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Endothelial disruptive pro-inflammatory effects of nicotine and e-cigarette vapor exposures

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Abstract

The increased use of inhaled nicotine via e-cigarettes has unknown risks to lung health. Having previously shown that cigarette smoke (CS) extract disrupts the lung microvasculature barrier function by endothelial cell activation and cytoskeletal rearrangement, we investigated the contribution of nicotine in CS or e-cigarettes (e-Cig) to lung endothelial injury. Primary lung microvascular endothelial cells were exposed to nicotine, e-Cig solution, or condensed e-Cig vapor (1-20 mM nicotine) or to nicotine-free CS extract or e-Cig solutions. Compared with nicotine-containing extract, nicotine free-CS extract (10-20%) caused significantly less endothelial permeability as measured with electric cell-substrate impedance sensing. Nicotine exposures triggered dose-dependent loss of endothelial barrier in cultured cell monolayers and rapidly increased lung inflammation and oxidative stress in mice. The endothelial barrier disruptive effects were associated with increased intracellular ceramides, p38 MAPK activation, and myosin light chain (MLC) phosphorylation, and was critically mediated by Rho-activated kinase via inhibition of MLC-phosphatase unit MYPT1. Although nicotine at sufficient concentrations to cause endothelial barrier loss did not trigger cell necrosis, it markedly inhibited cell proliferation. Augmentation of sphingosine-1-phosphate (S1P) signaling via S1P1 improved both endothelial cell proliferation and barrier function during nicotine exposures. Nicotine-independent effects of e-Cig solutions were noted, which may be attributable to acrolein, detected along with propylene glycol, glycerol, and nicotine by NMR, mass spectrometry, and gas chromatography, in both e-Cig solutions and vapor. These results suggest that soluble components of e-Cig, including nicotine, cause dose-dependent loss of lung endothelial barrier function, which is associated with oxidative stress and brisk inflammation.
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Endothelial disruptive pro-inflammatory effects of nicotine and e-cigarette vapor exposures 1
Kelly S. Schweitzer1, Steven X. Chen1, Sarah Law1, Mary Van Demark1, Christophe Poirier1, 2
Matthew J. Justice1, Walter C. Hubbard2, Elena S. Kim1, Xianyin Lai3, Mu Wang3, William 3
D. Kranz4, Clinton J. Carroll4, Bruce D. Ray5, Robert Bittman6*, John Goodpaster4, and 4
Irina Petrache1,3,8 5
*deceased 6
1Department of Medicine, Indiana University School of Medicine, Indianapolis, Indiana; 7
2Department of Clinical Pharmacology, The Johns Hopkins University, Baltimore, Maryland; 8
3Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, 9
Indianapolis, Indiana; 4Department of Chemistry and Chemical Biology; Indiana University – 10
Purdue University, Indianapolis, Indiana, USA; 5Department of Physics, Indiana University 11
Purdue University, Indianapolis, Indiana; 6Queens College of the City University, Flushing 12
NY; 8Richard L. Roudebush Veterans Affairs Medical Center, Indianapolis, Indiana 13
Running title: Nicotine-induced signaling in lung endothelial cells 14
Address correspondence to: Irina Petrache, MD, Indiana University, Division of Pulmonary, 15
Allergy, Critical Care and Occupational Medicine, Walther Hall-R3 C400, 980 W. Walnut 16
Street, Indianapolis, IN 46202-5120. Phone: 317-278-2891, Fax 317-988-3976, Email: 17
ipetrach@iu.edu 18
Keywords: tobacco, permeability, cell proliferation, sphingosine 1-P, inflammation 19
Acknowledgements: Project SEED program, for sponsoring JL. We thank Mrs. Margie 20
Albrecht Ms. Alayna Hutchinson, and Mr. Yuan Gu for expert technical assistance. NMR 21
spectra were acquired at the IUPUI School of Science NMR Center. 22
Funding sources: RO1HL077328 (IP); R21DA029249 (IP). 23
Articles in PresS. Am J Physiol Lung Cell Mol Physiol (May 15, 2015). doi:10.1152/ajplung.00411.2014
Copyright © 2015 by the American Physiological Society.
2
Abstract 24
The increased use of inhaled nicotine via e-cigarettes has unknown risks to lung health. Having 25
previously shown that cigarette smoke (CS) extract disrupts the lung microvasculature barrier 26
function by endothelial cell activation and cytoskeletal rearrangement, we investigated the 27
contribution of nicotine in CS or e-cigarette extracts (e-Cig) to lung endothelial injury. Primary lung 28
microvascular endothelial cells were exposed to nicotine, e-Cig solution, or condensed e-Cig vapor 29
(1-20 mM nicotine) or to nicotine-free CS extract or e-Cig solutions. Compared to nicotine-30
containing, nicotine free-CS extract (10-20%) caused significantly less endothelial permeability, as 31
measured with electric cell-substrate impedance sensing. Nicotine exposures triggered dose-32
dependent loss of endothelial barrier in cultured cell monolayers and rapidly increased lung 33
inflammation and oxidative stress in mice. The endothelial barrier disruptive effects were associated 34
with increased intracellular ceramides, p38 MAPK activation, myosin light chain (MLC) 35
phosphorylation and was critically mediated by Rho-activated kinase via inhibition of MLC-36
phosphatase unit MYPT1. Although nicotine at sufficient concentrations to cause endothelial barrier 37
loss did not trigger cell necrosis, it markedly inhibited cell proliferation. Augmentation of 38
sphingosine-1 phosphate (S1P) signaling via S1P1 improved both endothelial cell proliferation and 39
barrier function during nicotine exposures. Additional nicotine-independent effects of e-Cig may be 40
attributable to acrolein, which was detected, along with propylene glycol, glycerol, and nicotine, by 41
NMR, mass spectrometry, and gas chromatography, in both e-Cig solutions and vapor. These results 42
suggest that soluble components of e-Cig, including nicotine cause dose-dependent loss of lung 43
endothelial barrier function, associated with oxidative stress and brisk inflammation. 44
45
46
3
Introduction 47
Cigarette smoking (CS) is the primary causative factor for chronic obstructive pulmonary 48
disease (COPD), the third leading cause of death worldwide. We have shown that in addition to 49
injuring the lung epithelium, soluble components of CS can be directly injurious to lung endothelial 50
cells, disrupting the lung endothelial barrier function (22). It is not known if nicotine, the main 51
component of CS could be responsible for this effect. Furthermore, it is not known if inhalation of 52
the vapor released by electronic cigarette (e-Cig) has similar effects as CS on lung endothelium. 53
Given the increasing use of e-Cig, which results in inhalation of vapor produced by heating nicotine-54
containing liquid, it is important to define their biological effects on the lung endothelial barrier 55
function. 56
Previous studies of nicotine in endothelial cells have generated diverse results, showing 57
inhibition of human umbilical vein endothelial cell (HUVEC) proliferation (1), but enhanced 58
proliferation of endothelial progenitor cells (35). Further, high doses of nicotine were shown to 59
