Notch: a molecular hub controlling the metabolic adaptations responsible for redox homeostasis during endothelial quiescence

Blood vessels ensure nutrient and oxygen supply to all cells via transport through the endothelium. This endothelial cell (EC) layer lining the luminal side of blood vessels forms a selective interface between the circulating blood and the surrounding tissue. ECs display high plasticity: i.e. in health, ECs in established blood vessels are quiescent and rarely divide, but upon stimulation in pathological conditions (ischemic, inflammatory or malignant diseases), they switch to a proliferative state and can migrate to form new sprouts. In relation to their involvement in pathological conditions, these activated ECs are subject of intense research in the cardiovascular field. Recent findings demonstrate the existence of a metabolic switch coinciding with the angiogenic switch. While most studies focus on targeting the angiogenic switch and preventing activation and sprouting, we sought to study the switch to quiescence with the aim to obtain valuable insight into the metabolic adaptation associated with this switch. Given their distinct environmental conditions, functional role and physiology, quiescent and angiogenic ECs are expected to have different metabolic needs. Previous data of the host lab showed that angiogenic ECs use the majority of metabolic energy for biosynthesis reactions to synthesize DNA, proteins and lipids, and to facilitate migration. Conversely, since quiescent cells are exempted from these laborious tasks, we hypothesized that reprogramming of basal endothelial metabolism is necessary to facilitate this ‘pro-quiescent switch’ from rapid growth to quiescence. In this thesis work, diverse metabolic assays and measurements combined with biochemical and molecular techniques were applied to characterize the nature and mechanisms of this pro-quiescent switch. This revealed that quiescent ECs are not generally hypometabolic, but display an activated oxidative pentose phosphate pathway (oxPPP) as well as increased fatty acid b-oxidation (FAO). These findings contradict the commonly held opinion that decreased metabolism is a hallmark of quiescence. This metabolic switch does not result in increased ATP or biomass synthesis, but instead promotes NADPH production, providing the necessary reducing power needed to lower the oxidative status of the cell. This level of redox control is most likely needed to prevent high levels of reactive oxygen species (ROS) since EC reside in an oxygen rich and oxidative environment. This prevents ROS sensitive activation of ECs and to avert prolonged oxidative stress associated with EC dysfunction (as is observed in various cardiovascular diseases). Furthermore, the data indicates that FAO, which in contrast to the oxPPP is not able to directly produce NADPH, likely stimulates NADPH formation in ECs via the mitochondrial NADPH producing malic enzyme 3 (ME3), isocitrate dehydrogenase 1 (IDH1) and nicotinamide nucleotide transhydrogenase (NNT). Functionally, I demonstrate that FAO in quiescent ECs is essential to constrain ROS sensitive induction of endothelial permeability and counteracts permeability defects and EC dysfunction as observed in cardiovascular diseases. I further show that Dll4-Notch signaling serves as a molecular hub controlling the metabolic adaptations associated with the induction of EC quiescence. Interestingly, Dll4-Notch directly modulates the FAO flux through binding of the Notch intracellular domain (NICD) in complex with the transcription factor CSL to the promoter of carnitine palmitoyl transferase 1a (CPT1a), a key enzyme of FAO, and subsequent transcriptional activation. Taken together, I provide valuable information regarding the metabolic profile of quiescent ECs and present a molecular mechanism responsible for the metabolic switch associated with the reverse angiogenic switch.status: publishe

The reciprocal function and regulation of tumor vessels and immune cells offers new therapeutic opportunities in cancer

Tumor angiogenesis and escape of immunosurveillance are two cancer hallmarks that are tightly linked and reciprocally regulated by paracrine signaling cues of cell constituents from both compartments. Formation and remodeling of new blood vessels in tumors is abnormal and facilitates immune evasion. In turn, immune cells in the tumor, specifically in context with an acidic and hypoxic environment, can promote neovascularization. Immunotherapy has emerged as a major therapeutic modality in cancer but is often hampered by the low influx of activated cytotoxic T-cells. On the other hand, anti-angiogenic therapy has been shown to transiently normalize the tumor vasculature and enhance infiltration of T lymphocytes, providing a rationale for a combination of these two therapeutic approaches to sustain and improve therapeutic efficacy in cancer. In this review, we discuss how the tumor vasculature facilitates an immunosuppressive phenotype and vice versa how innate and adaptive immune cells regulate angiogenesis during tumor progression. We further highlight recent results of antiangiogenic immunotherapies in experimental models and the clinic to evaluate the concept that targeting both the tumor vessels and immune cells increases the effectiveness in cancer patients.status: publishe

Metabolic control of the cell cycle

Cell division is a metabolically demanding process, requiring the production of large amounts of energy and biomass. Not surprisingly therefore, a cell's decision to initiate division is co-determined by its metabolic status and the availability of nutrients. Emerging evidence reveals that metabolism is not only undergoing substantial changes during the cell cycle, but it is becoming equally clear that metabolism regulates cell cycle progression. Here, we overview the emerging role of those metabolic pathways that have been best characterized to change during or influence cell cycle progression. We then studied how Notch signaling, a key angiogenic pathway that inhibits endothelial cell (EC) proliferation, controls EC metabolism (glycolysis) during the cell cycle.peerreview_statement: The publishing and review policy for this title is described in its Aims & Scope. aims_and_scope_url: http://www.tandfonline.com/action/journalInformation?show=aimsScope&journalCode=kccy20status: publishe

