Oxidized LDL–Induced Microvascular Dysfunction
Dependence on Oxidation Procedure
Abstract Human LDLs oxidized with Cu2+ are known to promote leukocyte–endothelial cell adhesion (LECA) and albumin leakage in postcapillary venules. The objective of this study was to compare the ability of LDL oxidized with Cu2+ (Cu-LDL), phospholipase A2 plus lipoxygenase (PLA2-LDL), horseradish peroxidase plus H2O2 (HRP-LDL), or −OCl (−OCl-LDL) to promote (1)neutrophil–endothelial cell adhesion (NECA) in vitro and (2)LECA and albumin leakage in rat mesenteric venules. In vitro adhesion assays revealed that only Cu-LDL elicited a dose-dependent NECA response, whereas PLA2-LDL but not normal (N-LDL), HRP-LDL, or −OCl-LDL increased NECA at the highest concentration studied (670 μg/mL). The magnitude of the NECA responses elicited by the different forms of oxidized LDL was related to the degree of lipid peroxidation but unrelated to the level of protein oxidation. Local intra-arterial infusion of Cu-LDL, PLA2-LDL, or −OCl-LDL but not N-LDL elicited significant increases in leukocyte adherence and emigration, mast cell degranulation, and albumin leakage in rat mesenteric venules. The LECA induced by all forms of oxidized LDL was not accompanied by significant alterations of venular shear rate.
- lipoproteins, low-density
- adhesion, leukocyte–endothelial cell
- permeability, vascular
- degranulation, mast cell
- Received July 7, 1995.
- Accepted October 2, 1995.
Oxidatively modified lipoproteins, primarily LDLs, have been implicated in the pathogenesis of atherosclerosis.1 2 The vascular injury associated with atherosclerosis has been proposed to be a consequence of leukocyte–endothelial cell interactions elicited by ox-LDL.3 Lehr and colleagues3 showed that systemic administration of ox-LDL (in the form of Cu-LDL) promotes the adhesion of leukocytes in arterioles and postcapillary venules of the hamster dorsal skin fold. We made similar observations in rat mesenteric venules and found that the LECA elicited by Cu-LDL is accompanied by degranulation of mast cells and increased albumin extravasation.4 While these intravital microscopic analyses and a number of in vitro studies5 6 clearly demonstrate the potential for ox-LDL to induce LECA, the precise mechanism underlying this response as well as its dependence on the nature and extent of oxidative modification of lipoprotein components remains unclear.
While several biochemical procedures have been used to mimic the lipoprotein oxidation that is associated with atherosclerosis, the most extensively used form of ox-LDL is Cu-LDL. However, it has been demonstrated that −OCl, an oxidant generated by myeloperoxidase released from activated neutrophils, can react with LDL to promote LDL uptake by macrophages, even in the absence of lipid oxidation in the lipoprotein.7 It also has been demonstrated that enzymatic modification of LDL by the combination of phospholipase A2 and purified lipoxygenase mimics cell-mediated oxidative modification of the lipoprotein.8 Hence, there is growing evidence that events associated with acute and chronic inflammation (activation of lipoxygenase and phospholipase A2 as well as HOCl production) can result in LDL oxidation. Nonetheless, it is uncertain whether LDLs exposed to these physiologically relevant modes of oxidation also are able to elicit the leukocyte−endothelial cell interactions and albumin extravasation observed in microvessels exposed to Cu-LDL. Thus, the overall objective of this study was to assess and compare the ability of different forms of ox-LDL, ie, Cu-LDL, PLA2-LDL, HRP-LDL, or −OCl-LDL, to promote (1) NECA in vitro, and (2) LECA and albumin leakage in rat mesenteric venules.
