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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:1968-1974.)
© 1995 American Heart Association, Inc.

Articles

Lipoproteins Regulate C-Type Natriuretic Peptide Secretion From Cultured Vascular Endothelial Cells

Seigo Sugiyama; Kiyotaka Kugiyama; Toshiyuki Matsumura; Shin-ichi Suga; Hiroshi Itoh; Kazuwa Nakao; Hirofumi Yasue

From the Division of Cardiology, Kumamoto University School of Medicine, Kumamoto (S.S., K.K., T.M., H.Y.), and Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine, Kyoto (S-i.S., H.I., K.N.), Japan.

Correspondence to Kiyotaka Kugiyama, MD, Division of Cardiology, Kumamoto University School of Medicine, Honjo 1-1-1, Kumamoto 860, Japan.

	Abstract

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Abstract We have shown that oxidized low-density lipoprotein (Ox-LDL) modulates various endothelial cell (EC) functions. C-type natriuretic peptide (CNP), the third member of the natriuretic peptide family to be discovered, is secreted from peripheral vascular ECs and regulates body fluid homeostasis, vascular tone, and vascular growth. This study was designed to investigate the effects of lipoproteins on CNP secretion from cultured ECs. Treatment of bovine carotid ECs with Ox-LDL and its extracted lipids resulted in a concentration-dependent suppression of the spontaneous and transforming growth factor-ß1–stimulated secretion of CNP. Native LDL, its extracted lipids, and acetylated LDL were inactive. Ox-LDL depleted of its amphiphilic lipids, which was prepared by incubation with defatted albumin, lost its suppressive effect on CNP secretion. 7-Ketocholesterol, one of the amphiphilic lipids in Ox-LDL that is transferable from Ox-LDL to defatted albumin, suppressed CNP secretion by ECs, thus mimicking the effect of Ox-LDL. Coincubation with high-density lipoprotein (HDL), which alone had no effect on CNP release, significantly prevented Ox-LDL–induced inhibition of CNP secretion by ECs. Analysis by thin-layer chromatography demonstrated that oxysterols, including 7-ketocholesterol, in Ox-LDL were transferred from Ox-LDL to HDL during coincubation of these two lipoproteins. These results indicate that Ox-LDL suppresses CNP secretion from ECs by 7-ketocholesterol or other transferable hydrophilic lipids in Ox-LDL, and the suppressive effect of Ox-LDL is reversed by HDL. Lipoproteins thus may regulate CNP secretion from the endothelium of atherosclerotic arteries.

Key Words: C-type natriuretic peptide • endothelial cells • 7-ketocholesterol • oxidized LDL • oxysterols

	Introduction

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Atherosclerosis and hypercholesterolemia have been implicated in the pathogenesis of vasospasm and many other cardiovascular diseases. It is well known that atherosclerosis is associated with alterations in various endothelial functions^{1 2} and that ECs regulate vascular tone, thrombogenesis, leukocyte adhesion, and smooth muscle cell growth by releasing several factors.^{3 4} Ox-LDL, which is abundant in atherosclerotic arterial walls,⁵ has also been shown to cause alterations in various endothelial functions^{6 7} and could play an important role in atherogenesis.⁸ On the other hand, epidemiological studies have shown an inverse relation between the serum HDL level and the incidence of cardiovascular diseases.⁹ Thus, HDL has come to be considered an antiatherogenic lipoprotein, but its precise antiatherogenic mechanism(s) remain(s) unknown. Recent reports show that HDL can reverse the effects of Ox-LDL in monocytes/macrophages¹⁰ and ECs,¹¹ which may be one mechanism for the antiatherogenic action of HDL.

