Previous studies showed that transplantation of MSCs in the heart after MI leads to small but significant functional improvements . Understanding the molecular mechanisms by which MSCs promote cardiac function, especially in the oxidative microenvironment after MI, will greatly aid in improving efficacy of stem cell-based therapies. After myocardial infarction, elevated levels of ROS have been found at the infarct site , suggesting that ROS such as H2O2 might influence the differentiation and function of implanted MSCs. As substantial amounts of ROS have been found in the area at risk after MI , and as ROS have been identified to play a critical role in the differentiation of other stem cell types [24, 25], we chose to study the effect of H2O2 on MSC differentiation in vitro.
Here we showed that MSCs that were subjected to pulses of pathophysiologic levels of H2O2 for 1 week or continuous H2O2 produced by oxidation of glucose in the extracellular media by GOX for 48 hours increased the expression of early cardiac and endothelial genes with decreased expression of early smooth muscle genes.
Although only a twofold increase in cardiac markers is observed with H2O2 treatment, addition of GOX results in a more robust increase of 30-fold for αMHC and 75-fold for nkx2-5, which are comparable to neonatal rat ventricular cardiomyocytes, with 40-fold and 100-fold higher expression of αMHC and nkx2-5, respectively, when compared with untreated MSCs. These results were in agreement with those reported for human embryonic stem cells (ESCs) . Similar results were obtained when adult heart-resident cardiac progenitor cells (CPCs) were treated with GOX. These data demonstrate redox-sensitive alteration in cardiogenic gene expression in MSCs and CPCs.
Our results also demonstrate that only high levels of exogenous H2O2 (100 μM) and high concentrations of GOX (5 mU/ml) were able to regulate expression of Notch1 and cardiogenic genes. We believe this very narrow threshold effect may be due to a combination of factors, such as presence of basal H2O2 and constitutive expression of antioxidant enzymes by cells. Unpublished data from our laboratory demonstrate basal H2O2 levels of 1 μM in cultured stem cells, as measured with electron paramagnetic spin resonance. Recent reports demonstrate that many stem cells, including MSCs, contain higher levels of antioxidants [26, 27]. We measured H2O2 levels in stem cells after addition of 100 μM H2O2 and found that the concentration reduced to 12 μM, within an hour, indicating rapid scavenging of exogenous oxidants.
Finally, recent data from human MSCs determined higher levels of catalase and glutathione peroxidase, with no changes in superoxide dismutase compared with other stem cells and fully differentiated cells . In that study, a threshold response with human MSCs demonstrated almost 80% survival at 4 mM H2O2, decreasing to <10% at 8 mM. Our studies indicate robust survival at 100 μM H2O2 and 5 mU/ml GOX with cellular responses, but showed higher concentrations to be potentially cytotoxic. Taken together, these data demonstrate that many cells, especially stem and progenitor, have threshold responses with small windows of dose-responses.
Next, we investigated whether H2O2 regulates any signaling pathway involved in stem cell differentiation. One of the signaling pathways that greatly influence stem cell differentiation is the Notch signaling pathway . Therefore, we investigated whether interplay existed between H2O2 and Notch1 signaling pathways. Interestingly, the mRNA level of Notch1 as well as proteolytic cleavage of the Notch1 intracellular domain (NICD) was upregulated by treatment with 100 μM either H2O2 or GOX, suggesting that high levels of H2O2 affect expression of both the mRNA and protein activity of Notch1. Although only a 1.5-fold increase in mRNA and twofold increase in NICD protein is observed, reports suggest that very small changes in Notch1 activation are sufficient to induce Notch1 signaling . Furthermore, this increase in Notch1 also significantly increased mRNA expression of downstream targets of Notch1. As MSCs were pulsed with H2O2 for 1 week, discontinuous oxidative stress resulted in small fold changes in cardiac gene expression. Although these changes may not represent true differentiation, they suggest that H2O2 levels influence cardiac gene expression in MSCs.
Although upregulation of cardiac and endothelial genes by Notch1 signaling may appear to be counterintuitive, given the role of Notch signaling in suppressing cardiomyogenesis in ESCs , upregulation of nkx2-5 and vWF is consistent with reports indicating involvement of Notch1 signaling in regulating these genes in cardiac progenitor cells and bone marrow stromal cells, respectively [18, 29]. Our observation that treatment with 100 μM H2O2 or 5 mU/ml GOX decreased expression of the Notch1 ligand Jagged1 in MSCs is consistent with previous reports of an inverse relation between expression levels of Notch1 and Jagged 1 in other cell types .
