The early events in the pathogenesis of ANFH are incompletely understood due to a typically late diagnosis after fracture and collapse of the femoral head. Besides bone marrow changes, evidence has shown that apoptosis is involved in the early stages of steroid-induced osteonecrosis [26
]. Weinstein et al.
reported that the number of apoptotic bone cells increased significantly in mice after steroid administration [28
]. Recent studies have shown apoptotic cells in clinical and animal models of GC-induced ANFH [26
In previous studies, we characterized an inbred rat (WKY) susceptible to develop steroid-induced osteonecrosis [31
]. It is possible that this strain of rats has genetically predisposing factors to develop ANFH and additional risk exposures (GC) will facilitate the development of the disease. In our animal model, prednisone administration enhanced the incidence of the disease in up to 75% (6/8) of the male WKY rats, suggesting it is a suitable model. In the literature, 5 to 15 week-old rats have been used to study non-traumatic ANFH [23
]. In the current study, WKY rats started to receive continuous steroid dosage released from the pellets at the age of five weeks for 25 weeks. Harvest at six months showed classical histological signs of early ANFH.
For the Affymetrix GeneChip findings, comparison of G2 versus G1 indicated that multiple pathological reactions occurred. According to the functional annotation tool (DAVID), modulated genes in the comparison of G2 and G1 (Table ) were grouped mainly into skeletal development, ossification and bone remodelling. Functional clusters of genes were significantly represented by steroid stimulus response, apoptosis, blood vessel morphogenesis, vasculature development, coagulation-related, cell growth, proliferation and differentiation associated genes.
The expression of steroid stimulus response genes (A2M, alkaline phosphatase, tissue-nonspecific, transforming growth factor beta 2
and potassium large conductance calcium-activated channel, subfamily m, alpha member 1
) were, as predicted, altered significantly. Previous in vivo
and in vitro
models as well as clinical studies showed that steroids induce apoptosis in osteoblasts and osteocytes [30
]. Amongst the 51 differentially regulated genes identified in our gene array analysis (Table ), five genes (S100 protein-beta polypeptide, transforming growth factor-beta 2, vitamin D receptor, unc-5 homolog c (C. elegans) and growth hormone receptor
) are in fact components of the apoptosis pathway.
The process of apoptosis can be directly induced by steroids but is also related to thrombosis in the blood vessels of the femoral head. In fact, the vascular hypothesis (regional endothelial bed dysfunction) appears to be relevant in the pathogenesis of ANFH. Damage or activation of femoral head endothelial cells results in abnormal blood coagulation and thrombi formation [36
]. Due to heterogeneity of the phenotype expression between endothelial cells in the body, a local endothelial cell dysfunction can occur where the femoral head endothelial cells react differently to the ANFH risk factors (GCs) than other endothelial cells in the body. In keeping with the theory of endothelial cell activation having a role in ANFH, coagulation-related gene expression in particular serine (or cysteine) peptidase inhibitor, clade E, member 1
also named plasminogen activator inhibitor 1 (PAI-1)
, a serine protease inhibitor that is synthesized and released by endothelial cells in the blood, was shown to be significantly over-expressed in this study. An increase in PAI-1
suppresses the generation of plasmin resulting in hypofibrinolysis and a relative hypercoagulable state [1
]. Decreased fibrinolytic activity, which may be a consequence of increased PAI-1
, has been described in patients with ANFH [37
], although a few studies have reported that there were no significant differences in the levels of thrombotic and fibrinolytic factors [18
Similarly, our findings demonstrate that several genes involved in the dynamic remodelling structure of the femoral head are also shown to be differentially expressed in ANFH (Table ). Clinically this may be relevant in that if the balance between degradation and repair (bone remodelling) becomes shifted to degradation and bone loss by the effect of GC, a failure of structural integrity at the subchondral region of bone with collapse could occur.
In the present study, results showed A2M
gene expression to be the most significantly upregulated gene when comparing G2 to G1. Correlation was obtained at the microarray, RT-PCR as well as the protein level as demonstrated by IHC study results. Most importantly, A2M
was not significantly upregulated when comparing G3 to G1. A2M
is a plasma-derived matrix metalloproteinase inhibitor which obstructs cartilage degradation induced by matrix metalloproteinases [38
]. The literature supports the role of corticosteroids in the modulation of A2M
]. In both reports, corticosteroids were shown to enhance A2M
is reported as being implicated in cartilage degradation [41
], and as an osteogenic growth peptide (OGP) - binding protein. Activated A2M
may thus participate in the removal of OGP from the system [42
]. Additional reports suggest inhibition of BMP-1 (bone morphogenic protein-1) by A2M
has been identified on the luminal surface of endothelial cells in sections of normal human arteries and veins [44
has also been implicated in hemostasis as a regulator of thrombin [45
] and in the development of thromboembolism in children [46
]. Together, all these findings suggest that A2M
shares haemostatic, cartilaginous and osteogenic properties and may have a potential role in the development of early steroid-induced ANFH. Determination of whether A2M
over-expression in our study is either the result or the cause of the apoptosis found in our rats developing early ANFH following administration of steroids, will require further study.
Two other genes of interest, Col2A1 and MIA, were also shown to be over-expressed significantly by microarray analysis and RT-PCR results but immunohistochemical study failed to show an increased cell surface expression of these genes.
Comparing the gene profiling of G3 versus G2, six genes stood out in our analyses (Table ). Although G3 animals have not developed ANFH, their gene profile reflects inhibition of osteoblast proliferation, differentiation and osteoclast activation. Perhaps most osteogenic cells in this group have not gone through the apoptotic phase and there are more viable cells expressing these molecules in comparison to G2. Differences could also be explained in that gene expression analysis findings are supportive of a result effect indicating steroid treatment and a disease effect affecting the apoptotic process are involved in the early stages of ANFH. Secondly, a genetic variation based on differences in transcription and translation could provide an explanation for the phenotypic differences found in our study. Thirdly, epigenetic variation, resulting from the interaction between the genotype and the environment, is also a potential process that could explain the findings that not all treated animals developed early ANFH when submitted to the same experimental conditions. Also, any of the genes listed in the comparison of G3 to G2 (Table ) with the exception of MIA, could have a protective effect against the development of steroid-induced early AVN. Similarly, the absence of A2M over-expression in that same group comparison G3 to G2, and in group comparison G3 to G1 is consistent with the phenotypic absence of early ANFH in rats representing G3.