van Wijnen, Andre J

Significance Statement The arteriovenous fistula (AVF) is the preferred vascular access for hemodialysis, but several studies have shown that the primary patency rate at 1 year is approximately 60%. Percutaneous transluminal angioplasty (PTA) is the first line of treatment for AVF stenosis. However, the rate of restenosis after angioplasty is high and the primary patency rate after PTA treatment in AVFs at 6, 12, and 24 months is 61%, 42%, and 35%, respectively. Currently, no durable therapies prolong patency after angioplasty. This paper describes the use of adipose-derived mesenchymal stem cells to reduce stenosis after PTA treatment in a murine AVF model and provides rationale for potential clinical use. Background Percutaneous transluminal angioplasty (PTA) is the first line of treatment for stenosis in the arteriovenous fistula (AVF) created to provide access for hemodialysis, but resenosis still occurs. Transplants of adipose-derived mesenchymal stem cells (AMSCs) labeled with green fluorescent protein (GFP) to the adventitia could reduce pro-inflammatory gene expression, possibly restoring patency in a murine model of PTA for venous stenosis. Methods Partial nephrectomy of male C57BL/6J mice induced CKD. Placement of the AVF was 28 days later and, 14 days after that, PTA of the stenotic outflow vein was performed with delivery of either vehicle control or AMSCs (5×10 5 ) to the adventitia of the vein. Mice were euthanized 3 days later and gene expression for interleukin-1 beta (IL-1 β ) and tumor necrosis factor-alpha TNF- α ) analyzed, and histopathologic analysis performed on day 14 and 28. GFP (+) AMSCs were tracked after transplantation for up to 28 days and Doppler ultrasound performed weekly after AVF creation. Results Gene and protein expression of IL-1 β and TNF- α, fibrosis, proliferation, apoptosis and smooth muscle actin decreased, and the proportions of macrophage types (M2/M1) shifted in a manner consistent with less inflammation in AMSC-transplanted vessels compared to controls. After PTA, AMSC-treated vessels had significantly higher wall shear stress, average peak, and mean velocity, with increased lumen vessel area and decreased neointima/media area ratio compared to the control group. At 28 days after delivery, GFP (+) AMSC were present in the adventitia of the outflow vein. Conclusions AMSC-treated vessels had improved vascular remodeling with decreased proinflammatory gene expression, inflammation, and fibrotic staining compared to untreated vessels.

AbstractScapholunate interosseous ligament tears are a common wrist injury in young and active patients that can lead to suboptimal outcomes after repair. This research aims to assess a multiphasic scaffold using 3D‐printing for reconstruction of the dorsal scapholunate interosseous ligament. The scaffold was surgically implanted in vivo in the position of the native rabbit medial collateral ligament. Two branches of treatment were implemented in the study. In the first group, the rabbits (n = 8) had the knee joint fixed in flexion for 4 weeks using 1.4 mm K‐wires prior to sample harvesting. The second group (n = 8) had the rabbit knee joint immobilized for 4 weeks prior to K‐wire removal and mobilization for an additional 4 weeks prior to sample harvesting. Overall, samples were harvested at 4 weeks post‐surgery (immobilized group) and eight weeks post‐surgery (mobilized group). Mechanical tensile testing (n = 5/group) and histology (n = 3/group) of the constructs were conducted. Tissue integration and maturation were observed resulting in increased mechanical strength of the operated joint at 8 weeks (P < .05). Bone and ligament tissues were regenerated in their respective compartments with structural and mechanical properties approaching those reported for the human dorsal SLIL ligament. Clinical Significance: This proof of concept study has demonstrated that the synthetic multiphasic scaffold was capable of regenerating both bone and ligament while also withstanding the physiological load once implanted in the rabbit knee. The artificial scaffold may provide an alternative to current techniques for reconstruction of scapholunate instability or other ligament injuries in the hand and wrist.

