Effectiveness and safety of baricitinib in patients with moderate-to-severe refractory alopecia areata in real world: An open-label, single-center study.

PURPOSE: Baricitinib is a small-molecular drug that selectively inhibits the Janus Kinase (JAK) 1 and 2. However, it showed various efficiency and safety in treating moderate-to-severe alopecia areata (AA). This study was to describe the real-world effectiveness of baricitinib in treating moderate-to-severe refractory AA. METHODS: Patients who were affected by moderate-to-severe AA and reported no shrinkage in the alopecia area after 6 months of conventional treatment were enrolled in the retrospective study. The patients were treated with baricitinib orally for at least 24 weeks. The severity of alopecia was evaluated at the end of 4, 12, and 24 weeks of treatment. RESULTS: The 32 patients included 23 females and nine males, with a median duration of AA of 14.5 months. Among them, 28 patients received baricitinib 2 mg per day for 24 weeks while the other four patients increased the daily dose from 2 to 4 mg after the first 12 weeks due to the unobvious hair restoration. SALT value showed a significant decrease from baseline at week 12 and 24 (64.45 [44.68-100.00] vs. 26.80 [13.40-62.32], p < 0.0001 and 64.45 [44.68-100] vs. 9.40 [4.85-34.95], p < 0.0001). After 24 weeks of treatment, 50% of patients had an improvement of ≥2 points in IGA scores from the baseline, and IGA scores of 68.75% of patients were less than 2. CONCLUSION: This 24-week research showed that baricitinib had favorable clinical efficacy and safety in treating moderate-to-severe AA, which is worthy of attention and expectation.

The role of telomerase in hair growth and relevant disorders: A review.

BACKGROUND: Hair diseases may present with hair loss, hirsutism, hair melanin abnormalities and other manifestations. Hair follicles are known as mini-organs that undergo periodic remodeling, and their constant regeneration in vivo reflects interesting anti-aging functions. Telomerase prevents cellular senescence by maintaining telomere length, but its excessive proliferation in cancer cells may also induce cancer. However, the effects of telomerase in hair growth have rarely been reported. METHODS: In this study, we reviewed the role of telomerase in hair growth and the effects of hair disorders through literature search and analysis. RESULTS: There is growing evidence that telomerase plays an important role in maintaining hair follicle function and proliferation. Changes in telomerase levels in hair follicles have also been found in a variety of hair disorders. CONCLUSION: Telomerase plays a positive role in hair growth and is expected to become a new target for the treatment of alopecia or other hair diseases in the future.

The Efficacy and Safety of Oral and Topical Spironolactone in Androgenetic Alopecia Treatment: A Systematic Review.

Introduction: Androgenetic alopecia (AGA) has negative impacts on both men and women in terms of appearance and mental stress. Spironolactone is a synthetic aldosterone receptor antagonist known to stimulate hair growth and has been widely used by dermatologists to treat AGA. Objective: To conduct a systematic review evaluating the efficacy and safety of topical and oral spironolactone in AGA treatment. Methods: We searched PubMed, Embase, the Cochrane Library, and the Web of Science until October 23rd, 2022, for human studies evaluating the efficacy of spironolactone for the treatment of AGA, regardless of doses and routes. Results: We retrieved 784 papers and ultimately 7 articles matched our inclusion criteria and comprised 618 AGA patients (65 men, 553 women), 414 of them received spironolactone treatment. Oral spironolactone doses ranged from 25mg to 200mg daily, with the vast majority between 80mg and 110 mg. Dosage forms for topical spironolactone use include gels of 1% and solutions of 5% twice daily. Both oral and topical spironolactone have been shown efficacy for alopecia recovery, but topical use has significantly fewer side effects and is suitable for any gender. It showed better efficacy in combination with other therapies such as oral or topical minoxidil compared with monotherapy. Conclusion: Spironolactone is an effective and safe treatment of androgenic alopecia which can enhance the efficacy when combined with other conventional treatments such as minoxidil. Topical spironolactone is safer than oral administration and is suitable for both male and female patients, and is expected to become a common drug for those who do not have a good response to minoxidil. Furthermore, more high-quality clinical randomized controlled studies should be performed.

