In the present study, we showed an increased cardiovascular vulnerability of diabetic mice to particulate air pollution. We found an aggravation of the impact of acute exposure to DEP in diabetic mice substantiated by increase of systemic inflammation (leukocytosis and CRP), oxidative stress (8-isoprostane), hypoxemia, hepatotoxicity and acceleration of coagulation comprising thrombosis in vivo, platelet aggregation in vitro, and the increase in plasma concentrations of PAI-1 and vWF.
In the present study, we used a pertinent animal model of type 1 diabetes, i.e. STZ-induced diabetes in mice [11, 13] and assessed the acute effects of a relevant type of pollutant particles, namely DEP. The dose of DEP used here 0.4 mg/kg (10 μg/mouse) is lower than the dose previously tested, i.e. 0.5 mg/kg (15 μg/mouse) or 1 mg/kg (30 μg/mouse) because we hypothesized that the effects of DEP would be aggravated in STZ-induced type 1 diabetes in mice. DEP was given to mice by i.t. instillation because it provides more accurate dosing, given that mice are nose breathers that filter most inhaled particles . In 2002, the United States Environmental Protection Agency reported a range of maximal city PM10 concentrations between 26 and 534 μg/m3
. Several large cities in the world have much higher levels of PM10, with annual averages of 200 to 600 μg/m3 and peak concentrations frequently exceeding 1,000 μg/m3
. Using the highest value in the United States and assuming a minute ventilation of 6 l/min (~8.6 m3 over 24 hours) for a healthy adult at rest, the total dose of PM inhaled over 24 hours would be 4,614 μg . Exposure of a human to a daily dose of 4,614 μg of PM would correspond to more than 35 μg of PM exposure for a mouse (25 grams in size) with minute ventilation of 35–50 ml/min . The dose we tested here (10 μg/mouse) is lower than the comparative human dose of ± 35 μg/mouse reported by Mutlu et al. .
Our data show that in non-diabetic mice, at the dose and regimen studied, DEP did not affect the number of leukocytes or the CRP concentration in plasma. Previously, 24 h post-exposure to higher doses of DEP, i.e. 15 μg/mouse (0.5 mg/kg) or 30 μg/mouse (1 mg/kg), we found no increase in the number of leukocytes [18, 19]. No significant differences were observed between control diabetic and non-diabetic mice. Interestingly, DEP exposure induced a leukocytosis and a significant increase of CRP, indicating the occurrence of systemic inflammation. Our finding corroborate epidemiological studies that have reported positive associations between air pollution and indicators of systemic inflammation such as leukocyte numbers, interleukin 6 and CRP [3, 20]. Remarkably, it has been reported that these associations were stronger and most consistent in individuals with diabetes . We have recently reported that repeated exposure to DEP causes an increase in CRP concentration and that the pre-treatment with the anti-inflammatory and antioxidant curcumin returned the CRP concentrations to control levels .
We have recently demonstrated that 24 h following their i.t. instillation, DEP (0.5 and 1 mg/kg) caused pulmonary and systemic oxidative stress responsible for systemic inflammation, and that the pretreatment with a cysteine prodrug L-2-oxothiazolidine-4-carboxylic acid abrogated these effects through its ability to balance oxidant-antioxidant status . In the present study, as a marker for oxidative stress, we selected to measure the plasma concentrations of 8-isoprostane. Isoprostanes are a family of eicosanoids of nonenzymatic origin, produced by the random oxidation of tissue phospholipids by oxygen radicals. Elevated levels of isoprostanes have been found in serum, plasma, and urine of heavy smokers  and lung tissue of mice expose to carbon nanoparticles . Here, we found that plasma 8-isoprostane concentrations were significantly increased after the pulmonary exposure to DEP in diabetic mice versus diabetic mice exposed to saline or non-diabetic mice exposed to DEP. It is well-established that oxidative stress plays a key role in the pathogenesis of of diabetes mellitus . Diabetic patients usually have significantly elevated concentrations of 8-OHdG in their serum and decreased levels of glutathione . Our data are in agreement with previous findings which reported that PM2.5 exposure causes aggravation of plasma oxidative stress in STZ-diabetic rats compared to nondiabetic rats .
