This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Morishita, R. Articles by Ogihara, T. Search for Related Content PubMed PubMed Citation Articles by Morishita, R. Articles by Ogihara, T.

(Hypertension. 1998;32:215-222.)
© 1998 American Heart Association, Inc.

Scientific Contributions

Conditioned Medium From HepG2 Cells Transfected With Human Apolipoprotein(a) Gene Stimulates Growth of Human Vascular Smooth Muscle Cells

Effects of Overexpression of Human Apolipoprotein(a) Gene

Ryuichi Morishita; Shingo Yamada; Jitsuo Higaki; Naruya Tomita; Iwao Kida; Motokuni Aoki; Atsushi Moriguchi; Shin-ichiro Hayashi; Ikunosuke Sakurabayashi; Yasufumi Kaneda; ; Toshio Ogihara

From the Department of Geriatric Medicine (R.M., J.H., N.T., I.K., M.A., A.M., S.H., T.O.) and the Institute for Molecular and Cellular Biology (Y.K.), Osaka University Medical School, Suita; Shinotest Co, Tokyo (S.Y.); and the Ohmiya Medical Center, Jichi Medical School, Omiya (I.S.), Japan.

Correspondence to Toshio Ogihara, MD, PhD, Department of Geriatric Medicine, Osaka University Medical School, Suita 565, Japan.

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Abstract—Lipoprotein(a) [Lp(a)] is well known to stimulate growth of vascular smooth muscle cells (VSMCs), resulting in atherosclerosis. Its mechanism is postulated to be decreased in active transforming growth factor (TGF)-ß. However, the exact mechanisms and cellular processing from apolipoprotein(a) [apo(a)] to Lp(a) have not yet been clarified because no cultured cells producing apo(a) are available. Therefore, it is necessary to establish apo(a)-producing cells to study the role of apo(a). We evaluated the effects of overexpression of human apo(a) gene on human aortic VSMC growth. First, we tested whether transfection of apo(a) gene into human hepatoma cells, HepG2 cells, producing human apoB resulted in the formation of Lp(a). Transfection of apo(a) gene into HepG2 cells resulted in detectable levels of Lp(a) in the medium, as assessed by ELISA and Western blot, whereas no Lp(a) was detected in the medium of HepG2 cells transfected with control vector and untransfected HepG2 cells. Expression of apo(a) mRNA was also confirmed by reverse transcription–polymerase chain reaction. In contrast, Western blotting showed a single band detected by specific anti-apo(a) antibody, but not anti-apoB antibody, in the medium of apo(a)-transfected VSMCs. These results demonstrate that Lp(a) can be formed from apo(a) on HepG2 cells, whereas transfection of apo(a) gene into VSMCs resulted in the production of apo(a) alone but not Lp(a). Next, we examined the biological effects of overexpression of apo(a) gene on growth of VSMCs and endothelial cells. Incubation of cultured medium of HepG2 cells transfected with apo(a) gene with human VSMCs or endothelial cells resulted in a significant increase in cell number compared with the conditioned medium of HepG2 cells transfected with control vector. In contrast, transfection of apo(a) gene directly into VSMCs caused no significant effect on VSMC growth. Therefore, we measured TGF-ß concentration in the conditioned medium of VSMCs. However, using ELISA, only latent but not active TGF-ß was detected in the medium of VSMCs. Moreover, addition of neutralizing anti–TGF-ß antibody did not alter VSMC growth. These results suggest that Lp(a) could stimulate growth of VSMCs via the independent mechanisms from the inhibition of TGF-ß activation. Overall, these data demonstrate that overexpression of apo(a) gene in cells producing apoB results in formation of Lp(a), resulting in a mitogenic action on human endothelial cells and VSMCs. These results provide new information to understand the mechanisms of the mitogenic action of Lp(a) and suggest the role of Lp(a) in the pathogenesis of atherosclerosis.

Key Words: atherosclerosis • transforming growth factors • hyperplasia • liposomes • gene transfer

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Ahigh concentration of serum Lp(a) is a risk factor for atherosclerosis, restenosis after angioplasty, ischemic heart disease, and cerebral stroke.^{1 2 3 4 5} Lp(a) consists of LDL with an additional protein component, apo(a), a homologue of plasminogen.⁶ Lp(a) and apo(a) have been thought to enhance proliferation of human VSMCs in culture by inhibiting activation of plasminogen to plasmin, thus blocking the proteolytic activation of TGF-ß.^{7 8 9} On the other hand, Lp(a) has been postulated to bind to endothelial cells and macrophages and to extracellular components such as fibrin and to inhibit cell-associated plasminogen activation.^{10 11} Because the apo(a) gene is absent in rodents and nearly all subprimate species, there are limitations to available animal models. Currently, transgenic technology is attractive because it provides the means to isolate the effects of a single gene product on the complex pathophysiological processes that underlie thrombosis and atherosclerosis. In apo(a) transgenic mice, accumulation of apo(a) near the luminal surface of the aortic wall and the presence of apo(a) throughout the media of the vessel were observed.¹² The activation of TGF-ß is inhibited in the aortic wall and serum of mice expressing apo(a), possibly resulting in the development of atherosclerosis.⁹ Together with previous reports that in human VSMCs producing latent and active TGF-ß,⁸ both apo(a) and Lp(a) showed mitogenic activities, probably through the inhibition of activation of TGF-ß, the molecular mechanism of the mitogenic actions of apo(a) and Lp(a) has been postulated to inhibit activation of TGF-ß. However, it is still unclear whether the pathological role of Lp(a) and apo(a) is only limited to interference with TGF-ß activation. In addition to transgenic technology, another attractive approach to study a targeted gene is the gene transfer method.¹³ which we used to study the pathological roles of apo(a) and Lp(a) in this study. Highly efficient and nontoxic gene transfer using the Sendai virus (HVJ) has been reported in cultured VSMCs.^{14 15} Using apo(a) gene overexpression in cultured VSMCs and HepG2 cells, we addressed the following specific questions: (1) Can locally synthesized apo(a) be converted to Lp(a)? (2) Does overexpression of the apo(a) gene have biochemical and physiological effects on VSMC growth? (3) Is the mitogenic action of overexpressed apo(a) gene dependent on the inhibition of TGF-ß activation?

