Kerstin Foitzik

Mast cells are required for normal healing of skin wounds in mice

Mast cells (MCs) have recently been reported to play a pivotal role in the elicitation of inflammatory reactions that are beneficial to the host, e.g., during innate immune responses to bacteria. To explore whether MCs also contribute to wound repair, we studied experimentally induced skin wounds in MC‐deficient KitW/KitW‐v mice, normal Kit/ mice, and MC‐reconstituted KitW/KitW‐v mice. Wound closure was significantly impaired in the absence of MCs during the first 6 days of wound healing and histomorphometric analyses of MC degranulation at the wound edges revealed distance‐dependent MC activation, i.e., MC degranulation was most prominent directly adjacent to the wound. In addition, KitW/KitW‐v mice showed impaired extravasation and recruitment of neutrophils to the wounded areas. Notably, wound closure, extravasation, and neutrophil recruitment were found to be normal in MC‐reconstituted KitW/KitW‐v mice. Therefore, we examined whether MCs promote wound healing by releasing histamine or TNF‐. Interestingly, wound closure was reduced in mice treated with an H1‐receptor antagonist but not after treatment with an H2‐receptor antagonist or in the absence of TNF‐. Taken together, our findings indicate that MC activation and histamine release are required for normal cutaneous wound healing.—Weller, K., Foitzik, K., Paus, R., Syska, W., Maurer, M. Mast cells are required for normal healing of skin wounds in mice. FASEB J. 20, E1628 –E1635 (2006)

The Lysosomal Protease Cathepsin L Is an Important Regulator of Keratinocyte and Melanocyte Differentiation During Hair Follicle Morphogenesis and Cycling

We have previously shown that the ubiquitously expressed lysosomal cysteine protease, cathepsin L (CTSL), is essential for skin and hair follicle homeostasis. Here we examine the effect of CTSL deficiency on hair follicle development and cycling in ctsl(-/-) mice by light and electron microscopy, Ki67/terminal dUTP nick-end labeling, and trichohyalin immunofluorescence. Hair follicle morphogenesis in ctsl(-/-) mice was associated with several abnormalities. Defective terminal differentiation of keratinocytes occurred during the formation of the hair canal, resulting in disruption of hair shaft outgrowth. Both proliferation and apoptosis levels in keratinocytes and melanocytes were higher in ctsl(-/-) than in ctsl(+/+) hair follicles. The development of the hair follicle pigmentary unit was disrupted by vacuolation of differentiating melanocytes. Hair cycling was also abnormal in ctsl(-/-) mice. Final stages of hair follicle morphogenesis and the induction of hair follicle cycling were retarded. Thereafter, these follicles exhibited a truncated resting phase (telogen) and a premature entry into the first growth phase. Further abnormalities of telogen development included the defective anchoring of club hairs in the skin, which resulted in their abnormal shedding. Melanocyte vacuolation was again apparent during the hair cycle-associated reconstruction of the hair pigmentary unit. A hallmark of these ctsl(-/-) mice was the severe disruption in the exiting of hair shafts to the skin surface. This was mostly because of a failure of the inner root sheath (keratinocyte layer next to the hair shaft) to fully desquamate. These changes resulted in a massive dilation of the hair canal and the abnormal routing of sebaceous gland products to the skin surface. In summary, this study suggests novel roles for cathepsin proteases in skin, hair, and pigment biology. Principal target tissues that may contain protein substrate(s) for this cysteine protease include the developing hair cone, inner root sheath, anchoring apparatus of the telogen club, and organelles of lysosomal origin (eg, melanosomes).

Prolactin and Its Receptor Are Expressed in Murine Hair Follicle Epithelium, Show Hair Cycle-Dependent Expression, and Induce Catagen

Here, we provide the first study of prolactin (PRL) and prolactin receptor (PRLR) expression during the nonseasonal murine hair cycle, which is, in contrast to sheep, comparable with the human scalp and report that both PRL and PRLR are stringently restricted to the hair follicle epithelium and are strongly hair cycle-dependent. In addition we show that PRL exerts functional effects on anagen hair follicles in murine skin organ culture by down-regulation of proliferation in follicular keratinocytes. In telogen follicles, PRL-like immunoreactivity was detected in outer root sheath (ORS) keratinocytes. During early anagen (III to IV), the developing inner root sheath (IRS) and the surrounding ORS were positive for PRL. In later anagen stages, PRL could be detected in the proximal IRS and the inner layer of the ORS. The regressing (catagen) follicle showed a strong expression of PRL in the proximal ORS. In early anagen, PRLR immunoreactivity occurred in the distal part of the ORS around the developing IRS, and subsequently to a restricted area of the more distal ORS during later anagen stages and during early catagen. The dermal papilla (DP) stayed negative for both PRL and PRLR throughout the cycle. Telogen follicles showed only a very weak PRLR staining of ORS keratinocytes. The long-form PRLR transcript was shown by real-time polymerase chain reaction to be transiently down-regulated during early anagen, whereas PRL transcripts were up-regulated during mid anagen. Addition of PRL (400 ng/ml) to anagen hair follicles in murine skin organ culture for 72 hours induced premature catagen development in vitro along with a decline in the number of proliferating hair bulb keratinocytes. These data support the intriguing concept that PRL is generated locally in the hair follicle epithelium and acts directly in an autocrine or paracrine manner to modulate the hair cycle.

