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Vasculogenic mimicry (VM) describes the ability of highly aggressive tumor cells to develop pseudovascular structures without the participation of endothelial cells. PARP1 is implicated in the activation of hypoxia-inducible factors, which are crucial in tumor neovascularization. We have explored the role of hypoxia and PARP inhibition in VM. In uveal melanoma xenografts, the PARP inhibitor olaparib improved in vivo pericyte coverage specifically of VM channels. This was concomitant with reduced metastasis in olaparib-treated VM+ tumors. PARP inhibition and hypoxia modulated melanoma tube formation in vitro, inducing a more sparse and regular tubular architecture. Wholetranscriptome profiling revealed that olaparib treatment under hypoxic conditions modulated the expression of genes implicated in vasculogenesis during tube formation, enhancing the endothelial-like phenotype of VM+ uveal melanoma cells. PARP inhibition, especially during hypoxia, upregulated PDGFβ, which is essential for pericyte recruitment. Our study indicates that PARP inhibitors may enhance the endothelial characteristics of VM+ cells, modulate pericyte coverage, and reduce metastatic spread in VM+ melanoma., Spanish Government Ministry of Science and Innovation, Spain (MICINN) Spanish Government SAF2015-70520-R RTI2018-098968-B-I00 RTICC RD12/0036/0026, CIBER Cancer ISCIII CB16/12/00421, Junta de Andalucia PY20_01179, Fundacion Domingo Martinez

Intrinsic and adaptive resistance hampers the success of antiangiogenic therapies (AAT), especially in breast cancer where this treatment modality has proven largely ineffective. Therefore, novel strategies to improve the efficacy of AAT are warranted. Solid tumors such as breast cancer are characterized by a high infiltration of myeloid-derived suppressor cells (MDSC), which are key drivers of resistance to AAT. Therefore, we hypothesized that all-trans retinoic acid (ATRA), which induces differentiation of MDSC into mature cells, could improve the therapeutic effect of AAT. ATRA increased the efficacy of anti–VEGFR2 antibodies alone and in combination with chemotherapy in preclinical breast cancer models. ATRA reverted the anti–VEGFR2-induced accumulation of intratumoral MDSC, alleviated hypoxia, and counteracted the disorganization of tumor microvessels. Mechanistic studies indicate that ATRA treatment blocked the AAT-induced expansion of MDSC secreting high levels of vessel-destabilizing S100A8. Thus, concomitant treatment with ATRA holds the potential to improve AAT in breast cancer and possibly other tumor types.Significance: Increasing the therapeutic efficiency of antiangiogenic drugs by reducing resistance-conferring myeloid-derived suppressor cells might improve breast cancer treatment.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/12/3220/F1.large.jpg. Cancer Res; 78(12); 3220–32. ©2018 AACR.

Increasing evidence suggests that functional impairments at the level of the neurovascular unit (NVU) underlie many neurodegenerative and neuroinflammatory diseases. While being part of the NVU, astrocytes have been largely overlooked in this context and only recently, tightening of the glia limitans has been put forward as an important neuroprotective response to limit these injurious processes. In this study, using the retina as a central nervous system (CNS) model organ, we investigated the structure and function of the glia limitans, and reveal that the blood-retina barrier and glia limitans function as a coordinated double barrier to limit infiltration of leukocytes and immune molecules. We provide in vitro and in vivo evidence for a protective response at the NVU upon CNS injury, which evokes inflammation-induced glia limitans tightening. Matrix metalloproteinase-3 (MMP-3) was found to be a crucial regulator of this process, thereby revealing its beneficial and immunomodulatory role in the CNS. in vivo experiments in which MMP-3 activity was deleted via genetic and pharmacological approaches, combined with a comprehensive study of tight junction molecules, glial end feet markers, myeloid cell infiltration, cytokine expression and neurodegeneration, show that MMP-3 attenuates neuroinflammation and neurodegeneration by tightening the glia limitans, thereby pointing to a prominent role of MMP-3 in preserving the integrity of the NVU upon injury. Finally, we gathered promising evidence to suggest that IL1b, which is also regulated by MMP-3, is at least one of the molecular messengers that induces glia limitans tightening in the injured CNS. ispartof: Glia vol:68 issue:12 pages:2643-2660 ispartof: location:United States status: Published online

Retinal degeneration is a common feature in peroxisomal disorders leading to blindness. Peroxisomes are present in the different cell types of the retina; however, their precise contribution to retinal integrity is still unclear. We previously showed that mice lacking the central peroxisomal β-oxidation enzyme, multifunctional protein 2 (MFP2), develop an early onset retinal decay including photoreceptor cell death. To decipher the function of peroxisomal β-oxidation in photoreceptors, we generated cell type selective Mfp2 knockout mice, using the Crx promotor targeting photoreceptors and bipolar cells. Surprisingly, Crx-Mfp2−/− mice maintained photoreceptor length and number until the age of 1 year. A negative electroretinogram was indicative of preserved photoreceptor phototransduction, but impaired downstream bipolar cell signaling from the age of 6 months. The photoreceptor ribbon synapse was affected, containing free-floating ribbons and vesicles with altered size and density. The bipolar cell interneurons sprouted into the ONL and died. Whereas docosahexaenoic acid levels were normal in the neural retina, levels of lipids containing very long chain polyunsaturated fatty acids were highly increased. Crx-Pex5−/− mice, in which all peroxisomal functions are inactivated in photoreceptors and bipolar cells, developed the same phenotype as Crx-Mfp2−/− mice. In conclusion, the early photoreceptor death in global Mfp2−/− mice is not driven cell autonomously. However, peroxisomal β-oxidation is essential for the integrity of photoreceptor ribbon synapses and of bipolar cells.

