DEVELOPMENT and DISEASE a Critical Role for Elastin Signaling in Vascular Morphogenesis and Disease

Development 130, 411-423 411 © 2003 The Company of Biologists Ltd doi:10.1242/dev.00223 DEVELOPMENT AND DISEASE A critical role for elastin signaling in vascular morphogenesis and disease Satyajit K. Karnik1,2,*, Benjamin S. Brooke1,2,*, Antonio Bayes-Genis3,*, Lise Sorensen1, Joshua D. Wythe1,2, Robert S. Schwartz4, Mark T. Keating5 and Dean Y. Li1,2,† 1Program in Human Molecular Biology and Genetics, University of Utah, Salt Lake City, UT, USA 2Department of Medicine and Oncological Science, University of Utah, Salt Lake City, UT, USA 3Hospital Sant Pau, Barcelona, Spain 4Minnesota Cardiovascular Research Institute, Minneapolis, MN, USA 5Howard Hughes Medical Institute, Department of Cell Biology, Harvard Medical School, Department of Cardiology, Children’s Hospital, Boston, MA, USA *These authors contributed equally to the work †Author for correspondence (e-mail: [email protected]) Accepted 19 October 2002 SUMMARY Vascular proliferative diseases such as atherosclerosis and elastin induces actin stress fiber organization, inhibits coronary restenosis are leading causes of morbidity and proliferation, regulates migration and signals via a non- mortality in developed nations. Common features integrin, heterotrimeric G-protein-coupled pathway. In a associated with these heterogeneous disorders involve porcine coronary model of restenosis, the therapeutic phenotypic modulation and subsequent abnormal delivery of exogenous elastin to injured vessels in vivo proliferation and migration of vascular smooth muscle cells significantly reduces neointimal formation. These findings into the arterial lumen, leading to neointimal formation indicate that elastin stabilizes the arterial structure by and vascular stenosis. This fibrocellular response has inducing a quiescent contractile state in vascular smooth largely been attributed to the release of multiple cytokines muscle cells. Together, this work demonstrates that and growth factors by inflammatory cells. Previously, we signaling pathways crucial for arterial morphogenesis can demonstrated that the disruption of the elastin matrix leads play an important role in the pathogenesis and treatment to defective arterial morphogenesis. Here, we propose that of vascular disease. elastin is a potent autocrine regulator of vascular smooth muscle cell activity and that this regulation is important for preventing fibrocellular pathology. Using vascular smooth Key words: Elastin matrix, Vascular smooth muscle, Morphogenesis, muscle cells from mice lacking elastin (Eln–/–), we show that G-protein signaling, Vascular proliferative diseases INTRODUCTION Schwartz, 1997). Understanding the molecular mechanisms that underlie vascular smooth muscle cell regulation is crucial Vascular proliferative diseases are a heterogeneous group of for developing strategies to prevent and treat vascular disorders that include atherosclerosis, coronary restenosis, proliferative disorders. bypass-graft failure and transplant arteriopathy that lead to Vascular smooth muscle cells are not terminally arterial narrowing, restriction of blood flow and tissue differentiated and can alternate between a quiescent, ischemia. These diseases arise from a complex contractile state and a proliferative non-contractile state pathophysiological response to injury by multiple factors, (Raines and Ross, 1993; Owens, 1998; Thyberg, 1998). In a including hypercholesterolemia, diabetes, smoking, healthy artery, vascular smooth muscle cells are quiescent and hypertension and mechanical injury (Ross, 1995; Lusis, largely comprise a contractile apparatus that functions to dilate 2000). Although their etiologies are diverse, these disorders and constrict the lumen as required by physiological demands. all share a common pathological feature: the accumulation of Actin stress fibers serve as the scaffold for the contractile vascular smooth muscle cells and fibrous connective tissue apparatus, and are a hallmark of mature and quiescent vascular within the intima between the endothelium and medial layer smooth muscle cells (Burridge and Chrzanowska-Wodnicka, of the vessel wall (Lusis, 2000; Raines and Ross, 1993). This 1996; Small and Gimona, 1998). Under circumstances of fibrocellular pathology, known as neointimal formation, injury, repair and regeneration, vascular smooth muscle cells results from the activation, proliferation and migration of lose their contractile apparatus and dedifferentiate into an vascular smooth muscle cells (Raines and Ross, 1993; immature phenotype capable of proliferating and depositing 412 S. K. Karnik and others extracellular matrix (Owens, 1998; Thyberg, 1998). This MATERIALS AND METHODS fibrocellular response plays an important role in all forms of vascular proliferative diseases. In