Key words KIAA1199 - Power Doppler Ultrasound (PDUS) - Rheumatoid Arthritis (RA)
Schlüsselwörter KIAA1199 - Power-Doppler-Ultraschall (PDUS) - Rheumatoide Arthritis (RA)
Introduction
Rheumatoid arthritis (RA) is an autoimmune, systemic inflammatory arthritis
associated with both articular and extra-articular manifestations. The commonly
affected joints are those of hands, wrists, and knees. While some of the rheumatoid
patients have a mild self-limited disease, many suffer from joint destruction that
can cause chronic pain, unsteadiness, deformity, disability and multiple
co-morbidities [1 ].
Although the pathogenesis of RA is complex and is thought to be multifactorial, the
interaction of pro-inflammatory mediators is a likely mechanism. The inflammation
of
the synovial lining of joints leads to its hyperplasia and influx of leukocytes and
inflammatory cells [2 ]
[3 ]. This excessive migration of circulating
leukocytes into the inflamed joint demands formation of new blood vessels to provide
nutrients and oxygen to the hypertrophic joint. On this base, it is thought that one
of the earliest histological observations in early RA is angiogenesis. [4 ]
[5 ]. In addition, inflammatory associated
angiogenesis, like in RA is now recognized as a key event in the formation and
maintenance of the pannus, and without angiogenesis, leukocyte ingress could not
occur [6 ]
[7 ].
One of the identified angiogenic biomarkers are KIAA1199 (a 150-kDa protein).
KIAA1199 expression was high in RA patients by fibroblast-like synoviocytes (FLS)
derived from the lining layer of the synovial membrane. FLS are among the dominant
cell types involved in pannus formation [8 ].
KIAA1199 with other angiogenic biomarkers may accelerate the proliferation of FLS
cells and activate the downstream angiogenic signaling pathways, leading to the
formation of the pannus, and erosion of the cartilage and bone in the progression
of
RA [9 ]. KIA1199 was first described as an
inner ear-specific protein [10 ]. It was found
to be accused of cancer progression, metastasis and poor prognosis of patients with
cancer as determined in many clinical studies [11 ]
[12 ].
Musculoskeletal ultrasound (MSUS) is found to be an ideal modality to detect
subclinical joint inflammation and joint injury in patients with RA [13 ]
[14 ]
[15 ]. It is more sensitive than physical
examination, and radiographic imaging. MSUS is also noninvasive and inexpensive in
comparison to magnetic resonance imaging, and laboratory parameters [13 ]
[16 ]
[17 ]. In addition, MSUS Power Doppler (PD)
imaging of synovitis has been suggested to reflect the pathology of synovial
inflammation in patients with RA better than in grey scale imaging. So MSUS is a
more sensitive reliable method for assessment of inflammatory activity [13 ]
[18 ]. As it directly visualizes the
synovial-membrane vessels, which is important in providing very early information
on
the changes in synovitis activity during the course of inflammatory joint disease
[19 ]
[20 ].
The hypothesis of this study was that KIAA1199 biomarker could be used as a tool to
detect the disease activity in RA patients, so the aim of our study is to assess the
serum level of angiogenic biomarker KIAA1199 in RA patients and its correlation with
MSUS, PDUS findings, and the disease activity.
Patients and methods
Subjects
Fifty patients fulfilling the 2010 ACR-EULAR classification criteria for RA [21 ] were recruited from the inpatients and
outpatients’ clinic of the Rheumatology, Rehabilitation and Physical
Medicine Department of Assiut University hospitals, in the period from April
2016 to August 2017. Exclusion criteria comprised patients’ refusal,
patients aged less than 18 years old, a patient with other connective tissue
diseases, chronic kidney and liver disease. Full history taking including age,
sex, disease duration, and duration of morning stiffness, general and
musculoskeletal examination were done to RA patients group.
The disease activity in RA patients was assessed by the 28 joint count Disease
Activity Score (DAS 28) [22 ]. Modified
health assessment questionnaire (mHAQ), patient global assessment, physician
global assessment, clinical disease activity index (CDAI) performed to all
patients.