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Construction of Plasmids
A recombinant apo(a) (r-apo) molecule containing 17 kringle 4-like domains, as well as the kringle 5-like and protease-like domains, was assembled from apo(a) cDNA clones as described in Reference 17¹⁷ (kindly donated by Dr Richard Lawn, Stanford University, Calif). An apo(a) expression vector, containing the 5-untranslated region, the signal sequence, the first 5-kringle 4-like repeats, and 291 bp of the size kringle repeat of apo(a), was driven by the cytomegalovirus promoter.¹⁶ Koschinsky et al¹⁶ reported that the engineered protein (predicted mass of 250 kDa) contains 17 copies of the apo(a) domain, which resembles kringle 4 of plasminogen, followed by the plasminogen-like kringle 5 and protease-like domain of apo(a). Atherogenesis in transgenic mice expressing this human apo(a) mini-gene has been reported.^{9 12}

Cell Culture
Human aortic endothelial cells (passage 5) and human aortic VSMCs (passage 5) were obtained from Clonetics Corp and cultured in modified MCDB131 medium supplemented with 5% fetal calf serum, 100 U/mL penicillin, 100 mg/mL streptomycin, 10 ng/mL epidermal growth factor, 2 ng/mL basic fibroblast growth factor, and 1 µmol/L dexamethasone in the standard fashion.^{17 18} Cells were incubated at 37°C in a humidified atmosphere of 95% air-5% CO₂ with medium changes every 2 days. These cells showed the specific characteristics of endothelial cells and VSMCs by immunohistochemical examination and morphological observation. Briefly, human aortic endothelial cells tested positive for factor VIII antigen and for uptake of di-acetylated LDL. In contrast, human aortic VSMCs also tested positive for -actin and negative for expression of factor VIII antigen. All the cells were used within passages 5 and 6. HepG2 cells were obtained from American Tissue Culture Collection.

Preparation of HVJ-Liposomes
We have previously reported the high-efficiency transfection of cells in culture using HVJ-coated liposomes.^{14 15 19} Briefly, phosphatidylserine, phosphatidylcholine, and cholesterol were mixed in a weight ratio of 1:4.8:2 in tetrahydrofuran. The lipid mixture (10 mg) was deposited on the sides of a flask by removal of the solvent in a rotary evaporator. Then, 96 µg high mobility group 1 (HMG 1) nuclear protein, purified from calf thymus, was mixed with plasmid DNA (300 µg) in 200 µL BSS (137 mmol/L NaCl, 5.4 mmol/L KCl, 10 mmol/L Tris-HCl, pH 7.6) at 20°C for 1 hour and added to the dried lipid. Liposome-DNA-HMG 1 complex suspension was (1) mixed by vortex, (2) sonicated for 3 seconds, and (3) shaken for 30 minutes. Purified HVJ (Z strain) was inactivated by UV irradiation (110 erg/mm² per second) for 3 minutes immediately before use. The liposome suspension (0.5 mL, containing 10 mg lipid) was mixed with HVJ (20 000 hemagglutinating units) in a total volume of 4 mL BSS. The mixture was incubated at 4°C for 10 minutes and then for 30 minutes with gentle shaking at 37°C. Free HVJ was removed from the HVJ-liposomes by sucrose density gradient centrifugation. The top layer of the sucrose gradient containing the HVJ-liposome-DNA complex was collected and used immediately.

Measurement of Apo(a) and Lp(a) Concentration in Cultured Cells
To document the successful transfection of the cells, we examined the production of apo(a) and Lp(a). HepG2 cells and VSMCs were seeded onto 6-well plates (Corning) at a density of 5x10⁴ cells/cm² and cultured for 24 hours. VSMCs or HepG2 cells were seeded onto plates and grown to confluence. Cells were washed 3 times with BSS containing 2 mmol/L CaCl₂ and then incubated with 1 mL HVJ-liposomes-DNA complex (2.5 mg lipid and 10 µg encapsulated DNA) at 4°C for 5 minutes followed by 37°C for 30 minutes (total 35 minutes). The cells were then washed and fed fresh medium containing 10% fetal calf serum and placed in a CO₂ incubator. Twenty-four hours after transfection, the medium was changed, and the cells were incubated for an additional 48 hours. To study the release of apo(a) and Lp(a), transfected cells (48 hours after transfection) were washed and fed with 1 mL DSF containing medium supplemented with insulin (5x10^-7 mol/L), transferrin (5 mg/mL), and ascorbate (0.2 mmol/L).²⁰ Forty-eight hours later, conditioned medium was collected, centrifuged at 600g for 10 minutes, and stored at -20°C.²¹ The concentration of apo(a) and Lp(a) in the medium was determined by enzyme immunoassay using anti-apo(a) antibody.²² Briefly, the antibodies [anti-apo(a) polyclonal antibody, sheep anti-human apoB polyclonal antibody, mouse anti-human apoB monoclonal antibody; 15 µg/mL] were coated on 96-well plates (Corning) at 37°C for 2 hours, respectively. Mouse monoclonal anti-human apoB antibody was prepared according to the conventional method by fusion system using mouse myelomal cells. Both polyclonal and monoclonal anti-apoB antibodies recognize only apoB. Medium supernatants were diluted 3-fold [apo(a), plasminogen] or 30-fold (LDL) with 10 mmol/L Tris-HCl (pH 8.0) containing 0.85% sodium chloride and 1% BSA. After blocking with 1% BSA in PBS, conditioned medium was added to each well, and the preparation was incubated for 2 hours at room temperature. Wells were washed 3 times with PBS containing 0.025% Tween 20 (PBS-Tween), conjugated anti-apo(a) polyclonal antibody or anti-apoB polyclonal antibody (diluted 500- to 6000-fold) was added, and the preparation was incubated for 2 hours at room temperature. After a washing with PBS-Tween, wells were incubated with color reagent (3,3',5,5'-tetramethylbenzidine) in 24 mmol/L citric acid buffer, pH 5.0, containing 0.03% hydrogen peroxide. The enzyme reaction was halted by adding 0.5 mol/L H₂SO₄, and absorbance at 450 nm was measured.

