Wijnen, André J. van

Mesenchymal stem/stromal cells (MSCs) facilitate repair in experimental diabetic kidney disease (DKD). However, the hyperglycemic and uremic milieu may diminish regenerative capacity of patient-derived therapy. We hypothesized that DKD reduces human MSC paracrine function. Adipose-derived MSC from 38 participants with DKD and 16 control subjects were assessed for cell surface markers, trilineage differentiation, RNA sequencing (RNA-seq), in vitro function (coculture or conditioned medium experiments with T cells and human kidney cells [HK-2]), secretome profile, and cellular senescence abundance. The direction of association between MSC function and patient characteristics were also tested. RNA-seq analysis identified 353 differentially expressed genes and downregulation of several immunomodulatory genes/pathways in DKD-MSC versus Control-MSC. DKD-MSC phenotype, differentiation, and tube formation capacity were preserved, but migration was reduced. DKD-MSC with and without interferon-γ priming inhibited T-cell proliferation greater than Control-MSC. DKD-MSC medium contained higher levels of anti-inflammatory cytokines (indoleamine 2,3-deoxygenase 1 and prostaglandin-E2) and prorepair factors (hepatocyte growth factor and stromal cell–derived factor 1) but lower IL-6 versus control-MSC medium. DKD-MSC medium protected high glucose plus transforming growth factor-β–exposed HK-2 cells by reducing apoptotic, fibrotic, and inflammatory marker expression. Few DKD-MSC functions were affected by patient characteristics, including age, sex, BMI, hemoglobin A1c, kidney function, and urine albumin excretion. However, senescence-associated β-galactosidase activity was lower in DKD-MSC from participants on metformin therapy. Therefore, while DKD altered the transcriptome and migratory function of culture-expanded MSCs, DKD-MSC functionality, trophic factor secretion, and immunomodulatory activities contributing to repair remained intact. These observations support testing of patient-derived MSC therapy and may inform preconditioning regimens in DKD clinical trials.

An efficient musculoskeletal system depends on the precise assembly and coordinated growth and function of muscles, skeleton, and tendons. However, the mechanisms that drive integrated musculoskeletal development and coordinated growth and differentiation of each of these tissues are still being uncovered. Epigenetic modifiers have emerged as critical regulators of cell fate differentiation, but so far almost nothing is known about their roles in tendon biology. Previous studies have shown that epigenetic modifications driven by Enhancer of zeste homolog 2 (EZH2), a major histone methyltransferase, have significant roles in vertebrate development including skeletal patterning and bone formation. We now find that targeting Ezh2 through the limb mesenchyme also has significant effects on tendon and muscle patterning, likely reflecting the essential roles of early mesenchymal cues mediated by Ezh2 for coordinated patterning and development of all tissues of the musculoskeletal system. Conversely, loss of Ezh2 in the tendon cells did not disrupt overall tendon structure or collagen organization suggesting that tendon differentiation and maturation are independent of Ezh2 signaling.

Abstract ObjectiveChondrocyte apoptosis plays a vital role in osteoarthritis (OA) progression. Angelica sinensis polysaccharide (ASP), a traditional chinese medicine, possesses anti-inflammatory and anti-apoptotic properties in chondrocytes. This study aimed to determine the protective role of ASP on sodium nitroprusside (SNP)-induced chondrocyte apoptosis, and explore the underlying mechanism.MethodHuman primary chondrocytes isolated from articular cartilage of OA patients were treated with SNP alone or in combination with different doses of ASP. Cell viability and apoptosis were assessed, and apoptosis-related proteins including Bcl-2 and Bax were detected. Autophagy levels were evaluated by light chain 3 (LC3)II immunofluorescence staining, mRFP-GFP-LC3 fluorescence localisation and western blot (LC3II, p62, Beclin-1, Atg5). Meanwhile, activation of ERK 1/2 pathway was determined by western blot. The autophagy inhibitors, 3-Methyladenine (3-MA), chloroquine (CQ) and a specific inhibitor of ERK1/2, SCH772984, were used respectively to confirm the autophagic effect of ASP.ResultsThe results showed that SNP-induced chondrocyte apoptosis was significantly rescued by ASP, whereas ASP alone promoted chondrocytes proliferation. The anti-apoptotic effect of ASP was related to the enhanced autophagy and depended on the activation of ERK1/2 pathway.ConclusionASP markedly rescued SNP-induced apoptosis by activating ERK1/2-dependent autophagy in chondrocytes, and it made ASP a potential therapeutic supplementation for OA treatment.