inhibit cytokines required for neovascularization during bone healing (14, 26) and to affect the 60
vasoreactivity of various vascular networks (8, 9, 16, 17). However, the effects of nicotine on lung 61
barrier function are not known and, given that the loss of endothelial integrity contributes to lung 62
inflammation and injury, they are important to define. 63
The mechanisms by which nicotine triggers systemic endothelial cell responses have been 64
shown to involve increases in NO signaling molecules (18) and reactive oxygen species (ROS) (16), 65
as well as generation of pro-apoptotic metabolites (28), events which would be expected to also 66
impair lung endothelial barrier function. However, nicotine has been shown to have discrepant 67
effects, either decreasing or increasing the expression of intracellular adhesion molecules in HUVEC 68
(25), (24), via signaling pathways involving PKC, p38 and ERK1/2 MAPK (29, 33). Little is known 69
4
about the direct effect of nicotine on the lung cell endothelial barrier and the mechanisms by which 70
nicotine would exert such effects. 71
Endothelial cellular barrier is tightly regulated by the actomyosin cytoskeleton, whose 72
contraction is governed by myosin light chain kinase MLCK and Rho kinase enzymatic activities. 73
We have previously shown that CS extracts caused endothelial barrier dysfunction via oxidative 74
stress, p38 MAPK activation and ceramide release, generated upstream of Rho kinase activation and 75
cellular contraction (22). In addition, CS-induced barrier dysfunction may be attributed to loss of 76
inter-cellular tethering forces (22), which are typically reinforced by sphingosine-1 phosphate (S1P) 77
signaling (10). We investigated if nicotine, one of the hundreds of molecules present in CS extracts, 78
is sufficient to alter lung endothelial barrier function by affecting cytoskeletal regulation. 79
Using measurements of endothelial monolayer barrier function in cultured primary cells via 80
trans-cellular electrical cellular impedance sensing (ECIS) (27), and in vivo assessment of oxidative 81
stress and extravasated inflammatory cells in the bronchoalveolar lavage, we show that nicotine and 82
e-cig solutions or vapor condensates cause dose-dependent cell injury manifested by decreased 83
barrier function and decreased cell proliferation, via specific signaling pathways. 84
85
Materials and Methods 86
Reagents and pharmacological inhibitors. Unless otherwise stated, all chemical reagents were 87
purchased from Sigma-Aldrich (St. Louis, MO). Free radical scavenger and precursor to glutathione 88
N-acetylcysteine (NAC; 0.5 mM); and p38 MAPK inhibitor SB203580 (5 µM) were from Santa 89
Cruz Biotechnology (Dallas, TX). ERK1/2 inhibitor PD98059 (50 µM) and JNK inhibitor SP600125 90
(50 µM) were from Calbiochem (San Diego, CA). The S1P analogs (S)-FTY720 phosphonate (1S); 91
(S)-FTY720 enephosphonate (2S); (R)-FTY720 phosphonate (1R); and (R)-FTY720 92
5
enephosphonate (2R) (all solubilized in MeOH) were synthesized as previously described (13). 93
Ceramide synthase inhibitor, fumonisin (FB1; 10 μM) was from Cayman Chemical (Ann Arbor, MI) 94
and the serine palmitoyl transferase inhibitor, myriocin (Myr; 50 nM) was from Biomol International 95
(Plymouth Meeting, PA). The neutral sphingomyelinase (nSMase) inhibitor, GW4869 (GW; 20 96
µM), acid sphingomyelinase (aSMase) inhibitor, imipramine (Imi; 50 μM) were from Calbiochem. 97
Nicotine solutions were obtained from Sigma. E-Cig solutions for vaporization were purchased 98
from World of Vapor (Indianapolis, IN). 99
100
Cells. Primary rat lung endothelial cells (RLEC; from Dr. Troy Stevens; University of Southern 101
Alabama) and human bronchial epithelial cell line Beas-2B (ATCC) were cultured in Dulbecco’s 102
Modified Eagle Media with high glucose (DMEM-HG) from Invitrogen (Hercules, CA) containing 103
10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Primary mouse lung endothelial cells 104
(MLEC; from Dr. Patty Lee; Yale University) were grown as RLEC but with 20% FBS. Primary 105
human microvascular cells-lung derived (HMVEC-LBl, Lonza) were grown in EBM-2 media with 106
supplements. All cultures were maintained at 37°C with 5% CO2. Prior to treatments (2 hr), culture 107
medium was replaced with basal media containing 2% FBS. 108
109
Transcellular electrical resistance (TER) measurements. Electrical resistance across cell 110
monolayers was measured using ECIS (Applied Biophysics, Inc., Troy, NY) as previously described 111
(20). Cells were cultured on gold microelectrodes and the total resistance was measured in real time 112
across monolayers and recorded every 2-4 minutes, continuously, over 24 hours. Shown in figures 113
are select (e.g. 5h and 20h) time-points as representative of changes induced by exposures studied. 114
Experiments were conducted after cells were confluent (TER achieved a steady state). TER values 115
6
(ohm) for each timepoint were normalized to the initial resistance value (at the beginning of the 116
recording) and plotted as normalized TER. 117
118
Animal experiments. All experiments were performed according to IACUC guidelines and 119
approved protocols. C57Bl/6 mice (4 month old females) were nebulized (Aerogen; Galway, Ireland; 120
nebulizer unit 2.5-4.0 VMD) using either one dose of nicotine (2 µg) and harvested immediately, or 121
two doses of e-cig extract (1 µg each) and harvested after either 30 min or 24 hr. Controls were 122
nebulized with saline and harvested at similar time points. 123
124
Oxidative stress measurements. 125
Mouse plasma and BALF: 8-hydroxydeoxyguanosine (8-OHdG), a marker of oxidative damage, was 126
quantified in plasma (1:10 dilution) or BALF, using OxiSelect Oxidative DNA Damage ELISA kit 127
(Cell Biolabs; San Diego, CA). Total nitrotyrosine was determined in plasma (1:10) using 128
competitive ELISA (Hycult; Uden, Netherlands), following manufacturer’s specifications. 129
RLEC: cells were grown on gelatin-coated coverslips, pretreated with nicotine (10 mM; 30 min) 130
with or without NAC (0.5 M). ROS were detected using Image-iT LIVE Green Reactive Oxygen 131
Species Detection Kit (Invitrogen) following manufacturer’s instructions. Nuclei were stained using 132
DAPI (Invitrogen). Pictures were captured using a Nikon 80i fluorescent microscope. 133
134
CS extract and e-Cig vapor condensate. Aqueous CS extract was obtained from filtered research-135
grade cigarettes (2R4F) or nicotine-free cigarettes (1R5F) from the Kentucky Tobacco Research and 136
Development Center (University of Kentucky, Lexington, KY), as previously described (22). A 137
stock CS extract (100%) was prepared by bubbling smoke from 2 cigarettes into 20 ml of PBS at a 138
7
rate of 1 cigarette/min to 0.5 cm above the filter, followed by pH adjustment to 7.4 and 0.2 μm 139
filtration. A similar procedure was followed for air control (AC) extract preparation, bubbling 140
ambient air. Treatments were performed with CS or AC extract concentrations ranging from 1-20% 141
(vol:vol). Condensed e-Cig vapor was collected in a 25 ml side-armed Erlenmeyer flask placed 142
under vacuum while connected to the e-cigarette via Tygon tubing. A vacuum trap was created to 143
