Benzene is among the most produced chemicals in the United States, and epidemiological evidence links benzene exposure to impaired pulmonary function; yet, the influence of pre-existing metabolic disease on susceptibility remains poorly defined. We investigated the effects of inhaled benzene on pulmonary mechanics and surfactant homeostasis in diabetic mice with varying glycemic severity. Mildly hyperglycemic KK and severely hyperglycemic, insulin-resistant KK-Ay mice were exposed to filtered air or 50 ppm benzene (6 h/d, 5 d/wk) for 2 wk. Pulmonary function was assessed by the forced-oscillation technique, and bronchoalveolar lavage fluid was analyzed for surfactant proteins and lipid composition by lipidomics; whole-lung transcriptomes were profiled by RNA sequencing. Benzene increased respiratory system resistance and Newtonian resistance in KK-Ay mice without altering tissue elastance or compliance in either strain. Methacholine challenge revealed exaggerated bronchoconstriction in benzene-exposed KK-Ay mice, with significant increases in Rn and tissue damping absent in benzene-exposed KK controls. Surfactant protein B was reduced by benzene in both strains, whereas SP-C was diminished in benzene-exposed KK-Ay mice compared with benzene-exposed KK controls. Lipidomic profiling demonstrated benzene-induced surfactant lipid remodeling characterized by elevated triradylglycerols, increased phosphatidylcholine-to-phosphatidylethanolamine ratio, and reduced phosphatidylserine, with specific lipid classes correlating with airway resistance. Transcriptomic analysis identified enrichment of neurotransmitter transport, ion-channel, and calcium-regulated exocytosis pathways, alongside upregulation of phase II detoxification enzymes. These findings demonstrate that in severe hyperglycemic KK-Ay mice, benzene induced airway hyperresponsiveness through convergent disruption of surfactant protein/lipid homeostasis, identifying poorly controlled diabetes as a risk factor for benzene-related respiratory toxicity.

Background:Benzene is a ubiquitous environmental pollutant generated by a variety of natural and anthropological sources. It is a known carcinogen and hematopoietic toxin; however, little is known about benzene's potential atherogenicity. Hypothesis:Inhaled benzene induces atherogenesis by increasing vascular inflammation in LDL receptor Knockout (LDLR-KO) mice. Methods:Male LDLR-KO mice were exposed to HEPA-filtered air or benzene (1 ppm, 6h/day, 5days/week) for 24 weeks. For the last 12 weeks of exposure, the mice were maintained on a western diet. The single nuclei RNA sequencing (snRNAseq) of aortae was performed at Novogene. For in vitro experiments, splenic naïve T cells were exposed to 1 μM of hydroquinone (HQ) for 24 hours, and intracellular ROR-gamma levels were measured by flow cytometry. Results:Benzene inhalation increased the aortic valve lesion area by more than 25% (P<0.05) in LDLR-KO mice. Using snRNAseq, eleven major cell types were detected, including T cells and vascular smooth muscle cells (VSMC). Benzene increased the number of T cells by 2.5-fold, proliferating T-cells by 5.8-fold, and VSMC by 1.6-fold, suggesting increased cellularity and reduced plaque stability. In addition, benzene upregulated Th17 polarization marker Rorc and negative regulators of apoptosis Rag1 and Bcl11b while significantly attenuating the expression of proliferation inhibitor Ms4a4b in T cells. In VSMC, benzene downregulated extracellular matrix organization genes and upregulated platelet degranulation pathways. Polarization of T cells into Th17 was confirmed by HQ-dependent upregulation of ROR-gamma in vitro. Conclusion:Our data suggest that inhaled benzene exposure compromises plaque cellularity and stability by facilitating T-cell proliferation and polarization, which coincides with the degradation of smooth muscle extracellular matrix and platelet activation.

