Epithelial polarity-driven membrane separation but not cavitation regulates lumen formation of rat eccrine sweat glands.

BACKGROUND: Each eccrine sweat gland (ESG) is a single-tubular structure with a central lumen, and the formation of hollow lumen in the initial solid cell mass is a key developmental process. To date, there are no reports on the mechanism of native ESG lumen formation. METHODS: To investigate the lumen morphogenesis and the lumen formation mechanisms of Sprague-Dawley (SD) rat ESGs, SD rat hind-footpads at E20.5, P1-P5, P7, P9, P12, P21, P28 and P56 were obtained. The lumen morphogenesis of ESGs was examined by HE staining and immunofluorescence staining for polarity markers. The possible mechanisms of lumen formation were detected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) apoptosis assay and autophagy marker LC3B immunofluorescence staining, and further explored by ouabain intervention experiment. RESULTS: In SD rat ESGs, the microlumen was formed at P1, and the small intact lumen with apical-basal polarity appeared at P3. The expression of apical marker F-actin, basal marker Laminin, basolateral marker E-cadherin was consistent with the timing of lumen formation of SD rat ESGs. During rat ESG development, apoptosis and autophagy were not detected. However, inhibition of Na+-K+-ATPase (NKA) with ouabain resulted in decreased lumen size, although neither the timing of lumen formation nor the expression of polarity proteins was altered. CONCLUSIONS: Epithelial polarity-driven membrane separation but not cavitation regulates lumen formation of SD rat ESGs. NKA-regulated fluid accumulation drives lumen expansion.

Differential antigen expression between human apocrine sweat glands and eccrine sweat glands.

Bromhidrosis has a great negative impact on personal occupation and social psychology. It is not yet clear whether bromhidrosis is caused by apocrine sweat glands or the co-action of apocrine sweat glands and eccrine sweat glands. To distinguish between apocrine sweat glands and eccrine sweat glands, specific antigen markers for apocrine sweat glands and eccrine sweat glands must be found first. In the study, we detected the expression of K7, K18, K19, Na+-K+-2Cl- cotransporter 1 (NKCC1), carbonic anhydrase II (CAII), Forkhead transcription factor a1 (Foxa1), homeobox transcription factor engrailed homeobox1 (En1), gross cystic disease fluid protein-15 (GCDFP-15), mucin-1 (MUC-1), cluster of differentiation 15 (CD15) and apolipoprotein (APOD) in eccrine sweat glands and apocrine sweat glands by immunofluorescence staining. The results showed that K7, K18, K19, Foxa1, GCDFP-15 and MUC-1 were expressed in both apocrine and eccrine sweat glands, CD15 and APOD were only expressed in apocrine sweat glands, and CAII, NKCC1 and En1 were only expressed in eccrine sweat glands. We conclude that CD15 and APOD can serve as specific markers for apocrine sweat glands, while CAII, NKCC1 and En1 can serve as specific markers for eccrine sweat glands to differentiate the two sweat glands.

Differential distribution and genetic determination of eccrine sweat glands and hair follicles in the volar skin of C57BL/6 mice and SD rats.

BACKGROUND: Eccrine sweat glands (ESGs) and hair follicles (HFs) are the prominent skin appendages regulating human body temperature. C57BL/6 mice and Sprague-Dawley (SD) rats are the most commonly used model animals for studying ESGs and HFs. Previous studies have shown the distribution of ESGs and HFs in volar hindfeet of C57BL/6 mice, but there are few or no reports on the distribution of ESGs and HFs in volar forefeet of C57BL/6 mice and volar feet of SD rats. Here, we investigated the differential distribution and genetic determination of ESGs and HFs in the volar skin of C57BL/6 mice and SD rats through gross observation, iodine-starch sweat test, double staining with Nile Blue A and Oil Red O, hematoxylin and eosin (HE) staining, double immunofluorescence staining of LIM Homeobox 2 (LHX2)/Na+-K+-ATPase α1(NKA) or LHX2/Na+-K+-2Cl－ cotransporter 1 (NKCC1), and qRT-PCR detection of ESG-related gene Engrailed 1 (En1) and HF-related gene LHX2. RESULTS: The results showed ESGs but no HFs in the footpads of C57BL/6 mice and SD rats, both ESGs and HFs in the inter-footpads (IFPs) of C57BL/6 mice, and neither ESGs nor HFs in the IFPs of SD rats. The relative quantitative change in En1 was consistent with the differential distribution of ESGs, and the relative quantitative change of LHX2 was consistent with the differential distribution of HFs. CONCLUSION: C57BL/6 mice and SD rats had their own characteristics in the distribution of ESGs and HFs in the volar skin, and researchers should choose mice or rats, and even forefeet or hindfeet as their research object according to different purposes. The study provides a basis for selection of optimal animal models to study development, wound healing and regeneration of skin appendages.

