teatro 11 10 16 00 arthur burnett subs trinity bivalacqua · • strong presence of schawnn cells...
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Future Targets
Trinity J. Bivalacqua MD PhDJohns Hopkins Hospital
The James Buchanan Brady Urological InstituteBaltimore, MD USA
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Pathways activating cGMP
cGMP
GMP
sGuanylatecyclase
NO
Relaxation
Smooth Muscle
CO
HO‐1
Membrane
NEP
Opiorphin
xCNP
pGuanylatecyclase
Natriuretic peptides
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Arginase/ NOS Pathway
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cAMP signaling pathways
ATP cAMP
Ca2+PKA
Relaxation
Adenylatecyclase
PGE1R
Alprostadil
A2br
Adenosine
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AdenosineAdenosineDeaminase(ADA)
S‐adenosyl‐homocysteineHydrolase (SAHH)
Inosine S‐adenosyl‐homocysteine
AMP
ADP
ATP
ADK
CD39CD73
ATPAdenosine ADP
Adenosine
Adenosine is generated both intra‐ and extracellularly.
PDE5
Hif1
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Endothelial Cell
Smooth Muscle Cell
K+
K-ATP-Channels
cystathionineβ‐synthase (CBS) and CSE
Nerve
H2S
CSE
L‐cysteineL‐cysteine
Cystathionine γ‐lyase (CSE)
L‐cysteine
Ca2+
cAMP/cGMP?
H2S involvement in erectile physiology
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Vasoconstrictive Pathways
MLC
KC
a2+-C
aM SMM
P
RELAXATIONMyosinLC20
MyosinLC20.PCONTRACTION
ROCK
RhoA‐GTP
Endothelin‐1NorepinepherineSerotoninAngiotensin
DAGPLC
PKC
CPI‐17
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Future Targets
• Rho‐kinase• Arginase• Adenosine• sGC activators• Stem Cells/Tissue engineering
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Future Targets
• Rho‐kinase *• Arginase• Adenosine• sGC activators
• Stem Cells/Tissue engineering *
* Most promising targets at this time.
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MLC PHOSPHATASE(ACTIVE)
MLC PHOSPHATASE~P(INACTIVE)
RHO-KINASE
RHOA~GDP(INACTIVE)
RHOA~GTP(ACTIVE)
++ATP
HTN, DM, Hypoxia, NE, ET-1STIMULATE
MLC ~P(CONTRACTION)
MLC(RELAXATION)
Fibrosis/Apoptosis
MLC-KINASE
BINDS GTP
MIGRATES TO MEMBRANE
MODIFICATIONS
PHOSPHATASEPHOSPHATASE (?)
eNOS ProteineNOS mRNA stability
Activation of RhoA / Rho‐Kinase Inhibits Corporal Relaxation
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MLC PHOSPHATASE(ACTIVE)
MLC PHOSPHATASE~P(INACTIVE)
RHO-KINASE
RHOA~GDP(INACTIVE)
RHOA~GTP(ACTIVE)
NO/cGMP/PKGINHIBIT
MLC ~P(CONTRACTION)
MLC(RELAXATION)
ERECTION)
MLC-KINASE
PHOSPHATASEPHOSPHATASE (?)
eNOS ProteineNOS mRNA stability
Inhibition of RhoA / Rho‐Kinase Enables Corporal Relaxation
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Y-27632(30 nmol)
160
140
120
100
80
60
40
20
0
Pres
sure
(mm
Hg)
ICP
SAP
Time (min)0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Effect of Intracavernous Injection of the Rho-kinase Inhibitor, Y-27632
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Rho‐KINASE
Human CC
Sinusoid
DAPI
ROCK1
αSMA
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Diabetes 40%
Vascular Disease30%
Spinal Cord Injury8%
Radical Surgery13%
Endocrine Disorders6%
MultipleSclerosis3%
Zonszein, Urol Clin North Am, 1995.
