Activation of Dopamine D1 Receptors in Dermal Fibroblasts Restores Vascular Endothelial Growth Factor-A Production by These Cells and Subsequent Angiogenesis in Diabetic Cutaneous Wound Tissues

Abstract

In wound beds, fibroblasts are rich sources of vascular endothelial growth factor A, a cytokine necessary for promoting angiogenesis and thereby the healing of wound tissues. However, in diabetes mellitus, these cells are functionally impaired and produce reduced amounts of vascular endothelial growth factor A, resulting in deficient angiogenesis and delayed wound healing. We here for the first time demonstrate that stimulation of D1 dopamine receptors present in dermal fibroblasts restores vascular endothelial growth factor A production by these cells, resulting in adequate angiogenesis and subsequent healing of cutaneous wounds in both type 1 and type 2 diabetic mice. This action of D1 dopamine receptors was mediated through the protein kinase A pathway. As delayed wound healing or chronic wounds are one of the major health problems in diabetic patients, D1 dopamine receptor agonists, which are already in clinical use for the treatment of other disorders, may be of translational value in the treatment of chronic, nonhealing diabetic wounds.

Figure 1

Effect of D₁ dopamine receptor agonist treatment on full-thickness skin wound closure in streptozotocin (STZ)-induced diabetic mice. A: Treatment with a specific D₁ dopamine receptor agonist, SKF39383 or SKF81297, significantly accelerates the rate of wound healing compared to that in untreated controls, with complete closure of wounds by day 11. On the contrary, SKF39383 or SKF81297 treatment does not accelerate wound closure in diabetic D₁ dopamine receptor knockout mice (KOD1). B: Percentage wound closure, calculated as [(Area of original wound) − (Area of actual wound)] × 100/(Area of original wound), was measured by analyzing images using ImageJ software package version 1.49V (NIH, Bethesda, MD; http://imagej.nih.gov.ij). C: Significant increase in vascular endothelial growth factor (VEGF)-A and CD31 expression levels in wound tissues after treatment with SKF38393 or SKF81297. VEGF-A and CD31 expression levels in wound tissues from KOD1STZ diabetic mice were similar to those in nontreated diabetic control mice. Arrows indicate CD31⁺ vessels. D: Numbers of CD31⁺ microvessels in individual high-power microscopic fields (HPF). Data are expressed as means ± SEM. n = 11. ^∗P < 0.05 versus control. Scale bars = 100 μm.

Figure 2

Effect of D₁ dopamine receptor agonist treatment on full-thickness skin wound closure in genetically diabetic mice. A: Treatment with a specific D₁ dopamine receptor agonist, SKF38393 or SKF81297, significantly accelerates the rate of wound healing compared to that in nontreated controls (Con), with complete closure of wounds by day 11. B: Percentage wound closure. C: Significant increase in vascular endothelial growth factor (VEGF)-A and CD31 expressions in wound tissues after treatment with SKF38393 or SKF81297. Arrows indicate CD31⁺ vessels. D: Numbers of CD31⁺ microvessels in individual microscopic high-power fields (HPF). Data are expressed as means ± SEM. n = 11. ^∗P < 0.05 versus control. Scale bars = 100 μm.

Figure 3

D₁ dopamine receptor agonists have no direct effects on endothelial cells in vitro. A: Exogenous administration of SKF38393 or SKF81297 has no direct effect on human umbilical vein endothelial cell (HUVEC) migration, as determined by scratch wound assay. Eighteen hours after the creation of wound, complete wound closure is observed in vascular endothelial growth factor (VEGF)-A (positive control)–treated well. B: Exogenous administration of SKF38393 or SKF81297 also has no effect on HUVEC proliferation, as determined by MTT cell proliferation. C: Western blot analysis shows the presence of D₁ dopamine receptor (DA D1) in HUVECs, normal human fibroblasts, diabetic human fibroblasts, and mouse kidney lysate (positive control). Figures are representative of three separate experiments. Data are expressed as means ± SEM. n = 9. ^∗P < 0.05 versus control cells. Scale bars = 200 μm. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HDF, human dermal fibroblasts; M, mouse; O.D., optical density.

Figure 4

D₁ dopamine receptor agonist treatment increases the synthesis of vascular endothelial growth factor (VEGF)-A in diabetic human dermal fibroblasts through phosphorylation (p) of cAMP response element–binding protein (CREB). A: Confocal microscopy images show the presence of D₁ dopamine receptor (D1DR; red) in fibroblasts [smooth muscle actin (α-SMA); green] in normal, streptozotocin-induced type 1 diabetic mice (STZ-Diab) and db/db mouse wound tissues as well as in normal and diabetic human skin. B: Semiquantitative RT-PCR analysis shows that synthesis of VEGF-A is significantly decreased in diabetic human fibroblasts (control) when compared to normal human fibroblasts (normal). Treatment of diabetic human fibroblasts with SKF38393, SKF81297, or forskolin significantly increases VEGF-A synthesis after 24 hours. Treatment of diabetic fibroblasts with a specific D₁ dopamine receptor antagonist, SCH23390 or H89, before treatment with SKF38393 or SKF81297, abrogates their effects. C: Enzyme-linked immunosorbent assay for human VEGF-A. Treatment with SKF38393, SKF81297, or forskolin increases VEGF-A secretion by diabetic fibroblasts at 48 hours. However, treatment of diabetic fibroblasts with SCH23390 or H89 before treatment with SKF38393 or SKF81297 abrogates their effects. D: Western blot analysis shows that treatment of diabetic fibroblasts with SKF38393, SKF81297, or forskolin for 1 hour significantly increases phosphorylation of CREB, but that prior treatment with SCH23390 or H89 abrogates the effect of SKF38393 or SKF81297. Figures are representative of three separate experiments. Data are expressed as means ± SEM. n = 11. ^∗P < 0.05 versus control cells; ^†P < 0.05 versus D₁ dopamine receptor agonist– and forskolin-treated groups. Scale bars = 50 μm. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.