ASDIN 7th Annual Scientific Meeting
From Pathogenesis to Device
Development: How can we best use
animal models?
Outline
• Why interventional nephrologists need animal models
• Describe standard large and small animal models of AVF and AVG stenosis
Prabir Roy-Chaudhury MD, PhD, FACP
U i
University
it off Cincinnati
Ci i
ti and
d Cincinnati
Ci i
ti VAMC
¾Elucidate underlying mechanisms of stenosis
¾Elucidate underlying mechanisms of stenosis
¾Develop novel devices to reduce dialysis access stenosis
Vein
• Why animal models need interventional nephrologists
Artery
Why do we need animal models in
interventional nephrology
A vein is not an artery
≠
• Dialysis access stenosis is a unique situation!!!
• No other specialty deals creates an “iatrogenic” connection between an artery and vein!! • Focus on venous rather than arterial stenosis
• Uremia is a complicating factor
• Complete lack of effective therapies (need to elucidate mechanisms and to test out novel devices) • Anatomical: Veins have a poorly defined internal elastic A
i l V i h
l d fi d i
l l i
lamina
• Physiological: Veins release less nitric oxide and prostacyclin
• Molecular: Significant differential expression of gene products between normal vein and artery Size Matters!!
Animal models of AVF and AVG stenosis
LARGE
• Pig AVF/AVG
• Goat AVF/AVG
• Sheep AVG
• Dog AVG
SMALL
• Mouse AVF
• Rat AVF
LARGE
SMALL
• Surgical techniques
• Devices
• Grafts
• Bioengineered
• Mechanisms
• Knock out or
• Surgical
• COST!!!!
vessels
configurations
transgenic animals
• Easy availability of
antibodies and gene
sequences
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ASDIN 7th Annual Scientific Meeting
Development of a pig model of venous
neointimal hyperplasia in PTFE
dialysis access grafts
Specimen Preparation
GRAFT
VEIN
A
Suitable vascular tree
Response of vessel wall to injury is similar to Man
Pig Model (Day 2)
Significant venous neointimal
hyperplasia at 28 days
V
A
C
B
Validation Time Points: Days 2, 4, 7, 14, 28
Pig Model (Day 14)
Human/Pig Similarities
D 28
Human GVA
Pig GVA
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ASDIN 7th Annual Scientific Meeting
Placement of Perivascular Paclitaxel Polymers
into a Pig Model of Venous Stenosis
External radiation attenuates
neointimal hyperplasia
•
•
•
Single dose of 16 Gy
5 x 6 cm area
Within 24 hrs of surgery
% Luminal S
Stenosis
% Luminall Stenosis
Venous Neointimal Hyperplasia
60
50.5%
50
*
38.4%
40
30
20
10
0
Controls
Radiated
Vein: 50.5% vs 38.4%
p = 0.021
24% reduction
A
V
Kelly et al., Nephrol Dial Transplant 2006
The pig is ideally suited for devices (Sutureless
anastomosis graft)
Sutureless Anastomosis Hybrid Graft
Converts an end to side anastomosis into an end to end venous anastomosis
OptiflowTM is a sutureless anastomotic conduit for AVF creation
Conduit
Optiflow anastomotic conduit
Vein
Conduit
Artery
Flange
Flange
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ASDIN 7th Annual Scientific Meeting
Optiflow Angiography
AV Fistula Model Development (Dissection)
Femoral Vein
???
Femoral Artery
Wang et al. NDT 2008
AV Fistula Model Development (Arteriotomy)
AV Fistula Model Development (Anastomosis)
Wang et al. NDT 2008
AV Fistula Model Development
Wang et al. NDT 2008
Neointimal budding in the 7d AVF
7d AVF, PV, SMA x 400
7d AVF, PV, SMA x 100
7d AVF, PV, BrdU x 400
Krishnamoorthy et al. P-PO1054, ASN 2006
Wang et al. NDT 2008
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ASDIN 7th Annual Scientific Meeting
Aggressive stenosis at 42d
Alternative Origins for Neointimal Cells: Role of the Adventitia 42d AVF, PV, SMA x 400
BrDU x 400
A
BrdU -ve
M
2d
SMA x 400
N
Have we been targeting the wrong
part of the vessel wall?
Adventitia
Media
2d
Krishnamoorthy et al., F‐FC157, ASN 2007
Eccentric macrophage infiltration at 2d
AV Fistula Model Development (Shear Stress Analysis)
Neointima: NO
Adventitia: YES
Shi et al. Circ. 1996;7:1655
Eccentric neointimal hyperplasia at 42d
Eccentric shear stress to
macrophages to stenosis
Proximal artery
Proximal vein
Inflammatory (macrophage) response AV anast.
Different patterns of
shear on opposite walls
Chemokines and cytokines
Intima‐media thickening
Krishnamoorthy et al. Kidney International 2008
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ASDIN 7th Annual Scientific Meeting
The Pig is a good large animal model
Can we make a uremic pig??
