TY - JOUR A1 - Weng, Andreas M. A1 - Köstler, Herbert A1 - Bley, Thorsten A. A1 - Ritter, Christian O. T1 - Effect of short-term smoking & L-arginine on coronary endothelial function assessed by cardiac magnetic resonance cold pressor testing: a pilot study JF - BMC Cardiovascular Disorders N2 - Background The effect of smoking on coronary vasomotion has been investigated in the past with various imaging techniques in both short- and long-term smokers. Additionally, coronary vasomotion has been shown to be normalized in long-term smokers by L-Arginine acting as a substrate for NO synthase, revealing the coronary endothelium as the major site of abnormal vasomotor response. Aim of the prospective cohort study was to investigate coronary vasomotion of young healthy short-term smokers via magnetic resonance cold pressor test with and without the administration of L-Arginine and compare obtained results with the ones from nonsmokers. Methods Myocardial blood flow (MBF) was quantified with first-pass perfusion MRI on a 1.5 T scanner in healthy short-term smokers (N = 10, age: 25.0 ± 2.8 years, 5.0 ± 2.9 pack years) and nonsmokers (N = 10, age: 34.3 ± 13.6) both at rest and during cold pressor test (CPT). Smokers underwent an additional examination after administration of L-Arginine within a median of 7 days of the naïve examination. Results MBF at rest turned out to be 0.77 ± 0.30 (smokers with no L-Arginine; mean ± standard deviation), 0.66 ± 0.21 (smokers L-Arginine) and 0.84 ± 0.08 (nonsmokers). Values under CPT were 1.21 ± 0.42 (smokers no L-Arginine), 1.09 ± 0.35 (smokers L-Arginine) and 1.63 ± 0.33 (nonsmokers). In all groups, MBF was significantly increased under CPT compared to the corresponding rest examination (p < 0.05 in all cases). Additionally, MBF under CPT was significantly different between the smokers and the nonsmokers (p = 0.002). MBF at rest was significantly different between the smokers when L-Arginine was given and the nonsmokers (p = 0.035). Conclusion Short-term smokers showed a reduced response to cold both with and without the administration of L-Arginine. However, absolute MBF values under CPT were lower compared to nonsmokers independently of L-Arginine administration. KW - MRI KW - myocardial perfusion KW - cold pressor test KW - endothelium KW - L-arginine KW - smoking Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-260559 VL - 21 ER - TY - JOUR A1 - Paudel, Rupesh A1 - Fusi, Lorenza A1 - Schmidt, Marc T1 - The MEK5/ERK5 pathway in health and disease JF - International Journal of Molecular Sciences N2 - The MEK5/ERK5 mitogen-activated protein kinases (MAPK) cascade is a unique signaling module activated by both mitogens and stress stimuli, including cytokines, fluid shear stress, high osmolarity, and oxidative stress. Physiologically, it is mainly known as a mechanoreceptive pathway in the endothelium, where it transduces the various vasoprotective effects of laminar blood flow. However, it also maintains integrity in other tissues exposed to mechanical stress, including bone, cartilage, and muscle, where it exerts a key function as a survival and differentiation pathway. Beyond its diverse physiological roles, the MEK5/ERK5 pathway has also been implicated in various diseases, including cancer, where it has recently emerged as a major escape route, sustaining tumor cell survival and proliferation under drug stress. In addition, MEK5/ERK5 dysfunction may foster cardiovascular diseases such as atherosclerosis. Here, we highlight the importance of the MEK5/ERK5 pathway in health and disease, focusing on its role as a protective cascade in mechanical stress-exposed healthy tissues and its function as a therapy resistance pathway in cancers. We discuss the perspective of targeting this cascade for cancer treatment and weigh its chances and potential risks when considering its emerging role as a protective stress response pathway. KW - atherosclerosis KW - bone KW - cartilage KW - endothelium KW - extracellular-regulated kinase 5 KW - Krüppel-like factor KW - mechanotransduction KW - mitogen-activated protein kinase KW - stress signaling KW - tumor Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-261638 SN - 1422-0067 VL - 22 IS - 14 ER - TY - THES A1 - Dirimanov, Stoyan Dinkov T1 - Molecular Effects of Polyphenols in Experimental Type 2 Diabetes Mellitus and Metabolic Syndrome T1 - Molekulare Effekte von Polyphenolen bei experimentellem Diabetes mellitus Typ 2 und metabolischem Syndrom N2 - The growing prevalence of type 2 diabetes mellitus (T2DM) demands novel therapeutic and adjuvant strategies. Polyphenols (PPs) are plant secondary metabolites. Epidemiological studies demonstrate an inverse relationship between their increased intake and the risk of development of T2DM and cardiovascular complications. However, the PPs’ mechanism of action remains largely unknown. The present work aimed to expand knowledge regarding the effects of PPs on diabetes relevant molecular targets. Pycnogenol® (PYC) is a standardized pine bark extract which consists of oligomeric and monomeric PPs. Its anti-diabetic effects have been demonstrated in clinical trials. As a part of a human study involving 20 healthy volunteers, the extract’s effects on dipeptidyl peptidase IV (DPP IV) were investigated. This protease terminates the insulin secretagogue action of incretins. Its inhibition is a promising strategy in T2DM treatment. This study uncovered that PYC-intake of 100 mg daily over 14 days statistically significantly reduced DPP IV serum concentrations by 8.2 % (n= 38, p= 0.032). Contrary to expectations, this decrease was not paralleled by a reduction in the serum DPP IV enzymatic activity. To the best of our knowledge, the present study was the first investigating the effects of PPs on DPP IV serum concentrations and activities in humans. The finding that PYC is capable of reducing DPP IV serum concentrations might be important with regard to diabetes, where DPP IV levels are increased. Screenings for PPs’ in vitro effects on DPP IV activity were performed employing a purified enzyme. The effects of tested PPs (among which PYC ingredients) at a physiologically relevant concentration of 5 µM were weak (< 10 %) and too small compared to the reference compound sitagliptin, and thus not likely to be clinically relevant. This result is in discordance with some published data, but consistent with the outcome from the present human study. In addition, fluorescence interactions with the experimental setup were registered: under certain conditions urolithin B exhibited an autofluorescence which might mask eventual inhibitory activity. Quercetin quenched the fluorescence slightly which might contribute to false positive results. No statistically significant effects of selected constituents and metabolites of PYC on the total DPP IV protein expression were observed in 3T3-L1 adipocytes. Thus, the lower DPP IV in vivo concentrations after intake of PYC cannot be explained with down-regulation of the DPP IV expression in adipocytes. Akt kinase is responsible for the transmission of insulin signals and its dysregulation is related to insulin resistance and plays an important role in development of cardiovascular complications in T2DM. Thus, the modulation of the phosphorylation status of endothelial Akt-kinase, respectively its activity, might be a promising strategy in the management of these pathologies. This work aimed to uncover the effects of PPs from different structural subclasses on Akt-phosphorylation (pAkt) in endothelial cells (Ea.hy926). Short-term effects (5 – 30 min) were investigated at a concentration of 10 µM. In a pilot study two model PPs induced a moderate, but reproducible inhibition of pAkt Ser473 of 52.37 ± 21.01 % (quercetin; p= 0.006, n= 3) and 37.79 ± 7.14 % (resveratrol; p= 0.021, n= 4) compared to the negative control. A primary screening with Western blot analysis investigated the effects of eight compounds from different subclasses on pAkt Ser473 and Thr308 to reveal whether the observed inhibition PPs a group effect or specific to certain compounds. In addition to resveratrol and quercetin, statistically significant inhibitions of pAkt Ser473 were induced by luteolin (29.96 ± 11.06 %, p< 0.01, n= 6) and apigenin (22.57 ± 10.30 %, p< 