Nicholas Rufaut

Destruction of the arrector pili muscle and fat infiltration in androgenic alopecia.

Androgenic alopecia (AGA) is the most common hair loss condition in men and women. Hair loss is caused by follicle miniaturization, which is largely irreversible beyond a certain degree of follicular regression. In contrast, hair loss in telogen effluvium (TE) is readily reversible. The arrector pili muscle (APM) connects the follicle to the surrounding skin.

The proteomic profile of hair damage.

Background  Monilethrix is a congenital hair shaft disorder with associated fragility. Many of the changes seen in monilethrix hair on light microscopy and scanning electron microscopy are also seen in hair weathering and cosmetic damage to hair.

Beyond goosebumps: Does the arrector pili muscle have a role in hair loss?

The arrector pili muscle (APM) consists of a small band of smooth muscle that connects the hair follicle to the connective tissue of the basement membrane. The APM mediates thermoregulation by contracting to increase air-trapping, but was thought to be vestigial in humans. The APM attaches proximally to the hair follicle at the bulge, a known stem cell niche. Recent studies have been directed toward this muscles possible role in maintaining the follicular integrity and stability. This review summarizes APM anatomy and physiology and then discusses the relationship between the follicular unit and the APM. The potential role of the APM in hair loss disorders is also described, and a model explaining APM changes in hair loss is proposed.

Primary cicatricial alopecia: diagnosis and treatment

See also practice article by Aslam and Harries on page [1591][1] and at [www.cmaj.ca/lookup/doi/10.1503/cmaj.130305][2] Hair loss is common, the most prevalent disorders being androgenetic alopecia (male pattern baldness) and alopecia areata. Alopecia areata and androgenetic alopecia are

Miniaturized hairs maintain contact with the arrector pili muscle in alopecia areata but not in androgenetic alopecia: A model for reversible miniaturization and potential for hair regrowth

Background: Hair follicle miniaturization is the hallmark of male pattern hair loss (MPHL), female pattern hair loss (FPHL), and alopecia areata (AA). AA has the potential for complete hair regrowth and reversal of miniaturization. MPHL and FPHL are either irreversible or show only partial regrowth and minimal reversal of miniaturization. Hypothesis: The arrector pili muscle (APM) attachment to the hair follicle bulge, a recognized repository of stem cells may be necessary for reversal of hair follicle miniaturization. Materials and Methods: Sequential histological sections from MPHL, FPHL, AA, and telogen effluvium were used to create three-dimensional images to compare the relationship between the APM and bulge. Results: In AA, contact was maintained between the APM and the bulge of miniaturized follicles while in MPHL and FPHL contact was lost. Discussion: Contact between the APM and the bulge in AA may be required for reversal of hair follicle miniaturization. Maintenance of contact between miniaturized follicles in AA could explain the complete hair regrowth while loss of contact between the APM and the bulge in MPHL and FPHL may explain why the hair loss is largely irreversible. This loss of contact may reflect changes in stem cell biology that also underlie irreversible miniaturization.

The arrector pili muscle, the bridge between the follicular stem cell niche and the interfollicular epidermis

Proximally, the arrector pili muscle (APM) attaches to the follicular stem cell niche in the bulge, but its distal properties are comparatively unclear. In this work, a novel method employing an F-actin probe, phalloidin, was employed to visualize the APM anatomy. Phalloidin staining of the APM was validated by comparison with conventional antibodies/stains and by generating three-dimensional reconstructions. The proximal attachment of the APM to the bulge in 8 patients with androgenic alopecia was studied using Masson’s trichrome stain. Phalloidin visualized extensive branching of the APM. The distal end of the human APM exhibits a unique “C”-shaped structure connecting to the dermal–epidermal junction. The proximal APM attachment was observed to be lost or extremely miniaturized in androgenic alopecia. The unique shape, location, and attachment sites of the APM suggest a significant role for this muscle in maintaining follicular integrity. Proximally, the APM encircles the follicular unit and only attaches to the primary hair follicle in the bulge; this attachment is lost in irreversible hair loss. The APM exhibits an arborized morphology as it ascends toward the epidermis, and anchors to the basement membrane.

