Marked increase in the histamine content of neointima after stent implantation of pig coronary artery and growth-promoting effects of histamine in cultured smooth muscle cells

After coronary stent implantation, the unfavorable in-stent restenosis often occurs by the formation of neointima due to the proliferation of smooth muscle cells. Platelet-derived growth factor (PDGF) and other peptide growth factors contribute to this process, but little is known about the role of non-peptide factors in this process. In the present study, the role of histamine, a non-peptide factor, in the formation of neointima was investigated using a pig coronary model of in-stent restenosis and a culture system of coronary smooth muscle cells. A Palmaz-Schatz stent was implanted in the left anterior descending coronary artery of male pigs. At 1, 2 and 4 weeks after stenting, the histamine content of neointima was determined to be 326 +/- 82, 1427 +/- 280 and 440 +/- 69 pmol/mg protein, respectively, by HPLC fluorometry. In contrast, the histamine content of arterial media from the untreated control arteries was only 15.3 +/- 1.6 pmol/mg protein. These results demonstrate that the histamine content of neointima is about 20 to 90-fold that of the normal media. In vitro, histamine by itself did not stimulate the proliferation of cultured smooth muscle cells, but potentiated the PDGF-stimulated proliferation of the cultured cells via a mechanism independent of H1 and H2 histamine receptors. Thus, histamine may be an important non-peptide factor in the pathogenesis of in-stent restenosis.     

Yttrium-90 delivered via a centering catheter and afterloader, given both before and after stent implantation, inhibits neointima formation in porcine coronary arteries

Background: Intracoronary radiation (IR) studies have shown reduction of neointima formation (NF). Extrapolation of animal studies with beta-radiation to clinical trials have shown variable results, which may be related to dosimetry, centering issues, and/or shielding of beta-rays by the stent metal. We examined the effect of yttrium-90 (90Y), a pure beta-emitter delivered via an automatic afterloader to a centering catheter, on the inhibition of NF in balloon-injured (BI) porcine coronary arteries as well as in arteries receiving 90Y either prior to or following stent implantation (SI).Methods: Twenty-three swine (44 coronary arteries) were studied. In the first study, IR (18 Gy at 1.2 mm from the balloon surface) was administered in 17 arteries following BI, while eight control arteries were subjected to BI only. In the second study, 10 swine (19 coronary arteries) underwent SI. IR (18 Gy) was administered in six arteries before and in eight arteries after SI, while five control arteries received SI only. The animals were sacrificed 2 weeks after BI and 4 weeks after SI. Their coronaries were perfusion fixed and stained, and vessel parameters (intimal area [IA] and medial fracture length [FL]) were analyzed by computer-aided histomorphometry.Results: Arteries subjected to IR following BI had less NF compared to controls (IA/FL=0.14+/-0.2 mm vs. 0.49+/-0.2 mm; P=0.003). IA was reduced significantly in the arteries receiving radiation before and after SI compared to controls (0.92+/-0.98 and 0.00+/-0/00 vs. 2.72+/-1.2 mm(2); P=0.014), despite similar SI in all groups.Conclusions: IR with 90Y delivered via a centering catheter is safe and effective with complete and homogenous inhibition of NF in the context of BI or SI in the porcine coronary model.     

Leukocyte antigen-related deficiency enhances insulin-like growth factor-1 signaling in vascular smooth muscle cells and promotes neointima formation in response to vascular injury

Increase in the expression of leukocyte antigen-related (LAR) protein causes insulin resistance, an important contributor to atherosclerosis. However, the function of LAR in atherosclerosis is not known. To address whether LAR is important in the response of vascular cells to atherogenic stimuli, we investigated cell proliferation, migration, and insulin-like growth factor-1 receptor (IGF-1R) signaling in wild-type and LAR(-/-) mouse vascular smooth muscle cells (VSMC) treated with IGF-1. Absence of LAR significantly enhanced proliferation and migration of VSMC compared with wild-type cells after IGF-1 treatment. U0126 and LY249002, specific inhibitors of MAPK/ERK kinase (MEK) and phosphoinositide 3-kinase, respectively, inhibited IGF-1-induced DNA synthesis and migration in both wild-type and LAR(-/-) VSMC. IGF-1 markedly enhanced IGF-1R phosphorylation in both wild-type and LAR(-/-) VSMC, but the phosphorylation was 90% higher in knock-out cells compared with wild-type cells. Absence of LAR enhanced phosphorylation of insulin receptor substrate-1 and insulin receptor substrate-1-associated phosphoinositide 3-kinase activity in VSMC treated with IGF-1. IGF-1-induced phosphorylation of ERK1/2 also increased significantly in LAR(-/-) VSMC compared with wild-type cells. Furthermore, LAR directly binds to IGF-1R in glutathione S-transferase-LAR pull-down and IGF-1R immunoprecipitation experiments and recombinant LAR dephosphorylates IGF-1R in vitro. Neointima formation in response to arterial injury and IGF-1R phosphorylation in neointima increased significantly in LAR(-/-) mice compared with wild-type mice. A significant decrease in body weight, fasting insulin, and IGF-1 levels were observed in LAR(-/-) mice compared with wild-type mice. Together, these data indicate that LAR regulates IGF-1R signaling in VSMC and dysregulation of this phosphatase may lead to VSMC hyperplasia.     

