Regenerative Potential of Dental Pulp Stem Cells in Response to a Bioceramic Dental Sealer and Photobiomodulation: An In Vitro Study.

AIMS: This study aims to assess the synergistic effect of utilizing a bioceramic sealer, NeoPutty, with photobiomodulation (PBM) on dental pulp stem cells (DPSCs) for odontogenesis. MATERIALS AND METHODS: Dental pulp stem cells were collected from 10 premolars extracted from healthy individuals. Dental pulp stem cells were characterized using an inverted-phase microscope to detect cell shape and flow cytometry to detect stem cell-specific surface antigens. Three experimental groups were examined: the NP group, the PBM group, and the combined NP and PBM group. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) experiment was conducted to assess the viability of DPSCs. The odontogenic differentiation potential was analyzed using Alizarin red staining, RT-qPCR analysis of odontogenic genes DMP-1, DSPP, and alkaline phosphatase (ALP), and western blot analysis for detecting BMP-2 and RUNX-2 protein expression. An analysis of variance (ANOVA) followed by a post hoc t-test was employed to examine and compare the mean values of the results. RESULTS: The study showed a notable rise in cell viability when NP and PBM were used together. Odontogenic gene expression and the protein expression of BMP-2 and RUNX-2 were notably increased in the combined group. The combined effect of NeoPutty and PBM was significant in enhancing the odontogenic differentiation capability of DPSCs. CONCLUSION: The synergistic effect of NeoPutty and PBM produced the most positive effect on the cytocompatibility and odontogenic differentiation potential of DPSCs. CLINICAL SIGNIFICANCE: Creating innovative regenerative treatments to efficiently and durably repair injured dental tissues. How to cite this article: Alshawkani HA, Mansy M, Al Ankily M, et al. Regenerative Potential of Dental Pulp Stem Cells in Response to a Bioceramic Dental Sealer and Photobiomodulation: An In Vitro Study. J Contemp Dent Pract 2024;25(4):313-319.

Mechanism of Vaginal Epithelial Cell Pyroptosis Induced by the NLRP3 Inflammasome in Vulvovaginal Candidiasis.

PROBLEM: Vulvovaginal candidiasis (VVC) is a common mucosal fungal infection, and Candida albicans is the main causative agent. The NLRP3 inflammasome plays an important role in VVC, but the underlying mechanism is unknown. METHOD OF STUDY: Vaginal epithelial cells were divided into three groups: control, C. albicans strain SC5314 (wild-type, WT), and WT+ Matt Cooper Compound 950 (MCC950, a specific NLRP3 inhibitor). After human vaginal epithelial cells were pretreated with 1 µmol/L MCC950 for 2 h, C. albicans (MOI = 1) was cocultured with the human vaginal epithelial cells for 12 h. The cell supernatants were collected, LDH was detected, and the IL-1ß and IL-18 levels were determined by ELISA. The expression of the pyroptosis-related proteins NLRP3, Caspase-1 p20 and GSDMD was measured by Western blotting analysis. The protein expression of the pyroptosis-related N-terminus of GSDMD (GSDMD-N) was detected by immunofluorescence. RESULTS: In this study, we showed that the WT C. albicans strain induced pyroptosis in vaginal epithelial cells, as indicated by the LDH and proinflammatory cytokine levels and the upregulated levels of the pyroptosis-related proteins NLRP3, Caspase-1 p20, and GSDMD-N. MCC950 reversed the changes in the expression of these proteins and proinflammatory cytokines in vaginal epithelial cells. CONCLUSION: C. albicans activated the NLRP3 inflammasome to induce vaginal epithelial cell pyroptosis. MCC950 inhibited the NLRP3 inflammasome, reduced vaginal epithelial cell pyroptosis, and decreased the release of inflammatory cytokines.

Exploration of the miR-187-3p/CNR2 pathway in modulating osteoblast differentiation and treating postmenopausal osteoporosis through mechanical stress.