An In Vitro Model for the Morphogenesis of Hair Follicle Dermal Papillae

TO THE EDITOR Hair follicle size is correlated with the size of the dermal papilla (DP) (Ibrahim and Wright, 1982; Elliott et al., 1999). DP size is dynamically regulated during the follicle growth cycle. Cells emigrate from the DP during catagen and then repopulate it in anagen (Tobin et al., 2003; Chi et al., 2010). The follicle miniaturization seen in androgenetic alopecia is thought to be driven by dysfunctional DP cell (DPC) movement, whereas hypertrichosis is caused by excessive DPC migration or proliferation, producing an abnormally large DP (Jahoda, 1998). The mechanisms that determine DP size are poorly understood. An in vitro model for DP morphogenesis would facilitate their investigation. DPCs are well known to aggregate in culture, which is likely to be an expression of their morphogenetic behavior. However, DPC aggregation is variable and is typically lost after a period of culture ex vivo (Horne et al., 1986; Song et al., 2005). In contrast, we have found that ovine wool follicle DPCs exhibit particularly robust and stable aggregation. We have optimized culture conditions for these cells to establish a model for DP morphogenesis and a quantitative assay for aggregate size. Cultures of ovine DPCs were initiated by microdissecting papillae and explanting them in culture medium (Supplementary Methods online). The cells showed a fibroblastic morphology (Figure 1a), and formed whorl patterns on reaching confluence (Figure 1b). Localized variations in density then began to appear (Figure 1c and d). The highdensity patches continued to condense, eventually forming three-dimensional spheroids projecting up from the culture substrate (Figure 1e–g). The spheroids could be stained with Van Gieson’s solution (Figure 1h and i), allowing measurement of their size by image analysis. Alkaline phosphatase (AP) is expressed in the DP in vivo (Rendl et al., 2005). AP staining was observed in most ovine DPCs composing aggregates (Figure 1j–l). Versican, a proteoglycan also associated with DP formation (Kishimoto et al., 1999), was expressed only in aggregating cells (Figure 1m and p). Of the 19 cell strains from 12 sheep, 11 consistently aggregated as described above, for at least 5 passages. Among three strains (from two sheep), there was no loss of aggregative behavior before replicative senescence at 80.3–93.0 population doublings (22–27 passages, Supplementary Figure S1 online). Another 5 of the 19 strains aggregated in early passages but were not further tested. Three strains did not form discrete aggregates suitable for size determination. We established a standardized assay for quantifying the effect of bioactive compounds on aggregate size (Supplementary Methods online, Supplementary Figure S2 online). The addition of 10–30 mM lithium chloride (LiCl) induced a dose-dependent reduction in size (Figure 2a and b). Aggregation was abolished at a concentration of 40 mM. Dorsomorphin (a BMPR-1 and VEGFR-2 inhibitor) and SU5402 (a FGFR-1 and VEGFR-2 inhibitor) also reduced the aggregate size (Figure 2c and d). LiCl has pleiotropic effects on intracellular signaling, including action as a Wnt mimetic and an inhibitor of inositol phospholipid signaling (Chiu and Chuang, 2010). In light of the broad specificity of these compounds, the molecular mechanisms underlying aggregate miniaturization remain to be determined. The effects of additional compounds are shown in Supplementary Figure S3 online. The hair-loss drug, minoxidil, reversed the aggregate miniaturization induced by LiCl (Figure 2e). The effects of LiCl on aggregation persisted after it was removed from the cells (Supplementary Figure S4a online). We investigated the effect of LiCl pretreatment on the in vivo induction of hair follicles by ovine DPCs (Supplementary Figure S4b–f online). LiCl at a concentration of 40 mM blocked follicle induction in vivo, as well as aggregation in vitro (Supplementary Table S1 online). To our knowledge, a DPC culture system that permits quantitative measurement of aggregate size has not previously been reported. Although human and rodent DPCs exhibit aggregative behavior (Messenger, 1984; Horne et al., 1986; Song et al., 2005), the aggregates are less well formed, with less well-defined boundaries that preclude size measurement. Aggregation typically diminishes and then disappears as the cells continue to be propagated ex vivo. We found that robust aggregation of ovine DPCs continued after extensive growth in culture, allowing numerous assays to be performed with the same cell population. DPC aggregates expressed AP and versican, whereas monolayers did not. AP and versican are expressed in DP in vivo and are markers of follicleinducing activity (Kishimoto et al., 1999; Rendl et al., 2005). The localized expression of these markers LETTER TO THE EDITOR

