Hypoxia-induced pulmonary endothelin-1 expression is unaltered by nitric oxide.

Nitric oxide (NO) attenuates hypoxia-induced endothelin (ET)-1 expression in cultured umbilical vein endothelial cells. We hypothesized that NO similarly attenuates hypoxia-induced increases in ET-1 expression in the lungs of intact animals and reasoned that potentially reduced ET-1 levels may contribute to the protective effects of NO against the development of pulmonary hypertension during chronic hypoxia. As expected, hypoxic exposure (24 h, 10% O(2)) increased rat lung ET-1 peptide and prepro-ET-1 mRNA levels. Contrary to our hypothesis, inhaled NO (iNO) did not attenuate hypoxia-induced increases in pulmonary ET-1 peptide or prepro-ET-1 mRNA levels. Because of this surprising finding, we also examined the effects of NO on hypoxia-induced increases in ET peptide levels in cultured cell experiments. Consistent with the results of iNO experiments, administration of the NO donor S-nitroso-N-acetyl-penicillamine to cultured bovine pulmonary endothelial cells did not attenuate increases in ET peptide levels resulting from hypoxic (24 h, 3% O(2)) exposure. In additional experiments, we examined the effects of NO on the activity of a cloned ET-1 promoter fragment containing a functional hypoxia inducible factor-1 binding site in reporter gene experiments. Whereas moderate hypoxia (24 h, 3% O(2)) had no effect on ET-1 promoter activity, activity was increased by severe hypoxic (24 h, 0.5% O(2)) exposure. ET-1 promoter activity after S-nitroso-N-acetyl-penicillamine administration during severe hypoxia was greater than that in normoxic controls, although activity was reduced compared with that in hypoxic controls. These findings suggest that hypoxia-induced pulmonary ET-1 expression is unaffected by NO.     

Insulin regulates its own delivery to skeletal muscle by feed-forward actions on the vasculature

Insulin, at physiological concentrations, regulates the volume of microvasculature perfused within skeletal and cardiac muscle. It can also, by relaxing the larger resistance vessels, increase total muscle blood flow. Both of these effects require endothelial cell nitric oxide generation and smooth muscle cell relaxation, and each could increase delivery of insulin and nutrients to muscle. The capillary microvasculature possesses the greatest endothelial surface area of the body. Yet, whether insulin acts on the capillary endothelial cell is not known. Here, we review insulin's actions at each of three levels of the arterial vasculature as well as recent data suggesting that insulin can regulate a vesicular transport system within the endothelial cell. This latter action, if it occurs at the capillary level, could enhance insulin delivery to muscle interstitium and thereby complement insulin's actions on arteriolar endothelium to increase insulin delivery. We also review work that suggests that this action of insulin on vesicle transport depends on endothelial cell nitric oxide generation and that insulin's ability to regulate this vesicular transport system is impaired by inflammatory cytokines that provoke insulin resistance.     

Intravenous nanosomes of quercetin improve brain function and hemodynamic instability after severe hypoxia in newborn piglets

Perinatal asphyxia is a major cause of death and neurological morbidity in newborns and oxidative stress is one of the critical mechanisms leading to permanent brain lesions in this pathology. In this context we have chosen quercetin, a natural antioxidant, known also by its brain protective effects to study its potential as a therapy for brain pathology provoked by severe hypoxia in the brain. To overcame the difficulties of quercetin to access the brain, we have developed lecithin/cholesterol/cyclodextrin nanosomes as a safe and protective vehicle.We have applied the nanosomal preparation intravenously to newborn piglets submitted to a severe hypoxic or ischemic/hypoxic episode and followed them for 8 or 72 h, respectively. Either towards the end of 8 h after hypoxia or up to 72 h after, electroencephalographic amplitude records in animals that received the nanosomes improved significantly. Animals receiving quercetin also stabilized blood pressure and recovered spontaneous breathing. In this experimental group mechanical ventilation assistance was withdrawn in the first 24 h while the hypoxic and vehicle groups required more than 24 h of mechanical ventilation. Three days after the hypoxia the suckling and walking capacity in the group that received quercetin recovered significantly compared with the hypoxic groups. Pathological studies did not show significant differences in the brain of newborn piglets treated with nanosomes compared with hypoxic groups. The beneficial effects of quercetin nanosomal preparation after experimental perinatal asphyxia show it as a promising putative treatment for the damaged brain in development.     

