The plasminogen-plasmin system has potential beneficial or deleterious effects in the context of renal fibrosis. Recent studies have implicated plasminogen activators or their inhibitors in this process.

Cranial neural crest (CNC) is a multipotent migratory cell population that gives rise to most of the craniofacial bones. An intricate network mediates CNC formation, epithelial-mesenchymal transition, migration along distinct paths, and differentiation. Errors in these processes lead to craniofacial abnormalities, including cleft lip and palate. Clefts are the most common congenital craniofacial defects. Patients have complications with feeding, speech, hearing, and dental and psychological development. Affected by both genetic predisposition and environmental factors, the complex etiology of clefts remains largely unknown. Here we show that Fas-associated factor-1 (FAF1) is disrupted and that its expression is decreased in a Pierre Robin family with an inherited translocation. Furthermore, the locus is strongly associated with cleft palate and shows an increased relative risk. Expression studies show that faf1 is highly expressed in zebrafish cartilages during embryogenesis. Knockdown of zebrafish faf1 leads to pharyngeal cartilage defects and jaw abnormality as a result of a failure of CNC to differentiate into and express cartilage-specific markers, such as sox9a and col2a1. Administration of faf1 mRNA rescues this phenotype. Our findings therefore identify FAF1 as a regulator of CNC differentiation and show that it predisposes humans to cleft palate and is necessary for lower jaw development in zebrafish.

Oxidative phosphorylation in mitochondria is responsible for 90% of ATP synthesis in most cells. This essential housekeeping function is mediated by nuclear and mitochondrial genes encoding subunits of complex I to V of the respiratory chain. Although complex IV is the best studied of these complexes, the exact function of the striated muscle-specific subunit COX6A2 is still poorly understood. In this study, we show that Cox6a2-deficient mice are protected against high-fat diet-induced obesity, insulin resistance and glucose intolerance. This phenotype results from elevated energy expenditure and a skeletal muscle fiber type switch towards more oxidative fibers. At the molecular level we observe increased formation of reactive oxygen species, constitutive activation of AMP-activated protein kinase, and enhanced expression of uncoupling proteins. Our data indicate that COX6A2 is a regulator of respiratory uncoupling in muscle and we demonstrate that a novel and direct link exists between muscle respiratory chain activity and diet-induced obesity/insulin resistance.

The delivery of blood-borne molecules conveying metabolic information to neural networks that regulate energy homeostasis is restricted by brain barriers. The fenestrated endothelium of median eminence microvessels and tight junctions between tanycytes together compose one of these. Here, we show that the decrease in blood glucose levels during fasting alters the structural organization of this blood-hypothalamus barrier, resulting in the improved access of metabolic substrates to the arcuate nucleus. These changes are mimicked by 2-deoxyglucose-induced glucoprivation and reversed by raising blood glucose levels after fasting. Furthermore, we show that VEGF-A expression in tanycytes modulates these barrier properties. The neutralization of VEGF signaling blocks fasting-induced barrier remodeling and significantly impairs the physiological response to refeeding. These results implicate glucose in the control of blood-hypothalamus exchanges through a VEGF-dependent mechanism and demonstrate a hitherto unappreciated role for tanycytes and the permeable microvessels associated with them in the adaptive metabolic response to fasting.

