Tongxinluo modulates cytokine secretion by cardiac microvascular endothelial cells in ischemia/reperfusion injury

Abstract

Cardiac microvascular endothelial cells (CMECs) extensively secrete cytokines during myocardial ischemia/reperfusion injury (MIRI). Tongxinluo (TXL) has been demonstrated to preserve the function of the endothelium and myocardium against MIRI. This study was designed to identify alterations in the paracrine function of CMECs under hypoxia/reoxygenation (H/R) conditions and assess its modulation by TXL. CMECs were exposed to different concentrations of TXL for 30 min and then subjected to hypoxia and reoxygenation for 12 and 2 h, respectively. Apoptosis was measured to determine the optimal TXL concentration. Protein antibody arrays were used to assess changes in cytokines secreted into conditioned medium by CMECs. A Gene Ontology (GO) analysis was applied to interpret the functional implications of changes in cytokines. TXL inhibited CMEC apoptosis in a concentration-dependent manner after H/R, reaching peak efficacy at a concentration of 800 μg/ml. H/R significantly altered 33 cytokines, and TXL (800 μg/ml) changed the levels of 121 different cytokines compared with the H/R group. Among these cytokines, 10 that were increased by H/R were decreased by TXL, five that were decreased by H/R were increased by TXL, and eight that were attenuated by H/R were further decreased by TXL. Insulin-like growth factor binding protein-1 was up-regulated by H/R and was further increased by TXL. Significantly altered factors were found to be involved in cell proliferation, growth and differentiation, as well as chemotaxis and transport. TXL inhibited the apoptosis of CMECs and modulated their paracrine function in MIRI.

Keywords: Cytokine; Tongxinluo; cardiac microvascular endothelial cells; myocardial ischemia/reperfusion injury; paracrine.

Figure 1

TXL inhibits H/R-induced apoptosis of CMECs in a concentration-dependent manner. A. Representative scatterplots of flow cytometry results showing apoptotic quadrants. B. Histogram of CMEC apoptosis rates at different TXL concentrations. The values represent means ± SEM. Each group was biologically repeated three times (#P < 0.05 versus the normal group; *P < 0.05 versus the H/R group).

Figure 2

Results of a cluster analysis based on samples and all significantly altered factors (fold-change > 1.5; P < 0.05). The color gradient represents the range of cytokine levels, as indicated on the right. Each group was biologically repeated three times.

Figure 3

Cytokines that were significantly differentially expressed in pairwise comparisons. (fold-change > 1.5; P < 0.05). A. Histogram of fold-changes of factors in the H/R group compared with the normal group. B. Histogram of fold-changes of factors in the TXL group compared with the H/R group.

Figure 4

Notable differentially expressed factors revealed by pairwise comparisons. A. Color gradient representation of 24 factors that were significantly changed in comparisons of the H/R group versus the control group and of the TXL group versus the H/R group. B. Boxplots of ANGPTL-4, G-CSF, VEGF, and NRG-1β expression in different groups.