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THE ROLE OF CIRCULAR RNA CDR1AS IN MACROPHAGE MEDIATED CARDIAC INJURY AND REPAIR
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2024-08
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Biomedical Sciences
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http://dx.doi.org/10.34944/dspace/10598
Abstract
Introduction: Myocardial infarction is the most common form of acute cardiac injury attributed to heart failure. Despite advancements in prognosis and treatment, acute MI (AMI) still bears a considerable mortality rate within the initial year, with a significant portion of patients succumbing within the initial 30 days post-MI. The overall prognosis hinges on factors such as the extent of heart muscle damage, duration of the inflammatory response, and the efficacy of administered treatments in mitigating myocardial cell death and injury. This underscores the need for deeper mechanistic understanding and development of targeted therapies for cardiovascular diseases. In response to cardiac injury, macrophages are initially recruited to the infarcted myocardium and undergo phenotypic change from pro-inflammatory (M1) macrophages in the early stage to an anti-inflammatory (M2) macrophages phenotype in the later stage, orchestrating the initiation, maintenance, and eventual resolution of the inflammatory response. However, in chronic ischemia or severe infarction, continuous cardiomyocyte death prolongs pro-inflammatory macrophage activation resulting in robust secretion of pro-inflammatory cytokines perpetuating the inflammatory response and resulting in increased myocardial damage. Despite some understanding, further research is needed on the mechanisms and factors influencing macrophage function during injury. Circular RNAs are newly discovered non-coding RNA generated from protein-coding genes ubiquitously expressed in mammalian tissue, highly conserved among species, and recently implicated in the possible regulation of macrophage activation. However, their role in immunomodulation during cardiovascular injury remains unknown. Objective: This study focused on determining the specific role of circ-cdr1as in phenotypic switching between pro-, and anti-inflammatory macrophages and to determine whether functional regulation of circ-cdr1as modulates macrophage function post-cardiac injury. Methods and Results: We performed circular RNA microarray analyses to assess circular RNA transcriptome changes using RNA isolated from bone marrow derived macrophages (BMDM) polarized to a M1 phenotype by INFγ and TNFα or a M2 phenotype by IL-10, IL-4, and TGF-β. Following RNA isolation, samples are treated with RNaseR for enrichment of circular RNA and removal of linear RNA. We identified circRNAs differentially expressed in pro-and anti-inflammatory macrophages, including circRNA cdr1as (circ-cdr1as). RT-qPCR analysis revealed circ-cdr1as was one of the most downregulated in pro-inflammatory macrophages and significantly upregulated in anti-inflammatory macrophages in vitro. We established a circ-cdr1as overexpression system by generating a circ-cdr1as plasmid using pc3.1 plasmid with flanking regions that allows circularization of specified sequence for in vitro studies. For knockdown of circ-cdr1as, we used small hairpin RNA targeting the splicing junction found only in circular RNA. RT-qPCR and fluorescence activated cell sorting (FACS) analyses showed that overexpression of circ-Cdr1as increased transcription of anti-inflammatory markers and percentage of CD206+ (M2 macrophage marker) cells in naïve and pro-inflammatory macrophages in vitro. Meanwhile, knockdown decreased transcription of anti-inflammatory markers and increased the percentage of CD86+ (M1 macrophage marker) cells in naïve and anti-inflammatory macrophages in vitro. Disease enrichment analysis based on IPA system of the diseases associated with circular RNA involved in macrophage polarization indicated that cardiac fibrosis and cardiac enlargement as the top diseases. Therefore, we investigated the expression levels of circ-cdr1as in the heart after myocardial infarction (MI) injury in a mouse model. RT-qPCR analysis revealed downregulation of circ-cdr1as in the heart 3 days post MI, suggesting a possible physiological role for circ-cdr1as in MI pathophysiology. We isolated fibroblast, cardiomyocytes, CD31+ endothelial cells, and F4/80+ macrophages and investigated the transcriptional changes of circ-cdr1as to determine if it is cell-type specific. RT-qPCR analysis showed no significant difference in transcription of circ-cdr1as in fibroblast and endothelial cells. However, in cardiomyocytes and macrophages there was a significant downregulation of circ-cdr1as. To understand the role of circ-cdr1asmodulated macrophages in post-MI cardiac repair in vivo, we overexpressed circ-cdr1as in fluorescently labeled BMDMs and directly injected them into the ischemic myocardium immediately following MI surgery. FACS and immunohistochemistry analyses showed that these macrophages retained their anti-inflammatory phenotype during the initial stages of cardiac injury, and in the later stages improved cardiac left ventricular (LV) functions and reduced infarct size. Since circ-cdr1as was also decreased in cardiomyocytes post-MI, we additionally generated circ-cdr1as adeno associated virus 9 (circ-cdr1as-AAV-9) vectors to overexpress circ-cdr1as in mouse hearts. We performed tail vein injections of circ-cdr1as-AAV9 vectors 14 days prior to MI and conducted physiological and histological studies. Administration of circ-cdr1as-AAV9 significantly improved post-MI LV functions including ejection fraction (%EF) and fractional shortening (%FS) at 21-28D post MI, decreased infarct size, and improved angiogenesis. Interestingly, in the initial stages of cardiac injury, overexpression of circ-cdr1as reduced cardiomyocyte apoptosis and increased percentage of anti-inflammatory macrophages at injury site. Lastly, emerging evidence suggests that some circular RNAs function as microRNA (miR) sponges. Therefore, we investigated this mechanism to assess whether circular cdr1as invokes phenotypic changes in macrophages in both the steady-state and injured heart by acting as a sponge for miRNA to inhibit its function. Circ-cdr1as contains over 70 binding sites for miR-7, a microRNA known to exacerbate inflammation in lung related diseases through inhibition of KLF4. Pull-down assay indicated that circ-cdr1as directly interacts with miR-7. We found a reciprocal relationship between circ-cdr1as and miR-7 in macrophages and in the heart 3 days post-MI. Overexpression of miR-7 by miR-7-5p mimic increased the percentage of pro-inflammatory marker CD86 in naïve, pro-, and anti-inflammatory macrophages and upregulated transcription of pro-inflammatory markers. Meanwhile, inhibition of miR-7 had the opposite effect. We also found that expression of miR-7 target gene, KLF4, was reduced when macrophages were treated with miR-7 and increased when miR-7 was inhibited. Conclusions: In summary, this study suggests that circ-cdr1as plays a key role in regulating anti-inflammatory phenotype of macrophages through the modulation of miR-7 and its targets and exogenous delivery of circ-cdr1as may improve LV function over time. Therefore, circ-cdr1as may potentially be developed as an anti-inflammatory regulator in tissue inflammation after cardiac injury.
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