Shrookmousa s, Laymouna A, Mohamed N. E. G, Wadie M, Philip M. V. Serum Osteoprotegerin Level as a Risk Factor for Atherosclerosis in Systemic Lupus Erythematosus Patients: A Cross Sectional Study. Biomed Pharmacol J 2021;14(3)

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Manuscript received on :07-04-2021
Manuscript accepted on :09-08-2021
Published online on: 18-08-2021

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Plagiarism Check: Yes
Reviewed by: Dr. Salman Ahmed Pharmacognosy
Second Review by: Dr. Sabu Thomas
Final Approval by: Dr Anton R Kiselev  

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Shrookmousa¹, Ahmed Laymouna² , NagwaAbd El Ghaffar Mohamed³, Mary Wadie⁴ and Mariana V Philip¹.

¹Department of internal medicine, faculty of medicine, Cairo University.

²Department of internal medicine department, rheumatology and immunology subdivision, faculty of medicine, Cairo University.

³Department of Clinical and Chemical Pathology, National Research Center, Egypt.

⁴Department of internal medicine, rheumatology and immunology subdivision, faculty of medicine, Cairo University.

Corresponding Author E-mail: ahmedlaymouna@gmail.com

DOI : https://dx.doi.org/10.13005/bpj/2246

Abstract

Background: Prediction ofthe risk of atherosclerosis in Egyptian patients with systemic lupus erythematosus (SLE) by measuring serum osteoprotegerin (OPG) level and correlation between OPG levels and SLE disease activity (assessed by Systemic Lupus Erythematosus Disease Activity Index [SLEDAI] score), is the aim of this study. Methods:A cross sectional study in which all patients were subjected to full medical history, full clinical examination (with special emphasis to SLE symptoms and signs e.g. photosensitivity, arthralgia, arthritis, malar rash, blood pressure and lower limb edema), laboratory investigations (CBC, random blood sugar [RBS], ESR, albumin/creatinine ratio, serum urea and creatinine, serum albumin, C3, total cholesterol, LDL-cholesterol, HDL-cholesterol, triglycerides, calcium and phosphorus). Electrocardiography (ECG) was done to calculate QTc interval using Bazett’s formula: QT_C = QT / √ RR. Serum osteoprotegerin level was measured using ELISA technique. All participants were subjected to imaging in the form of: Carotid duplex: to assess intima-media thickness just two cm before carotid bifurcation, and to assess any plaques, if present. Also brachial artery flow mediated dilatation method: for detection of subclinical atherosclerosis. Results: There was a statistically significant negative correlation between serum levels of osteoprotegerin and brachial flow mediated dilation percentage (FMD %) values (P value > 0.001) which means a significant correlation between high serum OPG levels and subclinical atherosclerosis in SLE patients. There were statistically significant differences between SLE cases and controls in right and left carotid intima-media thickness (P value: 0.034 & 0.036 respectively), serum osteoprotegerin levels (P value: > 0.001) and QTc values (P value: 0.011) which were all statistically significantly higher in SLE patients, while brachial FMD % was statistically significantly lower in SLE patients than in controls (P value: > 0.001). Also, there was a statistically significant positive correlation between serum OPG levels and QTc interval (P value: 0.006). Conclusion: We have concluded that serum OPG level has a significant correlation with subclinical atherosclerosis and endothelial dysfunction, which was measured by CIMT and brachial mediated flow dilation method.

Keywords

Atherosclerosis; Carotid Intima-Media Thickness; Osteoprotegerin; SLE

Introduction

Osteoprotegerin (OPG) is a tumor necrosis factor (TNF) receptor family member. It is a regulator of bone resorption (1). OPG is produced by a variety of organs and tissues, including the cardiovascular system (heart, arteries, veins), lung, kidneys, bone and immune tissues ¹. The expression and production of OPG is complicated and is regulated by various cytokines and hormones ². Osteoprotegerin is a soluble protein that seems to exert proatherogenic effect and serum OPG has been involved in the development of atherosclerosis in general population ³. High levels were considered as a novel marker for stroke in women ⁴.

Atherosclerosis prevalence is higher in patients with SLE than in the general population, becoming a leading cause of morbidity and mortality in these patients ⁵. Carotid ultrasound is animaging modality that allow non-invasive assessment of vascular anatomy and function ⁶. Use of this technique allows measurement of a variety of parameters including intima-media thickness (IMT), arterial diameter and presence of plaques ⁶. Corrected QT interval (QTc), measured by electrocardiogram, is prolonged in SLE patients and can predict subclinical atherosclerosis ⁷.

