Adducin and cortactin influence on regulating endothelial adherens and tight junction integrity via cAMP signaling pathways

Moztarzadeh, Sina (2026): Adducin and cortactin influence on regulating endothelial adherens and tight junction integrity via cAMP signaling pathways. Dissertation, LMU München: Faculty of Biology

Abstract

Life-threatening diseases such as diabetes, atherosclerosis, cancer and acute inflammatory events such as sepsis, lung and kidney injury often trigger vascular endothelial dysfunction. The latter is characterized by reduced vasodilation and increased permeability, results of compromised endothelial monolayer due to disrupted endothelial cell contacts [1, 2]. The intercellular endothelial junctions are established and maintained by various intercellular junctional complexes mainly including the tight junctions (TJ) consisting of claudin-5, occludin, junctional adhesion molecules (JAMs) and the adherens junctions (AJ) mainly composed of vascular endothelial cadherin (VE-cadherin), both crucial for maintaining endothelial barrier integrity [3-5]. These junctional complexes are bound to the actin cytoskeleton through adaptor molecules such as zonula occludens-1 (ZO-1) and the members of the catenin family (α, β and γ- catenin) [4, 6, 7]. In addition, various signaling molecules like the secondary messenger cyclic adenosine monophosphate (cAMP) and the small Rho GTPases (Guanosine Triphosphatase), also participate in modulation of endothelial barrier integrity [8]. Thus, any molecule or protein influencing the dynamics of the actin cytoskeleton can directly or indirectly impact endothelial barrier function [9]. An emerging group of proteins fitting this role are the actin-binding proteins (ABPs) [10]. For example, cortactin (Cttn) is known for its involvement in actin nucleation and branching [11]. In addition, adducin (Add) was shown to play a critical role on stabilizing actin-spectrin networks [12]. However, independent of their different functions, both ABPs were confirmed to contribute to endothelial barrier regulation through a shared regulatory pathway, which may explain their similar effect on barrier integrity. Nevertheless, the association of these ABPs with endothelial barrier regulation is not well understood. This project aimed to explore the interplay among of Cttn and Add with various endothelial junctional components and important signaling molecules known to be involved in endothelial barrier integrity, thereby identifying new possibilities for therapeutic strategies with potential to prevent junctional breakdown and vascular permeability. To this end, myocardial endothelial cells from Cttn- and Add-knockout mice as well as respective wild type animals were used. Various analyses, including Western blot, ELISA (Enzyme-Linked Immunosorbent Assay), immunoprecipitation, PCR (Polymerase Chain Reaction), G-ELISA and immunostainings were employed to study endothelial intercellular contacts and junctional complexes as well as enzymatic activity or cAMP intracellular content. To assess endothelial barrier dynamics, real-time transendothelial electrical resistance (TEER) measurements were performed. Our data showed that in endothelial cells, Add is crucial for maintaining the integrity of AJ and TJ since the lack of this protein disturbed the localization of key junctional components at the membrane as well as their expression levels. Moreover, Add is required for cAMP-mediated barrier improvement and regulation of small Rho GTPases. Cttn was previously reported to be indispensable to preserve endothelial barrier stability by controlling actomyosin contractility [11]. However, the impact of Cttn on endothelial junctions was not explored in detail and an association with cAMP regulation was only briefly discussed. For this reason, in the current project, we aimed to provide a comprehensive perspective about the role of Cttn in endothelial junctional integrity and cAMP-mediated barrier enhancement. We found that Cttn exists within the VE-cadherin-based adherens junctional complex. Moreover, lack of Cttn compromised barrier integrity through disruption of cell junctions and by slowing down barrier recovery after Ca2+ repletion [13]. The underlying mechanism involved hindered Rap1 and Rac1 activation upon elevation of intracellular cAMP levels. Thus, the data indicate that Cttn acts as a structural and signaling hub, where junctional proteins and cAMP signaling effectors converge to modulate endothelial barrier integrity.

• Item Type: Theses (Dissertation, LMU Munich)
• Keywords: Cell biology, Cell adhesion, Adherens junctions, Cardiovascular biology
• Subjects: 500 Natural sciences and mathematics; 500 Natural sciences and mathematics > 570 Life sciences
• Faculties: Faculty of Biology
• Language: English
• Date of oral examination: 24. March 2026
• 1. Referee: Heß, Martin
• Signature of the printed copy: 0001/UMC 31911