https://orcid.org/0000-0002-7224-0949
Abstract
With the explosive development of nanotechnology, engineered nanomaterials are currently being used in various industries, including food and medicine. Concern about the health effects of nanomaterials has been raised, and available research indicates that the relative surface area of nanomaterials seems to correlate with the severity of their toxicity. With regard to engineered nanomaterials, the scope of their acute and chronic toxicities and their mechanisms are not fully understood. Studies suggest that exposure to certain nanomaterials can generate reactive oxidant species and enhance permeability of the phagolysosomal membrane, which leads to inflammasome activation, causing oxidative stress and inflammation. Since the latter 2 are implicated in the development of cardiovascular diseases, such as hypertension and atherosclerosis, it can be presumed that exposure to engineered nanomaterials could significantly impact cardiovascular function. In this review, I raise issues that should be considered in the assessment of the effects of engineered nanomaterials on cardiovascular function, and evaluate their cardiovascular toxicity as described in various in vitro and/or in vivo toxicological studies and industrial investigations.
Introduction
The successful manipulation of the size of materials to nano sizes (less than 100 nm) near the end of the last century and subsequent commercialization of this technology resulted in the spread of use of nanomaterials in various fields of industry. However, this rapid explosion in the use of nanomaterials has raised concerns about the potential harmful effects on humans of materials such as carbon nanotubes and some metal oxide nanoparticles, based on their minute physical properties, and the lack of accurate in vivo quantitative assessment. This gap between widespread commercial use and potential risks to humans calls for the urgent need to gather information on the safety and potential hazards of nanomaterials.
The toxic effect of nanomaterials is reported to correlate with the relative surface area of these materials. Furthermore, certain nanomaterials are known to generate reactive oxygen species that can subsequently cause oxidative stress and inflammation.1 Recent studies have shown that exposure of laboratory animals to engineered nanoparticles can induce pulmonary inflammation and, especially with long-term exposure to multi-walled carbon nanotubes (MWCNTs), can cause lung cancer.2 Furthermore, epidemiological and toxicological studies3 have concluded that exposure to high concentrations of particulate matter smaller than 2.5 μm (PM2.5) increases cardiovascular events and death, in addition to pulmonary complications, raising concern that nanomaterials may have major effects on the cardiovascular system.
Direct and indirect cardiovascular effects of nanomaterials
In humans, exposure to engineered nanomaterials could harm the respiratory system through inhalation, the digestive tract through ingestion, skin through direct contact, and blood circulation through injection.4 Pathologically, inhalation of nanomaterials can induce oxidative stress and cause inflammation, which may indirectly lead to endothelial dysfunction and atherosclerosis.5 Nanomaterials can also promote blood coagulation through the activation of platelets and induce autonomic nervous system abnormalities via pulmonary reflexes, and hence indirectly cause cardiovascular dysfunction. The schematic diagram in Figure 1 illustrates the routes and mechanisms by which engineered nanomaterials affect the cardiovascular system. Other reported pathological effects of inhaled nanomaterials are their ability to cross the blood–air barrier in the lung and enter the bloodstream.6 Furthermore, once in the circulation, nanomaterials can be taken up by vascular endothelial cells to induce endothelial dysfunction and inflammation of major vasculature. The results of experimental alveolar translocation of nanomaterials showed that a similar pathway exists in humans; however, the extent to which nanomaterials reach organs outside the lungs is highly dependent on their surface/chemical properties as well as their size. The translocation of nanomaterials into the peripheral circulation raises the question of how such materials can directly affect the cardiovascular system.
Effects of engineered nanomaterials on the cardiovascular system and their routes and mechanisms.
Illustration of the effect of nanomaterials on the progression of arteriosclerosis.
