Arhero.sc/ero.si.~,90 ( 199 1) IO 1- 108 (2 1991 Elsevier Scientific Publishers AL)ONIS 0021915091001695
ATHERO
101 Ireland,
Ltd. All rights reserved
0021-~lSO/~1/$03.SO
04696
Vascular smooth muscle cells from genetically hyperlipidemic rabbit (WHHL rabbit) exhibit decreased growth response Asahiko Oguchi ‘, Uichi Ikeda ‘, Hiroshi Miyashita I, Tomoko Ohara ‘, Yoshio Tsuruya ‘, Takashi Natsume ‘, Toshio Yaginuma 2 and Kazuyuki Shimada
’
’Department of Cardiology. Jichi Medical School, Minamikawachi, Tochigi 329-04 (Japan). and ’ Department of Medicine, Omiya Medical Center Jichi Medical School. Omiya. Saitama 330 (Japan) (Received I1 October, 1990) (Revised, received 3 June, 1991) (Accepted 17 June, 1991)
Summary The Watanabe Heritable Hyperlipidemic (WHHL) rabbit is a widely studied animal model for the human genetic disorder familial hypercholesterolemia, and spontaneously develops atherosclerotic disease. We studied the growth characteristics of cultured vascular smooth muscle cells (VSMC) from WHHL rabbits compared with VSMC from Japanese white rabbits. We measured cell proliferation, DNA synthesis, and c-myc proto-oncogene expression, in response to growth stimuli such as fetal bovine serum (FBS) and platelet-derived growth factor (PDGF). VSMC from Japanese white rabbits exhibited a 4-fold increase in cell numbers during a 5-day incubation period compared with those from WHHL rabbits, FBS and PDGF stimulated DNA synthesis, as measured by thymidine incorporation into VSMC, in both Japanese white rabbits and WHHL rabbits, however the response was significantly higher in the former strain. The intracellular pH value of VSMC determined using the pH-sensitive fluorescence dye 2’,7’-bis-carboxyethyl-carboxyfluorescein was significantly higher in WHHL rabbits than in Japanese white rabbits. Proto-oncogene c-myc was induced by exposure of VSMC to FBS, however there was no significant difference in c-myc mRNA levels between the two strains. These results suggest that VSMC from WHHL rabbits are not genetically growth accelerated, but show decreased growth response to growth stimuli.
Key words:
Atherosclerosis;
WHHL
rabbit;
Smooth
muscle
cell; DNA
synthesis;
c-myc
Introduction Correspondence to: Uichi Ikeda, MD, Department of Cardiology, Jichi Medical School, Minamikawachi, Tochigi 329-04, Japan. Tel.: X1-285-44-21 II ext. 3557; Fax 81-285-44-5317.
Atherogenesis involves the migration and proliferation of vascular smooth muscle cells (VSMC)
102 in the intima of vessels, and the increase in wall thickness in the intima may be accounted for by cellular proliferation or hypertrophy of VSMC. The Watanabe Heritable Hyperlipidemic (WHHL) rabbit is a widely studied animal model for the human genetic disorder familial hypercholesterolemia (FH). WHHL rabbits have elevated serum lipids; predominantly low density lipoproteins (LDL) and intermediate density lipoproteins (IDL). They develop spontaneous atherosclerotic disease that mimics the accelerated atherosclerosis seen in familial hypercholesterolemia [l]. To find out whether the growth of VSMC from WHHL rabbits is genetically accelerated or not, we have studied the growth characteristics of VSMC from WHHL rabbits and normal Japanese white rabbits. We report here that the growth responses of VSMC to various growth stimuli are suppressed in WHHL rabbits compared with normal rabbits. Methods VSMC culture
Primary cultures of aortic VSMC were obtained by enzymatic dissociation of the media of thoracic aortae from lo-month-old male WHHL rabbits (n = 4) and age-matched Japanese white rabbits (n = 41, as previously described [2,3]. Cells were grown in Dulbecco’s Minimum Essential Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin, and 100 kg/ml streptomycin. The cultures were harvested twice a week with 0.125% trypsin and passaged at a 1: 3 ratio in lOO-mm culture dishes (Falcon, NJ). The typical growth experiment was performed with VSMC at passage levels of 5-10. Cells were plated at 1.5 x lo4 cells/well in 24-well dishes (Falcon, NJ) in DMEM supplemented as described above and allowed to grow subconfluent for 24-48 h. They were then made “quiescent” by a 24-h incubation in serum-free DMEM supplemented with insulin (5 pg/ml) and transferrin (5 pg/ml). Electron microscopic study
For electron microscopic study, VSMC were prepared in the same way as those used in the in
vitro experiments described above. VSMC were cultured on glass slides immersed in lOO-mm culture dishes (Falcon, NJ) containing the culture medium. The cultured cells adhering on the glass slides were fixed with 2% glutaraldehyde in 0.1 M cacodylate buffer containing 6% sucrose (pH 7.4) for 2 h at 4 o C. The fixed cells were washed with the same buffer and postfixed with 1% osmium tetraoxide for 1 h at ,4 o C. They were dehydrated in an ethanol series followed by propylene oxide and subsequently embedded in Epon 812. After polymerization was completed, the embedded cells were detached from the glass slides with plastic capsules filled with the resin. Ultrathin sections were cut on a LKB ultramicrotome, and examined after electron staining with uranyl acetate and lead citrate, with an electron microscope (Hitachi H-7000, Japan) operating at 75 kV. Determination of DNA synthesis
After 24-h preincubation in serum-free DMEM, FBS or platelet-derived growth factor (PDGF) was added to the culture medium. After 24 h, 1.0 &i/ml tritiated thymidine (2.0 Ci/mmol, Amersham, England) was added to the medium and incubated for 24 h. The medium was aspirated and cells were washed twice with phosphatebuffered saline (PBS), fiied by washing with icecold 10% trichloroacetic acid, and extracted with 0.2 N NaOH over several h. Aliquots were counted in a liquid scintillation counter (Aloka, Japan). Protein content was measured by the Bradford method [4]. Measurements of intracellular pH
