10th German Pharm-Tox Summit / 91. Jahrestagung der Deutschen Gesellschaft für Experimentelle und Klinische Pharmakologie und Toxikologie (DGPT)

Prof. Dr. med.

Chair

Prof. Dr.

Prof.

Thrombin receptor PAR4 regulates the NLRP3 inflammasome/GRP78 signaling tandem in obese atria

Cardiac pharmacology

AP01

Background:Obese patients are highly prone to atrial cardiomyopathy, which provides the critical substrate for the development of atrial fibrillation (AF). We recently identified a functional connection between the NLRP3 inflammasome and the ER stress chaperone GRP78 in this context. Druggable upstream regulators of this signaling tandem are not known. One candidate is protease-activated receptor PAR4, which is upregulated in obesity and mediates coagulation-independent inflammatory signaling. Atrial fibroblasts are an emerging nexus for NLRP3 inflammasome activity in AF, an interaction with PAR4 and GRP78 especially in obesity is not known.Methods:Right atrial appendages (RAA) were collected from obese and non-obese patients undergoing cardiac surgery. Human primary atrial fibroblasts were stimulated with the PAR4 agonist peptide AYPGKF-NH2. Pooled left and right atria were isolated from wildtype and PAR4-/- mice fed chow or high fat diet (HFD, 16 weeks). Tissues, cell lysates and supernatants were assayed by qPCR, immunoblot and ELISA.Results:Obese human atria showed higher abundance of activated NLRP3 inflammasome-associated caspase-1, interleukin (IL)-1β, PAR4 and GRP78, compared to non-obese atria. Other markers of a triggered ER stress response (HSP70, ATG7, cleaved ATF6, spliced XBP1) did not significantly differ. Obese atria also displayed higher phosphorylation of the master regulator mTOR and more fibrotic markers. In human atrial fibroblasts, PAR4 activation increased mTOR phosphorylation and the transcriptional upregulation and secretion of GRP78 and IL-1β. Other ER stress mediators were not regulated. Atria of HFD vs. chow mice contained more GRP78, but not other ER stress markers, together with increased caspase-1, IL-1β, phospho-mTOR and Acta2. All of these signals, with the unexpected exception of GRP78, were abrogated in HFD-fed PAR4-/- mouse atria.Conclusion:Upregulated atrial abundance of the thrombin receptor PAR4 in obesity is causally linked with mTOR/NLRP3 inflammasome activation and an increased expression and mobilization of the ER stress chaperone GRP78. At least in part this convergence of multiple signaling platforms occurs at the level of atrial fibroblasts and likely supports fibrotic remodeling, independently of a full ER stress response. The emerging PAR4 antagonists provide new therapeutic perspective to modify obesity-associated atrial cardiomyopathy.

Applied Pharmacology

Short talk

Telmisartan improves hyperlipidemia-induced microvascular dysfunction, cerebral blood flow reduction and impaired remote memory

AP04

By using the PCSK9DY mouse model we have recently demonstrated a link between obesity/hyperlipidemia and cognitive/microvascular dysfunction. We firstly aimed to elucidate whether the AT1 receptor blocker Telmisartan (TEL) improves cerebral microvascular dysfunction, hippocampal blood flow (HBF) and cognitive dysfunction. We secondly investigated whether a protective TEL effect is attributed to an ACE/AngII/AT1R-axis or/and Ang(1-7)/ACE2/Mas-axis mediated mechanism, as we recently found that the anti-obesity effect of TEL is also based on a Mas-dependent mechanism, while its anti-atherosclerotic effect is not.C57BL/6N (wt) or Mas-ko mice received AAV-PCSK9DY injections (2×1011 VG) or AAV and were fed with Western diet (WD) or chow. Mice were treated with TEL (8 mg/kg) or vehicle. Learning and memory behavior of mice was determined by Barnes maze (BM) tests. HBF was evaluated by ASL-MRI. Brain immunofluorescence was performed on 20μm-thick frozen sections.Compared to AAV/chow controls, PCSK9DY/WD wt mice made more "primary errors" on day 17 of BM testing and also showed a less "direct" and quite "random" search strategy to find the escape hole, both indicative of impaired remote memory. Moreover, the HBF was lower in PCSK9DY/WD than in AAV/chow wt mice. Strikingly, TEL normalized these declines in remote memory and in HBF, despite mice remained hyperlipidemic. However, these protective TEL effects were not observed in Mas-ko mice. The CD31/Col IV ratio was lowered upon PCSK9DY/WD intervention in hippocampal vessels of both mice strain. TEL normalized this vascular dysfunction in both strains. Evaluation of the CD31/Col IV-stained images additionally revealed more pericytes in both strains upon PCSK9DY/WD intervention and normalization by TEL.We conclude that hyperlipidemia impairs remote memory and HBF and both are consolidated by TEL via a Mas-dependent mechanism. We furthermore suggest, that the TEL-dependent normalization of the PCSK9DY/WD-induced increase of pericytes can contribute to the favorable TEL-effects regarding HBF and memory consolidation, since in pathophysiological conditions such as Alzheimer's disease pericytes have been shown to contribute to vasoconstriction of cerebral capillaries and consequently to a reduction in BF. However, we also suspect that other microvascular effects (which are currently being investigated) must also cause these functional changes, since this pericyte regulation was also observed in the Mas-ko mice.

