Smooth-muscle progenitor cells isolated from patients with moyamoya disease: novel experimental cell model

Laboratory investigation

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Object

Moyamoya disease (MMD) is a cerebrovascular occlusive disease affecting bilateral internal carotid termini. Smooth-muscle cells are one of the major cell types involved in this disease process. The characteristics of circulating smooth-muscle progenitor cells (SPCs) in MMD are poorly understood. The authors purified SPCs from the peripheral blood of patients with MMD and sought to identify differentially expressed genes (DEGs) in SPCs from these patients.

Methods

The authors cultured and isolated SPCs from the peripheral blood of patients with MMD (n = 25) and healthy control volunteers (n = 22). After confirmation of the cellular phenotype, RNA was extracted from the cells and DEGs were identified using a commercially available gene chip. Real-time quantitative reverse transcription polymerase chain reaction was performed to confirm the putative pathogenetic DEGs.

Results

The SPC-type outgrowth cells in patients with MMD invariably showed a hill-and-valley appearance under microscopic examination, and demonstrated high α–smooth muscle actin, myosin heavy chain, and calponin expression (96.5% ± 2.1%, 42.8% ± 18.6%, and 87.1% ± 8.2%, respectively), and minimal CD31 expression (less than 1%) on fluorescence-activated cell sorter analysis. The SPCs in the MMD group tended to make more irregularly arranged and thickened tubules on the tube formation assay. In the SPCs from patients with MMD, 286 genes (124 upregulated and 162 downregulated) were differentially expressed; they were related to cell adhesion, cell migration, immune response, and vascular development.

Conclusions

With adequate culture conditions, SPCs could be established from the peripheral blood of patients with MMD. These cells showed specific DEGs compared with healthy control volunteers. This study provides a novel experimental cell model for further research of MMD.

Abbreviations used in this paper:α-SMA = α–smooth muscle actin; DEG = differentially expressed gene; EPC = endothelial progenitor cell; FACS = fluorescence-activated cell sorter; FSP-1 = fibroblast-specific protein–1; GO = gene ontology; GTPase = guanosine 5′-triphosphatase; KEGG = Kyoto Encyclopedia of Genes and Genomes; MCAM = melanoma cell adhesion molecule; MHC = myosin heavy chain; MMD = moyamoya disease; PBS = phosphate-buffered saline; PDGF, PDGFR = platelet-derived growth factor, PDGF receptor; qRT-PCR = quantitative reverse transcription polymerase chain reaction; SMC = smooth-muscle cell; SPC = smooth-muscle progenitor cell; VEGF, VEGFR = vascular endothelial growth factor, VEGF receptor.

Abstract

Object

Moyamoya disease (MMD) is a cerebrovascular occlusive disease affecting bilateral internal carotid termini. Smooth-muscle cells are one of the major cell types involved in this disease process. The characteristics of circulating smooth-muscle progenitor cells (SPCs) in MMD are poorly understood. The authors purified SPCs from the peripheral blood of patients with MMD and sought to identify differentially expressed genes (DEGs) in SPCs from these patients.

Methods

The authors cultured and isolated SPCs from the peripheral blood of patients with MMD (n = 25) and healthy control volunteers (n = 22). After confirmation of the cellular phenotype, RNA was extracted from the cells and DEGs were identified using a commercially available gene chip. Real-time quantitative reverse transcription polymerase chain reaction was performed to confirm the putative pathogenetic DEGs.

Results

The SPC-type outgrowth cells in patients with MMD invariably showed a hill-and-valley appearance under microscopic examination, and demonstrated high α–smooth muscle actin, myosin heavy chain, and calponin expression (96.5% ± 2.1%, 42.8% ± 18.6%, and 87.1% ± 8.2%, respectively), and minimal CD31 expression (less than 1%) on fluorescence-activated cell sorter analysis. The SPCs in the MMD group tended to make more irregularly arranged and thickened tubules on the tube formation assay. In the SPCs from patients with MMD, 286 genes (124 upregulated and 162 downregulated) were differentially expressed; they were related to cell adhesion, cell migration, immune response, and vascular development.

Conclusions

With adequate culture conditions, SPCs could be established from the peripheral blood of patients with MMD. These cells showed specific DEGs compared with healthy control volunteers. This study provides a novel experimental cell model for further research of MMD.

Moyamoya disease (MMD) is a unique cerebrovascular disease characterized by progressive stenoocclusive alterations in the terminal portion of the internal carotid artery and fine collateral network, the so-called moyamoya vessels. The clinical presentation of pediatric MMD usually includes repeated transient ischemic attacks, and the benefit of surgery for the ischemic type of MMD has been generally accepted.13,17

There have been expression studies of growth factors and cytokines in the intracranial artery, extracranial artery (especially the superficial temporal artery), serum, plasma, and CSF in patients with MMD. Notable examples included fibroblast growth factor,7 transforming growth factor β,6 platelet-derived growth factor (PDGF),2 hepatocyte growth factor,21 soluble adhesion molecules,33 matrix metalloproteinases,11 and cellular retinoic acid-binding protein.14 However, an integrative understanding of disease pathogenesis has not been attained due to the paucity of samples from the affected vessels of patients with MMD and the lack of experimental models.

Recently, endothelial progenitor cells (EPCs) obtained in patients with MMD have been valuable for understanding the disease, despite some controversial issues.10,12,29 The main histopathological finding in MMD is fibrocellular thickening of the intima that results from the proliferation of smooth-muscle cells (SMCs), which suggests the involvement of such cells in this disease.8 However, smooth-muscle progenitor cells (SPCs) have never been studied in regard to MMD.

In this study we purified SPCs from the peripheral blood of patients with MMD as well as healthy control volunteers, and identified differentially expressed genes (DEGs) by measuring mRNA expression. The study provides an experimental cell model of MMD and insight into the pathophysiological mechanisms of this disease.

Methods

Study Participants

We obtained blood samples from 25 patients with MMD prior to their first surgical treatment and from 22 healthy volunteers, after obtaining approval from the Seoul National University Hospital's institutional review board and informed consent from the participants. All patients underwent digital subtraction angiography to confirm the diagnosis. Patients with moyamoya syndrome associated with another condition, such as Down syndrome or neurofibromatosis, were excluded from the study. There were 17 female and 8 male patients, and their ages ranged from 7 to 48 years (median 19 years). Healthy volunteers were 12 women and 10 men, and their ages ranged from 21 to 26 years (median 22 years). They had no history of stroke, hypertension, or smoking.

Cell Culture

Buffy coat preparation and progenitor cell culture were performed following a previously described protocol, with some modifications.31 Blood samples (40 m l) were processed immediately after collection. Peripheral blood was diluted 1:1 with phosphate-buffered saline (PBS), and mononuclear cells were isolated from the buffy coat in Ficoll-1077 (Histopaque-1077; Sigma) for 25 minutes at 2300 rpm. After washing 3 times with PBS, the cells were suspended in an endothelial cell growth medium (EGM-2; Clonetics) and were seeded in 6-well plates coated with collagen Type I (BD BioCoat; BD Biosciences) at a density of 2 × 106 cells per well. The final concentration of the fetal bovine serum in cell culture medium was 10%. After 5 days, fresh culture medium was applied after removing nonadherent cells. At 1 week, PDGF-BB (5 ng/ml; R&D Systems) was added to the culture medium to facilitate differentiation into the SMC lineage. Thereafter, medium was changed every 3 days. Cells were maintained at 37°C in a humidified atmosphere containing 5% CO2.