collect the post-vaporized condensate of e-Cig solutions, using a gel-loading tip as a constriction 144
point. A total of 125 µl of condensate was collected from vaporization of 600 µl of e-cigarette 145
solution and applied to cell cultures in indicated concentrations (vol:vol). 146
147
Ceramide determination. Following treatments, culture media was washed with PBS and cells 148
were collected by scraping in methanol, followed by lipid extraction utilizing a modified Bligh and 149
Dyer method and sphingolipid analyses were performed via combined liquid chromatography-150
tandem mass spectrometry using AB-Sciex API4000 triple quadrupole mass spectrometer (Foster 151
City, CA) interfaced with an Agilent 1100 series liquid chromatograph (Agilent Technologies, 152
Wilmington, DE), as previously described (19). Ceramide analytes were ionized via positive ion 153
electrospray ionization. Elution of the ceramides was detected by multiple reaction monitoring 154
characteristic for 14:0, 16:0, 18:0, 18:1, 20:0, 24:0 and 24:1 ceramides. C17:0-ceramide was 155
employed as internal standard. All ceramide measurements were normalized by lipid phosphorus (Pi) 156
(19). 157
158
Cell proliferation and toxicity assays. MTT assay (Invitrogen) was used to determine cell 159
proliferation/metabolic activity, following manufacturer’s instructions. RLEC were plated in 160
triplicate at 2500 cells/ml for 18 hr and then medium was replaced with 2% FBS-containing medium 161
8
with inhibitors or their vehicle controls for 2 hr, followed by addition of nicotine and overnight 162
incubation before assay. Cell Counting Kit-8 (CCK-8) assay (Dojindo, Rockville, MD) was 163
performed on 1.6 X 104 cells/ well, plated on 96-well tissue culture plates and treated the next day as 164
indicated. The kit’s CCK-8 solution was added and the plate was read using a spectrophotometer at 165
450 nm. Cytotoxicity /LDH assay (Roche, Indianapolis, IN) was performed in HLMVEC plated in 166
triplicate onto 96-well dishes at 20 x 105 or 50 x 105 cells/well. 167
168
Immunoblotting. Cells were grown in 6-well dishes, washed with ice-cold PBS immediately after 169
treatment, and collected by centrifugation. Cells were lysed in standard RIPA buffer with protease 170
and phosphatase inhibitors (Complete and Phostop, respectively; Roche). Samples containing equal 171
protein amount, as determined by BCA protein analysis (Pierce; Rockford, IL), were resolved using 172
SDS-PAGE, transferred onto PVDF using semi-dry transfer (Bio-Rad; Hercules, CA), and were 173
probed with the following primary antibodies (all from Cell Signaling, Beverly, MA, unless 174
otherwise stated): phospho-p38 (1:500); total p38 (1:1000); phospho-MLC (1:500), phospho-myosin 175
phosphatase target subunit 1 (phos-MYPT1; 1:500). HRP-conjugated secondary antibodies to rabbit, 176
rat, or mouse were from Amersham (Piscataway, NJ). Protein expression was detected using 177
enhanced chemilluminescence ECL-plus (Amersham), quantified by densitometry, and normalized 178
by a housekeeping protein, vinculin (1:10,000; Calbiochem). 179
180
Nuclear Magnetic Resonance (NMR). Samples for NMR spectroscopy were prepared by 181
dissolving 25 µl e-Cig solution (World of Vapor, Indianapolis) in 675 µl [2H4] methanol from 182
Cambridge Isotope Laboratories, Andover, MA. 1H NMR spectra were acquired at 25°C on a Varian 183
9
Inova 500 equipped with a 5 mm triple resonance pulsed field gradient probe. All spectra were 184
acquired with 90° pulse with 8192 complex points, 64 transients, and 3 s recycle delay. 185
186
Mass Spectrometry (MS). One µl of each sample was diluted with 199 µl of 50% acetone nitrile in 187
water with 0.1% formic acid. The samples were analyzed using a Thermo Scientific Orbitrap Velos 188
Pro hybrid ion trap-Orbitrap mass spectrometer through direct infusion using a syringe pump. The 189
flow rate was 3 µl/min and the resolution was 60,000. 190
191
Gas Chromatography-Mass Spectrometry (GC-MS). All experiments used an Agilent 6890N gas 192
chromatograph coupled with an Agilent 5975 mass spectrometer. The method utilized an oven 193
program with an initial temperature of 40°C held for 1 minute, a ramp of 20°C/minute, and a final 194
temperature of 300°C held for 1 minute. The carrier gas was hydrogen, with a flow rate of 2.5 195
mL/minute and a split ratio of 20:1. The inlet was set at 250°C. The mass spectrometer operated in 196
electron ionization mode, with a scan range of m/z 50-550, and a solvent delay of 2.00 minutes. In an 197
initial experiment to determine the ingredients of each sample, 25mg of nicotine, nicotine-containing 198
and nicotine-free e-Cig solutions, and e-Cig condensed vapor were placed in a 25 mL volumetric 199
flask and diluted to the mark with dichloromethane. The samples were filtered with a 200
polytetrafluoroethylene syringe filter and analyzed. In a separate quantitation experiment, nicotine 201
and quinoline were diluted with dichloromethane to produce four standard solutions: a 100 mg/mL 202
nicotine, 1 mg/mL quinoline solution; a 10 mg/mL nicotine, 1 mg/mL quinoline solution; a 1 mg/mL 203
nicotine, 1 mg/mL quinoline solution; and a 0.1 mg/mL nicotine, 1 mg/mL quinoline solution. The 204
ratio of nicotine to quinoline in each standard was determined by peak integration, and this 205
information was used to create a calibration curve. Approximately 50 mg of three condensed vapor 206
10
samples were transferred into a 2 mL volumetric flask, spiked with 2 mg quinolone, and diluted to 207
the mark with dichloromethane. These were also analyzed via GC-MS using the same method and 208
compared against the calibration curve to determine the amount of nicotine in each sample. 209
210
Statistical Analysis. SigmaStat 3.5 (San Jose, CA) or Prism 6 (San Diego, CA) software was 211
utilized for comparisons among groups by ANOVA, as indicated, followed by inter-group 212
comparisons with Tukey post-hoc testing. For experiments in which two conditions were being 213
compared, a two-tailed Student's t-test was used. All data are expressed as mean + SEM and 214
statistically significant differences were considered if p < 0.05. 215
11
Results 216
To investigate the contribution of nicotine in CS extract to the loss of lung endothelial barrier 217
function, we compared the effect of soluble extract from nicotine-containing and nicotine-free 218
cigarettes. Primary rat lung endothelial cells (RLECs) exposed to nicotine-containing CS extract 219
(10% v:v), exhibited increased monolayer permeability as measured by ECIS, in a time dependent-220
manner, with ~ 40% decrease in trans-endothelial resistance (TER) at 5 hr and ~50 % at 20 hr 221
(Figure 1A), consistent with our previous report (22). In contrast, similar concentrations of nicotine-222
free CS extract had a markedly diminished effect, with no loss of barrier function at 5 hr and only ~ 223
30% loss of TER at 20 hr (Figure 1A). These results suggest that nicotine directly contributes to the 224
damaging effect of soluble CS extract on lung endothelial barrier. We next tested whether exposure 225
