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(Circulation Research. 2000;86:854.)
© 2000 American Heart Association, Inc.

Integrative Physiology

Phenotypic and Functional Changes in Regenerated Porcine Coronary Endothelial Cells

Increased Uptake of Modified LDL and Reduced Production of NO

Marie-Pierre Fournet-Bourguignon, Maria Castedo-Delrieu, Jean-Pierre Bidouard, Stephane Leonce, Delphine Saboureau, Isabelle Delescluse, Jean-Paul Vilaine, Paul M. Vanhoutte

From the Institut de Recherches Servier, Suresnes, France.

Correspondence to Paul M. Vanhoutte, Institut de Recherches Internationales Servier, 6 Place des Pléiades, 92415 Courbevoie, France. E-mail vanhoutt{at}servier.fr

	Abstract

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Abstract—Porcine coronary arteries with regenerated endothelium exhibit impaired endothelium-dependent relaxations. Experiments were designed to analyze the structural and functional changes occurring in regenerated endothelial cells. Primary cultures from regenerated endothelium contained giant endothelial cells, with an increased number of cells with diameter >14.5 µm, a reduced ability to proliferate, and signs of apoptosis. The uptake of fluorescent acetylated LDL was increased 2-fold in cultures from regenerated endothelium. The increased uptake of acetylated LDL was confirmed ex vivo in injured coronary arteries. In cultures from regenerated endothelium, cGMP production was decreased under basal conditions and during stimulation with serotonin, bradykinin, and A23187. Thus, during regeneration, there is accelerated senescence of endothelial cells accompanied by increased incorporation of modified LDL and reduction of NO production without decrease in endothelial NO synthase expression. These alterations help to explain the altered endothelium-dependent responses 28 days after balloon injury.

Key Words: endothelial dysfunction • modified LDL • cGMP • endothelial NO synthase • senescence

	Introduction

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In porcine coronary arteries, removal of the endothelium in vivo leads to proliferation of vascular smooth muscle. The adjacent endothelial cells migrate and proliferate to reline the vascular wall. Twenty-eight days after injury, coronary arteries with regenerated endothelium exhibit impaired endothelium-dependent relaxations to serotonin or ₂-adrenergic agonists, whereas those to ADP or bradykinin are maintained.^{1 2} This selective dysfunction of the pertussis toxin–sensitive signaling pathway is accompanied by a morphological heterogeneity of the regenerated endothelial cells,³ as is also observed with atherosclerosis and senescence.^{4 5 6} The present study was designed to further compare cells derived from control and regenerated endothelium.

	Materials and Methods

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Denudation
These experiments were carried out in accordance with the guidelines of the French Ministry of Agriculture for the use and care of animals. Large White pigs (18 to 25 kg) were anesthetized by intramuscular injection of tiletamine and zolazepan (15 mg/kg) containing atropine sulfate (50 µg/kg). The animals were intubated and ventilated with a respirator. Part of the left anterior coronary artery was denuded by inflating a balloon catheter.³ Twenty-eight days later, the animals were sedated (intramuscular zoletil) and euthanized by exsanguination.

Histology
Control and previously denuded coronary arteries were immersed in cold physiological salt solution containing (in mmol/L) CaCl₂ 2.5, EDTA 0.016, NaCl 118, NaHCO₃ 24.8, KH₂PO₄ 1.18, KCL 4.7, MgSO₄ 1.2, and glucose 11. A ring of each artery was fixed (4% formaldehyde). Different nonserial cross sections (5 mm in length, 200 mm apart) were prepared from paraffin blocks and stained with hematoxylin-eosin-safran for light microscopy.

Primary Cultures
Endothelial cells from native and injured coronary arteries were cultured as described.³

Cell Size
Light Microscopy
Cells on 24-well plates were fixed (ethanol), stained (Hemacolor reagents; Merck)⁷ and examined with a computerized image-analysis system (Histo Software, Biocom).

Flow Cytometry
Cells were washed with EBSS and treated with trypsin-EDTA to obtain a single-cell suspension. Diameter was analyzed with a flow cytometer (EPICS XL/MCL (Beckman Coulter, Villepinte, France).

