Metformin Attenuates the Inflammatory Response via the Regulation of Synovial M1 Macrophage in Osteoarthritis.

Osteoarthritis (OA), the most common chronic inflammatory joint disease, is characterized by progressive cartilage degeneration, subchondral bone sclerosis, synovitis, and osteophyte formation. Metformin, a hypoglycemic agent used in the treatment of type 2 diabetes, has been evidenced to have anti-inflammatory properties to treat OA. It hampers the M1 polarization of synovial sublining macrophages, which promotes synovitis and exacerbates OA, thus lessening cartilage loss. In this study, metformin prevented the pro-inflammatory cytokines secreted by M1 macrophages, suppressed the inflammatory response of chondrocytes cultured with conditional medium (CM) from M1 macrophages, and mitigated the migration of M1 macrophages induced by interleukin-1ß (IL-1ß)-treated chondrocytes in vitro. In the meantime, metformin reduced the invasion of M1 macrophages in synovial regions brought about by the destabilization of medial meniscus (DMM) surgery in mice, and alleviated cartilage degeneration. Mechanistically, metformin regulated PI3K/AKT and downstream pathways in M1 macrophages. Overall, we demonstrated the therapeutic potential of metformin targeting synovial M1 macrophages in OA.

MRL/MpJ Mice Resist to Age-Related and Long-Term Ovariectomy-Induced Bone Loss: Implications for Bone Regeneration and Repair.

Osteoporosis and age-related bone loss increase bone fracture risk and impair bone healing. The need for identifying new factors to prevent or treat bone loss is critical. Previously, we reported that young MRL/MpJ mice have superior bone microarchitecture and biomechanical properties as compared to wild-type (WT) mice. In this study, MRL/MpJ mice were tested for resistance to age-related and long-term ovariectomy-induced bone loss to uncover potential beneficial factors for bone regeneration and repair. Bone tissues collected from 14-month-old MRL/MpJ and C57BL/6J (WT) mice were analyzed using micro-CT, histology, and immunohistochemistry, and serum protein markers were characterized using ELISAs or multiplex assays. Furthermore, 4-month-old MRL/MpJ and WT mice were subjected to ovariectomy (OV) or sham surgery and bone loss was monitored continuously using micro-CT at 1, 2, 4, and 6 months (M) after surgery with histology and immunohistochemistry performed at 6 M post-surgery. Sera were collected for biomarker detection using ELISA and multiplex assays at 6 M after surgery. Our results indicated that MRL/MpJ mice maintained better bone microarchitecture and higher bone mass than WT mice during aging and long-term ovariectomy. This resistance of bone loss observed in MRL/MpJ mice correlated with the maintenance of higher OSX+ osteoprogenitor cell pools, higher activation of the pSMAD5 signaling pathway, more PCNA+ cells, and a lower number of osteoclasts. Systemically, lower serum RANKL and DKK1 with higher serum IGF1 and OPG in MRL/MpJ mice relative to WT mice may also contribute to the maintenance of higher bone microarchitecture during aging and less severe bone loss after long-term ovariectomy. These findings may be used to develop therapeutic approaches to maintain bone mass and improve bone regeneration and repair due to injury, disease, and aging.

Two reactive behaviors of chondrocytes in an IL-1ß-induced inflammatory environment revealed by the single-cell RNA sequencing.

OBJECTIVE: To investigate the heterogeneous responses of in vitro expanded chondrocytes, which were cultured in an interleukin (IL)-1ß -induced inflammatory environment. METHOD: Human articular chondrocytes were expanded, in vitro, for 13 days and treated with IL-1ß for 0, 24, and 48 h. Cells were collected and subjected to single-cell RNA sequencing. Multiple bioinformatics tools were used to determine the signatures that define chondrocyte physiology. RESULTS: Two major cell clusters with distinct expression patterns were identified at the initial phase and were with heterogeneous variation that coincides with inflammation progress. They transformed into two terminal cell clusters one of which exhibited OA-phenotype and proinflammatory characteristics through two paths, "response-to-inflammation" and "atypical response-to-inflammation", respectively. The involved cell clusters exhibited intrinsic relationship with cell types within native cartilage from OA patients. Genes controlling cell transformation to OA-phenotype were relating to the tumor necrosis factor (TNF) signaling pathway via NFKB, up-regulated KRAS signaling and the IL2/STAT5 signaling pathway and pathways relating to apoptosis and reactive oxygen species. CONCLUSION: The in vitro expanded chondrocytes under IL-1ß-induced inflammatory progression behave heterogeneously. One of the initial cell clusters could transform into a proinflammatory subpopulation through a termed response-to-inflammation path, which may serve as the core target to alleviate OA progression.

