Balloon Endothelial Denudation Research Articles

Beneficial effect of colchicine in an animal model of atherosclerosis

Abstract Background Atherosclerosis is a chronic inflammatory disease and colchicine demonstrated clinical benefits in the treatment of stable coronary artery disease. Purpose Our aim was to assess the anti-inflammatory effects of colchicine on atherosclerotic vascular disease. Methods Atherosclerosis was induced in the abdominal aorta of 20 rabbits with high-cholesterol diet and balloon endothelial denudation. The study protocol lasted 36 weeks. After 18 weeks rabbits were randomized to receive either colchicine or placebo. Two animals died in each group. At randomization and at the end of the study, all animals underwent MRI and positron 18F-FDG PET/CT. In this post-hoc subgroup analyses, animals of each group in the first quartile of cholesterol levels were excluded. For MRI plaque volume values were expressed as Normalized Wall Index (NWI) and for 18F-FDG PET/CT values were expressed as mean maximum standard uptake (meanSUVmax). Statistical comparisons were made by using the Mann-Whitney U test for unpaired data and Wilcoxon signed-rank test for paired data. Results Results are summarized in Table 1. Animals with higher levels of cholesterol (6 per group) showed significant differences in favor to colchicine group, both as NWI at the end of the protocol 0.47 (IQR 0.05) in the colchicine group versus 0.52 (IQR 0.03) in the placebo; p=0.01] and as relative increase in meanSUVmax [−4.0% (IQR 30.1) in the colchicine group versus 35.1 (IQR 59.0) in the placebo; p 0.041] (Figure 1). Conclusions Colchicine may stabilize atherosclerotic plaque by reducing inflammatory activity and plaque burden. Figure 1 Funding Acknowledgement Type of funding source: Public grant(s) – EU funding. Main funding source(s): This work was supported by a grant from the Sociedad Española de Cardiología and a grant from the Carlos III Institute of Health of Spain and Fondo Europeo de Desarrollo Regional (FEDER, “Una manera de hacer Europa”)

Effects of Colchicine on Atherosclerotic Plaque Stabilization: a Multimodality Imaging Study in an Animal Model.

Colchicine demonstrated clinical benefits in the treatment of stable coronary artery disease. Our aim was to evaluate the effects of colchicine on atherosclerotic plaque stabilization. Atherosclerosis was induced in the abdominal aorta of 20 rabbits with high-cholesterol diet and balloon endothelial denudation. Rabbits were randomized to receive either colchicine or placebo. All animals underwent MRI, 18F-FDG PET/CT, optical coherence tomography (OCT), and histology. Similar progression of atherosclerotic burden was observed in the two groups as relative increase of normalized wall index (NWI). Maximum 18F-FDG standardized uptake value (meanSUVmax) decreased after colchicine treatment, while it increased in the placebo group with a trend toward significance. Animals with higher levels of cholesterol showed significant differences in favor to colchicine group, both as NWI at the end of the protocol and as relative increase in meanSUVmax. Colchicine may stabilize atherosclerotic plaque by reducing inflammatory activity and plaque burden, without altering macrophage infiltration or plaque typology.

A Metabolic Intravascular Platform to Study FDG Uptake in Vascular Injury.

