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CCM2 expression during prenatal development and adult human neocortex
Int. J. Devl Neuroscience 29 (2011) 509–514
Contents lists available at ScienceDirect
International Journal of Developmental Neuroscience journal homepage: www.elsevier.com/locate/ijdevneu
CCM2 expression during prenatal development and adult human neocortex Gamze Tanriover a,∗ , Berna Sozen a , Murat Gunel b , Necdet Demir a a b
Department of Histology and Embryology, Faculty of Medicine, Akdeniz University, 07070, Antalya, Turkey Department of Neurosurgery, Yale University School of Medicine, 333 Cedar Street, 06510 New Haven, CT, USA
a r t i c l e
i n f o
Article history: Received 11 November 2010 Received in revised form 9 March 2011 Accepted 21 April 2011 Keywords: CCM2 Human neocortex Immunohistochemistry Western blotting
a b s t r a c t Cerebral cavernous malformation (CCM) is one of the most common types of vascular malformations of the central nervous system, affecting nearly one in 200 people. CCM lesions are characterized by grossly dilated vascular channels lined by a single layer of endothelium. Genetic linkage analyses have mapped three CCM loci to CCM1, CCM2 and CCM3. All three causative genes have now been identified allowing new insights into CCM pathophysiology. We focused on the CCM2 protein that might take place in blood vessel formation; we report here the expression patterns of CCM2 in prenatal development and adult human neocortex by means of immunohistochemistry and Western blot analysis. CCM2 was obviously detected in vascular endothelium and neuroglial precursor cells during development and also observed in arterial endothelium, neurons, some of the glial cells in adult neocortex. The expression patterns suggest that it could be one of the arterial markers whether this is a cause or a consequence of an altered vascular identity. CCM2 might play a role during vasculogenesis and angiogenesis during human brain development. Furthermore, with this study, CCM2 have been described for the first time in developing human neocortex. © 2011 ISDN. Published by Elsevier Ltd. All rights reserved.
1. Introduction Cerebral cavernous malformations (CCMs) are vascular malformations, mostly located in the central nervous system. Patients may have single or multiple malformations leading to focal neurologic signs, hemorrhagic strokes, seizures, or sometimes death (Rigamonti et al., 1988). CCM lesions are characterized by grossly dilated vascular channels lined by a single layer of endothelium (Russell and Rubinstein, 1989). They lack normal vessel wall elements such as smooth muscles and are also devoid of intervening normal parenchyma (Clatterbuck et al., 2001). CCM occurs sporadically but may also be inherited dominantly with incomplete penetrance. The pattern of inheritance of the familial form is autosomal dominant. The proportion of familial cases has been estimated to be as high as 50% in Hispanic-American patients and close to 10–40% in other populations (Gunel et al., 1996; Pozzati et al., 1996). Familial forms have been linked to three chromosomal loci, and loss of function mutations have been identified in the KRIT1/Ccm1 (Marchuk et al., 1995), MGC4607/Ccm2 (Craig et al., 1998), and PDCD10/Ccm3 (Dubovsky et al., 1995) genes. The early development and the structural organization of the human neocortex is divided into a number of histogenetic fields (Thiery, 1984). Marginal zone (MZ), is formed below the pial surface
∗ Corresponding author. Tel.: +90 242 249 6876; fax: +90 242 2274486. E-mail address: [email protected] (G. Tanriover). 0736-5748/$36.00 © 2011 ISDN. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijdevneu.2011.04.006
and the area is supposed to be an active zone of the developing human cortex. This zone promotes the maturation of early generated neurons and also marks the beginning of the cortical neurogenesis. Thus, the superficial lamina containing the first differentiated neuronal elements is called the primordial plexiform layer (PPL) (Marin-Padilla, 1998). It is already known that the neuroblasts from the VZ migrate upward and the arrival of the first migratory neurons splits the PPL in two regions. One, close to the pial surface, becomes the MZ and thereafter is called subplate (SP). At the end of the first trimester, the neocortex is comprised of six discrete zones MZ, CP (cortical plate), SP (subplate), IZ (intermediate zone), SVZ (subventricular zone), VZ (ventricular) (Chan et al., 2002). During further developmental stages, six layers are formed and the SP disappeared. The SP neurons destined to die later on through a process of programmed cell death, and the fibers will be free to reach their appropriate zone. So, SP transforms into white matter in adult cortex (Rakic, 1982). Also, the thickness of the CP progressively contributes to formation of layers II through VI in adult cortex (Bentivoglio et al., 2003). During the development, there are two processes involved in blood vessel formation. The first process, vasculogenesis, occurs when a primitive vascular network is constructed from pluripotent mesenchymal progenitors. The second process, angiogenesis, follows vasculogenesis, and it is characterized by capillary sprouts arising from pre-existing vasculogenic centers (Risau and Flamme, 1995; Risau, 1997). Endothelial cells play a dynamic role in both angiogenesis and vasculogenesis. Differentiation of multipotential
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mesencymal cells, migration and proliferation of endothelial cells and formation of cell–cell connections are important step, for successful vasculogenesis (Hanahan, 1997). The mechanisms involved in cerebral blood vessel angiogenesis during development are still poorly defined. Therefore, understanding of the mechanisms of angiogenesis is the most important step in cerebrovascular malformations. These three proteins leading to the CCM pathology suggest that there are new players in vascular morphogenesis and remodeling and also contributing to a better understanding of normal and pathological angiogenesis (Guzeloglu-Kayisli et al., 2004; Bergametti et al., 2005; Plummer et al., 2005; Tanriover et al., 2009). Moreover, Guzeloglu-Kayisli et al. (2004) and our previous study pointed out that CCM1, CCM2 and CCM3 may play a key role in vessel formation and development during early angiogenesis (Tanriover et al., 2009). And also, Seker et al. (2006) characterized the messenger ribonucleic acid (mRNA) distribution of Ccm1 and Ccm2 in the embryonic and postnatal periods of central nervous system of mice. Ccm1 expression parallels that of Ccm2 and is primarily observed on the arterial side of the cerebral vasculature, neurons and astrocytes in mouse. Thus, in the present study we analyzed the cell specific expression pattern of CCM2 protein in human neocortex. To this end, we performed immunohistochemical analysis using a novel antibody generated against the CCM2 protein expression during adult and developmental period of human neocortex. Also, the present study focuses on the CCM2 expression pattern to clarify the neuronal orientation in human cortical developmental period. 