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Moya, M; Campana, V; Gavotto, A; Spitale, L;
Simes, J; Palma, J
Cátedra de Física Biomédica,
Facultad de Ciencias Médicas,
UNC, Córdoba, Argentina
Plasma fibrinogen concentration was positively correlated with atherosclerosis disease. We studied if plasma fibrinogen modifications are important in the atherogenesis and its association with the presence or absence of thoracic aorta endothelial lesions. Rats were divided into: tissue injury in 8 ds (control), multiple tissue injury during 30 ds. and 60 ds. Tissue injury was induced hiperfibrinogenemia and was produced by laparotomies. Histopathology was examinated with a 10X objective. Plasma fibrinogen was significantly elevated in the rats with multiple tissue injury (60 ds) compared with control (p<0.001) and in the rats with tissue injury in 30 ds (P<0.001) and provoked in thoracic aorta important endothelial denudation areas and enlarged intimal (p<0.003), in the last group.
When multiple tissue injury 60 days produced vascular lumen damage, aorta's wall distortion and red globule aggregations, pavement and extensive areas of endothelial denudation, myxoid and collagenous changes in the adventitia level, disruption of internal elastic layer, dystrophic calcifications areas and extracellular matrix increase (p<0.001).
Hiperfibrinogenemia associated with histopathological changes in thoracic aorta from rats could reflect vascular endothelial dysfunction present in early stages of atherosclerosis disease. Endothelial dysfunction could produce a disbalance between relaxing and contracting factors, between procoagulant and anticoagulant mediators or growth inhibiting and growth promoting substances, witch would generate an increase in the influx of haemostatic factors into intimae. Intimae enlargement would induce migration, proliferation and elaboration of extracellular matrix by smooth muscle cells witch have changed contractile phenotype to synthetic proliferate phenotype.
We conclude that fibrinogen increase presents an individual atherogenic capacity, demonstrated by histopathological lesions and may reflect acute phase reactions, risk factor, subclinical degeneration of endothelium and inflammatory responses present in cardiovascular disease. From a preventive-medicine point of view, the measurement of fibrinogen could contribute anything beyond traditional risk factor in the prediction of atherosclerosis disease.
In atherosclerosis, initial changes in endothelium may be modulated by cytokines, which permit the interaction between endothelial cells, smooth- muscle cells and lipids (18). Injury or activation of the endothelium modifies its regulatory functions and results in abnormal endothelial cell function (17). Considerable epidemiological evidence indicates that the hemostatic system plays an important role in the pathogenesis of atherosclerotic vascular disease (5,6,14). Recently, the ARIC (Atherosclerosis Risk in Communities Study) found atherosclerotic vascular disease incidence to be associated with Plasma Fibrinogen (PF) levels, which suggests that PF could mediate some of the effects of other risk factors (7).
PF primary functions in hemostasis are to support the platelet aggregation and also to develop fibrin clot formation at the site of vessel injury (4). PF is an acute phase protein, although recent studies have shown that PF levels increase in response to IL1 and IL6 induction, cytokines are present in vascular dysfunction (20). In spite of previous investigations, it has not yet been established whether increased concentrations of PF can contribute to the pathogenesis and/or progression of the ischemic vascular disease or whether these concentrations are only present in complication mechanisms (2). In a previous work in our laboratory, we demonstrated that PF increased significantly by tissue injury induced by laparotomy or chemical mediators (8). In this work, we have determined the variability of PF and its relationship with the thoracic aortic endothelial lesions in rats in different periods. Furthermore, we investigated PF and probable anatomopathological lesions in thoracic aorta of rats in order to show how the presence of isolated or persistent hyperfibrinogenemia may reveal early signs of cardiovascular disease.
MATERIALS AND METHODS
54 male rats, Suquía strain, weighing 280-300 g were used. Animals were divided into 3 groups. In group a: 2 laparotomy in 8 days; in group b: one laparotomy/week along 30 days and in group c: one laparotomy/2 weeks along 60 days. Different injured-rat group by 8, 30 and 60 days, previous to ether anesthesia inhalation, were made to bleed 72 hs after last surgery. PF was determined by the Rattnoff and Menzie method (16) and results were expressed in mg/dl. Thoracic aorta artery of each animal was fixed in buffered 10% formalin, embedded in paraffin and histological slices were stained with hematoxylin-eosin. Thirty parallel cefalocaudal thoracic aorta artery (Ao) of 4 um thickness cross-sliced incisions were performed every 1 mm. A total of 300 slices per group were studied and the quantification of lesions was made on a blinded manner and examined by light low, middle and high power microscopy. All the obtained slices were examined with a 10 X objective.
