Glycoprotein biosynthesis in porcine aortic endothelial cells and changes in the apoptotic cell population

Inka Brockhausen1,2, Michael Lehotay2, Ji-Mao Yang2, Wensheng Qin2, David Young3, Jamie Lucien4, John Coles4 and Hans Paulsen5

2Department of Medicine, Division of Rheumatology, and Department of Biochemistry, Queen’s University, Kingston, K7L 3N6, Ontario, Canada; 3Arius Research, 55 York Street, 16th Floor, Toronto, M5J 1R7, Ontario, Canada; 4Hospital for Sick Children, 555 University Avenue, Toronto, M5G 1X8, Ontario, Canada; and 5Institut für Org. Chemie, Martin-Luther-King Platz 6, 20146 Hamburg, Germany

Received on May 17, 2001; revised on July 24, 2001; accepted on August 17, 2001.


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Porcine aortic endothelial cells (PAECs) produce glycoproteins with important biological functions, such as the control of cell adhesion, blood clotting, blood pressure, the immune system, and apoptosis. Cell surface glycoproteins play important roles in these biological activities. To understand the control of cell surface glycosylation, we elucidated biosynthetic pathways leading to N- and O-glycans in PAECs. Based on the enzyme activities, PAECs should be rich in complex biantennary N-glycans. In addition, the enzymes synthesizing complex O-glycans with core 1 and 2 structures are present in PAECs. The first enzyme of the O-glycosylation pathway, polypeptide GalNAc-transferase, was particularly active. Its specificity toward synthetic peptide substrates was found to be similar to that of purified bovine colostrum enzyme T1. A significant fraction of PAECs treated with tumour necrosis factor {alpha} or human serum detached from the culture plate, and most of these cells were apoptotic. The apoptotic cell population exhibited decreased core 2 ß6-GlcNAc-transferase activity. In contrast, the activities of core 1 ß3-Gal-transferase, which synthesizes O-glycan core 1, and of {alpha}3-sialyltransferase (O), which sialylates core 1, were increased in apoptotic PAECs. Thus, apoptotic PAECs are predicted to have fewer complex O-glycans and a higher proportion of short, sialylated core 1 chains.

Key words: apoptosis/glycosyltransferases/pig aortic endothelial cells/polypeptide GalNAc-transferase/synthetic substrates


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
The blood vessel endothelium regulates blood coagulation, vascular tone and permeability, blood pressure, leukocyte trafficking, the immune response, as well as vessel growth and angiogenesis. A great number of both soluble and cell surface–bound glycoproteins participate in these processes (Handin and Wagner, 1989Go; Harris and Spellman, 1993Go; Cruz et al., 1993Go; Dowbenko et al., 1993Go; Adams and Shaw, 1994Go; Moore et al., 1994Go). The endothelium is also involved in the rejection of xenotransplants (Galili et al., 1988Go; Platt et al., 1990Go; Robson et al., 1999Go; Cozzi et al., 2000Go). In particular, porcine aortic endothelial cells (PAECs) would be the target of human anti-porcine responses based on the presence of the linear B determinant, Gal {alpha}1-3Gal-, and other xenoantigens on pig cells (Oriol et al., 1993Go, 1999; Platt and Holzknecht, 1994Go; Rollins et al., 1994Go).

We previously reported that PAECs detach from the culture plate and become apoptotic when treated with human serum (Young et al., 1996Go). We have therefore proposed that xenograft rejection may involve apoptosis (Young et al., 1996Go). Apoptosis, or programmed cell death, is an important factor for tissue homeostasis that occurs during embryonic development and also in the immune system during cell selection. Pathological conditions, such as cancer, inflammatory disease, and arthritis, are associated with abnormal apoptosis. The expression of glycoproteins and specific cell surface carbohydrate structures have been shown to regulate apoptosis. In skin cells (Minamide et al., 1995Go), as well as in normal and tumor tissues (Hiraishi et al., 1993Go), the expression levels of Ley antigen (Fuc{alpha}1-2Galß1-4 [Fuc{alpha}1-3]GlcNAc-) were shown to be higher in the apoptotic cell populations. The activity of one of the enzymes synthesizing the Ley structure, {alpha}3-Fuc-transferase, correlated with apoptosis in human colon cancer cells HT29 (Akamatsu et al., 1996Go). By contrast, in rat colon cancer cells, {alpha}2-Fuc-transferase transfection conferred resistance to serum deprivation–induced apoptosis (Goupille et al., 2000Go).

Studies using glycosylation inhibitors have also demonstrated the importance of carbohydrates in apoptosis. For example, inhibition of N-glycosylation by tunicamycin in HL-60 cells is associated with early apoptosis (Pérez-Sala and Mollideno, 1996Go). Apoptosis of activated human T cells may be mediated by galectin-1 and thus depends on the exposure of terminal Gal residues (Perillo et al., 1995Go). Inhibition of O-glycan extension by GalNAc{alpha}-benzyl potentiates apoptosis induced by galectin-1, suggesting a role for O-glycans in apoptosis (Baum et al., 1995Go).

Preliminary studies showed that Gal- and GalNAc-binding lectins bound to PAECs and could induce apoptosis (unpublished data), suggesting that these sugar residues are exposed on cell surfaces and are of vital importance. However, to date only a preliminary study of the biosynthesis of glycoproteins in PAECs has been carried out in which nonspecific GlcNAc-, Gal-, Fuc-, and sialyltransferases were measured (Vischer and Buddecke, 1985Go). The combination of different peptide acceptor specificities and activities of various polypeptide GalNAc-transferases expressed in a particular cell determines the site specificity and extent of glycoprotein O-glycosylation. At least nine different enzymes (T1 to T9) of the polypeptide GalNAc-transferase family exist to catalyze the first step of O-glycosylation (Clausen and Bennett, 1996Go; Ten Hagen et al., 2001Go). Polypeptide GalNAc-transferase T1 has been cloned from several species including bovine (Hagen et al., 1993Go; Homa et al., 1993Go) and is widely expressed. Previous studies have shown that T1 acts mainly on Thr residues of peptide substrates but has low or no activity toward Ser residues in vitro. T1 activity is also controlled by the peptide structure and the existence of GalNAc-Thr or Gal ß1-3GalNAc-Thr in the substrate (Brockhausen et al., 1996Go; Iida et al., 1999Go).

