Detecting cathepsin activity in human osteoarthritis via activity-based probes

IntroductionLysosomal cathepsins have been reported to contribute to Osteoarthritis (OA) pathophysiology due to their increase in pro-inflammatory conditions. Given the causal role of cathepsins in OA, monitoring their specific activity could provide means for assessing OA severity. To this end, we herein sought to assess a cathepsin activity-based probe (ABP), GB123, in vitro and in vivo.MethodsProtein levels and activity of cathepsins B and S were monitored by immunoblot analysis and GB123 labeling in cultured primary chondrocytes and conditioned media, following stimuli with tumor necrosis factor alpha (TNFα) and/or Interleukin 1 beta (IL-1β). Similarly, cathepsin activity was examined in sections of intact cartilage (IC) and degraded cartilage (DC) regions of OA. Finally, synovial fluid (SF) and serum from donors with no signs of diseases, early OA, late OA and rheumatoid arthritis (RA) patients were analyzed with GB123 to detect distinct activity levels of cathepsin B and S.ResultsCathepsin activity in cell lysates, conditioned media explants and DC sections showed enhanced enzymatic activity of cathepsins B and S. Further histological analysis revealed that cathepsin activity was found higher in superficial zones of DC than in IC. Examining serum and SF revealed that cathepsin B is significantly elevated with OA severity in serum and SF, yet levels of cathepsin S are more correlated with synovitis and RA.ConclusionsBased on our data, cathepsin activity monitored by ABPs correlated well with OA severity and joint inflammation, directing towards a novel etiological target for OA, which possesses significant translational potential in developing means for non-invasive detection of early signs of OA.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0586-5) contains supplementary material, which is available to authorized users.     

Dioxo-triazines as a novel series of cathepsin K inhibitors

A novel dioxo-triazine series of cathepsin K inhibitors was identified from HTS. A rapid exploratory programme led to the discovery of potent and selective cathepsin K inhibitors, typified by compound 24 which displayed IC₅₀ values of 17 nM against catK and >10,000 nM in catL, catB and catS assays.     

Design and optimization of a series of novel 2-cyano-pyrimidines as cathepsin K inhibitors

Morphing structural features of HTS-derived chemotypes led to the discovery of novel 2-cyano-pyrimidine inhibitors of cathepsin K with good pharmacokinetic profiles, for example, compound 20 showed high catK potency (IC₅₀ = 4 nM), >580-fold selectivity over catL and catB, and oral bioavailability in the rat of 52%.     

Plasma prekallikrein/kallikrein processing by lysosomal cysteine proteases

Plasma kallikrein plays a role in coagulation, fibrinolysis and inflammation. Cathepsins B and L participate in (patho)physiological processes such as peptide antigen processing, tissue remodeling events, protein turnover in cells, hormone processing and tumor invasion. The present work analyzes the processing of prekallikrein/kallikrein by lysosomal cathepsins. Prekallikrein is not hydrolyzed by catB, and catL generates an inactive fragment of prekallikrein. Both kallikrein chains are hydrolyzed by catL and the light chain is mainly hydrolyzed by catB; kallikrein activity is lower after incubation with catL compared to catB. Our data suggest that the plasma kallikrein/ kinin system can be controlled by cathepsins.     

Effects of Cysteine Proteases on the Structural and Mechanical Properties of Collagen Fibers

Excessive cathepsin K (catK)-mediated turnover of fibrillar type I and II collagens in bone and cartilage leads to osteoporosis and osteoarthritis. However, little is known about how catK degrades compact collagen macromolecules. The present study is aimed to explore the structural and mechanical consequences of collagen fiber degradation by catK. Mouse tail type I collagen fibers were incubated with either catK or non-collagenase cathepsins. Methods used include scanning electron microscopy, protein electrophoresis, atomic force microscopy, and tensile strength testing. Our study revealed evidence of proteoglycan network degradation, followed by the progressive disassembly of macroscopic collagen fibers into primary structural elements by catK. Proteolytically released GAGs are involved in the generation of collagenolytically active catK-GAG complexes as shown by AFM. In addition to their structural disintegration, a decrease in the tensile properties of fibers was observed due to the action of catK. The Young''s moduli of untreated collagen fibers versus catK-treated fibers in dehydrated conditions were 3.2 ± 0.68 GPa and 1.9 ± 0.65 GPa, respectively. In contrast, cathepsin L, V, B, and S revealed no collagenase activity, except the disruption of proteoglycan-GAG interfibrillar bridges, which slightly decreased the tensile strength of fibers.     

