Relationship: 1702: Interaction with the lung cell membrane leads to Increased proinflammatory mediators

AOPs Referencing Relationship

Key Event Relationship Description

Innate immune response is the first line of defence in any organism against invading infectious pathogens and toxic substances. It involves tissue triggered startle response to cellular stress and is described by a complex set of interactions between the toxic stimuli, soluble macromolecules and cells (reviewed in Nathan, 2002). The process culminates in a functional change defined as inflammation, purpose of which is to resolve infection and promote healing. In lungs, the interaction of toxic substances with resident cells results in cellular stress, death or necrosis (Pouwels et al., 2016) leading to release of intracellular components such as alarmins (Damage associated molecular patterns [DAMPs], Interleukin (IL)-1α, High mobility group box 1 [HMGB1]). Released alarmins (danger sensors) bind cell surface receptors such as Interleukin 1 Receptor 1 (IL-1R1), Toll Like Receptors (TLRs) or others leading to activation of innate immune response signalling.

For example, binding of IL-1α to IL-1R1 can release Nuclear factor kappa B (NF-κB) resulting in its translocation to nucleus and transactivation of pro-inflammatory genes including cytokines, growth factors and acute phase genes. The signalling also stimulates secretion of a variety of pro-inflammatory mediators. Overexpression of IL-1α in cells induces increased secretion of pro-inflammatory mediators. Products of necrotic cells are shown to stimulate the immune system in an IL-1R1-dependent manner (Chen et al., 2007).

The secreted alarmins activate resident cells pre-stationed in the tissues such as mast cells or macrophages leading to propagation of the already initiated immune response by releasing more eicosanoids, cytokines, chemokines and other pro-inflammatory mediators. Thus, secreted mediators signal the recruitment of neutrophils, which are the first cell types to be recruited in acute inflammatory conditions. Neutrophil influx in sterile inflammation is driven mainly by IL-1α (Rider P, 2011). IL-1 mediated signalling regulates neutrophil influx in silica-induced acute lung inflammation (Hornung et al., 2008). IL-1 signalling also mediates neutrophil influx in other tissues and organs including liver and peritoneum. Other types of cells including macrophages, eosinophils, and lymphocytes are also recruited in a signal-specific manner. Recruitment of leukocytes induces critical cytokines associated with the T helper type 2 immune response, including Tumor necrosis factor alpha (TNF-α), IL-1β, and IL-13.

Evidence Supporting this KER

The biological plausibility of this relationship is high. There is a mechanistic relationship between the MIE (Event 1495) and KE1 (Event 1496) which has been evidenced in a number of both in vitro and in vivo model systems in response to stressors such as, asbestos, silica, bleomycin, carbon nanotubes, and metal oxide nanoparticles (NPs) (Behzadi et al., 2017; Denholm & Phan 1990; Dostert et al., 2008; Mossman & Churg 1998).

Increased expression of IL-1α or IL-1β following lung exposure to multi-walled carbon nanotubes (MWCNTs), bleomycin, micro silica particles, silica crystals, and polyhexamethylene guanidine phosphate has been shown to be associated with neutrophil influx in rodents (Gasse et al., 2007; Girtsman et al., 2014; Hornung et al., 2008; Nikota et al., 2017; Rabolli et al., 2014; Suwara et al., 2014). Inhibition of IL-1 function by knocking out the expression of IL-1R1 using IL-1R1 knockout mice or via treatment with IL-1α or IL-1β neutralising antibodies results in complete abrogation of lung neutrophilic influx following exposure to MWCNTs (Nikota et al, 2017), cigarette smoke (CS) (Halappanavar et al., 2013), silica crystals (Rabolli et al., 2014) and bleomycin (Gasse et al., 2007). IL1-R1, Myeloid differentiation primary response protein (Myd88) or the IL-33/St2 signaling are involved in pulmonary fibrosis induced by bleomycin (Gasse et al., 2007; Xu et al., 2016).

Empirical support for this KER is moderate. There are limited in vitro studies, which show a temporal and dose-dependent relationship between these two events, using the upregulation of specific surface receptors as a proxy for direct membrane interaction (Chan et al., 2018; Denholm & Phan, 1990; Roy et al., 2014). There are also studies that provide general support for the idea that an interaction with the lung resident cell membrane components leads to increased, secretion of pro-inflammatory and pro-fibrotic mediators (Table 1).

Dose-Response Evidence:

There are a few studies which provide evidence for a dose-response relationship in this KER. An in vitro study demonstrated a concentration-response relationship, in which silica exposure induced increases in pro-inflammatory cytokines through scavenger receptors in cultured bone marrow-derived murine mast cells. Cells were exposed to 6.25, 12.5, 25 or 50 µg/cm² silica dioxide (SiO₂) for 24 h. Macrophage scavenger receptor (MSR2) expression increased over time at 50 µg/cm² and in a concentration-dependent relationship. Moreover, Tumor necrosis factor alpha (TNF-α), IL-13 and Monocyte chemoattractant protein-1 (MCP-1) increased in a concentration-dependent manner (Brown et al., 2007). This provides indications that at higher concentrations of the stressor, the interaction with the lung resident cell membrane components (Event 1495) leads to an increased secretion of pro-inflammatory mediators (Event 1496).

Temporal Evidence:

In vitro and in vivo studies have demonstrated temporal concordance of the KEs.

TLR4 signal pathway was evaluated in differentiated macrophages exposed to silica at 2.5 µg/cm². After 16 and 24 h, the mRNA expression level of TLR4 increased. Moreover, the protein expression level of TLR-4 and related MyD88/Toll-interleukin-1 receptor domain containing adaptor protein (TIRAP) pathway increased at 24 h. Release of  IL-1β, IL-6, IL-10, and TNF-α was induced by silica exposure at 24 h. Pre-treatment with resatorvid (TAK-242), an inhibitor of TLR4 signaling, suppressed the release of the cytokines (Chan et al., 2018).

Macrophages exposed to zinc oxide (ZnO) NPs at 2.5 µg/mL for 24 h increased the expression level of TLR6 and MyD88, TNF receptor-associated factor (TRAF), and IL-1 receptor-associated kinase (IRAK). At 24 h, they also observed an increase in the mRNA and protein levels of the pro-inflammatory cytokines IL-1β, IL-6, and TNF-α. These results demonstrated that ZnO NPs induced pro-inflammatory mediators by TLR stimulation and Mitogen-activated protein kinases (MAPKs) activation (Roy et al., 2014).

The pro-inflammatory IL-1β induced granulocyte migration and can be produced as a result of cellular detection of pathogen associated molecular patterns (PAMPs). Mice exposed to 2.5 mg/mouse of silica by instillation showed an increase of mRNA expression of pro-IL-1β in bronchoalveolar lavage fluid (BALF) at 6, 12, and 24 h post-exposure in a time-dependent manner. At early time points (1 h, 3 h, 6 h), there was an increase in the release of an alarmin (IL-1α) which indicates that the alarmin was released due to cell damage leading to cytokine production and an inflammatory reaction. Moreover, at 24 h, the levels of mature IL-1β and neutrophil accumulation in BALF increased. Neutralization or deletion of IL-1α reduced the observed responses (Rabolli et al., 2014).

Epithelial damage can lead to the release of alarmins. In this stead, conditioned media from primary human bronchial epithelial cells (PBECs) exposed to thapsigargin was able to induce a pro-inflammatory response in primary human lung fibroblasts. PBECs were exposed to thapsigargin (a tumor promoter in mammalian cells) 20 µM for 2 h. After that, the cell culture medium was replaced, and cells were incubated for 24 h. At this time, the medium was recovered and used to culture lung fibroblast for 5 h. This conditioned media from epithelial cell damage contains the alarmin IL-1α, which induced increased gene expression of IL-6, IL-8, MCP-1, and Granulocyte-macrophage colony-stimulating factor (GM-CSF) in fibroblasts. These responses were reduced with anti-IL-1α treatment (Suwara et al., 2014).

Heijink et al. 2015 conducted a similar strategy to identify the relationship between DAMPs and pro-inflammatory mediator release after exposure to CS. Neutrophils treated with CS bubbled for 1 min, released high levels of HMGB1 as a consequence of necrotic cell death. The cell-free supernatant, which contains HMGB1, was used to culture human bronchial epithelial cells, and after 24 h it promoted the production of the C-X-C motif chemokine ligand (CXCL)8 or IL-8 by lung epithelial cells. Pharmacological inhibitors, such as 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (OxPAPC) and Receptor for advanced glycation endproducts (RAGE) antagonist peptide (RAP), reduced the effect of CXCL8 release.

HMGB1 and Heat shock protein 70 (HSP-70) can be released by damaged hepatocytes. In a study, mice were treated with acetaminophen 350 mg/Kg for 3 and 6 h. At these time points, the liver perfusate was obtained and an increase in HSP-70 and HMGB1 protein levels was observed. RAW 264.7 cells (a macrophage cell line) treated with the liver perfusate exhibited increased mRNA expression levels of MCP-1 and IL-1β (Martin-Murphy et al. 2010).

Female mice were intratracheally administered with bleomycin at 5 mg/kg to represent idiopathic pulmonary fibrosis. IL-33, a molecule that can act as a DAMP, increased in lungs after 3 and 7 days of treatment. In serum, at 7-, 14- and 28-days post-exposure, IL-4 and IL-13 increased. It was concluded that IL-33/ST2 signaling pathway is involved in pulmonary fibrosis by bleomycin (Xu et al., 2016).

