Cardiomyocyte-derived small extracellular vesicles can signal eNOS activation in cardiac microvascular endothelial cells to protect against Ischemia/Reperfusion injury

Abstract

Rationale: The crosstalk between cardiac microvascular endothelial cells (CMECs) and cardiomyocytes (CMs) has emerged as a key component in the development of, and protection against, cardiac diseases. For example, activation of endothelial nitric oxide synthase (eNOS) in CMECs, by therapeutic strategies such as ischemic preconditioning, plays a critical role in the protection against myocardial ischemia/reperfusion (I/R) injury. However, much less is known about the signals produced by CMs that are able to regulate CMEC biology. Here we uncovered one such mechanism using Tongxinluo (TXL), a traditional Chinese medicine, that alleviates myocardial ischemia/reperfusion (I/R) injury by activating CMEC eNOS. The aim of our study is to identify the signals produced by CMs that can regulate CMEC biology during I/R. Methods: Ex vivo, in vivo, and in vitro settings of ischemia-reperfusion were used in our study, with the protective signaling pathways activated in CMECs identified using genetic inhibition (p70s6k1 siRNA, miR-145-5p mimics, etc.), chemical inhibitors (the eNOS inhibitor, L-NNA, and the small extracellular vesicles (sEVs) inhibitor, GW4869) and Western blot analyses. TritonX-100 at a dose of 0.125% was utilized to inactivate the eNOS activity in endothelium to investigate the role of CMEC-derived eNOS in TXL-induced cardioprotection. Results: We found that while CMEC-derived eNOS activity was required for the cardioprotection of TXL, activation of eNOS in CMECs by TXL did not occur directly. Instead, eNOS activation in CMECs required a crosstalk between CMs and CMECs through the uptake of CM-derived sEVs. We further demonstrate that TXL induced CM-sEVs contain increased levels of Long Intergenic Non-Protein Coding RNA, Regulator Of Reprogramming (Linc-ROR). Upon uptake into CMECs, linc-ROR downregulates its target miR-145-5p leading to activation of the eNOS pathway by facilitating the expression of p70s6k1 in these cells. The activation of CMEC-derived eNOS works to increase survival in both the CMECs and the CMs themselves. Conclusions: These data uncover a mechanism by which the crosstalk between CMs and CMECs leads to the increased survival of the heart after I/R injury and point to a new therapeutic target for the blunting of myocardial I/R injury.

Network Pharmacology-Based Prediction of Active Ingredients and Mechanisms of Tongxinluo Against Acute Myocardial Infarction

“…In our recent experiment where ex vivo, in vivo, and in vitro settings of I/R were used, we identi ed the signals produced by cardiomyocytes that can regulate cardiac microvascular endothelial cells biology during myocardial I/R injury. We found that cardiac microvascular endothelial cells-derived eNOS activity was required for the cardioprotection of TXL, [44] which again con rmed the mechanism of TXL in the treatment of AMI.…”

Stem Cell-Derived Exosomes Potential Therapeutic Roles in Cardiovascular Diseases

“…The studies conclude that MSC-derived exosomes harbor all necessary enzymes that are required for the generation of ATP ( 165 , 166 ). The report also suggest that exosome treatment leads to the reduction in systematic inflammation in mice model after treatment with myocardial I/R ( 167 ) ( Figure 3 ). Thus, MSC-derived exosomes could be a powerful therapeutic solution for patients diagnosed with acute myocardial infarction ( 167 ).…”

“…The report also suggest that exosome treatment leads to the reduction in systematic inflammation in mice model after treatment with myocardial I/R (167) (Figure 3). Thus, MSCderived exosomes could be a powerful therapeutic solution for patients diagnosed with acute myocardial infarction (167). The cardiac progenitor cells plays crucial role in angiogenic and cardiogenic properties in high-glucose conditions in both in vitro and animal models (168).…”

Integrin Regulation in Immunological and Cancerous Cells and Exosomes

“…Upon transfer to cardiac microvascular endothelial cells, the internalized exosomes augment glucose uptake, glycolytic activity and pyruvate synthesis in endothelial cells [ 132 ]. In addition, cardiomyocyte-derived exosomes activate eNOS, which is responsible for nitric oxide production in endothelial cells, conferring protection against ischemia/reperfusion injuries [ 133 ]. This metabolic cross-talk also occurs between endothelial cells and immune cells.…”