In the context of fibrosis development in conventional cancer therapy, including radio- and chemotherapy, we studied here the effects of SU9518, a receptor tyrosine kinase inhibitor of PDGF signaling on human primary dermal fibroblasts and endothelial cells. We had previously shown that using this class of molecular targeted compounds – including SU9518 and Imatinib (Gleevec) as well as SU11657 (similar to SU11248, a compound currently in several clinical phase I and II anticancer trials) – can attenuate ionizing radiation-induced lung fibrosis in vivo if it is given either prior to or after radiation insult . Here we investigate in vitro whether primary human endothelial and fibroblast cells are an important primary target of the RTK inhibitor in the context of PDGF signaling. We found that, in vitro, irradiation of fibroblasts and endothelium induced autocrine and paracrine PDGF expression. Functionally, the isolated irradiation of primary human endothelial cells stimulated proliferation in human fibroblasts in a co-culture model. Thus irradiation has effects which can be described as an indirect promotion of fibroblast survival and proliferation. Importantly, RTK inhibitors could effectively attenuate this paracrine radiation-induced activation by blocking autophosphorylation of PDGFR and by downregulation of PDGFR expression. Since fibroblasts are most likely key players in radiotherapy related fibrosis, and given the close correlation between PDGF signaling and fibroblasts activity (e.g. survival and proliferation), along with results in vivo  we conclude that fibroblasts may as well be a target of RTK inhibitors in the process of attenuating radiation-induced fibrogenesis in vivo.
This conclusion is in agreement with earlier reports showing that PDGF is a potent mitogen and chemoattractant for mesenchymal cells and also a chemoattractant for neutrophils and monocytes [16–18]. Similarly, several studies had reported that PDGF isoforms and their receptors were upregulated during pathologic fibroproliferative diseases in humans and in murine models by different types of impairment (e.g. radiation- [19, 20]/bleomycin-  induced fibrosis, idiopathic pulmonary fibrosis ) vindicating the importance of the PDGF/PDGFR system in proliferative diseases.
PDGFR-β has been suggested to be predominantly expressed in fibroblasts  in agreement with our data presented here. In addition, we found that PDGFR-β was more upregulated after radiation in fibroblasts compared with PDGFR-α in both western blotting and immunocytochemistry assays. Others had shown that fibroblasts isolated from rat lungs over expressed PDGFR-β with higher chemotactic response to PDGF-BB in vivo . Although the ultimate mechanism of PDGFR overexpression remains unclear, we found ionizing radiation can upregulate PDGFR in fibroblasts. More importantly, for the ultimate goal of treating or preventing fibrosis in patients, we found that SU9518 attenuated the radiation-induced upregulation of PDGFR.
The best characterized mechanisms by which PDGF down-streaming signaling mediates cellular responses involve the activation of the ras/MAPK pathway, which can functionally increase cellular proliferation, and the PI3k/Akt pathway, which promotes cell survival in general tumor biology . PDGF has also been shown to stimulate mitogenicity and chemotaxis of fibroblasts and smooth muscle cells . In our fibroblast clonogenic and proliferation assays, inhibition of PDGF signaling by SU9518 significantly reduced the clonogenic survival fraction and the proliferation of fibroblasts, thus supporting the important role of PDGF signaling for fibroblasts survival. Moreover, PDGF has been shown to stimulate the production of several matrix molecules, such as collagen, fibronectin, and proteoglycan [23–25]. The resulting close correlation between PDGF/PDGFR and fibroblasts may explain the inhibitory effect of SU9518 to radiation-induced fibrosis .
To further evaluate the specificity of SU9518, its ability to inhibit cell proliferation stimulated by PDGF (-AB isoform) or combined VEGF and bFGF was measured, because these growth factors have been shown to induce similar signaling pathways, changes in gene expression, and cellular responses in mesenchymal cells .
Unresolved issues in fibrosis research inlclude the relative contribution of different cell types to the fibrotic development and the usefulness of acute anti-inflammatory drugs such as corticosteroids. A Cochrane review of corticosteroids in idiopathic lung fibrosis found no evidence that steroid treatment was of any benefit, in spite of repression of early inflammation . Savikko et al demonstrated that cyclosporin A treatment cannot inhibit the expression of PDGF ligands and receptors, although it ameliorated the extent of inflammation . This suggests the involvement of other cells besides acute inflammatory cells in the development of fibrosis. In vivo in lungs, the radiation-induced fibroproliferative process occurs within the microenvironment of the distal gas exchange units consisting of fibroblasts, epithelial cells [29, 30] and endothelial cells . In agreement with our co-culture results, endothelial cells are described as potential sources of PDGF after radiation . Epithelial cells do not express PDGF and PDGFR under physiological conditions. But expression of c-sis mRNA in epithelial cells has been reported in certain pathologic fibrotic states, such as in patients with idiopathic lung fibrosis .
SU9518 exerts its inhibitory effect by ATP-competitive inhibition of the catalytic activity of the tyrosine kinase after PDGFR activation . Under normal conditions PDGF-AA induces α/α receptor dimers, PDGF-AB induces α/α and α/β receptor dimers and PDGF-BB induces all three receptor dimer combinations. Since SU9518 inhibited fibroblast proliferation activated by all three isoforms, it is conceivable that SU9518 can inhibit both PDGFR-α and -β.
Despite many investigations, the relative roles of most cytokines/growth factors in fibroproliferative diseases are not completely defined. For some of the putative key players such as TGF-β, IL-1, TNF-α and thrombin it has been reported that they may exert their profibrotic activities through a PDGF-dependent pattern [33–36]. One should keep in mind however that in the case of ionizing radiation the signaling cascades in cells are complex and the resulting regulations may well be described as a network of signaling [36–40]. Nevertheless, PDGF may be an important node in this network and thus a key cytokine responsible for the fibrotic development, suggesting that perturbation of the PDGF/PDGFR system could provide an effective means of inhibiting fibrosis.