to nicotine itself decreases the endothelial barrier function. Upon incubation of primary RLECs with 226
increasing concentrations of nicotine (up to 50 mM for up to 15 hr), we noted significant time- and 227
dose-dependent decrease in TER (Figure 1B). Using a similar setup, monolayers of primary mouse 228
lung endothelial cells or human lung microvascular endothelial cells challenged with nicotine 229
exhibited similar time and dose-dependent decreases in transcellular resistance (Figure 1C-D), 230
indicating that nicotine effects are not species-specific. 231
Similar to pure, analytical-grade nicotine solutions tested above, two separate nicotine-232
containing solutions used for vaporization in commercially available electronic cigarettes (e-Cig) 233
also triggered barrier dysfunction in RLEC, consistent with their purported nicotine concentration 234
(Figure 2A). Unexpectedly, barrier dysfunction was also induced by exposures to similar volumes 235
of an e-Cig solution (e-Cig 2) that lacked nicotine (Figure 2A). Of note the nicotine-free e-Cig 236
solution 2 was marketed as having the same flavor as nicotine-containing e-Cig solution 2 and 237
shared the same manufacturer. Since vaporization of e-Cig solutions may generate different 238
12
metabolites than the original solution due to heating, we investigated if the condensed vapor isolated 239
from an e-Cig affected the endothelial barrier. For similar volumes as nicotine solutions, the e-Cig 240
vapor condensate was less potent on RLECs barrier function (Figure 2A). The barrier disruptive 241
effect of e-Cig solutions on human lung endothelial cells was nicotine dose-dependent (Figure 2B) 242
and of similar magnitude to that caused by exposures to 3% CS extract, a relatively low 243
concentration we have previously shown to not cause cell death (22). The vapor condensate of e-Cig 244
also caused a dose-related loss of endothelial barrier, that required higher volume compared to non-245
vaporized solutions of e-Cig (Figure 2C). 246
These results suggested that although nicotine in CS extracts is sufficient to trigger 247
endothelial barrier dysfunction, the effects of e-Cig solutions and vapors are only in part nicotine-248
dependent. Since commercially available e-cig extracts and vapors are not well regulated and 249
biochemically defined, we determined the composition of these solutions, compared to that of CS 250
extract and analytical-grade nicotine solutions. Nuclear magnetic resonance (NMR) confirmed the 251
presence of nicotine in e-Cig solutions marked as nicotine-containing and confirmed the lack of 252
nicotine in nicotine-free e-Cig solutions tested (Figure 3A). In addition, NMR detected the 253
propylene glycol (antifreeze) and glycerol in e-Cig solutions, and acrolein in e-Cig vapor condensate. 254
These compounds, in particular nicotine, acrolein, and glycerol, were confirmed using high 255
resolution mass spectrometry (MS) (Figure 3B), by comparing the monoisotopic mass of each 256
compound compared to its theoretical monoisotopic mass (Table 1). MS could not detect propylene 257
glycol, likely because of its poor ionization, but confirmed the lack of nicotine in nicotine-free e-Cig 258
solutions and, demonstrating increased sensitivity compared to NMR, detected acrolein not only in 259
condensed e-Cig vapor, but also in all e-Cig solutions tested. This finding suggested heating of e-Cig 260
solutions to produce vapor was not a necessary step to produce acrolein. We confirmed these results 261
13
using a third complementary method, gas chromatography (GC) (Figure 3C). Quantitative GC 262
analysis determined that the condensed e-Cig vapor generated for our experiments lost up to 4-times 263
the nicotine compared the e-Cig (stock) solutions used for vaporization. These measurements 264
suggested that the concentrations of nicotine used in these experiments are within the range of 265
nicotine that is inhaled by the average smoker per cigarette (1-2mg total nicotine). 266
Given the relatively high concentrations of nicotine applied to cells in cultures, we ensured 267
that the nicotine effect on the endothelium was not due to cell toxicity/necrosis, as determined by 268
LDH release (data not shown). However, nicotine exposure of RLEC significantly and dose-269
dependently decreased MTT activity, a test that reflects cellular metabolic activity, and decreased 270
CCK8 activity, a marker of cellular proliferation (Figure 4A-B). Nicotine-containing e-Cig 271
solutions had similar inhibitory effect on endothelial proliferation, as measured by CCK8 activity 272
(Figure 4C). These results indicated that nicotine and e-Cig trigger intracellular responses, at least in 273
vitro, in relevant primary cell cultures. 274
To investigate if inhalation of nicotine and e-Cig solutions also trigger short-term 275
(immediate; 30 min; or 24 hr) pulmonary responses in vivo, we administered 1 µg of nicotine or 2 µg 276
of e-Cig to mice via nebulization. These doses are equivalent to smoking 1 or 2 cigarettes, 277
respectively. There was a trend towards a rapid increase in PMN in the BALF at 24h (Table 2) 278
indirectly reflecting a permissive endothelial barrier for inflammatory cell extravasation. In addition, 279
there was evidence of systemic oxidative and nitroxidative stress, indicated by increased 8-OHdG 280
and nitrotyrosine levels in plasma in response to inhalation analytic-grade nicotine (Figure 5A-B). 281
These changes were paralleled by increases in the oxidative stress marker 8-OHdG levels in the 282
BALF (Figure 5C). Oxidative stress tended to increase by ~15% and ~10% compared to saline 283
vehicle in mice exposed to e-Cig solutions, as measured by 8-OHdG levels in plasma and BALF, 284
14
respectively (data not shown). Overall, these studies indicate that even brief exposures of lungs to 285
nicotine via inhalation are associated with pulmonary responses such as inflammation and oxidative 286
stress, which may cause or be the result of altered lung endothelial barrier function. A direct 287
oxidative stress-inducing effect of nicotine exposure was confirmed in cell cultures, using a 288
fluorescently-labeled ROS indicator, as well as the ROS scavenger N-acetylcysteine (NAC) (Figure 289
5D). 290
To define the signaling pathways by which nicotine impairs lung endothelial barrier function, 291
we focused on the mechanisms previously shown to be important in CS extract -induced endothelial 292
permeability, such as ROS, MAPK, and sphingolipid pathways, as well as cytoskeletal/cellular 293
contractility effectors (22). ROS played a critical role in the upstream activation of signaling 294
pathways induced by CS extract that decreased lung endothelial barrier in cell culture models (22). 295
Despite increased ROS induced by nicotine in vivo and in vitro, treatment of RLECs with NAC, a 296
potent ROS scavenger that attenuates CS extract -induced barrier dysfunction (Figure 6A), failed to 297
reduce the nicotine-induced loss of barrier in these cells. This result suggested that the mechanism 298
by which high concentrations of nicotine-induced barrier dysfunction may be distinct from those 299
engaged by whole CS extract (containing lower nicotine concentrations). 300