Intracellular pH (pHiI of cultured VSMC was determined using the pH-sensitive fluorescent dye BCECF (2’,7’-bis-carboxyethyl-carboxyfluorescein). Cells grown on glass coverslips were loaded with 5 PM BCECF-AM in serum-free culture medium at 37 o C for 60 min, washed 3 times with PBS. The coverslip was then inserted into a cuvette containing 2 ml of HCO;-free solution of the following composition (mM): NaCl 130, KC1 5, CaCI, 1, MgCl,, KH,PO, 2, Hepes 10, glucose 5; pH 7.4. The cuvette was placed in the thermostated holder of a fluorescence spectrophotometer (Shimazu RF 5000, Japan). The fluores-
103 cence intensity (FI) of BCECF was determined at excitation wavelengths (Ex) of 495 and 450 nm, and an emission wavelength (Em) of 530 nm using slits of 5 and 10 nm, respectively. At the end of the experimental procedure, pH, and outside pH (PH.) were equilibrated by permeating the cells with 10 kg/ml nigericin added to the incubation solution. The solution was then titrated with either 1 N HCl or 1 N NaOH over the range of fluorescence values obtained during the experiment. By measuring the pH of the solution after each addition of acid or alkali, a calibration curve of FI vs. pH was developed to compare each experimental sample. BCECF exhibited a nearly linear relationship between FI and pHi, at least over the pH range 6.4-7.6 (data not shown). Rh!A preparation and hybridization
After 24-h preincubation in serum-free DMEM, VSMC were exposed to 10% FBS for l-4 h. Total RNA was extracted from VSMC by the guanidinium isothiocynate/ cesium chloride (GITC/CsCl) procedure. 10 pg of total RNA was size-fractionated by electrophoresis on 1.0% agarose gels, and transferred to nylon membranes (Hybond N+, Amersham, U.K.). Filters were hybridized with a 32P-labeled 0.97-kb v-myc cDNA probe (specific activity > 1 X lo8 cpm/pg DNA) at 60 o C for 24 h. At the end of hybridization, the filters were washed for 30 min in 2 X SSC at 50 o C (1 x SSC contains 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0). Filters were exposed to Kodak XARS film overnight at - 80 o C with one intensifying screen. Autoradiography was quantitated by densitometric scanning (Immunomedica Image Analyzer TIF-64, Japan). Miscellaneous
Statistical analysis was performed using the Student’s t-test. P values < 0.05 were considered to indicate a statistically significant difference. Chemicals were of the highest grade commercially available.
the growth of VSMC from normal Japanese White rabbits and WHHL rabbits. VSMC from the former grew confluently, forming a “hill and valley” structure, whereas VSMC cultures from the latter showed less orderly growth (data not shown). VSMC from Japanese white and WHHL rabbits seemed to have a similar ultrastructure, i.e. the cytoplasm was rich in rough endoplasmic reticulum and free ribosomes, mitochondria was always observed in the perinuclear region, myofilaments were predominantly recognized peripherally, and the nucleus had evident nucleoli (Fig. 1). No apparent differences were noted in shapes or amounts of other ultrastructural components except lysosomes. Lysosomes containing lipofuscin pigments or residual bodies were apparently more numerous in VSMC from WHHL rabbits than in those from normal rabbits. Cell number
Fig. 2 illustrates the growth of VSMC in the presence of 10% FBS. In normal Japanese white rabbit, an approximate 4-fold increase in cell numbers was observed during a 5-day incubation, while in WHHL rabbits the increase was significantly lower than that of normal rabbits. DNA synthesis
FBS and PDGF caused a significant increase in DNA synthesis as measured by [3H]thymidine TABLE 1 EFFECTS OF FETAL BOVINE SERUM (FBS) AND PLATELET-DERIVED GROWTH FACTOR (PDGF) ON DNA SYNTHESIS OF VASCULAR SMOOTH MUSCLE CELLS (VSMC) FROM NORMAL JAPANESE WHITE RABBITS AND WATANABE HERITABLE HYPERLIPIDEMIC (WHHL) RABBITS VSMC were made quiescent by pretreatment with serum-free DMEM medium for 24 h, and then exposed to 10% FBS or PDGF (lo-’ Ml. After 24 h, 1.0 pCi/ml tritiated thymidine (2.0 Ci/mmol, Amersham) was added to the medium. Aliquots were counted in a liquid scintillation counter. Thymidine incorporation was converted per mg protein. Data are mean & SE of 4 separate dishes. * P < 0.05, * * P < 0.02 compared with control.
Results Morphological study
During cultivation, from the primary culture to the 5th subculture, differences were observed in
Control FBS PDGF
Normal (cpm/mg)
WHHL kpm/mg)
41126 + 1127 1260 104 + 371923 * 189649+ 64663 *
14227+ 1579 36302+5140 ** 24929+7691
Fig. 1. Ultrastructure of vascular smooth muscle cells (VSMC) from normal Japanese white rabbits and Watanal be Ileri table for 2 h at 4 o C, and pc3stfi xed with hype ,rli pidemic (WHHL) rabbits. The cells on the glass slides were fiied with 2% glutaraldehyde 1% t39 nium tetraoxide for 1 h at 4 o C. Ultrathin sections were examined with a Hitachi H-7000 electron microscope. (A) Nc Irma1 rabb its (X 12000), (B) WHHL rabbits (X 10000). Arrow indicates lipofuscin pigments and residual bodies in lysosomes. N: nut :leus. M: mitochondria.
105
. & 0
Normal
0
WHHL
f.
;1
P
0
I
I
I
1
3
5 days
a
INTRACELLULAR pH (pHi) OF CULTURED VASCULAR SMOOTH MUSCLE CELLS OBTAINED FROM NORMAL JAPANESE WHITE RABBITS AND WATANABE HERITABLE HYPERLIPIDEMIC (WHHL) RABBITS Cells were loaded with BCECF/AM and pHi was determined from the fluorescence intensity as described in the text. Data are mean i_ SE. *P < 0.01 compared with normal rabbits.
