Eccrine Sweat Gland Cells Research Articles

Molecular Characterization of Aged Human Eccrine Sweat Gland Cells Using Single-Cell Transcriptomic Analysis

Molecular Characterization of Aged Human Eccrine Sweat Gland Cells Using Single-Cell Transcriptomic Analysis

Epidermal and dermal cells from adult rat eccrine sweat gland-containing skin can reconstruct the three-dimensional structure of eccrine sweat glands

BackgroundPreviously, we have demonstrated that eccrine sweat gland cells (ESGCs) can reconstruct the three-dimensional (3D) structure of eccrine sweat glands (ESGs). However, there is still a need to explore source cells capable of regenerating ESG to address the issue of ESG regeneration in ESGC-deficient conditions, such as severe burns. MethodsThe epidermal cells and dermal cells in adult rat ventral foot skin (ESG-bearing) were isolated. The isolated single epidermal cells and dermal cells were mixed with Matrigel, and then the mixture was implanted into the axillary/inguinal fat pads of nude mice. Five weeks after implantation, the Matrigel plugs were harvested and the morphology and differentiation of the cells were examined by H&E staining and fluorescent immunohistochemical staining for ESG markers, such as Na+ -K+ -2Cl- cotransporter 1 (NKCC1), Na+ -K+ -ATPase (NKA), Foxa1 and K14. ResultsThe epidermal cells and dermal cells of adult rat ventral foot skin can reconstruct 3D structure and express specific markers of ESGs in skin, such as NKCC1, NKA and Foxa1, indicating the ESG-phenotypic differentiation of the 3D structures. Double immunofluorescence staining showed that some 3D structures expressed both the myoepithelial cell marker alpha-SMA and the common marker K14 of duct cells and myoepithelial cells, while some 3D structures expressed only K14, indicating that ESG-like 3D structures differentiated into duct-like and secretory coiled cells. ConclusionEpidermal and dermal cells from adult ESG-bearing skin can be used as a cell source for ESG regeneration.

Acquired Idiopathic Generalized Anhidrosis (AIGA) and Its Complications: Implications for AIGA as an Autoimmune Disease.

Acquired idiopathic generalized anhidrosis (AIGA) is a rare disorder in which systemic anhidrosis/hypohidrosis occurs without causative dermatological, metabolic or neurological disorder. Most cases of AIGA have been reported in Asia, especially in Japan, but there have been only a few reports in Europe and the United States. Severe AIGA may result in heatstroke and can reduce quality of life due to restriction of exercise and outdoor works. AIGA is often accompanied by cholinergic urticaria (CholU), and it is thought that AIGA and CholU with anhidrosis/hypohidrosis belong to the same spectrum of the disease. However, the pathophysiology of AIGA has not yet been clarified. Decreased expression of cholinergic receptor M3 on the epithelial cells of eccrine sweat glands is often accompanied by T cell infiltration around eccrine apparatus, suggesting an immunological mechanism of disordered perspiration. AIGA is occasionally associated with various complications indicative of autoimmune disorders. The association of autoimmune complications further suggests that AIGA is an autoimmune disorder. Studies on complications may lead to a better understanding of the pathophysiology of AIGA.

Quantitative ion determination in eccrine sweat gland cells correlates to sweat reduction of antiperspirant actives.

