Neointima formation in a restenosis model is suppressed in midkine-deficient mice.
ABSTRACT: Neointima formation is a common feature of atherosclerosis and restenosis after balloon angioplasty. To find a new target to suppress neointima formation, we investigated the possible role of midkine (MK), a heparin-binding growth factor with neurotrophic and chemotactic activities, in neointima formation. MK expression increased during neointima formation caused by intraluminal balloon injury of the rat carotid artery. Neointima formation in a restenosis model was strongly suppressed in MK-deficient mice. Continuous administration of MK protein to MK-deficient mice restored neointima formation. Leukocyte recruitment to the vascular walls after injury was markedly decreased in MK-deficient mice. Soluble MK as well as that bound to the substratum induced migration of macrophages in vitro. These results indicate that MK plays a critical role in neointima formation at least in part owing to its ability to mediate leukocyte recruitment.
Project description:Core2 1 to 6-N-glucosaminyltransferase-I (C2GlcNAcT-I) plays an important role in optimizing the binding functions of several selectin ligands, including P-selectin glycoprotein ligand. We used apolipoprotein E (ApoE)-deficient atherosclerotic mice to investigate the role of C2GlcNAcT-I in platelet and leukocyte interactions with injured arterial walls, in endothelial regeneration at injured sites, and in the formation of arterial neointima.Arterial neointima induced by wire injury was smaller in C2GlcNAcT-I-deficient apoE(-/-) mice than in control apoE(-/-) mice (a 79% reduction in size). Compared to controls, apoE(-/-) mice deficient in C2GlcNAcT-I also demonstrated less leukocyte adhesion on activated platelets in microflow chambers (a 75% reduction), and accumulation of leukocytes at injured areas of mouse carotid arteries was eliminated. Additionally, endothelial regeneration in injured lumenal areas was substantially faster in C2GlcNAcT-I-deficient apoE(-/-) mice than in control apoE(-/-) mice. Endothelial regeneration was associated with reduced accumulation of platelet factor 4 (PF4) at injured sites. PF4 deficiency accelerated endothelial regeneration and protected mice from neointima formation after arterial injury.C2GlcNAcT-I deficiency suppresses injury-induced arterial neointima formation, and this effect is attributable to decreased leukocyte recruitment to injured vascular walls and increased endothelial regeneration. Both C2GlcNAcT-I and PF4 are promising targets for the treatment of arterial restenosis.
Project description:On the luminal surface of injured arteries, platelet activation and leukocyte-platelet interactions are critical for the initiation and progression of arterial restenosis. The transcription factor nuclear factor-?B is a critical molecule in platelet activation. Here, we investigated the role of the platelet nuclear factor-?B pathway in forming arterial neointima after arterial injury.We performed carotid artery wire injuries in low-density lipoprotein receptor-deficient (LDLR(-/-)) mice with a platelet-specific deletion of I?B kinase-? (IKK?) (IKK?(fl/fl)/PF4(cre)/LDLR(-/-)) and in control mice (IKK?(fl/fl)/LDLR(-/-)). The size of the arterial neointima was 61% larger in the IKK?(fl/fl)/PF4(cre)/LDLR(-/-) mice compared with the littermate control IKK?(fl/fl)/LDLR(-/-) mice. Compared with the control mice, the IKK?(fl/fl)/PF4(cre)/LDLR(-/-) mice exhibited more leukocyte adhesion at the injured area. The extent of glycoprotein Ib? shedding after platelet activation was compromised in the IKK?-deficient platelets. This effect was associated with a low level of the active form of A Disintegrin And Metalloproteinase 17, the key enzyme involved in mediating glycoprotein Ib? shedding in activated IKK?-deficient platelets.Platelet IKK? deficiency increases the formation of injury-induced arterial neointima formation. Thus, nuclear factor-?B-related inhibitors should be carefully evaluated for use in patients after an arterial intervention.
Project description:The role of platelets in the development of thrombosis and abrupt closure after angioplasty is well recognized. However, the direct impact of platelets on neointima formation after arterial injury remains undetermined. Herein, we show that neointima formation after carotid artery wire injury reduces markedly in CD40-/- apolipoprotein E-deficient (apoE-/-) mice but only slightly in CD40 ligand-/-apoE-/- mice, compared with apoE-/- mice. Wild-type and CD40-deficient platelets were isolated from blood of apoE-/- and CD40-/-apoE-/- mice, respectively. The i.v. injection of thrombin-activated platelets into CD40-/-apoE-/- mice was performed every 5 days, starting at 2 days before wire injury. Injection of wild-type platelets promoted neointima formation, which was associated with increased inflammation by stimulating leukocyte recruitment via up-regulation of circulating platelet surface P-selectin expression and the formation of platelet-leukocyte aggregates. It was also associated with further promoting the luminal deposition of platelet-derived regulated on activation normal T cell expressed and secreted/chemokine (C-C motif) ligand 5 and expression of monocyte chemoattractant protein-1 and vascular cell adhesion molecule 1 in wire-injured carotid arteries. Remarkably, all these inflammatory actions by activated platelets were abrogated by lack of CD40 on injected platelets. Moreover, injection of wild-type platelets inhibited endothelial recovery in wire-injured carotid arteries, but this effect was also abrogated by lack of CD40 on injected platelets. Results suggest that platelet CD40 plays a pivotal role in neointima formation after arterial injury and might represent an attractive target to prevent restenosis after vascular interventions.
