# Proteolytic Potential of the MSC Exosome Proteome: Implications for an Exosome-Mediated Delivery of Therapeutic Proteasome

Authors: Ruenn Chai Lai; Soon Sim Tan; Bao Ju Teh; Siu Kwan Sze; Fatih Arslan; Dominique P. de Kleijn; Andre Choo; Sai Kiang Lim
Journal: International Journal of Proteomics
 (2012)

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## Abstract

Mesenchymal stem cells (MSCs) are used in many of the current stem cell-based clinical trials and their therapeutic efficacy has increasingly been attributed to secretion of paracrine factors. We have previously demonstrated that a therapeutic constituent of this secretion is exosome, a secreted bilipid membrane vesicle of~50–100 nm with a complex cargo that is readily internalized by H9C2 cardiomyocytes. It reduces infarct size in a mouse model of myocardial ischemia/reperfusion (MI/R) injury. We postulate that this therapeutic efficacy is derived from the synergy of a select permutation of individual exosome components. To identify protein candidates in this permutation, the proteome was profiled and here we identified 20S proteasome as a protein candidate. Mass spectrometry analysis detected all seven α and seven β chains of the 20S proteasome, and also the three beta subunits of “immunoproteasome” with a very high confidence level. We demonstrated that a functional proteasome copurified with MSC exosomes with a density of 1.10–1.18 g/mL, and its presence correlated with a modest but significant reduction in oligomerized protein in a mouse model of myocardial infarction. Circulating proteasomes in human blood also copurified with exosomes. Therefore, 20S proteasome is a candidate exosome protein that could synergize with other constituents to ameliorate tissue damage.


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## Body

## 1. Introduction

Mesenchymal stem cells (MSCs) are multipotent fibroblast-like cells that reside in many adult tissues such as bone marrow adipose tissue [1, 2], liver [3], muscle connective tissue [4], amniotic fluid [5], placenta [6, 7], umbilical cord blood [1], and dental pulp [8, 9]. Although their differentiation potentials are primarily osteogenesis, chondrogenesis, and adipogenesis, MSCs have been reported to have the potential to differentiate into an amazing array of cell types that include nearly every major cell types in the adult body [10, 11]. MSCs are currently the most evaluated experimental stem cells with more than 100 clinical trials in 2010 to test their efficacy in treating a myriad of diseases such as cardiovascular diseases (e.g., acute myocardial infarction, endstage ischemic heart disease, or prevention of vascular restenosis), osteogenesis imperfecta (OI) or brittle bone disease, amyotrophic lateral sclerosis (ALS), lysosomal storage diseases (e.g., Hurler syndrome), steroid refractory graft versus host disease (GVHD), periodontitis and bone fractures [12].The use of MSCs as therapeutics was initially predicated on the hypothesis that transplanted MSCs home and engraft in injured tissues, and then differentiate into cells to replace damaged cells. However, it has been estimated that <1% of transplanted cells actually reached the target tissue with most of the cells being trapped in the liver, spleen, and lung [13], and reported evidence for differentiation of transplanted MSCs at the site of injury often cannot eliminate the possibility of cell fusion [14–16]. It has also been increasingly observed that the therapeutic efficacy of MSC therapy is not dependent on the engraftment of MSC at the site of injury or differentiation capability of the transplanted MSC [17–20], essentially eliminating the need for MSCs to be in the vicinity of their target tissue or to differentiate to exert a therapeutic effect. To reconcile this discrepancy between the therapeutic efficacy of MSC and the lack of MSC engraftment or differentiation at the site of injury, it was proposed that MSCs exert their therapeutic effects through secreted trophic mediators. The general acceptance of this proposal is reflected in the MSC clinical trials of 2010 in which 65 of the 101 clinical trials were rationalized on the trophic secretion of MSCs while only 36 were based on the differentiation potential of MSCs [21]. This paradigm shift in the therapeutic mechanism of MSC from one based on cell engraftment, differentiation and replacement to one based on secretion and paracrine signaling could potentially engender the development of biologic instead of cell-based therapeutics.In 2008, our group demonstrated that intravenous administration of a single bolus of culture medium conditioned by human embryonic stem cell-derived MSCs (hESC-MSCs) reduced relative infarct size in a pig and mouse model of ischemia/reperfusion injury [22]. By molecular weight fractionation of this conditioned medium (CM), we demonstrated that the active component had a presumptive size of 50–200 nm [22]. Using size exclusion high performance liquid chromatography (HPLC), we purified a population of homogenously sized particles that have the biophysical parameters of exosomes, namely, a hydrodynamic radius of 55–65 nm and a flotation density in sucrose of 1.10–1.18 g/mL. We subsequently demonstrated that this exosome population alone could reduce infarct size by ~40% in a mouse model of myocardial ischemia/reperfusion injury and therefore was the therapeutic agent in the secretion of mesenchymal stem cells [23]. Therapeutic exosomes are also found to be secreted by primary MSC cultures [24] and myc-immortalized hESC-MSCs [25]. The exosomes have exosome-associated proteins such as the tetraspanin proteins, CD9 and CD81, Alix, Tsg101, and RNA that consists primarily of short RNAs of less than 300 nt. Some of these RNAs are microRNAs that are predominantly premicroRNAs [26].As cells for example, H9C2 cardiomyocytes [26] readily internalized MSC exosomes, possibly by endocytosis, the exosome cargo of protein and RNA could be delivered across plasma membranes into cells. This diverse exosome cargo could, in principle provide a molecular basis for the therapeutic efficacy of MSC secretion in treating a diverse range of diseases (e.g., acute myocardial infarction, endstage ischemic heart disease, or prevention of vascular restenosis), osteogenesis imperfecta (OI) or brittle bone disease, amyotrophic lateral sclerosis (ALS), lysosomal storage diseases (e.g., Hurler syndrome), steroid refractory graft versus host disease (GVHD), periodontitis and bone fractures [12]. The cargo complexity of exosome could also provide a rationale for the efficacy of exosomes in treating complex tissue injuries such as myocardial ischemia/reperfusion injury that involves multiple tissues and targets.We believe that the individual components in exosome cargo are not equally or sufficiently efficacious in ameliorating tissue injury. We hypothesize that the therapeutic efficacy of MSC exosome against a specific injury is derived from the synergy of a select permutation of individual exosome components. To identify optimal permutation of biochemical activities for effective amelioration of myocardial ischemia/reperfusion injury and other injury, we are now systematically analyzing each of the candidate biochemical activities in MSC exosomes to first assess each activity and its potential to ameliorate injury.In this paper, we focused on the proteome of exosomes to identify candidate proteins or protein complexes that could drive their efficacy against diverse disease targets, the proteome of these purified exosomes was profiled here by mass spectrometry and antibody array, and found to contain 857 unique gene products (http://www.exocarta.org/). These proteins were distributed over a wide array of biochemical and cellular processes such as communication, structure and mechanics, inflammation, exosome biogenesis, tissue repair and regeneration and metabolism (Lai et al., submitted) consistent with the reported proteome complexity of MSC exosomes.A predominant feature of MSC exosome proteome was the presence of all sevenα and seven β chains of the 20S proteasome, and the three beta subunits of “immunoproteasome” which were identified with very high confidence level by mass spectrometry. These observations were indicative of fully assembled and possibly functional 20S proteasome and immunoproteasome which constitute the catalytic core of the 26S proteasome. The presence of 20S proteasome in exosomes also provides a mechanism for cellular extrusion of the relatively large intact 20S proteasomes as extracellular, circulating proteasome [27–31]. Circulating proteasomes are functionally active and could degrade small peptides. They have been shown to correlate with disease state or progression demonstrating that extracellular proteasomes have important physiological and pathological functions [27–31]. It has been postulated that these extracellular proteasomes are important in degrading soluble peptides in extracellular fluids and modulating potentially pathogenic protein aggregation such as Alzheimer’s amyloid plaques [32]. A key to understanding the functions of these extracellular proteasomes will be to determine how and when cells extrude proteasome. As the level of circulating proteasome does not correlate with lactate dehydrogenase it is unlikely that extracellular proteasomes are products of cell lysis [33].In this paper, we demonstrated that MSC exosomes contained functional 20S proteasomes and its presence correlated with a modest but significant reduction in oligomerized protein in a mouse model of myocardial infarction. Therefore, 20S proteasome is a candidate exosome protein that could synergize with other constituents to ameliorate tissue damage.


## 2. Materials and Methods

### 2.1. Preparation of Exosomes

Exosomes were purified from culture medium conditioned by huES9.E1, human ESC-derived mesenchymal stem cells [34] by HPLC as previously described [24, 35]. Briefly, CM collected from MSCs culture was concentrated 50x by tangential flow filtration (TFF) using a membrane with a 100 kDa MWCO (Sartorius, Goettingen, Germany). CM was fractionated by high performance liquid chromatography (HPLC) (TSK Guard column SWXL, 6 × 40 mm and TSK gel G4000 SWXL, 7.8 × 300 mm, Tosoh Corp., Tokyo, Japan). Exosomes were collected from the first peak of the elution and concentrated using 100 kDa MWCO filter (Sartorius). Exosomes were filtered with a 0.22 μm filter and stored in −20°C freezer until use.


### 2.2. LC MS/MS

Three independent preparations (biological replicates) of HPLC-purified exosomes were analyzed by LC-MS/MS over nine months using three different methods of tryptic digestion. In each preparation, about 2 mL proteins of dialyzed exosomes were used. In the first preparation, the sample was in-solution trypsin digested, that is, reduced, alkylated and tryptic digested as described [36]. After desalting using Sep-Pak C-18 SPE cartridge (Waters, Milford, MA, USA), the tryptic digest was directly analyzed with LC-MS/MS twice (two injections) without further fractionation. In the second preparation, the sample was separated by SDS-PAGE and the gel lane was cut into 8 slices for in gel digestion. Tryptic peptides from each gel slice were analyzed by LC-MS/MS. In the third preparation, the sample was tryptic digested and desalted. To reduce sample complexity, offline peptide fractionation was carried out with a HPLC system (Shimadzu, Japan) through a Polysulfoethyl SCX column (200 mm × 4.6 mm) (PolyLC, USA). Mobile phase A (5 mM KH4PO4 + 30% acetonitrile) and mobile phase B (5 mM KH4PO4 + 30% acetonitrile + 350 mM KCl) at 1 mL/min. Eight fractions were collected, desalted, and dried with a vacuum centrifuge. All three independent replicates were analyzed by a LC-MS/MS system including a Shimadzu micro HPLC system coupled online to a LTQ-FT Ultra linear ion trap mass spectrometer (Thermo Electron, Bremem, Germany) fitted with a nanospray source. Injected peptides were trapped and desalted in a Zorvax 300SB-C18 enrichment column (5 mm × 03 mm, Agilent Technologies, Germany) and eluted into a nano-bored C18 packed column (75 μm × 100 Å, Michrom Bioresources, Auburn, CA USA). A 90 minute gradient at a constant flow rate of 20 μL/min with a splitter to an effective flow rate of 300 nl/min was used to elute the peptides into the mass spectrometer. The LTQ was operated in a data-dependent mode by performing MS/MS scans for 8 of the most intense peaks from each MS scan in the FTMS. For each experiment, all MS/MS (dta) spectra of each replicate were combined into a single mascot generic file by a home-written program. Protein identification was achieved by searching the combined data against the IPI human protein database (version 3.34; 69,164 sequences, 29,064,825 residues) via an in-house Mascot server (Version 2.2.04, Matrix Science, UK). The search parameters were a maximum of 2 missed cleavages using trypsin; fixed modification was carbamidomethylation of cysteine and variable modification was oxidation of methionine. The mass tolerances were set to 10 ppm and 0.8 Da for peptide precursor and fragment ions, respectively. Protein identification was accepted as true positive if two different peptides were found to have scores greater than the homology scores.