Nitric oxide mediates hypoxia-induced changes in paracellular permeability of cerebral microvasculature

Ischemic stroke from a reduction in blood flow to the brain microvasculature results in a subsequent decreased delivery of oxygen (i.e., hypoxia) and vital nutrients to endothelial, neuronal, and glial cells. Hypoxia associated with stroke has been shown to increase paracellular permeability of the blood-brain barrier, leading to the release of cellular mediators and brain tissue injury. Whereas reperfusion does not occur in all ischemic strokes, increased permeability has been seen in posthypoxic reoxygenation. Currently, it is unknown whether these deleterious effects result from cellular mechanisms stimulated by decreased oxygen during stroke or posthypoxic reoxygenation stress. This study used primary bovine brain microvessel endothelial cells (BBMECs) to examine the involvement of nitric oxide (NO) as a mediator in hypoxia-induced permeability changes. Hypoxia-induced increased transport of [14C]sucrose across BBMEC monolayers compared with normoxia was attenuated by either posthypoxic reoxygenation or inhibition of NO synthase (NOS). The hypoxia-induced permeability effect was further reduced when NOS inhibition was combined with posthypoxic reoxygenation. Additionally, a significant increase in total NO was seen in BBMECs after hypoxic exposure. This correlation was supported by the increased [14C]sucrose permeability observed when BBMECs were exposed to the NO donor diethylenetriaamine NONOate. Western blot analyses of NOS isoforms showed a significant increase in the inducible isoform after hypoxic exposure with a subsequent reduction in expression on reoxygenation. Results from this study suggest that hypoxia-induced blood-brain barrier breakdown can be diminished by inhibition of NO synthesis, decreased concentration of NO metabolites, and/or reoxygenation.     

Hyperthermia inhibits angiogenesis

Since in vitro studies have demonstrated that capillary endothelial cells are thermosensitive, experiments were performed to determine the (in vivo) heat sensitivity of blood capillaries and their endothelial cells. Angiogenesis discs were implanted subcutaneously in mice, and vascular growth was stimulated by slow release of epidermal growth factor placed in the center of each disc. After 5 days of growth the discs were subjected to radiofrequency-induced hyperthermia. Heat exposures were 41, 42, 43, and 44 degrees C for 30 min. Control discs were sham treated. Seven days after heating the discs were extracted and paraffin embedded. Centripetal (radial) vessel growth was measured in magnified medial planar sections. An inverse relationship was demonstrated between vessel growth and exposure temperature. The extent of the fibroblastic growth was also inversely proportional to temperature. Thus, at least in this system, the microvasculature shows dose-dependent damage by hyperthermia, consistent with preceding in vitro observations. This inhibition of angiogenesis may result from endothelial cell killing, interference with cell replication, inhibition of cell migration, or a combination of these mechanisms.     

Dehydroepiandrosterone protects the microcirculation of muscle flaps from ischemia-reperfusion injury by reducing the expression of adhesion molecules

Adhesion molecules contribute to ischemia-reperfusion injury by increasing the endothelial adhesion and extravasation of leukocytes. Scientific evidence suggests that presurgical treatment with dehydroepiandrosterone may protect the microvasculature against this damage, but the exact mechanism is not known. The purpose of this study was to investigate the effects of presurgical dehydroepiandrosterone treatment on microcirculatory hemodynamic parameters and the expression of adhesion molecules in a rat cremaster muscle flap model. Twenty male rats were randomly assigned to three experimental groups. In group I (n = 5), the muscle flaps did not receive presurgical treatment. In group II (n = 6), propylene glycol (30 mg/kg), the vehicle for dehydroepiandrosterone, was injected intravenously before ischemia was induced. In group III (n = 9), dehydroepiandrosterone (30 mg/kg) was injected intravenously before ischemia was induced. All flaps were subjected to 6 hours of ischemia and 90 minutes of reperfusion. Microcirculatory variables (functional capillary density, red blood cell velocity in the main flap arteriole, and numbers of rolling, sticking, and transmigrating leukocytes), blood levels of three adhesion molecules (L-selectin, Mac-1 integrin, and CD44), and the numbers of leukocytes expressing those molecules were analyzed. Analysis of the microcirculatory parameters revealed that dehydroepiandrosterone treatment before ischemia had significant preservative effects on the red blood cell velocity and functional capillary density 30 and 90 minutes after reperfusion, compared with the control and vehicle-treated groups. Leukocyte-endothelial cell interactions were also affected by dehydroepiandrosterone treatment, as reflected by significant decreases in the numbers of sticking and transmigrating leukocytes 30 and 90 minutes after reperfusion. In dehydroepiandrosterone-treated animals, leukocytes exhibited lower levels of expression of adhesion molecules after the onset of ischemia, compared with the control groups. In this study, intravenous dehydroepiandrosterone administration reduced the activation of leukocytes and improved red blood cell velocity and capillary perfusion in the muscle flap microcirculation during ischemia-reperfusion injury. This protective effect was most likely the result of delayed expression of Mac-1 integrin, L-selectin, and CD44 molecules on leukocytes.     