In the present study we conducted a comprehensive analysis of the biosynthetic pathways of N- and O-glycans in PAECs to understand the enzymes involved in the regulation of cell surface glycans in these cells. We characterized the specificity of PAEC polypeptide GalNAc-transferase by comparison to T1 from bovine colostrum using synthetic peptide derivatives. We also studied the alterations of glycosyltransferase activities in the apoptotic cell population. A preliminary report has been presented (Brockhausen et al., 2000Go).


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Biosynthesis of N- and O-glycans in PAECs
PAECs with a cobblestone morphology were grown in culture and harvested immediately when confluency was reached. The number of passages were kept between 5 and 7. The biosynthesis of complex glycans was studied in a number of PAEC cultures by assaying the activities of enzymes that synthesize N-glycans and/or O-glycans (Table I). Based on previous studies of these enzymes we chose the conditions and substrate concentrations that measure near-maximal initial velocities (Möller et al., 1992Go; Vavasseur et al., 1994Go; Yang et al., 1994Go; Brockhausen et al., 1995Go).


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Table I. Enzymes involved in glycoprotein biosynthesis in PAECs
 
Biosynthesis of N-glycans.
GlcNAc-transferase I, which initializes the synthesis of complex N-glycans by adding a GlcNAc ß1-2 residue to the Man {alpha}1-3 residue of the N-glycan core (Figure 1), was active in all PAEC cultures tested (12–158 nmol/h/mg protein, Table I). GlcNAc-transferase II was also active (12–35 nmol/h/mg). In contrast, GlcNAc-transferase V was only detectable in a small number of cell cultures (up to 0.4 nmol/h/mg). Using specific substrates for GlcNAc-transferases III and IV, these activities were 1.0 nmol/h/mg and 0.8 nmol/h/mg, respectively (Table I). The biantennary substrate GlcNAc ß1-2Man {alpha}1-6 (GlcNAc ß1-2Man {alpha}1-3) Man ß-octyl, which is a substrate for GlcNAc-transferases III, IV, and V, yielded an activity of 2.6 nmol/h/mg.



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Fig. 1. Biosynthesis of N-glycans in PAECs. The enzymes synthesizing the first two antennae of N-glycans, GlcNAc-transferase I and II (GnT I and GnT II), are very active in PAECs, which therefore are expected to make biantennary N-glycan chains. The antennae may be elongated by poly N-acetyllactosamine chains and sialylated and fucosylated. The linear B {alpha}3-Gal-transferase may add terminal linear B determinants. There are also low activities of GlcNAc-transferases III and IV (GnTIII and GnTIV). GlcNAc-transferase V (GnTV) is barely detectable. G, Gal; Gn, GlcNAc; M, Man. Blocked arrow denotes inactive pathway.

 
The enzymes involved in the elongation of N- and O-glycans, forming poly-N-acetyllactosamine chains were present in all PAEC cultures. i ß3-GlcNAc-transferase synthesizing the i antigen was active up to 1.1 nmol/h/mg, whereas ß4-Gal-transferase activities, measured by Dowex and high-performance liquid chromatography (HPLC) assays, were 30–247 nmol/h/mg. The {alpha}3- and {alpha}6-sialyltransferase activities acting on the termini of N-glycans were assayed by high-voltage electrophoresis using Gal ß1-4GlcNAc as a substrate. The combined activities were low, up to 2.7 nmol/h/mg. In some PAEC cultures, these sialyltransferase activities were not detectable. Blood group A {alpha}3-GalNAc-transferase was also not detectable in PAECs. However, the activity of the {alpha}3-Gal-transferase synthesizing the linear B determinant was high (5.9–15.7 nmol/h/mg). The Gal{alpha}1-3Gal linkage in the enzyme product of linear B {alpha}3-Gal-transferase in PAECs was confirmed by proton nuclear magnetic resonance (NMR). Diagnostic signals were the presence of the doublet at 5.146 ppm with a 3.8 Hz coupling constant due to H-1 of the Gal{alpha} residue and a shift in the H-4 signal of Galß1-4 from 3.928 ppm in the substrate to 4.184 ppm in the product. A summary of the N-glycosylation pathways active in PAECs are shown in Figure 1.

Polypeptide GalNAc-transferase activities in PAECs and from bovine colostrum
Because GalNAc appeared to be an important cell surface determinant in PAEC, we focused on the first enzyme of the O-glycosylation pathway, polypeptide {alpha}-GalNAc-transferase. The enzyme was assayed using a panel of novel synthetic peptide substrates containing Ser and Thr residues as potential acceptor sites and either free amino and carboxyl termini or amino termini protected with an acetyl group and the carboxyl group protected with an amide group (Figure 2). Using MUC2 mucin tandem repeat sequence derived peptides, the activities varied significantly between substrates but were generally very high (Figure 2). The exception was the MUC2 mucin peptide Ac-PSSSPIST-NH2 which has previously been shown to be a poor acceptor for bovine polypeptide {alpha}-GalNAc-T1 (Brockhausen et al., 1996Go). In contrast, Ac-PTSSPIST-NH2 and Ac-PTTTPIST-NH2, which are excellent substrates for purified T1 (Brockhausen et al., 1996Go), were also good substrates for PAEC (Figure 2). This indicates that the PAEC enzyme, like bovine T1, acts mainly on Thr residues in vitro. The MUC1 tandem repeat sequence VTSAPDTRPAPGST (compound 314) was a good substrate (68–101 nmol/h/mg) and was used as a control, with its activity set to 100% (Figure 2). The best substrate of the MUC2 series was TTTVTPTPTG (557 nmol/h/mg for the PAEC enzyme). Surprisingly, TPTGTQTPTG and TPTPTGTQTG substrates showed relatively low activities (5.7 and 1.7 nmol/h/mg, respectively, for the PAEC enzyme).