• Response to injury similar to humans
• Animal model of choice for cardiovascular
• Yes
• Misra et al. use embolization of one
research (comparisons)
• Better availability of antibodies and gene
sequences as compared to dogs and sheep
• More aggressive response to injury is an
advantage in intervention studies
• Lack of thrombosis could be a disadvantage
kidney followed by selective renal
artery infarction
• We have not made pigs uremic
• Remember you don’t want them uremic
enough to require HD
(Dog)
Mouse Model of AVF stenosis (14d)
Mouse Model Creation
b
Arteryy
Vein
Uremic mouse model of AVF stenosis
• Cauterize right kidney
• Nephrectomy left kidney 2 wks
Uremic mice have increased AV fistula stenosis
Non-uremic
Uremic
X 2-3
later
• Wait 6 weeks (BUN = 50-70)
• Place AVF
Neointimal Volume
AV
anastomosis
Avoid morbidity and mortality due to uremia
Non-uremic
CKD
Choi et al.
JASN. 2008
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ASDIN 7th Annual Scientific Meeting
The Mouse is a good small animal model
• Knock outs
• Transgenics
Do we really need animal models
• Gene sequences
• Molecular tools
A Mouse is not a Man
not a Man
and neither is a Pig!
Surgery is NOT easy
A good human surgeon is not always a
good mouse surgeon!!
Do we need animal models?
Do we need animal models?
•Yes for safety
•Yes for technique and
•Proof of final human
efficacy can only
come from human
clinical trials!!
feasibility
•Yes for mechanisms
•Yes for an efficacy signal
Animal models and interventional nephrology:
a win-win situation
Animal models and interventional nephrology:
a win-win situation
• Animal models need
• Increases innovation
• Develop and test out
intervention skills
• New devices will
???
mean new
angiographic
appearances
• Animal model work
gives you a heads up
new devices
???
• Intellectual property
• Starting a company!
• Go from an idea to a
product!!
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ASDIN 7th Annual Scientific Meeting
Paclitaxel Polymers Inhibit Neointimal Hyperplasia
AXZ-100
Paclitaxel
Control
Kelly et al., Nephrol Dial Transplant 2006
Periadventitial injection
Bullfrog Catheter Injections
• Endovascular device such as the “Bullfrog”
microinfusion catheter (Mercator-Med)
Sheathed needle
Tailor therapies to the biological
course of vascular stenosis
Extruded needle
Drug A initially followed by
Drug B at 6 monthly intervals
Bullfrog Catheter Injections
Bullfrog Catheter Injections
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ASDIN 7th Annual Scientific Meeting
PTFE grafts coated with Cu-II coatings generate a
significant NO flux POST-IMPLANT
Bullfrog Catheter Injections
No Generating
Control
•2 weeks
post implant
Is there a difference in graft thrombosis?
Surgery – 10/3/07
Sacrifice – 10/22/07
CONTROL
@ 3 weeks
Hemodynamics and
Histology
V
A
Surgery – 10/2/07
Sacrifice – 10/22/07
NITRIC OXIDE
@ 3 weeks
A
V
AV Fistula Model (CT Angiography)
3D Mesh Framework
Proximal Artery
AV Anastomosis
Proximal Vein
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ASDIN 7th Annual Scientific Meeting
AV Fistula Model (Flow and Pressure
Analysis)
3D Wall Shear Stress Profile
Proximal Artery
AV Anastomosis
Proximal Vein
Flow
Pressure
Wall Shear Stress Alterations in Systole and Diastole
AV Fistula Model Development (Shear Stress Analysis)
Proximal
Artery
Low Shear Stress
AV
anastomosis
Proximal
Vein
Krishnamoorthy et al. In Revision Kidney International 2007
AV Fistula Model Development (Shear Stress Analysis)
Implant Design
Proximal artery
Venous Flow
Proximal vein
Conduit
(Vein)
Flange
(Artery)
AV anast.
Proximity of high
and low shear stress
“Heel Flange”
“Toe Flange”
“Side Flange”
Confidential
Krishnamoorthy et al. In Revision Kidney Int 2007
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ASDIN 7th Annual Scientific Meeting
Do we really need animal models
A Mouse
Is not
A Man!
Do we need animal models
•Yes for safety
•Yes for technique and
feasibility
•? for efficacy
Optiflow Angiography
AV Fistula Model Development (Curved)
PA
DA
PV
Wang et al. NDT 2008
OptiflowTM is a sutureless anastomotic conduit for AVF creation
Conduit
Vein
Conduit
Artery
Flange
Flange
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ASDIN 7th Annual Scientific Meeting
OptiFlow Sutureless Anastomotic Device
1. Mobilize Vessels
6. Insert flanges inside arteriotomy
2. Venotomy
5. Arteriotomy
4. Suture the vein and mark arteriotomy
3. Insert device into vein
The adventitia is important!
2d AVF, PV, SMA x 200
2d AVF, PV, SMA x 200
2d AVF, PV, BrdU x 200
Normal Pig Vein, SMA x 200
Krishnamoorthy et al.
F-PO1054,
ASN 2006
Mouse model of AVF stenosis
• End of jugular vein to side of carotid artery
• 11/0 suture; finer than a human hair; barely
visible
• Significant neointimal hyperplasia by 2 wks
• Some thrombus formation also
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