PH,
WHHL
(n = 10)
7.37 & 0.01
WHHL
(n = 10)
7.45 * 0.03 *
FBS
1
PDGF
Fig. 3. DNA synthesis of VSMC from Japanese white rabbits and WHHL rabbits. VSMC were made quiescent by pretreatment with serum-free DMEM medium for 24 h. and then exposed to 10% fetal bovine serum (FBS) or platelet-derived growth factor (PDGF, IO-’ M). After 24 h, 1.0 FCi/ml tritiated thymidine (2.0 Ci/mmol, Amersham) was added to the medium and incubated for 24 h. Cells were washed with phosphate-buffered saline and extracted with 0.2 N NaOH. Aliquots were counted in a liquid scintillation counter. The increase of thymidine incorporation is shown as a relative increase to the control. Data are mean*SE of 4 separate dishes. * P < 0.01 compared with normal rabbits.
incorporation (Table 1). In Japanese white rabbits, 10% FBS stimulated DNA synthesis of VSMC about 30-fold over the unstimulated, control level. PDGF (lo-’ M) stimulated DNA synthesis of VSMC approximately 4.6-fold over the control level. However, in WHHL rabbits, FBS and PDGF stimulated DNA synthesis of VSMC
Normal 0
Oh
Control
2
Normal
Normal
‘i
Fig. 2. Cell proliferation of cultured VSMC. VSMC from normal Japanese white rabbits (0) and WHHL rabbits (0) were incubated in 10% FBS containing DMEM medium for 5 days. Cell number in each well was counted with a hemocytometer. The data are mean +SE of four separate dishes. * P < 0.02. * * P < 0.001 compared with normal rabbits.
TABLE
0 m
WHHL 4h
0
1
4h
28S-
Fig. 4. Induction of c-myc mRNA levels by FBS. Quiescent cells were exposed to 10% FBS for l-4 h. Total RNA was extracted from VSMC by the GITC/CsCI method. 10 Fg of total RNA was size-fractionated by electrophoresis on 1.0% agarose gels, and transferred to nylon membranes. Membranes were directly visualized for 28s ribosomal RNA by UV transillumination (28 S), or hybridized with the “‘P-labeled v-tnyc cDNA probe (c-myc) at 60°C for 24 h, washed for 30 min in 2 x SSC at 50 o C, and exposed to Kodak XAR-5 film overnight at - 70 o C with one intensifying screen. Data are representative of 3 separate experiments.
106
approximately 3-fold and 2-fold over the control level, respectively. FBS and PDGF stimulated DNA synthesis of VSMC in both normal rabbits and WHHL rabbits, but the growth response of VSMC to the growth stimuli was significantly lower in WHHL rabbits compared with normal rabbits (Fig. 3). Intracellular pH levels
After cells grown on coverslips were placed in the cuvette containing HCO;-free Hepes buffered solution (pH 7.401, pH, spontaneously declined by 0.1-0.2 pH units before stabilizing after - 10 min. The pH, values determined after initial decline were 7.37 k 0.01 (mean + SE, n = 10) and 7.45 + 0.03 (n = 10) for Japanese white rabbits and WHHL rabbits, respectively (P < 0.01, Table 2). c-myc mRNA levels
The product of c-myc proto-oncogene is a protein associated with cell nucleus that may be involved in signaling the early events in commitment of cell growth. Northern blotting experiments revealed almost no c-myc transcript in RNA isolated from unstimulated VSMC, however, a significant increase in c-myc mRNA levels was achieved after l-4 h exposure of quiescent VSMC to 10% FBS in both Japanese white rabbits and WHHL rabbits (Fig. 4). Despite the prominent DNA synthesis in normal rabbits after exposure to FBS (Table l), the increase in the c-myc mRNA levels in normal rabbits in response to FBS was similar to that of WHHL rabbits with a ratio of 1.1 k 0.2 (mean k SE, n = 3) after 4 h exposure. Discussion
At least five cell types participate in the formation of an atherosclerotic plaque; endothelial cells, tissue macrophages (derived from blood monocytes), T cells, platelets, and VSMC. VSMC modulation of phenotype from the contractile to synthetic state, and its proliferation in the intima are crucial processes in atherosclerosis [5,6]. The thickened intima mainly consists of proliferated VSMC. Growth factors produced from the endothelial cells [7], macrophages [S], T cells 191,
platelets [lo], and VSMC themselves [ll], can cause migration and proliferation of VSMC. In this study, we have shown that the proliferative capacities of VSMC derived from WHHL rabbits were less than those from normal rabbits. The recent discovery of WHHL rabbits has provided a genetically determined animal model of hypercholesterolemia [12]. WHHL rabbits exhibit clinical, metabolic, and biochemical features that are strikingly similar to those observed in human familial hypercholesterolemia (FH) which can develop into severe atherosclerosis. As in human FH heterozygosity, LDL accumulation in WHHL rabbits arises from a defect of the LDL receptor [13], and cultured VSMC from WHHL rabbits display low rates (< 10% of normal) of LDL binding and uptake. As shown in Fig. 2 and Table 1, the increase in cell numbers and thymidine incorporation were significantly suppressed in VSMC from WHHL rabbits compared with those from normal rabbits. Even when the serum obtained from WHHL rabbits was added to the serum-free culture medium, instead of FBS, the growth response was still significantly lower in WHHL rabbits compared with normal rabbits (data not shown). We could find only one previous report on the growth characteristics of VSMC from WHHL rabbits. Gabor et al. [14] demonstrated that total DNA content of VSMC from WHHL rabbits was about half of that of VSMC from normal rabbits, while the former showed 1Zfold higher thymidine incorporation into DNA than the latter. They speculated that the rate of DNA destruction was much higher in VSMC from WHHL rabbits than normal rabbits. We can not explain the discrepancy of the results, however, one possible explanation is that they used VSMC of intima-media origin, while we used VSMC only from the media origin. VSMC which migrate into the intima from the media are known to alter their characteristics and obtain higher autocrine mitogenic activity than VSMC in the media [11,153. The basal pH, in various vertebrate cells is principally regulated by the plasma membrane Na+/H+ exchanger [19]. In our study, the basal pH, value of WHHL rabbit VSMC was significantly higher than that of Japanese white rabbit