Inhibiting Monoacylglycerol Lipase Suppresses RANKL-Induced Osteoclastogenesis and Alleviates Ovariectomy-Induced Bone Loss.

Osteoporosis is a common chronic metabolic bone disease characterized by reduced trabecular bone and increased bone fragility. Monoacylglycerol lipase (MAGL) is a lipolytic enzyme to catalyze the hydrolysis of monoglycerides and specifically degrades the 2-arachidonoyl glycerol (2-AG). Previous studies have identified that 2-AG is the mainly source for arachidonic acid and the most abundant endogenous agonist of cannabinoid receptors. Considering the close relationship between inflammatory mediators/cannabinoid receptors and bone metabolism, we speculated that MAGL may play a role in the osteoclast differentiation. In the present study, we found that MAGL protein expression increased during osteoclast differentiation. MAGL knockdown by adenovirus-mediated shRNA in bone marrow-derived macrophages demonstrated the suppressive effects of MAGL on osteoclast formation and bone resorption. In addition, pharmacological inhibition of MAGL by JZL184 suppressed osteoclast differentiation, bone resorption, and osteoclast-specific gene expression. Activation of the Mitogen-activated protein kinase (MAPK) and nuclear factor κB (NF-κB) pathways was inhibited by JZL184 and deletion of MAGL. Our in vivo study indicated that JZL184 ameliorated bone loss in an ovariectomized mouse model. Furthermore, overexpressing H1 calponin partially alleviated the inhibition caused by JZL184 or MAGL deletion on osteoclastogenesis. Therefore, we conclude that targeting MAGL may be a novel therapeutic strategy for osteoporosis.

Gender differences in tibial fractures healing in normal and muscular dystrophic mice.

Duchenne muscular dystrophy (DMD) patients have a high fracture risk and poor fracture healing. The dystrophin-/- (mdx) mouse is a murine model of DMD and exhibits delayed bone fracture healing. Since our research team has shown that adult stem cells, such as muscle-derived stem cells, display a gender difference in their osteogenic potential with the male cells being more osteogenic, we hypothesize that a potential gender differences may exist during bone healing in normal and mdx mice. To test this hypothesis, wild-type (WT) and mdx mice underwent tibial fracture surgery and microCT live scanning biweekly. The mice were sacrificed at 6 weeks post-surgery and the calluses were collected for histological analysis. To further investigate the mechanism, another two sets of mice were sacrificed at 10 days after fracture for RNA extraction and gene expression analysis and histology. MicroCT results showed, at 6 weeks post- surgery, the calluses were larger but showed less remodeling in both normal and mdx male mice when compared to females, at the same time point. However, females had higher callus bone volume density and an increase in osteoclast (OCs) number. At 10 days after fracture surgery, male mice had formed larger calluses, whereas females formed well-remodeled calluses with more osteoblasts and a greater bone area for both WT and mdx mice. Higher IGF-1 expression was observed in male mdx mice when compared to their female counterparts, whereas female WT mice had higher BMP-9 expression when compared to WT males. In conclusion, male mice formed larger bone calluses than females during tibial fracture healing for both WT and mdx mice. This may be attributed to higher IGF-1 expression, activation of Wnt/ß-catennin signaling pathway and greater OB numbers during callus formation. Female mice achieved better bone remodeling in the regenerated bone with higher bone quality due to increased OC numbers that promote faster remodeling of the fracture calluses, and higher BMP-9 expression levels. Therefore, gender is one of many factors that need to be considered for both animal and human bone research.

Association between abdominal obesity and liver steatosis and fibrosis among patients with chronic hepatitis B measured by Fibroscan.

The present cross-sectional study aimed to assess hepatic fibrosis in chronic hepatitis B (CHB) patients with abdominal obesity and to explore the associated indicators. A total of 615 CHB patients were enrolled and 287 of them had abdominal obesity. The liver stiffness value was measured using Fibroscan. The diagnosis of liver fibrosis was confirmed by a liver stiffness value of >7.4 kPa, and a value of >10.6 kPa was considered to indicate advanced liver fibrosis. The Fibroscan results suggested that the liver stiffness value in patients with abdominal obesity was significantly higher than that in patients without abdominal obesity (9.94±11.59 vs. 7.47±7.58 kPa; P=0.002). The proportions of patients with liver fibrosis and advanced liver fibrosis among patients with abdominal obesity were significantly higher than those among patients without abdominal obesity (P=0.011). Multivariate logistic regression analysis indicated that a high aspartate aminotransferase (AST) level [odds ratio (OR)=2.991; P<0.001], smoking (OR=2.002; P=0.019) and diabetes mellitus (OR=2.047; P=0.029) were independent indicators for liver fibrosis in CHB patients with abdominal obesity. Furthermore, a high AST level (OR=1.024; P<0.001), alcohol consumption (OR=1.994; P=0.032) and diabetes mellitus (OR=1.977; P=0.045) were independent indicators for advanced hepatic fibrosis. The indicators associated with liver steatosis included high body weight (OR=1.113; P<0.001) and high diastolic blood pressure (OR=1.079; P=0.002). In conclusion, the present study indicated that abdominal obesity significantly exacerbates liver fibrosis in CHB patients. For CHB patients with abdominal obesity and a risk of developing liver fibrosis, priority screening and timely intervention should be provided.

