Diesel exhaust particles exposure exacerbates pro-thrombogenic plasma features ex-vivo after cerebral ischemia and accelerates tPA-induced clot-lysis in hypertensive subjects

Abstract

The combustion of fossil fuels, mainly by diesel engines, generates Diesel Exhaust Particles (DEP) which are the main source of Particulate Matter (PM), a major air pollutant in urban areas. These particles are a risk factor for stroke with 5.6% of cases attributed to PM exposure. Our aim was to evaluate the effect of DEP exposure on clot formation and lysis in the context of stroke. An ex-vivo clot formation and lysis turbidimetric assay has been conducted in human and mouse plasma samples from ischemic stroke or control subjects exposed to DEP or control conditions. Experimental DEP exposure was achieved by nasal instillation in mice, or by ex-vivo exposure in human plasma. Results show consistent pro-thrombogenic features in plasma after human ischemic stroke and mouse cerebral ischemia (distal MCAo), boosted by the presence of DEP. Otherwise, thrombolysis times were increased after ischemia in chronically exposed mice but not in the DEP exposed group. Finally, subjects living in areas with high PM levels presented accelerated thrombolysis compared to those living in low polluted areas. Overall, our results point at a disbalance of the thrombogenic/lytic system in presence of DEP which could impact on ischemic stroke onset, clot size and thrombolytic treatment.

Keywords

Stroke air pollution diesel exhaust particles clot thrombogenicity

Introduction

Air pollution in urban areas is a major concern for human health and, among its constituents, particulate matter (PM) accounts for the most toxic effects together with Ozone, sulfur dioxide and Nitric Oxide.^1,2 In this regard, a recent study has attributed an excess of 1.8 million deaths in 2019 in urban areas that exceeded the 10 μg/m³ annual average of PM_2.5.³ An important anthropogenic source of PM is the combustion of fossil fuels used in vehicles, including diesel vehicle emissions resulting in Diesel Exhaust Particles (DEP). According to their aerodynamic diameter, PM can be classified as: UltraFine Particulate Matter (below 0.1 µm), PM_2.5 (<2.5 µm) and PM₁₀ (<10 µm).^4,5 There are numerous studies associating PM air pollution exposure with high morbidity and mortality from cardiovascular diseases including stroke, myocardial infarction and ischemic heart disease.^6
–12 In this context, in 2005 the WHO established limits to these pollutants in order to safeguard human health¹³ and, more recently, they have been lowered to 5 µg/m³ for PM_2.5 and to 15 µg/m³ for PM₁₀ annual exposure limits,² although the European Environmental Agency (EEA) has set less restrictive annual limits for both PM_2.5 (25 µg/m³) and PM₁₀ (40 µg/m³).¹⁴

One of the cardiovascular diseases affected by PM is stroke, a prevalent disease with multifactorial causes that is positioned nowadays as the second cause of death globally with around 6 million people dying each year due to stroke.¹⁵ Furthermore, 90.5% of the stroke burden has been attributed to modifiable risk factors, among which, PM from air pollution accounts for a 5.6%.^16,17 Regarding stroke, the ischemic presentation accounts for 85% of all strokes¹⁷ and occurs after the occlusion of a blood vessel supplying the brain causing a sudden drop in cerebral blood flow (CBF) with subsequent focal tissue damage.¹⁸ The two main causes leading to ischemic stroke are atherosclerosis and cardioembolisms,¹⁹ both leading to the formation of a thrombus developed in the vessel or formed elsewhere (mainly in the heart) and reaching the brain circulation.²⁰ Ischemic stroke treatment consists of the intravenous thrombolytic drugs, mainly tissue plasminogen activator (tPA), that enhance clot lysis aiming at restoring the blood flow,^21,22 however it should be administered within 4.5 hours of symptoms onset.^23,24 More recently, mechanical thrombectomy has also been approved for large vessel occlusions and it can be conducted up to 24 hours following stroke onset.^25,26

In this context, the balance between thrombogenesis and fibrinolysis (endogenous or induced) is critical for the development of stroke. Previous research has suggested the interaction of PM/DEP exposure with increased pro-coagulation markers or deficient fibrinolysis. For example, increases in ambient PM_2.5 have been associated with shortened pro-thrombin time and increased sCD40L (present in platelets), relating PM with enhanced clot formation factors in healthy men.²⁷ Also, exposure to DEP (300 µg/m³) from a car engine for 1 hour during intermittent exercise suppressed the plasmatic increase of tPA induced by bradikynine, thus impairing the endogenous fibrinolytic capacity.²⁸ In a subsequent study, in men with previous well-controlled myocardial infarction, exposure to DEP, reduced the acute release of endogenous tPA, and aggravated the ST-segment depression induced by exercise, pointing to a greater myocardial ischemic burden.²⁹ Additionally, pre-clinical models have reported that mice exposed to DEP showed a reduced pro-thrombin time in an ex-vivo setting,³⁰ increased plasmatic fibrinogen in a thrombosis mouse model,³¹ and reduced the time to occlusion in an in-vivo arterial thrombosis rat model.³² Also, in a thrombosis hamster model, intratracheal DEP instillation enhanced arterial and venous platelet rich-thrombus formation after photochemical femoral injury model.³³

With this background, there is supporting evidence pointing to a role of DEP unbalancing the coagulation/fibrinolytic system towards a hypercoagulability stage, but some research has revealed opposite findings. For example, in healthy humans exposed to ambient PM_2.5, plasmatic fibrinogen concentrations were not associated with DEP exposure,³⁴ in elderly patients with cardiovascular disease no effect was observed in plasmatic fibrinogen with increases in ambient fine PM,³⁵ or in patients with type II diabetes increases in UFP concentrations were associated with a decrease in platelet CD40L, a platelet surface marker of activation.³⁶

Thus, the aim of the present study is to describe and explore potential changes in mouse and human plasma samples exposed to DEP in the context of ischemic stroke in a clot formation and tPA-induced thrombolysis ex-vivo assay.

Materials and methods

Physical characterization of diesel exhaust particles

DEP were purchased at the National Institute of Standards and Technology (NIST, Gaithersburg, USA), as Standard Reference Material (SRM): SRM2975 and SRM1650b being representative of different diesel exhaust engines. During their handling extreme security measures were taken: manipulation took place under a laboratory fume hood, double gloves, a filtering face piece-3 mask (FFP-3, Kimberly-Clark, Jackson safety, India) and safety glasses were used.

For physical characterization, DEP were diluted in Phosphate Buffered Saline (PBS, 1x) at different concentrations and studied by laser diffraction (Mastersizer 2000, Small Volume Sample Dispersion Unit, Malvern Panalytical, Netherlands and UK), Dynamic Light Scattering (Nano Zetasizer, Malvern Panalytical, Netherlands and UK) and electron microscopy imaging (Zeiss Merlin Field Emission Scanning Microscope) to determine the optimal protocol for the intranasal instillation considering the fractions of PM₁₀ and PM_2.5 contained in the fresh/thawn preparations and different sonication times. In brief, optical properties of the particles SRM1650b (Refractive Index, RI: 1.5, Absorption Index, AI: 0.1), SRM 2975 (RI: 2.45, AI: 0.5) and the dispersant: PBS (viscosity (cP): 0.8882, RI: 1.33, Dielectric constant (DC): 79) were set for the Mastersizer and Zetasizer analyzers. And DEP suspensions were further imaged by Field Emission SEM as follows: 3 μl of fresh and frozen SRM2975 and SRM1650b (7.5 mg/ml) sonicated for 10 minutes, were deposited on a silicon chip and let dry overnight. Uncoated samples were observed using a Zeiss Merlin Field Emission Scanning Microscope at 3Kv acceleration voltage and images were obtained with the secondary electron detector.

