The gluconeogenesis enzyme PCK2 has a non-enzymatic role in proteostasis in endothelial cells.

Endothelial cells (ECs) are highly glycolytic, but whether they generate glycolytic intermediates via gluconeogenesis (GNG) in glucose-deprived conditions remains unknown. Here, we report that glucose-deprived ECs upregulate the GNG enzyme PCK2 and rely on a PCK2-dependent truncated GNG, whereby lactate and glutamine are used for the synthesis of lower glycolytic intermediates that enter the serine and glycerophospholipid biosynthesis pathways, which can play key roles in redox homeostasis and phospholipid synthesis, respectively. Unexpectedly, however, even in normal glucose conditions, and independent of its enzymatic activity, PCK2 silencing perturbs proteostasis, beyond its traditional GNG role. Indeed, PCK2-silenced ECs have an impaired unfolded protein response, leading to accumulation of misfolded proteins, which due to defective proteasomes and impaired autophagy, results in the accumulation of protein aggregates in lysosomes and EC demise. Ultimately, loss of PCK2 in ECs impaired vessel sprouting. This study identifies a role for PCK2 in proteostasis beyond GNG.

Prioritization and functional validation of target genes from single-cell transcriptomics studies.

Translation of academic results into clinical practice is a formidable unmet medical need. Single-cell RNA-sequencing (scRNA-seq) studies generate long descriptive ranks of markers with predicted biological function, but without functional validation, it remains challenging to know which markers truly exert the putative function. Given the lengthy/costly nature of validation studies, gene prioritization is required to select candidates. We address these issues by studying tip endothelial cell (EC) marker genes because of their importance for angiogenesis. Here, by tailoring Guidelines On Target Assessment for Innovative Therapeutics, we in silico prioritize previously unreported/poorly described, high-ranking tip EC markers. Notably, functional validation reveals that four of six candidates behave as tip EC genes. We even discover a tip EC function for a gene lacking in-depth functional annotation. Thus, validating prioritized genes from scRNA-seq studies offers opportunities for identifying targets to be considered for possible translation, but not all top-ranked scRNA-seq markers exert the predicted function.

Role and therapeutic potential of dietary ketone bodies in lymph vessel growth.

Lymphatic vessels (LVs), lined by lymphatic endothelial cells (LECs), are indispensable for life1. However, the role of metabolism in LECs has been incompletely elucidated. In the present study, it is reported that LEC-specific loss of OXCT1, a key enzyme of ketone body oxidation2, reduces LEC proliferation, migration and vessel sprouting in vitro and impairs lymphangiogenesis in development and disease in Prox1ΔOXCT1 mice. Mechanistically, OXCT1 silencing lowers acetyl-CoA levels, tricarboxylic acid cycle metabolite pools, and nucleotide precursor and deoxynucleotide triphosphate levels required for LEC proliferation. Ketone body supplementation to LECs induces the opposite effects. Notably, elevation of lymph ketone body levels by a high-fat, low-carbohydrate ketogenic diet or by administration of the ketone body ß-hydroxybutyrate increases lymphangiogenesis after corneal injury and myocardial infarction. Intriguingly, in a mouse model of microsurgical ablation of LVs in the tail, which repeats features of acquired lymphoedema in humans, the ketogenic diet improves LV function and growth, reduces infiltration of anti-lymphangiogenic immune cells and decreases oedema, suggesting a novel dietary therapeutic opportunity.

Role of glutamine synthetase in angiogenesis beyond glutamine synthesis.

Glutamine synthetase, encoded by the gene GLUL, is an enzyme that converts glutamate and ammonia to glutamine. It is expressed by endothelial cells, but surprisingly shows negligible glutamine-synthesizing activity in these cells at physiological glutamine levels. Here we show in mice that genetic deletion of Glul in endothelial cells impairs vessel sprouting during vascular development, whereas pharmacological blockade of glutamine synthetase suppresses angiogenesis in ocular and inflammatory skin disease while only minimally affecting healthy adult quiescent endothelial cells. This relies on the inhibition of endothelial cell migration but not proliferation. Mechanistically we show that in human umbilical vein endothelial cells GLUL knockdown reduces membrane localization and activation of the GTPase RHOJ while activating other Rho GTPases and Rho kinase, thereby inducing actin stress fibres and impeding endothelial cell motility. Inhibition of Rho kinase rescues the defect in endothelial cell migration that is induced by GLUL knockdown. Notably, glutamine synthetase palmitoylates itself and interacts with RHOJ to sustain RHOJ palmitoylation, membrane localization and activation. These findings reveal that, in addition to the known formation of glutamine, the enzyme glutamine synthetase shows unknown activity in endothelial cell migration during pathological angiogenesis through RHOJ palmitoylation.

Serine Synthesis via PHGDH Is Essential for Heme Production in Endothelial Cells.

