Summary
Vascular smooth muscle cells were isolated from the aortas of spontaneously hypertensive rats and normotensive Wistar-Kyoto rats by use of the explant method on collagen gels. Clonal cell lines derived from these enriched populations possessed ultrastructural characteristics of vascular smooth muscle cells in culture; they grew in hill and valley configuration, immunostained with the muscle actin antibody HHF35, and failed to react with von Willebrand Factor VIII antibody. Fourteen clonal cell lines were characterized for growth and ligand binding characteristics. Large variations in growth rate and cell density at saturation were exhibited by clones of both strains. Similar variability was noted for specific binding of endothelial 1 and Sar1,Ile8-angiotensin II to their receptors, indicating considerable phenotypic heterogeneity among the clonal cell lines. Six selected clones were further characterized for angiotensin II receptor linkage to G proteins. Cells of both strains exhibited comparable affinity shifts in the presence of GTPγS. These clonal cell lines should be useful for a variety of analyses of the comparative biology of aortic cells. It is possible that the diversity of phenotypic traits exhibited by these clones reflects the heterogeneity of vascular smooth muscle tissue found in vivo.
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Absher, M.; Woodcock-Mitchell, J.; Mitchell, J., et al. Characterization of vascular smooth muscle cell phenotype in long-term culture. In Vitro Cell. Dev. Biol. 25:183–192; 1989.
Berecek, K. H.; Schwertschlag, U.; Gross, F. Alterations in renal vascular resistance and reactivity in spontaneous hypertension of rats. Am. J. Physiol. 238:H287-H293; 1980.
Berk, B. C.; Vallega, G.; Muslin, A. J., et al. Spontaneously hypertensive rat vascular smooth muscle cells in culture exhibit increased growth and Na+/H+ exchange. J. Clin. Invest. 83:822–829; 1989.
Björkerud, S.; Gustavsson, K.; Hasselgren, M.In vitro cultivation of rabbit aortic media and the development of the cultures in relation to heterogeneity. Acta Pathol. Microbiol. Immunol. Scand. A. 92:113–124; 1984.
Bolzon, B. J.; Cheung, D. W. Isolation and characterization of single vascular smooth muscle cells from spontaneously hypertensive rats. Hypertension 14:137–144; 1989.
Chamley-Campbell, J.; Campbell, G. R.; Ross, R. The smooth muscle cell in culture. Physiol. Rev. 59:1–61; 1979.
Chen, C. H.; Chen, S. C. Cell growth factor activity. Exp. Cell. Res. 136:43–51; 1981.
Clegg, K.; Eggena, P.; Barrett, J. D., et al. Smooth muscle cell growth ratein vitro and hyperplasia in the aorta of the spontaneously hypertensive rat. J. Hypertens. 4(suppl 3):S101-S103; 1986.
Cole, O. F.; Fan, T. P. D.; Lewis, G. P. Isolation, characterization, growth and culture of endothelial cells from the rat aorta. Cell Biol. Intl. Rep. 10:399–405; 1986.
Collis, M. G.; Vanhoutte, P. M. Vascular reactivity of isolated perfused kidneys from male and female spontaneously hypertensive rats. Circ. Res. 41:759–767; 1977.
De Lean, A.; Ong, H.; Gutkowska, J., et al. Evidence for agonist-induced interaction of angiotensin receptor with a guanine nucleotidebinding protein in bovine adrenal zona glomerulosa. Mol. Pharmacol. 26:498–508; 1984.
Geisterfer, A. T.; Peach, M. J.; Owens, G. K. Angiotensin II induces hypertrophy, not hyperplasia, of cultured rat aortic smooth muscle cells. Circ. Res. 62:749–756; 1988.
Hadrava, V.; Tremblay, J.; Hamet, P. Abnormalities in growth characteristics of aortic smooth muscle cells in spontaneously hypertensive rats. Hypertension 13:589–597; 1989.
Hall, M. M.; Khosla, M. C.; Khairallah, P. A., et al. Angiotensin analogs: the influence of sarcosine substituted in position 1. J. Pharmacol. Exp. Ther. 188:222–228; 1974.
Jazayeri, A.; Meyer, W. J. Beta-adrenergic receptor differences in cultured arterial smooth muscle cells between spontaneously hypertensive and Wistar-Kyoto rats. J. Hypertens. 7:895–900; 1989.
