Article Figures & Data
Figures
- Figure 1. Differential Notch pathway activities in fibroblasts of chronic diabetic skin wounds versus non-diabetic skin and wounds.
(A) High Notch pathway activity in diabetic foot ulcer fibroblasts (DFUF) versus low Notch pathway activity in normal foot fibroblasts (NFF). Expression of Notch pathway components in three DFUF and three NFF were assessed by immunoblot. β-actin was used as a loading control. The band of each molecule is shown. (B) Inhibition of the Notch pathway activity, reflected by decreased levels of N1IC and Hey-1, in DFUF by DAPT and Jag 1 neutralizing Ab. Compared with DAPT, Jag 1 neutralizing Ab only achieved a partial inhibition. (C) Representative immunostaining images show that fibroblasts (green) express higher levels of Hes-1 (red) in skin at the edge of diabetic foot ulcer tissue than that in non-diabetic foot skin. Highlighted areas show fibroblasts in reticular layers. (D) Representative immunostaining images show that fibroblasts (green) express higher levels of Hes-1 (red) in wounds of diabetic mice (db/db and NOD) but not in non-diabetic acute wound and ischemic chronic wounds in C57 BL6 mice. Wound tissues were harvested at day 7. Highlighted areas show fibroblasts in granulation tissues.
- Figure S1. Immunofluorescence images.
Individual and combined color of immunofluorescence images of highlighted reticular layers in skin tissue sections stained with anti–Hes-1 (Alexa Fluor 594–conjugated 2nd Ab), anti–FSP-1 (Alexa Fluor 488–conjugated 2nd Ab) and DAPI shown in Fig 1C. The ratio of Hes-1:FSP-1 in highlighted reticular layers is shown.
- Figure S2. Immunofluorescence images.
Individual and combined color of immunofluorescence images of highlighted papillary layers in skin tissue sections stained with Alexa Fluor 594–conjugated 2nd Ab, anti–FSP-1 Alexa Fluor 488–conjugated 2nd Ab, and DAPI shown in Fig 1C. The ratio of Hes-1:FSP-1 in highlighted papillary layers is shown.
- Figure S3. Immunofluorescence images.
Individual and combined color of immunofluorescence images of highlighted reticular layers in skin tissue sections of human non-diabetic foot skin versus diabetic foot ulcer stained with anti–Hey-1 (Alexa Fluor 594–conjugated 2nd Ab), anti–FSP-1 (Alexa Fluor 488–conjugated 2nd Ab), and DAPI. The ratio of Hes-1:FSP-1 in highlighted reticular layers is shown.
- Figure S4. Immunofluorescence images.
Individual and combined color of immunofluorescence images of highlighted areas in mouse skin tissue sections stained with Alexa Fluor 594–conjugated 2nd Ab, anti–FSP-1 (Alexa Fluor 488–conjugated 2nd Ab), and DAPI shown in Fig 1D. The ratio of Hes-1:FSP-1 is shown.
- Figure S5. Dynamic alteration of the Notch pathway activities in fibroblasts presented in mouse non-diabetic wounds versus diabetic skin wounds at various time points.
Wound tissues were harvested at day 1 (early time point), day 7 (middle time point) post-wounding in all mice, and at day 9/13/14/15 (late time point, when wounds are healed) in C57 BL6 (normal non-ischemic wounds)/C57 BL6 (ischemic wounds)/db/db/NOD mice, respectively. Levels of Hes-1 in fibroblasts presented in diabetic wounds (db/db and NOD mice) were detectable at day 1, peaked in day 7, and reduced to a very low level when the wounds were healed in diabetic mice (NOD and db/db), but remained undetectable throughout wound healing process in both normal non-ischemic acute and non-diabetic ischemic chronic wounds (C57 BL6 mice). Immunostaining images show that fibroblasts (green) express higher levels of Hes-1 (red) in wounds of diabetic mice (db/db and NOD) but not in non-diabetic acute wound and ischemic chronic wounds in C57 BL6 mice at day 7. Highlighted areas show fibroblasts in granulation tissues.
- Figure S6. Dynamic alteration of the Notch pathway activities in fibroblasts presented in mouse non-diabetic wounds versus diabetic skin wounds at various time points.
