Photobiomodulation of human dermal fibroblasts in vitro: decisive role of cell culture conditions and treatment protocols on experimental outcome.

Photobiomodulation-based (LLLT) therapies show tantalizing promise for treatment of skin diseases. Confidence in this approach is blighted however by lamentable inconsistency in published experimental designs, and so complicates interpretation. Here we interrogate the appropriateness of a range of previously-reported treatment parameters, including light wavelength, irradiance and radiant exposure, as well as cell culture conditions (e.g., serum concentration, cell confluency, medium refreshment, direct/indirect treatment, oxygen concentration, etc.), in primary cultures of normal human dermal fibroblasts exposed to visible and near infra-red (NIR) light. Apart from irradiance, all study parameters impacted significantly on fibroblast metabolic activity. Moreover, when cells were grown at atmospheric O2 levels (i.e. 20%) short wavelength light inhibited cell metabolism, while negligible effects were seen with long visible and NIR wavelength. By contrast, NIR stimulated cells when exposed to dermal tissue oxygen levels (approx. 2%). The impact of culture conditions was further seen when inhibitory effects of short wavelength light were reduced with increasing serum concentration and cell confluency. We conclude that a significant source of problematic interpretations in photobiomodulation reports derives from poor optimization of study design. Further development of this field using in vitro/ex vivo models should embrace significant standardization of study design, ideally within a design-of-experiment setting.

Noggin is required for induction of the hair follicle growth phase in postnatal skin.

During postnatal development, the hair follicle (HF) shows cyclic activity with periods of relative resting, active growth (anagen), and regression. We demonstrate that similar to the HF induction in embryonic skin, initiation of a new hair growth phase in postnatal skin requires neutralization of the inhibitory activity of bone morphogenetic protein 4 (BMP4) by the BMP antagonist noggin. In the resting HF, BMP4 mRNA predominates over noggin in the epithelium and mesenchyme, and the BMP receptor IA is prominently expressed in the follicular germ. Anagen development is accompanied by down-regulation of the BMP4 and increased noggin mRNA in the HF. Furthermore, administration of noggin protein induces new hair growth phase in postnatal telogen skin in vivo. In contrast, BMP4 induces selective arrest of anagen development in the non-tylotrich (secondary) HF. As a hair growth inducer, noggin increases Shh mRNA in the HF whereas BMP4 down-regulates Shh. This suggests that modulation of BMP4 signaling by noggin is essential for hair growth phase induction in postnatal skin and that the hair growth-inducing effect of noggin is mediated, at least in part, by Shh.

p53 Involvement in the control of murine hair follicle regression.

p53 is a transcription factor mediating a variety of biological responses including apoptotic cell death. p53 was recently shown to control apoptosis in the hair follicle induced by ionizing radiation and chemotherapy, but its role in the apoptosis-driven physiological hair follicle regression (catagen) remains to be elucidated. Here, we show that p53 protein is strongly expressed and co-localized with apoptotic markers in the regressing hair follicle compartments during catagen. In contrast to wild-type mice, p53 knockout mice show significant retardation of catagen accompanied by significant decrease in the number of apoptotic cells in the hair matrix. Furthermore, p53 null hair follicles are characterized by alterations in the expression of markers that are encoded by p53 target genes and are implicated in the control of catagen (Bax, Bcl-2, insulin-like growth factor binding protein-3). These data suggest that p53 is involved in the control of apoptosis in the hair follicle during physiological regression and imply that p53 antagonists may be useful for the management of hair growth disorders characterized by premature entry into catagen, such as androgenetic alopecia, alopecia areata, and telogen effluvium.

SCF/c-kit signaling is required for cyclic regeneration of the hair pigmentation unit.