Methods

Design: A cross sectional study

Population of the study were divided into 2 separate groups

Group A

Forty five SLE patients: were selected from the outpatient clinic of rheumatology and immunology at Kasr Al-Ainy Hospital, Cairo university who are fulfilling the American College of Rheumatology (ACR) revised classification criteria (1982) for SLE ⁸.

All patients were subjected to

Full medical history

Thorough clinical examination (with special emphasis to SLE symptoms and signs e.g. photosensitivity, arthralgia, arthritis, malar rash, blood pressure and lower limb edema … etc.).

Laboratory investigations in the form of: CBC, RBS, ESR, albumin/creatinine ratio, serum urea and creatinine, serum albumin, C3, calcium and phosphorus. Total cholesterol, LDL-cholesterol, HDL-cholesterol, triglycerides after fasting for 12 hours.

Serum osteoprotegerin level was be measured using ELISA technique.

ECG using 12-lead-ECG to calculate QTc interval using Bazett’s formula: QT_C= QT / √ RR ⁹.

All patients were subjected to imaging in the form of

Carotid duplex: was done using a linear probe 7.7 MHz using HDI 5000 machine to assess intima-media thickness of both common carotid arteries  just two cm before carotid bifurcation by the same operator while the patient is lying flat in bed, and to assess the presence of any plaques, if present.

Brachial artery flow mediated dilatation method: by a linear probe, 7.5 MHz using a machine. The diameter of the brachial artery was measured while the patient is lying flat in bed and then it was re-measured after in response to an increase in blood flow during reactive hyperemia (induced by cuff inflation and then deflation). The cuff inflation period of 5 minutes is decided to produce adequate hyperemia to allow flow-mediated dilatation, but not to compromise patient comfort. The usual scanning period used in our laboratory is 30 seconds before and 90 seconds after the cuff deflation. Then, the percentage of dilation was calculated by doing this equation:

(BA diameter after deflation – BA diameter before deflation) X 100 / BA diameter before deflation

Group B

Forty five subjects, who are age and sex matched, as a control group.

They were subjected to: Serum OPG level was measured by ELISA technique. ECG for measuring QTc interval. Imaging in the form of carotid artery duplex and brachial artery flow mediated dilation method.

Data were coded and entered using the statistical package SPSS (Statistical Package for the Social Sciences) version 25. Data was summarized using mean, standard deviation, median, minimum and maximum in quantitative data and using frequency (count) and relative frequency (percentage) for categorical data. Comparisons between groups were done using unpaired test in normally distributed quantitative variables while non-parametric Mann-Whitney test was used for non-normally distributed quantitative variables ^10,11. For comparing categorical data, Chi square (c2) test was performed. Exact test was used instead when the expected frequency is less than 5 ¹¹. Correlations between quantitative variables were done using Spearman correlation coefficient ¹². P-values less than 0.05 were considered as statistically significant.

Results

In our study, 90 participants were involved, 45 systemic lupus erythematosus (SLE) cases and 45 controls. Forty three SLE cases (95.6%) were females, while only two cases (4.4%) were males (table). Disease duration mean was 4.2 years with standard deviation (SD): 3.99 (table). Four cases only (8.9%) were known to have hypertension. Twenty five SLE cases (55.6%) had lupus nephritis. Twenty one SLE cases (46.7%) have hypocalcaemia, while twenty four cases (53.3%) have normal calcium levels. Twenty four cases (53.3%) are dyslipidemic (table 1).

Table 1: Percentages of some parameters in SLE cases.

Regarding SLE cases included in our study, their age mean was 28.56 years (SD: 6.91), both right and left carotid intima-media thicknesses (CIMTs) mean values were 0.8 mm (SD: 0.02). Random blood sugar mean was 129.47 mg/dl (SD: 50.24). Regarding brachial flow mediated dilation percentage (FMD %), mean was 6.66 % (SD: 1.54). Mean steoprotegerin level was 10.98 (SD: 1.96) table 2.

Table 2: Statistical analysis of different parameters in SLE cases.

