the role of nitric oxide in cellular aging: telomeres, mitochondria and stem cells nathan s. bryan,...
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The Role of Nitric Oxide in Cellular Aging:Telomeres, Mitochondria and Stem Cells
Nathan S. Bryan, Ph.D.Texas Therapeutics Institute
ACIM October 24-25, 2013
Structure of Presentation
What is Regenerative Medicine
Current Aging/Disease Hypotheses
Brief Overview of Nitric Oxide (NO)
NO effects on telomerase, mitochondria and stem cells
Strategies to diagnose and replete NO
Disclosure: N.S. Bryan is the Founder and Chief Science Officer of NeoGenis Labs, Inc.
Good Medicine – Applied Physiology
Bad Medicine – Applied Pharmacology
Chronic Diseasesaccount for 61% of deaths worldwide.
Most of these preventableby diet and lifestylemodifications.
Regenerative Medicine
process of replacing, engineering or regenerating human cells, tissues or organs to restore or establish normal function. This field holds the promise of engineering damaged tissues and organs via stimulating the body's own repair mechanisms to functionally heal previously irreparable tissues or organs.
What is Aging?
Aging is the biological consequence of our body’s inability to make new cells that work properly.
Jerry Tennant MD
1. What is needed to make new cells?
2. What makes them work properly?
What is needed to make new cells?
1. Fats and cholesterol for cell membrane20% of your body is fat (need the right fats)
2. Amino acidsneeded to make the protein machinery inside the cells
3. Vitamins and mineralsallow the body to make the fats and proteins work
What causes aging and is involvedIn regenerative medicine?
Three main hypotheses:
1. Telomere shortening2. Mitochondrial dysfunction3. Loss of stem cell function and repair
Unified Theory of Aging
Nitric Oxide controls and regulates1. Telomerase activity2. Mitochondrial biogenesis and function3. Mobilization of resident stem cells
Telomere Theory of Aging
A decrease in telomerase activity precedes telomere shortening andintroduction of telomerase into normal human cells extends life-span .Bodnar et al. Science 1998 Jan 16;279(5349):349-52.
On the cellular level, senescence, chromosome stability, and cell viability are regulated by the telomeres and their associated proteins, deoxyribonucleic acid-protein complexes located at both ends of eukaryotic chromosomes Blasco Nature Reviews Genetics 6, 611-622 (August 2005)
Shortening of the telomeres has been shown to be associated with increased mortality rate from age related diseases. Individuals with shorter telomeres had a mortality rate nearly twice that of those with longer telomeresCawthon et al Lancet 2003; 361: 393–95
Nitric Oxide Activates Telomerase and Delays Endothelial Cell Senescence
NO interferes with telomerase activity thereby inhibiting telomere shortening. The mechanism by which NO stimulates telomerase activity remains to be determined. Vasa et al Circulation Research. 2000; 87: 540-542
eNOS activity is required for hTERT expression and is dependent uponNO production. eNOS knockout mice lose regulation of telomerase activitythat is rescued by exogenous NO donorsGrasselli et al Circulation Research. 2008 Jul 3;103(1):34-42
NO is the master signaling molecule in the regulation of telomerase activityand provides the opportunity for innovative therapeutic approaches based onthe use of NO active compoundsFarsetti et al J. Appl Physiol. 2009 Jan;106(1):333-7
Schematic illustration of the mechanism involved in estrogen receptor- and endothelial nitric oxide synthase (eNOS)-induced hTERT transcription.
Farsetti A et al. J Appl Physiol 2009;106:333-337
Mitochondrial Theory of Aging
1. Free radicals play a major role in aging and most are mitochondrially produced.
2. Mitochondrial DNA lacks the protective DNA-binding protein (called histones), has less efficient DNA repair, and is close to the free radical-producing ETC, resulting in high levels of mitochondrial DNA damage compared to nuclear DNA.
3. Caloric restriction, the only known treatment to increase the mammalian lifespan, reduces mitochondrial free radical production and mitochondrial DNA oxidative damage.
4. Fibroblasts injected with mitochondria from old rats degenerate more rapidly than fibroblasts injected with mitochondria from young animals.
