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Exosomal miR-223 Contributes to Mesenchymal Stem Cell-Elicited Cardioprotection in Polymicrobial Sepsis.

Wang X, Gu H, Qin D, Yang L, Huang W, Essandoh K, Wang Y, Caldwell CC, Peng T, Zingarelli B, Fan GC - Sci Rep (2015)

Bottom Line: However, WT-MSCs were able to provide protection which was associated with exosome release.Conversely, WT-MSC-derived-exosomes displayed protective effects.By contrast, WT-exosomes encased higher levels of miR-223, which could be delivered to cardiomyocytes, resulting in down-regulation of Sema3A and Stat3.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Cincinnati, OH, USA.

ABSTRACT
Mesenchymal stem cells (MSCs) have been shown to elicit cardio-protective effects in sepsis. However, the underlying mechanism remains obscure. While recent studies have indicated that miR-223 is highly enriched in MSC-derived exosomes, whether exosomal miR-223 contributes to MSC-mediated cardio-protection in sepsis is unknown. In this study, loss-of-function approach was utilized, and sepsis was induced by cecal ligation and puncture (CLP). We observed that injection of miR-223-KO MSCs at 1 h post-CLP did not confer protection against CLP-triggered cardiac dysfunction, apoptosis and inflammatory response. However, WT-MSCs were able to provide protection which was associated with exosome release. Next, treatment of CLP mice with exosomes released from miR-223-KO MSCs significantly exaggerated sepsis-induced injury. Conversely, WT-MSC-derived-exosomes displayed protective effects. Mechanistically, we identified that miR-223-KO exosomes contained higher levels of Sema3A and Stat3, two known targets of miR-223 (5p &3p), than WT-exosomes. Accordingly, these exosomal proteins were transferred to cardiomyocytes, leading to increased inflammation and cell death. By contrast, WT-exosomes encased higher levels of miR-223, which could be delivered to cardiomyocytes, resulting in down-regulation of Sema3A and Stat3. These data for the first time indicate that exosomal miR-223 plays an essential role for MSC-induced cardio-protection in sepsis.

No MeSH data available.


Related in: MedlinePlus

miR-223-KO exosomes can deliver Sema3A and Stat3, whereas WT-exosomes can deliver miR-223 to the myocardium in vivo.(A) A diagram shows that both strands of pre-miR-223 can be processed to be mature miRNA and target Sema3A and Stat3, respectively. (B,C) Sema3A and Stat3 both were up-regulated in miR-223-KO MSCs (n = 4, *p < 0.05 vs. WTs). (D,E) Sema3A and Stat3 both proteins were highly enriched in exosomes released from miR-223-KO MSCs (n = 4, *p < 0.05 vs. WTs). (F–H) Red dye PKH26-labeled WT- and KO-exosomes both were detected in the myocardium after i.v. injection. Cardiomyocytes were stained with Alexa-Fluor 488 labeled α–actinin antibody. (I,J) Higher levels of Sema3A and Stat3 encased in miR-223-KO exosomes were effectively transported to the heart, whereas WT-exosome-treated hearts displayed lower levels of Sema3A and Stat3, compared to PBS-injected control hearts, respectively (n = 4, *p < 0.05 vs. PBS-treated samples). α-actin was used as a loading control. AU: arbitrary unit. The gel had been run under the same experimental conditions. The full-length blots are shown in Supplementary Figure S3. (K) The levels of miR-223-5p and -3p were significantly increased in WT-exosome-treated myocardium, whereas they were not altered in KO-exosome-treated hearts. U6 snRNA was used as an internal control for qRT-PCR analysis (n = 4, *p < 0.05 vs. PBS controls).
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f7: miR-223-KO exosomes can deliver Sema3A and Stat3, whereas WT-exosomes can deliver miR-223 to the myocardium in vivo.(A) A diagram shows that both strands of pre-miR-223 can be processed to be mature miRNA and target Sema3A and Stat3, respectively. (B,C) Sema3A and Stat3 both were up-regulated in miR-223-KO MSCs (n = 4, *p < 0.05 vs. WTs). (D,E) Sema3A and Stat3 both proteins were highly enriched in exosomes released from miR-223-KO MSCs (n = 4, *p < 0.05 vs. WTs). (F–H) Red dye PKH26-labeled WT- and KO-exosomes both were detected in the myocardium after i.v. injection. Cardiomyocytes were stained with Alexa-Fluor 488 labeled α–actinin antibody. (I,J) Higher levels of Sema3A and Stat3 encased in miR-223-KO exosomes were effectively transported to the heart, whereas WT-exosome-treated hearts displayed lower levels of Sema3A and Stat3, compared to PBS-injected control hearts, respectively (n = 4, *p < 0.05 vs. PBS-treated samples). α-actin was used as a loading control. AU: arbitrary unit. The gel had been run under the same experimental conditions. The full-length blots are shown in Supplementary Figure S3. (K) The levels of miR-223-5p and -3p were significantly increased in WT-exosome-treated myocardium, whereas they were not altered in KO-exosome-treated hearts. U6 snRNA was used as an internal control for qRT-PCR analysis (n = 4, *p < 0.05 vs. PBS controls).