Jones, W.; Hosick, H. L. Collagen concentration as a significant variable for growth and morphology of mouse mammary parenchyma in collagen lattice culture. Cell Biol. Intl. Rep. 10:277–286; 1986.
Kimes, B. W.; Brandt, B. L. Characterization of two putative smooth muscle cell lines from rat thoracic aorta. Exp. Cell Res. 98:349–366; 1976.
Lees, M. B.; Paxman, S. Modification of the Lowry procedure for the analysis of proteolipid protein. Anal. Biochem. 47:184–192; 1972.
McGuire, P. G.; Orkin, R. W. Isolation of rat aortic endothelial cells by primary explant techniques and their phenotypic modulation by defined substrata. Lab. Invest. 57:94–105; 1987.
Miyauchi, T.; Ishikawa, T.; Ttomobe, T., et al. Characteristics of pressor response to endothelin in spontaneously hypertensive and Wistar-Kyoto rats. Hypertension 14:427–434; 1989.
Munson, P. J.; Rodbard, D. Ligand: a versatile computerized approach to characterization of ligand binding systems. Anal. Biochem. 107:220–221; 1980.
Owens, G. K. Influence of blood pressure on development of aortic medial smooth muscle hypertrophy in spontaneously hypertensive rats. Hypertension 9:178–187; 1987.
Paglin, S.; Stukenbrok, H.; Joyce, N. C., et al. Interaction of angiotensin II with functional smooth muscle cells in culture. Am. J. Physiol. 253:C872-C882; 1987.
Peterson, G. L. A simplification of the protein assay method of Lowry,et al. which is more generally applicable. Anal. Biochem. 83:346–356; 1977.
Resnik, T. J.; Scott-Burden, T.; Baur, U., et al. Enhanced responsiveness to angiotensin II in vascular smooth muscle cells from spontaneously hypertensive rats is not associated with alterations in protein kinase C. Hypertension 14:293–303; 1989.
Scanlon, M. N.; Koziarz, P.; Moore, G. J. The relationship between homotropic and heterotropic cooperativity for angiotensin receptors in smooth muscle. Gen. Pharmacol. 21:59–65; 1990.
Schiffrin, E. L.; Thome, F. S.; Genest, J. Vascular angiotensin II receptors in SHR. Hypertension 6:682–688; 1984.
Schor, S. L.; Schor, A. M. Clonal heterogeneity in fibroblast phenotype: implications for the control of epithelial-mesenchymal interactions. Bioessays 7:200–204; 1987.
Socorro, L.; Vallega, G.; Nunn, A., et al. Vascular smooth muscle cells from the milan hypertensive rat exhibit decreased functional angiotensin II receptors. Hypertension 15:591–599; 1990.
Somlyo, A. P.; Somlyo, A. V. Vascular smooth muscle. I. Normal structure, pathology, biochemistry, and biophysics. Pharmacol. Rev. 24:197–272; 1968.
Tsukada, T.; Tippens, D.; Gordon, D., et al. HHF35, a muscle-actin-specific monoclonal antibody. I. Immunocytochemical and biochemical characterization. Am. J. Pathol. 126:51–60; 1987.
Wagner, D. D.; Olmsted, J. B.; Marder, V. J. Immunolocalization of von Willebrand protein in Weibel-Palade bodies of human endothelial cells. J. Cell Biol. 95:355–360; 1982.
Wright, G. B.; Alexander, R. W.; Ekstein, L. S., et al. Sodium, divalent cations, and guanine nucleotides regulate the affinity of the rat mesenteric artery angiotensin II receptor. Circ. Res. 50:462–469; 1982.
Wright, J. W.; Jensen, L. L.; Cushing, L. L., et al. Heightened blood pressure responsiveness to intracarotid infusion of angiotensins in the spontaneously hypertensive rat. Pharmacol. Biochem. Behav. 30:343–346; 1988.
Yamori, Y.; Igawa, T.; Kanbe, T., et al. Mechanisms of structural vascular changes in genetic hypertension: analyses on cultured vascular smooth muscle cells from spontaneously hypertensive rats. Clin. Sci. 61:121s-123s; 1981.
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Hall, K.L., Harding, J.W. & Hosick, H.L. Isolation and characterization of clonal vascular smooth muscle cell lines from spontaneously hypertensive and normotensive rat aortas. In Vitro Cell Dev Biol - Animal 27, 791–798 (1991). https://doi.org/10.1007/BF02631245
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DOI: https://doi.org/10.1007/BF02631245