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Fig. 2. Specificities of polypeptide GalNAc-transferases from PAEC and bovine colostrum. Polypeptide GalNAc-transferase activities were assayed with PAEC homogenates and with purified T1 by Dowex assays as described in Materials and methods. All compounds were measured at 1 mM concentration in the assay, with the exception of compound 294, Ac-PTTTPIST-NH2. Compound 294 was measured in PAECs by the Dowex assay at 0.5 mM concentration with an activity of 68 nmol/h/mg. Compounds 378, 383, 399, and 407 were assayed with PAEC homogenates by HPLC assays.

 
The specificities of purified bovine T1 and the enzyme in PAEC homogenates were found to be remarkably similar (Figure 2). Based on previous assays using peptides (Iida et al., 1999Go) or glycopeptides (Brockhausen et al., 1996Go) as substrates and purified polypeptide GalNAc-transferase T1, a near-saturating concentration of 1 mM was used in the assays. Bovine T1 was inactive or had very low activity toward peptides and glycopeptides derived from the MUC3 and MUC4 tandem repeat sequences containing multiple Ser and Thr residues (compounds 370–372, 377, 413–432, Table II) and from human choriogonadotropin (compound 433, Table II). Glycopeptides corresponding to MUC4 mucin sequences, containing GalNAc -linked to one of the Thr residues, showed very low activities (less than 6% of compound 314), which were always less than the activity with the corresponding unglycosylated peptides (Table II). This suggests that the two Thr residues within this MUC4 sequence were not favorable as substrates, and that the presence of GalNAc further reduces the activity. Amino acids Gly, Ser, and Asp in the +1 position of Thr, Ser, Ala, Val, and Asp in the –1 position appeared to be unfavorable for GalNAc transfer.


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Table II. Activity of bovine polypeptide GalNAc-transferase T1 toward synthetic peptide and glycopeptide acceptor substrates
 
Several acetyl-amide protected hexapeptides (compounds 395–398, Table II) were synthesized with multiple Pro residues and Thr at the N-terminus. None of these compounds were active (Table II), confirming that Thr in the N-terminal position is not a substrate. It also confirmed that Ser was not a substrate. Many of the decapeptides derived from MUC2, containing five to seven Thr residues were good substrates for purified T1 (Table II, Figure 2). The activities did not correlate with the number of Thr or Pro residues or with specific sequences. The best substrate in this series, compound 362, TTTVTPTPTG, as well as compounds 361, 368, and 369, produced at least two major enzyme products separable by HPLC, whereas three enzyme products could be separated using compound 364, TPTPTPTGTG, as a substrate. One or more minor products were also seen with most of the MUC2 series peptide substrates containing multiple Thr residues. This shows that several of the Thr residues within a peptide are utilized as substrate for GalNAc transfer.

Series of acetyl-amides with five amino acids and benzoylphenylalanine (Bpa) and 4-benzoyl-benzoylornithine groups (Bbo), as well as Thr in positions other than the N-terminal position were synthesized to produce stable and highly active polypeptide GalNAc-transferase substrates. Many of these compounds were very potent substrates for bovine T1 (Table II). Compounds 382 and 388 contained Glu within the peptide sequence and bound 100% to AG1x8. The products formed with these compounds were therefore evaluated after binding and elution from Sep-Pak C18 columns (Table II). The bulky hydrophobic Bbo and Bpa groups had variable effects on the potency of the substrate, and the effect was not related to their position within the peptide or their proximity to Thr residues. Ac-BpaPTPPP-NH2 (compound 383) and Ac-TPPPBpaT-NH2 (compound 378), were also very good substrates (187 and 137 nmol/h/mg, respectively) with the PAEC GalNAc-transferase (Figure 2).

Biosynthesis of complex O-glycans
Several glycosyltransferases specifically synthesizing O-glycans were very active in PAEC. These cells are therefore expected to efficiently O-glycosylate proteins (Figure 3). The synthesis of O-glycan core 1 (Figure 3) was measured by assaying core 1 ß3-Gal-transferase using GalNAc {alpha}-benzyl (Bzl) substrate. The enzyme was active in all batches of PAEC (7.7–25.7 nmol/h/mg). Core 2 synthesis was also active, measured with Gal ß1-3GalNAc-Bzl or p-nitrophenol (pnp) substrates, and varied from 2.6 to 17.5 nmol/h/mg. Using HPLC assays, the activities of core 3 ß3-GlcNAc-transferase and core 4 ß6-GlcNAc-transferase synthesizing core 3 and core 4, respectively, were not detectable. In addition, the subterminally acting I ß6-GlcNAc-transferase using GlcNAc ß1-3Gal-methyl substrate was not detectable. This indicates that core 2 ß6-GlcNAc-transferase L, but not M, is expressed in PAECs (Kuhns et al., 1993Go). The elongation ß3-GlcNAc-transferase that elongates O-glycan core 1 and core 2 structures showed activities up to 2.6 nmol/h/mg using core 2-Bzl substrate.



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Fig. 3. Biosynthesis of O-glycans in PAECs. The first step of O-glycosylation is catalyzed by a family of polypeptide {alpha}-GalNAc-transferases (path a). These enzyme(s) are particularly active in PAEC. Core 1 is synthesized by core 1 ß3-Gal-transferase (path b) and core 2 ß6-GlcNAc-transferase (path i). Core 3 ß3-GlcNAc-transferase (path c) and core 4 ß6-GlcNAc-transferase (path h) are not detectable. GalNAc{alpha}-Thr/Ser may be sialylated by {alpha}6-sialyltransferase (O) (path d). Cores 1 and 2 may be elongated by elongation ß3-GlcNAc-transferase (path k), core 1 may also be sulfated (path e), or sialylated by {alpha}3-sialyltransferase (O) (path f). Poly N-acetyllactosamine chains may be added by ß4-Gal-transferase (path l), and possibly by ß3-Gal-transferase (path m), and i ß3-GlcNAc-transferase (path o). These chains may be fucosylated by {alpha}3-Fuc-transferase (path n) and {alpha}4-Fuc-transferase (path q). Subterminal I antigenic branches may not be added (path p). {alpha}2-Fuc-transferase activity (path g) is not detectable in PAECs. SA, sialic acid. Blocked arrows denote inactive pathways.

