Pulmonary and Vascular Effects of Acute Ozone Exposure in Diabetic Rats Fed an Atherogenic Diet

Abstract

Air pollutants may increase risk for cardiopulmonary disease, particularly in susceptible populations with metabolic stressors such as diabetes and unhealthy diet. We investigated effects of inhaled ozone exposure and high-cholesterol diet (HCD) in healthy Wistar and Wistar-derived Goto-Kakizaki (GK) rats, a non-obese model of type 2 diabetes. Male rats (4-week old) were fed normal diet (ND) or HCD for 12 weeks and then exposed to filtered air or 1.0 ppm ozone (6hrs/day) for 1 or 2 days. We examined pulmonary, vascular, hematology, and inflammatory responses after each exposure plus an 18-hr recovery period. In both strains, ozone induced acute bronchiolar epithelial necrosis and inflammation on histopathology and pulmonary protein leakage and neutrophilia; the protein leakage was more rapid and persistent in GK compared to Wistar rats. Ozone also decreased lymphocytes after day 1 in both strains consuming ND (~50%), while HCD increased circulating leukocytes. Ozone increased plasma thrombin/antithrombin complexes and platelet disaggregation in Wistar rats on HCD and exacerbated diet effects on serum IFN-γ, IL-6, KC-GRO, IL-13, and TNF-α, which were higher with HCD (Wistar>GK). Ex vivo aortic contractility to phenylephrine was lower in GK versus Wistar rats at baseline(~30%); ozone enhanced this effect in Wistar rats on ND. GK rats on HCD had higher aortic e-NOS and tPA expression compared to Wistar rats. Ozone increased e-NOS in GK rats on ND (~3-fold) and Wistar rats on HCD (~2-fold). These findings demonstrate ways in which underlying diabetes and HCD may exacerbate pulmonary, systemic, and vascular effects of inhaled pollutants.

INTRODUCTION

Air pollution is a major cause of adverse health, accounting for an estimated 4.2 million annual premature deaths worldwide (Ritchie and Roser 2020; Landrigan et al., 2018). Risk factors that may exacerbate the health effects of air pollution include chronic pulmonary and cardiovascular diseases, diabetes, obesity, and poor diet (Al-Kindi et al., 2020; Jaganathan et al., 2019; Peters et al., 2019). Exposure to air pollution has also been associated with higher incidence of type 2 diabetes (Brook et al., 2017; Alderete et al., 2018). However, the mechanisms by which these host conditions exacerbate air pollutant-induced insulin resistance and diabetes remain unclear. The understanding of pathobiological mechanisms is important for better disease prevention approaches.

Type 2 diabetes represent over 90% of all human cases, which is characterized by chronic peripheral insulin resistance (Saeedi et al., 2019; 2020). At an advanced stage of diabetes, insulin resistance is associated with insulin insufficiency resulting from impairment of pancreatic beta cell function. Chronic insulin resistance at an advanced stage of diabetes is associated with many secondary health complications, including cardiovascular dysfunction, systemic inflammation, diminished pancreatic insulin production, and neuropathy (Wysham and Shubrook, 2020). In addition to genetic determinants, type 2 diabetes may also be caused by lifestyle, diet, and environmental factors. Diet-induced obesity, for example, increases risk of type 2 diabetes, although co-factors and underlying mechanistic drivers are often unclear (Yaribeygi et al., 2020). However, few experimental studies have examined air pollution effects using animal models of diabetes (Zhong et al., 2016; Nemmar et al., 2013). These studies have shown that diabetes increases the incidence of cardiopulmonary disease, and that air pollution may further accelerate this process.

Unhealthy high-cholesterol/high-fat diets may increase susceptibility to cardiovascular and lung diseases, including atherosclerosis (Huff, 2003) and asthma (Guilleminault et al., 2017). Epidemiologic studies have shown that particulate matter (PM) pollution may contribute to increased risk of cardiovascular morbidity and mortality through short-term increases in systemic arterial vascular narrowing and peripheral blood pressure in patients with preexisting cardiac disease (Zanobetti et al., 2004). Experimental models have further shown that chronic PM exposure or other air pollutants may enhance the atherogenic effects of a high-cholesterol diet (Soares et al., 2009) and interact with lipid-rich diets to directly affect the lung, especially the type 2 pneumocytes, which synthesize lipid-rich surfactants (Snow et al., 2018). However, it is not known how a high-cholesterol diet in healthy and diabetic settings can alter acute air pollutant-induced pulmonary injury and vascular function.

Our goal in this study was to investigate the pulmonary and vascular effects of acute ozone inhalation in context of non-obese type 2 diabetes, with or without a high-cholesterol atherogenic diet. Ozone was selected as a prototypic air pollutant, as oppose to ambient or other combustion source particulate matter mixtures, to enable the assessment of susceptibility differences associated with diabetes and unhealthy diet. The biological effects of ozone are extensively investigated in rodents and humans, including our recent studies demonstrating the role of neuroendocrine system (Miller et al., 2016; Henriquez et al., 2018). We used Wistar rats as a reference strain and Wistar-derived Goto Kakizaki (GK) rats as a genetic model of non-obese type 2 diabetes mellitus. The major genetic factor in GK rats leading to type 2 diabetes early in life involves defects in pancreatic insulin production and secretion mechanisms ultimately leading to insulin resistance (Nobrega 2009). Alterations in cholesterol/lipid metabolism pathways have also been reported in this congenic strain (Wallis et al., 2004), allowing us to examine high-cholesterol diet interactions with diabetes. To investigate these effects, Wistar and GK rats were fed either a standard rat chow or high-cholesterol atherogenic diet for a period of 12 weeks starting at 1 month of age. Rats were then exposed to clean air or ozone, and pulmonary toxicity and vascular effects were examined. We hypothesized that pulmonary and vascular responses to ozone would be exacerbated by diabetes and high-cholesterol diet.