Hair graying, an age-associated process of unknown etiology, is characterized by a reduced number and activity of hair follicle (HF) melanocytes. Stem cell factor (SCF) and its receptor c-kit are important for melanocyte survival during development, and mutations in these genes result in unpigmented hairs. Here we show that during cyclic HF regeneration in C57BL/6 mice, proliferating, differentiating, and melanin-producing melanocytes express c-kit, whereas presumptive melanocyte precursors do not. SCF overexpression in HF epithelium significantly increases the number and proliferative activity of melanocytes. During the induced hair cycle in C57BL/6 mice, administration of anti-c-kit antibody dose-dependently decreases hair pigmentation and leads to partially depigmented (gray) or fully depigmented (white) hairs, associated with significant decreases in melanocyte proliferation and differentiation, as determined by immunostaining and confocal microscopy. However, in the next hair cycle, the previously treated animals grow fully pigmented hairs with the normal number and distribution of melanocytes. This suggests that melanocyte stem cells are not dependent on SCF/c-kit and when appropriately stimulated can generate melanogenically active melanocytes. Therefore, the blockade of c-kit signaling offers a fully reversible model for hair depigmentation, which might be used for the studies of hair pigmentation disorders.

Hair-cycle-associated remodeling of the peptidergic innervation of murine skin, and hair growth modulation by neuropeptides.

As the neuropeptide substance P can manipulate murine hair growth in vivo, we here further studied the role of sensory neuropeptides in hair follicle biology by determining the distribution and hair-cycle-dependent remodeling of the sensory innervation in C57BL/6 mouse back skin. Calcitonin-gene-related peptide, substance P, and peptide histidine methionine (employed as vasoactive intestinal peptide marker) were identified by immunohistochemistry. All of these markers immunolocalized to bundles of nerve fibers and to single nerve fibers, with distinct distribution patterns and major hair-cycle-associated changes. In the epidermis and around the distal hair follicle and the arrector pili muscle, only calcitonin-gene-related peptide immunoreactive nerve fibers were visualized, whereas substance P and peptide histidine methionine immunoreactive nerve fibers were largely restricted to the dermis and subcutis. Compared to telogen skin, the number of calcitonin-gene-related peptide, substance P, and peptide histidine methionine immunoreactive single nerve fibers increased significantly (p < 0.01) during anagen, including around the bulge region (the seat of epithelial stem cells). Substance P significantly accelerated anagen progression in murine skin organ culture, whereas calcitonin-gene-related peptide and a substance-P-inhibitory peptide inhibited anagen (p < 0.05). The inhibitory effect of calcitonin-gene-related peptide could be antagonized by coadministrating substance P. In contrast to substance P, calcitonin-gene-related peptide failed to induce anagen when released from subcutaneous implants. This might reflect a differential functional assignment of the neuropeptides calcitonin-gene-related peptide and substance P in hair growth control, and invites the use of neuropeptide receptor agonists and antagonists as novel pharmacologic tools for therapeutic hair growth manipulation.

p53 is essential for chemotherapy-induced hair loss.

Anticancer drugs stimulate apoptosis in the hair follicles (HF) and cause hair loss, the most common side effect of chemotherapy. In a mouse model for chemotherapy-induced hair loss, we demonstrate that p53 is essential for this process: in contrast to wild-type mice, p53-deficient mice show neither hair loss nor apoptosis in the HF keratinocytes that maintained active proliferation after cyclophosphamide treatment. HF in p53 mutants are characterized by down-regulation of Fas and insulin-like growth factor-binding protein 3 and by increased expression of Bcl-2. These observations indicate that local pharmacological inhibition of p53 may be useful to prevent chemotherapy-associated hair loss.

A role for p75 neurotrophin receptor in the control of apoptosis-driven hair follicle regression.

