International Journal of Developmental Neuroscience
j our na l ho me p age: www.elsev ier .com/ locate / i jdevneu
CM2 expression during prenatal development and adult human neocortex
amze Tanriovera,∗, Berna Sozena, Murat Gunelb, Necdet Demira
Department of Histology and Embryology, Faculty of Medicine, Akdeniz University, 07070, Antalya, TurkeyDepartment of Neurosurgery, Yale University School of Medicine, 333 Cedar Street, 06510 New Haven, CT, USA
r t i c l e i n f o
rticle history:eceived 11 November 2010eceived in revised form 9 March 2011ccepted 21 April 2011
eywords:CM2uman neocortex
a b s t r a c t
Cerebral cavernous malformation (CCM) is one of the most common types of vascular malformations ofthe central nervous system, affecting nearly one in 200 people. CCM lesions are characterized by grosslydilated vascular channels lined by a single layer of endothelium. Genetic linkage analyses have mappedthree CCM loci to CCM1, CCM2 and CCM3. All three causative genes have now been identified allowingnew insights into CCM pathophysiology. We focused on the CCM2 protein that might take place in bloodvessel formation; we report here the expression patterns of CCM2 in prenatal development and adulthuman neocortex by means of immunohistochemistry and Western blot analysis. CCM2 was obviously
mmunohistochemistryestern blotting
detected in vascular endothelium and neuroglial precursor cells during development and also observedin arterial endothelium, neurons, some of the glial cells in adult neocortex. The expression patternssuggest that it could be one of the arterial markers whether this is a cause or a consequence of an alteredvascular identity. CCM2 might play a role during vasculogenesis and angiogenesis during human braindevelopment. Furthermore, with this study, CCM2 have been described for the first time in developinghuman neocortex.
. Introduction
Cerebral cavernous malformations (CCMs) are vascular malfor-ations, mostly located in the central nervous system. Patientsay have single or multiple malformations leading to focal neu-
ologic signs, hemorrhagic strokes, seizures, or sometimes deathRigamonti et al., 1988). CCM lesions are characterized by grosslyilated vascular channels lined by a single layer of endotheliumRussell and Rubinstein, 1989). They lack normal vessel wall ele-
ents such as smooth muscles and are also devoid of interveningormal parenchyma (Clatterbuck et al., 2001). CCM occurs spo-adically but may also be inherited dominantly with incompleteenetrance. The pattern of inheritance of the familial form isutosomal dominant. The proportion of familial cases has beenstimated to be as high as 50% in Hispanic-American patients andlose to 10–40% in other populations (Gunel et al., 1996; Pozzatit al., 1996). Familial forms have been linked to three chromoso-al loci, and loss of function mutations have been identified in the
RIT1/Ccm1 (Marchuk et al., 1995), MGC4607/Ccm2 (Craig et al.,998), and PDCD10/Ccm3 (Dubovsky et al., 1995) genes.
The early development and the structural organization of theuman neocortex is divided into a number of histogenetic fieldsThiery, 1984). Marginal zone (MZ), is formed below the pial surface
and the area is supposed to be an active zone of the develo-ping human cortex. This zone promotes the maturation of earlygenerated neurons and also marks the beginning of the corticalneurogenesis. Thus, the superficial lamina containing the first dif-ferentiated neuronal elements is called the primordial plexiformlayer (PPL) (Marin-Padilla, 1998). It is already known that the neu-roblasts from the VZ migrate upward and the arrival of the firstmigratory neurons splits the PPL in two regions. One, close to thepial surface, becomes the MZ and thereafter is called subplate (SP).At the end of the first trimester, the neocortex is comprised of sixdiscrete zones MZ, CP (cortical plate), SP (subplate), IZ (interme-diate zone), SVZ (subventricular zone), VZ (ventricular) (Chan et al.,2002). During further developmental stages, six layers are formedand the SP disappeared. The SP neurons destined to die later onthrough a process of programmed cell death, and the fibers will befree to reach their appropriate zone. So, SP transforms into whitematter in adult cortex (Rakic, 1982). Also, the thickness of the CPprogressively contributes to formation of layers II through VI inadult cortex (Bentivoglio et al., 2003).
