Identification of intimal subendothelial cells from human aorta in primary culture

Subendothelial cells (SEC) were obtained from the inner intimal layer of adult human aorta by collagenase treatment. SEC were identified in primary culture either as smooth muscle cells by staining with FITC-labeled antisera against human smooth muscle myosin or as macrophages, foam cells and contaminating endothelial cells by their uptake of malondialdehyde treated low density lipoproteins labeled with fluorescent dye 3,3'-dioctadecylindocarbocyanine. Between 1 and 5 days in culture, along with smooth muscle cells (SMC, 38-82%), endothelial cells (0-9%), macrophages and foam cells (2-32%), one more type of cell was found. This cell type resembled SMC in size and shape, but was not stained by antisera to SMC myosin. By ultrastructural criteria these cells were characterized as modulated SMC for they contained prominent rough endoplastic reticulum and Golgi complex together with basement membrane and a large number of plasmalemmal vesicles. Like SMC they reacted with phalloidin and were stained by anti-vimentin but not by anti-desmin monoclonal antibodies. The proportion of such cells varied from 5 to 33% of total cell number and increased in parallel to macrophages and foam cells in vessels with well developed atherosclerotic lesions. We conclude that the applied technique may be used for identification of cultured vascular cells including modulated SMC.     

The induction of smooth muscle cell proliferation in vitro using an organ culture system

Agents which promote vascular smooth muscle cell (SMC) proliferation are still unknown and cannot be readily defined in vivo. To examine this problem, we have developed a technique to maintain arteries in culture for more than two weeks without loss of endothelium and without loss of contractility. These vessel segments were maintained in 30% calf serum, and yet SMC replication rate in the media was found to be below 0.1% per day. No loss of medial cells nor medial necrosis was observed. Gentle endothelial denudation did not cause intimal thickening. If, in addition to denudation, direct mechanical injury was applied, then the replication rate of intimal SMC was found to be 60% per day during the first week. These cells were confirmed to be of SMC origin using cell specific antibodies. Since SMC start to proliferate after mechanical injury and not after application of serum, we conclude in our experimental model that exogenous growth factors play a minor role in the process of induction of proliferation of these cells.     

Rapamycin reduces neointima formation during vascular injury

BACKGROUND: Proliferation and migration of vascular smooth muscle cells (SMCs) mark the key processes in the development of bypass graft disease and during neointima formation in restenosis after angioplasty. Growth factors are potent SMC mitogens as they are involved in SMC proliferation and in extracellular matrix (ECM) synthesis. Based on these premises, we examined the effect of the proliferation inhibitor rapamycin in human SMC culture and in a rabbit vascular injury model. MATERIALS AND METHODS: Injection of rapamycin or its vehicle was performed with an infusion-balloon catheter directly into the vessel wall during vascular injury. The intima/media ratio was determined histologically whereas the protein expression was analysed using the powerful two-dimensional gel electrophoresis (2D page) technique. Inhibition of proliferation after rapamycin application was estimated in a human SMC culture for time and dose dependent effects. RESULTS: Rapamycin treatment resulted in a significant reduction of intima media ratio compared to vehicle treated animals after three weeks (0.65 +/- 0.1 vs. 1.2 +/- 0.2 intima-media-ratio, p < 0.05). 2D electrophoresis analysis proved increased ECM synthesis following angioplasty (i.e., lamin, vimentin) in vehicle treated animals. Local rapamycin administration resulted in profound reduction of ECM synthesis after vascular injury. In in-vitro experiments exposure of cultured human SMCs to rapamycin resulted in a significant and dose-dependent (1 nm-100 nm) reduction of human smooth muscle cell proliferation measured by cell counting. CONCLUSION: These above mentioned results suggest that protein synthesis in addition to reduction of cellular proliferation plays an important role following vascular injury, since application of rapamycin resulted in the reduction of SMC proliferation and ECM-synthesis.     

Phenotype related changes of intimal smooth muscle cells from human aorta in primary culture.

