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MEDICAL SPECIALITIES

Received: October 22^nd 2014

Accepted: March 6^th 2015

Carotid and brachial intima-media thickness of the allelic variants G894T and T-789C of eNOS in systemic sclerosis patients

Ángeles Martínez-Godínez,^a Ángel Miliar-García,^b María Itandehui Bautista-Jiménez,^c Fernando Jandete-Rivera,^d Olga Lidia Vera-Lastra,^e Luis J. Jara,f Héctor Bizueto-Rosas,^g María del Pilar Cruz-Domínguez^h

^aEscuela Superior de Medicina

^bJefatura de Laboratorio e Investigación de Biología Molecular y Genómica/Escuela Superior de Medicina

^cDepartamento de Medicina Interna ex residente de UMAE Especialidades La Raza

^dServicio de Radiología

^eDirección de Educación e Investigación en Salud

^fServicio de Angiología

^gDivisión en Investigación en Salud

^a,bInstituto Politécnico Nacional

^c,d,e,f,gHospital de Especialidades, Centro Médico Nacional La Raza, Instituto Mexicano del Seguro Social

Distrito Federal, México

Communication with: María del Pilar Cruz-Domínguez

Telephone: (55) 5724 5900, extensión 23015

Email: drapilarcd@prodigy.net.mx

Objective: To evaluate comparatively the carotid intima media thickness index (IMT) and brachial index with Doppler ultrasound, and pulmonary artery pressure with echocardiography in allelic variation of G894T and T-789C eNOS (endothelial nitric oxide synthase) in patients with systemic sclerosis.

Methods: This is a cross-sectional study in patients with scleroderma. The inclusion criteria were: age 18-70 years, scleroderma diagnosed patients with a prior informed consent. Polymorphism G894T and T-789C of eNOS was measured in blood, and IMT by carotid and brachial Doppler. We analyzed with descriptive statistics, Student’s t test or chi square for association of variables and Spearman to correlate Doppler parameters.

Results: We found abnormally high IMT in carotid and brachial arteries. We also observed low correlation between both brachial arteries, good correlation between carotids and no correlation between carotid and brachial arteries. The left IMT > 0.65 mm in brachial artery corresponds to pulmonary pressure > 30 mmHg by echocardiography. G894T gene polymorphism was associated with increased IMT in right carotid.

Conclusion: The G894T eNOS polymorphism was associated with increased IMT in right carotid. The one side carotid IMT is consistent with its contralateral. The left brachial IMT above to 0.65 mm, suggests the possibility of pulmonary arterial hypertension.

Keywords: Atherosclerosis, Carotid and brachial IMT, Systemic sclerosis.

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The association between autoimmune diseases and atherosclerosis is well-described in connective tissue diseases. Proposed mechanisms in systemic sclerosis (SS) are: chronic inflammation, dyslipidemia, autoantibodies, and endothelial dysfunction.¹ SS is characterized by endothelial damage, autoimmunity, and fibrosis, where the microvascular disease has been distinctive, and the macrovascular disease has been documented as accelerated atherosclerosis.^2,3

Intima-media thickness (IMT) by Doppler echo is the imaging modality most widely used to assess atherosclerosis. Carotid IMT correlates with traditional risk factors for atherosclerosis and it is an independent predictor of cardiovascular events.⁴ Carotid IMT in patients with scleroderma has been reported on average higher (0.8 to 0.96 mm) than expected in the general population (0.44-0.86 mm).^1,5-7

The macrovascular impact of atherosclerosis in 865 patients with scleroderma showed an adjusted hazard ratio of 1.80 (CI: 05%, 1.07-3.05) for myocardial infarction and 2.61 (95% CI, 1.54-4.44) for cerebrovascular disease. Incidence rates for peripheral arterial disease in scleroderma was 7.6 versus 1.9 per 1000.⁸ Patients with systemic sclerosis also have a higher frequency of cerebral calcifications with tomography and cerebral microinfarcts with magnetic resonance.^9,10

Genetic variants of nitric oxide without endothelial rate (eNOS) determine the structure and function of the eNOS, as well as abnormal NO synthesis and imbalance in vascular tone. Polymorphism of the G894T gene and the T-786C gene of eNOS apparently does not confer increased risk of SS, but its role in the vascular pathology that characterizes it remains to be evaluated.¹¹

Methods

Comparative cross-sectional study was performed in Servicios de Medicina Interna y Radiología at the Hospital de Especialidades of the Centro Médico Nacional La Raza from January to June 2013. Inclusion criteria were any gender, age 18-70 years, treated at the Servicio de Medicina Interna, diagnosed with scleroderma according to the ACR classification criteria, and who had blood determination of T-786C and G894T eNOS genes, as determined by RT-PCR.¹¹ We did not include subjects with autoimmune vascular comorbidity (vasculitis or other autoimmune diseases).

