Transient MCAo Animal Model

The use of animals in this study was approved by the Animal Care and Use Committee of Ajou University, and all procedures were carried out in accordance with institutional guidelines. Male Sprague–Dawley rats (250–300 g) were anesthetized with 4% isoflurane and maintained with 1.5% isoflurane in 70% N2O and 30% O2 using a face mask. Rectal temperature was maintained at 37.0–37.5°C with heating pads. Transient MCAo was induced using a method of intraluminal vascular occlusion that has been modified in our laboratory (Chen et al., 1992). A 4–0 surgical monofilament nylon suture with a rounded tip was moved from the left common carotid artery into the lumen of the internal carotid artery to block the origin of the MCAo. Two hours after MCAo, reperfusion was performed by the withdrawal of the suture to the tip of the common carotid artery.

2. Experimental Groups

One day post MCAo or sham, animals were divided into three groups: sham operation + phosphate buffered saline (PBS) injection (n=3), MCAo+ PBS injection (n=3), and MCAo +

hMSCs were obtained from 20 mL aspirates from the iliac crest of normal human donors (Bang et al., 2005) as part of a protocol approved by the Scientific-Ethical Review Board of Ajou University Medical Center. Briefly, cells were incubated in 150 cm² rectangular canted neck cell culture flasks (Corning Incorporated Life Sciences, USA) at 37°C in 5% CO2 for 1 day and non-adherent cells were removed by replacing the medium. The medium was then changed every 2 to 3 days until the adherent cells became 80% confluent. The cells were harvested with 0.05% trypsin and 0.53 mmol/L EDTA (Gibco) for 5 min at 37°C, replated in a flask, and cultured for an additional 3 to 5 days, before they were harvested. Cells used in these experiments were harvested after six passages. The expression levels of the MSC surface markers CD105 and CD73 in hMSC were evaluated using flow cytometry (n=3;

FACScan; Becton-Dickinson, Rurtherford, NJ). MSCs showed high levels of expression of stem cell markers CD105 and CD73 (data not shown).

4. Preparation of Rat Brain Tissues Samples

Frozen ischemia brain hemisphere tissues were homogenized within a detergent lysis buffer containing 7M urea, 2 M thiourea, 4% (w/v) Chaps, 0.5% (v/v) Triton X-100, 0.5%

(v/v) Pharmalytes pH 4-7(Amersham Biosciences, NJ), 100 mM DTT, and 1.5 mg/mL complete Protease Inhibitor Cocktail for mammalian tissues (Sigma–Aldrich, MO), sonicated, and incubated for 1 h at room temperature in an orbital shaker. The lysate was then centrifuged at 13,000 rpm for 30 min. The total protein concentration of each sample was analyzed by Bio-Rad Protein Assay Dye Reagent Concentrate (Bio-Rad Laboratories,

5. Two-dimensional Electrophoresis (2-DE)

For isoelectric focussing (IEF), IPG strips were used according to (Gorg et al., 2000) the supplier’s instructions. 200 μg of total proteins was mixed in a rehydration buffer (7 M urea, 2M thiourea, 2% Chaps, 0.5% Triton X-100, 100 mM DTT, 0.6% Pharmalytes pH 4-7, and bromophenol blue) in total volume of 340 μL and loaded on 18 cm pH 4-7 NL Immobiline DryStrip (an IPG strip, Amersham biosciences, NJ). After IPG strip rehydration,IEF was done initially at 250 V for 15 min, and then the voltage was increased to 10,000 V within 3 h, and maintained at 10,000 V for 7 h All IEF steps were carried out at 20℃ using pHaser Isoelectric Focusing System (Genomic Solutions, MI). After the first-dimensional IEF, IPG gel strips were placed in an equilibration solution (6 M urea,2% SDS, 30% glycerol, 50 mM Tris-HCl, pH 8.8) containing 1% DTT for 10 min with shaking at 50 rpm on an orbital shaker. The gels were then transferred to the equilibration solution containing 2.5%

iodoacetamide and shaken for a further 10 min before placing them on a 7.5% - 17.5%

gradient polyacrylamide gel slab (20x 20cm).Separation in the second dimension was carried out using Protean II xi electrophoresis equipment and Tris-glycine buffer (25 mM Tris, 192 mM glycine) containing 0.1% SDS, at a current setting of 5 mA/gel for the initial 1 h and 10