Common Systemic Diseases Associated with ED
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Rho-Kinase Inhibition Reduces Apoptosis and Preserves SMC content in Diabetic Rats
WJ Li et al., JSM 2011
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Rho-Kinase Inhibition Reduces Intimal Thicking in Internal Iliac Artery
K Park, et al. JSM 2006
Control Atherosclerosis
Fasudil
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K Park, et al. JSM 2006
Rho-Kinase Inhibition Improves Endothelial NOS and Prevents ED
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Peripheral Nerve Injuries
Cell Body
Basement Membrane
Myelin Sheath
Axon
Normal Compressed/Stretch
Sheath Loss Disconnection Degeneration
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Strategies to promote axon regeneration
3. Neutralization of the inhibitory factors in the injured PNS A therapeutic vaccine approach Anti-scarring treatment (inhibition of fibroblast
proliferation) Anti-inflammatory / Antioxidants Immune modulators Anti-apoptotic agents Axonal outgrowth inhibitory neutralizers
1. Stimulation of axon regeneration by modulating the neuronalsignaling responses:
- Treatment with neurotrophic factors (NGF, BDNF, NT-3,GDNF, LIF, FGF-2)
2. Cell transplantation: e.g. embryonic stem cells, adipose-muscle-, bone marrow derived -mesenchymal stem cells.
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• RhoA mediates growth inhibitor signals which stiffens the actincytoskeleton, thereby inhibiting axonal elongation.
• RhoA is a key inhibitory regulator of axonal regeneration in the CNS, however, little is known in the PNS.
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Bilateral Cavernous Nerve Injury (BCNI)
Right CN
Major PelvicGanglion (MPG)
Major PelvicGanglion (MPG)
Cavernous Nerve Injury with forceps 15 sec x 3
http://www.pelvipharm.com
Left CN
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Cellular responses to peripheral nerve injury
Major Pelvic
Ganglion (MPG)
Penis
Erectile Dysfunction
0.0 2.0 4.0 6.00.0
0.2
0.4
0.6
0.8
1.0
ShamBCNI 48hBCNI 7d
BCNI 14dBCNI 30dBCNI 60d
Volts
****
*
***** **
***
ICP/
MA
P
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Major Pelvic
Ganglion (MPG)
PenisInflammatory
Cytokines upregulate
RhoA/Rho-kinase
↑ Apoptosis(via caspase)
↓ neuronal nitric oxide synthase
WallerianDegeneration
Erectile Dysfunction
Hypothesis
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BCNI significantly elevated RhoA/Rho‐kinase
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Y-27632 → Rho-kinase (ROCK) inhibitor
5 mg/kg i.p. BID (dose selective for ROCK isoforms)
Effect of ROCK Inhibition on the MPG?
Day 0: Sham/BCNI Y-27632
Day 14: ICP, tissue collection
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ROCK inhibition preserves cavernous nerve structure
Magnification 40x
• Increased myelinated axons • Strong presence of Schawnn cells
Sham BCNI BCNI+Y-27632
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223.52 µm
275.35 µm
314.11 µm
179.21 µm
185.25 µm
Incubation 24h
10xIncubation 48h
10x
Incubation 72h
10x
A
C
B
D
Hannan JL et al., Molecular Neuroscience 2014
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48h incubation
Sham BCNI14d BCNI14d+Y276320
100
200
300
400
500
Neu
rite
Len
gth
( m
) **
Inhibition of ROCK: Effect onMPG Neuritogenesis
Sham Y27632
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1.0 2.0 4.0 8.0 16.0 32.0 48.0 64.00
50
100
150
200
250
SHAM (n=6)BCNI (n=6)BCNI + Y-27632 (n=8)
* * *
*
Frequency (Hz)
Con
trac
tion
(%M
ax K
Cl)
Improved nerve-mediated responses after ROCK inhibition
1.0 2.0 4.0 8.0 16.0 32.0 48.0 64.00
25
50
75
100
** *
* **
*
Frequency (Hz)
Rel
axat
ion
(%M
ax P
E)
* p<0.05 vs Sham; Ψ p<0.05 vs Sham and BCNI,;δ P<0.05 vs BCNI
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Sham BCNI BCNI + Y-276320.0
0.5
1.0
1.5
*
n=6
**
nNO
S/G
APD
H
Sham BCNI BCNI + Y-276320.0
0.5
1.0
1.5
*
n=4-6
m-e
NO
S/G
APD
H
m‐eNOS
GAPDH
a b
Figure 3
nNOS
GAPDH
Sham BCNIBCNI +Y‐27632 Sham BCNI
BCNI +Y‐27632
†
†
J Hannan et al, J Urol 2012
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2.0 4.0 6.0 8.00
25
50
75
100
SHAM (n=8)CNI (n=8)CNI + Y-27632 (n=7)
**
**
**
Voltage
Peak
ICP
(mm
Hg)
1 2 3 40.0
0.2
0.4
0.6
0.8
1.0
2.0 4.0 6.0 8.0
**
**
Voltage
ICP/
MAP
1 2 3 40
2000
4000
6000
*
2.0 4.0 6.0 8.0
**
*
*
Voltage
Tota
l IC
P(A
rea
Und
er th
e C
urve
; mm
Hg*
s)
2.0 4.0 6.0 8.00
10
20
30
40
* * *
*
Voltage
T80
(s)
a b
c d
Figure 1
†
† †† ††
†
†
† †† †
†
†† †
J Hannan et al, J Urol 2012
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Stem cells
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• Clinical need• Types of stem cells• Efficacy and mechanisms of action
– ED• cavernous nerve injury• (aging)• (diabetes and metabolic syndrome)
– Peyronie’s disease
STEM CELL TREATMENT FOR ERECTILE DYSFUNCTION
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Medline Search –Stem Cells and Erectile Dysfunction
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Stem Cells and Erectile Dysfunction
• Animal Models used to study stem cell-based therapies.– Aging– Hypogonadism– Hypercholesterolemia, Arterial insufficiency.– Diabetes mellitus type 1 and 2 (Metabolic Syndrome)– Cavernous nerve injury and resection.
