Atherosclerotic lesions in the coronary arteries of hyperlipidemic swine. Part 1. Cell increases, divisions, losses and cells of origin in first 90 days on diet

The intima of the proximal portion of the coronary arteries of young swine is normally thickened by accumulations of cells about 90% of which are smooth muscle cells (SMC) and about 10% are of probable monocyte origin. Extracellular components such as collagen and elastic tissue are also present but we have chosen to emphasize their cellular nature by calling the regions of thickened intima, intimal cell masses (ICM). We have previously shown that atherosclerotic lesions produced in the coronary arteries of swine by 90 days of feeding a hyperlipidemic (HL) diet arise almost exclusively in the normally occurring ICM. We are reporting here a study of the pathogenesis of these lesions following killing at 0, 14, 49 and 90 HL diet days with comparisons between ICM in control mash-fed swine and ICM-lesions in the HL swine. We found that in the ICM: lipid accumulation was present by 14 days and increased thereafter; the lipid was mostly in SMC but percentage wise the monocyte-macrophages were involved as much or more, cell division activity was increased 3-4-fold by 49 days, cell numbers in ICM were similar in HL and control swine at 49 days but were about 6-fold greater in the HL swine at 90 days, (now in ICM-lesions), at 90 days, circa 90% of the cells appeared to be of SMC and circa 10% of monocyte origin both in the ICM-lesions of the HL swine and in the normal ICM of the controls. The data suggest but do not prove that early lipid accumulation precedes increased cell divisions especially among the SMC component and this in turn precedes increased numbers of cells in the ICM. Although SMC constitute the major cell component of the ICM-lesion at 90 days, the monocyte-macrophage-like cells also increase in number as a result of the HL diet and constitute a small but definite minor component. One possible explanation for the increased cell division activity is that one of the lipid constituents is acting as mitogen; another possibility is that the effect of a well known mitogen such as platelet-derived growth factor is enhanced by the lipid; another is that the monocytes are being stimulated to produce monocyte-derived growth factor. In any event in the very early stage of atherogenesis in the coronary arteries in these experiments excessive proliferation of resident SMC in the ICM appears to be the predominant feature.     

Apoptosis and regulation of Bax and Bcl-X proteins during human neonatal vascular remodeling

To verify that apoptosis is one of the possible mechanisms of neonatal vascular remodeling during the transition from fetal to neonatal circulation, we assayed for apoptosis and evaluated the expression of apoptosis-regulatory proteins in umbilical vessel versus ascending aorta, ductus arteriosus (DA) versus adjacent pulmonary artery and aorta, or aorta versus its branching arteries. Twenty-two umbilical cords (UCs), 6 DAs with adjacent aortas and pulmonary arteries, and 4 aortic arches with their branching great arteries were obtained from neonates. Smooth muscle cell (SMC) apoptosis in umbilical vessels was identified in all UCs. The expressions of Bax and Bcl-X were stronger in umbilical artery than in the neonatal aorta, but Bcl-2 was weak in both arteries in immunohistochemistry. In the immunoblot analysis of UCs, the expression of the proapoptotic short isoform of Bcl-X was stronger than in other tissue, and caspase-3 was selectively activated, whereas it was not in the other components of the cardiovascular system. In contrast, the expression patterns of the FasAg and Fas ligand were similar in umbilical artery and aorta. Regulation of Bcl-2 family proteins was also observed in other vascular sites at which SMCs undergo apoptosis on hemodynamic changes during birth, such as the DA and the branching points of the great arteries from the aortic arch. Apoptosis is involved in the regression of human umbilical vessels and the DA and in the remodeling of the branching great arteries during the neonatal period, when Bcl-2 family proteins are likely to play a key role.     

Developmental changes in the expression of voltage-gated potassium channels in the ductus arteriosus of the fetal rat

Oxygen-sensitive, voltage-gated potassium channels (Kv) may contribute to the determination of the membrane potential in smooth muscle cells of the ductus arteriosus (DA), and thus to regulation of contractile tone in response to oxygen. Developmental changes in Kv during gestation may be related to closure of the DA after birth. This study investigated developmental changes in the expression of Kv in the DA and compared it with that of the pulmonary artery (PA) and the aorta (Ao). The DA, PA, and Ao were isolated from fetal rats at days 19 and 21 of gestation (term: 21.5 days). The expression of Kv1.2, Kv1.5, Kv2.1, and Kv3.1, putative oxygen-sensitive Kv channels that open in response to oxygen, was evaluated at both the mRNA and protein levels, using quantitative real-time polymerase chain reaction and immunohistochemistry. In the Kv family studied, Kv1.5 mRNA was expressed most abundantly in the DA, PA, and Ao in both day-19 and day-21 fetuses. Although the expression levels of Kv1.2, Kv1.5, Kv2.1, and Kv3.1 did not change much with development in the PA and Ao, in the DA they decreased with development. The decrease in the expression of Kv channels may enhance DA closure after birth by eliminating the opening of Kv channels when oxygen increases.     