Attenuation or complete abrogation of KE1 (Event 1496) and KE2 (Event 1497) following inflammogenic stimuli is observed in rodents lacking functional IL-1R1 or other cell surface receptors that engage innate immune response upon stimulation. However, following exposure to MWCNTs, it has been shown that absence of IL-1R1 signalling is compensated for eventually and neutrophil influx is observed at a later post-exposure time point (Nikota et al., 2017). In another study, acute neutrophilic inflammation induced by MWCNTs was suppressed at 24 h in mice deficient in IL-1R1 signalling; however, these mice showed exacerbated neutrophilic influx and fibrotic response at 28 days post-exposure (Girtsman et al., 2014). The early defence mechanisms involving DAMPs is fundamental for survival, which may necessitate activation of compensatory signaling pathways. As a result, inhibition of a single biological pathway mediated by an individual cell surface receptor may not be sufficient to completely abrogate the lung inflammatory response. Forced suppression of pro-inflammatory and immune responses early after exposure to substances that cannot be effectively cleared from lungs, may enhance the injury and initiate other pathways leading to exacerbated response.

Quantitative Understanding of the Linkage

A majority of the in vivo studies are conducted with only one dose and thus, it is difficult to derive quantitative dose-response relationships based on the existing data. However, it is clear from the studies referenced above that greater concentrations or doses of pro-fibrotic substances result in higher release of alarmins, and consequently, higher pro-inflammatory signalling. The above studies also demonstrate strong temporal relationships between the individual KEs.

One study has demonstrated a response-response relationship for this KER.

Human intervertebral disc cells were treated with 0, 0.5, 1, or 2 mg/ml of recombinant HMGB1 for 24 h. Protein levels were determined in cell medium supernatant by enzyme-linked immunosorbent assay (ELISA). HMGB1 stimulates the expression of IL-6 and Matrix metalloproteinase 1 (MMP-1) in a response-response relationship. A strong correlation was observed by Spearman’s rank correlation coefficient between HMGB1 treatment and IL-6 or MMP-1 levels (Shah et al., 2019).

Other reports have studied both KEs, but they do not indicate if the response-response relationship was linear or not (coefficient or correlation is not shown) (Chakraborty et al., 2017; Fukuda et al. 2017; Kim et al., 2020, Piazza et al., 2013; Yang et al., 2012;).

Some studies have described how long after a change in the MIE (Event 1495; interaction substance and components), KE1 (Event 1496; pro-inflammatory mediators are secreted) is impacted (Table 2).

Table 2. Time-scale related studies relevant to the MIE (Event 1495) - KE1 (Event 1496) relationship.

Pancreatic cancer cells stimulated with S100 calcium-binding protein A8 (S100A8) and S100 calcium-binding protein A9 (S100A9) released pro-inflammatory cytokines IL-8, TNF-α, and Fibroblast growth factor (FGF). Cancer cell-derived conditioned media and the individual cytokines (TNF-α and Transforming growth factor beta [TGF-β]) induced the protein expression of S100A8 and S100A9 in HL-60 monocytic cell line and primary human monocytes (Nedjadi et al. 2018).

References

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2. Brown JM, Swindle EJ, Kushnir-Sukhov NM, Holian A, Metcalfe DD. Silica-directed mast cell activation is enhanced by scavenger receptors. Am J Respir Cell Mol Biol. 2007 Jan;36(1):43-52. doi: 10.1165/rcmb.2006-0197OC. 

3. Chakraborty D, Zenker S, Rossaint J, Hölscher A, Pohlen M, Zarbock A, Roth J, Vogl T. Alarmin S100A8 Activates Alveolar Epithelial Cells in the Context of Acute Lung Injury in a TLR4-Dependent Manner. Front Immunol. 2017 Nov 13;8:1493. doi: 10.3389/fimmu.2017.01493.

4. Chan JYW, Tsui JCC, Law PTW, So WKW, Leung DYP, Sham MMK, Tsui SKW, Chan CWH. Regulation of TLR4 in silica-induced inflammation: An underlying mechanism of silicosis. Int J Med Sci. 2018 Jun 14;15(10):986-991. doi: 10.7150/ijms.24715. 

5. Chen CJ, Kono H, Golenbock D, Reed G, Akira S, Rock KL. Identification of a key pathway required for the sterile inflammatory response triggered by dying cells. Nat Med. 2007 Jul;13(7):851-6. doi: 10.1038/nm1603. 

6. Denholm EM, Phan SH. Bleomycin binding sites on alveolar macrophages. J Leukoc Biol. 1990 Dec;48(6):519-23. doi: 10.1002/jlb.48.6.519.

7. Dostert C, Pétrilli V, Van Bruggen R, Steele C, Mossman BT, Tschopp J. Innate immune activation through Nalp3 inflammasome sensing of asbestos and silica. Science. 2008 May 2;320(5876):674-7. doi: 10.1126/science.1156995. 

8. Fukuda K, Ishida W, Miura Y, Kishimoto T, Fukushima A. Cytokine expression and barrier disruption in human corneal epithelial cells induced by alarmin released from necrotic cells. Jpn J Ophthalmol. 2017 Sep;61(5):415-422. doi: 10.1007/s10384-017-0528-7.

9. Gasse P, Mary C, Guenon I, Noulin N, Charron S, Schnyder-Candrian S, Schnyder B, Akira S, Quesniaux VF, Lagente V, Ryffel B, Couillin I. IL-1R1/MyD88 signaling and the inflammasome are essential in pulmonary inflammation and fibrosis in mice. J Clin Invest. 2007 Dec;117(12):3786-99. doi: 10.1172/JCI32285. 

10. Girtsman TA, Beamer CA, Wu N, Buford M, Holian A. IL-1R signalling is critical for regulation of multi-walled carbon nanotubes-induced acute lung inflammation in C57Bl/6 mice. Nanotoxicology. 2014 Feb;8(1):17-27. doi: 10.3109/17435390.2012.744110.

11. Halappanavar S, Nikota J, Wu D, Williams A, Yauk CL, Stampfli M. IL-1 receptor regulates microRNA-135b expression in a negative feedback mechanism during cigarette smoke-induced inflammation. J Immunol. 2013 Apr 1;190(7):3679-86. doi: 10.4049/jimmunol.1202456.

12. Heijink IH, Pouwels SD, Leijendekker C, de Bruin HG, Zijlstra GJ, van der Vaart H, ten Hacken NH, van Oosterhout AJ, Nawijn MC, van der Toorn M. Cigarette smoke-induced damage-associated molecular pattern release from necrotic neutrophils triggers proinflammatory mediator release. Am J Respir Cell Mol Biol. 2015 May;52(5):554-62. doi: 10.1165/rcmb.2013-0505OC. 

13. Hornung V, Bauernfeind F, Halle A, Samstad EO, Kono H, Rock KL, Fitzgerald KA, Latz E. Silica crystals and aluminum salts activate the NALP3 inflammasome through phagosomal destabilization. Nat Immunol. 2008 Aug;9(8):847-56. doi: 10.1038/ni.1631.

14. Kim DH, Gu A, Lee JS, Yang EJ, Kashif A, Hong MH, Kim G, Park BS, Lee SJ, Kim IS. Suppressive effects of S100A8 and S100A9 on neutrophil apoptosis by cytokine release of human bronchial epithelial cells in asthma. Int J Med Sci. 2020 Feb 4;17(4):498-509. doi: 10.7150/ijms.37833. 

15. Martin-Murphy BV, Holt MP, Ju C. The role of damage associated molecular pattern molecules in acetaminophen-induced liver injury in mice. Toxicol Lett. 2010 Feb 15;192(3):387-94. doi: 10.1016/j.toxlet.2009.11.016.

16. Mossman BT, Churg A. Mechanisms in the pathogenesis of asbestosis and silicosis. Am J Respir Crit Care Med. 1998 May;157(5 Pt 1):1666-80. doi: 10.1164/ajrccm.157.5.9707141.

17. Nathan C. Points of control in inflammation. Nature. 2002 Dec 19-26;420(6917):846-52. doi: 10.1038/nature01320. 

18. Nedjadi T, Evans A, Sheikh A, Barerra L, Al-Ghamdi S, Oldfield L, Greenhalf W, Neoptolemos JP, Costello E. S100A8 and S100A9 proteins form part of a paracrine feedback loop between pancreatic cancer cells and monocytes. BMC Cancer. 2018 Dec 17;18(1):1255. doi: 10.1186/s12885-018-5161-4. 

19. Nikota J, Banville A, Goodwin LR, Wu D, Williams A, Yauk CL, Wallin H, Vogel U, Halappanavar S. Stat-6 signaling pathway and not Interleukin-1 mediates multi-walled carbon nanotube-induced lung fibrosis in mice: insights from an adverse outcome pathway framework. Part Fibre Toxicol. 2017 Sep 13;14(1):37. doi: 10.1186/s12989-017-0218-0.

20. Piazza O, Leggiero E, De Benedictis G, Pastore L, Salvatore F, Tufano R, De Robertis E. S100B induces the release of pro-inflammatory cytokines in alveolar type I-like cells. Int J Immunopathol Pharmacol. 2013 Apr-Jun;26(2):383-91. doi: 10.1177/039463201302600211.

21. Pouwels SD, Zijlstra GJ, van der Toorn M, Hesse L, Gras R, Ten Hacken NH, Krysko DV, Vandenabeele P, de Vries M, van Oosterhout AJ, Heijink IH, Nawijn MC. Cigarette smoke-induced necroptosis and DAMP release trigger neutrophilic airway inflammation in mice. Am J Physiol Lung Cell Mol Physiol. 2016 Feb 15;310(4):L377-86. doi: 10.1152/ajplung.00174.2015. 