Evaluation of Outgrowth Cell Phenotypes

Microscopic morphological evaluation (“hill-and-valley” appearance), fluorescence-activated cell sorter (FACS) analysis, and indirect immunofluorescence were performed to confirm the SPC phenotype.

The FACS analysis was performed when the outgrowth cells were confluent, showing a distinct morphological phenotype; cells were used at the third passage. Primary antibodies against CD31 (MiltenyiBiotec), CD34 (MiltenyiBiotec), α–smooth muscle actin (α-SMA; Dako), smooth-muscle myosin heavy chain (MHC; Dako), calponin (Dako), fibroblast-specific protein–1 (FSP-1; Abcam), vascular endothelial growth factor receptor (VEGFR) 1 (Flt-1; R&D Systems), VEGFR 2 (KDR; R&D Systems), CD140a (PDGFR alpha chain; BD Biosciences), and CD140b (PDGFR beta chain; BD Biosciences) were used. For intracellular antigen detection, cells were fixed with 4% paraformaldehyde in PBS and permeabilized with 0.5% saponin in PBS. An isotypematched IgG control was used for immunofluorescence analysis. The binding of primary antibodies to progenitor cells was detected with Alexa Fluor 488–conjugated anti–mouse IgG (Life Technologies). For FACS analysis, 104 cells were acquired and scored with a FACScan flow cytometer (Becton Dickinson) and CellQuest software (Becton Dickinson).

We also determined the expression of CD31, CD34, α-SMA, MHC, and calponin by using immunofluorescence microscopy. We included an isotype-matched control IgG for each antibody. Cells were grown on chamber slides (Lab-Tech; Poly Labo) and fixed with 1% paraformaldehyde solution. Nonspecific antibody binding was blocked by incubation with a 10% normal goat serum solution (Dako). The primary antibodies used included a polyclonal rabbit anti–human CD31 antibody (Dako), a polyclonal rabbit anti–human CD34 antibody (Abcam), a monoclonal mouse anti–human α-SMA antibody (clone 1A4; Dako), a monoclonal mouse anti–human MHC antibody (Dako), and a monoclonal mouse anti–human calponin antibody (Dako). After nuclear staining with DAPI, cells were mounted for fluorescence microscopy (Olympus BX-UCB).

In addition to showing a characteristic hill-and-valley appearance, we characterized the SPCs as those showing less than 1% CD31 and FSP-1 expression, more than 90% expression for at least one of three SMC markers (α-SMA, MHC, and calponin), predominant PDGFR expression, and minimal VEGFR expression.

In Vitro Tubule Formation

The function of SPCs was examined by capillary tubule formation on the Matrigel (BD Biosciences). Cells (2 × 104 cells/well) were labeled with red fluorescent dye (PKH26; Sigma) and grown in a Matrigel-coated 48-well plate for 24 hours. The tube formations were visualized using an inverted fluorescence microscope (Olympus BXUCB). The data were quantified by counting tube formations and measuring tube thickness with ImageJ software in 4 random microscopic fields per subject.

Extraction of RNA and Affymetrix Gene Chip Processing

After confirmation of the cellular phenotype, SPCs from 5 patients with MMD and 5 healthy control volunteers were used for gene expression analysis. The MMD group included 3 female and 2 male patients, and their ages ranged from 7 to 26 years (median 17 years). The control group included 4 women and 1 man, and their ages ranged from 21 to 26 years (median 22 years). The outgrowth cells grown to confluence in cell culture plates were washed twice with RPMI-1640 medium. Total RNA was extracted using TRIzol reagent (Invitrogen) and collected RNA was purified using the RNeasy mini kit (Qiagen). The purity and concentration of RNA were determined by a spectrophotometer (NanoDrop Technologies) and bioanalyzer (Agilent Technologies), respectively. The RNA was amplified and labeled according to the Affymetrix GeneChip Whole Transcript (WT) Sense Target Labeling protocol. The resulting labeled cDNA was hybridized to Affymetrix Human Gene 1.0 ST arrays and scanned, as described previously.28 Raw expression data were background corrected, normalized, and summarized using the robust multiarray average approach.9 The resulting log2-transformed data were used for further analysis.

Identification of DEGs and GO

Nonspecific filtering was performed to exclude noninformative probe sets with low variability; we excluded the probe sets with an SD below the value of the “shorth” (that is, the mean of the shortest interval containing half of the data).5 Differentially expressed probe sets were identified using a linear model applying moderated t-statistics based on an empirical Bayes approach.32 The DAVID bioinformatics resource (http://david.abcc.ncifcrf.gov) was used to detect overrepresented gene ontology (GO) categories and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways among the differentially regulated genes. Microarray data analysis was performed using R statistical software (http://www.R-project.org).

Data Validation by Real-Time qRT-PCR

The DEGs with possible pathogenetic roles in SPCs from patients with MMD were reexamined by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) to confirm the microarray data. Real-time qRT-PCR validation was performed on the same samples used for microarray analysis. The cDNA was generated from 1 μg of total RNA, and PCR cycling was performed on a 7000 Sequence Detection System (Applied Biosystems) in a 20-ml TaqMan Universal PCR Master Mix (Applied Biosystems) using 50 ng of cDNA. Predesigned Assays-on-Demand TaqMan probes and primer pairs were obtained from Applied Biosystems [Assay ID Hs01076873_m1 for integrin alpha 3 (ITGA3), Hs00989192_m1 for BAI1-associated protein 2–like 1 (BAIAP2L1), Hs00983056_m1 for N-cadherin (CDH2), Hs00300724_m1 for EPH receptor A5 (EPHA5), Hs00174838_m1 for melanoma cell adhesion molecule (MCAM), Hs00324432_m1 for guanosine 5′-triphosphatase (GTPase)-activating Rap/RanGAP domain–like 4 (GARNL4), Hs00955621_m1 for Plexin C1 (PLXNC1), Hs00170143_m1 for Neogenin homolog 1 (NEO1), and Hs00229108_m1 for Rho GTPase-activating protein 28 (ARHGAP28)]. Glyceraldehyde 3-phosphate dehydrogenase was used as an endogenous reference to control for expression-independent sample-to-sample variability. Relative gene expression was determined from the Ct (threshold cycle) values obtained using the 2−ΔΔCt method.18

Statistical Analysis

Data are presented as the mean ± SEM. Statistical analysis of the data was done using the Mann-Whitney test or t-test; p < 0.05 was considered significant.

Results

Cell Culture, Immunophenotyping of Outgrowth Cells, and Tube Formation

Isolation and maintenance of vascular progenitor cells was possible for 47 participants (25 patients with MMD, 22 control volunteers). The first appearance of outgrowth cells after initial blood sampling and plating of mononuclear cells occurred from Day 11 to Day 37. On average, SPCs started to appear on Day 27 and EPCs on Day 24 after plating. The SPCs from patients with MMD tended to appear later than those from healthy control volunteers (Day 34 vs Day 20; p = 0.0556).

