Promoter In Endothelial Cells Research Articles

Statins improve cardiac endothelial function to prevent heart failure with preserved ejection fraction through upregulating circRNA-RBCK1

Heart failure with preserved ejection fraction (HFpEF) is associated with endothelial dysfunction. We have previously reported that statins prevent endothelial dysfunction through inhibition of microRNA-133a (miR-133a). This study is to investigate the effects and the underlying mechanisms of statins on HFpEF. Here, we show that statins upregulate the expression of a circular RNA (circRNA-RBCK1) which is co-transcripted with the ring-B-box-coiled-coil protein interacting with protein kinase C-1 (RBCK1) gene. Simultaneously, statins increase activator protein 2 alpha (AP-2α) transcriptional activity and the interaction between circRNA-RBCK1 and miR-133a. Furthermore, AP-2α directly interacts with RBCK1 gene promoter in endothelial cells. In vivo, lovastatin improves diastolic function in male mice under HFpEF, which is abolished by loss function of endothelial AP-2α or circRNA-RBCK1. This study suggests that statins upregulate the AP-2α/circRNA-RBCK1 signaling to suppress miR-133a in cardiac endothelial cells and prevent diastolic dysfunction in HFpEF.

510-P: Tissue Nanotransfection–Based Endothelial-Targeted Epigenetic Gene Editing In Vivo to Rescue Diabetic Ischemic Wounds

Our previous work (PMID: 35192691) has identified that genetically silenced phospholipase Cγ2 (PLCγ2) hinders VEGF therapy of the diabetic ischemic limb. Hyperglycemia caused hypermethylation of endothelial specific gene promoter. Given that epigenetic changes are reversible, this work tests the significance of gene-targeted therapeutic DNA demethylation in improving blood flow to diabetic ischemic wounds. Bipedicle ischemic wounds were placed in streptozotocin (STZ) induced acute diabetic C57BL6 mice. Diabetic VWF+ dermal endothelial cells were isolated using flow sorting. In wound tissue, PLCγ2 promoter CpG methylation levels were analyzed using bisulfite sequencing. Next, to specifically demethylate PLCγ2 promoter in endothelial cells, a demethylation cocktail was designed: (i) (scFv)-TET1 catalytic domain (TET1CD) system capable of inducing targeted DNA methylation, and (ii) endothelial promoter driven gene specific guide RNAs. This demethylation cocktail was delivered at the diabetic ischemic wound-edge employing non-viral topical tissue nano-transfection (TNT) technology. PLCγ2 protein expression outcomes were assessed using flow cytometry. Functional outcome of such demethylation was assessed using Laser Speckle Perfusion imaging (Perimed Inc.) and ultrasonography (Vevo 2100) at days 3, 7&10. Overall DNA hyper methylation was prominent in murine ischemic flaps as demonstrated by the increased ratio of 5-methylcytosine (methylation mark) to 5-hydroxymethylcytosine (demethylation mark) (n=5). Specifically, the PLCγ2 promoter was hypermethylated (n=5). TNT mediated endothelial-targeted demethylation of the PLCγ2 promoter increased its expression (n=4). Such demethylation-based upregulation of PLCγ2 improved wound tissue blood flow with increased abundance of VWF+/PLCG2+ vascular elements (n=4). TNT-based endothelial demethylation of the PLCγ2 gene promoter improved perfusion of cutaneous diabetic wounds. Disclosure S.S.Verma: None. S.Kacar: None. S.C.Gnyawali: None. L.S.Fehme: None. C.K.Sen: n/a. K.Singh: None. Funding U.S. Department of Defense (W81XWH-22-1-0146)

SOX7 suppresses endothelial-to-mesenchymal transitions by enhancing VE-cadherin expression during outflow tract development.