ABSTRACT Use of the selective estrogen receptor modulator Tamoxifen (TAM) is a mainstay to induce conditional expression of Cre recombinase in transgenic laboratory mice. To excise β‐catenin fl/fl in 28‐day‐old male and female Prrx1‐CreER/β‐catenin fl/fl mice (C57BL/6), we utilized TAM at 150 mg/kg; despite β‐catenin knockout in MSC, we found a significant increase in trabecular and cortical bone volume in all genders. Because TAM was similarly anabolic in KO and control mice, we investigated a dose effect on bone formation by treating wild‐type mice (WT C57BL/6, 4 weeks) with TAM (total dose 0, 20, 40, 200 mg/kg via four injections). TAM increased bone in a dose‐dependent manner analyzed by micro–computed tomography (μCT), which showed that, compared to control, 20 mg/kg TAM increased femoral bone volume fraction (bone volume/total volume [BV/TV]) (21.6% ± 1.5% to 33% ± 2.5%; 153%, p < 0.005). With TAM 40 mg/kg and 200 mg/kg, BV/TV increased to 48.1% ± 4.4% (223%, p < 0.0005) and 58% ± 3.8% (269%, p < 0.0001) respectively, compared to control. Osteoblast markers increased with 200 mg/kg TAM: Dlx5 (224%, p < 0.0001), Alp (166%, p < 0.0001), Bglap (223%, p < 0.0001), and Sp7 (228%, p < 0.0001). Osteoclasts per bone surface (Oc#/BS) nearly doubled at the lowest TAM dose (20 mg/kg), but decreased to <20% control with 200 mg/kg TAM. Our data establish that use of TAM at even very low doses to excise a floxed target in postnatal mice has profound effects on trabecular and cortical bone formation. As such, TAM treatment is a major confounder in the interpretation of bone phenotypes in conditional gene knockout mouse models. © 2020 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research.

High concentrations of bone morphogenetic protein 2 (BMP2) in bone regeneration cause adverse events (e.g, heterotopic bone formation and acute inflammation). This study examines novel epigenetic strategies (i.e., EZH2 inhibition) for augmenting osteogenesis, thereby aiming to reduce the required BMP2 dose in vivo for bone regeneration and minimize these adverse effects. Human bone marrow-derived mesenchymal stem cells (BMSCs) were grown on three-dimensional (3D)-printed medical-grade polycaprolactone scaffolds and incubated in osteogenic media containing 50 ng/mL BMP2 and/or 5 μM GSK126 (EZH2 inhibitor) for 6 days ( n = 3 per group and timepoint). Constructs were harvested for realtime quantitative polymerase chain reaction analysis at Day 10 and immunofluorescence (IF) microscopy at Day 21. After pretreating for 6 days and maintaining in osteogenic media for 4 days, BMSC-seeded scaffolds were also implanted in an immunocompromised subcutaneous murine model ( n = 39; 3/group/donor and 3 control scaffolds) for histological analysis at 8 weeks. Pretreatment of BMSCs with BMP2 and BMP2/GSK126 costimulated expression of osteoblast-related genes (e.g., IBSP , SP7 , RUNX2 , and DLX5 ), as well as protein accumulation (e.g., collagen type 1/ COL1A1 and osteocalcin/ BGLAP ) based on IF staining. While in vivo implantation for 8 weeks did not result in bone formation, increased angiogenesis was observed in BMP2 and BMP2/GSK126 groups. This study finds that BMP2 and GSK126 costimulate osteogenic differentiation of MSCs on 3D scaffolds in vitro and may contribute to enhanced vascularization when implanted in vivo to support bone formation. Thus, epigenetic priming with EZH2 inhibitors may have translational potential in bone healing by permitting a reduction of BMP2 dosing in vivo to mitigate its side effects. Impact statement While autografts are still the gold standard for bone reconstruction, tissue availability and donor morbidity are significant limitations. Previous attempts to use high concentrations of bone morphogenetic protein 2 (BMP2) have been shown to cause adverse events such as excessive bone formation and acute inflammation. Overall, the utilization of EZH2 inhibitors to modulate gene expression in favor of bone healing has been demonstrated in vitro in a tissue engineering strategy. Our study will pave the way to developing tissue engineering strategies involving GSK126 as an adjuvant to increase the effects of BMP2 for stimulating cells of interest on a three-dimensional scaffold for bone regeneration.