### 2.3. Antibody Array

500μL of non-conditioned media and exosomes were assayed for the presence of cytokines andother proteins using RayBio Biotin Label-based Human Antibody Array I according to manufacturer’s instructions (RayBio, Norcross, GA). The cytokines and other proteins were considered to be present in the exosomes if the signal intensity was 2 fold higher (P<0.05) than that in non-conditioned medium.


### 2.4. Western Blot Hybridization

3μg of conditioned medium or exosomes were separated on 4–12% SDS-polyacrylamide gels and electroblotted onto a nitrocellulose membrane. The membrane was transferred to the membrane holder of SNAP i.d. system (Millipore, Billerica, MA), blocked and incubated with 1 : 200 diluted mouse anti-20S proteasome α1–7. The blot was then incubated with a 1 : 1250 diluted horseradish peroxidase-coupled goat anti-mouse IgG. All antibodies were obtained from Santa Cruz Biotechnology, Santa Cruz, CA. The blot was then incubated with HRP-enhanced chemiluminescent substrate (Thermo Fisher Scientific Inc., Waltham, MA, USA) and then exposed to X-ray film.


### 2.5. 20S Proteasome Enzymatic Assay

The proteasome activity was measured using a 20S proteasome activity assay kit (Millipore) based on detection of the fluorophore 7-Amino-4 methylcoumarin (AMC) after cleavage from the labeled substrate LLVY-AMC by 20S proteasome in the presence or absence of lactacystin, a specific 20S proteasome inhibitor. Briefly, 4μg of exosome was incubated with a reaction buffer containing LLVY-AMC in the presence or absence of 25 μM lactacystin. The samples and AMC standards were incubated at 37°C and fluorescence intensity at Ex/Em = 380/460 nm was monitored for 2 hours.


### 2.6. Animals and Experiments

Male C57Bl6/J (10–12 wks, 25–30 g) mice were obtained from Jackson Laboratory (Bar Harbor, USA). Mice received standard diet and water ad libitum. Myocardial infarction was induced between 8 am and 1 pm by left coronary artery ligation, just below the left atrial appendage. All animal experiments are performed in accordance with the national guidelines on animal care and with prior approval by the Animal Experimentation Committee of Utrecht University, The Netherlands.


### 2.7. Myocardial InfarctionIn Vivo

Mice were anesthetized with a mixture of Fentanyl (Jansen-Cilag) 0.05 mg/kg, Dormicum (Roche) 5 mg/kg and medetomidine 0.5 mg/kg through an intraperitoneal injection. Core body temperature was maintained around 37°C during surgery by continuous monitoring with a rectal thermometer connected to an automatic heating blanket. Mice were intubated and ventilated (Harvard Apparatus Inc.) with 100% oxygen. The left coronary artery (LCA) was ligated for 30 minutes using an 8-0 Ethilon (Ethicon) with a section of polyethylene-10 tubing placed over the LCA. Ischemia was confirmed by bleaching of the myocardium and ventricular tachyarrhythmia. Five minutes before reperfusion, mice were intravenously infused with 200μL saline-diluted exosome containing 0.4 μg protein via the tail vein. Control animals were infused with 200 μL saline. Reperfusion was initiated by releasing the ligature and removing the polyethylene-10 tubing. Reperfusion of the endangered myocardium was characterized by typical hyperemia in the first few minutes. The chest wall was closed and the animals received subcutaneously atipamezole (Antisedan, Pfizer) 2.5 mg/kg, flumazenil (Anexate, Roche) 0.5 mg/kg and buprenorphine (Temgesic, Schering-Plough) 0.1 mg/kg. At 15 min, 1 hour, 24 hours, or 3 days after reperfusion (n=4/group/time point), animals were sacrificed and the area at risk was excised for protein extraction. LCA ligation in mice results in anterior wall ischemia, while the septal wall is perfused by a septal coronary artery that originates separately from the sinus valsalva. The entire anterior wall of the left ventricle below the level of ligation was excised for area at risk protein extraction.


### 2.8. Antioligomer Dot Plot

Protein was extracted from area at risk of 30 min LCA ligated mouse heart treated with exosome or saline after 15 minutes, 1 hour, 1 day, and 3 days reperfusion using a cell extraction buffer from Biovision, Mountain View, CA, USA according to manufacturer’s instruction. Four animals were used at each time point in the exosome or saline treatment arm. Oneμg of each sample was applied on a nitrocellulose membrane, air dried, stained with Ponceau S, quantified using Image Lab (Bio-rab Laboratories) and destained. The membrane was then blocked with 10% non-fat dry milk TBST solution, incubated with a 1 : 1000 diluted rabbit anti-oligomer antibody (Invitrogen, Carlsbad, CA) for 2 hours and washed 3 times with TBST. This was followed by one hour incubation with a 1 : 2000 diluted horseradish peroxidase-coupled donkey anti-rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA). 3 washes with TBST and incubation with HRP-enhanced chemiluminescent substrate (Thermo Fisher Scientific Inc., Waltham, MA USA). The signal was scanned using ChemiDoc System (Bio-rab Laboratories, Philadelphia, PA) and quantified using Image Lab (Bio-rab Laboratories). The signal was normalized against the intensity of the Ponceu S stain.


### 2.9. Sucrose Density Gradient Equilibrium Centrifugation

To generate the sucrose density gradient for centrifugation, 14 sucrose solutions with concentrations from 22.8 to 60% were prepared and layered sequentially in an ultracentrifuge tube (Beckman Coulter Inc., CA, USA) starting with the most concentrated solution. Exosome or human plasma was loaded on top before ultracentrifugation for 16.5 h at 200 000 g, 4°C in a SW60Ti rotor (Beckman Coulter Inc.). After centrifugation, 13 fractions of 330μL each were collected sequentially starting from the top of the gradient. The densities of each fraction were determined by weighing a fixed volume. For pretreatment with detergent-based lysis buffer (Cell Extraction Buffer, Biovision), exosome was incubated with an equal volume of the lysis buffer containing protease inhibitors (Halt Protease Inhibitor Cocktail, Thermo Fisher Scientific) for 30 min on ice before loading on the sucrose density gradient. For subsequent western blot hybridization, 20 μL samples from each fraction were used. The mouse anti-CD63, -CD9, -CD81, and -CD59 antibodies, were obtained from Santa Cruz Biotechnology, Santa Cruz, CA, USA (1 : 60 dilution).


### 2.10. Liquid Phase Isoelectric Focusing

Liquid phase isoelectric focusing was performed using MicroRotofor, Liquid-Phase IEF Cell, from Bio-Rad (Bio-Rad Laboratories). 80μg exosome was added to 3 mL of 0.5% Bio-Lyte 3/10 Ampholyte (Bio-Rad Laboratories, diluted with PBS). The sample was loaded into the focusing chamber with 0.1 M phosphoric acid on anode and 0.1 M Sodium Hydroxide on cathode. Isoelectric focusing was performed for 215 min at 1 W constant power. The samples were then collected using vacuum suction into 10 fractions. The pH of each fraction was determined using pH indicator paper (Macherey-Nagel, Bethlehem, PA, USA) before concentrating to 25 μL using 100 kDa MWCO filter (Millipore).


### 2.11. Cholera Toxin B Precipitation

50μL of each human plasma fraction from sucrose gradient density equilibrium ultracentrifugation as described above was incubated with 0.005 μg biotinylated-cholera toxin B conjugated to streptavidin-conjugated magnetic beads (Invitrogen Corporation) for 30 min with gentle shaking followed by 30 min incubation The supernatant was then discarded and the magnetic beads were washed thrice with 0.1% BSA/PBS before incubating with 50 μL of detergent-based lysis buffer (Cell Extraction Buffer, Biovision) on ice for 10 min. The lysate was then collected and biotinylated by incubating with 4 μL 2.5 mM EZ-Link Sulfo-NHS-LC-LC-Biotin (Thermo Fisher Scientific) for 30 min with gentle shaking. The remaining reactive biotin was then quenched by adding 100 mM glycine buffer. The biotinylated lysate was then incubated with 5 μL anti-CD81 or -20S α1–7 conjugated magnetic beads (Invitrogen Corporation) for 30 min with gentle shaking. The supernatant was then discarded and the magnetic beads were washed thrice with 0.1% BSA/PBS before incubating with 50 μL of 1 : 5000 diluted HRP-conjugated streptavidin (Biolegend, San Diego, CA, USA) for 15 min at room temperature with gentle shaking. Finally the supernatant was discarded and the magnetic beads were washed thrice with 0.1% BSA/PBS. The amount of bound HRP was determined using Amplex UltraRed Reagent (Invitrogen Corporation) according to manufacturer’s instruction.


## 2.1. Preparation of Exosomes

Exosomes were purified from culture medium conditioned by huES9.E1, human ESC-derived mesenchymal stem cells [34] by HPLC as previously described [24, 35]. Briefly, CM collected from MSCs culture was concentrated 50x by tangential flow filtration (TFF) using a membrane with a 100 kDa MWCO (Sartorius, Goettingen, Germany). CM was fractionated by high performance liquid chromatography (HPLC) (TSK Guard column SWXL, 6 × 40 mm and TSK gel G4000 SWXL, 7.8 × 300 mm, Tosoh Corp., Tokyo, Japan). Exosomes were collected from the first peak of the elution and concentrated using 100 kDa MWCO filter (Sartorius). Exosomes were filtered with a 0.22 μm filter and stored in −20°C freezer until use.


## 2.2. LC MS/MS

Three independent preparations (biological replicates) of HPLC-purified exosomes were analyzed by LC-MS/MS over nine months using three different methods of tryptic digestion. In each preparation, about 2 mL proteins of dialyzed exosomes were used. In the first preparation, the sample was in-solution trypsin digested, that is, reduced, alkylated and tryptic digested as described [36]. After desalting using Sep-Pak C-18 SPE cartridge (Waters, Milford, MA, USA), the tryptic digest was directly analyzed with LC-MS/MS twice (two injections) without further fractionation. In the second preparation, the sample was separated by SDS-PAGE and the gel lane was cut into 8 slices for in gel digestion. Tryptic peptides from each gel slice were analyzed by LC-MS/MS. In the third preparation, the sample was tryptic digested and desalted. To reduce sample complexity, offline peptide fractionation was carried out with a HPLC system (Shimadzu, Japan) through a Polysulfoethyl SCX column (200 mm × 4.6 mm) (PolyLC, USA). Mobile phase A (5 mM KH4PO4 + 30% acetonitrile) and mobile phase B (5 mM KH4PO4 + 30% acetonitrile + 350 mM KCl) at 1 mL/min. Eight fractions were collected, desalted, and dried with a vacuum centrifuge. All three independent replicates were analyzed by a LC-MS/MS system including a Shimadzu micro HPLC system coupled online to a LTQ-FT Ultra linear ion trap mass spectrometer (Thermo Electron, Bremem, Germany) fitted with a nanospray source. Injected peptides were trapped and desalted in a Zorvax 300SB-C18 enrichment column (5 mm × 03 mm, Agilent Technologies, Germany) and eluted into a nano-bored C18 packed column (75 μm × 100 Å, Michrom Bioresources, Auburn, CA USA). A 90 minute gradient at a constant flow rate of 20 μL/min with a splitter to an effective flow rate of 300 nl/min was used to elute the peptides into the mass spectrometer. The LTQ was operated in a data-dependent mode by performing MS/MS scans for 8 of the most intense peaks from each MS scan in the FTMS. For each experiment, all MS/MS (dta) spectra of each replicate were combined into a single mascot generic file by a home-written program. Protein identification was achieved by searching the combined data against the IPI human protein database (version 3.34; 69,164 sequences, 29,064,825 residues) via an in-house Mascot server (Version 2.2.04, Matrix Science, UK). The search parameters were a maximum of 2 missed cleavages using trypsin; fixed modification was carbamidomethylation of cysteine and variable modification was oxidation of methionine. The mass tolerances were set to 10 ppm and 0.8 Da for peptide precursor and fragment ions, respectively. Protein identification was accepted as true positive if two different peptides were found to have scores greater than the homology scores.