There were statistically significant differences between SLE cases and controls in (table 3) right and left carotid intima-media thickness, serum osteoprotegerin levels and QTc values which were all significantly higher in SLE patients (P values 0.034, 0.036, > 0.001, and 0.011 respectively), while flow mediated dilation percentage (FMD %) was statistically significantly lower in SLE patients than in controls (P value: > 0.001) table3.

Table 3: Statistical comparison of certain parameters between SLE cases and controls. Click here to view table

There was a statistically significant negative correlation between serum levels of osteoprotegerin and brachial flow mediated dilation percentage (FMD %) values which means a significant correlation between high serum OPG levels and subclinical atherosclerosis in SLE patients (P value: > 0.001) table 4. Also, there was a statistically significant positive correlation between serum OPG levels and QTc interval (P value: 0.006) table 4.

There was also a statistically significant positive correlation between serum OPG values and dyslipidemia (P value: 0.003 for total cholesterol, 0.002 for LDL-cholesterol), right and left carotid intima-media thicknesses were not statistically significantly affected by higher serumosteoprotegerin levels (table 4).

Table 4: Statistical correlation between OPG and other parameters in SLE cases and controls.

We did a comparison between SLEDAI score of SLE patients and other parameters (table 5). It showed a significant positive correlation of SLEDAI score with ESR and a statistically significant negative correlation with C3 levels (P values: 0.002 and 0.005 respectively). There was also a statistically positive correlation between SLEDAI score and A/C ratio (P value: 0.00). There was no significant correlation between SLEDAI score and OPG, right and left CIMT, FMD % or QTc interval (P values: 0.328, 0.115, 0.722, 0.097, 0.930 respectively) table 5.

Table 5: Statistical correlation between SLEDAI score and other parameters in SLE cases.

We also compared right and left CIMT and all other parameters. It didn’t show any statistically significant correlation between CIMT and any other parameters studied including serum OPG, % FMD, SLEDAI score and QTc interval (table 6).

Table 6: Statistical comparison between different parameters in SLE cases and both right and left CIMT

In our study, we made a comparison between SLE patients with and without lupus nephritis (LN). There was a statistically significant positive difference between LN and OPG levels (P value: > 0.001), while there is no statistically significant difference between the presence of LN and FMD% (P value: 0.610). Right and left CIMTs have also no significant correlation with the presence of LN. LDL-cholesterol has significant correlation with LN. QTc interval has no significant correlation with LN (table 7).

Table 7: Statistical comparison of different parameters in SLE patients with and without LN. Click here to view table

Regarding dyslipidemia, serum OPG levels were significantly higher in dyslipidemic SLE patients. Right and left carotid IMTs are not different between both dyslipidemics and those patients with normal lipid profile values (table 8).

Table 8: Statistical comparison of different parameters in SLE patients with and without dyslipidemia. Click here to view table

Discussion

Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disease that has a wide range of clinical and serological manifestations that can affect any organ in the body ¹³.

Osteoprotegerin (OPG) is a member of the tumor necrosis factor (TNF) receptor family ¹⁴. It has been identified as a regulator of bone resorption (14). OPG is produced by a variety of organs and tissues, including the cardiovascular system (heart, arteries, and veins), lung, kidney, immune tissues, and bone ¹⁴. The expression and production of OPG is complicated and is regulated by various hormones and cytokines ¹⁵.

Elevated serum OPG level has been associated with the progression of vascular calcification ¹⁶.High OPG levels were not only associated with overall cardiovascular mortality but were also a novel marker for stroke in women ¹⁷.  In our study, we found that OPG was statistically significantly higher in SLE patients than in controls. This is in line with Park et al., 2014¹⁸ who described a statistically significantly higher levels of OPG in SLE patients than controls. Also Kwork et al., 2009 ¹⁹ concluded that OPG was statistically significantly higher in SLE patients than in control subjects. By contrast, Carmona-Fernandes et al., 2011 ²⁰ reported reduced serum OPG levels in SLE patients than in controls. This difference may be related to different ethnicities.

A prolonged QT interval on the electrocardiogram (ECG) is an easily measurable, reproducible parameter that has been linked with early-onset atherosclerosis in the general population and some subpopulations with high cardiovascular risk ²¹. SLE patients also appear to have a more prolonged QT interval, although clinical relevance of this has not been studied ²².

