At least in vitro the arterial smooth muscle cell population appears to include two cell types that have stable phenotypes in vitro. Existence of similar distinct lineage is suggested by the developmental pattern of expression of elastin, a gene whose expression is greatly enhanced in pup or intimal smooth muscle cells. Importantly for this hypothesis, elastin synthesis has been noted by others to be abnormal in the atherosclerotic plaque and we find enriched elastin synthesis in the neointima formed after balloon injury.
For atherosclerosis, the second cell type could explain the ontogeny of the intimal cell mass and might contribute to monoclonality.
For hypertension, the second cell type may represent a developmental stage that could be important in the ontogeny of early structural changes in hypertension.

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With increase in the elderly population, cerebral atherosclerosis has become one of the major medical problems in Japan, particularly diseases related to cerebral infarction. I report in this paper the relation between cerebrovascular diseases and various vascular factors from clinical as well as pathoanatomical aspects.
1) Moderate or severe atherosclerosis appeared as early as in the twenties, thereafter with an increase in the frequency and degree of the pathology in linear form, as shown from results of the nationwide autopsy study sponsored by the Ministry of Education (Chairman: S. Okinaka; Pathologists: Ohtsu, S., et al.).
2) The incidence of subjects with severe atherosclerosis in the aorta and middle cerebral artery, increased after 60 years of age, but this tendency was not seen in the coronary and basilar arteries. I call the former the “Living Artery” and the later the “Life Artery”. This distinction may be useful for discussing progression of atherosclerosis of visceral organs.
3) More than 50% stenosis on cross-sectioned areas of the internal carotid artery was found in 25% and in the vertebral artery in 23%, in a routine 400 autopsy series of subjects aged 60 years or more; complete occlusion occurred in 2% in the former and in 6% in the latter.
4) In diabetics, atherosclerosis of the cervical and intracranial arteries was more pronounced than in non-diabetics. Within the skull, atherosclerosis was more advanced in the posterior circulation in diabetics. The subjects with combined diabetes mellitus, hypercholesterolemia and hypertension almost always showed cerebral infarcts at autopsy. This finding was confirmed by MRI in the clinical level.
5) Severe atherosclerosis was significantly frequently distributed in patients with myocardial infarction and/or hypertension. In patients with pulmonary emphysema, malignancy or liver cirrhosis, cerebral atherosclerosis was significantly slighter than in the other diseases, the reasons for which were considered in the discussion.
6) In patients showing carotid bruits in their clinical courses, cerebral infarction at autopsy was significantly more frequent than in those without bruits.
7) Important risks of cortical type infarction were higher LDL- and lower HDL-cholesterol value than in patients with infarctions in the perforating arterial system. In the latter there was a high risk of hypertension.
8) Arteries with advential hyperplasia were common in the infarcted areas of the brain. The pathogenesis of such a secondary change of the arteries was considered in the discussion.
9) Familial hypercholesterolemia of the homoor heterozygote type showed a relatively high cerebral infarction on MRI.
10) Lp(a) seemed to be a strong indicator for coronary heart disease and cerebral infarction, irrespective of other blood lipid data.
11) In patients with vascular dementia, blood pressure should be maintained on more or less higher levels. Greater attention should be paid to the microcirculation of the brain.
12) In the future, molecular biological common factors including both atherogenesis and thrombogenesis should be investigated, and special patterns of cerebral circulation should be carefully considered.

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Thrombin and other proaggregators have been reported to induce endothelium dependent relaxation in porcine coronary arteries and LDL blocked the relaxation rapidly and reversibly (Tomita et al., Circ. Res. 1990, 66, 18-27). This inhibitory mechanism of LDL was investigated. Lysolecithin acyltransferase (LAT) activity was dose depenedently enhanced by LDL within the range of LDL concentrations in which it exerted an inhibitory effect on the relaxation. This enzyme activity was demonstrated in cultured endothelial cells from porcine aortas, and the activity in the intact cells was profoundly enhanced in the presence of thrombin and LDL in the medium. Heat-treatment (60°, 10min), or acid-treatment (pH 2, 30min at room temp) of LDL, resulted in a complete loss of the stimulatory effect on LAT activity as it caused a loss of LDL-inhibitory effects on the relaxation. In contrast, the DFP-treatment did not alter either of the effects of LDL. Thimerosal (10^-8-10^-4 M) induced endothelium-dependent relaxation, while the activation by LDL of LAT was blocked in the presence of thimerosal (10^-4 M). Thus, the effective deactivation mechanism of LDL inhibition of the endothelium-dependent relaxation coincided well with that for the enhancement of LAT activity. These results suggest that the profound enhancing effect of LDL on LAT activity in the endothelium was possibly involved in the inhibition of endothelium-dependent relaxation by LDL.