The endothelial-to-mesenchymal transition (EndMT) is a critical process that occurs during the development of the outflow tract (OFT). Malformations of the OFT can lead to the occurrence of conotruncal defect (CTD). SOX7 duplication has been reported in patients with congenital CTD, but its specific role in OFT development remains poorly understood. To decipher this, histological analysis showed that SRY-related HMG-box 7 (SOX7) was regionally expressed in the endocardial endothelial cells and in the mesenchymal cells of the OFT, where EndMT occurs. Experiments, using in vitro collagen gel culture system, revealed that SOX7 was a negative regulator of EndMT that inhibited endocardial cell (EC) migration and resulted in decreased number of mesenchymal cells. Forced expression of SOX7 in endothelial cells blocked further migration and improved the expression of the adhesion protein vascular endothelial (VE)-cadherin (VE-cadherin). Moreover, a VE-cadherin knockdown could partly reverse the SOX7-mediated repression of cell migration. Luciferase and electrophoretic mobility shift assay (EMSA) demonstrated that SOX7 up-regulated VE-cadherin by directly binding to the gene's promoter in endothelial cells. The coding exons and splicing regions of the SOX7 gene were also scanned in the 536 sporadic CTD patients and in 300 unaffected controls, which revealed four heterozygous SOX7 mutations. Luciferase assays revealed that two SOX7 variants weakened the transactivation of the VE-cadherin promoter. In conclusion, SOX7 inhibited EndMT during OFT development by directly up-regulating the endothelial-specific adhesion molecule VE-cadherin. SOX7 mutations can lead to impaired EndMT by regulating VE-cadherin, which may give rise to the molecular mechanisms associated with SOX7 in CTD pathogenesis.

Restoration of FVIII expression by targeted gene insertion in the FVIII locus in hemophilia A patient-derived iPSCs

Target-specific genome editing, using engineered nucleases zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and type II clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), is considered a promising approach to correct disease-causing mutations in various human diseases. In particular, hemophilia A can be considered an ideal target for gene modification via engineered nucleases because it is a monogenic disease caused by a mutation in coagulation factor VIII (FVIII), and a mild restoration of FVIII levels in plasma can prevent disease symptoms in patients with severe hemophilia A. In this study, we describe a universal genome correction strategy to restore FVIII expression in induced pluripotent stem cells (iPSCs) derived from a patient with hemophilia A by the human elongation factor 1 alpha (EF1α)-mediated normal FVIII gene expression in the FVIII locus of the patient. We used the CRISPR/Cas9-mediated homology-directed repair (HDR) system to insert the B-domain deleted from the FVIII gene with the human EF1α promoter. After gene targeting, the FVIII gene was correctly inserted into iPSC lines at a high frequency (81.81%), and these cell lines retained pluripotency after knock-in and neomycin resistance cassette removal. More importantly, we confirmed that endothelial cells from the gene-corrected iPSCs could generate functionally active FVIII protein from the inserted FVIII gene. This is the first demonstration that the FVIII locus is a suitable site for integration of the normal FVIII gene and can restore FVIII expression by the EF1α promoter in endothelial cells differentiated from the hemophilia A patient-derived gene-corrected iPSCs.

Class II transactivator (CIITA) mediates IFN-γ induced eNOS repression by enlisting SUV39H1

Endothelial nitric oxide synthase (eNOS), selectively expressed in vascular endothelial cells, plays important roles in a range of biological and pathological processes. eNOS levels can be altered by extrinsic and intrinsic cues at the transcriptional level. Here we examined the epigenetic mechanism whereby the pro-inflammatory cytokine interferon gamma (IFN-γ) represses eNOS transcription. In response to IFN-γ treatment, there was a simultaneous down-regulation of eNOS expression and up-regulation of class II trans-activator (CIITA). Over-expression of CIITA directly repressed eNOS promoter while CIITA knockdown attenuated IFN-γ induced eNOS repression. Chromatin immunoprecipitation (ChIP) assay revealed that IFN-γ stimulation promoted CIITA occupancy on the proximal eNOS (−430/−168). Coincidently, CIITA recruitment to the eNOS promoter was paralleled by the disappearance of trimethylated histone H3K4 (H3K4Me3) and the enrichment of trimethylated H3K9 (H3K9Me3) with no significant changes in the levels of trimethylated H3K27 (H3K27Me3) or trimethylated H4K20 (H4K20Me3). In accordance, CIITA depletion was associated with the normalization of H3K4Me3 and H3K9Me3 on the eNOS promoter. Mechanistically, CIITA interacted with and enlisted the histone H3K9 trimethyltransferase SUV39H1 to the eNOS promoter to repress transcription. IFN-γ treatment augmented SUV39H1 expression and promoted SUV39H1 recruitment to the eNOS promoter in endothelial cells. Silencing of SUV39H1 abrogated eNOS repression by IFN-γ by erasing H3K9Me3 from the eNOS promoter. In conclusion, our data reveal a novel role for CIITA in endothelial cells and present SUV39H1 as a druggable target in the intervention of endothelial dysfunction.

