Apelin Alleviates Meniscus Endothelial Cell Apoptosis in Osteoarthritis

Osteoarthritis (OA) is a degenerative disease characterized by articular cartilage and/or chondrocyte destruction, and although it has long been considered as a primary disease, the importance of meniscus endothelial cell modulation in the subchondral microenvironment has recently drawn attention. Previous studies have shown that apelin could potentially inhibit cellular apoptosis; however, it remains unclear whether apelin could play a protective role in protecting the endothelium in the OA meniscus. In this study, with the advantages of single-cell RNA sequencing (scRNA-seq) data, in combination with flow cytometry, we identified two endothelial subclusters in the meniscus, featured by high expression of Homeobox A13 (HOXA13) and Ras Protein-Specific Guanine Nucleotide Releasing Factor 2 (RASGRF2), respectively. Compared with control patients, both subclusters decreased in absolute cell numbers and exhibited downregulated APJ endogenous ligand (APLN, coding for apelin) and upregulated apelin receptor (APLNR, coding apelin receptor). Furthermore, we confirmed that in OA, decreased endothelial cell numbers, including both subclusters, were related to intrinsic apoptosis factors: one more relevant to caspase 3 (CASP3) and the other to BH3-Interacting Domain Death agonist (BID). In vitro culturing of meniscal endothelial cells purified from patients proved that apelin could significantly inhibit apoptosis by downregulating these two factors in endothelial cell subclusters, suggesting that apelin could potentially serve as a therapeutic target for patients with OA.

Axin2+ PDL Cells Directly Contribute to New Alveolar Bone Formation in Response to Orthodontic Tension Force

Wnt–β-catenin signaling plays a key role in orthodontic tooth movement (OTM), a common clinical practice for malocclusion correction. However, its targeted periodontal ligament (PDL) progenitor cells remain largely unclear. In this study, we first showed a synchronized increase in Wnt–β-catenin levels and Axin2+ PDL progenitor cell numbers during OTM using immunostaining of β-catenin in wild-type mice and X-gal staining in the Axin2-LacZ knock-in line. Next, we demonstrated time-dependent increases in Axin2+ PDL progenitors and their progeny cell numbers within PDL and alveolar bones during OTM using a one-time tamoxifen-induced Axin2 tracing line ( Axin2CreERT2/+; R26RtdTomato/+). Coimmunostaining images displayed both early and late bone markers (such as RUNX2 and DMP1) in the Axin2Lin PDL cells. Conversely, ablation of Axin2+ PDL cells via one-time tamoxifen-induced diphtheria toxin subunit A (DTA) led to a drastic decrease in osteogenic activity (as reflected by alkaline phosphatase) in PDL and alveolar bone. There was also a decrease in new bone mass and a significant reduction in the mineral apposition rate on both the control side (to a moderate degree) and the OTM side (to a severe degree). Thus, we conclude that the Axin2+ PDL cells (the Wnt-targeted key cells) are highly sensitive to orthodontic tension force and play a critical role in OTM-induced PDL expansion and alveolar bone formation. Future drug development targeting the Axin2+ PDL progenitor cells may accelerate alveolar bone formation during orthodontic treatment.

Hyaluronidase treatment of synovial fluid is required for accurate detection of inflammatory cells and soluble mediators

