Resemblance of osteophytes in experimental osteoarthritis to transforming growth factor β–induced osteophytes: Limited role of bone morphogenetic protein in early osteoarthritic osteophyte formation

Objective

Osteoarthritis (OA) is characterized by cartilage damage, synovial fibrosis, and osteophyte formation. Both transforming growth factor β (TGFβ) and bone morphogenetic protein 2 (BMP‐2) can induce the formation of osteophytes during OA, but their specific role in this process is unclear. The purpose of this study was to investigate the respective contributions of TGFβ and BMP‐2 to OA.

Methods

Mouse knee joints injected with adenovirus (Ad‐TGFβ or Ad‐BMP‐2) were compared histologically with knee joints from murine models of OA (joints injected with collagenase and joints from STR/Ort mice with spontaneous OA). To further investigate the role of BMP during osteophyte formation, adenovirus Ad‐Gremlin was injected into knee joints that had previously been injected with Ad‐TGFβ or collagenase.

Results

BMP‐2 induced early osteophytes, which bulged from the growth plates on the femur and grew on top of the patella, whereas TGFβ induced early osteophyte formation on the bone shaft beneath the collateral ligament on the femur as well as on top of the patella. The pattern of osteophyte formation during experimental OA closely resembled that of TGFβ‐induced osteophyte formation, but differed from the pattern induced by BMP‐2. Ad‐Gremlin proved to be able to totally block BMP‐2–induced osteophyte formation. However, blocking BMP activity inhibited neither TGFβ‐induced nor experimental OA–associated osteophyte formation.

Conclusion

Our findings demonstrate that the role of BMP during the onset of TGFβ‐induced and experimental OA–induced osteophyte formation is limited. The latter finding does not rule out a role of BMP during osteophyte maturation.

     

Evidence for a role of the genomic region of the gene encoding for the α1 chain of type IX collagen (COL9A1) in hip osteoarthritis: A population‐based study

Objective

Type IX collagen proteoglycan is an important protein in collagen networks and has been implicated in hip osteoarthritis (OA). We studied 2 COL9A1 markers (509‐8B2 and 509‐12B1) in relation to radiographic OA, within the framework of the Rotterdam Study, a population‐based study of 7,983 subjects ages 55 years and older.

Methods

We used 2 different designs, as follows: 1) a linkage study of 83 probands with multiple joints affected with radiographic OA and their 221 siblings, yielding 445 sibpairs who participated in the study, and 2) an association study in a series of 71 patients with radiographic hip OA and 269 controls without radiographic OA. All subjects were characterized for the 2 COL9A1 markers, 509‐8B2 and 509‐12B1. The mean test was used to assess the proportion of alleles shared in concordantly affected and unaffected sibpairs. The chi‐square test was used to compare the allele distributions in patients and controls.

Results

Affected sibpairs with radiographic hip OA shared alleles identical by descent at markers 8B2 and 12B1 significantly more often than expected (mean ± SD 0.66 ± 0.07 and 0.65 ± 0.08, respectively; P < 0.05). No excess sharing for radiographic OA was observed at other joint sites. When comparing the frequency of marker 8B2 and 12B1 alleles in subjects with radiographic OA and controls, the frequency of 8B2 alleles in subjects with radiographic OA differed significantly(P = 0.01) from that in controls.

Conclusion

Our data suggest that susceptibility for hip OA is conferred within or close to the COL9A1 gene in linkage disequilibrium with the COL9A1 509‐8B2 marker.

     

Interleukin‐1β induces differentiation of human mesenchymal stem cells into osteoblasts via the Wnt‐5a/receptor tyrosine kinase–like orphan receptor 2 pathway

Objective

Mesenchymal stem cells (MSCs) are considered to be a novel tool for the treatment of rheumatoid arthritis (RA) because of their multipotency to differentiate into osteoblasts and chondrocytes, their immunosuppressive effects, and availability. The aim of this study was to assess the mechanisms of human MSC differentiation into osteoblasts under inflammatory conditions.

Methods

Human MSCs were cultured in commercialized osteogenic induction medium with inflammatory cytokines for up to 10 days. Osteoblast differentiation was detected by alkaline phosphatase staining and messenger RNA (mRNA) expression of multiple osteoblast markers. Mineralization was assessed by alizarin red S staining.