22. Rabolli V, Badissi AA, Devosse R, Uwambayinema F, Yakoub Y, Palmai-Pallag M, Lebrun A, De Gussem V, Couillin I, Ryffel B, Marbaix E, Lison D, Huaux F. The alarmin IL-1α is a master cytokine in acute lung inflammation induced by silica micro- and nanoparticles. Part Fibre Toxicol. 2014 Dec 13;11:69. doi: 10.1186/s12989-014-0069-x.

23. Rider P, Carmi Y, Guttman O, Braiman A, Cohen I, Voronov E, White MR, Dinarello CA, Apte RN. IL-1α and IL-1β recruit different myeloid cells and promote different stages of sterile inflammation. J Immunol. 2011 Nov 1;187(9):4835-43. doi: 10.4049/jimmunol.1102048. 

24. Roy R, Singh SK, Das M, Tripathi A, Dwivedi PD. Toll-like receptor 6 mediated inflammatory and functional responses of zinc oxide nanoparticles primed macrophages. Immunology. 2014 Jul;142(3):453-64. doi: 10.1111/imm.12276.

25. Shah BS, Burt KG, Jacobsen T, Fernandes TD, Alipui DO, Weber KT, Levine M, Chavan SS, Yang H, Tracey KJ, Chahine NO. High mobility group box-1 induces pro-inflammatory signaling in human nucleus pulposus cells via toll-like receptor 4-dependent pathway. J Orthop Res. 2019 Jan;37(1):220-231. doi: 10.1002/jor.24154. 

26. Suwara MI, Green NJ, Borthwick LA, Mann J, Mayer-Barber KD, Barron L, Corris PA, Farrow SN, Wynn TA, Fisher AJ, Mann DA. IL-1α released from damaged epithelial cells is sufficient and essential to trigger inflammatory responses in human lung fibroblasts. Mucosal Immunol. 2014 May;7(3):684-93. doi: 10.1038/mi.2013.87.

27. Xu J, Zheng J, Song P, Zhou Y, Guan S. IL‑33/ST2 pathway in a bleomycin‑induced pulmonary fibrosis model. Mol Med Rep. 2016 Aug;14(2):1704-8. doi: 10.3892/mmr.2016.5446.

28. Yang D, Postnikov YV, Li Y, Tewary P, de la Rosa G, Wei F, Klinman D, Gioannini T, Weiss JP, Furusawa T, Bustin M, Oppenheim JJ. High-mobility group nucleosome-binding protein 1 acts as an alarmin and is critical for lipopolysaccharide-induced immune responses. J Exp Med. 2012 Jan 16;209(1):157-71. doi: 10.1084/jem.20101354.

Relationship: 2053: Increased proinflammatory mediators leads to Increased transcription of APP

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

Acute phase response is present in vertebrate species ¹⁴. In addition, serum amyloid A, one of the major acute phase proteins, has been conserved in mammals throughout evolution and has been described in humans, mice, dogs, horses, among others ⁴.

Key Event Relationship Description

This KER presents the association between the secretion of pro-inflammatory mediators and transcription of acute phase protein in different tissues, mainly lungs and liver. The evidence of the KER presented is based on animal studies (mice), human studies and in vitro studies.

Evidence Supporting this KER

The biological plausibility is high. It is known that acute phase proteins are induced by pro-inflammatory cytokines, primary interleukin (IL)-6, IL-1β, and tumor necrosis factor α (TNF-α). These cytokines are produced a sites of inflammation mainly by monocytes and macrophages ^1-4. Following cytokine release, signaling cascades and transcription factors are activated, regulating the expression of acute phase reaction genes ³.

Immune cells are recruited to inflammatory sites by inflammatory mediators (i.e. cytokines and chemokines)⁵. Pulmonary inflammation in mice is commonly assessed as the number or fraction of neutrophils in the broncheoalveolar lavage fluid (BALF) ⁶ and can be used as indirect marker of the release of pro-inflammatory factors.

• Il-1 (IL-1α and IL-1β, 10 ng/mL each) and IL-6 (500 units/mL), both in presence of 1µM dexamethasone, increased the relative levels of serum amyloid a (SAA) mRNA in cultured human adult aortic smooth muscle cells ⁷.
• Human hepatoma cells exposed to IL-6, IL-1β and TNF-α for 20 h showed a reduced synthesis of albumin and increased synthesis of the acute phase proteins C3 and ceruloplasmin. In addition, mice exposed to IL-1β and TNF-α showed an increase of Saa mRNA in liver tissue ⁸.
• After pulmonary exposure to lipopolysaccharide (LPS) (300 µg/mL), lung tissue from female C57BL/6 mice showed upregulation of several cytokines and chemokines genes and upregulation of the acute phase proteins genes serum amyloid A and α₁-protease inhibitor ⁹.
• Mice presenting IL-6 gene disruption (IL-6^-/-) shown a reduced response in liver mRNA levels of acute phase proteins haptoglobin, α1-acid glycoprotein and SAA, after challenged by turpentine, lipopolysaccharide and bacterial infection ¹⁰.
• After repeated instillation of carbon black nanoparticles, female C57BL/6BomTac mice showed increased expression of chemokine genes along with increased Saa3 gene expression in lung tissue. In addition, there were dose-response relationships with several cytokine proteins in lung tissue¹¹.
• Intratracheal instillation of titanium dioxide in female C57BL/6 showed that 28 days after exposure, several genes of cytokines, chemokines and acute phase proteins were upregulated. Additionally, there were significant increases in inflammatory mediators in lung tissue ¹².

The table in the following link presents evidence of the relationship using neutrophil numbers in BALF as indirect measurement of the release of pro-inflammatory mediators: Empirical evidence KER2.

Quantitative Understanding of the Linkage

A Pearson’s correlation coefficient of 0.82 (p<0.001) has been calculated between log-transformed neutrophil numbers in brochoalveolar lavage fluid and log-transformed Saa3 mRNA levels in lung tissue, in female C57BL/6J mice 1 and 28 days after intratracheal instillation of metal oxide nanomaterials ¹³ (Figure 1).

Figure 1. Correlations between neutrophil numbers and Saa3 mRNA levels in lung tissue, including data from 1 and 28 days after exposure to nanomaterials. Reproduced from Gutierrez et al. (2023)¹³.

It has been shown that pro-inflammatory mediators concentrations increase before acute phase proteins:

• Upregulation of cytokine genes (IL-1α, IL-1β, IL-6 and TNF- α) was shown to peak around 2h after pulmonary exposure to LPS in female C57BL/6J mice, while upregulation SAA genes showed their highest upregulation at 8-12h after exposure ⁹.

Some acute phase proteins (f. ex. C-reactive protein, serum amyloid A and complement components) have pro-inflammatory functions, including induction of inflammatory cytokines, chemotaxis and activation of immune cells. On the other hand, other acute phase proteins present anti-inflammatory functions (f. ex. Haptoglobin and fibrinogen) as antioxidative and tissue repair inducer¹.

References

1            Gabay, C. & Kushner, I. Acute-phase proteins and other systemic responses to inflammation. N Engl J Med 340, 448-454, doi:10.1056/NEJM199902113400607 (1999).

2            Mantovani, A. & Garlanda, C. Humoral Innate Immunity and Acute-Phase Proteins. N Engl J Med 388, 439-452, doi:10.1056/NEJMra2206346 (2023).

3            Venteclef, N., Jakobsson, T., Steffensen, K. R. & Treuter, E. Metabolic nuclear receptor signaling and the inflammatory acute phase response. Trends Endocrinol Metab 22, 333-343, doi:10.1016/j.tem.2011.04.004 (2011).

4            Uhlar, C. M. & Whitehead, A. S. Serum amyloid A, the major vertebrate acute-phase reactant. Eur J Biochem 265, 501-523, doi:10.1046/j.1432-1327.1999.00657.x (1999).

5            Janeway, C., Murphy, K. P., Travers, P. & Walport, M. Janeway's immunobiology. 7. ed. / Kenneth Murphy, Paul Travers, Mark Walport. edn,  (Garland Science, 2008).

6            Van Hoecke, L., Job, E. R., Saelens, X. & Roose, K. Bronchoalveolar Lavage of Murine Lungs to Analyze Inflammatory Cell Infiltration. J Vis Exp, doi:10.3791/55398 (2017).

7            Meek, R. L., Urieli-Shoval, S. & Benditt, E. P. Expression of apolipoprotein serum amyloid A mRNA in human atherosclerotic lesions and cultured vascular cells: implications for serum amyloid A function. Proc Natl Acad Sci U S A 91, 3186-3190, doi:10.1073/pnas.91.8.3186 (1994).

8            Ramadori, G., Van Damme, J., Rieder, H. & Meyer zum Buschenfelde, K. H. Interleukin 6, the third mediator of acute-phase reaction, modulates hepatic protein synthesis in human and mouse. Comparison with interleukin 1 beta and tumor necrosis factor-alpha. Eur J Immunol 18, 1259-1264, doi:10.1002/eji.1830180817 (1988).

9            Jeyaseelan, S., Chu, H. W., Young, S. K. & Worthen, G. S. Transcriptional profiling of lipopolysaccharide-induced acute lung injury. Infect Immun 72, 7247-7256, doi:10.1128/IAI.72.12.7247-7256.2004 (2004).

10          Kopf, M. et al. Impaired immune and acute-phase responses in interleukin-6-deficient mice. Nature 368, 339-342, doi:10.1038/368339a0 (1994).

