Sugar uptake, metabolism, and chloride secretion in the rectal gland of the spiny dogfish Squalus acanthias.

The rectal gland of the spiny dogfish Squalus acanthias secretes a salt solution isosmotic with plasma that maintains the salt homeostasis of the fish. It secretes salt against an electrochemical gradient that requires the expenditure of energy. Isolated rectal glands perfused without glucose secrete salt, albeit at a rate about 30% of glands perfused with 5 mM glucose. Gradually reducing the glucose concentration is associated with a progressive decrease in the secretion of chloride. The apparent Km for the exogenous glucose-dependent chloride secretion is around 2 mM. Phloretin and cytochalasin B, agents that inhibit facilitated glucose carriers of the solute carrier 2 (Slc2) family such as glucose transporter 2 (GLUT2), do not inhibit the secretion of chloride by the perfused rectal glands. Phloridzin, which inhibits Slc5 family of glucose symporters, or α-methyl-d-glucoside, which competitively inhibits the uptake of glucose through Slc5 symporters, inhibit the secretion of chloride. Thus the movement of glucose into the rectal gland cells appears to be mediated by a sodium-glucose symporter. Sodium-glucose cotransporter 1 (SGLT1), the first member of the Slc5 family of sodium-linked glucose symporters, was cloned from the rectal gland. No evidence of GLUT2 was found. The persistence of secretion of chloride in the absence of glucose in the perfusate suggests that there is an additional source of energy within the cells. The use of 2-mercapto-acetate did not result in any change in the secretion of chloride, suggesting that the oxidation of fatty acids is not the source of energy for the secretion of chloride. Perfusion of isolated glands with KCN in the absence of glucose further reduces the secretion of chloride but does not abolish it, again suggesting that there is another source of energy within the cells. Glucose was measured in the rectal gland cells and found to be at concentrations in the range of that in the perfusate. Glycogen measurements indicated that there are significant stores of glucose in the rectal gland. Moreover, glycogen synthase was partially cloned from rectal gland cells. The open reading frame of glycogen phosphorylase was also cloned from rectal gland cells. Measurements of glycogen phosphorylase showed that the enzyme is mostly in its active form in the cells. The cells of the rectal gland of the spiny dogfish require exogenous glucose to fully support the active secretion of salt. They have the means to transport glucose into the cells in the form of SGLT1. The cells also have an endogenous supply of glucose as glycogen and have the necessary elements to synthesize, store, and hydrolyze it.

Identification of extant vertebrate Myxine glutinosa VWF: evolutionary conservation of primary hemostasis.

Hemostasis in vertebrates involves both a cellular and a protein component. Previous studies in jawless vertebrates (cyclostomes) suggest that the protein response, which involves thrombin-catalyzed conversion of a soluble plasma protein, fibrinogen, into a polymeric fibrin clot, is conserved in all vertebrates. However, similar data are lacking for the cellular response, which in gnathostomes is regulated by von Willebrand factor (VWF), a glycoprotein that mediates the adhesion of platelets to the subendothelial matrix of injured blood vessels. To gain evolutionary insights into the cellular phase of coagulation, we asked whether a functional vwf gene is present in the Atlantic hagfish, Myxine glutinosa We found a single vwf transcript that encodes a simpler protein compared with higher vertebrates, the most striking difference being the absence of an A3 domain, which otherwise binds collagen under high-flow conditions. Immunohistochemical analyses of hagfish tissues and blood revealed Vwf expression in endothelial cells and thrombocytes. Electron microscopic studies of hagfish tissues demonstrated the presence of Weibel-Palade bodies in the endothelium. Hagfish Vwf formed high-molecular-weight multimers in hagfish plasma and in stably transfected CHO cells. In functional assays, botrocetin promoted VWF-dependent thrombocyte aggregation. A search for vwf sequences in the genome of sea squirts, the closest invertebrate relatives of hagfish, failed to reveal evidence of an intact vwf gene. Together, our findings suggest that VWF evolved in the ancestral vertebrate following the divergence of the urochordates some 500 million years ago and that it acquired increasing complexity though sequential insertion of functional modules.

