Identifying novel strategies for treating human hair loss disorders: Cyclosporine A suppresses the Wnt inhibitor, SFRP1, in the dermal papilla of human scalp hair follicles.

Hair growth disorders often carry a major psychological burden. Therefore, more effective human hair growth-modulatory agents urgently need to be developed. Here, we used the hypertrichosis-inducing immunosuppressant, Cyclosporine A (CsA), as a lead compound to identify new hair growth-promoting molecular targets. Through microarray analysis we identified the Wnt inhibitor, secreted frizzled related protein 1 (SFRP1), as being down-regulated in the dermal papilla (DP) of CsA-treated human scalp hair follicles (HFs) ex vivo. Therefore, we further investigated the function of SFRP1 using a pharmacological approach and found that SFRP1 regulates intrafollicular canonical Wnt/ß-catenin activity through inhibition of Wnt ligands in the human hair bulb. Conversely, inhibiting SFRP1 activity through the SFRP1 antagonist, WAY-316606, enhanced hair shaft production, hair shaft keratin expression, and inhibited spontaneous HF regression (catagen) ex vivo. Collectively, these data (a) identify Wnt signalling as a novel, non-immune-inhibitory CsA target; (b) introduce SFRP1 as a physiologically important regulator of canonical ß-catenin activity in a human (mini-)organ; and (c) demonstrate WAY-316606 to be a promising new promoter of human hair growth. Since inhibiting SFRP1 only facilitates Wnt signalling through ligands that are already present, this 'ligand-limited' therapeutic strategy for promoting human hair growth may circumvent potential oncological risks associated with chronic Wnt over-activation.

Axin2 marks quiescent hair follicle bulge stem cells that are maintained by autocrine Wnt/ß-catenin signaling.

How stem cells maintain their identity and potency as tissues change during growth is not well understood. In mammalian hair, it is unclear how hair follicle stem cells can enter an extended period of quiescence during the resting phase but retain stem cell potential and be subsequently activated for growth. Here, we use lineage tracing and gene expression mapping to show that the Wnt target gene Axin2 is constantly expressed throughout the hair cycle quiescent phase in outer bulge stem cells that produce their own Wnt signals. Ablating Wnt signaling in the bulge cells causes them to lose their stem cell potency to contribute to hair growth and undergo premature differentiation instead. Bulge cells express secreted Wnt inhibitors, including Dickkopf (Dkk) and secreted frizzled-related protein 1 (Sfrp1). However, the Dickkopf 3 (Dkk3) protein becomes localized to the Wnt-inactive inner bulge that contains differentiated cells. We find that Axin2 expression remains confined to the outer bulge, whereas Dkk3 continues to be localized to the inner bulge during the hair cycle growth phase. Our data suggest that autocrine Wnt signaling in the outer bulge maintains stem cell potency throughout hair cycle quiescence and growth, whereas paracrine Wnt inhibition of inner bulge cells reinforces differentiation.

Real-Time Imaging of Dynamic Cell Reprogramming with Nanosensors.

Cellular reprogramming, the process by which somatic cells regain pluripotency, is relevant in many disease modeling, therapeutic, and drug discovery applications. Molecular evaluation of reprogramming (e.g., polymerase chain reaction, immunostaining) is typically disruptive, and only provides snapshots of phenotypic traits. Gene reporter constructs facilitate live-cell evaluation but is labor intensive and may risk insertional mutagenesis during viral transfection. Herein, the utilization of a non-integrative nanosensor is demonstrated to visualize key reprogramming events in situ within live cells. Principally based on sustained intracellular release of encapsulated molecular probes, nanosensors successfully monitored mesenchymal-epithelial transition, pluripotency acquisition, and transdifferentiation events. Tracking the dynamic expression of four pivotal biomarkers (i.e., THY1, E-CADHERIN, OCT4, and GATA4 mRNA), nanosensor signal showed great agreement with polymerase chain reaction and gene reporter imaging (R2 > 0.9). Overall, such facile, versatile nanosensor enables real-time monitoring of low-frequency reprogramming events, thereby useful for high-throughput assessment, optimization, and biomarker-specific cell enrichment.