Background Synovial fluid (SF) is commonly used for diagnostic and research purposes, as it is believed to reflect the local inflammatory environment. Owing to its complex composition and especially the presence of hyaluronic acid, SF is usually viscous and non-homogeneous. In this study, we investigated the importance of homogenization of the total SF sample before subsequent analysis. Methods SF was obtained from the knee of 29 arthritis patients (26 rheumatoid arthritis, 2 osteoarthritis, and 1 juvenile idiopathic arthritis patient) as part of standard clinical care. Synovial fluid was either treated with hyaluronidase as a whole or after aliquoting to determine whether the concentration of soluble mediators is evenly distributed in the viscous synovial fluid. Cytokine and IgG levels were measured by ELISA or Luminex and a total of seven fatty acid and oxylipin levels were determined using LC-MS/MS in all aliquots. For cell analysis, synovial fluid was first centrifuged and the pellet was separated from the fluid. The fluid was subsequently treated with hyaluronidase and centrifuged to isolate remaining cells. Cell numbers and phenotype were determined using flow cytometry. Results In all patients, there was less variation in IgG, 17-HDHA, leukotriene B4 (LTB4), and prostaglandin E2 (PGE2) levels when homogenization was performed before aliquoting the SF sample. There was no difference in variation for cytokines, 15-HETE, and fatty acids arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA). Between 0.8 and 70% of immune cells (median 5%) remained in suspension and were missing in subsequent analyses when the cells were isolated from untreated SF. This percentage was higher for T and B cells: 7–85% (median 22%) and 7–88% (median 23 %), respectively. Conclusions Homogenization of the entire SF sample leads to less variability in IgG and oxylipin levels and prevents erroneous conclusions based on incomplete isolation of synovial fluid cells.

Metformin and Calcitriol Enhance 5-Fluorouracil Tumoricidal Effects in Colon Cancer By Modulating The PI3K/Akt/PTEN/mTOR Network

Purpose: Chemoresistance to 5-Fluorouracil (5-FU) is common during colorectal cancer (CRC) treatment. This study measured the chemotherapeutic effects of 5-FU, calcitriol, and/or metformin single/dual/triple regimens as complementary/alternative therapies. Methods: Ninety male mice were divided into: negative and positive (PC) controls, 5-FU, Cal, Met, 5-FU/Cal, 5-FU/Met, Cal/Met, and 5-FU/Cal/Met groups. Treatments lasted four weeks following CRC induction by azoxymethane. The therapeutic regimens were also applied in the SW480 and SW620 CRC cell lines. Results: The PC mice had abundant tumours, markedly elevated proliferation markers (survivin/CCND1) and PI3K/Akt/mTOR alongside reduced p21/PTEN/Cytochrome-C/Caspase-3 and apoptosis. All therapies reduced tumour numbers, with 5-FU/Cal/Met most prominent regimen. All protocols also decreased cell proliferation markers, inhibited PI3K/Akt/mTOR molecules, increased pro-apoptotic molecules with apoptosis index, and 5-FU/Cal/Met revealed the strongest anti-cancer effects. In vitro, all therapies equally induced G1-phase arrest in SW480 cells, whereas metformin-alone showed maximal SW620 cell numbers in G0/G1-phase. 5-FU/Met co-therapy also showed the highest apoptotic SW480 cell numbers (13%), whilst 5-FU/Cal/Met disclosed the lowest percentage (81%) of viable SW620 cells. Moreover, 5-FU/Cal/Met revealed maximal inhibitions of cell cycle inducers (CCND1/CCND3), cell survival (BCL2) and the PI3K/Akt/mTOR molecules alongside highest expression of cell cycle inhibitors (p21/p27), pro-apoptotic markers (BAX/Cytochrome-C/Caspase-3), and PTEN in both cell lines. Conclusions: Metformin monotherapy was superior to calcitriol, whereas the 5-FU/metformin protocol showed better anti-cancer effects relative to the other dual therapies. However, the 5-FU/Cal/Met approach displayed the best in vivo and in vitro tumoricidal effects related to cell cycle arrest and apoptosis, justifiably by enhanced modulations of the PI3K/PTEN/Akt/mTOR pathway.