This study aimed to explore how mechanical stress affects osteogenic differentiation via the miR-187-3p/CNR2 pathway. To conduct this study, 24 female C57BL/6 mice, aged 8 weeks, were used and divided into four groups. The Sham and OVX groups did not undergo treadmill exercise, while the Sham + EX and OVX + EX groups received a 8-week treadmill exercise. Post-training, bone marrow and fresh femur samples were collected for further analysis. Molecular biology analysis, histomorphology analysis, and micro-CT analysis were conducted on these samples. Moreover, primary osteoblasts were cultured under osteogenic conditions and divided into GM group and CTS group. The cells in the CTS group underwent a sinusoidal stretching regimen for either 3 or 7 days. The expression of early osteoblast markers (Runx2, OPN, and ALP) was measured to assess differentiation. The study findings revealed that mechanical stress has a regulatory impact on osteoblast differentiation. The expression of miR-187-3p was observed to decrease, facilitating osteogenic differentiation, while the expression of CNR2 increased significantly. These observations suggest that mechanical stress, miR-187-3p, and CNR2 play crucial roles in regulating osteogenic differentiation. Both in vivo and in vitro experiments have confirmed that mechanical stress downregulates miR-187-3p and upregulates CNR2, which leads to the restoration of distal femoral bone mass and enhancement of osteoblast differentiation. Therefore, mechanical stress promotes osteoblasts, resulting in improved osteoporosis through the miR-187-3p/CNR2 signaling pathway. These findings have broad prospect and provide molecular biology guidance for the basic research and clinical application of exercise in the prevention and treatment of PMOP.

Functional variation among mesenchymal stem cells derived from different tissue sources.

Background: Mesenchymal stem cells (MSCs) are increasingly recognized for their regenerative potential. However, their clinical application is hindered by their inherent variability, which is influenced by various factors, such as the tissue source, culture conditions, and passage number. Methods: MSCs were sourced from clinically relevant tissues, including adipose tissue-derived MSCs (ADMSCs, n = 2), chorionic villi-derived MSCs (CMMSCs, n = 2), amniotic membrane-derived MSCs (AMMSCs, n = 3), and umbilical cord-derived MSCs (UCMSCs, n = 3). Passages included the umbilical cord at P0 (UCMSCP0, n = 2), P3 (UCMSCP3, n = 2), and P5 (UCMSCP5, n = 2) as well as the umbilical cord at P5 cultured under low-oxygen conditions (UCMSCP5L, n = 2). Results: We observed that MSCs from different tissue origins clustered into six distinct functional subpopulations, each with varying proportions. Notably, ADMSCs exhibited a higher proportion of subpopulations associated with vascular regeneration, suggesting that they are beneficial for applications in vascular regeneration. Additionally, CMMSCs had a high proportion of subpopulations associated with reproductive processes. UCMSCP5 and UCMSCP5L had higher proportions of subpopulations related to female reproductive function than those for earlier passages. Furthermore, UCMSCP5L, cultured under low-oxygen (hypoxic) conditions, had a high proportion of subpopulations associated with pro-angiogenic characteristics, with implications for optimizing vascular regeneration. Conclusions: This study revealed variation in the distribution of MSC subpopulations among different tissue sources, passages, and culture conditions, including differences in functions related to vascular and reproductive system regeneration. These findings hold promise for personalized regenerative medicine and may lead to more effective clinical treatments across a spectrum of medical conditions.

Amorfrutin B Compromises Hypoxia/Ischemia-induced Activation of Human Microglia in a PPARγ-dependent Manner: Effects on Inflammation, Proliferation Potential, and Mitochondrial Status.

Amorfrutin B is a selective PPARγ modulator that we demonstrated to be a promising neuroprotective compound in cellular models of stroke and perinatal asphyxia. Although neuronal mechanisms of amorfrutin B-evoked neuroprotection have been identified, none of them reflects the actions of the compound on microglia, which play a pivotal role in brain response to hypoxia/ischemia. Here, we provide evidence for amorfrutin B-induced effects on human microglia subjected to hypoxia/ischemia; the compound counteracts inflammation, and influences mitochondrial status and proliferation potential in a PPARγ-dependent manner. Post-treatment with amorfrutin B decreased the IBA1 fluorescence intensity, reduced caspase-1 activity, and downregulated IL1B/IL-1ß and TNFA but not IL10/IL-10 expression, which was upregulated. Amorfrutin B also stimulated PPARγ signaling, as evidenced by increased mRNA and/or protein levels of PPARγ and PGC1α. In addition, amorfrutin B reversed the hypoxia/ischemia-evoked effects on mitochondria-related parameters, such as mitochondrial membrane potential, BCL2/BCL2 expression and metabolic activity, which were correlated with diminished proliferation potential of microglia. Interestingly, the inhibitory effect of amorfrutin B on the proliferation potential and mitochondrial function of microglia is opposite to the stimulatory effect of amorfrutin B on mouse neuronal survival, as evidenced by increased neuronal viability and reduced neurodegeneration. In summary, this study showed for the first time that amorfrutin B compromises hypoxia/ischemia-induced activation of human microglia in a PPARγ-dependent manner, which involves inhibiting inflammation, normalizing mitochondrial status, and controlling proliferation potential. These data extend the protective potential of amorfrutin B in the pharmacotherapy of hypoxic/ischemic brain injury, targeting not only neurons but also activated microglia.

