Mechanisms and regulation of endothelial VEGF receptor signalling.

Vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs) are uniquely required to balance the formation of new blood vessels with the maintenance and remodelling of existing ones, during development and in adult tissues. Recent advances have greatly expanded our understanding of the tight and multi-level regulation of VEGFR2 signalling, which is the primary focus of this Review. Important insights have been gained into the regulatory roles of VEGFR-interacting proteins (such as neuropilins, proteoglycans, integrins and protein tyrosine phosphatases); the dynamics of VEGFR2 endocytosis, trafficking and signalling; and the crosstalk between VEGF-induced signalling and other endothelial signalling cascades. A clear understanding of this multifaceted signalling web is key to successful therapeutic suppression or stimulation of vascular growth.

Endothelium-derived lactate is required for pericyte function and blood-brain barrier maintenance.

Endothelial cells differ from other cell types responsible for the formation of the vascular wall in their unusual reliance on glycolysis for most energy needs, which results in extensive production of lactate. We find that endothelium-derived lactate is taken up by pericytes, and contributes substantially to pericyte metabolism including energy generation and amino acid biosynthesis. Endothelial-pericyte proximity is required to facilitate the transport of endothelium-derived lactate into pericytes. Inhibition of lactate production in the endothelium by deletion of the glucose transporter-1 (GLUT1) in mice results in loss of pericyte coverage in the retina and brain vasculatures, leading to the blood-brain barrier breakdown and increased permeability. These abnormalities can be largely restored by oral lactate administration. Our studies demonstrate an unexpected link between endothelial and pericyte metabolisms and the role of endothelial lactate production in the maintenance of the blood-brain barrier integrity. In addition, our observations indicate that lactate supplementation could be a useful therapeutic approach for GLUT1 deficiency metabolic syndrome patients.

SUMOylation Fine-Tunes Endothelial HEY1 in the Regulation of Angiogenesis.

BACKGROUND: Angiogenesis, which plays a critical role in embryonic development and tissue repair, is controlled by a set of angiogenic signaling pathways. As a TF (transcription factor) belonging to the basic helix-loop-helix family, HEY (hairy/enhancer of split related with YRPW motif)-1 (YRPW motif, abbreviation of 4 highly conserved amino acids in the motif) has been identified as a key player in developmental angiogenesis. However, the precise mechanisms underlying HEY1's actions in angiogenesis remain largely unknown. Our previous studies have suggested a potential role for posttranslational SUMOylation in the dynamic regulation of vascular development and organization. METHODS: Immunoprecipitation, mass spectrometry, and bioinformatics analysis were used to determine the biochemical characteristics of HEY1 SUMOylation. The promoter-binding capability of HEY1 was determined by chromatin immunoprecipitation, dual luciferase, and electrophoretic mobility shift assays. The dimerization pattern of HEY1 was determined by coimmunoprecipitation. The angiogenic capabilities of endothelial cells were assessed by CCK-8 (cell counting kit-8), 5-ethynyl-2-deoxyuridine staining, wound healing, transwell, and sprouting assays. Embryonic and postnatal vascular growth in mouse tissues, matrigel plug assay, cutaneous wound healing model, oxygen-induced retinopathy model, and tumor angiogenesis model were used to investigate the angiogenesis in vivo. RESULTS: We identified intrinsic endothelial HEY1 SUMOylation at conserved lysines by TRIM28 (tripartite motif containing 28) as the unique E3 ligase. Functionally, SUMOylation facilitated HEY1-mediated suppression of angiogenic RTK (receptor tyrosine kinase) signaling and angiogenesis in primary human endothelial cells and mice with endothelial cell-specific expression of wild-type HEY1 or a SUMOylation-deficient HEY1 mutant. Mechanistically, SUMOylation facilitates HEY1 homodimer formation, which in turn preserves HEY1's DNA-binding capability via recognition of E-box promoter elements. Therefore, SUMOylation maintains HEY1's function as a repressive TF controlling numerous angiogenic genes, including RTKs and Notch pathway components. Proangiogenic stimuli induce HEY1 deSUMOylation, leading to heterodimerization of HEY1 with HES (hairy and enhancer of split)-1, which results in ineffective DNA binding and loss of HEY1's angiogenesis-suppressive activity. CONCLUSIONS: Our findings demonstrate that reversible HEY1 SUMOylation is a molecular mechanism that coordinates endothelial angiogenic signaling and angiogenesis, both in physiological and pathological milieus, by fine-tuning the transcriptional activity of HEY1. Specifically, SUMOylation facilitates the formation of the HEY1 transcriptional complex and enhances its DNA-binding capability in endothelial cells.

