17:00 - 18:30 Uhr
Löwengebäude HS XIII
Vorsitz: Altschmied, Joachim (Düsseldorf)
Cellular / Vascular Aging
O. Mece; N. Kryeziu; R. Heller; T. Grune1
Institut für Molekulare Zellbiologie, 1 Lehrstuhl für Ernährungstoxikologie, Institut für Ernährungswissenschaften, Friedrich-Schiller-Universität Jena, Jena;
Endothelial cells underlie senescence in vivo, which is believed to contribute to endothelial dysfunction and vascular diseases. Senescence may be triggered by stress-induced pathways and may especially take place in certain inflammatory or oxidative microenvironments. One way through which senescence may be elicited or aggravated is the induction of a functional decline in protein degradation systems leading to the accumulation of protein aggregates. The current project aims to characterize protein degradation pathways in endothelial cells undergoing oxidative stress-induced premature senescence and to investigate the effect of proteasomal inhibition on endothelial senescence.
Our results show that H2O2 (100-200 µM, 8 days) induces oxidative stress in endothelial cells as verified by protein carbonylation and led to senescence and growth arrest (increase of SA-?-gal positive cells and reduction of Ki-67-positive cells, respectively). At the same time, proteasomal activities, especially the chymotrypsin-like activity, declined while the expression levels of the proteasomal subunits remained unchanged. On the other hand, transient inhibition of the proteasome by MG132 led to an accumulation of carbonylated proteins, which persisted when proteasome activity was recovered. In parallel the development of senescence was observed.
Taken together, our data show that chronic oxidative stress leads to an induction of premature senescence in endothelial cells and to a decline of proteolytic activities. Proteasomal inhibition alone is able to aggravate senescence suggesting a causal relationship between catabolic insufficiency and senescence. Future studies will be required to understand whether and how premature senescence and proteolytic decline are linked and to characterize responsible mechanisms.
P. Rolewska; A. Simm1; R.-E. Silber; B. Bartling
Universitätsklinik und Poliklinik für Innere Medizin, Herz- und Thoraxchirurgie, Universitätsklinikum, 1 Zentrum für Medizinische Grundlagenforschung (ZMG), Medizinische Fakultät, Martin-Luther-Universität Halle-Wittenberg, Halle (Saale);
It is believed that senescence of cells contributes to lung aging and age-related diseases of the lung, including chronic obstructive pulmonary disease, lung fibrosis and pneumonia. Several markers are reported in senescent cells in vitro, such as irreversible loss of cell proliferation, acid beta-galactosidase activity, telomere shortening or activation of compounds of the DNA damage response. Since these markers are not absolutely specific or even non-detectable in situ, the simultaneous detection of senescence-related changes can clearly identify the presence of senescent cells in situ only. Therefore, the identification of specific markers indicating for cellular senescence is still needed. In our work we identified the reduction of the transcription factor cyclic AMP response element-binding protein (CREB) as a senescence-related marker in fibroblasts derived from adult human lung tissue. Reduction of CREB protein was mainly based on posttranslational processes as indicated by an increased CREB ubiquitination. CREB down-regulation in lung fibroblasts was accompanied by the reduced expression of many genes out of which some genes also showed a reduced expression in lung tissues of old mice. In particular, CREB-dependent reduction in the Ras-related protein Rab27A seems to be important for cellular senescence in lung. Since chronic down-regulation of CREB in human lung fibroblasts by siRNA transfection caused an increased number of cells with senescent phenotype, CREB reduction seems to play a pivotal role in lung aging and the progression of age-related lung diseases.
K. Spengler; S. Lindenmüller; N. Kryeziu; R. Heller
Institut für Molekulare Zellbiologie, Friedrich-Schiller-Universität Jena, Jena;
5’AMP-activated protein kinase (AMPK), an important intracellular energy sensor, is contributing to cellular signalling and homeostasis. Recent studies have shown that AMPK may be involved in the regulation of autophagy, which plays a role in the degradation of long-lived or damaged proteins and organelles and contributes to cell survival in an adverse environment. The present study was aimed at investigating the effects of the AMPK/autophagy pathway on endothelial cell function and stress protection.