2. Material and methods Thirteen human developing brains from spontaneous abortions (n = 5, n = 4 and n = 4 from second, third trimesters and adult) were used in the study. Written informed consent was obtained from each woman before the operation using consent forms and protocols approved by the Human Investigation Committee of Akdeniz University. None of the specimens had cerebral malformation or cerebral hemorrhagic abnormalities. All specimens used were in normal neurological conditions. The fetuses and adult brain materials had no pathological defect at the macroscopic and microscopic levels, as evaluated by the Department of Pathology, Akdeniz University, Medical Faculty (Tanriover et al., 2004; Tanriover et al., 2005). 2.1. Immunohistochemistry For CCM2 immunohistochemistry, sections were deparaffinized and rehydrated by standard methods then endogenous peroxidase activity was blocked with methanol containing 3% H2 O2 for 20 min, at room temperature. Rabbit polyclonal anti-CCM2 (made in Zymed) primary antibody (1/250) was applied for 2 h at room temperature. Negative controls were performed by replacing the primary antibodies with normal rabbit serum at the same concentration. After several rinses in PBS, biotinylated goat anti-rabbit IgG (1/400 dilution Vector Lab. Burlingame, CA, USA) was applied for 30 min. Following several PBS rinses, slides were incubated with streptavidin–peroxidase complex for the appropriate time by using Dako LSAB kit (Dako, Carpinteria, CA, USA). Antibody complex was visualized by incubation with diaminobenzidine (DAB) chromogen (BioGenex) prepared according to the manufacturer’s instructions. Slides were counterstained slightly with Mayer’s hematoxylin (Dako, Glostrup, Denmark) prior to permanent mounting and then evaluated under a light microscope.SDS polyacrylamide gel electrophoresis and Western blotting Total protein from the tissues was extracted in a lysis buffer (10 mM TrisHCL, 1 mM EDTA, 2.5% SDS, 1 mM phenylmethylsulfonylfluoride, 1 g/ml leupeptin) supplemented with CompleteR protease inhibitor cocktail (Boehringer, Mannheim, Germany). The protein concentration was determined using a standard BCA assay (Wiechelman et al., 1988) and 50 g proteins were applied per lane. Prior to electrophoresis, samples were heated for 5 min at 95 ◦ C. Samples were then subjected to SDS polyacrylamide gel electrophoresis under standard conditions and then transferred onto PVDF membrane (BioRad, Hercules, CA, USA) in a buffer containing 0.2 mol/l glycine, 25 mM Tris and 20% methanol. The membrane was blocked for 1 h with 5% nonfat dry milk (BioRad, Hercules, CA, USA) in TBS-T to decrease nonspecific binding. Afterwards, the membrane was incubated with rabbit polyclonal antibody against human CCM2 (dilution 1/1000 in 5% nonfat dry milk in TBS-T) for 1 h. The membrane was then incubated with horse peroxidase-labelled anti-rabbit IgG (dilution 1/10,000; Vector Laboratories) for 1 h. Immunolabelling was visualized using the chemiluminescence based SuperSignal CL HRP Substrate System (Pierce, Rockford, IL, USA) and the membrane was exposed to Hyperfilm (Amersham, Piscataway, NJ, USA).
Fig. 1. Representative pictures of CCM2 staining in developing neocortex during first trimester. The cytoplasmic reaction of neocortex cells and fibers show a strong immunoreactivity 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 loading of proteins in each lane was confirmed by re-probing the membrane with mouse monoclonal anti-human -actin (Abcam, Cambridge, UK).
3. Results 3.1. CCM2 expression in normal human neocortex during prenatal development and adult samples Using immunohistochemical staining, we studied the expression patterns of CCM2 in human neocortex tissue during developmental period and also adult samples. During the first trimester, the fibers in the developing human neocortex revealed a strong CCM2 protein immunoreactivity in PPL. In addition, the cytoplasm of the cells was also immunoreactive for CCM2 in SVZ and VZ (Fig. 1). During the second trimester, CCM2 immunoreactivity was detected throughout the neocortex layers (Fig. 2A–G). Additionally, there was a strong CCM2 immunoreaction localized in the vascular endothelium during second trimester (Fig. 2C–G, arrowheads). The moderate localization of CCM2 in some of the cells was specific to MZ, CP and SP zones (Fig. 2B–D arrows). The strong CCM2 immunolabelling was shown in the surface of the MZ and VZ. In comparison with the first trimester neocortex immunoreactivity was higher and occasionally nuclear in the specific zones. CCM2 immunoreactivity in the third trimester was stronger than that in the second and first trimesters, not only in the cells and fibers but also in the vascular endothelium. In the neocortex of the third trimester, CCM2 was observed in a strong immunoreactivity in some of the vascular endothelium (Fig. 3D, E and K, arrowheads). On the other hand, some of the vascular endothelium revealed a weak CCM2 immunolabelling during the third trimester (Fig. 3E, arrowhead). In the adult brain samples, the strongest CCM2 immunoreactivity was obtained in the neocortex layers from trimesters. The expression of CCM2 was increased in the arterial endothelium as referred in our previous study (Tanriover et al., 2008). On the other hand, the expression of CCM2 was detected in a subset of neuronal cells, some glial cells (Fig. 4B arrows) and, at very low levels in the venous endothelium (Fig. 4D, arrowheads). Moreover, the staining pattern for CCM2 protein was strongly restricted to the arterial endothelium in adult brain samples (Fig. 4C, E and F, arrowheads). No immunoreactivity was observed on the slides where primary antibody was replaced with their normal rabbit IgG (data not shown). 3.2. Confirmation of CCM2 expression by Western blot analysis CCM2 expression was analyzed by Western blotting from normal human developing and adult brain tissues. The blots clearly
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Fig. 2. Representative pictures of CCM2 staining in developing human neocortex during second trimester. The heterogeneous immunoreactivity was seen in second trimesters and 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. (F) Subventricular zone. (G) Ventricular zone. Vascular endothelium (C–G; arrowheads) and some of the cells (arrows) have a moderate immunoreactivity. Scale bar: 50 m.
