The Effects of Cigarette Smoke Extract on Ovulation,Oocyte Morphology and Ovarian Gene Expression in Mice

Cigarette smoking can harm fertility, but the existing research has targeted primarily on ovarian follicles, embryos or sex hormone. In this study, we tested cigarette smoke extract on ovulation, oocyte morphology and ovarian gene expression associated with inhibition of oxidative stress using C57BL/6 mice. Mice in the experimental group were administered a cigarette smoke extract (CSE) solution (2 mg/ml) orally daily, while the blank control group was given dimethylsulfoxide (DMSO). A positive control group (menadione) was used that received an intraperitoneal injection of 15 mg/kg menadione in oil solution daily. We found that the CSE group manifested a reduced diameter of zona pellucida-free oocyte (ZP-free OD) and a morphologically misshapen first polar body (PB). Our results suggest that CSE exposure is associated with a shrink size and poor quality of oocytes. Quitting smoking is a wise choice to ensure good fertility.     

Cigarette Smoke Exposure during Pregnancy Alters Fetomaternal Cell Trafficking Leading to Retention of Microchimeric Cells in the Maternal Lung

Cigarette smoke exposure causes chronic oxidative lung damage. During pregnancy, fetal microchimeric cells traffic to the mother. Their numbers are increased at the site of acute injury. We hypothesized that milder chronic diffuse smoke injury would attract fetal cells to maternal lungs. We used a green-fluorescent-protein (GFP) mouse model to study the effects of cigarette smoke exposure on fetomaternal cell trafficking. Wild-type female mice were exposed to cigarette smoke for about 4 weeks and bred with homozygote GFP males. Cigarette smoke exposure continued until lungs were harvested and analyzed. Exposure to cigarette smoke led to macrophage accumulation in the maternal lung and significantly lower fetal weights. Cigarette smoke exposure influenced fetomaternal cell trafficking. It was associated with retention of GFP-positive fetal cells in the maternal lung and a significant reduction of fetal cells in maternal livers at gestational day 18, when fetomaternal cell trafficking peaks in the mouse model. Cells quickly clear postpartum, leaving only a few, difficult to detect, persisting microchimeric cells behind. In our study, we confirmed the postpartum clearance of cells in the maternal lungs, with no significant difference in both groups. We conclude that in the mouse model, cigarette smoke exposure during pregnancy leads to a retention of fetal microchimeric cells in the maternal lung, the site of injury. Further studies will be needed to elucidate the effect of cigarette smoke exposure on the phenotypic characteristics and function of these fetal microchimeric cells, and confirm its course in cigarette smoke exposure in humans.     

Reduced expression of let‐7f activates TGF‐β/ALK5 pathway and leads to impaired ischaemia‐induced neovascularization after cigarette smoke exposure

This study sought to determine the potential role of microRNAs (miRNAs) in the detrimental effects of cigarette smoke on angiogenesis and neovascularization. Using large‐scale miRNA profiling and qRT‐PCR analyses, we identified let‐7f as a pro‐angiogenic miRNA which expression is significantly reduced in HUVECs treated with cigarette smoke extracts (CSE), and in the ischemic muscles of mice that are exposed to cigarette smoke (MES). In a mouse model of hindlimb ischaemia, intramuscular injection of let‐7f mimic restored ischaemia‐induced neovascularization in MES. Doppler flow ratios and capillary density in ischemic muscles were significantly improved in MES treated with let‐7f mimic. Clinically, this was associated with reduced ambulatory impairment and hindlimb ischaemic damage. Treatment with let‐7f mimic could also rescue pro‐angiogenic cell (PAC) number and function (attachment, proliferation, migration) in MES. ALK5 (TGF‐βR1), an important modulator of angiogenesis, is a target of let‐7f. Here we show that ALK5 is increased in HUVECs exposed to CSE and in the ischaemic muscles of MES. This is associated with a downstream activation of the anti‐angiogenic factors SMAD2/3 and PAI‐1. Importantly, treatment with let‐7f mimic reduces the expression of ALK5, SMAD2/3 and PAI‐1 both in vitro and in vivo. Moreover, let‐7f overexpression or ALK5 inhibition can rescue angiogenesis in HUVECs exposed to CSE. Cigarette smoke exposure is associated with reduced expression of let‐7f and activation of the anti‐angiogenic TGF‐β/ALK5 pathway. Overexpression of let‐7f using a miRNA mimic could constitute a novel therapeutic strategy to improve ischaemia‐induced neovascularization in pathological conditions.     