Experiments were performed in human umbilical vein endothelial cells (HUVEC). Our data demonstrate that AMPK activation induces autophagy. In addition, we show that autophagy is critical for maintaining endothelial function. Impairment of autophagy by siRNA-mediated downregulation of the regulatory proteins ULK1 and Beclin1 led to a clear inhibition of VEGF-induced angiogenesis. In parallel, cell proliferation was decreased and the amount of apoptotic cells was increased. The effect of autophagy on endothelial function may be related to its role in providing energy sources for cellular metabolism in conditions of cellular activation and/or stress. In line with this we show that autophagy is necessary for the recovery of intracellular ATP levels in response to metabolic stress.
Taken together, our results demonstrate that the AMPK/autophagy axis represents an important pathway to maintain endothelial function. Inhibition of this pathway decreases adaptation to metabolic stress and impairs important endothelial functions such as angiogenesis while its activation may mediate stress protection and cellular homeostasis.
C. Goy; J. Altschmied; J. Haendeler
Molekulare Alternsforschung, an der Universität Düsseldorf gGmbH, Leibniz-Institut für Umweltmedizinische Forschung, Düsseldorf;
Environmental stressors as well as genetic modifications are known to enhance oxidative stress and aging processes. Mitochondrial and nuclear dysfunctions contribute to the onset of aging.
One of the most important redox regulators in primary human endothelial cells is Thioredoxin-1 (Trx-1), a 12kD protein with additional anti-apoptotic properties. Cellular generators of reactive oxygen species are NADPH oxidases (NOXs), of which NOX4 shows highest expression levels in endothelial cells. Therefore, the aim of the study was to investigate how Trx-1 and NOX4 are regulated during stress-induced premature senescence in endothelial cells. We treated primary human endothelial cells for two weeks with H2O2 to generate stress-induced premature senescence in these cells. In this model senescence-associated ß-Galactosidase and nuclear p21 as senescence markers are increased.
Moreover, total and mitochondrial reactive oxygen species formation is enhanced. An imbalanced redox homeostasis is detected by elevated NOX4 and decreased Trx-1 levels. This can be rescued by lentiviral expression of Trx-1. Moreover, the lysosomal protease Cathepsin D is over-activated, which results in reduced Trx-1 protein levels. Inhibition of "over-active" Cathepsin D by the specific, cell permeable inhibitor pepstatin A abolishes increase in nuclear p21 protein, ROS formation and degradation of Trx-1 protein, thus leading to blockade of stress-induced premature senescence by stabilizing the cellular redox homeostasis. Aortic Trx-1 levels are decreased and Cathepsin D activity is increased in NOX4 transgenic mice exclusively expressing NOX4 in the endothelium when compared to their wildtype littermates. Thus, loss of Trx-1 and upregulation of NOX4 importantly contribute to the imbalance in the redoxstatus of senescent endothelial cells ex vivo and in vivo.
M. Bretschneider; B. Busch1; D. Müller; M. Gekle; C. Grossmann
Julius-Bernstein-Institut für Physiologie, Medizinische Fakultät, 1 Institut für Molekulare Medizin, Martin-Luther-Universität Halle-Wittenberg, Halle (Saale);
Vascular aging is associated with remodeling processes with enhanced production of extracellular matrix components, proliferation and migration of vascular smooth muscle cells (VSMC). Simultaneously, the expression and signaling of the mineralocorticoid receptor (MR) that binds aldosterone have been shown to be augmented in VSMCs of aged compared to young rats, coinciding with a proinflammatory phenotype. Additionally, VSMC-specific MR knockout mice lack an age-associated increase in vasoconstriction in response to angiotensin II and a rise in blood pressure. As one potential mechanism leading to aldosterone/MR-induced vascular aging, we studied posttranscriptional gene control through microRNAs. MicroRNAs are small non-coding RNA molecules that bind to the 3’-end of mRNAs and lead to their degradation or silencing. To investigate the involvement of micoRNAs in aging-related effects of aldosterone, we searched for MR-dependently regulated microRNAs with a microarray screening approach followed by Taqman validation experiments. We could identify mir-29b as a negatively aldosterone-regulated microRNA. Further investigations revealed that the inhibitory effect of aldosterone is achieved by enhanced degradation of miR-29b in the cytoplasm. Functional consequence of the decrease in the mir-29b level was an enhanced production of extracellular matrix components and a shift in the necrosis/apoptosis ratio towards the more damaging proinflammatory necrosis. Furthermore, we found that aldosterone via a decrease in mir-29b abundance enhances VSMC migration. Overall, our results support the hypothesis that aging-related vascular remodeling induced by aldosterone is at least partially mediated by a decrease in mir-29b.