Magnolol inhibits migration of vascular smooth muscle cells via cytoskeletal remodeling pathway to attenuate neointima formation

BackgroundIncreased proliferation and migration of vascular smooth muscle cells (VSMCs) contribute importantly to the formation of both atherosclerotic and restenotic lesions. The objective of this study was to investigate the effect of magnolol on VSMC migration.MethodsThe proteolytic activity of matrix metalloproteinases (MMPs) in tumor necrosis factor alpha (TNF-α) stimulated VSMCs was performed by gelatin zymography. VSMC migration was assessed by wound healing and Boyden chamber methods. Collagen induced VSMC adhesion was determined by spectrofluorimeter and stress fibers formation was evaluated by fluorescence microscope. The expression of signaling molecules involved in stress fibers formation was determined by western blot. The phosphorylation of myosin light chain (MLC20) was determined by urea-glycerol polyacrylamide gel electrophoresis. Immunohistochemistry was performed to determine the expression of β1-integrin and collagen type I in the injured carotid arteries of rats on day 35 after vascular injury.ResultsVSMC migration was strongly inhibited by magnolol without affecting MMPs expression. Also, magnolol inhibited β1-integrin expression, FAK phosphorylation and RhoA and Cdc42 activation to inhibit the collagen induced stress fibers formation. Moreover, magnolol inhibited the phosphorylation of MLC20. Our in vivo results showed that magnolol inhibited β1-integrin expression, collagen type I deposition and FAK phosphorylation in injured carotid arteries without affecting MMP-2 activity.ConclusionsMagnolol inhibited VSMC migration via inhibition of cytoskeletal remodeling pathway to attenuate neointima formation.General significanceThis study provides a rationale for further evaluation of magnolol for the management of atherosclerosis and restenosis.     

PLD1 promotes reactive oxygen species production in vascular smooth muscle cells and injury-induced neointima formation

Phospholipase D (PLD) generates the signaling lipid phosphatidic acid (PA) and has been known to mediate proliferation signal in vascular smooth muscle cells (VSMCs). However, it remains unclear how PLD contributes to vascular diseases. VSMC proliferation directly contributes to the development and progression of cardiovascular disease, such as atherosclerosis and restenosis after angioplasty. Using the mouse carotid artery ligation model, we find that deletion of Pld1 gene inhibits neointima formation of the injuried blood vessels. PLD1 deficiency reduces the proliferation of VSMCs in both injured artery and primary cultures through the inhibition of ERK1/2 and AKT signals. Immunohistochemical staining of injured artery and flow cytometry analysis of VSMCs shows a reduction of the levels of reactive oxygen species (ROS) in Pld1^?/? VSMCs. An increase of intracellular ROS by hydrogen peroxide stimulation restored the reduced activities of ERK and AKT in Pld1^?/? VSMCs, whereas a reduction of ROS by N-acetyl-l-cysteine (NAC) scavenger lowered their activity in wild-type VSMCs. These results indicate that PLD1 plays a critical role in neointima, and that PLD1 mediates VSMC proliferation signal through promoting the production of ROS. Therefore, inhibition of PLD1 may be used as a therapeutic approach to suppress neointimal formation in atherosclerosis and restenosis after angioplasty.     