MATERIALS AND METHODS

Animals and diets

Male Wistar and Wistar-derived Goto-Kakizaki (GK) rats were purchased from Charles River Laboratories, Inc. (Kingston, NY) at 3 weeks of age and housed two/cage in polycarbonate cages with hardwood chip bedding in an AAALAC-approved animal facility (12 h light/dark cycle, 23±1°C) for 1 week before starting the dietary regimen. All experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of the U.S. Environmental Protection Agency (EPA). Starting at 4 weeks of age and lasting until 16 weeks of age, half of the Wistar and GK rats continued on the normal diet (ND; Purina 5001; Ralston Purina Laboratories, St. Louis, MO), while the other half began receiving an atherogenic high-cholesterol diet (HCD, Teklad Custom Research Diet- TD.02028; Harlan Laboratories, Inc., Indianapolis, IN). The TD.02028 is a purified rodent diet with modestly higher levels of protein (23.9% vs. 17.3%), carbohydrates (48.7% vs. 46.9%), and fiber (5.3% vs. 0%) compared to ND, in addition to higher levels of cholesterol/fat (21.2% relative to 5.7% in ND). More information on diet composition can be found in our companion paper (Snow et al., 2021). We chose these two diets in order to compare a standard low-fat rodent chow-based diet with a high-fat/cholesterol atherogenic diet. Food and water were provided ad libitum unless otherwise stated.

Ozone exposure

After 12 weeks of the dietary protocol, rats were exposed to filtered air or 1.0 ppm ozone, 6 hrs/day, for one or two consecutive days (n=6/group). An additional group of rats was exposed to air or ozone for 2 consecutive days and allowed 18 hrs recovery. Ozone was generated using a silent arc discharge generator (OREC, Phoenix, AZ) and transported to Rochester-style “Hinners” chambers using mass flow controllers (Coastal Instruments Inc., Burgaw, NC). The chamber temperature, relative humidity, chamber air flow, and ozone concentrations were assessed continuously during exposure, as shown in our previous studies (Bass et al., 2013; Snow et al., 2018). The measured average chamber data for ozone concentration was 1.0±0.02 ppm. The average (mean ± standard deviation) temperature (^oF), relative humidity (RH), and chamber air flows (L/min) were 71.8±0.5, 52.8±2.7, and 265±35 for the air chamber and 72.6±0.6, 51.7±2.3, and 258±2 for the ozone chamber, respectively. Ozone concentration of 1 ppm is several folds higher than what may be encountered in tropical industrial locations (US. EPA 2020). However, the concentration of 1 ppm may be comparable to the ozone inhalation during human clinical studies, based on the evidence that resting rats inhaling ozone during inactivity retain <1/4^th the dose in the lung when compared to humans exposed to ozone during intermittent exercise (Hatch et al., 2013). Ozone concentration of 1 ppm was employed in this study to produce a clear pulmonary injury and inflammation response with limited number of animals per group, that allows for assessing rat strain differences and changes induced by HCD.

Necropsy, sample collection, and lung lavage

Rats exposed to air or ozone for 1 or 2 consecutive days were necropsied within 2 hrs of exposure. Additional group of rats exposed to air or ozone for 2 consecutive days and allowed 18 hour recovery, were necropsied after their glucose tolerance testing (GTT; reported in a companion paper, Snow et al., 2021) at 18 hour. All rats were euthanized using sodium pentobarbital (Virbac AH, Inc., Fort Worth, TX, diluted with saline to 200 mg/mL; >200 mg/kg, or as required; intraperitoneally). In each case, rats were fasted for 6–8 hrs prior to necropsy. Blood samples were collected from the abdominal aorta directly into three vacutainer tubes: citrated tube for platelet aggregation and blood clotting assay, EDTA tube for complete blood count and serum separator tubes for circulating cytokine assay.

The trachea was cannulated, the left lung was tied, and the right lung was lavaged 3 times using the same aliquot of Ca²⁺- and Mg²⁺-free PBS (pH 7.4, 37°C) to total lung capacity of 28 mL/kg body weight and 60% total lung weight. The left lung was tracheally-fixed with 10% neutral formalin, tied and submerged in a formalin-filled specimen cup for later histological assessment. Cytospin slides were prepared by spinning whole bronchoalveolar lavage fluid (BALF) to collect cells on slides. Dried slides were stained with Diff-quick and cell differentials were performed under light microscopy (300 cells/slide, one slide/animal). Whole BALF (0.5 mL) was diluted to 10 mL using isotone and spiked with 0.2 mL saponin to lyse cells. Nuclei were counted using a Z1 Coulter Counter (Coulter Inc., Miami, FL). The remaining BALF samples were centrifuged (1500 x g for 5 min) and cell-free BALF aliquots were analyzed for lung injury markers.

Assessment of lung injury markers

Total protein in BALF was analyzed to assess vascular leakage using Coomassie Plus Protein Reagent from Thermo Fisher Diagnostics (Rockford, IL) and albumin standards from Sigma-Aldrich (St. Louis, MO). The kits from Sekisui Diagnostics (Lexington, MA) were used for assessing BALF albumin levels. Activity of BALF N-acetylglucosaminidase (NAG) was assessed using reagents and controls from Sigma-Aldrich Diagnostics (St. Louis, MO). BALF gamma-glutamyl transpeptidase activity (GGT) was determined using Thermo Fisher Diagnostics kits (Middletown, VA). These assays were modified for use on the Konelab Arena 30 clinical analyzer (Thermo Chemical Lab Systems, Espoo, Finland).

Histopathological assessment of lung

Formalin-fixed lung tissues were paraffin-embedded, sectioned, and stained with hematoxylin and eosin (H&E) using standard histologic procedures. One section of lung was evaluated for each animal. Stained sections were evaluated via light microscopy by a board-certified pathologist in a blinded manner using established pathologic criteria for inflammatory, degenerative, metaplastic, and proliferative changes (Renne et al., 2009).

