Assessment of vascular endothelial growth factor a serum level in pediatric hemophilic arthropathy
Egyptian Rheumatology and Rehabilitation volume 50, Article number: 6 (2023)
Children with hemophilia have serious, recurrent joint bleeding that causes disabilities. Regular factor VIII concentrate replacements have not yet completely stopped intra-articular subclinical hemorrhages and permanent joint damage. The prevention of joint damage requires the early detection and management of this hemorrhage. Neoangiogenesis is crucial in the development of synovitis after recurrent hemophilic joint bleeding. This study assessed the level of serum Vascular endothelial growth factor A (VEGF-A) as a vascular biomarker in children with hemophilia A to determine its possible role as a diagnostic biomarker for hemophilic arthropathy.
A case–control study with 30 male children who had hemophilia A and 30 healthy controls was conducted. Patients had significantly higher serum VEGF-A levels than the control group (specificity was 70.0% and sensitivity was 83.3%). Hemophilia severity and Hemophilia Joint Health Score had a significant positive correlation with VEGF-A.
Children with hemophilia A had significantly higher levels of VEGF-A in their serum. Additionally, VEGF-A had a significant positive correlation with Hemophilia Joint Health Score as well as the severity of the disease. In children with hemophilia A, VEGF-A can be used as a marker for early hemophilic arthropathy detection.
Hemophilia A is an X-linked recessive blood clotting disorder caused by a deficiency or decreased activity of clotting factor VIII (hemophilia A) or IX (hemophilia B). This deficiency increases spontaneous joint bleeding, especially in larger hinged joints. As a result, synovial inflammation and cartilage damage occur, eventually leading to hemophilic arthropathy (HA) . The quantity of the factor determines the severity of the disease, which is classified as mild (factor level 5–40 IU/dL, or 5–40% of normal), moderate (1–5 IU/dL, or 1–5% of normal), and severe (1% of normal) . Recurrent joint bleeding in hemophilia A causes irreversible joint damage, known as HA, which causes significant morbidity in hemophilia A patients. Hemophilia causes radiographic changes such as subchondral cysts, osteoporosis, and accelerated degenerative changes. Edema of the bone marrow, joint effusion or hemorrhage, synovial hypertrophy, and concurrent ligament tears are all possible complications . Clinical examination and imaging are used to detect the progression of arthropathy; however, joint outcome measurements are insufficient for identifying early and subclinical joint damage. Biochemical markers that represent joint turnover have the ability to provide this important information about the joint status . Furthermore, angiogenic factors were discovered to be elevated and involved in the pathogenesis of HA. Vascular endothelial growth factor A (VEGF-A) is an angiogenic factor that has anti-apoptotic and mitogenic effects on endothelial cells, increases vascular permeability, and enhances cell migration. It actively contributes to the regulation of normal and pathological angiogenic processes . This study assessed the level of serum VEGF-A as a vascular biomarker in children with hemophilia A to determine its possible role as a diagnostic biomarker for hemophilic arthropathy (HA).
Subjects and methods
A case-control study included 30 hemophilic A children and 30 age and gender matched healthy participants as controls. We recruited the patients from the author's unit between August 2021 and May 2022. Inclusion criteria included boys (aged 4 to 18) diagnosed with Hemophilia A according to World Federation of Hemophilia guidelines for hemophilia management (2020) . Patients who had undergone arthroscopic or chemical synovectomy within the previous 2 years and combined bleeding with other organs were all excluded. The local ethics committee for the Faculty of Medicine authorized the research after receiving informed consent in writing from the participants’ legal guardians (FMAUS, MS454/2021). Registered 18 Augest 2021—prospectively registered.
Every patient was subjected to the following:
Medical history and clinical examination with particular attention paid to the number of bleeding episodes in 6 months, the severity of hemophilia, joint pain, and swelling duration.
Clinical evaluation of the joint with the Hemophilia Joint Health Score (HJHS): The HJHS assesses both elbows, knees, and ankles by evaluating and scoring 11 items which include swelling, its duration, axial alignment, muscle atrophy, crepitus, loss of flexion, loss of extension, instability, joint pain, strength, and gait. The highest grade permitted was 148 (total elbow score from 0 to 20, total ankle and knee score from 0 to 26, gait score is separately assessed from 0 to 4 ), and high values proposed more severe joint damage .
Functional assessment using the Functional Independence Score in Hemophilia (FISH): eight activities, including eating, grooming, dressing, chair transfers, squatting, walking, and step-climbing, were evaluated as part of an objective assessment of the participant’s ability to perform various tasks. Each activity was given a grade between 1 and 4 based on how much assistance was needed to complete it. The lowest score, 8, denotes the worst function, and the highest score, 32, denotes the best function .
