Pierre Gillet

Mutations in ANKH Cause Chondrocalcinosis

Chondrocalcinosis (CC) is a common cause of joint pain and arthritis that is caused by the deposition of calcium-containing crystals within articular cartilage. Although most cases are sporadic, rare familial forms have been linked to human chromosomes 8 (CCAL1) or 5p (CCAL2) (Baldwin et al. 1995; Hughes et al. 1995; Andrew et al. 1999). Here, we show that two previously described families with CCAL2 have mutations in the human homolog of the mouse progressive ankylosis gene (ANKH). One of the human mutations results in the substitution of a highly conserved amino acid residue within a predicted transmembrane segment. The other creates a new ATG start site that adds four additional residues to the ANKH protein. Both mutations segregate completely with disease status and are not found in control subjects. In addition, 1 of 95 U.K. patients with sporadic CC showed a deletion of a single codon in the ANKH gene. The same change was found in a sister who had bilateral knee replacement for osteoarthritis. Each of the three human mutations was reconstructed in a full-length ANK expression construct previously shown to regulate pyrophosphate levels in cultured cells in vitro. All three of the human mutations showed significantly more activity than a previously described nonsense mutation that causes severe hydroxyapatite mineral deposition and widespread joint ankylosis in mice. These results suggest that small sequence changes in ANKH are one cause of CC and joint disease in humans. Increased ANK activity may explain the different types of crystals commonly deposited in human CCAL2 families and mutant mice and may provide a useful pharmacological target for treating some forms of human CC.

Assessment of articular cartilage and subchondral bone : Subtle and progressive changes in experimental osteoarthritis using 50 MHz echography in vitro

The main objectives of this work were to demonstrate the potential of 50 MHz echography for assessing initial and progressive morphological and structural changes of articular cartilage and bone developed in an experimental model of osteoarthritis (OA). Degenerative lesions were induced in rat knees by the unilateral intra‐articular injection of a 3 mg dose of mono‐iodo‐acetic acid. To assess the lesion progression, the animals (n = 30) were sacrificed at different time intervals up to 8 weeks after the injection. Three‐dimensional echographic data were acquired in vitro on patellar cartilage and bone at various stages of the remodeling process using a scanning ultrasound microscope. Changes involving the OA cartilage characteristics are discussed relative to those of the contralateral control joint which received a placebo. Images of control cartilage showed a smooth hyperechoic articular surface and an echoic matrix. The cartilage thickness was 266 ± 44 μm (mean ± SD) in the central region of the tissue. The precision of ultrasonic thickness measurements was better than 1.3%. First changes in cartilage internal structure and subchondral bone appeared on ultrasound images 3 days after the injection and were even more evident by day 7. They resulted in a slight thinning of the cartilage, a 30% increase of its internal structure echogenicity, and the appearance of echoic zones in subchondral bone. Histologic findings confirmed chondrocyte depletion and degeneration, decrease of matrix proteoglycans, and fibrovascular connective tissue proliferation at the subchondral plate. Progressive and severe lesions at both bone and cartilage surface and internal structure were assessed and correlated to histologic features. These results show that high resolution echography is sensitive to subtle and progressive osteochondral remodeling. This technique has the potential to be used for intra‐articular quantitative imaging and assessment of early changes in bone and cartilage structure associated with natural human disease.

Relations between functional, inflammatory, and degenerative parameters during adjuvant arthritis in rats

