Protein profiling of human lung telocytes and microvascular endothelial cells using iTRAQ quantitative proteomics

Telocytes (TCs) are described as a particular type of cells of the interstitial space (www.telocytes.com). Their main characteristics are the very long telopodes with alternating podoms and podomers. Recently, we performed a comparative proteomic analysis of human lung TCs with fibroblasts, demonstrating that TCs are clearly a distinct cell type. Therefore, the present study aims to reinforce this idea by comparing lung TCs with endothelial cells (ECs), since TCs and ECs share immunopositivity for CD34. We applied isobaric tag for relative and absolute quantification (iTRAQ) combined with automated 2-D nano-ESI LC-MS/MS to analyse proteins extracted from TCs and ECs in primary cell cultures. In total, 1609 proteins were identified in cell cultures. 98 proteins (the 5th day), and 82 proteins (10th day) were confidently quantified (screened by two-sample t-test, P < 0.05) as up- or down-regulated (fold change >2). We found that in TCs there are 38 up-regulated proteins at the 5th day and 26 up-regulated proteins at the 10th day. Bioinformatics analysis using Panther revealed that the 38 proteins associated with TCs represented cellular functions such as intercellular communication (via vesicle mediated transport) and structure morphogenesis, being mainly cytoskeletal proteins and oxidoreductases. In addition, we found 60 up-regulated proteins in ECs e.g.: cell surface glycoprotein MUC18 (15.54-fold) and von Willebrand factor (5.74-fold). The 26 up-regulated proteins in TCs at 10th day, were also analysed and confirmed the same major cellular functions, while the 56 down-regulated proteins confirmed again their specificity for ECs. In conclusion, we report here the first extensive comparison of proteins from TCs and ECs using a quantitative proteomics approach. Our data show that TCs are completely different from ECs. Protein expression profile showed that TCs play specific roles in intercellular communication and intercellular signalling. Moreover, they might inhibit the oxidative stress and cellular ageing and may have pro-proliferative effects through the inhibition of apoptosis. The group of proteins identified in this study needs to be explored further for the role in pathogenesis of lung disease.

Recently, electrophysiological properties were described for TCs [28][29][30], their microarray-based gene expression analysis and micr-oRNA signature were established [31,32] and some of their genomic features were revealed [33]. In a previous study, we reported the proteomic profile differences between TCs and fibroblasts [34].
Since their description, it became clear that TCs develop a 3D network through the organ interstitial space and are frequently detected in close relationships with organ-specific structures, blood capillaries, nerve endings and even with stem cells niches and immune cells [11,16,29]. Numerous studies have described the unusual immunophenotype of the TCs providing a list of molecular markers such as CD34, PDGFR a and b, CD117 [20,25,[35][36][37]. Some of these markers are also expressed on endothelial cells (low level of CD34) and on pericytes (PDGFR a and b). However ECs co-express CD31 and pericytes co-express a-SMA, while TCs do not [25]. These similarities might be suggestive for a common mesodermal pre-cursor for TCs, ECs and for a perivascular origin of mesenchymal stem cells (for reviews see [38][39][40][41][42]).
It is largely accepted that ECs in culture are subjected to phenotypic drift because of the lack of in vivo typical conditions [43], mainly oxygen exposure which is higher in vitro. These aspects together with the fact that proteomic studies also point out the differences between venous and arterial ECs [44] should lead us to the idea that in vitro culture studies should be viewed with circumspection without outlooking in vivo physiological influences. A study by Nguyen et al., regarding differential proteomic analysis of lymphatic, venous and arterial endothelial cells extracted from bovine mesenteric vessels underline the lack of substantial overlap between results from different research groups [45].
The present study shows the proteomic analysis of the TCs, by comparing them with ECs using iTRAQ labelling to identify the differentially expressed proteins. We think that the identification of a panel of 98 proteins at 5th day, and 82 proteins at 10th day in cell cultures, may represent the most differentially expressed proteins between TCs and ECs. We found that 38 proteins were overexpressed in TCs compared to ECs (at 5th day) and that 26 proteins were overexpressed in TCs compared to ECs (at 10th day). Bioinformatics analysis of the upregulated proteins came again to confirm the involvement of TCs in intercellular communication, oxidative stress and cellular ageing. Also, TCs may have pro-proliferative effects through the inhibition of apoptosis.