USP10 exacerbates neointima formation by stabilizing Skp2 protein in vascular smooth muscle cells

The underlying mechanism of neointima formation remains unclear. Ubiquitin-specific peptidase 10 (USP10) is a deubiquitinase that plays a major role in cancer development and progression. However, the function of USP10 in arterial restenosis is unknown. Herein, USP10 expression was detected in mouse arteries and increased after carotid ligation. The inhibition of USP10 exhibited thinner neointima in the model of mouse carotid ligation. In vitro data showed that USP10 deficiency reduced proliferation and migration of rat thoracic aorta smooth muscle cells (A7r5) and human aortic smooth muscle cells (HASMCs). Mechanically, USP10 can bind to Skp2 and stabilize its protein level by removing polyubiquitin on Skp2 in the cytoplasm. The overexpression of Skp2 abrogated cell cycle arrest induced by USP10 inhibition. Overall, the current study demonstrated that USP10 is involved in vascular remodeling by directly promoting VSMC proliferation and migration via stabilization of Skp2 protein expression.     

Contribution of the endoplasmic reticulum to peroxisome formation

How peroxisomes are formed in eukaryotic cells is unknown but important for insight into a variety of diseases. Both human and yeast cells lacking peroxisomes due to mutations in PEX3 or PEX19 genes regenerate the organelles upon reintroduction of the corresponding wild-type version. To evaluate how and from where new peroxisomes are formed, we followed the trafficking route of newly made YFP-tagged Pex3 and Pex19 proteins by real-time fluorescence microscopy in Saccharomyces cerevisiae. Remarkably, Pex3 (an integral membrane protein) could first be observed in the endoplasmic reticulum (ER), where it concentrates in foci that then bud off in a Pex19-dependent manner and mature into fully functional peroxisomes. Pex19 (a farnesylated, mostly cytosolic protein) enriches first at the Pex3 foci on the ER and then on the maturing peroxisomes. This trafficking route of Pex3-YFP is the same in wild-type cells. These results demonstrate that peroxisomes are generated from domains in the ER.     

Genistein suppresses leptin‐induced proliferation and migration of vascular smooth muscle cells and neointima formation

Obesity is a strong risk factor for the development of cardiovascular diseases and is associated with a marked increase in circulating leptin concentration. Leptin is a peptide hormone mainly produced by adipose tissue and is regulated by energy level, hormones and various inflammatory mediators. Genistein is an isoflavone that exhibits diverse health‐promoting effects. Here, we investigated whether genistein suppressed the atherogenic effect induced by leptin. The A10 cells were treated with leptin and/or genistein, and then the cell proliferation and migration were analysed. The reactive oxygen species (ROS) and proteins levels were also measured, such as p44/42MAPK, cell cycle‐related protein (cyclin D1 and p21) and matrix metalloproteinase‐2 (MMP‐2). Immunohistochemistry and morphometric analysis were used for the neointima formation in a rat carotid artery injury model. Genistein (5 μM) significantly inhibited both the proliferation and migration of leptin (10 ng/ml)‐stimulated A10 cells. In accordance with these finding, genistein decreased the leptin‐stimulated ROS production and phosphorylation of the p44/42MAPK signal transduction pathway. Meanwhile, genistein reversed the leptin‐induced expression of cyclin D1, and cyclin‐dependent kinase inhibitor, p21. Genistein attenuated leptin‐induced A10 cell migration by inhibiting MMP‐2 activity. Furthermore, the leptin (0.25 mg/kg)‐augmented neointima formation in a rat carotid artery injury model was attenuated in the genistein (5 mg/kg body weight)‐treated group when compared with the balloon injury plus leptin group. Genistein was capable of suppressing the atherogenic effects of leptin in vitro and in vivo, and may be a promising candidate drug in the clinical setting.     

Manganese superoxide dismutase inhibits neointima formation through attenuation of migration and proliferation of vascular smooth muscle cells

Superoxide anion is elevated during neointima development and is essential for neointimal vascular smooth muscle cell (VSMC) proliferation. However, little is known about the role of manganese superoxide dismutase (MnSOD, SOD2) in the neointima formation following vascular injury. SOD2 in the mitochondria plays an important role in cellular defense against oxidative damage. Because of its subcellular localization, SOD2 is considered the first line of defense against oxidative stress and plays a central role in metabolizing superoxide. Because mitochondria are the most important sources of superoxide anion, we speculated that SOD2 may have therapeutic benefits in preventing vascular remodeling. In this study, we used a rat carotid artery balloon-injury model and an adenoviral gene delivery approach to test the hypothesis that SOD2 suppresses vascular lesion formation. SOD2 was activated along with the progression of neointima formation in balloon-injured rat carotid arteries. Depletion of SOD2 by RNA interference markedly promoted the lesion formation, whereas SOD2 overexpression suppressed the injury-induced neointima formation via attenuation of migration and proliferation of VSMCs. SOD2 exerts its inhibitory effect on VSMC migration induced by angiotensin II by scavenging superoxide anion and suppressing the phosphorylation of Akt. Our data indicate that SOD2 is a negative modulator of vascular lesion formation after injury. Therefore, SOD2 augmentation may be a promising therapeutic strategy for the prevention of lesion formation in proliferative vascular diseases such as restenosis.     

