The Experiment Study Of ApoA-I And D-4F Have An Effect On Lung Inflammation And Airway Hyperresponsiveness

Part One Genetic Deletion of Apolipoprotein A-?Increases Airway Hyperresponsiveness, Inflammation and Collagen Deposition in the Lung1. BackgroundAlthough apolipoprotein A-?(apoA-?), the major anti-atherogenic apolipoprotein of high-density lipoprotein (HDL) is well recognized for protecting the heart against vascular disease, it also protects other vascular beds and organs. Recent studies provide new evidence supporting the notion that HDL plays a protective role in the lung. Earlier ABCA1, which interacts with lipid poor apoA-?, was shown to be essential for maintaining normal lipid composition and architecture of the lung as well as respiratory physiology. More recently, proteomic studies revealed that homozygous sickle cell anemia patients with pulmonary arterial hypertension (PAH) consistently had lower apoA-?levels than sickle patients without PAH. Interestingly, genetic deletion of endothelial lipase resulted in a nearly 2-fold increase in HDL, which was credited with decreasing airway hyperresponsiveness and pulmonary inflammation in ovalbumin (OVA)-sensitized BALB/c mice. Although these reports provide some support for the idea that HDL helps maintain healthy lungs, no studies have directly determined the effects of apoA-?, or lack thereof, on pulmonary inflammation, vasodilatation and airway hyperresponsiveness.Increasing evidence suggests that elevated levels of HDL are not always atheroprotective. Indeed, chronic states of inflammation and oxidative stress have been shown to convert HDL from an anti-inflammatory and anti-atherogenic lipoprotein into a pro-inflammatory and pro-atherogenic lipoprotein, making it useless for protecting the vessel wall against the effects of atherogenic concentrations of LDL. Asthma also increases inflammation and oxidative stress. Such inflammatory changes may explain why adult-onset asthma is associated with significant increases in carotid artery intimal-medial thickness in women. In this report we examine the effects of genetic deletion of apoA-?on pulmonary inflammation, vasodilatation, collagen deposition and airway hyperresponsiveness. Our findings suggest apoA-?plays a critical role in protecting pulmonary artery and airway function as well as preventing inflammation and collagen deposition..2. Methods2.1 Plasma Total Cholesterol, HDL Cholesterol and Proinflammatory HDLTotal cholesterol (TC) was quantified using a cholesterol oxidase/esterase kit from Wako Chemical, Inc. (Richmond, VA). HDL cholesterol was quantified using a HDL Cholesterol E kit from Wako Diagnostics. Proinflammatory HDL (p-HDL) was determined using a modified method of a previously published cell-free assay with CuCl2 and H2DCF.2.2 Plasma PON1 Arylesterase ActivityArylesterase activity of PON1 was performed on whole plasma using phenyl acetate as the substrate as described. Initial rates of hydrolysis were determined spectrophotometrically at 270 nm on DU(?) 640 spectrophotometer. An aliquot of 20?L of 30X diluted mouse plasma was added to a final reaction volume of 500?L (phenyl acetate [100 mmol/L] and CaCl2 [100 mmol/L] in Tris-HCI [40 mmol/L] buffer, pH 8.0) for 6 min at 25?. One unit of arylesterase activity equals 1?mole of phenyl acetate hydrolyzed per mL per min.2.3 Quantification of Plasma Nitrite+Nitrate Nitrite+nitrate concentrations were determined by ozone chemiluminescence using the NO Analyzer (Model 280i, GE Analytical-Sievers, Boulder, CO) as described. An aliquot of 30?L was injected (plasma was diluted by 1:30) into a sealed glass reaction chamber at 95?containing VCI3. Nitric oxide chemiluminescence signals were quantified and peak areas compared to the areas of external nitrate standards.2.4 Estimates of Plasma 3-NitrotyrosineAn aliquot of plasma from C57BL/6J and apoA-?-/- mice was pipetted onto nitrocellulose membranes and allowed to bind. Membranes were blocked with 5% non-fat dry milk dissolved in fresh PBS-Tween (0.1%) and then incubated overnight at 4?with antibodies for 3-NT (1:5000;Millipore, Billerica, MA). The next day, the membranes were washed and incubated with the appropriate HRP-conjugated secondary antibody for 1 h. Bands of identity were visualized with ECL chemiluminescence following the manufacturer's recommendations. Autoradiograms were scanned with a laser densitometer or a UMax scanner.2.5 Effects of Genetic Deletion of ApoA-?on VasodilatationFacialis and pulmonary arteries were isolated from C57BL/6J and apoA-?