Transcription factor FOXC1 positively regulates SFRP1 expression in androgenetic alopecia.

Androgenetic alopecia (AGA) is the most common type of hair loss dysfunction. Secreted frizzled related protein 1 (SFRP1) is found to be associated with hair loss, but its role in AGA and the regulation mechanism of its transcription level is unclear. The aim of our study is to explore the expression of SFRP1 in AGA samples and its transcriptional mechanism. Male frontal and occipital scalp hair follicles from AGA patients were collected, and human dermal papilla cells (DPCs) were isolated and cultured. SFRP1 gene was cloned and constructed into recombinant plasmids to perform dual-luciferase reporter assay. Transcription factor binding sites were predicted through the Jaspar website and further confirmed by the chromatin immunoprecipitation (ChIP) assay. Expression of genes in DPCs was determined by immunofluorescence (IF) staining, quantitative real-time PCR (qRT-PCR) and western blotting. Our findings showed that SFRP1 was highly expressed in DPCs of AGA patients. The core promoter region of SFRP1 was from -100 to +50 bp and was found to be positively regulated by forkhead box C1 (FOXC1), a transcription factor related to hair growth, both at mRNA and protein level in DPCs. Our study suggests that FOXC1 plays an important role in regulating SFRP1 transcription, which may provide new insights into the development of therapeutic strategies for the treatment of AGA.

LOXL2 from human amniotic mesenchymal stem cells accelerates wound epithelialization by promoting differentiation and migration of keratinocytes.

In this study, we identified wound healing-related proteins secreted by human amniotic epithelial cells (hAECs) and human amniotic mesenchymal stem cells (hAMSCs). We observed increased migration and reduced proliferation and differentiation when keratinocytes were co-cultured in media conditioned by hAECs (hAECs-CM) and hAMSCs (hAMSCs-CM). Label-free mass spectrometry and bioinformatic analyses of the hAECs-CM and hAMSCs-CM proteome revealed several proteins associated with wound healing, angiogenesis, cellular differentiation, immune response and cell motility. The levels of the proteins related to wound healing, including CTHRC1, LOXL2 and LGALS1, were significantly higher in hAMSCs-CM than hAECs-CM. LOXL2 significantly enhanced in vitro keratinocyte migration and differentiation compared to CTHRC1 and LGALS1. Moreover, LOXL2 enhanced keratinocyte migration and differentiation by activating the JNK signaling pathway. We observed significant reduction in the in vitro migration and differentiation of keratinocytes when co-cultured with medium conditioned by LOXL2-silenced hAMSCs and when treated with 10 µM SP600125, a specific JNK inhibitor. Treatment with hAMSCs-CM and LOXL2 significantly accelerated wound healing in the murine skin wound model. These findings show that LOXL2 promotes wound healing by inducing keratinocyte migration and differentiation via a JNK signaling pathway.

Novel Mouse miRNA Chr13_novelMiR7354-5p Improves Bone-Marrow-Derived Mesenchymal Stem Cell Differentiation into Insulin-Producing Cells.

MicroRNAs (miRNAs) that play key roles in the generation of insulin-producing cells from stem cells provide a cell-based approach for insulin replacement therapy. In this study, we used next-generation sequencing to detect the miRNA expression profile of normal mouse pancreatic ß cells, non-ß cells, bone marrow mesenchymal stem cells (BM-MSCs), and adipose-derived stem cells (ADSCs) and determined relative miRNA expression levels in mouse pancreatic ß cells. After the novel mouse miRNA candidates were identified using miRDeep 2.0, we found that Chr13_novelMiR7354-5p, a novel miRNA candidate, significantly promoted the differentiation of BM-MSCs into insulin-producing cells in vitro. Furthermore, Chr13_novelMiR7354-5p-transfected BM-MSCs reversed hyperglycemia in streptozotocin (STZ)-treated diabetic mice. In addition, bioinformatics analyses, a luciferase reporter assay, and western blotting demonstrated that Chr13_novelMiR7354-5p targeted Notch1 and Rbpj. Our results provide compelling evidence of the existence of 65 novel mouse miRNA candidates and present a new treatment strategy to generate insulin-producing cells from stem cells.