Assessment of nanomaterials’ effects on blood vessels
In vitro evaluation of nanomaterials’ effects on vascular endothelial cells
Since nanomaterials can be taken up by vascular endothelial cells and directly cause endothelial dysfunction, these cells have been used to examine the effects of nanomaterials on endothelial function, particularly the cellular mechanisms of such toxicity. Nanomaterials tend to easily aggregate into micro-sized structures and therefore exhibit different biological effects depending on their aggregation state. Therefore, choosing an appropriate dispersion method in such experiments is critically important for accurate evaluation. We have previously provided detailed information on the methods used to prepare nanomaterial suspensions and the most suitable dispersion method for evaluation of toxicity.7
In a series of articles, our group reported previously that exposure to zinc oxide (ZnO) nanoparticles induced transmigration of monocytes into endothelial cells in co-cultures of human umbilical vein endothelial cells (HUVECs) and human monocytic leukemia (THP-1) cells.8 Further analysis showed that ZnO nanoparticles upregulated the expression of monocyte chemotactic protein-1 (MCP-1), which is known to play an essential role in monocyte recruitment in atherosclerotic lesions, and induced monocyte migration. Intercellular adhesion molecule-1 (ICAM-1) plays an important role in the adhesion of monocytes, macrophages, T lymphocytes, and platelets to vascular endothelial cells, which ultimately triggers the development of atherosclerosis. Our studies also showed that ZnO nanoparticles increased ICAM-1 expression in HUVECs, and adhesion assays confirmed the adhesion of THP-1 monocytes to HUVECs under the above exposure conditions.8 In another study, we also reported that ZnO nanoparticles suppressed vasculogenesis by downregulation of the receptors related to vasculogenesis in human endothelial colony-forming cells.9
Other in vitro studies reported that exposure to iron oxide (Fe2O3, Fe3O4) and aluminum oxide (Al2O3) nanoparticles also induced overexpression of ICAM-1, vascular inflammation, and endothelial dysfunction.10 Furthermore, studies using carbon nanotubes (CNTs) showed the translocation of these structures into the bloodstream, reaching distant organs, whereas exposure to MWCNTs induced cytotoxicity and genotoxicity in HUVECs.11 Studies from our laboratories showed that exposure to either single-walled carbon nanotubes (SWCNTs) or double-walled carbon nanotubes (DWCNTs) increased ICAM-1 expression and promoted monocyte adhesion to endothelial cells.12 Considered together, the above studies suggest that certain CNTs can cause endothelial cell damage. Figure 2 is a schematic diagram showing the mechanism of nanomaterials-induced arteriosclerosis.
In vivo evaluation of nanomaterials-related cardiovascular toxicity
The effects of nanomaterials on the cardiovascular system are being evaluated in experimental animals, particularly the apolipoprotein E knockout (ApoE KO) mouse, a well-known animal model of atherosclerosis (Table 1). Exposure of Wistar rats to ZnO nanoparticles induced inflammatory changes, which ultimately led to a pro-atherosclerotic state, including vascular wall thickening and hyperproliferation of smooth muscle cells in the aorta.13 In another study, intratracheal instillation of copper oxide (CuO) nanoparticles caused vascular endothelial injury through induction of oxidative stress and impairment of autophagy.14 Our group also reported that CuO nanoparticles reduced vasculogenesis through the induction of apoptosis and underexpression of vascular endothelial growth factor in transgenic zebrafish.15 Previous studies also concluded that exposure of mice to titanium dioxide (TiO2) nanoparticles slightly increased the progression of plaque in the aorta.16 Our study with HUVECs and ApoE KO mice found that anatase, but not rutile, TiO2 nanoparticles enhanced monocyte adhesion to endothelial cells and facilitated atherosclerosis formation in ApoE KO mice, a susceptible animal model.17 These effects were associated with macrophage infiltration and ICAM-1 upregulation in the aorta.17
ApoE KO mice exposed to MWCNTs showed accelerated progression of atherosclerosis through increased adhesion of monocytes to the endothelium and the transformation of monocytes into foam cells due to oxidative stress.18 Other studies using the Sprague-Dawley rats demonstrated that inhalation of MWCNTs increased blood pressure, which was associated with changes in the sympathetic and parasympathetic nervous system.19 Our study also found that exposure of ApoE KO mice to SWCNTs resulted in a slight increase in the aortic plaque area compared with the control mice, in part due to the induced dysfunction of endothelial progenitor cells (EPCs).12
Experimental studies on the effects on blood vessels of exposure to nanomaterials.
