Mitochondrial dysfunction results in enhanced adrenal androgen production in H295R cells.

The role of mitochondria in steroidogenesis is well established. However, the specific effects of mitochondrial dysfunction on androgen synthesis are not fully understood. In this study, we investigate the effects of various mitochondrial and metabolic inhibitors in H295R adrenal cells and perform a comprehensive analysis of steroid and metabolite profiling. We report that mitochondrial complex I inhibition by rotenone shifts cells toward anaerobic metabolism with a concomitant hyperandrogenic phenotype characterized by rapid stimulation of dehydroepiandrosterone (DHEA, 2 h) and slower accumulation of androstenedione and testosterone (24 h). Screening of metabolic inhibitors confirmed DHEA stimulation, which included mitochondrial complex III and mitochondrial pyruvate carrier inhibition. Metabolomic studies revealed truncated tricarboxylic acid cycle with an inverse correlation between citric acid and DHEA production as a common metabolic marker of hyperandrogenic inhibitors. The current study sheds light on a direct interplay between energy metabolism and androgen biosynthesis that could be further explored to identify novel molecular targets for efficient treatment of androgen excess disorders.

An evolutionarily conserved mechanism controls reversible amyloids of pyruvate kinase via pH-sensing regions.

Amyloids are known as irreversible aggregates associated with neurodegenerative diseases. However, recent evidence shows that a subset of amyloids can form reversibly and fulfill essential cellular functions. Yet, the molecular mechanisms regulating functional amyloids and distinguishing them from pathological aggregates remain unclear. Here, we investigate the conserved principles of amyloid reversibility by studying the essential metabolic enzyme pyruvate kinase (PK) in yeast and human cells. We demonstrate that yeast PK (Cdc19) and human PK (PKM2) form reversible amyloids through a pH-sensitive amyloid core. Stress-induced cytosolic acidification promotes aggregation via protonation of specific glutamate (yeast) or histidine (human) residues within the amyloid core. Mutations mimicking protonation cause constitutive PK aggregation, while non-protonatable PK mutants remain soluble even upon stress. Physiological PK aggregation is coupled to metabolic rewiring and glycolysis arrest, causing severe growth defects when misregulated. Our work thus identifies an evolutionarily conserved, potentially widespread mechanism regulating functional amyloids during stress.

Ancestral allele of DNA polymerase gamma modifies antiviral tolerance.

Mitochondria are critical modulators of antiviral tolerance through the release of mitochondrial RNA and DNA (mtDNA and mtRNA) fragments into the cytoplasm after infection, activating virus sensors and type-I interferon (IFN-I) response1-4. The relevance of these mechanisms for mitochondrial diseases remains understudied. Here we investigated mitochondrial recessive ataxia syndrome (MIRAS), which is caused by a common European founder mutation in DNA polymerase gamma (POLG1)5. Patients homozygous for the MIRAS variant p.W748S show exceptionally variable ages of onset and symptoms5, indicating that unknown modifying factors contribute to disease manifestation. We report that the mtDNA replicase POLG1 has a role in antiviral defence mechanisms to double-stranded DNA and positive-strand RNA virus infections (HSV-1, TBEV and SARS-CoV-2), and its p.W748S variant dampens innate immune responses. Our patient and knock-in mouse data show that p.W748S compromises mtDNA replisome stability, causing mtDNA depletion, aggravated by virus infection. Low mtDNA and mtRNA release into the cytoplasm and a slow IFN response in MIRAS offer viruses an early replicative advantage, leading to an augmented pro-inflammatory response, a subacute loss of GABAergic neurons and liver inflammation and necrosis. A population databank of around 300,000 Finnish individuals6 demonstrates enrichment of immunodeficient traits in carriers of the POLG1 p.W748S mutation. Our evidence suggests that POLG1 defects compromise antiviral tolerance, triggering epilepsy and liver disease. The finding has important implications for the mitochondrial disease spectrum, including epilepsy, ataxia and parkinsonism.

Glucose-derived glutamate drives neuronal terminal differentiation in vitro.

