An increased deposition of fibrous connective tissue is one of the main features of atherosclerosis. Elevated activity of prolyl hydroxylase, an enzyme involved in collagen biosynthesis, has been reported in the aorta of atherosclerotic rabbit. In an attempt to achieve an Immunohistochemical localization, we have produced monoclonal antibodies that specifically react with either prolyl hydroxylase or each type of collagen molecule (type I, III, IV, V and VI).
The specificity of monoclonal antibodies was determined by an inhibition ELISA and/or immunoblot. Prolyl hydroxylase was localized in the cytoplasm of intimal smooth muscle cells proliferating in the sclerotic plaque. An enhanced immunoreactivity was noted in the sclerotic plaque as compared to normal blood vessels. Type I and III collagens were co-localized and distributed in thick and thin collagenous bundles. Type IV was localized in the basement membrane surrounding smooth muscle cells. Localization of type V was quite unique in that a positive reaction was restricted to the cytoplasm and pericellular region of intimal smooth muscle cells. A proportional increase of type V collagen was evident and consistent with the biochemical data reported previously by one of the authors. Type VI was distributed in the extracellular matrix, including the vicinity of the basement membrane. When a monoclonal antibody to type I, III or IV was injected intravenously into a 1% cholesterol-loaded rabbit, the antibody reacted with collagen deposited in the sclerotic plaque. This may be explained by the fact that the permeability of endothelial cells was increased in the sclerotic plaque. When radiolabeled anti-collagen to type III was intravenously injected, a positive reaction as revealed by an autoradiography was found to coincide with atherosclerotic plaque. These preliminary results show the possibility of visualizing sclerotic plaque by immunoradioscanning in the future.

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The extracellular matrix components produced by cultured human vascular endothelial cells and smooth muscle cells were analyzed by the indirect immunofluorescent method. The extracellular matrix of the cultured smooth muscle cells was mainly composed of type I, IV and V collagen, elastin, and fibronectin. On the other hand, the cultured endothelial cells produced mainly type I, IV, and V collagen. Moreover, the endothelial cells were found to have newly formed type I collagen fibrils on the cell surface when the cells were stained by the indirect immunofluorescent method with an anti-type I collagen antibody. The fibrillar structure was formed at the 3rd day after subcultivation and had expanded like a knitting ball at the 7th day after subcultivation. The fibril formation was stimulated by the addition of a basic fibroblast growth factor (5 μg/ml), and completely blocked by the addition of β-aminopro-pionitrile (50 μg/ml). The fibrils were eliminated after the treatment of collagenase (50 μg/ml, 37°C, 60 min) or 0.5% Triton-X (4°C, 5 min). The significance of this type I collagen fibril formation of vascular endothelial cells in vascular diseases has been also discussed.

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Human arteries have areas that are preferential or resistant to atherosclerosis. The preferential sites include the proximal and outer walls of the branchings which are subject to low shear stress, while the resistant areas include an apical portion of the flow dividers of the branchings, which is subject to laminar high shear stress.
Electron microscopic studies revealed that synthetic intimal smooth muscle cells (I-SMC) were distributed in the intima of the preferential site which is rich in GAGs. However, most of the I-SMC in the resistant areas was located in the contractile, which is found in the collagenous intima even in youth.
In order to clarify the mechanisms of phenotypic changes in the I-SMC of both regions, the following in vitro studies were carried out.
1) Endothelial cells (EC) were exposed to a constant laminar shear stress of 10 dynes/cm² for up to 24 hours. Conditioning media (CM) of the EC exposed to shear stress were applied to the SMC which were cultivated in other dishes. Collagen synthesis of SMCs under CM was studied. CM for 2 to 8 hours augmented collagen synthesis in proportion to the exposure time of EC to shear stress. The collagen types produced were I, III and V. The production of type III by SMC increased in proportional to the amount of time EC was exposed to shear stress.
2) The effects of collagen types I and III on phenotype and the proliferation of SMCs were assessed for the respective collagen gels. Type III collagen caused a change in phenotype (from synthetic to contractile) and suppressed the DNA synthesis of SMC, while type I collagen caused no changes.
These results suggest that a laminar high shear stress can stimulate EC to secrete some effective substances and promote collagen synthesis of the underlying SMC. The growth of SMC surrounded by collagen fibers in the intima (particularly type III) was suppressed. These experimental results may explain the mechanisms by which laminar shear stress makes the arterial wall resistant to atherogenesis.