Wound tissues were harvested at day 1 (early time point), day 7 (middle time point) post-wounding in all mice, and at day 9/13/14/15 (late time point, when wounds are healed) in C57 BL6 (normal non-ischemic wounds)/C57 BL6 (ischemic wounds)/db/db/NOD mice, respectively. Levels of N1IC in fibroblasts presented in diabetic wounds (db/db and NOD mice) were detectable at day 1, peaked in day 7 and reduced to a very low level when the wounds were healed in diabetic mice (NOD and db/db), but remained undetectable throughout wound healing process in both normal non-ischemic acute and non-diabetic ischemic chronic wounds (C57 BL6 mice). Immunostaining images show that fibroblasts (green) express higher levels of N1IC (red) in wounds of diabetic mice (db/db and NOD) but not in non-diabetic acute wound and ischemic chronic wounds in C57 BL6 mice at day 7. Highlighted areas show fibroblasts in granulation tissues.
- Figure S7. Immunofluorescence images show expression of N1IC in FSP-1+ skin fibroblasts of GOFNotch1 versus GOFCtrl mice, and LOFNotch1 versus LOFctrl mice.
Tissue sections are stained with Alexa Fluor 594–conjugated 2nd Ab, anti–FSP-1 (Alexa Fluor 488–conjugated 2nd Ab), and DAPI. Skin fibroblasts in GOFNotch1 express increased Hes-1 in nuclei (pink color in overlay images) and/or cytoplasm (orange color in overlay images), whereas skin fibroblasts in LOFNotch1 express undetectable levels of Hes-1 in either nuclei or cytoplasm.
- Figure S8. Immunostaining of lymphocytes in skin of GOFNotch1 versus GOFctrl mice.
Lymphocytes are stained with anti-CD3 Ab (Alexa Fluor 594–conjugated 2nd Ab), fibroblasts are stained with anti–FSP-1 (Alexa Fluor 488–conjugated 2nd Ab), and nuclei were stained with DAPI. No lymphocytes are detectable. Highlighted areas are loose connective tissues where some fibroblasts present.
- Figure S9. Skin structure and collagen deposition.
Masson’s trichrome staining images show normal skin structure and cellular morphology in GOFNotch1 versus GOFCtrl mice, except less collagen deposition in the skin of GOFNotch1 mice compared with GOFCtrl mice.
- Figure 2. Activation of Notch1 pathway in fibroblasts delays skin wound healing in mouse models.
(A) Mouse skin wound healing was delayed in GOFNotch1 mice compared with GOFCtrl mice. Top: six representative images of wounds in each group at Day 0 and Day 9 were shown. Bottom: Wound healing curves. Numbers of mice in each group is listed. (B) Mouse skin wound healing rates were comparable between LOFNotch1 and LOFCtrl mice. Top: six representative images of wounds in each group at Day 0 and Day 8 were shown. Bottom: Wound healing curves. Numbers of mice in each group is listed. All data are analyzed by two way ANOVA followed by post-hoc tests and presented as percentage wound closure (recovery), mean ± SD from each group.
- Figure S10. Immunofluorescence images of generated GOFNotch1-(DF) dermal fibroblast, GOFCtrl-DF, LOFNotch1-DF, and LOFCtrl-DF.
Cells were stained with anti-N1IC (Alexa Fluor 594–conjugated 2nd Ab), anti–α-smooth muscle actin (Alexa Fluor 488–conjugated 2nd Ab), and DAPI. GOFNotch1-DF expressed elevated levels of N1IC in nuclei.
- Figure 3. Activation of Notch1 pathway in fibroblasts suppresses cellular proliferation and migration.
(A) GOFNotch1-(DF) dermal fibroblast grew slower than GOFCtrl-DF, whereas growth rates of LOFNotch1-DF and LOFCtrl-DF were comparable. Data of mean ± SD are based on results of three experiments of total six wells/group (cells grew in 96-well plate) and analyzed by t test. (B) Expression of cell proliferation marker Ki67 (red) is lower in fibroblasts (FSP-1, green) at wound granulation tissue of GOFNotch1 mice than that in GOFCtrl mice, yet no obvious difference between LOFNotch1 and LOFCtrl mice. Quantitative data are calculated based on three sections/wound and mean ± SD are analyzed by t test. (C) GOFNotch1-DF migrate and proliferate were slower than those from GOFCtrl-DF, whereas migration and proliferation of LOFNotch1-DF and GOFCtrl-DF were comparable as assessed by in vitro wound healing assay. Data of mean ± SD are based on results of three experiments of total six pseudo-wounds/group and analyzed by t test.
- Figure 4. Activation of the Notch1 pathway inhibits differentiation of fibroblasts into myofibroblasts.
