Endothelial Tip Cells Research Articles

Loss of apelin blocks the emergence of sprouting angiogenesis in experimental tumors.

Angiogenesis inhibitor drugs targeting vascular endothelial growth factor (VEGF) signaling to the endothelial cell (EC) are used to treat various cancer types. However, primary or secondary resistance to therapy is common. Clinical and pre-clinical studies suggest that alternative pro-angiogenic factors are upregulated after VEGF pathway inhibition. Therefore, identification of alternative pro-angiogenic pathway(s) is critical for the development of more effective anti-angiogenic therapy. Here we study the role of apelin as a pro-angiogenic G-protein-coupled receptor ligand in tumor growth and angiogenesis. We found that loss of apelin in mice delayed the primary tumor growth of Lewis lung carcinoma 1 and B16F10 melanoma when combined with the VEGF receptor tyrosine kinase inhibitor, sunitinib. Targeting apelin in combination with sunitinib markedly reduced the tumor vessel density, and decreased microvessel remodeling. Apelin loss reduced angiogenic sprouting and tip cell marker gene expression in comparison to the sunitinib-alone-treated mice. Single-cell RNA sequencing of tumor EC demonstrated that the loss of apelin prevented EC tip cell differentiation. Thus, apelin is a potent pro-angiogenic cue that supports initiation of tumor neovascularization. Together, our data suggest that targeting apelin may be useful as adjuvant therapy in combination with VEGF signaling inhibition to inhibit the growth of advanced tumors.

Ablation of Sphingosine Kinase 1 Protects Cornea from Neovascularization in a Mouse Corneal Injury Model.

The purpose of this study was to investigate the role of sphingosine kinase 1 (SphK1), which generates sphingosine-1-phosphate (S1P), in corneal neovascularization (NV). Wild-type (WT) and Sphk1 knockout (Sphk1−/−) mice received corneal alkali-burn treatment to induce corneal NV by placing a 2 mm round piece of Whatman No. 1 filter paper soaked in 1N NaOH on the center of the cornea for 20 s. Corneal sphingolipid species were extracted and identified using liquid chromatography/mass spectrometry (LC/MS). The total number of tip cells and those positive for ethynyl deoxy uridine (EdU) were quantified. Immunocytochemistry was done to examine whether pericytes were present on newly forming blood vessels. Cytokine signaling and angiogenic markers were compared between the two groups using multiplex assays. Data were analyzed using appropriate statistical tests. Here, we show that ablation of SphK1 can significantly reduce NV invasion in the cornea following injury. Corneal sphingolipid analysis showed that total levels of ceramides, monohexosyl ceramides (HexCer), and sphingomyelin were significantly elevated in Sphk−/− corneas compared to WT corneas, with a comparable level of sphingosine among the two genotypes. The numbers of total and proliferating endothelial tip cells were also lower in the Sphk1−/− corneas following injury. This study underscores the role of S1P in post-injury corneal NV and raises further questions about the roles played by ceramide, HexCer, and sphingomyelin in regulating corneal NV. Further studies are needed to unravel the role played by bioactive sphingolipids in maintenance of corneal transparency and clear vision.

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.

Estimation of shear stress values along endothelial tip cells past the lumen of capillary sprouts.

Shear stress is recognized as a regulator of angiogenesis. However, the shear stress experienced by the endothelial cells of capillary sprouts remains unknown. The objective of this study was to estimate shear stress due to local interstitial flow along endothelial tip cells at the end of the capillary sprout lumen. Computational fluid dynamics were used to model flow within a blind-ended vessel, transendothelial flow across the vessel wall, and flow within the surrounding perivascular/interstitial space. Shear stress along the wall of the tip cells was calculated while varying sprout length, perivascular space channel width, and vessel wall hydraulic conductivity. Increasing sprout length, increasing wall hydraulic conductivity, and decreasing perivascular space width increased shear stress magnitude. Wall shear stress magnitude within the lumen ranged from 0.015 to 0.55dyne/cm2 at the sprout entrance and linearly decreased to near zero at the base of the tip cells. Tip cell wall shear stress magnitude due to interstitial flow ranged from 0.009 to 4.65dyne/cm2. In 3 out of 8 cases, shear stress magnitude was above 1dyne/cm2 and considered physiologically relevant. The results provide a framework for discussing the role of local mechanical cues in regulating endothelial cell dynamics involved in angiogenesis. Mainly, interstitial flows may generate physiologically relevant shear stresses on tip cells in certain scenarios. This source of tip cell shear stress has not been previously considered or modeled.

Identification of Genes Related to 5-Fluorouracil Based Chemotherapy for Colorectal Cancer.

BackgroundColorectal cancer (CRC) is one of the most common malignancies and its incidence and mortality are increasing yearly. 5-Fluorouracil (5-FU) has long been used as a standard first-line treatment for CRC patients. Although 5-FU-based chemotherapy is effective for advanced CRC, the consequent resistance remains a key problem and causes the poor prognosis of CRC patients. Thus, there is an urgent need to identify new biomarkers to predict the response to 5-FU-based chemotherapy.MethodsCRC samples were retrieved from Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA). The immune-related genes were retrieved from the ImmPort database. Single-cell sequencing results from colorectal cancer were obtained by the ArrayExpress database. 5-FU resistance-related genes were filtered and validated by R packages. ESTIMATE algorithms were used to assess the tumor microenvironment (TME). KEGG and GO analysis were performed to explore the biological signaling pathway for resistant-response patients and sensitive-response patients in the tumor microenvironment. pRRophetic algorithms were used to predict 5-FU sensitivity. GSEA and GSVA analysis was performed to excavate the biological signaling pathway of the RBP7 gene.ResultsNine immune-related genes were identified to be associated with 5-FU resistance and poor disease-free survival (DFS) of CRC patients and the signature of these genes was developed in a DFS-prognostic model. Four immune-related genes were determined to be associated with 5-FU resistance and overall survival (OS) of CRC patients. The signature of these genes was developed an OS-prognostic model. ESTIMATE scores showed a significant difference between 5-FU resistant and 5-FU sensitive CRC patients. Resistant-response patients and sensitive-response patients to 5-FU based chemotherapy showed different GO and KEGG enrichment on the tumor microenvironment. RBP7, as a tumor immune microenvironment (TIME) related gene, was found to have the potential of predicting chemotherapy resistance and poor prognosis of CRC patients. GSEA analysis showed multiple signaling differences between the high and low expression of RBP7 in CRC patients. Hypoxia and TNFα signaling via NFκB gene sets were significantly different between chemotherapy resistant (RBP7High) and chemotherapy sensitive (RBP7Low) patients. Single-cell RNA-seq suggested RBP7 was centrally distributed in endothelial stalk cells, endothelial tip cells, and myeloid cells.ConclusionsImmune-related genes will hopefully be potential prognostic biomarkers to predict chemotherapy resistance for CRC. RBP7 may function as a tumor microenvironment regulator to induce 5-FU resistance, thereby affecting the prognosis of CRC patients.

Abstract 3648: Hedgehog represses angiogenesis in PDAC through a paracrine cascade mediated by Wif1

Abstract Pancreatic Ductal Adenocarcinoma (PDAC) is characterized by an extremely heterogeneous tumor microenvironment (TME) paired with a sparse and compromised vascular network. These features of the TME shape drug responses and contribute to PDAC’s distinctive chemotherapeutic resistance. Prior work demonstrated that inhibition of the Hedgehog (Hh) signaling pathway using Smoothened (Smo) inhibitors led to increased angiogenesis. However, the precise mechanisms through which the Hh pathway regulates angiogenesis has remained elusive and subsequent studies with different agents and timepoints yielded conflicting data. Frustratingly, the phenotype could not be recreated in simple co-culture models. In order to maintain the cellular heterogeneity and spatial organization that characterizes PDAC, we developed a novel tumor explant system for short-term cultures of both murine and human PDAC. Briefly, we cultured intact 300 μm slices of PDAC tissue on a modified gelatin platform system with a novel culture medium informed by the metabolic composition of PDAC tumor interstitial fluid. This system replicates some of the nutrient/waste gradient of PDAC and enables tumors to be cultured up to one week maintaining the representation of multiple cell types. Studies in explants were complemented by work in the Kras-LSL.G12D/+, p53-LSL.R172H/+, Pdx1-Cre (KPC) genetically engineered model of PDAC. Comparison of short versus long-term Smo inhibition in KPC mouse pancreatic tumors revealed a robust increase in vessel density in short-term treatment that is lost at later timepoints. Studies in murine and human PDAC explants employing different Smo inhibitors demonstrated increased endothelial tip cell formation upon two days of treatment, resulting in increased vessel density after four days. Computational analyses of human PDAC single cell RNAseq data identified candidate cellular subsets potentially involved in the signaling cascade. RNAseq of pre- and on-treatment tumor samples from KPC mice identified Wif1 as a candidate Hh pathway target gene that effected these programs. Mechanistic studies in tumor explants reveal a complex, multi-step mechanism involving several cell types that led to suppression of angiogenesis in PDAC. These studies both provide a candidate mechanism for Hh-mediated angiosuppression and also demonstrated how the tumor explant platform can elucidate complex intercellular signaling cascades in both murine and human PDAC. Citation Format: Marie C. Hasselluhn, Amanda R. Decker, Alvaro Curiel Garcia, Carlo Maurer, Dafydd H. Thomas, Kenneth P. Olive. Hedgehog represses angiogenesis in PDAC through a paracrine cascade mediated by Wif1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3648.

Abstract 247: Targeting Notch4 with a novel neutralizing antibody inhibits tumor growth and alters immune cell populations within the tumor microenvironment

Abstract Endothelial Notch signaling is critical for tumor angiogenesis and growth. Inhibition of Notch1 or its ligand, Dll4, can induce tumor vessel hypersprouting, which interferes with perfusion and reduces tumor development. Unfortunately, blocking Notch1 in endothelial cells of normal tissue can lead to tissue hypoxia and vascular neoplasms. Notch4, another member of the Notch family, is upregulated and increased in the endothelium of certain tumor types compared to normal vasculature. Reducing Notch4, unlike Notch1, causes reduction in developmental and tumor angiogenesis. Using a newly developed anti-Notch4 antibody, E7011, we assessed the effects on Notch4 inhibition on developmental retinal angiogenesis. E7011 treatment did not alter capillary density within the vascular plexus or endothelial tip cell number, but did decrease radial outgrowth and vein diameter, supporting the hypothesis that Notch4 functions as a pro-angiogenic signal. These findings suggest that the targeting of Notch4 could interfere with tumor angiogenesis and offer a novel therapeutic avenue. To establish the role of Notch4 within the tumor microenvironment, we analyzed its expression in both human and murine tumor samples. Immunofluorescent staining revealed a range of Notch4 expression in both tumor cells and endothelium in human triple negative breast cancers. Several murine syngeneic tumors examined expressed Notch4 primarily in the endothelium with only rare tumor expression. We treated orthotopically implanted Py8119 murine breast carcinomas and B16F10 melanomas with E7011, which significantly reduced tumor size compared to IgG treated controls, indicating that suppression of Notch4 is anti-tumorigenic. To determine mechanistically how E7011 delays tumor growth, we performed single cell RNA sequencing on E7011 and control treated Py8119 tumors. Surprisingly, we found that while Notch4 transcripts were present only in endothelial cells, E7011 administration dramatically altered macrophage and monocyte populations within the tumor. Taken together, these findings indicate that inhibition of tumor endothelial Notch4 by E7011 can regulate the presence of immune cells within the tumor microenvironment and improve tumor control. Citation Format: Jason Eng, L.A. Naiche, Bhairavi Swaminathan, Yu Kato, Krishna Thakkar, Rahul Vadakath, James Herts, Rajyasree Emmadi, Junji Matsui, Jan Kitajewski. Targeting Notch4 with a novel neutralizing antibody inhibits tumor growth and alters immune cell populations within the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 247.

Cellular heterogeneity and transcriptomic profiles during intrahepatic cholangiocarcinoma initiation and progression.

Intrahepatic cholangiocarcinoma (ICC) is not fully investigated, and how stromal cells contribute to ICC formation is poorly understood. We aimed to uncover ICC origin, cellular heterogeneity, and critical modulators during ICC initiation/progression, and to decipher how fibroblast and endothelial cells in the stromal compartment favor ICC progression. We performed single-cell RNA sequencing (scRNA-seq) using AKT/Notch intracellular domain-induced mouse ICC tissues at early, middle, and late stages. We analyzed the transcriptomic landscape, cellular classification and evolution, and intercellular communication during ICC initiation/progression. We confirmed the findings using quantitative real-time PCR, western blotting, immunohistochemistry or immunofluorescence, and gene knockout/knockdown analysis. We identified stress-responding and proliferating subpopulations in late-stage mouse ICC tissues and validated them using human scRNA-seq data sets. By integrating weighted correlation network analysis and protein-protein interaction through least absolute shrinkage and selection operator regression, we identified zinc finger, MIZ-type containing 1 (Zmiz1) and Y box protein 1 (Ybx1) as core transcription factors required by stress-responding and proliferating ICC cells, respectively. Knockout of either one led to the blockade of ICC initiation/progression. Using two other ICC mouse models (YAP/AKT, KRAS/p19) and human ICC scRNA-seq data sets, we confirmed the orchestrating roles of Zmiz1 and Ybx1 in ICC occurrence and development. In addition, hes family bHLH transcription factor 1, cofilin 1, and inhibitor of DNA binding 1 were identified as driver genes for ICC. Moreover, periportal liver sinusoidal endothelial cells could differentiate into tip endothelial cells to promote ICC development, and this was Dll4-Notch4-Efnb2 signaling-dependent. Stress-responding and ICC proliferating subtypes were identified, and Zmiz1 and Ybx1 were revealed as core transcription factors in these subtypes. Fibroblast-endothelial cell interaction promotes ICC development.

A simple agent-based model to simulate 3D tumor-induced angiogenesis considering the evolution of the hypoxic conditions of the cells

In tumors, angiogenesis (conformation of a new vasculature from another primal one) is produced with the releasing of tumor angiogenic factors from hypoxic cells. These angiogenic substances are distributed around the tumor micro-environment by diffusion. When they reach the primal blood vessel bed, the sprouting and branching of a new micro-vascular network is produced. These new capillaries will supply oxygen to cells so that their hypoxic state is overcome. In this work, a new and simple 3D agent-based model to simulate tumor-induced angiogenesis is presented. In this approach, the evolution of the hypoxic conditions in cells along the related conformation of the new micro-vessels is considered. The importance that the relative position of the primal vasculature and tumor structure takes in the final distribution of the new micro-vasculature has also been addressed. The diffusion of angiogenic factors and oxygen has been modelled at the targets by numerical convolution superposition of the analytical solution from the sources. Qualitative and quantitative results show the importance of tip endothelial cells in overcoming hypoxic conditions in cells at early stages of angiogenesis. At final stages, anastomosis plays an important role in the reduction of hypoxia in cells.

Targeting circular RNA-MET for anti-angiogenesis treatment via inhibiting endothelial tip cell specialization

Endothelial tip cell specialization plays an essential role in angiogenesis, which is tightly regulated by the complicated gene regulatory network. Circular RNA (circRNA) is a type of covalently closed non-coding RNA that regulates gene expression in eukaryotes. Here, we report that the levels of circMET expression are significantly upregulated in the retinas of mice with oxygen-induced retinopathy, choroidal neovascularization, and diabetic retinopathy. circMET silencing significantly reduces pathological angiogenesis and inhibits tip cell specialization invivo. circMET silencing also decreases endothelial migration and sprouting invitro. Mechanistically, circMET regulates endothelial sprouting and pathological angiogenesis by acting as a scaffold to enhance the interaction between IGF2BP2 and NRARP/ESM1. Clinically, circMET is significantly upregulated in the clinical samples of the patients of diabetic retinopathy. circMET silencing could reduce diabetic vitreous-induced endothelial sprouting and retinal angiogenesis invivo. Collectively, these data identify a circRNA-mediated mechanism that coordinates tip cell specialization and pathological angiogenesis. circMET silencing is an exploitable therapeutic approach for the treatment of neovascular diseases.

ENOS controls angiogenic sprouting and retinal neovascularization through the regulation of endothelial cell polarity

The roles of nitric oxide (NO) and endothelial NO synthase (eNOS) in the regulation of angiogenesis are well documented. However, the involvement of eNOS in the sprouting of endothelial tip-cells at the vascular front during sprouting angiogenesis remains poorly defined. In this study, we show that downregulation of eNOS markedly inhibits VEGF-stimulated migration of endothelial cells but increases their polarization, as evidenced by the reorientation of the Golgi in migrating monolayers and by the fewer filopodia on tip cells at ends of sprouts in endothelial cell spheroids. The effect of eNOS inhibition on EC polarization was prevented in Par3-depleted cells. Importantly, downregulation of eNOS increased the expression of polarity genes, such as PARD3B, PARD6A, PARD6B, PKCΖ, TJP3, and CRB1 in endothelial cells. In retinas of eNOS knockout mice, vascular development is retarded with decreased vessel density and vascular branching. Furthermore, tip cells at the extremities of the vascular front have a marked reduction in the number of filopodia per cell and are more oriented. In a model of oxygen-induced retinopathy (OIR), eNOS deficient mice are protected during the initial vaso-obliterative phase, have reduced pathological neovascularization, and retinal endothelial tip cells have fewer filopodia. Single-cell RNA sequencing of endothelial cells from OIR retinas revealed enrichment of genes related to cell polarity in the endothelial tip-cell subtype of eNOS deficient mice. These results indicate that inhibition of eNOS alters the polarity program of endothelial cells, which increases cell polarization, regulates sprouting angiogenesis and normalizes pathological neovascularization during retinopathy.

FGD5 regulates endothelial cell PI3 kinase-β to promote neo-angiogenesis.

Angiogenesis is required in embryonic development and tissue repair in the adult. Vascular endothelial growth factor (VEGF) initiates angiogenesis, and VEGF or its receptor is targeted therapeutically to block pathological angiogenesis. Additional pro-angiogenic cues, such as CXCL12 acting via the CXCR4 receptor, co-operate with VEGF/VEGFR2 to cue vascular patterning. We studied the role of FGD5, an endothelial Rho GTP/GDP exchange factor (RhoGEF), to regulate CXCR4-dependent signals in the endothelial cell (EC). Patient-derived renal cell carcinomas produce a complex milieu of growth factors that stimulated sprouting angiogenesis and endothelial tip cell differentiation ex vivo that was blocked by EC FGD5loss. In a simplified model, CXCL12 augmented sprouting and tip gene expression under conditions where VEGF was limiting. CXCL12-stimulated tip cell differentiation was dependent on PI3kinase (PI3K)-β activity. Knockdown of EC FGD5 abolished CXCR4signaling to PI3K-β and Akt. Further, inhibition of Rac1, a Rho GTPase required for PI3K-β activity, recapitulated the signaling defects of FGD5 deficiency, suggesting that FGD5may regulate PI3K-β activity through Rac1. Overexpression of a RhoGEF deficient, Dbl domain-deleted FGD5mutant reduced CXCL12-stimulated Akt phosphorylation and failed to rescue PI3K signaling in native FGD5-deficient EC, indicating that FGD5 RhoGEF activity is required for FDG5 function. Endothelial expression of mutant PI3K-β with an inactivated Rho binding domain confirmed that CXCL12-stimulated PI3K activity in EC requires Rac1-GTP co-regulation. Together, this data identify the role of FGD5 to generate Rac1-GTP to regulate pro-angiogenic CXCR4-dependent PI3K-β signaling in EC. Inhibition of FGD5 activity may complement current angiogenesis inhibitor drugs.

Abstract PR-014: Hedgehog represses angiogenesis in PDAC through a paracrine cascade mediated by Wif1

Abstract Pancreatic Ductal Adenocarcinoma (PDAC) is characterized by an extremely heterogeneous tumor microenvironment (TME) paired with a sparse and compromised vascular network. These features of the TME shape drug responses and contributes to PDAC’s distinctive chemotherapeutic resistance. Prior work demonstrated that inhibition of the Hedgehog (Hh) signaling using Smoothened (Smo) inhibitors relieved some of the local angiogenic suppression. However, the precise mechanisms through which the Hh pathway regulates angiogenesis has remained elusive. Subsequent studies evaluating different timepoints yielded conflicting data on the role of Hedgehog signaling in angiogenesis. In order to replicate the heterogeneity and spatial patterning of cells in pancreatic tumors, we developed a novel tumor explant system for short-term cultures of both murine and human PDAC. Briefly, we cultured intact 300 μm slices of PDAC tissues in a modified gelatin platform system with a novel culture medium informed by the metabolic composition of PDAC tumor interstitial fluid. This system replicates some of the nutrient/waste gradient of PDAC and enables tumors to be culture up to one week with good representation of multiple cell types. Studies in explants were complemented by work in the KPC genetically engineered model of PDAC. Comparison of short versus long-term Smo inhibition in KPC mouse pancreatic tumors revealed a robust increase in vessel density in short-term treatment, which is lost at later timepoints. Studies in murine and human PDAC explants employing different Smo inhibitors demonstrated increased endothelial tip cell formation upon two days of treatment, resulting in increased vessel density after four days. Computational analyses of human PDAC single cell RNAseq data identified candidate cellular subsets potentially involved in the signaling cascade. RNAseq of pre- and on-treatment tumor samples from KPC mice identified Wif1 as a candidate Hh target gene that effected these programs. Mechanistic studies in tumor explants reveal a complex, multi-step mechanism involving multiple cell types that led to suppression of angiogenesis in PDAC. These studies demonstrate how the tumor explant platform can elucidate complex intercellular signaling cascades and provide a candidate mechanism for the Hh-mediated suppression of angiogenesis in PDAC. Citation Format: Marie C. Hasselluhn, Amanda R. Decker, Alvaro Curiel Garcia, Carlo Maurer, Dafydd Thomas, Kenneth P. Olive. Hedgehog represses angiogenesis in PDAC through a paracrine cascade mediated by Wif1 [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PR-014.

Translational control in neurovascular brain development.

The human brain carries out complex tasks and higher functions and is crucial for organismal survival, as it senses both intrinsic and extrinsic environments. Proper brain development relies on the orchestrated development of different precursor cells, which will give rise to the plethora of mature brain cell-types. Within this process, neuronal cells develop closely to and in coordination with vascular cells (endothelial cells (ECs), pericytes) in a bilateral communication process that relies on neuronal activity, attractive or repulsive guidance cues for both cell types and on tight-regulation of gene expression. Translational control is a master regulator of the gene-expression pathway and in particular for neuronal and ECs, it can be localized in developmentally relevant (axon growth cone, endothelial tip cell) and mature compartments (synapses, axons). Herein, we will review mechanisms of translational control relevant to brain development in neurons and ECs in health and disease.

Specialized endothelial tip cells guide neuroretina vascularization and blood-retina-barrier formation.

