Alcohol use, and misuse, has been a part of human culture for thousands of years. In the modern medical era a great deal of attention has been justifiably focused on elucidating the mechanisms underlying the psychological and biological addiction to alcohol. However, a significant percentage, if not the majority, of alcohol-related morbidity and mortality occurs in individuals who do not meet the formal diagnostic criteria for alcohol use disorders. For example, many serious medical consequences of chronic alcohol ingestion can occur in individuals who do not have signs or symptoms of alcohol dependence. There is now clear evidence that even in otherwise healthy-appearing individuals who chronically consume excessive amounts of alcohol that alveolar macrophage immune capacity is impaired and, as a consequence, these individuals are at significantly increased risk of pneumonia. This brief review summarizes some of the key mechanisms underlying this phenomenon and proposes a hypothetical scheme by which alcohol interferes with zinc bioavailability within the alveolar space and thereby dampens macrophage function.
pneumonia; ARDS; acute lung injury; glutathione; zinc
Alcohol abuse and HIV-1 infection frequently co-exist and these individuals are at high risk for serious lung infections and respiratory failure. Although alcohol ingestion and HIV-1 transgene expression have been shown to independently cause oxidative stress and disrupt alveolar epithelial barrier function in experimental models, their interactive effects have not been examined.
Methods and Results
In this study we determined that chronic alcohol ingestion (12 wks) exacerbated the already significant defects in alveolar epithelial paracellular permeability and lung liquid clearance in HIV-1 transgenic rats. Further, immunocytochemical analyses of tight junction protein expression in primary alveolar epithelial cells showed that occludin and zonula occludens-1 (ZO-1) localization within the plasma membrane was more disrupted than in either condition alone, consistent with the observed defects in epithelial barrier function. Interestingly, expression of Nrf2, the transcription factor required to activate the antioxidant response element, was decreased in primary alveolar epithelial cells isolated from HIV-1 transgenic rats. In parallel, exposing lung epithelial cells in vitro to either alcohol or the HIV-related protein gp120 also decreased Nrf2 expression. Importantly, treatment with procysteine, which increases thiol antioxidants including glutathione, improved tight junction protein localization in the plasma membrane and restored alveolar epithelial barrier function in alcohol-fed HIV-1 transgenic rats.
These results provide novel evidence that HIV-related proteins and alcohol together causes more barrier dysfunction in the lung epithelium than either stress alone. However, these significant effects on the alveolar barrier can be mitigated by augmenting the thiol antioxidant pool, a strategy with potential clinical applications in subjects who are highly vulnerable to lung disease because of co-existent alcohol abuse and HIV infection.
alveolar barrier function; tight junction proteins; Nrf2; procysteine; glutathione
Chronic alcohol abuse causes oxidative stress, impairs alveolar macrophage immune function, and increases the risk of pneumonia and acute lung injury. Recently we determined that chronic alcohol ingestion in rats decreases zinc levels and macrophage function in the alveolar space; provocative findings in that zinc is essential for normal immune and antioxidant defenses. Alveolar macrophage immune function depends on stimulation by GM-CSF, which signals via the transcription factor PU.1. In parallel, the antioxidant response element signals via the transcription factor Nrf2. However, the role of zinc bioavailability on these signaling pathways within the alveolar space is unknown.
To determine the efficacy of dietary zinc supplementation on lung bacterial clearance and oxidative stress, we tested three different groups of rats: control-fed, alcohol-fed, and alcohol-fed with zinc supplementation. Rats were then inoculated with intratracheal Klebsiella pneumoniae and lung bacterial clearance was determined 24 hrs later. Isolated alveolar macrophages were isolated from uninfected animals and evaluated for oxidative stress and signaling through PU.1 and Nrf2.
Alcohol-fed rats had a 5-fold decrease in lung bacterial clearance compared to control-fed rats. Dietary zinc supplementation of alcohol-fed rats normalized bacterial clearance and mitigated oxidative stress in the alveolar space, as reflected by the relative balance of the thiol redox pair cysteine and cystine, and increased nuclear binding of both PU.1 and Nrf2 in alveolar macrophages from alcohol-fed rats.