Endothelial-derived angiocrine signals induce and sustain regenerative lung alveolarization.

To identify pathways involved in adult lung regeneration, we employ a unilateral pneumonectomy (PNX) model that promotes regenerative alveolarization in the remaining intact lung. We show that PNX stimulates pulmonary capillary endothelial cells (PCECs) to produce angiocrine growth factors that induce proliferation of epithelial progenitor cells supporting alveologenesis. Endothelial cells trigger expansion of cocultured epithelial cells, forming three-dimensional angiospheres reminiscent of alveolar-capillary sacs. After PNX, endothelial-specific inducible genetic ablation of Vegfr2 and Fgfr1 in mice inhibits production of MMP14, impairing alveolarization. MMP14 promotes expansion of epithelial progenitor cells by unmasking cryptic EGF-like ectodomains that activate the EGF receptor (EGFR). Consistent with this, neutralization of MMP14 impairs EGFR-mediated alveolar regeneration, whereas administration of EGF or intravascular transplantation of MMP14(+) PCECs into pneumonectomized Vegfr2/Fgfr1-deficient mice restores alveologenesis and lung inspiratory volume and compliance function. VEGFR2 and FGFR1 activation in PCECs therefore increases MMP14-dependent bioavailability of EGFR ligands to initiate and sustain alveologenesis.

TNFα increases the degradation of pyruvate dehydrogenase kinase 4 by the Lon protease to support proinflammatory genes.

The endothelium is a major target of the proinflammatory cytokine, tumor necrosis factor alpha (TNFα). Exposure of endothelial cells (EC) to proinflammatory stimuli leads to an increase in mitochondrial metabolism; however, the function and regulation of elevated mitochondrial metabolism in EC in response to proinflammatory cytokines remain unclear. Studies using high-resolution metabolomics and 13C-glucose and 13C-glutamine labeling flux techniques showed that pyruvate dehydrogenase activity (PDH) and oxidative tricarboxylic acid cycle (TCA) flux are elevated in human umbilical vein ECs in response to overnight (16 h) treatment with TNFα (10 ng/mL). Mechanistic studies indicated that TNFα mediated these metabolic changes via mitochondrial-specific protein degradation of pyruvate dehydrogenase kinase 4 (PDK4, inhibitor of PDH) by the Lon protease via an NF-κB-dependent mechanism. Using RNA sequencing following siRNA-mediated knockdown of the catalytically active subunit of PDH, PDHE1α (PDHA1 gene), we show that PDH flux controls the transcription of approximately one-third of the genes that are up-regulated by TNFα stimulation. Notably, TNFα-induced PDH flux regulates a unique signature of proinflammatory mediators (cytokines and chemokines) but not inducible adhesion molecules. Metabolomics and ChIP sequencing for acetylated modification on lysine 27 of histone 3 (H3K27ac) showed that TNFα-induced PDH flux promotes histone acetylation of specific gene loci via citrate accumulation and ATP-citrate lyase-mediated generation of acetyl CoA. Together, these results uncover a mechanism by which TNFα signaling increases oxidative TCA flux of glucose to support TNFα-induced gene transcription through extramitochondrial acetyl CoA generation and histone acetylation.

Chylomicrons Regulate Lacteal Permeability and Intestinal Lipid Absorption.

BACKGROUND: Lymphatic vessels are responsible for tissue drainage, and their malfunction is associated with chronic diseases. Lymph uptake occurs via specialized open cell-cell junctions between capillary lymphatic endothelial cells (LECs), whereas closed junctions in collecting LECs prevent lymph leakage. LEC junctions are known to dynamically remodel in development and disease, but how lymphatic permeability is regulated remains poorly understood. METHODS: We used various genetically engineered mouse models in combination with cellular, biochemical, and molecular biology approaches to elucidate the signaling pathways regulating junction morphology and function in lymphatic capillaries. RESULTS: By studying the permeability of intestinal lacteal capillaries to lipoprotein particles known as chylomicrons, we show that ROCK (Rho-associated kinase)-dependent cytoskeletal contractility is a fundamental mechanism of LEC permeability regulation. We show that chylomicron-derived lipids trigger neonatal lacteal junction opening via ROCK-dependent contraction of junction-anchored stress fibers. LEC-specific ROCK deletion abolished junction opening and plasma lipid uptake. Chylomicrons additionally inhibited VEGF (vascular endothelial growth factor)-A signaling. We show that VEGF-A antagonizes LEC junction opening via VEGFR (VEGF receptor) 2 and VEGFR3-dependent PI3K (phosphatidylinositol 3-kinase)/AKT (protein kinase B) activation of the small GTPase RAC1 (Rac family small GTPase 1), thereby restricting RhoA (Ras homolog family member A)/ROCK-mediated cytoskeleton contraction. CONCLUSIONS: Our results reveal that antagonistic inputs into ROCK-dependent cytoskeleton contractions regulate the interconversion of lymphatic junctions in the intestine and in other tissues, providing a tunable mechanism to control the lymphatic barrier.

Endothelial FIS1 DeSUMOylation Protects Against Hypoxic Pulmonary Hypertension.

BACKGROUND: Hypoxia is a major cause and promoter of pulmonary hypertension (PH), a representative vascular remodeling disease with poor prognosis and high mortality. However, the mechanism underlying how pulmonary arterial system responds to hypoxic stress during PH remains unclear. Endothelial mitochondria are considered signaling organelles on oxygen tension. Results from previous clinical research and our studies suggested a potential role of posttranslational SUMOylation (small ubiquitin-like modifier modification) in endothelial mitochondria in hypoxia-related vasculopathy. METHODS: Chronic hypoxia mouse model and Sugen/hypoxia rat model were employed as PH animal models. Mitochondrial morphology and subcellular structure were determined by transmission electron and immunofluorescent microscopies. Mitochondrial metabolism was determined by mitochondrial oxygen consumption rate and extracellular acidification rate. SUMOylation and protein interaction were determined by immunoprecipitation. RESULTS: The involvement of SENP1 (sentrin-specific protease 1)-mediated SUMOylation in mitochondrial remodeling in the pulmonary endothelium was identified in clinical specimens of hypoxia-related PH and was verified in human pulmonary artery endothelial cells under hypoxia. Further analyses in clinical specimens, hypoxic rat and mouse PH models, and human pulmonary artery endothelial cells and human embryonic stem cell-derived endothelial cells revealed that short-term hypoxia-induced SENP1 translocation to endothelial mitochondria to regulate deSUMOylation (the reversible process of SUMOylation) of mitochondrial fission protein FIS1 (mitochondrial fission 1), which facilitated FIS1 assembling with fusion protein MFN2 (mitofusin 2) and mitochondrial gatekeeper VDAC1 (voltage-dependent anion channel 1), and the membrane tethering activity of MFN2 by enhancing its oligomerization. Consequently, FIS1 deSUMOylation maintained the mitochondrial integrity and endoplasmic reticulum-mitochondria calcium communication across mitochondrial-associated membranes, subsequently preserving pulmonary endothelial function and vascular homeostasis. In contrast, prolonged hypoxia disabled the FIS1 deSUMOylation by diminishing the availability of SENP1 in mitochondria via inducing miR (micro RNA)-138 and consequently resulted in mitochondrial dysfunction and metabolic reprogramming in pulmonary endothelium. Functionally, introduction of viral-packaged deSUMOylated FIS1 within pulmonary endothelium in mice improved pulmonary endothelial dysfunction and hypoxic PH development, while knock-in of SUMO (small ubiquitin-like modifier)-conjugated FIS1 in mice exaggerated the diseased cellular and tissue phenotypes. CONCLUSIONS: By maintaining endothelial mitochondrial homeostasis, deSUMOylation of FIS1 adaptively preserves pulmonary endothelial function against hypoxic stress and consequently protects against PH. The FIS1 deSUMOylation-SUMOylation transition in pulmonary endothelium is an intrinsic pathogenesis of hypoxic PH.

FGFR1 SUMOylation coordinates endothelial angiogenic signaling in angiogenesis.