Mentions: We next tried to figure out the possible mechanisms underlying the beneficial effects of WT-exosomes and detrimental effects of miR-223-KO-exosomes in sepsis. It has been shown that: 1) miR-223 is highly enriched in not only bone marrow stem cells but also their released exosomes (Fig. 5D and Ref. 20); 2) miR-223-5p and -3p could negatively regulate the expression of Sema3A andStat3, respectively22 (Fig. 7A); and 3) Sema3A and Stat3 both contribute to inflammatory response29303132. We therefore speculated that the presence or absence of miR-223 in MSCs might affect cellular levels of Sema3A and Stat3, which were consequently incorporated into exosomes at different concentrations. To test this hypothesis, we firstly determined the expression levels of Sema3A and Stat3, two major inflammatory targets of miR-223, in MSCs collected from miR-223-KO mice and WT (miR-223+/+) mice. The results of Western-blotting revealed that the levels of Sema3A and Stat3 were increased in miR-223-KO MSCs by 1.4-fold and 1.9-fold, respectively, compared with WT-MSCs (Fig. 7B,C, p < 0.05). Of importance, we observed that exosomes released from miR-223-KO MSCs contained higher levels of Sema3A (2.1-fold) and Stat3 (2.8-fold) than WT-exosomes (Fig. 7D,E, p < 0.05). These data implicate that ablation of miR-223 in stem cells is able to reprogram the protein contents of exosomes.


Exosomal miR-223 Contributes to Mesenchymal Stem Cell-Elicited Cardioprotection in Polymicrobial Sepsis.

Wang X, Gu H, Qin D, Yang L, Huang W, Essandoh K, Wang Y, Caldwell CC, Peng T, Zingarelli B, Fan GC - Sci Rep (2015)

