Single-Cell Transcriptomics of Endothelial Cells in Upper and Lower Human Esophageal Squamous Cell Carcinoma.

Esophageal squamous cell carcinoma (ESCC) is a type of progressive and distant metastatic tumor. Targeting anti-angiogenic genes could effectively hinder ESCC development and metastasis, whereas ESCC locating on the upper or the lower esophagus showed different response to the same clinical treatment, suggesting ESCC location should be taken into account when exploring new therapeutic targets. In the current study, to find novel anti-angiogenic therapeutic targets, we identified endothelial cell subsets in upper and lower human ESCC using single-cell RNA sequencing (scRNA-seq), screened differentially expressed genes (DEGs), and performed gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. The results showed that common DEGs shared in the upper and the lower endothelial cells mainly are involved in vessel development, angiogenesis, and cell motility of endothelial cells by regulating PI3K-AKT, Rap1, Ras, TGF-beta, and Apelin signaling pathways. The critical regulatory genes were identified as ITGB1, Col4A1, Col4A2, ITGA6, LAMA4, LAMB1, LAMC1, VWF, ITGA5, THBS1, PDGFB, PGF, RHOC, and CTNNB1. Cell metabolism-relevant genes, e.g., MGST3, PNP, UPP1, and HYAL2 might be the prospective therapeutic targets. Furthermore, we found that DEGs only in the upper endothelial cells, such as MAPK3, STAT3, RHOA, MAPK11, HIF1A, FGFR1, GNG5, GNB1, and ARHGEF12, mainly regulated cell adhesion, structure morphogenesis, and motility through Phospholipase D, Apelin, and VEGF signaling pathways. Moreover, DEGs only in the lower endothelial cells, for instance PLCG2, EFNA1, CALM1, and RALA, mainly regulated cell apoptosis and survival by targeting calcium ion transport through Rap1, Ras, cAMP, Phospholipase D, and Phosphatidylinositol signaling pathways. In addition, the upper endothelial cells showed significant functional diversity such as cytokine-responsive, migratory, and proliferative capacity, presenting a better angiogenic capacity and making it more sensitive to anti-angiogenic therapy compared with the lower endothelial cells. Our study has identified the potential targeted genes for anti-angiogenic therapy for both upper and lower ESCC, and further indicated that anti-angiogenic therapy might be more effective for upper ESCC, which still need to be further examined in the future.

Dehydrocorydaline Accelerates Cell Proliferation and Extracellular Matrix Synthesis of TNFα-Treated Human Chondrocytes by Targeting Cox2 through JAK1-STAT3 Signaling Pathway.

Osteoarthritis (OA) causes severe degeneration of the meniscus and cartilage layer in the knee and endangers joint integrity and function. In this study, we utilized tumor necrosis factor α (TNFα) to establish in vitro OA models and analyzed the effects of dehydrocorydaline (DHC) on cell proliferation and extracellular matrix (ECM) synthesis in human chondrocytes with TNFα treatment. We found that TNFα treatment significantly reduced cell proliferation and mRNA and protein expression levels of aggrecan and type II collagen, but caused an increase in mRNA and protein expression levels of type I collagen, matrix metalloproteinase 1/13 (MMP1/13), and prostaglandin-endoperoxide synthase 2 (PTGS2, also known as Cox2) in human chondrocytes. DHC significantly promoted the cell activity of normal human chondrocytes without showing cytotoxity. Moreover, 10 and 20 µM DHC clearly restored cell proliferation, inhibited mRNA and protein expression levels of type I collagen, MMP 1/13, and Cox2, and further increased those of aggrecan and type II collagen in the TNFα-treated human chondrocytes. RNA transcriptome sequencing indicated that DHC could improve TNFα-induced metabolic abnormalities and inflammation reactions and inhibit the expression of TNFα-induced inflammatory factors. Furthermore, we found that the JAK1-STAT3 signaling pathway was confirmed to be involved in the regulatory effects of DHC on cell proliferation and ECM metabolism of the TNFα-treated human chondrocytes. Lastly, to explore the effects of DHC in vivo, we established an anterior cruciate ligament transection (ACLT)-stimulated rat OA model and found that DHC administration significantly attenuated OA development, inhibited the enzymatic hydrolysis of ECM, and reduced phosphorylated JAK1 and STAT3 protein expression in vivo after ACLT for 6 weeks. These results suggest that DHC can effectively relieve OA progression, and it has a potential to be utilized for the clinical prevention and therapy of OA as a natural small molecular drug.