New Insights into Flavonoid Binding to the Thromboxane A2 Receptor

G-protein coupled receptors

BR01

Introduction: Thromboxane A2 (TxA2) is a critical platelet agonist involved in hemostasis and thrombosis, interacting with the thromboxane A2/prostaglandin H2 (TP) receptor. While mutations in the extracellular loop (ECL) 1 region of the TP receptor do not affect TxA2 binding, mutations in ECL2 significantly reduce binding affinity. TxA2 also interacts with transmembrane region 7 (TM7) of the TP receptor, where specific amino acid substitutions lead to complete binding loss. Flavonoids effectively inhibit TxA2-mediated platelet signaling by directly binding to the TP receptor and blocking cyclooxygenase activity, thereby reducing TxA2 production. They consist of two phenyl rings and a heterocyclic pyrone ring, which influence their biological activity. Hydroxyl groups are crucial for their antithrombotic effects, as they significantly inhibit TP receptor function. In contrast, methylated or glycosylated flavonoids exhibit diminished inhibitory potential due to steric and structural constraints.Objective: To investigate the structural regions of the human TP receptor involved in TxA2 binding and to evaluate the inhibitory effects of flavonoids on TxA2-mediated platelet activation.Materials and Methods: The structures of the TP-receptor were retrieved from the Protein Data Bank and the AlphaFold Protein Structure Database. The compounds were docked in the generated grid by Glide (v8.7) in Maestro.Results: ECL1 mutations did not impact TxA2 binding, while ECL2 mutations significantly diminished it, underscoring its critical role. Mutations in TM7 resulted in the complete loss of receptor binding. Flavonoids effectively inhibited TxA2-mediated signaling by binding to the TP receptor and inhibiting COX activity, leading to reduced TxA2 production. Hydroxylated flavonoids showed the strongest inhibition, whereas methylated and glycosylated forms exhibited reduced efficacy due to structural constraints.Conclusion: Our study indicates that flavonoids occupy the ligand binding pocket in the ECL1/ECL2 and TM7 regions, competitively blocking TxA2 binding We propose that hydrogen bonds may alter the shape of the binding pocket and the distance between ECL1 and ECL2, inhibiting TxA2 binding. These findings provide molecular details of flavonoid binding to the TP receptor, facilitating the development of novel synthetic flavonoids that can inhibit TP receptor activity as alternative therapies for patients with aspirin resistance or bleeding complications.

Basic Research

Is there a role for the KATP channel as a Signal Transducer in Pancreatic Alpha-Cells? A Comparison with Pancreatic Beta-Cells