revealed bands for CCM2 corresponding to 47 kDa, in developing and adult normal brain tissue samples. Equivalent amounts of total proteins were loaded per lane as indicative by the immunoexpression of -actin (43 kDa). According to the Western blot results, CCM2 expression was detected at all trimesters and adult neocortex. Furthermore, it gradually increased from first to third trimester and adult neocortex. The protein level of CCM2 molecule was statistically higher in adult vs. second and third trimesters (P = <0.001). However, there were no differences between second vs. third trimesters for the expression of CCM2 (Fig. 5). 4. Discussion The present study was designed to investigate the presence of CCM2 in development of the neocortex by immunohistochemistry and Western blot analysis for the first time. Our previous study of the expression of CCM2 protein was reported in adult brain samples (Tanriover et al., 2008). But now, in this study the expression pattern of CCM2 protein is associated with the prenatal and adult neocortex and blood vessel formations. Since CCM2 is expressed in the vascular endothelial cells (Seker et al., 2006; Tanriover et al., 2008); and also this gene could possibly be related in endothelial cell functions during brain vascular development. Endothelial cells are the main cellular unit of vascular structures. Their assem-
bly into a well organized and functional structure is essential for organ growth during fetal development. Thus, development of the vascular tree involves two processes: vasculogenesis and angiogenesis (Kubis and Levy, 2004). Meanwhile, the arterial and venous endothelial cells are molecularly distinct from the earliest stage of angiogenesis. Differentiation into arteries or veins is not only determined by the direction and the importance of flow but endothelial cell linings appear to be different and determined by the presence or the absence of molecules (Rocha and Adams, 2009). Our results suggested that CCM2 immunoreactivity was obtained in vascular endothelium during prenatal development. While some of the vascular endothelial cells revealed a strong immunoreactivity, some showed weak immunoreactivity during the third trimester. We speculate that CCM2 is probably one of the molecules which determined the difference between arteries and veins difference in the development of brain vasculature. It might be CCM2 gene which disrupted the vein and artery remodeling whereby suggesting that a reciprocal interaction is necessary for brain angiogenesis. We already know that CCM molecules such as CCM1, CCM2 and CCM3 were detected in an arterial endothelium but weak or no immunoreactivity was observed in venous endothelium (Guzeloglu-Kayisli et al., 2004; Tanriover et al., 2008). Consistently, CCM2 immunoreaction was detected in the arterial endothelium during adult neocortex. Therefore, CCM2
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Fig. 3. Representative pictures of CCM2 staining in developing neocortex during third trimester. (A) An increasing and expanding immunoreactivity for CCM2 in developing neocortex is seen for CCM2. (D and E) The arterial endothelium shows strong immunoreactivity (D, E, K, arrowheads) while a weak immunoreactivity for venous endothelium (G; arrowheads) for CCM2. Scale bar: 50 m.
expression might be useful for the determination of arterial and venous systems in the third trimester samples. Our proposal is that CCM2 represents an arterial phenotype and it is important in establishing an arterial identity in developing brain vasculature. Alteration of the CCM2 expression could be responsible for the vascular remodeling. Boulday et al. (2009) showed that constitutive deletion of CCM2 leads to early embryonic death. Deletion of CCM2 from endothelial cells severely affects angiogenesis, leading to morphogenic defects in the major arterial and venous blood vessels and in the heart, and it results in fetal lethality at midgestation (Boulday et al., 2009). Our results were compatible with these findings establishing the essential role of endothelial CCM2 for proper vascular development. While the function of the CCM2
protein is unknown in the endothelial cells, it has been found that they may have a possible role in angiogenesis. Thus, it is possible to speculate that the loss-of-function mutations in the CCM2 gene in humans lead to cerebrovascular malformations, causing recurrent brain hemorrhages (Boulday et al., 2009). In addition to vascular endothelial cells, our results showed that CCM2 protein was also localized in cells during development and adult brain samples. It has been shown that CCM2 gene is also expressed in neurons (Seker et al., 2006; Tanriover et al., 2008) in pre and postnatal mouse brain. Also, our results confirmed as in previous studies that the immunoreactivity was observed in cells that probably consist of neurons and glias during prenatal development. So, the localization of CCM2 in neuroglial precursor cells might play a role in coordination with neuronal differentiation and orien-
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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 glial cells reveal a strong CCM2 immmunoreactivity in other CP layers which are Layers 3–6 (arrows). (C, E and F) The arterial endothelium shows a strong immunoreactivity (arrowheads). (D) The venous endothelium shows a weak immunoreaction for CCM2 (arrowheads). Scale bar: 50 m.
tation in addition to regulating neuronal migration. But, Boulday et al. (2009) demonstratedthat the deletion of CCM2 from neuroglial precursor cells does not lead to cerebrovascular defects though CCM2 is predominantly expressed in the neuronal layers within the central nervous system. While the function of the CCM2 protein is unknown, a specific interplay between the neuron, glia and endothelium at the level of the neurovascular unit might be crucial for the development of the brain.