m6A Modifications Play Crucial Roles in Glial Cell Development and Brain Tumorigenesis.

RNA methylation is a reversible post-transcriptional modification to RNA and has a significant impact on numerous biological processes. N 6-methyladenosine (m6A) is known as one of the most common types of eukaryotic mRNA methylation modifications, and exists in a wide variety of organisms, including viruses, yeast, plants, mice, and humans. Widespread and dynamic m6A methylation is identified in distinct developmental stages in the brain, and controls development of neural stem cells and their differentiation into neurons, glial cells such as oligodendrocytes and astrocytes. Here we summarize recent advances in our understanding of RNA methylation regulation in brain development, neurogenesis, gliogenesis, and its dysregulation in brain tumors. This review will highlight biological roles of RNA methylation in development and function of neurons and glial cells, and provide insights into brain tumor formation, and diagnostic and treatment strategies.

Lysyl oxidase suppresses the inflammatory response in anterior cruciate ligament fibroblasts and promotes tissue regeneration by targeting myotrophin via the nuclear factor-kappa B pathway.

Anterior cruciate ligament (ACL) regeneration is severely affected by the injury-induced overexpression of matrix metalloproteinases (MMPs) and downregulation of lysyl oxidase (LOX). Previous studies have focused on how the expression of MMPs and downregulation of LOX are physiologically balanced at injured sites for regenerating the ACL tissue, but the role of LOX in regulating cellular functions has not been investigated yet. Herein, we conducted an in vitro cellular experiment and unexpectedly found that exogenous LOX inhibited the expression of MMPs and inflammatory factors and recovered the cell growth; thus, LOX strongly inhibited the tumor necrosis factor-alpha (TNF-α)-induced inflammatory responses. In an in vivo animal model, LOX supplementation suppressed the expression of TNF-α in injured ACLs and promoted the recovery of the damaged tissues. RNA-sequencing-identified differentially expressed genes (DEGs) were highly enriched in the nuclear factor-kappa B (NF-κB), chemokine, cytokine-cytokine receptor interaction, Toll-like receptor, and TNF signaling pathways. Immunofluorescence tracing was employed to localise the exogenous LOX in the cell nucleus; the exogenous LOX indirectly suggests that it has other biological roles apart from the cross-linking of the extracellular matrix. Protein-protein interaction network analysis revealed the anti-inflammatory effect of LOX was alleviated by silencing the myotrophin (MTPN) expression, suggesting that LOX might interact with MTPN and regulate inflammation. Finally, this study suggests that LOX can inhibit the inflammatory response of ACL fibroblasts (ACLfs) and promote the recovery of the damaged ACL tissue through the MTPN-mediated NF-κB signaling pathway.

Effects of Bakuchiol on chondrocyte proliferation via the PI3K-Akt and ERK1/2 pathways mediated by the estrogen receptor for promotion of the regeneration of knee articular cartilage defects.

