lawrence hightower
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TRANSCRIPT
Hyperbaric Oxygen Therapy: cytoprotection and
stress conditioning
Larry Hightower, University of Connecticut
Oxygen Delivery Oxygen Delivery
Hyperbaric Hyperbaric EffectEffect
O2 Bound to
Hemoglobin
Dissolved O2
30
20
10
Blo
od
OB
loo
d O
22C
on
ten
t (m
L/d
L)
Co
nte
nt
(mL
/dL
)
100 660 1320 1980
PaOPaO2 2 (mmHg)(mmHg)
3 ATA2 ATA1 ATA
= 5 vol%
= 1,584
2.4ATA
x
Hemoglobin
HBOT Pre-treatments Decrease Post-Coronary Artery By-pass Graft
Cognitive Decline
Alex, J. et al. Jor Thorac Cardiovas Surg 2005;130:1623.
60 min
24, 12, 4 hrs
before CABG
48hrs post op
p = 0.05
0
10
20
30
40
50
60
Control HBOT
% C
og
nit
ive
de
clin
e
Cell Culture Model:
Human Microvascular Endothelial Cell Line: HMEC-1
Control HBOT
1X
2X
HBOT stimulates vascular tube formation on Matrigel
HBOT protects against lethal heat shock
0 200 400 600 800 1000Size of Cells
100
101
102
103
104
PI
40.2
59.8
47C
0 200 400 600 800 1000Size of Cells
100
101
102
103
104
PI
94.8
5.24
HBOT+ 47C
47C PI staining
Flow Cytometry
24 hrs� 1 hr
HBOT
24 hrs�
Recovery
37C
Protection Against Oxidative Stress
48h 16h 4h
Cells
plated
HBOT/
100% O2
t-bOOH MTT
HBOT Project
UCONN-OxyHeal
OxyCure 3000 DNA Microarray Technology
Human Microvascular Endothelial cell line (HMEC-1)
HBOT 1h, 2.4 atm,
100% O2
No Treatment Control
24h Recovery 37C, 5% CO2
Immediate RNA
Extraction
Illumina Microarray Analysis
RNA Extraction
EXPERIMENTAL
APPROACH
RESULTS 38,275 transcripts
on Microarray
8,181 regulated genes
Statistical Analysis
Dr. Dong-Guk Shin
OFFICIAL
SYMBOL GeneID HBOT0:CNTL0
FOSB 2354 38.7
MT1X 4501 34.69
MT1G 4495 29.5
FOS 2353 24.57
NR4A2 4929 22.72
RASD1 51655 16.72
EGR1 1958 11.74
EGR2 1959 11.25
SERTAD1 29950 10.6
SNF1LK 54018 10.23
MT1H 4496 10.2
MT1M 4499 9.25
JUNB 3726 8.92
MT1F 4494 8.63
AXUD1 64651 8.5
HES1 3280 7.81
SLC30A1 7779 7.43
BHLHB2 8553 7.42
MT1E 4493 6.42
KLF10 7071 6.34
MT1P3 140851 6.28
IL11 3589 5.69
ACTC1 70 5.17
OFFICIAL
SYMBOL GeneID HBOT0:CNTL0
GDF15 9518 -8.87
KLHL24 54800 -6.7
C5orf26 114915 -5.76
ABCA1 19 -5.43
PSG4 5672 -5.36
HSPA5 3309 -5.07
OFFICIAL
SYMBOL GeneID HBOT24: CNTL24
MT1H 4496 50
MT1X 4501 16.67
MT1F 4494 12.5
LIPG 9388 12.5
MT1P3 140851 8.33
MT1E 4493 7.14
ENDOGL1 9941 5.88
SLCO2A1 6578 5.26
GAL 51083 5
Top-responding genes immediately
up-regulated after HBOT Top-responding genes up-
regulated 24-hours after HBOT
Immediate early genes: FOSB, FOS, JUNB
regulate cell proliferation
Metallothioneins (MT1X, MT1G) help protect cells
from oxidative damage
Down-regulated
HSPA5 (Bip) is the ER form of HSP70
involved in protein folding
Gene Symbol GeneID HBOT24:CNTL24
DNAJC11 55735 2.04
HSPA14 51182 1.96
HSPE1 3336 1.96
HSPH2 3308 1.92
HSPH1 10808 1.82
HSPC3 3326 1.72
DNAJC7 7266 1.59
DNAJC17 55192 1.54
DNAJC3 5611 1.54
DNAJC5 80331 1.47
DNAJC19 131118 1.45
HSPD1 3329 1.45
DNAJA1 3301 1.39
HSPA8 3312 1.27
HSPA1A 3303 -1.39
DNAJB11 51726 -1.4
HSPA2 3306 -1.4
HSPBAP1 79663 -1.58
DNAJC18 202052 -1.61
HSP90B1 7184 -1.69
DNAJB9 4189 -3.4
HSPA5 3309 -5.07
Gene Symbol GeneID HBOT0:CNTL0
DNAJB1 3337 1.83
HSPA1B 3304 1.81
HSPA1A 3303 1.73
HSPH1 10808 1.49
HSPA2 3306 1.34
DNAJA4 55466 1.32
Molecular Chaperones Affected by HBOT
Immediately after HBOT 24-hours after HBOT
•Majority induced by protein damage
•Small fold-increases and
•No induction of HSPA6 indicates minor protein damage
•DNAJC proteins involved in Mt protein-import
induced
•Mt matrix chaperonins induced (HSPE1, HSPD1)
•HSPA1A (HSP70) transient induction
•Down-regulation of ER/Golgi pathway (DNAJB11,
DNAJB9, HSPA5, HSP90B1)
Keap1
SH SH
Nrf2
Cul3 Nrf2
Ub
Ub
Ub
Ub
Ub
Proteasome
ARE
sMAF
CBP/p300
TARGET GENES •Antioxidants •Xenobiotic metabolism •Glutathione homeostasis •DNA damage recognition •Proteasome function •Inhibition of inflammation
Nrf2 Signaling pathway
nucleus
cytoplasm
HBOT increases cell proliferation
2 Hrs post-HBOT 6 Hrs post-HBOT
In vitro wound closure using HMEC-1 cells
Summary
•HBOT may be useful as a pre-conditioning treatment before major surgeries
•HBOT increases/decreases gene expression in HMEC-1 cells
•HBOT as a promoter of rapid recovery from injury:
•Stimulates protection against oxidative stress
•Anti-inflammatory gene activation
•HBOT stimulates angiogenesis, cell proliferation and migration
in vivo and in vitro: a likely mechanism for wound healing by HBOT
Acknowledgements:
Dr. Cassandra Tierney
Dr. Charles Giardina
Dr. George Perdrizet