Changes In Chondrocyte Phenotype Research Articles

Is Osteoarthritis a Vascular Disease?

Osteoarthritis (OA) is now considered as a multifaceted disease affecting various articular tissues, including cartilage, bone, synovium, and surrounding ligaments. The pathophysiology strongly implicates intricate chemical communication, primarily through cytokines, leading to the production of degradative enzymes in cartilage, inflammatory peptides in synovium, and structural changes in bone, resulting in characteristic clinical features such as joint deformities and loss of cartilage space seen on X-rays. Recent studies highlight the previously underestimated role of subchondral bone in OA, revealing its permeability to cytokines and raising questions about the influence of abnormal perfusion on OA pathophysiology, suggesting a vascular component in the disease's etiology. In essence, alterations in bone perfusion, including reduced venous outflow and intraosseous hypertension, play a crucial role in influencing the physicochemical environment of subchondral bone, impacting osteoblast cytokine expression and contributing to trabecular remodeling, changes in chondrocyte phenotype, and ultimately cartilage matrix degeneration in OA. Dynamic contrast (gadolinium) enhanced magnetic resonance imaging (DCE-MRI) was used to quantify perfusion kinetics in normal and osteoarthritic subchondral bone, demonstrating that decreased perfusion temporally precedes and spatially correlates with cartilage lesions in both young Dunkin-Hartley (D-H) guinea pigs and humans with osteoarthritis. Pharmacokinetic analysis of DCE-MRI generated data reveals decreased tracer clearance and outflow obstruction in the medial tibial plateau of osteoarthritic guinea pigs, coinciding with progressive cartilage degradation, loss of Safranin O staining, and increased expression of matrix metalloproteinases and interleukin-1. Positron emission tomographic (PET) scanning using 18F-Fluoride reveals a relationship among bone blood flow, cartilage lesions, and 18F-Fluoride influx rate in OA, highlighting the intricate relationships between decreased perfusion, altered bone metabolism, and the progression of osteoarthritis. These findings, supported by 18F-Fluoride PET data, suggest the presence of venous stasis associated with outflow obstruction, emphasizing the role of decreased subchondral bone perfusion in the pathophysiology of OA and its association with reduced osteoblast activity and advanced cartilage degeneration.

Concentration-dependent effects of leptin on osteoarthritis-associated changes in phenotype of human chondrocytes

ABSTRACT Metabolic syndrome is a risk factor for osteoarthritis. Elevated leptin levels have been implicated as a potential cause of this association. Previous studies have shown that supra-physiological leptin concentrations can induce osteoarthritis-like changes in chondrocyte phenotype. Here, we tested the effects of leptin in the concentration range found in synovial fluid on chondrocyte phenotype. Chondrocytes isolated from macroscopically normal regions of cartilage within osteoarthritic joints from patients undergoing knee arthroplasty, all with body mass index >30 kg/m2 were treated with 2-40 ng/ml leptin for 24 h. Chondrocyte phenotype marker expression was measured by RT-qPCR and western blot. The role of HES1 in mediating the effects of leptin was determined by gene knockdown using RNAi and over-expression using adenoviral-mediated gene delivery. Treatment of chondrocytes with 20 or 40 ng/ml leptin resulted in decreased SOX9 levels and decreased levels of the SOX9-target genes COL2A1 and ACAN. Levels of HES1 were lower and ADAMTS5 higher in chondrocytes treated with 20 or 40 ng/ml leptin. HES1 knockdown resulted in increased ADAMTS5 expression whereas over-expression of HES1 prevented the leptin-induced increase in ADAMTS5. An increase in MMP13 expression was only evident in chondrocytes treated with 40 ng/ml leptin and was not mediated by HES1 activity. High concentrations of leptin can cause changes in chondrocyte phenotype consistent with those seen in osteoarthritis. Synovial fluid leptin concentrations of this level are typically observed in patients with metabolic syndrome and/or women, suggesting elevated leptin levels may form part of the multifactorial network that leads to osteoarthritis development in these patients.

MicroRNA-1 Modulates Chondrocyte Phenotype by Regulating FZD7 of Wnt/ β-Catenin Signaling Pathway.