Aims Arthrofibrosis is a relatively common complication after joint injuries and surgery, particularly in the knee. The present study used a previously described and validated rabbit model to assess the biomechanical, histopathological, and molecular effects of the mast cell stabilizer ketotifen on surgically induced knee joint contractures in female rabbits. Methods A group of 12 skeletally mature rabbits were randomly divided into two groups. One group received subcutaneous (SQ) saline, and a second group received SQ ketotifen injections. Biomechanical data were collected at eight, ten, 16, and 24 weeks. At the time of necropsy, posterior capsule tissue was collected for histopathological and gene expression analyses (messenger RNA (mRNA) and protein). Results At the 24-week timepoint, there was a statistically significant increase in passive extension among rabbits treated with ketotifen compared to those treated with saline (p = 0.03). However, no difference in capsular stiffness was detected. Histopathological data failed to demonstrate a decrease in the density of fibrous tissue or a decrease in α-smooth muscle actin (α-SMA) staining with ketotifen treatment. In contrast, tryptase and α-SMA protein expression in the ketotifen group were decreased when compared to saline controls (p = 0.007 and p = 0.01, respectively). Furthermore, there was a significant decrease in α-SMA (ACTA2) gene expression in the ketotifen group compared to the control group (p < 0.001). Conclusion Collectively, these data suggest that ketotifen mitigates the severity of contracture formation in a rabbit model of arthrofibrosis. Cite this article: Bone Joint Res 2020;9(6):302–310.

Skeletal development is controlled by epigenetic mechanisms that regulate chromatin compaction and suppress access to gene regulatory sequences. Post‐translational modifications in the N‐termini of histone 3 (H3), including tri‐methylated H3 lysine 9 (H3K9Me3) or lysine 27 (H3K27Me3) represent repressive chromatin marks that are recognized by a family of heterochromatin‐associated Chromobox (Cbx) proteins (i.e., Cbx1 to Cbx8). We show using RNA‐seq analysis that each of these Cbx isoforms is actively expressed in bone and osteoblasts with variable expression of distinct members (between 1 and 100 reads per million). The most prominently expressed Cbx member is Cbx3, which encodes a member of the heterochromatin protein 1 (HP1) family that interacts with H3K9me3. Cbx3 is known to be important for normal development while promoting neurogenesis, as well as renal and smooth muscle development. Therefore, we postulated that CBx3 may perform a similar role in osteoblastogenesis. We tested this hypothesis by both transient and stable knock‐down of Cbx3 by respectively siRNA or shRNA. RNA interference in MC3T3 osteoblasts effectively decreases Cbx3 mRNA levels by 2 to 5 fold, while increasing expression of bone‐related markers by RT‐qPCR (e.g., Alpl/alkaline phosphatase, Ibsp/bone sialoprotein, Bglap/osteocalcin, and Col1a1/Collagen Type I α1 chain). In addition, Cbx3 depletion increases mineral deposition as measured by Alizarin Red staining. Thus, Cbx3 normally appears to suppress osteoblastic differentiation, unlike its essential function in the neuronal differentiation. Because Cbx3 recognizes H3K9me3, we examined whether Cbx3 controls the levels of this histone mark. Loss of Cbx3 did not affect total H3K9me3 levels unless Cbx1 and Cbx5 were simultaneously depleted by siRNA. We propose that Cbx3 may suppress osteoblastogenesis by controlling select loci with H3K9me3 marks.

AbstractThe dense formation of abnormal scar tissue after total knee arthroplasty results in arthrofibrosis, an unfortunate sequela of inflammation. The purpose of this study was to use a validated rabbit model to assess the effects on surgically‐induced knee joint contractures of two combined pharmacological interventions: celecoxib (CXB) loaded on an implanted collagen membrane, and subcutaneously (SQ) injected ketotifen. Thirty rabbits were randomly divided into five groups. The first group received no intervention after the index surgery. The remaining four groups underwent intra‐articular implantation of collagen membranes loaded with or without CXB at the time of the index surgery; two of which were also treated with SQ ketotifen. Biomechanical joint contracture data were collected at 8, 10, 16, and 24 weeks. At the time of necropsy (24 weeks), posterior capsule tissue was collected for messenger RNA and histopathologic analyses. At 24 weeks, there was a statistically significant increase in passive extension among rabbits in all groups treated with CXB and/or ketotifen compared to those in the contracture control group. There was a statistically significant decrease in COL3A1, COL6A1, and ACTA2 gene expression in the treatment groups compared to the contracture control group (P < .001). Histopathologic data also demonstrated a trend towards decreased fibrous tissue density in the CXB membrane group compared to the vehicle membrane group. The present data suggest that intra‐articular placement of a treated collagen membrane blunts the severity of contracture development in a rabbit model of arthrofibrosis, and that ketotifen and CXB may independently contribute to the prevention of arthrofibrosis. Statement of clinical significance: Current literature has demonstrated that arthrofibrosis may affect up to 5% of primary total knee arthroplasty patients. For that reason, novel pharmacologic prophylaxis and treatment modalities are critical to mitigating reoperations and revisions while improving the quality of life for patients with this debilitating condition.