For silver staining, following second-dimensional SDS-PAGE, analytical gels were immersed in methanol: acetic acid: water (50:12:38) for 1.5 h, followed by washing twice in 50% ethanol for 20 min. Gels were pretreated for 1 min in a solution of 0.02% Na2S2O3. This was followed by three 1 min washes in deionized water. Proteins were stained in a solution containing 0.2% AgNO3 and 0.075% v/v formalin (37% formaldehyde in water) for 20 min, and washed twice in deionized water for 1 min. Subsequently, gels were developed in a solution of 0.06% v/v formalin, 2% Na2CO3, and 0.0004% Na2S2O3. When the desired intensity was attained, the developer was discarded and stopped by 1% acetic acid. Gel image matching was done with PDQuest software (Version 7.3; Bio-Rad). Scanned gel images were processed to remove backgrounds, staining on the gel borders and to automatically detect spots. For all spot intensity calculations, normalized values were used.

Normalization of spot intensity was done so that the total sum of intensities in a gel would be equal to 1,000,000, and normalized spot intensities were expressed in ppm.

7. In-gel Digestion

In-gel digestion of protein spots on silver stained gels was performed essentially as described (Jensen et al., 1999). After the completion of staining, the gel slab was washed twice with water for 10 min. The spots of interest were excised with a scalpel, cut into pieces, and put into 1.5 mL microtubes. The particles were washed twice with water for 15 min, and then twice with water/acetonitrile (1:1 v/v) for 15 min. The solvent volumes were about twice the gel volume. Liquid was removed, acetonitrile was added to the gel particles and the

NH4HCO3 for 5 min. Acetonitrile was added to give a 1:1 v/v mixture of 0.1 M NH4HCO3/acetonitrile and the mixture was incubated for 15 min. All liquid was removed and gel particles were dried in a vacuum centrifuge (Heto-Holten, Allerød, Denmark), reswelled in 10 mM DTT/0.1 M NH4HCO3, and incubated for 45 min at 56 ℃ to reduce the peptides. After chilling tubes to room temperature and removing the liquid, 55 mM iodoacetamide in 0.1 M NH4HCO3 was added, the tubes were incubated for 30 min at room temperature in the dark to S-alkylate the peptides. Iodoacetamide solution was removed, the gel particles were washed with 0.1 M NH4HCO3 and acetonitrile, dried in a vacuum centrifuge, rehydrated on ice in digestion buffer containing 50 mM NH4HCO3, 5 mM CaCl2, and 12.5 ng /μL of trypsin, and incubated for 45 min on ice. Excess liquid was removed and about 20 μL of digestion buffer without trypsin was added. After overnight digestion at

37 ℃, 25 mM NH4HCO3 was added, and the tube was incubated for 15 min. Acetonitrile was added and the tube was incubated for a further 15 min. The supernatant was recovered, and the extraction was repeated twice with 5% formic acid/acetonitrile (1:1 v/v). The three extracts were pooled and dried in a vacuum centrifuge.

