O sistema colinérgico não-neuronal na artéria coronária humana e sua relação com a aterosclerose

Abstract

The discovery that several tissues of non-neuronal origin express components related to the synthesis, release, and degradation of acetylcholine (ACh) changed the paradigm of ACh acting merely as a neurotransmitter and supports the existence of a “non-neuronal cholinergic system (NNCS).” The importance of this system is an expanding field. However, it is described that the paracrine/autocrine action of ACh via the alpha 7 nicotinic acetylcholine receptor (α7nAChR) has a potential role in inflammatory diseases, such as atherosclerosis. Coronary arteries are often affected by atherosclerosis, the main cause of coronary artery disease (CAD). The existence of NNCS in human coronary arteries, its physiological relevance, and the involvement of α7nAChR in human atherosclerosis are unknown. Our hypothesis is that the human coronary artery has its own NNCS and that this system plays a role in the pathogenesis of atherosclerosis. Human coronary artery samples obtained from hearts discarded after heart transplant surgery were used. The expression of NNCS components was investigated by analyzing public single-cell RNA sequencing (scRNA-seq), immunofluorescence (IF), and Western blot (WB) data. Choline uptake and ACh production was assessed using a commercial kit. Hematoxylin/eosin, oil red staining, and immunohistochemistry (IHC) assays were used to stage atherosclerotic plaque. α7nAChR expression, cellular colocalization, correlation with atherosclerosis, and plaque inflammatory profile were assessed by IF and IHC assays. The effects of α7nAChR activation were evaluated in ex vivo culture of coronary artery segments. To this end, the samples were incubated for 8 hours in RPMI medium, supplemented with fetal bovine serum and antibiotic, containing or not PNU-282987 (selective α7nAChR agonist), in a CO2 oven (5%) at 37º C. IF and TUNEL, dihydroethidium (DHE) probe labeling and WB were performed to evaluate inflammatory cytokines, apoptosis, production of reactive oxygen species (ROS) and expression of enzymes involved in oxidative stress, respectively. Our results showed that human coronary artery endothelial and smooth muscle cells (SMCs) express carnitine acetyltransferase (CarT), organic cation transporter 3 (OCT3), acetylcholinesterase (AChE), butyrylcholinesterase (BuChE), enzymes related to the synthesis, transport, and degradation of ACh, respectively. Choline uptake and ACh synthesis increased when vessels were incubated with choline, indicating that the cholinergic machinery of the coronary artery is functional. Furthermore, it was identified that the coronary artery expresses the α7nAChR and secreted lymphocyte antigen-6/urokinase-type plasminogen activator peptide-1 (SLURP-1), an endogenous negative allosteric modulator of the α7nAChR. In advanced atherosclerotic plaques, it was found that there is a greater expression of CarT, BuChE, α7nAChR, and SLURP-1, suggesting that NNCS/α7nAChR may play a role in human atherosclerosis. In atherosclerotic plaques, α7nAChR expression positively correlates with macrophage infiltration and smooth muscle cells proliferation. Activation of the α7nAChR in the coronary artery increased the expression of lectin-like oxidized low-density lipoprotein receptor 1 (LOX-1), MCP-1, and the production of IL-6 and IL-1β, molecules described as pro-inflammatory and pro-atherosclerotic. Furthermore, activation of the α7nAChR increased the production of ROS and the expression of NOX-5, suggesting that this enzyme contributes to the elevation of vascular oxidative stress. Finally, an increase in the number of apoptotic cells in the vascular wall and cleavage of caspases 8 and 3 was observed, indicating that activation of the α7nAChR contributes to vascular cell death by activating the extrinsic apoptosis signaling pathway. Our results show that the human coronary artery has its own functional NNSC and that this system, via activation of the α7nAChR, has pro-atherosclerotic effects. These data highlight a potential role for the α7nAChR in the pathogenesis of human atherosclerosis and place this receptor as a potential target for therapeutic interventions.