OBJECTIVES: Cartilaginous tissue degradation occurs because of the lack of survival of chondrocytes. Here, we ascertained whether bakuchiol (BAK) has the capability of activating chondrocyte proliferation. MATERIALS AND METHODS: The effect of BAK on the proliferation of rat chondrocytes at a concentration of 10 and 20 µmol/L was investigated. The molecular mechanisms involving target binding and signalling pathways were elucidated by RNA-sequencing, qPCR, molecular docking and Western blotting. Matrigel mixed with bakuchiol was implanted locally into rat knee articular cartilage defects to verify the activation of chondrocytes due to bakuchiol in vivo. RESULTS: Bakuchiol implantation resulted in the activation of rat chondrocyte proliferation in a dose-dependent manner. RNA-sequencing revealed 107 differentially expressed genes (DEGs) with 75 that were up-regulated and 32 that were down-regulated, indicating increased activation of the PI3K-Akt and cell cycle pathways. Activation of the phosphorylation of Akt, ERK1/2 and their inhibitors blocked the proliferative effect of bakuchiol treatment, confirming its direct involvement in these signal transduction pathways. Molecular docking and siRNA silencing revealed that estrogen receptor-α (ERα) was the target of bakuchiol in terms of its cell proliferative effect via PI3K activation. Two weeks after implantation of bakuchiol, the appearance and physiological structure of the articular cartilage was more integrated with abundant chondrocytes and cartilage matrix compared to that of the control. CONCLUSIONS: Bakuchiol demonstrated significant bioactivity towards chondrocyte proliferation via the PI3K-Akt and ERK1/2 pathways mediated by estrogen receptor activation and exhibited enhanced promotion of the remodelling of injured cartilage.

The Upregulation of Trophinin-Associated Protein (TROAP) Predicts a Poor Prognosis in Hepatocellular Carcinoma.

Purpose: Trophinin-associated protein (TROAP) is a cytoplasmic protein that plays a significant role in the processes of embryo transplantation and microtubule regulation. However, the relevant survival analysis and cancer progression analysis have not yet been reported. Methods: Eighteen matched pairs of tumor and adjacent non-tumor samples were evaluated to detect the TROAP mRNA level. Immunohistochemistry (IHC) was used to evaluate the TROAP expression in 108 hepatocellular carcinoma patients who underwent surgical resection. Meanwhile, data from the TCGA database was statistically evaluated. Results: In the present study, we detected a significant increase in the TROAP mRNA level in tumor tissues when compared with adjacent non-tumor tissues. Moreover, the upregulation of TROAP was associated with increased serum AFP and GGT; the greater the tumor number was, the larger the tumor size, differentiation grade, and cancer embolus in clinical analysis. In HCC patients, elevated TROAP expression in the primary tumor was positively related to clinical severity, such as poor overall survival and disease-free survival. In addition, both univariate and multivariate survival analysis validated that TROAP expression was a promising independent risk factor for overall survival and disease-free survival in HCC patients. Furthermore, the results derived from the analysis of data from the TCGA database were consistent with previous results. Altogether, our results show that TROAP is a novel crucial regulator of HCC progression and is a potential therapeutic biomarker for HCC patients. Conclusions: Elevated TROAP expression predicted a poor prognosis, and TROAP may serve as a potential biomarker for application in oncotherapy.

Psoralen Protects Chondrocytes, Exhibits Anti-Inflammatory Effects on Synoviocytes, and Attenuates Monosodium Iodoacetate-Induced Osteoarthritis.

Current study examined whether psoralen (PSO) exhibits anti-inflammatory responses, protection and activation of chondrocytes, and relieve osteoarthritis (OA). Rats chondrocytes and human synoviocytes were cultured in tumor necrosis factor-α (TNF-α) conditioned culture medium with/without PSO to test the cell morphologies and cytotoxicities in vitro. Cartilaginous extracellular matrix (ECM) and proliferative gene/protein expression levels were evaluated in chondrocytes. Meanwhile, matrix metalloproteinases (MMPs) and interleukins (ILs) gene/protein expression were analyzed in synoviocytes. SD rats of monosodium iodoacetate (MIA) induced OA model were used in order to assess the effects of PSO on attenuating degeneration of the articular cartilage in vivo. Results showed TNF-α conditioned culturing with/without PSO (1-100 µM) had no any toxicity on both the cell lines. PSO (10 µM) activated cartilaginous specific ECM expression along with up-regulation of proliferative genes at transcriptional levels. Interestingly, PSO significantly reversed TNF-α induced up-regulation of MMP13 and ILs synoviocytes in a dose-dependent manner (1 to 20 µM), while down-regulated cartilaginous ECM production. Following six weeks of PSO treatments to articular cartilage osteoarthritis, compared to MIA-induced group, the appearance and physiological structure of articular cartilage was more integrated with greatly organized chondrocytes and abundant cartilage matrix. In conclusion, PSO protects and activates chondrocytes, antagonizing the expression of MMPs and ILs secreted by synovial cells, and effectively attenuates MIA-induced OA.

