Abstract
Vascular endothelial cell (EC)-derived factors play an important role in endothelial–cardiomyocyte crosstalk and could save cardiomyocytes (CMs) from injury. The manipulation of endothelial cells to secrete protective factors could enhance cardioprotection. Secretory leukocyte protease inhibitor (SLPI) has been known to protect the heart. The goal of this study was to evaluate the in vitro paracrine protective effect and mechanisms of EC-derived human SLPI on cardiomyocytes subjected to hypoxia/reoxygenation (H/R) injury. Stable endothelial cells overexpressing human SLPI were generated from an endothelial cell line (EA.hy926). The cytoprotective effect was determined by cell survival assay. The results showed that endothelial-derived recombinant human SLPI (rhSLPI) reduced simulated ischemia/reperfusion (I/R)-(81.75% ± 1.42% vs. 60.27% ± 2.52%, p < 0.05) and hypoxia/reoxygenation (H/R)-induced EC injury (83.57% ± 1.78% vs. 63.07% ± 1.93%, p < 0.05). Moreover, co-culture of ECs overexpressing rhSLPI with CMs at ratios 1:1 and 1:3 or treatment with conditioned medium enhanced cell viability by 10.51–16.7% (co-culture) and 15.25–20.45% (conditioned medium) by reducing intracellular reactive oxygen species (ROS) production, the Bax/Bcl-2 expression ratio, caspase-3, and caspase-8, and in preconditioned CMs by activation of p38 MAPK and Akt survival kinase. In conclusion, this study showed for the first time that EC-derived rhSLPI provided cardio-vasculoprotective effects against I/R injury as a possible alternative therapeutic strategy for cardioprotection.
Highlights
Myocardial ischemia/reperfusion (I/R) is a pathological process of cardiomyocytes involving inflammatory responses [1]
The results showed that the G3 clone secreted the highest level of recombinant human SLPI (rhSLPI) compared to the others, and this was used in further experiments
The results showed that the population doubling time (PDT) of these cell lines were not significantly different (Figure 2C), while the cell morphologies of EA-WT cells and EA-secretory leukocyte protease inhibitor (SLPI) cells were similar in terms of size and shape
Summary
Myocardial ischemia/reperfusion (I/R) is a pathological process of cardiomyocytes involving inflammatory responses [1]. Reperfusion injury is caused by oxidative damage which stimulates infiltration of leukocytes to the injured area. I/R injury affects and harms surrounding cells, especially endothelial cells (ECs). This causes endothelial dysfunction [2,3] which aggravates CM injury and results in expansion of the damage and possible death [4]. Infiltrated leukocytes produce and secrete various protease enzymes which aggravate resident cell injury [2,8], especially in ECs and CMs. any strategy to lower oxidative stress and provide anti-protease has the potential to reduce the expansion of cardiac cell injury, reduce the severity of the outcome, and save patients’ lives
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