8. MALDI-TOF-MS and Database Search

mixture of the α-cyano-4-hydroxycinnamic acid solution, nitrocellulose solution, and

2-propanol was prepared at a ratio of 2:1:1. Peptide calibrants (50–200 fmol of each), des-Arg-bradykinin (monoisotopic mass, 904.4681) and neurotensin (1672.9715), were added and the mixture solution was then spotted on the target and dried. Dried samples were washed with 5 μL of 5% formic acid for 10 s, followed by 5 μL of Milli-Q water for 10 s, and then dried

spots were analyzed in a Voyager-DE STR MALDI-TOF mass spectrometer (PerSeptive Biosystems, Framingham, NA, USA). The spectrometer was run in positive ion mode and in reflector mode with the settings: accelerating voltage, 20 kV; grid voltage, 76%; guide wire voltage, 0.01%; and a delay of 150 ns. The low mass gate was set at 500 m/z. Proteins were identified by peptide mass fingerprinting with the search programs MS-FIT (http://prospe-ctor.ucsf.edu/ ucsfhtml3.4/ msfit.htm). The following search parameters were applied:

SWISS-PROT and NCBI were used as the protein sequence databases; a mass tolerance of 50 ppm and one incomplete cleavage were allowed; acetylation of the N-terminus, alkylation of cysteine by carbamidomethylation, oxidation of methionine, and pyroGlu formation of N-terminal Gln were considered as possible modifications.

9. Western Blot Analysis

The initial sample for immunoblotting was prepared in the same way as for silver staining. After a run on the 10% SDS-PAGE, the separated proteins were transferred to polyvinylidene fluoride membranes, washed with 0.1% Tween-20 in Tris-buffered saline (T-TBS), blocked with 5% non-fat milk/T-TBS, membranes were incubated with polyclonal

and β-actin(1:500; Santa Cruz, CA) as primary antibody overnight at 4°C, horseradish peroxidase-conjugated (HRP-conjugated,) goat anti-rabbit or anti-goat IgG as secondary antibodies (all used at a dilution of 1:10,000; Amersham Bioscience). The membranes were washed in TBS-T three times and processed with a chemiluminescent detection system (NEN Life Science Products, Boston, MA, USA) according to the manufacturer`s instructions. Chemiluminescence was detected for 5min in a Bio-Rad Fluor-S Multi Imager and the band density determined using Bio-Rad Quantity One software.

10. Statistical Analysis

The protein spots intensity were using to t-tests to evaluate difference between three groups. Statistical differences of western blot band density between the three groups were evaluated using one way analysis of variance. The statistical significance is given as P <0.05.

1. Spot Detection and Matching

A similar proteomic pattern of distribution was apparent in the three groups of brain tissue, representative images from each group were compared. Each two-dimensional gel contained proteins in the pH 4 to 7 range, using PDQuest^TM analysis of the nine gel images, between 1,184 and 1,638 spots were detected. An overall homology of 68%~96% was noted between the Matchset Standard and nine other samples. A matching summary is provided for each sample in table 1.

Table 1. Spot matching summary

Sample Spots detected Spots matched

Sham 1 1306 1280 (75%)

SDS-PAGE gel images were obtained from sham, tMCAo group and hMSC treated tMCAo group (Fig 1). The images were analyzed by PDQuest^TM software, and averages of 1,347 spots were found in the images of tMCAo brain tissue. There were 65 spots with significantly increased intensity and 110 spots with significantly decreased intensity in tMCAo brain tissue compared to sham brain tissue. 1,306 spots were found in the images of hSMC treated tMCAo brain tissue. There were 34 spots with significantly increased intensity and 57 spots with significantly decreased intensity (p<0.05) compared to tMCAo brain tissue.

After Coomasie staining of SDS-PAGE gels of brain tissues, 30 spots had enough size and intensity for protein identification, and the spots underwent an identification process using MALDI-TOF-MS and the search program MS-FIT. Among the 30 spots, 14 spots were identified and listed in Table 2. Among the 14 identified proteins, 11 proteins were up regulated or down regulated in hMSC treated group compared tMCAo group, 3 proteins were recovered to normal condition after hMSC treated group.