Osteoarthritis (OA) is an incurable joint disease characterized by pronounced pain. MicroRNAs constitute epigenetic mechanisms that may affect OA progression by contributing to changes in chondrocyte phenotype. This study investigates for the first time whether there is a link between miRNA-1 (miR-1) and OA pathogenesis, and the molecular mechanisms involved. OA-associated gene expression, including MMP-13, ADAMTS5, and COL2A1 was compared in chondrocytes from non-OA and OA cartilage, and in SW1353 cells over- and underexpressing miR-1. Bioinformatics and luciferase reporter assay were conducted to confirm whether FZD7 was a target of miR-1. The effects of miR-1 on FZD7 expression and downstream Wnt/β-catenin signalling were investigated. Non-OA and OA chondrocytes differed significantly in the expression of miR-1 and OA-associated genes. MiR-1 over- and underexpression in SW1353 cells, respectively, reduced and enhanced gene expression associated with cartilage catabolism. FZD7, which has an important role in the Wnt/β-catenin signaling pathway, was shown to be a potential target of miR-1. MiR-1 binding to FZD7 increased the levels of phosphorylated (inactivated) β-catenin, thereby preventing downstream β-catenin signaling. Inhibition of Wnt/β-catenin signaling by miR-1 in chondrocytes may attenuate the expression of genes that regulate the activity of catabolic enzymes. This finding may be useful for future investigations of molecular targets for OA treatment.

Impaired chondrocyte U3 snoRNA expression in osteoarthritis impacts the chondrocyte protein translation apparatus

Although pathways controlling ribosome activity have been described to regulate chondrocyte homeostasis in osteoarthritis, ribosome biogenesis in osteoarthritis is unexplored. We hypothesized that U3 snoRNA, a non-coding RNA involved in ribosomal RNA maturation, is critical for chondrocyte protein translation capacity in osteoarthritis. U3 snoRNA was one of a number of snoRNAs with decreased expression in osteoarthritic cartilage and osteoarthritic chondrocytes. OA synovial fluid impacted U3 snoRNA expression by affecting U3 snoRNA gene promoter activity, while BMP7 was able to increase its expression. Altering U3 snoRNA expression resulted in changes in chondrocyte phenotype. Interference with U3 snoRNA expression led to reduction of rRNA levels and translational capacity, whilst induced expression of U3 snoRNA was accompanied by increased 18S and 28S rRNA levels and elevated protein translation. Whole proteome analysis revealed a global impact of reduced U3 snoRNA expression on protein translational processes and inflammatory pathways. For the first time we demonstrate implications of a snoRNA in osteoarthritis chondrocyte biology and investigated its role in the chondrocyte differentiation status, rRNA levels and protein translational capacity.

Regional gene expression analysis of multiple tissues in an experimental animal model of post-traumatic osteoarthritis

To characterize local disease progression of the medial meniscus transection (MMT) model of post-traumatic osteoarthritis (OA) at the molecular level, in order to establish a baseline for therapeutic testing at the preclinical stage. Weight-matched male Lewis rats underwent MMT or sham surgery on the left limb with the right leg as contralateral control. At 1 and 3 weeks post-surgery, tissues were harvested from different areas of the articular cartilage (medial and lateral tibial plateaus, and medial osteophyte region) and synovium (medial and lateral), and analyzed separately. RNA was extracted and used for microarray (RT-PCR) analysis. Gene expression changes due to surgery were isolated to the medial side of the joint. Gene changes in chondrocyte phenotype of the medial tibial plateau cartilage preceded changes in tissue composition genes. Differences in inflammatory markers were only observed at the osteophyte region at 3 weeks post-surgery. There was surgical noise in the synovium at week 1, which dissipated at week 3. At this later timepoint, meniscal instability resulted in elevated expression of matrix degradation proteins and osteogenic markers in the synovium and cartilage. These results suggest feedback interactions between joint tissues during disease progression. Regional tissue expression differences found in MMT joints indicated similar pathophysiology to human OA, and provided novel insights about this degeneration model. The examination of gene expression at a localized level in multiple tissues provides a well-characterized baseline to evaluate mechanistic effects of potential therapeutic agents on OA disease progression in the MMT model.

Enhancing chondrogenic phenotype for cartilage tissue engineering: monoculture and coculture of articular chondrocytes and mesenchymal stem cells.

