Elly L. Vitters

Inhibition of endogenous TGF-beta during experimental osteoarthritis prevents osteophyte formation and impairs cartilage repair.

Osteoarthritis has as main characteristics the degradation of articular cartilage and the formation of new bone at the joint edges, so-called osteophytes. In this study enhanced expression of TGF-β1 and -β3 was detected in developing osteophytes and articular cartilage during murine experimental osteoarthritis. To determine the role of endogenous TGF-β on osteophyte formation and articular cartilage, TGF-β activity was blocked via a scavenging soluble TGF-β-RII. Our results clearly show that inhibition of endogenous TGF-β nearly completely prevented osteophyte formation. In contrast, treatment with recombinant soluble TGF-β-RII markedly enhanced articular cartilage proteoglycan loss and reduced the thickness of articular cartilage. In conclusion, we show for the first time that endogenous TGF-β is a crucial factor in the process of osteophyte formation and has an important function in protection against cartilage loss.

Reduced transforming growth factor-beta signaling in cartilage of old mice: role in impaired repair capacity

Osteoarthritis (OA) is a common joint disease, mainly effecting the elderly population. The cause of OA seems to be an imbalance in catabolic and anabolic factors that develops with age. IL-1 is a catabolic factor known to induce cartilage damage, and transforming growth factor (TGF)-beta is an anabolic factor that can counteract many IL-1-induced effects. In old mice, we observed reduced responsiveness to TGF-beta-induced IL-1 counteraction. We investigated whether expression of TGF-beta and its signaling molecules altered with age. To mimic the TGF-beta deprived conditions in aged mice, we assessed the functional consequence of TGF-beta blocking. We isolated knee joints of mice aged 5 months or 2 years, half of which were exposed to IL-1 by intra-articular injection 24 h prior to knee joint isolation. Immunohistochemistry was performed, staining for TGF-beta1, -2 or -3, TGF-betaRI or -RII, Smad2, -3, -4, -6 and -7 and Smad-2P. The percentage of cells staining positive was determined in tibial cartilage. To mimic the lack of TGF-beta signaling in old mice, young mice were injected with IL-1 and after 2 days Ad-LAP (TGF-beta inhibitor) or a control virus were injected. Proteoglycan (PG) synthesis (35S-sulfate incorporation) and PG content of the cartilage were determined. Our experiments revealed that TGF-beta2 and -3 expression decreased with age, as did the TGF-beta receptors. Although the number of cells positive for the Smad proteins was not altered, the number of cells expressing Smad2P strongly dropped in old mice. IL-1 did not alter the expression patterns. We mimicked the lack of TGF-beta signaling in old mice by TGF-beta inhibition with LAP. This resulted in a reduced level of PG synthesis and aggravation of PG depletion. The limited response of old mice to TGF-beta induced-IL-1 counteraction is not due to a diminished level of intracellular signaling molecules or an upregulation of intracellular inhibitors, but is likely due to an intrinsic absence of sufficient TGF-beta receptor expression. Blocking TGF-beta distorted the natural repair response after IL-1 injection. In conclusion, TGF-beta appears to play an important role in repair of cartilage and a lack of TGF-beta responsiveness in old mice might be at the root of OA development.

Expression of TGF-beta and the TGF-beta signaling molecule SMAD-2P in spontaneous and instability-induced osteoarthritis Role in cartilage degradation, chondrogenesis and osteophyte formation

Background: The primary feature of osteoarthritis is cartilage loss. In addition, osteophytes can frequently be observed. Transforming growth factor-β (TGFβ) has been suggested to be associated with protection against cartilage damage and new cartilage formation as seen in osteophytes. Objective: To study TGFβ and TGFβ signalling in experimental osteoarthritis to gain insight into the role of TGFβ in cartilage degradation and osteophyte formation during osteoarthritis progression. Methods: Histological sections of murine knee joints were stained immunohistochemically for TGFβ3 and phosphorylated SMAD-2 (SMAD-2P). Expression patterns were studied in two murine osteoarthritis models, representing spontaneous (STR/ort model) and instability-associated osteoarthritis (collagenase-induced instability model). Results: TGFβ3 and SMAD-2P staining was increasingly reduced in cartilage during osteoarthritis progression in both models. Severely damaged cartilage was negative for TGFβ3. In contrast, bone morphogenetic protein-2 (BMP-2) expression was increased. In chondrocyte clusters, preceding osteophyte formation, TGFβ3 and SMAD-2P were strongly expressed. In early osteophytes, TGFβ3 was found in the outer fibrous layer, in the peripheral chondroblasts and in the core. Late osteophytes expressed TGFβ3 only in the fibrous layer. SMAD-2P was found throughout the osteophyte at all stages. In the late-stage osteophytes, BMP-2 was strongly expressed. Conclusion: Data show that lack of TGFβ3 is associated with cartilage damage, suggesting loss of the protective effect of TGFβ3 during osteoarthritis progression. Additionally, our results indicate that TGFβ3 is involved in early osteophyte development, whereas BMP might be involved in late osteophyte development.

TGF β-induced cartilage repair is maintained but fibrosis is blocked in the presence of Smad7

Cartilage damage in osteoarthritis (OA) is considered an imbalance between catabolic and anabolic factors, favoring the catabolic side. We assessed whether adenoviral overexpression of transforming growth factor-β (TGFβ) enhanced cartilage repair and whether TGFβ-induced fibrosis was blocked by local expression of the intracellular TGFβ inhibitor Smad7. We inflicted cartilage damage by injection of interleukin-1 (IL-1) into murine knee joints. After 2 days, we injected an adenovirus encoding TGFβ. On day 4, we measured proteoglycan (PG) synthesis and content. To examine whether we could block TGFβ-induced fibrosis and stimulate cartilage repair simultaneously, we injected Ad-TGFβ and Ad-Smad7. This was performed both after IL-1-induced damage and in a model of primary OA. In addition to PG in cartilage, synovial fibrosis was measured by determining the synovial width and the number of procollagen I-expressing cells. Adenoviral overexpression of TGFβ restored the IL-1-induced reduction in PG content and increased PG synthesis. TGFβ-induced an elevation in PG content in cartilage of the OA model. TGFβ-induced synovial fibrosis was strongly diminished by simultaneous synovial overexpression of Smad7 in the synovial lining. Of great interest, overexpression of Smad7 did not reduce the repair-stimulating effect of TGFβ on cartilage. Adenoviral overexpression of TGFβ stimulated repair of IL-1- and OA-damaged cartilage. TGFβ-induced synovial fibrosis was blocked by locally inhibiting TGFβ signaling in the synovial lining by simultaneously transfecting it with an adenovirus overexpressing Smad7.

Gene Expression Analysis of Murine and Human Osteoarthritis Synovium Reveals Elevation of Transforming Growth Factor beta-Responsive Genes in Osteoarthritis-Related Fibrosis

Synovial fibrosis is a major contributor to joint stiffness in osteoarthritis (OA). Transforming growth factor β (TGFβ), which is elevated in OA, plays a key role in the onset and persistence of synovial fibrosis. However, blocking of TGFβ in OA as a therapeutic intervention for fibrosis is not an option since TGFβ is crucial for cartilage maintenance and repair. Therefore, we undertook the present study to seek targets downstream of TGFβ for preventing OA‐related fibrosis without interfering with joint homeostasis.

Reduced Euchromatin histone methyltransferase 1 causes developmental delay, hypotonia, and cranial abnormalities associated with increased bone gene expression in Kleefstra syndrome mice

Haploinsufficiency of Euchromatin histone methyltransferase 1 (EHMT1), a chromatin modifying enzyme, is the cause of Kleefstra syndrome (KS). KS is an intellectual disability (ID) syndrome, with general developmental delay, hypotonia, and craniofacial dysmorphisms as additional core features. Recent studies have been focused on the role of EHMT1 in learning and memory, linked to the ID phenotype of KS patients. In this study we used the Ehmt1(+/-) mouse model, and investigated whether the core features of KS were mimicked in these mice. When comparing Ehmt1(+/-) mice to wildtype littermates we observed delayed postnatal growth, eye opening, ear opening, and upper incisor eruption, indicating a delayed postnatal development. Furthermore, tests for muscular strength and motor coordination showed features of hypotonia in young Ehmt1(+/-) mice. Lastly, we found that Ehmt1(+/-) mice showed brachycephalic crania, a shorter or bent nose, and hypertelorism, reminiscent of the craniofacial dysmorphisms seen in KS. In addition, gene expression analysis revealed a significant upregulation of the mRNA levels of Runx2 and several other bone tissue related genes in P28 Ehmt1(+/-) mice. Runx2 immunostaining also appeared to be increased. The mRNA upregulation was associated with decreased histone H3 lysine 9 dimethylation (H3K9me2) levels, the epigenetic mark deposited by Ehmt1, in the promoter region of these genes. Together, Ehmt1(+/-) mice indeed recapitulate KS core features and can be used as an animal model for Kleefstra syndrome. The increased expression of bone developmental genes in the Ehmt1(+/-) mice likely contributes to their cranial dysmorphisms and might be explained by diminished Ehmt1-induced H3K9 dimethylation.

Induction of osteoarthritis by intra-articular injection of collagenase in mice. Strain and sex related differences

To study the effects of strain and sex on the development of injury-induced osteoarthritis (OA) in murine knee joints, two doses of highly purified bacterial collagenase (10 units and 30 units) were injected into male and female mice of two closely related strains, C57BL6 and C57BL10. Frontal histological sections of whole knee joints were made late in the disease process and examined for osteoarthritic lesions. Differences in prevalence of cartilage damage between strains and sexes were observed. Prevalence was higher in C57BL10 (male: almost 100%) than in C57BL6 (male: about 25%), and the prevalence was twice as high in males as in females in both strains. The amount of collagenase (10 or 30 units) did not affect the prevalence of lesions, however, it did influence the severity of the damage. The site of the damage appeared to be dose and strain dependent. Male C57BL6 always showed damage on the medial tibial plateau, independent of dose. In male C57BL10 damage almost always appeared on the lateral tibial plateau with 10 units, while with 30 units the medial plateau also became strongly involved. Since it is known that male mice are more prone to spontaneous OA than female mice and C57BL10 are more prone han C57BL6 mice, it can be concluded that predisposition to spontaneous osteoarthritis increases the risk of developing injury-induced osteoarthritis. Location and severity of the changes will probably be related to joint loading.