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The basic functions of the matrix receptors integrated in the plasma membrane of the cells are to recognize and link the matrix and to guide the cells to an adhesive state or spreading state. As adhesion and spreading take place, the cells undergo various functional expressions such as phenotypic change, migration and proliferation. Detachment of the cells from the tissue stabilized by the matrix receptor can also trigger phenotypic change in cells. Matrix receptors are 1) the integrin family which recognizes RGD-and other unknown sequences in the matrix with a rather low affinity of Km 10^-6 M and 2) Elastin receptors with a molecular weight of 67 KDa which recognize VGVAPG with a higher affinity of Km 10^-9 M. Integrin may be involved in the binding of the cells with the basement membrane. Elastin receptors may play an important role in stabilizing the phenotype of smooth muscle cells.

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To assess the effect of fish oil supplementation on the incidence of angiographic restenosis after successful percutaneous transluminal coronary angioplasty (PTCA), 36 male patients with stable angina received 5.4 g/day of fish oil (1.6 g eicosapentaenoic acid: EPA, 0.6 g docosahexaenoic acid: DHA), beginning 7 days before PTCA, in addition to the treatment with antiplatelet drugs, nitrates and calcium antagonists. All 34 successful patients were tolerant of the study medication. Of these, 2 patients with poor compliance and 2 asymptomatic patients without a follow-up (F/U) angiography were excluded from the analysis. Thirty patients (mean age 57±8 yrs, 41 lesions: fish oil group) were compared with 43 male patients (mean age 58±8 yrs, 66 lesions: control group) treated by conventional medical therapy. Despite of a greater percentage of left anterior descending artery lesions in the fish oil group, there was no difference in the baseline characteristics between the two groups. No significant changes in serum lipids after fish oil supplementation were observed in the fish oil group. The composition of plasma fatty acids was measured in 15 patients of the fish oil group at baseline, 1 week after (at PTCA), and at F/U angiography (an average of 3.8 months after PTCA). EPA was increased from 109±39 μg/ml at baseline to 241±68 μg/ml and 244±62 μg/ml after 1 week and at F/U angiography, respectively (p<0.01). DHA was also increased significantly. Arachidonic acid (AA), however, remained unchanged, and EPA/AA ratio increased from 0.7±0.3 before fish oil supplementation to 1.4±0.3 and 1.7±0.5 after 1 week and at F/U angiography, respectively (p<0.01). Angiographic restenosis was defined as a loss of more than 50% of the gain at PTCA in luminal diameter. Restenosis occurred in 6 of the 30 patients (20%) in the fish oil group and in 16 of the 43 patients (37%) in the control group (p=0.188). Restenosis occurred in 6 of 41 lesions (15%) in the fish oil group, which was significantly lower than in the control group (23 of 66 lesions, 35%) (p=0.037). Multiple logistic regression analysis using both fish oil and control groups also demonstrated that no use of fish oil was the highest independent risk factor for restenosis (p=0.066).
These results suggest that a dietary supplementation of fish oil, administered from 1 week before PTCA, reduces the rate of restenosis after successful PTCA in male patients and that EPA plays a major role in the prevention of restenosis.