大鼠主动脉内皮剥脱后平滑肌细胞增殖与c-myc基因表达的研究

用Fogarty导管对25只大鼠行主动脉内皮细胞剥脱，在剥脱后的24h,5天，10天，15天时观察，发现主动脉平滑肌细胞增殖和内膜增厚随时间延长而降低，20天接近基础水平，结果表明，c-myc基因表达可能在启动平滑肌细胞增殖和内膜增厚中起重要作用。     

大鼠主动脉内皮剥脱后平滑肌细胞增殖与c-myc基因表达的研究

用Ｆｏｇａｒｔｙ导管对２５只大鼠行主动脉内皮细胞剥脱。在剥脱后的２４ｈ、５天、１０天、１５天时观察，发现主动脉平滑肌细胞增殖和内膜增厚随时间延长而降低，２０天接近基础水平。结果表明，ｃ－ｍｙｃ基因表达可能在启动平滑肌细胞增殖和内膜增厚中起重要作用。     

Blood flow regulates the development of vascular hypertrophy, smooth muscle cell proliferation, and endothelial cell nitric oxide synthase in hypertension

Blood flow participates in vascular remodeling during development and growth by regulating cell apoptosis and proliferation. However, its significance in the development of vascular hypertrophy and vascular remodeling in hypertensive patients is not known. We investigated how changing blood flow through the common carotid artery (CA) of young adult rats rendered hypertensive via aortic coarctation affects CA hypertrophy and/or remodeling responses to hypertension. Blood flow was reduced by approximately 50% as a result of ligation of the external CA immediately after hypertension was induced, and the effects of that procedure were compared with those in similarly treated normotensive rats. Reducing blood flow in the hypertensive animals markedly augmented the development of CA hypertrophy over the ensuing 14 days by increasing the vessel wall cross-sectional area. In those animals, CA lumen size was unaltered by reducing blood flow, as was CA structure in normotensive animals. The greater hypertrophy in the hypertensive animals with reduced blood flow was associated with enhanced smooth muscle cell (SMC) proliferation 3 days after the hemodynamic changes were induced. There also appeared to be more extensive remodeling of the endothelium in the hypertensive animals with normal flow; this was indicated by the greater frequency of apoptotic endothelial cells at that time. This reduction in blood flow also attenuated endothelial cell nitric oxide synthase expression in hypertensive animals but not in normotensive animals. Severe reductions in blood flow ( approximately 90%) were required to reduce endothelial cell nitric oxide synthase in the normotensive animals. Increasing CA nitric oxide levels by perivascular application of S-nitroso-N-acetylpenicillamine (SNAP) to the CAs of hypertensive animals with reduced endothelial cell nitric oxide synthase attenuated the greater SMC proliferation. Thus, reduced blood flow in hypertensive animals promotes hypertrophy by enhancing SMC proliferation via mechanisms that reduce the inhibitory effects of nitric oxide on SMC proliferation.     

CD44-v6 expression in smooth muscle cells in the postnatal remodeling process of ductus arteriosus

Closure of the ductus arteriosus (DA) is due to functional constriction followed by wall remodeling, with neointimal formation caused by proliferation and migration of smooth muscle cells (SMCs) from the media to subendothelium. CD44 is a surface cell proteoglycan family. Its isoform, CD44-v6, is only minimally expressed in SMCs in the media of normal arteries, but is highly expressed in SMCs in the intima and media of injured arteries (e.g., atherosclerosis). Twenty-two autopsy DA specimens, 11 from full-term babies (age range 2 days to 5 months) and 11 from premature babies (age range 3 days to 5 months), with varying degrees of ductal wall remodeling, were evaluated by immunohistochemistry using antiactin, antifibronectin-extradomain A, anti-leukocyte common antigen, anti-CD44, and anti-CD44-v6. In DA with wall remodeling, synthetic antifibronectin-extradomain A-positive SMCs were evident at the neointimal mounds, and the SMCs were highly positive for the CD44-v6 isoform, irrespective of gestational age at birth. Conversely, SMCs of either closed DAs or persistently patent DAs were CD44-v6 negative. In conclusion, the present data provide evidence that closure of DA involves synthetic SMCs highly positive for CD44-v6, and patent or closed DAs are populated by CD44-v6-negative SMCs.     

