Vascular endothelial growth factor mediates the therapeutic efficacy of mesenchymal stem cell-derived extracellular vesicles against neonatal hyperoxic lung injury

Characterization of MSC-derived EVsCell demise was not indicated by terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick-end labeling (TUNEL) assays in cultured cells previous to EV isolation (knowledge not proven). Scanning and transmission electron microscopy pictures (Fig. 1a, b, and Supplemental Fig. 1) indicated that remoted EVs had been spheroidal in form with a peak dimension distribution of 50–70 nm in diameter, as measured by nanoparticle monitoring evaluation (Fig. 1c), and the EVs had been optimistic for EV-specific CD63 and CD9 markers, as decided by western blotting (Fig. 1d). Furthermore, mitochondrial, nuclear, and Golgi equipment markers had been noticed in cells however not in EVs (Fig. 1d).Fig. 1: Affirmation of extracellular vesicles (EVs).a Scanning electron microscopy pictures of the EVs derived from mesenchymal stem cells (MSCs). b Transmission electron microscopy pictures of EVs derived from mesenchymal stem cells (MSCs). c Particle dimension distribution (number-weighted dimension distribution) of MSC-derived EVs. d Western blot assays of MSCs and MSC-derived EVs; cytochrome C (mitochondria marker), fibrillarin (nucleus marker), GM130 (Golgi equipment marker), CD63, and CD9 (exosome marker)VEGF knockdown and human and rat VEGF levelsVEGF protein ranges had been measured by enzyme-linked immunosorbent assay (ELISA) in EVs from VEGF siRNA-transfected MSCs or fibroblasts and had been considerably diminished in comparison with these within the non-transfected naive or scramble siRNA-transfected MSC-derived EVs (Fig. 2a).Fig. 2: Induction of VEGF expression in rat lung epithelial L2 cells by the VEGF from MSC-derived extracellular vesicles (EVs) rescues oxidative damage in vitro.a The VEGF ranges had been measured within the EVs derived from naive mesenchymal stem cells (MSCs), the EVs from scramble siRNA-transfected MSCs, the EVs from VEGF siRNA-transfected MSCs, and the EVs from fibroblasts. The EVs from VEGF siRNA-transfected MSCs or fibroblasts offered with considerably decreased VEGF ranges in comparison with the degrees in EVs from naive MSCs or scramble siRNA-transfected MSCs. Rat lung epithelial (L2) cells had been handled with H2O2 for 1 h to induce oxidative stress. L2 cells had been co-treated with naive MSCs, EVs from naive MSCs, EVs from scramble siRNA-transfected MSCs, EVs from VEGF siRNA-transfected MSCs, or EVs from fibroblasts. Within the tradition medium, the expression of human VEGF protein (b), human VEGF mRNA (c), rat VEGF protein (d), and rat VEGF mRNA (e) was measured. f The cell survival fee in every group was evaluated by Three-(four,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. The in vitro L2 cell tradition teams are as follows: a, normoxic management; b, H2O2 management; c, H2O2 + human MSCs; d, H2O2 + EVs from human MSCs; e, H2O2 + EVs from scramble siRNA-transfected human MSCs; f, H2O2 + EVs from VEGF siRNA-transfected human MSCs; and g, H2O2 + EVs from human fibroblast (MRC5). Knowledge are offered because the means ± SEM. *P < zero.05 in comparison with the normoxic management, †P < zero.05 in comparison with the H2O2 management, ‡P < zero.05 in comparison with H2O2 + human MSCs, §P < zero.05 in comparison with H2O2 + EVs from human MSCsHuman- and rat-specific VEGF protein and mRNA expression ranges had been measured by ELISA and PCR, respectively, in cultured rat L2 cells after H2O2 publicity with or with out human MSCs or MSC-derived EVs.No human-specific VEGF protein or mRNA was detected within the supernatant of the normoxic or H2O2-exposed rat L2 cells cultured alone or co-cultured with fibroblast-derived EVs (Fig. 2b, c). The human VEGF protein ranges within the H2O2-exposed rat L2 cell supernatant had been highest when co-cultured with