Endothelial Cell Metabolism Research Articles

Abeta peptides disrupt the barrier integrity and glucose metabolism of human induced pluripotent stem cell-derived brain microvascular endothelial cells

Amyloid β (Aβ) peptides are key components of Alzheimer’s disease and cerebral amyloid angiopathy and have been associated with detrimental effects at the blood-brain barrier (BBB) in vivo. Yet, the cellular and molecular mechanisms by which such peptides exert their effect on the brain vasculature remain unclear. This study aimed to assess the cellular response of induced pluripotent stem cell (iPSC)-derived brain microvascular endothelial cells (BMECs) to Aβ peptides. Changes in the barrier function, efflux transporters activity, glucose uptake, and metabolism were assessed in such model. Although iPSC-derived BMECs sustained prolonged exposure (<72 h) to a high level of Aβ peptides including Aβ42, such cells also suffered from a loss of barrier integrity, coupled with reduced glucose uptake and impaired bioenergetic activity. Taken together, this study shows the ability of iPSC-derived BMECs to reproduce features observed in other models and suggests that Aβ peptides may compromise the BBB via different targets.

Gap Junction–Mediated Transport of Metabolites Between Stem Cells and Vascular Endothelial Cells

We have previously demonstrated that small molecular transfer, such as glucose, between hematopoietic stem cells (HSCs) or mesenchymal stem cells (MSCs) and vascular endothelial cells via gap junctions constitutes an important mechanism of stem cell therapy. Cell metabolites are high-potential small-molecule candidates that can be transferred to small molecules between stem cells and vascular endothelial cells. Here, we investigated the differences in metabolite levels between stem cells (HSCs and MSCs), vascular endothelial cells, and the levels of circulating non-hematopoietic white blood cells (WBCs). The results showed remarkable differences in metabolite concentrations between cells. Significantly higher concentrations of adenosine triphosphate (ATP), guanosine triphosphate (GTP), total adenylate or guanylate levels, glycolytic intermediates, and amino acids were found in HSCs compared with vascular endothelial cells. In contrast, there was no significant difference in the metabolism of MSCs and vascular endothelial cells. From the results of this study, it became clear that HSCs and MSCs differ in their metabolites. That is, metabolites that transfer between stem cells and vascular endothelial cells differ between HSCs and MSCs. HSCs may donate various metabolites, several glycolytic and tricarboxylic acid cycle metabolites, and amino acids to damaged vascular endothelial cells as energy sources and activate the energy metabolism of vascular endothelial cells. In contrast, MSCs and vascular endothelial cells regulate each other under normal conditions. As the existing MSCs cannot ameliorate the dysregulation during insult, exogenous MSCs administered by cell therapy may help restore normal metabolic function in the vascular endothelial cells by taking up excess energy sources from the lumens of blood vessels. Results of this study suggested that the appropriate timing of cell therapy is different between HSCs and MSCs.

Liver Endothelial Cells Induce Hepatocellular Carcinoma via HGF Secretion

Introduction: Liver sinusoidal endothelial cells (LSEC) control organ functions, metabolism and development. LSEC express hepatocyte growth factor (HGF), which is involved in prenatal development, metabolic homeostasis and liver regeneration. This study aimed to elucidate the precise contribution of LSEC-derived HGF in liver cancer. Method: Stab2-iCretg/wt;Hgffl/fl (HgfΔLSEC) mice and Hgffl/fl control mice were used to investigate the role of HGF in HCC development. Mice were injected with DEN to induce liver cancer. Liver changes in DEN-treated mice closely resemble chronic liver disease in humans, up to progression to HCC. After 12 months, macroscopic cancer development was assessed by magnetic resonance imaging (MRI). In addition, histopathologic and molecular examination of liver specimens was performed. Results: Magnetic resonance imaging and histologic evaluation showed significantly fewer malignant lesions in the HgfΔLSEC mice. Additionally, these lesions were found to be significantly smaller. On average, the tumors of Hgffl/fl were 1.5 cm and those of HgfΔLSEC were only 0.5cm in diameter. Further histological workup revealed a higher grade of proliferation in HgfΔLSEC mice compared to wildtypes. This was further confirmed by analysis for DNA damage pathways. Conclusion: In summary, our results indicate that LSECs may play a central role in HCC development through the production of HGF. Loss of HGF production in LSECs reduced the number of tumors as well as the size and progression of tumors. Further evaluation is needed to elucidate the complete role of LSECs in liver cancer development to find new therapeutic targets.

