Methotrexate carried in lipid core nanoparticles reduces myocardial infarction size and improves cardiac function in rats

Raul C Maranhão, Maria C Guido, Aline D de Lima, Elaine R Tavares, Alyne F Marques, Marcelo D Tavares de Melo, Jose C Nicolau, Vera Mc Salemi, Roberto Kalil-Filho, Raul C Maranhão, Maria C Guido, Aline D de Lima, Elaine R Tavares, Alyne F Marques, Marcelo D Tavares de Melo, Jose C Nicolau, Vera Mc Salemi, Roberto Kalil-Filho

Abstract

Purpose: Acute myocardial infarction (MI) is accompanied by myocardial inflammation, fibrosis, and ventricular remodeling that, when excessive or not properly regulated, may lead to heart failure. Previously, lipid core nanoparticles (LDE) used as carriers of the anti-inflammatory drug methotrexate (MTX) produced an 80-fold increase in the cell uptake of MTX. LDE-MTX treatment reduced vessel inflammation and atheromatous lesions induced in rabbits by cholesterol feeding. The aim of the study was to investigate the effects of LDE-MTX on rats with MI, compared with commercial MTX treatment.

Materials and methods: Thirty-eight Wistar rats underwent left coronary artery ligation and were treated with LDE-MTX, or with MTX (1 mg/kg intraperitoneally, once/week, starting 24 hours after surgery) or with LDE without drug (MI-controls). A sham-surgery group (n=12) was also included. Echocardiography was performed 24 hours and 6 weeks after surgery. The animals were euthanized and their hearts were analyzed for morphometry, protein expression, and confocal microscopy.

Results: LDE-MTX treatment achieved a 40% improvement in left ventricular (LV) systolic function and reduced cardiac dilation and LV mass, as shown by echocardiography. LDE-MTX reduced the infarction size, myocyte hypertrophy and necrosis, number of inflammatory cells, and myocardial fibrosis, as shown by morphometric analysis. LDE-MTX increased antioxidant enzymes; decreased apoptosis, macrophages, reactive oxygen species production; and tissue hypoxia in non-infarcted myocardium. LDE-MTX increased adenosine bioavailability in the LV by increasing adenosine receptors and modulating adenosine catabolic enzymes. LDE-MTX increased the expression of myocardial vascular endothelium growth factor (VEGF) associated with adenosine release; this correlated not only with an increase in angiogenesis, but also with other parameters improved by LDE-MTX, suggesting that VEGF increase played an important role in the beneficial effects of LDE-MTX. Overall effects of commercial MTX were minor, and did not improve LV function or infarction size. Both treatments did not induce any toxicity.

Conclusion: The remarkable improvement in heart function and reduction in infarction size achieved by LDE-MTX supports future clinical trials.

Keywords: VEGF; adenosine; drug delivery; lipid particle; myocardial infarction.

Conflict of interest statement

Disclosure The authors report no conflicts of interest in this work.