A role of stochastic phenotype switching in generating mosaic endothelial cell heterogeneity.

Previous studies have shown that biological noise may drive dynamic phenotypic mosaicism in isogenic unicellular organisms. However, there is no evidence for a similar mechanism operating in metazoans. Here we show that the endothelial-restricted gene, von Willebrand factor (VWF), is expressed in a mosaic pattern in the capillaries of many vascular beds and in the aorta. In capillaries, the mosaicism is dynamically regulated, with VWF switching between ON and OFF states during the lifetime of the animal. Clonal analysis of cultured endothelial cells reveals that dynamic mosaic heterogeneity is controlled by a low-barrier, noise-sensitive bistable switch that involves random transitions in the DNA methylation status of the VWF promoter. Finally, the hearts of VWF-null mice demonstrate an abnormal endothelial phenotype as well as cardiac dysfunction. Together, these findings suggest a novel stochastic phenotype switching strategy for adaptive homoeostasis in the adult vasculature.

PGC-1α induces SPP1 to activate macrophages and orchestrate functional angiogenesis in skeletal muscle.

RATIONALE: Mechanisms of angiogenesis in skeletal muscle remain poorly understood. Efforts to induce physiological angiogenesis hold promise for the treatment of diabetic microvascular disease and peripheral artery disease but are hindered by the complexity of physiological angiogenesis and by the poor angiogenic response of aged and patients with diabetes mellitus. To date, the best therapy for diabetic vascular disease remains exercise, often a challenging option for patients with leg pain. Peroxisome proliferation activator receptor-γ coactivator-1α (PGC-1α), a powerful regulator of metabolism, mediates exercise-induced angiogenesis in skeletal muscle. OBJECTIVE: To test whether, and how, PGC-1α can induce functional angiogenesis in adult skeletal muscle. METHODS AND RESULTS: Here, we show that muscle PGC-1α robustly induces functional angiogenesis in adult, aged, and diabetic mice. The process involves the orchestration of numerous cell types and leads to patent, nonleaky, properly organized, and functional nascent vessels. These findings contrast sharply with the disorganized vasculature elicited by induction of vascular endothelial growth factor alone. Bioinformatic analyses revealed that PGC-1α induces the secretion of secreted phosphoprotein 1 and the recruitment of macrophages. Secreted phosphoprotein 1 stimulates macrophages to secrete monocyte chemoattractant protein-1, which then activates adjacent endothelial cells, pericytes, and smooth muscle cells. In contrast, induction of PGC-1α in secreted phosphoprotein 1(-/-) mice leads to immature capillarization and blunted arteriolarization. Finally, adenoviral delivery of PGC-1α into skeletal muscle of either young or old and diabetic mice improved the recovery of blood flow in the murine hindlimb ischemia model of peripheral artery disease. CONCLUSIONS: PGC-1α drives functional angiogenesis in skeletal muscle and likely recapitulates the complex physiological angiogenesis elicited by exercise.

FOXO1-mediated activation of Akt plays a critical role in vascular homeostasis.

RATIONALE: Forkhead box-O transcription factors (FoxOs) transduce a wide range of extracellular signals, resulting in changes in cell survival, cell cycle progression, and several cell type-specific responses. FoxO1 is expressed in many cell types, including endothelial cells (ECs). Previous studies have shown that Foxo1 knockout in mice results in embryonic lethality at E11 because of impaired vascular development. In contrast, somatic deletion of Foxo1 is associated with hyperproliferation of ECs. Thus, the precise role of FoxO1 in the endothelium remains enigmatic. OBJECTIVE: To determine the effect of endothelial-specific knockout and overexpression of FoxO1 on vascular homeostasis. METHODS AND RESULTS: We show that EC-specific disruption of Foxo1 in mice phenocopies the full knockout. Although endothelial expression of FoxO1 rescued otherwise Foxo1-null animals, overexpression of constitutively active FoxO1 resulted in increased EC size, occlusion of capillaries, elevated peripheral resistance, heart failure, and death. Knockdown of FoxO1 in ECs resulted in marked inhibition of basal and vascular endothelial growth factor-induced Akt-mammalian target of rapamycin complex 1 (mTORC1) signaling. CONCLUSIONS: Our findings suggest that in mice, endothelial expression of FoxO1 is both necessary and sufficient for embryonic development. Moreover, FoxO1-mediated feedback activation of Akt maintains growth factor responsive Akt/mTORC1 activity within a homeostatic range.