107 VSMC, suggesting the contribution of the altered Na+/H+ exchanger activity to suppressed growth of VSMC in WHHL rabbits. Recently, Berk et al. [20] have reported that the pH, of cultured VSMC obtained from spontaneously hypertensive rats (SHR) was significantly higher than that of normotensive Wistar-Kyoto rats (WKY), and speculated that increased Na+/H+ exchanger contributes to enhanced growth of VSMC in SHR. However, Kobayashi et al. [21] have reported that the basal pH, of VSMC was significantly lower in stroke-prone SHR than in WKY rats. LDL has been shown to cause transient acidification followed by alkalization of VSMC due to stimulation of Na+/H+ exchanger through LDL-receptor pathway [24]. We speculate that the increase in pH, in WHHL rabbits might be due to the lack of LDL receptor activity on VSMC membranes in this animal model [23]. In recent years evidence has been accumulating suggesting that the proto-oncogenes are important regulators of cell growth. Numerous investigations have demonstrated that, when quiescent cells in culture are stimulated to grow by the addition of serum or specific growth factors, rapid and transient induction of the proto-oncogene c-myc occurs [ 16,171. In our study, FBS induced the expression of c-myc in quiescent VSMC from both Japanese white rabbits and WHHL rabbits to the same degree, although the growth response to FBS was much higher in the former than the latter. c-myc induction is not entirely associated with events leading to DNA synthesis and cellular proliferation. Banskota et al. [18] demonstrated that monoclonal antibody to the insulin-like growth factor 1 (IGF-1) receptor, alfa-IR3, inhibited both the insulin and IGF-1 effects on stimulating DNA synthesis of human VSMC or cellular proliferation, while alfa-IR3 itself stimulated cmyc mRNA expression. LDL-derived cholesterol is an essential component of cell membranes [22]. VSMC obtain cholesterol through the LDL receptor pathway [221, and the cultured VSMC from WHHL rabbits can not internalize and degradate LDL [23], causing a deficit of a substrate for membrane synthsis. We therefore speculate that the lack of cholesterol incorporation and/or the altered Na’/H+ exchanger activity might contribute to
the poor DNA and protein synthesis in WHHL rabbits, although surface receptor systems to the growth stimuli are intact judging from the c-myc mRNA expression (Fig. 4). In conclusion, VSMC from WHHL rabbits are not genetically growth accelerated, but showed a tendency to lower mitogenic activity than those from Japanese white rabbits. In the process of formation of atherosclerotic lesions, we suppose that factors other than the mitogenicity of VSMC play important roles in the abnormal proliferation of VSMC and hence the development of atherosclerotic lesions in WHHL rabbits. Acknowledgments
The authors thank Etsuko Chiku for her technical assistance. We also thank Drs. Yoshio Watanabe and Masahi Shiomi (Kobe University School of Medicine) for donating the WHHL rabbits. This study was supported by the Uehara Memorial Foundation, the Kowa Life Science Foundation, and the Takeda Medical Research Foundation (Japan). References Goldstein, J.L., Kita, T., Brown, MS., Defective lipoprotein receptors and atherosclerosis. Lessons from an animal counterpart of familial hypercholesterolemia. N. Engl. J. Ned., 309 (1983) 288. Charnley, J.H., Campbell, G.R., McConell, J.D., Groschel-Stewart, 1J., Comparison of vascular smooth muscle cells from adult human, monkey and rabbit in primary culture and in subculture. Cell Tissue Res., 177 (1977) 503. Ikeda, U., Ikeda, M., Oohara, T., Kano. S., Yaginuma, T., Mitogenic action of interleukin-1 on vascular smooth muscle cells mediated by PDGF. Atherosclerosis, 84 (1990) 183. Bradford, M.M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem., 72 (1976) 248. Charnley-Campbell, J.H., Campbell, G.R., What controls smooth muscle phenotype? Atherosclerosis, 40 (1981) 347. Campbell, G.R., Campbell, J.H., Recent advances in molecular pathology. Smooth muscle phenotypic changes in arterial wall homeostasis: implications for the pathogenesis of atherosclerosis. Exp. Mol. Pathol., 42 (1985) 139. Majack, R.A., Cook, S.C., Bornstein, P., Control of smooth muscle cell growth by components of the extracellular
108
8
9
10 11
12
13
14
15
16
