Symposium Biologie
Donnerstag, 08.09.2016
10:45 - 12:15
Studio A
Freie Vorträge - Molecular aspects in aging: from mechanism to disease
Caffeine improves cardiovascular functions in a p27-dependent manner

C. Goy, K. Aufenvenne, F. von Ameln, J. Altschmied, J. Haendeler; Düsseldorf, Duesseldorf

Background and Aims: Cardiovascular functionality decreases with age. Interestingly, recent studies have shown a protective effect of caffeine on the cardiovascular system. We have shown that concentrations of caffeine detectable in serum after moderate coffee consumption enhanced the migratory capacity of endothelial cells (EC), which critically depends on mitochondrial function. Therefore, we wanted to identify the molecular link between caffeine, mitochondrial energy metabolism and migration.
Methods: EC were transfected with standard procedures and migration was determined in a scratch wound assay. To specifically analyze mitochondrial and nuclear p27 we designed lentiviruses expressing these organelle-targeted proteins and transduced different cardiovascular cells. ATP production and mitochondrial membrane potential were measured in mitochondria isolated from mouse hearts. Microarray analyses were performed with RNA from mouse hearts.
Results: Surprisingly, we found that caffeine induces the translocation of p27/Kip1 (p27) into the mitochondria. Reducing p27 levels with siRNA inhibited caffeine induced migration. To investigate the effects of p27 localization on mitochondrial energy metabolism and migration we expressed mitochondrially and nuclear targeted p27 (mito p27/nuc p27) in EC. While expression of nuc p27 decreased basal migration, mito p27 increased migration, mitochondrial ATP production and the mitochondrial membrane potential. Similarly, only overexpression of mito p27, but not nuc p27, rescued the complete loss of migratory capacity induced by knockdown of p27.
To investigate the link between caffeine and p27 in vivo, we performed microarray analysis of hearts from wildtype and p27-deficient mice treated with caffeine in their drinking water. Caffeine induced expression of genes involved in mitochondrial energy metabolism and biogenesis only in wildtype mice demonstrating a crucial role for p27 in enhanced mitochondrial function.
Conclusion: Caffeine seems to have a dual function explaining its protective functions in the cardiovascular system, a short term translocation of p27 to the mitochondria improving their function and in the long run a change in gene expression leading to mitochondrial biogenesis.

Expression and modification of the transcription factor grainyhead-like 3 in the vascular wall: implications for age-associated cardiovascular diseases

S. Kohlgrüber, N. Dyballa-Rukes, S. Metzger, J. Haendeler, J. Altschmied; Düsseldorf

Background and Aims: We have previously shown that the transcription factor grainyhead-like 3 (GRHL3) and its splice variants have a profound impact on endothelial function, which is impaired in many age-related cardiovascular diseases like e.g. atherosclerosis. As many transcription factors are regulated not only via their expression, but also on the post-transcriptional level, the aim of this study was to analyze GRHL3 phosphorylation in endothelial cells as so far nothing is know about this modification of GRHL3.
Furthermore, using a murine model for the development and progression of atherosclerosis we examined GRHL3 expression in the vascular wall in a pathological setting.
Methods: We established procedures to use a new GRHL3 antibody for western blotting, immunofluorescence and immunoprecipitation to be able to study posttranslational modifications of GRHL3 in endothelial cells and its expression in vessel sections of ApoE knockout mice fed a high fat diet.
Results: Using the new GRHL3 antibody for immunoprecipitations followed by detection with a general anti-phospho-tyrosine antibody we could demonstrate tyrosine phosphorylation of GRHL3 in endothelial cells. This phosphorylation was enhanced by treatment of the cells with an NO-donor and this effect was blunted with the Src-kinase inhibitor PP2 indicating that this modification is Src-kinase dependent. Analysis of GRHL3 expression in aortic sections of ApoE knockout mice fed a high fat diet for 16 weeks, a model for atherosclerosis development and progression, revealed decreased expression in the endothelium compared to the control animals. Interestingly, a strong GRHL3 signal was detectable within the atherosclerotic lesions.
Conclusion: Taken together these findings indicate a potential, Src-kinase-mediated, phosphorylation of GRHL3. Using mass spectrometry we will identify the phosphorylated residue(s) to subsequently study their impact on endothelial function. Moreover, the immunofluorescence data revealed GRHL3 expression changes during atherosclerosis development. Functional analyses in surrogate ex vivo models will help to understand the functions of GRHL3 in other cell types than the endothelium in the development of this age-related disease.