Endocrine pharmacology

AP05

Like beta-cells, pancreatic alpha-cells express glucokinase and KATP channels, however, the increase of glucose inhibits glucagon secretion. It is still unclear whether the secretion is regulated by alpha-cell intrinsic mechanisms and whether oxidative phosphorylation and subsequent closure of KATP channels play a role in it. Pancreatic islets from NMRI mice were isolated and dispersed into single beta-and alpha cells. The membrane potential of the single cells was measured using the perforated patch-clamp technique, with voltage recordings taken by an EPC-7 patch-clamp amplifier. In addition to the cellular capacitance the beta- and alpha-cell identity was checked by immunofluorescence after the experiment. Lowering the glucose concentration from 5 to 1 mM did not significantly affect the membrane potential in both alpha- and beta-cells. In the presence of 1 mM glucose single beta-cells had a membrane potential of -61mV and reacted within 10 min to stimulation with either 30 mM glucose, 10 mM alpha-ketoisocaproic acid (KIC) or glutamine plus BCH (10 mM each) with a depolarization by 28 mV, 21 mV or 14 mV, respectively. The depolarization by glutamine plus BCH was very slow and the maximal effect was only attained during wash-out. The reaction of the alpha-cells was much more heterogeneous. When only those alpha-cells which responded to arginine and stained positive for glucagon were evaluated, glucose, KIC or glutamine plus BCH led to a depolarisation of 3 mV, 8 mV and 4 mV, respectively, starting from a resting value of about -49 mV. The 8 mV depolarization by KIC, which was the only significant effect, was already complete after 2 min, the same time as required by the KATP channel-blocking sulfonylurea tolbutamide to depolarize by ca. 15 mV. To check whether KIC directly affects KATP channels, we measured the membrane potential of beta and alpha cells from SUR1KO mice, which have no functional KATP channels and are partially depolarized. At 1 mM glucose, 10 mM KIC had only very weak, non-significant depolarizing effect on either cell type. In conclusion, compounds known to act as nutrient stimulators of insulin secretion depolarize beta-cells with kinetics which reflect their effect on the oxidative phosphorylation. The modest or even lacking depolarizing effect on alpha-cells suggests that alpha-cell-intrinsic recognition of nutrients, if existent at all, does not proceed via the cytosolic ATP/ADP ratio and the ensuing changes of KATP channel function.

Amlodipine reduces cortical stiffness and pulse wave velocity in atherosclerotic mice

Calcium channel blockers (CCBs) such as amlodipine (AMLO) are established drugs for treating hypertension. Despite numerous contradictory publications, experimental and clinical studies also indicate an anti-atherosclerotic effect of CCBs. These studies differ with regard to the atherosclerosis model used and are mostly limited to histological read outs. Thus, this project aims to identify the impact of AMLO on vascular function in atherosclerosis.PCSKY9DY-injected mice on Western diet (WD) which were treated with either AMLO (10 mg/kg) or vehicle (VEH) for 12 weeks. Controls received vector (AVV) plus chow. As hallmarks of endothelial dysfunction, we determined pulse wave velocity (PWV) by ultrasound and stiffness of the endothelial cortex ex vivo by atomic force microscopy (AFM). Additional AFM measurements were carried out on HUVEC cells to investigate glycocalyx as a feature of vascular inflammation.PWV was higher in the PCSK9DY+WDVEH (1.267 m/s) than in AAV+chowVEH mice (0.908 m/s) and almost normalized by AMLO (0.987 m/s). In thoracic aortic segments we determined a PCSK9DY+WD effect as this intervention increased endothelial stiffness compared to AAV+chow controls (1.412 vs. 0.992 pN/nm), while it was reduced by AMLO (1.181 pN/nm). AMLO reduced PWV and stiffness also in AAV+chow mice. Plasma lipids were higher in PCSK9DY+WDVEH than in AVV+chowVEH mice but remained unaffected by AMLO. The plaque burden of the PCSK9DY+WDVEH mice was halved by AMLO. Increased plasma levels of TNFa and IL-6 indicate systemic inflammation in PCSK9DY+WDVEH mice, with AMLO having no effect. Plasma NO (serving as a surrogate parameter for vasodilatation) was lowered by 43% in PCSK9DY+WDVEH compared to AAV+chowVEH mice but was normalized upon AMLO.We confirm that the PCSK9DY+WD mouse model is a very suitable for studying atherosclerosis. We further conclude that AMLO reduces PWV maybe caused by a decrease of endothelial stiffness. The improvement in PWV and endothelial function in AMLO-treated AAV+chow controls as well of the height of the endothelial glycocalyx in HUVEC cells suggest a mechanism that occurs independently of atherosclerotic impairment. This conclusion is strengthened as the AMLO effects are independent on lipids as these still remained high. As it is established that arterial stiffness is regulated by the release of NO, it is convincing that a correlation between stiffness and NO bioavailability was also found in our experimental setting.

Hörsaal C

Short talk 12

Cardiovascular, metabolic and molecular pharmacology

Short talks

Short talk 12
Cardiovascular, metabolic and molecular pharmacology

Termin

Chair

Programm

Short talk 12Cardiovascular, metabolic and molecular pharmacology

Termin

Chair

Programm

Short talk 12
Cardiovascular, metabolic and molecular pharmacology