Cardiovascular adaptations to particle inhalation exposure: molecular mechanisms of the toxicology

Abstract

Ambient air, occupational settings, and the use and distribution of consumer products all serve as conduits for toxicant exposure through inhalation. While the pulmonary system remains a primary target following inhalation exposure, cardiovascular implications are exceptionally culpable for increased morbidity and mortality. The epidemiological evidence for cardiovascular dysfunction resulting from acute or chronic inhalation exposure to particulate matter has been well documented, but the mechanisms driving the resulting disturbances remain elusive. In the current review, we aim to summarize the cellular and molecular mechanisms that are directly linked to cardiovascular health following exposure to a variety of inhaled toxicants. The purpose of this review is to provide a comprehensive overview of the biochemical changes in the cardiovascular system following particle inhalation exposure and to highlight potential biomarkers that exist across multiple exposure paradigms. We attempt to integrate these molecular signatures in an effort to provide direction for future investigations. This review also characterizes how molecular responses are modified in at-risk populations, specifically the impact of environmental exposure during critical windows of development. Maternal exposure to particulate matter during gestation can lead to fetal epigenetic reprogramming, resulting in long-term deficits to the cardiovascular system. In both direct and indirect (gestational) exposures, connecting the biochemical mechanisms with functional deficits outlines pathways that can be targeted for future therapeutic intervention. Ultimately, future investigations integrating “omics”-based approaches will better elucidate the mechanisms that are altered by xenobiotic inhalation exposure, identify biomarkers, and guide in clinical decision making.

INTRODUCTION

Irrespective of diet, physical fitness, or other controllable risk factors, the quality and contents of the air we breathe are often unavoidable. In industrial cities and large metropolitan areas, the most pervasive mode of exposure to aerosolized toxicants is through the ambient air. Long-term exposure to ambient air pollution contributes to 7.6% of global mortality, with 91% of the world’s population residing in places where air quality exceeds the guideline limits put forth by the World Health Organization (WHO) (30, 166, 167). The number of annual premature deaths as a result of outdoor air pollution are staggering, with projections indicating six to nine million deaths in 2060 (17, 116). Subsequently, the predicted economic costs are increasing substantially as a repercussion of the illnesses resulting from exposure (116). The importance of lowering ambient fine particulate matter exposure is highlighted specifically by the notion that if all countries met the WHO Air Quality Guidelines, life expectancy could increase by more than 7 mo (2). This becomes a daunting task with increasing urbanization (166) and implementation of nanoparticles for their advantageous physical, chemical, and biological properties in industrial, commercial, and medical sectors (131).

There are numerous factors that contribute to the diminishing air quality, including industrial sources, energy power plants, traffic, agriculture, and fires (36, 76, 137). Particulate matter (PM) is the sum of all organic and inorganic compounds dispersed in the solid and liquid phases that can be carried by air (21, 77). PM components are typically categorized by particle size, including coarse (PM₁₀; ≤10 µm in diameter), fine (PM_2.5; ≤2.5 µm in diameter), and ultrafine particles (UFP; PM_0.1; ≤0.1 µm in diameter). However, the surface area, chemical composition, number, solubility, and reactivity are some of the other characteristics that must be considered (15, 21, 102, 143). Decreasing the size of particulates from PM₁₀ to PM_2.5 increases the capacity for the material to travel further into the bronchoalveolar airways and allows for a broader interaction area (51, 170). Similarly to PM_2.5, UFP also have the capacity to penetrate deeply into the alveoli of the tracheobronchial airways (112, 145), but UFP exhibit an increased capacity for interstitialization and can transport systemically throughout the body due to their high surface-to-volume ratios (21, 39, 102, 112, 143). Because the bulk of cardiovascular effects originates from fine PM and nanoparticles (51, 139), this review will focus on PM_2.5, UFP, and engineered nanomaterials (ENM).

ENM (≥1 dimension ≤100 nm in diameter), which are similar in size to UFP, are anthropogenic materials that pose additional risk to public cardiovascular health (78, 120, 131, 141). Nanomaterials represent an ever-increasing vehicle for inhalation toxicology (81). Whether during the manufacturing of nanomaterials (81) or in consumer use (108), production and rate of application continue to rise (7, 64). Although inhalation studies examining ENMs and UFP typically differ in the number and standardization of compounds found within the mixtures, both share commonality in the nanometer scale that permits similar dispersion within the lungs during inhalation (143). Nanomaterials, similarly to UFP, exist exclusively at a biological scale that alters the dynamics of interactions with organic matter (107, 152), potentially altering toxicities compared with their micrometer-sized equivalents (21, 51, 139, 143). By integrating data from PM_2.5, UFP, and ENM inhalation exposures, the capacity to delineate unique or common mechanisms altered during multicomponent exposures can be achieved.

According to the WHO, more than four million people die every year from outdoor air pollution, with cardiovascular disease accounting for ∼40% of those deaths (2, 87, 167). Although the pulmonary system is considered the primary site of interaction with these particles, secondary target organs, such as the heart and circulatory system, may be subject to the most detrimental long-term effects. The overt epidemiological risk of cardiovascular injury following chronic and acute inhalation exposure to PM_2.5, UFP, and ENM has been well documented and includes increased propensity for ischemic heart disease, heart failure, out-of-hospital cardiac arrests, arrhythmias, atherosclerosis, and other cardiovascular complications (15, 27, 56, 123–125, 129, 147, 151, 182). The effects of PM inhalation exposure on the heart function of individuals who are susceptible to cardiovascular disease (CVD) have also gained recent interest. One of the main points of concern emanates from the increasing rate of patients with diet-induced obesity and type 2 diabetes (23). These populations with metabolic disorders can have further modifications to insulin-signaling pathways that arise from inhalation exposure, ultimately increasing susceptibility and aggravating the underlying pathophysiology (5, 14, 25, 70, 117, 119, 125).

Among those whom are most vulnerable to the repercussions of inhaled PM are those exposed at a critical point in development, such as during gestation. Although the main focus has been on the effects of toxicant inhalation exposure directly to an individual, recent studies have highlighted the importance of understanding how exposure during gestation impacts future progeny (12, 13, 50, 83, 96, 100, 103). As per the developmental origins of health and disease (DOHaD) hypothesis, a baleful gestational environment results in epigenetic alterations and has a notable influence on the health of the offspring (11, 47). Currently, there are limited studies defining the longitudinal cardiovascular effects of offspring exposed to PM during gestation and whether exposure increases susceptibility to metabolic diseases at a later point in life due to alterations in overlapping pathways (16, 68). Delineating the long-term effects of PM exposure on cardiovascular function, particularly in progeny, along with the molecular mechanisms governing predisposition to CVD, is critical for limiting premature mortality rates.

There are three prevailing theories on the mechanism by which inhaled pollutants result in deleterious effects on the cardiovascular system (lung inflammation, particle translocation, and autonomic regulation), which have been extensively reviewed elsewhere (102, 110, 112). Regardless of the initial response within the lungs, understanding the cardiovascular molecular pathways that exposure mediates is essential for identifying appropriate prophylactic and/or therapeutic strategies. In this review, we examine the molecular pathways that are implicated in cardiovascular adaptions to PM_2.5, UFP, ENM, and coexposures and attempt to provide a cohesive framework of how each of these molecular pathways has been evaluated across multiple models. Summarized findings of these molecular mechanisms for various exposures and exposure models are presented in Table 1. Furthermore, there are limited studies examining the cardiovascular consequences of gestational PM exposure on progeny. Therefore, this review will also focus on the effects of PM exposure during gestation on the cardiovascular system in progeny and highlight the molecular pathways that PM exposure alters during this critical window of fetal development. The following sections are divided into categories, with each section interweaving similar molecular pathways/responses to PM inhalation exposure.

Table 1.