Axillary wetness represents an unwanted effect of the physiologically vital sweating mechanism, especially when it becomes excessive. Cosmetic products reducing sweat secretion rely on aluminium salts as the active ingredient acting by physically blocking the sweat gland. Driven by the interest to better understand the sweat mechanism and to develop alternative technologies against excessive sweating a search for an effective testing approach started as up to now, cost- and time-consuming in vivo studies represent the standard procedure for testing and identifying these alternatives. The herein described in vitro test system is based on the measurement of intracellular changes of the ion equilibrium in cultured eccrine sweat gland cells. Subsequently, in vivo studies on the back of volunteers were conducted to verify the sweat-reducing effect of in vitro newly discovered substance. In this study, we describe an effective cell-based in vitro method as a potent tool for a more targeted screening of alternatives to aluminium salts. Testing the commonly used aluminium chlorohydrate as one example of an aluminium-based active in this screening procedure, we discovered a distinct influence on the ion equilibrium: Intracellular levels of sodium ions were decreased while those of chloride increased. Screening of various substances revealed a polyethyleneimine, adjusted to pH 3.5 with hydrochloric acid, to evoke the same alterations in the ion equilibrium as aluminium chlorohydrate. Subsequent in vivo studies showed its substantial antiperspirant action and confirmed the high efficiency of the polyethyleneimine solution in vivo. Further, specific investigations connecting the chloride content of the tested substances with the resulting sweat reduction pointed towards a substantial impact of the chloride ions on sweating. The newly described in vitro cell-based screening method represents an effective means for identifying new antiperspirant actives and suggests an additional biological mechanism of action of sweat-reducing ingredients which is directed towards unbalancing of the ion equilibrium inside eccrine sweat gland cells.

EGFR inhibitor AG1478 blocks the formation of 3D structures mainly through ERK signaling pathway in Matrigel-induced 3D reconstruction of eccrine sweat gland-like structures.

EGFR signaling plays important roles in the development of eccrine sweat glands. We previously demonstrate that Matrigel induces eccrine sweat gland cells to reconstruct the three-dimensional (3D) structures of eccrine sweat glands, but the mechanisms are still unknown. In the study, eccrine sweat gland cells were cultured within a 3D Matrigel, and EGFR inhibitor AG1478, or MEK1/2 inhibitor U0126, were added to the medium respectively. The morphology of the 3D-reconstructed eccrine sweat gland-like structures was observed, the localization of phospho-EGFR was detected, and protein levels of EGFR, phospho-EGFR, phospho-JAK, phospho-AKT and phospho-ERK were examined. The results showed that cells treatment with AG1478 from Day 0 of 3D cultures blocked formation of spheroid-like structures. AG1478 administration caused reduced phospho-EGFR, concomitant with downregulation of phospho-ERK1/2, but not phospho-JAK or phospho-AKT. Phospho-EGFR and phospho-ERK were reduced, and only a small number of 3D-structures were formed following treatment with U0126. We conclude that EGFR plays important roles in Matrigel-induced 3D structures of eccrine sweat gland-like structures, and ERK1/2 signaling is responsible, at least in part, for the effect of EGFR.

K31 as a novel marker for clear secretory cells in human eccrine sweat glands

K31 was previously considered as one of the hair keratins. During a study on differential markers between hair follicles and eccrine sweat glands, we observed that K31 was expressed in eccrine sweat gland cells in a scattered pattern, similar to the distribution of dark or clear secretory cells. To investigate the precise cell localization of K31 in human eccrine sweat glands and find new marker for eccrine sweat gland cells, human skin samples were fixed, paraffined and sectioned. The serial sections were stained for K31, dark secretory cell marker gross cystic disease fluid protein 15 (GCDFP15) and clear secretory cell marker carbonic anhydrase II (CAII). The exact cell localization of K31 was detected by double immunofluorescence staining of K31 and a serial of cell-specific markers, and further by dual stain using a combination of periodic acid-Schiff (PAS) and immunofluorescence for K31 and GCDFP15. The expression pattern of K31-positive cells was similar to that of CAII-positive cells but was different from that of GCDFP15-positive staining in serial sections. Double immunofluorescent staining showed that K31-positive cells co-expressed K7 and CAII, but not S100P, α-SMA or GCDFP15. Dual stain by combined PAS and immunofluorescence showed that K31-positive cells are negative for PAS staining. We conclude that K31 is a previously unreported eccrine clear cell marker that allows for distinction between clear and dark secretory cells, as well as between secretory coils and ducts of eccrine sweat glands in human eccrine sweat glands.