Project description:The 13?kDa heparin-binding growth factor midkine (MK) was originally identified as a molecule involved in the orchestration of embryonic development. Recent studies provided evidence for a new role of MK in acute and chronic inflammatory processes. Accordingly, several inflammatory diseases including nephritis, arthritis, atherosclerosis, colitis, and autoimmune encephalitis have been shown to be alleviated in the absence of MK in animal models. Reduced leukocyte recruitment to the sites of inflammation was found to be one important mechanism attenuating chronic inflammation when MK was absent. Furthermore, MK was found to modulate expression of proinflammatory cytokines and the expansion of regulatory T-cells. Here, we review the current understanding of the role of MK in different inflammatory disorders and summarize the knowledge of MK biology.
Project description:Abnormal growth of the intimal layer of blood vessels (neointima formation) contributes to the progression of atherosclerosis and in-stent restenosis. Recent evidence shows that the 18-kDa translocator protein (TSPO), a mitochondrial membrane protein, is involved in diverse cardiovascular diseases. In this study we investigated the role of endogenous TSPO in neointima formation after angioplasty in vitro and in vivo. We established a vascular injury model in vitro by using platelet-derived growth factor-BB (PDGF-BB) to stimulate rat thoracic aortic smooth muscle cells (A10 cells). We found that treatment with PDGF-BB (1-20?ng/mL) dose-dependently increased TSPO expression in A10 cells, which was blocked in the presence of PKC inhibitor or MAPK inhibitor. Overexpression of TSPO significantly promoted the proliferation and migration in A10 cells, whereas downregulation of TSPO expression by siRNA or treatment with TSPO ligands PK11195 or Ro5-4864 (104?nM) produced the opposite effects. Furthermore, we found that PK11195 (10-104?nM) dose-dependently activated AMPK in A10 cells. PK11195-induced inhibition on the proliferation and migration of PDGF-BB-treated A10 cells were abolished by compound C (an AMPK-specific inhibitor, 103?nM). In rats with balloon-injured carotid arteries, TSPO expression was markedly upregulated in the carotid arteries. Administration of PK11195 (3?mg/kg every 3 days, ip), starting from the initial balloon injury and lasting for 2 weeks, greatly attenuated carotid neointima formation by suppressing balloon injury-induced phenotype switching of VSMCs (increased ?-SMA expression). These results suggest that TSPO is a vascular injury-response molecule that promotes VSMC proliferation and migration and is responsible for the neointima formation after vascular injury, which provides a novel therapeutic target for various cardiovascular diseases including atherosclerosis and restenosis.
Project description:The effects of endothelium-derived hyperpolarizing factors have been attributed to cytochrome P450-derived epoxyeicosatrienoic acids (EETs), but the regulation and role of EETs in endothelial dysfunction remain largely unexplored. Hypertension is a primary risk factor for renal dysfunction, which is frequently accompanied by various systemic diseases induced by endothelial dysfunction in the microcirculation. We previously reported that the endothelial growth factor midkine (MK) enhances hypertension in a model of CKD. Here, we investigated the hypothesis that MK regulates EET activity and thereby BP. MK gene-deleted mice were resistant to hypertension and developed less glomerulosclerosis and proteinuria after administration of a nitric oxide synthase (NOS) inhibitor in the setting of uninephrectomy. The hypertension observed in uninephrectomized wild-type mice after NOS inhibition was ameliorated by anti-MK antibody. MK-deficient mice produced higher amounts of EETs, and EETs dominantly regulated BP in these mice. Furthermore, MK administration to MK-deficient mice recapitulated the BP control observed in wild-type mice. EETs also dominantly regulated renal blood flow, which may influence renal function, in MK-deficient mice. Taken together, these results suggest that the MK/EET pathway is physiologically engaged in BP control and could be a target for the treatment of hypertension complicated by endothelial dysfunction.