CNP, the third member of the natriuretic peptide family to be discovered, was first isolated from porcine brain,¹² and we and others have demonstrated that CNP is synthesized in peripheral vascular ECs¹³ and, as a paracrine factor, regulates vascular smooth muscle tone¹⁴ and vascular growth¹⁵ . Furthermore, we have shown that a specific receptor of CNP, atrial natriuretic peptide B (ie, particulate guanylate cyclase B) receptor, exists in the human vascular wall¹⁶ and smooth muscle cells,¹⁷ suggesting the existence of a vascular natriuretic peptide system¹⁶ that could counteract the vascular renin-angiotensin system. Therefore, CNP may play some role and could participate in vascular remodeling,¹⁸ which contributes to the pathophysiology of atherosclerosis and other cardiovascular diseases. Recently, we have demonstrated that circulating CNP can be detected in human plasma and is markedly elevated during septic shock¹⁹ in humans and that endothelial secretion of CNP is regulated by several cytokines, such as TGF-ß1, interleukin-1, interleukin-1ß, and tumor necrosis factor–.²⁰ However, the regulatory mechanisms of CNP secretion from the endothelium of atherosclerotic arteries remain unknown. It is possible that Ox-LDL influences CNP release from the endothelium of atherosclerotic vessels. Therefore, the present study was designed to determine whether Ox-LDL modulates the secretion of CNP-LI from cultured bovine ECs and to define which component(s) in Ox-LDL may affect CNP secretion. Furthermore, we examined whether HDL could reverse the effects of Ox-LDL on CNP secretion.

	Methods

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Cell Culture
ECs were isolated from adult bovine carotid artery by scraping the intimal surface with a scalpel and cultured in DMEM with 10% (vol/vol) FCS, 100 U/mL penicillin, and 100 µg/mL streptomycin at 37°C in a humidified atmosphere of 95% air–5% CO_2.¹³ ECs at passages 19 through 25 were used in the present study. Confluent cultures of bovine ECs exhibited the typical "cobblestone" morphology.

Preparation of Conditioned Medium
ECs were grown to confluence in 60-mm culture dishes. The cells were washed twice with serum-free DMEM, and the medium was replaced with 2 mL DMEM containing 0.5% (vol/vol) FCS and antibiotics 24 hours before the experiments were begun. Immediately before the experiments were started, the medium was replaced with fresh DMEM containing 0.5% (vol/vol) FCS. Thereafter, lipoprotein or lipid preparations were added to the culture medium, and the cells were incubated for the indicated times. In some experiments, ECs were incubated with Ox-LDL and HDL. After incubation, the conditioned medium was collected and centrifuged at 600g for 10 minutes to remove cell debris. The supernatants were stored at -80°C until assay. The number of cells was counted three times with a hemocytometer after the ECs were detached by treatment with 0.05% (vol/vol) trypsin–EDTA.²¹

Lipoprotein and Lipid Preparations
LDL (d=1.019 to 1.063 g/mL) and HDL (d=1.063 to 1.210 g/mL) were isolated by sequential ultracentrifugation from pooled, fresh, normal human plasma treated with EDTA (1 mg/mL). Ox-LDL was prepared by incubation of N-LDL (100 µg protein per milliliter) with Cu²⁺ (5 µmol/L) in PBS under sterile conditions at 37°C for 24 hours as reported.^{7 21 22} Thiobarbituric acid–reactive substances in the incubation mixture averaged 4.8±0.4 nmol malondialdehyde equivalents per milliliter of incubation mixture. The electrophoretic mobility of Ox-LDL relative to that of N-LDL was 3.1±0.3. Ox-LDL was reisolated from the incubation mixture by ultracentrifugation (d=1.21 g/mL) for 24 hours at 4°C. Before ultracentrifugation, some aliquots of the incubation mixture that contained Ox-LDL were used for preparing Ox-LDL depleted of hydrophilic lipid or LysoPC.^{21 22} Aliquots of the incubation mixture that contained Ox-LDL were incubated under sterile conditions with a 100-fold excess of defatted albumin (0.1 mg LDL protein per 10 mg albumin), an acceptor of hydrophilic lipids, for 3 hours at 37°C. To determine whether LysoPC might be responsible for the biologic action of Ox-LDL, Ox-LDL depleted of LysoPC was prepared by treatment of aliquots of the incubation mixture that contained Ox-LDL with PlB (4 U/mL) for 2 hours at 37°C.^{21 22} After treatment, the modified Ox-LDLs were recovered from the supernatants by ultracentrifugation (d=1.21 g/mL) for 24 hours at 4°C (Alb-Ox-LDL and PlB-Ox-LDL, respectively).^{21 22} At the same time, the incubated albumin with Ox-LDL was also recovered from the infranatant albumin fraction after ultracentrifugation ("treated albumin") as described.^{21 22} During recovery of Alb-Ox-LDL and treated albumin, care was taken to avoid cross-contamination, the lack of which was confirmed by gel electrophoresis. "Control LDL" was prepared by incubation of N-LDL in copper-free incubation mixture; otherwise, control LDL received treatment identical to that for Ox-LDL. In some experiments oxidative modification of LDL was performed by incubation of N-LDL with cultured porcine ECs in HAM's F-10 medium, yielding EC-LDL, as in our previous reports.^{7 21} EC-LDL without ultracentrifugation and dialysis (which contained 25±5 nmol malondialdehyde equivalents per milligram of LDL protein) was used directly in the bioassay experiments. Acetylation of LDL was performed by the method of Basu et al.²³ N-LDL and HDL from storage and the variously modified LDLs were extensively dialyzed against PBS containing 20 µmol/L BHT and 50 µmol/L EDTA for 24 hours under an N₂ stream at 4°C just before use in the bioassay experiments. Some lipid peroxides and hydrophilic products in Ox-LDL may be lost during dialysis, which is designed to remove Cu²⁺ and potassium bromide. The extent of LDL oxidation has been reported to be variable, although it was very reproducible in our experiments. Therefore, the effects of Alb-Ox-LDL and PlB-Ox-LDL were compared with those from the same stock of Ox-LDL used for preparation of Alb-Ox-LDL and PlB-Ox-LDL. Lipoprotein-free incubation mixtures that were subjected to the same preparative manipulations as the lipoprotein-containing mixtures were used as controls. Lipids were extracted from N-LDL, Ox-LDL, treated albumin, and "control albumin" (albumin recovered from the lipoprotein-free incubation mixture containing defatted albumin and subjected to the same preparative manipulation as treated albumin; the former served as a control) with chloroform-methanol (2:1, vol/vol)²⁴ and dried under N₂. After dispersion in PBS by sonication just before use, lipid extracts were used for the bioassay experiments. 7-Ketocholesterol was dissolved in ethanol, the total volume of which (as a drug vehicle in the final solution) was less than 0.1% (vol/vol). The medium containing 7-ketocholesterol was sonicated just before use. Cholesterol content was measured by a calorimetric procedure, and protein content was determined by the method of Lowry et al,²⁵ with BSA as the standard. Endotoxin levels in the LDL preparations and lipids were <10 pg/100 µg LDL protein, as measured by the chromogenic Limulus test.^{21 22}