Endothelial cells (ECs) are plastic cells that can switch between growth states with different bioenergetic and biosynthetic requirements. Although quiescent in most healthy tissues, ECs divide and migrate rapidly upon proangiogenic stimulation. Adjusting endothelial metabolism to the growth state is central to normal vessel growth and function, yet it is poorly understood at the molecular level. Here we report that the forkhead box O (FOXO) transcription factor FOXO1 is an essential regulator of vascular growth that couples metabolic and proliferative activities in ECs. Endothelial-restricted deletion of FOXO1 in mice induces a profound increase in EC proliferation that interferes with coordinated sprouting, thereby causing hyperplasia and vessel enlargement. Conversely, forced expression of FOXO1 restricts vascular expansion and leads to vessel thinning and hypobranching. We find that FOXO1 acts as a gatekeeper of endothelial quiescence, which decelerates metabolic activity by reducing glycolysis and mitochondrial respiration. Mechanistically, FOXO1 suppresses signalling by MYC (also known as c-MYC), a powerful driver of anabolic metabolism and growth. MYC ablation impairs glycolysis, mitochondrial function and proliferation of ECs while its EC-specific overexpression fuels these processes. Moreover, restoration of MYC signalling in FOXO1-overexpressing endothelium normalizes metabolic activity and branching behaviour. Our findings identify FOXO1 as a critical rheostat of vascular expansion and define the FOXO1-MYC transcriptional network as a novel metabolic checkpoint during endothelial growth and proliferation.

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related mortality. We previously showed that the inhibition of placental growth factor (PlGF) exerts antitumour effects and induces vessel normalisation, possibly reducing hypoxia. However, the exact mechanism underlying these effects remains unclear. Because hypoxia and endoplasmic reticulum stress, which activates the unfolded protein response (UPR), have been implicated in HCC progression, we assessed the interactions between PlGF and these microenvironmental stresses.

During vessel sprouting, endothelial cells (ECs) dynamically rearrange positions in the sprout to compete for the tip position. We recently identified a key role for the glycolytic activator PFKFB3 in vessel sprouting by regulating cytoskeleton remodelling, migration and tip cell competitiveness. It is, however, unknown how glycolysis regulates EC rearrangement during vessel sprouting. Here we report that computational simulations, validated by experimentation, predict that glycolytic production of ATP drives EC rearrangement by promoting filopodia formation and reducing intercellular adhesion. Notably, the simulations correctly predicted that blocking PFKFB3 normalizes the disturbed EC rearrangement in high VEGF conditions, as occurs during pathological angiogenesis. This interdisciplinary study integrates EC metabolism in vessel sprouting, yielding mechanistic insight in the control of vessel sprouting by glycolysis, and suggesting anti-glycolytic therapy for vessel normalization in cancer and non-malignant diseases.

Hypermethylation of the promoters of tumour suppressor genes represses transcription of these genes, conferring growth advantages to cancer cells. How these changes arise is poorly understood. Here we show that the activity of oxygen-dependent ten-eleven translocation (TET) enzymes is reduced by tumour hypoxia in human and mouse cells. TET enzymes catalyse DNA demethylation through 5-methylcytosine oxidation. This reduction in activity occurs independently of hypoxia-associated alterations in TET expression, proliferation, metabolism, hypoxia-inducible factor activity or reactive oxygen species, and depends directly on oxygen shortage. Hypoxia-induced loss of TET activity increases hypermethylation at gene promoters in vitro. In patients, tumour suppressor gene promoters are markedly more methylated in hypoxic tumour tissue, independent of proliferation, stromal cell infiltration and tumour characteristics. Our data suggest that up to half of hypermethylation events are due to hypoxia, with these events conferring a selective advantage. Accordingly, increased hypoxia in mouse breast tumours increases hypermethylation, while restoration of tumour oxygenation abrogates this effect. Tumour hypoxia therefore acts as a novel regulator of DNA methylation.

Cancer cell metabolism has received increasing attention. Despite a boost in the application of clinical metabolic profiling (CMP) in cancer patients, a meta-analysis has not been performed. The primary goal of this study was to assess whether public accessibility of metabolomics data and identification and reporting of metabolites were sufficient to assess which metabolites were consistently altered in cancer patients. We therefore retrospectively curated data from CMP studies in cancer patients published during 5 recent years and used an established vote-counting method to perform a semiquantitative meta-analysis of metabolites in tumor tissue and blood. This analysis confirmed well-known increases in glycolytic metabolites, but also unveiled unprecedented changes in other metabolites such as ketone bodies and amino acids (histidine, tryptophan). However, this study also highlighted that insufficient public accessibility of metabolomics data, and inadequate metabolite identification and reporting hamper the discovery potential of meta-analyses of CMP studies, calling for improved standardization of metabolomics studies.

