The major findings from this study are the following: (1) nano-TiO2
exposure increases coronary arteriolar ROS, (2) nano-TiO2
exposure impairs endothelium-dependent vasodilation, and this impairment is restored by incubation with ROS scavengers, (3) inhibition of NOS or COX inhibits arteriolar vasodilation in response to ACh in arterioles from sham-control rats, but not from nano-TiO2
-exposed rats, and (4) arachidonic acid causes slight vasoconstriction in coronary arterioles from rats exposed to nano-TiO2
, but dilation of microvessels from sham-control rats. These particle-dependent findings relate to human studies in that they have similarly shown an increase in free radical generation in coronary dysfunction during pathological states such as ischemia and MI [4
]. Considered together, it is attractive to speculate that the net result would be an augmentation of cardiac dysfunction.
The first critical finding in the current study is that intravascular ROS, as visualized by DHE, is greater in coronary arterioles from rats exposed to nano-TiO2
(). This is in agreement with previous reports from our laboratory which showed that inhalation of either fine or nano-TiO2
caused an enhanced ROS fluorescence in arterioles from the spinotrapezius muscle [21
]. As proposed by Okayama et al. [30
], one possible mechanism for particle-induced cardiac functional changes is an altered role of ROS. Specifically, these investigators showed that exposure to diesel exhaust particles (DEP) led to increased O2 −
production in rat cardiac myocytes in a concentration and time-dependent manner. Several other studies have concluded that particle exposure can induce ROS generation, specifically O2 −
, causing significant dysfunction in aortic rings in vitro [31
]. Therefore, it is plausible that pharmacologically scavenging ROS with TEMPOL and catalase would ameliorate the impairment of endothelium-dependent arteriolar vasodilation associated with nanoparticle inhalation.
The second major finding from this study was that incubation of coronary arterioles with ROS scavengers, TEMPOL and catalase, restored endothelium-dependent dilation in rats exposed to nano-TiO2
(). Our laboratory has previously shown that incubation with TEMPOL, a membrane soluble SOD mimetic which converts O2 −
; and catalase, which converts H2
to water and oxygen, partially restores both arteriolar endothelium-dependent dilation and NO production in the rat spinotrapezius muscle microcirculation after nanoparticle inhalation [21
]. It has been proposed that particle-induced oxidative stress is imposed by unfettered NO/NOS production and activity and subsequent conversion to O2 −
and eventually H2
]. Indeed, Knuckles et al. [35
] have suggested that an uncoupling of eNOS is the most likely mechanism for increased vasoconstriction after mesenteric arteries and veins were exposed to whole diesel exhaust. H2
can be generated as quickly as one minute after the treatment of human pulmonary artery endothelial cells with particles and can cause significant pulmonary artery vasoconstriction [32
]. However, Miller et al. [36
] recently showed that coincubation with SOD could completely reverse the inhibitory effect of 10 µg/mL DEP on ACh-induced vasodilation in aortic rings. Additionally, TEMPOL administration during inhalation exposure to gasoline-exhaust emissions attenuates increases in oxidative stress, as determined by the thiobarbituric acid reactive substances (TBARS) assay in the mouse aorta [37
]. Likewise, SOD and catalase pretreatment can significantly decrease DEP-induced cell damage in cardiac myocytes compared to DEP-treatment in the absence of these antioxidant enzymes [30
]. Collectively, these studies indicate that particle exposure induces vascular dysfunction via ROS generation, and this can be diminished by incubation with ROS scavengers.
The third major finding from the present study was that ACh-induced vasodilation in coronary arterioles from sham-control rats was significantly impaired in the presence of NOS or COX inhibitors, but this effect was not present in rats exposed to nano-TiO2
. This indicates that nanoparticle exposure impairs both NO and PG-dependent vasodilator mechanisms (). This is consistent with previous studies from our laboratory and others which report that particle exposure impairs NO-dependent vasodilation in a variety of vascular locations, such as pulmonary [38
], coronary [39
], and skeletal muscle [8
]. Cherng et al. [39
] proposed that the impaired vasodilator capacity in septal arteries after DE exposure was NO-dependent, which was suggested to normally oppose potent vasoconstriction by endothelin-1 in healthy vessels. However, the contribution of a COX-derived vasodilator was not evaluated. The impairment of endogenous NO after nanoparticle exposure is further corroborated in the present study owing to the heightened spontaneous tone in arterioles from sham-control after L-NMMA incubation compared to rats exposed to nano-TiO2
(). This indicates that NO contributes significantly not only to the coronary microvascular reactivity, but also to the establishment of basal tone in these vessels. Moreover, these contributions are compromised after nanoparticle inhalation. Because indomethacin treatment increased spontaneous arteriolar tone in both groups (), but decreased ACh-induced dilation only in arterioles from sham-control rats (); it is important to reiterate that the mechanisms that govern basal tone do not implicitly alter arteriolar vasoactive function in response to a given stimuli.
