Hair Follicle Transit-Amplifying Cells Phagocytose Dead Cells after Radiotherapeutic and Chemotherapeutic Injuries for Timely Regeneration.

Efficient clearance of dead cells is critical for tissue regeneration after injuries. How dead cells are removed from the skin after radiotherapy and chemotherapy is unclear. In this study, we found that radiotherapeutic and chemotherapeutic damage induced extensive apoptosis of highly proliferative transit-amplifying cells in hair follicles. These apoptotic cells disappeared rapidly in the early stage of regenerative attempts, and the lost structures were regenerated with transient and low-level inflammation. Without the recruitment of macrophages as scavengers, the dying cells were engulfed directly by adjacent surviving transit-amplifying cells, which produced mature phagosomes through fusion with lysosomes in a manner similar to professional phagocytosis and remained active in proliferation. Autophagy did not contribute significantly to the clearance of engulfed cell debris. Perturbing phagocytosis in the transit-amplifying cells hindered apoptotic cell removal, delayed structural recovery, and aggravated hair loss. Therefore, transit-amplifying cells are capacitated with both proliferative and efferocytic functions that facilitate tissue regeneration after tissue injury.

Delayed Collagen Production without Myofibroblast Formation Contributes to Reduced Scarring in Adult Skin Microwounds.

In adult mammals, wound healing predominantly follows a fibrotic pathway, culminating in scar formation. However, cutaneous microwounds generated through fractional photothermolysis, a modality that produces a constellation of microthermal zones, exhibit a markedly different healing trajectory. Our study delineates the cellular attributes of these microthermal zones, underscoring a temporally limited, subclinical inflammatory milieu concomitant with rapid re-epithelialization within 24 hours. This wound closure is facilitated by the activation of genes associated with keratinocyte migration and differentiation. In contrast to macrothermal wounds, which predominantly heal through a robust myofibroblast-mediated collagen deposition, microthermal zones are characterized by absence of wound contraction and feature delayed collagen remodeling, initiating 5-6 weeks after injury. This distinct wound healing is characterized by a rapid re-epithelialization process and a muted inflammatory response, which collectively serve to mitigate excessive myofibroblast activation. Furthermore, we identify an initial reparative phase characterized by a heterogeneous extracellular matrix protein composition, which precedes the delayed collagen remodeling. These findings extend our understanding of cutaneous wound healing and may have significant implications for the optimization of therapeutic strategies aimed at mitigating scar formation.

Single-Cell Transcriptomics Reveals Cellular Heterogeneity and Complex Cell-Cell Communication Networks in the Mouse Cornea.

Purpose: To generate a single-cell RNA-sequencing (scRNA-seq) map and construct cell-cell communication networks of mouse corneas. Methods: C57BL/6 mouse corneas were dissociated to single cells and subjected to scRNA-seq. Cell populations were clustered and annotated for bioinformatic analysis using the R package "Seurat." Differential expression patterns were validated and spatially mapped with whole-mount immunofluorescence staining. Global intercellular signaling networks were constructed using CellChat. Results: Unbiased clustering of scRNA-seq transcriptomes of 14,732 cells from 40 corneas revealed 17 cell clusters of six major cell types: nine epithelial cell, three keratocyte, two corneal endothelial cell, and one each of immune cell, vascular endothelial cell, and fibroblast clusters. The nine epithelial cell subtypes included quiescent limbal stem cells, transit-amplifying cells, and differentiated cells from corneas and two minor conjunctival epithelial clusters. CellChat analysis provided an atlas of the complex intercellular signaling communications among all cell types. Conclusions: We constructed a complete single-cell transcriptomic map and the complex signaling cross-talk among all cell types of the cornea, which can be used as a foundation atlas for further research on the cornea. This study also deepens the understanding of the cellular heterogeneity and heterotypic cell-cell interaction within corneas.

The cellular responses of corneal fibroblasts to cyclic stretching loads.