Dietary zinc supplementation prevents alcohol-induced alveolar macrophage immune dysfunction and oxidative stress in a relevant experimental model, suggesting that such a strategy could decrease the risk of pneumonia and lung injury in individuals with alcohol use disorders.
rat; macrophages; GM-CSF; oxidative stress; lung; bacterial infections; zinc
Highly effective antiviral treatment can suppress HIV-1 infection, but the chronic effects of HIV-1-related viral proteins, including gp120 and Tat, on organs such as the lungs can be damaging. HIV-1 transgenic rodent models are useful for studying the systemic effects of these proteins independently of viral infection. We have previously shown that HIV-1 transgene expression (and therefore, HIV-1-related protein expression) in rats decreases alveolar macrophage zinc levels and phagocytic capacity by unknown mechanisms. We hypothesized that HIV-1 transgene expression induces chronic inflammation and zinc sequestration within the liver and thereby decreases zinc bioavailability in the lung. We examined the expression of the pro-inflammatory cytokine, tumor necrosis factor alpha (TNFα), the zinc storage protein, metallothionein (MT1), and the zinc exporter, ZNT1 in the livers and the lungs of wild type and HIV-1 transgenic rats ± dietary zinc supplementation. In addition, we measured zinc levels, the zinc importing protein ZIP1, and the phagocytic capacity in the alveolar macrophages.
HIV-1 transgene expression increased the liver-specific expression of TNFα, suggesting a chronic inflammatory response within the liver in response to HIV-1-related protein expression. In parallel, HIV-1 transgene expression significantly increased MT1 and ZNT1 expression in the liver as compared to the lung, a pattern that is consistent with zinc sequestration in the liver as occurs during systemic inflammation. Further, HIV-1 transgene expression decreased intracellular zinc levels and increased expression of ZIP1 in the alveolar macrophages, a pattern consistent with zinc deficiency, and decreased their bacterial phagocytic capacity. Interestingly, dietary zinc supplementation in HIV-1 transgenic rats decreased gene expression of TNFα, MT1, and ZNT1 in the liver while simultaneously increasing their expression in the lung. In parallel, zinc supplementation increased alveolar macrophage intracellular zinc levels and bacterial phagocytic capacity in HIV-1 transgenic rats.
Taken together, these findings suggest that chronic HIV-1-related protein expression causes liver inflammation and zinc sequestration, which in turn limits zinc bioavailability in the lung and thereby impairs alveolar macrophage phagocytic function. Importantly, dietary zinc supplementation decreases liver inflammation and zinc sequestration and restores alveolar macrophage phagocytic function in HIV-1 transgenic rats, a result with potential clinical implications for improving lung health in HIV-1-infected individuals.
pulmonary; alveolar macrophages; AIDS; rodent; inflammation; micronutrients
Chronic alcohol consumption perturbs cellular function in a variety of organ systems. Previous studies have suggested that moderate alcohol consumption reduces vascular disease, whereas heavier alcohol consumption may worsen it. The mechanisms for these vascular effects of chronic alcohol ingestion continue to be defined and constitute the focus of this study.
Male Sprague Dawley rats were fed an isocaloric, Lieber-Decarli liquid diet containing either ethanol (36% calories) or Maltose–Dextrin (substituted for ethanol) for 6 weeks. Telemetric blood pressure measurements were taken before and after ethanol feeding. After the rats were killed, the aortas were analyzed for endothelial nitric oxide (NO) synthase expression and NO production.
Chronic ethanol ingestion decreased mean arterial pressure and increased aortic NO production as demonstrated by direct ex vivo measurements using iron diethyldithio-carbamic acid as well as analysis of nitrosyl-hemoglobin (NO-Hb) levels. Consistent with these assays of vascular NO production, endothelium-dependent relaxation responses to acetycholine (Ach) were enhanced in ethanol-fed animals. Aortic endothelial nitric oxide synthase expression was also increased by chronic ethanol ingestion.
These findings demonstrate that a regimen of chronic alcohol ingestion in the rat produced generally salutary effects in the systemic vasculature following a 6-week treatment regimen. These findings extend previous in vitro studies to demonstrate that alcohol has potent effects on vascular endothelial nitric oxide synthase expression, NO production, and vascular function. Consistent with previous reports, these findings confirm that alcohol-induced alterations in the production of reactive nitrogen species play an important role in the pathogenesis of alcohol-mediated tissue effects.
