Dataset Information

Densification of Type I Collagen Matrices as a Model for Cardiac Fibrosis.

ABSTRACT: Cardiac fibrosis is a disease state characterized by excessive collagenous matrix accumulation within the myocardium that can lead to ventricular dilation and systolic failure. Current treatment options are severely lacking due in part to the poor understanding of the complexity of molecular pathways involved in cardiac fibrosis. To close this gap, in vitro model systems that recapitulate the defining features of the fibrotic cellular environment are in need. Type I collagen, a major cardiac extracellular matrix protein and the defining component of fibrotic depositions, is an attractive choice for a fibrosis model, but demonstrates poor mechanical strength due to solubility limits. However, plastic compression of collagen matrices is shown to significantly increase its mechanical properties. Here, confined compression of oligomeric, type I collagen matrices is utilized to resemble defining hallmarks seen in fibrotic tissue such as increased collagen content, fibril thickness, and bulk compressive modulus. Cardiomyocytes seeded on compressed matrices show a strong beating abrogation as observed in cardiac fibrosis. Gene expression analysis of selected fibrosis markers indicates fibrotic activation and cardiomyocyte maturation with regard to the existing literature. With these results, a promising first step toward a facile heart-on-chip model is presented to study cardiac fibrosis.

SUBMITTER: Worke LJ

PROVIDER: S-EPMC5844700 | biostudies-literature | 2017 Nov

REPOSITORIES: biostudies-literature

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Densification of Type I Collagen Matrices as a Model for Cardiac Fibrosis.

Worke Logan J LJ Barthold Jeanne E JE Seelbinder Benjamin B Novak Tyler T Main Russell P RP Harbin Sherry L SL Neu Corey P CP

Advanced healthcare materials 20170907 22

Cardiac fibrosis is a disease state characterized by excessive collagenous matrix accumulation within the myocardium that can lead to ventricular dilation and systolic failure. Current treatment options are severely lacking due in part to the poor understanding of the complexity of molecular pathways involved in cardiac fibrosis. To close this gap, in vitro model systems that recapitulate the defining features of the fibrotic cellular environment are in need. Type I collagen, a major cardiac ext ...[more]

PMID: 28881428

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Project description:Background Cardiac fibrosis is the excessive deposition of extracellular matrix in the heart, triggered by a cardiac insult, aging, genetics, or environmental factors. Molecular imaging of the cardiac extracellular matrix with targeted probes could improve diagnosis and treatment of heart disease. However, although this technology has been used to demonstrate focal scarring arising from myocardial infarction, its capacity to demonstrate extracellular matrix expansion and diffuse cardiac fibrosis has not been assessed. Methods and Results Here, we report the use of collagen-targeted peptides labeled with near-infrared fluorophores for the detection of diffuse cardiac fibrosis in the β2-AR (β-2-adrenergic receptor) overexpressing mouse model and in ischemic human hearts. Two approaches were evaluated, the first based on a T peptide that binds matrix metalloproteinase-2-proteolyzed collagen IV, and the second on the cyclic peptide EP-3533, which targets collagen I. The systemic and cardiac uptakes of both peptides (intravenously administered) were quantified ex vivo by near-infrared imaging of whole organs, tissue sections, and heart lysates. The peptide accumulation profiles corresponded to an immunohistochemically-validated increase in collagen types I and IV in hearts of transgenic mice versus littermate controls. The T peptide could encouragingly demonstrate both the intermediate (7 months old) and severe (11 months old) cardiomyopathic phenotypes. Co-immunostainings of fluorescent peptides and collagens, as well as reduced collagen binding of a control peptide, confirmed the collagen specificity of the tracers. Qualitative analysis of heart samples from patients with ischemic cardiomyopathy compared with nondiseased donors supported the collagen-enhancement capabilities of these peptides also in the clinical settings. Conclusions Together, these observations demonstrate the feasibility and translation potential of molecular imaging with collagen-binding peptides for noninvasive imaging of diffuse cardiac fibrosis.

Project description:The fibroblast-populated 3D collagen matrix has been used to study the effect of mechanical stress on cell fate; this process is relevant to the fields of wound healing and tissue engineering. Gene array data was generated from mechanically stressed vs. stress-released matrices. The parameters of the collagen matrix model were: collagen type = bovine type I; collagen concentration = 1.5 mg/mL; initial matrix volume = 0.2 mL; initial matrix diameter = 11 mm (cultured in 24-well plates); cell type = human foreskin fibroblast, passage <10; initial matrix cell concentration = 1,000,000 cell/mL (200,000 cell/matrix); culture medium = 5% FBS in DMEM with 1 M-BM-5g/mL ascorbate. Matrices (n = 6 per experimental group) were incubated for 24 hr in the attached state; the released groups then underwent matrix detachment from the culture plate (defined as t = 0), while the attached groups were left undisturbed. RNA was isolated from attached and released matrices 6 and 24 hr after t = 0. Gene expression in the attached vs. released condition at 6 or 24 hr then was analyzed by hybridizing the anti-sense RNA derived from attached and released matrices at a given time point onto a single chip. Refer to the attached Figure 1 for the experimental design. The index experiment was defined as the comparison of gene expression in attached vs. released collagen matrices in a single strain of human foreskin fibroblasts at 6 and 24 hr after stress-release (i.e., after t = 0). Each experiment utilized two mechanical conditions (attached and released) at two time points (6 and 24 hr). So with each condition utilizing 6 matrices, each index experiment required a total of 24 matrices. In each index experiment, the chip hybridizations were: (i) 6 hr attached vs. 6 hr released, and (ii) 24 hr attached vs. 24 hr released (i.e., two gene chips per index experiment). Each hybridization was done using a 10K spotted gene chip manufactured in the UNMC Microarray Core Facility. The index experiment was performed on three fibroblast strains, meaning that expressional data was derived from three foreskin donors (nonpooled samples). Dye-swap was not performed; dye assigned to attached vs. released remained constant among all chips. Since the index experiment was performed three times, the total number of gene chips used for this entire dataset was six.

Dataset Information

Densification of Type I Collagen Matrices as a Model for Cardiac Fibrosis.

Publications

Densification of Type I Collagen Matrices as a Model for Cardiac Fibrosis.

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