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 Diseases
account for 61% of
deaths worldwide.
Most of these preventable
by diet and lifestyle
modifications.
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 membrane
20% of your body is fat (need the right fats)
2. Amino acids
needed to make the protein machinery inside the cells
3. Vitamins and minerals
allow the body to make the fats and proteins work
What causes aging and is involved
In regenerative medicine?
Three main hypotheses:
1. Telomere shortening
2. Mitochondrial dysfunction
3. Loss of stem cell function and repair
Unified Theory of Aging
Nitric Oxide controls and regulates
1. Telomerase activity
2. Mitochondrial biogenesis and function
3. Mobilization of resident stem cells
Telomere Theory of Aging
A decrease in telomerase activity precedes telomere shortening and
introduction 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 telomeres
Cawthon 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 upon
NO production. eNOS knockout mice lose regulation of telomerase activity
that is rescued by exogenous NO donors
Grasselli et al Circulation Research. 2008 Jul 3;103(1):34-42
NO is the master signaling molecule in the regulation of telomerase activity
and provides the opportunity for innovative therapeutic approaches based on
the use of NO active compounds
Farsetti 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 signaling
Heat Production
Cellular metabolism
Cellular differentiation
Cell death
Control of the cell cycle (cancer)
Cell growth
Steroid synthesis
Mitochondria have been implicated
in many human diseases and are
critical in the aging process.
Nitric Oxide Controls and Regulates
Mitochondrial:
ATP synthesis
Reactive Oxygen Species
Cell Signaling
Apoptosis (Cell Cycle)
Biogenesis
Metabolism/Bioenergetics
Nitric Oxide and Mitochondrial Biogenesis
Nisoli E et al. Circulation Research 2007;100:795-806
Copyright © American Heart Association
Extension of Tissue Oxygen Gradients
and Modulation of Exercise Capacity by
Nitrite Reduction to NO
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).
Shiva S. Nitric Oxide 2010 Feb 15;22(2):64-74
Nitrite Regulates Mitochondrial Function
At Different Stages of ETC
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.
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 cells
Aicher 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.
Nitric Oxide Plays a Key Role in the Regulation of
Numerous Vital Biological Functions
Immunology
Gastrointestinal/
Urogenital Tract
Unspecific Immunity
Inhibition of Viral Replication
Transplant Rejection
Penile Erection
Pre-term Labour
Respiratory Tract
Bronchodilatation
Asthma, ARDS
Cardiovascular System
NO
Peripheral
Nervous System
NANC nerve-mediated
Relaxation
Cell Proliferation
Apoptosis
Angiogenesis
Tumor Cell Growth
Vasorelaxation
Blood Cell Regulation
Myocardial Contractility
Microvascular Permeability
Central Nervous System
Learning and Memory
Pain Sensitization
Epilepsy
Neurodegeneration
Central BP Control
Regeneration
Mobilization of resident stem cells
Targeted differentiation
ACH
Shear Stress
M
NOS
L-arginine
L-citrulline
ENDOTHELIUM
NO
NO
SMOOTH
MUSCLE
guanylyl
cyclase (inactive) guanylyl
cyclase (active)
cGMP
GTP
PD5
inhibitors
+
Relaxation
The L-Arginine-Nitric Oxide Pathway
Health
Oxidation
Antioxidants
Urea Cycle
Diet
L-Arg
L-Arg
Disease
Ca/Cam
FMN
O2
GSH
NOS
L-Arg
Arginase
ADMA
BH4
FAD+
NADPH
Heme iron
Transport
Uncoupling
Reduced Oxygen
Reduced Cofactor + Substrate
Oxidative Stress
NO
+
Mitochondria
XO
NADPH oxidase
O2-٠
ONOO-
NO2
NO3
Bacterial Reduction
% Decline in NO Production
Humans lose ability
to produce NO with aging
men
women
100
80
60
40
20
0
10
20
30
40
50
Age in years
60
70
Gerhard et al Hypertension 1996
Celermajer et al JACC 1994
Taddei et al Hypertension 2001
Egashira et al Circulation 1993
What if 50% or more of NO
bioactivity was determined and
dictated by foods and diets containing
nitrite and nitrate?
Atmospheric Nitrogen Cycle
The 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.
New Paradigm - Human Nitrogen Cycle
One-electron reduction is favorable to five-electron oxidation
Dietary nitrate is rapidly absorbed
into the bloodstream, where it mixes
with 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 other
biologically 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.