At concentrations shown to cause barrier dysfunction, nicotine significantly activated p38 301
MAPK in RLEC, similar to CS extract, whereas nicotine-free CS extract (in similar concentrations) 302
failed to induce phospho-p38 (Figure 6B). Despite nicotine-dependent activation of p38 MAPK, 303
treatment of RLEC with p38 inhibitor SB203580 (5 µM) did not reduce nicotine-induced endothelial 304
permeability (Figure 6C). Since neither the ERK inhibitor PD98059 (at 50 µM), nor the JNK 305
inhibitor SP600125 (at 50 µM) attenuated nicotine-induced endothelial cell permeability (data not 306
shown), we conclude that nicotine induces MAPK-independent alterations of lung endothelial barrier. 307
15
Nicotine activated the actin-myosin apparatus, as measured by increased phosphorylation of 308
myosin light chains (MLC) (Figure 6D). Nicotine-induced MLC phosphorylation was partially 309
inhibited by the ROS scavenger NAC and abolished by p38 inhibitor (Figure 6D). MLC 310
phosphorylation occurs by activation of the MLC kinase (MLCK), or by inhibition of MLC 311
phosphatase. We investigated if the myosin phosphatase target subunit 1, MYPT1 is inhibited (via 312
phosphorylation) during nicotine exposure. Nicotine increased MYPT1 phosphorylation within 15 313
min of application, for up to 60 min (Figure 6E). Nicotine-induced MYPT1 phosphorylation was 314
prevented by treatment with the Rho kinase inhibitor Y27632 (Figure 6E). Unlike NAC or p38 315
inhibition, Rho kinase inhibition significantly attenuated nicotine-induced barrier dysfunction in 316
RLEC (Figure 6F), implicating a critical role of Rho kinase induced MYPT1 inhibition for 317
nicotine’s effect on endothelial permeability. 318
We have previously shown that Rho kinase was also a key mediator of endothelial 319
permeability induced by CS extract, and that ceramides were involved in the upstream signaling 320
leading to Rho kinase activation. We therefore interrogated the role of sphingolipid pathway in 321
nicotine-induced endothelial effects. First, nicotine exposure significantly increased Cer 16:0, total 322
ceramides, and total dihydroceramides (precursors of ceramides in the de novo sphingolipid 323
pathway) in RLEC (Figure 7A). This effect was not cell type- or host species-specific, as incubation 324
of human bronchial cell line Beas-2B cells with varying concentrations of nicotine also caused 325
significant and dose-dependent increase in total ceramides (Figure 7B) and dihydroceramides (data 326
not shown). Pharmacological inhibition of neutral sphingomyelinase, or any of the other enzymes 327
involved in ceramide production such as acid sphingomyelinase, serine palmitoyltransferase, and 328
ceramide synthase (with imipramine, myriocin, and fumonisin B1, respectively) did not attenuate the 329
nicotine-induced decrease in TER (Figure 7C). In contrast, treatment with analogs of S1P, a barrier 330
16
enhancing downstream metabolite of ceramide, significantly attenuated nicotine-induced endothelial 331
permeability in RLEC (Figure 7D). We tested S1P analogs, because the S1P molecule has a short 332
duration of action and it is impractical to use with exposures that lead to a relatively slow onset of 333
increased permeability. Indeed, concomitant treatment of S1P (5 µM) with nicotine did not attenuate 334
permeability responses to nicotine (data not shown). In contrast, FTY720 mono-(1) or bi-(2) 335
phosphonate enantiomers (R or S) of the S1P analog FTY720, significantly inhibited the decrease in 336
lung endothelial permeability triggered by nicotine exposure (Figure 7E). This effect was 337
associated with decreased nicotine-induced MYPT1 and MLC phosphorylation (Figure 7F), 338
suggesting FTY720 analogs affected, at least in part, actin cytoskeletal contraction. Since S1P is also 339
a pro-proliferative signaling molecule, we investigated if increased endothelial cell proliferation 340
could explain improvement in endothelial barrier induced by the S1P agonists. Interestingly, only the 341
FTY-2S agonist significantly increased cell proliferation, as measured by MTT assay (Figure 7G), 342
indirectly suggesting that cell proliferation is not the main mechanism by which S1P agonists exert 343
barrier protective effects in response to nicotine. Since it is not known if S1P could also ameliorate 344
CS extract induced permeability, we interrogated the effect of S1P agonists in primary human lung 345
microvascular endothelial cells, along with its dependence on S1P receptor 1 (S1P1) signaling. All 346
FTY agonists, with the exception of FTY-1S, significantly improved CS extract-induced endothelial 347
permeability; this effect was abolished by knockdown of S1P1 receptor with specific siRNA (Figure 348
7H). These studies revealed that nicotine triggers selective signaling pathways partially overlapping 349
to those engaged by CS extract to disrupt endothelial cells’ barrier and proliferative functions 350
(Schematic in Figure 8). 351
352
353
17
DISCUSSION 354
The results presented indicate that nicotine has dose-dependent deleterious pulmonary effects 355
that result in loss of lung endothelial barrier function, acute lung inflammation, and decreased lung 356
endothelial cell proliferation. These findings enhance our understanding of how CS exposure causes 357
inflammation and define pulmonary effects of nicotine inhalation. 358
The preservation of an intact endothelial barrier is determined by a balance of contracting 359
cytoskeletal forces and the integrity of cell-cell contacts, both of which can be affected by exposure 360
to soluble components of CS extract (22). In this work, we identified that nicotine, which can be 361
absorbed in the circulation as a component of CS or e-Cig disrupts endothelial barrier by increasing 362
acto-myosin contractile signaling, primarily by Rho kinase-dependent phosphorylation and therefore 363
inhibition of endothelial myosin phosphatase, causing increased MLC phosphorylation. Interestingly, 364
although nicotine caused oxidative stress and activated p38 MAPK, similar to CS extract (22), 365
neither p38 MAPK inhibition nor the ROS scavenger NAC were sufficient to restore barrier function 366
following nicotine exposure, in contrast to their remarkable effect on CS-induced barrier dysfunction. 367
These results suggest several possible explanations that include a threshold of MLC phosphorylation 368
that is needed for barrier dysfunction which is achievable by Rho kinase activation but not by p38 369
MAPK alone. Such a concept is supported by a recent report in which Rho kinase was found, in 370
certain conditions, to activate p38, but not vice versa (34). Alternatively, nicotine-activated Rho 371
kinase may have additional targets that cause barrier dysfunction besides MLC phosphorylation, as 372
supported by the recent finding of a critical role for Rho kinase isoform 2 in regulating cellular 373
junctional tension (2). Finally, at least theoretically, nicotine-activated p38 MAPK may have 374
additional unexpected barrier enhancing activities that counteract its MLC phosphorylation effects. 375
Either inhibition of Rho kinase, or enhancement of S1P to S1P1 signaling significantly counteracted 376
