Control of hair growth and follicle size by VEGF-mediated angiogenesis (2024)

Association of hair cycling with cyclic perifollicular angiogenesis. We first studied angiogenesis during depilation-induced, synchronized adult hair cycling (22) by computer-assisted morphometric analyses of back skin sections stained for CD31, an endothelial junction molecule (29). Tissue samples were obtained over a 22-day period that covered all phases of the murine hair cycle from early anagen to telogen (2, 23, 24). A more than fourfold increase in perifollicular vessel size was observed during the late anagen growth phase of the adult hair cycle, as compared with early anagen (Figure 1a), followed by complete reduction to pre-anagen sizes during the catagen and telogen phases (Figure 1, b and f). In contrast, the vessel density, defined as the number of vessels per area unit, was not significantly altered during the hair cycle (Figure 1g). The percentage of perifollicular area covered by vessels increased from 4.1% to 15.9% during the anagen phase and was reduced to 2.9% in telogen (Figure 1e). Similar findings were observed during the physiological first postnatal hair cycle (data not shown). On postnatal day 1 (P1), early anagen follicles were surrounded by small blood vessels with an average size of 148 μm², covering 5.9% of the surface area. During late anagen (P12), large, elongated, and dilated vessels were observed surrounding the elongated hair follicles, covering 17.1% of the surface area. A significant reduction in perifollicular vessel sizes to 343 μm² was detected during the catagen involution phase (P18) with a concomitant reduction in the surface area covered by vessels to 8.8%. The average vessel size was further reduced to 108 μm² in the telogen resting phase with vessels covering only 3.5% of the surface area. These results reveal an identical pattern of perifollicular vascularization during the induced adult hair cycle and the physiological postnatal hair development. Double immunofluorescence stainings for CD31 and BrdU detected proliferating perifollicular endothelial cells throughout the anagen growth phase of the induced hair cycle (Figure 1c), whereas no endothelial proliferation was observed during catagen and telogen. Conversely, apoptotic endothelial cells were selectively detected in perifollicular vessels during the catagen involution phase (Figure 1d). These results establish the murine hair cycle as an easily accessible in vivo system where physiological modulation of angiogenesis can be studied.

Temporal association of perifollicular angiogenesis with cyclic changes of follicle size and cutaneous thickness. We next examined whether the vascular remodeling observed during the anagen growth phase was temporally associated with increases in the size of hair follicles and in the thickness of the overlying epidermis or of the dermis and subcutis. The thickness of interfollicular epidermis significantly increased from day 1 to day 3 (early anagen) of the induced adult hair cycle, and reached a maximum thickness of 54.5 μm on day 5, followed by a steep decline during mid- to late anagen with a further reduction during telogen (Figure 1h). In contrast, cutaneous thickness (dermis and subcutis) steadily increased from early anagen to reach a peak in late anagen, followed by a decline during catagen and telogen (Figure 1j), coinciding with cyclic changes of hair follicle size (Figure 1i). Similar results were obtained during the first postnatal hair cycle. The exact temporal coincidence of changes in follicle size, cutaneous (but not epidermal) thickness, and perifollicular vascularization suggested that the angiogenic stimulus was derived from either the hair follicle itself or the surrounding dermis and subcutis, but not from the interfollicular epidermis.

Upregulation of follicular VEGF expression during the anagen growth phase. Because we had previously identified VEGF as an important mediator of skin angiogenesis (17, 30), we next examined, by in situ hybridization, the spatiotemporal expression pattern of VEGF mRNA during induced hair cycling. Whereas little VEGF mRNA expression was detected in early anagen (day 1) hair follicles or in interfollicular epidermal keratinocytes, VEGF mRNA was highly expressed in follicular keratinocytes during mid-anagen (days 5 and 8), predominantly in the middle third of hair follicles (Figure 2a) where perifollicular angiogenesis was most prominent. In contrast, little or no VEGF mRNA expression was detected in the surrounding dermis and subcutis and in dermal papilla cells. When we correlated VEGF mRNA expression levels with the cyclic changes of perifollicular vascularization, we found that modulations of VEGF expression preceded vascular changes by an interval of approximately 3 days with a marked decrease of VEGF expression during late anagen, catagen, and telogen (Figure 2b). Identical results were obtained during the physiological first postnatal hair cycle (Figure 2c). Thus, the close spatiotemporal association of follicular VEGF expression and perifollicular angiogenesis strongly suggested an important role of follicle-derived VEGF in the control of hair vascularization.