## 2.3. Antibody Array

500μL of non-conditioned media and exosomes were assayed for the presence of cytokines andother proteins using RayBio Biotin Label-based Human Antibody Array I according to manufacturer’s instructions (RayBio, Norcross, GA). The cytokines and other proteins were considered to be present in the exosomes if the signal intensity was 2 fold higher (P<0.05) than that in non-conditioned medium.


## 2.4. Western Blot Hybridization

3μg of conditioned medium or exosomes were separated on 4–12% SDS-polyacrylamide gels and electroblotted onto a nitrocellulose membrane. The membrane was transferred to the membrane holder of SNAP i.d. system (Millipore, Billerica, MA), blocked and incubated with 1 : 200 diluted mouse anti-20S proteasome α1–7. The blot was then incubated with a 1 : 1250 diluted horseradish peroxidase-coupled goat anti-mouse IgG. All antibodies were obtained from Santa Cruz Biotechnology, Santa Cruz, CA. The blot was then incubated with HRP-enhanced chemiluminescent substrate (Thermo Fisher Scientific Inc., Waltham, MA, USA) and then exposed to X-ray film.


## 2.5. 20S Proteasome Enzymatic Assay

The proteasome activity was measured using a 20S proteasome activity assay kit (Millipore) based on detection of the fluorophore 7-Amino-4 methylcoumarin (AMC) after cleavage from the labeled substrate LLVY-AMC by 20S proteasome in the presence or absence of lactacystin, a specific 20S proteasome inhibitor. Briefly, 4μg of exosome was incubated with a reaction buffer containing LLVY-AMC in the presence or absence of 25 μM lactacystin. The samples and AMC standards were incubated at 37°C and fluorescence intensity at Ex/Em = 380/460 nm was monitored for 2 hours.


## 2.6. Animals and Experiments

Male C57Bl6/J (10–12 wks, 25–30 g) mice were obtained from Jackson Laboratory (Bar Harbor, USA). Mice received standard diet and water ad libitum. Myocardial infarction was induced between 8 am and 1 pm by left coronary artery ligation, just below the left atrial appendage. All animal experiments are performed in accordance with the national guidelines on animal care and with prior approval by the Animal Experimentation Committee of Utrecht University, The Netherlands.


## 2.7. Myocardial InfarctionIn Vivo

Mice were anesthetized with a mixture of Fentanyl (Jansen-Cilag) 0.05 mg/kg, Dormicum (Roche) 5 mg/kg and medetomidine 0.5 mg/kg through an intraperitoneal injection. Core body temperature was maintained around 37°C during surgery by continuous monitoring with a rectal thermometer connected to an automatic heating blanket. Mice were intubated and ventilated (Harvard Apparatus Inc.) with 100% oxygen. The left coronary artery (LCA) was ligated for 30 minutes using an 8-0 Ethilon (Ethicon) with a section of polyethylene-10 tubing placed over the LCA. Ischemia was confirmed by bleaching of the myocardium and ventricular tachyarrhythmia. Five minutes before reperfusion, mice were intravenously infused with 200μL saline-diluted exosome containing 0.4 μg protein via the tail vein. Control animals were infused with 200 μL saline. Reperfusion was initiated by releasing the ligature and removing the polyethylene-10 tubing. Reperfusion of the endangered myocardium was characterized by typical hyperemia in the first few minutes. The chest wall was closed and the animals received subcutaneously atipamezole (Antisedan, Pfizer) 2.5 mg/kg, flumazenil (Anexate, Roche) 0.5 mg/kg and buprenorphine (Temgesic, Schering-Plough) 0.1 mg/kg. At 15 min, 1 hour, 24 hours, or 3 days after reperfusion (n=4/group/time point), animals were sacrificed and the area at risk was excised for protein extraction. LCA ligation in mice results in anterior wall ischemia, while the septal wall is perfused by a septal coronary artery that originates separately from the sinus valsalva. The entire anterior wall of the left ventricle below the level of ligation was excised for area at risk protein extraction.


## 2.8. Antioligomer Dot Plot

Protein was extracted from area at risk of 30 min LCA ligated mouse heart treated with exosome or saline after 15 minutes, 1 hour, 1 day, and 3 days reperfusion using a cell extraction buffer from Biovision, Mountain View, CA, USA according to manufacturer’s instruction. Four animals were used at each time point in the exosome or saline treatment arm. Oneμg of each sample was applied on a nitrocellulose membrane, air dried, stained with Ponceau S, quantified using Image Lab (Bio-rab Laboratories) and destained. The membrane was then blocked with 10% non-fat dry milk TBST solution, incubated with a 1 : 1000 diluted rabbit anti-oligomer antibody (Invitrogen, Carlsbad, CA) for 2 hours and washed 3 times with TBST. This was followed by one hour incubation with a 1 : 2000 diluted horseradish peroxidase-coupled donkey anti-rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA). 3 washes with TBST and incubation with HRP-enhanced chemiluminescent substrate (Thermo Fisher Scientific Inc., Waltham, MA USA). The signal was scanned using ChemiDoc System (Bio-rab Laboratories, Philadelphia, PA) and quantified using Image Lab (Bio-rab Laboratories). The signal was normalized against the intensity of the Ponceu S stain.


## 2.9. Sucrose Density Gradient Equilibrium Centrifugation

To generate the sucrose density gradient for centrifugation, 14 sucrose solutions with concentrations from 22.8 to 60% were prepared and layered sequentially in an ultracentrifuge tube (Beckman Coulter Inc., CA, USA) starting with the most concentrated solution. Exosome or human plasma was loaded on top before ultracentrifugation for 16.5 h at 200 000 g, 4°C in a SW60Ti rotor (Beckman Coulter Inc.). After centrifugation, 13 fractions of 330μL each were collected sequentially starting from the top of the gradient. The densities of each fraction were determined by weighing a fixed volume. For pretreatment with detergent-based lysis buffer (Cell Extraction Buffer, Biovision), exosome was incubated with an equal volume of the lysis buffer containing protease inhibitors (Halt Protease Inhibitor Cocktail, Thermo Fisher Scientific) for 30 min on ice before loading on the sucrose density gradient. For subsequent western blot hybridization, 20 μL samples from each fraction were used. The mouse anti-CD63, -CD9, -CD81, and -CD59 antibodies, were obtained from Santa Cruz Biotechnology, Santa Cruz, CA, USA (1 : 60 dilution).


## 2.10. Liquid Phase Isoelectric Focusing

Liquid phase isoelectric focusing was performed using MicroRotofor, Liquid-Phase IEF Cell, from Bio-Rad (Bio-Rad Laboratories). 80μg exosome was added to 3 mL of 0.5% Bio-Lyte 3/10 Ampholyte (Bio-Rad Laboratories, diluted with PBS). The sample was loaded into the focusing chamber with 0.1 M phosphoric acid on anode and 0.1 M Sodium Hydroxide on cathode. Isoelectric focusing was performed for 215 min at 1 W constant power. The samples were then collected using vacuum suction into 10 fractions. The pH of each fraction was determined using pH indicator paper (Macherey-Nagel, Bethlehem, PA, USA) before concentrating to 25 μL using 100 kDa MWCO filter (Millipore).


## 2.11. Cholera Toxin B Precipitation

50μL of each human plasma fraction from sucrose gradient density equilibrium ultracentrifugation as described above was incubated with 0.005 μg biotinylated-cholera toxin B conjugated to streptavidin-conjugated magnetic beads (Invitrogen Corporation) for 30 min with gentle shaking followed by 30 min incubation The supernatant was then discarded and the magnetic beads were washed thrice with 0.1% BSA/PBS before incubating with 50 μL of detergent-based lysis buffer (Cell Extraction Buffer, Biovision) on ice for 10 min. The lysate was then collected and biotinylated by incubating with 4 μL 2.5 mM EZ-Link Sulfo-NHS-LC-LC-Biotin (Thermo Fisher Scientific) for 30 min with gentle shaking. The remaining reactive biotin was then quenched by adding 100 mM glycine buffer. The biotinylated lysate was then incubated with 5 μL anti-CD81 or -20S α1–7 conjugated magnetic beads (Invitrogen Corporation) for 30 min with gentle shaking. The supernatant was then discarded and the magnetic beads were washed thrice with 0.1% BSA/PBS before incubating with 50 μL of 1 : 5000 diluted HRP-conjugated streptavidin (Biolegend, San Diego, CA, USA) for 15 min at room temperature with gentle shaking. Finally the supernatant was discarded and the magnetic beads were washed thrice with 0.1% BSA/PBS. The amount of bound HRP was determined using Amplex UltraRed Reagent (Invitrogen Corporation) according to manufacturer’s instruction.


