The roles of platelet-derived growth factors and their receptors in brain radiation necrosis

Abstract

Background: Brain radiation necrosis (RN) occurring after radiotherapy is a serious complication. We and others have performed several treatments for RN, using anticoagulants, corticosteroids, surgical resection and bevacizumab. However, the mechanisms underlying RN have not yet been completely elucidated. For more than a decade, platelet-derived growth factors (PDGFs) and their receptors (PDGFRs) have been extensively studied in many biological processes. These proteins influence a wide range of biological responses and participate in many normal and pathological conditions. In this study, we demonstrated that PDGF isoforms (PDGF-A, B, C, and D) and PDGFRs (PDGFR-α and β) are involved in the pathogenesis of human brain RN. We speculated on their roles, with a focus on their potential involvement in angiogenesis and inflammation in RN.

Methods: Seven surgical specimens of RN, obtained from 2006 to 2013 at our department, were subjected to histopathological analyses and stained with hematoxylin and eosin. We qualitatively analyzed the protein expression of each isoform of PDGF by immunohistochemistry. We also examined their expression with double immunofluorescence.

Results: All PDGFs were expressed in macrophages, microglia, and endothelial cells in the boundary of the core of RN, namely, the perinecrotic area (PN), as well as in undamaged brain tissue (UB). PDGF-C, D and PDGFR-α were also expressed in reactive astrocytes in PN. PDGFs and PDGFR-α were scarcely detected in UB, but PDGFR-β was specifically expressed in endothelial cells not only in PN but also in UB.

Conclusions: PDGFs/PDGFRs play critical roles in angiogenesis and possibly in inflammation, and they contribute to the pathogenesis of RN, irrespective of the original tumor pathology and applied radiation modality. Treatments for the inhibition of PDGF-C, PDGF-D, and PDGFR-α may provide new approaches for the treatment of RN induced by common radiation therapies.

Figure 1

Results of hematoxylin and eosin staining (H&E) and immunohistochemistry from case 1. H&E staining (A) revealed a necrotic core (NC) and perinecrotic area (PN), including micro bleeding (A, arrowhead) and abnormal angiogenesis (A, arrow). Immunostaining results for PDGF-C are presented as a representative example (B). PDGF-C (C and D), D (E and F) and PDGFR-α (G) were produced by monocytic cells (C, E, G, arrow) and reactive astrocytic cells (D, F, G, arrowhead) in PN. On the other hand, PDGFR-β (H and I) was expressed mainly in endothelial cells (H and I*). There was partially nonspecific staining in NC (B) or around blood vessels (I). Original magnification, A, B and H × 40, C, D, E, F, G and I × 200.

Figure 2

Representative results of immunostaining of undamaged brain tissue (UB). PDGF-A, B, C, D and PDGFR-α were scarcely detectable in UB (A through E). PDGFR-β (F) was specifically expressed in endothelial cells in UB. Many normal cerebral blood vessels stained with PDGFR-β (F *) were detected in UB. Original magnification, ×200.

Figure 3

Frequency of expression. We assessed the frequency of expression of PDGFs semi-quantitatively by the following method. Five fields of each PDGF isoform, in which abnormal angiogenesis were detected, were randomly selected with a microscope. The PDGF-positive mononuclear cells were counted. We observed all 7 cases and performed the counting using two observers to reduce bias. One observer, who was blind to the patients’ clinical and pathological information, evaluated the results of the immunohistochemical staining. The ratios of PDGF-positive cells to total cells in each field were calculated and were statistically analyzed using Steel-Dwass tests with JMP Pro 10 (SAS Institute Inc., Cary, NC, USA). Statistical analysis revealed that PDGF-C and D showed higher frequency of expression in the PN specimens than did PDGF-A and B. The difference was statistically significant (*p < 0.0001, Steel-Dwass test).

Figure 4

Double immunofluorescence staining. The results of double immunofluorescence staining from case 1 revealed that PDGF-C or D-positive cells were merged with many CD68 (A, E), GFAP (B, F), hGLUT5 (C, G), and CD45 (D, H) -positive cells in PN. Some PDGF-C or D-positive cells did not express CD68, GFAP, hGLUT5 or CD45 and vice versa. Endothelial cells (*) were nonspecifically stained with secondary fluorescence antibody. The scale bar represents 50 μm.

Figure 5

Double immunofluorescence staining. Double immunofluorescence staining from case 1 revealed that PDGFR-α and β were strongly expressed in CD31-positive cells in PN (D and I). PDGFR-α positive cells were merged with many cells positive for CD68 (A), GFAP (B), hGLUT5 (C), and CD45 (E). PDGFR-β-positive cells merged specifically with endothelial cells (F, G, H, I and J, *). Endothelial cells (*) were nonspecifically stained with secondary fluorescence antibody. The scale bar represents 50 μm.