Inhibition of glycosaminoglycan synthesis in anatomically intact rat patellar cartilage by paracetamol-induced serum sulfate depletion

We have studied the effect of low sulfate concentrations on the glycosaminoglycan synthesis in rat patellar cartilage in vivo as well as in vitro. The oral administration of 200 mg/kg paracetamol to male Wistar rats resulted in a significant reduction of the serum sulfate concentration. Reduced serum sulfate availability resulted in a 34% decrease of glycosaminoglycan synthesis in patellar cartilage. This is due to sulfate depletion since paracetamol had no direct effects on glycosaminoglycan synthesis and a slight but significant inhibitory effect on the catabolism of radiolabeled glycosaminoglycans in vitro. The glycosaminoglycans synthesized at low sulfate concentrations in vivo were similar to the glycosaminoglycans synthesized at physiological sulfate concentrations. Studying the effect of sulfate availability in vitro on glycosaminoglycan synthesis in patellar cartilage we found that incubation of rat patellae in medium containing less than 0.5 mM inorganic sulfate led to a decreased sulfate incorporation. The use of potential sulfate decreasing drugs can lead to inhibition of glycosaminoglycan synthesis. This argues for a reconsideration of the use of these drugs in patients with already dysfunctioning cartilage metabolism as in rheumatoid arthritis and osteoarthrosis.

TGF-β is a potent inducer of Nerve Growth Factor in articular cartilage via the ALK5-Smad2/3 pathway. Potential role in OA related pain?

OBJECTIVEnPain is the main problem for patients with osteoarthritis (OA). Pain is linked to inflammation, but in OA a subset of patients suffers from pain without inflammation, indicating an alternative source of pain. Nerve Growth Factor (NGF) inhibition is very efficient in blocking pain during OA, but the source of NGF is unclear. We hypothesize that damaged cartilage in OA releases Transforming Growth Factor-β (TGF-β), which in turn stimulates chondrocytes to produce NGF.nnnDESIGNnMurine and human chondrocyte cell lines, primary bovine and human chondrocytes, and cartilage explants from bovine metacarpal joints and human OA joints were stimulated with TGF-β1 and/or Interleukin-1 (IL-1)β. We analyzed NGF expression on mRNA level with QPCR and stained human OA cartilage for NGF immunohistochemically. Cultures were additionally pre-incubated with inhibitors for TAK1, Smad2/3 or Smad1/5/8 signaling to identify the TGF-β pathway inducing NGF.nnnRESULTSnNGF expression was consistently induced in higher levels by TGF-β than IL-1 in all of our experiments: murine, bovine and human origin, in cell lines, primary chondrocytes and explants cultures. TAK1 inhibition consistently reduced TGF-β-induced NGF whereas it fully blocked IL-1β-induced NGF expression. In contrast, ALK5-Smad2/3 inhibition fully blocked TGF-β-induced NGF expression. Despite the large variation in basal NGF in human OA samples (mRNA and histology), TGF-β exposure led to a consistent high level of NGF induction.nnnCONCLUSIONnWe show for the first time that TGF-β induces NGF expression in chondrocytes, in a ALK5-Smad2/3 dependent manner. This reveals a potential alternative non-inflammatory source of pain in OA.

Analysis of changes in proteoglycan content in murine articular cartilage using image analysis

The extracellular matrix of articular cartilage consists mainly of type II collagen and large aggregating proteoglycan (aggrecan). During arthritis and other joint diseases, the proteoglycan (PG) level of cartilage matrix is diminished, leading to impairment of normal joint function. A new method is described for measuring the changes in PG content of murine articular cartilage. The method is based on the automated densitometric analysis of patellar cartilage of standard, safranin O-stained sections of whole murine knee joints. It appeared to be possible to measure optical density in parallel layers of articular cartilage with high reproducibility. Approximately 25 sections can be evaluated within 1 h. Measuring a single section 10 times resulted in a coefficient of variation (CV) of 0.1-1.4%. A mean CV of 5-14% was calculated when a group of 18 sections was analyzed in quintuplicate. To validate the procedure, changes in PG content induced by arthritis or by intra-articular injection of TGFbeta-1 were analyzed by the image analysis method, the dimethylmethylene blue (DMB) assay and by visual grading. Although not a quantitive method, the newly developed image analysis method appeared to be more sensitive in detecting significant change in PG content of murine articular cartilage than the DMB method or visual grading. The image analysis method makes it possible to measure changes in PG content of specific areas of articular cartilage with higher sensitivity than the DMB method and eliminating the bias inherent to visual grading by human observers.