Nitric Oxide; Ethanol; Endothelial Nitric Oxide Synthase
Alcohol-related chronic myopathy is characterized by severe biochemical and structural changes to skeletal muscle. Our goals were to: (1) identify early regulatory elements that precede the overt manifestation of plantaris atrophy; and (2) circumvent these derangements by supplementing alcohol-fed rats with the glutathione precursor, procysteine. After 6 weeks of daily ingestion, before the development of overt atrophy of the plantaris muscle, alcohol increased several markers of oxidative stress and increased gene expressions of atrogin-1 and transforming growth factor-β1 (TGF-β1) by ~60- and ~65-fold, respectively, which were attenuated by procysteine supplementation. Interestingly, after 28 weeks of alcohol ingestion, when overt plantaris atrophy had developed, atrogin-1 and TGF-β1 gene expression had returned to baseline levels. Together, these findings suggest that alcohol-induced, redox-sensitive alterations drive pro-atrophy signaling pathways that precede muscle atrophy. Therefore, targeted anti-oxidant treatments such as procysteine supplementation may benefit individuals with chronic alcohol abuse, particularly if given prior to the development of clinically significant myopathy.
alcoholic myopathy; atrogin-1; glutathione; oxidative stress; transforming growth factor-β1
Alcohol abuse independently increases the risk of developing the acute respiratory distress syndrome (ARDS), a disease characterized by diffuse alveolar epithelial damage, lung edema, and consequent severe hypoxemia. Chronic alcohol abuse increases alveolar epithelial permeability both in vitro and in vivo, in part due to altered tight junction formation. However, both alcohol-fed animals and otherwise healthy alcoholic humans do not have pulmonary edema at baseline, even though their lungs are highly susceptible to acute edematous injury in response to inflammatory stresses. This suggests that active fluid transport by the alveolar epithelium is preserved or even augmented in the alcoholic lung. Chronic alcohol ingestion increases expression of apical sodium channels in the alveolar epithelium; however, its effects on the Na,K-ATPase complex that drives sodium and fluid transport out of the alveolar space have not been examined.
Age- and gender-matched Sprague–Dawley rats were fed the Lieber–DeCarli liquid diet containing either alcohol or an isocaloric substitution (control diet) for 6 weeks. Gene and protein expression of lung Na,K-ATPase α1, α2, and β1 subunits were quantified via real-time PCR and immunobiological analyses, respectively. Alcohol-induced, Na,K-ATPase-dependent epithelial barrier dysfunction was determined by calculating lung tissue wet:dry ratios following an ex vivo buffer-perfused challenge for 2 hours in the presence of ouabain (10−4 M), a Na, K-ATPase inhibitor.
Chronic alcohol ingestion significantly increased gene and protein expression of each Na,K-ATPase subunit in rat lungs. Immunohistochemical analyses of the alcoholic lung also revealed that protein expression of the Na,K-ATPase α1 subunit was increased throughout the alveolar epithelium. Additionally, lungs isolated from alcohol-fed rats developed more edema than comparably treated lungs from control-fed rats, as reflected by increased lung tissue wet:dry ratios.
These findings indicate that chronic alcohol ingestion, which is known to increase alveolar epithelial paracellular permeability, actually increases the expression of Na,K-ATPase in the lung as a compensatory mechanism. This provides a potential explanation as to why the otherwise healthy alcoholic does not have evidence of pulmonary edema at baseline.