Metabolic alterations underlie many pathophysiological conditions, and their understanding is critical for the development of novel therapies. Although the assessment of metabolic changes in vivo has been historically challenging, recent developments in molecular imaging have allowed us to study novel metabolic research concepts directly in the living subject, bringing us closer to patients. However, in many instances, there is need for sensors that are in close proximity to the organ under investigation, for example to study vascular metabolism. In this study, we developed and validated a metabolic detection platform directly in the living subject under an inflammatory condition. The signal collected by a scintillating fiber is amplified using a photomultiplier tube and decodified by an in-house tunable analysis platform. For in vivo testing, we based our experiments on the metabolic characteristics of macrophages, cells closely linked to inflammation and avid for glucose and its analog 18F-fluorodeoxyglucose (18F-FDG). The sensor was validated in New Zealand rabbits, in which inflammation was induced by either a) high cholesterol (HC) diet for 16weeks or b) vascular balloon endothelial denudation followed by HC diet. There was no difference in weight, hemodynamics, blood pressure, or heart rate between the groups. Vascular inflammation was detected by the metabolic sensor (Inflammation: 0.60 ± 0.03AU vs. control: 0.48 ± 0.03AU, p = 0.01), even though no significant inflammation/atherosclerosis was detected by intravascular ultrasound, underscoring the high sensitivity of the system. These findings were confirmed by the presence of macrophages on ex vivo aortic tissue staining. In this study, we validated a tunable very sensitive metabolic sensor platform that can be used for the detection of vascular metabolism, such as inflammation. This sensor can be used not only for the detection of macrophage activity but, with alternative probes, it could allow the detection of other pathophysiological processes.

Effects of Noradrenaline and Adenosine Triphosphate on the Degree on Contrast Enhancement in a Rabbit Model of Atherosclerosis during Contrast-Enhanced Ultrasonography

The aim of the study is to assess the effects of vasoactive agents on the degree of contrast enhancement in experimental atherosclerotic plaque during contrast-enhanced ultrasonography (CEUS). Abdominal aortic atherosclerosis was induced in 25 New Zealand white rabbits by a combination of cholesterol-rich diet and balloon endothelial denudation. Standard ultrasonography and CEUS were performed at baseline and during intravenous infusion of noradrenaline or adenosine triphosphate (ATP). The degree of contrast enhancement of the plaque after injection of contrast material was quantified by calculating the enhanced intensity in the plaque. The infusion of noradrenaline induced significant increase in systolic blood pressure (84 ± 13 mm Hg vs. 112 ± 20 mm Hg, p = 0.011) and significant decrease in the enhanced intensity in the plaque (7.52 ± 1.32 dB vs. 5.88 ± 1.33 dB, p < 0.001) during CEUS. The infusion of ATP resulted in the significant decrease in systolic blood pressure (80 ± 13 mm Hg vs. 65 ± 11 mm Hg, p = 0.005) and increase in the enhanced intensity in the plaque (7.52 ± 1.32 dB vs. 8.84 ± 1.55 dB, p < 0.001) during CEUS. The degree of contrast enhancement within an experimental atherosclerotic plaque during CEUS can be influenced by vasoactive agents and hemodynamic status.

Noninvasive Assessment of Hypoxia in Rabbit Advanced Atherosclerosis Using 18 F-fluoromisonidazole Positron Emission Tomographic Imaging

Background— Hypoxia is an important microenvironmental factor influencing atherosclerosis progression by inducing foam-cell formation, metabolic adaptation of infiltrated macrophages, and plaque neovascularization. Therefore, imaging plaque hypoxia could serve as a marker of lesions at risk. Methods and Results— Advanced aortic atherosclerosis was induced in 18 rabbits by atherogenic diet and double balloon endothelial denudation. Animals underwent 18 F-fluoromisonidazole positron emission tomographic and 18 F-fluorodeoxyglucose positron emission tomographic imaging after 6 to 8 months (atherosclerosis induction) and 12 to 16 months (progression) of diet initiation. Four rabbits fed standard chow served as controls. Radiotracer uptake of the abdominal aorta was measured using standardized uptake values. After imaging, plaque hypoxia (pimonidazole), macrophages (RAM-11), neovessels (CD31), and hypoxia-inducible factor-1α were assessed by immunohistochemistry. 18 F-fluoromisonidazole uptake increased with time on diet (standardized uptake value mean, 0.10±0.01 in nonatherosclerotic animals versus 0.20±0.03 [ P =0.002] at induction and 0.25±0.03 [ P &lt;0.001] at progression). Ex vivo positron emission tomographic imaging corroborated the 18 F-fluoromisonidazole uptake by the aorta of atherosclerotic rabbits. 18 F-fluorodeoxyglucose uptake also augmented in atherosclerotic animals, with an standardized uptake value mean of 0.43±0.02 at induction versus 0.35±0.02 in nonatherosclerotic animals ( P =0.031) and no further increase at progression. By immunohistochemistry, hypoxia was mainly located in the macrophage-rich areas within the atheromatous core, whereas the macrophages close to the lumen were hypoxia-negative. Intraplaque neovessels were found predominantly in macrophage-rich hypoxic regions (pimonidazole + /hypoxia-inducible factor-1α + /RAM-11 + ). Conclusions— Plaque hypoxia increases with disease progression and is present in macrophage-rich areas associated with neovascularization. 18 F-fluoromisonidazole positron emission tomographic imaging emerges as a new tool for the detection of atherosclerotic lesions.

