Toward designing human intervention studies to prevent osteoarthritis after knee injury: A report from an interdisciplinary OARSI 2023 workshop.

Objective: The global impact of osteoarthritis is growing. Currently no disease modifying osteoarthritis drugs/therapies exist, increasing the need for preventative strategies. Knee injuries have a high prevalence, distinct onset, and strong independent association with post-traumatic osteoarthritis (PTOA). Numerous groups are embarking upon research that will culminate in clinical trials to assess the effect of interventions to prevent knee PTOA despite challenges and lack of consensus about trial design in this population. Our objectives were to improve awareness of knee PTOA prevention trial design and discuss state-of-the art methods to address the unique opportunities and challenges of these studies. Design: An international interdisciplinary group developed a workshop, hosted at the 2023 Osteoarthritis Research Society International Congress. Here we summarize the workshop content and outputs, with the goal of moving the field of PTOA prevention trial design forward. Results: Workshop highlights included discussions about target population (considering risk, homogeneity, and possibility of modifying osteoarthritis outcome); target treatment (considering delivery, timing, feasibility and effectiveness); comparators (usual care, placebo), and primary symptomatic outcomes considering surrogates and the importance of knee function and symptoms other than pain to this population. Conclusions: Opportunities to test multimodal PTOA prevention interventions across preclinical models and clinical trials exist. As improving symptomatic outcomes aligns with patient and regulator priorities, co-primary symptomatic (single or aggregate/multidimensional outcome considering function and symptoms beyond pain) and structural/physiological outcomes may be appropriate for these trials. To ensure PTOA prevention trials are relevant and acceptable to all stakeholders, future research should address critical knowledge gaps and challenges.

Angiopoietin-like 3-derivative LNA043 for cartilage regeneration in osteoarthritis: a randomized phase 1 trial.

Osteoarthritis (OA) is a common, debilitating, chronic disease with no disease-modifying drug approved to date. We discovered LNA043-a derivative of angiopoietin-like 3 (ANGPTL3)-as a potent chondrogenesis inducer using a phenotypic screen with human mesenchymal stem cells. We show that LNA043 promotes chondrogenesis and cartilage matrix synthesis in vitro and regenerates hyaline articular cartilage in preclinical OA and cartilage injury models in vivo. LNA043 exerts at least part of these effects through binding to the fibronectin receptor, integrin α5ß1 on mesenchymal stem cells and chondrocytes. In a first-in-human (phase 1), randomized, double-blinded, placebo-controlled, single ascending dose, single-center trial ( NCT02491281 ; sponsored by Novartis Pharmaceuticals), 28 patients with knee OA were injected intra-articularly with LNA043 or placebo (3:1 ratio) either 2 h, 7 d or 21 d before total knee replacement. LNA043 met its primary safety endpoint and showed short serum pharmacokinetics, cartilage penetration and a lack of immunogenicity (secondary endpoints). Post-hoc transcriptomics profiling of cartilage revealed that a single LNA043 injection reverses the OA transcriptome signature over at least 21 d, inducing the expression of hyaline cartilage matrix components and anabolic signaling pathways, while suppressing mediators of OA progression. LNA043 is a novel disease-modifying OA drug candidate that is currently in a phase 2b trial ( NCT04864392 ) in patients with knee OA.

High Throughput Random Mutagenesis and Single Molecule Real Time Sequencing of the Muscle Nicotinic Acetylcholine Receptor.

High throughput random mutagenesis is a powerful tool to identify which residues are important for the function of a protein, and gain insight into its structure-function relation. The human muscle nicotinic acetylcholine receptor was used to test whether this technique previously used for monomeric receptors can be applied to a pentameric ligand-gated ion channel. A mutant library for the α1 subunit of the channel was generated by error-prone PCR, and full length sequences of all 2816 mutants were retrieved using single molecule real time sequencing. Each α1 mutant was co-transfected with wildtype ß1, δ, and ε subunits, and the channel function characterized by an ion flux assay. To test whether the strategy could map the structure-function relation of this receptor, we attempted to identify mutations that conferred resistance to competitive antagonists. Mutant hits were defined as receptors that responded to the nicotinic agonist epibatidine, but were not inhibited by either α-bungarotoxin or tubocurarine. Eight α1 subunit mutant hits were identified, six of which contained mutations at position Y233 or V275 in the transmembrane domain. Three single point mutations (Y233N, Y233H, and V275M) were studied further, and found to enhance the potencies of five channel agonists tested. This suggests that the mutations made the channel resistant to the antagonists, not by impairing antagonist binding, but rather by producing a gain-of-function phenotype, e.g. increased agonist sensitivity. Our data show that random high throughput mutagenesis is applicable to multimeric proteins to discover novel functional mutants, and outlines the benefits of using single molecule real time sequencing with regards to quality control of the mutant library as well as downstream mutant data interpretation.