Articular cartilage exhibits an inherently low rate of regeneration. Consequently, damage to articular cartilage often requires surgical intervention. However, existing treatments generally result in the formation of fibrocartilage tissue, which is inferior to native articular cartilage. As a result, cartilage engineering strategies seek to repair or replace damaged cartilage with an engineered tissue that restores full functionality to the impaired joint. These strategies often involve the use of chondrocytes, yet in vitro expansion and culture can lead to undesirable changes in chondrocyte phenotype. This review focuses on the use of articular chondrocytes and mesenchymal stem cells (MSCs) in either monoculture or coculture for the enhancement of chondrogenesis. Coculture strategies increasingly outperform their monoculture counterparts with regard to chondrogenesis and present unique opportunities to attain chondrocyte phenotype stability in vitro. Methods to prevent chondrocyte dedifferentiation and promote chondrocyte redifferentiation as well as to promote the chondrogenic differentiation of MSCs while preventing MSC hypertrophy are discussed.

A novel transgenic mouse model of growth plate dysplasia reveals that decreased chondrocyte proliferation due to chronic ER stress is a key factor in reduced bone growth.

SUMMARYDisease mechanisms leading to different forms of chondrodysplasia include extracellular matrix (ECM) alterations and intracellular stress resulting in abnormal changes to chondrocyte proliferation and survival. Delineating the relative contribution of these two disease mechanisms is a major challenge in understanding disease pathophysiology in genetic skeletal diseases and a prerequisite for developing effective therapies. To determine the influence of intracellular stress and changes in chondrocyte phenotype to the development of chondrodysplasia, we targeted the expression of the G2320R mutant form of thyroglobulin to the endoplasmic reticulum (ER) of resting and proliferating chondrocytes. Previous studies on this mutant protein have shown that it induces intracellular aggregates and causes cell stress and death in the thyroid gland. The expression and retention of this exogenous mutant protein in resting and proliferating chondrocytes resulted in a chronic cell stress response, growth plate dysplasia and reduced bone growth, without inducing any alterations to the architecture and organization of the cartilage ECM. More significantly, the decreased bone growth seemed to be the direct result of reduced chondrocyte proliferation in the proliferative zone of growth plates in transgenic mice, without transcriptional activation of a classical unfolded protein response (UPR) or apoptosis. Overall, these data show that mutant protein retention in the ER of resting and proliferative zone chondrocytes is sufficient to cause disrupted bone growth. The specific disease pathways triggered by mutant protein retention do not necessarily involve a prototypic UPR, but all pathways impact upon chondrocyte proliferation in the cartilage growth plate.

Small GTPase protein Rac-1 is activated with maturation and regulates cell morphology and function in chondrocytes

During maturation, chondrocytes undergo changes in morphology, matrix production, and gene expression; however, it remains unclear whether these are interrelated. In this study, we examined whether Rho GTPases were involved in these regulatory interplays. Levels of active Rho GTPases were assayed in immature and mature primary chondrocytes. We found that activation of Rac-1 and Cdc42 increased with maturation, whereas RhoA levels remained unchanged. GFP-tagged Rho GTPases tracked cellular localization. Rac-1 was enriched at the cell membrane where it co-localized with cortical actin, while RhoA and Cdc42 were cytoplasmic. To test the roles of Rac-1 in chondrocyte maturation, we force-expressed constitutively active or dominant negative forms of Rac-1 and assessed phenotypic consequences in primary chondrocytes. Activated Rac-1 expression induced chondrocyte enlargement and increased matrix metalloproteinase expression, which are characteristic of mature chondrocytes. Conversely, Rac-1 inactivation diminished adhesion, decreased alkaline phosphatase activity, and stimulated functions typical of immature chondrocytes. Exposure to a pro-maturation factor, Wnt3A, induced a flattened and enlarged morphology accompanied by peripheral Rac-1 re-arrangement. Wnt3A stimulated Tiam1 expression and Rac-1 activation, while DN-Rac-1 inhibited Wnt3A-induced cell spreading. Our data provide strong evidence that Rac-1 coordinates changes in chondrocyte phenotype and function and stimulates the maturation process essential for skeletal development.