Enhancement of endothelial function and attenuation of portal vein injury using mesenchymal stem cells carrying miRNA-25-3p.

The aims of this study were to determine whether human umbilical cord mesenchymal stem cells (hucMSCs) modified by miRNA-25-3p (miR-25-3p) overexpression could promote venous endothelial cell proliferation and attenuate portal endothelial cell injury. HucMSCs and human umbilical vein endothelial cells (HUVEC) were isolated and cultured from human umbilical cord and characterized. Lentiviral vectors expressing miRNA-25-3p were transfected into hucMSCs and confirmed by PCR. We verified the effect of miR-25-3p-modified hucMSCs on HUVEC by cell co-culture and cell supernatant experiments. Subsequently, exosomes of miR-25-3p-modified hucMSCs were isolated from cell culture supernatants and characterized by WB, NTA and TEM. We verified the effects of miR-25-3p-modified exosomes derived from hucMSCs on HUVEC proliferation, migration, and angiogenesis by in vitro cellular function experiments. Meanwhile, we further examined the downstream target genes and signaling pathways potentially affected by miR-25-3p-modified hucMSC-derived exosomes in HUVEC. Finally, we established a rat portal vein venous thrombosis model by injecting CM-DiR-labeled hucMSCs intravenously into rats and examining the homing of cells in the portal vein by fluorescence microscopy. Histological and immunohistochemical experiments were used to examine the effects of miRNA-25-3p-modified hucMSCs on the proliferation and damage of portal vein endothelial cells. Primary hucMSCs and HUVECs were successfully isolated, cultured and characterized. Primary hucMSCs were modified with a lentiviral vector carrying miR-25-3p at MOI 80. Co-culture and cell supernatant intervention experiments showed that overexpression of miRNA-25-3p in hucMSCs enhanced HUVEC proliferation, migration and tube formation in vitro. We successfully isolated and characterized exosomes of miR-25-3p-modified hucMSCs, and exosome intervention experiments demonstrated that miR-25-3p-modified exosomes derived from hucMSCs similarly enhanced the proliferation, migration, and angiogenesis of HUVECs. Subsequent PCR and WB analyses indicated PTEN/KLF4/AKT/ERK1/2 as potential pathways of action. Analysis in a rat portal vein thrombosis model showed that miR-25-3p-modified hucMSCs could homing to damaged portal veins. Subsequent histological and immunohistochemical examinations demonstrated that intervention with miR-25-3p overexpression-modified hucMSCs significantly reduced damage and attenuated thrombosis in rat portal veins. The above findings indicate suggest that hucMSCs based on miR-25-3p modification may be a promising therapeutic approach for use in venous thrombotic diseases.

Autophagy modulation effect on homotypic transfer of intracellular components via tunneling nanotubes in mesenchymal stem cells.

BACKGROUND: Recent studies have proved the role of autophagy in mesenchymal stem cell (MSCs) function and regenerative properties. How and by which mechanism autophagy modulation can affect the juxtacrine interaction of MSCs should be addressed. Here, the role of autophagy was investigated in the formation of tunneling nanotubes (TNTs) and homotypic mitochondrial donation. METHODS: MSCs were incubated with 15 µM Metformin (Met) and/or 3 µM 3-methyladenine (3-MA) for 48 h. The formation of TNTs was assessed using bright-field and SEM images. The mitochondria density and ΔΨ values were monitored using flow cytometry analysis. Using RT-PCR and protein array, the close interaction and shared mediators between autophagy, apoptosis, and Wnt signaling pathways were also monitored. The total fatty acid profile was assessed using gas chromatography. RESULT: Data indicated the increase of TNT length and number, along with other cell projections after the induction of autophagy while these features were blunted in 3-MA-treated MSCs (p < 0.05). Western blotting revealed the significant reduction of Rab8 and p-FAK in 3-MA-treated MSCs (p < 0.05), indicating the inhibition of TNT assembly and vesicle transport. Likewise, the stimulation of autophagy increased autophagic flux and mitochondrial membrane integrity compared to 3-MA-treated MSCs. Despite these findings, protein levels of mitochondrial membrane Miro1 and 2 were unchanged after autophagy inhibition/stimulation (p > 0.05). We found that the inhibition/stimulation of autophagy can affect the protein, and transcription levels of several mediators related to Wnt and apoptosis signaling pathways involved in different cell bioactivities. Data confirmed the profound increase of mono and polyunsaturated/saturated fatty acid ratio in MSCs exposed to autophagy stimulator. CONCLUSIONS: In summary, autophagy modulation could affect TNT formation which is required for homotypic mitochondrial donation. Thus, the modulation of autophagy creates a promising perspective to increase the efficiency of cell-based therapies.