The flow dependency of Tie2 expression in endotoxemia.

RATIONALE: Tie2 is predominantly expressed by endothelial cells and is involved in vascular integrity control during sepsis. Changes in Tie2 expression during sepsis development may contribute to microvascular dysfunction. Understanding the kinetics and molecular basis of these changes may assist in the development of therapeutic intervention to counteract microvascular dysfunction. OBJECTIVE: To investigate the molecular mechanisms underlying the changes in Tie2 expression upon lipopolysaccharide (LPS) challenge. METHODS AND RESULTS: Studies were performed in LPS and pro-inflammatory cytokine challenged mice as well as in mice subjected to hemorrhagic shock, primary endothelial cells were used for in vitro experiments in static and flow conditions. Eight hours after LPS challenge, Tie2 mRNA loss was observed in all major organs, while loss of Tie2 protein was predominantly observed in lungs and kidneys, in the capillaries. A similar loss could be induced by secondary cytokines TNF-α and IL-1ß. Ang2 protein administration did not affect Tie2 protein expression nor was Tie2 protein rescued in LPS-challenged Ang2-deficient mice, excluding a major role for Ang2 in Tie2 down regulation. In vitro, endothelial loss of Tie2 was observed upon lowering of shear stress, not upon LPS and TNF-α stimulation, suggesting that inflammation related haemodynamic changes play a major role in loss of Tie2 in vivo, as also hemorrhagic shock induced Tie2 mRNA loss. In vitro, this loss was partially counteracted by pre-incubation with a pharmacologically NF-кB inhibitor (BAY11-7082), an effect further substantiated in vivo by pre-treatment of mice with the NF-кB inhibitor prior to the inflammatory challenge. CONCLUSIONS: Microvascular bed specific loss of Tie2 mRNA and protein in vivo upon LPS, TNFα, IL-1ß challenge, as well as in response to hemorrhagic shock, is likely an indirect effect caused by a change in endothelial shear stress. This loss of Tie2 mRNA, but not Tie2 protein, induced by TNFα exposure was shown to be controlled by NF-кB signaling. Drugs aiming at restoring vascular integrity in sepsis could focus on preventing the Tie2 loss.

Role of RNA splicing in mediating lineage-specific expression of the von Willebrand factor gene in the endothelium.

We previously demonstrated that the first intron of the human von Willebrand factor (vWF) is required for gene expression in the endothelium of transgenic mice. Based on this finding, we hypothesized that RNA splicing plays a role in mediating vWF expression in the vasculature. To address this question, we used transient transfection assays in human endothelial cells and megakaryocytes with intron-containing and intronless human vWF promoter-luciferase constructs. Next, we generated knockin mice in which LacZ was targeted to the endogenous mouse vWF locus in the absence or presence of the native first intron or heterologous introns from the human ß-globin, mouse Down syndrome critical region 1, or hagfish coagulation factor X genes. In both the in vitro assays and the knockin mice, the loss of the first intron of vWF resulted in a significant reduction of reporter gene expression in endothelial cells but not megakaryocytes. This effect was rescued to varying degrees by the introduction of a heterologous intron. Intron-mediated enhancement of expression was mediated at a posttranscriptional level. Together, these findings implicate a role for intronic splicing in mediating lineage-specific expression of vWF in the endothelium.

Vascular bed-specific regulation of the von Willebrand factor promoter in the heart and skeletal muscle.

A region of the human von Willebrand factor (VWF) gene between -2812 and the end of the first intron (termed vWF2) 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 delineate the DNA sequences responsible for this effect. A series of constructs containing deletions or mutations of vWF2 coupled to LacZ were targeted to the Hprt locus of mice, and the resulting animals were analyzed for reporter gene expression. The findings demonstrate that DNA sequences between -843 and -620 are necessary for expression in capillary but not large vessel endothelium in heart and skeletal muscle. Further, expression of VWF in capillaries and larger vessels of both tissues required the presence of a native or heterologous intron. In vitro assays implicated a role for ERG-binding ETS motif at -56 in mediating basal expression of VWF. In Hprt-targeted mice, mutation of the ETS consensus motif resulted in loss of LacZ expression in the endothelium of the heart and skeletal muscle. Together, these data indicate that distinct DNA modules regulate vascular bed-specific expression of VWF.

