Mercedes Camacho

Rate of Vasoconstrictor Prostanoids Released by Endothelial Cells Depends on Cyclooxygenase-2 Expression and Prostaglandin I Synthase Activity

This study was undertaken to investigate the enzymatic regulation of the biosynthesis of vasoconstrictor prostanoids by resting and interleukin (IL)-1(beta)stimulated human umbilical vein endothelial cells (HUVECs). Biosynthesis of eicosanoids in response to IL-1beta, exogenous labeled arachidonic acid (AA), or histamine, as well as their spontaneous release, was evaluated by means of HPLC and RIA. HUVECs exposed to IL-1beta produced prostaglandin (PG) I2 for no longer than 30 seconds after the substrate was added irrespective of the cyclooxygenase (COX) activity, whereas the time course of PGE2 and PGD2 formation was parallel to the COX activity. The ratio of PGE2 to PGD2 produced by HUVECs was similar to that obtained by purified COX-1 and COX-2. Production of PGF2alpha from exogenous AA was limited and similar in both resting and IL-1beta-treated cells. PGF2alpha was the main prostanoid released into the medium during exposure to IL-1beta, whereas when HUVECs treated with IL-1beta were stimulated with histamine or exogenous AA, PGE2 was released in a higher quantity than PGF2alpha. PGF2alpha released into the medium during treatment with IL-1beta and the biosynthesis of PGE2 and PGD2 in response to exogenous AA or histamine increased with COX-2 expression, whereas this did not occur in the case of PGI2. We observed that PGI synthase (PGIS) mRNA levels were not modified by the exposure to IL-1beta, but the enzyme was partially inactivated. When SnCl2 was added to the incubation medium, the transformation of exogenous AA-derived PGH2 into PGE2 and PGD2 was totally diverted toward PGF2alpha. Overall, these results support the conclusions that PGE2 and PGD2 (and also probably PGF2alpha) were nonenzymatically derived from PGH2 in HUVECs. The concept that a high ratio of PGH2 was released by the IL-1beta-treated HUVECs and isomerized outside the cell into PGE2 and PGD2 was supported by the biosynthesis of thromboxane B2 by COX-inactivated platelets, indicating the uptake by platelets of HUVEC-derived PGH2. The IL-1beta-induced increase in the release of PGH2 by HUVECs was suppressed by the COX-2-selective inhibitor SC-58125 and correlated with both COX-2 expression and PGIS inactivation. An approach to the mechanism of inactivation of PGIS by the exposure to IL-1beta was performed by using labeled endoperoxides as substrate. The involvement of HO. in the PGIS inactivation was supported by the fact that deferoxamine, pyrrolidinedithiocarbamate, DMSO, mannitol, and captopril antagonized the effect of IL-1beta on PGIS to different degrees. The NO synthase inhibitor NG-monomethyl-L-arginine also antagonized the PGIS inhibitory effect of IL-1beta, indicating that NO. was also involved. NO. reacts with O2-. to form peroxynitrite, which has been reported to inactivate PGIS. Homolytic fission of the O-O bond of peroxynitrite yields NO2. and HO.. The fact that 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO), which reacts with NO. to form NO2., dramatically potentiated the IL-1beta effect suggests that NO2. could be a species implicated in the inactivation of PGIS. Cooperation of HO. was supported by the fact that DMSO partially antagonized the effect of carboxy-PTIO. Although our results on the exact mechanism of the inactivation of PGIS caused by IL-1beta were not conclusive, they strongly suggest that both NO. and HO. were involved.

Electronegative LDL of FH subjects: chemical characterization and induction of chemokine release from human endothelial cells.

Electronegative LDL (LDL(-)) constitutes a plasma subfraction of LDL with proinflammatory properties. Its proportion is increased in familial hypercholesterolemia (FH); however, the characteristics of LDL(-) isolated from FH subjects have not been previously studied. In this work, the composition, oxidative status, and inflammatory capacity on endothelial cells of LDL(-) from FH and normolipemic (NL) subjects were evaluated. LDL(-) from FH was relatively enriched in esterified and free cholesterol and triglyceride, and had lower apoB and phospholipid content compared with the non-electronegative fraction (LDL(+)). LDL(-) also contained increased amounts of apoE, apoC-III, sialic acid, and non-esterified fatty acids (NEFAs). The same was observed in NL subjects, except that esterified cholesterol and phospholipid were similar in LDL(-) and LDL(+). No difference was observed between the two fractions concerning malondialdehyde, fatty acid hydroxides, and antioxidants, thereby indicating the absence of increased oxidation of LDL(-) compared with LDL(+). When LDL(-) (100 mg/l) from NL and FH subjects was incubated for 24 h with human umbilical vein endothelial cells (HUVECs), interleukin 8 (IL-8) and monocyte chemotactic protein 1 (MCP-1) increased twofold in the culture medium compared with LDL(+). Vascular cell adhesion molecule 1 (VCAM-1) expression was not increased by LDL(-). Our data indicate that LDL(-) from FH or NL subjects shows no evidence of increased oxidative modification compared to LDL(+); however, LDL(-) induces twofold the release of chemokines by endothelial cells. This effect, which may contribute to leukocyte recruitment and promote atherogenesis, may be greater in FH subjects in which LDL(-) can be up to eightfold higher than in NL subjects.

