High Glucose, But Not Testosterone, Increases Platelet Aggregation Mediated by Endothelial Cells

Endothelial cells inhibit platelet aggregation by releasing thromboregulators, such as prostacyclin and nitric oxide. Male subject is a traditional risk factor for cardiovascular diseases. Platelet hyperreactivity has been frequently found in patient with diabetes mellitus. To examine whether testosterone and high glucose modify platelet aggregation through endothelial cells, we did an in vitro study using endothelial cells culture from human umbilical vein (HUVEC). Treatments were performed in HUVEC sub culture as either normoglucose (5.6 mM) or high glucose (22.4 mM) medium, with or without testosterone (0, 1, 10, 100 nM), for 24 hours. HUVEC were trypsinized, resuspended, and then incubated with platelet rich plasma from healthy male donors with ratio 1:104 for 3 minutes. Platelet aggregation measured by turbidimetry methode. This study showed that testosterone did not significantly influence platelet aggregation through endothelial cells in normoglucose (p = 0.144) or high glucose (p = 0.916) medium. There was no main effect of testosterone (p = 0.73) as well as no interaction between testosterone and glucose (p = 0.69), but there was a main effect of glucose (p = 0.004), to platelet aggregation through endothelial cells. In conclusion, high glucose, but not testosterone, inhibits platelet aggregation mediated by endothelial cells

Isolation of equine endothelial cells and life cell angiogenesis assay

Arterial or venous thromboses are frequent clinical complications with the risk of fatal progression. Recent studies suggest the disruption of angiogenesis in the course of thrombus resolution as the underlying pathomechanism. Very similar to the situation in human patients, equine vessels have been described to be particularly susceptible to thrombosis. In contrast to humans, equine donors are readily available to obtain organs and tissues for isolation of endothelial cells. Objective of this study was to isolate equine endothelial cells and develop an angiogenesis assay from primary cultures. Macrovascular endothelial cells were obtained from jugular veins and carotid arteries of nine horses, one of which suffered from inflammatory processes. After enzymatic isolation, the cells were incubated in different selective primary media. Phenotypic identification of endothelial cells was accomplished by morphology and positive staining to von Willebrand factor. The reliable, inexpensive, and standardized combination of methods presented here resulted in pure endothelial cultures for angiogenesis assays that can be used in any cell culture laboratory. Inverted phase microscopy and life cell imaging was used to characterize the stages of the angiogenic cascade of the endothelial cells. Life cell imaging gave new insights into the in vitro formation of capillary like structures including exocytosis of microparticles from endothelial cells before integration into the three-dimensional structure. We hypothesize that a specific population of endothelial cells showing a highly active migration pattern in life cell imaging might play a role in the resolution of thrombosis

Surface modification of hydrophobic polymers for improvement of endothelial cell-surface interactions

The aim of this study is to improve the interaction of endothelial cells with polymers used in vascular prostheses. Polytetrafluoroethylene (PTFE; Teflon) films were treated by means of nitrogen and oxygen plasmas. Depending on the plasma exposure time, modified PTFE surfaces showed water-contact angles of 15¿58° versus 96° for unmodified PTFE. Electron spectroscopy in chemical analysis (ESCA) measurements revealed incorporation of both nitrogenand oxygen-containing groups into the PTFE surfaces, dependent on the plasma composition and exposure time. In-vitro biological evaluation of unmodified and modified PTFE surfaces showed that human endothelial cells, seeded from 20% human serum-containing culture medium, adhered well on to modified PTFE surfaces, but not on to unmodified films. Adhesion of endothelial cells on to expanded PTFE graft material (Gore-Tex) was also stimulated by plasma treatment of this substrate. On plasma-treated expanded PTFE, the adhering endothelial cells formed a monolayer, which covered the textured surface. The latter observation is important in view of the hemocompatibility of vascular grafts seeded with endothelial cells before implantation

Layered Long Term Co-Culture of Hepatocytes and Endothelial Cells on a Transwell Membrane: Toward Engineering the Liver Sinusoid

This paper presents a novel liver model that mimics the liver sinusoid where most liver activities occur. A key aspect of our current liver model is a layered co-culture of primary rat hepatocytes (PRHs) and primary rat liver sinusoidal endothelial cells (LSECs) or bovine aortic endothelial cells (BAECs) on a transwell membrane. When a layered co-culture was attempted with a thin matrigel layer placed between hepatocytes and endothelial cells to mimic the Space of Disse, the cells did not form completely separated monolayers. However, when hepatocytes and endothelial cells were cultured on the opposite sides of a transwell membrane, PRHs co-cultured with LSECs or BAECs maintained their viability and normal morphology for 39 and 57 days, respectively. We assessed the presence of hepatocyte-specific differentiation markers to verify that PRHs remained differentiated in the long-term co-culture and analyzed hepatocyte function by monitoring urea synthesis. We also noted that the expression of cytochrome P-450 remained similar in the cocultured system from Day 13 to Day 48. Thus, our novel liver model system demonstrated that primary hepatocytes can be cultured for extended times and retain their hepatocyte-specific functions when layered with endothelial cells

Lactate Oxidation in Endothelial Cells: A Feature of All Endothelial Cells?