The sample size of 45 patients was calculated. In the Doppler variables for carotid and brachial artery we evaluated IMT, resistance index (RI) and pulsatility index (PI). 30% of the study population underwent a second measurement of carotid and brachial Doppler ultrasound to determine the inter-observer variability, which had 0.85% concordance by kappa test.

As for demographic and disease variables, we analyzed age, gender, type of scleroderma, duration of disease, pulmonary arterial hypertension (PAH) echocardiography, severity of Raynaud's phenomenon with the Medsger Scale,¹² lipid profile (HDL cholesterol, LDL cholesterol, triglycerides, and total cholesterol), and C-reactive protein (CRP).

To determine eNOS G894T and T-789C polymorphism the following was done:

Extraction of genomic DNA from peripheral blood was done using the extraction protocol for genomic DNA by enzymatic digestion (organic solvents: phenol, chloroform, or both). Each patient had 3 ml peripheral blood extracted, which was stored at -70 °C in falcon tubes with EDTA.

A spectrophotometer (JENWAY 6305) was used for quantitation of DNA. Wavelengths were between 260 nm and 280 nm.

PCR in real time: specific probes were designed for the analysis and identification of allelic variants to be studied by real-time PCR.

Each patient was scheduled three visits. At the first visit, data capture, physical examination, and classification of severity of Raynaud's phenomenon according to Medsger Severity Scale were done. At the second visit arterial Doppler ultrasound was performed ensuring measurement of PI, RI, and IMT of the bilateral carotid arteries at bulb and brachial levels. At the third visit blood samples were taken for PCR, HDL cholesterol, LDL cholesterol, triglycerides, and total cholesterol.

All information was captured and analyzed in SPSS (Statistical Package for Social Sciences) version 20. Descriptive statistics were done on the population characteristics with average, standard deviation, frequency, and percentage. We used chi-squared to compare qualitative variables such as variety of disease, and Student’s t test to compare surrogate parameters in Doppler for atherosclerosis in allele variants of the T-786C and G894T polymorphisms. The Pearson or Spearman correlation was calculated according to normal distribution of the data from all ultrasound parameters, severity of Raynaud syndrome, and pulmonary arterial pressure.

Results

In our study, 57 patients met the inclusion criteria; 55 women (96.5%) and 2 men (3.5%), of which 20 (35.1%) had the limited variety, and 37 (64.9%) the diffuse variety. The age was 50.85 ± 10.76 years, the duration of the disease was 10.58 ± 7.81 years; 54 were right-handed. They had the following comorbidities: ​​hypertension 12.5%, diabetes mellitus 3.6%, dyslipidemia 8.9%, hypothyroidism 16.1%, and 53.6% of patients had no associated comorbidities. We found no difference between the limited and diffuse variety in age or duration of disease (Table I). The severity of Raynaud's phenomenon according to the Medsger Scale was 1.40 ± 1.16 (0.76 ± 1.85 limited variety versus 083 ± 1.1 diffuse variety, p ≤ 0.001). Age showed significant correlation with carotid IMT (r = 0.34, p = 0.009) but not with the duration of disease. Triglycerides 152 ± 73.22 mg/dl were higher in the limited form in than in the diffuse (p ≤ 0.05). C-reactive protein was found at 6.85 ± 11.85 mg/l (range 3 to 83), significantly higher in the diffuse form of the disease. Total cholesterol was 177 ± 46.84 mg/dl, HDL cholesterol was 41.89 ± 11.69 mg/dl, LDL cholesterol was 111.24 ± 48.56 mg/dl with no difference between limited and diffuse subtypes. Total cholesterol was positively correlated with bilateral carotid IMT (right carotid r = 0.268, p ≤ 0.05; left carotid r = 0.324, p ≤ 0.01), and LDL cholesterol was positively correlated with left carotid IMT (r = 0.301, p ≤ 0.05).

