Involvement of notch signaling in wound healing

Abstract

The Notch signaling pathway is critically involved in cell fate decisions during development of many tissues and organs. In the present study we employed in vivo and cell culture models to elucidate the role of Notch signaling in wound healing. The healing of full-thickness dermal wounds was significantly delayed in Notch antisense transgenic mice and in normal mice treated with gamma-secretase inhibitors that block proteolytic cleavage and activation of Notch. In contrast, mice treated with a Notch ligand Jagged peptide showed significantly enhanced wound healing compared to controls. Activation or inhibition of Notch signaling altered the behaviors of cultured vascular endothelial cells, keratinocytes and fibroblasts in a scratch wound healing model in ways consistent with roles for Notch signaling in wound healing functions all three cell types. These results suggest that Notch signaling plays important roles in wound healing and tissue repair, and that targeting the Notch pathway might provide a novel strategy for treatment of wounds and for modulation of angiogenesis in other pathological conditions.

Figure 1. Genetic and pharmacological inhibition of Notch impairs wound healing.

Two full-thickness dermal wounds were induced in NAS mice and nontransgenic mice, vehicle-treated control mice, g-secretase inhibitor (GSI)-treated (100 µM DAPT) mice and 15 µM mouse Jagged peptide-treated mice. a. Images of a representative mouse from each group taken on post-injury days 1, 3, 5, 8 and 13 are shown. b. Wound sizes at the indicated time points in NAS and wild-type control mice (WT). c. Wound sizes at the indicated time points in control, GSI-treated and Jagged peptide-treated mice. Values are the mean and SEM (n = 6 mice per group). *p<0.001, #p<0.01 compared to the control value. Scale bar = 4 mm.

Figure 2. Histological features of wound healing in mice with decreased or increased Notch activity.

Images of skin tissue sections stained with hematoxylin and eosin showing histological changes during the wound healing process in non-transgenic control mice, NAS mice, GSI (100 µM DAPT) treated mice and 15 µM Jagged peptide-treated mice at post-injury days 1, 3, 5, 8 and 13. NAS and DAPT-treated mice exhibited delayed wound healing, and Jagged peptide-treated exhibited enhanced wound healing, compared to control mice. See Supplementary histological evaluation (Text S2) for a detailed description of histological changes in the different groups of mice. Scale bar = 0.5 mm

Figure 3. Notch activity in skin cells is decreased in NAS mice and in response to γ-secretase inhibition (10 µM), and is increased in response to Jagged peptide treatment (15 µM).

a and b. Representative immunoblot (a), and results of densitometric analysis of blots (b), showing relative levels of NICD (80 kDa) and actin (42 kDa) in lysates (40 µg/lane) of cultured human vascular endothelial cells (HUVEC), mouse keratinocytes (KC) and in mouse fibroblasts (MF) that had been treated for 6 h with vehicle or Jagged peptide. c and d. Representative immunoblot (c), and results of densitometric analysis of blots (d), showing relative levels of NICD in skin tissue samples from NAS and control mice taken at the indicated post-wounding time points. *p<0.01 compared to the control value. e and f. Representative immunoblot (e), and results of densitometric analysis of blots (f), showing relative levels of NICD in skin tissue samples from control and γ-secretase inhibitor (GSI)-treated mice taken at the indicated post-wounding time points. g and h. Representative immunoblot (g), and results of densitometric analysis of blots (h), showing relative levels of NICD in skin tissue samples from control and Jagged peptide-treated mice taken at the indicated post-wounding time points. *p<0.01 compared to the control value (ANOVA p = and Newman-Keuls post hoc tests for pairwise comparisons).

Figure 4. Notch signaling regulates the motility and proliferation of vascular endothelial cells.

a and b. Cultured human vascular endothelial cells (HUVEC) were treated with either vehicle (Control), g-secretase inhibitor (GSI; 10 µM DAPT) or Jagged peptide (15 µM) in conditioned medium and plated into chemo-attractant medium consisting regular growth medium with 10% FBS and other growth factors and cell migration was evaluated using a 24 well Transwell chamber assay. Representative images are shown in panel and a and quantitative data are shown in panel b (values are the mean±SEM for cells per 100×field; n = 3-4). *p<0.001 compared to control values. #p<0.001 compared to SCP values. c–f. HUVEC monolayers were mechanically wounded with the sterile tip of a 20–200 µl pipette tip following treatment with vehicle, GSI (10 µM DAPT), scrambled control peptide (SCP; 15 µM) or Jagged peptide (15 µM). Representative images are shown in c and e, and quantitative data on cell migration in d and f. Values are the mean and SEM (n = 3 separate experiments). *p<0.001. g and h. HUVEC were seeded on Matrigel-precoated wells and cultured in the presence of low-serum medium with GSI (10 µM DAPT), SCP (15 µM) or Jagged peptide (15 µM). Tube formation, designated as the number of branch points/100×field) was evaluated 18h after cell plating. Representative images are shown in g and quantitative data in h. Values are the mean and SEM (n = 12–16 cultures). *p<0.001. i. Cultured HUVEC were treated with GSI (10 µM DAPT), scrambled peptide (SCP; 15 µM) or Jagged peptide (15 µM) for the indicated time periods and relative cell numbers were estimated (n = 4–6 experiments). *p<0.001, #p<0.01 compared to the control cultures.

Figure 5. Evidence that Notch signaling regulates the behaviors of keratinocytes and skin fibroblasts.

a–h. Monolayers of cultured keratinocytes (a–d) or fibroblasts (e–h) were treated with vehicle (Control), GSI (10 µM DAPT), 15 µM SCP or 15 µM Jagged peptide, and were then subjected to scratch wounding. Eighteen hours after wounding, images of the wound area were acquired and the number of cells per field that had migrated into the cell-free wound zone were determined for each culture. Representative images (a, c, e, g) and quantitative data (b, d, f, h) are shown. Values are the mean and SEM (n = 3 separate experiments). *p<0.001. i and j. Cultured keratinocytes (i) and fibroblasts (j) were treated with vehicle (Control), GSI (10 µM DAPT), 15 µM SCP or 15 µM Jagged peptide for the indicated time periods and relative cell numbers were estimated. Values are the mean and SEM (n = 4–6 experiments).