Oxidative pentose phosphate pathway controls vascular mural cell coverage by regulating extracellular matrix composition

Vascular mural cells (vMC) play an essential role in the development and maturation of the vasculature by promoting vessel stabilization through their interactions with endothelial cells (EC). Whether endothelial metabolism influences mural cell recruitment and differentiation is unknown. Here, we show that the oxidative Pentose Phosphate Pathway (oxPPP) in EC is required for establishing vMC coverage of the dorsal aorta (DA) during early vertebrate development in zebrafish and mice. We demonstrate that laminar shear stress (LSS) and blood flow maintain oxPPP activity, which, in turn, promotes Elastin (Eln) expression in blood vessels through production of ribose-5-phosphate (R5P). Eln is both necessary and sufficient to drive vMC recruitment and maintenance when the oxPPP is active. In summary, our work demonstrates that endothelial cell metabolism regulates blood vessel maturation by controlling vascular matrix composition and vMC recruitment.

Redox balance in human skeletal muscle-derived endothelial cells - Effect of exercise training

Aerobic training can improve vascular endothelial function in-vivo. The aim of this study was to elucidate the mechanisms underlying this improvement in isolated human microvascular endothelial cells. Sedentary males, aged 57 ± 6 years completed 8 weeks of intense aerobic training. Resting muscle biopsies were obtained from the thigh muscle and used for isolation of endothelial cells (pre n = 23, post n = 16). The cells were analyzed for mitochondrial respiration, H2O2 emission, glycolysis, protein levels of antioxidants, NADPH oxidase, endothelial nitric oxide (NO) synthase and prostacyclin synthase (PGI2S). In-vivo microvascular function, assessed by acetylcholine infusion and arterial blood pressure were also determined. Endothelial mitochondrial respiration and H2O2 formation were similar before and after training whereas the expression of superoxide dismutase and the expression of glutathione peroxidase were 2.4-fold (p = 0.012) and 2.3–fold (p = 0.006) higher, respectively, after training. In-vivo microvascular function was increased by 1.4-fold (p = 0.036) in parallel with a 2.1-fold increase in endothelial PGI2S expression (p = 0.041). Endothelial cell glycolysis was reduced after training, as indicated by a 65% lower basal production of lactate (p = 0.003) and a 30% lower expression of phosphofructokinase (p = 0.011). Subdivision of the participants according to blood pressure at base-line (n = 23), revealed a 2-fold higher (p = 0.049) rate of H2O2 production in endothelial cells from hypertensive participants. Our data show that exercise training increases skeletal muscle microvascular endothelial cell metabolism, antioxidant capacity and the capacity to form prostacyclin. Moreover, elevated blood pressure is associated with increased endothelial mitochondrial ROS formation.

CD63+ and MHC Class I+ Subsets of Extracellular Vesicles Produced by Wild-Type and CD47-Deficient Jurkat T Cells Have Divergent Functional Effects on Endothelial Cell Gene Expression.

T cells and endothelial cells engage in bidirectional communication that regulates angiogenesis and T cell transmigration. Extracellular vesicles (EVs) mediate intercellular communication by the transfer of bioactive molecules including RNAs. EVs produced by a given cell type are heterogeneous in their RNA content, but it is unclear how specific EV surface markers relate to their functional effects on target cells. Our previous work established that Jurkat T cell EVs bearing CD63, MHC-I, or CD47 surface markers contain distinct noncoding RNA populations. The present study reveals that CD63+ and MHC-I+ EVs from CD47-deficient Jurkat T cells are enriched in small non-coding RNAs relative to EVs from wild-type Jurkat T cells. CD47-deficient Jurkat T cells secrete more CD63+ and MHC-I+ EVs, but MHC-I+ EVs are selectively taken up more by human umbilical vein endothelial cells. Transcriptomics analysis of endothelial cells treated with CD63+ or MHC-I+ EVs showed surface marker- and CD47-dependent changes in gene expression in the target cells. Gene set enrichment analysis identified CD47-dependent, and surface marker-dependent effects of T cell EVs on VEGF and inflammatory signaling, cell cycle, and lipid and cholesterol metabolism. Thus, subsets of T cell EVs differentially regulate endothelial cell metabolism and inflammatory and angiogenic responses.