According to the FACS analysis, SPCs were identified in 11 participants (5 patients with MMD and 6 control volunteers; Fig. 1), EPCs in 25 (17 patients and 8 controls), and cells with mixed characteristics in 11 (3 patients and 8 controls; Table 1). The SPCs invariably showed a hill-and-valley appearance under microscopic examination. As assessed by FACS analysis, α-SMA and CD31 were expressed in 94.2% ± 1.8% and 0.9% ± 0.2% of the SPCs, respectively. The CD34-positive cells, which are considered primitive SPCs, accounted for 9.6% ± 4.2%. T he EPCs had a cobblestone appearance and invariably showed high expression of CD31 (98.9% ± 0.4%). The EPCs had more CD34-positive cells compared with SPCs (29.7% ± 5.8% vs 9.6% ± 4.2%; p = 0.0370). The FACS analysis demonstrated lower expression of MHC (42.8% ± 18.6% vs 96% ± 1.8%; p = 0.0087) and calponin (87.1% ± 8.2% vs 99.8% ± 0.1%; p = 0.0519) in the SPCs obtained in patients with MMD compared with the control volunteers.

Fig. 1.
Fig. 1.

Findings related to SPCs obtained in a 7-year-old boy in whom MMD was diagnosed. A: Photomicrograph showing confluent cells that have a hill-and-valley morphology at Day 31. Bar = 500 μm. B–D: Graphs showing results of FACS analysis of SPCs obtained in this patient. The black curves represent the isotype-matched control IgG antibodies, and the green curves represent the test antibodies (anti–α-SMA, anti-CD31, and anti-CD34). Values on the y axis quantify the intensity of fluorescein isothiocyanate staining (FITC log), and on the x axis the values represent the number of cells counted. The SPCs showed 98.9% α-SMA and 0.7% CD31 expression. The CD34-positive cells, considered primitive SPCs, accounted for 13.7% of the SPCs. M1 = marker line for positive cells.

TABLE 1:

Results of FACS analysis according to type of outgrowth cells*

MarkersNo. of PtsCell Type (%)
α-SMAMHCCalponinCD31CD34FSP-1
SPC
 total1194.2 ± 1.871.8 ± 11.694.0 ± 4.00.9 ± 0.29.6 ± 4.20.28 ± 0.03
 MMD596.5 ± 2.142.8 ± 18.687.1 ± 8.20.6 ± 0.212.7 ± 7.20.30 ± 0.04
 control692.3 ± 2.696.0 ± 1.899.8 ± 0.11.2 ± 0.37.1 ± 5.20.26 ± 0.03
EPC
 total2530.7 ± 4.5NDND98.9 ± 0.429.7 ± 5.80.92 ± 0.13
 MMD1730.4 ± 6.2NDND98.8 ± 0.531.0 ± 6.80.96 ± 0.19
 control831.4 ± 5.7NDND99.0 ± 0.926.9 ± 11.50.85 ± 0.11

Values are expressed as the mean ± SEM. ND = not done; pts = patients.

Expression of α-SMA and calponin was observed in the SPCs by using immunofluorescence microscopy (Fig. 2). In addition, coexpression of α-SMA and CD34 was confirmed in some of the SPCs as well as coexpression of calponin and CD34; however, coexpression of CD31 and calponin was not observed.

Fig. 2.
Fig. 2.

Photomicrographs showing immunofluorescence staining of SPCs labeled with antibodies against α-SMA, calponin, CD34, and CD31. The coexpression of α-SMA and CD34 as well as calponin and CD34 was also observed in SPCs obtained in patients with MMD. Cell nuclei in the last column were counterstained with DAPI. Bar = 500 μm.

In healthy control volunteers, SPCs formed numerous smaller microtubule networks in comparison with EPCs. In patients with MMD, SPCs did make microtubules, whereas EPCs produced poorly organized tube formations. The networks formed by SPCs obtained in patients with MMD were rather sparse, but were thicker than those formed by SPCs from control volunteers (Fig. 3).

Fig. 3.
Fig. 3.

Tube formation assay. A and B: Photomicrographs showing immunofluorescence staining of SPCs. Compared with SPCs obtained in a healthy volunteer (A), SPCs obtained in a patient with MMD made rather irregularly arranged tubules of varying sizes (B). In some areas, thickened tubules were noted (arrows). Bars = 500 μm. C and D: Bar graphs showing that SPCs obtained in patients with MMD formed thickened tubules of fewer numbers compared with controls (SPCs were obtained in 3 patients and 3 control volunteers). Asterisks = p < 0.01.

Angiogenic Factor Enrichment and Receptor Expression in SPCs

We could not obtain SPCs from the mononuclear cells of 4 patients with MMD by using VEGF enrichment. Without VEGF enrichment, we obtained SPCs from 5 of the 10 patients. We then reintroduced VEGF, but did not obtain SPCs in 11 participants. Thus, the absence of VEGF enrichment is essential for the acquisition of SPCs from patients with MMD, with an OR of 31 (95% CI 1.4–657.8, p = 0.0047) without VEGF enrichment. In contrast, we obtained SPCs from 4 of the 6 cases with VEGF and from 2 of 16 cases without VEGF among healthy control volunteers (OR 14 [95% CI 1.5–133.3], p = 0.0254).

The SPC outgrowth cells showed minimal expression of VEGFRs, Flt-1, and KDR, and the receptor expression was not significantly different between patients with MMD and control volunteers. With regard to PDGFRs (Fig. 4), PDGFRα (CD140a) expression was significantly lower in patients with MMD compared with healthy control volunteers (control vs MMD, 66.1% ± 8 .2% v s 38.5% ± 2.6%; p = 0.0286), whereas PDGFRβ (CD140b) expression was not significantly different (96.6% ± 1.2% vs 87.7% ± 3.1%; p = 0.1143).

Fig. 4.
Fig. 4.

Boxplots demonstrating PDGFR expression in SPCs obtained in patients with MMD and control volunteers (left, PDGFRα; right, PDGFRβ). The SPCs obtained in patients with MMD show lower PDGFRα (CD140a, p = 0.0286) and similar PDGFRβ (CD140b, p = 0.1143) expression compared with controls. Percentages of positive cells are shown on each y axis.

Differentially Expressed Genes in SPCs Obtained in Patients With MMD

When we compared SPC gene expression in patients with MMD and healthy control volunteers, 286 genes were differentially expressed with a > 1.5-fold change and a t-test p value < 0.01. The heat map and principal component plot of the 286 DEGs showed clear differences in the expression profile of the patients with MMD and the control volunteers (Fig. 5). The GO analysis for 124 upregulated transcripts identified 19 terms (p < 0.05, Table 2), which included responses to endogenous stimulus (GO:0009719; p = 0 .016), a nterior/posterior p attern formation (GO:0009952; p = 0.033), and ephrin receptor signaling (GO:0048013; p = 0.035). In the KEGG pathway analysis, 4 genes (N-cadherin, claudin 1, major histocompatibility complex class II DP α1, and neural cell adhesion molecule) were associated with cell adhesion.

Fig. 5.
Fig. 5.

Heat map and principal component plot of 286 DEGs. Left: Heat map and 1-way hierarchical clustering of 286 DEGs. Right: Principal component plot of 286 DEGs.

