effects of small extracellular vesicles derived from miRNA-486 overexpressed dental pulp stem cells on HAPE.