Moderation: U. Müller-Werdan, Berlin
Development of age-associated diseases like atherosclerosis depends not only on genetic disposition but also on environmental influences. The Aryl Hydrocarbon Receptor (AhR) is a ubiquitously expressed transcription factor activated by ligands like 2,3,7,8-tetrachlorodibenzo-p-dioxin and benzo[a]pyrene. Epidemiological studies on populations heavily exposed to dioxin like 1976 in Seveso indicate increasing development of cardiovascular diseases in these populations. Furthermore, dioxin-exposed workers display higher incidence in atherosclerotic plaques and increased intima-media wall thickness, a risk factor and predictor for stroke and myocardial infarction. However, a direct role for AhR in cardiovascular and organismal aging has never been demonstrated across species from invertebrates to humans. To investigate the role of the AhR with respect to aging and age-related vascular processes, we analyzed vessel stiffness in AhR-deficient mice, functional parameters in primary human endothelial cells and AhR expression in blood cells of healthy human volunteers. Old (17-20 months) wt mice showed increased pulse wave velocity (PWV), which is indicative of impaired vessel function. Interestingly, in AhR-deficient mice, we observed reduced PWV in both old and young (3-6 months) mice, suggesting reduced vessel stiffness. Analysis of eNOS phosphorylation by Western Blot showed increased eNOS activity in thoracic aortas of AhR-deficient mice and a concomitant increase in NO production. In line with this finding, AhR activation and/or overexpression reduced the migratory capacity of primary human endothelial cells without affecting proliferation and apoptosis, impaired eNOS activation and reduced the S-NO content, which are all signs of endothelial dysfunction. Furthermore, AhR expression in blood cells of healthy human volunteers positively correlated with vessel stiffness, suggesting that AhR expression levels could serve as an additional new predictor of vessel aging. Detailed characterization of AhR-functions in aging and age-related disease might help to uncover the ancient functions of this protein and to develop strategies for healthier aging in the future.
Combustion-derived carbon nanoparticles, a fraction of particulate air pollution, are of particular toxicological relevance. Sources of this kind of air pollution are industrial and domestic emissions. But also intentionally produced carbon black particles, which are used for multiple technological applications including daily life products like copier and printer toner can contribute to human exposure.
Particulate air pollution enter the human body primarly via the airways by inhalation. Of note, unlike bigger size fractions, nanoparticles (<100 nm) are not specifically cleared by macrophages in the lower airways. Moreover, nanoparticles are able to penetrate the air blood barrier and enter the circulatory system. Association between particle exposure and adverse health effects in the cardiovascular system indicate the relevance of this kind of exposure for human health. Endothelial dysfunction is characterized by endothelial cell (EC) senescence, reduced migratory capacity, reduced NO bioavailability and increased sensitivity against apoptotic events. However, the molecular impact of combustion-derived nanoparticles on EC and vascular function is poorly understood. To investigate this, we used pure carbon nanoparticles (CNP) as a model substance for combustion-derived nanoparticles in non-cytotoxic doses and analyzed functional parameters ex vivo in human EC and in vivo in mice exposed to repetitive doses of CNP. We could show that treatment of EC with 1 µg/cm2 CNP for 4 h induced intracellular oxidative stress. Interestingly, this was accompanied by significant reduction of mitochondrial telomerase activity, which is mediated by a redox-dependent activation of the Src kinase. In line with that, we could observe for the first time, that CNP exposure for two weeks, induced a senescent phenotype in EC. Since NO bioavailability is reduced during the process of EC aging, we analyzed eNOS activation in endothelial cells and eNOS levels in aorta tissue of mice repetitively exposed to CNP via pharyngeal aspiration. This treatment reduced eNOS activation and expression. Our results give hints for molecular mechanisms of CNP-induced vascular aging and endothelial dysfunction, contributing to the development of age-related diseases like atherosclerosis.
The APEX nuclease (multifunctional DNA repair enzyme) 1 (APEX1) has a DNA repair and a redox domain. It can modulate transcription factor activities probably by recruiting other reducing molecules like Thioredoxin-1. APEX1 exerts anti-apoptotic properties in different tumor cell lines. As protection against apoptosis is a hallmark of vessel integrity, which decreases with age, we wanted to elucidate whether APEX1 acts anti-apoptotic in primary human endothelial cells (EC) and if, what the underlying mechanisms are.
