Kevin P. Mouillesseaux

Identification of Prostaglandin E2 Receptor Subtype 2 As a Receptor Activated by OxPAPC

Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC), which has been shown to accumulate in atherosclerotic lesions and other sites of chronic inflammation, activates endothelial cells (EC) to bind monocytes by activation of endothelial &bgr;1 integrin and subsequent deposition of fibronectin on the apical surface. Our previous studies suggest this function of OxPAPC is mediated via a Gs protein–coupled receptor (GPCR). PEIPC (1-palmitoyl-2-epoxyisoprostane E2-sn-glycero-3-phosphorylcholine) is the most active lipid in OxPAPC that activates this pathway. We screened a number of candidate GPCRs for their interaction with OxPAPC and PEIPC, using a reporter gene assay; we identified prostaglandin E2 receptor EP2 and prostaglandin D2 receptor DP as responsive to OxPAPC. We focused on EP2, which is expressed in ECs, monocytes, and macrophages. OxPAPC component PEIPC, but not POVPC, activated EP2 with an EC50 of 108.6 nmol/L. OxPAPC and PEIPC were also able to compete with PGE2 for binding to EP2 in a ligand-binding assay. The EP2 specific agonist butaprost was shown to mimic the effect of OxPAPC on the activation of &bgr;1 integrin and the stimulation of monocyte binding to endothelial cells. Butaprost also mimicked the effect of OxPAPC on the regulation of tumor necrosis factor-&agr; and interleukin-10 in monocyte-derived cells. EP2 antagonist AH6809 blocked the activation of EP2 by OxPAPC in HEK293 cells and blocked the interleukin-10 response to PEIPC in monocytic THP-1 cells. These results suggest that EP2 functions as a receptor for OxPAPC and PEIPC, either as the phospholipid ester or the released fatty acid, in both endothelial cells and macrophages.

Role of the JAK/STAT Pathway in the Regulation of Interleukin-8 Transcription by Oxidized Phospholipids in Vitro and in Atherosclerosis in Vivo

Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (Ox-PAPC) and its component phospholipid, 1-palmitoyl-2-epoxyisoprostane-sn-glycero-3-phosphorylcholine, induce endothelial cells (EC) to synthesize chemotactic factors, such as interleukin 8 (IL-8). Previously, we demonstrated a role for c-Src kinase activation in Ox-PAPC-induced IL-8 transcription. In this study, we have examined the mechanism regulating IL-8 transcription by Ox-PAPC downstream of c-Src. Our findings demonstrate an important role for JAK2 in the regulation of IL-8 transcription by Ox-PAPC. Treatment of human aortic EC with Ox-PAPC and 1-palmitoyl-2-epoxyisoprostane-sn-glycero-3-phosphorylcholine induced a rapid yet sustained activation of JAK2; activation of JAK2 by Ox-PAPC was dependent on c-Src kinase activity. Furthermore, pretreatment with selective JAK2 inhibitors significantly reduced Ox-PAPC-induced IL-8 transcription. In previous studies, we also demonstrated activation of STAT3 by Ox-PAPC. Here we provide evidence that STAT3 activation by Ox-PAPC is dependent on JAK2 activation and that STAT3 activation regulates IL-8 transcription by Ox-PAPC in human EC. Transfection with small interfering RNA against STAT3 significantly reduced Ox-PAPC-induced IL-8 transcription. Using chromatin immunoprecipitation assays, we demonstrated binding of activated STAT3 to the sequence flanking the consensus γ-interferon activation sequence (GAS) in the IL-8 promoter; site-directed mutagenesis of GAS inhibited IL-8 transcription by Ox-PAPC. Finally, these studies demonstrate a role for STAT3 activation in atherosclerosis in vivo. We found increased staining for activated STAT3 in the inflammatory regions of human atherosclerotic lesions and reduced fatty streak formation in EC-specific STAT3 knock-out mice on the atherogenic diet. Taken together, these data demonstrate an important role for the JAK2/STAT3 pathway in Ox-PAPC-induced IL-8 transcription in vitro and in atherosclerosis in vivo.

