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Proteomics Characterization of Extracellular Space Components in the Human Aorta

2010; Elsevier BV; Volume: 9; Issue: 9 Linguagem: Inglês

10.1074/mcp.m110.001693

1535-9484

Athanasios Didangelos, Xiaoke Yin, Kaushik Mandal, Mark Baumert, Marjan Jahangiri, Manuel Mayr,

Peptidase Inhibition and Analysis

The vascular extracellular matrix (ECM) is essential for the structural integrity of the vessel wall and also serves as a substrate for the binding and retention of secreted products of vascular cells as well as molecules coming from the circulation. Although proteomics has been previously applied to vascular tissues, few studies have specifically targeted the vascular ECM and its associated proteins. Thus, its detailed composition remains to be characterized. In this study, we describe a methodology for the extraction of extracellular proteins from human aortas and their identification by proteomics. The approach is based on (a) effective decellularization to enrich for scarce extracellular proteins, (b) successful solubilization and deglycosylation of ECM proteins, and (c) relative estimation of protein abundance using spectral counting. Our three-step extraction approach resulted in the identification of 103 extracellular proteins of which one-third have never been reported in the proteomics literature of vascular tissues. In particular, three glycoproteins (podocan, sclerostin, and agrin) were identified for the first time in human aortas at the protein level. We also identified extracellular adipocyte enhancer-binding protein 1, the cartilage glycoprotein asporin, and a previously hypothetical protein, retinal pigment epithelium (RPE) spondin. Moreover, our methodology allowed us to screen for proteolysis in the aortic samples based on the identification of proteolytic enzymes and their corresponding degradation products. For instance, we were able to detect matrix metalloproteinase-9 by mass spectrometry and relate its presence to degradation of fibronectin in a clinical specimen. We expect this proteomics methodology to further our understanding of the composition of the vascular extracellular environment, shed light on ECM remodeling and degradation, and provide insights into important pathological processes, such as plaque rupture, aneurysm formation, and restenosis. The vascular extracellular matrix (ECM) is essential for the structural integrity of the vessel wall and also serves as a substrate for the binding and retention of secreted products of vascular cells as well as molecules coming from the circulation. Although proteomics has been previously applied to vascular tissues, few studies have specifically targeted the vascular ECM and its associated proteins. Thus, its detailed composition remains to be characterized. In this study, we describe a methodology for the extraction of extracellular proteins from human aortas and their identification by proteomics. The approach is based on (a) effective decellularization to enrich for scarce extracellular proteins, (b) successful solubilization and deglycosylation of ECM proteins, and (c) relative estimation of protein abundance using spectral counting. Our three-step extraction approach resulted in the identification of 103 extracellular proteins of which one-third have never been reported in the proteomics literature of vascular tissues. In particular, three glycoproteins (podocan, sclerostin, and agrin) were identified for the first time in human aortas at the protein level. We also identified extracellular adipocyte enhancer-binding protein 1, the cartilage glycoprotein asporin, and a previously hypothetical protein, retinal pigment epithelium (RPE) spondin. Moreover, our methodology allowed us to screen for proteolysis in the aortic samples based on the identification of proteolytic enzymes and their corresponding degradation products. For instance, we were able to detect matrix metalloproteinase-9 by mass spectrometry and relate its presence to degradation of fibronectin in a clinical specimen. We expect this proteomics methodology to further our understanding of the composition of the vascular extracellular environment, shed light on ECM remodeling and degradation, and provide insights into important pathological