Hurpin is a selective inhibitor of lysosomal cathepsin L and protects keratinocytes from ultraviolet-induced apoptosis

Hurpin (headpin/PI13/serpinB13) is an intracellular, differentially spliced member of the serpin superfamily that has been linked to differentiation and apoptosis of human keratinocytes. It is transiently downregulated by UV light and overexpressed in psoriatic skin lesions. Although it has all of the features of an inhibitory serpin, a productive interaction between hurpin and a proteinase has not yet been reported. Here we demonstrate that hurpin is a potent and selective inhibitor of the archetypal lysosomal cysteine proteinase cathepsin L (catL). Recombinant hurpin inhibits human catL with a stoichiometry of inhibition (SI) of 1.7 and a rate constant k(assoc) of (4.6 +/- 0.14) x 10(5) M(-1) s(-1). It inefficiently inhibits catV and does not inhibit papain, catB, or catK. To investigate the inhibitory mechanism, we determined the P1-P1' bond in the reactive center loop cleaved by catL ((356)Thr-(357)Ser) and expressed variants in which the proximal hinge, P1 residue, or differentially spliced CD loop was mutated. The results of assays using these proteins suggest that inhibition of catL by hurpin occurs via the conventional serpin inhibitory mechanism and that the CD loop plays no role in the process. Finally, it was found that the majority of hurpin is cytosolic and that its overexpression in human keratinocytes confers resistance to UV-induced apoptosis. Given that lysosomal disruption, release of catL, and catL-mediated caspase activation are known to occur in response to cellular stress, we propose that a physiological role of hurpin is to protect epithelial cells from ectopic catL.     

Human cathepsins K,L, and S: Related proteases,but unique fibrinolytic activity

Background

Fibrin formation and dissolution are attributed to cascades of protease activation concluding with thrombin activation, and plasmin proteolysis for fibrin breakdown. Cysteine cathepsins are powerful proteases secreted by endothelial cells and others during cardiovascular disease and diabetes. Their fibrinolytic activity and putative role in hemostasis has not been well described.

Studies on the cathepsins in elastic cartilage

1. The presence of several enzymes in rabbit ear cartilage was examined by a quantitative method that permits the incubation of a fixed weight of cartilage sections (18mum.) with an appropriate exogeneous substrate. 2. As the presence of cathepsins B and D in cartilage has already been established, evidence is now provided to show that cathepsins A and C are also present and are maximally active at pH5. 3. Cathepsin A was recognized by its hydrolysis of benzyloxycarbonyl-glutamyl-tyrosine and cathepsin C by its hydrolysis of glycyl-tyrosine amide; the cartilage also hydrolysed benzyloxycarbonyl-glutamyl-phenylalanine and benzoyl-dl-phenylalanine 2-naphthyl ester at pH5. 4. The acid phosphatase activity and the DNA content of cartilage have also been measured to provide a basis for comparison with the cathepsin activity of cartilage obtained from other sites and species.     

Disrupting the Indian hedgehog signaling pathway in vivo attenuates surgically induced osteoarthritis progression in Col2a1-CreERT2; Ihhfl/fl mice

Introduction

Previous observations implicate Indian hedgehog (Ihh) signaling in osteoarthritis (OA) development because it regulates chondrocyte hypertrophy and matrix metallopeptidase 13 (MMP-13) expression. However, there is no direct genetic evidence for the role of Ihh in OA, because mice with cartilage or other tissue-specific deletion of the Ihh gene die shortly after birth. We evaluated the role of Ihh in vivo via a Cre-loxP-mediated approach to circumvent the early death caused by Ihh deficiency.

