Figure 1.
Treatment with dopamine (DA) D2 receptor specific antagonist significantly increased the number of circulating MSCs in peripheral blood of wound bearing mice.
(A) Effect of eticlopride (DA D2 receptor specific antagonist) on the number of circulating MSCs in peripheral blood of wound bearing mice. Blood was collected from both the control and eticlopride treated wound bearing mice at different time intervals after injury, mixed with EDTA, treated with RBC lysis buffer and incubated with anti-CD45, anti-CD34 and anti-CD105 antibodies. Cells were initially gated to exclude dead cells and debris and then the CD34− CD45− cell population was selected and evaluated for presence of CD105, a specific surface marker of MSCs. Results are representative of six separate experiments each yielding similar results. (B) Graph showing that DA D2 receptor antagonist treatment have significant positive regulatory effect on MSC mobilization as significantly higher number of MSCs (CD34− CD45− CD105+ cells) was evident in peripheral blood of mice compared to vehicle treated controls at all time points after wounding. (*, P<0.05). (C) Characterization of MSCs by flow cytometry. Analysis with flow cytometer showed that over 95% cells of the total 3rd passage murine bone marrow cell populations were positive for both SSEA-4 and CD105 (specific surface markers of MSCs) and negative for CD34 and CD45, thus confirming their identity as MSCs. BM-MSCs were cultured in appropriate induction media to evaluate osteogenic and adipogenic differentiation potential of these stem cells and were confirmed by von Kossa staining (D) and Oil-red O staining (E) respectively. (D) To confirm osteogenic differentiation, mineralized deposits in the extracellular matrix were visualized by von Kossa staining. Differentiated cells were fixed with 4% paraformaldehyde, washed and then stained with 5% silver nitrate (Sigma) solution in the absence of light at room temperature for 30 minutes. Cells were then exposed to sunlight for 5 minutes and excess silver staining was removed by washing 2–3 times with a 5% sodium thiosulfate solution and ultimately washed with distilled water. (E) Adipogenic differentiation of cultured MSCs was confirmed by Oil-red O staining. Cells were fixed with 4% paraformaldehyde and washed with 60% isopropanol. After completely drying the flasks Oil Red O working solution were added and stained for 15 minutes. Then all Oil Red O were removed and distilled water was added immediately to wash 2 times. Finally, the flasks were viewed under microscope and photographed. Original magnifications, ×200.
Figure 2.
Treatment with dopamine (DA) D2 receptor antagonist significantly accelerated mobilization of exogenously transplanted MSCs towards wound bed.
(A) Flow cytometric analysis of DA D2 receptors in murine MSCs. To confirm that CD34− CD45− CD105+ SSEA-4+ cells express DA D2 receptors, in vitro expanded Linneg bone marrow cell population (containing CD45−, CD11b− cell populations) were initially gated to exclude dead cells and debris and then the CD34− cell population were selected and these CD34− CD45− cells were evaluated for presence of CD105, SSEA-4 and DA D2 receptors. Results showed that almost 86% cells of the total MSC population (both CD34− CD45− CD105+ cells and CD34− CD45− SSEA-4+ cells) express DA D2 receptors on their surfaces. (B) Western blot analysis of DA D2 receptors in murine BM-MSCs. H4 neuroglioma cell line and sarcoma-180 (S-180) tumor cells were used as positive and negative controls, respectively. (C) Effect of DA D2 receptor antagonist treatment on mobilization of exogenously transplanted MSCs towards wound site. MSCs were labeled with BrdU in culture and injected into the tail vein of both control and eticlopride treated back skin-injured mice. After completion of eticlopride treatment, at 6th day the skin from the wound area was collected and analyzed by immunohistochemistry using BrdU labeling and detection kit (Roche Applied Science). Significantly higher number of BrdU positive transplanted MSCs are located in the wound bed of eticlopride treated group than vehicle treated control group, showing DA D2 receptor antagonist treatment has significant positive effect on mobilization of exogenously transplanted MSCs towards wound site. Original magnifications, ×200. Results are representative of six separate experiments each yielding similar results. (D) Graphical representation showing significantly higher number of exogenously transplanted MSCs (BrdU positive cells) in wound bed of eticlopride treated groups compared to vehicle treated controls at day 6 post wounding (*, P<0.05). Number of transplanted MSCs cells was measured by counting the number of BrdU positive cells in 10 randomly chosen high power microscopic fields within the sections.
Figure 3.
Dopamine (DA) D2 receptor antagonist treatment stimulate incorporation of MSCs into neovessels of wound bed and subsequent angiogenesis in wound bed.
(A) Effect of eticlopride treatment on incorporation of transplanted MSCs into newly formed blood vessels in wound bed. MSCs were labeled with CM-Dil and injected into the tail vein of both control and eticlopride treated back skin-injured mice. At day 6, the skin from the wound area was collected and sectioned with cryomicrotome. Frozen sections were immunostained with anti-CD31 antibody and FITC-conjugated secondary antibody and analyzed to determine the extent of incorporation of transplanted MSCs into blood vessels. Here co-localization study showed that exogenously transplanted MSCs, labeled with fluorescent dye CM-Dil (red in colour), had integrated into the newly formed blood vessels (green in colour) in wound site in much greater number following DA D2 receptor antagonist treatment than vehicle treated controls. Original magnifications, ×200. (B) Effect of MSC transplantation on formation of new blood vessels in wound bed. Immunohistochemical 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 ×200. (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.
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 37°C 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 µM eticlopride (a specific DA D2 receptor antagonist), the inhibitory effect of 1 µM 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 CD34− CD45− CD105+ cells express VEGFR-2 receptors, CD34− cells were selected from in vitro expanded Linneg bone marrow cell population (containing CD45−, CD11b− cell populations) and these CD34− CD45− cells were evaluated for presence of CD105 and VEGFR-2 receptors. Results showed that almost 89% cells of the total MSC population (CD34− CD45− CD105+ cells) express VEGFR-2 receptors on their surfaces.
Figure 5.
Dopamine (DA) through its D2 receptors inhibits VEGF induced migration of BM-MSCs by regulating phosphorylation of VEGFR-2 and Akt and actin polymerization.
(A and B) Effect of DA on VEGF induced phosphorylation of VEGFR-2 and Akt in murine MSCs. Lane 1: Cells stimulated with VEGF (10 ng/ml). Lane 2: Cells pretreated with 1 µM DA before being exposed to VEGF (10 ng/ml). Lane 3: Cells treated with 100 µM eticlopride followed by DA and VEGF. Addition of DA significantly inhibited VEGF induced phosphorylation of both VEGFR-2 receptors and its downstream target Akt when compared with VEGF treated control. Pre-treatment with eticlopride, abrogated DA induced dephosphorylation of both VEGFR-2 and Akt. However, expression of total VEGFR-2 and total Akt remained unchanged. Results are representative of six separate experiments each yielding similar results. (C) In actin polymerization assay, VEGF induced MSCs showed significant polymerization of actin cytoskeleton leading to formation of F-actin. However, no such alterations were observed in case of Akt silenced MSCs which showed strikingly lower migratory activity against VEGF than normal MSCs. (D) Effect of DA on VEGF induced actin polymerization in MSCs. Treatment with 1 µM DA had significant inhibitory effect on actin polymerization dynamics in MSCs when compared to VEGF treated control. However, pre-treatment with specific DA D2 receptor antagonist eticlopride (100 µM) abrogated the DA induced changes in the actin polymerization dynamics of the VEGF induced MSCs. Original magnifications, ×1000.