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1.
Antioxidants (Basel) ; 13(2)2024 Jan 30.
Artículo en Inglés | MEDLINE | ID: mdl-38397773

RESUMEN

Coronavirus disease 2019 (COVID-19) is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). While recent studies have demonstrated that SARS-CoV-2 may enter kidney and colon epithelial cells by inducing receptor-independent macropinocytosis, it remains unknown whether this process also occurs in cell types directly relevant to SARS-CoV-2-associated lung pneumonia, such as alveolar epithelial cells and macrophages. The goal of our study was to investigate the ability of SARS-CoV-2 spike protein subunits to stimulate macropinocytosis in human alveolar epithelial cells and primary human and murine macrophages. Flow cytometry analysis of fluid-phase marker internalization demonstrated that SARS-CoV-2 spike protein subunits S1, the receptor-binding domain (RBD) of S1, and S2 stimulate macropinocytosis in both human and murine macrophages in an angiotensin-converting enzyme 2 (ACE2)-independent manner. Pharmacological and genetic inhibition of macropinocytosis substantially decreased spike-protein-induced fluid-phase marker internalization in macrophages both in vitro and in vivo. High-resolution scanning electron microscopy (SEM) imaging confirmed that spike protein subunits promote the formation of membrane ruffles on the dorsal surface of macrophages. Mechanistic studies demonstrated that SARS-CoV-2 spike protein stimulated macropinocytosis via NADPH oxidase 2 (Nox2)-derived reactive oxygen species (ROS) generation. In addition, inhibition of protein kinase C (PKC) and phosphoinositide 3-kinase (PI3K) in macrophages blocked SARS-CoV-2 spike-protein-induced macropinocytosis. To our knowledge, these results demonstrate for the first time that SARS-CoV-2 spike protein subunits stimulate macropinocytosis in macrophages. These results may contribute to a better understanding of SARS-CoV-2 infection and COVID-19 pathogenesis.

2.
Int J Mol Sci ; 24(22)2023 Nov 16.
Artículo en Inglés | MEDLINE | ID: mdl-38003584

RESUMEN

Diabetics are more vulnerable to SARS-CoV-2 neurological manifestations. The molecular mechanisms of SARS-CoV-2-induced cerebrovascular dysfunction in diabetes are unclear. We hypothesize that SARS-CoV-2 exacerbates diabetes-induced cerebrovascular oxidative stress and inflammation via activation of the destructive arm of the renin-angiotensin-aldosterone system (RAAS) and Toll-like receptor (TLR) signaling. SARS-CoV-2 spike protein was injected in humanized ACE2 transgenic knock-in mice. Cognitive functions, cerebral blood flow, cerebrovascular architecture, RAAS, and TLR signaling were used to determine the effect of SARS-CoV-2 spike protein in diabetes. Studies were mirrored in vitro using human brain microvascular endothelial cells treated with high glucose-conditioned media to mimic diabetic conditions. Spike protein exacerbated diabetes-induced cerebrovascular oxidative stress, inflammation, and endothelial cell death resulting in an increase in vascular rarefaction and diminished cerebral blood flow. SARS-CoV-2 spike protein worsened cognitive dysfunction in diabetes compared to control mice. Spike protein enhanced the destructive RAAS arm at the expense of the RAAS protective arm. In parallel, spike protein significantly exacerbated TLR signaling in diabetes, aggravating inflammation and cellular apoptosis vicious circle. Our study illustrated that SAR-CoV-2 spike protein intensified RAAS and TLR signaling in diabetes, increasing cerebrovascular damage and cognitive dysfunction.


Asunto(s)
COVID-19 , Diabetes Mellitus , Humanos , Ratones , Animales , Sistema Renina-Angiotensina , Glicoproteína de la Espiga del Coronavirus/metabolismo , SARS-CoV-2/metabolismo , COVID-19/complicaciones , Células Endoteliales/metabolismo , Enzima Convertidora de Angiotensina 2/metabolismo , Inflamación , Receptores Toll-Like/metabolismo , Ratones Transgénicos
3.
Diabetologia ; 65(9): 1541-1554, 2022 09.
Artículo en Inglés | MEDLINE | ID: mdl-35687178

RESUMEN

AIMS/HYPOTHESIS: We have previously shown that diabetes causes pericyte dysfunction, leading to loss of vascular integrity and vascular cognitive impairment and dementia (VCID). Glucagon-like peptide-1 (GLP-1) receptor agonists (GLP-1 RAs), used in managing type 2 diabetes mellitus, improve the cognitive function of diabetic individuals beyond glycaemic control, yet the mechanism is not fully understood. In the present study, we hypothesise that GLP-1 RAs improve VCID by preventing diabetes-induced pericyte dysfunction. METHODS: Mice with streptozotocin-induced diabetes and non-diabetic control mice received either saline (NaCl 154 mmol/l) or exendin-4, a GLP-1 RA, through an osmotic pump over 28 days. Vascular integrity was assessed by measuring cerebrovascular neovascularisation indices (vascular density, tortuosity and branching density). Cognitive function was evaluated with Barnes maze and Morris water maze. Human brain microvascular pericytes (HBMPCs), were grown in high glucose (25 mmol/l) and sodium palmitate (200 µmol/l) to mimic diabetic conditions. HBMPCs were treated with/without exendin-4 and assessed for nitrative and oxidative stress, and angiogenic and blood-brain barrier functions. RESULTS: Diabetic mice treated with exendin-4 showed a significant reduction in all cerebral pathological neovascularisation indices and an improved blood-brain barrier (p<0.05). The vascular protective effects were accompanied by significant improvement in the learning and memory functions of diabetic mice compared with control mice (p<0.05). Our results showed that HBMPCs expressed the GLP-1 receptor. Diabetes increased GLP-1 receptor expression and receptor nitration in HBMPCs. Stimulation of HBMPCs with exendin-4 under diabetic conditions decreased diabetes-induced vascular inflammation and oxidative stress, and restored pericyte function (p<0.05). CONCLUSIONS/INTERPRETATION: This study provides novel evidence that brain pericytes express the GLP-1 receptor, which is nitrated under diabetic conditions. GLP-1 receptor activation improves brain pericyte function resulting in restoration of vascular integrity and BBB functions in diabetes. Furthermore, the GLP-1 RA exendin-4 alleviates diabetes-induced cognitive impairment in mice. Restoration of pericyte function in diabetes represents a novel therapeutic target for diabetes-induced cerebrovascular microangiopathy and VCID.


Asunto(s)
Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 2 , Receptor del Péptido 1 Similar al Glucagón , Pericitos , Animales , Encéfalo/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Modelos Animales de Enfermedad , Exenatida/uso terapéutico , Péptido 1 Similar al Glucagón/metabolismo , Receptor del Péptido 1 Similar al Glucagón/metabolismo , Humanos , Ratones , Pericitos/metabolismo
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