Expression of the mannose receptor (MRC1/CD206) identifies macrophage subtypes, such as alternatively activated macrophages (AAMs) and M2-polarized tumor-associated macrophages (TAMs), which are endowed with tissue-remodeling, proangiogenic, and protumoral activity. However, the significance of MRC1 expression for TAM's protumoral activity is unclear. Here, we describe and characterize miR-511-3p, an intronic microRNA (miRNA) encoded by both mouse and human MRC1 genes. By using sensitive miRNA reporter vectors, we demonstrate robust expression and bioactivity of miR-511-3p in MRC1(+) AAMs and TAMs. Unexpectedly, enforced expression of miR-511-3p tuned down the protumoral gene signature of MRC1(+) TAMs and inhibited tumor growth. Our findings suggest that transcriptional activation of Mrc1 in TAMs evokes a genetic program orchestrated by miR-511-3p, which limits rather than enhances their protumoral functions. Besides uncovering a role for MRC1 as gatekeeper of TAM's protumoral genetic programs, these observations suggest that endogenous miRNAs may operate to establish thresholds for inflammatory cell activation in tumors.

Increased copy number alterations (CNAs) indicative of chromosomal instability (CIN) have been associated with poor cancer outcome. Here, we study CNAs as potential biomarkers of bevacizumab (BVZ) response in metastatic colorectal cancer (mCRC). We cluster 409 mCRCs in three subclusters characterized by different degrees of CIN. Tumors belonging to intermediate-to-high instability clusters have improved outcome following chemotherapy plus BVZ versus chemotherapy alone. In contrast, low instability tumors, which amongst others consist of POLE-mutated and microsatellite-instable tumors, derive no further benefit from BVZ. This is confirmed in 81 mCRC tumors from the phase 2 MoMa study involving BVZ. CNA clusters overlap with CRC consensus molecular subtypes (CMS); CMS2/4 xenografts correspond to intermediate-to-high instability clusters and respond to FOLFOX chemotherapy plus mouse avastin (B20), while CMS1/3 xenografts match with low instability clusters and fail to respond. Overall, we identify copy number load as a novel potential predictive biomarker of BVZ combination therapy.

The secretome of cancer and stromal cells generates a microenvironment that contributes to tumour cell invasion and angiogenesis. Here we compare the secretome of human mammary normal and cancer-associated fibroblasts (CAFs). We discover that the chloride intracellular channel protein 3 (CLIC3) is an abundant component of the CAF secretome. Secreted CLIC3 promotes invasive behaviour of endothelial cells to drive angiogenesis and increases invasiveness of cancer cells both in vivo and in 3D cell culture models, and this requires active transglutaminase-2 (TGM2). CLIC3 acts as a glutathione-dependent oxidoreductase that reduces TGM2 and regulates TGM2 binding to its cofactors. Finally, CLIC3 is also secreted by cancer cells, is abundant in the stromal and tumour compartments of aggressive ovarian cancers and its levels correlate with poor clinical outcome. This work reveals a previously undescribed invasive mechanism whereby the secretion of a glutathione-dependent oxidoreductase drives angiogenesis and cancer progression by promoting TGM2-dependent invasion.

Control of energy homeostasis and metabolism is achieved by integrating numerous pathways, and miRNAs are involved in this process by regulating expression of multiple target genes. However, relatively little is known about the posttranscriptional processing of miRNAs and a potential role for the precursors they derive from. Here, we demonstrate that mature miRNA-22 is more abundant in muscle from male mice relative to females and that this enables sex-specific regulation of muscular lipid metabolism and body weight by repressing estrogen receptor alpha (ERα) expression. We found that the ERα adjusts its own activity by preventing processing of miR-22 via direct binding to a conserved ERα-binding element within the primary miR-22 precursor. Mutation of the ERα binding site within the pri-miR-22 in vivo eliminates sex-specific differences in miR-22 expression. We reason that the resulting tissue selective negative feedback regulation is essential to establish sex-specific differences in muscle metabolism and body weight development.

