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Macromolecularly crowded in vitro microenvironments accelerate the production of extracellular matrix-rich supramolecular assemblies.

ABSTRACT: Therapeutic strategies based on the principles of tissue engineering by self-assembly put forward the notion that functional regeneration can be achieved by utilising the inherent capacity of cells to create highly sophisticated supramolecular assemblies. However, in dilute ex vivo microenvironments, prolonged culture time is required to develop an extracellular matrix-rich implantable device. Herein, we assessed the influence of macromolecular crowding, a biophysical phenomenon that regulates intra- and extra-cellular activities in multicellular organisms, in human corneal fibroblast culture. In the presence of macromolecules, abundant extracellular matrix deposition was evidenced as fast as 48 h in culture, even at low serum concentration. Temperature responsive copolymers allowed the detachment of dense and cohesive supramolecularly assembled living substitutes within 6 days in culture. Morphological, histological, gene and protein analysis assays demonstrated maintenance of tissue-specific function. Macromolecular crowding opens new avenues for a more rational design in engineering of clinically relevant tissue modules in vitro.

SUBMITTER: Kumar P

PROVIDER: S-EPMC4348624 | biostudies-literature | 2015 Mar

REPOSITORIES: biostudies-literature

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Macromolecularly crowded in vitro microenvironments accelerate the production of extracellular matrix-rich supramolecular assemblies.

Kumar Pramod P Satyam Abhigyan A Fan Xingliang X Collin Estelle E Rochev Yury Y Rodriguez Brian J BJ Gorelov Alexander A Dillon Simon S Joshi Lokesh L Raghunath Michael M Pandit Abhay A Zeugolis Dimitrios I DI

Scientific reports 20150304

Therapeutic strategies based on the principles of tissue engineering by self-assembly put forward the notion that functional regeneration can be achieved by utilising the inherent capacity of cells to create highly sophisticated supramolecular assemblies. However, in dilute ex vivo microenvironments, prolonged culture time is required to develop an extracellular matrix-rich implantable device. Herein, we assessed the influence of macromolecular crowding, a biophysical phenomenon that regulates i ...[more]

PMID: 25736020

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Project description:In contrast to the dynamic intracellular environment, structural extracellular matrix (ECM) proteins with half-lives measured in decades, are susceptible to accumulating damage. Whilst conventional approaches such as histology, immunohistochemistry and mass spectrometry are able to identify age- and disease-related changes in protein abundance or distribution, these techniques are poorly suited to characterising molecular damage. We have previously shown that mass spectrometry can detect tissue-specific differences in the proteolytic susceptibility of protein regions within fibrillin-1 and collagen VI alpha-3. Here, we present a novel proteomic approach to detect damage-induced "peptide fingerprints" within complex multi-component ECM assemblies (fibrillin and collagen VI microfibrils) following their exposure to ultraviolet radiation (UVR) by broadband UVB or solar simulated radiation (SSR). These assemblies were chosen because, in chronically photoaged skin, fibrillin and collagen VI microfibril architectures are differentially susceptible to UVR. In this study, atomic force microscopy revealed that fibrillin microfibril ultrastructure was significantly altered by UVR exposure whereas the ultrastructure of collagen VI microfibrils was resistant. UVR-induced molecular damage was further characterised by proteolytic peptide generation with elastase followed by liquid chromatography tandem mass spectrometry (LC-MS/MS). Peptide mapping revealed that UVR exposure increased regional proteolytic susceptibility within the protein structures of fibrillin-1 and collagen VI alpha-3. This allowed the identification of UVR-induced molecular changes within these two key ECM assemblies. Additionally, similar changes were observed within protein regions of co-purifying, microfibril-associated receptors integrins αv and β1. This study demonstrates that LC-MS/MS mapping of peptides enables the characterisation of molecular post-translational damage (via direct irradiation and radiation-induced oxidative mechanisms) within a complex in vitro model system. This peptide fingerprinting approach reliably allows both the identification of UVR-induced molecular damage in and between proteins and the identification of specific protein domains with increased proteolytic susceptibility as a result of photo-denaturation. This has the potential to serve as a sensitive method of identifying accumulated molecular damage in vivo using conventional mass spectrometry data-sets.

Project description:Normal epithelium exists within a dynamic extracellular matrix (ECM) that is tuned to regulate tissue specific epithelial cell function. As such, ECM contributes to tissue homeostasis, differentiation, and disease, including cancer. Though it is now recognized that the functional unit of normal and transformed epithelium is the epithelial cell and its adjacent ECM, we lack a basic understanding of tissue-specific ECM composition and abundance, as well as how physiologic changes in ECM impact cancer risk and outcomes. While traditional proteomic techniques have advanced to robustly identify ECM proteins within tissues, methods to determine absolute abundance have lagged. Here, with a focus on tissues relevant to breast cancer, we utilize mass spectrometry methods optimized for absolute quantitative ECM analysis. Employing an extensive protein extraction and digestion method, combined with stable isotope labeled Quantitative conCATamer (QconCAT) peptides that serve as internal standards for absolute quantification of protein, we quantify 98 ECM, ECM-associated, and cellular proteins in a single analytical run. In rodent models, we applied this approach to the primary site of breast cancer, the normal mammary gland, as well as a common and particularly deadly site of breast cancer metastasis, the liver. We find that mammary gland and liver have distinct ECM abundance and relative composition. Further, we show mammary gland ECM abundance and relative compositions differ across the reproductive cycle, with the most dramatic changes occurring during the pro-tumorigenic window of weaning-induced involution. Combined, this work suggests ECM candidates for investigation of breast cancer progression and metastasis, particularly in postpartum breast cancers that are characterized by high metastatic rates. Finally, we suggest that with use of absolute quantitative ECM proteomics to characterize tissues of interest, it will be possible to reconstruct more relevant in vitro models to investigate tumor-ECM dynamics at higher resolution.

Dataset Information

Macromolecularly crowded in vitro microenvironments accelerate the production of extracellular matrix-rich supramolecular assemblies.

Publications

Macromolecularly crowded in vitro microenvironments accelerate the production of extracellular matrix-rich supramolecular assemblies.

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