Transient and Persistent Metabolomic Changes in Plasma following Chronic Cigarette Smoke Exposure in a Mouse Model

Cigarette smoke exposure is linked to the development of a variety of chronic lung and systemic diseases in susceptible individuals. Metabolomics approaches may aid in defining disease phenotypes, may help predict responses to treatment, and could identify biomarkers of risk for developing disease. Using a mouse model of chronic cigarette smoke exposure sufficient to cause mild emphysema, we investigated whether cigarette smoke induces distinct metabolic profiles and determined their persistence following smoking cessation. Metabolites were extracted from plasma and fractionated based on chemical class using liquid-liquid and solid-phase extraction prior to performing liquid chromatography mass spectrometry-based metabolomics. Metabolites were evaluated for statistically significant differences among group means (p-value≤0.05) and fold change ≥1.5). Cigarette smoke exposure was associated with significant differences in amino acid, purine, lipid, fatty acid, and steroid metabolite levels compared to air exposed animals. Whereas 60% of the metabolite changes were reversible, 40% of metabolites remained persistently altered even following 2 months of smoking cessation, including nicotine metabolites. Validation of metabolite species and translation of these findings to human plasma metabolite signatures induced by cigarette smoking may lead to the discovery of biomarkers or pathogenic pathways of smoking-induced disease.     

Cigarette smoke alters the secretome of lung epithelial cells

Cigarette smoke is the most relevant risk factor for the development of lung cancer and chronic obstructive pulmonary disease. Many of its more than 4500 chemicals are highly reactive, thereby altering protein structure and function. Here, we used subcellular fractionation coupled to label‐free quantitative MS to globally assess alterations in the proteome of different compartments of lung epithelial cells upon exposure to cigarette smoke extract. Proteomic profiling of the human alveolar derived cell line A549 revealed the most pronounced changes within the cellular secretome with preferential downregulation of proteins involved in wound healing and extracellular matrix organization. In particular, secretion of secreted protein acidic and rich in cysteine, a matricellular protein that functions in tissue response to injury, was consistently diminished by cigarette smoke extract in various pulmonary epithelial cell lines and primary cells of human and mouse origin as well as in mouse ex vivo lung tissue cultures. Our study reveals a previously unrecognized acute response of lung epithelial cells to cigarette smoke that includes altered secretion of proteins involved in extracellular matrix organization and wound healing. This may contribute to sustained alterations in tissue remodeling as observed in lung cancer and chronic obstructive pulmonary disease.     

Cigarette smoke impairs NK cell-dependent tumor immune surveillance

In this study, we investigated the impact of cigarette smoke on tumor immune surveillance and its consequences to lung tumor burden in a murine lung metastasis model. Cigarette smoke exposure significantly increased the numbers of lung metastases following B16-MO5 melanoma challenge. This effect was reversible; we observed significantly fewer tumor nodules following smoking cessation. Using RAG2(-/-) and RAG2(-/-)gamma(c)(-/-) mice, we provide strong evidence that increased tumor incidence was NK cell dependent. Furthermore, we show that cigarette smoke suppressed NK activation and attenuated NK CTL activity, without apparent effect on activating or inhibitory receptor expression. Finally, activation of NK cells through bone marrow-derived dendritic cells conferred protection against lung metastases in smoke-exposed mice; however, protection was not as efficacious as in sham-exposed mice. To our knowledge, this is the first experimental evidence showing that cigarette smoke impairs NK cell-dependent tumor immune surveillance and that altered immunity is associated with increased tumor burden. Our findings suggest that altered innate immunity may contribute to the increased risk of cancer in smokers.     

microRNA expression profile in porcine oocytes with different developmental competence derived from large or small follicles

