Rapid neutrophil mobilization by VCAM-1+ endothelial cell-derived extracellular vesicles.

AIMS: Acute myocardial infarction rapidly increases blood neutrophils (<2 h). Release from bone marrow, in response to chemokine elevation, has been considered their source, but chemokine levels peak up to 24 h after injury, and after neutrophil elevation. This suggests that additional non-chemokine-dependent processes may be involved. Endothelial cell (EC) activation promotes the rapid (<30 min) release of extracellular vesicles (EVs), which have emerged as an important means of cell-cell signalling and are thus a potential mechanism for communicating with remote tissues. METHODS AND RESULTS: Here, we show that injury to the myocardium rapidly mobilizes neutrophils from the spleen to peripheral blood and induces their transcriptional activation prior to arrival at the injured tissue. Time course analysis of plasma-EV composition revealed a rapid and selective increase in EVs bearing VCAM-1. These EVs, which were also enriched for miRNA-126, accumulated preferentially in the spleen where they induced local inflammatory gene and chemokine protein expression, and mobilized splenic-neutrophils to peripheral blood. Using CRISPR/Cas9 genome editing, we generated VCAM-1-deficient EC-EVs and showed that its deletion removed the ability of EC-EVs to provoke the mobilization of neutrophils. Furthermore, inhibition of miRNA-126 in vivo reduced myocardial infarction size in a mouse model. CONCLUSIONS: Our findings show a novel EV-dependent mechanism for the rapid mobilization of neutrophils to peripheral blood from a splenic reserve and establish a proof of concept for functional manipulation of EV-communications through genetic alteration of parent cells.

A Refined Protocol for Coronary Artery Ligation in the Neonatal Mouse.

The neonatal mouse heart can regenerate following myocardial infarction (MI), a capacity that is lost after 7 days, providing a model system to study tissue regeneration and the transition to adult wound healing. MI can be induced in neonatal mice surgically by coronary artery ligation. In this protocol, neonates are anesthetized using a combination of inhaled isoflurane anesthesia and induced hypothermia, a significant ethical refinement over previous protocols. A lateral thoracotomy is performed, and an 8-0 suture is used to ligate an area below the left atrium to induce MI. The ribs and skin are closed using 7-0 sutures, and the pup is rapidly rewarmed with a supply of oxygen. Once recovered, the pup is cleaned and rolled in a fecal bath to mask the smell of surgical interference before being returned to the mother. The surgical procedure is expected to take 10 to 15 min per pup, with a further 5 min for recovery. A dedicated assistant for recovery of pups is recommended to streamline the procedure. © 2021 Wiley Periodicals LLC.

Mouse models of myocardial infarction: comparing permanent ligation and ischaemia-reperfusion.

Myocardial infarction (MI) is a disease of major consequence in the modern world, causing permanent, irreversible damage to the heart. Survivors are at risk for developing further cardiovascular pathologies such as heart failure. Further study of MI injury is crucial to improve the understanding and treatment of the post-MI heart. The most commonly used model for MI in vivo is surgical ligation of the left anterior descending coronary artery (LAD). There are two predominant approaches: permanent ligation (PL), where the LAD is permanently occluded with a suture, or ischaemia-reperfusion (IR), where the LAD is temporarily occluded before removing the suture to restore blood flow and tissue reperfusion. PL results in the majority of the area at risk becoming infarcted, leading to significant apoptotic cell death and a large scar. Conversely, IR salvages some of the area at risk; thus, the scar is smaller and includes reperfusion injury, an additional, albeit smaller, second wave of necrotic damage. PL may be a more appropriate model choice for studies of heart tissue injury and wound healing, owing to the larger, more consistent infarcts, while IR enables the study of reperfusion injury. Both are clinically relevant, and the choice of model depends upon the precise pre-clinical research questions to be addressed.