Wnt signalling in smooth muscle cells and its role in cardiovascular disorders

Vascular smooth muscle cells (SMCs) are the major cell type within blood vessels. SMCs exhibit low rates of proliferation, migration, and apoptosis in normal blood vessels. However, increased SMC proliferation, migration, and apoptosis rates radically alter the composition and structure of the blood vessel wall and contribute to cardiovascular diseases, such as atherosclerosis, and restenosis that occur after coronary artery vein grafting and stent implantation. Consequently, therapies that modulate SMC proliferation, migration, and apoptosis may be useful for treating cardiovascular diseases. The family of Wnt proteins, which were first identified in the wingless drosophila, has a well-established role in embryogenesis and development. It is now emerging that Wnt proteins also regulate SMC proliferation, migration, and survival. In this review article, we discuss recently emerging research that has revealed that Wnt proteins are important regulators of SMC behaviour via activation of β-catenin-dependent and β-catenin-independent Wnt signalling pathways.     

BMP9 and BMP10 are necessary for proper closure of the ductus arteriosus

The transition to pulmonary respiration after birth requires rapid alterations in the structure of the mammalian cardiovascular system. One dramatic change that occurs is the closure of the ductus arteriosus (DA), an arterial connection in the fetus that directs blood flow away from the pulmonary circulation. Two members of the TGFβ family, bone morphogenetic protein 9 (BMP9) and BMP10, have been recently involved in postnatal angiogenesis, both being necessary for remodeling of newly formed microvascular beds. The aim of the present work was to study whether BMP9 and BMP10 could be involved in closure of the DA. We found that Bmp9 knockout in mice led to an imperfect closure of the DA. Further, addition of a neutralizing anti-BMP10 antibody at postnatal day 1 (P1) and P3 in these pups exacerbated the remodeling defect and led to a reopening of the DA at P4. Transmission electron microscopy images and immunofluorescence stainings suggested that this effect could be due to a defect in intimal cell differentiation from endothelial to mesenchymal cells, associated with a lack of extracellular matrix deposition within the center of the DA. This result was supported by the identification of the regulation by BMP9 and BMP10 of several genes known to be involved in this process. The involvement of these BMPs was further supported by human genomic data because we could define a critical region in chromosome 2 encoding eight genes including BMP10 that correlated with the presence of a patent DA. Together, these data establish roles for BMP9 and BMP10 in DA closure.The ductus arteriosus (DA) is a large blood vessel whose obstruction is essential for the transition from fetal to neonatal circulation. It is a fetal arterial shunt connecting the pulmonary artery with the aortic arch. During fetal life, the DA directs deoxygenated blood away from the pulmonary circulation and toward the descending aorta, bypassing the nonventilated fetal lungs. After birth, the DA closes spontaneously within 1–3 h in small rodents or within 24–48 h in human newborns (1, 2). Although an open DA is required for fetal survival, the persistence of a patent DA (PDA) after birth is a major cause of morbidity and mortality, particularly in preterm neonates, leading to severe complications, including pulmonary hypertension, right ventricular dysfunction, postnatal infections, and respiratory failure. The incidence of PDA has been estimated to be one in 500 in-term newborns and accounts for the majority of all cases of congenital heart disease in preterm newborns. It is currently believed that DA closure involves a two-step process (3, 4). The first, provisional closure, also called functional closure, occurs at birth and is accomplished by smooth muscle cell contraction and DA constriction. The second step, named anatomical closure, involves a profound remodeling of cells within the former DA lumen and permits permanent closure of the DA. Although several factors have been implicated in DA closure (oxygen tension, prostaglandin E2, laminin, growth hormone, and platelets), the precise molecular and cellular signals that promote the transition from initial constriction to definitive DA closure are not yet fully understood.Bone morphogenetic protein 9 (BMP9) and BMP10 are two members of the BMP family that have been demonstrated to play major roles in vascular development (5). In 2007, it was demonstrated that BMP9 and BMP10 bind with high affinity to the endothelial-specific receptor activin receptor-like kinase 1 (ALK1), a type 1 receptor of the TGFβ family (6) whose mutations are involved in vascular diseases (5). Both BMP9 and BMP10 are present in blood, and their circulating levels are particularly elevated in mice around birth (7, 8), suggesting that they could play a role in pre- and postnatal development. In the present work, we addressed whether BMP9 and BMP10 could be involved in DA closure. For this purpose, we used Bmp9-KO mice and neutralizing anti-BMP10 antibody. Herein, we show that injection of a neutralizing anti-BMP10 antibody into Bmp9-KO pups led to an open DA and identified several targets that may be involved in this closure defect. This work is further supported by human genomic data, based on the definition of a 700-kb minimal critical region in chromosome 2 encoding eight genes, including BMP10, that correlated with the presence of a PDA in two patients. This work thus identifies a previously unidentified signaling pathway, the BMP9/10 pathway, in the anatomical closure of the DA.     

