Requirement for p38 mitogen-activated protein kinase activity in neointima formation after vascular injury.
ABSTRACT: Angioplasty and stent delivery are performed to treat atherosclerotic vascular disease but often cause deleterious neointimal lesion formation. Previously, growth factor receptor-bound protein 2 (Grb2), an intracellular linker protein, was shown to be essential for neointima formation and for p38 mitogen-activated protein kinase (MAPK) activation in vascular smooth muscle cells (SMCs). In this study, the role of vascular SMC p38alpha MAPK in neointimal development was examined.Compound transgenic mice were generated with doxycycline-inducible SMC-specific expression of dominant-negative p38alpha MAPK (DN-p38alpha). Doxycycline treatment resulted in the expression of DN-p38alpha mRNA and protein in transgenic arteries. Doxycycline-treated compound transgenic mice were resistant to neointima formation 21 days after carotid injury and showed reduced arterial p38 MAPK activation. To explore the mechanism by which p38alpha MAPK promotes neointima formation, an in vitro SMC culture system was used. Inhibition of p38alpha MAPK in cultured SMCs by treatment with SB202190 or small interfering RNA blocked platelet-derived growth factor-induced SMC proliferation, DNA replication, phosphorylation of the retinoblastoma protein, and induction of minichromosome maintenance protein 6.SMC p38alpha MAPK activation is required for neointima formation, perhaps because of its ability to promote retinoblastoma protein phosphorylation and minichromosome maintenance protein 6 expression.
Project description:OBJECTIVE:Smooth muscle cells (SMCs) contribute to neointima formation after vascular injury. Although ?-catenin expression is induced after injury, whether its function is essential in SMCs for neointimal growth is unknown. Moreover, although inhibitors of ?-catenin have been developed, their effects on SMC growth have not been tested. We assessed the requirement for SMC ?-catenin in short-term vascular homeostasis and in response to arterial injury and investigated the effects of ?-catenin inhibitors on vascular SMC growth. APPROACH AND RESULTS:We used an inducible, conditional genetic deletion of ?-catenin in SMCs of adult mice. Uninjured arteries from adult mice lacking SMC ?-catenin were indistinguishable from controls in terms of structure and SMC marker gene expression. After carotid artery ligation, however, vessels from mice lacking SMC ?-catenin developed smaller neointimas, with lower neointimal cell proliferation and increased apoptosis. SMCs lacking ?-catenin showed decreased mRNA expression of Mmp2, Mmp9, Sphk1, and S1pr1 (genes that promote neointima formation), higher levels of Jag1 and Gja1 (genes that inhibit neointima formation), decreased Mmp2 protein expression and secretion, and reduced cell invasion in vitro. Moreover, ?-catenin inhibitors PKF118-310 and ICG-001 limited growth of mouse and human vascular SMCs in a dose-dependent manner. CONCLUSIONS:SMC ?-catenin is dispensable for maintenance of the structure and state of differentiation of uninjured adult arteries, but is required for neointima formation after vascular injury. Pharmacological ?-catenin inhibitors hinder growth of human vascular SMCs. Thus, inhibiting ?-catenin has potential as a therapy to limit SMC accumulation and vascular obstruction.
Project description:c-Kit+ progenitor smooth muscle cells (P-SMCs) can develop into SMCs that contribute to injury-induced neointimal thickening. Here, we investigated whether adenosine reduces P-SMC migration and proliferation and whether this contributes to adenosine's inhibitory actions on neointima formation. In human P-SMCs, 2-chloroadenosine (stable adenosine analogue) and BAY60-6583 (A<sub>2B</sub> agonist) inhibited P-SMC proliferation and migration. Likewise, increasing endogenous adenosine by blocking adenosine metabolism with erythro-9-(2-hydroxy-3-nonyl) adenine (inhibits adenosine deaminase) and 5-iodotubercidin (inhibits adenosine kinase) attenuated P-SMC proliferation and migration. Neither N<sup>6</sup>-cyclopentyladenosine (A<sub>1</sub> agonist), CGS21680 (A<sub>2A</sub> agonist), nor N<sup>6</sup>-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (A<sub>3</sub> agonist) affected P-SMC proliferation or migration. 2-Chloroadenosine increased cyclic AMP, reduced Akt phosphorylation (activates cyclin D expression), and reduced levels of cyclin D1 (promotes cell-cycle progression). Moreover, 2-chloroadenosine inhibited expression of Skp2 (promotes proteolysis of p27<sup>Kip1</sup>) and upregulated levels of p27<sup>Kip1</sup> (negative cell-cycle regulator). A<sub>2B</sub> receptor knockdown prevented the effects of 2-chloroadenosine on cyclic AMP production and P-SMC proliferation and migration. Likewise, inhibition of adenylyl cyclase and protein kinase A rescued P-SMCs from the inhibitory effects of 2-chloroadenosine. The inhibitory effects of adenosine were similar in male and female P-SMCs. In vivo, peri-arterial (rat carotid artery) 2-chloroadenosine (20 ?mol/L for 7 days) reduced neointimal hyperplasia by 64.5% (<i>P</i><0.05; intima/media ratio: control, 1.4±0.02; treated, 0.53±0.012) and reduced neointimal c-Kit+ cells. Adenosine inhibits P-SMC migration and proliferation via the A<sub>2B</sub> receptor/cyclic AMP/protein kinase A axis, which reduces cyclin D1 expression and activity via inhibiting Akt phosphorylation and Skp2 expression and upregulating p27<sup>kip1</sup> levels. Adenosine attenuates neointima formation in part by inhibiting infiltration and proliferation of c-Kit+ P-SMCs.