Conditioned medium derived from human amniotic stem cells delays H2O2­induced premature senescence in human dermal fibroblasts.

Stem cells derived from human amniotic membrane (hAM) are promising targets in regenerative medicine. A previous study focused on human amniotic stem cells in skin wound and scar­free healing. The present study aimed to investigate whether hydrogen peroxide (H2O2)­induced senescence of human dermal fibroblasts (hDFs) was influenced by the anti­aging effect of conditioned medium (CdM) derived from human amniotic stem cells. First, the biological function of two types of amniotic stem cells, namely human amniotic epithelial cells (hAECs) and human amniotic mesenchymal stem cells (hAMSCs), on hDFs was compared. The results of cell proliferation and wound healing assays showed that CdM promoted cell proliferation and migration. In addition, CdM from hAECs and hAMSCs significantly promoted proliferation of senescent hDFs induced by H2O2. These results indicated that CdM protects cells from damage caused by H2O2. Treatment with CdM decreased senescence­associated ß­galactosidase activity and improved the entry of proliferating cells into the S phase. Simultaneously, it was found that CdM increased the activity of superoxide dismutase and catalase and decreased malondialdehyde by reducing H2O2­induced intracellular reactive oxygen species production. It was found that CdM downregulated H2O2­stimulated 8­hydroxydeoxyguanosine and γ­H2AX levels and decreased the expression of the senescence­associated proteins p21 and p16. In conclusion, the findings indicated that the paracrine effects derived from human amniotic stem cells aided delaying oxidative stress­induced premature senescence.

Human Novel MicroRNA Seq-915_x4024 in Keratinocytes Contributes to Skin Regeneration by Suppressing Scar Formation.

Early in gestation, wounds in fetal skin heal by regeneration, in which microRNAs play key roles. Seq-915_x4024 is a novel microRNA candidate confirmed by deep sequencing and mirTools 2.0. It is highly expressed in fetal keratinocytes during early gestation. Using an in vitro wound-healing assay, Transwell cell migration assay, and MTS proliferation assay, we demonstrated that keratinocytes overexpressing seq-915_x4024 exhibited higher proliferative activity and the ability to promote fibroblast migration and fibroblast proliferation. These characteristics of keratinocytes are the same biological behaviors as those of fetal keratinocytes, which contribute to skin regeneration. In addition, seq-915_x4024 suppressed the expression of the pro-inflammatory markers TNF-α, IL-6, and IL-8 and the pro-inflammatory chemokines CXCL1 and CXCL5. We also demonstrated that seq-915_x4024 regulates TGF-ß isoforms and the extracellular matrix. Moreover, using an in vivo wound-healing model, we demonstrated that overexpression of seq-915_x4024 in keratinocytes suppresses inflammatory cell infiltration and scar formation. Using bioinformatics analyses, luciferase reporter assays, and western blotting, we further demonstrated that Sar1A, Smad2, TNF-α, and IL-8 are direct targets of seq-915_x4024. Furthermore, the expression of phosphorylated Smad2 and Smad3 was reduced by seq-915_x4024. Seq-915_x4024 could be used as an anti-fibrotic factor for the treatment of wound healing.

The differentiation of human MSCs derived from adipose and amniotic tissues into insulin-producing cells, induced by PEI@Fe3O4 nanoparticles-mediated NRSF and SHH silencing.