Prolactin—a novel neuroendocrine regulator of human keratin expression in situ

The controls of human keratin expression in situ remain to be fully elucidated. Here, we have investigated the effects of the neurohormone prolactin (PRL) on keratin expression in a physiologically and clinically relevant test system: organ‐cultured normal human hair follicles (HFs). Not only do HFs express a wide range of keratins, but they are also a source and target of PRL. Microarray analysis revealed that PRL differentially regulated a defined subset of keratins and keratin‐associated proteins. Quantitative immunohistomorphometry and quantitative PCR confirmed that PRL up‐regulated expression of keratins K5 and K14 and the epithelial stem cell‐associated keratins K15 and K19 in organ‐cultured HFs and/or isolated HF keratinocytes. PRL also up‐regulated K15 promoter activity and K15 protein expression in situ, whereas it inhibited K6 and K31 expression. These regulatory effects were reversed by a pure competitive PRL receptor antagonist. Antagonist alone also modulated keratin expression, suggesting that “tonic stimulation” by endogenous PRL is required for normal expression levels of selected keratins. Therefore, our study identifies PRL as a major, clinically relevant, novel neuroendocrine regulator of both human keratin expression and human epithelial stem cell biology in situ. —Ramot, Y., Bíro´, T., Tiede, S. To´th, B. I., Langan, E. A., Sugawara, K., Foitzik, K., Ingber, A., Goffin, V., Langbein, L., Paus, R. Prolactin—a novel neuroendocrine regulator of human keratin expression in situ. FASEB J. 24, 1768–1779 (2010). www.fasebj.org

Limitations of human occipital scalp hair follicle organ culture for studying the effects of minoxidil as a hair growth enhancer

Abstract:  Minoxidil induces new hair growth in approximately one‐third of patients with androgenetic alopecia after 1 year of treatment. With several conflicting reports in the literature based on small‐scale studies, the current study aimed to clarify whether organ culture of human scalp anagen VI hair follicles is a suitable in vitro test system for reproducing, and experimentally dissecting, the recognized in vivo hair‐growth‐promoting capacity of minoxidil. Hair shaft elongation was studied in terminal anagen VI hair follicles microdissected from the occipital scalp of 36 healthy adults. A total of 2300 hair follicles, approximately 65 per individual, were tested using modifications of a basic organ culture protocol. It is shown here that minoxidil does not significantly increase hair shaft elongation or the duration of anagen VI in ex vivo culture despite several enhancements on the conventional methodology. This disparity to what is seen clinically in minoxidil responders may be explained by the following: (i) use of occipital (rather than frontotemporal or vertex) hair follicles; (ii) use of, already maximally growing, anagen VI hair follicles; (iii) a predominance of hair follicles from minoxidil unresponsive‐donors; (iv) use of minoxidil rather than its sulfate metabolite; and/or (v) use of a suboptimal minoxidil dosage. This disparity questions the usefulness of standard human hair follicle organ culture in minoxidil research. Unexpectedly, minoxidil even inhibited hair shaft elongation in the absence of insulin, which may indicate that the actual hair‐growth‐modulatory effects of minoxidil depend on the concomitant local presence/absence of other growth modulators.

L‐Carnitine–L‐tartrate promotes human hair growth in vitro

Abstract:  The trimethylated amino acid l‐carnitine plays a key role in the intramitochondrial transport of fatty acids for β‐oxidation and thus serves important functions in energy metabolism. Here, we have tested the hypothesis that l‐carnitine, a frequently employed dietary supplement, may also stimulate hair growth by increasing energy supply to the massively proliferating and energy‐consuming anagen hair matrix. Hair follicles (HFs) in the anagen VI stage of the hair cycle were cultured in the presence of 0.5–50 μm of l‐carnitine–l‐tartrate (CT) for 9 days. At day 9, HFs treated with 5 μm or 0.5 μm of CT showed a moderate, but significant stimulation of hair shaft elongation compared with vehicle‐treated controls (P < 0.05). Also, CT prolonged the duration of anagen VI, down regulated apoptosis (as measured by TUNEL assay) and up regulated proliferation (as measured by Ki67 immunohistology) of hair matrix keratinocytes (P < 0.5). By immunohistology, intrafollicular immunoreactivity for TGFβ2, a key catagen‐promoting growth factor, in the dermal papilla and TGF‐β II receptor protein in the outer root sheath and dermal papilla was down regulated. As shown by caspase activity assay, caspase 3 and 7, which are known to initiate apoptosis, are down regulated at day 2 and day 4 after treatment of HFs with CT compared with vehicle‐treated control indicating that CT has an immediate protective effect on HFs to undergo programmed cell death. Our findings suggest that l‐carnitine stimulates human scalp hair growth by up regulation of proliferation and down regulation of apoptosis in follicular keratinocytes in vitro. They further encourage one to explore topical and nutraceutical administration of l‐carnitine as a well‐tolerated, relatively safe adjuvant treatment in the management of androgenetic alopecia and other forms of hair loss.