processes, such as plaque rupture, aneurysm formation, and restenosis. Vascular cells, in particular vascular smooth muscle cells, produce and maintain a complex meshwork of ECM. 1The abbreviations used are:ECMextracellular matrixMMPmatrix metalloproteinaseGAGglycosaminoglycanBis-Tris2-[bis(2- hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diolANOVAanalysis of varianceAEBP1adipocyte enhancer-binding protein 1RPEretinal pigment epitheliumLTQlinear trap quadrupole. The ECM is not only the scaffold for the anchorage and mobility of residing cells but also absorbs and transduces the shear and strain forces of the blood flow. It is primarily composed of elastin, collagen, proteoglycans, and glycoproteins. The elastin fibers and type I and III fibrillar collagens form a rigid network of highly cross-linked interstitial matrix. They offer elasticity (elastin) and tensile strength (collagens). Proteoglycans, because of their negative charge, attract water and confer resistance to compression. Finally, glycoproteins participate in matrix organization and are essential for cell attachment.The vascular ECM also serves as a substrate for the binding and retention of secreted, soluble proteins of vascular cells as well as molecules coming from the circulation, including lipoproteins, growth factors, cytokines, proteases, and protease inhibitors. These components are invariably associated with ECM proteins, especially proteoglycans. Together they comprise the vascular extracellular environment and are pivotal for disease processes, such as atherosclerosis and aneurysm formation (1.Didangelos A. Simper D. Monaco C. Mayr M. Proteomics of acute coronary syndromes.Curr. Atheroscler. Rep. 2009; 11: 188-195Crossref PubMed Scopus (34) Google Scholar).Although proteomics has been previously applied to vascular tissues, only one study has specifically targeted the extracellular vascular environment (2.Talusan P. Bedri S. Yang S. Kattapuram T. Silva N. Roughley P.J. Stone J.R. Analysis of intimal proteoglycans in atherosclerosis-prone and atherosclerosis-resistant human arteries by mass spectrometry.Mol. Cell. Proteomics. 2005; 4: 1350-1357Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). This study was focused on the isolation of intimal proteoglycans from human carotid arteries. Moreover, most proteomics studies use whole tissue lysates, which are rich in cellular proteins that inevitably mask the identification of the less abundant proteins of the vascular extracellular environment (3.Mayr M. Chung Y.L. Mayr U. Yin X. Ly L. Troy H. Fredericks S. Hu Y. Griffiths J.R. Xu Q. Proteomic and metabolomic analyses of atherosclerotic vessels from apolipoprotein E-deficient mice reveal alterations in inflammation, oxidative stress, and energy metabolism.Arterioscler. Thromb. Vasc. Biol. 2005; 25: 2135-2142Crossref PubMed Scopus (151) Google Scholar, 4.Mayr M. Zampetaki A. Sidibe A. Mayr U. Yin X. De Souza A.I. Chung Y.L. Madhu B. Quax P.H. Hu Y. Griffiths J.R. Xu Q. Proteomic and metabolomic analysis of smooth muscle cells derived from the arterial media and adventitial progenitors of apolipoprotein E-deficient mice.Circ. Res. 2008; 102: 1046-1056Crossref PubMed Scopus (47) Google Scholar, 5.Wu J. Liu W. Sousa E. Qiu Y. Pittman D.D. Maganti V. Feldman J. Gill D. Lu Z. Dorner A.J. Schaub R. Tan X.Y. Proteomic identification of endothelial proteins isolated in situ from atherosclerotic aorta via systemic perfusion.J. Proteome Res. 2007; 6: 4728-4736Crossref PubMed Scopus (25) Google Scholar). Thus, the composition of the vascular ECM and its associated proteins remains poorly defined. In the present study, we used morphologically normal human aortic samples to develop a method for the extraction of proteins present in the extracellular environment, including ECM proteins and proteins attached to the ECM. We had three specific aims: first, to reduce the contamination with cellular proteins, thereby increasing the chance of identifying scarce extracellular proteins; second, to efficiently solubilize and deglycosylate ECM proteins to improve their analysis by liquid chromatography tandem mass spectrometry (LC-MS/MS); and third, to interface the nanoflow LC system to a recently developed injection device, which splits the flow from the analytical