To study the functional characteristics of smooth muscle cell (SMC) phenotypes, we have investigated myosin expression, cell proliferation, collagen production and low-density lipoprotein (LDL) receptor activity in intimal SMCs of normal human aorta during their growth in primary culture. By staining with rabbit antibodies to smooth muscle myosin (ASMM) 3 cell types could be distinguished in culture: homogeneously stained cells, cells with discontinuous myosin fibrils and myosin-negative cells. The ratio of cell types greatly changed with culture growth: on days 5, 7 and 14 it was 82:1:17%, 70:5:25% and 10:30:60%, respectively. After 5-6 days of culture intimal SMCs began to proliferate and DNA-synthesizing nuclei were seen 1.5-4.3 times more frequently in myosin-negative cells than in cells with homogeneous myosin distribution. At that time the number of cells reacted with monoclonal antibody (MAb) to an epitope shared collagen types I and III started to increase. By double immunofluorescence staining it was shown that the cultured cells containing both ASMM and MAb markers were found 2.0-4.8 times more rarely than MAb-positive staining in myosin-negative cells. During the first 5 days in culture LDL binding and uptake were diminished in intimal cells with intercellular lipid inclusions independently of their myosin staining pattern, but their activity increased with culture growth. Thus, SMCs from human aortic intima change their phenotype on days 6 and 7 in primary culture as manifested by alteration of myosin expression, increased cell proliferation, collagen production and LDL receptor activity. Changes in myosin expression, however, are not an essential prerequisite for cell proliferation and collagen production.     

Effect of platelet factors on migration of cultured bovine aortic endothelial and smooth muscle cells

Endothelial cell (EC) injury and the response of EC and smooth muscle cells (SMCs) to injury contribute to the pathophysiology in patients with vascular disease and atherosclerosis. Since platelets have been suggested to play an important role in modulating vascular injury, the present study was undertaken to examine the influence and mechanism of action of individual platelet factors on bovine aortic EC and SMC migration using an in vitro wound assay system. Serotonin decreased EC proliferation and reduced EC migration 21 +/- 1% (p less than 0.005), which was attenuated by imipramine. Transforming growth factor-beta reduced EC proliferation and decreased EC migration 52 +/- 3% (p less than 0.005). Norepinephrine increased EC proliferation but decreased EC migration 26 +/- 2% (p less than 0.005), which was abolished by phenoxybenzamine. Histamine increased EC proliferation but reduced EC migration 29 +/- 2% (p less than 0.005), which was attenuated by diphenhydramine. Platelet-derived growth factor decreased EC proliferation and decreased EC migration 40 +/- 2% (p less than 0.005). In contrast, serotonin increased SMC proliferation and increased SMC migration 31 +/- 2% (p less than 0.005), which was abolished by ketanserin. Transforming growth factor-beta increased SMC migration 35 +/- 5% (p less than 0.005). Norepinephrine increased SMC proliferation and increased SMC migration 43 +/- 4% (p less than 0.005), which was abolished by propranolol. Histamine increased SMC proliferation and increased SMC migration 38 +/- 3% (p less than 0.005), which was abolished by cimetidine. Platelet-derived growth factor increased SMC proliferation and increased SMC migration 40 +/- 3% (p less than 0.005).(ABSTRACT TRUNCATED AT 250 WORDS)     

Phenotypic expression of surface antigens of rabbit aortic smooth muscle cells in culture. Monoclonal antibody, 2P1A2, characteristic of smooth muscle cells present in atherosclerotic plaque, is not correlated with cell proliferation

The expression of smooth muscle cell (SMC) antigens was studied in culture by immunofluorescence and immunoelectron microscopy. As specific SMC markers, we used 2 monoclonal antibodies (MAb), 1PC1 and 2P1A2 which are able to detect atherosclerotic plaques in the rabbit. MAb 1PC1 recognizes an antigen expressed on the cell surface, starting on the 7th day in primary culture after serum activation, and then secreted. On a confluent SMC monolayers this antigen appears outside the cell as an important filamentous network. The kinetics of secretion of this external protein recognized by 1PC1 corresponds to the kinetics of the secretory phenotype described by Chamley-Campbell and Campbell (Atherosclerosis, 40 (1981) 347). 2P1A2 MAb is specific for SMCs exclusively present in the rabbit atherosclerotic plaque. We studied the degree of reactivity of 2P1A2 with SMCs during primary cell culture. This "atherosclerotic" antigen of SMCs recognized by 2P1A2 is expressed in culture conditions by SMCs from rabbit normal media. This antigen appears after 3 days of serum activation, and heparin growth inhibition does not interfere with its expression. 2P1A2 recognized antigen is expressed during all cell cycle phases without amplification. 3 days after fetal calf serum (FCS) stimulation of cells which are in G0/G1, 89% are labelled by 2P1A2, 4 days later G0/G1 positive cells constitute 49%. We conclude that 2P1A2 immunolabelling on the SMC surface reflects an activated state which is not correlated with SMC proliferation.     