MSCs, excessive when co-cultured with non-transfected or scramble siRNA-transfected MSC-derived EVs, and lowest when co-cultured with VEGF siRNA-transfected MSC-derived EVs, respectively. Human VEGF mRNA was detectable solely following co-culture with MSCs, with non-transfected MSC-derived EVs or with scramble siRNA-transfected MSC-derived EVs however not with VEGF siRNA-transfected MSC-derived EVs.The numerous lower within the rat VEGF protein and mRNA ranges within the H2O2-exposed management group in comparison with the normoxic management group was considerably improved by co-culture with MSCs, with non-transfected MSC-derived EVs, or with scramble siRNA-transfected MSC-derived EVs however not with VEGF siRNA-transfected MSC-derived or fibroblast-derived EVs (Fig. second, e).Safety of MSC-derived EVs in vitroIn the cytotoxicity assay of cultured rat L2 cells, the H2O2 publicity considerably diminished cell survival in comparison with that below normoxic circumstances (Fig. 2f). H2O2-induced cell demise was considerably improved by co-culture with MSCs, with non-transfected MSC-derived EVs or with scramble siRNA-transfected MSC-derived EVs however not with VEGF siRNA-transfected MSC-derived or fibroblast-derived EVs.To find out whether or not MSC-derived EVs present a dose-dependent sample of protecting results, the in vitro mannequin described above was handled with totally different quantities of MSC- derived EVs (1, 5, 10, 15, and 20 µg). The oxidative stress-induced improve in cell demise was diminished within the MSC- derived EV-treated teams in any respect doses, besides within the 1-µg MSC-derived EV group (Supplemental Fig. 2). In comparison with the 5-µg MSC-derived EV group, the 10-, 15-, and 20-µg MSC-derived EV teams offered with considerably improved protecting results, and there have been no variations among the many teams (Supplemental Fig. 2).Normally, MSCs are believed to launch each soluble VEGF and EV-associated VEGF. Right here we tried to evaluate the quantity of VEGF launched in EV-associated kind and tried to check the protecting efficiency in opposition to oxidative stress-induced cell demise between the soluble and EV-associated VEGF types. First, we assessed the ratio of EV-associated VEGF to the full quantity of VEGF launched from MSCs within the tradition media. The EV-associated VEGF kind comprised 46.eight% (Supplemental Fig. 3a) of the full VEGF current, and we due to this fact recommend that roughly half of the VEGF is launched within the EV-associated kind and the remaining half of the VEGF is launched within the soluble kind from MSCs. As well as, the VEGF quantity was measured within the MSC-derived EVs. Within the 10-µg MSC-derived EV group, the measured VEGF degree was 1.9 ng/ml. Within the cytotoxicity assay of cultured rat L2 cells uncovered to H2O2, the cells had been handled with 10 µg of MSC-derived EVs and 1.9 ng of VEGF. The extent of oxidative stress-induced cell demise was considerably improved with the MSC-derived EV and VEGF therapies (Supplemental Fig. 3b). Furthermore, in contrast with the recombinant VEGF-treated group, the MSC-derived EV-treated group offered with higher protecting results (Supplemental Fig. 3b).Safety of MSC-derived EVs in vivoIn the rat lung tissue, the imply linear index and alveolar quantity had been considerably elevated (Fig. Three), indicating impaired alveolarization, and the expression of von Willebrand issue (vWF) was considerably diminished (Fig. four), indicating impaired angiogenesis, within the hyperoxic group in comparison with the normoxic group. The hyperoxia-induced impaired alveolarization and angiogenesis had been considerably improved by the intratracheal transplantation of MSCs and non-transfected or scramble siRNA-transfected MSC-derived EVs however not by VEGF siRNA-transfected MSC-derived or fibroblast-derived EV transplantation.Fig. Three: Extracellular vesicles (EVs) launched from MSCs