Cell lines and tissue sampling
Human lung samples were obtained from the patients undergoing surgery for lung cancer. Lung fragments were removed from normal tissue area located at least at 15 cm from the tumour tissue. All tissue samples were obtained in accordance with a protocol approved by the Ethical Evaluation Committee of Zhongshan Hospital, Fudan University, Shanghai, China. Samples were processed within 30 min. from surgery. Cells were cultured using the protocol previously described [34].
Human pulmonary microvascular endothelial cell line was obtained from ScienCell Research Laboratories (Cat. no. 3000; Carlsbad, CA, USA).

Cell culture and lysis
Cells from primary culture were used for the experiments. Cells (1 9 10 5 ) were placed in 10-cm dishes with 10 ml high glucose DMEM (Gibco, Grand Island, NY, USA) complete medium, including 10% foetal calf serum (Gibco, Grand Island, NY, USA), 100 UI/ml penicillin and 0.1 mg/ml streptomycin (Sigma Chemical, St. Louis, MO, USA) in a humidified atmosphere of 5% CO 2 at 370°C. Confluent cells were trypsinized at day 5 and day 10 respectively. Approximately 10 6 cells from day 5 or day 10 were re-suspended in a solution of 9.5 moles/l urea, 1% dithiothreitol, 40 mg/ml protease inhibitor cocktail, 0.2 mmoles/l Na 2 VO 3 and 1 mmole/l NaF. The mixture was incubated and stirred by end-over-end rotation at 4°C for 60 min. The resultant suspension was centrifuged at 40,000 9 g for 1 hr at 15°C. The supernatant was stored in small aliquots at À80°C, and the protein concentration was determined using a modified Bradford method.

Automated 2-D nano-ESI LC-MS/MS analysis of peptides
Proteins extracted from primary cultures of TCs and EC were analysed by automated 2-dimensional nano-electrospray ionization liquid chromatography tandem mass spectrometry as was previously described [46,47].

Sample preparation
The samples were ground in liquid nitrogen. One millilitre of lysis buffer (8 M urea, 19 Protease Inhibitor Cocktail (Roche Ltd. Basel, Switzerland)) was added to sample, followed by sonication on ice and centrifugation at 29,000 9 g for 10 min. at 4°C. The supernatant was transferred to a fresh tube, and stored at À80°C until needed.

iTRAQ labelling and protein digestion
For each sample, proteins were precipitated with ice-cold acetone, and then were redissolved in the dissolution buffer (0.5 M triethylammonium bicarbonate, 0.1% SDS). Then proteins were quantified by BCA protein assay, and 100 lg of protein was tryptically digested and the resultant peptide mixture was labelled using chemicals from the iTRAQ reagent kit (Applied Biosystems, Foster City, CA, USA). Disulphide bonds were reduced in 5 mM Tris-(2-carboxyethy) phosphine for 1 hr at 60°C, followed by blocking cysteine residues in 10 mM methyl methanethiosulfonate for 30 min. at room temperature, before digestion with sequence-grade modified trypsin (Promega, Madison, WI, USA). For labelling, each iTRAQ reagent was dissolved in 50 ll of isopropanol and added to the respective peptide mixture.
Proteins were labelled with the iTRAQ tags as follows: Pulmonary microvascular endothelial cells (5 days) -113 isobaric tag, TCs (5 days) -116 isobaric tag, Pulmonary microvascular endothelial cells ( High pH reverse phase separation Using a described protocol [48], the peptide mixture was redissolved in the buffer A (buffer A: 20 mM ammonium formate in water, pH 10.0, adjusted with ammonium hydroxide), and then fractionated by high pH separation using a Aquity UPLC system (Waters Corporation) connected to a reverse phase column (XBridge C18 column, 2.1 mm 9 150 mm, 3.5 lm, 300 A; Waters Corporation). High pH separation was performed with a linear gradient. Starting from 2% B to 40% B in 45 min. (B: 20 mM ammonium formate in 90% ACN, pH 10.0, adjusted with ammonium hydroxide). The column was re-equilibrated at initial conditions for 15 min. The column flow rate was maintained at 200 ll/min. and column temperature was maintained at room temperature. Fourteen fractions were collected, and each fraction was dried in a vacuum concentrator for the next step.