Expression and secretion of SPARC, FGF-21 and DCN in bovine muscle cells: Effects of age and differentiation.

Skeletal muscle growth is an economically important trait in the cattle industry. Secreted muscle-derived proteins, referred to as myokines, have important roles in regulating the growth, metabolism, and health of skeletal muscle in human and biomedical research models. Accumulating evidence supports the importance of myokines in skeletal muscle and whole-body health, though little is known about the potential presence and functional significance of these proteins in cattle. This study evaluates and confirms that secreted proteins acidic and rich in cysteine (SPARC), fibroblast growth factor 21 (FGF-21), myostatin (MSTN), and decorin (DCN) are expressed and SPARC, FGF-21, and DCN are secreted by primary bovine satellite cells from 3- (BSC3; n = 3) and 11- (BSC11; n = 3) month -old commercial angus steers. Cells were cultured and collected at zero, 12, 24, and 48 hours to characterize temporal expression and secretion from undifferentiated and differentiated cells. The expression of SPARC was higher in the undifferentiated (p = 0.04) and differentiated (p = 0.07) BSC11 than BSC3. The same was observed with protein secretion from undifferentiated (p <0.0001) BSC11 compared to BSC3. Protein secretion of FGF-21 was higher in undifferentiated BSC11 (p < 0.0001) vs. BSC3. DCN expression was higher in differentiated BSC11 (p = 0.006) vs. BSC3. Comparing undifferentiated vs. differentiated BSC, MSTN expression was higher in differentiated BSC3 (p ≤ 0.001) for 0, 12, and 24 hours and in BSC11 (p ≤ 0.03) for 0, 12, 24, and 48 hours. There is also a change over time for SPARC expression (p ≤ 0.03) in undifferentiated and differentiated BSC and protein secretion (p < 0.0001) in undifferentiated BSC, as well as FGF-21 expression (p = 0.007) in differentiated BSC. This study confirms SPARC, FGF-21, and DCN are secreted, and SPARC, FGF-21, MSTN, and DCN are expressed in primary bovine muscle cells with age and temporal differences.

Protective effect of Sasa veitchii extract against all-trans-retinoic acid-induced inhibition of proliferation of cultured human palate cells.

Cleft palate is the most common facial birth defect worldwide. It is caused by environmental factors or genetic mutations. Environmental factors such as pharmaceutical exposure in women are known to induce cleft palate. The aim of the present study was to investigate the protective effect of Sasa veitchii extract against medicine-induced inhibition of proliferation of human embryonic palatal mesenchymal cells. We demonstrated that all-trans-retinoic acid inhibited human embryonic palatal mesenchymal cell proliferation in a dose-dependent manner, whereas dexamethasone treatment had no effect on cell proliferation. Cotreatment with Sasa veitchii extract repressed all-trans-retinoic acid-induced toxicity in human embryonic palatal mesenchymal cells. We found that cotreatment with Sasa veitchii extract protected all-trans-retinoic acid-induced cyclin D1 downregulation in human embryonic palatal mesenchymal cells. Furthermore, Sasa veitchii extract suppressed all-trans-retinoic acid-induced miR-4680-3p expression. Additionally, the expression levels of the genes that function downstream of the target genes ( ERBB2 and JADE1 ) of miR-4680-3p in signaling pathways were enhanced by cotreatment with Sasa veitchii extract and all-trans-retinoic acid compared to all-trans-retinoic acid treatment. These results suggest that Sasa veitchii extract suppresses all-trans-retinoic acid-induced inhibition of cell proliferation via modulation of miR-4680-3p expression.

Effects of Injectable Solutions on the Quality of Monocyte-Derived Dendritic Cells for Immunotherapy.

Therapeutic vaccines based on monocyte-derived dendritic cells have been shown to be promising strategies and may act as complementary treatments for viral infections, cancers, and, more recently, autoimmune diseases. Alpha-type-1-polarized dendritic cells (aDC1s) have been shown to induce type-1 immunity with a high capacity to produce interleukin-12p70 (IL-12p70). In the clinical use of cell-based therapeutics, injectable solutions can affect the morphology, immunophenotypic profile, and viability of cells before delivery and their survival after injection. In this sense, preparing a cell suspension that maintains the quality of aDC1s is essential to ensure effective immunotherapy. In the present study, monocytes were differentiated into aDC1s in the presence of IL-4 and GM-CSF. On day 5, the cells were matured by the addition of a cytokine cocktail consisting of IFN-α, IFN-γ, IL-1ß, TNF-α, and Poly I:C. After 48 hr, mature aDC1s were harvested and suspended in two different solutions: normal saline and Ringer's lactate. The maintenance of cells in suspension was evaluated after 4, 6, and 8 hr of storage. Cell viability, immunophenotyping, and apoptosis analyses were performed by flow cytometry. Cellular morphology was observed by electron microscopy, and the production of IL-12p70 by aDC1s was evaluated by ELISA. Compared with normal saline, Ringer's lactate solution was more effective at maintaining DC viability for up to 8 hr of incubation at 4 or 22°C.