During the development, there are two processes involved inblood vessel formation. The first process, vasculogenesis, occurswhen a primitive vascular network is constructed from pluripotentmesenchymal progenitors. The second process, angiogenesis, fol-
lows vasculogenesis, and it is characterized by capillary sproutsarising from pre-existing vasculogenic centers (Risau and Flamme,1995; Risau, 1997). Endothelial cells play a dynamic role in bothangiogenesis and vasculogenesis. Differentiation of multipotential
Fig. 1. Representative pictures of CCM2 staining in developing neocortex during
10 G. Tanriover et al. / Int. J. Dev
esencymal cells, migration and proliferation of endothelial cellsnd formation of cell–cell connections are important step, for suc-essful vasculogenesis (Hanahan, 1997). The mechanisms involvedn cerebral blood vessel angiogenesis during development are stilloorly defined. Therefore, understanding of the mechanisms ofngiogenesis is the most important step in cerebrovascular mal-ormations. These three proteins leading to the CCM pathologyuggest that there are new players in vascular morphogenesis andemodeling and also contributing to a better understanding of nor-al and pathological angiogenesis (Guzeloglu-Kayisli et al., 2004;
ergametti et al., 2005; Plummer et al., 2005; Tanriover et al.,009). Moreover, Guzeloglu-Kayisli et al. (2004) and our previoustudy pointed out that CCM1, CCM2 and CCM3 may play a key rolen vessel formation and development during early angiogenesisTanriover et al., 2009). And also, Seker et al. (2006) characteri-ed the messenger ribonucleic acid (mRNA) distribution of Ccm1nd Ccm2 in the embryonic and postnatal periods of central ner-ous system of mice. Ccm1 expression parallels that of Ccm2 and isrimarily observed on the arterial side of the cerebral vasculature,eurons and astrocytes in mouse. Thus, in the present study we ana-
yzed the cell specific expression pattern of CCM2 protein in humaneocortex. To this end, we performed immunohistochemical ana-
ysis using a novel antibody generated against the CCM2 proteinxpression during adult and developmental period of human neo-ortex. Also, the present study focuses on the CCM2 expressionattern to clarify the neuronal orientation in human cortical deve-
opmental period.
. Material and methods
Thirteen human developing brains from spontaneous abortions (n = 5, n = 4 and = 4 from second, third trimesters and adult) were used in the study. Written infor-ed consent was obtained from each woman before the operation using consent
orms and protocols approved by the Human Investigation Committee of Akdenizniversity. None of the specimens had cerebral malformation or cerebral hemor-
hagic abnormalities. All specimens used were in normal neurological conditions.he fetuses and adult brain materials had no pathological defect at the macrosco-ic and microscopic levels, as evaluated by the Department of Pathology, Akdenizniversity, Medical Faculty (Tanriover et al., 2004; Tanriover et al., 2005).