To examine the mechanisms that underlie the neurotrophin-induced, apoptosis-driven hair follicle involution (catagen), the expression and function of p75 neurotrophin receptor (p75NTR), which is implicated in apoptosis control, were studied during spontaneous catagen development in murine skin. By RT-PCR, high steady-state p75NTR mRNA skin levels were found during the anagen-catagen transition of the hair follicle. By immunohistochemistry, p75NTR alone was strongly expressed in TUNEL+/Bcl2- keratinocytes of the regressing outer root sheath, but both p75NTR and TrkB and/or TrkC were expressed by the nonregressing TUNEL-/Bcl2+ secondary hair germ keratinocytes. To determine whether p75NTR is functionally involved in catagen control, spontaneous catagen development was compared in vivo between p75NTR knockout (-/-) and wild-type mice. There was significant catagen retardation in p75NTR knockout mice as compared to wild-type controls (P<0.05). Instead, transgenic mice-overexpressing NGF (promoter: K14) showed substantial acceleration of catagen (P<0.001). Although NGF, brain-derived neurotrophic factor (BDNF), and neurotrophin 3 (NT-3) accelerated catagen in the organ-cultured skin of C57BL/6 mice, these neurotrophins failed to promote catagen development in the organ-cultured p75NTR null skin. These findings suggest that p75NTR signaling is involved in the control of kerotinocyte apoptosis during catagen and that pharmacological manipulation of p75NTR signaling may prove useful for the treatment of hair disorders that display premature entry into catagen.

Control of murine hair follicle regression (catagen) by TGF-beta1 in vivo.

The regression phase of the hair cycle (catagen) is an apoptosis-driven process accompanied by terminal differentiation, proteolysis, and matrix remodeling. As an inhibitor of keratinocyte proliferation and inductor of keratinocyte apoptosis, transforming growth factor beta1 (TGF-beta1) has been proposed to play an important role in catagen regulation. This is suggested, for example, by maximal expression of TGF-beta1 and its receptors during late anagen and the onset of catagen of the hair cycle. We examined the potential involvement of TGF-beta1 in catagen control. We compared the first spontaneous entry of hair follicles into catagen between TGF-beta1 null mice and age-matched wild-type littermates, and assessed the effects of TGF-beta1 injection on murine anagen hair follicles in vivo. At day 18 p.p., hair follicles in TGF-beta1 -/- mice were still in early catagen, whereas hair follicles of +/+ littermates had already entered the subsequent resting phase (telogen). TGF-beta1-/- mice displayed more Ki-67-positive cells and fewer apoptotic cells than comparable catagen follicles from +/+ mice. In contrast, injection of TGF-beta1 into the back skin of mice induced premature catagen development. In addition, the number of proliferating follicle keratinocytes was reduced and the number of TUNEL + cells was increased in the TGF-beta1-treated mice compared to controls. Double visualization of TGF-beta type II receptor (TGFRII) and TUNEL reactivity revealed colocalization of apoptotic nuclei and TGFRII in catagen follicles. These data strongly support that TGF-beta1 ranks among the elusive endogenous regulators of catagen induction in vivo, possibly via the inhibition of keratinocyte proliferation and induction of apoptosis. Thus, TGF-betaRII agonists and antagonists may provide useful therapeutic tools for human hair growth disorders based on premature or retarded catagen development (effluvium, alopecia, hirsutism).

New roles for glial cell line-derived neurotrophic factor and neurturin: involvement in hair cycle control.

Glial cell line-derived neurotrophic factor (GDNF), neurturin (NTN), and their receptors, GDNF family receptor alpha-1 (GFRalpha-1) and GDNF family receptor alpha-2 (GFRalpha-2), are critically important for kidney and nervous system development. However, their role in skin biology, specifically in hair growth control, is as yet unknown. We have studied expression and function of GDNF, neurturin, GFRalpha-1, and GFRalpha-2 in murine skin during the cyclic transformation of the hair follicle (HF) from its resting state (telogen) to active growth (anagen) and then through regression (catagen) back to telogen. GDNF protein and GFRalpha-1 messenger RNA are prominently expressed in telogen skin, which lacks NTN and GFRalpha-2 transcripts. Early anagen development is accompanied by a significant decline in the skin content of GDNF protein and GFRalpha-1 transcripts. During the anagen-catagen transition, GDNF, GFRalpha-1, NTN, and GFRalpha-2 transcripts reach maximal levels. Compared with wild-type controls, GFRalpha-1 (+/-) and GFRalpha-2 (-/-) knockout mice show a significantly accelerated catagen development. Furthermore, GDNF or NTN administration significantly retards HF regression in organ-cultured mouse skin. This suggests important, previously unrecognized roles for GDNF/GFRalpha-1 and NTN/GFRalpha-2 signaling in skin biology, specifically in the control of apoptosis-driven HF involution, and raises the possibility that GFRalpha-1/GFRalpha-2 agonists/antagonists might become exploitable for the treatment of hair growth disorders that are related to abnormalities in catagen development.