Combined antiangiogenic and anti–PD-L1 therapy stimulates tumor immunity through HEV formation

Inhibitors of VEGF (vascular endothelial growth factor)/VEGFR2 (vascular endothelial growth factor receptor 2) are commonly used in the clinic, but their beneficial effects are only observed in a subset of patients and limited by induction of diverse relapse mechanisms. We describe the up-regulation of an adaptive immunosuppressive pathway during antiangiogenic therapy, by which PD-L1 (programmed cell death ligand 1), the ligand of the negative immune checkpoint regulator PD-1 (programmed cell death protein 1), is enhanced by interferon-γ-expressing T cells in distinct intratumoral cell types in refractory pancreatic, breast, and brain tumor mouse models. Successful treatment with a combination of anti-VEGFR2 and anti-PD-L1 antibodies induced high endothelial venules (HEVs) in PyMT (polyoma middle T oncoprotein) breast cancer and RT2-PNET (Rip1-Tag2 pancreatic neuroendocrine tumors), but not in glioblastoma (GBM). These HEVs promoted lymphocyte infiltration and activity through activation of lymphotoxin β receptor (LTβR) signaling. Further activation of LTβR signaling in tumor vessels using an agonistic antibody enhanced HEV formation, immunity, and subsequent apoptosis and necrosis in pancreatic and mammary tumors. Finally, LTβR agonists induced HEVs in recalcitrant GBM, enhanced cytotoxic T cell (CTL) activity, and thereby sensitized tumors to antiangiogenic/anti-PD-L1 therapy. Together, our preclinical studies provide evidence that anti-PD-L1 therapy can sensitize tumors to antiangiogenic therapy and prolong its efficacy, and conversely, antiangiogenic therapy can improve anti-PD-L1 treatment specifically when it generates intratumoral HEVs that facilitate enhanced CTL infiltration, activity, and tumor cell destruction

Fatty acid carbon is essential for dNTP synthesis in endothelial cells

The metabolism of endothelial cells during vessel sprouting remains poorly studied. Here we report that endothelial loss of CPT1A, a rate-limiting enzyme of fatty acid oxidation (FAO), causes vascular sprouting defects due to impaired proliferation, not migration, of human and murine endothelial cells. Reduction of FAO in endothelial cells did not cause energy depletion or disturb redox homeostasis, but impaired de novo nucleotide synthesis for DNA replication. Isotope labelling studies in control endothelial cells showed that fatty acid carbons substantially replenished the Krebs cycle, and were incorporated into aspartate (a nucleotide precursor), uridine monophosphate (a precursor of pyrimidine nucleoside triphosphates) and DNA. CPT1A silencing reduced these processes and depleted endothelial cell stores of aspartate and deoxyribonucleoside triphosphates. Acetate (metabolized to acetyl-CoA, thereby substituting for the depleted FAO-derived acetyl-CoA) or a nucleoside mix rescued the phenotype of CPT1A-silenced endothelial cells. Finally, CPT1 blockade inhibited pathological ocular angiogenesis in mice, suggesting a novel strategy for blocking angiogenesis.status: publishe

The role of fatty acid beta-oxidation in lymphangiogenesis

Lymphatic vessels are lined by lymphatic endothelial cells (LECs), and are critical for health. However, the role of metabolism in lymphatic development has not yet been elucidated. Here we report that in transgenic mouse models, LEC-specific loss of CPT1A, a rate-controlling enzyme in fatty acid β-oxidation, impairs lymphatic development. LECs use fatty acid β-oxidation to proliferate and for epigenetic regulation of lymphatic marker expression during LEC differentiation. Mechanistically, the transcription factor PROX1 upregulates CPT1A expression, which increases acetyl coenzyme A production dependent on fatty acid β-oxidation. Acetyl coenzyme A is used by the histone acetyltransferase p300 to acetylate histones at lymphangiogenic genes. PROX1-p300 interaction facilitates preferential histone acetylation at PROX1-target genes. Through this metabolism-dependent mechanism, PROX1 mediates epigenetic changes that promote lymphangiogenesis. Notably, blockade of CPT1 enzymes inhibits injury-induced lymphangiogenesis, and replenishing acetyl coenzyme A by supplementing acetate rescues this process in vivo.status: publishe

The role of fatty acid beta-oxidation in lymphangiogenesis.

Lymphatic vessels are lined by lymphatic endothelial cells (LECs), and are critical for health. However, the role of metabolism in lymphatic development has not yet been elucidated. Here we report that in transgenic mouse models, LEC-specific loss of CPT1A, a rate-controlling enzyme in fatty acid beta-oxidation, impairs lymphatic development. LECs use fatty acid beta-oxidation to proliferate and for epigenetic regulation of lymphatic marker expression during LEC differentiation. Mechanistically, the transcription factor PROX1 upregulates CPT1A expression, which increases acetyl coenzyme A production dependent on fatty acid beta-oxidation. Acetyl coenzyme A is used by the histone acetyltransferase p300 to acetylate histones at lymphangiogenic genes. PROX1-p300 interaction facilitates preferential histone acetylation at PROX1-target genes. Through this metabolism-dependent mechanism, PROX1 mediates epigenetic changes that promote lymphangiogenesis. Notably, blockade of CPT1 enzymes inhibits injury-induced lymphangiogenesis, and replenishing acetyl coenzyme A by supplementing acetate rescues this process in vivo