Alessandra Bini

Identification and distribution of fibrinogen, fibrin, and fibrin(ogen) degradation products in atherosclerosis. Use of monoclonal antibodies.

Samples of normal and atherosclerotic vessels obtained from vascular and cardlothoraclc surgery were examined for the distribution of flbrlnogen/flbrln I, fibrin II, and flbrin(ogen) degradation products (Fragment D/DD) by using recently characterized monoclonal antibodies that recognize and distinguish the three molecular forms (MAbs I8C6, T2G1, and GC4, respectively) with the ABC-immunoperoxidase technique. In normal aortas, little flbrlnogen/flbrln I or fibrin II was present and no ftbrln(ogen) degradation products could be detected. In early lesions and In fibrous plaques, flbrlnogen/flbrln I and fibrin II were distributed in long threads and surrounding vessel wall cells and macrophages. Rbrln(ogen) degradation products were not seen In early lesions. In fibrous and advanced plaques, flbrlnogen/flbrin I, fibrin II, and flbrin(ogen) degradation products were detected In areas of loose connective tissue, In thrombus, and around cholesterol crystals. The results of this study suggest that Increased fibrin formation and degradation may be associated with progression of atherosclerotic disease. The observed distribution of the different molecular forms of fibrinogen also suggests the possibility that the cells present In the lesions actively participate In the flbrinogen-to-fibrln transition within the vessel wall.

Noncollagenous Bone Matrix Proteins, Calcification, and Thrombosis in Carotid Artery Atherosclerosis

Advanced atherosclerosis is often associated with dystrophic calcification, which may contribute to plaque rupture and thrombosis. In this work, the localization and association of the noncollagenous bone matrix proteins osteonectin, osteopontin, and osteocalcin with calcification, lipoproteins, thrombus/hemorrhage (T/H), and matrix metalloproteinases (MMPs) in human carotid arteries from endarterectomy samples have been determined. According to the recent American Heart Association classification, 6 of the advanced lesions studied were type V (fibroatheroma) and 16 type VI (complicated). Osteonectin, osteocalcin, and osteopontin were identified by monoclonal antibodies IIIA(3)A(8), G12, and MPIIIB10(1) and antiserum LF-123. Apolipoprotein (apo) AI, B, and E; lipoprotein(a); fibrinogen; fibrin; fragment D/D-dimer; MMP-2 (gelatinase A); and MMP-3 (stromelysin-1) were identified with previously characterized antibodies. Calcium phosphate deposits (von Kossas stain) were present in 82% of samples (3 type V and 15 type VI). Osteonectin was localized in endothelial cells, SMCs, and macrophages and was associated with calcium deposits in 33% of type V and 88% of type VI lesions. Osteopontin was distributed similarly to osteonectin and was associated with calcium deposits in 50% of type V and 94% of type VI lesions. Osteocalcin was localized in large calcified areas only (in 17% of type V and 38% of type VI lesions). ApoB colocalized with cholesterol crystals and calcium deposits. Lipoprotein(a) was localized in the intima, subintima, and plaque shoulder. Fibrin (T/H) colocalized with bone matrix proteins in 33% of type V and 69% of type VI lesions. MMP-3 was cytoplasmic in most cells and colocalized with calcium and fibrin deposits. MMP-2 was less often associated with calcification. The results of this study show that osteonectin, osteopontin, and osteocalcin colocalized with calcium deposits with apoB, fibrin, and MMP-3 in advanced, symptomatic carotid lesions. These data suggest that the occurrence of T/H might contribute to dystrophic arterial calcification in the progression and complications of atherosclerosis.

Fibrinogen in Human Atherosclerosis

Clinical, pathological, and experimental evidence of the involvement of components of the hemostatic system in human atherosclerosis has been increasingly accumulating in the past few years. Some of the most recent work in thrombosis and atherosclerosis has come to regard the occlusion of a coronary artery, e.g., in myocardial infarction, as a dynamic event involving vessel wall and blood cellular and protein components, more than just a passive consequence of the presence of atherosclerotic plaques. Increased plasma fibrinogen concentration has been established as an independent risk factor in patients with unstable angina, myocardial infarction, and ischemic stroke. Moreover, numerous in vitro studies have shown that fibrinogen, fibrin, and fibrin(ogen)-degradation products affect a number of biological functions in endothelial cells, smooth muscle cells, and macrophages, some of which might occur in vivo and contribute to plaque growth and development. Fibrin(ogen)-degradation products and secreted cellular mediators in atherosclerotic plaques might be among the factors affecting circulating fibrinogen level. Moreover, recent studies have shown that fibrinogen concentration is affected by life-style and dietary habits.