Pre-clinical regulation

All experimental procedures were approved and monitored by the Ethics Committee of Animal Experimentation of the Vall d’Hebron Research Institute (CEEA 57/17) with posterior Governmental approval and conducted in compliance with the Spanish and European Union directives (2010/63/EU). All personnel participating in the animal experimentation procedures were qualified with the corresponding training and certificates. All in vivo experiments were conducted and reported considering the ARRIVE 2.0 guidelines checklist.

In vivo exposure to diesel exhaust particles, experimental mouse model of cerebral ischemia and sample processing

Preparation of DEP and experimental design

SRM2975 and SRM1650b DEP were suspended in PBS at 7.5 mg/ml. The resulting aliquots of DEP or PBS (vehicle) were sonicated for 10 minutes using an ultrasonic bath (Branson 5800 Ultrasonic Cleaner, 2.5-gallon, 40 kHz) and frozen at −20°C until use. Before use, aliquots were thawed, vortexed and sonicated for 10 minutes.

A total of 64 male BALB/cAnNRj mice (6 to 9 weeks old) were purchased from Janvier Labs (Saint Berthevin, France). All animals were housed in groups of 3 to 5 in plastic cages (Innovive, San Diego, USA) where food and water were given ad libitum. Prior to the experimental procedure, mice were acclimatized to the housing facility (temperature: 24 °C, relative humidity: 30–70% and 12 hours light-dark cycle) for several days. For this exploratory study, mice were randomly allocated to acute or chronic intranasal instillation with SRM1650b-DEP or PBS for 3 days or 3 weeks protocols (Figure 1(a)): n = 10 for naïve acute PBS/DEP groups and n = 10 for naïve chronic PBS/DEP groups. A subset of animals underwent Middle Cerebral Artery occlusion (MCAo) surgery after the instillation protocol (Figure 1(b)): n = 6 for MCAo acute PBS/DEP groups, n = 6 for MCAo chronic PBS/DEP groups. Instillation was performed using a micropipette, with mice in supine position after 5% inhalative anesthesia (Isofluorane, Abbot Laboratories, Spain) in medicinal air (79%N₂, 21%O₂). All mice received 20 µl of PBS or DEP (total dose of SRM1650b was 150 µg) in small drops distributed in each nostril. The chosen dose was calculated assuming a mouse ventilating capacity of 0.06 m³ per day in a highly polluted city (30–60 μg/m³) during its lifetime following previous publications.^37,38 Afterwards, animals recovered from anesthesia in a recovery cage with a heating pad and under visual supervision. Body weight was recorded periodically to assess a possible detrimental effect of DEP instillation in the animals’ wellbeing.

Figure 1.

Experimental protocol to study the effects of DEP exposure in plasma thrombogenesis and thrombolysis in naïve and ischemic mice. (a) Naïve mice were acute or chronically exposed to PBS or DEP (SRM1650b), twenty-four hours later they were euthanized and plasma was obtained. (b) Mice were exposed to PBS or DEP (SRM1650b), 24 hours after the last instillation they underwent Middle Cerebral Artery occlusion (MCAo) and 24 hours after surgery they were euthanized, when plasma was obtained and cerebral infarct confirmed. (c) Cerebral blood flow reduction achieved after MCAo in the vehicle or DEP-exposed mice being below 80% of the individual baseline flow and (d) Schematic representation of the turbidimetric clot/lysis assay and the obtained Optical Density (OD) curve with the 9 parameters analyzed: 1. Lag time, 2. Clot formation rate (CRc), 3. Maximum Absorbance (MaxAbs), 4. Lysis Rate (LR), 5. Lysis time, 6. Clot area, 7. Clot time, 8. Lysis 50 from time 0 (Lys50_t0) and 9. Lysis 50 from time of MaxAbs (Lysis 50t_Max). D (day), X (euthanasia), ● Naïve ▲ Ischemic, ns (non-significant).

Permanent focal cerebral ischemia model

Twenty-four hours after the last exposure, a subset of animals underwent distal permanent MCAo surgery (Figure 1(b) and (c)) which causes a cortical ischemic injury.³⁹ Briefly, anesthesia was induced with 5% isofluorane (Abbot Laboratories, Spain) in medicinal air (79%N₂, 21%O₂) and maintained at 2%. Body temperature was controlled between 36–37°C using a heating pad, and an ophthalmic ointment (LipolacTM, Angelini Farmaceutica, Spain) was applied in the eyes during surgery. Later on, the temporal muscle was exposed, cut and retracted with a 6-0 silk suture (TB10, Suturas Aragó, Spain). The MCA was identified through the translucent skull with a microscope (Leica MS5, Leica, Heerburg, Switzerland) and the laser Doppler probe (Moor Instruments, UK) was positioned distally in a thinned skull spot onto the posterior branch of the MCA to monitor transcranial cortical blood flow. Next, a small craniotomy in the frontotemporal bone was performed using a high-speed microdrill (FST, 19007-05) to expose the MCA. Before the bifurcation of the anterior branch, the MCA was compressed by a 30 G blunted needle using a micromanipulator and after ensuring an 80% decrease in the cerebral blood flow (considering 100% the pre-occlusion value, Figure 1(c)) the MCA was electro-coagulated indirectly through the needle with the use of a cauterizer (Change-A-Tip^TM, Aaron Medical) and cut distally to the occluded site. After occlusion, muscle and skin were placed to their original position, the skin was sutured with a 5-0 silk (TB12, Suturas Aragó, Spain) and disinfected. Buprenorphine (0.1 mg/Kg, Divasa Farma-Vic S.A.) was administered subcutaneously to avoid post-surgical pain. Finally, mice were allowed to recover in a nursing cage with a heating pad under visual supervision.

Grip strength

The forelimb force was assessed with a grip strength meter apparatus which displays the maximum force exerted by both forelimb/paws (Harvard Apparatus) when grasping a grid, as a result of both motor and somatosensory functions. Since cortical infarcts induced by distal MCAo directly affect both primary and secondary somatosensory and motor areas, this test was conducted to assess post-stroke deficits. Briefly, at the end of the instillation protocol and additionally at 24 h after MCAo, mice were allowed to grasp the grip with the forepaws and pulled backwards from the tail in the horitzontal plane thus exerting a force in the grid. The recorded score (in grams), of each animal, was determined by averaging 6 trials, then data is represented individually versus the baseline force.

Euthanasia and sample processing

Twenty-four hours after last exposure (naïve, Figure 1(a)) or surgery (ischemic, Figure 1(b)), animals were deeply anesthetized (4% isoflurane), the chest was exposed and a cardiac puncture was performed in the left ventricle to obtain blood which was transferred into 3.2% citrate microtubes (0.5 ml, Sarstedt, Spain), centrifuged at 1,500 rcf for 10 minutes at 4 °C to obtain the plasma fraction and stored at −80°C. Finally, mice were euthanized by exsanguination with transcardiac perfusion with 20 ml of ice-cold saline and brains were removed to verify the presence of a cortical infarct.