Intracellular Ca2+ can compensate for the lack of NADPH oxidase-derived ROS in endothelial cells.

The aim of the present study is to determine the role of intracellular Ca(2+) in VEGF signaling. We demonstrate that reduction in Ca(2+) by chelating compound BAPTA-AM or by IP(3)-endoplasmic reticulum blocker 2-APB selectively inhibited VEGF-induced activation of c-Src-PI3K-Akt but not ERK1/2 in human coronary artery endothelial cells (HCAEC). We also show that the selective inhibitory effects of NADPH oxidase knockdown on VEGF-mediated activation of c-Src-PI3K-Akt signaling and cell proliferation in HCAEC can be reversed by increase in intracellular Ca(2+). These results suggest an essential role for Ca(2+) in redox-dependent selective activation of c-Src-PI3K-Akt and endothelial cell proliferation.

Differential roles for ETS, CREB, and EGR binding sites in mediating VEGF receptor 1 expression in vivo.

Vascular endothelial growth factor receptor 1 (VEGFR1) is a marker for endothelial-specific gene expression. We previously reported that the human VEGFR1 promoter (between -748 and +284) contains information for expression in the intact endothelium of transgenic mice. The objective of this study was to dissect the cis-regulatory elements underlying VEGFR1 promoter activity in vitro and in vivo. In primary endothelial cells, binding sites for E74-like factor 1 (ELF-1; between -49 and -52), cyclic adenosine monophosphate response element binding (CREB; between -74 and -81), and early growth response factor 1/3 (EGR-1/3; between -16 to -25) were shown to play a positive role in gene transcription, whereas a putative E26 transformation-specificsequence (ETS) motif between -36 and -39 had a net negative effect on promoter activity. When targeted to the Hprt locus of mice, mutations of the ELF-1 binding site and the CRE element reduced promoter activity in the embryonic vasculature and resulted in a virtual loss of expression in adult endothelium. Postnatally, the EGR binding site mutant displayed significantly reduced promoter activity in a subset of vascular beds. In contrast, mutation of the -39 ETS site resulted in increased LacZ staining in multiple vascular beds. Together, these results provide new insights into the transcriptional regulatory mechanisms of VEGFR1.

Hepatocyte growth factor inhibits VEGF-forkhead-dependent gene expression in endothelial cells.

OBJECTIVE: Recently, we reported that the forkhead transcription factor, FKHR/FOXO1, is required for vascular endothelial growth factor (VEGF)-mediated upregulation of a number of genes in endothelial cells. Here, we tested the hypothesis that hepatocyte growth factor (HGF), a potent activator of PI3K-Akt in endothelial cells, is capable of depleting the nucleus of FKHR/FOXO1 and thus inhibiting VEGF induction of this class of genes. METHODS AND RESULTS: Incubation of human coronary artery endothelial cells with HGF induced prolonged PI3K/Akt-dependent phosphorylation and nuclear exclusion of FKHR/FOXO1. HGF-mediated inhibition of FKHR/FOXO1 activity resulted in secondary attenuation of VEGF-induced expression of FKHR/FOXO1-dependent genes including vascular cell adhesion molecule-1, manganese superoxide dismutase, endothelial specific molecule-1, CBP/p300 interacting transactivator with ED-rich tail-2, bone morphogenetic protein-2, matrix metalloproteinase (MMP)-10, and MGC5618. At a functional level, preincubation of HGF resulted in inhibition of VEGF-induced vascular cell adhesion molecule (VCAM)-1-mediated monocyte adhesion to endothelial cells. HGF-mediated inhibition of VEGF-inducible VCAM-1 expression and monocyte adhesion was reversed by overexpression of constitutively active phosphorylation-resistant triple mutant (TM)-FKHR. CONCLUSIONS: These findings suggest that physiological agonists of PI3K-Akt signaling pathway may modulate VEGF-FKHR/FOXO1-dependent gene expression in endothelial cells. The data underscore the importance of the "set point" of the endothelial cell when considering mechanisms of signal transduction.