Serum cytokines assessment

Blood samples were collected in serum separator tubes and centrifuged at 3500 × g for 10 min. Serum samples were aliquoted and stored at −80°C until assayed for cytokines. Diluted serum samples from day 1 and day 2 groups were used to quantify cytokine proteins (tumor necrosis factor-α [TNF-α], interleukin-6 [IL-6], IL-1β, KC/GRO, and interferon-γ [IFN-γ]) using the V-PLEX proinflammatory panel 2 (rat) kit according to the manufacturer’s protocol (Meso Scale Discovery, Gaithersburg, MD). The electrochemiluminescence signals for each cytokine protein were detected using the MESO QuickPlex SQ 120 (Mesoscale Discovery Inc., Rockville, MD).

Complete blood count and platelet aggregation assessment

Complete blood counts were run on EDTA-preserved blood using a Beckman-Coulter AcT blood analyzer (Beckman-Coulter Inc., Fullerton, CA), which included lymphocytes and platelet numbers. Citrated blood sample tubes were used to assess blood coagulation. Platelet aggregation profiler (Platelet Aggregation Profiler model PAP-8E, Bio/Data Corp., Horsham, PA) was used to assess platelet aggregation. Briefly, citrated blood was centrifuged at 200 × g for 30 seconds, and the resulting platelet-rich plasma was collected. After separating an aliquot for aggregation determination, the remaining platelet-rich plasma was centrifuged at 2000 x g for 120 seconds to collect platelet poor plasma to use as blank. Adenosine diphosphate (ADP)-induced primary aggregation, rate of aggregation, and disaggregation were measured by adding 25 μl of ADP (2 × 10⁻⁴ M) to the platelet-rich plasma fraction at 37 ^oC in a platelet aggregation profiler. The absorbance of the platelet poor plasma fraction was used to blank each individual sample. Each blood sample was run in duplicate, and an average of these duplicates was used for statistical analysis. An aliquot of citrated platelet poor plasma was used to assess thromboplastin time using Enzygnost® TAT micro ELISA kit (Siemens Medical Solutions USA, Inc., Malvern, PA) following kit instructions.

Aortic ring contractility assessment ex vivo

Animals from the day 1 time point were used for the aortic ring ex vivo protocol, which is described in Snow et al. (2018). Briefly, aortic segments ~2 mm in length (with no associated connective tissues) were mounted on pins in 8 mL chambers of a multi-wire myograph system (model 620M, Danish Myo Technology, Aarhus, Denmark) filled with 7 mL Krebs-Henseleit buffer (KHB; 120 mm NaCl, 25 mm NaHCO₃, 11 mm glucose, 4.7 mm KCL, 1.2 mm MgSO₄, 1.2 mm KH₂PO₄, and 1 mm CaCl₂ at pH 7.4), warmed at 37˚C, and continuously oxygenated during the experiment (95% O₂ and 5% CO₂).

The aorta rings were allowed to equilibrate at a tension of 40 mN for 30 min and then a tension of 20 mN was set. Tissue viability was assessed using 60 mM of KCl for 10 min. After washing, endothelial integrity was assessed by pre-contracting vessels with 1 µm phenylephrine (PE) for 10 min followed by 1 µm acetylcholine (ACh) for 3 min. After washing with KHB, aortic ring segments were evaluated for vasocontraction (PE), endothelial-dependent vasorelaxation (ACh), and endothelial-independent vasorelaxation (sodium nitroprusside, SNP) to obtain cumulative concentration-response curves (1 nm–10 µm) using modified experimental protocols (Thompson et al., 2014). Myograph data were recorded in mN and acquired using LabChart 8 Pro software (AD Instruments, Colorado Springs, CO). Data for each aortic segment were normalized to the vessel surface area to produce segment stress (mN/mm²), as described previously (Thompson et al., 2014).

Thoracic aorta RNA isolation and real-time quantitative PCR

Animals from the day 1 time point were used to test aortic function and expression of genes related to vascular function and inflammation. At the time of necropsy, half of each thoracic aorta was quick frozen in liquid nitrogen for later RNA extraction. Total RNA was isolated from thoracic aortas of animals using fibrous tissue RNeasy mini kit (Qiagen, Valencia, CA) utilizing a proteinase-K digestion. One-step real-time polymerase chain reaction (RT-PCR) was carried out using the Platinum Quantitative RT-PCR ThermoScript One-Step System (Invitrogen, Carlsbad, CA). The kit protocol was used for PCR amplification. Primers for β-actin (control), endothelial nitric oxide synthase (eNOS), endothelin-1 (ET-1), tissue plasminogen activator (tPA), and tissue factor (TF) were obtained from Applied Biosystems, Inc. (Foster City, CA). Relative expression was calculated using the ΔΔCT method, with Wistar ND air-exposed animals as referent controls.

Data analysis

GraphPad Prism v8.4.2 software (San Diego, CA) was used to graph and analyze BALF injury/inflammation markers, circulating cells count and cytokine concentration, aortic RNA levels, CBC, and other data. For each endpoint, three independent Two-way ANOVA tests were performed to determine ozone effect for a given strain/diet, rat strain effect for given exposure/diet and diet effect for given exposure/strain. Holm-Sidak’s multiple comparison test was used to establish significant differences and a p-value <0.05 was considered statistically significant. Vascular response curves were compared using a repeated measure Two-Way ANOVA with non-linear regression analysis to compare the four-parameter best-fit values. The Holm-Sidak’s post-hoc test was used to correct for all multiple comparisons for vascular contractility data (p ≤ 0.05 as statistically significant). For histopathological data, differences in lesion incidence between groups were evaluated using a Fisher’s Exact Test.