-/- mice by microdissection as previously described. Vasodilatation of pressurized (60 cm of H2O) facialis arteries was examined in the absence and presence of L-nitroargininemethylester (L-NAME-200?M, final concentration) as previously described.(14) Changes in pulmonary artery tension in response to acetylcholine (ACh) were recorded on a DMT wire-myograph using protocols similar to that which we have previously described.2.6 Measurements of Lung MechanicsMice were tracheostomized with an 18-gauge cannula and mechanically ventilated in a quasi-sinusoidal fashion with a small animal ventilator at 150 breaths per minute(19) and a tidal volume (VT) of 10 mL·kg-1 body weight. Basal respiratory system impedance (Zrs) was then assessed using the forced oscillation technique (FOT) applied over 2 sec (a Prime-2 perturbation) using the flexiVent system. Each level of PEEP (0,3,6, and 9 cm H2O) was held for 80 sec with the Prime-2 perturbation repeated twice, once at 35 sec and the other at 70 sec. Measurements at each PEEP were Rn, G and H.2.7 Methacholine challengeMice were challenged with 70 L of MCh (Sigma-Aldrich) of increasing concentrations (3.125 to 100 mg/mL). MCh aerosols were generated using an ultrasonic nebulizer and delivered to the inspiratory line of the ventilator using room air. Each aerosol was delivered for 30 sec, during which time ventilation was maintained mechanically by the flexiVent. Then for the next 3 min, each aerosol was delivered for 30 sec, during which time regular ventilation was maintained and measurements(Rn,G and H) were assessed as a curve, whch pick value were picked up for calculation.2.8 HistologyA 0.5mL aliquot of zinc-formalin was used to inflate the lung prior to removal. The lung was fixed in zinc-formalin, embedded in paraffin, sectioned and then stained with hematoxylin and eosin (H&E) for histology or with McLetchies' trichrome to assess collagen deposition.2.9 Isolation of BALF and AnalysisBALF was obtained by flushing the lung with PBS, first 1mL followed by 0.5mL. The rinses were combined and the BALF was centrifuged at 2000 rpm for 10 min at 4?. The supernatant was removed, stored at -80?. The cell pellet was gently resuspended in 1 mL PBS. An aliquot of 500?L was used to prepare slides using a Cytospin. Differential cell counts were made from slides stained with Diff-Quick. Five hundred cells were counted and identified (magnification, X40) for each mouse by a pathologist who had no prior knowledge of slide identities.2.10 ImmunofluorescenceImmunofluorescence was performed on 5?m sections of paraffin-embedded, PBS-zinc-formalin-fixed lungs. Two sections were present on each slide. The sections were incubated separately with antibodies against either 3-nitrotyrosine anti-T15 autoantibodies as previously described, or with anti-4-HNE Michael's adducts and with TGF?-1 for co-localization studies. Slides were washed with PBS (3X) and then incubated with the appropriate secondary antibodies. The slides were washed, sealed under cover slips and images captured using a krypton argon laser Nikon Eclipse TE2000U confocal microscope with 10x/0.17 aperture objective. Total magnification was 100 with Ex/Em at 488/580 nm for Alexa 488 and 633/661 nm forTO-PRO-3.2.11Western Blot AnalysisProteins were separated by SDS-PAGE (4-20%) and transferred onto nitrocellulose membranes. Membranes were blocked with 5% non-fat dry milk dissolved in fresh PBS-Tween (0.1%) and then incubated overnight at 4?with antibodies for XO (1:500), MPO (1:100) or eNOS (1:10,000). The next day, the membranes were washed and incubated with the appropriate HRP-conjugated secondary antibody for 1 h. Bands of identity were visualized with ECL chemiluminescence following the manufacturer's recommendations. Autoradiograms were scanned with a laser densitometer or an UMax scanner.2.12 BALF DCF-detectable OxidantsOxidation of H2DCF was used to obtain an index of the levels of oxidants in BALF. An aliquot of BALF (75?L) was mixed with H2DCF (100?L PBS+25?L H2DCF [a 1x10 dilution of 2 mg/mL] in a total volume of 200?L) and this mixture incubated for 2 h at 37?. Absolute changes in fluorescence (Ex 485 nm;Em 530 nm) were determined at the end of the 2 h incubation period using Spectra Max Gemini EM fluorescence plate reader.3. Statistical AnalysisData are presented as mean±SEM. Results were analyzed by Student's t-test, Mann-Whitney test or Fisher's exact test where appropriate. Airway data were analyzed by 2-way ANOVA to determine significance between curves and a Bonferroni post-test to determine significance of points between the curves. All statistical analysis was performed using GraphPad Prism Software (version 4.0).4. Results4.1 Effects of ApoA-?Deficiency on Lipids and Oxidative StressApoA-?