.1. Immunohistochemistry
For CCM2 immunohistochemistry, sections were deparaffinized and rehydra-ed by standard methods then endogenous peroxidase activity was blocked with
ethanol containing 3% H2O2 for 20 min, at room temperature. Rabbit polyclonalnti-CCM2 (made in Zymed) primary antibody (1/250) was applied for 2 h at roomemperature. Negative controls were performed by replacing the primary antibo-ies with normal rabbit serum at the same concentration. After several rinses inBS, biotinylated goat anti-rabbit IgG (1/400 dilution Vector Lab. Burlingame, CA,SA) was applied for 30 min. Following several PBS rinses, slides were incuba-
ed with streptavidin–peroxidase complex for the appropriate time by using DakoSAB kit (Dako, Carpinteria, CA, USA). Antibody complex was visualized by incu-ation with diaminobenzidine (DAB) chromogen (BioGenex) prepared according tohe manufacturer’s instructions. Slides were counterstained slightly with Mayer’sematoxylin (Dako, Glostrup, Denmark) prior to permanent mounting and thenvaluated under a light microscope.SDS polyacrylamide gel electrophoresis andestern blotting
Total protein from the tissues was extracted in a lysis buffer (10 mM Tris-CL, 1 mM EDTA, 2.5% SDS, 1 mM phenylmethylsulfonylfluoride, 1 �g/ml leupeptin)
upplemented with CompleteR protease inhibitor cocktail (Boehringer, Mannheim,ermany). The protein concentration was determined using a standard BCA assay
Wiechelman et al., 1988) and 50 �g proteins were applied per lane. Prior to electro-horesis, samples were heated for 5 min at 95 ◦C. Samples were then subjected toDS polyacrylamide gel electrophoresis under standard conditions and then trans-erred onto PVDF membrane (BioRad, Hercules, CA, USA) in a buffer containing.2 mol/l glycine, 25 mM Tris and 20% methanol. The membrane was blocked for 1 hith 5% nonfat dry milk (BioRad, Hercules, CA, USA) in TBS-T to decrease nonspecific
inding. Afterwards, the membrane was incubated with rabbit polyclonal antibodygainst human CCM2 (dilution 1/1000 in 5% nonfat dry milk in TBS-T) for 1 h. The
embrane was then incubated with horse peroxidase-labelled anti-rabbit IgG (dilu-
ion 1/10,000; Vector Laboratories) for 1 h. Immunolabelling was visualized usinghe chemiluminescence based SuperSignal CL HRP Substrate System (Pierce, Rock-ord, IL, USA) and the membrane was exposed to Hyperfilm (Amersham, Piscataway,J, USA).
first trimester. The cytoplasmic reaction of neocortex cells and fibers show a strongimmunoreactivity for CCM2. MZ: marginal zone, PPL: primordial plexiform layer,SVZ: subventricular zone, VZ: ventricular zone. Scale bar: 50 �m.
After the membrane was stripped using Stripping solution (Pierce), equal loa-ding of proteins in each lane was confirmed by re-probing the membrane withmouse monoclonal anti-human �-actin (Abcam, Cambridge, UK).
3. Results
3.1. CCM2 expression in normal human neocortex duringprenatal development and adult samples
Using immunohistochemical staining, we studied the expres-sion patterns of CCM2 in human neocortex tissue duringdevelopmental period and also adult samples. During the first tri-mester, the fibers in the developing human neocortex revealed astrong CCM2 protein immunoreactivity in PPL. In addition, the cyto-plasm of the cells was also immunoreactive for CCM2 in SVZ andVZ (Fig. 1).
During the second trimester, CCM2 immunoreactivity wasdetected throughout the neocortex layers (Fig. 2A–G). Additionally,there was a strong CCM2 immunoreaction localized in the vascu-lar endothelium during second trimester (Fig. 2C–G, arrowheads).The moderate localization of CCM2 in some of the cells was spe-cific to MZ, CP and SP zones (Fig. 2B–D arrows). The strong CCM2immunolabelling was shown in the surface of the MZ and VZ. Incomparison with the first trimester neocortex immunoreactivitywas higher and occasionally nuclear in the specific zones.
CCM2 immunoreactivity in the third trimester was strongerthan that in the second and first trimesters, not only in the cellsand fibers but also in the vascular endothelium. In the neocortexof the third trimester, CCM2 was observed in a strong immuno-reactivity in some of the vascular endothelium (Fig. 3D, E and K,arrowheads). On the other hand, some of the vascular endotheliumrevealed a weak CCM2 immunolabelling during the third trimester(Fig. 3E, arrowhead).
In the adult brain samples, the strongest CCM2 immunoreac-tivity was obtained in the neocortex layers from trimesters. Theexpression of CCM2 was increased in the arterial endothelium asreferred in our previous study (Tanriover et al., 2008). On the otherhand, the expression of CCM2 was detected in a subset of neuro-nal cells, some glial cells (Fig. 4B arrows) and, at very low levels inthe venous endothelium (Fig. 4D, arrowheads). Moreover, the stai-ning pattern for CCM2 protein was strongly restricted to the arterialendothelium in adult brain samples (Fig. 4C, E and F, arrowheads).