Oocyte developmental competence is acquired during folliculogenesis and regulated by complex molecular mechanisms. Several molecules are involved in these mechanisms, including microRNAs (miRNAs) that are essential for oocyte‐specific processes throughout the development. The objective of this study was to identify the expression profile of miRNAs in porcine oocytes derived from follicles of different sizes using RNA deep sequencing. Oocytes were aspirated from large (LO; 3–6 mm) or small (SO; 1.5–1.9 mm) follicles and tested for developmental competence and chromatin configurations. Small RNA libraries were constructed from both groups and then sequenced in an Illumina NextSeq. 500. Oocytes from the LO group exhibited higher developmental competence and different chromatin configuration compared with oocytes from the SO group. In total, 167 and 162 known miRNAs were detected in the LO and SO groups, respectively. MiR‐205, miR‐16, miR‐148a‐3p, and miR‐125b were among the top 10 highly expressed miRNAs in both groups. Eight miRNAs were differentially expressed (DE) between both groups. Target gene prediction and pathway analysis revealed 46 pathways that were enriched with miRNA‐target genes. The oocyte meiosis pathway and signaling pathways including FoxO, PI3K‐Akt, and cAMP were predictably targeted by DE miRNAs. These results give more insights into the potential role of miRNAs in regulating the oocyte development.     

TLR4 protein contributes to cigarette smoke-induced matrix metalloproteinase-1 (MMP-1) expression in chronic obstructive pulmonary disease

Cigarette smoke is the major risk factor associated with the development of chronic obstructive pulmonary disease and alters expression of proteolytic enzymes that contribute to disease pathology. Previously, we reported that smoke exposure leads to the induction of matrix metalloproteinase-1 (MMP-1) through the activation of ERK1/2, which is critical to the development of emphysema. To date, the upstream signaling pathway by which cigarette smoke induces MMP-1 expression has been undefined. This study demonstrates that cigarette smoke mediates MMP-1 expression via activation of the TLR4 signaling cascade. In vitro cell culture studies demonstrated that cigarette smoke-induced MMP-1 was regulated by TLR4 via MyD88/IRAK1. Blockade of TLR4 or inhibition of IRAK1 prevented cigarette smoke induction of MMP-1. Mice exposed to acute levels of cigarette smoke exhibited increased TLR4 expression. To further confirm the in vivo relevance of this signaling pathway, rabbits exposed to acute cigarette smoke were found to have elevated TLR4 signaling and subsequent MMP-1 expression. Additionally, lungs from smokers exhibited elevated TLR4 and MMP-1 levels. Therefore, our data indicate that TLR4 signaling, through MyD88 and IRAK1, plays a predominant role in MMP-1 induction by cigarette smoke. The identification of the TLR4 pathway as a regulator of smoke-induced protease production presents a series of novel targets for future therapy in chronic obstructive pulmonary disease.     

Mouse Protocadherin-1 Gene Expression Is Regulated by Cigarette Smoke Exposure In Vivo

Protocadherin-1 (PCDH1) is a novel susceptibility gene for airway hyperresponsiveness, first identified in families exposed to cigarette smoke and is expressed in bronchial epithelial cells. Here, we asked how mouse Pcdh1 expression is regulated in lung structural cells in vivo under physiological conditions, and in both short-term cigarette smoke exposure models characterized by airway inflammation and hyperresponsiveness and chronic cigarette smoke exposure models. Pcdh1 gene-structure was investigated by Rapid Amplification of cDNA Ends. Pcdh1 mRNA and protein expression was investigated by qRT-PCR, western blotting using isoform-specific antibodies. We observed 87% conservation of the Pcdh1 nucleotide sequence, and 96% conservation of the Pcdh1 protein sequence between men and mice. We identified a novel Pcdh1 isoform encoding only the intracellular signalling motifs. Cigarette smoke exposure for 4 consecutive days markedly reduced Pcdh1 mRNA expression in lung tissue (3 to 4-fold), while neutrophilia and airway hyperresponsiveness was induced. Moreover, Pcdh1 mRNA expression in lung tissue was reduced already 6 hours after an acute cigarette-smoke exposure in mice. Chronic exposure to cigarette smoke induced loss of Pcdh1 protein in lung tissue after 2 months, while Pcdh1 protein levels were no longer reduced after 9 months of cigarette smoke exposure. We conclude that Pcdh1 is highly homologous to human PCDH1, encodes two transmembrane proteins and one intracellular protein, and is regulated by cigarette smoke exposure in vivo.     