-/- mice had reduced levels of plasma total cholesterol and HDL cholesterol compared with control mice (Figure 1A and 1B). Although apoA-?-/-mice contained less HDL than control mice, their HDL oxidized at a faster rate than HDL from control mice, indicating that HDL in apoA-?-/- mice was proinflammatory (Figure 1C). PON1 plasma protein in apoA-?-/- mice was increased compared with protein levels in C57BL/6J mice (Figure 2A and 2B). However, PON1 activity was decreased (?34%,p<0.001) compared with controls (Figure 2C). Although the concentration of plasma nitrite+nitrate was decreased in apoA-?-/- mice, plasma 3-NT levels were increased compared with the levels in control mice (Figure 3A and 3B, respectively).4.2 Effects of ApoA-?Deficiency on Vasodilatation.Studies on ACh-dependent vasodilatation of isolated and pressurized facialis arteries revealed that genetic loss of apoA-?had no effect on relaxation responses in these vessels (Figure 4A vs.4B). These observations are consistent with previous studies showing that apoA-?-/- mice are not more susceptible to atherosclerosis.(27,28) However, relaxation responses of pulmonary artery rings isolated from apoA-?-/- mice were impaired at the highest ACh concentration (Figure 4C). Pulmonary artery rings from apoA-?-/-mice actually constricted when treated with the highest ACh concentration. In contrast, the pulmonary artery rings from C57BL/6J mice continued to relax. Such changes in the physiological responses of pulmonary arteries in apoA-?-/- mice are consistent with the idea that chronic exposure to inflammation and oxidative stress impair vasodilatation. 4.3 Effects of ApoA-?Deficiency on Airway HyperresponsivenessThe flexiVent provides quantitative information regarding the mechanical properties of the entire airway tree. In the large airways it assesses Newtonian resistance (RN), while in the smaller airways it is able to determine changes in tissue damping (G) and tissue elastance (H). In both apoA-?-/- and C57BL/6J mice, methacholine (MCh) induced dose-dependent increases in airway resistance associated with tissue dampening (G, Figure 5B; p<0.01) and tissue elastance (H, Figure 5C;p<0.001), but not RN (Figure 5A). Effects of increasing PEEP on airway mechanical parameters, RN, G and H, are shown in Figure 5D,4E and 4F, respectively. G was increased in apoA-?-/- mice compared with control mice at baseline PEEP=0 (p<0.01) as well as throughout the entire PEEP curve (p<0.001). H was increased in apoA-?-/- mice compared with controls throughout the entire PEEP range (p<0.001).4.4 Effects of apoA-?Deficiency on HistologyH&E sections of lungs harvested from apoA-?-/- mice (two lower left images, Figure 6A) contained more inflammatory cells than lungs from C57BL/6J mice (one upper left image, Figure 6A). Although inflammatory cells in perialveolar and perivascular regions of apoA-?-/- mice were increased more than two fold compared with C57BL/6J mice (Figure 6B, p<0.025 2-tailed t-test), the number of white blood cell infiltrating the lungs did not achieve the numbers that are achieved in ovalbumin-sensitized C57BL/6J mice, which can approach 25-50 cells PHF. Sections of lungs harvested from apoA-?-/- mice also revealed increased collagen deposition (two lower right images, Figure 6A). Trichrome staining is stronger and more extensive in apoA-?-/- sections than in sections of lungs from C57BL/6J mice (upper right image, Figure 6A). Cytospins of BALF isolated from apoA-?-/- and C57BL/6J mice showed that the BALF contained predominately alveolar macrophages(>98%). Cytospins of BALF isolated from C57BL/6J and apoA-?-/- mice reveal that the genetic loss of apoA-?did not have significant effect on lymphocyte infiltration in the bronchoalveolar space (Figure 6C).4.5 Effects of apoA-?Deficiency on Biomarkers of Oxidative Stress and InflammationImmunofluorescence studies revealed that lung sections from apoA-?-/-mice stained stronger for 3-NT and 4-HNE Michael's adducts than sections from controls (Figure 7A and 7B) but not T15 autoantibodies (Figure 7A). Genetic loss of apoA-?increases co-localization of 4-HNE-derived Michael's adducts with active TGF?