MGF E peptide improves anterior cruciate ligament repair by inhibiting hypoxia-induced cell apoptosis and accelerating angiogenesis.

Severe hypoxic microenvironment endangers cell survival of anterior cruciate ligament (ACL) fibroblasts and is harmful to ACL repair and regeneration. In the current study, we explored the effects of mechanogrowth factor (MGF) E peptide on the hypoxia-induced apoptosis of ACL fibroblasts and relevant mechanisms. It demonstrated that severe hypoxia promoted hypoxia-inducible factor-1α (HIF-1α) expression and caused cell apoptosis of ACL fibroblasts through increasing caspase 3/7/9 messenger RNA (mRNA), cleaved caspase 3 and proapoptotic proteins expression levels but decreasing antiapoptotic proteins expression levels. Fortunately, MGF E peptide effectively protected ACL fibroblasts against hypoxia-induced apoptosis through regulating caspase 3/7/9 mRNA, cleaved caspase 3 and apoptosis-relevant proteins expression levels. Simultaneously, mitochondrial, @@@MEK-ERK1/2 (extracellular-signal-regulated kinase 1/2), and phosphoinositide-3-kinase-protein kinase B (PI3K-Akt) pathways were involved in MGF E peptide regulating hypoxia-induced apoptosis of ACL fibroblasts. In rabbit ACL rupture model, MGF E peptide also decreased HIF-1α expression levels, cell apoptosis, and facilitated cell proliferation. In addition, MGF could accelerate angiogenesis after ACL injury probably owing to its recruitment of proangiogenesis cells by stromal cell-derived factor 1α/CXCR4 axis and stimulation of vascular endothelial growth factor α expression level. In conclusion, our findings suggested that MGF E peptide could be utilized for ACL repair and regeneration and supplied experimental support for its application in clinical ACL treatment as a potential strategy.

ERK1/2 and Akt phosphorylation were essential for MGF E peptide regulating cell morphology and mobility but not proangiogenic capacity of BMSCs under severe hypoxia.

Severe hypoxia inhibits the adhesion and mobility of bone marrow-derived mesenchymal stem cells (BMSCs) and limits their application in bone tissue engineering. In this study, CoCl2 was used to simulate severe hypoxia and the effects of mechano-growth factor (MGF) E peptide on the morphology, adhesion, migration, and proangiogenic capacity of BMSCs under hypoxia were measured. It was demonstrated that severe hypoxia (500-µM CoCl2 ) significantly caused cell contraction and reduced cell area, roundness, adhesion, and migration of BMSCs. RhoA and ROCK1 expression levels were upregulated by severe hypoxia, but p-RhoA and mobility-relevant protein (integrin ß1, p-FAK and fibronectin) expression levels in BMSCs were inhibited. Fortunately, MGF E peptide could restore all abovementioned indexes except RhoA expression. MEK-ERK1/2 pathway was involved in MGF E peptide regulating cell morphological changes, mobility, and relevant proteins (except p-FAK). PI3K-Akt pathway was involved in MGF E peptide regulating cell area, mobility, and relevant proteins. Besides, severe hypoxia upregulated vascular endothelial growth factor α expression but was harmful for proangiogenic capacity of BMSCs. Our study suggested that MGF E peptide might be helpful for the clinical application of tissue engineering strategy in bone defect repair. SIGNIFICANCE OF THE STUDY: Sever hypoxia impairs bone defect repair with bone marrow-derived mesenchymal stem cells (BMSCs). This study proved that mechano-growth factor E (MGF E) peptide could improve the severe hypoxia-induced cell contraction and decline of cell adhesion and migration of BMSCs. Besides, MGF E peptide weakened the effects of severe hypoxia on the cytoskeleton arrangement- and mobility-relevant protein expression levels in BMSCs. The underlying molecular mechanism was also verified. Finally, it was confirmed that MGF E peptide showed an adverse effect on the expression level of vascular endothelial growth factor α in BMSCs under severe hypoxia but could make up for this deficiency through accelerating cell proliferation.

