Zinc oxide nanoparticle-induced atherosclerotic alterations in vitro and in vivo

Abstract

Engineered zinc oxide nanoparticles (ZnO-NPs) are currently being produced in high tonnage. Exposure to ZnO-NPs presents potential risks to cardiovascular system. Thus far, the toxicological effects of ZnO-NPs on cardiovascular system have not been well characterized. In this study, human coronary artery endothelial cells (HCAECs) were exposed to ZnO-NPs directly or indirectly using a transwell coculture system with human alveolar epithelial cell line A549 to mimic the lung/circulation interaction. It was shown that levels of proinflammatory mediators (interleukin-8 [IL-8] and tumor necrosis factor-α [TNF-α]) and biomarkers of atherosclerogenesis (heme oxygenase-1 [HO-1] and platelet endothelial cell adhesion molecules-1 [PECAM-1]) in the supernatants of culture media were significantly increased. Pretreatment of A549 cells on the apical side of the coculture system with the phagocytosis inhibitor cytochalasin B (CB) blocked ZnO-NP-induced HO-1 and PECAM-1 expression in HCAEC, indicating that endocytosis of ZnO-NPs by alveolar epithelial cells was involved in ZnO-NP-induced HO-1 or PECAM-1 expression in endothelial cells. Moreover, Wistar rats were intratracheally instilled with ZnO-NP suspension and high fat diet (positive control). ZnO-NP treatment induced lung and systemic inflammation, dyslipidemia, increased levels of serum HO-1 and PECAM-1, and aortic pathological damage. Taken together, exposure to ZnO-NPs could induce atherosclerotic alterations, which might involve phagocytosis of nanoparticles and inflammation in the lung.

Keywords: atherosclerosis; heme oxygenase-1; lung inflammation; platelet endothelial cell adhesion molecules-1; zinc oxide nanoparticles.

Figure 1

Effect of ZnO-NPs on cell viability and expression of IL-8 and TNF-α in coculture. Notes: (A) HCAEC and A549 cells were incubated with ZnO-NPs, respectively. Cell viability was assessed by MTT assay. A549 cells in coculture were exposed to ZnO-NPs for 24 h. The basolateral media were collected for analysis of IL-8 (B) and TNF-α (C) expression by ELISA. Data shown were representative of three separate experiments (*compared to control group, P<0.05; ^▲compared to 10 μg/mL group, P<0.05; and ^#compared to 20 μg/mL group, P<0.05). Abbreviations: ZnO-NPs, zinc oxide nanoparticles; IL-8, interleukin-8; TNF-α, tumor necrosis factor-α; HCAEC, human coronary artery endothelial cell; A549, human alveolar type II epithelial cells; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; ELISA, enzyme-linked immunosorbent assay.

Figure 2

Effect of ZnO-NPs on HO-1 and PECAM-1 expression in HCAEC. Notes: HCAECs were cultured in the presence (coculture) and in the absence (monoculture) of A549 cells on transwell membranes. HCAEC monoculture and A549 cells in coculture were incubated with ZnO-NPs for 24 h, respectively. The expression of HO-1 (A) and PECAM-1 (B) from HCAEC in the supernatant was measured using ELISA (*compared to control group, P<0.05; ^▲compared to 10 μg/mL group, P<0.05; ^#compared to 20 μg/mL group, P<0.05). Abbreviations: ZnO-NPs, zinc oxide nanoparticles; HO-1, heme oxygenase-1; PECAM-1, platelet endothelial cell adhesion molecule-1; HCAECs, human coronary artery endothelial cells; A549, human alveolar type II epithelial cells; ELISA, enzyme-linked immunosorbent assay.

Figure 3

Effects of CB on ZnO-NP-induced HO-1 and PECAM-1 expression of HCAEC in coculture. Notes: (A) HCAECs in monoculture and A549 cells in coculture were incubated with ZnO-NPs for 24 h. Levels of intracellular zinc contents in HEAEC were measured using flame atomic absorption spectrometry (*compared to control group, P<0.05). A549 cells in coculture were preincubated with CB or vehicle control, prior to ZnO-NP (40 μg/mL) treatment for 24 h. HO-1 (B) and PECAM-1 (C) expression of the supernatant in basolateral chamber were measured using ELISA (*compared to the vehicle control group, P<0.05; ^▲compared to ZnO-NP-treated cells without CB treatment, P<0.05). Abbreviations: CB, cytochalasin B; ZnO-NPs, zinc oxide nanoparticles; HO-1, heme oxygenase-1; PECAM-1, platelet endothelial cell adhesion molecules-1; HCAEC, human coronary artery endothelial cells; A549, human alveolar type II epithelial cells; ELISA, enzyme-linked immunosorbent assay.

Figure 4

ZnO-NPs treatment induced dyslipidemia in rats. Notes: (A) Body mass of rats was measured weekly during the experiment. After last instillation, rats were anesthetized and sacrificed. (B) Epididymal adipose tissue was removed and weighed, and its ratio to body weight was calculated. The blood samples collected from heart were tested for levels of serum TC (C), HDL (D), and LDL (E) using ELISA (*compared to control group, P<0.05; ^▲compared to 2.5 mg/kg group, P<0.05; ^#compared to 5 mg/kg group, P<0.05). Abbreviations: ZnO-NPs, zinc oxide nanoparticles; TC, total cholesterol; HDL, high-density lipoprotein; LDL, low-density lipoprotein; ELISA, enzyme-linked immunosorbent assay.

Figure 5

ZnO-NP induces lung inflammation and systemic inflammation. Notes: After last instillation, rats were anesthetized and sacrificed. BALF was collected for the measurement of total proteins (A) and LDH (B) activity to represent the lung inflammation. The blood samples collected from heart were tested for levels of serum IL-8 (C) and TNF-α (D) using ELISA (*compared to control group, P<0.05; ^▲compared to 1.25 mg/kg group, P<0.05; ^#compared to 2.5 mg/kg group, P<0.05; ^◊compared to 5 mg/kg group, P<0.05). Abbreviations: ZnO-NPs, zinc oxide nanoparticles; BALF, bronchoalveolar lavage fluid; LDH, lactate dehydrogenase; IL-8, interleukin-8; TNF-α, tumor necrosis factor-α; ELISA, enzyme-linked immunosorbent assay.

Figure 6

ZnO-NP-induced atherosclerotic alterations in rats. Notes: After last instillation, rats were anesthetized and the blood samples collected from heart were tested for levels of serum HO-1 (A) and PECAM-1 (B) using ELISA. The aortas were separated from the anesthetized rats. Light microscopy was used to analyze the aortic sections stained with H&E at gross (C, 20×) and microscopic (D, 40×) levels (*compared to control group, P<0.05; ^▲compared to 1.25 mg/kg group, P<0.05; ^#compared to 2.5 mg/kg group, P<0.05; ^◊compared to 5 mg/kg group, P<0.05). Abbreviations: ZnO-NPs, zinc oxide nanoparticles; HO-1, heme oxygenase-1; PECAM-1, platelet endothelial cell adhesion molecules-1; ELISA, enzyme-linked immuno-sorbent assay; H&E, hematoxylin and eosin.

Scheme 1

Schematic diagram of noncontacting coculture of HCAEC and A549 cells. Abbreviations: HCAEC, human coronary artery endothelial cells; A549, human alveolar type II epithelial cells.