SP1 promotes high glucose-induced lens epithelial cell viability, migration and epithelial-mesenchymal transition via regulating FGF7 and PI3K/AKT pathway.

BACKGROUND: Diabetic cataract (DC) is a common complication of diabetes and its etiology and progression are multi-factorial. In this study, the roles of specific protein 1 (SP1) and fibroblast growth factor 7 (FGF7) in DC development were explored. METHODS: DC cell model was established by treating SRA01/04 cells with high glucose (HG). MTT assay was conducted to evaluate cell viability. Transwell assay and wound-healing assay were performed to assess cell migration and invasion. Western blot assay and qRT-PCR assay were conducted to measure the expression of N-cadherin, E-cadherin, Collagen I, Fibronectin, SP1 and FGF7 expression. CHIP assay and dual-luciferase reporter assay were conducted to analyze the combination between FGF7 and SP1. RESULTS: FGF7 was upregulated in DC patients and HG-induced SRA01/04 cells. HG treatment promoted SRA01/04 cell viability, migration, invasion and epithelial-mesenchymal transition (EMT), while FGF7 knockdown abated the effects. Transcription factor SP1 activated the transcription level of FGF7 and SP1 overexpression aggravated HG-induced SRA01/04 cell injury. SP1 silencing repressed HG-induced SRA01/04 cell viability, migration, invasion and EMT, but these effects were ameliorated by upregulating FGF7. Additionally, SP1 knockdown inhibited the PI3K/AKT pathway by regulating the transcription level of FGF7. CONCLUSION: Transcription factor SP1 activated the transcription level of FGF7 and the PI3K/AKT pathway to regulate HG-induced SRA01/04 cell viability, migration, invasion and EMT.

Plakophilin 4 controls the spatio-temporal activity of RhoA at adherens junctions to promote cortical actin ring formation and tissue tension.

Plakophilin 4 (PKP4) is a component of cell-cell junctions that regulates intercellular adhesion and Rho-signaling during cytokinesis with an unknown function during epidermal differentiation. Here we show that keratinocytes lacking PKP4 fail to develop a cortical actin ring, preventing adherens junction maturation and generation of tissue tension. Instead, PKP4-depleted cells display increased stress fibers. PKP4-dependent RhoA localization at AJs was required to activate a RhoA-ROCK2-MLCK-MLC2 axis and organize actin into a cortical ring. AJ-associated PKP4 provided a scaffold for the Rho activator ARHGEF2 and the RhoA effectors MLCK and MLC2, facilitating the spatio-temporal activation of RhoA signaling at cell junctions to allow cortical ring formation and actomyosin contraction. In contrast, association of PKP4 with the Rho suppressor ARHGAP23 reduced ARHGAP23 binding to RhoA which prevented RhoA activation in the cytoplasm and stress fiber formation. These data identify PKP4 as an AJ component that transduces mechanical signals into cytoskeletal organization.

Human cytomegalovirus infection impairs neural differentiation via repressing sterol regulatory element binding protein 2-mediated cholesterol biosynthesis.

Congenital human cytomegalovirus (HCMV) infection is a major cause of abnormalities and disorders in the central nervous system (CNS) and/or the peripheral nervous system (PNS). However, the complete pathogenesis of neural differentiation disorders caused by HCMV infection remains to be fully elucidated. Stem cells from human exfoliated deciduous teeth (SHEDs) are mesenchymal stem cells (MSCs) with a high proliferation and neurogenic differentiation capacity. Since SHEDs originate from the neural crest of the early embryonic ectoderm, SHEDs were hypothesized to serve as a promising cell line for investigating the pathogenesis of neural differentiation disorders in the PNS caused by congenital HCMV infection. In this work, SHEDs were demonstrated to be fully permissive to HCMV infection and the virus was able to complete its life cycle in SHEDs. Under neurogenic inductive conditions, HCMV infection of SHEDs caused an abnormal neural morphology. The expression of stem/neural cell markers was also disturbed by HCMV infection. The impairment of neural differentiation was mainly due to a reduction of intracellular cholesterol levels caused by HCMV infection. Sterol regulatory element binding protein-2 (SREBP2) is a critical transcription regulator that guides cholesterol synthesis. HCMV infection was shown to hinder the migration of SREBP2 into nucleus and resulted in perinuclear aggregations of SREBP2 during neural differentiation. Our findings provide new insights into the prevention and treatment of nervous system diseases caused by congenital HCMV infection.