For results of PF concentrations, a variance analysis linear model was used. For all the possible media pairs combinations comparison, the REWGQ multiple ranks test (Ryan-Einot-Gabriel-Welsch) was used. Histopathology was analysed by the Chi Square test. A value of p<0.05 was regarded as significant.
Fibrinogen levels obtained in different groups of the rats studied are shown in .
Multiple tissue injury induced by laparotomy in 30 days, produced a significantly levels increased of fibrinogen (336.6±7.5) compared with tissue injury in 8 days (306.6±5.4) (p<0.01) and with tissue injury in 60 days (358.7±9.97) (p<0.001).
When injured group along 60 days was compared with tissue injury in 8 days, fibrinogen showed a similar behaviour that the observed in injured along 30 days, (p<0.001).
Histopathological Evaluations - They are shown in .
No microscopically changes were observed in any of the thoracic aorta layers of injury group in 8 days. Both lots kept their structure in 100% of the observations ().
|Figure 2- Group a: Endothelial integrity and indemnity in thoracic aortic layers without inflammatory signs on the wall (arrow) (10X H-E)|
540 histopathological slices were studied of injured group in 30 days (), they presented an important and extensive endothelial denudations areas and enlarged intimal, in 90% of slices studied compared with injured group in 60 days (p<0.003).
|Figure 3- Group b: Microscopic view was showed endothelial denudation areas and enlarged intimal (arrow), discruption of internal elastic layers (star). (H-E X 10)|
In the last group (), 95% of 540 slices presented vascular lumen damage, aorta thoracic's wall distortion, extensive areas of endothelial denudation and pavement, mixoid and collagenous change in adventitia level, disruption of internal elastic layer, extracellular matrix increase and red globule aggregations.
|Figure 4- Group c: Microscopic view was showed extensive areas of endotelial denudation and pavement (arrow), mixoid and collagenous in adventitia levels (asterisk), aorta thoracic's wall distortion and red globule aggregations. (H-E X10)|
PF increment was produced 72 hours after each laparotomy and hyperfibrinogenemia was a response to tissue injury, which induced an inflammatory process that resulted in upregulation of hepatic fibrinogen synthesis and secretion (9,13). PF increment produced an imbalance in the protein C system inhibitors, a natural anticoagulant present in vascular endothelium and it results in the conversion of PF to fibrin modulated by thrombin (12). Hiperfibrinogenemia associated with histopathological changes in thoracic aorta from rats could reflect vascular endothelial dysfunction present in early stages of atherosclerosis disease. Endothelial dysfunction could produce a disbalance between relaxing and contracting factors, between procoagulant and anticoagulant mediators or growth inhibiting and growth promoting substances, witch would generate an increase in the influx of hemostatic factors into intima (11). Intimae enlargement would induce migration, proliferation and elaboration of extracellular matrix by smooth muscle cells witch have changed contractile to synthetic proliferative phenotype (1). Hyperfibrinogenemia could induce functional alterations like hemodynamic modifications for viscosity increase and demonstrate the inflammatory process that may occur in endothelial dysfunctions. Modifications in thoracic Ao endothelium may show the injury of endothelial cells and could reflect a disturbed blood flow that promotes erithropoietic cell agglomeration next to the vessel wall, as seen in our results (19). However, PF participation mechanism has not been determined, yet (10), endothelial intact cells presented few or no TNF activity, but after injury or stimulation it produced a significantly expressive increase and led to coagulation system activity (3,15). Presence of endothelial denudation in the wall vessel exposes the underlying collagen, thus facilitating the platelet aggregation and progression of the vascular disease. Perhaps fibrinogen derived from plasma due to increased vascular permeability may be incorporated to the provisional matrix, independently of conversion to fibrin and promote endothelial dysfunction. This situation could also play a role in the recruitment of inflammatory cells. Yet, the mechanism of fibrinogen assembly into matrix is not known at present (10). The most probable hypothesis that accounts for the development of atherosclerosis has been proposed by Ross (17) and it involves the endothelial disruption in response to lesions by lipid aggression, contributing to the modification of risk factors such as PF.