attenuated hyperoxia-induced impaired alveolarization within the lung tissue partly by transferring VEGF.a Consultant photomicrographs of hematoxylin and eosin staining within the lungs of P14 rats from every group (scale bar = 25 μm). The diploma of alveolarization was assessed by the (b) imply linear intercept and (c) imply alveolar quantity. The EV injection dose per rat was 20 µg. The experimental in vivo teams are as follows: a, normoxic management; b, hyperoxic management; c, hyperoxia + MSCs; d, hyperoxia + MSC-derived EVs; e, hyperoxia + scramble siRNA-transfected human MSC-derived EVs; f, in vivo hyperoxia + VEGF siRNA-transfected human MSCs EVs; and g, in vivo hyperoxia human fibroblast (MRC5)-derived EVs. Knowledge are offered because the means ± SEM. *P < zero.05 in comparison with the normoxic management, †P < zero.05 in comparison with the hyperoxic management, ‡P < zero.05 in comparison with hyperoxia + human MSCs, §P < zero.05 in comparison with hyperoxia + EVs derived from human MSCsFig. four: Extracellular vesicles (EVs) launched from MSCs improved the hyperoxia-induced impaired pulmonary vascularization partly by way of transferring VEGF. Pulmonary angiogenesis was decided by staining for vWF in histological sections of P14 rat lungs.a Consultant immunofluorescence photomicrographs of vWF staining within the lungs of P14 rats in every group. Nuclei had been labeled with four′,6-diamidino-2-phenylindole (DAPI, blue), and vWF was labeled with the fluorescent marker 5(6)-carboxyfluorescein diacetate N-succinimidyl ester (CFSE, pink) (×200, scale bar = 50 μm). b The imply mild depth of vWF immunofluorescence per excessive energy area (HPF) in every group. The EV injection dose was 20 µg per rat. The experimental in vivo teams are as follows: a, normoxic management; b, hyperoxic management; c, hyperoxia + MSCs; d, hyperoxia + MSC-derived EVs; e, hyperoxia + scramble siRNA-transfected human MSC-derived EVs; f, in vivo hyperoxia + VEGF siRNA-transfected human MSCs-derived EVs; and g, in vivo hyperoxia human fibroblast (MRC5)-derived EVs. Knowledge are offered because the means ± SEM. *P < zero.05 in comparison with the normoxic management, †P < zero.05 in comparison with the hyperoxic management, ‡P < zero.05 in comparison with hyperoxia + human MSCs, §P < zero.05 in comparison with hyperoxia + EVs derived from human MSCsThe hyperoxia-induced improve within the variety of TUNEL-positive cells within the rat lung tissue was considerably attenuated by the intratracheal transplantation of MSCs and non-transfected or scramble siRNA-transfected MSC-derived EVs however not by VEGF siRNA-transfected MSC-derived or fibroblast-derived EV transplantation (Fig. 5).Fig. 5: Extracellular vesicles (EVs) launched from MSCs improved hyperoxia-induced cell demise partly by way of VEGF.a Consultant optical microscopy photomicrographs of TUNEL-stained lung histological sections from P14 rats in every group. The nuclei are labeled with DAPI (blue), and the TUNEL-positive cells are labeled with FITC (inexperienced) (×200, scale bar = 50 μm). b The common variety of TUNEL-positive cells per high-power area (HPF) in every group. The EV injection dose was 20 µg per rat. The experimental in vivo teams are as follows: a, normoxic management; b, hyperoxic management; c, hyperoxia + MSCs; d, hyperoxia + MSC-derived EVs; e, hyperoxia + scramble siRNA-transfected human MSC-derived EVs; f, in vivo hyperoxia + VEGF siRNA-transfected human MSCs-derived EVs; and g, in vivo hyperoxia human fibroblast (MRC5)-derived EVs. Knowledge are offered because the means ± SEM. *P < zero.05 in comparison with the normoxic management, †P < zero.05 in comparison with the hyperoxic management, ‡P < zero.05 in comparison with hyperoxia + human MSCs, §P < zero.05 in comparison with hyperoxia + EVs derived from human MSCsEffects of MSC-derived EVs on the inflammatory response in vivoIn the rat lung tissue, the degrees