Mitochondrial uncouplers induce CpG methylation of the ICAM1 gene promoter in endothelial cells

Mitochondrial uncouplers induce CpG methylation of the ICAM1 gene promoter in endothelial cells

The transcriptional regulator CCCTC-binding factor limits oxidative stress in endothelial cells

The CCCTC-binding factor (CTCF) is a versatile transcriptional regulator required for embryogenesis, but its function in vascular development or in diseases with a vascular component is poorly understood. Here, we found that endothelial Ctcf is essential for mouse vascular development and limits accumulation of reactive oxygen species (ROS). Conditional knockout of Ctcf in endothelial progenitors and their descendants affected embryonic growth, and caused lethality at embryonic day 10.5 because of defective yolk sac and placental vascular development. Analysis of global gene expression revealed Frataxin (Fxn), the gene mutated in Friedreich's ataxia (FRDA), as the most strongly down-regulated gene in Ctcf-deficient placental endothelial cells. Moreover, in vitro reporter assays showed that Ctcf activates the Fxn promoter in endothelial cells. ROS are known to accumulate in the endothelium of FRDA patients. Importantly, Ctcf deficiency induced ROS-mediated DNA damage in endothelial cells in vitro, and in placental endothelium in vivo Taken together, our findings indicate that Ctcf promotes vascular development and limits oxidative stress in endothelial cells. These results reveal a function for Ctcf in vascular development, and suggest a potential mechanism for endothelial dysfunction in FRDA.

Enhancer-associated long non-coding RNA LEENE regulates endothelial nitric oxide synthase and endothelial function

The optimal expression of endothelial nitric oxide synthase (eNOS), the hallmark of endothelial homeostasis, is vital to vascular function. Dynamically regulated by various stimuli, eNOS expression is modulated at transcriptional, post-transcriptional, and post-translational levels. However, epigenetic modulations of eNOS, particularly through long non-coding RNAs (lncRNAs) and chromatin remodeling, remain to be explored. Here we identify an enhancer-associated lncRNA that enhances eNOS expression (LEENE). Combining RNA-sequencing and chromatin conformation capture methods, we demonstrate that LEENE is co-regulated with eNOS and that its enhancer resides in proximity to eNOS promoter in endothelial cells (ECs). Gain- and Loss-of-function of LEENE differentially regulate eNOS expression and EC function. Mechanistically, LEENE facilitates the recruitment of RNA Pol II to the eNOS promoter to enhance eNOS nascent RNA transcription. Our findings unravel a new layer in eNOS regulation and provide novel insights into cardiovascular regulation involving endothelial function.

Tumor Necrosis Factor α Induces the Expression of the Endothelial Cell-Specific Receptor Roundabout4 through the Nuclear Factor-κB Pathway.