RESULTS

Body weight, markers of pulmonary injury and inflammation

At the time of necropsy, Wistars on ND weighed nearly 150 grams heavier than GK rats (Wistar, 491 ± 7 grams; GK, 342 ± 3 grams). With HFD, GK gained 57 grams weight but Wistar rats gained only 33 grams (Wistar, 524 ± 7 grams; GK 399 ± 4 grams). The time course of weight gain and body fat accumulation in relation to HFD are explained in detail in Snow et al (2021) publication in this issue. Markers of pulmonary protein leakage and cell injury were assessed in the BALF after air or ozone exposure (day 1 and 2) and the 18-hr recovery period (day 2) (Figure 1A-F). As reported in several of our previous studies (Bass et al., 2013; Kodavanti et al., 2015; Miller et al., 2015), ozone exposure led to marked increases in lavage fluid protein and albumin in both strains and dietary groups. Protein and albumin leakage were higher in GK relative to Wistar rats on ND and HCD after day 1 and the 18-hr recovery period, but not day 2 (Figure 1A-F), suggesting earlier and longer vascular leakage response. Interestingly, this effect was less prominent in the HCD groups relative to ND. The activity of GGT, a BALF marker for airway cell injury, increased after day 2 and the 18-hr recovery period in ozone-exposed rats in both strains independent of diet (Figure 1G-I). The activity of NAG, a BALF marker of macrophage activation, increased after ozone exposure at day 1 and day 2 regardless of rat strain or diet and then partially subsided after the 18-hr recovery period (Figure 1J-L).

Figure 1.

Ozone-induced changes in BALF markers of lung injury in Wistar and GK rats maintained on a normal diet (ND) or high-cholesterol diet (HCD). Samples were collected within 2 hrs after each time point. The data show mean ± standard error (n=6/group). Significant (p ≤ 0.05) ozone effect is shown by “*” for matching strain/diet groups; strain effect by “†” for matching diet/exposure group; and diet effect by “‡” for matching strain/exposure group. GK, Goto-Kakizaki; BALF, bronchoalveolar lavage fluid; GGT, γ-glutamyl transpeptidase activity; NAG, N-acetyl glucosaminidase activity.

Immune cells including macrophages and neutrophils were assessed in the BALF as another indicator of lung inflammation. After day 1, ozone exposure was associated with higher macrophage numbers in Wistar and GK rats on HCD and ND. Similar ozone effects were observed with with each diet at day 2 and 18-hr recovery time point, although the effects were significant only for Wistar rats (Figure 2A-C). Neutrophils increased after ozone exposure at all time points in both strains regardless of diet; this increase was more pronounced in Wistar rats on HCD after the 18-hr recovery period (Figure 2D-F).

Figure 2.

Ozone-induced changes in BALF markers of lung inflammation in Wistar and GK rats maintained on a normal diet (ND) or high-cholesterol diet (HCD). Samples were collected within 2 hrs after each time point. The data show mean ± standard error (n=6/group). Significant (p ≤ 0.05) ozone effect is shown by “*” for matching strain/diet groups; strain effect by “†” for matching diet/exposure group; and diet effect by “‡” for matching strain/exposure group. GK, Goto-Kakizaki; BALF, bronchoalveolar lavage fluid.

Histopathological findings in lung

Lung tissues collected after day 1 of air or ozone exposure were processed, sectioned, stained by H&E, and examined by light microscopy. Ozone exposure resulted in mild to moderate acute bronchiolar epithelial necrosis and inflammation (Table 1). These changes were most prominent in and around the terminal bronchioles (Figure 3). Effects were multifocal and discrete, with normal bronchioles often intermixed with affected ones in the same section. Affected bronchioles contained sloughed epithelial cells admixed with macrophages and scattered neutrophils, erythrocytes, mucin, fibrin, and cell debris. Intact bronchiolar epithelial cells were often found with loss of ciliation. Minimal to moderate amounts of edema were present within peribronchiolar and perivascular areas in ozone-exposed groups, often with small numbers of neutrophils. Pulmonary effects of ozone were not influenced by rat strain or diet, suggesting that the differences in ozone-induced peripheral effects are not proportional to the degree of lung injury. The incidence (number of animals showing a lesion/total number of animals evaluated) and severity (the mean severity score assigned to the group) for lung lesions are shown in Table 1.

Table 1.

Histological findings in the lung after acute ozone exposure in Wistar and GK diabetic rats receiving normal diet (ND) or high-cholesterol diet (HCD).

Histologic change:			Alveolar macrophage aggregation	Focal mixed cell infiltrate	Bronchiolar acute mixed cell inflammation	Intra-bronchiolar mucus or fibrin	Bronchiolar epithelial necrosis	Peribronchiolar/perivascular edema
Rat model	Ozone	HCD	incidence (severity score)	incidence (severity score)	incidence (severity score)	incidence (severity score)	incidence (severity score)	incidence (severity score)
Wistar	n	n	0/6 (0.0)	2/6 (0.3)	0/6 (0.0)	0/6 (0.0)	0/6 (0.0)	1/6 (0.2)
GK	n	n	2/6 (0.3)	1/6 (0.2)	0/6 (0.0)	0/6 (0.0)	0/6 (0.0)	0/6 (0.0)
Wistar	y	n	2/6 (0.3)	0/6 (0.0)	6/6 (1.5)*	5/6 (0.8)*	6/6 (2.3)*	6/6 (1.8)*
GK	y	n	0/6 (0.0)	1/6 (0.2)	6/6 (1.2)*	6/6 (1.2)*	6/6 (1.8)*	6/6 (2.2)*
Wistar	n	y	1/6 (0.2)	2/6 (0.3)	0/6 (0.0)	0/6 (0.0)	0/6 (0.0)	1/6 (0.2)
GK	n	y	2/6 (0.3)	2/6 (0.3)	2/6 (0.3)	2/6 (0.3)	1/6 (0.2)	1/6 (0.2)
Wistar	y	y	2/6 (0.3)	1/6 (0.2)	6/6 (1.3)*	4/6 (0.6)	6/6 (2.2)*	6/6 (2.2)*
GK	y	y	0/6 (0.0)	0/6 (0.0)	6/6 (1.3)	6/6 (1.3)	6/6 (1.8)*	6/6 (1.7)*

Values represent incidence of each finding with the average severity score (across all animals in the group) in parentheses. Severity scores were based on a qualitative 0–4 scale assessment (0=absent, 1=minimal, 2=mild, 3=moderate, 4=severe). Alveolar macrophage aggregation represents foci of macrophages within air spaces of alveoli and/or terminal bronchioles (alveolar histiocytosis). Focal mixed cell infiltrate refers to localized aggregates of lymphocytes, histiocytes, and neutrophils within alveoli. See Figure 3 for representative images.