Intrajoint comparisons of gene expression patterns in human osteoarthritis suggest a change in chondrocyte phenotype

Osteoarthritis (OA) is a degenerative cartilage disease with varying degrees of severity within a given joint. The purpose of this study was to define a sampling procedure for comparing human minimal and advanced OA cartilage in the same patient and to determine basic patterns of gene expression in these regions. A specific hypothesis under study was that the expression level of Bcl-2 would correlate with Sox9 and aggrecan mRNA expression in vivo as has been demonstrated in vitro. Femoral condylar advanced OA cartilage was located within 1 cm of overt lesions, and minimal cartilage was taken from areas with no obvious surface defects. Histological sections were scored for disease severity and chondroitin sulfate and hydroxyproline content was determined. The expression level of nine specific genes (aggrecan, collagen type II, Bcl-2, Sox9, Link protein, osteopontin, and MMP-13, -3, and -9) was determined by quantitative real time PCR. The scores for fibrillation, chondrocyte cloning, and proteoglycan depletion were significantly different between advanced and minimal OA cartilage. The advanced OA cartilage had significantly less chondroitin sulfate than the minimal OA cartilage. Osteopontin mRNA expression showed a 3.6-fold increase in advanced compared to minimal OA cartilage. In contrast, the level of mRNA coding for aggrecan, link protein, Bcl-2, Sox9 and MMP-3, -9, -13 were all decreased in advanced compared to minimal cartilage in the majority of the patients studied. Collagen type II mRNA expression displayed a wide-range of variation. A statistically significant correlation was observed both between Bcl-2 and Sox9 mRNA level, and between Bcl-2 and aggrecan mRNA expression. The patient matched comparison of minimal and advanced OA cartilage revealed differences in cellular and tissue characteristics, and changes in gene expression that may be involved in OA progression. In addition, Bcl-2 may also play a role in regulating the expression of aggrecan through Sox9 in vivo as well as in vitro.

Immunohistochemical Analysis of Type X-Collagen Expression in Osteoarthritis of the Hip Joint

Conflicting data have been reported on the spatial distribution of type X-collagen expression in osteoarthritis, and no concise data exist on a possible correlation between type X-collagen expression and clinical and radiological alterations. Well defined clinical and radiological data were compared with histopathological and immunohistochemical findings to investigate the expression of type-X collagen in osteoarthritis of the hip joint. Femoral heads were obtained in toto from 11 patients undergoing routine hip arthroplasty for femoral neck fractures (n = 3) or osteoarthritis (n = 8) and from 13 patients (age: 12 days to 69 years) without any evidence of hip-joint pathology. Whole coronal sections from the femoral head were decalcified for routine histology and immunohistochemical analysis with use of type-specific monoclonal antibodies to type-X collagen. Our results demonstrate that type-X collagen is consistently found in osteoarthritic cartilage and is absent from normal adult cartilage (including the region of calcified cartilage). Except for the occurrence of type-X collagen in the middle zone of articular cartilage in advanced stages of osteoarthritis, there is no specific change in the staining pattern or intensity for the collagen during osteoarthritis, particularly when the staining is related to clinical and radiological parameters. Hardly more than 20% of the extracellular matrix stained for type-X collagen; therefore, we suggest that, in most cases, this type of collagen may not play a direct biomechanical role in the weakening of osteoarthritic cartilage but rather may contribute indirectly to a disturbance of the disc biomechanics by altering matrix-molecule interaction. However, expression of type-X collagen may indicate a change in chondrocyte phenotype that consistently coincides with the formation of chondrocyte clusters, one of the first alterations in osteoarthritis visible on histologic examination.

Immunohistochemical analysis of type-X-collagen expression in osteoarthritis of the hip joint.

Conflicting data have been reported on the spatial distribution of type X-collagen expression in osteoarthritis, and no concise data exist on a possible correlation between type X-collagen expression and clinical and radiological alterations. Well defined clinical and radiological data were compared with histopathological and immunohistochemical findings to investigate the expression of type-X collagen in osteoarthritis of the hip joint. Femoral heads were obtained in toto from 11 patients undergoing routine hip arthroplasty for femoral neck fractures (n = 3) or osteoarthritis (n = 8) and from 13 patients (age: 12 days to 69 years) without any evidence of hip-joint pathology. Whole coronal sections from the femoral head were decalcified for routine histology and immunohistochemical analysis with use of type-specific monoclonal antibodies to type-X collagen. Our results demonstrate that type-X collagen is consistently found in osteoarthritic cartilage and is absent from normal adult cartilage (including the region of calcified cartilage). Except for the occurrence of type-X collagen in the middle zone of articular cartilage in advanced stages of osteoarthritis, there is no specific change in the staining pattern or intensity for the collagen during osteoarthritis, particularly when the staining is related to clinical and radiological parameters. Hardly more than 20% of the extracellular matrix stained for type-X collagen; therefore, we suggest that, in most cases, this type of collagen may not play a direct biomechanical role in the weakening of osteoarthritic cartilage but rather may contribute indirectly to a disturbance of the disc biomechanics by altering matrix-molecule interaction. However, expression of type-X collagen may indicate a change in chondrocyte phenotype that consistently coincides with the formation of chondrocyte clusters, one of the first alterations in osteoarthritis visible on histologic examination.

