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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

Characterizations of exosomes derived from MSCs and their functional roles in macrophages and cardiomyocytes upon LPS challenge.(A,B) The size of exosomes derived from (A) WT-MSCs and (B) miR-223 KO-MSCs, measured using a Zetasizer Nano ZS instrument. (C) Protein levels of CD63 and CD81 were similarly encased in WT-exosomes and KO-exosomes. Figure represents truncated western blot images for simplicity. Whole membrane images are shown in Supplementary Figure S2. (D) Both strands of miR-223 were included in WT-exosomes and  in KO-exosomes, 100 bp-DNA lander was used as a gel loading marker. MiR-320 was used as an internal control for RT-PCR. n = 3 independent experiments for A–D. (E–G) Addition of WT-exosomes (20 μg/ml) to cultured RAW264.7 cells significantly inhibited LPS-triggered secretion of TNF-α (E), IL-1β (F), and IL-6 (G). Remarkably, KO-exosomes (20 μg/ml) promoted RAW264.7 cell secretion of TNF-α (E), IL-1β (F), and IL-6 (G) upon LPS challenge (100 ng/ml). n = 3 wells for each group; *p < 0.05, vs. medium controls; #p < 0.05, vs. medium controls. Similar results were observed in other two additional, independent experiments. (H–J) LPS-induced cardiomyocyte death/apoptosis was significantly mitigated by treatment with WT-exosomes (20 μg/ml) and remarkably promoted by addition of KO-exosomes (20 μg/ml). Representative images of cardiomyocytes in the absent and present of LPS plus WT-exosomes or KO-exosomes were shown in (H). Survival rate was determined by MTS incorporation (I), and cardiomyocyte apoptosis (DNA fragmentation) was determined using an ELISA kit (J). n = 3 wells for each group; *p < 0.05, vs. medium controls; #p < 0.05, vs. medium controls. Similar results were observed in other three additional, independent experiments.
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f5: Characterizations of exosomes derived from MSCs and their functional roles in macrophages and cardiomyocytes upon LPS challenge.(A,B) The size of exosomes derived from (A) WT-MSCs and (B) miR-223 KO-MSCs, measured using a Zetasizer Nano ZS instrument. (C) Protein levels of CD63 and CD81 were similarly encased in WT-exosomes and KO-exosomes. Figure represents truncated western blot images for simplicity. Whole membrane images are shown in Supplementary Figure S2. (D) Both strands of miR-223 were included in WT-exosomes and in KO-exosomes, 100 bp-DNA lander was used as a gel loading marker. MiR-320 was used as an internal control for RT-PCR. n = 3 independent experiments for A–D. (E–G) Addition of WT-exosomes (20 μg/ml) to cultured RAW264.7 cells significantly inhibited LPS-triggered secretion of TNF-α (E), IL-1β (F), and IL-6 (G). Remarkably, KO-exosomes (20 μg/ml) promoted RAW264.7 cell secretion of TNF-α (E), IL-1β (F), and IL-6 (G) upon LPS challenge (100 ng/ml). n = 3 wells for each group; *p < 0.05, vs. medium controls; #p < 0.05, vs. medium controls. Similar results were observed in other two additional, independent experiments. (H–J) LPS-induced cardiomyocyte death/apoptosis was significantly mitigated by treatment with WT-exosomes (20 μg/ml) and remarkably promoted by addition of KO-exosomes (20 μg/ml). Representative images of cardiomyocytes in the absent and present of LPS plus WT-exosomes or KO-exosomes were shown in (H). Survival rate was determined by MTS incorporation (I), and cardiomyocyte apoptosis (DNA fragmentation) was determined using an ELISA kit (J). n = 3 wells for each group; *p < 0.05, vs. medium controls; #p < 0.05, vs. medium controls. Similar results were observed in other three additional, independent experiments.

Mentions: To determine whether MSC-derived exosomes have the capacity of anti-inflammation and promoting cell survival, we purified exosomes from the culture supernatants of MSCs by serial differential centrifugation plus ultracentrifugation27. Using a particle size analyzer, we observed that the size of exosomes released from WT-MSCs (WT-Exo) ranged between 10–100 nm with an average of 34.68 nm, which was similar to that of miR-223-KO MSC-derived exosomes (KO-Exo: 35.52 nm) (Fig. 5A,B). In addition, both WT-Exo and KO-Exo contained similar levels of CD63 and CD81 (Fig. 5C), two widely recognized molecular markers for exosomes10111213. Using RT-PCR assays, we also confirmed that both strands of miR-223 were encased in WT-exosomes, which were absent in KO-exosomes (Fig. 5D). Notably, we utilized sucrose gradient ultracentrifugation to further purify exosomes from culture supernatants of MSCs, as described previously28, and also confirmed that miR-223 was included in exosomes derived WT-MSCs rather than KO-MSCs (Supplemental Fig. S1). To determine the effects of these MSC-derived exosomes on macrophage inflammatory response, we added either WT-exosomes or KO-exosomes to cultured RAW264.7 cells. One hour later, LPS (100 ng/ml) was added to these cultures for 12 h, and then supernatants were collected for cytokine measurements. The results of ELISA analysis revealed that addition of WT-exosomes significantly inhibited the secretion of TNF-α, IL-1β, and IL-6 from macrophages upon LPS stimulation, evidenced by lower levels of TNF-α, IL-1β, and IL-6 in culture supernatants of WT-exosome-treated macrophages than those of medium-treated controls (Fig. 5E–G). By contrast, LPS-stimulated production of TNF-α, IL-1β, and IL-6 was remarkably increased in miR-223-KO exosome-treated macrophages, compared with medium-treated samples (Fig. 5E–G).