The electrotonic architecture of the retinal microvasculature: Diabetes-induced alteration

Although microvascular cell death is a well established hallmark of diabetic retinopathy, which is a major cause of vision loss, much remains to be learned about the functional changes that precede the onset of morphological damage to retinal blood vessels. Early alterations of function are of interest since they may contribute to the development of irreversible pathological events. Because one of the earliest retinal effects of diabetes is the dysregulation of blood flow, we asked whether diabetes alters the functional organization of the capillary/arteriolar complex, which is the operational unit that plays an important role in regulating local perfusion. In this study, the effect of diabetes on the electrotonic architecture of the retinal microvasculature was characterized. To do this, we quantified the efficacy by which voltages are transmitted between pairs perforated-patch pipettes sealed onto abluminal cells located at well defined locations in capillary/arteriolar complexes freshly isolated from the retinas of rats made diabetic by streptozotocin. Results of these dual recording experiments were compared with data from similar experiments performed on non-diabetic retinal microvessels. These experiments revealed that diabetes caused a ∼5-fold increase in the rate at which a voltage decays as it axially spreads through the retinal microvasculature. In contrast, the efficacy of radial abluminal cell/endothelial cell transmission was not significantly affected by diabetes. Based on the results of this study, which is the first to characterize how diabetes affects voltage transmission in capillary/arteriolar complexes of any tissue, we concluded that by selectively inhibiting axial transmission, diabetes alters the electrotonic architecture of the retinal microvasculature. This diabetes-induced alteration in the functional organization of the capillary/arteriolar unit is likely to impair its ability to efficiently and effectively regulate blood flow and thereby, may contribute to the progression of sight-threatening complications of diabetic retinopathy.     

Intercommunications between brain capillary endothelial cells and glial cells increase the transcellular permeability of the blood-brain barrier during ischaemia

Increased cerebrovascular permeability is an important factor in the development of cerebral oedema after stroke, implicating the blood-brain barrier (BBB). To investigate the effect of hypoxia on the permeability changes, we used a cell culture model of the BBB consisting of a co-culture of brain capillary endothelial cells and glial cells. When endothelial cells from this co-culture model were submitted alone to hypoxic conditions, long exposures (48 h) were necessary to result in an increase in endothelial cell monolayer permeability to [3H]inulin. When endothelial cells were incubated in presence of glial cells, a huge increase in permeability occurred after 9 h of hypoxic conditions. Oxygen glucose deprivation (OGD) resulted in a much shorter time (i.e. 2 h) required for an increase in permeability. We have demonstrated that this OGD-induced permeability increase involves a transcellular rather than a paracellular pathway. Conditioned medium experiments showed that glial cells secrete soluble permeability factors during OGD. However, endothelial cells have to be made sensitive by OGD in order to respond to these glial soluble factors. This work shows that an early cross-talk between glial and endothelial cells occurs during ischaemic stroke and alters BBB transcellular transport by means of glial factor secretions.     

Temporal effects of 5.0 Gy radiation in healing subcutaneous microvasculature of a dorsal flap window chamber

The temporal effects of 5.0 Gy of radiation on healing subcutaneous microvasculature were studied using a window chamber in the dorsal flap of the Fischer-344 rat. Microvascular function was assessed by morphometric and dynamic flow measurements which were made prior to and at 24 and 72 h after exposure. A comparison was made between chamber preps that were 3 and 14 days postsurgery. The hypothesis of the study was that the older preparation would be more refractory to damage by radiation. Both unirradiated preparations showed an increase in capillary numbers over the period of observation, while irradiated preps had a reduction, especially in vessels less than 50 microns in diameter. Red cells velocities increased by 20-100% in those vessels which survived the radiation exposure, indicating that tissue oxygen tensions might be preserved in spite of a loss of vasculature. These results explain the need for both morphologic and dynamic flow measurements when assessing the effect of therapeutic intervention on microcirculatory function. Further studies are underway to identify a fully mature capillary bed in this model, since it is apparent that capillary growth is continuing in the 14-day preparation.     

