THE IMPACT OF VITAMIN D ON THE ENDOTHELIUM OF RHEUMATOID ARTHRITIS PATIENTS

THE IMPACT OF VITAMIN D ON THE
ENDOTHELIUM OF RHEUMATOID ARTHRITIS
PATIENTS
Mashael F. Alotaibi1 ; Sawsan O. Khoja1 ; Wesam A. Alhejily2 ;
Sami M. Bahlas3 ; Alia M. Aldahlawi4,5 ;
Mahmoud E . Gadalla6 and Mohamed F. Elshal1,7
1Department of Biochemistry, Faculty of Science, King Abdulaziz University, Jeddah,
Saudi Arabia.
2 Cardiovascular medicine and interventional Cardiology, Faculty of medicine, King
Abdulaziz University, Jeddah, Saudi Arabia.
3Department of Internal Medicine, College of Medicine, King Abdulaziz University,
Jeddah, Saudi Arabia.
4Department of Biological Sciences, Faculty of Science, King Abdulaziz University,
Jeddah, Saudi Arabia.
5Immunology Unit, King Fahd Medical Research Center, King Abdulaziz University,
Jeddah, Saudi Arabia.
6Department of Molecular Diagnosis, Genetic Engineering and Biotechnology Institute,
University of Sadat City, Sadat city, Egypt.
7Department of Molecular Biology, Genetic Engineering and Biotechnology Institute,
University of Sadat City, Sadat city, Egypt.
Running Title: Vitamin-D Impact on rheumatoid arthritis patients
Corresponding Author:
Professor Dr. Mohamed Elshal
Current address: Molecular Biology Depatment,
Genetic Engineering and Biotechnology Institute,
University of Sadat City,
Sadat City, Egypt.
Email : Mohamed.elshal@gebri.usc.edu.eg
ORCID No. : 0000-0001-8973-6847
Researcher ID : H-7953-2012
Scopus ID : 6506130470
Key Words: Rheumatoid arthritis, vitamin D3, cardiovascular diseases,
flow-mediated dilation, circulating endothelial cells,
adhesion molecules, endothelial growth factor.
ABSTRACT
Background: Vitamin D is a steroid hormone that affects the expression of
many genes and regulates a wide variety of cell types and organ systems,
including the cardiovascular system. Patients affected by rheumatoid
arthritis (RA) present with accelerated atherosclerosis and increased risk of
cardiovascular disease (CVD). Aim: The aim is to evaluate the impact of
vitamin-D intake on markers of vascular function in patients with RA.
Methods: Forty RA with vitamin-D deficient patients were included and
were supplemented with oral doses of cholecalciferol (50,000 IU) weekly
Egypt. J. of Appl. Sci., 35 (11) 2020 200-219
for 8 weeks. Conventional rheumatoid measures such as DAS-28, RF, CRP
and anti-MCV as well as measures of vascular functions flow-mediated
dilatation (FMD) and circulating endothelial cells (CECs) and serum
adhesion markers ICAM and VCAM were assessed at baseline and 8 weeks
post-treatment. Forty healthy individuals were included as controls. Results:
RA patients showed impaired FMD as compared with controls (4.28% vs
10.10%, p<0 .05). Significant inverse relationships were detected between
DAS-28 score and both vitamin D and FMD (r=0.437; P=0.001 and
r=0.315; P=0.002 respectively). Vitamin-D intake was associated with
significant improvement in FMD and lower CECs numbers. Intervention
also led to significant favorable effects on inflammatory (CRP and ESR),
rheumatic (RF, anti-MCV) and on circulating adhesion molecules levels
(ICAM and VCAM). Conclusion: The present data indicate that
supplementing RA with VD3 significantly improves vascular functions as
indicated by enhanced FMD through a mechanism that involves regulation
of adhesion molecules and inflammatory mediators.
INTRODUCTION
Rheumatoid arthritis (RA) is a systemic chronic autoimmune
inflammatory disorder that affects approximately 1 percent of the population
worldwide 1. It is characterized by variable autoreactivities immune cells
and autoantibodies against immunoglobulin G, cartilage components, stress
proteins, enzymes, nuclear proteins and citrullinated proteins causing
inflammation of the lining of the joints, in a symmetric pattern 2, 3. Although
primarily involves the joints, RA can have a significant impact on other
organ systems specially the heart and vascular system 4, 5. In addition,
numerous epidemiological investigations have revealed that risk of
developing cardiovascular diseases (CVD) in RA patients RA is more than
double that of the general population 6, 7.
Clinical and experimental data support that inflammatory responses
that accompanied immune-mediated disorders cause activation of endothelium
and overexpression of key adhesion molecules involved in the recruitment of
leukocytes to the endothelium. Adherence of leukocytes to endothelial cells
leads to disruption of the endothelial glycocalyx and to detachment of
endothelial cells into the circulation 8-10. Recently, circulating endothelial cells
(CECs) have emerged as hallmarks of vascular dysfunction 11, 12. CECs are
present in very small numbers in healthy individuals, whereas their number
increases dramatically in diseases with vascular damage, such as CVD, sepsis,
vasculitis, and autoimmune diseases 13-18. Several adhesion molecules involved
in this process including vascular cell adhesion molecule-1 (VCAM-1) and
intracellular cell adhesion molecule-1 (ICAM-1) 8. Leukocytes adhere to
endothelium then transmigrate through it to the vascular and extravascular
spaces that are central to endothelial dysfunction and the development of
atherosclerotic plaques and vascular inflammatory disorders 19, 20. In contrast to
201 Egypt. J. of Appl. Sci., 35 (11) 2020
healthy endothelium, dysfunctional endothelium loses its capacity of relaxation
in response to mediators such as acetylcholine or to increased blood flow.
Therefore, endothelial dysfunction could be detected by impaired flowmediated
dilation (FMD) 21, 22. FMD has been suggested as a marker of the
grade of atherosclerosis and may be used as a surrogate marker of
cardiovascular outcomes 23.