Role of Endothelial Nitric Oxide Synthase in the Regulation of SREBP Activation by Oxidized Phospholipids

Oxidized-1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (Ox-PAPC), found in atherosclerotic lesions and other sites of chronic inflammation, activates endothelial cells (EC) to synthesize chemotactic factors, such as interleukin (IL)-8. Previously, we demonstrated that the sustained induction of IL-8 transcription by Ox-PAPC was mediated through the activation of sterol regulatory element–binding protein (SREBP). We now present evidence for the role of endothelial nitric oxide synthase (eNOS) in the activation of SREBP by Ox-PAPC. Ox-PAPC treatment of EC induced a dose- and time-dependent activation of eNOS, as measured by phosphorylation of serine 1177, dephosphorylation of threonine 495, and the conversion of l-arginine to l-citrulline. Activation of eNOS by Ox-PAPC was regulated through a phosphatidylinositol-3-kinase/Akt-mediated mechanism. These studies also demonstrated that pretreatment of EC with NOS inhibitor, N&ohgr;-nitro-l-arginine-methyl ester (L-NAME), significantly inhibited Ox-PAPC–induced IL-8 synthesis. Because SREBP activation had been previously shown to regulate IL-8 transcription by Ox-PAPC, we examined the effects of L-NAME on Ox-PAPC–induced SREBP activation. Our data demonstrated that Ox-PAPC–induced SREBP activation and expression of SREBP target genes were significantly reduced by pretreatment with L-NAME. Interestingly, treatment of EC with NO donor, S-nitroso-N-acetylpenicillamine, did not activate SREBP, suggesting that NO alone was not sufficient for SREBP activation. Rather, our findings indicated that superoxide (O2·−), in combination with NO, regulated SREBP activation by Ox-PAPC. We found that Ox-PAPC treatment generated O2·− through an eNOS-mediated mechanism and that mercaptoethylguanidine, a peroxynitrite scavenger, reduced SREBP activation by Ox-PAPC. Taken together, these findings propose a novel role for eNOS in the activation of SREBP and SREBP-mediated inflammatory processes.

Vascular Endothelial Growth Factor Receptor 2 Plays a Role in the Activation of Aortic Endothelial Cells by Oxidized Phospholipids

Objective—Previous studies have shown that oxidized products of PAPC (Ox-PAPC) regulate cell transcription of interleukin-8, LDL receptor, and tissue factor. This upregulation takes place in part through the activation of sterol regulatory element-binding protein (SREBP) and Erk 1/2. The present studies identify vascular endothelial growth factor receptor 2 (VEGFR2) as a major regulator in the activation of SREBP and Erk 1/2 in endothelial cells activated by Ox-PAPC. Methods and Results—Ox-PAPC induced the phosphorylation of VEGFR2 at Tyr1175 in human aortic endothelial cells. Inhibitors and siRNA for VEGFR2 decreased the transcription of interleukin-8, LDL receptor, and tissue factor in response to Ox-PAPC and the activation of SREBP and Erk 1/2, which mediate this transcription. We provide evidence that the activation of VEGFR2 is rapid, sustained, and c-Src–dependent. Conclusions—These data point to a major role of VEGFR2 in endothelial regulation by oxidized phospholipids which accumulate in atherosclerotic lesions and apoptotic cells.

Protein targets of oxidized phospholipids in endothelial cells

Oxidation products of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphatidylcholine (Ox-PAPC) are found in atherosclerotic lesions, apoptotic cells, and oxidized LDL and stimulate human aortic endothelial cells (HAECs) to produce inflammatory cytokines, leukocyte chemoattractants, and coagulation factors. This regulation is thought to be a receptor-mediated process in which oxidized phospholipids activate specific receptors on HAECs to evoke an inflammatory response. To characterize the HAEC proteins with which oxidized phospholipids interact, a biotinylated PAPC analog, 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphatidyl-(N-biotinylethanolamine) (PAPE-N-biotin), was synthesized. Oxidation of PAPE-N-biotin in air generated a mixture of biotin-labeled oxidized lipids analogous to Ox-PAPC. Ox-PAPE-N-biotin, like Ox-PAPC, induced interleukin-8 (IL-8) protein synthesis and stimulated IL-8, low density lipoprotein receptor, heme oxygenase-1, and activating transcription factor-3 mRNA expression in HAECs. After treatment of HAECs with Ox-PAPE-N-biotin, the cellular proteins were isolated and separated by SDS-PAGE. Western analysis with streptavidin-HRP demonstrated at least 20 different biotinylated HAEC proteins to which the Ox-PAPE-N-biotin was associated, which were not detected with unoxidized PAPE-N-biotin treatment. This work suggests that oxidized phospholipids, such as those found in oxidized LDL, apoptotic cells, and atherosclerotic lesions, form tight interactions with specific endothelial cell proteins, which may be responsible for the inflammatory response. Identification of these putative oxidized phospholipid targets may reveal therapeutic targets to modulate inflammation and atherosclerosis.