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)

Characterizations of exosomes derived from MSCs and their functional roles in macrophages and cardiomyocytes upon LPS challenge.(A,B) The size of exosomes derived from (A) WT-MSCs and (B) miR-223 KO-MSCs, measured using a Zetasizer Nano ZS instrument. (C) Protein levels of CD63 and CD81 were similarly encased in WT-exosomes and KO-exosomes. Figure represents truncated western blot images for simplicity. Whole membrane images are shown in Supplementary Figure S2. (D) Both strands of miR-223 were included in WT-exosomes and  in KO-exosomes, 100 bp-DNA lander was used as a gel loading marker. MiR-320 was used as an internal control for RT-PCR. n = 3 independent experiments for A–D. (E–G) Addition of WT-exosomes (20 μg/ml) to cultured RAW264.7 cells significantly inhibited LPS-triggered secretion of TNF-α (E), IL-1β (F), and IL-6 (G). Remarkably, KO-exosomes (20 μg/ml) promoted RAW264.7 cell secretion of TNF-α (E), IL-1β (F), and IL-6 (G) upon LPS challenge (100 ng/ml). n = 3 wells for each group; *p < 0.05, vs. medium controls; #p < 0.05, vs. medium controls. Similar results were observed in other two additional, independent experiments. (H–J) LPS-induced cardiomyocyte death/apoptosis was significantly mitigated by treatment with WT-exosomes (20 μg/ml) and remarkably promoted by addition of KO-exosomes (20 μg/ml). Representative images of cardiomyocytes in the absent and present of LPS plus WT-exosomes or KO-exosomes were shown in (H). Survival rate was determined by MTS incorporation (I), and cardiomyocyte apoptosis (DNA fragmentation) was determined using an ELISA kit (J). n = 3 wells for each group; *p < 0.05, vs. medium controls; #p < 0.05, vs. medium controls. Similar results were observed in other three additional, independent experiments.
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f5: Characterizations of exosomes derived from MSCs and their functional roles in macrophages and cardiomyocytes upon LPS challenge.(A,B) The size of exosomes derived from (A) WT-MSCs and (B) miR-223 KO-MSCs, measured using a Zetasizer Nano ZS instrument. (C) Protein levels of CD63 and CD81 were similarly encased in WT-exosomes and KO-exosomes. Figure represents truncated western blot images for simplicity. Whole membrane images are shown in Supplementary Figure S2. (D) Both strands of miR-223 were included in WT-exosomes and in KO-exosomes, 100 bp-DNA lander was used as a gel loading marker. MiR-320 was used as an internal control for RT-PCR. n = 3 independent experiments for A–D. (E–G) Addition of WT-exosomes (20 μg/ml) to cultured RAW264.7 cells significantly inhibited LPS-triggered secretion of TNF-α (E), IL-1β (F), and IL-6 (G). Remarkably, KO-exosomes (20 μg/ml) promoted RAW264.7 cell secretion of TNF-α (E), IL-1β (F), and IL-6 (G) upon LPS challenge (100 ng/ml). n = 3 wells for each group; *p < 0.05, vs. medium controls; #p < 0.05, vs. medium controls. Similar results were observed in other two additional, independent experiments. (H–J) LPS-induced cardiomyocyte death/apoptosis was significantly mitigated by treatment with WT-exosomes (20 μg/ml) and remarkably promoted by addition of KO-exosomes (20 μg/ml). Representative images of cardiomyocytes in the absent and present of LPS plus WT-exosomes or KO-exosomes were shown in (H). Survival rate was determined by MTS incorporation (I), and cardiomyocyte apoptosis (DNA fragmentation) was determined using an ELISA kit (J). n = 3 wells for each group; *p < 0.05, vs. medium controls; #p < 0.05, vs. medium controls. Similar results were observed in other three additional, independent experiments.
Mentions: To determine whether MSC-derived exosomes have the capacity of anti-inflammation and promoting cell survival, we purified exosomes from the culture supernatants of MSCs by serial differential centrifugation plus ultracentrifugation27. Using a particle size analyzer, we observed that the size of exosomes released from WT-MSCs (WT-Exo) ranged between 10–100 nm with an average of 34.68 nm, which was similar to that of miR-223-KO MSC-derived exosomes (KO-Exo: 35.52 nm) (Fig. 5A,B). In addition, both WT-Exo and KO-Exo contained similar levels of CD63 and CD81 (Fig. 5C), two widely recognized molecular markers for exosomes10111213. Using RT-PCR assays, we also confirmed that both strands of miR-223 were encased in WT-exosomes, which were absent in KO-exosomes (Fig. 5D). Notably, we utilized sucrose gradient ultracentrifugation to further purify exosomes from culture supernatants of MSCs, as described previously28, and also confirmed that miR-223 was included in exosomes derived WT-MSCs rather than KO-MSCs (Supplemental Fig. S1). To determine the effects of these MSC-derived exosomes on macrophage inflammatory response, we added either WT-exosomes or KO-exosomes to cultured RAW264.7 cells. One hour later, LPS (100 ng/ml) was added to these cultures for 12 h, and then supernatants were collected for cytokine measurements. The results of ELISA analysis revealed that addition of WT-exosomes significantly inhibited the secretion of TNF-α, IL-1β, and IL-6 from macrophages upon LPS stimulation, evidenced by lower levels of TNF-α, IL-1β, and IL-6 in culture supernatants of WT-exosome-treated macrophages than those of medium-treated controls (Fig. 5E–G). By contrast, LPS-stimulated production of TNF-α, IL-1β, and IL-6 was remarkably increased in miR-223-KO exosome-treated macrophages, compared with medium-treated samples (Fig. 5E–G).

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