Identification of TAK-756, A Potent TAK1 Inhibitor for the Treatment of Osteoarthritis through Intra-Articular Administration.

Osteoarthritis (OA) is a chronic and degenerative joint disease affecting more than 500 million patients worldwide with no disease-modifying treatment approved to date. Several publications report on the transforming growth factor β-activated kinase 1 (TAK1) as a potential molecular target for OA, with complementary anti-catabolic and anti-inflammatory effects. We report herein on the development of TAK1 inhibitors with physicochemical properties suitable for intra-articular injection, with the aim to achieve high drug concentration at the affected joint, while avoiding severe toxicity associated with systemic inhibition.

Degenerative joint disease induced by repeated intra-articular injections of monosodium urate crystals in rats as investigated by translational imaging.

The objective of this work was to assess the consequences of repeated intra-articular injection of monosodium urate (MSU) crystals with inflammasome priming by lipopolysaccharide (LPS) in order to simulate recurrent bouts of gout in rats. Translational imaging was applied to simultaneously detect and quantify injury in different areas of the knee joint. MSU/LPS induced joint swelling, synovial membrane thickening, fibrosis of the infrapatellar fat pad, tidemark breaching, and cartilage invasion by inflammatory cells.

Role of HTRA1 in bone formation and regeneration: In vitro and in vivo evaluation.

The role of mammalian high temperature requirement protease A1 (HTRA1) in somatic stem cell differentiation and mineralized matrix formation remains controversial, having been demonstrated to impart either anti- or pro-osteogenic effects, depending on the in vitro cell model used. The aim of this study was therefore to further evaluate the role of HTRA1 in regulating the differentiation potential and lineage commitment of murine mesenchymal stem cells in vitro, and to assess its influence on bone structure and regeneration in vivo. Our results demonstrated that short hairpin RNA-mediated ablation of Htra1 in the murine mesenchymal cell line C3H10T1/2 increased the expression of several osteogenic gene markers, and significantly enhanced matrix mineralization in response to BMP-2 stimulation.

Prostaglandin E inhibits matrix mineralization by human bone marrow stromal cell-derived osteoblasts via Epac-dependent cAMP signaling.

The osteoinductive properties of prostaglandin E (PGE) and its signaling pathways have led to suggestions that it may serve as a potential therapeutic strategy for bone loss. However, the prominence of PGE as an inducer of bone formation is attributed primarily to findings from studies using rodent models. In the current study, we investigated the effects of PGE on human bone marrow stromal cell (hBMSC) lineage commitment and determined its mode of action.

Tailored Ahp-cyclodepsipeptides as Potent Non-covalent Serine Protease Inhibitors.

The S1 serine protease family is one of the largest and most biologically important protease families. Despite their biomedical significance, generic approaches to generate potent, class-specific, bioactive non-covalent inhibitors for these enzymes are still limited. In this work, we demonstrate that Ahp-cyclodepsipeptides represent a suitable scaffold for generating target-tailored inhibitors of serine proteases.

Identification of Novel Equine (Equus caballus) Tendon Markers Using RNA Sequencing.

Although several tendon-selective genes exist, they are also expressed in other musculoskeletal tissues. As cell and tissue engineering is reliant on specific molecular markers to discriminate between cell types, tendon-specific genes need to be identified. In order to accomplish this, we have used RNA sequencing (RNA-seq) to compare gene expression between tendon, bone, cartilage and ligament from horses.

Loss-of-Function of HtrA1 Abrogates All-Trans Retinoic Acid-Induced Osteogenic Differentiation of Mouse Adipose-Derived Stromal Cells Through Deficiencies in p70S6K Activation.

