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Role of Reactive Oxygen Species in Septic Shock Richard E. Richard E. Klabunde Klabunde , Ph.D. , Ph.D. Associate Professor of Physiology Associate Professor of Physiology OUCOM OUCOM 27 August 2002 27 August 2002

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Page 1: Role of Reactive Oxygen Species in Septic Shock Sepsis 082702.p… · Summary of Role of NO in Septic Shock • Increased NO production decreases SVR and leads to hypotension. •

Role of Reactive Oxygen Species in Septic Shock

Richard E.Richard E. KlabundeKlabunde, Ph.D., Ph.D.Associate Professor of PhysiologyAssociate Professor of Physiology

OUCOMOUCOM27 August 200227 August 2002

Page 2: Role of Reactive Oxygen Species in Septic Shock Sepsis 082702.p… · Summary of Role of NO in Septic Shock • Increased NO production decreases SVR and leads to hypotension. •

Outline

• General introduction and definitions• Pathophysiology of shock• Pathogenesis of shock• Current research and potential new

therapies– Nitric oxide – Reactive oxygen species– Peroxynitrite

Page 3: Role of Reactive Oxygen Species in Septic Shock Sepsis 082702.p… · Summary of Role of NO in Septic Shock • Increased NO production decreases SVR and leads to hypotension. •

Sepsis

• Systemic inflammatory response to a confirmed infectious process (most commonly caused by bacterial products (e.g., endotoxin)

Page 4: Role of Reactive Oxygen Species in Septic Shock Sepsis 082702.p… · Summary of Role of NO in Septic Shock • Increased NO production decreases SVR and leads to hypotension. •

Severe Sepsis

• Sepsis with either hypotension or systemic manifestations ofhypoperfusion– Lactic acidosis, oliguria, altered mental

status

Page 5: Role of Reactive Oxygen Species in Septic Shock Sepsis 082702.p… · Summary of Role of NO in Septic Shock • Increased NO production decreases SVR and leads to hypotension. •

Septic Shock

• Cardiovascular dysfunction associated with sepsis resulting in hypotension and organ hypoperfusion despite adequate fluid resuscitation

Page 6: Role of Reactive Oxygen Species in Septic Shock Sepsis 082702.p… · Summary of Role of NO in Septic Shock • Increased NO production decreases SVR and leads to hypotension. •

Endotoxemia & Endotoxic Shock

• Endotoxemia– Elevated levels of bacterial endotoxins (e.g.,

lipopolysaccharide, LPS) in the blood.

• Endotoxic Shock– Similar to septic shock (hypotension and

organ hypoperfusion despite fluids), but with specific involvement of bacterial endotoxins.

New Slide

Page 7: Role of Reactive Oxygen Species in Septic Shock Sepsis 082702.p… · Summary of Role of NO in Septic Shock • Increased NO production decreases SVR and leads to hypotension. •

Systemic Inflammatory Response Syndrome (SIRS)

• A more general, inclusive term• Systemic inflammatory response to a variety

of severe clinical insults (e.g., infection, burns, trauma, pancreatitis)

Page 8: Role of Reactive Oxygen Species in Septic Shock Sepsis 082702.p… · Summary of Role of NO in Septic Shock • Increased NO production decreases SVR and leads to hypotension. •

Multiple Organ Dysfunction Syndrome (MODS)

• Progressive distant organ failure following severe infectious or noninfectious insults

Page 9: Role of Reactive Oxygen Species in Septic Shock Sepsis 082702.p… · Summary of Role of NO in Septic Shock • Increased NO production decreases SVR and leads to hypotension. •

Morbidity/Mortality of Sepsis and Septic Shock

• Most common cause of death in ICUs• U.S. cases per year:

– Sepsis = 400,000– Septic Shock = 200,000– Death = 100,000

J.E. Parrillo, N. Eng. J. Med. 328:1471-1477, 1993

Page 10: Role of Reactive Oxygen Species in Septic Shock Sepsis 082702.p… · Summary of Role of NO in Septic Shock • Increased NO production decreases SVR and leads to hypotension. •

Pathophysiology of Septic Shock

General Clinical Signs

• Flu-like symptoms– chills followed by fever– general malaise, irritability, lethargy, mental

confusion• Warm skin (early sign)• Site of infection may or may not be evident

Page 11: Role of Reactive Oxygen Species in Septic Shock Sepsis 082702.p… · Summary of Role of NO in Septic Shock • Increased NO production decreases SVR and leads to hypotension. •

Pathophysiology Cont.