DNA Content
DNA content was determined in permeabilized fixed cells with propidium iodide.⁸ Subconfluent cultures were trypsinized, washed, fixed/permeabilized (ice-cold 70% ethanol overnight at -20°C), incubated with 50 µg/mL propidium iodide (Sigma Chemical Co) and 100 µg/mL RNase in isotonic phosphate buffer for 1 hour (20°C), and analyzed by flow cytometry. Cells in the different phases of the cell cycle were measured. Apoptotic cells were identified by reduced staining after loss of DNA (hypodiploid or sub-G₁ peak in a DNA histogram).

Mitochondrial Alterations
Transmembrane mitochondrial potential and oxidative capacity were measured with 3,3'-dihexylocarbocyanine iodide (40 nmol/L) and dihydroethidium (20 µmol/L), respectively (Molecular Probes).^{9 10}

LDL
LDL (d=1.019 to 1.063) from plasma of healthy, normolipidic volunteers was oxidized by exposure to 5 µmol/L CuSO₄ at 37°C for 24 hours. Oxidation was arrested with 200 µmol/L EDTA. Oxidized LDL was dialyzed against PBS-200 µmol/L EDTA at 4°C. The protein concentration was determined.¹¹ The amount of thiobarbituric acid–reactive lipid peroxides was checked using a colorimetric assay and malondialdehyde as a standard. Thiobarbituric acid reactivity of 24-hour–oxidized LDL after dialysis was 5.1±1.1 nmol of malondialdehyde/mg of protein (starting LDL, 0.8±0.1 nmol/mg).

Uptake of Fluorescent LDL
Confluent monolayers (passage 1) were incubated with 5 µg/mL fluorescence-conjugated acetylated LDL labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (Dil; 37°C in a CO₂ incubator). Then, cells were trypsinized to obtain a single-cell suspension. The incorporation of Dil-LDL was quantified by flow cytometry. Cells were incubated with Dil-acetylated LDL for 4 hours or with Dil-native LDL for 5 hours. The concentration of acetylated LDL needed for half-maximal incorporation (EC₅₀) was determined using Micropharm software (LOGINSERM).

For competition studies, cells were incubated with Dil-acetylated LDL (1 µg/mL) and 50 µg/mL of native LDL, modified LDL, or polyinosinic acid (2 hours at 37°C). Coronary arteries with control or regenerated endothelium were incubated ex vivo with Dil-acetylated LDL (5 µg/mL, for 4 hours, at 37°C in a CO₂ incubator). For microscopy, the coronary arteries were fixed with 4% formaldehyde and frozen. To perform flow cytometry, cells were harvested and trypsinized.

cGMP
Endothelial cells seeded in 96-well plates were placed in HEPES-Tris buffer (20 mmol/L) containing (in mmol/L) NaCl 140, KCl 5.4, CaCl₂ 2.4, and MgSO₄ 0.7, including 10 µmol/L indomethacin and 100 µmol/L isobutyl methylxanthine. cGMP production was determined under basal conditions and after 1 minute of stimulation with agonists under agitation (37°C). Agonists were the following (in µmol/L): bradykinin 0.1, serotonin 1, and A23187 1. The level of cGMP was measured by radioimmunoassay (Amersham, Amerlex method). Cellular density in each well was defined (Hemacolor method). Results are expressed as fmol per million cells. Cells from control and regenerated endothelium were studied in parallel.

NO Synthase (NOS) Activity
After trypsinization, cells were collected in buffer containing 50 mmol/L Tris base, EGTA (100 µmol/L), DTT (100 µmol/L), and a mixture of protease inhibitors, pH 7.4 at 4°C. To obtain cell membranes, cells were sonicated (20 strokes) on ice and centrifuged (120g for 10 minutes). The supernatant was centrifuged at 100 000g (60 minutes). The protein content of the pellet was measured.¹¹

The assay was conducted at 37°C for 1 hour under agitation in a reaction mixture of (in µmol/L) [¹⁴C]arginine (11 GBq/mmol) 1.7, calmodulin 1, flavin adenine dinucleotide and flavin mononucleotide 1, and tetrahydrobiopterin 50, as well as (in mmol/L) DTT 1, NADPH 1, and CaCl₂ 2, with 100 µg of cell membrane protein. The assay was terminated with HClO₄ (11.6 mol/L; 4°C). The mixture was centrifuged (1000g, 10 minutes). The supernatant was analyzed by HPLC. [¹⁴C]Arginine, [¹⁴C]ornithine, and [¹⁴C]citrulline were separated using a 150x4.6–mm Hypersil BDS C18 column isocratically eluted at 1 mL/min with 4 mL heptafluorobutyric acid, 125 mL acetonitrile, and 900 mL water. The radioactivity of the effluent was recorded. NOS activity was expressed as citrulline formed per minute and per mg of protein.