Weaker and advanced circadian endogenous rhythm of distal skin temperature in older compared with younger women under natural living conditions

M. Dittmar, T. Hollerbuhl, S. Anders; Kiel

Background and objective: Sleep is coupled with the thermoregulatory system, and age-related changes in distal skin temperature (DST) may influence sleep initiation. This study aimed (1) to analyze reproducibility of circadian DST rhythm in older people beyond laboratory conditions, (2) to characterize their circadian DST rhythm and (3) to relate DST rhythm to sleep and environmental variables.
Subjects and methods: Healthy participants were 35 older (68.9 ± 4.9 yrs) and 30 younger women (25.6 ± 3.6 yrs). Their DST rhythm (hand, foot) was recorded using portable wireless temperature sensors. Data on sleep, physical activity and light exposure were collected using actigraphy with integrated light sensor. All data were collected under natural living conditions and averaged over seven consecutive days. Circadian rhythm characteristics, amplitude and acrophase, describing intensity and timing of DST rhythm, respectively, were calculated using cosinor analysis. Reproducibility of DST rhythm was tested by repeated measures ANOVA. For group comparisons, independent samples’ t-tests were applied.
Results: Circadian amplitude and acrophase of DST were reproducible over seven days in both age groups. Older women exhibited a weaker circadian amplitude for DST rhythm (hand, 1.16 vs. 1.44°C, p<0.050; foot, 1.80 vs. 2.20°C, p<0.050) and an earlier acrophase (hand, 05:55 vs. 07:01 h, p<0.010; foot, 02:48 vs. 04:01 h, p<0.001) than younger women. This indicates a flatter and advanced circadian rhythm for DST in older women, being more pronounced for foot than hand DST rhythm. Amplitude and acrophase were not related to sleep-onset latency, sleep efficiency, total sleep time or overall physical activity in older women (p>0.050). By contrast, amplitude correlated negatively with average daylight exposition in older women.
Conclusions: Findings suggest that aging is associated with flatter and earlier phasing rhythm of DST, being consistent with laboratory studies on core body temperature. An explanation for the flatter amplitude of DST in older people may be daytime light exposure as well as the diminished ability of aged skin to vasoconstrict, thereby affecting temperature regulation. The phase-advance in older people could be due to a shortened circadian period.

Endothelial cell dysfunction caused by diabetes and age related reactive dicarbonyls

A. Simm, K. Jacobs, A. Navarrete Santos, H. Treede, B. Hofmann; Halle (Saale)

Background: Advanced Glycation Endproducts (AGEs) as well as AGE inducing Dicarbonyls are involved in cardiovascular aging. Circulating AGE-modified proteins and dicarbonyls like Glyoxal (GO) and Methylglyoxal (MGO) are directly in contact with endothelial cells (ECs) and are able to modulate fetal EC function. In contrast, little is known about the interaction with primary ECs isolated from the vessel of elderly patients.
Methods: Primary culture ECs were isolated by enzymatic digestion from residual bypass graft material (HSVECs, human saphenous vein endothelial cells) of coronary heart disease patients. HSVECs were stimulated with different concentrations of low and high in vivo glycated BSA, GO and MGO. The cells were chronically stimulated 3-4 days and then analyzed according to cell senescence and function.
Results: Our preliminary results show that GO leads to cell senescence associated changes in HSVECs. MGO alone has no effect, only in combination with GO further morphological changes of HSVECs were induced. On the molecular level, GO in combination with MGO induce cellular senescence, demonstrated by increased p21 protein expression and senescence-associated ß-galactosidase activity. Both dicarbonyls decreased mRNA expression of cell-adhesion molecules and affect the expression of different receptors for AGEs (RAGE ; SR-AII ; AGE-R complex ; SR-AI ; FEEL-1 ). In addition, the permeability of HSVEC monolayer decreased after chronic treatment with GO/MGO or with high in vivo-modified BSA.
Conclusion: Our findings suggest that elevated levels of diabetes / age related dicarbonyls and AGEs can damage the endothelium by inducing cell senescence and thus promote the development of vascular dysfunction. The elucidation of the mechanism by which GO/MGO act is ongoing.