Forty apparently healthy volunteers, age and sex-matched were also included as a
control group. The protocol of the study supported by Ethical Committee for
Scientific Research at Faculty of Medicine at Assiut University and informed
written consents was taken from all patients.
Laboratory
Eight ml of venous blood samples were collected from all subjects under complete
aseptic condition by clean venipuncture then dispensed into three tubes: For
rheumatoid factor (RF) and C reactive protein tests on the same day of blood
collection, complete blood count, ESR measurement, and 3 ml of collected
blood in the plane tube were allowed to clot, centrifuged at 3,000 rpm
for 20 min and plasma was then collected, aliquoted, and kept frozen at
−70°C until batch analysis.
Enzyme-linked immunosorbent assay for human KIAA1199
KIAA1199-level were detected in serum from patients with RA and controls.
Standards and samples were incubated at room temperature in a micro ELISA
plate coated with KIAA1199 monoclonal antibody (sc-164775, Santa Cruz, CA,
USA). After incubation, washing, and addition of a specific detection
antibody coupled to horseradish peroxidase, the substrate was added, and
incubation was performed after the stop solution was added. The optical
density was measured and different KIAA1199 concentrations were then
determined.
MSUS assessment of joints
MSUS equipment used was General Electric (GE) LOGIQ 3 digital US scanner
using a 7.5–10 MHz transducer. Contact gel was applied to
the skin to provide an acoustic interface. A detailed MSUS examination was
done for wrists, elbows, and knees directly after blood sample collection.
Both wrists were assessed in dorsal longitudinal radial and ulnar scans.
Both elbows were assessed in the ventral longitudinal scan (humero-radial
and humero ulnar) and dorsal longitudinal scan on olecranon fossa. Both
knees were assessed in the suprapatellar longitudinal scan, medial and
lateral longitudinal scans. The examined joints were evaluated
systematically for the presence of synovial hypertrophy and Power Doppler
signal. One Joint ultrasound findings were defined according to published
OMERACT definitions [23 ]: A
semi-quantitative scale for quantification of synovial thickening in B-mode
MSUS was used, based on 4 point scales (0-3) as: Grade 0=absence of
synovial thickening, Grade 1=mild synovial hypertrophy, Grade
2=moderate synovial hypertrophy, and Grade 3=severe synovial
hypertrophy.
PDUS: grade 0=no flow in the synovium, 1=single vessel
signals, 2=confluent vessel signals in less than half the area of
the synovium, 3=vessel signals in more than half the area of the
synovium.
To ensure a stringent definition of synovitis by ultrasound, only patients
with synovial hypertrophy (SH) grade≥2 plus PDUS signal were
classified as having active synovitis. If any assessed joint met these
criteria, the patient was classified as having active synovitis.
We calculated the total signal score of Power Doppler (TSS) for all RA
patients by summation of the PD signal score in six joints both elbows,
wrists, and knees in all views.
Statistical analysis
Results were collected, tabulated, statistically analyzed by statistical
package SPSS version 23. Two types of statistics were done; descriptive
(e. g., no, percentage (%), mean, standard deviation (SD)
and range) and analytical one (student’s t-test and
Pearson’s correlation analysis). The Student’s t-test was to
collectively indicate the presence of any significant difference between 2
groups for a normally distributed quantitative variable. Pearson’s
correlation analysis was used to show strength and direction of the
association between 2 quantitative variables. P-value, was considered
non-significant if p>0.05, significant difference if p<0.05,
and highly significant if p<0.01.
Results
Demographic characteristic of patients and control groups
Fifty RA patients (90% females and 10% males) were compared with
40 healthy control persons (80% females and 20% males) included
in this study (P=0.198), with a range & mean of age ±SD
(18-63) 41.16±12.74, (20-66) 40.08±12.21 years respectively
(P=0.968).
[Table 1 ] shows the clinical and
laboratory data; the mean of disease duration was 7.43±0.87 years,
morning stiffness was 0.97±0.10 h, and mean of the DAS-28 score
was 5.09±1.95.