No immunoreactivity was observed on the slides where pri-mary antibody was replaced with their normal rabbit IgG (data notshown).
3.2. Confirmation of CCM2 expression by Western blot analysis
CCM2 expression was analyzed by Western blotting from nor-mal human developing and adult brain tissues. The blots clearly
G. Tanriover et al. / Int. J. Devl Neuroscience 29 (2011) 509–514 511
Fig. 2. Representative pictures of CCM2 staining in developing human neocortex during second trimester. The heterogeneous immunoreactivity was seen in second trimestersand neocortex for CCM2. (A) Strong to moderate immunoreactivity for CCM2 in different layers marginal zone (MZ), cortical plate (CP), subplate (SP), intermediate zone (IZ),subvetricular zone (SVZ), and ventricular zone (VZ) of neocortex were seen in the second trimester. (B) Marginal zone. (C) Cortical plate. (D) Subplate. (E) Intermediate zone.( ads) an
evealed bands for CCM2 corresponding to 47 kDa, in developingnd adult normal brain tissue samples. Equivalent amounts ofotal proteins were loaded per lane as indicative by the immuno-xpression of �-actin (43 kDa). According to the Western blotesults, CCM2 expression was detected at all trimesters and adulteocortex. Furthermore, it gradually increased from first to thirdrimester and adult neocortex. The protein level of CCM2 mole-ule was statistically higher in adult vs. second and third trimestersP = <0.001). However, there were no differences between seconds. third trimesters for the expression of CCM2 (Fig. 5).
. Discussion
The present study was designed to investigate the presence ofCM2 in development of the neocortex by immunohistochemistrynd Western blot analysis for the first time. Our previous study ofhe expression of CCM2 protein was reported in adult brain samplesTanriover et al., 2008). But now, in this study the expression pat-ern of CCM2 protein is associated with the prenatal and adulteocortex and blood vessel formations. Since CCM2 is expressed in
he vascular endothelial cells (Seker et al., 2006; Tanriover et al.,008); and also this gene could possibly be related in endothe-
ial cell functions during brain vascular development. Endothelialells are the main cellular unit of vascular structures. Their assem-
d some of the cells (arrows) have a moderate immunoreactivity. Scale bar: 50 �m.
bly into a well organized and functional structure is essential fororgan growth during fetal development. Thus, development of thevascular tree involves two processes: vasculogenesis and angioge-nesis (Kubis and Levy, 2004). Meanwhile, the arterial and venousendothelial cells are molecularly distinct from the earliest stage ofangiogenesis. Differentiation into arteries or veins is not only deter-mined by the direction and the importance of flow but endothelialcell linings appear to be different and determined by the presenceor the absence of molecules (Rocha and Adams, 2009).
Our results suggested that CCM2 immunoreactivity was obtai-ned in vascular endothelium during prenatal development. Whilesome of the vascular endothelial cells revealed a strong immu-noreactivity, some showed weak immunoreactivity during thethird trimester. We speculate that CCM2 is probably one of themolecules which determined the difference between arteries andveins difference in the development of brain vasculature. It mightbe CCM2 gene which disrupted the vein and artery remode-ling whereby suggesting that a reciprocal interaction is necessaryfor brain angiogenesis. We already know that CCM moleculessuch as CCM1, CCM2 and CCM3 were detected in an arterial
endothelium but weak or no immunoreactivity was observed invenous endothelium (Guzeloglu-Kayisli et al., 2004; Tanrioveret al., 2008). Consistently, CCM2 immunoreaction was detected inthe arterial endothelium during adult neocortex. Therefore, CCM2
512 G. Tanriover et al. / Int. J. Devl Neuroscience 29 (2011) 509–514
Fig. 3. Representative pictures of CCM2 staining in developing neocortex during third trimester. (A) An increasing and expanding immunoreactivity for CCM2 in developingneocortex is seen for CCM2. (D and E) The arterial endothelium shows strong immunoreactivity (D, E, K, arrowheads) while a weak immunoreactivity for venous endothelium(
evtit
vtCtvgtf
G; arrowheads) for CCM2. Scale bar: 50 �m.
xpression might be useful for the determination of arterial andenous systems in the third trimester samples. Our proposal ishat CCM2 represents an arterial phenotype and it is importantn establishing an arterial identity in developing brain vascula-ure.