Embryo Quality Conditions the Secretory Profile of Tolerogenic Dendritic Cell DC-10 During the Peri-Implantation Period.

PROBLEM: The decidualization process conditions monocytes to the immunosuppressive and tolerogenic dendritic cell (DC)-10 profile, a DC subset with high IL-10 production. Since the implantation process implies an embryo-endometrium-immune crosstalk, here we focused on the ability of embryonic soluble factors to modify decidual DC conditioning accordingly with its quality. METHOD OF STUDY: Human endometrial stromal cell line (HESC) decidualized with medroxyprogesterone and dibutyryl-cAMP (Dec) was stimulated with human embryo-conditioned media (ECM), classified as normal (ND) or impaired developed (ID) for 48 h (n = 18/group). Monocytes isolated from six healthy women were differentiated to DCs with rhGM-CSF+rhIL-4 in the presence/absence of conditioned media (CM) from decidualized cells stimulated with ECM or nontreated. RESULTS: We found that decidualized cells stimulated with ECM sustain a myeloid regulatory cell profile on monocyte-derived culture with increased frequency of CD1a-CD14+ and CD83+CD86low cells. ND-Dec sustained the higher expression of the DC-10 markers, HLA-G and IL-10 whereas ID-Dec diminished IL-10 production (ID-Dec: 135 ± 37.4 vs. Dec: 223.3 ± 49.9 pg/mL, p < 0.05). The treatment with ECM-Dec sustained a higher IL-10 production and prevented the increase of CD83/CD86 after LPS challenge regardless of embryo quality. Notably, TNF-α production increased in ID-Dec cultures (ID-Dec: 475.1 ± 134.7 vs. Dec: 347.5 ± 98 pg/mL, p < 0.05). CONCLUSIONS: Although remaining in a tolerogenic profile compatible with DC-10, DCs can differentially respond to decidual secreted factors based on embryo quality, changing their secretome. These results suggest that in the presence of arrested embryo, DCs could differentially shape the immunological microenvironment, contributing to arrested embryo clearance during the menstrual phase.

Protective Effect of Nicotinamide Riboside on Glucocorticoid-Induced Glaucoma: Mitigating Mitochondrial Damage and Extracellular Matrix Deposition.

Purpose: Glucocorticoid-induced glaucoma (GIG) is a prevalent complication associated with glucocorticoids (GCs), resulting in irreversible blindness. GIG is characterized by the abnormal deposition of extracellular matrix (ECM) in the trabecular meshwork (TM), elevation of intraocular pressure (IOP), and loss of retinal ganglion cells (RGCs). The objective of this study is to investigate the effects of nicotinamide riboside (NR) on TM in GIG. Methods: Primary human TM cells (pHTMs) and C57BL/6J mice responsive to GCs were utilized to establish in vitro and in vivo GIG models, respectively. The study assessed the expression of ECM-related proteins in TM and the functions of pHTMs to reflect the effects of NR. Mitochondrial morphology and function were also examined in the GIG cell model. GIG progression was monitored through IOP, RGCs, and mitochondrial morphology. Intracellular nicotinamide adenine dinucleotide (NAD+) levels of pHTMs were enzymatically assayed. Results: NR significantly prevented the expression of ECM-related proteins and alleviated dysfunction in pHTMs after dexamethasone treatment. Importantly, NR protected damaged ATP synthesis, preventing overexpression of mitochondrial reactive oxygen species (ROS), and also protect against decreased mitochondrial membrane potential induced by GCs in vitro. In the GIG mouse model, NR partially prevented the elevation of IOP and the loss of RGCs. Furthermore, NR effectively suppressed the excessive expression of ECM-associated proteins and mitigated mitochondrial damage in vivo. Conclusions: Based on the results, NR effectively enhances intracellular levels of NAD+, thereby mitigating abnormal ECM deposition and TM dysfunction in GIG by attenuating mitochondrial damage induced by GCs. Thus, NR has promising potential as a therapeutic candidate for GIG treatment.

GDF-15 Attenuates the Epithelium-Mesenchymal Transition and Alleviates TGFß2-Induced Lens Opacity.