Project description:<h4>Objective</h4>Vascular injury causes neointimal hypertrophy, which is characterized by redox-mediated matrix degradation and smooth muscle cell (SMC) migration and proliferation. We hypothesized that, as compared to the adjacent medial SMCs, neointimal SMCs produce increased superoxide via NADPH oxidase, which induces redox-sensitive intracellular signaling to activate matrix metalloproteinase-9 (MMP-9).<h4>Methods and results</h4>Two weeks after balloon injury, rat aorta developed a prominent neointima, containing increased expression of NADPH oxidase and reactive oxygen species (ROS) as compared to the medial layer. Next, SMCs were isolated from either the neointima or the media and studied in culture. Neointimal-derived SMCs exhibited increased Nox1 expression and ROS levels as compared to medial SMCs. Neointimal SMCs had higher cell growth rates than medial SMCs. ROS-dependent ERK1/2 phosphorylation was greater in neointimal SMCs. MMP-9 activity, as detected by gel zymography, was greater in neointimal SMCs under resting and stimulated conditions and was prevented by expression of an antisense to Nox1 or treatment with an ERK1/2 inhibitor.<h4>Conclusions</h4>Following vascular injury, the increased expression of Nox1 in SMCs within the neointima initiates redox-dependent phosphorylation of ERK1/2 and subsequent MMP-9 activation.
Project description:Recent studies have shown that Sca-1(+) (stem cell antigen-1) stem/progenitor cells within blood vessel walls may contribute to neointima formation, but the mechanism behind their recruitment has not been explored. In this work Sca-1(+) progenitor cells were cultivated from mouse vein graft tissue and found to exhibit increased migration when cocultured with smooth muscle cells (SMCs) or when treated with SMC-derived conditioned medium. This migration was associated with elevated levels of chemokines, CCL2 (chemokine (C-C motif) ligand 2) and CXCL1 (chemokine (C-X-C motif) ligand 1), and their corresponding receptors on Sca-1(+) progenitors, CCR2 (chemokine (C-C motif) receptor 2) and CXCR2 (chemokine (C-X-C motif) receptor 2), which were also upregulated following SMC conditioned medium treatment. Knockdown of either receptor in Sca-1(+) progenitors significantly inhibited cell migration. The GTPases Cdc42 and Rac1 were activated by both CCL2 and CXCL1 stimulation and p38 phosphorylation was increased. However, only Rac1 inhibition significantly reduced migration and p38 phosphorylation. After Sca-1(+) progenitors labeled with green fluorescent protein (GFP) were applied to the adventitial side of wire-injured mouse femoral arteries, a large proportion of GFP-Sca-1(+) -cells were observed in neointimal lesions, and a marked increase in neointimal lesion formation was seen 1 week post-operation. Interestingly, Sca-1(+) progenitor migration from the adventitia to the neointima was abrogated and neointima formation diminished in a wire injury model using CCL2(-/-) mice. These findings suggest vascular stem/progenitor cell migration from the adventitia to the neointima can be induced by SMC release of chemokines which act via CCR2/Rac1/p38 and CXCR2/Rac1/p38 signaling pathways. Stem Cells 2016;34:2368-2380.