matrix. Autocrine role for thrombospondin. Proc. Natl. Acad. Sci. USA, 83 (1986) 9050. Gerrity, R.G., The role of monocyte in atherogenesis. Migration of foam cells from atherosclerotic lesions, Am. J. Pathol., 103 (1981) 191. Hansson, G.K., Helm, J., Jonasson, L., Detection of activated T lymphocytes in the human atherosclerotic plaque. Am. J. Physiol., 135 (1989) 169. Ross, R., Raines, E.W., Bowen-Pope, D.F., The biology of platelet-derived growth factor. Cell, 46 (1986) 155. Morisaki, N., Kanzaki, T., Koshikawa, T., Saito, Y., Yosida, S., Secretion of a new growth factor, smooth muscle cell derived growth factor, distinct from platelet derived growth factor by cultured rabbit aortic smooth muscle cells. FEBS Lett., 230 (1988) 186. Tanzawa, K., Shimada, Y., Kuroda, M., Tsujita, Y., Arai, M., Watanabe, Y., WHHL rabbit: A low density lipoprotein receptor-deficient animal model for familial hypercholesterolemia. FEBS Lett., 118 (1980) 81. Kita, T., Brown, M.S., Watanabe, Y., Goldstein, J.L., Deficiency of low density lipoprotein receptors in liver and adrenal gland of the WHHL rabbit, an animal model of familial hypercholesterolemia. Proc. Natl. Acad. Sci. USA, 78 (1981) 2268. Gabor, G., Ecsedi, Sandor, V., Accumulation of cholesterol ester in cultured smooth muscle cells of congenital hypercholesterolemic (WHHL) rabbits treated with normal and WHHL rabbit plasma LDL. Methods Find. Exp. Clin. Pharmacol., 8 (1986) 535. Saito, Y., Bujo, H., Morisaki N., Shirai, K., Yosida, S., Proliferation and LDL binding of cultured intimal smooth muscle cells from rabbits. Atherosclerosis, 69 (1988) 161. Kelly, K., Cochran, B.H., Stiles, C.D., Leder, P., Cellspecific regulation of the c-myc gene by lymphocyte mito-
17
18
19 20
21
22
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24
gens and platelet-derived growth factor. Cell, 35 (1983) 603. Armelin, H.A., Armelin, M.C.S., Kelly, K., Leder, P., Cochran, B.H., Stiles, CD., Functional role for c-myc in mitogenic response to platelet-derived growth factor. Nature, 310 (1984) 655. Banskota, N.K., Taub, R., Zellner, K., Olsen, P., King, G.L., Characterization of induction of protooncogene cmyc and cellular growth in human vascular smooth cells by insulin and IGF-1. Diabetes, 38 (1989) 123. Mahnesmith, R.L., Aronson, P.S., Plasma membrane Na/H exchanger. Circ. Res., 56 (1985) 777. Berk, B.C., Vallega, G., Muslin, A.J., Gordon, H.M., Canessa, M., Alexander, R.W., Spontaneously hypertensive rat vascular smooth muscle cells in culture exhibit increased growth and Nat/H+ exchanger. J. Clin. Invest. 83 (1989) 822. Kobayashi, A., Nara, Y., Nishio, T., Mori, C., Yamori, Y., NaC-H+ exchange activity and cytoplasmic pH in cultured vascular smooth muscle cells from stroke-prone spontaneously hypertensive rats. Jpn. Circ. J., 53 (1989) 528. Brown, M.S., Goldstein, J.L., Receptormediated endocytosis: Insights from the lipoprotein receptor system. Proc. Natl. Acad. Sci. USA, 76 (1979) 3330. Tsukitani, M., Okamoto, R., Suehiro, A., Hatani, M., Fujino, M., Imai, P., Takano, S., Watanabe, Y., Fukuzaki, H., LDL receptor activity of cultured arterial smooth muscle cells from genetically hyperlipidemic rabbits. Kobe J. Med. Sci., 28 (1982) 171. Sachinidis, A., Mengden, T., Lecher, R., Brunner, C., Vetter, W., Novel cellular activities for low density lipoprotein in vascular smooth muscle cells. Hypertension, 15 (1990) 704.
ATHERO
101 Ireland,
Ltd. All rights reserved
0021-~lSO/~1/$03.SO
04696
Vascular smooth muscle cells from genetically hyperlipidemic rabbit (WHHL rabbit) exhibit decreased growth response Asahiko Oguchi ‘, Uichi Ikeda ‘, Hiroshi Miyashita I, Tomoko Ohara ‘, Yoshio Tsuruya ‘, Takashi Natsume ‘, Toshio Yaginuma 2 and Kazuyuki Shimada
’
’Department of Cardiology. Jichi Medical School, Minamikawachi, Tochigi 329-04 (Japan). and ’ Department of Medicine, Omiya Medical Center Jichi Medical School. Omiya. Saitama 330 (Japan) (Received I1 October, 1990) (Revised, received 3 June, 1991) (Accepted 17 June, 1991)
Summary The Watanabe Heritable Hyperlipidemic (WHHL) rabbit is a widely studied animal model for the human genetic disorder familial hypercholesterolemia, and spontaneously develops atherosclerotic disease. We studied the growth characteristics of cultured vascular smooth muscle cells (VSMC) from WHHL rabbits compared with VSMC from Japanese white rabbits. We measured cell proliferation, DNA synthesis, and c-myc proto-oncogene expression, in response to growth stimuli such as fetal bovine serum (FBS) and platelet-derived growth factor (PDGF). VSMC from Japanese white rabbits exhibited a 4-fold increase in cell numbers during a 5-day incubation period compared with those from WHHL rabbits, FBS and PDGF stimulated DNA synthesis, as measured by thymidine incorporation into VSMC, in both Japanese white rabbits and WHHL rabbits, however the response was significantly higher in the former strain. The intracellular pH value of VSMC determined using the pH-sensitive fluorescence dye 2’,7’-bis-carboxyethyl-carboxyfluorescein was significantly higher in WHHL rabbits than in Japanese white rabbits. Proto-oncogene c-myc was induced by exposure of VSMC to FBS, however there was no significant difference in c-myc mRNA levels between the two strains. These results suggest that VSMC from WHHL rabbits are not genetically growth accelerated, but show decreased growth response to growth stimuli.
Key words:
Atherosclerosis;
WHHL
rabbit;
Smooth
muscle
cell; DNA
synthesis;
c-myc
Introduction Correspondence to: Uichi Ikeda, MD, Department of Cardiology, Jichi Medical School, Minamikawachi, Tochigi 329-04, Japan. Tel.: X1-285-44-21 II ext. 3557; Fax 81-285-44-5317.