miR-223-KO exosomes can deliver Sema3A and Stat3, whereas WT-exosomes can deliver miR-223 to the myocardium in vivo.(A) A diagram shows that both strands of pre-miR-223 can be processed to be mature miRNA and target Sema3A and Stat3, respectively. (B,C) Sema3A and Stat3 both were up-regulated in miR-223-KO MSCs (n = 4, *p < 0.05 vs. WTs). (D,E) Sema3A and Stat3 both proteins were highly enriched in exosomes released from miR-223-KO MSCs (n = 4, *p < 0.05 vs. WTs). (F–H) Red dye PKH26-labeled WT- and KO-exosomes both were detected in the myocardium after i.v. injection. Cardiomyocytes were stained with Alexa-Fluor 488 labeled α–actinin antibody. (I,J) Higher levels of Sema3A and Stat3 encased in miR-223-KO exosomes were effectively transported to the heart, whereas WT-exosome-treated hearts displayed lower levels of Sema3A and Stat3, compared to PBS-injected control hearts, respectively (n = 4, *p < 0.05 vs. PBS-treated samples). α-actin was used as a loading control. AU: arbitrary unit. The gel had been run under the same experimental conditions. The full-length blots are shown in Supplementary Figure S3. (K) The levels of miR-223-5p and -3p were significantly increased in WT-exosome-treated myocardium, whereas they were not altered in KO-exosome-treated hearts. U6 snRNA was used as an internal control for qRT-PCR analysis (n = 4, *p < 0.05 vs. PBS controls).
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f7: miR-223-KO exosomes can deliver Sema3A and Stat3, whereas WT-exosomes can deliver miR-223 to the myocardium in vivo.(A) A diagram shows that both strands of pre-miR-223 can be processed to be mature miRNA and target Sema3A and Stat3, respectively. (B,C) Sema3A and Stat3 both were up-regulated in miR-223-KO MSCs (n = 4, *p < 0.05 vs. WTs). (D,E) Sema3A and Stat3 both proteins were highly enriched in exosomes released from miR-223-KO MSCs (n = 4, *p < 0.05 vs. WTs). (F–H) Red dye PKH26-labeled WT- and KO-exosomes both were detected in the myocardium after i.v. injection. Cardiomyocytes were stained with Alexa-Fluor 488 labeled α–actinin antibody. (I,J) Higher levels of Sema3A and Stat3 encased in miR-223-KO exosomes were effectively transported to the heart, whereas WT-exosome-treated hearts displayed lower levels of Sema3A and Stat3, compared to PBS-injected control hearts, respectively (n = 4, *p < 0.05 vs. PBS-treated samples). α-actin was used as a loading control. AU: arbitrary unit. The gel had been run under the same experimental conditions. The full-length blots are shown in Supplementary Figure S3. (K) The levels of miR-223-5p and -3p were significantly increased in WT-exosome-treated myocardium, whereas they were not altered in KO-exosome-treated hearts. U6 snRNA was used as an internal control for qRT-PCR analysis (n = 4, *p < 0.05 vs. PBS controls).
Mentions: We next tried to figure out the possible mechanisms underlying the beneficial effects of WT-exosomes and detrimental effects of miR-223-KO-exosomes in sepsis. It has been shown that: 1) miR-223 is highly enriched in not only bone marrow stem cells but also their released exosomes (Fig. 5D and Ref. 20); 2) miR-223-5p and -3p could negatively regulate the expression of Sema3A andStat3, respectively22 (Fig. 7A); and 3) Sema3A and Stat3 both contribute to inflammatory response29303132. We therefore speculated that the presence or absence of miR-223 in MSCs might affect cellular levels of Sema3A and Stat3, which were consequently incorporated into exosomes at different concentrations. To test this hypothesis, we firstly determined the expression levels of Sema3A and Stat3, two major inflammatory targets of miR-223, in MSCs collected from miR-223-KO mice and WT (miR-223+/+) mice. The results of Western-blotting revealed that the levels of Sema3A and Stat3 were increased in miR-223-KO MSCs by 1.4-fold and 1.9-fold, respectively, compared with WT-MSCs (Fig. 7B,C, p < 0.05). Of importance, we observed that exosomes released from miR-223-KO MSCs contained higher levels of Sema3A (2.1-fold) and Stat3 (2.8-fold) than WT-exosomes (Fig. 7D,E, p < 0.05). These data implicate that ablation of miR-223 in stem cells is able to reprogram the protein contents of exosomes.

Bottom Line: However, WT-MSCs were able to provide protection which was associated with exosome release.Conversely, WT-MSC-derived-exosomes displayed protective effects.By contrast, WT-exosomes encased higher levels of miR-223, which could be delivered to cardiomyocytes, resulting in down-regulation of Sema3A and Stat3.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Cincinnati, OH, USA.

ABSTRACT
Mesenchymal stem cells (MSCs) have been shown to elicit cardio-protective effects in sepsis. However, the underlying mechanism remains obscure. While recent studies have indicated that miR-223 is highly enriched in MSC-derived exosomes, whether exosomal miR-223 contributes to MSC-mediated cardio-protection in sepsis is unknown. In this study, loss-of-function approach was utilized, and sepsis was induced by cecal ligation and puncture (CLP). We observed that injection of miR-223-KO MSCs at 1 h post-CLP did not confer protection against CLP-triggered cardiac dysfunction, apoptosis and inflammatory response. However, WT-MSCs were able to provide protection which was associated with exosome release. Next, treatment of CLP mice with exosomes released from miR-223-KO MSCs significantly exaggerated sepsis-induced injury. Conversely, WT-MSC-derived-exosomes displayed protective effects. Mechanistically, we identified that miR-223-KO exosomes contained higher levels of Sema3A and Stat3, two known targets of miR-223 (5p &3p), than WT-exosomes. Accordingly, these exosomal proteins were transferred to cardiomyocytes, leading to increased inflammation and cell death. By contrast, WT-exosomes encased higher levels of miR-223, which could be delivered to cardiomyocytes, resulting in down-regulation of Sema3A and Stat3. These data for the first time indicate that exosomal miR-223 plays an essential role for MSC-induced cardio-protection in sepsis.

No MeSH data available.


Related in: MedlinePlus