Arterial Neointima Research Articles

Deletion of angiotensin-converting enzyme 2 promotes the development of atherosclerosis and arterial neointima formation

Angiotensin-converting enzyme 2 (ACE2) is known as a negative regulator of the renin-angiotensin system. We aimed to determine the roles of ACE2 on the development of vascular diseases. Using two diversely different models of vascular diseases, hyperlipidaemia-induced atherosclerosis in apolipoprotein E knockout (KO) mice and mechanical injury-induced arterial neointimal hyperplasia in C57Bl6 mice, we examined whether ACE2 deficiency could affect formation of the vascular lesions. ACE2 deficiency resulted in significantly larger vascular lesions in both aortic atherosclerotic plaques and arterial neointima formation, compared with ACE2(+) control. These ACE2-deficient vascular lesions exhibited enhanced accumulation of macrophages into the lesions and proliferation of vascular smooth muscle cells (VSMCs), accompanied with increased angiotensin-II (Ang-II) levels and enhanced expression of vascular inflammation-related genes, including vascular cell adhesion molecule (VCAM)-1, monocyte chemoattractant protein (MCP)-1, and matrix metalloproteinase (MMP)9 in aorta/artery tissues. Primary bone marrow macrophages and aortic VSMCs isolated from ACE2 KO mice also displayed enhanced pro-inflammatory responsiveness such as up-regulated gene/protein expression of VCAM-1, MCP-1, and MMP9 to stimulation with tumour necrosis factor-α and Ang-II. The similar phenotype was shown in human macrophages and aortic VSMCs that were transfected with ACE2-specific siRNA. In ACE2-deficient VSMCs, inhibition of c-Jun N-terminal kinase (JNK) by pharmacological blockade with SP600125 or genetic knockdown with JNK-specific siRNA significantly attenuated their pro-inflammatory phenotype. ACE2 deficiency promotes the development of vascular diseases associated with Ang-II-mediated vascular inflammation and activation of the JNK signalling, leading to the notion that ACE2 potentially confers protection against vascular diseases.

Renal Heparan Sulfate Proteoglycans Modulate Fibroblast Growth Factor 2 Signaling in Experimental Chronic Transplant Dysfunction

Depending on the glycan structure, proteoglycans can act as coreceptors for growth factors. We hypothesized that proteoglycans and their growth factor ligands orchestrate tissue remodeling in chronic transplant dysfunction. We have previously shown perlecan to be selectively up-regulated in the glomeruli and arteries in a rat renal transplantation model. Using the same model, here we present quantitative RT-PCR profiling data on proteoglycans and growth factors from laser-microdissected glomeruli, arterial tunicae mediae, and neointimae at 12 weeks after transplantation. In glomeruli and neointimae of allografts, selective induction of the matrix heparan sulfate proteoglycan perlecan was observed, along with massive accumulation of fibroblast growth factor 2 (FGF2). Profiling the heparan sulfate polysaccharide side chains revealed conversion from a non-FGF2-binding heparan sulfate phenotype in control and isografted kidneys toward a FGF2-binding phenotype in allografts. In vitro experiments with perlecan-positive rat mesangial cells showed that FGF2-induced proliferation is dependent on sulfation and can be inhibited by exogenously added heparan sulfate. These findings indicate that matrix proteoglycans such as perlecan serve as functional docking platforms for FGF2 in chronic transplant dysfunction. We speculate that heparin-like glycomimetics could be a promising intervention to retard development of glomerulosclerosis and neointima formation in chronic transplant dysfunction.

PI3K/p110alpha inhibition selectively interferes with arterial thrombosis and neointima formation, but not re-endothelialization: a new concept in drug-eluting stent design