Atherogenesis involves the migration and proliferation of vascular smooth muscle cells (VSMC)
102 in the intima of vessels, and the increase in wall thickness in the intima may be accounted for by cellular proliferation or hypertrophy of VSMC. The Watanabe Heritable Hyperlipidemic (WHHL) rabbit is a widely studied animal model for the human genetic disorder familial hypercholesterolemia (FH). WHHL rabbits have elevated serum lipids; predominantly low density lipoproteins (LDL) and intermediate density lipoproteins (IDL). They develop spontaneous atherosclerotic disease that mimics the accelerated atherosclerosis seen in familial hypercholesterolemia [l]. To find out whether the growth of VSMC from WHHL rabbits is genetically accelerated or not, we have studied the growth characteristics of VSMC from WHHL rabbits and normal Japanese white rabbits. We report here that the growth responses of VSMC to various growth stimuli are suppressed in WHHL rabbits compared with normal rabbits. Methods VSMC culture
Primary cultures of aortic VSMC were obtained by enzymatic dissociation of the media of thoracic aortae from lo-month-old male WHHL rabbits (n = 4) and age-matched Japanese white rabbits (n = 41, as previously described [2,3]. Cells were grown in Dulbecco’s Minimum Essential Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin, and 100 kg/ml streptomycin. The cultures were harvested twice a week with 0.125% trypsin and passaged at a 1: 3 ratio in lOO-mm culture dishes (Falcon, NJ). The typical growth experiment was performed with VSMC at passage levels of 5-10. Cells were plated at 1.5 x lo4 cells/well in 24-well dishes (Falcon, NJ) in DMEM supplemented as described above and allowed to grow subconfluent for 24-48 h. They were then made “quiescent” by a 24-h incubation in serum-free DMEM supplemented with insulin (5 pg/ml) and transferrin (5 pg/ml). Electron microscopic study
For electron microscopic study, VSMC were prepared in the same way as those used in the in
vitro experiments described above. VSMC were cultured on glass slides immersed in lOO-mm culture dishes (Falcon, NJ) containing the culture medium. The cultured cells adhering on the glass slides were fixed with 2% glutaraldehyde in 0.1 M cacodylate buffer containing 6% sucrose (pH 7.4) for 2 h at 4 o C. The fixed cells were washed with the same buffer and postfixed with 1% osmium tetraoxide for 1 h at ,4 o C. They were dehydrated in an ethanol series followed by propylene oxide and subsequently embedded in Epon 812. After polymerization was completed, the embedded cells were detached from the glass slides with plastic capsules filled with the resin. Ultrathin sections were cut on a LKB ultramicrotome, and examined after electron staining with uranyl acetate and lead citrate, with an electron microscope (Hitachi H-7000, Japan) operating at 75 kV. Determination of DNA synthesis
After 24-h preincubation in serum-free DMEM, FBS or platelet-derived growth factor (PDGF) was added to the culture medium. After 24 h, 1.0 &i/ml tritiated thymidine (2.0 Ci/mmol, Amersham, England) was added to the medium and incubated for 24 h. The medium was aspirated and cells were washed twice with phosphatebuffered saline (PBS), fiied by washing with icecold 10% trichloroacetic acid, and extracted with 0.2 N NaOH over several h. Aliquots were counted in a liquid scintillation counter (Aloka, Japan). Protein content was measured by the Bradford method [4]. Measurements of intracellular pH
Intracellular pH (pHiI of cultured VSMC was determined using the pH-sensitive fluorescent dye BCECF (2’,7’-bis-carboxyethyl-carboxyfluorescein). Cells grown on glass coverslips were loaded with 5 PM BCECF-AM in serum-free culture medium at 37 o C for 60 min, washed 3 times with PBS. The coverslip was then inserted into a cuvette containing 2 ml of HCO;-free solution of the following composition (mM): NaCl 130, KC1 5, CaCI, 1, MgCl,, KH,PO, 2, Hepes 10, glucose 5; pH 7.4. The cuvette was placed in the thermostated holder of a fluorescence spectrophotometer (Shimazu RF 5000, Japan). The fluores-
103 cence intensity (FI) of BCECF was determined at excitation wavelengths (Ex) of 495 and 450 nm, and an emission wavelength (Em) of 530 nm using slits of 5 and 10 nm, respectively. At the end of the experimental procedure, pH, and outside pH (PH.) were equilibrated by permeating the cells with 10 kg/ml nigericin added to the incubation solution. The solution was then titrated with either 1 N HCl or 1 N NaOH over the range of fluorescence values obtained during the experiment. By measuring the pH of the solution after each addition of acid or alkali, a calibration curve of FI vs. pH was developed to compare each experimental sample. BCECF exhibited a nearly linear relationship between FI and pHi, at least over the pH range 6.4-7.6 (data not shown). Rh!A preparation and hybridization
After 24-h preincubation in serum-free DMEM, VSMC were exposed to 10% FBS for l-4 h. Total RNA was extracted from VSMC by the guanidinium isothiocynate/ cesium chloride (GITC/CsCl) procedure. 10 pg of total RNA was size-fractionated by electrophoresis on 1.0% agarose gels, and transferred to nylon membranes (Hybond N+, Amersham, U.K.). Filters were hybridized with a 32P-labeled 0.97-kb v-myc cDNA probe (specific activity > 1 X lo8 cpm/pg DNA) at 60 o C for 24 h. At the end of hybridization, the filters were washed for 30 min in 2 X SSC at 50 o C (1 x SSC contains 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0). Filters were exposed to Kodak XARS film overnight at - 80 o C with one intensifying screen. Autoradiography was quantitated by densitometric scanning (Immunomedica Image Analyzer TIF-64, Japan). Miscellaneous
Statistical analysis was performed using the Student’s t-test. P values < 0.05 were considered to indicate a statistically significant difference. Chemicals were of the highest grade commercially available.
the growth of VSMC from normal Japanese White rabbits and WHHL rabbits. VSMC from the former grew confluently, forming a “hill and valley” structure, whereas VSMC cultures from the latter showed less orderly growth (data not shown). VSMC from Japanese white and WHHL rabbits seemed to have a similar ultrastructure, i.e. the cytoplasm was rich in rough endoplasmic reticulum and free ribosomes, mitochondria was always observed in the perinuclear region, myofilaments were predominantly recognized peripherally, and the nucleus had evident nucleoli (Fig. 1). No apparent differences were noted in shapes or amounts of other ultrastructural components except lysosomes. Lysosomes containing lipofuscin pigments or residual bodies were apparently more numerous in VSMC from WHHL rabbits than in those from normal rabbits. Cell number
Fig. 2 illustrates the growth of VSMC in the presence of 10% FBS. In normal Japanese white rabbit, an approximate 4-fold increase in cell numbers was observed during a 5-day incubation, while in WHHL rabbits the increase was significantly lower than that of normal rabbits. DNA synthesis
FBS and PDGF caused a significant increase in DNA synthesis as measured by [3H]thymidine TABLE 1 EFFECTS OF FETAL BOVINE SERUM (FBS) AND PLATELET-DERIVED GROWTH FACTOR (PDGF) ON DNA SYNTHESIS OF VASCULAR SMOOTH MUSCLE CELLS (VSMC) FROM NORMAL JAPANESE WHITE RABBITS AND WATANABE HERITABLE HYPERLIPIDEMIC (WHHL) RABBITS VSMC were made quiescent by pretreatment with serum-free DMEM medium for 24 h, and then exposed to 10% FBS or PDGF (lo-’ Ml. After 24 h, 1.0 pCi/ml tritiated thymidine (2.0 Ci/mmol, Amersham) was added to the medium. Aliquots were counted in a liquid scintillation counter. Thymidine incorporation was converted per mg protein. Data are mean & SE of 4 separate dishes. * P < 0.05, * * P < 0.02 compared with control.
