Adenoviral-mediated uteroglobin gene transfer inhibits neointimal hyperplasia after balloon injury in the rat carotid artery.
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OBJECTIVE: Uteroglobin is a protein with potent anti-inflammatory and immunomodulatory effects. We hypothesize that induction of uteroglobin expression in the artery wall by local adenoviral gene transfer will decrease neointimal hyperplasia in the rat carotid artery after balloon injury. METHODS: Seven male Sprague-Dawley rats underwent balloon injury of the common carotid artery. After the injury, with flow occluded, the artery was instilled with 50 microL of the adenoviral vector encoding uteroglobin gene (Ad.UG) at a concentration of 1.35 x 10(11) pfu/mL (n = 7) or 0.68 x 10(11) pfu/mL (n = 7) (n = 7). Control animals were similarly treated: either an adenovirus encoding for beta-galactosidase gene (Ad.LacZ) at 1 x 10(11) pfu/mL (n = 7) or the phosphate-buffered saline (PBS) vehicle (n = 6) was used. The solution was allowed to dwell for 20 minutes. The rats were humanely killed after 14 days by perfusion fixation, and the carotid arteries were sectioned for analysis with computerized planimetry. The intima-media area ratios were calculated for each artery and compared with analysis of variance with Bonferroni/Dunn post hoc testing. One additional rat from the PBS, Ad.LacZ, and Ad.UG (1.35 x 10(11) pfu/mL) groups was humanely killed 4 days after treatment for carotid artery protein extraction and Western blotting. RESULTS: Uteroglobin protein production was confirmed in the Ad.UG-treated arteries with Western blotting. Morphometric analysis showed that the Ad.UG group at 1.35 x 10(11) pfu/mL had a significantly lower intima-media area ratio than both the Ad.LacZ (P =.002) and PBS (P =.004) controls. The Ad.UG group at 0.68 x 10(11) pfu/mL was also significantly different from the Ad. LacZ (P =.003) and PBS (P =.006) controls. There was no statistical difference between the two control groups or between the two Ad.UG groups. CONCLUSION: Adenoviral gene transfer of uteroglobin, delivered intraluminally after arterial injury causes the production of uteroglobin protein and has an inhibitory effect on neointimal accumulation in the rat model. (+info)
Plasminogen activator inhibitor-1 deficiency protects against atherosclerosis progression in the mouse carotid artery.
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Dissolution of the fibrin blood clot is regulated in large part by plasminogen activator inhibitor-1 (PAI-1). Elevated levels of plasma PAI-1 may be an important risk factor for atherosclerotic vascular disease and are associated with premature myocardial infarction. The role of the endogenous plasminogen activation system in limiting thrombus formation following atherosclerotic plaque disruption is unknown. This study found that genetic deficiency for PAI-1, the primary physiologic regulator of tissue-type plasminogen activator (tPA), prolonged the time to occlusive thrombosis following photochemical injury to carotid atherosclerotic plaque in apolipoprotein E-deficient (apoE(-/-)) mice. However, anatomic analysis revealed a striking difference in the extent of atherosclerosis at the carotid artery bifurcation between apoE(-/-) mice and mice doubly deficient for apoE and PAI-1 (PAI-1(-/-)/apoE(-/-)). Consistent with a previous report, PAI-1(+/+)/apoE(-/-)and PAI-1(-/-)/apoE(-/-) mice developed similar atherosclerosis in the aortic arch. The marked protection from atherosclerosis progression at the carotid bifurcation conferred by PAI-1 deficiency suggests a critical role for PAI-1 in the pathogenesis of atherosclerosis at sites of turbulent flow, potentially through the inhibition of fibrin clearance. Consistent with this hypothesis, intense fibrinogen/fibrin staining was observed in atherosclerotic lesions at the carotid bifurcation compared to the aortic arch. These observations identify significant differences in the pathogenesis of atherosclerosis at varying sites in the vascular tree and suggest a previously unappreciated role for the plasminogen activation system in atherosclerosis progression at sites of turbulent flow. (Blood. 2000;96:4212-4215) (+info)
Role of p38 mitogen-activated protein kinase in neointimal hyperplasia after vascular injury.