Methods

To evaluate the role of Ihh in OA development, Ihh was specifically deleted in murine cartilage using an Ihh conditional deletion construct (Col2a1-CreER^T2; Ihh^fl/fl). The extent of cartilage degradation and OA progression after Ihh deletion was assessed by histological analysis, immunohistochemistry, real-time PCR and in vivo fluorescence molecular tomography (FMT) 2 months after OA was induced by partial medial meniscectomy. The effect of Ihh signaling on cartilage was compared between Ihh-deleted mice and their control littermates.

Results

Only mild OA changes were observed in Ihh-deleted mice, while control mice displayed significantly more cartilage damage. Typical OA markers such as type X collagen and MMP-13 were decreased in Ihh-deleted mice. In vivo FMT demonstrated decreased cathepsins and MMP activity in knee joints of animals with deletion of Ihh.

Conclusions

These findings support the protective role of Ihh deletion in surgically induced OA. Thus, our findings suggest the potential to develop new therapeutic strategies that can prevent and treat OA by inhibiting Ihh signaling in chondrocytes.     

Hyaluronan injection in murine osteoarthritis prevents TGFbeta 1-induced synovial neovascularization and fibrosis and maintains articular cartilage integrity by a CD44-dependent mechanism

Introduction

The mechanism by which intra-articular injection of hyaluronan (HA) ameliorates joint pathology is unknown. Animal studies have shown that HA can reduce synovial activation, periarticular fibrosis and cartilage erosion; however, its specific effects on the different cell types involved remain unclear. We have used the TTR (TGFbeta1 injection and Treadmill Running) model of murine osteoarthritis (OA), which exhibits many OA-like changes, including synovial activation, to examine in vivo tissue-specific effects of intra-articular HA.

Methods

The kinetics of clearance of fluorotagged HA from joints was examined with whole-body imaging. Naïve and treated knee joints were examined macroscopically for cartilage erosion, meniscal damage and fibrosis. Quantitative histopathology was done with Safranin O for cartilage and with Hematoxylin & Eosin for synovium. Gene expression in joint tissues for Acan, Col1a1, Col2a1, Col3a1, Col5a1, Col10a1, Adamts5 and Mmp13 was done by quantitative PCR. The abundance and distribution of aggrecan, collagen types I, II, III, V and X, ADAMTS5 and MMP13 were examined by immunohistochemistry.

Results

Injected HA showed a half-life of less than 2 h in the murine knee joint. At the tissue level, HA protected against neovascularization and fibrosis of the meniscus/synovium and maintained articular cartilage integrity in wild-type but not in Cd44 knockout mice. HA injection enhanced the expression of chondrogenic genes and proteins and blocked that of fibrogenic/degradative genes and proteins in cartilage/subchondral bone, whereas it blocked activation of both groups in meniscus/synovium. In all locations it reduced the expression/protein for Mmp13 and blocked Adamts5 expression but not its protein abundance in the synovial lining.

Conclusions

The injection of HA, 24 h after TGFbeta1 injection, inhibited the cascade of OA-like joint changes seen after treadmill use in the TTR model of OA. In terms of mechanism, tissue protection by HA injection was abrogated by Cd44 ablation, suggesting that interaction of the injected HA with CD44 is central to its protective effects on joint tissue remodeling and degeneration in OA progression.     

Spontaneous Differentiation of Human Mesenchymal Stem Cells on Poly-Lactic-Co-Glycolic Acid Nano-Fiber Scaffold

