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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

The different vesicle transport pathways and the functions of Rab GTPases identified from the hUCBMSC MVs. Rab proteins found in MVstim were related to deeper endosomal route (blue), whereas MVcrtl contained Rab proteins from the rapid loop located near the plasma membrane (red). RAB1, which is localized at the endoplasmic reticulum (ER), mediates ER–Golgi trafficking together with RAB2, which might also regulate Golgi–ER trafficking. The Golgi-localized RAB6 and RAB34 mediate intra-Golgi trafficking. RAB8 mediates constitutive biosynthetic trafficking from the trans-Golgi network (TGN) to the plasma membrane. RAB3, and RAB37 regulate the secretory pathway. RAB32 is involved in the biogenesis of melanosomes and other lysosome-related organelles (LRO) as well as the formation of lipid droplets from the early endosome (EE). RAB18 controls the formation of lipid droplets from the ER. RAB5 mediates endocytosis and endosome fusion of clathrin-coated vesicles (CCVs). RAB11 and RAB35 mediate slow endocytic recycling through recycling endosomes, (RE). RAB15 is involved in the trafficking from early endosomes (EE) to recycling endosomes. The late endosome-associated RAB7 mediates the maturation of late endosomes (LE) and their fusion with lysosomes. See Refs. (75, 79–81) for further information.
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Figure 0007: The different vesicle transport pathways and the functions of Rab GTPases identified from the hUCBMSC MVs. Rab proteins found in MVstim were related to deeper endosomal route (blue), whereas MVcrtl contained Rab proteins from the rapid loop located near the plasma membrane (red). RAB1, which is localized at the endoplasmic reticulum (ER), mediates ER–Golgi trafficking together with RAB2, which might also regulate Golgi–ER trafficking. The Golgi-localized RAB6 and RAB34 mediate intra-Golgi trafficking. RAB8 mediates constitutive biosynthetic trafficking from the trans-Golgi network (TGN) to the plasma membrane. RAB3, and RAB37 regulate the secretory pathway. RAB32 is involved in the biogenesis of melanosomes and other lysosome-related organelles (LRO) as well as the formation of lipid droplets from the early endosome (EE). RAB18 controls the formation of lipid droplets from the ER. RAB5 mediates endocytosis and endosome fusion of clathrin-coated vesicles (CCVs). RAB11 and RAB35 mediate slow endocytic recycling through recycling endosomes, (RE). RAB15 is involved in the trafficking from early endosomes (EE) to recycling endosomes. The late endosome-associated RAB7 mediates the maturation of late endosomes (LE) and their fusion with lysosomes. See Refs. (75, 79–81) for further information.

Mentions: There is on-going scientific debate over the cellular origin, characteristics and nomenclature of extracellular vesicles. In this study, we made a conscious decision not to separate the different sizes or densities MVs by ultracentrifugation or filtration processes. Instead, we wanted to collect the whole pool of secreted MVs from the supernatant or hUCBMSCs culture milieu and study the effect of the different external signals on the composition of the MVs. One of the most interesting findings in our study was the distinct set of Rab proteins found in the different MV pools produced. It is well established that Rab GTPases, members of the Ras superfamily, are important regulators of membrane transport, vesicle formation, movement and fusion and can be considered as labels defining vesicle routing (75), with also their role in extracellular vesicle formation and transport being studied (76, 77). This superfamily is known to consist of at least 60 members in humans (78). Altogether 24 Rab proteins were identified in our study. The MS analysis revealed that IFN-γ induced production of MVs richer in Rab proteins. Altogether 19 Rab proteins were detected in MVstim, 17 of which were unique. Instead, in MVctrls, only 7 Rab proteins were identified, 5 of which were unique. The Rab proteins found in MVstims were distinctive for exocytosis and deeper endosomal recycling, extending from ER all through the exocytotic pathway (Rab 1, 2, 6 and 8) as illustrated in Fig. 7. In contrast, MVctrls contained Rab proteins more restricted to the early endosomal recycling near plasma membrane (such as Rab 3, 5, 14 and 34). Our interpretation is that this strikingly different and specific subcellular localization of the Rab proteins indicates that for the first time, we have been able to make a distinction between two separate routes of extracellular vesicles from MSCs: cytokine-induced deeper loop and constantly produced rapid loop of MVs. The presence of MHCI molecules in MVstims accompanied by the more complete proteasome complex than in MVctrls further proves the effective IFN-γ activation and the induction of the delivery of MHCI molecules to of the cell surface and consequently into MVstims. Already in 2003, Willem Stoorvogel's group described proteomic analysis of B-cell-derived exosomes (82). They demonstrated that B-cell-derived exosomes contained major histocompatibility Class I and II (MHCI and II) as major components in addition to integrins and many other proteins also seen in MSC-derived MVs. On the contrary to B-cells, MSCs do not express MHCII molecules on their cell surface. Furthermore, MHCI molecules are expressed at low levels. Our results show that after 24 hours of IFN-γ activation of hUCBMSCs, the surface expression of MHCI is increased but not MHCII for which 48 hours of activation is required. Interestingly, we found that MSC-derived MVctrls do not contain either of these molecules but after IFN-γ activation, MHCI and also a complete set of proteasome complex were detected in MVstims.


