Cerium oxide nanoparticles attenuate monocrotaline induced right ventricular hypertrophy following pulmonary arterial hypertension

Abstract

Cerium oxide (CeO2) nanoparticles have been posited to exhibit potent anti-oxidant activity which may allow for the use of these materials in biomedical applications. Herein, we investigate whether CeO2 nanoparticle administration can diminish right ventricular (RV) hypertrophy following four weeks of monocrotaline (MCT)-induced pulmonary arterial hypertension (PAH). Male Sprague Dawley rats were randomly divided into three groups: control, MCT only (60 mg/kg), or MCT + CeO2 nanoparticle treatment (60 mg/kg; 0.1 mg/kg). Compared to the control group, the RV weight to body weight ratio was 45% and 22% higher in the MCT and MCT + CeO2 groups, respectively (p < 0.05). Doppler echocardiography demonstrated that CeO2 nanoparticle treatment attenuated monocrotaline-induced changes in pulmonary flow and RV wall thickness. Paralleling these changes in cardiac function, CeO2 nanoparticle treatment also diminished MCT-induced increases in right ventricular (RV) cardiomyocyte cross sectional area, β-myosin heavy chain, fibronectin expression, protein nitrosylation, protein carbonylation and cardiac superoxide levels. These changes with treatment were accompanied by a decrease in the ratio of Bax/Bcl2, diminished caspase-3 activation and reduction in serum inflammatory markers. Taken together, these data suggest that CeO2 nanoparticle administration may attenuate the hypertrophic response of the heart following PAH.

Keywords: Cerium oxide nanoparticles; Monocrotaline; Pulmonary arterial hypertension; Right ventricular hypertrophy.

Fig. 1. Characterization of CeO₂ nanoparticles

Nanoparticle size was determined by dynamic light scattering (DLS) (Panel A) and transmission electron microscopy (TEM) (Panel B). Corresponding scattered area diffraction is shown (Panel C). Energy dispersive X-ray spectroscopy shows the presence of cerium and oxygen in the nanoparticles (Panel D). X-ray photoelectron spectroscopy shows the relative amounts of Ce⁺³ and Ce⁺⁴ in the nanoparticles (Panel E). X-ray diffraction of the nanoparticles demonstrates the typical lattice structure (Panel F).

Fig. 2. CeO₂ nanoparticle treatment improves pulmonary flow and diminishes pulmonary artery remodeling

Pulsed-wave Doppler echocardiography demonstrating normal, round shaped flow profile at baseline (day 1) in the MCT rats (Panel A) and triangular flow profile with mid-systolic notching on day 28 (Panel B). Normal, round shaped flow profile at baseline (day 1) in the MCT + CeO₂ nanoparticle treated rats (Panel C) and the continuation of the normal, round shaped flow profile observed with CeO₂ nanoparticle treatment at day 28 of treatment (Panel D). Pulsed-wave Doppler echocardiography quantified data for pulmonary arterial pressure (Panel E), mean pulmonary arterial diameter (Panel F), mean pulmonary arterial area (Panel G), right ventricular outflow tract diameter (Panel H). Data are mean ± SEM (n = 6 rats/group). Grey columns indicate readings at day 1 while black columns denote readings at day 28. *Significantly different from base line values of MCT only group. † Significantly different from day 28 values of MCT only group (P < 0.05). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3. CeO₂ nanoparticle treatment attenuates MCT–induced increases in right ventricle wall thickness and cardiac remodeling

M-mode echocardiography images representing right ventricle wall thickness of the MCT only rats on day 1 (Panel A) and at day 28 (Panel B) or the MCT + CeO₂ nanoparticle treated group on day 1 (Panel C) and at day 28 (Panel D). Ventricle wall thickness (Panel E) and intra-ventricular septum diameter (Panel F) in the MCT only and MCT treated groups. Data are mean ± SEM, (n = 6 rats/group). Grey columns indicate readings at day 1 while black columns denote readings at day 28. *Significantly different from base line values of MCT only group. † Significantly different from day 28 values of MCT only group (P < 0.05). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4. CeO₂ nanoparticle administration attenuates monocrotaline–induced increases in cardiomyocyte cross sectional area and cardiac fibrosis

Dystrophin stained right ventricular sections from control (Panel A), MCT only (Panel B), and MCT + CeO₂ nanoparticle treatment group (Panel C). Quantification of cardiomyocyte cross sectional area (Panel D). Picrosirius red staining was used to evaluate cardiac fibrosis in the right ventricles of control (Panel E), MCT only (Panel F) and MCT + CeO2 nanoparticle treatment groups (Panel G). Scale bar = 50 μm. Data are mean ± SEM (n = 3 rats/group). * Significantly different from control. † Significantly different from the MCT only group (P < 0.05).

Fig. 5. MCT induced increases in RV fibronectin and MHC–beta are attenuated with cerium oxide nanoparticle treatment

Protein isolates from the right ventricle of control, MCT only, MCT + CeO₂ nanoparticle treated groups were analyzed by immunoblotting for changes in fibronectin (Panel A) and myosin heavy chain-beta (Panel B) protein levels. Protein abundance was normalized to the expression of GAPDH. n = 6 for each group. * Significantly different from control. † Significantly different from the MCT only group (P < 0.05).

Fig. 6. Histological analysis of RV tissue sections demonstrates attenuation of monocrotaline induced increase in superoxide levels by cerium oxide nanoparticles

Dihydroethidium stained right ventricular sections from control (Panel A), MCT only (Panel B), and MCT + CeO₂ nanoparticle treatment group (Panel C) at 28 days. Quantification of intensity of dihydroethidium staining (Panel D). Scale bar 50 μm n = 3 animals/group. * Significantly different from control. † Significantly different from the MCT only group (P < 0.05).

Fig. 7. Cerium oxide nanoparticle treatment decreases MCT–induced increases in protein nitration and carbonylation

Protein isolates obtained from the right ventricles of control, MCT only and MCT + CeO₂ nanoparticle treated groups were analyzed by immunoblotting for alterations in protein 3-nitrosylation (Panel A) and carbonylation (Panel B). * Significantly different from control. † Significantly different from the MCT only group (P<0.05). n = 6 animals/group.

Fig. 8. Immunoblot analysis of RV tissue demonstrates attenuation of monocrotaline induced increase in markers of oxidative stress by cerium oxide nanoparticles

Protein isolates of RV from control, MCT only and MCT + CeO₂ nanoparticles treated group was analyzed by immunoblotting to determine the amount of p-JNK, total JNK(Panel A), p-ERk1/2, and total ERK1/2 (Panel B) normalized to the expression of GAPDH. * Significantly different from control. † Significantly different from the MCT only group (P < 0.05). n = 6 animals/group.

Fig. 9. Cerium oxide nanoparticle treatment attenuates MCT–induced increases in markers of apoptosis

Protein isolates of RV from control, MCT only and MCT + CeO₂ nanoparticles treated groups was analyzed by immunoblotting to determine the amount of Bax and Bcl2 and normalized to the expression of GAPDH (Panel A). Amount of cleaved caspase 3, and total caspase 3 normalized to the expression of GAPDH (Panel B). * Significantly different from control. † Significantly different from the MCT only group (P < 0.05). n = 6 animals/group.