18
the barrier disruptive effects of nicotine (Figure 8) and CS extract. The novel finding of a protective 377
effect of S1P1 agonists on the CS/nicotine-disrupted endothelial barrier is not surprising, given 378
reports of similar S1P1-dependent protective effects of FTY phosphonates against lung endothelial 379
permeability during sepsis or acute lung injury (23, 31, 32), or during synergistic conditions of 380
CFTR inhibition and CS extract exposure (3). FTY phosphonates acted at least in part by activating 381
MYPT and inhibiting MLC phosphorylation, although additional effect on intercellular tethering 382
cannot be ruled out. The fact that S1P augmentation did not recapitulate the effects of FTY 383
phosphonates may be due to the short half-life of the molecule, or due to complementary, S1P-384
independent mechanisms of action of FTY phsosphonates (7). 385
Using various pharmacological inhibitors of nicotinic receptors to test their involvement in 386
nicotine’s effects on the pulmonary endothelium, we could not identify a protective effect against 387
barrier dysfunction (data not shown). However, it is possible that untested receptors and other 388
mediators may regulate nicotine-altered endothelial barrier function. This may be true especially in 389
response to high, cytotoxic nicotine exposure levels shown to inhibit prostaglandin and endothelin 390
expression in bovine pulmonary endothelial cells (25), but were not tested in our work. The 391
concentrations of nicotine used in our cell culture studies were derived from detailed dose response 392
testing, were non-cytotoxic, and induced significant effects at levels higher than those absorbed in 393
the circulation by smokers, but which may be achieved in tissue levels with high nicotine 394
concentrations, such as the lung (6, 30). The effect of nicotine on barrier function may be organ 395
dependent, since other studies have shown an improvement in the gut barrier function by cholinergic 396
actions of nicotine on enteric glial cells (4). 397
While many of the nicotine effects on the lung endothelium were dose-dependent, nicotine-398
independent deleterious effects of e-Cig solutions were also noted. We have identified acrolein as 399
19
putative mediator for nicotine-independent toxicity, based its presence in both e-Cig solution and 400
vapor and on a large body of literature showing adverse pulmonary effects of acrolein, including on 401
endothelial intercellular tethering molecules (12). The signaling effects on nicotine-free e-Cig vapors 402
on the lung endothelial barrier remain to be investigated. 403
The noted dose-dependent anti-proliferative effects of nicotine on lung endothelial cells may 404
have implications in angiogenesis and in lung injury repair. Our results on primary lung endothelial 405
cells are in contrast to pro-proliferative effects of nicotine on human umbilical vascular endothelial 406
cells (11), systemic vasculature, or on lung cancer cells (15), suggesting cell type-specific effects of 407
low-dose nicotine. 408
Intra-vital lung microcopy in animals with intact circulation (no pump-perfusion) 409
demonstrated that the in vitro finding of CS extracts causing decreased endothelial barrier function 410
was paralleled by increased lung inflammation in vivo, measured by increased adherence of 411
circulating leukocytes to the lung microvasculature within 20 minutes of CS inhalation without 412
pulmonary edema (21). This previous work led us to complement our investigations of nicotine in 413
cell culture models with in vivo studies of acute lung and systemic effects of nebulized nicotine and 414
e-Cig extracts, mimicking the inhalation of e-Cig vapors by humans. We found that nicotine and e-415
Cig extracts caused rapid oxidative and nitroxidative stress observed in the BALF and plasma as 416
well as a trend of increased neutrophil lung inflammation at 24h following inhalation, measured by 417
the relatively less sensitive method of BALF cytospins, rather than intravital microscopy. Although 418
future studies will determine how these acute inflammatory lung responses translate into long term 419
effects of recurrent e-Cig exposures, we anticipate these will include dose-dependent sustained 420
oxidative-stress and inflammatory lung damage with limitation of endothelial repair. In this context, 421
ceramide/S1P balance may serve as an important rheostat of alveolar integrity, as seen in 422
20
experimental models of COPD (5). By augmenting barrier enhancing and angiogenic S1P signaling 423
via S1P1, such as shown here with pharmacological agonists, one may be able to improve barrier 424
function in vivo and potentially attenuate the chronic damage caused by e-Cig inhalation. 425
The clinical implications of this work are related to the potential detrimental lung effects of 426
exposure to inhaled e-Cig which may be dose-dependent, although further studies are needed to 427
determine what are the usual levels of absorbed e-Cig vapor that are harmful to human lung health. 428
Extrapolation of our results in primary human and murine lung endothelial cells and in animal 429
models to human lungs, may indicate the need for further studies into the safety of e-Cig use. 430
431
21
432
433
434
Table 1. Theoretic and detected monoisotopic mass of compounds identified by mass 435
spectrometry in e-Cig. 436
Compound Formula Theoretic Monoisotopic
Mass ([M+H]+)
Detected Monoisotopic
Mass ([M+H]+)
Nicotine C10H14N2 163.12352 163.12157
Acrolein C3H4O 57.03403 57.03297
Glycerol C3H8O3 93.05516 93.05386
437
438
439
440
441
442
443
444
445
446
447
Table 2. Cells detected in the BALF of mice exposed to inhaled e-Cig or saline control and 448
collected at the indicated time; mean (SEM). 449
Treatment Time Macrophages Lymph PMN n
saline 30 min 33228 (9264) 106 (82) 0 (0) 3
e-Cig 1 30 min 28278 (6664) 56 (34) 0 (0) 3
saline 24 hr 87128 (21520) 1205 (399) 0 (0) 3
e-Cig 1 24 hr 62317 (13064) 2122 (1862) 561 (427) 3
450
22
FIGURE LEGENDS 451
Figure 1. Effect of nicotine on lung endothelial and epithelial barrier function. A. Transcellular 452
electrical resistance TER measured at the indicated time-point normalized to TER at baseline (at the 453
beginning of the measurement, prior to any treatment) in cells exposed to ambient air control extract 454
(AC), nicotine-containing cigarette smoke extract (CS) or nicotine-free cigarette smoke extract (all 455
solutions were 10% v:v) measured by ECIS in primary lung microvascular endothelial cells. Mean + 456
SEM, n=4-10, One-way ANOVA (with Tukey’s post-hoc testing for inter-group comparisons). B-E. 457
Normalized TER measured at the indicated time (hours) in primary lung rat microvascular 458
endothelial (RLEC, B), primary mouse lung endothelial cells (C), primary human lung 459
microvascular endothelial cells (D) exposed to the indicated concentrations of nicotine. Mean + 460
SEM, n=5-56, One-way ANOVA (Tukey’s). 461
462
Figure 2. Effect of commercial electronic cigarette (e-Cig) solutions on lung endothelial 463
barrier. A-B. Normalized TER measured in cells (RLEC in A and HLMVEC in B) exposed to 464