Fibrin and Its Derivatives in the Normal and Diseased Vessel Wall

Among the various theories of atherogenesis the interrelationship between atherosclerosis and thrombosis has long been controversial because the final event, i t . , presence of thrombi in atherosclerotic arteries, has been mainly regarded as a consequence. Research in atherosclerosis has developed in many different directions and this has provided fundamental information on several aspects of the pathogenesis of the disease regrding the role of lipids and lipoproteins, endothelial injury, smooth muscle cell proliferation, inflammation as well as the role of the major risk factors including hyperlipidemia, hypertension, diabetes, and smoking. All these aspects have recently been extensively reviewed and are beyond the scope of the present review.1-5 In parallel with these major research lines, a few early and more recent studies have suggested a more active role of the hemostatic system in the development and progression of atherosclerotic plaques. This article will review previous studies on the presence of fibrin(ogen) in normal arteries and atherosclerotic plaques. Furthermore, we will also present recent work from our group and others on the molecular characterization of fibrin(ogen)-derived protein in the walls of atherosclerotic vessels. A number of recent investigations have identified increased plasma fibrinogen concentration as an additional risk factor in patients with unstable angina, myocardial inh t i o n and ischemic stroke. Studies have shown that arterial thrombosis is present in more than 90% of patients with acute myocardial i n k t i o n . The correlation between elevated fibrinogen levels, lipids and diet in the progressiodregression of the atherosclerotic process will also be reviewed. Fibrinogen and fibrin affect a number of biological functions of vascular cells in cul-

Thrombosis in atherogenesis.

This review addresses the question of the involvement of fibrin in the development of atherosclerotic plaques. Numerous studies in the older literature demonstrated the presence of fibrinogen and/or fibrin in plaques, but the techniques that were available (mainly immunochemistry and immunohistochemistry with polyclonal antifibrinogen antibodies) did not clearly distinguish fibrinogen from fibrin or fibrinogen/fibrin degradation products. Some of these studies suggested that the fibrinogen-related protein within lesions resulted from incorporation of thrombi into lesions, while other studies suggested that fibrinogen itself entered the vessel wall. Newer studies by the authors and collaborators used specific antibodies for various fibrinopeptides to quantitate fibrinogen, fibrin I, fibrin II, and fragment X in thrombi and different histologic types of plaques. These studies showed that normal aortas contained fibrinogen and that fatty and fibrous plaques contained fibrinogen, fibrin I, and fibrin II, while complicated plaques contained fibrin II and fragment X, indicating a progression from fibrinogen to fibrin and fibrinogen/fibrin degradation products in parallel with increasing severity of the lesions. Later studies by the authors and collaborators used a sensitive immunohistochemical technique with monoclonal antibodies to demonstrate the distribution of fibrinogen-related antigens. Patterns suggesting incorporation of thrombi were seen, as were patterns suggesting formation of fibrin in association with arterial wall monocyte/macrophages and smooth muscle cells. The data from these various studies suggest the possibility that fibrin formation occurs within the arterial wall and contributes to plaque formation.

Fibrin and the Vessel Wall

Fibrin is a major component of atherosclerotic plaques, and there may also be situations in which intravascular fibrin is formed in contact with the endothelium. The studies to be presented describe the distribution of fibrinogen/fibrin I, fibrin II, and fragments D and D-dimer in normal vessels and atherosclerotic plaques of increasing severity and also describe some functional effects of fibrin on normal endothelium. Immunohistochemical studies using three specific monoclonal antibodies with the avidin-biotin complex immunoperoxidase technique demonstrated that little fibrinogen/fibrin I or fibrin II and no D/D-dimer were detected in normal aortas. In early lesions and in fibrous plaques, fibrinogen/fibrin I and fibrin II were distributed in long threads and around vessel wall cells. D/D-dimer was not seen in early lesions. In advanced plaques all three molecular forms were detected in areas of loose connective tissue, in thrombi, and around cholesterol crystals. Thus increased fibrin formation and degradation may be associated with progression of atherosclerotic disease. Additionally, the presence of fibrin II around vessel wall cells suggests that these cells may be involved in the fbgn to fibrin transition within the vessel wall. The second aspect of the work to be presented concerns effects of fibrin on vascular endothelium. Fibrin formed on the surface of cultured human umbilical vein endothelial cells stimulated production of prostacyclin and tissue plasminogen activator by the cells in a time- and dose-dependent manner. Stimulation of prostacyclin was completely inhibited by indomethacin and partially inhibited by actinomycin D, cycloheximide, and trifluoperazine, while stimulation of t-PA synthesis was completely inhibited by actinomycin D and cycloheximide and partially inhibited by cytochalasin D, vinblastine, and trifluoperazine.(ABSTRACT TRUNCATED AT 250 WORDS)

Use of Monoclonal Antibodies to Fibrinogen, Fibrin and Fibrin(Ogen) Degradation Products in Atherosclerosis

Fibrinogen is a soluble protein which is converted into an insoluble gel, fibrin, by thrombin. Fibrin formation is involved in the physiology of hemostasis and wound repair and in a number of pathological processes such as thrombosis, atherosclerosis, tumors, renal disease and inflammation.