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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

H19 lncRNA promoted host myoblasts survival and proliferation under hypoxia (1.5% O2).(a) Primary myoblasts co-cultured with PGI2-hMSCs or with 3.1-hMSCs+ILO for 24 h showed a significant increase in H19 lncRNA levels as compared with those co-cultured with 3.1-hMSCs. The increase in H19 lncRNA levels was transient; at 48 h, levels were comparable among all the groups. (b) No differences were seen in the total number of viable cells among myoblasts co-cultured with PGI2-hMSCs, 3.1-hMSCs+ILO or 3.1-hMSCs for 24 h. However, by 48 h, the number of viable myoblasts was significantly higher when cells were co-cultured with PGI2-hMSCs or 3.1-hMSCs+ILO than with 3.1-hMSCs. (c) H19 silencing reduced the paracrine effects of PGI2-hMSCs on myoblast survival. The total number of viable cells was significantly reduced in myoblasts with H19 deficiency (H19 KD) that were co-cultured with PGI2-hMSCs for 48 h as compared with control myoblasts co-cultured with PGI2-hMSCs in parallel. (d) The paracrine effects of PGI2-hMSCs on myoblast proliferation were also impaired with H19 silencing. Myoblasts with H19 deficiency that were co-cultured with PGI2-hMSCs for 48 h showed a significant reduction in cell proliferation as compared with control myoblasts co-cultured in parallel with PGI2-hMSCs. **P<0.01. Statistical significance was determined by one-way ANOVA with Newman–Keuls post hoc test (a,b) and a two-tailed t-test (c,d). Data are shown as mean±s.e.m. from three to four replicates and are representative of three independent experiments with similar results.
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f7: H19 lncRNA promoted host myoblasts survival and proliferation under hypoxia (1.5% O2).(a) Primary myoblasts co-cultured with PGI2-hMSCs or with 3.1-hMSCs+ILO for 24 h showed a significant increase in H19 lncRNA levels as compared with those co-cultured with 3.1-hMSCs. The increase in H19 lncRNA levels was transient; at 48 h, levels were comparable among all the groups. (b) No differences were seen in the total number of viable cells among myoblasts co-cultured with PGI2-hMSCs, 3.1-hMSCs+ILO or 3.1-hMSCs for 24 h. However, by 48 h, the number of viable myoblasts was significantly higher when cells were co-cultured with PGI2-hMSCs or 3.1-hMSCs+ILO than with 3.1-hMSCs. (c) H19 silencing reduced the paracrine effects of PGI2-hMSCs on myoblast survival. The total number of viable cells was significantly reduced in myoblasts with H19 deficiency (H19 KD) that were co-cultured with PGI2-hMSCs for 48 h as compared with control myoblasts co-cultured with PGI2-hMSCs in parallel. (d) The paracrine effects of PGI2-hMSCs on myoblast proliferation were also impaired with H19 silencing. Myoblasts with H19 deficiency that were co-cultured with PGI2-hMSCs for 48 h showed a significant reduction in cell proliferation as compared with control myoblasts co-cultured in parallel with PGI2-hMSCs. **P<0.01. Statistical significance was determined by one-way ANOVA with Newman–Keuls post hoc test (a,b) and a two-tailed t-test (c,d). Data are shown as mean±s.e.m. from three to four replicates and are representative of three independent experiments with similar results.