LDL Isolation and Oxidation
Fresh blood was obtained from nonfasted, healthy, normolipidemic male and female human subjects and placed into tubes containing 1.5 mg EDTA (Na2) per milliliter blood. LDL fractions (d=1.019 to 1.063 g/mL) were isolated by density gradient ultracentrifugation9 from pooled plasma. The LDL sample was dialyzed extensively against PBS at 4°C, without EDTA, at a final concentration of 1 mg LDL protein per milliliter. Protein was determined by the method of Lowry et al10 with bovine serum albumin as a standard. Cu-LDL was prepared by adding CuSO4 to a final concentration of 10 μmol/L and incubating at 37°C for 24 hours. The reaction was stopped by adding EDTA (final concentration, 0.01 mmol/L). For PLA2-LDL, LDL was incubated in 50 mmol/L borate with soybean lipoxygenase (20 μg/mL, Sigma Chemical Co) and snake venom phospholipase (0.1 μg/mL, Sigma) at 37°C for 24 hours. HRP-LDL was prepared by coincubating LDL with horseradish peroxidase (type XII, 0.5 U/mL, Sigma) and H2O2 (final concentration, 200 μmol/L) at 37°C for 24 hours. Oxidation of −OCl-LDL was performed by the addition of NaOCl (available chlorine, 5% minimum; Aldrich) to a final concentration of 0.6 mmol/L and coincubated with LDL for 24 hours at 37°C. All forms of ox-LDL were dialyzed against Hanks’ balanced salt solution in vitro or PBS in vivo containing EDTA 0.01 mmol/L. Normal LDL was prepared by dialyzing the sample against Hanks’ balanced salt solution or PBS with EDTA. All lipoprotein fractions were stored at 4°C and used within 3 weeks.
Agarose Gel Electrophoresis
LDL (1 μL) was loaded on prepacked agarose gels, and the gels were run at 90 V for 35 minutes. Thereafter, the gels were fixed in methanol and stained with fat red 7B.11 The relative electrophoretic mobility was estimated by use of N-LDL as a standard with a value of 1.0.
Assay for TBARS
TBARS were measured by mixing 0.5 mL LDL with 1.0 mL of a mixed solution consisting of 15% trichloroacetic acid, 0.375% thiobarbituric acid, and 0.25N hydrochloric acid. This mixture was boiled for 15 minutes, centrifuged, and the absorbance of the supernatant measured at 532 nm.12
HUVECs were harvested from umbilical cords by collagenase treatment as previously described.13 The cells were placed in medium M199 (GIBCO) supplemented with 10% heat-inactivated fetal calf serum (Hyclone Laboratories Inc), glutamine (230 mg/L, JRH Biosciences), thymidine (2.4 mg/L, Sigma), heparin sodium (10 IU/mL, Sigma), endothelial cell growth factor (80 μg/mL, Biomedical Technologies lnc), and antibiotics (100 IU/mL penicillin, 100 μg/mL streptomycin, and 0.125 amphotericin B). The cell cultures were incubated at 37°C in a humidified atmosphere with 5% CO2 and expanded by brief trypsinization (0.25% trypsin in PBS containing 0.02% EDTA). Primary through third passage HUVECs were seeded into gelatin (0.1%) and fibronectin-coated (25 μg/mL) 11-mm, 48-well tissue culture plates (GIBCO) and used when confluent. The cells were identified as endothelial cells by their cobblestone appearance and positive labeling with (1) acetylated LDL labeled with 1,11-dioctadecyl-1-3,3,31,313-tetramethylindocarbocyanine perchlorate (Dil-Ac-LDL; Biomedical Technologies, Inc) and (2) mouse anti-human factor VIII (Calbiochem).
Venous blood from healthy adults who did not receive any medication for at least 2 weeks was collected in 1/10 vol 3.8% trisodium citrate. Neutrophils were isolated using standard dextran sedimentation and gradient separation on Histopaque 1077 (Sigma).13 14 This procedure yields a polymorphonuclear leukocyte population that is 95% to 98% viable (by trypan blue exclusion) and 98% pure (acetic acid–crystal violet staining).
In Vitro Adhesion Assays
Isolated neutrophils were suspended in PBS and radiolabeled by incubating the cells at 2×107 cells/mL with 30 μCi Na51CrO4/mL neutrophil suspension at 37°C for 60 minutes. The cells were then washed twice with cold PBS at 250g for 8 minutes to remove unincorporated radioactivity and were resuspended in plasma-free Hanks’ balanced salt solution. Labeled neutrophils were added to HUVEC monolayers at a neutrophil-to–endothelial cell ratio of 10:1 together with either N-LDL or ox-LDL (final concentrations of 1, 10, 100, and 670 μg LDL per milliliter). After coincubation (20 minutes) at 37°C, the supernatant was removed, the monolayers were gently washed (3 times) with Hanks’ balanced salt solution to remove nonadherent cells, and the remaining bound cells were lysed with NaOH (2N). The supernatant, wash fluid, and lysate were assayed for 51Cr activity using a gamma counter.13 The percent of added neutrophils that adhered to HUVEC monolayers was quantified as (lysate [cpm] multiplied by 100) divided by (supernatant [cpm] plus wash [cpm] plus lysate [cpm]).