Human-specific loss of regulatory DNA and the evolution of human-specific traits.

Humans differ from other animals in many aspects of anatomy, physiology, and behaviour; however, the genotypic basis of most human-specific traits remains unknown. Recent whole-genome comparisons have made it possible to identify genes with elevated rates of amino acid change or divergent expression in humans, and non-coding sequences with accelerated base pair changes. Regulatory alterations may be particularly likely to produce phenotypic effects while preserving viability, and are known to underlie interesting evolutionary differences in other species. Here we identify molecular events particularly likely to produce significant regulatory changes in humans: complete deletion of sequences otherwise highly conserved between chimpanzees and other mammals. We confirm 510 such deletions in humans, which fall almost exclusively in non-coding regions and are enriched near genes involved in steroid hormone signalling and neural function. One deletion removes a sensory vibrissae and penile spine enhancer from the human androgen receptor (AR) gene, a molecular change correlated with anatomical loss of androgen-dependent sensory vibrissae and penile spines in the human lineage. Another deletion removes a forebrain subventricular zone enhancer near the tumour suppressor gene growth arrest and DNA-damage-inducible, gamma (GADD45G), a loss correlated with expansion of specific brain regions in humans. Deletions of tissue-specific enhancers may thus accompany both loss and gain traits in the human lineage, and provide specific examples of the kinds of regulatory alterations and inactivation events long proposed to have an important role in human evolutionary divergence.

Lrig1-expressing epidermal progenitors require SCD1 to maintain the dermal papilla niche.

Niche cells are widely known to regulate stem/progenitor cells in many mammalian tissues. In the hair, dermal papilla niche cells are well accepted to regulate hair stem/progenitor cells. However, how niche cells themselves are maintained is largely unknown. We present evidence implicating hair matrix progenitors and the lipid modifying enzyme, Stearoyl CoA Desaturase 1, in the regulation of the dermal papilla niche during the anagen-catagen transition of the mouse hair cycle. Our data suggest that this takes place via autocrine Wnt signalling and paracrine Hedgehog signalling. To our knowledge, this is the first report demonstrating a potential role for matrix progenitor cells in maintaining the dermal papilla niche.

Aberrant Wnt Signaling Induces Comedo-Like Changes in the Murine Upper Hair Follicle.

Stem cell proliferation and differentiation must be carefully balanced to support tissue maintenance and growth. Defective stem cell regulation may underpin diseases in many organs, including the skin. LRIG1-expressing stem cells residing in the hair follicle junction zone (JZ) support sebaceous gland homeostasis. An emerging hypothesis from observations in both mice and human holds that imbalances in key stem cell regulatory pathways such as Wnt signaling may lead to abnormal fate determination of these LRIG1+ve cells. They accumulate and form cystic structures in the JZ that are similar to the comedones found in human acne. To test the possible involvement of Wnt signals in this scenario, we used the Lrig1-CreERT2 mouse line to modulate Wnt signaling in JZ stem cells. We observed that persistent activation of Wnt signaling leads to JZ cyst formation, with associated sebaceous gland atrophy. The cysts strongly express stem cell markers and can be partially reduced by all-trans retinoic acid treatment as well as by Hedgehog signaling inhibition. Conversely, loss of Wnt signaling leads to enlargement of JZ, infundibulum, and sebaceous glands. These data implicate abnormal Wnt signaling in the generation of mouse pathologies that resemble human acne and respond to acne treatments.

Pituitary stem cells produce paracrine WNT signals to control the expansion of their descendant progenitor cells.