Radioimmunoassay for CNP
The radioimmunoassay for CNP was performed as reported.^{13 19} In brief, 50 µL of sample or standard CNP, 50 µL of antibody against CNP, and 100 µL of assay buffer were incubated for 24 hours at 4°C. After incubation 50 µL ¹²⁵I–Tyr^o-CNP (10 000 cpm) was added, and incubation proceeded for another 10 hours at 4°C. Bound and free ligands were separated by the dextran coated–charcoal method. The sensitivity of this method is 0.3 fmol per tube, and cross-reactivities with –atrial natriuretic peptide, rat brain natriuretic peptide, and CNP-53 were 0.2%, <0.01%, and 20%, respectively, on a molar basis.^{13 19} Lipoproteins and lipids in the assay tubes did not affect the radioimmunoassay system.

Analysis of Lipids in Ox-LDL and HDL
Ox-LDL (10 mg protein per milliliter) was incubated with HDL (10 mg protein per milliliter) in PBS containing EDTA (1 mmol/L) at 37°C for 2 hours. After incubation, this mixture was applied to a Sephacryl S-300 column and eluted by gel filtration chromatography with 1 mmol/L PBS-EDTA–20 µmol/L BHT.²⁶ The protein concentration in each elution fraction was determined by spectrophotometrically monitoring the absorbance at 280 nm (model UV-265FW, Shimadzu). Ox-LDL was eluted and reisolated from the first absorbance peak and HDL from the second peak. There was no cross-contamination between Ox-LDL and HDL, as confirmed by SDS-PAGE. Lipids were extracted from each lipoprotein (200 µg protein) with chloroform-methanol²⁴ and analyzed by TLC on Whatman K6 250-µm silica-gel G plates that were developed in hexane-acetone–acetic acid (80:20:1, vol/vol/vol). Spots were visualized by spraying the plates with 5% (vol/vol) aqueous sulfuric acid, 5% (vol/vol) acetic acid, and 0.05% (wt/vol) FeCl₂ and heating at 155°C for 10 minutes.²⁷

Determination of Cell Viability
The cytotoxicity of lipoproteins or lipids was assessed by the trypan blue exclusion test and counts of the remaining adherent cells. To accomplish this, at the end of incubation ECs were washed twice with PBS and then stained for 2 minutes with 0.25% (vol/vol) trypan blue dissolved in PBS. After the culture dishes containing EC monolayers were washed twice with PBS, the number of nonviable cells (ie, those cells that failed to exclude the dye) was determined by counting 10 000 cells per dish by phase-contrast microscopy (x200). In addition, after incubation and the two washes with PBS mentioned above, the remaining adherent ECs were detached by trypsinization (0.05% [vol/vol] trypsin–EDTA)²¹ and counted in triplicate with a hemocytometer. The percentage of trypan blue–negative ECs and the number of remaining adherent ECs were recorded.