Background: Impaired re-endothelialization and stent thrombosis are a safety concern associated with drug-eluting stents (DES) as mTOR inhibitors reduce restenosis rates, but impair endothelialization and facilitate thrombus formation. PI3K/p110α controls cellular wound healing pathways, thereby representing an emerging drug target to modulate vascular homeostasis after injury. However, its effect on arterial thrombosis and neointima formation remains unknown. Methods: PI3K/p110α was inhibited by treatment with the small molecule inhibitor PIK75 or a specific siRNA. Arterial thrombosis, neointima formation and re-endothelialization studies were performed in a murine carotid artery injury model. Proliferation and migration of VSMC and EC were assessed by cell number and Boyden chamber, respectively. Endothelial senescence was evaluated by β-galactosidase assay, endothelial dysfunction by organ chambers for isometric tension. Tissue factor (TF), plasminogen activator inhibitor type 1 (PAI-1), and eNOS expression was determined by Western blot and real-time PCR analysis. Results: Male C57Bl6 mice were either treated with PIK75 (10 mg/kg/d for 7 days) or vehicle. Arterial thrombus formation was delayed in mice treated with PIK75 as compared to controls (n=8; p<0.005). PIK 75 impaired arterial expression and activity of TF and PAI-1 as well as NFκB activity (n=8; p<0.05); in contrast, plasma clotting and tail bleeding times did not differ (n=8; p=NS). In human vascular smooth muscle and endothelial cells, PIK75 inhibited expression and activity of TF and PAI-1 (n=4; p<0.01). These effects occurred at the transcriptional level via the RhoA signaling cascade and the transcription factor NFkB. Furthermore, inhibition of PI3K/p110α with PIK75 or a specific siRNA selectively impaired proliferation and migration of VSMC while sparing EC completely. In line with this observation, treatment with PIK75 selectively inhibited neointima formation without affecting re-endothelialization following vascular injury. In contrast to rapamycin and paclitaxel, treatment with PIK75 did not induce endothelial senescence nor inhibit eNOS expression or endothelium-dependent relaxations. Conclusions: Following vascular injury, PI3K/p110α inhibition selectively interferes with arterial thrombosis and neointima formation, but not reendothelialization. Hence, PI3K/p110α represents an attractive new target in DES design.

20-Hydroxyeicosatetraenoic Acid Inhibition Attenuates Balloon Injury-Induced Neointima Formation and Vascular Remodeling in Rat Carotid Arteries

20-Hydroxyeicosatetraenoic acid (20-HETE) contributes to the migration and proliferation of vascular smooth muscle cells (VSMC) in vitro, but there are few studies that address its effects on vascular remodeling in vivo. The present study determined whether inhibition of 20-HETE production attenuates intimal hyperplasia (IH) and vascular remodeling after balloon injury (BI). Sprague Dawley rats underwent BI of the common carotid artery and were treated with vehicle, 1-aminobenzotriazole (ABT, 50 mg/kg i.p. once daily), or HET0016 (N-hydroxy-N'-(4-butyl-2-methylphenyl)-formamidine) (2 mg/kg s.c. twice daily) for 14 days. Fourteen days after BI and treatment, the animals underwent carotid angiography, and the arteries were harvested for morphometric, enzymatic and immunohistochemical analysis. There was a 96% reduction of angiographic stenosis in the rats treated with 1-ABT. There was a 61 and 66% reduction of the intima/media area ratios in the 1-ABT and HET0016 treated rats compared with the vehicle-treated group. 20-HETE levels were elevated in BI carotid arteries, and the levels were markedly suppressed in the groups treated with 1-ABT and HET0016 (P < 0.001). Immunostaining revealed that the expression of CYP4A enzyme was markedly increased in the neointima of BI arteries, and it colocalized with the expression of smooth muscle-specific actin, indicating increased proliferation of VSMC. An increase in the expression of CYP4A and the production of 20-HETE contributes to neointimal growth in BI rat carotid arteries. Systemic administration 1-ABT or HET0016 prevents the increase in 20-HETE levels and attenuates VSMC migration and proliferation, resulting in a marked reduction in IH and vascular remodeling after endothelial injury.

Platelet IκB Kinase-β Deficiency Increases Mouse Arterial Neointima Formation via Delayed Glycoprotein Ibα Shedding

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-κB is a critical molecule in platelet activation. Here, we investigated the role of the platelet nuclear factor-κB 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 IκB kinase-β (IKKβ) (IKKβ(fl/fl)/PF4(cre)/LDLR(-/-)) and in control mice (IKKβ(fl/fl)/LDLR(-/-)). The size of the arterial neointima was 61% larger in the IKKβ(fl/fl)/PF4(cre)/LDLR(-/-) mice compared with the littermate control IKKβ(fl/fl)/LDLR(-/-) mice. Compared with the control mice, the IKKβ(fl/fl)/PF4(cre)/LDLR(-/-) mice exhibited more leukocyte adhesion at the injured area. The extent of glycoprotein Ibα shedding after platelet activation was compromised in the IKKβ-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 Ibα shedding in activated IKKβ-deficient platelets. Platelet IKKβ deficiency increases the formation of injury-induced arterial neointima formation. Thus, nuclear factor-κB-related inhibitors should be carefully evaluated for use in patients after an arterial intervention.