Project description:In the previous report, Meox1 was found to promote SMCs phenotypic modulation and injury-induced vascular remodeling by regulating the FAK-ERK1/2-autophagy signaling cascade (Wu et al., 2017) . Here, we presented new original data on the involvement of Mesoderm/mesenchyme homeobox gene l (Meox1) in balloon-injury-induced neointima formation of rat. In rat carotid artery balloon injury model to induce vascular remodeling, Meox1 was induced in vascular smooth muscle cell (SMCs) of rat carotid arteries. Most proliferating cell nuclear antigen (PCNA)-positive cells also expressed Meox1. These data suggested that Meox1 may be involved in SMCs proliferation during injury-induced neointima formation. Furthermore, knocked down its expression in injured arteries by adenoviral delivery of Meox1 short hairpin RNA (shRNA) (shMeox1), neointima formation was significantly inhibited. Elastin staining also confirmed the reduction of neointima in Meox1 shRNA-transduced arteries. Moreover, knockdown of Meox1 decreased the collagen production/deposition that was significantly increased in neointima induced by balloon injury.
Project description:Vascular smooth muscle cell (VSMC) activation in response to injury plays an important role in the development of vascular proliferative diseases, including restenosis and atherosclerosis. The aims of this study were to ascertain the physiological functions of exchange proteins directly activated by cAMP isoform 1 (Epac1) in VSMC and to evaluate the potential of Epac1 as therapeutic targets for neointima formation during vascular remodeling. In a mouse carotid artery ligation model, genetic knockdown of the Epac1 gene led to a significant reduction in neointima obstruction in response to vascular injury. Pharmacologic inhibition of Epac1 with an Epac specific inhibitor, ESI-09, phenocopied the effects of Epac1 null by suppressing neointima formation and proliferative VSMC accumulation in neointima area. Mechanistically, Epac1 deficient VSMCs exhibited lower level of PI3K/AKT signaling and dampened response to PDGF-induced mitochondrial fission and reactive oxygen species levels. Our studies indicate that Epac1 plays important roles in promoting VSMC proliferation and phenotypic switch in response to vascular injury, therefore, representing a therapeutic target for vascular proliferative diseases.
Project description:To use the mice deficient in both adenosine receptor A(2A)(A(2A)R(-/-)) and apolipoprotein E (apoE(-/-)) to investigate the role of A(2A)R in mediating the interactions of leukocytes with injured arterial walls and the formation of arterial neointima induced by a guide wire.In apoE(-/-) mice, A(2A)R deficiency increased the size of the arterial neointima in injured carotid arteries by 83%. Arterial neointima formation was also enhanced in chimeric mice that underwent bone marrow transplantation (these mice lacked A(2A)R in their bone marrow-derived cells). Epifluorescence intravital microscopy showed that neutrophil rolling and adherence to the injured arterial area were enhanced by 80% and 110% in A(2A)R(-/-)/apoE(-/-) mice, respectively. This phenomenon occurred even though the protein levels of homing molecules on A(2A)R-deficient neutrophils were unchanged from those of wild-type neutrophils. A(2A)R-deficient neutrophils exhibited an increase in the phosphorylation of p38 mitogen-activated protein kinase, P-selectin glycoprotein ligand-1 (PSGL-1) clustering, and the affinity of b(2) integrins. The inhibition of p38 phosphorylation abrogated the increased PSGL-1 clustering and beta(2) integrin affinity, thus reversing the increased homing ability of A(2A)R-deficient leukocytes.A(2A)R plays a complex role in inflammation and tissue injury. The deficiency of A(2A)R enhances the homing ability of leukocytes and increases the formation of the arterial neointima after injury. A(2A)R antagonists are being tested for the treatment of neurodegenerative and other chronic diseases. An evaluation of the effect of A(2A)R antagonists on arterial restenosis after arterial angioplasty should be conducted.
Project description:Percutaneous coronary intervention is widely adopted to treat patients with coronary artery disease. However, restenosis remains an unsolved clinical problem after vascular interventions. The role of the systemic and local immune response in the development of restenosis is not fully understood. Hence, the aim of the current study was to investigate the role of the human immune system on subsequent neointima formation elicited by vascular injury in a humanized mouse model. Immunodeficient NOD.Cg-Prkdc(scid)IL2rg(tm1Wjl)(NSG) mice were reconstituted with human (h)PBMCs immediately after both carotid wire and femoral cuff injury were induced in order to identify how differences in the severity of injury influenced endothelial regeneration, neointima formation, and homing of human inflammatory and progenitor cells. In contrast to non-reconstituted mice, hPBMC reconstitution reduced neointima formation after femoral cuff injury whereas hPBMCs promoted neointima formation after carotid wire injury 4 weeks after induction of injury. Neointimal endothelium and smooth muscle cells in the injured arteries were of mouse origin. Our results indicate that the immune system may differentially respond to arterial injury depending on the severity of injury, which may also be influenced by the intrinsic properties of the arteries themselves, resulting in either minimal or aggravated neointima formation.