Drugs
All cell culture reagents were obtained from GIBCO. PlB (P-8914) and other chemicals were from Sigma Chemical Co. Sephacryl S-300 was from Pharmacia Biotech AB, and TGF-ß1 was from R&D Systems Inc. 7-Ketocholesterol and other oxysterols were from Steraloids Inc.

Data Analysis
Results are expressed as mean±SEM. Statistical evaluation of the data was performed by Student's t test for unpaired observations. When more than two groups were compared an ANOVA was used. Values were considered to be statistically different at P<.05.

	Results

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Basal and TGF-ß1–Stimulated CNP-LI Secretion
As shown in Fig 1, the levels of CNP-LI released spontaneously into EC-conditioned media increased in a time-dependent manner for as long as 24 hours. The basal level of CNP-LI in the EC-conditioned medium was 17.3±0.8 fmol/10⁶ cells per 24 hours. TGF-ß1 (100 pmol/L) significantly increased the CNP-LI level in the conditioned medium to 1380±98 fmol/10⁶ cells per 24 hours (data not shown).

View larger version (15K): [in this window] [in a new window]   Figure 1. Graph showing time course of CNP-LI levels in conditioned media of cultured ECs. ECs were incubated for as long as 24 hours with or without LDL (100 µg protein per milliliter). Each point represents the mean±SEM of six independent experiments. *Significant difference at P<.01 vs control (incubated without LDL).

Effects of LDLs and Their Associated Lipids on CNP-LI Secretion
Incubation of ECs with Ox-LDL (100 µg protein per milliliter) but not with N-LDL (100 µg protein per milliliter) significantly suppressed spontaneous CNP-LI levels in conditioned media from 6 to 24 hours (Fig 1). Ox-LDL but not N-LDL significantly decreased the spontaneous and TGF-ß1–stimulated CNP-LI level in conditioned media in a dose-dependent manner (Figs 2 and 3). As shown in Fig 4, Ox-LDL (100 µg protein per milliliter) and its extracted lipid (50 µg cholesterol per milliliter) significantly suppressed the CNP-LI level in conditioned media, whereas N-LDL (100 µg protein per milliliter) and its extracted lipid (50 µg cholesterol per milliliter) did not. Lipid extracted from Ox-LDL also suppressed spontaneous CNP-LI levels in a dose-dependent manner (control: 17.8±1.2 fmol/10⁶ cells per 24 hours; Lipid from Ox-LDL: 50 µg cholesterol per milliliter, 8.2±0.6*; 100 µg cholesterol per milliliter, 6.7±0.5*; 150 µg cholesterol per milliliter, 5.6±0.5*; *P<.01 versus control, n=6 through 9). EC-LDL (100 µg protein per milliliter), which contained much lipid peroxide, exerted effects on CNP-LI secretion that were similar and equivalent to those obtained with reisolated and dialyzed Ox-LDL (Fig 4). To determine whether scavenger receptor–mediated processes were involved in the effects of Ox-LDL on CNP-LI secretion, we incubated ECs with Acetyl-LDL (100 µg protein per milliliter), a ligand for the scavenger receptor. Acetyl-LDL did not affect the CNP-LI level in the conditioned medium (Fig 4).

View larger version (14K): [in this window] [in a new window]   Figure 2. Graph showing the effects of various concentrations of Ox-LDL and N-LDL on spontaneous levels of CNP-LI in conditioned media. ECs were incubated with Ox-LDL or N-LDL for 24 hours. Ox-LDL but not N-LDL suppressed CNP-LI levels in a dose-dependent manner. Each point represents the mean±SEM of six independent experiments. *Significant difference at P<.01 vs N-LDL.