Abstract 9817: Krüppel-Like Factor 4 Integrates Immunometabolism of Human Arterial Endothelial Cells

Introduction: Inflammation and metabolic alterations are two common hallmarks of cardiovascular diseases. Yet, the molecular mechanisms integrating inflammation and cell metabolism remain elusive. Objective: We investigated whether Krüppel-like factor 4 (KLF4) serves as a link between cellular glucose metabolism and inflammation in human arterial endothelial cells. Methods and Results: Primary human pulmonary artery and aortic endothelial cells (PAEC and AoEC, respectively) were stimulated with tumor necrosis factor α (TNFα) or lipopolysaccharide (LPS). Results from fluorescent microscopy and Seahorse extracellular flux analysis revealed that TNFα and LPS enhanced glucose uptake by 2.2-fold, glycolysis by 2.3-fold, and mitochondrial respiration by 1.6-fold in PAEC and AoEC. The increase in glycolysis correlated with a 2-4-fold upregulation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) and PFKFB4, two key regulatory enzymes of glycolysis. Silencing PFKFB3 but not PFKFB4 entirely prevented TNFα-induced enhancement of glycolysis with a concomitant metabolic shift to mitochondrial respiration. Mechanistically, TNFα and LPS stimulated KLF4 mRNA and protein expression (2-4-fold), which functions as a transcriptional repressor of PFKFB3 . KLF4 knockdown potentiated TNFα-induced stimulation of PFKFB3 expression by 1.6-fold and glycolysis by 2.2-fold, effects which were completely prevented by co-silencing of PFKFB3 . Functionally, KLF4 silencing exacerbated TNFα-induced vascular cell adhesion molecule 1 (VCAM1) expression (by 2-3-fold), E-selectin secretion (by 2.8-fold), and monocyte adhesion (by 1.6-fold) in PAEC, all of which were attenuated by over 50% when PFKFB3 was inhibited by siRNA or with 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3-PO). By contrast, KLF4 overexpression-mediated inhibition of inflammation was significantly counteracted in PAEC with co-overexpression of PFKFB3 . Conclusion: These results indicate that KLF4 inhibits inflammation by normalizing PFKFB3-mediated enhancement of glycolysis and that the KLF4-PFKFB3 signaling axis integrates immunometabolism in human arterial endothelial cells.

Abstract 10099: Loss of CD70 Impairs Nitric Oxide Homeostasis and Augments Intracellular Oxidative Stress in Endothelial Cells

Introduction: TNF superfamily ligands exert multiple effects on endothelial cell (EC) function, metabolism, and redox status. CD70 is a TNF superfamily member classically expressed on antigen-presenting cells and drives T memory cell polarization. Despite evidence of expression by ECs, the role of CD70 in endothelial biology has not been defined. We hypothesized that CD70 affects EC function through regulation of nitric oxide (NO) generation and cellular oxidative stress. Methods: Human aortic and pulmonary artery ECs (HAECs and HPAECs, respectively) were transfected with control and CD70-directed siRNA (siCD70). NO levels of individual cells were monitored in real-time after stimulation with ATP using the intracellular fluorescent biosensor c-geNOp. Similarly, intracellular hydrogen peroxide was assayed in real-time using the intracellular fluorescent biosensor Hyper7 following stimulation by auranofin and histamine. For both studies, 70-100 individual cells, averaged over 4 experiments, were analyzed using confocal microscopy. Control and siCD70-treated cell lysates (n = 4-6) were analyzed by Western blot and quantitative RT-PCR, as well as for glutathione peroxidase (GPx) enzyme activity. Results: CD70 knockdown was associated with a reduction in agonist-stimulated NO levels in HAECs and HPAECs by 30% and 40%, respectively. Protein levels of eNOS decreased after CD70 knockdown, with a reduction of 50% in HAECs and 75% in HPAECs. Phosphorylation at Ser1177 of eNOS in response to EC stimulation with ATP was reduced after siCD70 treatment, and expression of the key eNOS-stimulatory protein Hsp90 was decreased. Intracellular hydrogen peroxide was significantly increased (20% in HAECs and 80% in HPAECs) following ROS stimulation in siCD70-treated versus control cells. This change in redox status was associated with a decrease in mitochondrial superoxide dismutase protein and an increase in GPx-1 expression and enzyme activity. Conclusions: Loss of CD70 in ECs is associated with reduced eNOS expression and bioactivity as well as increased susceptibility to intracellular oxidative stress, potentially from a mitochondrial source. These results suggest an important homeostatic role for CD70 in endothelial function and redox status.