0.01, n= 6). In contrast, genistein, 3,4,5-trimethoxystilbene, taxifolin and (+)-catechin caused no inhibition. A strong positive and statistically significant correlation between the mean inhibitory effects of the tested PPs on both Akt-residues Ser473 and Thr308 (r= 0.9478, p= 0.0003) was determined. A comprehensive secondary screening via ELISA involving 44 compounds from nine structural groups quantified the effects of PPs on pAkt Ser473 to uncover potential structure-activity features. The most potent inhibitors were luteolin (44.31 ± 17.95 %), quercetin (35.71 ± 8.33 %), urolithin A (35.28 ± 11.80 %), apigenin (31.79 ± 6.16 %), fisetin (28.09 ± 9.09 %), and resveratrol (26.04 ± 5.58 %). These effects were statistically significant (p< 0.01, n= 3 to 6). Further lead structure optimization might be based on the fact that the effects of luteolin and resveratrol also differed statistically significantly from each other (p= 0.008). To the best of our knowledge, the present study is the first to compare quantitatively the short term effects of PPs from different subclasses on pAkt in endothelial cells. Basic structure-activity relationships revealed that for flavones and flavonols the presence of a C2=C3 double bond (ring C) was essential for inhibitory activity and hydroxylation on the m- and p- positions in the ring B contributed to it. For stilbenoids, three free OH-groups appeared to be optimal. The comparison of the inhibitory potentials of ellagic acid and its microbial metabolites showed that urolithin A was statistically significantly more effective than its progenitor compound. Despite their structural similarities, the only active compound among all urolithins tested was urolithin A, hydroxylated at the C3 and C8 positions. This suggested a specific effect for urolithin A. Based on the common structural determinants and molecular geometry of the most active PPs a pharmacophore model regarding Akt-inhibition was proposed. In summary, the effects of a wide variety of PPs from diverse structural subclasses on the in vitro phosphorylation of endothelial Akt were quantitatively analyzed for the first time, the effects of previously undescribed compounds were determined and structure activity relationships were elucidated. The inhibitory potential of individual PPs might be beneficial in cases of sustained over-activation of Akt-kinase and its substrates such as S6 kinase as reported for certain T2DM-related pathological states, such as insulin resistance, endothelial dysfunction, excessive angiogenesis, vascular calcification, and insulin triggered DNA-damage. The results of the present work suggest potential molecular mechanisms of action of PP involving Akt-inhibition and DPP IV-down-regulation and thus contribute to the understanding of anti-diabetic effects of these compounds on the molecular level. N2 - Diabetes mellitus Typ 2 (DMT2) und seine Spätkomplikationen sind ein maßgeblicher Grund für Morbidität und Mortalität. Die steigende Prävalenz der Krankheit erfordert die Entwicklung neuer therapeutischer und prophylaktischer Strategien. Publizierte Daten deuten darauf hin, dass diätetische Polyphenole (PP) sowohl zur Prävention des Diabetes beitragen als auch therapiebegleitend sinnvoll eingesetzt werden können. Allerdings sind ihre Wirkmechanismen nicht vollständig aufgeklärt. Das Ziel der Arbeit war die Charakterisierung zellulärer Wirkungen von PP, die eine Relevanz in der unterstützenden Behandlung von DMT2 und denen Spätkomplikationen haben könnten. Pycnogenol® (PYC) ist ein standardisierter Kiefernrindenextrakt, der oligomere und monomere PP enthält. PYC hat in klinischen Studien mit Diabetikern viele vorteilhafte anti-diabetische und protektive Effekte gezeigt. Die Wirkungen des Extraktes auf die Serumkonzentration und die enzymatische Aktivität der Dipeptidylpeptidase IV (DPP IV) wurden im Rahmen einer klinischen Studie mit 20 gesunden Probanden untersucht. DPP IV ist eine Serin-Exopeptidase, die Inkretinhormone spaltet und damit deren Wirkung auf die Insulin-Freisetzung beendet. Nach PYC-Einnahme zeigte sich im Vergleich zu dem Kontrollzustand eine statistisch