Electrophoresis and immunoblotting were also performed. Briefly, agarose gel (1%) electrophoresis for Lp(a) or SDS-PAGE (4%) for apo(a) was performed using the Titan GEL Lipoprotein Electrophoresis kit (Helena Laboratory). After transfer onto a nitrocellulose membrane (Bio-Rad) using a nova blot electrophoretic transfer kit (Pharmacia-LKB), membranes were soaked overnight in PBS containing 1% BSA at 4°C to effect blocking. These nitrocellulose membranes were washed and soaked in PBS containing 3% BSA with 500-fold diluted peroxidase-labeled anti-mouse IgG antibody or peroxidase-labeled anti-sheep IgG or peroxidase-labeled anti-goat IgG (Dako Co). After a washing, membranes then were again soaked in PBS containing 0.025% 3,3-diaminobenzidine tetrahydrochloride and 0.01% hydrogen peroxidase at room temperature for 20 minutes for color development.

RNA was extracted using RNAzol (Tel-Test Inc) from HepG2 cells transfected with apo(a) or control vector at 24 hours after transfection.²³ Expression of apo(a) mRNA was measured by RT-PCR. Aliquots of RNA (0.5 µg) were amplified simultaneously by PCR (35 cycles) and compared with a negative control (primers without RNA). The apo(a) primers were previously described.²⁴ GAPDH primers were purchased from Clontech. Extreme care was taken to avoid contamination of tissue samples with trace amounts of experimental RNA. Amplification products were electrophoresed through polyacrylamide gels and stained with ethidium bromide. To ensure that the RT-PCR amplified products reflected transcribed targeted RNAs without significant DNA contamination, RNA samples treated with RNase A or amplified without reverse transcriptase were amplified simultaneously as negative controls. These samples did not produce a visual band. Moreover, PCR products were cut by restriction enzymes, and the fragments were identical to the theoretical bands. At least 3 aliquots of each DNA and RNA sample were subjected to separate PCR amplification in all experiments.

Cell Counting Assay
In this study, we measured cell number using a WST-cell counting kit, which is similar to the MTT assay (Wako).^{18 25 26} Tetrazolium salt has been used to develop a quantitative colorimetric assay for cell growth. The assay detects living but not dead cells. For this purpose, MTT is widely used.²⁷ In this study, we used an alternative to MTT, ie, sulfonated tetrazolium salt, 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1), since this compound produces a highly water-soluble formazan dye that makes the assay procedure easier to perform.²⁶ Briefly, 16.3 mg WST-1 and 0.2 mmol/L 1-methoxy-5-methyl-phenazinium methylsulfate were dissolved in 20 mmol/L HEPES buffer (pH 7.4). Then, 10 µL of the reaction solution was immediately added to 100 µL culture medium per well, and the cells were incubated for an additional 15 minutes. The plates were read on a Bio-Rad model 3550 Microplate reader, using a test wavelength of 450 nm and a reference wavelength of 650 nm. We confirmed that serum-stimulated increase in cell number is associated with increased absorbance at 450 nm (data not shown).

Effect of Transfection of Apo(a) Gene Directly Into VSMCs on VSMC Growth
Human aortic VSMCs were seeded onto uncoated 96-well tissue culture plates (Corning). In the preparation of experiments for determination of cell count, the cells were grown to 70% confluence in culture dishes. After 70% confluence, the medium was changed to fresh DSF. The cells were then incubated for 48 hours to make them quiescent. Transfection of apo(a) or control vector into VSMCs was performed as described above. Briefly, cells were washed 3 times with BSS containing 2 mmol/L CaCl₂ and then incubated with 500 µL HVJ-liposomes (1.3 mg lipid and 5 µg encapsulated DNA). The cells were incubated at 4°C for 5 minutes and then at 37°C for 30 minutes. On day 1, the medium was again changed to fresh DSF containing 0.05% fetal bovine serum (Gibco). After 4 days, an index of cell proliferation was determined to study the effect of locally produced apo(a) in VSMCs on VSMC growth in an autocrine manner.²¹

Effect of Conditioned Medium of HepG2 Cells or VSMCs Transfected With Apo(a) or Control Vector on VSMCs and Endothelial Cell Growth
The ability of conditioned medium to increase cell growth in a paracrine manner was also examined. HepG2 cells and VSMCs for transfection were seeded onto 6-well plates (Corning) at a density of 5x10⁴ cells/cm², cultured for 24 hours, and grown to confluence. Cells were then transfected as described above. Twenty-four hours after transfection, the medium was changed, and the cells were incubated for an additional 48 hours. To study the biological effect of locally produced apo(a) and Lp(a) on growth of VSMCs and endothelial cells, transfected HepG2 cells or VSMCs (48 hours after transfection) were washed and fed with 1 mL DSF. Forty-eight hours later, conditioned medium was collected, centrifuged at 600g for 10 minutes, and stored at -20°C.²¹ The test endothelial cells and VSMCs were seeded onto 24-well tissue culture plates. Quiescent VSMCs or endothelial cells (placed in DSF with 0.05% fetal calf serum for 48 hours after 80% confluence to make them quiescent) were treated with the conditioned media collected from HepG2 cells or VSMCs transfected with either the apo(a) expression or control vector, diluted 1:0 to 1:9 with fresh medium. After 48 hours, the medium was changed to fresh DSF diluted 1:1 with the conditioned medium. Four days later, cell growth assay was performed as described above.

