Stretch affects the vascular smooth muscle cell phenotype and cytoskeleton: implications for vascular tissue engineering

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14th IVBM Abstracts

conductance in human aortic endothelial (HAECs) cells are inwardly-rectifying K+ channels (Kir). We have also shown that Kir channels are significantly suppressed by exposing the cells to hypercholesterolemic environment in vitro and in vivo and that removing cellular cholesterol rescues the currents. In this study, we show that hypercholesterolemia not only suppresses the basal activity of Kir but also strongly suppresses the shearstress sensitivity of these channels and virtually abolishes shear stress-induced hyperpolarization. The activity of endothelial Kir channels and the membrane potentials were measured electrophysiologically using whole cell voltage clamp or current clamp respectively. The shear stress sensitivity of the Kir channels was tested under well-defined flow conditions using a Minimally Invasive Flow device (MIF) that has been developed in our earlier studies to allow electrophysiological recordings to be performed in a controlled flow environment. In general, as expected, introduction of shear stress of 0.5–2 dyne/cm2 induced a reversible increase in the current amplitude ranging from ∼ 4% to ∼ 10% and a shift in membrane potential to the more negative values. However, enriching HAECs with cholesterol either by incubating the cells with cholesterolsaturated methyl-methyl-cyclodextrin or by exposing the cells to VLDL resulted in significant suppression of the shear stressinduced Kir component (KirSS). Specifically, exposing HAECs to 50 ug/ml VLDL for 24 h resulted in a ∼ 40% increase in cellular cholesterol level and in a more than 2-fold decrease in the amplitude of KirSS. doi:10.1016/j.vph.2006.08.151

A7.17

systems, we investigated the effect of strain inflicted upon SMC by culture in three-dimensional (3D) collagen matrix of different rigidity on their susceptibility to apoptosis. Culture of human SMC within relaxed/floating 3D-collagen gels served as an in vitro system of low rigidity. Under these conditions, SMC underwent apoptosis, which was preceded by downregulation of the IAPs survivin, xIAP and c-IAP1 at the protein and mRNA level. In comparison, SMC cultured in restrained/ non-floating 3D-collagen gels neither down-regulated IAPs nor underwent apoptosis. Thus the amount of tension caused by the degree of collagen matrix rigidity may be regulating IAP gene expression and apoptosis in SMC. Not only rigidity-mediated tension but also exogenously applied strain had an effect on IAPs: exposure of SMC cultured on flexible silicone membranes coated with monomeric type I collagen to 7.5% biaxial strain of 1 Hz lead to a dramatic induction of survivin, xIAP and c-IAP2 compared to SMC under static conditions. Thus IAPs may be novel mechano-responsive genes regulated by 3Dmatrix tension/rigidity as well as exogenous strain. This may be a novel mechanism of cytoprotection of SMC against apoptosis in the vessel wall. doi:10.1016/j.vph.2006.08.153

A7.19 Stretch affects the vascular smooth muscle cell phenotype and cytoskeleton: implications for vascular tissue engineering A.T. Halka1, N.J. Turner1, M.O. Murphy1, A. Carter1, J. Ghosh1, N. Khwaja1, C.M. Kielty2, M.G. Walker1

Withdrawn 1

doi:10.1016/j.vph.2006.08.152

Department of Vascular Surgery, Manchester Royal Infirmary, Manchester, United Kingdom 2 UK Centre for Tissue Engineering, University of Manchester, Manchester, United Kingdom

A7.18 Rigidity of collagen governs apoptosis and expression of inhibitor of apoptosis proteins (IAPS) in vascular smooth muscle cells Karin von Wnuck Lipinski, Bodo Levkau Institute of Pathophysiology, University Hospital, Essen, Germany Mechanical forces are crucial for the regulation of cell morphology and function in vascular homeostasis and, when deregulated, contribute to cardiovascular pathologies such as atherosclerosis and arterial remodeling. In the vessel wall, vascular smooth muscle cells (SMC) are normally subjected to cyclic mechanical strain due to the pulsative nature of blood flow. As mechanotransduction influences cytoskeletal organization, gene expression and survival in a number of cellular

