Reduced Bone Mineral Density in Factor VIII Deficient Mice and the Role of Inflammatory Cytokines

Low bone mineral density (BMD) and increased risk of fracture are increasingly being identified in patients with hemophilia. Multiple clinical studies have shown that both children and adults with hemophilia have significantly decreased BMD, with up to 70% of adult patients affected. Decreased BMD in this population has previously been attributed to inactivity due to hemophilic arthropathy and comorbid conditions including infection with HIV and hepatitis C.

The factor VIII (FVIII) knockout (KO) mouse model has been extensively characterized with respect to bleeding and the development of inhibitors. However, the skeletal status of these animals has not been described. Therefore, the purpose of this study is to examine the skeletal phenotype of FVIII deficient mice and measure serum markers of bone turnover and regulation.

We compared the skeletal phenotype of 11 male FVIII KO mice and 8 matched wild-type (WT) controls at 20 weeks of age. BMD was measured using dual energy x-ray absorptiometry (DXA), bone geometry was examined by μCT and, after sacrifice, femoral breaking strength was measured by three-point bending until failure (ultimate force).

In a second group of 24 mice (12 KO and 12 WT), serum was obtained under uniform conditions at 20 weeks of age via cardiac puncture. Serum levels of receptor activator of nuclear factor kappa-B ligand (RANK-L), osteoprotegerin (OPG), interleukin 1α (IL-1α) and osteocalcin were measured using commercially available ELISAs. Alkaline phosphatase (alk phos) activity was measured using a colorimetric assay. The RANK-L/OPG system regulates bone turnover, osteocalcin and alk phos are markers of bone turnover and IL-1α is an inflammatory cytokine.

No differences in body weight, length, percent fat, or femoral length were observed between FVIII KO and WT mice (data not shown). No spontaneous bleeding was observed in any animal. As shown in Table I, KO mice had significantly decreased BMD, cortical thickness and stiffness compared to WT mice. This resulted in decreased resistance to fracture as measured via ultimate force. The data presented here demonstrate that adult FVIII KO male mice exhibit lower cortical bone mass resulting in femora less resistant to fracture compared with WT controls. Because FVIII KO mice do not have spontaneous joint hemorrhages and exhibit normal behavior and activity levels, these data suggest that there is an underlying connection between the coagulation system and bone metabolism that results in lower BMD and bone strength.

To understand why KO mice have decreased BMD, we measured serum levels of regulators of bone metabolism, markers of bone turnover and inflammatory cytokines. There was no difference in RANK-L, OPG or the RANK-L/OPG ratio between the KO and WT mice. In addition, there was no observed difference in osteocalcin and alk phos. However, WT animals had significantly higher levels of IL-1α expression (P = 0.008). All of the KO animals had undetectable IL-1α levels under physiologic conditions whereas the mean IL-1α level in WT mice was 32.2 pg/ml. Thrombin production is reduced in hemophilia and thrombin has previously been shown to stimulate inflammatory cytokine production. Therefore, these data suggest that thrombin may be necessary for IL-1α production under non-stress conditions and that lower levels of IL-1α may be detrimental to bone health. Although increased inflammatory cytokines are typically associated with decreased BMD, prior research has shown that physiologic levels of cytokines are necessary to maintain bone health.

These data provide compelling evidence that the link between FVIII deficiency and impaired skeletal health is real and independent of differences in physical activity and other medical co-morbidities. In addition, the decreased levels of IL-1α observed in KO mice suggest that inflammatory cytokines are involved in this pathophysiology. Ongoing work in our laboratory is aimed at further exploring these connections.

No relevant conflicts of interest to declare.