Tumor vessel co-option is poorly understood, yet it is a resistance mechanism against anti-angiogenic therapy (AAT). The heterogeneity of co-opted endothelial cells (ECs) and pericytes, co-opting cancer and myeloid cells in tumors growing via vessel co-option, has not been investigated at the single-cell level. Here, we use a murine AAT-resistant lung tumor model, in which VEGF-targeting induces vessel co-option for continued growth. Single-cell RNA sequencing (scRNA-seq) of 31,964 cells reveals, unexpectedly, a largely similar transcriptome of co-opted tumor ECs (TECs) and pericytes as their healthy counterparts. Notably, we identify cell types that might contribute to vessel co-option, i.e., an invasive cancer-cell subtype, possibly assisted by a matrix-remodeling macrophage population, and another M1-like macrophage subtype, possibly involved in keeping or rendering vascular cells quiescent. ispartof: CELL REPORTS vol:35 issue:11 ispartof: location:United States status: published

Autophagy has an emerging vasculoprotective role but whether and how it regulates lymphatic endothelial cells (LEC) and lymphangiogenesis is unknown. Here, we show that genetic deficiency of autophagy in LEC impairs the responses to VEGF-C and injury-driven corneal lymphangiogenesis. Loss of autophagy compromises expression of lymphatic markers, affects mitochondrial dynamics and causes an accumulation of lipid droplets (LDs) in LEC and lymphatic vessels in vivo. When LDs accumulate because lipophagy is impaired, mitochondrial ATP production, fatty acid oxidation (FAO), acetyl-CoA/CoA ratio and expression of lymphangiogenic PROX1 target genes are dwindled. Enforcing mitochondria fusion by silencing dynamin-related-protein 1 (DRP1) in autophagy-deficient LEC fails to affect LDs turnover and lymphatic gene expression, whereas supplementing the acetyl-CoA precursor acetate rescues LEC identity and lymphangiogenesis in LEC-Atg5-/- mice. Our findings reveal that lipophagy in LEC by supporting FAO, preserves a mitochondrial-PROX1 gene expression circuit that ensures LEC identity, responsiveness to lymphangiogenic mediators and lymphangiogenesis.

Aims Hepatic capillaries are lined with specialized liver sinusoidal endothelial cells (LSECs) which support macromolecule passage to hepatocytes and prevent fibrosis by keeping hepatic stellate cells (HSCs) quiescent. LSEC specialization is co-determined by transcription factors. The zinc-finger E-box-binding homeobox (Zeb)2 transcription factor is enriched in LSECs. Here, we aimed to elucidate the endothelium-specific role of Zeb2 during maintenance of the liver and in liver fibrosis. Methods and results To study the role of Zeb2 in liver endothelium we generated EC-specific Zeb2 knock-out (ECKO) mice. Sequencing of liver EC RNA revealed that deficiency of Zeb2 results in prominent expression changes in angiogenesis-related genes. Accordingly, the vascular area was expanded and the presence of pillars inside ECKO liver vessels indicated that this was likely due to increased intussusceptive angiogenesis. LSEC marker expression was not profoundly affected and fenestrations were preserved upon Zeb2 deficiency. However, an increase in continuous EC markers suggested that Zeb2-deficient LSECs are more prone to dedifferentiation, a process called ‘capillarization’. Changes in the endothelial expression of ligands that may be involved in HSC quiescence together with significant changes in the expression profile of HSCs showed that Zeb2 regulates LSEC–HSC communication and HSC activation. Accordingly, upon exposure to the hepatotoxin carbon tetrachloride (CCl4), livers of ECKO mice showed increased capillarization, HSC activation, and fibrosis compared to livers from wild-type littermates. The vascular maintenance and anti-fibrotic role of endothelial Zeb2 was confirmed in mice with EC-specific overexpression of Zeb2, as the latter resulted in reduced vascularity and attenuated CCl4-induced liver fibrosis. Conclusion Endothelial Zeb2 preserves liver angioarchitecture and protects against liver fibrosis. Zeb2 and Zeb2-dependent genes in liver ECs may be exploited to design novel therapeutic strategies to attenuate hepatic fibrosis.