Mechanical signaling plays a crucial role in maintaining extracellular matrix (ECM) homeostasis in various structures. In this study, we investigated the responses of corneal fibroblasts to cyclic stretching loads using an in vitro cell culture system. Bovine corneal fibroblasts were cultured and subjected to equibiaxial cyclic strain of 15% for 72 h at a frequency of 0.25 Hz, with bovine skin fibroblasts used as a comparison. We explored various cellular behaviors, including morphological changes, cell proliferation, and metabolism in response to mechanical stretching loads. The expression of genes, protein secretion, and enzymatic activity for several major metalloproteinases was also determined through Q-PCR, Western blot, and gel zymography. Additionally, we investigated the involvement of mitogen-activated protein kinases (MAPKs) signaling pathways in the corneal fibroblasts when subjected to mechanical stimuli. Our findings revealed that, compared to skin fibroblasts, corneal fibroblasts were reluctant to morphological changes in response to a prolonged (72 h) and high-amplitude (15% of strain) cyclic stretching load. However, cyclic stretching loads stimulated the upregulation of MMP-2 expression in corneal fibroblasts via the MAPK signaling pathways involving extracellular signal-regulated kinase and p38. Together with a lack of upregulation in type I collagen expression, our results indicate the induction of the ECM degradation process in corneal fibroblasts in response to cyclic stretching. These findings emphasize the mechanoresponsive nature of corneal fibroblasts and shed light on the potential impact of intense mechanical stress on the cornea in both normal and pathological conditions such as keratoconus, providing valuable insights for understanding corneal mechanobiology.

Depilatory laser miniaturizes hair by inducing bystander dermal papilla cell necrosis through thermal diffusion.

OBJECTIVES: Depilatory laser targeting melanin has been widely applied for the treatment of hypertrichosis. Both selective photothermolysis and thermal diffusion have been proposed for its effect, but the exact mechanism of permanent hair reduction remains unclear. In this study, we explore the role of thermal diffusion in depilatory laser-induced permanent hair loss and determine whether nonpigmented cells are injured by thermal diffusion. MATERIALS AND METHODS: C57BL/6 mice in anagen and telogen were treated with alexandrite laser (wavelength 755 nm, pulse duration 3 milliseconds, fluence 12 J/cm2 , spot size 12 mm), respectively. Histological analysis, terminal deoxynucleotidyl transferase dUTP nick-end labeling assay, and transmission electron microscopic imaging were employed to evaluate the injury to hair follicle (HF) cells. The proliferation status of HF cells was examined by 5-bromo-2'-deoxyuridine pulse labeling. The number of HF stem cells was quantified by fluorescence-activated cell sorting. The size of the regenerated hair was determined by measuring its length and width. RESULTS: We found that irradiating C57BL/6 mice in anagen with alexandrite laser led to hair miniaturization in the next anagen. In addition to thermal disruption of melanin-containing cells in the precortex region, we also detected necrosis of the adjacent nonpigmented dermal papilla cells due to thermal diffusion. Dermal papilla cells decreased by 24% after laser injury, while the number of bulge stem cells remained unchanged. When the laser was delivered to telogen HFs where no melanin was present adjacent to the dermal papilla, thermal necrosis and cell reduction were not detected in the dermal papilla and no hair miniaturization was observed. CONCLUSION: Our results suggest that depilatory laser miniaturizes hair by inducing thermal necrosis of dermal papilla cells due to secondary thermal diffusion from melanin-containing precortex cells in the anagen hair bulbs.

Targeting ER protein TXNDC5 in hepatic stellate cell mitigates liver fibrosis by repressing non-canonical TGFß signalling.