Magnoliae Cortex inhibits intimal thickening of carotid artery through modulation of proliferation and migration of vascular smooth muscle cells

Endovascular injury leads to proliferation and migration of vascular smooth muscle cells from the media to the intima leading to the intimal thickening. The objective of this study was to examine the effect of Magnoliae Cortex extract (MOE) on intimal thickening of rat carotid artery injured by balloon endothelial denudation. MOE was administered orally using gastric sonde at three different doses MOE200 (200mg/kg), MOE400 (400mg/kg), and MOE800 (800mg/kg) for 14days from the day of balloon injury. Also, in vitro assays of proliferation, migration and expression of matrix metalloproteinase-2 (MMP-2) in human aortic smooth muscle cells (HASMCs) were carried out using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, transwell boyden chamber method and gelatin zymography, respectively. Oral administration of MOE400 and MOE800 for 14days significantly inhibited intimal area, intimal/medial ratio (I/M), stenosis rate, expression of proliferating cell nuclear antigen (PCNA), MMP-2, and -9 in denudated rat carotid artery. Our in vitro assays revealed that MOE dose dependently inhibited proliferation, migration and expression of MMP-2 in HASMCs. Thus, the results suggest that MOE can be considered as a therapeutic value in the prevention of atherosclerosis because restenosis after percutaneous transluminal coronary angioplasty (PTCA) is supposed to be ‘accelerated atherosclerosis’.

English

To investigate the protective effects of rosiglitazone on the endothelial cells in a rabbit iliac artery balloon injury, thirty male New Zealand white rabbits were randomly divided into control group (hypercholesterol diet), balloon injury group (hypercholesterol diet plus balloon endothelial denudation) and rosiglitazone group (balloon endothelial denudation plus hypercholesterol diet and rosiglitazone). Rabbits were fed for eight weeks, and balloon endothelial denudation was performed. Rosiglitazone was administrated at a dose of 0.5 mg&middot;kg-1&middot;d-1&nbsp;from three days before surgery, and lasted for 4 weeks. Four weeks later, blood was collected and serum lipids, glucose, hs-CRP, NO, NOS and ET-1 levels were measured. Then, local iliac arteries were obtained for morphological examination and immunohistochemistry for PCNA and apoptosis.&nbsp;When compared with balloon injury group, the levels of serum TC, TG, LDL-C, ET-1 were significantly decreased in the rosiglitazone group. Morphological examination showed the intimal area (IA), medial area (MA) and intimal proliferation index in rosiglitazone group were significantly lower than those in the balloon injury group. The PCNA expression was markedly decreased and the apoptosis rate significantly increased in rosiglitazone group when compared with the balloon injury group. Rosiglitazone could exert protective effects on the endothelial cells after balloon injury in which the lipid-lowering, inhibited proliferation and increased apoptosis of endothelial cells, and compromised NOS activity and secretion of NO and ET-1 played important roles. &nbsp; Key words:&nbsp;Rosiglitazone, apoptosis, nitric oxide synthase, endothelin, rabbit.