View larger version (15K): [in this window] [in a new window]   Figure 3. Graph showing the effects of various concentrations of Ox-LDL and N-LDL on TGF-ß1–stimulated CNP-LI levels in conditioned media. ECs were incubated with Ox-LDL or N-LDL in the presence of TGF-ß1 (100 pmol/L) for 24 hours. Ox-LDL but not N-LDL suppressed TGF-ß1–stimulated CNP-LI levels in a dose-dependent manner. Each point represents the mean±SEM of six independent experiments. *Significant difference at P<.01 vs N-LDL.

View larger version (43K): [in this window] [in a new window]   Figure 4. Bar graph showing the effects of incubation with lipoprotein or lipid preparations on CNP-LI levels in conditioned media. Control ECs were incubated without any lipoprotein or lipid; other ECs were incubated with lipoprotein (100 µg protein per milliliter) or lipid (50 µg cholesterol per milliliter) for 24 hours. *Significant difference at P<.01 vs control (n=6-9).

Ox-LDL contains a variety of amphiphiles, such as oxysterols, lipid peroxides, and LysoPC. Some of these compounds are transferable from Ox-LDL to defatted albumin, an acceptor for hydrophilic lipids, and the hydrophilic lipids in Ox-LDL may play an important role in endothelial function, as shown in our previous reports.^{7 21 22} To determine whether the transferable hydrophilic lipids were responsible for the effects of Ox-LDL on CNP secretion, we examined the effect of Alb-Ox-LDL that was depleted of transferable hydrophilic lipids. As shown in Fig 5, Alb-Ox-LDL after this treatment lost its inhibitory effect on CNP-LI level in the conditioned medium. The lipid extract from treated albumin, which accepted hydrophilic lipids from Ox-LDL, significantly suppressed the CNP-LI level, thus mimicking the effects of Ox-LDL, whereas lipid extracted from control albumin was inactive. LysoPC is one of the hydrophilic lipids in Ox-LDL and has been shown to modulate various endothelial functions.^{7 21 22} To determine whether LysoPC was responsible for the suppressive effects of Ox-LDL on CNP secretion, we examined the effect on CNP secretion of PlB-Ox-LDL that was depleted of its LysoPC^{21 22} . As shown in Fig 5, PlB-Ox-LDL suppressed the CNP-LI level in the conditioned medium at a magnitude similar to that due to Ox-LDL, suggesting that the active components for CNP suppression exerted by Ox-LDL still remained in PlB-Ox-LDL. These results indicate that the transferable hydrophilic lipids in Ox-LDL rather than its LysoPC are responsible for the inhibitory effects of Ox-LDL on CNP secretion from ECs.

View larger version (38K): [in this window] [in a new window]   Figure 5. Bar graph showing the effects of incubation with treated lipoprotein or lipid preparations on CNP-LI levels in conditioned media. Control ECs were incubated without any lipoprotein or lipid. The dose of treated-albumin lipid is equivalent to 150 µg protein per milliliter of Ox-LDL in the culture medium. ECs were incubated with LDLs (100 µg protein per milliliter), lipids (dose equivalent to 150 µg protein per milliliter of Ox-LDL), or 7-ketocholesterol (50 µmol/L) for 24 hours. *Significant difference at P<.01 vs control (n=6-9).

We have previously demonstrated that some oxysterols, such as 7-ketocholesterol, 25-hydroxycholesterol, 7-hydroxycholesterol, and 7ß-hydroxycholesterol, are generated in Ox-LDL, and some of them are transferable from Ox-LDL to defatted albumin.²¹ Therefore, we examined the effects of oxysterols on CNP secretion. As shown in Figs 5 and 6, incubation of ECs with 7-ketocholesterol (50 µmol/L) significantly decreased the CNP-LI level in the conditioned medium, and 7-ketocholesterol (but not cholesterol) at various concentrations (25 to 75 µmol/L) significantly suppressed CNP-LI levels. However, other oxysterols at 50 µmol/L, namely, 25-hydroxycholesterol, 7-hydroxycholesterol, and 7ß-hydroxycholesterol, did not significantly suppress CNP-LI levels in conditioned media (data not shown). Furthermore, 7-ketocholesterol (50 µmol/L) also suppressed TGF-ß1 (100 pmol/L)–stimulated CNP-LI levels in the conditioned medium (control, 1050±110 fmol/10⁶ cells per 24 hours; 7-ketocholesterol, 315±65 fmol/10⁶ cells per 24 hours; P<.01, n=6). These results indicate that 7-ketocholesterol may be at least partially responsible for the suppressive effects of Ox-LDL on CNP secretion from ECs.