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 β-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. ispartof: Nature Metabolism vol:1 issue:7 pages:666-675 ispartof: location:Germany status: Published online

Objectives Peroxisomes play a crucial role in lipid and reactive oxygen species metabolism, but their importance for pancreatic β-cell functioning is presently unknown. To examine the contribution of peroxisomal metabolism to β-cell homeostasis in mice, we inactivated PEX5, the import receptor for peroxisomal matrix proteins, in an inducible and β-cell restricted manner (Rip-Pex5−/− mice). Methods After tamoxifen-induced recombination of the Pex5 gene at the age of 6 weeks, mice were fed either normal chow or a high-fat diet for 12 weeks and were subsequently phenotyped. Results Increased levels of very long chain fatty acids and reduced levels of plasmalogens in islets confirmed impairment of peroxisomal fatty acid oxidation and ether lipid synthesis, respectively. The Rip-Pex5−/− mice fed on either diet exhibited glucose intolerance associated with impaired insulin secretion. Ultrastructural and biochemical analysis revealed a decrease in the density of mature insulin granules and total pancreatic insulin content, which was further accompanied by mitochondrial disruptions, reduced complex I activity and massive vacuole overload in β-cells. RNAseq analysis suggested that cell death pathways were affected in islets from HFD-fed Rip-Pex5−/− mice. Consistent with this change we observed increased β-cell apoptosis in islets and a decrease in β-cell mass. Conclusions Our data indicate that normal peroxisome metabolism in β-cells is crucial to preserve their structure and function., Graphical abstract Image 1, Highlights • Pex5 deletion in β-cells impairs glucose tolerance and reduces β-cell mass. • Pex5-deficient β-cells display increased apoptosis. • Peroxisomal loss causes mitochondrial deterioration and cytoplasmic vacuolization.

We have previously demonstrated that systemic AMP-activated protein kinase α1 (AMPKα1) invalidation enhanced adverse LV remodelling by increasing fibroblast proliferation, while myodifferentiation and scar maturation were impaired. We thus hypothesised that fibroblastic AMPKα1 was a key signalling element in regulating fibrosis in the infarcted myocardium and an attractive target for therapeutic intervention. The present study investigates the effects of myofibroblast (MF)-specific deletion of AMPKα1 on left ventricular (LV) adaptation following myocardial infarction (MI), and the underlying molecular mechanisms. MF-restricted AMPKα1 conditional knockout (cKO) mice were subjected to permanent ligation of the left anterior descending coronary artery. cKO hearts exhibit exacerbated post-MI adverse LV remodelling and are characterised by exaggerated fibrotic response, compared to wild-type (WT) hearts. Cardiac fibroblast proliferation and MF content significantly increase in cKO infarcted hearts, coincident with a significant reduction of connexin 43 (Cx43) expression in MFs. Mechanistically, AMPKα1 influences Cx43 expression by both a transcriptional and a post-transcriptional mechanism involving miR-125b-5p. Collectively, our data demonstrate that MF-AMPKα1 functions as a master regulator of cardiac fibrosis and remodelling and might constitute a novel potential target for pharmacological anti-fibrotic applications. Supplementary Information The online version contains supplementary material available at 10.1007/s00395-021-00846-y.

Since a detailed inventory of endothelial cell (EC) heterogeneity in breast cancer (BC) is lacking, here we perform single cell RNA-sequencing of 26,515 cells (including 8433 ECs) from 9 BC patients and compare them to published EC taxonomies from lung tumors. Angiogenic ECs are phenotypically similar, while other EC subtypes are different. Predictive interactome analysis reveals known but also previously unreported receptor-ligand interactions between ECs and immune cells, suggesting an involvement of breast EC subtypes in immune responses. We also identify a capillary EC subtype (LIPEC (Lipid Processing EC)), which expresses genes involved in lipid processing that are regulated by PPAR-gamma and is more abundant in peri-tumoral breast tissue. Retrospective analysis of 4648 BC patients reveals that treatment with metformin (an indirect PPAR-gamma signaling activator) provides long-lasting clinical benefit and is positively associated with LIPEC abundance. Our findings warrant further exploration of this LIPEC/PPAR-gamma link for BC treatment. Tumor blood vessels contribute to cancer growth, invasion and metastasis. Here, by using single cell transcriptomics, the authors report an inventory of endothelial cell heterogeneity in patients with breast cancer, including a subtype that expresses genes involved in lipid processing and is regulated by PPAR-gamma.

