BMAL1 regulates cell cycle progression and angiogenesis of endothelial cells

This editorial refers to ‘The circadian protein BMAL1 supports endothelial cell cycle during angiogenesis’ by M. Astone et al. https://doi.org/10.1093/cvr/cvad057. The core circadian protein brain and muscle aryl hydrocarbon receptor nuclear translocator-Like 1 (BMAL1) is a key regulator of cell and tissue homeostasis. Through its dimerization with the Clock (circadian locomotor output cycles protein kaput) protein BMAL1 binds to canonical E-box elements within its target gene promoters to active transcription.1,2 The role of BMAL1 has been widely established in tumorigenesis, apoptosis, DNA repair, cell proliferation, and angiogenesis.3–5 In the context of angiogenesis, BMAL1 was shown to activate vascular endothelial growth factor (VEGF) promoter activity through a mechanism dependent upon the critical E-box elements within the VEGF promoter. Conversely, suppressing BMAL1 activity abrogated Notch-dependent vascular sprouting.6 While these studies highlighted a tentative connection between the molecular clock protein BMAL1 and VEGF—mediated angiogenesis, this mechanism was largely worked out in a zebra fish model—raising the question, whether, similar BMAL1-VEGF regulated signalling pathways are universal and operationally conserved in higher organisms such as mammals. In this issue of Cardiovascular Research, Astone et al. address this question and uncover for the first time a novel role for BMAL1 in cell cycle re-entry and proliferation of endothelial cells (EC) in mammals.7 Herein, Astone et al. demonstrated that ECs exhibit circadian rhythmicity evidenced by daily, high-amplitude oscillations of several circadian core genes in blood vessels.7 Based on this observation, the authors examined whether the robust molecular clock activity observed in ECs was related to the regulation of key angiogenic properties of EC, such as cell survival, proliferation, and migration.7 Using a variety of methodologies, Astone et al. systematically tested the impact of single and combined core clock genes depletion on circadian regulation of EC and identified BMAL1 as the central component of the circadian clock in EC. This was substantiated by studies in human umbilical vein endothelial cells in which BMAL1 knock-down resulted in G0/G1 cell cycle exit, increased apoptosis, diminished wound healing response, impaired sprouting capacity, and cell migration. Taken together, these findings supported a functional link between the circadian core protein BMAL1 and EC-regulated angiogenesis.7 This finding was further tested by examining the involvement of BMAL1 in angiogenesis in EC-specific BMAL1 knockout mice (Bmal1iΔEC) in vivo. By immunohistochemical staining, Astone et al. demonstrated that retinal angiogenesis is severely impaired in Bmal1iΔEC mice compared to wild-type control, as evidenced by a decrease in radial vasculature growth, EC capacity, and number of branch points and vessel length—supporting a crucial role for BMAL1 in the regulation of EC-regulated angiogenesis in vivo. To address the underlying mechanisms of the angiogenic properties of BMAL1 in EC, the authors evaluated a previously published BMAL1 ChIP-seq database using gene ontology- bioinformatic analysis identifying the cell-cycle proteins Ccna1 and Cdk1 as two down-stream transcriptional target genes of BMAL1. In vivo analysis confirmed that BMAL1 transcriptionally regulates these genes as demonstrated by downregulated expression of both Ccna and Ckd1 in Bmal1iΔEC lung EC. Finally, the authors examined the clinical relevance of their findings by evaluating whether BMAL1 plays a role in tumour angiogenesis. Indeed, silencing BMAL1 resulted in decreased tumour growth coincident with reduced angiogenic properties in Bmal1iΔEC mice, supporting the notion that BMAL1 plays a key role in EC-driven tumour angiogenesis.7 Collectively, the work highlights a putative connection between the circadian clock via BMAL1 and the cell-cycle, suggesting that circadian-regulated G0/G1 progression plays an important role in EC-mediated angiogenesis. Furthermore, the study also reveals an important role for endothelial restricted expression of BMAL1 in tumour angiogenesis, thereby identifying BMAL1 as a novel therapeutic target for curtailing angiogenesis as an anti-cancer therapy (Figure 1). Scheme depicts BMAL1 regulation of cell-cycle, angiogenesis, and tumorigenesis in EC. Inhibition of BMAL1 in EC impairs promoter activity and gene transcription of the cell cycle regulators, Ccna1, and Cdk1 which promotes G1 exit and cell cycle arrest. This suppresses endothelial-driven angiogenesis and tumour formation. While the data presented by Astone et al. provides evidence for a central role of BMAL1 in the regulation of EC-driven angiogenesis, it also raises several important questions and limitations that need to be considered. One of the most intriguing questions arising from this study is that BMAL1 is one of the main regulators of the circadian clock, and as such, it holds many important roles in regulating a wide variety of physiological processes. Therefore, although BMAL1 is suggested as a tentative therapeutic candidate for cancer or other vascular diseases, the non-specific effects of targeting BMAL1 need to be evaluated. This view is supported by studies showing that global knockout of BMAL1 resulted had an opposing carcinogenic effect resulting in an increase in tumour burden.8 In addition, the transcriptional properties of BMAL1 are known to be mutually dependent and obligatorily linked to the Clock gene as the two genes heterodimerize in the nucleus and bind to canonical E-box elements on target gene promoters. For this reason, it remains unclear whether the angiogenic properties of BMAL1 are functionally independent of Clock and/or whether Clock is needed in some capacity for the effects mediated by BMAL1 on cell cycle activation and EC-mediated angiogenesis. Another point of consideration is related to Ccna1 and Cdk1. Although these genes which are important for cell cycle were identified as regulatory targets of BMAL1 in EC, additional studies are needed to understand the specific molecular role of these proteins in relation to BMAL1 and EC-mediated angiogenesis. It also remains unclear whether the observed effects of BMAL1 on angiogenesis play an equivalent role on EC function during pathological conditions such as atherosclerosis or other vasculopathies. Finally, angiogenesis and endothelial function is a multifaceted process that involves cytoskeleton EC reorganization, expression of cell surface adhesion proteins such as integrins and selectins, and secretion of various proteolytic enzymes that remodel the extracellular matrix.9,10 It is, therefore, important to further investigate whether the circadian machinery and especially BMAL1 are involved in these critical steps of angiogenesis and whether the effects of BMAL1 described herein are universally conserved or a restricted feature of ECs. Nevertheless, the study by Astone et al. provides a new insight regarding the involvement of the circadian clock in the interrelationship between cell cycle and EC—mediated angiogenesis by BMAL1. This study suggests that chronotherapy may be an important consideration when targeting angiogenic pathways as anti-tumorigenesis therapies.

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