In our study, corrected QT interval (QTc) was statistically significantly prolonged in SLE cases than in control subjects. This was in line with Cardoso et al., 2005 ²² who concluded that QTc interval is statistically significantly higher in SLE patients than in controls. Riveral-Lopez et al., 2016(7) found a statistically significant prolongation of QTc in SLE patients. This denotes that patients with SLE had higher incidence to develop early and premature atherosclerosis than age- and sex-matched control group and this highlights the importance of QT prolongation in SLE patients and that SLE patients should be cautious when they use drugs that cause QT prolongation.

The prevalence of atherosclerosis is higher in patients with SLE than in the general population, becoming a leading cause of morbidity and mortality in these patients ²³. Carotid ultrasound is one of the several imaging modalities that allow non- invasive assessment of vascular anatomy and function ²⁴. Use of this technique allows measurement of a variety of parameters including IMT and presence of plaques ²⁴. CIMT and presence of plaques have been shown to predict cardiovascular events in multiple large studies ²⁵.

In this study, we assessed subclinical atherosclerosis by measuring CIMT and we concluded that CIMT is statistically significantly higher in SLE patients than in controls, in spite of normal mean level of CIMT it is still higher than controls who are age- and sex-matched. This was in a line with a systematic review by Wu et al., 2016 ²⁶ who concluded that CIMT was statistically significantly higher in SLE patients than in controls. Roman et al., 2003 ²⁷, found that ultrasonographically demonstrated carotid artery plaques were present in 37 percent among 197 patients with SLE, but were present in only 15 percent of controls.

It has been noted that the carotid plaque prevalence increased from 31 percent at baseline to 40 percent at follow-up of 217 patients with SLE (disease duration mean: 10.5 years with SD 7.5 years) followed for an average of four years in a longitudinal study ²⁸. Our finding, together with prolongation of QTc interval confirm our hypothesis of premature atherosclerosis in SLE patients.

Flow- and nitrate-mediated vasodilation (endothelial dependent dilation) was measured to describe endothelial dysfunction in SLE patients when Lima et al., 2002 ²⁹ found a statistically significant lower values of flow mediated dilation (FMD) % in SLE patients.

In our study, we found that FMD % was significantly reduced in SLE patients compared to controls.

El-Magadmi et al., 2004 ³⁰ showed similar results of FMD % being significantly lower in SLE patients than in controls. Also, this was in a line with Kiss et al., 2006 ³¹ who concluded  significantly lower values of FMD % in SLE patients than in controls. Rivera-Lopez, 2016⁷  has found a statistically significant relation between SLE and subclinical atherosclerosis but using pulse wave velocity, another method to detect subclinical atherosclerosis.

The presence of prolonged QT interval, higher CIMT and lower brachial FMD in SLE patients compared to control subjects in this study confirm the presence of subclinical atherosclerosis in SLE patients, and endothelial dysfunction in those patients confirm the idea of considering SLE as a major risk factor for atherosclerosis and early cardiovascular morbidity.

OPG may mediate important and complex links between the bone and vascular systems ³². Thus, this molecule may play a central role in regulating the development of vascular calcification coincident with declines in bone mineralization with osteoporosis ³³.

In our study, we found that there is a significant correlation between high serum OPG level and subclinical atherosclerosis in SLE patients as it is inversely correlated with brachial flow mediated dilation.

This was in consistent with De Ciriza et al., 2014 ³⁴ who found a significant association between serum OPG level and carotid intima media thickness. Also Vik et al., 2010 ³⁵ found that Serum OPG was a significant predictor for CIMT in crude analysis and after adjustment for cardiovascular risk factors. In 2011, Vik et al.³⁶ performed a population based study with 12 years of follow up that found that serum OPG was significantly associated with incident myocardial infarction, ischemic stroke and total mortality independent of traditional cardiovascular risk factors and that serum OPG acts as a specific predictor for atherothrombotic cardiovascular diseases. All these studies were in contrast to our study as OPG didn’t statistically correlated to CIMT and this may be due to normal mean CIMT in our patients while in their study this wasn’t the case as mean values of right and left CIMT in our study were both 0.8 mm with SD: 0.2. This may be due to a relatively shorter disease duration in our study (mean 4.2 with SD: 3.99), while in a s study of Thompson et al., 2008(28) disease duration was higher (mean 10 years with SD 7.5).  Also we have suggested that changes in brachial FMD may precede changes in CIMT and it may be considered as an early indicator of subclinical atherosclerosis in SLE patients before CIMT changes and that OPG may play a role in the endothelial dysfunction more than it did in arterial atherosclerosis and we hypothesize that the starting process of premature atherosclerosis in SLE is endothelial dysfunction, and this may explain why dyslipidemia didn’t correlate with FMD and CIMT, however the absence of correlation between CIMT, FMD, and SLEDAI, C3 and ESR denotes that premature atherosclerosis is not totally preventable through disease activity control and raise the possibility of different underlying mechanisms in our study from which we highlighted elevated OPG serum levels, however triglycerides only is related to disease activity. So, controlling disease may minimally contribute to ameliorating premature atherosclerosis.

