Balloon-injured Arteries Research Articles

Suppression of Activation of Signal Transducer and Activator of Transcription-5B Signaling in the Vessel Wall Reduces Balloon Injury-Induced Neointima Formation

Previously, we have demonstrated that STAT-5B plays a role in thrombin-induced vascular smooth muscle cell (VSMC) growth and motility. To learn more about the role of STAT-5B in vessel wall remodeling, we examined its involvement in platelet-derived growth factor-BB (PDGF-BB)-stimulated VSMC growth and motility and balloon injury-induced neointima formation. PDGF-BB activated STAT-5B as measured by its tyrosine phosphorylation, DNA binding, and reporter gene activity. PDGF-BB induced cyclin D1 expression, CDK4 activity, and Rb protein phosphorylation, leading to VSMC growth and motility, and these responses were suppressed by the blockade of STAT-5B. Increased cyclin D1 levels, CDK4 activity, and Rb protein phosphorylation were observed in 1-week balloon-injured arteries compared with uninjured arteries, and these responses were also suppressed by adenovirus-mediated expression of dnSTAT-5B. In addition, adenovirus-mediated expression of dnSTAT-5B attenuated balloon injury-induced smooth muscle cell migration from media to intima and their proliferation in intima, resulting in reduced neointima formation. These observations indicate that STAT-5B plays an important role in PDGF-BB-induced VSMC growth and motility in vitro and balloon injury-induced neointima formation in vivo.

Inhibition of MMP-2 gene expression with small interfering RNA in rabbit vascular smooth muscle cells

Matrix metalloproteinase-2 (MMP-2) is constitutively expressed in vascular smooth muscle cells (VSMCs). Using small interfering RNA (siRNA), we evaluated the effect of MMP-2 inhibition in VSMCs in vitro and ex vivo. Rabbit VSMCs were transfected in vitro with 50 nmol/l MMP-2 siRNA or scramble siRNA. Flow cytometry and confocal microscopy showed cellular uptake of siRNA in approximately 80% of VSMCs. MMP-2 mRNA levels evaluated by real-time RT-PCR, pro-MMP-2 activity from conditioned culture media evaluated by gelatin zymography, and VSMC migration were reduced by 44 +/- 19%, 43 +/- 14%, and 36 +/- 14%, respectively, in MMP-2 siRNA-transfected compared with scramble siRNA-transfected VSMCs (P < 0.005 for all). Ex vivo MMP-2 siRNA transfection was performed 2 wk after balloon injury of hypercholesterolemic rabbit carotid arteries. Fluorescence microscopy showed circumferential siRNA uptake in neointimal cells. Gelatin zymography of carotid artery culture medium demonstrated a significant decrease of pro-MMP-2 activity in MMP-2 siRNA-transfected compared with scramble siRNA-transfected arteries (P < 0.01). Overall, our results demonstrate that in vitro MMP-2 siRNA transfection in VSMCs markedly inhibits MMP-2 gene expression and VSMC migration and that ex vivo delivery of MMP-2 siRNA in balloon-injured arteries reduces pro-MMP-2 activity in neointimal cells, suggesting that siRNA could be used to modify arterial biology in vivo.

Ubiquitin Carboxyl-Terminal Hydrolase L1, a Novel Deubiquitinating Enzyme in the Vasculature, Attenuates NF-κB Activation