column, to allow the reanalysis of the same sample during a single LC-MS/MS run (RePlay, Advion).Our methodology provides a detailed overview of the aortic ECM and its associated proteins, many reported for the first time in proteomics analysis of the vasculature. Most importantly, this method could be adapted for use with other tissues to further our understanding of the composition of extracellular environment and ECM turnover under various disease conditions.EXPERIMENTAL PROCEDURESTissue Processing, Decellularization, and Extraction of Extracellular Space ProteinsThe data presented were derived from three human aortic samples. The samples were obtained upon aortotomy performed during routine aortic valve replacement. They were collected from positions of the ascending aorta that were free of macroscopically evident vascular pathology, including atherosclerosis or aneurysm formation. The tissue was immediately snap frozen and subsequently kept in liquid nitrogen for later use. A fourth sample was also obtained upon aortotomy and processed as described below, but the proteomics analysis revealed proteolysis of ECM proteins. Therefore, we used it to examine the potential of our methodology to study proteolytic activity in the tissue. All procedures were approved by our local Regional Ethics Committee Board. Informed consent was obtained from all patients recruited into the study.Before extraction, the tissue pieces were partially thawed and weighed. Approximately 150 mg of tissue per aortic sample were immediately placed in ice-cold phosphate-buffered saline to remove plasma contaminants. Commercially available protease and phosphatase inhibitor mixtures (Sigma-Aldrich) were included according to the manufacturer's instructions to inhibit broad range proteinase activity, and 25 mm EDTA was included to ensure inhibition of metalloproteinases. While the tissue samples were immersed in the cold saline mixture, they were quickly diced with a scalpel into 8–10 smaller pieces. This would enhance (a) the initial removal of plasma contaminants and (b) the effective extraction of extracellular proteins, which is described below. The saline mixture was changed five times (∼30 ml total per sample).Then, the diced samples were incubated with 0.5 m NaCl, 10 mm Tris, pH 7.5 supplemented with proteinase/phosphatase inhibitor mixtures (Sigma-Aldrich) and 25 mm EDTA. The volume of the buffer was adjusted to 10:1 of the tissue weight (i.e. 100 mg in 1 ml), and the samples were mildly vortexed for 4 h at room temperature. The NaCl extracts were then desalted with centrifugation using desalting columns (Zeba Spin, Pierce). Following desalting, the extracts were mixed with 100% acetone (5:1 volume ratio) at −20 °C for 16 h. Proteins were precipitated with centrifugation (16,000 × g for 45 min), and the pellets were dried and redissolved in deglycosylation buffer (see below).Subsequently, the aortic samples were incubated with 0.08% SDS (10:1 buffer volume to tissue weight) supplemented with proteinase/phosphatase inhibitor mixtures and 25 mm EDTA. The samples were mildly vortexed for 4 h at room temperature to achieve removal of cellular components. From our experience with tissue extractions, we took particular care to ensure a low vortexing speed to decrease the possibility of mechanical disruption of the ECM. The SDS solution was then removed and stored frozen for later use.Finally, the samples were incubated in a 4 m guanidine HCl, 50 mm sodium acetate, pH 5.8 buffer (5:1 buffer volume to tissue weight) supplemented with proteinase/phosphatase inhibitor mixtures and 25 mm EDTA. Adjusting the pH to 5.8 is important; more than 30 years ago it was shown that at this pH guanidine is more effective in solubilizing vascular proteoglycans, whereas higher pH is required for the solubilization of the proteoglycans from other ECM-rich tissues, such as cartilage (6.Eisenstein R. Larsson S.E. Kuettner K.E. Sorgente N. Hascal V.C. The ground substance of the arterial wall. Part 1. Extractability of glycosaminoglycans and the isolation of a proteoglycan from bovine aorta.Atherosclerosis. 