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Background: The pumping function of corneal endothelial cells (CECs) plays a pivotal role in the maintenance of corneal water homeostasis. Corneal endothelial dysfunction (CED) leads to corneal edema and opacity, but with the exception of keratoplasty, no optimal therapeutic strategies have been established for CED. In this study, we aimed to investigate the ameliorative effect of ascorbic acid (AA) on CED and the underlying mechanism of action in the corneal endothelium., Methods: Rabbit corneal endothelial damage was induced by anterior chamber injection of benzalkonium chloride (BAK). AA was topically administered to the corneal surface, and the transparency and thickness of the cornea were assessed by external eye photography, slit-lamp photography, and ultrasonic pachymetry. To further analyze the mechanism, rabbit CECs and immortalized human CECs (B4G12 cells) were cultured. A ferric reducing/antioxidant and AA (FRASC) assay was performed to measure the AA concentration. Cell proliferation was evaluated by cell counting and bromodeoxyuridine (BrdU) labeling assays, and protein expression was examined by liquid chromatography-mass spectrometry (LC/MS) and immunoblotting. The involvement of glucose transporter 1 (GLUT1) and phospho-ERK was evaluated via GLUT1-siRNA and phospho-ERK inhibitor (PD98059) treatment., Interpretation: We observed that topical AA ameliorates BAK-induced rabbit corneal endothelial damage. Furthermore, we demonstrated that AA is transported into B4G12 cells via GLUT1, and afterward, AA increases ERK phosphorylation and promotes cell proliferation. Our findings indicate that CEC proliferation stimulated via the noncanonical AA-GLUT1-ERK axis contributes to AA-enhanced healing of CED., (Copyright © 2021 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

The major biological methyl donor, S-adenosylmethionine (adoMet) synthesis occurs mainly in the liver. Methionine adenosyltransferase 1A (MAT1A) and glycine N-methyltransferase (GNMT) are two key enzymes involved in the functional implications of that variation. We collected 42 RNA-seq data from paired hepatocellular carcinoma (HCC) and its adjacent normal liver tissue from the Cancer Genome Atlas (TCGA). There was no mutation found in MAT1A or GNMT RNA in the 42 HCC patients. The 11,799 genes were annotated in the RNA-Seq data, and their expression levels were used to investigate the phenotypes of low MAT1A and low GNMT by Gene Set Enrichment Analysis (GSEA). The REACTOME_TRANSLATION gene set was enriched and visualized in a heatmap along with corresponding differences in gene expression between low MAT1A versus high MAT1A and low GNMT versus high GNMT. We identified 43 genes of the REACTOME_TRANSLATION gene set that are powerful prognosis factors in HCC. The significantly predicted genes were referred into eukaryotic translation initiation (EIF3B, EIF3K), eukaryotic translation elongation (EEF1D), and ribosomal proteins (RPs). Cell models expressing various MAT1A and GNMT proved that simultaneous restoring the expression of MAT1A and GNMT decreased cell proliferation, invasion, as well as the REACTOME_TRANSLATION gene EEF1D, consistent with a better prognosis in human HCC. We demonstrated new findings that downregulation or defect in MAT1A and GNMT genes can enrich the protein-associated translation process that may account for poor HCC prognosis. This is the first study demonstrated that MAT1A and GNMT, the 2 key enzymes involved in methionine cycle, could attenuate the function of ribosome translation. We propose a potential novel mechanism by which the diminished GNMT and MAT1A expression may confer poor prognosis for HCC.

