The Role of Endoglin in the Immunomodulatory Capacities of Mesenchymal Stem Cells and the Relationship to Hyperbaric Oxygen Therapy

Mesenchymal stem cells (MSCs), a type of “adult” stem cell found in most organs, represent an emerging tool in the field of regenerative medicine. In the setting of myocardial injury, for example, MSCs have been shown to promote repair and recovery. Studies have been hampered, however, by variation in MSC phenotypes, as defined by cell surface marker expression, and the absence of a clear consensus of what MSC phenotypes are best able to support regeneration. Providing insight into the regenerative capabilities of varying MSCs phenotypes is crucial to their continued success in therapies. One cell surface marker that has been shown to have functional relevance to regenerative medicine is endoglin (CD105). Endoglin is a type I transmembrane protein that functions as an auxiliary receptor for the TGFβ receptor complex. The absence of endoglin on MSCs reportedly defines a population with a greater propensity for cardiovascular differentiation and greater capacity to support myocardial repair. However, despite their ability to improve post-infarct cardiac function and reduce the size of resultant scars, the retention of these cells in myocardial tissue varies from only 3%-6%. Thus, transdifferentiation seems an unlikely explanation for the regenerative effects. In addition to their capacity for multipotential differentiation, MSCs also have a reported ability to suppress various immune responses, including suppression of stimulated T-cell proliferation. I hypothesized that the absence of CD105 may identify a population of MSCs with an altered capacity to modulate the immunologic milieu at the time of myocardial injury, and this difference in immunomodulatory function accounts for the improved outcomes. To investigate this idea, MSCs that either express endoglin (CD105+) or not (CD105-) were co-cultured with T-cells and effects on stimulated T-cell proliferation examined. Surprisingly, neither CD105+ nor CD105- MSCs were able to suppress proliferation of either CD4+ or CD8+ T-cells. In light of this observation, effects of MSCs on T-cell differentiation were assessed. Notably, co-culture of both CD105+ and CD105- MSCs with CD4+ T-cells showed a striking effect on T-cell differentiation. Syngeneic MSCs induced Th2 skewing, with increased expression of IL-4 and IL-10 and a marked decrease in IL-17 expression. The presence of CD105 in the MSCs influenced this outcome, with a more pronounced decrease in IL-17 expression and increased IL-4 secretion. Effects of allogeneic MSCs on T-cell differentiation were also examined. Due to the reported “immune privilege” of MSCs, it has been proposed that allogeneic MSCs may be utilized to expand the pool of MSCs available for clinical use. In an allogeneic co-culture system, both CD105+ and CD105- MSCs significantly affected T-cell differentiation. Compared to syngeneic MSCs, allogeneic MSCs stimulated higher expression of IL-4, IL-5, and IL-10, as well as increased secretion of IL-4 and IL-10. There were fewer differences between CD105+ and CD105- MSCs. However, CD105- MSCs induced much less IL-2 and IFNγ compared to CD105+ MSCs. These results indicate that MSCs influence T-cell differentiation, resulting in a Th2 skewing with increased production of the immunosuppressive cytokine IL-10 and, in the case of syngeneic cells, diminished Th17 differentiation. This effect could be essential for the previously described MSC-induced cardiac functional preservation, and could explain differences between CD105+ and CD105- phenotypes. In addition to these mechanistic studies, I also examined potential clinical relevance of CD105 expression in MSCs in umbilical cord blood transplantation. This question arose from a clinical study was underway at the University of Kansas Cancer Center that focused on using hyperbaric oxygen (HBO) for improving clinical outcomes post umbilical cord blood (UCB) transplant. In the observation of post-transplant transfusion requirements, HBO-treated patients required less supportive blood products than historic UCB-recipients. Furthermore, they experienced decreased time to transfusion independence. However, upon examining the correlation between levels of erythropoietin in these HBO-patients to their transfusion requirements, the data did not match with animal studies, which showed a reduction in erythropoietin was the causative method by which HBO improved engraftment. Therefore, we examined the supportive role of MSCs in response to HBO in the hematopoietic niche. This included observing changes in CD105 on MSCs after exposure to HBO, as endoglin is a known hypoxia response gene. While the observation of immunomodulatory factors produced by MSCs as a result of HBO is still underway, a downregulation of CD105 12 hours after exposure to HBO was observed. This correlates to the homing window of hematopoietic stem cells in the context of transplant and may be indicative of changes in MSCs providing a more supportive bone marrow microenvironment after exposure to HBO-therapy

Correction: STAT3 balances myocyte hypertrophy vis-à-vis autophagy in response to Angiotensin II by modulating the AMPKα/mTOR axis.

[This corrects the article DOI: 10.1371/journal.pone.0179835.]