Effect of Calstabin1 depletion on calcium transients and energy utilization in muscle fibers and treatment opportunities with RyR1 stabilizers.

Depletion of calstabin1 (FKBP12) from the RyR1 channel and consequential calcium leakage from the sarcoplasmic reticulum (SR) is found in certain disease conditions such as dystrophy, aging or muscle overuse. Here, we first assessed the effect of calstabin1 depletion on resting Ca(2+) levels and transients. We found that depletion of calstabin1 with the calstabin1-dissociation compound FK506 increased the release of calcium from the SR by 14 % during tetanic stimulation (50 Hz, 300 ms) and delayed cytosolic calcium removal. However, we did not find a significant increase in resting cytosolic Ca(2+) levels. Therefore, we tested if increased SERCA activity could counterbalance calcium leakage. By measuring the energy utilization of muscle fibers with and without FK506 treatment, we observed that FK506-treatment increased oxygen consumption by 125% compared to baseline levels. Finally, we found that pretreatment of muscle fibers with the RyR1 stabilizer JTV-519 led to an almost complete normalization of calcium flux dynamics and energy utilization. We conclude that cytosolic calcium levels are mostly preserved in conditions with leaky RyR1 channels due to increased SERCA activity. Therefore, we suggest that RyR1 leakiness might lead to chronic metabolic stress, followed by cellular damage, and RyR1 stabilizers could potentially protect diseased muscle tissue.

Ubiquitous overexpression of Hey1 transcription factor leads to osteopenia and chondrocyte hypertrophy in bone.

The transcription factor Hey1, a known Notch target gene of the HES family, has recently been described as a target gene of bone morphogenetic protein-2 (BMP-2) during osteoblastic differentiation in vitro. As the role of Hey1 in skeletal physiology is unknown, we analyzed bones of mice ubiquitously lacking or overexpressing Hey1. This strategy enabled us to evaluate whether Hey1 modulation in the whole organism could serve as a drug or antibody target for therapy of diseases associated with bone loss. Hey1 deficiency resulted in modest osteopenia in vivo and increased number and activity of osteoclasts generated ex vivo. Hey1 overexpression resulted in distinct progressive osteopenia and inhibition of osteoblasts ex vivo, an effect apparently dominant to a mild inhibition of osteoclasts. In both Hey1 deficient and overexpressing mice, males were less affected than females and skeleton was not affected during development. Bone histomorphometry did not reveal major changes in animals at 20 weeks, suggesting that modulation had occurred before. Adult Hey1 transgenics also displayed increased type X collagen expression and an enlarged hypertrophic zone in the growth plate. Taken together, our data suggest that ubiquitous in vivo Hey1 regulation affects osteoblasts, osteoclasts and chondrocytes. Due to the complex role of Hey1 in bone, inhibition of Hey1 does not appear to be a straightforward therapeutic strategy to increase the bone mass.

Intraarticular drug delivery in osteoarthritis.

Osteoarthritis (OA) is a primarily non-inflammatory, degenerative joint disease characterized by progressive loss of articular cartilage, subchondral bone sclerosis, osteophyte formation, changes in the synovial membrane, and an increased volume of synovial fluid with reduced viscosity and hence changed lubrication properties. As OA is the most common type of arthritis and a leading cause of disability, there is a largely unmet medical need for disease-modifying and symptomatic treatment. Due to the localized nature of the disease, intraarticular (IA) drug injection is an attractive treatment approach for OA. The various glucocorticoid and hyaluronic acid (HA) formulations, which are currently available on the market for IA treatment, provide only short-term pain relief or/and often do not provide adequate pain relief. The available oral drugs for symptomatic treatment also have shortcomings, most notably side effects. Therefore, there is still a large unmet need for novel OA drugs, which provide effective long-term pain relief and/or have disease-modifying properties. To achieve long-term drug exposure, different established formulations such as suspensions and hydrogels, and also novel approaches such as lipid based formulations and nano- or microparticles are currently in development. The development of novel drugs in combination with new formulations for IA treatment of OA, represents a promising approach in this challenging area of research.