CRIF1 Deficiency Increased Homocysteine Production by Disrupting Dihydrofolate Reductase Expression in Vascular Endothelial Cells.

Elevated plasma homocysteine levels can induce vascular endothelial dysfunction; however, the mechanisms regulating homocysteine metabolism in impaired endothelial cells are currently unclear. In this study, we deleted the essential mitoribosomal gene CR6 interacting factor 1 (CRIF1) in human umbilical vein endothelial cells (HUVECs) and mice to induce endothelial cell dysfunction; then, we monitored homocysteine accumulation. We found that CRIF1 downregulation caused significant increases in intracellular and plasma concentrations of homocysteine, which were associated with decreased levels of folate cycle intermediates such as 5-methyltetrahydrofolate (MTHF) and tetrahydrofolate (THF). Moreover, dihydrofolate reductase (DHFR), a key enzyme in folate-mediated metabolism, exhibited impaired activity and decreased protein expression in CRIF1 knockdown endothelial cells. Supplementation with folic acid did not restore DHFR expression levels or MTHF and homocysteine concentrations in endothelial cells with a CRIF1 deletion or DHFR knockdown. However, the overexpression of DHFR in CRIF1 knockdown endothelial cells resulted in decreased accumulation of homocysteine. Taken together, our findings suggest that CRIF1-deleted endothelial cells accumulated more homocysteine, compared with control cells; this was primarily mediated by the disruption of DHFR expression.

CD226 deficiency promotes glutaminolysis and alleviates mitochondria damage in vascular endothelial cells under hemorrhagic shock.

Hemorrhagic shock (HS) is common in clinical emergencies, leading to millions of deaths each year globally. CD226 is a costimulatory adhesion molecule expressed on both immune cells and endothelial cells (ECs) to regulate their metabolic activity and function. As endothelial dysfunction occurs after HS, the roles CD226 plays in vascular EC metabolism were investigated. CD226fl/fl Tekcre mice were adopted to achieve vascular EC-specific knockout of CD226, and subjected to HS modelling. Serum levels of crucial intermediate metabolites were evaluated through liquid chromatography-mass spectrometry analysis. Human umbilical vein ECs (HUVECs) were used to study the effects of CD226 under hypoxia in vitro. Seahorse analysis evaluated the cellular glycolysis and mitochondria bioenergetics. Results showed that CD226 deficiency in vascular ECs alleviated HS-induced intestinal damage and inflammatory response in mice. Animal studies indicated an improved energy metabolism when CD226 was knocked out in ECs after HS, as evidenced by enhanced glutamine-glutamate metabolism and decreased lactic acid levels. Glut-1 was upregulated in mouse vascular ECs after HS and HUVECs under hypoxia, combined with decreased CD226. Moreover, HUVECs with CD226 knockdown exhibited relieved mitochondrial damage and early apoptosis under hypoxia, whereas CD226 overexpression showed opposite effects. Seahorse analysis showed that downregulated CD226 significantly increased mitochondrial ATP production and glucose uptake in HUVECs under hypoxia. Additionally, Erk/PHD2 signaling-mediated HIF-1α/Glut-1 and HIF-2α/ASCT2 pathways were involved in CD226 regulation on HUVEC glutaminolysis after hypoxia. Hence, CD226 deficiency promotes bypass energy supply to vascular ECs under ischemic or hypoxic stress, to ameliorate the stress-mediated metabolic disturbance.

