Expression of hypoxia-inducible factor (HIF)-1a-vascular endothelial growth factor (VEGF)-inhibitory growth factor (ING)-4- axis in sarcoidosis patients

Abstract

Background: Sarcoidosis is a granulomatous disorder of unknown etiology. The term of immunoangiostasis has been addressed by various studies as potentially involved in the disease pathogenesis. The aim of the study was to investigate the expression of the master regulator of angiogenesis hypoxia inducible factor (HIF)-1a - vascular endothelial growth factor (VEGF)- inhibitor of growth factor 4-(ING4) - axis within sarcoid granuloma.

Methods: A total of 37 patients with sarcoidosis stages II-III were recruited in our study. Tissue microarray technology coupled with immunohistochemistry analysis were applied to video-assisted thoracoscopic surgery (VATS) lung biopsy samples collected from 37 sarcoidosis patients and 24 controls underwent surgery for benign lesions of the lung. Computerized image analysis was used to quantify immunohistochemistry results. qRT-PCR was used to assess HIF-1a and ING4 expression in 10 sarcoidosis mediastinal lymph node and 10 control lung samples.

Results: HIF-1a and VEGF-ING4 expression, both in protein and mRNA level, was found to be downregulated and upregulated, respectively, in sarcoidosis samples compared to controls. Immunohistochemistry coupled with computerized image analysis revealed minimal expression of HIF-1a within sarcoid granulomas whereas an abundant staining of ING4 and VEGF in epithelioid cells was also visualized.

Conclusions: Our data suggest an impairment of the HIF-1a - VEGF axis, potentialy arising by ING4 overexpression and ultimately resulting in angiostasis and monocyte recruitment within granulomas. The concept of immunoangiostasis as a possible protection mechanism against antigens of infectious origin needs further research to be verified.

Figure 1

HIF-1a, Vegf and Ing4 mRNA expression levels in sarcoidosis lung samples. Hif-1a (A), Vegf (B) and Ing4 (C) gene expression levels quantified by qRT-PCR showed a statistically significant incline and decline in sarcoidosis lung tissue samples (n=10) compared to control lung samples (n=10). All values were normalized with the reference gene B2m. *p < 0.05.

Figure 2

HIF-1a immunohistochemical staining in sarcoidosis lung samples. Representative tissue microarray section immunostained with monoclonal antibody HIF-1a demonstrating diffuse cytoplasmic reaction of weak intensity in epithelioid cells within granulomas (arrows) derived from sarcoidosis patients (n=37) compared to control lung samples (n=24) (E). Scale bars in panel A: 100μm, B and C: 25 μm and D, E: 10 μm. Immunohistochemical findings were confirmed by computerized image analysis (F).

Figure 3

VEGF immunohistochemical staining in sarcoidosis lung samples. Representative immunohistochemically stained tissue microarray section with monoclonal antibody against VEGF demonstrates diffuse cytoplasmic stain of moderately strong intensity in epithelioid cells within granulomas (arrows) derived from sarcoidosis patients (n=37) compared to control lung samples (n=24). Scale bars in panel A: 100μm, B and C: 25 μm and D, E: 10 μm. Immunohistochemical findings were confirmed by computerized image analysis (F).

Figure 4

ING4 immunohistochemical staining in sarcoidosis lung samples. Representative immunohistochemical staining with monoclonal antibody against ING4 shows positively stained epithelioid cells within within granulomas (arrows) derived from sarcoidosis patients (n=37) compared to control lung samples (n=24). Scale bars in panel A: 100μm, B and C: 25 μm and D, E: 10 μm. Immunohistochemical findings were confirmed by computerized image analysis (F).