The most significant finding of the present study is the novel characterization of a ROS-dependent pathway that causes calpain-dependent endothelial ZO-1 degradation in response to PM. These data represent the first evidence that calpain signaling, via calcium leakage from activated TRPM2 by ROS, plays a critical role in modulating endothelial cell barrier function, resulting in tight junction protein ZO-1 degradation (Additional file 1 Figure S7). The consequence of ZO-1 degradation is sustained endothelial hyperpermeability and persistent lung inflammation, both of which contribute to variety of acute or chronic cardiovascular disorders [25, 26]. These effects were also observed with other types of PM samples (1648a from National Institute of Standards and Technology, fine PM collected from New York city or Baltimore, data not shown), indicating a selective pathogenesis pathway by PM pollution.
Previous studies report that PM triggers the generation of reactive oxygen species or ROS mainly from dysfunctional mitochondria [27, 28], and we also noticed the massive generation of ROS by this PM sample is also mainly from mitochondria (unpublished observation). The high iron level of this particular PM might also contribute to the ROS generated via Fenton reactions. ROS released endogenously, have been implicated in the pathophysiology of several lung diseases, including asthma and COPD, as the biochemical mechanisms underlying the urban PM-induced airway inflammation and toxicity . ROS are highly reactive and cause deleterious gene, protein, and tissue effects. ROS are increased in BAL or exhaled breath condensate from patients with inflammatory lung injuries and from people with cardiopulmonary disease who have been exposed to PM [30, 31]. This response may reflect the high oxidative potential of fine and ultrafine particulates. Residual oil fly ash (ROFA) and PM1.7-3.5 cause pulmonary inflammation mediated by oxidative stress [32, 33]. In vivo, exposing rats to PM leads to the formation of free radicals in the lung . Since cardiovascular disease is considered a risk factor of PM-related mortality and morbidity, it is interesting to note that spontaneously hypertensive rats, when exposed to PM, were more susceptible to pulmonary (inflammatory injury) and cardiovascular complications (acute depression of ECG activity) in an oxidant-dependent manner . Besides ROS, PM might trigger adverse outcomes via other potential mechanisms including nonselective phosphatase inhibition (by vanadium) or competitive ion channel inhibition (by nickel) due to the complex and variable chemical components.
In this study, we first define a novel pathway that mediates ROS-dependent tight junction disruption upon particulate matter challenge. Tight junctions, or zonula occludens, are the most apical component of the intercellular junctional complex, which also includes adherens junctions, desmosomes, and gap junctions . ZO-1 was the first tight junction protein to be identified, and ZO-2 and ZO-3 were later isolated as proteins that co-immunoprecipitated with ZO-1 [37, 38]. ZO-1 is a peripheral membrane-associated component of the cytoplasmic plaque of tight junctions and is found ubiquitously within tight junctions of epithelial and endothelial cells . ZO-1 interacts with many cellular proteins via its multiple protein-binding domains. ZO-1 has been reported to interact with other ZO family members or claudins via the PDZ domains [40, 41]. ZO-1 interacts with the C-terminus of occludin with its GuK domain and the acidic domain . The proline-rich C-terminus of ZO-1 mediates its binding to F-actin in vitro, and thus links it to the cytoskeleton . Clearly, ZO-1 interacts with a wide variety of cell skeleton components and plays a central role in orchestrating tight junction complexes. Any dysregulation of ZO-1 in endothelial cells by extracellular stimuli, such as virus shell proteins or alcohol, leads to persistent tight junction disruption and vascular hyperpermeability.
Calpain is a regulator of endothelial integrity which helps control fundamental cellular processes including cytoskeletal remodeling, membrane fusion, cell proliferation and differentiation, and activation of proteolytical cascades leading to apoptosis [44, 45]. Under oxidative stress, activated calpain cleaves eNOS and cytoskeletal proteins and induces apoptosis [21, 46–48]. Particulate matter induces endothelial cell intracellular oxidative stress, which leads to the activation of calpain, one of the major cytoskeletal regulators. Here we describe the cleavage of tight junction protein ZO-1 by activated calpain both in vitro and in vivo, indicating that calpain plays a central role in PM-induced endothelial barrier disruption and vascular hyperpermeability. In addition, as activated calpain cleaves other critical cytoskeletal proteins including ezrin and MARCKS protein, the contribution of the other cytoskeletal proteins to the EC hyperpermeability induced by PM needs to be further investigated.
Oxidative calcium influx is mediated by plasma membrane cation-permeable ion channels. The transient receptor potential protein (TRP) and its homologs are cation channels with a tetramer secondary structure which senses diverse stimuli from the extracellular and intracellular environments . Mammalian TRPs comprise six major subfamilies. TRPM2, a member of the TRP channel M2 subtype, is a calcium-permeable channel activated by intracellular messengers such as ADP-ribose . Massive ROS burden induced by PM contributes to DNA oxidation and damage, which activates poly-ADP ribose polymerase (PARP) to initiate DNA repair mechanisms. PARP binds to single-stranded and double-stranded DNA breaks and catalyses the breakdown of NAD into nicotinamide and ADP-ribose, the intracellular agonist of TRPM2 [22, 51, 52]. Oxidative stress-mediated activation of the PARP pathway serves as the major source of free ADP-ribose production in endothelial cells . Intracellular ADP-ribose activates TRPM2, allowing calcium ions to enter the cell, which in turn trigger numerous physiological and pathological processes.
An important limitation of our study is the high dose of PM that we employed. With 10–30 μg/m3 ambient PM level in the US or Europe, it is hardly to achieve a high level of acute PM exposure. While 100 μg/ml (in vitro) or 10 mg/kg (in vivo) are typical doses used in particulate matter toxicology studies [12, 13, 27, 54–56]. With an assumed ambient PM level of 20 μg/m3, one man with 70 kg body weight and 8 m3/minute respiration rate would receive a dose of 10 mg/kg corresponding to about 16 years of exposure with 50% deposition rate. As noted, a lot of cities in the developing countries still have high levels of ambient PM. A report by world bank  stated that in the year of 2006, extremely high PM10 levels still existed in a lot of cities (μg/m3): Nyala in Sudan (359), Kano in Nigeria (283), Hyderabad in Pakistan (239), Maroua in Cameroon (228), Muzaffarpur in India (218), N'DJAMENA in Chad (204), Segou in Mali (200), Erbil in Iraq (195), Shubra-El-Khema in Egypt (186), DHAKA in Bangladesh (174). Over 13 million people live with more than 200 μg/m3 ambient PM in Pakistan, which results over 20 mg inhaled per week, or 16 mg/kg per year.
Extensive epidemiologic and experimental evidence has demonstrated that particulate air pollution directly causes cardiopulmonary damage. Our observations demonstrate a novel mechanism of PM-mediated disruption of endothelial barrier function which is attributable to ZO-1 degradation by calpain, which is activated by extracellular calcium leakage through oxidant-sensitive TRPM2 channels. Therefore, inhibition of ROS/TRPM2/calpain/ZO-1 degradation may provide useful therapeutic strategies for the treatment of endothelial barrier dysfunction and lung inflammation.