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We investigated the possibility of the existence of hepatic Chylomicron Remnant (CR) receptors with HepG₂ cells. In HepG₂ cells incubated with LPDS or LPDS+25 OH cholesterol for 24 hours, the binding and internalization of ¹²⁵I-CR were inhibited about 79.0% and 78.8% respectively by the incubation with unlabelled CR. On the other hand, in HepG₂ cells incubated with LPDS or LPDS+25 OH cholesterol for 24 hours, the binding and internalization of ¹²⁵I-CR were inhibited about 80.8% and 52.7% respectively by the incubation with unlabelled LDL. In human fibroblasts incubated with LPDS or LPDS+25 OH cholesterol for 24 hours, the bining and internalization of ¹²⁵I-CR were inhibited about 79.7-82.4% and 73.7-78.6% by the incubation with unlabelled CR or unlabelled LDL. These results suggest that HepG₂ cells may have functional remnant receptors which are not regulated by cholesterol loading.

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Apo A-I has been known to have five isoforms which can be separated by two-dimensional gel electrophoresis. The three acidic isoforms (A-I₀, A-I_-1, A-I_-2) are mature apo A-I consisting of 243 amino acids. The other two basic isoforms (A-I₊₂, A-I₊₁) are proapo A-I, having a six-amino-acid extension to the NH₂ terminal of the mature apo A-I.
In the present study, the patterns of apo A-I isoforms under three different conditions were examined. Firstly, the effect of weight reduction on the apo A-I isoforms of 11 normolipidemic obese women was investigated. Secondly, the effect of Probucol administration on the apo A-I isoforms of 15 hypercholesterolemic subjects was investigated. Finally, the apo A-I isoform pattern of 9 trained swimmers was compared with that of 7 sedentary controls.
Weight reduction of obese women caused proapo A-I decrease not only in relative percentage, but in the total amount. (significant only in HDL₃-A-I₊₂, 3.1±1.2 vs 2.3±0.5mg/dl, p>0.05) Mature apo A-I also tended to decrease in amount. Probucol administration caused significant increase of HDL₂-A-I₊₂ in relative percentage. (5.4±1.4 vs 6.6±1.7%, p>0.02). Mature apo A-I tended to decrease both in relative percentage and total amount. Swimmers' apo A-I isoform was high in the pro/mature ratio in HDL₃. (0.10±0.02) Significance was seen only in HDL₃-A-I₊₂. (5.9±1.0 vs 3.9±1.0%, p<0.01)
Apo A-I isoform pattern is thought to be influenced by at least three factors: synthesis and degradation of apo A-I and proapo A-I protease activity. The results of the present study suggest that the determination of apo A-I isoforms might provide crucial information about apo A-I metabolism.

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Morphometric studies of endothelial cells on the aortic surface of flow dividers of aortic branchings to brachiocephalic (BC) and subclavian (SA) arteries were performed in either normo- or cholesterol-fed rabbits using scanning electron microscopy.
The cells of BC in the sudanophilic region were variously ellipsoidal, and somewhat round. All of the ellipsoidal cells were arranged multidirectionally, presumably caused by separation of the blood flow. The region was located just downstream from the leading edge covered by elongated fusiform endothelial cells arranged in alignment and parallel to blood flow. The apex of the flow divider, or stagnation point was covered by round cells. SA had also the sudanophilic region covered by ellipsoidal endothelial cells, which was downstream from the stagnation point which was not coincidental with the apex of the flow divider of SA. The areas covered by either round or elongated fusiform cells were free from lipid deposition.
It can be concluded that the sudanophilic lesions in hyperlipidemic rabbits would evolve where endothelial cells might be exposed to moderately mean low shear stress, either laminar (SA) or turbulent (BC). Hence the high shear theory of atherogenesis of animals should be reexamined.

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A monoclonal antibody, PGF007, specifically recognizing human platelet-derived growth factor (hPDGF)-B chain was generated from a hybridoma derived from the spleen cells of a mouse immunized with synthetic peptide corresponding to No. 73-97 amino acid residues of the hPDGF-B chain. A sandwich-type enzyme immunoassay (EIA) for measuring hPDGF level was successfully developed using enzyme-labelled goat antihPDGF polyclonal antibody and PGF007 as the immobilized antibody. Human PDGF even in human serum was specifically detected with the resulting EIA with a sensitivity of 2ng/ml, which is almost equivalent to that of radioimmunoassay. With this EIA we compard hPDGF levels in the sera obtained from 12 healthy aged male volunteers (mean age: 59.2 years) and from 9 healthy young male volunteers (mean age: 25.2 years). As a result, we found the tendency for the serum levels of hPDGF and the calculated hPDGF/platelet values in the older group to be lower than those in the younger group.