In conclusion, our results showed that recently identified new critical gene, CCM2 is involved in vascular morphogenesis and studies in the molecular pathways underlying vasculogenesis and angiogenesis. This protein might be leading to the better understanding of associated several vascular complications. Further studies should aim to find other molecules interacting with CCM2 in the endothelial cells and to identify which signaling pathways are affected by the protein. Furthermore, it is likely that the interaction
Table 1 Summary of the staining intensities of the CCM2 antibody in the staining regions. 1st trimester
Staining intensities in the vascular endoth.
PPL ++ +
Staining intensities in the fibers
++
Staining intensities in cortical zones
SVZ +
2nd trimester VZ +
MZ ++a ++ ++
CP ++
3rd trimester SP ++
IZ ++
SVZ ++
VZ ++a
L1–6 +++ +++ +++
SP +++
Adult WM +++
L1–6 +++ A ++ +++
WM +++ V +/−
PPL: primordial plexiform layer, SVZ: subventricular zone, VZ: ventricular zone, MZ: marginal zone, CP: cortical plate, IZ: intermediate zone, SP: subplate zone, L1–6: layer 1–6, WM: white matter, A: arterial endothelium, V: venous endothelium. a The strong CCM2 immunolabelling was shown in the surface of the MZ and VZ.
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Fig. 5. (A) Western blotting analysis of CCM2 in second, third trimesters and adult neocortex tissues. Immunoblots of cortical extracts by using anti-CCM2: a 47 kDa band has been detected in trimesters and adult human neocortex samples. The immunoexpression of -actin (43 kDa) was used to show prospective equivalent amounts of total proteins loaded per lane. (B) The immunoblot bands were quantified by an optical densitometer. The OD (optical density) values of CCM2 bands were normalized to the OD values of -actin bands. The protein level of CCM2 molecule was statistically higher in adult vs. second and third trimesters (P = <0.001).
among neurons, glia and endothelia would lead to better understanding the CCM lesions in patients (Table 1). Acknowledgments The authors would like to thank Dr. E.I. Gurer from Department of Pathology, Akdeniz University School of Medicine, Antalya, Turkey for her excellent cooperation to evaluate normal or pathologic brain material differences. This study was partially supported by the Akdeniz University Research Foundation, Antalya, Turkey. References Bentivoglio, M., Tassi, L., Pech, E., Costa, C., Fabene, P.F., Spreafico, R., 2003. Cortical development and focal cortical dysplasia. Epileptic Disord. 5 (Suppl. 2), S27–S34. Bergametti, 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 the programmed cell death 10 gene cause cerebral cavernous malformations. Am. J. Hum. Genet. 76, 42–51. Boulday, G., Blecon, A., Petit, N., Chareyre, F., Garcia, L.A., Niwa-Kawakita, M., Giovannini, M., Tournier-Lasserve, E., 2009. Tissue-specific conditional CCM2 knockout mice establish the essential role of endothelial CCM2 in angiogenesis: implications for human cerebral cavernous malformations. Dis. Model Mech. 2, 168–177. Chan, W.Y., Lorke, D.E., Tiu, S.C., Yew, D.T., 2002. Proliferation and apoptosis in the developing human neocortex. Anat. Rec. 267, 261–276.
Clatterbuck, R.E., Eberhart, C.G., Crain, B.J., Rigamonti, D., 2001. Ultrastructural and immunocytochemical evidence that an incompetent blood-brain barrier is related 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., Kennedy, C.T., Mettler, G., Beis, M.J., Fryer, A., Awad, I.A., Lifton, R.P., 1998. Multilocus linkage identifies two new loci for a mendelian form of stroke, cerebral cavernous 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 to chromosome 7q. Hum. Mol. Genet. 4, 453–458. Gunel, M., Awad, I.A., Finberg, K., Anson, J.A., Steinberg, G.K., Batjer, H.H., Kopitnik, T.A., Morrison, L., Giannotta, S.L., Nelson-Williams, C., Lifton, R.P., 1996. A founder mutation as a cause of cerebral cavernous malformation in Hispanic Americans. 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 interaction trapped-1/cerebral cavernous malformation-1 protein expression during early angiogenesis. 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 understanding vascular 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 cavernous 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. Plummer, N.W., Zawistowski, J.S., Marchuk, D.A., 2005. Genetics of cerebral cavernous malformations. Curr. Neurol. Neurosci. Rep. 5, 391–396. Pozzati, E., Acciarri, N., Tognetti, F., Marliani, F., Giangaspero, F., 1996. Growth, subsequent 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 neuronal positions, 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 familial 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 vascular 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 expression 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 expressed in the neurovascular unit. Neurosurgery 62, 930–938 (discussion 938). Tanriover, G., Demir, N., Pestereli, E., Demir, R., Kayisli, U.A., 2005. PTEN-mediated Akt 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. Distribution 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 proteins contribute to vasculogenesis and angiogenesis in human placenta. Histol. Histopathol. 24, 1287–1294. Thiery, J.P., 1984. Mechanisms of cell migration in the vertebrate embryo. Cell Differ. 15, 1–15. Wiechelman, K.J., Braun, R.D., Fitzpatrick, J.D., 1988. Investigation of the bicinchoninic acid protein assay: identification of the groups responsible for color formation. Anal. Biochem. 175, 231–237.
