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ASCORBIC ACID 2-PHOSPHATE PROMOTES HAIR GROWTH Kim MK, Kim JC, Sung YK Department of Immunology, Kyungpook National University School of Medicine, Daegu, Korea

"In summary, we, first time to our knowledge, report that Asc 2-P stimulates the growth of dermal papilla cells and promotes the elongation of hair shafts in isolated hair follicles in culture. These data, together with induction of early conversion from a telogen phase to an anagen phase by Asc 2-P in animal model, suggest that Vitamin C or its derivatives may regulate mesenchymal-epithelial interactions in the hair follicle."

Cultured dermal papilla cells exhibit normal fibroblast-like morphology at early passage. In contrast to the dermal fibroblast, cultured dermal papilla cells can induce hair follicle growth in vivo, although their inductivity is gradually lost during subculture. It is known that ascorbic acid 2-phosphate (Asc 2-P) stimulates growth of dermal fibroblasts by enhanced production of collagen synthesis. However, it is not known whether Asc 2-P stimulates growth of dermal papilla cells and promotes hair follicle growth. In this study, Asc 2-P stimulated growth of dermal papilla cells and promoted hair follicle growth in organ culture model. On the other hand, Asc 2-P did not significantly promoted the growth of outer root sheath keratinocytes. The mRNA level of IGF-1 was increased 3.8-folds with Asc 2-P treatment while that of HGF, VEGF and KGF were not affected in dermal papilla cells. Versican expression in dermal papilla was also increased by Asc 2-P. However, the mRNA level of collagen types I and III was not affected by Asc 2-P in dermal papilla cells. These data, first time to our knowledge, demonstrate that Asc 2-P stimulates growth of dermal papilla cells and promote hair follicle growth in vitro. The growth stimulation of dermal papilla cells and induction of hair follicle growth seems to be, at least in part, mediated by IGF-1 over-expression from dermal papilla cells by Asc 2-P. In addition, these data suggests that signalling pathway that leads to versican expression is activated by Asc 2-P and Asc 2-P may keep dermal papilla cells to maintain hair-inducing activity by regulating versican.

The mammalian hair follicle contains dermal papilla and dermal sheath cells derived from the mesenchyme <1>. It also contains epithelial cells of outer and inner root sheaths, matrix, and hair shaft, derived from the epithelium <1>. The post-natal hair follicle undergoes a cycle of growth (anagen), regression (catagen) and rest (telogen). The reciprocal interactions between the epithelium and mesenchyme are essential for post-natal hair growth as well as embryonic formation of hair follicle <1>. Dermal papilla, a cluster of specialized fibroblasts, is known to play a key role in the regulation of hair growth. Dermal papilla is encapsulated by the overlying epithelial matrix cells during hair follicle growth phase (anagen) and growth factors from dermal papilla including insulin-like growth factor 1 (IGF- 1) are believed to cause epithelial cells to proliferate and differentiate to produce hair shaft <2,3>.

L-Ascorbic acid 2-phosphate magnesium salt (Asc 2-P; Fig. 1A), a derivative of L-ascorbic acid (Vitamin C), is more stable than ascorbic acid and is known to stimulate growth of human dermal fibroblasts and osteoblast-like cells <4,5>. However, it is not known whether Asc 2-P stimulates growth of dermal papilla cells. Here, we investigated the biological effects of Asc 2-P for hair apparatus.

Fig. 1 (A) Chemical structure of L-ascorbic acid-2-phosphatemagnesiumsalt (Asc 2-P). (B) Effect of Asc 2-P on the growth of cultured dermal papilla cells (top) and ORS keratinocytes (bottom). Dermal papilla cells were incubated in serum-free or 1% FBS-supplemented DMEM for 5 days. ORS cells were incubated in MCDB 153 medium or KGM for 5 days. (C) Effect of Asc 2-P on the growth of human hair follicles cultured in vitro for 9 days. Data are means +- standard errors of five to six determinations per experiment in four independent experiments (*P < 0.05). (D) Effect of Asc 2-P on the expression of mRNA levels (top) and quantification of IGF-1 mRNA level (bottom; 40 cycles were employed in RT-PCR analysis). Dermal papilla cells were treated with 0.25 mM Asc 2-P for 5 days. RT-PCR conditions and primer sequences are same as described <10>.

Hair biopsy specimens were obtained from the non-balding occipital scalp region of patients with androgenic alopecia during hair transplantation at Kyungpook National University Hospital (Daegu, Korea) with patients’ consents. Hair follicles were isolated and cultured by the method described previously <6,7>. Dermal papilla was isolated from the bulbs of dissected anagen hair follicles and cultured as described previously <8>. Cells from the 2-3 passage were used in this study.