In response to physiological demand, the pituitary gland generates new hormone-secreting cells from committed progenitor cells throughout life. It remains unclear to what extent pituitary stem cells (PSCs), which uniquely express SOX2, contribute to pituitary growth and renewal. Moreover, neither the signals that drive proliferation nor their sources have been elucidated. We have used genetic approaches in the mouse, showing that the WNT pathway is essential for proliferation of all lineages in the gland. We reveal that SOX2+ stem cells are a key source of WNT ligands. By blocking secretion of WNTs from SOX2+ PSCs in vivo, we demonstrate that proliferation of neighbouring committed progenitor cells declines, demonstrating that progenitor multiplication depends on the paracrine WNT secretion from SOX2+ PSCs. Our results indicate that stem cells can hold additional roles in tissue expansion and homeostasis, acting as paracrine signalling centres to coordinate the proliferation of neighbouring cells.

Neuroendocrinology and neurobiology of sebaceous glands.

The nervous system communicates with peripheral tissues through nerve fibres and the systemic release of hypothalamic and pituitary neurohormones. Communication between the nervous system and the largest human organ, skin, has traditionally received little attention. In particular, the neuro-regulation of sebaceous glands (SGs), a major skin appendage, is rarely considered. Yet, it is clear that the SG is under stringent pituitary control, and forms a fascinating, clinically relevant peripheral target organ in which to study the neuroendocrine and neural regulation of epithelia. Sebum, the major secretory product of the SG, is composed of a complex mixture of lipids resulting from the holocrine secretion of specialised epithelial cells (sebocytes). It is indicative of a role of the neuroendocrine system in SG function that excess circulating levels of growth hormone, thyroxine or prolactin result in increased sebum production (seborrhoea). Conversely, growth hormone deficiency, hypothyroidism, and adrenal insufficiency result in reduced sebum production and dry skin. Furthermore, the androgen sensitivity of SGs appears to be under neuroendocrine control, as hypophysectomy (removal of the pituitary) renders SGs largely insensitive to stimulation by testosterone, which is crucial for maintaining SG homeostasis. However, several neurohormones, such as adrenocorticotropic hormone and α-melanocyte-stimulating hormone, can stimulate sebum production independently of either the testes or the adrenal glands, further underscoring the importance of neuroendocrine control in SG biology. Moreover, sebocytes synthesise several neurohormones and express their receptors, suggestive of the presence of neuro-autocrine mechanisms of sebocyte modulation. Aside from the neuroendocrine system, it is conceivable that secretion of neuropeptides and neurotransmitters from cutaneous nerve endings may also act on sebocytes or their progenitors, given that the skin is richly innervated. However, to date, the neural controls of SG development and function remain poorly investigated and incompletely understood. Botulinum toxin-mediated or facial paresis-associated reduction of human sebum secretion suggests that cutaneous nerve-derived substances modulate lipid and inflammatory cytokine synthesis by sebocytes, possibly implicating the nervous system in acne pathogenesis. Additionally, evidence suggests that cutaneous denervation in mice alters the expression of key regulators of SG homeostasis. In this review, we examine the current evidence regarding neuroendocrine and neurobiological regulation of human SG function in physiology and pathology. We further call attention to this line of research as an instructive model for probing and therapeutically manipulating the mechanistic links between the nervous system and mammalian skin.

Changes in cytokine signaling and extracellular matrix production induced by inflammatory factors in cultured vocal fold fibroblasts.