Results

Among the various cytokines tested, interleukin‐1β (IL‐1β) induced differentiation of human MSCs into osteoblasts, which was confirmed by alkaline phosphatase activity, expression of RUNX2 mRNA, and strong alizarin red S staining. Among various molecules of the Wnt family, Wnt‐5a and receptor tyrosine kinase–like orphan receptor 2 (Ror2), a major receptor of Wnt‐5a, were significantly induced in human MSCs by IL‐1β. Silencing of either WNT5A or ROR2 by small interfering RNA with 2 different sequences reduced alkaline phosphatase activity, RUNX2 expression, and alizarin red S staining of human MSCs induced by IL‐1β.

Conclusion

IL‐1β effectively and rapidly induced human MSC differentiation into osteoblasts and mineralization, mainly through the noncanonical Wnt‐5a/Ror2 pathway. These results suggest potential benefits of IL‐1β–treated human MSCs in the treatment of damaged bone as well as in the induction of self‐renewal and self‐repair of damaged tissue, including osseous tissue.

     

Reduction of osteophyte formation and synovial thickening by adenoviral overexpression of transforming growth factor β/bone morphogenetic protein inhibitors during experimental osteoarthritis

Objective

Osteoarthritis (OA) is a joint disease characterized by osteophyte development, fibrosis, and articular cartilage damage. Effects of exogenous transforming growth factor β (TGFβ) isoforms and bone morphogenetic proteins (BMPs) suggest a role for these growth factors in the pathogenesis of OA. The aim of this study was to elucidate the role of endogenous TGFβ and BMP during papain‐induced OA‐like changes in mice.

Methods

We used adenoviral overexpression of TGFβ and BMP antagonists to block growth factor signaling. An adenovirus expressing a secreted, pan–specific TGFβ antagonist called murine latency‐associated peptide 1 (mLAP‐1) was used. In addition, we used intracellular inhibitory Smad6 as a BMP antagonist and Smad7 as a TGFβ/BMP inhibitor. Papain was injected into the knee joints of C57BL/6 mice to induce osteophyte development, synovial thickening, and articular cartilage proteoglycan (PG) loss.

Results

Intraarticular injection of papain caused increased protein expression of several TGFβ and BMP isoforms in synovium and cartilage. Adenovirus transfection into the joint resulted in a strong expression of the transgenes in the synovial lining. Overexpression of mLAP‐1, Smad6, and Smad7 led to a significant reduction in osteophyte formation compared with that in controls. Smad6 and Smad7 overexpression also significantly decreased synovial thickening. Furthermore, the secreted TGFβ inhibitor mLAP‐1 increased articular cartilage PG loss.

Conclusion

Our results indicate a pivotal role of endogenous TGFβ in the development of osteophytes and synovial thickening, implicating endogenous TGFβ in the pathogenesis of OA. In contrast, the prevention of cartilage damage by endogenous TGFβ signifies the protective role of TGFβ in articular cartilage. This is the first study to demonstrate that endogenous BMPs are involved in osteophyte formation and synovial thickening in experimental OA.

     

MicroRNA‐140 is expressed in differentiated human articular chondrocytes and modulates interleukin‐1 responses

Objective

MicroRNA (miRNA) are a class of noncoding small RNAs that act as negative regulators of gene expression. MiRNA exhibit tissue‐specific expression patterns, and changes in their expression may contribute to pathogenesis. The objectives of this study were to identify miRNA expressed in articular chondrocytes, to determine changes in osteoarthritic (OA) cartilage, and to address the function of miRNA‐140 (miR‐140).

Methods

To identify miRNA specifically expressed in chondrocytes, we performed gene expression profiling using miRNA microarrays and quantitative polymerase chain reaction with human articular chondrocytes compared with human mesenchymal stem cells (MSCs). The expression pattern of miR‐140 was monitored during chondrogenic differentiation of human MSCs in pellet cultures and in human articular cartilage from normal and OA knee joints. We tested the effects of interleukin‐1β (IL‐1β) on miR‐140 expression. Double‐stranded miR‐140 (ds–miR‐140) was transfected into chondrocytes to analyze changes in the expression of genes associated with OA.