## 3. Results

### 3.1. Proteomic Profiling of Exosome

Proteomic profiling using mass spectrometry and antibody array approaches were performed as previously described [24, 35, 37] on three or one independent preparations of HPLC-purified exosomes, respectively. Mass spectrometric analysis of the three exosome preparations identified 379, 432, and 420 proteins, respectively (please refer to Supplementary File 1 for the LC MS/MS raw data). Of these, 154 (~20%) were present in all three preparations, 157 (~20%) were present in two while 455 (~59%) were present in only one (Figure 1(a)). These analyses were subsequently validated by western blot hybridization or biochemical assays using either the three exosome preparations used in the mass spectrometric analysis or other independently prepared exosomes. Several proteins determined by mass spectrometry to be present in two or one of the three preparations were found to be present in these or other subsequent preparations by either western blot hybridization or biochemical assays. For example, PFKFB3 and PGK were detected in only one and two of the three batches, respectively. However, when tested by western blot hybridization or biochemical assays, both proteins were found to be present in two or more of the three preparations and also subsequent exosome preparations (data not shown). This indicated that the three independent exosome preparations analyzed by LC-MS/MS within nine months interval using three different sample preparation methods were complementary to each other in identifying different sets of low abundant proteins or our selection criteria were too stringent. To ensure a more comprehensive coverage of the exosome proteome, we combined the 3 sets of mass spectrometry data. The analysis of the exosome proteome by antibody array identified 101 proteins of which 10 were also detected by mass spectrometry analysis (Figure 1(b)). By combining the results from mass spectrometry and antibody array, we determined that the exosome proteome has a total of 857 proteins (Table 1) and this dataset has been deposited at http://www.exocarta.org/. In Table 1, the regular font symbols represent the proteins identified by LC MS/MS; the underline symbols represent the proteins identified by antibody arrays; the bold and underline symbols represent the proteins identified by both LC MS/MS and antibody arrays; and lastly the grey shaded symbols represent the proteins that were found to be present in at least 50% of exosomes characterized [38]. Of these 857 proteins, 320 were found in the 739 proteins previously identified in the unfractionated conditioned medium [24, 35] (Figure 1(c)). The remaining 537 proteins in the exosome were detected only when HPLC-purified exosomes were used. Since exosomes constituted about 10% of the total proteins in the conditioned medium [35], we postulated that many of the exosome proteins were masked by the more abundant nonexosome proteins in the conditioned medium.Table 1
Proteomic profile of 3 independently prepared exosomes as determined by LC MS/MS and antibody arrays.
A2M
C11orf59
COPS4
FBXW8
HNRNPA1
KRT16
MMP3
PRR4
RPS3
TGFB2
ABI3BP
C1orf78
COPS8
FEN1
HP
KRT17
MOS
PRSS23
RPS4X
TGFBI
ACAA2
C1R
CPS1
FER1L3
HPX
KRT18
MPO
PSMA1
RPS5
TGM2
ACAT2
C1S
CREG1
FGA
HRSP12
KRT19
MPZL1
PSMA2
RPSA
TGOLN2
ACLY
C20orf114
CRIPT
FGB
HSP90AA1
KRT2
MRC2
PSMA3
RRAS2
THBS1
ACSL1
C3
CRTAP
FGF16
HSP90AB1
KRT27
MSN
PSMA4
RTN4
THBS2
ACTA1
C5orf24
CSF1
FGF18
HSP90B1
KRT28
MXRA5
PSMA5
RUVBL1
THY1
ACTA2
C9orf19
CSF2
FGF19
HSPA1A
KRT3
MYADM
PSMA6
S100A11
TIMP1
ACTB
C9orf91
CSF3
FGFRL1
HSPA1L
KRT4
MYCBPAP
PSMA7
S100A13
TIMP2
ACTG2
CACNA2D1
CSPG4
FGG
HSPA5
KRT5
MYH14
PSMB1
S100A8
TIMP3
ACTN1
CACNA2D4
CST4
FLG2
HSPA6
KRT6A
MYH9
PSMB10
S100P
TKT
ACTN2
CALR
CTA-221G9.4
FLJ13197
HSPA8
KRT6B
MYL6B
PSMB2
SAA4
TLN1
ACTN3
CAND1
CTBP2
FLJ22184
HSPB1
KRT6C
MYO1C
PSMB3
SASS6
TMBIM1
ACTN4
CAP1
CTNNA1
FLJ32784
HSPD1
KRT7
NBL1
PSMB4
SCAMP3
TMED10
ACTR1A
CAPNS1
CTNNA2
FLNA
HSPG2
KRT72
NEFH
PSMB5
SCGB2A1
TMED9
ACTR2
CAPZA1
CTNNB1
FLNB
HTRA1
KRT73
NEK10
PSMB6
SCYE1
TMEM16B
ACTR3
CASP14
CTNND1
FLNC
HYI
KRT74
NID1
PSMB7
SDC1
TMEM2
ADAM10
CAT
CTSG
FLOT1
ICAM1
KRT76
NLRP8
PSMB8
SDC2
TMEM47
ADAM9
CAV1
CXCL16
FLOT2
ICAM5
KRT77
NME1
PSMB9
SDC4
TMEM51
ADAMTS12
CCDC129
CXCL2
FLT1
IDH3B
KRT78
NOMO1
PSMC5
SDCBP
TNC
AEBP1
CCDC64B
CXorf39
FN1
IFITM2
KRT79
NRAS
PSMD11
SEC14L4
TNFRSF11B
AFM
CCL2
CYBRD1
FREM3
IFNG
KRT8
NRG2
PSMD14
SEMA5A
TNFRSF12A
AGRN
CCL20
DBF4B
FST
IFRD1
KRT80
NRLN1
PSMD6
SEPT2
TNFRSF1A
AHCY
CCL28
DCD
FTL
IFT140
KRT84
NRP1
PSMD7
SEPT7
TNFSF18
AHNAK2
CCL7
DCHS2
FUCA2
IGF2R
KRT9
NT5E
PTGFRN
SERINC5
TNFSF5
AHSG
CCR4
DCLK2
GALNT5
IGFBP3
LACRT
NTF5
PTK7
SERPINA1
TPBG
AKR1B1
CCR5
DCN
GANAB
IGFBP4
LAMA4
NUSAP1
PTPRK
SERPINB3
TPI1
AKR7A2
CCT5
DCTN1
GAPDH
IGFBP6
LAMB1
OBFC1
PTRF
SERPINE1
TRAP1
ALB
CCT6A
DECR1
GAPDHS
IGFBP7
LAMC1
ODZ3
PTTG1IP
SERPINE2
TREM1
ALCAM
CD109
DEFA1
GARS
IGHA1
LAMP1
OFD1
PTX3
SERPINF1
TREML2P
ALDH2
CD151
DIP2B
GAS6
IGHA2
LAMP2
OPRM1
PXDN
SFN
TRIM40
ALDH3A2
CD248
DIRAS2
GDF1
IGHG1
LAP3
OSM
PZP
SFRP1
TRIM41
ALDH6A1
CD276
DKFZp686D0972
GDF11
IGHG2
LCN1
OTC
QPCTL
SFRP4
TSN
ALDH7A1
CD44
DKK1
GDF3
IGHG4
LCN2
OXNAD1
QSOX1
SHANK3
TSNAX
ALDH9A1
CD47
DKK3
GDF5
IGHM
LDHA
OXTR
RAB10
SLAIN1
TSPAN14
ALDOA
CD59
DMBT1
GDF8
IGJ
LDHAL6B
P4HB
RAB11B
SLC16A1
TSPAN4
ALDOB
CD63
DNASE1L1
GDF9
IGKC
LDHB
PAICS
RAB14
SLC16A3
TSPAN6
ALDOC
CD81
DNPEP
GDI1
IGKV1-5
LEPRE1
PAN3
RAB15
SLC1A4
TSPAN9
ALOX12P2
CD82
DPYS
GDI2
IGL@
LGALS1
PAPPA
RAB1A
SLC1A5
TSTA3
ANG
CD9
DPYSL2
GFRA3
IGLV4-3
LGALS3
PARP10
RAB1B
SLC22A2
TTLL3
ANGPTL2
CDC2L5
DSP
GLDC
IGSF8
LGALS3BP
PARP16
RAB2A
SLC25A10
TTN
ANPEP
CDC42
DULLARD
GLUD1
IL10
LGALS8
PARVG
RAB33B
SLC25A13
TTYH3
ANXA1
CDH13
ECM1
GNA13
IL11
LGR6
PC
RAB35
SLC2A1
TUBA1A
ANXA11
CDIPT
ED1
GNAI2
IL13
LIF
PCOLCE
RAB39B
SLC2A3
TUBA1B
ANXA2
CDK5R2
EDG2
GNAL
IL15RA
LMNA
PDCD6
RAB5A
SLC38A2
TUBA1C
ANXA2P1
CEACAM8
EDIL3
GNAS
IL17B
LOC124220
PDCD6IP
RAB5B
SLC38A3
TUBB
ANXA3
CFB
EEA1
GNAT3
IL17R
LOC283523
PDGFA
RAB5C
SLC39A14
TUBB2A
ANXA4
CFI
EEF1A1
GNB1
IL19
LOC284297
PDGFC
RAB6A
SLC3A2
TUBB2C
ANXA5
CFL1
EEF1G
GNB2
IL1F9
LOC388344
PDGFRB
RAB7A
SLC44A1
TUBB3
ANXA6
CFL2
EEF2
GNB4
IL1RAP
LOC389827
PDIA3
RAB8A
SLC44A2
TUBB6
ANXA7
CFTR
EFEMP2
GNG12
IL1RAPL1
LOC442497
PEBP1
RAB8B
SLC7A10
UBA52
AP1S1
CHMP2A
EHD1
GNPDA1
IL1RL2
LOC653269
PFAS
RAC1
SLC7A5
UBB
APEH
CHST12
EHD2
GOT2
IL22RA1
LOC727942
PFKFB3
RAC2
SMARCA4
UBE1
APOA1
CITED1
EHD4
GPC1
IL23A
LOC728320
PFN1
RAD21
SMC1A
UBE2N
APOE
CLASP2
EIF4A1
GPC5
IL3
LOC728378
PFN2
RALA
SORT1
UGP2
APP
CLDN1
EMILIN1
GPI
IL5
LOC730013
PGAM2
RAN
SPACA1
UNC13B
ARF1
CLEC11A
ENG
GPR112
IL6ST
LRP1
PGD
RAP1A
SPARC
UNC45A
ARF4
CLIC1
ENO1
GREM1
IL7
LRP6
PGK1
RAP1B
SPOCK1
VAMP3
ARF5
CLIC6
ENO2
GRM2
IL8
LRRFIP2
PGLYRP2
RAP1B
SPRY4
VANGL1
ARHGAP18
CLPX
ENO3
GRM3
INHBA
LTBP1
PIGR
RAP2C
SPTAN1
VASN
ARHGAP23
CLSTN1
ENTPD4
GRM7
INHBB
LTBP2
PIP
RARRES1
SPTBN1
VAT1
ARHGDIA
CLTA
ENTPD4;LOXL2
GSN
INSR
LTF
PKM2
RASA1
SPTBN4
VCAN
ARHGEF1
CLTC
EPB41L3
GSTM1
IQGAP1
LYAR
PLAB
RASA4
SRGN
VCL
ARL6IP5
CLTCL1
EPHA2
GSTM2
ITGA11
LYZ
PLAU
RB1CC1
SRI
VCP
ARMS2
CLU
EPO
GSTM5
ITGA2
MADH4
PLEC1
RCOR2
SRPX2
VEGFC
ARPC3
CMIP
ESM1
GSTO1
ITGA3
MAMDC2
PLEKHG3
RDH5
ST6GALNAC6
VIL1
ARPC4
CNGB1
ETFB
GSTP1
ITGA4
MAP1A
PLOD1
RFTN1
STAT1
VIL2
ARPC5
COL12A1
F2R
GTPBP2
ITGA5
MAP2K6
PLOD2
RGN
STC1
VIM
ASH1L
COL14A1
F3
GYLTL1B
ITGAL
MAP3K1
PLOD3
RHOC
STC2
VTI1A
ASL
COL18A1
F8
GZMA
ITGAV
MARCKS
PLP2
RMND5A
STOM
VTN
ATP1A1
COL1A1
FADD
H2AFV
ITGB1
MARCKSL1
PLSCR3
RNF123
STOML3
WDR49
ATP1B3
COL1A2
FAH
H2AFX
ITGB5
MAT1A
PLTP
RNF40
STX12
WDR52
ATP2B1
COL2A1
FAM108A1
HBB
ITIH2
MBD3
PLUNC
RPL10A
STX2
WNT5A
ATP2B4
COL3A1
FAM129B
HBE1
ITIH4
MCC
PNO1
RPL12
SURF4
YBX1
ATP5A1
COL4A1
FAM29A
HDAC5
ITPR2
MCM10
PODN
RPL15
SVEP1
YWHAB
ATP5B
COL4A2
FAM3B
HERC5
JUP
MDH1
POLN
RPL18
SYT1
YWHAE
ATP8B3
COL4A3
FAM64A
HGF
KIAA0146
MDH2
POSTN
RPL23
SYT9
YWHAG
ATRN
COL5A1
FAM71F1
HGFR
KIAA0256
ME1
POTE2
RPL29
TAAR2
YWHAQ
ATXN1
COL5A2
FAP
HISPPD2A
KIAA0467
MECP2
PPIA
RPL35A
TAGLN
YWHAZ
AXL
COL6A1
FASN
HIST1H2AE
KIAA1881
MFAP4
PPIB
RPLP0
TALDO1
ZBTB4
BASP1
COL6A2
FAT
HIST1H2BA
KPNB1
MFGE8
PPME1
RPS10
TAS2R60
ZNF134
BDNF
COL6A3
FAT2
HIST1H2BL
KRT1
MFSD2
PPP1CC
RPS16
TCN1
ZNF503
BGN
COL7A1
FAT4
HIST1H4H
KRT10
MIF
PRDM16
RPS18
TF
ZNF614
BHMT2
COMP
FBLN1
HIST2H2BE
KRT13
MMP1
PRDX1
RPS2
TFG
BRMS1
COPB1
FBN1
HLAA
KRT14
MMP10
PRDX6
RPS24
TFRC
BSG
COPS3
FBN2
HMGCS2
KRT15
MMP2
PRNP
RPS27A
TGFB1
Regular font: identified by LC MS/MS.
Underline:
identified by antibody arrays.
Bold and Underline:
identified by both LC MS/MS and antibody arrays.
Bold:
identified by LC MS/MS and was found to be present in at least 50% of exosomes characterized.Figure 1
Exosome proteins. (a) Venn diagram of number of proteins detected by LC-MS/MS in 3 independent batches of exosomes, CM15, CM17, and CM18. There are 379, 432, and 420 proteins detected in CM15, CM17 and CM18, respectively. The combined protein number is 766. (b) Venn diagram shows the overlap of proteins detected by LC-MS/MS and antibody array. There are 101 proteins detected by antibody array in CM15. 10 of the 101 proteins detected are overlapping with proteins detected by LC-MS/MS. (c) Intersection of the 739 proteins previously identified in MSC conditioned medium versus the 857 proteins identified in purified exosomes.Based on 15 proteomic analyses carried out on exosomes purified from cultured cells and from biological fluids by different groups, Thery et al. had observed that a set of about 17 proteins, namely glyceraldehyde 3-phosphate dehydrogenase (GAPDH), pyruvate kinase (PK), eukaryotic translation elongation factor 1A1 (EEF1A1), milk fat globule EGF factor 8 protein (MFGE8), tetraspanins, 14-3-3 proteins, Gα proteins, clathrin, Alix (PDCD6IP), MHC class1, annexins (ANX), Rab proteins, ezrin(VIL2), radixin(RDX) and moesin (MSN)(ERM), actin, tubulin, HSP70, and HSP90 were found to be present in at least 50% of the exosomes that were characterized [38]. Not unexpectedly, most of these proteins were also found in the proteome of the HPLC-purified MSC exosomes (Table 1). Also consistent with the endosomal origin of exosomes, we detected the presence of endosome-associated proteins such as Alix (PDCD6IP) and Rab (Table 1).To better understand the biological significance of the proteins in the exosomes, we had previously performed functional clustering of the 857 proteins into biological processes using PANTHER (protein analysis through evolutionary relationships) analytical software [39, 40] (Lai et al.; submitted) The observed frequency of genes from the exosome proteome in each biological process was compared with the reference frequency of 25431 Homo sapiensgene list in the NCBI database for that biological process. The 857 gene products could be clustered into 32 biological processes that were over-represented (P<0.001) and 3 that were under-represented (P<0.001) (Lai et al.; submitted). Many of these biological processes are consistent with activities associated with exosome biology for example, communication, cellular motility, inflammation, and exosome biogenesis. One predominant biological process that could not be definitively associated with exosome biology is proteolysis (P=3.76×10-10) and of the unique genes clustered in this process, ~22% (17 out of 78) encode for peptides in the 20S proteasome or immunoproteasome. To ensure the robustness of this observation, examination of the 3 sets of mass spectrometry datasets revealed that with only one exception of PSMB5 in the first exosome preparation, all the 14 peptides of 20S proteasome were detected with very high confidence in all three exosome preparations as demonstrated by the Mascot search results, number of identified peptides with score higher than homology or identity scores, as well as the MS/MS spectra and fragment ions assignment of unique identified peptides (Supplementary data 1–7).