Mentions: To gain insight into the endogenous regenerative capability mediated by PGI2-hMSCs in a hostile microenvironment, we conducted in vitro co-culture mechanistic studies under hypoxia (1.5% O2). Myogenic progenitor cells (myoblasts) were used to assess the paracrine effects of PGI2-hMSCs because we observed muscle regeneration at day 14 and muscle mass gain at day 30 in PGI2-hMSC-treated mice. We chose to use transwell co-culturing of PGI2-hMSCs (resulting in a constant release of PGI2) and myoblasts rather than conditioned media from PGI2-hMSCs to study the paracrine effects because PGI2 is not stable, with a half-life of 1–2 min (ref. 16). Emerging evidence has shown that long noncoding RNAs contribute significantly to diverse biological functions such as cell proliferation, survival and differentiation17. Specifically, H19 lncRNA has been identified as an important factor in regulating skeletal muscle development1819. Dey et al. showed that H19 lncRNA encodes miR-675-3p and miR-675-5p, which stimulate myogenesis. H19 deficiency in skeletal muscle resulted in impaired regeneration and smaller myofiber size after injury18. Thus, we sought to determine whether H19 lncRNA was involved in PGI2-hMSC-induced myogenesis. To this end, we first assessed whether PGI2-hMSCs regulate H19 transcript levels in myoblasts under hypoxic stress by co-culturing proliferating primary myoblasts (isolated from the same strain of NOD/SCID mice used in in vivo functional assessments) with PGI2-hMSCs, 3.1-hMSCs+ILO or 3.1-hMSCs. We found a significant transient increase (more than twofold) in H19 transcript levels in myoblasts co-cultured with PGI2-hMSCs as compared with those co-cultured with 3.1-hMSCs (P<0.01; one-way ANOVA; Fig. 7a). This early increase in H19 lncRNA seen at 24 h was not maintained at 48 h (Fig. 7a). There was no difference in the total viable cells at 24 h; however, at 48 h, the number of viable myoblasts co-cultured with PGI2-hMSCs increased significantly as compared with those co-cultured with 3.1-hMSCs (7.67±0.10 × 104 versus 6.47±0.10 × 104 cells, P<0.01; one-way ANOVA; Fig. 7b). Co-culturing myoblasts with 3.1-hMSCs+ILO had similar effects on H19 upregulation and cell survival as did co-culturing with PGI2-hMSCs (Fig. 7a,b). Co-culture of PGI2-hMSCs significantly reduced the number of nonviable myoblasts at both 24 and 48 h (one-way ANOVA; Supplementary Fig. 3a). However, the total number of nonviable cells under all treatment conditions was 10-fold less than the number of viable cells (Fig. 7b; Supplementary Fig. 3a). Therefore, the increase in viable myoblasts at 48 h, after co-culture with PGI2-hMSCs or 3.1-hMSCs+ILO, is likely due mainly to increased cell proliferation, with a minor component due to cell survival.


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)

H19 lncRNA promoted host myoblasts survival and proliferation under hypoxia (1.5% O2).(a) Primary myoblasts co-cultured with PGI2-hMSCs or with 3.1-hMSCs+ILO for 24 h showed a significant increase in H19 lncRNA levels as compared with those co-cultured with 3.1-hMSCs. The increase in H19 lncRNA levels was transient; at 48 h, levels were comparable among all the groups. (b) No differences were seen in the total number of viable cells among myoblasts co-cultured with PGI2-hMSCs, 3.1-hMSCs+ILO or 3.1-hMSCs for 24 h. However, by 48 h, the number of viable myoblasts was significantly higher when cells were co-cultured with PGI2-hMSCs or 3.1-hMSCs+ILO than with 3.1-hMSCs. (c) H19 silencing reduced the paracrine effects of PGI2-hMSCs on myoblast survival. The total number of viable cells was significantly reduced in myoblasts with H19 deficiency (H19 KD) that were co-cultured with PGI2-hMSCs for 48 h as compared with control myoblasts co-cultured with PGI2-hMSCs in parallel. (d) The paracrine effects of PGI2-hMSCs on myoblast proliferation were also impaired with H19 silencing. Myoblasts with H19 deficiency that were co-cultured with PGI2-hMSCs for 48 h showed a significant reduction in cell proliferation as compared with control myoblasts co-cultured in parallel with PGI2-hMSCs. **P<0.01. Statistical significance was determined by one-way ANOVA with Newman–Keuls post hoc test (a,b) and a two-tailed t-test (c,d). Data are shown as mean±s.e.m. from three to four replicates and are representative of three independent experiments with similar results.
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Related In: Results  -  Collection