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p38 mitogen-activated protein kinase (MAPK) is involved in intracellular signals that regulate a variety of cellular responses during inflammation. However, the role of p38 MAPK in atherosclerosis, a chronic inflammatory disorder, remains uncertain. The aim of the present study was to examine the role of p38 MAPK in the development of neointimal hyperplasia in balloon-injured rat carotid arteries. Immunohistochemical studies indicated that p38 MAPK was rapidly activated in the majority of medial cells in injured arterial walls. Rats treated with FR167653, a selective inhibitor of p38 MAPK, at a dosage of 10 mg x kg(-1) x d(-1), had a 29.4% lower intima-to-media ratio than the untreated controls at 14 days after balloon injury (P:<0.05). The percentage of proliferating nuclear antigen-positive cells in the media at 48 hours was significantly lower in the FR167653-treated group than in the control group. Quantitative competitive reverse transcription-polymerase chain reaction analysis revealed that interleukin-1beta mRNA expression in arteries was significantly inhibited by FR167653 (to 18.1% of control, P:<0.05) at 8 hours after balloon injury. Moreover, p38 MAPK activation and interleukin-1beta production by lipopolysaccharide-stimulated vascular smooth muscle cells were inhibited by FR167653 in a concentration-dependent manner in vitro. These results indicate that p38 MAPK is activated in vascular walls after injury and promotes neointimal formation and suggest that selective inhibition of p38 MAPK may be effective in the prevention of restenosis after percutaneous transluminal coronary angioplasty. (+info)
Soluble guanylate cyclase alpha(1) and beta(1) gene transfer increases NO responsiveness and reduces neointima formation after balloon injury in rats via antiproliferative and antimigratory effects.
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In vascular smooth muscle cells, NO stimulates the synthesis of cGMP by soluble guanylate cyclase (sGC), a heterodimer composed of alpha(1) and beta(1) subunits. NO/cGMP signal transduction affects multiple cell functions that contribute to neointima formation after vascular injury. Balloon-induced vascular injury was found to decrease sGC subunit expression and enzyme activity in rat carotid arteries. The effect of restoring sGC enzyme activity on neointima formation was investigated using recombinant adenoviruses specifying sGC alpha(1) and beta(1) subunits (Adalpha1 and Adbeta1). Coinfection of cultured rat aortic smooth muscle cells with Adalpha1 and Adbeta1 increased NO-stimulated intracellular cGMP levels 60-fold and decreased DNA synthesis and migration by 16% and 48%, respectively. Immunoreactivity for alpha(1) and beta(1) subunits colocalized in carotid arteries infected with Adalpha1 and Adbeta1. Molsidomine-stimulated carotid tissue cGMP levels were greater after coinfection with Adalpha1 and Adbeta1 than after infection with a control virus, AdRR5 (0.53+/-0.09 pmol/mg protein, mean+/-SEM, versus 0.23+/-0.09, P<0.05). Mean intima/media ratio, 2 weeks after balloon injury and twice-daily administration of 5 mg/kg molsidomine, was less in rats coinfected with Adalpha1 and Adss1 than in rats infected with AdRR5 or in uninfected rats (0.36+/-0.11 versus 0. 81+/-0.13 and 0.75+/-0.25, respectively, P<0.05). Thus, Adalpha1 and Adbeta1 gene transfer to balloon-injured rat carotid arteries increases NO responsiveness and attenuates neointima formation via a direct antiproliferative and antimigratory effect on vascular smooth muscle cells. (+info)
Low blood flow after angioplasty augments mechanisms of restenosis: inward vessel remodeling, cell migration, and activity of genes regulating migration.
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-The predominant cause of restenosis after angioplasty is now thought to be inward remodeling, but the mechanisms responsible are unknown. Remodeling in normal vessels is regulated by the endothelium in response to altered shear stress. Although the endothelium is often damaged by angioplasty, restenosis rates after angioplasty have been correlated with impaired coronary flow. Thus, we examined how increases or decreases in blood flow through balloon catheter-injured rat carotid arteries affect vessel morphometry (4, 10, and 28 days), cell migration (4 days), and levels of promigratory mRNAs (2 and 10 days). After 28 days, the luminal area in vessels with low blood flow was significantly less than in those with normal and high blood flow (0.17+/-0.01 [low] versus 0.24+/-0.06 [normal] versus 0.30+/-0.02 [high] mm(2), P:<0.01), predominantly because of accentuated inward remodeling (or reduced area within the external elastic lamina; 0.42+/-0.02 [low] versus 0.54+/-0.07 [normal] versus 0.53+/-0.04 [high] mm(2), P:<0.05). Low flow also enhanced smooth muscle cell migration 4 days after injury by 90% above normal and high flows (P:<0.01). Two days after injury, low flow significantly increased levels of mRNAs encoding promigratory peptides (integrin alpha(v)ss(3), transforming growth factor-ss(1), CD44v6, MDC9, urokinase plasminogen activator receptor, and ss-inducible gene h3); these changes persisted 10 days after injury and were localized to the neointima. Low blood flow may promote restenosis after angioplasty because of its adverse effect on vessel remodeling, and it is associated with the augmented expression of multiple genes central to cell migration and restenosis. (+info)
Cellular senescence after single and repeated balloon catheter denudations of rabbit carotid arteries.
