Hyperreactivity to vasoconstrictors in aorta from ApoE -/- mice on high fat diet is rescued by apoptotic ablation of vascular smooth muscle cells

1 J.J. Maguire, 2M.C.H. Clarke, 2N. Figg, 2M.R Bennett & 1A.P. Davenport, 1Clinical Pharmacology Unit and 2Division of Cardiovascular Medicine, University of Cambridge, Level 6 Centre for Clinical Investigation, Box 110 Addenbrooke’s Hospital, Cambridge, CB2 2QQ, UK.

Apoptosis of vascular smooth muscle cells (VSMCs) occurs in human cardiovascular diseases such as atherosclerosis. We have previously shown that VSMC loss per se had no effect on vascular reactivity to constrictors in aorta from SM22αDTR ablated transgenic mice. This novel murine model of targeted VSMC ablation exhibited an ~50% loss of medial VSMCs in their large and medium arteries in response to diphtheria toxin (DT) treatment compared to untreated SM22αDTR controls (Clarke et al., 2006). We have now extended this investigation to determine vasoconstrictor responses in the ApoE-/- mouse model of atherosclerosis and to identify the consequence of VSMC loss in this model by generation of SM22αDTR/ApoE-/- mice.

Male or female ApoE-/- or SM22αDTR/ApoE-/- mice (20 weeks old) were placed on a high (21%) fat diet for 12 weeks. For the last nine weeks all animals received 1ng/g diphtheria toxin i.p. to trigger vascular smooth muscle apoptosis in the responsive SM22αDTR/ApoE-/- animals. Mice were killed by CO2 inhalation and adjacent segments of thoracic aorta (1-2mm) were set up in wire myographs for isometric tension recordings in Krebs’ solution (37°C). Following normalisation, cumulative concentration-response curves were constructed to phenylephrine (PE, 1x10 -9-3x10-5M) and the thromboxane mimetic U-46619 (1x10-10-1x10-6M). Constrictor responses were analysed using the curve-fitting programme FigSys (Biosoft, Cambridge, UK) with potency (pD2) and maximum response (Emax, mN/mm) expressed as mean±s.e.mean. Data for the two groups were compared using Student’s 2-tailed t-test. Significance was set at P<0.05. n-Values are the number of mice.

Normalised internal diameters and basal tension of aortic segments from ApoE-/- mice (1501.5±37.3μm; 5.89±0.10mN/mm n=6) were not different from SM22αDTR/ApoE-/- animals (1423.5±33.0μm; 5.69±0.16 mN/mm n=6; P>0.05). ApoE-/- mice showed enhanced sensitivity to PE and U46619 compared to previous data from ablated and non-ablated SM22αDTR mice. However, SM22αDTR/ApoE-/- exhibited a significantly (P<0.05) normalised agonist sensitivity compared to the ApoE-/- group (Table 1). There were no significant differences between Emax values.

Table 1. Potency (pD2) and maximum response (Emax) to constrictor agonists. *P<0.05 SM22αDTR/ApoE-/- compared to ApoE-/-, Student’s 2-tailed t-test.

These data demonstrate that vascular sensitivity to PE and U-46619 is increased in ApoE-/- mice compared to SM22αDTR non-ablated mice with normal vasculature. VSMC apoptosis alone had no effect, however, VSMC loss in SM22αDTR/ApoE-/- mice appears to compensate for the hyperreactivity observed in the ApoE-/- animals.

Clarke, M.C. et al., (2006) Nat Med., 12(9):1075-1080.