Human plasma samples

Plasma samples from two different human cohorts were studied. Firstly, retrospective samples from ischemic stroke patients (n = 19) and matching control subjects (n = 19) obtained from the SMARRTS study recruited in our laboratory between February 2014 to February 2015⁴⁰ were studied. Strokes correspond to patients who underwent intensive rehabilitation therapy, and the baseline citrate plasma sample obtained after stroke and before the rehabilitation started was used for the clot-lysis assay in this study. The control cohort consisted in stroke relatives with no known neurologic, malignant, infectious or inflammatory diseases. Demographic, risk factors and medication in use characteristics are shown in Table 1. For the clot-lysis assay, plasmas were tested with the exogenous addition of DEP (SRM2975) that was established at 0.1 µg/ml. A previous study was done to ensure that this DEP concentration was not altering the absorbance readings among several tested (0.1, 1 and 5 µg/ml; data not shown). Secondly, samples from control subjects exposed to different background air pollution levels were grouped as those living in low or high polluted areas. Blood was obtained in citrate tubes between November 2010 and May 2012 from the ISSYS cohort.⁴¹ These were hypertensive subjects (arterial systolic pressure >120 mm Hg) without stroke events at the baseline (confirmed by MRI) whose place of residence was geo-coded and their residential levels of PM₁₀ and PM_2.5 were estimated using a land-use-regression model.⁴² Two groups were established for the analysis of the clot-lysis assay parameters with PM levels distributed in the highest and the lowest decile of the distribution curve as follows: low PM-exposure group (n = 20; estimated mean PM_2.5 was 11 µg/m³) and high PM-exposure group (n = 20; estimated mean PM_2.5 was 26 µg/m³).

Table 1.

Principal characteristics of the control and stroke cohorts.

Demographics	Control (n = 17)	Ischemic (n = 15)	p-value
Sex (female)	47.1%⁸	20%³	0.147
Age (years)	64.1 ± 4.1	52.6 ± 5.2	0.001
Sample (days after stroke)	…	12.4 ± 3	…
Vascular risk factors
Alcohol	52.9%⁹	20%³	0.076
Active smoker	17.6%³	40%⁶	0.243
Physical activity	82.4%¹⁴	53.8%⁷	0.123
Obesity	64.7%¹¹	28.6%⁴	0.073
Atrial fibrilation	0%	13.3%²	0.212
Hypertension	88.2%¹⁵	60%⁹	0.106
Dyslipemia	70.6%¹²	40%⁶	0.153
Diabetes	23.5%⁴	14.3%²	0.664
Medication use
Antiplatelet	29.4%⁵	46.7%⁷	0.467
Anticoagulant	0%	0%	…
Statin	41.2%⁷	73.3%¹¹	0.087
Antihypertensive	82.4%¹⁴	60%⁹	0.243
Insulin	17.6%³	20%³	0.608

Note: Values are expressed as mean ± SD or as percentatge (%). All categorical variables were analyzed with a Chi-square test whereas continuous variable (age) with a t-test.

Turbidimetric clot assay

This assay was conducted to evaluate the influence of DEP on clot formation and lysis under thrombolytic conditions with recombinant tissue plasminogen activator (rtPA, Actilyse®), based on a turbidimetric assay published by Carter and colleagues with some modifications.⁴³ With this aim, citrate plasma samples preserved at −80°C from naïve and ischemic mice, from the SMARRTS cohort and the ISSYS cohort were thawed and the clot/lysis assay was run as follows: 25 µl of citrate plasma were added to a transparent 96-well microtiter plate and then, the activation of plasma coagulation was initiated by adding 50 µl of Activation Mix containing 7.5 mM CaCl₂ and 0.03 U/ml thrombin (Sigma T6884-100UN). To further induce the clot lysis, rtPA (Actylise®, Boehringer, 83 ng/mL) was added in 75 µl of Assay Buffer containing 0.05 M Tris-HCl (Sigma-Aldrich, USA), 1 M NaCl (Sigma-Aldrich) at pH = 7.4. Both assay buffer and activation mix were added consecutively and Optical Densities (OD) were immediately read at 405 nm every 40 seconds for 3 hours at 37 °C using a BIO-TEK SynergyMx microplate reader and Gen5 software. For each sample 2 conditions were tested: clot formation (−tPA) and clot formation+lysis (+tPA) (Figure 1(d)). Additionally, plasma from the SMARRTS stroke and control were tested with the exogenous addition of SRM2975 mixed in the 75 µl of Assay Buffer (final concentration 0.1 µg/ml).

For each experiment, 96-well plates were designed including samples from all tested groups, and the difference in pipetting time between columns was considered when analyzing the data. All samples were run per triplicate and the accepted CV for the OD values was <25%. Each independent experiment was run together with inter-assay control samples (n = 3) that showed a CV <30% between plates. Following these criteria, the final number of samples analyzed for each study was: n = 56 for the mouse experimental model (n = 9/8 for naïve acute PBS/DEP groups, n = 6/5 for MCAo acute PBS/DEP groups, n = 7/10 for naïve chronic PBS/DEP groups and n = 5/6 for MCAo chronic PBS/DEP groups), n = 32 for the SMARRTS cohort (17 control subjects and 15 stroke patients) and n = 31 for the ISSYS cohort (14 low PM and 17 High PM exposure).

The registered changes in OD along time correspond to the different phases of clot formation and lysis generating a turbidimetric curve in which 9 parameters related to clot formation (1–3) and lysis (4–9) were analyzed⁴³ (Figure 1(d)): 1. Lag time (Lagtime): a sharp rise in OD consistent with the time required for protofibrils to aggregate and ensure clot formation (given in minutes); 2. Clot formation rate (CRc): maximum change in clot formation OD, reflects the maximum rate of fibrin fiber assembly (given in OD per seconds); 3. Maximum Absorbance (MaxAbs): maximum OD minus baseline OD, reflects the structural density of the thrombus (given in OD); 4. Lysis Rate (LR): maximum rate of fibrin fibers disassembly (given in OD per seconds); 5. Lysis time: Fibrinolysis duration (given in hours or minutes); 6. Clot area: area under the curve, the balance between clot formation and lysis; 7. Clot time: clot lifetime (given in hours or minutes); 8. Lysis 50 from time 0 (Lysis 50 t₀): time to reach 50% clot lysis from clot formation initial time (given in hours or minutes); 9. Lysis 50 from time of Maximum Absorbance (Lysis 50 t_Max): time to reach 50% clot lysis from clot MaxAbs time point (given in hours or minutes).

Data processing and statistical analyses

All data was processed blindly to treatment group. Variables are represented as mean ± Standard Deviation (SD) or median ± Interquartile Range (IQR) using GraphPad Prism 9.0 according to their normal or non-normal distribution, respectively. Statistical analyses were performed with the SPSS 20.0 software. The normality of continuous variables was assessed by Shapiro-Wilk test (n < 30) or Kolmogorov-Smirnov (n ≥ 30). Normally-distributed variables were analyzed by Student t-tests or one-way ANOVA followed by Tukey post-hoc whereas non-normally distributed variables were analyzed by Mann-Whitney U-test or Kruskal Wallis followed by Dunn's or Bonferroni post-hoc tests as indicated in figure legends. Categorical variables were studied using contingency tables and a Chi-squared test using the Pearson or Fisher p-value. Results with p < 0.05 were considered statistically significant. Graphs also show statistical trend p values between 0.1–0.05.

Results

DEP suspensions contained a high proportion of PM_2.5 and PM₁₀

DEP suspensions (0.5 mg/ml) were sonicated 10, 30 and 60 minutes and compared to non-sonicated DEP suspensions. After 10 minutes, the relative volume distribution of the smaller-sized particles (UFPM, PM_2,5 and PM₁₀) increased significantly for both DEP materials, but by increasing sonication time did not enlarge these fractions (Supplementary Figure 1A). Moreover, the freezing/thawing cycle of the prepared suspensions for treatment (7.5 mg/ml) did not alter the resulting proportion of smaller-sized particles obtained after 10-minute sonication (Supplementary Figure 1B). SEM images allowed us to visualize the presence of aggregates and fine particles in both DEP materials (Supplementary Figure 1C). These results confirmed that fractions of PM₁₀ and PM_2.5 were used in our in vivo and ex vivo exposure protocols for both SRM-DEP materials.