Summarized recent findings

Study (Ref. No.)	Findings	Model	Exposure
PM2.5
6	Fine PM2.5 and coarse PM decreased Alu and TLR4 methylation, respectively	15 Local participants	PM2.5 collected in downtown Toronto, ON, Canada. Administered 250 and 200 μg/m3 per session
16	No changes seen in inflammatory markers related to PM2.5 exposure (monocytes, TNFα, IL-10, IL-6, IL-8, IL-1β)	25 Healthy male and female participants 18–50 yr old	PM2.5 data obtained from Dearborn, Tecumesh, and Dexter, MI. Three subacute integrated 5-day-long exposure periods in Dearborn, MI (mean concentration of 11.5 ± 4.8 µg/m−3)
18	PM2.5 exposure was associated with mtDNA D-loop methylation in peripheral blood leukocytes	48 Male participants	PM2.5 collected January 2007 to June 2012 in Boilermaker Union Local 29, Quincy, MA
29	Elemental carbon (48 genes), PM2.5 (49 genes), and organic carbon (260 genes) differentially affected the transcriptome	63 Pickup and delivery drivers and dock workers	PM2.5 collected February 2009 and October 2010 from 10 trucking terminals in the northeastern US (CT, MA, MD, NJ, NY, and PA)
32	PM2.5 increased STAT3, which promoted microRNA-21 expression, leading to decreased TIMP3 and increased MMP9	Male Sprague-Dawley rats, 6 wk old	PM2.5 collected in Beijing, China; trachea drip, 4 mg/kg body wt every 3 days for 36 days
37	PM2.5 reduced intracellular Ca2+ via decreased RYR2 and increased SERCA2a	Male Balb/c mice, 6–8 wk old	PM2.5 collected, Hebei, China; intratracheal instillation of 0.5 mg on days 0 and 2
38	PM2.5 exposure with vitamin E and ω-3 polyunsaturated fatty acid administration decreased inflammation (TNFα, IL-1β, IL-6)	Sprague-Dawley rats, 6–8 wk old	PM2.5 collected May–September 2015 in Shanghai, China; intratracheal instillation every other day for 6 days at 10 mg/kg body wt or 1.5 mL/kg body wt
42	Increased LINE-1 methylation with increasing dose of PM2.5	66 Male participants	PM2.5 collected (pre- and postwelding shift) January 2010–June 2012 in Boilermaker Union Local 29, Quincy, MA
49	PM2.5 increasing concentration was associated with decreasing levels of ET-1 (negatively) and PF-4 (positively).	15 Young, healthy, nonsmoking subjects	PM2.5 ambient air concentrations in Utah Valley from 2 monitoring sites (Lindon, North Provo) measured daily (January–March 2009); blood collections done during high (PM2.5 > 40 µg/m3), moderate (PM2.5 ∼20–40 µg/m3), and low (PM2.5 < 10 µg/m3) concentrations
54	Exercise during DE (PM2.5) exposure decreased ET-1 and increased NOx but was not modified by exercise intensity	18 Male participants	PM2.5 collected a 5.5-kW diesel engine under a constant 2.5 kW load; exposure of 300 μg/m3 over 6/30-min low- and high-intensity cycling periods
57	PM2.5 increased expression of ICAM-1, VCAM, and CRP in sedentary mice and increased VCAM in exercised mice	Male ob/ob mice, 12 wk old	PM2.5 collected in Columbus, OH; average 32 μg/m3 for 6 h/day, 5 days/wk for 9 mo through a whole body inhalation system
58	PM2.5 decreased VEGF-induced Akt/eNOS phosphorylation and circulating levels of Flk-1+/Sca-1+ cells (EPCs)	C57BL/6J male mice, 8–12 wk old	PM2.5 collected June 2009 and December 2010 in Louisville, KY; whole body exposure, 6 h/day for 4–30 days at 30–100 μg/m3
59	PM2.5 reduced insulin-stimulated Akt phosphorylation and circulating levels of Flk-1+/Sca-1+ cells (EPCs), elevated oxidative stress (SOD2, GST-P), and caused inflammasome activation (IL-1β, pro-IL-18 cleavage, activation of Casp-1)	C57BL/6J male mice, 8 wk old (control/high-fat diet), C57BL/6J male mice, 12 wk old (control diet, pre-treated with metformin/rosiglitazone	PM2.5 collected June 2009 and December 2010 in Louisville, KY; whole body exposure, 6 h/day for 9 or 30 days at 30–120 μg/m3
60	PM2.5 decreased insulin-stimulated Akt/eNOS phosphorylation and IκBα, antioxidant treatment attenuated vascular insulin resistance and inflammation	C57BL/6J male mice, 12 wk old (control/high-fat diet), pretreated with Tempol	PM2.5 collected June 2009 and December 2010 in Louisville, KY; whole body exposure, 6 h/day for 9 or 30 days at 30–120 μg/m3
75	PM2.5 increased phospho-EGFR (Tyr1068), phospho-Akt (Thr308), NLRP3, NF-κB-p52/p100, and NF-κB-p65 in heart, as well as CXCL1, IL-6, IL-18, and NLRP12 mRNA	BALB/c mice, 6–8 wk old	PM2.5 collected November 2014 in a major city in central China; intratracheal instillation of 4.0 mg/kg body wt for 5 consecutive days
89	PM2.5 decreased SOS1, CREB, GSK3b, and GRB2 expression; fucoidan treatment rescued all but CREB levels.	C57BL/6J mice, 8 wk old	PM2.5 collected November–December 2016 in Taipei, Taiwan; 100 μg/m3, 28 days, 6 h/day
91	PM2.5 caused mitochondrial size/cristae deformation (increased FIS1, MFN1, MFN2, DRP1, and OPA1), inflammation (increased TNF-α, IL-6, and IL-1β), decreased SOD, increased MDA, and iNOS	Sprague-Dawley rats	PM2.5 collected January 2013 in Taiyuan, China; intratracheal instillation of 0.375, 1.5, 6.0, and 24.0 mg/kg body wt performed 5 times
94	PM2.5 increased caspase-3, Bax, and Bcl-2 in heart and NF-κB in cardiac myocytes	Male C57/BL6 mice, 8 wk old	PM2.5 was collected in, Nanjing, China; 10 µg PM2.5 (10 µl) twice/wk per intranasal instillation.
95	Acute PM2.5 exposure decreased 5-mC methylation and DNMT1 mRNA expression in heart and blood; chronic PM2.5 exposure decreased these factors only in blood	C57BL/6J male mice, 7 wk old	PM2.5 (collection location not stated), whole body acute exposure (24 h; PM2.5 dose of 271.8 ± 86.8 µg/m3) and chronic exposure (140 days; PM2.5 dose of 271.8 ± 86.8 µg/m3)
99	PM2.5 increased inflammation; high-intensity exercise stimulated reduced inflammation (eHSP70)	Male B6.129SF2/J mice, 30 days old	PM2.5 collected in São Paulo, Brazil, 5 µg nasotropic instillation daily before exercise (12 wk)
109	DEP (PM2.5) increased PAI-1, fibrinogen, lipid peroxidation, and IL-6; Nootkatone pretreatment alleviated all and reduced thrombosis with increased Nrf2 and HO-1 activation.	BALB/C mice, 8 wk old	Diesel exhaust particles (DEP) with a geometric mean aerodynamic diameter of 215 nm from the National Institute of Standards and Technology; intratracheal instillation of 30 μg/mouse
118	PM2.5 exposure in atherosclerosis model increased MDA and NOX4 subunits (p22phox, p47phox) in cardiac tissue	Male ApoE−/− Tg mice, 8 wk old	PM2.5 collected June–October 2013 in Shanghai, China; intratracheal installation at 0–30 mg/kg body wt
122	PM2.5 exposure was associated with elevated levels of CD14+, CD16+, CD4+, CD8+, endothelial microparticles (annexin V+/CD41−/CD31+), antiangiogenic (TNFα, IP-10) and proinflammatory cytokines (MCP-1, MIP-1α/β, IL-6, IL-1β), and sICAM-1 and sVCAM-1, as well as decreased proangiogenic growth factors (EGF, sCD40L, PDGF, RANTES, GROα, and VEGF)	72 Young, healthy, nonsmoking subjects (24 individuals per 3 winter/spring time periods (2013, 2014, and 2015))	PM2.5 ambient air concentrations in Utah Valley from 3 monitoring sites (Lindon, North Provo, and Spanish Fork) measured daily; at Lindon and North Provo sites, hourly PM2.5 concentrations were used to estimate the average concentration for the ∼24-h period before each blood draw
126	PM2.5 increased fibrosis (Col1a1, Col3a1), oxidative stress (NOX4), and transcription factor binding (TGFβ1, SMAD3)	Female C57BL/6, 10 mo and 4 wk old	PM2.5 collected in 2012 and 2013 from Taiyuan, Northern China; 3 mg/kg body wt PM2.5 oropharyngeal aspiration every other day for 4 wk
128	CAPs exposure increased 7-KCh in plasma and aortic plaques and increased CD36 expression in plaque macrophages and CD36 uptake of oxidized lipids	ApoE−/− Tg and LDLR−/− Tg mice, 8 wk old	Concentrated ambient PM2.5 collected in Columbus, OH; mice exposed for 6 mo to 9.1 ± 7.3 μg/m3
130	12.4% Decrease in RHI for every 10 µg/m3 increase in PM2.5; IsoP, angiopoietin 1, VEGF, PlGF, MMP-9, and ICAM-1 positively associated with 10 µg/m3 increases in PM2.5, decreased VCAM-1	100 Male and female participants (44% had type 2 diabetes; 52% had diagnosis of hypertension)	PM2.5 levels obtained from 5 monitoring stations in Jefferson County, KY (June 2011–May 2013) with average concentration of 11.45µg/m3
135	PM2.5 increased Hsp-70, HO-1, and MPO in heart as well as laminin, collagen, and calcium signaling proteins (181 upregulated, 178 downregulated genes)	Male BALB/c mice, 7–8 wk old	PM2.5 with high PAH, nitrite, and organic carbon levels collected during winter 2008 in an urban site (Milano, Italy); intratracheal instillation on days 0, 3, and 6, at 0.3 mg/mouse
152	TRPA1 activation increases negative cardiovascular responses to CAPs exposure through myocardial dyssynchrony	Trpa1tm1Kykw/J (Trpa1−/−) Tg female mice	PM2.5 collected November–December of 2014 in Triangle Park, NC; 3 h/day, 2 days/wk, 8 total whole body exposures
156	PM2.5 exposure increased oxidative stress (PRNP) and endoplasmic reticulum stress (GRP78). Nanoparticles found in myocardial ER and in abnormal mitochondria	30 Children and young adults (∼20 yr old)	PM2.5 data obtained in Mexico City Metropolitan Area 1997–2012
160	PM2.5 increased fibrosis and collagen genes, and oxidative stress (8-OHdg and 4-HNE) reduced antioxidant genes (PRDX5)	Male AMPK α2−/− Tg mice, 6–8 wk old	PM2.5 collected April–October 2016 in Beijing, China; intratracheal instillation for 6 mo at 10 mg/kg (64 µg/m3).
162	PM2.5 exposure in hyperlipidemic rats increased CRP, JNK, and P53 phosphorylation, increased Bax and caspase-3 in the heart, and decreased SOD	Male Wistar rats, 8 wk old, on high-fat diet	PM2.5 collected October–December 2012 in Beijing, China; endotracheal instillation of suspension at 0, 4, and 40 mg/kg
165	Higher PM2.5 resulted in myosin heavy chain isoform switch (increased β-MHC) and decreased SERCA2a	C57BL/6 male mice, 8 wk old	Concentrated PM2.5 in Columbus, OH; mean daily concentration of PM2.5 in the exposure chamber was 85.3 μg/m3; mice were exposed for 6 h/day, 5 days/wk, for 9 mo.
168	PM2.5 increased MDA, iNOS activity, TNFα, IL-1β, along with NOX4 and NOX subunits as p67phox, p47phox, and p22phox in the heart and reduced SOD activity	C57BL/6 mice, 6–8 wk old	PM2.5 collected September 2013 in Zhengzhou, Taiwan; instilled with 1.5, 3.0, 6.0 mg/kg body wt 5 days/wk for 2 wk
176	PM2.5 decreased GSH-Px, increased MDA and sICAM-1; SeY treatment reduced inflammation markers (TNFα, IL-1β, sICM-1)	Male Sprague-Dawley rats, 7 wk old	PM2.5 collected September–November 2015 in Shanghai, China; intratracheal instillation at 40 mg/kg
180	PM2.5 exposure in diabetic model induced NF-κB, COX-2, MAPK in heart, IκB inhibitor restored function through NF-κB reduction	KKay Tg mice, 7 wk old	PM2.5 collected Polaris in Columbus, OH; intratracheal instillation for 6 h/day, 5 day/wk, 8 wk at dose of 1.6 mg/kg
181	PM2.5 increased DNA damage (increased OGG1 and GADD153, decreased MTH1and XRCC1) in the heart and decreased SOD	Male Wistar rats	PM2.5 containing PAHs collected Taiyuan, China; intratracheal instillation of 1.5 mg/kg body wt PM2.5, 1.6 × 10− 4 mg/kg body wt 1-NP, 1.2 × 10− 4 mg/kg body wt 9-NA
183	Higher PM2.5 correlated with increased blood TLR2 methylation, flavonoid intake decreased TLR2 methylation	Normative Aging Study, 573 participants	PM2.5 data collected from Harvard School of Public Health
Gestational
26	PM2.5 exposure increased GATA4, NKX2–5, and inflammatory markers (TNFα and IL-1β); homocysteine exacerbates these effects	Female Sprague-Dawley rats, 10 wk old, neonatal progeny	PM2.5 collected Fujian, China; exposed during gestation and lactation (∼42 days) to 36.5 μg/m3 through a whole body inhalation system
55	DE PM2.5 caused promoter methylation of microRNA-133a-2 in progeny, and cardiovascular stress decreased microRNA 133a-2 expression	Female C57BL/6 12–14 wk old, 12-wk-old progeny	PM2.5 collected a single cylinder Yanmar diesel engine, operating at 82% load ∼300 μg/m3 for 6 h/day for a total of 5 days during pregnancy
148	PM2.5 preconception exposure increased inflammatory markers (IL‐6, IL‐15, NF-κB, CRP, CD26E, CD26P, VCAM-1, and MCP-1), fibrosis (Col3a1), Ca2+ regulation (SERCA2a, p‐PLN), and altered epigenetics (DNMT1↓, SIRT1↑, SIRT2↑)	Paternal and maternal FVB mice exposure; male 12 wk-old progeny	PM2.5 collected in Columbus, OH; average 38.58 μg/m3 for 6 h/day, 5 days/wk, for 3 mo through a whole body inhalation system preconception
149	PM2.5 induced changes in calcium dynamics by decreasing NCX and CaV1.2	Female FVB mice, 12 wk old, 14-day-old progeny	PM2.5 collected in Columbus, OH; exposed 6 h/day, 5 days/wk, average 91.78 μg/m3, through a whole body inhalation system during pregnancy
150	PM2.5 decreased SIRT1 and SIRT2, increased DNMT1, DNMT3a, and DNMT3b, decreased Ca2+ markers (NCX, p-PLN, and SERCA2a), and increased IL-1 β, IL-6, Col-1, MMP9, and MMP13A	Female FVB mice, 12 wk old, 12-wk-old progeny	PM2.5 collected in Columbus, OH; 6 h/day, 7 days/wk, throughout pregnancy, average of 73.61 μg/m3 through a whole body inhalation system