We identified a ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) gene, which encodes a deubiquitinating enzyme and is expressed in the vasculature, by functional screening of a human endothelial cell (EC) cDNA library. UCHL1 is expressed in neurons, and abnormalities in UCHL1 are responsible for inherited Parkinson's disease via its effects on the ubiquitin-proteasome system. Therefore, the goal of present study was to clarify the role of the UCHL1 gene in vascular remodeling by evaluating nuclear factor-kappaB (NF-kappaB) inactivation in ECs and vascular smooth muscle cells (VSMCs). From Northern blot and immunohistochemical analysis, the UCHL1 gene was endogenously expressed in vascular ECs, VSMCs, and brain tissue. Expression of UCHL1 was markedly increased in the neointima of the balloon-injured carotid artery and was also present in atherosclerotic lesions from human carotid arteries. Overexpression of the UCHL1 gene significantly attenuated tumor necrosis factor (TNF)-alpha-induced NF-kappaB activity in vascular cells and increased inhibitor of kappa B-alpha (IkappaB-alpha), possibly through the attenuation of IkappaB-alpha ubiquitination, leading to decreased neointima in the balloon-injured artery. In contrast, knockdown of UCHL1 by small interfering RNA resulted in increased NF-kappaB activity in VSMCs. These data suggest that UCHL1 may partially attenuate vascular remodeling through inhibition of NF-kappaB activity.

Prolonged application of clopidogrel reduces inflammation after percutaneous coronary intervention in the porcine model

We determined the effect of prolonged treatment with clopidogrel on C-reactive protein (CRP) concentrations and blood thrombogenicity after percutaneous transluminal coronary angioplasty followed by intracoronary brachytherapy in the porcine model. ANIMAL MODEL: All 48 pigs received antiplatelet therapy, including aspirin (325 mg, daily) and clopidogrel (300 mg, loading dose) 1 day before PCI, followed by a daily dose of clopidogrel (75 mg/day) in addition to aspirin. During PCI, one of two balloon-injured arteries was randomly assigned to receive immediate radiation treatment. Animals were sacrificed after 24 h, 1 month, and 3 months post-PCI. The pigs, which were sacrificed 3 months post-PCI, were divided into two groups. The first group received clopidogrel in addition to aspirin for 3 months, and the second group received clopidogrel in addition to aspirin for only 1 month after PCI and then aspirin alone. Blood was taken from all pigs before intervention, immediately after intervention, and before sacrifice. Serum CRP was measured by enzyme-linked immunosorbent assay. To analyze the procoagulant effects of PCI on blood thrombogenicity, a one-stage clotting assay was performed. Clopidogrel treatment for 3 months reduced CRP levels more than did clopidogrel therapy for 1 month only at 3 months post-PCI (27.9+/-3.9 vs. 56.6+/-11.3 microg/ml; P=.019). Baseline CRP levels were found to be 50.4+/-4.8 microg/ml. Plasma clotting was not affected by prolonged clopidogrel therapy (322.8+/-59.3 s vs. 295.2+/-52.5 s; P=ns). Prolonged treatment with clopidogrel reduced CRP levels post-PCI.

The cyclolignan picropodophyllin attenuates intimal hyperplasia after rat carotid balloon injury by blocking insulin-like growth factor-1 receptor signaling

Smooth muscle cell proliferation (SMC) is a pivotal factor in the development of intimal hyperplasia after vascular injury. A number of growth factors, including insulin-like growth factor-1 (IGF-1), have been shown to be involved in SMC proliferation. We evaluated the effect of picropodophyllin (PPP), a new IGF-1 receptor inhibitor, in the prevention of SMC proliferation and development of intimal hyperplasia after vascular injury. The effects of systemic administration of PPP on intimal hyperplasia were studied in a balloon rat carotid injury model. Lesions were quantified by morphometry and SMC proliferation and apoptosis was studied by immunohistochemical staining for proliferating cell nuclear antigen (PCNA) and activated caspase 3, respectively. The effect of PPP on rat aortic SMC proliferation and apoptosis was studied in vitro by using cell counting, 3[H]-thymidine incorporation, and a flow cytometry assay for annexin V. Phosphorylation of the IGF-1 receptor, protein kinase B (Akt), and extracellular signal-regulated kinase 1/2 (ERK1/2) in vitro and in vivo were analyzed by using Western blotting. PPP inhibited IGF-1-mediated SMC proliferation in vitro but no significant increase in apoptosis was detected. In rats treated with PPP, a more than a twofold reduction in carotid intima area was observed 2 weeks after balloon injury, a significant decrease in PCNA staining was demonstrated in early lesions, but activated caspase 3 was not detected. In addition, PPP attenuated phosphorylation of the IGF-1 receptor, Akt, and ERK1/2 in IGF-1-stimulated SMCs in vitro, and a reduced phosphorylation of the IGF-1 receptor and Akt was found in balloon-injured carotid arteries in rats treated with PPP. These results show that PPP potently blocks IGF-1-mediated phosphorylation of the IGF-1 receptor in SMCs, decreases downstream Akt and ERK1/2 activation, inhibits SMC replication, and subsequently attenuates intimal hyperplasia after balloon injury of rat carotid arteries.