Many cardiovascular risk factors have been recognized. Among them,
vitamin D (VD) deficiency [25(OH) D <20 ng/mL] is emerging as a novel one
24, 25. VD is a steroid hormone that is photosynthesized mainly by skin upon the
exposure to sun light 26. VD can also be supplemented through diet where
gastrointestinal absorption takes it to blood circulation 27, 28. In addition to its
well-defined role in bone and calcium metabolism, vitamin D has been
identified as an vital factor in several physiological processes, including
immunity, cellular growth, and cellular differentiation 29. VD exerts these
diverse physiological functions upon binding to its nuclear receptor (VDR), a
ligand-responsive transcription factor that is ubiquitously expressed in a wide
variety of cell types and organ systems, including the cardiovascular system 30.
Recent studies reported the expression of VDR on vascular endothelial cells and
that its interaction with VD results in the expression of hundreds of gene targets
which regulate vascular cell growth, migration, and differentiation; immune
response modulation; cytokine expression; and inflammatory and fibrotic
pathways, all of which play a crucial role, starting from the early stage of
endothelial activation/dysfunction to the later stages of the plaque vulnerability
and rupture 31.
In Saudi Arabia, the current epidemiological data indicate that vitamin
D deficiency is prevalent in more than 80% of Saudi population, and it might be
related to lack of adequate exposure to sunlight and inadequate consumption of
dairy products 32. Deficiency of vitamin D has been acknowledged as an
independent predictor of CVD in patients with RA 33-35. In addition, several
studies have suggested that vitamin D deficiency in RA may affect
inflammatory responses and endothelial functions 36-38. These reports suggest
that maintaining normal serum vitamin D levels may protect against CVD in
patients with RA. To test this suggestion, and due to the lack of clinical studies
on the effects of Vitamin D supplementation, we designed the current study to
assess the impact of vitamin D intake on inflammatory markers, vascular
functions measures and circulating endothelial adhesion molecules.
MATERIALS AND METHODS
Study Design
The present randomized cross-sectional study was carried out during
the period from September 2014 and July 2015. All the patients met the
2010 American College of Rheumatology/European League Against
Rheumatism (EULAR) criteria for RA classification 39. All procedures
Egypt. J. of Appl. Sci., 35 (11) 2020 202
performed in the study were in accordance with the ethical standards of the
institutional and/or national research committee and with the 1964 Helsinki
Declaration and its later amendments or comparable ethical standards. All
patients who participated in the present study signed an informed consent
form per the Declaration of Helsinki (October 2008). The study included
two groups of subjects: group A, consists of 50 patients with RA and group
B, consists of 40 healthy sex- and age-matched controls. After initial clinical
assessment, patients with serum vitamin D (25(OH) D) <25 ng/mL were
supplemented with oral cholecalciferol 50,000 IU per week for 8 weeks.
Anthropometric data of the patients and healthy controls and clinical data
were collected from the questionnaire designed for each group. The main
clinical data that were collected are age, smoking, disease activity, disease
duration, DAS-28 (disease activity score of 28 joints), ability of movement,
vitamin D supplementations, dairy consumption, and sun’s exposure. Due to
incomplete follow-up data from the hospital and absence of patients at
follow-ups, the final number in group A became 40 patients.
Blood samples
Blood samples were withdrawn into two Ethylenediaminetetraacetic
acid (EDTA) tubes. Serum was separated from one tube by centrifugation at
1500 rpm for 5 minutes. Serum samples were aliquoted in eppendorf tubes
and were stored at -80 oC until analysis. The other blood tube was mixed
with 5ml of Hank’s Balanced Salt Solution (HBSS), and slowly layered on
top of Ficoll-Paque Premium solution (GE-Healthcare, USA) for leukocyte
separation. The tubes were centrifuged at 1400 rpm, 4oC with no break for
30 minutes. The developed buffy coat was then collected and washed twice
in HBSS. Leukocytes were then stored in frozen nutrient media (23% RPMI
1640 + 70% Fetal Bovine Serum + 7% Dimethylsulfoxide DEMSO).
Estimation of serum Vitamin D3
Vitamin D3 levels were determined in the serum using 25 Hydroxy
Vitamin D (25OHVD) ELISA kit (Cat. No. MBS722906, My BioSource,
Inc. San Diego, CA, USA) according to the manufacturer's instructions.
Determination of vascular function markers
a. FMD index assessments
FMD is the most widely used non-invasive method to assess the
endothelial function, reflecting the endothelial nitric oxide (NO) dependent
vasodilation in response to occlusion-induced reactive hyperemia. FMD
assessments were done using Philips iU22 high-end ultrasound machine at
the Ultrasound Vascular Lab, King Abdulaziz University Hospital. This
system is equipped with vascular software for two-dimensional (2D)
203 Egypt. J. of Appl. Sci., 35 (11) 2020
imaging, color and spectral Doppler, an internal electrocardiogram (ECG)
monitor and a high-frequency vascular transducer. A linear array transducer
with a minimum frequency of 7 MHz, attached to a high-quality mainframe
ultrasound system, was used to acquire images with sufficient resolution for
subsequent analysis. Images of the brachial artery were obtained for subjects
after they were positioned in supine with the arm in a comfortable position
for imaging. The brachial artery was imaged above the antecubital fossa in
the longitudinal plane. A segment with clear anterior and posterior intimal
interfaces between the lumen and vessel wall was selected for continuous
2D gray scale imaging. Percent of FMD was calculated as follows: [(post
deflation diameter - resting diameter) / resting diameter] X 100.
b. CECs using Flow Cytometry (FCM)
CECs were enumerated using Flow Cytometry (FCM) as described
previously 40. Briefly, stored BPMCs samples were thawed and washed
before the addition of Anti-CD3 FITC (Cat. No. ab34275, Abcam,
Cambridge, UK), CD146 [P1H12] Phycoerythrin (PE) (Cat. No. ab78488,
Abcam, Cambridge, UK) and CD45 Allophycocyanin (APC) (Cat. No.
ab28106, Abcam, Cambridge, UK) monoclonal antibodies antibodies. After
incubation for 20 min in the dark, 5 ul of 7-Aminoactinomycin D (7-AAD)
(Cat. No. ab142391, Abcam, Cambridge, UK) were added 5 minutes before
analysis for exclusion of dead cells. Stained samples were then analyzed
using Navios software on GalliosTM Flow Cytometer (Beckman Coulter,
Inc., Miami, FL, USA). A sequential flow cytometry strategy was used for
enumeration of CECs as illustrated in Figure 1.