159	PM2.5 caused a dose-dependent increase of OPA1, MFN1, DRP1, and FIS1 in progeny.	Female Wistar rats, 10–12 wk old, 1-day-old progeny	PM2.5 collected September 2014 to March 2016 in Harbin, China; administered dropwise, 3 doses (0.375 mg/kg, 1.5 mg/kg, and 6.0 mg/kg) during pregnancy
169	PM2.5 increased histone acetyltransferase (H3K9ac), GATA4, and MEF2c; exposure also increased histone acetylation of GATA4/MEF2c promoters	Female C57BL/6 mice, 12 wk old; 1-day- and 16-wk-old progeny	PM2.5 collected in Chongqing, China; gestational exposure by ultrasonic nebulization, 2 h/day during pregnancy, at ∼300 µg/m3
UFP
4	UFP exposure increased AT1R protein along with Acta1 and Col3a1 in the heart, with decreased HO-1 expression	Male Sprague-Dawley rats	UFP collected north of Mexico City (May–July 2009); acute (3 days, 5 h/day) and subchronic (8 wk, 4 days/wk, 5 h/day) exposure to varying concentrations
35	UCAP exposure decreased blood plasminogen and thrombomodulin and increased CRP and SAA; GSTM1-null participants had worse outcomes	34 Participants with metabolic syndrome, ∼48 yr old	UCAPs collected at EPA facility in Chapel Hill, NC; whole body exposure for 2 h at average particle concentration of 189,000 particles/cm3
66	UFP exposure cause mitochondrial mPTP deficits, cyclosporin A resulted in attenuation	Male Sprague-Dawley rats	UFP collected at EPA facility in Chapel Hill, NC; 100 μg of UFP laryngopharynx instillation
86	TAA-PNC of UFP was positively associated with CRP and TNFR2 in white non-Hispanic participants	408 individuals aged 40–91 yr old	UFP (near-highway, long-term exposure), Dorchester, South Boston, Chinatown (Boston, MA)
142	Elemental carbon UFP exposure increased platelet CD40L and vWF and decreased circulating CD40L	19 type 2 diabetics, 30–60 yr old, never smoked	UFP particles were generated at 32 nm; elemental carbon UFP through mouthpiece for 2 h at 50 μg/m3
154	UFCP exposure increased CRP and fibrinogen in systemic circulation	Male spontaneously hypertensive rats (SHRs), 12–13 mo old	UFCP exposure at 180 µg/m3 in a 24-h time frame
178	Increasing UFCP concentrations prevented the beneficial effects of captopril in reducing ANG II	Spontaneously hypertensive rats (SHRs), 10 wk old	UFP collected in Beijing, China; intratracheal instillation of UFCP (0.15 mg/kg, 0.45 mg/kg, and 1.35 mg/kg) and captopril administration
Gestational
104	UFP exposure induced placental HSD11B2 DNA methylation, elevated IL-1β, IL-6 and MCP-1, and caused increased activation of AT1R and ACE	Female C57BL/6J pun/pun mice, 6 wk old; gestational day 17.5 progeny	UFP collected in northern Mexico City, Mexico (April–June 2016); intratracheal instillation of 12 μg or 400 μg/kg during pregnancy
134	UFP exposure caused higher serum levels of IL-10 in C67BL/6 progeny	Female C57BL/6 and BALB/C mice; progeny 0–4 wk old	UFP generated with average mass concentration of 101.94 µg/m3; 24-h daily mean dose of 25 µg/m3 during pregnancy
Engineered nanomaterials and advanced materials
52	Carbon nanoparticle inhalation, compared with intra-arterial injection, was more detrimental to cardiac tissue with perturbation of inflammatory and endothelial/epithelial pathways	Female BALB/cJ mice, 10–12 wk old	Carbon nanoparticles with primary particle diameter of 10 ± 2 nm; administered through intra-arterial infusion (30 mm2/animal) and inhalation (lung deposited dose 10,000 mm2). Whole body exposure for 4 or 24 h
61	Nano-TiO2-exposed microRNA-378a Tg mice had higher MFN1 levels (fusion) and preserved metabolic profiles and ultrastructure in the heart	FVB and miRNA-378a knockout Tg mice aged 14–18 wk old	Nano-TiO2, particle size (21 nm)/surface area (48.08 m2/g);’ whole body inhalation exposure at a dose of 11.09 mg/m3 for 4 h
67	Nano-TiO2 exposure increased inflammatory markers (NF-κB, TNF-α, IL-4, IL-6, TGF-β, CK, CRP, and IFN-γ) and transcriptional regulators (STAT1, STAT3, STAT6, GATA3, and GATA4)	CD-1 (ICR) male mice, 4 wk old	Nano-TiO2 prepared through hydrolysis of Ti(OBu)4; administered via nasal instillation of mice every other day for 6 mo; treatment of 1.25–5 mg/kg TiO2 NPs
73	Nano-TiO2 exposure caused blood-specific activation of C3 and heart-specific activation of the complement cascade	Female C57BL/6 mice, 8 wk old	Nano-TiO2, UV-Titan L181; 6.0 mg of Ti/m3 – 10 mg TiO2/m3 administered through intratracheal instillation
111	Nano-TiO2-exposed mPHGPx Tg mice had reduced ROS and preserved electron transport chain complex constituents in the heart	Male FVB and male mPHGPx Tg mice, 10–12 wk	Nano-TiO2, particle size (21 nm)/surface area (48.08 m2/g); 11.58 ± 0.27 μg lung deposition, whole body inhalation exposure for 6 h
Gestational
62	Nano-TiO2 exposure increased cardiac mitochondrial proton leak (UCP2) and fatty acid metabolism (increased CPT1A and PDH phosphorylation)	Female Sprague-Dawley rats 10–12 wk,12-wk-old progeny	Nano-TiO2, particle size (21 nm)/surface area (48.08 m2/g); 7.79 ± 0.26 days, 5 h/day, 10.35 ± 0.13 mg/m3 through a whole body inhalation system during pregnancy
83	Nano-TiO2 exposure increased ROS (H2O2), global 5-mC methylation, DNMT1 protein expression, and HIF-1α activity and downregulated mPHGPx in the heart	Female FVB 12, wk old; gestational day 15 fetal progeny, 11-wk-old adult progeny	Nano-TiO2, particle size (21 nm)/surface area (48.08 m2/g); whole body inhalation exposure at a dose of 12.09 ± 0.26 mg/m3 for 6 days (over an 8-day period), 6 h/day, starting from gestational day 5
138	Nano-TiO2 exposure resulted in cardiac mitochondrial epigenomic remodeling (increased H3K4Me3) and decreased transcriptomic immune response	Female Sprague-Dawley rats 10–12 wk old; gestational day 20 fetal progeny	Nano-TiO2, particle size (21 nm)/surface area (48.08 m2/g); whole body inhalation exposure at a dose of 10.35 ± 0.13 mg/m3 for 7.79 ± 0.26 days during gestation
140	Nano-TiO2 exposure decreased state 3 and State 4 mitochondrial respiration	Female Sprague-Dawley rats, female 11- to 16-wk-old progeny	Nano-TiO2, particle size (21 nm)/surface area (48.08 m2/g); whole body inhalation exposure at a dose of 10.6 ± 0.3 mg/m3 for 6.8 ± 0.5 days during gestation
Multicomponent exposure
9	Higher PM2.5 BC and mass PM2.5 exacerbated hypomethylation of IFNγ and ICAM-1	Normative Aging Study, 777 elderly men	PM2.5 mass concentration and PM2.5 BC concentration measured hourly at the Harvard supersite, Boston, MA
10	Higher black carbon concentration associated with 12% reduction in F3 methylation	Normative Aging Study, 777 elderly men	PM2.5, BC, So4, and ozone concentrations of ambient air collected from Boston, MA, were measured hourly
31	Increases in PM2.5, BC, and UFP concentrations were positively associated with inflammation (CRP and fibrinogen) and coagulation, but not endothelial dysfunction; Delta-C increasing concentrations associated positively with fibrinogen and negatively with MPO	135 Patients undergoing cardiac catheterization	Ambient air pollution, ΔC (wood smoke), PM2.5, BC, and UFP collected from New York State Department of Environmental Conservation (DEC) site in Rochester, NY
41	PM2.5 levels, but not UFP, were positively associated with hsa-miR-197-3p and hsa-miR-99a-5p	59 volunteers (20 were healthy, 20 had COPD, and 19 had ischemic heart disease)	Ambient air pollution exposure (personal exposure level measurements of PM10 and PM2.5, UFP, nitrogen oxides, BC, and CO) during 2-h walk; Oxford Street or Hyde Park, London, UK
46	SiNP and Pb coexposure elevated markers of heart failure (ANP and BNP) and inflammation (CRP, IL-6, and TNFα) in heart and increased ANG II and ET-1 in serum; T-PA, TFPI, and ATIII decreased whereas fibrinogen and D2D increased with coexposure	Sprague-Dawley rats, 6 wk old	Stöber technique was used to synthesize SiNPs; intratracheal instillation of 2 mg/kg body SiNPs and/or 0.25 mg/kg of lead acetate (Pb) for 30 days
80	54 Circulating miRNAs were correlated with dose of exposure and pollution type	24 Nonsmoking adults	PM2.5, NO2, UFP, BC, and PM10 collected on Oxford Street in London, 2-h exposure
82	UFP, CO, and So2 exposure was associated with increased inflammatory markers (IL-6, IL-8, ET-1) in blood	52 men and women 18–34 yr old	UFP, CO, and So2 concentrations collected in Sault Ste. Marie, ON, Canada (Summer 2010); occupational air pollution
92	PM2.5 and So4 increases associated with CRP levels, NOx positively associated with IL-6, BC, sulfate, ozone positively associated with TNFR2	3,996 non-smoking participants Framingham Offspring cohort cycle 7 and 8 and Third Generation cohort 1	PM2.5, BC, $\text{S}{\text{O}}_4^{2-}$, nitrogen oxides, and ozone collected at the Boston Harvard Supersite and assessed as 7-day moving average
93	Higher PM2.5 and BC concentrations were associated with lower P-selectin levels	3,820 nonsmoking participants; Framingham Offspring cohort cycles 7 and 8 and third-generation cohort 1	PM2.5, BC, $\text{S}{\text{O}}_4^{2-}$, nitrogen oxides, and ozone collected at the Boston Harvard Supersite and assessed as 7-day moving average
97	Nickel, barium, and silver levels were positively correlated with VEGF, UCHL1, and cortisol	53 Healthy participants, 18–60 yr old	Particulate matter collected in downtown Toronto, ON, Canada; 130-min exposure to PM10 (213 µg/m3), PM2.5 (238 µg/m3), and/or concentrated UFP (213 µg/m3)
98	Higher BC is associated with decreased methylation of Alu, and both increased So4 and BC resulted in decreased methylation of LINE-1	Normative Aging Study, 706 individuals	PM2.5, BC, and So4 data collected from the Harvard School of Public Health from January 1995 to November 2007
105	PM2.5 concentration was positively associated with 69 differentially methylated regions and 13 CpG sites, including altered methylation of KNDC1 and FAM50B; UFP was also associated with 15 differentially methylated regions	157 Nonsmoking adults	PM2.5 and UFP levels collected in personal and ambient air pollution, measured in 24-h intervals, part of the EXPOsOMICS project conducted in 4 European countries (December 2013–February 2015)
113	BC was associated with sVCAM-1, sICAM-1, and vWF; PM2.5 was associated with sVCAM-1 and sICAM-1 in patients not taking statins; in smokers, PM2.5 and BC were positively associated with sVCAM-1	60 Male and 37 female participants with type 2 diabetes (smokers and nonsmokers)	PM2.5 (1998–2002) and BC (1999–2002) measured hourly and $\text{S}{\text{O}}_4^{2-}$ measured daily (1999–2002) at the Harvard School of Public Health (Boston, MA)
127	PM2.5 mass concentration shower positive trend with HDL-oxidant index (HOI); changes not seen with ozone coexposure;	30 participants, 19–37 yr old	PM2.5 (149 µg/m3), ozone (221 µg/m3), and PM2.5 and ozone data collected from Toronto, Canada as part of Clean Air Research Center Project.
153	PM2.5 and quasi-ultrafine particles <0.50 µm dysregulated parasympathetic response and increased IFNγ methylation in blood	12 Healthy participants	Particulate matter collected in Milan, Italy. Inhalation to mixture (PM10, PM2.5, PM1.0, and PM0.5µm) over 2 sessions
155	PM2.5 exposure was associated with higher inflammation (CRP) and coagulation (platelet count in blood)	3,275 Participants	Ambient air pollution PM10 and PM2.5 levels from the German Heinz Nixdorf Recall Study, 3 adjacent cities (Essen, Mülheim, and Bochum, Germany)
158	Coexposure potentiated increases in CRP, IL-6, CK, LDH, and MDA and decreased SOD	Male Wistar rats (age not defined)	PM2.5 (varying doses), ozone (0.81 ppm), PM2.5, and ozone collected from June to October 2011 in Shanghai, China, administered as whole body inhalation exposure (ozone) and intratracheal instillation (PM2.5)
164	BC, CO, NOx, and PAHs positively associated with IL-6 and TNFα, and quasi-ultrafine particles <0.25 µm were also associated with IL-6; associations were stronger for haplotype H than haplotype U	36 Participants, ∼84 yr old	Particulate matter (PM-varying sizes), nitrogen oxides (NOx), carbon monoxide (CO), and organic (OC) and elemental carbon (EC), collected from 4 retirement communities in the Los Angeles, CA, air basin
171	SiNP and MeHg coexposure caused cardiac injury through changes in serum biomarker activity, including increased SERCA2, cTnT, ANP, and BNP	Sprague-Dawley rats, 6 wk old	Silica nanoparticles (SiNPs) and methylmercury (MeHg) administered through intratracheal instillation 10 times over 30 days at doses of 0.25 mg/kg (MeHg) and 2 mg/kg (SiNPs)
179	C-exposure (PM2.5, So2, and NO2) causes endothelial dysfunction (increased ET-1 and decreased eNOS) and inflammation (increased COX-2, iNOS, TNFα, and IL-6)	Male C57BL/6 mice, 6–8 wk	PM2.5, So2, and NO2, collected in Taiyuan, China; intranasal instillation every other day for 6 h/28 days; 0.5/3.5 mg/m3 So2, 0.2/2 mg/m3 NO2, 1/10 mg/kg PM2.5