Table 1 Clinical and laboratory data of patients group
Clinical and laboratory data in patients group
Patient no=50
Disease duration “years”
(mean±SD)
(0.25–30) 7.43±0.87
Morning Stiffness
“hours”(mean±SD)
0.97±0.10
Subcutaneous nodule (%)
7(14.0%)
Pain (%)
43(86.0%)
Swelling (%)
35(70.0%)
Tender joint count “TJC”
(mean±SD)
8.28±1.07
Swollen joint count “SJC”
(mean±SD)
5.00±0.70
Visual analogue scale
"VAS"(mean±SD)
4.38±0.25
Patient global assessment
"PGA"(mean±SD)
4.38±0.25
Physician global assessment “Ph.GA”
(mean±SD)
3.56±0.29
Erythrocyte Sedimentation Rate “ESR”1
st
hour “mm/h”
(mean±SD)
45.88±3.21
C reactive protein “CRP” mg/L
(mean±SD)
19.25±2.10
Rheumatoid factor
“RF””IU/ml”
(mean±SD)
192.86±35.54
Number of patient with positive RF(%)
40(80.0%)
Complete Blood Count “CBC”
1-Hb “g/dl” (mean±SD)
11.30±0.21
2-Platelets “x10
3
uL” (mean±SD)
317.90±11.09
4-WBCs “x10
6
/uL” (mean±SD)
6.45±0.28
Clinical disease activity index
20.94±1.95
DAS28 (disease activity score) (mean±SD)
5.09±1.95
High(%)
4(8.0%)
Moderate(%)
21(42.0%)
Low(%)
25(50.0%)
Serum level of KIAA1199 and ROC curve
Serum KIAA1199 level was significantly higher among RA patients
4.36±1.22 ng/dl compared to control group
2.87±0.51 ng/dl (p<0.001). ([Table 2 ]). With the aim of verifying the
diagnostic role of KIAA1199 in RA, we conducted a receiver operator
characteristic (ROC) curve analysis of KIAA1199. Our result indicates that the
areas under the ROC curve for the serum KIAA1199 level from RA patients were
0.89 indicating good diagnostic value (80–90%). Cutoff values
corresponding to 3.55 ng/dl were able to detect active RA with a
sensitivity of 70% and specificity of 90.02% ([Fig. 1 ]). [Table 3 ]: shows the correlation between
serum level of KIAA1199 and history, clinical, laboratory data, and assessment
of the disease activity. There was a highly significant correlation between
serum level of KIAA1199 and morning stiffness, ESR, CDAI, CRP, DAS28, and mHAQ
(p=0.004, 0.009, 0.002, 0.000, 0.004, 0.007 respectively), and a
significant correlation with RF, Hb, and platelets (p=0.019, 0.032,
0.035 respectively), with no correlation with age, disease duration.
Fig. 1 shows the ROC curve analysis of KIAA1199 in serum of RA
patients. The area under the curve (AUC=0.888) for RA. A cutoff
value of 3.55 ng/dl detected in RA patients at the sensitivity
of 70.0% and the specificity of 99.02%.
Table 2 KIAA1199 serum level in patients & control
groups.
Item
Patients group
Control group
P-value
KIAA1199 in ng/dl (mean ± SD)
4.36±1.22
2.87±0.51
P<0.000***
Table 3 Correlation between serum KIAA1199 level with
clinical, laboratory data, and assessment of disease activity in
patients group.
Clinical &laboratory data
KIAA1199
r
p
Age
0.129-
0.371
Diseases duration
0.025
0.865
MS
0.121
0.004**
TJC
0.505
0.004**
SJC
0.798
0.001**
VAS
0.733
0.000**
ph.GA
0.716
0.001**
ESR
0.341
0.009**
CRP
0.114
0.002**
RF
0.037
0.019*
Hb
− 0.578
0.032*
Platelets.