Background: Tendons have limited regenerative potential, so healing of ruptured tendon tissue requires a prolonged period, and the prognosis is suboptimal. Although stem cell transplantation–based approaches show promise for accelerating tendon repair, the resultant therapeutic efficacy remains unsatisfactory. Hypothesis: The transplantation of stem cells preassembled as 3-dimensional spheroids achieves a superior therapeutic outcome compared with the transplantation of single-cell suspensions. Study Design: Controlled laboratory study. Methods: Adipose-derived stem cells (ADSCs) were assembled as spheroids using a methylcellulose hydrogel system. The secretome of ADSC suspensions or spheroids was collected and utilized to treat tenocytes and macrophages to evaluate their therapeutic potential and investigate the mechanisms underlying their effects. RNA sequencing was performed to investigate the global difference in gene expression between ADSC suspensions and spheroids in an in vitro inflammatory microenvironment. For the in vivo experiment, rabbits that underwent Achilles tendon transection, followed by stump suturing, were randomly assigned to 1 of 3 groups: intratendinous injection of saline, rabbit ADSCs as conventional single-cell suspensions, or preassembled ADSC spheroids. The tendons were harvested for biomechanical testing and histological analysis at 4 weeks postoperatively. Results: Our in vitro results demonstrated that the secretome of ADSCs assembled as spheroids exhibited enhanced modulatory activity in (1) tenocyte proliferation (P =.015) and migration (P =.001) by activating extracellular signal-regulated kinase (ERK) signaling and (2) the suppression of the secretion of interleukin-6 (P =.005) and interleukin-1α (P =.042) by M1 macrophages via the COX-2/PGE2/EP4 signaling axis. Gene expression profiling of cells exposed to an inflammatory milieu revealed significantly enriched terms that were associated with the immune response, cytokines, and tissue remodeling in preassembled ADSC spheroids. Ex vivo fluorescence imaging revealed that the engraftment efficiency of ADSCs in the form of spheroids was higher than that of ADSCs in single-cell suspensions (P =.003). Furthermore, the transplantation of ADSC spheroids showed superior therapeutic effects in promoting the healing of sutured stumps, as evidenced by improvements in the tensile strength (P =.019) and fiber alignment (P <.001) of the repaired tendons. Conclusion: The assembly of ADSCs as spheroids significantly advanced their potential to harness tenocytes and macrophages. As a proof of concept, this study clearly demonstrates the effectiveness of using ADSC spheroids to promote tendon regeneration. Clinical Relevance: The present study lays a foundation for future clinical applications of stem cell spheroid–based therapy for the management of tendon injuries. [ABSTRACT FROM AUTHOR]

After the discovery of endogenous dinitrosyl iron complexes (DNICs) as a potential biological equivalent of nitric oxide (NO), bioinorganic engineering of [Fe(NO)2] unit has emerged to develop biomimetic DNICs [(NO)2Fe(L)2] as a chemical biology tool for controlled delivery of NO. For example, water-soluble DNIC [Fe2(μ-SCH2CH2OH)2(NO)4] (DNIC-1) was explored for oral delivery of NO to the brain and for the activation of hippocampal neurogenesis. However, the kinetics and mechanism for cellular uptake and intracellular release of NO, as well as the biocompatibility of synthetic DNICs, remain elusive. Prompted by the potential application of NO to dermato-physiological regulations, in this study, cellular uptake and intracellular delivery of DNIC [Fe2(μ-SCH2CH2COOH)2(NO)4] (DNIC-2) and its regulatory effect/biocompatibility toward epidermal cells were investigated. Upon the treatment of DNIC-2 to human fibroblast cells, cellular uptake of DNIC-2 followed by transformation into protein-bound DNICs occur to trigger the intracellular release of NO with a half-life of 1.8 ± 0.2 h. As opposed to the burst release of extracellular NO from diethylamine NONOate (DEANO), the cell-penetrating nature of DNIC-2 rationalizes its overwhelming efficacy for intracellular delivery of NO. Moreover, NO-delivery DNIC-2 can regulate cell proliferation, accelerate wound healing, and enhance the deposition of collagen in human fibroblast cells. Based on the in vitro and in vivo biocompatibility evaluation, biocompatible DNIC-2 holds the potential to be a novel active ingredient for skincare products. [ABSTRACT FROM AUTHOR]