Contents lists available at ScienceDirect
International Journal of Developmental Neuroscience journal homepage: www.elsevier.com/locate/ijdevneu
CCM2 expression during prenatal development and adult human neocortex Gamze Tanriover a,∗ , Berna Sozen a , Murat Gunel b , Necdet Demir a a b
Department of Histology and Embryology, Faculty of Medicine, Akdeniz University, 07070, Antalya, Turkey Department of Neurosurgery, Yale University School of Medicine, 333 Cedar Street, 06510 New Haven, CT, USA
a r t i c l e
i n f o
Article history: Received 11 November 2010 Received in revised form 9 March 2011 Accepted 21 April 2011 Keywords: CCM2 Human neocortex Immunohistochemistry Western blotting
a b s t r a c t Cerebral cavernous malformation (CCM) is one of the most common types of vascular malformations of the central nervous system, affecting nearly one in 200 people. CCM lesions are characterized by grossly dilated vascular channels lined by a single layer of endothelium. Genetic linkage analyses have mapped three CCM loci to CCM1, CCM2 and CCM3. All three causative genes have now been identified allowing new insights into CCM pathophysiology. We focused on the CCM2 protein that might take place in blood vessel formation; we report here the expression patterns of CCM2 in prenatal development and adult human neocortex by means of immunohistochemistry and Western blot analysis. CCM2 was obviously detected in vascular endothelium and neuroglial precursor cells during development and also observed in arterial endothelium, neurons, some of the glial cells in adult neocortex. The expression patterns suggest that it could be one of the arterial markers whether this is a cause or a consequence of an altered vascular identity. CCM2 might play a role during vasculogenesis and angiogenesis during human brain development. Furthermore, with this study, CCM2 have been described for the first time in developing human neocortex. © 2011 ISDN. Published by Elsevier Ltd. All rights reserved.
1. Introduction Cerebral cavernous malformations (CCMs) are vascular malformations, mostly located in the central nervous system. Patients may have single or multiple malformations leading to focal neurologic signs, hemorrhagic strokes, seizures, or sometimes death (Rigamonti et al., 1988). CCM lesions are characterized by grossly dilated vascular channels lined by a single layer of endothelium (Russell and Rubinstein, 1989). They lack normal vessel wall elements such as smooth muscles and are also devoid of intervening normal parenchyma (Clatterbuck et al., 2001). CCM occurs sporadically but may also be inherited dominantly with incomplete penetrance. The pattern of inheritance of the familial form is autosomal dominant. The proportion of familial cases has been estimated to be as high as 50% in Hispanic-American patients and close to 10–40% in other populations (Gunel et al., 1996; Pozzati et al., 1996). Familial forms have been linked to three chromosomal loci, and loss of function mutations have been identified in the KRIT1/Ccm1 (Marchuk et al., 1995), MGC4607/Ccm2 (Craig et al., 1998), and PDCD10/Ccm3 (Dubovsky et al., 1995) genes. The early development and the structural organization of the human neocortex is divided into a number of histogenetic fields (Thiery, 1984). Marginal zone (MZ), is formed below the pial surface
∗ Corresponding author. Tel.: +90 242 249 6876; fax: +90 242 2274486. E-mail address: [email protected] (G. Tanriover). 0736-5748/$36.00 © 2011 ISDN. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijdevneu.2011.04.006
and the area is supposed to be an active zone of the developing human cortex. This zone promotes the maturation of early generated neurons and also marks the beginning of the cortical neurogenesis. Thus, the superficial lamina containing the first differentiated neuronal elements is called the primordial plexiform layer (PPL) (Marin-Padilla, 1998). It is already known that the neuroblasts from the VZ migrate upward and the arrival of the first migratory neurons splits the PPL in two regions. One, close to the pial surface, becomes the MZ and thereafter is called subplate (SP). At the end of the first trimester, the neocortex is comprised of six discrete zones MZ, CP (cortical plate), SP (subplate), IZ (intermediate zone), SVZ (subventricular zone), VZ (ventricular) (Chan et al., 2002). During further developmental stages, six layers are formed and the SP disappeared. The SP neurons destined to die later on through a process of programmed cell death, and the fibers will be free to reach their appropriate zone. So, SP transforms into white matter in adult cortex (Rakic, 1982). Also, the thickness of the CP progressively contributes to formation of layers II through VI in adult cortex (Bentivoglio et al., 2003). During the development, there are two processes involved in blood vessel formation. The first process, vasculogenesis, occurs when a primitive vascular network is constructed from pluripotent mesenchymal progenitors. The second process, angiogenesis, follows vasculogenesis, and it is characterized by capillary sprouts arising from pre-existing vasculogenic centers (Risau and Flamme, 1995; Risau, 1997). Endothelial cells play a dynamic role in both angiogenesis and vasculogenesis. Differentiation of multipotential
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mesencymal cells, migration and proliferation of endothelial cells and formation of cell–cell connections are important step, for successful vasculogenesis (Hanahan, 1997). The mechanisms involved in cerebral blood vessel angiogenesis during development are still poorly defined. Therefore, understanding of the mechanisms of angiogenesis is the most important step in cerebrovascular malformations. These three proteins leading to the CCM pathology suggest that there are new players in vascular morphogenesis and remodeling and also contributing to a better understanding of normal and pathological angiogenesis (Guzeloglu-Kayisli et al., 2004; Bergametti et al., 2005; Plummer et al., 2005; Tanriover et al., 2009). Moreover, Guzeloglu-Kayisli et al. (2004) and our previous study pointed out that CCM1, CCM2 and CCM3 may play a key role in vessel formation and development during early angiogenesis (Tanriover et al., 2009). And also, Seker et al. (2006) characterized the messenger ribonucleic acid (mRNA) distribution of Ccm1 and Ccm2 in the embryonic and postnatal periods of central nervous system of mice. Ccm1 expression parallels that of Ccm2 and is primarily observed on the arterial side of the cerebral vasculature, neurons and astrocytes in mouse. Thus, in the present study we analyzed the cell specific expression pattern of CCM2 protein in human neocortex. To this end, we performed immunohistochemical analysis using a novel antibody generated against the CCM2 protein expression during adult and developmental period of human neocortex. Also, the present study focuses on the CCM2 expression pattern to clarify the neuronal orientation in human cortical developmental period. 