0.299
0.035*
WBCs
0.226
0.123
CDAI
0.793
0.000**
DAS28
0.67
0.004**
mHAQ
0.094
0.007**
*Correlation is significant at the 0.05 level (2-tailed);
** Correlation is highly significant at the 0.01 level
(2-tailed); MS=Morning stiffness, TJC=tender joint
count, SJC=swollen joint count,, VAS=Visual, Ph G
A=physician global assessment, analogue
scale,,Hb=hemoglobin, ESR=erythrocyte sedimentation
rate, CRP=C- reactive protein,, RF=Rheumatoid factor,
WBCs=white blood cells, CDAI=clinical disease activity
index, DAS28=Disease activity score, mHAQ=modified
Health Assessment Questionnaire
Correlation between KIAA1199, PD, and disease activity
The correlation between serum KIAA1199 level and DAS 28 with PDUS finding and
synovial thickness score, for different joints among RA patients, is shown in
[Table 4 ]. There was a significant
positive correlation between MSUS (SH) with serum level of KIAA1199 and DAS28
(p=0.001, 0.004 respectively), and also between the PDUS with serum
level of KIAA1199 and DAS28 (p=0.001, 0.002 respectively) in wrist
joints only, while there was no positive correlation between both elbow and knee
joints. There was a positive correlation between TSS and DAS28 ESR, CDAI, mHAQ
(p<0.001) and serum level of KIAA1199 p<0.05 among RA group
shown in [Table 5 ].
Table 4 Correlation between serum KIAA1199 level and DAS28
ESR with Synovial hypertrophy “SH” and power Doppler
signal ‘PD’ among patients group.
Synovial hypertrophy
KIAA1199
DAS28 ESR
Wrist SH
r
0.556
0.733
P
0.001**
0.004**
Elbow SH
R
−0.075
0. 251
P
0.583
0. 554
Knee SH
R
−0.033-
0.217
P
0.720
0.251
Power Doppler signal (PD)
Wrist PD
R
0.656
0.493
P
0.001**
0.002**
Elbow PD
R
0.085
0.027
P
0.091
0.132
Knee PD
R
−0.033-
0.217
P
0.720
0.251
*Correlation is significant at the 0.05 level (2-tailed);
** Correlation is highly significant at the 0.01 level
(2-tailed); SH=Synovial hypertrophy, PD=power Doppler
signal
Table 5 Correlation between total signal score with serum
KIAA 1199 level, DAS 28-ESR, CDAI, m HAQ:.
Total signal score (TSS)
r=
P=
KIAA1199
0.037
0.04*
DAS-28 ESR
0.468
0.001**
CDAI
0.462
0.001**
mHAQ
0.497
0.000**
*Correlation is significant at the 0.05 level (2-tailed);
** Correlation is highly significant at the 0.01 level
(2-tailed); TSS=total signal score, DAS28=Disease
activity score, CDAI=clinical disease activity index,
mHAQ=modified Health Assessment Questionnaire
Discussion
As a basic mechanism in any inflammatory arthritis like RA, leukocytes migrate from
the bloodstream through the vessel wall into the synovium, which may also contain
a
number of cell adhesion molecules, as integrin, selectins and their respective
ligands [5 ]
[24 ]
[25 ]. This excessive migration of circulating
leukocytes into the inflamed joint demands formation of new blood vessels to provide
nutrients and oxygen to the hypertrophic joint. In addition, these inflammatory
mediators including pro-inflammatory cytokines and chemokines may promote both
endothelial cells adhesion, receptor expression and hence angiogenesis. Moreover,
some adhesion molecules themselves induce neovascularization [25 ]
[26 ]. One of the most evident signs of
synovitis is the increase in synovial vascularization due to angiogenesis, which is
crucial for synovial growth and invasiveness [27 ]
[28 ].
In our study, we clarified a significant increase in the serum level of KIAA1199 in
patients with RA, vs. controls. This is compatible with the results of a previous
study by Yang et al. 2015 as they found a significant increase in the expression of
KIAA1199 in plasma, synovial tissue, and synovial fluid in RA patients in comparison
to healthy controls [11 ]. Also, Wang et al.
2012 designed a comparative proteomics study of proteins in primary cultures of RA
and normal synoviocytes. They concluded that all angiogenesis-related proteins were
up-regulated in the synovial tissue of patients with RA and this may be the
essential cause of pannus formation. They also elucidated increase expression of
KIAA1199 by 5.19 fold in RA synoviocytes in comparison to healthy controls, which
may be one of the factors that lead to the development of vasculature in the
synovial membrane of patients with RA [10 ].
