Cigarette smoking still represents an important cause of cardiovascular and neoplastic diseases. The damage created by cigarette smoke does not only occur through direct inhalation of toxic substances but it also involves passively exposed people who inhale combustion elements emitted by the cigarette (second-hand smoke). This also occurs through inhalation, ingestion or transdermal contact of toxic substances deposited on the surfaces of closed environments such as apartments and vehicles (third-hand smoke).
Increased oxidative stress due to passive smoke can result in cardiovascular damage through the onset of endothelial dysfunction, increase in intimal-media thickening and platelet activation. NADPH oxidase activation has been recognized as the major reactive oxygen species source. NADPH oxidase plays a pivotal role in modulating arterial tone and in inducing platelet activation in humans. Passive smoke increases cardiovascular risk and atherosclerosis by endothelial dysfunction and platelet activation. Children are a frail population that is prevalently exposed to the damages derived from passive smoking. Experimental studies showed that passive smoking provokes endothelial dysfunction and atherosclerosis by NADPH oxidase activation. However, until now, there aren't studies in humans that have explored the role of NADPH oxidase in children exposed to passive smoking.
The aim of this research project will be to assess NADPH oxidase activation on endothelial function and platelet activation in 200 children exposed or not to passive smoke.
Flow mediated dilation and intima-media thickness assessment will be used as surrogate markers of atherosclerosis. Platelet activation will be assessed by markers of platelet activation such as p-selectin; furthermore the components of NETs (i.e. chromatin, histones and granular enzymes), that promote coagulation and platelet activation thereby increasing the risk of thrombosis and cardiovascular disease, will be evaluated.
The work laid out in this proposal is innovative for the following reasons:
1) Passive smoking is believed to exert cardiovascular damage by activating NADPH oxidase as shown by several experimental studies. Actually, there aren't studies in humans, and particularly in children, that analysed the role of passive smoking on NADPH activation and endothelial function, IMT and platelet activation. The results of this study could contribute to understand the pathophysiological mechanisms by which passive smoking damages arteries and develops atherosclerosis, so increasing the cardiovascular risk of exposed subjects.
Understanding the mechanisms related to cardiovascular damage oxidative-stress mediated is important to reduce the cardiovascular risk of children exposed to passive smoking in order to increase anti-smoking advertising campaigns.
2) Furthermore, this study want to assess any differences in oxidative stress levels, endothelial dysfunction, platelet activation according to the level (measured by serum cotinine levels) and type (second and third hand) of passive smoke exposition. Nowadays there aren't studies that explored this issue.
2) Employs state-of-the-art flow cytometry techniques that allow to gather a lot of information from small volumes of patient blood samples and in a short amount of time. Platelet functional studies in pediatric patients are limited because of difficulties obtaining adequate blood volume. Since there is often no time to analyse fresh platelets, platelet activation is usually estimated by measuring indirect plasma-soluble markers that fail to provide any mechanistic insight. The flow cytometry protocol described in this proposal allows to surpass these limitations by looking directly at platelets immediately after the blood is drawn using as little as 50µl of whole blood.
3) Platelet phenotypes in children are poorly understood. This project will generate a large dataset of high-resolution platelet phenotypes. The acquired knowledge will be used to improve the clinical management of pediatric patients at higher risk of cardiovascular disease.
4) Recent advances suggest that NADPH oxidase triggers NETosis, thus our studies could reveal whether NETs are the missing link between oxidative stress and increased risk of cardiovascular disease.