2. Material and methods Thirteen human developing brains from spontaneous abortions (n = 5, n = 4 and n = 4 from second, third trimesters and adult) were used in the study. Written informed consent was obtained from each woman before the operation using consent forms and protocols approved by the Human Investigation Committee of Akdeniz University. None of the specimens had cerebral malformation or cerebral hemorrhagic abnormalities. All specimens used were in normal neurological conditions. The fetuses and adult brain materials had no pathological defect at the macroscopic and microscopic levels, as evaluated by the Department of Pathology, Akdeniz University, Medical Faculty (Tanriover et al., 2004; Tanriover et al., 2005). 2.1. Immunohistochemistry For CCM2 immunohistochemistry, sections were deparaffinized and rehydrated by standard methods then endogenous peroxidase activity was blocked with methanol containing 3% H2 O2 for 20 min, at room temperature. Rabbit polyclonal anti-CCM2 (made in Zymed) primary antibody (1/250) was applied for 2 h at room temperature. Negative controls were performed by replacing the primary antibodies with normal rabbit serum at the same concentration. After several rinses in PBS, biotinylated goat anti-rabbit IgG (1/400 dilution Vector Lab. Burlingame, CA, USA) was applied for 30 min. Following several PBS rinses, slides were incubated with streptavidin–peroxidase complex for the appropriate time by using Dako LSAB kit (Dako, Carpinteria, CA, USA). Antibody complex was visualized by incubation with diaminobenzidine (DAB) chromogen (BioGenex) prepared according to the manufacturer’s instructions. Slides were counterstained slightly with Mayer’s hematoxylin (Dako, Glostrup, Denmark) prior to permanent mounting and then evaluated under a light microscope.SDS polyacrylamide gel electrophoresis and Western blotting Total protein from the tissues was extracted in a lysis buffer (10 mM TrisHCL, 1 mM EDTA, 2.5% SDS, 1 mM phenylmethylsulfonylfluoride, 1 g/ml leupeptin) supplemented with CompleteR protease inhibitor cocktail (Boehringer, Mannheim, Germany). The protein concentration was determined using a standard BCA assay (Wiechelman et al., 1988) and 50 g proteins were applied per lane. Prior to electrophoresis, samples were heated for 5 min at 95 ◦ C. Samples were then subjected to SDS polyacrylamide gel electrophoresis under standard conditions and then transferred onto PVDF membrane (BioRad, Hercules, CA, USA) in a buffer containing 0.2 mol/l glycine, 25 mM Tris and 20% methanol. The membrane was blocked for 1 h with 5% nonfat dry milk (BioRad, Hercules, CA, USA) in TBS-T to decrease nonspecific binding. Afterwards, the membrane was incubated with rabbit polyclonal antibody against human CCM2 (dilution 1/1000 in 5% nonfat dry milk in TBS-T) for 1 h. The membrane was then incubated with horse peroxidase-labelled anti-rabbit IgG (dilution 1/10,000; Vector Laboratories) for 1 h. Immunolabelling was visualized using the chemiluminescence based SuperSignal CL HRP Substrate System (Pierce, Rockford, IL, USA) and the membrane was exposed to Hyperfilm (Amersham, Piscataway, NJ, USA).
Fig. 1. Representative pictures of CCM2 staining in developing neocortex during first trimester. The cytoplasmic reaction of neocortex cells and fibers show a strong immunoreactivity 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 loading of proteins in each lane was confirmed by re-probing the membrane with mouse monoclonal anti-human -actin (Abcam, Cambridge, UK).
3. Results 3.1. CCM2 expression in normal human neocortex during prenatal development and adult samples Using immunohistochemical staining, we studied the expression patterns of CCM2 in human neocortex tissue during developmental period and also adult samples. During the first trimester, the fibers in the developing human neocortex revealed a strong CCM2 protein immunoreactivity in PPL. In addition, the cytoplasm of the cells was also immunoreactive for CCM2 in SVZ and VZ (Fig. 1). During the second trimester, CCM2 immunoreactivity was detected throughout the neocortex layers (Fig. 2A–G). Additionally, there was a strong CCM2 immunoreaction localized in the vascular endothelium during second trimester (Fig. 2C–G, arrowheads). The moderate localization of CCM2 in some of the cells was specific to MZ, CP and SP zones (Fig. 2B–D arrows). The strong CCM2 immunolabelling was shown in the surface of the MZ and VZ. In comparison with the first trimester neocortex immunoreactivity was higher and occasionally nuclear in the specific zones. CCM2 immunoreactivity in the third trimester was stronger than that in the second and first trimesters, not only in the cells and fibers but also in the vascular endothelium. In the neocortex of the third trimester, CCM2 was observed in a strong immunoreactivity in some of the vascular endothelium (Fig. 3D, E and K, arrowheads). On the other hand, some of the vascular endothelium revealed a weak CCM2 immunolabelling during the third trimester (Fig. 3E, arrowhead). In the adult brain samples, the strongest CCM2 immunoreactivity was obtained in the neocortex layers from trimesters. The expression of CCM2 was increased in the arterial endothelium as referred in our previous study (Tanriover et al., 2008). On the other hand, the expression of CCM2 was detected in a subset of neuronal cells, some glial cells (Fig. 4B arrows) and, at very low levels in the venous endothelium (Fig. 4D, arrowheads). Moreover, the staining pattern for CCM2 protein was strongly restricted to the arterial endothelium in adult brain samples (Fig. 4C, E and F, arrowheads). No immunoreactivity was observed on the slides where primary antibody was replaced with their normal rabbit IgG (data not shown). 3.2. Confirmation of CCM2 expression by Western blot analysis CCM2 expression was analyzed by Western blotting from normal human developing and adult brain tissues. The blots clearly
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Fig. 2. Representative pictures of CCM2 staining in developing human neocortex during second trimester. The heterogeneous immunoreactivity was seen in second trimesters and 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. (F) Subventricular zone. (G) Ventricular zone. Vascular endothelium (C–G; arrowheads) and some of the cells (arrows) have a moderate immunoreactivity. Scale bar: 50 m.