Proprotein convertase furin enhances survival and migration of vascular smooth muscle cells via processing of pro-nerve growth factor

Maturation of nerve growth factor (NGF) in neuronal cells requires endoproteolytic processing of the precursor protein proNGF to β-NGF by the proprotein convertase furin. Pro- and β-NGF elicit opposite biological functions by differential neurotrophin-receptor binding, leading to apoptosis via sortilin or survival via neurotrophic tyrosine kinase receptor type-1 (TrkA), respectively. The present study was done to investigate the impact of furin-dependent proNGF processing on vascular smooth muscle cell (VSMC) function. We found that β-NGF mRNA and protein expression was upregulated in platelet-derived growth factor-BB/transforming growth factor-β1-stimulated, proliferating rat aortic VSMCs. Although β-NGF itself did not affect VSMC proliferation, it promoted VSMC motility in an autocrine fashion via TrkA/Akt-dependent integrin inside-out signalling. The β-NGF-induced migration of VSMCs required proNGF processing by furin, which was co-regulated with NGF. Furin-inhibition increased proNGF and reduced β-NGF secretion, leading to apoptosis rather than migration. In line with our in vitro demonstration, we found co- and upregulation of NGF, its convertase furin and its high-affinity receptor TrkA in the neointima of balloon-injured rodent arteries. These results indicate that furin determines the balance between proNGF and β-NGF in proliferating VSMCs, thus impacting on VSMC survival and migration and is also important in neointima formation.

Effects of chemokine-like factor 1 on vascular smooth muscle cell migration and proliferation in vascular inflammation

ObjectiveVascular smooth muscle cell (VSMC) migration and proliferation are key components of vascular inflammation that may lead to atherosclerosis and restenosis, in which cytokines are considered as pivotal factors regarding recruitment of VSMC. A member of recently described family of chemokines, chemokine-like factor 1 (CKLF1), displays a wide spectrum of chemotaxis. This study investigated the role of CKLF1 in VSMC migration and proliferation during the process of vascular inflammation. Methods and results: The effects of CKLF1 on migration, proliferation and neointimal formation were investigated in cultured VSMCs, rat balloon injured arteries and human atherosclerotic plaques. CKLF1 overexpression greatly enhanced, whereas shRNA knockdown markedly retarded, VSMC migration and proliferation in vitro. In addition, CKLF1 protein accumulated preferentially in neointima of the injured rat arteries in vivo. CKLF1 overexpression resulted in a 2.5-fold increase in intimal thickness. In contrast, shRNA-mediated CKLF1 knockdown significantly suppressed neointima formation by 70% compared that in control group. Intriguingly, besides animal model, higher level of CKLF1 was observed in human atherosclerotic plaques than that in normal arteries. ConclusionCKLF1 plays an essential role in migration and proliferation of VSMCs, which in turn facilitated neointimal hyperplasia and atherosclerosis. Inhibition of CKLF1 activity provides a potential target for the prevention of atherosclerosis and restenosis.

β2-Integrin and Notch-1 differentially regulate CD34+CD31+ cell plasticity in vascular niches