TABLE 2:

Gene ontology terms in upregulated genes in SPCs obtained in patients with MMD

Termp Value
GO:0009719; response to endogenous stimulus0.016
GO:0003002; regionalization0.017
GO:0042493; response to drug0.023
GO:0009952; anterior/posterior pattern formation0.033
GO:0048013; ephrin receptor signaling pathway0.035
GO:0009725; response to hormone stimulus0.037
GO:0010243; response to organic nitrogen0.040
GO:0043434; response to peptide hormone stimulus0.042
GO:0007389; pattern specification process0.045
GO:0000002; mitochondrial genome maintenance0.054
GO:0030031; cell projection assembly0.065
GO:0030182; neuron differentiation0.069
GO:0010033; response to organic substance0.069
GO:0008202; steroid metabolic process0.081
GO:0001822; kidney development0.084
GO:0030324; lung development0.089
GO:0030323; respiratory tube development0.093
GO:0035295; tube development0.099
GO:0042127; regulation of cell proliferation0.099

Meanwhile, GO analysis for 162 downregulated transcripts identified 26 terms (p < 0.01, Table 3), including cell adhesion (GO:0007155; p = 0.0000385), regulation of cell migration (GO:0030334; p = 0.000122), innate immune response (GO:0045087; p = 0.000214), enzyme-linked receptor protein signaling pathway (GO:0007167; p = 0.000868), and vasculature development (GO:0001944; p = 0.0067). In the KEGG pathway analysis, 6 genes (acyl-CoA synthetase long-chain family member 5, acyl-CoA oxidase 2, diazepam binding inhibitor, matrix metallopeptidase 1, nuclear receptor subfamily 1H3, and stearoyl-CoA desaturase) were associated with peroxisome proliferator-activated receptor signaling; 5 genes (bradykinin receptor B1, bradykinin receptor B2, complement factor B, complement factor D, and serpin peptidase inhibitor) with complement and coagulation cascades; and 4 genes (integrin β8, laminin α2, lymphoid enhancer-binding factor 1, and sarcoglycan delta) with arrhythmogenic cardiomyopathy. The representative sets of DEGs related to each GO term are presented in Table 4.

Fig. 6.
Fig. 6.

Real-time qRT-PCR analysis showing differential expression of 9 selected genes: increased expression of integrin alpha 3 (ITGA3), BAI1-associated protein 2–like 1 (BAIAP2L1), N-cadherin (CDH2), EPH receptor A5 (EPHA5), melanoma cell adhesion molecule (MCAM), GTPase-activating Rap/RanGAP domain–like 4 (GARNL4), and decreased expression of Plexin C1 (PLXNC1), Neogenin homolog 1 (NEO1), and Rho GTPase-activating protein 28 (ARHGAP28).

TABLE 3:

Gene ontology terms in downregulated genes in SPCs obtained in patients with MMD

Termp Value
GO:0007155; cell adhesion0.0000385
GO:0022610; biological adhesion0.0000392
GO:0010033; response to organic substance0.0000565
GO:0030334; regulation of cell migration0.000122
GO:0045087; innate immune response0.000214
GO:0040012; regulation of locomotion0.000291
GO:0051270; regulation of cell motion0.000302
GO:0007167; enzyme-linked receptor protein signaling pathway0.000868
GO:0051605; protein maturation by peptide bond cleavage0.000956
GO:0002684; positive regulation of immune system process0.0012
GO:0003013; circulatory system process0.0013
GO:0008015; blood circulation0.0013
GO:0032535; regulation of cellular component size0.0027
GO:0016485; protein processing0.0031
GO:0051604; protein maturation0.0044
GO:0006959; humoral immune response0.0051
GO:0006956; complement activation0.0059
GO:0002541; activation of plasma proteins involved in acute inflammatory response0.0063
GO:0001944; vasculature development0.0067
GO:0009611; response to wounding0.0070
GO:0006955; immune response0.0076
GO:0030335; positive regulation of cell migration0.0078
GO:0007160; cell-matrix adhesion0.0078
GO:0006952; defense response0.0079
GO:0050778; positive regulation of immune response0.0091
GO:0002253; activation of immune response0.0095
TABLE 4:

Representative sets of DEGs belonging to each GO term in patients with MMD*

GO TermDEGs
upregulated
 response to endogenous stimulusCTTNBP2, CDKN1A, BTG2, PGF, HDAC9, DDIT3, VLDLR
 anterior/posterior pattern formationHOXC10, KIF3A, BTG2, HOXA5
 ephrin receptor signaling pathwayEPHA2, EPHB2
downregulated
 cell adhesionPLXNC1, PDPN, COL3A1, CNKSR3, LEF1, SPOCK1, NID1, NEO1, CXCL12, CDSN, LAMA2, ISLR, WISP2, SNED1, ITGB8, TEK, ROR2, BOC, SPON1
 regulation of cell migrationLAMA2, ACVRL1, PDPN, SMAD7, CXCL16, TEK, PDGFRA, BDKRB1, CXCL12
 innate immune responseTMEM173, CFB, CXCL16, CLU, TLR3, COLEC12, SERPING1, CFD
 enzyme-linked receptor protein signaling pathwayMSX2, FMOD, ACVRL1, SOCS2, SMAD7, STAT5A, TEK, COL3A1, PDGFRA, ROR2, BDKRB2
 vasculature developmentACVRL1, MEOX2, PDPN, SMAD7, COL3A1, HS6ST1, PPAP2B, CXCL12

ACVRL1 = activin A receptor type II–like 1; BDKRB1, BDKRB2 = bradykinin receptor B1, B2; BOC = brother of CDO precursor homolog; BTG2 = B-cell translocation gene family member 2; CDKN1A = cyclin-dependent kinase inhibitor 1A (p21); CDSN = corneodesmosin; CFB = complement factor B; CFD = complement factor D (adipsin); CNKSR3 = Connector enhancer of kinase suppressor of ras family member 3; CLU = clusterin; COLEC12 = collectin subfamily member 12; COL3A1 = collagen, type III, alpha 1; CTTNBP2 = cortactin-binding protein 2; CXCL12 = chemokine (C-X-C motif) ligand 12 (stromal cell–derived factor 1); CXCL16 = chemokine (C-X-C motif) ligand 16; DDIT3 = DNA-damage–inducible transcript 3; EPHA2, EPHB2 = EPH receptor A2, B2; FMOD = fibromodulin; HDAC9 = histone deacetylase 9; HOXA5, HOXC10 = homeobox A5, C10; HS6ST1 = heparan sulfate 6-O-sulfotransferase 1; ISLR = immunoglobulin superfamily containing leucine-rich repeat; ITGB8 = integrin beta 8; KIF3A = kinesin family member 3A; LAMA2 = laminin alpha 2; LEF1 = lymphoid enhancer–binding factor 1; MEOX2 = mesenchyme homeobox 2; MSX2 = msh homeobox 2; NEO1 = neogenin homolog 1; NID1 = nidogen 1; PDGFRA = platelet-derived growth factor receptor alpha polypeptide; PDPN = podoplanin; PGF = placental growth factor; PLXNC1 = plexin C1; PPAP2B = phosphatidic acid phosphatase type 2B; ROR2 = receptor tyrosine kinase-like orphan receptor 2; SERPING1 = serpin peptidase inhibitor clade G (C1 inhibitor) member 1; SMAD7 = mothers against decapentaplegic homolog 7; SNED1 = sushi, nidogen, and EGF-like domains 1; SOCS2 = suppressor of cytokine signaling 2; SPOCK1 = sparc/osteonectin, cwcv and kazal-like domains proteoglycan (testican) 1; SPON1 = spondin 1; STAT5A = signal transducer and activator of transcription 5A; TEK = endothelial TEK tyrosine kinase; TLR3 = toll-like receptor 3; TMEM173 = transmembrane protein 173; VLDLR = very low density lipoprotein receptor; WISP2 = WNT1-inducible signaling pathway protein 2.