-1 in pulmonary airways (Figure 7B). Lungs from apoA-?-/- mice express higher levels of XO, MPO and eNOS than lungs from C57BL/6 mice (Figure 8). These data are in contrast to nitrite+nitrate data showing that BALF isolated from apoA-?-/- mice contains less nitrite+nitrate than BALF isolated from C57BL/6J mice (Figure 9A). Finally, BALF from apoA-?-/- mice increased DCF fluorescence to a greater extent than BALF from C57BL/6J mice (Figure 9B). These data indicate that BALF from apoA-?-/- mice contains more pro-oxidant compounds than BALF from C57BL/6J mice.5. Conclusion(1) Deletiong of apoA-?makes HDL lost its protect fuction and turns it to proinflammatiory HDL, which aggregate a serial oxidatives stress and ucoupled eNOS.(2) ApoA-?plays an important protective role in preventing pulmonary inflammation, impaired vasodilatation and increased airway hyperresponsiveness. Loss of apoA-?and its associated anti-inflammatory and anti-atherogenic properties has a profound and severe negative impact on the lung with respect to vascular function and airway physiology. Paper Two D-4F attenuates oxidative stress and inflammation in T-bet and apoA-?double knock out mice1 Back groundHigh-density lipoprotein (HDL) is important in reverse cholesterol transport and is believed to play important anti-inflammatory and anti-atherogenic roles in vascular physiology. Increasing evidence suggests that elevated levels of HDL are not always atheroprotective. Indeed, chronic states of inflammation and oxidative stress have been shown to convert HDL from an anti-inflammatory and anti-atherogenic particle into a pro-inflammatory and pro-atherogenic particle, making it useless for protecting the vessel wall against the effects of atherogenic concentrations of LDL. Asthma also increases inflammation and oxidative stress. In our previous study, we observed that apoA-?plays an important protective role in preventing pulmonary inflammation and airway hyperresponsiveness.D-4F is an apoA-?mimetic made with all D-amino acids. D-4F is an amphipathic helix with a hydrophobic face that binds lipids in a manner similar to apolipoprotein A-?. D-4F appears to protect HDL from becoming proinflammatory by binding oxidized lipids with greater affinity than native lipids. This ability to preferentially bind oxidized lipids and remodel HDL may be the responsible way for decreasing production of inflammatory cytokines in the mouse influenza model.T-bet knock out mice, which develop airway hyperresponsiveness and are prone to spontaneous asthma, show lung inflammation and airway remodeling. T-bet deficient mice on Balb/c background have demonstrated dysfunctional HDL in lung inflammation and airway remodeling (unpublished abstractions). To examine the role of HDL in airway disease, we crossed T-bet-/- mice and apoA-?-/- mice to create double knock out T-bet-/-/ apoA-?-/- mice. To determine if D-4F could decrease airway disease we also treated T-bet-/-/apoA-?-/- mice with D-4F. We found that T-bet-/-/apoA-?-/- mice displayed higher airway hyperresponsiveness, pulmonary inflammation, collagen deposition in perivascular and peri alveolar region compared with T-bet-/-/apoA-?+/+ control mice. Intriguingly, D4F reduced airway responsiveness, pulmonary inflammation and collagen deposition in the T-bet-/-/ apoA-?-/- mice. Additionally, D4F attenuated oxidative stress in the T-bet-/-/apoA-?-/-mice lung.2 Methods2.1 MiceApoA-?-/- mice and T-bet-/- mice on a C57BL/6J background were purchased from Jackson Laboratory. The doubly heterozygous progeny were crossed to generate double knockout T-bet-/-/ apoA-?-/- mice. Animal genotype was identified by a PCR-based assay. There were no discernible differences in litter size or appearance of the T-bet-/-/ apoA-?-/- mice versus T-bet-/-/ apoA-?+/+ control mice. Mice of 10 weeks old were injected with PBS and Apolipoprotein-mimetic peptide (D-4F, AC-DWFKAFYDKVAEKFKEAF-NH2) by daily intraperitoneal (IP) injection (3 mg/kg) for 6 weeks.2.2 Plasma Total Cholesterol, HDL CholesterolTotal cholesterol (TC) was quantified using a cholesterol oxidase/esterase kit from Wako Chemical, Inc. (Richmond, VA). HDL cholesterol was quantified using a HDL Cholesterol E kit from Wako Diagnostics. Proinflammatory HDL (p-HDL) was determined using a modified method of a previously published cell-free assay with CuCl2 and H2DCF(14).2.3 Plasma PON1 Arylesterase ActivityArylesterase activity of PON1 was performed on whole plasma using phenyl acetate as the substrate as described.