^*indicates a significant group difference (p ≤ 0.05) based on a 2-tailed Fisher’s Exact Test for ozone vs. respective air control group (for same animal model and dietary group). No significant differences were observed for HCD vs. respective ND groups (for the same exposure group or animal model) or for Wistar vs. GK rats (for the same exposure condition or diet). GK = Goto-Kakizaki.

Figure 3.

Ozone effects on lung histopathology. Representative images of (A) a normal terminal bronchiole (Wistar, filtered air for 1 day) and (B) a terminal bronchiole with epithelial necrosis, intraluminal and peribronchiolar mixed cell inflammation, and peribronchiolar edema (Wistar, 1.0 ppm ozone for 1 day). Objective magnification = 20x.

Blood leukocytes

Previous reports have shown that ozone exposure may impact circulating lymphocytes (Henriquez et al., 2018, 2019) and that HCD may induce systemic inflammation and prothrombogenic effects (Subramanian and Chait, 2009). In this study, circulating white blood cells were increased by HCD in both rat strains (Wistar>GK) with non-significant ozone effect (Figure 4A-C). Circulating lymphocytes also increased with HCD but this effect was significant only for Wistar rats. A significant ozone-induced decline of lymphocytes was noted at day 1 in both Wistar and GK rats on ND (Figure 4D-F). On day 2 ozone-induced decrease of circulating lymphocytes was significant in GK on ND and Wistar on HCD. Ozone effects were not evident at recovery time point. Unlike humans, where circulating white blood cells comprise of 20–40% lymphocytes, 2–8% monocytes and 40–60% neutrophils (Valiathan et al., 2016), in rats nearly 75% of circulating white blood cells are lymphocytes, 20% neutrophils and only 3–4% monocytes (Faas et al., 2003). Thus, the decline in circulating lymphocytes of HCD animals after ozone-exposure but increases in overall while blood cells suggest increases in non-lymphocytic white blood cells such as monocytes and/or neutrophils. The involvement of these specific cell types needs to be determined in future studies.

Figure 4.

Circulating leukocytes and lymphocytes after air or ozone (1 ppm) exposure in Wistar and GK rats maintained on a normal diet (ND) or high-cholesterol diet (HCD). Samples were collected within 2 hrs after each time point. The data show mean ± standard error (n=6/group). Significant (p ≤ 0.05) ozone effect is shown by “*” for matching strain/diet groups; strain effect by “†” for matching diet/exposure group; and diet effect by “‡” for matching strain/exposure group. GK, Goto-Kakizaki.

Platelets, thrombin/antithrombin levels, and platelet aggregation

Circulating platelet numbers were determined as a part of the complete blood count to examine potential thrombogenic effects of diet and ozone in healthy and diabetic rats. Interestingly, GK rats had consistently lower number of platelets in the blood compared to Wistar rats. There were no diet-related changes in platelet numbers, and the small increases observed after ozone exposure were evident in Wistars on ND at day 2 and on HCD at 18-hr recovery time point (Figure 5A-C). Plasma thrombin-antithrombin (TAT) complex levels were measured as a complimentary marker of thrombogenicity. There were no clear differences between strains at any timepoint. After day 2, ozone resulted in higher TAT complex levels but only within HCD groups (Wistar>GK) (Figure 5D-F), suggesting a potential interaction between ozone and diet related to thrombogenicity.

Figure 5.

Ozone-induced changes in blood platelets and plasma thrombin/antithrombin (TAT) complex levels in Wistar and GK rats maintained on a normal diet (ND) or high-cholesterol diet (HCD). Samples were collected within 2 hrs after each time point. The data show mean ± standard error (n=6/group). Significant (p ≤ 0.05) ozone effect is shown by “*” for matching strain/diet groups; strain effect by “†” for matching diet/exposure group; and diet effect by “‡” for matching strain/exposure group. GK, Goto-Kakizaki.

Platelet aggregation may also serve as an indicator of enhanced blood coagulation. GK rats on HCD exposed to ozone had a small increase in ADP-induced primary aggregation at day 1. No other ozone, diet, or strain differences were noted (Figure 6A-C). The rate of aggregation was generally lower in GK rats when compared to Wistar rats, which may relate to lower platelet count in GK rats at baseline. Similar to primary aggregation, GK rats on HCD had higher rate of aggregation on day 1 after ozone exposure (Figure 6D-F). The rate of platelet disaggregation may indicate the stability of thrombi. Atherogenic diet was associated with increased platelet disaggregation in both Wistar and GK rats (Figure 6G-I). Ozone resulted in higher disaggregation at day 2 in Wistar rats on HCD, consistent with higher TAT complexes.

Figure 6.

Ozone-induced changes in platelet primary aggregation, aggregation rate, and disaggregation in Wistar and GK rats maintained on a normal diet (ND) or high-cholesterol diet (HCD). Samples were collected within 2 hrs after each time point. Platelet aggregation was determined immediately following sample collection using platelet-rich plasma obtained from blood samples collected in citrated anticoagulant vacutainer tubes. The data show mean ± standard error (n=6/group). Significant (p ≤ 0.05) ozone effect is shown by “*” for matching strain/diet groups; strain effect by “†” for matching diet/exposure group; and diet effect by “‡” for matching strain/exposure group. GK, Goto-Kakizaki.