In Vivo Reduction of the Nuclear Factor-κB Activity Using Synthetic cis-Element Decoy Oligonucleotides Suppresses Intimal Hyperplasia in the Injured Carotid Arteries in Rabbits

Nuclear factor-kappaB (NF-kappaB) plays a critical role in inflammation-related reactions, and is also found in the injured arterial wall. The purpose of this study was to introduce synthetic double-stranded cis-element "decoy" oligonucleotides (ODNs) into the arterial wall using the hemagglutinating virus of Japan (HVJ) liposome, and to investigate the inhibitory potential of decoy ODN against balloon injury-induced intimal hyperplasia by reducing NF-kappaB activity. Fluorescein isothiocyanate (FITC)-labeled decoy ODNs using the HVJ-liposome method were tranfected in balloon-injured rabbit carotid arteries. We then performed electrophoretic mobility shift assay to examine NF-kappaB activity using balloon-injured arteries, and we introduced NF-kappaB decoy into balloon-injured arteries. Transfection of FITC-labeled decoy ODNs by using the HVJ-liposome method demonstrated highly efficient protein expression with diffuse, frequent, and widespread nuclear signals over the entire medial layer, while the same amount of naked ODNs showed much less efficiency with scattered distribution of fluorescence in balloon-injured carotid arteries. Electrophoretic mobility shift assay showed activation of NF-kappaB in balloon-injured arteries. In vivo transfection of decoy ODNs mediated by HVJ liposome abolished the NF-kappaB activity in injured arteries with specific binding affinity to NF-kappaB protein. Intimal hyperplasia of carotid artery after balloon injury was reduced by approximately 50% by NF-kappaB decoy transfection compared with buffer treatment or scrambled decoy transfection. Our results demonstrated involvement of NF-kappaB in intimal formation after arterial injury, and indicated that NF-kappaB can be an appropriate molecular target for anti-restenosis therapy.

Pigment Epithelium-Derived Factor Inhibits Neointimal Hyperplasia after Vascular Injury by Blocking NADPH Oxidase-Mediated Reactive Oxygen Species Generation

Pigment epithelium-derived factor (PEDF) inhibits cytokine-induced endothelial cell activation through its antioxidative properties. However, the effect of PEDF on restenosis remains to be elucidated. Because the pathophysiological feature of restenosis is characterized by increased superoxide formation and accumulation of smooth muscle cells (SMCs), PEDF may inhibit this process via suppression of reactive oxygen species generation. We investigated here whether PEDF could prevent neointimal formation after balloon injury. PEDF levels were decreased in balloon-injured arteries. Adenoviral vector encoding human PEDF (Ad-PEDF) prevented neointimal formation. Expression and superoxide generation of the membrane components of NADPH oxidase, p22(phox) and gp91(phox), in the neointima were also suppressed by Ad-PEDF. Ad-PEDF reduced G(1) cyclin (cyclin D1 and E) expression and increased p27, a cyclin-dependent kinase inhibitor. In vitro, PEDF inhibited platelet-derived growth factor-BB-induced SMC proliferation and migration by blocking reactive oxygen species generation through suppression of NADPH oxidase activity via down-regulation of p22(PHOX) and gp91(PHOX). PEDF down-regulated G(1) cyclins and up-regulated p27 levels in platelet-derived growth factor-BB-exposed SMCs as well. These results demonstrate that PEDF could inhibit neointimal formation via suppression of NADPH oxidase-mediated reactive oxygen species generation. Our present study suggests that substitution of PEDF may be a novel therapeutic strategy for restenosis after balloon angioplasty.

