Reverse cholesterol transport is a pathway of cholesterol from the peripheral cells to the liver, and is thought to be a very important factor in the regression of atheromatous lesions. HDL has been thought to play an important role in the first step of this pathway. However, it is interstitial fluid instead of plasma that interferes directly with peripheral tissue cells. Recent studies have revealed that substantial amounts of discoidal HDL particles and lipid-unassociated (free) apo A-I are present in peripheral lymph.
We demonstrated that, in humans, the free apo A-I, A-II and E that are contained in HDL particles interact with cholesterol-loaded macrophages to decrease intracellularly accumulated cholesteryl ester, to remove free cholesterol and phospholipids from the cells, and to form HDL-like particles in the medium. The HDL-like particles which are formed through the interaction of apolipoproteins with macrophages contained free cholesterol and phospholipids released from the cells. They had a density of 1.1g/l and alpha-2 mobility on agarose gel electrophoresis. The K_m values of the reaction for apo A-I, A-II and E were 0.11μM, 0.14μM and 0.24μM, respectively. Because these values are as low as 1: 320-400, 1:140-160, and 1: 6-8 of the plasma concentrations of these apolipoproteins, respectively, the results suggest the physiological relevance of the function of free apolipoproteins in interstitial fluid.
Furthermore, human apo A-IV and apolipophorin III from Manduca sexta (a kind of insect) were shown to remove cholesterol from cholesterolloaded macrophages and to generate HDL-like particles. However, neither human apo C-III nor reduced-and-carboxymethylated (CM) apo A-II showed these functions. Apo A-I, A-II, E, A-IV and apo Lp III have more than four amphiphilic alpha-helixes. Apo C-III and CM apo A-II have two alpha-helixes. Thus, apolipoproteins must have multisegmentary amphiphilic helix structures in order to generate HDL-like particles.

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Recruitment of mononuclear cells and their localization in the arterial wall are considered to be initial reactions of atherogenesis. Monocyte migration from the blood compartment into the arterial wall should depend on the chemotactic signals derived from arterial wall cells. Cachectin/TNF has been reported to be one such chemotactic factors. In apparent contrast with these reports, several studies have shown that cachectin/TNF does not induce chemotactic migration of monocytes/macrophage. The present study employed the Boyden chamber method to reevaluate the chemotactic activity of cachectin/TNF. Cachectin/TNF induced migration of human monocytes across a filter membrane at a concentration of more than 500ng/ml. The effect was significant but not remarkable. More potent chemotactic activity, however., was detected in the conditioned medium from the culture of endothelial cells when the cells were stimulated with cachectin/TNF. The checkerboard analysis showed that chemotactic activity was mainly chemoattractant rather than chemokinetic. These results indicate that cachectin/TNF does elicit chemotaxis of human monocytes by its direct effect on these cells, but that the stimulation of chemotactic factor secretion by endothelial cells would be the more important mechanism by which cachectin/TNF recruit mononuclear cells in vascular walls in vivo. Cachectin/TNF has been shown to have a number of effects on blood leukocytes and vascular endothelial cells which are possibly relevant to the initiation of arteriosclerosis, including adhesion of leukocytes to endothelium, invasion of leukocytes into vascular walls, enhancement of blood coagulation, and stimulation of platelet derived growth factor (PDGF) production. The present study constitutes one more step foward understanding the role of cachectin/TNF in atherogenesis.

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Macrophages and T lymphocytes are ubiquitous in all stages of atherogenesis. It has been postulated that altering the endothelial function leads to the increased adhesion of monocytes and lymphocytes to the endothelium, followed by their chemoattraction into the artery wall, where the monocytes are converted to macrophages. The macrophages and their secretory products, including platelet-derived growth factor (PDGF), may then play a role in inducing smooth muscle migration and proliferation within the intima, ultimately leading to the fibroproliferative advanced lesions of atherosclerosis. Furthermore, we postulated that T lymphocytes may regulate the growth factor production and secretion from macrophages.
We examined developing lesions in nonhuman primates to study the levels of gene expression in the growth factors and cytokines and were positive for PDGF-A, PDGF-B, and IL-lβ. We also examined the lesions of both nonhuman primates and humans immunohistochemically using a monoclonal anti-peptide antibody (PGF-007) with residues 73-97 of the mature PDGF B-chain. Immunohistochemical staining of macrophages (using monoclonal antibody HAM56, specific for macrophages) and PGF-007 demonstrated co-localization of PDGF-B within the macrophages in a perinuclear region. Immuno-staining of PDGF-B was observed in macrophages in all phases of atherogenesis in both humans and nonhuman primates.
We also examined the regulatory effect of interferon-γ (IFN-γ), a cytokine secreted by activated T lymphocytes, on growth factor production and secretion from macrophages. THP-1 cells, a monocytic leukemic cell line, were activated by incubation with phorbol 12-myristate 13-acetate (PMA). After 24 hours, IFN-γ was added to the culture medium and the effect on the levels of PDGF mRNA expression in THP-1 cells and the amount of PDGF secreted into the culture medium were examined. The levels of mRNA for both the A- and B-chains of PDGF decreased in a dose- and time-dependent fashion. A culture medium collected 48 hours after the addition of IFN-γ demonstrated a decrease of PDGF-dependent mitogenic activity, estimated by preincubation with anti-PDGF antibody, as well as the values of radioimmunoassay for PDGF.
These results suggest that macrophages can serve as a source of PDGF, which can act both as a chemoattractant and as a potent mitogen for smooth muscle cells, and thus play a putative role in the proliferative lesions of atherosclerosis. Furthermore, IFN-γ modulates PDGF production and secretion from macrophages, and the concomitant appearance of T cells and macrophages in atherosclerotic lesions may imply the cellular interaction of the regulatory mechanism for growth factor secretion from macrophages through the action of INF-γ.

