Exacerbated vein graft arteriosclerosis in protein kinase Cδ–null mice
Michael Leitges, Manuel Mayr, Ursula Braun, Ursula Mayr, Chaohong Li, Gerald Pfister, Nassim Ghaffari-Tabrizi, Gottfried Baier, Yanhua Hu, Qingbo Xu
Michael Leitges, Manuel Mayr, Ursula Braun, Ursula Mayr, Chaohong Li, Gerald Pfister, Nassim Ghaffari-Tabrizi, Gottfried Baier, Yanhua Hu, Qingbo Xu
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Exacerbated vein graft arteriosclerosis in protein kinase Cδ–null mice

Abstract

Smooth muscle cell (SMC) accumulation is a key event in the development of atherosclerosis, including vein bypass graft arteriosclerosis. Because members of the protein kinase C (PKC) family signal cells to undergo proliferation, differentiation, or apoptosis, we generated PKCδ knockout mice and performed vein bypass grafts on these animals. PKCδ–/– mice developed normally and were fertile. Vein segments from PKCδ–/– mice isografted to carotid arteries of recipient mice of either genotype led to a more severe arteriosclerosis than was seen with PKCδ+/+ vein grafts. Arteriosclerotic lesions in PKCδ–/– mice showed a significantly higher number of SMCs than were found in wild-type animals; this was correlated with decreased SMC death in lesions of PKCδ–/– mice. SMCs derived from PKCδ–/– aortae were resistant to cell death induced by any of several stimuli, but they were similar to wild-type SMCs with respect to mitogen-stimulated cell proliferation in vitro. Furthermore, pro-apoptotic treatments led to diminished caspase-3 activation, poly(ADP-ribose) polymerase cleavage, and cytochrome c release in PKCδ–/– relative to wild-type SMCs, suggesting that their apoptotic resistance involves the loss of free radical generation and mitochondrial dysfunction in response to stress stimuli. Our data indicate that PKCδ maintains SMC homeostasis and that its function in the vessel wall per se is crucial in the development of vein graft arteriosclerosis.

Authors

Michael Leitges, Manuel Mayr, Ursula Braun, Ursula Mayr, Chaohong Li, Gerald Pfister, Nassim Ghaffari-Tabrizi, Gottfried Baier, Yanhua Hu, Qingbo Xu

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Immunofluorescence double labeling of vein graft sections (a and b). Cry...
Immunofluorescence double labeling of vein graft sections (a and b). Cryostat sections from mouse vein grafts 8 weeks after surgery were fixed with cold 5% acetone/methanol for 30 minutes, air-dried, and incubated with a rat monoclonal antibody to mouse MAC-1, and then visualized by anti-rat Ig-FITC-conjugated rabbit Ig. After washing, sections were incubated with mouse monoclonal antibody against α-actin conjugated with Cy3 for 30 minutes at room temperature. The sections were examined by laser confocal microscopy. (c and d) Statistical data are means ± SEM of percentage of positive cells. Sections were labeled with α-actin antibody/Cy3 or rabbit anti-PCNA antibodies. Nuclei were stained with Hoechst 33258 (1 μg/ml). Positive cells and total nuclei were counted by microscopy. (e–h) TUNEL staining of vein grafts of PKCδ+/+ (e–g) and PKCδ–/– (e and h) mice. Vein grafts were harvested 8 weeks after surgery, and frozen sections were prepared and stained with TUNEL as described in Methods. Arrows indicate typical examples of TUNEL+ cells in lesions. (e) Statistical data are means ± SEM of three independent experiments, *P < 0.05.