Mice mortality, weight and motor function were not altered by the DEP instillation

None of the animals died during DEP or vehicle instillations (Figure 2(a) and Figure 3(a)) . The presence of a cortical infarct was verified in all ischemic animals included in the study by proper CBF reduction (Figure 1(c)) and post-mortem cortical injury. Acute or chronic instillation of DEP did not alter mice weight, not in naïve or in ischemic animals (Figure 2(b) and Figure 3(b)). Regarding the forelimb strength after ischemia, the grip test showed a decrease in both vehicle and DEP-treated mice at 24 h, but no changes were observed between groups (Figure 2(c) and Figure 3(c)).

Figure 2.

Clot-lysis assay in plasma from acutely exposed naïve and ischemic mice. (a) Experimental design overview with representative turbidimetric curves. (b) Weight variation after instillations in naïve and ischemic (MCAo) mice before and after surgery. (c) Grip test performance of ischemic mice at 24 hours, individual data represent percentage versus baseline score (set at 100%) and (d) Clot formation parameters changing after ischemia: the lag time shortens, the slope formation accelerates (CRc) and the density of the thrombus (MaxAbs) increases, peaking in the DEP-exposed mice for the two last parameters. Data is represented as mean ± SD (B, C; T-test) or median (IQR) (d; Kruskal-Wallis and Dunn’s post-hoc). ns: not significant. *p < 0.05, **p < 0.01, ***p < 0.001. Naïve-PBS/SRM1650b (n = 9/8) and MCAo-PBS/SRM1650b (n = 6/5).

Figure 3.

Clot-lysis assay in plasma from chronically exposed naïve and ischemic mice. (a) Experimental design overview with representative turbidimetric curves. (b) Weight variation after the instillation protocol in naïve mice and in ischemic (MCAo) mice, before and after surgery. (c) Grip test performance of ischemic mice at 24 hours, individual data represent percentage versus baseline score (set at 100%) and (d) CRc and MaxAbs, clot formation parameters increased in the ischemic group and boosted after DEP exposure, while lysis parameters were delayed in ischemic mice but not in the DEP-exposed ischemic group. Data is represented as mean ± SD (b, c: T-test and D: Clot time, Lysis time, Lys50T0 and Lys50TMax One way ANOVA and Tukey’s post-hoc) or median (IQR) (d: CRc, MaxAbs; Kruskal-Wallis and Dunn’s post-hoc). ns: not significant. *p < 0.05, **p < 0.01. ***p < 0.001. Naïve-PBS/SRM1650b (n = 7/10) and MCAo-PBS/SRM1650b (n = 5/6).

In vivo DEP exposure enhances pro-thrombogenic plasma features after ischemia

Plasma of acutely (Figure 2(a)) and chronically (Figure 3(a)) exposed naïve and ischemic mice were first studied. In acutely exposed mice (Figure 2(d)), significant differences were observed in parameters of clot formation, but not in the clot lysis by tPA (data not shown). Specifically, a reduction in the clot formation time in plasma of ischemic mice compared to the naïve animals was observed (χ²(3) = 8.13, with a Median Lagtime: 1.3 vs. 4.9 mins, p = 0.007). Also, plasma from ischemic mice presented increased clot formation speed (CRc) and clot density (MaxAbs) compared to naïve mice (χ²(3) = 15.69 with a Median CRc: 43.1 vs. 12.6 OD/sec, p = 0.02; χ²(3) = 21.95 with a Median MaxAbs: 0.17 vs. 0.07 OD, p = 0.03). Importantly, the highest values in clot formation speed and clot density were observed in ischemic mice exposed to DEP compared to naïve vehicle (χ²(3) = 15.69 with a Median CRc: 55.5 vs. 12.6 OD/sec, p = 0.001; χ²(3) = 21.95 with a Median MaxAbs: 0.22 vs. 0.07 OD, p = 0.002), suggesting that the exposure to DEP could boost the formation of denser blood clots in the context of ischemia (Figure 2(d)).

Next, plasma of ischemic animals that received chronic vehicle instillations (Figure 3(d)), also formed faster and denser clots compared to naïve mice (χ²(3) = 20.00 with a Median CRc: 36.7 vs. 11.1 OD/sec, p = 0.01; χ²(3) = 20.66 with a Median MaxAbs: 0.17 vs. 0.06 OD, p = 0.02), peaking when exposed to DEP (χ²(3) = 20.00 with a Median CRc: 54.6 vs. 11.1 OD/sec, p = 0.001; χ²(3) = 20.66 with a Median MaxAbs: 0.2 vs. 0.06 OD, p = 0.01). Moreover, in chronically vehicle-exposed mice some clot lysis parameters were also modified in ischemic mice towards a slower lysis time compared to the naïve mice (F_(3,24) = 3.81 with a Mean Clot time: 1.7 vs. 1.2, p = 0.02; F_(3,24) = 3.45 with a Mean Lysis time 1.6 vs. 1.1 hours, p = 0.03; F_(3,24) = 4.15 with a Mean Lys 50t₀: 0.9 vs. 0.6 hours, p = 0.01 and F_(3,24) = 3.45 with a Mean Lys 50t_MAX: 0.8 vs. 0.55 hours, p = 0.03), but not when exposed to DEP chronically which presented similar lysis parameters as naïve groups (Figure 3(d)).

DEP exacerbates the pro-thrombogenic characteristics of plasma from ischemic stroke patients ex-vivo

The turbidimetric assay was conducted in human plasma from stroke subjects and controls exposed to DEP (0.1 µg/ml) or vehicle solutions during the assay, as represented in Figure 4(a), in order to determine changes in the thrombogenic/lysis characteristics. Data regarding demographic characteristics, vascular risk factors and medication in use is shown in Table 1. Plasma samples from the Ischemic Stroke cohort (IS) cohort were obtained, on average, at day 12 after the event. Control subjects (Co) were significantly older than strokes (64 vs. 52 average age, p = 0.001), tended to be more obese (64% vs. 28%, p = 0.07) and to consume more alcohol (52% vs. 20% p = 0.07) (Table 1).

Figure 4.

Clot-lysis assay in plasma from control subjects and ischemic stroke patients. (a) Representative turbidimetric curves and (b) Reduced latency time (lagtime), increased clot formation rate (CRc) and maximum absorbace (MaxAbs) were observed in clots of IS patients whose plasma was exposed to DEP during the assay, being significant in the CRc when compared to Controls. *p < 0.05. Data is represented as median (IQR) and tested with Kruskal-Wallis followed by Bonferroni post-hoc. Co: Control (n = 17), IS: ischemic stroke (n = 15).

The analysis showed pro-thrombogenic features only in the clot formation parameters in the stroke cohort (Figure 4(b)), similar to the previous results described in mice exposed to DEP. Specifically, plasma samples from stroke patients, exogenously exposed to DEP, presented a significant increase in the clot formation speed (χ²(3) = 9.88 Median CRc: 40.5 vs. 31.7 OD/sec, p = 0.02). In the clot latency to form and density parameters, we also observed slight differences between groups towards a more thrombogenic profile in DEP-exposed plasmas from stroke patients (χ²(3) = 6.88, p = 0.07 Median Lagtime and χ²(3) = 8.29, p = 0.04 Median MaxAbs), with the biggest difference between vehicle-exposed plasmas from controls and DEP-exposed plasmas from stroke patients (Median Lagtime: 16.2 vs. 10.5 mins and Median MaxAbs: 0.14 vs. 0.18 OD), although not significant; see Figure 4(b).

