96 Cardiovascular mechanics in newborn ELN+/+, +/- and-/- mice Jessica E. Wagenseil a , Attila Kovacs b , Robert P. Mecham a a Department of Cell Biology and Physiology, Washington University, St. Louis, MO, United States b Department of Internal Medicine, Washington University, St. Louis, MO, United States ELN+/- mice have increased blood pressure, smaller vessels and increased elastic lamellae. ELN-/- mice die soon after birth due to occlusive proliferation of smooth muscle cells (SMCs). In both cases, decreased elastin levels and altered mechanical signals may influence SMC phenotype. We have measured blood pressure, aortic wall structure and aortic mechanical properties of ELN+/+, +/- and-/- mice on postnatal day 0 (P0). Blood pressure in P0 ELN-/- mice is higher than ELN+/+ and +/-. Aortic inner diameter is significantly different between all genotypes, being smallest in ELN-/-, then ELN+/- and largest in ELN+/+. P0 ELN-/- aorta show disorganized, proliferating SMCs at the intimal surface, instead of the circumferentially organized layers seen in ELN+/+. ELN+/- aorta show elastin producing SMCs in the adventitia, instead of collagen producing fibroblasts as in ELN+/+. Adventitial progenitor cells can be stimulated to differentiate into elastin producing myofibroblasts after stretch induced injury. ELN-/- aorta have lower circumferential stresses at similar pressures compared to ELN+/+ and +/-, but almost identical stresses at physiologic pressure. ELN+/- aorta have increased circumferential stretch ratios at similar pressures compared to ELN+/+ and-/-. At P0, the characteristics of ELN-/- and +/- aorta suggest that circumferential stress induces SMCs to proliferate, while circumferential stretch induces progenitor cells to differentiate. This hypothesis will be investigated through similar studies at additional developmental time points. doi:10.1016/j.matbio.2008.09.311 97 HtrA1-a serine protease that regulates vascular calcification Colette A. Inkson, Kristen D. Hadfield, Fiona L. Wilkinson, Gillian A. Wallis, Ann E. Canfield Wellcome Trust Centre for Cell-Matrix Research, University of Manchester, UK Calcification of blood vessels is highly correlated with increased morbidity and mortality in patients with atherosclerosis, diabetes and chronic kidney disease. To identify factors involved in vascular calcification we performed differential analysis of mineralizing pericytes and identified HtrA1 (High Temperature Requirement protein A) a secreted protein with serine protease activity. Recent data from our lab and others suggests that HtrA1 is involved in the development, pathogenesis and mineralization of skeletal tissues. Therefore, this study tested the hypothesis that HtrA1 also regulates mineralization in the vasculature. Analysis of pericyte and vascular smooth muscle cell (VSMC) cultures revealed that HtrA1 is expressed by these cells and is down-regulated when mineralization occurs. Immunolocalization demonstrated that HtrA1 is expressed at sites of mineralization in human atherosclerotic arteries, co- localizing with matrix Gla protein (MGP) and osteopontin. To determine if HtrA1 regulates mineral deposition, VMSC were treated with recombinant HtrA1 (rHtrA1); these cells exhibited significantly less matrix mineralization compared to controls. To analyse the effect of HtrA1 protease activity on VSMC a Ser to Ala mutation of the active site triad was introduced; this protease inactive HtrA1 does not inhibit mineralization. Finally, we have shown that rHtrA1 cleaves elastin, decorin and matrix Gla protein. Together these results suggest that HtrA1 regulates mineral deposition by VSMC and experiments are currently in progress to determine the mechanism by which this occurs. doi:10.1016/j.matbio.2008.09.312 98 Vascular specific gene expression using the PDGFR-beta promoter Renee J. LeClair, Qiaozeng Wang, Lucy Liaw, Volkhard Lindner Center for Molecular Medicine, Maine Medical Center Research Institute, Scarborough, ME 04074, United States The platelet-derived growth factor (PDGF) signaling pathway is of particular relevance to vascular biology as it regulates mural cell recruitment and smooth muscle cell migration. Currently many promoters used to drive expression in vascular smooth muscle in vivo have decreased activity in dedifferentiated SMC of the neointima. To overcome these limitations we generated transgenic mice with Cre recombinase under the control of the PDGF receptor-β(PDGFR-β) promoter, which displays increased activity following vascular injury. 19 transgenic founder lines were generated in the ROSA26 background for characterization of transgene expression by LacZ staining in embryonic and postnatal tissues as well as after vascular injury. Remarkable differences between transgenic lines with respect to transgene activity in different tissues were observed. Strong expression was commonly observed in the cartilage, sub epidermal mesenchyme, vascular smooth muscle in several organ beds, kidney mesenchymal cells and in the brain. Unique lines were identified showing high expression only in the smooth muscle cells, vascular endothelium and inner and outer granular layers of the brain. Another line had strict expression in the vascular smooth muscle, collecting ducts and mesenchymal cells of the kidney. After vascular injury, promoter activity was either unchanged or enhanced relative to basal levels of activity in large vessels.Generation of these transgenic animals is an important tool for vascular bed specific targeting of gene deletion and over expression. These lines will be especially useful for directing gene expression or deletion during vascular remodeling by overcoming the limitations of current Cre driven vascular promoters. doi:10.1016/j.matbio.2008.09.313 99 Fbln-2 and fbln-5 cooperate to assemble and maintain the IEL Shelby L. Chapman a , F.-X. Sicot b , Elaine C. Davis c , Takako Sasaki d , Mon-Li Chu b , Hiromi Yanagisawa a a Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX, USA b Department of Dermatology and Cutaneous Biology, Thomas Jefferson University, Philadelphia, PA, USA c Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec, Canada d Shriners Hospital for Children Research Center, Portland, Oregon, USA Recent findings on the role of fibulin-5 have provided substantial progress in understanding the molecular mechanism of elastic fiber assembly in vitro. However, little is known about the differential roles of fibulin proteins in the formation of elastic fibers in blood vessels. Here, we test the involvement of fibulin-2 and fibulin-5 in the formation of the internal elastic lamina (IEL) in vivo. Fibulin-2 is distinctly expressed in the IEL, whereas fibulin-5 is observed throughout the vessel wall. Although fibulin-2 and fibulin-5 exhibited strong binding to tropoelastin, no interaction was observed between fibulin-2 and fibulin-5. We generated double knockout mice for the fibulin-2 and fibulin-5 genes (termed DKO) and found that the IEL was severely disorganized in DKO mice. Furthermore, DKO vessels displayed abnormal remodeling, thrombus formation and aneurysmal change after carotid artery ligation-injury, under- scoring the importance of the IEL in maintenance of the integrity of the vessel wall. In conclusion, fibulin-2 and fibulin-5 cooperatively function to form the IEL during postnatal development by directing assembly of elastic fibers, and are responsible for maintenance of adult vessel walls after injury. In additon, the DKO mouse will serve as a unique animal model to test the effect of vessel integrity during various pathological insults. doi:10.1016/j.matbio.2008.09.314 100 WITHDRAWN TG2, the missing link in the development of vascular stiffness? Simon Moreau a , Nicolas Chabot b , Jeffrey Wayne Keillor b , Pierre Moreau a University of Montreal, Faculty of Pharmacy, United States b University of Montreal, Department of Chemistry, United States Abstracts S33