Vasoactive responses in aorta from a novel murine model for the conditional ablation of vascular smooth muscle cells

1J.J. Maguire, 2M.C.H. Clarke, 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 and vascular remodelling (Stoneman & Bennett, 2004) resulting in a thinned media and predisposition to plaque rupture. We have investigated the consequence of VSMC loss on vascular reactivity of aorta from transgenic mice (SM22αDTR) expressing the human diphtheria toxin (DT) receptor under the minimal SM22α promoter. Exposure of these mice to DT results in specific loss of ~50% of medial VSMCs in large and medium arteries, with evidence of caspase-3 activation, nuclear condensation, but not inflammation; consistent with apoptotic cell death.

Female SM22αDTR mice (25-35g) were placed into two groups – control and ablated – the latter receiving 1ng/g DT i.p. for 42 days. Mice were killed by CO2 inhalation and four adjacent segments of thoracic aorta (1-2mm) were set up in wire myographs for isometric tension recordings in Krebs’ solution (37 ° C). Segments were also removed for histology. Following normalisation, cumulative concentration-response curves were constructed to phenylephrine (PE, 1x10-9-3x10-5M), the thromboxane mimetic U-46619 (1x10-11-3x10-6M), endothelin-1 (ET-1, 1x10-10-3x10-7M) and KCl (1x10-4–1x10-1M). Constrictor responses (mN/mm) were analysed using the curve fitting programme FigSys (Biosoft, Cambridge, UK) with potency (pD2) and maximum response (Emax) expressed as mean±s.e.mean. Data for the two groups were compared using Student’s two-tailed t-test with significance set at P<0.05. n-Values are the number of mice.

The aorta of SM22αDTR ablated mice had significantly fewer VSMCs (2539±132 cells/mm2, n=5) compared to controls (4252±241 cells/mm2, n=5; P<0.001,). However, the internal diameters of aortic segments from control mice (1684.0±87.9μm) were not significantly different from ablated mice (1524.6±26.0μm, P<0.05) nor were basal tensions determined from the normalisation procedure (control 5.98±0.37 vs ablated 5.69±0.07 mN/mm, P<0.05). Similarly there was no difference between potencies and maximum responses for the three agonists and KCl between the two groups (Table 1).

Table 1. Potency (pD2) and maximum response (Emax) to constrictor agonists in SM22αDTR control and ablated mice. n.d.- not determined.

Surprisingly, these data demonstrate that despite the significant loss of VSMCs, aorta from SM22αDTR mice maintain their responsiveness to both receptor-dependent and -independent vasoconstrictors in vitro. The mechanism by which this occurs remains to be clarified. Manipulation of this murine model of VSMC apoptosis e.g. when crossed onto an ApoE-/- background, may be of value in determining the mechanisms contributing to human cardiovascular disease.

Stoneman, V.E. & Bennett, M.R. (2004). Clin. Sci.(Lond.), 107(4), 343-354.