Prolonged exposure to PM_2.5 and PM₁₀ in hypertensive subjects alters the rtPA-mediated clot lysis

Finally, the clot formation and lysis characteristics were analyzed in human plasma of subjects exposed to different ambient levels of residential PM (Figure 5(a)). Demographic characteristics, relevant risk factors and medication in use, according to lower or higher exposure to PM, are detailed in Table 2. Groups matched in age and sex as well as in cardiovascular risk factors and medication use, but differed significantly in the PM_2.5 and PM₁₀ mean annual exposure according to the place of residence.

Figure 5.

Turbidimetric assay and clot lysis parameters in the low or high PM-exposed groups. (a) Representative turbidimetric curves and (b) Graphs showing reduced clot time, lysis time and Lysis 50t_max in the highly exposed group. Data is represented as mean ± SD and analyzed with a T-test. *p < 0.05. Low (n = 14), High (n = 17).

Table 2.

Principal characteristics of the studied cohorts living in low and high polluted areas.

Characteristics	Low PM exposure (n = 14)	High PM exposure (n = 17)	p-value
PM_2.5 Annual mean (µg/m³)	10.9 (10.6–11.3)	25.3 (23.5–30.1)	0.001
PM₁₀ Annual mean (µg/m³)	29.9 (28.0–31.4)	37.1 (29.3–45.3)	0.001
Demographics
Age (years)	60.1 ± 5.8	61.0 ± 4.9	0.660
Sex (female)	64.3%⁹	47.1%⁸	0.473
Arterial systolic pressure (mean)	142.1 ± 9.2	140.6 ± 7.7	0.797
Education
Primary uncompleted	35.7%⁵	35.3%⁶	0.726
Primary completed	35.7%⁵	47.1%⁸
Secondary or higher completed	28.6%⁴	17.6³
Vascular risk factors
Dyslipemia
Hypercholesterolemia	64.3%⁹	52.9%⁹	0.603
Hypertriglyceridemia	0% (0)	5.9%¹
Mixed	7.1%¹	5.9%¹
Unknown	7.1%¹	0% (0)
Ischemic cardiopathy	0%	17.6%³	0.098
Diabetes type II	28.6%⁴	41.2%⁷	0.707
Alcohol intake	64.3%⁹	47.1%⁸	0.473
Active smoker	21.4%³	17.6%³	0.139
Obesity	50%⁷	70.6 (12)	0.240
Medication use
Antiplatelet	0%	17.6%³	0.224
Antihypertensive	100%¹⁴	88.2%¹⁵	0.488
Statin	35.7%⁵	53.3%⁸	0.462

Note: Values are expressed as mean ± SD, median (IQR) or as percentatge (%). All categorical variables were analyzed with a Chi-square test whereas continuous variables were analyzed with a t-test (age) or a Mann-Whitney test (PM_2.5 and PM₁₀ Annual mean exposures).

The turbidimetric assay (Figure 5(a)) did not show differences in any of the clot formation parameters between high- and low-exposure groups (data not shown). On the contrary, lysis parameters (Figure 5(b)) showed significantly shortened lysis times in the highly-exposed compared to the lower-exposed group (Mean Clot time: 25.9 vs. 30.5 mins, p = 0.04; Mean Lysis time: 18.6 vs. 22.6 mins, p = 0.02; Mean Lys 50t_Max: 9.3 vs. 11.3 mins, p = 0.02), indicating that clots formed with plasma from subjects living in areas of high PM concentrations lysed significantly faster than those living in less polluted regions (Figure 5(b)).

Discussion

The aim of the present study was to evaluate the influence of DEP, as main PM air pollutant, on clot formation and tPA-induced lysis in an ex-vivo turbidimetric assay in human and mouse plasma samples related to stroke disease. In summary, we have shown that after ischemia pro-thrombogenic changes take place in both mouse and human plasma, which are exacerbated by DEP exposure. On the other hand, clot lysis parameters were also altered after long-term exposures showing a delayed lysis of the thrombus after ischemia but not after DEP exposure in mice plasma samples, and faster clot lysis in plasma from hypertensive subjects living in highly polluted areas. Overall, our results support the assumption of DEP being involved in stroke pathophysiology and point at a potential influence with thrombolytic treatments with unknown consequences.

To date, it has been suggested that DEP alter blood hemostasis in humans, mainly towards a pro-coagulant stage. In human cohort studies, healthy volunteers exposed to diesel exhaust for 1 hour during exercise, it was observed the suppression of the tPA activation.²⁸ In healthy young adults whose plasma components were analyzed before, during and after the 2008 Olympics in Beijing (when the air pollution was monitored and reduced), soluble P-selectin (a signalling molecule of leukocytes contributing to thrombosis) and Von-Willebrand Factor (vWF, a glycoprotein participating in platelets’ aggregation) levels were normalized when compared to pre-existing low-quality air pollution levels. They also observed that increases in PM_2.5 concentrations, among other air pollutants, were positively associated with increases in fibrinogen, vWF, sP-selectin and sCD40L concentrations.⁴⁴ However, other authors have described that fibrinogen in healthy subjects was not associated with ambient particulate matter increases.³⁴

The pro-thrombogenic activity of the air pollutant particles observed in humans has been also suggested by experimental exposures in in vitro and in vivo models: in vitro, blood clots significantly increased polymerization rates in the presence of PM,⁴⁵ and the ex-vivo addition of 0.5 µg/ml of DEP (SRM1650b) to hamsters’ blood induced fast platelet activation.³³ This same group also observed that the in vivo intratracheal instillation of DEP enhanced the risk of arterial and venous platelet rich-thrombus formation in a photothrombosis model, as early as 1 hour.³³ In intratracheal instilled mice with different types of particles (collected urban PM and DEP-generated by a diesel engine), it was observed that urban PM enhanced arterial but not venous thrombosis (DEP did not alter any), and both type of particles modestly increased factor VII, FVIII and fibrinogen, important elements of the coagulation cascade.³¹

In the present study, DEP particles did not show a substantial effect in the clot formation or lysis parameters in naïve instilled mice, and this was consistent for both acute and chronic exposures to DEP. However, we could observe an effect of the induced ischemic insult, at both exposure times, as well as in plasma from stroke patients, towards a pro-thrombogenic stage, which has already been suggested by other authors with the analysis of other parameters and techniques. For example, plasma from acute ischemic stroke patients formed in-vitro clots that lysed slower: they were more compact, showed increased fibre thickness and shorter lag time compared to those of control individuals measured by turbidity and D-dimer release.⁴⁶ Also, TAFI (Thrombin Activatable Fibrinolysis Inhibitor) levels in plasma were significantly elevated in ischemic stroke patients.⁴⁷ In those studies, it is demonstrated that pro-thrombogenic and anti-fibrinolytic features are developed in the plasma of ischemic patients compared to the plasma from control subjects during ex-vivo clot formation experiments and plasma analyses. Additionally, other authors have proposed dynamic changes in the plasma fibrinolytic system after stroke during different stages of the disease, describing molecular changes of (t-PA) and plasminogen activator inhibitor-1 (PAI-1) activities, and beta-thromboglobulin (β-TG), which could condition plasma features for blood clotting and lysis.⁴⁸

Our results in both human strokes and ischemic mice are in line with these observations, and provide novel insights on the influence of DEP in clot formation in the context of ischemic stroke. The underlying factors leading to dynamic coagulation and fibrinolytic changes in plasma at molecular or cellular level are not being investigated in our study, but deserve further investigations.