Protective Effect of Hydrogen Sulfide on Balloon Injury-Induced Neointima Hyperplasia in Rat Carotid Arteries

Endogenous hydrogen sulfide (H(2)S), generated from homocysteine metabolism mainly catalyzed by cystathionine gamma-lyase (CSE), possesses important functions in the cardiovascular system. In this study, we investigated the role of H(2)S during the pathogenesis of neointimal formation induced by balloon injury in rats. CSE mRNA levels were reduced by 86.5% at 1 week and 64.0% at 4 weeks after balloon injury compared with the uninjured controls. CSE activity was also correspondingly reduced. Endogenous production of H(2)S in the injured carotid artery was significantly inhibited at 1 week and 4 weeks after balloon injury. Treatment with NaHS (a donor of H(2)S) enhanced methacholine-induced vasorelaxation of balloon-injured artery. More importantly, treatment with NaHS significantly inhibited neointima formation (0.15 +/- 0.01 mm(2) versus 0.21 +/- 0.01 mm(2), P < 0.001) of the balloon-injured carotid arteries and reduced the intima/media ratio (1.05 +/- 0.07 versus 1.43 +/- 0.06, P < 0.001). A significant decrease in vascular smooth muscle cell proliferation was demonstrated by bromodeoxyuridine incorporation at day 7 after injury. In conclusion, CSE expression and H(2)S production are reduced during the development of balloon injury-induced neointimal hyperplasia, and treatment with NaHS significantly reduces neointimal lesion formation.

Nf-κb and AP-1 activation is associated with late lumen loss after porcine coronary angioplasty and antiproliferative beta-irradiation

Despite the success of antiproliferative therapies, restenosis remains a common problem after percutaneous transluminal coronary angioplasty (PTCA). Longer-term clinical results of brachytherapy (intracoronary radiation), the lack of long-term clinical results after implantation of drug eluting stents, and the occurrence of late thrombosis after both procedures leave room for skepticism. Neointimal proliferation is not substantially inhibited at late time points after brachytherapy, and late lumen loss with a "catch-up" proliferation can occur. We hypothesized that the transcription factors nuclear factor-{kappa}B (NF-kappaB) and activator protein-1 (AP-1) are involved in these processes. We addressed the role of these mediators in a porcine model of coronary restenosis. Thirty-nine pigs underwent PTCA in two major coronary arteries. One of the two balloon-injured arteries was randomly assigned to receive immediate 20 Gy beta-irradiation (Brachy group) using a noncentered source train ((90)Sr/Y Beta-Cath, Novoste). Animals were sacrificed after 1 day, 14 days, or 28 days. Proliferating cells were labeled prior to euthanasia. At late time points, lumen area was significantly smaller and the inflammatory response was more pronounced in the Brachy group than in the PTCA group. These findings coincided with sustained activation of MMP-9 and transcription factors like NF-kappaB and AP-1. Initially, cell proliferation was reduced in the Brachy group; however, at late time points, differences between the two treatment groups were no longer significant. Brachytherapy initially inhibits cell proliferation; however, cellular and molecular inflammatory processes (e.g. activation of NF-kappaB) are enhanced within the arterial wall. This proinflammatory side effect may be responsible for the observed delayed proliferation and the resulting lumen loss.