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Most hypolipidemic drugs can not normalize plasma cholesterol and LDL levels in type II hyperlipoproteinemia (HLP) when used alone. Furthermore, combinations of many hypolipidemic agents can have additive effects.
In this study, we investigated the effects of niceritrol-probucol combination therapy on serum lipid and apoprotein concentrations in type II HLP.
Thirty one type II HLP (28; IIa, 3; IIb) patients were treated with probucol (500mg/day) for more than 12 weeks, then niceritrol (500-1, 500mg/day) was added to the treatment regimen. In six patients, medication of niceritrol was ferminated after combined treatment.
Total cholesterol (TC) was reduced from 307mg/dl to 273mg/dl (10% of reduction) by probucol alone and further reduced to 235mg/dl (14% of further reduction) by combination therapy with niceritrol. Total reduction rate of TC was 23% by combination therapy. Additionally, LDL-c was reduced 9% by probucol alone and a further 14% by combination treatment. Total reduction rate of LDL-c was 23% by combination therapy. HDL-c was reduced 23% by probucol alone, but no further reduction was observed in the combination period. Hence, the atherogenic Index (AI) was reduced 16% by combination therapy. Serum TG did not show significant change by probucol alone, but was reduced 9% by combination therapy. Serum concentration of apoB was reduced 11% by combination therapy.
These data indicated that combined niceritrolprobucol therapy for type II HLP is very useful.

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In a previous study, we found that the human hepatoma cell line, HepG2, Produces an activity to inactivate platelet-activating factor (PAF: 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) by the hydrolysis of the sn-2 acetyl group. This the activity was found to be associated with lipoproteins. However, since HepG2 cells are known to secrete lecithin: cholesterol acyltransferase (LCAT), as a complex with lipoproteins, and PAF is also inactivated by phospholipase A₂, the activity secreted by HepG2 cells can be attributed, at least partly, to the phospholipase A₂ activity of LCAT. We addressed this possibility by examining the substrate specificity and immunological identification of the PAF-hydrolyzing activity in a HepG2-conditioned medium. HepG2 cells were cultured for 24 hours in a serum-free medium and this conditioned medium was assayed for PAF acetylhydrolase or LCAT activities according to the method of Stafforini et al and Chen et al, respectively.
HepG2 cells were found to secrete, in 24 hours, 12±14pmol/min/mg cell protein PAF acetylhydrolase (n=6) and 0.9±0.3nmol/hr/mg cell protein LCAT (n=3). Addition of a sufficient amount of egg yolk phosphatidylcholine (PC), egg yolk phosphatidylethanolamine, hexadecyl-oleyl-PC, or tributyrin to the reaction mixture did not interfere with the hydrolysis of PAF. PAF acetylhydrolase activity in HepG2-conditioned medium was almost completely precipitated by immunoprecipitation using anti-PAF acetylhydrolase antiserum, while LCAT remained in the supernatant. The immunoprecipitation using anti-LCAT monoclonal antibody disclosed that virtually all of the LCAT activity was precipitated whereas PAF acetylhydrolase was not. Thus, the PAF-hydrolyzing activity produced by HepG2 cells exhibits strict substrate specificity. Furthermore, it was identified immunologically as PAF acetylhydrolase. These results suggest the important role of the liver in regulating the blood levels of PAF acetylhydrolase.

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To determine the relationship between aging and the degree of coronary atherosclerosis in symptomatic ischemic heart disease (IHD), coronary angiograms of 197 patients with IHD (160 with myocardial infarction and 37 with angina pectoris) ranging between 34 and 75 years of age were evaluated. The degree of coronary atherosclerosis was evaluated in terms of the severity of stenosis (number of segments with more than 75% luminal reduction) as well as extent (number of segments with wall irregularity irrespective of degrees of luminal reduction), and was correlated with age and three major risk factors (smoking, hypertension and hypercholesterolemia).
The extent of coronary atherosclerosis was significantly related to age (r=0.62, p>0.01), while the degree of stenosis did not show any correlation with age. Neither the number of risk factors nor the degree of coronary atherosclerosis was related to age.
These results suggested that in IHD the extent of coronary atherosclerosis increases with age, but the severity of the coronary stenosis does not.