| Nanomaterials | Size, nm | Animal model | Exposure route | Dose | Duration | Mechanisms | Major findings | Authors |
|---|---|---|---|---|---|---|---|---|
| ZnO NPs | 30 | Wistar rats | Intratracheal instillation | 1.25, 2.5, 5.0 mg/kg | Once a week, 12 wk | Inflammatory responses; increased HO-1 and PECAM-1 | ZnO NPs exposure induced aortic pathological damage. | Yan et al (2017) [13] |
| CuO NPs | <50 | C57BL/6J mice | Intratracheal instillation | 5 mg/kg | 3 d | Oxidative stress, impaired autophagy | CuO NPs exposure induced vascular endothelial injury | Li et al (2022) [14] |
| CuO NPs | 40 | Zebrafish | Aqueous exposure | 0.01, 1, 100 μg/mL | From 1 to 5 dpf | Apoptosis, reduction of expression | CuO NPs exposure inhibited vasculogenesis | Chang et al (2015) [15] |
| TiO2NPs | 21.6 | ApoE-null mice | Intratracheal instillation | 0.5 mg/kg | Once a week, 4 wk | No association with inflammation and vasodilatory dysfunction | TiO2 NPs exposure slightly increased the progression of plaque in the aorta | Mikkelsen et al (2011) [16] |
| TiO2NPs | 20 | ApoE-null mice | Pharyngeal aspiration | 10, 40 μg/mouse | Once a week, 10 wk | Monocyte adhesion; ICAM-1 and F4/80 upregulation | Anatase TiO2 NPs exposure increased plaque formation in the aorta | Suzuki et al (2020) [17] |
| MWCNTs | — | ApoE-null mice | Intratracheal instillation | 25.6 μg/mouse | Once a week, 5 wk | Oxidative stress, monocyte adhesion; ICAM-1 and VECAM-1 upregulation | MWCNTs exposure accelerated the progression of plaque in the aorta | Cao et al (2014) [18] |
| MWCNTs | — | Sprague-Dawley rats | Inhalation | 5 mg/m3 | 5 h/d, 7 d | Altered the sympathetic and parasympathetic nervous system | MWCNTs exposure increased both systolic and diastolic blood pressure | Zheng et al (2018) [19] |
| SWCNTs | — | ApoE-null mice | Pharyngeal aspiration | 10, 40 μg/mouse | Once a week, 10 wk | Induction of endothelial progenitor cell dysfunction | SWCNT exposure slightly increased plaque progression in aorta | Suzuki et al (2016) [12] |
| Nanomaterials | Size, nm | Animal model | Exposure route | Dose | Duration | Mechanisms | Major findings | Authors |
|---|---|---|---|---|---|---|---|---|
| ZnO NPs | 30 | Wistar rats | Intratracheal instillation | 1.25, 2.5, 5.0 mg/kg | Once a week, 12 wk | Inflammatory responses; increased HO-1 and PECAM-1 | ZnO NPs exposure induced aortic pathological damage. | Yan et al (2017) [13] |
| CuO NPs | <50 | C57BL/6J mice | Intratracheal instillation | 5 mg/kg | 3 d | Oxidative stress, impaired autophagy | CuO NPs exposure induced vascular endothelial injury | Li et al (2022) [14] |
| CuO NPs | 40 | Zebrafish | Aqueous exposure | 0.01, 1, 100 μg/mL | From 1 to 5 dpf | Apoptosis, reduction of expression | CuO NPs exposure inhibited vasculogenesis | Chang et al (2015) [15] |
| TiO2NPs | 21.6 | ApoE-null mice | Intratracheal instillation | 0.5 mg/kg | Once a week, 4 wk | No association with inflammation and vasodilatory dysfunction | TiO2 NPs exposure slightly increased the progression of plaque in the aorta | Mikkelsen et al (2011) [16] |
| TiO2NPs | 20 | ApoE-null mice | Pharyngeal aspiration | 10, 40 μg/mouse | Once a week, 10 wk | Monocyte adhesion; ICAM-1 and F4/80 upregulation | Anatase TiO2 NPs exposure increased plaque formation in the aorta | Suzuki et al (2020) [17] |
| MWCNTs | — | ApoE-null mice | Intratracheal instillation | 25.6 μg/mouse | Once a week, 5 wk | Oxidative stress, monocyte adhesion; ICAM-1 and VECAM-1 upregulation | MWCNTs exposure accelerated the progression of plaque in the aorta | Cao et al (2014) [18] |
| MWCNTs | — | Sprague-Dawley rats | Inhalation | 5 mg/m3 | 5 h/d, 7 d | Altered the sympathetic and parasympathetic nervous system | MWCNTs exposure increased both systolic and diastolic blood pressure | Zheng et al (2018) [19] |
| SWCNTs | — | ApoE-null mice | Pharyngeal aspiration | 10, 40 μg/mouse | Once a week, 10 wk | Induction of endothelial progenitor cell dysfunction | SWCNT exposure slightly increased plaque progression in aorta | Suzuki et al (2016) [12] |
Abbreviations: ApoE, apolipoprotein E; dpf, days post-fertilization; HO-1, heme oxygenase-1; ICAM-1, intercellular adhesion molecule-1; MWCNT, multi-walled carbon nanotube; NP, nanoparticle; PECAM-1, platelet endothelial cell adhesion molecule-1; SWCNT, single-walled carbon nanotube; VECAM-1, vascular cell adhesion protein-1; VEGF, vascular endothelial growth factor.