revealed bands for CCM2 corresponding to 47 kDa, in developing and adult normal brain tissue samples. Equivalent amounts of total proteins were loaded per lane as indicative by the immunoexpression of -actin (43 kDa). According to the Western blot results, CCM2 expression was detected at all trimesters and adult neocortex. Furthermore, it gradually increased from first to third trimester and adult neocortex. The protein level of CCM2 molecule was statistically higher in adult vs. second and third trimesters (P = <0.001). However, there were no differences between second vs. third trimesters for the expression of CCM2 (Fig. 5). 4. Discussion The present study was designed to investigate the presence of CCM2 in development of the neocortex by immunohistochemistry and Western blot analysis for the first time. Our previous study of the expression of CCM2 protein was reported in adult brain samples (Tanriover et al., 2008). But now, in this study the expression pattern of CCM2 protein is associated with the prenatal and adult neocortex and blood vessel formations. Since CCM2 is expressed in the vascular endothelial cells (Seker et al., 2006; Tanriover et al., 2008); and also this gene could possibly be related in endothelial cell functions during brain vascular development. Endothelial cells are the main cellular unit of vascular structures. Their assem-
bly into a well organized and functional structure is essential for organ growth during fetal development. Thus, development of the vascular tree involves two processes: vasculogenesis and angiogenesis (Kubis and Levy, 2004). Meanwhile, the arterial and venous endothelial cells are molecularly distinct from the earliest stage of angiogenesis. Differentiation into arteries or veins is not only determined by the direction and the importance of flow but endothelial cell linings appear to be different and determined by the presence or the absence of molecules (Rocha and Adams, 2009). Our results suggested that CCM2 immunoreactivity was obtained in vascular endothelium during prenatal development. While some of the vascular endothelial cells revealed a strong immunoreactivity, some showed weak immunoreactivity during the third trimester. We speculate that CCM2 is probably one of the molecules which determined the difference between arteries and veins difference in the development of brain vasculature. It might be CCM2 gene which disrupted the vein and artery remodeling whereby suggesting that a reciprocal interaction is necessary for brain angiogenesis. We already know that CCM molecules such as CCM1, CCM2 and CCM3 were detected in an arterial endothelium but weak or no immunoreactivity was observed in venous endothelium (Guzeloglu-Kayisli et al., 2004; Tanriover et al., 2008). Consistently, CCM2 immunoreaction was detected in the arterial endothelium during adult neocortex. Therefore, CCM2
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Fig. 3. Representative pictures of CCM2 staining in developing neocortex during third trimester. (A) An increasing and expanding immunoreactivity for CCM2 in developing neocortex is seen for CCM2. (D and E) The arterial endothelium shows strong immunoreactivity (D, E, K, arrowheads) while a weak immunoreactivity for venous endothelium (G; arrowheads) for CCM2. Scale bar: 50 m.
expression might be useful for the determination of arterial and venous systems in the third trimester samples. Our proposal is that CCM2 represents an arterial phenotype and it is important in establishing an arterial identity in developing brain vasculature. Alteration of the CCM2 expression could be responsible for the vascular remodeling. Boulday et al. (2009) showed that constitutive deletion of CCM2 leads to early embryonic death. Deletion of CCM2 from endothelial cells severely affects angiogenesis, leading to morphogenic defects in the major arterial and venous blood vessels and in the heart, and it results in fetal lethality at midgestation (Boulday et al., 2009). Our results were compatible with these findings establishing the essential role of endothelial CCM2 for proper vascular development. While the function of the CCM2
protein is unknown in the endothelial cells, it has been found that they may have a possible role in angiogenesis. Thus, it is possible to speculate that the loss-of-function mutations in the CCM2 gene in humans lead to cerebrovascular malformations, causing recurrent brain hemorrhages (Boulday et al., 2009). In addition to vascular endothelial cells, our results showed that CCM2 protein was also localized in cells during development and adult brain samples. It has been shown that CCM2 gene is also expressed in neurons (Seker et al., 2006; Tanriover et al., 2008) in pre and postnatal mouse brain. Also, our results confirmed as in previous studies that the immunoreactivity was observed in cells that probably consist of neurons and glias during prenatal development. So, the localization of CCM2 in neuroglial precursor cells might play a role in coordination with neuronal differentiation and orien-
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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 glial cells reveal a strong CCM2 immmunoreactivity in other CP layers which are Layers 3–6 (arrows). (C, E and F) The arterial endothelium shows a strong immunoreactivity (arrowheads). (D) The venous endothelium shows a weak immunoreaction for CCM2 (arrowheads). Scale bar: 50 m.
tation in addition to regulating neuronal migration. But, Boulday et al. (2009) demonstratedthat the deletion of CCM2 from neuroglial precursor cells does not lead to cerebrovascular defects though CCM2 is predominantly expressed in the neuronal layers within the central nervous system. While the function of the CCM2 protein is unknown, a specific interplay between the neuron, glia and endothelium at the level of the neurovascular unit might be crucial for the development of the brain.