Alcohol; Acute Respiratory Distress Syndrome; Edema; Lung
Previously we determined that chronic alcohol ingestion (6 wks) in rats increases lung epithelial permeability in vivo ~5-6-fold and promotes flooding of the alveolar airspaces with proteinaceous fluid in response to stresses such as sepsis. In parallel, alveolar epithelial cells isolated from alcohol-fed rats fail to form tight monolayers in vitro, even when cultured for up to 8 days in the absence of alcohol. However, the molecular mechanisms underlying alcohol-induced permeability are unknown. Claudins are key components of tight junctions that restrict the paracellular movement of water, proteins, and solutes across cellular barriers including the alveolar epithelium. In this study, we examined the expression of multiple members of the claudin protein family in the lungs of alcohol-fed vs. control-fed rats (Lieber-DeCarli liquid diet with 36% of calories as alcohol vs. maltin-dextrin for 6 wks). We determined that chronic alcohol ingestion affected the expression of multiple claudins; most striking were decreases in claudin-1 and claudin-7, and an increase in claudin-5, in the whole lung and in alveolar epithelial monolayers derived from alcohol-fed rats. In parallel, immunocytochemistry of alveolar epithelial monolayers from alcohol-fed rats revealed abnormal intracellular accumulation of claudin-7 protein and relatively decreased localization to cell membranes. Claudin-1 and claudin-7 are relatively specific to alveolar epithelial type I pneumocytes that form the vast majority of the alveolar epithelial barrier in vivo, and increases in claudin-5 have been associated with increased epithelial permeability in other systems. Therefore, these findings suggest that changes in claudin expression in the alveolar epithelium produce a “leakier” phenotype that renders the alcoholic lung susceptible to alveolar flooding during acute inflammatory stresses.
claudin; ARDS; ethanol; type II pneumocyte; type I pneumocyte; tight junction
Aims: To assess the effectiveness of procysteine (PRO) supplementation provided during a period of abstinence (ABS) on alcohol-induced skeletal muscle atrophy and oxidant stress. Methods: Age- and gender-matched Sprague–Dawley rats were fed the Lieber–DeCarli liquid diet containing either alcohol or an isocaloric substitution (control diet) for 12 week. Next, subgroups of alcohol-fed rats were fed the control diet for 2 week (ABS) supplemented with either PRO (0.35%, w/v) or vehicle. Plantaris morphology was assessed by hematoxylin and eosin staining. Total, reduced and oxidized glutathione (GSH) levels and total antioxidant potential were determined by commercially available assay kits. Antibody arrays were used to determine cytokine levels. Real-time polymerase chain reaction was used to determine gene expressions of two E3 ubiquitin ligases, atrogin-1 and muscle ring finger protein-1 (MuRF-1). Results: Plantaris muscles from alcohol-fed rats displayed extensive atrophy, as well as decreased GSH levels, a trend for decreased total antioxidant potential and elevated atrogin-1 and MuRF-1 mRNA levels. GSH levels and total antioxidant potential continued to decrease during 2 weeks of ABS from alcohol, which were normalized in abstinent rats provided PRO. Gene levels of both E3 ligases returned to baseline during ABS. In parallel, plantaris cross-sectional area increased in both groups during ABS. Conclusions: PRO supplementation during ABS significantly attenuated alcohol-induced redox stress compared with untreated abstinent rats. Thus, our data may suggest that GSH restoration therapy may provide therapeutic benefits to the overall antioxidant state of skeletal muscle when prescribed in conjunction with an established detoxification program for recovering alcoholics.
Chronic alcohol abuse impairs both alveolar epithelial and macrophage function, and renders individuals susceptible to acute lung injury, pneumonia, and other serious lung diseases. Zinc deficiency, which is known to impact both epithelial and immune cell functions, is also associated with alcohol abuse. In this study, chronic alcohol ingestion (6 wk) in rats altered expression of key zinc transporters and storage proteins in the small intestine and the lung, and decreased zinc levels in the alveolar compartment. Zinc supplementation of alveolar epithelial monolayers derived from alcohol-fed rats in vitro, or of the diets of alcohol-fed rats in vivo, restored alveolar epithelial barrier function, and these improvements were associated with salutary changes in tight junction protein expression and membrane localization. In parallel, dietary zinc supplementation increased intracellular zinc levels, GM-CSF receptor expression, and bacterial phagocytic capacity in the alveolar macrophages of alcohol-fed rats. Together, these studies implicate zinc deficiency as a novel mechanism mediating alcohol-induced alveolar epithelial and macrophage dysfunction. Importantly, these findings argue that dietary supplementation can overcome alcohol-induced zinc deficiency and restore alveolar epithelial and macrophage function, and therefore could be an effective treatment for the susceptible alcoholic lung phenotype.