Effect of Neutralizing Anti–TGF-ß Antibody on VSMC Growth
Endogenously produced local TGF-ß production in human VSMCs was characterized as TGF-ß specific by a neutralization procedure, using rabbit anti-human TGF-ß (R&D Research). The IgG fraction (purified with Protein A-agarose) was able to neutralize the biological activity of TGF-ß. Normal rabbit serum IgG fraction was used as a control. After 96 hours of incubation, cell count assay was performed.

ELISA of TGF-ß
Conditioned medium was collected from VSMCs maintained in DSF 48 hours later, centrifuged at 600g for 10 minutes, and stored at -20°C. ELISA for immunoreactive TGF-ß₁ in the supernatant was performed using an ELISA kit (Amersham). The antibody against TGF-ß₁ cross-reacts with active rat TGF-ß₁ but not with latent rat TGF-ß₁, TGF-ß₂ or TGF-ß₃. After conversion of TGF-ß from the inactive to the active form by the addition of hydrochloride, measurement of latent TGF-ß was performed using ELISA.²⁸

Materials
Human recombinant apo(a) was purified from the culture medium of Chinese hamster ovary cells and transfected with expression plasmid containing human apo(a) cDNA.¹⁶ Lp(a) used as a positive control of Western blot was purified from plasma of donors with elevated Lp(a) concentration after 12 hours of fasting. Lp(a) was isolated according to the modified method of Albers and Hazzard.²⁹ Butyrated hydroxytoluene (10 µmol/L) was added during all procedures to avoid the oxidation of lipoprotein.

Statistical Analysis
All values are expressed as mean±SEM. All experiments were repeated at least 3 times. ANOVA with subsequent Bonferroni's test was used to determine differences in multiple comparisons. A value of P<0.05 was considered statistically significant.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Apo(a) Expression in Transfected HepG2 Cells
First, we transfected human apo(a) gene into human hepatic cells, HepG2 cells. As shown in Figure 1, top, transfection of human apo(a) gene into HepG2 cells resulted in a visual band detected by anti-apo(a) antibody (Figure 1, top) compared with HepG2 cells transfected with control vector and untransfected HepG2 cells. A small amount of apo(a) mRNA was found in untransfected HepG2 cells assessed by RT-PCR (Figure 1, bottom 2 panels). Increased apo(a) mRNA expression was also observed in HepG2 cells transfected with apo(a) vector compared with cells transfected with control vector (Figure 1, bottom). The presence of Lp(a) was also confirmed by ELISA, in which the first antibody was anti-apo(a) antibody and the second was anti-apo(a) antibody or 2 different anti-apoB antibodies (Table 1). These results demonstrated that transfection of apo(a) vector resulted in the formation of Lp(a) in the conditioned medium. The presence of apoB was also confirmed by ELISA where the first antibody was anti-apoB antibody and second antibodies were 2 different anti-apoB antibodies (Table 2). The size of bands was identical to those for Lp(a) and LDL, respectively, assessed by Western blot using anti-apoB antibody (data not shown).

View larger version (34K): [in this window] [in a new window]   Figure 1. Top, Western blot of Lp(a) in conditioned medium of cultured HepG2 cells transfected with apo(a) or control vector. Bottom, Expression of apo(a) mRNA in cultured HepG2 cells transfected with apo(a) or control vector detected by RT-PCR. CV indicates cells transfected with control vector; apo(a), cells transfected with apo(a) expression vector; UN, untreated cells; Lp(a), purified Lp(a) from serum; and NC, negative control (without RNA).

View this table: [in this window] [in a new window]   Table 1. Concentration of Apo(a) Assessed by ELISA Using Anti-apo(a) Antibody as First Antibody and Anti-apo(a) Antibody or Anti-apoB Antibody as Second Antibody in Conditioned Medium of Human Aortic VSCMs and HepG2 Cells Transfected With Control Vector or Apo(a) Vector

View this table: [in this window] [in a new window]   Table 2. Concentration of ApoB Assessed by ELISA Using Anti-apoB Antibody as First Antibody and Anti-apoB Antibody as Second Antibody in Conditioned Medium of Human Aortic VSCMs and HepG2 Cells Transfected With Control Vector or Apo(a) Vector

To study Lp(a) formation from apo(a) gene transfer further, we also measured Lp(a) and apoB production in HepG2 cells transfected with apo(a) or control vector using ELISA. As shown in Figure 2, Lp(a) concentration in conditioned medium of HepG2 cells transfected with apo(a) gene was significantly increased at 1 and 3 days after transfection in a time-dependent manner, whereas the presence of Lp(a) was not detected in those transfected with control vector and untransfected cells. Interestingly, apoB concentration in conditioned medium from HepG2 cells transfected with apo(a) gene significantly decreased compared with those transfected with control vector or basal production, as shown in Figure 3.

View larger version (12K): [in this window] [in a new window]   Figure 2. Time-dependent Lp(a) concentration in conditioned medium of cultured HepG2 cells transfected with apo(a) or control vector. Untreated indicates untreated cells; Control, cells transfected with control vector; and apo(a), cells transfected with apo(a) expression vector. n=6 per group. **P<0.01 vs untreated and control.

View larger version (12K): [in this window] [in a new window]   Figure 3. Time-dependent apoB concentration in conditioned medium of cultured HepG2 cells transfected with apo(a) or control vector. Control indicates cells transfected with control vector; apo(a), cells transfected with apo(a) expression vector. n=6 per group. **P<0.01 vs control.

