TD is the most common leg disease in broilers, and the incidence of TD can be up to 10% in some chicken farms, which has been the main cause of lameness in poultry raised for meat production (Mehmood et al. 2019). Mn is an essential element for bone growth (Defu Li 2020; Gajula et al. 2011), and its deficiency leads to the failure of endochondral osteogenesis in tibia of fast-growing broiler chickens (Liao et al. 2019). The National Research Council (NRC) recommends a dietary minimum of 60 mg Mn/kg for growing chicks (Mondal et al. 2010). Thus, Mn deficiency diet (21.8 ± 1.4 mg Mn/kg) was fed with growing chicks to establish Mn deficiency-induced TD in this study. We found that Mn deficiency inhibits angiogenesis in the proximal tibial metaphysis by down-regulating VEGF and MIF expression levels, while HIF-1α upregulation and autophagy activation in TGP play a protective role in this process.
During the elongation of the tibia, chondrocytes are transformed into TGP cartilage and bone tissue adjacent to the metaphysis through proliferation and differentiation to increase the tibia length. Metaphyseal vascularization is closely related to the chondrocyte maturation in TGP and tibial elongation (Herzog et al. 2011). The final event of endochondral ossification is the replacement of avascular cartilage tissue with highly vascularized bone tissue, and the failure of angiogenesis is liable to increase the incidence of TD (Sivaraj and Adams 2016). During this process, MIF and VEGF are secreted by chondrocytes in hypertrophic chondrocyte zone of TGP to induce the vascular invasion and osteogenesis, which play a crucial role in regulate angiogenesis and enhance endothelial cell adhesion, migration, proliferation and microtubule formation (Zhang et al. 2018). In this study, we found that the number of blood vessels in the Mn-D group was sharply reduced, which led to the transportation hurdles of nutrition to chondrocyte. Ultimately, cartilage in the zone of mineralization and calcium deposition fails to complete the calcification. This study showed that Mn deficiency inhibited the metaphysis vascularization and down-regulated the expression levels of VEGF and MIF in TGP of proximal tibia in chicks, which is consistent with the results of thiram-induced TD in chicks (Nabi et al. 2016). Our research group previously found that Mn deficiency induces an increase in apoptotic death of hypertrophic chondrocytes in TGP (Wang et al. 2015), while the present data further revealed that insufficient blood supply in TGP is another factor contributing to the death of hypertrophic chondrocytes. Given the above mentioned, we guess that down-regulation of VEGF and MIF expression in TGP may be responsible for the inhibition of metaphysis vascularization, resulting in inhibition of TGP development, but it needs to be further investigated.
TGP resides in a low oxygen environment and it is an avascular tissue that obtains nutrition and oxygen by diffusion from the metaphysis vascular (Alonso et al. 2019; Sivaraj and Adams 2016). Many previous studies have shown that HIF-1α, a master regulator of the cellular response to hypoxia, is essential for growth and survival of TGP chondrocytes in vivo (Jahejo and Tian 2021; Wang et al. 2018). HIF-1α is expressed in a hypoxia-independent manner under physiological conditions, mainly in hypertrophic zone of TGP, where it is necessary for chondrocyte maturation and differentiation (Hu et al. 2020) (Lee et al. 2012). It has been shown that inadequate blood supply to chondrocytes leads to a decrease in oxygen concentration and an up-regulation of HIF-1α expression in TGP of chicks with TD (Huang et al. 2018). Moreover, chondrocytes lacking functional HIF-1α undergo massive cell death in the TGP, which leads to the tibia narrow and exhibits less vascularization in metaphysis (Oda et al. 2008). Another hypoxia experiment using AA chicks has shown that tibial angiogenesis is enhanced by upregulation of pro-angiogenic factors, such as MIF and VEGF, contributing to oxygen and nutrition delivery to TGP (Huang et al. 2017). Data in this study showed that Mn deficiency induces the HIF-1α up-regulation and down-regulation of MIF and VEGF expression in TGP. This distinction may be explained that Mn deficiency suppresses normal hypertrophy of chondrocytes in TGP, resulting in decreased production of MIF and VEGF and subsequent inhibition of metaphyseal vascularization. Given the above mentioned, we speculated that HIF-1α up-regulation may mitigate the chondrocytes damage in TGP caused by Mn deficiency via antagonizing the decrease in angiogenesis.
Autophagy is an evolutionarily conserved process that plays an important role in maintaining intracellular homeostasis and degrading cytoplasmic components, such as damaged organelles and misfolded proteins, to provide energy for the body (Song et al. 2017) (Lu et al. 2018). Recent study has shown that hypoxia can activate autophagy and promote lysosomal degradation of proteins and organelles (Muz et al. 2015). Hypoxia-induced autophagy has been shown to be initiated by HIF-1α in a variety of cell lines (Zhang et al. 2008). In hypoxic condition, the degradation of HIF-1α is inhibited, resulting in increased accumulation of HIF-1α, which directly binds to the hypoxia response elements of the target genes that are involved in various events including angiogenesis, cell migration and tumor invasion (Hu et al. 2012). Previous studies have shown that inhibition of autophagy genes results in the death of chondrocytes, suggesting that autophagy functions as cytoprotective effect in tibial growth (Wang et al. 2021) (Yang et al. 2020). It is reported that HIF-1α up-regulation can induce the autophagy activation under stress conditions to ensure the survival of TGP chondrocytes (Hu et al. 2020; Singh et al. 2020). Data in this study showed that Mn deficiency induces the autophagy activation of TGP in chicks. This may be ascribed to the decreased angiogenesis in metaphyseal regions, which results in hypoxia environment in TGP and subsequent HIF-1α up-regulation. We speculate that autophagy activation may provide essential energy for chondrocytes and metaphyseal vessels to ensure normal secretion of MIF and VEGF and normal angiogenesis in metaphysis, further indicating that HIF-1α up-regulation protects against Mn deficiency-induced abnormal metaphysis angiogenesis in broilers. However, the role of autophagy in abnormal TGP development and anomalous metaphyseal angiogenesis induced by Mn deficiency has not been fully elucidated, which needs to be further investigated.
In summary, our findings demonstrate that HIF-1α up-regulation plays a protective role in Mn deficiency-induced TGP developmental disorder by antagonizing the angiogenesis inhibition and inducing autophagy activation, which provides us a new insight to the pathogenesis of Mn deficiency-induced TD.