Stéphanie Etienne

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)

Direct gene transfer into rat articular cartilage by in vivo electroporation

To establish a system for efficient direct in vivo gene targeting into rat joint, we have evaluated a strategy of gene transfer by means of the delivery of external electric pulses (EP) to the knee after intraarticular injection of a reporter gene (GFP). Rats were killed at various times after the electro gene‐therapy to analyze GFP gene expression by immunohistochemistry. GFP staining was detected in the superficial, middle, and deep zones of the patellar cartilage at days 2 and 9, and thereafter only in the deep zone (months 1 and 2). The average percentage of GFP‐positive cells was estimated at 30% both one and 2 months after the gene transfer. Moreover, no pathologic change caused by the EP was detected in the cartilage. The level and stability of the long‐term GFP expression found in this study demonstrate the feasibility of a treatment of joint disorders (inflammatory or degenerative, focal or diffuse) using electric gene transfer.—Grossin, L., Cournil‐Henrionnet, C., Mir, L. M., Liagre, B., Dumas, D., Etienne, S., Guingamp, C., Netter, P., Gillet, P. Direct gene transfer into rat articular cartilage by in vivo electroporation. FASEB J. 17, 829–835 (2003)

Structural evaluation of articular cartilage: Potential contribution of magnetic resonance techniques used in clinical practice

OBJECTIVE To determine whether routine magnetic resonance imaging (MRI) techniques can detect age-related structural modifications of bovine articular cartilage. METHODS The cartilage of 3-month-old, 3-year-old, and 13-year-old animals was studied. T1- and T2-weighted MR sequences were performed using a 1.5T clinical imager and a 3-inch surface coil. Histologic slices (5 microm) of cartilage specimens were stained with picrosirius red (for collagen) and toluidine blue (for glycosaminoglycans [GAGs]). A polarized light study was performed to determine the collagen network organization. Except for the 13-year-old animal cartilage, the biochemical content was studied on slices cut parallel to the surface to determine GAG and hydroxyproline (collagen) content. Cartilage profiles were performed to determine the MR pixel intensity and the histologic color intensity. RESULTS On T1-weighted images, the cartilage was homogeneous, with pixel intensity profiles presenting low variations. On T2-weighted images, the cartilage was laminar in the 3-month-old animals and became homogeneous thereafter. The pixel intensity varied through the cartilage depth with a profile that depended on the age of the animal. The collagen and GAG staining showed abrupt transitions in the 3-month-old animal, while in older animals the cartilage became more homogeneous with a mild gradient of matrix constituents with depth. These results were confirmed by findings of a biochemical study. In addition to these matrix content variations, the bovine cartilage presented modifications of its collagen network organization with aging. CONCLUSION The MR T2-weighted sequences depicted signal variations with age in bovine cartilage concomitant with modifications in its structure. If confirmed in clinics, these observations will reinforce the place of MRI in characterizing cartilage with aging and pathologic processes.

C-Propeptides of Procollagens Iα1 and II that Differentially Accumulate in Enchondromas versus Chondrosarcomas Regulate Tumor Cell Survival and Migration

Chondrogenic tumors that exhibit benign or malignant behaviors synthesize variable amounts of cartilage-like extracellular matrix. To define the regulators of these phenotypes, we performed a proteomic comparison of multiple human chondrogenic tumors, which revealed differential accumulation of the C-propeptides of procollagens Ialpha1 and II (PC1CP and PC2CP) in malignant versus benign tumors, respectively. Expression patterns of PC1CP correlated with levels of tumor vascularization, whereas expression patterns of PC2CP suggested its susceptibility to immobilization within the extracellular matrix. Prompted by these observations, we investigated the functions of recombinant PC1CP and PC2CP in the extracellular matrix in soluble or immobilized states. Each induced beta1 integrin-mediated chondrocyte adhesion by distinct domains and efficacies, suggesting that they initiated distinct signaling pathways. Indeed, immobilized PC2CP, but not PC1CP, induced apoptosis of primary chondrocytes and EAhy926 endothelial cells. In contrast, soluble PC1CP, but not PC2CP, induced the migration of EAhy926 cells and increased vascular endothelial growth factor (VEGF) and CXCR4 expression in chondrocytes. Soluble PC2CP also increased VEGF expression, but along with a more pronounced effect on CXCR4 and matrix metalloproteinase 13 expression. Our findings suggest that PC1CP favors angiogenesis and tumor progression, but that PC2CP acts in a more complex manner, exerting antitumor and antiangiogenic properties through apoptosis induction when immobilized, but progression and metastasis when soluble. In summary, the relative levels of PC1CP and PC2CP and their interactions within the extracellular matrix contribute to tumor progression, angiogenesis, and metastasis in chondrogenic tumors.