Data Validation by Real-Time qRT-PCR

We determined the relative expression of possible pathogenetic genes related to vascular patterning, polarity control, and adhesion-related molecules in the SPCs from patients with MMD and control volunteers. We found increased expression of integrin α3, BAI1-associated protein 2–like 1, N-cadherin, EPH receptor A5, MCAM, GTPase-activating Rap/RanGAP domain–like 4, and decreased expression of Plexin C1, Neogenin homolog 1, and Rho GTPase-activating protein 28 (Fig. 6). These data confirmed the microarray results.

Discussion

Smooth-Muscle Progenitor Cells in MMD

This is the first study of SPCs from the peripheral blood of patients with MMD. The SPCs were characterized by distinctive expression of the SMC markers and could be differentiated from the EPCs and fibroblasts by showing little expression of CD31 and FSP-1, respectively.31,34,36,39

We discovered distinct characteristics of the progenitor cells during expansion. First, the absence of VEGF in the media and the addition of PDGF-BB seemed to facilitate differentiation into SPCs in patients with MMD. This finding indicates that vascular progenitor cells from patients with MMD may be quite sensitive to VEGF and primed to differentiate into EPCs rather than SPCs, although why this occurs is not known. Second, the emergence of SPC outgrowth cells from patients with MMD occurred at a later time point than for the control volunteers (Day 34 vs Day 20). Similarly, the first appearance of cobblestone-shaped EPCs was delayed in the patients' samples when cultured in an endothelial cell growth medium (EGM-2) without PDGF-BB enrichment.12 The reduced capability of vascular progenitor cells to be recruited and differentiated in patients with MMD might result in inadequate repair of vascular injury, which may be a key component of the disease pathogenesis. Third, SPC outgrowth cells from the patients tended to show lower expression of MHC and calponin compared with the controls. The earliest marker of vascular SMC differentiation is α-SMA, and MHC and calponin are specific markers of differentiated SMCs.19,25 A defect in the cell maturation process may have occurred in the SPCs from patients with MMD. In previous experiments, SMCs from the superficial temporal arteries of patients with MMD proliferated less rapidly and responded poorly to PDGF compared with controls.1 In addition, downregulation of PDGFR expression is observed in those cells, although study of the receptor subtypes was not performed. Our study demonstrated that expression of PDGFRα was lower in SPC outgrowth cells from patients with MMD compared with that in cells from healthy controls. Last, SPCs from patients with MMD made tubes. In the previous tube formation assay with EPCs in MMD, the cells demonstrated a poor capability for forming tubes.10,12 Notably, the tubes made from SPCs obtained in patients with MMD were rather irregularly arranged in comparison with those of healthy controls, and frequently tended to be thickened. This was reminiscent of the disease pathology, intimal thickening by proliferative SMCs. We postulate that SPCs in patients with MMD seem to have a role in constituting the intimal lesion.39 However, this in vitro observation needs to be ascertained in some in vivo situations.

The SMCs seem to be primarily affected by the MMD process, and MMD may be regarded as a type of hyperplastic vasculomyopathy.20 In this respect, we believe that SPCs from the peripheral blood of patients with MMD would provide a novel experimental cell model of MMD. The characteristics of the outgrowth cells would be significantly influenced by the cell culture conditions in which they were grown. Thus, these findings in vitro could be an artifact of the cell culture, which necessitates well-designed in vivo studies.

Gene Expression Signatures of SPCs in MMD

Our results showed a differential expression pattern of various relevant genes in the SPCs obtained in the patients with MMD. The GO analysis demonstrated that numerous upregulated genes are related to various responses, including the response to endogenous stimuli, pharmaceutical agents, hormonal stimuli, organic nitrogen, and peptide hormones. Furthermore, genes related to organogenesis such as regionalization, anterior/posterior pattern formation, and pattern specification process were upregulated. In addition, the downregulated DEGs are primarily involved in cell adhesion and cell migration as well as the innate immune response. It is known that MMD is caused by both genetic and environmental factors, and the internal carotid artery terminus is primarily affected by this disease. Therefore, these features (for example, hyperresponsiveness to stimuli and abnormality in cell adhesion and migration) of the DEGs in the SPCs seem be relevant to the disease process.

Putative Pathogenetic DEGs in SPCs Related to MMD

This study showed differential expression of several adhesion-related molecules, including the upregulation of integrin α3, BAI1-associated protein 2–like 1, N-cadherin, Eph receptor A5, and MCAM. Integrins are cell surface receptors responsive to the extracellular matrix, and play a key role in mediating various signals from the extracellular environment into the intracellular compartment. The integrin α3 subunit regulates the activation of NG2, a transmembrane chondroitin sulfate proteoglycan that is expressed exclusively in mural cells during vascular morphogenesis.26 NG2 proteoglycan can induce endothelial cell migration and promote the assembly of endothelial cell networks. Thus, increased expression of integrin α3 in the SPCs of patients with MMD may be related to disease pathogenesis. Brain-specific angiogenesis inhibitor 1 is specifically expressed in the brain and is a p53-inducible antiangiogenic molecule.22 It suppresses endothelial cell proliferation by blocking alpha(v)beta(5) integrin signaling.15 Increased expression of BAI1-associated protein 2–like 1 in the SPCs of patients with MMD may be related to occlusion of the internal carotid artery terminus. N-cadherin forms adherens junctions in vascular SMCs and modulates cell spreading, motility, and growth. N-cadherin is important for pericyte coverage of endothelial outgrowth in embryonic stem cell–derived angiogenesis.35 Therefore, overexpression of N-cadherin in the SPCs of patients with MMD may influence the SMC phenotype and increase mural cell mobilization.

In contrast to the transient upregulation of N-cadherin in the injury model, its overexpression might be sustained in SPCs during MMD. The role of Eph receptors and ephrins in vasculogenesis and angiogenesis is well known.38 Recently, an emerging role of Eph-ephrin molecules in mural cells suggests that these molecules function in vessel wall assembly; however, the role of specific Eph receptors in the mural cells remains unclear.16 The effect of upregulation of Eph receptor A5 in SPCs during MMD remains to be elucidated. MCAM, also known as CD146 and MUC18, is related to vascular SMC proliferation.30 Expression of gicerin, which is the rodent homolog of CD146, is increased in the SMCs of the rat carotid artery after balloon injury and is related to neointimal formation.24 Therefore, overexpression of MCAM in SPCs during MMD is of interest.

Notably, numerous genes related to guiding cue (“vascular patterning”) and polarity control were differentially expressed in SPCs obtained from patients with MMD. The expression of GTPase-activating Rap/RanGAP domain–like 4 was upregulated, whereas plexin C1, neogenin homolog 1, and Rho GTPase-activating protein 28 were downregulated. Both Rap (Ras-associated protein) and Ran (Ras-related nuclear protein) are small GTPases; Ran is known to play a role in nucleocytoplasmic transport of macromolecules, whereas Rap regulates integrin affinity and endothelial cell migration, and thus is a key player in postnatal angiogenesis.23

Upregulated GTPase-activating Rap/RanGAP domain–like 4 in the SPCs of patients with MMD would result in significant impairment of postnatal angiogenesis.3 Plexin C1 is a receptor for semaphorine 7A and is related to the inhibition of integrin-mediated adhesion and chemokine-induced migration of dendritic cells.37 Downregulation of this molecule in SPCs of patients with MMD may result in the disruption of vascular tree formation, increased cellular migration, and intimal hyperplasia. The netrin system is important for guidance during neural and vascular development, and neogenin is an important receptor for netrin, regulating diverse developmental processes. Neogenin mediates netrin signaling in vascular SMCs, and promotes adhesion and migration of vascular SMCs during angiogenesis.27 Thus, downregulation of neogenin in SPCs of patients with MMD would result in interruption of vessel wall maturation. Rho GTPases are known for their pivotal role in the regulation of the actin cytoskeleton; cell polarity and morphology; and cell migration, contraction, and proliferation.4 In addition, Rho GTPase stimulates SMC contraction to control blood flow. Downregulation of Rho GTPase-activating protein 28 in SPCs of patients with MMD would result in sustained activation of Rho GTPases, which may be related to increased SMC migration and proliferation. Putative pathogenetic roles of these molecules are summarized in Table 5. Further research is required to substantiate these hypotheses.

