van Wijnen, Andre J

AbstractAutologous transplantation of mesenchymal stem cells (MSCs) is a viable option for the treatment of several diseases. Evidence indicates that MSCs release extracellular vesicles (EVs) and that EVs shuttle miRNAs to damaged parenchymal cells to activate an endogenous repair program. We hypothesize that comorbidities may interfere with the packaging of cargo in MSC‐derived EVs. Therefore, we examined whether metabolic syndrome (MetS) modulates the miRNA content packed within MSC‐derived EVs. MSCs were collected from swine abdominal adipose tissue after 16 weeks of lean or obese diet (n = 7 each). Next‐generation RNA sequencing of miRNAs (miRNA‐seq) was performed to identify miRNAs enriched in MSC‐derived EVs and their predicted target genes. Functional pathway analysis of the top 50 target genes of the top 4 miRNAs enriched in each group was performed using gene ontology analysis. Lean‐ and MetS‐EVs were enriched in, respectively, 14 and 8 distinct miRNAs. Target genes of miRNAs enriched in MetS‐EVs were implicated in the development of MetS and its complications, including diabetes‐related pathways, validated transcriptional targets of AP1 family members Fra1 and Fra2, Class A/1 (Rhodopsin‐like receptors), and Peptide ligand‐binding receptors. In contrast, miRNAs enriched in Lean EVs target primarily EphrinA‐EPHA and the Rho family of GTPases. MetS alters the miRNA content of EVs derived from porcine adipose tissue MSCs. These alterations could impair the efficacy and limit the therapeutic use of autologous MSCs in subjects with MetS. Our findings may assist in developing adequate regenerative strategies to preserve the reparative potency of MSCs in individuals with MetS. © 2017 International Society for Advancement of Cytometry

AbstractSubstance P (SP), a neurotransmitter released after injury, has been linked to deregulated tissue repair and fibrosis in musculoskeletal tissues and other organs. Although SP inhibition is an effective treatment for nausea, it has not been previously considered as an anti‐fibrotic therapy. Although there are extensive medical records of individuals who have used SP antagonists, our analysis of human registry data revealed that patients receiving these antagonists and arthroplasty are exceedingly rare, thus precluding a clinical evaluation of their potential effects in the context of arthrofibrosis. Therefore, we pursued in vivo studies to assess the effect of SP inhibition early after injury on pro‐fibrotic gene expression and contractures in an animal model of post‐traumatic joint stiffening. Skeletally mature rabbits (n = 24) underwent surgically induced severe joint contracture, while injected with either fosaprepitant (a selective SP antagonist) or saline (control) early after surgery (3, 6, 12, and 24 h). Biomechanical testing revealed that differences in mean contracture angles between the groups were not statistically significant (P = 0.27), suggesting that the drug neither mitigates nor exacerbates joint contracture. However, microarray gene expression analysis revealed that mRNA levels for proteins related to cell signaling, pro‐angiogenic, pro‐inflammatory, and collagen matrix production were significantly different between control and fosaprepitant treated rabbits (P < 0.05). Hence, our study demonstrates that inhibition of SP alters expression of pro‐fibrotic genes in vivo. This finding will motivate future studies to optimize interventions that target SP to reduce the formation of post‐traumatic joint contractures.

Alterations in nuclear morphology are common in cancer progression. However, the degree to which gross morphological abnormalities translate into compromised higher‐order chromatin organization is poorly understood. To explore the functional links between gene expression and chromatin structure in breast cancer, we performed RNA‐seq gene expression analysis on the basal breast cancer progression model based on human MCF10A cells. Positional gene enrichment identified the major histone gene cluster at chromosome 6p22 as one of the most significantly upregulated (and not amplified) clusters of genes from the normal‐like MCF10A to premalignant MCF10AT1 and metastatic MCF10CA1a cells. This cluster is subdivided into three sub‐clusters of histone genes that are organized into hierarchical topologically associating domains (TADs). Interestingly, the sub‐clusters of histone genes are located at TAD boundaries and interact more frequently with each other than the regions in‐between them, suggesting that the histone sub‐clusters form an active chromatin hub. The anchor sites of loops within this hub are occupied by CTCF, a known chromatin organizer. These histone genes are transcribed and processed at a specific sub‐nuclear microenvironment termed the major histone locus body (HLB). While the overall chromatin structure of the major HLB is maintained across breast cancer progression, we detected alterations in its structure that may relate to gene expression. Importantly, breast tumor specimens also exhibit a coordinate pattern of upregulation across the major histone gene cluster. Our results provide a novel insight into the connection between the higher‐order chromatin organization of the major HLB and its regulation during breast cancer progression.