Roundabout4 (Robo4) is an endothelial cell-specific receptor that regulates vascular stability. Recently, Robo4 has been shown to regulate vascular permeability in inflammation. However, the mechanisms regulating the Robo4 gene in the context of inflammation are poorly understood. In this study, we found that intravenous injection of tumor necrosis factor (TNF) α increased Robo4 expression in mouse organs. In vitro analyses showed that TNFα increased Robo4 expression in human primary endothelial cells, but not in cells pretreated with a nuclear factor (NF)-κB inhibitor. Reporter assays using wild-type and mutant Robo4 promoters indicated that TNFα activated the Robo4 promoter and that both the -2753 and -2220 NF-κB motifs were essential for this activation. Electrophoretic mobility shift assays demonstrated that the NF-κB p65-p50 heterodimer bound to these motifs. These findings were further supported by chromatin immunoprecipitation assays in endothelial cells. Taken together, these results indicated that TNFα induced Robo4 expression by facilitating NF-κB p65-p50 heterodimer binding to the -2753 and -2220 motifs in the Robo4 promoter in endothelial cells in the context of inflammation.

Contribution of endothelial injury and inflammation in early phase to vein graft failure: the causal factors impact on the development of intimal hyperplasia in murine models.

ObjectivesAutologous veins are preferred conduits in by-pass surgery. However, long-term results are hampered by limited patency due to intimal hyperplasia. Although mechanisms involved in development of intimal hyperplasia have been established, the role of inflammatory processes is still unclear. Here, we studied leukocyte recruitment and intimal hyperplasia in inferior vena cava grafts transferred to abdominal aorta in mice.Methods and ResultsSeveral microscopic techniques were used to study endothelium denudation and regeneration and leukocyte recruitment on endothelium. Scanning electron microscopy demonstrated denudation of vein graft endothelium 7 days post-transfer and complete endothelial regeneration by 28 days. Examination of vein grafts transferred to mice transgenic for green fluorescent protein under Tie2 promoter in endothelial cells showed regeneration of graft endothelium from the adjacent aorta. Intravital microscopy revealed recruitment of leukocytes in vein grafts at 7 days in wild type mice, which had tapered off by 28 days. At 28 and 63 days there was significant development of intimal hyperplasia. In contrast; no injury, leukocyte recruitment nor intimal hyperplasia occurred in arterial grafts. Leukocyte recruitment was reduced in vein grafts in mice deficient in E- and P-selectin. In parallel, intimal hyperplasia was reduced in vein grafts in mice deficient in E- and P-selectin and in wild type mice receiving P-selectin/E-selectin function-blocking antibodies.ConclusionThe results show that early phase endothelial injury and inflammation are crucial processes in intimal hyperplasia in murine vein grafts. The data implicate endothelial selectins as targets for intervention of vein graft disease.

Endorepellin Evokes Autophagy in Endothelial Cells

Endorepellin, the C-terminal fragment of the heparan sulfate proteoglycan perlecan, possesses angiostatic activity via dual receptor antagonism, through concurrent binding to the α2β1 integrin and vascular endothelial growth factor receptor 2 (VEGFR2). Here, we discovered that soluble endorepellin induced autophagy in endothelial cells by modulating the expression of Beclin 1, LC3, and p62, three established autophagic markers. Moreover, endorepellin evoked expression of the imprinted tumor suppressor gene Peg3 and its co-localization with Beclin 1 and LC3 in autophagosomes, suggesting a major role for this gene in endothelial cell autophagy. Mechanistically, endorepellin induced autophagy by down-regulating VEGFR2 via the two LG1/2 domains, whereas the C-terminal LG3 domain, the portion responsible for binding the α2β1 integrin, was ineffective. Endorepellin also induced transcriptional activity of the BECN1 promoter in endothelial cells, and the VEGFR2-specific tyrosine kinase inhibitor, SU5416, blocked this effect. Finally, we found a correlation between endorepellin-evoked inhibition of capillary morphogenesis and enhanced autophagy. Thus, we have identified a new role for this endogenous angiostatic fragment in inducing autophagy through a VEGFR2-dependent but α2β1 integrin-independent pathway. This novel mechanism specifically targets endothelial cells and could represent a promising new strategy to potentiate the angiostatic effect of endorepellin and perhaps other angiostatic matrix proteins.