TABLE 5:

Literature review of putative pathogenetic DEGs in SPCs related to MMD*

Genes & ExpressionPutative Pathogenetic RolesAuthors & Year
ITGA3defective vascular morphogenesisOzerdem et al., 2001
BAIAP2L1occlusion of internal carotid artery terminusKoh et al., 2004; Nishimori et al., 1997
CDH2SMC phenotype change & increasing mobilizationTillet et al., 2005
EPHA5defective vessel wall assemblyKuijper et al., 2007; Zhang & Hughes, 2006
MCAMvascular SMC proliferationOkumura et al., 2004; Shih & Kurman, 1996
GARNL4impairment of postnatal angiogenesisCarmona et al., 2009; Nishimoto, 2000
PLXNC1disruption of vascular tree formation & increased cellular migration& intimal hyperplasiaWalzer et al., 2005
NEO1interruption of vessel wall maturationPark et al., 2004
ARHGAP28sustained activation of Rho GTPases & increased SMC migration& proliferationEtienne-Manneville& Hall, 2002

ARHGAP28 = Rho GTPase-activating protein 28; BAIAP2L1 = BAI1-associated protein 2–like 1; CDH2 = N-cadherin; EPHA5 = Eph receptor A5; GARNL4 = GTPase-activating Rap/RanGAP domain–like 4; ITGA3 = integrin α3; NEO1 = neogenin homolog 1; PLXNC1 = plexin C1; ↑ = upregulated; ↓ = downregulated.

Conclusions

With adequate culture conditions we obtained SPCs with distinct characteristics from the peripheral blood of patients with MMD. We confirmed specific DEGs related to cell adhesion, cell migration, immune response, and vascular development in the SPCs obtained in patients with MMD. This study provides a novel experimental cell model and basis for future research of MMD.

Acknowledgments

This study was supported by a grant from the Korea Healthcare Technology Research & Development Project, Ministry for Health, Welfare & Family Affairs, Republic of Korea (A120099), and the Seoul National University Hospital research fund (04-2010-0510).

Disclosure

The authors have nothing to disclose, and there are no conflicts of interest.

Author contributions to the study and manuscript preparation include the following. Conception and design: SK Kim, Kang, Wang. Acquisition of data: Moon, YY Kim, WY Park, AK Park. Analysis and interpretation of data: Kang, Moon, WY Park, AK Park. Drafting the article: Kang. Critically revising the article: all authors. Reviewed submitted version of manuscript: all authors. Approved the final version of the manuscript on behalf of all authors: SK Kim. Statistical analysis: Kang. Administrative/technical/material support: Moon. Study supervision: SK Kim.

References

  • 1

    Aoyagi MFukai NMatsushima YYamamoto MYamamoto K: Kinetics of 125I-PDGF binding and down-regulation of PDGF receptor in arterial smooth muscle cells derived from patients with moyamoya disease. J Cell Physiol 154:2812881993

  • 2

    Aoyagi MFukai NSakamoto HShinkai TMatsushima YYamamoto M: Altered cellular responses to serum mitogens, including platelet-derived growth factor, in cultured smooth muscle cells derived from arteries of patients with moyamoya disease. J Cell Physiol 147:1911981991

  • 3

    Carmona GGöttig SOrlandi AScheele JBäuerle TJugold M: Role of the small GTPase Rap1 for integrin activity regulation in endothelial cells and angiogenesis. Blood 113:4884972009

  • 4

    Etienne-Manneville SHall A: Rho GTPases in cell biology. Nature 420:6296352002

  • 5

    Hahne FMehrle AArlt DPoustka AWiemann SBeissbarth T: Extending pathways based on gene lists using InterPro domain signatures. BMC Bioinformatics 9:32008

  • 6

    Hojo MHoshimaru MMiyamoto STaki WNagata IAsahi M: Role of transforming growth factor–β1 in the pathogenesis of moyamoya disease. J Neurosurg 89:6236291998

  • 7

    Hoshimaru MTakahashi JAKikuchi HNagata IHatanaka M: Possible roles of basic fibroblast growth factor in the pathogenesis of moyamoya disease: an immunohistochemical study. J Neurosurg 75:2672701991

  • 8

    Imamura HOhta TTsunetoshi KDoi KNozaki KTakagi Y: Transdifferentiation of bone marrow-derived endothelial progenitor cells into the smooth muscle cell lineage mediated by tansforming growth factor-β1. Atherosclerosis 211:1141212010

  • 9

    Irizarry RAHobbs BCollin FBeazer-Barclay YDAntonellis KJScherf U: Exploration, normalization, and summaries of high density oligonucleotide array probe level data. Biostatistics 4:2492642003

  • 10

    Jung KHChu KLee STPark HKKim DHKim JH: Circulating endothelial progenitor cells as a pathogenetic marker of moyamoya disease. J Cereb Blood Flow Metab 28:179518032008

  • 11

    Kang HSKim JHPhi JHKim YYKim JEWang KC: Plasma matrix metalloproteinases, cytokines and angiogenic factors in moyamoya disease. J Neurol Neurosurg Psychiatry 81:6736782010

  • 12

    Kim JHJung JHPhi JHKang HSKim JEChae JH: Decreased level and defective function of circulating endothelial progenitor cells in children with moyamoya disease. J Neurosci Res 88:5105182010

  • 13

    Kim SKCho BKPhi JHLee JYChae JHKim KJ: Pediatric moyamoya disease: an analysis of 410 consecutive cases. Ann Neurol 68:921012010

  • 14

    Kim SKYoo JICho BKHong SJKim YKMoon JA: Elevation of CRABP-I in the cerebrospinal fluid of patients with Moyamoya disease. Stroke 34:283528412003

  • 15

    Koh KKChung WJAhn JYHan SHKang WCSeo YH: Angiotensin II type 1 receptor blockers reduce tissue factor activity and plasminogen activator inhibitor type-1 antigen in hypertensive patients: a randomized, double-blind, placebo-controlled study. Atherosclerosis 177:1551602004

  • 16

    Kuijper STurner CJAdams RH: Regulation of angiogenesis by Eph-ephrin interactions. Trends Cardiovasc Med 17:1451512007

  • 17

    Lee JYPhi JHWang KCCho BKShin MSKim SK: Neurocognitive profiles of children with moyamoya disease before and after surgical intervention. Cerebrovasc Dis 31:2302372011

  • 18

    Livak KJSchmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25:4024082001

  • 19

    Miano JMCserjesi PLigon KLPeriasamy MOlson EN: Smooth muscle myosin heavy chain exclusively marks the smooth muscle lineage during mouse embryogenesis. Circ Res 75:8038121994