All-trans retinoic acid (ATRA) is a potent inducer of osteogenic differentiation in mouse adipose-derived stromal cells (mASCs), although the underlying mechanisms responsible for its mode of action have yet to be completely elucidated. High temperature requirement protease A1 (HtrA1) is a newly recognized modulator of human multipotent stromal cell (MSC) osteogenesis and as such, may play a role in regulating ATRA-dependent osteogenic differentiation of mASCs. In this study, we assessed the influence of small interfering RNA (siRNA)-induced repression of HtrA1 production on mASC osteogenesis and examined its effects on ATRA-mediated mammalian target of rapamycin (mTOR) signaling.

ARTD1 regulates osteoclastogenesis and bone homeostasis by dampening NF-κB-dependent transcription of IL-1β.

While ADP-ribosyltransferase diphtheria toxin-like 1 (ARTD1, formerly PARP1) and its enzymatic activity have been shown to be important for reprogramming and differentiation of cells, such as during adipogenesis, their role and mechanism in regulating osteoclastogenesis and bone homeostasis are largely unknown. Here, in cell culture-based RANKL-induced osteoclastogenesis models, we show that silencing of ARTD1 or inhibition of its enzymatic activity enhances osteoclast differentiation and function. As a consequence of ARTD1 silencing or inhibition, the recruitment of p65/RelA to the IL-1β promoter, which is associated with transcriptionally active histone marks, IL-1β expression and inflammasome-dependent secretion of IL-1β are enhanced.

Novel Function of Serine Protease HTRA1 in Inhibiting Adipogenic Differentiation of Human Mesenchymal Stem Cells via MAP Kinase-Mediated MMP Upregulation.

Adipogenesis is the process by which mesenchymal stem cells (MSCs) develop into lipid-laden adipocytes. Being the dominant cell type within adipose tissue, adipocytes play a central role in regulating circulating fatty acid levels, which is considered to be of critical importance in maintaining insulin sensitivity. High temperature requirement protease A1 (HTRA1) is a newly recognized regulator of MSC differentiation, although its role as a mediator of adipogenesis has not yet been defined.

Use of biomimetic microtissue spheroids and specific growth factor supplementation to improve tenocyte differentiation and adaptation to a collagen-based scaffold in vitro.

Tenocytes represent a valuable source of cells for the purposes of tendon tissue engineering and regenerative medicine and as such, should possess a high degree of tenogenic differentiation prior to their use in vivo in order to achieve maximal efficacy. In the current report, we identify an efficient means by which to maintain differentiated tenocytes in vitro by employing the hanging drop technique in combination with defined growth media supplements. Equine tenocytes retained a more differentiated state when cultured as scaffold-free microtissue spheroids in low serum-containing medium supplemented with L-ascorbic acid 2-phosphate, insulin and transforming growth factor (TGF)-β1.

Hypoxia attenuates the proinflammatory response in colon cancer cells by regulating IκB.

Two main features common to all solid tumors are tissue hypoxia and inflammation, both of which cause tumor progression, metastasis, therapy resistance and increased mortality. Chronic inflammation is associated with increased cancer risk, as demonstrated for inflammatory bowel disease patients developing colon cancer. However, the interplay between hypoxia and inflammation on the molecular level remains to be elucidated.

ARTD1-induced poly-ADP-ribose formation enhances PPARγ ligand binding and co-factor exchange.

PPARγ-dependent gene expression during adipogenesis is facilitated by ADP-ribosyltransferase D-type 1 (ARTD1; PARP1)-catalyzed poly-ADP-ribose (PAR) formation. Adipogenesis is accompanied by a dynamic modulation of the chromatin landscape at PPARγ target genes by ligand-dependent co-factor exchange. However, how endogenous PPARγ ligands, which have a low affinity for the receptor and are present at low levels in the cell, can induce sufficient co-factor exchange is unknown.

Therapeutic potential of adipose-derived stromal cells in age-related osteoporosis.