5. The mitochondria of older mammals are often larger and less efficient than those from younger mammals.
6. Targeted increased mitochondrial catalase (an enzymatic anti-oxidant) expression increases the mouse lifespan by around 20%.
7. Studies between different species have demonstrated that longer-lived species typically have lower mitochondrial DNA oxidative damage and lower free radical production.
8. Targeted mutation of the mitochondrial DNA polymerase-g, which causes an increased mitochondrial mutation rate with aging, results in a premature aging phenotype.
Mitochondria – Cellular Power Plants
Mitochondria are found in nearly all eukaryotes. They vary in number and location according to cell type. A single mitochondrion is often found in unicellular organisms. Conversely, numerous mitochondria are found in human liver cells, with about 1000–2000 mitochondria per cell, making up 1/5 of the cell volume.
Generate ATP, used as a source of chemical energy
Involved in cell signalingHeat ProductionCellular metabolismCellular differentiationCell deathControl of the cell cycle (cancer)Cell growthSteroid synthesis
Mitochondria have been implicated in many human diseases and arecritical in the aging process.
Nitric Oxide Controls and RegulatesMitochondrial:
ATP synthesisReactive Oxygen SpeciesCell SignalingApoptosis (Cell Cycle)BiogenesisMetabolism/Bioenergetics
Nitric Oxide and Mitochondrial Biogenesis
Nisoli E et al. Circulation Research 2007;100:795-806
Copyright © American Heart Association
Nitrite-dependent extension of oxygen gradients. (A) During normoxia, NOS is functional, myoglobin is oxygenated, and sufficient oxygen is available to diffuse from the source of oxygen through the tissue. (B) In hypoxic conditions, NOS is substrate limited and cannot make NO and myoglobin becomes deoxygenated. The majority of oxygen present is consumed by mitochondria close to the oxygen source, leading to a shortened oxygen gradient. (C) If nitrite is present during hypoxic conditions, it can be reduced by deoxygenated myoglobin. The NO generated can then partially inhibit mitochondrial oxygen consumption, allowing more oxygen to diffuse past these mitochondria and further into the tissue (elongation of oxygen gradient).
Extension of Tissue Oxygen Gradients and Modulation of Exercise Capacity by
Nitrite Reduction to NO
Shiva S. Nitric Oxide 2010 Feb 15;22(2):64-74
Nitrite regulates mitochondrial function. During hypoxia, nitrite is reduced to NO by deoxygenated myoglobin and nitrosylates the binuclear center of complex IV. This results in the inhibition of oxygen consumption which may contribute to the regulation of oxygen gradients and the modulation of exercise efficiency. During ischemia/reperfusion, nitrite is converted to a nitrosating species (possibly N2O3 through its reductive anhydrase reaction with heme) and S-nitrosates complex I at reperfusion. This leads to decreased ROS generation and inhibition of cytochrome c release, which contribute to cytoprotection after I/R.
Nitrite Regulates Mitochondrial FunctionAt Different Stages of ETC
Stem Cell Theory of Aging
Aging is the result of the inability of various types of stem cells to continue to replenish the tissues of an organism with functional differentiated cells capable of maintaining that tissue’s original function.
The number of stem cells in young people is very much higher than older people and this cause a better and more efficient replacement mechanism in the young contrary to the old.
Aging is not a matter of the increase of damage, but a matter of failure to replace it due to decreased number of stem cells. They decrease in number and tend to loose the ability to differentiate
Nitric Oxide is the requisite signal for stem cell mobilization and differentiation
into target cell types
The bioavailability of NO in patients may predict stem cell therapy success or failure
Essential role of endothelial nitric oxide synthase for mobilization of stem and progenitor cellsAicher et al Nature Medicine 9, 1370 - 1376 (2003)
Nitric oxide-cyclic GMP signaling in stem cell differentiation.Free Radic Biol Med. 2011 Dec 15;51(12):2150-7
Role of nitric oxide signaling components in differentiation of embryonic stem cells into myocardial cells.
Mujoo K, Sharin VG, Bryan NS, Krumenacker JS, Sloan C, Parveen S, Nikonoff LE, Kots AY, Murad F.
