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Thromboangiitis obliterans (TAO) is an episodic, sharply segmental, inflammatory and thrombotic-occlusive peripheral vascular disease with unknown etiology that can lead to gangrene and tissue loss. It particularly affects young males from low socioeconomic situations and its progression and outcome have a close relationship with tobacco consumption. Although TAO is more prevalent in the Middle East, Far East and Eastern Europe, it is now being reported more often in Western developed countries, which might reflect an immigration phenomenon.
One enduring question remains regarding TAO: namely, whether it is a form of vasculitis. It is unclear if thrombus formation is the first event in TAO and vascular inflammation is secondary to thrombosis or whether the first event is vascular inflammation, which then increases the risk of thrombotic events. Interestingly, Leo Buerger had believed that “external influences, as well as possible toxic condition of the blood” might be responsible for pathological features of TAO.
However, some publications consider TAO to be a type of vasculitis and indicate that an autoimmune or an autoinflammatory vascular reaction underlies the thrombotic events for the following reasons: (i) it is episodic, with relapsing-remitting phases; (ii) it affects a young population; (iii) several auto-antibodies against endothelial cells and collagens type I and III occur in blood samples of patients with TAO; (iv) the pathology of the disease shows vascular inflammation as well as non-ischemic neural involvement, with an inflammatory thrombus; and (v) up-regulation of endothelial cell adhesion molecules has also been detected in the vascular tissues of non-diseased limbs in TAO patients.
However, the Chapel Hill Consensus Conference did not recognized TAO as a form of vasculitis but instead considered it to be a kind of vasculopathy. The opinion was that TAO patients are susceptible to thrombosis and that secondary vascular inflammation occurs at the site of thrombus formation, for the following reasons: (i) the outcome of the disease has a close relationship with cigarette smoking; (ii) the pathology shows that the internal elastic lamina remains intact; (iii) patients do not develop systemic manifestations of other kinds of vasculitis and most show normal sedimentation rate and C-reactive protein (CRP); and (iv) several studies could not demonstrate effectiveness of immunosuppressive therapies in TAO patients.
Developing a research plan to find an answer to this old question is important because the management of thrombotic events, which are limb-threatening events in TAO, would change depending on its underlying mechanism. For instance, TAO patients might benefit from plasma exchange in the acute phase of their disease, for transient removal of autoantibodies, cytokines and complement associated with the inflammatory process. Consequently, thrombotic events secondary to inflammation might be diminished after plasma exchange.
This study represents the first step in evaluating this hypothesis and presents an examination of the effects of sera from TAO patients on activation of primary cultures of human umbilical vein endothelial cells (HUVECs). The two main adhesion molecules, ICAM-1 (intercellular adhesion molecule), which mediates adhesion and extravasation of leukocytes to the inflammation site, and VCAM-1 (vascular adhesion molecule), which mediates the attachment of lymphocytes to the vascular endothelium, were selected as markers of vascular endothelial cell activation.[7, 8] Their gene expressions after incubation with TAO sera were evaluated.
Almost all TAO patients are tobacco users and since the role of tobacco components in activation of endothelial cells have been reported, we used two control groups: healthy smokers and nonsmokers.
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A total of 21 patients clinically diagnosed with TAO in 2009 and 2010 were selected for this study at the referral vascular and endovascular department of Emam Reza Hospital in the north-east of Iran. For each subject 10 mL of blood samples were taken after obtaining informed consent. The study protocol was approved by the Ethics Committee for Clinical Research of the Mashhad University of Medical Sciences (No: 900133).
The clinical diagnostic criteria for inclusion were Shionoya's criteria: age of disease onset before 50 years, history of cigarette smoking, upper-limb involvement or thrombophlebitis migrans, infrapopliteal arterial occlusion and absence of other atherosclerotic risk factors other than smoking. We assessed traditional atherosclerosis risk factors including blood pressure, fasting blood sugar and total cholesterol in this study. Systolic blood pressure below 12 mmHg, diastolic blood pressure below 8 mmHg, fasting blood sugar < 90 mg/dL, and total cholesterol < 180 mg/dL were considered as the upper limits for this assessment. Patients who were in acute phases of their disease were included in this study. Suffering from burning pain at rest, with or without gangrene, was the key sign for consideration of the acute phase of the disease.