Characterization of articular cartilage homeostasis and the mechanism of superior cartilage regeneration of MRL/MpJ mice.

This study investigated articular cartilage (AC) homeostasis and different signaling pathways involved in the superior cartilage regeneration of Murphy Roths large (MRL/MpJ) mice previously reported. We collected uninjured and destabilized medial meniscus (DMM)-injured knees from 8-wk-old C57BL/6J and MRL/MpJ mice. We used micro-computed tomography (microCT), histology, and immunohistochemistry to evaluate AC homeostasis and repair. We used the ear punch model to investigate the role of angiogenesis and inflammation in the superior healing of MRL/MpJ mice. We found fewer ß-catenin and more pSMAD5 positive cells in the uninjured AC of MRL/MpJ mice than that from C57BL/6J mice. MRL/MpJ mice exhibited better AC repair in DMM-induced OA, as indicated by microCT results, Alcian blue, and Safranin O staining. Mechanistically, fewer ß-catenin, pSMAD2-, pSMAD3-, a disintegrin and metalloproteinase with thrombospondin motifs 4-, matrix metalloproteinase (MMP) 9-, and MMP13-positive cells and more proliferating cell nuclear antigen- and pSMAD5-positive cells were found in the DMM-injured AC in MRL/MpJ mice than in normal mice. The accelerated ear wound healing of MRL/MpJ mice correlated with enhanced angiogenesis and macrophage polarization toward the M2a phenotype through elevated IL-10 and IL-4 expressing cells. Collectively, our study revealed that down-regulation of pSMAD2/3, ß-catenin, and MMPs and up-regulation of pSMAD5 and M2a macrophage polarization contribute to the enhanced cartilage repair observed in MRL/MpJ mice.-Deng, Z., Gao, X., Sun, X., Amra, S., Lu, A., Cui, Y., Eltzschig, H. K., Lei, G., Huard, J. Characterization of articular cartilage homeostasis and the mechanism of superior cartilage regeneration of MRL/MpJ mice.

Dynamic Changes of the Aspartate Aminotransferase-to-Platelet Ratio and Transient Elastography in Predicting a Histologic Response in Patients With Chronic Hepatitis B After Entecavir Treatment.

OBJECTIVES: To evaluate the dynamic changes of the aspartate aminotransferase (AST)-to-platelet ratio and transient elastography (FibroScan; Echosens, Paris, France) in predicting a histologic response in patients with chronic hepatitis B (CHB) after entecavir treatment. METHODS: A total of 148 patients with CHB were enrolled. Patient information was collected. All patients received liver biopsy and FibroScan before and after 96 weeks of entecavir treatment. RESULTS: Baseline liver biopsy results showed that there were 7 patients without liver fibrosis (fibrosis stage F0; 4.7%), 51 patients with mild liver fibrosis (F1; 34.5%), and 90 patients with advanced liver fibrosis (>F1; 60.9%). The liver stiffness value and AST-to-platelet ratio increased significantly as the METAVIR score of the patients increased from F0 to F4 (P < .001). After antiviral therapy for 96 weeks, the average liver stiffness value measured by FibroScan and the AST-to-platelet ratio showed a significant decrease. When we use a decreased liver stiffness value to predict a histologic response, the area under the receiver operating characteristic curve was 0.70 (95% confidence interval, 0.61-0.79; P < .001), and the sensitivity and specificity were 74.3% and 68.8%, respectively. The decrease of the AST-to-platelet ratio also could predict the histologic response of patients with CHB; the area under the receiver operating characteristic curve was 0.77 (95% confidence interval, 0.69-0.86; P < .001) with sensitivity of 76.2% and specificity of 70.2%. A multivariate analysis indicated that a high hepatitis B virus DNA viral load (odds ratio, 1.44; P = .04) and high METAVIR score (odds ratio, 1.38; P = .02) were independent risk factors for the histologic response. CONCLUSIONS: Both the AST-to-platelet ratio and FibroScan value can effectively predict a histologic response in patients with CHB during entecavir treatment. Therefore, they can be used to monitor these patients during antiviral treatment to avoid multiple liver biopsies.

