Induction of Fenestrae in Human Induced Pluripotent Stem Cell-Derived Endothelial Cells for Disease Modeling.

The endothelial linings of capillaries, such as those in the kidney and small intestines, possess fenestrae that facilitate fluid and exchange of small molecules. Alterations in the size and number of endothelial fenestrae have been implicated in the pathogenesis of various diseases. The re-creation of fenestrated endothelium using human induced pluripotent stem cells (hiPSCs) provides a promising avenue to investigate the involvement of fenestrae in disease mechanisms and pharmacodynamics. In this project, we aim to induce the formation of fenestrae in nonfenestrated hiPSCs-derived endothelial cells (hiPSC-ECs). Vascular endothelial growth factor A (VEGFA) and phorbol myristate acetate (PMA) were used as inducers of fenestrae in hiPSC-ECs. The assessment of fenestrae formation included gene-expression analysis, scanning electron microscopy (SEM), transmission electron microscopy (TEM), and immunofluorescent staining. Endothelial monolayer functionality was evaluated by dextran permeability assays. Stimulation with VEGFA and PMA significantly induced expression of the diaphragmed fenestrae-associated marker, plasmalemmal vesicle-associated protein (PLVAP), in hiPSC-ECs at the mRNA, and protein levels. SEM analysis revealed VEGFA- and PMA-induced fenestrae structures on the cell membrane of hiPSC-ECs. The increased membrane localization of PLVAP visualized by TEM and immunofluorescent staining supported these findings. The induced fenestrated endothelium in hiPSC-ECs demonstrated selective passage of small solutes across a confluent monolayer with intact cell junctions, confirming functional competence. In conclusion, we present a novel methodology for inducing and regulating fenestrated endothelium in hiPSC-ECs. This innovative approach paves the way for the development of fenestrated microvasculature in human organ-on-a-chip systems, enabling complex disease modeling and physiologically relevant investigations of pharmacodynamics.

CMTM4 regulates angiogenesis by promoting cell surface recycling of VE-cadherin to endothelial adherens junctions.

Vascular endothelial (VE) cadherin is a key component of endothelial adherens junctions (AJs) and plays an important role in maintaining vascular integrity. Endocytosis of VE-cadherin regulates junctional strength and a decrease of surface VE-cadherin reduces vascular stability. However, disruption of AJs is also a requirement for vascular sprouting. Identifying novel regulators of endothelial endocytosis could enhance our understanding of angiogenesis. Here, we evaluated the angiogenic potential of (CKLF-like MARVEL transmembrane domain 4) CMTM4 and assessed in which molecular pathway CMTM4 is involved during angiogenesis. Using a 3D vascular assay composed of GFP-labeled HUVECs and dsRED-labeled pericytes, we demonstrated in vitro that siRNA-mediated CMTM4 silencing impairs vascular sprouting. In vivo, CMTM4 silencing by morpholino injection in zebrafish larvae inhibits intersomitic vessel growth. Intracellular staining revealed that CMTM4 colocalizes with Rab4+ and Rab7+ vesicles, both markers of the endocytic trafficking pathway. CMTM4 colocalizes with both membrane-bound and internalized VE-cadherin. Adenovirus-mediated CMTM4 overexpression enhances the endothelial endocytic pathway, in particular the rapid recycling pathway, shown by an increase in early endosomal antigen-1 positive (EEA1+), Rab4+, Rab11+ , and Rab7+ vesicles. CMTM4 overexpression enhances membrane-bound VE-cadherin internalization, whereas CMTM4 knockdown decreases internalization of VE-cadherin. CMTM4 overexpression promotes endothelial barrier function, shown by an increase in recovery of transendothelial electrical resistance (TEER) after thrombin stimulation. We have identified in this study a novel regulatory function for CMTM4 in angiogenesis. CMTM4 plays an important role in the turnover of membrane-bound VE-cadherin at AJs, mediating endothelial barrier function and controlling vascular sprouting.

Comparative proteomic analysis of cat eye syndrome critical region protein 1- function in tumor-associated macrophages and immune response regulation of glial tumors.