Prostacyclin synthase gene transfer inhibits neointimal formation by suppressing PPARδ expression

Objective Prostacyclin (PGI 2) is a potent ligand of peroxisome proliferator-activated receptor δ (PPARδ) that regulates cell growth and differentiation. The aim of this study was to elucidate how endogenous PGI 2 overexpression affects the expressions of PPARδ and mitogen-activated protein kinases (MAPKs) in the development of neointimal formation in experimental angioplasty with adenovirus-mediated PGI 2 synthase (Ad-PGIS) gene transfer. Methods and results In human aortic smooth muscle cells, protein blotting analysis showed that PGI 2 overproduction decreased the levels of phosphorylated p38 MAPK (P-p38 MAPK) (2.0-fold versus 0.83-fold relative to control). Immunohistochemical analysis in balloon-injured arteries revealed diffuse expression of PPARδ in the neointima of control vessels, with no expression in uninjured vessels. The level of PPARδ expression was lower in Ad-PGIS-treated arteries than in control vessels, with the PPARδ localized in the neointima adjacent to endothelium. Staining of P-p38 MAPK showed a similar pattern to PPARδ among the three groups. Morphometric analysis at day 14 revealed that Ad-PGIS reduced the intima-to-media ratio by up to 59%. Conclusions Ad-PGIS gene transfer reduced PPARδ expression and inhibited neointimal formation after balloon injury in accordance with the reduction in the phosphorylation of p38 MAPK.

Poly(vinyl alcohol)-graft-poly(lactide-co-glycolide) nanoparticles for local delivery of paclitaxel for restenosis treatment

Catheter-based local delivery of biodegradable nanoparticles (NP) with sustained release characteristics represents a therapeutic approach to reduce restenosis. Paclitaxel-loaded NP consisting of poly(vinyl alcohol)-graft-poly(lactide-co-glycolide) (PVA-g-PLGA) with varying PLGA chain length as well as poly(lactide-co-glycolide) (PLGA), were prepared by a solvent evaporation technique. NP of < 180 nm in diameter characterized by photon correlation spectroscopy (PCS), scanning electron microscopy (SEM), and atomic force microscopy (AFM) are spherical and show smooth surfaces. Yields typically range from 80 to 95% with encapsulation efficiencies between 77 and 87%. The extent of initial in vitro paclitaxel release was affected by the PVA-g-PLGA composition. Blank nanoparticles from PVA 300-g-PLGA(30) and PVA 300-g-PLGA(15) showed excellent biocompatibility in rabbit vascular smooth muscle cells (RbVSMC) at polymer concentrations of 0.37 mg/ml. Paclitaxel-loaded NP have an increased antiproliferative effect on cells in comparison to free drug. Confocal laser scanning microscopy of RbVSMC confirmed cellular uptake of nanoparticles composed of fluorescently labeled PVA 300-g-PLGA(15) loaded with Oregon Green labeled paclitaxel. Cells showed a clearly increased fluorescence activity with a co-localization of paclitaxel and polymer nanoparticles during incubation with particle suspension. To evaluate the antirestenotic effect in vivo, paclitaxel-loaded nanoparticles were administered locally to the wall of balloon-injured rabbit iliac arteries using a porous balloon catheter. As a result a 50% reduction in neointimal area in vessel segments treated with paclitaxel-loaded nanoparticles compared to control vessel segments could be observed (local paclitaxel nanoparticle treated segments 0.80 ± 0.19 mm 2, control segments 1.58 ± 0.6 mm 2; p < 0.05).

The Effects of Chymase on Matrix Metalloproteinase-2 Activation in Neointimal Hyperplasia after Balloon Injury in Dogs

Chymase is known to generate angiotensin II in the vascular wall. In this study we investigated a novel role for chymase other than angiotensin II production in vascular proliferation after balloon injury. Chymase promoted the migration of vascular smooth muscle cells in the matrix-coated invasion chambers and activated promatrix metalloproteinase-2 obtained from the culture medium of vascular smooth muscle cells. Two weeks after balloon injury, significant neointimal formation was found in dog carotid arteries. After injury, active matrix metalloproteinase-2 was increased in parallel with the augmentation of chymase activity that was seen in the proliferating region of the vascular wall. The oral administration of NK3201 (1 mg/kg per day), a chymase inhibitor, prevented neointimal formation and significantly suppressed both active matrix metalloproteinase-2 and chymase activities 2 weeks after injury. These results suggest that chymase inhibitors can prevent the development of intimal hyperplasia via the inhibition of matrix metalloproteinase-2 activation in balloon-injured arteries.

