Intraperitoneal injection of MSC-derived exosomes prevent experimental bronchopulmonary dysplasia

Abstract

Mesenchymal stromal cell (MSC) derived exosomes mediate tissue protection and regeneration in many injuries and diseases by modulating cell protein production, protecting from apoptosis, inhibiting inflammation, and increasing angiogenesis. In the present study, daily intraperitoneal injection of MSC-derived exosomes protected alveolarization and angiogenesis in a newborn rat model of bronchopulmonary dysplasia (BPD) induced by 14 days of neonatal hyperoxia exposure (85% O₂). Exosome treatment during hyperoxia prevented disruption of alveolar growth, increased small blood vessel number, and inhibited right heart hypertrophy at P14, P21, and P56. In vitro, exosomes significantly increased tube-like network formation by HUVEC, in part through a VEGF mediated mechanism. In summary, daily intraperitoneal injection of exosomes increased blood vessel number and size in the lung through pro-angiogenic mechanisms. MSC-derived exosomes therefore have both anti-inflammatory and pro-angiogenic mechanism to protect the lung from hyperoxia induced lung and heart disease associated with BPD.

Keywords: Bronchopulmonary dysplasia; Chronic lung disease; Exosomes; Lung prematurity; Mesenchymal stromal cells.

Figure 1.. Exosome Characterization.

Exosomes concentration and size were analyzed by the Nanosight particle tracking (A) and size and shape were analyzed by Electron Transmission Microscopy (B). C) Western Blot analysis for exosomal markers: CD9, CD63 and CD81 in the isolated exosomes. D) Schematic representation of exosome-treatment. Exosomes (Exo); mean linear intercept (MLI); Fulton index (FI).

Figure 2.. MSC-derived Exosomes alleviates hyperoxia induced lung destruction.

A) Lungs were collected, processed and sectioned as described in Materials and Methods. Representative micrographs of H&E stained lung sections are shown. Normoxia exposed saline treated (Norm/Sal); hyperoxia exposed saline treated (Hyp/Sal); hyperoxia exposed exosome treated (Hyp/Exo). Solid bar represents 100μm. B&C) Morphometric analysis of free distance in air spaces in the lung, mean linear intercept (MLI) was performed as described in Materials and Methods. Exosome injections prevented loss of alveolarization at days P14, P21, and P56 comparing three treatment groups: Normoxia exposed saline treated (Norm/Sal; n=4), hyperoxia exposed saline treated (Hyp/Sal; n=4; at P21 n=3) and hyperoxia exposed exosomes-treated (Hyp/Exo; n=4). Two-way ANOVA was used to analyze the treatment and time interaction (B). Effect of exosome depleted medium (Edm) at day P21 comparing Hyp/Sal, Hyp/Exo, and Hyp/Edm. One-way ANOVA was used to analyze the effect of Edm (C). * indicates a significant difference between the indicated data sets (p<0.05).

Figure 3.. A&B) MSC-derived Exosomes improve pulmonary vasculature in hyperoxia-injured lung.

Lung sections were analyzed for small blood vessel number and density by immunohistochemical analysis as described in materials and methods. The presence of small blood vessels in the lung parenchyma was visualized by DAB staining. Antibodies for Von Willebrand Factor (vWF) at day P14 and CD31 at days P21 and P56 were used. solid bar represents 20 μm. Also see supplemental figure 2 (A). The number of blood vessels of 20 – 100μm diameter per field, from at least 12 microscopic fields/group, were counted for each time point. Normoxia exposed saline treated (Norm/Sal), hyperoxia exposed saline treated (Hyp/Sal), and hyperoxia exposed exosomes-treated (Hyp/Exo) (B). *indicates a significant difference between the indicated data sets (p<0.05). (p<0.05). Multiple T-test was used to analyze the data. C&D) MSC-derived Exosomes prevented hyperoxia induced right ventricle hypertrophy (RV). Foulton index (FI) was calculated as described in Materials and Methods. Normoxia exposed saline treated (Norm/Sal); hyperoxia exposed saline treated (Hyp/Sal); and hyperoxia exposed exosomes-treated (Hyp/Exo. Two-way ANOVA was used to analyze the treatment and time interaction (C). Effect of exosomes depleted medium (Edm) on RV hypertrophy at day P21 comparing Norm/Sal, Hyp/Sal, Hyp/Exo, and Hyp/Edm. One-way ANOVA was used to analyze the effect of Edm (D). *indicates a significant difference between the indicated data sets (p<0.05).

Figure 4.. HUVEC tube formation induced by MSC-derived exosomes.

Tube formation assay was performed as described in Materials and Methods. A) Tube-like structures induced significantly higher in exosomes treated cells compared to controls. B) The degree of angiogenic induction was quantified for the numerical value of the product of the relative capillary length per microscopic field. Average tube length was determined from 5 different fields and shown as arbitrary units (A.U.). Exosomes (Exo), exosomes + isotype antibody (Exo+isotype) and exosomes + anti-VEGF antibody (Exo+anti-VEGF) * indicates a significant difference between the indicated data sets (p≤0.05). T-test was used to compare the data. C) Equal amounts of proteins from exosomes and exosome-depleted medium (Supernatant) were analyzed by Western blot analysis for VEGF content. CD63 used as a control.