### 3.2. Presence of 20S Proteasome in MSC Exosome

Mass spectrometry analysis of MSC exosomes not only detected the presence of all sevenα- (PSMA1–7) and all seven β-subunits (PSMB1–7) of the 20S core particle with very high confidence (See Supplementary Material available online at doi:10.1155/2012/971907 for the representative spectra, number of peptide detected, and the peptide score of them), but also the three beta subunits of “immunoproteasome,” PSMB8 (β5i or LMP7), PSMB9 (β1i or LMP2), PSMB10 (β2i or LMP10) gene product [41]. To form the 26S proteasome, the 20S core particle must be complemented by other peptides. Although some of these peptides such as PSMC5, PSMD6, PSMD7, PSMD11, and PSMD14 were present, they were not sufficient to form the 26S proteasome. The presence of some of the 20S proteasome peptides was further confirmed by western blot hybridization (Figure 2(a)). As exosomes are known to have a typical density range of 1.10 to 1.18 gmL−1 that could be resolved on sucrose gradients [42], we checked whether these 20S proteasome subunits have the typical exosome flotation density and postulated that the flotation densities of the 20S proteasome subunits would be different before and after release from such vesicles by a detergent-based buffer. Fractionation of exosomes or exosomes pretreated with a detergent-based lysis buffer on a sucrose density gradient by equilibrium ultracentrifugation revealed that 20S proteasome subunits had a similar flotation density as that of exosome-associated proteins, namely, CD9, CD81, CD59, and CD63. All of them had an exosomes-like flotation density of 1.10–1.18 g/mL (Figure 2(b)). Pretreatment with a detergent-based cell lysis buffer decreased the apparent flotation densities of these proteins. Liquid phase isoelectric focusing of exosomes further revealed that 20S proteasome had a similar protein isoelectric point (pI) as that of exosome-associated CD9 (Figure 2(c)). Together the exosome-like flotation density and pI of the 20S proteasome suggested that the 20S proteasome was associated with exosomes and not a contaminant of exosome preparation.Figure 2
20S proteasome in MSC exosome. (a) Western blot analysis of MSC conditioned medium (CM) and exosome (Exo) using an antibody specific for PMSA 1–7 peptides. (b) Protein analysis of exosome fractionated on a sucrose gradient density. Exosome or exosome pretreated with lysis buffer was loaded on a sucrose density gradient prepared by layering 14 sucrose solutions of concentrations from 22.8 to 60% (w/v) in a SW60Ti centrifuge tube and then ultracentrifuged for 16.5 h at 200 000 g, 4°C, in a SW60Ti rotor. The gradients were removed from the top and the density of each fraction was calculated by weighing a fixed volume of each fraction. The fractions were analyzed by western blot analysis for CD9, PSMA1–7, CD81, CD59, and CD63 in exosome (upper panel) and pretreated exosomes (lower panel). (c) pI of 20S proteasome and CD9 in exosome. Exosome sample was separated by liquid-phase isoelectric focusing into 10 fractions from pH1–14. Each fraction was then concentrated and analyzed by western blot analysis for CD9, PSMA1–7. (d) Proteasome activity in MSC exosome was determined using a commercially available proteasome activity assay kit as described in the Materials and Methods section. Proteasome activity was measured by the rate of degradation of a fluorogenic peptide in the absence or presence of lactacystin, a proteasome inhibitor. One unit (U) enzyme activity is defined as the activity to generate 1μmole product per minute at 37°C. Each bar represents mean ± SEM of 2 independent assays with each assay performed in triplicate. *P=0.00023.


### 3.3. Enzymatic Activity of 20S Proteasome in MSC Exosome

The presence of all sevenα- and all seven β-subunits of the 20S core particle indicates that MSC exosomes contain intact 20S proteasome complexes and therefore 20S proteasome enzymatic activity. To test if exosomes contain functional 20S proteasome, purified MSC exosomes were incubated with a fluorophore-labeled peptide, LLVY-AMC (a substrate for the chymotrypsin-like activity; AMC, 7-amido 4-methylcoumarin) [43], that upon cleavage by 20S proteasome released fluorescent AMC. The fluorescence produced by the released AMC would be directly proportional to the proteasome activity. Based on this assay, the 20S proteasome enzymatic activity in MSC exosomes was determined to be 5.00 μU/μg protein where one unit (U) enzyme activity is defined as the activity to generate 1 μmole product per minute at 37°C. This degradation was inhibited by lactacystin, a proteasome-specific inhibitor (Figure 2(d)).


### 3.4. Exosome Proteasome Reduced Misfolded ProteinsIn Vivo

To determine if exosome proteasome can reduce misfolded or aberrant proteinsin vivo in a disease model and possibly contribute to a therapeutic outcome, we measured the accumulation of misfolded proteins in heart tissue of a mouse model of myocardial ischemia/reperfusion injury with and without exosome treatment.This model is most appropriate for our aim as accumulation of misfolded proteins has been recognized as an important contributor to tissue damage during myocardial ischemia/reperfusion injury [44–48] (reviewed [49]) and we have previously shown that exosome treatment resulted in extensive cardioprotection [23]. Furthermore, in our hands, the area at risk in this mouse model is also highly reproducible [23, 24, 50–52].To determine if exosome could reduce the amount of misfolded proteins, we used a conformation-dependent antibody [53] to quantify the amount of misfolded proteins in the area at risk of ischemic mouse heart after 15 min, one hour, one day and three days reperfusion. The level of denatured or misfolded protein was assayed using an antibody against oligomers. Denatured or partially unfolded proteins tend to self-associate to form high molecular aggregates such as oligomers and these oligomers have a common structure that is independent of amino acid sequence [54]. An antibody known as A11 that was raised against synthetic Aβ oligomers was found to specifically recognize this common structure present in misfolded protein oligomers but did not recognize monomers or mature fibers of proteins or peptides [54]. It will react with soluble Aβ40 oligomers and does not react with soluble low molecular weight Aβ40 or Aβ40 fibrils.Using this antibody, we found that those mice treated with exosome had a modest but significantly lower level of misfolded proteins (P=0.037) (n=4 per time point) 1 day after reperfusion compared with those saline treated controls (n=4 per time point) (Figure 3). This demonstrated that MSC exosome has the potential to reduce misfolded proteins in vivo and may contribute to the exosome-mediated cardioprotection.Exosome reduced oxidized proteinin vivo. Myocardial ischemia/reperfusion injury was induced by ligation of the left coronary artery (LCA) for 30 min and subsequent reperfusion by releasing the ligation. Five minutes before reperfusion, mice (n=4/group/time point) were intravenously infused with exosome or saline. At 15 min, 1 hour, 1 day, or 3 days after reperfusion, the animal was scarified and the area at risk (AAR) was excised and extracted for protein. One μg of each sample was applied onto a nitrocellulose membrane, stained with Ponceau S, destained and probed with rabbit antioligomer antibody and a HRP reporting system as described in material and method. The intensity of the antibody binding was quantified using Image Lab and normalized against the intensity of Ponceau S staining. (a) Representative dot plots. (b) Each bar represented mean ± SEM of 4 animal.
(a)
(b)


### 3.5. Association of Plasma 20S Proteasome with Exosome

Although our experimental observations strongly supported the presence of functional 20S proteasome in MSC exosomes, the association of 20S proteasome with exosomes could be a culture artifact contaminant. To test this possibility, we determined if circulating 20S proteasome in human plasma is associated with exosomes. 20S proteasomes are widely reported to be present in plasma [27–31] and we confirmed this by immunoblotting plasma for the presence of proteasome subunits (Figure 4(a)). Fractionation of plasma on a sucrose density gradient revealed that the plasma 20S proteasome fractionated at the flotation density of exosome that is, 1.10–1.18 g/mL (Figure 4(a)). CD81, a tetraspanin protein commonly found on exosome membrane also floated at this density. Each of the fractions was then extracted with cholera toxin B chain (CTB) which binds GM1 ganglioside, a glycolipid found to be enriched in the membrane of exosomes [55] and also MSC exosomes (unpublished data). CTB together with cholera toxin A chain forms the cholera toxin. Cholera toxin enters the cytosol by binding GM1 ganglioside through CTB [56]. This cellular entry of cholera toxin is a well characterized highly specific receptor mediated pathway [57]. The CTB extract was then assayed by ELISA for 20S proteasome subunits and CD81. 20S proteasome subunits and CD81 were both found to be present in the CTB extraction (Figure 4(b)). These observations demonstrated that plasma 20S proteasome colocalized with CD81 in GM1 ganglioside-enriched complex that floats at 1.10–1.18 g/mL and that at least some of the circulating proteasomes in the plasma are associated with exosomes. The presence of 20S proteasome in MSC exosomes and plasma exosomes together implicated exosome as a normal physiological conduit for cellular extrusion of 20S proteasome.Human plasma contained exosome-associated 20S proteasome. (a) Human plasma was loaded on a sucrose density gradient prepared by layering 14 sucrose solutions of concentrations from 22.8 to 60% (w/v) in a SW60Ti centrifuge tube and then ultracentrifuged for 16.5 h at 200 000 g, 4°C, in a SW60Ti rotor. 330μL fractions were removed sequentially from the top and the density of each fraction was calculated by weighing a fixed volume of each fraction. The fractions were analyzed by Western blot analysis for PSMA1–7. (b) Relative amount of CD81 and PSMA1–7 in plasma exosome. Exosomes which are known to have membrane enriched in GM1 gangliosides were isolated from the sucrose density gradient by the high affinity of GM1 gangliosides for cholera toxin B (CTB) chains conjugated to magnetic beads. The CTB extract was assayed by ELISA for the amount of PSMA1–7 and CD81. Each point represents mean ± SEM (n=2).
(a)
(b)


## 3.1. Proteomic Profiling of Exosome

Proteomic profiling using mass spectrometry and antibody array approaches were performed as previously described [24, 35, 37] on three or one independent preparations of HPLC-purified exosomes, respectively. Mass spectrometric analysis of the three exosome preparations identified 379, 432, and 420 proteins, respectively (please refer to Supplementary File 1 for the LC MS/MS raw data). Of these, 154 (~20%) were present in all three preparations, 157 (~20%) were present in two while 455 (~59%) were present in only one (Figure 1(a)). These analyses were subsequently validated by western blot hybridization or biochemical assays using either the three exosome preparations used in the mass spectrometric analysis or other independently prepared exosomes. Several proteins determined by mass spectrometry to be present in two or one of the three preparations were found to be present in these or other subsequent preparations by either western blot hybridization or biochemical assays. For example, PFKFB3 and PGK were detected in only one and two of the three batches, respectively. However, when tested by western blot hybridization or biochemical assays, both proteins were found to be present in two or more of the three preparations and also subsequent exosome preparations (data not shown). This indicated that the three independent exosome preparations analyzed by LC-MS/MS within nine months interval using three different sample preparation methods were complementary to each other in identifying different sets of low abundant proteins or our selection criteria were too stringent. To ensure a more comprehensive coverage of the exosome proteome, we combined the 3 sets of mass spectrometry data. The analysis of the exosome proteome by antibody array identified 101 proteins of which 10 were also detected by mass spectrometry analysis (Figure 1(b)). By combining the results from mass spectrometry and antibody array, we determined that the exosome proteome has a total of 857 proteins (Table 1) and this dataset has been deposited at http://www.exocarta.org/. In Table 1, the regular font symbols represent the proteins identified by LC MS/MS; the underline symbols represent the proteins identified by antibody arrays; the bold and underline symbols represent the proteins identified by both LC MS/MS and antibody arrays; and lastly the grey shaded symbols represent the proteins that were found to be present in at least 50% of exosomes characterized [38]. Of these 857 proteins, 320 were found in the 739 proteins previously identified in the unfractionated conditioned medium [24, 35] (Figure 1(c)). The remaining 537 proteins in the exosome were detected only when HPLC-purified exosomes were used. Since exosomes constituted about 10% of the total proteins in the conditioned medium [35], we postulated that many of the exosome proteins were masked by the more abundant nonexosome proteins in the conditioned medium.Table 1
Proteomic profile of 3 independently prepared exosomes as determined by LC MS/MS and antibody arrays.
A2M
C11orf59
COPS4
FBXW8
HNRNPA1
KRT16
MMP3
PRR4
RPS3
TGFB2
ABI3BP
C1orf78
COPS8
FEN1
HP
KRT17
MOS
PRSS23
RPS4X
TGFBI
ACAA2
C1R
CPS1
FER1L3
HPX
KRT18
MPO
PSMA1
RPS5
TGM2
ACAT2
C1S
CREG1
FGA
HRSP12
KRT19
MPZL1
PSMA2
RPSA
TGOLN2
ACLY
C20orf114
CRIPT
FGB
HSP90AA1
KRT2
MRC2
PSMA3
RRAS2
THBS1
ACSL1
C3
CRTAP
FGF16
HSP90AB1
KRT27
MSN
PSMA4
RTN4
THBS2
ACTA1
C5orf24
CSF1
FGF18
HSP90B1
KRT28
MXRA5
PSMA5
RUVBL1
THY1
ACTA2
C9orf19
CSF2
FGF19
HSPA1A
KRT3
MYADM
PSMA6
S100A11
TIMP1
ACTB
C9orf91
CSF3
FGFRL1
HSPA1L
KRT4
MYCBPAP
PSMA7
S100A13
TIMP2
ACTG2
CACNA2D1
CSPG4
FGG
HSPA5
KRT5
MYH14
PSMB1
S100A8
TIMP3
ACTN1
CACNA2D4
CST4
FLG2
HSPA6
KRT6A
MYH9
PSMB10
S100P
TKT
ACTN2
CALR
CTA-221G9.4
FLJ13197
HSPA8
KRT6B
MYL6B
PSMB2
SAA4
TLN1
ACTN3
CAND1
CTBP2
FLJ22184
HSPB1
KRT6C
MYO1C
PSMB3
SASS6
TMBIM1
ACTN4
CAP1
CTNNA1
FLJ32784
HSPD1
KRT7
NBL1
PSMB4
SCAMP3
TMED10
ACTR1A
CAPNS1
CTNNA2
FLNA
HSPG2
KRT72
NEFH
PSMB5
SCGB2A1
TMED9
ACTR2
CAPZA1
CTNNB1
FLNB
HTRA1
KRT73
NEK10
PSMB6
SCYE1
TMEM16B
ACTR3
CASP14
CTNND1
FLNC
HYI
KRT74
NID1
PSMB7
SDC1
TMEM2
ADAM10
CAT
CTSG
FLOT1
ICAM1
KRT76
NLRP8
PSMB8
SDC2
TMEM47
ADAM9
CAV1
CXCL16
FLOT2
ICAM5
KRT77
NME1
PSMB9
SDC4
TMEM51
ADAMTS12
CCDC129
CXCL2
FLT1
IDH3B
KRT78
NOMO1
PSMC5
SDCBP
TNC
AEBP1
CCDC64B
CXorf39
FN1
IFITM2
KRT79
NRAS
PSMD11
SEC14L4
TNFRSF11B
AFM
CCL2
CYBRD1
FREM3
IFNG
KRT8
NRG2
PSMD14
SEMA5A
TNFRSF12A
AGRN
CCL20
DBF4B
FST
IFRD1
KRT80
NRLN1
PSMD6
SEPT2
TNFRSF1A
AHCY
CCL28
DCD
FTL
IFT140
KRT84
NRP1
PSMD7
SEPT7
TNFSF18
AHNAK2
CCL7
DCHS2
FUCA2
IGF2R
KRT9
NT5E
PTGFRN
SERINC5
TNFSF5
AHSG
CCR4
DCLK2
GALNT5
IGFBP3
LACRT
NTF5
PTK7
SERPINA1
TPBG
AKR1B1
CCR5
DCN
GANAB
IGFBP4
LAMA4
NUSAP1
PTPRK
SERPINB3
TPI1
AKR7A2
CCT5
DCTN1
GAPDH
IGFBP6
LAMB1
OBFC1
PTRF
SERPINE1
TRAP1
ALB
CCT6A
DECR1
GAPDHS
IGFBP7
LAMC1
ODZ3
PTTG1IP
SERPINE2
TREM1
ALCAM
CD109
DEFA1
GARS
IGHA1
LAMP1
OFD1
PTX3
SERPINF1
TREML2P
ALDH2
CD151
DIP2B
GAS6
IGHA2
LAMP2
OPRM1
PXDN
SFN
TRIM40
ALDH3A2
CD248
DIRAS2
GDF1
IGHG1
LAP3
OSM
PZP
SFRP1
TRIM41
ALDH6A1
CD276
DKFZp686D0972
GDF11
IGHG2
LCN1
OTC
QPCTL
SFRP4
TSN
ALDH7A1
CD44
DKK1
GDF3
IGHG4
LCN2
OXNAD1
QSOX1
SHANK3
TSNAX
ALDH9A1
CD47
DKK3
GDF5
IGHM
LDHA
OXTR
RAB10
SLAIN1
TSPAN14
ALDOA
CD59
DMBT1
GDF8
IGJ
LDHAL6B
P4HB
RAB11B
SLC16A1
TSPAN4
ALDOB
CD63
DNASE1L1
GDF9
IGKC
LDHB
PAICS
RAB14
SLC16A3
TSPAN6
ALDOC
CD81
DNPEP
GDI1
IGKV1-5
LEPRE1
PAN3
RAB15
SLC1A4
TSPAN9
ALOX12P2
CD82
DPYS
GDI2
IGL@
LGALS1
PAPPA
RAB1A
SLC1A5
TSTA3
ANG
CD9
DPYSL2
GFRA3
IGLV4-3
LGALS3
PARP10
RAB1B
SLC22A2
TTLL3
ANGPTL2
CDC2L5
DSP
GLDC
IGSF8
LGALS3BP
PARP16
RAB2A
SLC25A10
TTN
ANPEP
CDC42
DULLARD
GLUD1
IL10
LGALS8
PARVG
RAB33B
SLC25A13
TTYH3
ANXA1
CDH13
ECM1
GNA13
IL11
LGR6
PC
RAB35
SLC2A1
TUBA1A
ANXA11
CDIPT
ED1
GNAI2
IL13
LIF
PCOLCE
RAB39B
SLC2A3
TUBA1B
ANXA2
CDK5R2
EDG2
GNAL
IL15RA
LMNA
PDCD6
RAB5A
SLC38A2
TUBA1C
ANXA2P1
CEACAM8
EDIL3
GNAS
IL17B
LOC124220
PDCD6IP
RAB5B
SLC38A3
TUBB
ANXA3
CFB
EEA1
GNAT3
IL17R
LOC283523
PDGFA
RAB5C
SLC39A14
TUBB2A
ANXA4
CFI
EEF1A1
GNB1
IL19
LOC284297
PDGFC
RAB6A
SLC3A2
TUBB2C
ANXA5
CFL1
EEF1G
GNB2
IL1F9
LOC388344
PDGFRB
RAB7A
SLC44A1
TUBB3
ANXA6
CFL2
EEF2
GNB4
IL1RAP
LOC389827
PDIA3
RAB8A
SLC44A2
TUBB6
ANXA7
CFTR
EFEMP2
GNG12
IL1RAPL1
LOC442497
PEBP1
RAB8B
SLC7A10
UBA52
AP1S1
CHMP2A
EHD1
GNPDA1
IL1RL2
LOC653269
PFAS
RAC1
SLC7A5
UBB
APEH
CHST12
EHD2
GOT2
IL22RA1
LOC727942
PFKFB3
RAC2
SMARCA4
UBE1
APOA1
CITED1
EHD4
GPC1
IL23A
LOC728320
PFN1
RAD21
SMC1A
UBE2N
APOE
CLASP2
EIF4A1
GPC5
IL3
LOC728378
PFN2
RALA
SORT1
UGP2
APP
CLDN1
EMILIN1
GPI
IL5
LOC730013
PGAM2
RAN
SPACA1
UNC13B
ARF1
CLEC11A
ENG
GPR112
IL6ST
LRP1
PGD
RAP1A
SPARC
UNC45A
ARF4
CLIC1
ENO1
GREM1
IL7
LRP6
PGK1
RAP1B
SPOCK1
VAMP3
ARF5
CLIC6
ENO2
GRM2
IL8
LRRFIP2
PGLYRP2
RAP1B
SPRY4
VANGL1
ARHGAP18
CLPX
ENO3
GRM3
INHBA
LTBP1
PIGR
RAP2C
SPTAN1
VASN
ARHGAP23
CLSTN1
ENTPD4
GRM7
INHBB
LTBP2
PIP
RARRES1
SPTBN1
VAT1
ARHGDIA
CLTA
ENTPD4;LOXL2
GSN
INSR
LTF
PKM2
RASA1
SPTBN4
VCAN
ARHGEF1
CLTC
EPB41L3
GSTM1
IQGAP1
LYAR
PLAB
RASA4
SRGN
VCL
ARL6IP5
CLTCL1
EPHA2
GSTM2
ITGA11
LYZ
PLAU
RB1CC1
SRI
VCP
ARMS2
CLU
EPO
GSTM5
ITGA2
MADH4
PLEC1
RCOR2
SRPX2
VEGFC
ARPC3
CMIP
ESM1
GSTO1
ITGA3
MAMDC2
PLEKHG3
RDH5
ST6GALNAC6
VIL1
ARPC4
CNGB1
ETFB
GSTP1
ITGA4
MAP1A
PLOD1
RFTN1
STAT1
VIL2
ARPC5
COL12A1
F2R
GTPBP2
ITGA5
MAP2K6
PLOD2
RGN
STC1
VIM
ASH1L
COL14A1
F3
GYLTL1B
ITGAL
MAP3K1
PLOD3
RHOC
STC2
VTI1A
ASL
COL18A1
F8
GZMA
ITGAV
MARCKS
PLP2
RMND5A
STOM
VTN
ATP1A1
COL1A1
FADD
H2AFV
ITGB1
MARCKSL1
PLSCR3
RNF123
STOML3
WDR49
ATP1B3
COL1A2
FAH
H2AFX
ITGB5
MAT1A
PLTP
RNF40
STX12
WDR52
ATP2B1
COL2A1
FAM108A1
HBB
ITIH2
MBD3
PLUNC
RPL10A
STX2
WNT5A
ATP2B4
COL3A1
FAM129B
HBE1
ITIH4
MCC
PNO1
RPL12
SURF4
YBX1
ATP5A1
COL4A1
FAM29A
HDAC5
ITPR2
MCM10
PODN
RPL15
SVEP1
YWHAB
ATP5B
COL4A2
FAM3B
HERC5
JUP
MDH1
POLN
RPL18
SYT1
YWHAE
ATP8B3
COL4A3
FAM64A
HGF
KIAA0146
MDH2
POSTN
RPL23
SYT9
YWHAG
ATRN
COL5A1
FAM71F1
HGFR
KIAA0256
ME1
POTE2
RPL29
TAAR2
YWHAQ
ATXN1
COL5A2
FAP
HISPPD2A
KIAA0467
MECP2
PPIA
RPL35A
TAGLN
YWHAZ
AXL
COL6A1
FASN
HIST1H2AE
KIAA1881
MFAP4
PPIB
RPLP0
TALDO1
ZBTB4
BASP1
COL6A2
FAT
HIST1H2BA
KPNB1
MFGE8
PPME1
RPS10
TAS2R60
ZNF134
BDNF
COL6A3
FAT2
HIST1H2BL
KRT1
MFSD2
PPP1CC
RPS16
TCN1
ZNF503
BGN
COL7A1
FAT4
HIST1H4H
KRT10
MIF
PRDM16
RPS18
TF
ZNF614
BHMT2
COMP
FBLN1
HIST2H2BE
KRT13
MMP1
PRDX1
RPS2
TFG
BRMS1
COPB1
FBN1
HLAA
KRT14
MMP10
PRDX6
RPS24
TFRC
BSG
COPS3
FBN2
HMGCS2
KRT15
MMP2
PRNP
RPS27A
TGFB1
Regular font: identified by LC MS/MS.
Underline:
identified by antibody arrays.
Bold and Underline:
identified by both LC MS/MS and antibody arrays.
Bold:
identified by LC MS/MS and was found to be present in at least 50% of exosomes characterized.Figure 1
Exosome proteins. (a) Venn diagram of number of proteins detected by LC-MS/MS in 3 independent batches of exosomes, CM15, CM17, and CM18. There are 379, 432, and 420 proteins detected in CM15, CM17 and CM18, respectively. The combined protein number is 766. (b) Venn diagram shows the overlap of proteins detected by LC-MS/MS and antibody array. There are 101 proteins detected by antibody array in CM15. 10 of the 101 proteins detected are overlapping with proteins detected by LC-MS/MS. (c) Intersection of the 739 proteins previously identified in MSC conditioned medium versus the 857 proteins identified in purified exosomes.Based on 15 proteomic analyses carried out on exosomes purified from cultured cells and from biological fluids by different groups, Thery et al. had observed that a set of about 17 proteins, namely glyceraldehyde 3-phosphate dehydrogenase (GAPDH), pyruvate kinase (PK), eukaryotic translation elongation factor 1A1 (EEF1A1), milk fat globule EGF factor 8 protein (MFGE8), tetraspanins, 14-3-3 proteins, Gα proteins, clathrin, Alix (PDCD6IP), MHC class1, annexins (ANX), Rab proteins, ezrin(VIL2), radixin(RDX) and moesin (MSN)(ERM), actin, tubulin, HSP70, and HSP90 were found to be present in at least 50% of the exosomes that were characterized [38]. Not unexpectedly, most of these proteins were also found in the proteome of the HPLC-purified MSC exosomes (Table 1). Also consistent with the endosomal origin of exosomes, we detected the presence of endosome-associated proteins such as Alix (PDCD6IP) and Rab (Table 1).To better understand the biological significance of the proteins in the exosomes, we had previously performed functional clustering of the 857 proteins into biological processes using PANTHER (protein analysis through evolutionary relationships) analytical software [39, 40] (Lai et al.; submitted) The observed frequency of genes from the exosome proteome in each biological process was compared with the reference frequency of 25431 Homo sapiensgene list in the NCBI database for that biological process. The 857 gene products could be clustered into 32 biological processes that were over-represented (P<0.001) and 3 that were under-represented (P<0.001) (Lai et al.; submitted). Many of these biological processes are consistent with activities associated with exosome biology for example, communication, cellular motility, inflammation, and exosome biogenesis. One predominant biological process that could not be definitively associated with exosome biology is proteolysis (P=3.76×10-10) and of the unique genes clustered in this process, ~22% (17 out of 78) encode for peptides in the 20S proteasome or immunoproteasome. To ensure the robustness of this observation, examination of the 3 sets of mass spectrometry datasets revealed that with only one exception of PSMB5 in the first exosome preparation, all the 14 peptides of 20S proteasome were detected with very high confidence in all three exosome preparations as demonstrated by the Mascot search results, number of identified peptides with score higher than homology or identity scores, as well as the MS/MS spectra and fragment ions assignment of unique identified peptides (Supplementary data 1–7).


