Jorge A. Roman-Blas

Osteoarthritis associated with estrogen deficiency

Osteoarthritis (OA) affects all articular tissues and finally leads to joint failure. Although articular tissues have long been considered unresponsive to estrogens or their deficiency, there is now increasing evidence that estrogens influence the activity of joint tissues through complex molecular pathways that act at multiple levels. Indeed, we are only just beginning to understand the effects of estrogen deficiency on articular tissues during OA development and progression, as well as on the association between OA and osteoporosis. Estrogen replacement therapy and current selective estrogen receptor modulators have mixed effectiveness in preserving and/or restoring joint tissue in OA. Thus, a better understanding of how estrogen acts on joints and other tissues in OA will aid the development of specific and safe estrogen ligands as novel therapeutic agents targeting the OA joint as a whole organ.

Subchondral bone microstructural damage by increased remodelling aggravates experimental osteoarthritis preceded by osteoporosis

IntroductionOsteoporosis (OP) increases cartilage damage in a combined rabbit model of OP and osteoarthritis (OA). Accordingly, we assessed whether microstructure impairment at subchondral bone aggravates cartilage damage in this experimental model.MethodsOP was induced in 20 female rabbits, by ovariectomy and intramuscular injections of methylprednisolone hemisuccinate for four weeks. Ten healthy animals were used as controls. At week 7, OA was surgically induced in left knees of all rabbits. At 22 weeks, after sacrifice, microstructure parameters were assessed by micro-computed tomography, and osteoprotegerin (OPG), receptor activator of nuclear factor-κB ligand (RANKL), alkaline phosphatase (ALP) and metalloproteinase 9 (MMP9) protein expressions were evaluated by Western Blot at subchondral bone. In addition, cartilage damage was estimated using the histopathological Mankin score. Mann-Whitney and Spearman statistical tests were performed as appropriate, using SPSS software v 11.0. Significant difference was established at P < 0.05.ResultsSubchondral bone area/tissue area, trabecular thickness and polar moment of inertia were diminished in OPOA knees compared with control or OA knees (P < 0.05). A decrease of plate thickness, ALP expression and OPG/RANKL ratio as well as an increased fractal dimension and MMP9 expression occurred at subchondral bone of OA, OP and OPOA knees vs. controls (P < 0.05). In addition, the severity of cartilage damage was increased in OPOA knees vs. controls (P < 0.05). Remarkably, good correlations were observed between structural and remodelling parameters at subchondral bone, and furthermore, between subchondral structural parameters and cartilage Mankin score.ConclusionsMicrostructure impairment at subchondral bone associated with an increased remodelling aggravated cartilage damage in OA rabbits with previous OP. Our results suggest that an increased subchondral bone resorption may account for the exacerbation of cartilage damage when early OA and OP coexist simultaneously in same individuals.

Subchondral bone as a key target for osteoarthritis treatment.

Osteoarthritis (OA), the most common form of arthritis, is a debilitating and progressive disease that has become a major cause of disability and impaired quality of life in the elderly. OA is considered an organ disease that affects the whole joint, where the subchondral bone (SB) plays a crucial role. Regardless of whether SB alterations precede cartilage damage or appear during the evolution of the disease, SB is currently recognised as a key target in OA treatment. In fact, bone abnormalities, especially increased bone turnover, have been detected in the early evolution of some forms of OA. Systemic osteoporosis (OP) and OA share a paradoxical relationship in which both high and low bone mass conditions may result in induction and/or OA progression. Recent findings suggest that some drugs may be useful in treating both processes simultaneously, at least in a subgroup of patients with OA and OP. This review focuses on the role of SB in OA pathogenesis, describing relevant underlying mechanisms involved in the process and examining the potential activity as disease-modifying anti-osteoarthritic drugs (DMOADs) of certain SB-targeting agents currently under study.

Improving subchondral bone integrity reduces progression of cartilage damage in experimental osteoarthritis preceded by osteoporosis

PURPOSE Impairment of subchondral bone density and quality aggravates cartilage damage in osteoarthritis (OA). Accordingly, we assessed whether improving microstructure and quality at subchondral bone by the bone-forming agent parathyroid hormone (PTH) [1-34] prevent cartilage damage progression in a rabbit model of OA preceded by osteoporosis (OP). METHODS OP was induced in 20 female rabbits. At week 7, these rabbits underwent knee surgery to induce OA and, at week 12, they started either saline vehicle (n=10) or PTH (n=10) for 10 weeks. Ten healthy animals were used as controls. At week 22, microstructure was assessed by micro-computed tomography and bone remodelling by protein expression of alkaline phosphatase (ALP), metalloproteinase-9 (MMP9), osteoprotegerin (OPG) and receptor activator of nuclear factor-κB ligand (RANKL) at subchondral bone. Cartilage damage was evaluated using Mankin score. RESULTS PTH reversed the decrease of bone area/tissue area, trabecular thickness, plate thickness, polar moment of inertia, ALP expression and OPG/RANKL ratio, as well as counteracted the increase of fractal dimension and MMP9 expression at subchondral bone of osteoarthritis preceded by osteoporosis (OPOA) rabbits compared to vehicle administration (P<0.05). Likewise, PTH decreased cartilage damage severity in OPOA rabbits. Good correlations were observed between subchondral bone structure or remodelling parameters, and cartilage Mankin score. CONCLUSIONS Improvement of microstructural and remodelling parameters at subchondral bone by PTH [1-34] contributed to prevent cartilage damage progression in rabbits with early OPOA. These findings support the role of subchondral bone in OA. Further studies are warranted to establish the place of bone-forming agents as potential treatment in OA.