Exogenous heat shock protein-70 inhibits cigarette smoke-induced intimal thickening

Cigarette smoke is associated with increased carotid intimal thickening or stroke. Preliminary work showed that exposure to smoke resulted in a 4.5-fold reduction of heat shock protein-70 (HSP70) expression in spleens of mice using gene microarray analysis. In the current study, we investigated the role of extracellular HSP70 in carotid intimal thickening of mice exposed to cigarette smoke. Intimal thickening was induced by placement of a cuff around the right carotid artery of mice. Cuff injury resulted in increased HSP70 mRNA expression in carotid arteries that persisted for 21 days. Cigarette smoke exposure decreased arterial HSP70 expression and significantly increased intimal thickening compared with mice exposed to air. Treatment of mice exposed to cigarette smoke with intravenous recombinant HSP70 attenuated intimal thickening through reduced phosphorylated extracellular signal-regulated kinase (pERK) expression in the arterial wall. In vitro experiments with rat aortic smooth muscle cells confirmed that recombinant HSP70 decreases pERK and proliferating cell nuclear antigen (PCNA) expression in cells exposed to cigarette smoke extract and H(2)O(2). Our study suggests that decreased expression of arterial HSP70 is an important mechanism by which exposure to cigarette smoke augments intimal thickening. The effects of recombinant HSP70 suggest a role for extracellular HSP70.     

Caspase-12 Silencing Attenuates Inhibitory Effects of Cigarette Smoke Extract on NOD1 Signaling and hBDs Expression in Human Oral Mucosal Epithelial Cells

Cigarette smoke exposure is associated with increased risk of various diseases. Epithelial cells-mediated innate immune responses to infectious pathogens are compromised by cigarette smoke. Although many studies have established that cigarette smoke exposure affects the expression of Toll-liked receptor (TLR), it remains unknown whether the nucleotide-binding oligomerization domain-containing protein 1 (NOD1) expression is affected by cigarette smoke exposure. In the study, we investigated effects of cigarette smoke extract (CSE) on NOD1 signaling in an immortalized human oral mucosal epithelial (Leuk-1) cell line. We first found that CSE inhibited NOD1 expression in a dose-dependent manner. Moreover, CSE modulated the expression of other crucial molecules in NOD1 signaling and human β defensin (hBD) 1, 2 and 3. We found that RNA interference-induced Caspase-12 silencing increased NOD1 and phospho-NF-κB (p-NF-κB) expression and down-regulated RIP2 expression. The inhibitory effects of CSE on NOD1 signaling can be attenuated partially through Caspase-12 silencing. Intriguingly, Caspase-12 silencing abrogated inhibitory effects of CSE on hBD1, 3 expression and augmented induced effect of CSE on hBD2 expression. Caspase-12 could play a vital role in the inhibitory effects of cigarette smoke on NOD1 signaling and hBDs expression in oral mucosal epithelial cells.     

Differential protease, innate immunity, and NF-kappaB induction profiles during lung inflammation induced by subchronic cigarette smoke exposure in mice

Cigarette smoke exposure is a major determinant of adverse lung health, but the molecular processes underlying its effects on inflammation and immunity remain poorly understood. Therefore, we sought to understand whether inflammatory and host defense determinants are affected during subchronic cigarette smoke exposure. Dose-response and time course studies of lungs from Balb/c mice exposed to smoke generated from 3, 6, and 9 cigarettes/day for 4 days showed macrophage- and S100A8-positive neutrophil-rich inflammation in lung tissue and bronchoalveolar lavage (BAL) fluid, matrix metalloproteinase (MMP) and serine protease induction, sustained NF-kappaB translocation and binding, and mucus cell induction but very small numbers of CD3+CD4+ and CD3+CD8+ lymphocytes. Cigarette smoke had no effect on phospho-Akt but caused a small upregulation of phospho-Erk1/2. Activator protein-1 and phospho-p38 MAPK could not be detected. Quantitative real-time PCR showed upregulation of chemokines (macrophage inflammatory protein-2, monocyte chemoattractant protein-1), inflammatory mediators (TNF-alpha, IL-1beta), leukocyte growth and survival factors [granulocyte-macrophage colony-stimulating factor, colony-stimulating factor (CSF)-1, CSF-1 receptor], transforming growth factor-beta, matrix-degrading MMP-9 and MMP-12, and Toll-like receptor (TLR)2, broadly mirroring NF-kappaB activation. No upregulation was observed for MMP-2, urokinase-type plasminogen activator, tissue-type plasminogen activator, and TLRs 3, 4, and 9. In mouse strain comparisons the rank order of susceptibility was Balb/c > C3H/HeJ > 129SvJ > C57BL6. Partition of responses into BAL macrophages vs. lavaged lung strongly implicated macrophages in the inflammatory responses. Strikingly, except for IL-10 and MMP-12, macrophage and lung gene profiles in Balb/c and C57BL/6 mice were very similar. The response pattern we observed suggests that subchronic cigarette smoke exposure may be useful to understand pathogenic mechanisms triggered by cigarette smoke in the lungs including inflammation and alteration of host defense.     