## 3.2. Presence of 20S Proteasome in MSC Exosome

Mass spectrometry analysis of MSC exosomes not only detected the presence of all sevenα- (PSMA1–7) and all seven β-subunits (PSMB1–7) of the 20S core particle with very high confidence (See Supplementary Material available online at doi:10.1155/2012/971907 for the representative spectra, number of peptide detected, and the peptide score of them), but also the three beta subunits of “immunoproteasome,” PSMB8 (β5i or LMP7), PSMB9 (β1i or LMP2), PSMB10 (β2i or LMP10) gene product [41]. To form the 26S proteasome, the 20S core particle must be complemented by other peptides. Although some of these peptides such as PSMC5, PSMD6, PSMD7, PSMD11, and PSMD14 were present, they were not sufficient to form the 26S proteasome. The presence of some of the 20S proteasome peptides was further confirmed by western blot hybridization (Figure 2(a)). As exosomes are known to have a typical density range of 1.10 to 1.18 gmL−1 that could be resolved on sucrose gradients [42], we checked whether these 20S proteasome subunits have the typical exosome flotation density and postulated that the flotation densities of the 20S proteasome subunits would be different before and after release from such vesicles by a detergent-based buffer. Fractionation of exosomes or exosomes pretreated with a detergent-based lysis buffer on a sucrose density gradient by equilibrium ultracentrifugation revealed that 20S proteasome subunits had a similar flotation density as that of exosome-associated proteins, namely, CD9, CD81, CD59, and CD63. All of them had an exosomes-like flotation density of 1.10–1.18 g/mL (Figure 2(b)). Pretreatment with a detergent-based cell lysis buffer decreased the apparent flotation densities of these proteins. Liquid phase isoelectric focusing of exosomes further revealed that 20S proteasome had a similar protein isoelectric point (pI) as that of exosome-associated CD9 (Figure 2(c)). Together the exosome-like flotation density and pI of the 20S proteasome suggested that the 20S proteasome was associated with exosomes and not a contaminant of exosome preparation.Figure 2
20S proteasome in MSC exosome. (a) Western blot analysis of MSC conditioned medium (CM) and exosome (Exo) using an antibody specific for PMSA 1–7 peptides. (b) Protein analysis of exosome fractionated on a sucrose gradient density. Exosome or exosome pretreated with lysis buffer was loaded on a sucrose density gradient prepared by layering 14 sucrose solutions of concentrations from 22.8 to 60% (w/v) in a SW60Ti centrifuge tube and then ultracentrifuged for 16.5 h at 200 000 g, 4°C, in a SW60Ti rotor. The gradients were removed from the top and the density of each fraction was calculated by weighing a fixed volume of each fraction. The fractions were analyzed by western blot analysis for CD9, PSMA1–7, CD81, CD59, and CD63 in exosome (upper panel) and pretreated exosomes (lower panel). (c) pI of 20S proteasome and CD9 in exosome. Exosome sample was separated by liquid-phase isoelectric focusing into 10 fractions from pH1–14. Each fraction was then concentrated and analyzed by western blot analysis for CD9, PSMA1–7. (d) Proteasome activity in MSC exosome was determined using a commercially available proteasome activity assay kit as described in the Materials and Methods section. Proteasome activity was measured by the rate of degradation of a fluorogenic peptide in the absence or presence of lactacystin, a proteasome inhibitor. One unit (U) enzyme activity is defined as the activity to generate 1μmole product per minute at 37°C. Each bar represents mean ± SEM of 2 independent assays with each assay performed in triplicate. *P=0.00023.


