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

Theranostics. 2020 Sep 23;10(25):11754-11774. doi: 10.7150/thno.43163. eCollection 2020.


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.

Keywords: Cardioprotection; cardiomyocytes; crosstalk; endothelial cells; tongxinluo.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aniline Compounds / pharmacology
  • Animals
  • Benzylidene Compounds / pharmacology
  • Cardiotonic Agents / pharmacology*
  • Cardiotonic Agents / therapeutic use
  • Cell Communication / drug effects
  • Cells, Cultured
  • Coronary Vessels / cytology
  • Disease Models, Animal
  • Drugs, Chinese Herbal / pharmacology*
  • Drugs, Chinese Herbal / therapeutic use
  • Endothelial Cells / metabolism
  • Endothelium, Vascular / cytology
  • Extracellular Vesicles / drug effects
  • Extracellular Vesicles / metabolism
  • Humans
  • Isolated Heart Preparation
  • Male
  • Microvessels / cytology
  • Myocardial Reperfusion Injury / pathology
  • Myocardial Reperfusion Injury / prevention & control*
  • Myocytes, Cardiac / drug effects*
  • Myocytes, Cardiac / metabolism
  • Nitric Oxide Synthase Type III / antagonists & inhibitors
  • Nitric Oxide Synthase Type III / metabolism*
  • Nitroarginine / pharmacology
  • Rats
  • Signal Transduction / drug effects


  • Aniline Compounds
  • Benzylidene Compounds
  • Cardiotonic Agents
  • Drugs, Chinese Herbal
  • GW 4869
  • tongxinluo
  • Nitroarginine
  • Nitric Oxide Synthase Type III
  • Nos3 protein, rat