IntroductionMesenchymal stem cells (MSCs) have immunosuppressive activity and can differentiate into bone and cartilage; and thus seem ideal for treatment of rheumatoid arthritis (RA). Here, we investigated the osteogenesis and chondrogenesis potentials of MSCs seeded onto nano-fiber scaffolds (NFs) in vitro and possible use for the repair of RA-affected joints.MethodsMSCs derived from healthy donors and patients with RA or osteoarthritis (OA) were seeded on poly-lactic-glycolic acid (PLGA) electrospun NFs and cultured in vitro.ResultsHealthy donor-derived MSCs seeded onto NFs stained positive with von Kossa at Day 14 post-stimulation for osteoblast differentiation. Similarly, MSCs stained positive with Safranin O at Day 14 post-stimulation for chondrocyte differentiation. Surprisingly, even cultured without any stimulation, MSCs expressed RUNX2 and SOX9 (master regulators of bone and cartilage differentiation) at Day 7. Moreover, MSCs stained positive for osteocalcin, a bone marker, and simultaneously also with Safranin O at Day 14. On Day 28, the cell morphology changed from a spindle-like to an osteocyte-like appearance with processes, along with the expression of dentin matrix protein-1 (DMP-1) and matrix extracellular phosphoglycoprotein (MEPE), suggesting possible differentiation of MSCs into osteocytes. Calcification was observed on Day 56. Expression of osteoblast and chondrocyte differentiation markers was also noted in MSCs derived from RA or OA patients seeded on NFs. Lactic acid present in NFs potentially induced MSC differentiation into osteoblasts.ConclusionsOur PLGA scaffold NFs induced MSC differentiation into bone and cartilage. NFs induction process resembled the procedure of endochondral ossification. This finding indicates that the combination of MSCs and NFs is a promising therapeutic technique for the repair of RA or OA joints affected by bone and cartilage destruction.     

Properties of a fructose 1,6-bisphosphatase converting enzyme in rat liver lysosomes.

An enzyme present in rat liver lysosomes catalyzes the conversion of neutral rabbit liver fructose 1,6-bisphosphatase (Fru-P₂ase, EC 3.1.3.11) to a form having maximum activity at pH 9.2. The converting enzyme is partly released when lysosomes are subjected to a single freeze-thaw cycle, but a significant fraction tends to remain with the lysosomal membrane fraction even after repeated freezing and thawing. After repeated freezing and thawing hexosaminidase and cathepsin D are also partly membrane-bound, but cathepsins A, B, and C are completely solubilized. The membrane-bound enzymes, unlike those in intact lysosomes, are not cryptic. The converting enzyme activity is inactivated by phenylmethanesulfonyl fluoride, and is almost completely inactive after exposure to iodoacetic acid or tosylamido-2-phenylethyl and N-α-tosyl lysyl chloromethyl ketones. Unlike cathepsin B, it is not inhibited by leupeptin. Converting enzyme is unstable above pH 6.5, and this property also serves to distinguish it from cathepsins B and D. The results suggest that the converting enzyme is not identical to any of the well-characterized cathepsins.     

Three-Dimensional Quantitative Morphometric Analysis (QMA) for In Situ Joint and Tissue Assessment of Osteoarthritis in a Preclinical Rabbit Disease Model

This work utilises advances in multi-tissue imaging, and incorporates new metrics which define in situ joint changes and individual tissue changes in osteoarthritis (OA). The aims are to (1) demonstrate a protocol for processing intact animal joints for microCT to visualise relevant joint, bone and cartilage structures for understanding OA in a preclinical rabbit model, and (2) introduce a comprehensive three-dimensional (3D) quantitative morphometric analysis (QMA), including an assessment of reproducibility. Sixteen rabbit joints with and without transection of the anterior cruciate ligament were scanned with microCT and contrast agents, and processed for histology. Semi-quantitative evaluation was performed on matching two-dimensional (2D) histology and microCT images. Subsequently, 3D QMA was performed; including measures of cartilage, subchondral cortical and epiphyseal bone, and novel tibio-femoral joint metrics. Reproducibility of the QMA was tested on seven additional joints. A significant correlation was observed in cartilage thickness from matching histology-microCT pairs. The lateral compartment of operated joints had larger joint space width, thicker femoral cartilage and reduced bone volume, while osteophytes could be detected quantitatively. Measures between the in situ tibia and femur indicated an altered loading scenario. High measurement reproducibility was observed for all new parameters; with ICC ranging from 0.754 to 0.998. In conclusion, this study provides a novel 3D QMA to quantify macro and micro tissue measures in the joint of a rabbit OA model. New metrics were established consisting of: an angle to quantitatively measure osteophytes (σ), an angle to indicate erosion between the lateral and medial femoral condyles (ρ), a vector defining altered angulation (λ, α, β, γ) and a twist angle (τ) measuring instability and tissue degeneration between the femur and tibia, a length measure of joint space width (JSW), and a slope and intercept (m, Χ) of joint contact to demonstrate altered loading with disease progression, as well as traditional bone and cartilage and histo-morphometry measures. We demonstrate correlation of microCT and histology, sensitive discrimination of OA change and robust reproducibility.     

