Abstract
The use of murine cerebrovascular endothelial and smooth muscle cells has not been widely employed as a cell culture model for the investigation of cellular mechanisms involved in cerebral amyloid angiopathy (CAA). Difficulties in isolation and propagation of murine cerebrovascular cells and insufficient yields for molecular and cell culture studies have deterred investigators from using mice as a source for cerebrovascular cells in culture. Instead, cerebrovascular cells from larger mammals are preferred and several methods describing the isolation of endothelial and smooth muscle cells from human, canine, rat, and guinea pig have been published. In recent years, several transgenic mouse lines showing CAA pathology have been established; consequently murine cerebrovascular cells derived from these animals can serve as a key cellular model to study CAA. Here, we describe a procedure for isolating murine microvessels that yields healthy smooth muscle and endothelial cell populations and produce sufficient material for experimental purposes. Murine smooth muscle cells isolated using this protocol exhibit the classic “hill and valley” morphology and are immunoreactive for the smooth muscle cell marker α-actin. Endothelial cells display a “cobblestone” pattern phenotype and show the characteristic immunostaining for the von Willebrand factor and the factor VIII-related antigen. In addition, we describe methods designed to preserve these cells by storage in liquid nitrogen and reestablishing viable cell cultures. Finally, we compare our methods with protocols designed to isolate and maintain human cerebrovascular cell cultures.
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References
Kalaria, R. N. (2001) Advances in molecular genetics and pathology of cerebrovascular disorders. Trends Neurosci 24, 392–400.
Vinters, H. V. (1987) Cerebral amyloid angiopathy. A critical review. Stroke 18, 311–324.
Greenberg, S. (2002) Cerebral amyloid angiopathy and dementia - Two amyloids are worse than one. Neurology 58, 1587–1588.
Pfeifer, L. A., White, L. R., Ross, G. W., Petrovitch, H., and Launer, L. J. (2002) Cerebral amyloid angiopathy and cognitive function: the HAAS autopsy study, Neurology 58. 1629–1634.
Levy, E., Carman, M. D., Fernandez-Madrid, I. J., Power, M. D., Lieberburg, I., van Duinen, S. G., Bots, G. T. A. M., Luyendijk, W., and Frangione, B. (1990) Mutation of the Alzheimer’s disease amyloid gene in hereditary cerebral hemorrhage, Dutch type. Science 248, 1124–1126.
Grabowski, T. J., Cho, H. S., Vonsattel, J. P., Rebeck, G. W., and Greenberg, S. M. (2001) Novel amyloid precursor protein mutation in an Iowa family with dementia and severe cerebral amyloid angiopathy. Ann Neurol 49, 697–705.
Tsubuki, S., Takaki, Y., and Saido, T. C. (2003) Dutch, Flemish, Italian, and Arctic mutations of APP and resistance of Aβ to physiologically relevant proteolytic degradation. Lancet 361, 1957–1958.
Gudmundsson, G., Hallgrimsson, J., Jonasson, T. A., and Bjarnason, O. (1972) Hereditary cerebral haemorrhage with amyloidosis. Brain 95, 387–404.
Olafsson, I., Thorsteinsson, L., and Jensson, O. (1996) The molecular pathology of hereditary cystatin C amyloid angiopathy causing brain hemorrhage. Brain Pathol 6, 121–126.
Cohen, D. H., Feiner, H., Jensson, O., and Frangione, B. (1983) Amyloid fibril in hereditary cerebral hemorrhage with amyloidosis (HCHWA) is related to the gastroentero-pancreatic neuroendocrine protein, γ trace. J Exp Med 158, 623–628.
Ghiso, J., Jensson, O., and Frangione, B. (1986) Amyloid fibrils in hereditary cerebral hemorrhage with amyloidosis of Icelandic type is a variant of γ-trace basic protein (cystatin C). Proc Natl Acad Sci USA 83, 2974–2978.
Vidal, R., Frangione, B., Rostagno, A., Mead, S., Revesz, T., Plant, G., and Ghiso, J. (1999) A stop-codon mutation in the BRI gene associated with familial British dementia. Nature 399, 776–781.
Vidal, R., Revesz, T., Rostagno, A., Kim, E., Holton, J. L., Bek, T., Bojsen-Moller, M., Braendgaard, H., Plant, G., Ghiso, J., and Frangione, B. (2000) A decamer duplication in the 3′ region of the BRI gene originates an amyloid peptide that is associated with dementia in a Danish kindred. Proc Natl Acad Sci USA 97, 4920–4925.
Kiuru, S., Salonen, O., and Haltia, M. (1999) Gelsolin-related spinal and cerebral amyloid angiopathy. Ann Neurol 45, 305–311.
Sturchler-Pierrat, C., Abramowski, D., Duke, M., Wiederhold, K. H., Mistl, C., Rothacher, S., Ledermann, B., Burki, K., Frey, P., Paganetti, P. A., Waridel, C., Calhoun, M. E., Jucker, M., Probst, A., Staufenbiel, M., and Sommer, B. (1997) Two amyloid precursor protein transgenic mouse models with Alzheimer disease-like pathology. Proc Natl Acad Sci USA 94, 13287–13292.
Calhoun, M. E., Burgermeister, P., Phinney, A. L., Stalder, M., Tolnay, M., Wiederhold, K. H., Abramowski, D., Sturchler-Pierrat, C., Sommer, B., Staufenbiel, M., and Jucker, M. (1999) Neuronal overexpression of mutant amyloid precursor protein results in prominent deposition of cerebrovascular amyloid. Proc Natl Acad Sci USA 96, 14088–14093.