Neuronal maturation is the phase during which neurons acquire their final characteristics in terms of morphology, electrical activity, and metabolism. However, little is known about the metabolic pathways governing neuronal maturation. Here, we investigate the contribution of the main metabolic pathways, namely glucose, glutamine, and fatty acid oxidation, during the maturation of primary rat hippocampal neurons. Blunting glucose oxidation through the genetic and chemical inhibition of the mitochondrial pyruvate transporter reveals that this protein is critical for the production of glutamate, which is required for neuronal arborization, proper dendritic elongation, and spine formation. Glutamate supplementation in the early phase of differentiation restores morphological defects and synaptic function in mitochondrial pyruvate transporter-inhibited cells. Furthermore, the selective activation of metabotropic glutamate receptors restores the impairment of neuronal differentiation due to the reduced generation of glucose-derived glutamate and rescues synaptic local translation. Fatty acid oxidation does not impact neuronal maturation. Whereas glutamine metabolism is important for mitochondria, it is not for endogenous glutamate production. Our results provide insights into the role of glucose-derived glutamate as a key player in neuronal terminal differentiation.

Effect of high-dose glucocorticoid treatment on human brown adipose tissue activity: a randomised, double-blinded, placebo-controlled cross-over trial in healthy men.

BACKGROUND: Glucocorticoids (GCs) are widely applied anti-inflammatory drugs that are associated with adverse metabolic effects including insulin resistance and weight gain. Previous research indicates that GCs may negatively impact brown adipose tissue (BAT) activity in rodents and humans. METHODS: We performed a randomised, double-blinded cross-over trial in 16 healthy men (clinicaltrials.govNCT03269747). Participants received 40 mg of prednisone per day for one week or placebo. After a washout period of four weeks, participants crossed-over to the other treatment arm. Primary endpoint was the increase in resting energy expenditure (EE) in response to a mild-cold stimulus (cold-induced thermogenesis, CIT). Secondary outcomes comprised mean 18F-FDG uptake into supraclavicular BAT (SUVmean) as determined by FDG-PET/CT, volume of the BAT depot as well as fat content determined by MRI. The plasma metabolome and the transcriptome of supraclavicular BAT and of skeletal muscle biopsies after each treatment period were analysed. FINDINGS: Sixteen participants were recruited to the trial and completed it successfully per protocol. After prednisone treatment resting EE was higher both during warm and cold conditions. However, CIT was similar, 153 kcal/24 h (95% CI 40-266 kcal/24 h) after placebo and 186 kcal/24 h (95% CI 94-277 kcal/24 h, p = 0.38) after prednisone. SUVmean of BAT after cold exposure was not significantly affected by prednisone (3.36 g/ml, 95% CI 2.69-4.02 g/ml, vs 3.07 g/ml, 95% CI 2.52-3.62 g/ml, p = 0.28). Results of plasma metabolomics and BAT transcriptomics corroborated these findings. RNA sequencing of muscle biopsies revealed higher expression of genes involved in calcium cycling. No serious adverse events were reported and adverse events were evenly distributed between the two treatments. INTERPRETATION: Prednisone increased EE in healthy men possibly by altering skeletal muscle calcium cycling. Cold-induced BAT activity was not affected by GC treatment, which indicates that the unfavourable metabolic effects of GCs are independent from thermogenic adipocytes. FUNDING: Grants from Swiss National Science Foundation (PZ00P3_167823), Bangerter-Rhyner Foundation and from Nora van der Meeuwen-Häfliger Foundation to MJB. A fellowship-grant from the Swiss National Science Foundation (SNF211053) to WS. Grants from German Research Foundation (project number: 314061271-TRR 205) and Else Kröner-Fresenius (grant support 2012_A103 and 2015_A228) to MR.

Structure of the connexin-43 gap junction channel in a putative closed state.

Gap junction channels (GJCs) mediate intercellular communication by connecting two neighbouring cells and enabling direct exchange of ions and small molecules. Cell coupling via connexin-43 (Cx43) GJCs is important in a wide range of cellular processes in health and disease (Churko and Laird, 2013; Liang et al., 2020; Poelzing and Rosenbaum, 2004), yet the structural basis of Cx43 function and regulation has not been determined until now. Here, we describe the structure of a human Cx43 GJC solved by cryo-EM and single particle analysis at 2.26 Å resolution. The pore region of Cx43 GJC features several lipid-like densities per Cx43 monomer, located close to a putative lateral access site at the monomer boundary. We found a previously undescribed conformation on the cytosolic side of the pore, formed by the N-terminal domain and the transmembrane helix 2 of Cx43 and stabilized by a small molecule. Structures of the Cx43 GJC and hemichannels (HCs) in nanodiscs reveal a similar gate arrangement. The features of the Cx43 GJC and HC cryo-EM maps and the channel properties revealed by molecular dynamics simulations suggest that the captured states of Cx43 are consistent with a closed state.