Angiogenesis contributes fundamentally to embryonic development, tissue homeostasis, and wound healing. Basic fibroblast growth factor (FGF2) is recognized as the first proangiogenic molecule discovered, and it facilitates angiogenesis by activating FGF receptor 1 (FGFR1) signaling in endothelial cells. However, the precise roles of FGFR and the FGF/FGFR signaling axis in angiogenesis remain unclear, especially because of the contradictory phenotypes of in vivo FGF and FGFR gene deficiency models. Our previous study results suggested a potential role of posttranslational small ubiquitin-like modifier modification (SUMOylation), with highly dynamic regulatory features, in vascular development and disorder. Here, we identified SENP1-regulated endothelial FGFR1 SUMOylation at conserved lysines responding to proangiogenic stimuli, while SENP1 functioned as the deSUMOylase. Hypoxia-enhanced FGFR1 SUMOylation restricted the tyrosine kinase activation of FGFR1 by modulating the dimerization of FGFR1 and FGFR1 binding with its phosphatase PTPRG. Consequently, it facilitated the recruitment of FRS2α to VEGFR2 but limited additional recruitment of FRS2α to FGFR1, supporting the activation of VEGFA/VEGFR2 signaling in endothelial cells. Furthermore, SUMOylation-defective mutation of FGFR1 resulted in exaggerated FGF2/FGFR1 signaling but suppressed VEGFA/VEGFR2 signaling and the angiogenic capabilities of endothelial cells, which were rescued by FRS2α overexpression. Reduced angiogenesis and endothelial sprouting in mice bearing an endothelial-specific, FGFR1 SUMOylation-defective mutant confirmed the functional significance of endothelial FGFR1 SUMOylation in vivo. Our findings identify the reversible SUMOylation of FGFR1 as an intrinsic fine-tuned mechanism in coordinating endothelial angiogenic signaling during neovascularization; SENP1-regulated FGFR1 SUMOylation and deSUMOylation controls the competitive recruitment of FRS2α by FGFR1 and VEGFR2 to switch receptor-complex formation responding to hypoxia and normoxia angiogenic environments.

Heterozygous deficiency of PHD2 restores tumor oxygenation and inhibits metastasis via endothelial normalization.

A key function of blood vessels, to supply oxygen, is impaired in tumors because of abnormalities in their endothelial lining. PHD proteins serve as oxygen sensors and may regulate oxygen delivery. We therefore studied the role of endothelial PHD2 in vessel shaping by implanting tumors in PHD2(+/-) mice. Haplodeficiency of PHD2 did not affect tumor vessel density or lumen size, but normalized the endothelial lining and vessel maturation. This resulted in improved tumor perfusion and oxygenation and inhibited tumor cell invasion, intravasation, and metastasis. Haplodeficiency of PHD2 redirected the specification of endothelial tip cells to a more quiescent cell type, lacking filopodia and arrayed in a phalanx formation. This transition relied on HIF-driven upregulation of (soluble) VEGFR-1 and VE-cadherin. Thus, decreased activity of an oxygen sensor in hypoxic conditions prompts endothelial cells to readjust their shape and phenotype to restore oxygen supply. Inhibition of PHD2 may offer alternative therapeutic opportunities for anticancer therapy.

MEKK3-TGFß crosstalk regulates inward arterial remodeling.

Arterial remodeling is an important adaptive mechanism that maintains normal fluid shear stress in a variety of physiologic and pathologic conditions. Inward remodeling, a process that leads to reduction in arterial diameter, plays a critical role in progression of such common diseases as hypertension and atherosclerosis. Yet, despite its pathogenic importance, molecular mechanisms controlling inward remodeling remain undefined. Mitogen-activated protein kinases (MAPKs) perform a number of functions ranging from control of proliferation to migration and cell-fate transitions. While the MAPK ERK1/2 signaling pathway has been extensively examined in the endothelium, less is known about the role of the MEKK3/ERK5 pathway in vascular remodeling. To better define the role played by this signaling cascade, we studied the effect of endothelial-specific deletion of its key upstream MAP3K, MEKK3, in adult mice. The gene's deletion resulted in a gradual inward remodeling of both pulmonary and systematic arteries, leading to spontaneous hypertension in both vascular circuits and accelerated progression of atherosclerosis in hyperlipidemic mice. Molecular analysis revealed activation of TGFß-signaling both in vitro and in vivo. Endothelial-specific TGFßR1 knockout prevented inward arterial remodeling in MEKK3 endothelial knockout mice. These data point to the unexpected participation of endothelial MEKK3 in regulation of TGFßR1-Smad2/3 signaling and inward arterial remodeling in artery diseases.