  • 20

    Milewicz DMKwartler CSPapke CLRegalado ESCao JReid AJ: Genetic variants promoting smooth muscle cell proliferation can result in diffuse and diverse vascular diseases: evidence for a hyperplastic vasculomyopathy. Genet Med 12:1962032010

  • 21

    Nanba RKuroda SIshikawa THoukin KIwasaki Y: Increased expression of hepatocyte growth factor in cerebrospinal fluid and intracranial artery in moyamoya disease. Stroke 35:283728422004

  • 22

    Nishimori HShiratsuchi TUrano TKimura YKiyono KTatsumi K: A novel brain-specific p53-target gene, BAI1, containing thrombospondin type 1 repeats inhibits experimental angiogenesis. Oncogene 15:214521501997

  • 23

    Nishimoto T: Upstream and downstream of ran GTPase. Biol Chem 381:3974052000

  • 24

    Okumura SKohama KKim SIwao HMiki NTaira E: Induction of gicerin/CD146 in the rat carotid artery after balloon injury. Biochem Biophys Res Commun 313:9029062004

  • 25

    Owens GKKumar MSWamhoff BR: Molecular regulation of vascular smooth muscle cell differentiation in development and disease. Physiol Rev 84:7678012004

  • 26

    Ozerdem UGrako KADahlin-Huppe KMonosov EStallcup WB: NG2 proteoglycan is expressed exclusively by mural cells during vascular morphogenesis. Dev Dyn 222:2182272001

  • 27

    Park KWCrouse DLee MKarnik SKSorensen LKMurphy KJ: The axonal attractant Netrin-1 is an angiogenic factor. Proc Natl Acad Sci U S A 101:16210162152004

  • 28

    Park WYHwang CIIm CNKang MJWoo JHKim JH: Identification of radiation-specific responses from gene expression profile. Oncogene 21:852185282002

  • 29

    Rafat NBeck GChPeña-Tapia PGSchmiedek PVajkoczy P: Increased levels of circulating endothelial progenitor cells in patients with Moyamoya disease. Stroke 40:4324382009

  • 30

    Shih IMKurman RJ: Expression of melanoma cell adhesion molecule in intermediate trophoblast. Lab Invest 75:3773881996

  • 31

    Simper DStalboerger PGPanetta CJWang SHCaplice NM: Smooth muscle progenitor cells in human blood. Circulation 106:119912042002

  • 32

    Smyth GK: Linear models and empirical bayes methods for assessing differential expression in microarray experiments. Stat Appl Genet Mol Biol 3:Article 32004

  • 33

    Soriano SGCowan DBProctor MRScott RM: Levels of soluble adhesion molecules are elevated in the cerebrospinal fluid of children with moyamoya syndrome. Neurosurgery 50:5445492002

  • 34

    Sugiyama SKugiyama KNakamura SKataoka KAikawa MShimizu K: Characterization of smooth muscle-like cells in circulating human peripheral blood. Atherosclerosis 187:3513622006

  • 35

    Tillet EVittet DFéraud OMoore RKemler RHuber P: N-cadherin deficiency impairs pericyte recruitment, and not endothelial differentiation or sprouting, in embryonic stem cell-derived angiogenesis. Exp Cell Res 310:3924002005

  • 36

    van Oostrom OFledderus JOde Kleijn DPasterkamp GVerhaar MC: Smooth muscle progenitor cells: friend or foe in vascular disease?. Curr Stem Cell Res Ther 4:1311402009

  • 37

    Walzer TDalod MRobbins SHZitvogel LVivier E: Natural-killer cells and dendritic cells: “l'union fait la force.”. Blood 106:225222582005

  • 38

    Zhang JHughes S: Role of the ephrin and Eph receptor tyrosine kinase families in angiogenesis and development of the cardiovascular system. J Pathol 208:4534612006

  • 39

    Zoll JFontaine VGourdy PBarateau VVilar JLeroyer A: Role of human smooth muscle cell progenitors in atherosclerotic plaque development and composition. Cardiovasc Res 77:4714802008

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Article Information

Address correspondence to: Seung-Ki Kim, M.D., Ph.D., Division of Pediatric Neurosurgery, Seoul National University Children's Hospital, 101 Daehak-ro, Jongno-gu, Seoul 110-744, Korea. email: nsthomas@snu.ac.kr.

Please include this information when citing this paper: published online October 25, 2013; DOI: 10.3171/2013.9.JNS131000.

© AANS, except where prohibited by US copyright law.

Headings

Figures

  • View in gallery

    Findings related to SPCs obtained in a 7-year-old boy in whom MMD was diagnosed. A: Photomicrograph showing confluent cells that have a hill-and-valley morphology at Day 31. Bar = 500 μm. B–D: Graphs showing results of FACS analysis of SPCs obtained in this patient. The black curves represent the isotype-matched control IgG antibodies, and the green curves represent the test antibodies (anti–α-SMA, anti-CD31, and anti-CD34). Values on the y axis quantify the intensity of fluorescein isothiocyanate staining (FITC log), and on the x axis the values represent the number of cells counted. The SPCs showed 98.9% α-SMA and 0.7% CD31 expression. The CD34-positive cells, considered primitive SPCs, accounted for 13.7% of the SPCs. M1 = marker line for positive cells.

  • View in gallery

    Photomicrographs showing immunofluorescence staining of SPCs labeled with antibodies against α-SMA, calponin, CD34, and CD31. The coexpression of α-SMA and CD34 as well as calponin and CD34 was also observed in SPCs obtained in patients with MMD. Cell nuclei in the last column were counterstained with DAPI. Bar = 500 μm.

  • View in gallery

    Tube formation assay. A and B: Photomicrographs showing immunofluorescence staining of SPCs. Compared with SPCs obtained in a healthy volunteer (A), SPCs obtained in a patient with MMD made rather irregularly arranged tubules of varying sizes (B). In some areas, thickened tubules were noted (arrows). Bars = 500 μm. C and D: Bar graphs showing that SPCs obtained in patients with MMD formed thickened tubules of fewer numbers compared with controls (SPCs were obtained in 3 patients and 3 control volunteers). Asterisks = p < 0.01.

  • View in gallery

    Boxplots demonstrating PDGFR expression in SPCs obtained in patients with MMD and control volunteers (left, PDGFRα; right, PDGFRβ). The SPCs obtained in patients with MMD show lower PDGFRα (CD140a, p = 0.0286) and similar PDGFRβ (CD140b, p = 0.1143) expression compared with controls. Percentages of positive cells are shown on each y axis.

  • View in gallery

    Heat map and principal component plot of 286 DEGs. Left: Heat map and 1-way hierarchical clustering of 286 DEGs. Right: Principal component plot of 286 DEGs.

  • View in gallery

    Real-time qRT-PCR analysis showing differential expression of 9 selected genes: increased expression of integrin alpha 3 (ITGA3), BAI1-associated protein 2–like 1 (BAIAP2L1), N-cadherin (CDH2), EPH receptor A5 (EPHA5), melanoma cell adhesion molecule (MCAM), GTPase-activating Rap/RanGAP domain–like 4 (GARNL4), and decreased expression of Plexin C1 (PLXNC1), Neogenin homolog 1 (NEO1), and Rho GTPase-activating protein 28 (ARHGAP28).