A group of 20 age-matched male healthy smokers and a group of 17 age-matched male healthy non-smokers were also chosen as a healthy smoker and non-smoker control group, respectively. The number of daily smoked cigarettes and duration of smoking were also matched between the TAO and smoker control groups. All of the patients and control smokers smoked filtered cigarettes. Those patients who had given up smoking at the time of blood sampling were excluded from the study. Since all TAO patients in this study were from low socioeconomic status, based on our suggestive indices, including poverty line, hygiene, education, professional occupation and long-term unemployment, the two control groups were also matched based on their socioeconomic status. For this reason, the numbers in our control groups became limited. Precise clinical examination, including vascular examination, was performed for each control subject before blood sampling in order to rule out any site of inflammation or any type of peripheral vascular disease. All of the TAO patients and the control group participants were Caucasian males.
Pooled HUVECs were purchased from Pasteur Institute (Tehran, Iran). For all other experiments, primary endothelial cells (ECs) were harvested from human umbilical veins from different donors as per a previous protocol. The HUVEC cells were initially seeded in 25-cm2 ventilated flasks (SPL Life Science, Pocheon-si, South Korea), in Dulbecco's modified Eagle's medium (DMEM)-F12 medium (Sigma-Aldrich, St Louis, MO, USA) with 20% fetal bovine serum (Gibco, Grand Island, NY, USA) and then incubated at 37°C in 95% air and 5% CO2.
Antibiotics and antifungal agents were not added to the medium in order to avoid any interruption in expression of the adhesion molecules.[13, 14] When the flasks became confluent, the endothelial cells were passaged with 10 × (0.5%) trypsin-ethylenediaminetetraacetic acid (EDTA) (Gibco) into 75-cm2 and then 150-cm2 ventilated flasks (SPL Life Science) and finally, after three passages, the cells were seeded in 60 12.5 cm2 ventilated flasks (JET BIOFIL, Guangzhou, China) to yield confluent monolayers within 48 h. All experiments were conducted on HUVECs at passage four to diminish in vitro confounding factors. After 48 h, HUVECs were incubated with sera from the case and control groups for 2 h. The sera were heat inactivated (decomplemented) and then titrated down to 20% in the culture medium. After 2 h, the cells were dissolved in 5 cc Tripure Reagent (Roche, Mannheim, Germany) and total RNA was isolated following the manufacturer's instructions.
Real-time quantitative PCR
ICAM-1 and VCAM-1 messenger RNA (mRNA) were quantified by TaqMan® real-time polymerase chain reaction (PCR) using a Q-6000 machine (Qiagen, Hilden, Germany). In brief, total RNA was isolated using Tripure Reagent (Roche) according to the manufacturer's instructions. The mRNA was reverse-transcribed using complementary DNA (cDNA) synthesis kit (Fermentas, St leon-Rot, Germany).
Primers and probes for ICAM-1, VCAM-1 genes, and also for beta 2 microglobulin as the reference gene, were designed by Beacon Designer 7 software (PREMIER Biosoft International, Palo Alto, CA, USA). Beta 2 microglobulin which is a low molecular weight protein and is present on all nucleated cells, was used as a housekeeping gene to normalize the mRNA expression levels and control error between samples. The sequences (5′ to 3′) of the specific PCR primers and probes are shown in Table 1. The real-time PCR assay was conducted according to the manufacturer's instructions.
Table 1. Designed primers and probes for semi-quantitative taqman real-time polymerase chain reaction used in this study. The conditions were as follows: 45 cycles at 95°C for 10 s and 60°C for 45 s
Measuring oxidative stress
According to the direct effect of oxidative stress on activating endothelial cells, we also evaluated oxidative stress in the sera of the patients and control groups using pro-oxidant–antioxidant balance (PAB) assay protocol. The PAB value from 0–25 HK (Hamidi and Koliakos), 25–50 HK, 50–75 HK, and 75–100 HK was considered as low, moderate, high and very high oxidative stress, respectively.
The Statistical Package for the Social Sciences version 16.0 (SPSS Inc., Chicago, IL, USA) was used for statistical analysis. The descriptive data were presented as means ± standard error (mean ± SE) and frequency tables. The Kolmogrov–Smirnov test indicated that the obtained data did not have a normal distribution. Hence, statistical procedures for comparing the gene expression of ICAM-1 and VCAM-1 between groups were performed with the Kruskal–Wallis and Mann–Whitney tests. In addition, Spearman correlations between the number of smoked cigarettes, duration of smoking, and gene expression of ICAM-1 and VCAM-1 were calculated to evaluate the potential effects of smoking habit on the gene expression of targeted molecules. The significance level was considered < 0.05 with confidence interval of 95%.