Glucagon, the principal hyperglycemic hormone, is secreted from pancreatic islet α cells as part of the counter-regulatory response to hypoglycemia. Hence, secretory output from α cells is under high demand in conditions of low glucose supply. Many tissues oxidize fat as an alternate energy substrate. Here, we show that glucagon secretion in low glucose conditions is maintained by fatty acid metabolism in both mouse and human islets, and that inhibiting this metabolic pathway profoundly decreases glucagon output by depolarizing α cell membrane potential and decreasing action potential amplitude. We demonstrate, by using experimental and computational approaches, that this is not mediated by the KATP channel, but instead due to reduced operation of the Na+-K+ pump. These data suggest that counter-regulatory secretion of glucagon is driven by fatty acid metabolism, and that the Na+-K+ pump is an important ATP-dependent regulator of α cell function.

Offspring of preeclamptic pregnancies have cognitive alterations. Placental growth factor (PGF), is low in preeclampsia; reduced levels may affect brain development. PGF-null mice differ from normal congenic controls in cerebrovasculature, neuroanatomy and behavior. Using brain imaging and behavioral testing, we asked whether developmentally asynchronous (i.e. neonatal) PGF supplementation alters the vascular, neuroanatomic and/or behavioral status of Pgf-/- mice at adulthood. C57BL/6-Pgf-/- pups were treated intraperitoneally on postnatal days 1-10 with vehicle or PGF at 10 pg/g, 70 pg/g or 700 pg/g. These mice underwent behavioral testing and perfusion for MRI and analysis of retinal vasculature. A second cohort of vehicle- or PGF-treated mice was perfused for micro-CT imaging. 10 pg/g PGF-treated mice exhibited less locomotor activity and greater anxiety-like behavior relative to vehicle-treated mice. Depressive-like behavior showed a sex-specific, dose-dependent decrease and was lowest in 700 pg/g PGF-treated females relative to vehicle-treated females. Spatial learning did not differ. MRI revealed smaller volume of three structures in the 10 pg/g group, larger volume of seven structures in the 70 pg/g group and smaller volume of one structure in the 700 pg/g group. No cerebral or retinal vascular differences were detected. Overall, neonatal PGF replacement altered behavior and neuroanatomy of adult Pgf-/- mice.

Angiogenesis, the formation of new blood vessels from pre-existing ones, occurs in pathophysiological contexts such as wound healing, cancer, and chronic inflammatory disease. During sprouting angiogenesis, endothelial tip and stalk cells coordinately remodel their cell-cell junctions to allow collective migration and extension of the sprout while maintaining barrier integrity. All these processes require energy, and the predominant ATP generation route in endothelial cells is glycolysis. However, it remains unclear how ATP reaches the plasma membrane and intercellular junctions. In this study, we demonstrate that the glycolytic enzyme pyruvate kinase 2 (PKM2) is required for sprouting angiogenesis in vitro and in vivo through the regulation of endothelial cell-junction dynamics and collective migration. We show that PKM2-silencing decreases ATP required for proper VE-cadherin internalization/traffic at endothelial cell-cell junctions. Our study provides fresh insight into the role of ATP subcellular compartmentalization in endothelial cells during angiogenesis. Since manipulation of EC glycolysis constitutes a potential therapeutic intervention route, particularly in tumors and chronic inflammatory disease, these findings may help to refine the targeting of endothelial glycolytic activity in disease.

