The preferential homing of a platelet derived growth factor receptor-recognizing macromolecule to fibroblast-like cells in fibrotic tissue
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.
Section snippets
Synthesis and characterization of pPB-HSA
The cyclic peptide C∗SRNLIDC∗ was prepared by Ansynth Service BV and covalently coupled to HSA (obtained from Sanquin, Central Laboratory of Blood Transfusion Services), as described previously [21]. pPB-HSA was characterized with polyacrylamide gel electrophoresis (7.5%), fast protein liquid chromatography (Superdex-200 column (Pharmacia)), and circular dichroism [21].
Characterization of pPB-HSA
The cyclic peptides C∗SRNLIDC∗ were prepared and coupled to HSA. The molecular weight of pPB-HSA was increased as compared to HSA, as detected with polyacryl amide gel electrophoresis. The increase in molecular weight indicated that pPB-HSA consisted of HSA substituted with a mean of 15 peptide groups. Additionally, electrophoretic and chromatographic analysis revealed that the major part of the pPB-HSA preparation consisted of monomeric protein. Furthermore, using circular dichroism analysis,
Discussion
A new approach to treat fibrotic diseases is proposed in the present study, that is, drug targeting to the proliferating fibroblast-like cells. A common feature in fibrotic processes is the upregulation of the PDGF-β receptor on fibroblasts. Therefore, we decided to design a drug carrier directed to this particular receptor. Although PDGF itself may display the most optimal interaction with the receptor, the use of this growth factor as a carrier molecule is not feasible because of its
Acknowledgements
This study was supported by a grant from the Foundation of Technical Sciences (STW), which is part of the Dutch organization for Scientific Research (NWO) (Grant: GFA-33.3072). We thank G. Molema for her valuable scientific discussions and in particular for the participation in the []-thymidine incorporation studies (performed by I. Veen-Hof, both Department of Pathology and Laboratory Medicine, Med. Biology Section, Tumor Immunology Lab., Groningen, The Netherlands). Furthermore, we thank
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