Adipose-derived stromal cells (ASCs) are increasingly being used for orthopedic-based tissue engineering approaches due to their ability to readily undergo osteogenic differentiation. In the present study, we used in vitro and in vivo approaches to evaluate the use of ASCs as a treatment strategy for age-related osteoporosis. Molecular, histological and micro-computed tomography (micro-CT) based approaches confirmed that ASCs isolated from 18-week-old osteoporotic senescence-accelerated mice (SAMP6) were capable of undergoing osteogenesis when cultured in either silk fibroin (SF) scaffolds or scaffold-free microtissues (ASC-MT).

Preparation and osteogenic differentiation of scaffold-free mouse adipose-derived stromal cell microtissue spheroids (ASC-MT).

In this unit, previously described methods are expanded upon, where procedures relating to the preparation, culturing, and osteogenic differentiation of scaffold-free mouse adipose-derived stromal cell microtissue spheroids (ASC-MT) are outlined. Not only is a detailed methodology of how to engineer such spheroids are presented, but a full account of how to induce and analyze osteogenesis in these ASC-MT constructs is given along with relevant figures to help better illustrate the methods described.

Hyaluronic acid fragments enhance the inflammatory and catabolic response in human intervertebral disc cells through modulation of toll-like receptor 2 signalling pathways.

Introduction: Intervertebral disc (IVD) degeneration is characterized by extracellular matrix breakdown and is considered to be a primary cause of discogenic back pain. Although increases in pro-inflammatory cytokine levels within degenerating discs are associated with discogenic back pain, the mechanisms leading to their overproduction have not yet been elucidated. As fragmentation of matrix components occurs during IVD degeneration, we assessed the potential involvement of hyaluronic acid fragments (fHAs) in the induction of inflammatory and catabolic mediators.

Assessment of the matrix degenerative effects of MMP-3, ADAMTS-4, and HTRA1, injected into a bovine intervertebral disc organ culture model.

Study Design: In vitro study to develop an intervertebral disc degeneration organ culture model, using coccygeal bovine intervertebral discs (IVDs) and injection of proteolytic enzymes MMP-3, ADAMTS-4, and HTRA1.

Objective: This study aimed to develop an in vitro model of enzyme-mediated intervertebral disc degeneration to mimic the clinical outcome in humans for investigation of therapeutic treatment options.

Summary Of Background Data: Bovine IVDs are comparable with human IVDs in terms of cell composition and biomechanical behavior.

The emerging roles of HTRA1 in musculoskeletal disease.

High-temperature requirement serine protease A1 (HTRA1) is one of four known proteases belonging to the broadly conserved family of HTRA proteins. Although it was originally considered as representing an important modulator of tumorigenesis, an increasing number of reports have suggested that its influence on human disease may extend beyond cancer. HTRA1 has the capacity to degrade numerous extracellular matrix proteins, and as such, its potential involvement in diseases of the musculoskeletal system has been gaining increased attention.

Human serine protease HTRA1 positively regulates osteogenesis of human bone marrow-derived mesenchymal stem cells and mineralization of differentiating bone-forming cells through the modulation of extracellular matrix protein.

Mammalian high-temperature requirement serine protease A1 (HTRA1) is a secreted member of the trypsin family of serine proteases which can degrade a variety of bone matrix proteins and as such has been implicated in musculoskeletal development. In this study, we have investigated the role of HTRA1 in mesenchymal stem cell (MSC) osteogenesis and suggest a potential mechanism through which it controls matrix mineralization by differentiating bone-forming cells. Osteogenic induction resulted in a significant elevation in the expression and secretion of HTRA1 in MSCs isolated from human bone marrow-derived MSCs (hBMSCs), mouse adipose-derived stromal cells (mASCs), and mouse embryonic stem cells.

Detrimental role for human high temperature requirement serine protease A1 (HTRA1) in the pathogenesis of intervertebral disc (IVD) degeneration.