Summarized findings of the molecular mechanisms contributing to cardiovascular complications following particle inhalation exposure. Studies are organized by exposure (PM_2.5, UFP, ENM, and multicomponent), including subsections for gestational and preconception exposures. All abbreviations are defined in glossary.

CARDIAC REMODELING

Whereas inhalation exposure directly impacts the lungs, the subsequent downstream adaptations of the heart can lead to sustained cardiac remodeling. This remodeling can be presented in the form of fibrosis, hypertrophy, and other structural changes to both the cardiomyocyte and the surrounding connective tissue of the heart. In understanding how this process occurs, examining the biochemical signature linked to cardiac remodeling changes associated with toxicant inhalation exposure can provide insight for future investigations into prevention and treatment. The reported cardiovascular adaptations that occur as a result of toxicant inhalation exposure are depicted in Fig. 1.

Fig. 1.

Summary of the cardiovascular changes following particulate inhalation exposures and the associated studies detailing these findings. Dia, diastolic; LV, left ventricular; MPI, myocardial performance index; Sys, systolic.

Cardiac fibrosis involves the imbalance of extracellular matrix production and degradation, which results in accumulation of scar tissue. Therefore, cardiac fibrosis, which occurs as a result of PM exposure, decreases compliance, impairing the ability of the heart to contract and relax properly. PM_2.5 exposure results in elevation of several genes linked to collagen deposition [collagen type I α1 (Col1a1) and collagen type III α1 (Col3a1) and the fibrogenic growth factor transforming growth factor-β1 (TGFβ1)] (126). Simultaneously, NADPH oxidase (NOX4) is increased, matching the resultant cardiac dysfunction and prooxidative environment found in the heart as a result of PM exposure. Of note is that Qin et al. (126) also further demonstrates that the effects of PM_2.5 are worse in older mice, making them more susceptible to prolonged cardiac systolic dysfunction marked by decreased ejection fraction and increased E/E′ ratio when compared with juveniles. PM_2.5-induced fibrosis is also related to cardiac inflammation in the hearts of mice, which show elevated expression of inflammatory genes, including interleukin (IL)-6, IL-18, and C-X-C motif chemokine ligand 1 (CXCL1) (75). Inflammation and fibrosis in the heart may be attributed to increased phosphorylation and, therefore, activation of epidermal growth factor receptor (EGFR)/Akt signaling and increased expression of nuclear factor-κβ (NF-κB) following inhalation exposure. NF-κB expression in the heart is likewise increased as a result of PM_2.5 exposure in diabetic mice, along with cyclooxygenase-2 (COX-2) and mitogen-activated protein kinase (MAPK), indicating high oxidative stress and inflammation (180). Intracerebroventricular injection of an inhibitor of IκB kinase-2 (IKK) analog (IMD-0354), which regulates the nuclear translocation of NF-κB, can remediate the inflammatory response that occurs following PM exposure. Whereas NF-κB plays an important role in the cardiovascular effects of PM inhalation exposure, other inflammatory markers contribute to cardiac remodeling. Cardiac injury and inflammation that occurred following PM_2.5 exposure were attenuated with supplementation of vitamin E and ω-3 polyunsaturated fatty acids before the exposure in rats by decreasing expression of tumor necrosis factor-α (TNFα), IL-1β, and IL-6 (38). In addition to TNFα and IL-1β, PM_2.5 exposure also induces soluble intercellular adhesion molecule 1 (ICAM-1) (176). Together, these inflammatory markers can form a positive feedback loop with NF-κB, amplifying PM-induced cardiac fibrosis and remodeling. Selenium yeast (SeY) supplementation can similarly serve as a pretreatment protective strategy, as reported in Sprague-Dawley rats that were subsequently exposed to PM_2.5 (176). PM-exposed rats that were administered SeY pretreatment had lower levels of proinflammatory markers and significantly higher total antioxidant capacity, total superoxide dismutase (SOD), and total glutathione peroxidase (GSH-Px) compared with the PM_2.5-exposed group without pretreatment (176). The ability of antioxidant therapy to thwart the perpetuation of inflammatory signaling associated with cardiac remodeling highlights the essential role of oxidative stress in the development of cardiac pathology following ambient air exposure. Understanding the proteins involved in the regulation of reactive oxygen species (ROS) may provide insight into long-term remodeling of the heart. Whereas most population-derived analyses assess cardiovascular impact in blood samples, a study of the effects of UFP urban air pollution used postmortem ventricular autopsies of 30 children and young adults to substantiate that early and prolonged cardiac stress can result in irreversible consequences (156). Compared with clean air controls, long-term UFP exposure results in significant upregulation of proteins involved in oxidative [prion protein (PRNP)] and endoplasmic reticulum (ER) [glucose regulated protein 78 (GRP78)] stress. These response markers of stress were disproportionately upregulated in the left ventricle compared with the right ventricle. Along with abnormal left-ventricular histopathology, increased oxidative and ER stress markers suggest a compensation by the heart to attenuate the inflammatory effects of air pollution in response to UFP exposure (156). GRP78 elevation in cardiomyocytes is sufficient to promote myocyte growth, potentially through stimulation of cardiac-specific transcriptional factor GATA sequence-binding protein 4 (GATA4) (177). Similarly to UFP, nano-TiO₂ exposure in outbred mice can lead to an increase in cardiac lesions and a significant change in inflammation markers, including changes in transcription factors [signal transducer and activator of transcription (STAT) 1/3/6 and GATA 3/4] in the heart (67). Therefore, the ability for oxidative and inflammatory proteins to activate transcription factors such as GATA4 is notable, particularly following UFP/ENM exposure, as they appear to have strong correlations with cardiac hypertrophy. Fine PM exposures can also lead to sustained detrimental effects in the heart by altering the transcription factor profile. One example of this has been shown previously in a cohort of PM_2.5-exposed mice (89). Cardiac remodeling through hypertrophy, QT interval prolongation, and fibrosis were correlated with elevated cAMP response element-binding protein (CREB) as well as SOS Ras/Rac guanine nucleotide exchange factor 1 (SOS1) and glycogen synthase kinase-3β (GSK3β) expression. Measuring biomarkers associated with myocardial infarction can also provide information on the extent of cardiac tissue dysfunction and death. An examination of the cardiovascular effects of a coexposure model using silica nanoparticles (SiNP) and methylmercury (MeHg) has shown increases in myocardial edema, myocardial gap expansion, and myofibril disorder as well as increases in activities of myocardial enzymes, including cardiac troponin T (cTnT), atrial natriuretic peptide (ANP), and brain natriuretic peptide (BNP) compared with the serum of single-exposure and sham-exposed rats (171). These authors also report increased sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA2), a potential marker of endoplasmic reticulum stress (65), in the coexposure cohort along with oxidative stress. Similar to the preceding studies mentioned, coexposures can induce cardiac remodeling by increasing oxidative and ER stress and elevating prominent markers of heart failure. PM exposures, including UFP, ENM, and multicomponent exposure, all have the ability to alter the transcription factor profile in the heart and lead to sustained detrimental effects on cardiac structure. The molecular adaptations to particle exposures that result in cardiac remodeling center around a theme of inflammation and oxidative stress, which may ultimately be regulated by a greater transcriptional and epigenetic adaptation following toxicant inhalation exposure.

CARDIAC HEMODYNAMICS

The performance of the heart can highlight both the acute and chronic effects of toxicant inhalation exposure. Although cardiac remodeling provides an informative diagnostic standard for assessing the impacts of PM exposure, more immediate changes are captured in measures of heart function. Whether there is a sustained or transient perturbation to the heart is likely determined by the molecular cascades involved, such as transcriptional or epigenetic reprogramming.

Controlled studies on human exposure to PM allow for the most relevant simulation of pollutant exposure while also limiting interference of confounding factors. Measurements of blood methylation levels in 15 healthy participants revealed that both fine and coarse concentrated ambient particle (CAP) exposures induce hypomethylation of the Alu-repetitive transposable element and Toll-like receptor 4 (TLR4) (6). Decreased methylation of the short, interspersed nucleotide element Alu and TLR4 was also associated with higher systolic blood pressure postexposure, which is similar to previous findings that have shown a role for hypomethylation in atherosclerosis (22). Other studies have reported similar findings with specific components of PM_2.5, including with black carbon (BC). Over a 90-day period, increased BC levels correlated with decreased methylation of Alu, whereas both carbon black and sulfate (So₄) were associated with decreased methylation of long interspersed nuclear emlement-1 (LINE-1) (98). Interestingly, in a study examining occupational exposure to PM_2.5, methylation of LINE-1 was significantly increased in peripheral blood leukocytes of participants but was not directly associated with declining heart rate variability (HRV) (42). When DNA methylation is altered, it has the potential to activate proinflammatory factors or repress anti-inflammatory factors, and therefore, it can activate pathways that are prevalent in the development of cardiovascular diseases. The discrepancy of whether toxicant exposure results in hypo- or hypermethylation can likely be attributed to different exposure environments (controlled or ambient), duration, and concentration. The concentration of PM_2.5 during exposure was positively associated with Toll‐like receptor 2 (TLR2) methylation in older adults, further suggesting potentiation of the inflammatory response (183). Flavonoid supplementation can potentially limit TLR2 methylation, as correlations indicate and ameliorate PM_2.5-induced low frequency HRV. Another study that aimed to address the epigenetic regulation of inflammatory markers by PM exposure used young, healthy subjects who inhaled an array of PM compositions (PM₁₀, PM_2.5, PM_1.0, and PM_0.5) over two sessions (153). These subjects presented with decreased HRV, indicating a stress response, which is consistent with previous studies of PM exposure. Furthermore, PM_2.5 as well as quasi-ultrafine particles <0.50 µm were significantly associated with increased proinflammatory cytokine [interferon-γ (IFNγ)] methylation, correlating with alterations in the parasympathetic nervous system as an adaptive response (153). However, Bind et al. (9) reported that both PM_2.5 BC and PM_2.5 mass concentrations were associated with significantly lower methylation of immunoregulatory genes, IFNγ, and intercellular adhesion molecule 1 (ICAM-1) in individuals with low methylation levels before exposure. The main contributing factor that is likely responsible for these contradictory findings is the population that was studied. Whereas Tobaldini et al. (153) conducted a more controlled experiment with healthy, young males (n = 12), Bind et al. (9) used a large cohort (n = 777) of elderly men with varying health conditions. Other studies address epigenetic alterations of the cardiovascular system following PM inhalation exposure as well (95, 105), albeit without direct functional associations. Although the studies outlined provide a link between epigenetic mechanisms and cardiac function, specifically HRV, it remains unclear whether epigenetic reprogramming is a primary mechanism for sustaining adaptations to PM exposures or whether it is transiently changed with exposure. MicroRNA-378A and other microRNAs are potential molecular mediators of metabolic and cardiac dysfunction that ensue following ENM exposure (61). Nano-TiO₂ inhalation exposure induced diastolic dysfunction and E-to-A ratio elevation and decreased myocardial performance indices, which were preserved in a microRNA-knockout transgenic mouse model (microRNA-378A). These authors point to regulation of mitofusin 1 (Mfn1) expression through microRNA-378A as a mechanism for controlling mitochondrial dynamics following exposure (61). Although the preceding studies examined epigenetic changes, the mechanisms of action were not elucidated. Multifaceted assessments of how pollutants impact the epigenome and inflammation-associated genes are critical, as both are implicated in processes mediating cardiovascular function impairment.