nicotine (15 mM, 5 hr), to cigarette smoke extract (CSE with similar nicotine content), or to e-Cig 465
extracts or condensed vapor (commercial preparation with the indicated nicotine content; 5 hr). 466
Mean + SEM, n=4-10, One-way ANOVA (Tukey’s). C. Normalized TER measured in HLMVEC 467
exposed to the indicated volume (μL) of e-Cig or condensed e-Cig vapor. Mean + SEM, n=4-10, 468
One-way ANOVA (Tukey’s). 469
470
Figure 3. Composition of e-Cig and condensed e-Cig vapor. A Spectra from indicated solutions 471
analyzed with nuclear magnetic resonance (NMR; Resonances are ±0.05 ppm), which detected 472
methanol solvent OH 4.87, s; 3.30, quintet; nicotine, H2 8.50, d ; H6 8.44, dd; H4 7.85, dt; H5 7.42, 473
23
dd; H9a 3.24, t, H7 3.20, dd; H9b 2.37, dd; H11a 2.26, m; HN-methyl 2.17, s; H10a 1.98, m; H10b 1.88, m; 474
H11b 1.77, m; propylene glycol H2, 3.78, m; H1 3.42, d; H3 1.15, d; glycerol, H2 3.66, tt; H1,3 3.57, 475
dm. In some spectra, a small aldehyde singlet (presumed acrolein) is visible at 9.77 ppm. Noted 476
molecules’ spectra obtained from high resolution mass spectrometry (MS, B) or gas chromatography 477
(GC, C) analyses of indicated solutions. 478
479
Figure 4. Effect of nicotine on lung endothelial cells proliferation. Cell proliferation determined 480
with the metabolic activity indicator MTT (A) or the cell division marker CCK-8 (B) in primary lung 481
microvascular endothelial cells (RLEC) exposed to increasing concentrations of nicotine or e-Cig 482
(C) solutions. Mean + SEM, n=3; One-way ANOVA (Tukey’s). 483
484
Figure 5. Oxidative stress induced by nicotine. A. Nitrotyrosine levels from the plasma of 485
C57Bl/6 mice nebulized with 1 dose of nicotine and harvested immediately. B-C. 8-OHdG levels in 486
plasma (B) or bronchoalveolar lavage fluid (BALF, C) of C57Bl/6 mice nebulized with 1 dose of 487
nicotine and collected immediately. Mean + SEM, n=3/ group, Student’s t test. D. ROS detection 488
(green) in RLEC exposed to nicotine (10mM; 30 min) with or without NAC (0.5 M) using Image-iT 489
LIVE Green Reactive Oxygen Species Detection Kit and DAPI staining of nuclei (blue). 490
491
Figure 6. Signaling in nicotine-induced endothelial barrier dysfunction. A. Normalized TER 492
measured in rat lung endothelial cells exposed to CS (10%) or to nicotine (15 mM) for the indicated 493
time (hours), and effect of the antioxidant N-acetylcysteine (NAC, 0.5 M, Mean + SEM, n = 2-12). 494
B. p38 MAPK activation by nicotine in lung endothelium detected by Western blotting for phospho- 495
and total p38 (α,β,γ,δ isoforms) in RLEC exposed to ambient air control extract (AC), CS extract 496
24
(CS), or nicotine solution at the indicated concentrations and time-points. Blot representative of n=3. 497
C. Normalized TER measured in RLEC exposed to nicotine (15 mM) for the indicated time (hr), and 498
effect of a p38 inhibitor (SB203580, 30 μM, Mean + SEM, n = 6-50) One-way ANOVA (Tukey’s). 499
D. MLCK activation detected by immunoblotting for phospho-MLC (Ser19) of RLEC following 500
exposure to nicotine solution (15 mM; 1 hr) in the absence or presence of the antioxidant NAC (0.5 501
M), the ERK-MAPK inhibitor PD98059 (PD; 50 µM), or the p38 inhibitor SB203580 (SB; 30 µM). 502
E. Myosin phosphatase inhibition detected by phosphorylation of myosin phosphatase target subunit 503
1 (MYPT1) in RLEC exposed to nicotine (15 mM) for the indicated time-points in the presence of 504
Rho kinase inhibitor Y29632 (Rho kinase inh), 3 µM. F. Normalized TER measured in RLEC 505
exposed to nicotine (10 mM) for the indicated time, and effect of a Rho kinase inhibitor (Y29632, 3 506
μM, Mean + SEM, n= 10. One-way ANOVA (Tukey’s). 507
508
Figure 7. Role of sphingolipids in cellular responses to nicotine. Ceramide and dihydroceramide 509
levels in RLEC following exposure to nicotine (15 mM; 4 hr; A-B) and in human lung epithelial 510
cells Beas-2B (C) following exposure to the indicated nicotine concentrations (mM) for the 511
indicated time. Mean + SEM, n=3, Student’s t-test. D. Effect of ceramide synthesis inhibitors 512
including that of ASMase with imipramine (Imi; 50 µM), of nSMase with GW4869 (GW; 15 μM), 513
of the de novo pathway with myriocin (My; 50 nM); or of ceramide synthases in the recycling 514
pathway with fumonisin B1 (FB1; 5 µM); or their respective vehicle controls dH2O (for FB1, Imi, 515
My) or DMSO (for GW). E. Normalized TER of RLEC exposed to nicotine (15 mM; 5 hr) and 516
impact of S1P receptor agonists (FTY phosphonate analogs 1S, 1R, 2S, 2R, 10 μM) or vehicle 517
(methanol). Mean+ SEM, n=15-45, Student’s t-test. F. Myosin phosphatase inhibition MLCK 518
activation and MLCK activation detected by phospho-Mypt1 (Thr 696) and phospho-MLC (Ser19) 519
25
immunoblotting followed by densitometry in RLEC exposed to nicotine (15 mM; 20 min) and 520
vehicle (methanol) or the indicated FTY720-analog (10 µM). Mean + SEM, n=3, One-way ANOVA 521
(Tukey’s). G. Cell proliferation measured with MTT in RLEC exposed to nicotine (15 mM) in the 522
presence or absence of S1P receptor agonists (FTY phosphonate analogs 1S, 1R, 2S, 2R, 10 μM) or 523
vehicle (methanol). Mean+SEM, n=3. H. TER of primary human lung microvascular cells exposed 524
to CS (3%) or air extract (3%) and attenuated with S1P receptor agonists (FTY phosphonate analogs 525
1S, 1R, 2S, 2R, 10 μM), in the presence or absence of S1PR1-specific siRNA. Mean+SEM, n=4-14; 526
ANOVA (Tukey’s). 527
528
Figure 8. Schematic of signaling events detected in lung endothelial cells exposed to nicotine. 529
Arrows indicate activation and blocked lines indicate inhibition. Nicotine activates Rho kinase, 530
which in turn inhibits the myosin phosphatase target subunit 1, MYPT1, enhancing phosphorylation 531
of myosin light chains (MLC-P) to increase endothelial permeability. Rho kinase may have other 532
targets in the cell to increase endothelial permeability, since nicotine-induced oxidative stress 533
(ROS)-dependent p38 MAPK activation also contributed to MLC phosphorylation, but not 534
sufficiently to alone increase permeability. Nicotine also increases the ceramide/sphingosine-1 535
phosphate (S1P) ratios, which may inhibit lung endothelial cell proliferation. Enhancing S1P 536
signaling opposes the decreased cell proliferation and the increase in permeability induced by 537
nicotine in part by inhibiting MLC phosphorylation, restoring the lung endothelial barrier function. 538
539
540
541
542
543
544
545
26
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... As previously reported by Hamza and El-shenawy (2017) the results showed that intraperitoneal nicotine injection induced a significant increase in serum MDA concentrations while decreasing GPx activity compared to the control group, giving the impression of induced oxidative stress. This result is in agreement with (Budzynska et al. 2013;Chattopadhyay 2016;Hamza and El-shenawy 2017;Schweitzer et al. 2021). Oxidative stress is characterized by the lower activity of antioxidant enzymes such as GPx. ...
... Enhanced ROS has resulted an increase in levels of MDA. A number of reports have shown that the administration of nicotine increases ROS generation within the lungs (Ahmad, et al. 2019;Schweitzer et al. 2021). ...