Cardiovascular• Systemic vasodilation and hypotension (Psys < 90

mmHg); increased SVR in late stages• Tachycardia (>100 beats/min)• Increased cardiac output (hyperdynamic),

although contractility is depressed; hypodynamic in late shock

• Ventricular dilation; decreased ejection fraction • Loss of sympathetic responsiveness

Page 12: Role of Reactive Oxygen Species in Septic Shock Sepsis 082702.p… · Summary of Role of NO in Septic Shock • Increased NO production decreases SVR and leads to hypotension. •

Pathophysiology Cont.

CardiovascularCont.

• Hypovolemia due to vascular leakage; central venous pressure may be decreased or increased depending upon fluid resuscitation

• Compromised nutrient blood flow to organs; decreased organ oxygen extraction

Page 13: Role of Reactive Oxygen Species in Septic Shock Sepsis 082702.p… · Summary of Role of NO in Septic Shock • Increased NO production decreases SVR and leads to hypotension. •

Cardiogenic Shock

Hemorrhagic Shock

Septic Shock

Primary CV Origin

Cardiac Volume Vascular

Cardiac Output

↓ ↓ ↑↓

Vascular Resistance

↑ ↑ ↓↑

Blood Volume

↑ ↓ ↓

Management Mechanical Inotropes

Vasopressors Vasodilators

IV Fluids/Blood Vasopressors

IV Fluids Antibiotics

Vasopressors Inotropes

Page 14: Role of Reactive Oxygen Species in Septic Shock Sepsis 082702.p… · Summary of Role of NO in Septic Shock • Increased NO production decreases SVR and leads to hypotension. •

Pathophysiology Cont.

Pulmonary & Renal

• Hyperventilation with respiratory alkalosis• Pulmonary hypertension and edema• Hypoxemia (arterial pO2 < 50 mmHg) • Reduced pulmonary compliance; increased work• Respiratory muscle failure • Renal hypoperfusion and oliguria despite

elevated cardiac output• Acute tubular necrosis and renal failure

Page 15: Role of Reactive Oxygen Species in Septic Shock Sepsis 082702.p… · Summary of Role of NO in Septic Shock • Increased NO production decreases SVR and leads to hypotension. •

Pathophysiology Cont.

Other

• Disseminated intravascular coagulation (DIC)• Blood dyscrasias

– leukopenia– thrombocytopenia– polycythemia

• Central and peripheral nervous dysfunction• Increased lactate occurs early

Page 16: Role of Reactive Oxygen Species in Septic Shock Sepsis 082702.p… · Summary of Role of NO in Septic Shock • Increased NO production decreases SVR and leads to hypotension. •

Pathogenesis of Septic Shock

Infectious triggers

Cytokine and inflammatory mediator cascade

Cardiac dysfunction and microvascular injury

Hypotension and shock

Revised Slide

Page 17: Role of Reactive Oxygen Species in Septic Shock Sepsis 082702.p… · Summary of Role of NO in Septic Shock • Increased NO production decreases SVR and leads to hypotension. •

Bacterial-Mediated Sepsis(Gram-negative)

Bacteria MacrophageLPS + LBP

+death

Mediatorsof

InflammationLPS = LipopolysaccharideLPS = Lipopolysaccharide

LBP = LPSLBP = LPS--Binding ProteinBinding Protein

CD14

Page 18: Role of Reactive Oxygen Species in Septic Shock Sepsis 082702.p… · Summary of Role of NO in Septic Shock • Increased NO production decreases SVR and leads to hypotension. •

VasodilationCardiac DepressionMicrovascular LeakagePlatelet AggregationLeukocyte Adhesion

ENDOTOXIN

Tumor Necrosis FactorTumor Necrosis FactorInterleukinsInterleukins

Gamma InterferonGamma InterferonPlatelet Activating FactorPlatelet Activating Factor