Flt-1 (Vascular Endothelial Growth Factor Receptor-1) Is Essential for the Vascular Endothelial Growth Factor–Notch Feedback Loop During Angiogenesis

Objective—Vascular endothelial growth factor (VEGF) signaling induces Notch signaling during angiogenesis. Flt-1/VEGF receptor-1 negatively modulates VEGF signaling. Therefore, we tested the hypothesis that disrupted Flt-1 regulation of VEGF signaling causes Notch pathway defects that contribute to dysmorphogenesis of Flt-1 mutant vessels. Approach and Results—Wild-type and flt-1−/− mouse embryonic stem cell–derived vessels were exposed to pharmacological and protein-based Notch inhibitors with and without added VEGF. Vessel morphology, endothelial cell proliferation, and Notch target gene expression levels were assessed. Similar pathway manipulations were performed in developing vessels of zebrafish embryos. Notch inhibition reduced flt-1−/− embryonic stem cell–derived vessel branching dysmorphogenesis and endothelial hyperproliferation, and rescue of flt-1−/− vessels was accompanied by a reduction in elevated Notch targets. Surprisingly, wild-type vessel morphogenesis and proliferation were unaffected by Notch suppression, Notch targets in wild-type endothelium were unchanged, and Notch suppression perturbed zebrafish intersegmental vessels but not caudal vein plexuses. In contrast, exogenous VEGF caused wild-type embryonic stem cell–derived vessel and zebrafish intersegmental vessel dysmorphogenesis that was rescued by Notch blockade. Conclusions—Elevated Notch signaling downstream of perturbed VEGF signaling contributes to aberrant flt-1−/− blood vessel formation. Notch signaling may be dispensable for blood vessel formation when VEGF signaling is below a critical threshold.

Notch regulates BMP responsiveness and lateral branching in vessel networks via SMAD6

Functional blood vessel growth depends on generation of distinct but coordinated responses from endothelial cells. Bone morphogenetic proteins (BMP), part of the TGFβ superfamily, bind receptors to induce phosphorylation and nuclear translocation of SMAD transcription factors (R-SMAD1/5/8) and regulate vessel growth. However, SMAD1/5/8 signalling results in both pro- and anti-angiogenic outputs, highlighting a poor understanding of the complexities of BMP signalling in the vasculature. Here we show that BMP6 and BMP2 ligands are pro-angiogenic in vitro and in vivo, and that lateral vessel branching requires threshold levels of R-SMAD phosphorylation. Endothelial cell responsiveness to these pro-angiogenic BMP ligands is regulated by Notch status and Notch sets responsiveness by regulating a cell-intrinsic BMP inhibitor, SMAD6, which affects BMP responses upstream of target gene expression. Thus, we reveal a paradigm for Notch-dependent regulation of angiogenesis: Notch regulates SMAD6 expression to affect BMP responsiveness of endothelial cells and new vessel branch formation.

Flt-1 (VEGFR-1) coordinates discrete stages of blood vessel formation.