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f7: H19 lncRNA promoted host myoblasts survival and proliferation under hypoxia (1.5% O2).(a) Primary myoblasts co-cultured with PGI2-hMSCs or with 3.1-hMSCs+ILO for 24 h showed a significant increase in H19 lncRNA levels as compared with those co-cultured with 3.1-hMSCs. The increase in H19 lncRNA levels was transient; at 48 h, levels were comparable among all the groups. (b) No differences were seen in the total number of viable cells among myoblasts co-cultured with PGI2-hMSCs, 3.1-hMSCs+ILO or 3.1-hMSCs for 24 h. However, by 48 h, the number of viable myoblasts was significantly higher when cells were co-cultured with PGI2-hMSCs or 3.1-hMSCs+ILO than with 3.1-hMSCs. (c) H19 silencing reduced the paracrine effects of PGI2-hMSCs on myoblast survival. The total number of viable cells was significantly reduced in myoblasts with H19 deficiency (H19 KD) that were co-cultured with PGI2-hMSCs for 48 h as compared with control myoblasts co-cultured with PGI2-hMSCs in parallel. (d) The paracrine effects of PGI2-hMSCs on myoblast proliferation were also impaired with H19 silencing. Myoblasts with H19 deficiency that were co-cultured with PGI2-hMSCs for 48 h showed a significant reduction in cell proliferation as compared with control myoblasts co-cultured in parallel with PGI2-hMSCs. **P<0.01. Statistical significance was determined by one-way ANOVA with Newman–Keuls post hoc test (a,b) and a two-tailed t-test (c,d). Data are shown as mean±s.e.m. from three to four replicates and are representative of three independent experiments with similar results.
Mentions: To gain insight into the endogenous regenerative capability mediated by PGI2-hMSCs in a hostile microenvironment, we conducted in vitro co-culture mechanistic studies under hypoxia (1.5% O2). Myogenic progenitor cells (myoblasts) were used to assess the paracrine effects of PGI2-hMSCs because we observed muscle regeneration at day 14 and muscle mass gain at day 30 in PGI2-hMSC-treated mice. We chose to use transwell co-culturing of PGI2-hMSCs (resulting in a constant release of PGI2) and myoblasts rather than conditioned media from PGI2-hMSCs to study the paracrine effects because PGI2 is not stable, with a half-life of 1–2 min (ref. 16). Emerging evidence has shown that long noncoding RNAs contribute significantly to diverse biological functions such as cell proliferation, survival and differentiation17. Specifically, H19 lncRNA has been identified as an important factor in regulating skeletal muscle development1819. Dey et al. showed that H19 lncRNA encodes miR-675-3p and miR-675-5p, which stimulate myogenesis. H19 deficiency in skeletal muscle resulted in impaired regeneration and smaller myofiber size after injury18. Thus, we sought to determine whether H19 lncRNA was involved in PGI2-hMSC-induced myogenesis. To this end, we first assessed whether PGI2-hMSCs regulate H19 transcript levels in myoblasts under hypoxic stress by co-culturing proliferating primary myoblasts (isolated from the same strain of NOD/SCID mice used in in vivo functional assessments) with PGI2-hMSCs, 3.1-hMSCs+ILO or 3.1-hMSCs. We found a significant transient increase (more than twofold) in H19 transcript levels in myoblasts co-cultured with PGI2-hMSCs as compared with those co-cultured with 3.1-hMSCs (P<0.01; one-way ANOVA; Fig. 7a). This early increase in H19 lncRNA seen at 24 h was not maintained at 48 h (Fig. 7a). There was no difference in the total viable cells at 24 h; however, at 48 h, the number of viable myoblasts co-cultured with PGI2-hMSCs increased significantly as compared with those co-cultured with 3.1-hMSCs (7.67±0.10 × 104 versus 6.47±0.10 × 104 cells, P<0.01; one-way ANOVA; Fig. 7b). Co-culturing myoblasts with 3.1-hMSCs+ILO had similar effects on H19 upregulation and cell survival as did co-culturing with PGI2-hMSCs (Fig. 7a,b). Co-culture of PGI2-hMSCs significantly reduced the number of nonviable myoblasts at both 24 and 48 h (one-way ANOVA; Supplementary Fig. 3a). However, the total number of nonviable cells under all treatment conditions was 10-fold less than the number of viable cells (Fig. 7b; Supplementary Fig. 3a). Therefore, the increase in viable myoblasts at 48 h, after co-culture with PGI2-hMSCs or 3.1-hMSCs+ILO, is likely due mainly to increased cell proliferation, with a minor component due to cell survival.

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