LeukotrienesLeukotrienesThromboxanesThromboxanesProstaglandinsProstaglandins

HistamineHistamineNitric OxideNitric Oxide

Oxygen Free RadicalsOxygen Free Radicals

Page 19: Role of Reactive Oxygen Species in Septic Shock Sepsis 082702.p… · Summary of Role of NO in Septic Shock • Increased NO production decreases SVR and leads to hypotension. •

Nitric Oxide Formation

EndotoxinCytokines

NO + citrulline

AcetylcholineBradykininSubstance-PInsulin

iNOS cNOS

L-arg

NO

L-arg

++

cGMP

GTP

GC+

Ca++

+ R

Page 20: Role of Reactive Oxygen Species in Septic Shock Sepsis 082702.p… · Summary of Role of NO in Septic Shock • Increased NO production decreases SVR and leads to hypotension. •

Actions of Nitric Oxide

• Vasodilation (direct via cGMP and indirect via inhibition of NE and ET-1 release)

• Inhibits leukocyte-endothelial cell adhesion

• Inhibits platelet adhesion/aggregation• Modulates vascular permeability• Scavenges superoxide radicals• High concentrations are cytotoxic

Page 21: Role of Reactive Oxygen Species in Septic Shock Sepsis 082702.p… · Summary of Role of NO in Septic Shock • Increased NO production decreases SVR and leads to hypotension. •

What role does NO play inseptic shock?

Page 22: Role of Reactive Oxygen Species in Septic Shock Sepsis 082702.p… · Summary of Role of NO in Septic Shock • Increased NO production decreases SVR and leads to hypotension. •

NO Formation in Septic ShockN

O P

rodu

ctio

n

0 12 24Time (hours) Post-Endotoxemia

cNOS

iNOS

Page 23: Role of Reactive Oxygen Species in Septic Shock Sepsis 082702.p… · Summary of Role of NO in Septic Shock • Increased NO production decreases SVR and leads to hypotension. •

Pretreatment with NOS Inhibitors

• Hypothesis: – Increased NO production during sepsis causes

systemic vasodilation, cardiac depression, increased capillary permeability (leading to decreased blood volume) and hypotension.

• Therefore:– Pretreatment with a NOS inhibitor in animal models

of sepsis should prevent NO-induced hypotension and edema.

– Treatment with a NOS inhibitor during sepsis should reverse cardiovascular changes.

Page 24: Role of Reactive Oxygen Species in Septic Shock Sepsis 082702.p… · Summary of Role of NO in Septic Shock • Increased NO production decreases SVR and leads to hypotension. •

80

100

120

140

160M

ean

Arte

rial P

ress

ure

(mm

Hg)

Effects of Pretreatment with NOS Inhibitor

Saline

Saline

Saline

SalineLPS LPS LPS LPSLPS

NNA_H NNA_L AG DEX NNA_H

8

7

6 5

5

4

*

*6 Hours Post-LPS

8

1 2 3 4 5 6 7

Klabunde and Coston. Shock 3:73-78, 1995.

Page 25: Role of Reactive Oxygen Species in Septic Shock Sepsis 082702.p… · Summary of Role of NO in Septic Shock • Increased NO production decreases SVR and leads to hypotension. •

NO and Microvascular Leakage

0 30 60 90 120Time (min) Post-LPS

-40

0

40

80

120

160

200FI

TC L

eaka

ge (%

Cha

nge)

Control (5)

LPS (14)

DEX+LPS (4)

L-NAME+LPS (7)

PAF Antg + LPS (4)

Laniyonu, Coston and Klabunde. Shock 7:49-54, 1997.

Page 26: Role of Reactive Oxygen Species in Septic Shock Sepsis 082702.p… · Summary of Role of NO in Septic Shock • Increased NO production decreases SVR and leads to hypotension. •

Other Studies

• Pretreatment with NOS inhibitors:– Prevents fall in systemic vascular resistance– Prevents arteriolar hyporesponsiveness to

catecholamines

Page 27: Role of Reactive Oxygen Species in Septic Shock Sepsis 082702.p… · Summary of Role of NO in Septic Shock • Increased NO production decreases SVR and leads to hypotension. •

Therapeutic Efficacy of NOS Inhibitors in Endotoxic Shock

0 50 100 150 200 250

Time (min)

-100

-50

0

50

100

150Pe

rcen

t Cha

nge

MAPCOSVRFBF

NMA30 mg/kg

LPS (E. coli), 250 ng/kg/min

Klabunde and Ritger. Biophys Biochem Res Commun 178:1135-1140, 1991.