GM-CSF; phagocytosis; tight junctions; zinc transporters; metallothionein
Using an experimental model of airway fibrosis following lung transplantation, we recently showed that chronic alcohol ingestion by donor rats amplifies airway fibrosis in the recipient. Associated with alcohol-mediated amplification of airway fibrosis is increased transforming growth factor beta-1(TGFβ1) and alpha-smooth muscle actin (α–SMA) expression. Other studies have shown that interleukin-13 (IL-13) modulates TGFβ1 signaling during experimentally-induced airway fibrosis. Therefore, we hypothesized that IL-13 is a component of alcohol-mediated amplification of pro-fibrotic mediators in the alcoholic lung.
To test this hypothesis, we analyzed tracheal epithelial cells and type II alveolar cells from control- or alcohol-fed rats, alcohol-treated mouse lung fibroblasts and human bronchial epithelial cells in vitro for expression of various components of the IL-13 signaling pathway. Signaling via the IL-13 pathway was assessed by measuring levels of phosphorylated signal transducers and activators of transcription-6 (STAT6). In addition, we performed heterotopic tracheal transplantation using control-fed and alcohol-fed donor rats and analyzed tracheal allografts for expression of components of the IL-13 signaling pathway by RT-PCR and immunocytochemical analyses.
IL-13 expression was detected in type II alveolar epithelial cells and human bronchial epithelial cells, but not in lung fibroblasts. IL-13 expression was decreased in whole lung and type II cells in response to alcohol exposure. In all cell types analyzed, expression of IL-13 signaling receptor (IL-13Rα1) mRNA was markedly increased. In contrast, mRNA and protein expression of the IL-13 decoy receptor (IL-13Rα2) were decreased in all cells analyzed. Exposure to alcohol also increased STAT6 phosphorylation in response to IL-13 and lipopolysaccharide.
Data from multiple cell types in the pulmonary system suggest that IL-13 and its receptors play a role in alcohol-mediated activation of pro-fibrotic pathways. Taken together, these data suggest that alcohol primes the airway for increased IL-13 signaling and subsequent tissue remodeling upon injury such as transplantation.
alcohol; airway fibrosis; obliterative airway disease; IL-13; IL-13 receptor
Long term alcohol ingestion may produce severe oxidant stress and lead to skeletal muscle dysfunction. Emerging evidence has suggested that members of the interleukin-6 (IL-6) family of cytokines play diverse roles in the regulation of skeletal muscle mass. Thus, our goals were (1) to minimize the degree of oxidant stress and attenuate atrophy by supplementing the diets of alcohol-fed rats with the glutathione precursor, procysteine, and (2) to identify the roles of IL-6 family members in alcoholic myopathy.
Age- and gender-matched Sprague-Dawley rats were fed the Lieber-DeCarli liquid diet containing either alcohol or an isocaloric substitution (control diet) for 35 wk. Subgroups of alcohol-fed rats received procysteine (0.35%, w/v) for the final 12 wk. Plantaris morphology was assessed by hematoxylin and eosin staining. Major components of glutathione metabolism were determined by assay kits. Real time PCR was used to determine expression levels of several genes.
Plantaris muscles from alcohol-fed rats displayed extensive atrophy, as well as decreased glutathione levels, decreased activities of glutathione reductase and glutathione peroxidase, decreased superoxide dismutase (SOD)-2 (Mn-SOD2), and increased NADPH oxidase-1 gene expression - each indicative of significant oxidant stress. Alcohol also induced gene expression of catabolic factors including IL-6, oncostatin M, atrogin-1, muscle ring finger protein-1, and IGFBP-1. Procysteine treatment attenuated plantaris atrophy, restored glutathione levels, and increased catalase, Cu/Zn-SOD1, and Mn-SOD2 mRNA expression, but did not reduce other markers of oxidant stress or levels of these catabolic factors. Instead, procysteine stimulated gene expression of anabolic factors such as insulin-like growth factor-1, ciliary neurotrophic factor and cardiotrophin-1.