Figures

Figure 1
Figure 1
MTX morphological structure. Note: MTX was obtained by esterification of the α- and γ-carbonyl groups of the glutamic acid residue in the presence of cesium carbonate and dodecyl bromide. Abbreviation: MTX, methotrexate.
Figure 2
Figure 2
LV uptake of radioactively labeled LDE in MI (n=8) and sham (n=6) groups and LV systolic function, infarction size, cardiac hypertrophy and the presence of inflammatory cells in MI-LDE-MTX (n=13), MI-MTX (n=12), MI-control (n=13), and sham (n=12) groups. Notes: (A) The uptake of LDE labeled with 3H-cholesteryl oleate ether (%) by the myocardial tissues of the LV of sham group and non-infarcted area of MI groups 6 weeks after the induction surgeries. aP<0.05 vs sham. (B) Ejection fraction (%) obtained by transthoracic echocardiography 24 hours and 6 weeks after the sham and MI induction surgeries. aP<0.01 vs sham 24 hours; bP<0.001 vs sham 6 weeks; cP<0.01 vs MI-control 24 hours; dP<0.05 vs MI-control 6 weeks; eP<0.01 vs MI-MTX 24 hours; fP<0.05 vs MI-MTX 6 weeks; gP<0.01 vs MI-LDE-MTX 24 hours. (C) Infarction size measured 6 weeks after the infarction induction surgery: bars are % of infarcted area relative to LV total area. The transversal heart sections on top are representative Masson’s trichrome-stained tissues of MI scar and non-infarcted myocardium identified in blue and red, respectively. aP<0.001 vs MI-control; bP<0.05 vs MI-MTX. (D) Relative heart weight index (heart weight/total body weight) measured 6 weeks after the infarction induction or sham surgery. aP<0.001 vs sham; bP<0.05 vs MI-control. (E) Diameter of myocytes (mm) in the non-infarcted areas of LV measured 6 weeks after the infarction induction or sham surgery in HE-stained sections under 400× magnification. aP<0.001 vs sham; bP<0.01 vs MI-control; cP<0.001 vs MI-MTX. (F–G) Total number inflammatory cells in the LV section areas. Cells were counted in non-infarcted SEN (F) and INT (G) areas in HE-stained sections under 400× magnification. aP<0.001 vs sham; bP<0.01 vs MI-control; cP<0.001 vs MI-MTX; (H, I) Protein expression of CD68 (H) and CD3 (I) measured in the LV non-infarcted areas was performed 6 weeks after the infarction induction or sham surgery (in % difference from sham). aP<0.001 vs sham; bP<0.01 vs MI-control; cP<0.001 vs MI-MTX. Data are expressed in mean ± SEM in all plots. Abbreviations: INT, interstitium; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HE, hematoxylin and eosin; LDE, lipid core nanoparticles; LV, left ventricular; MI, myocardial infarction; MTX, methotrexate; SEN, subendocardium; VEGF, vascular endothelium growth factor.
Figure 3
Figure 3
Pro- and anti-inflammatory cytokines in LV non-infarcted areas in MI-LDE-MTX (n=5), MI-MTX (n=5), MI-control (n=5), and sham (n=5) groups. Notes: Protein expression of TNF-α (A), IL-1β (B), IL-6 (C), and IL-10 (D) measured in the LV non-infarcted areas was performed 6 weeks after the infarction induction or sham surgery (in % difference from sham). aP<0.001 vs sham; bP<0.01 vs MI-control; cP<0.01 vs MI-MTX. Data presented as mean ± standard error of mean. Abbreviations: GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IL, interleukin; LDE, lipid core nanoparticles; LV, left ventricular; MI, myocardial infarction; MTX, methotrexate; TNF, tumor necrosis factor.
Figure 4
Figure 4