Although it is well established that tumor-associated macrophages take part in each step of cancer progression, less is known about the distinct role of the so-called metastasis-associated macrophages (MAMs) at the metastatic site. Previous studies reported that Caveolin-1 (Cav1) has both tumor-promoting and tumor-suppressive functions. However, the role of Cav1 in bone-marrow-derived cells is unknown. Here, we describe Cav1 as an anti-metastatic regulator in mouse models of lung and breast cancer pulmonary metastasis. Among all the recruited inflammatory cell populations, we show that MAMs uniquely express abundant levels of Cav1. Using clodronate depletion of macrophages, we demonstrate that macrophage Cav1 signaling is critical for metastasis and not for primary tumor growth. In particular, Cav1 inhibition does not affect MAM recruitment to the metastatic site but, in turn, favors angiogenesis. We describe a mechanism by which Cav1 in MAMs specifically restrains vascular endothelial growth factor A/vascular endothelial growth factor receptor 1 (VEGF-A/VEGFR1) signaling and its downstream effectors, matrix metallopeptidase 9 (MMP9) and colony-stimulating factor 1 (CSF1).

Oncogenic transformation triggers reprogramming of cell metabolism, as part of the tumorigenic process. However, metabolic reprogramming may also increase the sensitivity of transformed cells to microenvironmental stress, at the early stages of tumor development. Herein, we show that transformation of human breast epithelial cells by the p95HER2/611CTF oncogene markedly sensitizes these cells to metabolic stress induced by the simultaneous inhibition of glucose and glutamine metabolism. In p95HER2/611CTF-transformed cells, metabolic stress activates a TNF related apoptosis-inducing ligand (TRAIL)-R and caspase-8-dependent apoptotic process that requires prior down-regulation of cellular FLICE-like inhibitor protein (c-FLIP) levels. Importantly, sustained mTOR activation is involved in FLIP down-regulation and apoptosis induced by metabolic stress. In vivo experiments in immunodeficient mice demonstrate a requirement for caspase-8 in restraining primary tumor growth of xenografts with p95HER2/611CTF-transformed cells. Collectively, these data define a critical role of the extrinsic pathway of apoptosis in the control of tumor initiation by microenvironmental cues.

Glioma growth and progression are characterized by abundant development of blood vessels that are highly aberrant and poorly functional, with detrimental consequences for drug delivery efficacy. The mechanisms driving this vessel dysmorphia during tumor progression are poorly understood. Using longitudinal intravital imaging in a mouse glioma model, we identify that dynamic sprouting and functional morphogenesis of a highly branched vessel network characterize the initial tumor growth, dramatically changing to vessel expansion, leakage, and loss of branching complexity in the later stages. This vascular phenotype transition was accompanied by recruitment of predominantly pro-inflammatory M1-like macrophages in the early stages, followed by in situ repolarization to M2-like macrophages, which produced VEGF-A and relocate to perivascular areas. A similar enrichment and perivascular accumulation of M2 versus M1 macrophages correlated with vessel dilation and malignancy in human glioma samples of different WHO malignancy grade. Targeting macrophages using anti-CSF1 treatment restored normal blood vessel patterning and function. Combination treatment with chemotherapy showed survival benefit, suggesting that targeting macrophages as the key driver of blood vessel dysmorphia in glioma progression presents opportunities to improve efficacy of chemotherapeutic agents. We propose that vessel dysfunction is not simply a general feature of tumor vessel formation, but rather an emergent property resulting from a dynamic and functional reorganization of the tumor stroma and its angiogenic influences.

Tumor progression alters the composition and physical properties of the extracellular matrix. Particularly, increased matrix stiffness has profound effects on tumor growth and metastasis. While endothelial cells are key players in cancer progression, the influence of tumor stiffness on the endothelium and the impact on metastasis is unknown. Through quantitative mass spectrometry, we find that the matricellular protein CCN1/CYR61 is highly regulated by stiffness in endothelial cells. We show that stiffness-induced CCN1 activates β-catenin nuclear translocation and signaling and that this contributes to upregulate N-cadherin levels on the surface of the endothelium, in vitro This facilitates N-cadherin-dependent cancer cell-endothelium interaction. Using intravital imaging, we show that knockout of Ccn1 in endothelial cells inhibits melanoma cancer cell binding to the blood vessels, a critical step in cancer cell transit through the vasculature to metastasize. Targeting stiffness-induced changes in the vasculature, such as CCN1, is therefore a potential yet unappreciated mechanism to impair metastasis.