Wijnen, André J. van

Breast cancer is the most common cancer in women, and accounts for ~30% of new cancer cases and 15% of cancer‐related deaths. Tumor relapse and metastasis are primary factors contributing to breast cancer‐related deaths. Therefore, the challenge for breast cancer treatment is to sustain remission. A driving force behind tumor relapse is breast cancer heterogeneity (both intertumor, between different patients, and intratumor, within the same tumor). Understanding breast cancer heterogeneity is necessary to develop preventive interventions and targeted therapies. A recently emerging concept is that intratumor heterogeneity is driven by cancer stem cells (CSCs) that are capable of giving rise to a multitude of different cells within a tumor. Studies have highlighted linkage of CSC formation with epithelial‐to‐mesenchymal transition (EMT). In this review, we summarize the current understanding of breast cancer heterogeneity, links between EMT and CSCs, regulation of EMT by Runx transcription factors, and potential therapeutic strategies targeting these processes.

Background: Extracellular vesicles (EVs) released from mesenchymal stem/stromal cells (MSCs) mediate their paracrine effect, but their efficacy to protect the microcirculation of the kidney is unknown. Using a novel swine model of unilateral renovascular disease (RVD) complicated by metabolic syndrome (MetS), we tested the hypothesis that EVs would attenuate renal microvascular loss. Methods: Four groups of pigs ( n = 7 each) were studied after 16 weeks of diet-induced MetS and RVD (MetS+RVD), MetS+RVD treated 4 weeks earlier with a single intra-renal delivery of EVs harvested from autologous adipose tissue-derived MSCs, and Lean and MetS Sham controls. Stenotic-kidney renal blood flow (RBF) and glomerular filtration rate (GFR) were measured in-vivo (fast CT), whereas EV characteristics, renal microvascular architecture (micro-CT), and injury pathways were studied ex-vivo. Results: mRNA sequencing and proteomic analysis revealed that EVs are packed with several pro-angiogenic genes and proteins, such as vascular endothelial growth factor. Labeled EVs were detected in the stenotic kidney 4 weeks after injection internalized by tubular and endothelial cells. EVs restored renal expression of angiogenic factors and improved cortical microvascular and peritubular capillary density. Renal apoptosis, oxidative stress, tubular injury, and fibrosis were also attenuated in EV-treated pigs. RBF and GFR decreased in MetS+RVD compared with MetS, but normalized in MetS+RVD+EVs. Conclusions: Intra-renal delivery of MSC-derived EVs bearing pro-angiogenic properties restored the renal microcirculation and in turn hemodynamics and function in chronic experimental MetS+RVD. Our study suggests a novel therapeutic potential for MSC-derived EVs in restoring renal hemodynamics in experimental MetS+RVD.

Elevated levels of follistatin‐like protein 1 (FSTL1) have been found both in mouse models for human rheumatoid arthritis (RA) and collagen‐induced arthritis (CIA). In this study, we elucidated the potential mechanisms by which FSTL1 contributes to the pathogenesis of RA. Fibroblast‐like synoviocytes (FLSs) were established from synovial tissues of RA patients and stimulated with human recombinant FSTL1. Protein and mRNA expression levels of select matrix metalloproteinases (i.e., MMP1, MMP3, MMP13) in FLS were measured by, respectively, real‐time RT‐qPCR and ELISA. Activation of MAPK and other pathways that affect MMPs were evaluated by Western blotting. We also compared concentrations of MMPs in plasma in RA patients versus healthy controls (HC). Expression levels of MMP1, MMP3, and MMP13 were clearly stimulated by FSTL1 in vitro. FSTL1 activated the inflammation‐related NF‐κB signaling pathway, as well as all three mitogen‐activated protein kinase (MAPK) pathways and the JAK/STAT3 pathway. Moreover, select chemical inhibitors that target p38 (SB203580), Erk1/2 (SP600125), JNK (SCH772984), STAT3 (AG490), and NF‐κB (BAY 11–7082) significantly attenuated MMP expression. Inhibition of Toll‐like receptor 4 by compound TAK‐242 significantly abolished those effects of FSTL1. Importantly, elevated plasma concentrations of MMP3 were found to correlate with plasma FSTL1 levels in RA patients. These findings suggest that FSTL1 accelerates RA progression by activating MAPK, JAK/STAT3, and NF‐κB pathways to enhance secretion of different MMPs and this enhancement is via TLR4. Targeting FSTL1 may provide a promising pharmacological drug therapy to ameliorate RA symptoms and perhaps reverse disease progression.

