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Extracellular membrane vesicles from umbilical cord blood-derived MSC protect against ischemic acute kidney injury, a feature that is lost after inflammatory conditioning.

Kilpinen L, Impola U, Sankkila L, Ritamo I, Aatonen M, Kilpinen S, Tuimala J, Valmu L, Levijoki J, Finckenberg P, Siljander P, Kankuri E, Mervaala E, Laitinen S - J Extracell Vesicles (2013)

Bottom Line: Currently the therapeutic effect of MSCs is considered to be mediated via paracrine interactions with immune cells.Complement factors (C3, C4A, C5) and lipid binding proteins (i.e apolipoproteins) were only found in the MVctrls, whereas the MVstim contained tetraspanins (CD9, CD63, CD81) and more complete proteasome complex accompanied with MHCI.We demonstrate by both in vitro and in vivo models accompanied with a detailed analysis of molecular characteristics that inflammatory conditioning of MSCs influence on the protein content and functional properties of MVs revealing the complexity of the MSC paracrine regulation.

View Article: PubMed Central - PubMed

Affiliation: Finnish Red Cross Blood Service, Helsinki, Finland.

ABSTRACT

Background: Mesenchymal stromal cells (MSC) are shown to have a great therapeutic potential in many immunological disorders. Currently the therapeutic effect of MSCs is considered to be mediated via paracrine interactions with immune cells. Umbilical cord blood is an attractive but still less studied source of MSCs. We investigated the production of extracellular membrane vesicles (MVs) from human umbilical cord blood derived MSCs (hUCBMSC) in the presence (MVstim) or absence (MVctrl) of inflammatory stimulus.

Methods: hUCBMSCs were cultured in serum free media with or without IFN-γ and MVs were collected from conditioned media by ultracentrifugation. The protein content of MVs were analyzed by mass spectrometry. Hypoxia induced acute kidney injury rat model was used to analyze the in vivo therapeutic potential of MVs and T-cell proliferation and induction of regulatory T cells were analyzed by co-culture assays.

Results: Both MVstim and MVctrl showed similar T-cell modulation activity in vitro, but only MVctrls were able to protect rat kidneys from reperfusion injury in vivo. To clarify this difference in functionality we made a comparative mass spectrometric analysis of the MV protein contents. The IFN-γ stimulation induced dramatic changes in the protein content of the MVs. Complement factors (C3, C4A, C5) and lipid binding proteins (i.e apolipoproteins) were only found in the MVctrls, whereas the MVstim contained tetraspanins (CD9, CD63, CD81) and more complete proteasome complex accompanied with MHCI. We further discovered that differently produced MV pools contained specific Rab proteins suggesting that same cells, depending on external signals, produce vesicles originating from different intracellular locations.

Conclusions: We demonstrate by both in vitro and in vivo models accompanied with a detailed analysis of molecular characteristics that inflammatory conditioning of MSCs influence on the protein content and functional properties of MVs revealing the complexity of the MSC paracrine regulation.

No MeSH data available.


Related in: MedlinePlus

Effects of MV treatment on acute tubular necrosis (ATN) in rats with renal ischemia-reperfusion injury. (A) Representative photomicrographs from untreated rats with I/R injury. (B) Representative photomicrographs from rats with I/R injury treated with MVctrl. (C) Representative photomicrographs from rats with I/R injury treated with MVstim. Magnification ×200, scale bar 100 µm. (D) Quantification of ATN with a scoring system. RIR denotes rats with renal I/R injury; RIR+MVctrl, rats with I/R injury treated with control microvesicles after reperfusion; RIR+MVstim, rats with I/R injury treated with interferon-γ microvesicles after reperfusion; *p<0.05 RIR+MVctrl vs. RIR; #p<0.05 RIR+MVctrl vs. RIR+MVstim.
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Figure 0006: Effects of MV treatment on acute tubular necrosis (ATN) in rats with renal ischemia-reperfusion injury. (A) Representative photomicrographs from untreated rats with I/R injury. (B) Representative photomicrographs from rats with I/R injury treated with MVctrl. (C) Representative photomicrographs from rats with I/R injury treated with MVstim. Magnification ×200, scale bar 100 µm. (D) Quantification of ATN with a scoring system. RIR denotes rats with renal I/R injury; RIR+MVctrl, rats with I/R injury treated with control microvesicles after reperfusion; RIR+MVstim, rats with I/R injury treated with interferon-γ microvesicles after reperfusion; *p<0.05 RIR+MVctrl vs. RIR; #p<0.05 RIR+MVctrl vs. RIR+MVstim.

Mentions: Acute kidney I/R injury was associated with an 8.4-fold increase in serum creatinine concentration as compared to pre-operative baseline values (Fig. 5A). The concentrations of serum urea also had an 8-fold increase (Fig. 5B) and liver enzyme aspartate aminotransferase (ASAT) had a 6.6-fold increase (Fig. 5C) and gamma glutamyltransferase (γ-GT) had an 18.9-fold increase (Fig. 5D) in 24 hours after operation. Histopathological analysis of the kidneys harvested 48 hours after I/R showed marked injury of the renal parenchyma comprising vast necrosis of the tubuloepithelial cells, tubular dilatation and cast formation (Fig. 6A and D).


