Abstract
Cardiac fibrosis is a major hallmark of cardiac diseases. For evaluation of cardiac fibrosis, the development of highly specific and preferably noninvasive methods is desired. Our aim was to evaluate CNA35, a protein known to specifically bind to collagen, as a specific marker of cardiac fibrosis. Fluorescently labeled CNA35 was applied ex vivo on tissue sections of fibrotic rat, mouse, and canine myocardium. After quantification of CNA35, sections were examined with picrosirius red (PSR) and compared to CNA35. Furthermore, fluorescently labeled CNA35 was administered in vivo in mice. Hearts were isolated, and CNA35 labeling was examined in tissue sections. Serial sections were histologically examined with PSR. Ex vivo application of CNA35 showed specific binding to collagen and a high correlation with PSR (Pearson r = .86 for mice/rats and r = .98 for canine; both p < .001). After in vivo administration, CNA35 labeling was observed around individual cardiomyocytes, indicating its ability to penetrate cardiac endothelium. High correlation was observed between CNA35 and PSR (r = .91, p < .001). CNA35 specifically binds to cardiac collagen and can cross the endothelial barrier. Therefore, labeled CNA35 is useful to specifically detect collagen both ex vivo and in vivo and potentially can be converted to a noninvasive method to detect cardiac fibrosis.
Highlights
Cardiac fibrosis is a major hallmark of cardiac diseases
Ex vivo analysis of collagen labeling by CNA35-fluorescein isothiocyanate (FITC) showed that whole heart sections labeled with CNA35 corresponded to the picrosirius red (PSR) staining of serial de Jong et al sections (Figure 1, A–D)
When collagen concentration was determined with PSR, comparable results were observed: 9.8 6 1.6% and 21.5 6 4.2% in mouse and rat sections, respectively
Summary
Cardiac fibrosis is a major hallmark of cardiac diseases. For evaluation of cardiac fibrosis, the development of highly specific and preferably noninvasive methods is desired. Ex vivo application of CNA35 showed specific binding to collagen and a high correlation with PSR (Pearson r 5 .86 for mice/rats and r 5 .98 for canine; both p , .001). Labeled CNA35 is useful to detect collagen both ex vivo and in vivo and potentially can be converted to a noninvasive method to detect cardiac fibrosis. Cardiac tissue can be histologically examined ex vivo for fibrosis with dye stainings such as picrosirius red (PSR) or with specific fluorescently labeled antibodies.[11] Due to the highly noncentrosymmetrical characteristics of fibrillar collagen, collagen can be examined with second harmonic generation (SHG) microscopy.[12] the acquirement of ventricular cardiac tissue in patients is not without risk; histology or SHG imaging of biopsies is not preferred in the clinical setting to examine fibrosis. In animal studies, histology is the most commonly used technique for collagen detection in cardiac tissue
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