AbstractOsteoblast differentiation is controlled by transcription factor RUNX2 which temporally activates or represses several bone‐related genes, including those encoding extracellular matrix proteins or factors that control cell‐cell, and cell‐matrix interactions. Cell‐cell communication in the many skeletal pericellular micro‐niches is critical for bone development and involves paracrine secretion of growth factors and morphogens. This paracrine signaling is in part regulated by “A Disintegrin And Metalloproteinase” (ADAM) proteins. These cell membrane‐associated metalloproteinases support proteolytic release (“shedding”) of protein ectodomains residing at the cell surface. We analyzed microarray and RNA‐sequencing data for Adam genes and show that Adam17, Adam10, and Adam9 are stimulated during BMP2 mediated induction of osteogenic differentiation and are robustly expressed in human osteoblastic cells. ADAM17, which was initially identified as a tumor necrosis factor alpha (TNFα) converting enzyme also called (TACE), regulates TNFα‐signaling pathway, which inhibits osteoblast differentiation. We demonstrate that Adam17 expression is suppressed by RUNX2 during osteoblast differentiation through the proximal Adam17 promoter region (−0.4 kb) containing two functional RUNX2 binding motifs. Adam17 downregulation during osteoblast differentiation is paralleled by increased RUNX2 expression, cytoplasmic‐nuclear translocation and enhanced binding to the Adam17 proximal promoter. Forced expression of Adam17 reduces Runx2 and Alpl expression, indicating that Adam17 may negatively modulate osteoblast differentiation. These findings suggest a novel regulatory mechanism involving a reciprocal Runx2‐Adam17 negative feedback loop to regulate progression through osteoblast differentiation. Our results suggest that RUNX2 may control paracrine signaling through regulation of ectodomain shedding at the cell surface of osteoblasts by directly suppressing Adam17 expression.

Abstract The bone marrow microenvironment harbors and protects leukemic cells from apoptosis-inducing agents via mechanisms that are incompletely understood. We previously showed SDF-1 (CXCL-12), a chemokine readily abundant within the bone marrow microenvironment, induces apoptosis in acute myeloid leukemia (AML) cells that express high levels of the SDF-1 receptor CXCR4. However, differentiating osteoblasts found within this niche protect cocultured AML cells from apoptosis. Additionally, this protection was abrogated upon treatment of the differentiating osteoblasts with histone deacetylase inhibitors (HDACi). In this study, we begin to characterize and target the molecular mechanisms that mediate this osteoblast protection. Quantitative RT-PCR revealed that HDACi treatment of differentiating osteoblasts (mouse MC3T3 osteoblast cell line) reduced expression of multiple genes required for osteoblast differentiation, including genes important for producing mineralized bone matrix. Interestingly, pretreating differentiating osteoblasts with cyclosporine A, a drug known to inhibit osteoblast differentiation, similarly impaired osteoblast-mediated protection of cocultured AML cells (KG1a and U937 human AML cell lines). Both HDACi and cyclosporine A reduced osteoblast expression of the key mineralization enzyme tissue-nonspecific alkaline phosphatase (TNAP; encoded by Alpl). Moreover, specifically reducing TNAP expression or activity in differentiating osteoblasts significantly impaired the ability of the osteoblasts to protect cocultured AML cells. Together, our results indicate that inhibiting osteoblast matrix mineralization by specifically targeting TNAP is sufficient to significantly impair osteoblast-mediated protection of AML cells. Therefore, designing combination therapies that additionally target the osteoblast-produced mineralized bone matrix may improve treatment of AML by reducing the protection of leukemic cells within the bone marrow microenvironment.

ABSTRACTTrauma, surgery, and other inflammatory conditions can lead to debilitating joint contractures. Adjunct pharmacologic modalities may permit clinical prevention and treatment of recalcitrant joint contractures. We investigated the therapeutic potential of rosiglitazone by intra‐articular delivery via oligo[poly(ethylene glycol)fumarate] (OPF) hydrogels in an established rabbit model of arthrofibrosis. OPF hydrogels loaded with rosiglitazone were characterized for drug elution properties upon soaking in minimum essential media (MEM) with 10% fetal bovine serum and measurements of drug concentrations via High Performance Liquid Chromatography (HPLC). Drug‐loaded scaffolds were surgically implanted into 24 skeletally mature female New Zealand White rabbits that were divided into equal groups receiving OPF hydrogels loaded with rosiglitazone (1.67 mg), or vehicle control (10 µl DMSO). After 8 weeks of joint immobilization, rabbits were allowed unrestricted cage activity for 16 weeks. Contracture angles of rabbit limbs treated with rosiglitazone showed statistically significant improvements in flexion compared to control animals (mean angles, respectively, 64.4° vs. 53.3°, p < 0.03). At time of sacrifice (week 24), animals in the rosiglitazone group continued to exhibit less joint contracture than controls (119.0° vs. 99.5°, p = 0.014). The intra‐articular delivery of rosiglitazone using implanted OPF hydrogels decreases flexion contractures in a rabbit model of arthrofibrosis without causing adverse effects (e.g., gross inflammation or arthritis). Statement of Clinical Significance: Post‐traumatic joint contractures are common and debilitating, with limited available treatment options. Pharmacologic interventions can potentially prevent and treat such contractures. This study is translational in that a commercially approved medication has been repurposed through a novel delivery device. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2949–2955, 2018.