To characterize the anti-apoptotic properties of APEX1, whose overexpression inhibited apoptosis in EC, we generated mutants lacking the DNA repair domain (APEX1ΔC(1-127)) or parts of the redox domain (APEX1ΔN(21-318)). Only the mutant with the intact redox domain prevented basal and stimulus-dependent apoptosis similar to APEX1wt, while the one retaining the DNA repair domain significantly aggravated apoptosis. Thus, the anti-apoptotic properties of APEX1 are independent of its DNA repair function. As Thioredoxin-1 (Trx-1) is crucial for apoptosis-protection in EC, we investigated the impact of the pro-apoptotic mutant APEX1ΔN(21-318) on Thioredoxin-1. While Trx-1 mRNA levels were unaffected in EC overexpressing APEX1ΔN(21-318), the amount of Trx-1 protein was significantly reduced. Since the lysosomal protease Cathepsin D is known to degrade Trx-1, we measured total and active Cathepsin D protein levels and activity. In APEX1ΔN(21-318) transfected cells Cathepsin D activity and the proteolytically activated protein were increased. We next used a mouse model, in which restenosis is induced by ligation of the carotid, that is characterized by increased oxidative stress in the vessels. Indeed, we found decreased Trx-1 protein levels in the carotids. Thus, these data demonstrate that a redox active APEX1 protein protects Trx-1 from degradation and inhibits apoptosis. Oxidative stress induces Trx-1 degradation in vivo probably via an inactivation of APEX1. To test whether normalization of Trx-1 levels rescues endothelial cells from APEX1ΔN(21-318)-induced apoptosis we will reexpress Trx-1 via lentiviral transduction and investigate whether endothelial cells will be protected.
Background and Aims: Age-associated cardiovascular disorders are associated with mitochondrial dysfunction, which causes reduced respiratory chain activity, decreased mitochondrial ATP production, an increase in reactive oxygen species (ROS) and cell death. We could show that the catalytic subunit of Telomerase, Telomerase Reverse Transcriptase (TERT), has a beneficial effect on heart function. Furthermore, we recently demonstrated that TERT is also localized within the mitochondria and belongs to the group of dual-targeted proteins with additional functions outside the nucleus. Therefore, the aim of this study was to characterize the functions of mitochondrial TERT in cardiovascular aging and diseases.
Methods: To analyze specifically the role of mitochondrial TERT we designed lentiviruses expressing mitochondrial TERT and transduced different cardiovascular cells. Apoptosis as well as cytosolic and mitochondrial ROS were measured by flow cytometry. Furthermore, we created mice containing TERT exclusively in the mitochondria (mitoTERT mice). Respiratory chain activity was measured by oxygraphy in isolated heart mitochondria. Murine cardiac fibroblasts were isolated by collagenase digestion. Protein levels and localization were assessed by immunofluorescence and immunoblotting.
Results: To study the impact of mitochondrial TERT on respiratory chain activity we isolated mitochondria from hearts of TERT-deficient mice, which showed a significantly lower state 3 respiration compared to mitochondria from hearts of wt littermates, whereas the mitochondria isolated from hearts of mitoTERT mice had a significant higher state 3 respiration. Furthermore, we could demonstrate that this effect is not dependent on the Telomerase RNA subunit TERC. Moreover, mitoTERT mice showed elevated ATP levels in heart mitochondria when compared to TERT-deficient animals. On the other hand, overexpression of mitoTERT prevented hydrogen peroxide induced apoptosis in endothelial cells and cardiomyocytes. We could also observe, that transduction of fibroblasts isolated from TERT-deficient mice with mitoTERT reduced mitochondrial ROS.
Conclusion: We could show for the first time the compartment-specific roles of TERT in the cardiovascular system ex vivo and in vivo.
Exposure to particulate air pollution is correlated with age-associated degenerative diseases of the airways. Particularly emphysema, a main factor of chronic obstructive pulmonary disease (COPD), may be induced by inhaled environmental particles. However, the mechanistic background of this pathogenic effect is so far not well understood. Besides inflammatory reactions triggered by the particles, a direct interaction of inhaled particles with cells of the airway epithelium may cause cellular reactions responsible for these adverse health effects. In previous studies we demonstrated that carbon nanoparticles as model particles for environmental combustion-derived particles induce stress induced senescence accompanied by cell cycle arrest in lung epithelial and endothelial cells. Cell cycle arrest in lung epithelial cells has been suggested to be a cause of age-associated airway diseases due to potential loss of regenerative capacity. We therefore aimed to investigate if the regenerative capacity of lung epithelial cells is impaired by carbon nanoparticles.
For this purpose, the cell cycle of lung epithelial cells repetitively exposed to low, non-cytotoxic doses of carbon nanoparticles was monitored by flow cytometry. Moreover, the histone deacetylase SIRT-1, a known cell cycle regulator and marker of lung aging, and Thioredoxin-1, which is reduced in stress-induced senescence, were analyzed. Furthermore, Connexin 43 was investigated because intercellular communication is decreased in lung diseases like COPD.
Exposure of lung epithelial cells to carbon nanoparticles led to cell cycle arrest. Analyses of apoptosis rates at the level of DNA content and of caspase-3 activation demonstrated that neither programmed cell death nor necrosis are responsible for this effect. Moreover, at the protein level, significant reduction of SIRT-1, Thioredoxin-1 and active Connexin 43 were observed upon particle exposure. Accordingly, in rats the repetitive application of carbon nanoparticles diminished SIRT-1 and active Connexin 43 in lung tissue.
These results demonstrate that the regenerative capacity of lung epithelial cells is impaired by carbon nanoparticles. Moreover, application of these particles in vivo evoked signs of age-associated disease parameters in the lung.