Perspectives for future research

The data presented in this study and in resulting publications (MECKLENBURG et al.

2000) establishes that the vascular system in mouse skin is far from being quiescent.

Instead it undergoes substantial cyclic remodeling processes, that are associated with and governed by the activity of hair follicles. Furthermore, we present evidence for the involvement of VEGF and angiopoietins in the regulation of these remodeling processes. However, many open questions remain that have to be adressed by subsequent experimental studies:

Which further investigations could contribute to the validity of the presented concept?

In the current study, determination of mRNA levels of VEGF and angiopoietins was limited to four time-points of the depilation-induced murine hair growth cycle. In order to obtain a continuous time-profile of the expression of these important growth factors during the hair growth cycle, more points of measurement would be necessary. In addition, investigation of a larger population of animals would facilitate the confirmation of the statistical significance of observed differences. Cell capture by laser microdissection would allow to study a clearly definable population of cells, i.e.

outer root sheath keratinocytes of the bulge region, and would probably overcome the problem of normalization that we faced during our quantitative RT-PCR analyses.

More detailed investigation of Ang-1- and Ang-2–gene expression could help to clarify the mechanism of their interaction at each point of time during the hair cycle.

Transgenic mice that express reporter genes under the control of either angiopoietin-promoter, e.g. Ang-2/LacZ-mice (YUAN et al. 2000), can serve as suitable models to answer this question, and are currently under investigation.

Furthermore, the effects of Ang-1 and Ang-2 on the regulation of remodeling of the perifollicular vasculature could be investigated in mice that overexpress Ang-1 in outer root sheath keratinocytes (MAISONPIERRE et al. 1997) and in mice, overexpressing Ang-2 in blood vessels (SURI et al. 1998). Overexpression of Ang-1

should lead to prolonged stabilization of the vasculature and would thus disturb both anagen-associated angiogenesis and catagen-associated vascular regression. In contrast, mice that overexpress Ang-2 in blood vessels should exhibit a destabilized vasculature, possibly leading to disturbed maintenance of the perifollicular blood vessels in mature anagen skin. Mice with targeted gene ablation, similar to those that lack VEGF in keratin 5-expressing cells, would provide further insight in the role of hair follicle-derived angiopoietins, but have not been generated so far.

Which other factors are involved in the hair cycle-dependent vascular remodeling in the skin?

As is demonstrated in Table 1.1, a multitude of factors participate in the regulation of vascular remodeling. Therefore, it is rather likely that, beside VEGF and the angiopoietins, other factors are also involved in regulating the hair cycle-depdendent remodeling of the perifollicular vasculature.

Most of these factors are pleitrop, i.e. they mediate different biological effects in various tissues. PDGF-B, TGF-β1, FGF, HGF and prostaglandins (generated by (cyclooxigenase-2 activity) are not only involved in angiogenesis but are also part of the epithelial-mesenchymal interactions that participate in hair follicle morphogenesis/cycling (MCELWEE and HOFFMANN 2000).

Furthermore, degradation of the extracellular matrix occurs both during anagen- and catagen-development of hair follicles (MESSENGER 1991, PAUS et al. 1994ab). An array of proteinases, e.g. proteinases of the plasminogen activator family and matrix metalloproteinases, is activated and makes it possible for the hair follicle to extend into and retract from the hypodermis (WEINBERG et al. 1990, GOODMAN and LEDBETTER, KARELINA et al. 1994, PAUS et al. 1994ab, SCANDURRO et al.

1995, YAMAZAKI et al. 1999, JENSEN et al. 2000, JAROUSSE et al. 2001). These proteinases might not only influence the mesenchymal remodeling during the hair growth cycle but also effect angiogenesis directly or indirectly via the liberation of several growth factors that are sequestered in the extracellular matrix, e.g. fibroblast

growth factor, angiostatin, endostatin, and thrombospondins (O´REILLY 1997, O´REILLY et al. 1997, DETMAR 2000).

Is the long-term deprival of VEGF a pathogenetic mechanism of hair loss?

It has been claimed that a reduced blood supply is a pathogenic factor of hair loss (CHIALE 1927, ELLIS 1958, MONTAGNA and PARAKKAL 1974). By pharmacological inhibition of angiogenesis we could indeed show that there is a reduction of the microvessel density in the skin, which leads to inhibition of anagen-development. However, nothing is known so far about the long-term effect of a reduced cutaneous vascularization on hair growth. We have demonstrated that short-time deprival of VEGF is one mechanism that leads to a reduction of the cutaneous blood supply, and it has been reported that lack of VEGF-bioactivity results in a decreased diameter of hair shafts (YANO et al. 2001).

Therefore, it is interesting to study the long-term effect of lack of VEGF on hair folicles. We started with studies in transgenic mice that lack endogenous VEGF in the follicular outer root sheath. In these mice, a reduced hair growth after repeated depilation has been reported (ROSSITER et al., unpubl. oberservation), but not carefully studied so far. Corresponding studies are on their way.

Can hair growth be influenced by manipulating the perifollicular vasculature?

Based on our findings that the lack of endogenous VEGF results in a destabilized cutaneous vasculature which regresses faster during catagen, it would be interesting to investigate whether maintenance of the vasculature (e.g. by prolonged administration of VEGF or Ang-1) would prolong-anagen and delay catagen-development of the hair follicles. Conversely, a reduction of the perifollicular vasculature might be able to induce catagen-development and would thus be exploitable as a therapeutical approach to treat excessive hair growth.