Dataset Information

Attachment-regulated signaling networks in the fibroblast-populated 3D collagen matrix.

ABSTRACT: Fibroblasts in the attached collagen matrix are in a pro-survival, pro-proliferative state relative to fibroblasts in the released collagen matrix, such that matrix cell number increases in the former over time. Gene array data from attached vs. released matrices were analyzed for putative networks that regulated matrix cell number. Select networks then underwent augmentation and/or inhibition in order to determine their biologic relevance. Matrix stress-release was associated with modulation of signaling networks that involved IL6, IL8, NF-κB, TGF-β1, p53, interferon-γ, and other entities as central participants. Perturbation of select networks in multiple fibroblast strains suggested that IL6 and IL8 secretion may have been involved in preservation of matrix cell population in the released matrix, though there was variability in testing results among the strains. NF-κB activation may have contributed to the induction of population regression after matrix release.

SUBMITTER: Carlson MA

PROVIDER: S-EPMC6504840 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Attachment-regulated signaling networks in the fibroblast-populated 3D collagen matrix.

Carlson Mark A MA Smith Lynette M LM Cordes Crystal M CM Chao Jie J Eudy James D JD

Scientific reports 20130101

Fibroblasts in the attached collagen matrix are in a pro-survival, pro-proliferative state relative to fibroblasts in the released collagen matrix, such that matrix cell number increases in the former over time. Gene array data from attached vs. released matrices were analyzed for putative networks that regulated matrix cell number. Select networks then underwent augmentation and/or inhibition in order to determine their biologic relevance. Matrix stress-release was associated with modulation of ...[more]

PMID: 23697962

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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.

Dataset Information

Attachment-regulated signaling networks in the fibroblast-populated 3D collagen matrix.

Publications

Attachment-regulated signaling networks in the fibroblast-populated 3D collagen matrix.

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