1975; 22: 1-17Abstract Full Text PDF PubMed Scopus (33) Google Scholar). The samples were then incubated for 48 h at room temperature and vortexed vigorously to enhance mechanical disruption of the ECM components. Subsequently, the guanidine extracts were mixed with 100% ethanol (5:1 volume ratio) at −20 °C for 16 h to ensure removal of guanidine (the presence of guanidine would hinder further biochemical processing). Proteins were precipitated with centrifugation (16,000 × g for 45 min), and the pellets were washed with 90% ethanol, dried, and redissolved in deglycosylation buffer (see below).Deglycosylation (removal of glycosaminoglycan side chains) of the different extracts was achieved in a 150 mm NaCl, 50 mm sodium acetate, pH 6.8 buffer supplemented with proteinase/phosphatase inhibitors and 10 mm EDTA for 16 h at 37 °C. The deglycosylation enzymes (0.05 unit) used were chondroitinase ABC from Proteus vulgaris (It catalyzes the removal of polysaccharides containing 1→4-β-d-hexosaminyl and 1→3-β-d-glucuronosyl or 1→3-α-l-iduronosyl linkages to disaccharides containing 4-deoxy-β-d-gluc-4-enuronosyl groups. It acts on chondroitin 4-sulfate, chondroitin 6-sulfate, and dermatan sulfate glycosaminoglycan side chains.), keratanase from Bacteroides fragilis (cleaves internal 1→4-β-galactose linkages in unbranched, repeating poly-N-acetyl-lactosamine and keratan sulfate), and heparitinase II from Flavobacterium heparinum (cleaves heparan sulfate), all purchased from Sigma-Aldrich. Following deglycosylation, the solutions were clarified again with centrifugation (16,000 × g for 10 min) to ensure that the samples were free of turbidity, and protein concentration was estimated by UV absorbance at 280 nm using extinction coefficient of 1.1 of 0.1% mg/ml solution calculated on the basis of the frequency of tyrosine and tryptophan, which are the main UV light-absorbing amino acids at 280 nm in mammalian proteins (7.Zhuang Y. Ma F. Li-Ling J. Xu X. Li Y. Comparative analysis of amino acid usage and protein length distribution between alternatively and non-alternatively spliced genes across six eukaryotic genomes.Mol. Biol. Evol. 2003; 20: 1978-1985Crossref PubMed Scopus (19) Google Scholar).One-dimensional ElectrophoresisThe NaCl, guanidine, and SDS extracts were denatured and reduced in sample buffer containing 100 mm Tris, pH 6.8, 40% glycerol, 0.2% SDS, 2% β-mercaptoethanol, and 0.02% bromphenol blue and boiled at 96 °C for 10 min. 35 μg of protein per sample were loaded and separated on Bis-Tris discontinuous 4–12% polyacrylamide gradient gels (NuPAGE, Invitrogen). Prestained protein standards were run alongside the samples to allow molecular mass estimation of proteins (All Blue, Precision Plus, Bio-Rad).Nanoflow LC-MS/MS for NaCl and Guanidine ExtractsAfter electrophoresis, gels were stained using the PlusOne Silver staining kit (GE Healthcare). Silver staining was used for band excision to avoid cross-contamination with fainter gel bands. (Coomassie staining will predominantly stain the abundant proteins; hence, fainter gel bands could be missed.). The gel bands were excised in identical parallel positions across lanes, and no "empty" gel pieces were left behind. Subsequently, all gel bands were subjected to in-gel digestion with trypsin using an Investigator ProGest (Genomic Solutions) robotic digestion system. Tryptic peptides from the NaCl and guanidine extracts were separated on a nanoflow LC system (Dionex UltiMate 3000) and eluted with a 40-min gradient (10–25% B in 35 min, 25–40% B in 5 min, 90% B in 10 min, and 2% B in 30min where A is 2% ACN, 0.1% formic acid in HPLC H2O and B is 90% ACN, 0.1% formic acid in HPLC H2O). The column (Dionex PepMap C18, 25-cm length, 75-μm internal diameter, 3-μm particle size) was coupled to a nanospray source (Picoview) using RePlay (Advion). After the direct LC-MS run, the flow was switched, and the portion stored in the capillary of the RePlay device was reanalyzed ("replay run" ) (8.Waanders L.F. Almeida R. Prosser S. Cox J. Eikel D. Allen M.H. Schultz G.A. Mann M. A novel chromatographic method allows on-line reanalysis of the proteome.Mol. Cell. Proteomics. 