Baseline angiopoietin-2/angiopoietin-1 (Ang2/Ang1) ratio is correlated with the synovial vascularity measured 1 month later in rheumatoid arthritis

Introduction The inflammatory process in rheumatoid arthritis (RA) is characterized by excessive, tumor-like proliferation of synoviocytes, which is associated with an increase in the vasculature needed to support metabolic requirements. Neovascularization is a complex process in which new blood vessels develop from the existing microvascular bed; it involves endothelial cell division, selective degradation of vascular basement membranes and the surrounding extracellular matrix, and endothelial cell migration [1]. This process is driven by a combination of upregulation of angiogenesis promoters and downregulation of inhibitors [2].


Introduction
The inflammatory process in rheumatoid arthritis (RA) is characterized by excessive, tumor-like proliferation of synoviocytes, which is associated with an increase in the vasculature needed to support metabolic requirements. Neovascularization is a complex process in which new blood vessels develop from the existing microvascular bed; it involves endothelial cell division, selective degradation of vascular basement membranes and the surrounding extracellular matrix, and endothelial cell migration [1]. This process is driven by a combination of upregulation of angiogenesis promoters and downregulation of inhibitors [2].
The angiogenic factor angiopoietin-1 (Ang1) is an agonist ligand of the endothelial receptor tyrosine kinase Tie-2. In adults, the Ang1/Tie-2 signaling system is essential for the maturation of vessels. Experiments in transgenic mice have shown that vascular endothelial growth factor (VEGF) induces angiogenesis, whereas Ang1 induces vessels stabilization by maximizing the interactions between endothelial cells and their surrounding support cells and matrix [3].
Angiopoietin-2 (Ang2) is the natural antagonist of Ang1 [4]. By inhibiting Tie-2 signaling, Ang2 leads to a loosening of cell matrix and cell-cell interactions [5][6][7]. This antagonistic effect is considered to be a requirement for the sensitivity of endothelial cells to other angiogenic factors such as VEGF. In the absence of angiogenic growth or survival signals, Ang2 action results in destabilization and finally regression of vessels [5].
Because angiogenesis and formation of new blood vessels is a component of pannus in RA, we hypothesized that the balance between Ang1 and Ang2 is in favor of Ang2 (represented by Ang2/Ang1 ratio) in RA pannus.
Baseline angiopoietin-2/angiopoietin-1 (Ang2/Ang1) ratio is correlated with the synovial vascularity measured 1 month later in rheumatoid arthritis Mohammed Kamal Senna a , Shereen Aly Machaly a , Manal Foda b , Nermeen Eid c Background Synovial angiogenesis is a component of rheumatoid pannus and considered to be an important early step in the pathogenesis of rheumatoid arthritis (RA).

Objectives
The aim of this study was to evaluate the serum levels of angiopoietin-1 (Ang1), angiopoietin-2 (Ang2), as well as the significance of the Ang2/Ang1 ratio in RA in relation to the joints' blood flow signals and RA activity parameters. In addition, we studied their relevance in predicting the state of angiogenesis.

Patients and methods
This study was carried out on 65 consecutive very early RA patients. Power Doppler ultrasonography was performed in a total of 10 joints. The blood flow signals of each joint were scored on a three-grade scaling system. The total of the scores of the 10 joints was defined as the total signal score (TSS). On the same day, serum variables including vascular endothelial growth factor (VEGF), Ang1 and Ang2 levels, and clinical disease activity were evaluated. All the parameters measured were reassessed after 1 month.

Results
Serum VEGF, Ang2 levels, and Ang2/Ang1 ratio were significantly correlated with C-reactive protein, DAS28-CRP, and TSS at baseline and after 1 month. Moreover, significant correlations were observed between serum VEGF and Ang2 levels and Ang2/Ang1 ratio both at baseline and 1 month later. Interestingly, baseline serum VEGF level and Ang2/Ang1 ratio were significantly correlated with TSS after 1 month.