Purpose: We sought to evaluate the efficacy of growth differentiation factor (GDF)-15 treatment for suppressing epithelial-mesenchymal transition (EMT) and alleviating transforming growth factor ß2 (TGFß2)-induced lens opacity. Methods: To test whether GDF-15 is a molecule that prevents EMT, we pretreated the culture with GDF-15 in neural progenitor cells, retinal pigment epithelial cells, and lens epithelial cells and then treated with factors that promote EMT, GDF-11, and TGFß2, respectively. To further investigate the efficacy of GDF-15 on alleviating lens opacity, we used mouse lens explant culture to mimic secondary cataracts. We pretreated the lens culture with GDF-15 and then added TGFß2 to develop lens opacity (n = 3 for each group). Western blot and quantitative reverse transcription polymerase chain reaction (qRT-PCR) were used to measure EMT protein and gene expression, respectively. Results: In cell culture, GDF-15 pretreatment significantly attenuated EMT marker expression in cultured cells induced by treatment with GDF-11 or TGFß2. In the lens explant culture, GDF-15 pretreatment also reduced mouse lens opacity induced by exposure to TGFß2. Conclusions: Our results indicate that GDF-15 could alleviate TGFß2-induced EMT and is a potential therapeutic agent to slow or prevent posterior capsular opacification (PCO) progression after cataract surgery. Translational Relevance: Cataracts are the leading cause of blindness worldwide, with the only current treatment involving surgical removal of the lens and replacement with an artificial lens. However, PCO, also known as secondary cataract, is a common complication after cataract surgery. The development of an adjuvant that slows the progression of PCO will be beneficial to the field of anterior complications.

Production and stability of cultured red blood cells depends on the concentration of cholesterol in culture medium.

The production of cultured red blood cells (cRBC) for transfusion purposes requires large scale cultures and downstream processes to purify enucleated cRBC. The membrane composition, and cholesterol content in particular, are important during proliferation of (pro)erythroblasts and for cRBC quality. Therefore, we tested the requirement for cholesterol in the culture medium during expansion and differentiation of erythroid cultures with respect to proliferation, enucleation and purification by filtration. The low cholesterol level (22 µg/dl) in serum free medium was sufficient to expand (pro)erythroblast cultures. Addition of 2.0 or 5.0 mg/dL of free cholesterol at the start of differentiation induction inhibited enucleation compared to the default condition containing 3.3 mg/dl total cholesterol derived from the addition of Omniplasma to serum free medium. Addition of 5.0 mg/dl cholesterol at day 5 of differentiation did not affect the enucleation process but significantly increased recovery of enucleated cRBC following filtration over leukodepletion filters. The addition of cholesterol at day 5 increased the osmotic resistance of cRBC. In conclusion, cholesterol supplementation after the onset of enucleation improved the robustness of cRBC and increased the yield of enucleated cRBC in the purification process.

Therapeutic application of nicotinamide: As a potential target for inhibiting fibrotic scar formation following spinal cord injury.

AIM: We aimed to confirm the inhibitory effect of nicotinamide on fibrotic scar formation following spinal cord injury in mice using functional metabolomics. METHODS: We proposed a novel functional metabolomics strategy to establish correlations between gene expression changes and metabolic phenotypes using integrated multi-omics analysis. Through the integration of quantitative metabolites analysis and assessments of differential gene expression, we identified nicotinamide as a functional metabolite capable of inhibiting fibrotic scar formation and confirmed the effect in vivo using a mouse model of spinal cord injury. Furthermore, to mimic fibrosis models in vitro, primary mouse embryonic fibroblasts and spinal cord fibroblasts were stimulated by TGFß, and the influence of nicotinamide on TGFß-induced fibrosis-associated genes and its underlying mechanism were examined. RESULTS: Administration of nicotinamide led to a reduction in fibrotic lesion area and promoted functional rehabilitation following spinal cord injury. Nicotinamide effectively downregulated the expression of fibrosis genes, including Col1α1, Vimentin, Col4α1, Col1α2, Fn1, and Acta2, by repressing the TGFß/SMADs pathway. CONCLUSION: Our functional metabolomics strategy identified nicotinamide as a metabolite with the potential to inhibit fibrotic scar formation following SCI by suppressing the TGFß/SMADs signaling. This finding provides new therapeutic strategies and new ideas for clinical treatment.

Inflammation Can Be a High-Risk Factor for Mucosal Nonunion of MRONJ by Regulating SIRT1 Signaling When Treated with an Oncologic Dose of Zoledronate.