INTRODUCTION: Tumor associated macrophages (TAMs) promote tumor development, angiogenesis and distal metastasis. In previous studies, we showed that Cat Eye Syndrome Critical Region Protein 1 (CECR1) is expressed by M2-like TAMs in human glioma samples. CECR1 promoted M2 TAMs differentiation and affected glioma cell proliferation and migration. Here we investigated the proteomic profile of TAMs expressing CECR1 in absence or presence of glioma cells. RESULTS: CECR1 siRNA transfection upregulated 67 proteins in THP-1-derived Macrophages (MQs). Pathway annotation mapped this set to 3 major pathways relevant for MQ function, including 'MHC-I antigen presentation', 'phagosome maturation' and 'endocytosis'. Co-culture of siCECR1 THP-1-derived MQs with U87 glioma cells attenuated the changes observed on protein and mRNA level in response to MQ CECR1 silencing. SiCECR1 in U87 co-cultured MQs was associated with an IL-10low, IL-12high M1-like phenotype. In U87 co-culture conditions, SiCECR1 also downregulated S20 proteasome complex proteins PSMA5, PSMA7, PSMC6 and PSMD8. This protein profile was linked to a low proliferation rate of siCECR1 MQs. Overlap analysis identified S100A9 and PLAU as CECR1-related proteins that were significantly correlated with expression of CECR1 and macrophage lineage markers in three large public GBM datasets. CONCLUSION: This study reports the molecular pathways and key molecules that are mediated by CECR1 function in THP- 1-derived MQs and TAMs in glioma. METHODS: PMA-treated THP-1 cells (MQs) were siRNA transfected for CECR1 in vitro, with or without stimulation of the primary glioma cell line U87. Lysates were analyzed by (nano)LC-MS. Significant altered protein levels were identified (P < 0.05), followed by pathway annotation.

Endothelial loss of Fzd5 stimulates PKC/Ets1-mediated transcription of Angpt2 and Flt1.

AIMS: Formation of a functional vascular system is essential and its formation is a highly regulated process initiated during embryogenesis, which continues to play important roles throughout life in both health and disease. In previous studies, Fzd5 was shown to be critically involved in this process and here we investigated the molecular mechanism by which endothelial loss of this receptor attenuates angiogenesis. METHODS AND RESULTS: Using short interference RNA-mediated loss-of-function assays, the function and mechanism of signaling via Fzd5 was studied in human endothelial cells (ECs). Our findings indicate that Fzd5 signaling promotes neovessel formation in vitro in a collagen matrix-based 3D co-culture of primary vascular cells. Silencing of Fzd5 reduced EC proliferation, as a result of G0/G1 cell cycle arrest, and decreased cell migration. Furthermore, Fzd5 knockdown resulted in enhanced expression of the factors Angpt2 and Flt1, which are mainly known for their destabilizing effects on the vasculature. In Fzd5-silenced ECs, Angpt2 and Flt1 upregulation was induced by enhanced PKC signaling, without the involvement of canonical Wnt signaling, non-canonical Wnt/Ca2+-mediated activation of NFAT, and non-canonical Wnt/PCP-mediated activation of JNK. We demonstrated that PKC-induced transcription of Angpt2 and Flt1 involved the transcription factor Ets1. CONCLUSIONS: The current study demonstrates a pro-angiogenic role of Fzd5, which was shown to be involved in endothelial tubule formation, cell cycle progression and migration, and partly does so by repression of PKC/Ets1-mediated transcription of Flt1 and Angpt2.

Folic acid reduces doxorubicin-induced cardiomyopathy by modulating endothelial nitric oxide synthase.

The use of doxorubicin (DOXO) as a chemotherapeutic drug has been hampered by cardiotoxicity leading to cardiomyopathy and heart failure. Folic acid (FA) is a modulator of endothelial nitric oxide (NO) synthase (eNOS), which in turn is an important player in diseases associated with NO insufficiency or NOS dysregulation, such as pressure overload and myocardial infarction. However, the role of FA in DOXO-induced cardiomyopathy is poorly understood. The aim of this study was to test the hypothesis that FA prevents DOXO-induced cardiomyopathy by modulating eNOS and mitochondrial structure and function. Male C57BL/6 mice were randomized to a single dose of DOXO (20 mg/kg intraperitoneal) or sham. FA supplementation (10 mg/day per oral) was started 7 days before DOXO injection and continued thereafter. DOXO resulted in 70% mortality after 10 days, with the surviving mice demonstrating a 30% reduction in stroke volume compared with sham groups. Pre-treatment with FA reduced mortality to 45% and improved stroke volume (both P < 0.05 versus DOXO). These effects of FA were underlain by blunting of DOXO-induced cardiomyocyte atrophy, apoptosis, interstitial fibrosis and impairment of mitochondrial function. Mechanistically, pre-treatment with FA prevented DOXO-induced increases in superoxide anion production by reducing the eNOS monomer:dimer ratio and eNOS S-glutathionylation, and attenuated DOXO-induced decreases in superoxide dismutase, eNOS phosphorylation and NO production. Enhancing eNOS function by restoring its coupling and subsequently reducing oxidative stress with FA may be a novel therapeutic approach to attenuate DOXO-induced cardiomyopathy.