Endothelial NOS (eNOS) Expression
For immunocytochemistry staining of eNOS, monoclonal antibodies (20 µg/mL) recognizing particulate eNOS were applied (room temperature) for 1 hour. Two types of secondary antibodies were used, Alexa 488 rabbit anti-mouse (0.2 µg/mL) and ¹²⁵I-labeled sheep anti-mouse (0.02 µg/mL). All washes were performed with PBS–1% BSA.

Statistical Analysis
Data are expressed as mean±SEM; n refers to the number of animals. Statistical evaluation was performed by paired Student t test. Multiple comparisons of kinetic curves are based on the Newman-Keuls test. Differences were considered to be significant at P<0.05.

An expanded Materials and Methods section is available online at http://www.circresaha.org.

	Results

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Histology
Twenty-eight days after balloon injury, porcine coronary arteries were covered with regenerated endothelium and exhibited myointimal thickening (Figure 1).

View larger version (148K): [in this window] [in a new window]   Figure 1. A through D, Light microscopy of porcine coronary arteries. Shown are cross sections of control arteries (A) and arteries denuded 28 days previously (B) (hematoxylin-eosin, x40). C and D, Larger magnification of panels A and B, respectively, showing the presence of endothelial cells (x200). E and F, Phase-contrast pictures of primary cultures of native (E) and regenerated (F) endothelial cells. Colorimetric labeling using Hemacolor reagents of fixed and permeabilized endothelial cells (x400).

Cell Size
In primary culture, control cells were mostly uniform in size and displayed a cobblestone pattern (Figure 1). Regenerated cells were heterogenous with the presence of sparse giant cells. Such cells incorporated Dil-acetylated LDLs and were not stained by antibodies against anti-smooth muscle -actin (not shown). Flow cytometry (Figure 2) revealed that in control and regenerated cultures, the majority of endothelial cells had a diameter of 11 µm. Cells with a diameter >14.5 µm were significantly more numerous in cultures from regenerated than those from control endothelium. Counting of giant cells confirmed a significant increase in the number of enlarged cells after denudation (4.17±1.69 and 9.88±3.3 per 1000 cells for control and regenerated cells, respectively).

View larger version (19K): [in this window] [in a new window]   Figure 2. Flow cytometric analysis of cell size in primary culture. Top, Histogram of control endothelial cells. Bottom, Quantification of cells with a diameter >14.5 µm (n=15). Data are mean±SEM. *Statistically significant difference (P<0.001).

Proliferation
The percentage of cells in S and G₂/M phases was significantly lower in cultures from regenerated than control endothelium (11.6±0.9 versus 18.9±2.7%, respectively). The reduction in proliferating cells was associated with a significant increase in the sub-G₁ population relative to apoptotic cells in cultures from regenerated endothelium (2.6±0.7% versus 1.4±0.4%).

Mitochondrial Dysfunction
An increased oxidation of dihydroethidium associated with a decreased mitochondrial transmembrane potential was revealed by the biparametric analysis of cultures from regenerated cells. A greater number of cells with altered mitochondrial function was observed in regenerated endothelial cells (Figure 3).

View larger version (36K): [in this window] [in a new window]   Figure 3. Apoptosis-associated mitochondrial dysfunction in cultured endothelial cells. Top, Typical biparametric analysis of mitochondrial transmembrane potential (DiOC6[3]) and ability to oxidize dihydroethidium (HE) related to the production of reactive oxygen species in cultured regenerated endothelial cells. Cells were incubated with 3,3'-dihexylocarbocyanine iodide [DiOC(3)] and dihydroethidium followed by flow cytometric analysis. Bottom, Percentage of cells with reduced mitochondrial transmembrane potential and producing reactive oxygen species (n=13). Data are mean±SEM. *Statistically significant differences (P<0.05). A.U. indicates arbitrary units.

Acetylated LDL
Cultured endothelial cells from control and regenerated endothelium incorporated fluorescent acetylated LDL (Figure 4). Smooth muscle cells remained unstained under the same conditions (not shown). The incorporation of acetylated LDL was time and concentration dependent (Figure 5). It was significantly lower for control than for regenerated cells (Figure 5). The EC₅₀ was similar for the 2 populations of cells (8 to 10 µg/mL). No significant difference in native LDL incorporation was detected between control and regenerated cells (Figure 5).