Page 28: Role of Reactive Oxygen Species in Septic Shock Sepsis 082702.p… · Summary of Role of NO in Septic Shock • Increased NO production decreases SVR and leads to hypotension. •

Summary of Role of NO in Septic Shock

• Increased NO production decreases SVR and leads to hypotension.

• Non-specific NOS inhibition restores arterial pressure, but reduces cardiac output and organ perfusion

• NO is involved in microvascular leakage during endotoxemia

• Other studies:– NOS inhibition causes pulmonary dysfunction and

increases mortality.– Selective iNOS inhibition being investigated for

therapeutic potential; non-selective inhibitors increase mortality

– Clinical studies show no benefit on survival

Page 29: Role of Reactive Oxygen Species in Septic Shock Sepsis 082702.p… · Summary of Role of NO in Septic Shock • Increased NO production decreases SVR and leads to hypotension. •

Are nitric oxide inhibitors good or bad in septic shock?

• Bad – NOS inhibitors increase– Cardiac depression and organ hypoperfusion– Thrombosis– Leukocyte-endothelial adhesion and inflammation– Superoxide anion (NO normally scavenges superoxide)– Increase mortality in many studies

• Good – NOS inhibitors prevent– Excessive vasodilation and hypotension– Possible cardiac depression– Formation of new, damaging free radical species (e.g.,

peroxynitrite)

New Slide

Page 30: Role of Reactive Oxygen Species in Septic Shock Sepsis 082702.p… · Summary of Role of NO in Septic Shock • Increased NO production decreases SVR and leads to hypotension. •

Formation of Reactive Oxygen Species

CytokinesEndotoxins

⋅⋅OO22--

H2O2 ⋅OH-H2O

SOD

CatalaseCellular

Damage &Dysfunction

VasodilationNegative inotropy

Microvascular leakage

NAD(P)H oxidasesCyclooxygenase

Xanthine OxidaseNitric Oxide Synthase

HOCl

MPO

GSHpx

MacrophagesNeutrophils

Endothelial CellsMyocytes

etc.

Page 31: Role of Reactive Oxygen Species in Septic Shock Sepsis 082702.p… · Summary of Role of NO in Septic Shock • Increased NO production decreases SVR and leads to hypotension. •

Free Radicals in Septic Shock

• Reactive oxygen species (ROS) are elevated in sepsis– In rat models of acute endotoxemia,

superoxide anion increases 3-fold within 60 min and plateaus after about 4 hours (Brovkovych et al, J Physiol Pharmacol 48:633, 1997)

Page 32: Role of Reactive Oxygen Species in Septic Shock Sepsis 082702.p… · Summary of Role of NO in Septic Shock • Increased NO production decreases SVR and leads to hypotension. •

Free Radicals in Septic Shock cont.

• Endogenous antioxidant and scavenging systems can become depleted in severe sepsis, particularly– Alpha-tocopherol– Ascorbic acid– β-carotene

• Survival in septic patients with organ dysfunction is inversely correlated with the reduction in plasma antioxidant potential (Cowley et al., Crit Care Med 24:1179, 1996)

Page 33: Role of Reactive Oxygen Species in Septic Shock Sepsis 082702.p… · Summary of Role of NO in Septic Shock • Increased NO production decreases SVR and leads to hypotension. •

Free Radicals in Septic Shock cont.