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Pod-based electronic (e-) cigarettes more efficiently deliver nicotine using a protonated formulation. The cardiovascular effects associated with these devices are poorly understood. We evaluated whether pod-based e-liquids and their individual components impair endothelial cell function. We isolated endothelial cells from people who are pod users (n = 10), tobacco never users (n = 7), and combustible cigarette users (n = 6). After a structured use, pod users had lower acetylcholine-mediated endothelial nitric oxide synthase (eNOS) activation compared with never users and was similar to levels from combustible cigarette users (overall P = 0.008, P = 0.01 pod vs never; P = 0.96 pod vs combustible cigarette). The effects of pod-based e-cigarettes and their constituents on vascular cell function were further studied in commercially available human aortic endothelial cells (HAECs) incubated with flavored JUUL e-liquids or propylene glycol (PG):vegetable glycerol (VG) at 30:70 ratio with or without 60 mg/mL nicotine salt for 90 min. A progressive increase in cell death with JUUL e-liquid exposure was observed across 0.0001–1% dilutions; PG:VG vehicle with and without nicotine salt induced cell death. A23187-stimulated nitric oxide production was decreased with all JUUL e-liquid flavors, PG:VG and nicotine salt exposures. Aerosols generated by JUUL e-liquid heating similarly decreased stimulated nitric oxide production. Only mint flavored e-liquids increased inflammation and menthol flavored e-liquids enhanced oxidative stress in HAECs. In conclusion, pod e-liquids and their individual components appear to impair endothelial cell function. These findings indicate the potential harm of pod-based devices on endothelial cell function and thus may be relevant to cardiovascular injury in pod type e-cigarette users.
... Nikotin, salah satu senyawa aktif utama asap rokok, juga telah terbukti me miliki efek buruk pada sistem kardiovaskular dan telah di dokumentasikan bahwa nikotin dengan konsentrasi yang sama dengan yang ditemukan dalam darah perokok dapat mengubah metabolisme lipid dan merusak fungsi endotel pada hewan (Liu et al., 2017). Mekanisme nikotin memicu respon sel endotel sistemik dan melibatkan peningkatan molekul pensinyalan nitrit oksida (NO) dan spesies oksigen reaktif (ROS) serta pembentukan metabolit proapoptosis, (Schweitzer et al., 2015). ...
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Novel therapeutic strategies are needed to reverse the loss of endothelial cell (EC) barrier integrity that occurs during inflammatory disease states such as acute lung injury. We previously demonstrated potent EC barrier augmentation in vivo and in vitro by the platelet-derived phospholipid, sphingosine 1-phosphate (S1P) via ligation of the S1P1 receptor. The S1P analogue, FTY720, similarly exerts barrier-protective vascular effects via presumed S1P1 receptor ligation. We examined the role of the S1P1 receptor in sphingolipid-mediated human lung EC barrier enhancement. Both S1P and FTY-induced sustained, dose-dependent barrier enhancement, reflected by increases in transendothelial electrical resistance (TER), which was abolished by pertussis toxin indicating Gi-coupled receptor activation. FTY-mediated increases in TER exhibited significantly delayed onset and intensity relative to the S1P response. Reduction of S1P1R expression (via siRNA) attenuated S1P-induced TER elevations whereas the TER response to FTY was unaffected. Both S1P and FTY rapidly (within 5 min) induced S1P1R accumulation in membrane lipid rafts, but only S1P stimulated S1P1R phosphorylation on threonine residues. Inhibition of PI3 kinase activity attenuated S1P-mediated TER increases but failed to alter FTY-induced TER elevation. Finally, S1P, but not FTY, induced significant myosin light chain phosphorylation and dramatic actin cytoskeletal rearrangement whereas reduced expression of the cytoskeletal effectors, Rac1 and cortactin (via siRNA), attenuated S1P-, but not FTY-induced TER elevations. These results mechanistically characterize pulmonary vascular barrier regulation by FTY720, suggesting a novel barrier-enhancing pathway for modulating vascular permeability.
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Lung inflammation and alterations in endothelial cell (EC) micro- and macrovascular permeability are key events to development of acute lung injury. Using ECs derived from human pulmonary artery and lung microvasculature, we investigated the interplay between p38 stress mitogen-activated protein kinase (MAPK) and Rho guanosine triphosphatase signaling in inflammatory and hyperpermeability responses. Both cell types were treated with Staphylococcus aureus-derived peptidoglycan (PepG) and lipoteichoic acid (LTA) with or without pretreatment with p38 MAPK or Rho kinase inhibitors. LTA and PepG increased permeability markedly in both pulmonary macrovascular and microvascular ECs. Agonist-induced hyperpermeability was accompanied by cytoskeletal remodeling, disruption of cell-cell contacts, formation of paracellular gaps, and activation of p38 MAPK, nuclear factor kappa-B (NFκB), and Rho/Rho kinase signaling. In macrovascular ECs, pharmacologic inhibition of Rho kinase with Y27632 suppressed p38 MAP kinase cascade activation significantly, whereas inhibition of p38 MAPK with SB203580 had no effect on Rho activation. In contrast, inhibition of p38 MAPK in microvascular ECs suppressed LTA/PepG-induced activation of Rho, whereas the Rho inhibitor suppressed activation of p38 MAPK. Inhibition of either p38 MAPK or Rho kinase attenuated activation of NFκB signaling substantially. These results demonstrate cell-type-specific differences in signaling induced by Staphylococcus aureus-derived pathogens in pulmonary endothelium. Thus, although Gram-positive bacterial compounds caused barrier dysfunction in both EC types, it was induced by a different pattern of crosstalk between Rho, p38 MAPK, and NFκB signaling. These observations may have important implications in defining microvasculature-specific therapeutic strategies aimed at the treatment of sepsis and acute lung injury induced by Gram-positive bacterial pathogens.
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A sensitive and specific ultra performance liquid chromatography-tandem mass spectrometry method for the simultaneous quantification of nicotine, its metabolites cotinine and trans-3'-hydroxycotinine and varenicline in human plasma was developed and validated. Sample preparation was realized by solid phase extraction of the target compounds and of the internal standards (nicotine-d4, cotinine-d3, trans-3'-hydroxycotinine-d3 and CP-533,633, a structural analog of varenicline) from 0.5 mL of plasma, using a mixed-mode cation exchange support. Chromatographic separations were performed on a hydrophilic interaction liquid chromatography column (HILIC BEH 2.1×100 mm, 1.7 μm). A gradient program was used, with a 10 mM ammonium formate buffer pH 3/acetonitrile mobile phase at a flow of 0.4 mL/min. The compounds were detected on a triple quadrupole mass spectrometer, operated with an electrospray interface in positive ionization mode and quantification was performed using multiple reaction monitoring. Matrix effects were quantitatively evaluated with success, with coefficients of variation inferior to 8%. The procedure was fully validated according to Food and Drug Administration guidelines and to Société Française des Sciences et Techniques Pharmaceutiques. The concentration range was 2-500 ng/mL for nicotine, 1-1000 ng/mL for cotinine, 2-1000 ng/mL for trans-3'-hydroxycotinine and 1-500 ng/mL for varenicline, according to levels usually measured in plasma. Trueness (86.2-113.6%), repeatability (1.9-12.3%) and intermediate precision (4.4-15.9%) were found to be satisfactory, as well as stability in plasma. The procedure was successfully used to quantify nicotine, its metabolites and varenicline in more than 400 plasma samples from participants in a clinical study on smoking cessation.