Abstract Osteolineage cell‐derived extracellular vesicles (EVs) play a regulatory role in hematopoiesis and have been shown to promote the ex vivo expansion of human hematopoietic stem and progenitor cells (HSPCs). Here, we demonstrate that EVs from different human osteolineage sources do not have the same HSPC expansion promoting potential. Comparison of stimulatory and non‐stimulatory osteolineage EVs by next‐generation sequencing and mass spectrometry analyses revealed distinct microRNA and protein signatures identifying EV‐derived candidate regulators of ex vivo HSPC expansion. Accordingly, the treatment of umbilical cord blood‐derived CD34 + HSPCs with stimulatory EVs‐altered HSPC transcriptome, including genes with known roles in cell proliferation. An integrative bioinformatics approach, which connects the HSPC gene expression data with the candidate cargo in stimulatory EVs, delineated the potentially targeted biological functions and pathways during hematopoietic cell expansion and development. In conclusion, our study gives novel insights into the complex biological role of EVs in osteolineage cell‐HSPC crosstalk and promotes the utility of EVs and their cargo as therapeutic agents in regenerative medicine.

ABSTRACT During bone marrow stromal cell (BMSC) differentiation, both Wnt signaling and the development of a rigid cytoskeleton promote commitment to the osteoblastic over adipogenic lineage. β-catenin plays a critical role in the Wnt signaling pathway to facilitate downstream effects on gene expression. We show that β-catenin was additive with cytoskeletal signals to prevent adipogenesis, and β-catenin knockdown promoted adipogenesis even when the actin cytoskeleton was depolymerized. β-catenin also prevented osteoblast commitment in a cytoskeletal-independent manner, with β-catenin knockdown enhancing lineage commitment. Chromatin immunoprecipitation (ChIP)-sequencing demonstrated binding of β-catenin to the promoter of enhancer of zeste homolog 2 (EZH2), a key component of the polycomb repressive complex 2 (PRC2) complex that catalyzes histone methylation. Knockdown of β-catenin reduced EZH2 protein levels and decreased methylated histone 3 (H3K27me3) at osteogenic loci. Further, when EZH2 was inhibited, β-catenin's anti-differentiation effects were lost. These results indicate that regulating EZH2 activity is key to β-catenin's effects on BMSCs to preserve multipotentiality. © 2020 American Society for Bone and Mineral Research.

The liver is an immunologically active organ with a tolerogenic microenvironment at a quiescent state. The immunoregulatory properties of the liver appear to be retained after transplantation because liver allografts can reduce alloresponses against other organs that are simultaneously transplanted. Mechanisms of this phenomenon remain unknown. Given the known immunomodulatory properties of mesenchymal stromal cells (MSCs), we hypothesized that liver mesenchymal stromal cells (L‐MSCs) are superior immunomodulators and contribute to liver‐mediated tolerance. L‐MSCs, generated from human liver allograft biopsies, were compared with adipose mesenchymal stromal cells (A‐MSCs) and bone marrow mesenchymal stromal cells (BM‐MSCs). Trilineage differentiation of L‐MSCs was confirmed by immunohistochemistry. Comparative phenotypic analyses were done by flow cytometry and transcriptome analyses by RNA sequencing in unaltered cell cultures. The in vitro functional analyses were performed using alloreactive T cell proliferation assays. The transcriptome analysis showed that the L‐MSCs are different than the A‐MSCs and BM‐MSCs, with significant enrichment of genes and gene sets associated with immunoregulation. Compared with the others, L‐MSCs were found to express higher cell surface levels of several select immunomodulatory molecules. L‐MSCs (versus A‐MSCs/BM‐MSCs) inhibited alloreactive T cell proliferation (22.7% versus 56.4%/58.7%, respectively; P < 0.05) and reduced the frequency of interferon ɤ–producing T cells better than other MSCs (52.8% versus 94.4%/155.4%; P < 0.05). The antiproliferative impact of L‐MSCs was not dependent on cell‐to‐cell contact, could be reversed incompletely by blocking programmed death ligand 1, and required a higher concentration of the competitive inhibitor of indoleamine 2,3‐dioxygenase for complete reversal. In conclusion, L‐MSCs appear to be uniquely well‐equipped immunomodulatory cells, and they are more potent than A‐MSCs and BM‐MSCs in that capacity, which suggests that they may contribute to liver‐induced systemic tolerance.