VEGF-A serum level assessment (performed on both the patients and control groups):
The concentrations of VEGF-A in serum were determined using an Enzyme-Linked Immunosorbent Assay kit (Shanghai Korean Biotech Co., LTD.). In this test, a 96-well plate with an antibody that is specific for human VEGF-A
Version 20.0 of the statistical package for social sciences was used to analyze the recorded data (SPSS Inc., Chicago, IL, USA). The mean and standard deviation were used to express quantitative data (SD). Frequency and percentage were used to express qualitative data. The statistical significance of the difference between more than two means was evaluated using the independent-samples t test of significance. The statistical significance of the difference between more than two means was evaluated using the ANOVA test. The strength of the correlation between two sets of variables was evaluated using Pearson’s correlation coefficient (r) test. ROC curve analysis was used to determine the overall predictiveness of parameters as well as the best cut-off value with the detection of sensitivity and specificity at this cut-off value. The confidence interval was set to 95%, and the acceptable margin of error was set to 5%. As a result, the P value was considered significant at the level of ≤ 0.05.
Results of HJHS and FISH score
Total HJHS for the patients was 0 to 28, with a mean and standard deviation of (7.033 ± 7.005). Their total FISH score ranged from 8 to 32, with a mean and standard deviation of 30.767 and 4.423, respectively (Table 3).
Results of VEGF-A level assessment and its correlation with other parameters
The patients with hemophilia A had a significantly higher serum level of VEGF A (ng/L) than the control group (patients were 326 ± 198.192, vs the control group 110.267 ± 100.755, P < 0.001) (Table 4).
Serum VEGF-A was found to be higher in more severe types of hemophilia (P < 0.001) (Table 5).
VEGF-A had a significant positive correlation with total HJHS (p = 0.008) but a non-significant negative correlation with FISH score (p = 0.800) (Table 3). A non-significant negative correlation between VEGF-A and the type of treatment regimen (p = 0.921) (Table 6).
ROC curve analysis
In order to distinguish between patients with hemophilia A and healthy boys using serum VEGF-A, a cut-off point level of 65 pg/mL had a sensitivity of 83.33% and a specificity of 70% (Fig. 1 and Table 7).
Hemophilia A is a common hereditary blood disorder. Children born with hemophilia have frequent bleeding episodes, particularly in the muscles and joints. Repeated hemarthroses cause hemophilic arthropathy, which is characterized by the damage of joint cartilage, pain, and a reduced range of motion, as well as severe limitations in physical activity , neo-angiogenesis contributes to the progression of hemophilic joint diseases .
Biomarkers are helpful for measuring joint turnover as they reveal the pathogenic mechanisms resulting from joint bleeds. In the early stages, these markers could monitor the underline joint pathology more precisely and closely than diagnostic ultrasound and Magnetic resonance imaging . The aim of this study is to assess the level of serum VEGF-A as a vascular biomarker in children with hemophilia A to determine its possible role as a diagnostic biomarker for hemophilic arthropathy (HA).
Angiogenesis is the initial cause of joint pathology that opens the synovium up to more ingredients and inflammatory agents. Therefore, it is important in the development and progression of joint pathology following recurrent joint bleeding . Increasing the level of VEGF-A encourages cell migration and division of vascular endothelial cells, and serves as a key regulator of angiogenesis .
In our study, the serum level of VEGF-A in hemophilic A children was statistically significantly higher than in controls. This study reinforces a previous study by Andrawes et al. , who noticed a rise in serum VEGF levels in their hemophilic A patients compared to controls. They came to the conclusion that VEGF was a key mediator of either the inflammatory process or neoangiogenesis in HA. In the synovium, stimulated macrophages and neutrophils produce more VEGF, resulting in neoangiogenesis. Neoangiogenesis allows inflammatory cells to be transported to the inflammatory area. Also, it provides oxygen and nutrients to the inflammatory cells, causing more chemokines and metalloproteinases to be produced, resulting in more inflammation. They recommended the use of VEGF for the early prediction of joint affection in boys with severe hemophilia A .
Our results coincide with those of Xu et al. , who revealed that hemophilic patients had significantly higher VEGF levels than healthy participants. Patients with joint bleeding had significantly higher levels of VEGF than patients without joint bleeding. Joint bleeding was related to both CRP and VEGF levels in patients with severe hemophilia, which could be used as diagnostic markers to predict joint bleeding . In contrast to Zetterberg et al. , who found no significant difference in VEGF levels in plasma and platelets between hemophilic patients and controls, hemophilic synovial tissue had a significant increase in VEGF-positive cells compared to controls. This disparity, we believe, stems from the fact that we measure VEGF serum levels rather than plasma and platelet levels. Furthermore, the age range of our patients’ means ± SD was (10.83 ± 3.364), whereas it was 32 ± 17 years in their study.