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Monocyte Colony Stimulating Factor (M-CSF) is a growth factor specific to monocyte-macrophage lineage, which functions during the growth and differentiation of these cells. In addition to its action on hematopoietic cells, we found that M-CSF has a plasma cholesterol lowering activity in NZW and WHHL rabbits through enhanced clearance of plasma lipoproteins containing apo B100 in the reticuloendothelial system. On the other hand, M-CSF acts on cultured macrophages and stimulates both the uptake of acetyl-LDL into the cells and the excretion of cholesterol from the cells. We finally found that sustained injection of M-CSF inhibits the progression of atheroma in the aortae of WHHL rabbits. The mechanism of the anti-atherogenic effect of M-CSF was complex because many factors might be involved in vivo. Our results indicate that M-CSF has a profound effect on lipoprotein metabolism and atherogenesis, and might shed a new light on the unresolved question: Are macrophages good guys or bad guys in atherogenesis?

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Percutaneous transluminal coronary angioscopy was performed in 180 consecutive patients anatomically suitable angioscopy to investigate the pathogenesis of ischemic heart disease. Good pictures for analysis were obtained in 134 patients (22 with an acute myocardial infarction, 28 with a recent myocardial infarction, 37 with an old myocardial infarction, 18 with an unstable angina, 23 with a stable angina, and 6 with a vasospastic angina). Thrombi were observed in most patients with an acute myocardial infarction, a postinfarction angina, and an unstable angina. Yellow atheromas were observed more frequently in patients with an acute myocardial infarction, an unstable angina, and a recent myocardial infarction. The frequency of an intimal damage was significantly higher in patients with an acute myocardial infarction, an unstable angina, a recent myocardial infarction, and a vasospastic angina.
The progression of stenosis in coronary lesions of 10 patients who had previously undergone arteriography and were later hospitalized for an episode of unstable angina, and compared them to the findings in 10-matched stable angina patients who had undergone prior coronary arteriography. Stenotic progression occurred in 9 of 10 patients with an unstable angina but in 5 of 10 patients with a stable angina. And there was a significant difference in the degree of stenotic progression between 2 groups.
Relationships between the progression of the coronary stenosis and the plasma lipid levels in the stable angina were studied in 102 patients. Forty-four patients were revealed as showing a stenotic progression of their coronary artery disease and 56 patients did not. At both the first and the second arteriography, the values of the total cholesterol, Apo B, Apo C-II, Apo E and Apo B/A-I were significantly higher in the stenotic progression group than in the group showing no such progression.
In conclusion, the presence of thrombi may be important factor in the pathogenesis of acute coronary disease and in the stenotic progression of a coronary obstruction in patients with an unstable angina. Further, the plasma lipid levels and the apolipoproteins appear to play an important role in the progression of coronary artery stenosis in patients with a stable angina.

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We studied 37 patients to investigate whether the findings of the coronary angiograms can predict the onset of acute myocardial infarction. A second coronary angiogram was performed after the onset of acute myocardial infarction in 37 patients with organic coronary artery disease who had received medical treatment and had previously undergone cardiac catheterization. The interval between the two angiograms was 38±31 months.
At the second coronary angiogram, 18 (49%) of the 37 infarct-related arteries showed no or only minimal irregular lesions, and 28 (76%) showed less than 70% stenoses. At the second angiogram after infarction, however, most cases (81-100%) showed lesions with more than 90% stenoses in infarct-related arteries.
We also investigated 43 patients who underwent repeated coronary angiograms due to unstable angina with an interval of 49±37 months. In contrast to myocardial infarction, 25 (58%) of the 43 lesions which were responsible for unstable angina at the second angiogram had already had more than 70% stenoses at the first examination.
In addition, among 46 patients with vasospastic angina, repeated angiograms at an interval of 46±36 months revealed that the progression of atherosclerotic stenosis occurs more frequently in coronary arteries with vasospasm than in those without it.
We conclude that the pathogenesis causing acute myocardial infarction differs from that of unstable angina and that the future onset of myocardial infarction is not predictable from a coronary angiogram. In addition, coronary vasospasm may contribute to the progression of coronary atherosclerotic lesions.