Benzene is a ubiquitous environmental pollutant, abundant in both the outdoor and indoor air. Chronic exposure is associated with increased risk for cardiovascular disease; however, the underlying mechanisms remain unknown. We examined the effect of bioactive benzene metabolites on endothelial integrity. In vitro, highly reactive benzene metabolites, specifically trans, trans-muconaldehyde (MA, 10 µM), decreased the impedance of murine cardiac microvascular endothelial cells (MCMVEC) in a time- and dose-dependent manner and increased the endothelial permeability to 70 kDa dextran. Intradermal injection of MA (400 pmol) increased the vascular leakage by 54% (P < 0.0001) in adult male C57BL/6J mice. This was accompanied by increased levels of endothelial microparticles in the circulation. RNA sequencing of MA-treated MCMVEC and human aortic endothelial cells revealed the robust induction of heat shock proteins (HSPs), particularly members of the HSP70 and HSP90 families. Reactome pathway enrichment analyses suggested that MA dysregulates pathways associated with G protein-coupled receptor and heat shock factor-1-dependent transactivation. Pharmacological inhibition of HSP70s and HSP90s prevented an MA-induced increase in MCMVEC monolayer permeability. Similarly, pharmacological inhibition of Rho-associated coiled-coil-containing protein kinase (ROCK) attenuated MA-induced endothelial permeability in MCMVEC, accompanied by a dose-dependent activation of Rac1 GTPase. To assess the contribution of HSPs to MA-induced endothelial function impairment, we generated a transgenic mouse overexpressing HSPA1B (a member of the HSP70 family; HSPA1B-TGEC). MA exposure increased the vascular leakage by 15% (P < 0.05) in HSPA1B-TGEC mice as compared with the littermate controls. Collectively, our data suggest that MA increases vascular permeability by activating HSP and GTPase signaling pathways.

Background: Benzene is a ubiquitous environmental pollutant abundant in automobile exhaust, cigarette smoke, forest fires, and present in several household products. It is ranked # 6 on Agency for Toxic Substances and Disease Registry (ATSDR) priority list, however, little is known about its effect on cardiovascular disease, especially atherosclerosis. Hypothesis: Inhaled benzene induces atherogenesis by increasing vascular inflammation in LDL receptor Knockout (LDLR-KO) mice. Methods: Male LDLR-KO or apoE-KO mice were exposed to HEPA-filtered air or benzene (1.0 or 10.0 ppm, 6h/day, 5 days/week) for 24 weeks and maintained on Western diet for the last 12 weeks of exposure. Atherosclerotic lesion composition and nature were determined by liquid chromatography-mass spectrometry (LC-MS), single nuclei RNA sequencing (snRNAseq), and Cytometry by time of flight (CyTOF). Results: Inhaled benzene exposure significantly increased lesions in the aortic valves (>25%, P<0.05). Single cell RNA sequencing of the aortae of benzene-exposed mice revealed 11 major cell types (e.g., vascular smooth muscle cells (VSMCs), endothelial cells, macrophages, T cells, etc.) categorized into 23 sub-populations. snRNAseq and CyTOF analysis showed that benzene exposure robustly increased (P<0.05) CD4 + and CD8 + T-cells, and B-cells in the lesions. Clustering of T cells showed significantly higher expression (P<0.05) of CD4, CD8, and Th17 marker RAR Related Orphan Receptor C (RORc). snRNAseq data also suggested increased cell proliferation and >1.5-fold increase in VSMC number following benzene exposure. LC-MS analysis revealed that benzene exposure differentially regulated the abundance of 930 proteins (at least 2 two unique peptides) in innominate plaque. Importantly, several of the proteins involved in an extracellular matrix organization and elastic fibre formation (e.g. Elastin, Fibronectin , and Latent transforming growth factor beta binding protein 1 ) were downregulated in benzene-exposed mice. These data agree with the snRNAseq data. In vitro , benzene metabolite hydroquinone (HQ, 1 μM) induced ROR-gamma, the transcription factor that regulates Th17, in spleen-derived CD4 + T cells (P<0.05). HQ (0.1 μM) also increased protein kinase C activity and proliferation of human aortic smooth muscle cells (P<0.001). Conclusion: Benzene exposure exacerbates atherosclerosis and compromises plaque cellularity and stability.