Apo(a) Expression in Transfected VSMCs
We next examined overexpression of apo(a) gene in human aortic VSMCs. There was no detectable level of Lp(a) in the medium of untransfected VSMCs. In VSMCs transfected with apo(a), but not control vector, a single band was detected by Western blot using anti-apo(a) antibody in SDS-PAGE (Figure 4). However, Western blot using anti-apoB antibody failed to show any bands in the conditioned medium of VSMCs transfected with apo(a) and control vector, and untransfected VSMC both in agarose gel (1%) and SDS-PAGE (4%) (data not shown). These results suggested that the conditioned medium of VSMCs transfected with apo(a) vector contains apo(a) but not Lp(a). Indeed, this hypothesis was confirmed by an ELISA that distinguishes apo(a) and Lp(a) (Tables 1 and 2). Overall, transfection of apo(a) gene into VSMCs resulted in the secretion and production of only free apo(a) that was not bound to apoB. Similar results were obtained in rat VSMCs transfected with apo(a) and control vector (data not shown).

View larger version (70K): [in this window] [in a new window]   Figure 4. Western blot of apo(a) in conditioned medium of cultured VSMCs transfected with apo(a) or control vector. Apo(a) indicates cells transfected with apo(a) expression vector; CV, cells transfected with control vector; UN, untreated cells; and r-apo(a), recombinant apo(a) purified from Chinese hamster ovary cells expressing apo(a) vector.

Mitogenic Action of Overexpression of Apo(a) Gene
Finally, we examined the biological effects of overexpression of apo(a) gene on VSMC and endothelial cell growth. Incubation of conditioned medium of HepG2 cells transfected with apo(a) gene with human VSMCs resulted in a significant increase in cell number compared with conditioned medium of transfected cells with control vector or untransfected cells, according to the diluted ratio of the conditioned medium from HepG2 cells transfected with apo(a) or control vector (Figure 5). Mitogenic activity of the conditioned medium from HepG2 cells transfected with apo(a) vector is almost equivalent to the effects of purified Lp(a) added to the media with a final concentration of 10 µg/mL, as shown in Figure 5c. Similarly, incubation of conditioned medium of HepG2 cells transfected with apo(a) gene, but not control vector, with human endothelial cells resulted in a significant increase in cell number (Figure 6). In contrast, transfection of apo(a) gene directly into VSMCs resulted in no significant change in VSMC number compared with control vector (Figure 7). We also examined the mitogenic activity of conditioned medium of VSMCs transfected with apo(a) or control vector on quiescent subconfluent untreated VSMCs. There was no significant difference in number of VSMCs between the conditioned medium of cells transfected with apo(a) and control vector (data not shown). In rat VSMCs, similar to human VSMCs, transfection of apo(a) vector into VSMCs resulted in no significant change in cell number (cell growth at OD 450 nm: apo(a), 0.200±0.006; control, 0.181±0.005; NS).

View larger version (23K): [in this window] [in a new window]   Figure 5. a and b, Effect of conditioned medium of cultured HepG2 cells transfected with apo(a) or control vector on test human aortic VSMCs on day 4 (a) and day 6 (b). Control indicates conditioned medium from cells transfected with control vector; apo(a), conditioned medium from cells transfected with apo(a) expression vector; and apo(a):control, dilution ratio of conditioned medium. *P<0.05, **P<0.01 vs control. n=8 per group. c, Comparison of VSMC growth stimulated by conditioned medium from HepG2 cells transfected with apo(a) or control vector with authentic Lp(a) purified from plasma. CM-apo(a) indicates conditioned medium from HepG2 cells transfected with apo(a) vector (1:1 dilution); CM-control, conditioned medium from HepG2 cells transfected with control vector (1:1 dilution). n=8 per group. Authentic Lp(a) was isolated according to the modified method of Albers and Hazzard.29

View larger version (35K): [in this window] [in a new window]   Figure 6. Effect of conditioned medium of cultured HepG2 cells transfected with apo(a) or control vector on test human aortic endothelial cells. CV indicates cells transfected with control vector; apo(a), cells transfected with apo(a) expression vector. n=8 per group. P<0.01 vs untreated and control.

View larger version (45K): [in this window] [in a new window]   Figure 7. Effect of direct transfection of apo(a) or control vector into human aortic VSMCs on VSMC growth. Untransfected indicates untransfected cells; CV, cells transfected with control vector; and apo(a), cells transfected with apo(a) expression vector. n=8 per group.

To clarify the cellular mechanisms of the mitogenic action of overexpression of apo(a) gene, we measured TGF-ß in the conditioned medium of VSMCs using ELISA. Using ELISA, active TGF-ß could not be detected in the conditioned medium of VSMCs, whereas latent TGF-ß was readily detected in VSMCs (latent, 0.904±0.044 ng/mL; active, not detected). Moreover, we examined the effect of addition of neutralizing anti–TGF-ß antibody into the conditioned medium of VSMCs on VSMC growth to clarify the role of TGF-ß in the mitogenic action of Lp(a). No significant difference in cell number in human VSMCs was observed between anti–TGF-ß antibody and control IgG (cell growth at OD 450 nm: vehicle, 0.346±0.011; IgG control, 0.373±0.018; anti–TGF-ß, 0.370±0.013; NS).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Lp(a) has been of interest in vascular biology since epidemiological studies indicated it to be an independent risk factor for cardiovascular disease (eg, atherosclerosis and ischemic heart disease).^{1 2 3 4 5} Recent studies using transgenic technology showing that mice expressing the apo(a) gene develop atherosclerosis have focused on the mitogenic action of apo(a) and Lp(a).^{9 12} Recently, the possible mechanism of apo(a) as a mitogen has been thought to be inhibition of the activation of TGF-ß, an autocrine inhibitor of VSMC growth. Beside the action of apo(a) on TGF-ß activation, little is known about the molecular mechanisms of the mitogenic actions of apo(a) and Lp(a). As mentioned earlier, in this study we used gene transfer to address the following specific questions: (1) Can locally synthesized apo(a) be converted to Lp(a)? (2) Does overexpression of apo(a) gene have biochemical and physiological effects on VSMC growth? (3) Does overexpression of apo(a) gene have an effect on VSMC growth independent from the inhibition of TGF-ß activation? Studying the effect of autocrine-paracrine vasoactive modulators on VSMCs is very difficult in vivo because in vivo studies are limited by (1) the multiplicity of coexisting variables, (2) difficulties in manipulation of individual components, and (3) methodological limitations in studying the function of a locally produced modulator in the absence of any contribution by the circulatory system. Cell culture and gene transfer technologies have provided us with the opportunity to study cellular responses to the manipulation of individual components (ie, by overexpression or inhibition).

