neuronal synapse [40] inhibits VEGF induced migration of MSCs. Furthermore, this in vitro result also correlated well with our previous in vivo observations of eticlopride, a DA D2 receptor antagonist- mediated increased mobilization of MSCs into wound bed. DA regulates VEGF induced migration of murine MSCs by inhibiting Akt phosphorylation Because neurotransmitter DA by acting through its D2 receptors significantly inhibited VEGF induced migration of murine MSCs in vitro (Fig. 4A), we next elucidated the regulatory role of DA in influencing the key signaling pathway for VEGF induced mobilization of MSCs into the wound site. Since recent experi- mental results have shown that activation of Akt increases the migratory activity of MSCs [41–44], experiments were therefore designed to dissect the regulatory role of Akt in VEGF induced migration of MSCs in mice. siRNA transfection was utilized to knock-down (silence) Akt in MSCs. After 48 hours, western blot analysis was performed to confirm silencing in Akt siRNA transfected cells (iAkt) (Fig. 4B). The role of Akt kinase on growth factor induced mobilization of murine MSCs was then evaluated by in vitro migration assay of siRNA transfected (Akt-silenced) MSCs against chemotactic activity of 10 ng/ml VEGF. Silencing of Akt significantly inhibited VEGF induced migration of MSCs (Fig. 4C). Furthermore, flow cytometry analysis showed that murine BM-MSCs also express VEGFR-2 receptors on their surfaces (Fig. 4D), which corrobo- rated well with previous observations [45–46] thus showing that VEGF considerably influences mobilization of murine MSCs by activating Akt kinase through its VEGFR-2 receptors present on these cells. We next elucidated whether DA exerted its inhibitory effects on the mobilization of MSCs by suppresing the activation of Akt in these cells. The activation of DA receptors in MSCs by 1 mM of DA significantly inhibited VEGF (10 ng/ml) induced phosphor- ylation of both VEGFR-2 and its downstream signaling molecule Akt (Fig. 5A and 5B). However, these actions of DA were abrogated following treatment with 100 mM eticlopride, a DA D2 receptor antagonist (Fig. 5A and 5B). In contrast, no changes were observed in the expression of total VEGFR-2 and total Akt. These results indicated that DA through its D2 receptors significantly inhibited VEGF induced migration of murine MSCs by depho- sphorylating both VEGFR-2 receptors and its downstream target Akt kinase in these stem cells. DA regulates polymerization of actin filaments in migrating MSCs Reorganization and active regulation of actin cytoskeleton is an important event in the migratory response of cells to growth factors, and phosphorylation of Akt had been implicated to induce this actin polymerization [43–44]. In our actin polymerization assay, VEGF stimulated MSCs which showed high migratory activities [Fig. 4A], was also associated with changes in the polymerization dynamics of this protein leading to formation of F-actin [Fig. 5C]. However, no such alterations were observed in Akt silenced MSCs [Fig. 5C], which also showed strikingly lower migratory activity against VEGF in comparison to normal MSCs [Fig. 4C]. These results indicated that Akt regulated the mobilization of MSCs by changing polymerization dynamics of actin cytoskeleton. Furthermore, treatment with 1 mM of DA, which significantly inhibited VEGF induced migration of MSCs by suppressing phophorylation of both VEGFR-2 and its downstream signaling molecule Akt, was also associated with significant actin depolymer- Dopamine and Mesenchymal Stem Cells ization effect leading to the inhibition MSCs migration (Fig. 5D). In contrast, pre-treatment with specific DA D2 receptor antagonist eticlopride abolished this DA mediated inhibitory changes in the actin polymerization dynamics of VEGF treated MSCs. These results indicated that DA regulated inhibition of VEGF induced actin polymerization and migration of MSCs were specifically mediated through its D2 receptors. Discussion Angiogenesis is necessary for successful wound healing and defects in this process leads to delayed healing as observed in different types of chronic non-healing wounds. During cutaneous wound repair, the neovessels provide nutrition and oxygen to the growing tissues and aids in the formation of the provisional wound matrix. This dynamic physiological process is temporally and spatially regulated by interactions between different cell types and other factors present in the wound microenvironment [1–2]. Among the various cellular effectors, the roles of endothelial cells and endothelial progenitor cells are well established in the process of angiogenesis [1–5]. In pathological conditions like malignant tumors, DA can significantly inhibit tumor angiogenesis by inhibiting VEGF induced proliferation and migration of both adult and progenitor endothelial cells [21–24]. We have recently demonstrated that DA can inhibit angiogenesis in normal wound tissue by inhibiting the expression of HoxD3 and its target a5b1 integrin in adult endothelial cells [25]. DA acted through its D2 receptors [21–24]. However, current attention has been drawn to the role of MSCs in the formation of neovessels in wound tissues. Mesenchymal stem cells are either absent or present in significantly low numbers in the steady state conditions in circulation. However, their numbers considerably increase after trauma or injury and this is associated with increased level of plasma VEGF [14–16]. These circulating cells then migrate to the site of the injury by the process of chemotaxis and subsequently incorporate into the newly formed vessels to be transdifferentiated into endothelial cells [8,10]. Several studies have indicated that MSCs can differentiate into endothelial cells and are capable of inducing neoangiogenesis [8,10,47–48]. In addition, these stem cells also release several pro-angiogenic molecules like VEGF to recruit inflammatory and progenitor cells to make the wound tissue microenvironment conducive for neovessel formation and subsequent healing [9,13,17,19–20,49]. These reports thus clearly indicate the importance of MSCs in wound tissue angiogenesis. The bone marrow and the adipose tissue are considered to be the principal sources of MSCs [11–13,17–20] and both these tissues are richly innervated by the sympathetic nerves [50–53]. Furthermore, substantial amount of dopamine is present in the bone marrow niche as well as adipocytes [24,54]. We have previously reported that DA by acting through its DA D2 receptors can inhibit the activity of MMP-9, a potent regulator cell mobilization from bone marrow to peripheral circulation [24]. Therefore, in the present study, the increased numbers of MSCs in the circulation of specific DA D2 receptor antagonist treated wound bearing mice may be due to inhibition of DA induced suppression of adipose tissue and/or bone marrow MMP-9 activity. Our study also for the first time demonstrates the presence of DA D2 receptors in the bone marrow- derived MSCs. In the present investigation, in vivo results showed increased incorporation of exogenously transplanted MSCs into wound bed after eticlopride treatment. Furthermore, it was well associated with significantly enhanced angiogenesis. These results corrobo- rate well with other reports that indicate significant pro-angiogenic effect of these stem cells [6–10,48–49]. This present study also PLoS ONE | www.plosone.org 7 February 2012 | Volume 7 | Issue 2 | e31682