Evidence for TRPV4 channel induced skin vasodilatation through NOS, COX, and KCa channel mechanisms with no effect on sweat rate in humans

Transient receptor potential vanilloid 4 (TRPV4) channels exist in the endothelial cells of cutaneous blood vessels and the secretory cells of eccrine sweat glands. We assessed if exogenous TRPV4 channel activation elicits cutaneous vasodilatation and sweating in humans in vivo, and if so, whether this response is mediated by nitric oxide synthase (NOS)- cyclooxygenase (COX)- and/or Ca2+-sensitive K+ (KCa) channel-related mechanisms. In ten healthy young adults (24±2 years, 5 women), cutaneous vascular conductance and sweat rate were assessed at four dorsal forearm skin sites continuously treated with either: 1) lactated Ringer's solution (Control), 2) 20 mM L-NAME, a non-selective NOS inhibitor, 3) 10 mM ketorolac, a non-selective COX inhibitor, or 4) 50 mM TEA, a non-selective KCa channel blocker. A potent and selective TRPV4 channel agonist, GSK1016790 A (GSK101), was administered to each skin site in a dose-dependent manner (1, 10, 100, 1000 μM each for ≥30min) via intradermal microdialysis. Administration of 100 and 1000 μM GSK101 increased cutaneous vascular conductance from pre-infusion level at the Control site (48±12 and 57±9%max, respectively, P≤0.004). This response was markedly (53–83%) attenuated by NOS inhibition, COX inhibition, or KCa channel blockade (all P≤0.037), except KCa channel blockade had no effect during 1000 μM GSK101 administration. GSK101 did not influence sweat rate regardless of skin site. We showed that in human skin in vivo, exogenous activation of TRPV4 channels mediates cutaneous vasodilatation, but not sweating through NOS, COX, and KCa channel mechanisms.

867 Contraction of myoepithelial cells in human eccrine sweat glands, revealed by 3D live imaging

Sweat glands are single tubular organs subdivided into two parts: excretory and secretory ducts, both of which form a complicated coiled structure in the deep dermis. Secretory ducts are surrounded by contractile myoepithelial cells. Contraction of these myoepithelial cells is thought to contribute to the secretion of sweat. However, it remains unknown whether sweat gland myoepithelial cells contract. We previously revealed that myoepithelial cells were aligned in a well-oriented manner, enabling efficient contraction of secretory ducts, which likely facilitates sweat excretion. Human sweat gland dynamics were captured using a live-imaging system with actin, nuclear, and cell membrane markers. The contraction of myoepithelial cells was induced by pilocarpine, an acetylcholine analogue. Live-imaging data revealed that a portion of the secretory duct began to contract in the direction of narrowing tube diameter, immediately after pilocarpine addition; this was followed by gradual distention of two distal excretory ducts. The contractile motion of the coiled tubular components of the sweat gland showed sequential contraction, enabling transportation of sweat. Moreover, this contraction was inhibited by the sweating antagonist, atropine. These results suggest that myoepithelial cell contraction may be necessary to generate the driving force for transporting the sweat. We plan to further analyze the kinetics of myoepithelial contracting motion, in combination with pharmacological intervention, to identify the specific regulatory mechanisms involved.

Detection of fluid secretion of three-dimensional reconstructed eccrine sweat glands by magnetic resonance imaging.

We previously showed three-dimensional (3D) reconstructed eccrine sweat glands have similar structures as native eccrine sweat glands, but whether the 3D reconstructed sweat glands appropriately secrete fluid is still unknown. In this study, Matrigel-embedded human eccrine sweat gland cells or Matrigel alone were implanted into the groin subcutis of the nude mice. Ten weeks post-implantation, images of the subcutaneously formed plugs, as well as footpads of rats, pre- and post-pilocarpine/normal saline (NS) injection were acquired using a fat-suppressed proton density-weighted magnetic resonance imaging (MRI) sequence at 7.0T, and the regions of interest (ROIs) in plugs and rat footpads were analysed and graphed. A significant increase in the ROI mean proton intensity occurred in both 3D reconstructed and native eccrine sweat glands after pilocarpine injection. The mean proton intensity had no noticeable changes in ROIs of Matrigel plugs between pre- and post-pilocarpine injection, and in ROIs of rat footpads between pre- and post-NS injection. In conclusion, the 3D reconstructed sweat glands possess fluid secretion, which is detectable by fat-suppressed proton density-weighted MRI.