Determination of serum adhesion markers
Adhesion molecules were determined in serum samples using
ELISA kits for vascular cell adhesion molecule 1 (VCAM1, Cat. No.
ab187393, Abcam, Cambridge, UK), intercellular adhesion molecule 1
(ICAM1, Cat. No. ab187403, Abcam, Cambridge, UK), vascular
endothelial growth factor (VEGF, Cat. No. ab100662, Abcam,
Cambridge, UK), and vascular endothelial growth factor receptor1
(VEGFR1, Cat. No. ab119567, Abcam, Cambridge, UK) according to the
manufacturer's instructions.
Statistical Analysis
Statistical analysis was performed using SPSS software (version
20; SPSS Inc., Chicago, IL, USA). Values are expressed as mean ±
standard deviation. The Student’s t-test was used to assess differences in
tested parameters between RA patients and the control group. Pearson’s
Chi-square test was used to evaluate correlations between parameters. A
level of 0.05 (two-sided) was chosen to indicate statistical significance.
Egypt. J. of Appl. Sci., 35 (11) 2020 204
Figure 1: Sequential flow cytometry strategy for enumeration of CECs. CECs are
defined as CD45 − /CD3 - /CD146 + using CD3-FITC, CD146-PE, CD45-
APC conjugated antibodies. CECs: (A) Forward (FSC) and side scatter (SSC)
plot of white blood cells and gating region G1 to include all mononuclear cell
events while excluding platelets, dead cells, and microparticles. (B) Live
cells were selected as 7AAD- events in region G2 on an Intensity 7AAD
versus Intensity SSC plot gated on G1. (C) CD3 negative events were
selected on cellular events from gated region G2. (D) CD146+CD45- CECs
were selected in a plot gated on live single CD3- cells from G3.
RESULTS
Forty patients had completed the follow-up visit at 8 weeks after
treatment. The demographic data of the patients and healthy controls were
collected from the questionnaire designed for the current study and were
presented. The age range was 17–86 years. Eleven out of the 51 RA patients
have been excluded from the study due to incomplete follow-up data.
Disease duration ranged from 6 months to 12 years with mean of
89.62±83.88 months. Three (7.5%) RA patients were smokers and 3 other
patients have significant movement difficulties. No significant difference in
dairy consumption or sun exposure between patients and controls. All
patients have received corticosteroids previously and more than 35% of
205 Egypt. J. of Appl. Sci., 35 (11) 2020
patients were under treatment with Methotrexate and Plaquenil (DMARDs;
Disease-modifying antirheumatic drugs).
Figure 2 shows that RA patients had significantly higher DAS-28
before treatment compared with controls (P<0.001). After treatment, mean
DAS-28 value was lower in comparison to baseline values, however the
difference was not statistically significant (P>0.05). Demonstrates the
biochemical measurements and inflammatory markers of the RA patients at
baseline and post-treatment with oral VD3 as compared to the reference
group. At baseline, RA patients showed significantly (P<0.05) lower serum
total vitamin D and serum VD3 concentrations compared with controls Both
at P<0.05). Whereas after treatment with oral 50,000 IU vitamin D3 per
week for 8 weeks; RA patients showed significantly higher levels of total
vitamin D in comparison to their baseline values and to that of controls
(p<0.05; p<0.001 respectively). After treatment, levels of serum vitamin D3
were slightly increased (p>0.05) compared with RA before treatment. The
inflammatory markers CRP and ESR were found significantly lower in
patients received VD3 than their baseline levels. Treatment with VD3 also
reduced the rheumatic markers RF and Anti-MCV compared than that
measured at baseline.
Figure 2: Mean DAS-28 levels in RA patients before and after vitamin D treatment
in comparison to controls.
Egypt. J. of Appl. Sci., 35 (11) 2020 206
FMD of the brachial artery is an accepted procedure for noninvasive
assessment of systemic endothelial function. The mean value of
FMD in controls was 15.13±11.32% (median, 8.9%; IQR, 4.0–8.0%;
range, 7.87–21.9%) (Figure 3A). Baseline FMD was significantly lower
in RA group than in controls (6.33±6.58 vs 15.13±11.32 %, p<0.01). Post
Treatment, RA group showed significant improvement in FMD
compared with baseline measurements (6.33±6.58 vs 12.92±7.18 mm,
p<0.05).
CECs were enumerated before and post treatment using flow
cytometry as illustrated in Figure 1. Baseline CECs numbers were found
significantly higher in RA group (p<0.001). CECs numbers also
significantly reduced post vitamin D3 treatment compared with pretreatment
numbers. However, post-treatment CECs numbers were still
significantly higher than in controls (Figure 3B).
Figure 3: Endothelial biomarkers FMD% (A) and number of CECs as measured
by flow cytometry (B) in RA patients in Comparisons to controls.
207 Egypt. J. of Appl. Sci., 35 (11) 2020
The concentrations of serum endothelial growth factors and
adhesion molecules of the RA patients in comparisons controls are
demonstrated. Serum VEGF showed significant increase (P<0.01) at
baseline and then decreased significantly (P<0.05) after 8 weeks of
treatment. The soluble receptors of VEGF type 1 (sVEGFR-1) also
significantly (P<0.05) decreased after treatment. Serum concentrations of
adhesion molecules VCAM1 and ICAM1 also showed significant increase
at baseline (both at p<0.01) in comparison to controls. After treatment a
significant decrease were only recorded in VCAM1 (P<0.01) compared with
their baseline values. In case of ICAM1, our patients did not show
significant decrease post-treatment in comparison to baseline values.
