BubR1 insufficiency impairs angiogenesis in aging and in experimental critical limb ischemic mice

Objectives: Budding uninhibited by benzimidazole-related 1 (BubR1), a cell cycle-related protein, is an essential component of the spindle checkpoint that regulates cell division. Mice in which BubR1 expression is reduced to 10% of the normal level display the phenotypic features of progeria. However, the role of BubR1 in vascular diseases and angiogenesis remains unknown. To investigate the influence of BubR1 on angiogenesis, we generated a low-null-BubR1-expressing (BubR1(L/-)) mouse strain with reduced BubR1 expression as low as 15% of the normal level without any abnormalities in appearance. Methods: To elucidate the role of BubR1 in angiogenesis, we used a hind limb ischemia model induced in BubR1(L/-) mice and age-matched wild-type (WT) littermates. To evaluate the pathologic influence of BubR1 on angiogenesis, we measured the blood flow before and after hind limb ischemia surgery, and the expression of typical angiogenic factors in vivo and in vitro. Results: In WT mice, blood flow in the ischemic left limb gradually recovered to approximately 80%, 14 days after surgery. Conversely, in the BubR1(L/-) group, blood flow in the left ischemic limb recovered to at most 30% (14 days after surgery, P <.01; immediately after the operation, and 5 and 9 days after surgery, P <.05). In adductor and calf muscles from BubR1(L/-) mice, regenerated muscle bundles, granulation tissue, and inflammatory cell invasion were more evident than in calf muscles from WT mice at 14 days after surgery. All WT mice at 14 days after surgery had complete limb salvage, but loss of limbs was observed in approximately 70% of BubR1(L/-) mice (P <.05). The vascular endothelial growth factor protein increase in ischemic hind limb muscles was lower in BubR1(L/-) mice compared with WT mice (P <.05), and vascular endothelial growth factor levels in human aortic smooth muscle cells treated with BubR1 knockdown siRNA were lower compared with scramble siRNA under hypoxic conditions (P <.01). HIF1 alpha protein levels in the muscles after hind limb ischemia surgery were also significantly lower in BubR1(L/-) mice compared with WT mice (P <.05). Conclusions: BubR1 insufficiency impairs angiogenesis and results in limb loss in ischemic hind limbs. BubR1 may be a crucial angiogenic factor and might be beneficial for the treatment of limb ischemia.