Circulating cytokines

HCD has been associated with increased systemic inflammation (Subramanian and Chait, 2009). Here, we wanted to determine if circulating cytokines were influenced by diabetes, diet, and ozone exposure in our models. Cytokine levels were measured in serum samples collected following day 1 and 2 of air or ozone exposure. HCD resulted in higher IFN-γ in ozone-exposed rats after day 1 in both strains (Figure 7A-B). Circulating IL-4 levels were lower in GK relative to Wistar rats, but no diet or ozone exposure-related effects were noted (Figure 7C-D). In contrast, IL-6 levels increased after day 1 of ozone exposure in both Wistar and GK rats irrespective of diet and at day 2, this effect was significant only in ozone-exposed Wistar rats (Wistar>GK) (Figure 7E-F). There were no consistent changes related to ozone exposure, diet, or rat strain on IL-10 (Figure 7G-H). IL-13 levels were higher in Wistar relative to GK rats. HCD was associated with increases in IL-13 in both strains, but no significant ozone-related changes were noted (Figure 7I-J). KC-GRO levels were generally higher in all rats receiving HCD; ozone further exacerbated this response in rats on HCD (Wistar>GK) (Figure 7K-L). HCD resulted in markedly higher TNF-α in both strains (Wistar>GK) and ozone further increased circulating levels at day 2 in Wistars on HCD (Figure 7M-N).

Figure 7.

Ozone-induced changes in serum cytokines in Wistar and GK rats maintained on a normal diet (ND) or high-cholesterol diet (HCD). Samples were collected within 2 hrs after each time point. The data show mean ± standard error (n=6/group). Significant (p ≤ 0.05) ozone effect is shown by “*” for matching strain/diet groups; strain effect by “†” for matching diet/exposure group; and diet effect by “‡” for matching strain/exposure group. GK, Goto-Kakizaki; INF-γ, interferon-gamma; IL-4, interleukin-4; IL-6, interleukin-6; IL-10, interleukin-10; IL-13, interleukin-13; KC-GRO, keratinocyte chemoattractant (KC)/human growth-regulated oncogene (GRO); TNF-α, tumor necrosis factor-alpha.

Aortic contractile response to diet and ozone in healthy and diabetic rats

Thoracic aorta segments from animals exposed to air or ozone for 1 day were isolated for myographic assessment of vascular reactivity. There were significant strain-, diet-, and ozone-related differences in the vasocontraction response induced by PE. Baseline vasocontraction response to increasing concentrations of PE was more pronounced in Wistar relative to GK regardless of diet (Figure 8A and B). Ozone exposure led to marked increase in PE-induced vasocontraction in a dose-dependent manner, but only in Wistar rats receiving ND. In contrast, no ozone effect on vasocontraction was observed in GK rats on ND (Figure 8A). In Wistar rats, HCD nearly completely abolished the ozone-induced contractile response to PE seen in ND rats (Figure 8B).

Figure 8.

Ozone-induced changes in aortic contractility after day 1 in Wistar and GK rats on a normal diet (ND) or high-cholesterol diet (HCD). Samples were collected within 2 hrs after exposure to air or 1.0 ppm ozone for 6 hrs for 1 day. The data show mean ± standard error (n=6/group). * indicates a significant ozone effect (p ≤ 0.05) in matched strain/diet groups. Significant difference between rat strains within the same diet and exposure groups are indicated by † (p ≤ 0.05) for Wistar and †† (p=0.05) for GK rats. GK, Goto-Kakizaki; PE, phenylephrine; ACh, acetylcholine; SNP, sodium nitroprusside.

The addition of increasing concentrations of ACh to PE-contracted aortic rings was used to determine endothelial-dependent vasorelaxation responses. Aortic rings of GK rats on ND and HCD relaxed more efficiently in response to ACh than the rings from Wistar rats; there were no significant ozone- or diet-related differences in either strain (Figure 8C). To assess endothelial-independent vasorelaxation, graded concentrations of SNP were added to the PE-contracted aortic rings. Although there were a few strain-related differences that were found to be significant, the changes appear very small and might not be biologically meaningful. No ozone or diet effects were evident in SNP-induced vasorelaxation (Figure 8E-F).

Aortic expression of genes involved in vascular function and thrombosis

Changes in inflammatory and prothrombotic markers expression in the thoracic aorta can occur following exposure to inhaled pollutants or other inflammatory conditions (Kodavanti et al., 2011). Ozone exposure increased e-NOS expression in GK but not Wistar rats on ND, while HCD increased e-NOS expression in GK rats exposed to air or ozone and Wistar rats exposed to ozone (Figure 9A). Thus, ozone-induced contractile response in Wistar on ND coincided with the lack of increase in e-NOS expression. There were no significant changes in tissue factor (TF) expression in the aorta due to ozone or diet in Wistar rats, however air-exposed but not ozone-exposed GK rats fed HCD had increased expression (Figure 9B). Tissue plasminogen activator (tPA) expression was significantly increased in GK rats on HCD exposed ozone relative to ND (Figure 9C). The expression of endothelin-1 (ET-1), a vasoconstrictor, was not different between strains, diet groups, or air/ozone exposure groups (Figure 9D).

Figure 9.

Ozone-induced changes in mRNA of gene markers for vascular function and inflammation in the aorta in Wistar and GK rats maintained on normal diet (ND) or high-cholesterol diet (HCD). Samples were collected within 2 hrs after exposure to air or 1.0 ppm ozone for 6 hrs for 1 day. The data show mean ± standard error (n=6/group).. Significant (p ≤ 0.05) ozone effect is shown by “*” for matching strain/diet groups; strain effect by “†” for matching diet/exposure group; and diet effect by “‡” for matching strain/exposure group. GK, Goto-Kakizaki; eNOS, endothelial nitric oxide synthase; TF, tissue factor; tPA, tissue plasminogen activator; ET-1, endothelin-1.

DISCUSSION

Two important contributors to vascular disease, diabetes mellitus and vascular disease induced by atherogenic diet, are also potential risk factors of air pollution-induced cardiovascular effects. The goal of this study was to examine how these underlying conditions in rodent models may exacerbate pulmonary and vascular responses to a prototypic air pollutant, ozone. Using healthy Wistar and diabetic GK rats, we examined ozone-induced lung injury, thrombogenic potential, systemic inflammation, and vascular contractility in context of a standard or atherogenic diet. We found that GK rats had a more prolonged vascular leakage response compared to Wistar rats, irrespective of diet. The atherogenic diet increased circulating total leukocytes in both strains and increased several inflammatory cytokines, suggesting increased systemic inflammation. This effect on cytokines was more pronounced in Wistar rats and was modestly exacerbated by ozone. GK rats fed ND had impaired vasocontraction response to PE and no response to ozone, whereas Wistar rats had marked vasocontraction following ozone exposure. The HCD nearly abolished this response to ozone in Wistar rats. Changes in vascular contractility differences could be explained by aortic eNOS expression differences. These data demonstrate that both diabetes and HCD may independently exacerbate specific effects of inhaled pollutants and contribute to pulmonary and vascular disease susceptibility.