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Injured vascular walls suffer from fibrous intimal hyperplasia induced by the progression of elastin and collagen fibers, which causes vascular stenosis or obstruction at anastomotic and other portions. If this problem can be solved, the success rate of vascular surgery would improve markedly to a high clinical value.
Presently, almost no drugs are available to prevent the progress of intimal hyperplasia in injured vascular walls. However, a recent report suggested the possibility that the swine pancreatic elastase, already in clinical applications as a hypolipidemic agent, may have a suppressive effect on fibrous intimal hyperplasia in injured vascular walls, as well as its known effect of improving serum lipid metabolism.
In the present study, the antiatherosclerotic action of elastase on normal and injured arterial walls of abdominal aorta in rabbits was investigated experimentally.
The results obtained are summarized as follows:
1. Hypercholesterolemia was found to promote the hyperplasia of injured arterial walls. This wall hyperplasia is afferent.
2. The hyperplasia of injured arterial wall noted with hypercholesterolemia was due mainly to intimal hyperplasia, with the tunica media tending to be thinner.
3. The administration of elastase lowered the level of serum cholesterol, but its action was weak.
4. The administration of elastase suppressed lipid deposition in the liver.
5. Elastase has an inhibitory effect on lipid deposition of normal arterial walls. This inhibitory action on lipid deposition in normal arterial walls was attributable to its antiatherosclerotic action, based on a mechanism different from that of hypolipidemic action.
6. The administration of elastase significantly suppressed this intimal hyperplasia in injured arterial walls. The suppressive effect on the intimal hyperplasia of injured arterial walls was also attributable to the antiatherosclerotic action of elastase.
7. The group of elastase administration had a lower degree of medial elastic fiber reduction and fissuring.
8. Judging from these findings, it is evident that elastase administration is effective in preventing the progress of intimal hyperplasia in injured vascular walls.
Injured vascular walls suffer from fibrous intimal hyperplasia induced by the progression of elastin and collagen fibers, which causes vascular stenosis or obstruction at anastomotic and other portions. If this problem can be solved, the success rate of vascular surgery would improve markedly to a high clinical value.
Presently, almost no drugs are available to prevent the progress of intimal hyperplasia in injured vascular walls. However, a recent report suggested the possibility that the swine pancreatic elastase, already in clinical applications as a hypolipidemic agent, may have a suppressive effect on fibrous intimal hyperplasia in injured vascular walls, as well as its known effect of improving serum lipid metabolism.
In the present study, the antiatherosclerotic action of elastase on normal and injured arterial walls of abdominal aorta in rabbits was investigated experimentally.
The results obtained are summarized as follows:
1. Hypercholesterolemia was found to promote the hyperplasia of injured arterial walls. This wall hyperplasia is afferent.
2. The hyperplasia of injured arterial wall noted with hypercholesterolemia was due mainly to intimal hyperplasia, with the tunica media tending to be thinner.
3. The administration of elastase lowered the level of serum cholesterol, but its action was weak.
4. The administration of elastase suppressed lipid deposition in the liver.
5. Elastase has an inhibitory effect on lipid deposition of normal arterial walls. This inhibitory action on lipid deposition in normal arterial walls was attributable to its antiatherosclerotic action, based on a mechanism different from that of hypolipidemic action.
6. The administration of elastase significantly suppressed

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There are two major types of occlusion following vascular surgery using artificial prosthetic grafts. One is thromboembolic occlusion in the early postoperative stages, and the other is latestage occlusion. The major cause of late-occlusion is hyperplasia of the neointima at the site of artificial inosculation. This hyperplasia precludes the clinical application of artificial prosthesis grafts with an inside diameter of less than 4mm. The fact is, however, that the prospective demand for prosthhetic grafts is mainly for those with inside diameters between 3-5mm. Therefore, it is important to find a solution to the problem of neointimal hyperplasia of the artificial inosculation site.
In the first paper of this series, the authors reported the promotive effect of hypercholesterolemia and the suppressive effect of elastase on neointimal hyperplasia of injured arterial walls. In this paper, we report our experiments on the effects of hyperlipidemia on artificial inosculation and that of elastase in preventing neointimal hyperplasia as well. The following results were obtained.
1. Hypercholesterolemia was found to enhance the development of neointimal hyperplasia of the host's arterial wall at the site of artificial inosculation.
2. Hypercholesterolemia was found to enhance the development of neointima hyperplasia at the site of artificial inosculation.
3. The administration of elastase inhibited neointimal hyperplasia of the host's arterial wall at the site of artificial inosculation.
4. The administration of elastase inhibited neointimal hyperplasia at the site of artificial inosculation.
5. A 1% cholesterol diet to induce hypercholesterolemia had an enhancing effect on neointimal hyperplasia that was more potent than the inhibitory effect of elastase.
6. In conclusion, we observed an inhibitory effect of elastase on neointimal hypertrophy at the inosculation site, showing that elastase may be used clinically to improve the patency rate of artificial prosthetic grafts.
7. The abdominal aorta substitution model in rabbits, suggested by the author in the present report, is an appropriate model for studies of neointima in artificial prosthetic grafts.
8. Use of the color image analyzer to determine graft wall thickness and host arterial wall thickness permits quantitative determination of the neointima.

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