Background: The addition of adipose-derived mesenchymal stromal cells to decellularized nerve allografts may improve outcomes of nerve reconstruction. Prior techniques used for cell seeding are traumatic to both the mesenchymal stromal cells and nerve graft. An adequate, reliable, and validated cell seeding technique is an essential step for evaluating the translational utility of mesenchymal stromal cell–enhanced decellularized nerve grafts. The purpose of this study was to develop a simple seeding strategy with an optimal seeding duration. Methods: A dynamic bioreactor was used to seed rat and human mesenchymal stromal cells separately onto rat and human decellularized nerve allografts. Cell viability was evaluated by MTS assays and cellular topology after seeding was determined by scanning electron microscopy. Cell density and distribution were determined by Live/Dead assays and Hoechst staining at four different time points (6, 12, 24, and 72 hours). The validity and reliability of the seeding method were calculated. Results: Cells remained viable at all time points, and mesenchymal stromal cells exhibited exponential growth in the first 12 hours of seeding. Seeding efficiency increased significantly from 79.5 percent at 6 hours to 89.2 percent after 12 hours of seeding (p = 0.004). Both intrarater reliability (r = 0.97) and interrater reliability (r = 0.92) of the technique were high. Conclusions: This study describes and validates a new method of effectively seeding decellularized nerve allografts with mesenchymal stromal cells. This method is reproducible, distributes cells homogenously over the graft, and does not traumatize the intraneural architecture of the allograft. Use of this validated seeding technique will permit critical comparison of graft outcomes.

BackgroundTransplantation of mesenchymal stem cells (MSCs) is a promising therapy for degenerative spine conditions. However, cell therapy for painful spine degeneration presently requires use of contrast agents during fluoroscopy‐guided injections, and the effects of these agents on MSCs represents a gap in knowledge.ObjectiveTo investigate the biological effects of contrast media (CM) that are coinjected with MSCs.DesignProspective observational study.SettingAcademic medical center.ParticipantsPatient‐derived clinical‐grade culture expanded MSCs.InterventionsIohexol (Omnipaque300) was reduced to 12.5%, 25%, 50%, and 100% of the stock solution and incubated with MSCs for 30 minutes, 4 hours, and 48 hours. We also used complete media and 12.5%, 25%, 50%, 100% of phosphate‐buffered saline as a control group.Main Outcome MeasuresWe examined cytotoxicity of iohexol at different concentrations and exposure duration, as well as the potential for recovery over time. Cell counts, mitochondrial activity, and quantitative real time reverse‐transcriptase polymerase chain reaction of related genes were analyzed immediately after exposure (day 0) and after 2 days of exposure (day 2).ResultsHuman MSCs exhibit a time‐ and concentration‐dependent cytotoxic response to iodinated CM. A brief, 30‐minute exposure did not affect MSCs function and viability. However, extended treatment with iohexol for 4 hours at 50% or higher concentration had a significant impact on both viability and gene expression in MSCs.ConclusionsIohexol is cytotoxic to MSCs in a time‐and concentration‐dependent manner. Hence, the concentration of CM that accompanies MSC injections should be carefully considered during MSC therapy for disk‐degenerative diseases.Level of EvidenceIII.

Fidelity of histone gene regulation, and ultimately of histone protein biosynthesis, is obligatory for packaging of newly replicated DNA into chromatin. Control of histone gene expression within the 3‐dimensional context of nuclear organization is reflected by two well documented observations. DNA replication‐dependent histone mRNAs are synthesized at specialized subnuclear domains designated histone locus bodies (HLBs), in response to activation of the growth factor dependent Cyclin E/CDK2/HINFP/NPAT pathway at the G1/S transition in mammalian cells. Complete loss of the histone gene regulatory factors HINFP or NPAT disrupts HLB integrity that is necessary for coordinate control of DNA replication and histone gene transcription. Here we review the molecular histone‐related requirements for G1/S‐phase progression during the cell cycle. Recently developed experimental strategies, now enable us to explore mechanisms involved in dynamic control of histone gene expression in the context of the temporal (cell cycle) and spatial (HLBs) remodeling of the histone gene loci.