In conclusion, our results showed that recently identified new critical gene, CCM2 is involved in vascular morphogenesis and studies in the molecular pathways underlying vasculogenesis and angiogenesis. This protein might be leading to the better understanding of associated several vascular complications. Further studies should aim to find other molecules interacting with CCM2 in the endothelial cells and to identify which signaling pathways are affected by the protein. Furthermore, it is likely that the interaction
Table 1 Summary of the staining intensities of the CCM2 antibody in the staining regions. 1st trimester
Staining intensities in the vascular endoth.
PPL ++ +
Staining intensities in the fibers
++
Staining intensities in cortical zones
SVZ +
2nd trimester VZ +
MZ ++a ++ ++
CP ++
3rd trimester SP ++
IZ ++
SVZ ++
VZ ++a
L1–6 +++ +++ +++
SP +++
Adult WM +++
L1–6 +++ A ++ +++
WM +++ V +/−
PPL: primordial plexiform layer, SVZ: subventricular zone, VZ: ventricular zone, MZ: marginal zone, CP: cortical plate, IZ: intermediate zone, SP: subplate zone, L1–6: layer 1–6, WM: white matter, A: arterial endothelium, V: venous endothelium. a The strong CCM2 immunolabelling was shown in the surface of the MZ and VZ.
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Fig. 5. (A) Western blotting analysis of CCM2 in second, third trimesters and adult neocortex tissues. Immunoblots of cortical extracts by using anti-CCM2: a 47 kDa band has been detected in trimesters and adult human neocortex samples. The immunoexpression of -actin (43 kDa) was used to show prospective equivalent amounts of total proteins loaded per lane. (B) The immunoblot bands were quantified by an optical densitometer. The OD (optical density) values of CCM2 bands were normalized to the OD values of -actin bands. The protein level of CCM2 molecule was statistically higher in adult vs. second and third trimesters (P = <0.001).
among neurons, glia and endothelia would lead to better understanding the CCM lesions in patients (Table 1). Acknowledgments The authors would like to thank Dr. E.I. Gurer from Department of Pathology, Akdeniz University School of Medicine, Antalya, Turkey for her excellent cooperation to evaluate normal or pathologic brain material differences. This study was partially supported by the Akdeniz University Research Foundation, Antalya, Turkey. References Bentivoglio, M., Tassi, L., Pech, E., Costa, C., Fabene, P.F., Spreafico, R., 2003. Cortical development and focal cortical dysplasia. Epileptic Disord. 5 (Suppl. 2), S27–S34. Bergametti, 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 the programmed cell death 10 gene cause cerebral cavernous malformations. Am. J. Hum. Genet. 76, 42–51. Boulday, G., Blecon, A., Petit, N., Chareyre, F., Garcia, L.A., Niwa-Kawakita, M., Giovannini, M., Tournier-Lasserve, E., 2009. Tissue-specific conditional CCM2 knockout mice establish the essential role of endothelial CCM2 in angiogenesis: implications for human cerebral cavernous malformations. Dis. Model Mech. 2, 168–177. Chan, W.Y., Lorke, D.E., Tiu, S.C., Yew, D.T., 2002. Proliferation and apoptosis in the developing human neocortex. Anat. Rec. 267, 261–276.
Clatterbuck, R.E., Eberhart, C.G., Crain, B.J., Rigamonti, D., 2001. Ultrastructural and immunocytochemical evidence that an incompetent blood-brain barrier is related 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., Kennedy, C.T., Mettler, G., Beis, M.J., Fryer, A., Awad, I.A., Lifton, R.P., 1998. Multilocus linkage identifies two new loci for a mendelian form of stroke, cerebral cavernous 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 to chromosome 7q. Hum. Mol. Genet. 4, 453–458. Gunel, M., Awad, I.A., Finberg, K., Anson, J.A., Steinberg, G.K., Batjer, H.H., Kopitnik, T.A., Morrison, L., Giannotta, S.L., Nelson-Williams, C., Lifton, R.P., 1996. A founder mutation as a cause of cerebral cavernous malformation in Hispanic Americans. 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 interaction trapped-1/cerebral cavernous malformation-1 protein expression during early angiogenesis. 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 understanding vascular 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 cavernous 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. Plummer, N.W., Zawistowski, J.S., Marchuk, D.A., 2005. Genetics of cerebral cavernous malformations. Curr. Neurol. Neurosci. Rep. 5, 391–396. Pozzati, E., Acciarri, N., Tognetti, F., Marliani, F., Giangaspero, F., 1996. Growth, subsequent 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 neuronal positions, 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 familial 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 vascular 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 expression 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 expressed in the neurovascular unit. Neurosurgery 62, 930–938 (discussion 938). Tanriover, G., Demir, N., Pestereli, E., Demir, R., Kayisli, U.A., 2005. PTEN-mediated Akt 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. Distribution 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 proteins contribute to vasculogenesis and angiogenesis in human placenta. Histol. Histopathol. 24, 1287–1294. Thiery, J.P., 1984. Mechanisms of cell migration in the vertebrate embryo. Cell Differ. 15, 1–15. Wiechelman, K.J., Braun, R.D., Fitzpatrick, J.D., 1988. Investigation of the bicinchoninic acid protein assay: identification of the groups responsible for color formation. Anal. Biochem. 175, 231–237.