We assessed the time-course of adjuvant arthritis (AA) in Lewis rats, using biotelemetry to monitor the rats spontaneous locomotor activity and body temperature, and studied the evolution of the arthritic index, circulating concentrations of inflammation-promoting cytokines, cartilage proteoglycan synthesis, and the effect of indomethacin as a cyclooxygenase inhibitor to evaluate prostaglandin (PG) contribution in AA. The injection of complete Freunds adjuvant on day 0( D0) induced a marked, transient loss of locomotor activity ( D1- D4; initial phase) and then a second phase of hypomobility peaking on D15 and thereafter irreversible ( D16- D20; arthritic phase). Fever peaked first on D1 and again between D13 and D17. The primary hyperthermia was associated with increases in plasma interleukin-6 and tumor necrosis factor-α concentrations and seemed to be partly PG dependent. Proteoglycan synthesis inhibition in the patellar cartilage increased gradually, spreading from the injected paw to the contralateral paw. It was corrected on D20 by preventive and curative indomethacin treatments. Indomethacin also greatly relieved hypomobility during the systemic phase of AA ( D10- D15). The combination of information about cartilage metabolism, body temperature, locomotor activity, and cytokine in this study permits analysis of analgesic, antipyretic, anti-inflammatory, and chondroprotective properties of drugs in the various phases of AA. Thus, using a new methodology, we have discriminated the different phases of the disease and confirmed the symptomatic and systemic inhibitory effect of indomethacin on fever, activity, and cartilage metabolism.

Magnetic resonance imaging in osteoarthritis: which method best reflects synovial membrane inflammation? Correlations with clinical, macroscopic and microscopic features

OBJECTIVES : To study synovial membrane (SM) inflammation near the patella with different magnetic resonance imaging (MRI) approaches performed using a T1-injected sequence in knee osteoarthritis (OA), and to compare MRI results with macroscopic, microscopic and clinical findings. METHODS Fifteen patients fulfilling American College of Rheumatology (ACR) criteria for knee OA and requiring joint lavage completed a functional index (Lequesnes functional index) and a pain visual analog scale (VAS). SM inflammation near the patella was assessed on axial fat saturation post-injected T1 MRI images using three different methods: (1) semi-quantitative score=MRI synovitis score; (2) synovial membrane volume (SMV) analysis; (3) SMV with low (SMVL) (<0.3%/s(-1)), intermediate (SMVI) (0.3%/s(-1) to 1%/s(-1)) and high (SMVH) (> or =1%/s(-1)) speed of enhancement. Chondral lesions and SM inflammation were macroscopically graded and SM biopsies performed for microscopic scoring. RESULTS All MRI approaches exhibited excellent intra- and inter-observer reproducibility. MRI synovitis score correlated well with macroscopic (r=0.61, P=0.003) and total microscopic scores (r=0.55, P=0.03). Correlations between SMV and macroscopic (r=0.60, P=0.02) and microscopic congestion (r=0.63, P=0.01) were good. SMVH was correlated only with microscopic congestion (r=0.79, P=0.01). Low SMV was associated with neither macroscopic nor microscopic scores. However, it did correlate well with pain-VAS score (r=0.61, P=0.03) and moderately with a functional index (r=0.46, P=0.10). CONCLUSION The three MRI approaches used here provided highly reproducible information on SM inflammation near the patella in knee OA. Compared to SMV, MRI synovitis score seems sufficient to assess synovial inflammation but high SMV is an appropriate indicator of vascular congestion, and low SMV reflects pain in knee OA.

Pefloxacin-Induced Achilles Tendon Toxicity in Rodents: Biochemical Changes in Proteoglycan Synthesis and Oxidative Damage to Collagen

ABSTRACT Despite a relatively low incidence of serious side effects, fluoroquinolones and the fluoroquinolone pefloxacin have been reported to occasionally promote tendinopathy that might result in the complication of spontaneous rupture of tendons. In the present study, we investigated in rodents the intrinsic deleterious effect of pefloxacin (400 mg/kg of body weight) on Achilles tendon proteoglycans and collagen. Proteoglycan synthesis was determined by measurement of in vivo and ex vivo radiosulfate incorporation in mice. Collagen oxidative modifications were measured by carbonyl derivative detection by Western blotting. An experimental model of tendinous ischemia (2 h) and reperfusion (3 days) was achieved in rats. Biphasic changes in proteoglycan synthesis were observed after a single administration of pefloxacin, consisting of an early inhibition followed by a repair-like phase. The depletion phase was accompanied by a marked decrease in the endogenous serum sulfate level and a concomitant increase in the level of sulfate excretion in urine. Studies of ex vivo proteoglycan synthesis confirmed the in vivo results that were obtained. The decrease in proteoglycan anabolism seemed to be a direct effect of pefloxacin on tissue metabolism rather than a consequence of the low concentration of sulfate. Pefloxacin treatment for several days induced oxidative damage of type I collagen, with the alterations being identical to those observed in the experimental tendinous ischemia and reperfusion model. Oxidative damage was prevented by coadministration of N-acetylcysteine (150 mg/kg) to the mice. These results provide the first experimental evidence of a pefloxacin-induced oxidative stress in the Achilles tendon that altered proteoglycan anabolism and oxidized collagen.

