Vascular endothelial growth factor in hypersensitivity pneumonitis and connective tissue disease‑associated interstitial lung disease

Background A growing evidence on the role of vascular endothelial growth factor (VEGF) in the pathogenesis of interstitial lung diseases accumulated over the past decade; with the development of nintedanib for the treatment of fibrotic interstitial lung diseases, our aim was to quantify serum levels of VEGF in patients’ hypersensitivity pneu‑ monitis (HP) and connective tissue disease‑associated interstitial lung diseases (CTD‑ILD) with an assessment of its relationship with functional status parameters and echocardiographic findings. Methods Spirometry, 6‑min walking test, echocardiography, and serum VEGF levels were assessed in HP and CTD‑ ILD patients. Results The study included 31 HP patients, 30 CTD‑ILD patients, and 29 control subjects. VEGF level was significantly higher in HP patients than in patients with CTD‑ILD and control subjects. VEGF level showed positive correlation with 6‑min walk distance and forced vital capacity percent predicted and inverse correlation with percent desatura‑ tion in 6‑min walk test, dyspnea score, and echocardiographic findings in both groups. Conclusion Serum VEGF is higher in HP patients than in patients with CTD‑ILD and control.


Introduction
Biomarkers are of great interest in interstitial lung diseases (ILD), as they can help in the diagnosis, the assessment of severity, and predicting the prognosis of the disease.They are simple, easier, and more rapid than invasive procedures for diagnosis.Vascular endothelial growth factor (VEGF) is a tyrosine kinase glycoprotein important in the regulation of endothelial function, capillary permeability, and angiogenesis, playing an important role in maintaining normal lung, angiogenesis of endothelial cells, and restoration of pulmonary circulation.Given that inflammation, abnormal angiogenesis, and altered fibrosis are involved in the pathogenesis of ILD, VEGF is believed to have a role [1].Hypersensitivity pneumonitis (HP) showed an upregulation of alveolar epithelial apoptosis markers suggesting an important role for alveolar epithelial cell integrity in this disease [2].
Previous studies raised a question about the diagnostic utility of VEGF in HP as they found an elevated serum level in HP patients when compared with the control group [3,4].
VEGF is believed to contribute in the progression of connective tissue disease-associated interstitial lung diseases (CTD-ILD), as it plays a dual role in encouraging and inhibiting pulmonary fibrosis.Also, VEGF subtype expression imbalance can contribute to the development of the disease, in which proliferation of endothelial cells and fibroblasts can be induced by VEGF [5].Our aim was to compare serum VEGF level in HP and CTD-ILD patients in relation to control and to assess the relationship between serum level of VEGF, functional severity, and presence of pulmonary hypertension.

Methods
This prospective case control study was conducted from February 2023 and December 2023.The study was conducted in accordance with the Helsinki Declaration and was approved by the research ethics committee of our institute (No: N-144-2023).A written informed consent was obtained from all patients.
Patients with HP and CTD-ILD aging 18 years or more were included.HP was diagnosed according to ATS/ JRS/ALAT clinical practice guidelines for the diagnosis of hypersensitivity pneumonitis in adults [6].Connective tissue diseases were diagnosed according to relevant guidelines [7][8][9].

Data collection
Medical history included comorbidities, smoking history, and modified Medical Research Council (MMRC) dyspnea score.High-resolution computed tomography (HRCT) to determine the type and extent of ILD, 6-min walking test (6MWT), spirometry to assess the functional capacity, and transthoracic echocardiography to assess the probability of pulmonary hypertension and right heart affection were done.Quantification of serum VEGF level was done using enzyme-linked immunosorbent assay (human VEGF Elisa kit, Cloud-Clone Corp, Houston, USA).

Statistical methods
The program used for statistical analysis was IBM SPSS (Statistical Package for the Social Science; IBM Corp, Armonk, NY, USA) release 22 for Microsoft Windows.Data were statistically described as mean ± standard deviation (SD) or frequencies (number of cases) and percentages when indicated.The following tests were used: Kolmogorov-Smirnov test, Mann-Whitney U test, and Spearman rank correlation equation.Two-sided P values were considered statistically significant if less than 0.05.

Results
The study included 90 subjects: 31 patients with HP, 30 patients with CTD-ILD, and 29 age-and sex-matched healthy control subjects.
The mean value of serum VEGF was 664.58 ± 375.95 pg/ml, and the mean value of serum VEGF in the control group was 205.69 ± 44.89 pg/ml (P value < 0.0001).
Table 1 shows the correlation between serum VEGF level, functional assessment parameters, and echocardiographic findings in the HP group.Table 2 shows the relationship between serum VEGF level and radiological findings in the HP group.
Table 3 shows the correlation between percent desaturation during 6MWT and FVC% and MMRC score in the HP group.
Table 4 shows the relationship between percent desaturation during 6MWT and radiological findings in the HP group.

CTD-ILD patients
This group of patients comprised 24 females (80%) and 6 males (20%).The mean age was 46.9 ± 11.96 years.Only 7 patients (23.3%) had comorbidities in the form of systemic hypertension and diabetes mellitus.In our study group, connective tissue diseases were systemic sclerosis, rheumatoid arthritis, dermatomyositis/polymyositis, and mixed connective tissue disease.
The mean value of serum VEGF was 209.33 ± 57.83 pg/ ml (P value 0.789 between CTD-ILD and control groups).
Table 5 shows the correlation between serum VEGF level, functional assessment parameters, and echocardiographic findings in the CTD-ILD group.
Table 6 shows the relationship between serum VEGF level and radiological findings in the CTD-ILD group.
Table 7 shows the correlation between percent desaturation during 6MWT and FVC% and MMRC score in the CTD-ILD group.
Table 8 shows the relationship between percent desaturation during 6MWT and radiological findings in the CTD-ILD group.