Nitric Oxide Generation by NO Donors Is Enhanced Following Balloon Injury in the Porcine Coronary Artery

Vasospasm is a complication of cardiological procedures such as balloon angioplasty and may be related to vascular oxidant stress. Although nitric oxide donor drugs are often administered to prevent vasospasm, the response to these drugs in balloon-injured arteries has not been studied. Pig coronary arteries were balloon-injured in vitro and relaxations to nitric oxide (NO)-donating and NO-independent vasodilators studied. Generation of superoxide in response to injury was assayed using dihydroethidium. NO formation on addition of the NO donor drugs was studied using an amperometric sensor. Expression of nitrotyrosine, a peroxynitrite marker, was probed using immunocytochemistry. In vitro injury enhanced sensitivity to the NO donors SNAP and SpermineNONOate but blunted the response to isoprenaline or chromakalim. With both donors, NO formation was significantly enhanced in the presence of an injured vessel. Vascular superoxide generation was also increased throughout the vessel wall and a small increase in nitrotyrosine was detected in the injured vessel media following addition of SNAP. In conclusion, injured vessels were more sensitive to NO donors and this appears to be due to enhanced NO generation by the donor molecule. Increased formation of peroxynitrite within the injured vessel may contribute to the enhanced relaxation in injured vessels.

Gender difference of atorvastatin's vasoprotective effect in balloon-injured rat carotid arteries

The effect of estrogen on neointimal formation in injured rat arteries has been reported to be a sexual dimorphic effect. Recently, it has been reported that 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) exhibit vasoprotective effects, which are independent of their cholesterol-lowering effects. In this study, we examined the gender differences of atorvastatin's effect on neointimal formation in balloon-injured rat arteries. Male and female Sprague Dawley rats underwent gonadectomy and balloon injury of the carotid artery. Ovariectomized female, as well as intact and castrated male, rats exhibited marked neointimal formation. Treatment with atorvastatin significantly reduced neointimal formation at day 14 (14 days after injury) and NADPH oxidase-dependent superoxide production at day 2 in ovariectomy, but not in intact and castrated males. In ovariectomized rats, 7 days of atorvastatin treatment from days − 3 to 3 but not from days 7 to 14 suppressed neointimal formation at day 14. In this study, we showed that atorvastatin's effect on neointimal formation was female-specific and was more marked in ovariectomized female rats. NADPH oxidase-dependent superoxide production may be involved in the mechanism of the sexual dimorphic response seen in response to atorvastatin treatment. Furthermore, the results suggest the importance of treatment in the early phase after vascular injury.

Sequential patterns of chemokine- and chemokine receptor-synthesis following vessel wall injury in porcine coronary arteries

Inflammation plays a central role in vascular repair, and spreads into perivascular tissue (PVT) following angioplasty. Chemokines (CK) and chemokine receptors (CKR) are key determinants of inflammatory chemotaxis. We sought to assess the arterial and perivascular expression of the CK CCL2 and CXCL2, and the CKR CCR2, CCR5, and CXCR4 in balloon-injured porcine coronary arteries. Vascular cells that express specific CK and CKR mRNA during post-angioplasty time course were detected by in situ hybridization (ISH), and expression was quantified by real time RT-PCR in PVT. CCL2 was maximal in PVT from 2 to 24 h post injury, coincident with local macrophage-activation. Expression was upregulated in media and adventitia from 24 h to 3 days, and in neointima at 7 days. CXCL2 was detected in media at 2 and 4 h, and also in some neointimal cells. CCR2 and CCR5 were maximal in PVT at 24 h and 3 days, respectively. Expression shifted to media and adventitia at 2 and 3 days, and to neointima and adventitia at 7 days, and was low at 14 days. CXCR4 was low in PVT, but was upregulated in media and adventitia at 2 and 3 days, as well as in neointima and adventitia at 7 days. In conclusion, PVT is the primary source of inflammatory CK and CKR early post-angioplasty. Specific sequential patterns of CK- and CKR-synthesis are identified that may regulate phase-specific chemotaxis by spatio-temporally differential expression during coronary response to injury.