References

1

Aoyagi MFukai NMatsushima YYamamoto MYamamoto K: Kinetics of 125I-PDGF binding and down-regulation of PDGF receptor in arterial smooth muscle cells derived from patients with moyamoya disease. J Cell Physiol 154:2812881993

2

Aoyagi MFukai NSakamoto HShinkai TMatsushima YYamamoto M: Altered cellular responses to serum mitogens, including platelet-derived growth factor, in cultured smooth muscle cells derived from arteries of patients with moyamoya disease. J Cell Physiol 147:1911981991

3

Carmona GGöttig SOrlandi AScheele JBäuerle TJugold M: Role of the small GTPase Rap1 for integrin activity regulation in endothelial cells and angiogenesis. Blood 113:4884972009

4

Etienne-Manneville SHall A: Rho GTPases in cell biology. Nature 420:6296352002

5

Hahne FMehrle AArlt DPoustka AWiemann SBeissbarth T: Extending pathways based on gene lists using InterPro domain signatures. BMC Bioinformatics 9:32008

6

Hojo MHoshimaru MMiyamoto STaki WNagata IAsahi M: Role of transforming growth factor–β1 in the pathogenesis of moyamoya disease. J Neurosurg 89:6236291998

7

Hoshimaru MTakahashi JAKikuchi HNagata IHatanaka M: Possible roles of basic fibroblast growth factor in the pathogenesis of moyamoya disease: an immunohistochemical study. J Neurosurg 75:2672701991

8

Imamura HOhta TTsunetoshi KDoi KNozaki KTakagi Y: Transdifferentiation of bone marrow-derived endothelial progenitor cells into the smooth muscle cell lineage mediated by tansforming growth factor-β1. Atherosclerosis 211:1141212010

9

Irizarry RAHobbs BCollin FBeazer-Barclay YDAntonellis KJScherf U: Exploration, normalization, and summaries of high density oligonucleotide array probe level data. Biostatistics 4:2492642003

10

Jung KHChu KLee STPark HKKim DHKim JH: Circulating endothelial progenitor cells as a pathogenetic marker of moyamoya disease. J Cereb Blood Flow Metab 28:179518032008

11

Kang HSKim JHPhi JHKim YYKim JEWang KC: Plasma matrix metalloproteinases, cytokines and angiogenic factors in moyamoya disease. J Neurol Neurosurg Psychiatry 81:6736782010

12

Kim JHJung JHPhi JHKang HSKim JEChae JH: Decreased level and defective function of circulating endothelial progenitor cells in children with moyamoya disease. J Neurosci Res 88:5105182010

13

Kim SKCho BKPhi JHLee JYChae JHKim KJ: Pediatric moyamoya disease: an analysis of 410 consecutive cases. Ann Neurol 68:921012010

14

Kim SKYoo JICho BKHong SJKim YKMoon JA: Elevation of CRABP-I in the cerebrospinal fluid of patients with Moyamoya disease. Stroke 34:283528412003

15

Koh KKChung WJAhn JYHan SHKang WCSeo YH: Angiotensin II type 1 receptor blockers reduce tissue factor activity and plasminogen activator inhibitor type-1 antigen in hypertensive patients: a randomized, double-blind, placebo-controlled study. Atherosclerosis 177:1551602004

16

Kuijper STurner CJAdams RH: Regulation of angiogenesis by Eph-ephrin interactions. Trends Cardiovasc Med 17:1451512007

17

Lee JYPhi JHWang KCCho BKShin MSKim SK: Neurocognitive profiles of children with moyamoya disease before and after surgical intervention. Cerebrovasc Dis 31:2302372011

18

Livak KJSchmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25:4024082001

19

Miano JMCserjesi PLigon KLPeriasamy MOlson EN: Smooth muscle myosin heavy chain exclusively marks the smooth muscle lineage during mouse embryogenesis. Circ Res 75:8038121994

20

Milewicz DMKwartler CSPapke CLRegalado ESCao JReid AJ: Genetic variants promoting smooth muscle cell proliferation can result in diffuse and diverse vascular diseases: evidence for a hyperplastic vasculomyopathy. Genet Med 12:1962032010

21

Nanba RKuroda SIshikawa THoukin KIwasaki Y: Increased expression of hepatocyte growth factor in cerebrospinal fluid and intracranial artery in moyamoya disease. Stroke 35:283728422004

22

Nishimori HShiratsuchi TUrano TKimura YKiyono KTatsumi K: A novel brain-specific p53-target gene, BAI1, containing thrombospondin type 1 repeats inhibits experimental angiogenesis. Oncogene 15:214521501997

23

Nishimoto T: Upstream and downstream of ran GTPase. Biol Chem 381:3974052000

24

Okumura SKohama KKim SIwao HMiki NTaira E: Induction of gicerin/CD146 in the rat carotid artery after balloon injury. Biochem Biophys Res Commun 313:9029062004

25

Owens GKKumar MSWamhoff BR: Molecular regulation of vascular smooth muscle cell differentiation in development and disease. Physiol Rev 84:7678012004

26

Ozerdem UGrako KADahlin-Huppe KMonosov EStallcup WB: NG2 proteoglycan is expressed exclusively by mural cells during vascular morphogenesis. Dev Dyn 222:2182272001

27

Park KWCrouse DLee MKarnik SKSorensen LKMurphy KJ: The axonal attractant Netrin-1 is an angiogenic factor. Proc Natl Acad Sci U S A 101:16210162152004

28

Park WYHwang CIIm CNKang MJWoo JHKim JH: Identification of radiation-specific responses from gene expression profile. Oncogene 21:852185282002

29

Rafat NBeck GChPeña-Tapia PGSchmiedek PVajkoczy P: Increased levels of circulating endothelial progenitor cells in patients with Moyamoya disease. Stroke 40:4324382009

30

Shih IMKurman RJ: Expression of melanoma cell adhesion molecule in intermediate trophoblast. Lab Invest 75:3773881996

31

Simper DStalboerger PGPanetta CJWang SHCaplice NM: Smooth muscle progenitor cells in human blood. Circulation 106:119912042002

32

Smyth GK: Linear models and empirical bayes methods for assessing differential expression in microarray experiments. Stat Appl Genet Mol Biol 3:Article 32004

33

Soriano SGCowan DBProctor MRScott RM: Levels of soluble adhesion molecules are elevated in the cerebrospinal fluid of children with moyamoya syndrome. Neurosurgery 50:5445492002

34

Sugiyama SKugiyama KNakamura SKataoka KAikawa MShimizu K: Characterization of smooth muscle-like cells in circulating human peripheral blood. Atherosclerosis 187:3513622006

35

Tillet EVittet DFéraud OMoore RKemler RHuber P: N-cadherin deficiency impairs pericyte recruitment, and not endothelial differentiation or sprouting, in embryonic stem cell-derived angiogenesis. Exp Cell Res 310:3924002005

36

van Oostrom OFledderus JOde Kleijn DPasterkamp GVerhaar MC: Smooth muscle progenitor cells: friend or foe in vascular disease?. Curr Stem Cell Res Ther 4:1311402009

37

Walzer TDalod MRobbins SHZitvogel LVivier E: Natural-killer cells and dendritic cells: “l'union fait la force.”. Blood 106:225222582005

38

Zhang JHughes S: Role of the ephrin and Eph receptor tyrosine kinase families in angiogenesis and development of the cardiovascular system. J Pathol 208:4534612006

39

Zoll JFontaine VGourdy PBarateau VVilar JLeroyer A: Role of human smooth muscle cell progenitors in atherosclerotic plaque development and composition. Cardiovasc Res 77:4714802008

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