Gene transfer with HSP 70 in rat chondrocytes confers cytoprotection in vitro and during experimental osteoarthritis

Osteoarthritis is characterized by a gradual degradation of extracellular matrix, resulting from an excess of chondrocyte cell death, mainly due to an increase in apoptotis. Recent studies have revealed the essential role of HSP70 in protecting cells from stressful stimuli. Therefore, overexpressing HSP70 in chondrocytes could represent a good strategy to prevent extracellular matrix destruction. To this end, we have developed a vector carrying HSP70/GFP, and transduced chondrocytes were thus more resistant to cell death induced by mono‐iodoacetate (MIA). To overcome the barrier‐effect of matrix, we investigated the efficacy of plasmid delivery by electroporation (EP) in rat patellar cartilage. Two days after EP, 50% of patellar chondrocytes were HSP/GFP+. After 3 months, long‐term expression of transgene was only depicted in the deep layer (20–30% positive cells). HSP70 overexpression inhibited the natural endochondral ossification in the deep layer, thus leading to a lesser decrease in chondrocyte distribution. Moreover, overexpression of HSP70, after a preventive EP transfer in rat patella, was sufficient to decrease the severity of osteoarthritis‐induced lesions, as demonstrated histologically and biochemically. In conclusion, intracellular overexpression of HSP70, through EP delivery, could protect chondrocytes from cellular injuries and thus might be a novel chondroprotective modality in rat OA.—Grossin, L., Cournil‐Henrionnet, C., Pinzano, A., Gaborit, N., Dumas, D., Etienne, S., Stoltz, J. F., Terlain, B., Netter, P., Mir, L. M., Gillet, P. Gene transfer with HSP 70 in rat chondrocytes confers cytoprotection in vitro and during experimental osteoarthritis. FASEB J. 20, 65–75 (2006)

Evaluation of cartilage repair tissue after biomaterial implantation in rat patella by using T2 mapping

To evaluate the ability of MR T2 mapping (8.5 T) to characterize ex vivo longitudinally, morphologically and quantitatively, alginate-based tissue engineering in a rat model of patellar cartilage chondral focal defect. Calibrated rat patellar cartilage defects (1.3 mm) were created at day 0 (D0) and alginate sponge with (Sp/C+) or without (Sp/C−) autologous chondrocytes were implanted. Animals were sacrificed sequentially at D20, D40 and D60 after surgery and dissected patellae underwent MRI exploration (8.5 T). T2 values were calculated from eight SE images by using nonlinear least-squares curve fitting on a pixel-by-pixel basis (constant repetition time of 1.5 s, eight different echo times: 5.5, 7.5, 10.5, 12.5, 15.0, 20.0, 25.0 and 30.0 ms). On the T2 map, acquired in a transversal plane through the repair zone, global T2 values and zonal variation of T2 values of repair tissue were evaluated versus control group and compared with macroscopic score and histological studies (toluidine blue, sirius red and hematoxylin-eosin). “Partial”, “total” and “hypertrophic” repair patterns were identified. At D40 and D60, Sp/C+ group was characterized by a higher proportion of “total” repair in comparison to Sp/C− group. At D60, the proportion of “hypertrophic” repair was two fold in Sp/C− group versus Sp/C+ group. As confirmed morphologically and histologically, the T2 map also permitted the distinction of three types of repair tissue: “total”, “partial” and “hypertrophic”. “Total” repair tissue was characterized by high T2 values versus normal cartilage (p<0.05). Zonal variation, reflecting the collagen network organization, appeared only at D60 for Sp/C+ group (p<0.05). “Hypertrophic” tissue, mainly observed at D60, presented high T2 global values without zonal variation with cartilage depth. These results confirm the potency of the MR T2 map (8.5 T) to characterize macroscopically and microscopically the patterns of the scaffold guided-tissue repair of a focal chondral lesion in the rat patella (“total”, “partial” and “hypertrophic”). On T2 map, three parameters (i.e. MRI macroscopic pattern, T2 global values and zonal variation of T2 values) permit to characterize chondral repair tissue, as a virtual biopsy.

