The structure of fibrin plays an important role in the organization of thrombi, the development of atherosclerosis, and restenosis after PTCA. In this study, we examined the mechanisms of the migration of vascular smooth muscle cells (SMCs) into fibrin gels, using an in vitro assay system. Cultured SMCs from bovine fetal aortic media migrated into fibrin gels prepared with thrombin, which cleaves both fibrinopeptides A and B from fibrinogen, without other chemotactic stimuli. Both desA fibrin gels prepared with batroxobin, which cleaves only fibrinopeptide A, and desB fibrin gels prepared with Agkistrodon contortrix thrombin-like enzyme (ACTE), which cleaves only fibrinopeptide B, similarly induced the migration of SMCs compared to fibrin gels prepared with thrombin. These results suggest that the cleavage of fibrinopeptides is not necessary, but rather that the three-dimensional structure of the gel may be important for the migration of SMCs. Furthermore, gels prepared with protamine sulfate, which forms fibrin-like gels non-enzymatically, similarly induced the migration of SMCs compared to the gels prepared with thrombin. Both anti-fibrin(ogen) fragment D and anti-fibrin(ogen) E antibodies inhibited the migration of SMCs into fibrin gels, suggesting that both the D and E domains of fibrin(ogen) are involved in the migration of SMCs into fibrin gels. The addition of GRGDS, a synthetic RGD-containing peptide, but not that of GRGES, a control peptide, partially inhibited the migration of SMCs into fibrin gels, suggesting that the migration of SMCs into fibrin gels is at least in part dependent on the RGD-containing region of the α chain. The migration of SMCs into fibrin gels was also inhibited by a monoclonal antibody for integrin αvβ3 and α5β1, indicating that migration is dependent on these integrins. Furthermore, both fibrin(ogen) fragments D and E inhibited the migration of SMCs into fibrin gels, suggesting that these fragments, generated during fibrino(geno)lysis, may be relevant in the regulation of SMC migration into fibrin gels.

“Insoluble” lipid accumulates in normal intima with aging, and in some (but not all) atherosclerotic lesions, this paper I want to raise some questions about the source this lipid, and the factors involved in its deposition.

The simple concept that endothelium acts as a barrier to plasma macromolecules is no longer tenable. Instead, endothelium appears to provide a sophisticated transport system, and the main barrier is situated at the internal elastic lamina (IEL). In experimental animals the endothelium virtually lies on the IEL, making accurate study of their separate functions extremely difficult. Endothelium and IEL are separated by the diffusely thickened intima of the adult human aorta and coronary arteries. Unfortunately, in human subjects it is usually only possible to measure the steady state concentration of plasma macromolecules, which represents the resultant of influx, efflux, reversible and irreversible binding and destruction.

Fibrinogen and fibrinogen/fibrin-related antigen (total FRA) was measured in human normal intima and different types of atherosclerotic lesions and mural thrombi. The amount showed marked variation between groups of tissue samples, but within each group there was a significant correlation between levels of total FRA and low density lipoprotein (LDL), suggesting that some common factor must influence their influx or retention. The total FRA were analyzed by gradient sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotting with antisera to whole fibrinogen and fragments D and E, and fibrinopeptide A (FPA). All intimal samples (but not thrombi) contained fragment X, the first product of plasmin digestion of fibrinogen, but fragment Y was present in only half the samples, and no core-fragment E containing FPA was detected in any sample, suggesting that fibrinogenolysis is limited. By contrast, all samples contained fragment E, which was negative for FPA, so presumably derived from fibrin; they also contained fragments D-dimer and DY, which are characteristic degradation products of cross-linked fibrin. There were no differences between samples obtained during reconstructive vascular surgery and samples obtained at autopsy, so the patterns appear to represent the steady state. This implies that within the intima there is continuous formation of cross-linked fibrin and continuous fibrinolysis, both processes generating fragments that may have atherogenic properties.

