Bmal1 Downregulation Worsens Critical Limb Ischemia by Promoting Inflammation and Impairing Angiogenesis

Abstract

Critical limb ischemia (CLI) is the most advanced clinical stage of peripheral vascular disease with high mobility and mortality. CLI patients suffer from lower extremity rest pain, ulceration, and gangrene caused by insufficient blood and oxygen supply. Seeking for effective biomarkers and therapeutic targets is of great significance for improving the life quality of CLI patients. The circadian clock has been reported to be involved in the progression of kinds of cardiovascular diseases. Whether and how circadian genes play a role in CLI remains unknown. In this study, by collecting femoral artery and muscle specimens of CLI patients who underwent amputation, we confirmed that the circadian gene Bmal1 is downregulated in the CLI femoral artery and ischemic distal lower limb muscle. Furthermore, we verified that Bmal1 affects CLI by regulating lipid metabolism, inflammation, and angiogenesis. A hindlimb ischemia model performed in wild-type and Bmal1−/− mice confirmed that Bmal1 disruption would lead to impaired angiogenesis. In vitro experiments indicated that the decreased expression of Bmal1 would increase ox-LDL uptake and impair endothelial cell functions, including proliferation, migration, and tube formation. As for mechanisms, Bmal1 represses inflammation by inhibiting lipid uptake and by activating IL-10 transcription and promotes angiogenesis by transcriptionally regulating VEGF expression. In conclusion, we provide evidence that the circadian gene Bmal1 plays an important role in CLI by inhibiting inflammation and promoting angiogenesis. Thus, Bmal1 may be an effective biomarker and a potential therapeutic target in CLI.

Importance of Bmal1 in Alzheimer's disease and associated aging‐related diseases: Mechanisms and interventions

“…Second, evidence showed that Bmal1 was associated with other ischemia‐related diseases such as myocardial infarction (Liu, Xiao, et al, 2021 ), stroke (Beker et al, 2018 ), and peripheral vascular disease (Xu et al, 2021 ), which are highly prevalent in aging populations (Hadjipanayi & Schilling, 2013 ); and some of these diseases have a close relationship with hypertension and atherosclerosis and may promote AD (Vijayan & Reddy, 2016 ; Zhang & Luo, 2020 ). There is an interesting link between vascular damage and Bmal1 deficiency when putting these diseases together, suggesting that a mechanism related to vascular pathology may be a common trigger.…”

The Circadian Clock is Essential for the Crosstalk of VEGF-Notch-mediated Endothelial Angiogenesis in Ischemic Stroke

“…As the core transcriptional factor, Bmal1 regulates the circadian pattern of downstream gene expression [49]. Besides this, Bmal1 has been shown to promote angiogenesis in ischemia-injured HUVECs (human umbilical vein endothelial cells) [50]. However, there is little evidence about the transcription function and epigenetic regulation of Bmal1.…”

New insight into ischemic stroke: Circadian rhythm in post-stroke angiogenesis

“…As known downstream targets of the circadian clock network, VEGF protein displays the oscillation expression pattern, which is regulated by the core circadian component Bmal1, Clock, Per, and Cry genes ( Koyanagi et al, 2003 ). The overexpression of Bmal1 promotes the luciferase activity of VEGF; however, knocking down the VEGF expression reversed the promoting effects of Bmal1 in pro-angiogenesis in HUVECs Xu et al, (2021 ). The human VEGF promoter activity follows a 24-h circadian pattern under the 12 h light–dark (LD) cycle, and the core circadian gene Bmal1 plays a transcriptional role by targeting the region of the VEGF promoter ( Jensen et al, 2012 ).…”

“…In HUVECs, overexpression of Bmal1 promotes angiogenic activity, including proliferation, migration, and tube formation ability. Furthermore, in vitro research, Bmal1 −/− mice impaired angiogenesis by labeling CD31 and measuring the decreased VEGF protein level after peripheral ischemic injury ( Xu et al, 2021 ). Similarly, endothelial isolated from Per2 −/− mice substantially lost the vascular networks and proliferation ability, further contributing to increasing senescence ( Wang et al, 2008 ).…”

“…In a clinical study of 55 stroke patients, the result showed that core clock gene Bmal1 methylation induced by PM2.5 exposure was negatively associated with the National Institutes of Health Stroke Scale (NIHSS) score ( Cantone et al, 2020 ). More than this, Bmal1 contributes to revascularization after ischemic injury, Bmal1 −/− mice displayed an impaired angiogenesis ability after ischemic injury, anti-CD31-positive capillary density and VEGF protein levels arereduced in Bmal1 −/− mice ( Xu et al, 2021 ). Even so, the exact mechanism of the circadian in angiogenesis after ischemic stroke remains unclear.…”