ICAM-2 mediates neutrophil transmigration in vivo: evidence for stimulus specificity and a role in PECAM-1-independent transmigration.

ICAM-2 has been implicated in leukocyte transmigration in vitro, but there is little in vivo evidence to support this. To address this, neutrophil migration was investigated in ICAM-2-deficient mice (KO) and in wild-type (WT) mice treated with an anti-ICAM-2 blocking monoclonal antibody (mAb) (3C4). In a peritonitis model, IL-1beta-induced accumulation of neutrophils was significantly reduced in mice treated with 3C4 (51% inhibition) and in KO mice (41% inhibition). In contrast, TNF-alpha- or thioglycolate-induced responses were not suppressed in KO mice. Analysis of IL-1beta-induced leukocyte responses in cremasteric venules of KO animals by intravital microscopy indicated a defect in transmigration (44% inhibition) but not rolling or adhesion. As found before, TNF-alpha-induced leukocyte transmigration was unaltered in the KO mice. WT mice treated with the anti-ICAM-2 mAb also exhibited a selective reduction in leukocyte transmigration in response to IL-1beta while an anti-ICAM-1 mAb inhibited both leukocyte adhesion and transmigration. Interestingly, mAb 3C4 significantly suppressed IL-1beta-induced neutrophil transmigration in PE-CAM-1 KO animals in the peritonitis model but not in the cremaster muscle. The findings provide direct evidence for the involvement of ICAM-2 in neutrophil transmigration in vivo, though this role appears to be stimulus specific. Furthermore, ICAM-2 appears capable of mediating PECAM-1-independent leukocyte transmigration.

Validation of a diffusion chamber as in vitro system for the analysis of compound diffusibility through cartilage tissue.

The validation of a diffusion chamber comprising a donor and a receptor side separated by a cartilage membrane was undertaken according to the basic principles described by Peng et al. (1998). The study had three targets: first to evaluate the chamber as in vitro system by the examination of the diffusibility of compound through bovine cartilage samples; second the analysis of the affinity of compound (RS-130830) to cartilage; third to test the influence of two pre-incubation periods (one or three nights) of the cartilage samples. The validation of the chamber as in vitro system for the analysis of compound diffusibility and affinity to cartilage was performed using membrane slices of fresh bovine cartilage and a hydroxamic acid derivative (RS-130830) known as matrix metalloproteinase inhibitor (MMPI). The influence of the pre-incubation of cartilage was also examined. Compound concentrations in donor, receptor and membrane were determined by high performance liquid chromatography-mass spectrometry (HPLC-MS). Diffusion could be demonstrated after 6 h and finally 24 h incubation: the compound concentration in the receptor increased from 0 to 35 microM (mean) while it decreased in the donor from 200 to 144 microM (mean). We also found compound in the cartilage membrane (approximately 1.2 nmol (mean)). Pre-incubation of cartilage samples in culture buffer is suitable as a storage procedure, since the results on the donor side only were influenced significantly but not for the receptor and the cartilage affinity. Thus, the system could clearly reflect relevant properties of the tested compound with regard to its diffusibility and affinity to cartilage tissue.

Endothelial intercellular adhesion molecule (ICAM)-2 regulates angiogenesis.