Metabolic landscape of the male mouse gut identifies different niches determined by microbial activities.

Distinct niches of the mammalian gut are populated by diverse microbiota, but the contribution of spatial variation to intestinal metabolism remains unclear. Here we present a map of the longitudinal metabolome along the gut of healthy colonized and germ-free male mice. With this map, we reveal a general shift from amino acids in the small intestine to organic acids, vitamins and nucleotides in the large intestine. We compare the metabolic landscapes in colonized versus germ-free mice to disentangle the origin of many metabolites in different niches, which in some cases allows us to infer the underlying processes or identify the producing species. Beyond the known impact of diet on the small intestinal metabolic niche, distinct spatial patterns suggest specific microbial influence on the metabolome in the small intestine. Thus, we present a map of intestinal metabolism and identify metabolite-microbe associations, which provide a basis to connect the spatial occurrence of bioactive compounds to host or microorganism metabolism.

Mitochondrial pyruvate metabolism regulates the activation of quiescent adult neural stem cells.

Cellular metabolism is important for adult neural stem/progenitor cell (NSPC) behavior. However, its role in the transition from quiescence to proliferation is not fully understood. We here show that the mitochondrial pyruvate carrier (MPC) plays a crucial and unexpected part in this process. MPC transports pyruvate into mitochondria, linking cytosolic glycolysis to mitochondrial tricarboxylic acid cycle and oxidative phosphorylation. Despite its metabolic key function, the role of MPC in NSPCs has not been addressed. We show that quiescent NSPCs have an active mitochondrial metabolism and express high levels of MPC. Pharmacological MPC inhibition increases aspartate and triggers NSPC activation. Furthermore, genetic Mpc1 ablation in vitro and in vivo also activates NSPCs, which differentiate into mature neurons, leading to overall increased hippocampal neurogenesis in adult and aged mice. These findings highlight the importance of metabolism for NSPC regulation and identify an important pathway through which mitochondrial pyruvate import controls NSPC quiescence and activation.

ROS Induction Targets Persister Cancer Cells with Low Metabolic Activity in NRAS-Mutated Melanoma.

Clinical management of melanomas with NRAS mutations is challenging. Targeting MAPK signaling is only beneficial to a small subset of patients due to resistance that arises through genetic, transcriptional, and metabolic adaptation. Identification of targetable vulnerabilities in NRAS-mutated melanoma could help improve patient treatment. Here, we used multiomics analyses to reveal that NRAS-mutated melanoma cells adopt a mesenchymal phenotype with a quiescent metabolic program to resist cellular stress induced by MEK inhibition. The metabolic alterations elevated baseline reactive oxygen species (ROS) levels, leading these cells to become highly sensitive to ROS induction. In vivo xenograft experiments and single-cell RNA sequencing demonstrated that intratumor heterogeneity necessitates the combination of a ROS inducer and a MEK inhibitor to inhibit both tumor growth and metastasis. Ex vivo pharmacoscopy of 62 human metastatic melanomas confirmed that MEK inhibitor-resistant tumors significantly benefited from the combination therapy. Finally, oxidative stress response and translational suppression corresponded with ROS-inducer sensitivity in 486 cancer cell lines, independent of cancer type. These findings link transcriptional plasticity to a metabolic phenotype that can be inhibited by ROS inducers in melanoma and other cancers. SIGNIFICANCE: Metabolic reprogramming in drug-resistant NRAS-mutated melanoma cells confers sensitivity to ROS induction, which suppresses tumor growth and metastasis in combination with MAPK pathway inhibitors.

Basal re-esterification finetunes mitochondrial fatty acid utilization.