11          Jackson, P. et al. Exposure of pregnant mice to carbon black by intratracheal instillation: toxicogenomic effects in dams and offspring. Mutat Res 745, 73-83, doi:10.1016/j.mrgentox.2011.09.018 (2012).

12          Husain, M. et al. Pulmonary instillation of low doses of titanium dioxide nanoparticles in mice leads to particle retention and gene expression changes in the absence of inflammation. Toxicol Appl Pharmacol 269, 250-262, doi:10.1016/j.taap.2013.03.018 (2013).

13          Gutierrez, C. T. et al. Acute phase response following pulmonary exposure to soluble and insoluble metal oxide nanomaterials in mice. Part Fibre Toxicol 20, 4, doi:10.1186/s12989-023-00514-0 (2023).

14          Cray, C., Zaias, J. & Altman, N. H. Acute phase response in animals: a review. Comp Med 59, 517-526 (2009).

Relationship: 1589: Increased transcription of APP leads to Systemic APR

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

Acute phase response is present in vertebrate species ¹⁷. In addition, serum amyloid A, one of the major acute phase proteins, has been conserved in mammals throughout evolution and has been described in humans, mice, dogs, horses, among others ¹⁸.

Key Event Relationship Description

This KER presents the association between the transcription of acute phase protein genes in different tissues and induction of systemic acute phase response. The evidence of the KER presented is based on animal studies (mice).

Evidence Supporting this KER

The biological plausibility is high. After gene expression of acute phase proteins in tissues during inflammatory conditions, mRNA is translated and folded into proteins ¹. These proteins are then release to the systemic circulation ².

Although it is suggested that acute phase proteins are mainly produced in the liver ¹³, it has been shown that in mice, the liver has little upregulation of Saa genes after exposure to ultrafine carbon particles or diesel exhaust particle, while it is in the lung where there is a marked expression of Saa3 mRNA ^14,15.

It has been observed in some studies that the increase of Saa genes in lung or liver tissue does not translate into an increase in plasma SAA concentration ^6,8,9. This might be due to a protein concentration below the methods detection levels⁹, while measuring gene expression provides a larger dynamic range.

Quantitative Understanding of the Linkage

A Pearson’s correlation coefficient of 0.89 (p<0.001) has been calculated between log-transformed Saa3 mRNA levels in lung tissue and log-transformed SAA3 plasma protein levels, in female C57BL/6J mice 1 day after intratracheal instillation of metal oxide nanomaterials ¹⁰ (Figure 1).

Figure 1. Correlations between Saa3 mRNA levels in lung tissue and SAA3 plasma protein levels, including data from 1 day after exposure to nanomaterials. Reproduced from Gutierrez et al. (2023)¹⁰.

After exposure to titanium dioxide nanoparticles in mice, expression of Saa1 mRNA in the liver is short lasting, while expression of Saa3 mRNA in lung tissue is longer lasting, as it has been observed 28 day after exposure ¹⁶.

After exposure to multiwalled carbonanotubes, it has been observed that expression of Saa1 and Saa3 in liver and lung tissue can be elevated 28 days after exposure, however in most cases there is no increase in plasma SAA1/2 nor SAA3 levels past day 1 after exposure ⁷.

References

1            Alberts, B. Molecular biology of the cell. Sixth edition. edn,  (CRC Press, an imprint of Garland Science, 2017).

2            Van Eeden, S., Leipsic, J., Paul Man, S. F. & Sin, D. D. The relationship between lung inflammation and cardiovascular disease. Am J Respir Crit Care Med 186, 11-16, doi:10.1164/rccm.201203-0455PP (2012).

3            Bourdon, J. A. et al. Hepatic and pulmonary toxicogenomic profiles in mice intratracheally instilled with carbon black nanoparticles reveal pulmonary inflammation, acute phase response, and alterations in lipid homeostasis. Toxicol Sci 127, 474-484, doi:10.1093/toxsci/kfs119 (2012).

4            Poulsen, S. S. et al. Changes in cholesterol homeostasis and acute phase response link pulmonary exposure to multi-walled carbon nanotubes to risk of cardiovascular disease. Toxicol Appl Pharmacol 283, 210-222, doi:10.1016/j.taap.2015.01.011 (2015).

5            Poulsen, S. S. et al. MWCNTs of different physicochemical properties cause similar inflammatory responses, but differences in transcriptional and histological markers of fibrosis in mouse lungs. Toxicol Appl Pharmacol 284, 16-32, doi:10.1016/j.taap.2014.12.011 (2015).

6            Bengtson, S. et al. Differences in inflammation and acute phase response but similar genotoxicity in mice following pulmonary exposure to graphene oxide and reduced graphene oxide. PLoS One 12, e0178355, doi:10.1371/journal.pone.0178355 (2017).

7            Poulsen, S. S. et al. Multi-walled carbon nanotube-physicochemical properties predict the systemic acute phase response following pulmonary exposure in mice. PLoS One 12, e0174167, doi:10.1371/journal.pone.0174167 (2017).

8            Bendtsen, K. M. et al. Airport emission particles: exposure characterization and toxicity following intratracheal instillation in mice. Part Fibre Toxicol 16, 23, doi:10.1186/s12989-019-0305-5 (2019).

9            Hadrup, N. et al. Acute phase response and inflammation following pulmonary exposure to low doses of zinc oxide nanoparticles in mice. Nanotoxicology 13, 1275-1292, doi:10.1080/17435390.2019.1654004 (2019).

10          Gutierrez, C. T. et al. Acute phase response following pulmonary exposure to soluble and insoluble metal oxide nanomaterials in mice. Part Fibre Toxicol 20, 4, doi:10.1186/s12989-023-00514-0 (2023).

11          Erdely, A. et al. Identification of systemic markers from a pulmonary carbon nanotube exposure. J Occup Environ Med 53, S80-86, doi:10.1097/JOM.0b013e31821ad724 (2011).

12          Christophersen, D. V. et al. Accelerated atherosclerosis caused by serum amyloid A response in lungs of ApoE(-/-) mice. FASEB J 35, e21307, doi:10.1096/fj.202002017R (2021).

13          Gabay, C. & Kushner, I. Acute-phase proteins and other systemic responses to inflammation. N Engl J Med 340, 448-454, doi:10.1056/NEJM199902113400607 (1999).

14          Saber, A. T. et al. Lack of acute phase response in the livers of mice exposed to diesel exhaust particles or carbon black by inhalation. Part Fibre Toxicol 6, 12, doi:10.1186/1743-8977-6-12 (2009).

15          Saber, A. T. et al. Particle-induced pulmonary acute phase response correlates with neutrophil influx linking inhaled particles and cardiovascular risk. PLoS One 8, e69020, doi:10.1371/journal.pone.0069020 (2013).

16          Wallin, H. et al. Surface modification does not influence the genotoxic and inflammatory effects of TiO2 nanoparticles after pulmonary exposure by instillation in mice. Mutagenesis 32, 47-57, doi:10.1093/mutage/gew046 (2017).

17          Cray, C., Zaias, J. & Altman, N. H. Acute phase response in animals: a review. Comp Med 59, 517-526 (2009).

18          Uhlar, C. M. & Whitehead, A. S. Serum amyloid A, the major vertebrate acute-phase reactant. Eur J Biochem 265, 501-523, doi:10.1046/j.1432-1327.1999.00657.x (1999).

Relationship: 2860: Systemic APR leads to Atherosclerosis

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

Although atherosclerosis is mostly observed in adult humans, this condition begins early in life, and progresses through adulthood ^23,24. Children with chronic inflammation diseases have shown to develop atherosclerosis in early childhood. ^25,26. In addition, atherosclerosis is manifested in males and females ²⁷.

Key Event Relationship Description

This KER presents the association between systemic acute phase response and atherosclerosis as adverse outcome. The evidence of the KER presented is based on animal studies (mice), epidemiological studies and in vitro studies.

Evidence Supporting this KER

The biological plausibility is high. During acute phase response, serum amyloid A (SAA), one of the major acute phase proteins, replaces Apolipoprotein A-1 from high density lipoprotein (HDL). This replacement obstructs the reverse transport of cholesterol to the liver, allowing the accumulation of cholesterol in cells, denominated foam cells ^1-3. Foam cells are early markers of atherosclerotic lesions ⁴, and it has been shown that macrophages have a higher uptake of HDL containing SAA than HDL alone ².

The two major human acute phase response, SAA and C-reactive protein (CRP), have been shown to be correlated in humans ^5-7, and both are predictors of future cardiovascular event risks ⁷.

• SAA was moderately associated with angiographic coronary artery disease in women (21-86 years old) suspected on having myocardial ischemia ⁸.
• High levels of CRP were associated with an increased risk of coronary heart disease in men and women ⁹.
• In mouse model of periodontal disease, ApoE^-/- mice were infected with a polymicrobial consortium (Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia). 16-weeks after infection, the infected mice presented elevated levels of SAA in comparison to control mice, in addition of increased plaque progression ¹⁰.
• Male ApoE^-/- mice overexpressing SAA1 presented higher levels of plasma SAA and an increase in atherosclerotic lesions (plaques) than non-SAA1 overexpressing ApoE^-/- mice ¹¹.
• After one injection of adenoviral vector enconding human SAA1, ApoE^-/- mice presented elevated and transient levels of human SAA along with an increase in atherosclerotic lesions ¹².
• Overexpression of SAA3 led to increased levels of SAA3 and atherosclerosis lesions in ApoE^-/- mice in comparison to control mice. In addition, when SAA3 was suppressed in ApoE^−/− × SAA1.1/2.1-DKO (ApoE^-/- mice deficient in SAA1 and SAA2), there was a significant decreases in atherosclerotic lesions ¹³.
• In an in vitro study, Increasing concentrations of SAA (0 – 2 µM) produced a dose-response relationship of foam cells in RAW264.7 cells ¹⁴.
• Intratracheal instillation of human serum amyloid A once a week for 10 weeks in ApoE^-/- mice (on Western-type diet) induced an increase in plasma SAA3 and atherosclerotic plaque progression ¹⁵.