Targeting NF-kappaB: a promising molecular therapy in inflammatory arthritis.

The nuclear factor-κ B family of transcription factors is intimately involved in the regulation of the inflammatory responses that play a fundamental role in the damage of articular tissues. Thus, many studies have examined the important contributions of components of the NF-κB signaling pathways to the pathogenesis of various rheumatic diseases and their pharmacologic modulation. Currently available therapeutic agents including nonsteroidal anti-inflammatory drugs, corticosteroids, nutraceuticals, and disease-modifying antirheumatic drugs, as well as novel specific small-molecule inhibitors have been employed. In addition, promising nucleic acid–based strategies have shown encouraging results. However, further research will be needed before NF-κB–aimed strategies become an effective therapy for inflammatory arthritis.

Bone mineral density and joint cartilage: four clinical settings of a complex relationship in osteoarthritis

Experimental and clinical data support the hypothesis that both high and low bone mineral density (BMD) conditions, including osteoporosis, may induce osteoarthritis. However, these conditions do not always predispose to osteoarthritis progression. Four clinical settings could arise from this relationship, and furthermore two phenotypes may be identified whether early osteoarthritis coexists with high or low BMD.

RANKL synthesized by articular chondrocytes contributes to juxta-articular bone loss in chronic arthritis

IntroductionThe receptor activator nuclear factor-kappaB ligand (RANKL) diffuses from articular cartilage to subchondral bone. However, the role of chondrocyte-synthesized RANKL in rheumatoid arthritis-associated juxta-articular bone loss has not yet been explored. This study aimed to determine whether RANKL produced by chondrocytes induces osteoclastogenesis and juxta-articular bone loss associated with chronic arthritis.MethodsChronic antigen-induced arthritis (AIA) was induced in New Zealand (NZ) rabbits. Osteoarthritis (OA) and control groups were simultaneously studied. Dual X-ray absorptiometry of subchondral knee bone was performed before sacrifice. Histological analysis and protein expression of RANKL and osteoprotegerin (OPG) were evaluated in joint tissues. Co-cultures of human OA articular chondrocytes with peripheral blood mononuclear cells (PBMCs) from healthy donors were stimulated with macrophage-colony stimulating factor (M-CSF) and prostaglandin E2 (PGE2), then further stained with tartrate-resistant acid phosphatase.ResultsSubchondral bone loss was confirmed in AIA rabbits when compared with controls. The expression of RANKL, OPG and RANKL/OPG ratio in cartilage were increased in AIA compared to control animals, although this pattern was not seen in synovium. Furthermore, RANKL expression and RANKL/OPG ratio were inversely related to subchondral bone mineral density. RANKL expression was observed throughout all cartilage zones of rabbits and was specially increased in the calcified cartilage of AIA animals. Co-cultures demonstrated that PGE2-stimulated human chondrocytes, which produce RANKL, also induce osteoclasts differentiation from PBMCs.ConclusionsChondrocyte-synthesized RANKL may contribute to the development of juxta-articular osteoporosis associated with chronic arthritis, by enhancing osteoclastogenesis. These results point out a new mechanism of bone loss in patients with rheumatoid arthritis.

N-cadherin mediated distribution of β-catenin alters MAP kinase and BMP-2 signaling on chondrogenesis-related gene expression†

We have examined the effect of calcium‐dependent adhesion, mediated by N‐cadherin, on cell signaling during chondrogenesis of multipotential embryonic mouse C3H10T1/2 cells. The activity of chondrogenic genes, type II collagen, aggrecan, and Sox9 were examined in monolayer (non‐chondrogenic), and micromass (chondrogenic) cultures of parental C3H10T1/2 cells and altered C3H10T1/2 cell lines that express a dominant negative form of N‐cadherin (Δ390‐T1/2) or overexpress normal N‐cadherin (MNCD2‐T1/2). Our findings show that missexpression or inhibition of N‐cadherin in C3H10T1/2 cells results in temporal and spatial changes in expression of the chondrogenic genes Sox9, aggrecan, and collagen type II. We have also analyzed activity of the serum response factor (SRF), a nuclear target of MAP kinase signaling implicated in chondrogenesis. In semi‐confluent monolayer cultures (minimum cell–cell contact) of C3H10T1/2, MNCD2‐T1/2, or Δ390‐T1/2 cells, there was no significant change in the pattern of MAP kinase or bone morphogenetic protein‐2 (BMP‐2) regulation of SRF. However, in micromass cultures, the effect of MAP kinase and BMP‐2 on SRF activity was proportional to the nuclear localization of β‐catenin, a Wnt stabilized cytoplasmic factor that can associate with lymphoid enhancer‐binding factor (LEF) to serve as a transcription factor. Our findings suggest that the extent of adherens junction formation mediated by N‐cadherin can modulate the potential Wnt‐induced nuclear activity of β‐catenin. Published 2005 Wiley‐Liss, Inc.