Park et al., 2014¹⁸ also had found a statistically significant association between OPG and carotid intima media thickness even after adjustment for age, hypertension, LDL and creatinine, OPG levels (1086 versus 517 pg/ml, p < 0.001) and CIMT (0.63 versus 0.45 mm, p < 0.001) compared with control subjects. Similarly, Kiechl et al., 2004 ³⁷ also found a statistically significant association between OPG and carotid atherosclerosis, progressive atherosclerosis and cardiovascular risk. Also, in our study, OPG correlates with QTc interval which also may be an earlier sign of atherosclerosis together with low FMD % before CIMT changes.

OPG has been implicated diabetes mellitus, myocardial ischemia and left ventricular dysfunction ³⁸.

In our study, there was a statistically significant positive correlation between OPG and blood sugar and this may raise the possibility of secondary diabetes mellitus (DM) in SLE patients other than steroid induced DM, so we suggest follow up of blood sugar in SLE patients even if they are off steroids.

This was in a line with Jono et al., 2002 ⁴⁰ andSchoppet et al., 2003⁴⁰ who found that OPG was significantly higher in diabetics and those with poor glycemic control.

Musialik et al., 2017 ⁴¹ also found a statistically significant correlation between OPG and body mass index independent on age, systolic blood pressure or glucose. This was also in line with Bernardi et al., 2014⁴² who concluded that OPG, after adjustment of other risk factors of metabolic syndrome, is still significantly associated with metabolic syndrome.

More recent evidence has linked OPG to kidney injury ⁴³. Circulating OPG levels increase as estimated glomerular filtration rate (eGFR) decreases in diabetic and non-diabetic CKD patients ^44,45.

In our study, we found that OPG was significantly higher in SLE patients with lupus nephritis rather than in SLE patients without lupus nephritis.

Also, we found that OPG was statistically significantly higher in lupus patients with high urea than in those with normal urea. This was in a line with Mikami et al., 2008 (46) who found a significantly positive correlation between serum OPG and CKD. This also was supported with Jiang et al., 2011(47) who concluded also a significantly positive correlation between OPG and renal failure. Other studies also have similar results (48,49). So we concluded that OPG is higher in lupus nephritis together with increased LDL in LN compared to those without LN in spite of the absence of statistically significant difference in CIMT between the 2 groups raising the possibility that OPG may be one of the etiologies of dyslipidemia in SLE patients and that OPG may be used as an early marker impaired renal chemistry in SLE patients.

In our study, the presence of increased CIMT in SLE patients compared to controls and being not correlated to dyslipidemia raising the probability of underlying etiology to premature atherosclerosis in SLE patients rather than elevated lipid profile and the presence of negative correlation between OPG and brachial FMD together with the positive correlation of OPG and with cholesterol and LDL may highlight that OPG may be one of the underlying causes of secondary dyslipidemia in SLE patients and one of the main factors in premature atherosclerosis in SLE patients, also this was confirmed by its positive correlation with QTc interval. Also, in our study, the presence of prolonged QT interval in the absence of hypocalcemia highlights an underlying pathology of the conduction system in SLE patients.

The absence of correlation between of SLEDAI and OPG and also that there is no statistically significant differences in OPG in both dyslipidemic and non-dyslipidemic SLE patients may highlight that dyslipidemia in SLE isn’t related to disease activity and should be followed regularly and monitored to prevent an important contributor to premature atherosclerosis in SLE patient if the patient is in remission, but also the positive correlation SLEDAI and triglycerides raise the role of disease activity in dyslipidemia  and that controlling disease activity may guard against premature atherosclerosis and secondary dyslipidem

Conclusion

Serum OPG level has a significant correlation with subclinical atherosclerosis and endothelial dysfunction, which was measured by CIMT and brachial mediated flow dilation method.

Acknowledgement

Not applicable

Conflict of interests

Not applicable

Funding source

No funding received.

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