Inflammation plays a key role in pollutant exposure response in both individual particulate exposures and multicomponent exposures. Coexposure of sulfur dioxide (So₂), nitrogen dioxide (NO₂), and PM_2.5, typical air pollutants produced as a result of coal combustion, results in an enhanced inflammatory response with upregulation of COX‐2, inducible nitric oxide synthase (iNOS), TNFα, and IL‐6 (179). The coexposure contributed to decreased blood pressure and increased heart rate when compared with PM_2.5 alone. Similarly, IL-6, IL-8, and endothelin-1 (ET-1) were significantly associated with rises in carbon monoxide (CO), UFP, and So₂ concentrations in healthy individuals near a steel mill (82). The precursor for ET-1 (BET-1) was positively correlated with systolic blood pressure, whereas C-reactive protein (CRP) was correlated with increased heart rate. Protein ontology revealed a significant elevation of inflammation specific pathways (82). Combined high-dose PM_2.5 and ozone exposure also promote cardiovascular functional injuries that are presented as abnormal electrocardiogram (ECG) results (extended QRS complex and depression of ST segment) (158). The addition of ozone to the exposure paradigm propagated inflammatory and oxidative stress responses with elevated CRP, IL-6, creatine kinase (CK), lactate dehydrogenase (LDH), and malondialdehyde (MDA). These studies highlight the importance of controlled coexposure studies for delineating the risk of ambient pollutants and combustion materials on cardiovascular hemodynamics and the associated inflammatory and oxidative stress mechanisms.

Coexposure of CAPs and acrolein causes myocardial dyssynchrony, albeit through a less understood mechanism involving the activation of transient receptor potential cation channel A1 (TRPA1), which the authors report may act to increase cardiac risk, specifically in exposures with heterogeneous compositions (152). Along with channel proteins affecting cardiac conduction, modifications in the uptake and clearance of calcium can alter contractility within cardiomyocytes, further dysregulating the cardiac cycle. One of the more studied pathways is through SERCA2a. PM_2.5 exposure increased intracellular free calcium (Ca²⁺) that was associated with increased ryanodine receptor 2 (RYR2) and decreased SERCA2a, which are responsible for shuttling Ca²⁺ from the sarcoplasmic reticulum to the cytoplasm and back, respectively (37). The changes in calcium handling may be a direct result of PM_2.5-induced oxidative stress, thereby contributing to contractile dysfunction in cardiomyocytes. In response to long-term exposure to PM_2.5, others have reported decreased fractional shortening, impaired mitral inflow patterns, and depressed contractile reserve, along with decreased peak shortening and relengthening of isolated cardiomyocytes in C57BL/6 mice (165). Downregulation of SERCA2a in PM_2.5-exposed mice revealed abnormal Ca²⁺ cycling, as previously seen, but also elevation of β-myosin heavy chain (β-MHC) indicative of heart failure (165). Apart from contractility, tone of the vasculature within the heart can alter cardiac hemodynamics. A study of acute and subchronic inhalation exposure of Sprague-Dawley rats to PM_2.5 and PM_0.1 separately elevated angiotensin II (ANG II) type 1 receptor (AT₁R) mRNA (4). The subchronic exposure to both sizes of PM further increased AT₁R protein levels, expression of markers for myocardial adaptive response to damage [actin α1 (Acta1) and Col3a] and IL-6, while decreasing antioxidant heme-oxygenase 1 (HO-1). These authors suggest that angiotensin overexpression can promote coronary artery wall thickness, which is likely to influence blood pressure in exposure models (4). Another study aimed to elucidate the effects of toxicant exposure on the regulation of blood pressure using 12- to 13-mo-old spontaneously hypertensive rats (SHRs), which were exposed to ultrafine carbon particles (UFCP) (154). UFCP inhalation exposure resulted in increased blood pressure and heart rate, which were correlated with increased levels of serum CRP, plasma fibrinogen, and levels of ET-1 in the heart. UFCP exposure-induced hypertension in SHRs is also associated with elevated ANG II in the blood, with increasing doses of UFCP diminishing the effectiveness of captopril, an angiotensin-converting enzyme inhibitor (178). The diminished effectiveness of captopril suggests that increasing doses of UFP may activate a variety of pathways outside of angiotensin to induce changes in blood pressure. These data suggest that regulation of blood pressure, like other cardiovascular responses, is a multifactorial pathway that is influenced by other circumstances, including genetic makeup and various environmental influences.

One of these environmental influences, high-fat diet feeding, is often used to model hyperlipidemia. Hyperlipidemic rats exposed to PM_2.5 are susceptible to higher blood pressure, lower HRV, and higher levels of cardiomyocyte apoptosis, accompanied by decreased expression of antioxidant proteins, including SOD (162). Markedly, high-fat diet-exposed mice, compared with high-fat diet alone, also revealed increased myocardial death through increased levels of cardiac troponin I (cTnI), LDH, and CK. Animal models with a high propensity for metabolic dysfunction, such as AMP-activated protein kinase-α2 knockout (AMPKα2^−/−) mice that lack this crucial gene for fatty acid oxidation, are critical for understanding how PM_2.5 inhalation exposure impairs cardiovascular function in vulnerable populations (160). PM_2.5 exposure diminished left ventricular ejection fraction in AMPKα2^−/− mice and promoted an oxidative and inflammatory environment through decreased expression of peroxiredoxin 5 (PRDX5) and increased expression of NF-κB and TNFα. These studies highlight the importance of understanding the molecular changes that occur and the downstream effects on oxidative stress regulation in susceptible populations exposed to PM. UFP inhalation exposure in human subjects with metabolic syndrome can be specifically detrimental to cardiac repolarization, as demonstrated by the QRS complexity in those without the glutathione s-transferase mu 1 (GTSM1) protein, a pivotal antioxidant gene (35). In the complete cohort (participants with and without the GTSM1 mutation), UFP exposure elevated CRP and serum amyloid A (SAA) with subsequent decreases in plasminogen and thrombomodulin within blood, indicating an upregulation of inflammatory pathways and the critical role of antioxidant genes. On the contrary, subacute exposure to PM_2.5 in healthy adults was not correlated with increased inflammatory markers or altered vascular function (16). However, a 10 µg/m³ increase in exposure was associated with decreased HRV, correlating to increased homeostatic model assessment of insulin resistance (HOMA-IR). This suggests the possibility that chronic periods of exposure to PM_2.5 could reduce insulin sensitivity and thus potentiate susceptibility to diabetes mellitus, although inflammation may not be the etiology implicated by this study (16). Particularly concerning is the notion that in obese mice, chronic exposure to PM_2.5 can prevent the beneficial effects of exercise on cardiac function and anti-inflammatory pathways (57). Sedentary mice that were exposed to PM_2.5 presented with increased left ventricular diameter (systolic and diastolic) that was not ameliorated in the exercised group. Moreover, PM_2.5 exposure enhanced CRP, ICAM-1, and vascular cell adhesion protein 1 (VCAM-1), which were elevated in the PM_2.5 exercised group as well (57). These results are similar to those seen in a previous study that reported the inability of moderate aerobic exercise to provide anti-inflammatory protection in mice exposed to PM_2.5 (99), which may be linked to expression of heat shock protein 70 kilodalton (HSP70) (99, 135). The advantages of moderate exercise on cardiovascular function in obese mice (106, 121, 144), but not in obese mice exposed to PM_2.5 (57), emphasize the consequential results of PM_2.5 exposure in vulnerable populations. However, the use of a high-fat diet-induced obesity model may be more relevant in substantiating whether fine particulate exposure hinders the ability of exercise to mitigate the detrimental cardiovascular effects in diabetic models, as the ob/ob model may have impaired exercise capacity (57).

COAGULATION

Within the lungs, the circulatory system provides a necessary pathway for mediation of the inflammatory response. Due to this close association of the pulmonary and circulatory systems, changes arising in blood have the potential to alter homeostasis within blood vessels. Alterations in the coagulation pathway, specifically through fibrinogen, have been reported following PM exposure.

Levels of a pollutant from wood smoke, Delta-C, are positively correlated with fibrinogen and negatively correlated with myeloperoxidase (MPO), an enzyme involved in the inflammatory pathway and platelet activation (31). The study also demonstrated that PM_2.5 and UFP retain a positive correlation with fibrinogen as well as CRP, sharing a similarity in the effects on blood coagulation with effects of Delta-C. In a coexposure model of SiNPs and lead acetate (Pb), Feng et al. (46) reported alterations to the blood-clotting cascade evidenced by decreased tissue-type plasminogen activator (t-PA), tissue factor pathway inhibitor (TFPI), and antithrombin III (ATIII) as well as elevated fibrinogen and D-dimer (D2D). Following coexposure, serum analyses revealed leukocytosis and thrombocytopenia, concomitant with increased expression of inflammatory markers (CRP, IL-6, and TNFα) and markers of heart failure (ANP and BNP). Overlap found between studies of varying particle sizes can help identify critical biomarkers of increased blood clotting that are a product of multicomponent exposure in both human and animal models. Furthermore, others have shown that levels of fibrinogen can be increased by other ambient air pollution constituents (8). Specific associations between PM_2.5 BC and altered epigenetic modification status of tissue factor III (F3) were later reported by this group, indicating alterations to extrinsic blood coagulation (10). Decreased methylation of F3, which likely leads to increased transcription of the gene, can provide the initial stimulus for altering the response of thrombin and fibrinogen, thereby increasing coagulation (28). Similarly, in a meta-population study, including 3,275 participants, long-term rises in PM_2.5, but not PM_0.1, were matched with significantly increased CRP and platelet count (155), suggesting an increased propensity for blood clotting.

Interestingly, annual average UFP exposure levels and fibrinogen were negatively associated, albeit not significantly, in human blood samples (86), which may have been a result of microenvironment compared with ambient concentrations. To better elucidate the dynamic relationship between ambient exposure and the time/concentration of the exposure in terms of their effects on the cardiovascular system, Lane et al. (86) examined biomarkers in patients with varying levels of microenvironment exposures (time/activity adjusted) in addition to the annual average particle number concentration of UFPs. There were significant positive associations between time/concentration of exposure and inflammatory pathway genes in blood, including CRP and tumor necrosis factor receptor 2 (TNFR2) (86). These findings illustrate the significance of monitoring personal exposure levels, which may account for the differences seen in fibrinogen expression between studies that solely use annual outdoor particle concentrations and those that consider varying levels of individual microenvironment exposure. Modifying the coagulation pathway in individuals that are continuously exposed to high levels of pollutants through ambient air may provide a potential a potential prophylactic strategy to prevent the inevitable high risk of CVD. Nootkatone, a sesquiterpenoid in grapefruit, has potential as a pretreatment strategy for PM_2.5 diesel exhaust exposure through its ability to reduce blood clotting by decreasing plasminogen activator inhibitor-1 (PAI-1) and fibrinogen and restoring thrombotic occlusion time in arterioles (109). These authors suggest that the pretreatment strategy activated nuclear factor erythroid-derived 2-like 2 (NRF2), which plays a key role in initiating HO-1, to provide protection from oxidative injury (109). Although the studies discussed thus far have primarily used healthy cohorts, it is important to understand how the coagulation pathway is altered in at-risk populations as well. Elemental carbon UFP inhalation exposure modifies platelet activation in type 2 diabetic patients by increasing CD40 ligand (CD40L) expression in platelets, with a resulting decrease in soluble CD40 (sCD40) in blood (142). Higher CD40L levels, as a result of UFP exposure, exerted a stimulatory role on von Willebrand factor (vWF) and elevated its expression. Although this study revealed the adaptations of those with diabetes mellitus, it did not provide a control cohort to elucidate whether these effects of PM on coagulation are exaggerated in the at-risk population. Nonetheless, genes involved in the activation of pathways involving fibrinogen and platelet activation are potential early biomarkers of increased CVD risk in healthy and vulnerable populations and require further clarification.

VASCULAR DYSFUNCTION

Along with mediating the blood clotting cascade, particulate exposure has the capacity to modify the integrity of vascular tissue, thus interfering with blood flow and cellular metabolism directly within the vasculature. Through direct damage to the vasculature or mediation of the inflammatory response, particulate matter can cause blood vessels to become predisposed to ultrastructural changes. Atherosclerosis, which involves the buildup of fatty acids within the intima layer of arteries, is linked to PM exposure.