Winkler, D. T., Bondolfi, L., Herzig, M. C., Jann, L., Calhoun, M. E., Wiederhold, K. H., Tolnay, M., Staufenbiel, M., and Jucker, M. (2001) Spontaneous hemorrhagic stroke in a mouse model of cerebral amyloid angiopathy. J Neurosci 21, 1619–1627.
Herzig, M. C., Eisele, Y. S., Staufenbiel, M., and Jucker, M. (2009) E22Q-mutant Aβ peptide (AβDutch) increases vascular but reduces parenchymal Aβ deposition. Am J Pathol 174, 722–726.
Kumar-Singh, S. (2009) Hereditary and sporadic forms of Aβ-cerebrovascular amyloidosis and relevant transgenic mouse models. Int J Mol Sci 10, 1872–1895.
Pawlik, M., Sastre, M., Calero, M., Mathews, P. M., Schmidt, S. D., Nixon, R. A., and Levy, E. (2004) Overexpression of human cystatin C in transgenic mice does not affect levels of endogenous brain amyloid β peptide. J Mol Neurosci 22, 13–18.
Vidal, R., Barbeito, A. G., Miravalle, L., and Ghetti, B. (2009) Cerebral amyloid angiopathy and parenchymal amyloid deposition in transgenic mice expressing the Danish mutant form of human BRI2. Brain Pathol 19, 58–68.
Vinters, H. V., Reave, S., Costello, P., Girvin, J. P., and Moore, S. A. (1987) Isolation and culture of cells derived from human cerebral microvessels. Cell Tissue Res 249, 657–667.
Vinters, H. V. (1987) Cerebral amyloid angiopathy. A critical review. Stroke 18, 311–324.
Frackowiak, J., Mazur-Kolecka, B., Wisniewski, H. M., Potempska, A., Carroll, R. T., Emmerling, M. R., and Kim, K. S. (1995) Secretion and accumulation of Alzheimer’s β-protein by cultured vascular smooth muscle cells from old and young dogs. Brain Research 676, 225–230.
Mazur-Kolecka, B., Frackowiak, J., Krzeslowska, J., Ramakrishna, N., Haske, T., Emmerling, M. R., Zhang, W., Kim, K. S., and Wisniewski, H. M. (1999) Apolipoprotein E alters metabolism of AβPP in cells engaged in β-amyloidosis. Journal of Neuropathology and Experimental Neurology 58, 288–295.
Mazur-Kolecka, B., Frackowiak, J., and Wisniewski, H. M. (1995) Apolipoproteins E3 and E4 induce, and transthyretin prevents accumulation of the Alzheimer’s β-amyloid peptide in cultured vascular smooth muscle cells. Brain Research 698, 217–222.
Diglio, C. A., Grammas, P., Giacomelli, F., and Wiener, J. (1982) Primary culture of rat cerebral microvascular endothelial cells. Isolation, growth, and characterization. Lab Invest 46, 554–563.
Diglio, C. A., Grammas, P., Giacomelli, F., and Wiener, J. (1986) Rat cerebral microvascular smooth muscle cells in culture. J Cell Physiol 129, 131–141.
Diglio, C. A., Liu, W., Grammas, P., Giacomelli, F., and Wiener, J. (1993) Isolation and characterization of cerebral resistance vessel endothelium in culture. Tissue Cell 25, 833–846.
Reisner, A., Olson, J. J., Yang, J., Assietti, R., Klemm, J. M., and Girard, P. R. (1995) Isolation and culture of bovine intracranial arterial endothelial cells. Neurosurgery 36, 806–812; discussion 813.
Seidel, M. F., Simard, J. M., Hunter, S. F., and Campbell, G. A. (1991) Isolation of arteriolar microvessels and culture of smooth muscle cells from cerebral cortex of guinea pig. Cell Tissue Res 265, 579–587.
Tontsch, U., and Bauer, H. C. (1989) Isolation, characterization, and long-term cultivation of porcine and murine cerebral capillary endothelial cells. Microvasc Res 37, 148–161.
Van Nostrand, W. E., Rozemuller, A. J. M., Chung, R., Cotman, C. W., and Saporito-Irwin, S. M. (1994) Amyloid β-protein precursor in cultured leptomeningeal smooth muscle cells. Int.J.Exp.Clin.Invest. 1, 1–7.
Frackowiak, J., Miller, D. L., Potempska, A., Sukontasup, T., and Mazur-Kolecka, B. (2003) Secretion and accumulation of Aβ by brain vascular smooth muscle cells from AβPP-Swedish transgenic mice. J Neuropathol Exp Neurol 62, 685–696.
Grammas, P., Roher, A. E., and Ball, M. J. (1991) Decreased α-adrenergic receptors at the blood-brain barrier in Alzheimer’s disease, in Alzheimer’s Disease: Basic Mechanisms, Diagnosis and Therapeutic Strategies (Iqbal, K., McLachlan, D. R. C., Winblad, B., and Wisnieski, H. M., Eds.), pp 129–136, John Wiley & Sons Ltd., New York.
Jung, S. S., and Van Nostrand, W. E. (2002) Aβ does not induce oxidative stress in human cerebrovascular smooth muscle cells. Neuroreport 13, 1309–1312.
Acknowledgments
This work was supported by grants from the NINDS (NS42029), NIA (AG017617), and the Alzheimer’s Association (IIRG-07-59699).
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Gauthier, S.A., Sahoo, S., Jung, S.S., Levy, E. (2012). Murine Cerebrovascular Cells as a Cell Culture Model for Cerebral Amyloid Angiopathy: Isolation of Smooth Muscle and Endothelial Cells from Mouse Brain. In: Sigurdsson, E., Calero, M., Gasset, M. (eds) Amyloid Proteins. Methods in Molecular Biology, vol 849. Humana Press. https://doi.org/10.1007/978-1-61779-551-0_18
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DOI: https://doi.org/10.1007/978-1-61779-551-0_18
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