OBJECTIVES: Modulating cytokine signaling in vocal fold fibroblasts after injury may influence extracellular matrix (ECM) production and eventual fibrotic outcome. To evaluate previously established in vivo cytokine and ECM gene expression hypotheses, we examined in vitro vocal fold fibroblast responses to exogenous inflammatory factor stimulation. METHODS: Rat vocal fold fibroblast lines derived from explants were separately treated with interleukin-13 (IL-13), interferon gamma (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), transforming growth factor beta subtype 1 (TGF-beta1), or prostaglandin E2 (PGE2). We examined the in vitro messenger RNA expression profiles of IL-1beta, IFN-gamma, TNF-alpha, TGF-beta1, and cyclooxygenase 2 (COX-2), as well as those of hyaluronic acid synthase (HAS) 1, HAS-2, procollagen subtype 1, and procollagen subtype 3, at 1,4, 8, 16, 24, and 72 hours after treatment, and compared them to those of untreated fibroblasts and in vivo data, using real-time reverse transcription-polymerase chain reaction. RESULTS: IL-1beta and TNF-alpha induced each other and synergistically increased HAS-1 and HAS-2 expression. PGE2 also up-regulated HAS-1 and HAS-2 expression. IFN-gamma, IL-1beta, TNF-alpha, and TGF-beta1 up-regulated HAS expression alongside either transient up-regulation of, or no change in, procollagen 1 and 3 expression. Most treatments appeared to suppress procollagen expression, possibly through HAS up-regulation. All inflammatory factors attenuated TGF-beta1 expression. CONCLUSIONS: These results confirm several in vivo trends, identify potential cytokine pathways and therapeutic candidates, and suggest the utility of this in vitro setup for future studies.

Inflammatory factor profiles one hour following vocal fold injury.

OBJECTIVES: Inflammatory factors are key mediators of wound healing processes following injury, and their modulation may improve healing outcomes. The objective of this study was to characterize in vivo inflammatory factor and extracellular matrix (ECM) messenger RNA (mRNA) expression levels 1 hour after vocal fold injury. METHODS: Five Sprague-Dawley rats were subjected to bilateral vocal fold injury, 5 rats were reserved as uninjured controls, and 1 rat was subjected to unilateral vocal fold injury and reserved for histology. Tissue was harvested 1 hour after injury. Real-time reverse transcription-polymerase chain reaction was performed to examine the mRNA expression profiles of inflammatory factors nuclear factor kappa beta (NF-kappabeta), interferon gamma (IFN-gamma), cyclooxygenase 2 (COX-2), transforming growth factor beta isoform 1 (TGF-beta1), tumor necrosis factor alpha (TNF-alpha), and interleukin 1 beta (IL-1beta), as well as ECM genes hyaluronic acid synthase (HAS) 1, HAS-2, procollagen 1, procollagen 3, and elastin, in the injured samples compared with the uninjured controls. RESULTS: Injury resulted in subepithelial bleeding throughout the vocal fold. The COX-2, TNF-alpha, IL-1beta, and HAS-1 mRNA expression levels were significantly up-regulated 1 hour after injury compared with the uninjured controls. CONCLUSIONS: Inflammatory factor and ECM gene expression changes occur in vocal fold wound sites as early as 1 hour after injury. These results should inform future efforts to attenuate vocal fold scarring via the modulation of inflammatory factors.

Cell production in injured vocal folds: a rat study.

OBJECTIVES: Fibroblasts are reported to play an important role in producing the extracellular matrix of the vocal fold. However, no reports have focused on how and where these cells are generated in the vocal fold after injury. To reveal the characteristics of vocal fold cell production, we investigated cell proliferation in the acute phase of wound healing. METHODS: Using a telescope for guidance, we made an incision in the middle region of the vocal fold tissue in 24 rats and performed immunohistochemical staining for vimentin, alpha-smooth muscle actin, and 5-bromo-2-deoxyuridine. RESULTS: After injury, epithelialization occurred with a peak at day 1, and fibroblasts proliferated in the lamina propria with a peak at day 3, whereas those in the macula flava did not show any increased proliferation. CONCLUSIONS: It is suggested that the fibroblasts in the macula flava have functions different from those of fibroblasts in the lamina propria and that the macula flava does not serve as a cell source for the vocal fold in response to injury.

Immediate inflammatory response and scar formation in wounded vocal folds.

