Effect Of Inhibition Of HSP-90 On Extracellular Matrix Degradation In Avian Growth Plate In Thiram-induced Tibial Dyschondroplasia

Tibial dyschondroplasia(TD) is an avian bone disorder of different etiologies that may be associated with lameness. The disorder is characterized by focal disruption of endochondral bone formation, with a lack of matrix proteolysis and an accumulation of non-mineralized avascular cartilage.1. Differential expression of extracellular matrix metalloproteinase inducer(EMMPRIN/CD147) in avian tibial dyschondroplasiaThe extracellular matrix metalloproteinase inducer(EMMPRIN/CD147) is expressed on a wide variety of cell types and is best known as a potent inducer of matrix metalloproteinases(MMPs) directly in various biological processes. The aim of this study was to determine the expression of EMMPRIN/CD147 in normal, thiram-induced TD lesions and in the process of recovery from TD in broiler chickens. An extracellular matrix(ECM) degrading enzyme, matrix metalloproteinase-9(MMP-9), was selected to investigate the effects of CD147 in the degradation of ECM. Gene expression was analyzed by quantitative real-time polymerase chain reaction and protein levels by immunohistochemistry and western blotting. The birds were divided into three groups: thiram fed; recovery; and controls. Genes encoding CD147 and MMP-9 were downregulated during the development of the disease, and were up-regulated during recovery. Western blotting also showed lower protein levels of CD147 in TD, which increased during the recovery phase associated with ECM degradation and growth plate repair. The findings of this study suggest that ECM has a crucial role in the occurrence of TD and that CD147 appears to play a pivotal role in matrix proteolysis in the chicken, similar to that in other species.2. Effect of hsp-90 inhibition on extracellular matrix degradation in thiram-induced avian tibial dyschondroplasiaThis study was conducted to evaluate the effects of hsp-90 inhibition on ECMdegradation in thiram induced-TD. The gene expressions of EMMPRIN/CD147 and MMP-2 &-9 were used to mark the ECM degradation as described in previous study. The angiogenesis was spotted by determining the heat shock protein-90(hsp90), vascular endothelial growth factor(VEGF) and Flk-1(VEGF receptor) gene expressions. The administration of hsp90 inhibitor in thiram-induced TD birds resulted in the reduction of growth plate size and abrogated the lameness. This was occurred due to the up-regulation of Flk-1 gene expressions, one of the major rate limiting factors of vascularization. In addition, the ECM degradation, as characterized by the CD147 and MMP-2 &-9 were also restored. All these changes lead into the prevention of lameness. The inhibition of hsp90 activity not only increased the vascularization but also enhanced the ECM degradation which resulted into the reduced growth plate size and mitigated the lameness in TD chicks with established lesions. In summary, this is the first report describing the relation of hsp90 inhibition with ECM which resulted into the activation of angiogenic and ECM degradation switches “on” in TD-afflicted chicks and reinstated the normal growth plate morphology.3. Hsp-90 inhibitor geldanamycin attenuates liver oxidative stress and toxicity in thiraminduced tibial dyschondroplasiaAn experiment was conducted to study the effects of hsp90 inhibitor geldanamycin(17-DMAG) on liver functioning and its antioxidant ability in thiram-induced tibial dyschondroplasia. One hundred and twenty commercial chicken broilers were allocated into three groups: 1) control, 2) thiram-induced and 3) 17-DMAG treated. Serum samples were collected on day 11 and 14 post-hatch to determine the liver ALT, AST and ALP activity. The liver samples were collected at the end of trial to determine the activity of SOD(superoxide dismutase), GSH-Px(glutathione peroxidase) and MDA(malondialdehyde) contents. The results depicted that thiram increased the level of serum ALT, AST and liver MDA contents while decreased the serum ALP and liver antioxidant enzymes(SOD, GSH-Px); however, by administering 17-DMAG, these values were observed close to normal range as compared to control group. In conclusion, the oxidative imbalance and damage to liver caused by thiram can be restored by using 17-DMAG.4. Expression of genes encoding Matrilin-3 and Cyclin-I during the impairment and recovery of chicken growth plate in tibial dyschondroplasiaMatrilin-3, the smallest member of the Matrilins family, has high similarity across species between human and chicken. It is capable of interconnecting macromolecular networks and improves the coordination between cartilage cells and ECM. Cyclin-I, a member of the Cyclin family, controls the cell cycle progression by regulating the activity of cyclin-dependent kinases. It is involved in various biological processes, such as cell survival and differentiation. The aim of this study was to determine the expression of Matrilin-3 and Cyclin-I genes in chicken growth plate during impairment and recovery from TD. Gene expressions were analyzed by polymerase chain reaction and proteins by immunohistochemistry and in situ hybridizations. Expression of genes encoding Matrilin-3 and Cyclin-I were diminished with parallel decrease in proteins during TD, with fewer signs of cartilage cell differentiation. In contrast, there was an increase in m RNA expressions and protein levels of both genes during the recovery phase. These findings suggest that the Matrilin-3 and Cyclin-I genes also play a role in chondrocyte differentiation during the impairment and recovery of growth plate in TD.