The effects of THL (triphala churna) and chebulinic acid (CI) on vascular endothelial growth factor (VEGF) induced migration of human umbilical vein cells (HUVEC).

<p>Phase-contrast microphotographs of the wound area in HUVEC monolayer at 18 h after wounding (<b>A–D</b>) VEGF promotes complete wound closure or healing in 18vh. In contrast, this effect is lost when cells are treated either with THL (<b>A, C</b>) or CI (<b>B, D</b>). Wound healing is calculated as the distance covered by cells in relation to the initial wound distance at 0 h and is expressed as a percentage of the initial distance at 0 h. *, <i>P</i><0.05. All error bars represent SEM. Scale bars in <b>A</b> and <b>B</b>, 200 μm. Results shown are representative of six separate experiments.</p

The effects of Triphala churna (THL) and chebulinic acid (CI) on vascular endothelial growth factor (VEGF) induced angiogenesis in the CAM assay.

<p>(<b>A, E</b>) PBS used as a control does not induce blood vessel formation. (<b>B, E</b>) VEGF induces new blood vessel formation. (<b>C, E</b>) THL inhibits VEGF induced new blood vessel formation (<b>D, E</b>) CI inhibits VEGF mediated new blood vessel formation. Representative photographs of six separate experiments are shown.</p

The effects of THL (triphala churna) on <i>in vivo</i> matrigel angiogenesis assay.

<p>(<b>A</b>) Photographs of representative matrigel plugs show THL untreated red colored vascular endothelial growth factor (VEGF) containing plugs in comparison to the VEGF minus PBS containing controls. (<b>A</b>) In contrast, THL (triphala churna) treated VEGF containing matrigel plugs were pale. (<b>B</b>) Masson's trichrome staining (endothelial cells stain red and matrigel stains blue) and (<b>C, D</b>) CD31 immunohistochemistry of the matrigel plug sections show large numbers of endothelial cells in THL untreated VEGF containing plugs in comparison to controls (+, <i>p</i><0.05). In contrast, THL treated VEGF containing matrigel plug section has considerably low numbers of endothelial cells as detected by Masson's trichrome staining and CD31 staining (*, p<0.05). Scale bars in <b>B</b> and <b>C</b>, 50 μm. <i>n</i> =  six for each experimental group.</p

Effect of eticlopride treatment on expression of HoxD3 and its target α5β1 integrin in cutaneous wound tissue of mice.

<p>(<b>A</b>) Immunoprecipitation followed by immunoblot analysis of HoxD3 expression in wound tissues of both eticlopride and saline treated wound bearing mice at day 5 post wounding. The immunoblot analysis shows significantly higher expression of HoxD3 protein in wound tissues of eticlopride treated mice than vehicle treated controls at 5^th day after creation of wounds. (<b>B</b>) The bar graphs represent the density of each HoxD3 protein band relative to the IgG expression as quantified by ImageJ (NIH), *, P<0.05. (<b>C</b>) Immunohistochemical analysis of the expression of α5β1 integrin in cutaneous wound tissues of both control and eticlopride treated mice at day 5 post wounding. Frozen sections were immunostained with anti-α5β1 integrin antibodies followed by biotin conjugated secondary antibodies. The sections were stained using ABC staining kit and Nova-Red substrate solution to develop color. Significantly more areas of wound bed show positive staining (reddish brown color) for α5β1 integrin following eticlopride treatment. Original magnifications, x 100. Results are representative of six separate experiments each yielding similar results.</p

D₂ Dopamine receptors are present on the surface of human umbilical vein endothelial cells.

<p>(<b>A</b>) Immunoblot shows presence of D₂ dopamine receptors in HUVEC. (<b>B</b>) Flow cytometric analysis of D₂ dopamine Receptors in HUVEC. Over 81% cells of the total HUVEC population express D₂ dopamine receptors on their surfaces as evident from the lower right quadrant.</p

Effect of eticlopride treatment on macroscopic aspect of wound closure and formation of new blood vessels in wound bed.

<p>(<b>A</b>) The treatment with eticlopride, a specific D₂ dopamine receptor antagonist; 10 mg/kg/4 days/i.p. significantly accelerated the rate of wound healing with resurfacing occurring by 9 days whereas complete wound closure in controls occurred on day 14. The control group received similar volume of normal saline only. (<b>B</b>) Wound closure analysis. In treatment group of mice eticlopride significantly accelerated wound closure compared to vehicle treated control (each group, n = 6; *, P<0.05). Time to wound closure was defined as the time until the re-epithelialization process was complete and the wound bed was filled with new tissues. The percentage of wound closure was calculated as: (area of original wound − area of actual wound) x100/area of original wound and were measured by analyzing images using an image analysis program (ImageJ, NIH). (<b>C</b>) Immunohistochemical staining of CD31, a specific endothelial cell surface marker to enumerate the number of microvessels. The figure shows significantly greater number of microvessels (reddish brown in color) in wound tissue sections of eticlopride treated mice in comparison to vehicle treated controls. Original magnifications, × 100. (<b>D</b>) Graphical representation shows significantly higher number of microvessels in eticlopride treated groups when compared to vehicle treated controls at day 5, 24 hours after completion of treatment schedule. Microvessel density was measured by counting the number of microvessels in 10 randomly chosen high power microscopic fields within the sections,*, P<0.05). Results are representative of six separate experiments each yielding similar results.</p

Dopamine through its D₂ receptors can significantly downregulate VEGF induced expressions of HoxD3 and its target genes α5 and β1 integrins in HUVEC.

<p>(<b>A, B and C</b>) Western blot analysis of the effect of dopamine on VEGF induced expressions of HoxD3 and its target genes α5 and β1 integrins in HUVEC. Lane 1: Serum starved Human Umbilical Vein Endothelial Cells show no expression of HoxD3. However expression of both α5 and β1 integrins was observed. Lane 2: Cells stimulated with VEGF (10 ng/ml) show significant expression of HoxD3 and both the integrins after 8 hours of stimulation. Lane 3: Cells pretreated with 1 µM dopamine (concentration of DA found in synaptic clefts) 5 minutes before being exposed to VEGF (10 ng/ml) show significantly down-regulated VEGF induced expression of HoxD3 and both α5 and β1 integrins compared with VEGF treated controls. Lane 4: Cells treated with 100 µM eticlopride followed by dopamine and VEGF. Pre-treatment with eticlopride abrogated dopamine-induced down-regulation of HoxD3 and its target α5 and β1 integrin expression in HUVEC. β-actin was used as loading controls. Results are representative of six separate experiments each yielding similar results. (<b>D, E and F</b>) The bar graphs represent the density of each protein band relative to the β-actin expression. These have been quantified by ImageJ (NIH), *, P<0.05.</p