Proc Natl Acad Sci U S A. 2008 Dec 2;105(48):18924-9
What is Nitric Oxide?
The chemical compound nitric oxide is a gas with chemical formula NO٠.
It is an important signaling molecule in the body of mammals including humans, one of the few gaseous signaling molecules known.
It is also a toxic air pollutant produced by automobile engines and power plants.
NO should not be confused with nitrous oxide (N2O), a general anesthetic, or with nitrogen dioxide(NO2) which is another poisonous air pollutant.
The nitric oxide molecule is a free radical, which is relevant to understanding its high reactivity. It reacts with the oxygen in air to form nitrogen dioxide, signaled by the appearance of the reddish-brown color.
NO
Cardiovascular System
Respiratory Tract
Immunology
Cell Proliferation
Central Nervous System
Peripheral Nervous System
Gastrointestinal/Urogenital Tract
VasorelaxationBlood Cell RegulationMyocardial ContractilityMicrovascular Permeability
BronchodilatationAsthma, ARDS
NANC nerve-mediatedRelaxation
Learning and MemoryPain SensitizationEpilepsyNeurodegenerationCentral BP Control
ApoptosisAngiogenesisTumor Cell Growth
Unspecific ImmunityInhibition of Viral ReplicationTransplant Rejection
Penile ErectionPre-term Labour
Nitric Oxide Plays a Key Role in the Regulation of Numerous Vital Biological Functions
Regeneration
Mobilization of resident stem cellsTargeted differentiation
NOS
L-citrulline
L-arginine
NOENDOTHELIUM
Shear Stress ACH
M
SMOOTHMUSCLE
Relaxation
GTPcGMP
NO
guanylylcyclase (inactive) guanylyl
cyclase (active)
+PD5 inhibitors
L-Arg
Diet
L-Arg
ArginaseADMA
Transport
NOSUncouplingReduced OxygenReduced Cofactor + SubstrateOxidative Stress
Antioxidants
NO2
NO3
Oxidation
Bacterial Reduction
NO
+
O2-٠
ONOO-
Health
Disease
BH4 Ca/CamFAD+ FMNNADPH O2Heme iron GSH
MitochondriaXONADPH oxidase
The L-Arginine-Nitric Oxide Pathway
Urea Cycle
L-Arg
10 20 30 40 50 60 70
0
20
40
60
80
100
% D
eclin
e in
NO
Pro
duct
ion
Age in years
men women
Gerhard et al Hypertension 1996Celermajer et al JACC 1994Taddei et al Hypertension 2001Egashira et al Circulation 1993
Humans lose abilityto produce NO with aging
What if 50% or more of NObioactivity was determined and
dictated by foods and diets containingnitrite and nitrate?
Atmospheric Nitrogen CycleThe store of nitrogen found in the atmosphere, where it exists as a gas (mainly N2), plays an important role for life. Most plants can only take up nitrogen in two solid forms: ammonium ion (NH4+ ) and the nitrate ion (NO3- ). Most plants obtain the nitrogen they need as nitrate from the soil. When released, most of the ammonium is often chemically altered by a specific type of bacteria (genus Nitrosomonas) into nitrite (NO2- ). Further modification by another type of bacteria (genus Nitrobacter) converts the nitrite to nitrate. All nitrogen obtained by animals can be traced back to the eating of plants at some stage of the food chain.
Dietary nitrate is rapidly absorbed into the bloodstream, where it mixeswith endogenous nitrate from the NOS/NO pathway. A large portion of nitrate is taken up by the salivary glands, secreted with saliva and reduced to nitrite by symbiotic bacteria in the oral cavity. Salivary-derived nitrite is further reduced to NO and otherbiologically active nitrogen oxides in the acidic stomach. Remaining nitrite is rapidly absorbed and accumulates in tissues, where it serves to regulate cellular functions via reduction to NO or possibly by direct reactions with protein and lipids. NO and nitrite are ultimately oxidized to nitrate, which again enters the enterosalivary circulation or is excreted in urine.