Distinct roles for nerve growth factor and brain-derived neurotrophic factor in controlling the rate of hair follicle morphogenesis.

Increasing evidence suggests that neurotrophins play an important part in the control of the development of ectodermal derivatives, such as the hair follicle. Here, we show that, during hair follicle morphogenesis in C57BL/6 mice, nerve growth factor, brain-derived neurotrophic factor and their corresponding high-affinity tyrosine kinase receptors, TrkA and TrkB, show stringently controlled spatiotemporal expression patterns in the follicular epithelium and mesenchyme. Constitutive overexpression of nerve growth factor in mice is associated with a discrete, but significant acceleration of hair follicle morphogenesis, whereas this is not seen in brain-derived neurotrophic factor transgenic mice. In neonatal skin organ culture, nerve growth factor and brain-derived neurotrophic factor differentially influence hair follicle development: nerve growth factor accelerates late stages of hair follicle morphogenesis, whereas brain-derived neurotrophic factor does not show significant effects. This suggests that the morphogenetic properties of locally generated neurotrophins in the skin, similar to their classical neurotrophic functions, are quite distinct and depend on the response patterns of the corresponding neurotrophin target receptor-expressing cells in the developing hair follicle. These data further strengthen the concept that neurotrophin signaling is an important element in controlling the rate of hair follicle morphogenesis, yet also highlight the complexity of this signaling system.

A role for p75 neurotrophin receptor in the control of hair follicle morphogenesis.

During hair follicle (HF) morphogenesis, p75 neurotrophin receptor (p75NTR) reportedly is the first growth factor receptor found to be expressed by those fibroblasts that later develop into the dermal papilla (DP) of the HF. However, the functional role of p75NTR in HF morphogenesis is still unknown. Studying HF development in fetal and neonatal C57BL/6 murine back skin, we show that p75NTR-immunoreactivity (IR) is prominently expressed by DP fibroblasts as well as by skin nerves during the early steps of HF development. In contrast, p75NTR-IR disappears from the DP in the fully developed HF and it is expressed only in the epithelial outer root sheath of the HF. Compared to age-matched wild-type animals, p75NTR knockout (-/-) mice show significant acceleration of HF morphogenesis, and DP fibroblasts of p75NTR knockout mice show reduced proliferative activity in situ, indicating alterations in their transition from proliferation to differentiation. Although no significant differences in the expression of adhesion molecules (NCAM), selected morphogens (TGFbeta-2, HGF/SF, FGF-2, KGF), or their receptors (TGFbetaR-II, m-met, FGFR-1) were seen between DP of p75NTR knockout and wild-type mice, p75NTR mutants showed a prominent upregulation of FGFR-2, a high-affinity receptor for KGF, in both follicular DP and epithelium. Furthermore, the administration of anti-KGF neutralizing antibody significantly inhibited acceleration of HF morphogenesis in p75NTR knockout mice in vivo. These observations suggest that p75NTR plays an important role during HF morphogenesis, functioning as a receptor that negatively controls HF development, most likely via alterations in DP fibroblast proliferation/differentiation and via downregulation of KGF/FGFR-2 signaling in the HF.

Hair cycle-dependent changes in adrenergic skin innervation, and hair growth modulation by adrenergic drugs.