OBJECTIVE: Emerging evidence suggest the existence of constant basal lipolysis and re-esterification of a substantial fraction of thus liberated fatty acids. In stimulated lipolysis, the re-esterification is proposed to be a protective mechanism against lipotoxicity; however, the role of the lipolysis coupled to re-esterification under basal conditions has not been deciphered. METHODS: We used adipocytes (in vitro differentiated brown and white adipocytes derived from a cell line or primary SVF culture) to study the effect of inhibition of re-esterification by pharmacological DGAT1 and DGAT2 inhibitors alone or in combination. We then evaluated cellular energetics, lipolysis flux, and lipidomic parameters along with mitochondrial properties and fuel utilization. RESULTS: In adipocytes, DGAT1 and 2 mediated re-esterification is a moderator of fatty acid oxidation. Combined inhibition of both DGATs (D1+2i) increases oxygen consumption, which is largely due to enhanced mitochondrial respiration by lipolysis-derived fatty acids (FAs). Acute D1+2i selectively affects mitochondrial respiration without affecting the transcriptional homeostasis of genes relevant to mitochondrial health and lipid metabolism. D1+2i enhances the mitochondrial import of pyruvate and activates AMP Kinase to counteract CPT1 antagonism, thus facilitating the mitochondrial import of fatty acyl-CoA. CONCLUSIONS: These data implicate the process of re-esterification in the regulation of mitochondrial FA usage and uncover a mechanism of FAO regulation via crosstalk with FA re-esterification.

Recent advances in mass spectrometry-based computational metabolomics.

The computational metabolomics field brings together computer scientists, bioinformaticians, chemists, clinicians, and biologists to maximize the impact of metabolomics across a wide array of scientific and medical disciplines. The field continues to expand as modern instrumentation produces datasets with increasing complexity, resolution, and sensitivity. These datasets must be processed, annotated, modeled, and interpreted to enable biological insight. Techniques for visualization, integration (within or between omics), and interpretation of metabolomics data have evolved along with innovation in the databases and knowledge resources required to aid understanding. In this review, we highlight recent advances in the field and reflect on opportunities and innovations in response to the most pressing challenges. This review was compiled from discussions from the 2022 Dagstuhl seminar entitled "Computational Metabolomics: From Spectra to Knowledge".

Regularized adversarial learning for normalization of multi-batch untargeted metabolomics data.

MOTIVATION: Untargeted metabolomics by mass spectrometry is the method of choice for unbiased analysis of molecules in complex samples of biological, clinical or environmental relevance. The exceptional versatility and sensitivity of modern high-resolution instruments allows profiling of thousands of known and unknown molecules in parallel. Inter-batch differences constitute a common and unresolved problem in untargeted metabolomics, and hinder the analysis of multi-batch studies or the intercomparison of experiments. RESULTS: We present a new method, Regularized Adversarial Learning Preserving Similarity (RALPS), for the normalization of multi-batch untargeted metabolomics data. RALPS builds on deep adversarial learning with a three-term loss function that mitigates batch effects while preserving biological identity, spectral properties and coefficients of variation. Using two large metabolomics datasets, we showcase the superior performance of RALPS as compared with six state-of-the-art methods for batch correction. Further, we demonstrate that RALPS scales well, is robust, deals with missing values and can handle different experimental designs. AVAILABILITY AND IMPLEMENTATION: https://github.com/zamboni-lab/RALPS. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Integrated multi-omics reveals anaplerotic rewiring in methylmalonyl-CoA mutase deficiency.

Methylmalonic aciduria (MMA) is an inborn error of metabolism with multiple monogenic causes and a poorly understood pathogenesis, leading to the absence of effective causal treatments. Here we employ multi-layered omics profiling combined with biochemical and clinical features of individuals with MMA to reveal a molecular diagnosis for 177 out of 210 (84%) cases, the majority (148) of whom display pathogenic variants in methylmalonyl-CoA mutase (MMUT). Stratification of these data layers by disease severity shows dysregulation of the tricarboxylic acid cycle and its replenishment (anaplerosis) by glutamine. The relevance of these disturbances is evidenced by multi-organ metabolomics of a hemizygous Mmut mouse model as well as through identification of physical interactions between MMUT and glutamine anaplerotic enzymes. Using stable-isotope tracing, we find that treatment with dimethyl-oxoglutarate restores deficient tricarboxylic acid cycling. Our work highlights glutamine anaplerosis as a potential therapeutic intervention point in MMA.

A functional analysis of 180 cancer cell lines reveals conserved intrinsic metabolic programs.