Pearson correlations analyses revealed a significant negative
correlation between DAS-28 with serum vitamin D3 (r=0.437; P=0.001) and
with FMD% (r=0.315; P=0.002), while no correlation was found between
DAS-28 with CECs (Figure 4A, B).
Figure 4: Correlations of disease activity score (DAS-28) with (A) levels of serum
Vitamin D3 and (B) percent of flow mediated dilation
Egypt. J. of Appl. Sci., 35 (11) 2020 208
DISCUSSION
The pathogenesis of the chronic inflammation related rheumatic
autoimmune diseases diversified greatly based on the area, the way of living,
and social position suggesting that genetic and environmental factors play an
essential role in occurrence and advancement of rheumatic diseases 41. In
Saudi Arabia, the current epidemiological data indicate that overall Vitamin
D deficiency is 63.5% (95% CI: 53.3, 73.7), and it might be related to lack
of adequate exposure to sunlight and possibly inadequate consumption of
dairy products 42. Several researchers have confirmed a greater occurrence
of vitamin D deficiency in RA patients in comparison to healthy people 33.
Studies also show that prevalence of vitamin D deficiency in RA and that
there is an association with disease activity and cardiovascular risk factors 33-
35. Therefore, it has been proposed by several research groups that vitamin D
and its analogs not only can ameliorate autoimmune diseases, but also could
be utilized in the management of associated vascular disorders 43-45. In the
current study, the total vitamin D was measured before and after
supplementation of RA patients with VD3. Post treatment with 50000 U of
oral VD3 per week for 2 months, RA patients showed significant increase in
total vitamin D levels (VD2 plus VD3) compared with its levels before the
treatment.
Autoantibodies are frequent and characteristic aspect of rheumatic
autoimmune diseases. While the greater part of autoantibodies do not appear
to perform a significant pathogenetic function in these disorders, some of
them have found highly beneficial as diagnostic methods and signs of
disease activity. Our study revealed that the RF was significantly higher in
RA group compared with healthy group. Supplementation with vitamin D
significantly reduced RF quantities in both categories, suggesting Vitamin D
ability to ameliorate the increase in RF associated with RA. Mutated
citrullinated vimentin MCV autoantibodies are an indication of preceding
damage to the joints and extreme advancement of the disease 46. The anti-
MCV ELISA locates autoantibodies against MCV. It is a powerful and
effective serological analysis for rheumatoid arthritis as it detects the disease
at earliest stage, even in patients who has been recognized as negative for
RF, and to some extent even prior to the appearance of certain clinical signs
47. Based on our data, there was a significant reduction in MCV levels after
treatment with vitamin D3.
The level of disease activity in RA is frequently assessed with the
Disease Activity Score of 28 Joints (DAS-28). Higher disease activity has
been also involved with increased death rate, and experiments have shown
that death rate is decreased by intense treatments for RA 48. Song et al.
review the reverse relationship between disease activity in RA patients and
levels of serum vitamin D 49. The current study is in line with Song’s
209 Egypt. J. of Appl. Sci., 35 (11) 2020
findings that supplementation of RA patients with vitamin D3 reduces the
levels of DAS-28.
The studies in recent years revealed that vitamin D deficiency could
influence endothelial performance 35. FMD of brachial arteries is a validated
and useful biomarker of endothelial functionality 22, 23. Harris and colleagues
noticed that vitamin D3 supplements could enhance FMD in comparison to
placebo users 50. Additionally, Malik, Giri 51 reported that there were
significant increases in %FMD in patients of myocardial infarction after
vitamin D supplementation. In line with these studies, our data show
enhanced FMD after vitamin D supplementation. These finding may suggest
that vitamin D3 supplementations may improve endothelial performance.
Damage to endothelium has been associated with CVD morbidity
and mortality in young patients with RA 34, 52. Endothelial damage
correspond with disease development and estimates CV activities in the
normal population 53. CECs (CD45-CD146+) are angiogenic cells that are
truly uncommon in normal individual peripheral blood and seem to be
elevated in numbers in the peripheral circulation as a consequence of
vascular damage or in response to vasculogenic stimulating factors 12, 22, 54,
55. Several researches suggest that vitamin D may influence ECs indirectly
through regulating inflammatory mediators on the blood vessels 34, 56. In
agreement with these data, the present study demonstrates that
administration of VD3 was associated with significant decrease in CECs,
and that decrease was concomitant with significant decrease in the
inflammatory disease activity index DAS-28 and the other conventional
inflammatory markers ESR, CRP and anti-MCV. In addition, it has been
reported that vitamin D might reduce CVD and cardiovascular risk by
regulating the production of VEGF and protecting the endothelial cells from
oxidative stress through autophagic and survival pathways 56, 57. It has been
reported that increased levels of VEGF may result in alterations of
endothelial functions, which may subsequently trigger the valvular
remodelling or even damage of heart valves that may leads to rheumatic
heart diseases 57. Also, it has been reported that VEGF and its receptor
VEGFR-1 levels are positively correlated with disease activity in RA
patients 58-60. In agreement with these studies, we have observed that the
levels of VEGF were significantly reduced in patients after supplementation
with vitamin D3, confirming the role of VD3 in regulating VEGF
production. The integrins or the adhesion molecules (ICAM1 and VCAM1)
also appeared to have important roles in development of rheumatic heart
disease as it were found associated with valvular lesion, cardiac muscle cell
death and heart failure 61. ICAM and VCAM adhesion molecules belong to
a subgroup of Ig-like superfamily gene that is secreted from the
endothelium. The secretion of these molecules was found positively
associated with endothelial cell damage and endothelial activation and cause
Egypt. J. of Appl. Sci., 35 (11) 2020 210
adhesion of leukocytes to the vascular membrane and escalation of the
atherosclerotic process 9, 61. In the present study the concentrations of serum
integrins were found significantly higher in RA patients at baseline.