Time course of smooth muscle cell proliferation in the intima and media of arteries following experimental angioplasty.

Smooth muscle cell (SMC) proliferation is known to be an important factor for the development of restenosis after percutaneous transluminal coronary angioplasty. To determine the time course of intimal and medial SMC proliferation and morphological changes after experimental angioplasty, an intimal atheroma was produced with repeated weak electrical stimulations in the right carotid artery of 45 male New Zealand White rabbits. Angioplasty was subsequently performed in 35 rabbits, and the proliferative responses were analyzed with histomorphological and immunohistological criteria at 3, 7, 14, 21, 28, and 42 days after intervention. A hemodynamic relevant stenosis after angioplasty was found in eight (23%) of 35 dilated arteries. In five rabbits the stenosis was due to a mural thrombus, and in three animals restenosis was caused by intimal SMC proliferation. In all dilated arteries the intimal wall thickness increased from 13 +/- 5 intimal cell layers (after electrical stimulation) to 33 +/- 14 cell layers during 28 days after angioplasty (p less than 0.05). Later than 4 weeks after angioplasty, no additional increase of intimal thickening occurred. Application of bromodeoxyuridine 18 and 12 hours before excision of the vessels allowed determination of the percent of cells undergoing DNA synthesis in the intima and media using monoclonal antibody against bromodeoxyuridine. SMCs were identified by alpha-actin staining. Immunohistological quantification of intimal SMC proliferation showed a maximum of cells undergoing DNA synthesis within the first 7 days after angioplasty (p less than 0.01). In contrast, medial proliferation of SMCs was delayed and showed a small but significant increase 21 days after dilatation (p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Morphological changes and smooth muscle cell proliferation after experimental excimer laser treatment

BACKGROUND. Little is known about the mechanism(s) in the development of restenosis after excimer laser angioplasty. Thus, the rationale of this study was to determine the time course of intimal and medial smooth muscle cell (SMC) proliferation and histomorphological changes after experimental excimer laser treatment. METHODS AND RESULTS. Laser ablation was performed in the right carotid artery of 34 New Zealand White rabbits after development of a fibromuscular plaque by repeated weak electrical stimulations. The vessels were excised 3, 7, 14, 21, 28, and 42 days after excimer laser treatment. Staining of alpha-actin was used to identify SMCs. In five rabbits (15%), a stenosis of more than 50% of luminal area was due to intimal proliferation of SMCs, and in four other rabbits, a total occlusion was due to organized thrombi. After the initial ablation of the performed plaque (13 +/- 6 intimal SMC layers) a continuous increase of intimal wall thickness was found from 7 +/- 6 SMC layers at 7 days to 28 +/- 5 intimal SMC layers at 28 days after excimer laser ablation (p less than 0.01). After 42 days, no additional increase of intimal thickening occurred. After bromodeoxyuridine labeling, the extent of cell proliferation (percent of cells undergoing DNA synthesis) in the intima and media was determined using a monoclonal antibody against bromodeoxyuridine. Immunohistological quantification of SMC proliferation in the intima revealed a significant increase of cells undergoing DNA synthesis at 3 (p less than 0.05) and 14 (p less than 0.01) days after laser treatment. Medial proliferation of SMCs was delayed and had a significant increase 7 days (p less than 0.05) after intervention. Twenty-one days after laser treatment, SMC proliferation in the intima as well as in the media was normalized. CONCLUSIONS. The proliferative response of SMCs after experimental excimer laser treatment will occur as a dynamic process with a maximum of SMCs undergoing DNA synthesis during 14 days after laser ablation, resulting in an increase of intimal thickening within 4 weeks after laser treatment. The extent of intimal hyperplasia due to SMC proliferation after excimer laser treatment is comparable with the effect of transluminal balloon angioplasty in this experimental model.     