CMTM3 (CKLF-Like Marvel Transmembrane Domain 3) Mediates Angiogenesis by Regulating Cell Surface Availability of VE-Cadherin in Endothelial Adherens Junctions.

OBJECTIVE: Decrease in VE-cadherin adherens junctions reduces vascular stability, whereas disruption of adherens junctions is a requirement for neovessel sprouting during angiogenesis. Endocytosis plays a key role in regulating junctional strength by altering bioavailability of cell surface proteins, including VE-cadherin. Identification of new mediators of endothelial endocytosis could enhance our understanding of angiogenesis. Here, we assessed the function of CMTM3 (CKLF-like MARVEL transmembrane domain 3), which we have previously identified as highly expressed in Flk1+ endothelial progenitor cells during embryonic development. APPROACH AND RESULTS: Using a 3-dimensional coculture of human umbilical vein endothelial cells-GFP (green fluorescent protein) and pericytes-RFP (red fluorescent protein), we demonstrated that siRNA-mediated CMTM3 silencing in human umbilical vein endothelial cells impairs angiogenesis. In vivo CMTM3 inhibition by morpholino injection in developing zebrafish larvae confirmed that CMTM3 expression is required for vascular sprouting. CMTM3 knockdown in human umbilical vein endothelial cells does not affect proliferation or migration. Intracellular staining demonstrated that CMTM3 colocalizes with early endosome markers EEA1 (early endosome marker 1) and Clathrin+ vesicles and with cytosolic VE-cadherin in human umbilical vein endothelial cells. Adenovirus-mediated CMTM3 overexpression enhances endothelial endocytosis, shown by an increase in Clathrin+, EEA1+, Rab11+, Rab5+, and Rab7+ vesicles. CMTM3 overexpression enhances, whereas CMTM3 knockdown decreases internalization of cell surface VE-cadherin in vitro. CMTM3 promotes loss of endothelial barrier function in thrombin-induced responses, shown by transendothelial electric resistance measurements in vitro. CONCLUSIONS: In this study, we have identified a new regulatory function for CMTM3 in angiogenesis. CMTM3 is involved in VE-cadherin turnover and is a regulator of the cell surface pool of VE-cadherin. Therefore, CMTM3 mediates cell-cell adhesion at adherens junctions and contributes to the control of vascular sprouting.

Activation of CECR1 in M2-like TAMs promotes paracrine stimulation-mediated glial tumor progression.

Background: The majority of glioma-associated microglia/macrophages have been identified as M2-type macrophages with immune suppressive and tumor supportive action. Recently, the extracellular adenosine deaminase protein Cat Eye Syndrome Critical Region Protein 1 (CECR1) was shown to regulate macrophage maturation. In this study, we investigate the role of CECR1 in the regulation of the glioma-associated macrophage response. Methods: Expression of CECR1 was assessed in human glioma samples. CECR1-mediated macrophage response was studied in vitro, using donor derived CD14+ monocytes and the THP-1 monocytic cell line. The response of the human glioma cell line U87 to conditioned medium of macrophages preconditioned with recombinant human CECR1 or CECR1 silencing was also assessed. Results: CECR1 was strongly expressed in high-grade gliomas (P < .001) and correlated positively with the M2 phenotype markers in tumor-associated microglia/macrophages (TAMs) (overall, P < .05). In vitro studies confirmed the presence of a significantly higher level of CECR1 expression in M2-like macrophages exposed to U87 conditioned medium (P < .001). CECR1 knockdown or stimulation of macrophages affected differentiation toward the M2-like phenotype. Stimulation of U87 cells with conditioned medium of CECR1 knockdown or stimulated macrophages affected tumor cell proliferation and migration, coinciding with altered intracellular signaling of mitogen-activated protein kinase (MAPK). In glioma tissue samples, CECR1 expression correlated with Ki67 and MAPK signaling protein. Conclusions: CECR1 is a potent regulator of TAM polarization and is consistently highly expressed by M2-type TAMs, particularly in high-grade glioma. Paracrine effects induced by CECR1 in M2-like TAMs activate MAPK signaling and stimulate the proliferation and migration of glioma cells.

Uridine adenosine tetraphosphate acts as a proangiogenic factor in vitro through purinergic P2Y receptors.