Iron loading induces cholesterol synthesis and sensitizes endothelial cells to TNFα-mediated apoptosis

In plasma, iron is normally bound to transferrin, the principal protein in blood responsible for binding and transporting iron throughout the body. However, in conditions of iron overload when the iron-binding capacity of transferrin is exceeded, non–transferrin-bound iron (NTBI) appears in plasma. NTBI is taken up by hepatocytes and other parenchymal cells via NTBI transporters and can cause cellular damage by promoting the generation of reactive oxygen species. However, how NTBI affects endothelial cells, the most proximal cell type exposed to circulating NTBI, has not been explored. We modeled in vitro the effects of systemic iron overload on endothelial cells by treating primary human umbilical vein endothelial cells (HUVECs) with NTBI (ferric ammonium citrate [FAC]). We showed by RNA-Seq that iron loading alters lipid homeostasis in HUVECs by inducing sterol regulatory element-binding protein 2–mediated cholesterol biosynthesis. We also determined that FAC increased the susceptibility of HUVECs to apoptosis induced by tumor necrosis factor-α (TNFα). Moreover, we showed that cholesterol biosynthesis contributes to iron-potentiated apoptosis. Treating HUVECs with a cholesterol chelator hydroxypropyl-β-cyclodextrin demonstrated that depletion of cholesterol was sufficient to rescue HUVECs from TNFα-induced apoptosis, even in the presence of FAC. Finally, we showed that FAC or cholesterol treatment modulated the TNFα pathway by inducing novel proteolytic processing of TNFR1 to a short isoform that localizes to lipid rafts. Our study raises the possibility that iron-mediated toxicity in human iron overload disorders is at least in part dependent on alterations in cholesterol metabolism in endothelial cells, increasing their susceptibility to apoptosis.

Hormone metabolic pattern in the preclinical stage of preeclampsia

BACKGROUND: The imbalance of vascular endothelial cell metabolism determines the clinical manifestations of preeclampsia; however, the molecular mechanisms underlying the vessel destabilization are not fully understood. In recent years, researchers have focused on clarifying the role of dysmetabolic disorders in patients with obstetric pathology, including preeclampsia. This is due to the fact that pregnancy is accompanied by metabolic restructuring aimed at switching the energy supply of the pregnant womans body from the carbohydrate to the fat component in order to maintain an effective energoplastic supply of the developing fetus. Impairment of this evolutionary adaptation mechanism realized during pregnancy requires additional in-depth study.&#x0D; AIM: This study was aimed to identify and compare pathogenetic patterns that characterize early and late preeclampsia at the preclinical stage, based on dynamic clinical and laboratory examination of high-risk pregnant women.&#x0D; MATERIALS AND METHODS: A prospective clinical and laboratory examination of 180 pregnant women with independent factors of high risk of developing preeclampsia was carried out. Comparison groups were identified retrospectively, depending on the period of preeclampsia manifestation: Group I consisted of 31 pregnant women with early preeclampsia; Group II comprised 58 pregnant women with late preeclampsia; and Group III (control) included 30 healthy pregnant women with uncomplicated gestation. Pregnant women were examined twice at the preclinical stage of preeclampsia (11-14 and 18-21 weeks of gestation) and once at clinical manifestation of the disease (28-36 weeks of gestation). The markers of metabolic, hormonal, hemocirculatory, hemostasiological and placental disorders were evaluated.&#x0D; RESULTS: We found similar pathophysiological changes in pregnant women with both early and late PE, from early gestation periods. Those were characterized by pathological insulin resistance and hyperinsulinemia, as well as associated atherogenic changes in the lipid profile, hyperleptinemia, hyperuricemia, hypersympathicotonia, visceral fat deposition, and contra-insular hormonal deviations. The observed alterations reflected a single hormonal and metabolic pattern of the preclinical stage of preeclampsia. During pregnancy, there was shown an increase in clustering diabetogenic and atherogenic abnormalities and hormonal changes, which were supplemented by associated endothelial and hemostasiological dysfunction and, in early preeclampsia, placental dysfunction, thus accelerating the time of clinical implementation of preeclampsia.&#x0D; CONCLUSIONS: From the pathogenetic point of view, preeclampsia of various periods of manifestation is an indivisible category with a common basic developmental mechanism characterized by a hormone metabolic pattern from the early stages of pregnancy. These stable changes are the result of the pathologically transformed phylogenetic mechanism of energoplastic supply of the fetus. This transformation is realized via physiological insulin resistance and compensatory hyperinsulinemia development due to the contra-insular activity of placental hormones. The added structural and functional disorders of the embryo (feto) placental system potentiate basic mechanisms (pathological insulin resistance and hyperinsulinemia) and determine the period of preeclampsia clinical manifestation in each particular woman.