Thirty male Sprague-Dawley rats (200 to 250 g) were maintained on a purified laboratory diet and fasted for 24 hours before each experiment. The animals were initially anesthetized with an injection of 140 mg IP thiobutabarbital (Inactin) per kilogram body wt. A tracheotomy was performed on each rat to facilitate breathing throughout the experiment. The right carotid artery was cannulated, and systemic arterial pressure was measured with a Statham P23A pressure transducer connected to the carotid artery cannula. Systemic blood pressure and heart rate were continuously recorded with a Grass physiological recorder (Grass Instruments). A midline abdominal incision was made to allow a section of mesentery from the small intestine to be exteriorized. The aorta was cannulated with the tip of the cannula placed at the superior mesenteric artery bifurcation. All exposed tissue was moistened with saline-soaked gauze to minimize evaporation and tissue damage.
The intravital microscopy rat was placed in a supine position on an adjustable acrylic plastic (Plexiglas) microscope stage, and the mesentery was prepared for microscopic observation as described previously.15 Briefly, the mesentery was draped over a nonfluorescent coverslip that allowed for observation of a 2-cm2 segment of tissue. The exposed bowel wall was covered with clear plastic wrap (Saran Wrap, Dow Chemical Co), and then the mesentery was superfused (bathed at a constant rate) with bicarbonate-buffered saline (37°C, pH 7.4) that was bubbled with a mixture of 5%CO2/95% N2, which exposes mesenteric tissue to an oxygen tension of ≈40 mm Hg.
An inverted microscope (Diaphot 300, Nikon) with a ×40 magnification objective lens (Fluor, Nikon) was used to observe the mesenteric microcirculation. The mesentery was transilluminated with a 12-V, 100-W direct current–stabilized light source. A video camera (VK-C150, Hitachi) mounted on the microscope projected the image onto a color monitor (PMV-2030, Sony), and the image was recorded using a videocassette recorder (BR-S601MU, JVC). A video time-date generator (WJ810, Panasonic) projected the time, date, and stopwatch functions onto the monitor.
Single, unbranched venules with diameters ranging between 25 and 35 μm and length greater than 150 μm were selected for study. Venular diameter was measured either on-line or off-line using a video caliper (Microcirculation Research Institute, Texas A&M University). RBC centerline velocity was measured in venules with an optical Doppler velocimeter (Microcirculation Research Institute). The velocimeter was calibrated against a rotating glass disk coated with RBCs. Venular blood flow was calculated from the product of mean RBC velocity (Vmean, where Vmean=Centerline Velocity/1.6)16 and microvascular cross-sectional area, assuming cylindrical geometry. Wall shear rate (γ) was calculated on the basis of the newtonian definition γ=8(Vmean/D), where D is diameter.
The number of adherent leukocytes was determined off-line during playback of videotaped images. A leukocyte was considered to be adherent to venular endothelium if it remained stationary for a period ≥30 seconds.17 Adherent leukocytes were expressed as the number per 100-μm length of venule. The number of emigrated leukocytes was also determined off-line during playback of videotaped images. Any interstitial leukocytes present in the mesentery at the onset of experiment were subtracted from the total number of leukocytes that accumulated during the course of the experiment. Leukocyte emigration was expressed as the number per field of view surrounding the venule. To visualize mast cells surrounding the mesenteric microvasculature, 0.1 g% toluidine blue was added to the mesentery at the end of each experiment.18 The number of intact and degranulated mast cells was determined, and the percentage of degranulated mast cells was calculated.
To quantify albumin leakage across mesenteric venules, 50 mg/kg FITC-albumin (Sigma) was administered intravenously to the rats 15 minutes before each experiment.19 Fluorescence intensity (excitation wavelength, 420 to 490 nm; emission wavelength, 520 nm) was detected with a silicon-intensified target camera (C2400-08, Hamamatsu Photonics). The fluorescence intensity of FITC-albumin within three segments of the venule under study and in three contiguous areas of perivenular interstitium was measured, at various times after administration of FITC-albumin, with a computer-assisted digital imaging processor (NIH Image 1.35 on a Macintosh computer, Quadra 840AV). An index of vascular albumin leakage was determined from the ratio of interstitial to venular intensity of FITC-albumin fluorescence at specific intervals (10 minutes) after infusion of ox-LDL. All data presented for albumin leakage represent the ratio of interstitial to venular intensity of FITC-albumin fluorescence at 60 minutes after injection of ox-LDL.18 19
After all parameters measured on-line were in a steady state, images from the mesenteric preparation were recorded on videotape for 10 minutes. Immediately thereafter, either N-LDL or one of several forms of ox-LDL was infused into the superior mesenteric artery at a rate of 1 mg LDL protein per kilogram per minute for 5 minutes with the mesentery superfused with bicarbonate-buffered saline (2 mL/min), and repeated measurements were obtained at regular intervals (15 minutes) for 60 minutes.