Role of Src in angiopoietin 1-induced capillary morphogenesis of endothelial cells: Effect of chronic hypoxia on Src inhibition by PP2

Signal transduction pathways leading to angiopoietin 1 (Ang1)-induced capillary morphogenesis by endothelial cells remain poorly defined. Angiogenic cellular responses by endothelial cells may be modulated in vivo by chronic hypoxia, such as that induced by tumors. Here, we studied Ang1-induced capillary morphogenesis in human umbilical-vein endothelial cells (HUVECs) cultured chronically under normoxic (21% oxygen) or hypoxic (1.5% oxygen) conditions. Downregulation of Src using a small interfering RNA (siRNA) inhibited Ang1-induced capillary morphogenesis of HUVECs cultured under both conditions by blocking cell spreading and protrusion. Ang1 upregulated the Src-dependent secretion of vascular endothelial growth factor-A (VEGF-A). Blockade of endogenous VEGF-A also inhibited Ang1-induced capillary morphogenesis. Addition of exogenous VEGF-A restored cell spreading and protrusion, leading to Ang1-induced capillary morphogenesis of Src siRNA-treated HUVECs, suggesting that Ang1-induced VEGF-A secretion through Src was required for capillary morphogenesis. PP2 inhibited both Ang1-induced capillary morphogenesis and Src activation in HUVECs cultured under normoxic conditions, but the PP2 activity was significantly impaired in HUVECs cultured under hypoxic conditions. Expression of multidrug resistance-associated protein 1 (MRP 1) was upregulated in hypoxic HUVECs, and treatment with MRP 1 siRNA restored the inhibitory action of PP2. Taken together, our results suggest that Ang1 induces capillary morphogenesis in HUVECs through Src-dependent upregulation of endogenous VEGF-A. Conditions of chronic hypoxia impaired the effect of PP2, possibly via MRP 1.     

Hypoxic damage to the periventricular white matter in neonatal brain: role of vascular endothelial growth factor, nitric oxide and excitotoxicity

The present study examined factors that may be involved in the development of hypoxic periventricular white matter damage in the neonatal brain. Wistar rats (1-day old) were subjected to hypoxia and the periventricular white matter (corpus callosum) was examined for the mRNA and protein expression of hypoxia-inducible factor-1alpha (HIF-1alpha), endothelial, neuronal and inducible nitric oxide synthase (eNOS, nNOS and iNOS), vascular endothelial growth factor (VEGF) and N-methyl-D-aspartate receptor subunit 1 (NMDAR1) between 3 h and 14 days after hypoxic exposure by real-time RT-PCR, western blotting and immunohistochemistry. Up-regulated mRNA and protein expression of HIF-1alpha, VEGF, NMDAR1, eNOS, nNOS and iNOS in corpus callosum was observed in response to hypoxia. NMDAR1 and iNOS expression was found in the activated microglial cells, whereas VEGF was localized to astrocytes. An enzyme immunoassay showed that the VEGF concentration in corpus callosum was significantly higher up to 7 days after hypoxic exposure. NO levels, measured by colorimetric assay, were also significantly higher in hypoxic rats up to 14 days after hypoxic exposure as compared with the controls. A large number of axons undergoing degeneration were observed between 3 h and 7 days after the hypoxic exposure at electron-microscopic level. Our findings point towards the involvement of excitotoxicity, VEGF and NO in periventricular white matter damage in response to hypoxia.     

Light- and electron-microscopic study on the maturation of the primary portal plexus during the perinatal period in rats

The maturation of the capillaries of the primary portal plexus in rats during the perinatal period has been studied light- and electron-microscopically. The number of capillaries covering the median eminence and of those invading the nervous tissue (capillary loops) increases significantly with age. Capillary loops were observed as early as the 18th fetal day. The mitotic divisions of the endothelial cells within the preexisting capillaries seem to be the main reason for the vascular growth. Immature capillaries with a characteristic narrow lumen are surrounded by a fuzzy basal lamina; their wall is formed by a generally expanded endothelium with rather sparse organelles and inclusions, and by minute flattened areas. The maturation of the capillary results in a progressive spread of flattened endothelium followed by an enlargment of the capillary lumen. Moreover, a rising concentration of organelles and inclusions, relatively numerous luminal microvilli, and a dense and uniform basal lamina become noticeable as capillary differentiation proceeds. These data are thought to reflect the progressive increase in the metabolic activity of the endothelium as well as the establishment of capillary patency during the perinatal period of rats.     