Experimental studies on the effects on blood vessels of exposure to nanomaterials.
| Nanomaterials | Size, nm | Animal model | Exposure route | Dose | Duration | Mechanisms | Major findings | Authors |
|---|---|---|---|---|---|---|---|---|
| ZnO NPs | 30 | Wistar rats | Intratracheal instillation | 1.25, 2.5, 5.0 mg/kg | Once a week, 12 wk | Inflammatory responses; increased HO-1 and PECAM-1 | ZnO NPs exposure induced aortic pathological damage. | Yan et al (2017) [13] |
| CuO NPs | <50 | C57BL/6J mice | Intratracheal instillation | 5 mg/kg | 3 d | Oxidative stress, impaired autophagy | CuO NPs exposure induced vascular endothelial injury | Li et al (2022) [14] |
| CuO NPs | 40 | Zebrafish | Aqueous exposure | 0.01, 1, 100 μg/mL | From 1 to 5 dpf | Apoptosis, reduction of expression | CuO NPs exposure inhibited vasculogenesis | Chang et al (2015) [15] |
| TiO2NPs | 21.6 | ApoE-null mice | Intratracheal instillation | 0.5 mg/kg | Once a week, 4 wk | No association with inflammation and vasodilatory dysfunction | TiO2 NPs exposure slightly increased the progression of plaque in the aorta | Mikkelsen et al (2011) [16] |
| TiO2NPs | 20 | ApoE-null mice | Pharyngeal aspiration | 10, 40 μg/mouse | Once a week, 10 wk | Monocyte adhesion; ICAM-1 and F4/80 upregulation | Anatase TiO2 NPs exposure increased plaque formation in the aorta | Suzuki et al (2020) [17] |
| MWCNTs | — | ApoE-null mice | Intratracheal instillation | 25.6 μg/mouse | Once a week, 5 wk | Oxidative stress, monocyte adhesion; ICAM-1 and VECAM-1 upregulation | MWCNTs exposure accelerated the progression of plaque in the aorta | Cao et al (2014) [18] |
| MWCNTs | — | Sprague-Dawley rats | Inhalation | 5 mg/m3 | 5 h/d, 7 d | Altered the sympathetic and parasympathetic nervous system | MWCNTs exposure increased both systolic and diastolic blood pressure | Zheng et al (2018) [19] |
| SWCNTs | — | ApoE-null mice | Pharyngeal aspiration | 10, 40 μg/mouse | Once a week, 10 wk | Induction of endothelial progenitor cell dysfunction | SWCNT exposure slightly increased plaque progression in aorta | Suzuki et al (2016) [12] |
| Nanomaterials | Size, nm | Animal model | Exposure route | Dose | Duration | Mechanisms | Major findings | Authors |
|---|---|---|---|---|---|---|---|---|
| ZnO NPs | 30 | Wistar rats | Intratracheal instillation | 1.25, 2.5, 5.0 mg/kg | Once a week, 12 wk | Inflammatory responses; increased HO-1 and PECAM-1 | ZnO NPs exposure induced aortic pathological damage. | Yan et al (2017) [13] |
| CuO NPs | <50 | C57BL/6J mice | Intratracheal instillation | 5 mg/kg | 3 d | Oxidative stress, impaired autophagy | CuO NPs exposure induced vascular endothelial injury | Li et al (2022) [14] |
| CuO NPs | 40 | Zebrafish | Aqueous exposure | 0.01, 1, 100 μg/mL | From 1 to 5 dpf | Apoptosis, reduction of expression | CuO NPs exposure inhibited vasculogenesis | Chang et al (2015) [15] |
| TiO2NPs | 21.6 | ApoE-null mice | Intratracheal instillation | 0.5 mg/kg | Once a week, 4 wk | No association with inflammation and vasodilatory dysfunction | TiO2 NPs exposure slightly increased the progression of plaque in the aorta | Mikkelsen et al (2011) [16] |
| TiO2NPs | 20 | ApoE-null mice | Pharyngeal aspiration | 10, 40 μg/mouse | Once a week, 10 wk | Monocyte adhesion; ICAM-1 and F4/80 upregulation | Anatase TiO2 NPs exposure increased plaque formation in the aorta | Suzuki et al (2020) [17] |
| MWCNTs | — | ApoE-null mice | Intratracheal instillation | 25.6 μg/mouse | Once a week, 5 wk | Oxidative stress, monocyte adhesion; ICAM-1 and VECAM-1 upregulation | MWCNTs exposure accelerated the progression of plaque in the aorta | Cao et al (2014) [18] |
| MWCNTs | — | Sprague-Dawley rats | Inhalation | 5 mg/m3 | 5 h/d, 7 d | Altered the sympathetic and parasympathetic nervous system | MWCNTs exposure increased both systolic and diastolic blood pressure | Zheng et al (2018) [19] |