View larger version (28K): [in this window] [in a new window]   Figure 4. Uptake of acetylated LDL in cultured endothelial cells (passage 1) incubated with 5 µg/mL Dil-acetylated LDL. Top, Fluorescence microscopy of control endothelial cells (x200) incubated for 4 hours. Middle, Representative flow cytometric histogram of Dil fluorescence. Bottom, Incorporation of Dil-acetylated LDL by cells from control and regenerated endothelium analyzed by flow cytometry (n=4). Data are mean fluorescence intensity±SEM. *Statistically significant difference (P<0.01). Mean fluorescence intensity of control and regenerated cells incubated without fluorescent acetylated LDL was 0.44±0.05 and 0.57±0.1 arbitrary units (A.U.), respectively.

View larger version (17K): [in this window] [in a new window]   Figure 5. Top and Middle, Concentration dependence of Dil-acetylated or Dil-native LDL uptake in endothelial cells (n=4). Data are mean fluorescence intensity±SEM. *Statistically significant difference between control and regenerated cells from the first concentration of Dil-acetylated LDL (Ac-LDL; P<0.05). Bottom, Dil-acetylated LDL uptake in cells from control and regenerated endothelium. Cultured cells were incubated in the presence of 1 µg/mL Dil-acetylated LDL with or without 50 µg/mL oxidized LDL. Data are mean±SEM (n=5). *Statistically significant difference of Dil-acetylated LDL uptake (P<0.05).

Addition of a 50-fold excess of native LDL did not abolish the uptake of Dil-acetylated LDL, whereas nonlabeled acetylated LDL or polyinosinic acid had the same effect (95% and 99%, respectively). The nonspecific staining was comparable between control and regenerated cells (3.6±0.9 and 4.8±1.6%, respectively).

An excess of oxidized LDL partially inhibited acetylated LDL incorporation in control and regenerated cells. The remaining acetylated LDL uptake after incubation with oxidized LDL was identical in cultures from control and regenerated endothelium. The increased uptake of acetylated LDL observed in cultures from regenerated endothelium was abolished by oxidized LDL.

Incorporation of Acetylated LDL Ex Vivo
Sections of artery with regenerated (Figure 6) or native (not shown) endothelium demonstrated incorporation of acetylated LDL in endothelial cells, whereas the underlying smooth muscle cells remained unstained. Flow cytometry revealed that regenerated endothelium exhibited a significantly greater incorporation of acetylated LDL than of native endothelium (Figure 6).

View larger version (36K): [in this window] [in a new window]   Figure 6. Incorporation of Dil-acetylated LDL in coronary arteries ex vivo. Top, Photomicrograph of cryostat section of coronary artery with regenerated endothelium. Coronary artery was incubated with 5 µg/mL Dil-acetylated LDL in culture medium for 4 hours. Bottom, Flow cytometric analysis of Dil-acetylated LDL incorporated by endothelial cells. Data are mean fluorescence intensity±SEM (n=5). *Statistically significant difference between control and regenerated cells (P<0.01). A.U. indicates arbitrary units.

cGMP
N-Nitro-L arginine (NLA; 10 µmol/L) inhibited basal and bradykinin-induced cGMP production in endothelial cells (basal, 2273±258, and with NLA, 587±64 fmol/million cells; stimulated, 4360±134, and with NLA, 627±127 fmol/million cells). Oxadiazoloquinoxalin (ODQ, 1 µmol/L), a soluble guanylate cyclase inhibitor, also inhibited basal and bradykinin-stimulated production of cGMP in endothelial cells (basal, 1203±328, and with ODQ, 458±69 fmol/million cells; stimulated, 4095±1560, and with ODQ, 638±128 fmol/million cells; n=6). The basal cGMP production was decreased significantly (by 55%) in cultures from regenerated endothelium. cGMP production induced by bradykinin (0.1 µmol/L), serotonin (1 µmol/L), and calcium ionophore (A23187, 1 µmol/L) was also reduced (Table).