Musculoskeletal evaluation is an important part of integrated care for people who have hemophilia. The HJHS is a feasible and dependable method for detecting early joint changes in hemophiliac children and adolescents . In our study, the total HJHS range was 0 to 28, and the mean was (17.03 ± 7.005). Total HJHS and serum VEGF-A had a significant positive correlation, indicating that serum VEGF-A may play a role in HA as a biomarker for severity and was able to monitor joint affection. There was also a highly significant relationship between VEGF-A levels and the severity of hemophilia. There was an increase in HJHS among hemophiliac children with hemophiliac arthropathy in our study. Prasetyo et al.  discovered that hemophilic patients with HA had an increase in total HJHS, which supports our finding. Similarly, Andrawes et al. . Increase in HJHS among hemophiliac children in our study due to repeated joint bleeding, which results in hemophiliac arthropathy in target joints. This finding is supported by the result of Prasetyo et al.  who found that increase in the total HJHS of their hemophilic patients (ranged from 0 to 35) which indicated joint affection. Likewise, Andrawes et al.  used HJHS to assess the musculoskeletal condition in their study, with a mean ± score of 4.5 ± 3.0 and a range of 0 to 11. There was a significant positive correlation between total HJHS and serum VEGF-A, demonstrating the significance of neoangiogenesis as an independent mechanism involved in HA.
Our study did not reveal any significant correlation between VEGF and the method of factor treatment. Andrawes et al.  found a significant correlation between serum VEGF level and treatment type. This disparity may be due to the difference in sample size, as we had 30 patients while they had 50. Additionally, all of their patients were severe hemophilic patients, whereas our patients were mild, moderate, and severe. Furthermore, the majority of our patients (80%) were on regular therapy, whereas only 62% of their patients were on regular therapy.
The FISH score was established as a performance-based evaluation method to assess functional ability objectively. It can also be used to assess functional independence change after therapeutic intervention . In our research, we found that the total FISH score was (30.7 ± 4.4). In addition, we discovered a non-significant negative correlation with VEGF-A. Because no other studies had been conducted to evaluate the serum level of VEGF and its correlation with FISH score, we had to compare our results with those obtained using FISH by comparing the mean of the score. Our findings were in opposition to those of Kachooei et al. , who discovered that the total FISH score was (25.8 ± 3.6). Additionally, Tlacuilo et al.  disagreed with the findings of our study in terms of FISH score. Their study involved 60 pediatric patients with FISH scores ranging from (15 to 28) and a mean of (25.8 ± 3.6), all of whom were receiving on-demand treatment for hemophilia. Our rationale is that while 80% of the patients in our study were receiving regular hemophilia treatment, these studies were conducted on a majority of patients receiving on-demand treatment. In addition, their patients were larger than ours.
ROC curve analysis in our study revealed a cut-off point value of more than 65 ng/L with a sensitivity of 83.3% and specificity of 70%. This strongly suggests that serum VEGF-A has the ability to distinguish between the patients and controls. The sensitivities and specificities of VEGF-A in detecting hemophilic arthropathy were 78% and 88%, respectively in the study by Andrawes et al. , and similarly, Xu et al.  found that the ROC curve produced a sensitivity of 82.8% and a specificity of 68.3%, which was also close to our results.
The following are some of the study's limitations: First, we did not compare and correlate the serum VEGF A level with the radiological modality that is typically used to detect HA. Second, the small number of study participants.
Children with hemophilia A had significantly higher levels of VEGF-A in their serum. Additionally, VEGF-A had a significant positive correlation with HJHS as well as the severity of the disease. In children with hemophilia A, VEGF-A can be used as a marker for early HA detection.
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.
Faculty of Medicine, Ain Shams University
Functional Independence Score in Hemophilia
Hemophilia Joint Health Score
Receiver operating characteristic
Vascular endothelial growth factor A
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Ethics approval and consent to participate
This study was approved by Ain Shams University, Faculty of Medicine Research Ethics Committee (REC) FWA 000017585. (FMAUS, MS454/2021. A written informed consent was obtained from the participants' legal guardians sharing in the study.
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Saboony, O.A.M.N., Sebaie, M.A.E., Mikkawy, D.M.E.E.D.E. et al. Assessment of vascular endothelial growth factor a serum level in pediatric hemophilic arthropathy. Egypt Rheumatol Rehabil 50, 6 (2023). https://doi.org/10.1186/s43166-023-00172-2