EMMPRIN-Targeted Magnetic Nanoparticles for In Vivo Visualization and Regression of Acute Myocardial Infarction

Abstract

Inhibition of extracellular matrix (ECM) degradation may represent a mechanism for cardiac protection against ischemia. Extracellular matrix metalloproteinase inducer (EMMPRIN) is highly expressed in response to acute myocardial infarction (AMI), and induces activation of several matrix metalloproteinases (MMPs), including gelatinases MMP-2 and MMP-9. We targeted EMMPRIN with paramagnetic/fluorescent micellar nanoparticles conjugated with the EMMPRIN binding peptide AP-9 (NAP9), or an AP-9 scrambled peptide as a negative control (NAPSC). We found that NAP9 binds to endogenous EMMPRIN in cultured HL1 myocytes and in mouse hearts subjected to ischemia/reperfusion (IR). Injection of NAP9 at the time of or one day after IR, was enough to reduce progression of myocardial cell death when compared to CONTROL and NAPSC injected mice (infarct size in NAP9 injected mice: 32%±6.59 vs

Control: 46%±9.04 or NAPSC injected mice: 48%±7.64). In the same way, cardiac parameters were recovered to almost healthy levels (LVEF NAP9 63% ± 7.24 vs CONTROL 42% ± 4.74 or NAPSC 39% ± 6.44), whereas ECM degradation was also reduced as shown by inhibition of MMP-2 and MMP-9 activation. Cardiac magnetic resonance (CMR) scans have shown a signal enhancement in the left ventricle of NAP9 injected mice with respect to non-injected, and to mice injected with NAPSC. A positive correlation between CMR enhancement and Evans-Blue/TTC staining of infarct size was calculated (R:0.65). Taken together, these results point to EMMPRIN targeted nanoparticles as a new approach to the mitigation of ischemic/reperfusion injury.

Keywords: AP9.; EMMPRIN; Ischemia/Reperfusion; Matrix metalloproteinases; nanoparticles.

Figure 1

Structure and chemical properties of NAP9 and NAPSC. A. Aminoacid composition of AP9 and AP9 scrambled peptides. B. Composition of NAP9 and NAPSC micelles containing AP9 and AP9 scrambled peptides respectively. C. Structure of micelle components and conjugation of Cys-peptides to maleimide moiety. D. Physical and chemical properties of NAP9, NAPSC, and empty micelles.

Figure 2

NAP9 binds to EMMPRIN in HL1 myocyte cells. A. Percentage of healthy, necrotic, and apoptotic HL1 myocytes incubated with NAP9 at the dosages shown. B. Confocal microscopy visualization of NAP9, NAPSC, and EMMPRIN in HL1 myocytes. Cells were incubated 1 hour with 10nM NAP9 or NAPSC. Nanoprobe uptake was evaluated by detection of Rhodamine (NAP9, and NAPSC, red). Endogenous EMMPRIN was detected in green (anti-EMMPRIN primary antibodies and FITC-conjugated secondary antibodies (green)), Co-localization was detected by merging both fluorescing signals (Merged panels, yellow). Scale bars, 50 μm (n= 3 by triplicate).

Figure 3

In vivo toxicity of NAP9. A. Mice were injected with NAP9 at the dosages shown. After 10 days, plasma levels of hepatic alanine transaminase (ALT), aspartate transaminase (AST), creatinine (Crea) and creatinine kinase (CK-MB) were measured. (n=9 mice). B. Tissue distribution of 50 mg/Kg NAP9 in the same mice as before, as detected by confocal microscopy. Right. Confocal microscopy visualization of endogenous EMMPRIN (green) and NAP9 (red) in crossed sections of pulmonary tissue. Merged plane shows co-localization of both signals in yellow.