Skin nerves may exert "trophic" functions during hair follicle development, growth, and/or cycling. Here, we demonstrate hair cycle-related plasticity in the sympathetic innervation of skin and hair follicle in C57BL/6 mice. Compared with telogen skin, the number of nerve fibers containing norepinephrine or immunoreactive for tyrosine hydroxylase increased during the early growth phase of the hair cycle (anagen) in dermis and subcutis. The number of these fibers declined again during late anagen. beta2-adrenoreceptor-positive keratinocytes were transiently detectable in the noncycling hair follicle epithelium, especially in the isthmus and bulge region, but only during early anagen. In early anagen skin organ culture, the beta2-adrenoreceptor agonist isoproterenol promoted hair cycle progression from anagen III to anagen IV. The observed hair cycle-dependent changes in adrenergic skin innervation on the one hand, and hair growth modulation by isoproterenol, accompanied by changes in beta2-adrenoreceptor expression of selected regions of the hair follicle epithelium on the other, further support the concept that bi-directional interactions between the hair follicle and its innervation play a part in hair growth control. This invites one to systematically explore the neuropharmacologic manipulation of follicular neuroepithelial interactions as a novel therapeutic strategy for managing hair growth disorders.

Noggin is a mesenchymally derived stimulator of hair-follicle induction.

The induction of developmental structures derived from the ectoderm, such as the neural tube or tooth, occurs through neutralization of the inhibitory activity of members of the bone-morphogenetic protein (BMP) family by BMP antagonists. Here we show that, during hair-follicle development, the neural inducer and BMP-neutralizing protein Noggin is expressed in the follicular mesenchyme, that noggin-knockout mice show significant retardation of hair-follicle induction, and that Noggin neutralizes the inhibitory action of BMP-4 and stimulates hair-follicle induction in embryonic skin organ culture. As a crucial mesenchymal signal that stimulates hair-follicle induction, Noggin operates through antagonistic interactions with BMP-4, which result in upregulation of the transcription factor Lef-1 and the cell-adhesion molecule NCAM, as well as through BMP4-independent downregulation of the 75 kD neurotrophin receptor in the developing hair follicle.

Abundant production of brain-derived neurotrophic factor by adult visceral epithelia. Implications for paracrine and target-derived Neurotrophic functions.

Brain-derived neurotrophic factor (BDNF) plays a crucial role for the survival of visceral sensory neurons during development. However, the physiological sources and the function of BDNF in the adult viscera are poorly described. We have investigated the cellular sources and the potential role of BDNF in adult murine viscera. We found markedly different amounts of BDNF protein in different organs. Surprisingly, BDNF levels in the urinary bladder, lung, and colon were higher than those found in the brain or skin. In situ hybridization experiments revealed that BDNF mRNA was made by visceral epithelial cells, several types of smooth muscle, and neurons of the myenteric plexus. Epithelia that expressed BDNF lacked both the high- and low-affinity receptors for BDNF, trkB and p75(NTR). In contrast, both receptors were present on neurons of the peripheral nervous system. Studies with BDNF-/-mice demonstrated that epithelial and smooth muscle cells developed normally in the absence of BDNF. These data provide evidence that visceral epithelia are a major source, but not a target, of BDNF in the adult viscera. The abundance of BDNF protein in certain internal organs suggests that this neurotrophin may regulate the function of adult visceral sensory and motor neurons.

The role of the hairless (hr) gene in the regulation of hair follicle catagen transformation.

Mice that carry a mutation at the hairless (hr) locus develop seemingly normal hair follicles (HF) but shed their hairs completely soon after birth. Histologically, their HFs degenerate into characteristic utriculi and dermal cysts shortly after the entry of the HF into the first regression phase (catagen), during the initiation of HF cycling. Here, we show that at least nine distinct stages of HF disintegration can be distinguished in hr/hr mice. Toward the end of HF morphogenesis (day 15 postpartum) the proximal hair bulb in hr/hr skin undergoes premature and massive apoptosis. This is associated with a dyscoordination of cell proliferation in defined HF compartments, malpositioning of the proximal inner root sheath, striking atrophy of outer root sheath, and failure of trichilemmal keratinization in the developing club hair. Rather than undergoing their normal catagen-associated involution, the hair bulb and central outer root sheath disintegrate into separate cell clusters, thus disrupting all epithelial contact with the dermal papilla. Dermal papilla fibroblasts fail to migrate upward, and break up into clusters of shrunken cells stranded in the reticular dermis as dermal cyst precursors, while the upper HF epithelium transforms into utriculi. Some dermal papilla cells, which normally never undergo apoptosis, also become TUNEL+ in hr/hr skin, and their normally high expression of a key adhesion molecule, neural cell adhesion molecule, declines. Thus, loss of a functional hr gene product (a putative zinc finger transcription factor) initiates a premature, highly dysregulated catagen, which results in the destruction of the normal HF architecture and abrogates the HF's ability to cycle. This provides new insights into the pathobiology of the hr mutation, and suggests that the normal hr gene product is a crucial element of catagen control.

