Quantitative proteomics reveals altered expression

Authors: Stephan A Müller Anja van der Smissen Margarete von Feilitzsch Ulf Anderegg Stefan Kalkhof Martin von Bergen

Publish Date: 2012/09/19

Volume: 23, Issue: 12, Pages: 3053-3065

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Abstract

Fibroblasts are the main matrix producing cells of the dermis and are also strongly regulated by their matrix environment which can be used to improve and guide skin wound healing processes Here we systematically investigated the molecular effects on primary dermal fibroblasts in response to highsulfated hyaluronan HA hsHA by quantitative proteomics The comparison of non and highsulfated HA revealed regulation of 84 of more than 1200 quantified proteins Based on gene enrichment we found that sulfation of HA alters extracellular matrix remodeling The collagen degrading enzymes cathepsin K matrix metalloproteinases2 and 14 were found to be downregulated on hsHA Additionally protein expression of thrombospondin1 decorin collagen types I and XII were reduced whereas the expression of trophoblast glycoprotein and collagen type VI were slightly increased This study demonstrates that global proteomics provides a valuable tool for revealing proteins involved in molecular effects of growth substrates for further material optimizationThe skin is the largest organ of the human body It has many essential functions like body temperature regulation oxygen uptake pathogen defense and fluid loss prevention Thus dermal wounds can cause severe health problems by the restriction of these functions The therapeutic band width of skin wound treatment includes dressing with autografts allografts xenografts or tissueengineered skin substitutes TESS TESS have been proven to be a good alternative to conventional treatment by grafting of skin wounds 1 Clinical products from different companies are extensively reviewed by Eisenbud et al 2 and Damanhuri et al 3 while Metcalfe and Ferguson 4 have reviewed developments of bioengineered artificial skin The usage of cellfree scaffolds as matrix supports for selfregeneration of skin is an alternative to skin biopsies and dermal cell culturing Especially cellfree scaffolds based on biodegradable substances like polylactides collagens and/or glycosaminoglycans GAGs which mimic the extracellular matrix ECM are good alternatives to conventional skin grafting 5 6 7 8A promising approach for the development of new artificial ECMs aECMs for wound healing of skin tissue is the integration of chemically modified natural ECM components In particular sulfated GAG have been supposed to improve wound healing of skin tissue by the interaction of negatively charged sulfate groups with cytokines growth factors and dermal cells 9 10Sulfated derivatives of GAGs mimic the behavior of heparin the most biological active natural GAG compound which plays an important role in wound healing 11 Heparin interacts with a huge variety of different proteins like growth factors FGFs fibroblast growth factors1 2 and 7 12 or cytokines such as platelet factor 4 13 interleukin 8 IL8 10 14 or interferon gamma 15 Heparin further binds to adhesion proteins like selectins 16 the heparinbinding growth associated molecule 13 and fibronectin 17 Protein binding to heparin promotes different functions like protection from proteolysis ie FGFs1 2 and 7 12 18 or modification of biological activity shown for transforming growth factor β1 TGFβ1 13 Thus heparin and other sulfated GAG have an influence on key processes of wound healing like inflammation cell proliferation or cell–matrix interactions 13 Most interactions between sulfated GAG and proteins are governed by negatively charged sulfate groups which form ionic bonds with basic amino acid residues 10 12 13 15Hence cell studies with sulfated GAG can provide valuable information for the engineering of new skin substitutes We have chosen hyaluronan HA to investigate the effect of chemical sulfation HA is the most suited GAG for this study since naturally HA does not contain sulfate groups It has a regular sequence of alternating units of Nacetylglucosamine and glucuronic acid and is not covalently linked to proteins Additionally HA can be chemically modified without loss of structure 11 Since our research focus is on acquiring knowledge about the influence of synthetized aECMs for improved wound healing of skin tissue we have chosen dermal fibroblasts dFbs as model cells for investigation of our modified aECM They are crucial for wound healing of skin tissue and strongly regulated by their surrounding ECM 19 The previous work of van der Smissen et al 20 showed that sulfated GAGs improved initial cell adhesion and proliferation of dFbs in a sulfation dependent manner By testing a few selected mRNA of involved key proteins the expression levels of collagen type I α chain HA synthase 2 and matrix metalloproteinase1 MMP1 were found to be significantly reduced on highsulfated GAGs whereas lowsulfated GAG derivatives only slightly changed the mRNA expression of these componentsOn the basis of these data 20 the influence of HA sulfation on the expression of other proteins by a nontargeted approach is of great interest since this will allow detecting so far unrecognized signaling pathways in response to the tested biomaterials We analyzed the influence of aECMs consisting of collagen type I mixed with HA or its highsulfated derivative hsHA on protein level For that reason we have chosen stable isotope labeling by amino acids in cell culture SILAC which is a wellestablished method enabling accurate relative quantification of thousands of proteins in an untargeted approach 21 22 As long as primary cells can be cultivated for a sufficient time to obtain quantitative isotope labeling SILAC provides superior protein coverage and better quantitative reproducibility in comparison to the usage of cells or organs from different individuals or label free quantification 23 Especially relative quantification to a control of the same donor within one measurement reduces variabilityGlobal analyses provide a broader overview and higher protein coverage than targeted experiments Computational analyzes of regulated proteins according to databases like PANTHER Protein ANalysis THrough Evolutionary Relationships 24 reveal protein cluster enriched according to their molecular functions and biological processes Bioinformatics tools like DAVID 25 additionally calculate enrichment factors and determine statistical significance of these clusters

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