The data obtained in this study are expressed as mean±SEM. The data were analyzed by use of standard statistical analyses, ie, one-way ANOVA with Scheffé’s post hoc test and Student’s t test. Statistical significance was set at P<.05.
Incubation of LDLs (1 mg/mL) at 37°C for 24 hours in the presence of Cu2+ (10 μmol/L), lipoxygenase (20 μg/mL) and phospholipase A2 (0.1 μg/mL), or horseradish peroxidase (0.5 U/mL) and H2O2 (200 μmol) significantly increased the amount of lipid hydroperoxide products in LDL (14.9±1.6, 8.5±0.1, and 3.7±0.1 nmol malondialdehyde equivalents per mg of LDL protein, respectively) relative to N-LDL (0.4±0.1 nmol malondialdehyde equivalents per milligram LDLprotein) (Fig 1⇓). However, incubation of LDL with NaOCl (0.6 mmol/L) did not lead to significant lipid oxidation (0.4±0.1 nmol malondialdehyde equivalents per mg of LDL).
The results in Fig 2⇓ summarize the changes in REM of different forms of ox-LDL (relative to N-LDL) on 1% agarose gel. −OCl-LDL exhibited the highest REM (2.6) followed by PLA2-LDL (2.4) and Cu-LDL (1.8). HRP-LDL exhibited the smallest change in REM, ie, 1.2.
Fig 3⇓ illustrates the effects of different forms of ox-LDL on the adhesion of isolated human neutrophils to HUVEC monolayers. None of the ox-LDL increased polymorphonuclear cell adhesion at LDL concentrations of 1.0 or 10.0 μg LDL per milliliter. Cu-LDL significantly increased NECA at a concentration of 100 μg LDL per milliliter (Cu-LDL, 9.8±1.9% versus control, 3.9±1.1%;P<.05). Both Cu-LDL and PLA2-LDL (Cu-LDL, 22.7±4.1%; PLA2-LDL, 22.5±1.9% versus control, 5.2±0.9%; P<.05) but not N-LDL, HRP-LDL, or −OCl-LDL significantly increased NECA at highest concentration (670 μg/mL) studied.
Fig 4⇓ illustrates the dependence of the ox-LDL–induced NECA responses on the extent of lipid hydroperoxide product formation (Fig 4A⇓) and apolipoprotein oxidation (Fig 4B⇓). All values were derived from data already presented in Figs 1 through 3⇑⇑⇑. The NECA induced by the different forms of ox-LDL was positively correlated (r=.93, P<.05) with the extent of LDL lipid peroxidation but not with the level of protein oxidation (r=.3, P>.75). The tight coupling between the magnitude of ox-LDL–induced NECA and the formation of lipid hydroperoxide products suggests that the proadhesive effects of ox-LDL are related to the ability of lipid peroxidation products to activate neutrophils, endothelial cells, or both.
Fig 5⇓ illustrates changes in the number of adherent (Fig 5A⇓) and emigrated (Fig 5B⇓) leukocytes and albumin leakage (Fig 5C⇓) in rat mesenteric venules that are elicited by intra-arterial infusion of either N-LDL or different forms of ox-LDL. The number of adherent and emigrated leukocytes was significantly elevated at 30 minutes after ox-LDL infusion and increased progressively thereafter. In the control group (the group in which the infusion of PBS was administered), the number of adherent leukocytes was 2.7±1.5 per 100 μm, with 0.3±0.2 emigrated leukocytes per field and an albumin leakage index of 2.2±1.1%. Corresponding values for number of adherent leukocytes obtained 30 minutes after infusion of Cu-LDL, PLA2-LDL, or −OCl-LDL were 16.2±3.3, 7.2±1.1, or 10.0±1.8 per 100 μm, respectively, whereas emigrated leukocytes were 3.3±0.6, 3.0±0.4, or 3.0±0.5 per field. The albumin leakage responses observed 30 minutes after infusion of Cu-LDL, PLA2-LDL, or −OCl-LDL were 19.4±1.8%, 16.0±7.3%, or 21.9±2.7%, respectively. No significant changes in leukocyte adherence, emigration, and albumin leakage response were noted in preparations exposed to N-LDL (3.7±1.1 per 100 μm, 0.2±0.2 per field, 3.1±2.8%, respectively, 30 minutes after infusion).