Cancer cells reprogram their metabolism to support growth and invasion. While previous work has highlighted how single altered reactions and pathways can drive tumorigenesis, it remains unclear how individual changes propagate at the network level and eventually determine global metabolic activity. To characterize the metabolic lifestyle of cancer cells across pathways and genotypes, we profiled the intracellular metabolome of 180 pan-cancer cell lines grown in identical conditions. For each cell line, we estimated activity for 49 pathways spanning the entirety of the metabolic network. Upon clustering, we discovered a convergence into only two major metabolic types. These were functionally confirmed by 13 C-flux analysis, lipidomics, and analysis of sensitivity to perturbations. They revealed that the major differences in cancers are associated with lipid, TCA cycle, and carbohydrate metabolism. Thorough integration of these types with multiomics highlighted little association with genetic alterations but a strong association with markers of epithelial-mesenchymal transition. Our analysis indicates that in absence of variations imposed by the microenvironment, cancer cells adopt distinct metabolic programs which serve as vulnerabilities for therapy.

A novel function of the key nitrogen-fixation activator NifA in beta-rhizobia: Repression of bacterial auxin synthesis during symbiosis.

Rhizobia fix nitrogen within root nodules of host plants where nitrogenase expression is strictly controlled by its key regulator NifA. We recently discovered that in nodules infected by the beta-rhizobial strain Paraburkholderia phymatum STM815, NifA controls expression of two bacterial auxin synthesis genes. Both the iaaM and iaaH transcripts, as well as the metabolites indole-acetamide (IAM) and indole-3-acetic acid (IAA) showed increased abundance in nodules occupied by a nifA mutant compared to wild-type nodules. Here, we document the structural changes that a P. phymatum nifA mutant induces in common bean (Phaseolus vulgaris) nodules, eventually leading to hypernodulation. To investigate the role of the P. phymatum iaaMH genes during symbiosis, we monitored their expression in presence and absence of NifA over different stages of the symbiosis. The iaaMH genes were found to be under negative control of NifA in all symbiotic stages. While a P. phymatum iaaMH mutant produced the same number of nodules and nitrogenase activity as the wild-type strain, the nifA mutant produced more nodules than the wild-type that clustered into regularly-patterned root zones. Mutation of the iaaMH genes in a nifA mutant background reduced the presence of these nodule clusters on the root. We further show that the P. phymatum iaaMH genes are located in a region of the symbiotic plasmid with a significantly lower GC content and exhibit high similarity to two genes of the IAM pathway often used by bacterial phytopathogens to deploy IAA as a virulence factor. Overall, our data suggest that the increased abundance of rhizobial auxin in the non-fixing nifA mutant strain enables greater root infection rates and a role for bacterial auxin production in the control of early stage symbiotic interactions.

A system suitability testing platform for untargeted, high-resolution mass spectrometry.

The broad coverage of untargeted metabolomics poses fundamental challenges for the harmonization of measurements along time, even if they originate from the very same instrument. Internal isotopic standards can hardly cover the chemical complexity of study samples. Therefore, they are insufficient for normalizing data a posteriori as done for targeted metabolomics. Instead, it is crucial to verify instrument's performance a priori, that is, before samples are injected. Here, we propose a system suitability testing platform for time-of-flight mass spectrometers independent of liquid chromatography. It includes a chemically defined quality control mixture, a fast acquisition method, software for extracting ca. 3,000 numerical features from profile data, and a simple web service for monitoring. We ran a pilot for 21 months and present illustrative results for anomaly detection or learning causal relationships between the spectral features and machine settings. Beyond mere detection of anomalies, our results highlight several future applications such as 1) recommending instrument retuning strategies to achieve desired values of quality indicators, 2) driving preventive maintenance, and 3) using the obtained, detailed spectral features for posterior data harmonization.

Systematic multi-level analysis of an organelle proteome reveals new peroxisomal functions.

Seventy years following the discovery of peroxisomes, their complete proteome, the peroxi-ome, remains undefined. Uncovering the peroxi-ome is crucial for understanding peroxisomal activities and cellular metabolism. We used high-content microscopy to uncover peroxisomal proteins in the model eukaryote - Saccharomyces cerevisiae. This strategy enabled us to expand the known peroxi-ome by ~40% and paved the way for performing systematic, whole-organellar proteome assays. By characterizing the sub-organellar localization and protein targeting dependencies into the organelle, we unveiled non-canonical targeting routes. Metabolomic analysis of the peroxi-ome revealed the role of several newly identified resident enzymes. Importantly, we found a regulatory role of peroxisomes during gluconeogenesis, which is fundamental for understanding cellular metabolism. With the current recognition that peroxisomes play a crucial part in organismal physiology, our approach lays the foundation for deep characterization of peroxisome function in health and disease.

c-MAF coordinates enterocyte zonation and nutrient uptake transcriptional programs.

