The preferential homing of a platelet derived growth factor receptor-recognizing macromolecule to fibroblast-like cells in fibrotic tissue

Abstract

Platelet derived growth factor (PDGF) is a key factor in the induction and progression of fibrotic diseases with the activated fibroblast as its target cell. Drug targeting to the PDGF-receptor is explored as a new approach to treat this disease. Therefore, we constructed a macromolecule with affinity for the PDGF-β receptor by modification of albumin with a small peptide that recognises this PDGF-β receptor. The binding of the peptide-modified albumin (pPB-HSA) to the PDGF-β receptor was confirmed in competition studies with PDGF-BB using NIH/3T3-fibroblasts and activated hepatic stellate cells. Furthermore, pPB-HSA was able to reduce PDGF-BB-induced fibroblast proliferation in vitro, and proved to be devoid of proliferation-inducing activity itself. We assessed the distribution of pPB-HSA in vivo in two models of fibrosis and related the distribution of pPB-HSA to PDGF-β receptor density. In rats with liver fibrosis (bile duct ligation model), pPB-HSA quickly accumulated in the liver in contrast to unmodified HSA (P<0.001). The major part of pPB-HSA in the fibrotic liver was localized in hepatic stellate cells. In rats with renal fibrosis (anti-Thy1.1 model), pPB-HSA also homed to the cells that expressed the PDGF-β receptor, i.e. the mesangial cells in the glomeruli of the kidney. These results indicate that pPB-HSA may be applied as a macromolecular drug-carrier that accumulates specifically in cells expressing the PDGF-β receptor, thus allowing a selective delivery of anti-fibrotic agents to these cells.

Introduction

Fibrotic diseases can occur in many organs of the body and in all cases this disease is associated with a gradual loss of tissue architecture and normal cell activities, leading to end-stage organ dysfunction [1]. In general, no pharmacotherapeutical intervention is available to treat fibrotic processes, which warrants the search for effective anti-fibrotic drugs. The main features of fibrosis are proliferation of fibroblast-like cells and excessive deposition of extracellular matrix products. The application of many anti-fibrotic drugs is hampered by the fact that they do not accumulate sufficiently into the target cells or cause to many side effects elsewhere in the body. In particular fibrosis, which is part of the normal tissue rearrangement process in all organs, calls for a cell-specific delivery of drugs.

A prominent stimulator of fibroblast proliferation during fibrosis is PDGF [1]. PDGF is a dimeric molecule composed of A and/or B chains. The PDGF isoforms, -AA, -AB, or -BB, exert their effects on target cells by binding to two structurally different receptors. The PDGF-receptor α binds to both A- and B-chains with high affinity, whereas PDGF-receptor β only binds the B-chain [2], [3], [4]. Recently, two other isoforms were identified, i.e. PDGF-C and PDGF-D [5], [6], [7], [8]. Both forms require proteolytic activation before binding to and activation of, respectively, PDGF-α or -β receptors.

In liver fibrosis, PDGF is the most important mediator involved in the proliferation and activation of HSC, which transform subsequently into myofibroblasts. The HSC, being the major producers of extracellular matrix constituents, are crucial in the pathogenesis of liver fibrosis [9]. The local production of PDGF is increased during fibrosis, and the de novo expression of the PDGF-β receptor is strongly enhanced on activated HSC [10], [11], [12]. Similarly, proliferation of fibroblast-like cells also occurs in renal fibrosis (glomerulosclerosis or tubulointerstitial fibrosis). Again, the enhanced expression of PDGF receptors on the fibroblast-like cells (mesangial cells and interstitial fibroblasts) as well as an increased production of PDGF plays a key role in the development of fibrotic processes in the kidney [13], [14], [15], [16]. Therefore, HSC and mesangial cells are the major target cells for anti-fibrotic therapies in the liver and in the kidney, respectively.

The general event of the induction of PDGF-β receptors on cell membranes of fibroblasts after the onset of fibrosis prompted us to examine the possibility of developing a drug carrier that distributes to this receptor in order to obtain homing to the target cells in fibrotic tissues. This strategy finally aims at an improvement of pharmacological effects of drugs to treat fibrotic and sclerotic diseases and simultaneously decrease adverse effects of the chronically applied drugs. To create a drug carrier that specifically distributes to fibrogenic cells, we coupled a PDGF receptor-recognizing peptide to albumin. This novel peptide (C^∗SRNLIDC^∗) contains the amino acids arginine (R) and isoleucine (I), as being the receptor-binding moieties of the PDGF B-chain [17], together with the amino acids flanking these moieties in the native growth factor. Cyclization of the peptide, obtained by cys–cys interaction, was employed based on observations that cyclic peptides display stronger binding to their receptor than the linear analogues [18], [19]. The use of small peptides as specific receptor ligands has been described amongst others for targeting to integrin receptors [18], [20], [21]. This study is the first to describe a PDGF receptor-recognizing peptide, and its application for targeting purposes. We studied the binding of albumin modified with these PDGF receptor-recognizing peptides (pPB-HSA) to the PDGF receptor on fibroblasts in vitro, and in vivo in a rat model of liver fibrosis as well as in a model of renal fibrosis.