Angiogenic Potential and Secretome of Human Apical Papilla Mesenchymal Stem Cells in Various Stress Microenvironments

Abstract

Stem cells from the apical papilla (SCAP) of human adult teeth are considered an accessible source of cells with angiogenic properties. The aims of this study were to investigate the endothelial transdifferentiation of SCAP, the secretion of pro-and antiangiogenic factors from SCAP, and the paracrine effects of SCAP when exposed to environmental stress to stimulate tissue damage. SCAP were exposed to serum deprivation (SD), glucose deprivation (GD), and oxygen deprivation/hypoxia (OD) conditions, individually or in combination. Endothelial transdifferentiation was evaluated by in vitro capillary-like formation assays, real-time polymerase chain reaction, western blot, and flow cytometric analyses of angiogenesis-related markers; secretome by antibody arrays and enzyme-linked immunosorbent assays (ELISA); and paracrine impact on human umbilical vein endothelial cells (HUVECs) by in vitro transwell migration and capillary-like formation assays. The short-term exposure of SCAP to glucose/oxygen deprivation (GOD) in the presence, but mainly in deprivation, of serum (SGOD) elicited a proangiogenesis effect indicated by expression of angiogenesis-related genes involved in vascular endothelial growth factor (VEGF)/VEGFR and angiopoietins/Tie pathways. This effect was unachievable under SD in normoxia, suggesting that the critical microenvironmental condition inducing rapid endothelial shift of SCAP is the combination of SGOD. Interestingly, SCAP showed high adaptability to these adverse conditions, retaining cell viability and acquiring a capillary-forming phenotype. SCAP secreted higher numbers and amounts of pro-(angiogenin, IGFBP-3, VEGF) and lower amounts of antiangiogenic factors (serpin-E1, TIMP-1, TSP-1) under SGOD compared with SOD or SD alone. Finally, secretome obtained under SGOD was most effective in inducing migration and capillary-like formation by HUVECs. These data provide new evidence on the microenvironmental factors favoring endothelial transdifferentiation of SCAP, uncovering the molecular mechanisms regulating their fate. They also validate the angiogenic properties of their secretome giving insights into preconditioning strategies enhancing their therapeutic potential.

Stem Cells of Dental Origin: Current Research Trends and Key Milestones towards Clinical Application

“…Other, less commonly found markers, such as CD44, CD9, CD10, CD13, CD59, and MSCA-1, have also been reported as expressed in DPSCs [54], while CD44 and CD13 are also expressed in SHED [55]. Dental MSCs, including DPSCs [56], SHED [57], and SCAP [58], also show variable but increased expression of embryonic SC markers, such as Nanog, Oct3/4, SSEAs (-1, -3, -4, and -5), and to a less extent TRA-1-60 and TRA-1-81 [56], as compared to other MSC types [19], such as BMMSCs. Other pluripotency markers, such as SOX-2 and MYC, not normally found in other ASCs, have been reported in tooth germ-derived MSCs [59].…”

“…Encouraged by the exceptional “plasticity” of dental MSCs, a limited number of studies have attempted to investigate the endothelial transdifferentiation potential of DPSCs [51, 62, 115–118], SHED [119, 120], and SCAP [58, 121] in the presence of specialized angiogenesis-inductive media (summarized in Table 2). The endothelial shift of MSCs in these studies is mainly indicated by the upregulation of typical endothelial cell (EC) markers, such as PECAM-1, VEGFR-2, vWF, and VE-cadherin and further evidenced by functional assays, such as ability to form capillary-like structures on Matrigel or other matrices or by uptake of Acetylated-Low Density LipoProtein Lipase (Ac-LPL), but also by various in vivo assays, including mouse Matrigel assays and Chicken Chorioallantoic Membrane (CAM) assays [33, 34, 122].…”

“…Moreover, a VEGFR-2-dependent function of murine DPSCs as pericyte-like cells has been substantiated, since a shRNA knockdown of VEGFR-2 produced a decreased expression of VEGFA, VEGF receptors, and Ephrin B-2 and reduced vascular density of Matrigel plugs in vivo [118]. Finally, short-term exposure of SCAP to serum, glucose, and oxygen deprivation (SGOD) conditions has been shown to be potent in eliciting a proangiogenesis program, as evidenced by activation of VEGF/VEGFR and Angiopoietins/Tie pathways [58]. These results confirm that dental MSCs can actually show considerable adaptability to severely adverse microenvironmental conditions, by undergoing a rapid endothelial shift rather than activating apoptosis.…”

“…Dental MSCs (DPSCs and SCAP) have been shown by recent studies to secrete, under various stress conditions, several pro- and antiangiogenetic factors able to stimulate endothelial motility and function [58, 121]. In particular, it has been shown that DPSCs secrete several proangiogenic factors (VEGF, Monocyte Chemotactic Protein 1- (MCP-1), IL-8, Insulin-Like Growth Factor Binding Protein 3 (IGFBP-3), and Urokinase Plasminogen Activator (uPA)) and antiangiogenic factors (Tissue Inhibitor Of Metalloproteinase 1 (TIMP-1), Plasminogen Activator Inhibitor-1 (PAI-1), endostatin, and Thrombospondin-1 (TSP-1)), under serum deprivation conditions [117], while in a later study by the same group differential angiogenic secretome expression was observed among various dental MSC types, including DPSCs, SCAP, and DFSCs [121].…”

“…Interestingly, DPSCs and SCAP elicited a predominant proangiogenic effect in vitro and in vivo compared to DFSCs, which renders them an attractive cell source for angiogenesis applications. Subsequently, it has been shown that, under serum, glucose, and oxygen deprivation (SGOD) conditions, SCAP release higher numbers and amounts of proangiogenic factors (Angiogenin, IGFBP-3, and VEGF) and lower amounts of antiangiogenic factors (Serpin-E1, TIMP-1, and TSP-1) in comparison with SOD or SD alone, providing insights into the optimal preconditioning strategies for SC-based treatment of damaged/ischemic tissues [58]. Most recently, SCAP secretome has been extensively profiled [131]; it was found that a total of 2,046 proteins are released, including chemokines, angiogenic, immunomodulatory, antiapoptotic, and neuroprotective factors, and ECM proteins.…”