While PaCO2 was not affected by DEP in both diabetic and non-diabetic mice, the PaO2 was significantly decreased in diabetic mice exposed to DEP compared to diabetic mice exposed to saline or non-diabetic mice exposed to DEP. We recently demonstrated that DEP exposure in hypertensive mice significantly decreased the PaO2 compared with DEP-treated normotensive mice. Moreover, using a rat model of cisplatin-induced acute renal failure, we have recently shown a decrease in PaO2 following DEP exposure . Our findings are in agreement with epidemiological studies that suggested that pollution may result in hypoxemia and that these effects might be most relevant in older and sicker individuals [26, 27].
In non-diabetic mice, DEP administration did not affect the plasma activities of AST and ALT compared to saline-exposed mice. No difference in the enzyme activities was found between saline-treated diabetic and saline-treated non-diabetic mice. Remarkably, the AST and ALT activities were increased in DEP-exposed diabetic mice compared to diabetic mice exposed to saline or DEP-exposed non-diabetic mice, indicating that DEP causes tissue damage in diabetic mice. Exposure to PM2.5 in healthy mice did not affect AST and ALT activities . However, it has been reported that pulmonary exposure of obese diabetic mice to DEP causes an increase in the activities of AST, ALT, the ratio of liver weight, and the magnitude of fatty change of the liver in histology . Epidemiological and clinical studies are needed to verify the occurrence of liver injury following the exposure to particulate air pollution in susceptible population.
A strong epidemiologic association has been observed between increased levels of PM and hospitalizations for heart disease among those who had diabetes compared with those who did not . The risk of coronary heart disease, stroke, and peripheral arterial disease is increased in persons with diabetes . Several experimental studies have reported that exposure to particles causes prothrombotic effects in the ear vein of rats , femoral vein and artery of hamsters [32–35] carotid artery of mice  and pial venule or arterioles of mice [18, 36]. Our data confirms the occurrence of prothrombotic effects following the exposure to DEP in non-diabetic mice compared to saline-treated non-diabetic mice. Similarly, we found a shortening in the thrombotic occlusion time in diabetic mice exposed to DEP compared to those exposed to saline. Interestingly, the degree of shortening in the thrombotic occlusion time was significantly greater DEP-exposed diabetic mice compared to DEP-exposed non-diabetic mice. Recently, we reported an aggravation of thrombotic events in hypertensive mice .
Along with the potentiation of prothrombotic effect in diabetic mice exposed to DEP, we found a significant decrease in platelet numbers in DEP-exposed diabetic mice compared to DEP-exposed non-diabetic mice or saline-exposed diabetic mice, this is indicative of platelet activation in vivo. A decrease of platelet numbers following exposure to particles has been reported from experimental and clinical studies [18, 37].
It has been suggested that inhaled particles may lead to systemic inflammatory response through the release of inflammatory mediators and oxidative stress within the lungs and/or systemically [1, 38]. Additional experiments showed that air pollution exposure is associated with rapid changes in autonomic nervous system balance, favouring sympathetic nervous system activation and parasympathetic withdrawal [1, 38]. Other lines of evidence also suggest that nanoparticulate inhalation can rapidly translocate from through the alveolar capillary barrier and directly affect the cardiovascular system [1, 38–40]. Because arteriolar thrombosis measured in vivo in our model depends mainly on the intensity of the vascular lesion and subsequent platelet recruitment and aggregation, we wanted to test the direct effect of DEP on platelet aggregation in whole blood of diabetic and non-diabetic mice in vitro. We, and others, have previously reported that DEP cause platelet aggregation [36, 41]. Our in vitro observations confirmed the occurrence of platelet aggregation following the addition of DEP. Clearly, an aggravated effect was observed in diabetic mouse blood with dose-dependent and significant graded effects at 0.25, 0.5 and 1 μg/ml DEP. Interestingly, in diabetic blood, the effect observed at 1 μg/ml was statistically significant compared with the same dose in non-diabetic mouse blood. This in vitro finding corroborates our in vivo observation. Such observation has, as far as we are aware, never been reported before. Our data corroborate a recent human study which reported that PM exposure was associated with a change in platelet function toward a greater prothrombotic tendency in diabetic patients .