Fig. 1. Overview 2-DE maps of rat brain tissue. (A). Sham; (B). MCAo+PBS treated groups; (C). MCAo+hMSC treated groups. Protein (200ug) were separated on PH 4-7 nonlinear IPG strip in the first dimension and 7.5-17.5% linear-gradient SDS-PAGF in the second dimension. All labeled spots have been identified by peptide mass fingerprinting.

Identified spots are indicated by their Accession numbers proteins displaying different levels in threes groups.

Accession A. Up-regulated proteins in hMSC treated tMCAo Groups compared with tMCAo only groups

1 6740 Serum albumin precursor 8.806e+10 68719/6.1 18(47) 35.0

2 4637

4 2805 78KDa glucose-regulated

protein precursor (GRP 78) 5.418e+09 72348/5.1 16(32) 21.0

5 2531 Protein disulfide-isomerase

A6 precursor (EC 5.3.4.1) 25958 48174/5.0 6(22) 19.0 B. Down-regulated proteins in hMSC treated tMCAo Groups compared with tMCAo only groups

1 3318

3 3432 Actin, cytoplasmic

1(Beta-actin) 1000 41737/5.3 5(9) 19.0

4 3219 Pyridoxal phosphate

phosphatase 4.426e+08 33115/5.4 13(23) 48.0

5 1117 Lactoylglutathione lyase 295722 20689/5.1 8(29) 43.0

6 3005 Glia maturation factor beta 19832 16723/5.1 5(16) 31.0 C. Recovered to normal condition after hMSC treated groups

1 3812 Transitional endoplasmic

reticulum ATPase 9.678e+13 89218/5.1 26(41) 33.0 NADH-ubiquinone

Among the 15 identified proteins, the spot 6204 increased significantly in hMSC-treated groups over the sham operation group or the tMCAo-only group (p<0.05 in all cases). This spot was identified as annexin A3 (Fig. 2A) and markedly increased 1.71 times in tMCAo-only group and 3.43 times in hMSC-treated group compared with sham operation group.

While spot no 0105, represented in Fig. 2B, was 1.7-fold up-regulated in tMCAo-only group and recovered normal condition in hMSC-treated group. The spot was identified as Synaptosomal-associated protein 25 (SNAP 25). Identified proteins using MALDI-TOF-MS and the search program MS-FIT were shown in Fig. 3.

To confirm 2-DE results, Western blots were carried out two proteins expression patterns from two-dimensional gels by using antibodies specific to the candidate proteins (Fig. 4).

Anti-β actin antidody was used to normalize the optical density values. The analysis

performed on three brain preparation (sham, PBS-only group and MSC-only group) and immunoblots were replicated twice. Western blot results are shown in Fig. 4, that were significantly differently expressed in the sham operation, tMCAo-only group and hMSC-treated group with antibodies against annexin A3 and SNAP-25. Western blotting consistently confirmed the differential protein expression determined by two-dimensional gel electrophoresis.

Fig. 2. Distinguishing proteins on the cropped gel images. (A). Spot no. 6204, Annexin A3, gradually increased in sham (926.988 ), tMCAo group (1587.797) and hMSC treated tMCAo group (3180.678 ). (B). Spot no. 0105, SNAP25, expressed in lower levels in hMSC treated group (829.5883) than in sham group (1289.104) and tMCAo group (2227.795).

Fig. 3. MALDI-TOF mass spectra of tryptic digests of two protein spots resolved on 2-DE gel. (A). Identification of spots 6204 as annexin A3. (B). Identification of spots 0105 as SNAP25.

Fig. 4. Western blot analysis of two proteins showing the different levels among the three groups. (A). Film images. (B). Relative protein expression normalized to β-actin signal intensity as an internal control. (*p<0.05; **p<0.01)

In this study, I evaluated the protein expression patterns in hMSC-treated MCAo rats Through numerous ischemia animal experiments, it is well known that systemic injection of hMSC results in functional recovery and promotes angiogenesis and neurogenesis (Horita et al., 2006; Kurozumi et al., 2005; Kurozumi et al., 2004). The objective of this experiment was to identify at protein level through discovering proteins that participate in the therapy effect of hMSC.

