General mechanisms of nicotine-induced ... - The FASEB Journal

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General mechanisms of nicotine-induced fibrogenesis Kendal Jensen,* Damir Nizamutdinov,† Micheleine Guerrier,* Syeda Afroze,* David Dostal,† and Shannon Glaser*,‡,1 *Division of Gastroenterology and †Division of Molecular Cardiology, Department of Internal Medicine, Texas A&M Health Science Center, Central Texas Veterans Health Care System, Temple, Texas, USA; and ‡Scott and White Healthcare Digestive Disease Research Center, Temple, Texas, USA Cigarette smoking contributes to the development of cancer, and pathogenesis of other diseases. Many chemicals have been identified in cigarettes that have potent biological properties. Nicotine is especially known for its role in addiction and plays a role in other physiological effects of smoking and tobacco use. Recent studies have provided compelling evidence that, in addition to promoting cancer, nicotine also plays a pathogenic role in systems, such as the lung, kidney, heart, and liver. In many organ systems, nicotine modulates fibrosis by altering the functions of fibroblasts. Understanding the processes modulated by nicotine holds therapeutic potential and may guide future clinical and research decisions. This review discusses the role of nicotine in the general fibrogenic process that governs fibrosis and fibrosis-related diseases, focusing on the cellular mechanisms that have implications in multiple organ systems. Potential research directions for the management of nicotineinduced fibrosis, and potential clinical considerations with regard to nicotine-replacement therapy (NRT) are presented.—Jensen, K., Nizamutdinov, D., Guerrier, M., Afroze, S., Dostal, D., Glaser, S. General mechanisms of nicotine-induced fibrogenesis. FASEB J. 26, 4778 – 4787 (2012). www.fasebj.org

ABSTRACT

Key Words: cigarette smoking 䡠 fibroblast 䡠 reactive oxygen species 䡠 collagen 䡠 NRT Nicotine is an addictive substance in cigarette smoke (CS), chewing tobacco, and nicotine replacement therapy (NRT), which has effects on mood, appetite, and task performance through the central nervous system (CNS) (1, 2). Nicotine is a tertiary amine consisting of a pyridine and pyrrolidine ring that is rapidly absorbed through the skin, alveoli of the

Abbreviations: ␣-SMA, ␣-smooth muscle actin; CNS, central nervous system; CS, cigarette smoke; CTGF, connective tissue growth factor; C, dendritic cell; ECM, extracellular matrix; HSC, hepatic stellate cell; IL, interleukin, JNK, c-Jun N-terminal kinase; miR, microRNA; nAChR, nicotinic acetylcholine receptor; NADPH, nicotinamide adenine dinucleotide phosphate; NRT, nicotine-replacement therapy; ROS, reactive oxygen species; TGF-␤I, transforming growth factor-␤I; TGF␤RII, transforming growth factor-␤ receptor II 4778

lungs, and gastrointestinal epithelium prior to entering the bloodstream (2– 4). Nicotine is distributed throughout the body by the circulatory system and alters the physiological processes of cells that express its receptors (2– 4). The liver extensively metabolizes nicotine to form numerous metabolites. Typically, in humans, 7080% of nicotine is metabolized to form cotinine. This is catalyzed by cytochrome P-450 2A6 (CYP2A6) and results in aldehyde formation on the fourth carbon (carbon oxidation) of the pyrrolidine ring (5, 6). Cotinine itself may undergo further transformation into trans-3=-hydroxycotinine and be excreted into the urine by the kidneys, or, similar to nicotine, stimulate its own biological effects (7). Nicotine exerts its biological actions by binding to nicotinic acetylcholine receptors (nAChRs) located on the cell membrane. NAChRs are pentameric proteins formed by the assembly of nAChR subunits: ␣1–␣10, ␤1–␤4, ␥, ␦, and ε (8). The combination of the various subunits gives rise to nicotine’s differential effects on the activation of signaling mechanisms. NAChRs are nonselective ligand-gated ion channels that allow several different positively charged ions, such as potassium, sodium, and calcium, to cross the cell membrane (8). Recently, the ␣7-nAChR has been the primary focus of many studies due to its pronounced effects on proliferation and the pathogenesis of several disease states (9). The binding of nicotine to the ␣7-nAChR receptor triggers an influx of calcium across the plasma membrane, thereby activating a downstream signaling mechanism that contributes to dysregulated growth, angiogenesis, release of growth factors, and modulation of the microenvironment (10). Numerous studies have linked CS to a number of pathologies, such as cancer, and diseases of the cardiovascular and respiratory systems (11–13). However, it is not well understood to what extent nicotine alone contributes to these disease processes. Studies in various cell culture and animal models indicate that nicotine is a potent agent and stimulates biological re1 Correspondence: Department of Medicine, Texas A&M Health Science Center, Central Texas Veterans Health Care System, VA Bldg. 205, 1901 South 1st St., Temple, TX 76504, USA. E-mail: [email protected] doi: 10.1096/fj.12-206458