Human Apolipoprotein A-II Enrichment Displaces Paraoxonase From HDL and Impairs Its Antioxidant Properties: A New Mechanism Linking HDL Protein Composition and Antiatherogenic Potential

Apolipoprotein A-II (apoA-II), the second major high-density lipoprotein (HDL) apolipoprotein, has been linked to familial combined hyperlipidemia. Human apoA-II transgenic mice constitute an animal model for this proatherogenic disease. We studied the ability of human apoA-II transgenic mice HDL to protect against oxidative modification of apoB-containing lipoproteins. When challenged with an atherogenic diet, antigens related to low-density lipoprotein (LDL) oxidation were markedly increased in the aorta of 11.1 transgenic mice (high human apoA-II expressor). HDL from control mice and 11.1 transgenic mice were coincubated with autologous very LDL (VLDL) or LDL, or with human LDL under oxidative conditions. The degree of oxidative modification of apoB lipoproteins was then evaluated by measuring relative electrophoretic mobility, dichlorofluorescein fluorescence, 9- and 13-hydroxyoctadecadienoic acid content, and conjugated diene kinetics. In all these different approaches, and in contrast to control mice, HDL from 11.1 transgenic mice failed to protect LDL from oxidative modification. A decreased content of apoA-I, paraoxonase (PON1), and platelet-activated factor acetyl-hydrolase activities was found in HDL of 11.1 transgenic mice. Liver gene expression of these HDL-associated proteins did not differ from that of control mice. In contrast, incubation of isolated human apoA-II with control mouse plasma at 37°C decreased PON1 activity and displaced the enzyme from HDL. Thus, overexpression of human apoA-II in mice impairs the ability of HDL to protect apoB-containing lipoproteins from oxidation. Further, the displacement of PON1 by apoA-II could explain in part why PON1 is mostly found in HDL particles with apoA-I and without apoA-II, as well as the poor antiatherogenic properties of apoA-II–rich HDL.

Human Apolipoprotein A-II Enrichment Displaces Paraoxonase From High-Density Lipoprotein (HDL) and Impairs Its Antioxidant Properties. A New Mechanism Linking HDL Protein Composition and Antiatherogenic Potential

Apolipoprotein A-II (apoA-II), the second major high-density lipoprotein (HDL) apolipoprotein, has been linked to familial combined hyperlipidemia. Human apoA-II transgenic mice constitute an animal model for this proatherogenic disease. We studied the ability of human apoA-II transgenic mice HDL to protect against oxidative modification of apoB-containing lipoproteins. When challenged with an atherogenic diet, antigens related to low-density lipoprotein (LDL) oxidation were markedly increased in the aorta of 11.1 transgenic mice (high human apoA-II expressor). HDL from control mice and 11.1 transgenic mice were coincubated with autologous very LDL (VLDL) or LDL, or with human LDL under oxidative conditions. The degree of oxidative modification of apoB lipoproteins was then evaluated by measuring relative electrophoretic mobility, dichlorofluorescein fluorescence, 9- and 13-hydroxyoctadecadienoic acid content, and conjugated diene kinetics. In all these different approaches, and in contrast to control mice, HDL from 11.1 transgenic mice failed to protect LDL from oxidative modification. A decreased content of apoA-I, paraoxonase (PON1), and platelet-activated factor acetyl-hydrolase activities was found in HDL of 11.1 transgenic mice. Liver gene expression of these HDL-associated proteins did not differ from that of control mice. In contrast, incubation of isolated human apoA-II with control mouse plasma at 37°C decreased PON1 activity and displaced the enzyme from HDL. Thus, overexpression of human apoA-II in mice impairs the ability of HDL to protect apoB-containing lipoproteins from oxidation. Further, the displacement of PON1 by apoA-II could explain in part why PON1 is mostly found in HDL particles with apoA-I and without apoA-II, as well as the poor antiatherogenic properties of apoA-II–rich HDL.