Results Morphological study
During cultivation, from the primary culture to the 5th subculture, differences were observed in
Control FBS PDGF
Normal (cpm/mg)
WHHL kpm/mg)
41126 + 1127 1260 104 + 371923 * 189649+ 64663 *
14227+ 1579 36302+5140 ** 24929+7691
Fig. 1. Ultrastructure of vascular smooth muscle cells (VSMC) from normal Japanese white rabbits and Watanal be Ileri table for 2 h at 4 o C, and pc3stfi xed with hype ,rli pidemic (WHHL) rabbits. The cells on the glass slides were fiied with 2% glutaraldehyde 1% t39 nium tetraoxide for 1 h at 4 o C. Ultrathin sections were examined with a Hitachi H-7000 electron microscope. (A) Nc Irma1 rabb its (X 12000), (B) WHHL rabbits (X 10000). Arrow indicates lipofuscin pigments and residual bodies in lysosomes. N: nut :leus. M: mitochondria.
105
. & 0
Normal
0
WHHL
f.
;1
P
0
I
I
I
1
3
5 days
a
INTRACELLULAR pH (pHi) OF CULTURED VASCULAR SMOOTH MUSCLE CELLS OBTAINED FROM NORMAL JAPANESE WHITE RABBITS AND WATANABE HERITABLE HYPERLIPIDEMIC (WHHL) RABBITS Cells were loaded with BCECF/AM and pHi was determined from the fluorescence intensity as described in the text. Data are mean i_ SE. *P < 0.01 compared with normal rabbits.
PH,
WHHL
(n = 10)
7.37 & 0.01
WHHL
(n = 10)
7.45 * 0.03 *
FBS
1
PDGF
Fig. 3. DNA synthesis of VSMC from Japanese white rabbits and WHHL rabbits. VSMC were made quiescent by pretreatment with serum-free DMEM medium for 24 h. and then exposed to 10% fetal bovine serum (FBS) or platelet-derived growth factor (PDGF, IO-’ M). After 24 h, 1.0 FCi/ml tritiated thymidine (2.0 Ci/mmol, Amersham) was added to the medium and incubated for 24 h. Cells were washed with phosphate-buffered saline and extracted with 0.2 N NaOH. Aliquots were counted in a liquid scintillation counter. The increase of thymidine incorporation is shown as a relative increase to the control. Data are mean*SE of 4 separate dishes. * P < 0.01 compared with normal rabbits.
incorporation (Table 1). In Japanese white rabbits, 10% FBS stimulated DNA synthesis of VSMC about 30-fold over the unstimulated, control level. PDGF (lo-’ M) stimulated DNA synthesis of VSMC approximately 4.6-fold over the control level. However, in WHHL rabbits, FBS and PDGF stimulated DNA synthesis of VSMC
Normal 0
Oh
Control
2
Normal
Normal
‘i
Fig. 2. Cell proliferation of cultured VSMC. VSMC from normal Japanese white rabbits (0) and WHHL rabbits (0) were incubated in 10% FBS containing DMEM medium for 5 days. Cell number in each well was counted with a hemocytometer. The data are mean +SE of four separate dishes. * P < 0.02. * * P < 0.001 compared with normal rabbits.
TABLE
0 m
WHHL 4h
0
1
4h
28S-
Fig. 4. Induction of c-myc mRNA levels by FBS. Quiescent cells were exposed to 10% FBS for l-4 h. Total RNA was extracted from VSMC by the GITC/CsCI method. 10 Fg of total RNA was size-fractionated by electrophoresis on 1.0% agarose gels, and transferred to nylon membranes. Membranes were directly visualized for 28s ribosomal RNA by UV transillumination (28 S), or hybridized with the “‘P-labeled v-tnyc cDNA probe (c-myc) at 60°C for 24 h, washed for 30 min in 2 x SSC at 50 o C, and exposed to Kodak XAR-5 film overnight at - 70 o C with one intensifying screen. Data are representative of 3 separate experiments.
106
approximately 3-fold and 2-fold over the control level, respectively. FBS and PDGF stimulated DNA synthesis of VSMC in both normal rabbits and WHHL rabbits, but the growth response of VSMC to the growth stimuli was significantly lower in WHHL rabbits compared with normal rabbits (Fig. 3). Intracellular pH levels
After cells grown on coverslips were placed in the cuvette containing HCO;-free Hepes buffered solution (pH 7.401, pH, spontaneously declined by 0.1-0.2 pH units before stabilizing after - 10 min. The pH, values determined after initial decline were 7.37 k 0.01 (mean + SE, n = 10) and 7.45 + 0.03 (n = 10) for Japanese white rabbits and WHHL rabbits, respectively (P < 0.01, Table 2). c-myc mRNA levels
The product of c-myc proto-oncogene is a protein associated with cell nucleus that may be involved in signaling the early events in commitment of cell growth. Northern blotting experiments revealed almost no c-myc transcript in RNA isolated from unstimulated VSMC, however, a significant increase in c-myc mRNA levels was achieved after l-4 h exposure of quiescent VSMC to 10% FBS in both Japanese white rabbits and WHHL rabbits (Fig. 4). Despite the prominent DNA synthesis in normal rabbits after exposure to FBS (Table l), the increase in the c-myc mRNA levels in normal rabbits in response to FBS was similar to that of WHHL rabbits with a ratio of 1.1 k 0.2 (mean k SE, n = 3) after 4 h exposure. Discussion
At least five cell types participate in the formation of an atherosclerotic plaque; endothelial cells, tissue macrophages (derived from blood monocytes), T cells, platelets, and VSMC. VSMC modulation of phenotype from the contractile to synthetic state, and its proliferation in the intima are crucial processes in atherosclerosis [5,6]. The thickened intima mainly consists of proliferated VSMC. Growth factors produced from the endothelial cells [7], macrophages [S], T cells 191,