 
The {alpha}3-sialyltransferase (O) acting on O-glycan core 1, Gal ß1-3GalNAc {alpha}-pNp substrate, was measured in high voltage electrophoresis assays, and was active in all PAEC batches (5.5–7.1 nmol/h/mg). Surprisingly, the {alpha}6-sialyltransferase (O) acting on GalNAc-mucin was also detected in some batches (up to 5.2 nmol/h/mg). Sulfotransferase activities were relatively low, compared to the control rat colon homogenates. Of the four sulfotransferase substrates chosen, only Gal ß1-3GalNAc {alpha}-Bzl and Gal ß1-4GlcNAc showed low activities; activities with GlcNAc ß1-3Gal ß-methyl and Gal ß1-4(Fuc {alpha}1-3) GlcNAc were undetectable.

{alpha}2-Fuc-transferase activity, using several assay systems, and Galß-phenyl and Gal ß1–3GalNAc{alpha}-Bzl substrates for the hematopoietic cell type and the secretory cell type enzymes, respectively, could not be detected in PAECs with the exception of one batch where a low activity (0.7 nmol/h/mg) was found. However, {alpha}3-Fuc-transferase activities were present (1.2–3.8 nmol/h/mg) using the nonspecific substrate Gal ß1-4 GlcNAc and the specific substrate 2-O-methyl Gal ß1-4GlcNAc. {alpha}4-Fuc-transferase activities, using specific substrate 2-O-methyl Gal ß1-3GlcNAc ß-Bzl, were also found (5.9 nmol/h/mg), therefore at least {alpha}3/4-FucT III must be expressed (Table I).

Demonstration of PAEC apoptosis
On treatment with TNF{alpha} or pooled human AB serum PAEC randomly detached from the culture plates. This process was time-dependent and concentration-dependent. After 18 h treatment with 25% human serum about 12% of the cells detached (Figure 4), and this concentration was used in subsequent studies. Porcine serum did not induce this effect. The apparent decrease in detachment after 20 h does not represent reattachment but was due to the loss of intact cells resulting from cell death. Detachment was associated with the acquisition of a rounded shape and membrane blebbing as well as cell shrinkage, as demonstrated by fluorescence microscopy. PAECs treated with 25% human serum were stained with the fluorescent dye Hoechst 33259, calcein AM, and ethidium homodimer and revealed randomly distributed cells with the typical nuclear morphology of apoptotic cells. Nuclear condensation, budding of apoptotic bodies, and nuclear fragmentation was seen in more than 90% of the detached cells. In contrast, cells treated with porcine serum, heated human serum, or media alone showed very low cell detachment (Figure 4).



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Fig. 4. Time course of PAEC detachment. PAECs were treated with human serum for different time intervals. The percentage of PAECs that detach after treatment with 25% human serum in the cell medium (open squares) or porcine serum (closed squares) are shown.

 
Agarose gel electrophoresis of DNA extracted from detached cells (after 6–24 h of 25% human serum treatment) showed DNA ladders typical for endonuclease degradation of DNA of apoptotic cells (data not shown) (Arends et al., 1990Go). PAECs treated with porcine serum or heat-inactivated human serum or those killed by necrosis through several freeze/thawing cycles did not show DNA degradation.

Terminal deoxynucleotidyl transferase dUTP nicked end labeling (TUNEL) stains of apoptotic PAECs (Gavrieli et al., 1992Go) after 24 h of human serum treatment–labeled cells well (Figure 5). More than 90% of detached cells (Figure 5E) and 5% of attached cells (Figure 5A) were labeled and found to be apoptotic. Tumor necrosis factor {alpha} (TNF {alpha}) treatment resulted in numbers of apoptotic cells in the detached (not shown) and the attached fraction (Figure 5C) that were similar to those obtained with human serum. This was in contrast to cells treated with porcine serum or heat-inactivated human serum (Figure 5B), medium (Figure 5D), or freeze/thawed cells (Figure 5F), which showed minimal background labeling.



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Fig. 5. TUNEL of PAECs. Digoxigenin-labeled dUTP was detected by FITC anti-digoxigenin and fluorescence microscopy (100x) of PAECs; propidium iodide was used for counterstaining. Arrows show labels of apoptotic cells. (A) PAECs treated with 25% human serum for 24 h; (B) PAECs treated with heat-inactivated human serum; (C) TNF{alpha} treatment; (D) control cells in media only; (E) detached cells that were treated with 25% human serum; (F) freeze/thawed cells. The percentage of TUNEL-stained apoptotic cells was determined by counting more than 500 cells in triplicate experiments.

 
Electron microscopy of human serum treated cells showed ultrastructural features typical of apoptosis (Figure 6). DNA clumping and margination of clumped DNA to the nuclear membrane was seen (Figure 6A, C), as well as dilation of the endoplasmic reticulum and nuclear clefting. Some of the cell membranes showed blebbing (Figure 6B), and there were some examples of apoptotic cells undergoing phagocytosis by adjacent cells (Figure 6D). TNF{alpha}-treated detached PAECs showed similar features, but nonapoptotic attached cells had normal cellular and nuclear morphology.



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Fig. 6. Electron micrograph (10,000x) of detached PAECs. PAECs were treated with 25% human serum. In the initial stage of apoptosis there is condensation and margination of nuclear DNA (A, arrow). As apoptosis proceeds there is a decrease in cell volume, and cells lose their surface attachments and become round in shape. There is also endoplasmic reticulum dilation and cell membrane ruffling (B, arrow). In the later stages, there is marked condensation of DNA (C, arrow) with formation of apoptotic bodies (arrow). Sometimes, apoptotic cells are engulfed by neighboring phagocytic cells (D, arrow).

 
Alterations of glycosyltransferase activities in apoptotic cells
Cells that detached from the culture plates after treatment with human serum or TNF{alpha} were collected and analyzed for differences in glycosyltransferase activities, in comparison to those in attached cells (Table III). We assayed those glycosyltransferases that had previously been shown to be altered during changes of cellular growth, angiogenesis, differentiation, and cancer. Core 1 ß3-Gal-transferase activity was increased 2.4- to 5.5-fold in all batches of human serum–treated PAECs that detached, compared to the corresponding attached cells. This increase was even more prominent (12-fold) on TNF{alpha} treatment. TNF{alpha} treatment also caused an almost twofold increase of core 1 ß3-Gal-transferase activity in the attached cell population, compared to untreated cells. Similarly, on human serum treatment, {alpha}3-sialyltransferase (O) activity acting on O-glycan core 1 was increased 2.1- to 3.5-fold in the detached cell fraction compared to attached cells, but was only 1.3-fold increased with TNF{alpha} treatment. In contrast, detached human serum treated cells showed a 3.8- to 6.6-fold decrease in core 2 ß6-GlcNAc-transferase activity and a 45-fold decrease in TNF{alpha} treated cells, compared to the corresponding attached cells.