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sponses leading to disease. For example, there is increasing evidence that nicotine, through the ␣7nAChR pathway, triggers the release of growth factors that contribute to the pathogenesis of cancer (10, 14). In a nude mouse xenograft tumor model, nicotine promoted the growth of colon cancer (15). The researchers postulated that the ␤-adrenergic receptordependent growth-promoting effects of nicotine might be due to either nicotine-induced simulation of local catecholamine release by tumor cells or catecholamine release by the adrenal glands (16). These findings illustrate the fundamental concept that the release of neuroendocrine factors stimulated by nicotine may contribute to the activation of other pathogenic processes in animal models.

OVERVIEW OF FIBROGENIC MECHANISMS Fibrosis is the pathophysiological response to chronic injury that results in excessive deposition of extracellular-matrix (ECM) proteins and scarring. In each organ system, the fibrogenic process has unique characteristics related to the function of the organ and the microenvironment created by the organ-specific epithelium. Yet key steps are common to the majority of fibrotic diseases. Fibrogenesis has been described as a stepwise process, as follows: damage to epithelial/ endothelial barriers; release of transforming growth factor-␤1 (TGF-␤1); recruitment of inflammatory cells; production of reactive oxygen species (ROS); activation of collagen-producing cells; and ECM-dependent activation of myofibroblast cells (17). There has been long-standing interest in elucidating the process of fibrogenesis on a cellular level and identifying the environmental factors controlling the progression of fibrosis. Numerous studies over the past decade have provided compelling evidence for the involvement of nicotine in the modulation of fibroblast activation and function (18, 19). In this review, we critically discuss evidence indicating that nicotine plays a role in one or more profibrotic mechanisms. In general, collagen and related ECM molecules are the end product of these fibrogenic processes. Therefore, nicotine may stimulate the creation of a microenvironment that potentiates the activation of fibroblast function. Although the steps of fibrogenesis are often studied independently, these mechanisms are interrelated and may occur simultaneously, or in a different order. For example, some fibrotic diseases may rely more heavily on the activation of one process over another.

NICOTINE PROMOTES DAMAGE TO EPITHELIAL/ENDOTHELIAL BARRIERS Damage to epithelial and endothelial cells plays a pivotal role in the activation of fibrogenesis. In vascular disorders, such as systemic sclerosis, damage to the endothelial bed activates the production of proinflamMECHANISMS OF NICOTINE-INDUCED FIBROGENESIS

