Use of fibronectin binding peptides to measure mechanical strain of fibronectin fibers in tissue
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Date
2017Type
- Doctoral Thesis
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Abstract
The extracellular matrix (ECM) plays an important role in tissue homeostasis and as mechanical anchorage for cells. Especially the ECM protein fibronectin (Fn) represents a crucial factor in early development of organisms, in wound healing, but also in several pathologies, such as cancer or fibrotic disorders. These pathologies have frequently been reported to go along with an increase in tissue stiffness, mediated in part by an enhanced expression of collagen-I, an enhanced enzymatic crosslinking of ECM residues, as well as an increased number of highly contractile, activated fibroblasts, so called myofibroblasts. However, despite growing knowledge on alterations in tissue mechanics in cancer, it remains unclear whether and how changed tissue stiffness can impact the strain state of protein fibrils and thus the display and accessibility of potential binding sites on these fibers. Given the knowledge of mechanically induced opening up of cryptic motifs and destruction of binding epitopes on Fn and the sheer abundance of binding sites for a variety of growth factors, cytokines, integrins and other matrix proteins, the question arises, to what extent and which of these binding sites are affected by a changed mechanical strain state of fibronectin protein fibers in tissues. However, to address this important question in a satisfactory way, methods and probes for the measurement of Fn fiber strain in tissues are missing,
as previously proposed methods are either only applicable in vitro, or have unknown binding mechanism and affinities.
Therefore, the aim here is to introduce and establish the Fn binding peptide FnBPA5, originating from bacterium S. aureus, as a novel strain probe to specifically bind to relaxed Fn fibers in tissue and in vivo to ultimately find correlations with functional aspects triggering Fn relaxation in different systems. Utilization of this novel strain probe enables a first of its kind assessment and comparison of the mechanical strain of Fn fibers in different tissue. First, the strain sensitive binding of FnBPA5 peptide is assessed via binding capability to relaxed or stretched manually pulled Fn fibers and comparison of FnBPA5 binding with Fn-FRET ratio in fibroblast cell culture matrices. In a second step FnBPA5 is then utilized as a strain probe to visualize Fn fiber strain in tissue sections and in vivo, to investigate the physiological strain state in organs, and assess alterations of this parameter in pathologic situations.
The herein presented data reveal, that FnBPA5 shows preferential binding to relaxed Fn fibers in an in vitro assay of manually pulled Fn fibers, as well as in comparison with the Fn-FRET probe in human dermal fibroblast cell culture experiments. FnBPA5 is found to exhibit high affinity binding to both plasma Fn and relaxed Fn fibers with a dissociation constant in the nanomolar range, similar to currently used antibodies against fibronectin, and has an adequate plasma stability for in vivo applications. Ex vivo tissue staining of tumor tissue with FnBPA5 shows that areas of relaxed Fn fibers, exhibiting high FnBPA5 binding to Fn, coincide with presence of mature collagen fibers and alpha-smooth muscle actin (a-SMA) expressing cells. In vivo experiments using 111In-radiolabeled FnBPA5 derivative reveals specific uptake in tumors and other organs with enhanced retention in tumors compared to other organs in a mouse prostate cancer (PC-3) xenograft tumor model.
Tissue stainings of PC-3 tumor tissue sections and sections from healthy organs reveal no binding to tissue sections from healthy organs, with binding of FnBPA5 only observable in tumor tissue sections, suggesting a highly stretched state of Fn fibers in healthy organs and relaxation of Fn fibers only occuring in tumor tissue. Costaining of FnBPA5 with cell contractility marker a-SMA reveals higher FnBPA5 binding to Fn in areas adjacent to a-SMA expressing cells, suggesting a relaxation of Fn in these areas, triggered by yet unknown mechanisms. A model of lymph node swelling using lymph nodes from mice injected with lymphocytic choriomeningitis virus (LCMV) or CpG oligodeoxynucleotide adjuvant compared to untreated lymph nodes only shows binding of FnBPA5 to lymph node tissue sections from LCMV infected mice, compared to the other two experimental groups leading to the notion that infection and partial destruction of lymph node fibroblasts as previously reported for LCMV is responsible for Fn relaxation, with physiological lymph node swelling, as observed for CpG, leaving Fn fiber strain unchanged.
It was further investigated whether different glycosaminoglycans (GAGs), such as heparin, clinically used low molecular weight heparin derivatives, as well as hyaluronic acid and synthetically sulfated hyaluronic acid, bind to and are capable of changing Fn conformation. Additionally, given the fact that one major binding site for GAGs on Fn is overlapping with the bacterial binding site also used by FnBPA5, competitive binding studies with different GAGs and FnBPA5 were carried out revealing only little potential of these GAGs to efficiently block FnBPA5 binding to manually pulled Fn fibers.
In summary, this thesis introduces a novel class of peptide probes, showcased here using the S. aureus Fn binding peptide FnBPA5, being capable of visualizing relaxed Fn fibers in tissue sections and in vivo. The usage of such probes could lead to a better understanding of the dynamic interplay of matrix composition, cellular forces and mechanical strain of Fn fibers and how this is altered in pathologies, such as cancer or fibrotic disorders. The herein presented characterization of an in vivo tumor model and ex vivo tissue stainings from cancer and lymph node model gives first evidence that the FnBPA5 peptide can serve as an important novel tool to characterize alterations in Fn fiber strain upon pathological changes in tissues. Ultimately, it can be envisioned that bacterial peptide probes, after careful trial experiments can be clinically exploited for diagnosis of pathologic tissue changes affecting Fn strain state, or even for targeting such changes in vivo. Show more
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https://doi.org/10.3929/ethz-b-000199166Publication status
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ETH ZurichOrganisational unit
03640 - Vogel, Viola / Vogel, Viola
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