Lethal Weapon Against Angiotensin II–Induced Cardiac Injury
See related article, p 776–785
Chronic systemic angiotensin II (Ang II) administration causes cardiac fibrosis, inflammation, hypertrophy, and hypertension, which leads to cardiac dysfunction and failure. Although our knowledge of the molecular mechanisms that lead to cardiovascular disease states has significantly expanded during recent decades, there are still missing pieces to fully explain, and even more importantly to therapeutically modulate, cardiac disease. MicroRNAs are endogenous small noncoding RNAs that control gene expression by promoting degradation or translational repression of target mRNAs. In recent years, a continuously growing body of evidence has accumulated associating microRNAs with multiple health and disease states. The cardiovascular system is no exception, and microRNAs have been implicated in cardiac hypertrophy, fibrosis, inflammation, angiogenesis, and dysfunction.1
In this issue of Hypertension, Wang et al2 performed a parallel time-series whole-genome cardiac gene and microRNA expression analysis in mice treated with Ang II to identify microRNAs that mediate Ang II–induced cardiac injury and dysfunction. As expected for any screening, multiple candidates were identified, and prioritizing them is always a big dilemma. In this study, the authors applied an experimental and educated guess combination approach. They used gene and microRNA profiles to identify putative microRNA–mRNA targets with opposite regulation and gene ontology analysis to identify target mRNAs involved in cardiac pathophysiology. Let-7i, a member of the let-7 family, was identified as the cardiac microRNA targeting the highest number of mRNA targets known to play critical roles in cardiac injury and dysfunction.
The lethal-7 (let-7) gene was initially discovered as an essential developmental gene in C. elegans.3 During evolution, the let-7 gene has been conserved and amplified to generate the let-7 family. In humans, the let-7 family consists of 10 mature let-7 sequences that are produced from 13 precursor sequences distributed across 9 chromosomes. Multiple let-7 family members have been implicated in development, stem cell self-renewal and differentiation, and cancer. More recently, multiple let-7 family members have been associated with cardiovascular pathologies either as drivers, mediators, or markers of cardiac hypertrophy, fibrosis, endothelial-to-mesenchymal transition, myocardial infarction, heart failure, dilated cardiomyopathy, and angiogenesis.4 However, our knowledge of the role of let-7i in cardiac health and disease is limited, highlighting the importance of the study by Wang et al.2
Wang et al2 report that the time-dependent downregulation of let-7i in cardiac tissue by Ang II is paralleled by an upregulation of interleukin-6 (IL-6) and collagens (Col1a2, Col3a1, Col4a1, and Col5a2), markers of cardiac inflammation, and fibrosis, respectively. Besides let-7i, only another let-7 family member, let-7g, was regulated by Ang II in cardiac tissue in vivo. This is not surprising because the 13 let-7 family precursors originate from 9 independent transcriptional units. Multiple examples of differential expression regulation of let-7 family members have been reported in experimental models and human tissues with differential regulation between let-7 family members being more a rule than an exception.
Mature microRNAs contain a seed sequence that corresponds to nucleotides 2 to 8 of the ≈22-nucleotide long sequence, which is considered essential for binding to its target mRNA. Mature let-7 family members present 100% identity in their seed sequence, which is not surprising because the definition of the family is largely based on this seed sequence. However, the rest of the mature sequence is similar but not identical. This leads to the question of whether different let-7 family members target the same set of genes or if there is only partial overlap between the different let-7 family member targets. Most likely, the second option is the valid one. Genome-wide experimentally validated physical microRNA:mRNA interactions have shown that approximately half of them are noncanonical perfect seed matches.5 Moreover, seed interactions are generally accompanied by specific, nonseed base pairing.6 Furthermore, an alternative mode of microRNA target recognition involving G-bulge sites (at positions 5–6) is evolutionarily conserved and comprises ≈15% of all microRNA:mRNA interactions in mouse brain.7 Noncanonical microRNA target recognition would suggest that the evolutionary explosion of the let-7 family is not only because of differential regulation of its family members but also that each member targets a different and probably partially overlapping subset of target mRNAs.