Background : Muscle wasting is a serious complication in heart failure patients, and oxidative stress is involved in the pathogenesis of muscle wasting. Oxidative stress leads to the formation of toxic lipid peroxidation products, such as 4-hydroxy-2-nonenal (HNE) and acrolein, which causemuscle wasting. In tissues, these toxic aldehydes are metabolically removed by enzymes such asaldo keto reductases and endogenous nucleophiles, such as glutathione and carnosine. Whether these metabolic pathways could be affected in skeletal muscle during heart failure has never been studied. Methods : Male wild-type C57BL/6J mice were subjected to a pressure overload model of hypertrophy by transaortic constriction (TAC) surgery, and echocardiography was performed after 14 weeks. Different skeletal muscle beds were weighed and analyzed for atrophic and inflammatory markers, Atrogin1 and TRIM63, TNF-α and IL-6 , respectively, by RT‒PCR. Levels of acrolein and HNE-protein adducts, aldehyde-removing enzymes, aldose reductase (AKR1B1) and aldehyde dehydrogenase 2 (ALDH2) were measured by Western blotting, and histidyl dipeptides and histidyl dipeptide aldehyde conjugates were analyzed by LC/MS-MS in the gastrocnemius and soleus muscles of sham- and TAC-operated mice. Furthermore, histidyl dipeptide synthesizing enzyme carnosine synthase (CARNS) and amino acid transporters (PEPT2 and TAUT)wasmeasured in the gastrocnemius muscles of the sham and TAC-operated mice. Results : TAC-induced heart failure decreases body weight and gastrocnemius and soleus muscle weights. The expression of the atrophic and inflammatory markers Atrogin1 and TNF-α, respectively, wasincreased (~1.5-2-fold), and the formation of HNE and acrolein-protein adducts was increased in the gastrocnemius muscle of TAC-operated mice. The expression of AKR1B1 remained unchanged, whereas ALDH2 was decreased, in the gastrocnemius muscle of TAC mice. Similarly, in the atrophic gastrocnemius muscle, levels of total histidyl dipeptides (carnosine and anserine) and, in particular,carnosine were decreased. Depletion of histidyl dipeptides diminished the aldehyde removal capacity of the atrophic gastrocnemius muscle. Furthermore, the expression of CARNS and TAUT wasdecreased in the atrophic gastrocnemius muscle. Conclusions : Collectively, these results show that metabolic pathways involved in the removal of lipid peroxidation products and synthesis of histidyl dipeptides are diminished in atrophic skeletal muscle during heart failure, which could contribute to muscle atrophy.

Benzene is a ubiquitous environmental and occupational pollutant abundant in household products, petrochemicals, and cigarette smoke. It is also a well-known carcinogen and hematopoietic toxin. Population-based studies indicate an increased risk of heart failure in subjects exposed to inhaled benzene, which coincides with the infiltration of immune cells into the myocardium. However, the mechanisms of benzene-induced cardiovascular disease remain unknown. Our data suggests that benzene metabolites trans,trans-muconaldehyde (MA), and hydroquinone (HQ) propagate endothelial activation and apoptosis analyzed by endothelial-specific microparticles in C57BL/6J mice plasma. Subcutaneous injections of MA and HQ increased vascular permeability by 1.54 fold and 1.27 fold correspondingly. In addition, the exposure of primary cardiac microvascular endothelial cells to MA increased vascular permeability detected by transendothelial monolayer resistance and by fluorescently labeled dextrans diffusion. The bulk RNA sequencing of endothelial cells exposed to MA for 2, 6, and 24 hours showed MA-dependent upregulation of heat shock-related pathways at 2 and 6 hours, dysregulation of GTPases at 6 hours, and altered cytoskeleton organization at 24 hours of exposure. We found that the HSP70 protein induced by MA in endothelial cells is colocalized with F-actin foci. HSP70 inhibitor 17AAG and HSP90 inhibitor JG98 attenuated MA-induced endothelial permeability, while HSP activator TRC enhanced endothelial leakage. Moreover, MA induced Rac1 GTPase activity, while Rho GTPase inhibitor Y-27632 attenuated MA-induced endothelial permeability. We showed that benzene metabolites compromised the endothelial barrier by altering HSP- and GTPase-related signaling pathways.