Abstract Bone marrow adipocytes (BMAd) have recently been implicated in accelerating bone metastatic cancers, such as acute myelogenous leukemia and breast cancer. Importantly, bone marrow adipose tissue (BMAT) expands with aging and obesity, two key risk factors in multiple myeloma disease prevalence, suggesting that BMAds may influence and be influenced by myeloma cells in the marrow. Here, we provide evidence that reciprocal interactions and cross-regulation of myeloma cells and BMAds play a role in multiple myeloma pathogenesis and treatment response. Bone marrow biopsies from patients with multiple myeloma revealed significant loss of BMAT with myeloma cell infiltration of the marrow, whereas BMAT was restored after treatment for multiple myeloma. Myeloma cells reduced BMAT in different preclinical murine models of multiple myeloma and in vitro using myeloma cell-adipocyte cocultures. In addition, multiple myeloma cells altered adipocyte gene expression and cytokine secretory profiles, which were also associated with bioenergetic changes and induction of a senescent-like phenotype. In vivo, senescence markers were also increased in the bone marrow of tumor-burdened mice. BMAds, in turn, provided resistance to dexamethasone-induced cell-cycle arrest and apoptosis, illuminating a new possible driver of myeloma cell evolution in a drug-resistant clone. Our findings reveal that bidirectional interactions between BMAds and myeloma cells have significant implications for the pathogenesis and treatment of multiple myeloma. Targeting senescence in the BMAd or other bone marrow cells may represent a novel therapeutic approach for treatment of multiple myeloma. Significance: This study changes the foundational understanding of how cancer cells hijack the bone marrow microenvironment and demonstrates that tumor cells induce senescence and metabolic changes in adipocytes, potentially driving new therapeutic directions.

(1) Background: Arthrofibrosis is a common cause of patient debility and dissatisfaction after total knee arthroplasty (TKA). The diversity of molecular pathways involved in arthrofibrosis disease progression suggest that effective treatments for arthrofibrosis may require a multimodal approach to counter the complex cellular mechanisms that direct disease pathogenesis. In this study, we leveraged RNA-seq data to define genes that are suppressed in arthrofibrosis patients and identified adiponectin (ADIPOQ) as a potential candidate. We hypothesized that signaling pathways activated by ADIPOQ and the cognate receptors ADIPOR1 and ADIPOR2 may prevent fibrosis-related events that contribute to arthrofibrosis. (2) Methods: Therefore, ADIPOR1 and ADIPOR2 were analyzed in a TGFβ1 inducible cell model for human myofibroblastogenesis by both loss- and gain-of-function experiments. (3) Results: Treatment with AdipoRon, which is a small molecule agonist of ADIPOR1 and ADIPOR2, decreased expression of collagens (COL1A1, COL3A1, and COL6A1) and the myofibroblast marker smooth muscle α-actin (ACTA2) at both mRNA and protein levels in basal and TGFβ1-induced cells. (4) Conclusions: Thus, ADIPOR1 and ADIPOR2 represent potential drug targets that may attenuate the pathogenesis of arthrofibrosis by suppressing TGFβ-dependent induction of myofibroblasts. These findings also suggest that AdipoRon therapy may reduce the development of arthrofibrosis by mediating anti-fibrotic effects in joint capsular tissues.

Abstract ObjectiveChondrocyte apoptosis plays a vital role in osteoarthritis (OA) progression. Angelica sinensis polysaccharide (ASP), a traditional Chinese medicine, possesses anti-inflammatory and anti-apoptotic properties in chondrocytes. This study aimed to determine the protective role of ASP on sodium nitroprusside (SNP)-induced chondrocyte apoptosis, and explore the underlying mechanism.MethodHuman primary chondrocytes isolated from articular cartilage of OA patients were treated with SNP alone or in combination with different doses of ASP. Cell viability and apoptosis were assessed, and apoptosis-related proteins including Bcl-2 and Bax were detected. Autophagy levels were evaluated by light chain 3 (LC3)-II immunofluorescence staining, mRFP-GFP-LC3 fluorescence localisation and western blot (LC3II, p62, Beclin-1, Atg5). Meanwhile，activation of ERK 1/2 pathway was determined by western blot. The autophagy inhibitors 3-Methyladenine (3-MA), chloroquine (CQ) and a specific inhibitor of ERK1/2, SCH772984, were used respectively to confirm the autophagic effect of ASP.ResultsThe results showed that SNP-induced chondrocyte apoptosis was significantly rescued by ASP, whereas ASP alone promoted chondrocytes proliferation. The anti-apoptotic effect of ASP was related to the enhanced autophagy and depended on the activation of ERK1/2 pathway.ConclusionASP markedly rescued SNP-induced chondrocyte apoptosis by activating ERK1/2-dependent autophagy in chondrocytes, and it made ASP a potential therapeutic supplementation for OA treatment.