N-6-methyladenosine (m6A) is the most prevalent post-transcriptional RNA modification in eukaryotic cells. The modification is reversible and can be dynamically regulated by writer and eraser... Show moreN-6-methyladenosine (m6A) is the most prevalent post-transcriptional RNA modification in eukaryotic cells. The modification is reversible and can be dynamically regulated by writer and eraser enzymes. Alteration in the levels of these enzymes can lead to changes in mRNA stability, alternative splicing or microRNA processing, depending on the m6A-binding proteins. Dynamic regulation of mRNA m6A methylation after ischemia and hypoxia influences mRNA stability, alternative splicing and translation, contributing to heart failure. In this study, we studied vasoactive microRNA m6A methylation in fibroblasts and examined the effect of hypoxia on microRNAs methylation using m6A immunoprecipitation. Of the 19 microRNAs investigated, at least 16 contained m6A in both primary human fibroblasts and a human fibroblast cell line, suggesting vasoactive microRNAs are commonly m6A methylated in fibroblasts. More importantly, we found that mature microRNA m6A levels increased upon subjecting cells to hypoxia. By silencing different m6A writer and eraser enzymes followed by m6A immunoprecipitation, we identified METTL4, an snRNA m6A methyltransferase, to be predominantly responsible for the increase in m6A modification. Moreover, by using m6A-methylated microRNA mimics, we found that microRNA m6A directly affects downstream target mRNA repression efficacy. Our findings highlight the regulatory potential of the emerging field of microRNA modifications. Show less
Cardiovascular diseases (CVDs) remain the leading cause of death worldwide, and thus, novel therapies are required. CVDs generally result in local shortages in the blood supply, known as ischemia.... Show moreCardiovascular diseases (CVDs) remain the leading cause of death worldwide, and thus, novel therapies are required. CVDs generally result in local shortages in the blood supply, known as ischemia. Neovascularization is the body's innate response mechanism that stimulates the restoration of blood flow to ischemic tissues. During the last decade, microRNAs have emerged as critical regulators of both CVD and neovascularization. Recent studies demonstrated that microRNAs are altered in many ways; however, whether these microRNA modifications could be physiologically relevant remained unclear. We examined whether specific microRNAs with a known cardiovascular function are subject to particular microRNA-alterations and if they could be relevant in cardiovascular disease. Our experiments demonstrated that the level of specific microRNA alterations, including isomiR formation, adenosine-to-inosine editing, and N6-adenosine methylation, changed in response to cardiovascular pathology. Many of these alterations changed the microRNAs function, which had a direct effect on processes like neovascularization. For example, microRNA adenosine-to-inosine editing increased after ischemia in both mice and humans and promoted neovascularization. These findings suggest that microRNA modifications can potentially be harnessed as a biomarker for cardiovascular disease, or even a novel therapeutic target. Show less
Kwast, R.V.C.T. van der; Quax, P.H.A.; Nossent, A.Y. 2020
Therapeutic neovascularization can facilitate blood flow recovery in patients with ischemic cardiovascular disease, the leading cause of death worldwide. Neovascularization encompasses both... Show moreTherapeutic neovascularization can facilitate blood flow recovery in patients with ischemic cardiovascular disease, the leading cause of death worldwide. Neovascularization encompasses both angiogenesis, the sprouting of new capillaries from existing vessels, and arteriogenesis, the maturation of preexisting collateral arterioles into fully functional arteries. Both angiogenesis and arteriogenesis are highly multifactorial processes that require a multifactorial regulator to be stimulated simultaneously. MicroRNAs can regulate both angiogenesis and arteriogenesis due to their ability to modulate expression of many genes simultaneously. Recent studies have revealed that many microRNAs have variants with altered terminal sequences, known as isomiRs. Additionally, endogenous microRNAs have been identified that carry biochemically modified nucleotides, revealing a dynamic microRNA epitranscriptome. Both types of microRNA alterations were shown to be dynamically regulated in response to ischemia and are able to influence neovascularization by affecting the microRNA's biogenesis, or even its silencing activity. Therefore, these novel regulatory layers influence microRNA functioning and could provide new opportunities to stimulate neovascularization. In this review we will highlight the formation and function of isomiRs and various forms of microRNA modifications, and discuss recent findings that demonstrate that both isomiRs and microRNA modifications directly affect neovascularization and vascular remodeling. Show less