Mendelian randomization studies have shown that CRP genotypes are not associated with risk of coronary heart disease and that genetically elevated levels of CRP are not associated with coronary heart disease risk ^16,17.

Quantitative Understanding of the Linkage

The association between CRP and SAA levels and risk of nonfatal myocardial infarction or fatal coronary heart disease (i.e. acute events due to the progression of atherosclerosis) can be calculated from prospective, epidemiological studies ^7,9. This approach was used by the Dutch Expert Committee on Occupational Safety (DECOS) when establishing a health-based occupational exposure limit for diesel engine exhaust based on risk of lung cancer (https://www.healthcouncil.nl/documents/advisory-reports/2019/03/13/diesel-engine-exhaust).

The Nurses’ Health Study (NHS) and the Health Professionals Follow-up Study (HPFS) are prospective cohort investigations respectively involving 121,700 female U.S. registered nurses who were 30 to 55 years old at baseline in 1976 and 51,529 U.S. male health professionals who were 40 to 75 years old at baseline in 1986 ⁹. In the NHS, among women without cardiovascular disease or cancer before 1990, 249 women had a nonfatal myocardial infarction or fatal coronary heart disease between the date of blood drawing and follow-up in June 1998. In the HPFS, 266 men had a nonfatal myocardial infarction or fatal coronary heart disease between the date of blood drawing and the return of a follow-up questionnaire in year 2000.

In the NHS and HPFS studies, the associations between CRP in blood and risk of nonfatal myocardial infarction or fatal coronary heart disease for women and men were reported in Pai et al. (2004)⁹, whereas the association for both SAA and CRP in NHS was reported in Ridker et al. (2000)⁷.

The dose-response relationships are shown in Figure 1. Here, plasma levels of CRP and SAA were closely associated with future risk of coronary heart disease (CHD).

Figure 1. Association between the relative risk (RR) of CHD in NHS as function of quartiles of serum levels of CRP and SAA from Ridker et al. ⁷ and quintiles of CRP from the NHS and the HPFS studies from Pai et al.⁹. The trend lines are linear associations, as these gave the highest R² values.

According to the Danish Heart Foundation (https://hjerteforeningen.dk/alt-om-dit-hjerte/noegletal/), when a person reached the age of 55 years, the lifetime risk of a cardiovascular event is 67% in men and 66% in women. 56,379 Danes are diagnosed with a cardiovascular disease each year. Of these, 15,087 were diagnosed with are apoplexy and 16,050 with ischemic heart disease. These diagnoses are here regarded as manifestations of plaque progression. Thus, 55% of the cardiovascular diagnoses are relate to plaque progression. The lifetime risk of these diseases is thus 0.66X0.55= 0.363 = 36%.

The relative risk of 1:100 excess cardiovascular disease was calculated as

RR= (1 + 36)/36= 1.02778

The relative risk of 1:1000 excess cardiovascular disease was calculated as

RR= (1+360)/360= 1.00278

If the relative risk of 1.02778 excess is used in the equations obtained in Figure 1 and presented in the next table, it is observed that in the studies by Ridker et. al and Pai et al., 6-54% increases in blood levels of CRP or SAA were associated with 1% increased risk of cardiovascular disease.

⁽¹⁾ The biomarker level is calculated as 0.02778/slope. For example, for CRP level in women CRP = 0.02778/0.4025 = 0.07 mg/L.

Atherosclerosis is an inflammatory condition ^20,21, therefore there are increased levels of pro-inflammatory factors, including acute phase proteins, than can sustain the progression of atherosclerosis ²².

References

1            Meek, R. L., Urieli-Shoval, S. & Benditt, E. P. Expression of apolipoprotein serum amyloid A mRNA in human atherosclerotic lesions and cultured vascular cells: implications for serum amyloid A function. Proc Natl Acad Sci U S A 91, 3186-3190, doi:10.1073/pnas.91.8.3186 (1994).

2            Lindhorst, E., Young, D., Bagshaw, W., Hyland, M. & Kisilevsky, R. Acute inflammation, acute phase serum amyloid A and cholesterol metabolism in the mouse. Biochim Biophys Acta 1339, 143-154, doi:10.1016/s0167-4838(96)00227-0 (1997).

3            McGillicuddy, F. C. et al. Inflammation impairs reverse cholesterol transport in vivo. Circulation 119, 1135-1145, doi:10.1161/CIRCULATIONAHA.108.810721 (2009).

4            Libby, P. et al. Atherosclerosis. Nat Rev Dis Primers 5, 56, doi:10.1038/s41572-019-0106-z (2019).

5            Baumann, R. et al. Human nasal mucosal C-reactive protein responses after inhalation of ultrafine welding fume particles: positive correlation to systemic C-reactive protein responses. Nanotoxicology 12, 1130-1147, doi:10.1080/17435390.2018.1498930 (2018).

6            Monse, C. et al. Concentration-dependent systemic response after inhalation of nano-sized zinc oxide particles in human volunteers. Part Fibre Toxicol 15, 8, doi:10.1186/s12989-018-0246-4 (2018).

7            Ridker, P. M., Hennekens, C. H., Buring, J. E. & Rifai, N. C-reactive protein and other markers of inflammation in the prediction of cardiovascular disease in women. N Engl J Med 342, 836-843, doi:10.1056/NEJM200003233421202 (2000).

8            Johnson, B. D. et al. Serum amyloid A as a predictor of coronary artery disease and cardiovascular outcome in women: the National Heart, Lung, and Blood Institute-Sponsored Women's Ischemia Syndrome Evaluation (WISE). Circulation 109, 726-732, doi:10.1161/01.CIR.0000115516.54550.B1 (2004).

9            Pai, J. K. et al. Inflammatory markers and the risk of coronary heart disease in men and women. N Engl J Med 351, 2599-2610, doi:10.1056/NEJMoa040967 (2004).

10          Rivera, M. F. et al. Polymicrobial infection with major periodontal pathogens induced periodontal disease and aortic atherosclerosis in hyperlipidemic ApoE(null) mice. PLoS One 8, e57178, doi:10.1371/journal.pone.0057178 (2013).

11          Dong, Z. et al. Serum amyloid A directly accelerates the progression of atherosclerosis in apolipoprotein E-deficient mice. Mol Med 17, 1357-1364, doi:10.2119/molmed.2011.00186 (2011).

12          Thompson, J. C. et al. A brief elevation of serum amyloid A is sufficient to increase atherosclerosis. J Lipid Res 56, 286-293, doi:10.1194/jlr.M054015 (2015).

13          Thompson, J. C. et al. Serum amyloid A3 is pro-atherogenic. Atherosclerosis 268, 32-35, doi:10.1016/j.atherosclerosis.2017.11.011 (2018).

14          Lee, H. Y. et al. Serum amyloid A stimulates macrophage foam cell formation via lectin-like oxidized low-density lipoprotein receptor 1 upregulation. Biochem Biophys Res Commun 433, 18-23, doi:10.1016/j.bbrc.2013.02.077 (2013).

15          Christophersen, D. V. et al. Accelerated atherosclerosis caused by serum amyloid A response in lungs of ApoE(-/-) mice. FASEB J 35, e21307, doi:10.1096/fj.202002017R (2021).

16          Collaboration, C. R. P. C. H. D. G. et al. Association between C reactive protein and coronary heart disease: mendelian randomisation analysis based on individual participant data. BMJ 342, d548, doi:10.1136/bmj.d548 (2011).

17          Elliott, P. et al. Genetic Loci associated with C-reactive protein levels and risk of coronary heart disease. JAMA 302, 37-48, doi:10.1001/jama.2009.954 (2009).

18          Willeit, J. et al. Distinct risk profiles of early and advanced atherosclerosis: prospective results from the Bruneck Study. Arterioscler Thromb Vasc Biol 20, 529-537, doi:10.1161/01.atv.20.2.529 (2000).

19          Gabay, C. & Kushner, I. Acute-phase proteins and other systemic responses to inflammation. N Engl J Med 340, 448-454, doi:10.1056/NEJM199902113400607 (1999).

20          Ross, R. Atherosclerosis--an inflammatory disease. N Engl J Med 340, 115-126, doi:10.1056/NEJM199901143400207 (1999).

21          Balci, B. The modification of serum lipids after acute coronary syndrome and importance in clinical practice. Curr Cardiol Rev 7, 272-276, doi:10.2174/157340311799960690 (2011).

22          Kobiyama, K. & Ley, K. Atherosclerosis. Circ Res 123, 1118-1120, doi:10.1161/CIRCRESAHA.118.313816 (2018).

23          McGill, H. C., Jr., McMahan, C. A. & Gidding, S. S. Preventing heart disease in the 21st century: implications of the Pathobiological Determinants of Atherosclerosis in Youth (PDAY) study. Circulation 117, 1216-1227, doi:10.1161/CIRCULATIONAHA.107.717033 (2008).

24          McMahan, C. A. et al. Risk scores predict atherosclerotic lesions in young people. Arch Intern Med 165, 883-890, doi:10.1001/archinte.165.8.883 (2005).