Resumen de la comunicaciónMetabolism of endothelial cells is a topic that has gained an increasing interest in the last years. This is due to their role in the angiogenic process, which is pathologically upregulated in several diseases, such as retinopathies, diabetes and cancer. Glycolysis, among other metabolic routes, has been found to be essential for triggering the angiogenic switch. Additionally, it has been seen that endothelial cells are able to take up lactate from the extracellular media, for example in the case of the tumor microenvironment, where cancer cells would have secreted high amounts of this metabolite. Endothelial cells would oxidize this lactate for obtaining energy, but lactate can also act as a signaling molecule for the angiogenic process. However, experiments to determine the molecular fate of lactate have been performed using only macrovascular endothelial cells. The aim of the present work is to prove whether microvascular endothelial cells are also able to take up and oxidize lactate. For this purpose, fluorimetry, isotopic labeling and Seahorse experiments were used to study the metabolism of a human microvascular endothelial cell line (HMEC). The expression levels of transcripts and proteins of different enzymes and transporters related to lactate metabolism were estimated by qPCR and Western blotting. The results obtained indicate that these cells rely on glycolysis for their metabolism, while the oxidation of glucose and glutamine seems to be considerably low. On the other hand, no lactate oxidation could be detected. We then checked the mRNA expression of the two isoenzymes of lactate dehydrogenase (LDH) and the two main lactate transporters, MCT1 and MCT4, and found that levels of LDH-B and MCT1 were undetectable. We failed to measure any MCT1 mRNA or protein expression either in normoxia or hypoxia. Hence, we can conclude that at least this microvascular endothelial cell line cannot use extracellular lactate as a metabolic fuel.Our experimental work is supported by grants BIO2014-56092-R (MINECO and FEDER) and P12-CTS-1507 (Andalusian Government and FEDER) and funds from group BIO-267 (Andalusian Government). The "CIBER de Enfermedades Raras" is an initiative from the ISCIII (Spain). This communicaction has the support of a travel grant "Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech"

TSPO ligands promote cholesterol efflux and suppress oxidative stress and inflammation in choroidal endothelial cells

Choroidal endothelial cells supply oxygen and nutrients to retinal pigment epithelial (RPE) cells and photoreceptors, recycle metabolites, and dispose of metabolic waste through the choroidal blood circulation. Death of the endothelial cells of the choroid may cause abnormal deposits including unesterified and esterified cholesterol beneath RPE cells and within Bruch’s membrane that contribute to the progression of age-related macular degeneration (AMD), the most prevalent cause of blindness in older people. Translocator protein (TSPO) is a cholesterol-binding protein that is involved in mitochondrial cholesterol transport and other cellular functions. We have investigated the role of TSPO in choroidal endothelial cells. Immunocytochemistry showed that TSPO was localized to the mitochondria of choroidal endothelial cells. Choroidal endothelial cells exposed to TSPO ligands (Etifoxine or XBD-173) had significantly increased cholesterol efflux, higher expression of cholesterol homeostasis genes (LXRα, CYP27A1, CYP46A1, ABCA1 and ABCG1), and reduced biosynthesis of cholesterol and phospholipids from [14C]acetate, when compared to untreated controls. Treatment with TSPO ligands also resulted in reduced production of reactive oxygen species (ROS), increased antioxidant capacity, and reduced release of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α and VEGF) induced by oxidized LDL. These data suggest TSPO ligands may offer promise for the treatment of AMD

Profiling the mouse brain endothelial transcriptome in health and disease models reveals a core blood-brain barrier dysfunction module.

Blood vessels in the CNS form a specialized and critical structure, the blood-brain barrier (BBB). We present a resource to understand the molecular mechanisms that regulate BBB function in health and dysfunction during disease. Using endothelial cell enrichment and RNA sequencing, we analyzed the gene expression of endothelial cells in mice, comparing brain endothelial cells with peripheral endothelial cells. We also assessed the regulation of CNS endothelial gene expression in models of stroke, multiple sclerosis, traumatic brain injury and seizure, each having profound BBB disruption. We found that although each is caused by a distinct trigger, they exhibit strikingly similar endothelial gene expression changes during BBB disruption, comprising a core BBB dysfunction module that shifts the CNS endothelial cells into a peripheral endothelial cell-like state. The identification of a common pathway for BBB dysfunction suggests that targeting therapeutic agents to limit it may be effective across multiple neurological disorders

Crucial role of local peroxynitrite formation in neutrophil-induced endothelial cell activation

Introduction and methods: The reaction of superoxide anions and NO not only results in a decreased availability of NO, but also leads to the formation of peroxynitrite, the role of which in the cardiovascular system is still discussed controversially. In cultured human endothelial cells, we studied whether there is a significant interaction between endothelial NO and neutrophil-derived superoxide anions in terms of endothelial peroxynitrite formation. We particularly studied whether a significantly higher redox-stress can be found in those endothelial cells directly adjacent to an activated neutrophil. Results: A considerable part of the 2,7-dihydrodichlorofluoresceine signal in endothelial cells was due to oxidation by peroxynitrite. Providing superoxide radicals by enzymatic source or by the neutrophil respiratory burst increased the fluorescence, which was attenuated by blockade of endothelial NO-synthase, suggesting that peroxynitrite was formed from neutrophil- or extracellular enzyme-derived superoxide and endothelial NO. Considerably higher fluorescence intensity was observed in endothelial cells in direct neighborhood to a neutrophil. This was particularly pronounced in the presence of a NO-donor and was accompanied by a strong activation of NF-κB and increased expression of E-selectin in these cells. Conclusion: Endothelial cells adjacent to neutrophils may have elevated levels of peroxynitrite that result in an increased expression of adhesion molecules. Such cells might represent a preferential site for adhesion and migration of additional neutrophils when simultaneously high concentrations of NO and neutrophil-derived superoxide are present