Human endothelial cells in high glucose: New clues from culture in 3D microfluidic chips.

Several studies have demonstrated the role of high glucose in promoting endothelial dysfunction utilizing traditional two-dimensional (2D) culture systems, which, however, do not replicate the complex organization of the endothelium within a vessel constantly exposed to flow. Here we describe the response to high glucose of micro- and macro-vascular human endothelial cells (EC) cultured in biomimetic microchannels fabricated through soft lithography and perfused to generate shear stress. In 3D macrovascular EC exposed to a shear stress of 0.4 Pa respond to high glucose with cytoskeletal remodeling and alterations in cell shape. Under the same experimental conditions, these effects are more pronounced in microvascular cells that show massive cytoskeletal disassembly and apoptosis after culture in high glucose. However, when exposed to a shear stress of 4 Pa, which is physiological in the microvasculature, human dermal microvascular endothelial cells (HDMEC) show alterations of the cytoskeleton but no apoptosis. This result emphasizes the sensitivity of HDMEC to different regimens of flow. No significant variations in the thickness of glycocalyx were detected in both human endothelial cells from the umbilical vein and HDMEC exposed to high glucose in 3D, whereas clear differences emerge between cells cultured in static 2D versus microfluidic channels. We conclude that culture in microfluidic microchannels unveils unique insights into endothelial dysfunction by high glucose.

A Comparison of Doxorubicin-Resistant Colon Cancer LoVo and Leukemia HL60 Cells: Common Features, Different Underlying Mechanisms.

Chemoresistance causes cancer relapse and metastasis, thus remaining the major obstacle to cancer therapy. While some light has been shed on the underlying mechanisms, it is clear that chemoresistance is a multifaceted problem strictly interconnected with the high heterogeneity of neoplastic cells. We utilized two different human cell lines, i.e., LoVo colon cancer and promyelocytic leukemia HL60 cells sensitive and resistant to doxorubicin (DXR), largely used as a chemotherapeutic and frequently leading to chemoresistance. LoVo and HL60 resistant cells accumulate less reactive oxygen species by differently modulating the levels of some pro- and antioxidant proteins. Moreover, the content of intracellular magnesium, known to contribute to protect cells from oxidative stress, is increased in DXR-resistant LoVo through the upregulation of MagT1 and in DXR-resistant HL60 because of the overexpression of TRPM7. In addition, while no major differences in mitochondrial mass are observed in resistant HL60 and LoVo cells, fragmented mitochondria due to increased fission and decreased fusion are detected only in resistant LoVo cells. We conclude that DXR-resistant cells evolve adaptive mechanisms to survive DXR cytotoxicity by activating different molecular pathways.

Endothelial Hyper-Permeability Induced by T1D Sera Can be Reversed by iNOS Inactivation.

Type 1 Diabetes Mellitus (T1D) is associated with accelerated atherosclerosis that is responsible for high morbidity and mortality. Endothelial hyperpermeability, a feature of endothelial dysfunction, is an early step of atherogenesis since it favours intimal lipid uptake. Therefore, we tested endothelial leakage by loading the sera from T1D patients onto cultured human endothelial cells and found it increased by hyperglycaemic sera. These results were phenocopied in endothelial cells cultured in a medium containing high concentrations of glucose, which activates inducible nitric oxide synthase with a consequent increase of nitric oxide. Inhibition of the enzyme prevented high glucose-induced hyperpermeability, thus pointing to nitric oxide as the mediator involved in altering the endothelial barrier function. Since nitric oxide is much higher in sera from hyperglycaemic than normoglycaemic T1D patients, and the inhibition of inducible nitric oxide synthase prevents sera-dependent increased endothelial permeability, this enzyme might represent a promising biochemical marker to be monitored in T1D patients to predict alterations of the vascular wall, eventually promoting intimal lipid accumulation.

Ultrastructural features mirror metabolic derangement in human endothelial cells exposed to high glucose.

High glucose-induced endothelial dysfunction is the early event that initiates diabetes-induced vascular disease. Here we employed Cryo Soft X-ray Tomography to obtain three-dimensional maps of high D-glucose-treated endothelial cells and their controls at nanometric spatial resolution. We then correlated ultrastructural differences with metabolic rewiring. While the total mitochondrial mass does not change, high D-glucose promotes mitochondrial fragmentation, as confirmed by the modulation of fission-fusion markers, and dysfunction, as demonstrated by the drop of membrane potential, the decreased oxygen consumption and the increased production of reactive oxygen species. The 3D ultrastructural analysis also indicates the accumulation of lipid droplets in cells cultured in high D-glucose. Indeed, because of the decrease of fatty acid ß-oxidation induced by high D-glucose concentration, triglycerides are esterified into fatty acids and then stored into lipid droplets. We propose that the increase of lipid droplets represents an adaptive mechanism to cope with the overload of glucose and associated oxidative stress and metabolic dysregulation.