The implication of circulating haematopoietic CD34(+) progenitors in the vasculature is unclear due to the lack of understanding of their characteristics and plasticity mediated by their cellular microenvironment. We investigated how vascular smooth muscle cells (SMCs) and their interactions with endothelial cells (ECs) affect the behaviour and plasticity of CD34(+)CD31(+) progenitors and the underlying mechanisms. Human peripheral blood-derived CD34(+)CD31(+) cells were directly transplanted into injured arteries in vivo and co-cultured with ECs and SMCs in vitro. CD34(+)CD31(+) progenitors injected into wire-injured mouse arteries differentiate into ECs and macrophages in the neoendothelial layer and neointima, respectively. SMC-co-culture increases CD34(+)CD31(+) cell mobility and adhesion to and transmigration across ECs. Sorted CD34(+)CD31(+) progenitors that adhered to ECs co-cultured with SMCs have the capacity to form capillary-like structures in Matrigel and chimeric blood vessels in vivo. Sorted transmigrated progenitors give rise to macrophages with increased pro-angiogenic activity. These differentiations of CD34(+)CD31(+) progenitors into ECs and macrophages are mediated by β(2)-integrin and Notch-1, respectively. β(2)-Integrin and Notch-1 are activated by their counterligands, intercellular adhesion molecule-1 (ICAM-1) and jagged-1, which are highly expressed in the neoendothelium and neointima in injured arteries. Intra-arterial injection of β(2)-integrin-activated CD34(+)CD31(+) progenitors into wire-injured mouse arteries inhibits neointima formation. Our findings indicate that the peripheral vascular niches composed of ECs and SMCs may predispose haematopoietic CD34(+)CD31(+) progenitors to differentiate into ECs and macrophages through the activations of the ICAM-1/β(2)-integrin and jagged-1/Notch-1 cascades, respectively.

From Skeleton to Cytoskeleton

Rationale: The expression of osteocalcin is augmented in human atherosclerotic lesions. How osteocalcin triggers vascular pathogenesis and remodeling is unclear. Objective: To investigate whether osteocalcin promotes transformation of adventitial fibroblast to myofibroblasts and the molecular mechanism involved. Methods and Results: Immunohistochemistry indicated that osteocalcin was expressed in the neointima of renal arteries from diabetic patients. Western blotting and wound-healing assay showed that osteocalcin induced fibroblast transformation and migration, which were attenuated by blockers of the renin-angiotensin system and protein kinase Cδ (PKCδ), toll-like receptor 4 (TLR4) neutralizing antibody, and antagonist and inhibitors of free radical production and cyclooxygenase-2. Small interfering RNA silencing of TLR4 and PKCδ abolished fibroblast transformation. Angiotensin II level in the conditioned medium from the osteocalcin-treated fibroblasts was found elevated using enzyme immunoassay. Culturing of fibroblasts in conditioned medium collected from differentiated osteoblasts promoted fibroblast transformation. The expression of fibronectin, TLR4, and cyclooxygenase-2 is augmented in human mesenteric arteries after 5-day in vitro exposure to osteocalcin. Conclusions: Osteocalcin transforms adventitial fibroblasts to myofibroblasts through stimulating angiotensin II release and subsequent activation of PKCδ/TLR4/reactive oxygen species/cyclooxygenase-2 signaling cascade. This study reveals that the skeletal hormone osteocalcin cross-talks with vascular system and contributes to vascular remodeling.

Molecular Imaging of Vascular Endothelial Growth Factor Receptors in Graft Arteriosclerosis

Vascular endothelial growth factor (VEGF) signaling plays a key role in the pathogenesis of vascular remodeling, including graft arteriosclerosis. Graft arteriosclerosis is the major cause of late organ failure in cardiac transplantation. We used molecular near-infrared fluorescent imaging with an engineered Cy5.5-labeled single-chain VEGF tracer (scVEGF/Cy) to detect VEGF receptors and vascular remodeling in human coronary artery grafts by molecular imaging. VEGF receptor specificity of probe uptake was shown by flow cytometry in endothelial cells. In severe combined immunodeficiency mice, transplantation of human coronary artery segments into the aorta followed by adoptive transfer of allogeneic human peripheral blood mononuclear cells led to significant neointima formation in the grafts over a period of 4 weeks. Near-infrared fluorescent imaging of transplant recipients at 4 weeks demonstrated focal uptake of scVEGF/Cy in remodeling artery grafts. Uptake specificity was demonstrated using an inactive homolog of scVEGF/Cy. scVEGF/Cy uptake predominantly localized in the neointima of remodeling coronary arteries and correlated with VEGF receptor-1 but not VEGF receptor-2 expression. There was a significant correlation between scVEGF/Cy uptake and transplanted artery neointima area. Molecular imaging of VEGF receptors may provide a noninvasive tool for detection of graft arteriosclerosis in solid organ transplantation.