atherosclerotic lesions, the Isolation of vascular smooth muscle cells major components of the fibrous plaque are vascular smooth Newborn (postnatal day 0.5) Eln+/+ and Eln–/– pups were sacrificed muscle cells, the matrix products deposited by these cells, and by CO2 asphyxiation. Their ascending aortas were dissected free, extracellular cholesterol (Ross, 1995; Lusis, 2000; Raines and endothelium removed and placed into individual wells of a plastic 12- Ross, 1993). In restenosis, transplant arteriopathy and vascular well culture dish containing Amniomax C-100 growth medium graft disease; smooth muscle cells are the predominant (Gibco-BRL) supplemented with penicillin/streptomycin. Cultures of smooth muscle cells that formed around the tissue were trypsinized component of the occlusive lesion (Raines and Ross, 1993). (0.05% trypsin, 0.53 mM EDTA), passaged, expanded and genotyped The phenotypic modulation of vascular smooth muscle cells to confirm their identity. Cells were stained with SM α-actin (Clone offers a tempting target for preventing vascular proliferative 1A4, Sigma) and checked for classic ‘hill-and-valley’ morphology diseases. to confirm their smooth muscle status. In situ hybridization, Elastin is the dominant extracellular matrix protein immunostaining and RT/PCR analysis established that these cells deposited in the arterial wall and can contribute up to 50% of expressed the vascular smooth muscle cell markers SM α-actin, SM its dry weight (Parks et al., 1993). The protein product of the γ-actin, SM-22, calponin, desmin, ang-1/ang-2, and did not express elastin gene is synthesized by vascular smooth muscle cells the endothelial markers Flk-1 or Flt-1. and secreted as a monomer, tropoelastin. After post- Cellular assays translational modification, tropoelastin is crosslinked and Cellular experiments of proliferation, actin polymerization and organized into elastin polymers that form concentric rings of migration described below were performed on at least three elastic lamellae around the arterial lumen. Each elastic lamella independently isolated primary cell lines. Proliferation was assayed alternates with a ring of smooth muscle, and provides the by cell count and [3H]thymidine incorporation. Confluent cultures of compliance that arteries need to absorb and transmit Eln+/+ and Eln–/– vascular smooth muscle cells from the fourth hemodynamic force (Wolinski et al., 1967). There is a growing passage were seeded at a density of 2×103 cells/well on a plastic body of evidence that implicates elastin in vascular 24-well plate (Corning Costar, Corning, NY), and stimulated to proliferate in AmnioMAX C-100 growth medium (Gibco-BRL) development and disease. We previously demonstrated that µ loss-of-function mutations of one elastin allele cause treated with 100 g/ml recombinant tropoelastin or left untreated. Cell numbers for each culture were assayed by hemocytometer after 24, supravalvular aortic stenosis (SVAS) and Williams-Beuren 48 and 72 hours of incubation. For [3H]thymidine incorporation, Syndrome (Li et al., 1997; Curran et al., 1993; Ewart et al., Eln+/+ and Eln–/– cells were seeded at a density of 20,000 cells/well 1993). These disorders are characterized by discrete on a 24-well plate. After attachment, cells were starved in 0.1% BSA fibrocellular stenoses in the aorta, coronary arteries, carotid (Fisher) in Amniomax Basal Medium (Gibco) for 24 hours. Cells were arteries, pulmonary arteries and other peripheral arteries. then grown in whole Amniomax medium treated/untreated with 100 Often, individuals with these diseases are young children who µg/ml tropoelastin (Grosso et al., 1991a). Cells were assayed for are susceptible to peripheral vascular disease, myocardial [3H]thymidine incorporation using a scintillation counter after 24 infarctions or stroke in the absence of other risk factors such hours by precipitating with 5% TCA, followed by NaOH ± as high serum cholesterol, diabetes and cigarette smoking (van solubilization. Data were calculated as mean s.d. (n=6). Son et al., 1994). In subsequent experiments, we showed that Subconfluent cultures of vascular smooth muscle cells were evaluated and scored for the presence of actin stress fibers in the the deposition of elastin matrix in the arterial wall during late cytoplasm following immunofluorescent staining for SM α-actin fetal development is essential to arterial morphogenesis (Li et (Clone 1A4, Sigma), vinculin (Clone hVin-1, Sigma), desmin (Sigma) –/– al., 1998). Mice that lack elastin (Eln ) died from occlusive and tubulin (Clone DM-1A, Calbiochem). Cells were treated with fibrocellular pathology caused by subendothelial proliferation tropoelastin (1 µg/ml) (Grosso et al., 1991a) or not, and assays were and accumulation of vascular smooth muscle cells in early performed

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