Lactate transport facilitates neurite outgrowth.

How glia affect neurite outgrowth during neural development has not been well elucidated. In the present study, we found that disruption of lactate production using 1,4-dideoxy-1,4-imino-D-arabinitol (DAB) and isofagomine significantly interfered with neurite outgrowth and that exogenous application of L-lactate rescued neurite growth failure. Monocarboxylate transporter-2-knockout, which blocked the lactate shuttle in neurons, showed a remarkable decrease in the length of axons and dendrites. We further demonstrated that Akt activity was decreased while glycogen synthase kinase 3ß (GSK3ß) activity was increased after astrocytic glycogen phosphorylase blockade. Additionally, GSK3ßSer9 mutation reversed neurite growth failure caused by DAB and isofagomine. Our results suggested that lactate transportation played a critical role in neural development and disruption of the lactate shuttle in quiescent condition also affected neurite outgrowth in the central nervous system.

Cyclooxygenase-2 deficiency impairs muscle-derived stem cell-mediated bone regeneration via cellular autonomous and non-autonomous mechanisms.

This study investigated the role of cyclooxygenase-2 (COX-2) expression by donor and host cells in muscle-derived stem cell (MDSC)-mediated bone regeneration utilizing a critical size calvarial defect model. We found that BMP4/green fluorescent protein (GFP)-transduced MDSCs formed significantly less bone in COX-2 knock-out (Cox-2KO) than in COX-2 wild-type (WT) mice. BMP4/GFP-transduced Cox-2KO MDSCs also formed significantly less bone than transduced WT MDSCs when transplanted into calvarial defects created in CD-1 nude mice. The impaired bone regeneration in the Cox-2KO MDSCBMP4/GFP group is associated with downregulation of BMP4-pSMAD1/5 signaling, decreased osteogenic differentiation and lowered proliferation capacity after transplantation, compared with WT MDSCBMP4/GFP cells. The Cox-2KO MDSCBMP4/GFP group demonstrated a reduction in cell survival and direct osteogenic differentiation in vitro These effects were mediated in part by the downregulation of Igf1 and Igf2. In addition, the Cox-2KO MDSCBMP4/GFP cells recruited fewer macrophages than the WT MDSC/BMP4/GFP cells in the early phase after injury. We concluded that the bone regeneration capacity of Cox-2KO MDSCs was impaired because of a reduction in cell proliferation and survival capacities, reduction in osteogenic differentiation and a decrease in the ability of the cells to recruit host cells to the injury site.

Protein phosphatase 2A (PP2A) activation promotes axonal growth and recovery in the CNS.

Current treatments to restore neurological deficits caused by axonal disconnection following central nervous system (CNS) injury are extremely limited. Protein phosphatase 2A (PP2A), one of the main serine-threonine phosphatases in mammalian cells, dephosphorylates collapsin response mediator protein-2 (CRMP2) in the developing CNS. In our study, we found that the major CNS inhibiting substrates, including chondroitin sulfate proteoglycans (CSPGs) and myelin associated glycoproteins (MAG), activated epidermal growth factor receptor (EGFR), but inactivated PP2A and downstream CRMP2. Both EGFR inactivation and PP2A activation promoted axon elongation in vitro in the presence of inhibitory substrates. EGFR blockage by AG1478 selectively attenuated the inactive form of PP2A in pY307 phosphorylation, thus increasing PP2A activity. EGFR activation by EGF attenuated PP2A activity, whereas mutation of Y307 to phenylalanine abolished the effect. Furthermore, PP2A activity was down-regulated immediately after spinal cord injury (SCI) in rats. Chronic application of d-erythro-sphingosine (DES), the PP2A agonist, to spinal cord-lesioned rats enhanced the activity of this phosphatase and dephosphorylated CRMP2 around the lesion. PP2A activation induced significant axon sprouting in the lesioned spinal cord and promoted function recovery after SCI. These findings suggest that PP2A works downstream of EGFR and dephosphorylates CRMP2 after CNS injury. Therefore, therapies targeting PP2A may be effective following SCI.

Transdifferentiation via transcription factors or microRNAs: Current status and perspective.