Selective induction of cyclo‐oxygenase‐2 activity in the permanent human endothelial cell line HUV‐EC‐C: biochemical and pharmacological characterization

Cyclo‐oxygenase (COX), the enzyme responsible for the conversion of arachidonic acid (AA) to prostaglandin H2 (PGH2), exists in two forms, termed COX‐1 and COX‐2 which are encoded by different genes. COX‐1 is expressed constitutively and is known to be the site of action of aspirin and other non‐steroidal anti‐inflammatory drugs. COX‐2 may be induced by a series of pro‐inflammatory stimuli and its role in the development of inflammation has been claimed. Endothelial cells are an important physiological source of prostanoids and the selective induction of COX‐2 activity has been described for finite cultures of endothelial cells, but not for permanent endothelial cell lines. The HUV‐EC‐C line is a permanent endothelial cell line of human origin. We have determined the COX activity of these cells under basal conditions and after its exposure to two different stimuli, phorbol 12‐myristate 13‐acetate (PMA) and interleukin‐1β (IL‐1β). Both PMA and IL‐1β produced dose‐ and time‐dependent increases of the synthesis of the COX‐derived eicosanoids. These increases were maximal after the treatment with 10 nM PMA for 6 to 9 h. Under these conditions, the main eicosanoid produced by the cells was PGE2. The increase of COX activity by PMA or IL‐1β correlated with an increase of the enzymes apparent Vmax, whilst the affinity for the substrate, measured as apparent Km, remained unaffected. Treatment of the cells with PMA induced a time‐dependent increase in the expression of both COX‐1 and COX‐2 mRNAs. Nevertheless, this increase was reflected only as an increase of the COX‐2 isoenzyme at the protein level. The enzymatic activity of the PMA‐induced COX was measured in the presence of a panel of enzyme inhibitors, and the IC50 values obtained were compared with those obtained for the inhibition of human platelet COX activity, a COX‐1 selective assay. Classical non‐steroidal anti‐inflammatory drugs (NSAIDs) inhibited both enzymes with varying potencies but only the three compounds previously shown to be selective COX‐2 inhibitors (SC‐58125, NS‐398 and nimesulide) showed higher potency towards the COX of PMA‐treated HUV‐EC‐C. Overall, it appears that the stimulation of the HUV‐EC‐C line with PMA selectively induces the COX‐2 isoenzyme. This appears to be a suitable model for the study of the physiology and pharmacology of this important isoenzyme, with a permanent endothelial cell line of human origin.

Microsomal prostaglandin E synthase-1, which is not coupled to a particular cyclooxygenase isoenzyme, is essential for prostaglandin E2 biosynthesis in vascular smooth muscle cells

Summary.  Background: Prostaglandin (PG) E2 induces expression of matrix metalloproteinases and angiogenic factors, thereby contributing to plaque instability. Objective: To study the influence of cyclooxygenase (COX) and PGE synthase (PGES) isoenzyme expression on PGE2 and PGI2 biosynthesis in vascular smooth muscle cells (VSMC) in culture. Methods: Cells were treated with human recombinant IL‐1β over different periods of time. Expression of PGI synthase, and COX and PGES isoenzymes was determined by real‐time reverse transcriptase polymerase chain reaction and immunoblotting. Biosynthesis of prostanoids from exogenous or endogenous substrate was analyzed by high‐performance liquid chromatography or enzyme‐immunoassay after incubation of cells with labeled arachidonic acid or thrombin, respectively. Results: Cytosolic PGES and microsomal PGES (mPGES) ‐1 and ‐2 were expressed in VSMC. PGES activity was mainly linked to mPGES‐1. IL‐1β induced COX‐2 and mPGES‐1 with a different time course. VSMC ability to synthesize PGE2 and PGI2 fitted mPGES‐1 and COX‐2 expression, respectively. The ability of VSMC to produce PGI2 was downregulated by mPGES‐1 expression and was restored when mPGES‐1 expression was silenced. Results from COX‐1 and COX‐2 silencing and selective inhibition showed that both COX‐1 and COX‐2 were involved in the biosynthesis of PGE2 and their relative contribution depended on the time of incubation with IL‐1β. Conclusions: mPGES‐1 is the main PGES responsible for PGE2 biosynthesis by VSMC and its induction downregulates VSMC ability to produce PGI2. These results support the concept that under inflammatory conditions VSMC could significantly contribute to plaque instability and that mPGES‐1 may be a target for therapeutic intervention in patients with cardiovascular risk.