Purpose: Zoledronate (ZA) stands as a highly effective antiresorptive agent known to trigger medication-related osteonecrosis of the jaw (MRONJ). Its clinical dosages primarily encompass those used for oncologic and osteoporosis treatments. While inflammation is recognized as a potential disruptor of mucosal healing processes associated with ZA, prior research has overlooked the influence of varying ZA dosages on tissue adaptability. Therefore, a deeper understanding of the specific mechanisms by which inflammation exacerbates ZA-induced MRONJ, particularly when inflammation acts as a risk factor, remains crucial. Methods: Cell proliferation and migration of human oral keratinocytes (HOK) was analyzed after treatment with different doses of ZA and/or lipopolysaccharide (LPS) to assess their possible effect on mucosal healing of extraction wounds. Mouse periodontitis models were established using LPS, and histological changes in extraction wounds were observed after the administration of oncologic dose ZA. Hematoxylin and eosin (HE) staining and immunofluorescence were used to evaluate mucosal healing. Results: In vitro, LPS did not exacerbate the effects of osteoporosis therapeutic dose of ZA on the proliferation and migration of HOK cells, while aggravated these with the oncologic dose of ZA treatment by inducing mitochondrial dysfunction and oxidative stress via regulating SIRT1 expression. Furthermore, SIRT1 overexpression can alleviate this process. In vivo, local injection of LPS increased the nonunion of mucous membranes in MRONJ and decreased the expression of SIRT1, PGC-1α, and MnSOD. Conclusion: Inflammation aggravates oncologic dose of ZA-induced mitochondrial dysfunction and oxidative stress via a SIRT1-dependent pathway, enhancing the risk of impaired mucosal healing in MRONJ. Our study implies that inflammation becomes a critical risk factor for MRONJ development at higher ZA concentrations. Elucidating the mechanisms of inflammation as a risk factor for mucosal non-healing in MRONJ could inform the development of SIRT1-targeted therapies.

Anandamide modulation of monocyte-derived Langerhans cells: implications for immune homeostasis and skin inflammation.

Introduction: The endocannabinoid system (ECS), named after the chemical compounds found in the cannabis plant, is a regulatory network of neurotransmitters, receptors, and enzymes that plays crucial roles in skin health and disease. Endogenous ligands of the ECS, called endocannabinoids, have proven to be important regulators of immune responses. One of the most prevalent endocannabinoids, arachidonoylethanolamide (also known as anandamide), is known for its anti-inflammatory effects. Langerhans cells (LCs) are the sole antigen-presenting cells present in the human epidermis. They serve as the first line of defense against pathogens and are essential for the skin's specific immune responses and play a critical role in maintaining tissue homeostasis; however, little is known about the effect of endocannabinoids on these cells. Our research aimed to provide the connection between monocyte-derived Langerhans cells (moLCs) and the ECS, shedding light on their collaborative roles in immune homeostasis and inflammation. Methods: Human monocytes were differentiated into moLCs using established protocols. Anandamide was applied during the differentiation process to test its effect on the viability, marker expression, and cytokine production of the cells, as well as in short term treatments for intracellular calcium measurement. TLR ligands applied after the differentiation protocol were used to activate moLCs. The impact of anandamide on the functionality of moLCs was further assessed using differential gene expression analysis of bulk RNA-Seq data, moLC-T cell cocultures, while ELISpot was employed to determine polarization of T cells activated in the aforementioned cocultures. Results: Anandamide did not significantly affect the viability of moLCs up to 10 µM. When applied during the differentiation process it had only a negligible effect on CD207 expression, the prototypic marker of LCs; however, there was an observed reduction in CD1a expression by moLCs. Anandamide had no significant effects on the maturation status of moLCs, nor did it affect the maturation induced by TLR3 and TLR7/8 agonists. MoLCs differentiated in the presence of anandamide did however show decreased production of CXCL8, IL-6, IL-10 and IL-12 cytokines induced by TLR3 and TLR7/8 activation. Anandamide-treated moLCs showed an increased capability to activate naïve T cells; however, not to the level seen with combined TLR agonism. RNA sequencing analysis of moLCs differentiated with anandamide showed modest changes compared to control cells but did reveal an inhibitory effect on oxidative phosphorylation specifically in activated moLCs. Anandamide also promoted the polarization of naïve T cells towards a Th1 phenotype. Discussion: Our results show that anandamide has nuanced effects on the differentiation, maturation, cytokine secretion, metabolism and function of activated moLCs. Among these changes the decrease in CD1a expression on moLCs holds promise to selectively dampen inflammation induced by CD1a restricted T cells, which have been implicated as drivers of inflammation in common inflammatory skin conditions such as psoriasis, atopic dermatitis and contact dermatitis.