The present results also indicate that compared to the dilation observed in sham-control microvessels, nano-TiO2
exposure caused a slight vasoconstriction in coronary arterioles in response to arachidonic acid (). Therefore, we suggest that inhalation of nano-sized particles also induces a loss of arachidonic acid metabolite vasodilators. More precisely, it appears as though nanoparticle inhalation instigates a conversion to arachidonic acid-dependent vasoconstriction, while also initiating ROS generation in coronary arterioles. To better characterize the former possibility, the arachidonic acid metabolite thromboxane was evaluated in the present study. Although previous research shows that chronic exposure to the particulate phase of smoke induced elevates platelet thromboxane formation [40
], the present study found that nanoparticle exposure does not alter arteriolar vasoconstriction in response to the T×A2
analog U46619 (). Because T×A2
production was not evaluated, we cannot currently conclude that T×A2
activity is not altered by nanoparticle exposure. Ideally, a long-term inhalation study would be necessary to perturb the thromboxane pathway and subsequent vasoactive response. However, other possible sources of arachidonic acid-mediated vasoconstriction could contribute to nanoparticle-induced alterations in vasoreactivity. For example, the cytochrome p-450 pathway, specifically 20-HETE, and also certain prostaglandins (PGF2alpha
) can function as vasoconstrictors and are common products of arachidonic acid metabolism [19
]. Increases in coronary vascular resistance and subsequent reduction in myocardial perfusion have been shown to contribute to myocardial ischemia after particle inhalation [14
]. Therefore, the contributions of these arachidonic acid-mediated vasoconstrictors to coronary arteriolar function after nanoparticle exposure must be defined by further investigation.
As previously reported, we estimate that in the occupational setting, it would take a typical worker 5 years to achieve a similar pulmonary burden equivalent to the 10 µg burdens used herein [2
]. This alone may in first consideration appear unrealistic. However, given that: aerosol concentrations encountered in the workplace can easily exceed those used in the current study (6 mg/m3
); our calculation of 5 years was based upon sedentary pulmonary function; the bioretention of inhaled particles is quite variable; not all workers are young and/or healthy; and typical occupational careers exceed 5 years, the burdens used in this study may have not only occupational relevance, but also personal or environmental relevance.
We first characterized the particle-dependent impairments of endothelium-dependent arteriolar dilation in the systemic microcirculation in 2004 [41
]. These observations were also coupled to a large increase in rolling and adhering polymorphonuclear leukocytes in the venular circuit [42
]. While these initial observations were made with exposures to larger, fine TiO2
, and residual oil fly ash, we later showed that exposure to nano-TiO2
at similar mass depositions produced a far more robust impairment of microvascular reactivity [8
]. In all of these studies, pulmonary status was evaluated by bronchoalveolar lavage and histology. At lung burdens used in the present study, the common pulmonary response was focalized alveolitis with no change in lavage markers of gross inflammation or damage. Taken together, this suggests that subsequent systemic effects are not due to the inherent toxicity of a given particle or pulmonary overload. Most recently, we have identified mechanisms in the arteriolar wall that compromise microvascular reactivity after nanoparticle exposure. Specifically, endogenous NO bioavailability is compromised, and this occurs in the presence of elevated ROS and reactive nitrogen species associated with NADPH oxidase activity and/or myeloperoxidase activity [21
]. The logical extension of these serial studies is to determine whether a similar toxicity results in critical organs such as the heart (in effort to more directly link particle exposure with cardiovascular morbidity and mortality). This was first shown with subepicardial arterioles from rats exposed to nano-TiO2
]. In these studies, we were able to make such a link and verify that a similar level microvascular dysfunction occurs in the heart after pulmonary nanoparticle exposure. The current study strengthens our initial findings in the coronary microcirculation and offers potential mechanisms by which this effect manifests itself.