p21Cip1 modulates arterial wound repair through the stromal cell–derived factor-1/CXCR4 axis in mice
Michelle Olive, … , Jason C. Kovacic, Manfred Boehm
Michelle Olive, … , Jason C. Kovacic, Manfred Boehm
Published May 8, 2008
Citation Information: J Clin Invest. 2008;118(6):2050-2061. https://doi.org/10.1172/JCI31244.
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Research Article Vascular biology

p21Cip1 modulates arterial wound repair through the stromal cell–derived factor-1/CXCR4 axis in mice

Abstract

Cyclin-dependent kinase inhibitors, including p21Cip1, are implicated in cell turnover and are active players in cardiovascular wound repair. Here, we show that p21Cip1 orchestrates the complex interactions between local vascular and circulating immune cells during vascular wound repair. In response to femoral artery mechanical injury, mice with homozygous deletion of p21Cip1 displayed accelerated proliferation of VSMCs and increased immune cell infiltration. BM transplantation experiments indicated that local p21Cip1 plays a pivotal role in restraining excessive proliferation during vascular wound repair. Increased local vascular stromal cell–derived factor-1 (SDF-1) levels were observed after femoral artery injury in p21+/+ and p21–/– mice, although this was significantly greater in p21–/– animals. In addition, disruption of SDF-1/CXCR4 signaling inhibited the proliferative response during vascular remodeling in both p21+/+ and p21–/– mice. We provide evidence that the JAK/STAT signaling pathway is an important regulator of vascular SDF-1 levels and that p21Cip1 inhibits STAT3 binding to the STAT-binding site within the murine SDF-1 promoter. Collectively, these results suggest that p21Cip1 activity is essential for the regulation of cell proliferation and inflammation after arterial injury in local vascular cells and that the SDF-1/CXCR4 signaling system is a key mediator of vascular proliferation in response to injury.

Authors

Michelle Olive, Jason A. Mellad, Leilani E. Beltran, Mingchao Ma, Thomas Cimato, Audrey C. Noguchi, Hong San, Richard Childs, Jason C. Kovacic, Manfred Boehm

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Figure 8

p21Cip1 stimulates SDF-1 via STAT3 in VSMCs.

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SDF-1 inhibition prevents excessive proliferation during vascular wound ...
(A) SDF-1 promoter activity is increased in p21–/– compared with p21+/+ VSMCs (pGL3.0, backbone vector) (n = 3; ***P < 0.001). (B) Left: SDF-1 mRNA levels are elevated at baseline and in LIF-stimulated cells (40 ng/ml) in p21–/– compared with p21+/+ VSMCs. Results are expressed as fold activation compared with untreated (Co) p21+/+ VSMCs (n = 3; *P < 0.05, **P < 0.01 compared with p21+/+ at same time point). Right: Inhibition of STAT3 with PpYLKTK-mps at 1, 10, and 100 μM decreased SDF-1 mRNA expression. Results are presented as fold activation compared with untreated p21+/+ and p21–/– VSMCs (n = 3; *P < 0.05, **P < 0.01, ***P < 0.001). (C) Mutation of the STAT3 binding site in the SDF-1 promoter (SDF-mut) decreased luciferase activity compared with unmutated SDF-1 promoter (SDF) in p21+/+ and p21–/– VSMCs (n = 3; ***P < 0.001). (D) Far left: Western blot analysis of p-STAT3 in p21–/– compared with p21+/+ VSMC extracts. Middle (2 panels): p21Cip1 interacts with STAT3 in p21+/+ VSMC extracts. p21Cip1 is pulled down by anti-STAT3 antibody, and STAT3 is pulled down by anti-p21Cip1 antibody. Far right: p21Cip1 and STAT3 inputs for the IP experiments. (E) ChIP from p21+/+ and p21–/– VSMC chromatin extracts using anti-STAT3 antibody. Left: Quantification of STAT3 occupancy in the SDF-1 promoter region in p21+/+ and p21–/– VSMCs (n = 3; **P < 0.01 versus p21+/+). Right: PCR amplification of the SDF-1 (–576/–372 bp) and β-actin promoters after ChIP with control IgG or anti-STAT3 antibodies.