Monique Agrapart

Hypoxia-Induced Apelin Expression Regulates Endothelial Cell Proliferation and Regenerative Angiogenesis

Apelin has been identified as the endogenous ligand of the human orphan G protein-coupled receptor APJ. This peptide exerts a variety of cardiovascular effects and particularly acts as an activator of angiogenesis. Importantly, hypoxia has been reported to regulate apelin expression but the molecular mechanism underlying hypoxia-induced apelin expression and the relationship with the physiological response of the apelin/APJ system are still not established. Here, we demonstrate that apelin expression is induced by hypoxia in cultured endothelial and vascular smooth muscle cells as well as in lung from mice exposed to acute hypoxia. Transient transfection experiments show that hypoxia-inducible transcriptional activation of apelin requires an intact hypoxia-responsive element (+813/+826) located within the first intron of the human apelin gene. Chromatin immunoprecipitation assay reveals that hypoxia-inducible factor-1&agr; binds to the endogenous hypoxia-responsive element site of the apelin gene. Moreover, overexpression of hypoxia-inducible factor-1&agr; increases the transcriptional activity of a reporter construct containing this hypoxia-responsive element, whereas small interfering RNA-mediated hypoxia-inducible factor-1&agr; knockdown abolishes hypoxia-induced apelin expression. Finally, microinterfering RNA-mediated apelin or APJ receptor knockdown inhibits both hypoxia-induced endothelial cell proliferation in vitro and hypoxia-induced vessel regeneration in the caudal fin regeneration of Fli-1 transgenic zebrafish. The hypoxia-induced apelin expression may, thus, provide a new mechanism involved in adaptive physiological and pathophysiological response of vascular cells to low oxygen level.

Characterization of an Upstream Enhancer Region in the Promoter of the Human Endothelial Nitric-oxide Synthase Gene

The endothelial nitric-oxide synthase gene is constitutively expressed in endothelial cells. Several transcriptionally active regulatory elements have been identified in the proximal promoter, including a GATA-2 and an Sp-1 binding site. Because they cannot account for the constitutive expression of endothelial nitric-oxide synthase gene in a restricted number of cells, we have searched for other cell-specific regulatory elements. By DNase I hypersensitivity mapping and deletion studies we have identified a 269-base pair activator element located 4.9 kilobases upstream from the transcription start site that acts as an enhancer. DNase I footprinting and linker-scanning experiments showed that several regions within the 269-base pair enhancer are important for transcription factor binding and for full enhancer activity. The endothelial specificity of this activation seems partly due to interaction between this enhancer in its native configuration and the promoter in endothelial cells. EMSA experiments suggested the implication of MZF-like, AP-2, Sp-1-related, and Ets-related factors. Among Ets factors, Erg was the only one able to bind to cognate sites in the enhancer, as found by EMSA and supershift experiments, and to activate the transcriptional activity of the enhancer in cotransfection experiments. Therefore, multiple protein complexes involving Erg, other Ets-related factors, AP-2, Sp-1-related factor, and MZF-like factors are important for the function of this enhancer in endothelial cells.

Induction of Angiotensin I-converting Enzyme Transcription by a Protein Kinase C-dependent Mechanism in Human Endothelial Cells

Angiotensin I-converting enzyme (ACE) has been implicated in various cardiovascular diseases; however, little is known about the ACE gene regulation in endothelial cells. We have investigated the effect of the protein kinase C activator phorbol 12-myristate 13-acetate (PMA) on ACE activity and gene expression in human umbilical vein endothelial cells (HUVEC). Our results showed a 3- and 5-fold increase in ACE activity in the medium and in the cells, respectively, after 24-h stimulation by PMA. We also observed an increase in the cellular ACE mRNA content starting after 6 h and reaching a 10-fold increase at 24 h in response to 100 ng/ml PMA as measured by ribonuclease protection assay. This effect was mediated by an increased transcription of the ACE gene as demonstrated by nuclear run-on experiments and nearly abolished by the specific PKC inhibitor GF 109203X. Our results indicate that PMA-activated PKC strongly increases ACE mRNA level and ACE gene transcription in HUVEC, an effect associated with an increased ACE secretion. A role for early growth response factor-1 (Egr-1) as a factor regulating ACE gene expression is suggested by both the presence of an Egr-1-responsive element in the proximal portion of the ACE promoter and the kinetics of the Egr-1 mRNA increase in HUVEC treated with PMA.