We have previously utilized this unique rat model to investigate susceptibility factors to air pollutants (Snow et al., 2017; McGee Hargrove et al., 2018). These rats have impaired insulin secretion leading to insulin resistance, hyperglycemia, and glucose intolerance (Portha et al., 2012; Nagao et al., 2020), similar to advanced forms of human type 2 diabetes (e.g. Guest 2019); however, the pathogenesis of age-related diabetes in GK rats differs from that of humans. For example, impaired insulin secretion due to genetic defects is generally not a feature of type 2 diabetes pathogenesis in humans (Portha et al., 2012; Nagao et al., 2020). GK rats also develop obesity, endothelial dysfunction (Cheng et al., 2001), and impaired vascular and metabolic functions when fed a high-fat diet (Sena et al., 2008; Kengkoom et al., 2013). As shown in our companion paper to the current study, GK rats had increased relative body fat mass which was exacerbated by HCD and demonstrated key features of diabetes (Snow et al., 2021). We report herein that HCD also induced markers of systemic inflammation, including higher circulating cytokines. Collectively, these findings indicate that HCD may enhance susceptibility to vascular and pulmonary effects of ozone in the Wistar and GK rat models.

During acute exposure to an inhaled stressor such as ozone, the initial event may be direct injury to pulmonary epithelial cells and vessels, followed by activation of various secondary inflammatory and compensatory biological processes. The degree to which these processes occur depends on the underlying (patho)physiological condition of the host and specific biomarker being examined. We predicted that GK rats on an atherogenic diet would have the greatest degree of lung injury and inflammation resulting from acute ozone exposure, given underlying metabolic and vascular stress. However, no major strain- or diet-related differences were noted in histopathological effects or BALF biomarkers of lung injury in this study, except for the early and persistent protein leakage in GK relative to Wistar rats. The latter finding may relate to lung vascular integrity and a compromised ability to repair ozone-induced injury in GK rats. By comparison, acrolein exposure in this model did not exacerbate respiratory protein leakage relative to Wistar rats (Snow et al., 2017). We have also observed that in rat models with genetic predisposition to obesity, ozone exposure did not result in increased lung protein leakage and inflammation relative to healthy rat strains (Kodavanti et al., 2015; Snow et al., 2018). These findings contrast with studies in mouse models, which have reported exacerbated pulmonary response to ozone, such as pulmonary resistance, BALF neutrophils, and airway hyperresponsiveness in obese vs. lean mice (Shore 2017). Collectively, these studies highlight the complexity of interactions between metabolic status and lung injury and the importance of understanding physiological status and the model.

Since nearly 75% of circulating white blood cells are accounted by lymphocytes (Faas et al., 2003), the overall increase in white blood cells in both stains fed HCD suggests that increased blood leukocytes may be accounted by monocytes and/or neutrophils. It has been shown that HCD leads to accumulation of foamy monocytes in circulation (Foster et al., 2015). It is likely that extravasation of these cells to the lung in HCD groups might not be further exacerbated after ozone exposure, since the increases in alveolar macrophages were similar for ND and HCD. The increased levels of blood lipids due to HCD (Snow et al., 2021) might inhibit extravasation of white blood cells to the lung capillaries leading to a high number of leukocytes in the blood (Wu et al., 2009; Zhang et al., 2014). Exposure to ozone in Wistar Kyoto rats leads to lymphopenia (Henriquez et al., 2018; 2019). In this study we noted lymphopenia on day 1 in Wistar and GK that are on ND, but not HCD, suggesting that the ozone-induced re-distribution of circulating lymphocytes may also be affected by HCD. Underlying hyperglycemia might not play a significant role in this biological response (Proto et al., 2018), since no significant differences were noted between Wistar and GK rats. Nevertheless, leukocyte adhesion to pulmonary microvessels and extravasation to lung have been proposed to be involved in inflammation after air pollution exposure. While short term ozone exposure in humans increases pulmonary bronchiolar and microvascular P-selectin expression (Krishna et al., 1997); exposure to environmental tobacco smoke has been reported to increase leukocyte trafficking in the pulmonary microvessels (Rao et al., 2009).

A secondary objective of this study was to evaluate thrombogenic responses to ozone in context of diabetes and HCD. Although rats do not develop atherosclerotic lesions in the aorta similar to humans, aortic changes in prothrombotic and contractile markers have been noted after air pollutant exposures in our previous studies (Kodavanti et al., 2011; Bass et al., 2015). Thrombogenic potential has also been shown in other air pollution studies (Nemmar et al., 2003; Emmerechts et al., 2010). We predicted that ozone would alter platelet aggregation and markers of thrombosis specifically in HCD-fed GK rats. While the platelet number was significantly lower in GK relative to Wistar rats that were exposed to air and fed the ND, HCD-fed Wistar rats showed higher TAT complexes following day 2 of ozone exposure. We also noted that HCD, but not ozone or underlying diabetes, resulted in higher platelet disaggregation. It has been shown that tPA promotes disaggregation of platelets in the plasma (Loscalzo et al., 1987). We noted that tPA mRNA expression was increased in aortas of GK but not Wistar rats on atherogenic diet, whereas diet-induced disaggregation appeared to be more pronounced in Wistar than GK rats. Thus, limited effects of atherogenic diet and ozone in Wistar and GK rats were not consistent across all markers examined.