Liver sinusoidal endothelial cells (LSECs) are the first liver cells to encounter waste macromolecules, pathogens, and toxins in blood. LSECs are highly specialized to mediate the clearance of these substances via endocytic scavenger receptors and are equipped with fenestrae that mediate the passage of macromolecules toward hepatocytes. Although some transcription factors (TFs) are known to play a role in LSEC specialization, information about the specialized LSEC signature and its transcriptional determinants remains incomplete.Based on a comparison of liver, heart, and brain endothelial cells (ECs), we established a 30-gene LSEC signature comprising both established and newly identified markers, including 7 genes encoding TFs. To evaluate the LSEC TF regulatory network, we artificially increased the expression of the 7 LSEC-specific TFs in human umbilical vein ECs. Although Zinc finger E-box-binding protein 2, homeobox B5, Cut-like homolog 2, and transcription factor EC (TCFEC) had limited contributions, musculoaponeurotic fibrosarcoma (C-MAF), GATA binding protein 4 (GATA4), and MEIS homeobox 2 (MEIS2) emerged as stronger inducers of LSEC marker expression. Furthermore, a combination of C-MAF, GATA4, and MEIS2 showed a synergistic effect on the increase of LSEC signature genes, including liver/lymph node-specific ICAM-3 grabbing non-integrin (L-SIGN) (or C-type lectin domain family member M (CLEC4M)), mannose receptor C-Type 1 (MRC1), legumain (LGMN), G protein-coupled receptor 182 (GPR182), Plexin C1 (PLXNC1), and solute carrier organic anion transporter family member 2A1 (SLCO2A1). Accordingly, L-SIGN, MRC1, pro-LGMN, GPR182, PLXNC1, and SLCO2A1 protein levels were elevated by this combined overexpression. Although receptor-mediated endocytosis was not significantly induced by the triple TF combination, it enhanced binding to E2, the hepatitis C virus host-binding protein. We conclude that C-MAF, GATA4, and MEIS2 are important transcriptional regulators of the unique LSEC fingerprint and LSEC interaction with viruses. Additional factors are however required to fully recapitulate the molecular, morphological, and functional LSEC fingerprint.NEW & NOTEWORTHY Liver sinusoidal endothelial cells (LSECs) are the first liver cells to encounter waste macromolecules, pathogens, and toxins in the blood and are highly specialized. Although some transcription factors are known to play a role in LSEC specialization, information about the specialized LSEC signature and its transcriptional determinants remains incomplete. Here, we show that Musculoaponeurotic Fibrosarcoma (C-MAF), GATA binding protein 4 (GATA4), and Meis homeobox 2 (MEIS2) are important transcriptional regulators of the unique LSEC signature and that they affect the interaction of LSECs with viruses. ispartof: AMERICAN JOURNAL OF PHYSIOLOGY-GASTROINTESTINAL AND LIVER PHYSIOLOGY vol:318 issue:4 pages:G803-G815 ispartof: location:United States status: published

Cystic fibrosis (CF) is a life-threatening disorder characterised by decreased pulmonary mucociliary and pathogen clearance, and an exaggerated inflammatory response leading to progressive lung damage. CF is caused by bi-allelic pathogenic variants of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes a chloride channel. CFTR is expressed in endothelial cells (ECs) and EC dysfunction has been reported in CF patients, but a role for this ion channel in ECs regarding CF disease progression is poorly described.We used an unbiased RNA sequencing approach in complementary models of CFTR silencing and blockade (by the CFTR inhibitor CFTRinh-172) in human ECs to characterise the changes upon CFTR impairment. Key findings were further validatedin vitroandin vivoin CFTR-knockout mice andex vivoin CF patient-derived ECs.Both models of CFTR impairment revealed that EC proliferation, migration and autophagy were downregulated. Remarkably though, defective CFTR function led to EC activation and a persisting pro-inflammatory state of the endothelium with increased leukocyte adhesion. Further validation in CFTR-knockout mice revealed enhanced leukocyte extravasation in lung and liver parenchyma associated with increased levels of EC activation markers. In addition, CF patient-derived ECs displayed increased EC activation markers and leukocyte adhesion, which was partially rescued by the CFTR modulators VX-770 and VX-809.Our integrated analysis thus suggests that ECs are no innocent bystanders in CF pathology, but rather may contribute to the exaggerated inflammatory phenotype, raising the question of whether normalisation of vascular inflammation might be a novel therapeutic strategy to ameliorate the disease severity of CF.

BACKGROUND: Renal endothelial cells from glomerular, cortical, and medullary kidney compartments are exposed to different microenvironmental conditions and support specific kidney processes. However, the heterogeneous phenotypes of these cells remain incompletely inventoried. Osmotic homeostasis is vitally important for regulating cell volume and function, and in mammals, osmotic equilibrium is regulated through the countercurrent system in the renal medulla, where water exchange through endothelium occurs against an osmotic pressure gradient. Dehydration exposes medullary renal endothelial cells to extreme hyperosmolarity, and how these cells adapt to and survive in this hypertonic milieu is unknown. METHODS: We inventoried renal endothelial cell heterogeneity by single-cell RNA sequencing >40,000 mouse renal endothelial cells, and studied transcriptome changes during osmotic adaptation upon water deprivation. We validated our findings by immunostaining and functionally by targeting oxidative phosphorylation in a hyperosmolarity model in vitro and in dehydrated mice in vivo. RESULTS: We identified 24 renal endothelial cell phenotypes (of which eight were novel), highlighting extensive heterogeneity of these cells between and within the cortex, glomeruli, and medulla. In response to dehydration and hypertonicity, medullary renal endothelial cells upregulated the expression of genes involved in the hypoxia response, glycolysis, and-surprisingly-oxidative phosphorylation. Endothelial cells increased oxygen consumption when exposed to hyperosmolarity, whereas blocking oxidative phosphorylation compromised endothelial cell viability during hyperosmotic stress and impaired urine concentration during dehydration. CONCLUSIONS: This study provides a high-resolution atlas of the renal endothelium and highlights extensive renal endothelial cell phenotypic heterogeneity, as well as a previously unrecognized role of oxidative phosphorylation in the metabolic adaptation of medullary renal endothelial cells to water deprivation. ispartof: JOURNAL OF THE AMERICAN SOCIETY OF NEPHROLOGY vol:31 issue:1 pages:118-138 ispartof: location:United States status: published