Abstract Although degenerative disc disease (DDD) and related low back pain (LBP) are growing public health problems, the underlying disease mechanisms remain unclear. An increase in the vascular endothelial growth factor (VEGF) levels in DDD has been reported. This study aimed to examine the role of VEGF receptors (VEGFRs) in DDD, using a mouse model of DDD. Progressive DDD was induced by anterior stabbing of lumbar intervertebral discs in wild type (WT) and VEGFR‐1 tyrosine‐kinase deficient mice ( vegfr‐1 TK−/ − ). Pain assessments were performed weekly for 12 weeks. Histological and immunohistochemical assessments were made for discs, dorsal root ganglions, and spinal cord. Both vegfr‐1 TK − / − and WT mice presented with similar pathological changes in discs with an increased expression of inflammatory cytokines and matrix‐degrading enzymes. Despite the similar pathological patterns, vegfr‐1 TK − / − mice showed insensitivity to pain compared with WT mice. This insensitivity to discogenic pain was related to lower levels of pain factors in the discs and peripheral sensory neurons and lower spinal glial activation in the vegfr‐1 TK − / − mice than in the WT mice. Exogenous stimulation of bovine disc cells with VEGF increased inflammatory and cartilage degrading enzyme. Silencing vegfr‐1 by small‐interfering‐RNA decreased VEGF‐induced expression of pain markers, while silencing vegfr‐2 decreased VEGF‐induced expression of inflammatory and metabolic markers without changing pain markers. This suggests the involvement of VEGFR‐1 signaling specifically in pain transmission. Collectively, our results indicate that the VEGF signaling is involved in DDD. Particularly, VEGFR‐1 is critical for discogenic LBP transmission independent of the degree of disc pathology.

AbstractTranscription networks and epigenetic mechanisms including DNA methylation, histone modifications, and noncoding RNAs control lineage commitment of multipotent mesenchymal progenitor cells. Proteins that read, write, and erase histone tail modifications curate and interpret the highly intricate histone code. Epigenetic reader proteins that recognize and bind histone marks provide a crucial link between histone modifications and their downstream biological effects. Here, we investigate the role of bromodomain‐containing (BRD) proteins, which recognize acetylated histones, during osteogenic differentiation. Using RNA‐sequencing (RNA‐seq) analysis, we screened for BRD proteins (n = 40) that are robustly expressed in MC3T3 osteoblasts. We focused functional follow‐up studies on Brd2 and Brd4 which are highly expressed in MC3T3 preosteoblasts and represent “bromodomain and extra terminal domain” (BET) proteins that are sensitive to pharmacological agents (BET inhibitors). We show that small interfering RNA depletion of Brd4 has stronger inhibitory effects on osteoblast differentiation than Brd2 loss as measured by osteoblast‐related gene expression, extracellular matrix deposition, and alkaline phosphatase activity. Similar effects on osteoblast differentiation are seen with the BET inhibitor +JQ1, and this effect is reversible upon its removal indicating that this small molecule has no lasting effects on the differentiation capacity of MC3T3 cells. Mechanistically, we find that Brd4 binds at known Runx2 binding sites in promoters of bone‐related genes. Collectively, these findings suggest that Brd4 is recruited to osteoblast‐specific genes and may cooperate with bone‐related transcription factors to promote osteoblast lineage commitment and maturation.