**2. Smoke, oxidative stress and fibrosis in lung pathogenesis**

Cigarette smoking likely represents the single most significant risk factor for several lung conditions, and it is strongly associated with COPD and IPF, both familial and sporadic. Although observations about environmental risk factors have many biases and limitations [25], increasing knowledge on the underlying causes of lung diseases is evidencing how oxidative stress (OXS) and reactive oxygen species (ROS) play a crucial pathogenetic role (**Figure 1**).

The lungs are indeed highly susceptible to ROS-induced injuries. ROS are commonly thought to be a harmful by-product generated in cellular systems. However, recent studies have suggested that ROS physiological levels regulate important biological functions in cellular processes [2, 26]. Normally, ROS are tightly controlled by enzymes and antioxidant molecules. Nonetheless, excessive ROS accumulation may occur under certain conditions, thus making detoxification by the antioxidant system difficult. The result is indeed a condition called OXS that can affect cell proliferation, differentiation, aging, and death [27]. Cigarette smoke is responsible for significant oxidant burden and decreased antioxidant capacity even in plasma [28, 29]. ROS produced from cigarette smoke, combustion of organic matter and gases, like ozone and nitrogen dioxide, are featured on the lung epithelium [30], and could decrease antioxidant defenses, increasing OXS in the lungs [31].

**Figure 1.**

*Oxidative stress in lung pathogenesis. Intrinsic and extrinsic risk factors contribute to the progression of lung damage in the development of idiopathic pulmonary fibrosis (IPF) and obstructive pulmonary disease (COPD). In particular, cigarette smoking, environmental exposures, and air pollution induce an increase of reactive oxygen species (ROS) and oxidative stress condition, which play a crucial pathogenetic role in lung diseases. ROS, such as H2O2 and O2−, generated from NOX2 and NOX4, have a central role in the pathogenesis of pulmonary diseases. Indeed, ROS produced by these enzymes are involved in alveolar epithelial cell apoptosis, activation of inflammation, and induction of tissue fibrosis, that are all mechanisms underlying the progression of IPF and COPD. Figure was prepared using images from Servier Medical Art by Servier (https:// smart.servier.com), which are licensed under a Creative Commons Attribution 3.0 Unported License.*

A disrupted function of the redox system can consequentially impact on key cell signaling pathways involved in disease progression. Conversely, several signals can alter the oxidative state of lung cells. For example, the lung is constantly exposed to biomechanical forces, such as fluid shear stress, cyclic stretch, and pressure, due to the blood flowing through the pulmonary vessels, and the distension of the lungs during the breathing cycle. It is indeed known that cells within the lung respond to these changes by activating signal transduction pathways that can also alter their redox state with pathophysiological consequences [32]. Particularly in the vasculature, the two types of biomechanical stimuli, such as frictional force known as shear stress (SS), or wall shear stress (WSS) that acts tangentially to the vessel, could determinate dysregulation of the cellular redox status, that in turn could have effects on intracellular signaling pathways involved in disease progression [33]. For example, exposure of endothelial cells to laminar SS can induce a suppression of ROS levels [34, 35]. Conversely, exposure of endothelial cells to WSS using an irregular flow induces an increase of ROS levels and a reduced bioavailability of the vasodilator molecule NO [36], which is involved in preventing the activation and adhesion of platelets and leukocytes to the wall of the injured vessel [37].

A significant role in the pathogenesis of COPD is precisely the imbalance of ROS production and antioxidant capacity [38]. Changes in the redox balance in the lungs and circulatory system, genetic polymorphisms, and activation of transcription factors, such as the nuclear factor kappa B (NF-κB), lead to the molecular pathogenesis of COPD [39, 40]. Oxidized proteins and lipid products, such as isoprostanes and carbonylated proteins, can be identified in exhaled air, bronchoalveolar lavage fluid, and lung tissue from patients with fibrotic lung diseases and COPD [41, 42]. Furthermore, clinical worsening of COPD is often associated with down-regulation of the antioxidant system, thus a possible therapeutic method for COPD could be the administration of redox-protective antioxidants [38]. Finally, it is possible that maintaining a balance between oxidant and antioxidant species in COPD affected smokers may slow down disease progression [43].

As discussed, smoking, occupational exposures like asbestos or silica, and radiation are the principal sources of OXS with overproduction of ROS, that could lead

and contribute to pulmonary fibrosis [44]. Indeed, OXS is an important molecular mechanism underlying fibrosis in a variety of organs, including lungs. Bleomycininduced pulmonary fibrosis, the most commonly used experimental animal model, has been shown to be associated with marked increase in the level of ROS, oxidized proteins, DNA and lipids [45]. Following lung injury three main mechanisms (i.e. inflammation, coagulation disturbances, and OXS) are involved and alter the lung interstitial cell compartment and extracellular matrix (ECM) homeostasis, resulting overall in pulmonary fibrosis. ROS can be produced by several cellular types involved in fibrosis including alveolar macrophages [46–48] and lung epithelial cells [49]. In particular, ROS generated from the mitochondria of stressed or damaged epithelial cells are very important; their mitochondrial dysfunction results in the generation and release of ROS, such as H2O2 and O2−, further enhancing OXS and cell damage [50]. As previously discussed, NAD(P)H oxidase is the main source of ROS, and isoforms NOX1, NOX2, and NOX4 have a central role in the pathogenesis of pulmonary fibrosis [51] (**Figure 1**). For example, NOX4 is strongly expressed in the hyperplastic alveolar epithelium of IPF patients [52], and ROS produced by NOX4 are involved in alveolar epithelial cell apoptosis. Continuous epithelial apoptosis further supports activation of inflammatory processes and cytokine release, including myofibroblast activating molecules, such as TGF-β1, PDGF, IL-1, and TNFα (**Figure 1**). There is also evidence of direct pathogenetic involvement of these enzymes in IPF, for example for NOX2: in fact, supporting data have shown that mice genetically deficient in NOX2 do not develop IPF after bleomycin or carbon nanotubes exposure [51, 53]. Finally, the interplay between oxidative stress and TGF-β1 signaling is of great importance in promoting fibrosis. In fact, TGF-β1 is the most profibrogenic protein and can directly stimulate NOX-mediated ROS production, while OXS in turn can activate latent TGF-β1, setting up a vicious profibrogenic positive feedback loop [54].