signifikante (p= 0,032, n= 38) Abnahme der DPP-IV-Konzentration von 8.2 %. Allerdings wurden keine deutlichen Änderungen der mittleren DPP-IV-Aktivität durch die PYC-Behandlung hervorgerufen. Nach unserem Wissensstand war die klinische Studie die erste ihrer Art, welche die Effekte von PP auf die Serumkonzentration und die enzymatischen Aktivität von DPP IV im Menschen untersuchte. Da die Serumspiegel von DPP IV bei diabetischen Patienten im Vergleich zu gesunden Menschen erhöht sind, könnte der beobachtete Effekt vorteilhaft sein. Als Nächstes sollten die Effekte von einzelnen PP und ihren Metaboliten auf die enzymatische Aktivität der DPP IV in vitro untersucht werden. Die Ergebnisse zeigten nur eine geringe Hemmung (< 10 %) der DPP IV Aktivität durch die PP (auch Inhaltsstoffe von PYC und deren Metaboliten) in einer Konzentration von 5 µM, verglichen zur initialen 100 %-igen Enzymaktivität. Die Positivkontrolle (50 nM Sitagliptin) verursachte hingegen eine starke Senkung der Aktivität von DPP IV. Es ist daher unwahrscheinlich, dass die Effekte der PP eine klinische Relevanz haben. Die Ergebnisse stehen im Widerspruch zu bisher veröffentlichten Daten, unterstützen aber die Resultate aus der vorliegenden Humanstudie. Zusätzlich durchgeführte Fluoreszenzmessungen deuten darauf hin, dass Quercetin und Urolithin B unter definierten Bedingungen in der Lage sind, die Ergebnisse des DPP IV-Inhibitoren-Screening-Assays unspezifisch zu beeinflussen. In-vitro-Experimente in 3T3-L1 differenzierten Adipozyten zeigten zudem keine statistisch signifikanten Effekte auf die Proteinexpression von DPP IV – weder für einzelne Bestandteile noch für Metabolite von PYC. Somit konnte die beobachtete Abnahme der DPP-IV-Konzentrationen in vivo nach der Einnahme von PYC nicht durch eine Herunterregulierung der DPP-IV-Expression in Adipozyten erklärt werden. Die Akt-Kinase/PKB (Protein Kinase B) spielt eine wesentliche Rolle bei der Vermittlung der Effekte des Insulins auf intrazellulärer Ebene bzw. bei der Pathophysiologie von DMT2 sowie dessen vaskulären Spätfolgen. Die Modulation von Akt in Endothelzellen ist ein vielversprechender Ansatz, um pathophysiologische Veränderungen zu beeinflussen, die für diabetische kardiovaskuläre Spätkomplikationen verantwortlich sind. Daher sollte in der vorliegenden Arbeit der Einfluss von PP aus verschiedenen strukturellen Subklassen auf die Akt-Phosphorylierung (pAkt) an Ser 473 und Thr 308 in Endothelzellen (EA.hy926) in vitro untersucht werden (10 µM, 5 – 30 min). In der Pilotstudie hemmten beide Modellverbindungen die Akt-Phosphorylierung (pAkt Ser473) statistisch signifikant mit 52,37 ± 21,01 % (Quercetin; p= 0,006, n= 3) und 37,79 ± 7,14 % (Resveratrol; p= 0,021, n= 4). Im Primärscreening wurden acht Verbindungen verschiedener Subklassen einbezogen. Die Substanzen, welche neben Quercetin und Resveratrol die stärksten Hemmungsaktivitäten auf pAkt Ser473 (Mittelwert ± Standardabweichung) hatten, waren Luteolin (29,96 ± 11,06 %, p< 0,01, n= 6) und Apigenin (22,57 ± 10,30 %, p< 0,01, n= 6). Im Gegensatz dazu zeigten Genistein, 3,4,5-Trimethoxy-trans-stilben, Taxifolin und (+)-Catechin keine Hemmung. Die inhibierenden Effekte der PP auf pAkt Thr308 und pAkt Ser473 korrelierten positiv und statistisch signifikant miteinander (r= 0,9478, p= 0,0003). In einem sekundären Screening sollten die Effekte diverser PP auf die Phosphorylierung von Akt an Ser 473 in Endothelzellen mittels quantitativen ELISA detailliert untersucht werden. Die Verbindungen mit den größten Hemmungseffekte waren Luteolin (44,31 ± 17,95 %), Quercetin (35,71 ± 8,33 %), Urolithin A (35,28 ± 11,80 %), Apigenin (31,79 ± 6,16 %), Fisetin (28,09 ± 9,09 %), und Resveratrol (26,04 ± 5,58 %). Diese Hemmungen waren statistisch signifikant (p< 0,01, n= 3 - 6). Zusätzlich war die Wirkung von Luteolin und Resveratrol statistisch signifikant verschieden (p= 0,008), was einer weiteren Leitstrukturoptimierung dienen kann. Der quantitative Vergleich der Substanzaktivitäten war die Grundlage für nachfolgende Struktur-Wirkungsuntersuchungen (SAR). Die Doppelbindung C2=C3 (Ring C) war für die Inhibitionseffekte bei Flavonen