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Table III. Alterations of glycosyltransferase activities in apoptotic PAECs
 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Based on the activities of N-glycan banching GlcNAc-transferases, PAECs have the potential of synthesizing biantennary N-glycans, as well as a small amount of triantennary and bisected N-glycan chains (Figure 1). Tetraantennary chains are probably not present. The binding of ConA lectin to PAECs (unpublished data) confirms the presence of N-glycans that are not highly branched or bisected.

Enzyme activities in PAECs suggest that N-glycans, as well as O-glycans, may be elongated by linear poly-N-acetyllactosamine chains. Although the ß4-Gal-transferase is very active, the elongating ß3-GlcNAc-transferase shows a low activity, thereby limiting the amounts and lengths of poly-N-acetyllactosamine chains. The branching reactions to form the I antigenic branch at subterminal Gal residues were not detectable, but it remains to be verified if branches can be synthesized on centrally located Gal residues.

Poly-N-acetyllactosamine chains may be sialylated as well as sulfated. The {alpha}2-Fuc-transferase activities toward Gal ß-R or Gal ß1-3GalNAc-R acceptors were not detectable in most PAEC batches, indicating that H determinants cannot be made. However, blood groups H and A substances may be found in pig secretory tissues. The lack of processing of terminal Gal residues of N- and O-glycans to {alpha}2-fucosylated structures in PAECs promotes the addition of the linear B determinant, which therefore is expected to be a major terminal sugar. Lewisa and Lewisx determinants and sialylated termini as well as sulfate esters on Gal residues can also be synthesized and may play an important role in the cell adhesion properties and attachment of leukocytes to these endothelial cells via selectins.

In the O-glycosylation pathways (Figure 3) we established that core 1 and 2 structures can be synthesized in PAECs, but cores 3 and 4 cannot. These pathways are typical for a nonmucin–secreting cell type, such as leukocytes (Higgins et al., 1991Go; Brockhausen et al., 1991aGo; Brockhausen and Kuhns, 1997Go). Although PAECs lack some O-glycans that are found particularly on mucins (e.g., cores 3 and 4), these cells still retain a high capacity to synthesize mucin type O-glycan chains.

The first enzyme of the O-glycosylation pathway in PAECs, polypeptide {alpha}-GalNAc-transferase, was unusually active when assayed with a number of synthetic peptide substrates. The patterns of relative activities toward a panel of peptide substrates paralleled those of bovine T1. This similarity of specificity suggests that T1 may be the major enzyme expressed in PAECs. It is interesting that two of the peptide substrates with MUC2 sequences are poor acceptors for polypeptide {alpha}-GalNAc-transferase although five potential acceptor sites are present, whereas peptides with variations of this sequence are excellent substrates. Ser does not appear to be a substrate, and the enzyme requires Thr residues in specific nonamino terminal positions within the peptide to yield a good acceptor site. Some of the compounds containing bulky, hydrophobic benzoylphenylalanine groups form the best substrates tested with PAECs or purified polypeptide GalNAc-transferase. Thus the direct chemical environment, exposure, and flexibility of Thr residues within the peptide determine their potential as a substrate.

The attachment of GalNAc residues to the peptide of glycoproteins is much more active in PAECs compared to other cell types (Brockhausen et al., 1995Go, 2001) and compared to the enzymes processing GalNAc residues. It is thus conceivable that a number of GalNAc residues may not be further processed and thus may be exposed on PAEC surfaces. The binding of lectins (unpublished data) that recognize terminal GalNAc on PAEC concurs with these glycosyltransferase data.

The processing reactions of GalNAc-residues in PAEC include the conversions to core 1 and core 2 (Figure 3), and their elongation by N-acetyllactosamine chains. Both core 1 and 2 structures may be sialylated, which will restrict the size and complexity of these chains and make them more negatively charged. The two main enzymes competing for the core 1 substrate, core 2 ß6-GlcNAc-transferase and {alpha}3-sialyltransferase (O), are active in PAECs, suggesting that there are some short sialylated chains as well as more complex chains with core 2 structures. The relative activities of these two enzymes have been shown to control the cell surface exposure of glycans (Brockhausen et al., 1995Go; Brockhausen, 1999Go; Dalziel et al., 2000Go). In the apoptotic cell population, the sialyltransferase is relatively more active than the core 2 ß6-GlcNAc-transferase (Figure 7). This reversal of activities is expected to result in relatively fewer branched and complex O-glycans, and more sialylated core 1 structures in apoptotic PAECs. The consequence of this would be a major shift in the exposure and presentation of cell surface antigens, such as a reduction in selectin ligands for cell adhesion.



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Fig. 7. Competition between core 2 synthesis and sialylation. In the attached (nonapoptotic) PAECs, synthesis of core 2 and complex structures is active. In detached (apoptotic) cells, synthesis of core 1 by core 1 ß3-Gal-transferase is dramatically increased, sialylation of core 1 by {alpha}3-sialyltransferase is also increased, but the synthesis of core 2 by core 2 ß6-GlcNAc-transferase is decreased. Because core 2 ß6-GlcNAc-transferase competes with {alpha}3-sialyltransferase, core 1 chains are expected to be short and sialylated rather than branched and complex in the apoptotic cell population.

 
The {alpha}3-sialyltransferase (O) has previously been found to be increased in leukemia and breast cancer cells, as well as in colon cancer tissue (Brockhausen and Kuhns, 1997Go; Brockhausen et al., 1998Go), compared to the normal counterparts. The increase of this enzyme activity in apoptotic PAECs indicates a major shift in cell surface properties related to cell detachment. Thus the initial induction of the apoptotic process may be regulated by carbohydrate, but then results in a specifically changed pattern of cell surface glycans, and concomitant alterations of the biological properties of the cell.