Results

Microarray analysis showed that miR‐140 had the largest difference in expression between chondrocytes and MSCs. During chondrogenesis, miR‐140 expression in MSC cultures increased in parallel with the expression of SOX9 and COL2A1. Normal human articular cartilage expressed miR‐140, and this expression was significantly reduced in OA tissue. In vitro treatment of chondrocytes with IL‐1β suppressed miR‐140 expression. Transfection of chondrocytes with ds–miR‐140 down‐regulated IL‐1β–induced ADAMTS5 expression and rescued the IL‐1β–dependent repression of AGGRECAN gene expression.

Conclusion

This study shows that miR‐140 has a chondrocyte differentiation–related expression pattern. The reduction in miR‐140 expression in OA cartilage and in response to IL‐1β may contribute to the abnormal gene expression pattern characteristic of OA.

     

Potent induction of chondrocytic differentiation of human adipose‐derived adult stem cells by bone morphogenetic protein 6

Objective

Recent studies have identified an abundant source of multipotent progenitor cells in subcutaneous human adipose tissue, termed human adipose‐derived adult stem cells (ADAS cells). In response to specific media formulations, including transforming growth factor β1 (TGFβ1), these cells exhibit significant ability to differentiate into a chondrocyte‐like phenotype, expressing cartilage‐specific genes and proteins such as aggrecan and type II collagen. However, the influence of other growth factors on the chondrogenic differentiation of ADAS cells is not fully understood. This study was undertaken to investigate the effects of TGFβ1, TGFβ3, insulin‐like growth factor 1, bone morphogenetic protein 6 (BMP‐6), and dexamethasone, in various combinations, on the chondrogenic potential of ADAS cells in alginate beads.

Methods

The chondrogenic response of alginate‐encapsulated ADAS cells was measured by quantitative polymerase chain reaction, ³H‐proline and ³⁵S‐sulfate incorporation, and immunolabeling for specific extracellular matrix components.

Results

Significant differences in chondrogenesis were observed under the different culture conditions for all outcomes measured. Most notably, BMP‐6 up‐regulated AGC1 and COL2A1 expression by an average of 205‐fold and 38‐fold, respectively, over day‐0 controls, while down‐regulating COL10A1 expression by ∼2‐fold.

Conclusion

These findings suggest that BMP‐6 is a potent inducer of chondrogenesis in ADAS cells, in contrast to mesenchymal stem cells, which exhibit increased expression of type X collagen and a hypertrophic phenotype in response to BMP‐6. Combinations of growth factors containing BMP‐6 may provide a novel means of regulating the differentiation of ADAS cells for applications in the tissue‐engineered repair or regeneration of articular cartilage.

     

Type I collagen α1 Sp1 transcription factor binding site polymorphism is associated with reduced risk of hip osteoarthritis defined by severe joint space narrowing in elderly women

Objective

A common G/T substitution at an Sp1 binding site in intron 1 of the COL1A1 gene has been reported to be associated with reduced bone mineral density and increased risk of osteoporotic fracture. The purpose of this study was to examine whether there is an association between COL1A1 Sp1 polymorphism and radiographic osteoarthritis (OA) of the hip in elderly women in the Study of Osteoporotic Fractures.

Methods

Radiographic hip OA status of subjects was defined by the presence of 1 of the following criteria in either hip: a joint space narrowing (JSN) score of ≥3, a Croft summary grade of ≥3, or both definite (score ≥2) osteophytes and JSN in the same hip. Cases of radiographic OA of the hip were further subdivided into those with JSN score ≥3 and those with a femoral osteophyte score ≥2 and JSN score ≤2. The COL1A1 Sp1 polymorphism was genotyped using allele‐specific kinetic polymerase chain reaction in 4,746 women. Multivariate logistic regression was performed to estimate odds ratios (ORs) and 95% confidence intervals (95% CIs).