Synergistic promoting effects of bone morphogenetic protein 12/connective tissue growth factor on functional differentiation of tendon derived stem cells and patellar tendon window defect regeneration.

Current study investigated bone morphogenetic protein 12 (BMP12) and connective tissue growth factor (CTGF) activate tendon derived stem cells (TDSCs) tenogenic differentiation, and promotion of injured tendon regeneration. TDSCs were transfected with BMP12 and CTGF via recombinant adenovirus (Ad) infection. Gene transfection efficiency, cell viability and cytotoxicity, tenogenic gene expression, collagen I/III synthesis were evaluated in vitro. For the in vivo study, the transfected cells were transplanted into the rat patellar tendon window defect. At weeks 2 and 8 of post-surgery, the repaired tendon tissues were harvested for histological and biomechanical examinations. The transfected TDSCs revealed relatively stable transfection efficiency (80-90%) with active cell viability means while rare cytotoxicity in each group. During days 1 and 5, BMP12 and CTGF transfection caused tenogenic differentiation genes activation in TDSCs: type I/III collagen, tenascin-C, and scleraxis were all up-regulated, whereas osteogenic, adipogenic, and chondrogenic markers were all down-regulated respectively. In addition, BMP12 and CTGF overexpression significantly promote type I/III collagen synthesis. After in vivo transplantation, at 2 and 8 weeks post-surgery, BMP12, CTGF and co-transfection groups showed more integrated tendon tissue structure versus control, meanwhile, the ultimate failure loads and Young's were all higher than control. Remarkably, at 8 weeks post-surgery, the biomechanical properties of co-transfection group was approaching to normal rat patellar tendon, moreover, the ratio of type III/I collagen maintained about 20% in each transfection group, meanwhile, the type I collagen were significantly increased with co-transfection treatment. In conclusion, BMP12 and CTGF transfection stimulate tenogenic differentiation of TDSCs. The synergistic effects of simultaneous transfection of both may significantly promoted rat patellar tendon window defect regeneration.

Mechano growth factor E peptide inhibits invasion of melanoma cells and up-regulates CHOP expression via endoplasmic reticulum stress.

OBJECTIVE: To evaluate the effects of mechano growth factor E peptide (MGF) on the invasive properties of melanoma cells. RESULTS: Melanoma cells (GLL19) were treated with 10, 20 and 30 ng MGF/ml for 24 h. Their invasive properties were investigated by transwell assay. Cytoskeleton reorganization was assessed via staining with phalloidin-FITC; lysyl oxidase (LOX) family gene expression was tested by qRT-PCR, and western blotting was used to detect expression of the matrix metalloproteinases (MMPs) and endoplasmic reticulum (ER) stress. MGF decreased the invasive capabilities of melanoma cells and induced changes in cytoskeleton distribution. MGF also down-regulated the expression of MMPs and up-regulated the expression of the cell apoptosis-related protein CHOP by inducing ER stress. CONCLUSIONS: MGF can decrease the invasive properties of melanoma cells and induce ER stress, promoting cell apoptosis. Thus, MGF represents a novel strategy for the potential treatment of patients presenting with cutaneous melanoma.