Role of VEGF-B in the lung during development of chronic hypoxic pulmonary hypertension

Angiogenic factors exert protective effects on the lung. To investigate the effect of VEGF-B, a factor coexpressed in the lung with VEGF-A, we assessed chronic hypoxic pulmonary hypertension in VEGF-B knockout mice (VEGF-B-/-) and in rats with lung overexpression of VEGF-B induced by adenovirus transfer. No significant difference in pulmonary hemodynamics, right ventricular hypertrophy, distal vessel muscularization, or vascular density was found between VEGF-B-/- and control mice after 3 wk of hypoxia. When overexpressed, VEGF-B(167) or VEGF-B(186) had protective effects similar to those of human VEGF-A(165). Lung endothelial nitric oxide synthase (eNOS) expression was increased by 5 days of hypoxia or VEGF-A adenovirus vector (Ad.VEGF-A) overexpression, whereas VEGF-B(167) or VEGF-B(186) had no effect. With hypoxia or normoxia, the wet-to-dry lung weight ratio was increased 5 days after Ad.VEGF-A administration compared with control (Ad.nul), Ad.VEGF-B(167), or Ad.VEGF-B(186). Endogenous VEGF-B does not counteract the development of hypoxic pulmonary hypertension. However, when overexpressed in the lung, VEGF-B can be as potent as VEGF-A in attenuating pulmonary hypertension, although it has no effect on eNOS expression or vascular permeability.     

Effects of the calcium channel blocker flunarizine on the hemodynamics and oxygenation of tumor microvasculature.

Flunarizine is a diphenylpiperazine calcium entry blocker that has been shown previously to increase tumor blood flow and sensitivity to radiotherapy via reduction in the radiobiologically significant hypoxic fraction. Two mechanisms of action have been proposed previously (vasodilation, altered blood viscosity), but no studies have been performed to examine its mechanisms of action in vivo. Such information would be invaluable in determining the role of flunarizine in multimodality approaches to reduce tumor hypoxia. Fisher-344 rats bearing R3230Ac tumors transplanted into dorsal flap window chambers were used to examine microcirculatory changes after administration of flunarizine (1.0 mg/kg, iv). The drug increased the diameters of the microvasculature and red cell velocities specifically in central tumor regions (producing an average increase in vessel flow by a factor of 1.96), which was accompanied by an increase in perivascular pO2 of 12 mm Hg, on the average. The drug did not change the diameters of tumor "feeding" vessels, nor did it change vascular length densities. Thus the improvement in central tumor blood flow and oxygenation could not be attributed to dilation of feeding vessels. The oxygen-carrying capacity of the blood was not altered either since hemoglobin saturation (measured in vitro) and the hematocrits of the microvasculature were unchanged after drug administration. Therefore, by a process of elimination, the most likely explanation for the effect of the drug is modification of blood viscosity. Additional studies are under way in this laboratory to examine whether changes in viscosity occur after flunarizine administration.     

大鼠胃有腺区粘膜层微血管构筑的发育及衰老变化

应用扫描电镜、透射电镜及血管铸型等技术方法,对初生、离乳、成年和老年4个年龄段大鼠胃粘膜血管构筑及毛细血管内皮细胞超微结构的发育及衰老变化进行了研究。结果表明,初生大鼠胃粘膜形成大量雏襞,微血管正处于分化、发育的过程中,尚未形态典型的构型;从离乳到老年,胃粘膜毛细血管形成明显的分区,胃粘膜层微血管构型为“树型”;毛细血管内皮细胞的超微结构随发育、衰老呈明显的变化趋势,即毛细血管内皮细胞质膜突起的数量有增多的趋势,基底膜有增厚的趋势,而质膜小泡的数量没有明显的改变。     

Barrier dysfunction and RhoA activation are blunted by homocysteine and adenosine in pulmonary endothelium