View this table: [in this window] [in a new window]   Table 1. Effect of Bradykinin, Serotonin, and Calcium Ionophore on cGMP Production in Cultures From Control and Regenerated Endothelium

NOS Activity
In cell membranes from both control and regenerated endothelium, the citrulline production was abolished by LNA (10 µmol/L) without formation of ornithine (Figure 7). The NOS activity (V_max) and the Michaelis constant (K_m) of membranes from native endothelial cells were 1.12±0.10 pmol/minxmg protein^–1 and 0.61± 0.06 µmol/L (n=4), respectively. The basal NOS activity of membranes from regenerated endothelial cells was significantly lower than that measured in membranes of native endothelial cells (Figure 7).

View larger version (21K): [in this window] [in a new window]   Figure 7. NOS activity of endothelial cell membranes from control and regenerated endothelium of porcine coronary arteries. A, Representative HPLC chromatograms. B and C, Chromatograms of a sample containing the oxidation products of [14C]arginine induced by the presence of native endothelial cell membranes (NOS) either in the absence (B) or in the presence (C) of nitro-L-arginine (10 µmol/L). eNOS activity is expressed as citrulline formed per minute and per mg of protein. Data are mean±SEM of triplicate measurements (n=6). *Statistically significant difference (P<0.05).

eNOS Expression
The nonspecific fluorescence represented <14% whatever the origin of the cells, indicating the specificity of the staining. The fluorescence values for control and regenerated endothelial cells were not different (16.5±1.1 and 16.8±1.2 arbitrary units; n=4). The flow cytometric biparametric analysis, comparing the intensity of the fluorescence as a function of cell size, did not show differences in eNOS expression between normal and large endothelial cells.

Likewise, no significant difference in eNOS expression was found between control and regenerated endothelial cells using ¹²⁵I-labeled secondary antibodies (94 090±19 541 and 79 073±14 555 cpm/million cells, respectively; n=9).

	Discussion

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In primary culture, cells from regenerated endothelium were morphologically more heterogenous than those from control endothelium.³ Quantitative analysis of endothelial cell size variability revealed a significant increase in the number of large and giant cells. These atypical cells were characterized as endothelial cells, because they stained positively with von Willebrand factor antibody, incorporated Dil-acetylated LDLs, and did not stain with monoclonal antibodies specific for smooth muscle cells. Morphological similarities exist between cells from porcine regenerated endothelium and the giant cells contained in the endothelium taken from elderly patients,^{4 12} atheromatous plaques,^{4 5} or regions submitted to increased hemodynamic stress. Hence, even if the factor or factors contributing to their formation are not necessarily the same, the accumulation of enlarged endothelial cells represents a marker of injured endothelium.

The present findings suggest that cells from regenerated endothelium have a reduced capacity to proliferate. In vitro, increase in cell volume and decline in proliferative capacity characterize endothelium that becomes senescent after a large number of subcultures.⁶ Hence, the numerous endothelial cell doublings that the cells undergo in vivo to repopulate the luminal surface of the artery after balloon injury could be sufficient to induce an altered phenotype of cells. In mammals, cells damaged by age are eliminated by apoptosis. The presence of apoptotic events in control and regenerated endothelium was evaluated by measuring partial loss of nuclear DNA content⁸ and mitochondrial dysfunction that precedes nuclear DNA alteration.^{9 13 14 15} Apoptotic cells sequentially exhibit a reduction in mitochondrial transmembrane potential and then an increase in production of reactive oxygen species. In the present study, the number of cells with nuclear and mitochondrial alterations increased in regenerated compared with control cultures. This decrease in mitochondrial transmembrane potential is often associated with an increased ability of cells from regenerated endothelium to oxidize dihydroethidium, indicating a modification of their redox homeostasis, which could involve an increased production of reactive oxygen species. Human senescent endothelial cells maintained in culture for up to 5 passages display a greater rate of apoptosis than younger cells.¹⁶ Thus, in the present study, the increased number of apoptotic cells in regenerated cultures may be another sign of senescence. This tendency to increased apoptosis associated with a reduced proliferative potential may alter the integrity of the regenerated endothelial lining in the arterial wall. This could contribute to the progression of the impairment of endothelium-dependent relaxation after balloon injury.^{2 17}

A major finding of the present study is that all cells from regenerated endothelium in porcine coronary arteries exhibit an increased uptake of modified LDL. The global shift in fluorescence intensity of cells from regenerated endothelium demonstrates that both normal and enlarged cells present an increased ability to incorporate the modified LDL. In parallel, ex vivo arteries with regenerated endothelium exhibited a greater ability to incorporate modified LDL than those with native endothelium. Thus, the increased uptake of modified LDL was not due to the culture conditions but to an alteration of the phenotype of cells during the regeneration process.