New Paradigm - Human Nitrogen Cycle
One-electron reduction is favorable to five-electron oxidation
Nitrate, bacteria and human healthLundberg JO, Weitzberg E, Cole JA, Benjamin N.Nat Rev Microbiol. 2004 Jul;2(7):593-602
Acute blood pressure lowering, vasoprotective, and antiplatelet properties of dietary nitrate via bioconversion to nitrite.Webb AJ, Patel N, Loukogeorgakis S, Okorie M, Aboud Z, Misra S, Rashid R, Miall P, Deanfield J, Benjamin N, MacAllister R, Hobbs AJ, Ahluwalia A.Hypertension. 2008 Mar;51(3):784-90.
Dietary nitrate supplementation reduces the O2 cost of low-intensity exercise and enhances tolerance to high-intensity exercise in humans.Bailey SJ, Winyard P, Vanhatalo A, Blackwell JR, Dimenna FJ, Wilkerson DP, Tarr J, Benjamin N, Jones AM.J Appl Physiol. 2009 Oct;107(4):1144-55.
Physiological role for nitrate-reducing oral bacteria in blood pressure controlKapil V, Haydar SM, Pearl V, Lundberg JO, Weitzberg E, Ahluwalia A.Free Radic Biol Med. 2013 Feb;55:93-100.
Dietary Nitrate Can Be Metabolized to Nitrite and NO
NO 3-
NO 2-
NO
N 2O
N 2
NH 3
2e-
1e-
1e-
1e-
3e-
Bacteria
Increase NO 2-
NR
NiR
NOR
N 2OR
Ideal Community:• Higher Nitrate reduction efficacy• No NiR enzyme; Nitrite can accumulate, enrich saliva to form NO when swallowed.
Best
Intermediate
Worst
Hyde et al PLoS One (in press)
NEW PARADIGM FOR NO REGULATION
- There exists specific bacterial communities that provide the human body with continuous sources of nitrite and NO from dietary nitrate.
- Contributes to optimal cardiovascular health
- Absence of these oral bacterial communities affects NO homeostasis.
- Individuals deficient in these commensal bacteria would be NO deficient and perhaps at increased risk for cardiovascular disease
Disruption of Nitrate-Nitrite-NO Pathway
1. Insufficient dietary intake of nitrate/nitrite rich foods(green leafy vegetables, beets, etc)
2. Problems with nitrate uptake in duodenum (sialin (SLC17A5) transporter mutations – Salla Disease)
3. Insufficient saliva production (Sjogrens syndrome)
4. Lack of oral commensal bacteria to reduce nitrate to nitrite (use of antibiotics/antiseptic mouthwash, poor oral hygeine)
5. Insufficient stomach acid production – Achlorhydria(use of PPI’s, H. Pylori infection, iron overload)
6. Increased oxidative stress that scavenges NO
What might this mean?
• Absence of these select bacteria - a new risk factor for cardiovascular disease.
• Patients with periodontal disease , affecting the NO producing communities - possibly linking oral health to cardiovascular disease risk by disruption of NO production
• Use of antiseptic mouthwash or overuse antibiotics can disrupt nitrate reducing communities
• Patients taking proton pump inhibitors to suppress stomach acid production
• Develop this pathway as a primary therapeutic target to affect NO production
NO3-
NO2-
NO
Manipulating the NO System Through Diet and Nutrition
oxidation reduction
Facultative anaerobes5-8%Spiegelhalder 1976Lundberg 2004
Mammalian enzymes~ 0.01%Bryan Nat Chem Biol. 2005Feelisch JBC 2008
Beet, kale, etc
Oxygen,ceruloplasmin
Oxyhemeproteins
L-arginine
50-90%
Lowering blood pressure by 5 mmHgreduces risk of stroke by 34% and
Ischemic heart disease by 21%
Health Technol Assess. 2003;7(31):1-94.
Lowering blood pressure to prevent myocardial infarction and stroke: a new preventive strategy.
Law M, Wald N, Morris J.
What are the available strategiesFor enhancing NO?
