**8. Current research on the RAS in cardiac stem cells**

Regarding the intracellular signalling pathway of Ang II, RAS effect on cardiovascular stem/progenitor cell transplantation has largely been investigated. Among the regenerative medicine-based therapies in the cardiovascular system, induced pluripotent stem cells (iPSCs), which are artificially derived from an adult non-differentiated somatic cell, are a field of research study. In spite of different origin, they resemble ESCs in their growth and gene expression profile [57]. Also, Ang II receptors are expressed in iPSCs, which induce the proliferation and differentiation of pluripotent stem cells to several kinds of stem cells. As mentioned before, Ang II stimulates cell-signalling cascade through ROS production which in turn instigates stem cell proliferation [58]. In a study, the administration of Tempol (ROS generation-blocking agent) in Ang II-treated pluripotent stem cells has attenuated the proliferation of stem cells and DNA synthesis suggesting the role of oxidative-signalling pathway in RAS-associated cell proliferation. The other signalling pathway linked to the differentiation of iPSCs and Ang II is JAK/STAT pathway [59].

Ang II is also able to induce ESCs differentiation. In this context, the effect of AT1 receptor activation on collagen IV protein has been investigated [18]. Collagen IV is an extracellular matrix protein having a role in cell adhesion, growth, migration and differentiation. Collagen IV has been shown to be involved in the differentiation of ESCs to smooth muscle cell.

Up-regulation of several transcription factors such as egr-1, c-fos/c-jun, Stat91, NFk-B, which has a fundamental role in stem cell differentiation, is mediated through PI3/Akt pathway. Ang II is the upstream cascade of PI3/Akt82-84. NFk-B is markedly up-regulated in Ang II-treated cells, proposing that there is NFk-B involvement in ESC differentiation into the smooth muscle cells [60].

The TGF-β/Smad pathway plays a key role in the cellular responses to Ang II. Ang II activates TGF-β secretion in various tissues, such as fibroblasts and smooth muscle cells that induce interstitial fibrosis in the heart and kidney. Besides, TGF-β/Smad pathway is highly engaged to vascular fibrosis and arteriosclerosis [61] and gives rise to the differentiation of MSCs to smooth muscle cell. Furthermore, TGF-β secretion is connected with the MAPK/ERK cascade, and Ang II in this pathway interferes with TGF-β production, thus leading to the differentiation of MSCs to smooth muscle cells [62].

Regarding owning various paracrine effects, MSC transplantation has gained great importance in cardiovascular disease [63]. The supportive effects of vascular VEGF have been recognized in the migration, invasion of extracellular matrix, proliferation, survival of MSCs, and they contribute to MSCs' paracrine effects [64, 65]. In this context, all pathways increasing VEGF would give rise to the function of MSCs. Ang II increases VEGF mRNA and protein expression in MSCs [20], which is associated with Akt-signalling pathway. Pre-treatment of MSCs with the Akt inhibitor (LY292002) has been shown to reduce Ang II-induced VEGF expression. So, local Ang II, as a cytokine, might boost VEGF generation in MSC grafts and upgrade the transplantation effectiveness.

The excess RAS expression is detected in CVDs such as myocardial infarction, hypertension, heart failure and atherosclerosis [66]. On the other hand, RAS inhibition via ACE inhibitors (ACEIs) and angiotensin receptor blockers (ARBs) has been widely examined in cardiovascular disease beyond their effects in lowering the blood pressure [67]. Despite the daily increasing use of regenerative medicine in treating different disorders, the functionality of stem cell transplantation is not sufficient in animal models and clinical trials [68].

Therefore, clarifying the mechanisms that enhance the graft efficacy seems important. Researchers have investigated the effect of RAS inhibition on stem cell therapy of cardiovascular system [69].

The insulin-like growth factor 1-1 secreted from stem cells has a close relationship with the RAS and down-regulates the local RAS through the attenuation of the p53 gene [70]. The IGF-1 has an anti-apoptotic effect on cardiomyocytes in ischaemic heart disease and also enhances differentiation and survival of stem cells after transplantation [71]. In acute MI in cardiomyocytes, ACEIs up-regulate the IGF-1 receptors; thus, the concurrent use of perindopril in bone marrow stem cell transplantation increases the paracrine effects of the IGF-1, which abolishes apoptosis through increased Bcl2 expression and improves cardiac function [72]. Also, pretreatment of MSCs with ARBs before transplantation increases their trans-differentiation efficacy and also improves the systolic function of the heart [73].