View larger version (15K): [in this window] [in a new window]   Figure 6. Graph showing the effects of various concentrations of cholesterol and 7-ketocholesterol on spontaneous levels of CNP-LI in conditioned media. ECs were incubated with cholesterol or 7-ketocholesterol for 24 hours. 7-Ketocholesterol but not cholesterol at various concentrations (25-75 µmol/L) significantly suppressed CNP-LI levels. Each point represents the mean±SEM of six independent experiments. *Significant difference at P<.01 vs cholesterol.

Effects of HDL
HDL has been known to be antiatherogenic and interact with other lipoproteins.⁹ To determine whether HDL modulated EC secretion of CNP and influenced the Ox-LDL–induced suppression of CNP secretion, ECs were incubated with or without Ox-LDL (100 µg protein per milliliter) in combination with HDL (1.0 to 2.0 mg protein per milliliter). Incubation with HDL alone (1.0 to 2.0 mg protein per milliliter) did not affect CNP-LI levels in the conditioned medium (control: 17.9±1.1 fmol/10⁶ cells per 24 hours; HDL: 1.0 mg protein per milliliter, 17.3±1.2 fmol/10⁶ cells per 24 hours; 1.5 mg protein per milliliter, 17.0±1.0 fmol/10⁶ cells per 24 hours; 2.0 mg protein per milliliter, 17.6±1.3 fmol/10⁶ cells per 24 hours; n=6 through 8). HDL significantly reversed the inhibitory effects of Ox-LDL on CNP secretion in a dose-dependent manner, as shown in Fig 7.

View larger version (40K): [in this window] [in a new window]   Figure 7. Bar graph showing the effects of HDL on Ox-LDL–induced suppression of CNP-LI levels in conditioned media. ECs were incubated with Ox-LDL (100 µg protein per milliliter) in combination with HDL (1.0-2.0 mg protein per milliliter) for 24 hours. HDL significantly reversed the suppressive effects of Ox-LDL on CNP secretion in a dose-dependent manner. Significant difference at *P<.05 and **P<.01 versus Ox-LDL alone (n=6-8).

Analysis of Lipids by TLC
As shown in Fig 8, the mixture of Ox-LDL and HDL in the incubation medium could be clearly separated into Ox-LDL and HDL components by gel filtration chromatography. Ox-LDL was eluted and recovered from the first peak (fraction 20) and HDL from the second peak (fraction 33). After incubation of HDL in EDTA-PBS without Ox-LDL, which served as a control for HDL treated with Ox-LDL (OxLDL–treated HDL), it was recovered by the same gel filtration chromatography procedure as above. The lipids extracted from untreated HDL, Ox-LDL–treated HDL, untreated Ox-LDL, and Ox-LDL treated with HDL (HDL–treated Ox-LDL) were analyzed by TLC. As shown in Fig 9, TLC analysis demonstrated the appearance of "new" bands corresponding to oxysterols in Ox-LDL–treated HDL and reduced the intensity of these same bands in HDL-treated Ox-LDL, suggesting that some oxysterols were transferred from Ox-LDL to HDL. No oxysterol bands were observed in untreated HDL.

View larger version (13K): [in this window] [in a new window]   Figure 8. Redrawn chromatogram showing the separation of Ox-LDL and HDL in the incubation mixture by Sephacryl S-300 gel filtration chromatography. Ox-LDL and HDL (both 10 mg protein) were coincubated in PBS containing EDTA at 37°C for 2 hours. The mixture was applied to a Sephacryl S-300 gel filtration column and eluted with PBS-EDTA-BHT. Ox-LDL was eluted and recovered from the first peak (fraction 20) and HDL from the second peak (fraction 33).

View larger version (64K): [in this window] [in a new window]   Figure 9. TLC for lipid analysis of Ox-LDL, HDL, Ox-LDL treated with HDL, and HDL treated with Ox-LDL. Each lipoprotein was separated and collected by Sephacryl S-300 gel filtration chromatography as shown in Fig 7. Lipids were extracted from each lipoprotein (200 µg protein) and separated on silica-gel G plates. Cholesteryl oleate (CE), triolein (TG), cholesterol (Cho), and 7-ketocholesterol (7-keto) were run as standards (lane 1). Lane 2, Ox-LDL treated without lipoprotein; lane 3, HDL treated without lipoprotein; lane 4, Ox-LDL treated with HDL; lane 5, HDL treated with Ox-LDL. Bands corresponding to oxysterols were located between the starting point and the cholesterol spot. An asterisk indicates the oxysterols transferred from Ox-LDL to HDL after incubation.