Phorbol Ester Induction of Angiotensin-Converting Enzyme Transcription Is Mediated by Egr-1 and AP-1 in Human Endothelial Cells via ERK1/2 Pathway

Abstract— Angiotensin-converting enzyme (ACE) is an enzyme that plays a major role in vasoactive peptide metabolism, and it has been implicated in various cardiovascular diseases. Phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator, has been shown to increase ACE mRNA at the transcriptional level in human umbilical vein endothelial cells. We have investigated the transcriptional mechanism involved in protein kinase C induction of the ACE gene. Deletion and transfection analyses have revealed that two regions are required for PMA-inducible gene expression. The first is a G+C-rich region located in the proximal ACE promoter bearing overlapping consensus recognition sequences for stimulatory protein-1 (Sp1) and early growth response gene 1 (Egr-1). Electrophoretic mobility shift assay and supershift experiments have shown that Egr-1 is present in the specific nucleoprotein complex induced by PMA in human umbilical vein endothelial cells. The second region is located in the distal ACE promoter. DNase I footprinting analysis restricted this region to a 21-bp element containing a cAMP-responsive element/12-O-tetradecanoylphorbol 13-acetate–responsive element sequence. Electrophoretic mobility shift assays and supershift analyses have revealed that activating protein 1 (AP-1) is the transcription factor binding the cAMP-responsive element/12-O-tetradecanoylphorbol 13-acetate–responsive element located in the ACE promoter after PMA stimulation. Mutations of either Egr-1 or AP-1 binding sites partially abrogate ACE expression induced by PMA, whereas mutation of both sites totally abrogates PMA-induced ACE expression. Treatment of cells with PD98059, a mitogen-activated protein kinase kinase-1–specific inhibitor, inhibited PMA-induced ACE expression. Our results demonstrate that the two transcription factors, Egr-1 and AP-1, are involved in the PMA-induced ACE transcriptional activation in human endothelial cells via the activation of the extracellular signal–regulated kinase 1/2 signaling pathway.

Human lymphocyte responses to Plasmodium falciparum merozoite antigens. A functional assay of protective immunity

Merozoites obtained from cutaneous cultures of Plasmodium falciparum were used as antigen for an in vitro lymphocyte assay. Antigen specific proliferative responses were observed with lymphocytes from individuals with long-standing immunity to P. falciparum. Donors whose last P. falciparum challenge occurred within the year preceding the assay exhibited lymphocyte responses significantly higher than those from donors whose infection was more remote. This suggests that a lymphocyte dependent assay may relate to the protective status of the donor.

Plasmodium falciparum exoprotein stimulation of human T-lymphocytes unsensitized to malaria

The effects of Plasmodium falciparum proteins released in asexual blood stage culture supernatants on human T-lymphocytes from malaria non-immune donors were examined. Supernatants from several plasmodial strains stimulated both CD+4 and CD+8 T-lymphocytes to proliferate and secrete interferon gamma in vitro. Active moieties were predominantly released during the final stages of the parasite cycle. They were enriched by gel filtration and were further purified by anion-exchange and Superose 12 column fast protein liquid chromatography. Three active fractions of apparent 250, 70 and 18 kilodaltons were identified. The parasitic origin of the predominant 70-kilodaltons protein(s) was shown by biosynthesis experiments with radioactive amino acid precursors and was also demonstrated by in vitro translation of parasitic mRNA species. Interestingly, antibodies to the 70-kilodalton exoprotein(s) also reacted to a schizont protein of similar molecular weight.

Separation of precursor T cells and Ig-secreting B cells from the large lymphocytic cells of human tonsil☆

Abstract Large lymphocytic cells of human tonsils concentrating in the lighter fractions of a BSA gradient contained both precursors of E rosette-forming cells and lymphocytes carrying μ and γ determinants. These two subpopulations of cells could be separated by retaining the Ig-carrying cells on an anti-Fab column. Cells which did not bind to the column matured within 3–5 days into E rosette-forming cells. The Ig-carrying cells were eluted by medium supplemented with immunoglobulins and continued to secrete γ determinants in vitro . Both types of cells were also distinguished by different profiles of spontaneous [ 3 H]thymidine incorporation, the persisting capacity for division of the E-rosette-maturing cells suggesting that they represent an earlier stage of lymphocyte maturation (precursor T cells).