Effects of Nicotine on Angiogenesis and Restenosis in a Rabbit Model

Background and Aims: Nicotine is a major component of cigarette smoke and has been found to play an important role in angiogenesis. However, whether nicotine plays a role in restenosis has not been determined. Therefore, the aim of the present study was to determine the effects of nicotine on angiogenesis and restenosis in a rabbit model. Methods: Forty male New Zealand White rabbits were randomly divided into control and low-, middle-, and high-dose (0.005, 0.05 or 5 µg/kg, respectively) nicotine-treated groups. Balloon catheter denuding injury iliac artery and ligation of left anterior descending coronary artery were performed in all animals fed with a high-cholesterol diet (HCD) beginning 2 weeks before operation. Nicotine was administered daily by intramuscular injection in the ischemic hindlimb for 3 weeks. Control rabbits received an equal volume of phosphate-buffered saline alone. Collateral vessels of the ischemic hindlimb were observed by angiography of abdominal aorta, and the density of intramyocardial microvessels and proliferative activity of balloon-injured arteries were examined by immunohistochemistry. Serum levels of lipids and the indexes of hepatic or renal functions were also determined before HCD and after nicotine treatment. Results: One rabbit in control, two in low-, one in middle- and two in high-dose groups died during the experiment. The remaining 34 rabbits were included in the study. Two or five weeks after HCD, the levels of serum lipids were significantly increased in all groups, but there was no significant difference of the levels between control and nicotine-treated groups 3 weeks after treatment; the indexes of hepatic or renal functions showed no significant changes 3 weeks after nicotine treatment; there were no significant differences on collateral vessels shown by angiography in all four groups; the density of intramyocardial microvessels in three nicotine-treated groups was significantly higher than that in control group; but the intimal area and proliferative activity in the balloon-injured arteries in three nicotine-treated groups were also higher than those in control group. Conclusions: The present study shows that intramuscular administration of nicotine for 3 weeks could not increase arteriogenesis in ischemic hindlimb of rabbits, but is capable of significantly promoting intramyocardial angiogenesis. Nicotine can also accelerate intimal proliferation and thickening of balloon catheter denuding injury iliac artery, so it may contribute to the development of restenosis.

The flavonoid quercetin induces apoptosis and inhibits JNK activation in intimal vascular smooth muscle cells

Quercetin, the most abundant dietary flavonol, exerts vasodilator, anti-hypertensive, and anti-atherogenic effects and reduces the vascular remodelling associated with elevated blood pressure. Here, we have compared the effects of quercetin in intimal- and medial-type rat vascular smooth muscle cells (VSMC) in culture. After 48 h, quercetin reduced the viability of a polyclonal intimal-type cell line derived from neonatal aorta but not of a medial-type cell line derived from adult aorta. These differential effects were similar in both proliferating and quiescent VSMC. Quercetin also preferentially reduced the viability of intimal-type over medial-type VSMC in primary cultures derived from balloon-injured carotid arteries. The effects of quercetin on cell viability were mainly dependent upon induction of apoptosis, as demonstrated by nuclear condensation and fragmentation, and were unrelated to PPARγ, pro-oxidant effects or nitric oxide. The expression of MAPKs (ERK, p38, and JNK) and ERK phosphorylation were not different between intimal- and medial-type VSMC. p38 phosphorylation was negligible in both cell types. Medial-type showed a weak JNK phosphorylation while this was markedly increased in intimal-type cells. Quercetin reduced JNK phosphorylation but had no consistent effect on ERK phosphorylation. In conclusion, quercetin preferentially produced apoptosis in intimal-type compared to medial-type VSMC. This might play a role in the anti-atherogenic and anti-hypertensive effects of quercetin.