In vitro and ex vivo regulation of vascular smooth muscle cell growth and phenotypic modulation by sulphated polysaccharides

Heparin and pentosan polysulphate (PPS), a semi-synthetic sulphated polysaccharide, affected the phenotypic modulation of primary or subcultured rabbit smooth muscle cells (SMC) and at the same time inhibited their proliferation in culture. PPS was 5 fold more potent than heparin. Ex vivo, after subcutaneous administration of PPS or heparin (8 mg/kg/day for 13 days), SMC isolated from treated rabbits were growth-inhibited. At the same time, they reversed promptly to the contractile state as evidenced by immunofluorescent detection of intracellular myosin or electron microscopy. This ex vivo inhibitory effect was related to the dose and the duration of treatment, and was lost if 6 hours elapsed between the final dose and removal of the aorta. Such an effect was also observed after oral treatment with PPS (200 mg/kg/day for 7 days).     

Relationship between type IV collagen degradation,metalloproteinase activity and smooth muscle cell migration and proliferation in cultured human saphenous vein

The relationship between degradation of basement membranes, metalloproteinase (MMP) activity and smooth muscle cell (SMC) migration and proliferation has not been previously investigated in any intervention study. We used adenoviral overexpression of tissue inhibitors of metalloproteinases (TIMPs) in cultured human saphenous veins. By immunocytochemistry, the percentage of medial SMC surrounded by basement membrane type IV collagen (Coll-IV) decreased from 93+/-1 to 77+/-4% and 82+/-1% (n=18, both P<0.001) after 7 and 14 days of culture, respectively, while all SMC that migrated to the neointima lacked Coll-IV. Overexpression of TIMP-1 or TIMP-3 significantly increased the percentage of medial SMC surrounded by Coll-IV (94+/-2 or 98+/-2%, respectively, both P<0.01 vs. no treatment) and decreased the number of neointimal SMC. Some 44+/-18 and 30+/-6%, respectively, of BrdU or PCNA labeled medial SMC remained surrounded by type IV collagen and this was not affected by overexpression of TIMP-1 or TIMP-3. We conclude that MMPs mediate loss of basement membrane and this is closely related to SMC migration. SMC proliferation does not require complete basement membrane degradation, which itself does not require MMPs in proliferating SMC.     

Focal expression of angiotensin II type 1 receptor and smooth muscle cell proliferation in the neointima of experimental vein grafts: relation to eddy blood flow

Eddy flow has been shown to promote focal smooth muscle cell (SMC) proliferation and neointimal formation in experimental vein grafts. This study focuses on whether the angiotensin II type 1 (AT(1)) receptor mediates these events. Experimental vein grafts with and without eddy flow were created in the rat. Losartan was used to assess the influence of the AT(1) receptor on SMC proliferation. In vein grafts with eddy flow, apparent focal expression of AT(1) mRNA and protein was found in the leading region of the proximal focal neointima, where eddy flow occurred, but not in the trailing region, where eddy flow diminished, at days 5, 10, 20, and 30. The rate of SMC proliferation in the leading region (10.9+/-1.4%, 19.5+/-2.2%, 12.2+/-2.0%, and 6.9+/-1.3% at these times, respectively) was significantly higher than that in the trailing region (9.5+/-1.8%, 15.3+/-2.0%, 8.2+/-1.9%, and 3.2+/-0.7%) in these vein grafts. When eddy flow was prevented in engineered vein grafts, no apparent location difference was found in the distribution of AT(1) receptor mRNA and protein in the neointima, and the rate of SMC proliferation (5.3+/-1.0%, 5.8+/-0.9%, 3.4+/-1.0%, and 3.7+/-0.9% at days 5, 10, 20, and 30, respectively) was reduced significantly. In vein grafts with losartan, the rate of SMC proliferation in the leading region of the neointima (9.4+/-1.8%, 10.1+/-1.3%, 8.3+/-0.9%, and 4.2+/-0. 5% at days 5, 10, 20, and 30, respectively) was significantly lower than that in vein grafts without losartan. These results suggested that eddy flow upregulated the AT(1) receptor, which in turn mediated focal SMC proliferation in the neointima of experimental vein grafts.     