of inflammatory cytokines, reminiscent of interleukin (IL)-1α, IL-1ß, IL-6, and tumor necrosis factor-α (Fig. 6a), in addition to ED-1-positive alveolar macrophages (Fig. 6b, c) had been considerably elevated within the hyperoxic group in comparison with the normoxic group. These hyperoxia-induced will increase within the inflammatory cytokine and ED-1-positive alveolar macrophage ranges had been considerably attenuated by the intratracheal transplantation of MSCs, non-transfected MSCs, or scramble siRNA-transfected MSC-derived EVs however not by VEGF siRNA-transfected MSC-derived or fibroblast-derived EVs.Fig. 6: Extracellular vesicles (EVs) launched from MSCs attenuated the hyperoxia-induced improve within the inflammatory response in lung tissue partly by way of transferring VEGF.a The expression of interleukin (IL)-1α, IL-1β, IL-6, and TNF-α in P14 rat lungs from every group was measured with ELISA. b Consultant immunofluorescence photomicrographs of ED-1 staining, which signifies lively macrophages within the lungs of the P14 rats from every group. ED-1 optimistic alveolar macrophages had been labeled with CFSE (pink), and nuclei had been labeled with DAPI (blue) (×200, scale bar = 50 μm). c The common variety of ED-1 optimistic cells per HPF in every group. The EV injection dosage was 20 µg per rat. The experimental in vivo teams are as follows: a, normoxic management; b, hyperoxic management; c, hyperoxia + MSCs; d, hyperoxia + MSC-derived EVs; e, hyperoxia + scramble siRNA-transfected human MSC-derived EVs; f, in vivo hyperoxia + VEGF siRNA-transfected human MSCs-derived EVs; and g, in vivo hyperoxia human fibroblast (MRC5)-derived EVs. Knowledge are offered because the means ± SEM. *P < zero.05 in comparison with the normoxic management, †P < zero.05 in comparison with the hyperoxic management, ‡P < zero.05 in comparison with hyperoxia + human MSCs, §P < zero.05 in comparison with hyperoxia + EVs derived from human MSCsLocalization of donor MSC-derived EVs in vivoTo decide the distribution of transplanted MSC-derived EVs within the lung tissue in vivo, the co-localization of PKH67 inexperienced fluorescence with numerous lung cell markers was examined by immunofluorescence staining at P6 (Fig. 7). PKH67 inexperienced fluorescence most regularly co-localized with NG-2-positive pericytes (22.7%) and was additionally recognized with Iba-1-positive (21.Three%) and ED-1 (21.1%)-positive alveolar macrophages, SP-C-positive kind II pneumocytes (19.5%), and α-smooth muscle actin-positive muscle cells (four.four%) however not with vWF-positive vascular endothelial cells.Fig. 7: Distribution of the intratracheally delivered extracellular vesicles (EVs) in line with the pulmonary tissue cell kind. MSC-derived EVs displayed a cell-type dependent distribution within the pulmonary tissue.a Consultant immunofluorescence photomicrographs of donor EVs and host pulmonary cell staining within the lungs of P14 rats from every group. The nuclei are labeled with DAPI (blue), and the kind 2 alveolar cells, complete macrophages, activated macrophages, vascular muscle cells, vascular endothelial cells, and vascular pericytes had been every immunohistochemically stained with pro-surfactant protein C (SP-C), Iba-1, ED1, α-smooth muscle actin, von Willebrand issue (vWF), and NG2 (pink), respectively. The extracellular vesicles (EVs) had been prestained with PKH67 dye (inexperienced). The three-dimensional pictures of the co-localized donor EVs and recipient cells had been obtained utilizing confocal z-stack pictures. b The variety of every kind of host pulmonary cell was counted and evaluated in line with EVs co-localization. The speed of donor EVs incorporation into every kind of host pulmonary cell is offered as the proportion of double-labeled pulmonary cells with EVs among the many non-EVs-merged pulmonary cells. EVs: Extracellular vesicles

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