BACKGROUND AND OBJECTIVES: Liver fibrosis (LF) occurs following chronic liver injuries. Currently, there is no effective therapy for LF. Recently, we identified thioredoxin domain containing 5 (TXNDC5), an ER protein disulfide isomerase (PDI), as a critical mediator of cardiac and lung fibrosis. We aimed to determine if TXNDC5 also contributes to LF and its potential as a therapeutic target for LF. DESIGN: Histological and transcriptome analyses on human cirrhotic livers were performed. Col1a1-GFPTg , Alb-Cre;Rosa26-tdTomato and Tie2-Cre/ERT2;Rosa26-tdTomato mice were used to determine the cell type(s) where TXNDC5 was induced following liver injury. In vitro investigations were conducted in human hepatic stellate cells (HSCs). Col1a2-Cre/ERT2;Txndc5fl/fl (Txndc5cKO ) and Alb-Cre;Txndc5fl/fl (Txndc5Hep-cKO ) mice were generated to delete TXNDC5 in HSCs and hepatocytes, respectively. Carbon tetrachloride treatment and bile duct ligation surgery were employed to induce liver injury/fibrosis in mice. The extent of LF was quantified using histological, imaging and biochemical analyses. RESULTS: TXNDC5 was upregulated markedly in human and mouse fibrotic livers, particularly in activated HSC at the fibrotic foci. TXNDC5 was induced by transforming growth factor ß1 (TGFß1) in HSCs and it was both required and sufficient for the activation, proliferation, survival and extracellular matrix production of HSC. Mechanistically, TGFß1 induces TXNDC5 expression through increased ER stress and ATF6-mediated transcriptional regulation. In addition, TXNDC5 promotes LF by redox-dependent JNK and signal transducer and activator of transcription 3 activation in HSCs through its PDI activity, activating HSCs and making them resistant to apoptosis. HSC-specific deletion of Txndc5 reverted established LF in mice. CONCLUSIONS: ER protein TXNDC5 promotes LF through redox-dependent HSC activation, proliferation and excessive extracellular matrix production. Targeting TXNDC5, therefore, could be a potential novel therapeutic strategy to ameliorate LF.

Endoplasmic reticulum protein TXNDC5 promotes renal fibrosis by enforcing TGF-ß signaling in kidney fibroblasts.

Renal fibrosis, a common pathological manifestation of virtually all types of chronic kidney disease (CKD), often results in diffuse kidney scarring and predisposes to end-stage renal disease. Currently, there is no effective therapy against renal fibrosis. Recently, our laboratory identified an ER-resident protein, thioredoxin domain containing 5 (TXNDC5), as a critical mediator of cardiac fibrosis. Transcriptome analyses of renal biopsy specimens from patients with CKD revealed marked TXNDC5 upregulation in fibrotic kidneys, suggesting a potential role of TXNDC5 in renal fibrosis. Employing multiple fluorescence reporter mouse lines, we showed that TXNDC5 was specifically upregulated in collagen-secreting fibroblasts in fibrotic mouse kidneys. In addition, we showed that TXNDC5 was required for TGF-ß1-induced fibrogenic responses in human kidney fibroblasts (HKFs), whereas TXNDC5 overexpression was sufficient to promote HKF activation, proliferation, and collagen production. Mechanistically, we showed that TXNDC5, transcriptionally controlled by the ATF6-dependent ER stress pathway, mediated its profibrogenic effects by enforcing TGF-ß signaling activity through posttranslational stabilization and upregulation of type I TGF-ß receptor in kidney fibroblasts. Using a tamoxifen-inducible, fibroblast-specific Txndc5 knockout mouse line, we demonstrated that deletion of Txndc5 in kidney fibroblasts mitigated the progression of established kidney fibrosis, suggesting the therapeutic potential of TXNDC5 targeting for renal fibrosis and CKD.

Fibroblast-enriched endoplasmic reticulum protein TXNDC5 promotes pulmonary fibrosis by augmenting TGFß signaling through TGFBR1 stabilization.