External beam radiation after stent implantation increases neointimal hyperplasia by augmenting smooth muscle cell proliferation and extracellular matrix accumulation.

OBJECTIVES: We sought to examine the effects of high volume external beam radiation (EBR) after stent implantation on neointimal hyperplasia, smooth muscle cell (SMC) proliferation, presence of inflammatory cells and expression of extracellular matrix (ECM). BACKGROUND: Endovascular irradiation has been shown to reduce restenosis rates after angioplasty in preliminary trials, but conflicting results have been reported for the effects of external beam irradiation. METHODS: Forty-three Palmaz-Schatz stents were implanted into iliac arteries of New Zealand White rabbits. The arteries were externally irradiated after stent implantation with a single dose of 8 Gy (at day 3) or 16 Gy in two fractions (8 Gy at days 3 and 4) by means of a linear accelerator. In the control rabbits, no radiation was applied after stent implantation. Smooth muscle cells, macrophages and ECM were studied by immunohistochemistry at one and 12 weeks after stent implantation. Collagen type I and biglycan messenger ribonucleic acid (mRNA) levels were assessed by Northern blot analysis at one week. Neointimal cell densities and arterial lumen stenosis were measured by histomorphometry at 12 weeks. RESULTS: At 1 week, SMC proliferation at the site of stent implantation was increased after EBR with 8 and 16 Gy (26 +/- 5%, 32 +/- 3% vs. 17 +/- 8%; p < 0.01, 16 Gy vs. control). External beam radiation with 8 and 16 Gy augmented SMC proliferation proximal and distal to the angioplasty site (11 +/- 3%, 14 +/- 3 vs. 6 +/- 1%; p < 0.01, 16 Gy vs. control). Collagen type I and biglycan mRNA levels were elevated in stented arteries after EBR with 16 Gy. At 12 weeks, a marked decrease in neointimal cell density (248 +/- 97 vs. 498 +/- 117 SMCs/0.1 mm2 neointima; p < 0.005 vs. control) was noted after EBR with 16 Gy. Irradiation with 8 and 16 Gy increased arterial lumen stenosis compared with nonirradiated control rabbits (45 +/- 7%, 55 +/- 9% vs. 33 +/- 7%; p < 0.05, 8 Gy and p < 0.001, 16 Gy vs. control). CONCLUSIONS: High volume external beam radiation at doses of 8 or 16 Gy causes restenosis by augmenting proliferative activity at and adjacent to the site of stent implantation, and by dose-dependent up-regulation of extracellular matrix expression. The study suggests that excessive matrix accumulation is an important determinant of failure of radiation therapy to prevent restenosis.     

In vivo temperature heterogeneity is associated with plaque regions of increased MMP-9 activity.

AIMS: Plaque rupture has been associated with a high matrix metalloproteinase (MMP) activity. Recently, regional temperature variations have been observed in atherosclerotic plaques in vivo and ascribed to the presence of macrophages. As macrophages are a major source of MMPs, we examined whether regional temperature changes are related to local MMP activity and macrophage accumulation. METHODS AND RESULTS: Plaques were experimentally induced in rabbit (n=11) aortas, and at the day of sacrifice, a pull-back was performed with a thermography catheter. Hot (n=10), cold (n=10), and reference (n=11) regions were dissected and analysed for smooth muscle cell (SMC), lipids (L), collagen (COL), and macrophage (MPhi) cell densities (%); a vulnerability index (VI) was calculated as VI=MPhi+L/(SMC+COL). In addition, accumulation and activity of MMP-2 and MMP-9 were determined with zymography. Ten hot regions were identified with an average temperature of 0.40+/-0.03 degrees C (P<0.05 vs. reference) and 10 cold regions with 0.07+/-0.03 degrees C (P<0.05 vs. hot). In the hot regions, a higher macrophage density (173%), less SMC density (77%), and a higher VI (100%) were identified. In addition, MMP-9 (673%) activity was increased. A detailed regression analysis revealed that MMP-9 predicted hot regions better than macrophage accumulation alone. CONCLUSION: In vivo temperature measurements enable to detect plaques that contain more macrophages, less SMCs, and a higher MMP-9 activity.     