A prospective, observational study of soluble FLT-1 and vascular endothelial growth factor in sepsis.

Prior murine and human studies suggest that vascular endothelial growth factor (VEGF) contributes to endothelial cell activation and severity of illness in sepsis. Furthermore, circulating levels of soluble VEGF receptor 1 (sFLT) levels were found to increase as part of the early response to sepsis in mice. The objective of the study was to evaluate the blood levels of free VEGF-A and sFLT in patients presenting to the emergency department (ED) with suspected infection and to assess the relationship of these levels with severity of illness and inflammation. It was a prospective, observational study initiated in the ED of an urban, tertiary care, university hospital. Inclusion criteria were (1) ED patients aged 18 years or older and (2) clinical suspicion of infection. Eighty-three patients were enrolled in the study. The major findings were that (1) the mean VEGF and sFLT levels were increasingly higher across the following groups: noninfected control patients, infected patients without shock, and septic shock patients; (2) initial and 24-h VEGF levels had a significant correlation with the presence of septic shock at 24 h; (3) initial and 24-h sFLT levels correlated with Acute Physiology Age Chronic Health Evaluation II and Sepsis-related Organ/Failure Assessment scores initially and at 24 h; and (4) VEGF and sFLT levels correlated with inflammatory cascade activation. This is the first report of sFLT as a potential new marker of severity in patients with sepsis. Vascular endothelial cell growth factor and its signaling axis are important in the endothelial cell response to sepsis, and further elucidation of these mechanisms may lead to advances in future diagnostic and therapeutic opportunities.

Thrombin downregulates thrombomodulin expression and activity in primary human endothelial cells.

Thrombomodulin (TM) is a cell surface anticoagulant glycoprotein that plays a key role in the protein C pathway. TM expression in endothelial cells may be modulated by a variety of extracellular signals. Most notably, TM has been shown to be downregulated by inflammatory mediators, such as tumor necrosis factor-alpha and lipopolysaccharide. The objective of this study was to determine the effect of thrombin on TM expression and activity. Thrombin resulted in reduced TM in primary cultures of human endothelial cells by approximately 40% at the level of mRNA, protein, and activity. These effects were blocked by the thrombin inhibitor hirudin. These results suggest that activation of the coagulation cascade may result in a positive-feedback loop consisting of thrombin-mediated repression of TM-dependent protein C activation.

Priming effect of homocysteine on inducible vascular cell adhesion molecule-1 expression in endothelial cells.

Hyperhomocysteinemia is an independent risk factor for the development of atherosclerosis, as well as for arterial and venous thrombosis. However, the mechanisms through which elevated circulating levels of homocysteine cause vascular injury and promote thrombosis remain unclear. Here, we tested the hypothesis that homocysteine (Hcy) sensitizes endothelial cells to the effect of inflammatory mediators. Human umbilical vein endothelial cells (HUVEC) were incubated with Hcy 1.0 mM for varying time points, and then treated in the absence or presence of 1.5 U/ml thrombin or 10 mg/ml lipopolysaccharide (LPS). Hcy alone had no effect on the expression of vascular cell adhesion molecule (VCAM)-1. However, Hcy enhanced thrombin- and LPS-mediated induction of VCAM-1 mRNA and protein levels. Consistent with these results, pretreatment of HUVEC with Hcy resulted in a two-fold increase in LSP-mediated induction of leukocyte adhesion. The latter effect was significantly inhibited by anti-VCAM-1 antibodies. Together, these findings suggest that Hcy sensitizes HUVEC to the effect of inflammatory mediators thrombin and LPS, at least in part through VCAM-1 expression and function.

NADPH oxidase activity selectively modulates vascular endothelial growth factor signaling pathways.