STAT3 balances myocyte hypertrophy vis-à-vis autophagy in response to Angiotensin II by modulating the AMPKα/mTOR axis

<div><p>Signal transducers and activators of transcription 3 (STAT3) is known to participate in various cardiovascular signal transduction pathways, including those responsible for cardiac hypertrophy and cytoprotection. However, the role of STAT3 signaling in cardiomyocyte autophagy remains unclear. We tested the hypothesis that Angiotensin II (Ang II)-induced cardiomyocyte hypertrophy is effected, at least in part, through STAT3-mediated inhibition of cellular autophagy. In H9c2 cells, Ang II treatment resulted in STAT3 activation and cellular hypertrophy in a dose-dependent manner. Ang II enhanced autophagy, albeit without impacting AMPKα/mTOR signaling or cellular ADP/ATP ratio. Pharmacologic inhibition of STAT3 with WP1066 suppressed Ang II-induced myocyte hypertrophy and mRNA expression of hypertrophy-related genes ANP and β-MHC. These molecular events were recapitulated in cells with STAT3 knockdown. Genetic or pharmacologic inhibition of STAT3 significantly increased myocyte ADP/ATP ratio and enhanced autophagy through AMPKα/mTOR signaling. Pharmacologic activation and inhibition of AMPKα attenuated and exaggerated, respectively, the effects of Ang II on ANP and β-MHC gene expression, while concomitant inhibition of STAT3 accentuated the inhibition of hypertrophy. Together, these data indicate that novel nongenomic effects of STAT3 influence myocyte energy status and modulate AMPKα/mTOR signaling and autophagy to balance the transcriptional hypertrophic response to Ang II stimulation. These findings may have significant relevance for various cardiovascular pathological processes mediated by Ang II signaling.</p></div

Induction of myocyte autophagy by Ang II and STAT3 inhibition.

<p>(A) GFP-LC3 puncta (green) in H9c2 cells transfected with GFP-LC3 and treated with vehicle (Control) or Ang II and WP1066 alone or in combination. Scale bar = 10 μm. (B) Quantitation of GFP-LC3 autophagosomes (green puncta) in H9c2 cells treated with vehicle (Control) or Ang II and WP1066 alone or in combination. (C) Protein expression of autophagy markers p62 and LC3 in H9c2 cells treated with Ang II or/and WP1066. (D) Densitometric analysis of p62 and LC3 protein expression levels. Data represent mean ± SEM (n = 3). *<i>P</i><0.05 vs. control; ^#<i>P</i><0.05 vs. Ang II only.</p

AMPKα signaling is critical for the mediation of STAT3 influence on hypertrophy.

<p>(A) WP1066 and AICAR treatment for 48 h suppressed Ang II-induced ANP and β-MHC mRNA expression. Data represent mean ± SEM (n = 4); *<i>P</i><0.01 vs. Control; ^†<i>P</i><0.01 vs. Ang II alone; ^#<i>P</i><0.01 vs. Ang II + WP1066; ^§<i>P</i><0.05 vs. Ang II + AICAR. (B) Inhibition of AMPK with Compound C reversed the suppression of hypertrophy marker expression by WP1066 following Ang II treatment of H9c2 cells. Data represent mean ± SEM (n = 4); *<i>P</i><0.01 vs. Control; ^†<i>P</i><0.01 vs. Ang II alone; ^#<i>P</i><0.01 vs. Ang II + WP1066; and ^§<i>P</i><0.05 vs. Ang II + Compound C.</p

Exposure to Ang II induced myocyte hypertrophy and activated STAT3.

<p>(A) Representative photomicrographs of H9c2 cells stained with crystal violet, magnification: 20x; scale bar = 10 μm. (B) Cross-sectional cell surface area (n = 100 cells/group). (C) Time-course of Ang II-induced Tyr705 and Ser727 phosphorylation of STAT3 in H9c2 cells. (D) Densitometric data from Western immunoblotting. Levels of p-STAT3 (Y705), p-STAT3 (S727), STAT3 and p-JAK2 (Y1007/1008) were quantified and normalized relative to β-Actin. Data represent mean ± SEM (n = 4), *<i>P</i><0.05 vs. baseline (0h).</p

STAT3 modulates autophagy in H9c2 cells via AMPKα/mTOR pathway.

<p>(A) Representative Western immunoblots showing p-AMPKα, AMPKα, p-mTOR, mTOR, and β-actin protein expression in H9c2 cells treated with vehicle control, Ang II (5 μM), WP1066 (4μM) alone, and Ang II + WP1066 for 48 h. (B) The densitometric quantitation of p-AMPKα, AMPKα, p-mTOR and mTOR protein levels in H9c2 cells. (C) ADP/ATP ratio in H9c2 cells following indicated treatments. Data represent mean ± SEM (n = 3).*<i>P</i><0.05 vs. control; ^#<i>P</i><0.05 vs. Ang II only. (D) Representative Western immunoblots showing STAT3, p-AMPKα, AMPKα, p-mTOR, mTOR, and β-actin protein expression in H9c2 cells treated with scramble peptide control, STAT3 shRNA, scramble RNA + Ang II, and STAT3 shRNA + Ang II for 48 h.(E) The densitometric quantitation of p-AMPKα, AMPKα, p-mTOR and mTOR protein levels in H9c2 cells. (F) ADP/ATP ratio in STAT3-KD cells following Ang II treatment. Data represent mean ± SEM (n = 3).*<i>P</i><0.05 vs. control (scramble RNA); ^#<i>P</i><0.05 vs. STAT3-KD only.</p

Pharmacological inhibition of STAT3 attenuated Ang II-induced myocyte hypertrophy.

<p>(A) Representative photomicrographs of H9c2 cells stained with crystal violet. Magnification 20x; scale bar = 10 μm. (B) Cross-sectional cell surface area (n = 100 cells/group). (C and D) qRT-PCR analysis of ANP and β-MHC mRNA expression in Ang II-treated cells with or without STAT3 inhibition. Data represent mean ± SEM (n = 4), *<i>P</i><0.05 vs. control, ^#<i>P</i><0.05 vs. Ang II only.</p