Interestingly, our study shows that only mice receiving prolonged vehicle instillations for 3 weeks presented longer clot lysis times after ischemia than control naïve mice (which was not observed in the acute exposure protocol), pointing at a delayed clot-lysis in plasma. This difference between exposure protocols could be attributed to the different ages of the animals: 7 weeks (acutely exposed) vs. 10 weeks (chronically exposed). In this regard, a study examining the effects of urban roadside PM exposure in young (10 weeks) and older mice (20 weeks), saw that the older mice presented higher baseline levels of Factor VIII, sP-selectin and vWF (pro-thrombogenic factors) which could difficult the clot lysis, and that after exposure younger mice presented a sP-selectin increase while the older group increased platelet numbers, vWF and sP-selectin to the highest values, sustaining an elevated thrombogenicity after the exposure to PM larger in the older group.⁴⁹ Although the age difference in our study between acute and chronically exposed mice is much smaller, it should be considered. Interestingly, the observed delay in the tPA-induced clot lysis, was not significant in plasma from ischemic mice exposed to DEP for 3 weeks, indirectly pointing to a possible interaction of the particles with the fibrinolytic system in the context of tPA thrombolysis after longer exposure periods.

In line with this observation, in hypertensive subjects we have directly observed a significant acceleration in the same clot lysis parameters only in the group of subjects living in high polluted areas (above the European PM_2.5 limit), pointing out that prolonged exposures to high PM environments, where DEP represent a high proportion of its composition,⁵⁰ could influence the thrombus lysis by tPA by shortening the lysis time. Considering that both low- and high-exposed cohorts were similar in demographic characteristics, vascular risk factors or in-use medication, the differences in tPA thrombolysis responses among subjects may be explained by the differences in exposure to ambient PM. Although these were unexpected results they are in line with our observations in the mouse ischemia model exposed to chronic DEP, but should be examined including a model adjusting for potential confounding factors, which is limited here by the sample size.

In patients presenting known risk factors of ischemic stroke, different effects of PM exposure have been described supporting our findings. For example, Pan and colleagues found that PM₁₀ levels were associated with denser fibrin clot structure only in patients with deep vein thrombosis but not in controls, suggesting that air pollution may trigger differences in fibrin clot structure, to a more pro-thrombogenic stage, only in patients predisposed to thrombogenic disease.⁵¹ Similar to the pro-thrombogenic changes we have described in the clot-lysis assay in ischemic animals, but not in naïve mice after DEP exposures. Additionally, and highly matching our findings of hypertensive subjects, other authors have described that type II diabetes patients (another risk factor for suffering a stroke) exposed to UFPM presented a decrease in cell-surface markers of platelet activation such as sp-Selectin and platelet CD40L.³⁶ On the other hand, another study stated that in men with previous myocardial infarction, the exposure to DEP and moderate exercise did not aggravate the pre-existing vasomotor dysfunction, and it reduced the acute release of tPA, studied by intra-arterial agonist infusions.²⁹

Several limitations should be noted: First, we have not quantified the extension of the ischemic lesion in each individual but other actions have been taken to ensure the inclusion of animals with evidence of success of the MCA occlusion with cortical lesions such as the reduction in cortical cerebral blood flow after electrocoagulation,⁵² distal cut of the MCA post-cauterization, visual verification post-mortem of the cortical lesion and reduction in forelimb/paw force, although the last was not fulfilled in all animals probably due to the early assessment at 24 h and the possibility of worsening days after.⁵³ Second, there are three important factors in human stroke studies related with final outcome unexplored in the present study that should be considered in the future: first, the relationship between infarct extension and the magnitude of clot formation/lysis changes under DEP-exposure conditions, second the impact of sex-differences in an in vivo scenario of clot formation (vs. our ex vivo study) where clot formation and lysis would be influenced by metabolic, genetic and vascular characteristics intrinsically different between biological sexes^54
–57 and third, the influence of age as it has been recently reported that age increases DNA damage in DEP-exposed mice and it is known to be a key factor for stroke outcome and related to neuroprotectant or recanalization treatments.^52,58,59 Importantly, our results obtained from human cohorts match with those experimentally-produced in mouse models, but it should be noted that the sample size in the human cohorts is still small and results need further confirmatory studies in larger cohorts. In this regard, including other subjects representing other environmental urban areas such as green spaces or more heavily polluted areas would be desirable. Finally, confirmatory studies including sample size calculations based on the presented results would be needed to confirm the disbalance of the thrombogenic/lytic system in presence of DEP in stroke.

In conclusion, attending to our results and previous background, we have found that ischemia significantly alters plasma characteristics to a more pro-thrombogenic stage, which is boosted by DEP exposure. At the same time, blood clot lysis by tPA could be accelerated by prolonged PM exposures and should be considered in the presence of other stroke risk factors such as hypertension. The consequences of these observations in the clinical scenario are unknown but our results warrant further investigations in the context of acute ischemic stroke management focusing on the incidence of large vessel occlusions, recanalization rates, thrombolytic treatment, incidence of haemorrhagic transformations, or clot composition in larger cohorts. Finally, it is important to elucidate if acute or prolonged exposures to ambient PM or DEP could modify the thrombogenic/thrombolytic balance considering the existence of other co-morbidities such as hypertension, diabetes or dyslipidaemia, and how this impacts the final stroke outcome.

Supplemental Material

sj-pdf-1-jcb-10.1177_0271678X231214826 - Supplemental material for Diesel exhaust particles exposure exacerbates pro-thrombogenic plasma features ex-vivo after cerebral ischemia and accelerates tPA-induced clot-lysis in hypertensive subjects

Supplemental material, sj-pdf-1-jcb-10.1177_0271678X231214826 for Diesel exhaust particles exposure exacerbates pro-thrombogenic plasma features ex-vivo after cerebral ischemia and accelerates tPA-induced clot-lysis in hypertensive subjects by Mercedes Arrúe, Anna Penalba, Ane Rodriguez-Bodero, Amaia Elicegui, Miquel de Homdedeu, María-Jesús Cruz, Alba Simats, Susana Rodriguez, Xavier Buxó, Nicolás Garcia-Rodriguez, Jesús Pizarro, Michelle C Turner, Pilar Delgado and Anna Rosell in Journal of Cerebral Blood Flow & Metabolism

Footnotes

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work has been supported by a research grant from Fundació La MaratóTV3 (2017310) which also funded M.A fellowship. The SMARRTS and ISSYS human cohorts were funded by PI16/00981, PI19/00186 and PI14/1535), M.A, A.P, J.P, P.D and A.R take part of the STROKE-RICORS network (RD21/0006/0007), JP was supported by a Rio Hortega fellowship (CM20/00218) and MJC by the Miguel Servet programme (MSII17/00025), all from Instituto de Salud Carlos III with FEDER funds. MDH is supported by CIBER, AGAUR supports the Neurovascular Research Laboratory (SGR2021/0656) and MCT is funded by a Ramón y Cajal fellowship (RYC-2017-01892) from the Spanish Ministry of Science, Innovation and Universities, co-funded by the European Social Fund. ISGlobal acknowledges support from the Spanish Ministry of Science and Innovation through the “Centro de Excelencia Severo Ochoa 2019–2023” Program (CEX2018-000806-S), and support from the Generalitat de Catalunya through the CERCA Program.