Alteration of the CCM2 expression could be responsible for theascular remodeling. Boulday et al. (2009) showed that constitu-ive deletion of CCM2 leads to early embryonic death. Deletion ofCM2 from endothelial cells severely affects angiogenesis, leadingo morphogenic defects in the major arterial and venous blood
essels and in the heart, and it results in fetal lethality at mid-estation (Boulday et al., 2009). Our results were compatible withhese findings establishing the essential role of endothelial CCM2or proper vascular development. While the function of the CCM2
protein is unknown in the endothelial cells, it has been found thatthey may have a possible role in angiogenesis. Thus, it is possible tospeculate that the loss-of-function mutations in the CCM2 gene inhumans lead to cerebrovascular malformations, causing recurrentbrain hemorrhages (Boulday et al., 2009).
In addition to vascular endothelial cells, our results showed thatCCM2 protein was also localized in cells during development andadult brain samples. It has been shown that CCM2 gene is alsoexpressed in neurons (Seker et al., 2006; Tanriover et al., 2008) inpre and postnatal mouse brain. Also, our results confirmed as in pre-
vious studies that the immunoreactivity was observed in cells thatprobably consist of neurons and glias during prenatal development.So, the localization of CCM2 in neuroglial precursor cells mightplay a role in coordination with neuronal differentiation and orien-
G. Tanriover et al. / Int. J. Devl Neuroscience 29 (2011) 509–514 513
Fig. 4. Representative pictures of CCM2 staining in adult neocortex. (A) There are no glial and neuronal immunoreactivity detects in Layers 1 and 2. (B) Some of the glialc s 3–6
( 2 (arr
tegCcutt
TS
P1
ells reveal a strong CCM2 immmunoreactivity in other CP layers which are Layerarrowheads). (D) The venous endothelium shows a weak immunoreaction for CCM
ation in addition to regulating neuronal migration. But, Bouldayt al. (2009) demonstratedthat the deletion of CCM2 from neuro-lial precursor cells does not lead to cerebrovascular defects thoughCM2 is predominantly expressed in the neuronal layers within the
entral nervous system. While the function of the CCM2 protein isnknown, a specific interplay between the neuron, glia and endo-helium at the level of the neurovascular unit might be crucial forhe development of the brain.
able 1ummary of the staining intensities of the CCM2 antibody in the staining regions.
1st trimester 2nd trimeste
Staining intensities in cortical zones PPL SVZ VZ MZ CP
++ + + ++a ++
Staining intensities in the vascular endoth. + ++
Staining intensities in the fibers ++ ++
PL: primordial plexiform layer, SVZ: subventricular zone, VZ: ventricular zone, MZ: mar–6, WM: white matter, A: arterial endothelium, V: venous endothelium.a The strong CCM2 immunolabelling was shown in the surface of the MZ and VZ.
(arrows). (C, E and F) The arterial endothelium shows a strong immunoreactivityowheads). Scale bar: 50 �m.