We observed significant growth stimulation in dermal papilla cells when Asc 2-P was added into growth medium (Fig. 1B). Asc 2-P at 0.25 mM stimulated the growth of dermal papilla cells the most. In contrast, there was no growth stimulating effect on the ORS keratinocytes (Fig. 1B).We also observed that Asc 2-P treatment resulted in significant elongation of hair shafts in isolated hair follicles in culture (Fig. 1C). Probability values of 0.012 and 0.025 were obtained for hair follicles exposed to 0.05 and 0.25 mM Asc 2-P, respectively.

Fig. 2 (A) Early onset of anagen by Asc 2-P in C57BL/6 mice. The back skins were shaved at 7 weeks of age (telogen stage) and then vehicle (left) or Asc 2-P at 250 mM (right) were applied by iontophoresis. We adapted the unique iontophoresis system, which provides a constant electric current of 0.1-1.0 mA/cm2 areas. Iontophoresis was performed for 15 min at 0.2-0.4 mV constant currents daily for 4 weeks. (B) Representative hematoxylin and eosin stained sections of back skin of control (left) and Asc 2-P (250 mM) treated (right) mouse.

Growth stimulation of cultured dermal papilla cells and elongation of hair shafts in isolated hair follicles by Asc 2-P suggest that the hair growth promoting effect of Asc 2-P may be mediated through proliferative and anti-apoptotic effects on dermal papilla cells, thereby prolonging the growth phase. It is also possible that the elongation of hair shafts in culture is mediated through the expression of growth factors by Asc 2-P in dermal papilla. Indeed, we observed that the mRNA level of IGF-1 was increased 3.8-folds after treatment with Asc 2-P, while that of HGF, VEGF and KGF were not affected in dermal papilla cells (Fig. 1D). These data suggest that Asc 2-P induces secretion of growth factor(s), including IGF-1, which in turn proliferate and differentiate overlying keratinocytes to promote elongation of hair shafts. Our data is in line with the finding that IGF-1 proliferates epithelial cells and promotes hair follicle growth in cultured hair follicles <2,7> as well as in transgenic animals <9>.

We also observed that Asc 2-P at 250 mM induced earlier telogen to anagen conversion as compared with vehicle treated group (Fig. 2A). Histology at necropsy also showed that mice treated with Asc 2-P and control mice present anagen and telogen hair follicle stage, respectively (Fig. 2B).

In summary, we, first time to our knowledge, report that Asc 2-P stimulates the growth of dermal papilla cells and promotes the elongation of hair shafts in isolated hair follicles in culture. These data, together with induction of early conversion from a telogen phase to an anagen phase by Asc 2-P in animal model, suggest that Vitamin C or its derivatives may regulate mesenchymal-epithelial interactions in the hair follicle.

Acknowledgement:This study was supported by a grant (03-PJ1-PG1- CH13-0001) of the Korea Health 21 R&D project, Ministry of Health & Welfare, Republic of Korea.

References: <1> Botchkarev VA, Kishimoto J. Molecular control of epithelial-mesenchymal interactions during hair follicle cycling. J Investig Dermatol Symp Proc 2003;8:46-55. <2> Itami S, Kurata S, Takayasu S. Androgen induction of follicular epithelial cell growth is mediated via insulin-like growth factor-I from dermal papilla cells. Biochem Biophys Res Commun 1995;212:p988-94. <3> Stenn KS, Paus R. Controls of hair follicle cycling. Physiol Rev 2001;81:449-94. <4> Takamizawa S, Maehata Y, Imai K, Senoo H, Sato S, Hata R. Effects of ascorbic acid and ascorbic acid-2-phosphate, a long-acting Vitamin C derivative, on the proliferation and differentiation of human osteoblast-like cells. Cell Biol Int 2004;28:255-65. <5> Hata R, Senoo H. L-Ascorbic acid-2-phosphate stimulates collagen accumulation, cell proliferation, and formation of a three-dimensional tissuelike substance by skin fibroblasts. J Cell Physiol 1989;138:8-16. <6> Philpott MP, Green MR, Kealey T. Human hair growth in vitro. J Cell Sci 1990;97:463-71. <7> Philpott MP, Sanders DA, Kealey T. Effects of insulin and insulin-like growth factors on cultured human hair follicles: IGF-I at physiologic concentrations is an important regulator of hair follicle growth in vitro. J Invest Dermatol 1994;102: 857-61. <8> Magerl M, Kauser S, Paus R, Tobin DJ. Simple and rapid method to isolate and culture follicular papillae from human scalp hair follicles. Exp Dermatol 2002;11:381-5. <9> Damak S, Su H, Jay NP, Bullock DW. Improved wool production in transgenic sheep expressing insulin-like growth factor 1. Biotechnology 1996;14:185-8. <10> Roh SS, Kim CD, Lee MH, Hwang SL, Rang MJ, Yoon YK. The hair growth promoting effect of Sophora flavescens extract and its molecular regulation. J Dermatol Sci 2002;30: 43-9.

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