Another study by Yoshida et al. 2013 showed that KIAA1199 is involved in hyaluronan
(HA) degradation in normal skin fibroblasts and arthritic synovial
fibroblasts, which is independent of CD44 and HYAL enzymes. Their
findings provide that KIAA1199 is implicated in physiological and
pathological HA catabolism and suggest that therapeutic interventions targeting
KIAA1199 may be of clinical value. Collectively, these data suggesting that
increased level of KIAA1199 might play an important role in the pathogenesis of RA
by different pathways [29 ].
In this study, KIAA1199 ROC curve analysis showed that the AUC=0.888 for RA
patients with a sensitivity of 70.0% and the specificity of 99.02%.
In the study of Yang and his colleagues; 2015 [11 ] they found that the sensitivity of KIAA1199 biomarker by analysis of
ROC curve in plasma, synovial, and synovial tissue of RA patient was 72%,
84% and 80% respectively and specificity was 80%,
93.3% and 93.3% respectively. Our results indicate that KIAA1199 may
be a potential diagnostic biomarker of RA disease activity.
We noticed that the serum KIAA1199 levels were significantly correlated with morning
stiffness, ESR, RF, Hb, CDAI, CRP, DAS28, platelets, and mHAQ, with no correlation
with age, disease duration, and WBCs. This is in accordance with Yang et al., who
demonstrate a positive correlation between KIAA1199 and DAS28. These data indicate
that the level of KIAA1199 is not only increased in RA but also an index that
correlated closely with clinical parameters of RA disease activity [11 ].
In the current study, we found that there was a positive correlation between serum
KIAA1199 level, and SH &PD in wrist joint but not in elbow and knee joints.
This can be explained by that the wrist joint is the most commonly affected joint
in
RA for swelling and tenderness [30 ]. Serum
KIAA1199 level was also correlated with TSS. This confirms the role of serum
KIAA1199 level in synovitis and angiogenesis associated with RA. These results are
in accordance with studies that found a positive correlation between MSUS finding
and levels of Angiopiotein-2, and VEGF. [31 ].
Kelly et al. 2015 demonstrated that there is a relationship between PD signal and
angiogenic gene expression, they also found a correlation between SH and cellular
markers of synovial inflammation and pro-inflammatory cytokines [32 ]. In another study, they demonstrated that
there was a positive correlation between PDUS scores with disease activity, and with
plasma biomarkers such as VEGF, Matrix metalloproteinases-3, and tissue inhibitor
of
metalloproteinases-1. All these biomarkers have a role in the budding of endothelial
cells and which is an early step in angiogenesis [33 ]. The TSS was highly positively correlated with DAS28, CDAI, and mHAQ
in agreement with Naredo et al. 2008 and Dougados et al. 2010 studies [34 ]
[35 ].
In this study, PDUS has been regarded as an important tool for globally examining
the
degree of synovitis in RA. PDUS signal was assessed in 6 joints for each patient,
other studies used different numbers of joints [35 ]
[36 ]. It is difficult to determine the minimal
numbers of joints to be included in a global US score. There was a lack of a clear
definition of synovitis as well as varying validity data with respect to the
proposed scores. So, scoring systems comprise a wide range and number of joints.
More studies are needed in order to achieve optimal US scoring systems for
monitoring patients with RA in clinical trials and in clinical practice [23 ]
[35 ].
PDUS correlates significantly with disease activity and novel angiogenesis biomarker
along with synovial histopathology in patients with RA. Unlike clinical examination,
PDUS has the ability to discover subclinical synovitis making it a more reliable,
sensitive to change, and convenient investigational method. Therefore, it should
have a potential role in standard monitoring and follow-up of patients for response
to treatment as well as prediction of future structural damage [36 ]
[37 ]
[38 ]. The MSUS has some limitations because
its technique is operator dependent, not every rheumatologist is skilled in its use
and assessments are time consuming if large number of joints are examined.
Conclusion
Serum KIAA1199 level may be a useful angiogenic biomarker to detect RA disease
activity, and seems to be a potential therapeutic target in RA. Also, PDUS has the
ability to detect subclinical synovitis not easily detected by clinical examination
and correlate with serum KIAA1199 biomarker in our study.