In conclusion, our results showed that recently identified newcritical gene, CCM2 is involved in vascular morphogenesis andstudies in the molecular pathways underlying vasculogenesis andangiogenesis. This protein might be leading to the better understan-
ding of associated several vascular complications. Further studiesshould aim to find other molecules interacting with CCM2 in theendothelial cells and to identify which signaling pathways are affec-ted by the protein. Furthermore, it is likely that the interaction
Fig. 5. (A) Western blotting analysis of CCM2 in second, third trimesters and adultneocortex tissues. Immunoblots of cortical extracts by using anti-CCM2: a 47 kDaband has been detected in trimesters and adult human neocortex samples. Theimmunoexpression of �-actin (43 kDa) was used to show prospective equivalentamounts of total proteins loaded per lane. (B) The immunoblot bands were quanti-finw
at
A
mTlb
R
B
B
B
C
Thiery, J.P., 1984. Mechanisms of cell migration in the vertebrate embryo. Cell Differ.
ed by an optical densitometer. The OD (optical density) values of CCM2 bands wereormalized to the OD values of �-actin bands. The protein level of CCM2 moleculeas statistically higher in adult vs. second and third trimesters (P = <0.001).
mong neurons, glia and endothelia would lead to better unders-anding the CCM lesions in patients (Table 1).
cknowledgments
The authors would like to thank Dr. E.I. Gurer from Depart-ent of Pathology, Akdeniz University School of Medicine, Antalya,
urkey for her excellent cooperation to evaluate normal or patho-ogic brain material differences. This study was partially supportedy the Akdeniz University Research Foundation, Antalya, Turkey.
eferences
entivoglio, M., Tassi, L., Pech, E., Costa, C., Fabene, P.F., Spreafico, R., 2003. Corticaldevelopment and focal cortical dysplasia. Epileptic Disord. 5 (Suppl. 2), S27–S34.
ergametti, F., Denier, C., Labauge, P., Arnoult, M., Boetto, S., Clanet, M., Coubes, P.,Echenne, B., Ibrahim, R., Irthum, B., Jacquet, G., Lonjon, M., Moreau, J.J., Neau,J.P., Parker, F., Tremoulet, M., Tournier-Lasserve, E., 2005. Mutations within theprogrammed cell death 10 gene cause cerebral cavernous malformations. Am.J. Hum. Genet. 76, 42–51.
oulday, G., Blecon, A., Petit, N., Chareyre, F., Garcia, L.A., Niwa-Kawakita, M., Giovan-nini, M., Tournier-Lasserve, E., 2009. Tissue-specific conditional CCM2 knockout
mice establish the essential role of endothelial CCM2 in angiogenesis: impli-cations for human cerebral cavernous malformations. Dis. Model Mech. 2,168–177.
han, W.Y., Lorke, D.E., Tiu, S.C., Yew, D.T., 2002. Proliferation and apoptosis in thedeveloping human neocortex. Anat. Rec. 267, 261–276.
oscience 29 (2011) 509–514
Clatterbuck, R.E., Eberhart, C.G., Crain, B.J., Rigamonti, D., 2001. Ultrastructural andimmunocytochemical evidence that an incompetent blood-brain barrier is rela-ted to the pathophysiology of cavernous malformations. J. Neurol. Neurosurg.Psychiatry 71, 188–192.
Craig, H.D., Gunel, M., Cepeda, O., Johnson, E.W., Ptacek, L., Steinberg, G.K., Ogilvy,C.S., Berg, M.J., Crawford, S.C., Scott, R.M., Steichen-Gersdorf, E., Sabroe, R., Ken-nedy, C.T., Mettler, G., Beis, M.J., Fryer, A., Awad, I.A., Lifton, R.P., 1998. Multilocuslinkage identifies two new loci for a mendelian form of stroke, cerebral caver-nous malformation, at 7p15-13 and 3q25.2 27. Hum. Mol. Genet. 7, 1851–1858.
Dubovsky, J., Zabramski, J.M., Kurth, J., Spetzler, R.F., Rich, S.S., Orr, H.T., Weber, J.L.,1995. A gene responsible for cavernous malformations of the brain maps tochromosome 7q. Hum. Mol. Genet. 4, 453–458.
Gunel, M., Awad, I.A., Finberg, K., Anson, J.A., Steinberg, G.K., Batjer, H.H., Kopit-nik, T.A., Morrison, L., Giannotta, S.L., Nelson-Williams, C., Lifton, R.P., 1996. Afounder mutation as a cause of cerebral cavernous malformation in HispanicAmericans. N. Engl. J. Med. 334, 946–951.