Abstract Actin structure contributes to physiologic events within the nucleus to control mesenchymal stromal cell (MSC) differentiation. Continuous cytochalasin D (Cyto D) disruption of the MSC actin cytoskeleton leads to osteogenic or adipogenic differentiation, both requiring mass transfer of actin into the nucleus. Cyto D remains extranuclear, thus intranuclear actin polymerization is potentiated by actin transfer: we asked whether actin structure affects differentiation. We show that secondary actin filament branching via the Arp2/3 complex is required for osteogenesis and that preventing actin branching stimulates adipogenesis, as shown by expression profiling of osteogenic and adipogenic biomarkers and unbiased RNA-seq analysis. Mechanistically, Cyto D activates osteoblast master regulators (e.g., Runx2, Sp7, Dlx5) and novel coregulated genes (e.g., Atoh8, Nr4a3, Slfn5). Formin-induced primary actin filament formation is critical for Arp2/3 complex recruitment: osteogenesis is prevented by silencing of the formin mDia1, but not its paralog mDia2. Furthermore, while inhibition of actin, branching is a potent adipogenic stimulus, silencing of either mDia1 or mDia2 blocks adipogenic gene expression. We propose that mDia1, which localizes in the cytoplasm of multipotential MSCs and traffics into the nucleus after cytoskeletal disruption, joins intranuclear mDia2 to facilitate primary filament formation before mediating subsequent branching via Arp2/3 complex recruitment. The resulting intranuclear branched actin network specifies osteogenic differentiation, while actin polymerization in the absence of Arp2/3 complex-mediated secondary branching causes adipogenic differentiation.

ABSTRACTOsteosarcoma is the most common malignant bone tumor in children and adolescents. Metastasis and poor responsiveness to chemotherapy in osteosarcoma correlates with over‐expression of the runt‐related transcription factor RUNX2, which normally plays a key role in osteogenic lineage commitment, osteoblast differentiation, and bone formation. Furthermore, WNT/β‐catenin signaling is over‐activated in osteosarcoma and promotes tumor progression. Importantly, the WNT/β‐catenin pathway normally activates RUNX2 gene expression during osteogenic lineage commitment. Therefore, we examined whether the WNT/β‐catenin pathway controls the tumor‐related elevation of RUNX2 expression in osteosarcoma. We analyzed protein levels and nuclear localization of β‐catenin and RUNX2 in a panel of human osteosarcoma cell lines (SAOS, MG63, U2OS, HOS, G292, and 143B). In all six cell lines, β‐catenin and RUNX2 are expressed to different degrees and localized in the nucleus and/or cytoplasm. SAOS cells have the highest levels of RUNX2 protein that is localized in the nucleus, while MG63 cells have the lowest RUNX2 levels which is mostly localized in the cytoplasm. Levels of β‐catenin and RUNX2 protein are enhanced in HOS, G292, and 143B cells after treatment with the GSK3β inhibitor SB216763. Furthermore, small interfering RNA (siRNA)‐mediated depletion of β‐catenin inhibits RUNX2 expression in G292 cells. Thus, WNT/β‐catenin activation is required for RUNX2 expression in at least some osteosarcoma cell types, where RUNX2 is known to promote expression of metastasis related genes. J. Cell. Biochem. 118: 3662–3674, 2017. © 2017 Wiley Periodicals, Inc.

Objectives Sustained intra-articular delivery of pharmacological agents is an attractive modality but requires use of a safe carrier that would not induce cartilage damage or fibrosis. Collagen scaffolds are widely available and could be used intra-articularly, but no investigation has looked at the safety of collagen scaffolds within synovial joints. The aim of this study was to determine the safety of collagen scaffold implantation in a validated in vivo animal model of knee arthrofibrosis. Materials and Methods A total of 96 rabbits were randomly and equally assigned to four different groups: arthrotomy alone; arthrotomy and collagen scaffold placement; contracture surgery; and contracture surgery and collagen scaffold placement. Animals were killed in equal numbers at 72 hours, two weeks, eight weeks, and 24 weeks. Joint contracture was measured, and cartilage and synovial samples underwent histological analysis. Results Animals that underwent arthrotomy had equivalent joint contractures regardless of scaffold implantation (-13.9° versus -10.9°, equivalence limit 15°). Animals that underwent surgery to induce contracture did not demonstrate equivalent joint contractures with (41.8°) or without (53.9°) collagen scaffold implantation. Chondral damage occurred in similar rates with (11 of 48) and without (nine of 48) scaffold implantation. No significant difference in synovitis was noted between groups. Absorption of the collagen scaffold occurred within eight weeks in all animals Conclusion Our data suggest that intra-articular implantation of a collagen sponge does not induce synovitis or cartilage damage. Implantation in a native joint does not seem to induce contracture. Implantation of the collagen sponge in a rabbit knee model of contracture may decrease the severity of the contracture. Cite this article: J. A. Walker, T. J. Ewald, E. Lewallen, A. Van Wijnen, A. D. Hanssen, B. F. Morrey, M. E. Morrey, M. P. Abdel, J. Sanchez-Sotelo. Intra-articular implantation of collagen scaffold carriers is safe in both native and arthrofibrotic rabbit knee joints. Bone Joint Res 2016;6:162–171. DOI: 10.1302/2046-3758.63.BJR-2016-0193.