Small intestinal villi are structural and functional units present in higher vertebrates and uniquely adapted to nutrient absorption. Villus enterocytes are organized in transcriptional "zones" dedicated to specialized tasks such as absorption of specific nutrients. We report that the transcription factor c-MAF is expressed in differentiated lower and mid-villus enterocytes and is a target of BMP signaling. Maf inactivation perturbed the villus zonation program by increasing carbohydrate-related transcripts while suppressing transcripts linked to amino-acid and lipid absorption. The formation of cytoplasmic lipid droplets, shuttling dietary fat to chylomicrons, was impaired upon Maf loss indicating its role in dietary lipid handling. Maf inactivation under homeostatic conditions expanded tuft cells and led to compensatory gut lengthening, preventing weight loss. However, delayed Maf-/- enterocyte maturation impaired weight recovery after acute intestinal injury, resulting in reduced survival. Our results identify c-MAF as a regulator of the intestinal villus zonation program, while highlighting the importance of coordination between stem/progenitor and differentiation programs for intestinal regeneration.

Targeting lactate dehydrogenase B-dependent mitochondrial metabolism affects tumor initiating cells and inhibits tumorigenesis of non-small cell lung cancer by inducing mtDNA damage.

Once considered a waste product of anaerobic cellular metabolism, lactate has been identified as a critical regulator of tumorigenesis, maintenance, and progression. The putative primary function of lactate dehydrogenase B (LDHB) is to catalyze the conversion of lactate to pyruvate; however, its role in regulating metabolism during tumorigenesis is largely unknown. To determine whether LDHB plays a pivotal role in tumorigenesis, we performed 2D and 3D in vitro experiments, utilized a conventional xenograft tumor model, and developed a novel genetically engineered mouse model (GEMM) of non-small cell lung cancer (NSCLC), in which we combined an LDHB deletion allele with an inducible model of lung adenocarcinoma driven by the concomitant loss of p53 (also known as Trp53) and expression of oncogenic KRAS (G12D) (KP). Here, we show that epithelial-like, tumor-initiating NSCLC cells feature oxidative phosphorylation (OXPHOS) phenotype that is regulated by LDHB-mediated lactate metabolism. We show that silencing of LDHB induces persistent mitochondrial DNA damage, decreases mitochondrial respiratory complex activity and OXPHOS, resulting in reduced levels of mitochondria-dependent metabolites, e.g., TCA intermediates, amino acids, and nucleotides. Inhibition of LDHB dramatically reduced the survival of tumor-initiating cells and sphere formation in vitro, which can be partially restored by nucleotide supplementation. In addition, LDHB silencing reduced tumor initiation and growth of xenograft tumors. Furthermore, we report for the first time that homozygous deletion of LDHB significantly reduced lung tumorigenesis upon the concomitant loss of Tp53 and expression of oncogenic KRAS without considerably affecting the animal's health status, thereby identifying LDHB as a potential target for NSCLC therapy. In conclusion, our study shows for the first time that LDHB is essential for the maintenance of mitochondrial metabolism, especially nucleotide metabolism, demonstrating that LDHB is crucial for the survival and proliferation of NSCLC tumor-initiating cells and tumorigenesis.

ADAMTS18+ villus tip telocytes maintain a polarized VEGFA signaling domain and fenestrations in nutrient-absorbing intestinal blood vessels.

The small intestinal villus tip is the first point of contact for lumen-derived substances including nutrients and microbial products. Electron microscopy studies from the early 1970s uncovered unusual spatial organization of small intestinal villus tip blood vessels: their exterior, epithelial-facing side is fenestrated, while the side facing the villus stroma is non-fenestrated, covered by pericytes and harbors endothelial nuclei. Such organization optimizes the absorption process, however the molecular mechanisms maintaining this highly specialized structure remain unclear. Here we report that perivascular LGR5+ villus tip telocytes (VTTs) are necessary for maintenance of villus tip endothelial cell polarization and fenestration by sequestering VEGFA signaling. Mechanistically, unique VTT expression of the protease ADAMTS18 is necessary for VEGFA signaling sequestration through limiting fibronectin accumulation. Therefore, we propose a model in which LGR5+ ADAMTS18+ telocytes are necessary to maintain a "just-right" level and location of VEGFA signaling in intestinal villus blood vasculature to ensure on one hand the presence of sufficient endothelial fenestrae, while avoiding excessive leakiness of the vessels and destabilization of villus tip epithelial structures.