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Reduced serum content and increased matrix stiffness promote the cardiac myofibroblast transition in 3D collagen matrices.

ABSTRACT: The fibroblast-myofibroblast transition is an important event in the development of cardiac fibrosis and scar formation initiated after myocardial ischemia. The goals of the present study were to better understand the contribution of environmental factors to this transition and determine whether myofibroblasts provide equally important feedback to the surrounding environment.The influence of matrix stiffness and serum concentration on the myofibroblast transition was assessed by measuring message levels of a panel of cardiac fibroblast phenotype markers using quantitative reverse transcriptase polymerase chain reaction. Cell-mediated gel compaction measured the influence of environmental factors on cardiac fibroblast contractility. Immunohistochemistry characterized alpha-smooth muscle actin expression and cell morphology, while static and dynamic compression testing evaluated the effect of the cell response on the mechanical properties of the cell-seeded collagen hydrogels.Both reduced serum content and increased matrix stiffness contributed to the myofibroblast transition, as indicated by contractile compaction of the gels, increased message levels of col3?1 and alpha-smooth muscle actin, and a less stellate morphology. However, the effects of serum and matrix stiffness were not additive. Mechanical testing indicated that reduced serum content increased the initial elastic modulus of cell-seeded gels and that gels lost their viscous character with time.The results suggest that reduced serum and increased matrix stiffness promote the myofibroblast phenotype in the myocardium. This transition both enhances and is promoted by matrix stiffness, indicating the presence of positive feedback that may contribute to the pathogenesis of cardiac fibrosis.

SUBMITTER: Galie PA

PROVIDER: S-EPMC3110608 | biostudies-literature | 2011 Nov-Dec

REPOSITORIES: biostudies-literature

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Reduced serum content and increased matrix stiffness promote the cardiac myofibroblast transition in 3D collagen matrices.

Galie Peter A PA Westfall Margaret V MV Stegemann Jan P JP

Cardiovascular pathology : the official journal of the Society for Cardiovascular Pathology 20110208 6

<h4>Introduction</h4>The fibroblast-myofibroblast transition is an important event in the development of cardiac fibrosis and scar formation initiated after myocardial ischemia. The goals of the present study were to better understand the contribution of environmental factors to this transition and determine whether myofibroblasts provide equally important feedback to the surrounding environment.<h4>Methods</h4>The influence of matrix stiffness and serum concentration on the myofibroblast transi ...[more]

PMID: 21306921

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Project description:Cell motility under physiological and pathological conditions including malignant progression of cancer and subsequent metastasis are founded on environmental confinements. During the last two decades, three-dimensional cell migration has been studied mostly by utilizing biomimetic extracellular matrix models. In the majority of these studies, the in vitro collagen scaffolds are usually assumed to be homogenous, as they consist commonly of one specific type of collagen, such as collagen type I, isolated from one species. These collagen matrices should resemble in vivo extracellular matrix scaffolds physiologically, however, mechanical phenotype and functional reliability have been addressed poorly due to certain limitations based on the assumption of homogeneity. How local variations of extracellular matrix structure impact matrix mechanics and cell migration is largely unknown. Here, we hypothesize that local inhomogeneities alter cell movement due to alterations in matrix mechanics, as they frequently occur in in vivo tissue scaffolds and were even changed in diseased tissues. To analyze the effect of structural inhomogeneities on cell migration, we used a mixture of rat tail and bovine dermal collagen type I as well as pure rat and pure bovine collagens at four different concentrations to assess three-dimensional scaffold inhomogeneities. Collagen type I from rat self-assembled to elongated fibrils, whereas bovine collagen tended to build node-shaped inhomogeneous scaffolds. We have shown that the elastic modulus determined with atomic force microscopy in combination with pore size analysis using confocal laser scanning microscopy revealed distinct inhomogeneities within collagen matrices. We hypothesized that elastic modulus and pore size govern cancer cell invasion in three-dimensional collagen matrices. In fact, invasiveness of three breast cancer cell types is altered due to matrix-type and concentration indicating that these two factors are crucial for cellular invasiveness. Our findings revealed that local matrix scaffold inhomogeneity is another crucial parameter to explain differences in cell migration, which not solely depended on pore size and stiffness of the collagen matrices. With these three distinct biophysical parameters, characterizing structure and mechanics of the studied collagen matrices, we were able to explain differences in the invasion behavior of the studied cancer cell lines in dependence of the used collagen model.

Project description:BackgroundFibrosis plays an important role in both normal physiological and pathological phenomena as fibroblasts differentiate to myofibroblasts. The activation of fibroblasts is determined through interactions with the surrounding extracellular matrix (ECM). However, how this fibroblast-to-myofibroblast transition (FMT) is regulated and affected by elastin concentration in a three-dimensional (3D) microenvironment has not been investigated.MethodsWe developed an insoluble elastin-gradient 3D hydrogel system for long-lasting cell culture and studied the molecular mechanisms of the FMT in embedded cells by nanoflow LC-MS/MS analysis along with validation through real-time PCR and immunofluorescence staining.ResultsBy optimizing pH and temperature, four 3D hydrogels containing fibroblasts were successfully fabricated having elastin concentrations of 0, 20, 50, and 80% in collagen. At the low elastin level (20%), fibroblast proliferation was significantly increased compared to others, and in particular, the FMT was clearly observed in this condition. Moreover, through mass spectrometry of the hydrogel environment, it was confirmed that differentiation proceeded in two stages. In the early stage, calcium-dependent proteins including calmodulin and S100A4 were highly associated. On the other hand, in the late stage after several passages of cells, distinct markers of myofibroblasts were presented such as morphological changes, increased production of ECM, and increased α-SMA expression. We also demonstrated that the low level of elastin concentration induced some cancer-associated fibroblast (CAF) markers, including PDGFR-β, and fibrosis-related disease markers, including THY-1.ConclusionUsing our developed 3D elastin-gradient hydrogel system, we evaluated the effect of different elastin concentrations on the FMT. The FMT was induced even at a low concentration of elastin with increasing CAF level via calcium signaling. With this system, we were able to analyze varying protein expressions in the overall FMT process over several cellular passages. Our results suggest that the elastin-gradient system employing nonlinear optics imaging provides a good platform to study activated fibroblasts interacting with the microenvironment, where the ECM plays a pivotal role.

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Reduced serum content and increased matrix stiffness promote the cardiac myofibroblast transition in 3D collagen matrices.

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Reduced serum content and increased matrix stiffness promote the cardiac myofibroblast transition in 3D collagen matrices.

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