Dietary cholesterol withdrawal reduces vascular inflammation and induces coronary plaque stabilization in miniature pigs

OBJECTIVE: To study the effect of dietary cholesterol withdrawal on size and composition of LDL-hypercholesterolemia-induced coronary plaques in miniature pigs. METHODS: Pigs were on normal chow (control group), on a cholesterol-rich diet for 37 weeks (hypercholesterolemic group) or on a cholesterol-rich diet followed by normal chow for 26 weeks (cholesterol withdrawal group). Endothelial function was assessed with quantitative angiography after intracoronary infusion of acetylcholine, plaque load with intra-coronary ultrasound and plaque composition with image analysis of cross-sections. The effect of porcine serum on coronary smooth muscle cell (SMC) function was studied in vitro. RESULTS: Cholesterol-rich diet caused LDL-hypercholesterolemia, increased plasma levels of oxidized LDL (ox-LDL) and C-reactive protein (CRP), and induced endothelial dysfunction and coronary atherosclerosis. Dietary cholesterol withdrawal lowered LDL, ox-LDL and CRP. It restored endothelial function, did not affect plaque size but decreased lipid, ox-LDL and macrophage content. Smooth muscle cells and collagen accumulated within the plaque. Increased smoothelin-to-alpha-smooth muscle actin ratio indicated a more differentiated SMC phenotype. Cholesterol lowering reduced proliferation and apoptosis. In vitro, hypercholesterolemic serum increased SMC apoptosis and decreased SMC migration compared to non-hypercholesterolemic serum. CONCLUSIONS: Cholesterol lowering induced coronary plaque stabilization as evidenced by a decrease in lipids, ox-LDL, macrophages, apoptosis and cell proliferation, and an increase in differentiated SMC and collagen. Increased migration and decreased apoptosis of SMC may contribute to the disappearance of the a-cellular core after lipid lowering.     

大鼠主动脉内皮损伤后原生型一氧化氮合成酶和内皮素-1基因表达的改变

采用Ｆｏｇａｒｔｙ导管损伤大鼠胸主动脉段内皮细胞，观察１、５、１５和２０天后胸主动脉组织形态学改变；提取胸主动脉段组织ＲＮＡ采用反转录－多聚酶链反应方法测定原生型一氧化氨合成酶基因和内皮素－１基因ｍＲＮＡ的表达。结果表明，胸主动脉压血管平滑肌细胞在内皮损伤后增殖，内皮素－１基因表达增加，原生型一氧化氮合成酶基因表达减少，提示原生型一氧化氮合成酶基因和内皮素－１基因表达的改变可能在内皮损伤后的平滑肌细胞增殖中起重要作用。     

大鼠主动脉内皮损伤后原生型一氧化氮合成酶和内皮素-1基因表达的改变

采用Ｆｏｇａｒｔｙ导管损伤大鼠胸主动脉面内皮细胞，观察１、５、１５和２０天后胸主动脉组织形态学改变，提取胸主动脉段组织ＲＮＡ采用反转录－多聚酶链反应方法测定原生型一氧化氮合成酶基因和内皮素－１基因ｍＲＮＡ的表达，结果表明，胸主动脉段血管平滑肌细胞在内皮损伤后增殖，内皮素－１基因表达增加，原生型一氧化氮合成酶基因表达减少，提示原生型一氧化氮合成酶基因和内皮素－１基因表达的改变可能在内皮损伤后的平滑肌     

Early administration of keratinocyte growth factor improves {beta}-cell regeneration in rat with streptozotocin-induced diabetes