The role of phosphoglycerate dehydrogenase (PHGDH), a key enzyme of the serine synthesis pathway (SSP), in endothelial cells (ECs) remains poorly characterized. We report that mouse neonates with EC-specific PHGDH deficiency suffer lethal vascular defects within days of gene inactivation, due to reduced EC proliferation and survival. In addition to nucleotide synthesis impairment, PHGDH knockdown (PHGDHKD) caused oxidative stress, due not only to decreased glutathione and NADPH synthesis but also to mitochondrial dysfunction. Electron transport chain (ETC) enzyme activities were compromised upon PHGDHKD because of insufficient heme production due to cellular serine depletion, not observed in other cell types. As a result of heme depletion, elevated reactive oxygen species levels caused EC demise. Supplementation of hemin in PHGDHKD ECs restored ETC function and rescued the apoptosis and angiogenesis defects. These data argue that ECs die upon PHGDH inhibition, even without external serine deprivation, illustrating an unusual importance of serine synthesis for ECs.

Live imaging reveals a conserved role of fatty acid ß-oxidation in early lymphatic development in zebrafish.

During embryonic development, lymphatic endothelial cells (LECs) differentiate from venous endothelial cells (VECs), a process that is tightly regulated by several genetic signals. While the aquatic zebrafish model is regularly used for studying lymphangiogenesis and offers the unique advantage of time-lapse video-imaging of lymphatic development, some aspects of lymphatic development in this model differ from those in the mouse. It therefore remained to be determined whether fatty acid ß-oxidation (FAO), which we showed to regulate lymphatic formation in the mouse, also co-determines lymphatic development in this aquatic model. Here, we took advantage of the power of the zebrafish embryo model to visualize the earliest steps of lymphatic development through time-lapse video-imaging. By targeting zebrafish isoforms of carnitine palmitoyltransferase 1a (cpt1a), a rate controlling enzyme of FAO, with multiple morpholinos, we demonstrate that reducing CPT1A levels and FAO flux during zebrafish development impairs lymphangiogenic secondary sprouting, the initiation of lymphatic development in the zebrafish trunk, and the formation of the first lymphatic structures. These findings not only show evolutionary conservation of the importance of FAO for lymphatic development, but also suggest a role for FAO in co-regulating the process of VEC-to-LEC differentiation in zebrafish in vivo.

Notch3 Arg170Cys knock-in mice display pathologic and clinical features of the neurovascular disorder cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy.

OBJECTIVE: Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an adult-onset neurovascular disorder caused by stereotyped mutations in the NOTCH3 receptor. Elucidation of its pathobiology is still incomplete and remains a challenge, in part because the available preclinical mouse models to date do not reproduce the full spectrum of CADASIL pathology and clinical disease. METHODS AND RESULTS: Here, we report a novel knock-in mouse with Arg170Cys substitution in murine Notch3, corresponding to the prevalent Arg169Cys substitution in CADASIL. The Notch3(Arg170Cys) mice displayed late-onset, dominant CADASIL arteriopathy with typical granular osmiophilic material deposition and developed brain histopathology including thrombosis, microbleeds, gliosis, and microinfarction. Furthermore, Notch3(Arg170Cys) mice experienced neurological symptoms with motor defects such as staggering gait and limb paresis. CONCLUSIONS: This model, for the first time, phenocopies the arteriopathy and the histopathologic as well as clinical features of CADASIL and may offer novel opportunities to investigate disease pathogenesis.

VEGF-D deficiency in mice does not affect embryonic or postnatal lymphangiogenesis but reduces lymphatic metastasis.

Vascular endothelial growth factor-D (VEGF-D) is one of the two ligands of the VEGFR-3 receptor on lymphatic endothelial cells. Gene-silencing studies in mice and Xenopus tadpoles recently showed that the role of endogenous VEGF-D in lymphatic development is moderate. By contrast, exogenous VEGF-D is capable of stimulating lymphangiogenesis. Nonetheless, its endogenous role in pathological conditions remains largely unknown. Hence, we reassessed its role in disease, using Vegf-d(null) mice. Vegf-d(null) mice were generated that, under physiological conditions, displayed normal embryonic and postnatal lymphangiogenesis and lymphatic remodelling, efficient lymphatic functioning and normal health. Vegf-d(null) mice also reponded normally in models of skin wound healing and healing of infarcted myocardium, despite enhanced expression of VEGF-D in these models in wild-type mice. In contrast, Vegf-d(null) mice displayed reduced peritumoral lymphangiogenesis and lymph node metastasis in an orthotopic pancreatic tumour model. Together, our data indicate that endogenous VEGF-D in mice is dispensible for lymphangiogenesis during development, in postnatal and adult physiology and in several pathological conditions, but significantly contributes to lymphatic metastasis.