Characterization of ovine dermal papilla cell aggregation

Context: The dermal papilla (DP) is a condensation of mesenchymal cells at the proximal end of the hair follicle, which determines hair shaft size and regulates matrix cell proliferation and differentiation. DP cells have the ability to regenerate new hair follicles. These cells tend to aggregate both in vitro and in vivo. This tendency is associated with the ability of papilla cells to induce hair growth. However, human papilla cells lose their hair-inducing activity in later passage number. Ovine DP cells are different from human DP cells since they do not lose their aggregative behavior or hair-inducing activity in culture. Nonetheless, our understanding of ovine DP cells is still limited. Aim: The aim of this study was to observe the expression of established DP markers in ovine cells and their association with aggregation. Subjects and Methods: Ovine DP cells from three different sheep were compared. Histochemistry, immunoflourescence, and polymerase chain reaction experiments were done to analyze the DP markers. Results: We found that ovine DP aggregates expressed all the 16 markers evaluated, including alkaline phosphatase and versican. Expression of the versican V0 and V3 isoforms, neural cell adhesion molecule, and corin was increased significantly with aggregation, while hey-1 expression was significantly decreased. Conclusions: Overall, the stable expression of numerous markers suggests that aggregating ovine DP cells have a similar phenotype to papillae in vivo. The stability of their molecular phenotype is consistent with their robust aggregative behavior and retained follicle-inducing activity after prolonged culture. Their phenotypic stability in culture contrasts with DP cells from other species, and suggests that a better understanding of ovine DP cells might provide opportunities to improve the hair-inducing activity and therapeutic potential of human cells.

Laser Capture Microdissection Reveals Transcriptional Abnormalities in Alopecia Areata before, during, and after Active Hair Loss

TO THE EDITOR Alopecia areata (AA) is a common nonscarring autoimmune hair loss disorder postulated to occur due to hair follicle (HF) immune privilege collapse (Paus and Bertolini, 2013). AA predominantly affects the anagen hair bulb, where a dense infiltrate of T cells is seen in acute disease (Gilhar et al., 2012). The cause of AA is unknown. Recently it was discovered that IFN-IL15-JAK/Stat cytokine pathways are activated in AA, leading to promising new treatments that are undergoing clinical trials (Xing et al., 2014). Other potential therapeutic targets may be found in the local chemokine milieu responsible for inflammatory cell recruitment. In a recent microarray analysis, CCL5, CXCL1, CXCL10, and CX3CL1 were found to be upregulated in lesional compared with nonlesional skin in patients with patchy AA (Subramanya et al., 2010). Other studies in mouse and human AA have shown overexpression of CXCL9, CCL2, CCL17, CCL20, and CXCR3 (Gupta et al., 2006; Ito et al., 2013; McPhee et al., 2012). However, these reports mostly examined a limited selection of chemokines and receptors, and utilized patient serum or bulk tissue rather than targeting the bulb, which is the primary focus of disease activity. We addressed these issues by collecting skin biopsies from 25 AA volunteers and 23 healthy controls. Biopsies from patients with AA were obtained from (i) active regions of hair loss, (ii) regrown areas of AA remission, and (iii) unaffected areas that had never been affected. Laser capture microdissection was performed on HF

Hair transplantation in mice: Challenges and solutions

Hair follicle cells contribute to wound healing, skin circulation, and skin diseases including skin cancer, and hair transplantation is a useful technique to study the participation of hair follicle cells in skin homeostasis and wound healing. Although hair follicle transplantation is a well‐established human hair‐restoration procedure, follicular transplantation techniques in animals have a number of shortcomings and have not been well described or optimized. To facilitate the study of follicular stem and progenitor cells and their interaction with surrounding skin, we have established a new murine transplantation model, similar to follicular unit transplantation in humans. Vibrissae from GFP transgenic mice were harvested, flip‐side microdissected, and implanted individually into needle hole incisions in the back skin of immune‐deficient nude mice. Grafts were evaluated histologically and the growth of transplanted vibrissae was observed. Transplanted follicles cycled spontaneously and newly formed hair shafts emerged from the skin after 2 weeks. Ninety percent of grafted vibrissae produced a hair shaft at 6 weeks. After pluck‐induced follicle cycling, growth rates were equivalent to ungrafted vibrissae. Transplanted vibrissae with GFP‐positive cells were easily identified in histological sections. We established a follicular vibrissa transplantation method that recapitulates human follicular unit transplantation. This method has several advantages over current protocols for animal hair transplantation. The method requires no suturing and minimizes the damage to donor follicles and recipient skin. Vibrissae are easier to microdissect and transplant than pelage follicles and, once transplanted, are readily distinguished from host pelage hair. This facilitates measurement of hair growth. Flip‐side hair follicle microdissection precisely separates donor follicular tissue from interfollicular tissue and donor cells remain confined to hair follicles. This makes it possible to differentiate migration of hair follicle cells from interfollicular epidermis in lineage tracing wound experiments using genetically labeled donor follicles.