The effect of exogenous activation of protease-activated receptor 2 on cutaneous vasodilatation and sweating in young males during rest and exercise in the heat

ABSTRACTProtease-activated receptor 2 (PAR2) exists in the endothelial cells of skin vessels and eccrine sweat glands. We evaluated the hypothesis that exogeneous activation of PAR2 augments cutaneous vasodilatation and sweating during rest and exercise in the heat. In 10 young males (23 ± 5 y), cutaneous vascular conductance (CVC) and sweat rate were measured at four forearm skin sites treated with either 1) lactated Ringer (Control), 2) 0.05 mM, 3) 0.5 mM, or 4) 5 mM SLIGKV-NH2 (PAR2 agonist). Participants initially rested in a semi-recumbent posture under a normothermic ambient condition (25°C) for ~60 min. Thereafter, ambient temperature was increased to 35°C while the participants rested for an additional 60 min. Participants then performed a 50-min bout of cycling (~55% of their pre-determined peak oxygen uptake) followed by a 30-min recovery period. Administration of 5 mM SLIGKV-NH2 increased cutaneous vascular conductance relative to the Control site during normothermic resting (P ≤ 0.05). However, we showed that relative to the Control site, no effect on CVC was observed for any administered dose of SLIGKV-NH2 (0.05-5 mM) during rest (33–39%max CVC), end-exercise (68–70%max CVC), and postexercise recovery (49–53%max CVC) in the heat (all P > 0.05). There were no differences in sweat rate between the Control and all SLIGKV-NH2-treated sites throughout the protocol (0.21–0.23, 1.20–1.27, and 0.32–0.33 mg∙min−1∙cm−2 for rest, end-exercise, and postexercise in the heat, respectively, all P > 0.05). We show that while exogeneous PAR2 activation induces cutaneous vasodilatation during normothermic rest, it does not influence the cutaneous blood flow and sweating responses during rest, exercise or recovery in the heat.

Expression and localization of Forkhead transcription factor A1 in the three-dimensional reconstructed eccrine sweat glands

Previously studies showed that Forkhead transcription factor A1 (FoxA1) was associated with sweat secretion. To investigate the expression and localization of FoxA1 in the three-dimensional (3D) reconstructed eccrine sweat glands, eccrine sweat gland cells were transplanted subcutaneously into nude mice with Matrigel, and at 2, 3, 4, 5, 6, 8, 10 and 12 weeks post-transplantation, the reconstructed eccrine sweat glands were removed and immunostained for FoxA1 and co-immunostained for FoxA1 and eccrine sweat markers, K7, carbonic anhydrase II (CA Ⅱ), gross cystic disease fluid protein-15 (GCDFP-15) and α-smooth muscle actin (α-SMA), and FoxA1 and sweat secretion-related proteins, Na+-K+-ATPase α and Na+-K+-2Cl- cotransporter 1 (NKCC1). The results showed that FoxA1-positive cells weren’t detected until 3 weeks post-implantation, a time point of the differntiation of secretory coil-like structures. From the fourth week on, the number of FoxA1-positive cells increased and thereafter maintained at a high number. Double immunofluorescence staining showed that FoxA1-positive cells co-expressed dark cell marker GCDFP-15 and myoepithelial cell marker α-SMA, as well as secretion-related proteins, Na+-K+-ATPase α and NKCC1 in both the native and reconstructed eccrine sweat glands. In conclusion, FoxA1 might be related to the development and differentiation of secretory coil-like structures, as well as the secretory function of the 3D reconstructed eccrine sweat glands.