Vascular Normalization: A New Window Opened for Cancer Therapies.

Preclinical and clinical antiangiogenic approaches, with multiple side effects such as resistance, have not been proved to be very successful in treating tumor blood vessels which are important targets for tumor therapy. Meanwhile, restoring aberrant tumor blood vessels, known as tumor vascular normalization, has been shown not only capable of reducing tumor invasion and metastasis but also of enhancing the effectiveness of chemotherapy, radiation therapy, and immunotherapy. In addition to the introduction of such methods of promoting tumor vascular normalization such as maintaining the balance between proangiogenic and antiangiogenic factors and targeting endothelial cell metabolism, microRNAs, and the extracellular matrix, the latest molecular mechanisms and the potential connections between them were primarily explored. In particular, the immunotherapy-induced normalization of blood vessels further promotes infiltration of immune effector cells, which in turn improves immunotherapy, thus forming an enhanced loop. Thus, immunotherapy in combination with antiangiogenic agents is recommended. Finally, we introduce the imaging technologies and serum markers, which can be used to determine the window for tumor vascular normalization.

Metabolic effects of CoCr-enriched medium on shear-stressed endothelial cell and osteoblasts: A possible mechanism involving a hypoxic condition on bone healing

Cobalt-chromium (CoCr)-based alloys have emerged as an interesting biomaterial within biomedical field, mainly considering their biocompatibility, resistance to corrosion and absence of magnetism; however, its effect on cell metabolism is barely known and this prompted us better evaluating whether CoCr-enriched medium affects the metabolism of both osteoblast and endothelial cells, and also if there is a coupling between them. This is also considered here the already-known effect of Cobalt (Co) as a hypoxic element. Firstly, discs of CoCr [subjecting (W) or not (Wo) to dual acid-etched (DAE)] were incubated into FBS-free cell culture medium up to 24 h (37 °C). This CoCr-enriched medium was further used to treat shear-stressed endothelial cells cultures up to 72 h. Thereafter, the conditioned medium containing metabolites of shear-stressed endothelial cells in response to CoCr-enriched medium was further used to subject osteoblast's cultures, when the samples were properly harvested to allow the analysis of the molecular issues. Our data shows that CoCr-enriched medium contains 1.5 ng–2.0 ng/mL of Co, which was captured by endothelial cells and osteoblasts in about 30% in amount and it seems modulate their metabolic pathways: shear-stressed endothelial cells expressed higher profile of HIF1α, VEGF and nNOS genes, while their global profile of protein carbonylation was lower than the control cultures, suggesting lower oxidative stress commitment. Additionally, osteoblasts responding to metabolites of CoCr-challenged endothelial cells show dynamic expression of marker genes in osteogenic differentiation, with alkaline phosphatase (ALP), osteocalcin, and bone sialoprotein (BSP) genes being significantly increased. Additionally, tensional shear-stress forces decrease the stimulus for ColA1gene expression in osteoblasts responding to endothelial cells metabolites, as well as modifying the extracellular matrix remodeling related genes. Analyzing the activities of matrix metalloproteinases (MMPs), the data shows that shear-stressed endothelial cells metabolites increase the activities of both MMP9 and MMP2 in osteoblasts. Altogether, our data shows for the first time that shear-stressed endothelial metabolites responding to CoCr discs contribute to osteogenic phenotype in vitro, and this predicts an active crosstalk between angiogenesis and osteogenesis during osseointegration of CoCr alloy and bone healing, maybe guided by the Co-induced hypoxic condition.

Effect of Bevacizumab on the Viability and Metabolism of Human Corneal Epithelial and Endothelial Cells: An In Vitro Study.