Enhanced hair growth and follicle size in VEGF transgenic mice. To investigate whether follicular VEGF expression directly promotes hair growth and vascularization, we studied the hair cycle in transgenic mice with selective overexpression of VEGF in basal epidermal keratinocytes and in outer root sheath keratinocytes of hair follicles, targeted by a keratin 14 promoter cassette (17). As early as 10 days after depilation, VEGF-overexpressing mice showed accelerated hair regrowth as compared with wild-type littermates. After 11 days, these differences were more obvious, and the hair appeared both longer and thicker in VEGF transgenic mice (Figure 3, a and b). Histological analysis at days 12 and 15 (late anagen) of the induced hair cycle revealed that hair follicles in VEGF transgenic mice were larger than in wild-type controls, most prominently at the level of the hair bulb (Figure 3, c–f). Image analysis of hair bulbs at the level of the maximum bulb diameter revealed a more than 30% increase in diameter in VEGF transgenic mice at day 12 (Figure 3g) and at day 15 (Figure 3j). Importantly, the thickness of fully developed hair shafts was significantly increased (P < 0.001), by more than 30%, in VEGF transgenic mice (Figure 4, a and b), whereas no differences in length were observed. Hair follicles in VEGF transgenic mice were also enlarged during the growth phase (day 15) of the physiological first hair cycle (P < 0.05; data not shown). A more than 40% increase in vessel sizes (Figure 3, h and k) and total vascular mass (Figure 3, i and l) was detected surrounding VEGF-overexpressing hair follicles at days 12 and 15, suggesting that accelerated hair growth and increased hair size were a consequence of VEGF-mediated angiogenesis.

Blockade of VEGF activity inhibits hair growth. We next examined whether VEGF-mediated angiogenesis was essential for the timely growth of hair follicles during the anagen phase. Adult C57BL/6 mice were treated systemically with a neutralizing anti-VEGF antibody, and the hair cycle was induced by depilation 1 day after the first antibody application. The neutralizing activity of the anti-VEGF antibody at the concentrations used was confirmed by its ability to completely inhibit VEGF-induced microvascular leakage in the skin of guinea pigs and of mice, measured in a modified Miles assay (data not shown). During the first 8 days after depilation, no differences in the macroscopic skin appearance were observed in anti-VEGF–treated mice. Thereafter, delayed hair regrowth was apparent in anti-VEGF–treated mice. After 12 days, mice treated with the anti-VEGF antibody still showed bald spots and had an overall reduced hair growth (Figure 5b) as compared with normal hair regrowth in control antibody–treated mice (Figure 5a). Histological analysis revealed that anagen hair follicles in anti-VEGF–treated mice were thinner (Figure 5, c and d) with a more than 30% reduction in bulb diameters (Figure 5e), mainly due to reduced thickness of the follicular epithelium, associated with a significant, more than 40% decrease of perifollicular vascularization (Figure 5, f and g).

Absence of VEGF effects on hair growth and follicle size in vibrissa follicle cultures in vitro. Next we investigated whether VEGF might also directly act on hair follicle cells, in addition to its effects on perifollicular angiogenesis. Therefore, we isolated mouse vibrissae and quantitated hair growth and follicle size in organ cultures in vitro, in the absence of a functioning vascular system. Vibrissa follicles are capable of responding to VEGF, as indicated by the significantly increased (P < 0.01) diameter of vibrissa follicles in VEGF transgenic mice (Figure 6b) compared with wild-type mice (Figure 6a). In organ culture, untreated mouse vibrissae showed an average hair shaft outgrowth of approximately 2 mm over a period of 4 days (Figure 6, c and d). Addition of 5% FBS to vibrissa cultures, used as a positive control, resulted in a significant (P < 0.001) increase of hair shaft growth, confirming the inducibility of hair growth in this experimental system. In contrast, addition of murine VEGF did not affect the hair growth rate in organ culture. Similarly, incubation with a neutralizing goat anti–murine VEGF antibody did not modify in vitro hair growth, as compared with vibrissa cultures treated with equivalent concentrations of goat IgG (Figure 6, c and d). Moreover, treatment of isolated follicles with VEGF or with anti-VEGF antibody did not significantly modify the size of hair bulbs (data not shown). These results suggest that a functional perifollicular vascular system is necessary for the mediation of VEGF effects on follicle growth.