In an atherosclerotic transgenic mouse model (ApoE^−/−), PM_2.5 exposure causes cardiac autonomic nervous system dysfunction concomitant with oxidative stress (118). Of note is that the high-fat diet fed atherosclerotic mice displayed a greater proclivity for oxidative damage, suggesting, once again the impairment of cellular protective mechanisms in those who already have an altered metabolic profile. Similarly, concentrated PM_2.5 exposure significantly altered high-density lipoprotein (HDL) antioxidant and anti-inflammatory capacity [HDL oxidative index (HOI)], along with increased systolic blood pressure (127). However, ozone exposure in addition to the PM_2.5 exposure in this study did not exacerbate these effects, suggesting that short-term CAP and ozone coexposure may not induce overt changes that are suggestive of atherosclerosis progression. On the other hand, long-term CAP exposure does contribute to the progression of atherosclerosis. Rao, et al. (128) discovered that PM_2.5 exposure increased abnormal accumulation of an oxidized variant of cholesterol, 7-ketocholesterol (7-KCh), in macrophages and the aortic wall. PM_2.5-exposed mice also presented with increased CD36 expression in plaque macrophages, implicating CD36 in the accumulation of oxidized lipids, promoting atherogenesis. By reducing the number of CD36-positive macrophages, internalization of 7-KCh can be limited, and therefore, the oxidized lipids can be efficiently cleared from the vasculature (128). Along with altered macrophage clearance, other immune responses have been implicated in vascular maladaptation following particle exposure, including changes to the innate immune response. ENM inhalation exposure resulted in translocation of nanomaterials to the heart, which initiated the complement cascade, setting off a local immune response, indicated by global complement factor 3 (C3) upregulation in the blood (73). This study demonstrates that alterations to the innate immune response may be a direct effect of translocated particles, but both studies (73, 128) highlight the importance of the inflammatory response and part of its role in altering vascular function. PM_2.5 exposure can impact vascular endothelial cell permeability pathways by causing an inflammatory response, which is a hallmark of increased vascular permeability. A recent study has demonstrated that long-term changes in vascular permeability are promoted specifically by IL-6 and sustained in part by STAT3 phosphorylation (1). In rats, PM_2.5 exposure induced phosphorylation and subsequently expression of STAT3, which upregulated microRNA-21 expression (32). Increased microRNA-21 expression reduced tissue inhibitor of metalloproteinase 3 (TIMP3) expression and enhanced matrix metalloproteinase 9 (MMP9) expression. Ultimately, vascular endothelial dysfunction was linked to extracellular matrix remodeling propagated by STAT3 (32), which was likely promoted by the PM-induced inflammatory response. Changes in the composition of the vascular matrix could result in alterations to immune cell binding and further changes to diapedesis, leading to vascular dysfunction.

In a 3,820 noncurrent smoking patient population that was part of the Framingham Heart Study, acute PM_2.5 and BC exposure concentrations were inversely correlated with expression of P-selectin, suggesting decreased endothelial surface cell adhesion (93). Along with changes to vascular structure, the ability for the endothelium to regenerate and adapt to pollutant exposure can be compromised. Typically, this occurs through the production of endothelial progenitor cells (EPCs) in response to vascular injury (48). However, PM_2.5 exposure diminishes circulating EPC levels, which may occur as a result of vascular endothelial growth factor (VEGF)-induced Akt and endothelial nitric oxide synthase (eNOS) phosphorylation (48, 58). Short-term CAP exposure in mice fed a control diet remarkably induced vascular insulin resistance, indicated by decreased Akt phosphorylation, and suppressed circulating levels of EPCs through activation of the NF-κB inflammatory pathway (59). Increasing insulin sensitivity prevented activation of these pathways, substantiating that PM_2.5 exposure can lead to insulin resistance and, therefore, type 2 diabetes in healthy populations (48). Furthermore, CAP-induced vascular insulin resistance was accompanied by increased vascular oxidative stress marked by mitochondrial SOD2 and glutathione s-transferase-P (GST-P) mRNA levels and expression of protein-HNE adducts (59). Interestingly, 9-day CAP exposure did not exacerbate high-fat diet-induced changes in vascular insulin resistance (59), but a 30-day CAP exposure significantly aggravated systemic insulin resistance, elevating glucose intolerance and HOMA-IR in high-fat diet-fed mice (60). In aortic tissue, both 9-day and 30-day CAP exposures resulted in decreased insulin-stimulated phosphorylation of Akt and eNOS, subsequently suppressing inhibition of the transcription factor NF-κB (IκBα) and promoting an inflammatory response (60). By treating with an antioxidant, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (Tempol), CAP-induced inflammation and the associated vascular insulin resistance were alleviated in high-fat diet-fed mice, leading to the conclusion that short-term exposure to PM_2.5 triggers a pulmonary oxidative stress response that is able to elicit downstream vascular insulin resistance and inflammation (60).

Compounding environmental factors can further propagate the effects of particulate exposure. A study in type 2 diabetic residents of Boston, MA, provided evidence that both PM_2.5 and BC are significantly associated with increased VCAM-1 and vWF in participants who have smoked in the past (113). These data indicate that prior exposure to smoking-related particulates may exacerbate the inflammatory response seen with ambient PM_2.5 exposure, creating a greater risk for endothelial dysfunction and cardiovascular damage in type 2 diabetic patients. In an attempt to directly assess the physiological impact of PM_2.5 exposure on vasculature in a CVD at-risk population, Riggs et al. (130) assessed individuals’ reactive hyperemia index as a measure of endothelial function. Increasing concentrations of PM_2.5 significantly reduced the reactive hyperemia index and were associated with angiogenic signaling and inflammation markers, angiopoietin 1, placental growth factor (PlGF), VEGF, ICAM-1, and MMP9 and negatively associated with VCAM-1 and urinary F2-isoprostane (Isop), a measure of oxidative stress (130). Interestingly, incremental increases of PM_2.5 by 10 μg/m³ were also associated with increased proinflammatory cytokines, including monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein 1α/β (MIP-1α/β), and IFNγ-induced protein 10 (IP-10) in a cohort of young, healthy individuals with low CVD risk (122). Together, these studies indicate that PM_2.5 initiates changes in cytokines and growth factors, which lead to endothelial injury and an immune response that may result in acute cardiovascular events in both high-risk and low-risk CVD populations (122, 130). Furthermore, PM_2.5 exposure, in a subset of the same cohort, revealed an inverse association with ET-1 but a positive association with platelet factor 4 (PF-4) (49). Additionally, ET-1 was negatively associated with PF-4 following PM_2.5 exposure, which the authors conclude may be the result of an antiangiogenic effect of the exposure that could also suppress endothelial repair (49, 122). Although this is contrary to previous studies reporting positive correlations between PM and ET-1 (46, 82, 179), another acute exposure reported similar findings (54). Conversely, chronic exposure paradigms appear to cause elevated ET-1 levels (46, 82, 179). Current literature provides convincing evidence that particulate matter exposure causes a remarkable maladaptive vascular response that can lead to increased risk of CVD. However, understanding the genome-wide effects of exposure may provide a better understanding of the molecular mechanisms that are responsible for these alterations.

CELLULAR STRESS AND DEATH

PM inhalation exposure-induced changes to the cardiovascular system can eventually trigger cell death pathways through varying mechanisms, including oxidative stress (90). These pathways include apoptosis and necrosis, leading to programmed or uncontrolled cell death and subsequently cardiovascular hypertrophy.

One of the mechanisms that initiates the cell death pathways is damage to nuclear DNA. Assessment of blood from trucking industry participants revealed that 48 [elemental carbon (EC)], 260 [organic carbon (OC)], and 49 (PM_2.5) differentially regulated genes were associated with each of the exposures; these expression profiles implicate genes regulating apoptosis, DNA and metal binding, and chronic heart and lung disease pathways (29). Consistent with these findings, exposure to PM_2.5-containing polycyclic aromatic hydrocarbons (PAHs) increased DNA damage and DNA damage response genes in a rodent model (181). These genes included 8-oxoguanine DNA glycosylase (OGG1) and growth arrest and DNA damage 153 (GADD153). PM exposure also altered genes involved in ROS regulation [glutathione S-transferase (GST) and SOD] (181). An increased expression of GST and decreased expression of SOD may promote a high-oxidative environment with reduced ability to scavenge ROS, which may help propagate the effects of DNA damage. NOX4 is a prominent contributor of oxidative stress in the failing heart and is associated with initiating increased DNA damage through this mechanism (85). PM_2.5 exposure in mice enhances not only inflammatory markers, as previously reported (TNFα and IL-1β) but also myocardial apoptosis, NOX4, and NOX4-associated subunits, suggesting that the progression of cardiovascular disease may be dictated by ROS generation and the modification of antioxidant protein levels, which are initially regulated by PM_2.5 exposure (168). Whereas unregulated levels of ROS may function through multiple pathways to cause cell death, programmed cell death through apoptosis has been linked to PM inhalation exposure through other channels. Hyperlipidemic rats exposed to PM_2.5 are more prone to cardiac apoptosis (162), that is, mediated through c-Jun NH₂-terminal kinase (JNK), P53 phosphorylation, and downstream activation of (Bcl-2-associated X) Bax and caspase-3 (162). The expression of both GST and JNK is modified following PM exposure, which implies the presence of an overlap among these pathways that mediate apoptosis, as previously suggested (53). Similar results are seen in other vulnerable models, such as mice that were challenged with myocardial infarction (94). PM inhalation exposure exacerbated left ventricular dysfunction and increased infarct size, with increased myocardial apoptosis through caspase-3, Bax, and B cell lymphoma 2 (Bcl-2). Moreover, PM exposure may not immediately stimulate cell death but rather result in reprogramming of the cell, making it susceptible to future insult. One-way reprogramming may be occurring is through changes in microRNAs, which are associated with PM_2.5, but not UFP, in the blood of individuals residing in multiple urban regions (41). Specifically, PM_2.5 is correlated with increased hsa-miR-197-3p and hsa-miR-99a-5p, which play roles in cell death and cancer (41, 175). PM_2.5 inhalation exposure was also linked to microRNAs involved in cardiovascular function, including decreased expression of microRNA-133a, 145-5p, and 499a-5p in plasma (80). Overall, these studies outline some of the potential mechanisms that initiate myocardial apoptosis and eventually lead to cardiovascular abnormalities due to particulate exposure.

MITOCHONDRIAL BIOENERGETICS AND ULTRASTRUCTURE

In the cardiovascular system, slight perturbations to mitochondrial health could result in significant detriments and sustained pathology. The energy needed for the heart to contract is derived primarily through oxidative phosphorylation of mitochondrion (∼95%) (184). Therefore, alterations to mitochondrial function are implicated as a primary pathway for cardiovascular dysfunction and disease (112).

The integrity of mitochondrial DNA defines its respiratory potential, allowing for separation of individuals into a variety of haplogroups (133). Ambient air pollution has the potential to differentially affect individuals with diverse haplotype backgrounds (164). Haplogroup H (high ROS production) compared with haplogroup U (low ROS production) is more susceptible to quasi-ultrafine particle <0.25-µm exposure. Quasi-ultrafine particle exposure induced inflammation to a greater extent in haplogroup H than U, as demonstrated by increased IL-6 and TNFα (164). Furthermore, high doses of PM_2.5 result in cardiac mitochondrial ultrastructure (size and cristae formation) changes in addition to increased inflammation (91). These ultrastructural changes are supported by increases in mitochondrial fission 1 (FIS1), MFN1, MFN2, dynamin-related protein 1 (DRP1), and mitochondrial dynamin like GTPase (OPA1), which suggest alterations to the fission/fusion pathway (91). UFP exposure has also been linked to mitochondrial dysfunction (66). Exposure to UFP exacerbated ischemia-reperfusion damage in the isolated hearts of rats. Isolated cardiac mitochondria of UFP-exposed rats displayed decreased Ca²⁺ buffering before the mitochondrial permeability transition pore (mPTP) opening, which indicates that exposure can increase mPTP Ca²⁺ sensitization and, therefore, mitochondrial permeability (66). The alterations to fission/fusion proteins (91) and mitochondrial permeability (66) showcase changes to the composition and functionality of mitochondrion following exposure, which can propagate oxidative damage and initiate mPTP-associated cell death pathways to instigate cardiac damage. A single dose of ENM through inhalation exposure in mice has the ability to trigger cardiac functional changes and mitochondrial dysregulation (111). However, a transgenic mouse model for mitochondrial phospholipid hydroperoxide glutathione peroxidase (mPHGPx) was used to restore cardiac function and mitochondrial respiratory function and abrogate ROS levels following nano-TiO₂ exposure. By increasing antioxidant defense through mPHGPx overexpression, proteomics revealed that changes in mitochondrial composition, including individual electron transport chain complex constituents, were protected from the detrimental effects of exposure (111). This study further validates the importance of antioxidant protection in preservation of mitochondrial structure and function and, therefore, should be investigated as a potentially protective approach.