Delta/Jagged-mediated Notch signaling induces the differentiation of agr2-positive epidermal mucous cells in zebrafish embryos

Teleosts live in aquatic habitats, where they encounter ionic and acid-base fluctuations as well as infectious pathogens. To protect from these external challenges, the teleost epidermis is composed of living cells, including keratinocytes and ionocytes that maintain body fluid ionic homeostasis, and mucous cells that secret mucus. While ionocyte progenitors are known to be specified by Delta-Notch-mediated lateral inhibition during late gastrulation and early segmentation, it remains unclear how epidermal mucous cells (EMCs) are differentiated and maintained. Here, we show that Delta/Jagged-mediated activation of Notch signaling induces the differentiation of agr2-positive (agr2+) EMCs in zebrafish embryos during segmentation. We demonstrated that agr2+ EMCs contain cytoplasmic secretory granules and express muc5.1 and muc5.2. Reductions in agr2+ EMC number were observed in mib mutants and notch3 MOs-injected notch1a mutants, while increases in agr2+ cell number were detected in notch1a- and X-Su(H)/ANK-overexpressing embryos. Treatment with γ-secretase inhibitors further revealed that Notch signaling is required during bud to 15 hpf for the differentiation of agr2+ EMCs. Increased agr2+ EMC numbers were also observed in jag1a-, jag1b-, jag2a- and dlc-overexpressing, but not jag2b-overexpressing embryos. Meanwhile, reductions in agr2+ EMC numbers were detected in jag1a morphants, jag1b mutants, jag2a mutants and dlc morphants, but not jag2b mutants. Reduced numbers of pvalb8-positive epidermal cells were also observed in mib or jag2a mutants and jag1a or jag1b morphants, while increased pvalb8-positive epidermal cell numbers were detected in notch1a-overexpressing, but not dlc-overexpressing embryos. BrdU labeling further revealed that the agr2+ EMC population is maintained by proliferation. Cell lineage experiments showed that agr2+ EMCs are derived from the same ectodermal precursors as keratinocytes or ionocytes. Together, our results indicate that specification of agr2+ EMCs in zebrafish embryos is induced by DeltaC/Jagged-dependent activation of Notch1a/3 signaling, and the cell population is maintained by proliferation.

Fucoidan Independently Enhances Activity in Human Immune Cells and Has a Cytostatic Effect on Prostate Cancer Cells in the Presence of Nivolumab

Fucoidan compounds may increase immune activity and are known to have cancer inhibitory effects in vitro and in vivo. In this study, we aimed to investigate the effect of fucoidan compounds on ex vivo human peripheral blood mononuclear cells (PBMCs), and to determine their cancer cell killing activity both solely, and in combination with an immune-checkpoint inhibitor drug, Nivolumab. Proliferation of PBMCs and interferon gamma (IFNg) release were assessed in the presence of fucoidan compounds extracted from Fucus vesiculosus, Undaria pinnatifida and Macrocystis pyrifera. Total cell numbers and cell killing activity were assessed using a hormone resistant prostate cancer cell line, PC3. All fucoidan compounds activated PBMCs, and increased the effects of Nivolumab. All fucoidan compounds had significant direct cytostatic effects on PC3 cells, reducing cancer cell numbers, and PBMCs exhibited cell killing activity as measured by apoptosis. However, there was no fucoidan mediated increase in the cell killing activity. In conclusion, fucoidan compounds promoted proliferation and activity of PBMCs and added to the effects of Nivolumab. Fucoidan compounds all had a direct cytostatic effect on PC3 cells, as shown through their proliferation reduction, while their killing was not increased.

Leukemia Inhibitory Factor Stimulates Primitive Endoderm Expansion in the Bovine Inner Cell Mass

Previous work determined that bovine interleukin-6 (IL6) increases inner cell mass (ICM), primitive endoderm (PE), and total cell number in in vitro produced (IVP) bovine blastocysts. Another IL6 family member, leukemia inhibitory factor (LIF), has the potential to produce the same effects of IL6 due to the presence of its receptor in bovine blastocysts. We compared the abilities of LIF and IL6 to increase ICM cell numbers in day 7, 8, and 9 IVP bovine blastocysts. Supplementation with 100 ng/ml LIF from day 5 onward improved blastocyst formation rates on days 7 and 8 similar to what was observed when supplementing 100 ng/ml IL6. However, LIF supplementation did not cause an increase in ICM numbers like was observed after supplementing IL6. On day 9, increases in PE cell numbers were detected after LIF supplementation, but 300 ng/ml LIF was required to achieve the same effect on PE numbers that was observed by providing 100 ng/ml IL6. Collectively, these results show that LIF can mimic at least some of the effects of IL6 in bovine blastocyst.

