Abstract
Collagen, the major structural component of nearly all mammalian tissues, undergoes extensive proteolytic remodeling during developmental states and a variety of life-threatening diseases such as cancer, myocardial infarction, and fibrosis. While degraded collagen could be an important marker of tissue damage, it is difficult to detect and target using conventional tools. Here, we show that a designed peptide (collagen hybridizing peptide: CHP), which specifically hybridizes to the degraded, unfolded collagen chains, can be used to image degraded collagen and inform tissue remodeling activity in various tissues: labeled with 5-carboxyfluorescein and biotin, CHPs enabled direct localization and quantification of collagen degradation in isolated tissues within pathologic states ranging from osteoarthritis and myocardial infarction to glomerulonephritis and pulmonary fibrosis, as well as in normal tissues during developmental programs associated with embryonic bone formation and skin aging. The results indicate the general correlation between the level of collagen remodeling and the amount of denatured collagen in tissue and show that the CHP probes can be used across species and collagen types, providing a versatile tool for not only pathology and developmental biology research but also histology-based disease diagnosis, staging, and therapeutic screening. This study lays the foundation for further testing CHP as a targeting moiety for theranostic delivery in various animal models.
Highlights
While collagen synthesis and degradation are delicately coordinated during tissue development and homeostasis, excessive collagen remodeling has been implicated in numerous pathologic states.[3−5] For example, collagen degradation mediated by matrix metalloproteinases (MMPs) is responsible for cancer progression and metastasis.[6−8] In atherosclerosis, the thinning and weakening of the fibrous collagen cap by enzymatic degradation renders atherosclerotic plaques susceptible to rupture, resulting in myocardial infarction and sudden cardiac death.[9]
It remained to be shown that the presence of degraded collagen is a hallmark feature shared by almost every tissue type and numerous pathologic conditions beyond cancer and Marfan’s syndrome.[3−5] In this study, we used fluorescence microscopy to evaluate the binding of collagen hybridizing peptide (CHP) labeled with 5-carboxyfluorescein (5-FAM) or biotin on a series of animal and human tissue samples that have undergone remodeling during representative pathologic and physiologic events
We selected the following tissue samples to investigate the specifics of CHP binding because they represent a full spectrum of known scenarios involving extracellular matrix (ECM) remodeling and collagen degradation,[3] ranging from tissue degeneration, acute inflammation, and fibrotic remodeling to tissue development and aging
Summary
Collagen is the major structural component of the extracellular matrix (ECM) present in virtually all mammalian tissue and organs,[1] with an essential role in supporting cell attachment, proliferation, migration, and differentiation.[1,2] While collagen synthesis and degradation are delicately coordinated during tissue development and homeostasis, excessive collagen remodeling has been implicated in numerous pathologic states.[3−5] For example, collagen degradation mediated by matrix metalloproteinases (MMPs) is responsible for cancer progression and metastasis.[6−8] In atherosclerosis, the thinning and weakening of the fibrous collagen cap by enzymatic degradation renders atherosclerotic plaques susceptible to rupture, resulting in myocardial infarction and sudden cardiac death.[9]. Antibodies with higher specificity have been developed to recognize degraded collagen as a consequence of the generation of new N- or C-terminal epitopes or as soluble collagen fragments, but only for a handful of the 28 mammalian collagen subtypes.[14−19] More importantly, these antibodies fail to detect collagen fragments in tissues if specific epitopes are lost following more extensive proteolysis Microscopic methods, such as second-harmonic generation (SHG) and transmission electron microscopy (TEM), have been used to visualize fibrillar collagen[20] where a reduction of SHG signal[21] or the disruption of the periodic D-banding pattern in TEM22,23 can indicate alteration of collagen structure. Given the wide distribution of collagen in mammalian tissues,[1] our results suggest that CHP represents a versatile, but simple staining tool for monitoring many, if not all, remodeling events in isolated tissues and a potential delivery vehicle for targeting a wide range of tissues damaged by disease or injury
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