Reduced expression and function of aquaporin-3 in mouse metaphase-II oocytes induced by controlled ovarian hyperstimulation were associated with subsequent low fertilization rate.

BACKGROUND/AIMS: Aquaporin-3 (AQP3), one isoform of water channel family, has been found to be expressed in mouse oocytes. The present study aimed to investigate whether functional AQP3 was expressed in oocytes induced by controlled ovarian hyperstimulation (COH), and whether altered oocyte AQP3 expression was associated with changes in fertilization rate. METHODS: Sixty ICR female mice were divided into two groups: COH and control. AQP3 mRNA expression of mouse metaphase II (MII) oocytes was quantified by real-time RT-PCR. The water permeability of oocytes was assessed with cell swelling test. The fertilization profiles of oocytes were generated via in vitro fertilization. RESULTS: AQP3 mRNA was expressed in both natural and COH-induced mouse oocytes. COH significantly reduced AQP3 mRNA expression. The volume of oocytes was significantly increased after exposure to hypotonic medium and pretreatment with HgCl(2) attenuated hypotonic medium-induced increase in oocyte volume and water permeability coefficient (Pf). Furthermore, the expression of AQP3, Pf and the fertilization rate were significantly lower in COH oocytes than those in control. CONCLUSION: AQP3 might play an important role in controlling oocyte quality and a low in vitro fertilization rate of COH mice might, in part, result from reduced AQP3 expression and water permeability in mouse oocytes.     

Cigarette smoke exposure aggravates air space enlargement and alveolar cell apoptosis in Smad3 knockout mice

The concept of genetic susceptibility factors predisposing cigarette smokers to develop emphysema stems from the clinical observation that only a fraction of smokers develop clinically significant chronic obstructive pulmonary disease. We investigated whether Smad3 knockout mice, which develop spontaneous air space enlargement after birth because of a defect in transforming growth factor-β (TGF-β) signaling, develop enhanced alveolar cell apoptosis and air space enlargement following cigarette smoke exposure. We investigated Smad3(-/-) and Smad3(+/+) mice at different adult ages and determined air space enlargement, alveolar cell proliferation, and apoptosis. Furthermore, laser-capture microdissection and real-time PCR were used to measure compartment-specific gene expression. We then compared the effects of cigarette smoke exposure on Smad3(-/-) and littermate controls. Smad3 knockout resulted in the development of air space enlargement in the adult mouse and was associated with decreased alveolar VEGF levels and activity and increased alveolar cell apoptosis. Cigarette smoke exposure aggravated air space enlargement and alveolar cell apoptosis. We also found increased Smad2 protein expression and phosphorylation, which was enhanced following cigarette smoke exposure, in Smad3-knockout animals. Double immunofluorescence analysis revealed that endothelial apoptosis started before epithelial apoptosis. Our data indicate that balanced TGF-β signaling is not only important for regulation of extracellular matrix turnover, but also for alveolar cell homeostasis. Impaired signaling via the Smad3 pathway results in alveolar cell apoptosis and alveolar destruction, likely via increased Smad2 and reduced VEGF expression and might represent a predisposition for accelerated development of emphysema due to cigarette smoke exposure.     

Whole Cigarette Smoke Increased the Expression of TLRs,HBDs, and Proinflammory Cytokines by Human Gingival Epithelial Cells through Different Signaling Pathways