Procysteine significantly attenuated, but did not completely abrogate, alcohol-induced oxidant stress or catabolic factors. Rather, procysteine minimized the extent of plantaris atrophy by inducing components of several anabolic pathways. Therefore, anti-oxidant treatments such as procysteine supplementation may benefit individuals with alcoholic myopathy.
alcoholic myopathy; cardiotrophin-1; ciliary neurotrophic factor; interleukin-6; procysteine
HIV-1 infection impairs alveolar macrophage immune function and renders patients susceptible to pneumonia by poorly understood mechanisms. Alveolar macrophage maturation and function depends on granulocyte-macrophage colony–stimulating factor (GM-CSF), which is produced and secreted by the alveolar epithelium. Macrophages respond to GM-CSF through the GM-CSF receptor (GM-CSFR), which has a binding subunit (GM-CSFRα) and a signaling subunit (GM-CSFRβ). In this study, we measured GM-CSFR expression and alveolar macrophage function in a transgene HIV-1 rat model (NL4-3Δ gag/pol); this construct bears a pro-virus with gag and pol deleted, but other HIV-1–related proteins, such as gp120 and Tat, are expressed, and the rats develop an AIDS-like phenotype as they age. We first determined that HIV-1–transgenic expression selectively decreased alveolar macrophage expression of GM-CSFRβ and impaired bacterial phagocytosis in vitro. Next, we examined the role of zinc (Zn) deficiency as a potential mechanism underlying these effects, and determined that HIV-1–transgenic rats have significantly lower levels of Zn in the alveolar space and macrophages. To test the direct effect of Zn deficiency on macrophage dysfunction, we treated rat alveolar macrophage cell line with a Zn chelator, N,N,N′,N′-tetrakis-(2-pyridyl-methyl) ethylenediamine, and this decreased GM-CSFRβ expression and phagocytosis. In parallel, treatment with Zn acetate in vitro for 48 hours restored intracellular Zn levels and phagocytic function in alveolar macrophages from HIV-1–transgenic rats. Taken together, these data suggest that pulmonary Zn deficiency could be one of the mechanisms by which chronic HIV-1 infection impairs alveolar macrophage immune function and renders these individuals susceptible to serious lung infections.
AIDS; lung; monocyte/macrophages; phagocytosis; rodent
HIV-infected individuals are at increased risk for acute and chronic airway disease even though there is no evidence that the virus can infect the lung epithelium. Although HIV-related proteins including gp120 and Tat can directly cause oxidant stress and cellular dysfunction, their effects in the lung are unknown. The goal of this study was to determine the effects of HIV-1 transgene expression in rats on alveolar epithelial barrier function. Alveolar epithelial barrier function was assessed by determining lung liquid clearance in vivo and alveolar epithelial monolayer permeability in vitro. Oxidant stress in the alveolar space was determined by measuring the glutathione redox couple by high performance liquid chromatography, and the expression and membrane localization of key tight junction proteins were assessed. Finally, the direct effects of the HIV-related proteins gp120 and Tat on alveolar epithelial barrier formation and tight junction protein expression were determined.
HIV-1 transgene expression caused oxidant stress within the alveolar space and impaired epithelial barrier function even though there was no evidence of overt inflammation within the airways. The expression and membrane localization of the tight junction proteins zonula occludens-1 and occludin were decreased in alveolar epithelial cells from HIV-1 transgenic rats. Further, treating alveolar epithelial monolayers from wild type rats in vitro with recombinant gp120 or Tat for 24 hours reproduced many of the effects on zonula occludens-1 and occludin expression and membrane localization.
Taken together, these data indicate that HIV-related proteins cause oxidant stress and alter the expression of critical tight junction proteins in the alveolar epithelium, resulting in barrier dysfunction.
Rationale: Obliterative bronchiolitis (OB) after lung transplantation is triggered by alloimmunity, but is ultimately mediated by transforming growth factor (TGF)-β1–dependent airway fibrosis.
Objectives: Chronic alcohol use increases TGF-β1 expression and renders the lung susceptible to injury. Therefore, we hypothesized that donor alcohol abuse could prime the lung allograft for OB, as many organ donors have a history of alcohol abuse.
Methods: Tracheas from control and alcohol-fed rats (8 wk) were heterotopically transplanted into recipients with varying degrees of alloimmune mismatch and analyzed for obliterative airway disease severity on Postoperative Day 21.
Measurements and Main Results: Although donor alcohol ingestion did not increase the number of antigen-presenting cells or infiltrating lymphocytes, it nevertheless increased allograft lumenal collagen content fourfold compared with allografts from control donors. In parallel, alcohol increased TGF-β1 and α-smooth muscle actin expression in allografts. Alcohol amplified airway disease even in isografts with minor alloimmune mismatches. In contrast, it did not cause any airway disease in isografts in a pure isogenic background, suggesting that a minimal alloimmune response is necessary to trigger alcohol-induced airway fibrosis.