Local induction of heat shock protein 70 (Hsp70) by proteasome inhibition confers chondroprotection during surgically induced osteoarthritis in the rat knee

AIM to determine if chondrocytic Hsp70 induction, via intra-articular injections of a reversible proteasome inhibitor (MG132), can protect articular chondrocytes from cellular death in experimental rat OA knee induced surgically by anterior cruciate ligament transection (ACLT). MATERIALS AND METHODS ACLT was performed on D0. Histological lesions in naive (sham) controls (ACLT+saline) and treated (ACLT+MG132) rats were assessed according to Mankins score. Repeated intra-articular injections (1.5 muM MG132 or saline were performed on D1, D7, D14 and D21. Rats were sacrificed sequentially on D7, D14 and D28. Detection of active caspase-3 and protein expression of Hsp70 was also determined on D7, D14 and D28 by immunostaining methods. RESULTS MG132 significantly reduced OA lesions on D28 in the MG132 treated group. The expression of Hsp70 increased 11-fold in the MG132-treated group versus 2-3-fold in ACLT-control rats on D28. Concomitantly, cells expressing caspase-3 increased 4-fold in ACLT model and decreased 2-fold with MG132 treatment. CONCLUSIONS Intra-articular induction of Hsp70 by MG132 could be a safe and interesting tool in chondrocytes protection from cellular injuries and thus might be a novel chondroprotective modality in rat OA.

Activation of PPARs α, β/δ, and γ Impairs TGF-β1-Induced Collagens' Production and Modulates the TIMP-1/MMPs Balance in Three-Dimensional Cultured Chondrocytes.

Background and Purpose. We investigated the potency of Peroxisome Proliferators-Activated Receptors (PPARs) α, β/δ, and γ agonists to modulate Transforming Growth Factor-β1 (TGF-β1-) induced collagen production or changes in Tissue Inhibitor of Matrix Metalloproteinase- (TIMP-) 1/Matrix Metalloproteinase (MMP) balance in rat chondrocytes embedded in alginate beads. Experimental Approach. Collagen production was evaluated by quantitative Sirius red staining, while TIMP-1 protein levels and global MMP (-1, -2, -3, -7, and -9) or specific MMP-13 activities were measured by ELISA and fluorigenic assays in culture media, respectively. Levels of mRNA for type II collagen, TIMP-1, and MMP-3 & 13 were quantified by real-time PCR. Key Results. TGF-β1 increased collagen deposition and type II collagen mRNA levels, while inducing TIMP-1 mRNA and protein expression. In contrast, it decreased global MMP or specific MMP-13 activities, while decreasing MMP-3 or MMP-13 mRNA levels. PPAR agonists reduced most of the effects of TGF-β1 on changes in collagen metabolism and TIMP-1/MMP balance in rat in a PPAR-dependent manner, excepted for Wy14643 on MMP activities. Conclusions and Implications. PPAR agonists reduce TGF-β1-modulated ECM turnover and inhibit chondrocyte activities crucial for collagen biosynthesis, and display a different inhibitory profile depending on selectivity for PPAR isotypes.

In vivo rat knee cartilage volume measurement using quantitative high resolution MRI (7 T): feasibility and reproducibility.

OBJECTIVES to assess reliability and reproducibility of quantitative MRI (7 T) in assessing rat femoro-tibial cartilage volume. METHODS 5 healthy rat knees were scanned in vivo using a 7 T experimental imager. Sagittal high resolution 3D Gradient Echo with fat suppression sequences were performed with a dedicated home-made 2-elements array coil. 3D MRI sets were used to perform manual segmentation of the 3 cartilage compartments (femoral groove, medial and lateral tibial plateaus) by using a tactile screen. To evaluate inter- and intra-observer reproducibilities, the segmentation procedure was done blindly by two trained observers. One observer repeated the operation twice, with a period of 10 months between both readings. RESULTS the mean duration to manually segment all the slices covering the cartilaginous joint was 4 hours. On the one hand, the inter-observer root mean square of coefficients of variation was 9.1%, 6.2%, 9.6% for the femoral, medial and lateral tibial compartments respectively. On the other hand, the intra-observer reproducibility was 2.1%, 3.2%, 2.5% for these cartilage compartments cited above. CONCLUSION the image quality obtained at 7 Teslas with our dedicated coil allowed segmentation of the cartilage compartments with good reproducibility. This study demonstrated that MRI is a useful technology to provide a non-invasive and reliable assessment of rat knee cartilage volume.