The aim of our study was to investigate the ability of keratinocyte growth factor (KGF; palifermin) in regulating beta-cell growth in normal newborn rats and in rats with neonatal diabetes. Wistar rats were injected with streptozotocin (STZ) to induce diabetes on the dayof birth. From days 2 to 6 after birth, animals received a daily s.c. injection of KGF (STZ/KGF group) and at the dose of 3 mg/kg body weight or saline solution (STZ groups). A group of non-diabetic Wistar rats was treated either with saline (Wistar group) or with KGF from days 2 to 6 after birth at the dose of 3 mg/kg body weight (Wistar/KGF group). beta-cell mass was measured at day 7 after birth in all groups. beta- and ductal cells replication were measured in all groups and apoptosis was assessed in the pancreas of 2-, 4-, and 7-day-old STZ and STZ/KGF rats. The total beta-cell mass of the 7-day-old KGF/STZ neonates was significantly increased compared with that of age-matched STZ rats. beta-cell replication rate was decreased at day 2 in the STZ/KGF group and was similar in the 4- and 7-day-old rats from STZ and STZ/KGF groups. Duct cell replication was significantly increased in the pancreas of 2- and 4-day-old KGF/STZ neonates when compared with that of age-matched rats from STZ control group. The rate of apoptosis in the neonatal pancreases of STZ and KGF/STZ groups was not significantly different. In non-diabetic Wistar rats, KGF treatment led to a slight but significant increase in duct cell proliferation at day 2 without significant changes in the total beta-cell mass in the 7-day-old rats. We provide evidence for a growth-promoting effect of KGF during beta-cell regeneration in neonatal diabetic rats. KGF exerts strong mitogenic effect on the pancreatic duct cells, thus expanding the population of precursor cells that subsequently differentiate into insulin-producing beta-cells.     

Regression of neointimal lesions in the carotid artery of nifedipine-treated SHR and WKY rats: possible role of apoptosis

We previously observed that nifedipine reduces aortic hypertrophy in spontaneously hypertensive rats (SHR) partly by inducing smooth muscle cell (SMC) apoptosis. The present study examined nifedipine regulation of SMC apoptosis in carotids with or without a neointima. A neointima was produced by endothelial denudation of the left carotid of SHR and WKY rats. The contralateral carotid remained uninjured. Beginning at week 6 after injury, rats received nifedipine or placebo for 5 and 7 additional weeks. In situ terminal deoxynucleotidyl transferase (TdT)-mediated DNA labeling was used to mark apoptotic nuclei. Nifedipine reduced blood pressure in SHR but not WKY rats. Nifedipine had antihypertrophic effects in both SHR and WKY rats. In each strain, the greater reduction in cross-sectional area was seen in the neointima. In this tissue, nifedipine significantly increased TdT-positive SMC (4-fold at week 5 in SHR and 5-fold at week 7 in WKY rats) and reduced SMC number (70% in SHR and 29% in WKY rats) at week 7 compared to week 0. The effects were less pronounced in the injured and uninjured media. Thus, the antihypertrophic action of nifedipine is amplified in the neointima of SHR and WKY rat carotids, where histological evidence suggests SMC deletion via apoptosis.     

c-Myc antisense oligonucleotides preserve smooth muscle differentiation and reduce negative remodelling following rat carotid arteriotomy

OBJECTIVES: The vascular biology of restenosis is complex and not fully understood, thus explaining the lack of effective therapy for its prevention in clinical settings. The role of c-Myc in arteriotomy-induced stenosis, smooth muscle cell (SMC) differentiation and apoptosis was investigated in rat carotids applying full phosphorothioate antisense (AS) oligonucleotides (ODNs). METHODS: Carotid arteries from WKY rats were submitted to arteriotomy and to local application of ODNs through pluronic gel. Apoptosis (deoxynucleotidyl transferase-mediated dUTP nick end-labelling), SMC differentiation (SM22 immunofluorescence) and vessel morphology and morphometry (image analysis) were determined 2, 5 and 30 days after injury, respectively. RESULTS: AS ODNs induced a 60% decrease of target c-Myc mRNA 4 h after surgery in comparison to control sense (S) and scrambled ODN-treated carotids (p < 0.05). A significant 37 and 50% decrease in SM22 protein in the media of S ODN-treated and untreated carotids was detected when compared to uninjured contralateral arteries (p < 0.05). This reduction in SM22 expression was prevented in AS ODN-treated carotids. Stenosis was mainly due to adventitial constrictive remodelling. Lumen area in AS ODN-treated carotids was 35% greater than in control arteries 30 days after surgery (p < 0.05). TUNEL assay revealed increased apoptosis in AS ODN-treated carotids (p < 0.05). CONCLUSIONS: c-Myc AS ODNs reduce arteriotomy-induced negative remodelling. This is accompanied by maintained SMC differentiation and greater apoptosis. The combination of reduced c-Myc-induced proliferation and increased apoptosis may thus underlie the less severe remodelling upon treatment with c-Myc mRNA AS ODN.     

