Heterogeneous In Vivo Role of Clotting Factors FVIII and FIX in Atherogenesis
Authors: Fabri Daniela , Baila Stefano , Costa Devanira , Annichino-Bizzacchi Joyce M. , Arruda Valder R. 
Published: April 2020

Abstract
Complications of atherosclerosis are the most common causes of death in Western societies. The knowledge that atherosclerosis is an inflammatory disease and coagulation affects the disease’s complications offers new opportunities for prevention and treatment. Population studies demonstrated that the risk for myocardial infarction is reduced by 80% among hemophilia A (HA) men compared to age and gender-matched controls. However, early atherosclerotic lesions were readily identified in small cohorts of adults HA and hemophilia B (HB) by ultrasonography of carotid and femoral arteries in a similar fashion to male controls. Here we sought to determine the role of FVIII and FIX on the development of atherosclerosis in two different mouse models. We compared a group of FVIII (HA) or FIX deficient mice (HB) lacking the low-density lipoprotein receptor (LDLR−/−) or apolipoprotein E (apoE−/−) with hemostatically normal littermate controls lacking either LDLR (LDLR−/−) or apoE (apoE−/−). All mice were on C57Bl/6 background and all groups were matched for gender and age. When mice were 8 weeks old, they were placed on atherogenic diet. Analyzes of whole aortic (Sudan IV staining) or aortic sinus (Oil Red O) revealed that HA/apoE−/− mice exhibit less atherosclerotic lesions in a time-dependent manner. At weeks 22 (n=9) and 37 (n=9), the rate of atherosclerosis was 2-3-fold lower in HA/apoE when compared to age and gender-matched apoE (−/−) control group (p=0.001, and p&lt;0.001, respectively), but not at week 8. In contrast, no differences were noted in the rates of vascular lesions between HA/LDLR(−/−) (n=9) and controls (n=9) at all time points. Overall, HB/LDLR (−/−) developed atherosclerosis lesions at aortic sinus with similar rates as the controls at 8 and 22 weeks. However, at week 37, these mice exhibit more extensive tissue damage than the control (p=0.02). Surprisingly, the analyses of whole aorta revealed that atherosclerotic area was more wide-spread in FIX deficient mice in both models, i.e. HB/apoE(−/−) and HB/LDLR(−/−), compared to matched controls. At week 22, 16% and 11% (p=0.04) of the aorta (HB/LDLR[−/−] vs LDLR[−/−]) was covered by atherosclerosis, whereas the rates were 37% vs 21% (p=0.001) at week 37. Moreover, in the apoE(−/−) model the lack of FIX enhances the vascular lesions (14% vs 7%, p=0.02 at week 22) and (36% vs 22%, p=0.04 at week 37). In another model, we tested whether male transgenic (Tg+) mice for high levels of human FIX (∼ 200% of activity above the normal) would influence the development of atherosclerotic lesions in the apoE(−/−) model. After 20 weeks, the rates of arterial vascular lesions were not different among Tg+FIX mice and controls after weeks 20 or 40 on atherogenic diet. In addition, Tg+FIX mice kept on regular chow diet up to 52 weeks showed no enhanced atherosclerosis but they presented high risk for venous thrombosis, as documented in 3/6 Tg+FIX mice and none of controls (0/6). These data demonstrate that coagulation activation in atherogenesis exhibits a rather heterogenous role in the disease evolution which suggest that clotting factors may have functions other than hemostasis in the development of vascular disease. Further studies in hemophilia B subjects are warranted to define the FIX effect on the onset and progression of occlusive vascular disease which may raises concerns on the onset cardiovascular risks on an aging hemophilia population.