Intrinsic and adaptive resistance hampers the success of antiangiogenic therapies (AAT), especially in breast cancer where this treatment modality has proven largely ineffective. Therefore, novel strategies to improve the efficacy of AAT are warranted. Solid tumors such as breast cancer are characterized by a high infiltration of myeloid-derived suppressor cells (MDSC), which are key drivers of resistance to AAT. Therefore, we hypothesized that all-trans retinoic acid (ATRA), which induces differentiation of MDSC into mature cells, could improve the therapeutic effect of AAT. ATRA increased the efficacy of anti–VEGFR2 antibodies alone and in combination with chemotherapy in preclinical breast cancer models. ATRA reverted the anti–VEGFR2-induced accumulation of intratumoral MDSC, alleviated hypoxia, and counteracted the disorganization of tumor microvessels. Mechanistic studies indicate that ATRA treatment blocked the AAT-induced expansion of MDSC secreting high levels of vessel-destabilizing S100A8. Thus, concomitant treatment with ATRA holds the potential to improve AAT in breast cancer and possibly other tumor types. Significance: Increasing the therapeutic efficiency of antiangiogenic drugs by reducing resistance-conferring myeloid-derived suppressor cells might improve breast cancer treatment. Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/12/3220/F1.large.jpg. Cancer Res; 78(12); 3220–32. ©2018 AACR.

The structural disruption of the mitochondrial inner membrane in hepatocytes lacking functional peroxisomes along with selective impairment of respiratory complexes and depletion of mitochondrial DNA was previously reported. In search for the molecular origin of these mitochondrial alterations, we here show that these are tissue selective as they do neither occur in peroxisome deficient brain nor in peroxisome deficient striated muscle. Given the hepatocyte selectivity, we investigated the potential involvement of metabolites that are primarily handled by hepatic peroxisomes. Levels of these metabolites were manipulated in L-Pex5 knockout mice and/or compared with levels in different mouse models with a peroxisomal β-oxidation deficiency. We show that neither the deficiency of docosahexaenoic acid nor the accumulation of branched chain fatty acids, dicarboxylic acids or C27 bile acid intermediates are solely responsible for the mitochondrial anomalies. In conclusion, we demonstrate that peroxisomal inactivity differentially impacts mitochondria depending on the cell type but the cause of the mitochondrial destruction needs to be further explored. ispartof: Mitochondrion vol:39 pages:51-59 ispartof: location:Netherlands status: published

Blockade of the glycolytic activator PFKFB3 in cancer cells (using a maximum tolerable dose of 70 mg/kg of the PFKFB3 blocker 3PO) inhibits tumor growth in preclinical models and is currently being tested as a novel anticancer treatment in phase I clinical trials. However, a detailed preclinical analysis of the effects of such maximum tolerable dose of a PFKFB3 blocker on the tumor vasculature is lacking, even though tumor endothelial cells are hyper-glycolytic. We report here that a high dose of 3PO (70 mg/kg), which inhibits cancer cell proliferation and reduces primary tumor growth, causes tumor vessel disintegration, suppresses endothelial cell growth for protracted periods, (model-dependently) aggravates tumor hypoxia, and compromises vascular barrier integrity, thereby rendering tumor vessels more leaky and facilitating cancer cell intravasation and dissemination. These findings contrast to the effects of a low dose of 3PO (25 mg/kg), which induces tumor vessel normalization, characterized by vascular barrier tightening and maturation, but reduces cancer cell intravasation and metastasis. Our findings highlight the importance of adequately dosing a glycolytic inhibitor for anticancer treatment. ispartof: Angiogenesis vol:20 issue:4 pages:599-613 ispartof: location:Germany status: published

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. ispartof: Nature vol:542 issue:7639 pages:49-+ ispartof: location:England status: published

Endochondral ossification, an important process in vertebrate bone formation, is highly dependent on correct functioning of growth plate chondrocytes1. Proliferation of these cells determines longitudinal bone growth and the matrix deposited provides a scaffold for future bone formation. However, these two energy-dependent anabolic processes occur in an avascular environment1,2. In addition, the centre of the expanding growth plate becomes hypoxic, and local activation of the hypoxia-inducible transcription factor HIF-1α is necessary for chondrocyte survival by unidentified cell-intrinsic mechanisms3-6. It is unknown whether there is a requirement for restriction of HIF-1α signalling in the other regions of the growth plate and whether chondrocyte metabolism controls cell function. Here we show that prolonged HIF-1α signalling in chondrocytes leads to skeletal dysplasia by interfering with cellular bioenergetics and biosynthesis. Decreased glucose oxidation results in an energy deficit, which limits proliferation, activates the unfolded protein response and reduces collagen synthesis. However, enhanced glutamine flux increases α-ketoglutarate levels, which in turn increases proline and lysine hydroxylation on collagen. This metabolically regulated collagen modification renders the cartilaginous matrix more resistant to protease-mediated degradation and thereby increases bone mass. Thus, inappropriate HIF-1α signalling results in skeletal dysplasia caused by collagen overmodification, an effect that may also contribute to other diseases involving the extracellular matrix such as cancer and fibrosis. ispartof: NATURE vol:565 issue:7740 pages:511-+ ispartof: location:England status: published