Cell Viability
To determine the effects of lipoproteins and lipids on cell viability, ECs were stained with a 0.25% (vol/vol) trypan blue solution,²¹ and the number of remaining adherent ECs was counted after incubation. The number of ECs was not significantly changed (control, 2.6±0.2x10⁶ per dish; Ox-LDL at 100 µg protein per milliliter, 2.4±0.2x10⁶ per dish; and 7-ketocholesterol at 50 µmol/L, 2.4±0.2x10⁶ per dish; n=6 or 7, P=NS), and trypan blue staining showed that no significant EC death occurred after a 24-hour incubation for Ox-LDL at concentrations 100 µg protein per milliliter or for 7-ketocholesterol at concentrations 50 µmol/L (viable cells: Ox-LDL at 100 µg protein per milliliter, 94±3%; 7-ketocholesterol at 50 µmol/L, 93±4%; n=6 or 7).

	Discussion

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The present study demonstrated that treatment of ECs with Ox-LDL but not with N-LDL significantly suppressed spontaneous and TGF-ß1–stimulated secretion of CNP from cultured ECs. The inhibitory effects of Ox-LDL on CNP release were not mediated by scavenger receptor–mediated intracellular signaling, because Acetyl-LDL, a ligand for the scavenger receptor, had no effect on CNP secretion. However, we cannot exclude the possibility that Ox-LDL uptake through the scavenger receptor pathway may play a role in Ox-LDL–induced CNP suppression. LysoPC, which has been shown to contribute to the effects of Ox-LDL on various endothelial functions,^{7 21 22} does not seem to be responsible for the inhibition by Ox-LDL of CNP secretion from ECs, as the suppressive effect of Ox-LDL on CNP release remained in PlB-Ox-LDL that had been depleted of its LysoPC. The inhibition by Ox-LDL of CNP secretion was caused by the transfer of hydrophilic lipids from Ox-LDL, because Alb-Ox-LDL depleted of its transferable lipid was inactive, but the lipid extracted from treated albumin, which accepted the transferable lipid, gained an inhibitory effect on CNP secretion. Recently, we have shown that 7-ketocholesterol is generated in Ox-LDL (50 to 100 µg/mg protein in Ox-LDL but not detectable in N-LDL), can be transferred from Ox-LDL to defatted albumin, and modulates endothelial function.²¹ The present study has shown that 7-ketocholesterol suppresses EC secretion of CNP, thus mimicking the effects of Ox-LDL. In the present study the effective concentration of Ox-LDL for such suppression was >50 to 100 µg protein per milliliter and that of 7-ketocholesterol >25 to 50 µmol/L. The concentration of 7-ketocholesterol in our Ox-LDL was 125±50 µg (312±125 nmol) per milligram protein (n=4) and therefore sufficient to have contributed to the observed effects. These results indicate that 7-ketocholesterol could have participated in the partial suppressive effects of Ox-LDL on CNP secretion from ECs. The oxidation of lipids in LDL also yields a variety of fatty acid peroxides,²⁸ some of which are also capable of being transferred to albumin. The present study showed that EC-LDL, which contained much lipid peroxide, exerted very similar and equivalent effects on CNP secretion when compared with those obtained with reisolated and dialyzed Ox-LDL, which contained smaller amounts of lipid peroxides. Furthermore, the fatty acids (arachidonic and palmitic) and their autoxidation products had no significant inhibitory effect on CNP secretion (data not shown). Thus, it is unlikely that lipid peroxides per se played a major role in the observed effects of Ox-LDL on CNP release, although we cannot completely exclude the potential contribution of such peroxides to the actions of Ox-LDL.