The incorporation of acetylated LDL was saturable, with an identical apparent EC₅₀ in both types of cells. The maximal uptake was 2-fold higher in cultures from regenerated than those from native endothelium. The remaining nonspecific staining was comparable, demonstrating that the greater fluorescence intensity of regenerated cells is not due to a higher nonspecific staining. These findings suggest an increase in acetylated LDL (scavenger) receptor density in cells from denuded blood vessels. These receptors are present constitutively on endothelial cells and exhibit characteristic ligand specificity different from receptors for native LDL.^{18 19} The modifications of LDL that convert it into a high-affinity ligand for the scavenger receptors include acetylation and oxidation. These binding sites share the property of being completely inhibited by polyinosinic acid but not by native LDL.^{18 19} This is confirmed by the competition experiments performed in the present study in cells from both control and regenerated endothelium. Only a partial competition of acetylated LDL uptake by oxidized LDL was observed. Nonreciprocal cross-competition between modified lipoproteins does not necessarily imply the presence of receptors specific only for acetylated LDL rather than receptors that recognize acetylated and oxidized LDL.¹⁸ Different binding sites on the same receptor or the extent of oxidation of the modified LDL could be involved.^{20 21 22} The increased degree of competition of acetylated LDL uptake with oxidized LDL in cells from regenerated endothelium demonstrates an increased capacity of such cells to take up oxidized LDL through a scavenger receptor that recognizes both oxidized and acetylated LDL.

As in macrophages,²³ the uptake of acetylated LDL is increased in endothelial cells from older rats.²⁴ In bovine aortic endothelial cells, growth cessation acts as a signal for upregulation of scavenger receptor activity.²⁵ Certain inflammatory cytokines upregulate the expression of receptors for modified LDL in macrophages.²⁶ In the present study, the persisting increased uptake of acetylated LDL in cells from regenerated endothelium separated from components of regenerated lesions implies an intrinsic (genetic) modification of these cells. The numerous cell doublings could be implicated, given that an increased uptake of acetylated LDL has been observed after several passages in vitro of endothelial cells from control porcine coronary arteries (data not shown).

Arteries exposed to modified LDL exhibit impaired endothelium-dependent responses, a phenomenon closely resembling that observed in atherosclerotic arteries.²⁷ Oxidized LDL or its lipid constituents induce a selective alteration of endothelium-dependent relaxations similar to that observed 28 days after denudation.^{28 29 30 31 32} Transendothelial cholesterol transport takes place at caveolae, where endothelial signal transductions, such as G proteins and NOS, are situated.³³ This colocalization in the plasma membrane may facilitate the interaction of these transduction signals. A second major finding was that the production of NO is reduced in cultures from regenerated endothelium. Indeed, the formation of NO measured as increases in intracellular cGMP under basal conditions or that induced by various agonists was decreased. Likewise, the activity of eNOS, estimated by measuring the citrulline formed during the oxidation of arginine, demonstrated that indeed the enzyme activity is decreased in cultures from regenerated endothelium, which then explains a reduced production of cGMP. However, this abnormal enzyme activity cannot be explained by a decrease of eNOS expression in cultures from previously injured coronary arteries.

In arteries with regenerated endothelium, the relaxation in response to serotonin is impaired severely, whereas that due to bradykinin is mildly to moderately reduced and that due to the calcium ionophore remains unchanged^{2 3} ; this appears to be related to the ability of these agonists to stimulate the production of cGMP in the regenerated endothelium. Contrary to the relaxations induced by both bradykinin and the calcium ionophore for which the decreased production of NO can be compensated by the release of endothelium-derived hyperpolarizing factor,^{34 35} serotonin exclusively stimulated NO production and so fails to induce relaxation of coronary arteries with regenerated endothelium. The present findings permit us to propose an explanation for the endothelial dysfunction in which the pertussis toxin–sensitive G protein pathway is altered first.^{31 36} After balloon injury of the vascular wall, the endothelial cells that repopulate the denuded area exhibit characteristics of senescent cells with an increase in the uptake of modified LDL. It is tempting to propose that this phenomenon modifies the redox homeostasis of the endothelial cells and alters the function of G proteins and of the NOS involved in the endothelium-dependent relaxations.

	Acknowledgments

 
Funding for this research was provided by Servier Discovery Research.

Received January 26, 2000; accepted February 16, 2000.

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