Effects of transforming growth factor-β1 on human arterial smooth muscle cells in vitro

Abstract

Control of the thickness of the arterial wall is critical, as excessive overgrowth of constituent smooth muscle cells (SMCs) may interfere with blood flow. Effects on SMCs in vitro of several growth factors that are present in blood and/or that are produced endogenously in the arterial wall under certain conditions suggest that influences of endocrine, paracrine, and autocrine nature from stimulating and inhibiting factors may control the smooth muscle tissue mass in the artery. This possibility was explored further by investigating the degree of myodifferentiation in terms of the presence of differentiation-specific filamentous α-smooth muscle actin and growth, as measured by the synthesis of DNA and cell number, of SMCs as influenced by their exposure to the mitogens, platelet-derived growth factor and epidermal growth factor, and the bifunctional growth factor, transforming growth factor-β1 (TGF-β1). Exposure to TGF-β1 markedly enhanced differentiation-specific filamentous α-smooth muscle actin. This effect did not require arrest of growth, which speaks against a direct causal relation between loss of myodifferentiation (modulation) and multiplication. When quiescent cultures were exposed to TGF-β1, α-smooth muscle actin was further increased, indicating a more specific differentiation-promoting effect by TGF-β1 than mere inhibition of growth. Exposure to TGF-β1 also increased spreading, which occurred in parallel with increased filamentous α-smooth muscle actin and appearance of stress fibers. Exposure to platelet-derived growth factor under serum-free conditions and to epidermal growth factor in cultures exposed to serum markedly decreased the number of α-actin-positive SMCs, indicating a dedifferentiating effect by these mitogens. Exposure of SMCs to TGF-β1 under serum-free conditions had pronounced effects on growth, with a concentration-dependent inhibition of platelet-derived growth factor-induced DNA synthesis and cell multiplication. The basal synthesis of DNA in the absence of added growth factors was also greatly inhibited. With serum-free cultures, some loss of cells occurred even with very low concentrations of TGF-β1 (5 pg/ml), against which platelet-derived growth factor or a dense cultural state had a protective effect. Enhancement of cell multiplication was not detected for cultivated human SMCs exposed to TGF-β1, irrespective of culture density, in contrast to that reported for dense cultures of rat SMCs. TGF-β1 is present in and may be released from platelets in situations that promote platelet adherence such as endothelial injury; TGF-β1 may also be released from activated macrophages and T lymphocytes either during an immune reaction or inflammation or from the endothelium. The pronounced effects in vitro of this factor with promotion of myodifferentiation and inhibition of growth of SMCs could be consistent with a role for this factor of regulation of SMC growth and myodifferentiation in the arterial wall normally, during inflammation, and in atherosclerosis.