Systemic inflammation is a common feature of type 2 diabetes (Donath et al., 2019) and diet-induced atherosclerosis (Subramanian and Chait, 2009), and it has been variably associated with exposure to air pollution (Calderón-Garcidueñas and de la Monte, 2017). We hypothesized here that changes in circulating cytokines, as biomarkers of systemic inflammation, would provide insights into diabetes, HCD, and ozone interactions. Air-exposed diabetic GK rats on ND did not have higher levels of circulating cytokines relative to Wistar rats, in contrast to studies of human diabetics (Herder et al., 2019). Treatment with HCD resulted in higher levels of IFN-γ, IL-6, TNF-α, IL-13, and KC-GRO, which coincided with increases in circulating leukocytes and cholesterolemia (Snow et al., 2021). These effects were variably present in both strains but, notably, they were not exacerbated by underlying diabetes in GK rats, suggesting that other factors beyond hyperglycemia and insulin insufficiency, such as obesity, chronicity of insulin resistance, and age, may contribute to increased systemic inflammation. Ozone also induced higher circulating IL-6 and KC-GRO in both strains specifically in HCD groups, suggesting a potential interactive influence of diet and ozone exposure on cytokine responses. Collectively, these findings suggest that underlying metabolic stress associated with consumption of HCD may serve as a predisposing factor to systemic inflammation due to air pollution exposure.

Air pollution exposure has been associated with systemic inflammation and vascular dysfunction (Pope et al., 2016) We have previously shown that a single exposure to ozone increases the contractile response of the thoracic aorta in male Wistar Kyoto rats (Snow et al., 2018). Here we assessed the response in Wistar and GK rats on ND and HCD. As observed in our previous study (Snow et al., 2018), a single ozone exposure led to marked increase in ex vivo PE-induced aortic vasocontraction in Wistar rats on ND. While this response was inhibited in GK rats on ND or HCD relative to Wistar rats at baseline, ozone did not significantly change aortic contractility or relaxation in GK rats. Importantly, HCD in Wistar rats completely inhibited ozone-induced vasocontraction in response to PE, producing a GK-like phenotype. eNOS plays an important role in endothelial-dependent vasorelaxation by increasing NO levels (Dart and Chin-Dusting, 1999). HCD has been shown to induce vascular dysfunction associated with reduced vasorelaxation response by reducing NO availability (Zhang et al., 2020). In our study, the HCD-induced reversal of ozone effects in Wistar rats was associated with increases in aortic eNOS expression. Ethanol-induced vasocontraction brought about by inhibition of potassium channels of large conductance in rat cerebral artery smooth muscle was reversed by cholesterol enrichment (Bisen et al., 2016). It is interesting to note that although we did not measure NO, the eNOS mRNA expression was increased by ozone in GK rats on ND, which did not show vasocontraction to ozone unlike Wistar on ND (with no increase in eNOS after ozone exposure). Ozone-exposed Wistar on HCD and both air and ozone-exposed GK rats on ND and HCD had increased eNOS expression which did not show excessive vasocontraction in response to ozone. Thus, the lack of ozone response is associated with increased eNOS expression which supplies NO for vascular relaxation and signifies its contribution to differential vascular effects. It is interesting to note that in our previous study, Wistar Kyoto rats fed fish oil supplemented diet, but not coconut or olive oil-rich diets, resulted in reduced ozone-induced vasocontraction responses (Snow et al., 2018).

Availability of NO through increased eNOS activity plays an important role in inhibiting leukocyte endothelial adhesion acutely (Gao et al., 2018), the long-term inhibition of NO production may not contribute to leukocyte adhesion through NO-dependent manner (Kuhlencordt et al., 2004). The ability of Wistar rats on ND to induce eNOS expression and the lack of the ability to increase eNOS in GK and HCD-fed Wistar could contribute to differential leukocyte trafficking to the lung following ozone exposure. Although the precise contribution of eNOS activation could not be ascertained from our data, it has been shown that in muscle microvasculature the AKT/eNOS pathway was responsible for resveratrol-induced inhibition of leukocyte adhesion in high fat diet-fed mice (Huang et al., 2018). HCD-induced increases in the number of circulating leukocytes in Wistar and GK rats and ozone-induced lymphopenia may directly or indirectly involve effect on eNOS expression.

We should note several limitations of the current study. We did not use female rats because of the already complicated experimental design and the goal to follow up on our earlier work in male GK rats (Snow et al., 2017). Second, the GK diabetic model is non-obese, which is often a major contributing factor to type 2 diabetes pathogenesis. The GK rat strain develops insulin resistance via different mechanisms, potentially impacting underlying vascular disease. Further research is needed in understanding the mechanism by which eNOS may impact vasoconstriction response and transendothelial migration of immune cells in animal models of diabetes and HCD. Finally, this study did not address the susceptibility aspect with subchronic exposure scenario, which might culminate in greatly exacerbated cardiopulmonary effects of ozone.

In summary, we show that GK rats with non-obese type 2 diabetes demonstrate quicker and prolonged, but not necessarily heightened, pulmonary injury responses to ozone compared to Wistar rats. HCD caused systemic inflammation, as evidenced by increased leukocytes and circulating cytokine levels in both strains, and ozone exacerbated increases in some cytokines. Strain-related differences in platelet number and (dis)aggregation were noted, in which Wistar rats had higher number of platelets, rate of aggregation with HCD, and HCD-induced increases in disaggregation relative to GK rats. Large strain differences in ex vivo vascular contractility were also noted, including impaired vasocontraction at baseline in GK rats and reduced vasorelaxation responses in Wistar rats. Ozone exposure induced vasocontraction and impaired vasorelaxation, but only in Wistar rats on ND. Interestingly, HCD nearly abolished this ozone-induced response in Wistar rats. Ozone- and HCD-induced vasocontraction changes supported higher aortic eNOS expression in both strains. These data demonstrate ways in which underlying diabetes and HCD may independently exacerbate pulmonary, systemic, and vascular effects of inhaled pollutants. These findings also indicate that healthy and diabetic individuals may show different inflammatory and vascular contractility responses to HCD and acute exposure to air pollutants.