Conclusions: Although alloimmune inflammation is required to initiate airway disease, alcohol primes the allograft for greater TGF-β1 expression, myofibroblast transdifferentiation, and fibrosis than by alloimmune inflammation alone. This has serious clinical implications, as many lung donors have underlying alcohol abuse that may prime the allograft recipient for subsequent OB.
fibrosis; lung transplant; obliterative bronchiolitis; transforming growth factor-β1
Human immunodeficiency virus (HIV)-infected patients have a higher incidence of oxidative stress, endothelial dysfunction, and cardiovascular disease than uninfected individuals. Recent reports have demonstrated that viral proteins upregulate reactive oxygen species, which may contribute to elevated cardiovascular risk in HIV-1 patients. In this study we employed an HIV-1 transgenic rat model to investigate the physiological effects of viral protein expression on the vasculature. Markers of oxidative stress in wild-type and HIV-1 transgenic rats were measured using electron spin resonance, fluorescence microscopy, and various molecular techniques. Relaxation studies were completed on isolated aortic rings, and mRNA and protein were collected to measure changes in expression of nitric oxide (NO) and superoxide sources. HIV-1 transgenic rats displayed significantly less NO-hemoglobin, serum nitrite, serum S-nitrosothiols, aortic tissue NO, and impaired endothelium-dependent vasorelaxation than wild-type rats. NO reduction was not attributed to differences in endothelial NO synthase (eNOS) protein expression, eNOS-Ser1177 phosphorylation, or tetrahydrobiopterin availability. Aortas from HIV-1 transgenic rats had higher levels of superoxide and 3-nitrotyrosine but did not differ in expression of superoxide-generating sources NADPH oxidase or xanthine oxidase. However, transgenic aortas displayed decreased superoxide dismutase and glutathione. Administering the glutathione precursor procysteine decreased superoxide, restored aortic NO levels and NO-hemoglobin, and improved endothelium-dependent relaxation in HIV-1 transgenic rats. These results show that HIV-1 protein expression decreases NO and causes endothelial dysfunction. Diminished antioxidant capacity increases vascular superoxide levels, which reduce NO bioavailability and promote peroxynitrite generation. Restoring glutathione levels reverses HIV-1 protein-mediated effects on superoxide, NO, and vasorelaxation.
acquired immunodeficiency syndrome; antioxidants; superoxide
Acute lung injury affects close to 200,000 people in the U.S. annually and leads to death in 40–50% of affected patients. Chronic ethanol abuse is thought to contribute to up to 40–50% of subjects who develop acute lung injury. We previously demonstrated in a rat model that chronic ethanol ingestion promoted acute lung injury and associated with chronic oxidant stress, activated matrix metalloproteinases, increased release of transforming growth factor-β, as well as increased expression and deposition of fibronectin, a matrix glycoprotein implicated in lung injury and repair. Since fibronectin can activate monocytes to increase proinflammatory cytokine expression, we hypothesized that generation of fibronectin-enriched matrices during chronic ethanol ingestion might contribute to the development of acute lung injury by stimulating unopposed inflammation. To test this hypothesis, we harvested alveolar type II cells from rats fed the Lieber DiCarli diet (6 wk; 36% of calories from ethanol). After 96 hours of culture, the matrices deposited ex vivo by the type II cells derived from ethanol-fed rats showed increased amounts of fibronectin protein as demonstrated by ELISA. When monocytic U937 cells were plated atop these matrices, there was increased expression of interleukin-1β. This stimulation was inhibited by antibodies against α5β1, a receptor that mediates many of the biological effects of fibronectin. We then tested whether antioxidants ameliorated these effects. Dietary supplements of the antioxidants N-acetylcysteine and Procysteine normalized matrix production by type II cells. Furthermore, the newly derived matrices did not stimulate interleukin-1β expression over control cells. These studies suggest that chronic ethanol exposure induces oxidant stress and activates lung tissue remodeling characterized by increased expression of fibronectin by alveolar type II cells. The newly deposited fibronectin-enriched matrices may stimulate the expression of proinflammatory cytokines in monocytic cells recruited to the lung after injury thereby explaining the priming effects of ethanol.