The Impact of Different Concentrations of Sodium Tetradecyl Sulphate and Initial Balloon Denudation on Endothelial Cell Loss and Tunica Media Injury in a Model of Foam Sclerotherapy

Introduction Recanalisation rates (20–32%) 1–3 years after truncal vein foam sclerotherapy (FS) suggest thrombotic occlusion rather than irreversible vein wall injury. This study examines the injury inflicted by sodium tetradecyl sulphate (STD) foam before and after balloon endothelial denudation (BD). Methods In 20 patients undergoing great saphenous vein (GSV) stripping 1.5 cm proximal GSV were harvested (controls). The next 1.5 cm were harvested after in situ BD (n = 10) or no denudation (n = 10). These test segments were filled with 1% or 3% STD foam (5 min), flushed and fixed in formalin. Percentage endothelial cell loss (ECL) and tunica media injury were determined (H&E staining) and collagen structure assessed (transmission electron microscopy, TEM). Results Controls showed no injury. 1% and 3% STD foam caused 86.3% and 92.2% ECL ( p < 0.001 versus controls; 1% versus 3%, p = 0.55). Endothelial cells persisted in all sections. BD increased ECL (1%: 96.9%, 3%: 98.1%, p = 0.01) Tunica media injury (smooth muscle vacuolation) was minimal (8.9% (1% STD) and 12% (3% STD) of its depth) and not enhanced by BD (1%: 8.7%, p = 0.93; 3%: 11.3%; p = 0.86). No collagen disruption occurred (TEM). Conclusions Balloon denudation increased ECL but did not facilitate tunica media injury. Equivalent injury was inflicted by 1% and 3% STD.

Inhibitory Effects of Bezoar Bovis on Intimal Formation and Vascular Smooth Muscle Cell Proliferation in Rat

Intimal formation of animal carotid arteries induced by balloon endothelial denudation has been considered to be an "accelerated atherosclerosis" model and used in primary screening methods to evaluate natural drugs and chemical candidates. The aim of the present study was to examine whether intimal formation is prevented by Bezoar Bovis (dried cattle gallbladder stones: Niuhuang in Chinese and Go-o in Japanese), which has been used to prevent heart palpitation in patients with hypertension. The intimal-to-medial area ratio in rat carotid arteries 7 days after balloon endothelial denudation was significantly reduced by oral administration of Bezoar Bovis. Bezoar Bovis also suppressed vascular smooth muscle cells (VSMCs) proliferation, which is thought to play important roles in the intimal formation after endothelial damage and also atherosclerosis resulting from long-term inappropriate lifestyle. The present findings suggest that Bezoar Bovis may be useful for preventing atherosclerosis and for protection against restenosis after percutaneous coronary intervention, for which effective reduction method is not currently available.

Bofutsushosan, a Traditional Chinese Formulation, Prevents Intimal Thickening and Vascular Smooth Muscle Cell Proliferation Induced by Balloon Endothelial Denudation in Rats

Bofutsushosan (BOF), a traditional Chinese formulation (Kampo formulation in Japanese), is widely used for patients with obesity and hyperlipidemia resulting from long-term inappropriate lifestyles. Since atherosclerosis, a lifestyle-related disease, is accompanied by an abnormal accumulation of vascular smooth muscle cells (VSMCs) in the intimal area of the artery, we investigated the preventive effect of BOF on intimal thickening. Oral administration of BOF extracts 3 d before and 7 d after balloon endothelial denudation dose dependently suppressed the intimal thickening and proliferation of VSMCs in the intimal area in rat carotid arteries. This model has a similar pathologic process to atherosclerosis and is considered to be an "accelerated atherosclerosis" model. BOF extract also dose dependently inhibited the migration of cultured VSMCs. BOF extract suppressed serum lipid levels, which are a major risk factor for atherosclerosis. These findings clarified the usefulness of BOF in cardiovascular risk-reduction therapy.

Effects of traditional Chinese formulations on rat carotid artery injured by balloon endothelial denudation.