Project description:Dedifferentiation, migration, and proliferation of resident vascular smooth muscle cells (SMCs) are key components of neointima formation after vascular injury. Activation of signal transducer and activator of transcription-3 (STAT3) is suggested to be critically involved in this process, but the complex regulation of STAT3-dependent genes and the functional significance of inhibiting this pathway during the development of vascular proliferative diseases remain elusive. In this study, we demonstrate that STAT3 was activated in neointimal lesions following wire-induced injury in mice. Phosphorylation of STAT3 induced trans-activation of cyclin D1 and survivin in SMCs in vitro and in neointimal cells in vivo, thus promoting proliferation and migration of SMCs as well as reducing apoptotic cell death. WP1066, a highly potent inhibitor of STAT3 signaling, abrogated phosphorylation of STAT3 and dose-dependently inhibited the functional effects of activated STAT3 in stimulated SMCs. The local application of WP1066 via a thermosensitive pluronic F-127 gel around the dilated arteries significantly inhibited proliferation of neointimal cells and decreased the neointimal lesion size at 3 weeks after injury. Even though WP1066 application attenuated the injury-induced up-regulation of the chemokine RANTES at 6 h after injury, there was no significant effect on the accumulation of circulating cells at 1 week after injury. In conclusion, these data identify STAT3 as a key molecule for the proliferative response of SMC and neointima formation. Moreover, inhibition of STAT3 by the potent and specific compound WP1066 might represent a novel and attractive approach for the local treatment of vascular proliferative diseases.
Project description:Integrin-mediated interactions between smooth muscle cells (SMCs) and the extracellular matrix regulate cell migration and proliferation during neointimal hyperplasia. Integrin-linked kinase (ILK) is a serine-threonine kinase and scaffolding molecule that acts downstream of integrin receptors to modulate cell adhesion; therefore, we examined ILK function in SMCs during wound repair. Silencing of ILK expression with siRNA in vitro decreased cell adhesion to fibronectin and accelerated both cell proliferation and wound closure in the cell monolayer; it also resulted in the rearrangement of focal adhesions and diminished central actin stress fibers. Akt and GSK3beta are ILK substrates that are important in cell motility; however, ILK siRNA silencing did not attenuate injury-induced increases in Akt and GSK3beta phosphorylation. Following balloon catheter injury of the rat carotid artery in vivo, a dramatic decrease in ILK levels coincided with both the proliferation and migration of SMCs, which leads to the formation of a thickened neointima. Immunostaining revealed decreased ILK levels in the media and deep layers of the neointima, but increased ILK levels in the subluminal layers of the intima. Taken together, these results suggest that ILK functions to maintain SMC quiescence in the normal artery. A decrease in ILK levels after injury may permit SMC migration, proliferation, and neointimal thickening, and its re-expression at the luminal surface may attenuate this process during later stages of the injury response.
Project description:Interferon regulatory factor 8 (IRF8), a member of the IRF transcription factor family, was recently implicated in vascular diseases. In the present study, using the mouse left carotid artery wire injury model, we unexpectedly observed that the expression of IRF8 was greatly enhanced in smooth muscle cells (SMCs) by injury. Compared with the wild-type controls, IRF8 global knockout mice exhibited reduced neointimal lesions and maintained SMC marker gene expression. We further generated SMC-specific IRF8 transgenic mice using an SM22?-driven IRF8 plasmid construct. In contrast to the knockout mice, mice with SMC-overexpressing IRF8 exhibited a synthetic phenotype and enhanced neointima formation. Mechanistically, IRF8 inhibited SMC marker gene expression through regulating serum response factor (SRF) transactivation in a myocardin-dependent manner. Furthermore, a coimmunoprecipitation assay indicated a direct interaction of IRF8 with myocardin, in which a specific region of myocardin was essential for recruiting acetyltransferase p300. Altogether, IRF8 is crucial in modulating SMC phenotype switching and neointima formation in response to vascular injury via direct interaction with the SRF/myocardin complex.