The inflammatory properties of electronegative low-density lipoprotein from type 1 diabetic patients are related to increased platelet-activating factor acetylhydrolase activity

Aims/hypothesisChemical and biological characteristics of LDL(−) from type 1 diabetic subjects were analysed. The diabetic patients were studied during poor and optimised glycaemic control.Materials and methodsTotal LDL was subfractionated into electropositive LDL(+) and electronegative LDL(−) by anion exchange chromatography and the lipid and protein composition of the two determined.ResultsLDL(−) differed from LDL(+) in that it had higher triglyceride, non-esterified fatty acids, apoE, apoC-III and platelet-activating factor acetylhydrolase (PAF-AH), as well as lower apoB relative content. No evidence of increased oxidation was observed in LDL(−). LDL(−) increased two-fold the release of interleukin 8 (IL-8) and monocyte chemotactic protein 1 (MCP-1) in endothelial cells, suggesting an inflammatory role. Optimisation of glycaemic control after insulin therapy decreased the proportion of LDL(−), but did not modify the composition of LDL subfractions, except for a decrease in PAF-AH activity in LDL(−). The possibility that LDL(−) could be generated by non-enzymatic glycosylation was studied. Fructosamine and glycated LDL content in LDL subfractions from type 1 diabetic patients was greater than in LDL subfractions isolated from normoglycaemic subjects, and decreased after glycaemic optimisation in both subfractions. However, no difference was observed between LDL(+) and LDL(−) before and after insulin therapy.Conclusions/interpretationThese results provide evidence that LDL(−) is not produced by glycosylation. Nevertheless, LDL(−) from diabetic patients displays inflammatory potential reflected by the induction of chemokine release in endothelial cells. This proatherogenic effect could be related to the high PAF-AH activity in LDL(−).

Prostaglandin E(2) pathway in head and neck squamous cell carcinoma.

Prostaglandin E2 (PGE2) is involved in malignant growth. The objective was to study the PGE2 pathway in head and neck squamous cell carcinoma (HNSCC) patients.

Interaction between head and neck squamous cell carcinoma cells and fibroblasts in the biosynthesis of PGE2

Prostaglandin (PG)E2 is relevant in tumor biology, and interactions between tumor and stroma cells dramatically influence tumor progression. We tested the hypothesis that cross-talk between head and neck squamous cell carcinoma (HNSCC) cells and fibroblasts could substantially enhance PGE2 biosynthesis. We observed an enhanced production of PGE2 in cocultures of HNSCC cell lines and fibroblasts, which was consistent with an upregulation of COX-2 and microsomal PGE-synthase-1 (mPGES-1) in fibroblasts. In cultured endothelial cells, medium from fibroblasts treated with tumor cell-conditioned medium induced in vitro angiogenesis, and in tumor cell induced migration and proliferation, these effects were sensitive to PGs inhibition. Proteomic analysis shows that tumor cells released IL-1, and tumor cell-induced COX-2 and mPGES-1 were suppressed by the IL-1-receptor antagonist. IL-1α levels were higher than those of IL-1β in the tumor cell-conditioning medium and in the secretion from samples obtained from 20 patients with HNSCC. Fractionation of tumor cell-conditioning media indicated that tumor cells secreted mature and unprocessed forms of IL-1. Our results support the concept that tumor-associated fibroblasts are a relevant source of PGE2 in the tumor mass. Because mPGES-1 seems to be essential for a substantial biosynthesis of PGE2, these findings also strengthen the concept that mPGES-1 may be \a target for therapeutic intervention in patients with HNSCC.

Tumor cells induce COX-2 and mPGES-1 expression in microvascular endothelial cells mainly by means of IL-1 receptor activation

Prostaglandin (PG) E(2) plays a key role in immune response, tumor progression and metastasis. We previously showed that macrovessel-derived endothelial cells do not produce PGE(2) enzymatically because they do not express the inducible microsomal PGE-synthase-1 (mPGES-1). Nevertheless, differences between macro- and micro-vessel-derived endothelial cells regarding arachidonic acid (AAc) metabolism profile have been reported. The present work was conducted to evaluate the expression of PGE(2)-pathway-related enzymes in human microvascular endothelial cells (HMVEC) in culture and to test the hypothesis that the tumor cell-HMVEC cross talk could increase mPGES-1 expression in HMVEC. We treated HMVEC in culture with human recombinant IL-1β. IL-1β induced PGE(2) release and COX-2 and mPGES-1 expression in terms of mRNA and protein, determined by real-time PCR and immunoblotting, respectively. HMVEC constitutively expressed mPGES-2 and cytosolic PGES (cPGES) and the IL-1β treatment did not modify their expression. PGE(2) synthesized by HMVEC from exogenous AAc was linked to mPGES-1 expression. Immunohistochemistry analysis confirmed mPGES-1 expression in microvessels in vivo. COX-2 and mPGES-1 were also induced in HMVEC by the conditioned medium from two squamous head and neck carcinoma cell lines. Conditioned medium from tumor cell cultures contained several cytokines including the IL-1β and IL-1α. Tumor cell-induced COX-2 and mPGES-1 in HMVEC was strongly inhibited by the IL-1-receptor antagonist, indicating the important implication of IL-1 in this effect. HMVEC could therefore contribute directly to PGE(2) formed in the tumor. Our findings support the concept that mPGES-1 could be a target for therapeutic intervention in patients with cancer.