25          Yamamura, K. et al. Early progression of atherosclerosis in children with chronic infantile neurological cutaneous and articular syndrome. Rheumatology (Oxford) 53, 1783-1787, doi:10.1093/rheumatology/keu180 (2014).

26          Tyrrell, P. N. et al. Rheumatic disease and carotid intima-media thickness: a systematic review and meta-analysis. Arterioscler Thromb Vasc Biol 30, 1014-1026, doi:10.1161/ATVBAHA.109.198424 (2010).

27          Libby, P. The changing landscape of atherosclerosis. Nature 592, 524-533, doi:10.1038/s41586-021-03392-8 (2021).

Relationship: 2958: Interaction with the lung cell membrane leads to Increased transcription of APP

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

The expression of Saa mRNA in lung and liver tissue has been shown in mice after pulmonary exposure to a variety of nanomaterials (see Empirical evidence), and in humans in different tissues as lung, liver and arteries ^13,14.

Key Event Relationship Description

This KER presents the association between the interaction of stressors with the lungs cells and transcription of acute phase proteins in different tissues, mainly lungs and liver. The evidence of the KER presented is based on animal studies (mice).

Evidence Supporting this KER

The biological plausibility is high. Production of acute phase proteins is triggered by cellular pattern-recognition molecules after sensing pathogens, tissue damage or dysmetabolism, through a cytokine cascade ¹. In the lungs, this cytokine cascade is produced by epithelial cells and resident macrophages ². In the table below it is shown that a variety of stressors produced an increase in Serum amyloid A (SAA) gene isoforms in mice tissue.

For this KER, exposure through the respiratory system (inhalation or intratracheal instillation) of stressors is considered as interaction with lung resident cell membrane components. The table in the following link presents evidence of KER: EMPIRICAL EVIDENCE KER5.

Although it is suggested that acute phase proteins are mainly produced in the liver ³, it has been shown that in mice, the liver has little upregulation of Saa genes after exposure to ultrafine carbon particles or diesel exhaust particle, while it is in the lung where there is a marked expression of Saa3 mRNA ^4,5.

In the case of nanomaterials, it has been shown that physicochemical characteristics as size, surface area, surface functionalization, shape, composition, among others, affect the magnitude and duration of the expression of acute phase proteins in mice ^6-12.

In humans, measuring gene expression of acute phase proteins is not very common as a tissue sample is needed, while measuring acute phase protein in blood in more common. However, Saa mRNA has been shown expressed in different tissues including lung, liver and arteries ^13,14.

Quantitative Understanding of the Linkage

In the case of some insoluble nanomaterials, it has been observed that log-transformed dosed surface area (dosed mass multiply by specific surface area) and log-transformed Saa3 mRNA levels in mice lung tissue presented a Pearson’s correlation coefficient of 0.70 (p <0.001) 1 day post-exposure. The linear regression formula obtained was Log Saa3mRNA = 1.080*Log Dosed surface area + 0.9415 (p<0.001)⁷ (Figure 1).

Figure 1. Correlations between dosed surface area and Saa3 mRNA levels in lung tissue, 1 day after exposure to nanomaterials. Reproduced from Gutierrez et al. (2023)⁷.

After exposure to titanium dioxide nanoparticles in mice, expression of Saa1 mRNA in the liver is short lasting, while expression of Saa3 mRNA in lung tissue is longer lasting, as it has been observed 28 day after exposure ¹⁰.

References

1            Mantovani, A. & Garlanda, C. Humoral Innate Immunity and Acute-Phase Proteins. N Engl J Med 388, 439-452, doi:10.1056/NEJMra2206346 (2023).

2            Moldoveanu, B. et al. Inflammatory mechanisms in the lung. J Inflamm Res 2, 1-11 (2009).

3            Gabay, C. & Kushner, I. Acute-phase proteins and other systemic responses to inflammation. N Engl J Med 340, 448-454, doi:10.1056/NEJM199902113400607 (1999).

4            Saber, A. T. et al. Lack of acute phase response in the livers of mice exposed to diesel exhaust particles or carbon black by inhalation. Part Fibre Toxicol 6, 12, doi:10.1186/1743-8977-6-12 (2009).

5            Saber, A. T. et al. Particle-induced pulmonary acute phase response correlates with neutrophil influx linking inhaled particles and cardiovascular risk. PLoS One 8, e69020, doi:10.1371/journal.pone.0069020 (2013).

6            Poulsen, S. S. et al. Multi-walled carbon nanotube-physicochemical properties predict the systemic acute phase response following pulmonary exposure in mice. PLoS One 12, e0174167, doi:10.1371/journal.pone.0174167 (2017).

7            Gutierrez, C. T. et al. Acute phase response following pulmonary exposure to soluble and insoluble metal oxide nanomaterials in mice. Part Fibre Toxicol 20, 4, doi:10.1186/s12989-023-00514-0 (2023).

8            Barfod, K. K. et al. Increased surface area of halloysite nanotubes due to surface modification predicts lung inflammation and acute phase response after pulmonary exposure in mice. Environ Toxicol Pharmacol 73, 103266, doi:10.1016/j.etap.2019.103266 (2020).

9            Bengtson, S. et al. Differences in inflammation and acute phase response but similar genotoxicity in mice following pulmonary exposure to graphene oxide and reduced graphene oxide. PLoS One 12, e0178355, doi:10.1371/journal.pone.0178355 (2017).

10          Wallin, H. et al. Surface modification does not influence the genotoxic and inflammatory effects of TiO2 nanoparticles after pulmonary exposure by instillation in mice. Mutagenesis 32, 47-57, doi:10.1093/mutage/gew046 (2017).

11          Hadrup, N. et al. Acute phase response and inflammation following pulmonary exposure to low doses of zinc oxide nanoparticles in mice. Nanotoxicology 13, 1275-1292, doi:10.1080/17435390.2019.1654004 (2019).

12          Danielsen, P. H. et al. Effects of physicochemical properties of TiO(2) nanomaterials for pulmonary inflammation, acute phase response and alveolar proteinosis in intratracheally exposed mice. Toxicol Appl Pharmacol 386, 114830, doi:10.1016/j.taap.2019.114830 (2020).

13          Meek, R. L., Urieli-Shoval, S. & Benditt, E. P. Expression of apolipoprotein serum amyloid A mRNA in human atherosclerotic lesions and cultured vascular cells: implications for serum amyloid A function. Proc Natl Acad Sci U S A 91, 3186-3190, doi:10.1073/pnas.91.8.3186 (1994).

14          Urieli-Shoval, S., Cohen, P., Eisenberg, S. & Matzner, Y. Widespread expression of serum amyloid A in histologically normal human tissues. Predominant localization to the epithelium. J Histochem Cytochem 46, 1377-1384, doi:10.1177/002215549804601206 (1998).

Relationship: 2959: Interaction with the lung cell membrane leads to Systemic APR

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

Systemic acute phase response measured as elevation of CRP and SAA in humans, and SAA in mice has been shown after exposure to several stressors (see Empirical evidence).

Key Event Relationship Description

This KER presents the association between the interaction of stressors with the lungs and the induction of systematic acute phase response. The evidence of the KER presented is based on animal studies (mice), controlled human studies and epidemiological studies.

Evidence Supporting this KER

The biological plausibility is high. Pulmonary inflammation occurs when stressor interact with the airways ¹ and acute phase response is induced during inflammatory conditions ². It has been shown (see table below) that exposure to different stressors produces an increase of acute phase proteins in blood [i.e. C-reactive protein (CRP) and serum amyloid A (SAA)] in humans and mice.

For this KER, exposure through the respiratory system (inhalation or intratracheal instillation) of stressors is considered as interaction with lung resident cell membrane components. The table in the following link presents evidence of KER: EMPIRICAL EVIDENCE KER6.

In the case of nanomaterials, it has been shown that physicochemical characteristics as size, surface area, surface functionalization, shape, composition, among others, affect the magnitude and duration of acute phase response in mice ^3-5.

It has been observed that in most controlled human studies, an increase in CRP and/or SAA was observed after exposure to particulate matter ^6-10. However, in other human studies the exposure did not induce acute phase response^11,12, maybe due to a low level of exposure ¹³

Quantitative Understanding of the Linkage

In the case of some insoluble nanomaterials, it has been observed that log-transformed dosed surface area (dosed mass multiply by specific surface area) and log-transformed SAA3 plasma levels in mice presented a Pearson’s correlation coefficient of 0.92 (p <0.001) 1 day post-exposure ⁴ (Figure 1). The linear regression formula obtained was Log SAA3 = 0.9459 *Log Dosed surface area – 2.854 (p=0.01). The correlation coefficient between log-transformed dosed surface area and log-transformed SAA1/2 plasma levels was 0.83 (p<0.05) and the linear regression formula was Log SAA1/2 = 0.6368 *Log Dosed surface area +0.09524 (p=0.01) ⁴ (Figure 2).

Figure 1. Correlations between pulmonary dosed surface area and SAA3 protein in plasma, 1 day after exposure to nanomaterials. Reproduced from Gutierrez et al. (2023) ⁴.

Figure 2. Correlations between pulmonary dosed surface area and SAA1/2 protein in plasma, 1 day after exposure to nanomaterials. Reproduced from Gutierrez et al. (2023) ⁴.

In mice, increased SAA levels are observed 1 and 3 days after most exposures, however increased SAA levels are not frequently observed 28 or 90 days after exposure ^3,14-17.

In humans, increased SAA and CRP has been observed 22h and 2 days after exposure to zinc oxide, but not 3 days after exposure ⁷. After exposure to zinc oxide, copper oxide or a mix both, SAA levels were elevated 24h after exposure in humans, but not 6h after exposure ¹⁰.

