Endothelial cells (ECs) form the inner layer of all the blood vessels and, due to this strategic localization, they constantly face oscillating blood glucose concentrations in relation to the pre- and post- prandial cycles. They do not represent a passive barrier between blood and tissues, but they play a wide variety of pivotal roles to control vascular homeostasis. Uncontrolled hyperglycaemia elicits ECs to become dysfunctional, leading to the onset of endothelial dysfunction, defined as a shift of properties of the endothelium toward a proinflammatory and prothrombotic phenotype characterised by altered release of Nitric Oxide (NO) and overproduction of pro-inflammatory cytokines and Reactive Oxygen Species (ROS). Endothelial dysfunction is classically described in patients affected by diabetes and has a role in the pathogenesis of many cardiovascular complications associated with this pathology. In particular, diabetes is a group of life-long chronic metabolic disorders characterized by high levels of glucose in the blood and by a predisposition to premature atherosclerosis, the main reason for high morbidity and impaired life expectancy in patients. To investigate the effects of high glucose on ECs, Human Umbilical Vein Endothelial Cells (HUVEC) were cultured in the presence of either high glucose-containing medium or of blood sera collected from diabetic patients. Cells were seeded both in 2D cell culture systems on flat dishes, a method which has yielded major advances in our knowledge about endothelial pathophysiology, and in 3D microfluidic chips, which show a higher degree of structural complexity allowing perfusion, thus generating shear stress fundamental for endothelial homeostasis. In 2- and 3- D, the cells were cultured until they reach confluence to reproduce the physiological inner layer of a blood vessel as closely as possible. High glucose rapidly alters the cellular redox balance resulting in a higher production of ROS associated with a lower content of the antioxidant glutathione (GSH). ROS induce iNOS, which overproduces NO leading to endothelial hyperpermeability. In parallel, mitochondrial dysfunction occurs as a result of the imbalance in mitochondrial dynamics in favour of mitochondrial fission. This evidence is connected to the higher storage of triglycerides caused by both enhanced lipogenesis and reduced β-oxidation. The next step was to individuate a countermeasure that might prevent the detrimental effects of high glucose on HUVEC. Numerous observational studies demonstrated that the circulating levels of Vitamin D3 (VitD) are low in patients affected by diabetes. In these experimental models, VitD is capable of blocking the increase of ROS production, thus preventing all the harmful effects caused by high glucose on HUVEC. In summary, the results reveal that it is fundamental to keep glycaemia within the physiological range in diabetic patients, preventing hyperglycaemic peaks, and that VitD could represents a serviceable tool to control the redox equilibrium, thus re-establishing NO levels and permeability and mitochondrial fitness, to limit or at least delay the insurgence of endothelial dysfunction caused by high fasting glucose concentrations.

ENDOTHELIUM IN PETRI DISH (2D) OR ON-A-CHIP (3D): STUDIES ON ENDOTHELIAL FUNCTION / DYSFUNCTION / R. Scrimieri ; tutor: J.A.M. Maier ; director: N. Landsberger. - : . Dipartimento di Scienze Biomediche e Cliniche L. Sacco, 2022 Jan 17. ((34. ciclo, Anno Accademico 2021.

ENDOTHELIUM IN PETRI DISH (2D) OR ON-A-CHIP (3D): STUDIES ON ENDOTHELIAL FUNCTION / DYSFUNCTION

R. Scrimieri
2022-01-17

Abstract

Endothelial cells (ECs) form the inner layer of all the blood vessels and, due to this strategic localization, they constantly face oscillating blood glucose concentrations in relation to the pre- and post- prandial cycles. They do not represent a passive barrier between blood and tissues, but they play a wide variety of pivotal roles to control vascular homeostasis. Uncontrolled hyperglycaemia elicits ECs to become dysfunctional, leading to the onset of endothelial dysfunction, defined as a shift of properties of the endothelium toward a proinflammatory and prothrombotic phenotype characterised by altered release of Nitric Oxide (NO) and overproduction of pro-inflammatory cytokines and Reactive Oxygen Species (ROS). Endothelial dysfunction is classically described in patients affected by diabetes and has a role in the pathogenesis of many cardiovascular complications associated with this pathology. In particular, diabetes is a group of life-long chronic metabolic disorders characterized by high levels of glucose in the blood and by a predisposition to premature atherosclerosis, the main reason for high morbidity and impaired life expectancy in patients. To investigate the effects of high glucose on ECs, Human Umbilical Vein Endothelial Cells (HUVEC) were cultured in the presence of either high glucose-containing medium or of blood sera collected from diabetic patients. Cells were seeded both in 2D cell culture systems on flat dishes, a method which has yielded major advances in our knowledge about endothelial pathophysiology, and in 3D microfluidic chips, which show a higher degree of structural complexity allowing perfusion, thus generating shear stress fundamental for endothelial homeostasis. In 2- and 3- D, the cells were cultured until they reach confluence to reproduce the physiological inner layer of a blood vessel as closely as possible. High glucose rapidly alters the cellular redox balance resulting in a higher production of ROS associated with a lower content of the antioxidant glutathione (GSH). ROS induce iNOS, which overproduces NO leading to endothelial hyperpermeability. In parallel, mitochondrial dysfunction occurs as a result of the imbalance in mitochondrial dynamics in favour of mitochondrial fission. This evidence is connected to the higher storage of triglycerides caused by both enhanced lipogenesis and reduced β-oxidation. The next step was to individuate a countermeasure that might prevent the detrimental effects of high glucose on HUVEC. Numerous observational studies demonstrated that the circulating levels of Vitamin D3 (VitD) are low in patients affected by diabetes. In these experimental models, VitD is capable of blocking the increase of ROS production, thus preventing all the harmful effects caused by high glucose on HUVEC. In summary, the results reveal that it is fundamental to keep glycaemia within the physiological range in diabetic patients, preventing hyperglycaemic peaks, and that VitD could represents a serviceable tool to control the redox equilibrium, thus re-establishing NO levels and permeability and mitochondrial fitness, to limit or at least delay the insurgence of endothelial dysfunction caused by high fasting glucose concentrations.
MAIER, JEANETTE ANNE MARIE
LANDSBERGER, NICOLETTA
Diabetes; hyperglycaemia; endothelial cells; Vitamin D
Settore MED/04 - Patologia Generale
https://pubmed.ncbi.nlm.nih.gov/32316573/
https://pubmed.ncbi.nlm.nih.gov/32162606/
ENDOTHELIUM IN PETRI DISH (2D) OR ON-A-CHIP (3D): STUDIES ON ENDOTHELIAL FUNCTION / DYSFUNCTION / R. Scrimieri ; tutor: J.A.M. Maier ; director: N. Landsberger. - : . Dipartimento di Scienze Biomediche e Cliniche L. Sacco, 2022 Jan 17. ((34. ciclo, Anno Accademico 2021.
Doctoral Thesis
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2434/887953
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