The pollen morphology of 63 African Impatiens species (Balsaminaceae) is studied using scanning electron microscopy. In addition, the pollen wall structure of five African Impatiens species was investigated with transmission electron microscopy. Pollen of African balsams is medium to large in size. Oblate Impatiens pollen is most common yet peroblate and suboblate pollen are also well represented. African pollen of the genus Impatiens is mainly circular, rectangular or elliptic. Reticulate sexine ornamentation is characteristic for most of the species investigated. Both homobrochate and heterobrochate pollen is present within the genus. Occasionally, the lumina are not entirely demarcated by muri, resulting in coalescence between adjacent lumina. The muri vary from slender with a tapering top edge, to slender with a rounded edge, to broad, more or less flattened muri. The tectal muri are always supported by two or three rows of short and massive columellae. Orbicules are absent in all species investigated. Observations of the pollen wall stratification show that pollen of African Impatiens consists of a thin foot layer, a lamellated endexine (characterized by remnants of the white lines) and a one-layered intine. Foot layer and endexine are slightly intermingled and therefore sometimes difficult to distinguish from each other.

Purpose: To compensate for the lack of haptic feedback by surgical robots, limitation of exerted forces could be implemented. The limits should be based on the observed relationship between tissue load and induced damage. This study examines whether age-related changes influence this relationship. Methods: Descending thoracic aortas of male C57BL/6J mice of 10, 25 and 40 weeks were clamped in vivo (no clamp, 0.5N or 2.0N) for 2 min. Functional integrity was tested in vitro by studying endothelium-dependent and -independent vasoreactivity. Results: Endothelium-dependent relaxation deteriorated with increased clamping force at all ages. Clamping did not influence endothelium-independent vasodilation. Age (10, 25 and 40 weeks) did not significantly impact on the effect of clamping on endothelium-dependent and independent vasoreactivity. Conclusions: Within the tested conditions, mechanical clamping induces damage to the vascular endothelium, but not to the smooth muscle cells. Age has no effect on the obtained results in mice from 10 to 40 weeks old.

Endothelial cell (EC) metabolism is an emerging target for anti-angiogenic therapy in tumor angiogenesis and choroidal neovascularization (CNV), but little is known about individual EC metabolic transcriptomes. By single-cell RNA sequencing 28,337 murine choroidal ECs (CECs) and sprouting CNV-ECs, we constructed a taxonomy to characterize their heterogeneity. Comparison with murine lung tumor ECs (TECs) revealed congruent marker gene expression by distinct EC phenotypes across tissues and diseases, suggesting similar angiogenic mechanisms. Trajectory inference predicted that differentiation of venous to angiogenic ECs was accompanied by metabolic transcriptome plasticity. ECs displayed metabolic transcriptome heterogeneity during cellcycle progression and in quiescence. Hypothesizing that conserved genes are important, we used an integrated analysis, based on congruent transcriptome analysis, CEC-tailored genome-scale metabolic modeling, and gene expression meta-analysis in cross-species datasets, followed by in vitro and in vivo validation, to identify SQLE and ALDH18A1 as previously unknown metabolic angiogenic targets. ispartof: Cell Metabolism vol:31 issue:4 pages:862-877 ispartof: location:United States status: published

The heterogeneity of endothelial cells (ECs) across tissues remains incompletely inventoried. We constructed an atlas of >32,000 single-EC transcriptomes from 11 mouse tissues and identified 78 EC subclusters, including Aqp7+ intestinal capillaries and angiogenic ECs in healthy tissues. ECs from brain/testis, liver/spleen, small intestine/colon, and skeletal muscle/heart pairwise expressed partially overlapping marker genes. Arterial, venous, and lymphatic ECs shared more markers in more tissues than did heterogeneous capillary ECs. ECs from different vascular beds (arteries, capillaries, veins, lymphatics) exhibited transcriptome similarity across tissues, but the tissue (rather than the vessel) type contributed to the EC heterogeneity. Metabolic transcriptome analysis revealed a similar tissue-grouping phenomenon of ECs and heterogeneous metabolic gene signatures in ECs between tissues and between vascular beds within a single tissue in a tissue-type-dependent pattern. The EC atlas taxonomy enabled identification of EC subclusters in public scRNA-seq datasets and provides a powerful discovery tool and resource value. ispartof: CELL vol:180 issue:4 pages:764-+ ispartof: location:United States status: published

Heterogeneity of lung tumor endothelial cell (TEC) phenotypes across patients, species (human/mouse), and models (in vivo/in vitro) remains poorly inventoried at the single-cell level. We single-cell RNA (scRNA)-sequenced 56,771 endothelial cells from human/mouse (peri)-tumoral lung and cultured human lung TECs, and detected 17 known and 16 previously unrecognized phenotypes, including TECs putatively regulating immune surveillance. We resolved the canonical tip TECs into a known migratory tip and a putative basement-membrane remodeling breach phenotype. Tip TEC signatures correlated with patient survival, and tip/breach TECs were most sensitive to vascular endothelial growth factor blockade. Only tip TECs were congruent across species/models and shared conserved markers. Integrated analysis of the scRNA-sequenced data with orthogonal multi-omics and meta-analysis data across different human tumors, validated by functional analysis, identified collagen modification as a candidate angiogenic pathway.

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. ispartof: NATURE vol:561 issue:7721 pages:63-+ ispartof: location:England status: published

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 β-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 .