(15)2.4 Quantification of Plasma Nitrite+NitrateNitrite+nitrate concentrations were determined by ozone chemiluminescence using the NO Analyzer (Model 280i, GE Analytical-Sievers, Boulder, CO) as described.(16,17) An aliquot of 30?L was injected (plasma was diluted by 1:30) into a sealed glass reaction chamber at 95?containing VCl3.(17) Nitric oxide chemiluminescence signals were quantified and peak areas compared to the areas of external nitrate standards.2.5 Measurements of Lung MechanicsEach level of PEEP (0,3,6, and 9 cm H2O) was held for 80 sec with the Prime-2 perturbation repeated twice, once at 35 sec and the other at 70 sec. Measurements at each PEEP were Rn,G and H.2.6 Methacholine challengeMice were challenged with 70 L of MCh (Sigma-Aldrich) of increasing concentrations (3.125 to 100 mg/mL) measurements(Rn,G and H) were assessed as a curve, whch pick value were picked up for calculation.2.7 HistologyA 0.5 mL aliquot of zinc-formalin was used to inflate the lung prior to removal. The lung was fixed in zinc-formalin, embedded in paraffin, sectioned and then stained with hematoxylin and eosin (H&E) for histology or with McLetchies' trichrome to assess collagen deposition.2.8 Immunofluorescence4-HNE Michael's adducts and TGF?-1 expression were detected by immunofluorescence for co-localization studies.Images captured using a krypton argon laser Nikon Eclipse TE2000U confocal microscope (Melville, NY) with 10x/0.17 aperture objective.2.9 Western Blot AnalysisXO,MPO and eNOS protein levels were determined by western blot analysis. Autoradiograms were scanned with a laser densitometer or an UMax scanner. 3. Statistical AnalysisData are presented as mean±SEM. Results were analyzed by Student's t-test, Mann-Whitney test or Fisher's exact test where appropriate. Airway data were analyzed by 2-way ANOVA to determine significance between curves and a Bonferroni post-test to determine significance of points between the curves. All statistical analysis was performed using GraphPad Prism Software (version 4.0).4 Results4.1 Cholesterol ProfilesThe levels of total cholesterol and HDL cholesterol in plasma of T-bet-/-/ apoA-?-/- mice treated with either PBS or D-4F were significantly reduced compared with T-bet-/-/7apoA-?+/+ control mice (P < 0.001) . No difference in the total cholesterol or HDL cholesterol was observed between T-bet-/-/apoA-?-/-mice treated with PBS and D4F.4.2 Respiratory MechanicsThe effects of MCh on RN and G had no differences between groups. Lung tissue elastance was increased in the T-bet-/-/ apoA-?-/- mice treated with PBS compared with T-bet-/-/apoA-?-/- control mice (P < 0.05)4.3 TGF-?1and 4- HNE in Lung Tissue ImmunofluorescenceTGF-?and 4-HNE content in lungs is significantly reduced in T-bet-/-/ apoA-?-/- mice treated with D-4F.4.4 Lung histology4.5 Perivascular and peribronchiolar inflammation and collagen deposition were increased slightly in T-bet-/-/apoA-?+/+ control mice; and to a much greater extent in the T-bet-/-/apoA-?-/- mice treated with PBS. However D-4F treatments markedly decreased inflammation and collagen deposition in T-bet-/-/ apoA-?-/-mice.4.6 XO ExpressionThe 145-kDa band corresponding to full length XD expression in the lung was significantly increased in T-bet-/-/apoA-?-/- mice treated with D-4F compared with T-bet-/-/apoA-?+/+ control mice and T-bet-/-/apoA-?-/- mice treated with PBS(P<0.05). There was no difference between T-bef/7apoA-r/+ control mice and T-bet-/-/apoA-?-/- mice treated with PBS.4.7 Nitrate and Nitrite Level in BAL Fluid and PlasmaThe nitrate and nitrite concentration in BAL fluids, which indicates the in vivo generation of NO in the airways, from the T-bet-/-/ apoA-?-/- mice treated with PBS and D4F was significantly reduced compared with T-bet-/-/ apoA-?+/+ control mice. Compared with T-bet-/-/ apoA-?-/- mice treated with PBS, T-bet-/-/ apoA-?-/- mice treated with D4F had increased nitrate and nitrite levels in the plasma and no significant difference had been achieved.5 ConclusionTaken together, these data suggest that genetic loss of apoA-?on the background of T-bet deficient mice increases pulmonary inflammation and collagen deposition as well as airway hyperresponsiveness. D4F reduced airway responsiveness, pulmonary inflammation and collagen deposition in the T-bet-/-/ apoA-?-/- mice lung. D4F plays an important role in the mechanics of asthma.