However, their concentrations were reduced dramatically post-treatment
with oral VD3. Our results are comparable to that of Naeini, Moeinzadeh 62
who found that administration of 50,000 IU Vitamin D orally per week for 8
weeks in ESRD patients reduces serum levels of ICAM and VCAM which
might improve the vascular condition of these patients. In summary, despite
the limitations of small number of patients in this study, the preliminary
results demonstrated that treatment of patients with RA with vitamin D
50,000 IU per week improved the endothelial functions as indicated by
enhanced FMD and lowered numbers of CECs, VEGF and the levels of
adhesion molecules ICAM-1 and VCAM-1.
CONFLICT OF INTERESTS
None.
Ethics Approval
King Abdulaziz University Research Ethics Board, Jeddah, Saudi
Arabia.
Informed consent:
Informed consent was obtained from all individual participants
included in the study.
Acknowledgments
The authors would like to acknowledge the King Abdulaziz City for
Science and Technology KACST for funding this study.
REFERNCES
1. van der Woude, D. and van A.H.M. der Helm-van Mil (2018):.
Update on the epidemiology, risk factors, and disease outcomes
of rheumatoid arthritis. Best Pract Res Clin Rheumatol; 32:
174-187.
2. Yap, H.Y. ; S.Z. Tee ; M.M. Wong and et al.(2018): Pathogenic
Role of Immune Cells in Rheumatoid Arthritis: Implications in
Clinical Treatment and Biomarker Development. Cells; 7
2018/10/12. DOI: 10.3390/cells7100161.
3. Alivernini, S. ; B. Tolusso ; G. Ferraccioli and et al.(2018)
Driving chronicity in rheumatoid arthritis: perpetuating role of
myeloid cells. Clin Exp Immunol; 193: 13-23. 2018/01/10. DOI:
10.1111/cei.13098.
4. Urman, A. ; N.Taklalsingh ; C. Sorrento and et al.(2018):
Inflammation beyond the Joints: Rheumatoid Arthritis and
Cardiovascular Disease. Scifed J Cardiol; 2 2019/01/05.
211 Egypt. J. of Appl. Sci., 35 (11) 2020
5. Amigues, I. ; A. Tugcu ; C. Russo and et al.(2019): Myocardial
Inflammation, Measured Using 18-Fluorodeoxyglucose
Positron Emission Tomography With Computed Tomography,
Is Associated With Disease Activity in Rheumatoid Arthritis.
Arthritis Rheumatol; 71: 496-506. 2018/11/09. DOI:
10.1002/art.40771.
6. Blum, A. and M. Adawi (2019): Rheumatoid arthritis (RA) and
cardiovascular disease. Autoimmun Rev ., 2019/05/07. DOI:
10.1016/j.autrev.2019.05.005.
7. Agca, R. ; L. Hopman ; K.C.J.Laan and et al.(2019):
Cardiovascular Event Risk in Rheumatoid Arthritis is Higher
than in Type 2 Diabetes: a 15 Year Longitudinal Study. J
Rheumatol ,2019/05/17. DOI: 10.3899/jrheum.180726.
8. Moroni, G. ; N. Del Papa ; L.M. Moronetti and et al.(2005):
Increased levels of circulating endothelial cells in chronic
periaortitis as a marker of active disease. Kidney Int; 68: 562-
568. 2005/07/15. DOI: 10.1111/j.1523-1755.2005.00434.x.
9. Kluz, J. ; W. Kopec ; U. Jakobsche-Policht and et al. (2009):
Circulating endothelial cells, endothelial apoptosis and soluble
markers of endothelial dysfunction in patients with systemic
lupus erythematosus-related vasculitis. Int Angiol; 28: 192-201.
2009/06/10.
10. Dane, M. ; B. van den Berg and T. Rabelink (2014): The
endothelial glycocalyx: scratching the surface for
cardiovascular disease in kidney failure. Atherosclerosis; 235:
56-57. /05/13. DOI: 10.1016/j.atherosclerosis.2014.04.005.
11. Totoson, P. ; K. Maguin-Gate ; M. Nappey and et al.(2016):
Endothelial Dysfunction in Rheumatoid Arthritis: Mechanistic
Insights and Correlation with Circulating Markers of Systemic
Inflammation. PLoS One; 11: e0146744. 2016/01/14. DOI:
10.1371/journal.pone.0146744.
12. Farinacci, M. ; T. Krahn ; W. Dinh and et al.(2019): Circulating
endothelial cells as biomarker for cardiovascular diseases. Res
Pract Thromb Haemost; 3: 49-58. 2019/01/19. DOI:
10.1002/rth2.12158.
13. Erdbruegger, U. ; M. Haubitz and A.Woywodt (2006):
Circulating endothelial cells: a novel marker of endothelial
damage. Clin Chim Acta 2006; 373: 17-26. 2006/07/14. DOI:
10.1016/j.cca.2006.05.016.
Egypt. J. of Appl. Sci., 35 (11) 2020 212
14. Budzyn, M. ; B. Gryszczynka ; M. Boruczkowski and et al.
(2019): The endothelial status reflected by circulating
endothelial cells, circulating endothelial progenitor cells and
soluble thrombomodulin in patients with mild and resistant
hypertension. Vascul Pharmacol; 113: 77-85. 2018/12/28. DOI:
10.1016/j.vph.2018.12.005.
15. Moussa, M.D. ; C. Santonocito ; D. Fagnoul and et al. (2014):
Evaluation of endothelial damage in sepsis-related ARDS using
circulating endothelial cells. Intensive Care Med; 41: 231-238.
2014/12/17. DOI: 10.1007/s00134-014-3589-9.
16. Milardi, D. ; G. Grande ; A. Giampietro and et al.(2012):
Circulating endothelial cells as marker of endothelial damage in
male hypogonadism. J Androl; 33: 1291-1297. 2012/06/16.
