In Situ Imaging of Tissue Remodeling with Collagen Hybridizing Peptides

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.

Divergent Matrix-Remodeling Strategies Distinguish Developmental from Neoplastic Mammary Epithelial Cell Invasion Programs

“…S2A; upper panels). As protease- dependent invasion programs would be predicted to correlate closely with ECM remodeling, the proteolysis of native collagen fibers was assessed in situ using carboxyfluorescein-labeled collagen hybridizing peptide (CF-CHP) that binds specifically to degraded collagen by folding into a triple helix (Hwang et al, 2017; Li et al, 2012). Importantly, labeling of primary MMTV-PyMT tumor tissue reveals dense arrays of remodeled collagen at the carcinoma-stromal tissue interface, but not at sites distant to the carcinoma cells (Fig.…”

Microplate assay for denatured collagen using collagen hybridizing peptides

“…This protocol will allow researchers to rapidly quantify the percentage of collagen denatured in connective tissues. Given the prevalence and role of denatured collagen in injuries and pathologies such as osteoarthritis, cancer, and fibrosis, 12 the method can help provide insight on the onset and progression of these ailments.…”

“…23 Healthy unloaded cartilage stained with F-CHP have revealed the presence of denatured collagen. 12 This suggests that CHPs are more sensitive for detecting denatured collagen compared to immunological methods, and are able to detect denatured collagen prior to degradation of the collagen matrix. 24 Overall, the CHP microplate assay is more versatile than existing methods of absolutely quantifying denatured collagen, while being more efficient.…”

“…12 Therefore, this assay could potentially be used with any type of collagenous tissue. When applied to mechanically damaged RTT fascicles of varying strains, no statistical difference was found between the values calculated from the calibration curve and the T-H assay (Fig.…”

“…10,11 By conjugating CHPs with fluorescent or bioactive moieties such as biotin, collagen that has become denatured due to injury or disease can be identified and visualized. 4,12 Previously in our lab, CHPs with a (GPO) 9 sequence bound to carboxyfluorescein (F-CHP) were used to stain rat tail tendon (RTT) fascicles after they were mechanically loaded. The fascicles were imaged using widefield fluorescence microscopy to measure and quantify fluorescence intensity and by extension collagen damage.…”