Human HTRA1 is a highly conserved secreted serine protease that degrades numerous extracellular matrix proteins. We have previously identified HTRA1 as being up-regulated in osteoarthritic patients and as having the potential to regulate matrix metalloproteinase (MMP) expression in synovial fibroblasts through the generation of fibronectin fragments. In the present report, we have extended these studies and investigated the role of HTRA1 in the pathogenesis of intervertebral disc (IVD) degeneration.

ARTD1 deletion causes increased hepatic lipid accumulation in mice fed a high-fat diet and impairs adipocyte function and differentiation.

ADP-ribosyltransferase Diphtheria toxin-like 1 [ARTD1; formerly called poly-ADP-ribose polymerase 1 (PARP1)] is a chromatin-associated enzyme involved in regulating metabolic homeostasis. The liver is at the core of glucose and lipid metabolism and is significantly affected by obesity and the metabolic syndrome. Here, we show that when fed a high-fat diet (HFD), mice lacking ARTD1 developed exacerbated hepatic steatosis.

Telomere length, telomerase activity and osteogenic differentiation are maintained in adipose-derived stromal cells from senile osteoporotic SAMP6 mice.

Adipose tissue provides for a rich and easily accessible source of multipotent stromal cells and thus offers the potential for autologous cell-based therapy for a number of degenerative diseases. Senile osteoporosis is characterized by a reduction in bone quality, which is associated with inadequacies in bone marrow stromal cell (BMSC) differentiation. In the present study, we have characterized adipose-derived stromal cells (ASCs) isolated from aged osteoporotic mice and evaluated their suitability as a source of osteogenic precursor cells.

Interferon-gamma inhibits interleukin-1beta-induced matrix metalloproteinase production by synovial fibroblasts and protects articular cartilage in early arthritis.

Introduction: The first few months after symptom onset represents a pathologically distinct phase in rheumatoid arthritis (RA). We used relevant experimental models to define the pathological role of interferon-gamma (IFN-gamma) during early inflammatory arthritis.

Methods: We studied IFN-gamma's capacity to modulate interleukin-1beta (IL-1beta) induced degenerative responses using RA fibroblast-like synoviocytes (FLS), a bovine articular cartilage explant (BACE)/RA-FLS co-culture model and an experimental inflammatory arthritis model (murine antigen-induced arthritis (AIA)).

Influence of defective bone marrow osteogenesis on fracture repair in an experimental model of senile osteoporosis.

Bone marrow osteogenesis in senile osteoporotic bone is impaired and, as such, may have significant implications on the successful outcome of fracture repair. Here we utilize a well-established murine model of senile osteoporosis, the P6 strain of senescence-accelerated mice (SAMP6), to investigate fracture healing in aged osteoporotic bone. A femoral osteotomy was created in SAMP6 and in non-osteoporotic age-matched control R1 senescence-resistant mice (SAMR1).

Reduction in postlaminectomy epidural adhesions in sheep using a fibrin sealant-based medicated adhesion barrier.

Epidural adhesion formation is believed to be a central governing factor in the prevalence of pain after spinal surgery and is regarded as being the primary instigator of neural tethering, leading to complications during revision surgery. In this study, we assess the effectiveness and safety of fibrin sealant supplemented with tributyrin, termed Medicated Adhesion Barrier (MAB), as an alternative means of reducing the incidence of posterior spinal epidural adhesion formation. Laminectomy defects in sheep were treated with MAB, fibrin sealant alone, ADCONGel, or remained untreated.

Therapeutic targeting of IL-6 trans signaling counteracts STAT3 control of experimental inflammatory arthritis.

Cytokine control of the synovial infiltrate is a central process in the development of inflammatory arthritis. In this study, we combine genetic approaches and intervention strategies to describe a fundamental requirement for IL-6-mediated STAT3 signaling in orchestrating the inflammatory infiltrate in monoarticular and systemic models of experimental arthritis. STAT3 activation via the common gp130 signal-transducing receptor for all IL-6-related cytokines led to increased retention of neutrophils and T cells within the inflamed synovium, which included STAT3-regulated IL-17A-secreting T cells.