We conclude that fibrinogen increase presents an individual atherogenic capacity, demonstrated by histopathological lesions and may reflect acute phase reactions, risk factor, subclinical degeneration of endothelium and inflammatory responses present in cardiovascular disease (7). From a preventive-medicine point of view, the measurement of fibrinogen could contribute anything beyond traditional risk factor in the prediction of atherosclerosis disease.
1. Casscells W. Migration of smooth muscle cells and endothelial cells. Critical events in reestenosis. Circulation.86:723 (1997).
2. Ceriello A, Pirisi M, Giacomello R, Stel G, Falleti E, Motz E, Lizzio S, Gonano F & Bartoli, e. Fibrinogen plasma levels as a marker of thrombin activation: New insights on the role of fibrinogen as a cardiovascular factor. Thromb & Hemost. 71: 593-595 (1994).
3. Chorda C & Paramo J.A. Endotelio vascular: Fisiopatología y participación en la trombogénesis. Sangre. 40: 491-498 (1995).
4. Crabtree GR. The molecular biology of fibrinogen, in Stomatoyannopoulos. G., NienhuIs, A.W., Leder, P., Maperus., P.W (eds). The molecular basis of blood diseases. Philadelphia. P.A. Saunders. 631 (1987).
5. Cremer P, Nagel D et al. Fibrinogen: ein koronerer risikofaktor. Diagnose Labor. 42:28-35 (1992)
6. Ernst E. & Resch KL. Fibrinogen as a CVC risk factor: a meta-analysis and review of the literature. Ann. Intern. Med. 118: 956-963 (1993).
7. Folsom Aaron R, Wu Kenneth K, Rosamond W, Richey Sharrett A. & Chambless LE. Prospective Study of Hemostatic Factors and Incidence of Coronary Heart Disease. Circulation. 96: 1102-1108 (1997).
8. Gavotto A & Palma JA. Role of histamine on plasma fibrinogen levels in rats with surgical injury (laparotomy). Arch. Int. Physiol. Biochem. 93: 175-179, (1985).
9. Gouldie J, Baumman H: Cytokines and acute phase protein expression. En Kimball. ES(ed): Cytokines in inflammation. Boca Raton. FL CRC Press. 275-298 (1992).
10. Guadiz G, Sporn lA & Simpson-Haidaris P. Thrombin Cleavage-Independent Deposition of Fibrinogen in Extracellular Matrices. Blood. 90: 2644-2653 (1999).
11. Guido RY, De Meyer & Herman Arnold G. Vascular Endothelial Dysfunction. Progress in Cardiovascular Diseases. XXXIX: 325-342 (1997).
12. Hantgan RR, Francis CW. & Marder VJ. Fibrinogen structure and physiology, in Colman, R.W., Hirsch, J., Marder, V.J., Salzman EW (eds). Hemostasis & Thrombosis, Philadelphia, P.A., Lippincott. 227 (1994).
13. Koj A. Definition and classification of acute phase proteins, in Gordon, A., Koj. A (eds). Acute Phase Response to Injury and Infection. New York, N.Y., Elsevier. 139 (1985).
14. Möller L, Kristensen TS. Plasma fibrinogen and ischemic heart disease risk factors. Arterioscler. Thromb.11:344-350 (1999)
15. Moya M, Campana V, Gavotto A, Simes J, Spitale L, Palma J. Relación del fibrinógeno y del TNF-alfa con lesiones histopatológicas en aortas de ratas. Medicina 60:746 (2000)
16. Ratnoff O.D. & Menzie A.C. A method for the determination of fibrinogen in small samples of plasma. J.Lab.Clin.Med. 37: 316-320 (1951).
17. Ross R. Mechanisms of disease: Atherosclerosis- an Inflammatory Disease. N.Engl. J. Med. 340:115-126 (1999).
18. Vasse M, Paysant J, Soria J, Collet AP et al. Regulation of fibrinogen biosynthesis by cytokines, consequences on the vascular risk. Haemosthasis. 26:(Suppl.4) (1999).
19. Vogel RA. Coronary risk factors, endothelial function and atherosclerosis: a review. Clin Cardiol.20: 426 (1997)
20. Zhang Z, Fuentes NL, Fuller G.M. Characterization of the IL-6 responsive elements in the gamma fibrinogen gene promoter. J. Biol. Chem. 270: 24287(1995).
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2nd Virtual Congress of Cardiology
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