Abstract
Ischemia/reperfusion (I/R)-induced inflammation is associated with enhanced leukocyte rolling, adhesion and transmigration within the microcirculation. These steps are mediated by hypoxia-triggered signaling pathways, which upregulate adhesion molecule expression on endothelial cells and pericytes. We analyzed whether these cellular events are affected by maslinic acid (MA). Mitochondrial activity and viability of MA-exposed endothelial cells and pericytes were assessed by water-soluble tetrazolium (WST)-1 and lactate dehydrogenase (LDH) assays as well as Annexin V/propidium iodide (PI) stainings. Effects of MA on hypoxia and reoxygenation-induced expression of E-selectin, intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 were determined by flow cytometry. The subcellular localization of the NFκB subunit p65 was analyzed by immunofluorescence and Western blot. I/R-induced leukocytic inflammation was studied in MA- and vehicle-treated mouse dorsal skinfold chambers by intravital fluorescence microscopy and immunohistochemistry. MA did not affect viability, but suppressed the mitochondrial activity of endothelial cells. Furthermore, MA reduced adhesion molecule expression on endothelial cells and pericytes due to an inhibitory action on NFκB signaling. Numbers of adherent and transmigrated leukocytes were lower in post-ischemic tissue of MA-treated mice when compared to vehicle-treated controls. In addition, MA affected reactive oxygen species (ROS) formation, resulting in a diminished oxidative DNA damage. Hence, MA represents an attractive compound for the establishment of novel therapeutic approaches against I/R-induced inflammation.
Highlights
Ischemia/reperfusion (I/R) is associated with hypoxia-induced cytokine release, which promotes the binding of leukocytes to the microvascular endothelium and their transmigration across the endothelial lining into the surrounding tissue[1]
Thereafter, we performed a water-soluble tetrazolium (WST)-1 assay and found that concentrations of maslinic acid (MA) ranging from 20–40 μM reduce the mitochondrial activity of human dermal microvascular endothelial cells (HDMEC) when compared to vehicle-treated controls (Fig. 1b)
MA treatment did neither result in an increase of lactate dehydrogenase (LDH) release (Fig. 1c) nor in a higher number of Annexin V/propidium iodide (PI)-positive cells (Fig. 1d). This indicates that MA dose-dependently affects the mitochondrial activity of endothelial cells without inducing apoptotic or necrotic cell death
Summary
Ischemia/reperfusion (I/R) is associated with hypoxia-induced cytokine release, which promotes the binding of leukocytes to the microvascular endothelium and their transmigration across the endothelial lining into the surrounding tissue[1] This process is mediated by specific adhesion molecules on endothelial cells[2] and pericytes[3]. Several studies reported potent anti-tumor[15,16], anti-oxidant[17] and anti-inflammatory[18,19,20] effects of MA These are mediated by inhibitory actions of MA on the activity of protein kinase (PK)C and the NFκB pathway and stimulatory actions on heme oxygenase (HO)-1 and endothelial www.nature.com/scientificreports/. Nitric oxide synthase (eNOS), which suppresses the synthesis of important cytokines and reactive oxygen species (ROS)[13,21,22] These findings indicate that MA may be capable of alleviating I/R-induced inflammation by downregulation of NFκB-triggered endothelial and pericyte adhesion molecule expression. We analyzed in vivo leukocyte rolling, adhesion and transmigration in dorsal skinfold chambers of vehicle- and MA-treated mice, which were exposed to I/R
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