There are many examples where glycosylation changes and a shift in the tissue distribution of glycoforms can be linked to growth and differentiation (Brockhausen et al., 1998Go). Core 2 ß6-GlcNAc-transferase L present in PAECs (Kuhns et al., 1993Go; Schwientek et al., 2000Go) appears to be regulated during growth and differentiation, lymphocyte activation (Piller et al., 1988Go), in immunodeficiency syndrome (Higgins et al., 1991Go), differentiating cancer cells (Brockhausen et al., 1991bGo), and in leukemias (Brockhausen et al., 1991aGo; Saitoh et al., 1991Go). It is therefore not surprising that this enzyme activity is altered in apoptotic PAECs. The inflammatory cytokines TNF{alpha} and interleukin-1 have been shown to be effective in cultured human endothelial cells in inducing the expression of {alpha}6-sialyltransferase acting on N-glycans, along with endothelial glycoproteins bearing N-glycans with increased numbers of {alpha}2-6-linked sialic acid residues (Hanasaki et al., 1994Go). {alpha}6-Sialylation may also change during development and cell differentiation (Dall'Olio et al., 1992Go, 1996).

Because there was only a small amount of cellular material available of detached PAECs, a limited number of glycosyltransferases was examined in the apoptotic cell population. However, additional enzymes of the N- and O-glycosylation pathways should be examined to establish the effect of apoptotic agents on cellular glycosylation. It also remains to be shown if the activity changes observed in apoptotic cells are due to altered mRNA expression of glycosyltransferases and sulfotransferases. Alternative explanations include alterations of an inhibitor or alterations in the composition of membranes that influence the activities of membrane bound enzymes.

It is possible that human serum or TNF{alpha} treatment causes altered glycosylation that leads to different cell adhesion and causes cell detachment. Detached cells then may undergo accelerated apoptosis. Alternatively, the apoptotic cascade induced by TNF{alpha} or by human serum may also lead to altered glycosyltransferase activities. The third possibility is that the expression of transferases and their activities may be controlled by a mechanism that is unrelated to apoptosis but a consequence of the effects of TNF{alpha} or human serum components.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Materials
Human blood group AB sera were obtained from the Canadian Red Cross (Canadian Blood Services). Plasma from 4–10 donors was pooled and clotted by adding CaCl2 to make a 10-mM plasma solution. After the clot had formed it was removed by centrifugation, and sera were stored at –70°C. Heat-inactivated sera were produced by heating sera at 56°C for 30 min. Hoechst 33258 stain, propidium iodide, calcein AM, and ethidium homodimer were from Molecular Probes. Calcein AM and ethidium homodimer were stored at 1 mg/ml in dimethylsulfoxide at –20°C. Propidium iodide and Hoechst dye were stored at 10 mg/ml water at 4°C. GlcNAc, GalNAc{alpha}-Bzl, Galß1-3GalNAc{alpha}-Bzl, GlcNAcß1-3GalNAc{alpha}-pnp, Galß1-4GlcNAc, Galß-phenyl, and GlcNAcß1-3Galß-methyl were purchased from Sigma. Galß1-3GalNAc{alpha}-pnp was obtained from Toronto Research Chemicals. Other compounds were obtained as previously described (Möller et al., 1992Go; Paulsen et al., 1993Go; Vavasseur et al., 1994Go; Brockhausen et al., 1995Go, 1996). Culture media, trypsin–ethylenediamine tetraacetic acid (EDTA), phosphate buffered saline (PBS), and antibiotics were from Gibco (Grand Island, NY). Acetonitrile (UV grade) was obtained from Caledon Laboratories or Fisher Scientific, liquid scintillation fluid (Ready Safe) from Beckman. Dowex or AG1x8 beads (100–200 mesh, chloride form) were from Bio-Rad. Other reagents came from sources described previously (Möller et al., 1992Go; Yang et al., 1994Go; Brockhausen et al., 1995Go).

Polypeptide GalNAc-transferase substrates
Compound 314, VTSAPDTRPAPGST, was chemically synthesized and was a kind gift from J. Taylor-Papadimitriou (ICRF, London). Compounds 358 to 377, derived from MUC2 and MUC3 sequences, and hydrophobic and amino/carboxy terminus protected compounds 378 to 392 with Bbo and Bpa groups were synthesized according to standard multiple-column solid phase synthesis. All amino acids were coupled as their Fmoc-amino acid-pentafluorophenylesters with the addition of 3,4,-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazin. In the case of compounds 378 to 392, Fmoc-4-benzoylphenylalanine-pentafluorophenylester and Fmoc-p-benzoyl-benzoylornithine-pentafluorophenylester were used as building blocks. All glycosylated compounds 413 to 432 derived from MUC4 were synthesized by the method described by Mathieux et al. (1997)Go. Compound 433, an acetylated peptide derived from choriogonadotropin, was also synthesized by standard solid phase peptide synthesis using Fmoc amino acid-pentafluorophenylesters. Fast atom bombardment mass spectrometry (FAB-MS) analysis of all compounds showed the correct molecular weight. The proton-NMR data confirmed the expected structures.

Cell cultures
PAECs were isolated from pig aortas obtained from the abbatoir. Cells were cultured in 10% fetal bovine serum (Immunocorp, Montreal, Quebec) in M199 medium and passaged using trypsin-EDTA (Gibco) in 100-mm culture dishes. The cell type was confirmed by uptake of acetylated low-density lipoprotein and by the cobblestone morphology seen by phase contrast microscopy. Cell viability was examined by Trypan Blue exclusion.