matory cytokines, TGF-␤1 production, tissue hypoxia, and platelet aggregation, which contribute to the development and progression of fibrosis (20, 21). Chronic epithelial damage and/or apoptosis followed by tissue healing can lead to the release of inflammatory factors that trigger immune-cell infiltration and activation of local fibroblasts. Nicotine can promote both epithelial and endothelial cell damage (Fig. 1). In a recent in vivo study, nicotine directly affected the ionic homeostasis of lung and gastrointestinal tract epithelial cells and also provoked an inflammatory reaction that contributed to cellular damage (22). The researchers postulated that the epithelial damage observed in the nicotine-treated rats was due to alterations in Na⫹ and Cl⫺ conductance (22). Chronic treatment with nicotine also stimulated increased eosinophil number in the lamina propria, edema, and increased damage to basal cells in the trachea (22). These effects were found in both the trachea and gastrointestinal tract. However, they were more prominent in the trachea (22). Another group demonstrated that nicotine has toxic effects in the proximal tubules of the kidney and causes to renal damage (14, 23). In a mouse model of ischemia reperfusion injury, nicotine-induced epithelial damage was associated with morphological changes in the renal epithelium, increased expression kidney injury molecule-1, and elevated creatinine levels (23). Nicotine alone was sufficient to stimulate increased expression of markers of oxidative stress and augment fibrotic and inflammatory pathways (23). In addition, there was increased expression of the classic epithelialmesenchymal transition (EMT) markers vimentin, fibronectin, and ␣-smooth muscle actin (␣-SMA) in the renal epithelium (14). Taken together, these studies indicate that chronic nicotine exposure may facilitate progression from acute kidney injury to chronic kidney disease (14, 23). These in vivo studies were complemented by several in vitro studies that demonstrated nicotine-induced toxicity in epithelial cells of various organs, such as the lungs and oral mucosa (24 –26). The mechanisms regulating nicotine-induced endothelial cell damage are not well understood. A study in an in vivo model suggested that nicotine promoted the growth of atherosclerotic plaques through the activation of multiple mechanisms (27). Nicotine also regulated endothelial cell gene expression, which promoted plaque growth and angiogenesis in mice (27). Consistent with these findings, in vitro treatment of primary human coronary endothelial cells with nicotine for 24 h increased expression of nitric oxide synthase, angiotensin-I-converting enzyme, tissue-type plasminogen activator, and vascular cell adhesion molecule-1, which are factors known to promote the development of atherosclerotic plaques (28). NICOTINE STIMULATES THE PRODUCTION AND RELEASE OF TGF-␤1 TGF-␤1 is a critical cytokine involved in wound healing, repair, and differentiation, in many cell types and 4779

Figure 1. Nicotine causes damage to epithelial cells. In various systems, nicotine has been shown either to directly cause damage to epithelial barriers or to potentiate the effects of injury. This results in increased release of inflammatory factors, expression of epithelial-mesenchymal transition (EMT) markers, and a change in ion homeostasis representative of epithelial dysfunction. In the lung and intestine, increased eosinophils accompany these changes.

tissues (29). TGF-␤1 stimulates the production of ECM proteins (including collagen in parenchymal cells, fibroblasts, and inflammatory cells) and plays a key role in the regulation of fibrosis (30, 31). Various in vivo models of fibrosis have demonstrated that nicotine activates TGF-␤1 and stimulates fibroblast functions in an autocrine fashion (Fig. 2). In a canine model of atrial fibrosis, nicotine significantly up-regulated TGF-␤1 and TGF-␤ receptor II (TGF-␤RII) expression, and stimulated atrial fibroblast proliferation and collagen deposition (18). Introduction of microRNA-133 (miR-133) and miR-590 abolished the fibrogenic effects, or, conversely, siRNA knockdown of miR-133 and miR-590 increased fibrogenesis (18). In addition, nicotine decreased expression of miR-133 and miR-590, which was negated by pretreatment with a specific ␣7-nAChR antagonist. These findings suggest that nicotine-induced ␣7-nAChR signaling was responsible for the activation of atrial fibroblasts through microRNAdependent regulation of the TGF-␤1 pathway (18). Another in vivo study demonstrated that nicotine increased the expression of TGF-␤1, which was responsible for F-actin reorganization, and expression of vimentin, fibronectin, and ␣-SMA in the mouse kidney (14). Nicotine stimulates fibrogenesis and activates secretion of collagen from human primary liver cultures. An in vitro study on human hepatic stellate cells (HSCs) demonstrated that nicotine-activated collagen synthesis and TGF-␤1 (32). In this study, healthy human liver cells treated with nicotine exhibited increased expression of a variety of proteins, including collagen I and TGF-␤1 (32). A general antagonist for nAChRs abolished these effects, indicating a direct role for nicotine 4780

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in the activation of the expression of profibrotic proteins (32). Another key cytokine known for its involvement in fibrogenesis is connective tissue growth factor (CTGF). The functions of CTGF parallel those of TGF-␤1, including the stimulation of ECM remodeling and collagen synthesis. CTGF also plays a crucial role in the chemotaxis and mitogenesis of fibroblasts (33). In a recent study, nicotine increased CTGF in human gingival cells and collagen production in the periodontal ligament (34). The findings suggested that nicotine triggered direct cellular damage, induced secretion of CTGF, and activation of fibrogenesis in an autocrine fashion (34).