As we observed changes in cardiogenic gene expression at the mRNA level, we sought to determine whether these changes translated correspondingly at the protein level. Flow analysis of GOX-treated MSCs indicated that a small number of MSCs have high expression of αMHC, along with increased Flt1 expression and decreased sm α-actin expression. These results suggest that GOX treatment increases the frequency αMHC- and Flt1-positive cells while decreasing sm α-actin-positive cells.
The mechanism of upregulation of Notch1 activation by H2O2 may be due to activation of enzymes involved in Notch1 cleavage and processing. It is possible that H2O2 may increase Notch1 activation via γ-secretase activation, as H2O2-mediated increase in γ-secretase activation has been demonstrated in the pathogenesis of Alzheimer disease . Pharmacologic inhibition of γ-secretase activity by using DAPT inhibits Notch1 activation in different stem cells [29, 32]. Therefore, to determine whether H2O2 regulates cardiogenic gene expression in MSCs through Notch1 signaling, we blocked Notch1 activation daily by using DAPT and analyzed expression of the different cardiogenic markers in the presence and absence of 100 μM H2O2. Among the markers analyzed, the increase in expression of the high-affinity VEGF receptor Flt1 and the cardiac marker αMHC observed with 100 μM H2O2 was abrogated by co-treatment with DAPT, indicating that H2O2 regulates Flt1 and αMHC expression through Notch1 signaling. We attempted knockdown of Notch1 by using siRNA; however, this was not successful, as siRNAs that significantly reduced Notch1 gene expression greatly reduced cell survival over the 1-week period of treatment.
To determine whether glucose oxidase (GOX) mediated acute changes in cardiogenic gene expression through Notch1 signaling, MSCs were transfected with siNotch1 along with GOX for 48 hours. Treatment with siNotch1 showed a strong trend toward decreasing the GOX-mediated increase in αMHC and Flt1, whereas no effect was observed on smooth muscle gene expression by addition of GOX ± siNotch1. As ADAM17 is involved in Notch1 processing, MSCs were pretreated with an ADAM17 inhibitor. No effect was observed on H2O2-induced gene expression, nor was the expression or activity of ADAM17 altered by H2O2 treatment, suggesting the importance of the γ-secretase component of this pathway (Additional file 1, Figure S4). Further, treatment with GOX increased expression of enzymes involved in processing of Notch1 and Jagged1, such as Mfng, Lfng, and Neurl, indicating that H2O2 influences both notch1 cleavage and processing enzymes.
Expression of smooth muscle markers decreased significantly on treatment with 100 μM H2O2. Inhibition of Notch1 also decreased basal expression of smooth muscle markers, in keeping with prior findings . Interestingly, co-treatment of MSCs with H2O2 and DAPT resulted in a further decrease in smooth muscle markers. This indicates that H2O2 decreases smooth muscle gene expression through a parallel pathway, and that activation of Notch serves as a compensatory mechanism to stabilize smooth muscle gene expression.
Finally, expression of vimentin was also decreased by both H2O2 and GOX treatment. Although vimentin is expressed in many cell types, it is most prevalent in fibroblasts and is thought to be a partial marker of fibroblastic lineage ; lower expression could lead to decreased fibrosis.
To understand the mechanism by which oxidative stress mediated by GOX resulted in robust increases in cardiogenic gene expression, the expression of Notch1-related genes in GOX-treated MSCs was analyzed with a PCR array. Interestingly, Wnt11 expression was increased in GOX-treated MSCs. Wnt11 signaling has been shown to promote cardiomyogenic differentiation of human endothelial progenitor cells and mouse marrow mononuclear cells [35, 36]. Moreover, Wnt signaling has been identified as a downstream target of Notch1 that regulates expression of cardiac transcription factors during mouse cardiogenesis and is essential for cardiac development [37, 38]. MSCs overexpressing Wnt11 have been shown to be cardioprotective after oxidative stress in rats through increased cardiac gene expression and release of paracrine factors [39, 40].
Of note, MSCs used in this study were a heterogenous mix of cells present in the adult rat bone marrow. It is unclear whether one particular lineage in the heterogenous mix is most responsible for these changes or whether Notch1 is activated in all these cell types. Although published literature suggests that all these cells express Notch [18, 41], the optimal cell type must be determined.