Cell staining
The DNA staining Hoechst 33258 dye, which freely permeates membranes, was used to evaluate nuclear morphology. Cells were incubated with 500 µl of 1 µg/ml Hoechst dye in M199 medium for 30 min at 37°C. Excess dye was removed by washing with PBS; cells were mounted with 90% glycerol in PBS and examined with a fluorescence microscope. Cells were also fixed with 0.5% paraformaldehyde for 30 min at room temperature. Fixed cells were washed and examined with a fluorescence microscope (Olympus AH3) equipped with a mercury lamp and a 440–460 nm excitation filter. Two different emission filters were used, 420 nm for calcein AM–ethidium homodimer, and 570 nm for propidium iodide–Hoechst stain. To assess membrane integrity cells were stained with 6 µM ethidium homodimer in M199 medium (excluded by cells with intact membranes) or 4 µM calcein AM in M199 medium (which penetrates membranes) followed by phase contrast and fluorescence microscopy. Nuclear morphology was also examined by propidium iodide. Cells were photographed with a Nikon camera, using Kodak Ectachrome film. Slides were digitized using the Nikon scanner LS-10E and a Macintosh Quadra 840AV computer.

Quantitation of cells with damaged membranes was performed by counting cells that took up ethidium homodimer and released calcein. Apoptotic cells were identified as those that demonstrated features of apoptosis such as nuclear condensation, decrease in cell volume, and formation of apoptotic bodies.

TUNEL assay for detecting apoptotic DNA
DNA fragmentation was detected by 3'-end DNA labeling, using digoxigenin-labeled UTP and deoxynucleotidyl transferase, followed by binding of an anti-digoxigenin antibody-fluorescein conjugate. The TUNEL ApopTag Kit was from Oncor. Cells were washed in PBS and fixed in 1% paraformaldehyde at 4°C overnight, and permeabilized with 2:1 (v/v) ethanol/acetic acid. Cells were then incubated with deoxynucleotidyl transferase for 1 h at 37°C, followed by fluorescein-conjugated anti -digoxygenin antibody for 30 min at room temperature. Propidium iodide was used to stain DNA. Stained cells were examined under UV light using a green/yellow filter to screen for fluorescence-positive cells and a red filter to detect propidium iodide–stained cells. At least 500 adherent as well as detached nonadherent cells were examined and counted per experiment. The degree of apoptosis was determined by the ratio of fluorescence-labeled cells versus nonfluorescent cells.

Electron microscopy
Cells were fixed for 1 h in 2% glutaraldehyde in 0.1 M Sorensen’s phosphate buffer. Following rinsing with 0.1 M Sorensen’s phosphate buffer the cells were postfixed with 1% aqueous osmium tetroxide for 1 h and then briefly rinsed with water before dehydration through a series of graded acetone preparations. Cells were infiltrated by several changes of 100% acetone followed by a 1:1 mixture of acetone/epon araldite and by two changes of epon araldite. Cells were embedded in fresh epon araldite and polymerized overnight at 60°C. One-micron-thick sections were cut with a Reichert Ultracut E ultramicrotone and stained with 1% toluidine blue for examination under the light microscope. Ultrathin sections were then cut from a representative block. Sections were collected on copper grids and counterstained with uranylacetate and Sato’s lead citrate. Cells were examined at 60 kV on a Philips 400T transmission electron microscope and photographed.

DNA electrophoresis
DNA fragmentation was assessed by DNA agarose gel electrophoresis according to Herrmann et al. (1994)Go. Following treatment with human serum or pig serum cells were washed with PBS; cells detaching by the wash procedure were added to the fraction of nonadherent cells. Nonadherent cells were removed by centrifugation at 500 x g for 10 min at room temperature. Adherent and nonadherent cells were treated with 500 µl 1% NP-40/20 mM EDTA/50 mM Tris–HCl, pH 7.5, for 1 min and then centrifuged at 1600 x g for 10 min at 4°C. The supernatant was decanted and treated with 8 µl of 12 µg/ml RNase for 2 h at 56°C, and then 5 µl of 20 µg/ml proteinase A for 2h at 56°C (Pharmacia). DNA was precipitated with 100 µl of 5 M NaCl and 700 µl 70% isopropanol overnight at –20°C. DNA was recovered by centrifugation at 21,000 x g for 30 min at 4°C, and then solubilized in Tris-EDTA and analyzed by 1.8% agarose gel electrophoresis.

Induction of apoptosis
Confluent cells were washed with PBS. To induce apoptosis, M199 medium containing 40 ng/ml TNF{alpha} (Calbiochem, La Jolla, CA), 25% human AB sera, or pig serum control (obtained from the abbatoir) was added to washed, confluent cells, and incubated for 18 h. Under these conditions, cells detached, and most of the detached cells were apoptotic.

Preparation of cell homogenates
PAECs were cultured to confluence for five to eight passages. Detached cells were recovered from the culture medium by centrifugation. Attached cells were then removed from the dish with a rubber scraper, washed twice in PBS, and washed twice again with 0.9% NaCl. Cells were homogenized in 0.25 M sucrose with a hand homogenizer (approximately 1 ml sucrose/108 cells), and stored at –80°C until use.

Assays for glycosyl- and sulfotransferases
Assays for GlcNAc-transferases.
The standard assay mixture for GlcNAc-transferases contained the following ingredients in a total volume of 40 µl: 0.125 M MES, pH 7, 0.125% Triton X-100, 12.5 mM MnCl2, 1–1.4 mM UDP-N-[1-14C]-acetylglucosamine (2000–4000 dpm/nmol) or UDP-[G-3H]-GlcNAc (2000–10,200 dpm/nmol), 5–10 mM AMP, 0.125 M GlcNAc, acceptor substrates as indicated in the tables, and 10 µl of PAEC homogenate or control homogenates from rat colon or pig gastric mucosa (0.02–0.145 mg protein). Core 2, core 4, blood group I ß6-GlcNAc-transferases and GlcNAc-transferase V were measured in the absence of exogenous MnCl2.

Mixtures were incubated for 1 h at 37 °C. Reactions were stopped by the addition of 400 µl 20 mM sodium tetraborate/1 mM EDTA, pH 9, or with 100 µl of water (4°C) followed by freezing. Depending on the substrate, either Dowex, Sep-Pak, or HPLC assays were carried out, as described below and as indicated in the tables. Product formation was determined by subtracting radioactivity from control assays lacking the acceptor substrate.