We examined the inhibitory effects of traditional Chinese formulations (TCFs: Kampo formulation in Japanese) on intimal thickening of the carotid artery injured by balloon endothelial denudation in rats. Among the eight TCFs examined oren-gedoku-to (huanglian-jiedu-tang in Chinese), choto-san (diao-teng-san), saiko-ka-ryukotsu-borei-to (chaihujia-longgu-muli-tang) and dai-joki-to (da-cheng-qi-tang) significantly inhibited the intimal thickening 7 days after denudation. These four TCFs also inhibited proliferation of vascular smooth muscle cells (VSMC), which may play a central role in the development of restenosis after balloon endothelial denudation. The present results suggest that further evaluation of these four TCFs as inhibitors of VSMC proliferation to prevent arteriosclerosis is warranted.

Contribution of the Helix-Loop-Helix Factor Id2 to Regulation of Vascular Smooth Muscle Cell Proliferation

Smooth muscle cell (SMC) proliferation plays a key role in vascular proliferative disorders. The molecular mechanisms that control cell cycle entry of SMCs in response to vascular injury are not well understood. Id2 (inhibitor of DNA binding) is a member of the helix-loop-helix (HLH) family of transcription regulators that are known to promote cell cycle progression. Thus, we investigated the role of Id2 in SMC growth and cell cycle regulation. The results demonstrated that overexpression of Id2 resulted in a significant enhancement of SMC growth via increased S-phase entry. A possible mechanism of Id2-enhanced SMC growth is via regulation of p21 expression, as overexpression of Id2-inhibited transcriptional activity of a 2.3-kb p21 promoter/luciferase reporter construct as well as p21 protein levels. Id2 enhancement of SMC growth and inhibition of p21 expression were dependent on phosphorylation of Id2 by cyclin E/cdk2, as an Id2 cDNA containing a mutation in the cdk2 phosphorylation site (serine 5) failed to regulate SMC cell cycle progression or p21 promoter activity. The mechanism of cyclin E/cdk2 control of the Id2 effect may in part involve regulation of nuclear transport; unlike wild-type Id2, the Id2 mutant was not transported to the nucleus. Finally, in a rat carotid model of arterial injury, Id2 was expressed in a temporal pattern that parallels the kinetics of cellular proliferation. In summary, these results provide evidence that the Id2 protein is integrated into the cell cycle regulatory cascade that results in SMC proliferation following vascular injury and suggest that this effect is at least in part via a cdk2-dependent inhibition of p21 gene expression.

Neutrophil, not macrophage, infiltration precedes neointimal thickening in balloon-injured arteries.

Macrophages are abundant after stent-induced arterial injury. Inhibition of macrophage recruitment blocks neointimal growth in this model. In contrast, after superficial injury from balloon endothelial denudation, macrophages are sparse. However, many anti-inflammatory therapies remain effective against neointimal growth after balloon injury. To investigate further the role of leukocytes after injury, 41 New Zealand White rabbits underwent iliac artery balloon denudation. In 18, subcutaneous pumps were placed to deliver intravenous heparin (0.3 mg/kg per hour). Arteries were harvested at 6 hours and at 3, 7, and 14 days. In 8 animals, either M1/70 (a monoclonal antibody [mAb] against adhesion molecule Mac-1) or a nonspecific IgG was given (5 mg/kg IV bolus and then 1 mg/kg SC QOD), and arteries were harvested at 6 hours and 3 days. Computer-aided morphometry was performed as was immunohistochemistry to assess smooth muscle cell (SMC) proliferation (bromodeoxyuridine-positive cells), neutrophil content (RPN357, mAb against rabbit neutrophil/thymocyte), and macrophage content (RAM-11, mAb against rabbit macrophage). Heparin inhibited neointimal growth at 7 and 14 days (64% and 32.5% reduction, respectively; P:<0.05). Neutrophils were observed in the media early after balloon injury, and heparin and M1/70 inhibited this infiltration (82% and 83% reduction, respectively; P:<0.05 each) with a coincident inhibition of medial SMC proliferation at 3 days (49% and 84% reduction, respectively; P:<0.05 each). Macrophages were absent at all time points. Neutrophil, but not macrophage, infiltration occurs early after endothelial denudation. Inhibition of this process is associated with a reduction in medial SMC proliferation. These data suggest a central role for neutrophils in restenosis and help to explain prior reports of an inhibitory effect of anti-inflammatory therapies on neointimal growth after balloon injury.