DOI: 10.2164/jandrol.112.016600.
17. Strijbos, M.H. ; P.P.Landburg ; E. Nur and et al.(2009):
Circulating endothelial cells: a potential parameter of organ
damage in sickle cell anemia? Blood Cells Mol Dis; 43: 63-67.
2009/04/10. DOI: 10.1016/j.bcmd.2009.02.007.
18. Boos, C.J. ; G.Y. Lip and A.D. Blann (2006): Circulating
endothelial cells in cardiovascular disease. J Am Coll Cardiol;
48: 1538-1547. 2006/10/19. DOI: 10.1016/j.jacc.2006.02.078.
19. Issekutz, A.C. (1998):Adhesion molecules mediating neutrophil
migration to arthritis in vivo and across endothelium and
connective tissue barriers in vitro. Inflamm Res; 47 Suppl 3:
S123-132. 1998/11/27.
20. Weber, C. ; L. Fraemohs and E. Dejana (2007): The role of
junctional adhesion molecules in vascular inflammation. Nat
Rev Immunol; 7: 467-477. 2007/05/26. DOI: 10.1038/nri2096.
21. Lee, K.W. ; A.D. Blann and G.Y. Lip (2006): Inter-relationships
of indices of endothelial damage/dysfunction [circulating
endothelial cells, von Willebrand factor and flow-mediated
dilatation] to tissue factor and interleukin-6 in acute coronary
syndromes. Int J Cardiol; 111: 302-308. 2005/12/06. DOI:
10.1016/j.ijcard.2005.10.014.
22. Chong, A.Y. ; A.D. Blann ; J. Patel and et al.(2004): Endothelial
dysfunction and damage in congestive heart failure: relation of
flow-mediated dilation to circulating endothelial cells, plasma
indexes of endothelial damage, and brain natriuretic peptide.
213 Egypt. J. of Appl. Sci., 35 (11) 2020
Circulation; 110: 1794-1798. 2004/09/15. DOI:
10.1161/01.CIR.0000143073.60937.50.
23. Maruhashi, T. ; J. Soga ; N. Fujimura and et al.(2013):
Relationship between flow-mediated vasodilation and
cardiovascular risk factors in a large community-based study.
Heart; 99: 1837-1842. 2013/10/25. DOI: 10.1136/heartjnl-
2013-304739.
24. Chen, F.H. ; T. Liu ; L. Xu and et al.(2018): Association of Serum
Vitamin D Level and Carotid Atherosclerosis: A Systematic
Review and Meta-analysis. J Ultrasound Med; 37: 1293-1303.
2017/11/25. DOI: 10.1002/jum.14494.
25. Karakas, M. ; B. Thorand ; A. Zierer and et al.(2013): Low levels
of serum 25-hydroxyvitamin D are associated with increased risk
of myocardial infarction, especially in women: results from the
MONICA/KORA Augsburg case-cohort study. The Journal of
Clinical Endocrinology & Metabolism; 98: 272-280.
26. Perez-Lopez, F.R. (2007):Vitamin D: the secosteroid hormone and
human reproduction. Gynecol Endocrinol; 23: 13-24. 2007/05/09.
27. Brouwer-Brolsma, E.M. ; H.A. Bischoff-Ferrari ; R. Bouillon
and et al.(2013): Vitamin D: do we get enough? A discussion
between vitamin D experts in order to make a step towards the
harmonisation of dietary reference intakes for vitamin D across
Europe. Osteoporos Int; 24: 1567-1577. 2012/12/12. DOI:
10.1007/s00198-012-2231-3.
28. Wolpowitz, D. and B.A. Gilchrest (2006): The vitamin D
questions: how much do you need and how should you get it? J
Am Acad Dermatol; 54: 301-317. 2006/01/31. DOI:
10.1016/j.jaad.2005.11.1057.
29. DeLuca, H.F. (2004):Overview of general physiologic features and
functions of vitamin D. Am J Clin Nutr; 80: 1689S-1696S.
2004/12/09. DOI: 10.1093/ajcn/80.6.1689S.
30. Wu-Wong, J.R.(2007): The potential for vitamin D receptor activation
in cardiovascular research. Expert Opin Investig Drugs; 16: 407-
411. 2007/03/21. DOI: 10.1517/13543784.16.4.407.
31. Bozic, M. ; A. Alvarez ; C. de Pablo and et al.(2015): Impaired
Vitamin D Signaling in Endothelial Cell Leads to an Enhanced
Leukocyte-Endothelium Interplay: Implications for
Atherosclerosis Development. PLoS One; 10: e0136863.
2015/09/01. DOI: 10.1371/journal.pone.0136863.
Egypt. J. of Appl. Sci., 35 (11) 2020 214
32. Al-Daghri, N.M. (2018): Vitamin D in Saudi Arabia: Prevalence,
distribution and disease associations. J Steroid Biochem Mol
Biol; 175: 102-107.
33. Cecchetti, S. ; Z. Tatar ; P. Galan and et al.(2016): Prevalence of
vitamin D deficiency in rheumatoid arthritis and association with
disease activity and cardiovascular risk factors: data from the
COMEDRA study. Clin Exp Rheumatol; 34: 984-990.
34. Lo Gullo, A. ; J. Rodriguez-Carrio ; C.O. Aragona and et
al.(2018): Subclinical impairment of myocardial and
endothelial functionality in very early psoriatic and rheumatoid
arthritis patients: Association with vitamin D and inflammation.
Atherosclerosis; 271: 214-222. 2018/03/11. DOI:
10.1016/j.atherosclerosis.2018.03.004.
35. Caraba, A. ; V. Crisan ; I. Romosan and et al.(2017): Vitamin D
Status, Disease Activity, and Endothelial Dysfunction in Early
Rheumatoid Arthritis Patients. Dis Markers; 2017: 5241012.
2017/12/05. DOI: 10.1155/2017/5241012.
