Characterisation and application of antibodies specific for the long platelet-derived growth factor A and B chains

Abstract

The platelet-derived growth factor (PDGF) family comprises important mitogens for mesenchymal cells. The active dimeric form of PDGF consists of four structurally related A, B, C, and D chains. All PDGF-variants bind to PDGF-receptors. The A and B chains occur with and without basic C-terminal amino acid extensions as long (A_L and B_L) and short (A_S and B_S) isoforms. PDGF-A and -B form homo- or heterodimers. The biological relevance of short and long isoforms is unknown, although it may relate to different affinities for glycosaminoglycans of the cell glycocalix and intercellular matrix. Commercially available anti-PDGF-A and anti-PDGF-B antibodies cannot discriminate between the short and the long isoforms. Thus, to investigate the function of the long and short isoforms, we raised antibodies specific for the long A and B chain isoforms. The antibodies were affinity-purified and their properties analysed by surface plasmon resonance. Inhibition studies with different PDGF homodimers and dot–blot studies proved their high specificity for the respective isoforms. Both antibodies recognised the target PDGF homodimers complexed to the glycocalix of human arterial smooth muscle cells and human monocyte-derived macrophages. By using these specific antibodies, we were able to confirm at the protein level the synthesis of PDGF-A and -B during differentiation of human monocyte-derived macrophages and to demonstrate the presence of the PDGF-A_L and PDGF-B_L isoforms in human arterial tissue.

Introduction

The platelet-derived growth factor (PDGF) family is an important group of mitogens and chemoattractants for mesenchymal cells. PDGF is involved in normal embryonic development, wound healing, development of cancer, and in cardiovascular disease (Heldin, Eriksson, & Östman, 2002; Yu, Ustach, & Kim, 2003). Until recently, two homologous PDGF chains encoded by different genes, PDGF-A and PDGF-B, were identified (Betsholtz et al., 1986; DallaFavera, Gallo, Ciallongo, & Croce, 1982; Swan et al., 1982). The mature PDGF, homo- (PDGF-AA and PDGF-BB) or heterodimers (PDGF-AB) of these chains, signal through distinct PDGF-α or/and -β receptors (Heidaran et al., 1993; Heldin & Westermark, 1989; Krettek, Fager, Jernberg, Östergren-Lundén, & Lustig, 1997a).

Due to alternative splicing of exons 6 and 7, there are two isoforms of the PDGF-A chain (Betsholtz et al., 1986). The longer A-chain contains a basic amino acid C-terminal extension with a high proportion of lysines and arginines. The PDGF-B chain is produced and secreted only as the long isoform, and contains a basic C-terminal sequence similar to but distinct from the long A-chain (Heldin, 1992). This basic extension is subsequently proteolytically cleaved to produce the mature short PDGF-B chain (Heldin, 1992). Recently, two new PDGFs, PDGF-C (Li et al., 2000) and PDGF-D (Uutela et al., 2001), which bind to the PDGF-receptors, were identified. PDGF-C and -D lack basic C-terminal extensions and presumably do not interact with GAGs. The biological role of these new PDGFs is still unclear.

The expression of PDGF-A and -B isoforms in normal cells is regulated in response to various exogenous stimuli. In a number of transformed cell lines, PDGF is constitutively expressed causing auto- or paracrine stimulation. While PDGF production is essential in normal embryonic development (Levéen et al., 1994; Lindahl et al., 1997) and wound healing (Pierce et al., 1995), it may contribute to uncontrolled proliferation of tumour cells in cancer or of arterial smooth muscle cells in atherosclerotic cardiovascular disease. The mitogenic effect of PDGF may be attenuated by its interaction with glycosaminoglycans (GAGs) in the cell glycocalix and intercellular matrix. Indeed, it has been shown that the PDGF isoforms bind to heparin-like GAGs (Andersson, Östman, Westermark, & Heldin, 1994; Fager et al., 1995; Feyzi et al., 1997; Lustig et al., 1996, Lustig et al., 1999; Raines & Ross, 1992) and that this binding inhibits the mitogenic properties of PDGF. Moreover, the affinities of various PDGF isoforms for GAGs (heparan sulphate, dermatan sulphate, chondroitin sulphate, or heparin) are strikingly different (Feyzi et al., 1997, Garcia-Olivas et al., 2003, Lustig et al., 1996, Lustig et al., 1999).

Specific antibodies for isoforms of the PDGF-A and B-chains would facilitate the study of interactions between PDGF, cells and intercellular matrix components in situ. Commercially available antibodies against PDGF-A and -B chains are unable to discriminate between the short and the long isoforms. In this study, we describe the production of such antibodies using peptides corresponding to the unique basic C-terminal extensions of long PDGF-A and -B chain isoforms as immunogens. These antibodies were characterised using surface plasmon resonance. Their use as monospecific reagents was assessed by dot–blot analysis as well as by in situ staining of exogenously added PDGF complexed to the glycocalix of smooth muscle cells and endogenously secreted PDGF isoforms of both arterial smooth muscle cells and monocyte-derived macrophages during differentiation. Antibodies were also used to detect the long PDGF-isoforms in human arterial tissue.