Fig 6⇓ illustrates the percentage of degranulated mast cells adjacent to mesenteric venules at 60 minutes after an intra-arterial infusion of either N-LDL or different forms of ox-LDL. In control preparations, <1% (0.7±0.4%) of the total mast cell population situated along postcapillary venules was degranulated. Infusion of N-LDL did not increase the number of degranulated mast cells (1.0±0.7%), whereas infusion of Cu-LDL, PLA2-LDL, or −OCl-LDL significantly increased the number of degranulated mast cells to 20.3±3.4%, 9.7±1.4%, or 20.0±4.4%, respectively.
The dose-dependent effects of Cu-LDL on leukocyte adhesion and emigration, albumin leakage, and mast cell degranulation are summarized in Table 1⇓. Infusion of 0.25 mg LDL per kilogram per minute for 5 minutes elicited significant increases in leukocyte adherence and emigration, albumin leakage, and mast cell degranulation. The magnitude of these responses to Cu-LDL increased progressively when higher doses (0.5 and 1.0 mg LDL per kilogram per minute) were administered.
The effects of different forms of ox-LDL on leukocyte adhesion and emigration and albumin leakage 15 minutes after infusion are summarized in Table 2⇓. Both Cu-LDL and −OCl-LDL elicited significant increase in leukocyte adhesion, whereas PLA2-LDL had no effect. The leukocyte emigration response induced by different forms of ox-LDL at 15 minutes was minimal. Of the different forms of ox-LDL, only Cu-LDL caused a significant increase in albumin leakage across the postcapillary venules at 15 minutes.
Venular diameter, red blood cell velocity, and wall shear rate were not significantly altered by intra-arterial infusion of any form of LDL (Table 3⇓), indicating that the accompanying leukocyte–endothelial cell interactions were not a result of microhemodynamic changes. Systemic blood pressure also was unaffected by infusion of either N-LDL or ox-LDL.
One of the unifying hypotheses that have been proposed to explain the pathogenesis of atherosclerosis invokes a critical role for endothelial cell integrity and function in the initiation of the disease process.1 There is a large body of evidence that implicates LECA as a key determinant of the early vascular alterations associated with atherogenesis. As a consequence, there has been an intensive effort to define the factors that elicit the LECA response. Ox-LDLs, which are elevated in the plasma of atherosclerotic subjects, have been a major focal point of these investigative efforts. Data derived from both in vitro and in vivo models of LECA clearly demonstrate that ox-LDL promotes leukocyte adhesion by means of mechanisms that involve the participation of endothelial cell–derived oxygen radicals, inflammatory mediators (platelet activating factor and leukotriene B4 ), and nitric oxide.4 20 21 22 In vivo studies have revealed that Cu-LDL elicits the adherence and emigration of leukocytes in postcapillary venules,3 4 changes that are accompanied by degranulation of neighboring mast cells and an accelerated rate of albumin extravasation.4 18 The results of the present study confirm these observations and demonstrate that exposure of mesenteric venules to Cu-LDL results in a dose-dependent increase in leukocyte adhesion and emigration, mast cell degranulation, and albumin extravasation in rat mesentery. Similarly, we have shown that Cu-LDL promotes the adhesion of isolated human leukocytes to monolayers of HUVECs, a finding consistent with previously published reports.5 23 24
A major objective of the present study was to determine whether the nature and/or extent of oxidative modification of lipoprotein components influence the level of LECA and consequent microvascular dysfunction elicited by ox-LDLs. The results of our in vitro studies reveal that Cu-LDL and PLA2-LDL were the most effective in eliciting the adhesion of isolated human neutrophils to HUVEC monolayers, whereas −OCl-LDL was least effective. Correlation analyses of data obtained at 20 minutes after coincubation of neutrophils and HUVEC monolayers with different forms of ox-LDL suggest that the magnitude of the neutrophil adhesion response is more closely linked to the degree of lipid rather than protein oxidation. The observed dependence of neutrophil adhesion to HUVECs on the level of lipid peroxidation of LDL is consistent with in vitro studies demonstrating that oxygen radical–catalyzed peroxidation of phospholipids can result in the formation of potent platelet-activating factor–like compounds that promote leukocyte activation and adhesion.25 26 Furthermore, the data derived from our in vitro experiments are consistent with the notion that the circulating agent in atherosclerosis that causes both the expression of monocyte-binding proteins on endothelial cells and the endothelial production of a monocyte chemotactic protein is a lipid oxidation product of LDL particles.6 27