36. Lo Gullo, A. ; G. Mandraffino ; G. Bagnato and et al. (2015):
Vitamin D Status in Rheumatoid Arthritis: Inflammation, Arterial
Stiffness and Circulating Progenitor Cell Number. PLoS One; 10:
e0134602. 2015/08/05. DOI: 10.1371/journal.pone.0134602.
37. Delgado-Frias, E. ; R. Lopez-Mejias ; F. Genre and et al.(2015):
Relationship between endothelial dysfunction and osteoprotegerin,
vitamin D, and bone mineral density in patients with rheumatoid
arthritis. Clin Exp Rheumatol; 33: 241-249. 2015/03/24.
38. Ranganathan, P. ; S. Khalatbari ; S. Yalavarthi and et al.(2013):
Vitamin D deficiency, interleukin 17, and vascular function in
rheumatoid arthritis. J Rheumatol; 40: 1529-1534. 2013/07/03.
DOI: 10.3899/jrheum.130012.
39. Aletaha, D. ; T. Neogi ; A.J. Silman and et al. (2010): Rheumatoid
arthritis classification criteria: an American College of
Rheumatology/European League Against Rheumatism
collaborative initiative. Arthritis Rheum; 62: 2569-2581.
2010/09/28. DOI: 10.1002/art.27584.
40. Elshal, M. ; A. Abdelaziz ; A. Abbas and et al. (2009):
Quantification of circulating endothelial cells in peripheral
blood of systemic lupus erythematosus patients: a simple and
reproducible method of assessing endothelial injury and repair.
215 Egypt. J. of Appl. Sci., 35 (11) 2020
Nephrol Dial Transplant; 24: 1495-1499. 2008/11/29. DOI:
10.1093/ndt/gfn650.
41. Sparks, J.A. and K.H. Costenbader (2014): Genetics, environment,
and gene-environment interactions in the development of systemic
rheumatic diseases. Rheum Dis Clin North Am; 40: 637-657.
2014/12/02. DOI: 10.1016/j.rdc.2014.07.005.
42. Al-Alyani, H. ; H.A. Al-Turki ; O.N. Al-Essa and et al. (2018):
Vitamin D deficiency in Saudi Arabians: A reality or simply
hype: A meta-analysis (2008-2015). J Family Community Med;
25: 1-4. 2018/02/02. DOI: 10.4103/jfcm.JFCM_73_17.
43. Kheiri, B. ; A. Abdalla ; M. Osman and et al.(2018): Vitamin D
deficiency and risk of cardiovascular diseases: a narrative review.
Clin Hypertens; 24: 9. 2018/07/07. DOI: 10.1186/s40885-018-
0094-4.
44. Antico, A. ; M. Tampoia ; R. Tozzoli and et al.(2012): Can
supplementation with vitamin D reduce the risk or modify the
course of autoimmune diseases? A systematic review of the
literature. Autoimmun Rev; 12: 127-136. 2012/07/11. DOI:
10.1016/j.autrev.2012.07.007.
45. Franco, A.S. ; T.Q. Freitas ; W.M. Bernardo and et al.(2017):
Vitamin D supplementation and disease activity in patients with
immune-mediated rheumatic diseases: A systematic review and
meta-analysis. Medicine (Baltimore); 96: e7024. 2017/06/08.
DOI: 10.1097/MD.0000000000007024.
46. Innala, L. ; H. Kokkonen ; C. Eriksson and et al.(2008):
Antibodies against mutated citrullinated vimentin are a better
predictor of disease activity at 24 months in early rheumatoid
arthritis than antibodies against cyclic citrullinated peptides. J
Rheumatol; 35: 1002-1008. 2008/04/10.
47. Dejaco, C. ; W. Klotz ; H. Larcher and et al.(2006): Diagnostic
value of antibodies against a modified citrullinated vimentin in
rheumatoid arthritis. Arthritis Res Ther; 8: R119. 2006/07/25.
DOI: 10.1186/ar2008.
48. Listing, J. ; J. Kekow ; B. Manger and et al. (2015):Mortality in
rheumatoid arthritis: the impact of disease activity, treatment
with glucocorticoids, TNFalpha inhibitors and rituximab. Ann
Rheum Dis; 74: 415-421. 2013/12/03. DOI:
10.1136/annrheumdis-2013-204021.
Egypt. J. of Appl. Sci., 35 (11) 2020 216
49. Song, G.G. ; S.C. Bae and Y.H. Lee (2012): Association between
vitamin D intake and the risk of rheumatoid arthritis: a metaanalysis.
Clinical rheumatology; 31: 1733-1739.
50. Harris, S.S. ; A.G.Pittas and N.J.Palermo (2012): A randomized,
placebo-controlled trial of vitamin D supplementation to
improve glycaemia in overweight and obese African
Americans. Diabetes Obes Metab; 14: 789-794. 2012/04/11.
DOI: 10.1111/j.1463-1326.2012.01605.x.
51. Malik, S. ; S. Giri ; S.V. Madhu and et al. (2016):Relationship of
levels of Vitamin D with flow-mediated dilatation of brachial
artery in patients of myocardial infarction and healthy control:
A case-control study. Indian J Endocrinol Metab; 20: 684-689.
2016/10/13. DOI: 10.4103/2230-8210.190558.
52. Khan, F.(2010): Assessment of endothelial function as a marker of
cardiovascular risk in patients with rheumatoid arthritis. Int J
Rheum Dis 2010; 13: 189-195. 2010/08/14. DOI:
10.1111/j.1756-185X.2010.01480.x.
53. Boos, C.J. ; A.D. Blann and G.Y. Lip (2007): Assessment of
endothelial damage/dysfunction: a focus on circulating
endothelial cells. Methods Mol Med; 139: 211-224. 2008/02/22.
54. Hill, J.M. ; G. Zalos ; J.P. Halcox and et al. (2003):Circulating
endothelial progenitor cells, vascular function, and
cardiovascular risk. N Engl J Med; 348: 593-600. 2003/02/14.
DOI: 10.1056/NEJMoa022287.