In vitro studies by Wang et al2 show that let-7i is preferentially expressed in neonatal rat cardiac fibroblasts, and Ang II time dependently downregulates let-7i expression. Furthermore, they elegantly show that let-7i mimics attenuate and let-7i inhibitors exacerbate Ang II–induced upregulation of IL-6 and collagens, although let-7i mimics do not completely abolish Ang II–induced effects (Figure). Although the lack of complete reversal can be explained by a dose-dependent effect, it probably more closely reflects the fact that microRNAs:mRNAs form an interconnected network in which one microRNA can target multiple mRNAs, while also one mRNA can be targeted by multiple microRNAs. This combinatorial effect may explain why even modest regulation of particular target mRNAs by a specific microRNA can still have strong phenotypic effects as evidenced in in vivo experiments in the accompanying article. Furthermore, through a combination of in silico and mRNA fusion reporter assays, Wang et al2 show that let-7i has a direct effect on the mRNAs of IL-6 and collagens. Although direct experimental evidence through Argonaute cross-linking immunoprecipitation and its plethora of analytic modifications is needed to prove a physical interaction between let-7i and mRNAs of IL-6 and collagens mRNAs, these results strongly suggest that indeed these mRNAs are let-7i targets.
Finally, Wang et al2 present compelling evidence that let-7i mediates Ang II–induced cardiac inflammation and fibrosis in vivo. Pharmacological upregulation of let-7i not only partially reverted Ang II–induced mRNA upregulation of cardiac IL-6 and collagens but, more importantly, also significantly attenuated cardiac macrophage infiltration and fibrosis. Notably, let-7i pharmacological upregulation had no effect on Ang II–induced hypertension, cardiomyocyte apoptosis, and cardiac hypertrophy and dysfunction. Although this selective beneficial effect of let-7i upregulation exclusively in cardiac inflammation and fibrosis may suggest that let-7i is not the magic bullet to abolish all cardiovascular deleterious effects of excess Ang II, it does not diminish its potential clinical relevance as a cardiovascular therapy. However, further studies are needed in more clinically relevant models to elucidate whether let-7i supplementation would be a valid combination therapy to add to standard therapies (ie, renin–angiotensin–aldosterone system inhibitors, diuretics, β-blockers, etc.) for heart failure. Furthermore, this selective effect on cardiac inflammation and fibrosis strongly suggests that let-7i effects are blood pressure independent by directly and selectively targeting and modulating stressed cardiac fibroblasts. Although obviously not proposed as a therapeutic approach but still critically relevant to prove the role of let-7i in cardiac injury, the authors pharmacologically downregulated let-7i in vivo to show that indeed the reduction of let-7i exacerbates Ang II–mediated cardiac inflammation and fibrosis without significant effects on blood pressure or cardiac hypertrophy and function.2 These complementary experimental approaches make the case that let-7i is a mediator of Ang II–induced cardiac inflammation and fibrosis in vivo (Figure).
Lin28 family members, Lin28A and Lin28B, are small proteins implicated in RNA binding that selectively block let-7 biogenesis by inhibiting pri-/pre-microRNA by Drosha and Dicer, respectively.8 Because Lin28A and Lin28B mRNAs are let-7 targets themselves, this Lin28/let-7 axis establishes a double-negative feedback loop that indirectly regulates let-7 targets. It would be exciting to know whether the Lin28/let-7 autoregulatory loop holds true also for let-7i in Ang II–stimulated cardiac fibroblasts. This would be particularly important because it has been recently reported that let-7b is downregulated, whereas mRNAs of Lin28b and collagens are upregulated in glomeruli from diabetic mice.9 These studies would suggest that let-7 upregulation or Lin28 downregulation may be a valid therapy to mitigate target organ damage in multiple tissues.
The clinical relevance and potential therapeutic use of the findings of Wang et al2 are highlighted by the fact that microRNA mimics, and in particular let-7 mimics, are currently pursued by biopharmaceutical companies. MRX34 (Mirna Therapeutics, Austin, TX) is a chemically modified double-stranded RNA encapsulated in a liposomal formulation that mimics microRNA-34 and is targeted to patients with advanced primary liver cancer, other solid tumors with or without liver metastasis, and hematologic malignancies. It is in a multicenter open-labeled phase 1 clinical trial (ClinicalTrials.gov identifier: NCT01829971), which began in 2013, with encouraging preliminary safety results.10 MiR-Rxlet-7 (Mirna Therapeutics) is a let-7 microRNA mimic currently in preclinical stages that in combination with MRX34 has shown suppressed tumor growth leading to survival advantage in a mouse model of nonsmall cell lung cancer.11 The advanced stage of these microRNA mimic therapies holds promise that pharmacological upregulation of let-7i as a standalone or combinatorial therapy is not too far in the future.
In summary, although let-7i may not be the magic bullet to mitigate all of the Ang II–induced effects, it still has potential to be a lethal weapon against cardiac inflammation and fibrosis, with exciting possibilities of clinical use in the near future.
Sources of Funding
This work was supported by American Heart Association Scientist Development Grant 12SDG8980032 (D.G. Romero).
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
- © 2015 American Heart Association, Inc.
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