Doppler ultrasound found abnormally high IMT in the carotid artery (> 1.0 mm) in 57% and brachial artery (> 0.6) in 77.2% regardless of the variety of the disease. In 9 (15%) atherosclerosis plaque was identified, one ulcerated and two calcified; 4 of them with the limited cutaneous variety and 5 with the diffuse form. The right carotid IMT tended to be lower than the left, and the right brachial also showed a tendency to be less than the left brachial although not statistically significant, and similar between subtypes of the disease (Tables I and II). The resistance index (RI) between the brachial artery and the carotid was similar. The pulsatility index (PI) in brachial and carotid arteries was greater in the right than the left (Table II). We found no correlation between IMT in carotid arteries with the brachial, but between carotid arteries (r = 0.484, p < 0.01) and between brachial arteries (r = 0.289, p < 0.05). We included 26 patients (46%) with pulmonary artery pressure > 30 mmHg, 30 (52%) with normal pulmonary artery pressure, and one (2%) who did not have a good echocardiographic window. Performing the ROC analysis for patients with scleroderma and PAH with Doppler ultrasound findings revealed that IMT of the brachial artery on the left side predicts the existence of pulmonary hypertension measured by echocardiography (area under the curve 75%, specificity 46.2%, and sensitivity 81%), when the cutoff was set at 0.65.   

In the polymorphism of eNOS for the T-786C gene, we analyzed 30% T/C or CC (Non-Wild Type or NWT) and 70% T/T (Wild Type or WT), while 46.7% were G/T or T/T (NWT) and 53.3 (WT) for G894T. As for the findings by Doppler ultrasound in patients NWT for the G894T polymorphism, the right carotid IMT was significantly higher than for WT; in the left carotid and brachial arteries IMT was similar between the allelic variants (Table III). In the case of the T-786C polymorphism, left carotid IMT was significantly lower in the NWT variant, IMT of right carotid and right and left brachial was similar between allelic variants (Table IV).  

The resistance index (RI) showed a tendency to be higher in the right carotid artery compared with the left in the diffuse subtype of the disease, and there were no differences in left carotid and brachial between subtypes of the disease (Tables I and II). RI was significantly higher for NWT allelic variants of gene G894T and similar for the allelic variants of gene T-786C (Table III and IV).

Discussion

Atherosclerosis is an inflammatory disorder mediated by the immune system, magnified by the inflammatory condition present in autoimmune diseases beyond the traditional risk factors.¹³ In patients with systemic sclerosis, IMT is higher than expected in the general population. In this study 57% of our patients with scleroderma had increased carotid IMT measured by Doppler. 15% of them had evidence of an atherosclerotic plaque. We found that despite higher frequency of hypertriglyceridemia in the group of patients with the limited form of the disease and increased inflammatory state in the diffuse form, IMT, pulsatility index, and resistance index were similar between the two varieties of the disease. The NWT allelic variant of the gene G894T, however, was associated with increased right carotid IMT and therefore atherosclerosis. By contrast, the NWT variant of gene T-786C was significantly associated with lower IMT in the left carotid, suggesting a protective role in atherosclerosis, which concurs a proposal in a recent meta-analysis where the risk of stroke was significantly lower in NWT genetic models in African-Americans and Caucasians.¹⁴ The set of NWT allelic variants of the gene G894T was also associated with higher rates of resistance in the right brachial artery, suggesting greater arterial rigidity and likely risk of vascular disease, atherosclerosis, and vascular calcification.^15,16 The IMT of the left vessels was higher than that of the right, but this was not statistically significant. However, the pulsatility index in the right carotid and brachial was significantly higher when compared to the left side, which could be related to the fact that most of our patients are right-handed, rather than to stenosis.¹⁷ Vascular disorders stand out in systemic sclerosis, mainly by microvascular damage in the capillary system and arterioles leading to decreased blood flow and consequent tissue ischemia. In contrast to the microvascular disorders, the effects of atherosclerosis and damage to blood vessels have received less attention. According to Brewster’s classification for peripheral vascular disease, atherosclerosis is divided into 4 categories: type 1 and type 2 comprising the condition of the large central vessels (from the aorta to the vessels of the proximal end of the knee and the elbows); type 3 includes vascular disease in both proximal and distal ends; type 4 only involves distal vessels of the knees and elbows; according to this classification, vascular alterations in SS consist mainly of type 3 and 4, but deterioration is also present in the vessels of the upper extremities, an atypical site for the development of atherosclerosis.^2,18 According to the meta-analysis by Au et al.,¹ macrovascular disorder and the prevalence of atherosclerosis and are clearly increased in the carotid, coronary, cerebrovascular, and renal arteries and in the peripheral vascular system in patients with SS compared to people without the disease.