Activation of Smad2/3 signaling by low fluid shear stress mediates artery inward remodeling.

Endothelial cell (EC) sensing of wall fluid shear stress (FSS) from blood flow governs vessel remodeling to maintain FSS at a specific magnitude or set point in healthy vessels. Low FSS triggers inward remodeling to restore normal FSS but the regulatory mechanisms are unknown. In this paper, we describe the signaling network that governs inward artery remodeling. FSS induces Smad2/3 phosphorylation through the type I transforming growth factor (TGF)-ß family receptor Alk5 and the transmembrane protein Neuropilin-1, which together increase sensitivity to circulating bone morphogenetic protein (BMP)-9. Smad2/3 nuclear translocation and target gene expression but not phosphorylation are maximal at low FSS and suppressed at physiological high shear. Reducing flow by carotid ligation in rodents increases Smad2/3 nuclear localization, while the resultant inward remodeling is blocked by the EC-specific deletion of Alk5. The flow-activated MEKK3/Klf2 pathway mediates the suppression of Smad2/3 nuclear translocation at high FSS, mainly through the cyclin-dependent kinase (CDK)-2-dependent phosphosphorylation of the Smad linker region. Thus, low FSS activates Smad2/3, while higher FSS blocks nuclear translocation to induce inward artery remodeling, specifically at low FSS. These results are likely relevant to inward remodeling in atherosclerotic vessels, in which Smad2/3 is activated through TGF-ß signaling.

Feasibility, satisfaction, and goal attainment in routine telemedicine consultation in child and adolescent psychiatry and psychotherapy.

Telehealth services were rapidly adopted during the COVID-19 pandemic, but evidence regarding the effectiveness and feasibility of telehealth services in child and adolescent mental healthcare is sparse. This study aims to investigate feasibility, satisfaction, and goal attainment in video-delivered consultations in routine care child and adolescent psychiatry and psychotherapy. A total of 1046 patients from four university child and adolescent outpatient psychiatric clinics and one university outpatient unit for child and adolescent psychotherapy were screened for study participation. We examined a) the percentage of patients considered eligible for video-delivered consultation, b) clinicians', parents' and patients' satisfaction with video consultation, c) clinicians' ratings of goal attainment in video consultation, and d) factors associated with satisfaction and goal attainment. 59% of the screening sample (n = 621) fulfilled eligibility criteria and were considered eligible for video consultation. A total of 267 patients consented to participate in the study and received a video consultation. Clinicians reported high levels of satisfaction with video consultation and high levels of goal attainment in video consultations, especially for patients scheduled for initial patient assessments. Parents and patients were also highly satisfied with the video consultations, especially if patients had less severe emotional and behavioral problems. The present findings suggest that video consultations are a feasible and well-accepted alternative to in-person consultations in child and adolescent mental health care, especially for children with less severe symptoms and for children in early phases of assessment and treatment. Limitations include the lack of a control group. The study was registered at the German Clinical Trials Registry (DRKS00023525).

Translational opportunities of single-cell biology in atherosclerosis.

The advent of single-cell biology opens a new chapter for understanding human biological processes and for diagnosing, monitoring, and treating disease. This revolution now reaches the field of cardiovascular disease (CVD). New technologies to interrogate CVD samples at single-cell resolution are allowing the identification of novel cell communities that are important in shaping disease development and direct towards new therapeutic strategies. These approaches have begun to revolutionize atherosclerosis pathology and redraw our understanding of disease development. This review discusses the state-of-the-art of single-cell analysis of atherosclerotic plaques, with a particular focus on human lesions, and presents the current resolution of cellular subpopulations and their heterogeneity and plasticity in relation to clinically relevant features. Opportunities and pitfalls of current technologies as well as the clinical impact of single-cell technologies in CVD patient care are highlighted, advocating for multidisciplinary and international collaborative efforts to join the cellular dots of CVD.

FGF-dependent metabolic control of vascular development.