Endothelial tip cells guiding tissue vascularization are primary targets for angiogenic therapies. Whether tip cells require differential signals to develop their complex branching patterns remained unknown. Here, we show that diving tip cells invading the mouse neuroretina (D-tip cells) are distinct from tip cells guiding the superficial retinal vascular plexus (S-tip cells). D-tip cells have a unique transcriptional signature, including high TGF-β signaling, and they begin to acquire blood-retina barrier properties. Endothelial deletion of TGF-β receptor I (Alk5) inhibits D-tip cell identity acquisition and deep vascular plexus formation. Loss of endothelial ALK5, but not of the canonical SMAD effectors, leads to aberrant contractile pericyte differentiation and hemorrhagic vascular malformations. Oxygen-induced retinopathy vasculature exhibits S-like tip cells, and Alk5 deletion impedes retina revascularization. Our data reveal stage-specific tip cell heterogeneity as a requirement for retinal vascular development and suggest that non-canonical-TGF-β signaling could improve retinal revascularization and neural function in ischemic retinopathy.

Electrochemically derived nanographene oxide activates endothelial tip cells and promotes angiogenesis by binding endogenous lysophosphatidic acid

Graphene oxide (GO) exhibits good mechanical and physicochemical characteristics and has extensive application prospects in bone tissue engineering. However, its effect on angiogenesis is unclear, and its potential toxic effects are heavily disputed. Herein, we found that nanographene oxide (NGO) synthesized by one-step water electrolytic oxidation is smaller and shows superior biocompatibility. Moreover, NGO significantly enhanced angiogenesis in calvarial bone defect areas in vivo, providing a good microenvironment for bone regeneration. Endothelial tip cell differentiation is an important step in the initiation of angiogenesis. We verified that NGO activates endothelial tip cells by coupling with lysophosphatidic acid (LPA) in serum via strong hydrogen bonding interactions, which has not been reported. In addition, the mechanism by which NGO promotes angiogenesis was systematically studied. NGO-coupled LPA activates LPAR6 and facilitates the formation of migratory tip cells via Hippo/Yes-associated protein (YAP) independent of reactive oxygen species (ROS) stimulation or additional complex modifications. These results provide an effective strategy for the application of electrochemically derived NGO and more insight into NGO-mediated angiogenesis.

Defective Flow-Migration Coupling Causes Arteriovenous Malformations in Hereditary Hemorrhagic Telangiectasia.

Activin receptor-like kinase 1 (ALK1) is an endothelial transmembrane serine threonine kinase receptor for BMP family ligands that plays a critical role in cardiovascular development and pathology. Loss-of-function mutations in the ALK1 gene cause type 2 hereditary hemorrhagic telangiectasia, a devastating disorder that leads to arteriovenous malformations. Here, we show that ALK1 controls endothelial cell polarization against the direction of blood flow and flow-induced endothelial migration from veins through capillaries into arterioles. Using Cre lines that recombine in different subsets of arterial, capillary-venous, or endothelial tip cells, we show that capillary-venous Alk1 deletion was sufficient to induce arteriovenous malformation formation in the postnatal retina. ALK1 deletion impaired capillary-venous endothelial cell polarization against the direction of blood flow in vivo and in vitro. Mechanistically, ALK1-deficient cells exhibited increased integrin signaling interaction with vascular endothelial growth factor receptor 2, which enhanced downstream YAP/TAZ nuclear translocation. Pharmacologic inhibition of integrin or YAP/TAZ signaling rescued flow migration coupling and prevented vascular malformations in Alk1-deficient mice. Our study reveals ALK1 as an essential driver of flow-induced endothelial cell migration and identifies loss of flow-migration coupling as a driver of arteriovenous malformation formation in hereditary hemorrhagic telangiectasia disease. Integrin-YAP/TAZ signaling blockers are new potential targets to prevent vascular malformations in patients with hereditary hemorrhagic telangiectasia.

The ER membrane protein complex subunit Emc3 controls angiogenesis via the FZD4/WNT signaling axis.

