Biochim. pr., cerc.st.III, Simona Opris

Institutul National de Gerontologie si Geriatrie “Ana Aslan”, Bucuresti

REZUMAT

Concentraţiile crescute de fibrinogen in bolile cardiovasculare au condus la ideea ca fibrinogenul este un factor de risc independent pentru cresterea morbiditatii cardiovasculare si mortalitatii acestor boli. S-au luat in studiu 2 grupe de varsta: grupul 45-64 ani cu loturile:control C1 (n=8) si cu aterosclerozǎ carotidianǎ ATS1 (n=12) si grupul 65-84 ani cu loturile : control C2 (n=9) si cu aterosclerozǎ carotidianǎ ATS2 (n=15). In plus, s-a mai luat in studiu un lot- ATS3 de pacienti (45-84 ani, n=12) cu grad de stenozǎ avansatǎ (70-90%) evidentiate prin Doppler carotidian. Pentru fibrinogen s-a obtinut o crestere semnificativa statistic la ATS 1 vs. C1 (510.5 vs. 402.87 ; p<0.05) cat si la ATS 2 vs. C2 (523.3 vs. 360.89 ; p<0.001). Ecuatiile de regresie liniara au aratat o corelatie puternica intre nivelele de fibrinogen si aria ateroamelor la lotul ATS3 (p<0.05). Din punct de vedere al ariei totale de ateroame se observa o crestere inalt semnificativa la loturile ATS1 si ATS2 fata de ATS3 (p<0.005). Relatia dintre imbatranire si parametrii studiati, prin ecuatiile de regresie linara, arata o crestere nesemnificativa statistic pentru fibrinogen la cei cu ateroscleroza. Rezultatele studiului sugereaza ca posibilele alterari ale nivelelor de fibrinogen sunt asociate cu ateroscleroza.

ABSTRACT

Increased concentrations of fibrinogen in cardiovascular diseases have led to the idea that fibrinogen is an independent risk factor for developing cardiovascular morbidity and mortality. We have studied two age groups: group 45-64 years with lots: control C1 (n=8) and carotidian atherosclerosis ATS1 (n=12) and group 65-84 years with lots: control C2 (n=9) and ATS2 carotidian atherosclerosis (n=15). In addition, a group-ATS3 patients (45-84 years, n=12) with advanced stenosis (70-90%) highlighted by carotid Doppler. Data have shown for fibrinogen a statistically significant increase in ATS 1 vs. C1 (510.5 vs. 402.87, p <0.05) and ATS2 vs. C2 (523.3 vs. 360.89, p <0.001). The equations of the linear regression showed a strong correlation between the levels of fibrinogen and atheroma aria at ATS3 (p <0.05). In terms of total atheroma area we observed a highly significant increase in ATS2 and ATS1 vs. ATS3 (p <0.005). The relationship between aging and the studied parameters, by linear regression equation had shown a statistically non-significant increase of fibrinogen in ATS patients. Study results suggest that possible alterations in the levels of fibrinogen are associated with atherosclerosis.

Key words: nitric oxide, atherosclerosis, atheroma aria, aging

INTRODUCTION

The first correlation of fibrinogen with cardiovascular disease was made in 1950 when high levels were found in patients with cardiac ischemia. Fibrinogen is a liver protein whose changes in circulating levels have implications for various diseases. It also acts as an acute phase protein and increase after myocardial injury reaching a maximum 5 days after.

The role of elevated fibrinogen levels as an independent risk factor for coronary, cerebral, and peripheral vascular disease is well established on the basis of clinical and epidemiological studies. In cardiovascular disease, fibrinogen has been mainly considered as being involved in thrombotic occlusion and hence in the final stage of atherothrombosis. However, a number of investigators have suggested that fibrinogen may play a more active role in the development and progression of atherosclerotic plaque. The simultaneous presence of fibrinogen, its degradation products, and LDL cholesterol has been observed to influence atherogenesis in the arterial wall. Fibrinogen is involved in platelet aggregation and the formation of fibrin substrate. Fibrin appears to be a multi-potential component of atherogenesis, intervening at virtually all stages of lesion development. Fibrin and microthrombus deposition on normal intima is associated with endothelial disruption and intimal oedema, and oedema is a primary characteristic of early proliferative lesions. Fibrin strands on or in the intima encourage smooth muscle cell (SMC) migration and proliferation, and contribute to the growth of plaques. Fibrin also provides a continuing source of fibrin degradation products, and these have mitogenic activity which will sustain SMC proliferation in growing plaques, and act as chemoattractants for blood leucocytes.

Atherogenic factors include: elevated LDL cholesterol, low HDL cholesterol, elevated triglycerides, oxidized LDL, hypertension, elevated C-reactive protein, elevated Lp-PLA₂, elevated omega-6:omega-3 ratio, elevated glucose, excess insulin, elevated homocysteine, elevated fibrinogen, insufficient vitamin D, insufficient vitamin K, low testosterone and excess estrogen (in men), insufficient CoQ₁₀, nitric oxide deficit.

Elevated fibrinogen in obesity, diabetes and cardiovascular disease have led to the idea that fibrinogen is an independent risk factor (1,2) to increase cardiovascular morbidity and mortality of these diseases, which should be added to the profile cardiovascular risk factors.