Guzeloglu-Kayisli, O., Kayisli, U.A., Amankulor, N.M., Voorhees, J.R., Gokce, O.,DiLuna, M.L., Laurans, M.S., Luleci, G., Gunel, M., 2004. Krev1 interactiontrapped-1/cerebral cavernous malformation-1 protein expression during earlyangiogenesis. J. Neurosurg. 100, 481–487.
Hanahan, D., 1997. Signaling vascular morphogenesis and maintenance. Science 277,48–50.
Kubis, N., Levy, B.I., 2004. Understanding angiogenesis: a clue for understandingvascular malformations. J. Neuroradiol. 31, 365–368.
Marchuk, D.A., Gallione, C.J., Morrison, L.A., Clericuzio, C.L., Hart, B.L., Kosofsky, B.E.,Louis, D.N., Gusella, J.F., Davis, L.E., Prenger, V.L., 1995. A locus for cerebral caver-nous malformations maps to chromosome 7q in two families. Genomics 28,311–314.
Marin-Padilla, M., 1998. Cajal-Retzius cells and the development of the neocortex.Trends Neurosci. 21, 64–71.
Pozzati, E., Acciarri, N., Tognetti, F., Marliani, F., Giangaspero, F., 1996. Growth, sub-sequent bleeding, and de novo appearance of cerebral cavernous angiomas.Neurosurgery 38, 662–669 (discussion 669–670).
Rakic, P., 1982. Early developmental events: cell lineages, acquisition of neuronalpositions, and areal and laminar development. Neurosci. Res. Program Bull. 20,439–451.
Rigamonti, D., Hadley, M.N., Drayer, B.P., Johnson, P.C., Hoenig-Rigamonti, K., Knight,J.T., Spetzler, R.F., 1988. Cerebral cavernous malformations. Incidence and fami-lial occurrence. N. Engl. J. Med. 319, 343–347.
Risau, W., 1997. Mechanisms of angiogenesis. Nature 386, 671–674.Risau, W., Flamme, I., 1995. Vasculogenesis. Annu. Rev. Cell Dev. Biol. 11, 73–91.Rocha, S.F., Adams, R.H., 2009. Molecular differentiation and specialization of vas-
cular beds. Angiogenesis 12, 139–147.Russell, D.S., Rubinstein, L.J., 1989. Pathology of Tumors of the Nervous System.
Williams and Wilkins, Baltimore.Seker, A., Pricola, K.L., Guclu, B., Ozturk, A.K., Louvi, A., Gunel, M., 2006. CCM2 expres-
sion parallels that of CCM1. Stroke 37, 518–523.Tanriover, G., Boylan, A.J., Diluna, M.L., Pricola, K.L., Louvi, A., Gunel, M., 2008.
PDCD10, the gene mutated in cerebral cavernous malformation 3, is expressedin the neurovascular unit. Neurosurgery 62, 930–938 (discussion 938).
Tanriover, G., Demir, N., Pestereli, E., Demir, R., Kayisli, U.A., 2005. PTEN-mediatedAkt activation in human neocortex during prenatal development. Histochem.Cell Biol. 123, 393–406.
Tanriover, G., Kayisli, U.A., Demir, R., Pestereli, E., Karaveli, S., Demir, N., 2004. Dis-tribution of N-cadherin in human cerebral cortex during prenatal development.Histochem. Cell Biol. 122, 191–200.
Tanriover, G., Seval, Y., Sati, L., Gunel, M., Demir, N., 2009. CCM2 and CCM3 pro-teins contribute to vasculogenesis and angiogenesis in human placenta. Histol.Histopathol. 24, 1287–1294.
15, 1–15.Wiechelman, K.J., Braun, R.D., Fitzpatrick, J.D., 1988. Investigation of the bicin-
choninic acid protein assay: identification of the groups responsible for colorformation. Anal. Biochem. 175, 231–237.