Assessment using PDUS
Ultrasound was emitted using a linear array transducer (7.5-10 MHz, Esaote-GPX-color Doppler apparatus, Genoa, Italy). The pulse repetition frequency was set at the lowest level in the tolerated range to achieve the maximum sensitivity: 500-750 Hz. Low wall filters were used. The dynamic range was set at 20-40 dB. Power Doppler ultrasonography (PDUS) was performed in a total of 10 joints: the bilateral elbows, wrists, metacarpophalangeal, knees, and ankles. The probe was applied to the anterior and posterior recesses in the elbow, dorsal carpal recesses, extensor tendon sheaths and flexor tendon sheaths in the wrist, anterior and posterior recesses in the wrist, suprapatellar, medial parapatellar and lateral parapatellar recesses in the knee, and anterior, medial, and lateral tendon sheaths in the ankle. The degree of vascularity at various sites of the synovial membrane was quantified by counting color pixels of region of interest using a special computer software, followed by calculation of the color fraction (defined as the ratio of color pixels number to the total number of pixels in the region of interest [10]). The color fraction was scored as follows: score 0, no PDUS signals; score 1, color fraction less than 25%; score 2, color fraction from 25 to 50%; and score 3, color fraction greater than 50% [11]. The score at the site with the strongest finding in each joint was adopted as the score of the joint and the total of the scores of the 10 joints was defined as the total signal score (TSS).

Statistical analyses
All statistical analyses were carried out using SPSS for windows version 17.0 (SPSS, Chicago, Illinois, USA). Continuous data were expressed as mean±SD and median. The correlations between the angiogenic markers (VEGF, Ang1, Ang2, and Ang2/Ang1 ratio) and the parameters of inflammation (CRP and DAS28-CRP) were assessed by Spearman's r coefficient. Correlations among serum angiogenic markers as well as the Ang2/Ang1 ratio were also assessed using Spearman's r coefficient. P-values of less than 0.05 were considered to be statistically significant.

Results
Patient characteristics at baseline and after 1 month are summarized in Table 1. This study included 65 patients (59 women and six men); their mean age was 56.1 ± 13.7 years (age range from 24 to 85 years). The disease duration was 6.8 ± 1.2 months (ranging from 4 to 10 months). Serum VEGF concentrations were significantly higher in patients with early RA than in the controls.
The aim of this work is to study the serum levels of Ang1, Ang2, and the Ang2/Ang1 ratio in RA in relation to blood flow signals in a total of 10 joints and RA activity parameters. In addition, we aimed to study their relevance to predict the state of angiogenesis.

Patients and methods
This study was carried out on 65 consecutive very early RA patients (59 women and six men) who were attending the outpatient clinic of Rheumatology and Rehabilitation, Mansoura University Hospital, Egypt. All patients fulfilled the American College of Rheumatology (ACR)/European League Against Rheumatism (EULAR) 2010 classification criteria for RA [8]. Their age ranged from 24 to 85 years. Disease duration was less than 12 months. All patients were naïve for disease-modifying antirheumatic drugs. All patients underwent a standardized interview for assessment of history, followed by a physical examination. The disease activity was assessed using the disease activity score DAS28-CRP tool [9]. In addition, 15 apparently healthy individuals were enrolled as controls. Written consent was obtained from every participant in this study after approval of this study from the local Ethical Committee.

Collection and preparation of samples
After a 12 h fast, venous blood samples were collected from every participant by sterile venipuncture on the morning of the same day of assessment of history and clinical examination. The separated serum was kept frozen at -20°C until the time of analyses, which were carried out at the latest after 3 months. Samples were used for estimation of serum C-reactive protein (CRP) and the angiogenic factors.

Evaluation of angiogenic factors and calculation of Ang2/Ang1 ratio
Serum VEGF and Ang1 and Ang2 levels were determined using an ELISA kit (R&D Systems, Minneapolis, Nebraska, USA). The mean values of double measurements were calculated. Concentrations are reported as pg/ml. After obtaining Ang1 and Ang2, the Ang2/Ang1 ratio was calculated using the statistical program for each patient.

