Corticosteroid suppression of VEGF-A in infantile hemangioma-derived stem cells

Abstract

Background: Corticosteroids are commonly used to treat infantile hemangioma, but the mechanism of action of this therapy is unknown. We investigated the effect of corticosteroids in a previously described in vivo model of infantile hemangioma and in cultured hemangioma-derived cells.

Methods: We tested hemangioma-derived stem cells for vasculogenic activity in vivo after implantation into immune-deficient (nude) mice. We studied dexamethasone treatment of both the cells before implantation and the mice after implantation. We also tested hemangioma-derived stem cells for expression of vascular endothelial growth factor A (VEGF-A) in vitro and studied the inhibition of VEGF-A expression, using short hairpin RNA (shRNA) in vivo and in vitro.

Results: Systemic treatment with dexamethasone led to dose-dependent inhibition of tumor vasculogenesis in the murine model. Pretreatment of hemangioma-derived stem cells in vitro before implantation also inhibited vasculogenesis. Dexamethasone suppressed VEGF-A production by hemangioma-derived stem cells in vitro but not by hemangioma-derived endothelial cells or human umbilical-vein endothelial cells. Silencing VEGF-A in hemangioma-derived stem cells reduced vasculogenesis in vivo. VEGF-A was detected in hemangioma specimens in the proliferating phase but not in the involuting phase and was shown by immunostaining to reside outside of vessels. Corticosteroid treatment suppressed other proangiogenic factors in hemangioma-derived stem cells, including urokinase plasminogen activator receptor, interleukin-6, monocyte chemoattractant protein 1, and matrix metalloproteinase 1.

Conclusions: In a murine model, dexamethasone inhibited the vasculogenic potential of stem cells derived from human infantile hemangioma. The corticosteroid also inhibited the expression of VEGF-A by hemangioma-derived stem cells, and silencing of VEGF-A expression in these cells inhibited vasculogenesis in vivo.

Figure 1. Dose-Dependent Effect of Systemic Dexamethasone on a Murine Model of Infantile Hemangioma

Hemangioma-derived stem cells and endothelial progenitor cells isolated from human umbilical-cord blood were suspended in Matrigel and injected subcutaneously into nude mice. Dexamethasone, at the indicated doses, was injected intraperitoneally for 7 consecutive days. Panel A shows cell–Matrigel explants from mice that were treated with either dexamethasone (2 mg per kilogram of body weight) or saline at day 7 (scale bar, 1 cm). Panel B shows sections from paraffin-embedded explants shown in Panel A (scale bar, 100 µm; hematoxylin and eosin). Panel C shows the microvessel density of samples from mice that were treated with dexamethasone (in doses of 0.125, 0.5, or 2.0 mg per kilogram of body weight) or saline, according to the number of erythrocyte-filled vessels in the sections (microvessel density). Bars represent the mean microvessel density determined from all the explants, with five samples in each subgroup. The T bars indicate standard errors. Asterisks indicate that the comparison with the control group was significant. For details regarding the experimental design, see Figure 1A in the Supplementary Appendix.

Figure 2. Inhibition of Vasculogenesis by Pretreatment of Hemangioma-Derived Stem Cells with Dexamethasone

Hemangioma-derived stem cells (HemSC), cord-blood endothelial progenitor cells (cbEPC), or both types of cells were treated with 0.2 µM dexamethasone for 3 days. Dexamethasone was washed out, and the cells were implanted in nude mice. Pretreatment of hemangioma-derived stem cells with dexamethasone led to a significant decrease in the number of blood vessels in the murine tumors, as compared with no significant decrease in cord-blood endothelial progenitor cells. Plus symbols indicate cells that were treated with dexamethasone. The bars represent the mean microvessel density determined from all the explants, with four to six samples in each subgroup. The T bars indicate standard errors. Asterisks indicate that the comparison with the control group was significant. This experiment was repeated twice with similar results. For details regarding the experimental design, see Figure 2A in the Supplementary Appendix.

Figure 3. Down-Regulation of VEGF-A Production in Hemangioma-Derived Stem Cells Treated with Dexamethasone

Hemangioma-derived stem cells (HemSC) and hemangioma-derived endothelial progenitor cells (HemEPC) that were isolated from the same infantile hemangioma specimen, as well as normal human fibroblasts (NHDF) and human umbilical-vein endothelial cells (HUVEC), were treated with 0.2 µM or 2.0 µM dexamethasone (Dex) for 3 days in culture. Panel A shows the results of quantitative reverse-transcriptase–polymerase-chain-reaction assays for vascular endothelial growth factor A (VEGF-A) messenger RNA (mRNA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the normalization control. Panel B shows VEGF-A protein levels, as measured by enzyme-linked immunosorbent assay, in the conditioned medium of the cells. Results are representative of two different experiments performed with cells isolated from two different specimens of infantile hemangioma. The bars represent the mean values, and the T bars indicate standard deviations.

Figure 4. Inhibition of Vasculogenesis through Silencing of VEGF-A in Hemangioma-Derived Stem Cells

Hemangioma-derived stem cells (HemSC) were stably infected with short hairpin RNA (shRNA) from vascular endothelial growth factor A (VEGF-A) or with a nontargeting shRNA (control). Hemangioma-derived stem cells (4×10⁶) that were infected with either VEGF-A shRNA or nontargeting shRNA were suspended in Matrigel and implanted subcutaneously in nude mice. Panel A shows representative Matrigel explants at day 7, with 7 to 10 samples in each subgroup. Panel B shows sections from representative explants from the VEGF-A shRNA group and the nontargeting shRNA group (hematoxylin and eosin). Panel C shows microvessel-density analysis of explants containing VEGF-A shRNA or nontargeting shRNA after 7 days in vivo. Panel D shows microvessel-density analysis of explants with the use of the two-cell model, in which either VEGF-A shRNA cells or nontargeting shRNA cells were combined with cord-blood endothelial progenitor cells (cbEPC), with eight samples in each subgroup. In Panels C and D, asterisks indicate that the comparison with the control group was significant. The T bars indicate standard errors. For both one-cell and two-cell models, the experiment was repeated twice with similar results.

Figure 5. VEGF-A Expression in Infantile Hemangiomas in the Proliferating Phase but Not the Involuting Phase

In Panel A, tissue lysates from seven proliferating and five involuting hemangiomas were analyzed by Western blot with the use of antihuman VEGF-A antibody, antihuman CD31 antibody, and anti–glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (protein-loading control). CD31, an endothelial-cell adhesion molecule, was reduced in the involuting-phase samples to a level below detection by Western blot. Coomassie blue staining of the sodium dodecyl sulfate–polyacrylamide-gel electrophoresis analysis shows equivalent protein in each tissue lysate, and Ponceau S staining of the Western blot membrane verifies equivalent protein transfer. Proliferating hemangiomas are listed according to the coded number (e.g., 68, 88, etc.), and involuting hemangiomas are designated with “I” and the coded number (I-30, I-41, and so forth). Clinical data for patients from whom the individual specimens were obtained are provided in Table 1 in the Supplementary Appendix.