Using 2-DE PAGE proteomic analysis of an ischemic rat brain, I identified 14 proteins that were differentially expressed among the sham group, the tMCAo-only group, and the hMSC-treated tMCAo group (Table 2). Five of these proteins were upregulated in the hMSC-treated group compared to the PBS-treated tMCAo group and the sham group. For example, annexin A3 was up-regulated in the hMSC-treated ischemia rat. This study has also been observed by western blot analysis. Annexin A3, also called “lipocortin 3” or “placental anticoagulant protein 3” (PAP-III) (Crumpton and Dedman, 1990), is a member of the lipocortin/annexin family, and is also a calcium-dependent phospholipid binding protein.

Annexin A3 is known to be a mediator of angiogenesis, as it up-regulates the HIF-1 signaling pathway and induces VEGF production (Park et al., 2005). Furthermore, annexin A3 is an anticoagulant and anti-phospholipase, which are A2 properties in vitro (Tait et al., 1988). In this experiment, it was observed that the expression of annexin A3 increased in the hMSC-treated group. This result may be indicated that annexin A3 could be one of the proteins that participates in angiogenesis during hMSC therapy. Moreover, vaculor ATP

low levels in most cells and high levels in cells including osteoclasts, macrophages, and neurons (Nelson et al., 1992; Puopolo et al., 1992). In this regard, A and B subunits of V-ATPase alter conformation in response to ATP hydrolysis, which is coupled to proton transport across an associated membrane. Due to the distant location from the associated membrane, the B subunit is an attractive candidate to mediate interactions between V-ATPase and cytoskeletal elements (Holliday et al., 2000). In addition, calcium binding protein 1 (protein disulfide-isomerase A6 precursor) and serum albumin precursor were upregulated in the hMSC-treated group.

There is also a glucose-regulated protein (GRP) 78 or Bip which is upregulated in the hMSC-treated tMCAo group. As it is a member of heat shock protein 70 family and an endoplasmic reticulum (ER) lumen protein, this protein increases its expression through ER stress. Also, GRP78 is involved in polypeptide translocation across the ER membrane, and acts as protective role for GRP78 in preventing ER stress-induced cell death (Rao et al., 2002).

Transitional endoplasmic reticulum ATPase is also known to increase through ER stress just like apoptosis (Short et al., 2007). Transitional ER ATPase forms a ternary complex with ER-associated degradation proteins UFD1L, VCP and NPL4 to bind ubiquitinated proteins

ischemic rat (Li et al., 2002).

In addition, SNAP-25 and NADH-ubiquinone oxidoreductase 24 kDa subunit (mitochondrial precursor) were observed to increase in the tMCAo group, and decreased and recovered in the hMSC-treated group. SNAP-25 is a neuron-specific, developmentally regulated presynaptic protein. In this study, SNAP-25 expression has significantly increased in the ischemia group compared to the sham group, but the expression level in the hMSC-treated ischemia group is similar to that of the sham group. This result may be because of accumulated destruction of SNAP-25 axonal transport due to axonal degeneration caused by ischemia. As a similar experiment result, I can refer to the fact that SNAP-25 showed to increase the most on the 14^th day in the CCA occlusion ischemia model. SNAP-25 is a presynaptic protein that exists in the hippocampal CA1 area, and occurs at an early stage of the pathological changes after ischemia (Ishimaru et al., 2001). Moreover, it has been observed that SNAP-25 expresses and increases in the trimethyltin model that induces hippocampal neuron loss (Patanow et al., 1997). Also, SNAP-25 expression seemed to increase through chronic social stress as well (Abumaria et al., 2006). SNAP-25 expression due to axonal degeneration or neuron loss was observed to significantly decrease in the hMSC-treated group. This also relates to the result that it has neuroprotective effects such as reducing the infarct size (Kurozumi et al., 2005) and reducing apoptosis, with hMSC treatment. In this experiment, SNAP-25 as a neuroprotector at a hMSC-treated tMCAo group