| SWCNTs | — | ApoE-null mice | Pharyngeal aspiration | 10, 40 μg/mouse | Once a week, 10 wk | Induction of endothelial progenitor cell dysfunction | SWCNT exposure slightly increased plaque progression in aorta | Suzuki et al (2016) [12] |
Abbreviations: ApoE, apolipoprotein E; dpf, days post-fertilization; HO-1, heme oxygenase-1; ICAM-1, intercellular adhesion molecule-1; MWCNT, multi-walled carbon nanotube; NP, nanoparticle; PECAM-1, platelet endothelial cell adhesion molecule-1; SWCNT, single-walled carbon nanotube; VECAM-1, vascular cell adhesion protein-1; VEGF, vascular endothelial growth factor.
Assessment of nanoparticles-induced cardiovascular toxicity in factory workers
A few studies have also been conducted in factories that handle nanoparticles to examine the effects of the latter on the cardiovascular system. Zhao et al20 reported that occupational exposure to TiO2 nanoparticles was associated with high serum levels of cardiovascular disease markers (vascular endothelial growth factor: VCAM-1, ICAM-1, and total cholesterol). Another study analyzed exhaled breath condensates collected from workers involved in TiO2 production and reported increased markers of oxidative stress, such as lipid peroxidation.21 In addition, examination of workers with prolonged exposure to silver and silica nanoparticles showed overexpression of various inflammatory cytokines in peripheral blood samples.22 Our study involving workers in a factory that handled nano-sized TiO2 particles showed that exposure to particles less than 300 nm in diameter was associated with reduced heart rate variability (HRV), suggesting that exposure to engineered nanomaterials may affect the autonomic nervous system.23 Another study involving workers who handled nano-silver materials also reported the induction of autonomic dysfunction, which was associated with alteration of HRV and cardiac function.24
Summary and future research
Exposure to engineered nanomaterials, through the lungs, for example, can induce systemic inflammation via increased oxidative stress and activation of inflammasomes, leading to endothelial dysfunction and atherosclerogenesis. Additionally, nanomaterials may invade the air–blood barrier in the lung and enter the bloodstream where they are taken up by vascular endothelial cells, directly inducing endothelial cell damage. Although characterization of nanomaterials has allowed proper interpretation of results, the results of both in vitro and in vivo studies must be interpreted with caution. Almost all experimental studies have applied delivery methods routinely used in particles-exposure studies: intratracheal instillation or pharyngeal aspiration. However, since intratracheal instillation and aspiration are less physiological methods of exposure than inhalation, the results may differ from those obtained with inhalation exposure. The continuous inhalation experiments may reflect actual human exposure conditions. There is therefore a need for more inhalation-based experiments using natural delivery routes, in addition to epidemiological studies, in order to establish the true cardiovascular effects of nanomaterial exposure on acceleration of atherosclerogenesis. Furthermore, experimental studies are needed to determine the exact mechanisms of nanomaterials-induced changes in the cardiovascular system.
Funding
This work was supported by a grant (NEXT Program #LS056) from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) and grants (20H03935 and 24K02690) from the Japan Society for the Promotion of Science (JSPS), Japan.
Conflicts of interest
The author declare no competing interests, including direct and indirect support.