Dietary Nitrate Can Be Metabolized to
Nitrite and NO
Nitrate, bacteria and human health
Lundberg 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 control
Kapil V, Haydar SM, Pearl V, Lundberg JO, Weitzberg E, Ahluwalia A.
Free Radic Biol Med. 2013 Feb;55:93-100.
NO 32e-
Bacteria
NR
NO 2Increase NO
2
NiR
1e-
NO
NOR
1e-
N 2O
N 2OR
1e-
N2
3e-
NH 3
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
Manipulating the NO System
Through Diet and Nutrition
Beet, kale, etc
NO3
Oxyheme
proteins
NO2
Oxygen,
ceruloplasmin
oxidation
50-90%
NO
L-arginine
Facultative anaerobes
5-8%
Spiegelhalder 1976
Lundberg 2004
Mammalian enzymes
~ 0.01%
Bryan Nat Chem Biol. 2005
Feelisch JBC 2008
reduction
Lowering blood pressure by 5 mmHg
reduces 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 strategies
For enhancing NO?
NO based Clinical Trial Results
Strong & sustained Nitric Oxide activity
1.8
12000
1.6
Blood
NO Levels
[µM]
nitrite[µM]
Plasma
14000
NO [ppb]
10000
8000
6000
4000
2000
0
L-Arginine + antioxidants
Neo40 Daily
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
0
60 120 180 240 300 360 420 480 540 600
Time (seconds)
Zand et al Nutrition Research 2011
0
10
20
30
40
Time (min)
50
60
Clinical Trial Results
Significant reduction in patients
with elevated triglycerides
A
B
400
Triglycerides (mg/dL)
Triglycerides (mg/dL)
350
300
250
200
150
100
300
250
p = 0.02
200
*
150
100
50
0
50
Day 0
Day 30
Zand et al Nutrition Research 2011
Day 0
Day 30
Hypertension Study Protocol
Active
Ultrasound
BP
Pulse
wave
BP
30 min
60 min
Endopat
Blood pressure
Ultrasound
Pulse wave
Endopat
0
10 min 20 min
4 hours
3 week washout
Placebo
Ultrasound
BP
Pulse
wave
BP
30 min
60 min
Endopat
Blood pressure
Ultrasound
Pulse wave
Endopat
0
10 min 20 min
4 hours
Hypertension Trial – Vanderbilt Univ
dem o
dem o
dem o
dem o
Systolic Active
Diastolic Active
dem o
dem o
Systolic Placebo
Diastolic
d
e m o Placebo
dem o
dem o
dem o
dem o
145
140
135
*
3.0
2.5
dem o
dem o
dem o
dem o
dem o
dem o
dem o
dem o
dem o
dem o
dem o
dem o
dem o
dem o
d e@m o
dem o
dem o
*
#@
90
85
*
dem o
dem o
dem o
dem o
dem o
dem o
dem o
dem o
*
80
#@
Endoscore
Blood Pressure (mm Hg)
150
2.0
1.5
1.0
0.5
*
Endopat
dem o
dem o
dem o
dem o
dem o
dem o
dem o
dem o
dem o
dem o
dem o
dem o
dem o
dem o
dem o
dem o
dem o
dem o
dem o
dem o
dem o
dem o
dem o
dem o
dem o
dem o
dem o
dem o
dem o
dem o
dem o
dem o
dem o
dem o
dem o
dem o
0.0
0
10
20
30
40
Time (min)
Houston, Hays JCH 2014
50
60
Baseline
4 hour Neo
4 hour post active
n=30
160
Systolic
Diastolic
145
*
*
140
#
135
90
*
*#
85
80
0
10
20
30
40
50
Blood Pressure (mmHg)
Blood Pressure (mmHg)
150
Hypertensives n=17
155
*
150
140
135
90
*
135
80
0
10
Systolic
Diastolic
*
130
125
*
*
85
80
0
10
20
30
40
Time (min)
20
30
40
50
60
Time (min)
*
90
*
85
60
50
60
135
Blood Pressure (mmHg)
Blood Pressure (mmHg)
Pre-Hypertensives n=9
*
145
Time (min)
140
Systolic
Diastolic
Systolic
Diastolic
Normotensives n=5
130
125
120
115
90
85
80
0
10
20
30
40
Time (min)
50
60
Representative Ultrasound Before and
10 minutes after NO
13% increase in vessel diameter causes
a 34% increase in blood flow
N0 Dilates Carotid Artery within
90 Seconds
LCCA diameter (cm)