RAGE signaling mediates post-injury arterial neointima formation by suppression of liver kinase B1 and AMPK activity

ObjectiveIntima formation involves smooth muscle cell (SMC) proliferation and migration that ultimately drives arterial stenosis, thrombosis, and ischemia in atherosclerosis, hypertension, and arterial revascularization. Receptor for advanced glycation endproducts (RAGE) is a transmembrane signaling receptor implicated in diabetic renal and vascular complications, and post-injury intima formation, partly via Signal transducer and activator of transcription 3 (STAT3) activation. The metabolic super-regulator Adenosine monophosphate kinase (AMPK) inhibits SMC proliferation and intima formation. AMPK activation is promoted by liver kinase B1 (LKB1), and LKB1 inhibits STAT3 activation. Here, we tested the hypothesis that RAGE promotes arterial intima formation by modulating both LKB1 and AMPK. Methods and resultsRAGE ligands (the calgranulin S100A11, and glycated albumin) suppressed AMPK activation in conjunction with increased proliferation and migration of cultured SMCs. These effects were inhibited both by RAGE deficiency and by prior AMPK activation. In SMCs, RAGE ligands decreased LKB1 activity. Moreover, knockdown of both LKB1 and AMPK were associated with increased STAT3 phosphorylation levels. In response to murine carotid artery ligation, expression of RAGE and S100A11 increased, whereas AMPK and LKB1 activity decreased in situ. Conversely, LKB1 and AMPK activity increased in situ, and neointima formation was attenuated in Rage−/− mice. ConclusionThe linkage of decreased LKB1 and AMPK activity with increased STAT3 in SMCs mediates the capacity of RAGE ligand-induced signaling to promote neointima formation in response to arterial injury.

357 Comparative Effects of the Drugs Used for Pulmonary Hypertension Reversibility Testing: A Meta-Analysis

pyruvate dehydrogenase kinase (PDK) 2, catalyzing the phosphorylation of pyruvate dehydrogenase (PDH) and thus regulating the flux of pyruvate into the mitochondria, were investigated during SMC proliferation and the development of human IH. Methods and Materials: A novel human intimal hyperplasia (IH) model was established in which balloon-injured human internal mammary arteries (hMA) were transplanted into the abdominal aortic position of nude rats. Results: IH rapidly developed over 21 days and was morphologically and in immunohistochemistry identical to the neointima of human coronary arteries. Over 21 days, we found increased proliferative and decreased apoptotic activity within the IH lesion and luminal obliteration progressed to approx. 60%. Tissue PDK2 expression was significantly elevated and PDH was inhibited.PDGF was markedly elevated in the animal serum during neointima formation. Activation of SMC in vitro with PDGF caused increased PDK2 activity via both PI3K/Akt and Ras/Raf/Erkpathways. We observed an increase of the mitochondrial membrane potential ( m), a decreased release of mitochondrial reactive oxygen species (mROS), as well as enhanced cell proliferation. PDK2 inhibition using dichloroacetate(DCA) or PDK2 shRNA prevented PDGF-induced mitochondrial hyperpolarization and SMC proliferation.In vivo, oral DCAtreatment of rats or pre-implant PDK2 shRNA-transduction of the hMAs prevented the injuryinduced increase of SMC proliferation and significantly decreased IH development. Conclusions: PDK2 is the link between PDGF downstream pathways and mitochondrial metabolic modulation and its activation facilitates SMC proliferation. Pharmacological or genetic inhibition of PDK2 in SMC attenuates PDGF-induced increase in m, cell proliferation and the development of human IH. PDK2 may be a novel target for the prevention of restenosis.