MGF E peptide pretreatment improves the proliferation and osteogenic differentiation of BMSCs via MEK-ERK1/2 and PI3K-Akt pathway under severe hypoxia.

AIMS: Severe hypoxia always inhibits the cell proliferation, osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs), and hinders bone defect repair. Herein we explored the effects of mechano-growth factor (MGF) E peptide on the proliferation and osteogenic differentiation of BMSCs under severe hypoxia. MATERIALS AND METHODS: CoCl2 was utilized to simulate severe hypoxia. MTS was used to detect cell viability. Cell proliferation was verified through flow cytometry and EdU assay. Osteogenic differentiation of BMSCs and osteoblast-specific genes were detected through alkaline phosphatase (ALP) and Alizarin Red S staining, and quantitative real-time PCR, respectively. Hypoxia-inducible factor 1α (HIF-1α), p-ERK1/2 and p-Akt expression levels were detected through western blotting and immunofluorescence. KEY FINDINGS: Severe hypoxia induced HIF-1α accumulation and transferring into the nucleus, and reduced cell proliferation and osteogenic differentiation of BMSCs. The expression levels of osteoblast-specific genes were markedly decreased after differentiation culture for 0, 7 or 14days. Fortunately, MGF E peptide inhibited HIF-1α expression and transferring into the nucleus. Cell proliferation and osteogenic differentiation of BMSCs could be recovered by MGF E peptide pretreatment. MEK-ERK1/2 and PI3K-Akt signaling pathway were confirmed to be involved in MGF E peptide regulating the abovementioned indexes of BMSCs. What's more, short-time treatment with MGF E peptide alone promoted the osteogenic differentiation of BMSCs as well. SIGNIFICANCE: Our study provides new evidence for the role of MGF E peptide in regulating proliferation and osteogenic differentiation of BMSCs under severe hypoxia, which may potentially have therapeutic implication for bone defect repair.

Pretreatment with mechano-growth factor E peptide protects bone marrow mesenchymal cells against damage by fluid shear stress.

Improper fluid shear stress (FSS) can cause serious damages to bone marrow mesenchymal stem cells (MSCs). Mechano-growth factor (MGF) E peptide pretreatment was proposed to protect MSCs against FSS damage in this study. MSCs were exposed to FSS for 30 min after they were pretreated with MGF E peptide for 24 h. Then, the effects of MGF E peptide on the viability, proliferation and cell apoptosis of MSCs were investigated. MGF E peptide pretreatment could recover the cellular metabolic activity of MSCs reduced by 72 dyne cm(-2) FSS and had a synergistic effect with FSS on the cellular metabolic viability of MSCs under 24 and 72 dyne cm(-2) FSS. These results suggested that MGF E peptide pretreatment could be an effective method for the protection of FSS damage in bone tissue engineering.

Effects of low-intensity pulsed ultrasound on cell viability, proliferation and neural differentiation of induced pluripotent stem cells-derived neural crest stem cells.

Low-intensity pulsed ultrasound (LIPUS) acting on induced pluripotent stem cells-derived neural crest stem cells (iPSCs-NCSCs) is considered a promising therapy to improve the efficacy of injured peripheral nerve regeneration. Effects of LIPUS on cell viability, proliferation and neural differentiation of iPSCs-NCSCs were examined respectively in this study. LIPUS at 500 mW cm(-2) enhanced the viability and proliferation of iPSCs-NCSCs after 2 days and, after 4 days, up-regulated gene and protein expressions of NF-M, Tuj1, S100ß and GFAP in iPSCs-NCSCs whereas after 7 days expression of only NF-M, S100ß and GFAP were up-regulated. LIPUS treatment at an appropriate intensity can, therefore, be an efficient and cost-effective method to enhance cell viability, proliferation and neural differentiation of iPSCs-NCSCs in vitro for peripheral nerve tissue engineering.