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Isolation and culture of cells derived from human cerebral microvessels

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Summary

Microvessels were isolated from non-neoplastic human cerebral cortical fragments resected for treatment of intractable seizure disorder. The microvessels were incubated in modified Lewis medium with 20 or 30% fetal bovine serum. Within 1–2 weeks, two cell populations emerged from the isolates. One type of cells had polygonal morphology, showed density-dependent contact inhibition at confluence in vitro, showed lectin-binding characteristics of endothelium (but only moderate positivity for factor VIII antigen), demonstrated induction of γ-glutamyl trans-peptidase when exposed to astrocyte-conditioned media, and responded to insulin by a pronounced increase in DNA synthesis. The other variety of cells grew in vitro more slowly in irregular strands separated by clear zones, showed ultrastructural features of smooth muscle, and isoelectric focusing of cell proteins revealed the presence of smooth-musclespecific α-isoactin. Both types of cells could be serially subcultured. The ability to isolate and grow the two cell types, tentatively identified as human cerebral microvascular endothelium and smooth muscle, may facilitate studies of human blood-brain barrier function as well as the pathogenesis of cerebral microangiopathies unique to the human brain.

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References

  • Beck DW, Vinters HV, Hart MN, Henn FA, Cancilla PA (1983a) Uptake of adenosine into cultured cerebral endothelium. Brain Res 271:180–183

    Google Scholar 

  • Beck DW, Vinters HV, Moore SA, Hart MN, Cancilla PA (1983b) Uptake of adenosine by cultured cerebral vascular smooth muscle cells. J Neurochem 41:939–941

    Google Scholar 

  • Beck DW, Vinters HV, Hart MN, Cancilla PA (1984) Glial cells influence polarity of the blood-brain barrier. J Neuropathol Exp Neurol 43:219–224

    Google Scholar 

  • Berliner JA, Vinters HV, Karasic D, Cancilla PA, Frank HJL (1983) Insulin stimulation of amino-acid uptake in aortic and capillary endothelial cells. J Cell Biol 97:163a

    Google Scholar 

  • Böhling T, Paetau A, Ekblom P, Haltia M (1983) Distribution of endothelial and basement membrane markers in angiogenic tumors of the nervous system. Acta Neuropathol (Berl) 62:67–72

    Google Scholar 

  • Bowman PD, Betz AL, Ar D, Wolinsky JS, Penney JB, Shivers RR, Goldstein GW (1981) Primary culture of capillary endothelium from rat brain. In Vitro 17:353–362

    Google Scholar 

  • Bowman PD, Ennis SR, Rarey KE, Betz AL, Goldstein GW (1983) Brain microvessel endothelial cells in tissue culture: a model for study of blood-brain barrier permeability. Ann Neurol 14:396–402

    Google Scholar 

  • Bradbury MWB (1984) The structure and function of the bloodbrain barrier. Fed Proc 43:186–190

    Google Scholar 

  • Buzney SM, Massicotte SJ, Hetu N, Zetter BR (1983) Retinal vascular endothelial cells and pericytes. Differential growth characteristics in vitro. Invest Ophthalmol Vis Sci 24:470–480

    Google Scholar 

  • Chamley-Campbell J, Campbell GR, Ross R (1979) The smooth muscle cell in culture. Physiol Rev 59:1–61

    Google Scholar 

  • Davison PM, Bensch K, Karasek MA (1983) Isolation and longterm serial cultivation of endothelial cells from the microvessels of the adult human dermis. In Vitro 19:937–945

    Google Scholar 

  • De Bault LE (1982) Isolation and characterization of the cells of the cerebral microvessels. Adv Cell Neurobiol 3:339–371

    Google Scholar 

  • De Bault LE, Cancilla PA (1980a) Some properties of isolated endothelial cells in culture. Adv Exp Med Biol 131:69–78