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)

The different vesicle transport pathways and the functions of Rab GTPases identified from the hUCBMSC MVs. Rab proteins found in MVstim were related to deeper endosomal route (blue), whereas MVcrtl contained Rab proteins from the rapid loop located near the plasma membrane (red). RAB1, which is localized at the endoplasmic reticulum (ER), mediates ER–Golgi trafficking together with RAB2, which might also regulate Golgi–ER trafficking. The Golgi-localized RAB6 and RAB34 mediate intra-Golgi trafficking. RAB8 mediates constitutive biosynthetic trafficking from the trans-Golgi network (TGN) to the plasma membrane. RAB3, and RAB37 regulate the secretory pathway. RAB32 is involved in the biogenesis of melanosomes and other lysosome-related organelles (LRO) as well as the formation of lipid droplets from the early endosome (EE). RAB18 controls the formation of lipid droplets from the ER. RAB5 mediates endocytosis and endosome fusion of clathrin-coated vesicles (CCVs). RAB11 and RAB35 mediate slow endocytic recycling through recycling endosomes, (RE). RAB15 is involved in the trafficking from early endosomes (EE) to recycling endosomes. The late endosome-associated RAB7 mediates the maturation of late endosomes (LE) and their fusion with lysosomes. See Refs. (75, 79–81) for further information.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 0007: The different vesicle transport pathways and the functions of Rab GTPases identified from the hUCBMSC MVs. Rab proteins found in MVstim were related to deeper endosomal route (blue), whereas MVcrtl contained Rab proteins from the rapid loop located near the plasma membrane (red). RAB1, which is localized at the endoplasmic reticulum (ER), mediates ER–Golgi trafficking together with RAB2, which might also regulate Golgi–ER trafficking. The Golgi-localized RAB6 and RAB34 mediate intra-Golgi trafficking. RAB8 mediates constitutive biosynthetic trafficking from the trans-Golgi network (TGN) to the plasma membrane. RAB3, and RAB37 regulate the secretory pathway. RAB32 is involved in the biogenesis of melanosomes and other lysosome-related organelles (LRO) as well as the formation of lipid droplets from the early endosome (EE). RAB18 controls the formation of lipid droplets from the ER. RAB5 mediates endocytosis and endosome fusion of clathrin-coated vesicles (CCVs). RAB11 and RAB35 mediate slow endocytic recycling through recycling endosomes, (RE). RAB15 is involved in the trafficking from early endosomes (EE) to recycling endosomes. The late endosome-associated RAB7 mediates the maturation of late endosomes (LE) and their fusion with lysosomes. See Refs. (75, 79–81) for further information.
Mentions: There is on-going scientific debate over the cellular origin, characteristics and nomenclature of extracellular vesicles. In this study, we made a conscious decision not to separate the different sizes or densities MVs by ultracentrifugation or filtration processes. Instead, we wanted to collect the whole pool of secreted MVs from the supernatant or hUCBMSCs culture milieu and study the effect of the different external signals on the composition of the MVs. One of the most interesting findings in our study was the distinct set of Rab proteins found in the different MV pools produced. It is well established that Rab GTPases, members of the Ras superfamily, are important regulators of membrane transport, vesicle formation, movement and fusion and can be considered as labels defining vesicle routing (75), with also their role in extracellular vesicle formation and transport being studied (76, 77). This superfamily is known to consist of at least 60 members in humans (78). Altogether 24 Rab proteins were identified in our study. The MS analysis revealed that IFN-γ induced production of MVs richer in Rab proteins. Altogether 19 Rab proteins were detected in MVstim, 17 of which were unique. Instead, in MVctrls, only 7 Rab proteins were identified, 5 of which were unique. The Rab proteins found in MVstims were distinctive for exocytosis and deeper endosomal recycling, extending from ER all through the exocytotic pathway (Rab 1, 2, 6 and 8) as illustrated in Fig. 7. In contrast, MVctrls contained Rab proteins more restricted to the early endosomal recycling near plasma membrane (such as Rab 3, 5, 14 and 34). Our interpretation is that this strikingly different and specific subcellular localization of the Rab proteins indicates that for the first time, we have been able to make a distinction between two separate routes of extracellular vesicles from MSCs: cytokine-induced deeper loop and constantly produced rapid loop of MVs. The presence of MHCI molecules in MVstims accompanied by the more complete proteasome complex than in MVctrls further proves the effective IFN-γ activation and the induction of the delivery of MHCI molecules to of the cell surface and consequently into MVstims. Already in 2003, Willem Stoorvogel's group described proteomic analysis of B-cell-derived exosomes (82). They demonstrated that B-cell-derived exosomes contained major histocompatibility Class I and II (MHCI and II) as major components in addition to integrins and many other proteins also seen in MSC-derived MVs. On the contrary to B-cells, MSCs do not express MHCII molecules on their cell surface. Furthermore, MHCI molecules are expressed at low levels. Our results show that after 24 hours of IFN-γ activation of hUCBMSCs, the surface expression of MHCI is increased but not MHCII for which 48 hours of activation is required. Interestingly, we found that MSC-derived MVctrls do not contain either of these molecules but after IFN-γ activation, MHCI and also a complete set of proteasome complex were detected in MVstims.

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