CRP assay
Quantitative assay of the CRP kit was supplied by Turbox CRP (Orion Diagnostica, Espoo, Finland). The Turbox assay for CRP is a liquid-phase immunoprecipitation assay with nephelometric detection. Concentrations are reported as mg/dl.

Serum levels of angiogenic factors and Ang2/Ang1 ratio with indices of disease activity
synovial blood flow signals in the wrist. In the study of Strunk and colleagues, only the wrists were evaluated. Although the serum VEGF level is a systemic finding, the synovial blood flow signals of the wrist are local findings. Therefore, evaluation of various joints of the body would be more relevant to examination of the relationship between serum angiogenesis factors and synovial blood flow signals. Hence, we examined multiple joints. Moreover, Strunk and colleagues examined only 21 RA patients, whereas in the current study, 65 RA patients were examined. As a result, a correlation between the serum VEGF level and TSS could be confirmed. The increase in synovial blood flow signals has been observed histologically to be caused by an increase in the number of blood vessels in the synovial tissue, that is, angiogenesis [16]. Thus, the correlation between the serum level of VEGF, which is a major angiogenic factor, and TSS appears reasonable.
Ang1 and Ang2 are ligands of the tyrosine kinase receptor Tie-2 that contribute toward blood vessel formation during angiogenesis [17]. Stabilization of the vascular wall is regulated by Ang1 binding to the Tie-2 receptor. In contrast, Ang2 Tie-2 binding induces vessel destabilization, which leads to angiogenic sprouting [6,18]. The balance between these factors and the level of other angiogenic factors, particularly VEGF, determines whether new blood vessels form or blood vessels become stable [19].  levels, serum Ang2 levels, and Ang2/Ang1 ratio are correlated with CRP and DAS28-CRP and thus seem to reflect the RA activity.
The serum VEGF level and serum Ang2 level were correlated with the serum CRP levels, DAS28-CRP, and TSS. Conflicting data on the association of angiogenic factors with parameters of inflammation in RA have been reported by previous studies. In addition to our study, the serum VEGF level has been observed to be correlated with the serum CRP level [12], parameters of inflammation, and DAS28-CRP [13].
Kurosaka et al. [14] found that serum Ang2 levels are correlated with serum CRP but not with DAS28-CRP; they also reported a relationship between serum VEGF and Ang2 levels, but not Ang1, with synovial blood flow signals. However, Strunk et al. [15] did not observe a correlation between serum VEGF level and Baseline serum vascular endothelial growth factor (VEGF) level is correlated significantly with the total signal score (TSS) measured 1 month later (r = 0.452, P < 0.001).

Figure 2
As the TSS in the current study is correlated with serum VEGF and Ang2 levels but not with serum Ang1 levels, the synovial flow signals are considered to represent a phase of vigorous angiogenesis. Interestingly, our finding of a correlation between baseline serum VEGF level and baseline Ang2/Ang1 ratio with the TSS measured 1 month later seems to confirm this concept.
Among the angiogenic factors we examined, a correlation was observed between the serum levels of VEGF and Ang2 but not between serum levels of VEGF or Ang2 with Ang1. The absence of a correlation between the serum levels of VEGF and Ang1 levels is understandable on the basis of these data. The correlation of VEGF level with the level of Ang2 rather than the level of Ang1 may indicate the usefulness of both markers as an index of active arthritis.
Thus, as Ang1 stabilizes the blood vessels, whereas Ang2 induces angiogenesis, it is reasonable that the elevated Ang2/Ang1 ratio (in favor of Ang2) reflects the phase of vigorous angiogenesis. The correlation of the baseline VEGF and Ang2/Ang1 ratio with TSS measured 1 month later observed in the current study may be explained from this prospective. To our knowledge, this is the first study in which the ratio of Ang2 to Ang1 is determined in relation to synovial blood flow.

Conclusion
Serum concentrations of VEGF and Ang2 as well as Ang2/Ang1 ratio were correlated with parameters of inflammation in early RA. Our results show that the elevated serum VEGF and Ang2/Ang1 ratio (in favor of Ang2) reflect a phase of vigorous angiogenesis.