    Google Scholar 

  • De Bault LE, Cancilla PA (1980b) Gamma-glutamyl transpeptidase in isolated brain endothelial cells: induction by glial cells in vitro. Science 207:653–655

    Google Scholar 

  • De Bault LE, Kahn LE, Frommes SP, Cancilla PA (1979) Cerebral microvessels and derived cells in tissue culture: isolation and preliminary characterization. In Vitro 15:473–487

    Google Scholar 

  • De Bault LE, Henriquez E, Hart MN, Cancilla PA (1981) Cerebral microvessels and derived cells in tissue culture: II. Establishment, identification, and preliminary characterization of an endothelial cell line. In Vitro 17:480–494

    Google Scholar 

  • DiCorleto PE, Gajdusek CM, Schwartz SM, Ross R (1983) Biochemical properties of the endothelium-derived growth factor: comparison to other growth factors. J Cell Physiol 114:339–345

    Google Scholar 

  • Diglio CA, Grammas P, Giacomelli F, Wiener J (1982) Primary culture of rat cerebral microvascular endothelial cells. Isolation, growth, and characterization. Lab Invest 46:554–563

    Google Scholar 

  • Folkman J, Haudenschild CC, Zetter BR (1979) Long-term culture of capillary endothelial cells. Proc Natl Acad Sci USA 76:5217–5221

    Google Scholar 

  • Fry G, Parsons T, Hoak J, Sage H, Gingrich RD, Ercolani L, Nghiem D, Czervionke R (1984) Properties of cultured endothelium from adult human vessels. Arteriosclerosis 4:4–13

    Google Scholar 

  • Gajdusek CM, Schwartz SM (1982) Ability of endothelial cells to condition culture medium. J Cell Physiol 110:35–42

    Google Scholar 

  • Garrels JI, Gibson W (1976) Identification and characterization of multiple forms of actin. Cell 9:793–805

    Google Scholar 

  • Gimbrone MA Jr, Cotran RS, Folkman J (1974) Human vascular endothelial cells in culture. Growth and DNA synthesis. J Cell Biol 60:673–684

    Google Scholar 

  • Gimbrone MA Jr, Cotran RS (1975) Human vascular smooth muscle in culture. Growth and ultrastructure. Lab Invest 33:16–27

    Google Scholar 

  • Goetz IE, Warren J, Estrada C, Roberts E, Krause DN (1985) Longterm serial cultivation of arterial and capillary endothelium from adult bovine brain. In Vitro 21:172–180

    Google Scholar 

  • Goldstein GW, Betz AL (1983) Recent advances in understanding brain capillary function. Ann Neurol 14:389–395

    Google Scholar 

  • Goldstein GW, Betz AL, Bowman PD (1984) Use of isolated brain capillaries and cultured endothelial cells to study the bloodbrain barrier. Fed Proc 43:191–195

    Google Scholar 

  • Herman IM, D'Amore PA (1985) Microvascular pericytes contain muscle and nonmuscle actins. J Cell Biol 101:43–52

    Google Scholar 

  • Hormia M, Lehto V-P, Virtanen I (1983) Identification of UEA I-binding surface glycoproteins of cultured human endothelial cells. Cell Biol Int Rep 7:467–475

    Google Scholar 

  • Hunter EE (1984) Practical electron microscopy. Praeger Scientific, New York, 121 pp

    Google Scholar 

  • Joyce NC, DeCamilli P, Boyles J (1984) Pericytes, like vascular smooth muscle cells, are immunocytochemically positive for cyclic GMP-dependent protein kinase. Microvasc Res 28:206–219

    CAS  PubMed  Google Scholar 

  • Joyce NC, Haire MF, Palade GE (1985a) Contractile proteins in pericytes. I. Immunoperoxidase localization of tropomyosin. J Cell Biol 100:1379–1386