Refinement of the Chromosome 5p Locus for Familial Calcium Pyrophosphate Dihydrate Deposition Disease

Familial calcium pyrophosphate dihydrate deposition disease (CPPDD) is a disease of articular cartilage that is radiographically characterized by chondrocalcinosis due to the deposition of calcium-containing crystals in affected joints. We have documented the disease in an Argentinean kindred of northern Italian ancestry and in a French kindred from the Alsace region. Both families presented with a common phenotype including early age at onset and deposition of crystals of calcium pyrophosphate dihydrate in a similar pattern of affected joints. Affected family members were karyotypically normal. Linkage to the short arm of chromosome 5 was observed, consistent with a previous report of linkage of the CPPDD phenotype in a large British kindred to the 5p15 region. However, recombinants in the Argentinean kindred have enabled us to designate a region<1 cM in length between the markers D5S416 and D5S2114 as the CPPDD locus.

Evidence for neurogenic transmission inducing degenerative cartilage damage distant from local inflammation.

OBJECTIVE To investigate involvement of the nervous system in ipsilateral and contralateral joint inflammation. METHODS Freunds complete adjuvant (CFA; 1 mg or 1 microg) was injected unilaterally and the messages (a) from the hind paw to the ipsilateral and contralateral knees and (b) from one knee to the contralateral knee were analyzed. The degenerative impact of the local injury on distant cartilage was assessed using patellar proteoglycan synthesis as an indicator. Neurogenic mechanisms were blocked either by spinal cord compression or by injection of neurokinin 1 (NK-1) antagonist, or they were mimicked by intraarticular injection of substance P. The data were compared with those gathered in a model of systemic inflammation, characterized by fever and serum interleukin-6 production. RESULTS After unilateral subcutaneous injection of CFA, proteoglycan anabolism decreased bilaterally. Spinal cord compression and administration of the NK-1 antagonist inhibited the response in the contralateral limb. Following 1 mg CFA subcutaneous injection, the ipsilateral response implicated both neurogenic and systemic mechanisms, whereas the nervous system alone was implicated after 1 microg subcutaneous CFA injection. The 1 microg CFA intraarticular injection induced a degenerative contralateral signal, which was abolished by spinal cord compression and by pretreatment with the NK-1 antagonist. Intraarticular injection of 1 microg CFA also induced an ipsilateral increase of anabolism, which was enhanced by spinal cord compression. Similar results were obtained after intraarticular injections of substance P. These effects were not reproduced with turpentine treatment, a systemic model, in which spinal cord compression had no effect. CONCLUSION A unilateral inflammation can induce, by neurogenic mechanisms, distal bilateral degeneration of articular cartilage, implicating involvement of neuropeptides.

MAGNETIC RESONANCE IMAGING OF NORMAL AND OSTEOARTHRITIC CARTILAGE

(MRI) has been slow to gain acceptance for cartilage evaluation because of its limited spatial resolution and because of the poor contrast between cartilage and adjacent structures. Continual improvement in gradient performance and coil design and the development of more efficient pulse sequences have overcome many of the early limitations of MIU. These improvements make possible high-resolution multiplanar and 3-dimensional (3-D) images with a wide variety of contrast. In this article, we present recent refinements in MRI of cartilage and offer a key for interpreting the wide range of images that are produced. Technical factors in MRI The pattern of cartilage as seen on MRT depends on spatial resolution and contrast. These factors are related to the choice of sequence and can be modified in various ways by the addition of pulses and contrast agents. In some cases, artifacts can greatly affect the quality of the images of hyaline cartilage.