Legg-Calve-Perthes disease is a juvenile form of ischemic osteonecrosis of the femoral head that can produce femoral head deformity and premature osteoarthritis. It remains one of the most challenging conditions to treat due to our lack of understanding of the biology of the disease. Femoral head osteonecrosis occurs due to blood supply disruption which produces hypoxic injury to the femoral head. Our histological studies showed increased vessel formation in cartilage in the ischemic group compared to the control group in a pig model of femoral head osteonecrosis. Here we have presented evidence to indicate that there are coordinated expressions of HIF-1α and VEGF in the cartilage in animal model of femoral head ischemia and that upregulation of VEGF during hypoxia in chondrocyte is mediated partially through HIF-1α.
First, we identified coordinated upregulation of HIF-1α and VEGF under hypoxia in RNA expression level in the ischemic femoral heads. This is supported by the cartilage and primary chondrocyte analysis. Quantitative RT-PCR confirmed Microarray results to show that the RNA expression of VEGF was upregulated along with HIF-1α in pig ischemic cartilage (). Coordinated upregulation of HIF-1α and VEGF under hypoxia was also observed in primary chondrocytes isolated from femoral heads (). Our immunohistochemistry assay indicated that both HIF-1α and VEGF were upregulated in chondrocytes in the ischemic femoral heads also at the protein level in and . To investigate the mechanisms responsible for upregulation of VEGF by hypoxia, we tested the effects of HIF-1α activator, DFO, on VEGF expression in chondrocytes under hypoxia. Results from this study indicate that DFO enhanced VEGF expression under hypoxia (), suggesting HIF-1α is involved in mediating VEGF expression in chondrocytes under hypoxia.
Conditional HIF-1α knockout mice provide strong evidence for the involvement of HIF-1α in cartilage development. In these animals, HIF-1α deletion in chondrocytes in the interior of the developing growth plate induced premature programmed cell death. In addition, chondrocytes lacking HIF-1α exposed to hypoxia showed decreased expression of chondrocyte marker gene Col2a10
. This in vitro study provides partial molecular mechanisms for transcriptional activity of HIF-1α during hypoxia. VEGF is a well-characterized angiogenic factor that is activated by hypoxia. HIF-1α is a master regulator of cellular response to hypoxia. The loss of HIF-1α makes bone narrow and less vascularized. Nevertheless, VEGF was still expressed in HIF-1α null mice indicating that besides HIF-1α, other factors are also involved in VEGF regulation during embryonic development16
. Our observation in current study is consistent with this notion from the HIF-1α null mice data in that expression of VEGF was reduced significantly but not abolished after HIF-1α siRNA transfection as shown in . VEGF expression was reduced by 44% after HIF-1α knockdown by siRNA transfection, suggesting that 1) HIF-1α is involved in VEGF upregulation during hypoxia and that 2) other factors besides HIF-1α may also be responsible for VEGF regulation during hypoxia. HIF-1α upregulation of VEGF activity may be one of the mechanisms for angiogenesis response following femoral head ischemia.
It is still an open question what other factors control VEGF expression besides HIF-1α. Runx2 is an essential regulator of both endochondral and intramembranous ossification17
. Runx2 has been shown to control VEGF expression in chondrocytes during endochondral bone formation18
. There is no VEGF expression in the hypertrophic chondrocytes of Runx2 knockout mice. Overexpression of Runx2 in fibroblasts induces an increase in VEGF mRNA and protein levels by upregulating VEGF transcription18
. It is possible that Runx2 is also involved in VEGF regulation in the absence of HIF-1α. Osterix (Osx) is an osteoblast-specific transcription factor required for bone formation, and is considered a master regulator essential for the commitment of preosteoblast differentiation into mature osteoblasts19,20
. Osx directly targets VEGF expression, involving direct binding of Osx to sequence specific promoter elements to activate the VEGF expression in osteoblasts21
. It has been demonstrated that Osx regulation of VEGF is independent of HIF-1α expression level22
. Interestingly, Osx cooperates with HIF-1α to positively regulate VEGF expression22
. VEGF regulations by HIF-1α–independent mechanisms deserve further investigation.
In summary, we have demonstrated that upregulation of VEGF in chondrocytes following femoral head ischemia is mediated partially through HIF-1α. Ischemic osteonecrosis of the femoral head is a significant clinical problem that can lead to femoral head collapse and early degenerative arthritis in young patients23
. Our understanding of the pathogenesis and repair process is still much limited. Results in this study provide the possible mechanisms to explain the more vessel formation after ischemia induction in pig femoral head via VEGF regulation partially through HIF-1α. Further studies are needed to identify additional factors which may be responsible for more vessel formation after ischemia induction in this model.