Data availability statement

Data will be available upon reasonable request to the corresponding author.

Acknowledgements

The authors would also like to acknowledge Marta Cirach for preparation of the residential PM estimates, the ICTS NANBIOSIS PLATFORM-U6 supporting the Mastersizer 2000 and the VHIR Nanoparticles and Pharmacokinetic group for using their Nano ZetaSizer.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Authors’ contributions

MA, AP, MDH, MJC, PD and AR conducted the study design; MA, AR-B, AE, AP, SR, XB, NG-R, JP, MT and PD performed the data acquisition; MA, AS and AR conducted the data analysis; all authors participated in the article writing and revision.

Supplementary material

Supplemental material for this article is available online.

ORCID iDs

Amaia Elicegui

Alba Simats

Anna Rosell

References

Block

Calderon-Garcidueñas

Air pollution: mechanisms of neuroinflammation and CNS disease. Trends Neurosci 2009; 32: 506–516.

World Health Organization. Global air quality guidelines, particulate matter, ozone, nitrogen dioxide, sulfur dioxide and carbon monoxide. Geneva: WHO European Centre for Environment and Health, 2021, pp. 1–285.

Southerland

Brauer

Mohegh

, et al. Global urban temporal trends in fine particulate matter (PM2.5) and attributable health burdens: estimates from global datasets. Lancet Planet Health 2022; 6: 139–146.

Brook

Franklin

Cascio

, et al. Air pollution and cardiovascular disease: a statement for healthcare professionals from the expert panel on population and prevention science of the American Heart Association. Circulation 2004; 109: 2655–2671.

Chen

Liu

, et al. Beyond PM2,5 the role of ultrafine particles on adverse health effects of air pollution. Biochim Biophys Acta 2016; 1860: 2844–2855.

Bañeras

Ferreira-González

Marsal

, et al. Short-term exposure to air pollutants increases the risk of ST elevation myocardial infarction and of infarct-related ventricular arrhythmias and mortality. Int J Cardiol 2018; 250: 35–42.

Brook

Rajagopalan

Pope

, et al. Particulate matter air pollution and cardiovascular disease: an update to the scientific statement from the American Heart Association. Circulation 2010; 121: 2331–2378.

Kettunen

Lanki

Tiittanen

, et al. Associations of fine and ultrafine particulate air pollution with stroke mortality in an area of low air pollution levels. Stroke 2007; 38: 918–922.

Pope

Burnett

Thurston

, et al. Cardiovascular mortality and long-term exposure to particulate air pollution: epidemiological evidence of general pathophysiological pathways of disease. Circulation 2004; 109: 71–77.

10.

Pope

Turner

Burnett

, et al. Relationships between fine particulate air pollution, cardiometabolic disorders, and cardiovascular mortality. Circ Res 2015; 116: 108–115.

11.

Scheers

Jacobs

Casas

, et al. Long-term exposure to particulate matter air pollution is a risk factor for stroke: meta-analytical evidence. Stroke 2015; 46: 3058–3066.

12.

Tian

Liu

Xiang

, et al. Ambient coarse particulate matter and hospital admissions for ischemic stroke: a national analysis. Stroke 2019; 50: 813–819.

13.

World Health Organization. Global air quality guidelines, particulate matter, ozone, nitrogen dioxide, sulfur dioxide and carbon monoxide. Geneva: Author, 2005, pp. 1–285.

14.

González Ortiz

Soares

Air quality in Europe – 2020 report . Volume 1. Luxembourg: European Environmental Agency, 2020, p.162.

15.

Feigin

Stark

, et al. Global, regional, and national burden of stroke and its risk factors, 1990–2019: a systematic analysis for the global burden of disease study 2019. Lancet Neurol. 2021 20: 1–26.

16.

Abbafati

Abbas

Abbasi-Kangevari

, et al. Global burden of 87 risk factors in 204 countries and territories, 1990–2019: a systematic analysis for the global burden of disease study 2019. Lancet 2020; 396: 1223–1249.

17.

Johnson

Nguyen

Roth

, et al. Global, regional, and national burden of stroke, 1990–2016: a systematic analysis for the global burden of disease study 2016. Lancet Neurol 2019; 18: 439–458.

18.

Carmichael

ST.

The 3 Rs of stroke biology: radial, relayed, and regenerative. Neurotherapeutics 2016; 13: 348–359.

19.

Adams

Bendixen

Kappelle

, et al. Classification of subtype of acute ischemic stroke. Definitions for use in a multicenter clinical trial. Stroke 1993; 24: 35–41.

20.

Badimon

Vilahur

Thrombosis formation on atherosclerotic lesions and plaque rupture. J Intern Med 2014; 276: 618–632.

21.

Hacke

Kaste

Fieschi

, et al. Intravenous thrombolysis with recombinant tissue plasminogen activator for acute hemispheric stroke. The European Cooperative Acute Stroke Study (ECASS). JAMA 1995; 274: 1017–1025.

22.

Hacke

Kaste

Fieschi

, et al. Randomised double-blind placebo-controlled trial of thrombolytic therapy with intravenous alteplase in acute ischaemic stroke (ECASS II). Second European-Australasian acute stroke study investigators. Lancet Lond Lancet 1998; 352: 1245–1251.

23.

Adams

Del Zoppo

Alberts

, et al. Guidelines for the early management of adults with ischemic stroke: a guideline from the American Heart Association/American Stroke Association Stroke Council, Clinical Cardiology Council, Cardiovascular Radiology and Intervention Council, and the Atherosclerotic Peripheral Vascular Disease and Quality of Care Outcomes in Research Interdisciplinary Working Groups. Stroke 2007; 38: 1655–1711.

24.

Alonso de Leciñana

Egido

Casado

, et al. Guidelines for the treatment of acute ischaemic stroke. Neurologia 2014; 29: 102–122.

25.

Aver

Goyal

Bonafe

, et al. Stent-retriever thrombectomy after intravenous t-PA vs. t-PA alone in stroke. N Engl J Med 2015; 372: 2285–2295.

26.

Fischer

Kaesmacher

Pereira

, et al. Direct mechanical thrombectomy versus combined intravenous and mechanical thrombectomy in large-artery anterior circulation. Stroke 2017; 48: 2912–2918.

27.

Wang

Liu

, et al. Extreme levels of air pollution associated with changes in biomarkers of atherosclerotic plaque vulnerability and thrombogenicity in healthy adults: the Beijing AIRCHD study. Circ Res 2019; 124: e30–e43.

28.

Mills

Törnqvist

Robinson

, et al. Diesel exhaust inhalation causes vascular dysfunction and impaired endogenous fibrinolysis. Circulation 2005; 112: 3930–3936.

29.

Mills

Törnqvist

Gonzalez

, et al. Ischemic and thrombotic effects of dilute diesel-exhaust inhalation in men with coronary heart disease. N Engl J Med 2007; 357: 1075–1082.

30.

Budinger

GRS

McKell

Urich

, et al. Particulate matter-induced lung inflammation increases systemic levels of PAI-1 and activates coagulation through distinct mechanisms. PLoS ONE 2011; 6: e18525–9.

31.

Emmerechts

Alfaro-Moreno

Vanaudenaerde

, et al. Short-term exposure to particulate matter induces arterial but not venous thrombosis in healthy mice. J Thromb Haemost 2012; 32: 5–13.

32.