2008; 7: 1452-1459Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Spectra were collected from an ion trap mass analyzer (LTQ-Orbitrap XL, Thermo Fisher Scientific) using full ion scan mode over the mass-to-charge (m/z) range 450–1600. MS/MS was performed on the top six ions in each MS scan using the data-dependent acquisition mode with dynamic exclusion enabled. MS/MS peak lists were generated by extract_msn.exe and matched to a human database (UniProtKB/Swiss-Prot Release 14.6, 20,333 protein entries) using SEQUEST version 28 (revision 13) (Bioworks Browser 3.3.1 SP1, Thermo Fisher Scientific) and X! Tandem (version 2007.01.01.2). Carboxyamidomethylation of cysteine was chosen as a fixed modification, and oxidation of methionine was chosen as a variable modification. The mass tolerance was set at 1.5 amu for the precursor ions and at 1.0 amu for fragment ions. Two missed cleavages were allowed. Scaffold (version 2.0.5, Proteome Software Inc., Portland, OR) was used to calculate the spectral counts and to validate MS/MS-based peptide and protein identifications (9.Keller A. Nesvizhskii A.I. Kolker E. Aebersold R. Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search.Anal. Chem. 2002; 74: 5383-5392Crossref PubMed Scopus (3837) Google Scholar, 10.Nesvizhskii A.I. Keller A. Kolker E. Aebersold R. A statistical model for identifying proteins by tandem mass spectrometry.Anal. Chem. 2003; 75: 4646-4658Crossref PubMed Scopus (3566) Google Scholar). According to the default values in the Scaffold software, the following peptide thresholds were applied: X! Tandem: −log(Expect scores) > 2.0; SEQUEST: ΔCn > 0.10 and XCorr > 2.5 (2+), 3.5 (3+), and 3.5 (4+). Peptide identifications were accepted if they could be established at greater than 95.0% probability as specified by the Peptide Prophet algorithm (9.Keller A. Nesvizhskii A.I. Kolker E. Aebersold R. Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search.Anal. Chem. 2002; 74: 5383-5392Crossref PubMed Scopus (3837) Google Scholar). Protein identifications were accepted if they could be established at greater than 99.0% probability (10.Nesvizhskii A.I. Keller A. Kolker E. Aebersold R. A statistical model for identifying proteins by tandem mass spectrometry.Anal. Chem. 2003; 75: 4646-4658Crossref PubMed Scopus (3566) Google Scholar) with at least two independent peptides and a precursor ion mass accuracy ≤10 ppm.Nanoflow LC-MS/MS for SDS ExtractsThe protein concentration in the SDS extracts was estimated using the Groves equation, (A280 × 1.55) − (A260 × 0.76) (11.Groves W.E. Davis Jr., F.C. Sells B.H. Spectrophotometric determination of microgram quantities of protein without nucleic acid interference.Anal. Biochem. 1968; 22: 195-210Crossref PubMed Scopus (329) Google Scholar), which takes into account nucleic acid interference. 35 μg of protein per sample were loaded and separated on Bis-Tris discontinuous 4–12% polyacrylamide gradient gels as above. The gel was silver-stained, and protein bands were digested by trypsin as described above. Tryptic peptides were separated on a split-free, nanoflow LC system (EASY-nLC, Proxeon) on a C18 column (Easy-nLC C18, Proxeon, 10-cm length, 75-μm internal diameter, 3-μm particle size) and eluted with an 80-min gradient (10–25% B in 35 min, 25–40% B in 5 min, 90% B in 10 min, and 2% B in 30min where A is 2% ACN, 0.1% formic acid in HPLC H2O and B is 90% ACN, 0.1% formic acid in HPLC H2O). The nanoflow LC system was not coupled to a RePlay device. Spectra were collected from a dual pressure, linear ion trap (LTQ Velos, Thermo Fisher Scientific) using full ion scan mode over the m/z range 300–1800. MS/MS was performed on the top 20 ions in each MS scan using the data-dependent acquisition mode with dynamic exclusion enabled. MS/MS peak lists were generated by extract_msn.exe and matched to a human database (UniProtKB/Swiss-Prot Release 15.13, 20,276 protein entries) using SEQUEST version 28 (revision 13) (Proteome Discoverer 1.2 beta, Thermo Fisher Scientific). Carboxyamidomethylation of cysteine was chosen as a fixed modification, and oxidation of methionine was chosen as a variable modification. The mass tolerance was set at 0.5 amu for the precursor ions and at 0.5 amu for fragment ions. Two missed cleavages were allowed. Scaffold (version 3, Proteome Software Inc.) was used to calculate the spectral counts and to validate MS/MS-based peptide and protein identifications as before. According to the default values in the Scaffold software, the following peptide thresholds were applied: SEQUEST: ΔCn > 0.10 and XCorr > 2.5 (2+), 3.5 (3+), and 3.5 (4+). Peptide identifications were accepted if they could be established at greater than 95.0% probability as specified by the Peptide Prophet algorithm (9.Keller A. Nesvizhskii A.I. Kolker E. Aebersold R. Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search.Anal. Chem. 2002; 74: 5383-5392Crossref PubMed Scopus (3837) Google Scholar). Protein identifications were accepted if they could be established at greater than 99.0% probability (10.Nesvizhskii A.I. Keller A. Kolker E. Aebersold R. A statistical model for identifying proteins by tandem mass spectrometry.Anal. Chem. 2003; 75: 4646-4658Crossref PubMed Scopus (3566) Google Scholar) with at least two independent peptides.Western BlottingAliquots of the NaCl, guanidine, and SDS extracts were mixed with denaturing sample buffer and boiled. 20 μg of protein per sample were loaded and separated on 4–12% gradient gels as above. Proteins were then transferred on nitrocellulose membranes. Membranes were blocked in 5% fat-free milk powder in PBS and then probed for 16 h at 4 °C with primary antibodies to β-actin (sc-130656), periostin (sc-67233), and fibronectin (sc-56391). The antibodies were purchased from Santa Cruz Biotechnology and were used at a 1:500 dilution in 5% BSA. The membranes were then treated with the appropriate secondary, peroxidase-conjugated antibodies (Dako) at a 1:2000 dilution. Finally, the blots were imaged using enhanced chemiluminescence (ECL; GE Healthcare), and films were developed on a Xograph processor. The densitometry for the lanes from developed blots for periostin and asporin was performed using ImageJ software.Gelatinolytic ZymographyGelatinolytic zymograms were used to detect the presence of MMP-2 and -9 in the 0.5 m NaCl extracts. Extract aliquots (20 μg of protein) were mixed with non-reducing sample buffer containing 100 mm Tris, pH 6.8, 40% glycerol, 0.2% SDS, and 0.02% bromphenol blue and separated by 10% SDS-PAGE in gels containing 0.5 mg/ml gelatin (Sigma-Aldrich). Prestained protein standards were run alongside the samples as before. To allow in-gel MMP activity, SDS was removed from the gels with three washes in a buffer containing 2% Triton X-100, 50 mm Tris, pH 7.4, and 200 mm NaCl for 45 min. Gels were then incubated for 16 h to detect MMP-9 or 36 h to detect MMP-2 at 37 °C in a buffer containing 50 mm Tris, pH 7.4, 200 mm NaCl, and 10 mm CaCl2. Finally, gels were stained for 60 min with Coomassie Brilliant Blue (Sigma-Aldrich) and destained for 2 h to visualize the MMPs.HistochemistrySmall representative pieces from each aortic sample were placed in optimal cutting temperature (OCT) compound (BDH), which was subsequently frozen. The frozen tissue blocks were used to generate consecutive 7-μm-thick cross-sections. The sections were air-dried (30 min) and fixed in 100% acetone for 20 min. Subsequently, sections were used either to generate hematoxylin- and eosin-stained histological slides or for immunohistochemical analysis using antibodies to podocan used at 2 μg/ml (MAB4220) (R&D Systems), sclerostin (sc-130258) used at 1 μg/ml, and agrin (sc-25528) used at used at 2 μg/ml (Santa Cruz Biotechnology). Negative controls were generated with either mouse (podocan) or rabbit (sclerostin and agrin) isotype IgG antibodies (Sigma-Aldrich) used at 2 μg/ml. The immunostaining was generated using the DAB+ (diaminobenzidine) peroxidase-based chromogen system (Dako) according to the manufacturer's instructions. Images were taken by a Zeiss Axioplan 2ie microscope interfaced to AxioVision software (version 3.0.6).Statistical Analysis of NaCl and Guanidine ExtractsFor each clinical sample (n = 4) and biochemical fraction (n = 2) analyzed, we obtained a technical replicate by using the RePlay device. The reproducibility of the RePlay analysis was assessed by calculating the Pearson correlation coefficient for the spectral counts. All spectra from proteins identified with at least two unique peptides were included in the analysis, and no outliers were removed. The p value for the linear dependence between the RePlay runs was obtained using the Fisher transformation. To assess biological variability among the four clinical specimens, we computed the coefficient of variation for all identified proteins using Scaffold software (version 