Results

Radiographic OA of the hip was present in 571 women (12%). Of these patients, 325 (57%) had severe JSN (score ≥3), and 131 (23%) had moderate or moderate‐to‐severe femoral osteophytosis (score ≥2). There was no association of the T/T genotype with either radiographic hip OA or radiographic hip OA characterized by osteophytosis. For radiographic OA of the hip characterized by moderate‐to‐severe JSN, the odds of disease were significantly reduced among subjects with the T/T compared with the G/G genotype (OR 0.30, 95% CI 0.11–0.81, P = 0.02) and did not change after adjustment for potential confounders (OR 0.36, 95% CI 0.13–0.99, P = 0.048).

Conclusion

The T/T genotype of the COL1A1 Sp1 polymorphism was associated with a reduced risk of radiographic OA of the hip characterized by JSN. This association should be confirmed in other populations to determine if mechanistic studies are warranted.

     

Neutrophil cannibalism triggers transforming growth factor β1 production and self regulation of neutrophil inflammatory function in monosodium urate monohydrate crystal–induced inflammation in mice

Objective

To identify macrophage‐independent sources of transforming growth factor β1 (TGFβ1) production during monosodium urate monohydrate (MSU) crystal–induced inflammation and to determine how TGFβ1 alters MSU crystal–recruited neutrophil functions.

Methods

C57BL/6J mice were injected intraperitoneally with MSU crystals with or without TGFβ1‐neutralizing antibody. MSU crystal–recruited peritoneal and blood neutrophils were purified and cultured ex vivo. Peritoneal neutrophils were treated with the caspase inhibitor Q‐VD‐OPh, anti‐TGFβ1 antibody, or fluorochrome‐labeled apoptotic neutrophils. Neutrophils were analyzed for expression of annexin V, caspase 3, and TGFβ1 by flow cytometry or fluorescence microscopy, for superoxide production using the redox‐sensitive dye water‐soluble tetrazolium 1, and for TGFβ1 and interleukin‐1β (IL‐1β) production by enzyme‐linked immunosorbent assay.

Results

Eighteen hours after MSU crystal administration in vivo, TGFβ1 levels were elevated in peritoneal lavage fluids, and a significant number of peritoneal neutrophils were TGFβ1+. Purified blood or peritoneal neutrophils cultured ex vivo showed TGFβ1+ neutrophils coexpressing the apoptosis marker caspase 3 and increased TGFβ1 production, both of which dropped following inhibition of apoptosis. Live neutrophils that had phagocytosed apoptotic neutrophils showed greatest TGFβ1 expression. Superoxide production by purified MSU crystal–recruited neutrophils ex vivo was enhanced by anti‐TGFβ1 antibody treatment. Neutrophils purified from the peritoneum of MSU crystal–challenged mice treated with anti‐TGFβ1 antibody produced elevated levels of superoxide, but neutrophil IL‐1β production was unaffected.

Conclusion

Neutrophil cannibalism and TGFβ1 production have the potential to make a significant contribution to the controlled resolution of neutrophil‐driven inflammatory diseases such as gout.

     

Bone morphogenetic protein and transforming growth factor β inhibitory Smads 6 and 7 are expressed in human adult normal and osteoarthritic cartilage in vivo and are differentially regulated in vitro by interleukin‐1β

Objective

Bone morphogenetic protein (BMP) and transforming growth factor β (TGFβ) are potent anabolic factors in adult articular chondrocytes. In this study, we investigated whether intracellular inhibitors of BMP and TGFβ signaling, inhibitory Smad6 (I‐Smad6) and I‐Smad7, are expressed in articular chondrocytes in normal and osteoarthritic (OA) cartilage, and whether their expression shows a correlation with the anabolic activity of OA chondrocytes in vivo and after interleukin‐1β (IL‐1β) stimulation in vitro.

Methods

RNA isolated directly from normal and OA human knee cartilage as well as from cultured articular chondrocytes was analyzed by (quantitative) polymerase chain reaction technology. Immunolocalization of the I‐Smads was performed on tissue sections and compared with the anabolic cellular activity as documented by in situ hybridization experiments for aggrecan and type II collagen.

Results

Both Smad6 and Smad7 were expressed in all samples of normal and OA cartilage. Immunostaining (including confocal microscopy) confirmed the presence of Smad6 and Smad7 in the majority of normal and degenerated articular chondrocytes; localization was mostly cytoplasmic. No correlation between expression of the main anabolic genes and expression of the I‐Smads was found. In cultured articular chondrocytes, stimulation with IL‐1β showed up‐regulation of Smad7, whereas Smad6 was down‐regulated.