Synovial mesenchymal progenitor derived aggrecan regulates cartilage homeostasis and endogenous repair capacity

Aggrecan is a critical component of the extracellular matrix of all cartilages. One of the early hallmarks of osteoarthritis (OA) is the loss of aggrecan from articular cartilage followed by degeneration of the tissue. Mesenchymal progenitor cell (MPC) populations in joints, including those in the synovium, have been hypothesized to play a role in the maintenance and/or repair of cartilage, however, the mechanism by which this may occur is unknown. In the current study, we have uncovered that aggrecan is secreted by synovial MPCs from healthy joints yet accumulates inside synovial MPCs within OA joints. Using human synovial biopsies and a rat model of OA, we established that this observation in aggrecan metabolism also occurs in vivo. Moreover, the loss of the “anti-proteinase” molecule alpha-2 macroglobulin (A2M) inhibits aggrecan secretion in OA synovial MPCs, whereas overexpressing A2M rescues the normal secretion of aggrecan. Using mice models of OA and cartilage repair, we have demonstrated that intra-articular injection of aggrecan into OA joints inhibits cartilage degeneration and stimulates cartilage repair respectively. Furthermore, when synovial MPCs overexpressing aggrecan were transplanted into injured joints, increased cartilage regeneration was observed vs. wild-type MPCs or MPCs with diminished aggrecan expression. Overall, these results suggest that aggrecan secreted from joint-associated MPCs may play a role in tissue homeostasis and repair of synovial joints.Subject terms: Mesenchymal stem cells, Cartilage, Experimental models of disease     

Deficiency for the Cysteine Protease Cathepsin L Impairs Myc-Induced Tumorigenesis in a Mouse Model of Pancreatic Neuroendocrine Cancer

Motivated by the recent implication of cysteine protease cathepsin L as a potential target for anti-cancer drug development, we used a conditional MycER^TAM;Bcl-x_L model of pancreatic neuroendocrine tumorigenesis (PNET) to assess the role of cathepsin L in Myc-induced tumor progression. By employing a cysteine cathepsin activity probe in vivo and in vitro, we first established that cathepsin activity increases during the initial stages of MycER^TAM;Bcl-x_L tumor development. Among the cathepsin family members investigated, only cathepsin L was predominately produced by beta-tumor cells in neoplastic pancreata and, consistent with this, cathepsin L mRNA expression was rapidly upregulated following Myc activation in the beta cell compartment. By contrast, cathepsins B, S and C were highly enriched in tumor-infiltrating leukocytes. Genetic deletion of cathepsin L had no discernible effect on the initiation of neoplastic growth or concordant angiogenesis. However, the tumors that developed in the cathepsin L-deficient background were markedly reduced in size relative to their typical wild-type counterparts, indicative of a role for cathepsin L in enabling expansive tumor growth. Thus, genetic blockade of cathepsin L activity is inferred to retard Myc-driven tumor growth, encouraging the potential utility of pharmacological inhibitors of cysteine cathepsins in treating late stage tumors.     