OBJECTIVES: Vocal fold scarring is the major cause of voice disorders after voice surgery or laryngeal trauma. The role of inflammatory factors in vocal fold wound healing and fibrosis has not been adequately investigated. Scarless wound healing has been associated with decreased inflammatory responses. To understand scar formation and develop reliable treatments, it is necessary to control extracellular matrix production and inflammation. Thus, we examined the inflammation profile and extracellular matrix production in wounded vocal folds in the acute phase of wound healing. METHODS: Vocal fold stripping was performed on 30 Sprague-Dawley rats. Vocal fold tissue was collected at 5 time points (4, 8, 16, 24, and 72 hours). We examined the in vivo messenger RNA expression profile of inflammatory factors interleukin 1beta, interferon gamma, tumor necrosis factor alpha, nuclear factor kappa beta, transforming growth factor beta, and cyclooxygenase 2, as well as hyaluronic acid synthases 1 and 2, procollagen subtypes I and III, and elastin synthase in scarred vocal folds after injury, compared to normal vocal folds, using real-time reverse transcription-polymerase chain reaction. RESULTS: The inflammatory factors showed a time-dependent sequence of expression peaks, starting with interleukin 1beta, nuclear factor kappa beta, tumor necrosis factor alpha (4 and 8 hours), and transforming growth factor beta (72 hours). Interferon gamma decreased at 24 hours. Correspondingly, hyaluronic acid synthase 1 expression peaked first (4 and 8 hours), whereas hyaluronic acid synthase 2 expression peaked at 16 hours and again at 72 hours. Procollagen I expression peaked at 72 hours, whereas procollagen III decreased from 8 to 16 hours but peaked at 72 hours. Cyclooxygenase 2 expression was elevated, whereas elastin expression remained constant. CONCLUSIONS: The results show a clear profile of vocal fold inflammation with corresponding changes in extracellular matrix production.

Investigation of anti-hyaluronidase treatment on vocal fold wound healing.

SUMMARY: Phytochemical constituents of medicinal plants demonstrate inhibition of tissue and bacterial hyaluronidase. Echinacoside is a caffeoyl conjugate of Echinacea with known anti-hyaluronidase properties. The purpose of this study was to investigate the wound healing effects of Echinacea on vocal fold wound healing and functional voice outcomes. Pig animal model. METHODS: Vocal fold injury was induced in 18 pigs by unilateral vocal fold stripping. The uninjured vocal fold served as control. Three groups of six pigs randomly received a topical application of 300, 600, or 1,200 mg of standardized Echinacea on the injured side. Animals were euthanized after 3, 10, and 15 days of wound healing. Phonation threshold pressure and vocal economy measurements were obtained from excised larynges. Treatment outcomes were examined by comparing the animals receiving treatment with a set of 19 untreated and 5 historical controls. Treatment effects on wound healing were evaluated by histologic staining for hyaluronan and collagen. Treated larynges revealed improved vocal economy and phonation threshold pressure compared with untreated larynges. Histologically, treated vocal folds revealed stable hyaluronan content and no significant accumulation of collagen compared with control. Findings provide a favorable outcome of anti-hyaluronidase treatment on acute vocal fold wound healing and functional measures of voice.

In vivo clonal analysis reveals lineage-restricted progenitor characteristics in mammalian kidney development, maintenance, and regeneration.

The mechanism and magnitude by which the mammalian kidney generates and maintains its proximal tubules, distal tubules, and collecting ducts remain controversial. Here, we use long-term in vivo genetic lineage tracing and clonal analysis of individual cells from kidneys undergoing development, maintenance, and regeneration. We show that the adult mammalian kidney undergoes continuous tubulogenesis via expansions of fate-restricted clones. Kidneys recovering from damage undergo tubulogenesis through expansions of clones with segment-specific borders, and renal spheres developing in vitro from individual cells maintain distinct, segment-specific fates. Analysis of mice derived by transfer of color-marked embryonic stem cells (ESCs) into uncolored blastocysts demonstrates that nephrons are polyclonal, developing from expansions of singly fated clones. Finally, we show that adult renal clones are derived from Wnt-responsive precursors, and their tracing in vivo generates tubules that are segment specific. Collectively, these analyses demonstrate that fate-restricted precursors functioning as unipotent progenitors continuously maintain and self-preserve the mouse kidney throughout life.