First, we examined the formation of Lp(a) from apo(a) in VSMCs and HepG2 cells. It is not surprising that Lp(a) could not be formed from apo(a) in human VSMCs, which do not produce apoB. In contrast, in HepG2 cells, transfection of human apo(a) gene resulted in the formation of Lp(a) in the conditioned medium. These data are consistent with the reports that Lp(a) can be formed in mice expressing both apo(a) and apoB genes.^{29 30} Using this apo(a) mini-gene, assembly of apo(a) to apoB and LDL was previously reported.³¹ Moreover, production of apo(a) or Lp(a) from this apo(a) mini-gene showed the biological action, similar to mature Lp(a), using transgenic technology.^{9 12 30 31} Interestingly, the present data also revealed that apoB concentration decreased according to increase in Lp(a) concentration (Figure 3). Because there are few studies of apoB production in transgenic mice expressing apo(a) gene and in humans, the relation between apo(a) and apoB has not yet been clarified in vivo.

The potential importance of local apo(a) and Lp(a) in the pathogenesis of atherosclerosis has been reported^{32 33 34 35} as the presence of apo(a) was confirmed in the vascular wall of apo(a) transgenic mice. Moreover, Lp(a) is more highly concentrated in the arterial wall than in plasma. Plasma-derived Lp(a) is known to penetrate human arteries^{32 33 34 35} and the arteries of mice.²⁹ In this study, we evaluated the biological action of locally produced apo(a) on VSMC growth. In contrast to the previous observation that addition of recombinant apo(a) or Lp(a) stimulated human VSMC growth,⁸ we failed to show a stimulatory effect of apo(a) gene overexpression in human VSMCs. In our human VSMCs, only latent TGF-ß was detected in conditioned medium assessed by ELISA, different from the previous report that both active and latent TGF-ß were detected.⁸ Moreover, addition of neutralizing anti–TGF-ß antibody to human VSMCs also failed to alter cell number. In contrast, the conditioned medium of HepG2 cells transfected with apo(a) gene showed a strong mitogenic activity compared with medium of those transfected with control vector or untransfected (Figure 5a). Conditioned medium of HepG2 transfected with apo(a) gene contained Lp(a), possibly free apo(a) and free apoB, revealed mitogenic activity on VSMC growth. Because our human VSMCs secrete no active TGF-ß, the mitogenic activity of Lp(a) shown here may be independent from inhibition of TGF-ß activation. Miyata et al³⁶ previously reported that Lp(a) stimulated the proliferation of VSMCs through 2 pathways: apo(a)-induced inhibition of TGF-ß activation and stimulation of VSMCs by the LDL particle of Lp(a). However, their results could not exclude the potential contribution of plasma, since they used purified Lp(a) from plasma. Our data using gene transfer support the potential mitogenic mechanisms of Lp(a), in addition to the inhibition of TGF-ß activation pathway. Taken together, the mitogenic actions of apo(a) and Lp(a) on VSMCs may be different, although further studies are necessary. Similar mitogenic activity of the conditioned medium of HepG2 cells transfected with apo(a) gene was also observed in human endothelial cells. Given that plasma-derived Lp(a) penetrates human and mice arteries and Lp(a) accumulates in vascular lesions,^{29 32 33 34 35} plasma-derived Lp(a), rather than free apo(a) present in the vascular wall, may have an important role in the pathogenesis of atherosclerosis. Moreover, the combination of Lp(a) and hypertension may enhance the incidence of cardiovascular disease. Study of the molecular mechanisms of mitogenic actions of Lp(a) may help in the understanding of the pathogenesis of cardiovascular disease as complications of hypertension.

In conclusion, the present study demonstrated that (1) transfection of apo(a) gene into HepG2 cells caused formation of Lp(a) from apo(a), resulting in an increase in VSMC number; (2) overexpression of apo(a) gene in HepG2 cells resulted in a significant decrease in apoB concentration; and (3) transfection of apo(a) into human VSMCs resulted in the production of only free apo(a), but not Lp(a), and the direct transfection of apo(a) gene into VSMCs had no effect on VSMC growth. Overall, the mitogenic activity of the conditioned medium of HepG2 cells transfected with apo(a) gene containing Lp(a) shown may be mediated through a different pathway than inhibition of TGF-ß activation. These results provide new information to understand the molecular mechanisms of the mitogenic action of Lp(a).

	Selected Abbreviations and Acronyms

 

apo = apolipoprotein BSS = balanced salt solution DSF = defined serum-free medium ELISA = enzyme-linked immunosorbent assay HVJ = hemagglutinating virus of Japan Lp(a) = lipoprotein(a) RT-PCR = reverse transcription–polymerase chain reaction SDS-PAGE = SDS–polyacrylamide gel electrophoresis TGF-ß = transforming growth factor-ß VSMC = vascular smooth muscle cell

	Acknowledgments

 
This work was partially supported by grants from the Japan Society for the Promotion of Science, ONO Medical Research Foundation, the Japan Heart Foundation–Pfizer Pharmaceuticals grant for research on coronary artery disease, and the Uehara Memorial Foundation. Dr Morishita is the recipient of a Harry Goldblatt Award from the Council of High Blood Pressure, American Heart Association. Dr Hayashi is a Research Fellow of the Japan Society for the Promotion of Science. We wish to thank Chihiro Noguchi for excellent technical assistance.

Received May 12, 1997; first decision June 18, 1997; accepted March 17, 1998.