Exposure to DEP in both diabetic and non-diabetic mice caused a significant increase of PAI-1 concentration compared to their respective controls. However, PAI-1 was increased in a greater fashion in the non-diabetic + saline group versus non-diabetic + DEP (+35%) group compared to diabetic + saline versus diabetic + DEP (+21%) mice. This difference can be explained by the fact that the concentration of PAI-1 in diabetic + saline group was significantly increased compared to non-diabetic + saline group. This finding corroborates the study of Tagher et al.  who found that PAI-1 concentration was significantly higher in patient with type 1 diabetes compared to healthy controls. We found a significant increase of circulating PAI-1 in diabetic mice exposed to DEP compared to diabetic mice exposed to saline or non-diabetic mice exposed to DEP. Raised concentrations of circulating PAI-1 have been acknowledged as an independent risk factor for the development of ischemic cardiovascular events [44, 45]. The concurrent increase of plasma PAI-1 and decrease of PaO2 that we observed corroborate the finding of pinsky et al.  who demonstrated that enhanced expression of PAI-1 is an important mechanism suppressing fibrinolysis under conditions of low oxygen tension. We recently reported that repeated exposure to DEP in healthy mice caused an increase of plasma PAI-1 concentration, and another study showed an increase in PAI-1 mRNA and protein concentrations in lung and adipose tissue of mice treated with PM . Moreover, Erdely et al.  showed that pulmonary exposure to carbon nanotube increased the active form as well as total PAI-1 in the circulation. We also found an increase of vWF in DEP-treated diabetic mice compared to saline-treated diabetic mice or DEP-treated non-diabetic mice. vWF reflects endothelial cell release and probably vascular reactivity. Vascular reactivity could results from the oxidative stress or direct effects of DEP that have presumably translocated into the systemic circulation. Moreover, vWF can mediate platelet adhesion to damaged endothelium, this could explain at least partly the observed exaggerated prothrombotic effects of DEP in diabetic mice. Elevated levels of vWF were observed in association with increased concentrations of particulate matter in patients with coronary heart disease . In healthy mice, increased vWF expression on hepatic endothelium was detected after intraarterial administration of nanoparticles .
Collectively, our data show an aggravation of various systemic and coagulation endpoints in vivo and in vitro in diabetic mice acutely exposed to DEP compared to non-diabetic mice exposed to DEP or diabetic mice exposed to saline. These exacerbations could be ascribed to the increase of systemic oxidative stress and inflammation observed particularly in diabetic mice exposed to DEP (Figure 1). Indeed, both oxidative stress and inflammation were reported to play a critical role in the cardiovascular effects of particulate air pollution [1, 18, 36] and diabetes . Nevertheless, further studies are required clarify the mechanisms underlying the effect of type 1 diabetes and DEP on the cardiovascular system and whether the observed effects are strain-dependent. A murine strain differences in airway inflammation caused by DEP has been previously reported .
We conclude that systemic and coagulation events are aggravated in type 1 diabetic mice acutely exposed to DEP. Our findings provide possible plausible explanation for the exacerbation of cardiovascular morbidity accompanying particulate air pollution in diabetic patients. Additional experiments are needed to evaluate the chronic effect of DEP on type 1 diabetes and determine whether the observed effects are related to the DEP-associated components or by particles themselves.