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Nanoparticle Delivery of miRNA-21 mimic to Cardiac Macrophages Improves Myocardial Remodeling after Myocardial Infarction Tzlil Bejerano, Sharon Etzion, Sigal Elyagon, Yoram Etzion, and Smadar Cohen Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.8b02578 • Publication Date (Web): 24 Aug 2018 Downloaded from http://pubs.acs.org on August 25, 2018
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Nano Letters
Nanoparticle Delivery of miRNA-21 mimic to Cardiac Macrophages Improves Myocardial Remodeling after Myocardial Infarction Tzlil Bejerano1, Sharon Etzion2, Sigal Elyagon2,3, Yoram Etzion2,3, Smadar Cohen1,2,4 *
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Avram and Stella Goldstein-Goren Department of Biotechnology Engineering, Ben-Gurion University of the Negev, Beer-Sheva 8410501, Israel
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Regenerative Medicine and Stem Cell (RMSC) Research Center, Ben-Gurion University of the Negev, Beer-Sheva 8410501, Israel
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Department of Physiology and Cell Biology, Ben-Gurion University of the Negev, Beer-Sheva 8410501, Israel 4
The Ilse Katz Institute for Nanoscale Science and Technology, Ben-Gurion University of the Negev, Beer-Sheva 8410501, Israel
KEYWORDS miRNA-21, laser capture microdissection, macrophages, myocardial infarction, nanoparticles
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Abstract
MicroRNA-based therapy that targets cardiac macrophages holds great potential for treatment of myocardial infarction (MI). Here, we explored whether boosting the miRNA-21 transcript level in macrophage-enriched areas of the infarcted heart could switch their phenotype from proinflammatory to reparative, thus promoting resolution of inflammation and improving cardiac healing. We employed laser capture microdissection (LCM) to spatially monitor the response to this treatment in the macrophage-enriched zones. MiRNA-21 mimic was delivered to cardiac macrophages post MI by nanoparticles (NPs), spontaneously assembled due to the complexation of hyaluronan-sulfate with the nucleic acid mediated by calcium ion bridges, yielding slightly anionic NPs with a mean diameter of 130 nm. Following intravenous administration, the miRNA21 NPs were targeted to cardiac macrophages at the infarct zone, elicited their phenotype switch from pro-inflammatory to reparative, promoted angiogenesis, and reduced hypertrophy, fibrosis and cell apoptosis in the remote myocardium. Our work thus presents a new therapeutic strategy to manipulate macrophage phenotype using nanoparticle delivery of miRNA-21, with a potential for use to attenuate post-MI remodeling and heart failure.
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TEXT
Myocardial infarction (MI), commonly known as a 'heart attack' causes the formation of noncontractile scar tissue and left ventricular (LV) remodeling that further deteriorate cardiac function. MI is associated with excessive risk of heart failure and death1. In response to cardiomyocyte cell death and matrix degradation after MI, the innate immune system is robustly activated; myeloid cells, including neutrophils, monocytes and macrophages are accumulated within the injured myocardium2. These cells produce inflammatory and oxidative responses and contribute to scar expansion, LV remodeling and LV systolic dysfunction. Central to the cellular events after MI is the increase in macrophage number in the heart through the combined effects of massive recruitment of bone marrow–derived cells and local self-renewal of macrophages that predominately function to remove by phagocytosis the apoptotic/necrotic cardiomyocytes, and produce important mediators, such as tumor necrosis factor α (TNF-α), that establish crosstalk with other cardiac cell types3. During inflammation resolution within a few days, reparative macrophages replace and dominate the tissue, up-regulating signals that direct endothelial cells, fibroblasts, and local progenitor cells to rebuild damaged tissue4. The extent and coordination of these two macrophage responses is critical for the appropriate myocardial healing after MI; in cases where inflammation is persistent and/or resolution is delayed, the implications on infarct size and LV remodeling are devastating. Realizing this, we5 and others6,
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have suggested that
manipulation of innate immune responses may be sufficient to reduce cardiac tissue damage and improve outcomes. In the present study, we investigated whether targeting post-MI inflammation status could be achieved by nanoparticle delivered miRNA-21 mimic to cardiac macrophages post MI. Our strategy was prompted by recent findings showing miRNA-21 upregulation following the
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phagocytosis of apoptotic cells by peritoneal macrophages, consequently leading to inflammation resolution (reviewed by Sheedy et al.8). Thus, miRNA-21 mimic was delivered in nanoparticles (NPs), spontaneously assembled due to the complexation of hyaluronan-sulfate (HAS) with the nucleic acid mediated by calcium ion bridges9, 10. We locally monitored the therapeutic effect of the nanoparticle delivered miRNA-21 mimic to the macrophage-enriched area at the infarct border vs. its effect on the remote area, i.e., the LV posterior wall, by employing laser capture microdissection (LCM) system. The use of the LCM system enabled researching the cardiac macrophages in their natural microenvironment with no need for cell processing or in vitro culturing, which have been previously shown to greatly affect their activation11. At first, we aimed to quantify miRNA21 expression in the heart and specifically in cardiac macrophages following coronary ligation in mice. Previous reports on miRNA-21 expression in macrophages were mainly obtained in peritoneal and bone marrow derived cells; information on miRNA-21 expression in myocardial macrophages of infarcted hearts, however, is unavailable. Furthermore, various findings have reported conflicting effects of miRNA-21 on macrophage polarization, as reviewed by Essandoh et al12. Some studies reported that miRNA-21 is upregulated in macrophages after the engulfment of apoptotic cells3 augmenting inflammation resolution13, while others reported upregulation of pri-miR-21 to control the early stages of inflammation14. To address this knowledge gap, we used LCM to collect the macrophage-enriched tissue samples from the infarct border as well as from samples from the LV posterior wall (the remote area). Initially, the macrophage-enriched area was identified on cryo-sections from the infarcted hearts (Figure 1Ai) by laser scanning confocal microscope (LSCM) after immunostaining the cryosections for CD11b, a surface marker of macrophages (Figure 1Aii, red) and DAPI for nuclei
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(Figure 1Aiii, blue). Adjacent sections collected by LCM were further quantitatively analyzed by qRT-PCR for macrophage-related genes in the infarct border compared to the remote myocardium and healthy LV myocardial tissue, 3 and 7 days post-MI (Figure 1B).
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Figure 1 Isolation of macrophage-enriched area at the infarct border area, using LCM. MI was induced in mice, and heart samples were collected 3 and 7 days post MI. (A) Frozen sections
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of the cardiac tissue were stained with cresyl violet to histologically define the infarct (i) and with CD11b (red) and nuclei (blue) (ii) to define a macrophage enriched area (the infarct border area). The infarct border area and a remote area were cut and captured using LCM (iii), and RNA was extracted from these areas. (B) Relative levels of inflammatory gene expression in the MI border area and a remote area. Both areas were normalized to LV samples from healthy hearts. qPCR readout of inflammatory genes TNF-α (i) and iNOS (ii), and anti-inflammatory genes Arg1 (iii) and HO-1 (iv). (C) Relative miRNA-21 expression levels at the MI border area (i) and a remote area (ii). Both areas were normalized to healthy hearts. Data represent mean±SEM (n=3) *p