und Flavon-3-olen von wesentlicher Bedeutung. Die Anwesenheit einer meta- und einer para- OH-Gruppe (Ring B) trug sehr wahrscheinlich zu den Hemmeffekten bei. Bei den Stilbenoiden hingegen waren drei freie OH-Gruppen (bei Resveratrol) optimal für die Aktivität. Der mikrobielle Metabolit der Ellagsäure, Urolithin A, hemmte im Vergleich zur Muttersubstanz die Akt-Phosphorylierung statistisch signifikant. Im Gegensatz dazu zeigten die anderen Urolithine trotz der strukturellen Ähnlichkeit nur geringe Effekte. Dies deutet auf einen spezifischen Effekt von Urolithin A hin. Basierend auf den gemeinsamen Merkmalen, die für die Aktivität wichtig sein können und der molekularen Geometrie der aktivsten PP, wurde ein Pharmakophormodell für die Akt-Hemmung vorgeschlagen. Zusammengefasst ist die vorliegende Studie nach unserem Wissensstand die erste, welche die Auswirkungen einer Vielzahl von PP verschiedener struktureller Subklassen auf die Akt-Phosphorylierung in Endothelzellen quantitativ verglich. Es wurden Effekte von zuvor nicht beschriebenen Verbindungen und Struktur-Aktivitäts-Beziehungen ermittelt. Die hemmenden Eigenschaften einzelner PP auf die Akt-Kinase könnten im Fall einer anhaltenden Überaktivierung der Akt-Kinase und ihren Substraten wie die S6-Kinase vorteilhaft sein. Dieser Zustand wurde bei bestimmten DMT2-verwandten pathologischen Prozessen, wie Insulinresistenz, endotheliale Dysfunktion, übermäßige Angiogenese, vaskuläre Kalzifizierung und Insulin-induzierte-DNA-Schäden beobachtet. Die Ergebnisse der vorliegenden Arbeit deuten darauf hin, dass die Wirkmechanismen von PP die Akt-Inhibierung und DPP IV-Herunterregulierung umfassen könnten, was zum Verständnis der anti-diabetischen und protektiven Wirkungen dieser Verbindungen auf molekularer Ebene beitragen kann. KW - Polyphenole KW - Endothel KW - Struktur-Aktivitäts-Beziehung KW - Diabetes mellitus KW - in vitro KW - endothelium KW - screening KW - structure-activity relationship KW - DPP IV KW - Akt KW - Diabetes mellitus Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-185701 ER - TY - JOUR A1 - Dirimanov, Stoyan A1 - Högger, Petra T1 - Screening of inhibitory effects of polyphenols on Akt-phosphorylation in endothelial cells and determination of structure-activity features JF - Biomolecules N2 - Polyphenols exert beneficial effects in type 2 diabetes mellitus (T2DM). However, their mechanism of action remains largely unknown. Endothelial Akt-kinase plays a key role in the pathogenesis of cardiovascular complications in T2DM and therefore the modulation of its activity is of interest. This work aimed to characterize effects of structurally different polyphenols on Akt-phosphorylation (pAkt) in endothelial cells (Ea.hy926) and to describe structure-activity features. A comprehensive screening via ELISA quantified the effects of 44 polyphenols (10 µM) on pAkt Ser473. The most pronounced inhibitors were luteolin (44 ± 18%), quercetin (36 ± 8%), urolithin A (35 ± 12%), apigenin, fisetin, and resveratrol; (p < 0.01). The results were confirmed by Western blotting and complemented with corresponding experiments in HUVEC cells. A strong positive and statistically significant correlation between the mean inhibitory effects of the tested polyphenols on both Akt-residues Ser473 and Thr308 (r = 0.9478, p = 0.0003) was determined by immunoblotting. Interestingly, the structural characteristics favoring pAkt inhibition partially differed from structural features enhancing the compounds’ antioxidant activity. The present study is the first to quantitatively compare the influence of polyphenols from nine different structural subclasses on pAkt in endothelial cells. These effects might be advantageous in certain T2DM-complications involving over-activation of the Akt-pathway. The suggested molecular mode of action of polyphenols involving Akt-inhibition contributes to understanding their effects on the cellular level. KW - Akt/PKB KW - endothelium KW - diabetes KW - polyphenols KW - in vitro KW - structure-activity relationships KW - screening Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-197333 SN - 2218-273X VL - 9 IS - 6 ER -