Severe Gestational Low-Protein Intake Impacts Hippocampal Cellularity, Tau, and Amyloid-β Levels, and Memory Performance in Male Adult Offspring: An Alzheimer-Simile Disease Model?

Background: Maternal undernutrition has been associated with psychiatric and neurological disorders characterized by learning and memory impairment. Objective: Considering the lack of evidence, we aimed to analyze the effects of gestational protein restriction on learning and memory function associated with hippocampal cell numbers and neurodegenerative protein content later in life. Methods: Experiments were conducted in gestational low- (LP, 6% casein) or regular-protein (NP, 17% casein) diet intake offspring. Behavioral tests, isolated hippocampal isotropic fractionator cell studies, immunoblotting, and survival lifetime were observed. Results: The birthweight of LP males is significantly reduced relative to NP male progeny, and hippocampal mass increased in 88-week-old LP compared to age-matched NP offspring. The results showed an increased proximity measure in 87-week-old LP compared to NP offspring. Also, LP rats exhibited anxiety-like behaviors compared to NP rats at 48 and 86-wk of life. The estimated neuron number was unaltered in LP rats; however, non-neuron cell numbers increased compared to NP progeny. Here, we showed unprecedented hippocampal deposition of brain-derived neurotrophic factor, amyloid-β peptide (Aβ), and tau protein in 88-week-old LP relative to age-matched NP offspring. Conclusion: To date, no predicted studies showed changes in hippocampal morphological structure in maternal protein-restricted elderly offspring. The current data suggest that gestational protein restriction may accelerate hippocampal function loss, impacting learning/memory performance, and supposedly developing diseases similar to Alzheimer’s disease (AD) in elderly offspring. Thus, we propose that maternal protein restriction could be an elegant and novel method for constructing an AD-like model in adult male offspring.

The Effect of High-Fat Diet and Exercise Intervention on the TNF-α Level in Rat Spleen

High-fat diet (HFD) consumption can trigger chronic inflammation in some tissues. However, it remains unclear if HFD induces chronic inflammation in the spleen. This investigation aims to address the effect of HFD consumption and exercise intervention on the level of tumor necrosis factor alpha (TNF-α) in the spleen. Rats were subjected to HFD feeding and/or moderate-intensity treadmill running. The TNF-α levels in plasma and spleen were detected by ELISA. The mass and total cell numbers of the spleen were measured. In addition, the expression of TNF-α and its relevant gene mRNAs in macrophages from the spleen were analyzed by qRT-PCR. We found that HFD consumption did not significantly affect the mass and total cell numbers of the spleen. However, HFD consumption significantly increased splenic TNF-α level, the expression of TNF-α, toll-like receptor 4, and nuclear factor κB p65 mRNAs. In contrast, the expression of nicotinic acetylcholine receptor alpha 7 subunit (α7nAChR) mRNA in macrophages was downregulated. Additionally, exercise abolished the increase in splenic TNF-α level as well as the abnormal expression of TNF-α and related gene mRNAs in macrophages in HFD-fed rats. In conclusion, our results reveal that HFD consumption increases TNF-α level in the spleen, which is along with upregulation of the expression of TLR4 and NF-κB mRNAs as well as downregulation of the expression of α7nAChR mRNA in splenic macrophages in rats. Exercise abolished detrimental effects of HFD on TNF-α level in the spleen and prevented abnormal expression of these genes in the macrophages from rat spleen.