Smooth muscle Notch1 mediates neointimal formation after vascular injury - PubMed (original) (raw)

. 2009 May 26;119(20):2686-92.

doi: 10.1161/CIRCULATIONAHA.108.790485. Epub 2009 May 11.

Kyosuke Takeshita, Ping-Yen Liu, Minoru Satoh, Naotsugu Oyama, Yasushi Mukai, Michael T Chin, Luke Krebs, Michael I Kotlikoff, Freddy Radtke, Thomas Gridley, James K Liao

Affiliations

Smooth muscle Notch1 mediates neointimal formation after vascular injury

Yuxin Li et al. Circulation. 2009.

Abstract

Background: Notch1 regulates binary cell fate determination and is critical for angiogenesis and cardiovascular development. However, the pathophysiological role of Notch1 in the postnatal period is not known. We hypothesize that Notch1 signaling in vascular smooth muscle cells (SMCs) may contribute to neointimal formation after vascular injury.

Methods and results: We performed carotid artery ligation in wild-type, control (SMC-specific Cre recombinase transgenic [smCre-Tg]), general Notch1 heterozygous deficient (N1+/-), SMC-specific Notch1 heterozygous deficient (smN1+/-), and general Notch3 homozygous deficient (N3-/-) mice. Compared with wild-type or control mice, N1+/- and smN1+/- mice showed a 70% decrease in neointimal formation after carotid artery ligation. However, neointimal formation was similar between wild-type and N3-/- mice. Indeed, SMCs derived from explanted aortas of either N1(+/-)- or smN1+/- mice showed decreased chemotaxis and proliferation and increased apoptosis compared with control or N3-/- mice. This correlated with decreased staining of proliferating cell nuclear antigen-positive cells and increased staining of cleaved caspase-3 in the intima of N1(+/-)- or smN1+/- mice. In SMCs derived from CHF1/Hey2-/- mice, activation of Notch signaling did not lead to increased SMC proliferation or migration.

Conclusions: These findings indicate that Notch1, rather than Notch3, mediates SMC proliferation and neointimal formation after vascular injury through CHF1/Hey2 and suggest that therapies that target Notch1/CHF1/Hey2 in SMCs may be beneficial in preventing vascular proliferative diseases.

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Figures

Figure 1

Figure 1. Expression of Notch receptors in endothelial and smooth muscle cells

A. Western blot analysis shows that the expression of total Notch1 in EC isolated from hearts of WT, Notch1flox/+, smCre-Tg, N1+/-, and smN1+/- mice. B. Western blot analysis shows that the expression of total Notch1 and Notch3 in SMC isolated from aortas of WT, Notch1flox/+, smCre-Tg, N1+/-, and smN1+/- mice. C. Immunofluorescence staining of Notch1, Notch2, and Notch3 in uninjured carotid artery (left panel) and ligated injured carotid artery (14 days after ligation, right panel) of WT mice. Vessels were co-stained for smooth muscle actin (SMA). SMA is stained red; Notch receptors were stained green; and nuclei were stained blue (DAPI). Yellow indicates presence of both red and green staining. White bar was 20 μm. D. Immunofluorescence co-staining of smooth muscle actin (SMA) with activated Notch1 (NICD). Red was SMA, green was NICD, and blue was DAPI. White bar was 20 μm.

Figure 2

Figure 2. Neointimal formation in Notch mutant mice following vascular injury

Carotid arteries were collected 28 days after the procedure. HE staining was performed on sections from WT, smCre-Tg, N1+/-, N3-/- and smN1+/- mice. Arrows indicate internal elastic lamina. A. Representative sections are shown. Scale bar, 100 μm. B. Quantitative morphometric analysis of intimal area in WT, smCre-Tg, N1+/-, N3-/- and smN1+/- mice. _n_=10 in each group, *; P<0.01, vs. WT; †; P<0.01, vs. smCre-Tg C. Attenuated intima/media area ratio in Notch1 mutant, but not N3-/- mice compared with control mice. _n_=10 in each group, *; P<0.01, vs. WT; †; P<0.01, vs. smCre-Tg

Figure 3

Figure 3. Migration of SMC in Notch mutant mice

A. Micrographs of SMC migration from aortic explants from WT, N1+/-, smN1+/-, and N3-/- mice. B. Quantification of SMC migration from aortic explants of WT, N1+/-, smN1+/-, and N3-/- mice. _n_=8, *; _P<_0.05 C. SMC chemotaxis in response to serum stimulation (percent of migrated WT SMC). _n_=12, *; P<0.05, vs. WT

Figure 4

Figure 4. Proliferation of SMC in Notch mutant mice

A. SMC proliferation in response to serum after 3days (percent of WT SMC). _n_=10, *; P<0.05, vs. WT; †; P<0.05, vs. smCre-Tg B. [3H]Thymidine incorporation (percent of incorporation of WT SMC). _n_=6, *; P<0.05, vs. WT; †; P<0.05, vs. smCre-Tg C. Left panel: Co-staining for SMA (red) and PCNA (green) 14 days after ligation injury in WT and N1+/- mice. White scale bar was 20 μm. Right panel: Quantification of PCNA positive cells in neointima of WT and N1+/- mice. _n_=6; *; P<0.05, vs. WT

Figure 5

Figure 5. Apoptosis of SMC in Notch mutant mice

A. H2O2-induced apoptosis of SMC. _n_=10, *; P<0.01, vs. WT; †; P<0.01, vs. smCre-Tg. B. Left panel: Co-staining for SMA (red) and cleaved Caspase-3 (green) 14 days after ligation injury in WT and N1+/- mice. Right panel: Quantification of cleaved Caspase-3-positive cells in neointima from WT and N1+/- mice _n_=6, *; P<0.05, vs. WT

Figure 6

Figure 6. CHF1/Hey2 mediates SMC proliferation and migration

A. Effects of transfection with Ad.GFP or Ad.NICD on SMC proliferation (cell count) from WT or CHF1-/- mice. _n_=6 B. Effects of transfection with Ad.GFP or Ad.NICD on SMC proliferation (thymidine incorporation) from WT or CHF1-/- mice. _n_=6 C. Effects of transfection with Ad.GFP or Ad.NICD on SMC migration (% of WT) from WT or CHF1-/- mice. _n_=6

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