Studies on aorta during development. I. Fetal rabbit aorta under ex vivo and in vitro conditions: rapid changes in smooth muscle cell phenotype, cell proliferation and cholesterol content with organ culture

The structural development of the already well defined fetal rabbit aortic wall from 22 to 31 days of gestation in vivo consists of increasing aortic wall thickness, elastic lamina continuities, extracellular matrix deposition, and maturing of the fine structure of the medial smooth muscle cells. In vivo at term (31 days), the mature aortic smooth muscle cells demonstrated the characteristic thin, thick and intermediate filaments, dense plaques, endoplasmic reticulum, golgi, plasmalemma vesicles and an incomplete basal lamina. The fetal aorta rapidly responded to organ culture with various changes. Fetal smooth muscle cells modified their phenotype to the synthetic state when cultured in both serum-supplemented and serum-free media. This smooth muscle cell modification occurred after 3 days of culture in fetal explants. The synthetic type smooth muscle cells (fetal) began to proliferate after 6 days of culture. This proliferation resulted in a peripheral outgrowth after 9 days of 10-20 layers in fetal cultures from serum-supplemented media and of 2-4 layers in serum-free media. The orderly arrangement of the internal elastic lamina and alternating medial layers of smooth muscle cells and elastic lamina seen in vivo was disrupted along with increased matrix after 9 days of fetal explant culture. Significant numbers of 'modified' synthetic phenotype smooth muscle cells were not observed in adult aortic explants until after 15 days in culture in serum supplemented media. The mature contractile phenotype smooth muscle cell predominated in adult explants cultured in serum-free media. Significant synthetic phenotype smooth muscle cell proliferation only occurred in adult explants after 15 days culture in serum-supplemented media. When compared to aorta in vivo evidence for increases in cholesterol esterification were observed in both fetal (9 days) and adult (15 days) explants cultured in both serum-supplemented and serum-free media. The fetal aorta in organ culture appeared to be more susceptible than the adult aorta to (a) phenotypic modulation of smooth muscle cells to the synthetic state, (b) smooth muscle cell proliferation, and (c) early cholesteryl ester accumulation.     

Intraperitoneal infusion of homocysteine increases intimal hyperplasia in balloon-injured rat carotid arteries.

Hyperhomocysteinemia is a significant risk factor in atherosclerosis and thrombosis. However, its role in the development of intimal hyperplasia after arterial reconstructive procedures remains uncertain. We therefore studied the effect of homocysteine on intimal hyperplasia in a rat model of carotid artery balloon injury. Twenty-four Sprague-Dawley rats were divided into three groups: control (saline infusion), and low dose (0.14 mg/day) and high dose (0.71 mg/day) homocysteine delivered continuously via osmotic pumps implanted intraperitoneally. All animals underwent left common carotid artery balloon denudation with sacrifice after 14 days. Plasma homocysteine levels, intimal hyperplasia, and cell proliferation of rat carotid arteries were determined. In vitro rat smooth muscle cell (SMC) proliferation with homocysteine treatment was also performed. Plasma homocysteine levels at sacrifice were 1.80+/-0.35, 2.65+/-0.05 and 3.50+/-0.22 microM in three groups, respectively. Intimal hyperplasia developed in all balloon-injured arteries in both control and homocysteine-treated animals. The intimal area and intima/media area ratio were increased by 92% (P<0.05) and 105% (P<0.05), respectively, in the high dose-homocysteine-treated animals as compared to the control animals. Homocysteine (high dose) also significantly promoted the intimal cell proliferation (bromodeoxyuridine incorporation) by 2.2-fold as compared to controls. Furthermore, homocysteine treatment in the cell culture study showed a concentration-dependent increase of rat SMC proliferation. These data demonstrate that the continuous intraperitoneal administration of homocysteine significantly increases intimal hyperplasia and SMC proliferation after carotid artery balloon injury in the rat as well as in vitro SMC proliferation. This study suggests that, following arterial reconstructive procedures, elevated plasma homocysteine may increase the complications of clinical restenoses that are associated with intimal hyperplasia.     