While our studies on ox-LDL–induced NECA indicate profound differences in the adhesion responses to different forms of ox-LDL, such differences were not as readily apparent when similar experiments were performed with an in vivo model. Our evaluation of rat mesenteric venules exposed to different forms of ox-LDL revealed that Cu-LDL, PLA2-LDL, and −OCl-LDLs are all equally effective in promoting leukocyte adherence and emigration, albumin leakage, and mast cell degranulation. This observation suggests that the inflammatory responses elicited by ox-LDL in postcapillary venules do not exhibit a dependence on the extent of lipid (versus protein) oxidation, which was predicted from the in vitro experiments. Furthermore, the data derived from the studies of postcapillary venules indicate that while the use of Cu-LDL may be criticized on the basis of physiological relevance, the microvascular responses to this form of ox-LDL do not differ appreciably from those elicited by forms of ox-LDL that are presumed to be more physiological, ie, PLA2-LDL and −OCl-LDL.
Although a definitive explanation for the differences observed between the in vitro and in vivo models of ox-LDL-induced inflammation is not readily available, some possibilities warrant consideration. One explanation relates to the fact that the in vitro experiments used cells (neutrophils and endothelial cells) and LDLs derived from humans, whereas the in vivo experiments used human LDLs in the microcirculation of the rat. Conceivably, differences in leukocyte adhesion that are related to the nature and extent of lipoprotein oxidation may be demonstrated readily in a homologous experimental system. The indiscriminate responses of postcapillary venules to different forms of ox-LDL may also reflect the participation of another cell type (eg, platelets) that modulates the inflammatory reactions to ox-LDL under in vivo conditions. Platelets, which are known to form aggregates when exposed to Cu-LDL, may qualify as such a modulator. Mast cells, which degranulate in response to the different forms of ox-LDL, may also exert a modulating influence on the LECA and albumin leakage responses observed in vivo. Since neither platelets nor mast cells were present in the in vitro assays of neutrophil adhesion, the contribution of these potential modulators of the inflammatory response to different forms of ox-LDL cannot be assessed. Irrespective of the mechanisms that underlie the differences between the in vitro and in vivo inflammatory responses to various forms of ox-LDL, our findings raise the possibility that there are multiple physiological forms of ox-LDL that can mimic the macrovascular and microvascular dysfunction associated with atherosclerosis.
In the present study, we chose to use neutrophils rather than monocytes for our in vitro experiments because the in vivo LECA response observed in postcapillary venules exposed to ox-LDL represents the recruitment of granulocytes.21 While the relevance of ox-LDL–induced granulocyte recruitment to the pathobiology of atherosclerosis remains unclear, it is possible that ox-LDL in plasma may predispose the microvasculature of hypercholesterolemic individuals to neutrophil-mediated injury in conditions such as ischemia/reperfusion. Reperfusion of ischemic microvessels is associated with the recruitment of rolling and adherent leukocytes, platelet-leukocyte aggregation, mast cell degranulation, and enhanced albumin leakage.13 Our observation that different forms of ox-LDL elicit an acute inflammatory response similar to that seen in postischemic venules would support the view that the existence of ox-LDL in plasma should exacerbate ischemia/reperfusion–mediated inflammatory responses. The results of our in vitro experiments demonstrating ox-LDL–mediated adhesion of human neutrophils to HUVEC monolayers are also consistent with the enhanced neutrophil adhesion to HUVEC monolayers exposed to anoxia/reoxygenation.13
Selected Abbreviations and Acronyms
|FITC-albumin||=||fluorescein isothiocyanate–conjugated albumin|
|HRP-LDL||=||horseradish peroxidase–and-H2O2–oxidized LDL|
|HUVEC||=||human umbilical vein endothelial cell|
|LECA||=||leukocyte adhesion to (venular) endothelial cells (in vivo)|
|NECA||=||(isolated) neutrophil adhesion to (cultured) endothelial cells|
|PLA2-LDL||=||phospholipase A2–and-lipoxygenase–oxidized LDL|
|RBC||=||red blood cell|
|REM||=||relative electrophoretic mobility|
|TBARS||=||thiobarbituric acid–reacting substance(s)|
This work was supported by a grant (HL-26441) from the National Heart, Lung, and Blood Institute.