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The hypothesis that increased cellular proliferation in the vasculature may lead to replicative senescence has been tested in a model of neointima formation. We have used a biomarker of replicative senescence, senescence-associated beta-galactosidase (SA-beta-gal), to detect senescence in rabbit carotid arteries subjected to single and double balloon denudations. We found an accumulation of senescent cells in the neointima and media of all injured vessels, in contrast to the near absence of such cells in control vessels. The relative area occupied by SA-beta-gal-positive cells was higher in vessels subjected to double denudation than in those subjected to single denudation, both in the neointima (0.99% versus 0.06%, respectively; P:<0.001) and in the media (0.11% versus 0.01%, respectively; P:<0.02). The majority of SA-beta-gal-positive cells were vascular smooth muscle cells, and a minority were endothelial cells. SA-beta-gal-positive cells showed no evidence of apoptosis by use of terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling. Our results indicate that the proliferative response that follows intraluminal injury to the artery leads to the emergence of senescent endothelial and smooth muscle cells. The demonstration that vascular cell senescence can occur in vivo suggests that this process may be involved in cardiovascular pathologies that have a proliferative component. (+info)
Estradiol accelerates reendothelialization in mouse carotid artery through estrogen receptor-alpha but not estrogen receptor-beta.
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BACKGROUND: The atheroprotective effect of 17beta-estradiol (E(2)) has been suggested in women and clearly demonstrated in animals through both an effect on lipid metabolism and a direct effect on the cells of the arterial wall. It has been shown, for example, that E(2) promotes endothelium-dependent relaxation and accelerates reendothelialization in rats. Similar studies have been undertaken in mice to appreciate the molecular mechanism of this process. METHODS AND RESULTS: We report here a model of electric carotid injury adapted from that described by Carmeliet et al (1997) that allows us to precisely evaluate the reendothelialization process. We demonstrate that E(2) accelerates endothelial regeneration in castrated female wild-type mice. In ovariectomized transgenic mice in which either the estrogen receptor (ER)-alpha or ERbeta gene has been disrupted, E(2) accelerated reendothelialization in female ERbeta knockout mice, whereas this effect was abolished in female ERalpha knockout mice. CONCLUSIONS: This study demonstrates that ERalpha but not ERbeta mediates the beneficial effect of E(2) on reendothelialization and potentially the prevention of atherosclerosis. (+info)
Combination of a brief irrigation with tissue factor pathway inhibitor (TFPI) and adenovirus-mediated local TFPI gene transfer additively reduces neointima formation in balloon-injured rabbit carotid arteries.
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BACKGROUND: Tissue factor pathway inhibitor (TFPI) is a physiological antagonist of TF. We tested whether a brief irrigation with TFPI protein (rTFPI) or TFPI gene transfer into injured arteries would suppress TF activity and reduce fibroproliferative changes and investigated whether a combination of these methods would show an additive effect. METHODS AND RESULTS: We prepared adenoviruses expressing either TFPI (AdTFPI) or bacterial ss-galactosidase (AdLacZ). Rabbit carotid arteries were balloon-injured and either infected with AdTFPI (or AdLacZ) or irrigated briefly with rTFPI (or saline). After injury, TF activity in arteries increased and was sustained; however, it was suppressed during the initial 24 hours by rTFPI irrigation (but not by gene transfer) and for a substantial period of time by TFPI gene transfer (but not by rTFPI irrigation). Four weeks later, the ratio of the intimal to medial areas was 34.3+/-8.7% (mean+/-SD, n=14) in saline-treated arteries and 33.3+/-4.2% in AdLacZ-infected arteries (P:=NS versus saline). However, it was reduced to 25.5+/-8.5% in rTFPI-irrigated arteries (P:<0.01 versus saline) and to 20.7+/-5.3% in AdTFPI-infected arteries (P:<0.01 versus AdLacZ). With a combination of irrigation and gene transfer, the ratio was further reduced to 12.6+/-4.7% (P:<0.01 versus rTFPI, P:<0.05 versus AdTFPI). Systemic coagulation status was not affected in these animals. CONCLUSIONS: A combination of rTFPI irrigation and TFPI gene transfer overcomes the shortcomings shown by each method when used alone and achieves a full coverage of TF activity suppression, thereby enhancing their therapeutic effects without systemic side effects. This combination may be an effective strategy for the prevention of thrombosis and proliferative changes after angioplasty in humans. (+info)