Molecular probes for imaging tissue remodeling

The extracellular matrix (ECM) is a highly organized and complex network of glycoproteins and proteoglycans that surrounds all cells present in multicellular organisms. The ECM provides structural support for cells, but is also responsible for many cellular functions by means of cell-matrix interactions. Excessive remodeling is a characteristic feature of many disease processes, like atherosclerosis, angiogenesis and tumor metastasis. To obtain a more detailed understanding of these ECM related diseases, molecular imaging of the individual components of the ECM is required. Moreover, ECM imaging is also valuable for visualizing the structure and composition in tissue engineered constructs. This thesis describes a variety of strategies to obtain molecular probes that target structural components, but also dual-specific probes that combine collagen targeting and sensitivity for enzymes responsible for remodeling. Chapter 1 gives an overview of the structure and function of the ECM, remodeling during diseases and the currently available approaches to visualize ECM turnover. Since the development of some molecular probes that are described in this thesis is based on multivalency, this concept are explained and examples of synthetic multivalent peptide and protein constructs are discussed. Collagen is the most abundant protein present in the ECM. A significant part of the research performed focuses on collagen targeting using the collagen binding protein CNA35. Therefore the structures of collagen CNA35 are highlighted in this chapter as well as research that has been performed previously on collagen imaging. Finally, currently existing protease activatable probes and their limitations are discussed as an introduction to the work performed on the development of molecular probes that are sensitive to the enzymes responsible for collagen degradation during remodeling. Previously, a strategy was developed within our group to obtain specific high affinity probes for ECM proteins. Phage display was used to identify peptide binders for collagen type I and V. Subsequently, the multivalent architecture of the phages was translated into multivalent display of the selected peptides on a dendritic wedge. The resulting multivalent structures showed an impressive increase in target affinity compared to the monovalent selected peptides. In Chapter 2, this strategy to find specific probes for ECM proteins was tested for its general applicability. Phage display was used to find specific peptide binders for elastin, fibronectin, collagen type III, collagen type IV and fibrinogen. The selection procedure resulted in the identification of target binding peptides for fibronectin (GETRAPL) and fibrinogen (GPRP-containing sequences). No peptide binders were found for elastin, collagen type III and collagen type V, showing that phage display is certainly not a method that is successful in finding target binders for every ECM protein. Multivalent display of the peptides selected for fibronectin and fibrinogen on a dendritic wedge was shown to give a significant increase in affinity for the target protein, suggesting that multivalent interactions are a prerequisite to select target binding peptides using phage display. The pentavalent fibronectin binding peptide wedge was used in ex vivo imaging experiments to explore whether specific fibronectin structures could be stained in sections of human kidney. Unfortunately, the overall affinity of the dendritic imaging probe was probably too low to specifically visualize fibronectin, since background binding to epithelial cells was observed. This experiment demonstrates that the applicability of the dendritic structures resulting from multimerization of selected target binding peptides remains to be proven in each specific case. Having demonstrated the potential of multivalent peptide ligands to target ECM proteins with a significantly higher affinity than monovalent peptides, this concept was applied to proteins by multimerization of the collagen binding protein CNA35, as described in Chapter 3. Well-defined protein dendrimers were generated to increase the collagen binding affinity by coupling of CNA35 to divalent and tetravalent dendritic wedges using native chemical ligation. In addition, multivalent structures of a weakly binding variant of CNA35, CNA35(Y175K), were prepared. The affinities of the multivalent protein constructs were evaluated in surface plasmon resonance (SPR) experiments using chip surfaces immobilized with collagen, but also chip surfaces immobilized with a small, synthetic collagen mimic to be able to vary the target density. The dissociation kinetics of the multivalent CNA35 dendrimers was found to be strongly attenuated. Multimerization of the weakly binding CNA35(Y175K) resulted in an impressive increase in collagen affinity. The binding studies indicated that this restoration of the collagen binding capacity requires at least three simultaneous interactions, illustrating the strength of multiple weak interactions. Use of the collagen targeting ligands described in Chapter 3 is an effective approach to target structural ECM features with a high affinity. For imaging of ECM turnover enzymatic activity is an even more distinctive marker. Matrix metalloproteinases (MMPs) are the enzymes that are mainly responsible for the collagen degradation and MMP levels are often elevated in diseased tissue. Chapter 4 focuses on the development of a dual-specific MMP sensitive collagen probe based on the collagen binding protein CNA35. Binding of CNA35 to mature collagen was topologically inhibited by cyclization of the protein via an MMP cleavage site that prevents it from wrapping around the collagen triple helices. SPR binding experiments as well as tissue imaging experiments showed that cyclization of CNA35 resulted in a significant reduction in binding to native collagen. SPR experiments using a chip surface immobilized with a small, synthetic collagen mimic showed a collagen affinity for cyclic CNA35 (cCNA35) that is comparable to wt CNA35, indicating that the decreased affinity was due to topological inhibition and not a result of protein misfolding. The collagen binding capacity could be restored by the presence of MMP-1. Cyclization of CNA35 as a strategy to obtain an MMP activatable ligand was based on the specific binding mechanism of CNA35. In Chapter 5 a more general approach is described for the generation of dual-specific imaging probes, which is relying on intramolecular blocking of the target binding protein. A hybrid of CNA35 and a synthetic collagen mimic, connected via an MMP recognition site, was synthesized. CNA35 with a C-terminal thioester was generated and subsequently coupled to a trivalent wedge using native chemical ligation. The three cysteine end groups of the wedge were then functionalized with (GPO)5 using oxime chemistry for the formation of a templated triple-helical collagen peptide. Evaluation of the binding characteristics of the blocked CNA35 construct using SPR showed a 5-fold increase of the collagen binding capacity upon MMP-1 digestion. Activation of the CNA35 sensor could be visualized in a co-staining experiment on pig pericardial tissue using wild type CNA35 as a reference.