    Google Scholar 

  • Joyce NC, Haire MF, Palade GE (1985b) Contractile proteins in pericytes. II. Immunocytochemical evidence for the presence of two isomyosins in graded concentrations. J Cell Biol 100:1387–1395

    Google Scholar 

  • Kan M, Kato M, Yamane I (1985) Long-term serial cultivation and growth requirements for human umbilical vein endothelial cells. In Vitro 21:181–188

    Google Scholar 

  • Krause DN, Goetz IE, Warren J, Roberts E (1983) Cerebrovascular endothelium: specific properties studied in tissue culture. J Cereb Blood Flow Metab 3 (Suppl 1):S427-S428

    Google Scholar 

  • Lee M-C, Wu T-C, Wan Y-J, Damjanov I (1983) Pregnancy-related changes in the mouse oviduct and uterus revealed by differential binding of fluoresceinated lectins. Histochemistry 79:365–375

    Google Scholar 

  • Lewis LJ, Hoak JC, Maca RD, Fry GL (1973) Replication of human endothelial cells in culture. Science 181:453–454

    Google Scholar 

  • Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275

    CAS  PubMed  Google Scholar 

  • Maciag T, Hoover GA, Stemerman MB, Weinstein R (1981) Serial propagation of human endothelial cells in vitro. J Cell Biol 91:420–426

    Google Scholar 

  • Maxwell K, Berliner JA, Cancilla PA (1986) Induction of γ-GTP in cultured cerebral endothelial cells by a product released by astrocytes. Brain Res (in press)

  • McCarthy KD, de Vellis J (1980) Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue. J Cell Biol 85:890–902

    Google Scholar 

  • Miettinen M, Holthofer H, Lehto V-P, Miettinen A, Virtanen I (1983) Ulex europaeus I lectin as a marker for tumors derived from endothelial cells. Am J Clin Pathol 79:32–36

    Google Scholar 

  • Monteiro MLR, Swanson RA, Coppeto JR, Cuneo RA, DeArmond SJ, Prusiner SB (1985) A microangiopathic syndrome of encephalopathy, hearing loss, and retinal arteriolar occlusions. Neurology (NY) 35:1113–1121

    Google Scholar 

  • Moore SA, Strauch AR, Yoder EJ, Rubenstein PA, Hart MN (1984) Cerebral microvascular smooth muscle in tissue culture. In Vitro 20:512–520

    Google Scholar 

  • Pardridge WM (1983) Brain metabolism: a perspective from the blood-brain barrier. Physiol Rev 63:1481–1535

    Google Scholar 

  • Pardridge WM (1984) Transport of nutrients and hormones through the blood-brain barrier. Fed Proc 43:201–204

    Google Scholar 

  • Petito CK, Levy DE (1980) The importance of cerebral arterioles in alterations of the blood-brain barrier. Lab Invest 43:262–268

    Google Scholar 

  • Rcese TS, Karnovsky MJ (1967) Fine structural localization of a blood-brain barrier to exogenous peroxidase. J Cell Biol 34:207–217

    Article  CAS  PubMed  Google Scholar 

  • Ross R (1986) The pathogenesis of atherosclerosis — an update. N EnglJ Med 314:488–500

    Google Scholar 

  • Rubenstein PA, Spudich JA (1977) Actin microheterogeneity in chick embryo fibroblasts. Proc Natl Acad Sci USA 74:120–123

    Google Scholar 

  • Rubinstein LJ (1972) Tumors of the central nervous system. (Second series, fascicle 6). Armed Forces Institute of Pathology, Bethesda, Maryland, pp 400

    Google Scholar 

  • Ryan US, White LA (1985) Varicose veins as a source of adult human endothelial cells. Tissue Cell 17:171–176

    Google Scholar 

  • Sato E, Maruta K, Yonezawa S, Nakamura T (1984) Blood group H(O) antigen in normal, dysplastic and carcinomatous esophagcal epithelium. Gann 75:223–229

