Exosomes from Endothelial Progenitor Cells Improve the Outcome of a Murine Model of Sepsis

Abstract

Microvascular dysfunction leads to multi-organ failure and mortality in sepsis. Our previous studies demonstrated that administration of exogenous endothelial progenitor cells (EPCs) confers protection in sepsis as evidenced by reduced vascular leakage, improved organ function, and increased survival. We hypothesize that EPCs protect the microvasculature through the exosomes-mediated transfer of microRNAs (miRNAs). Mice were rendered septic by cecal ligation and puncture (CLP), and EPC exosomes were administered intravenously at 4 hr after CLP. EPC exosomes treatment improved survival, suppressing lung and renal vascular leakage, and reducing liver and kidney dysfunction in septic mice. EPC exosomes attenuated sepsis-induced increases in plasma levels of cytokines and chemokine. Moreover, we determined miRNA contents of EPC exosomes with next-generation sequencing and found abundant miR-126-3p and 5p. We demonstrated that exosomal miR-126-5p and 3p suppressed LPS-induced high mobility group box 1 (HMGB1) and vascular cell adhesion molecule 1 (VCAM1) levels, respectively, in human microvascular endothelial cells (HMVECs). Inhibition of microRNA-126-5p and 3p through transfection with microRNA-126-5p and 3p inhibitors abrogated the beneficial effect of EPC exosomes. The inhibition of exosomal microRNA-126 failed to block LPS-induced increase in HMGB1 and VCAM1 protein levels in HMVECs and negated the protective effect of exosomes on sepsis survival. Thus, EPC exosomes prevent microvascular dysfunction and improve sepsis outcomes potentially through the delivery of miR-126.

Keywords: barrier function; endothelial progenitor cells; exosomes; microRNA; sepsis.

Figure 1

Size Distribution and Total Particle Number of EPC-Exosomes The number of particles versus particle size was generated by nanoparticle tracking analysis with ZetaView. The results are represented as the mean of three independent experiments.

Figure 2

Effect of EPC-Exosomes on CLP-Induced Mortality CD-1 mice were subjected to CLP and treated with EPC exosomes (2 mg protein/kg body weight), control NIH 3T3-exosomes (2 mg protein/kg body weight), or PBS. Survival rate was monitored for a total of 168 hr (7 days). *p < 0.05 compared with CLP-PBS group; ^#p < 0.05 compared with CLP-NIH 3T3-exosomes group. n = 15–23 mice per group.

Figure 3

Effect of EPC-Exosomes on Organ Dysfunction, Vascular Leakage, and Lung Edema in CLP-Induced Sepsis Plasma levels of AST (A), ALT (B), and BUN (C) were measured at 24 hr after CLP. n = 3–6 mice per group. Lung (D) and kidney (E) sections were stained with H&E and examined histologically. The representative sections are shown at ×400 original magnification, and scale bars are 20 μm. For lung histology, the yellow arrow indicates alveolar wall thickening, the green arrow indicates infiltrated inflammatory cells in the alveoli, and the blue arrow indicates enlarged interstitial space. For kidney histology, the yellow arrow indicates a shrunken glomerulus, the green arrow indicates tubular injury including brush border loss and tubular luminal debris or obstruction, and the blue arrow indicates capillary congestion. Lung (F) and renal (G) injury scores were assessed. n = 3–4 mice per group. Vascular leakage in lung (H) and kidney (I) were measured via injecting Evans blue dye at 24 hr after CLP. Lung water content was determined by wet (W)/dry (D) lung tissue weight ratio (J). *p < 0.05 compared with the sham group; ^#p < 0.05 compared with the CLP group. n = 3–6 mice per group. Results are represented as mean ± SE.

Figure 4

Effect of EPC Exosomes on Plasma Cytokine/Chemokine Levels in Septic Mice Mice were subjected to sham or CLP and injected with EPC exosomes (2 mg protein/kg body weight) or control PBS (same volume) at 4 hr after CLP surgery. Plasma cytokine IL-6 (A), IFNγ (B), TNF-α (C), IL-10 (D), and chemokine MCP-1 (E) levels were determined by mouse cytokine and chemokine array at 24 hr after CLP. *p < 0.05 compared with sham group; ^#p < 0.05 compared with CLP group. n = 3–4 mice per group. Results are represented as mean ± SE.

Figure 5

Highly Expressed MicroRNAs Differ between EPC-Exosomes and NIH 3T3 Exosomes MicroRNA content in EPC exosomes (A) and NIH 3T3 cell exosomes (B) was analyzed by next generation sequencing. Each microRNA expression level was determined by unique molecular index (UMI) from three independent experiments. The results are represented as the mean of three independent experiments.

Figure 6

Effect of EPC-Exosomes on miRNA-126-5p and 3p and HMGB1 Protein Levels in Lung Tissue in CLP-Induced Sepsis Lung miRNA-126-5p (A) and 3p (B) expression were determined by real-time qPCR per group. Protein levels of HMGB1 (C) in lung tissue were measured by western blot. α-Tubulin served as an internal control. *p < 0.05 compared with the sham group; ^#p < 0.05 compared with the CLP group. n = 3–6 mice. Results are represented as mean ± SE.

Figure 7

Effect of EPC Exosomal miR-126-3p and 5p on LPS-Induced HMVEC Target Expression and CLP-Induced Mortality Protein levels of VCAM1 (A) and HMGB1 (B) in HMVECs were measured by western blot. α-Tubulin served as an internal control. *p < 0.05 compared with control group; ^#p < 0.05 compared with LPS group; **p < 0.05 compared with LPS+ EPC exosomes group. The results represent the means ± SE of three independent experiments. CD-1 mice were subjected to CLP and treated with EPC exosomes, miR-126-reduced EPC exosomes (2 mg protein/kg body weight), or PBS. Survival rate was monitored for a total of 168 hr (7 days) (C). n = 15–16 mice per group.