Three-dimensional reconstructed eccrine sweat glands with vascularization and cholinergic and adrenergic innervation.

Functional integrity of the regenerated tissues requires not only structural integrity but also vascularization and innervation. We previously demonstrated that the three-dimensional (3D) reconstructed eccrine sweat glands had similar structures as those of the native ones did, but whether the 3D reconstructed glands possessing vascularization and innervation was still unknown. In the study, Matrigel-embedded eccrine sweat gland cells were implanted under the inguinal skin. Ten weeks post-implantation, the vascularization, and innervation in the 10-week reconstructed eccrine sweat glands and native human eccrine sweat glands were detected by immunofluorescence staining. The results showed that the fluorescent signals of general neuronal marker protein gene product 9.5, adrenergic nerve fiber marker tyrosine hydroxylase, and cholinergic nerve fiber markers acetylcholinesterase and vasoactive intestinal peptide embraced the 3D reconstructed glands in circular patterns, as the signals appeared in native eccrine sweat glands. There were many CD31- and von Willebrand factor-positive vessels growing into the plugs. We demonstrated that the 3D reconstructed eccrine sweat glands were nourished by blood vessels, and we for the first time demonstrated that the engineering sweat glands were innervated by both cholinergic and adrenergic fibers. In conclusion, the 3D reconstructed eccrine sweat glands may have functions as the native ones do.

A novel organotypic 3D sweat gland model with physiological functionality.

Dysregulated human eccrine sweat glands can negatively impact the quality-of-life of people suffering from disorders like hyperhidrosis. Inability of sweating can even result in serious health effects in humans affected by anhidrosis. The underlying mechanisms must be elucidated and a reliable in vitro test system for drug screening must be developed. Here we describe a novel organotypic three-dimensional (3D) sweat gland model made of primary human eccrine sweat gland cells. Initial experiments revealed that eccrine sweat gland cells in a two-dimensional (2D) culture lose typical physiological markers. To resemble the in vivo situation as close as possible, we applied the hanging drop cultivation technology regaining most of the markers when cultured in its natural spherical environment. To compare the organotypic 3D sweat gland model versus human sweat glands in vivo, we compared markers relevant for the eccrine sweat gland using transcriptomic and proteomic analysis. Comparing the marker profile, a high in vitro-in vivo correlation was shown. Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5), muscarinic acetylcholine receptor M3 (CHRM3), Na+-K+-Cl- cotransporter 1 (NKCC1), calcium-activated chloride channel anoctamin-1 (ANO1/TMEM16A), and aquaporin-5 (AQP5) are found at significant expression levels in the 3D model. Moreover, cholinergic stimulation with acetylcholine or pilocarpine leads to calcium influx monitored in a calcium flux assay. Cholinergic stimulation cannot be achieved with the sweat gland cell line NCL-SG3 used as a sweat gland model system. Our results show clear benefits of the organotypic 3D sweat gland model versus 2D cultures in terms of the expression of essential eccrine sweat gland key regulators and in the physiological response to stimulation. Taken together, this novel organotypic 3D sweat gland model shows a good in vitro-in vivo correlation and is an appropriate alternative for screening of potential bioactives regulating the sweat mechanism.

Stage-specific embryonic antigen-4 as a novel marker of ductal cells of human eccrine sweat glands.