Pulmonary fibrosis (PF) is a major public health problem with limited therapeutic options. There is a clear need to identify novel mediators of PF to develop effective therapeutics. Here we show that an ER protein disulfide isomerase, thioredoxin domain containing 5 (TXNDC5), is highly upregulated in the lung tissues from both patients with idiopathic pulmonary fibrosis and a mouse model of bleomycin (BLM)-induced PF. Global deletion of Txndc5 markedly reduces the extent of PF and preserves lung function in mice following BLM treatment. Mechanistic investigations demonstrate that TXNDC5 promotes fibrogenesis by enhancing TGFß1 signaling through direct binding with and stabilization of TGFBR1 in lung fibroblasts. Moreover, TGFß1 stimulation is shown to upregulate TXNDC5 via ER stress/ATF6-dependent transcriptional control in lung fibroblasts. Inducing fibroblast-specific deletion of Txndc5 mitigates the progression of BLM-induced PF and lung function deterioration. Targeting TXNDC5, therefore, could be a novel therapeutic approach against PF.

A Custom Multiphoton Microscopy Platform for Live Imaging of Mouse Cornea and Conjunctiva.

Conventional histological analysis and cell culture systems are insufficient to simulate in vivo physiological and pathological dynamics completely. Multiphoton microscopy (MPM) has become one of the most popular imaging modalities for biomedical study at cellular levels in vivo, advantages include high resolution, deep tissue penetration and minimal phototoxicity. We have designed an MPM imaging platform with a customized mouse eye holder and a stereotaxic stage for imaging ocular surface in vivo. Dual fluorescent protein reporter mouse enables visualization of cell nuclei, cell membranes, nerve fibers, and capillaries within the ocular surface. In addition to multiphoton fluorescence signals, acquiring second harmonic generation (SHG) simultaneously allows for the characterization of collagenous stromal architecture. This platform can be employed for intravital imaging with accurate positioning across the entire ocular surface, including cornea and conjunctiva.

Long-Term Intravital Imaging of the Cornea, Skin, and Hair Follicle by Multiphoton Microscope.

Multiphoton microscopy allows long-term direct visualization of cells in live animals due to its low photodamage. When coupled with fluorescence protein targeting and second harmonic generation signals from natural collagen as contrast, multiphoton microscopy enables intravital tracing of cells while providing structural information from the extracellular matrix. Compared with conventional histological analysis, it can bring new insight into the cell dynamics in stem cell research. Here, we demonstrate cell imaging and tracing at a single cell resolution in the cornea, skin, and hair follicles using multiphoton microscopy in transgenic mice of which specific cell populations are tagged with fluorescent proteins.

Intravital multiphoton microscopic imaging platform for ocular surface imaging.

The purpose of this study is to provide an intravital noninvasive multiphoton microscopic platform for long-term ocular imaging in transgenic fluorescent mice with subcellular resolution. A multiphoton microscopic system with tunable laser output was employed. We designed a mouse holder incorporated with stereotaxic motorized stage for in vivo three-dimensional imaging of ocular surface in 3 transgenic mouse line with fluorescent protein (FP) expression to visualize distinct structures. With our imaging platform and the expression of FPs, we obtained the three-dimensional images across the whole cornea from epithelium to endothelium and in conjunctiva with subcellular resolution in vivo. Specified EGFP expression in corneal epithelium of K5-H2B-EGFP mice helped to identify both corneal and limbal epithelial cells while ubiquitous nuclear FP expression in R26R-GR mice allowed us to visualized nuclei of all cell types. Universal membrane-localized FP in mT/mG mice outlined all cell boundaries, nerve fibers, and capillaries. The simultaneously collected second harmonic generation signals from collagenous stroma provided architectural contrast. Time-lapsed recording enabled monitoring the mitotic activity of corneal epithelial cells and limbal epithelial cells. We developed an intravital multiphoton microscopic stereotaxic imaging platform and showed that, by incorporating FP-expressing transgenic mice, this platform enables in vivo 4-dimensional ophthalmic study at subcellular resolution.

Chidamide-induced ROS accumulation and miR-129-3p-dependent cell cycle arrest in non-small lung cancer cells.