Blood and lymphatic vasculatures are intimately involved in tissue oxygenation and fluid homeostasis maintenance. Assembly of these vascular networks involves sprouting, migration and proliferation of endothelial cells. Recent studies have suggested that changes in cellular metabolism are important to these processes. Although much is known about vascular endothelial growth factor (VEGF)-dependent regulation of vascular development and metabolism, little is understood about the role of fibroblast growth factors (FGFs) in this context. Here we identify FGF receptor (FGFR) signalling as a critical regulator of vascular development. This is achieved by FGF-dependent control of c-MYC (MYC) expression that, in turn, regulates expression of the glycolytic enzyme hexokinase 2 (HK2). A decrease in HK2 levels in the absence of FGF signalling inputs results in decreased glycolysis, leading to impaired endothelial cell proliferation and migration. Pan-endothelial- and lymphatic-specific Hk2 knockouts phenocopy blood and/or lymphatic vascular defects seen in Fgfr1/Fgfr3 double mutant mice, while HK2 overexpression partly rescues the defects caused by suppression of FGF signalling. Thus, FGF-dependent regulation of endothelial glycolysis is a pivotal process in developmental and adult vascular growth and development.

Delayed intubation associated with in-hospital mortality in patients with COVID-19 respiratory failure who fail heated and humified high flow nasal canula.

BACKGROUND: Advanced respiratory support modalities such as non-invasive positive pressure ventilation (NiPPV) and heated and humidified high flow nasal canula (HFNC) served as useful alternatives to invasive mechanical ventilatory support for acute respiratory failure (ARF) during the peak of the SARS-CoV-2/COVID-19 pandemic. Unlike NiPPV, HFNC is a newer modality and its role in the treatment of patients with severe ARF is not yet clearly defined. Furthermore, the characteristics of responders versus non-responders to HFNC have not been determined. Although recent evidence indicates that many patients with ARF treated with HFNC survive without needing intubation, those who fail and are subsequently intubated have worse outcomes. Given that prolonged use of HFNC in patients with ARF might exacerbate patient self-inflicted lung injury, we hypothesized that among those patients with ARF due to COVID-19 pneumonia, prolonged HFNC beyond 24 h before intubation would be associated with increased in-hospital mortality. METHODS: This was a retrospective, multicenter, observational cohort study of 2720 patients treated for ARF secondary to SARS-CoV-2/COVID-19 pneumonia and initially managed with HFNC within the Banner Health system during the period from March 1st, 2020, to July 31st, 2021. In the subgroup of patients for went from HFNC to IMV, we assessed the effect of the duration of HFNC prior to intubation on mortality. RESULTS: 1392 (51%) were successfully treated with HFNC alone and 1328 (49%) failed HFNC and were intubated (HFNC to IMV). When adjusted for the covariates, HFNC duration less than 24 h prior to intubation was significantly associated with reduced mortality. CONCLUSIONS: Among patients with ARF due to COVID-19 pneumonia who fail HFNC, delay of intubation beyond 24 h is associated with increased mortality.

Role of Venous Endothelial Cells in Developmental and Pathologic Angiogenesis.

BACKGROUND: Angiogenesis is a dynamic process that involves expansion of a preexisting vascular network that can occur in a number of physiological and pathological settings. Despite its importance, the origin of the new angiogenic vasculature is poorly defined. In particular, the primary subtype of endothelial cells (capillary, venous, arterial) driving this process remains undefined. METHODS: Endothelial cells were fate-mapped with the use of genetic markers specific to arterial and capillary cells. In addition, we identified a novel venous endothelial marker gene (Gm5127) and used it to generate inducible venous endothelium-specific Cre and Dre driver mouse lines. Contributions of these various types of endothelial cells to angiogenesis were examined during normal postnatal development and in disease-specific setting. RESULTS: Using a comprehensive set of endothelial subtype-specific inducible reporter mice, including tip, arterial, and venous endothelial reporter lines, we showed that venous endothelial cells are the primary endothelial subtype responsible for the expansion of an angiogenic vascular network. During physiological angiogenesis, venous endothelial cells proliferate, migrating against the blood flow and differentiating into tip, capillary, and arterial endothelial cells of the new vasculature. Using intravital 2-photon imaging, we observed venous endothelial cells migrating against the blood flow to form new blood vessels. Venous endothelial cell migration also plays a key role in pathological angiogenesis. This was observed both in formation of arteriovenous malformations in mice with inducible endothelium-specific Smad4 deletion mice and in pathological vessel growth seen in oxygen-induced retinopathy. CONCLUSIONS: Our studies establish that venous endothelial cells are the primary endothelial subtype responsible for normal expansion of vascular networks, formation of arteriovenous malformations, and pathological angiogenesis. These observations highlight the central role of the venous endothelium in normal development and disease pathogenesis.