Low pH nano-HPLC-MS/MS analysis
The peptides were re-suspended with 80 ll solvent C (C: water with 0.1% formic acid; D: ACN with 0.1% formic acid), separated by nanoLC and analysed by on-line electrospray tandem mass spectrometry. The experiments were performed on a Nano Aquity UPLC system (Waters Corporation) connected to an LTQ Orbitrap XL mass spectrometer (Thermo Electron Corp., Bremen, Germany) equipped with an online nanoelectrospray ion source (Michrom Bioresources, Auburn, CA, USA). 20 ll peptide sample was loaded onto the Thermo Scientific Acclaim PepMap C18 column (100 lm 9 2 cm, 3 lm particle size), with a flow of 10 ll/min. for 5 min. and subsequently separated on the analytical column (Acclaim PepMap C18, 75 lm 9 15 cm) with a linear gradient, from 5% D to 45% D in 165 min. The column was re-equilibrated at initial conditions for 15 min. The column flow rate was maintained at 300 nl/min. and column temperature was maintained at 35°C. The electrospray voltage of 1.9 kV versus the inlet of the mass spectrometer was used.
LTQ Orbitrap XL mass spectrometer was operated after a protocol previously described [34].

Database searching and criteria
Protein identification and quantification for the iTRAQ experiment was performed with the ProteinPilot software version 4.0 (Applied Biosystems). The database was the Human UniProtKB/Swiss-Prot database (Release 2011_10_15, with 20248 sequences). The Paragon Algorithm in ProteinPilot software was used for peptide identification and isoform specific quantification. The detailed method of ProteinPilot analysis was described previously [34]. For iTRAQ quantification, the peptide for quantification was automatically selected by Pro Group algorithm (at least two peptides with 99% confidence) to calculate the reporter peak area, error factor and P-value. For the selection of differentially expressed proteins, we considered the following situation: (1) the proteins must contain at least two unique high-scoring peptides; (2) the proteins must have a P < 0.05, and the proteins identified with mass tag changes ratio must be ≥1.3 or ≤0.75. Differentially expressed proteins were screened by two-sample t-test (P < 0.05) and fold change (>2), based on the bioinformatics analysis.
The biological interpretation of the results was aided by PANTHER (Protein ANalysis THrough Evolutionary Relationships) Classification System annotations (http://www.pantherdb.org/).
Heat maps were created after MS/MS fragmentation spectra were analysed using PEAKS search engine tool (PEAKS Studio 7; Bioinformatics Solutions Inc., Waterloo, ON, Canada).
We also used the Search Tool for the Retrieval of Interacting Genes/ Proteins (STRING) (http://www.string-db.org/) database of physical and functional interactions to evaluate the interactions among the up-regulated proteins of the TCs and ECs. Bonferroni correction was used as a conservative method to control the family wise error rate when highlighting proteins involved in different biological processes.

Results
Quantitative proteomics has been used to evaluate the differentially expressed proteins in TCs and ECs. We compared the protein expression profiles between those two cell types, at different moments in time (5th day and 10th day in cell culture). In particular, we identified a total of 1609 proteins of which 98 satisfied our filtering criteria of proteins that exhibited fold changes ≥2 in TCs versus ECs at day 5 (Table 1), and 82 proteins in TCs versus ECs at day 10 ( Table 2), respectively.