According to the British Heart Foundation cardiovascular disease causes 40% of all deaths in the United Kingdom; more than all types of cancer combined. Surgical intervention remains the mainstay of treatment but suitable bypass conduits are not always available hence the need for the development of grafts using vascular tissue engineering techniques. Vascular smooth muscle cells (VSMC) would be a crucial component of an engineered blood vessel as they would assist with extracellular matrix deposition and vessel stability. VSMC possess the ability to alter their phenotype depending on the stimuli to which they are exposed. In vivo the arterial wall is mainly exposed to circumferential and longitudinal stretch. Little is understood about the effects of various degrees of stretch on the VSMC phenotype and cytoskeleton. Human VSMC, cultured on silicone membranes pre-coated with type IV collagen or laminin, were manipulated for varying time periods with two magnitudes of stretch using the Flexercell System. VSMC were prepared for immunofluorescent examination or had their RNA

14th IVBM Abstracts

or protein extracted. RNA was analysed by reverse transcriptase polymerase chain reactions using primers for á-smooth muscle actin, smooth muscle myosin heavy chain and h1-calponin. Extracted protein was analysed by Western blotting using antibodies for the aforementioned VSMC markers. The coating on which the VSMC were cultured affected their morphology. When exposed to varying degrees of stretch the cells altered their orientation as well as the organisation of their α-smooth muscle actin. Protein analysis demonstrated down-regulation of VSMC markers, in particular α-smooth muscle actin, when VSMC were exposed to pathological magnitudes of stretch. The converse was the case for more physiological magnitudes of stretch. By stretching the VSMC in vitro we influenced the expression of markers which may signal the initiation of phenotypic modification. Mechanical forces could provide a mechanism of controlling VSMC plasticity which may influence the development of pathology such as neointimal hyperplasia and also contribute to the successful engineering of a vascular graft. doi:10.1016/j.vph.2006.08.154

A7.20 The role of c-jun in flow dependent and flow independent restenosis after angioplasty and stenting MJ Murrell1, L Khachigian2, MR Ward1 1 2

Royal North Shore Hospital, Sydney, Australia University of New South Wales, Sydney, Australia

Restenosis after balloon angioplasty and stenting is exacerbated by low flow in a redox-sensitive manner. We

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have previously shown that c-jun expression is enhanced by low flow and injury (stent > balloon > uninjured) and inhibited by the antioxidant PDTC. We investigated the role of c-jun in key events in restenosis, intimal hyperplasia and arterial remodeling, in the presence and absence of low flow. We performed over-sized (1.3–1.5:1) stenting (S) and balloon injury (B), in adjacent segments of the carotid arteries of cholesterol fed rabbits subjected to either low flow or normal flow, and then locally delivered DZ13, (a DNAzyme specific for c-jun) or scrambled DZ13 (inactive DNAzyme) via a microporous weeping catheter. Vessel areas were compared to those in uninjured segments (U) in the same artery and those from vessels undergoing injury without local delivery by histomorphometry at 28 days. Neointima formation occurred in all groups as the passage of the catheter through the proximal vessel showed evidence of injury to the U vessel. Low flow significantly increased intimal hyperplasia in B and S relative to normal flow (p < 0.05). Passage of the bulky local delivery catheter caused a proportional increase (p < 0.001) in intimal hyperplasia in both normal and low flow vessels. The active DNAzyme DZ13 markedly reduced intimal hyperplasia (p < 0.001) and increased lumen size (p < 0.05) in balloon injured vessels subject to low flow. Interestingly, however, in stented vessels there was no apparent effect of DZ13 on intimal hyperplasia. These studies show that that specific inhibition of c-jun with the DNAzyme DZ13 markedly inhibits intimal hyperplasia and restenosis in balloon injured but not stented vessels. The previously noted accentuation of restenosis with low flow is also abrogated by DZ13 in balloon injured but not stented vessels. DZ13 may be useful for restenosis after angioplasty but not after stenting. doi:10.1016/j.vph.2006.08.155