Rapid Lipid Modification of Endothelial Cell Membranes in Cardiac Ischemia/Reperfusion Injury: a Novel Therapeutic Strategy to Reduce Infarct Size

Abstract

Purpose: Plasma membranes constitute a gathering point for lipids and signaling proteins. Lipids are known to regulate the location and activity of signaling proteins under physiological and pathophysiological conditions. Membrane lipid therapies (MLTs) that gradually modify lipid content of plasma membranes have been developed to treat chronic disease; however, no MLTs have been developed to treat acute conditions such as reperfusion injury following myocardial infarction (MI) and percutaneous coronary intervention (PCI). A fusogenic nanoliposome (FNL) that rapidly incorporates exogenous unsaturated lipids into endothelial cell (EC) membranes was developed to attenuate reperfusion-induced protein signaling. We hypothesized that administration of intracoronary (IC) FNL-MLT interferes with EC membrane protein signaling, leading to reduced microvascular dysfunction and infarct size (IS).

Methods: Using a myocardial ischemia/reperfusion swine model, the efficacy of FNL-MLT in reducing IS following a 60-min coronary artery occlusion was tested. Animals were randomized to receive IC Ringer's lactate solution with or without 10 mg/mL/min of FNLs for 10 min prior to reperfusion (n = 6 per group).

Results: The IC FNL-MLT reduced IS (25.45 ± 16.4% vs. 49.7 ± 14.1%, P < 0.02) and enhanced regional myocardial blood flow (RMBF) in the ischemic zone at 15 min of reperfusion (2.13 ± 1.48 mL/min/g vs. 0.70 ± 0.43 mL/min/g, P < 0.001). The total cumulative plasma levels of the cardiac injury biomarker cardiac troponin I (cTnI) were trending downward but were not significant (999.3 ± 38.7 ng/mL vs. 1456.5 ± 64.8 ng/mL, P = 0.1867). However, plasma levels of heart-specific fatty acid binding protein (hFABP), another injury biomarker, were reduced at 2 h of reperfusion (70.3 ± 38.0 ng/mL vs. 137.3 ± 58.2 ng/mL, P = 0.0115). CONCLUSION: The IC FNL-MLT reduced IS compared to vehicle in this swine model. The FNL-MLT maybe a promising adjuvant to PCI in the treatment of acute MI.

Keywords: Endothelium; Ischemia/reperfusion; Lipid rafts; Liposomes; Membrane lipid therapy; Myocardial infarction; Percutaneous coronary intervention.

Fig. 1

Comparison of fusogenic characteristics of FNLs (PC/PA/PE-DHA/S1P) and nanoliposomes partially formulated with FNL lipid components (PC only or PC/PA/PE). MCAECs were incubated with FNLs or nanoliposomes labeled with DiR (a lipophilic infrared dye). The incorporation of DiR into cell membranes was quantified every 2.5 min using a microplate reader. (a) Fusion kinetic curves generated by FNLs and nanoliposomes; DiR incorporation was measured in terms of relative fluorescence units (RFU)/concentration of number of FNL particles (#particles). (b) Comparison of fusion rates calculated from the slope of best fit line through the data points for FNLs and nanoliposomes. Data are shown as mean ± SD; ***P < 0.001

Fig. 2

Representative images from Epi, FRET, and TIRF microscopy studies using live MAECs transfected to express GFP-flotillin-1 and incubated with rhodamine-labeled fusogenic nanoliposomes (PC/PA/Rho-DOGS). Epi shows a merged image of light microscopy, rhodamine, and GFP signals. FRET shows merged images of Rho-DOGS lipid and GFP-flotillin-1 interacting with each other (arrows). TIRF shows merged images demonstrating that Rho-DOGS and GFP-flotillin-1 are in close proximity to each other (yellowish dots) at the level of the cell membrane (pointers)

Fig. 3

Effect of FNL-MLT on the activation of MCAECs and RAW cells. (a) MCAEC ICAM-1 expression was quantified after pretreatment with FNLs (2.5, 5.0, and 10.0 mg/mL) and incubation with activated plasma (PCAP) for 2 h, followed by a 1-h incubation with growth medium. (b) RAW cell TNF production was quantified after pretreatment with FNLs (2.5 mg/mL) for 10, 20, and 30 min and incubation with LPS for 18 h. Values are shown as mean ± SD; *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 4

Hemodynamic and temperature data of vehicle (control) and FNL-treated swine throughout the study. There was no difference between groups for any of the variables. Data are shown as mean ± SD

Fig. 5

Infarct size analyses from swine hearts treated with IC vehicle (control) or IC FNLs following a 60-min LAD occlusion and 72 h of reperfusion. (a) Representative images of LV slices stained to distinguish the NIZ (phthalo blue dye) and the IZ (TTC red dye). The left panel shows a slice with transmural infarction with hemorrhage (white/beige color) from a vehicle-treated heart, and the right panel shows a slice with patchy infarction from an FNL-treated heart. (b) Illustrates that the AAR expressed as a percent of the LV was similar for both groups. (c) Demonstrates that the IS expressed as a percent of the AAR was statistically significant between groups. Data are shown as mean ± SD

Fig. 6

Change in plasma levels of the cardiac injury biomarkers cTnI and hFABP following a 60-min LAD occlusion. Swine hearts were treated with IC vehicle (control) or IC FNLs 10 min prior to myocardial reperfusion. (a) Serial cTnI plasma levels (line graph) and calculated cumulative levels of cTnI released (inset bar graph) are presented. (b) Levels of hFABP at baseline, 2, and 4 h of reperfusion are shown. Data are shown as mean ± SD

Fig. 7

RMBF data from swine hearts treated with IC vehicle (control) or IC FNLs before and after myocardial infarction. The mean RMBF was measured in the LV’s IZ and NIZ using four different neutron-activated microspheres administered at baseline, 45 min of occlusion (Occ), 15 min of reperfusion (Rep), and 72 h of reperfusion. At 15 min of reperfusion, RMBF increased by ~ threefold in the IZ of FNL-treated hearts suggesting a decrease in microvascular dysfunction. Data are shown as mean ± SD