RhoA GTPases modulate endothelial permeability. We have previously shown that adenosine and homocysteine enhance basal barrier function in pulmonary artery endothelial cells by a mechanism involving diminution of RhoA carboxyl methylation and activity. In the current study, we investigated the effects of adenosine and homocysteine on endothelial monolayer permeability in cultured monolayers. Adenosine and homocysteine significantly attenuated thrombin-induced endothelial barrier dysfunction and intercellular gap formation. We found significantly diminished RhoA associated with the membrane subcellular fraction in endothelial cells pretreated with adenosine and homocysteine, compared with vehicle-treated endothelial cells. Additionally, adenosine and homocysteine significantly blunted RhoA activation following thrombin exposure. Incubation with adenosine and homocysteine also enhanced in vitro interactions between RhoA and RhoGDI, as well as subcellular translocation of p190RhoGAP to the cytosol. These data demonstrate that elevated intracellular concentrations of homocysteine and adenosine enhance endothelial barrier function in cultured endothelial cells isolated from the main pulmonary artery and lung microvasculature, suggesting a potentially protective effect against pulmonary edema in response to lung injury. We speculate that homocysteine and adenosine modulate the level of endothelial barrier dysfunction through modulation of RhoA posttranslational processing resulting in diminished GTPase activity through altered interactions with modulators of RhoA activation.     

Morphological Differentiation of the Microvasculature During Follicular Development, Ovulation and Luteinization of Mouse Ovaries

The chronological changes of the microvasculature during follicular development, ovulation and luteinization of mouse ovaries were examined by observation of serial histological sections, lectin angiographs and resin-corrosion casts. Graafian follicles possessing oocytes with germinal vesicles were surrounded by a few layers of basket-like capillary wreath adjacent to the follicular basement membrane. Just before ovulation 11–12 hr after hCG administration, some theca cells differentiated into hypertrophic cells, and the follicular basement membrane underwent fragmentation. Then the capillaries within the theca interna became dilated, and hyperpermeable and appeared to be injured. The capillary wreath extended into the follicle via the hypertrophied theca interna. After ovulation, the follicular wall became markedly edematous. Capillary branches invaded the granulosa cell layer of the ruptured follicle from the region of extravasation to form an intricate capillary network. The capillary network occupied the whole corpus luteum until 24 hr after hCG administration.     

Perinatal history of hypoxia leads to lower vascular pressures and hyporeactivity to angiotensin II in isolated lungs of adult rats

The most dramatic changes in pulmonary circulation occur at the time of birth. We hypothesized that some of the effects of perinatal hypoxia on pulmonary vessels are permanent. We studied the consequences of perinatal exposure to hypoxia (12 % O2 one week before and one week after birth) in isolated lungs of adult male rats (approximately 12 weeks old) perfused with homologous blood. Perfusion pressure-flow relationship was tilted towards lower pressures in the perinatally hypoxic as compared to the control, perinatally normoxic rats. A non-linear, distensible vessel model analysis revealed that this was due to increased vascular distensibility in perinatally hypoxic rats (4.1 +/- 0.6 %/mm Hg vs. 2.3 +/- 0.4 %/mm Hg in controls, P = 0.03). Vascular occlusion techniques showed that lungs of the perinatally hypoxic rats had lower pressures at both the pre-capillary and post-capillary level. To assess its role, basal vascular tone was eliminated by a high dose of sodium nitroprusside (20 microM). This reduced perfusion pressures only in the lungs of rats born in hypoxia, indicating that perinatal hypoxia leads to a permanent increase in the basal tone of the pulmonary vessels. Pulmonary vasoconstrictor reactivity to angiotensin II (0.1-0.5 microg) was reduced in rats with the history of perinatal-hypoxia. These data show that perinatal hypoxia has permanent effects on the pulmonary circulation that may be beneficial and perhaps serve to offset the previously described adverse consequences.     

Reactive carbonyls from tobacco smoke increase arterial endothelial layer injury

We hypothesized that reactive carbonyls generated from smoke exposure cause increased arterial low-density lipoprotein (LDL) accumulation and endothelial layer permeability. In addition, we hypothesized that estrogen supplementation was protective against chronic environmental tobacco smoke (ETS) exposure to the artery wall. Quantitative fluorescence microscopy was used to determine artery injury after exposure. For our chronic studies, ovariectomized rats treated with subcutaneous placebo or 17beta-estradiol pellets were exposed to ETS or filtered air for 6 wk. ETS exposure increased carotid artery LDL accumulation more than fourfold compared with filtered air exposure, an effect largely mediated by increased permeability. No protective effect of estradiol was observed. Acute ETS exposure of a buffer solution containing LDL resulted in a more than sixfold increase in the highly reactive carbonyl glyoxal. Perfusion of this solution through carotid arteries resulted in a 105% increase in permeability. Moreover, perfusion of glyoxal alone caused a 50% increase in carotid artery permeability. This endothelial damage and changes in lipid accumulation may serve as an initiating event in atheroma formation in individuals exposed to ETS.