Rindert Missiaen

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.

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

Efficacy of antiangiogenic immunotherapy is associated with intratumoral formation of high endothelial venules. Antitumor attack on two fronts The use of immune checkpoint inhibitors and other immunotherapies for the treatment of cancer is continuing to expand as these drugs demonstrate effectiveness in progressively more cancer types and therapeutic contexts. At the same time, the drugs are not perfect, and not all patients respond to them, so a key subject of research in this field is determining optimal ways to combine immune checkpoint therapies with other cancer treatments. Schmittnaegel et al. and Allen et al. focused their studies on the combination of antiangiogenic treatments with checkpoint inhibitors. The authors demonstrated how inhibition of tumor angiogenesis can facilitate the access of cytotoxic T cells to tumors, while the checkpoint inhibitors protect these T cells from exhaustion, enhancing their antitumor effects. 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.

The role of fatty acid β-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.

Targeting endothelial metabolism for anti-angiogenesis therapy: A pharmacological perspective

Current anti-angiogenic therapies in malignant and ocular diseases target growth factor signaling in order to attenuate excessive vascular growth. Although initial responses are promising, overall therapeutic success is limited due to insufficient efficiency, tumor refractoriness and resistance. Emerging evidence suggests that diverse growth factor signaling pathways in endothelial cells (ECs) converge onto cellular metabolism, creating an attractive target for novel alternative anti-angiogenic therapies. Recent studies show that ECs rely on glycolysis for ATP and biomass synthesis, necessary for proliferation and migration, key processes of angiogenesis. In addition, fatty acid β-oxidation (FAO) is essential for de novo nucleotide synthesis during EC proliferation. Initial proof-of-evidence has been given that administration of pharmacological inhibitors of those metabolic pathways can be used to inhibit pathological angiogenesis in vivo. Deciphering the role of other metabolic pathways and exploring the therapeutic potential of blocking these pathways await further investigation.

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 cells 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.

Corrigendum: Fatty acid carbon is essential for dNTP synthesis in endothelial cells.

This corrects the article DOI: 10.1038/nature14362

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.

Quiescent Endothelial Cells Upregulate Fatty Acid β-Oxidation for Vasculoprotection via Redox Homeostasis

Little is known about the metabolism of quiescent endothelial cells (QECs). Nonetheless, when dysfunctional, QECs contribute to multiple diseases. Previously, we demonstrated that proliferating endothelial cells (PECs) use fatty acid β-oxidation (FAO) for de novo dNTP synthesis. We report now that QECs are not hypometabolic, but upregulate FAO >3-fold higher than PECs, not to support biomass or energy production but to sustain the tricarboxylic acid cycle for redox homeostasis through NADPH regeneration. Hence, endothelial loss of FAO-controlling CPT1A in CPT1AΔEC mice promotes EC dysfunction (leukocyte infiltration, barrier disruption) by increasing endothelial oxidative stress, rendering CPT1AΔEC mice more susceptible to LPS and inflammatory bowel disease. Mechanistically, Notch1 orchestrates the use of FAO for redox balance in QECs. Supplementation of acetate (metabolized to acetyl-coenzyme A) restores endothelial quiescence and counters oxidative stress-mediated EC dysfunction in CPT1AΔEC mice, offering therapeutic opportunities. Thus, QECs use FAO for vasculoprotection against oxidative stress-prone exposure.

Therapeutic induction of high endothelial venules (HEVs) to enhance T-cell infiltration in tumors

There has been an explosion of clinical trials in an increasing variety of tumors utilizing immunotherapies as the consequence of their striking efficacy in the treatment of certain cancers, albeit in a minor population of patients [1]. In parallel, years of clinical trials with antiangiogenics demonstrated encouraging results in a small subset of patients, but have failed to produce increased overall survival [2]. Accumulating evidence indicates that a crosstalk exists between angiogenesis and immunity an aberrant angiogenic tumor vasculature can thwart the immune response by reducing immune T effector cell infiltration into tumors, along with leukocyte-endothelial transmigration and extravasation [[3, 4] and references therein]. In line with these observations, our group and the DePalma group [3, 4] found that targeting the tumor vasculature can transiently prune and normalize tumor vessels during a response phase, leading to an angiostatic phenotype characterized by increased infiltration of immune-stimulatory cells and tumor stasis (Figure 1A) [5]. During relapse, however, tumors initiated an adaptive immune suppressive mechanism that limited the efficacy of antiangiogenic agents by upregulating the negative immune checkpoint regulator, programmed cell death ligand 1 (PD-L1) in tumor and stromal cells. This lead to immunosuppression triggered by PD-L1 binding PD-1 on the surface of activated T cells to produce T cell anergy or exhaustion [3, 4]. Combining immunotherapy using anti-PD-L1 with antiangiogenic therapy had reciprocally beneficial effects in that immunotherapy targeted evasion from antiangiogenic therapy, while vascular normalization elicited by antiangiogenic treatment could increase lymphocyte infiltration and activation [3, 4]. In addition, antiangiogenic immunotherapy produced an unanticipated response – it induced a specialized form of blood vessels in treated tumors, reminiscent of high endothelial venules (HEVs) [4], that are typically found in lymphoid tissue. HEVs are morphologically and functionally distinct postcapillary venules that mediate the adhesion and transendothelial migration of circulating Tand B-cells to secondary lymphoid organs, including lymph nodes, where they encounter antigens and become activated [[6] and references therein]. HEVs can also be induced at sites of chronic inflammation found in autoimmune diseases or infection and become an integral part of tertiary lymphoid structures (TLSs) when they are surrounded by compartimentalized immunestimulatory immune cells (naive and CD8+ GranzymeB+ T-cells and others). While under these circumstances, the development of TLSs is thought to exacerbate disease, the spontaneous development of HEVs observed in several cancers has been found to be associated with improved patient outcomes. [[7] and references therein]. Taken together, these results suggest that therapeutic HEV induction in tumors may enable potent T-cell infiltration in human tumors to overcome and reinvigorate a rate-limiting step in the cancer-immunity cycle. Several lines of evidence point to a critical role of lymphotoxin (LIGHT, LTαβ) /lymphotoxin β-receptor (LTβR) signaling in HEV formation, and recent studies revealing that EC-specific LTβR knockout animals lack fully functional HEVs, reinforce this notion [[7] and Editorial

Trimming the Vascular Tree in Tumors: Metabolic and Immune Adaptations

Angiogenesis, the formation of new blood vessels, has become a well-established hallmark of cancer. Its functional importance for the manifestation and progression of tumors has been further validated by the beneficial therapeutic effects of angiogenesis inhibitors, most notably ones targeting the vascular endothelial growth factor (VEGF) signaling pathways. However, with the transient and short-lived nature of the patient response, it has become evident that tumors have the ability to adapt to the pressures of vascular growth restriction. Several escape mechanisms have been described that adapt tumors to therapy-induced low-oxygen tension by either reinstating tumor growth by vascular rebound or by altering tumor behavior without the necessity to reinitiate revascularization. We review here two bypass mechanisms that either instigate angiogenic and immune-suppressive polarization of intratumoral innate immune cells to facilitate VEGF-independent angiogenesis or enable metabolic adaptation and reprogramming of endothelial cells and tumor cells to adapt to low-oxygen tension.