In this study, we showed that Ang II can promote the migration of MSCs in an AT2R-dependent manner. Moreover, we proved that Ang II-enhanced migration is mediated by FAK activation. On the one hand, FAK activation forms the focal contacts that enhance cell adhesion. On the other hand, RhoA and Cdc42 are activated by FAK to increase the cytoskeletal organization, thus promoting cell contraction. In addition, the formation of focal contacts and organization of the cytoskeleton are mediated through AT2R. Taken together, Ang II-AT2R mediates MSC migration through the FAK and RhoA/Cdc42 pathways in vitro ( Fig.
Schematic of Ang II-AT2R-increased MSC migration by signaling through the FAK and RhoA/Cdc42 pathways. Ang II-AT2R activates FAK, leading to the formation of focal contacts and organization of the cytoskeleton, which is mediated by RhoA and Cdc42, and increases the migration of MSCs. Ang II angiotensin II, AT1R angiotensin II type 1 receptor, AT2R angiotensin II type 2 receptor, FAK focal adhesion kinase
The utilization of MSC transplantation to enhance therapeutic effects has been reported previously . However, the invasive character of local transplantation might be not feasible for widespread clinical application. Thus, a successful systemic transplantation and the migratory ability of MSCs toward sites of injury are essential for enhancing the healing process . Most importantly, this process is controlled by several inflammatory factors that are released at the injured site, which promote the recruitment of MSCs [25, 26]. Undoubtedly, Ang II is involved in key events of the inflammatory process and contributes to the recruitment of inflammatory cells into the injured tissue ; this motivated our investigation of whether Ang II could promote the migration of MSCs. Our result demonstrated that Ang II significantly enhanced the migration of MSCs in a dose-dependent manner. This is the first time we have demonstrated that Ang II can promote the migration of human bone marrow MSCs.
Only a few studies have shown that human MSCs express Ang II receptors. It is known that these two receptors are expressed in the monkey and human HS-5 stromal cell line at the protein level . Nevertheless, AT2R mRNA was not detected in human MSCs . However, our study detected not only AT1R mRNA but also AT2R mRNA in cultured human bone marrow MSCs. In accordance with our findings, human MSCs express both Ang II receptors at the mRNA level [28, 29]. We did not analyze the protein expression of AT1R and AT2R because the commercially available AT1R [30, 31] and AT2R  antibodies are nonspecific. Thus, the determination of mRNA expression remains the only reliable approach to date for examining AT1R and AT2R expression. However, the different AT2R gene expression patterns in human MSCs can be attributed to the different sources of the cells or cell lineages. Furthermore, after stimulation using different concentrations of Ang II, the expression of both receptors was increased to different levels, which suggests that the expression level correlates with receptor function.
Previous studies have demonstrated that Ang II stimulates cellular proliferation of different types of cells, including smooth muscle cells , hepatic stellate cells , and cardiac fibroblasts . Additionally, Ang II is known to promote the proliferation of hematopoietic stem cells (HSCs) . To exclude the effect of Ang II-mediated stimulation of proliferation on the migration of human bone marrow MSCs, we investigated MSC proliferation using the same concentration of Ang II and found that Ang II had no effect on cell proliferation. However, Zhang et al.  reported that exogenous applications of Ang II could increase mouse MSC proliferation. This discrepancy might be explained by the different species origins of the MSCs. Based on these findings, we conclude that Ang II-induced human bone marrow MSC migration is not mediated through the effects of Ang II on proliferation.
The role of the Ang II receptors in cell migration is less clear. We found that PD123319 inhibits Ang II-induced migration of MSCs in both the scratch and Transwell culture assays. Meanwhile, the migration enhanced by Ang II is consistent with the AT2R expression. In accordance with our findings, a recent study showed that PD123319 inhibits the angiotensin-induced migration of porcine vascular smooth muscle cells . In contrast, PD123319 has been shown to enhance the Ang II-induced migration of keratinocytes, suggesting an inhibitory action of AT2R in cell migration . There are also several studies showing that AT2R does not contribute to the effect of Ang II on the migration of vascular smooth muscle cells  or monocytes . Intriguingly, Zhao et al.  showed that Ang II plays an important role in promoting human breast cancer cell migration via AT1R. These apparent discrepancies concerning the role of Ang II receptors on migration could be attributed to cell line differences.
We next investigated potential pathways that participate in Ang II-enhanced migration. In this study, we found that Ang II enhanced the expression of FAK in human bone marrow MSCs. Moreover, blocking FAK  using a small molecule inhibitor, PF-573228, effectively reduced Ang II-induced MSC migration to baseline levels, thus indicating that FAK plays an important role in Ang II-induced MSC migration.
Cell adhesion and cell contraction are crucial steps in cell migration and have been reviewed recently in detail by Nitzsche et al.  for the mechanisms of MSC migration. FAK not only affects the assembly or disassembly of focal contacts but also influences the activity of Rho-family GTPases. The regulation of the Rho family of small GTPases, which includes RhoA, Rac1, and Cdc42, is essential for controlling the dynamics of the actin cytoskeleton and actin-associated adhesions during polarized cell migration . Therefore, another important finding in our study showed that PF-573228 not only inhibits the expression of focal adhesion proteins which decrease cell adhesion but also inhibits the activity of RhoA and Cdc42, which decreases formation of the F-actin network. Furthermore, the RhoA and Cdc42 inhibitors can individually reverse the enhanced migration of MSCs that is induced by Ang II. These results suggest that Ang II enhances MSC migration by signaling through the FAK and RhoA/Cdc42 pathways. It has been shown that high targeted migration of human MSCs is associated with enhanced activation of RhoA , which is consistent with our results. Similarly, Cdc42 activation has also been demonstrated to enhance cell migration in human corneal endothelial cells . One unanticipated finding was that Rac1 had no role in Ang II-mediated migration. The reason for this is unclear, but may have something to do with differences in cell type.
Perhaps the most significant idea in this study is the suggestion by these findings of a novel and unique role for AT2R in mediating MSC migration in response to Ang II and the establishment of FAK and RhoA/Cdc42 as a downstream target of AT2R. However, we will need to verify the function of AT2R in MSCs using animal models in future studies. It should be noted that Ang II was shown to play a role in the osteogenesis of MSCs  and to enhance the paracrine production of VEGF in rat MSCs . Therefore, the effect of Ang II at the same concentration on the differentiating ability and paracrine action of MSCs warrants future studies.