Sera from HJD subjects induce expression of HIF-1α in synovial cells. (A) Synovial cells were incubated with HJD serum and serum from control groups A and B for 10, 30, and 60 minutes, after which mRNA was extracted and subjected to quantitative PCR. (A) Significantly increased mRNA expression for HIF-1α was observed at 60 minutes in cells incubated with HJD serum compared with control serum (CNTRLs; P < .0001). No significant difference was observed at the 10- or 30-minute time points. Results represent data from 3 independent experiments. (B) Immunofluorescence of hemophilic synovium expressing HIF-1α. Immunofluorescence using a monoclononal antibody against HIF-1α demonstrated abundant expression of HIF-1α in hemophilic synovium in the endothelial cells and lining cells (B) but not in control synovium (E). Similarly, immunohistochemical staining using DAB confirmed HIF-1α positivity in similar regions in hemophilic synovium (C, original magnification ×40) and expression of HIF-1α only in endothelial cells in normal synovium (D, original magnification ×40). Results were replicated in 8 different hemophilic samples and 6 control samples and represent data from 1 set of samples.