Tabor

Shaw

Robertson

, et al. Platelet activation independent of pulmonary inflammation contributes to diesel exhaust particulate-induced promotion of arterial thrombosis. Part Fibre Toxicol 2016; 13: 6.

33.

Nemmar

Hoet

PHM

Dinsdale

, et al. Diesel exhaust particles in lung acutely enhance experimental peripheral thrombosis. Circulation 2003; 107: 1202–1208.

34.

Thompson

AMS

Zanobetti

Silverman

, et al. Baseline repeated measures from controlled human exposure studies: associations between ambient air pollution exposure and the systemic inflammatory biomarkers IL-6 and fibrinogen. Environ Health Perspect 2010; 118: 120–124.

35.

Sullivan

Hubbard

Liu

SLJ

, et al. A community study of the effect of particulate matter on blood measures of inflammation and thrombosis in an elderly population. Environ Health 2007; 6: 3–7.

36.

Frampton

Bausch

Chalupa

, et al. Effects of outdoor air pollutants on platelet activation in people with type 2 diabetes. Inhal Toxicol 2012; 24: 831–838.

37.

Pöss

Lorenz

Werner

, et al. Diesel exhaust particles impair endothelial progenitor cells, compromise endothelial integrity, reduce neoangiogenesis, and increase atherogenesis in mice. Cardiovasc Toxicol 2013; 13: 290–300.

38.

Yoshizaki

Brito

Toledo

, et al. Subchronic effects of nasally instilled diesel exhaust particulates on the nasal and airway epithelia in mice. Inhal Toxicol 2010; 22: 610–617.

39.

Rosell

Agin

Rahman

, et al. Distal occlusion of the middle cerebral artery in mice: are we ready to assess long-term functional outcome? Transl Stroke Res 2013; 4: 297–307.

40.

Rodriguez

Buxo

, et al. Plasma matrix metalloproteinases in patients with stroke during intensive rehabilitation therapy. Arch Phys Med Rehabil 2016; 97: 1832–1840.

41.

Riba-Llena

Jarca

Mundet

, et al. Investigating silent strokes in hypertensives: a magnetic resonance imaging study (ISSYS): rationale and protocol design. BMC Neurol 2013; 13: 130–138.

42.

Eeftens

Beelen

De Hoogh

, et al. Development of land use regression models for PM2.5, PM 2.5 absorbance, PM10 and PMcoarse in 20 European study areas; results of the ESCAPE project. Environ Sci Technol 2012; 46: 11195–11205.

43.

Carter

Cymbalista

Spector

, et al. Heritability of clot formation, morphology, and lysis: the EuroCLOT study. Arterioscler Thromb Vasc Biol 2007; 27: 2783–2789.

44.

Rich

Kipen

Huang

, et al. Association between changes in air pollution levels during the Beijing Olympics and biomarkers of inflammation and thrombosis in healthy young adults. JAMA 2012; 307: 2068–2078.

45.

Metassan

Charlton

Routledge

, et al. Alteration of fibrin clot properties by ultrafine particulate matter. Thromb Haemost 2010; 103: 103–113.

46.

Undas

Slowik

Wolkow

, et al. Fibrin clot properties in acute ischemic stroke: relation to neurological deficit. Thromb Res 2010; 125: 357–361.

47.

Santamaria

Oliver

Borrell

, et al. Risk of ischemic stroke associated with functional thrombin-activatable fibrinolysis inhibitor plasma levels. Stroke 2003; 34: 2387–2391.

48.

Zhuang

, et al. Dynamic changes in plasma tissue plasminogen activator, plasminogen activator inhibitor-1 and beta-thromboglobulin content in ischemic stroke. J Clin Neurosci 2015; 22: 1123–1127. Clinical Study

49.

Emmerechts

De Vooght

Haenen

, et al. Thrombogenic changes in young and old mice upon subchronic exposure to air pollution in an urban roadside tunnel. Thromb Haemost 2012; 108: 756–768.

50.

Karagulian

Belis

Francisco

, et al. Contributions to cities’ ambient particulate matter (PM): a systematic review of local source contributions at global level. Atmos Environ 2015; 120: 475–483.

51.

Pan

Gong

Martinelli

, et al. Fibrin clot structure is affected by levels of particulate air pollution exposure in patients with venous thrombosis. Environ Int 2016; 92–93: 70–76.

52.

Lyden

Buchan

Boltze

, et al. Top priorities for cerebroprotective studies – a paradigm shift: report from STAIR XI. Stroke 2021; 52: 3063–3071.

53.

Martinez-San Segundo

Barceló

, et al. Matrix metalloproteinase-13 participates in neuroprotection and neurorepair after cerebral ischemia in mice. Neurobiol Dis 2016; 91: 236–246.

54.

Nimjee

Akhter

Zakeri

, et al. Sex differences in thrombosis as it affects acute ischemic stroke. Neurobiol Dis 2022; 165: 105647.

55.

Kopić

Benamara

Schuster

, et al. Coagulation phenotype of wild-type mice on different genetic backgrounds. Lab Anim 2019; 53: 43–52.

56.

Chandra

Cikic

Baddoo

, et al. Transcriptome analysis reveals sexual disparities in gene expression in rat brain microvessels. J Cereb Blood Flow Metab 2021; 41: 2311–2328.

57.

Cikic

Chandra

Harman

, et al. Sexual differences in mitochondrial and related proteins in rat cerebral microvessels: a proteomic approach. J Cereb Blood Flow Metab 2021; 41: 397–412.

58.

Lee

Kim

Lee

, et al. Age and gender effects on genotoxicity in diesel exhaust particles exposed C57BL/6 mice. Biomolecules 2021; 11: 374.

59.

Meyer

Schönfeld

Bechstein

, et al. Ischemic lesion water homeostasis after thrombectomy for large vessel occlusion stroke within the anterior circulation: the impact of age. J Cereb Blood Flow Metab 2021; 41: 45–52.

Supplementary Material

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Diesel exhaust particles exposure exacerbates pro-thrombogenic plasma features ex-vivo after cerebral ischemia and accelerates tPA-induced clot-lysis in hypertensive subjects

Abstract

Keywords

Introduction

Materials and methods

Physical characterization of diesel exhaust particles

Pre-clinical regulation

In vivo exposure to diesel exhaust particles, experimental mouse model of cerebral ischemia and sample processing

Preparation of DEP and experimental design

Permanent focal cerebral ischemia model

Grip strength

Euthanasia and sample processing

Human plasma samples

Turbidimetric clot assay

Data processing and statistical analyses

Results

DEP suspensions contained a high proportion of PM2.5 and PM10

Mice mortality, weight and motor function were not altered by the DEP instillation

In vivo DEP exposure enhances pro-thrombogenic plasma features after ischemia

DEP exacerbates the pro-thrombogenic characteristics of plasma from ischemic stroke patients ex-vivo

Prolonged exposure to PM2.5 and PM10 in hypertensive subjects alters the rtPA-mediated clot lysis

Discussion

Supplemental Material

sj-pdf-1-jcb-10.1177_0271678X231214826 - Supplemental material for Diesel exhaust particles exposure exacerbates pro-thrombogenic plasma features ex-vivo after cerebral ischemia and accelerates tPA-induced clot-lysis in hypertensive subjects

Footnotes

Funding

Data availability statement

Acknowledgements

Declaration of conflicting interests

Authors’ contributions

Supplementary material

ORCID iDs

References

Supplementary Material

DEP suspensions contained a high proportion of PM_2.5 and PM₁₀

Prolonged exposure to PM_2.5 and PM₁₀ in hypertensive subjects alters the rtPA-mediated clot lysis