Extracellular membrane vesicles from umbilical cord blood-derived MSC protect against ischemic acute kidney injury, a feature that is lost after inflammatory conditioning.

Kilpinen L, Impola U, Sankkila L, Ritamo I, Aatonen M, Kilpinen S, Tuimala J, Valmu L, Levijoki J, Finckenberg P, Siljander P, Kankuri E, Mervaala E, Laitinen S - J Extracell Vesicles (2013)

Effects of MV treatment on acute tubular necrosis (ATN) in rats with renal ischemia-reperfusion injury. (A) Representative photomicrographs from untreated rats with I/R injury. (B) Representative photomicrographs from rats with I/R injury treated with MVctrl. (C) Representative photomicrographs from rats with I/R injury treated with MVstim. Magnification ×200, scale bar 100 µm. (D) Quantification of ATN with a scoring system. RIR denotes rats with renal I/R injury; RIR+MVctrl, rats with I/R injury treated with control microvesicles after reperfusion; RIR+MVstim, rats with I/R injury treated with interferon-γ microvesicles after reperfusion; *p<0.05 RIR+MVctrl vs. RIR; #p<0.05 RIR+MVctrl vs. RIR+MVstim.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3860334&req=5

Figure 0006: Effects of MV treatment on acute tubular necrosis (ATN) in rats with renal ischemia-reperfusion injury. (A) Representative photomicrographs from untreated rats with I/R injury. (B) Representative photomicrographs from rats with I/R injury treated with MVctrl. (C) Representative photomicrographs from rats with I/R injury treated with MVstim. Magnification ×200, scale bar 100 µm. (D) Quantification of ATN with a scoring system. RIR denotes rats with renal I/R injury; RIR+MVctrl, rats with I/R injury treated with control microvesicles after reperfusion; RIR+MVstim, rats with I/R injury treated with interferon-γ microvesicles after reperfusion; *p<0.05 RIR+MVctrl vs. RIR; #p<0.05 RIR+MVctrl vs. RIR+MVstim.
Mentions: Acute kidney I/R injury was associated with an 8.4-fold increase in serum creatinine concentration as compared to pre-operative baseline values (Fig. 5A). The concentrations of serum urea also had an 8-fold increase (Fig. 5B) and liver enzyme aspartate aminotransferase (ASAT) had a 6.6-fold increase (Fig. 5C) and gamma glutamyltransferase (γ-GT) had an 18.9-fold increase (Fig. 5D) in 24 hours after operation. Histopathological analysis of the kidneys harvested 48 hours after I/R showed marked injury of the renal parenchyma comprising vast necrosis of the tubuloepithelial cells, tubular dilatation and cast formation (Fig. 6A and D).

Bottom Line: Currently the therapeutic effect of MSCs is considered to be mediated via paracrine interactions with immune cells.Complement factors (C3, C4A, C5) and lipid binding proteins (i.e apolipoproteins) were only found in the MVctrls, whereas the MVstim contained tetraspanins (CD9, CD63, CD81) and more complete proteasome complex accompanied with MHCI.We demonstrate by both in vitro and in vivo models accompanied with a detailed analysis of molecular characteristics that inflammatory conditioning of MSCs influence on the protein content and functional properties of MVs revealing the complexity of the MSC paracrine regulation.

View Article: PubMed Central - PubMed

Affiliation: Finnish Red Cross Blood Service, Helsinki, Finland.

ABSTRACT

Background: Mesenchymal stromal cells (MSC) are shown to have a great therapeutic potential in many immunological disorders. Currently the therapeutic effect of MSCs is considered to be mediated via paracrine interactions with immune cells. Umbilical cord blood is an attractive but still less studied source of MSCs. We investigated the production of extracellular membrane vesicles (MVs) from human umbilical cord blood derived MSCs (hUCBMSC) in the presence (MVstim) or absence (MVctrl) of inflammatory stimulus.

Methods: hUCBMSCs were cultured in serum free media with or without IFN-γ and MVs were collected from conditioned media by ultracentrifugation. The protein content of MVs were analyzed by mass spectrometry. Hypoxia induced acute kidney injury rat model was used to analyze the in vivo therapeutic potential of MVs and T-cell proliferation and induction of regulatory T cells were analyzed by co-culture assays.

Results: Both MVstim and MVctrl showed similar T-cell modulation activity in vitro, but only MVctrls were able to protect rat kidneys from reperfusion injury in vivo. To clarify this difference in functionality we made a comparative mass spectrometric analysis of the MV protein contents. The IFN-γ stimulation induced dramatic changes in the protein content of the MVs. Complement factors (C3, C4A, C5) and lipid binding proteins (i.e apolipoproteins) were only found in the MVctrls, whereas the MVstim contained tetraspanins (CD9, CD63, CD81) and more complete proteasome complex accompanied with MHCI. We further discovered that differently produced MV pools contained specific Rab proteins suggesting that same cells, depending on external signals, produce vesicles originating from different intracellular locations.

Conclusions: We demonstrate by both in vitro and in vivo models accompanied with a detailed analysis of molecular characteristics that inflammatory conditioning of MSCs influence on the protein content and functional properties of MVs revealing the complexity of the MSC paracrine regulation.

No MeSH data available.


Related in: MedlinePlus