Vitronectin is up-regulated after vascular injury and vitronectin blockade prevents neointima formation

OBJECTIVE: Smooth muscle cell (SMC) migration involves interactions with extracellular matrix (ECM) and is an important process in response to arterial wall injury. We investigated the expression and the functional role of vitronectin (VN) in the response after vascular injury. METHODS: VN and alpha v beta 3/beta 5 integrin expressions were investigated after balloon carotid injury of Sprague-Dawley rats. Adventitial delivery of blocking antibodies to VN, alpha v beta 5 and beta 3 integrins were performed to assess their roles in neointima formation. In vitro, migration assays were carried out on human SMC. RESULTS: Immunohistochemistry and in situ hybridization for VN showed an upregulation of VN during the early time points of intima formation. alpha v beta 3/beta 5 integrins expression correlated with VN expression. After 7 days, blocking antibodies to VN, alpha v beta 5 and beta 3 induced a significant decrease on intimal area associated with a decrease in intimal cell counts. A slight decrease in intimal cell proliferation without any effect on apoptosis was observed after VN blockade. In vitro, migrating SMC strongly expressed VN after injury and neutralizing anti-VN antibody inhibited SMC migration. Blocking experiment with anti-alpha v beta 5 and -alpha v beta 3 integrin antibodies showed that not only VN-alpha v beta 3 but also VN-alpha v beta 5 interactions are required for SMC migration. CONCLUSION: This study characterizes the VN-ECM interaction in SMC and supports the role of VN in mediating SMC migration and neointimal formation in response to injury.     

Aortic flow patterns in normal neonates with patent ductus arteriosus: evaluation by Doppler color flow imaging

Serial Doppler echocardiography was performed in 12 normal neonates (0.5-4.0 hrs after birth) to evaluate flow patterns through the ductus arteriosus, and in the aorta and brachiocephalic artery. At the initial examination, flow through the ductus arteriosus was bidirectional in eight of the 12 neonates and continuously left-to-right in the remaining four. The bidirectional ductal shunts became continuous left-to-right flows within 11-21 hrs after birth in seven of the eight neonates and resolved by 29-47 hrs after birth. In the remaining four neonates, the continuous left-to-right shunts disappeared 14-36 hrs after birth. Systolic ejection flow patterns in the aorta and brachiocephalic artery had a triangular shape with the peak velocity in early systole, followed by a minimal flow reversal in all sites examined. Diastolic flow patterns in each arterial site were as follows: 1. In the ascending aorta, there was slow and sustained diastolic forward flow, which did not change with increasing age. 2. In the brachiocephalic artery, there was a pan-diastolic flow reversal in the neonates with bidirectional ductal flow (7/8). This pattern changed to slow pan-diastolic forward flow when the ductal changed to continuous left-to-right flow or when the ductal closure was confirmed. Most (3/4) of the remaining four neonates with continuous left-to-right ductal flow exhibited pan-diastolic forward flow. Another showed a pan-diastolic flow reversal 2 hrs after birth, which changed to pan-diastolic forward flow in the second examination 6 hrs after birth. 3. In the distal aortic arch, there was a pan-diastolic forward flow in all the neonates, and the velocity decreased when a closure of the ductus was confirmed. 4. In the descending aorta, there was a pan-diastolic flow reversal in neonates with bidirectional ductal flow (7/8). This reversal changed to pan-diastolic forward flow, when the ductal flow changed to continuous left-to-right flow or when the ductal closure was confirmed. In the remaining four neonates with continuous left-to-right ductal flow, two showed a pan-diastolic flow reversal at the initial examinations 2 to 3 hrs after birth. This became a pan-diastolic forward flow at the second examinations 6 and 12 hrs after birth. In the other two, there was a pan-diastolic forward flow which did not change. This pan-diastolic flow reversal observed in the brachiocephalic artery and descending aorta was closely related to the bidirectional ductal flow.(ABSTRACT TRUNCATED AT 400 WORDS)