Liao L, Granger DN. Modulation of oxidized low-density lipoprotein-induced microvascular dysfunction by nitric oxide. Am J Physiol. 1995;268(Heart Circ Physiol.):H1643-H1650.
Berliner JA, Territo MC, Sevanian A, Ramin S, Kim JA, Bamshad B, Esterson M, Fogelman AM. Minimally modified low density-lipoprotein stimulates monocyte endothelial interactions. J Clin Invest.. 1990;85:1260-1266.
Hazzel L, Stocker R. Oxidation of low-density lipoprotein with hypochlorite causes transformation of the lipoprotein into a high-uptake form for macrophages. Biochem J.. 1993;290:165-172.
Sparrow CP, Parthasarathy S, Steinberg D. Enzymatic modification of low density lipoprotein by purified lipoxygenase plus phospholipase A2 mimics cell-mediated oxidative modification. J Lipid Res.. 1988;29:745-753.
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem.. 1951;193:265-275.
Buege JA, Aust SD. Microsomal lipid peroxidation. Methods Enzymol. 1978;30:302-310.
Yoshida N, Granger DN, Anderson DC, Rothlein R, Lane C, Kvietys PR. Anoxia/reoxygenation-induced neutrophil adherence to cultured endothelial cells. Am J Physiol. 1992;292:H1891-H1898.
Inauen W, Granger DN, Meininger CJ, Schelling ME, Granger HJ, Kvietys PR. An in vitro model of ischemia/reperfusion-induced microvascular injury. Am J Physiol. 1990;259:G134-G139.
Asako H, Kurose I, Wolf R, DeFree S, Zheng Z, Phillips ML, Paulson JP, Granger DN. Role of H1 receptors and P-selectin in histamine-induced leukocyte rolling and adhesion in postcapillary venules. J Clin Invest.. 1994;93:1508-1515.
Granger DN, Kubes P. The microcirculation and inflammation: modulation of leukocyte-endothelial cell adhesion. J Leukoc Biol. 1994;55:662-675.
Kurose I, Wolf R, Grisham MB, Granger DN. Modulation of ischemia/reperfusion-induced microvascular dysfunction by nitric oxide. Circ Res.. 1994;74:376-382.
Kurose I, Kubes P, Wolf R, Anderson DC, Paulson J, Miyasaka M, Granger DN. Inhibition of nitric oxide production: mechanisms of vascular albumin leakage. Circ Res. 1993;73:164-171.
Lehr HA, Becker M, Markland SL, Hubner C, Arfors KE, Kohlschutter A, Messmer K. Superoxide-dependent stimulation of leukocyte adhesion by oxidatively modified LDL in vivo. Arterioscler Thromb. 1992;12:824-829.
Lehr HA, Hubner C, Finckh B, Angermuller S, Nolte D, Beisiegel U, Kohlschutter A, Messmer K. Role of leukotrienes in leukocyte adhesion following systemic administration of oxidatively modified human low density lipoprotein in hamsters. J Clin Invest. 1991;88:9-14.
Lehr HA, Seemuller J, Hubner C, Menger MD, Messmer K. Oxidized LDL-induced leukocyte/endothelium interaction in vivo involves the receptor for platelet-activating factor. Arterioscler Thromb. 1993;13:1013-1018.
Noguchi N, Gotoh N, Niki E. Dynamics of the oxidation of low density lipoprotein induced by free radicals. Arch Biochem Biophys. 1993;1168:348-357.
Smiley PL, Stremler KE, Prescott SM, Zimmerman GA, McIntyre TM. Oxidatively fragmented phosphatidylcholines activate human neutrophils through the receptor for platelet-activating factor. J Biol Chem. l991;266:11104-11110.
Cushing SD, Berliner JA, Valente AJ, Territo MC, Navab M, Parhami F, Gerrity R, Schwartz CJ, Fogelman AM. Minimally modified low density lipoprotein induces monocyte chemotactic protein l in human endothelial cells and smooth muscle cells. Proc Natl Acad Sci U S A. 1990;87:5134-5138.