The precise mechanisms of 7-ketocholesterol–induced suppression of CNP secretion from ECs still remain to be determined. Amphiphilic lipids, such as LysoPC and oxysterols, can be transferred in the aqueous phase to accessible membranes or macromolecular acceptors.²⁹ It is possible that 7-ketocholesterol is directly incorporated into the EC surface membrane and modulates endothelial function, as does LysoPC,⁷ on the basis of the fact that amphiphilic lipids (including oxysterols) have been shown to exert various effects on membrane-associated cellular functions after insertion into the cellular surface membrane.^{30 31 32 33} In our previous report,¹³ we demonstrated that CNP is synthesized in ECs and constitutively released from ECs into the culture medium. We suspect that the CNP secretion in this study is also constitutive and that the CNP levels in conditioned media demonstrate CNP synthesis in ECs. Therefore, it seems reasonable that Ox-LDL could inhibit CNP synthesis. However, the possibility that Ox-LDL inhibits CNP secretion at the level of postsynthetic release from ECs cannot be excluded. It may be difficult to obtain conclusive answers to such questions because limited quantities of the basal steady-state level of CNP mRNA can be detected in ECs by Northern blot analysis.¹³ Ox-LDL and certain oxysterols are known to be cytotoxic to vascular cells.^{34 35} It is less likely that the cytotoxic effect is the one mainly responsible for the action of Ox-LDL and 7-ketocholesterol on CNP release, because the present study has shown that Ox-LDL (100 µg protein per milliliter) and 7-ketocholesterol (50 µmol/L) did not cause significant EC death, as we demonstrated previously.²¹ Furthermore, we showed that the inhibitory effect of Ox-LDL on CNP release was significantly reversed by coincubation with HDL. The curtailing effects of HDL on Ox-LDL–induced suppression of CNP secretion were associated with the transfer of oxysterols, including 7-ketocholesterol, from Ox-LDL to HDL, as demonstrated by the present TLC analysis. Therefore, HDL may "absorb" oxysterols that are released from Ox-LDL or possibly, the EC surface membrane,³⁶ resulting in a reduced transfer of oxysterols into the EC surface membrane. This may be one reason why HDL attenuated the inhibitory action of Ox-LDL on CNP secretion shown in this study.

CNP has been shown to be secreted from ECs¹³ and to cause vasodilatation,¹⁴ inhibit vascular smooth muscle cell proliferation,¹⁵ prevent intimal thickening after vascular injury,³⁷ and suppress endothelin release from ECs.³⁸ Thus, CNP secreted from the endothelium and acting as a paracrine factor may play an important role in vascular diseases by regulating vascular smooth muscle tone and growth. It is thought that Ox-LDL can significantly influence the pathogenesis and development of atherosclerosis.⁸ Therefore, Ox-LDL–induced inhibition of CNP secretion may be implicated in the increased vascular contractility and cell proliferation that are commonly observed in atherogenesis.

Some epidemiological studies have shown an inverse relation between serum HDL level and the incidence of cardiovascular disease.⁹ HDL has been considered to be antiatherogenic by promoting reverse cholesterol transport,¹⁰ preventing oxidation of LDL,³⁹ and counteracting the actions of Ox-LDL.¹¹ The finding that HDL can reverse the suppression of CNP release induced by Ox-LDL may thus partially explain the beneficial effects attributed to HDL for protection against atherosclerosis.

In conclusion, Ox-LDL suppresses CNP secretion from cultured ECs due to the action of 7-ketocholesterol or other transferable hydrophilic lipids, and these inhibitory effects of Ox-LDL appear to be reversed by HDL. Thus, lipoproteins may regulate secretion of CNP from the endothelium in atherosclerotic arterial walls.

	Selected Abbreviations and Acronyms

 

Acetyl-LDL = acetylated LDL Alb-Ox-LDL = Ox-LDL treated with albumin CNP = C-type natriuretic peptide CNP-LI = CNP-like immunoreactivity DMEM = Dulbecco's modified Eagle's medium EC(s) = endothelial cell(s) EC-LDL = LDL modified by ECs FCS = fetal calf serum LysoPC = lysophosphatidylcholine N-LDL = native LDL Ox-LDL = oxidized LDL PlB = phospholipase B PlB-Ox-LDL = Ox-LDL treated with phospholipase B SDS-PAGE = SDS–polyacrylamide gel electrophoresis TGF-ß1 = transforming growth factor–ß1 TLC = thin-layer chromatography<\/.>

	Acknowledgments

 
This work was supported in part by a grant-in-aid for Scientific Research on priority areas 03268107, B03454257, and C3670460 from the Ministry of Education, Science and Culture of Japan and by a Smoking Research Foundation Grant for Biomedical Research, Tokyo, Japan. We thank Yoshiko Suginohara, Masakazu Sakai, and Hideki Hakamata for their technical advice.

Received February 5, 1995; accepted June 28, 1995.

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