55. Erdbruegger, U. ; A. Dhaygude ; M. Haubitz and et al.(2010):
Circulating endothelial cells: markers and mediators of vascular
damage. Curr Stem Cell Res Ther; 5: 294-302. 2010/06/10.
56. Uberti, F. ; D. Lattuada ; V. Morsanuto and et al.(2014): Vitamin
D protects human endothelial cells from oxidative stress
through the autophagic and survival pathways. J Clin
Endocrinol Metab; 99: 1367-1374. 2013/11/29. DOI:
10.1210/jc.2013-2103.
57. Sarkar, S. ; S. Chopra ; M.K. Rohit and et al.(2016): Vitamin D
regulates the production of vascular endothelial growth factor:
A triggering cause in the pathogenesis of rheumatic heart
disease? Med Hypotheses; 95: 62-66. 2016/10/04. DOI:
10.1016/j.mehy.2016.09.001.
58. Ozgonenel, L. ; E. Cetin ; S. Tutun and et al. (2010);The relation
of serum vascular endothelial growth factor level with disease
217 Egypt. J. of Appl. Sci., 35 (11) 2020
duration and activity in patients with rheumatoid arthritis. Clin
Rheumatol; 29: 473-477. 2010/01/14. DOI: 10.1007/s10067-
009-1343-4.
59. Sone, H. ; M. Sakauchi ; A. Takahashi and et al.(2001): Elevated
levels of vascular endothelial growth factor in the sera of
patients with rheumatoid arthritis correlation with disease
activity. Life Sci; 69: 1861-1869. 2001/11/06.
60. Maeno, N. ; S. Takei ; H. Imanaka and et al.(1999): Increased
circulating vascular endothelial growth factor is correlated with
disease activity in polyarticular juvenile rheumatoid arthritis. J
Rheumatol; 26: 2244-2248. 1999/10/21.
61. Hafez, M. ; S. Yahia ; W. Eldars and et al.(2013): Prediction of
residual valvular lesions in rheumatic heart disease: role of
adhesion molecules. Pediatr Cardiol; 34: 583-590. 2012/09/19.
DOI: 10.1007/s00246-012-0501-7.
62. Naeini, A.E. ; F. Moeinzadeh ; S. Vahdat and et al.(2017): The
Effect of Vitamin D Administration on Intracellular Adhesion
Molecule-1 and Vascular Cell Adhesion Molecule-1 Levels in
Hemodialysis Patients: A Placebo-controlled, Double-blinded
Clinical Trial. J Res Pharm Pract; 6: 16-20. 2017/03/24. DOI:
10.4103/2279-042X.200994.
تأثير تناول فيتامين دال على مرضى التهاب المفاصل الروماتويدي
مشعل العتيبي ١، سوسن خوجه ١، وسام الحجيلي ٢، سامي بحلس ٣، علياء الدحلاوي ٤،٥
. ،محمود جاد الله ٦، محمد الشال ١،٧
١-قسم الکيمياء الحيوية، کمية العموم، جامعة الممک عبدالعزيز. جدة – المممکة العربية السعودية .
٢- قسم طب القمب و القمب التداخمي ، کمية الطب، جامعة الممک عبدالعزيز. جدة – المممکة العربية السعودية .
٣-قسم الباطنة العامة، کمية الطب ،جامعة الممک عبدالعزيز. جدة – المممکة العربية السعودية .
٤- قسم العموم الحيويو ، کمية العموم، جامعة الممک عبدالعزيز. جدة – المممکة العربية السعودية .
٥- وحدة المناعة ، مرکز الممک فهد لمبحوث الطبية ، جامعة الممک عبدالعزيز. جدة – المممکة العربية السعودية .
٦- قسم المشخصات الجزيئيو ، معهد الهندسة الو ا رثية و التکنولوجيا الحيوية ، جامعة مدينة السادات.
٧- قسم البيولوجيا الجزيئيو ، معهد الهندسة الو ا رثية و التکنولوجيا الحيوية ، جامعة مدينة السادات.
المقدمة
يؤثر فيتامين دال عمى التعبير الجيني لمعديد من الجينات وکذا ينظم عمل طائفو واسعو من
الخلايا بما فيها القمب والأوعية الدموية.
Egypt. J. of Appl. Sci., 35 (11) 2020 218
المرضي المصابون بالتهاب المفاصل الروماتويدي عادةً ما يعانون من تصمب الش ا ريين وخطر الإصابة
بأم ا رض القمب والأوعية الدموية.
أظهرت د ا رسات بيئيو في المممکة العربية السعوديو ان نحو ٨٨ ٪ مصابون بنقص فيتامين دال قد يکون
بسبب عدم التعرض لمشمس او لقمة تناول اطعمة ومنتجات تحتوي عمى فيتامين دال.
الهدف من الد ا رسة
تقييم اثر تناول فيتامين دال عمي وظائف الاوعيو الدمويو في مرضي الروماتويد.
المرضي والطرق المستخدمة
شممت الد ا رسة مجموعتين الأولى من ٥٨ مريضًا بالروماتويد يعانون من نقص فيتامين دال مقارنة
بمجموعة اخري ضابطة مکونة من ٤٨ شخصا صحيحا وذلک عن طريق إعطاء فيتامين دال بترکيز
٥٨٨٨٨ وحده بالفم لمدة ٨ أسابيع.
النتائج
أظهرت النتائج زيادة ممحوظة في عوامل النمو المبطنة للاوعية الدمويو وجزيئات الالتحام مقارنة
بالمجموعة الضابطة وذلک عن طريق الية تحفز وتنظم عمل وسائط الالتهاب
الخلاصة
تناول فيتامين دال في المرضي المصابون بالتهاب المفاصل الروماتويدي يؤدي الي تحسن
ممحوظ في وظائف الأوعية الدموية وذلک عن طريق الية تحفز وتنظم عمل وسائط الالتهاب
219 Egypt. J. of Appl. Sci., 35 (11) 2020