Regulation of Cardiomyocyte Differentiation, Angiogenesis, and Inflammation by the Delta Opioid Signaling in Human Mesenchymal Stem Cells
- 6 Downloads
Lack of angiogenesis, inflammation, and low differentiation of MSCs upon transplantation to ischemic site are major drawbacks in cell therapies for myocardial tissue regeneration. To overcome these problems, this study aimed to increase the angiogenesis, anti-inflammation, and differentiation of human umbilical cord MSCs (hMSCs) into cardiomyocytes by activating the delta opioid pathway by its agonist SNC80 with the combination of zebularine, activin A and oxytocin.
Materials and Methods
Human umbilical cord MSCs were seeded on gelatin-coated plates by using alpha MEM. MSCs were treated with SNC80 and the combination of zebularine, activin A and oxytocin. Media and inducers were changed every 3 days once until 21 days. Spent media collected from day 7 and day 21 was used to estimate angiogenic (VEGF) and anti- or pro-inflammatory cytokines (IL-10, IL1b, IL-6) levels by ELISA. The effect of spent media on in vitro endothelial tube formation and anti-inflammatory effect on inflammation induced with LPS on macrophages was also studied along with cytokine levels. Proteins that were isolated from day 7 and day 21 were used to study for cardiomyocyte markers (GATA4, MEF2C, connexin 43, and KCNJ2) and NOTCH1 involved in cardiomyocyte differentiation using Western blot.
Activation of DOR increases the VEGF secretion levels estimated by ELISA and increases the in vitro tube formation in terms of number of loops and total length. Delta opioid receptor (DOR)-activated conditioned media secreted anti-inflammatory cytokine IL-10, which could prevent the secretion of pro-inflammatory cytokines IL-1b and IL-6 from LPS induced macrophages. DOR activated MSCs upregulated the early and late cardiomyocytes markers (GATA4, MEF2C and connexin 43, KCNJ43) via NOTCH1 signaling.
Activation of DOR on hMSCs plays a vital role in angiogenesis, anti-inflammation, and cardiomyocyte differentiation by regulating the NOTCH1 signaling pathway.
Our results indicate that, upon activation of delta opioid receptors on human umbilical cord derived mesenchymal stem cells, they can differentiate into heart cells (by expressing specific markers GATA4, MEF2C, Connexin43), show enhanced formation of blood vessels and anti-inflammatory activity. This approach may help to treat pateints post heart attack.
KeywordsHuman umbilical cord mesenchymal stem cell SNC80 Angiogenesis Inflammation Cardiomyocytes NOTCH1 signaling
This study was funded by VIT SEED Fund awarded to DS.
- 3.Mullick M, Sen D. The delta opioid peptide DADLE represses hypoxia-reperfusion mimicked stress mediated apoptotic cell death in human mesenchymal stem cells in part by downregulating the unfolded protein response and ROS along with enhanced anti-inflammatory effect. Stem Cell Rev. 2018;14(4):558–73.CrossRefGoogle Scholar
- 6.Sinova R, Kudova J, Nesporova K, Karel S, Sulakova R, Velebny V, et al. Opioid receptors and opioid peptides in the cardiomyogenesis of mouse embryonic stem cells. J Cell Physiol 2018.Google Scholar
- 8.Shrivastava P, Cabrera MA, Chastain LG, Boyadjieva NI, Jabbar S, Franklin T, et al. Mu-opioid receptor and delta-opioid receptor differentially regulate microglial inflammatory response to control proopiomelanocortin neuronal apoptosis in the hypothalamus: effects of neonatal alcohol. J Neuroinflammation. 2017;14(1):83.CrossRefGoogle Scholar
- 16.Khanabdali R, Saadat A, Fazilah M, Bazli KF, Qazi RE, Khalid RS, et al. Promoting effect of small molecules in cardiomyogenic and neurogenic differentiation of rat bone marrow-derived mesenchymal stem cells. Drug Des Devel Ther. 2015;10:81–91.Google Scholar
- 22.Yang Y, Yan X, Xue J, Zheng Y, Chen M, Sun Z, Liu T., Wang C., You H., Luo D. Connexin43 dephosphorylation at serine 282 is associated with connexin43-mediated cardiomyocyte apoptosis. Cell Death Differ 2019.Google Scholar
- 28.Iwaszkiewicz K, Schneider J, Hua S. Targeting peripheral opioid receptors to promote analgesic and anti-inflammatory actions. Front Pharmacol [Mini Review]. 2013;4:132.Google Scholar