PM exposure can modify the transcriptional capacity of the mitochondrion and likewise incite dysfunction through this approach. Boilermakers exposed to PM_2.5 were used in a study that aimed to elucidate the exposure-induced changes in methylation of mitochondrial DNA (18). Methylation of specific, mitochondrially transcribed genes involved in ATP synthesis, including mitochondrially encoded tRNA phenylalanine (MT‐TF) and mitochondrial 12s RNA (MT‐RNR1), were not associated with PM_2.5 exposure. However, methylation of the mitochondrial promoter D-loop was associated with PM_2.5 exposure. Participants with mitochondrial DNA hypermethylation were more vulnerable to the effects of PM_2.5 exposure on HRV (18). Inhalation of toxicant particles may exert its effects on overall mitochondrial structure and function, and thus cardiovascular function, through ROS-regulated and inflammatory pathways, as the preceding studies indicate. Both increased inflammation and ROS have the ability to recruit methyltransferases, such as DNA methyltransferase 1 (DNMT1), and alter the methylome of various genes implicated in CVD, causing repression of the associated genes (79).

Although the molecular mechanisms behind cardiovascular impairment associated with particulate matter exposure vary based on the particle size, constituents, and physiochemical properties, there are common overlapping genes that appear to be modulated by multiple toxicants present in air pollution. Figure 2, created using ingenuity pathway analysis (IPA), provides an interaction network of genes that are dysregulated following particulate inhalation exposure. IPA included genes that were upregulated or downregulated or had diverse expression following more than one type of particulate exposure mentioned throughout this review. These genes can serve as potential early biomarkers of cardiovascular damage as a result of inhaled toxicant exposure.

Fig. 2.

Ingenuity pathway depicting the interaction network of genes that are dysregulated following particulate inhalation exposure. The genes included in the interaction network were associated with more than 1 type of particulate exposure. The interactions represent direct, validated experimental associations between genes that were increased (red), decreased (green), or found to have a diverse expression profile (yellow) following exposures. All abbreviations are defined in glossary.

GESTATIONAL EXPOSURES

There has been an increasing volume of literature investigating how particulate exposure can affect vulnerable populations, including women during pregnancy. Because of the coinciding metabolic changes prompted by both diabetes/obesity and air pollution, particularly related to oxidative stress and inflammation, understanding the dynamics of both pathologies is critical in determining how pollutant exposure can alter the metabolome (112). Particulate exposure during gestation is a major risk factor for gestational diabetes (101, 174). However, the effects of pollutant insult in utero on the offspring remain a critical gap in knowledge (11, 74, 96). This is particularly concerning for fine and ultrafine particles that have the ability to enter circulation and reach the fetal side of the human placenta (12). Recent studies indicate that maternal toxicant inhalation exposure during gestation is a predisposing factor for CVD in offspring.

OXIDATIVE STRESS AND INFLAMMATION

PM₂ exposure results in activation of oxidative and inflammatory pathways, as has been described in the previous sections. Preconception toxicant exposure predisposes future offspring to the development of cardiac dysfunction (148). PM_2.5 preconception exposure in both parents decreases ejection fraction, fractional shortening, and cardiomyocyte contractility in 3-mo-old progeny (148). Inflammatory and oxidative pathways are thereby activated, resulting in increased IL‐6, IL‐15, NF-κB, and CRP, along with elevated fibrosis (Col3a1), similar to the results found in many direct exposure studies. Notably, calcium-handling proteins SERCA2a and phosphorylated phospholamban (p‐PLN) are elevated in preconception PM_2.5 exposed offspring, as increased p-PLN loses its ability to inhibit SERCA2a. Whereas PM-induced inflammation and oxidative stress are common in both direct and preconception exposures, SERCA2a is contrarily decreased in direct exposures (37, 165). One potential reason for this discrepancy may be that preconception exposure results in an adaptive response. Similar to direct and preconception exposure, PM_2.5 exposure during gestation also results in cardiac remodeling with associated inflammation (increased IL‐6 and IL‐1β) (150). Furthermore, extracellular matrix remodeling, marked by increased collagen‐1, MMP9, and MMP13 expression, is also present in neonates following gestational exposure but was not assessed into adulthood. Nevertheless, long-term maternal exposure to PM_2.5 throughout gestation and lactation enhanced activation of the inflammatory markers and myocardial apoptosis in both neonatal and weanling Sprague-Dawley rats (26). UFP exposure to pregnant C57BL/6J dams led to fetal reabsorption and increased blood pressure in adult progeny (104). Increases in maternal serum inflammation during gestation, along with AT₁R and angiotensin I-converting enzyme (ACE) activation in the lungs, are suggested to contribute to progeny cardiovascular dysfunction both short and long term. Therefore, the notion that maternal and paternal exposure activates an inflammatory response in progeny from early life into adulthood is well established, but whether the changing maternal environment (104), particle translocation, or a combination of such factors is culpable in altering progeny development and metabolic function remains elusive.

In addition to inflammatory pathways, immune responses and oxidative stress pathways are changed in progeny who are exposed during gestation. Challenging progeny immune systems with house dust mite (HDM) allergen revealed a decreased immune response through lower IL-13 and IL-17, with higher serum levels of IL-10 following UFP exposure (134). This has also been demonstrated with UFP containing persistent free radicals (MCP230), which suppressed the T helper and T regulatory cells found within the lungs of offspring (161). Maternal ENM exposure during gestation may, however, modulate a regulatory axis involving enhanced hypoxia-inducible factor (HIF)-1α activity and decreased mPHGPx expression, which are critical in the regulation of ROS in fetal and young adult cardiac development (83). Understanding how gestational exposure, as opposed to direct exposure, affects individuals will contribute in the development of preventative measures for vulnerable populations, including pregnant women and children, and promote safer limits of exposure, thereby mitigating susceptibility to metabolic diseases and CVD in offspring.

CELLULAR FUNCTION

Calcium-regulated contraction is significantly affected throughout development in progeny exposed to particulate matter during gestation. Assessment of 14-day-old progeny whose dams were exposed to PM_2.5 during gestation revealed decreased cardiomyocyte contractility through calcium dynamics, with changes to proteins involved in calcium flux in the heart, SERCA-2a, p-PLN, Na⁺/Ca²⁺ exchanger (NCX), calcium channel, voltage-dependent L type, and α1C subunit (CaV1.2) (149, 150). Offspring whose dams were exposed during gestation also present with major cardiac anomalies present into adulthood as a result of ENM-induced mitochondrial dysfunction. Nano-TiO₂ gestational exposure decreased diastolic cardiac function, marked by a 15% increase in E-to-A ratio, and diminished mitochondrial respiration in young adult progeny (62). These results were simultaneous with mitochondrial proton leak and increased uncoupling protein 2 (UCP2) as well as upregulation of mitochondrial metabolism proteins, carnitine palmitoyltransferase 1A (CPT1A), and pyruvate dehydrogenase (PDH) phosphorylation. ENM exposure during gestation also affects mitochondrial bioenergetics by limiting state 3 respiration in the left ventricle of young adult progeny, coinciding with impaired endothelium-dependent dilation in coronary and uterine arterioles (140). PM_2.5 gestational exposure can trigger myocardial apoptosis in a dose-dependent manner, as has been revealed in 1-day-old neonatal offspring from Wistar rats (159). Myocardial cell death likely occurred due to alterations in mitochondrial fission/fusion through OPA1, MFN1, DRP1, and FIS1 following PM_2.5 exposure during gestation (159). Using functional genomics in addition to mitochondrial structural and bioenergetic assessment may provide insight into the mechanisms driving the deficits observed in progeny whose dams were exposed to particulate matter during gestation.

EPIGENETICS AND TRANSCRIPTIONAL REGULATION

Studies in progeny exposed to particulate exposure during a critical point in development, such as gestation, reveal that cellular regulatory networks are substantially altered, likely contributing to sustained detrimental effects into adulthood. These regulatory systems include epigenetic remodeling as well as noncoding RNA networks, which can act to reprogram the basal cellular function in progeny and cause predisposition to metabolic disorders that modulate analogous pathways. Maternal PM_2.5 exposure during gestation can promote long-term cardiac remodeling and causes cardiac hypertrophy in young adult progeny, with an increase in both total histone acetylation and promoter acetylation of hypertrophy genes GATA4 and myocyte enhancer factor 2C (Mef2c) (169). This further suggests the ability of gestational PM exposure to induce global epigenetic remodeling through transcriptional activation of acetylated regions in offspring. Supporting these findings, multiple additional studies have reported a cardiac hypertrophic/fibrotic phenotype, cardiac functional changes, and epigenetic reprogramming in early development due to gestational PM exposure (148, 150, 169). Preconception exposure in both parents to PM_2.5 resulted in progeny with decreased ejection fraction, fractional shortening, and cardiomyocyte contractility at 12 wk of age (148). Epigenetic assessments suggested changes that were indicative of reprogramming induced by the particulate exposure, with decreased expression of the maintenance methyltransferase DNMT1 but increased histone deacetylase sirtuin (SIRT) 1/2. This pattern of expression may be explained by the ability of SIRT to regulate DNMTs and alter their activity. Similarly to preconception exposure, PM_2.5 exposure during gestation also impaired cardiac function of progeny at 12 wk of age, revealed by decreased fractional shortening and left ventricular posterior wall thickness and increased left ventricular systolic/diastolic diameters and collagen deposition in cardiac tissue (150). Epigenetic regulation likely contributed substantially to persistent cardiac detriments, as seen in the preceding study. In contrast with preconception PM exposure (148), gestational exposure decreased SIRT1/2 and increased DNMT1, DNMT3a, and DNMT3b expression (150). Although there are differences in expression profiles of these epigenetic regulators that likely stem from the time of exposure (preconception versus gestation), both are representative of profiles that can cause sustained biological detriments and/or susceptibility to pathological insult. ENM inhalation exposure during gestation causes similar changes to DNA methylation machinery (83). Maternal exposure to nano-TiO₂ during gestation decreases cardiac output and increases left ventricular mass, with a subsequent decrease in fractional shortening in progeny. These functional alterations were linked to elevated levels of 5-methylcytosine (5-mC), a marker of global DNA methylation, DNMT1 protein expression, and HIF-1α activity (83). Along with DNA methylation, changes in the histone composition of DNA can contribute to modified gene expression profiles. Chromatin immunoprecipitation sequencing for histone 3 lysine 4/27 trimethyaltion (H3K4me3 and H3K27me3), coupled with the transcriptomic profiling of fetal cardiac tissue from dams exposed to ENM during gestation, has revealed significant changes to major pathways, including immune adaption and organismal growth (138). These authors suggest that cardiovascular outcomes at the young adult stage may actually be linked to liver and fetal development, as evidenced through IPA protein ontology. MicroRNAs have also been implicated in metabolic changes in offspring as a result of gestational particulate exposure (55). Diesel exhaust exposure in utero decreased methylation of microRNA133a-2 at the promoter region in isolated neonatal cardiomyocytes (55). As the progeny aged, introducing stress, such as transverse aortic constriction, enhanced microRNA-133a-2 and decreased protein tyrosine phosphatase receptor type F (PTPRF) and peptidase domain-containing associated with muscle regeneration 1 (PAMR1) expression, which mediates adult sensitivity to heart failure by inducing pathological hypertrophic signaling or preventing antihypertrophic signaling (55). Currently, longitudinal studies on the effects of maternal exposure to various pollutants on offspring cardiovascular function are limited. Figure 3 provides a summary of the factors involved in epigenetic regulation that have been discussed in this review, in the context of preconception/gestational particulate inhalation exposure. Functional genomics studies that combine transcriptomic, proteomic, and metabolomic data in cohorts presenting with particulate-induced cardiovascular alterations will provide a more thorough understanding of early epigenetic remodeling and its involvement in contributing to CVD predisposition later in life.