Brain-derived neurotrophic factor, neurotrophin-3, and neurotrophin-4 act as "epitheliotrophins" in murine skin.

Nerve growth factor (NGF) is produced by keratinocytes and modulates their proliferation and apoptosis. However, it is as yet unknown whether other members of the NGF family of neurotrophins, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4), also modulate keratinocyte proliferation in situ. We determined by ELISA and reverse transcriptase-PCR that BDNF, NT-3, and NT-4 are expressed in C57BL/6 mouse skin. By immunofluorescence, the subcutaneous panniculus carnosus muscle and arrector pili muscle showed strong NT-3 immunoreactivity, whereas BDNF-IR was found only in skin nerve bundles. NT-4 immunoreactivity was noted in single epidermal keratinocytes. The high affinity receptor for both BDNF and NT-4, TrkB, was detected in basal and suprabasal epidermal keratinocytes, whereas the high affinity NT-3 receptor, TrkC, was observed in skin nerve bundles. Compared with the corresponding age-matched wild-type mice, BDNF or NT-3-overexpressing transgenic mice showed a significantly increased epidermal thickness and enhanced number of Ki-67-positive (ie, proliferating) epidermal keratinocytes in vivo, whereas the number of these cells was substantially reduced in BDNF knockout mice. In skin organ culture of C57BL/6 mice, BDNF, NT-3, and NT-4 all significantly increased 5-bromo-2'-deoxyuridine incorporation into epidermal keratinocytes. Co-administration of NGF neutralizing antibody failed to abrogate the stimulatory effect of NT-3 on keratinocyte proliferation in skin organ culture. This demonstrates that normal murine epidermal keratinocytes in situ are direct or indirect target cells for these neurotrophins. Therefore, BDNF, NT-3, and NT-4 can also act as "epitheliotrophins" and may thus be intimately involved in the control of epidermal homeostasis.

A new role for neurotrophins: involvement of brain-derived neurotrophic factor and neurotrophin-4 in hair cycle control.

Neurotrophins exert many biological effects not directly targeted at neurons, including modulation of keratinocyte proliferation and apoptosis in vitro. Here we exploit the cyclic growth and regression activity of the murine hair follicle to explore potential nonneuronal functions of neurotrophins in the skin, and analyze the follicular expression and hair growth-modulatory function of BDNF, NT-4, and their high-affinity receptor, TrkB. The cutaneous expression of BDNF and NT-4 mRNA was strikingly hair cycle dependent and peaked during the spontaneous, apoptosis-driven hair follicle regression (catagen). During catagen, BDNF mRNA and immunoreactivity, as well as NT-4-immunoreactivity, were expressed in the regressing hair follicle compartments, whereas TrkB mRNA and immunoreactivity were seen in dermal papilla fibroblasts, epithelial strand, and hair germ. BDNF or NT-4 knockout mice showed significant catagen retardation, whereas BDNF-overexpressing mice displayed acceleration of catagen and significant shortening of hair length. Finally, BDNF and NT-4 accelerated catagen development in murine skin organ culture. Together, our data suggest that BDNF and NT-4 play a previously unrecognized role in skin physiology as agents of hair growth control. Thus, TrkB agonists and antagonists deserve exploration as novel hair growth-modulatory drugs for the management of common hair growth disorders.