Takotsubo patient plasma induce endothelial eNOS-NO/ROS imbalance promoting monocyte and platelet adhesion: Protective effects of beta2-adrenergic receptor stimulation and SGLT2 inhibition

Abstract Background Acute reversible apical dysfunction in Takotsubo syndrome (TTS) has been associated with dysregulation of the β-adrenergic signaling pathway, coronary microcirculation dysfunction and oxidative stress. Although SGLT2 inhibition (SGLT2i) and activation of endothelial β2-adrenergic receptors (β2AR) have been shown to improve endothelial function in major heart diseases, their roles in TTS remain unclear. Purpose This study aims to evaluate the impact of TTS plasma on endothelial function and to assess the potential protective effects of SGLT2i and β2AR stimulation. Methods and results Plasma of 20 TTS patients was collected, and levels of soluble adhesion molecules and proinflammatory cytokines were measured by ELISA. Human microvascular endothelial cells (HMEC-1) were exposed to 10% TTS plasma for 30 min or 24 h, followed by assessment of gene expression via RT-qPCR, platelet and monocyte adhesion, and thrombin generation. Nitric oxide (NO) and reactive oxygen species (ROS) formation were evaluated using DAF-FM and dihydroethidium fluorescence assays, respectively. TTS plasma showed elevated levels of markers of sVCAM-1, sICAM-1 as well as IL-1β, IL-6, and TNFα. Both short-term and long-term exposure of HMEC-1 to TTS plasma increased the formation ROS. The short-term response was mitigated by metoprolol (β1AR antagonist) and ICI 115,881 (β2AR antagonist) and the long-term by L-NAME (NOS inhibitor), fenoterol (β2AR agonist), empagliflozin (SGLT2i) and a mixture of neutralizing antibodies against IL-6, IL-1β, TNF-α. The protective effect of fenoterol was blocked by H-89 (PKA inhibitor) and mimicked by forskolin (adenylyl cyclase activator). The most active TTS samples (upper mean induction of ROS formation and sICAM-1 levels) were further characterized. Long-term stimulation with TTS plasma for 24 h increased basal levels of NO, markedly reduced bradykinin-stimulated NO formation, enhanced platelet and monocyte adhesion, and thrombin generation. These effects were prevented by empagliflozin, silencing of SGLT2 and by fenoterol. Long-term TTS plasma treatment increased mRNA expression levels of SGLT2, β1 and β2AR, markers of endothelial activation (ICAM-1, VCAM-1), pro-inflammatory cytokines (IL-1β, IL-6, TNFα), eNOS and iNOS in HMEC-1. Conclusion Plasma from TTS patients activate endothelial cells via β1/β2 adrenergic receptors, causing eNOS-NO/ROS imbalance with uncoupled eNOS and induction of iNOS, pro-inflammatory, pro-adhesive and pro-thrombotic responses. The protective effects of β2AR stimulation, mediated by the cAMP/PKA pathway, and SGLT2i suggest potential therapeutic strategies to mitigate endothelial dysfunction in TTS.