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We made a preliminary study on the possibility of reducing restenosis after PTCA by administering lipid lowering therapy prior to PTCA. The 73 cases studied included 18 cases who received Probucol 750 mg/day for more than 2 weeks (Group P-1), 17 cases who received Pravastatin 10 mg/day for more than 2 weeks (Group M-1), 23 cases who received Probucol 750 mg/day for less than 2 weeks (Group M-2), and 15 cases who re ceived Pravastatin 10 mg/day for less than 2 weeks (Group M-2). We also treated 9 cases who have a serum cholesterol level of 230 mg/dl or more despite taking Probucol by LDL-apheresis 3 times prior to and after PTCA (Group A).
PTCA was indicated in cases with 90% or more coronary stenosis according to the AHA severity classification: We regarded cases with less than 50% lesion stenosis as successful PTCA, and cases with 75% or more lesion stenosis at CAG examination 5 months later as restenosis.
There were no significant differences in these 5 groups in the target vessel to be dilated, number of target vessels, severity of stenosis before PTCA, residual stenosis after PTCA, and coronary risk factors such as age, hypertension, diabetes, and smoking.
The rate of restenosis after PTCA showed 2 of 18 cases (11.1%) in group P-1, 5 of 17 cases (29%) in group M-1, 9 of 23 cases (39.1%) in group P-2, 1 of 15 cases (7%) in group M-2, and 1 of 9 cases (11%) in group A. In contrast to failure reducing restenosis rate in group M-1, lipid parameters such as total cholesterol, LDL-cholesterol, apoprotein B, ratio of apoprotein B to apoprotein AI, Atherogenic Index (LDL/HDL) had been much more improved in this group than group P-1. However, serum Lp(a) level of group M-1 showed 27.2 mg/dl which was significantly elevated than those of group P-1 and group M-2, where it showed 13.8 mg/dl and 17.1 mg/dl respectively.
In Group A serum cholesterol level showed 359 mg/dl at first, 274 mg/dl after diet control and taking probucol, and came to be 161 mg/dl after LDL-apheresis which could to be thought almost equal to the level at the time of PTCA.
Then we can conclude that (1) combined lipid lowering therapy by Probucol and LDL-apheresis may contribute to reducing the restenosis rate after PTCA. (2) To reducing the restenosis rate after PTCA we had better consider to choose lipid lowering drug which acts not only to lower serum lipid level but also to improve interrelation to endothelium and intimal cells of coronary artery. (3) In cases of restenosis who got lowered serum cholesterol level at the time of PTCA they may have elevated serum Lp(a) level.

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The late restenosis of coronary artery after percutaneous transluminal coronary angioplasty (PTCA) may be histologically analogous to severe coronary atherosclerosis from the perspective of “response to injury hypothesis” (R. Ross). To clarify this analogy, we light-and electron microscopes to examine 5 sutopsy cases who died with acute myocardial infarction caused by late restenosis after PTCA, and 3 autopsy cases who had successful PTCA and died with extracardiac diseases. The following results were obtained.
1. Morphological cause of the late restenosis of coronary artery after PTCA was determined on the basis of the localized, excessive hyperplasia of smooth muscle cells at the intima that had been damaged, showing rupture of media by PTCA. Histological examination of successful cases of PTCA, however, did not reveal any scar of the deeply ruptured media in the portion of PTCA.
2. Excessive hyperplasia of intimal smooth muscle cells at the portion of PTCA showed no insudation or incorporation of lipids, regardless of the presence of severe atheromatous plaque before PTCA.
3. Excessive intimal cell hyperplasia at the portion of PTCA was accompanied by considerable smooth muscle cell necrosis, probably used to provide local cellular maintenance for homeostasis.
In conclusion, the morphological events which occurred at the late restenosis of coronary artery after PTCA were not similar to those originally found with human coronary atherosclerosis.

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Proliferation of acortic smooth muscle cells (SMC) is one of the key events in restenosis after percutaneous transluminal coronary angioplasty (PTCA). We studied the growth behavior of SMC after rat aortic injury, and the effect of polyamine synthesis inhibitors on the growth of SMC.
³H-thymidine incorporation into SMC in aorta denuded with a balloon catheter started at 25 hours after injury, and maximal incorporation occurred 33 to 37 hours after injury. Transient increase in ODC activity was observed within 8 hours after injury. The polyamine level increased and were maximal at 48 hours after injury. Increased levels of spermidine continued until 7 days after injury. Intimal thickening started 7 days after injury and peaked at 21 days.
Administration of α-difluoromethylornithine (DFMO), a specific inhibitor of ODC, prevented the enhancement of ODC activity stimulated by aortic injury. Combined administration of DFMO and methyglyoxal bis (guanylhydrazone) (MGBG), a specific inhibitor of S-adenosylmethionine decarboxylase (SAMDC), inhibited the increase in DNA synthesis as well as the polyamine level in the injured aorta, but had no effect on intimal thickening.

