moyamoya disease ( mmd ) is a specific cerebrovascular disease affecting the termini of the internal carotid arteries , typically bilaterally9 ) .
thus , a natural approach is to investigate the vessel wall cells of mmd patients in search of significant findings that are relevant to mmd pathophysiology and treatment .
we propose that this research interest must be driven in two directions , endothelial - lineage cells and smooth muscle - type ( mural ) cells , as both cells are indispensable to completely healthy vasculature . in reality , it is not easy to obtain these cells directly from patients , except for tiny specimens that are obtained during direct bypass surgery and tissues from autopsy cases . therefore , vascular progenitor cells seem to be a valuable cell type for disease research because they can be derived from patient peripheral blood .
endothelial progenitor cells ( epcs ) have been an important topic in research evaluating several vascular diseases , including heart disease and ischemic cerebrovascular disease ; however , the concept of smooth muscle progenitor cells ( spcs ) has only recently been introduced . in this review
, we present the up - to - date accomplishments with regard to vascular progenitor cell studies in moyamoya disease .
circulating epcs originate from the bone marrow , and they seem to take part in both postnatal vasculogenesis and vascular homeostasis7 ) .
epcs are usually characterized by the surface protein expression of cd31 [ an endothelial cell marker , also known as pecam-1 ( platelet endothelial cell adhesion molecule-1 ) ] , cd34 ( a hematopoietic or progenitor marker ) , and vascular endothelial growth factor receptor-2 [ vegfr-2 , also known as kdr [ kinase insert domain receptor ) ] .
several investigators consider the stem cell marker cd133 to be a more precise epc marker .
cd133/cd34/vegfr-2 cells are considered to represent a more primitive epc type with high proliferative potential . in identifying epcs , the usefulness of other markers such as cd45 ( the leukocyte common antigen ) and cd14 ( a myelomonocytic cell marker ) is controversial3 ) .
the expression of von willebrand factor , uptake of acetylated low - density lipoprotein , and enhanced endothelial nitric oxide synthase expression after shear - stress exposure can also be demonstrated14 ) .
functionally , epcs exhibit tremendous proliferative capacity and can be mobilized to vasculogenesis sites1 ) .
spcs from human peripheral blood were first isolated and cultured by simper et al.12 ) in platelet - derived growth factor ( pdgf ) bb - enriched media .
spc - type outgrowth cells show a " hill - and - valley " appearance , while epc - type outgrowth cells show a " cobblestone " appearance ( fig . 1)1214 ) .
in addition to the striking morphological differences in the outgrowth cells , they are characterized by positive staining with smooth muscle actin- , smooth muscle myosin heavy chain ( mhc ) and calponin , which are smooth muscle - specific . in a previous experiment , these cells had 4- to 5-fold greater proliferative potential compared with endothelial outgrowth cells12 ) . for epc culture from peripheral blood ,
the cells are plated in culture dishes that are coated with fibronectin and are cultured in endothelial cell growth medium that is enriched with vascular endothelial growth factor ( vegf ) .
for spc cultures , pdgf bb - enriched media seems to be essential . in a previous experiment ,
pdgf - bb was added at a concentration of 5 ng / ml since the seventh day of culturing6 ) .
two studies present opposing opinions on the relation between epc circulation and moyamoya disease810 ) .
rafat et al.10 ) demonstrated increased circulating hematopoietic stem cell and epc levels ( healthy controls vs. moyamoya disease subjects , 0.09% vs. 0.29% for cd34cd133 cells ; 0.02% vs. 0.11% for cd34cd133vegfr-2 cells ) from an adult moyamoya disease population ( mean age , 35.715.9 years ) .
in contrast , kim et al.8 ) studied circulating epcs from a pediatric moyamoya disease population ( mean age , 7.5 years ; age range , 2 - 13 years ) . on the day when the blood was collected ( day 0 ) , kim et al.8 ) showed that the percentages of circulating cells that were positive for cd34 , cd133 , and kdr were significantly lower in the study subject population ( normal controls vs. subjects , 8.48% vs. 2.97% for cd34 ; 0.56% vs. 0.09% for cd133 ; and 8.40% vs. 1.04% for kdr ; all p less than 0.05 ) .
rafat et al.10 ) presented data regarding double ( cd34cd133 cells ) and triple positive cells ( cd34cd133vegfr-2 cells ) , while kim et al.8 ) provided the numbers of cells that were positive for the respective markers .
thus , the differences in the datasets and ages of the study subjects make direct comparison of the two studies difficult .
we acknowledge that the cd133 cells made up less than 1% of the peripheral blood mononuclear cells in both studies . in general , there are more driving cues for vascular progenitor cell recruitment in patients with ischemic cerebrovascular diseases . failure to show such an adequate response might be responsible for early and severe manifestations in pediatric mmd patients .
jung et al.5 ) compared the numbers of colony - forming units ( cfu , also called as ' cell clusters ' ) of epcs and outgrowth cells between mmd patients and healthy controls , and they demonstrated that the cfu numbers decreased ( mmd patients vs. controls , 32.426.7 vs. 65.218.3 , p<0.001 ) and the outgrowth cells were more frequently isolated in the mmd patients ( 33.3% vs. 10.4% , p=0.025 ) .