## 3.3. Enzymatic Activity of 20S Proteasome in MSC Exosome

The presence of all sevenα- and all seven β-subunits of the 20S core particle indicates that MSC exosomes contain intact 20S proteasome complexes and therefore 20S proteasome enzymatic activity. To test if exosomes contain functional 20S proteasome, purified MSC exosomes were incubated with a fluorophore-labeled peptide, LLVY-AMC (a substrate for the chymotrypsin-like activity; AMC, 7-amido 4-methylcoumarin) [43], that upon cleavage by 20S proteasome released fluorescent AMC. The fluorescence produced by the released AMC would be directly proportional to the proteasome activity. Based on this assay, the 20S proteasome enzymatic activity in MSC exosomes was determined to be 5.00 μU/μg protein where one unit (U) enzyme activity is defined as the activity to generate 1 μmole product per minute at 37°C. This degradation was inhibited by lactacystin, a proteasome-specific inhibitor (Figure 2(d)).


## 3.4. Exosome Proteasome Reduced Misfolded ProteinsIn Vivo

To determine if exosome proteasome can reduce misfolded or aberrant proteinsin vivo in a disease model and possibly contribute to a therapeutic outcome, we measured the accumulation of misfolded proteins in heart tissue of a mouse model of myocardial ischemia/reperfusion injury with and without exosome treatment.This model is most appropriate for our aim as accumulation of misfolded proteins has been recognized as an important contributor to tissue damage during myocardial ischemia/reperfusion injury [44–48] (reviewed [49]) and we have previously shown that exosome treatment resulted in extensive cardioprotection [23]. Furthermore, in our hands, the area at risk in this mouse model is also highly reproducible [23, 24, 50–52].To determine if exosome could reduce the amount of misfolded proteins, we used a conformation-dependent antibody [53] to quantify the amount of misfolded proteins in the area at risk of ischemic mouse heart after 15 min, one hour, one day and three days reperfusion. The level of denatured or misfolded protein was assayed using an antibody against oligomers. Denatured or partially unfolded proteins tend to self-associate to form high molecular aggregates such as oligomers and these oligomers have a common structure that is independent of amino acid sequence [54]. An antibody known as A11 that was raised against synthetic Aβ oligomers was found to specifically recognize this common structure present in misfolded protein oligomers but did not recognize monomers or mature fibers of proteins or peptides [54]. It will react with soluble Aβ40 oligomers and does not react with soluble low molecular weight Aβ40 or Aβ40 fibrils.Using this antibody, we found that those mice treated with exosome had a modest but significantly lower level of misfolded proteins (P=0.037) (n=4 per time point) 1 day after reperfusion compared with those saline treated controls (n=4 per time point) (Figure 3). This demonstrated that MSC exosome has the potential to reduce misfolded proteins in vivo and may contribute to the exosome-mediated cardioprotection.Exosome reduced oxidized proteinin vivo. Myocardial ischemia/reperfusion injury was induced by ligation of the left coronary artery (LCA) for 30 min and subsequent reperfusion by releasing the ligation. Five minutes before reperfusion, mice (n=4/group/time point) were intravenously infused with exosome or saline. At 15 min, 1 hour, 1 day, or 3 days after reperfusion, the animal was scarified and the area at risk (AAR) was excised and extracted for protein. One μg of each sample was applied onto a nitrocellulose membrane, stained with Ponceau S, destained and probed with rabbit antioligomer antibody and a HRP reporting system as described in material and method. The intensity of the antibody binding was quantified using Image Lab and normalized against the intensity of Ponceau S staining. (a) Representative dot plots. (b) Each bar represented mean ± SEM of 4 animal.
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## 3.5. Association of Plasma 20S Proteasome with Exosome

Although our experimental observations strongly supported the presence of functional 20S proteasome in MSC exosomes, the association of 20S proteasome with exosomes could be a culture artifact contaminant. To test this possibility, we determined if circulating 20S proteasome in human plasma is associated with exosomes. 20S proteasomes are widely reported to be present in plasma [27–31] and we confirmed this by immunoblotting plasma for the presence of proteasome subunits (Figure 4(a)). Fractionation of plasma on a sucrose density gradient revealed that the plasma 20S proteasome fractionated at the flotation density of exosome that is, 1.10–1.18 g/mL (Figure 4(a)). CD81, a tetraspanin protein commonly found on exosome membrane also floated at this density. Each of the fractions was then extracted with cholera toxin B chain (CTB) which binds GM1 ganglioside, a glycolipid found to be enriched in the membrane of exosomes [55] and also MSC exosomes (unpublished data). CTB together with cholera toxin A chain forms the cholera toxin. Cholera toxin enters the cytosol by binding GM1 ganglioside through CTB [56]. This cellular entry of cholera toxin is a well characterized highly specific receptor mediated pathway [57]. The CTB extract was then assayed by ELISA for 20S proteasome subunits and CD81. 20S proteasome subunits and CD81 were both found to be present in the CTB extraction (Figure 4(b)). These observations demonstrated that plasma 20S proteasome colocalized with CD81 in GM1 ganglioside-enriched complex that floats at 1.10–1.18 g/mL and that at least some of the circulating proteasomes in the plasma are associated with exosomes. The presence of 20S proteasome in MSC exosomes and plasma exosomes together implicated exosome as a normal physiological conduit for cellular extrusion of 20S proteasome.Human plasma contained exosome-associated 20S proteasome. (a) Human plasma was loaded on a sucrose density gradient prepared by layering 14 sucrose solutions of concentrations from 22.8 to 60% (w/v) in a SW60Ti centrifuge tube and then ultracentrifuged for 16.5 h at 200 000 g, 4°C, in a SW60Ti rotor. 330μL fractions were removed sequentially from the top and the density of each fraction was calculated by weighing a fixed volume of each fraction. The fractions were analyzed by Western blot analysis for PSMA1–7. (b) Relative amount of CD81 and PSMA1–7 in plasma exosome. Exosomes which are known to have membrane enriched in GM1 gangliosides were isolated from the sucrose density gradient by the high affinity of GM1 gangliosides for cholera toxin B (CTB) chains conjugated to magnetic beads. The CTB extract was assayed by ELISA for the amount of PSMA1–7 and CD81. Each point represents mean ± SEM (n=2).
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## 4. Discussion

In this paper, we investigated the proteome of MSC exosome to identify candidate proteins or protein complexes that could contribute to their therapeutic efficacy in ameliorating myocardial ischemia/reperfusion injury or other pathological conditions. We hypothesize that this therapeutic efficacy against a complex injury is likely to be derived from the synergy of a select permutation of individual exosome components rather than a single component. To elucidate this permutation which may include proteins, RNA and other molecules, we focused first on the proteins by elucidating the exosome proteome to identify candidate proteins or protein complexes that have the biochemical potential to elicit a therapeutic response.The proteome of 3 independently prepared, HPLC-purified ESC-derived MSC exosome using mass spectrometry and cytokine array were analyzed and 857 proteins were identified. We have previously reported that 739 proteins were profiled in hESC-MSC conditioned medium using LTQ-FT mass spectrometer. These proteins included many proteins commonly found in other exosomes. The proteome of MSC exosomes contained a diverse array of proteins. Clustering of these proteins according to their functions suggested that the exosome has the potential to drive many biological processes (Lai et al., submitted). This is consistent with the reported efficacy of MSCs in treating a myriad of diseases such as cardiovascular diseases (e.g., acute myocardial infarction, endstage ischemic heart disease, or prevention of vascular restenosis), osteogenesis imperfecta (OI) or brittle bone disease, amyotrophic lateral sclerosis (ALS), lysosomal storage diseases (e.g., Hurler syndrome), steroid refractory Graft versus Host Disease (GVHD), and periodontitis and bone fractures [12].A significant fraction of proteins in MSC exosomes are involved in the highly regulated and complex intracellular membrane trafficking and sorting through the biosynthetic and endocytotic pathways [58], and these processes probably reflect the biogenesis of exosomes. In fact, many of the proteins in these processes constitute a common set of proteins found in exosomes from different cell sources [59]. However, the biological significance of the other proteins remains to be uncovered. Exosomes have been implicated in an increasing number of important physiological and pathological processes such as disposal of unwanted protein [60], antigen presentation [42], genetic exchange [61], immune responses [38, 62] and tumor metastasis [38, 62–66]. Whether the proteome of MSC exosomes could sustain such processes remains to be determined.Proteasome subunits have been previously reported to be present in exosomes [61, 67–69] but this is the first time that mass spectrometry analysis of exosome proteome had detected the entire protein complement of a 20S proteasome with very high confidence. In addition, we also demonstrated that exosome had the lactacystin-sensitive proteolytic activity of 20S proteasome. Since decreased proteasome activity resulting in accumulation of misfolded proteins has been recognized as an important contributor to tissue damage during myocardial ischemia/reperfusion injury [44–48] (reviewed [49]) and MSC exosomes reproducibly reduce IS/AAR by ~40% in a mouse model of myocardial ischemia/reperfusion injury [23, 24, 52] we tested if cardioprotective MSC exosomes exert some of their therapeutic effects through proteolytic degradation of misfolded proteins. The level of denatured or misfolded protein was assayed using an antibody against oligomers. Using this antibody, we demonstrated that accumulation of misfolded proteins or oligomers was reduced in the heart tissues of a mouse model of myocardial ischemia/reperfusion injury that had been treated with MSC exosomes. This positive correlation between exosome-mediated decreased accumulation of misfolded proteins and exosome-mediated reduction in IS/AAR supports a therapeutic role the 20S proteasome in MSC exosome.20S proteasome is responsible for the degradation of about 90% of all intracellular oxidatively damaged proteins [70] and reduced proteasomal activity has been postulated to be a contributing factor in the pathogenesis of aging-related neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease [71, 72] or cardiovascular disease [73–75]. The presence of all seven α and β subunits of the 20S proteasome, the three beta subunits of “immunoproteasome” and validation of 20S proteasome enzymatic activity in the exosomes in vitro and in vivo suggested that the cardioprotective activity of MSC exosomes [35] could be partly attributed to the presence of 20S proteasome. This further implied that MSC exosomes have the potential to therapeutically correct for proteasome insufficiency. Many debilitating neurodegenerative diseases such as Alzheimer’s, Parkinson’s or prion disease that are caused by accumulation of denatured or misfolded proteins could thus be alleviated by MSC exosomes carrying functional proteasomes. The use of exosomes as a delivery vehicle for proteasome has the added advantage of being able to transcend the blood-brain-barrier. This barrier is the main cause in the intractability of neurodegenerative diseases to medical intervention. Unlike most conventional drugs, exosomes could cross the blood-brain-barrier as demonstrated by the recent knockdown of the gene target in the brain by exosomes loaded with exogenous siRNA [76].The presence of 20S proteasome in MSC exosomes also suggested that cells extruded 20S proteasome through exosomes. Consistent with this, we observed that plasma proteasomes had the flotation density (1.10–1.19 g/mL) and pI of exosomes, and were associated with GM1 gangliosides, a glycolipid found to be enriched in the membrane of exosome [77]. This finding supported our hypothesis that cellular extrusion of 20S proteasomes via exosomes is a normal physiological process.Together this study demonstrated that MSC and plasma exosomes contain functional 20S proteasome, and cells secrete proteasome via the exosome pathway. MSC exosomes could reduce misfolded proteins in the heart after ischemia/reperfusion injury suggesting that they could also be used to alleviate diseases precipitated by accumulation of denatured proteins such as Alzheimer’s disease, Parkinson’s disease or prion disease.In conclusion, MSC exosome has functional 20S proteamsomes and its presence correlated with a modest but significant reduction in oligomerized protein in a mouse model of myocardial infarction. Therefore, 20S proteasome is a candidate exosome protein that could synergize with other constituents in the exosome to ameliorate tissue damage.



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*Source: 971907-2012-07-18.xml*