The gingival epithelium is becoming known as a regulator of the oral innate immune responses to a variety of insults such as bacteria and chemicals, including those chemicals found in cigarette smoke. We investigated the effects of whole cigarette smoke on cell-surface-expressed Toll-like receptors (TLR)-2, −4 and −6, human β-defensin (HBD) and proinflammatory cytokine expression and production in primary human gingival epithelial cells. Whole cigarette smoke was shown to increase TLR2, TLR4 and TLR6 expression. Cigarette smoke led to ERK1/2, p38 and JNK phosphorylation in conjunction with nuclear factor-κB (NFκB) translocation into the nucleus. TLR expression following cigarette smoke exposure was down regulated by the use of ERK1/2, p38, JNK MAP kinases, and NFκB inhibitors, suggesting the involvement of these signaling pathways in the cellular response against cigarette smoke. Cigarette smoke also promoted HBD2, HBD3, IL-1β, and IL-6 expression through the ERK1/2 and NFκB pathways. Interestingly, the modulation of TLR, HBD, and cytokine expression was maintained long after the gingival epithelial cells were exposed to smoke. By promoting TLR, HBDs, and proinflammatory cytokine expression and production, cigarette smoke may contribute to innate immunity dysregulation, which may have a negative effect on human health.     

Cigarette smoke increases progesterone receptor and homeobox A10 expression in human endometrium and endometrial cells: a potential role in the decreased prevalence of endometrial pathology in smokers

Cigarette smoking has long been tied to a multitude of poor health outcomes; however, in reproductive biology, smoking has shown several unintuitive findings. Smoking is associated with significantly decreased rates of endometriosis and endometrial cancer. Here, we show that treatment with cigarette smoke extract leads to increased mRNA and protein expression of homeobox A10 (HOXA10) and progesterone receptor (PGR) as well as more rapid decidualization of endometrial stromal cells in vitro. In vivo, mice exposed to cigarette smoke similarly showed increased expression of HOXA10 and PGR in the endometrium. Both HOXA10 and PGR drive endometrial differentiation and are suppressed in endometrial tumors and in endometriosis. The increased expression found upon exposure to cigarette smoke may provide a protective effect, mediating the decreased incidence of endometrial disease among smokers. This mechanism contrasts with the accepted paradigm that the effects of smoking on the uterus are secondary to ovarian alterations rather than direct effects on endometrium as demonstrated here.     

PAF mediates cigarette smoke-induced goblet cell metaplasia in guinea pig airways

Goblet cell metaplasia is an important morphological feature in the airways of patients with chronic airway diseases; however, the precise mechanisms that cause this feature are unknown. We investigated the role of endogenous platelet-activating factor (PAF) in airway goblet cell metaplasia induced by cigarette smoke in vivo. Guinea pigs were exposed repeatedly to cigarette smoke for 14 consecutive days. The number of goblet cells in each trachea was determined with Alcian blue-periodic acid-Schiff staining. Differential cell counts and PAF levels in bronchoalveolar lavage fluid were also evaluated. Cigarette smoke exposure significantly increased the number of goblet cells. Eosinophils, neutrophils, and PAF levels in bronchoalveolar lavage fluid were also significantly increased after cigarette smoke. Treatment with a specific PAF receptor antagonist, E-6123, significantly attenuated the increases in the number of airway goblet cells, eosinophils, and neutrophils observed after cigarette smoke exposure. These results suggest that endogenous PAF may play a key role in goblet cell metaplasia induced by cigarette smoke and that potential roles exist for inhibitors of PAF receptor in the treatment of hypersecretory airway diseases.     

Reduction of EGF is associated with the delay of ulcer healing by cigarette smoking

Cigarette smoking is associated with peptic ulcer diseases. Smokers have lower levels of salivary epidermal growth factor (EGF) than nonsmokers. We investigated whether reduction of EGF is involved in the delay of gastric ulcer healing by cigarette smoking. Rats with acetic acid-induced ulcers were exposed to cigarette smoke (0, 2, or 4% vol/vol) 1 day after ulcer induction. EGF level was elevated 1 day after ulcer induction in salivary glands and serum, and 4 days after ulcer induction in the gastric mucosa. However, cigarette smoke depressed these beneficial effects and EGF mRNA expression in salivary glands and gastric mucosa. Cigarette smoke delayed gastric ulcer healing and reduced cell proliferation, angiogenesis, and mucus synthesis. Exogenous EGF (10 and 20 microg/kg i.v.) before smoke exposure reversed the adverse effects of cigarette smoke, whereas vascular endothelial growth factor level and nitric oxide synthase activity were unaffected. It is concluded that the detrimental effect of cigarette smoke on ulcer healing is a consequence of reduction of angiogenesis, cell proliferation, and mucus secretion through the depressive action on EGF biosynthesis and its mRNA expression in salivary glands and gastric mucosa.