Background: Benzene is a ubiquitous environmental pollutant generated by a variety of natural and anthropological sources. It is a known carcinogen and hematopoietic toxin; however, little is known about benzene's potential atherogenicity. Hypothesis: Inhaled benzene induces atherogenesis by increasing vascular inflammation in LDL receptor Knockout (LDLR-KO) mice. Methods: Male LDLR-KO mice were exposed to HEPA-filtered air or benzene (1 ppm, 6h/day, 5days/week) for 24 weeks. For the last 12 weeks of exposure, the mice were maintained on a western diet. The single nuclei RNA sequencing (snRNAseq) of aortae was performed at Novogene. In an independent experiment, male ApoE-KO mice were maintained on a western diet for 12 weeks and then exposed to benzene for 2 weeks. Cytometry by time of flight (CyTOF) analysis was used for the plaque immunophenotyping. For in vitro experiments, splenic naïve T cells were exposed to 1 μM of hydroquinone (HQ) for 24 hours, and intracellular ROR-gamma levels were measured by flow cytometry. Results: Benzene inhalation increased the aortic valve lesion area by more than 25% (P<0.05) in LDLR-KO mice. Using snRNAseq, eleven major cell types were detected, including T cells and vascular smooth muscle cells (VSMC). Benzene increased the number of T cells by 2.5-fold, proliferating T-cells by 5.8-fold, and VSMC by 1.6-fold, suggesting increased cellularity and reduced plaque stability. In addition, benzene upregulated Th17 polarization marker Rorc and negative regulators of apoptosis Rag1 and Bcl11b while significantly attenuating the expression of proliferation inhibitor Ms4a4b in T cells. In VSMC, benzene downregulated extracellular matrix organization genes and upregulated platelet degranulation pathways. CyTOF analysis of the plaques of ApoE-KO mice showed that even short-term benzene exposure increased T-cell abundance in pre-existing lesions. Polarization of T cells into Th17 was confirmed by HQ-dependent upregulation of ROR-gamma in vitro . Conclusion: Our data suggest that inhaled benzene exposure compromises plaque cellularity and stability by facilitating T-cell proliferation and polarization, which coincides with the degradation of smooth muscle extracellular matrix and platelet activation.

Environmental pollution accounts for nine million premature deaths worldwide every year and cardiovascular disease is the leading cause environmental pollution-associated deaths. Our previous studies have shown that exposure to combustion-derived chemicals (e.g., acrolein), petroleum products-derived chemicals (e.g., benzene) and metalloid such as arsenic exacerbates atherosclerosis in mice. In vitro studies suggest that these chemicals endothelial activation and injury, at least in part, by inducing endoplasmic reticulum stress. To examine how endothelial cells cope with environmental chemicals-induced toxicity, we performed transcriptomic analyses of human aortic endothelial cells (HAEC) exposed to acrolein (10 μM), t,t -muconaldehyde (MA, reactive metabolite of benzene, 10 μM), and arsenic (20 μM). Our data show that exposure of HAEC to these chemicals significantly induced the expression (log 2FC = 2-13-fold) of heat shock proteins - Hspa1a, Hspa1b, Hspa6 , and Hspa7 , the molecular chaperones which keep the proteins in their native structures. MA-induced transcription of HSPs in HAEC was accompanied by the induction (log 2FC = 1-3) of oxidative stress-responsive genes superoxide dismutase 1, heme oxygenase-1, cyclooxygenase-2, and Akr1b10 ; ER-stress responsive gene Atf3; and adhesion molecules Icam-1 and P-selectin; and downregulation of endothelial nitric oxide synthase ( Nos 3, log 2FC = -0.4). Like HAEC, MA also significantly induced Hspa1b1 and Atf3 (log 2FC= 2.3 and 0.7 respectively). siRNA-mediated knockdown of Hspa1b in HAEC significantly increased MA-induced transcription of Icam-1 and leukocyte adhesion to endothelial cells, abundance of ATF3 protein, endothelial cell apoptosis, and attenuated MA-induced downregulation of Nos 3 gene. The reactome analysis of MA-treated HAEC suggested that heat shock factor-1 (HSF-1) is the transcriptional regulator of Hspa1a and Hspa1b . Western blotting of MA-treated HAEC showed that MA upregulates as well as phosphorylates HSF-1, which attenuates IL-6 expression by activating ATF3. Collectively, these data suggest that HSPs prevent environmental chemicals-induced endothelial toxicity by preventing oxidative stress, ER-stress, inflammation, and depletion of Nos3 .