Transdifferentiation as a new approach for obtaining the ideal cells for transplantation has gradually become a hot research topic. Compared with the induced pluripotent stem cells technique, transdifferentiation may have better efficiency and safety. Although the mechanism of transdifferentiation is still unknown, many studies have achieved transformation of one cell type to another through transcription factors or microRNA. The current major strategy for transdifferentiation is via transcription factors; however, there are some safety issues with the use of transcription factors. In contrast, microRNA as a novel tool for inducing transdifferentiation through post-transcriptional regulation may be more safe and efficient. In addition, the present transdifferentiation strategies involve obtaining the terminal cell directly, so the amount of cells produced may not be sufficient and they may have low capacity for cell immigration and integration. Therefore, an indirect transdifferentiation strategy for producing unipotent cells is ideal as it can preserve the proliferation capacity and differentiation pathway.

MicroRNA-34c Downregulation Ameliorates Amyloid-ß-Induced Synaptic Failure and Memory Deficits by Targeting VAMP2.

MicroRNAs (miRNAs) are small (∼22-nucleotide [nt]) noncoding RNAs that regulate biological processes at the post-transcriptional level. Dysregulation of specific miRNAs leads to impaired synaptic plasticity resulting in Alzheimer's disease (AD). Amyloid-ß (Aß) accumulation is the most important pathogenic factor for AD development. Therefore, focusing on Aß-targeted miRNAs may have therapeutic implications for AD. We found that miR-34c, a miRNA that was previously reported to be upregulated in a transgenic AD model and patients, was significantly increased in hippocampal neurons exposed to Aß. Western blots and luciferase assay confirmed that increased miR-34c was closely related to VAMP2 reduction. Furthermore, miR-34c blockade upregulated VAMP2 expression and rescued synaptic failure as well as learning and memory deficits caused by Aß. The Aß-miR-34c-VAMP2 pathway mediates the sustained VAMP2 reduction in AD patients and provides a novel underlying epigenetic mechanism for attenuation of Aß toxicity in AD.

Helicobacter pylori filtrate induces Alzheimer-like tau hyperphosphorylation by activating glycogen synthase kinase-3ß.

Abnormal hyperphosphorylation of microtubule-associated protein tau is involved in the pathogenesis of several neurodegenerative disorders including Alzheimer's disease (AD). Helicobacter pylori (H. pylori) infection has been reported to be related with a high risk of AD, but the direct laboratory evidence is lacking. Here we explored the effect of H. pylori infection on tau phosphorylation. The results showed that H. pylori filtrate induced significant tau hyperphosphorylation at several AD-related tau phosphorylation sites, such as Thr205, Thr231, and Ser404, both in mouse neuroblastoma N2a cells and rat brains with activation of glycogen synthase kinase-3ß (GSK-3ß). Application of GSK-3 inhibitors efficiently attenuated the H. pylori-induced tau hyperphosphorylation. Our data provide evidence supporting the role of H. pylori infection in AD-like tau pathology, suggesting that H. pylori eradication may be beneficial in the prevention of tauopathy.

Zinc induces protein phosphatase 2A inactivation and tau hyperphosphorylation through Src dependent PP2A (tyrosine 307) phosphorylation.

The activity of protein phosphatase (PP) 2A is downregulated and promotes the hyperphosphorylation of tau in the brains of Alzheimer's disease (AD), but the mechanism for PP2A inactivation has not been elucidated. We have reported that PP2A phosphorylation at tyrosine 307 (Y307) is involved in PP2A inactivation. Here, we further studied the upstream mechanisms for PP2A phosphorylation and inactivation. We found that zinc, a heavy metal ion that is widely distributed in the normal brain and accumulated in the susceptible regions of AD brain, could induce PP2A inhibition, phosphorylation of PP2A at Y307 and tau hyperphosphorylation both in rat brains and cultured N2a cells, while zinc chelating prevented these changes completely. Upregulation of PP2A chemically or genetically attenuated zinc-induced tau hyperphosphorylation, whereas mutation of Y307 to phenylalanine abolished the zinc-induced tyrosine phosphorylation and inactivation of PP2A. Zinc could activate Src, while PP2, a specific Src family kinases inhibitor, attenuated zinc-induced PP2A phosphorylation and inactivation, indicating that zinc induces PP2A Y307 phosphorylation and inactivation through Src activation. In human tau transgenic mice, zinc chelator rescued PP2A activity, prevented Src activation, and reduced hyperphosphorylated and insoluble tau levels. We concluded that zinc induces PP2A inactivation and tau hyperphosphorylation through Src-dependent pathway, regulation of zinc homeostasis may be a promising therapeutic for AD and the related tauopathies.