notably , the outgrowth cells were isolated in all patients ( n=3 ) who underwent revascularization surgery .
additionally , they found that low epc - cfu number ( less than 30 ) was more prevalent in the patients with more advanced mmd ( p=0.001 ) .
they also showed that conditioned media from epcs of mmd patients significantly induced less tube formation , which reflected impaired paracrine function in the patients ' epcs .
three - day cultures of mononuclear cells were used to produce the conditioned media , and human umbilical vein endothelial cells were employed for the tubule formation assay .
pediatric mmd patients also showed reduced epc cell cluster formation8 ) . from a different perspective ,
kim et al.8 ) conducted a tubule formation assay with late epcs ( i.e. , cells with cobblestone morphology ) from mmd patients and normal controls , and they showed that the patient cells had reduced tubule formation capability .
a senescence - associated -galactosidase assay revealed a higher percentage of senescent cells among the epcs from the patients .
recently , cells expressing cd34 and vegfr2 were found in the thickened intima of supraclinoid internal carotid arteries that were collected from adult mmd patients13 ) .
these findings suggest that bone marrow - derived circulating epcs may participate in development of the occlusive arterial lesions in mmd .
the origin of the smooth muscle cells is one of the key questions regarding mmd pathophysiology .
we established spcs from the peripheral blood of mmd patients6 ) . for cell culture , well plates that were coated with type i collagen were used .
after 5 days , non - adherent cells were removed , and fresh culture medium was applied . at 1 week , pdgf - bb was added to the culture medium .
after the initial cell cluster formation , two patterns of outgrowth cells appeared , which included spcs ( hill - and - valley pattern ) and epcs ( cobblestone appearance ) .
fluorescence - activated cell sorter ( facs ) analysis demonstrated that spcs were present in 20% of the observed mmd patients , epcs were present in 68% of the patients , and a mixed pattern was observed in 12% of the patients . among the normal controls ,
facs analysis revealed that spcs were present in 30% , epcs were present in 40% , and a mixed population was present in 40% of the subjects
spcs from the patients had lower pdgf receptor  expression ( mmd patients vs. controls , 38.5%2.6% vs. 66.1%8.2% , p=0.0286)6 ) .
pdgf receptor  expression was not significantly different between the groups ( 87.73.1% vs. 96.61.2% , p= 0.1143 ) .
spcs from the patients also had lower mhc ( 42.818.6% vs. 961.8% ; p=0.0087 ) and calponin expression levels ( 87.1 8.2% vs. 99.80.1% ; p=0.0519 ) .
it was presumed that a defect in the cell maturation process might have occurred in the spcs from the mmd patients .
interestingly , the tubule formation assay identified more irregularly arranged and thickened tubules with the patient spcs , which is reminiscent of the pathologic findings of the mmd patient cerebral arteries ( fig .
an affymetrix gene chip mrna microarray revealed 286 differentially expressed genes in spcs from mmd patients with a > 1.5-fold change and a t - test p value<0.016 ) .
gene ontology ( go ) analysis for the 124 up - regulated transcripts identified 19 terms ( p<0.05 ) , which included responses to endogenous stimulus , anterior / posterior pattern formation , and ephrin receptor signaling .
meanwhile , the go analysis for the 162 down - regulated transcripts identified 26 terms ( p<0.01 ) , including cell adhesion , cell migration regulation , innate immune response , enzyme - linked receptor protein signaling pathways , and vasculature development .
it is known that circulating epc number increases significantly after various injury types , including myocardial infarction , coronary artery bypass grafting , and stroke2411 ) .
studies evaluating epcs from mmd patients showed differences in epc mobilization between adult and pediatric patients810 ) , which might explain differences in the disease presentation and severity between the two age groups .
additionally , epcs from mmd patients were found to be functionally impaired , whereby they failed to form tubules and showed defects in paracrine functions510 ) .
further studies are under way to define the specific causes and possible solutions for these functional impairments .
it can be hypothesized that the thickened intima in mmd patients ' cerebral arteries may ( at least partly ) result from malfunctioning epcs and spcs during the vascular repair and maintenance process .
although studies on spcs are scarce , a study revealed some distinctive characteristics in relation to the cell culture finding , immaturity in terms of expression markers , defective tubule formation , and differential gene expression that were relevant to vascular development .
further studies are required to specify these characteristics in the setting of disease pathogenesis . moreover , in vivo studies and animal models that mimic the disease are also required .
still , there is much to learn in regard to mmd and vascular progenitor cells .
thus , research that utilizes these particular cells seems to be promising , although the establishment and maintenance of progenitor cells requires significant manpower , facilities , and funds .
 various approaches have been attempted in translational moyamoya disease research . one promising material for modeling and treating this disease is vascular progenitor cells , which can be acquired and expanded from patient peripheral blood . 
 these cells may provide a novel experimental model and enable us to obtain insights regarding moyamoya disease pathogenesis . 
 we briefly present the recent accomplishments in regard to the studies of vascular progenitor cells in moyamoya disease . 
INTRODUCTION
VASCULAR PROGENITOR CELLS
EPCS AND MOYAMOYA DISEASE
VASCULAR SPCS AND MOYAMOYA DISEASE
FUTURE PROSPECTS
CONCLUSION