References

1            Moldoveanu, B. et al. Inflammatory mechanisms in the lung. J Inflamm Res 2, 1-11 (2009).

2            Gabay, C. & Kushner, I. Acute-phase proteins and other systemic responses to inflammation. N Engl J Med 340, 448-454, doi:10.1056/NEJM199902113400607 (1999).

3            Poulsen, S. S. et al. Multi-walled carbon nanotube-physicochemical properties predict the systemic acute phase response following pulmonary exposure in mice. PLoS One 12, e0174167, doi:10.1371/journal.pone.0174167 (2017).

4            Gutierrez, C. T. et al. Acute phase response following pulmonary exposure to soluble and insoluble metal oxide nanomaterials in mice. Part Fibre Toxicol 20, 4, doi:10.1186/s12989-023-00514-0 (2023).

5            Bengtson, S. et al. Differences in inflammation and acute phase response but similar genotoxicity in mice following pulmonary exposure to graphene oxide and reduced graphene oxide. PLoS One 12, e0178355, doi:10.1371/journal.pone.0178355 (2017).

6            Monse, C. et al. Concentration-dependent systemic response after inhalation of nano-sized zinc oxide particles in human volunteers. Part Fibre Toxicol 15, 8, doi:10.1186/s12989-018-0246-4 (2018).

7            Monse, C. et al. Health effects after inhalation of micro- and nano-sized zinc oxide particles in human volunteers. Arch Toxicol 95, 53-65, doi:10.1007/s00204-020-02923-y (2021).

8            Walker, E. S. et al. Acute differences in blood lipids and inflammatory biomarkers following controlled exposures to cookstove air pollution in the STOVES study. Int J Environ Health Res 32, 565-578, doi:10.1080/09603123.2020.1785402 (2022).

9            Wyatt, L. H., Devlin, R. B., Rappold, A. G., Case, M. W. & Diaz-Sanchez, D. Low levels of fine particulate matter increase vascular damage and reduce pulmonary function in young healthy adults. Part Fibre Toxicol 17, 58, doi:10.1186/s12989-020-00389-5 (2020).

10          Baumann, R. et al. Systemic serum amyloid A as a biomarker for exposure to zinc and/or copper-containing metal fumes. J Expo Sci Environ Epidemiol 28, 84-91, doi:10.1038/jes.2016.86 (2018).

11          Andersen, M. H. G. et al. Association between polycyclic aromatic hydrocarbon exposure and peripheral blood mononuclear cell DNA damage in human volunteers during fire extinction exercises. Mutagenesis 33, 105-115, doi:10.1093/mutage/gex021 (2018).

12          Andersen, M. H. G. et al. Assessment of polycyclic aromatic hydrocarbon exposure, lung function, systemic inflammation, and genotoxicity in peripheral blood mononuclear cells from firefighters before and after a work shift. Environ Mol Mutagen 59, 539-548, doi:10.1002/em.22193 (2018).

13          Andersen, M. H. G. et al. Health effects of exposure to diesel exhaust in diesel-powered trains. Part Fibre Toxicol 16, 21, doi:10.1186/s12989-019-0306-4 (2019).

14          Bourdon, J. A. et al. Hepatic and pulmonary toxicogenomic profiles in mice intratracheally instilled with carbon black nanoparticles reveal pulmonary inflammation, acute phase response, and alterations in lipid homeostasis. Toxicol Sci 127, 474-484, doi:10.1093/toxsci/kfs119 (2012).

15          Poulsen, S. S. et al. Changes in cholesterol homeostasis and acute phase response link pulmonary exposure to multi-walled carbon nanotubes to risk of cardiovascular disease. Toxicol Appl Pharmacol 283, 210-222, doi:10.1016/j.taap.2015.01.011 (2015).

16          Poulsen, S. S. et al. MWCNTs of different physicochemical properties cause similar inflammatory responses, but differences in transcriptional and histological markers of fibrosis in mouse lungs. Toxicol Appl Pharmacol 284, 16-32, doi:10.1016/j.taap.2014.12.011 (2015).

17          Hadrup, N. et al. Pulmonary effects of nanofibrillated celluloses in mice suggest that carboxylation lowers the inflammatory and acute phase responses. Environ Toxicol Pharmacol 66, 116-125, doi:10.1016/j.etap.2019.01.003 (2019).

Relationship: 3052: Increased proinflammatory mediators leads to Systemic APR

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

Acute phase response is conserved in vertebrate species ¹³.

Key Event Relationship Description

This KER presents the association between the secretion of pro-inflammatory mediators and induction of systemic acute phase response. The evidence of the KER presented is based on animal studies (mice), controlled human studies and epidemiological studies.

Evidence Supporting this KER

The biological plausibility is high. The production of acute phase proteins during acute phase response is induced by the release of pro-inflammatory markers as interleukin (IL)-6, IL-1β, and tumor necrosis factor α (TNF-α) at inflammatory sites ^1,2. The release of inflammatory markers also induces the recruitment of immune cells to inflammation sites ³.

Neutrophils in the broncheoalveolar lavage fluid (BALF) are frequently used to measure pulmonary inflammation in mice ⁴ and can be utilized as an indirect indicator of the release of pro-inflammatory factors.

Evidence of the secretion of pro-inflammatory mediators is presented as change in concentration of pro-inflammatory markers in blood, or increase neutrophil numbers in blood or BALF. The table in the following link presents evidence of KER: EMPIRICAL EVIDENCE KER8.

Wyatt et al. observed a decrease in blood neutrophil numbers in humans after exposure to ambient particulate matter although an increase in SAA and CRP was observed. It was mentioned this might be due to the translocation of neutrophil from major vessels to smaller arteries ⁵.

In the study by Meier et al., the authors obtained a negative association between PM_2.5 exposure and blood levels of TNF-α and IL-6, while SAA and CRP were positive associated with the exposure. The authors mentioned these results might be due the time point where the samples were taken ⁶.

Barregard et al. also observed that IL-6 levels were lower after exposure to wood smoke than after exposure to clean air. The discussed this response as a possible sequestering of cytokines in the pulmonary capillary bed ⁷.

Quantitative Understanding of the Linkage

A Pearson’s correlation coefficient of 0.79 (p<0.001) has been calculated between log-transformed neutrophil number in BALF and log-transformed SAA3 plasma protein levels, in female C57BL/6J mice 1 day after intratracheal instillation of metal oxide nanomaterials ⁸ (Figure 1).

Figure 1. Correlations between neutrophil numbers and SAA3 plasma protein levels, including data from 1 day after exposure to nanomaterials. Reproduced from Gutierrez et al. (2023)⁸.

A linear dose-response has also been found between log10-transformed neutrophil numbers and log2-transformes SAA3 plasma protein levels in mice, 1 day after exposure to multiwalled carbon nanotubes (Figure 2) ⁹.

Figure 2. Transformed SAA3 protein vs. transformed neutrophil influx. Reproduced from Poulsen et al. (2017) ⁹.

It has been shown that pro-inflammatory mediators concentrations increase before acute phase proteins:

• In humans patients with atherosclerotic renal stenosis, blood IL-6 increased in the first hour after renal artery stenting and reach its highest concentration at 6h, while C-reactive protein (CRP) increased 6h after the treatment, peaking at 24h after treatment ¹⁰.
• In human infants undergoing cardiopulmonary bypass, it has been observed that blood concentrations of IL-6 significantly increased after cessation of the procedure and remained elevated 24h later, while CRP started increased 6h after bypass and kept increasing at 12h and 24h after bypass ¹¹.

IL-1, IL-6 and TNF- α can decrease acute phase response by decreasing their own production through the induction of corticosteroids ¹².

References

1            Gabay, C. & Kushner, I. Acute-phase proteins and other systemic responses to inflammation. N Engl J Med 340, 448-454, doi:10.1056/NEJM199902113400607 (1999).

2            Mantovani, A. & Garlanda, C. Humoral Innate Immunity and Acute-Phase Proteins. N Engl J Med 388, 439-452, doi:10.1056/NEJMra2206346 (2023).

3            Janeway, C., Murphy, K. P., Travers, P. & Walport, M. Janeway's immunobiology. 7. ed. / Kenneth Murphy, Paul Travers, Mark Walport. edn,  (Garland Science, 2008).

4            Van Hoecke, L., Job, E. R., Saelens, X. & Roose, K. Bronchoalveolar Lavage of Murine Lungs to Analyze Inflammatory Cell Infiltration. J Vis Exp, doi:10.3791/55398 (2017).

5            Wyatt, L. H., Devlin, R. B., Rappold, A. G., Case, M. W. & Diaz-Sanchez, D. Low levels of fine particulate matter increase vascular damage and reduce pulmonary function in young healthy adults. Part Fibre Toxicol 17, 58, doi:10.1186/s12989-020-00389-5 (2020).

6            Meier, R. et al. Associations of short-term particle and noise exposures with markers of cardiovascular and respiratory health among highway maintenance workers. Environ Health Perspect 122, 726-732, doi:10.1289/ehp.1307100 (2014).

7            Barregard, L. et al. Experimental exposure to wood-smoke particles in healthy humans: effects on markers of inflammation, coagulation, and lipid peroxidation. Inhal Toxicol 18, 845-853, doi:10.1080/08958370600685798 (2006).

8            Gutierrez, C. T. et al. Acute phase response following pulmonary exposure to soluble and insoluble metal oxide nanomaterials in mice. Part Fibre Toxicol 20, 4, doi:10.1186/s12989-023-00514-0 (2023).

