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Prostacyclin-producing human mesenchymal cells target H19 lncRNA to augment endogenous progenitor function in hindlimb ischaemia.

Deng Y, Yang Z, Terry T, Pan S, Woodside DG, Wang J, Ruan K, Willerson JT, Dixon RA, Liu Q - Nat Commun (2016)

Bottom Line: Here we develop an innovative strategy to enhance the paracrine effects of hMSCs.Transplanted PGI2-hMSCs do not incorporate long term into host tissue, but rather they mediate host regeneration and muscle mass gain in a paracrine manner.Mechanistically, this involves long noncoding RNA H19 in promoting PGI2-hMSC-associated survival and proliferation of host progenitor cells under hypoxic conditions.

View Article: PubMed Central - PubMed

Affiliation: Wafic Said Molecular Cardiology Research Laboratory, Texas Heart Institute, P.O. Box 20345, MC 2-255, Houston, Texas 77225-0345, USA.

ABSTRACT
Promoting the paracrine effects of human mesenchymal stem cell (hMSC) therapy may contribute to improvements in patient outcomes. Here we develop an innovative strategy to enhance the paracrine effects of hMSCs. In a mouse hindlimb ischaemia model, we examine the effects of hMSCs in which a novel triple-catalytic enzyme is introduced to stably produce prostacyclin (PGI2-hMSCs). We show that PGI2-hMSCs facilitate perfusion recovery and enhance running capability as compared with control hMSCs or iloprost (a stable PGI2 analogue). Transplanted PGI2-hMSCs do not incorporate long term into host tissue, but rather they mediate host regeneration and muscle mass gain in a paracrine manner. Mechanistically, this involves long noncoding RNA H19 in promoting PGI2-hMSC-associated survival and proliferation of host progenitor cells under hypoxic conditions. Together, our data reveal the novel ability of PGI2-hMSCs to stimulate host regenerative processes and improve physical function by regulating long noncoding RNA in resident progenitor cells.

No MeSH data available.


Related in: MedlinePlus

Upregulation of H19 lncRNA in ischaemic hindlimbs treated with PGI2-hMSCs.(a) Representative images of H19 RNA fluorescence in situ hybridization in gastrocnemius muscle sections at 3 days after 3.1-hMSC, 3.1-hMSC+ILO or PG2-hMSC injections. We used a fluorescein-tagged H19 FISH probe that specifically targets endogenous H19 lncRNA, resulting in intense intracellular green fluorescent particles. We found a higher expression of host H19 lncRNA in PGI2-hMSC- and in 3.1-hMSC+ILO-treated muscles than in 3.1-hMSC-treated muscles (n=3 mice per group). All the sections were counterstained with 4,6-diamidino-2-phenylindole (DAPI) to localize nuclei. (b) Quantitative RT–PCR showed a significant increase in H19 lncRNA levels in ischaemic gastrocnemius muscle 5 days after injections with 3.1-hMSCs+ILO or PG2-hMSCs as compared with tissue samples injected with 3.1-hMSCs. *P<0.05 by one-way ANOVA with Newman–Keuls post hoc test. Data are shown as mean±s.e.m. from three independent assays. N=3 mice (male and female) per group. Mice were sex matched among the groups. Scale bar, 10 μm.
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f8: Upregulation of H19 lncRNA in ischaemic hindlimbs treated with PGI2-hMSCs.(a) Representative images of H19 RNA fluorescence in situ hybridization in gastrocnemius muscle sections at 3 days after 3.1-hMSC, 3.1-hMSC+ILO or PG2-hMSC injections. We used a fluorescein-tagged H19 FISH probe that specifically targets endogenous H19 lncRNA, resulting in intense intracellular green fluorescent particles. We found a higher expression of host H19 lncRNA in PGI2-hMSC- and in 3.1-hMSC+ILO-treated muscles than in 3.1-hMSC-treated muscles (n=3 mice per group). All the sections were counterstained with 4,6-diamidino-2-phenylindole (DAPI) to localize nuclei. (b) Quantitative RT–PCR showed a significant increase in H19 lncRNA levels in ischaemic gastrocnemius muscle 5 days after injections with 3.1-hMSCs+ILO or PG2-hMSCs as compared with tissue samples injected with 3.1-hMSCs. *P<0.05 by one-way ANOVA with Newman–Keuls post hoc test. Data are shown as mean±s.e.m. from three independent assays. N=3 mice (male and female) per group. Mice were sex matched among the groups. Scale bar, 10 μm.

Mentions: After confirming that PGI2-hMSCs trigger H19 lncRNA upregulation in myoblasts in vitro during low-oxygen tension, we examined the effect of PGI2-hMSCs on endogenous H19 lncRNA expression in ischaemic hindlimbs using RNA fluorescence in situ hybridization (RNA-FISH). A mouse H19 fluorescent oligonucleotide probe was used to detect single H19 lncRNA molecules. H19 sequences that contain miR-675-3p and miR-675-5p were excluded in the probe design to avoid non-specific binding20. RNA-FISH studies showed an increase in H19 lncRNA levels in the cytoplasm of endogenous cells surrounding PGI2-hMSC injection sites as compared with 3.1-hMSC injection sites at 3 days after cell administration (Fig. 8a). Quantitative PCR with reverse transcription (RT–qPCR) further showed that levels of H19 RNA were significantly higher in gastrocnemius muscle treated with PGI2-hMSCs than in that treated with 3.1-hMSCs, suggesting that PGI2-hMSCs induce an upregulation of endogenous H19 lncRNA in ischaemic hindlimbs (one-way ANOVA; Fig. 8b).


Prostacyclin-producing human mesenchymal cells target H19 lncRNA to augment endogenous progenitor function in hindlimb ischaemia.

Deng Y, Yang Z, Terry T, Pan S, Woodside DG, Wang J, Ruan K, Willerson JT, Dixon RA, Liu Q - Nat Commun (2016)

Upregulation of H19 lncRNA in ischaemic hindlimbs treated with PGI2-hMSCs.(a) Representative images of H19 RNA fluorescence in situ hybridization in gastrocnemius muscle sections at 3 days after 3.1-hMSC, 3.1-hMSC+ILO or PG2-hMSC injections. We used a fluorescein-tagged H19 FISH probe that specifically targets endogenous H19 lncRNA, resulting in intense intracellular green fluorescent particles. We found a higher expression of host H19 lncRNA in PGI2-hMSC- and in 3.1-hMSC+ILO-treated muscles than in 3.1-hMSC-treated muscles (n=3 mice per group). All the sections were counterstained with 4,6-diamidino-2-phenylindole (DAPI) to localize nuclei. (b) Quantitative RT–PCR showed a significant increase in H19 lncRNA levels in ischaemic gastrocnemius muscle 5 days after injections with 3.1-hMSCs+ILO or PG2-hMSCs as compared with tissue samples injected with 3.1-hMSCs. *P<0.05 by one-way ANOVA with Newman–Keuls post hoc test. Data are shown as mean±s.e.m. from three independent assays. N=3 mice (male and female) per group. Mice were sex matched among the groups. Scale bar, 10 μm.
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f8: Upregulation of H19 lncRNA in ischaemic hindlimbs treated with PGI2-hMSCs.(a) Representative images of H19 RNA fluorescence in situ hybridization in gastrocnemius muscle sections at 3 days after 3.1-hMSC, 3.1-hMSC+ILO or PG2-hMSC injections. We used a fluorescein-tagged H19 FISH probe that specifically targets endogenous H19 lncRNA, resulting in intense intracellular green fluorescent particles. We found a higher expression of host H19 lncRNA in PGI2-hMSC- and in 3.1-hMSC+ILO-treated muscles than in 3.1-hMSC-treated muscles (n=3 mice per group). All the sections were counterstained with 4,6-diamidino-2-phenylindole (DAPI) to localize nuclei. (b) Quantitative RT–PCR showed a significant increase in H19 lncRNA levels in ischaemic gastrocnemius muscle 5 days after injections with 3.1-hMSCs+ILO or PG2-hMSCs as compared with tissue samples injected with 3.1-hMSCs. *P<0.05 by one-way ANOVA with Newman–Keuls post hoc test. Data are shown as mean±s.e.m. from three independent assays. N=3 mice (male and female) per group. Mice were sex matched among the groups. Scale bar, 10 μm.
Mentions: After confirming that PGI2-hMSCs trigger H19 lncRNA upregulation in myoblasts in vitro during low-oxygen tension, we examined the effect of PGI2-hMSCs on endogenous H19 lncRNA expression in ischaemic hindlimbs using RNA fluorescence in situ hybridization (RNA-FISH). A mouse H19 fluorescent oligonucleotide probe was used to detect single H19 lncRNA molecules. H19 sequences that contain miR-675-3p and miR-675-5p were excluded in the probe design to avoid non-specific binding20. RNA-FISH studies showed an increase in H19 lncRNA levels in the cytoplasm of endogenous cells surrounding PGI2-hMSC injection sites as compared with 3.1-hMSC injection sites at 3 days after cell administration (Fig. 8a). Quantitative PCR with reverse transcription (RT–qPCR) further showed that levels of H19 RNA were significantly higher in gastrocnemius muscle treated with PGI2-hMSCs than in that treated with 3.1-hMSCs, suggesting that PGI2-hMSCs induce an upregulation of endogenous H19 lncRNA in ischaemic hindlimbs (one-way ANOVA; Fig. 8b).

Bottom Line: Here we develop an innovative strategy to enhance the paracrine effects of hMSCs.Transplanted PGI2-hMSCs do not incorporate long term into host tissue, but rather they mediate host regeneration and muscle mass gain in a paracrine manner.Mechanistically, this involves long noncoding RNA H19 in promoting PGI2-hMSC-associated survival and proliferation of host progenitor cells under hypoxic conditions.

View Article: PubMed Central - PubMed

Affiliation: Wafic Said Molecular Cardiology Research Laboratory, Texas Heart Institute, P.O. Box 20345, MC 2-255, Houston, Texas 77225-0345, USA.

ABSTRACT
Promoting the paracrine effects of human mesenchymal stem cell (hMSC) therapy may contribute to improvements in patient outcomes. Here we develop an innovative strategy to enhance the paracrine effects of hMSCs. In a mouse hindlimb ischaemia model, we examine the effects of hMSCs in which a novel triple-catalytic enzyme is introduced to stably produce prostacyclin (PGI2-hMSCs). We show that PGI2-hMSCs facilitate perfusion recovery and enhance running capability as compared with control hMSCs or iloprost (a stable PGI2 analogue). Transplanted PGI2-hMSCs do not incorporate long term into host tissue, but rather they mediate host regeneration and muscle mass gain in a paracrine manner. Mechanistically, this involves long noncoding RNA H19 in promoting PGI2-hMSC-associated survival and proliferation of host progenitor cells under hypoxic conditions. Together, our data reveal the novel ability of PGI2-hMSCs to stimulate host regenerative processes and improve physical function by regulating long noncoding RNA in resident progenitor cells.

No MeSH data available.


Related in: MedlinePlus