Consistency of interneuronal group formation in response to stimulation of identified cells

In crayfish stimulation of abdominal positioning interneurons (APIs) recruits other interneurons producing various abdominal movements. We investigated whether: (1) the same API from different preparations activated a similar number or group of interneurons, (2) different APIs activated different groups, and (3) repeated stimulation of an API consistently affected a similar set of interneurons. To quantify the similarities and differences of the recruited interneuronal groups we compared the number of interneurons affected, their firing frequencies, and motor outputs. Three types of APIs (Curly Q, L and T) were identified and each type was stimulated in three preparations. Our results showed that for the Curly Q and L cells, each cell type activated interneuronal groups that were statistically similar in number and firing frequency. The T cell activated interneuronal groups that were more variable. Some APIs generally provided a repeatable motor output; all did not. The interneuronal groups activated by the Curly Q, L and T cells were very different from each other. Repeated stimulation of one Curly Q cell affected similar although not identical sets of interneurons. These data suggest that repeated motor outputs could be produced by a similar but not identical group of cells. Accepted: 29 September 1997     

Contribution of the plasma membrane and central vacuole in the formation of autolysosomes in cultured tobacco cells

Autolysosomes accumulate in tobacco cells cultured under sucrose starvation conditions in the presence of a cysteine protease inhibitor. We characterized these plant autolysosomes using fluorescent dyes and green fluorescent protein (GFP). Observation using the endocytosis markers, FM4-64 and Lucifer Yellow CH, suggested that there is a membrane flow from the plasma membrane to autolysosomes. Using these dyes as well as GFP-AtVam3p, sporamin-GFP and gamma-VM23-GFP fusion proteins as markers of the central vacuole, we found transport of components of the central vacuole to autolysosomes. Thus endocytosis and the supply from the central vacuole may contribute to the formation of autolysosomes.     

The role of extracellular vesicles in neointima formation post vascular injury

Pathological neointimal growth can develop in patients as a result of vascular injury following percutaneous coronary intervention and coronary artery bypass grafting using autologous saphenous vein, leading to arterial or vein graft occlusion. Neointima formation driven by intimal hyperplasia occurs as a result of a complex interplay between molecular and cellular processes involving different cell types including endothelial cells, vascular smooth muscle cells and various inflammatory cells. Therefore, understanding the intercellular communication mechanisms underlying this process remains of fundamental importance in order to develop therapeutic strategies to preserve endothelial integrity and vascular health post coronary interventions. Extracellular vesicles (EVs), including microvesicles and exosomes, are membrane-bound particles secreted by cells which mediate intercellular signalling in physiological and pathophysiological states, however their role in neointima formation is not fully understood. The purification and characterization techniques currently used in the field are associated with many limitations which significantly hinder the ability to comprehensively study the role of specific EV types and make direct functional comparisons between EV subpopulations. In this review, the current knowledge focusing on EV signalling in neointima formation post vascular injury is discussed.     

Antisense oligodeoxyribonucleotide as to the growth factor midkine suppresses neointima formation induced by balloon injury

Restenosis is the major clinical problem of angioplasty. We have previously shown that neointima formation is strikingly suppressed in midkine (MK)-deficient mice. Neointima formation is restored if MK protein is administrated to the deficient mice. MK is a heparin-binding growth factor and implicated in the migration of inflammatory cells and vascular smooth muscle cells. Consistently, the suppression of neointima formation in the deficient mice is accompanied by suppression of recruitment of inflammatory cells into the vascular wall. Here, we evaluated the potential of MK antisense oligodeoxyribonucleotide (ODN) for the prevention of restenosis. We cloned the cDNA of rabbit MK, which showed a strongly conserved sequence in mammals. The balloon injury induced MK expression, with the maximum level occurring 7-14 days after angioplasty, in the rabbit carotid artery. Two antisense ODNs suppressed the production of MK in a rabbit kidney cell line, RK13 cells, one of which was then transfected into the arterial wall by means of lipofection immediately after balloon treatment. The antisense ODN suppressed MK induction in vivo and consequently suppressed neointima formation to 60% of the control level. These results suggest that MK is a candidate molecular target for the therapy for vascular restenosis.