PurposeTo examine the cytotoxic effects of bevacizumab on the viability and metabolism of human corneal epithelial cells (HCEpCs) and human corneal endothelial cells (HCEnCs), as well as human retinal pigment epithelial (ARPE-19) cells for comparison.MethodsImmortalized cell lines of HCEpCs, HCEnCs, and ARPE-19 cells were exposed to clinically relevant concentrations of bevacizumab (0.313–5.00 mg/mL). The ApoTox-Glo Triplex Assay was used to assess cell viability, cytotoxicity, and apoptosis, and the Mitochondrial ToxGlo Assay was used to assess cell membrane integrity and adenosine triphosphate (ATP) levels after a 24-hour treatment period.ResultsAcross all three cell types, we observed similar results of a decrease in cell viability at 5.00 mg/mL (P < 0.05) and an increase in cytotoxicity at 5.00 mg/mL (P < 0.05), whereas apoptotic activity remained unchanged (P > 0.05), which is a profile consistent with cells undergoing primary necrosis at high concentrations. Additionally, cell membrane integrity was compromised at 5.00 mg/mL (P < 0.05), whereas no decrease in ATP levels were observed (P > 0.05). Thus, no interference with mitochondrial oxidative phosphorylation in ATP production was seen, and the cells were able to maintain normal metabolic levels at high concentrations of bevacizumab.ConclusionsHCEpCs, HCEnCs, and ARPE-19 cells experience a decrease in viability and undergo primary necrosis when exposed to bevacizumab at a concentration of 5.00 mg/mL; however, they are able to maintain normal metabolism and mitochondrial function at the high concentrations used for the treatment of corneal neovascularization.Translational RelevanceThis study provides safety data on the concentrations of bevacizumab injected intravitreally and complements clinical data showing toxicity of topical bevacizumab on corneal epithelial and endothelial cells.

Endothelial Dysfunction in Atherosclerotic Cardiovascular Diseases and Beyond: From Mechanism to Pharmacotherapies.

The endothelium, a cellular monolayer lining the blood vessel wall, plays a critical role in maintaining multiorgan health and homeostasis. Endothelial functions in health include dynamic maintenance of vascular tone, angiogenesis, hemostasis, and the provision of an antioxidant, anti-inflammatory, and antithrombotic interface. Dysfunction of the vascular endothelium presents with impaired endothelium-dependent vasodilation, heightened oxidative stress, chronic inflammation, leukocyte adhesion and hyperpermeability, and endothelial cell senescence. Recent studies have implicated altered endothelial cell metabolism and endothelial-to-mesenchymal transition as new features of endothelial dysfunction. Endothelial dysfunction is regarded as a hallmark of many diverse human panvascular diseases, including atherosclerosis, hypertension, and diabetes. Endothelial dysfunction has also been implicated in severe coronavirus disease 2019. Many clinically used pharmacotherapies, ranging from traditional lipid-lowering drugs, antihypertensive drugs, and antidiabetic drugs to proprotein convertase subtilisin/kexin type 9 inhibitors and interleukin 1β monoclonal antibodies, counter endothelial dysfunction as part of their clinical benefits. The regulation of endothelial dysfunction by noncoding RNAs has provided novel insights into these newly described regulators of endothelial dysfunction, thus yielding potential new therapeutic approaches. Altogether, a better understanding of the versatile (dys)functions of endothelial cells will not only deepen our comprehension of human diseases but also accelerate effective therapeutic drug discovery. In this review, we provide a timely overview of the multiple layers of endothelial function, describe the consequences and mechanisms of endothelial dysfunction, and identify pathways to effective targeted therapies. SIGNIFICANCE STATEMENT: The endothelium was initially considered to be a semipermeable biomechanical barrier and gatekeeper of vascular health. In recent decades, a deepened understanding of the biological functions of the endothelium has led to its recognition as a ubiquitous tissue regulating vascular tone, cell behavior, innate immunity, cell-cell interactions, and cell metabolism in the vessel wall. Endothelial dysfunction is the hallmark of cardiovascular, metabolic, and emerging infectious diseases. Pharmacotherapies targeting endothelial dysfunction have potential for treatment of cardiovascular and many other diseases.

The PFKFB3 Inhibitor AZ67 Inhibits Angiogenesis Independently of Glycolysis Inhibition.