Pulmonary mechanosensory receptors provide important inputs to the respiratory center for control of breathing. However, what is known about their structure-function relationship is still limited. In these studies, we explored this relationship comparing bronchopulmonary slowly adapting receptor (SAR) units in rabbits and rats. In morphological studies, sensory units in tracheobronchial smooth muscle labeled with anti-Na+ /K+ -ATPase (α3 subunit) were found to be larger in the rabbit. Since larger structures may result from increased receptor size or more numerous receptors, further examination showed receptor size was the same in both species, but more receptors in a structure in rabbits than rats, accounting for their larger structure. In functional studies, SAR units were recorded electrically in anesthetized, open-chest, and artificially ventilated animals and responses to lung inflation were compared at three different constant airway pressures (10, 20, and 30 cmH2 O). At each level of the inflation, SAR discharge frequencies were found to be higher in rabbits than rats. We conclude that a relatively larger number of receptors in a sensory unit may be responsible for higher SAR activities in rabbit SAR units.

Vinyl chloride (VC) is an organochlorine mainly used to manufacture its polymer polyvinyl chloride, which is extensively used in the manufacturing of consumer products. Recent studies suggest that chronic low dose VC exposure affects glucose homeostasis in high fat diet-fed mice. Our data suggest that even in the absence of high fat diet, exposure to VC (0.8 ppm, 6 h/day, 5 day/week, for 12 weeks) induces glucose intolerance (1.0 g/kg, i.p.) in male C57BL/6 mice. This was accompanied with the depletion of hepatic glutathione and a modest increase in lung interstitial macrophages. VC exposure did not affect the levels of circulating immune cells, endothelial progenitor cells, platelet-immune cell aggregates, and cytokines and chemokines. The acute challenge of VC-exposed mice with LPS did not affect lung immune cell composition or plasma IL-6. To examine the effect of VC exposure on vascular inflammation and atherosclerosis, LDL receptor-KO mice on C57BL/6 background maintained on western diet were exposed to VC for 12 weeks (0.8 ppm, 6 h/day, 5 day/week). Unlike the WT C57BL/6 mice, VC exposure did not affect glucose tolerance in the LDL receptor-KO mice. Plasma cytokines, lesion area in the aortic valve, and markers of lesional inflammation in VC-exposed LDL receptor-KO mice were comparable with the air-exposed controls. Collectively, despite impaired glucose tolerance and modest pulmonary inflammation, chronic low dose VC exposure does not affect surrogate markers of cardiovascular injury, LPS-induced acute inflammation in C57BL/6 mice, and chronic inflammation and atherosclerosis in the LDL receptor-KO mice.