Vascular endothelial growth factor (VEGF) and reactive oxygen species (ROS) play critical roles in vascular physiology and pathophysiology. We have demonstrated previously that NADPH oxidase-derived ROS are required for VEGF-mediated migration and proliferation of endothelial cells. The goal of this study was to determine the extent to which VEGF signaling is coupled to NADPH oxidase activity. Human umbilical vein endothelial cells and/or human coronary artery endothelial cells were transfected with short interfering RNA against the p47(phox) subunit of NADPH oxidase, treated in the absence or presence of VEGF, and assayed for signaling, gene expression, and function. We show that NADPH oxidase activity is required for VEGF activation of phosphoinositide 3-kinase-Akt-forkhead, and p38 MAPK, but not ERK1/2 or JNK. The permissive role of NADPH oxidase on phosphoinositide 3-kinase-Akt-forkhead signaling is mediated at post-VEGF receptor levels and involves the nonreceptor tyrosine kinase Src. DNA microarrays revealed the existence of two distinct classes of VEGF-responsive genes, one that is ROS-dependent and another that is independent of ROS levels. VEGF-induced, thrombomodulin-dependent activation of protein C was dependent on NADPH oxidase activity, whereas VEGF-induced decay-accelerating factor-mediated protection of endothelial cells against complement-mediated lysis was not. Taken together, these findings suggest that NADPH oxidase-derived ROS selectively modulate some but not all the effects of VEGF on endothelial cell phenotypes.

A novel class of vascular endothelial growth factor-responsive genes that require forkhead activity for expression.

Recently, we have shown that transient phosphorylation and inhibition of the pro-apoptotic transcription factor, forkhead, by vascular endothelial growth factor (VEGF) is essential for endothelial cell (EC) survival and proliferation. The goal of the present study was to determine whether forkhead (FKHR) also plays a positive role in agonist-mediated gene induction. Human coronary artery ECs were transduced with adenovirus overexpressing constitutively active phosphorylation-resistant triple mutant FKHR or transfected with small interference RNA (siRNA) against FKHR. The cells were then treated in the absence or presence of VEGF and assayed for gene expression using quantitative real-time PCR and Northern blots analyses. The data revealed a novel set of VEGF-responsive genes that require FKHR activity for optimal expression in ECs, including bone morphogenic protein 2, cbp/p300-interacting transactivator 2, decay accelerating factor (DAF), vascular cell adhesion molecule-1 (VCAM-1), manganese superoxide dismutase, endothelial-specific molecule-1, RING1 and YY1-binding protein, and matrix metalloproteinase-10. Consistent with a positive role for FKHR in mediating VEGF induction of DAF and VCAM-1 mRNA, siRNA against FKHR attenuated the effect of VEGF on complement-mediated EC lysis and monocyte adhesion, respectively. VEGF induction of the forkhead-dependent genes was down-regulated by the NF-kappaB inhibitor, constitutively active Ad-IkappaB, and in some cases by the nuclear factor of activated T-cells (NF-AT) inhibitor, cyclosporin. Together, these findings suggest that the VEGF-forkhead signaling axis plays an important functional role in ECs beyond the regulation of cell survival/apoptosis and cell cycle.

Forkhead transcription factors inhibit vascular smooth muscle cell proliferation and neointimal hyperplasia.

Vascular smooth muscle cell (VSMC) proliferation and migration contribute significantly to atherosclerosis, postangioplasty restenosis, and transplant vasculopathy. Forkhead transcription factors belonging to the FoxO subfamily have been shown to inhibit growth and cell cycle progression in a variety of cell types. We hypothesized that forkhead proteins may play a role in VSMC biology. Under in vitro conditions, platelet-derived growth factor (PDGF)-BB, tumor necrosis factor-alpha, and insulin-like growth factor 1 stimulated phosphorylation of FoxO in human coronary artery smooth muscle cells via MEK1/2 and/or phosphatidylinositol 3-kinase-dependent signaling pathways. PDGF-BB, tumor necrosis factor-alpha, and insulin-like growth factor 1 treatment resulted in the nuclear exclusion of FoxO, whereas PDGF-BB alone down-regulated the FoxO target gene, p27(kip1), and enhanced cell survival and progression through the cell cycle. These effects were abrogated by overexpression of a constitutively active, phosphorylation-resistant mutant of the FoxO family member, TM-FKHRL1. The anti-proliferative effect of TM-FKHRL1 was partially reversed by small interfering RNA against p27(kip1). In a rat balloon carotid arterial injury model, adenovirus-mediated gene transfer of FKHRL1 caused an increase in the expression of p27(kip1) in the VSMC and inhibition of neointimal hyperplasia. These data suggest that FoxO activity inhibits VSMC proliferation and activation and that this signaling axis may represent a therapeutic target in vasculopathic disease states.