Also, an age-related increase in fibrinogen has been reported in many epidemiological studies (3). However, little is known about the biochemical mechanism of this increase in fibrinogen with age. Measurement of circulating fibrinogen could give important information about a potential association between elevated plasma levels of this protein and aging.

Under these conditions we intend to study in patients with carotid atherosclerosis pathological changes in the levels of fibrinogen with aging and the correlations between them and total atheroma area.

MATERIALS AND METHODS

Subjects

Patients distributed in 4 groups:

• control (without ATS) C1 – 45-64 years (n=8) ;
• with carotidian atherosclerosis ATS1 – 45-64 years (n=9);
• control C2 -  60-75 years (n=9);
• ATS2 – 60-75 years (n=15) were studied.

Additionally, a group –ATS3 (45-85 years, n=12) with advanced stenosis (70-90%). Patients (diabetes mellitus, acute and chronic inflammatory state, neoplasie were excluded)  with significant atherosclerotic  injury, evidenced by carotidian Doppler (minimum total atheroma aria considered as 6 mm²) were compared with elderly subjects with the same age, apparently healthy, without significant atherosclerosis injury. For accurate evaluation of atherosclerosis process, clinical and laboratory exams were performed: vascular Doppler, total cholesterol, HDL, LDL, triglycerides, creatinine, urea, uric acid.

Doppler extracranian ultrasonography was performed with an ultrasound probe Interspec pencil Apogee Cx 5-MHz for spectral Doppler and sectorial pencil with variable frequency for ecotomografie.

Samples processing

For fibrinogen assay, blood samples were drawn by venopuncture into the tubes containing sodium citrate, centrifuged, diluted 1/10 and then determinated with a cuagulometer (excluded lipemic and hemolisated samples).

Fibrinogen assay

Was performed with a 1-channel optical cuagulometru COATRON M1, which measures the basic parameters of haemostasis – stage II using the Clauss method and clotting time obtained is inversely proportional to the amount of fibrinogen. Diluted serum samples are coagulated by trombine and clotting time was obtained by extrapolation of the sample on the standard curve, the results were expressed in mg/dl (normal values: 200-400 mg/dL).

Statistical analysis

Results are presented as means ± S.D. Statistical analyses were done by Student’s “t” test and p<0.05 was considered to be statistically significant. The relationship between age, fibrinogen and total atheroma aria was assessed using a linear regression model.

RESULTS AND DISCUSSIONS

Our data (Table 1) revealed a statistically significant increase for fibrinogen at ATS1 vs. C1 (510.5 vs. 402.87, p <0.05) and ATS2 vs. C2 (523.3 vs. 360.89, p <0.001). The association between increased levels of fibrinogen and atherosclerosis observed in the present study are in agreement with other studies (4,5,6). Other authors (7) too, have shown that plasma fibrinogen levels are increased in patients with coronary atherosclerosis. Some researchers (8) have shown a positive association of high levels of fibrinogen and atheroma area. Regression analysis of our data also showed a strong correlation (Fig. 2) between fibrinogen levels and atheroma area at ATS3 (p <0.05).

Tabel 1. Studied parameters at ATS vs. Control

Results are presented as means ±D.S.; p^*<0.05 vs. C1, p^**<0.001 vs. C2,  p^t<0.005 vs. ATS3, p^tt<0.005 vs. ATS3

Fig 1 Fibrinogen levels variation at all pacients for ATS vs. Control

Fig. 2 Correlation between fibrinogen and total atheroma aria at ATS3

Curve fitting was by linear regression. r = correlation coefficient

The presence of an inflammatory status increases plasma levels of fibrinogen. The tissues reaction to these stimuli lead to degradation products of fibrinogen which in turn acts on macrophages to release of the hepatocyte-stimulating factor and thereby increases hepatic synthesis of fibrinogen (9). This increase reflects hypercoagulability, hyperviscosity and increased platelet aggregation, suggesting that plasma fibrinogen penetrate the vascular wall (10). Therefore, the more plasma levels of fibrinogen increases, more fibrinogen penetrate the vessel wall, leading to the progression of atherosclerosis.

In terms of total atheroma area (Table 1) there is a highly significant increase in ATS2 and ATS1 vs. ATS3 (p <0.005). This underlines the importance of early examination for taking preventive measures before installation and progression of atherosclerosis.

Normal aging is characterized by specific age-related changes in cardiovascular structure and function. These changes can be accelerated by diseases like atherosclerosis, mainly in the elderly population (11). In addition, aging of the other systems (neuroendocrine, respiratory) may change the aging cardiovascular process and contribute to the total clinical phenotype.

The relationship between aging and the studied parameters by linear regression equations show a statistically insignificant increase in fibrinogen in patients with atherosclerosis. Other authors have found too, high concentrations of fibrinogen in the elderly patients (12).

Fig 3. Correlation between age and fibrinogen at ATS group

Curve fitting was by linear regression. r = correlation coefficient

CONCLUSIONS

Ageing is an independent risk factor in the development of atherosclerosis and is associated with a progressive decline in endothelial-dependent vasodilation. Study results suggest that the possible alterations in the levels of fibrinogen are associated with atherosclerosis, but detailed studies are needed to evaluate the mechanisms and interactions of these biochemical changes that lead to disease. If fibrinogen is accepted as an independent risk factor and marker for chronic inflammatory processes that reflect atherogenesis would be very impotant to know what factors influence its levels.

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