There is increasing evidence that a hypercoaguable state is a significant risk factor for myocardial infarction (MI), and at autopsy occlusive thrombus has been found in 95

The concentration of plasma proteins was examined in interstitial fluid collected from human aortas, obtained at autopsy, from patients from whom a blood sample had been taken for routine analysis shortly before death. The interstitial fluid was absorbed onto small, preweighed pieces of filter paper which were inserted into natural strip planes in the tunica intima and inner tunica media. After equilibration the papers were removed and weighed to measure the amount of interstitial fluid collected, then analysed by quantitative immunoelectrophoresis for three plasma proteins covering a range of molecular masses ( M r ): low density lipoprotein (LDL) with molecular mass 2.4 x 10 6 , α 2 -macroglobulin ( M r 720 000) and serum albumin ( M r 68 000). In interstitial fluid obtained from normal intima of 20 men and women, the mean levels of the proteins were LDL 215%, α 2 -macroglobulin 115% and albumin 54% of the concentration in the patient’s own plasma. Concentrations were not influenced by depth within the intima, or by distance down the aorta. The patients covered the age range 31-96 years; relative concentrations of LDL, α 2 -macroglobulin and albumin increased in parallel by about 10% per decade. Large samples (up to 7 μl) of interstitial fluid were collected from inner media but they contained no measurable LDL; the levels of α 2 -macroglobulin and albumin were respectively 11 and 18% of plasma concentration. Analysis of interstitial fluid from one sample of normal intima obtained at vascular surgery, and from two freshly killed pigs, suggested that results were not invalidated by the use of autopsy material. Direct comparison of whole intimal tissue and interstitial fluid provided no evidence of preferential binding of LDL in tissue. There was no evidence of preferential adsorbtion of LDL by the filter paper. Interstitial fluid of six samples of normal intima from four patients was compared with normal plasma by two-dimensional immunoelectrophoresis. The peaks produced in the antiserum to LDL were identical in mobility, shape and staining properties. Thus the concentration of LDL found in interstitial fluid from normal aortic intima was more than twice the plasma concentration, and the relation between concentration and molecular mass was the inverse of that reported for peripheral interstitial fluid and lymph. Endothelium appears to trap LDL in the intima instead of keeping it out, and this raises fundamental questions about the function of arterial endothelium.

In this Advanced Study Institute other speakers will discuss endothelial prostaglandin metabolism, factors controlling smooth muscle and endothelial cell proliferation, and aspects of lipid metabolism. I will confine my discussion to three other areas of arterial wall metabolism — energy production and the consequences of hypoxia, the intimal and medial cellular environment, and collagen synthesis — and will try to show how these may be inter-related in terms of atherogenesis.

Juvenile-type fatty streaks are the earliest lesions that can be recognized by macroscopic inspection of aortas of children. They characteristically appear as small yellow/white dots most frequently in longitudinal lines between the intercostal branches, they stain brilliantly red with macroscopic sudan staining, and Holman reported that they were already present in all children aged more than 3, increased in area rapidly between ages 8–15, and reached a maximum at about age 20 (1).

Unlike most organs, arteries have two distinct characteristics: they are tubes that are subjected to external haemodynamic stresses and pulsatile bombardment with plasma constituents under pressure, and they are also metabolizing tissues that may face special problems in the supply of nutrients and oxygen and in changes in cellular environment resulting from accumulation of abnormal quantities of plasma macromolecules. In this workshop we have experts in both these areas, and I hope that this will lead to a stimulating interchange of ideas. I would like to start the discussion by considering some of the information available on transport of plasma proteins and lipoproteins into and across arterial wall.

It is now accepted that large amounts of plasma low density lipoprotein (LDL), and cholesterol ester which must be derived from it, accumulate in atherosclerotic lesions, but it is not clear if this is the result of increased endothelial permeability or of increased retention within the intima. In this paper I will try to draw together some of the available biochemical evidence.

This chapter reviews the current state of knowledge on optimal levels of blood lipids based on research in the laboratory, including work with human tissues, cells, and lipoproteins, and investigations using experimental animal models of atherosclerosis. It presents a summary of report of the laboratory–experimental section examined in the workshop. The workshop examined the experimental results on optimal plasma lipid and lipoprotein profile that are expected to favor prevention, retardation, and/or regression of clinically important atherosclerosis. It examined many of the nutritional variables that appear to influence the development of atherosclerosis, including the amount of food fat and the degree of saturation or randomization of fatty acids, the type and amount of dietary fiber, the presence of dietary saponins, the amount and source of dietary protein, the amount of lysine in the diet, and the type of dietary carbohydrate. All of these dietary factors seem to mediate their effects by affecting serum low-density lipoproteins (LDL) levels or the high-density lipoproteins (HDL)/LDL ratios.

A quantitative assay for fibrin or other insoluble fibrin-like antigens (“fibrin”) in small samples of intima is described. Tissue samples were subjected to electrophoresis directly from the intima into an antibody-containing gel to remove and measure fibrinogen and other soluble fibrin reactive antigens (FRA). The residual tissue was then exhaustively incubated with plasmin, and the soluble fragments generated from the insoluble “fibrin” were measured by quantitative immunoelectrophoresis.