Foxa1 mediates eccrine sweat gland development through transcriptional regulation of Na-K-ATPase expression.

Eccrine sweat glands (ESGs) perform critical functions in temperature regulation in humans. Foxa1 plays an important role in ESG maturation and sweat secretion. Its molecular mechanism, however, remains unknown. This study investigated the expression of Foxa1 and Na-K-ATPase (NKA) in rat footpads at different development stages using immunofluorescence staining, qRT-PCR, and immunoblotting. Also, bioinformatics analysis and Foxa1 overexpression and silencing were employed to evaluate Foxa1 regulation of NKA. The results demonstrated that Foxa1 was consistently expressed during the late stages of ESGs and had a significant role in secretory coil maturation during sweat secretion. Furthermore, the mRNA abundance and protein expression of NKA had similar accumulation trends to those of Foxa1, confirming their underlying connections. Bioinformatics analysis revealed that Foxa1 may interact with these two proteins via binding to conserved motifs in their promoter regions. Foxa1 gain-of-function and loss-of-function experiments in Foxa1-modified cells demonstrated that the activities of NKA were dependent on the presence of Foxa1. Collectively, these data provided evidence that Foxa1 may influence ESG development through transcriptional regulation of NKA expression.

Autophagy, not apoptosis, plays a role in lumen formation of eccrine gland organoids.

BACKGROUND: Sweat secreted by eccrine sweat glands is transported to the skin surface through the lumen. The eccrine sweat gland develops from the initial solid bud to the final gland structure with a lumen, but how the lumen is formed and the mechanism of lumen formation have not yet been fully elucidated. This study aimed to investigate the mechanism of lumen formation of eccrine gland organoids (EGOs). METHODS: Human eccrine sweat glands were isolated from the skin for tissue culture, and the primary cultured cells were collected and cultured in Matrigel for 14 days in vitro. EGOs at different development days were collected for hematoxylin and eosin (H&E) staining to observe morphological changes and for immunofluorescence staining of proliferation marker Ki67, cellular motility marker filamentous actin (F-actin), and autophagy marker LC3B. Western blotting was used to detect the expression of Ki67, F-actin, and LC3B. Moreover, apoptosis was detected using a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) apoptosis assay kit, and the expression of poly (ADP-ribose) polymerase and Caspase-3 was detected by Western blot. In addition, 3-methyladenine (3MA) was used as an autophagy inhibitor to detect whether the formation of sweat glands can be effectively inhibited. RESULTS: The results showed that a single gland cell proliferated rapidly and formed EGOs on day 4. The earliest lumen formation was observed on day 6. From day 8 to day 14, the rate of lumen formation in EGOs increased significantly. The immunofluorescence and Western blot analyses showed that the expression of Ki67 gradually decreased with the increase in days, while the F-actin expression level did not change. Notably, the expression of autophagy marker LC3B was detected in the interior cells of EGOs as the apoptosis signal of EGOs was negative. Compared with the control group, the autophagy inhibitor 3MA can effectively limit the formation rate of the lumen and reduce the inner diameter of EGOs. CONCLUSION: Using our model of eccrine gland 3D-reconstruction in Matrigel, we determined that autophagy rather than apoptosis plays a role in the lumen formation of EGOs.

Local autologous platelet rich plasma injection combined with platelet rich fibrin filling as the main treatment for refractory wounds: A case series.