Figure 4

NAP9 binds to EMMPRIN in mouse hearts subjected to IR. A. Study design. B. Confocal microscopy visualization of NAP9, or NAPSC (rhodamine, red), and endogenous EMMPRIN (FITC, green) in healthy hearts (upper panels) and in hearts subjected to IR (lower panels). Scale bars: 50 μm (n=9 mice/condition). C. Confocal microscopy visualization of NAP9 (rhodamine, red) and the macrophage marker CD68 (FlTC, green) or the muscle cell myosin heavy chain (MHC, FITC, green) in hearts subjected to IR (n=9 mice).

Figure 5

NAP9 induces cardiac protection in mice subjected to IR. A. LVEF values from non ischemic (healthy) mice and from mice who underwent IR for 24 hours and were injected with 50 mg/Kg NAP9, NAPSC, or saline (Control) right after ischemia (n=9 mice/group. Mean ± SD; *p <0.05, NAP9 vs Control). B. Measurement of LV necrotic area as percentage with respect to the area at risk, detected by double Evans Blue/TTC staining (n = 18 mice/group; mean ± SD; *p <0.05, NAP9 vs Control). C. Immunoblot detection of MMP9 (upper panel), and MMP-2 (lower panel) in non ischemic (Healthy) mice, and in mice injected with 50 mg/Kg NAP9, NAPSC, or saline (Control) right after reperfusion. GAPDH was used as loading control (n=9 mice/group; mean ± SD; *p <0.05, NAP9 vs Control and NAPSC). D. Detection of MMP-9 by Gelatin zymography in the same hearts as in C. The two bands of 92 kDa and 87kDa correspond with the sizes of proMMP-9 and active-MMP-9 respectively. LMW: low molecular weight. HMG: high molecular weight (n=9 mice/group; mean ± SD; *p <0.05, NAP9 vs Control and NAPSC).

Figure 6

Non invasive CMR visualization of NAP9 in mice subjected to IR, correlates with ex vivo Evans Blue/TTC measures taken in the same mice. A. CMR study design. B. Parasternal short axis view of hearts from healthy mice (upper), or mice who underwent IR for 24 hours (IR, lower), and were injected with 50 mg/Kg NAP9, NAPSC, or saline (Control). Bottom. Detailed magnification of NAP9-injected healthy mice, or mice under IR. Green lines show Gadolinium enhanced areas corresponding to nanoprobe uptake. Blue and red dotted lines are marking external and internal LV walls respectively. Right graph shows the result of 3 independent assays (n=9 mice/condition; mean ± SD; *p <0.05, NAP9 vs Control and NAPSC in AMI). C. Scattered plot representing the extension of NAP9 uptake as detected by CMR (Y axis) with respect to the extension of infarct size, estimated by Evans Blue/TTC staining (X-axis) (R=0.69; p<0.05).

Figure 7

NAP9 has no effect before IR. A. Study design. B. LVEF of healthy hearts or hearts from mice subjected to IR and injected one day before (Group A: T-1d) at the same time (Group B: T0d) or one day after (Group C: T+1d) with 50 mg/Kg NAP9, NAPSC, or saline (Control), and measured 24 hours after injection (see schematic representation of the assay) (n=10 mice/condition; mean ± SD; *p <0.05, NAP9 t0 vs NAP9 t-1d; p <0.05, NAP9 t+1d vs NAP9 t-1d). B Immunoblot detection of MMP-9 in the same hearts as before. GADP was used as loading control (n=9 mice/group *p <0.05, NAP9 vs Control).

Figure 8

NAP9 is detected in mouse hearts within the first 30 minutes of injection. A. Study design and timeline of imaging assessments. B. Parasternal shot axis view of mouse hearts at the times indicated after injection of 50mg/Kg NAP9 or NAPSC. White arrows point to gadolinium areas of enhancement. Bottom left: Normalized enhancement ratios. (n=9 mice/condition. mean ± SD; *p <0.05, NAP9 vs No lipid and NAPSC). Bottom right, magnification of CMR images recorded 120 minutes after injection. The area of signal enhancement is represented in red.