A Comparison of the Acute Phase Proteins in Chronic Aortic Occlusion versus Diffuse Aortoiliac Occlusive Disease

Methods Sixty patients with the aortoiliac occlusive disease were divided in two groups. First group consisted of thirty patients with the angiographic signs of diffuse aortoiliac occlusive disease, Leriche type III (AIOD group). Second (COA) group consisted of 30 patients characterized by either bifurcated aortic occlusion or juxtarenal aortic occlusion. Those two groups were compared according to symptomatology, ankle-brachial index (ABI) values, traditional risk factors and some acute phase proteins (high-sensitivity C-reactive protein – hs-CRP; homocysteine) taken at inclusion.


INTRODUCTION
Increasing evidence suggests that elevated levels of the acute-phase proteins occur in the acute coronary syndromes, carotid disease and aortoiliac occlusive disease and may also predict future cardiovascular events [1][2][3][4].On the other hand, there are many controversial reports as to whether inflammatory markers are the cause or the result of atherosclerosis.Consequently, circulating factors related to inflammation may be potential predictors of atherosclerosis.It has been shown that the only acute phase protein independently associated with cerebrovascular diseases was fibrinogen [5].On the other hand, high-sensitivity C-reactive protein (hs-CRP) does seem to be associated with plaque density, especially at the femoral level, which supports the previously described link between C-reactive protein (CRP) and peripheral arterial disease while plasma homocysteine levels are related to the extent of atherosclerosis in coronary and peripheral arteries.Measurements of CRP and homocysteine with high sensitive techniques, with low detection levels, have attracted a lot of attention and those two acute-phase proteins are more often connected with the extent of peripheral disease.Chronic aortic occlusion (COA) is a rare condition, confined to the aortic bifurcation or juxtarenal position with usually spared distal vascular tree [6].Contrary, diffuse aortoiliac occlusive disease (AIOD) is more pronounced, affecting infrainguinal segment additionally.The only connection between these two variants of disease is the same degenerative etiology.To the best of our knowledge, studies which considered formal assessment of similarities in inflammatory markers between COA and AIOD have not been previously undertaken.
The purpose of the present study is to evaluate if there is a specific relationship of the acute phase proteins between those two variants of the same disease and to investigate if there is a connection between the acute phase proteins and the extent of disease.

MATERIAL AND METHODS
This study is a part of academic (noncommercial) phase IV prospective study, carried out in compliance with the EU Directives of clinical trials [7].Informed consent was obtained from each subject enrolled in the study.The study was approved by the Institutional Ethics Board of the Clinical Center of Serbia.
From January 2009 to January 2011, 351 patients with symptomatic peripheral arterial disease were enrolled at Clinic of Vascular and Endovascular Surgery, Clinical Center of Serbia, Belgrade, Serbia.All patients underwent translumbar aortography since multi slice computed tomography (MSCT) is not always available due to technical reasons.Out of them, 60 patients with the aortoiliac occlusive disease were divided into two groups.First AIOD group consisted of 30 patients with the angiographic signs of diffuse aortoiliac occlusive disease, Leriche type III with the affected femoropopliteal segment.These patients had to fulfill following criteria: 1) diminished but present femoral pulses confirmed by spectral analysis measurements; 2) angiographic signs of diffuse aortoiliac occlusive disease but without occlusion.Second COA group consisted of 30 patients characterized by either bifurcated aortic occlusion or juxtarenal aortic occlusion, based on the level of proximal extension of the chronic athero-thrombotic material and level of disruption of the contrast column on standard translumbar aortography.All patients but six from COA group had a patent femoropopliteal segment.Iliac artery segment was spared in all patients from COA group.Patients were excluded from this study if they had: 1) open or endovascular lower limb revascularization; 2) acute abdominal aortic occlusion; 3) inflammatory etiology of COA; 4) type I or II Leriche disease.At the time of the aortogram, the presence of classic atherosclerotic risk factors, smoking, hypertension, dyslipidemia and diabetes as well as inflammatory markers were assessed.Those two groups were compared according to symptomatology, ankle-brachial index (ABI) values, traditional risk factors and some acute phase proteins (hs-CRP, homocysteine).All patients diagnosed with any chronic inflammatory disease or acute infectious process and those who had fever physical trauma or had undergone surgery in the previous 45 days were excluded from the sample.Aortic reconstruction included aortobifemoral using gelatin sealed polyester prosthesis.16×8 mm bifurcated grafts were employed in all operations.

Inflammatory markers level
For blood collection, a 21-guage butterfly ne edle was inserted into an antecubital vein, and the tourniquet was removed immediately.Blood was spun at 3000 rpm for 20 minutes at 4°C in a refrigerated centrifuge.Within 90 minutes off collection, processing and storage in a -70°C freezer was accomplished.
Homocysteine was measured in serum, using HPLC method with fluorescence detection (Varian, Inc.).
Hs-CRP levels were determined using an immunotechnique on the Olympus AU 680 system with the Olympus reagents.

