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

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Abstract

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 (BubR1L/−) 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 BubR1L/− 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 BubR1L/− 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 BubR1L/− 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 BubR1L/− mice (P < .05). The vascular endothelial growth factor protein increase in ischemic hind limb muscles was lower in BubR1L/− 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). HIF1α protein levels in the muscles after hind limb ischemia surgery were also significantly lower in BubR1L/− 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.

Clinical Relevance:

Aging impairs angiogenesis, mediates endothelial dysfunction, and leads to increased prevalence of both cardiovascular disease and adverse sequelae in the elderly. These pathologies lead to higher mortality and an elevated risk of limb amputation. BubR1, a cell cycle-related protein, is a crucial factor in aging. This study presents novel findings indicating that BubR1 insufficiency impairs angiogenesis and results in limb amputation in the critical limb ischemic mouse model. We also showed that the decrease in BubR1 expression was associated with a reduction in vascular endothelial growth factor expression and hypoxia inducible factor-1α instability. Therefore, BubR1 insufficiency delayed angiogenesis, which eventually resulted in limb loss.

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