A Potential Interplay between HDLs and Adiponectin in Promoting Endothelial Dysfunction in Obesity.

Obesity is an epidemic public health problem that has progressively worsened in recent decades and is associated with low-grade chronic inflammation (LGCI) in metabolic tissues and an increased risk of several diseases. In particular, LGCI alters metabolism and increases cardiovascular risk by impairing endothelial function and altering the functions of adiponectin and high-density lipoproteins (HDLs). Adiponectin is an adipokine involved in regulating energy metabolism and body composition. Serum adiponectin levels are reduced in obese individuals and negatively correlate with chronic sub-clinical inflammatory markers. HDLs are a heterogeneous and complex class of lipoproteins that can be dysfunctional in obesity. Adiponectin and HDLs are strictly interdependent, and the maintenance of their interplay is essential for vascular function. Since such a complex network of interactions is still overlooked in clinical settings, this review aims to highlight the mechanisms involved in the impairment of the HDLs/adiponectin axis in obese patients to predict the risk of cardiovascular diseases and activate preventive countermeasures. Here, we provide a narrative review of the role of LGCI in altering HDLs, adiponectin and endothelial functions in obesity to encourage new studies about their synergic effects on cardiovascular health and disease.

Vitamin D Prevents High Glucose-Induced Lipid Droplets Accumulation in Cultured Endothelial Cells: The Role of Thioredoxin Interacting Protein.

Vitamin D (VitD) exerts protective effects on the endothelium, which is fundamental for vascular integrity, partly by inhibiting free radical formation. We found that VitD prevents high glucose-induced Thioredoxin Interacting Protein (TXNIP) upregulation. Increased amounts of TXNIP are responsible for the accumulation of reactive oxygen species and, as a consequence, of lipid droplets. This is associated with increased amounts of triglycerides as the result of increased lipogenesis and reduced fatty acid oxidation. Remarkably, VitD rebalances the redox equilibrium, restores normal lipid content, and prevents the accumulation of lipid droplets. Our results highlight TXNIP as one of the targets of VitD in high glucose-cultured endothelial cells and shed some light on the protective effect of VitD on the endothelium.

Unveiling the basis of antiretroviral therapy-induced osteopenia: the effects of Dolutegravir, Darunavir and Atazanavir on osteogenesis.

OBJECTIVES: Osteopenia is frequent in HIV-infected patients treated with antiretroviral therapy (ART) and has been linked to increased osteoclastogenesis. Little is known about the effects of ART on osteogenesis. DESIGN: We investigated the effect on human mesenchymal stem cells (hMSC) and osteoblasts of Darunavir and Dolutegravir, the most highly used as anchor drugs within a three-drug regimen, and Atazanavir, which was widely utilized in the past. RESULTS: We found that Atazanavir and Dolutegravir delay the osteogenic differentiation of hMSC, impair the activity of osteoblasts and inhibit their conversion into osteocytes, whereas Darunavir exerts no effect. CONCLUSION: Atazanavir and Dolutegravir impair osteogenesis. It is essential to diagnose impaired osteogenesis early and to devise effective therapeutic interventions to preserve bone health in ART-treated HIV patients, putting it in the context of a correct antiretroviral combination.

Reactive oxygen species are implicated in altering magnesium homeostasis in endothelial cells exposed to high glucose.

Transient Receptor Potential Melastatin (TRPM)7 is important in maintaining the intracellular homeostasis of magnesium (Mg), which is instrumental for vital cellular functions. Since the upregulation of TRPM7 has been proposed as a marker of endothelial dysfunction, we evaluated the effects of high glucose, which markedly impacts endothelial performance, on TRPM7 and intracellular Mg homeostasis in human macrovascular endothelial cells. We show that glucose-induced free radicals increase the amounts of TRPM7 as well as total intracellular magnesium. On the contrary, the highly selective Mg transporter MagT1 is not modulated by high glucose, hydrogen peroxide and low extracellular magnesium. We conclude that in endothelial cells high glucose alters Mg homeostasis through the upregulation of TRPM7.