Characterization of proximal pulmonary arterial cells from chronic thromboembolic pulmonary hypertension patients

BackgroundChronic thromboembolic pulmonary hypertension (CTEPH) is associated with proximal pulmonary artery obstruction and vascular remodeling. We hypothesized that pulmonary arterial smooth muscle (PASMC) and endothelial cells (PAEC) may actively contribute to remodeling of the proximal pulmonary vascular wall in CTEPH. Our present objective was to characterize PASMC and PAEC from large arteries of CTEPH patients and investigate their potential involvement in vascular remodeling.MethodsPrimary cultures of proximal PAEC and PASMC from patients with CTEPH, with non-thromboembolic pulmonary hypertension (PH) and lung donors have been established. PAEC and PASMC have been characterized by immunofluorescence using specific markers. Expression of smooth muscle specific markers within the pulmonary vascular wall has been studied by immunofluorescence and Western blotting. Mitogenic activity and migratory capacity of PASMC and PAEC have been investigated in vitro.ResultsPAEC express CD31 on their surface, von Willebrand factor in Weibel-Palade bodies and take up acetylated LDL. PASMC express various differentiation markers including α-smooth muscle actin (α-SMA), desmin and smooth muscle myosin heavy chain (SMMHC). In vascular tissue from CTEPH and non-thromboembolic PH patients, expression of α-SMA and desmin is down-regulated compared to lung donors; desmin expression is also down-regulated in vascular tissue from CTEPH compared to non-thromboembolic PH patients. A low proportion of α-SMA positive cells express desmin and SMMHC in the neointima of proximal pulmonary arteries from CTEPH patients. Serum-induced mitogenic activity of PAEC and PASMC, as well as migratory capacity of PASMC, were increased in CTEPH only.ConclusionsModified proliferative and/or migratory responses of PASMC and PAEC in vitro, associated to a proliferative phenotype of PASMC suggest that PASMC and PAEC could contribute to proximal vascular remodeling in CTEPH.

Increased neointimal thickening in dystrophin-deficient mdx mice.

BackgroundThe dystrophin gene, which is mutated in Duchenne muscular dystrophy (DMD), encodes a large cytoskeletal protein present in muscle fibers. While dystrophin in skeletal muscle has been extensively studied, the function of dystrophin in vascular smooth muscle is less clear. Here, we have analyzed the role of dystrophin in injury-induced arterial neointima formation.Methodology/Principal FindingsWe detected a down-regulation of dystrophin, dystroglycan and β-sarcoglycan mRNA expression when vascular smooth muscle cells de-differentiate in vitro. To further mimic development of intimal lesions, we performed a collar-induced injury of the carotid artery in the mdx mouse, a model for DMD. As compared with control mice, mdx mice develop larger lesions with increased numbers of proliferating cells. In vitro experiments demonstrate increased migration of vascular smooth muscle cells from mdx mice whereas the rate of proliferation was similar in cells isolated from wild-type and mdx mice.Conclusions/SignificanceThese results show that dystrophin deficiency stimulates neointima formation and suggest that expression of dystrophin in vascular smooth muscle cells may protect the artery wall against injury-induced intimal thickening.

Bivalirudin Inhibits Periprocedural Platelet Function and Tissue Factor Expression of Human Smooth Muscle Cells

A major concern of stent implantation after percutaneous coronary intervention (PCI) is acute stent thrombosis. Effective inhibition of periprocedural platelet function in patients with coronary artery disease (CAD) leads to an improved outcome. In this study, we examined the periprocedural platelet reactivity after administrating bivalirudin during PCI compared to unfractionated heparin (UFH) administration. Further, the effect of bivalirudin on induced tissue factor (TF) expression in smooth muscle cells (SMC) was determined. Patients with CAD (n = 58) and double antithrombotic medication were treated intraprocedural with UFH (n = 30) or bivalirudin (n = 28). Platelet activation markers were flow cytometrically measured before and after stenting. The expression of TF in SMC was determined by real-time PCR and Western blotting. The thrombogenicity of platelet-derived microparticles and SMC was assessed via a TF activity assay. Bivalirudin significantly diminished the agonist-induced platelet reactivity post-PCI. Compared to UFH treatment, the adenosine diphosphate (ADP) and thrombin receptor-activating peptide (TRAP)-induced thrombospondin expression post-PCI was reduced when bivalirudin was administrated during intervention. In contrast to UFH, bivalirudin reduced the P-selectin expression of unstimulated and ADP-induced platelets post-PCI. Moreover, bivalirudin inhibited the thrombin-, but not FVIIa- or FVIIa/FX-induced TF expression and pro-coagulant TF activity of SMC. Moreover, bivalirudin reduced the TF activity of platelet-derived microparticles postinduction with TRAP or ADP. Bivalirudin is better than UFH in reducing periprocedural platelet activation. Moreover, thrombin-induced TF expression is inhibited by bivalirudin. Thus, bivalirudin seems to be a better anticoagulant during PCI than UFH.