Intrinsic and adaptive resistance hampers the success of antiangiogenic therapies (AAT), especially in breast cancer where this treatment modality has proven largely ineffective. Therefore, novel strategies to improve the efficacy of AAT are warranted. Solid tumors such as breast cancer are characterized by a high infiltration of myeloid-derived suppressor cells (MDSC), which are key drivers of resistance to AAT. Therefore, we hypothesized that all

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. ispartof: Nature vol:520 issue:7546 pages:192-7 ispartof: location:England status: published

Endothelial cell (EC) metabolism is emerging as a regulator of angiogenesis, but the precise role of glutamine metabolism in ECs is unknown. Here, we show that depriving ECs of glutamine or inhibiting glutaminase 1 (GLS1) caused vessel sprouting defects due to impaired proliferation and migration, and reduced pathological ocular angiogenesis. Inhibition of glutamine metabolism in ECs did not cause energy distress, but impaired tricarboxylic acid (TCA) cycle anaplerosis, macromolecule production, and redox homeostasis. Only the combination of TCA cycle replenishment plus asparagine supplementation restored the metabolic aberrations and proliferation defect caused by glutamine deprivation. Mechanistically, glutamine provided nitrogen for asparagine synthesis to sustain cellular homeostasis. While ECs can take up asparagine, silencing asparagine synthetase (ASNS, which converts glutamine-derived nitrogen and aspartate to asparagine) impaired EC sprouting even in the presence of glutamine and asparagine. Asparagine further proved crucial in glutamine-deprived ECs to restore protein synthesis, suppress ER stress, and reactivate mTOR signaling. These findings reveal a novel link between endothelial glutamine and asparagine metabolism in vessel sprouting. ispartof: EMBO Journal vol:36 issue:16 pages:2334-2352 ispartof: location:England status: published

Whether new neurons are added in the postnatal cerebral cortex is still debated. Here, we report that the meninges of perinatal mice contain a population of neurogenic progenitors formed during embryonic development that migrate to the caudal cortex and differentiate into Satb2+ neurons in cortical layers IIIV. The resulting neurons are electrically functional and integrated into local microcircuits. Single-cell RNA sequencing identified meningeal cells with distinct transcriptome signatures characteristic of (1) neurogenic radial glia-like cells (resembling neural stem cells in the SVZ), (2) neuronal cells, and (3) a cell type with an intermediate phenotype, possibly representing radial glia-like meningeal cells differentiating to neuronal cells. Thus, we have identified a pool of embryonically derived radial glia-like cells present in the meninges that migrate and differentiate into functional neurons in the neonatal cerebral cortex.

Background: Cardiovascular diseases remain the predominant cause of death worldwide, with the prevalence of heart failure continuing to increase. Despite increased knowledge of the metabolic alterations that occur in heart failure, novel therapies to treat the observed metabolic disturbances are still lacking. Methods: Mice were subjected to pressure overload by means of angiotensin-II infusion or transversal aortic constriction. MicroRNA-146a was either genetically or pharmacologically knocked out or genetically overexpressed in cardiomyocytes. Furthermore, overexpression of dihydrolipoyl succinyltransferase (DLST) in the murine heart was performed by means of an adeno-associated virus. Results: MicroRNA-146a was upregulated in whole heart tissue in multiple murine pressure overload models. Also, microRNA-146a levels were moderately increased in left ventricular biopsies of patients with aortic stenosis. Overexpression of microRNA-146a in cardiomyocytes provoked cardiac hypertrophy and left ventricular dysfunction in vivo, whereas genetic knockdown or pharmacological blockade of microRNA-146a blunted the hypertrophic response and attenuated cardiac dysfunction in vivo. Mechanistically, microRNA-146a reduced its target DLST—the E2 subcomponent of the α-ketoglutarate dehydrogenase complex, a rate-controlling tricarboxylic acid cycle enzyme. DLST protein levels significantly decreased on pressure overload in wild-type mice, paralleling a decreased oxidative metabolism, whereas DLST protein levels and hence oxidative metabolism were partially maintained in microRNA-146a knockout mice. Moreover, overexpression of DLST in wild-type mice protected against cardiac hypertrophy and dysfunction in vivo. Conclusions: Altogether we show that the microRNA-146a and its target DLST are important metabolic players in left ventricular dysfunction.

Background Edema represents a key feature of nasal polyp (NP) disease. Members of the vascular endothelial growth factor (VEGF) family may be involved, but the precise role of VEGF-A, VEGF-B, placental growth factor (PlGF), and their receptors VEGFR1 and VEGFR2 in NP edema formation remains elusive. Objective Exploring the expression of VEGF family members and their receptors and their correlation with clinical, radiological, and edema markers in NP. Methods The expression of VEGF-A, VEGF-B, PlGF, VEGFR1, and VEGFR2 was measured in NP (n = 23) and control tissue (n = 22) at mRNA and protein level. Edema was evaluated by measuring albumin levels and wet/dry ratios. Computed tomography (CT) scans were scored using the Lund-Mackay scoring system. IL-5 mRNA expression was determined by real-time RT-PCR. Cell suspensions from NP (n = 10) and control tissue (n = 12) were stimulated in vitro with IL-1β or TNFα. Results mRNA expression of VEGFR1 and VEGF-B was significantly higher in NP compared with control tissue. Expression levels of VEGF-B and VEGFR1 significantly correlated with NP albumin content (VEGF-B: P = 0.0208; VEGFR1: P = 0.0293), CT scan scores (VEGF-B: P = 0.0075; VEGFR1: P = 0.0068), and IL-5 mRNA (VEGF-B: P = 0.0027; VEGFR1: P = 0.0001). In vitro stimulation of control and NP tissue cell suspensions with IL-1β or TNFα significantly reduced the expression of VEGFR2 in control tissue, without altering VEGFR1 and VEGF-B expression. hVEGF-B induced nitric oxide production in NP macrophages (P