Discussion
Very few studies examined the role of VEGF in HP.In the small numbers studied, serum VEGF levels were elevated compared with controls [2].In our study, the mean value    of serum VEGF in the HP group was significantly higher than that of the CTD-ILD group (however, the 2 groups were not matched regarding functional status) and control group (P value < 0.0001) (Table 9); this agrees with previous studies by Navarro et al. [3] and Yamashita et al. [4].
Serum VEGF level showed a positive correlation with 6MWD, FVC%, and inverse correlation with percent desaturation during 6MWT, MMRC score, presence of pulmonary hypertension, and right-sided cardiac affection, but this was statistically insignificant.Yamashita et al. [4] stated that the serum levels of VEGF were not correlated with the pulmonary function tests in HP patients.
As far as we know, no previous studies assessed the relationship between serum VEGF level and results of 6MWT, MMRC score, radiological findings, or echocardiographic findings in patients with HP.Zhong and Luo [10] found that serum VEGF was significantly and positively correlated with HRCT scores in IPF patients, suggesting an association with disease severity.In addition, elevated serum VEGF levels were closely associated with impairment of lung function.Ando et al. [11] reported no correlation between serum VEGF level and the results of pulmonary function tests in IPF patients.Another study by Ventetuolo et al. [12] showed that VEGF levels were not correlated with FVC, 6MWD, or New York Heart Association functional class in patients with IPF and that there was no significant correlation between VEGF levels and hemodynamics.
In our CTD-ILD group, there was no statistically significant difference between the mean value of serum VEGF and that of control group (P value 0.789).Previous studies by Hashimoto et al. [13] and Kikuchi et al. [14] found that serum VEGF levels were significantly higher in patients with rheumatic diseases compared with healthy controls, and VEGF levels were correlated with the presence of ILD.Saranya et al. [15] reported that median serum VEGF in systemic sclerosis patients was significantly higher than in controls; also, De Santis et al. [16] found that serum VEGF was higher in systemic sclerosis patients versus healthy controls with lower VEGF levels in the serum of patients with evidence of ILD.
In this study, serum VEGF levels showed positive correlation with 6MWD, and a significant positive correlation with FVC %, but showed inverse correlation with percent desaturation during 6MWT, MMRC score, presence of pulmonary hypertension, tricuspid annular plane systolic excursion, and right-sided cardiac dilatation, and this was statistically insignificant.Saranya et al. [15] found that serum VEGF levels were inversely correlated with FVC and that there was a significant positive correlation with the MMRC dyspnea score.
Our results showed that there was no statistically significant relationship between serum VEGF levels and radiological findings, while De Santis et al. [16] reported that among systemic sclerosis cases, serum VEGF was directly correlated with ground glass and reticular pattern extent on HRCT, and Lv et al. [5] showed that VEGF levels were positively correlated with CTD-ILD severity by HRCT.
Limited exercise tolerance is a major symptom of ILD, resulting in reduced ability to perform daily activities and poor quality of life [17].Exercise-induced desaturation is an index of the severity of interstitial lung disease [18].The 6MWT is a simple test of exercise capacity that is commonly used to assess the functional status and follow-up treatment responses in ILD patients [19].
In our study, percent desaturation during 6MWT showed a significant inverse correlation with FVC% and a significant positive correlation with MMRC dyspnea score in both groups but showed a statistically insignificant relationship with radiological findings on HRCT in both groups.A relationship between desaturation during 6MWT and parameters of pulmonary function was confirmed by Rosa et al. [20] and Aktan et al. [21], while Seema et al. [22] found no statistically significant correlation between percent desaturation during 6MWT and  spirometry results in CTD-ILD patients.Villalba et al. [23] found that in patients with scleroderma-associated ILD, a statistical association was found between percent desaturation and dyspnea score, fibrosis on chest radiograph, FVC < 80% of the predicted value, and presence of ground glass or reticular opacities on HRCT.

Conclusion
From our results, we could conclude that serum VEGF levels were significantly higher in the HP group than the control group, but in CTD-ILD patients, the level was almost the same as control group.However, serum VEGF level could not be correlated to functional status or disease severity in both groups.

Table 1
Correlation between serum VEGF level, functional assessment parameters, and echocardiographic findings in the HP group

Table 2
Relationship between serum VEGF level and radiological findings in the HP group

Table 3
Correlation between percent desaturation during 6MWT and FVC% and MMRC score in the HP group

Table 4
Relationship between percent desaturation during 6MWT and radiological findings in the HP group

Table 5
Correlation between serum VEGF level, functional assessment parameters, and echocardiographic findings in the CTD-ILD group

Table 6
Relationship between serum VEGF level and radiological findings in the CTD-ILD group

Table 7
Correlation between percent desaturation during 6MWT and FVC% and MMRC score in the CTD-ILD group

Table 8
Relationship between percent desaturation during 6MWT and radiological findings in the CTD-ILD group

Table 9
Mean value of serum VEGF in study groups