NICOTINE RECRUITS INFLAMMATORY CELLS Recruitment of infiltrating inflammatory cells is a key event leading to fibrosis, and a pathogenic result of acute and chronic tissue injury. Changes in the activation status and the number of inflammatory cells play a vital role in a number of diseases associated with smoking. Therefore, it is relevant to discern the effects of nicotine on both the recruitment and activation of inflammatory cells and to identify inflammatory mediators/cytokines involved. In several systems, the effects of nicotine are controversial and appear concentrationdependent. Furthermore, this illustrates the concept that nicotine’s actions are pleiotropic and variable. Inflammatory cytokines, including the prime neutrophil chemoattractant interleukin 8 (IL-8), leukotriene B4 (LTB4), monocyte chemotactic protein-1 (MCP-1),

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Figure 2. Nicotine-stimulated release of TGF-␤1 and CTGF. Nicotine stimulates the release of TGF-␤1 either directly or through the ␣7-nAChR. Nicotine is postulated to trigger TGF-␤1 release in other cell types, such as hepatocytes and other epithelial cells, in a similar way.

and a number of other factors attract inflammatory cells of the neutrophil and monocyte-macrophage lineage (31). In tobacco-induced emphysema, these cells pool in the pulmonary circulation of cigarette smokers and secrete proteases that cause irreversible destruction of the alveolar walls of the lungs (35, 36). A number of in vitro and in vivo studies have described nicotine’s profound effects on neutrophils during emphysema (Fig. 3). Yet the precise mechanism whereby nicotine induces emphysema is controversial. Nearly 30 years ago, Totti et al. (37) reported that nicotine was a chemotactic for neutrophils in vitro. In this study, neutrophils obtained from human blood exhibited a dose-dependent migratory response to nicotine (37). In a subsequent study, high concentrations of nicotine suppressed neutrophil chemotaxis. Interestingly, nicotine at this high dose increased neutrophil degranulation and eicosanoid generation (38). Despite contradictory findings, it is notable that both studies agreed that nicotine contributes to profound changes in neutrophil function that lead to emphysema. Recent in vitro studies have focused on evaluating the mechanisms whereby nicotine stimulates neutrophil activity. In one study, nicotine activated F-actin formation and intracellular Ca2⫹ release in neutrophils and was postulated to play a role in neutrophil migration and/or MECHANISMS OF NICOTINE-INDUCED FIBROGENESIS

degranulation during pulmonary disease (39). Another in vitro study in a human myeloblast/promyelocyte cell line demonstrated that nicotine at both low and high doses caused increased elastase gene and protein expression (40). In human bronchial epithelial cells, nicotine up-regulated the IL-8 gene (and 260 other genes) downstream of Ca2⫹ signaling (41). Inhibition of extracellular signal-related kinase (ERK) and c-Jun N-terminal kinase (JNK) mitogen-associated protein kinases (MAPKs) significantly inhibited the nicotineinduced IL-8 response (41). Although the findings of this study have not been supported by in vivo data, another in vitro study has reported that nicotine increases IL-8 expression in gingival cells following the activation of Ca2⫹ signaling (42). Recently, several in vivo studies have sought to confirm that nicotine stimulated the activation of neutrophilic recruitment. In a mouse model of lung inflammation, nicotine enhanced the localization of neutrophils to inflammatory sites created by treatment with zymosan (activator of Tolllike receptor 2; ref. 43). On the other hand, nicotine alone triggered only a weak response. Together, these in vivo data explain, in part, the clinical finding that neutrophil numbers increase in the lower airways of cigarette smokers, where an elastase/antielastase imbalance is more prevalent (43). The mechanism responsi4781

Figure 3. Nicotine stimulates neutrophilic functions in emphysema. Nicotine increases the recruitment of neutrophils to the alveolar space and release of granules.

ble for neutrophilic recruitment is poorly understood, and it should be mentioned that nicotine’s relationship to IL-8 secretion levels was reversed in an in vitro arthritis model (44). Here, nicotine decreased IL-8 secretion in response to TNF-␣ in synoviocytes of the joint (44). These findings clearly indicate that nicotine has pleiotropic effects depending on the cell type and disease process (44). In addition to neutrophils, other inflammatory cells types, such as macrophages, dendritic cells (DCs), and lymphocytes, respond to nicotine. Macrophage activation by nicotine has been assessed in both in vitro and in vivo models. In one study, peritoneal macrophages from mice were treated with increasing concentrations of nicotine to evaluate its effects on superoxide anion generation, lipid peroxidation, protein oxidation, and antioxidant activity (45). Superoxide radical and lipid/ protein peroxidation increased with increasing doses of nicotine (45). Conversely, the antioxidant status decreased (45). This suggests a ROS-mediated activation of an inflammatory reaction by macrophages (45). In vivo, a decrease in the antioxidants glutathione, catalase, superoxide dismutase, glutathione peroxidase, and glutathione reductase in the liver and spleen was observed after intraperitoneal administration of nicotine (45). Several similar reports published by this group of scientists have demonstrated macrophage activation by nicotine and is discussed in the following section. Reports on the effect of nicotine on DCs are conflicting. Most studies tend to agree that nicotine has potent effects on DC regulation. However, nicotine has been observed to both activate and suppress DCs. In a mouse model of atherosclerosis, nicotine strongly activated DCs and the cell-mediated T-helper 1 (TH1) cell response (46). However, other studies have presented data demonstrating the opposite effect for nicotine (47, 48). The concentration of nicotine, and costimulation 4782

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by other factors, could be responsible for these differences. Clearly, more studies are required to understand the exact mechanisms triggered by nicotine in DCs and during cell-mediated immunity.

NICOTINE ACTIVATES ROS PRODUCTION ROS production has a significant effect on different organs and plays roles in physiological (i.e., immune cells digest foreign particles using ROS) and pathological (i.e., excess ROS can lead to cell damage) conditions (49). ROS are strong modulators of inflammatory processes in the CNS, development of atherosclerosis in vessels, stroke injury, renal epithelial damage, pulmonary cell damage, and critical factors in cancer development (50). The key step in the synthesis of ROS is the formation of superoxide by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (51). In some organs, this function is carried out by NADPH homologs (51). Antioxidants play a role in reducing lipid peroxides throughout the body. In certain systems such as the liver, endogenous production of antioxidants is a powerful tool for defense against toxin exposure. Nicotine induces oxidative stress in a number of cell types, including epithelial cells, macrophages, fatty liver cells, and mesangial cells (Fig. 4). Nicotine-stimulated ROS production has been linked to the damage of epithelial cells in an in vivo model of chronic kidney disease (CKD; ref. 23). In this model, nicotine exposure produced ROS through NADPH oxidase in the proximal tubules of the kidney. Chronic nicotine administration not only increased ROS by itself, but also exacerbated acute renal injury, and stimulated the progression into CKD (23). The activation of JNK and activator protein (AP-1) were shown to be involved in this process (23). Since pJNK and AP-1 gene targets are


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Figure 4. Proposed mechanisms for the production of ROS in various cells types. Nicotine causes epithelial cell damage that results in the production of ROS. Nicotine also triggers the production of ROS by the up-regulation of NADPH in macrophages. Nicotine causes increases in lipid peroxides and decreased GSH in fatty liver disease. Nicotine also triggers the production of ROS through injury and up-regulation of NADPH and NOX4 in mesangial cells. Nicotine is implicated in ROS generation in other cell types and future research directions. Overlap of these mechanisms (not shown) likely occurs. Some of these mechanisms (i.e., decreased GSH) are suspected in many systems.

increased in smokers with CKD, nicotine is thought contribute to CKD through this mechanism (23). In an in vitro study, nicotine-induced injury and ROS in mesangial cells of the kidney via NADPH oxidase (52). In a mouse model of type 2 diabetes, nicotine worsened diabetic nephropathy through increased NADPH oxidase homologue-NOX4 activity and production of ROS (53). However, these effects could be reduced by the inclusion of exercise (53). ROS production by inflammatory cells is an essential mediator of nicotine toxicity. Nicotine induces ROS production in macrophages in vitro and in vivo. Elevation of nicotine concentration correlated with increased lipid peroxidation and decreased antioxidant activity of peritoneal macrophages (45). These antioxidants included glutathione, catalase, superoxide dismutase, glutathione peroxidase, and glutathione reductase (45). These effects were assessed in vivo by examining the antioxidant status of the liver and spleen after intraperitoneal administration of nicotine (45). This same group demonstrated that nicotine induces activation of macrophages and superoxide-mediated oxidative damage in several studies that aimed to identify compounds that reduce the oxidative stress caused by nicotine (54, 55). Finally, nicotine increased the expression of markers for oxidative damage in the systemic circulation of rats, as well as in lymphocytes in culture (56, 57). Nicotine has also been shown to induce ROS production in the liver. Physiologically, the liver deals with toxins that produce lipid peroxidation in the pathogenesis of several liver diseases. For example, lipid peroxidation plays a key role in alcoholic liver disease (58). Diets depleted in unsaturated fats and high in saturated fats lower the peroxidation of lipids and alcohol-inMECHANISMS OF NICOTINE-INDUCED FIBROGENESIS

duced damage (59, 60). In vivo models have provided pertinent information about nicotine’s effects on lipid peroxidation. In addition, nicotine exacerbated lipid peroxidation and reduced ROS-scavenging enzymes in rats fed a high-fat diet (61). In addition, nicotine decreased the overall activity of scavenging enzymes (61). Interestingly, in vivo studies have shown that the antioxidants glutathione (GTH), S-allyl cysteine sulfoxide, and vitamin E protect against nicotine-induced lipid peroxidation in the liver, kidney and lung (62, 63). The effects of nicotine on lipid peroxide levels were further enhanced by nicotine’s ability to reduce the levels of antioxidants. Nicotine by itself or in combination with ethanol decreased GSH activity in the liver and lungs (64). Furthermore, the combination of nicotine and ethanol also increased lipid peroxidation (64). Helen et al. (65) suggested that antioxidant depletion occurs via the high levels of superoxide anion and hydrogen peroxide production induced by nicotine.

NICOTINE ACTIVATES COLLAGEN-PRODUCING CELLS Collagen, a chief component of the ECM, is synthesized primarily by resident fibroblasts during fibrogenesis (17). In general, fibroblasts require a stimulus such as TGF-␤1 to activate the secretion of collagen. In fibroblasts, increased collagen synthesis is accompanied by a rapid myofibroblastic change, which can be recognized by the elevation of ␣-smooth muscle actin (␣-SMA), vimentin, and ECM protein expression levels (66). Several studies have shown that CS contributes to cardiac fibrosis by increasing the deposition of collagen 4783

(11, 67–70). Specifically, collagen accumulation in the atrial myocardium correlates with pack-year history of cigarette abuse and nicotine base concentration (70). A number of in vitro and in vivo studies have demonstrated that nicotine induces changes in collagen deposition via fibroblasts (Fig. 5). In vitro, stimulation with nicotine for 24 h caused a 7-fold increase in collagen III production in primary cultures of human atrial myocardial cells (70). Another group has shown in multiple reports that nicotine alters the differentiation and activation status of lung fibroblasts in vitro (19, 71–73). Exposure of embryonic lung fibroblasts in culture to nicotine for 7 d resulted in decreased expression of the key lipogenic markers parathyroid hormone-related peptide (PTHrP) and peroxisomal proliferator-activated receptor ␥ (PPAR␥), and it increased expression of the myogenic marker ␣-SMA (71). This together with a decrease in triglyceride uptake suggested differentiation of normal lung fibroblasts to a myofibroblast phenotype during lung disease (71). On the signaling level, nicotine altered PTHrP-receptor binding, resulting in decreased cyclic AMP (cAMP)/PKA signaling, which contributed to the change in fibroblast phenotype (71). Lipogenic transdifferentiation into a myofibroblastic phenotype has been hypothesized to contribute to increased collagen production by fibroblasts (71). A number of reports in vivo have demonstrated that nicotine increases collagen and fibroblast activation in various systems, such as the heart, lung, gingiva, prostate, and joints (18, 34, 72, 74, 75). Collectively, it is not surprising that nicotine alters the morphogenesis and differentiation of fibroblasts, since nicotine stimulates the transdifferentiation process in other disease models, such as gastric cancer (76). The phagocytosis of collagen is another fibroblast function altered by nicotine (77). An in vitro study demonstrated that fibroblasts from patient gingiva and cultures stimulated with nicotine had reduced internalization of collagen-coated beads (77). In vivo, nicotine

Figure 5. Nicotine increases collagen production through its actions on fibroblasts. Nicotine increases collagen production in fibroblasts and reduces the phagocytosis of collagen. Nicotine has a proliferative effect on fibroblasts. Nicotine stimulates the morphogenesis of fibroblasts into a myofibroblastic phenotype. 4784

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inhibited the phagocytosis of collagen dose dependently, and areas of oral submucosal fibrosis exhibited less phagocytosis than normal gingiva from the same patient (77). While nicotine promotes fibroblast activation and collagen synthesis in many organs, there are some exceptions. For example, nicotine decreased collagen expression and ECM synthesis in two in vivo models of bone remodeling and tendon-bone healing (78, 79). In this setting, inflammation may be responsible for the decreased healing observed during nicotine treatment (79). Another study demonstrated in vivo that scar tissue healing was unaffected by nicotine (80). These processes are not well understood and require further investigation into the signaling mechanisms induced by nicotine.

SUMMARY AND FUTURE DIRECTIONS The understanding of the processes activated by nicotine has considerably expanded to include the activation of fibrogenesis. The growing volume of data from different systems suggests activation of general components of the fibrogenic process and calls for more focus on the subject. To date, the data support nicotine’s fibrogenic actions in cardiac, renal, lung, oral, and other organs and is currently under evaluation in the liver. A deeper understanding of the specific, and/or differential mechanisms by which nicotine stimulates the fibrosis of various organ systems, will provide a foundation for the development of novel therapeutic paradigms. The effects of nicotine that have been reported from in vitro studies should be confirmed in animal models. In particular, nicotine dramatically increases the levels of circulating catecholamines and other hormones that could have serious consequences on various organ systems. At the same time, there is much to be gained from in vitro cell culture models, from the standpoint that elucidating the intracellular signaling mechanisms and cellular processes may have utility in multiple organ systems. Since nicotine is metabolized to cotinine in vivo, studies in animal models may have a confounding contribution due to the effects of circulating cotinine and other nicotine metabolites. In addition, there is a clear need for extensive studies regarding the effects of nicotine in the kidney and vascular system, since smoking is a risk factor for diseases in these organ systems. While smoking is prohibited for patients with a number of diseases, including primary biliary cirrhosis, little is known about nicotine’s contribution to their pathogenesis. Liver cirrhosis is a disease that is potentiated by smoking. An understanding of the protective mechanisms activated in the liver during nicotine injury would be essential, especially since the liver is the site for nicotine metabolism. Furthermore, the liver has a high capacity to adapt to increased loads of oxidative stress by increasing antioxidants and wound healing. Activation of HSCs plays a key role in the progression of


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liver diseases. Preliminary in vitro studies in HSCs suggest that nicotine is fibrogenic and plays a role in HSC activation through nAChRs (32). The ability of the liver to adapt to injury and activate repair mechanisms to return to its normal function is not shared by all organs. For example, the lung easily reacts to nicotine to produce the irreversible pathology seen in emphysema.

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8. 9. 10. 11.

CLINICAL PERSPECTIVES 12.

Cigarette smoking is responsible for over 435,000 deaths each year in the United States (81). Cigarette smoke contains many biologically active components. However, nicotine is responsible for its addictive properties, and an estimated 21% of Americans smoke (81, 82). NRT is the most widely used treatment for promoting smoking cessation (83). NRT has been approved by the Food and Drug Administration and is considered a safe and effective aid in quitting smoking. Epidemiological studies of the nicotine patch primarily reported non-life-threatening side effects (84, 85). At firstglance, this appears to contradict the large number of cell culture and animal studies reporting nicotine’s disease-causing mechanisms. However, consideration should be given to the fact that the long-term nicotine patch utilization has not been studied. That being said there is a critical need for epidemiological research to evaluate long-term NRT usage. Clinical investigators should monitor patients using NRT for extended periods of time, especially if they are at risk for cancer or fibrotic/inflammatory diseases.

13. 14.

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Portions of this work were supported by the Scott and White Hospital, Department of Internal Medicine; a Scott and White Research Advancement award; a U.S. Department of Veteran’s Affairs Career Development Award-2; and a U.S. National Institutes of Health RO1 grant (DK081442) to S.G. The authors thank Dr. Gianfranco Alpini for his kind suggestions and comments on the manuscript.

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