Fig 1.
Lymphatic vessels are localized in close proximity to the HF.
(A) Representative image of the back skin of a Prox1-tdTomato mouse (red, lymphatic vasculature). Autofluorescence from the hair fiber (green). The dotted area on the left is magnified in the right panel. Scale bars: 200 μm (left panel), 100 μm (right panel). (B-F) Immunofluorescent staining of 10-μm frozen sections of back skin (anagen phase, postnatal day 8) for CD31 (B; panendothelial marker, red), LYVE-1 (B-F; lymphatic marker, green), CD68 (C; macrophage marker, red), podoplanin (D; lymphatic marker, red) and cytokeratin 15 (E and F; marker of HF stem cells, red). White arrows indicate the bulge area (Bu). (G) Staining of 50-μm cryosections for Prox1 (lymphatic-specific transcription factor, red). Maximum intensity projection of a Z stacks of images acquired using a Zeiss LSM 710 confocal microscope. (H) Immunofluorescent staining of back skin (anagen phase, postnatal day 8) for Prox1 (red). (B-H) Nuclear staining with Hoechst 33342 (blue). Scale bars: 100 μm (B); 50 μm (C-E, H); 20 μm (F and G). DP = dermal papilla, Bu = bulge area.
Fig 2.
Prolongation of anagen HF growth in K14-VEGF-C transgenic mice and early entry into the catagen phase in K14-sVEGFR3-Ig transgenic mice.
(A and E) Immunofluorescent staining of 10-μm frozen sections of back skin (anagen phase, postnatal day 8) for LYVE-1 (lymphatic marker, red). Nuclear staining with Hoechst 33342 (blue). (B and F) In H&E stained paraffin sections, 3 images/mouse were acquired and the bulb diameter was measured at the level of the largest diameter (“Auber’s line”). Data were analyzed using the two-tailed unpaired t-test for each time point. Results are presented as mean ± standard error of the mean (SEM). ***P < 0.001, **P < 0.01 versus control group. (C-D and G-H) After depilation-induced HF regeneration, back skin samples were obtained at days 15 (C, WT: n = 6, K14-VEGF-C transgenic mice: n = 4), 18 (D, WT: n = 8, K14-VEGF-C transgenic mice: n = 6), days 15 (G, WT: n = 5, K14-sVEGFR3 transgenic mice: n = 4), 18 (H, WT: n = 5, K14-sVEGFR3 transgenic mice: n = 4). The paraffin sections were stained with H&E. Scale bar: 100 μm.
Fig 3.
Intradermal delivery of VEGF-C promotes anagen hair growth.
(A) Back skin samples were obtained at postnatal days 25 (A and B, P25, WT: n = 4, transgenic: n = 9) and paraffin sections were stained with H&E. (B) 3 images/mouse were acquired and differences in the hair cycle phase of WT and K14-VEGF-C transgenic mice were compared using Fisher’s exact test. Scale bars: 100 μm. (C) 8-week-old C57BL/6 female mice in the telogen phase were shaved on the back skin with a clipper, and were intradermally injected daily for 40 days with vehicle (n = 4), VEGF-C (n = 5) or minoxidil (MNX; n = 5). Black arrows indicate the site of intradermal injection. (D) Back skin samples were obtained at the site of intradermal injection and paraffin sections were stained with H&E. Scale bars: 100 μm. (E) Grading the hair cycle phases was performed using H&E-stained paraffin sections.
Fig 4.
LEC conditioned media promote DPC proliferation, enhance their expression of IGF-1 and ALP, and inhibit BMP-2 and BMP-4 expression.
(A) DPCs were incubated with control CM (CON-CM) or LEC-CM (10, 30, 50 or 90%), or 100 ng/ml IGF-1 as a positive control for 72 h. Cell proliferation was assessed with the CCK-8 assay. Differences in relative absorbance levels were analyzed by the two-tailed unpaired t-tests. One representative experiment is presented as mean ± standard deviation (SD). **P<0.01 versus control group. (B) DPCs were incubated with 50% LEC-CM or CON-CM for 72 h. Cells were lysed and western blotting was performed, using antibodies for total Akt (T-Akt), phosphorylated Akt (P-Akt), or beta-actin. (C) DPCs were treated with the indicated concentrations of recombinant human VEGF-C for 72 h. Cell proliferation was assessed with the CCK-8 assay. Differences in relative absorbance levels were analyzed with the two-tailed unpaired t-test. Results are presented as mean±SD. (D-I) DPCs were incubated with CON-CM or LEC-CM (10, 30, 50 or 90%), BEC-CM (50%), dermal fibroblast-CM (DF-CM; 50%), or 100 ng/ml IGF-1 for 72 h. Total RNA was isolated and qRT-PCR was performed. mRNA expression levels were analyzed using the two-tailed paired t-test. Pooled data from 4 independent experiments are presented as mean±SEM. *P<0.05, **P<0.01 versus control group.