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In total, 58 volunteers completed the study (21 TAO patients, 20 young male smokers, and 17 young male nonsmokers). The mean age was 39 ± 2 years for the TAO patients, 36 ± 2 years for the smokers and 36 ± 1 years for the nonsmokers. The number of cigarettes smoked daily was 24 ± 3 cigarettes for the TAO group and 20 ± 2 cigarettes for control smoker group. The duration of smoking was 19 ± 2 years for the TAO group and 15 ± 1 year for the smoker control group. All of the TAO patients complained of burning pain at rest. Of the 21 TAO patients, 19 were new cases of TAO, and two had a history of several admissions. All of the TAO patients were current smokers at the time of blood sampling. One TAO patient underwent below-knee amputation and three patients underwent toe amputations.
The PAB values of the TAO, smoker and non-smoker groups were 67.9 ± 14.8 HK, 51.6 ± 16.2 HK and 42.8 ± 8.1 HK, respectively.
The mean index of ICAM-1 mRNA expression in HUVECs after incubation with TAO sera showed 18.26 ± 6.9; for smokers and nonsmokers, the means showed 2.76 ± 0.73 and 2.59 ± 0.5, respectively. Based on Kruskal–Wallis test, no significant difference was observed in ICAM-1 gene expression among the studied groups (P = 0.08). The gene expression of ICAM-1 in HUVECs after incubation with TAO sera was significantly increased when compared to expression following treatment with sera of healthy smokers (P = 0.04) or non-smokers (P = 0.028). However, no significant differences were found in the expression of ICAM-1 between the smokers and non-smokers (P = 0.76).
The mean index of VCAM-1 mRNA expression in HUVECs following incubation in TAO sera showed 1.58 ± 0.59; for the smokers and the nonsmokers, these values were 0.11 ± 0.03 and 0.08 ± 0.07, respectively. Significant differences in VCAM-1 gene expression were found among the studied groups (P < 0.001). The expression of VCAM-1 in HUVECs following incubation in TAO sera was also significantly increased when compared to expression in cells treated with sera from healthy smokers (P = 0.001) and nonsmokers (P < 0.001). VCAM-1 gene expression was also significantly increased in smokers compared to nonsmokers (P = 0.006). Based on the Spearman test, a significant correlation was noted between the gene expression of ICAM-1 and number of cigarettes smoked daily (P = 0.04, R = 0.421), but expression of VCAM-1 did not correlate with the number of cigarettes smoked per day (P = 0.68). Interestingly, a significant correlation was found between duration of smoking and expression of VCAM-1 (P < 0.001, R = 0.672) (Fig. 1), but no correlation was demonstrated between the expression of ICAM-1 and duration of smoking (P = 0.44).
Figure 1. The probability plot of regression showing correlation between expression of vascular adhesion molecule (VCAM-1) and duration of smoking (P < 0.001, R = 0.672).
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This study showed that TAO sera could activate HUVECs and that a significantly greater gene expression of cell adhesion molecules occurred after incubation of HUVECs for 2 h with TAO sera than with control sera (Table 2). We used a smoking habit-matched control group for this study to omit the bias of oxidative stress induced by smoking, which might enhance the up-regulation of endothelial cell adhesion molecules, particularly ICAM-1.
Table 2. General characteristics of all the subjects included in this study. The descriptive data are presented as means ± standard error
|Characteristics||TAO patients (n = 21)||Healthy smokers (n = 20)||Healthy non-smokers (n = 17)|
|Age in years||39.5 ± 1.6||35.6 ± 1.4||35.9 ± 1.3|
|No. of daily smoked cigarettes||24 ± 3||20 ± 2||0|
|Duration of smoking in years||19.14 ± 2.04||14.4 ± 1.28||0|
|PAB assay value||67.9 ± 14.8 HK||51.6 ± 16.2 HK||42.8 ± 8.1 HK|
|ICAM-1 expression (expression index)||18.26 ± 6.9 ei||2.76 ± 0.73 ei||2.59 ± 0.5 ei|
|VCAM-1 expression (expression index)||1.58 ± 0.59 ei||0.11 ± 0.03 ei||0.08 ± 0.07 ei|
We also endeavored to match the socioeconomic class of the TAO subjects with that of the two control groups. This was done to omit possible bias due to an influence of the lack of nutritional anti-oxidants, arising from a less nutritious diet, on the oxidative stress level in endothelial cells. Notably, the number of cigarettes smoked daily had a significant relationship with ICAM-1 gene expression. Therefore, cigarette smoke (CS) might induce ICAM-1 expression due to its role in promoting systemic oxidative stress.[17, 19] This might explain why the gene expression of ICAM-1 was significantly higher than in smoker group, but the P-value was near the significance level (P = 0.04). The difference between the smoker and TAO group might reflect the existence of pro-inflammatory cytokines such as tumor necrosis factor (TNF)-α and interleukin 1 (IL-1), the presence of which in TAO has been reported in several studies. The mean level of ICAM-1 gene expression was higher in the smoker group than in the nonsmokers, but this difference was not statistically significant. Therefore, we checked the pro-oxidant–antioxidant balance in the sera of TAO and control groups. We found that oxidative stress was significantly higher in the TAO group than in either of the two control groups, while no significant difference was observed in the oxidative stress levels of the control smokers and nonsmokers. This might explain the lack of a significant difference in ICAM-1 gene expression between the smoker and nonsmoker groups. However, the limited numbers in the control groups might also be a confounding reason. Our insistence in matching our subjects for socioeconomic class and for smoking habit limited our recruitment of control subjects.