BACKGROUND: Epigenetic therapy is a promising popular treatment modality for various cancers. Histone modification and miRNA should not be underestimated in lung cancer. This study aimed to investigate whether chidamide, a histone deacetylase inhibitor (HDACi), which inhibits telomerase activity and induces cell cycle arrest, influences ROS and miRNA production in non-small cell lung cancer (NSCLC) cells. METHODS: H1355 and A549 were treated with chidamide. The analysis of DNA content was measured by FACSCalibur equipped with a 488 nm laser. H1355 cells were transfected with miR-129-3p mimic by Lipofectamine2000. Telomerase activity was performed on the telomeric repeat amplification protocol (TRAP) assay. Detection of thymidylate synthase (TS), p21, p53, pRB, and ß-actin, were performed by western blot analysis. RESULTS: Our data showed that expression of TS, p21, and pRB were altered in the presence of chidamide by PCR and western blot. Using BrdU-incorporation analysis, we found that chidamide induced G1 arrest through the regulation of the TS gene by miR-129-3p. Chidamide was shown to suppress telomerase activity in the TRAP assay and reduced the expression of human telomerase reverse transcriptase (hTERT) by PCR and q-PCR in H1355 and A549 cells. Chidamide increased the generation of reactive oxygen species (ROS) by flow cytometry. N-acetyl cysteine (NAC), a ROS scavenger, attenuated chidamide-induced telomerase activity inhibition. CONCLUSION: Chidamide repressed telomerase activity through ROS accumulation and cell cycle arrest by miR-129-3p upregulation in both H1355 and A549 cells. This is the first study to demonstrate that chidamide induces miR-129-3p upregulation and ROS accumulation, leading to cell cycle arrest.

Chidamide alleviates TGF-ß-induced epithelial-mesenchymal transition in lung cancer cell lines.

Transforming growth factor-ß (TGF-ß)-induced epithelial-mesenchymal transition is a critical process in the initiation of metastasis of various types of cancer. Chidamide is a class I histone deacetylase inhibitor with anti-tumor activity. This study investigated the effects of chidamide on TGF-ß-mediated suppression of E-cadherin expression in adenocarcinomic lung epithelial cells and the molecular mechanisms involved in these effects. Western blot analysis, confocal microscopy, Quantitative methyl-specific PCR and bisulfite sequencing were used to evaluate the effects of different treatments on chidamide ameliorating TGF-ß induced-E-cadherin loss. H3 acetylation binding to the promoter of E-cadherin was detected by chromatin immunoprecipitations (CHIP). We found that chidamide reduced the level of lung cancer cell migration observed using a Boyden chamber assay (as an indicator of metastatic potential). Chidamide inhibited TGF-ß-induced SMAD2 phosphorylation and attenuated TGF-ß-induced loss of E-cadherin expression in lung cancer cells by Western blotting and confocal microscopy, respectively. Quantitative methyl-specific PCR and bisulfite sequencing revealed that TGF-ß-enhanced E-cadherin promoter methylation was ameliorated in cells treated with chidamide. We demonstrated that histone H3 deacetylation within the E-cadherin promoter was required for TGF-ß-induced E-cadherin loss; cell treatment with chidamide increased the H3 acetylation detected by CHIP. Taken together, our results demonstrate that TGF-ß suppressed E-cadherin expression by regulating promoter methylation and histone H3 acetylation. Chidamide significantly enhanced E-cadherin expression in TGF-ß-treated cells and inhibited lung cancer cell migration. These findings indicate that chidamide has a potential therapeutic use due to its capacity to prevent cancer cell metastasis.

Enhancing hair follicle regeneration by nonablative fractional laser: Assessment of irradiation parameters and tissue response.