	References

Top Abstract Introduction Methods Results Discussion References

 
1. Jurgens G, Taddei-Peters WC, Koltringer P, Petek W, Chen Q, Greilberger J, Macomber PF, Butman BT, Stead AG, Ransom JH. Lipoprotein(a) serum concentration and apolipoprotein(a) phenotype correlate with severity and presence of ischemic cerebrovascular disease. Stroke. 1995;26:1841–1848.[Abstract/Free Full Text]

2. Willeit S, Kiechl P, Santer F, Oberhollenzer G, Egger E, Jarosch A, Mair A. Lipoprotein(a) and asymptomatic carotid artery disease: evidence of a prominent role in the evolution of advanced carotid plaques: the Bruneck Study. Stroke. 1995;26:1582–1587.[Abstract/Free Full Text]

3. Loscalzo J. Lipoprotein(a): a unique risk factor for atherothrombotic disease. Arteriosclerosis. 1990;10:672–679.[Free Full Text]

4. Sandkamp M, Funke H, Schulte H, Kohler E, Assmann G. Lipoprotein(a) is an independent risk factor for myocardial infarction at a young age. Clin Chem. 1990;36:20–23.[Abstract/Free Full Text]

5. Rosengren A, Wilhelmsen L, Eriksson E, Risberg B, Wedel H. Lipoprotein(a) and coronary heart disease: a prospective case-control study in a general population sample of middle aged men. BMJ. 1990;301:1248–1251.

6. McLean JW, Tomlinson JE, Kuang WJ, Eaton DL, Chen EY, Fless GM, Scanu AM, Lawn RM. cDNA sequence of human apolipoprotein(a) is homologous to plasminogen. Nature. 1987;330:132–137.[Medline] [Order article via Infotrieve]

7. Kojima S, Harpel PC, Rifkin DB. Lipoprotein(a) inhibits the generation of transforming growth factor ß: an endogenous inhibitor of smooth muscle cell migration. J Cell Biol. 1991;113:1439–1445.[Abstract/Free Full Text]

8. Grainger DJ, Kirschenlohr HL, Metcalfe JC, Weissberg PL, Wade DP, Lawn RM. Proliferation of human smooth muscle cells promoted by lipoprotein(a). Science. 1993;260:1655–1658.[Abstract/Free Full Text]

9. Grainger DJ, Kemp PR, Liu AC, Lawn RM, Metcalfe JC. Activation of transforming growth factor-ß is inhibited in transgenic apolipoprotein(a) mice. Nature. 1994;370:460–462.[Medline] [Order article via Infotrieve]

10. Hajjar KA, Gavish D, Breslow JL, Nachman RL. Lipoprotein(a) modulation of endothelial cell surface fibrinolysis and its potential role in atherosclerosis. Nature. 1989;339:303–305.[Medline] [Order article via Infotrieve]

11. Bihari-Varga M, Gruber E, Rotheneder M, Zechner R, Kostner GM. Interaction of lipoprotein Lp(a) and low density lipoprotein with glycosaminoglycans from human aorta. Arteriosclerosis. 1988;8:851–857.[Abstract/Free Full Text]

12. Lawn RM, Wade DP, Hammer RE, Chiesa G, Verstuyft JG, Rubin EM. Atherogenesis in transgenic mice expressing human apolipoprotein(a). Nature. 1992;360:670–672.[Medline] [Order article via Infotrieve]

13. Dzau VJ, Gibbons GH, Morishita R, Pratt E. New perspectives in hypertension research: potentials of vascular biology. Hypertension. 1994;23:1132–1140.[Abstract/Free Full Text]

14. Morishita R, Gibbons GH, Kaneda Y, Ogihara T, Dzau VJ. Novel in vitro gene transfer method for study of local modulators in vascular smooth muscle cells. Hypertension. 1993;21:894–899.[Abstract/Free Full Text]

15. Morishita R, Gibbons GH, Kaneda Y, Ogihara T, Dzau VJ. Novel and effective gene transfer method for study of vascular renin angiotensin system. J Clin Invest. 1993;91:2580–2585.

16. Koschinsky ML, Tomlinson JE, Zioncheck TF, Schwartz K, Eaton DL, Lawn RM. Apolipoprotein(a): expression and characterization of a recombinant form of the protein in mammalian cells. Biochemistry. 1991;30:5044–5051.[Medline] [Order article via Infotrieve]

17. Bonin PD, Leadley RJ, Erickson LA. Growth factor-induced modulation of endothelial-1 binding to human smooth muscle cells. J Cardiovasc Pharmacol. 1993;22:S125–S127.

18. Morishita R, Nakamura S, Nakamura Y, Aoki M, Moriguchi A, Kida I, Yo Y, Matsumoto K, Nakamura T, Higaki J, Ogihara T. Potential role of endothelium-specific growth factor, hepatocyte growth factor, on endothelial damage in diabetes mellitus. Diabetes. 1997;46:138–142.[Abstract]

19. Kaneda Y, Iwai K, Uchida T. Increased expression of DNA co-introduced with nuclear protein in adult rat liver. Science. 1989;243:375–378.[Abstract/Free Full Text]

20. Libby P, O'Brien KV. Culture of quiescent arterial smooth muscle cells in a defined serum-free medium. J Cell Physiol. 1983;115:217–223.[Medline] [Order article via Infotrieve]

21. Morishita R, Gibbons GH, Pratt RE, Tomita N, Kaneda Y, Ogihara T, Dzau VJ. Autocrine and paracrine effects of atrial natriuretic peptide gene transfer on vascular smooth muscle and endothelial cellular growth. J Clin Invest. 1994;94:824–829.