Benzene is a ubiquitous environmental pollutant. Recent population-based studies suggest that benzene exposure is associated with an increased risk for cardiovascular disease. However, it is unclear whether benzene exposure by itself is sufficient to induce cardiovascular toxicity. We examined the effects of benzene inhalation (50 ppm, 6 h/day, 5 days/week, 6 weeks) or HEPA-filtered air exposure on the biomarkers of cardiovascular toxicity in male C57BL/6J mice. Benzene inhalation significantly increased the biomarkers of endothelial activation and injury including endothelial microparticles, activated endothelial microparticles, endothelial progenitor cell microparticles, lung endothelial microparticles, and activated lung and endothelial microparticles while having no effect on circulating levels of endothelial adhesion molecules, endothelial selectins, and biomarkers of angiogenesis. To understand how benzene may induce endothelial injury, we exposed human aortic endothelial cells to benzene metabolites. Of the metabolites tested, trans,trans-mucondialdehyde (10 μM, 18h) was the most toxic. It induced caspases-3, -7 and -9 (intrinsic pathway) activation and enhanced microparticle formation by 2.4-fold. Levels of platelet-leukocyte aggregates, platelet macroparticles, and a proportion of CD4+ and CD8+ T-cells were also significantly elevated in the blood of the benzene-exposed mice. We also found that benzene exposure increased the transcription of genes associated with endothelial cell and platelet activation in the liver; and induced inflammatory genes and suppressed cytochrome P450s in the lungs and the liver. Together, these data suggest that benzene exposure induces endothelial injury, enhances platelet activation and inflammatory processes; and circulatory levels of endothelial cell and platelet-derived microparticles and platelet-leukocyte aggregates are excellent biomarkers of cardiovascular toxicity of benzene.

Benzene is a ubiquitous environmental pollutant abundant in household products, petrochemicals, and cigarette smoke. Benzene is a well-known carcinogen in humans and experimental animals; however, little is known about the cardiovascular toxicity of benzene. Recent population-based studies indicate that benzene exposure is associated with an increased risk for heart failure. Nonetheless, it is unclear whether benzene exposure is sufficient to induce and/or exacerbate heart failure. We examined the effects of benzene (50 ppm, 6 h/day, 5 days/week, and 6 weeks) or high-efficiency particulate absorbing-filtered air exposure on transverse aortic constriction (TAC)-induced pressure overload in male C57BL/6J mice. Our data show that benzene exposure had no effect on cardiac function in the Sham group; however, it significantly compromised cardiac function as depicted by a significant decrease in fractional shortening and ejection fraction, as compared with TAC/Air-exposed mice. RNA-seq analysis of the cardiac tissue from the TAC/benzene-exposed mice showed a significant increase in several genes associated with adhesion molecules, cell-cell adhesion, inflammation, and stress response. In particular, neutrophils were implicated in our unbiased analyses. Indeed, immunofluorescence studies showed that TAC/benzene exposure promotes infiltration of CD11b(+)/S100A8(+)/myeloperoxidase(+)-positive neutrophils in the hearts by 3-fold. In vitro, the benzene metabolites, hydroquinone, and catechol, induced the expression of P-selectin in cardiac microvascular endothelial cells by 5-fold and increased the adhesion of neutrophils to these endothelial cells by 1.5- to 2.0-fold. Benzene metabolite-induced adhesion of neutrophils to the endothelial cells was attenuated by anti-P-selectin antibody. Together, these data suggest that benzene exacerbates heart failure by promoting endothelial activation and neutrophil recruitment.

Recent studies point to the important role of in utero malnutrition in gene programming and in the development of vascular diseases. We hypothesize that maternal undernutrition affects vascular function in the offspring by promoting epigenetic changes that drive the differential expression of genes involved in endothelial function. To test this, we exposed mice to nutrient deprivation in utero and analyzed its effect on global DNA methylation and expression of endothelium-specific genes in the pulmonary endothelium of the adult progeny. Mice were kept either on ad libitum (AL) or energy-restricted (ER) diet during the second and third trimesters of gestation. Mice in the ER group received 65% of energy compared to mice in the AL diet group. Pulmonary endothelial cells were isolated from 6-week-old male offspring mice (AL-F1 and ER-F1). The expression of genes in the pulmonary endothelium was analyzed using quantitative reverse-transcription polymerase chain reaction array and confirmed by qRT-PCR. Several genes including fibronectin 1 and plasminogen activator inhibitor 1 were upregulated in the endothelium of male ER-F1 mice, whereas the expression of genes involved in regulation of histone acetylation was significantly attenuated. At the same time, the global DNA methylation did not change in pulmonary endothelial cells of ER-F1 mice compared to AL-F1 mice. Overall, we found that maternal undernutrition during pregnancy affects the expression of genes involved in regulation of endothelial cell function in the pulmonary vasculature of male progeny, which could potentially promote pulmonary vascular remodeling.