Abstract 17094: “Trojan Horse” Monocytes: a New Approach for Gene Delivery to Angioplastied Arteries

Introduction: Monocyte-derived macrophages are implicated in sustained inflammation typical of atherosclerotic and restenotic arteries. Since the role of macrophage-mediated inflammatory reactions in the progression of atherosclerosis and post-angioplasty restenosis is firmly established, multiple approaches have been devised to decrease the number and/or functionality of mononuclear phagocytic cells residing in atherosclerotic and stented arteries. However, no prior research has exploited the capability of monocytes to target sites of vascular injury in order to introduce pro-healing “therapeutic” genes into diseased vasculature. Here we report preliminary results demonstrating feasibility of this concept. Methods and Results: Monocytes were isolated from heparinized rat blood by sequential Ficoll and Percoll gradient centrifugation. The cells labeled with CM-DiI dye (5x10 5 cells/ml) were suspended in complete RPMI1640 medium and administered via tail vein to rats that underwent balloon denudation injury of carotid arteries 4 days prior to cell therapy. The animals were euthanized on day 8 after balloon injury (day 4 after monocyte delivery). The CM-DiI-labeled cells were found in the nascent neointima and adventitia of balloon-injured arteries (373±90 and 77±37 per mm 2 of tissue, respectively). Comparison of CM-DiI labeling with immunohistochemical staining results (CD 68) has demonstrated that up to 30% of all macrophages in the neointimal tissue of rats treated with ex vivo modified monocytes derive from the delivered cells. To verify functional competence of delivered cells, monocytes isolated as above were transduced with adenovectors driving synthesis of firefly luciferase and administered to rats that underwent Fogarty balloon injury 4 days prior to the cell delivery. One day later, luciferase expression in balloon injured arteries was confirmed by bioluminescence imaging. Conclusions: These pilot studies demonstrate the feasibility of using ex vivo transfected autologous or syngeneic monocytes as a means to introduce prohealing factors to diseased vasculature.

Cilostazol Promotes Vascular Smooth Muscles Cell Differentiation Through the cAMP Response Element-Binding Protein-Dependent Pathway

Cilostazol, a potent type 3 phosphodiesterase inhibitor, has recently been found to reduce neointimal formation by inhibiting vascular smooth muscle cell (VSMC) proliferation. The aim of this study is to investigate whether cilostazol exerts an action on phenotypic modulation of VSMCs, another important process in the pathogenesis of neointimal formation. Cilostazol may convert VSMCs from a serum-induced dedifferentiation state to a differentiated state, as indicated by a spindle-shaped morphology and an increase in the expression of smooth muscle cell differentiation marker contractile proteins. The upregulation of contractile proteins by cilostazol involves the cAMP/protein kinase A (PKA) signaling pathway, because the cAMP analog mimicked and specific cAMP/PKA inhibitors opposed the effect of cilostazol. Furthermore, cilostazol-activated cAMP response element (CRE)-binding protein (CREB), including phosphorylation at Ser133 and its nuclear translocation. Deletion and mutational analysis of the contractile protein promoters along with chromatin immunoprecipitation using anti-CREB antibody showed that CRE is essential for cilostazol-induced contractile protein expression. Transfection of dominant-negative CREB (mutated Ser133) plasmid in VSMCs blocked cilostazol-stimulated contractile protein expression. In vivo, cilostazol upregulated contractile proteins and induced the activation of CREB in the neointima of balloon-injured arteries. Cilostazol promotes VSMC differentiation through the cAMP/PKA/CREB signaling cascade.