Mechanotransduction-induced glycolysis epigenetically regulates a CXCL1-dominant angiocrine signaling program in liver sinusoidal endothelial cells in vitro and in vivo.

BACKGROUND & AIMS: Liver sinusoidal endothelial cells (LSECs) are ideally situated to sense stiffness and generate angiocrine programs that potentially regulate liver fibrosis and portal hypertension. We explored how specific focal adhesion (FA) proteins parlay LSEC mechanotransduction into stiffness-induced angiocrine signaling in vitro and in vivo. METHODS: Primary human and murine LSECs were placed on gels with incremental stiffness (0.2 kPa vs. 32 kPa). Cell response was studied by FA isolation, actin polymerization assay, RNA-sequencing and electron microscopy. Glycolysis was assessed using radioactive tracers. Epigenetic regulation of stiffness-induced genes was analyzed by chromatin-immunoprecipitation (ChIP) analysis of histone activation marks, ChIP sequencing and circularized chromosome conformation capture (4C). Mice with LSEC-selective deletion of glycolytic enzymes (Hk2fl/fl/Cdh5cre-ERT2) or treatment with the glycolysis inhibitor 3PO were studied in portal hypertension (partial ligation of the inferior vena cava, pIVCL) and early liver fibrosis (CCl4) models. RESULTS: Glycolytic enzymes, particularly phosphofructokinase 1 isoform P (PFKP), are enriched in isolated FAs from LSECs on gels with incremental stiffness. Stiffness resulted in PFKP recruitment to FAs, which paralleled an increase in glycolysis. Glycolysis was associated with expansion of actin dynamics and was attenuated by inhibition of integrin ß1. Inhibition of glycolysis attenuated a stiffness-induced CXCL1-dominant angiocrine program. Mechanistically, glycolysis promoted CXCL1 expression through nuclear pore changes and increases in NF-kB translocation. Biochemically, this CXCL1 expression was mediated through spatial re-organization of nuclear chromatin resulting in formation of super-enhancers, histone acetylation and NF-kB interaction with the CXCL1 promoter. Hk2fl/fl/Cdh5cre-ERT2 mice showed attenuated neutrophil infiltration and portal hypertension after pIVCL. 3PO treatment attenuated liver fibrosis in a CCl4 model. CONCLUSION: Glycolytic enzymes are involved in stiffness-induced angiocrine signaling in LSECs and represent druggable targets in early liver disease. LAY SUMMARY: Treatment options for liver fibrosis and portal hypertension still represent an unmet need. Herein, we uncovered a novel role for glycolytic enzymes in promoting stiffness-induced angiocrine signaling, which resulted in inflammation, fibrosis and portal hypertension. This work has revealed new targets that could be used in the prevention and treatment of liver fibrosis and portal hypertension.

FRS2α-dependent cell fate transition during endocardial cushion morphogenesis.

Atrioventricular valve development requires endothelial-to-mesenchymal transition (EndMT) that induces cushion endocardial cells to give rise to mesenchymal cells crucial to valve formation. In the adult endothelium, deletion of the docking protein FRS2α induces EndMT by activating TGFß signaling in a miRNA let-7-dependent manner. To study the role of endothelial FRS2α during embryonic development, we generated mice with an inducible endothelial-specific deletion of Frs2α (FRS2αiECKO). Analysis of the FRS2αiECKO embryos uncovered a combination of impaired EndMT in AV cushions and defective maturation of AV valves leading to development of thickened, abnormal valves when Frs2α was deleted early (E7.5) in development. At the same time, no AV valve developmental abnormalities were observed after late (E10.5) deletion. These observations identify FRS2α as a pivotal controller of cell fate transition during both EndMT and post-EndMT valvulogenesis.