Ambivalent properties of hyaluronate and hylan during post-traumatic OA in the rat knee.

AIM To determine whether viscosupplementation with intra-articular (i.a.) low- or high-molecular-weight hyaluronate (HA) injections influenced both chondral and synovial lesions in rats with surgically-induced OA knee. METHODS On D0, rats underwent anterior cruciate ligament transection (ACLX) and were divided in 4 groups: sham group, ACLX-saline control group, ACLX-hyaluronate group, ACLX-hylan group. IA injections were performed on D7, D14 and D21. Histological grading of chondral and synovial lesions were performed on D28. Concomitant immunostainings of Caspase3a and Hsp70 were also performed. RESULTS Articular damages were significantly reduced in both HAs-treated knee joints. In contrast, a significant increase of histological score of synovial inflammation was noted in both ACLX + HAs groups. Apoptotic events significantly decreased as anti-apoptotic Hsp70 expression increased significantly in both HAs groups. CONCLUSION HAs may exert, independently of its molecular weight, ambivalent properties on articular structures, simultaneously exerting chondroprotective properties and promoting long-term subacute synovitis.

88 Étude du genou arthrosique de rat en IRM 3D haute résolution et en histologie conventionnelle : analyse comparative

Introduction L’IRM champs eleves 3 dimensions (3D) a haute resolution (HR) permettant d’optimiser la qualite des images est un outil de recherche interessant pour etudier le cartilage articulaire des petites articulations de facon non-invasive. A partir des donnees 3D de l’IRM, nous avons realise des mesures quantitatives de volumes et d’epaisseurs du cartilage qui furent confrontees aux donnees histologiques classiques. Materiel et Methodes 48 rats Wistar mâles ont subi une section du ligament croise anterieur du genou droit selon un protocole standardise, le genou gauche servant de temoin. Les genoux etaient explores par IRM in vivo a 7T (sequence 3D echo de gradient, suppression du signal de la graisse, antenne specifique a deux elements en reseau) a J7, J14, J28, J42 et J56. Apres l’acquisition, les animaux etaient sacrifies pour l’etude histologique. Des coupes histologiques sagittales colorees par l’hematoxyline-eosine-safran etaient realisees, correspondant aux zones articulaires portantes selectionnees en IRM. Pour les deux techniques d’imagerie, les cartilages femoraux et tibiaux etaient segmentes manuellement. Les masques binaires resultants permettaient de determiner les volumes et epaisseurs (confrontations IRM-histologie) du cartilage articulaire. Resultats Concernant l’epaisseur du cartilage, il existait une correlation significative p = 0,02 entre l’IRM et l’histologie, au niveau femoral et tibial. Les valeurs des scores histologiques (membrane synoviale et cartilage) sont significativement differentes entre les genoux operes et temoins. Le suivi de la progression arthrosique en IRM montre d’importantes lesions degeneratives avec pertes cartilagineuses predominant au niveau du compartiment femoral, le compartiment tibial etant plutot le siege de phenomenes inflammatoires et oedemateux. Une correlation significative existait entre les mesures d’epaisseur relevees en IRM et en histologie (femur : r = 0,45 ; p = 0,02 ; tibia : r = 0,47 ; p = 0,01). Conclusion L’IRM 3D HR a 7T permet l’analyse in vivo de la progression arthrosique chez le rat. Les valeurs quantitatives obtenues en IRM sont compatibles avec les variations d’epaisseurs relevees en histologie. Une correlation significative est etablie entre ces deux techniques. L’IRM 3D HR a 7T est un outil non-invasif prometteur pour l’evaluation et le suivi in vivo de reponses therapeutiques chez le rongeur arthrosique.