Glucose and Vascular Inflammation

Background: Endothelial cells form a highly dynamic barrier between blood and tissues and can be activated in response to angiogenic and inflammatory stimuli. Endothelial cells rely on glycolysis for ATP production, and it was recently demonstrated that inhibition of endothelial glycolysis attenuates angiogenic activation. We wanted to examine the metabolic profile of inflammatory activated endothelial cells and explore the role of glycolysis in endothelial inflammatory activation. Methods: Human umbilical vein endothelial cells were stimulated with IL-1β and the rates of glucose uptake, glycolysis and fatty acid oxidation were determined using radioactively labelled substrates. The expression of the metabolic enzymes PFKFB3 and CPT1A were determined by qPCR and confirmed on the protein level by immunoblotting. Glycolysis was partially inhibited using 3PO, a small molecule inhibitor of PFKFB3, and IL-1β induced adhesion molecule expression was assessed using cell ELISA and qPCR. Activation of NFκB was assessed using a NFκB luciferase assay kit and immunoblotting against phosphorylated IκBα, IKKα/β and JNK following stimulation with IL-1β and TNF. The levels of pan-O-GlcNAcylation in whole cell lysates were determined by immunoblotting. Results: The endothelial glucose uptake was increased by approximately 60% after stimulation with IL-1β (P<0.001), correlating with a steady increase in the rate of glycolysis and a decrease in fatty acid oxidation. The increase of glycolysis was associated with increased expression of the glycolytic enzyme PFKFB3, while CPT1A, the rate limiting enzyme of fatty acid oxidation was not significantly affected by inflammatory activation. Inhibition of glycolysis attenuated endothelial upregulation of the adhesion molecules E-selectin and VCAM-1, correlating with reduced phosphorylation of key signalling molecules in both the NFκB and the JNK pathways. Conclusion: We have shown that endothelial cells upregulate their rate of glycolysis in response to IL-1β stimulation and that this correlates with an increased expression of the rate limiting glycolytic enzyme PFKFB3. Pharmacological inhibition of glycolysis reduced the upregulation of adhesion molecules involved in in leukocyte recruitment by attenuating activation of inflammatory signalling cascades, raising the question whether PFKFB3 could be a potential therapeutic target in inflammatory disease. The exact molecular mechanisms by which 3PO inhibits phosphorylation of signalling molecules has not yet been determined and will be targeted in future experiments

Metabolic characterization of endothelial and CD4+ T cells in immune activation

Inflammation is a part of the immune response towards harmful stimuli, including infection or damage. Inflammation is characterized by activation of several signaling pathways, and recently by a range of metabolic adaptations. The primary aim of this thesis was to study the how metabolism in endothelial and T helper cells (Th) is changed upon immune activation. Measuring uptake and metabolism of radiolabeled glucose revealed that interleukin-1β (IL-1 β)-stimulation increased glucose uptake and combustion despite decreasing proliferation in endothelial cells. This was correlated with increased extracellular acidification and expression of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3). Despite this, lactic acid secretion was unaffected. As oxygen consumption was increased, this indicates that IL-1β-stimulation increases mitochondrial pyruvate metabolism. Notably, donor heterogeneity was observed, which may be important for therapeutic efficacy of metabolic inhibitors. It was further revealed that the PFKFB3 inhibitor 3PO reduced IL-1β-induced activation of inflammatory signaling pathways and expression of adhesion molecules in endothelial cells. However, this effect was not reproduced by alternative methods of inhibiting PFKFB3, indicating that this effect was not mediated by PFKFB3 inhibition. Lastly, Th-cell proliferation and viability was shown to require the presence of glutamine in both normoxia and hypoxia. It was demonstrated by mass spectrometry that depletion of glutamine or blocking glutamine metabolism was shown to cause depletion of several endogenous amino acids, as well as reduce oxygen consumption. Blocking glutamine metabolism also resulted in reduced rate of glycolysis, mediated by reduced activity of hexokinase. Overall, these results show that metabolic reprograming is crucial for the immune response, indicating that metabolism may offer a novel strategy for targeting pathological inflammation

Identification and characterization of a novel glutaminase inhibitor

In humans, there are two forms of glutaminase (GLS), designated GLS1 and GLS2. These enzymes catalyse the conversion of glutamine to glutamate. GLS1 exists as two isozymes: kidney glutaminase (KGA) and glutaminase C (GAC). Several GLS inhibitors have been identified, of which DON (6-diazo-5-oxonorleucine), BPTES (bis-2-(5-phenylacetamido-1, 3, 4-thiadiazol-2-yl) ethyl sulphide), 968 (5-(3-Bromo-4-(dimethylamino)phenyl)-2,2-dimethyl-2,3,5,6-tetrahydrobenzo[a]phenanthridin-4(1H)-one) and CB839 (Telaglenastat) are the most widely used. However, these inhibitors have variable efficacy, specificity and bioavailability in research and clinical settings, implying the need for novel and improved GLS inhibitors. Based on this need, a diverse library of 28,000 compounds from Enamine was screened for inhibition of recombinant, purified GAC. From this library, one inhibitor designated compound 19 (C19) was identified with kinetic features revealing allosteric inhibition of GAC in the µm range. Moreover, C19 inhibits anti-CD3/CD28-induced CD4+ T-cell proliferation and cytokine production with similar or greater potency as compared to BPTES. Taken together, our data suggest that C19 has the potential to modulate GLS1 activity and alter metabolic activity of T cells