Rabbit ear model of injury-induced arterial smooth muscle cell proliferation. Kinetics, reproducibility, and implications

Recently, considerable interest has focused on the vascular smooth muscle cell (SMC) response to injury, particularly as it relates to restenosis after angioplasty. In an effort to find an optimal experimental model of arterial SMC proliferation after injury, we examined the effects of external injury to the central artery of the rabbit ear and assessed the reproducibility, morphological changes, and time course of cellular proliferation after such an injury. With rabbits under general anesthesia, direct pressure was applied at two sites along the central artery of the ears of 19 New Zealand White rabbits. Rabbits were maintained on a diet of 2.4% fat and 0.001% cholesterol throughout the experiment. In seven rabbits examined after 21 days, marked SMC proliferation with neointimal formation was observed at all 28 sites (100%). Mean neointimal area, expressed as a percentage of the area of the tunica media, was 82 +/- 40% (range, 21-203%). Compared with the uninvolved artery displaced 2 mm from the injury site, mechanical crush caused a 38% increase in total vessel area (p less than 0.001), a 40% decrease in luminal area (p less than 0.002), and no change in the area of the media. Serial histological studies were performed 1-42 days after injury, using light and electron microscopy and bromodeoxyuridine immunohistochemistry. Beginning at day 3, activated medial SMCs were noted to migrate through defects in the internal elastic membrane, with a gradual increase in neointimal area between days 5 and 12. Peak DNA synthesis was identified in the media 5 days after injury, with proliferative activity shifting almost exclusively to the neointima thereafter.(ABSTRACT TRUNCATED AT 250 WORDS)     

不要低估血管平滑肌细胞在动脉粥样硬化斑块形成中的作用

血管内皮细胞、平滑肌细胞(SMC)和巨噬细胞共同参与动脉粥样硬化(As)斑块形成。近年研究表明,SMC来源的细胞占As斑块中细胞总数的70%以上。As斑块中的SMC通过自分泌细胞因子促进自身的增殖、迁移和炎症反应,通过旁分泌激活单核/巨噬细胞并将其募集到As损伤部位,同时通过其细胞膜表面表达的脂蛋白受体摄取脂质形成泡沫细胞。SMC在As斑块形成中扮演十分重要的角色,应进一步深化对SMC在As发生发展中的作用及作用机制的研究。     

Activated platelets and leucocytes cooperatively stimulate smooth muscle cell proliferation and proto-oncogene expression via release of soluble growth factors.

BACKGROUND: Previous studies indicate that platelets and leucocytes might contribute to the development of neointimal hyperplasia following arterial injury. The present study was aimed at further investigating the role of platelets and leucocytes, alone or in combination, in promoting vascular smooth muscle cell (SMC) proliferation in vitro, focusing on the relative contribution of different soluble growth factors released by these cells, and on the ability to induce proto-oncogene expression, such as c-fos. METHODS: SMCs from rabbit aortas, made quiescent by serum deprivation, were stimulated with either activated platelets, leucocytes, or both, separated from SMCs by a membrane insert. SMC proliferation was evaluated by measuring the incorporation of 3H-thymidine. The relative contribution of different platelet-derived mediators to SMC growth was evaluated by adding either ketanserin, a 5-HT2 receptor antagonist, R68070, a TxA2 receptor antagonist, BN52021, a platelet activating factor (PAF) receptor antagonist, and trapidil, a platelet derived growth factor (PDGF) receptor antagonist. The role of different leucocyte sub-populations (neutrophils and monocytes + lymphocytes) was also determined in additional experiments. RESULTS: SMC proliferation was significantly increased by activated platelets to 360 +/- 9% of control values (P < 0.05). This effect was reduced by ketanserin, R68070, BN 52021 or trapidil. Whole leucocytes, neutrophils or lymphocytes + monocytes also increased SMC proliferation with respect to control experiments. Simultaneous stimulation of SMCs by platelets and whole leucocytes was associated with a significant greater increase in SMC proliferation as compared to SMC stimulated with platelets or leucocytes alone. c-fos expression, almost undetectable in unstimulated SMCs, was markedly increased by activated platelets or leucocytes. CONCLUSIONS: Activated platelets promote SMC proliferation in vitro via release of soluble mediators, including serotonin, thromboxane A2 PAF and PDGF; activated leucocytes also induce a significant SMC proliferation and exert an additive effect when activated together with platelets; SMCs stimulated with activated platelets and leucocytes show an early expression of the proto-oncogene c-fos.