0.80
0.75
0.70
0.65
0.60
0.55
0.50
0
2
4
6
Time (min)
Houston, Hays JCH 2014
8
10
Average Changes in 10 subjects
After 10 minutes
Thermographic Images
Before NO
10 min After NO
49 yof chronic smoker with Raynauds
Pre-hypertension trial
Cedars Sinai Medical Center
PI: Ernst Schwarz MD, PhD
Baseline Demographics
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
138 ± 21.37 (systole), 80 ± 7.68
(diastole)
(diastole)
Heart Rate (bpm)
74.7 ± 9.24
80.4 ± 10.44
Orthostatics (mmHg)
139 ± 10.95 (systole), 86 ± 4.12
134 ± 19.22 (systole), 82 ± 7.79
(diastole)
(diastole)
Pre-Hypertension Trial – Cedars Sinai
School of Medicine
Blood Pressure
Figure 1
160
160
135
135
Systole
mm Hg
mm Hg
Systole
90
90
Diastole
Diastole
45
45
Baseline
Follow Up
Group 1
Biswas et al (in press) JCPT
Baseline
Follow Up
Group 2
30 Day Placebo controlled Trial
Group 1 (mean ± SD)
Follow-Up
∆
138±12;
84±5
126±12; 78±4
75±9
Baseline
BP
(mmHg,
systole;
diastole)
Heart
Rate
(bpm)
6-Minute
Walk Test
(meters)
SF-36v2
(PCS;
MCS)
Group 2 (mean ± SD)
∆
Baseline:
NO vs
placebo
(p-value)
FollowUp: NO vs
placebo
(p-value)
Baseline
Follow-Up
12; 6 reduction
(p<0.001)
138±21;
80±8
135±17;
82±8
N.S.
0.19;
0.012
0.26; 0.25
76±8
N.S.
80±10
79±8
N.S.
0.14
0.33
596±214
650±197
55
improvement
(p<0.005)
590±8
606±225
N.S.
0.25
0.35
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
Biswas et al (in press) JCPT
NO Leads to Plaque Regression
850
CIMT (microns)
800
750
700
650
600
550
500
Baseline
Edwin Lee MD – case report
6 months
NO Supplementation Rescues
Inborn Error in Metabolism
The Urea Cycle converts ammonia
to urea for excretion
ASL deficiency is an Inborn
error in metabolism
• Hyperammonemia
• In addition:
– Progressive liver dysfunction and cirrhosis
– Coagulopathy
– Neurological dysfunction independent of
recurrent hyperammonemia
– Hypertension
– Renal dysfunction
• More than hyperammonemia?
NO
3/22—116/75
3/23—122/78
3/24—133/80
3/25—106/80
3/28—120/75
3/29—114/77
3/30—124/72
3/31—126/73
4/1—109/80
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 effect
on all 3 theories of aging
Restoring NO production can lead to better mitochondrial function, increased
telomerase activity and improved mobilization of stem cells for tissue repair
Strategies to restore NO production/homeostasis will have a profound
impact 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 products
work
2. Make them show you it works
3. Demand published research on the product
If they cannot provide you these 3
simple requests, then RUN
Nitroso/Nitrosyl
Products
RSNO
RSH
Thiols
Cellular Targets
Nitrosating Agents
Nitrosation
+
[NO ]
R2NNO
Mex+NO
R2NH
Mex+
Amines
Metals
Hypoxia / H+
H2O
NO2-
ox.
NO3-
H2O
ox.
N2O4
N2O3
Transnitrosation
NO-Generation/
Detection
red.
NO
Formation of
Nitroso/Nitrosyl Species
Biochemical Pathways of NO-Target Interactions in vivo
NO2
ONOO -
- OH•
-NO2-
O2• -
O2
RS•
NO
L-NIO
RSH
Bryan et al., PNAS 2004
L-Arg
Formation of
Nitric Oxide
+H+
Nitrosylation
Oxidative
Nitrosylation
1:1
Book Highlights:
Restoring nitric oxide
production in the
body thereby
combating:
•High blood pressure
•Heart attack
•Stroke
•Diabetes
•Arthritis
•Kidney disease
•Memory loss
•Osteoporosis