Wnt signaling in skin development, homeostasis, and disease.

The skin and its appendages constitute the largest organ of the body. Its stratified epithelia offer protection from environmental stresses such as dehydration, irradiation, mechanical trauma, and pathogenic infection, whereas its appendages, like hair and sebaceous glands, help regulate body temperature as well as influence animal interaction and social behavior through camouflage and sexual signaling. To respond to and function effectively in a dynamic external environment, the skin and its appendages possess a remarkable ability to regenerate in a carefully controlled fashion. When this finely tuned homeostatic process is disrupted, skin diseases such as cancers may result. At present, the molecular signals that orchestrate cell proliferation, differentiation, and patterning in the skin remain incompletely understood. It is increasingly apparent that many morphogenetic pathways with key roles in development are also important in regulating skin biology. Of these, Wnt signaling has emerged as the dominant pathway controlling the patterning of skin and influencing the decisions of embryonic and adult stem cells to adopt the various cell lineages of the skin and its appendages, as well as subsequently controlling the function of differentiated skin cells. Here we will review established concepts and present recent advances in our understanding of the diverse roles that Wnt signaling plays in skin development, homeostasis, and disease.

Interfollicular epidermal stem cells self-renew via autocrine Wnt signaling.

The skin is a classical example of a tissue maintained by stem cells. However, the identity of the stem cells that maintain the interfollicular epidermis and the source of the signals that control their activity remain unclear. Using mouse lineage tracing and quantitative clonal analyses, we showed that the Wnt target gene Axin2 marks interfollicular epidermal stem cells. These Axin2-expressing cells constitute the majority of the basal epidermal layer, compete neutrally, and require Wnt/ß-catenin signaling to proliferate. The same cells contribute robustly to wound healing, with no requirement for a quiescent stem cell subpopulation. By means of double-labeling RNA in situ hybridization in mice, we showed that the Axin2-expressing cells themselves produce Wnt signals as well as long-range secreted Wnt inhibitors, suggesting an autocrine mechanism of stem cell self-renewal.

Heparan sulfate mediates the proliferation and differentiation of rat mesenchymal stem cells.

The growth and differentiation of mesenchymal stem cells (MSCs) is controlled by various growth factors, the activities of which can be modulated by heparan sulfates (HSs). We have previously noted the necessity of sulfated glycosaminoglycans for the fibroblast growth factor type 2 (FGF-2)-stimulated differentiation of osteoprogenitor cells. Here we show that exogenous application of HS to cultures of primary rat MSCs stimulates their proliferation, leading to increased expression of osteogenic markers and enhanced bone nodule formation. FGF-2 can also increase the proliferation, and osteogenic differentiation of rat bone marrow stem cells (rMSCs) when applied exogenously during their linear growth. However, as opposed to exogenous HS, the continuous use of FGF-2 during in vitro differentiation completely blocked rMSC mineralization. We show that the effects of both FGF-2 and HS are mediated through FGF receptor 1 (FGFR1) and that inhibition of signaling through this receptor arrests cell growth, resulting in the cells being unable to reach the critical density necessary to induce differentiation. Blocking FGFR1 signaling in postconfluent osteogenic cultures significantly increased calcium deposition. Taken together our data suggest that FGFR1 signaling plays an important role during osteogenic differentiation, first by stimulating cell growth that is closely followed by an inhibitory effect once the cells have reached confluence. It also confirms the importance of HS as a coreceptor for the signaling of endogenous FGF-2 and suggests that purified glycosaminoglycans may be attractive alternatives to growth factors for improved ex vivo growth and differentiation of MSCs.