2.0.5, Proteome Software Inc.). A coefficient of variation exceeding 100% was applied as a cutoff. Statistical significance was determined by using analysis of variance (ANOVA). A p value <0.05 was considered significant.RESULTSBiochemical Extraction of Proteins in Extracellular EnvironmentOur extraction methodology was developed on human aortic samples obtained during cardiac surgery for aortic valve replacement of degenerative aortic valve stenosis. Fig. 1A is a schematic summary of the methodology used in this study. The samples (n = 3) were macroscopically classified as normal during surgery, and upon histological examination, they showed no pathological signs. Extraction of the proteins of the vascular extracellular environment and reduction of the contamination with cellular proteins were achieved by a three-step extraction procedure. First, the tissue samples were treated with 0.5 m NaCl. The salt ions induce displacement of polyionic interactions between proteins, thus enabling the extraction of loosely bound extracellular proteins, including newly synthesized ECM proteins and degradation products (12.Mason R.M. Mayes R.W. Extraction of cartilage protein-polysaccharides with inorganic salt solutions.Biochem. J. 1973; 131: 535-540Crossref PubMed Scopus (21) Google Scholar), providing an overview of tissue turnover. The tissue was then decellularized using SDS. In contrast to other decellularization reagents, such as non-ionic detergents (Triton X-100) (13.Dahl S.L. Koh J. Prabhakar V. Niklason L.E. Decellularized native and engineered arterial scaffolds for transplantation.Cell Transplant. 2003; 12: 659-666Crossref PubMed Scopus (295) Google Scholar), 0.1% SDS has been previously shown to be very effective in solubilizing cytoplasmic and nuclear membranes, thereby removing cellular components from tissues (14.Korossis S.A. Wilcox H.E. Watterson K.G. Kearney J.N. Ingham E. Fisher J. In-vitro assessment of the functional performance of the decellularized intact porcine aortic root.J. Heart Valve Dis. 2005; 14: 408-422PubMed Google Scholar). However, the ionic detergent above its critical micelle concentration can also cause protein denaturation and disrupt native ECM proteins (15.Gilbert T.W. Sellaro T.L. Badylak S.F. Decellularization of tissues and organs.Biomaterials. 2006; 27: 3675-3683Crossref PubMed Scopus (1689) Google Scholar).To substantially remove the cellular material while preserving the ECM and its associated proteins as much as possible, we used 2.8 mm (0.08%) SDS in distilled water. This concentration is well below the critical micelle concentration of SDS in distilled water, which is 8 mm (16.Izano E.A. Wang H. Ragunath C. Ramasubbu N. Kaplan J.B. Detachment and killing of Aggregatibacter actinomycetemcomitans biofilms by dispersin B and SDS.J. Dent. Res. 2007; 86: 618-622Crossref PubMed Scopus (92) Google Scholar). In addition, at this concentration, SDS is effective in solubilizing cell membrane lipid bilayers (17.Tan A. Ziegler A. Steinbauer B. Seelig J. Thermodynamics of sodium dodecyl sulfate partitioning into lipid membranes.Biophys. J. 2002; 83: 1547-1556Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Treatment with 0.08% SDS was sufficient to remove most cellular components as shown by the marked reduction of nuclear staining (hematoxylin; Fig. 1B, blue). The cytoplasmic staining (eosin; pink) was also reduced, but other eosinophilic structures, such as collagen and elastin, were preserved in the tissue. Finally, the specimens were subjected to denaturing extraction in a 4 m guanidine HCl buffer. Guanidine HCl extractions were developed more than 40 years ago as a very effective way to solubilize most of the strongly bound ECM components, including large aggregating proteoglycans (versican, aggrecan, etc.), small proteoglycans (decorin, biglycan, etc.), cell attachment matrix glycoproteins (such as type VI collagen, fibronectins, and laminins), and basement membrane components (perlecan, type IV collagen, etc.). Guanidine induces disaggregation of ECM components by destabilizing the ionic, disulfide-dependent protein conformations (18.Sajdera S.W. Hascall V.C. Proteinpolysaccharide complex from bovine nasal cartilage. A comparison of low and high shear extraction procedures.J. Biol. Chem. 1969; 244: