In Vitro Ceil.Dev.Biol.~Anima135:647-654, November-December 1999 © 1999 Society for In Vitro Biology 1071-2690/99 $05.00 + 0.00 HEPARIN/ENDOTHELIAL CELL GROWTH SUPPLEMENT REGULATES MATRIX GENE EXPRESSION AND PROLONGS LIFE SPAN OF VASCULAR SMOOTH MUSCLE CELLS THROUGH MODULATION OF INTERLEUKIN-1 JU-YU HSU, 1 MEI-YU HSU, THOMAS SORGER/MEENHARD HERLYN, ANDELLIOT M. LEVINE 3 The Wistar Institute, 3601 Spruce Street, Philadelphia, Pennsylvania 19104-4268 (Received 26 December 1998; accepted 12 June 1999) SUMMARY Vascular smooth muscle cells produce and respond to interleukin-1, a cytokine which modifies inflammation-associated vascular activities including the synthesis of extracellular matrix proteins. We have established vascular smooth muscle ceils culture conditions in which heparin, in the presence of endothelial cell growth supplement, promotes cell proliferation and inhibits interleukin-1 and matrix protein expression. To test whether interleukin-1 mediates growth and matrix mod- ulation by heparin/endothelial cell growth supplement, vascular smooth muscle cells were transfected with an Epstein-Barr virus-derived expression vector designed to express interleukin-1 antisense transcripts. RNase protection and ELISA assays demonstrated a complete block of interleukin-1 transcription and protein synthesis. Northern blot analysis also showed that interleukin-1 antisense decreased the expression of matrix genes such as type I collagen, fibronectin, and decorin similar to downregulation after heparin/endothelial cell growth supplement treatment. In contrast, the expression of versican was not affected, indicating a selective suppression of matrix proteins. In addition, interlenkin-1 antisense significantly prolonged the life span of vascular smooth muscle cells in culture. Our data suggest that heparin/endothelial cell growth supplement induces matrix remodeling and controls growth and senescence of vascular smooth muscle cells through down- regulation of interleukin-1. Key words: matrix; antisense; vascular smooth muscle; heparin. INTRODUCTION Atherosclerosis involves an aberrant proliferation of vascular smooth muscle ceils (VSMC) and inappropriate accumulation of ex- tracellular matrix (ECM). The identification of factors regulating this pathophysiological process is essential for the design of rational ap- proaches for prevention and therapy. Heparin, a highly charged gly- cosaminoglycan synthesized by VSMC (58) exerts diverse effects on VSMC properties, such as migration (19,39), proliferation (10,20), protease activity (23,24) and elaboration of ECM proteins including collagen (38,50-52), fibronectin (51), decorin (51), thrombospondin (34,35), elastin (55) and proteoglycans (9,52,59). Some regulatory actions, such as on ECM composition, vary according to cell culture characteristics including species of origin, cell density, state of con- iluence, serum concentration, and prior cultivation in the presence of mitogens (22,25,40,48,51,52). Other effects, for example on pro- liferation, are either stimulatory or inhibitory depending on the mix of interacting mitogens, such as fibroblast growth factors (FGFs) and cytokines (12). Consistent with this, we have shown previously that heparin promotes proliferation, alters matrix production, and extends the life span of human VSMC cultures in the presence of endothelial Tresent address: Sidney KimmelCancer Center, 10835 Altman Row, Suite 240, San Diego, California 92121. 2present address: Department of Biology, Roger Williams University, One OId Fel~ Road, Bristol, Rhode Island 02809. ZTowhom correspondence should be addressed. cell growth supplement (ECGS), composed mostly of acidic FGF (aFGF) and basic FGF (bFGF) (48,49). Nevertheless, it remained unclear whether heparin functions directly or through a mediator. In the present study, we attempted to clarify the underlying mechanisms by which heparin, in combination with FGFs, exerts its effects upon VSMC. The pleiotropic inflammatory cytokine interleukin-1 (IL-1) is a critical mediator of vascular pathophysiology (13,14). Human VSMC are not only the targets but also the sources of IL-1 (30,56). IL-1 regulates proliferation (6,21,29), contractility (3), expression of cy- tokines (32,60), and extracellular matrix (ECM) synthesis (1) in hu- man VSMC. In our study, we report a coordinate downregulation of IL-1 and ECM in heparin/ECGS-treated VSMC, suggesting that hep- arin in the presence of ECGS modulates matrix gene expression via IL-1 downregulation. To investigate this hypothesis, we downregu- lated IL-1 expression in VSMC by antisense RNA generated in IL- l antisense stable transfectants. These transfectants exhibited a pro- longed life span and constitutive inhibition of selected ECM proteins, an expression pattern paralleling the combined effect of heparin and ECGS in untransfected VSMC. Therefore, we conclude that IL-1 me- diates heparin/ECGS-induced matrix remodeling and induces de- layed quiescence in VSMC. MATERIALSAND METHODS Cells and .u~dia. Human VSMC (HIVS 125) were isolated from the iliac vein of a 21-yr-old male Caucasian cadaver donor (48). Cells were cultured 647