[The mental burden of children, adolescents, and their families during the COVID-19 pandemic and associations with emotional and behavioral problems]. / Die psychische Belastung von Kindern, Jugendlichen und ihren Familien während der COVID-19-Pandemie und der Zusammenhang mit emotionalen und Verhaltensauffälligkeiten.

BACKGROUND AND OBJECTIVE: The infection protection measures adopted as part of the COVID-19 pandemic led to profound restrictions and changes in the social, (pre-) school, family, and leisure areas. The objective of the current study was to examine the mental burden of children and adolescents and their families during the COVID-19 pandemic. Furthermore, this study aimed to identify possible factors that influence the mental burden. MATERIALS AND METHODS: The examinations were carried out between autumn 2020 and spring 2021 in a clinical sample (n = 280 patients aged 4-17 years) and a community sample (n = 1958 children and adolescents aged 4-19 years recruited via schools and preschools). Ratings of parents as well as children and adolescents via questionnaires were assessed. RESULTS: Mental burden due to the corona pandemic was assessed as slightly to moderately increased across both rating perspectives and both samples. Overall, around 60 to 70% of the parents, children, and adolescents describe an increase in mental burden; in contrast, up to 12% of parents as well as children and adolescents describe relief. When comparing both samples, a slightly higher burden on children and adolescents can only be seen in the self-assessment of the clinical sample. None of the socio-demographic factors analyzed influences the mental burden statistically significant. However, low to moderate correlations between the subjectively experienced deterioration in the family and social situation and an increased level of stress is found. DISCUSSION: Targeted interventions for exposed subgroups should be offered during a pandemic. Universal interventions are not indicated.

Divergent angiocrine signals from vascular niche balance liver regeneration and fibrosis.

Chemical or traumatic damage to the liver is frequently associated with aberrant healing (fibrosis) that overrides liver regeneration. The mechanism by which hepatic niche cells differentially modulate regeneration and fibrosis during liver repair remains to be defined. Hepatic vascular niche predominantly represented by liver sinusoidal endothelial cells deploys paracrine trophogens, known as angiocrine factors, to stimulate regeneration. Nevertheless, it is not known how pro-regenerative angiocrine signals from liver sinusoidal endothelial cells is subverted to promote fibrosis. Here, by combining an inducible endothelial-cell-specific mouse gene deletion strategy and complementary models of acute and chronic liver injury, we show that divergent angiocrine signals from liver sinusoidal endothelial cells stimulate regeneration after immediate injury and provoke fibrosis after chronic insult. The pro-fibrotic transition of vascular niche results from differential expression of stromal-derived factor-1 receptors, CXCR7 and CXCR4 (refs 18, 19, 20, 21), in liver sinusoidal endothelial cells. After acute injury, CXCR7 upregulation in liver sinusoidal endothelial cells acts with CXCR4 to induce transcription factor Id1, deploying pro-regenerative angiocrine factors and triggering regeneration. Inducible deletion of Cxcr7 in sinusoidal endothelial cells (Cxcr7(iΔEC/iΔEC)) from the adult mouse liver impaired liver regeneration by diminishing Id1-mediated production of angiocrine factors. By contrast, after chronic injury inflicted by iterative hepatotoxin (carbon tetrachloride) injection and bile duct ligation, constitutive FGFR1 signalling in liver sinusoidal endothelial cells counterbalanced CXCR7-dependent pro-regenerative response and augmented CXCR4 expression. This predominance of CXCR4 over CXCR7 expression shifted angiocrine response of liver sinusoidal endothelial cells, stimulating proliferation of desmin(+) hepatic stellate-like cells and enforcing a pro-fibrotic vascular niche. Endothelial-cell-specific ablation of either Fgfr1 (Fgfr1(iΔEC/iΔEC)) or Cxcr4 (Cxcr4(iΔEC/iΔEC)) in mice restored the pro-regenerative pathway and prevented FGFR1-mediated maladaptive subversion of angiocrine factors. Similarly, selective CXCR7 activation in liver sinusoidal endothelial cells abrogated fibrogenesis. Thus, we demonstrate that in response to liver injury, differential recruitment of pro-regenerative CXCR7-Id1 versus pro-fibrotic FGFR1-CXCR4 angiocrine pathways in vascular niche balances regeneration and fibrosis. These results provide a therapeutic roadmap to achieve hepatic regeneration without provoking fibrosis.