Assays for Gal-transferases.
Assays for Gal-transferases were carried out as described for GlcNAc-transferases except that the assay mixtures contained 0.9 mM UDP-[3H]-Gal (6100 dpm/nmol) instead of UDP-N-[14C]-acetylglucosamine. GlcNAc (0.125 M) was not added, but some of the assay mixtures contained 5 mM {gamma}-galactonolactone. Product formation was measured by Dowex and HPLC assays. Acceptor substrates are listed in the tables.

Assays for GalNAc-transferases.
Assays for PAEC polypeptide GalNAc-transferases were performed as described for GlcNAc-transferases except that the assay mixtures contained 0.8–1 mM UDP-[14C]-GalNAc (2000–6000 dpm/nmol) instead of UDP-[14C]GlcNAc and no GlcNAc. Product formation was measured by Dowex and HPLC assays. Polypeptide GalNAc-transferase T1 purified from bovine colostrum was kindly donated by Fred Hagen and Lawrence Tabak, University of Rochester (Hagen et al., 1993Go), and diluted 1:10 with 50 mM sodium cacodylate, pH 5, 100 mM NaCl, and 50% glycerol for use in enzyme assays, and stored at –20°C. This T1 enzyme was assayed as described (Brockhausen et al., 1996Go).

Assays for Fuc-transferases.
Assays for Fuc-transferases were carried out as described for GlcNAc-transferases except that the assay mixtures contained 1 mM GDP-[3H]Fuc (2400 dpm/nmol) instead of UDP-[14C]GlcNAc and no GlcNAc. Product formation was determined by Dowex, HPLC, and Sep-Pak assays.

Assays for sialyltransferases.
The standard assay mixtures for sialyl-transferases contained the following ingredients in a total volume of 40 µl: 0.1 M Tris–HCl, pH 7.0, 0.25% Triton X-100, 5 mM AMP, 0.6–1 mM CMP-[14C] NeuAc (800–2000 dpm/nmol), acceptor substrates as indicated in the tables, and 10 µl of PAECs or control homogenate (0.02–0.145 mg protein). Mixtures were incubated for 1 h at 37°C. Reactions were stopped by the addition of 10 µl 20 mM EDTA/1% borate, and the product was separated by high-voltage electrophoresis as described below.

Assays for sulfotransferases.
The standard assay mixtures for sulfotransferases contained the following ingredients in a total volume of 40 µl: 40 mM Tris–HCl, pH 6.3, 2.5 mM ATP, 10 mM NaF, 2.5 mM Mg-acetate, 10 mM 2,3-mercaptopropanol, 1% Triton X-100, 6.7 or 7.5 µM 3'-phospo-adenosine-5'-phospho-[35S] sulfate (2200–2400 dpm/nmol), acceptor substrates as indicated in Table I, and 10 µl PAECs or control homogenate (0.02–0.145 mg protein). The reaction was incubated for 1 h at 37°C and stopped by the addition of 10 µl 20 mM EDTA/1% sodium tetraborate, pH 9. Mixtures were separated by high-voltage electrophoresis.

Dowex assays
For Dowex assays, 100–600 µl cold water were added to the assay mixtures following the incubations. The solutions were then passed through Pasteur pipettes containing 0.4 or 0.8 ml AG I-X8, 100–200 mesh, Cl–2 form, and the columns were washed with 1.8 ml or 2.6 ml water, respectively. The eluates were then lyophilized for HPLC assays, or else counted directly in 5 ml scintillation fluid with an LKB or Beckman scintillation counter.

HPLC assays
Lyophilized samples after AG1x8 chromatography were taken up in 120 or 200 µl water and stored frozen. Aliquots (one-half or two-thirds of total volume) were injected into the HPLC. Standard oligosaccharides, substrates and enzyme products were separated on either an Alltech Econosil C18 10-micron column with acetonitrile-water mixtures as the mobile phase as indicated in Table I (for substrates with large hydrophobic groups), a mixed amino-cyano (PAC) column, or an Alltech Econosil amine column (for oligosaccharide substrates without large hydrophobic groups) with acetonitrile-water mixtures as the mobile phase at a flow rate of 1 ml/min. Two-minute fractions were collected, the absorbance at 195 nm was measured, and activity counted after the addition of 5 ml scintillation fluid.

Sep-Pak assays
Sep-Pak assays were used to isolate products containing hydrophobic aglycon groups. The Sep-Pak assays consisted of passing assay mixtures (after the addition of about 600 µl water) slowly through methanol- and water-washed Sep-Pak C18 cartridges (Waters). Cartridges were then washed three times each with 1 ml water. The wash contained all the radioactivity of the unreacted nucleotide sugar, and charged and nonhydrophobic neutral breakdown products. Glycosyltransferase products were eluted with 5 ml methanol and collected and counted in three fractions.

High-voltage electrophoresis assays
For high-voltage electrophoresis assays, reactions were stopped with 10 µl 20 mM EDTA/1% sodium tetraborate. Mixtures were applied to Whatman No. 1 paper and separated by electrophoresis in 1% sodium-tetraborate buffer at 150 mA and 1.5–2 kV for 60 or 90 min. Papers were dried and cut in 2-cm strips, which were counted in 5 ml scintillation fluid.

Protein assays
Protein concentrations were determined with the Bio-Rad protein assay kit according to the manufacturer’s instructions using bovine serum albumin as the standard.


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
The authors gratefully acknowledge support from the Canadian Cystic Fibrosis Foundation (to I.B.), the Heart and Stroke Foundation of Ontario (to I.B.), the Medical Research Council (to I.B. and J.C.), and a European Community Science Project (to H.P.). We thank Fred Hagen and Lawrence Tabak for providing the bovine T1, Darinka Sakac and Philip Dennis for help with the experimental work, and the Arthritis Society for a Research Scientist Award to I.B.


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Bbo, 4-benzoyl-benzoylornithine; Bpa, benzoylphenylalanine; Bzl, benzyl; EDTA, ethylenediamine tetraacetic acid; FAB-MS, fast atom bombardment mass spectrometry; HPLC, high-performance liquid chromatography; NMR, nuclear magnetic resonance; pnp, p-nitrophenyl; PAECs, porcine aortic endothelial cells; PBS, phospho-buffered saline; TNF, tumor necrosis factor; TUNEL, terminal deoxynucleotidyl transferase dUTP nicked end labeling.


    Footnotes
 
1 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
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