Abstract 195: Characterization of DNA Modules in Regulating VWF Vascular Bed--Specific Expression

Expression of von Willebrand Factor (vWF) in the endothelium is regulated by vascular bed-specific pathways. A region of the human vWF gene between -843 and -620 was previously shown to direct expression in the endothelium of capillaries and a subset of larger blood vessels in the heart and skeletal muscle. Here, our goal was to further delineate the responsible DNA sequences. A series of constructs containing 5’-deletions, internal deletions or point mutations of the vWF promoter coupled to LacZ were targeted to the Hprt locus of mice using homologous recombination, and the resulting animals were analyzed for reporter gene expression. The findings demonstrate that a three-base-pair fragment within vWF promoter is necessary for expression in capillary but not large vessel endothelium in heart and skeletal muscle. Although the sequence does not conform to established DNA binding sites, it bound to nuclear protein as determined by elecrophoretic mobility shift assay. Mass spectrometry of the DNA-protein complex revealed several potential binding transcription factors. The heart- and skeletal muscle-specific promoter region did not response to shear stress or mechanical stress. Bisulfite sequencing revealed differential methylation of the human vWF promoter in endothelial cells from expressing and non-expressing vascular beds. These differences were altered by the mutation at the short cis-element. Together, these data support a model of modular gene regulation in which distinct DNA sequences in the vWF promoter direct expression in different vascular beds.

Inhibition of endothelial ERK signalling by Smad1/5 is essential for haematopoietic stem cell emergence

The earliest HSCs are derived from haemogenic endothelium via endothelial-to-haematopoietic transition during vertebrate embryogenesis; however, the underlying mechanism is largely unclear. Here we show that interplay of Smad1/5 and ERK signalling is essential for haemogenic endothelium-based HSC emergence. Smad1/5 directly inhibits erk expression through recruiting HDAC1 to and inducing de-acetylation of the erk promoter in endothelial cells. Over-activated ERK signalling conferred by inhibition of Smad1/5 promotes the arterial endothelial cell fate and constitutively strengthens the tight junction between endothelial cells, thereby repressing the specification of haemogenic endothelium and the following endothelial-to-haematopoietic transition process. These findings provide new insights into the in vitro generation of transplantable HSCs for potential clinical applications.

Histone and DNA Methylation–Mediated Epigenetic Downregulation of Endothelial Kruppel-Like Factor 2 by Low-Density Lipoprotein Cholesterol

Low-density lipoprotein (LDL) cholesterol induces endothelial dysfunction and is a major modifiable risk factor for coronary heart disease. Endothelial Kruppel-like Factor 2 (KLF2) is a transcription factor that is vital to endothelium-dependent vascular homeostasis. The purpose of this study is to determine whether and how LDL affects endothelial KLF2 expression. LDL downregulates KLF2 expression and promoter activity in endothelial cells. LDL-induced decrease in KLF2 parallels changes in endothelial KLF2 target genes thrombomodulin, endothelial NO synthase, and plasminogen activator inhibitor-1. Pharmacological inhibition of DNA methyltransferases or knockdown of DNA methyltransferase 1 prevents downregulation of endothelial KLF2 by LDL. LDL induces endothelial DNA methyltransferase 1 expression and DNA methyltransferase activity. LDL stimulates binding of the DNA methyl-CpG-binding protein-2 and histone methyltransferase enhancer of zeste homolog 2, whereas decreases binding of the KLF2 transcriptional activator myocyte enhancing factor-2, to the KLF2 promoter in endothelial cells. Knockdown of myocyte enhancing factor-2, or mutation of the myocyte enhancing factor-2 site in the KLF2 promoter, abrogates LDL-induced downregulation of endothelial KLF2 and thrombomodulin, and KLF2 promoter activity. Similarly, knockdown of enhancer of zeste homolog 2 negates LDL-induced downregulation of KLF2 and thrombomodulin in endothelial cells. Finally, overexpression of KLF2 rescues LDL-induced clotting of platelet-rich plasma on endothelial cells. LDL represses endothelial KLF2 expression via DNA and histone methylation. Downregulation of KLF2 by LDL leads to a dysfunctional, hypercoagulable endothelium.

