Dopamine and Mesenchymal Stem Cells staining of CD31, a specific endothelial cell surface marker, shows significantly greater number of microvessels in wound tissue sections of DA D2 receptor antagonist treated mice in comparison to vehicle treated controls at day 6 post wounding. Treatment with MSCs significantly increases microvessel density in wound bed in comparison to saline treated controls. However, eticlopride treatment along with MSCs transplantation is most effective in increasing angiogenesis in wound tissue. Original magnifications 6200. (C) Graphical representation showing microvessels density in wound tissue sections of different experimental groups at day 6 after post wounding (*, P,0.05). Microvessel density (CD31 positive cells) in wound bed was measured by counting the number of microvessels in 10 randomly chosen high power microscopic fields within the sections. doi:10.1371/journal.pone.0031682.g003 Dermal tissues are richly innervated by sympathetic nerve fibers in which DA is a major catecholamine neurotransmitter found in the extracellular fluid surrounding neuronal synapses [38–39]. Therefore in the present investigation, the effect of physiological concentration of DA (1 mM as observed in neuronal synapses) [40] was evaluated in regulating VEGF induced migration of MSCs in vitro. Addition of 1 mM DA in the medium significantly inhibited VEGF induced migration of MSCs, whereas, when these cells were pre-treated with 100 mM eticlopride (a specific DA D2 receptor antagonist), the inhibitory effect of DA on this growth factor mediated mobilization was abrogated (Fig. 4A). Thus, the in vitro results showed that physiological concentration of DA as observed in Figure 4. VEGF regulates migration of murine BM-MSCs through activation of Akt. (A) Effects of VEGF on in vitro migration of MSCs in transwell chambers. After incubation with MSCs at 37uC for 24 hours, VEGF was added. After overnight incubation cells remaining on the upper face of the filters were removed with a cotton wool swab and migrated cells that remained on the lower face of the filters were stained with Gill’s Haematoxylin, counted and photographed. Numbers of migrated cells were counted in 10 high-power fields (HPFs) after subtraction of the basal migration observed in the presence of DMEM alone (without presence of any growth factor). Dopamine (DA) significantly inhibits VEGF induced migration of MSCs (*, P,0.05), whereas when cells were pre-treated with 100 mM eticlopride (a specific DA D2 receptor antagonist), the inhibitory effect of 1 mM DA on VEGF mediated mobilization was abrogated. Results are representative of six separate experiments each yielding similar results. (B) Silencing of Akt in MSCs by siRNA transfection. After 48 hours of transfection, no expression of Akt was found in MSCs transfected with Akt siRNA (iAkt) whereas control MSCs in which control siRNA containing scrambled sequence (iScr) was transfected showed similar expression of Akt as normal non-transfected MSCs. (C) Effect of Akt silencing on VEGF induced migration of MSCs in a chemotaxis assay. Numbers of migrated cells were counted in 10 high-power fields (HPFs) after subtraction of the basal migration observed in the presence of DMEM alone (without presence of any growth factor). Silencing of Akt significantly inhibits VEGF induced migration of MSCs (*, P,0.05). Results are representative of six separate experiments each yielding similar results. (D) Flow cytometric analysis of VEGFR-2 receptors in murine MSCs. To confirm that CD342 CD452 CD105+ cells express VEGFR-2 receptors, CD342 cells were selected from in vitro expanded Linneg bone marrow cell population (containing CD452, CD11b2 cell populations) and these CD342 CD452 cells were evaluated for presence of CD105 and VEGFR-2 receptors. Results showed that almost 89% cells of the total MSC population (CD342 CD452 CD105+ cells) express VEGFR-2 receptors on their surfaces. doi:10.1371/journal.pone.0031682.g004 PLoS ONE | www.plosone.org 6 February 2012 | Volume 7 | Issue 2 | e31682