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Hyperlipidemia has not been established as a risk factor of cerebral infarction. Recently, apolipoprotein abnormalities in cases of cerebral infarction have been reported. At the same time, there have been many patients with normolipidemic cerebral infarction. We examined serum the apolipoprotein (apo A-I, B, E) and lipid (Total cholesterol, TC; Triglyceride, TG; HDL-cholesterol; HDL-C) levels in patients with normolipidemic acute cerebral infarction. The subjects were 14 patients with cerebral infarction in the are a of the cortical arteries (CIC) (9 males, 5 females, mean age: 73.5±9.6 years old), 28 patients with cerebral infarction in the area of the perforating arteries (CIP) (15 males, 13 females, mean age: 67.3±9.0 years old) and 27 normal healthy controls (NC) (15 males, 12 females, mean age: 65.0±6.6 years old). The definition of normolipidemia applies the criteria of the Consensus Conference of Japan Atherosclerosis Society in 1987. In CIC and CIP subjects, the levels of HDL-C and apo A-I were significantly lower than the levels of NC (p<0.01), and the atherogenic index (TC—HDL-C/HDL-C), and the apo B/A-I ratio was significantly higher than that of NC (p<0.01). There were no significant differences between CIC and CIP in the levels of lipids and other apolipoproteins. These results suggest that apo A-I and HDL-C are useful indicators of lipid metabolism abnormalities in cases of cerebral infarction.

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In a female case of familial homozygous hypercholesterolemia, we have carried out selective plasma LDL apheresis (apheresis) for five years, in order to reduce total plasma cholesterol to the scheduled level (80 mg/dl in our case). As already reported, there have been no adverse reactions through apheresis. CAG showed 90% stenosis on LAD seg. 7 and 75% on RCA seg. 1, respectively. Deterioration of the coronary arteriosclerotic lesions was not found by the follow-up study of CAG. However, her complaints of anginal pain (effort angina) increased, and we decided to conduct PTCA to prevent the occurrence of myocardial infarction. We succeeded in the improvement of coronary stenosis and at the same time observed the disappearance of anginal pain accompanied by the increment of daily living activity. From this result, we conclude that the combined therapy of long-term apheresis and PICA can benefit cardiac function and daily activity in addition to preventing myocardial infarction.

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This study was designed to clarify the relationship between aerobic power level and ECG Abnormality in Japanese Females.
Oxygen uptake per kilogram of body weight (VO₂ max/wt) and ECG abnormality were examined in 274 middle-aged females.
The findings were as follows.
1) When we compared VO₂ max/wt classes in terms of proportion of ECG abnormality expressed as percentage, percentage of ECG abnormality was significantly higher in classes less than 30 ml/kg/min of VO₂ max/wt as compared with the higher classes (p<0.01).
2) The proportion of ECG abnormality expressed as percentage in fifty years old class was 3.4 times higher than that in the forty-year class (p<0.01).
3) Both average VO₂ max/wt in the normal ECG class and that in the mild abnormal ECG class were significantly higher than that in the abnormal ECG group after adjustment for the effects of age and %fat (p<0.01).
From these results, it was concluded that VO₂ max/wt may be related to ECG abnormality independently of age and %fat and that it may be necessary to maintain the VO₂ max/wt level at more than 30 ml/kg/min for health pormotion. Especially in post menopause women.

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With the participation of 77 patients (55 men and 22 women) suffering from ischemic heart disease, we studied the relationship between coronary atherosclerosis, carotid atherosclerosis and cerebral atherosclerosis (arteriolosclerosis). At the same time we studied the risk factors behind each type of atherosclerosis. Coronary atherosclerosis was assessed by the number of significant stenotic major vessels and its Gensini score, carotid atherosclerosis was assessed by its carotid ultrasonographic score, while cerebral arteriolosclerosis was assessed by its brain CT score. To detect the significant risk factors of each type of atherosclerosis, various risk factors (aging, hyperlipidemia, hypertension, diabetes mellitus, smoking and obesity) were analized by applying the multivalidation technique. Our results were as follows:
1) As coronary atherosclerosis progressed, carotid atherosclerosis and cerebral arteriolosclerosis also tended to progress.
2) Age and hyperlipidemia were significant risk factors of coronary atherosclerosis, and so it is important to control hyperlipidemia to prevent ischemic heart disease.
3) Age was the only significant risk factor of carotid atherosclerosis.
4) Age and hypertension were significant risk factors of cerebral arteriolosclerosis. It is, therefore, important to control blood pressure to prevent cerebral arteriolosclerosis.

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