9            Poulsen, S. S. et al. Multi-walled carbon nanotube-physicochemical properties predict the systemic acute phase response following pulmonary exposure in mice. PLoS One 12, e0174167, doi:10.1371/journal.pone.0174167 (2017).

10          Li, J. J. et al. Time course of inflammatory response after renal artery stenting in patients with atherosclerotic renal stenosis. Clin Chim Acta 350, 115-121, doi:10.1016/j.cccn.2004.07.013 (2004).

11          Allan, C. K. et al. The relationship between inflammatory activation and clinical outcome after infant cardiopulmonary bypass. Anesth Analg 111, 1244-1251, doi:10.1213/ANE.0b013e3181f333aa (2010).

12          Uhlar, C. M. & Whitehead, A. S. Serum amyloid A, the major vertebrate acute-phase reactant. Eur J Biochem 265, 501-523, doi:10.1046/j.1432-1327.1999.00657.x (1999).

13          Cray, C., Zaias, J. & Altman, N. H. Acute phase response in animals: a review. Comp Med 59, 517-526 (2009).

Relationship: 2960: Interaction with the lung cell membrane leads to Atherosclerosis

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

Mouse models of human atherosclerosis has been shown to present atherosclerotic lesion progression after exposure to concentrated ambient particles, welding fumes and diesel exhaust particles ^3,5,7.

In humans, epidemiological studies have shown that air pollution, as a stressor that interacts with the lungs, is a risk factor for cardiovascular diseases ¹⁴.

Key Event Relationship Description

This KER presents the association between the interaction of stressors with the lungs and atherosclerosis as the outcome. The evidence of the KER presented is based on mouse models of human atherosclerosis.

Evidence Supporting this KER

The biological plausibility is moderate. Exposure to different stressors have been shown to induce the progression of atherosclerotic in mouse models of human atherosclerosis (see below). In humans, it has been hypothesized that air pollution, an example of stressor that interacts with the lungs, and cardiovascular diseases are linked by three pathways: i) translocation of inflammatory mediators from the lungs to the systemic circulation, ii) activation of alveolar receptors that results in the alteration of autonomic response and changes in cardiovascular function, and iii) translocation of particles (stressors) from the lungs to the systemic circulation ^1,2.

For this KER, exposure through the respiratory system (inhalation or intratracheal instillation) of stressors is considered as interaction with lung resident cell membrane components.

• ApoE^-/- and double knockout ApoE-/-LDLr^-/- mice exposed to concentrated ambient particles (110 µg/m³) for 6h/d, 5d/week for 5 months develop severe atherosclerosis. ApoE^-/- mice exposed to concentrated ambient particles presented a 57% increase in aortic intima surface coverage by atherosclerotic lesion than mice exposed to air ³.
• Intrapharyngeal aspiration of singlewalled carbon nanotubes into ApoE^-/- mice (20 µg/mouse every 2 weeks for 8 weeks) induced a significant increase in plaque progression ⁴.
• ApoE^-/- mice on a Western diet showed an increase in atherosclerotic lesion area after exposure to fumes from gas metal arc-stainless steel welding (40 mg/m³) for 3h/day for 10 days ⁵.
• A modest increase in atherosclerotic plaque area was observed in ApoE^-/- mice after intratracheal instillation of titanium dioxide nanoparticles (0.5 mg/kg) once a week for four weeks ⁶.
• ApoE^-/- mice, fed a Western diet and exposed to diesel exhaust particles through oropharyngeal aspiration (35 µg) twice a week for four weeks, presented an increased atherosclerotic lesions area ⁷.
• Intratracheal instillation of human serum amyloid A once a week for 10 weeks in ApoE^-/- mice (on Western-type diet) induced atherosclerotic plaque progression ⁸.

In addition, several epidemiological studies have shown that exposure to particulate matter from air pollution is associated to cardiovascular diseases:

• A prospective study in six cities from USA showed that air pollution was associated with death from cardiopulmonary diseases ⁹.
• In Dublin, there was a decrease in black smoke concentration in air, along with a significant decrease in the number of cardiovascular deaths, after the 1990 ban on coal sales ¹⁰.

ApoE^-/- mice seem to have a moderate plaque progression when feed a normal diet, instead of high-fat diet, and exposed to the stressor for a short period ⁶.

Quantitative Understanding of the Linkage

• Following the ban of coal in Dublin, a decrease of 70% of black smoke (35 μg/m³) was observed along with a 10.3% decrease (p<0.0001) in cardiovascular deaths ¹⁰.
• A prospective study following postmenopausal women from USA for 6 years observed that an increase of 10 μg of PM_2.5 (particulate matter with a diameter of less than 2.5 μm) was associated with 24% increased risk of cardiovascular event and a 76% increased risk of death from a cardiovascular disease ¹¹.
• Beelen et al. analyzed data from 22 European cohort studies on long-term exposure to air pollution and associations with cardiovascular diseases mortality. It was obtained that a PM_2.5 increase of 5 μg/m³ was associated with 21% increased risk of death from cerebrovascular disease, while an increase of 10 μg/m³ of PM₁₀ (particulate matter with a diameter of less than 10 μm) was associated with an 22% increased risk of death from cerebrovascular disease ¹².
• Results from 11 cohort studies on long-term exposure to air pollution and incidence of acute coronary events showed a 13% increased risk of coronary events associated to 5 μg/m³ increase of PM_2.5, and a 12% increased risk of coronary events associated to 10 μg/m³ increase of PM₁₀ ¹³.
• A cohort study of population living in Denmark between 2005 and 2017, and aged more than 50 years old, showed that a 5 μg/m³ increase of PM_2.5 was associated to a 22% increased risk of stroke, while an increase of 1.85 μg/m³ increase of PM_2.5 was associated to a 5.3% increased risk of myocardial infarction ^14,15.

References

1            Miller, M. R. & Newby, D. E. Air pollution and cardiovascular disease: car sick. Cardiovasc Res 116, 279-294, doi:10.1093/cvr/cvz228 (2020).

2            Van Eeden, S., Leipsic, J., Paul Man, S. F. & Sin, D. D. The relationship between lung inflammation and cardiovascular disease. Am J Respir Crit Care Med 186, 11-16, doi:10.1164/rccm.201203-0455PP (2012).

3            Chen, L. C. & Nadziejko, C. Effects of subchronic exposures to concentrated ambient particles (CAPs) in mice. V. CAPs exacerbate aortic plaque development in hyperlipidemic mice. Inhal Toxicol 17, 217-224, doi:10.1080/08958370590912815 (2005).

4            Li, Z. et al. Cardiovascular effects of pulmonary exposure to single-wall carbon nanotubes. Environ Health Perspect 115, 377-382, doi:10.1289/ehp.9688 (2007).

5            Erdely, A. et al. Inhalation exposure of gas-metal arc stainless steel welding fume increased atherosclerotic lesions in apolipoprotein E knockout mice. Toxicol Lett 204, 12-16, doi:10.1016/j.toxlet.2011.03.030 (2011).

6            Mikkelsen, L. et al. Modest effect on plaque progression and vasodilatory function in atherosclerosis-prone mice exposed to nanosized TiO(2). Part Fibre Toxicol 8, 32, doi:10.1186/1743-8977-8-32 (2011).

7            Miller, M. R. et al. Diesel exhaust particulate increases the size and complexity of lesions in atherosclerotic mice. Part Fibre Toxicol 10, 61, doi:10.1186/1743-8977-10-61 (2013).

8            Christophersen, D. V. et al. Accelerated atherosclerosis caused by serum amyloid A response in lungs of ApoE(-/-) mice. FASEB J 35, e21307, doi:10.1096/fj.202002017R (2021).

9            Dockery, D. W. et al. An association between air pollution and mortality in six U.S. cities. N Engl J Med 329, 1753-1759, doi:10.1056/NEJM199312093292401 (1993).

10          Clancy, L., Goodman, P., Sinclair, H. & Dockery, D. W. Effect of air-pollution control on death rates in Dublin, Ireland: an intervention study. Lancet 360, 1210-1214, doi:10.1016/S0140-6736(02)11281-5 (2002).

11          Miller, K. A. et al. Long-term exposure to air pollution and incidence of cardiovascular events in women. N Engl J Med 356, 447-458, doi:10.1056/NEJMoa054409 (2007).

12          Beelen, R. et al. Long-term exposure to air pollution and cardiovascular mortality: an analysis of 22 European cohorts. Epidemiology 25, 368-378, doi:10.1097/EDE.0000000000000076 (2014).

13          Cesaroni, G. et al. Long term exposure to ambient air pollution and incidence of acute coronary events: prospective cohort study and meta-analysis in 11 European cohorts from the ESCAPE Project. BMJ 348, f7412, doi:10.1136/bmj.f7412 (2014).

14          Poulsen, A. H. et al. 'Source-specific' air pollution and risk of stroke in Denmark. Int J Epidemiol 52, 727-737, doi:10.1093/ije/dyad030 (2023).

15          Poulsen, A. H. et al. Source-Specific Air Pollution Including Ultrafine Particles and Risk of Myocardial Infarction: A Nationwide Cohort Study from Denmark. Environ Health Perspect 131, 57010, doi:10.1289/EHP10556 (2023).

16          Vaduganathan, M., Mensah, G. A., Turco, J. V., Fuster, V. & Roth, G. A. The Global Burden of Cardiovascular Diseases and Risk: A Compass for Future Health. J Am Coll Cardiol 80, 2361-2371, doi:10.1016/j.jacc.2022.11.005 (2022).