The thickness of the carotid has been variable depending on the population included. Among them, the IMT of the common carotid in 12 Greek patients with diffuse scleroderma was 0.83 ± 0.46 mm vs. 0.3 ± 0.2 mm in its general population.⁵ Carotid IMT in 35 Italian patients with systemic sclerosis was 0.93 ± 0.29 mm vs. 0.77 ± 0.13 mm in controls.⁴ This is similar to a second study in 66 Italian patients with scleroderma, where the IMT was 0.90 ± 0.037 mm compared to controls with 0.69 ± 0.013 mm. In 44 Israelis with scleroderma, IMT > 0.9 mm was significantly higher in patients with systemic sclerosis than in controls (43 versus 28%).6 In our study population we found increased carotid intima-media thickness on average above 1.15 mm bilateral, higher than that in all of the populations before mentioned.^1,18 Although it is a weakness of our study that it failed to include controls, in studies of Mexicans from 35 to 64, the maximum IMT is expected at 0.76 mm for men and 0.71 mm for women.¹⁹ Similarly, the literature has considered an IMT ≥ 0.65 mm abnormal for medium-sized arteries in upper extremities;²⁰ we find in both brachial arteries average IMT above 0.67 mm, associated with increase in the resistance index for both arms in patients with SS. Previous studies have observed upper extremity vascular disease transcending to larger vessels than those involved in Raynaud's phenomenon, which characterizes the disease.¹³ Another of the data obtained in the study by Doppler ultrasound is PI, which normally decreases from the proximal arterial vessels to the distal vessels, so this index is higher in the brachial region of both sides with respect to the carotid arteries, suggesting increased peripheral resistance of patients with SS.¹⁵   

Autoimmune diseases such as scleroderma have been associated with high morbidity and mortality from cardiovascular disease and accelerated atherosclerosis, where drugs such as corticosteroids may aggravate it. Advanced subclinical disease should be identified and should be a therapeutic target to limit cerebrovascular and coronary complications.¹³ We found advanced disease in 15% of our patients, in whom we observed atherosclerotic carotid plaques, one with ulceration and turbulent flow; however, these changes were not found in the arms.

We found no linear correlation between the brachial and carotid arteries, this means that the patients with increased brachial IMT do not necessarily have disorder in the carotid IMT, as they can individually have brachial and/or carotid disorder. However, if abnormal values ​​are found in a brachial artery, disorder is very likely to be found on the contralateral side, this also applies in carotid location. Another important aspect of this study is that when we found brachial IMT greater than 0.67 mm, we also found pulmonary hypertension evaluated by echocardiography, a major cause of mortality in this population and that should be corroborated by cardiac catheterization. Another important aspect to note is that there was correlation with traditional cardiovascular risk factors with the presence of an altered carotid IMT, mainly for elevated total cholesterol and LDL cholesterol, however, in the logistic regression analysis we found no increased risk by presenting abnormal IMT, as other studies have reported.¹⁷

For this study, we intentionally included patients who had genetic analysis for allelic variants of eNOS in scleroderma, and of whom almost half were the NWT variants, in order to compare them, since the actual prevalence is much lower. In evaluating the eNOS polymorphisms T-786C and G894T in the NWT and WT groups, we found that there is increased IMT in the NWT group for eNOS G894T very significantly in the right carotid and in the right brachial resistance index, suggesting that the expression of allelic variants of gene G894T is strongly associated with increased macrovascular damage in patients with SS. PI was also higher in the NWT group of gene G894T, but it did not reach statistical significance because, in general, all patients had an abnormally high PI, indicating arterial stiffness, possibly due to vasculopathy, which requires further study. There are no previous studies on the gene T-786C which seems to have a protective effect for this disease, so it will be necessary to expand the sample to perform a multivariate analysis to identify whether it is a risk factor independent from those already known, and what degree of risk is conferred. However, a meta-analysis has made it clear that the presence of allelic variants of non-wild type gene G894T in Asian individuals, increases the risk of myocardial infarction.²¹

The clinical relevance of this study is to sustain that in the Mexican population with SS macrovascular disorder is found translated as carotid atherosclerosis and increased brachial arterial rigidity, which may be associated with increased risk of cardiovascular morbidity and mortality and pulmonary hypertension. These data show us the necessity of dietary and pharmacological intervention to reverse the accelerated progression of atherosclerosis, prognosis and survival of patients with systemic sclerosis.

Conclusions

The age of patients with SS is in direct correlation with carotid IMT, and increased carotid IMT is associated with the presence of the non-wild type allelic variant of gene G894. The abnormal increase of carotid IMT, considered surrogate marker of atherosclerosis and independent risk factor for cardiovascular disease, suggests the presence of accelerated atherosclerosis in patients with scleroderma.

This study was funded by the Fondo de Investigación en salud of IMSS, registered as FIS/IMSS/PROTMD11/1001.

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