Inflammatory activation of endothelial cells increases glycolysis and oxygen consumption despite inhibiting cell proliferation

Endothelial cell function and metabolism are closely linked to differential use of energy substrate sources and combustion. While endothelial cell migration is promoted by 2-phosphofructokinase-6/fructose-2,6-bisphosphatase (PFKFB3)-driven glycolysis, proliferation also depends on fatty acid oxidation for dNTP synthesis. We show that inflammatory activation of human umbilical vein endothelial cells (HUVECs) by interleukin-1β (IL-1β), despite inhibiting proliferation, promotes a shift toward more metabolically active phenotype. This was reflected in increased cellular glucose uptake and consumption, which was preceded by an increase in PFKFB3 mRNA and protein expression. However, despite a modest increase in extracellular acidification rates, the increase in glycolysis did not correlate with extracellular lactate accumulation. Accordingly, IL-1β stimulation also increased oxygen consumption rate, but without a concomitant rise in fatty acid oxidation. Together, this suggests that the IL-1β-stimulated energy shift is driven by shunting of glucose-derived pyruvate into mitochondria to maintain elevated oxygen consumption in HUVECs. We also revealed a marked donor-dependent variation in the amplitude of the metabolic response to IL-1β and postulate that the donor-specific response should be taken into account when considering targeting dysregulated endothelial cell metabolism

3PO inhibits inflammatory NFκB and stress-activated kinase signaling in primary human endothelial cells independently of its target PFKFB3

Inhibition of the key glycolytic activator 6-phosphofructokinase 2/fructose-2,6-bisphosphatase-3 (PFKFB3) by 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO) strongly attenuates pathological angiogenesis in cancer and inflammation. In addition to modulating endothelial proliferation and migration, 3PO also dampens proinflammatory activation of endothelial cells and experimental inflammation in vivo, suggesting a potential for 3PO in the treatment of chronic inflammation. The aim of our study was to explore if the anti-inflammatory action of 3PO in human endothelial cells was mediated by inhibition of PFKFB3 and glycolysis and assess if other means of PFKFB3 inhibition reduced inflammatory activation in a similar manner. We found that 3PO caused a rapid and transient reduction in IL-1β- and TNF-induced phosphorylation of both IKKα/β and JNK, thus inhibiting signaling through the NFκB and the stress-activated kinase pathways. However, in contrast to 3PO-treatment, neither shRNA-mediated silencing of PFKFB3 nor treatment with the alternative PFKFB3 inhibitor 7,8-dihydroxy-3-(4-hydroxy-phenyl)-chromen-4-one (YN1) prevented cytokine-induced NFκB signaling and upregulation of the adhesion molecules VCAM-1 and E-selectin, implying off target effects of 3PO. Collectively, our results suggest that the anti-inflammatory action of 3PO in human endothelial cells is not limited to inhibition of PFKFB3 and cellular glycolysis

Cellular metabolism dictates T cell effector function in health and disease

In a healthy person, metabolically quiescent T lymphocytes (T cells) circulate between lymph nodes and peripheral tissues in search of antigens. Upon infection, some T cells will encounter cognate antigens followed by proliferation and clonal expansion in a context‐dependent manner, to become effector T cells. These events are accompanied by changes in cellular metabolism, known as metabolic reprogramming. The magnitude and variation of metabolic reprogramming are, in addition to antigens, dependent on factors such as nutrients and oxygen to ensure host survival during various diseases. Herein, we describe how metabolic programmes define T cell subset identity and effector functions. In addition, we will discuss how metabolic programs can be modulated and affect T cell activity in health and disease using cancer and autoimmunity as examples

Antibody blockade of Jagged1 attenuates choroidal neovascularization

Abstract Antibody-based blocking of vascular endothelial growth factor (VEGF) reduces choroidal neovascularization (CNV) and retinal edema, rescuing vision in patients with neovascular age-related macular degeneration (nAMD). However, poor response and resistance to anti-VEGF treatment occurs. We report that targeting the Notch ligand Jagged1 by a monoclonal antibody reduces neovascular lesion size, number of activated phagocytes and inflammatory markers and vascular leakage in an experimental CNV mouse model. Additionally, we demonstrate that Jagged1 is expressed in mouse and human eyes, and that Jagged1 expression is independent of VEGF signaling in human endothelial cells. When anti-Jagged1 was combined with anti-VEGF in mice, the decrease in lesion size exceeded that of either antibody alone. The therapeutic effect was solely dependent on blocking, as engineering antibodies to abolish effector functions did not impair the therapeutic effect. Targeting of Jagged1 alone or in combination with anti-VEGF may thus be an attractive strategy to attenuate CNV-bearing diseases