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In order to elucidate the pathogenesis and triggers of acute myocardial infarction, 152 autopsied cases were examined clinically and histopathologically. Twenty patients with unstable angina and 18 patients with stable angina in whom coronary angiography was performed at the time of angina pectoris were investigated to compare the angiographic morphology and post mortem histopathological findings of coronary arteries.
The results of this study are as follows:
1) Sixty-one cases (40.1%) had no history of angina pectoris one month prior to infarction. Seventy-six cases (50%) had a history of unstable angina. Fifteen cases (9.9%) had a history of stable angina one month prior to infarction.
2) Acute myocardial infarction occurred during sleep in 18.4% of all cases and at rest in 21% of all cases. A definite trigger, such as heavy exercise or emotional stress, was identified in less than 12% of the entire series.
3) Type II eccentric lesions were significantly more frequent in patients with unstable angina, whereas Type I eccentric lesions were seen more frequently in patients with stable angina. Angiographically demonstrated Type II eccentric lesions revealed coronary stenosis due to atheromatous plaque histopathologically.
4) A high incidence (88.2%) of thrombus formation, corresponding to the site of the infarction, was observed.
5) Fresh occluding thrombi were formed at the site of the ruptured atheromatous plaque in 91% of all cases. A high incidence of thrombus formation was detected in cases in which coronary stenosis was more than 75%; however, thrombus formation was detected in cases in which coronary stenosis was less than 50%. Potential triggers may play an important role in the latter case.

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The serum Lp (a) level was assessed in 831 healthy Japanese males to clarify its distribution and normal range, as well as to analyze the relationship of Lp (a) taking into account age, serum lipids, and other clinical parameters. The mean serum Lp (a) level (±SD) was 18.3±16.6mg/dl, while the frequency distribution was skewed to the lower value. The median, the 90th and the 95th percentile values were 14.2, 39.9 and 51.5mg/dl, respectively. On the basis of these results, we have set the upper limit of the normal range for Lp (a) at 40mg/dl tentatively. Multivariate analysis demonstrated a positive correlation of the Lp (a) level with TC and LDL-C levels, while no correlation was found between the serum Lp (a) level and age or the other clinical parameters investigated in this study. Although Lp (a) is considered to be an independent risk factor for arteriosclerotic diseases, we obtained a significant correlation of the Lp (a) level and the TC and LDL-C levels simultaneously. Lp (a) may therefore play an important role by itself or together with hyperlipidemia in the acceleration of arteriosclerotic diseases.

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In view of the fact that diabetes mellitus (DM) is a major risk factor in ischemic heart disease, we conducted a study on the relationship between DM and coronary arteriosclerosis. Previous reports have addressed patients with concurrent coronary artery disease (CAD), DM, hyperlipidemia, and significant coronary artery stenosis (>50%). There have also been reports correlating etiologic data with patient gender. However, to the best of our knowledge, there have not been any published reports that consider patients with CAD and DM exhibiting normolipidemia, data on patients with mild stenosis (>50%), or reports relating gender with CAG confirmed CAD data. Therefore, we conducted studies on 220 patients who had undergone coronary arteriography at our institution, and we analyzed the relationship between the severity of stenosis in CAD and the interaction of DM with hyperlipidemia and normolipidemia, how the influence of DM on CAD differed by gender, and the relationship between DM and mild coronary arteriosclerosis. The incidence of DM in CAD was high and proportionally increased with the severity of stenosis of the CAD. The male/female ratio in CAD was different for the DM and non-DM groups. In both normolipidemia and hyperlipidemia the severity of CAD was greater in the DM group than in the non-DM group. Females with DM and CAD exhibited a greater body mass index (BMI), Apo B, B/Al, than non-DM with CAD females; however, no differences were observed in the males. Both the DM and non-DM groups with mild coronary arteriosclerosis exhibited a much higher level of total cholesterol (TC), atherosclerosis index (AI), TC/HDL-C, LDL-C/HDL-C, LDL-C/Apo Al, and lower HDL-C than the DM and non-DM normal coronary groups. This finding indicates that the effect of DM is less pronounced in the early stages of coronary arteriosclerosis. Coupled with previous data where the effect of DM was seen to increase proportionally with the CAD severity, the results suggest that DM acts as a geometrically progressive coronary arteriosclerosis promoting factor. The results of our study also suggest that its mechanism includes other factors in addition to abnormal lipid metabolism, and that its manifestation is affected by gender.

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Oxygenated derivatives of cholesterol (oxysterol) have demonstrated a wide variety of biological properties and have been evaluated for their ability to inhibit cholesterol biosynthesis. We developed a method to analyze copper-catalyzed oxidation products of human plasma cholesterol. After incubation with copper ions for 24hr at 37°C, oxidation products of free and esterified cholesterol were separated with a silica column and analyzed by gas-liquid chromatography. The oxysterols produced were mainly 7-keto-cholesterol, although small amounts of 7β-hydroxy- and 5, 6α-epoxy-cholesterols were also identified. Esterified (fatty-acylated) cholesterol suffered significantly less oxidation compared with the free form. This finding suggests that the cholesterol nucleus of the ester form is more resistant to oxidative stress than the free form. We also demonstrated that the addition of probucol inhibits this copper-catalyzed oxidation of cholesterol.

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