P337	Conditional ablation of vascular smooth muscle cells: direct effects of cell death on normal arteries and in atherosclerosis.
1M.Clarke, 1N.Figg, 2J.Maguire, 2A.Davenport, 3M.Goddard, 1T.Littlewood, 1M.Bennett
1Cardiovascular Medicine, Cambridge, GB; 2Clinical Pharmacology Unit, Cambridge, GB; 3Papworth Hospital, Cambridge, GB.

Apoptosis of vascular smooth muscle cells (VSMCs) is evident in normal artery development, and disease states such as aneurysm formation, restenosis after injury and atherosclerosis, and may promote vessel remodelling, plaque instability, coagulation and inflammation. However, the precise contribution of VSMC apoptosis to disease is unknown since cell death accompanies other pathological changes, such as alteration to flow or vessel injury. To examine the effects of VSMC apoptosis in vivo, we generated transgenic mice expressing the human diphtheria toxin receptor (hDTR) from a VSMC-specific murine minimal SM22α promoter. Administration of DT induced apoptosis in VSMCs in vitro and in vivo with a loss of VSMCs in large and medium-sized arteries. Vessel Control 28 d. DT 28 d. DT + 14 d. Thoracic Aorta 4976 ± 90 1806 ± 687 2220 ± 578 Abdominal Aorta 4899 ± 767 2477 ± 532 2387 ± 551 Aorta Bifurcation 4626 ± 591 3154 ± 265 2628 ± 1161 Carotid 5045 ± 144 3150 ± 449 2805 ± 461 Brachiocephalic 5182 ± 57 2413 ± 321 2835 ± 824 Table 1. Treatment of SM22α-DTR mice with DT at 5ng/g for 28d, or 28d with a 14d respite. Cellularity is expressed as total cells per mm2 of vessel wall ± S.D., n=7. Difference between control and both ablated groups is significant (p=0.001), difference between ablated groups is not (p=0.95). Surprisingly, despite a loss of 50-70% of VSMCs, vessels showed no inflammatory infiltrate, reactive proliferation, thrombosis or coagulation, and no evidence of vessel remodelling or aneurysm formation. Apoptotic bodies were rapidly cleared and vessels showed no medial repopulation on stopping DT treatment. In addition, passive mechanical and active constrictor responses both to receptor-dependent and independent stimuli were unaffected. To examine the effect of VSMC apoptosis on atherosclerosis, SM22α-DTR/ApoE-/- mice were generated, fed a Western diet for 8 weeks, and subsequently received DT. DT induced VSMC loss from the fibrous caps of plaques, accumulation of cell debris, and localised inflammation. Importantly, all of the SM22α-DTR/ApoE-/- mice died within a 24h time period 3 weeks after DT administration, whilst none of the control mice were affected. We conclude that in the absence of other stimuli VSMC apoptosis is a silent phenomenon, whereby normal arteries demonstrate a remarkable capacity to withstand cell loss and compensatory mechanisms to minimise its effect. In contrast, VSMC apoptosis in atherosclerosis induces features of vulnerable plaques.

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