O-43 - Role of iNOS during the course of neointimal formation in the rabbit carotid artery after balloon endothelial denudation

O-43 - Role of iNOS during the course of neointimal formation in the rabbit carotid artery after balloon endothelial denudation

Alteration of endothelium-dependent hyperpolarizations in porcine coronary arteries with regenerated endothelium.

The present study was designed to test the ability of regenerated endothelium to evoke endothelium-dependent hyperpolarizations. Hyperpolarizations induced by serotonin and bradykinin were compared in isolated porcine coronary arteries with native or regenerated endothelium, 4 weeks after balloon endothelial denudation. The experiments were performed in the presence of inhibitors of nitric oxide synthase (Nomega-nitro-L-arginine) and cyclooxygenase (indomethacin). The transmembrane potential was measured using conventional glass microelectrodes. Smooth muscle cells from coronary arteries with regenerated endothelium were depolarized in comparison with control coronary arteries from the same hearts. Spontaneous membrane potential oscillations of small amplitude or spikes were observed in some of these arteries but never in arteries with native endothelium. In coronary arteries from control pigs, both serotonin and bradykinin induced concentration-dependent hyperpolarizations. In the presence of ketanserin, 10 micromol/L serotonin induced a transient hyperpolarization in control coronary arteries. Four weeks after balloon denudation, the response to serotonin was normal in arteries with native endothelium, but the hyperpolarization was significantly lower in coronary arteries with regenerated endothelium. In control arteries, the endothelium-dependent hyperpolarization obtained with bradykinin (30 nmol/L) was reproducible. Four weeks after balloon denudation, comparable hyperpolarizations were obtained in coronary arteries with native endothelium. By contrast, in arteries with regenerated endothelium, the hyperpolarization to bradykinin became voltage-dependent. In the most depolarized cells, the hyperpolarization to bradykinin was augmented. The changes in resting membrane potential and the alteration in endothelium-dependent hyperpolarizations observed in the coronary arteries with regenerated endothelium may contribute to the reduced response to serotonin and the unchanged relaxation to bradykinin described previously.

Differential regulation of somatostatin receptor types 1-5 in rat aorta after angioplasty.

Treatment of restenosis after angioplasty with octapeptide somatostatin (SST) analogs has met with variable success. These analogs bind with high affinity to only two SST receptor (SSTR) subtypes (2 and 5), display moderate affinity for SSTR3, and low affinity for SSTR1 and 4. To optimize the vasculoprotective effect of SST, we have investigated the pattern of expression of all five SSTRs in rat thoracic aorta in the resting state and at 15 min, 3, 7, and 14 days after balloon endothelial denudation. SSTR1-5 were analyzed as mRNA by semiquantitative reverse transcriptase-polymerase chain reaction and as protein by immunocytochemistry. All five SSTRs were expressed in rat aorta both as mRNA and protein and displayed a time-dependent, subtype-selective response to endothelial denudation. mRNA for SSTR1 and 2 increased acutely (SSTR1 > SSTR2) on days 3 and 7, coincident with smooth muscle cell (SMC) proliferation, and declined to basal levels by day 14. SSTR3 and 4 displayed a different pattern with a delayed, more gradual increase in mRNA beginning at days 3-7 and continued to increase thereafter. SSTR5 mRNA was constitutively expressed at a low level and showed no change during the 2 wk postinjury period. By immunohistochemistry, SSTR1-5 antigens were localized predominantly in SMC that were present in the media or had migrated into the intima; antigen expression correlated with receptor mRNA expression. Notably, only SSTR1,3,4 were expressed in the intima: SSTR1 and 4 during the proliferative burst and SSTR3 and 4 after proliferation, when SMC migration into the intima continues. These results demonstrate dynamic changes in SSTR1-5 expression after vascular trauma localized to areas of vascular SMC migration and replication. In view of their early and prominent induction, SSTR1 may be the optimal subtype to target for inhibition of myointimal proliferation, and SSTR3 and 4 for migration and remodeling.