Different populations of unipotent or multipotent stem cells have been identified in human epidermis and its appendages. It is well documented that these cells maintain tissue homeostasis and actively participate in epidermal regeneration after injury. However, there is no evidence of the presence of pluripotent stem cells in human epidermis. In this study we investigated whether cells positive for embryonic stem cell marker stage-specific embryonic antigen-4 (SSEA-4) are present in adult human epidermis and, if so, whether they are pluripotent and correspond to the population of primitive stem cells. The expressions of SSEA-4 and pluripotency transcription factors were analysed using flow cytometry. By means of immunohistochemical staining, we studied the exact localization of these cells in sections of human skin. We show that a population of SSEA-4+ cells is present in human epidermis. In contrast to the commonly accepted belief, the expression of SSEA-4 is not connected with the pluripotent character of isolated cells. We found that these SSEA-4+ cells are localized in the ducts of eccrine sweat glands. Our results indicate that SSEA-4 is a novel marker identifying the ductal cells of human sweat glands. The surface character of the antigen provides for a simple method of isolating this cell population and suggests applications of SSEA-4 for future cell therapy research.

Activation of Protease‐Activated Receptor 2 Directly Mediates Cutaneous Vasodilatation but Not Sweating in Humans In Vivo: Roles of Nitric Oxide Synthase and Cyclooxygenase

Protease‐activated receptor 2 (PAR2) is present in the endothelial cells of skin microvessels and eccrine sweat glands in humans. Notably, this receptor has been shown to play a role in the vascular control of larger arteries and the control of epidermal permeability. However, it remains unclear if PAR2 functionally contributes to the regulation of cutaneous blood flow and sweating in humans. Therefore, the objective of the current study was to determine whether the activation of PAR2 directly stimulates cutaneous vasodilation and sweating through nitric oxide synthase (NOS) and cyclooxygenase (COX) mediated mechanisms. In twelve young males (29±5 years), cutaneous vascular conductance (CVC, measured via laser‐Doppler perfusion units/mean arterial pressure) and sweat rate (measured via ventilated capsule) were evaluated at four intradermal microdialysis forearm skin sites that were constantly perfused with either 1) lactated Ringer (Control), 2) 10 mM Nω‐nitro‐L‐arginine (NOS inhibitor), 3) 10 mM ketorolac (COX inhibitor), or 4) a combination of 10 mM Nω‐nitro‐L‐arginine + 10 mM ketorolac. The PAR2 agonist (SLIGKV‐NH2) was co‐administered at all sites in a dose‐dependent fashion (0.06, 0.18, 0.55, 1.66, and 5 mM, each for 25 min). Throughout the protocol, participants rested in a semi‐recumbent position in a thermoneutral room (23°C). Responses were compared at baseline and the last 5 min of each dose administered. The highest dose of SLIGKV‐NH2 (5 mM) increased CVC from baseline (BL) at the Control site (BL: 15 ± 8% CVCmax, 5 mM: 37 ± 22% CVCmax; P ≤ 0.01). This increase in CVC associated with PAR2 activation was attenuated by NOS inhibition with or without simultaneous COX inhibition (Control: 37 ± 22%CVCmax, NOS inhibition: 25 ± 16% CVCmax, NOS+COX inhibition: 23 ± 16% CVCmax; both P ≤ 0.05). However, COX inhibition alone had no effect on the PAR2 mediated increase in CVC at all doses (all P > 0.05). No increase in sweat rate was measured at any administered dose of SLIGKV‐NH2 irrespective of the agent co‐administered (all P > 0.05). In conclusion, we show that in normothermic resting humans, in vivo activation of PAR2 directly mediates cutaneous vasodilation through NOS‐dependent mechanisms without any contribution of COX mediated pathways. Alternatively, the same activation of PAR2 does not lead to increases in forearm sweat rate.Support or Funding InformationThis study was supported by the Natural Sciences and Engineering Research Council of Canada (Discovery grant, RGPIN‐06313‐2014; Discovery Grants Program ‐ Accelerator Supplement, RGPAS‐462252‐2014; funds held by Dr. Glen P. Kenny).

Expression of S100A2 and S100P in human eccrine sweat glands and their application in differentiating secretory coil-like from duct-like structures in the 3D reconstituted eccrine sweat spheroids.