Project description:The ubiquitous enzyme protein kinase C (PKC) has been linked to the pathogenesis of vascular injury, but the cell-specific and discrete functions of the betaII isoform have yet to be discovered in this setting. Our previous findings demonstrated significantly increased PKCbetaII in the membrane fraction of injured femoral arteries in wild type (WT) mice and revealed reduction of neointimal expansion in PKCbeta(-/-) mice after acute vascular injury. As PKCbeta(-/-) mice are globally devoid of PKCbeta, we established novel transgenic (Tg) mice to test the hypothesis that the action of PKCbetaII specifically in smooth muscle cells (SMCs) mediates the formation of neointimal lesions in response to arterial injury.Tg mice expressing SM22alpha promoter-targeted mouse carboxyl-terminal deletion mutant PKCbetaII were produced using standard techniques, subjected to femoral artery injury and compared with littermate controls. Smooth muscle cells (SMCs) were isolated from wild type (WT) and Tg mice and exposed to a prototypic stimulus, tumor necrosis factor (TNF)-alpha. Multiple strategies were employed in vivo and in vitro to examine the molecular mechanisms underlying the specific effects of SMC PKCbetaII in neointimal expansion.In vivo and in vitro analyses demonstrated that PKCbetaII activity in SMCs was critical for neointimal expansion in response to arterial injury, at least in part via regulation of ERK1/2, Egr-1 and induction of MMP-9.These data identify the SMC-specific regulatory role of PKCbetaII in neointimal expansion in response to acute arterial injury, and suggest that targeted inactivation of PKCbetaII may be beneficial in limiting restenosis via suppression of the neointima-mediating effects of Egr-1 and MMP-9.
Project description:Neointima formation is the leading cause of arteriovenous fistula (AVF) failure. We have shown that CKD accelerates this process by transforming the vascular smooth muscle cells (SMCs) lining the AVF from a contractile to the synthetic phenotype. However, the underlying mechanisms affecting this transformation are not clear. Previous studies have shown that the ?-class glutathione transferase isozymes have an important role in regulating 4-hydroxynonenal (4-HNE)-mediated proliferative signaling of cells. Here, using both the loss- and gain-of-function approaches, we investigated the role of glutathione S-transferase ?4 (GSTA4) in modulating cellular 4-HNE levels for the transformation and proliferation of SMCs. Compared with non-CKD controls, mice with CKD had downregulated expression of GSTA4 at the mRNA and protein levels, with concomitant increase in 4-HNE in arteries and veins. This effect was associated with upregulated phosphorylation of MAPK signaling pathway proteins in proliferating SMCs. Overexpressing GSTA4 blocked 4-HNE-induced SMC proliferation. Additionally, inhibitors of MAPK signaling inhibited the 4-HNE-induced responses. Compared with wild-type mice, mice lacking GSTA4 exhibited increased CKD-induced neointima formation in AVF. Transient expression of an activated form of GSTA4, achieved using a combined Tet-On/Cre induction system in mice, lowered levels of 4-HNE and reduced the proliferation of SMCs. Together, these results demonstrate the critical role of GSTA4 in blocking CKD-induced neointima formation and AVF failure.
Project description:<h4>Aims</h4>Emerging evidence has suggested that adventitia stem/progenitor cells (AdSPCs) migrate into the intima of arteries in response to injury, where they differentiate towards smooth muscle cells (SMCs) and participate in neointimal hyperplasia. We have previously identified matrix metalloproteinase-8 (MMP8) as a key player in atherogenesis. In this study, we aimed to investigate the functional roles of macrophage-derived MMP8 in AdSPC differentiation and injury-induced arterial remodelling.<h4>Methods and results</h4>We first observed an important role for MMP8 in SMC differentiation from embryonic stem cells, but this effect was not seen in AdSPCs. Instead, through macrophages/AdSPCs co-culture and macrophage conditional culture medium studies, we have demonstrated that the MMP8 protein secreted from macrophages promotes SMC differentiation from AdSPCs. Mechanistically, we showed that macrophage-derived MMP8 promotes SMC differentiation from AdSPCs through modulating transforming growth factor-? activity and a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10)/Notch1 signalling. We further demonstrated that the binding site for CBF1, Suppressor of Hairless, and Lag-1 (CSL) within SMC gene promoters is responsible for Notch1 mediated SMC differentiation. Finally, we demonstrated that macrophage-derived MMP8 increased injury-induced neointimal SMC hyperplasia by activating ADAM10/Notch1 signalling.<h4>Conclusions</h4>We have identified macrophage-derived MMP8 as a regulator in SMC differentiation from AdSPCs and neointimal SMC hyperplasia in response to injury. Our data provide new insights into the roles of MMP8 in AdSPC differentiation and the pathogenesis of neointima formation in the context of angiographic restenosis, and therefore may aid in the development of novel therapeutic agents for the prevention of this disease.