Uridine adenosine tetraphosphate (Up4A), a dinucleotide, exerts vascular influence via purinergic receptors (PR). We investigated the effects of Up4A on angiogenesis and the putative PR involved. Tubule formation assay was performed in a three-dimensional system, in which human endothelial cells were cocultured with pericytes with various Up4A concentrations for 5 days. Expression of PR subtypes and angiogenic factors was assessed in human endothelial cells with and without P2Y6R antagonist. No difference in initial tubule formation was detected between Up4A stimulation and control conditions at day 2 In contrast, a significant increase in vascular density in response to Up4A was observed at day 5 Up4A at an optimal concentration of 5 µM promoted total tubule length, number of tubules, and number of junctions, all of which were inhibited by the P2Y6R antagonist MRS2578. Higher concentrations of Up4A (10 µM) had no effects on angiogenesis parameters. Up4A increased mRNA level of P2YRs (P2Y2R, P2Y4R, and P2Y6R) but not P2XR (P2X4R and P2X7R) or P1R (A2AR and A2BR), while Up4A upregulated VEGFA and ANGPT1, but not VEGFR2, ANGPT2, Tie1, and Tie2. In addition, Up4A increased VEGFA protein levels. Transcriptional upregulation of P2YRs by Up4A was inhibited by MRS2578. In conclusion, Up4A is functionally capable of promoting tubule formation in an in vitro coculture system, which is likely mediated by pyrimidine-favored P2YRs but not P2XRs or P1Rs, and involves upregulation of angiogenic factors.

PDGF-induced migration of vascular smooth muscle cells is inhibited by heme oxygenase-1 via VEGFR2 upregulation and subsequent assembly of inactive VEGFR2/PDGFRß heterodimers.

OBJECTIVE: In cardiovascular regulation, heme oxygenase-1 (HO-1) activity has been shown to inhibit vascular smooth muscle cell (VSMC) proliferation by promoting cell cycle arrest at the G1/S phase. However, the effect of HO-1 on VSMC migration remains unclear. We aim to elucidate the mechanism by which HO-1 regulates PDGFBB-induced VSMC migration. METHODS AND RESULTS: Transduction of HO-1 cDNA adenoviral vector severely impeded human VSMC migration in a scratch, transmembrane, and directional migration assay in response to PDGFBB stimulation. Similarly, HO-1 overexpression in the remodeling process during murine retinal vasculature development attenuated VSMC coverage over the major arterial branches as compared with sham vector-transduced eyes. HO-1 expression in VSMCs significantly upregulated VEGFA and VEGFR2 expression, which subsequently promoted the formation of inactive PDGFRß/VEGFR2 complexes. This compromised PDGFRß phosphorylation and impeded the downstream cascade of FAK-p38 signaling. siRNA-mediated silencing of VEGFA or VEGFR2 could reverse the inhibitory effect of HO-1 on VSMC migration. CONCLUSIONS: These findings identify a potent antimigratory function of HO-1 in VSMCs, a mechanism that involves VEGFA and VEGFR2 upregulation, followed by assembly of inactive VEGFR2/PDGFRß complexes that attenuates effective PDGFRß signaling.

Ets2 determines the inflammatory state of endothelial cells in advanced atherosclerotic lesions.

RATIONALE: Neovascularization is required for embryonic development and plays a central role in diseases in adults. In atherosclerosis, the role of neovascularization remains to be elucidated. In a genome-wide microarray-screen of Flk1+ angioblasts during murine embryogenesis, the v-ets erythroblastosis virus E26 oncogene homolog 2 (Ets2) transcription factor was identified as a potential angiogenic factor. OBJECTIVES: We assessed the role of Ets2 in endothelial cells during atherosclerotic lesion progression toward plaque instability. METHODS AND RESULTS: In 91 patients treated for carotid artery disease, Ets2 levels showed modest correlations with capillary growth, thrombogenicity, and rising levels of tumor necrosis factor-α (TNFα), monocyte chemoattractant protein 1, and interleukin-6 in the atherosclerotic lesions. Experiments in ApoE(-/-) mice, using a vulnerable plaque model, showed that Ets2 expression was increased under atherogenic conditions and was augmented specifically in the vulnerable versus stable lesions. In endothelial cell cultures, Ets2 expression and activation was responsive to the atherogenic cytokine TNFα. In the murine vulnerable plaque model, overexpression of Ets2 promoted lesion growth with neovessel formation, hemorrhaging, and plaque destabilization. In contrast, Ets2 silencing, using a lentiviral shRNA construct, promoted lesion stabilization. In vitro studies showed that Ets2 was crucial for TNFα-induced expression of monocyte chemoattractant protein 1, interleukin-6, and vascular cell adhesion molecule 1 in endothelial cells. In addition, Ets2 promoted tube formation and amplified TNFα-induced loss of vascular endothelial integrity. Evaluation in a murine retina model further validated the role of Ets2 in regulating vessel inflammation and endothelial leakage. CONCLUSIONS: We provide the first evidence for the plaque-destabilizing role of Ets2 in atherosclerosis development by induction of an intraplaque proinflammatory phenotype in endothelial cells.