Functional analysis of the identified proteins
The protein expression profiles were analysed with the aid of PAN-THER Classification System and depicted in Figures 1-3 (details are given in Tables S1 and S2). The highly expressed proteins in TCs are involved in important molecular functions such as: catalytic activity (17 proteins), structural molecule activity (13 proteins) as seen in Figure 1A compared to ECs where significantly more proteins are involved in catalytic activity (30 proteins) and 29 proteins have molecular binding function (Fig. 1B). In addition, in ECs, two upregulated proteins are involved in nucleic acid-binding transcription and one has anti-oxidant activity.
Interestingly to note, there are 6 up-regulated proteins in TCs compared to ECs involved in biological regulation and 5 related to response to stimulus.
The heat map showing the differentially expressed proteins between TCs and ECs, in cell culture after 5 days, can be observed in Figure 4.

Functional analysis of the identified proteins
The 26 up-regulated proteins found in TCs were assigned to the following biological processes (according to PANTHER): metabolic processes (16 proteins), cellular processes (11 proteins), developmental processes (8 proteins), cellular component organization (8 proteins), etc. - (Fig. 6A). The only cellular process which involve TCs up-regulated proteins is cell communication - Figure 6C. Eight up-regulated proteins in TCs are involved in one developmental process -anatomical structure morphogenesis - Figure 6E. Two proteins in TCs are related to localization processes such as vesicle mediated transport (1 protein) and protein transport (2 proteins) - Figure 6G.
The protein classes of the TCs enclose oxidoreductase (12 proteins), cytoskeletal proteins (8 proteins Figure 7F.
The heat map presenting the differentially expressed proteins between TCs and ECs is showed in Figure 8 and demonstrate that the differences between this two cell types are still preserved in cell culture after 10 days. Figures 9 and 10 use radar-chart representation of differentially expressed proteins between TCs and ECs at 5th day and at 10th day in cell cultures. Radar charts were chosen because they allow the visualization of a large numbers of proteins at the same time.
A String Network analysis was also performed to study the relation among differentially expressed proteins. In the global STRING-generated protein-protein network, several complexes and cellular functions formed prominent, tightly connected clusters as assessed by means of molecular complex detection (see Figs S1-S4). Figures 11 and 12 quantify protein-interaction properties of the TCs and ECs, respectively where the confidence view is presented and stronger associations are represented by thicker lines. These results indicate that while TCs are involved mainly in oxidation-reduction processes (Fig. 11A), ECs are involved (as expected) in haemostasis (Fig. 12A). Both cell types release extracellular vesicles (exosomes) [29,49], however their content is different as indicated in Figures 11B and 12B.

Discussion
Previously, we performed a proteomic analysis of human lung TCs compared to fibroblasts, at different time-points (the 5th and 10th day in primary cell culture) and we demonstrated that TCs protein expression profile is different [34]. The results were suggestive for specific roles of TCs in mechanical sensing and mechanic-chemical conversion task, tissue homoeostasis and remodelling/renewal. In addition, the presence of some proteins, specific for extracellular vesicles, emphasize TCs roles in intercellular signalling and stem cell niche modulation [19,34,50].
Beyond scientific interest in general, the comparison of TCs with ECs has a specific purpose. Both TCs and ECs are immunohistochemically CD34 positive, but while ECs are CD31 positive, TCs are CD31 negative. The present proteomic comparison confirms these immunohistochemical differences.

