Limits...
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

PGI2-hMSC therapy concurrently improved distal perfusion and treadmill performance after hindlimb ischaemia.(a) Schematic cartoon illustration of the COX-1-10aa-PGIS fusion protein. A His–Ala–Ile–Met–Gly–Val–Ala–Phe–Thr–Trp peptide linker (from a helical transmembrane domain of bovine rhodopsin) was used to connect the chimeric protein and to maintain the topology of both COX-1 and PGIS proteins. Structures used for generation of the schematic included PDB 3N8Z (COX-1)33, PDB 2IAG (PGIS)34 and PDB 1GZM (rhodopsin)35. (b) Quantitative analysis of perfusion from ankle to toe showed different rates of blood flow recovery among the five treatment groups over the 14-day observation period. Notably, at day 14 after cell administration, perfusion in the PGI2-hMSC group exceeded that in the four other treatment groups, leading to significantly better perfusion recovery. Day 5: *P<0.05 for 3.1-hMSCs+ILO versus ILO or versus PGI2-hMSCs; ***P<0.001 for 3.1-hMSCs+ILO versus 3.1-hMSCs or versus PBS. Day 7: **P<0.01 for 3.1-hMSCs+ILO versus 3.1-hMSCs; PGI2-hMSCs versus 3.1-hMSCs. ***P<0.001 for 3.1-hMSCs+ILO versus PBS; PGI2-hMSCs versus PBS. Day 14, *P<0.05 for 3.1-hMSCs versus PBS, ILO versus 3.1-hMSCs; **P<0.01 for 3.1-hMSCs+ILO versus ILO, PGI2-hMSCs versus 3.1-hMSCs+ILO; ***P<0.001 for ILO versus PBS, 3.1-hMSCs+ILO versus PBS or versus 3.1-hMSCs, PGI2-hMSCs versus PBS or versus ILO or versus 3.1-hMSCs. (c) In run-to-exhaustion tests, mice treated with PGI2-hMSCs, daily injections of ILO or 3.1-hMSCs+ILO had a significantly longer maximal running distance than those treated with 3.1-hMSCs or vehicle (PBS) at 21 days. *P<0.05 for PGI2-hMSCs versus 3.1-hMSCs; 3.1-hMSCs+ILO versus 3.1-hMSCs. **P<0.01 for ILO versus 3.1-hMSCs; ***P<0.001 for ILO versus PBS; 3.1-hMSCs+ILO versus PBS; PGI2-hMSCs versus PBS. (d) At 28 days, the performance enhancement in PGI2-hMSC-treated mice was similar to that seen at day 21. Meanwhile, 3.1-hMSCs+ILO outperformed ILO treatment when compared with 3.1-hMSC treatment (one-way ANOVA followed by Dunnett's multiple comparison test). *P<0.05 for PGI2-hMSCs versus ILO or versus 3.1-hMSCs+ILO. **P<0.01 for ILO versus PBS, 3.1-hMSCs+ILO versus PBS, PGI2-hMSCs versus 3.1-hMSCs. ***P<0.001 for PGI2-hMSCs versus PBS. Statistical significance was determined by one-way ANOVA followed by Newman–Keuls post hoc test. Data are shown as mean±s.e.m. N=16 of sex-matched mice in vehicle (PBS) group; N=10 sex-matched mice each in the other four treatment groups.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4835554&req=5

f1: PGI2-hMSC therapy concurrently improved distal perfusion and treadmill performance after hindlimb ischaemia.(a) Schematic cartoon illustration of the COX-1-10aa-PGIS fusion protein. A His–Ala–Ile–Met–Gly–Val–Ala–Phe–Thr–Trp peptide linker (from a helical transmembrane domain of bovine rhodopsin) was used to connect the chimeric protein and to maintain the topology of both COX-1 and PGIS proteins. Structures used for generation of the schematic included PDB 3N8Z (COX-1)33, PDB 2IAG (PGIS)34 and PDB 1GZM (rhodopsin)35. (b) Quantitative analysis of perfusion from ankle to toe showed different rates of blood flow recovery among the five treatment groups over the 14-day observation period. Notably, at day 14 after cell administration, perfusion in the PGI2-hMSC group exceeded that in the four other treatment groups, leading to significantly better perfusion recovery. Day 5: *P<0.05 for 3.1-hMSCs+ILO versus ILO or versus PGI2-hMSCs; ***P<0.001 for 3.1-hMSCs+ILO versus 3.1-hMSCs or versus PBS. Day 7: **P<0.01 for 3.1-hMSCs+ILO versus 3.1-hMSCs; PGI2-hMSCs versus 3.1-hMSCs. ***P<0.001 for 3.1-hMSCs+ILO versus PBS; PGI2-hMSCs versus PBS. Day 14, *P<0.05 for 3.1-hMSCs versus PBS, ILO versus 3.1-hMSCs; **P<0.01 for 3.1-hMSCs+ILO versus ILO, PGI2-hMSCs versus 3.1-hMSCs+ILO; ***P<0.001 for ILO versus PBS, 3.1-hMSCs+ILO versus PBS or versus 3.1-hMSCs, PGI2-hMSCs versus PBS or versus ILO or versus 3.1-hMSCs. (c) In run-to-exhaustion tests, mice treated with PGI2-hMSCs, daily injections of ILO or 3.1-hMSCs+ILO had a significantly longer maximal running distance than those treated with 3.1-hMSCs or vehicle (PBS) at 21 days. *P<0.05 for PGI2-hMSCs versus 3.1-hMSCs; 3.1-hMSCs+ILO versus 3.1-hMSCs. **P<0.01 for ILO versus 3.1-hMSCs; ***P<0.001 for ILO versus PBS; 3.1-hMSCs+ILO versus PBS; PGI2-hMSCs versus PBS. (d) At 28 days, the performance enhancement in PGI2-hMSC-treated mice was similar to that seen at day 21. Meanwhile, 3.1-hMSCs+ILO outperformed ILO treatment when compared with 3.1-hMSC treatment (one-way ANOVA followed by Dunnett's multiple comparison test). *P<0.05 for PGI2-hMSCs versus ILO or versus 3.1-hMSCs+ILO. **P<0.01 for ILO versus PBS, 3.1-hMSCs+ILO versus PBS, PGI2-hMSCs versus 3.1-hMSCs. ***P<0.001 for PGI2-hMSCs versus PBS. Statistical significance was determined by one-way ANOVA followed by Newman–Keuls post hoc test. Data are shown as mean±s.e.m. N=16 of sex-matched mice in vehicle (PBS) group; N=10 sex-matched mice each in the other four treatment groups.

Mentions: We created PGI2-overexpressing hMSCs by introducing an active triple-catalytic enzyme that links cyclooxygenase-1 to prostacyclin synthase (COX-1-10aa-PGIS) based on our previous biochemical and structural studies of COX-1 and PGIS (Fig. 1a)7. This triple-catalytic enzyme catalyses three key reactions that allow the production of PGI2 from arachidonic acid as previously described8. We confirmed the stable expression of the transgene in PGI2-hMSCs by genomic PCR and western blot (a 130-kDa protein; Supplementary Fig. 1a,b). To assess the production of PGI2, we used an enzyme immunoassay to measure the metabolite 6-keto PGF1α. Compared with hMSCs (native hMSCs containing no vector) and 3.1-hMSCs (hMSCs containing pcDNA3.1 [vector used to construct pcDNA COX-1-10aa-PGIS]), PGI2-hMSCs were capable of secreting a fivefold higher concentration of 6-keto PGF1α in the supernatant after incubating cells with arachidonic acid for 20 min (Supplementary Fig. 1c; P<0.01; one-way analysis of variance [ANOVA]).


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)

PGI2-hMSC therapy concurrently improved distal perfusion and treadmill performance after hindlimb ischaemia.(a) Schematic cartoon illustration of the COX-1-10aa-PGIS fusion protein. A His–Ala–Ile–Met–Gly–Val–Ala–Phe–Thr–Trp peptide linker (from a helical transmembrane domain of bovine rhodopsin) was used to connect the chimeric protein and to maintain the topology of both COX-1 and PGIS proteins. Structures used for generation of the schematic included PDB 3N8Z (COX-1)33, PDB 2IAG (PGIS)34 and PDB 1GZM (rhodopsin)35. (b) Quantitative analysis of perfusion from ankle to toe showed different rates of blood flow recovery among the five treatment groups over the 14-day observation period. Notably, at day 14 after cell administration, perfusion in the PGI2-hMSC group exceeded that in the four other treatment groups, leading to significantly better perfusion recovery. Day 5: *P<0.05 for 3.1-hMSCs+ILO versus ILO or versus PGI2-hMSCs; ***P<0.001 for 3.1-hMSCs+ILO versus 3.1-hMSCs or versus PBS. Day 7: **P<0.01 for 3.1-hMSCs+ILO versus 3.1-hMSCs; PGI2-hMSCs versus 3.1-hMSCs. ***P<0.001 for 3.1-hMSCs+ILO versus PBS; PGI2-hMSCs versus PBS. Day 14, *P<0.05 for 3.1-hMSCs versus PBS, ILO versus 3.1-hMSCs; **P<0.01 for 3.1-hMSCs+ILO versus ILO, PGI2-hMSCs versus 3.1-hMSCs+ILO; ***P<0.001 for ILO versus PBS, 3.1-hMSCs+ILO versus PBS or versus 3.1-hMSCs, PGI2-hMSCs versus PBS or versus ILO or versus 3.1-hMSCs. (c) In run-to-exhaustion tests, mice treated with PGI2-hMSCs, daily injections of ILO or 3.1-hMSCs+ILO had a significantly longer maximal running distance than those treated with 3.1-hMSCs or vehicle (PBS) at 21 days. *P<0.05 for PGI2-hMSCs versus 3.1-hMSCs; 3.1-hMSCs+ILO versus 3.1-hMSCs. **P<0.01 for ILO versus 3.1-hMSCs; ***P<0.001 for ILO versus PBS; 3.1-hMSCs+ILO versus PBS; PGI2-hMSCs versus PBS. (d) At 28 days, the performance enhancement in PGI2-hMSC-treated mice was similar to that seen at day 21. Meanwhile, 3.1-hMSCs+ILO outperformed ILO treatment when compared with 3.1-hMSC treatment (one-way ANOVA followed by Dunnett's multiple comparison test). *P<0.05 for PGI2-hMSCs versus ILO or versus 3.1-hMSCs+ILO. **P<0.01 for ILO versus PBS, 3.1-hMSCs+ILO versus PBS, PGI2-hMSCs versus 3.1-hMSCs. ***P<0.001 for PGI2-hMSCs versus PBS. Statistical significance was determined by one-way ANOVA followed by Newman–Keuls post hoc test. Data are shown as mean±s.e.m. N=16 of sex-matched mice in vehicle (PBS) group; N=10 sex-matched mice each in the other four treatment groups.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4835554&req=5

f1: PGI2-hMSC therapy concurrently improved distal perfusion and treadmill performance after hindlimb ischaemia.(a) Schematic cartoon illustration of the COX-1-10aa-PGIS fusion protein. A His–Ala–Ile–Met–Gly–Val–Ala–Phe–Thr–Trp peptide linker (from a helical transmembrane domain of bovine rhodopsin) was used to connect the chimeric protein and to maintain the topology of both COX-1 and PGIS proteins. Structures used for generation of the schematic included PDB 3N8Z (COX-1)33, PDB 2IAG (PGIS)34 and PDB 1GZM (rhodopsin)35. (b) Quantitative analysis of perfusion from ankle to toe showed different rates of blood flow recovery among the five treatment groups over the 14-day observation period. Notably, at day 14 after cell administration, perfusion in the PGI2-hMSC group exceeded that in the four other treatment groups, leading to significantly better perfusion recovery. Day 5: *P<0.05 for 3.1-hMSCs+ILO versus ILO or versus PGI2-hMSCs; ***P<0.001 for 3.1-hMSCs+ILO versus 3.1-hMSCs or versus PBS. Day 7: **P<0.01 for 3.1-hMSCs+ILO versus 3.1-hMSCs; PGI2-hMSCs versus 3.1-hMSCs. ***P<0.001 for 3.1-hMSCs+ILO versus PBS; PGI2-hMSCs versus PBS. Day 14, *P<0.05 for 3.1-hMSCs versus PBS, ILO versus 3.1-hMSCs; **P<0.01 for 3.1-hMSCs+ILO versus ILO, PGI2-hMSCs versus 3.1-hMSCs+ILO; ***P<0.001 for ILO versus PBS, 3.1-hMSCs+ILO versus PBS or versus 3.1-hMSCs, PGI2-hMSCs versus PBS or versus ILO or versus 3.1-hMSCs. (c) In run-to-exhaustion tests, mice treated with PGI2-hMSCs, daily injections of ILO or 3.1-hMSCs+ILO had a significantly longer maximal running distance than those treated with 3.1-hMSCs or vehicle (PBS) at 21 days. *P<0.05 for PGI2-hMSCs versus 3.1-hMSCs; 3.1-hMSCs+ILO versus 3.1-hMSCs. **P<0.01 for ILO versus 3.1-hMSCs; ***P<0.001 for ILO versus PBS; 3.1-hMSCs+ILO versus PBS; PGI2-hMSCs versus PBS. (d) At 28 days, the performance enhancement in PGI2-hMSC-treated mice was similar to that seen at day 21. Meanwhile, 3.1-hMSCs+ILO outperformed ILO treatment when compared with 3.1-hMSC treatment (one-way ANOVA followed by Dunnett's multiple comparison test). *P<0.05 for PGI2-hMSCs versus ILO or versus 3.1-hMSCs+ILO. **P<0.01 for ILO versus PBS, 3.1-hMSCs+ILO versus PBS, PGI2-hMSCs versus 3.1-hMSCs. ***P<0.001 for PGI2-hMSCs versus PBS. Statistical significance was determined by one-way ANOVA followed by Newman–Keuls post hoc test. Data are shown as mean±s.e.m. N=16 of sex-matched mice in vehicle (PBS) group; N=10 sex-matched mice each in the other four treatment groups.
Mentions: We created PGI2-overexpressing hMSCs by introducing an active triple-catalytic enzyme that links cyclooxygenase-1 to prostacyclin synthase (COX-1-10aa-PGIS) based on our previous biochemical and structural studies of COX-1 and PGIS (Fig. 1a)7. This triple-catalytic enzyme catalyses three key reactions that allow the production of PGI2 from arachidonic acid as previously described8. We confirmed the stable expression of the transgene in PGI2-hMSCs by genomic PCR and western blot (a 130-kDa protein; Supplementary Fig. 1a,b). To assess the production of PGI2, we used an enzyme immunoassay to measure the metabolite 6-keto PGF1α. Compared with hMSCs (native hMSCs containing no vector) and 3.1-hMSCs (hMSCs containing pcDNA3.1 [vector used to construct pcDNA COX-1-10aa-PGIS]), PGI2-hMSCs were capable of secreting a fivefold higher concentration of 6-keto PGF1α in the supernatant after incubating cells with arachidonic acid for 20 min (Supplementary Fig. 1c; P<0.01; one-way analysis of variance [ANOVA]).

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