Secretory coils and ducts are two components of eccrine sweat glands with different structures and functions. In our previous study, we combined keratins and α-SMA to distinguish between secretory coils and ducts. However, the key deficiency of the method was that none of the antibodies used was specific for ducts. In this study, we first examined the co-localization of K5/K7, α-SMA/K14, K7/S100P and α-SMA/S100A2 by double-immunofluorescence staining to confirm the localization of S100P and S100A2 in native human eccrine sweat glands, and second we identified secretory coil-like and duct-like structures in the 3D reconstituted eccrine sweat gland spheroids by double-immunofluorescence staining for K7/S100P and α-SMA/S100A2. In native human eccrine sweat glands, S100A2 immunoreactivity was confined to the outer layer and S100P to the inner layer of the duct. In 12-week Matrigel plugs containing eccrine sweat gland cells, double-immunofluorescence staining for K7/S100P and α-SMA/S100A2 could easily distinguish duct-like structures from secretory coil-like structures. We conclude that S100A2 and S100P can be used as specific duct markers in eccrine sweat glands, and combined use of S100P or S100A2 with keratins enables easy to distinction between secretory coils and ducts.

Serum is an indispensable factor in the maintenance of the biological characteristics of sweat gland cells.

The tolerance of sweat gland cells for in vitro amplification and subcultivation is low as they are somatic cells. The present study aimed to formulate an optimal medium for the culture of human eccrine sweat gland cells (HESGCs) and to establish a method for induction of HESGCs proliferation, whilst maintaining the characteristics of sweat gland cells. HESGCs cultured in sweat gland (SG):keratinocyte growth medium-2 (KGM-2) (1:1) medium had a higher proliferation rate and a stable morphology compared with cells cultured in SG and KGM-2 medium only. Reverse transcription-quantitative polymerase chain reaction indicated that cells cultured in the SG:KGM-2 (1:1) medium exhibited higher expression levels of α-smooth muscle actin, keratin (K)77, carcinoembryonic antigen, K8, K18, ectodysplasin A receptor, c-Myc, Kruppel-like factor 4 and octamer-binding transcription factor 4 compared with cells cultured in SG only or KGM-2 only medium. Three-dimensional culture analysis revealed that HESGCs cultured in SG:KGM-2 1:1 medium differentiated into sweat gland-like structures, whereas cells cultured in KGM-2 only medium underwent cornification. The present study also determined that the maintenance of the biological characteristics of HESGCs occurred due to the presence of fetal bovine serum (FBS). Cells cultured in medium without FBS differentiated into keratinocytes. Therefore, the SG:KGM-2 (1:1) medium may be a suitable culture medium for HESGCs. In conclusion, this mixed medium is a valuable compound and should be considered to be a potential supplemental medium for HESGCs.

Activation of protease-activated receptor 2 mediates cutaneous vasodilatation but not sweating: roles of nitric oxide synthase and cyclo-oxygenase.

What is the central question of this study? Protease-activated receptor 2 (PAR2) is located in the endothelial cells of skin vessels and eccrine sweat glands. However, a functional role of PAR2 in the control of cutaneous blood flow and sweating remains to be assessed in humans in vivo. What is the main finding and its importance? Our results demonstrate that in normothermic resting humans in vivo, activation of PAR2 elicits cutaneous vasodilatation partly through nitric oxide synthase-dependent mechanisms, but does not mediate sweating. These results provide important new insights into the physiological significance of PAR2 in human skin. Protease-activated receptor 2 (PAR2) is present in human skin, including keratinocytes, endothelial cells of skin microvessels and eccrine sweat glands. However, whether PAR2 contributes functionally to the regulation of cutaneous blood flow and sweating remains entirely unclear in humans in vivo. We hypothesized that activation of PAR2 directly stimulates cutaneous vasodilatation and sweating via actions of nitric oxide synthase (NOS) and cyclo-oxygenase (COX). In 12 physically active young men (29±5years old), cutaneous vascular conductance (CVC) and sweat rate were measured at four intradermal microdialysis forearm skin sites that were treated with the following: (i)lactated Ringer's solution (control); (ii)10mm NG -nitro-l-arginine (NOS inhibitor); (iii)10mm ketorolac (COX inhibitor); or (iv) a combination of both inhibitors. At all sites, a PAR2 agonist (SLIGKV-NH2 ) was co-administered in a dose-dependent fashion (0.06, 0.18, 0.55, 1.66 and 5mm, each for 25min). The highest dose of SLIGKV-NH2 (5mm) increased CVC from baseline at the control site (P≤0.05). This increase in CVC associated with PAR2 activation was attenuated by NOS inhibition regardless of the presence or absence of simultaneous COX inhibition (both P≤0.05). However, COX inhibition alone did not affect the PAR2-mediated increase in CVC (P>0.05). No increase in sweat rate was measured at any administered dose of SLIGKV-NH2 (all P>0.05). We show that in normothermic resting humans in vivo, PAR2 activation does not increase sweat rate, whereas it does modulate cutaneous vasodilatation through NOS-dependent mechanisms.