The endoplasmic reticulum (ER) membrane protein complex (EMC) regulates the synthesis and quality control of membrane proteins with multiple transmembrane domains. One of the membrane spanning subunits, EMC3, is a core member of the EMC complex that provides essential hydrophilic vestibule for substrate insertion. Here, we show that the EMC subunit Emc3 plays critical roles in the retinal vascular angiogenesis by regulating Norrin/Wnt signaling. Postnatal endothelial cell (EC)-specific deletion of Emc3 led to retarded retinal vascular development with a hyperpruned vascular network, the appearance of blunt-ended, aneurysm-like tip endothelial cells (ECs) with reduced numbers of filopodia and leakage of erythrocytes at the vascular front. Diminished tube formation and cell proliferation were also observed in EMC3 depleted human retinal endothelial cells (HRECs). We then discovered a critical role for EMC3 in expression of FZD4 receptor of β-catenin signaling using RNA sequencing, real-time quantitative PCR (RT-qPCR) and luciferase reporter assay. Moreover, augmentation of Wnt activity via lithium chloride (LiCl) treatment remarkably enhanced β-catenin signaling and cell proliferation of HRECs. Additionally, LiCl partially reversed the angiogenesis defects in Emc3-cKO mice. Our data reveal that Emc3 plays essential roles in angiogenesis through direct control of FZD4 expression and Norrin/β-catenin signaling.

Matrix stiffness modulates tip cell formation through the p-PXN-Rac1-YAP signaling axis

Endothelial tip cell outgrowth of blood-vessel sprouts marks the initiation of angiogenesis which is critical in physiological and pathophysiological procedures. However, how mechanical characteristics of extracellular matrix (ECM) modulates tip cell formation has been largely neglected. In this study, we found enhanced CD31 expression in the stiffening outer layer of hepatocellular carcinoma than in surrounding soft tissues. Stiffened matrix promoted sprouting from endothelial cell (EC) spheroids and upregulated expressions of tip cell-enriched genes in vitro. Moreover, tip cells showed increased cellular stiffness, more actin cytoskeleton organization and enhanced YAP nuclear transfer than stalk and phalanx ECs. We further uncovered that substrate stiffness regulates FAK and Paxillin phosphorylation in focal adhesion of ECs promoting Rac1 transition from inactive to active state. YAP is subsequently activated and translocated into nucleus, leading to increased tip cell specification. p-Paxillin can also loosen the intercellular connection which also facilitates tip cell specification. Collectively our present study shows that matrix stiffness modulates tip cell formation through p-PXN-Rac1-YAP signaling axis, shedding light on the role of mechanotransduction in tip cell formation. This is of special significance in biomaterial design and treatment of some pathological situations.

The Role of HIF1α-PFKFB3 Pathway in Diabetic Retinopathy.

Diabetic retinopathy (DR) is the leading cause of blindness for adults in developed countries. Both microvasculopathy and neurodegeneration are implicated in mechanisms of DR development, with neuronal impairment preceding microvascular abnormalities, which is often underappreciated in the clinic. Most current therapeutic strategies, including anti-vascular endothelial growth factor (anti-VEGF)-antibodies, aim at treating the advanced stages (diabetic macular edema and proliferative diabetic retinopathy) and fail to target the neuronal deterioration. Hence, new therapeutic approach(es) intended to address both vascular and neuronal impairment are urgently needed.The hypoxia-inducible factor 1α (HIF1α)–6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) pathway is critically implicated in the islet pathology of diabetes. Recent evidence highlighted the pathway relevance for pathologic angiogenesis and neurodegeneration, two key aspects in DR. PFKFB3 is key to the sprouting angiogenesis, along with VEGF, by determining the endothelial tip-cell competition. Also, PFKFB3-driven glycolysis compromises the antioxidative capacity of neurons leading to neuronal loss and reactive gliosis. Therefore, the HIF1α-PFKFB3 signaling pathway is unique as being a pervasive pathological component across multiple cell types in the retina in the early as well as late stages of DR. A metabolic point-of-intervention based on HIF1α-PFKFB3 targeting thus deserves further consideration in DR.