Strong & sustained Nitric Oxide activity
NO based Clinical Trial Results
Zand et al Nutrition Research 2011
0 60 120 180 240 300 360 420 480 540 600
0
2000
4000
6000
8000
10000
12000
14000
NO
[ppb
]
Time (seconds)
0 10 20 30 40 50 60
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
Blo
od N
O L
evel
s [µ
M]
Time (min)
L-Arginine + antioxidants Neo40 Daily
Pla
sma
nitr
ite [µ
M]
Day 0 Day 30
50
100
150
200
250
300
350
400
Tri
gly
ceri
de
s (m
g/d
L)
A
Day 0 Day 30
0
50
100
150
200
250
300
Tri
gly
ceri
de
s (m
g/d
L)
*p = 0.02
B
Significant reduction in patients with elevated triglycerides
Zand et al Nutrition Research 2011
Clinical Trial Results
Blood pressureUltrasoundPulse waveEndopat
0 10 min 20 min 30 min 60 min 4 hours
Ultra-sound BP
Pulsewave BP Endopat
Active
Placebo
Blood pressureUltrasoundPulse waveEndopat
0 10 min 20 min 30 min 60 min 4 hours
Ultra-sound BP
Pulsewave BP Endopat
3 week washout
Hypertension Study Protocol
Baseline 4 hour Neo0.0
0.5
1.0
1.5
2.0
2.5
3.0
End
osco
re
Endopat *d e m o d e m o d e m o d e m o
d e m o d e m o d e m o d e m o
d e m o d e m o d e m o d e m o
d e m o d e m o d e m o d e m o
d e m o d e m o d e m o d e m o
d e m o d e m o d e m o d e m o
d e m o d e m o d e m o d e m o
d e m o d e m o d e m o d e m o
d e m o d e m o d e m o d e m o
Hypertension Trial – Vanderbilt Univ
0 10 20 30 40 50 6080
85
90
135
140
145
150
*
*
Blo
od P
ress
ure
(mm
Hg)
Time (min)
Systolic Active Diastolic Active Systolic Placebo Diastolic Placebo
#* @
#* @
@
d e m o d e m o d e m o d e m o
d e m o d e m o d e m o d e m o
d e m o d e m o d e m o d e m o
d e m o d e m o d e m o d e m o
d e m o d e m o d e m o d e m o
d e m o d e m o d e m o d e m o
d e m o d e m o d e m o d e m o
d e m o d e m o d e m o d e m o
d e m o d e m o d e m o d e m o
Houston, Hays JCH 2014
4 hour post active
0 10 20 30 40 50 60
80
85
90
135
140
145
150
155
160
*
*
*
*
Blo
od P
ress
ure
(mm
Hg)
Time (min)
Systolic Diastolic
Hypertensives n=17
0 10 20 30 40 50 60
80
85
90
125
130
135
140 Pre-Hypertensives n=9
*
*
*
*
Blo
od P
ress
ure
(mm
Hg)
Time (min)
Systolic Diastolic
0 10 20 30 40 50 60
80
85
90
115
120
125
130
135Normotensives n=5
Blo
od P
ress
ure
(mm
Hg)
Time (min)
Systolic Diastolic
0 10 20 30 40 50 60
80
85
90
135
140
145
150
#*
*
*
Blo
od P
ress
ure
(mm
Hg)
Time (min)
Systolic Diastolic
* #
n=30
Representative Ultrasound Before and10 minutes after NO
13% increase in vessel diameter causesa 34% increase in blood flow
0 2 4 6 8 10
0.50
0.55
0.60
0.65
0.70
0.75
0.80
LCCA d
iam
eter
(cm
)
Time (min)
N0 Dilates Carotid Artery within 90 Seconds
Houston, Hays JCH 2014
Average Changes in 10 subjectsAfter 10 minutes
Thermographic Images
Before NO 10 min After NO
49 yof chronic smoker with Raynauds
Pre-hypertension trialCedars Sinai Medical CenterPI: Ernst Schwarz MD, PhD
Group 1 (n=17) Group 2 (n=12)
Males 10 5
Females 7 7
Age 39 ± 11.77 43 ± 10.68
Blood Pressure (mmHg) 138 ± 11.66 (systole), 84 ± 5.09
(diastole)
138 ± 21.37 (systole), 80 ± 7.68
(diastole)
Heart Rate (bpm) 74.7 ± 9.24 80.4 ± 10.44