Statistical analysis
Summarizing data were displayed as mean and standard deviation (SD) or median (range).Continuous normally distributed variables were compared using Student's t-test or the Mann-Whitney U-test for non-normally distributed variables.Differences among categorical variables were analyzed using the Chi-square test or the two-tailed Fisher's exact test, as appropriate.P-values less than 0.05 were considered statistically significant.Statistical analysis was carried out using the SPSS 15 software (SPSS, Chicago, IL, USA).

RESULTS
These two groups were homogenous except patients from COA group who were younger, while some traditional risk factors were (p<0.001)more pronounced in AIOD group.Demographic characteristics, comorbid conditions and traditional risk factors were shown in Table 1.
There were highly significant differences in ABIs between the AIOD and COA patients (p=0.013)(Table 2).Furthermore, claudications were dominant symptomatology in COA group, while severe ischemia (grade III and IV) was more pronounced in AIOD group (p=0.001)(Table 2).

DISCUSSION
This study evaluated the inflammatory profile of patients with the aortoiliac occlusive disease by comparing two groups of patients with the same degenerative etiology and assessed the association between the acute phase proteins and the extent of atherosclerotic disease.As demonstrated by our data and those of other investigators, COA tends to occur in relatively young patients who have a history of tobacco abuse [6,8].Hypertension and cholesterol individually have been shown to be significant clinical hemodynamic risk factors of atherosclerosis-related disease as we confirmed in our study as well.Both risk factors dominated in AIOD group but, to our opinion, a possible reason for higher number of patients with hypertension in AIOD group may be fairly older population which had more time to develop it.Our data showed that patients with the aortoilac occlusive disease had increased serum hs-CRP but significantly more pronounced in COA group.This is a new and interesting finding which may suggest that high baseline serum hs-CRP may identify patients who are most likely to develop COA or experience accelerated atherosclerosis of the native lower limb arteries.On the other hand, someone might argue that it is equally plausible that total aortic occlusive process is what caused the finding of high hs-CRP level.However, some previous studies have indicated an association between the elevated systemic hs-CRP levels and the impairment of endothelium-dependent vasorelaxation [9,10,11].Contrary to initial thoughts, generated by previous trials, the novel study showed that hs-CRP did not impair an endothelium-dependent vasorelaxation and, fur-thermore, that CRP was not a vasoconstrictor but an endothelium-independent vasodilator [12].It is interesting that high concentrations of hs-CRP are detected in COA group where distal arterial tree is usually spared of atherosclerosis, so it might be speculated than that the direct vasorelaxing effect of hs-CRP in patients with COA might be compensated response for massive thrombotic occlusion, thus allowing the hyperemic response, keeping distal vasculature patent and free of atherosclerosis.Furthermore, hs-CRP was inversely related to ABI.This is in agreement with previous studies showing that hs-CRP is associated with hemodynamic [13] and functional [14] outcomes and with the clinical severity of peripheral arterial disease [15].These patients should be considered to be at high risk of future complications according to guidelines from the American Heart Association and the Centers for Disease Control and Prevention [16].Similar to other investigations [17,18], this study shows hyperhomocysteinaemia in both groups, but significantly higher mean plasma homocysteine concentration in patients with the advanced ischemia.These data suggest that hyperhomocysteinaemia may either be a marker of AIOD, or etiologically implicated in development of AIOD.Thus, this study does not establish elevated plasma homocysteine as a risk factor of atherosclerosis but there is a significant correlation with the extent of disease which is compatible with other studies.Additionally, the data herein confirm elevated plasma homocysteine as a risk factor of symptomatic atherosclerosis.It is not known how a moderate increase in plasma homocysteine is related atherogenic process in man.Several mechanisms could be involved: hyperhomocysteinaemia may cause endothelial damage and proliferation of smooth muscle cells in the intima [19].This may lead to lipid accumulation in the arterial wall under hypercholesterolaemic conditions [20].The association of elevated CRP levels in patients with distal aortic occlusion comparing with AIOD is a novelty, and it may be affected

CONCLUSIONS
In conclusion, our data demonstrate significant difference of hs-CRP and homocysteine concentrations among two variants of aortoiliac occlusive disease.In this particular pathology as COA, high hs-CRP concentration does not associate, at least with angiographic extent of disease.

Table 1 .
Demographic, comorbid data and traditional risk factors

Table 2 .
Symptomatology and preoperative ABI values Values are expressed as mean ± SD, or number of patients (%).

Table 3 .
Acute phase proteins between two groups of patients by patient selection and very small sample size.That is one of the major limitations of our study.