Cell proliferation and differentiation during the three dimensional reconstitution of eccrine sweat glands.

The aim of this study is to characterize the cell proliferation and proliferating cell types during three-dimensional reconstitution of eccrine sweat glands. Eccrine sweat gland cells suspended in Matrigel were injected subcutaneously into the inguinal regions of nude mice. At 1, 2, 4, 6, 8, 14, 21, 28, 35 and 42days post-implantation, Matrigel plugs were immunostained for Ki67, to detect cycling cells, and the Ki67 labeling index at different time points was calculated. Three pairs of antibodies, Ki67/K7, Ki67/K14 and Ki67/α-SMA, were used to identify proliferating cell types in the plugs, on days 28, 35 and 42, by immunofluorescence double staining. The Ki67 labeling index on the first day of implantation was 30.53%, rapidly reached a peak value of 81.43% at 2days post-implantation, and then decreased gradually to a low of 2.87% at 42days. Double immunofluorescence staining showed that K14/Ki67 double-stained cells accounted for 80% of the Ki67-positive cells, whereas K7/Ki67 and α-SMA/Ki67 double-stained cells each accounted for 10% of the Ki67-positive population on days 28, 35, or 42 post-implantation. We conclude that eccrine sweat gland cells rapidly enter the cell cycle after implantation, but quickly show decreased cell proliferation and increased cell differentiation.

CD99 Is Strongly Expressed in Basal Cells of the Normal Adult Epidermis and Some Subpopulations of Appendages: Comparison with Developing Fetal Skin.

BackgroundCD99 is a cell surface transmembrane glycoprotein expressed in various tissues. CD99 is differentially expressed between subpopulations of each tissue and is highly expressed in certain hematopoietic and precursor cells. However, there has been no comprehensive study of CD99 expression in normal skin. We evaluated CD99 expression in normal human skin and developing fetal skin. MethodsSeventy-five adult skin samples containing normal skin and eight fetal skin samples of different gestational ages were collected. CD99 immunohistochemical staining was performed to evaluate expression pattern in adult and fetal skin samples. CD99 and CD34 expression were compared by double immunofluorescence. ResultsIn normal adult skin, CD99 was strongly expressed in the membrane of epidermal basal keratinocytes, hair follicle bulges and outer root sheaths, and inner secretory cells of eccrine sweat glands. In fetal skin, CD99 was not expressed on the periderm at 16 weeks of gestation but was expressed in basal cells of fetal skin at around 19 weeks of gestation. CD99 expression became comparable to that of the adult skin after 20 weeks of gestation. CD99 and CD34 were co-expressed in hair follicle outer root sheaths, as seen by double immunofluorescence study. ConclusionsThis is the first study examining CD99 expression pattern in normal adult and fetal skin. CD99 tends to be expressed in the basal/precursor cells of epidermis and in hair follicles. These results provide a basis for future investigation on functions of CD99 in the skin and provide a novel potential target for the treatment of dermatologic lesions.