The importance of the blood- and lymph vessels in the transport of essential fluids, gases, macromolecules and cells in vertebrates warrants optimal insight into the regulatory mechanisms underlying their development. Mouse and zebrafish models of lymphatic development are instrumental for gene discovery and gene characterization but are challenging for certain aspects, e.g. no direct accessibility of embryonic stages, or non-straightforward visualization of early lymphatic sprouting, respectively. We previously demonstrated that the Xenopus tadpole is a valuable model to study the processes of lymphatic development. However, a fluorescent Xenopus reporter directly visualizing the lymph vessels was lacking. Here, we created transgenic Tg(Flk1:eGFP) Xenopus laevis reporter lines expressing green fluorescent protein (GFP) in blood- and lymph vessels driven by the Flk1 (VEGFR-2) promoter. We also established a high-resolution fluorescent dye labeling technique selectively and persistently visualizing lymphatic endothelial cells, even in conditions of impaired lymph vessel formation or drainage function upon silencing of lymphangiogenic factors. Next, we applied the model to dynamically document blood and lymphatic sprouting and patterning of the initially avascular tadpole fin. Furthermore, quantifiable models of spontaneous or induced lymphatic sprouting into the tadpole fin were developed for dynamic analysis of loss-of-function and gain-of-function phenotypes using pharmacologic or genetic manipulation. Together with angiography and lymphangiography to assess functionality, Tg(Flk1:eGFP) reporter tadpoles readily allowed detailed lymphatic phenotyping of live tadpoles by fluorescence microscopy. The Tg(Flk1:eGFP) tadpoles represent a versatile model for functional lymph/angiogenomics and drug screening. ispartof: Biology Open vol:2 issue:9 pages:882-90 ispartof: location:England status: published

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., Glycolytic regulator PFKFB3 is a key player in vessel sprouting. Here the authors develop a computational model predicting that PFKFB3 drives endothelial cell rearrangement during vessel sprouting by promoting filopodia formation and reducing intercellular adhesion, and empirically validate this prediction.

Microsporogenesis in Impatiens parviflora was studied with special attention to the mode of exine deposition and tapetum development. Our results are based on light, scanning electron and transmission electron microscopic observations of developing anthers of I. parviflora, from the microspore mother cell stage to anther dehiscence. In I. parviflora, the secretory tapetal cell layer is extended into the sporogenous tissue forming protrusions. The tapetal cytoplasm undergoes considerable changes during all stages of microsporogenesis such as fluctuations in volume densities of endoplasmic reticulum and plastids, occasional presence of two nuclei and the occurrence of a single very pronounced lipid droplet, which are indications that the tapetal cells are subject to cycles of different metabolic activity. Microsporogenesis in I. parviflora is simultaneous. Mature pollen is oblate, medium sized, 4-colpate, with a duplicolumellate reticulate sexine ornamentation. Anther dehiscence of I. parviflora in...

SummaryGrowing axons are guided to their targets by attractive and repulsive cues. In the developing spinal cord, Netrin-1 and Shh guide commissural axons toward the midline. However, the combined inhibition of their activity in commissural axon turning assays does not completely abrogate turning toward floor plate tissue, suggesting that additional guidance cues are present. Here we show that the prototypic angiogenic factor VEGF is secreted by the floor plate and is a chemoattractant for commissural axons in vitro and in vivo. Inactivation of Vegf in the floor plate or of its receptor Flk1 in commissural neurons causes axon guidance defects, whereas Flk1 blockade inhibits turning of axons to VEGF in vitro. Similar to Shh and Netrin-1, VEGF-mediated commissural axon guidance requires the activity of Src family kinases. Our results identify VEGF and Flk1 as a novel ligand/receptor pair controlling commissural axon guidance.

SummaryPolarization of tumor-associated macrophages (TAMs) to a proangiogenic/immune-suppressive (M2-like) phenotype and abnormal, hypoperfused vessels are hallmarks of malignancy, but their molecular basis and interrelationship remains enigmatic. We report that the host-produced histidine-rich glycoprotein (HRG) inhibits tumor growth and metastasis, while improving chemotherapy. By skewing TAM polarization away from the M2- to a tumor-inhibiting M1-like phenotype, HRG promotes antitumor immune responses and vessel normalization, effects known to decrease tumor growth and metastasis and to enhance chemotherapy. Skewing of TAM polarization by HRG relies substantially on downregulation of placental growth factor (PlGF). Besides unveiling an important role for TAM polarization in tumor vessel abnormalization, and its regulation by HRG/PlGF, these findings offer therapeutic opportunities for anticancer and antiangiogenic treatment.