Six proteins were down-regulated in the hMSC-treated group compared with the tMCAo-only group. Broadly, these proteins can be grouped into: a calcium-binding protein

or Lactoylglutathione lyase (EC4.4.1.5)); involved as a modulator or transducer in various transmembrane signaling systems (guanine nucleotide-binding protein G(I)/G(S)/G(T) beta subunit 1); On the other hand, pyridoxal phosphate(PLP) phosphatase(EC 3.1.3.74) also showed a decrease in expression in the hMSC-treated group. PLP phosphatase conducts regulation of PLP concentration through catabolism of PLP (Jang et al., 2003). However PLP is the coenzymatically active form of vitamin B6 and plays an important role in maintaining biochemical homeostasis (Meister, 1990; Nelson et al., 1992). There have been exceptional cases when it was hard to analyze the hMSC effect. For instance, glia maturation factor beta (GMFB) was initially isolated as a growth/differentiation factor which is released from brain tissue after injury (Nieto-Sampedro et al., 1988). Overexpression of GMFB in primary astrocytes caused secretion of neurotrophic factors such as brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) through activation of the p38 MAPK pathway (Zaheer et al., 1999). Some reports suggest that the effects of GMFB lead to neuroprotection against adverse environmental conditions. According to my experiment results, GMFB showed a considerable decrease in the hMSC-treated tMCAo group compared to the tMCAo group, and the level of expression was much less in the hMSC-treated group in comparison to the tMCAo-only group. In other report, GMFB protein

for the stem cell therapy in ischemia.

Using 2-DE and matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS), I was able to identify 14 proteins in hMSC treated tMCAo rat brains. Among the 14 identified proteins, 11 proteins were up-regulated and down-regulated in the hMSC-treated group compared to the tMCAo group; three proteins recovered to their normal condition after hMSC treatment. In future, this study might be useful for elucidation of therapeutic effects in rat MCAo model after transplantation with hMSC.

1. Abumaria N, Rygula R, Havemann-Reinecke U, Ruther E, Bodemer W, Roos C.

Flugge G: Identification of genes regulated by chronic social stress in the rat dorsal raphe nucleus. Cell Mol Neurobiol 26: 145-162, 2006

2. Bang OY, Lee JS, Lee PH. Lee G: Autologous mesenchymal stem cell transplantation in stroke patients. Ann Neurol 57: 874-882, 2005

3. Bays NW. Hampton RY: Cdc48-Ufd1-Npl4: stuck in the middle with Ub. Curr Biol 12:

R366-371, 2002

4. Borlongan CV, Lind JG, Dillon-Carter O, Yu G, Hadman M, Cheng C, Carroll J, Hess DC: Bone marrow grafts restore cerebral blood flow and blood brain barrier in stroke rats. Brain Res 1010: 108-116, 2004

5. Cecconi D, Mion S, Astner H, Domenici E, Righetti PG, Carboni L: Proteomic analysis of rat cortical neurons after fluoxetine treatment. Brain Res 1135: 41-51, 2007

6. Chen H, Chopp M, Zhang ZG, Garcia JH: The effect of hypothermia on transient middle cerebral artery occlusion in the rat. J Cereb Blood Flow Metab 12: 621-628, 1992

92-100, 2002

8. Chopp M, Zhang XH, Li Y, Wang L, Chen J, Lu D, Lu M, Rosenblum M: Spinal cord injury in rat: treatment with bone marrow stromal cell transplantation. Neuroreport 11:

3001-3005, 2000

9. Crumpton MJ, Dedman JR: Protein terminology tangle. Nature 345: 212, 1990

9. Crumpton MJ, Dedman JR: Protein terminology tangle. Nature 345: 212, 1990