(A) Representative Masson’s trichrome staining images show decreased collagen deposition in wound tissues of GOFNotch1 mice compared to GOFCtrl mice. No obvious difference in the wounds of LOFNotch1 versus LOFCtrl mice was found. Collagen levels in highlighted areas were quantified by ImageJ. Quantitative data of mean ± SD are based on results from three sections/wound (see N of wounds in Fig 3) and analyzed by t test. (B) Immunostaining shows robustly decreased expression of α-smooth muscle actin (α-SMA) in GOFNotch1-(DF) dermal fibroblast compared with GOFCtrl-DF, but no obvious difference in LOFNotch1-DF and LOFCtrl-DF was found. Quantitative data are mean ± SD of intensity of green fluorescence of α-SMA/cell based on total 100 cells in each group. (C) GOFNotch1-DF exhibited weak ability to contract the collagen gel. Top: representative three gel images/group of gel contraction assay in 0 and 5 h. Bottom: data are mean ± SD of sizes of six gels in each group compared to initial size (set as 100%) at 0 h. Experiments were repeated three times. (D) Immunostaining shows the levels of α-SMA in myofibroblasts at wound granulation tissues of GOFNotch1 versus GOFCtrl and LOFNotch1 versus LOFCtrl mice. There were fewer myofibroblasts in a given area at wound granulation tissue of the GOFNotch1 mice than GOFCtrl mice. Also, (myo)fibroblasts at wound granulation tissue of GOFNotch1 express lower amounts of α-SMA than that in GOFCtrl mice. No obvious difference between LOFNotch1 and LOFCtrl mice was found. Data are mean ± SD of numbers of myofibroblasts in selected given area with equal size and intensity of green fluorescence of α-SMA/cell based on total 100 cells in each group (ANOVA). Intensity of green fluorescence is adjusted by blue signal intensity (DAPI signal) of each cell.
- Figure 5. Activation of the Notch1 pathway in fibroblasts inhibits the angiogenic response of endothelial cells.
(A) Decreased neovascularization in the wounds of GOFNotch1 mice compared with the wounds of GOFCtrl mice. Top: representative three images of capillary networks developed in the wound beds. Centers of wound beds are highlighted by dash circles. Bottom: quantitative data are mean ± SD of intensity of red fluorescence signals of Dil in a given area in the center of wound bed in each group (n = 6/group). Ratio of peripheral/center reflects immature leaky vessels in the center of wound beds. (B) No significant difference in wound angiogenesis between LOFNotch1 and LOFCtrl mice was found. (A) The same displays in Top and Bottom as that in (A). (C) Inhibition of vascular network formation by GOFNotch1-DF. Left: representative images of capillary networks developed in 3D gel of fibroblasts-modulated in vitro angiogenesis assay. Right: quantitative data are mean ± SD of number of branches in a low power field (×0) of 3D gel (n = 6/group, ANOVA). Experiments were repeated three times.
- Figure 6. Down-regulation of IL-6 production by activation of Notch1 pathway in fibroblasts.
(A) Immunoblotting data show expression of N1IC protein in HDF transduced with N1IC-GFP/Lentiviral and GFP/Lentiviral vectors, respectively. (B) Protein Array analysis displays that IL-6 production is down-regulated in HDF expressing N1IC-GFP compared with HDF expressing GFP. White arrows point to the spots of IL-6 on the array membrane. (C) Quantitative data of ELISA analysis of IL-6 production (mean ± SD) of three independent experiments (t test). (D) ELISA shows decreased levels of IL-6 in cell lysates of three diabetic foot ulcer fibroblast compared with three NFF. Levels of IL-6 in NFF are set as 100%. Relative amount of IL-6 in diabetic foot ulcer fibroblast were calculated. (E) Immunostaining shows decreased levels of IL-6 (red) in myofibroblasts (FSP-1, green) at wound granulation tissues of GOFNotch1 compared with the GOFCtrl mice. Top: representative images of immunostaining. Bottom: Quantitative data of IL-6 production are calculated based on three sections/wound and mean ± SD are analyzed by t test.
- Figure 7. Rescue of capillary network formation with supplemental IL-6.
(A) Left: representative images of capillary networks in in vitro 3D angiogenesis assay. Exogenous supplemental γhIL-6 can partially rescue the vascular network formation modulated by N1IC–GFP/HDF. Right: quantitative data of vascular network formation by endothelial cells in 3D angiogenesis assay. Data are analyzed by one-way ANOVA followed by post-hoc test and presented as mean ± SD of three independently performed experiments. (B) Supplemental γhIL-6 in Matrigel reverses the inhibitory effects of N1IC–GFP/HDF on angiogenesis in mouse Matrigel plug model. Left: representative images of IHC. Blood vessels are brown color (DAB) Right: quantitative data of vessel density. Data are analyzed by one-way ANOVA followed by post-hoc test and presented as mean ± SD of five randomly selected fields from each mouse/Matrigel plug in a given group (5 mice/group) (×40).
Supplementary Materials
