Fibroblast Growth Factor-2 Expression Is Altered in Lambs With Increased Pulmonary Blood Flow and Pulmonary Hypertension

0031-3998/07/6101-0032 PEDIATRIC RESEARCH Copyright © 2006 International Pediatric Research Foundation, Inc. Vol. 61, No. 1, 2007 Printed in U.S.A. Fibroblast Growth Factor-2 Expression Is Altered in Lambs With Increased Pulmonary Blood Flow and Pulmonary Hypertension STEPHEN WEDGWOOD, JENNIFER M. DEVOL, ALBERT GROBE, EILEEN BENAVIDEZ, ANTHONY AZAKIE, JEFFREY R. FINEMAN, AND STEPHEN M. BLACK Department of Pediatrics [S.W., J.M.D.], Northwestern University, Chicago, Illinois 60611; Department of Biomedical and Pharmaceutical Sciences [A.G., E.B., S.M.B.], University of Montana, Missoula, Montana 59802; Departments of Surgery [A.A.] and Pediatrics [J.R.F.], University of California–San Francisco, San Francisco, California 94143 Recent studies have demonstrated that shunt lambs display abnormal signaling by several growth factors mitogenic for vascular smooth muscle, including ET-1 (6), transforming growth factor ␤-1 (TGF ␤-1) (8), and vascular endothelial growth factor (VEGF) (9). Another potential contributor to vascular remodeling is FGF-2. FGF-2 displays mitogenic effects in the early proliferation of SMC (10,11) and in neointimal thickening (12) following vascular injury. Furthermore, a progressive increase in FGF-2 protein within the smooth muscle layer of pulmonary arteries was demonstrated in a rat model of monocrotaline-induced pulmonary hypertension (13). In addition, elevated FGF-2 protein levels were detected in the urine and plasma of patients with pulmonary arterial hypertension, suggesting the involvement of FGF-2 in the SMC proliferation characteristic of this disease (14). Vascular cells within arterial walls are subjected to biomechanical forces, including cyclic stretch generated by pulsatile blood flow. EC are also subjected to shear stress due to blood flow, and fluid dynamic models of intact blood vessels suggest that SMC also experience shear stress as a result of interstitial flow (15). Cyclic stretch increased FGF-2 mRNA in PASMC (16) and shear stress increased the release of FGF-2 from aortic SMC (17) and aortic EC (18). Since these biomechanical forces are increased in shunt pulmonary arteries due to elevated pulmonary blood flow, we hypothesized that similar to monocrotaline-induced and adult pulmonary hypertension, increased FGF-2 signaling may be associated with pulmonary vascular remodeling in shunt lambs. Thus, the purpose of this study was to monitor FGF-2 expression in shunt lambs, to determine the effects of cyclic stretch and shear stress on FGF-2 expression in PASMC and PAEC, and to determine the effects of exogenous FGF-2 on PASMC proliferation. ABSTRACT: A lamb model of pulmonary hypertension, developed by inserting an aortopulmonary vascular graft (shunt), displays vascular remodeling and increased pulmonary blood flow characteristic of children with congenital heart disease. The purpose of this study was to determine whether expression of fibroblast growth factor-2 (FGF-2), a smooth muscle cell mitogen, is altered in shunt lambs. FGF-2 mRNA and protein levels were increased in lung tissue extracts from shunt lambs at 4 wk of age relative to age-matched controls (p ⬍ 0.05). FGF-2 protein levels were also increased in the pulmonary arteries and serum of shunt lambs (p ⬍ 0.05). Pulmonary arterial smooth muscle cells (PASMC) and endothelial cells (PAEC) were isolated from 4 wk-old lambs and subjected to cyclic stretch and laminar shear stress to mimic increased pulmonary blood flow. Stretch and shear increased FGF-2 promoter activity, and intracellular and extracellular FGF-2 protein levels in both cell types (p ⬍ 0.05). Exogenous FGF-2 stimulated PASMC proliferation at levels detected in the extracellular medium of sheared cells (p ⬍ 0.05). Elevated FGF-2 signaling by PASMC and PAEC exposed to increased pulmonary blood flow may play a role in the pulmonary vascular remodeling associated with the shunt model of pulmonary hypertension secondary to congenital heart disease. (Pediatr Res 61: 32–36, 2007) C HD with increased pulmonary blood flow is often associated with the development of pulmonary hypertension (1). After birth, as pulmonary vascular resistance normally decreases, the presence of a systemic to pulmonary communication generates an increase in pulmonary blood flow. This abnormal postnatal hemodynamic state results in progressive structural and functional abnormalities of the pulmonary vascular bed (2,3). Our animal model of pulmonary hypertension, developed by inserting an aortopulmonary vascular graft in the late-gestational fetal lamb (4 – 6), may help elucidate the mechanisms involved. Postnatally, these shunt lambs have increased pulmonary blood flow and pressure (4). In addition, they display vascular remodeling typical of pulmonary hypertension secondary to CHD, characterized by increased medial wall thickness of the small pulmonary arteries and abnormal extension of muscle to peripheral pulmonary arteries (1,4,7). MATERIALS AND METHODS Surgical preparation and care. Twenty mixed-breed Western pregnant ewes (137–141 d gestation, term ⫽ 145 d) were operated on under sterile conditions to insert an 8.0-mm Gore-tex vascular graft (2 mm length; W.L. Gore, Milpitas, CA) between the ascending aorta and main pulmonary artery of the fetus as previously described (4). Unoperated twin fetuses served as controls, because we have previously shown that unoperated and sham- Received June 20, 2006; accepted August 17, 2006. Correspondence: Stephen Wedgwood, Ph.D., Ward 12-189, 303 E. Chicago Ave., Chicago, IL 60611; e-mail: Supported, in part, by grant 0330292N from the American Heart Association National Office (SW) and grants HL60190 (SMB), HL67841 (SMB), HL072123 (SMB), HL070061 (SMB), and HL61284 (JRF) from the National Institutes of Health. Abbreviations: CHD, congenital heart disease; EC, endothelial cells; ET-1, endothelin-1; ETA, endothelin A receptor; FGF-2, fibroblast growth factor-2; PAEC, pulmonary arterial endothelial cells; PASMC, pulmonary arterial smooth muscle cells; SMC, smooth muscle cells DOI: 10.1203/01.pdr.0000250013.77008.28 32 33 ELEVATED FGF-2 IN PULMONARY HYPERTENSION operated control lambs have similar physiology and morphology (4). Lambs were delivered spontaneously, and at 4 wk of age were killed by an intravenous injection of pentobarbital sodium (Euthanasia CII; Central City Medical, Union City, CA) followed by bilateral thoracotomy. All procedures and protocols were approved by the Committee on Animal Research of the University of California, San Francisco. Tissue/RNA/protein preparation. The heart and lungs were removed en bloc. Two to three gram sections from each lobe of the lung and sections from third- to fifth-generation intralobar pulmonary arteries and veins, with inside diameters of 0.5–2.0 mm, were removed. These tissues were snap-frozen in liquid nitrogen and stored at – 80°C until used. RNA and protein were isolated from snap-frozen lung tissue and analyzed by RNAase protect (...truncated)