Type 1 diabetes involves the immunologically mediated destruction of insulin­producing cells (IPCs) in the pancreatic islet. Mesenchymal stem cells (MSCs) have the ability to differentiate into IPCs and have become the most promising means for diabetes therapy. The present study demonstrated that human adipose­derived stem cells (hADSCs) and human amniotic MSCs (hAMSCs) are able to differentiate into functional IPCs by knocking down neuronal restrictive silencing factor (NRSF) and Sonic hedgehog (SHH). In the current study, PEI@Fe3O4 nanoparticles (NPs) were used to deliver NRSF small interfering (si)RNA and SHH siRNA to hADSCs and hAMSCs. Following infection with PEI@Fe3O4 NPs containing NRSF siRNA and SHH siRNA, the MSCs were induced to differentiate into IPCs. Four specific genes for islet cells were expressed in the differentiated cells. These cells also produced and released insulin in a glucose­responsive manner. These findings indicated that hADSCs and hAMSCs may be induced to differentiate into IPCs via PEI@Fe3O4 NP­mediated NRSF and SHH silencing.

The Novel miRNA N-72 Regulates EGF-Induced Migration of Human Amnion Mesenchymal Stem Cells by Targeting MMP2.

Human amnion mesenchymal stem cells (hAMSCs) are promising sources of stem cells in regenerative medicine. The migration stimulated by cytokines is critical for mesenchymal stem cells (MSCs)-based cytotherapy, while the regulatory mechanisms of EGF (epidermal growth factor)-induced hAMSC migration are largely unclear. Here, a novel miRNA N-72 (GenBank accession number: MH269369) has been discovered, and its function on EGF-induced migration in hAMSCs was investigated. High-purity hAMSCs were isolated and cultured in vitro, which were characterized by flow cytometry and trilineage differentiation. The N-72 located on chromosome three was conserved, and pri-N-72 owned the ability to form a stem-loop secondary structure, which was predicated by bioinformatic programs. The expression of mature N-72 was verified in several human cells including hAMSC by real-time PCR. In EGF-stimulated hAMSC, N-72 showed a significant reduction in a PI3K and p38 MAPK-dependent manner, and N-72 mimics transfection-inhibited EGF-induced migration, which was verified by scratch assay and transwell assay. Further, the predicated target gene MMP2 was proved to be a direct target of N-72 via luciferase reporter assay, real-time PCR, and Western blotting. The results that MMP2 silencing repressed hAMSC migration suggested MMP2 as a functional downstream target of N-72. In summary, we have discovered the novel N-72, and it was crucial for EGF-induced migration by targeting MMP2 in hAMSCs.

Repression of COUP-TFI Improves Bone Marrow-Derived Mesenchymal Stem Cell Differentiation into Insulin-Producing Cells.

Identifying molecular mechanisms that regulate insulin expression in bone marrow-derived mesenchymal stem cells (bmMSCs) can provide clues on how to stimulate the differentiation of bmMSCs into insulin-producing cells (IPCs), which can be used as a therapeutic approach against type 1 diabetes (T1D). As repression factors may inhibit differentiation, the efficiency of this process is insufficient for cell transplantation. In this study, we used the mouse insulin 2 (Ins2) promoter sequence and performed a DNA affinity precipitation assay combined with liquid chromatography-mass spectrometry to identify the transcription factor, chicken ovalbumin upstream promoter transcriptional factor I (COUP-TFI). Functionally, bmMSCs were reprogrammed into IPCs via COUP-TFI suppression and MafA overexpression. The differentiated cells expressed higher levels of genes specific for islet endocrine cells, and they released C-peptide and insulin in response to glucose stimulation. Transplantation of IPCs into streptozotocin-induced diabetic mice caused a reduction in hyperglycemia. Mechanistically, COUP-TFI bound to the DR1 (direct repeats with 1 spacer) element in the Ins2 promoter, thereby negatively regulating promoter activity. Taken together, the data provide a novel mechanism by which COUP-TFI acts as a negative regulator in the Ins2 promoter. The differentiation of bmMSCs into IPCs could be improved by knockdown of COUP-TFI, which may provide a novel stem cell-based therapy for T1D.

Upregulated unique long 16 binding protein 1 detected in preeclamptic placenta affects human extravillous trophoblast cell line (HTR-8/SVneo) invasion by modulating the function of uterine natural killer cells.