Fig. 3.

Epigenetic alterations observed following gestational and/or preconception particulate inhalation exposures. Gene expression of GATA4 and MEF2C is increased through promoter acetylation at H3K9. MicroRNA-133a-2 expression is repressed through elevated 5mC. DNA methyl transferases (DNMT1, DNMT3a, and DNMT3b) are all increased following exposure, along with increased expression of transcription factors (STAT3 and HIF-1α). H3K4me3 and H3K27me3 alterations are observed at promoter regions throughout the genome. The histone deacetylases SIRT1/2 reveal a dysregulated expression profile in progeny exposed during gestation. All abbreviations are defined in glossary.

FUTURE DIRECTIONS

From a morning commute, to an occupational setting, to our very homes, exposure to particulates remains a central issue for debate and therapeutic/lifestyle interventions. Industrialization and increased urbanization will continue to exacerbate issues concerning air quality and human health unless better-defined guidelines and limits are introduced. Although we provide a summary of the molecular mechanisms propagating cardiovascular decline following exposure in human and animal models, areas of research that are crucial in driving the field of molecular inhalation cardiovascular toxicology forward still remain uncharted. Future investigations should aim to address understudied populations.

There are limited studies investigating whether increased particulate matter exposure is associated with higher rates of cardiac arrest. One study that aimed to assess this association in New York City reported that a 10 µg/m³ increase in PM_2.5 1 day before and on the day of the event significantly increased the relative risk for a cardiac arrest in the warm season (136). Similar results were seen in Melbourne (34), Copenhagen (163), Indianapolis, IN (132), and Houston, TX (40). Contrarily, a study done in Stockholm (129) and several studies by a group in Washington, DC (24, 88, 146), report no association between PM exposure and out-of-hospital cardiac arrests. Nonetheless, because of the high mortality rate associated with cardiac arrests, future studies should attempt to further clarify these associations through assessment of molecular markers.

There has been a growing body of literature exploring the adverse outcomes of PM exposure during gestation, including associations with metabolic dysregulation and cardiovascular deficits. In addition to a necessity for a better understanding of how particles induce specific changes during embryonic and fetal development, evaluation of the long-term effects is essential. This is particularly important for determining whether particulate exposure during gestation elicits alterations to the epigenome of offspring that can cause increased susceptibility to prevalent metabolic disorders, including diabetes mellitus (96). Notably, recent research has revealed the ability for prenatal and perinatal PM exposure to alter glucose metabolism (103) and elevate low-density lipoprotein cholesterol (LDL-C) (100), suggesting an increased risk of diabetes and atherosclerosis. However, the molecular mechanisms were not evaluated. To address these gaps in knowledge, focus must turn to the epigenetic effects following particle exposure during intrauterine and early childhood development concomitantly with metabolic assessments, which are currently meager (47, 74).

As we continue to shift the paradigm of cardiovascular research in inhalation toxicology, much of the cellular and molecular mechanisms are yet to be discerned. Cell culture models of exposure interactions can help provide a foundational understanding of cell and organelle function that cannot be fully captured through in vivo inhalation studies. Although it remains outside the scope of this review, in vitro models can provide a very granular and controlled approach to mechanistic evaluations. Of note, some authors have recently investigated the effects of PM_2.5 exposure (19, 33, 44, 69, 172, 173, 179) as well as the toxicology of nanomaterials and advanced materials (45, 71, 72, 171) in cells. Additionally, in vitro studies can provide preliminary data for determining how complex and dynamic mixtures of particulate and gaseous substances affect cardiomyocytes, establishing potential molecular mechanisms and thus setting a path for future in vivo experiments (3, 63, 115).

Most of the current research is focused on ambient PM_2.5 exposure, but with standards being set based on mass, UFP concentration is not accounted for thoroughly due to their low mass. However, the high surface area of UFP and ENM makes them considerably detrimental to health, potentially even more than fine and coarse PM (143). Because most ambient PM exposures are performed by collection and monitoring of PM in different regions, the problem of variable PM composition in these regions also arises. Integration of ENM exposure with UFP and PM multicomponent studies on both humans and mice will allow for a more thorough interpretation of the direct molecular mechanisms by which pollutants induce toxicity while also allowing the most relevant elucidation of overall health effects (143). Though coexposure studies are becoming more common in exploring the effects of present-day air pollution constituents on cardiac function in healthy and metabolically challenged systems (20, 43, 84, 114, 157), there is limited research assessing both cardiac function and the associated molecular mechanisms, leaving a critical gap in knowledge (3). This is one of the main limitations in many studies that could be addressed with the identification of altered candidate genes via functional genomic approaches.

A clear necessity exists for more encompassing “omics”-based approaches. Although investigators have begun to examine molecular modulators of the responses to inhalation exposure in the heart, including protein and gene markers for inflammation, few studies have examined the complete cellular protein, gene, or metabolic regulatory networks that coincide with the functional and structural changes. The integration of transcriptomics, epigenomics, proteomics, and other holistic approaches will provide a comprehensive understanding of cellular reprogramming, which can be impacted by the size, source, and duration of particle inhalation exposure (Fig. 4). The implementation of these approaches will outline early biomarkers induced by particulate exposure, ultimately encouraging exposure limits and standards that more precisely prevent the adverse effects of inhaled toxicants.

Fig. 4.

An overview of various contributing factors that can differentially alter the cardiovascular responses to toxicant inhalation exposure is provided. Current gaps in knowledge are outlined, including the lack of systemic integration of omics data across varying inhalation exposure paradigms.

GRANTS

This work was supported by National Heart, Lung, and Blood Institute Grant R01-HL-128485 (to J.M.H.), American Heart Association Grants AHA-20PRE35080170 (to A.K.) and AHA-17PRE33660333 (to Q.A.H.), and the Community Foundation for the Ohio Valley Whipkey Trust.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

A.K., Q.A.H., and J.M.H. conceived and designed research; A.K. and Q.A.H. prepared figures; A.K., Q.A.H., and M.V.P. drafted manuscript; A.K., Q.A.H., M.V.P., A.D.T., and J.M.H. edited and revised manuscript; A.K., Q.A.H., M.V.P., A.D.T., and J.M.H. approved final version of manuscript.

GLOSSARY

5-mC

5-Methylcytosine

7-KCh

7-Ketocholesterol

ACE

Angiotensin I-converting enzyme

Acta1

Actin α1 (ACTA1)

AMPKα2^−/−

AMP-activated protein kinase α2 knockout

ANG II

Angiotensin II (ANG)

ANP

Atrial natriuretic peptide (NPPA)

ATIII

Antithrombin III (SERPINC1)

AT₁R

Angiotensin II type I receptor (ATGR1)

Bax

Bcl-2-associated X protein

BC

Black carbon

Bcl-2

B cell lymphoma 2

BNP

Brain natriuretic peptide (NPPB)

β-MHC

β-myosin heavy chain

C3

Complement factor 3

Ca²⁺

Calcium

CAP

Concentrated ambient particles

CaV1.2

Calcium channel, voltage-dependent, L type, alpha 1C subunit

CK

Creatine kinase

CO

Carbon monoxide

Col1a1

Collagen type I α1

Col3a1

Collagen type III α1

COX-2

Cyclooxygenase-2 (PTGS2)

CPT1A

Carnitine palmitoyltransferase

CREB

cAMP response element binding protein

CRP

C-reactive protein

CsA

Cyclosporin A

cTnI

Cardiac troponin I

cTnT

Cardiac troponin T

CVD

Cardiovascular disease

CXCL1

C-X-C motif chemokine ligand 1

D2D

D-dimer

DNMT

DNA methyltransferase

DRP1

Dynamin-related protein 1

EC

Elemental carbon

EGFR

Epidermal growth factor receptor

ENM

Engineered nanomaterials

eNOS

Endothelial nitric oxide synthase

EPC

Endothelial progenitor cells

ER

Endoplasmic reticulum

ET-1

Endothelin-1(EDN1)

F3

Tissue factor III

FBA/B/G

Fibrinogen genes

FIS1

Fission 1

GADD153

Growth arrest and DNA damage 153

GATA

GATA sequence binding protein

GRP78

Glucose-regulated protein 78

GSH-Px

Glutathione peroxidase

GSK3β

Glycogen synthase kinase-3β

GST

Glutathione S-transferase

GST-P

Glutathione s-transferase P

GTSM1

Glutathione s-transferase mu 1

H3K27me3

Histone 3 lysine 27 trimethyaltion

H3K4me3

Histone 3 lysine 4 trimethyaltion

HDL

High-density lipoprotein

HDM

House dust mite

HIF

Hypoxia inducible factor

HO-1

Heme oxygenase-1(HMOX)

HOI

HDL oxidative index

HOMA-IR

Homeostatic Model Assessment of Insulin Resistance

HRV

Heart rate variability

HSP70

Heat-shock protein 70 kD

ICAM-1

Intercellular adhesion molecule 1

IFNγ

Interferon-γ (IFNG)

IKK

Inhibitor of IκB kinase-2

IL

Interleukin

CXCL8

Interleukin 8

iNOS

Inducible nitric oxide (NOS2)

IP-10

IFNγ-induced protein 10

IPA

Ingenuity Pathway Analysis

Isop

F2-isoprostane

JNK

C-Jun NH₂-terminal kinase

LDH

Lactate dehydrogenase (LDHB)

LDL-C

Low-density lipoprotein cholesterol

LINE-1

Long interspersed nuclear element-1

MAPK

Mitogen activated protein kinase

MCP-1

Monocyte chemoattractant protein 1

MDA

Malondialdehyde

Mef2c

Myocyte enhancer factor

MeHg

Methylmercury

Mfn1

Mitofusin 1

MIP-1

Macrophage inflammatory protein

MMP9

Matrix metalloproteinase

mPHGPx

Mitochondrial phospholipid hydroperoxide glutathione peroxidase

MPO

Myeloperoxidase

mPTP

Mitochondrial permeability transition pore

MT‐RNR1

Mitochondrial 12s RNA

MT‐TF

Mitochondrially encoded tRNA phenylalanine

NCX

Na⁺/Ca²⁺ exchanger

NF-κB

Nuclear factor-κβ (NFKB1)

NO₂

Nitrogen dioxide

NOX4

NADPH oxidase 4

NRF2

Nuclear factor erythroid-derived 2-like 2

OC

Organic carbon

OGG1

8-oxoguanine DNA glycosylase

OPA1

Mitochondrial dynamin like GTPase

PAH

Polycyclic aromatic hydrocarbons

PAI-1

Plasminogen activator inhibitor-1

PAMR1

Peptidase domain containing associated with muscle regeneration 1

Pb

Lead acetate

PDH

Pyruvate dehydrogenase

PF-4

Platelet factor 4

PlGF

Placental growth factor

PM

Particulate Matter

p-PLN

Phosphorylated phospholamban

PRDX5

Peroxiredoxin 5

PRNP

Prion protein

PTPRF

Protein tyrosine phosphatase receptor type F

ROS

Reactive oxygen species

RYR2

Ryanodine receptor 2

SAA

Serum amyloid A

SELP

P-selectin

SERCA2

Sarco/endoplasmic reticulum Ca²⁺-ATPase (ATP2A2)

SHR

Spontaneously hypertensive rats

SiNP

Silica nanoparticles

SIRT

Sirtuin

So₂

Sulfur dioxide

So₄

Sulfate

SOD

Superoxide dismutase

SOS1

SOS Ras/Rac Guanine Nucleotide Exchange Factor 1

STAT

Signal transducer and activator of transcription

TFPI

Tissue factor pathway inhibitor

TGFβ1

Transforming growth factor beta 1

TIMP3

Tissue inhibitor of metalloproteinase 3

TiO₂

Titanium dioxide

TLR2

Toll-like receptor 2

TLR4

Toll-like receptor 4

TNFR2

Tumor necrosis factor receptor 2 (LTBR)

TNFα

Tumor necrosis factor-α

t-PA

Tissue-type plasminogen activator (PLAT)

TRPA1

Transient receptor potential cation channel A1

UCAP

Concentrated ambient ultrafine particles

UCP2

Uncoupling protein 2

UFCP

Ultrafine carbon particles

UFP

Ultrafine particles

VCAM-1

Vascular Cell Adhesion Protein 1

VEGF

Vascular endothelial growth factor (VEGFA)

vWF

Von Willebrand factor