Work-place exposure to silica dust may lead to progressive lung inflammation culminating in the development of silicosis, an irreversible condition that can be complicated by onset of pulmonary hypertension (PH). The molecular mechanisms leading to the development of PH and lung fibrosis in response to silica are not well understood. Oxidant/antioxidant imbalance in the lung may promote fibroproliferation and vascular smooth muscle proliferation, ultimately leading to the development of PH. Herein, we analyze the development of PH and lung fibrosis in mice deficient in extracellular superoxide dismutase (SOD3), an enzyme with anti-oxidant activity.

Lung disorders characterized by fibroproliferation and excessive deposition of extracellular matrices occur in late adulthood, and their pathological manifestations become more prominent with aging. The exact mechanisms linking aging and fibroproliferative disorders are unknown, but increased oxidative stress resulting in the accumulation of damaged proteins, DNA, and lipids is considered a major factor. In the lung, and especially in the pulmonary fibroblasts, the extracellular superoxide dismutase (EC-SOD) is a major antioxidant enzyme that has been implicated in pulmonary fibrosing disorders, among others. Here, we investigate the regulation of EC-SOD in pulmonary lung fibroblasts derived from young (up to 3 month) and old (24 month) C57BL6 mice. We found that old fibroblasts have marginally elevated levels of reactive oxidant species (ROS), which coincides with attenuated expression a number of antioxidant enzymes including EC-SOD. Exposure of old fibroblasts to the DNA methyltransferase inhibitor 5-aza-dC did not restore expression of EC-SOD. On the other hand, repression of EC-SOD expression was associated with deacetylation of lysine 9 on histone H3 and lysines 5, 8, 12 and 16 on histone H4 located at the gene promoter. Interestingly, the repressive tri-methylation of lysine 27 on histone H3 was elevated in old compared to young fibroblasts. In addition, exposure of old lung fibroblasts to HDAC class 1 and class 2 inhibitors restored EC-SOD expression to the level observed in young fibroblasts. While the exact mechanism of age-dependent downregulation of EC-SOD is yet to be defined, our studies indicate a potential role of epigenetic mechanisms including histone deacetylation in this process.

Occupational and environmental exposure to crystalline silica may lead to the development of silicosis, which is characterized by inflammation and progressive fibrosis. A substantial number of patients diagnosed with silicosis develop pulmonary hypertension. Pulmonary hypertension associated with silicosis and with related restrictive lung diseases significantly reduces survival in affected subjects. An animal model of silicosis has been described previously however, the magnitude of vascular remodeling and hemodynamic effects of inhaled silica are largely unknown. Considering the importance of such information, this study investigated whether mice exposed to silica develop pulmonary hypertension and vascular remodeling.

Aging is associated with progressive oxidation of plasma cysteine (Cys)/cystine (CySS) redox state, expressed as EhCySS. Cultured cells condition their media to reproduce physiological EhCySS, but it is unknown whether aged cells produce a more oxidized extracellular environment reflective of that seen in vivo. In the current study, we isolated primary lung fibroblasts from young and old female mice and measured the media EhCySS before and after challenge with Cys or CySS. We also measured expression of genes related to redox regulation and fibroblast function. These studies revealed that old fibroblasts produced a more oxidizing extracellular EhCySS than young fibroblasts and that old fibroblasts had a decreased capacity to recover from an oxidative challenge due to a slower rate of reduction of CySS to Cys. These defects were associated with 10-fold lower expression of the Slc7a11 subunit of the xCT cystine-glutamate transporter. Extracellular superoxide dismutase (Sod3) was the only antioxidant or thiol-disulfide regulating enzyme among 36 examined that was downregulated in old fibroblasts by more than 2-fold, but there were numerous changes in extracellular matrix components. Thus, aging fibroblasts not only contribute to remodeling of the extracellular matrix but also have a profound effect on the extracellular redox environment.