In Vitro Targeted Gene Electrotransfer to Endothelial Cells with Plasmid DNA Containing Human Endothelin-1 Promoter

Development of recombinant DNA technologies has allowed us to create new delivery systems that target specific cell types and that can be used in gene therapy. One of these targets is vascular endothelium because of its important role in tumor angiogenesis. For tumor endothelium-specific targeting, we prepared plasmid DNA encoding green fluorescent protein under the control of human endothelin-1 promoter (pENDO-EGFP), which is specific for endothelial cells. First we determined gene electrotransfer parameters for improved transfection of endothelial cells evaluating different osmolarity of electroporation buffer, voltages of applied electric pulses, and addition of fetal bovine serum immediately after electroporation to the cells for improved transfection and survival. Transfection efficacy of pENDO-EGFP in different endothelial and nonendothelial cell lines was determined next. Gene electrotransfer efficacy was evaluated using three different methods: fluorescence microscopy, fluorescence microplate reader, and flow cytometry. Our results showed that transfection efficacy was higher when cells were prepared in hypoosmolar compared to isoosmolar electroporation buffer. Furthermore, immediate addition of fetal bovine serum to the cells after pulsing also improved gene electrotransfer into target cells. We proved expression of EGFP under the control of human endothelin-1 promoter in endothelial cells, which was also significantly higher compared to nonendothelial cells. Taken together, we successfully constructed pENDO-EGFP, which was specifically expressed in endothelial cells using improved gene electrotransfer parameters.

Arterial flow reduces oxidative stress via an antioxidant response element and Oct-1 binding site within the NADPH oxidase 4 promoter in endothelial cells

The main sources of oxidative stress in the vessel wall are nicotine adenine dinucleotide phosphate (NADPH) oxidase (Nox) complexes. The endothelium mainly expresses the Nox4-containing complex; however, the mechanism by which shear stress in endothelial cells regulates Nox4 is not well understood. This study demonstrates that long-term application of arterial laminar shear stress using a cone-and-plate viscometer reduces endothelial superoxide anion formation and Nox4 expression. In primary human endothelial cells, we identified a 47bp 5'-untranslated region of Nox4 mRNA by 5'-rapid amplification of cDNA ends (5'-RACE) PCR. Cloning and functional analysis of human Nox4 promoter revealed a range between -1,490 and -1,310bp responsible for flow-dependent downregulation. Mutation of an overlapping antioxidative response element (ARE)-like and Oct-1 binding site at -1,376bp eliminated shear stress-dependent Nox4 downregulation. Consistent with these observations, electrophoretic mobility shift assays (EMSA) demonstrated an enhanced shear stress-dependent binding of Nox4 oligonucleotide containing the ARE-like/Oct-1 binding site, which could be inhibited by specific antibodies against the transcription factors nuclear factor erythroid 2-related factor 2 (Nrf2) and octamer transcription factor 1 (Oct-1). Furthermore, shear stress caused the translocation of Nrf2 and Oct-1 from the cytoplasm to the nucleus. Knockdown of Nrf2 by short hairpin RNA (shRNA) increased Nox4 expression twofold, indicating a direct cross-talk between Nrf2 and Nox4. In conclusion, an ARE-like/Oct-1 binding site was noticed to be essential for shear stress-dependent downregulation of Nox4. This novel mechanism may be involved in the flow-dependent downregulation of endothelial superoxide anion formation.

Aryl Hydrocarbon Receptor Is Activated by Glucose and Regulates the Thrombospondin-1 Gene Promoter in Endothelial Cells