Doxycycline Inhibits Collagen Synthesis by Bovine Chondrocytes Cultured in Alginate

Doxycycline is known for its ability to inhibit matrix metalloproteinases (MMPs), a family of enzymes that play a role in cartilage breakdown in arthritides. Its prophylactic effect in reducing joint degradation in osteoarthritis is mainly attributed to this property. In this study, we show that doxycycline exhibits a profound inhibition of collagen synthesis by bovine articular chondrocytes cultured in alginate. At 25 μM doxycycline, collagen synthesis was decreased by 50 %; no effect on cell proliferation (DNA levels) or general protein synthesis (35S-Met and35S-Cys incorporation) was observed. Messenger RNA levels of type II collagen were also reduced, indicating an effect of doxycycline at the transcriptional level. The concentration of doxycycline needed to downregulate collagen synthesis was > 10-fold lower than that needed to inhibit most of the MMPs. Inasmuch as differentiated chondrocytes in the early stages of osteoarthritis exhibit increased collagen synthesis, the beneficial effect of doxycyclinein vivomay involve prevention of changes in chondrocyte phenotype.

Increased proteoglycan synthesis in cartilage in experimental canine osteoarthritis does not reflect a permanent change in chondrocyte phenotype.

To determine whether chondrocytes in early experimental osteoarthritic (OA) cartilage continue to show increased synthesis and turnover of proteoglycans (PGs) during explant culture. A comparison was also made between the responsiveness of experimental OA and control cartilage to interleukin-1 beta (IL-1 beta) and tumor necrosis factor alpha (TNF alpha) after 1 day and 3 days in culture. OA was induced in mature animals by sectioning of the anterior cruciate ligament followed by 3 months of normal exercise. PG synthesis in the articular cartilage was determined by measuring 35S-sulfate incorporation during explant culture over 1-3 days. Inhibition of PG synthesis was also determined with various concentrations of IL-1 beta and TNF alpha after 1 and 3 days in culture. PGs extracted from the articular cartilage over 1-3 days in culture were examined by agarose-polyacrylamide gel electrophoresis. Up to 24 hours after excision from the joint, PG synthesis was higher in experimental OA cartilage than in control cartilage. It was also less sensitive to inhibition by TNF alpha. These differences were no longer detected after 48-72 hours in culture. There were no changes in the relative proportions of aggrecan and decorin/biglycan extracted from and synthesized by control and experimental OA cartilage over the 3 days in culture. Previous results indicated that PG synthesis and turnover in articular cartilage was increased for many months after induction of experimental OA. Our present results show that the enhanced rate of PG synthesis and turnover were evident in freshly explanted tissue, but the differences were lost over 3 days in culture. A decreased responsiveness to TNF alpha was also lost. The hypermetabolic activity of experimental OA chondrocytes was thus reversible and not a permanent change in chondrocyte phenotype.

Fibronectin, cartilage, and osteoarthritis

Fibronectin is a multifunctional glycoprotein present at low levels in the extracellular matrix of normal cartilage. In this tissue, as in others, it may be a component of a cell matrix adhesion complex together with cell-surface proteoglycans but also may play a role in the organization of the extracellular matrix. In osteoarthritis (OA), fibronectin content is markedly increased in the altered matrix because of an increased synthesis by the chondrocytes and accumulation in the extracellular matrix. At least part of the fibronectins synthesized in one degenerated cartilage is composed of isoforms more sensitive to proteolytic cleavage that are absent in normal cartilage. This increased content of fibronectin during osteoarthritic processes might entail several consequences related to the multiple functions of fibronectin and its generated fragments, namely a change in chondrocyte phenotype, a switch in synthesis of collagen type, an increased activity of locally secreted metalloproteases, and induction of self-proteolytic activities against gelatin and fibronectin. However, it is not yet clearly understood whether the early increased synthesis of fibronectin in OA acts as an "agent" in an attempt to repair cartilage by the chondrocytes or whether it acts as a deleterious "agent."