Structural and functional changes of the articular surface in a post-traumatic model of early osteoarthritis measured by atomic force microscopy

The functional integrity of the articulating cartilage surface is a critical determinant of joint health. Although a variety of techniques exist to characterize the structural changes in the tissue with osteoarthritis (OA), some with extremely high resolution, most lack the ability to detect and monitor the functional changes that accompany the structural deterioration of this essential bearing surface. Atomic force microscopy (AFM) enables the acquisition of both structural and mechanical properties of the articular cartilage surface, with up to nanoscale resolution, making it particularly useful for evaluating the functional behavior of the macromolecular network forming the cartilage surface, which disintegrates in OA.In the present study, AFM was applied to the articular cartilage surfaces from six pairs of canine knee joints with post-traumatic OA. Microstructure (RMS roughness) and micromechanics (dynamic indentation modulus, E^?) of medial femoral condyle cartilages were compared between contralateral controls and cruciate-transected knee joints, which develop early signs of OA by three months after surgery.Results reveal a significant increase in RMS roughness and a significant four-fold decrease in E^? in cartilages from cruciate-transected joints versus contralateral controls. Compared to previous reports of changes in bulk mechanics, AFM was considerably more sensitive at detecting early cartilage changes due to cruciate-deficiency. The use of AFM in this study provides important new information on early changes in the natural history of OA because of its ability to sensitively detect and measure local structural and functional changes of the articular cartilage surface, the presumptive site of osteoarthritic initiation.     

Cathepsin M: a lysosomal proteinase with aldolase-inactivating activity

A proteinase, designated cathepsin M, that catalyzes the limited modification and inactivation of fructose 1,6-bisphosphate aldolase (EC 4.1.2.13) and fructose 1,6-bisphosphatase (EC 3.1.3.11) has been partially purified from rabbit liver. On the basis of its molecular size (M_r = 30,000), activation by sulfhydryl compounds and inhibition by leupeptin it has been characterized as a B-type cathepsin, but other properties distinguish it from cathepsins B, L, and H. Approximately 50% of the total cathepsin M activity is associated with membranes prepared from disrupted lysosomes; this fraction of the activity is also expressed by intact lysosomes. In the membrane-bound form the enzyme is active at neutral pH, but the soluble enzyme and the activity eluted from the membranes are maximally active at pH 5.0. Fasting increases the activity of cathepsin M; the increase is almost entirely in the membrane-bound fraction.     

Bevacizumab,an anti-vascular endothelial growth factor antibody,inhibits osteoarthritis

Introduction

Angiogenesis is an important factor in the development of osteoarthritis (OA). We investigated the efficacy of bevacizumab, an antibody against vascular endothelial growth factor and an inhibitor of angiogenesis, in the treatment of OA using a rabbit model of anterior cruciate ligament transection.

Methods

First, we evaluated the response of gene expression and histology of the normal joint to bevacizumab treatment. Next, in a rabbit model of OA induced by anterior cruciate ligament transection, we used macroscopic and histological evaluations and real-time polymerase chain reaction (PCR) to examine the responses to intravenous (systemic) administration of bevacizumab (OAB IV group). We also investigated the efficacy of intra-articular (local) administration of bevacizumab in OA-induced rabbits (OAB IA group).

Results

Histologically, bevacizumab had no negative effect in normal joints. Bevacizumab did not increase the expression of genes for catabolic factors in the synovium, subchondral bone, or articular cartilage, but it increased the expression of collagen type 2 in the articular cartilage. Macroscopically and histologically, the OAB IV group exhibited a reduction in articular cartilage degeneration and less osteophyte formation and synovitis compared with the control group (no bevacizumab; OA group). Real-time PCR showed significantly lower expression of catabolic factors in the synovium in the OAB IV group compared with the OA group. In articular cartilage, expression levels of aggrecan, collagen type 2, and chondromodulin-1 were higher in the OAB IV group than in the OA group. Histological evaluation and assessment of pain behaviour showed a superior effect in the OAB IA group compared with the OAB IV group 12 weeks after administration of bevacizumab, even though the total dosage given to the OAB IA group was half that received by the OAB IV group.

Conclusions

Considering the dosage and potential adverse effects of bevacizumab, the local administration of bevacizumab is a more advantageous approach than systemic administration. Our results suggest that intra-articular bevacizumab may offer a new therapeutic approach for patients with post-traumatic OA.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-014-0427-y) contains supplementary material, which is available to authorized users.