Hyperglycemia is an independent risk factor for development of diabetic vascular complications. The molecular mechanisms that are activated by glucose in vascular cells and could explain the development of vascular complications are still poorly understood. A putative binding site for the transcription factor aryl hydrocarbon receptor (AhR) was identified in the glucose-responsive fragment of the promoter of thrombospondin-1, a potent antiangiogenic and proatherogenic protein involved in development of diabetic vascular complications. AhR was expressed in aortic endothelial cells (ECs), activated, and bound to the promoter in response to high glucose stimulation of ECs. The constitutively active form of AhR induced activation of the thrombospondin-1 gene promoter. In response to high glucose stimulation, AhR was found in complex with Egr-1 and activator protein-2, which are 2 other nuclear transcription factors activated by glucose in ECs that have not been previously detected in complex with AhR. The activity of the DNA-binding complex was regulated by glucose through the activation of hexosamine pathway and intracellular glycosylation. This is the first report of activation of AhR (a receptor for xenobiotic compounds) by a physiological stimulus. This report links the activation of AhR to the pathological effects of hyperglycemia in the vasculature.

Aryl Hydrocarbon Receptor is Activated by High Glucose and Regulates the Thrombospondin‐1 Gene Promoter in Endothelial Cells

Glucose levels correlate directly with the diabetic vascular complications, and the earliest sign of vascular complications is endothelial dysfunction. However, the transcriptional mechanisms activated by glucose in endothelial cells (EC) are poorly understood. Aryl Hydrocarbon Receptor (AhR), a receptor for xenobiotic compounds with unknown physiological function, was activated in EC by high glucose and, as shown by chromatin immunoprecipitation, bound the promoter of thrombospondin‐1, a potent anti‐angiogenic and pro‐atherogenic protein involved in development of diabetic vascular complications. A binding site for AhR was identified in the glucose‐responsive fragment of the promoter (−280/+66), and the deletion of 15 base pairs containing the binding site abrogated the response. The constitutively active rAhR induced activation of the TSP‐1 gene promoter more than 4 fold. In response to high glucose stimulation, AhR formed a complex with Egr‐1 and AP‐2, two other transcription factors activated by glucose in EC that have not been previously detected in complex with AhR. The activity of the DNA‐binding complex was regulated by glucose through the activation of the hexosamine pathway and intracellular glycosylation. This is the first report of activation of AhR by a physiological stimulus. This report links the activation of AhR to the pathological effects of hyperglycemia in the vasculature.

Forkhead transcription factors regulate expression of the chemokine receptor CXCR4 in endothelial cells and CXCL12-induced cell migration

Foxc1 and Foxc2 transcription factors are required for vascular development. However, the molecular mechanisms by which Foxc1 and Foxc2 control angiogenesis, the growth of new blood vessels from pre-existing vessels and capillaries, remain unknown. CXC chemokine ligand 12 (CXCL12) and its receptor, CXCR4, are critical for the process of angiogenesis, including the migration and tube formation of endothelial cells. Here we show that Foxc1 and Foxc2 directly induce CXCR4 expression by activating its promoter in endothelial cells. Furthermore, Foxc1-deficient endothelial cells show a significant reduction in CXCR4 expression as well as CXCL12-stimulated migration. Taken together, these results provide novel evidence that Foxc transcription factors are important regulators of the chemotactic motility of endothelial cells through the induction of CXCR4 expression.

HESR1/CHF2 suppresses VEGFR2 transcription independent of binding to E-boxes

The bHLH transcription factor HESR1 (CHF2) acts downstream of notch to regulate cardiovascular development and angiogenesis, at least in part through down-regulation of the VEGF receptor, VEGFR2. Surprisingly, we find that HESR1 interacts with the promoter in endothelial cells (EC) not through direct binding to the E-boxes, but through intermediary interactions with GC-box-binding proteins. The bHLH and orange domains of HESR1 are sufficient for repression in EC, likely through recruitment of co-repressors, however, the C-terminal YRPW motif is not required. The VEGFR2 promoter contains a functional initiator element but no TATA box, however, addition of a TATA sequence renders the promoter resistant to inhibition by HESR1. In agreement with this finding, the NrCAM, TK, and CMV promoters, which have TATA boxes, cannot be repressed. Thus, HESR1 represses VEGFR2 through interactions with SP-1-like factors and requires an Inr element in the absence of a TATA box. Our findings illuminate an important mechanism for notch/HESR1 regulation of VEGF-induced angiogenesis.