Pathophysiological and Pharmacological Relevance of NO/cGMP Signaling in Atherosclerosis

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URI: http://hdl.handle.net/10900/153358
http://nbn-resolving.de/urn:nbn:de:bsz:21-dspace-1533581
http://dx.doi.org/10.15496/publikation-94697
Dokumentart: PhDThesis
Date: 2026-04-11
Language: English
Faculty: 7 Mathematisch-Naturwissenschaftliche Fakultät
Department: Biochemie
Advisor: Feil, Susanne (Dr.)
Day of Oral Examination: 2024-04-11
DDC Classifikation: 500 - Natural sciences and mathematics
Keywords: Cyclo-GMP
Other Keywords:
Atherosclerosis
NO-GC Modulator
Nitric oxide (NO)-sensitive guanylyl cyclase
Vascular smooth muscle cell
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Inhaltszusammenfassung:

Die Dissertation ist gesperrt bis zum 11. April 2026 !

Abstract:

Atherosclerosis leads to heart attack and stroke, the most common causes of death worldwide. This disease is characterized by chronic inflammation of the blood vessel walls, accompanied by the formation of atherosclerotic plaques. Vascular smooth muscle cells (VSMCs) play a crucial role in this process. VSMCs significantly contribute to the plaque formation through migration, clonal expansion, and phenotypic modulation. The molecular mechanisms leading to the alteration of VSMC behavior in atherogenesis are not fully understood. The cyclic gua-nosine monophosphate (cGMP) signaling pathway, synthesized by the nitric oxide (NO)-sen-sitive guanylyl cyclase (NO-GC) is a key factor in the (patho-)physiology of VSMCs. It is assumed that a disruption of the NO/cGMP signaling pathway in VSMCs impact phenotypic mod-ulation and plaque growth. However, it remains unclear whether pharmacological stimulation/activation of the NO/cGMP pathway in VSMCs exerts pro- or anti-atherogenic effects. This study aimed to further elucidate the pathophysiological and pharmacological relevance of the NO/cGMP pathway in VSMCs in a mouse model of atherosclerosis and investigate its potential effects on atherosclerotic plaque growth and composition. To modulate the activity of the NO/cGMP signaling pathway in vivo, NO-GC modulating drugs or cell specific genetic in-activation of NO-GC in VSMCs (NO-GCsmko) were used. A novel aspect of this study was the comparison of the effects of a NO-GC activator (BAY Activator) and a NO-GC stimulator (Vericiguat). Both compounds induce cGMP generation by binding to NO-GC but differ in their mode of action. NO-GC stimulators enhance NO-GC activity independently or synergistically with endogenous NO. In contrast, NO-GC activators activate the dysfunctional heme-oxidized NO-GC. Particularly under conditions of increased oxidative stress, as observed in atheroscle-rosis, NO-GC activators are assumed to have high therapeutic potential. Various biochemical, immunological, and histochemical methods were combined for analysis. Initially, the expression of NO-GC as a potential drug target was demonstrated by in situ im-munofluorescence staining in VSMCs and macrophage antigen-2 (MAC2)-positive cells in ath-erosclerotic plaques, indicating the possibility of pharmacological modulation of the enzyme. MAC2 was used as a cellular marker for VSMC-derived transdifferentiated macrophage-like cells or macrophages. Interestingly, en face analysis of atherosclerotic aortas showed that BAY Activator treatment led to a significant increase in the atherosclerotic lesion area, while Vericiguat did not exhibit this pro-atherosclerotic effect. Comprehensive analyses revealed that the administered drugs were detectable in the blood plasma and that various parameters that could influence atherosclerosis, such as blood pressure, plasma lipid profile and complete blood count, were not significantly changed by the BAY Activator treatment. To investigate the functional relevance of the NO/cGMP signaling pathway in VSMCs for the pro-atherosclerotic effect of the BAY Activator, in vitro experiments were performed with primary VSMCs isolated from mouse aortas. First, the effects of NO-GC modulators on intracellular cGMP concentration were visualized in real-time under normal and oxidative conditions using a Förster/fluorescence resonance energy transfer (FRET)-based cGMP biosensor. Additionally, the effects on VSMC growth were examined using the xCELLigence system. These experiments demon-strated that both the BAY Activator and Vericiguat increased cGMP levels and promoted VSMC growth. As expected, the BAY Activator showed higher efficacy in increasing intracellular cGMP levels under conditions of oxidative stress, which may also imply an enhanced efficacy under atherosclerotic conditions. Based on the results of the present study and the existing literature, we postulate that the growth-promoting effects of NO-GC modulators are likely as-sociated with the role of NO-GC in the phenotypic modulation of VSMCs. Interestingly, our in vivo study revealed that the increased atherosclerotic lesion area caused by the BAY Activator treatment was accompanied by an accumulation of MAC2-positive cells. This indicates that activation of the NO/cGMP signaling pathway led to an enrichment of VSMC-derived transdifferentiated macrophage-like cells or macrophages. It was also planned to validate the target specificity of the effects of NO-GC modulators and the pathophysiological role of the NO/cGMP signaling cascade in VSMCs using an NO-GCsmko mouse model. Although we successfully generated the NO-GCsmko mouse model, the preliminary data suggested that the induction of NO-GCsmko led to a hypertensive phenotype and elevated plasma lipid levels, which complicated the interpretation of the data regarding the effects on atherosclerosis. Thus, no further conclusions were drawn from these data at this time. In summary, our results suggest a pro-atherosclerotic effect of activation of the NO/cGMP sig-naling pathway by BAY Activator treatment. We hypothesize that this is closely related to the increased efficacy of BAY Activator under the oxidative conditions prevalent in atherosclerotic plaques and the impact on the phenotypic modulation of VSMCs in the plaque. These findings provide the basis for further research to deepen our understanding of the pathophysiological and pharmacological relevance of the NO/cGMP signaling pathway in atherosclerosis.

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