Orthostatics (mmHg) 139 ± 10.95 (systole), 86 ± 4.12
(diastole)
134 ± 19.22 (systole), 82 ± 7.79
(diastole)
Baseline Demographics
Pre-Hypertension Trial – Cedars SinaiSchool of Medicine
160 160
135 135
90 90
45 45
Group 1Baseline Follow Up
Group 2Baseline Follow Up
mm
Hg
mm
Hg
Blood PressureFigure 1
Systole
Diastole Diastole
Systole
Biswas et al (in press) JCPT
Group 1 (mean ± SD) Group 2 (mean ± SD) Baseline: NO vs
placebo (p-value)
Follow-Up: NO vs placebo (p-value)
Baseline Follow-Up ∆ Baseline Follow-Up ∆
BP (mmHg, systole; diastole)
138±12; 84±5
126±12; 78±4 12; 6 reduction (p<0.001)
138±21; 80±8
135±17; 82±8
N.S. 0.19; 0.012
0.26; 0.25
Heart Rate
(bpm)
75±9 76±8 N.S. 80±10 79±8 N.S. 0.14 0.33
6-Minute Walk Test (meters)
596±214 650±197 55 improvement (p<0.005)
590±8 606±225 N.S. 0.25 0.35
SF-36v2 (PCS; MCS)
48±10; 40±9
50±8; 45±7 p<0.05 43±10; 37±9
37±11; 37±7
significant worsening (p<0.05)
0.08; 0.06 0.08; 0.03
30 Day Placebo controlled Trial
Biswas et al (in press) JCPT
Baseline 6 months
500
550
600
650
700
750
800
850
CIM
T (m
icro
ns)
Edwin Lee MD – case report
NO Leads to Plaque Regression
NO Supplementation Rescues Inborn Error in Metabolism
The Urea Cycle converts ammonia to urea for excretion
• Hyperammonemia • In addition:
– Progressive liver dysfunction and cirrhosis– Coagulopathy– Neurological dysfunction independent of
recurrent hyperammonemia– Hypertension– Renal dysfunction
• More than hyperammonemia?
ASL deficiency is an Inborn error in metabolism
3/22—116/753/23—122/783/24—133/803/25—106/803/28—120/753/29—114/773/30—124/723/31—126/73 4/1—109/80
NO
NO
NO
CONCLUSIONS
Nitric oxide controls and regulates telomeres, mitochondria and stem cell function
There is an age-related decline in NO production that asserts its effecton all 3 theories of aging
Restoring NO production can lead to better mitochondrial function, increasedtelomerase activity and improved mobilization of stem cells for tissue repair
Strategies to restore NO production/homeostasis will have a profoundimpact on public health and on the aging process
Any anti-aging strategy should include NO as a first line of defense.
Beware of Pretenders!!!
1. Ask for clinical evidence that NO productswork
2. Make them show you it works
3. Demand published research on the product
If they cannot provide you these 3simple requests, then RUN
Nitrosating Agents
Mex+NO
NO2
L-Arg
O2
L-NIO
Nitrosation
Nitr
osy
latio
n
Cellular Targets
Nitroso/NitrosylProducts
For
mat
ion
of
Nitr
oso/
Nitr
osyl
Spe
cies
NO
-Gen
erat
ion/
Det
ectio
nNO
ox.red.
Transnitrosation
For
mat
ion
ofN
itric
Oxi
deO2• -
ONOO -
NO3-
+H+
- OH•
NO2-
H2Oox
.
ox.
[NO+] Amines Metals
RSNO R2NNO
NO
Hypoxia / H+
Thiols
red.
N2O3
H2O
1:1
RSH R2NH Mex+
RSH
N2O4
RS•Oxi
da
tive
Nitr
osy
latio
n
-NO2-
Biochemical Pathways of NO-Target Interactions in vivo
Bryan et al., PNAS 2004
Book Highlights:Restoring nitric oxide
production in the body thereby combating:
• High blood pressure• Heart attack
• Stroke• Diabetes• Arthritis
• Kidney disease• Memory loss• Osteoporosis