BACKGROUND AND OBJECTIVE: Identification of methods to enhance anagen entry can be helpful for alopecia. Recently, nonablative laser has been proposed as a potential treatment for alopecia. However, how the laser parameters affect stem cell activity, hair cycles and the associated side effects have not been well characterized. Here we examine the effects of irradiation parameters of 1,550-nm fractional laser on hair cycles. STUDY DESIGN/MATERIALS AND METHODS: The dorsal skin of eight-week-old female C57BL/6 mice with hair follicles in synchronized telogen was shaved and irradiated with a 1,550-nm fractional erbium-glass laser (Fraxel RE:STORE (SR1500) Laser System, Solta Medical, U.S.A.) with varied beam energies (5-35 mJ) and beam densities (500-3500 microthermal zones/cm(2) ). The cutaneous changes were evaluated both grossly and histologically. Hair follicle stem cell activity was detected by BrdU incorporation and changes in gene expression were quantified by real-time PCR. RESULTS: Direct thermal injury to hair follicles could be observed early after irradiation, especially at higher beam energy. Anagen induction in the irradiated skin showed an all-or-non change. Anagen induction and ulcer formation were affected by the combination of beam energy and density. The lowest beam energy of 5 mJ failed to promote anagen entry at all beam densities tested. As beam energy increased from 10 mJ to 35 mJ, we found a decreasing trend of beam density that could induce anagen entry within 7-9 days with activation of hair follicle stem cells. Beam density above the pro-regeneration density could lead to ulcers and scarring followed by anagen entry in adjacent skin. Analysis of inflammatory cytokines, including TNF-α, IL-1ß, and IL-6, revealed that transient moderate inflammation was associated with anagen induction and intense prolonged inflammation preceded ulcer formation. CONCLUSION: To avoid side effects of hair follicle injury and scarring, appropriate combination of beam energy and density is required. Parameters outside the therapeutic window can result in either no anagen promotion or ulcer formation.

Visible red light enhances physiological anagen entry in vivo and has direct and indirect stimulative effects in vitro.

BACKGROUND AND OBJECTIVES: Hair follicles are located at the interface of the external and internal environments and their cycling has been shown to be regulated by intra- and extra-follicular factors. The aim of this study is to examine whether or how hair follicles respond to visible light. STUDY DESIGN/MATERIALS AND METHODS: We examined the effect of 3 mW red (630 nm, 1 J/cm(2)), 2 mW green (522 nm, 1 J/cm(2)), and 2 mW blue light (463 nm, 1 J/cm(2)) on telogen in mice for 3 weeks. The photobiologic effects of red light on cell proliferation of outer root sheath keratinocytes and dermal papilla cells were studied in vitro. RESULTS: We found that red light accelerated anagen entry faster than green and blue light in mice. Red light irradiation stimulated the proliferation of both outer root sheath keratinocytes and dermal papilla cells in a dose-dependent manner by promoting cell cycle progression. This stimulative effect was mediated via extracellular signal-regulated kinase phosphorylation in both cells. In a co-culture condition, dermal papilla cells irradiated by red light further enhanced keratinocyte proliferation, suggesting enhanced epithelial-mesenchymal interaction. In search for factors that mediated this paracrine effect, we found fibroblast growth factor 7 was upregulated in both mRNA and protein levels. The stimulative paracrine effect on keratinocytes was significantly inhibited by neutralizing antibody against fibroblast growth factor 7. CONCLUSIONS: These results suggest that hair follicles respond to visible light in vivo. Red light may promote physiological telogen to anagen transition by directly stimulating outer root sheath keratinocytes and indirectly by enhancing epithelial-mesenchymal interaction in vitro.

Air embolism complicating computed tomography-guided core needle biopsy of the lung.

Computed tomography-guided transthoracic lung biopsy is a common clinical procedure for the diagnosis of a broad range of pulmonary pathological conditions. Mild self-limiting pneumothorax and haemoptysis are common complications of this procedure. Air embolism is a potentially life-threatening but extremely rare complication. We report a case of an air embolism in the left ventricle of the heart that developed after a computed tomography-guided percutaneous needle biopsy of the lung. The patient did not exhibit cardiac or cerebral symptoms after conservative treatment. The patient underwent a successful left thoracotomy with a lobectomy of the left lower lobe of the lung 5 days after the biopsy and recovered uneventfully.