Refractory wounds are a major global health problem that not only affects the quality of life, but also causes significant physical, psychological and economic burdens. How to promote wound healing has become the main goal of clinicians. To evaluate the safety and efficacy of local autologous platelet rich plasma (PRP) injection combined with platelet rich fibrin (PRF) filling as the main treatment for refractory wounds. In the study, autologous PRP and PRF were prepared from whole blood. Twelve patients, each having a refractory wound, were included. The wounds were debrided first to remove necrotic and infected tissues, and then were given once or twice local PRP injection combined with PRF filling treatment. The total healing time (the time from admission to wound healing) and the healing time after PRP/PRF combined treatment (the time from PRP treatment to wound healing), as well as the adverse events were recorded. The results showed that the wound duration before the combination treatment was 10.48 ± 3.66 weeks, and the mean area/volume (for sinus or fistula) of the wounds was 8.23 ± 2.67 cm2/9.54 ± 2.13 cm3 (for sinus or fistula). All wounds healed after once or twice PRP/PRF combined treatment. The total healing time was 26.91 ± 8.01 days, and the healing time after the combined treatment was 16.36 ± 7.47 days. No adverse events were reported during the treatment and follow-up period. Our case series demonstrate the safety and synergistic effectiveness of local autologous PRP injection combined with PRF filling as the main treatment for refractory wounds. Platelet concentrates is not only an adjuvant treatment for chronic wounds, but a potential substitute for chronic wounds, especially in sinuses and fistulas.

Foxa1 mediates eccrine sweat gland development through transcriptional regulation of Na-K-ATPase expression

Eccrine sweat glands (ESGs) perform critical functions in temperature regulation in humans. Foxa1 plays an important role in ESG maturation and sweat secretion. Its molecular mechanism, however, remains unknown. This study investigated the expression of Foxa1 and Na-K-ATPase (NKA) in rat footpads at different development stages using immunofluorescence staining, qRT-PCR, and immunoblotting. Also, bioinformatics analysis and Foxa1 overexpression and silencing were employed to evaluate Foxa1 regulation of NKA. The results demonstrated that Foxa1 was consistently expressed during the late stages of ESGs and had a significant role in secretory coil maturation during sweat secretion. Furthermore, the mRNA abundance and protein expression of NKA had similar accumulation trends to those of Foxa1, confirming their underlying connections. Bioinformatics analysis revealed that Foxa1 may interact with these two proteins via binding to conserved motifs in their promoter regions. Foxa1 gain-of-function and loss-of-function experiments in Foxa1-modified cells demonstrated that the activities of NKA were dependent on the presence of Foxa1. Collectively, these data provided evidence that Foxa1 may influence ESG development through transcriptional regulation of NKA expression.

The combination of hair follicle-specific marker LHX2 and co-expressed marker can distinguish between sweat gland placodes and hair placodes in rat.

Eccrine sweat gland (ESG) and hair follicle (HF) are different skin appendages but share many common development characteristics. Although the morphology of adult ESG and HF is obviously different, it is difficult to distinguish ESG placodes from HFs placodes morphologically. To study the fate determination of ESG and HF, specific antigen markers for ESG placodes and HF placodes must be found first to distinguish them. In the study, we detected the expression of commonly used keratins 4, 5, 7-10, 12, 14, 15, 17-20, 27 and 73, and the reported ESG and HF specific markers, P-cadherin, Lymphoid enhancer factor 1 (LEF1), LIM Homeobox gene 2 (LHX2), Na+/K+-ATPase (NKA) and Na+-K+-2Cl- cotransporter 1 (NKCC1) in ESG and HF placodes by single-immunofluorescence staining and double-immunofluorescence staining. To further verify the results of immunofluorescence staining, Western blot (WB) was used to detect the differential antigen and some co-expressed antigens of ESG and HF placodes. The results showed that both ESG and HF placodes expressed K4/5/14/1517/18, P-cadherin and LEF1, neither expressed K7/8/9/10/12/19/20/27/73, NKA or NKCC1. HF placodes specifically expressed LHX2. Combination of LHX2 and co-expressed antigen K14, can distinguish ESG placodes from HF placodes. We conclude that LHX2 is a specific marker for HF placodes, and ESG placodes and HF placodes can be distinguished by double immunofluorescence staining of the specific marker LHX2 and the co-expressed markers, such as K4, K5, K14, K15, K17, K18, P-cadherin and LEF1.