platelets [lo], and VSMC themselves [ll], can cause migration and proliferation of VSMC. In this study, we have shown that the proliferative capacities of VSMC derived from WHHL rabbits were less than those from normal rabbits. The recent discovery of WHHL rabbits has provided a genetically determined animal model of hypercholesterolemia [12]. WHHL rabbits exhibit clinical, metabolic, and biochemical features that are strikingly similar to those observed in human familial hypercholesterolemia (FH) which can develop into severe atherosclerosis. As in human FH heterozygosity, LDL accumulation in WHHL rabbits arises from a defect of the LDL receptor [13], and cultured VSMC from WHHL rabbits display low rates (< 10% of normal) of LDL binding and uptake. As shown in Fig. 2 and Table 1, the increase in cell numbers and thymidine incorporation were significantly suppressed in VSMC from WHHL rabbits compared with those from normal rabbits. Even when the serum obtained from WHHL rabbits was added to the serum-free culture medium, instead of FBS, the growth response was still significantly lower in WHHL rabbits compared with normal rabbits (data not shown). We could find only one previous report on the growth characteristics of VSMC from WHHL rabbits. Gabor et al. [14] demonstrated that total DNA content of VSMC from WHHL rabbits was about half of that of VSMC from normal rabbits, while the former showed 1Zfold higher thymidine incorporation into DNA than the latter. They speculated that the rate of DNA destruction was much higher in VSMC from WHHL rabbits than normal rabbits. We can not explain the discrepancy of the results, however, one possible explanation is that they used VSMC of intima-media origin, while we used VSMC only from the media origin. VSMC which migrate into the intima from the media are known to alter their characteristics and obtain higher autocrine mitogenic activity than VSMC in the media [11,153. The basal pH, in various vertebrate cells is principally regulated by the plasma membrane Na+/H+ exchanger [19]. In our study, the basal pH, value of WHHL rabbit VSMC was significantly higher than that of Japanese white rabbit
107 VSMC, suggesting the contribution of the altered Na+/H+ exchanger activity to suppressed growth of VSMC in WHHL rabbits. Recently, Berk et al. [20] have reported that the pH, of cultured VSMC obtained from spontaneously hypertensive rats (SHR) was significantly higher than that of normotensive Wistar-Kyoto rats (WKY), and speculated that increased Na+/H+ exchanger contributes to enhanced growth of VSMC in SHR. However, Kobayashi et al. [21] have reported that the basal pH, of VSMC was significantly lower in stroke-prone SHR than in WKY rats. LDL has been shown to cause transient acidification followed by alkalization of VSMC due to stimulation of Na+/H+ exchanger through LDL-receptor pathway [24]. We speculate that the increase in pH, in WHHL rabbits might be due to the lack of LDL receptor activity on VSMC membranes in this animal model [23]. In recent years evidence has been accumulating suggesting that the proto-oncogenes are important regulators of cell growth. Numerous investigations have demonstrated that, when quiescent cells in culture are stimulated to grow by the addition of serum or specific growth factors, rapid and transient induction of the proto-oncogene c-myc occurs [ 16,171. In our study, FBS induced the expression of c-myc in quiescent VSMC from both Japanese white rabbits and WHHL rabbits to the same degree, although the growth response to FBS was much higher in the former than the latter. c-myc induction is not entirely associated with events leading to DNA synthesis and cellular proliferation. Banskota et al. [18] demonstrated that monoclonal antibody to the insulin-like growth factor 1 (IGF-1) receptor, alfa-IR3, inhibited both the insulin and IGF-1 effects on stimulating DNA synthesis of human VSMC or cellular proliferation, while alfa-IR3 itself stimulated cmyc mRNA expression. LDL-derived cholesterol is an essential component of cell membranes [22]. VSMC obtain cholesterol through the LDL receptor pathway [221, and the cultured VSMC from WHHL rabbits can not internalize and degradate LDL [23], causing a deficit of a substrate for membrane synthsis. We therefore speculate that the lack of cholesterol incorporation and/or the altered Na’/H+ exchanger activity might contribute to
the poor DNA and protein synthesis in WHHL rabbits, although surface receptor systems to the growth stimuli are intact judging from the c-myc mRNA expression (Fig. 4). In conclusion, VSMC from WHHL rabbits are not genetically growth accelerated, but showed a tendency to lower mitogenic activity than those from Japanese white rabbits. In the process of formation of atherosclerotic lesions, we suppose that factors other than the mitogenicity of VSMC play important roles in the abnormal proliferation of VSMC and hence the development of atherosclerotic lesions in WHHL rabbits. Acknowledgments
The authors thank Etsuko Chiku for her technical assistance. We also thank Drs. Yoshio Watanabe and Masahi Shiomi (Kobe University School of Medicine) for donating the WHHL rabbits. This study was supported by the Uehara Memorial Foundation, the Kowa Life Science Foundation, and the Takeda Medical Research Foundation (Japan). References Goldstein, J.L., Kita, T., Brown, MS., Defective lipoprotein receptors and atherosclerosis. Lessons from an animal counterpart of familial hypercholesterolemia. N. Engl. J. Ned., 309 (1983) 288. Charnley, J.H., Campbell, G.R., McConell, J.D., Groschel-Stewart, 1J., Comparison of vascular smooth muscle cells from adult human, monkey and rabbit in primary culture and in subculture. Cell Tissue Res., 177 (1977) 503. Ikeda, U., Ikeda, M., Oohara, T., Kano. S., Yaginuma, T., Mitogenic action of interleukin-1 on vascular smooth muscle cells mediated by PDGF. Atherosclerosis, 84 (1990) 183. Bradford, M.M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem., 72 (1976) 248. Charnley-Campbell, J.H., Campbell, G.R., What controls smooth muscle phenotype? Atherosclerosis, 40 (1981) 347. Campbell, G.R., Campbell, J.H., Recent advances in molecular pathology. Smooth muscle phenotypic changes in arterial wall homeostasis: implications for the pathogenesis of atherosclerosis. Exp. Mol. Pathol., 42 (1985) 139. Majack, R.A., Cook, S.C., Bornstein, P., Control of smooth muscle cell growth by components of the extracellular
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