The Actin-Binding Protein Girdin and Its Akt-Mediated Phosphorylation Regulate Neointima Formation After Vascular Injury

It is well established that the migration and proliferation of vascular smooth muscle cells (VSMCs) have major roles in the vascular remodeling process. Our previous study showed that the Akt substrate Girdin, which is expressed in VSMCs and endothelial cells, is essential for postnatal angiogenesis. However, the function of Girdin and its Akt-mediated phosphorylation in VSMCs and their in vivo roles in vascular remodeling remain to be elucidated. We investigated the function of Girdin and its Akt-mediated phosphorylation using cultured VSMCs and animal models of vascular remodeling. The depletion of Girdin by RNA interference disrupted the rearrangement of the actin cytoskeleton in VSMCs, resulting in impaired cell migration. The depletion of Girdin also inhibited VSMC proliferation. Girdin expression was highly upregulated and its serine at position 1416 was phosphorylated in the neointima of carotid arteries after balloon injury in a rat model. The introduction of an adenovirus harboring short hairpin RNA against Girdin attenuated the proliferation of VSMCs and neointima formation without affecting reendothelialization. Furthermore, we found that neointima formation after femoral wire injury was significantly attenuated in Girdin S1416A knock-in mice, in which the Akt phosphorylation site of Girdin was mutated, thus indicating a major role for Girdin phosphorylation in vascular remodeling. These findings indicate that Girdin and its Akt-mediated phosphorylation have major roles in the migration and proliferation of VSMCs and vascular remodeling, making the Akt/Girdin signaling pathway a potential target for the development of new therapeutics for vascular diseases.

Protective role of Gipie, a Girdin family protein, in endoplasmic reticulum stress responses in endothelial cells

Continued exposure of endothelial cells to mechanical/shear stress elicits the unfolded protein response (UPR), which enhances intracellular homeostasis and protect cells against the accumulation of improperly folded proteins. Cells commit to apoptosis when subjected to continuous and high endoplasmic reticulum (ER) stress unless homeostasis is maintained. It is unknown how endothelial cells differentially regulate the UPR. Here we show that a novel Girdin family protein, Gipie (78 kDa glucose-regulated protein [GRP78]-interacting protein induced by ER stress), is expressed in endothelial cells, where it interacts with GRP78, a master regulator of the UPR. Gipie stabilizes the interaction between GRP78 and the ER stress sensor inositol-requiring protein 1 (IRE1) at the ER, leading to the attenuation of IRE1-induced c-Jun N-terminal kinase (JNK) activation. Gipie expression is induced upon ER stress and suppresses the IRE1-JNK pathway and ER stress-induced apoptosis. Furthermore we found that Gipie expression is up-regulated in the neointima of carotid arteries after balloon injury in a rat model that is known to result in the induction of the UPR. Thus our data indicate that Gipie/GRP78 interaction controls the IRE1-JNK signaling pathway. That interaction appears to protect endothelial cells against ER stress-induced apoptosis in pathological contexts such as atherosclerosis and vascular endothelial dysfunction.

Transfusion of autologous late-outgrowth endothelial cells reduces arterial neointima formation after injury

Late-outgrowth endothelial cells (OECs) exist in blood and other organs. We aimed to explore whether and how OECs participate in re-endothelialization and prevent vascular neointima formation after injury. Rabbit bone marrow OECs were cultured for 4 weeks to increase their numbers. Transfusion of autologous OECs (2 × 10⁶-1 × 10⁷/kg) soon after rabbit ear central artery injury reduced the increase in intima area and the decrease in lumen area observed at days 14 and 28. Transfusion of autologous OECs (1 × 10⁷/kg) ameliorated some early (days 2 and 7) inflammatory and angiogenic responses (local and systemic) to the injury. Red fluorescence was seen within 7 days after transfusion of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine-labelled acetylated low-density lipoprotein (Dil-acLDL)-incorporated OECs, and 1 h after perfusion of the isolated rabbit ear with Ringer-Locke solution containing Dil-acLDL-incorporated OECs, in the injured rabbit ear central artery. After transfusion of 5-bromo-2'-deoxyuridine (BrdU) incorporated autologous OECs, BrdU-positive cells appeared in the injured artery intima at day 3 and were present in the rescued artery endothelium at day 28. The OECs, ranging from 5%-15% of vascular smooth muscle cells (VSMCs), and the OEC-conditioned medium (5-15%) both inhibited VSMC proliferation and migration in vitro and regulated the arrangement of VSMCs. The VSMCs were helpful for OECs to form tubes in vitro. Circulating OECs participate in re-endothelialization directly and inhibit VSMC migration and proliferation by a paracrine pathway; transfusion of large numbers of autologous OECs soon after vascular injury may prevent neointima formation.