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Stem Cell Biology
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Stem cell research in ED
LacZ (gene)GFP (gene)BrdU (DNA-incorporation)EdU (DNA-incorporation)CM-DiI (cytoplasm)PKH26 (cell membrane)
Ex-Vivo Gene Modifications (eNOS, VEGF)
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Stem cells1) diseased/lost cell replacement2) paracrine modification of natural coarse of disease
or paracrine stimulation of trophic processes
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Dual effects: neural tissues vs penile tissues
Primary:
CAVERNOUS NERVE INJURYAxonotmesis
neuro-inflammation
Wallerian degeneration
NO bioavailability
TNF-αTGF-β1
chemokines
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Dual effects: neural tissues vs penile tissues
Secondary:
CAVERNOSAL DENERVATION
hypoxia
smooth muscle apoptosiscollagen deposition (fibrosis)
veno-occlusive dysfunction
TGF-β1
Sham
BC
NI 1
4dB
CN
I 30d
2x 10x
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adipose tissue-derived stem cells (ADSC)
bone marrow derived stem cells (BMDSC)
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J Sex Med 2010;7:3331–3340
ADSC recovers erectile function in cavernous nerve injured rats
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J Sex Med 2010;7:3331–3340
Preserved nNOS expression in dorsal penile nerve
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Proteomic profile angiogenesis array analysis of human ADSCs
Guihua Liu et al, PLOS One 8: 2013
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Mesenchymal Stem Cells (ADSC) demonstrate neurotrophic effects on
cavernous nerve regeneration
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Nerve Grafts to replace CN
Hannan JL, Mao M, Bivalacqua TJ
Biomaterials with Schwann cells,Growth factors, Rho-kinase inhibitors,Neurotrophic factors, stem cells
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Stem Cell Based Clinical Trials• Treatment of Diabetic Impotence with Umbilical Cord
Blood Stem Cell Intracavernosal Transplant –negative trial (Jong Yoon Bahk et al. Experimental and Clinical Transplantation 8:150-160, 2010)
• Clinical trial in France using IC MSCs – ongoing.• Industry sponsored IC ADSCs for post-RP ED.
5/7 clinicalresponse
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Stem Cell Based ED Clinical Trial at Johns Hopkins: PRIMES 2014
• PRIMES (PReventing Impotence with MEsenchymalStem Cells) trial for post-radical prostatectomy ED.
• PI: Medical Oncology – Sam Denmeade M.D. Ph.D. and Johns Isaacs Ph.D
• PI: Urology – Trinity J. Bivalacqua M.D. Ph.DAlan Partwin M.D. Ph.D.
• Protocol – Intravenous injection of MSCs prior to RP to prevent degeneration of cavernous nerves and identification in prostate cancer.
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Conclusion
• Development of pharmacological agents targeting a number of molecular pathways are necessary for treatment of PDE5 inhibitor non‐responders.– However, is there interest to invest in their development for treatment of ED.
• Stem cell based therapies have support in pre‐clinical animal models and clinical trials results are forthcoming to determine efficacy in men with ED.
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AcknowledgmentsArthur L. Burnett, M.D. MBAJohanna Hannan Ph.D.Ahmet Hoke, M.D.Maarten Albersen MD PhD.Xiaopu Liu, B.S.Joseph WatsonHotaka Matsui M.D.