Targeting Vascular Injury Using Hantavirus-Pseudotyped Lentiviral Vectors

Restenosis is a pathological condition involving intimal hyperplasia and negative arterial remodeling. Gene therapy vectors have shown modest therapeutic effects, but the level of infectivity has been relatively poor. In the present study we have designed a modified lentiviral vector (LV) pseudotyped with a strain of Hantavirus (HTNV) to improve the transduction efficiency into vascular smooth muscle and endothelial cells in vitro and in vivo. In vivo studies using adult New Zealand White rabbits demonstrated that local delivery of HTNV-pseudotyped LV (2 × 107 TU) into balloon-injured carotid arteries led to highly efficient transduction into endothelial and smooth muscle cells more effectively than VSV-G-pseudotyped LV (2 × 107 TU) or replication-defective adenoviral vectors (1–1.5 × 109 pfu) as determined by β-gal immunohistochemistry. Overexpression of extracellular superoxide dismutase in balloon-injured carotid arteries 6 weeks after LV administration resulted in a significant reduction (P = 0.0024) of the intima/media ratio (0.18 ± 0.09; n = 4) compared to vehicle-infused carotid arteries (0.69 ± 0.08; n = 7). No β-gal immunostaining was detected in other systemic organs, including the spleen, liver, heart, lung, kidneys, and brain. Moreover, no changes in plasma alanine aminotransferase or aspartate aminotransferase were detected following LV administration. In all, these data show that LV pseudotyped with Hantaviral glycoproteins can be a useful vector for targeting therapeutic genes to the vasculature in vivo.

Anti-Oxidant Gene Therapy by NFkB Decoy Oligodeoxynucleotide

Oxidative stress to cardiovascular cells induced by an interaction of multiple cytokines and adhesion molecules has been postulated to be responsible for cardiovascular disease. Since nuclear factor-kappaB (NFkappaB) also plays a pivotal role in the coordinated transactivation of cytokine and adhesion molecule genes, we utilized oligodeoxynucleotides (ODNs) as "decoy" cis-elements that block the binding of nuclear factors to promoter regions of targeted genes, resulting in the inhibition of gene transactivation. Indeed, transfection of NFkappaB decoy ODNs into coronary artery effectively prevented transactivation of essential cytokine and adhesion molecule protein expression, and thereby protected the myocardium from infarction. Transfection of NFkappaB decoy ODNs into balloon-injured carotid artery or porcine coronary artery markedly reduced neointimal formation. Thus, a clinical trial using NFkappaB decoy ODNs to treat restenosis was started in 2002. Recently, the therapeutic target utilizing NFkappaB decoy has been expanded to glomerulonephritis, rheumatoid arthritis, atopic dermatitis and osteoporosis. Moreover, the clinical trials to treat RA patients were initiated in 2003 and a Phase I/IIa human clinical trial using NFkappaB decoy ODNs to treat atopic dermatitis was initiated in December 2001. Topical application of NFkappaB decoy ODNs exhibited marked therapeutic effects on the facial skin condition of patients with atopic dermatitis. The covalently modified ODNs were developed by enzymatically ligating two identical molecules, thereby preventing their degradation by exonucleases. Indeed, the modified decoy ODNs possess increased nuclease resistance and are transported more efficiently into cells. Although there are still unresolved issues, decoy ODN drugs should become a reality.

Long-term inhibition of intimal hyperplasia using vascular photodynamic therapy in balloon-injured carotid arteries

Long-term inhibition of intimal hyperplasia using vascular photodynamic therapy in balloon-injured carotid arteries