Well-controlled trophoblast invasion at the maternal-fetal interface is crucial for normal placentation and successful pregnancy, otherwise pathological conditions of pregnancy occur, such as preeclampsia. In previous studies, it has been demonstrated that unique long 16 binding protein (ULBP)1, a ligand for the natural-killer group (NKG)2D receptor on uterine natural killer (uNK) cells, is upregulated in the placenta in patients with preeclampsia. As they are present on the majority of the decidua, uNK have an important role in pregnancy. The aim of the present study was to determine the role of ULBP1 in trophoblast cell invasion, which is closely associated with the occurrence of preeclampsia. In the present study, ULBP1 expression levels in placentas collected after cesarean section from women with preeclampsia and normal pregnant women were determined by immunohistochemistry, reverse transcription-quantitative polymerase chain reaction and western blotting. The effects of ULBP1 on extravillous trophoblast cell line (HTR-8/SVneo) invasion mediated via uNK cells and the underlying mechanisms were investigated. mRNA and protein expression levels of ULBP1 were significantly upregulated (P<0.05) in preeclamptic placentas compared with normal controls. ULBP1 inhibited HTR-8/SVneo cells via the regulation of biological functions of uNK cells, including the downregulation of NKG2D expression on uNK cells and the stimulation of production of cytokines and chemokines that affect extravillous cytotrophoblast invasion by uNK cells. ULBP1 may have an important role in the pathophysiology of preeclampsia through the modification of biological functions of uNK cells, which may affect trophoblast invasion.

In vitro reprogramming of rat bmMSCs into pancreatic endocrine-like cells.

Islet transplantation provides curative treatments to patients with type 1 diabetes, but donor shortage restricts the broad use of this therapy. Thus, generation of alternative transplantable cell sources is intensively investigated worldwide. We previously showed that bone marrow-derived mesenchymal stem cells (bmMSCs) can be reprogrammed to pancreatic-like cells through simultaneously forced suppression of Rest/Nrsf (repressor element-1 silencing transcription factor/neuronal restrictive silencing factor) and Shh (sonic hedgehog) and activation of Pdx1 (pancreas and duodenal transcription factor 1). We here aimed to reprogram bmMSCs further along the developmental pathway towards the islet lineages by improving our previous strategy and by overexpression of Ngn3 (neurogenin 3) and NeuroD1 (neurogenic differentiation 1), critical regulators of the development of endocrine pancreas. We showed that compared to the previous protocol, the overexpression of only Pdx1 and Ngn3 reprogrammed bmMSCs into cells with more characteristics of islet endocrine lineages verified with bioinformatic analyses of our RNA-Seq datasets. These analyses indicated 2325 differentially expressed genes including those involved in the pancreas and islet development. We validated with qRT-PCR analysis selective genes identified from the RNA-Seq datasets. Thus, we reprogrammed bmMSCs into islet endocrine-like cells and advanced the endeavor to generate surrogate functional insulin-secreting cells.

MiR-378b Promotes Differentiation of Keratinocytes through NKX3.1.

MicroRNA (miRNA) is a kind of short non-coding RNA, involved in various cellular processes. During keratinocyte differentiation, miRNAs act as important regulators. In this study, we demonstrated by microarray assay that the expression of miR-378b significantly increased during keratinocytes differentiation. Our findings showed that miR-378b could inhibit proliferation, migration and differentiation in keratinocytes. Luciferase reporter assays showed that miR-378b directly target NKX3.1. Silencing of NKX3.1 could coincide with the effects of miR-24 overexpression. In conclusion, our results demonstrate miR-378b promote keratinocytes differentiation by targeting NKX3.1. Manipulation of miR-378b may afford a new strategy to clinic treatment of skin injury and repair.

miR-136 modulates TGF-ß1-induced proliferation arrest by targeting PPP2R2A in keratinocytes.