ATZ1993, an orally active and novel nonpeptide antagonist for endothelin receptors and inhibition of intimal hyperplasia after balloon denudation of the rabbit carotid artery.

The present experiments were designed to investigate the effect of ATZ1993 [3-carboxy-4,5-dihydro-1-[1-(3-ethoxyphenyl)propyl]-7-(5-pyrimidinyl)met hoxy-[1H]-benz[g]indazole] on the intimal hyperplasia after balloon endothelial denudation of the rabbit carotid artery. ATZ1993 inhibited the specific [125I]endothelin (ET)-1 binding not only to ET-receptor subtype A (ET(A)) with a pKi value of 8.69+/-0.02, but also to ET-receptor subtype B (ET(B)) with a pKi value of 7.20+/-0.03. Counterscreening in the binding assay (30 different receptors) confirmed that ATZ1993 had a high selectivity for ET receptors. Increases in intima:media ratio and DNA content in the vessel wall were significantly (P < 0.005) inhibited by ATZ1993 in a daily dose of 30 mg x 200 ml(-1) x kg(-1) for 1 week before and 6 weeks after balloon denudation. Inhibition of the intimal hyperplasia with ATZ1993 was determined as approximately 77% for increases in intima:media ratio and DNA content. Plasma concentrations of ATZ1993 ranged between 121.6+/-26.6 and 131.7+/-20.9 nM throughout experimental periods. Mean arterial blood pressure, heart rate and body weight gain remained unaffected by administering ATZ1993. These results demonstrate that ATZ1993 is a novel nonpeptide and nonselective ET(A)/ET(B)-receptor antagonist, and the agent when administered orally inhibits effectively intimal hyperplasia after balloon denudation of the rabbit carotid artery.

Effect of Aloe emodin on proliferation of vascular smooth muscle cells after arterial injury

To study the effect of Aloe-emodin (AE), an active ingredient of Rhubarb, on the kinetics of proliferation of smooth muscular cells (SMCs) cultured in vitro after rabbit iliac arterial injury. Forty-eight hours after de-endothelialization (balloon endothelial denudation), the iliac arteries of the Japanese white rabbits were isolated and the smooth muscle cells were cultured primarily. AE was added to culture medium containing 10% fetal calf serum (FCS). The cultures were pulse-labeled with 3H-TdR and TdR uptake into VSMC were measured and the cell cycle of the cultures were analyzed by using flow cytometer. Compared with control, when the concentration gradient ranged from 10(-1) to 10(-5) g/L, the amount (cpm, count per minute) of 3H-TdR uptake into SMCs has significant differences (P < 0.05) and 10(-1) and 10(-2) g/L AE showed strong inhibitory effects on TdR uptake into VSMC and the percentage of inhibition [% inhibition = (cpm without AE - cpm with AE)/cpm without AE x 100%] was more than 90%. AE displayed concentration dependent inhibitory effects. The percentage of cells in G0/G1 phase was increased, but the percentage of cells in S phase was decreased in AE group, the transition of SMC cycle phase from G0 to S was blocked. AE is a strong inhibitor to the proliferation of SMCs and the pharmacological action of AE might reduce SMC proliferation in vivo and decrease intimal hyperplasia of restenosis.

Expression of inducible nitric oxide synthase after endothelial denudation of the rat carotid artery: role of platelets.