Vascular endothelial growth factor-mediated induction of manganese superoxide dismutase occurs through redox-dependent regulation of forkhead and IkappaB/NF-kappaB.

The mitochondrial antioxidant manganese superoxide dismutase (Mn-SOD) plays a critical cytoprotective role against oxidative stress. Vascular endothelial growth factor (VEGF) was shown previously to induce expression of Mn-SOD in endothelial cells by a NADPH oxidase-dependent mechanism. The goal of the current study was to determine the transcriptional mechanisms underlying this phenomenon. VEGF resulted in protein kinase C-dependent phosphorylation of IkappaB and subsequent translocation of p65 NF-kappaB into the nucleus. Overexpression of constitutively active IkappaB blocked VEGF stimulation of Mn-SOD. In transient transfection assays, VEGF increased Mn-SOD promoter activity, an effect that was dependent on a second intronic NF-kappaB consensus motif. In contrast, VEGF-mediated induction of Mn-SOD was enhanced by the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 and by dominant negative Akt and was decreased by constitutively active Akt. Overexpression of a constitutively active (phosphorylation-resistant) form of FKHRL1 (TMFKHRL1) resulted in increased Mn-SOD expression, suggesting that the negative effect of PI3K-Akt involves attenuation of forkhead activity. In co-transfection assays, the Mn-SOD promoter was transactivated by TMFKHRL1. Flavoenzyme inhibitor, diphenyleneiodonium (DPI), and antisense oligonucleotides against p47phox (AS-p47phox) inhibited VEGF stimulation of IkappaB/NF-kappaB and forkhead phosphorylation, supporting a role for NADPH oxidase activity in both signaling pathways. Like VEGF, hepatocyte growth factor (HGF) activated the PI3K-Akt-forkhead pathway. However, HGF-PI3K-Akt-forkhead signaling was insensitive to diphenyleneiodonium and AS-p47phox. Moreover, HGF failed to induce phosphorylation of IkappaB/NF-kappaB or nuclear translocation of NF-kappaB and had no effect on Mn-SOD expression. Together, these data suggest that VEGF is uniquely coupled to Mn-SOD expression through growth factor-specific reactive oxygen species (ROS)-sensitive positive (protein kinase C-NF-kappaB) and negative (PI3K-Akt-forkhead) signaling pathways.

Vascular endothelial growth factor activates PI3K/Akt/forkhead signaling in endothelial cells.

OBJECTIVE: Vascular endothelial growth factor (VEGF) is a potent angiogenic growth factor that promotes endothelial cell (EC) survival, migration, and permeability. The forkhead transcription factors FKHR, FKHRL1, and AFX are mammalian orthologues of DAF-16, a forkhead protein that controls longevity in Caenorhabditis elegans. In this study, we examined whether VEGF is coupled to phosphatidyl inositol 3-kinase (PI3K)/Akt/forkhead in ECs. METHODS AND RESULTS: We demonstrate that human ECs express members of the forkhead family (FKHR, FKHRL1, and AFX) and that VEGF modulates the phosphorylation, subcellular localization, and transcriptional activity of one or more of these isoforms by a PI3K/Akt signaling pathway. VEGF inhibited EC apoptosis, promoted DNA synthesis and the G(1)-to-S transition, and reduced expression of the cyclin-dependent kinase inhibitor p27(kip1). Each of these effects was blocked by the PI3K inhibitor LY294002 or by a phosphorylation-resistant mutant of FKHRL1, but not by wild-type FKHRL1. CONCLUSIONS: These results suggest that VEGF signaling in ECs is coupled to forkhead transcription factors through a PI3K/Akt-dependent pathway.