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.

Keywords: bone formation; fibrosis; inflammation; matrix metalloproteinase; targeted delivery; triple helix.

Figure 1

Schematic of a CHP strand (labeled with X) hybridizing to denatured collagen chains and forming a collagen triple helix. During disease progression, tissue development, or aging, collagen can be extensively degraded by collagenolytic proteases, causing its triple helix to unfold at the physiological temperature due to reduced thermal stability. X represents the biotin or fluorescent tag.

Figure 2

Osteoarthritis. Micrographs of articular cartilage tissue within the osteoarthritic or normal region from an OA patient. Sections from the same tissue samples were stained with Safranin O/fast green (A) or with F-CHP (B). Images of the Safranin O staining (A) were provided by the tissue supplier with permission (Copyright OriGene Technologies). In panel (B), Hoechst 33342-stained cell nuclei are shown in blue, and each image is representative of similar results from three stained sections. Scale bars: 500 μm.

Figure 3

Myocardial infarction. (A) Representative fluorescence scans of axial cross sections of the mouse hearts before (normal) or at three time points after myocardial infarction (MI), stained with Hoechst 33342 (blue) and B-CHP (detected with AlexaFluor647-streptavidin, red-yellow). (B) Digitally quantified total CHP fluorescence signals in the whole-section scans of the three hearts in each group are shown (a.u. = arbitrary unit). The means of the paired groups labeled under the graph are significantly different (one-way ANOVA with post hoc Tukey HSD test, P < 0.05). (C) Fluorescence scans of a heart harvested 7 days after MI show close spatial similarity between signals from B-CHP and macrophages as detected with an anti-CD68 antibody. (D) A magnified view of the infarcted region within the red box in panel (C) with merged fluorescence signals shows that high level of degraded collagen is in areas crowded with macrophages. Scale bars: 1 mm (A, C), 50 μm (D).

Figure 4

Glomerulonephritis. Representative micrographs of kidney cryosections from anti-Thy-1 nephritic and normal control rats stained with PAS (A) or F-CHP and an anti-collagen IV antibody (B). Images are representative of similar results from three animals within each group, three stained sections per animal. Scale bar: 100 μm.

Figure 5

Pulmonary fibrosis. (A) Representative fluorescence micrographs of the central and subpleural areas of lung cryosections obtained from mice dosed with bleomycin through minipumps for varying time periods versus control mice dosed with PBS for 1 week, and stained with F-CHP and Hoechst 33342. Selected micrographs are representative of images collected from 3 animals per group. (B) Quantified signals showing the time course and spatial difference of F-CHP signal levels. Numbers are presented as the mean + standard error and analyzed using one-way ANOVA with post hoc Tukey HSD test. * indicates significant difference in means (P < 0.05). (C) Micrographs showing multiple views of lung tissue harvested from mice treated with bleomycin for 3 weeks. The tissues were double stained with B-CHP and an anti-MMP2 antibody, which were visualized using AlexaFluor647-labeled streptavidin and AlexaFluor555-labeled donkey anti-rabbit IgG H&L, respectively. Yellow arrows mark the locations where high MMP2 signals overlap with decreased CHP signals. Additional examples are provided in Figure S7. Scale bars: 200 μm (A), 50 μm (C).

Figure 6

Endochondral ossification. (A) Localization of CHP binding in a sagittal section of an 18 d.p.c. mouse embryo (E18) double stained with B-CHP (detected by AlexaFluor647-streptavidin, orange) and an anti-collagen I antibody (detected by AlexaFluor555-labeled donkey anti-rabbit IgG H&L, cyan). mx, maxilla; md, mandibular bone; bp, basisphenoid bone; bo, basioccipital bone; vc, vertebral column; rb, rib; h, hipbone; d, digital bones. (B) Magnified views of the basioccipital bone (bo) beside the C1 vertebra (v) (top images) and the manubrium sterni (ms, bottom images) in the sagittal section of a 17 d.p.c. mouse embryo (E17) stained in the same fashion. High levels of CHP binding are found in the hypertrophic zone surrounding the newly deposited collagen I bone matrix (yellow arrow heads), which is visualized by the anti-collagen I antibody. Scale bars: 3 mm (A), 0.5 mm (B).

Figure 7

Chronological skin aging. (A) Representative fluorescence micrographs of formalin-fixed paraffin-embedded skin sections from 21-day (young) versus 9-month-old mice (aged), stained with Hoechst 33342 (blue) and B-CHP (detected by AlexaFluor647-streptavidin, yellow). (B) Representative views from simultaneous multiphoton imaging of frozen skin sections from the same group of mice, stained by F-CHP, showing the distinct fiber morphologies (via SHG, white) and degraded collagen content (via F-CHP, green). Remaining hair shafts (white arrows) are strongly autofluorescent. Both experiments were performed on skin samples obtained from 3 mice at each age with similar results. Scale bars: 100 μm (A), 150 μm (B).