Adult mesenchymal cells are multipotent cells and can be differentiated into different types of specialized mesenchymal cells such as osteoblasts, chondrocytes, adipocytes, and other cells (Caplan, 1991). Mesenchymal stem cells have been shown to be actively involved in tissue repair and transplantation. These cells perform this role through their potential to regulate immune cells (Aggarwal & Pittenger, 2005). Another important feature of mesenchymal cells is their ability to inhibit immune responses by inhibiting dendritic cells, and B and T lymphocytes (Jiang et al., 2005). This has made mesenchymal stem cells attractive candidates for tissue repair in medicine (S.-H. Yang et al., 2009). Some studies have shown the role of MSCs in the treatment of certain types of diseases in both in vivo and in vitro conditions. For example, Lei Chen et al. in 2014 referred to the role of mesenchymal cells in hypoxic conditions for wound healing and stated that the factors secreted by mesenchymal stem cells, especially VEGF-A and bFGF, could play an effective role in wound healing (Chen et al., 2014a).
One of the most common elements of tissue injury is the presence of hypoxia. It is known that reduction in oxygen tension in a variety of tissues leads to increased levels of the hypoxia inducible factor (HIF-1), which induces transcription of a wide variety of genes including angiogenic genes such as vascular endothelial growth factor (VEGF) (Ahluwalia & S Tarnawski, 2012; Berniakovich & Giorgio, 2013), as well as the MSC chemoattractant stromal cell-derived factor 1 (SDF-1) (Youn et al., 2011).
HIF-1 is a transcription factor that is present in almost all cell types and is regulated by oxygen levels, causing hundreds of genes to be up-regulated. HIF-1 is a heterodimer and consists of α and β subunits. HIF-1β constitutively expressed in both normoxia and hypoxia, whereas HIF-1α is present in very small amounts under normoxia. HIF-1 acts by binding to regulatory elements called Hypoxia Responsive Elements (HREs) within target genes. To do this, HIF-1α must first dimerise with HIF-1β. When oxygen levels are high, levels of HIF-1α are limiting as it is degraded in a process involving oxygen-dependent hydroxylation and subsequent ubiquitination by the Von Hippel-Lindau protein (VHL) and therefore cannot bind to HIF-1β (Lofstedt & Nilsson, 2006). The results of the present study showed the HIF-1α protein was abundant in MSC in hypoxia-mimetic conditions, but was present at very low levels in normoxia. Research by Ying-Wei Lan et al. has shown similar results (Lan et al., 2015) .
Several groups have demonstrated the relevance of hypoxia to MSC growth factor production in vitro. For example, exposure of bone marrow (BM)-MSC to 24 hours of hypoxia (1% oxygen) resulted in marked induction of VEGF, Fibroblast growth factor 2 (FGF-2), Hepatocyte growth factor (HGF), and Insulin like growth factor 1 (IGF-1) production, in an NF-kappa β dependent manner (Crisostomo et al., 2008). The stimulation of growth factor production by hypoxia is not specific to BM-MSC and has also been demonstrated in MSC derived from adipose tissue (Rasmussen et al., 2011), placenta (Yust-Katz et al., 2012), and dental pulp (Iida et al., 2010). Furthermore, hypoxic stimulation of angiogenic and anti-apoptotic factors such as VEGF, FGF-2, HGF and IGF-1 has been reported to also occur in MSC from aged animals, supporting clinical utility (Efimenko, Starostina, Kalinina, & Stolzing, 2011).
The results of the present study showed that the expression of IL-10, TGF-β, VEGF and versican genes in hypoxia-mimetic conditions was significantly higher than normoxia. Our study also found that the fold change of IL-10 in mesenchymal cells derived from adipose tissue was significantly higher than in bone marrow, but the fold change of TGF-β, VEGF and versican in bone marrow MSCs was significantly higher. Nasef et al. have shown that the production and secretion of IL-10 by mesenchymal stem cells can inhibit the proliferation and activity of T lymphocyte cells (Nasef et al., 2006). In 2017, Volchenkov et al. showed that Th17 cells increase the expression of IL-10 gene under hypoxia (Volchenkov, Nygaard, Sener, & Skålhegg, 2017). In 2014, Lei Chen et al. reported that the production of anti-inflammatory factors such as IL-10 and TGF-β in MSCs under hypoxia was significantly higher than innormoxia, and these factors could activate regulatory T cells (Treg), which leads to a reduction of inflammation and cytotoxic T lymphocyte activity (Chen et al., 2014b). Rehman J et al. in 2004 showed that adipose tissue-derived mesenchymal cells expressed factors such as HGF, VEGF, TGF-β, fibroblast growth factor (bFGF and FGF2) and macrophage-granulocyte colony stimulating factor (GM-CSF) and the expression of these molecules increases under hypoxia. In particular, the increase in VEGF expression in hypoxia is higher than other factors (Rehman et al., 2004). Studies conducted by Tischer et al. in 1991 showed that the VEGF promoter has a binding site for the HIF-1 transcription factor (Wang, Jiang, Rue, & Semenza, 1995). Forsythe et al. showed that transcription of the VEGF gene under hypoxia was increased due to the stabilization of HIF-1α (Forsythe et al., 1996). The VEGF gene is one of the genes regulated by the HIF-1 transcription factor. Interestingly, various studies have shown that VEGF can increase the expression of the TGF-β gene in a variety of cells (Shih & Claffey, 2001).
We also investigated the regulation of versican in MSCs by hypoxia-mimetic conditions which induce HIF-1. Versican is an extracellular matrix proteoglycan and is one of the genes that respond to hypoxia (Sotoodehnejadnematalahi et al., 2015). Research showed that versican can play a role in the repair of various tissues, including lung, skin ,and other tissues (Andersson-Sjöland et al., 2014; W. Yang & Yee, 2014). Several reports highlighted the role of versican in wound healing (Theocharis, 2002) and in vascular disease, especially atherosclerosis (Rahmani, McDonald, Wong, & McManus, 2004; Seidelmann et al., 2008). Versican binds low-density lipoprotein particles, and accumulation of versican in blood vessel walls is believed to promote extracellular lipoprotein retention and uptake leading to foam cell formation (Wight & Merrilees, 2004). In one study hypoxic induction of Versican was reported and suggested to be regulated, at least in part, by HIF-1 (Asplund et al., 2010).