Differential effects of the antioxidant n-acetylcysteine on the production of catabolic mediators in IL-1β-stimulated human osteoarthritic synoviocytes and chondrocytes

Oxidative stress may play a relevant role in synovial inflammation and subsequently on cartilage damage during osteoarthritis development. We have assessed how the antioxidant N-acetylcysteine (NAC) affects the expression of different proinflammatory and structural mediators in human stimulated osteoarthritic synoviocytes and chondrocytes. Synovial membrane and articular cartilage were obtained from the osteoarthritis knees of patients who underwent joint replacement surgery. In cells stimulated with IL-1beta (10U/mL), the effects of NAC (2mmol/L) were tested at the mean peak plasma level following oral administration of therapeutic doses and its influence on prostaglandin E2 (PGE(2)) production, cyclooxigenase-2 (COX-2) expression, nitric oxide (NO) synthesis, nuclear factor kappa B (NF-kappaB) activation, and metalloproteinase-1 (MMP-1) and metalloproteinase-13 (MMP-13) expression were evaluated. While NAC significantly diminished PGE(2) release and the expression of both COX-2 and MMP-13 protein in IL-1beta-stimulated synoviocytes, it failed to modify their production in stimulated chondrocytes. Likewise, NAC only inhibited IL-1beta-stimulated NF-kappaB activation in synoviocytes. No inhibition of IL-1beta-induced NO synthesis by chondrocytes was observed following NAC incubation, while synoviocytes did not release detectable levels of NO. NAC did not induce changes in MMP-1 protein expression in either IL-1beta-stimulated synoviocytes or chondrocytes. Thus, NAC decreases the synthesis of several catabolic mediators by osteoarthritic synoviocytes, whereas, it failed to produce the same effects in osteoarthritic chondrocytes. Our results suggest that NAC inhibits some oxygen-dependent mechanisms in synoviocytes but not in chondrocytes during osteoarthritis. Therefore, NAC might have a symptomatic effect on the synovium rather than a structural effect on the cartilage in osteoarthritis.

Progression of chondrogenesis in C3H10T1/2 cells is associated with prolonged and tight regulation of ERK1/2†

Close contact of mesenchymal cells in vivo and also in super dense micromass cultures in vitro results in cellular condensation and alteration of existing cellular signaling required for initiation and progression of chondrogenesis. To investigate chondrogenesis related changes in the activity of ubiquitous cell signaling mediated by mitogen‐activated protein kinases (MAP kinase), we have compared the effect of cell seeding of pluripotent C3H10T1/2 mesenchymal cells as monolayers (non‐chondrogenic culture) or high density micromass cultures (chondrogenic) on the regulation and phosphorylation state of extracellular signal‐regulated kinase 1 and 2 (ERK1/2) and also on regulation of ERK1/2 nuclear targets, namely, activation protein‐1 (AP‐1) and serum response factor (SRF). Increasing cell density resulted in reduced DNA binding as well as activity of AP‐1. SRF activity, on the other hand, was up‐regulated in confluent monolayer cultures but like AP‐1 was inhibited in micromass cultures. Low levels of PD 98059 (5 μM), a specific inhibitor of ERK1/2, resulted in delayed induction of AP‐1 and SRF activity whereas higher concentrations of this inhibitor (10–50 μM) conferred an opposite effect. Increasing concentrations of the PD 98059 inhibitor in long term monolayer or micromass cultures (2.5 day) resulted in differential regulation of c‐Fos and c‐Jun protein levels as well as total expression and phosphorylation levels of ERK1/2. PD 98059 treatment of C3H10T1/2 micromass cultures also resulted in up‐regulation of type IIB collagen and Sox9 gene expression. While high expression of aggrecan and type IIB collagen genes were dependent on BMP‐2 signaling, ERK inhibition of BMP‐2 treated micromass cultures resulted in reduced activity of both genes. Our findings show that the activity of ERK1/2 in chondrogenic cultures of C3H10T1/2 cells is tightly controlled and can cross interact with other signaling activities mediated by BMP‐2 to positively regulate chondrogensis. J. Cell. Biochem. 88: 1129–1144, 2003. Published 2003 Wiley‐Liss, Inc.