Hair follicles continuously cycle through anagen (growth), catagen (regression), and telogen (resting) stages (Schneider
Chemotherapy or radiation therapy, which targets rapidly dividing cancer cells, frequently leads to visibly distressing side effects such as hair loss. Chemotherapy-induced alopecia, ranging from mild to severe, often causes patients to refuse chemotherapy (Yeager and Olsen, 2011; Dunnill
5-Fluorouracil (5-FU) is an antimetabolite anticancer drug widely used to treat gastrointestinal, breast, gynecological, and head and neck cancers (Grem, 1997). However, patients receiving 5-FU treatment commonly display endovascular injury in various organs (Jensen and Sørensen, 2012; Focaccetti
Synchronized anagen was induced in the dorsal skin of approximately 7-week-old (P50-53) C57BL/6 mice by depilation, as previously described (Paus
P50-53 wild-type C57BL/6 mice and Tie2cre;
Lentivirus was produced as previously reported (Park
P50-53 C57BL/6 mice were depilated and intraperitoneally injected with 250 mg/kg 5-FU or PBS. Lentiviral
In brief, the dorsal skin was excised at day 13 (late anagen) after depilation and fixed using 4% paraformaldehyde in PBS. The skins were then frozen in optimal cutting temperature compound (Tissue-Tek, Sakura Finetk, Torrance, CA, USA) at −20°C, and cut into 10-µm thick sections using a Cryostat Cryocut Microtome (CM1850, Leica, Nussloch, Germany). For CD31 staining, tissue sections were incubated overnight at 4°C with rat anti-platelet endothelial cell adhesion molecule (CD31, PECAM-1) (1:25) antibody (BD Bioscience, San Jose, CA, USA), followed by staining with Alexa Fluor 594-labeled goat anti-rat (1:200) for 1 h at room temperature (Molecular Probes, Grand Island, NY, USA). Nuclei were counterstained with 4,6 diamidino-2-phenylindole (DAPI, Vector Laboratories, Burlingame, CA, USA) and examined using a fluorescence microscope (Axiovert 200M, ZEISS, Göttingen, Germany). At least eight images were randomly selected from each slide, and fluorescent vessels were counted to quantify CD31 positive vessels in the skin.
The dorsal skins were excised on day 13 after depilation, fixed in 4% paraformaldehyde in PBS, dehydrated in a graded ethanol series, and embedded in paraffin. Tissue sections (4-μm thick) were stained with hematoxylin and eosin. Five images were randomly selected from each slide (n=1). The skin thickness was measured in at least five different regions and averaged per image using ImageJ (National Institutes of Health, Bethesda, MD, USA).
Mice were injected intravenously with 70 kDa rhodamine-dextran (50 μL) on day 13 after depilation. A two-photon laser tuned to 890 nm was used to excite dextran. In each experiment, a total of 50 two-dimensional (2D) slices were obtained at 2.5 μm steps and extrapolated into a three-dimensional (3D) volume using the Imaris software. Blood vessel diameters and volumes were measured from 3D rendering using Imaris’s FilamentTracer module (OXFORD Instruments, Zurich, Switzerland). The fractal dimension of the vasculature was calculated in MATLAB using an iterative box-counting algorithm on the maximum intensity projection of the 3D volume. Data are presented as the mean ± standard error (SE) (n=7-8, z-stack images).
Statistical analysis of blood vessel density measurements and quantification of anagen induction was performed using analysis of variance (ANOVA). For 2P image quantification, repeated measures analysis of covariance (ANCOVA) was performed using SAS statistical software (Campus Drive, Cary, NC, USA) with proc mixed. Multiple images per mouse were obtained, and model-fitting statistics, such as AIC and log-likelihood, suggested that a diagonal variance component matrix best fits the repeated measures effect. Outcome variables were adjusted for covariance from naturally occurring predictor variables (e.g., longer vessels have higher curvature regardless of treatment). Statistical contrasts were performed to assess within-group differences, and a
Previous studies have revealed that depilation (shaving) induces anagen in telogenic mice (Yano
In a mouse hindlimb ischemic injury model, lentiviral
Moreover, the 5-FU treatment decreased the thickness of the dermis and subcutaneous tissue of the dorsal skin, and
Approximately 80% of patients with cancer have reported that chemotherapy-induced alopecia is a severe problem associated with cancer treatment after nausea and vomiting (Yeager and Olsen, 2011). Accordingly, several patients refuse to undergo cancer chemotherapy (Yeager and Olsen, 2011). Understanding hair loss mechanisms and hair regeneration after chemotherapy can improve the quality of life in patients undergoing cancer treatment.
Chemotherapeutic agents reportedly cause vascular damage to hair follicles (Amoh
Etv2 is critical for vascular development and regeneration (Lee
In the present study, we were unable to confirm the effect of Etv2 on the regulation of the normal hair cycle; however, Etv2 activation might also enhance hair regeneration under normal conditions. ER71 promotes FLK1+ mesoderm formation by directly activating
In conclusion, our data demonstrated that Etv2 deficiency in endothelial cells reduces hair regeneration. Conversely, enforced Etv2 expression led to improved angiogenesis and hair regeneration after 5-FU treatment. Our data support the notion that Etv2-mediated enhanced vascularization may offer a novel approach for hair regeneration after chemotherapy. Our studies further support the idea that vessel regeneration strategies may be integrated into future tissue regeneration therapies.
We thank the lab members for constructive criticism and discussions of this work. This work was supported by the NIH grants HL119291 (C.P), HL149954 and HL55337 (K.C), and the 2020 Research Grant from Kangwon National University (520200067) (T. L.).
The authors have declared that no competing interest exists.