Angiogenesis is the process of new blood vessel formation. In this complex orchestrated growth, many factors are included. Lately, focus has shifted to endothelial cell metabolism, particularly to the PFKFB3 protein, a key regulatory enzyme of the glycolytic pathway. A variety of inhibitors of this important target have been studied, and a plethora of biological effects related to the process of angiogenesis have been reported. However, recent studies have disputed their mechanism of action, questioning whether all the effects are indeed due to PFKFB3 inhibition. Remarkably, the most well-studied inhibitor, 3PO, does not bind to PFKFB3, raising questions about this target. In our study, we aimed to elucidate the effects of PFKFB3 inhibition in angiogenesis by using the small molecule AZ67. We used isothermal titration calorimetry and confirmed binding to PFKFB3. In vitro, AZ67 did not decrease lactate production in endothelial cells (ECs), nor ATP levels, but exhibited good inhibitory efficacy in the tube-formation assay. Surprisingly, this was independent of EC migratory and proliferative abilities, as this was not diminished upon treatment. Strikingly however, even the lowest dose of AZ67 demonstrated significant inhibition of angiogenesis in vivo. To our knowledge, this is the first study to demonstrate that the process of angiogenesis can be disrupted by targeting PFKFB3 independently of glycolysis inhibition.

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.

Conjugated Bile Acids Activate Lymphangiogenic Pathways, Induce Chemokine Production and Significantly Alter Cellular Metabolism in Lymphatic Endothelial Cells

Elevated levels of conjugated bile acids (BA) such as taurochenodeoxycholic acid (TCDCA) and taurocholic acid (TCA) are found in serum of patients with chronic inflammatory liver diseases and liver cancers. Increased lymphangiogenesis has been documented in several chronic liver diseases, as lymphatics are integral to the hepatic microcirculation and also plays an important role in early metastasis of liver cancers like hepatocellular and cholangiocarcinoma. High levels of BAs have been shown in metastatic tumor-bearing LNs compared to naive LNs, however, the effects of BAs on lymphatic endothelial cells (LECs) remain completely undefined. The present study focuses on investigating the effects of TCDCA and TCA on inducing molecular and metabolic changes in LECs. LECs were treated with TCDCA (100 uM) and TCA (100 uM) for 24hrs in presence or absence of specific BA receptor inhibitor (FXR or TGR5: 50uM) and the changes in proliferation, migration, tube formation was determined. Effects of BAs on LEC cellular metabolism was assessed by Seahorse analysis and lactate levels, ATP production and ROS was measured by fluorometric assays. mRNA levels of bile acid receptors- FXR, TGR5, VDR as well as receptors for VEGF and FGF, chemokines and chemokine receptors were analyzed by real time quantitative PCR. Inhibitor for TGR5 was used to test the role of TGR5. Our results showed that LECs express the BA receptors FXR, TGR5 and VDR with TGR5 showing highest basal level expression. Both TCDCA and TCA induced significant induction in migration rates as well as increased LEC tube formation. Among the two BA used, TCDCA induced more metabolic changes in LECs than TCA with significant increase in maximal glycolytic capacity as well as basal rate of ATP production. These alterations were abrogated in presence of the TGR5 inhibitor. BAs, also increased levels of Yes-associated protein (YAP) (associated with increased lymphangiogenesis and tumor metastasis) and several downstream mediators in the LECs. Further, TCDCA elevated several lymphangiogenic molecules such as VEGFR1, VEGFR3, FGFR2 and FGFR4, and other inflammatory chemokines and chemokine receptors that play an important role in LEC immune cell crosstalk such as CCL19, CXCL5, CCR4, CCR5. TCA on the other hand, significantly increased mRNA expression of VEGFR3, MCP1 and CCR4 indicating specific roles for the different BAs on LECs. From these data we conclude that TCDCA and TCA alters morphological and phenotypic characteristics of LECs, induces lymphangiogenesis and promotes inflammatory signaling. We also predict that these activities of BA are initiated through the FXR and TGR5 receptors and targeting these pathways could suppress lymphangiogenesis and progression of inflammation.

Immunometabolic Endothelial Phenotypes: Integrating Inflammation and Glucose Metabolism.

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