Dickkopf Homolog 3 Induces Stem Cell Differentiation into Smooth Muscle Lineage via ATF6 Signalling

Smooth muscle cells (SMCs) are a key component of healthy and tissue engineered vessels and play a crucial role in vascular development and the pathogenic events of vascular remodeling i.e. restenosis. However, the cell source from which they can be isolated is limited. Embryonic stem (ES) cells that have the remarkable capability to differentiate into vascular SMCs in response to specific stimuli provide a useful model for studying SMC differentiation. Previous studies suggested that dickkopf homolog 3 (DKK3) has a role in human partially induced pluripotent stem cell to SMC differentiation. Here, we demonstrate that the expression of DKK3 is essential for the expression of SMC markers and myocardin at both the mRNA and protein levels during mouse ES cell differentiation into SMCs (ESC-SMC differentiation). Overexpression of DKK3 leads to further up-regulation of the aforementioned markers. Further investigation indicates that DKK3 added as a cytokine activates activating transcription factor 6 (ATF6), leading to the increased binding of ATF6 on the myocardin promoter and increased its expression. In addition, inhibition of extracellular signal-regulated kinases 1/2 (ERK1/2) promotes the expression of ATF6 and leads to further increase of myocardin transcription. Our findings offer a novel mechanism by which DKK3 regulates ESC-SMC differentiation by activating ATF6 and promoting myocardin expression

Origin and differentiation of vascular smooth muscle cells

Vascular smooth muscle cells (SMCs), a major structural component of the vessel wall, not only play a key role in maintaining vascular structure but also perform various functions. During embryogenesis, SMC recruitment from their progenitors is an important step in the formation of the embryonic vascular system. SMCs in the arterial wall are mostly quiescent but can display a contractile phenotype in adults. Under pathophysiological conditions, i.e. vascular remodelling after endothelial dysfunction or damage, contractile SMCs found in the media switch to a secretory type, which will facilitate their ability to migrate to the intima and proliferate to contribute to neointimal lesions. However, recent evidence suggests that the mobilization and recruitment of abundant stem/progenitor cells present in the vessel wall are largely responsible for SMC accumulation in the intima during vascular remodelling such as neointimal hyperplasia and arteriosclerosis. Therefore, understanding the regulatory mechanisms that control SMC differentiation from vascular progenitors is essential for exploring therapeutic targets for potential clinical applications. In this article, we review the origin and differentiation of SMCs from stem/progenitor cells during cardiovascular development and in the adult, highlighting the environmental cues and signalling pathways that control phenotypic modulation within the vasculature

Generation and grafting of tissue-engineered vessels in a mouse model

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. The current protocol presents an efficient and reproducible strategy in which functional tissue engineered vessel grafts can be generated using partially induced pluripotent stem cell (PiPSC) from human fibroblasts. We designed a decellularized vessel scaffold bioreactor, which closely mimics the matrix protein structure and blood flow that exists within a native vessel, for seeding of PiPSC-endothelial cells or smooth muscle cells prior to grafting into mice. This approach was demonstrated to be advantageous because immune-deficient mice engrafted with the PiPSC-derived grafts presented with markedly increased survival rate 3 weeks after surgery. This protocol represents a valuable tool for regenerative medicine, tissue engineering and potentially patient-specific cell-therapy in the near future

Endothelial lineage differentiation from induced pluripotent stem cells is regulated by microRNA-21 and transforming growth factor β2 (TGF-β2) pathways.

Finding a suitable cell source for endothelial cells (ECs) for cardiovascular regeneration is a challenging issue for regenerative medicine. In this paper, we describe a novel mechanism regulating induced pluripotent stem cells (iPSC) differentiation into ECs, with a particular focus on miRNAs and their targets. We first established a protocol using collagen IV and VEGF to drive the functional differentiation of iPSCs into ECs and compared the miRNA signature of differentiated and undifferentiated cells. Among the miRNAs overrepresented in differentiated cells, we focused on microRNA-21 (miR-21) and studied its role in iPSC differentiation. Overexpression of miR-21 in predifferentiated iPSCs induced EC marker up-regulation and in vitro and in vivo capillary formation; accordingly, inhibition of miR-21 produced the opposite effects. Importantly, miR-21 overexpression increased TGF-2 mRNA and secreted protein level, consistent with the strong up-regulation of TGF-2 during iPSC differentiation. Indeed, treatment of iPSCs with TGF-2 induced EC marker expression and in vitro tube formation. Inhibition of SMAD3, a downstream effector of TGF-2, strongly decreased VE-cadherin expression. Furthermore,TGF-2 neutralization and knockdown inhibited miR-21-induced EC marker expression. Finally, we confirmed the PTEN/Akt pathway as a direct target of miR-21, and we showed that PTEN knockdown is required for miR-21-mediated endothelial differentiation. In conclusion, we elucidated a novel signaling pathway that promotes the differentiation of iPSC into functional ECs suitable for regenerative medicine applications

Sirolimus Stimulates Vascular Stem/Progenitor Cell Migration and Differentiation Into Smooth Muscle Cells via Epidermal Growth Factor Receptor/Extracellular Signal-Regulated Kinase/β-Catenin Signaling Pathway

Objective— Sirolimus-eluting stent therapy has achieved considerable success in overcoming coronary artery restenosis. However, there remain a large number of patients presenting with restenosis after the treatment, and the source of its persistence remains unclarified. Although recent evidence supports the contribution of vascular stem/progenitor cells in restenosis formation, their functional and molecular responses to sirolimus are largely unknown. Approach and Results— Using an established technique, vascular progenitor cells were isolated from adventitial tissues of mouse vessel grafts and purified with microbeads specific for stem cell antigen-1. We provide evidence that vascular progenitor cells treated with sirolimus resulted in an induction of their migration in both transwell and wound healing models, clearly mediated by CXCR4 activation. We confirmed the sirolimus-mediated increase of migration from the adventitial into the intima side using an ex vivo decellularized vessel scaffold, where they form neointima-like lesions that expressed high levels of smooth muscle cell (SMC) markers (SM-22α and calponin). Subsequent in vitro studies confirmed that sirolimus can induce SMC but not endothelial cell differentiation of progenitor cells. Mechanistically, we showed that sirolimus-induced progenitor-SMC differentiation was mediated via epidermal growth factor receptor and extracellular signal–regulated kinase 1/2 activation that lead to β-catenin nuclear translocation. The ablation of epidermal growth factor receptor, extracellular signal–regulated kinase 1/2, or β-catenin attenuated sirolimus-induced SM-22α promoter activation and SMC differentiation. Conclusions— These findings provide direct evidence of sirolimus-induced progenitor cell migration and differentiation into SMC via CXCR4 and epidermal growth factor receptor/extracellular signal–regulated kinase/β-catenin signal pathways, thus implicating a novel mechanism of restenosis formation after sirolimus-eluting stent treatment. </jats:sec