Control of hair growth and follicle size by VEGF-mediated angiogenesis

Abstract

The murine hair follicle undergoes pronounced cyclic expansion and regression, leading to rapidly changing demands for its vascular support. Our study aimed to quantify the cyclic changes of perifollicular vascularization and to characterize the biological role of VEGF for hair growth, angiogenesis, and follicle cycling. We found a significant increase in perifollicular vascularization during the growth phase (anagen) of the hair cycle, followed by regression of angiogenic blood vessels during the involution (catagen) and the resting (telogen) phase. Perifollicular angiogenesis was temporally and spatially correlated with upregulation of VEGF mRNA expression by follicular keratinocytes of the outer root sheath, but not by dermal papilla cells. Transgenic overexpression of VEGF in outer root sheath keratinocytes of hair follicles strongly induced perifollicular vascularization, resulting in accelerated hair regrowth after depilation and in increased size of hair follicles and hair shafts. Conversely, systemic treatment with a neutralizing anti-VEGF antibody led to hair growth retardation and reduced hair follicle size. No effects of VEGF treatment or VEGF blockade were observed in mouse vibrissa organ cultures, which lack a functional vascular system. These results identify VEGF as a major mediator of hair follicle growth and cycling and provide the first direct evidence that improved follicle vascularization promotes hair growth and increases hair follicle and hair size.

Figure 1

Pronounced vascular changes during the induced murine hair cycle. (a and b) CD31 immunostains demonstrate a marked increase in perifollicular vascularization during late anagen (day 12, a) with subsequent regression of blood vessels during catagen and telogen (day 22, b). (c) Detection of proliferating endothelial cells (arrowheads) in perifollicular vessels during the anagen growth phase. Proliferating cells are depicted in red (BrdU), endothelial cells in green (CD31). (d) Endothelial cell apoptosis (arrowheads) was detectable in perifollicular vessels during the catagen involution phase. Apoptotic cells are depicted in green, endothelial cells in red. Asterisks indicate the location of hair bulbs. Scale bars = 100 μm. Computer-assisted image analysis revealed significant cyclic changes of relative areas covered by vessels (e) and of the average vessel size (f), with a more than fourfold increase during anagen (P < 0.001) and a decrease to early anagen levels during catagen and telogen. (g) Vessel densities were not significantly changed during the hair cycle. Vascular changes were temporally associated with cyclic changes of hair follicle size (i) and dermal thickness (j), but not of epidermal thickness (h). H&E-stained sections were evaluated as described in Methods. Data are expressed as means ± SD of three independent experiments. ^AP < 0.05; ^BP < 0.01; ^CP < 0.001 (increase over early anagen). EA, early anagen; MA, mid-anagen; LA, late anagen; C, catagen; T, telogen; D, day.

Figure 2

(a) In situ hybridization demonstrates strong VEGF mRNA expression in follicular keratinocytes (arrows) during mid-anagen of the induced hair cycle. Scale bar = 50 μm. (b and c) Temporal correlation of follicular VEGF mRNA expression levels (filled circles) and perifollicular angiogenesis during the induced adult hair cycle (b) and the physiological first postnatal hair cycle (c). Relative vessel area (open circles) is expressed as percentage of the maximum vessel area detected during late anagen (day 12; compare with Figure 1e).

Figure 3

Accelerated hair regrowth and increased follicle size in VEGF transgenic mice. VEGF transgenic mice showed more and thicker hair at day 11 after depilation (b), as compared with wild-type littermates (a). Histological analysis of H&E-stained paraffin sections demonstrates increased size of hair bulbs in VEGF transgenic mice at day 12 (d) and day 15 (f) after depilation, as compared with wild-type (WT) mice at day 12 (c) and day 15 (e). Scale bars = 100 μm. Hair bulbs in VEGF transgenic mice, measured at the level of the largest diameter, were more than 35% thicker than in wild-type mice at day 12 (g) and day 15 (j) after depilation. Quantitative analysis of CD31 stains revealed that perifollicular vascularization, assessed as average vessel size (h, day 12; k, day 15) or relative vessel area (i, day 12; l, day 15), was significantly increased in VEGF transgenic mice during late anagen. Data are expressed as means ± SD. ^AP < 0.01, ^BP < 0.001, two-sided unpaired Student’s t test.

Figure 4

Increased diameter of hair shafts in 12-week-old VEGF transgenic mice (VEGF TG) during late anagen, as compared with wild-type (WT) littermates. (a) Light microscopy of unstained plucked awl hair; scale bar = 30 μm. (b) Quantitative analysis of hair shafts, measured at the level of their greatest width, revealed a significantly increased hair diameter in VEGF transgenic mice. Data are expressed as means ± SD (n = 50 for each genotype). ^AP < 0.001, two-sided unpaired Student’s t test.

Figure 5

Delayed hair regrowth in C57BL/6 mice after treatment with a neutralizing anti-VEGF antibody. (a) At day 12, hair regrowth was complete in control-treated mice, whereas anti-VEGF–treated mice still showed bald spots (b, arrowheads). (c and d) Histological analysis demonstrates diminished thickness of hair bulbs in anti-VEGF–treated mice at day 12 after depilation (d), as compared with control mice (c). Scale bars = 100 μm. (e) Hair bulbs in anti-VEGF–treated mice, measured at the level of the largest diameter, were more than 30% thinner at day 12 after depilation. (f and g) Quantitative analysis of CD31 stains revealed that perifollicular vascularization, assessed as average vessel size (f) or relative vessel area (g), was significantly diminished in anti-VEGF–treated mice during late anagen. Data are expressed as means ± SD. ^AP < 0.001, two-sided unpaired Student’s t test.

Figure 6

(a and b) Representative photomicrographs of hematoxylin-stained paraffin sections depict increased size of vibrissa follicles in 8-week-old VEGF transgenic mice (b), as compared with age-matched wild-type littermates (a). Scale bars = 100 μm. (c) Representative photomicrographs of mouse vibrissa organ cultures demonstrate absence of effects of VEGF treatment (V) on the in vitro hair growth rate, as compared with untreated controls (C1). Addition of 5% FBS (P), used as a positive control, resulted in a more than 15% increase in hair growth. Treatment with a neutralizing anti-VEGF antibody (Vab) did not influence in vitro hair growth, as compared with control antibody–treated follicles (C2). (d) Quantitative analysis demonstrates significant induction of in vitro hair growth by 5% FBS (P) (P < 0.001) but lack of efficiency of VEGF (V) or anti-VEGF antibody (Vab) treatment. Data are expressed as means ± SD. NS, no significant differences between the groups compared. ^AP < 0.001, two-sided unpaired Student’s t test.