An important role of cutaneous lymphatic vessels in coordinating and promoting anagen hair follicle growth

Abstract

The lymphatic vascular system plays important roles in the control of tissue fluid homeostasis and immune responses. While VEGF-A-induced angiogenesis promotes hair follicle (HF) growth, the potential role of lymphatic vessels (LVs) in HF cycling has remained unknown. In this study, we found that LVs are localized in close proximity to the HF bulge area throughout the postnatal and depilation-induced hair cycle in mice and that a network of LVs directly connects the individual HFs. Increased LV density in the skin of K14-VEGF-C transgenic mice was associated with prolongation of anagen HF growth. Conversely, HF entry into the catagen phase was accelerated in K14-sVEGFR3 transgenic mice that lack cutaneous LVs. Importantly, repeated intradermal injections of VEGF-C promoted hair growth in mice. Conditioned media from lymphatic endothelial cells promoted human dermal papilla cell (DPC) growth and expression of IGF-1 and alkaline phosphatase, both activators of DPCs. Our results reveal an unexpected role of LVs in coordinating and promoting HF growth and identify potential new therapeutic strategies for hair loss-associated conditions.

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.