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