    Google Scholar 

  • Schwartz SM (1984) Molecular biology and the vascular wall. Proceedings of a meeting. Arteriosclerosis 4:647–656

    Google Scholar 

  • Spatz M, Dodson RF, Bembry J (1983) Cerebrovascular muscle cultures. I. Isolation, growth and morphological characterization. Brain Res 280:387–391

    Google Scholar 

  • Striker GE, Harlan JM, Schwartz SM (1980) Human endothelial cells in vitro. Methods Cell Biol 21 A:135–151

    Google Scholar 

  • Thornton SC, Mueller SN, Levine EM (1983) Human endothelial cells: use of heparin in cloning and long-term serial cultivation. Science 222:623–625

    Google Scholar 

  • Vandekerckhove J, Weber K (1978) At least six different actins are expressed in a higher animal: an analysis based on the amino acid sequence of the amino-terminal tryptic peptide. J Mol Biol 126:783–802

    Google Scholar 

  • Vandekerckhove J, Weber K (1981) Actin typing on total cellular extracts. Eur J Biochem 113:595–603

    Google Scholar 

  • Vinters HV, Beck DW, Bready JV, Maxwell K, Berliner JA, Hart MN, Cancilla PA (1985a) Uptake of glucose analogues into cultured cerebral microvessel endothelium. J Neuropathol Exp Neurol 44:445–458

    Google Scholar 

  • Vinters HV, Berliner JA, Beck DW, Maxwell K, Bready JV, Cancilla PA (1985b) Insulin stimulates DNA synthesis in cerebral microvessel endothelium and smooth muscle. Diabetes 34:964–969

    Google Scholar 

  • Vinters HV, Reave S, Costello P, Girvin JP, Moore SA (1985e) Isolation and culture of cells derived from human cerebral microvessels. J Neuropathol Exp Neurol 44:338

    Google Scholar 

  • Vinters HV, Gilbert JJ (1983) Cerebral amyloid angiopathy: incidence and complications in the aging brain. II. The distribution of amyloid vascular changes. Stroke 14:924–928

    Google Scholar 

  • Voyta JC, Via DP, Butterfield CE, Zetter BR (1984) Identification and isolation of endothelial cells based on their increased uptake of acetylated-low density lipoprotein. J Cell Biol 99:2034–2040

    Google Scholar 

  • Weber T, Seitz RJ, Liebert UG, Gallasch E, Wechsler W (1985) Affinity cytochemistry of vascular endothelia in brain tumors by biotinylated Ulex europaeus type I lectin (UEAI). Acta Neuropathol (Berl) 67:128–135

    Google Scholar 

  • Weibel ER, Palade GE (1964) New cytoplasmic components in arterial endothelia. J Cell Biol 23:101–112

    Google Scholar 

  • Weinstein R, Stemerman MB, Maciag T (1981) Hormonal requireents for growth of arterial smooth muscle cells in vitro: an endocrine approach to atherosclerosis. Science 212:818–820

    Google Scholar 

  • Wion KL, Kelly D, Summerfield JA, Tuddenham EGD, Lawn RM (1985) Distribution of factor VIII mRNA and antigen in human liver and other tissues. Nature 317:726–729

    Google Scholar 

  • Zelechowska MG, van Mourik JA, Brodniewicz-Proba T (1985) Ultrastructural localization of factor VIII procoagulant antigen in human liver hepatocytes. Nature 317:729–730

    Google Scholar 

  • Zetter BR (1981) The endothelial cells of large and small blood vessels. Diabetes 30 (Suppl 2):24–28

    Google Scholar 

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Funded by Canadian Heart Foundation, Heart and Stroke Foundation of Ontario and UCLA Biomedical Research Support Grant

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Vinters, H.V., Reave, S., Costello, P. et al. Isolation and culture of cells derived from human cerebral microvessels. Cell Tissue Res. 249, 657–667 (1987). https://doi.org/10.1007/BF00217338

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