Keratinocytes proliferation is critical for the capacity to heal wounds and accumulating evidences have proved that dysregulation of microRNAs is involved in proliferation of keratinocytes. However, the molecular mechanisms remain to be completely elucidated. Here, we show that miR-136 was significantly decreased by TGF-ß1 treatment in HaCaT cells and normal human epidermal keratinocytes (NHEK), and it was a Smad3-dependent manner. By cell proliferation assay and cell cycle analysis, we found that reintroduction of miR-136 by transfection, as well as PPP2R2A silencing, counteracted TGF-ß-induced proliferation arrest in HaCaT cells. Further, PPP2R2A was verified as a direct target of miR-136 by dual-luciferase reporter assays and Western blotting. These data suggest that miR-136 may play an important role during TGF-ß1-induced proliferation arrest by targeting PPP2R2A in keratinocytes, which might represent a potential target for improving skin wound healing.

Phenotypic and functional modulation of 20-30 year old dermal fibroblasts by mid- and late-gestational keratinocytes in vitro.

Fetal wound healing occurs rapidly and without scar formation early in gestation, but the mechanisms underlying this scarless healing are poorly understood. This study explores the phenotypic and functional modulation of 20-30 year old dermal fibroblasts by mid- and late-gestational keratinocytes (KCs) in vitro. Human KCs of different gestational ages were isolated, characterized, and co-cultured with human 20-30 year old fibroblasts. Gene expression and protein levels of TGF-ß family members, precollagen, collagen, matrix metalloproteinases (MMPs), and the tissue inhibitors of metalloproteinases (TIMPs) were measured in the fibroblasts. Mid-gestational KCs promoted faster proliferation and migration of fibroblasts than late-gestational KCs. Additionally, significant differences in gene expression and protein levels of some markers were observed in fibroblasts co-cultured with mid- or late-gestational KCs. Fibroblasts co-cultured with mid-gestational KCs for 48 h exhibited downregulated gene expression of precollagen 1, collagen 1, TGF-ß1, TGF-ß2, TIMP-2 and TIMP-3, while precollagen 3, collagen 3, TGF-ß3, and MMP-1, -2, -3, -9 and -14 were upregulated. In contrast, late-gestational KCs exhibited downregulated TIMP-1, TIMP-2 and TIMP-3 levels, while collagen 1, TGF-ß2, TGF-ß3, and MMP-2, -3, -9 and -14 were upregulated. Moreover, statistically significant differences in expression levels of precollagen 1, precollagen 3, collagen 1, TGF-ß1, -ß2, and -ß3, MMP-1, -3 and MMP-14, TIMP-1 and TIMP-2 were found between fibroblasts co-cultured with mid- and late-gestational KCs. Furthermore, cytokine levels of IL-1a and HB-EGF were found to be statistically different between conditioned medium from mid- and late-gestational KCs. Therefore, the gestational age of KCs appears to have an important effect on scarless wound healing in the human fetus.

In vitro reprogramming of rat bone marrow-derived mesenchymal stem cells into insulin-producing cells by genetically manipulating negative and positive regulators.

Islet cell replacement therapy represents the most promising approach for the cure of type 1 diabetes if autoimmunity to ß cells is under control. However, this potential is limited by a shortage of pancreas donors. To address the donor shortage problem, we determined whether bone marrow-derived mesenchymal stem cells (bmMSCs) can be directly reprogrammed to islet lineages by simultaneously forced suppression and over-expression of key regulator genes that play critical roles during pancreas development. Here, we report that rat bmMSCs were converted in vitro into insulin-producing cells by suppressing two-repressor genes repressor element-1 silencing transcription factor/neuronal restrictive silencing factor (Rest/Nrsf) and sonic hedgehog (Shh) and by over-expressing pancreas and duodenal transcription factor 1 (Pdx1). The reprogrammed bmMSCs expressed both genes and proteins specific for islet cells. These converted cells were capable of releasing insulin in a glucose-responsive manner. Our study suggests that bmMSCs may ultimately be reprogrammed to functional insulin-secreting cells.