• In animal studies, treatment with ROS scavengers can improve clinical status and decrease mortality

• Animal studies suggest that the following treatments may improve outcome:– N-acetyl-L-cysteine (scavenges hydroxyl radical and

hypochlorus acid; replenishes glutathione)

– Alpha-tocopherol– Ascorbic acid (treatment during bacteremia in rats prevents

hypotension - Armour et al., J Appl Physiol 90:795, 2001)

– TEMPO (SOD mimic)

Page 34: Role of Reactive Oxygen Species in Septic Shock Sepsis 082702.p… · Summary of Role of NO in Septic Shock • Increased NO production decreases SVR and leads to hypotension. •

-10 0 10 20 30 40 50 60 70 80

Time (hr)

0

1

2

3

4

5

Sum

med

Mor

bidi

ty In

dex

Combined Morbidity* of Endotoxic Rats(LPS = 15 mg/kg, ip)

(9/15 Survived)

(8/16 Survived)

Control

Alpha-tocopherolAscorbic acidN-acetyl-cysteine

*Responses Included:Uprighting

LocomotoryToe Pinch

Page 35: Role of Reactive Oxygen Species in Septic Shock Sepsis 082702.p… · Summary of Role of NO in Septic Shock • Increased NO production decreases SVR and leads to hypotension. •

Effects of ROS Scavenging on LPS-Induced Microvascular Leakage

Page 36: Role of Reactive Oxygen Species in Septic Shock Sepsis 082702.p… · Summary of Role of NO in Septic Shock • Increased NO production decreases SVR and leads to hypotension. •

Unresolved Issues with Antioxidant Therapy

• Is there one antioxidant that is best or should a cocktail be use?

• What is the therapeutic window for antioxidant therapy?

• Do antioxidants increase survival?• Is there a therapeutic role for inhibitors on ROS

production? e.g., xanthine oxididase inhibitors (allopurinol), NAD(P)H oxidase inhibitors

Page 37: Role of Reactive Oxygen Species in Septic Shock Sepsis 082702.p… · Summary of Role of NO in Septic Shock • Increased NO production decreases SVR and leads to hypotension. •

Nitric Oxide and Superoxide Anion Interactions

⋅⋅OO22--

ONOO-

L-Arg

H2O2

··NONO

⋅OH-H2O

+

SOD

Catalase

NOS

HOCl

MPO

O2

NAD(P)H oxidase,XO, COX, NOS

Sepsis(Peroxynitrite)

Page 38: Role of Reactive Oxygen Species in Septic Shock Sepsis 082702.p… · Summary of Role of NO in Septic Shock • Increased NO production decreases SVR and leads to hypotension. •

Superoxide Scavenging of NO and the Formation of Peroxynitrite

• Increased ROS decreases NO, which may contribute to:– Leukocyte and platelet adhesion (leading to vascular

plugging)– Vasoconstriction (leading to impaired perfusion)

• Peroxynitrite formation can cause vasodilation and cardiac depression, as well as cellular damage in general

• Decreasing ROS by antioxidants, for example, may enhance hypotension by increasing NO bioavailability.– Therefore, therapy may need to be directed against both

ROS and NO.

Page 39: Role of Reactive Oxygen Species in Septic Shock Sepsis 082702.p… · Summary of Role of NO in Septic Shock • Increased NO production decreases SVR and leads to hypotension. •

Nitric Oxide Contributes to ROS-Induced Microvascular Leakage

0

20

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60

80

100M

ax L

eaka

ge (%

TPI)

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Klabunde and Anderson, J Vasc Res 39:338, 2002.

Page 40: Role of Reactive Oxygen Species in Septic Shock Sepsis 082702.p… · Summary of Role of NO in Septic Shock • Increased NO production decreases SVR and leads to hypotension. •

Peroxynitrite Formation Contributes to PAF-Induced Microvascular

Leakage

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ax L

eaka

ge (%

TPI)

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Klabunde and Anderson, J Vasc Res 39:338, 2002.

Page 41: Role of Reactive Oxygen Species in Septic Shock Sepsis 082702.p… · Summary of Role of NO in Septic Shock • Increased NO production decreases SVR and leads to hypotension. •

Summary

• Cardiovascular manifestations of septic shock include:– Cardiac depression– Systemic vasodilation, hypovolemia and hypotension– Reduced organ perfusion

• NO and ROS are implicated in contributing to these cardiovascular changes

• ROS scavengers and selective iNOS inhibitors may provide a new therapeutic approach to improving survival in septic patients