extracellular matrix; lung injury; oxidant stress; tissue remodeling
Human immunodeficiency virus type 1 (HIV-1) infection and the consequent acquired immunodeficiency syndrome (AIDS) has protean manifestations, including muscle wasting and cardiomyopathy, which contribute to its high morbidity. The pathogenesis of these myopathies remains partially understood, and may include nutritional deficiencies, biochemical abnormalities, inflammation, and other mechanisms due to viral infection and replication. Growing evidence has suggested that HIV-1-related proteins expressed by the host in response to viral infection, including Tat and gp120, may also be involved in the pathophysiology of AIDS, particularly in cells or tissues that are not directly infected with HIV-1. To explore the potentially independent effects of HIV-1-related proteins on heart and skeletal muscles, we used a transgenic rat model that expresses several HIV-1-related proteins (e.g., Tat, gp120, and Nef). Outcome measures included basic heart and skeletal muscle morphology, glutathione metabolism and oxidative stress, and gene expressions of atrogin-1, muscle ring finger protein-1 (MuRF-1) and Transforming Growth Factor-β1 (TGFβ1), three factors associated with muscle catabolism.
Consistent with HIV-1 associated myopathies in humans, HIV-1 transgenic rats had increased relative heart masses, decreased relative masses of soleus, plantaris and gastrocnemius muscles, and decreased total and myosin heavy chain type-specific plantaris muscle fiber areas. In both tissues, the levels of cystine (Cyss), the oxidized form of the anti-oxidant cysteine (Cys), and Cyss:Cys ratios were significantly elevated, and cardiac tissue from HIV-1 transgenic rats had altered glutathione metabolism, all reflective of significant oxidative stress. In HIV-1 transgenic rat hearts, MuRF-1 gene expression was increased. Further, HIV-1-related protein expression also increased atrogin-1 (~14- and ~3-fold) and TGFβ1 (~5-fold and ~3-fold) in heart and plantaris muscle tissues, respectively.
We provide compelling experimental evidence that HIV-1-related proteins can lead to significant cardiac and skeletal muscle complications independently of viral infection or replication. Our data support the concept that HIV-1-related proteins are not merely disease markers, but rather have significant biological activity that may lead to increased oxidative stress, the stimulation of redox-sensitive pathways, and altered muscle morphologies. If correct, this pathophysiological scheme suggests that the use of dietary thiol supplements could reduce skeletal and cardiac muscle dysfunction in HIV-1-infected individuals.
Alcohol abuse increases the incidence of acute respiratory distress syndrome and causes oxidative stress and cellular dysfunction in the lung. The mechanisms of ethanol (EtOH)-induced oxidative stress in the lung remain to be defined. Chronic alcohol ingestion has been associated with increased renin-angiotensin system (RAS) activity. Therefore, the current study investigated the ability of lisinopril, an angiotensin-converting enzyme (ACE) inhibitor, to modulate oxidative stress in the lung after chronic EtOH ingestion in a well-established rat model. Male Sprague-Dawley rats were fed liquid diets containing EtOH (36% of calories) or maltose-dextrin as an isocaloric substitution for EtOH (Control) for 6 wk. Selected animals were also treated with lisinopril (3 mg/liter) for 6 wk. Chronic EtOH ingestion increased bronchoalveolar lavage fluid glutathione disulfide levels and superoxide formation in lung parenchyma. These effects of EtOH were attenuated by lisinopril treatment. Chronic EtOH ingestion failed to increase ACE expression or angiotensin II levels in lung homogenates, but increased angiotensinogen, angiotensin II type 1 and type 2 receptor levels, and ACE activity. Chronic EtOH ingestion also increased the levels of the NADPH oxidase subunit, gp91phox, an effect that was attenuated by lisinopril, but had no effect on lung p22phox or p47phox levels. These findings suggest that EtOH-mediated RAS activation plays an important role in pulmonary oxidative stress and provide new insights into mechanisms by which EtOH causes oxidative stress in the lung and potential strategies of lung protection through ACE inhibition.
ARDS; chronic ethanol ingestion; lung; NADPH oxidase; superoxide