22. Rainwater DL, Lanford RE. Production of lipoprotein(a) by primary baboon hepatocytes. Biochim Biophys Acta. 1989;1003:30–35.[Medline] [Order article via Infotrieve]

23. Morishita R, Gibbons GH, Ellison KE, Nakajima M, Zhang L, Kaneda Y, Ogihara T, Dzau VJ. Single intraluminal delivery of antisense cdc 2 kinase and PCNA oligonucleotides results in chronic inhibition of neointimal hyperplasia. Proc Natl Acad Sci U S A. 1993;90:8474–8478.[Abstract/Free Full Text]

24. Kraft HG, Haibach C, Lingenhel A, Brunner C, Trommsdorff M, Kronenberg F, Muller HJ, Utermann, G. Sequence polymorphism in kringle IV 37 in linkage disequilibrium with the apolipoprotein(a) size polymorphism. Hum Genet. 1995;95:275–282.[Medline] [Order article via Infotrieve]

25. Ishiyama M, Shiga M, Sasamoto K, Mizoguchi M, He P. A new sulfonated tetrazolium salt that produces a highly water-soluble formazan dye. Chem Pharm Bull. 1993;41:1118–1122.

26. Shirahata S, Watanabe J, Teruya K, Yano T, Osada K, Ohashi H, Tachibana H, Kim EH, Murakami H. E1A and ras oncogenes synergistically enhance recombinant protein production under control of the cytomegalovirus promoter in BHK-21 cells. Biosci Biotech Biochem. 1995;59:345–347.[Medline] [Order article via Infotrieve]

27. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983;65:55–63.[Medline] [Order article via Infotrieve]

28. Gibbons GH, Pratt RE, Dzau VJ. Vascular smooth muscle cell hypertrophy vs hyperplasia: autocrine transforming growth factor beta 1 expression determines growth response to angiotensin II. J Clin Invest. 1992;90:456–461.

29. Albers JJ, Hazzard WR. Immunochemical quantification of human plasma Lp(a) lipoprotein. Lipids. 1974;9:15–26.[Medline] [Order article via Infotrieve]

30. Callow MJ, Verstuyft J, Tangirala R, Palinski W, Rubin EM. Atherogenesis in transgenic mice with human apolipoprotein B and lipoprotein(a). J Clin Invest. 1995;96:1639–1646.

31. Bonen DK, Hausman AML, Hadjiagapiou C, Skarosi SF, Davidson NO. Expression of a recombinant apolipoprotein(a) in HepG2 cells: evidence for intracellular assembly of lipoprotein(a). J Biol Chem. 1997;272:5659–5667.[Abstract/Free Full Text]

32. Callow MJ, Stoltzfus LJ, Lawn RM, Rubin EM. Expression of human apolipoprotein B and assembly of lipoprotein(a) in transgenic mice. Proc Natl Acad Sci U S A. 1994;91:2130–2134.[Abstract/Free Full Text]

33. Rath M, Niendorf A, Reblin T, Dietel M, Krebber HJ, Beisiegel U. Detection and quantification of lipoprotein(a) in the arterial wall of 107 coronary bypass patients. Arteriosclerosis. 1989;9:579–592.[Abstract/Free Full Text]

34. Niendorf A, Rath M, Wolf K, Peters S, Arps H, Beisiegel U, Dietel M. Morphological detection and quantification of lipoprotein(a) deposition in atheromatous lesions of human aorta and coronary arteries. Virchows Arch. 1990;417:105–111.

35. Cushing GL, Gaubatz JW, Nava ML, Burdick BJ, Bocan TM, Guyton JR, Weilbaecher D, DeBakey ME, Lawrie GM, Morrisett JD. Quantitation and localization of apolipoproteins [a] and B in coronary artery bypass vein grafts resected at re-operation. Arteriosclerosis. 1989;9:593–603.[Abstract/Free Full Text]

36. Miyata M, Biro S, Kaieda H, Tanaka H. Lipoprotein(a) stimulates the proliferation of cultured human arterial smooth muscle cells through two pathways. FEBS Lett. 1995;377:493–496.[Medline] [Order article via Infotrieve]

This article has been cited by other articles:

				C. H. O'Neil, M. B. Boffa, M. A. Hancock, J. G. Pickering, and M. L. Koschinsky Stimulation of Vascular Smooth Muscle Cell Proliferation and Migration by Apolipoprotein(a) Is Dependent on Inhibition of Transforming Growth Factor-{beta} Activation and on the Presence of Kringle IV Type 9 J. Biol. Chem., December 31, 2004; 279(53): 55187 - 55195. [Abstract] [Full Text] [PDF]

				N. Komai, R. Morishita, S. Yamada, M. Oishi, S. Iguchi, M. Aoki, M. Sasaki, I. Sakurabayashi, J. Higaki, and T. Ogihara Mitogenic Activity of Oxidized Lipoprotein (a) on Human Vascular Smooth Muscle Cells Hypertension, September 1, 2002; 40(3): 310 - 314. [Abstract] [Full Text] [PDF]

				T. Ichikawa, H. Unoki, H. Sun, H. Shimoyamada, S. Marcovina, H. Shikama, T. Watanabe, and J. Fan Lipoprotein(a) Promotes Smooth Muscle Cell Proliferation and Dedifferentiation in Atherosclerotic Lesions of Human Apo(a) Transgenic Rabbits Am. J. Pathol., January 1, 2002; 160(1): 227 - 236. [Abstract] [Full Text] [PDF]

				G. Lippi, E. Arosio, M. Prior, and G. Guidi Biochemical Risk Factors for Cardiovascular Disease in an Aged Male Population: Emerging Vascular Pathogens Angiology, October 1, 2001; 52(10): 681 - 687. [Abstract] [PDF]

				R. Morishita, M. Sakaki, K. Yamamoto, S. Iguchi, M. Aoki, K. Yamasaki, K. Matsumoto, T. Nakamura, R. Lawn, T. Ogihara, et al. Impairment of Collateral Formation in Lipoprotein(a) Transgenic Mice: Therapeutic Angiogenesis Induced by Human Hepatocyte Growth Factor Gene Circulation, March 26, 2002; 105(12): 1491 - 1496. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Morishita, R. Articles by Ogihara, T. Search for Related Content PubMed PubMed Citation Articles by Morishita, R. Articles by Ogihara, T.