Necrosis and apoptosis in LV non-infarcted areas in MI-LDE-MTX (n=13), MI-MTX (n=12), MI-control (n=13), and sham (n=12) groups. Notes: (A) Representative photomicrographs of myocardial necrosis in SEN and INT non-infarcted areas of HE-stained sections under 400× magnification. Arrows indicate nuclear pyknosis and karyolysis as well as cytoplasmic changes, including vacuolization, contraction bands, and hypereosinophilia. Bar in photomicrographs stands for 50 µm. (B–D) Protein expression of pro-apoptotic, caspase 3 (B) and BAX (C), and anti-apoptotic, Bcl-2 (D) measured in the LV non-infarcted areas was performed 6 weeks after the infarction induction or sham surgery (in % difference from sham). aP<0.001 vs sham; bP<0.05 vs MI-control; cP<0.01 vs MI-MTX. Data are expressed in mean ± SEM. Abbreviations: BAX, BCL2 associated X protein; INT, interstitium; LDE, lipid core nanoparticles; LV, left ventricular; MI, myocardial infarction; MTX, methotrexate; SEN, subendocardium.
Figure 5
Figure 5
Oxidative stress and antioxidant enzymes in LV non-infarcted areas in MI-LDE-MTX (n=5), MI-MTX (n=5), MI-control (n=5) and sham (n=5) groups. Notes: (A–C) Microfluorotopography of DHE oxidation products in MI (A), SEN (B), and INT (C) areas was performed 6 weeks after the infarction induction or sham surgery. (D) Representative photomicrograph of MI, SEN, and INT areas, showing microfluorotopography of DHE oxidation products. Red staining indicates fluorescence by DHE under 400× magnification. Bar in photomicrographs stands for 50 µm. (E, F) Protein expression of antioxidant enzymes, SOD1 (E) and catalase (F), measured in the LV non-infarcted areas was performed 6 weeks after the infarction induction or sham surgery (in % difference from sham). aP<0.05 vs sham; bP<0.05 vs MI-control; cP<0.05 vs MI-MTX. Data expressed in mean ± SEM in all plots. Abbreviations: DHE, dihydroethidium; INT, interstitium; LDE, lipid core nanoparticles; LV, left ventricular; MI, myocardial infarction; MTX, methotrexate; SEN, subendocardium; SOD1, superoxide dismutase 1.
Figure 5
Figure 5
Oxidative stress and antioxidant enzymes in LV non-infarcted areas in MI-LDE-MTX (n=5), MI-MTX (n=5), MI-control (n=5) and sham (n=5) groups. Notes: (A–C) Microfluorotopography of DHE oxidation products in MI (A), SEN (B), and INT (C) areas was performed 6 weeks after the infarction induction or sham surgery. (D) Representative photomicrograph of MI, SEN, and INT areas, showing microfluorotopography of DHE oxidation products. Red staining indicates fluorescence by DHE under 400× magnification. Bar in photomicrographs stands for 50 µm. (E, F) Protein expression of antioxidant enzymes, SOD1 (E) and catalase (F), measured in the LV non-infarcted areas was performed 6 weeks after the infarction induction or sham surgery (in % difference from sham). aP<0.05 vs sham; bP<0.05 vs MI-control; cP<0.05 vs MI-MTX. Data expressed in mean ± SEM in all plots. Abbreviations: DHE, dihydroethidium; INT, interstitium; LDE, lipid core nanoparticles; LV, left ventricular; MI, myocardial infarction; MTX, methotrexate; SEN, subendocardium; SOD1, superoxide dismutase 1.
Figure 6
Figure 6
Myocardial fibrosis and types of collagen in LV non-infarcted areas in MI-LDE-MTX (n=13), MI-MTX (n=12), MI-control (n=13) and sham (n=12) groups. Notes: (A–C) Collagen volume fraction in MI (A), SEN (B), and INT (C) areas was performed 6 weeks after the infarction induction or sham surgery. (D) Representative photomicrographs of myocardial fibrosis in MI, SEN, and INT areas of Masson’s trichrome-stain sections under 200× magnification. Bar in photomicrographs stands for 100 µm. (E, F) Protein expression of collagen I (E) and collagen III (F) measured in the LV non-infarcted areas was performed 6 weeks after the infarction induction or sham surgery (in % difference from sham). aP<0.01 vs sham; bP<0.01 vs MI-control; cP<0.01 vs MI-MTX. Data expressed in mean ± SEM. Abbreviations: GAPDH, glyceraldehyde 3-phosphate dehydrogenase; INT, interstitium; LDE, lipid core nanoparticles; LV, left ventricular; MI, myocardial infarction; MTX, methotrexate; SEN, subendocardium; SEM, standard error of mean.
