Thiram-induced tibial dyschondroplasia (TD) and vitamin-D deficiency rickets are avian bone disorders of different etiologies characterized by abnormal chondrocyte differentiation, enlarged and unvascularized growth plates, and lameness. Heat-shock protein 90 (Hsp90) is a proangiogenic factor in mammalian tissues and in tumors; therefore, Hsp90 inhibitors were developed as antiangiogenic factors. In this study, we evaluated the association between Hsp90, hypoxia, and angiogenesis in the chick growth plate. Administration of the Hsp90 inhibitor to TD- and rickets-afflicted chicks at the time of induction resulted in reduction in growth-plate size and, contrary to its antiangiogenic effect in tumors, a major invasion of blood vessels occurred in the growth plates. This was the result of upregulation of the VEGF receptor Flk-1, the major rate-limiting factor of vascularization in TD and rickets. In addition, the abnormal chondrocyte differentiation, as characterized by collagen type II expression and alkaline phosphatase activity, and the changes in hypoxia-inducible factor-1α (HIF-1α) in both disorders were restored. All these changes resulted in prevention of lameness. Inhibition of Hsp90 activity reduced growth-plate size, increased vascularization, and mitigated lameness also in TD chicks with established lesions. In summary, this is the first reported demonstration of involvement of Hsp90 in chondrocyte differentiation and growth-plate vascularization. In contrast to the antiangiogenic effect of Hsp90 inhibitors observed in mammals, inhibition of Hsp90 activity in the unvascularized TD- and rickets-afflicted chicks resulted in activation of the angiogenic switch and reinstated normal growth-plate morphology.

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Tibial dyschondroplasia (TD) is a skeletal abnormality that can cause economic losses and animal welfare concerns. Thiram-induced TD is characterized by enlarged, unvascularized growth plates, low levels of the vascular endothelial growth factor receptor Flk-1, abnormal chondrocyte differentiation, and lameness. Recently we reported the involvement of heat-shock protein 90 (Hsp90) in chondrocyte differentiation and growth-plate vascularization. Inhibition of Hsp90 activity in thiram-induced TD resulted in increased Flk-1 levels, re-instated normal growth-plate angiogenesis and morphology, and abrogated lameness. In the present study, we evaluated the efficacy of various concentrations of 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG), an inhibitor of Hsp90 activity, in preventing growth-plate histopathology and lameness in TD-affected chicks. Low doses of 17-DMAG (2 injections, each of 100 or 300 μg) did not prevent TD development even though Flk-1 levels were restored, which suggests that Flk-1 is not the only rate-limiting factor in growth-plate angiogenesis. High doses of 17-DMAG (2 injections, each of 600 or 900 μg) prevented BW loss, decreased the TD score, reduced lesion width, restored proper chondrocyte differentiation, increased blood vessel invasion, and eliminated lameness. To assess the specificity of Hsp90, we evaluated the efficacy of the flavonoid quercetin, an inhibitor of Hsp70 synthesis, in preventing TD development; it decreased Hsp70 levels but not those of Hsp90 in the control growth plates and prevented upregulation of Hsp70 in the TD-affected growth plates. Dietary quercetin (at 100 or 500 ppm) did not prevent the hypoxia that is characteristic of the TD-affected growth plate or development of thiram-induced TD and lameness. The present results demonstrate the specificity and the major role of Hsp90 in chondrocyte differentiation and growth-plate vascularization. In contrast to the anti-angiogenic effect of 17-DMAG observed in mammals, inhibition of Hsp90 activity in the unvascularized TD-affected growth plates resulted in activation of the angiogenic switch and restored normal growth-plate morphology.

Tibial dyschondroplasia (TD) is one of the most prevalent skeletal abnormalities in avian species; it causes economic losses and is an animal welfare problem. It has been hypothesized that the absence of vasculature in the lesion of the TD growth plates at the ends of the long bones is involved in the etiology of the disease. We evaluated the hypoxia status of normal and thiram-induced TD growth plates by immunostaining the protein adducts after pimonidazole hydrochloride administration. In addition, we evaluated the expression of hypoxia-inducible factor-1alpha (HIF-1alpha), the major regulator of the hypoxic response that is essential for chondrogenesis, and that of heat-shock proteins (Hsp) downstream from HIF-1alpha. We demonstrated that, in contrast to the normal growth plates, those afflicted by TD were hypoxic. A major increase in hypoxia was observed in the proliferative, hypertrophic, and calcified zones. In the normal growth plate, HIF-1alpha was expressed in chondrocytes of the articular cartilage and of the maturation zone, whereas in cases of TD, HIF-1alpha was also expressed in chondrocytes below the lesion. The expression level of HIF-1alpha was related to the severity of the disease, but was independent of its cause; the same pattern of expression was observed in growth plates of chicks selected for a high incidence of TD. No differentiation-dependent expression of HIF-1alpha was observed in response to hypoxia, as demonstrated by the use of primary cultures of growth plate chondrocytes. In the normal growth plates, Hsp90 and Hsp70 were localized to the maturation zone. More cells expressed both Hsp in the TD lesion. In conclusion, we demonstrated that the TD growth plate, in contrast to the normal one, is hypoxic, probably because of the lack of vascularization. Hypoxia leads to an increase in the transcription factor HIF-1alpha, causing increases in the levels of Hsp90 and Hsp70.

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