AIMS In developing blood vessel networks, the overall level of vessel branching often correlates with angiogenic sprout initiations, but in some pathological situations, increased sprout initiations paradoxically lead to reduced vessel branching and impaired vascular function. We examine the hypothesis that defects in the discrete stages of angiogenesis can uniquely contribute to vessel branching outcomes. METHODS AND RESULTS Time-lapse movies of mammalian blood vessel development were used to define and quantify the dynamics of angiogenic sprouting. We characterized the formation of new functional conduits by classifying discrete sequential stages-sprout initiation, extension, connection, and stability-that are differentially affected by manipulation of vascular endothelial growth factor-A (VEGF-A) signalling via genetic loss of the receptor flt-1 (vegfr1). In mouse embryonic stem cell-derived vessels genetically lacking flt-1, overall branching is significantly decreased while sprout initiations are significantly increased. Flt-1(-/-) mutant sprouts are less likely to retract, and they form increased numbers of connections with other vessels. However, loss of flt-1 also leads to vessel collapse, which reduces the number of new stable conduits. Computational simulations predict that loss of flt-1 results in ectopic Flk-1 signalling in connecting sprouts post-fusion, causing protrusion of cell processes into avascular gaps and collapse of branches. Thus, defects in stabilization of new vessel connections offset increased sprout initiations and connectivity in flt-1(-/-) vascular networks, with an overall outcome of reduced numbers of new conduits. CONCLUSIONS These results show that VEGF-A signalling has stage-specific effects on vascular morphogenesis, and that understanding these effects on dynamic stages of angiogenesis and how they integrate to expand a vessel network may suggest new therapeutic strategies.

Systems proteomics of cardiac chromatin identifies nucleolin as a regulator of growth and cellular plasticity in cardiomyocytes

Myocyte hypertrophy antecedent to heart failure involves changes in global gene expression, although the preceding mechanisms to coordinate DNA accessibility on a genomic scale are unknown. Chromatin-associated proteins alter chromatin structure by changing their association with DNA, thereby altering the gene expression profile. Little is known about the global changes in chromatin subproteomes that accompany heart failure, and the mechanisms by which these proteins alter chromatin structure. The present study tests the fundamental hypothesis that cardiac growth and plasticity in the setting of disease recapitulates conserved developmental chromatin remodeling events. We used quantitative proteomics to identify chromatin-associated proteins extracted via detergent and to quantify changes in their abundance during disease. Our study identified 321 proteins in this subproteome, demonstrating it to have modest conservation (37%) with that revealed using strong acid. Of these proteins, 176 exhibited altered expression during cardiac hypertrophy and failure; we conducted extensive functional characterization of one of these proteins, Nucleolin. Morpholino-based knockdown of nucleolin nearly abolished protein expression but surprisingly had little impact on gross morphological development. However, hearts of fish lacking Nucleolin displayed severe developmental impairment, abnormal chamber patterning and functional deficits, ostensibly due to defects in cardiac looping and myocyte differentiation. The mechanisms underlying these defects involve perturbed bone morphogenetic protein 4 expression, decreased rRNA transcription, and a shift to more heterochromatic chromatin. This study reports the quantitative analysis of a new chromatin subproteome in the normal and diseased mouse heart. Validation studies in the complementary model system of zebrafish examine the role of Nucleolin to orchestrate genomic reprogramming events shared between development and disease.

Mutation in utp15 Disrupts Vascular Patterning in a p53-Dependent Manner in Zebrafish Embryos

Background Angiogenesis is the process by which the highly branched and functional vasculature arises from the major vessels, providing developing tissues with nutrients, oxygen, and removing metabolic waste. During embryogenesis, vascular patterning is dependent on a tightly regulated balance between pro- and anti-angiogenic signals, and failure of angiogenesis leads to embryonic lethality. Using the zebrafish as a model organism, we sought to identify genes that influence normal vascular patterning. Methodology and Principal Findings In a forward genetic screen, we identified mutant LA1908, which manifests massive apoptosis during early embryogenesis, abnormal expression of several markers of arterial-venous specification, delayed angiogenic sprouting of the intersegmental vessels (ISV), and malformation of the caudal vein plexus (CVP), indicating a critical role for LA1908 in cell survival and angiogenesis. Genetic mapping and sequencing identified a G to A transition in the splice site preceding exon 11 of utp15 in LA1908 mutant embryos. Overexpression of wild type utp15 mRNA suppresses all observed mutant phenotypes, demonstrating a causative relationship between utp15 and LA1908. Furthermore, we found that injecting morpholino oligonucleotides inhibiting p53 translation prevents cell death and rescues the vascular abnormalities, indicating that p53 is downstream of Utp15 deficiency in mediating the LA1908 phenotypes. Conclusions and Significance Taken together, our data demonstrate an early embryonic effect of Utp15 deficiency on cell survival and the normal patterning of the vasculature and highlight an anti-angiogenic role of p53 in developing embryos.