Endothelial junctions maintain endothelial integrity and vascular homeostasis. They modulate cell trafficking into tissues, mediate cell-cell contact and regulate endothelial survival and apoptosis. Junctional adhesion molecules such as vascular endothelial (VE)-cadherin and CD31/platelet endothelial cell adhesion molecule (PECAM) mediate contact between adjacent endothelial cells and regulate leukocyte transmigration and angiogenesis. The leukocyte adhesion molecule intercellular adhesion molecule 2 (ICAM-2) is expressed at the endothelial junctions. In this study we demonstrate that endothelial ICAM-2 also mediates angiogenesis. Using ICAM-2-deficient mice and ICAM-2-deficient endothelial cells, we show that the lack of ICAM-2 expression results in impaired angiogenesis both in vitro and in vivo. We show that ICAM-2 supports homophilic interaction, and that this may be involved in tube formation. ICAM-2-deficient cells show defective in vitro migration, as well as increased apoptosis in response to serum deprivation, anti-Fas antibody, or staurosporine. ICAM-2 signaling in human umbilical vein endothelial cells (HUVECs) was found to activate the small guanosine triphosphatase (GTPase) Rac, which is required for endothelial tube formation and migration. These data indicate that ICAM-2 may regulate angiogenesis via several mechanisms including survival, cell migration, and Rac activation. Our findings identify a novel pathway regulating angiogenesis through ICAM-2 and a novel mechanism for Rac activation during angiogenesis.

ADAMTS-1, a gene product of articular chondrocytes in vivo and in vitro, is downregulated by interleukin 1beta.

OBJECTIVE: Osteoarthritic (OA) cartilage degeneration and cartilage destruction in rheumatoid arthritis depends significantly on enzymatic degradation of cartilage proteoglycan aggrecan. A member of the ADAMTS family of proteases, ADAMTS-1 was described to have "aggrecanase" activity. We investigated the quantitative expression and distribution of ADAMTS-1 in healthy and OA cartilage and in cultured articular chondrocytes with and without stimulation by interleukin 1beta (IL-1beta) and insulin-like growth factor-I (IGF-I). METHODS: Conventional and online polymerase chain reaction (PCR) technology was used to determine ADAMTS-1 mRNA expression levels of ADAMTS-1. Protein was localized using immunostaining with different polyclonal antibodies. RESULTS: Conventional and online semiquantitative PCR showed significant levels of ADAMTS-1 mRNA expression in normal and OA chondrocytes in vivo and in vitro. Only a slight increase was observed in OA cartilage. After stimulation with IL-1beta a downregulation of ADAMTS-1 was observed, whereas IGF did not appear to change mRNA expression levels in vitro. The in vivo mRNA expression results were confirmed by the presence of significant protein staining with antibodies for ADAMTS-1 in normal and OA chondrocytes as well as Western blotting analysis. Whereas a significantly stronger stain was seen in normal articular cartilage in the upper zones, in OA cartilage as well the middle zone showed enhanced staining. CONCLUSION: Our results confirm the expression and presence of ADAMTS-1 in articular cartilage. However, they also point out that ADAMTS-1 appears to be constitutively expressed by adult articular chondrocytes and overall is not strongly upregulated in OA. Thus our data suggest that ADAMTS-1 is the first matrix-degrading enzyme downregulated by the catabolic factor IL-1beta in vitro.

T-cell interaction with ICAM-1/ICAM-2 double-deficient brain endothelium in vitro: the cytoplasmic tail of endothelial ICAM-1 is necessary for transendothelial migration of T cells.

Endothelial intercellular adhesion molecule 1 (ICAM-1) and ICAM-2 are both involved in lymphocyte extravasation during immunosurveillance and inflammation. To define their exact role during T-cell extravasation, we used mouse T cells and ICAM-1-/-ICAM-2-/- brain endothelioma cells. ICAM-1-/-ICAM-2-/- brain endothelioma cells did not support transendothelial migration (TEM) of T cells in vitro. Re-expression of different ICAM-1 mutants in the ICAM-1-/-ICAM-2-/- endothelioma line bEndI1/2.1 or in the ICAM-1-/- endothelioma line bEndI1.1 demonstrated that the extracellular domain of ICAM-1 suffices to support T-cell adhesion while the presence of the cytoplasmic tail was strictly required for TEM. Surprisingly, tyrosine phosphorylation of endothelial ICAM-1 was not necessary for TEM of T cells or for Rho guanosine triphosphatase (RhoGTPase) activation. Furthermore, cytoplasmic deletion mutants of ICAM-1 were unable to mediate RhoGTPase activation. Thus, our data demonstrate that the cytoplasmic tail of endothelial ICAM-1-independently from tyrosine phosphorylation-is essential for supporting TEM of T lymphocytes, while Rho signaling is involved in endothelial cells.