Transforming Growth Factor-J3-induced Growth Inhibition and Cellular Hypertrophy in Cultured Vascular Smooth Muscle Cells

Abstract

We have explored the hypothesis that hypertrophy of vascular smooth muscle cells may be regulated, in part, by growth inhibitory factors that alter the pattern of the growth response to serum mitogens by characterizing the effects of the potent growth inhibitor, transforming growth factor-[I (TGF-13), on both hyperplastic and hypertrophic growth of cultured rat aortic smooth muscle cells. TGF-13 inhibited seruminduced proliferation of rat aortic smooth muscle cells (EDs0 = 2 pM); this is consistent with previously reported observations in bovine aortic smooth muscle cells (Assoian et al. 1982. J. Biol. Chem. 258:71557160). Growth inhibition was due in part to a greater than twofold increase in the cell cycle transit time in cells that continued to proliferate in the presence of TGF-13. TGF-I~ concurrently induced cellular hypertrophy as assessed by flow cytometric analysis of cellular protein content (47 % increase) and forward angle light scatter (32-50% increase), an index of cell size. In addition to being time and concentration dependent, this hypertrophy was reversible. Simultaneous flow cytometric evaluation of forward angle light scatter and cellular DNA content demonstrated that TGF-I~-induced hypertrophy was not dependent on withdrawal of cells from the cell cycle nor was it dependent on growth arrest of cells at a particular point in the cell cycle in that both cycling cells in the G2 phase of the cell cycle and those in Gm were hypertrophied with respect to the corresponding ceils in vehicle-treated controls. Chronic treatment with TGF-I~ (100 pM, 9 d) was associated with accumulation of cells in the G2 phase of the cell cycle in the virtual absence of cells in S phase, whereas subsequent removal of TGF-I~ from these cultures was associated with the appearance of a significant fraction of cycling cells with >4c DNA content, consistent with development of tetraploidy. Results of these studies support a role for TGF-I~ in the control of smooth muscle cell growth and suggest that at least one mechanism whereby hypertrophy and hyperploidy may occur in this, as well as other cell types, is by alterations in the response to serum mitogens by potent growth inhibitors such as TGF-I~. C ELLULAR enlargement or hypertrophy plays a prominent role in the postnatal growth of many tissues, as well as in physiological and pathological hypertrophy of a variety of tissues (4). However, relatively little is known regarding the mechanisms that control cell size. Even less is known concerning the control mechanisms for the DNA endoreduplication and polyploidy that often accompanies cellular hypertrophy, although its occurrence is widespread in eukaryotic cells in vivo, occurring in terminally differentiated cardiac myocytes (14, 15, 29), and neurons (22), as well as in nonterminally differentiated hepatocytes (8), smooth muscle cells (10, 23, 30), and other cell types (see review by Brodsky and Uryvaeva [8]). Our laboratory has been particularly interested in the meDr. Komoriya's present address is Food and Drug Administration, Center for Biologic Evaluation and Research, Bethesda, MD 20892. Address reprint requests to Dr. Owens, Box 449, Department of Physiology, University of Virginia School of Medicine, Charlottesville, VA 22908. dial hypertrophy of smooth muscle that develops in large atmiles of hypertensive animals and humans (17, 23, 30) and has been implicated in the pathogenesis of this disease. Aortic hypertrophy is characterized by enlargement of existing smooth muscle cells with little or no change in cell number and is accompanied by development of polyploidy in a large fraction of smooth muscle cells (10, 23, 24, 30). Development of polyploidy in these cells does not appear to be due to an inherent loss in the capacity of cells to divide since tetraploid cells can be induced to proliferate in vitro in serum containing medium after isolation from intact vessels and separation on the basis of DNA content using a fluorescence activated cell sorter (19) or in vivo by subjecting vessels that contain polyploid cells to balloon embolectomy-induced injury (Owens, G. K., manuscript in preparation). This suggests that the growth response of smooth muscle cells, i.e., hypertrophy vs. hyperplasia, is a function of the nature of the growth stimulus. However, the precise factors that control hypertrophic vs. hyperplastic growth of smooth muscle cells © The Rockefeller University Press, 0021-9525/88/08/771/10 $2.00 The Journal of Cell Biology, Volume 107, August 1988 771-780 771 are not known and virtually nothing is known regarding the mechanisms responsible for development of polyploidy in this cell. There are several possible mechanisms to explain development of cellular hypertrophy. One hypothesis is that hypertrophy results from incomplete growth stimulation whereby the cell receives signals for the increased cell mass and DNA replication associated with cell cycle progression but not for cell division (5, 18). An alternative hypothesis, and the principal focus of the present study, is that cellular hypertrophy may be regulated, in part, by growth inhibitory factors that alter the pattern of the growth response to mitogens resulting in incomplete growth stimulation. An attractive candidate for a substance that might elicit the latter response in smooth muscle cells is transforming growth factor-13 (TGF-13). ~ This 25-kD polypeptide homodimer (2) has been shown to be a potent growth inhibitor for vascular smooth muscle cells (3) as well as a variety of other anchorage-dependent cells (32), and it has been suggested that it may be a mediator of smooth muscle cell growth in vivo during wound repair and atherogenesis (3, 12). Of particular interest, treatment of primary cultures of renal proximal tubule cells with BSC-1 growth inhibitor, which has similar biological properties as TGF-[I, is associated with development of cellular hypertrophy (16). The principal aim of the present study was to explore the possible mechanisms for development of hypertrophy and hyperploidy in vascular smooth muscle cells by characterizing the role of TGF-13 in control of hyperplastic growth of cultured rat aortic smooth muscle cells, and determining whether TGF-13-induced growth inhibition was associated with development of cellular hypertrophy and/or accumulation of cells with tetraploid DNA content. Materials and Methods

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@inproceedings{Owens2002TransformingGF, title={Transforming Growth Factor-J3-induced Growth Inhibition and Cellular Hypertrophy in Cultured Vascular Smooth Muscle Cells}, author={Kenneth Owens and A . A . T . Geisterfer and Yvonne Wei - Hwa Yang and Akira}, year={2002} }