In contrast to the results for ICAM-1, the gene expression of VCAM-1 was significantly higher in HUVECs treated with TAO sera than with sera from smokers or nonsmokers. This gene expression was also significantly higher in the smoker group than in the nonsmoker group. Notably, the duration of smoking, but not the number of cigarettes smoked, had a significant correlation with VCAM-1 gene expression. A recent study has demonstrated that smoking more than 20 years would significantly enhance platelet activation in comparison to smoking < 20 years. It is noteworthy that activated platelets could IL-1α which can induce the expression of VCAM-1 on endothelial cells. Additionally, platelet activation has also been reported in TAO. Moreover, IL-1α can up-regulate the expression of Very Late Antigen-4 (VLA-4) ligand on the surface of erythrocytes, enabling them to attach to the VCAM-1 of endothelial cells and enhance thrombus formation.
In addition, chronic smoking might also contribute to endothelial cell dysfunction and nitric oxide (NO) bioavailability. NO has an important role in regulating the expression of adhesion molecules on endothelial cells. Of note, polymorphism in the promoter region of endothelial NO synthase gene (eNOS-768C), and subsequent NO expression impairment, have also been detected in TAO patients. Therefore, decreasing the systemic NO levels might promote up-regulation of endothelial cell adhesion molecules, in particular VCAM-1.[24-26] This might explain the significant difference observed here in VCAM-1 gene expression between TAO cases and healthy smokers and also between smokers and nonsmokers. NO is also a strong anti-oxidant, so a lack of systemic NO bioavailability would lead to high oxidative stress.
In 2001, a study demonstrated that monocytes which adhere to VCAM-1 on endothelial cells release tissue factor, a protein which is necessary for the initiation of thrombin formation from prothrombin, and therefore induces a hypercoagulative state.
In addition, our previous study pointed to a significant relationship between duration of smoking and further need for amputation and we found that smoking for more than 20 years was a risk factor for greater limb loss. In contrast, the number of cigarettes smoked daily was not associated with the degree of limb loss in TAO patients. In the current study we demonstrated the close relationship between duration of smoking and VCAM-1 expression, whereas number of daily smoked cigarettes had a significant relationship with ICAM-1 expression. Therefore, up-regulation of VCAM-1 might be more important than that of ICAM-1 for the outcome of TAO, since it enhances thrombus formation and consequently might lead to limb ischemia.[2, 23] This finding might explain why immunosuppressive treatments that predominantly down-regulate ICAM-1 expression do not have any noticeable effect on recovery in TAO patients.
In conclusion, TAO sera can activate in vitro cultured HUVECs and the same activation might also occur in vivo. This would explain the observed up-regulation of endothelial cell adhesion molecules in non-diseased limbs of TAO patients. On the other hand, vascular inflammation and activated endothelial cells accompanying platelet activation might reflect a background of thrombotic events in TAO. Therefore, plasma exchange might be beneficial for TAO patients in the acute phase of their disease as it would transiently remove inflammatory mediators, oxygen free radicals and activated platelets, thereby down-regulating the expression of endothelial cell adhesion molecules. This might reduce the risk of thrombotic events and limb loss in the acute phase of this disease.
However, the use of plasma exchange in the acute phase of TAO remains debatable. Plasma exchange might be no more effective than the combined effects of anti-oxidant therapy and administration of exogenous NO or NO inducers, together with smoking cessation, for down-regulation of endothelial cell adhesion molecules. However, plasma exchange might be helpful for those heavy-smoking patients who are unable to stop smoking in the acute phase of their disease. Additionally, for long-term management of TAO, administration of NO inducers such as cilostazol and anti-oxidants such as zinc or selenium, might be helpful in reducing the risk of limb loss.