Figure 6
Figure 6
Myocardial fibrosis and types of collagen in LV non-infarcted areas in MI-LDE-MTX (n=13), MI-MTX (n=12), MI-control (n=13) and sham (n=12) groups. Notes: (A–C) Collagen volume fraction in MI (A), SEN (B), and INT (C) areas was performed 6 weeks after the infarction induction or sham surgery. (D) Representative photomicrographs of myocardial fibrosis in MI, SEN, and INT areas of Masson’s trichrome-stain sections under 200× magnification. Bar in photomicrographs stands for 100 µm. (E, F) Protein expression of collagen I (E) and collagen III (F) measured in the LV non-infarcted areas was performed 6 weeks after the infarction induction or sham surgery (in % difference from sham). aP<0.01 vs sham; bP<0.01 vs MI-control; cP<0.01 vs MI-MTX. Data expressed in mean ± SEM. Abbreviations: GAPDH, glyceraldehyde 3-phosphate dehydrogenase; INT, interstitium; LDE, lipid core nanoparticles; LV, left ventricular; MI, myocardial infarction; MTX, methotrexate; SEN, subendocardium; SEM, standard error of mean.
Figure 7
Figure 7
Hypoxia, angiogenesis, adenosine receptors and adenosine catabolic enzymes in LV non-infarcted areas in MI-LDE-MTX (n=5), MI-MTX (n=5), MI-control (n=5), and sham (n=5) groups. Notes: (A–I) Protein expression of HIF-1α (A), HIF-2α (B), VEGF (C), A1 (D), A2a (E), A2b (F), and A3 (G) adenosine receptors, adenosine deaminase (H), and adenosine kinase (I) measured in the LV non-infarcted areas was performed 6 weeks after the infarction induction or sham surgery (in % difference from sham). aP<0.001 vs sham; bP<0.001 vs MI-control; cP<0.001 vs MI-MTX. Data expressed in mean ± SEM. Abbreviations: HIF, hipoxia-inducible factor; LDE, lipid core nanoparticles; LV, left ventricular; MI, myocardial infarction; MTX, methotrexate; SEM, standard error of mean; VEGF, vascular endothelium growth factor.
Figure 7
Figure 7
Hypoxia, angiogenesis, adenosine receptors and adenosine catabolic enzymes in LV non-infarcted areas in MI-LDE-MTX (n=5), MI-MTX (n=5), MI-control (n=5), and sham (n=5) groups. Notes: (A–I) Protein expression of HIF-1α (A), HIF-2α (B), VEGF (C), A1 (D), A2a (E), A2b (F), and A3 (G) adenosine receptors, adenosine deaminase (H), and adenosine kinase (I) measured in the LV non-infarcted areas was performed 6 weeks after the infarction induction or sham surgery (in % difference from sham). aP<0.001 vs sham; bP<0.001 vs MI-control; cP<0.001 vs MI-MTX. Data expressed in mean ± SEM. Abbreviations: HIF, hipoxia-inducible factor; LDE, lipid core nanoparticles; LV, left ventricular; MI, myocardial infarction; MTX, methotrexate; SEM, standard error of mean; VEGF, vascular endothelium growth factor.
Figure 8
Figure 8
Colocalization of VEGF and A2a and A2b adenosine receptors, CD68 and troponin I in LV non-infarcted areas in MI-LDE-MTX (n=5), MI-MTX (n=5), MI-control (n=5) and sham (n=5) groups. Notes: Representative photomicrograph of confocal microscopy of VEGF and A2a adenosine receptor (A), VEGF and A2b adenosine receptor (B), VEGF and CD68 (C), and VEGF and troponin I (D). Immunofluorescence staining of merged images is shown in the Figure. Nuclei are shown in blue, VEGF in red, and A2a and A2b adenosine receptors, CD68 and troponin I, in green. Colocalization is shown in yellow under 400× magnification. Bar in photomicrographs stands for 50 µm. Abbreviations: LDE, lipid core nanoparticles; LV, left ventricular; MI, myocardial infarction; MTX, methotrexate; VEGF, vascular endothelium growth factor.

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