Conclusion

Both Smad6 and Smad7 are expressed in adult human articular chondrocytes. The primarily cytoplasmic localization suggests permanent activation of the I‐Smads in articular cartilage in vivo. No evidence was found that up‐regulation or down‐regulation of I‐Smads in OA cartilage correlates directly with the anabolic (or catabolic) activity of articular chondrocytes. The regulation in chondrocytes of Smad6 and Smad7 expression by IL‐1β suggests a potentially important role of IL‐1β signaling in chondrocytes, via indirect influencing of the BMP/TGFβ signaling cascade.

     

Hepatocyte growth factor and transforming growth factor β1 ratio at baseline can predict early response to cyclophosphamide in systemic lupus erythematosus nephritis

Objective

To determine whether the ratio of hepatocyte growth factor (HGF) to transforming growth factor β1 (TGFβ1) in systemic lupus erythematosus (SLE) nephritis could be a prognostic factor for response to therapy with cyclophosphamide (CYC) and steroids at 6 months, and to examine whether the molecular ratio of HGF to TGFβ1 correlates with the activity index (AI) and chronicity index (CI) and has predictive value for remission at the sixth month.

Methods

Thirty‐six SLE patients with new‐onset nephritis, 25 of whom were treated with CYC and steroids, entered into a prospective observational cohort trial at a tertiary university referral center. Renal biopsy findings and clinical parameters were recorded for all patients. Histopathologic, clinical, and immunohistochemical data at baseline served to define the predictive value for the outcome at 6 months.

Results

AI and CI at baseline did not distinguish patients who had achieved remission from those who had not achieved remission after receiving CYC plus steroids. HGF and TGFβ1 were expressed in the tubuli, not in the glomeruli. The CI correlated directly with the TGFβ1 extension score (TGFβ1‐ES) (r = 0.43, P = 0.008), but correlated indirectly with the HGF intensity score (HGF‐IS) (r = −0.39, P = 0.02) and the HGF‐ES (r = −0.45, P = 0.006). An HGF‐ES:TGFβ1‐ES ratio of ≥1 at baseline distinguished patients who had achieved remission from those who had not achieved remission, with a predictive value of 94%.

Conclusion

These findings indicate that baseline expression of renal HGF and TGFβ1 predicts short‐term renal outcome after therapy with CYC and steroids.

     

Female mice are more susceptible to developing inflammatory disorders due to impaired transforming growth factor β signaling in salivary glands

Objective

Transforming growth factor β (TGFβ) plays a key role in the onset and resolution of autoimmune diseases and chronic inflammation. The aim of this study was to delineate the precise function of TGFβ signaling in salivary gland inflammation.

Methods

We impaired TGFβ signaling in mouse salivary glands by conditionally inactivating expression of TGFβ receptor type I (TGFβRI), either by using mouse mammary tumor virus–Cre mice or by delivering adenoviral vector containing Cre to mouse salivary glands via retrograde infusion of the cannulated main excretory ducts of submandibular glands.

Results

TGFβRI–conditional knockout (TGFβRI‐coko) mice were born normal; however, female TGFβRI‐coko mice developed severe multifocal inflammation in salivary and mammary glands and in the heart. The inflammatory disorder affected normal growth and resulted in the death of the mice at ages 4–5 weeks. Interestingly, male TGFβRI‐coko mice did not exhibit any signs of inflammation. The female TGFβRI‐coko mice also showed an increase in Th1 proinflammatory cytokines in salivary glands and exhibited an up‐regulation of peripheral T cells. In addition, these mice showed an atypical distribution of aquaporin 5 in their salivary glands, suggesting likely secretory impairment. Administration of an adenoviral vector encoding Cre recombinase into the salivary glands resulted in inflammatory foci only in the glands of female TGFβRI–loxP‐flanked (floxed) mice (TGFβRI‐f/f mice), but not in those of male and female wild‐type mice or male TGFβRI‐f/f mice.

Conclusion

These results suggest that female mice are uniquely more susceptible to developing inflammatory disorders due to impaired TGFβ signaling in their salivary glands.