There is functional evidence suggesting that endothelial denudation stimulates inducible nitric oxide synthase (iNOS) activity in the vascular wall. In vitro studies have shown that iNOS expression in smooth muscle cells is reduced by endothelial cells. In the present study we have analyzed the time course of iNOS protein expression in the arterial wall after in vivo deendothelialization. Endothelial denudation was performed in the left carotid artery of Wistar rats, and the right carotid artery was used as control. Whereas iNOS protein was weakly expressed 6, 24, and 48 hours after endothelial denudation, a marked iNOS expression was found 7, 14, and 30 days after vascular damage. Because platelet adhesion and aggregation occur early after endothelial damage, we studied the role of activated platelets in the negative modulation of iNOS protein expression during the first 2 days after endothelial denudation. Early after in vivo endothelial injury, platelet-depleted rats showed a marked iNOS protein expression in the vascular wall. Similar results were obtained by blocking the platelet glycoprotein (GP) IIb/IIIa. Although iNOS protein is present in the arterial wall several days after endothelial denudation, early after arterial wall injury iNOS protein is weakly expressed. Platelets play a crucial role in preventing iNOS protein expression early after endothelial damage, an effect that can be avoided with GP IIb/IIIa blockers. Although iNOS protein was weakly expressed in vivo in the rat carotid artery wall 6, 24, and 48 hours after balloon endothelial denudation, a marked iNOS expression was found 7, 14, and 30 days after arterial damage. iNOS expression could be increased early after endothelial injury by removing circulating platelets and by an antibody against the GP IIb/IIIa. In conclusion, platelets prevent iNOS protein expression early after endothelial balloon damage, an effect that can be avoided with GP IIb/IIIa blocking agents.

Morphological and functional changes in coronary vessel evoked by repeated endothelial injury in pigs.

We examined the morphological changes induced by repeated endothelial denudation in coronary artery (CA), as well as functional changes in the endothelium-dependent and smooth muscle responses to various vasoactive agents during the process of intimal thickening. We observed vascular responses in denuded and non-denuded portions of pig CA while being fed a normal diet (n = 11, N group) or 2% cholesterol diet (n = 25, C group) to intracoronary acetylcholine (ACh), 5-hydroxytryptamine (5-HT), substance P (SP), and isosorbide dinitrate (ISDN) with and without the nitric oxide synthesis inhibitor N omega-nitro-L-arginine methyl ester (L-NAME, 10 mg/kg i.v.) over a period of 8 weeks. Balloon endothelial denudation of the left anterior descending CA was carried out every 2 weeks. In N group, maximum vasoconstriction was obtained with ACh 2 weeks after the first denudation [26 +/- 5% vs. 1 +/- 1% pre-denudation, p < 0.05]. L-NAME did not affect ACh-induced CA diameter changes. Thereafter, the response to ACh was attenuated by repeated denudation in N groups. However, the degree of 5-HT-induced CA narrowing at the denuded portion increased from 7 +/- 4% (0 week) to 88 +/- 8% (8 weeks) (p < 0.05). The changes resulted in severe myocardial ischaemia, and suggested that endothelium-dependent vasodilation was progressively attenuated while hyperreactivity of vascular smooth muscle simultaneously increased. Vasodilation induced by SP was attenuated somewhat, but ISDN-induced vasodilation was preserved. Although mild hypercholesterolaemia was induced in C group, the vascular responses to these vasoactive agents did not differ from those of N group. Repeated CA endothelial injury and regeneration induce the change of morphology and vascular reactivity in the denuded portion regardless of atherogenic diet. This study strongly suggests that intimal thickening caused by repeated endothelial injury and regeneration induces specific vascular responses to vasoactive agents. Moreover, it is also suggested that during the progression of intimal thickening, increased vascular smooth muscle contraction and decreased endothelium-dependent dilation appear in a stimulus-dependent manner, often leading to severe coronary vasoconstriction accompanied with definitive ECG ST change.