Putative roles of differentially expressed proteins
We previously showed that myosin-14 which is the main up-regulated protein in TCs make these cells candidates for a mechanical sensing and mechanochemical conversion task [34].
Telocytes proteome revealed the presence of SOD2 (SODM), a tetrameric anti-oxidative enzyme located in the mitochondrial matrix, encoded by genes located on chromosome 6 (6q25.3). The enzyme has manganese in its reactive centre, and catalyse the dismutation of superoxide (O 2 À ) into oxygen and hydrogen peroxide. SOD2 act as a cytoprotective enzyme proved to be essential for the survival of aerobic organisms [51]. It also serves as key anti-oxidant being considered a tumour suppressor protein via modulating redox-related transcriptional factors [52].
Acid ceramidase, (an enzyme encoded by the ASAH1 gene) which was found to be up-regulated in TCs, is located in lysosomes and active at acidic pH [53]. It was shown to have a noteworthy position in cancer biology: high AC activity leads to an enhanced cell growth, while low AC activity leads to reduced cell growth through an enhanced ceramide response [54]. Also, AC has been shown to play important roles in tumour pathogenesis, and in resistance to therapy having a key role in controlling the ceramide-sphingosine-sphingosine-1-phosphate (S1P) balance that regulates cellular homoeostasis [55]. Therefore, we can hypothesize that TCs might have pro-proliferative effects through the inhibition of apoptosis through the regulation of inter-conversion of ceramide, sphingosine and S1P.
Envoplakin is a protein that in humans is encoded by the EVPL gene, and it is a member of a family of large multi-domain molecules [56]. Periplakin (195 kD) and envoplakin (210 kD) are closely related  and have various functions to link cytoskeletal elements together and to connect them to junctional complexes. As we previously suggested, the presence of plakins in TCs is related to their homo and heterocellular junctions and it might be related to mechanical sensing and mechanochemical conversion task [34]. Plakins may also have additional roles in signal transduction [56].
Endothelial cells proteomic analysis revealed that proteins like microtubule-associated protein RP/EB family member 1, MUC18, cysteine-rich protein 2, von Willebrand factor (15.89-fold) and platelet endothelial cell adhesion molecule were found to be upregulated at 5th day and also at 10th day in ECs culture. Microtubule-associated protein RP/EB family member 1 is a ubiquitously expressed protein which binds to the plus end of microtubules and regulates the dynamics of the microtubule cytoskeleton, probably playing a role in cell migration [57]. MUC18 (CD146) is a glycoprotein detected in endothelial cells as a surface receptor that triggers a transient increase in the intracellular calcium concentration [58]. Cysteine-rich protein (CRP) 2 is a member of the LIMonly CRP family, also expressed in vascular smooth muscle cells (VSMCs) of blood vessels [59]. Its role is to repress VSMC migration and vascular remodelling, because it was demonstrated that the absence of CRP2 increases neointima formation, correlating with increased VSMC migration [60]. von Willebrand factor is a haemostatic protein stored in Weibel Palade bodies (considered as a hallmark of endothelial cells) until release [61]. In addition, we identified Ras-interacting protein 1 (RAIN) as being overexpressed in ECs. We can consider RAIN -known to be essential for endothelial cell morphogenesis and blood vessel tubulogenes -as being a part of the specific signature for ECs , in consistency with other recent proteomic study [62].
We found no significant differences between protein expression, in ECs, at 5 days and at 10 days. Our present results suggest that TCs are cells relatively rich in mitochondria, which correlates with previous findings [34]. The primary functions of mitochondria include: generating energy by oxidative phosphorylation, creating reactive oxygen species (ROS) and regulating apoptosis. It is also known that cellular ageing is influenced by oxidative phosphorylation, ROS and telomeres. Therefore, this study enabled us to suggest TCs involvement in the modulation of oxidative stress levels which might lead to a rigorous control in apoptosis activation. This finding is also in agreement with the fact that TCs are decreasing during ageing of myocardium (work in progress).
This study provides a comprehensive approach to analyse the comparative proteome between TCs and ECs and we can conclude that the significant discriminative power of each of the proteins mentioned above supports the case for TCs as distinctive cells, while ECs are characterized by the already known marker molecules such as MUC18 and von Willebrand factor. Also, it supports once more the idea of TCs involvement in tissue homoeostasis and in stem cell activity, as previously suggested by our group.
Moreover, it stands for a recent perspective suggested by Smithies and Edelstein considering TCs as a very primitive nervous system at the cellular level which can play a major role in morphogenetic bioelectrical signalling [63,64].
In conclusion, the current proteomic analysis presented here, clearly depicts that TCs are completely different from ECs. Protein expression profile demonstrates that TCs might play specific roles in intercellular signaling and also as physical and/or chemical sensors. Their close relationships with stem cells should not be overlooked.

Supporting information
Additional Supporting Information may be found in the online version of this article: