project 1: biocompatible heterograft biomaterials robert levy, pi
DESCRIPTION
Project 1: Biocompatible heterograft biomaterials Robert Levy, PI. Aims Chemistry and crosslinking using triglycidyl amine (TGA) to prepare bioprosthetic heart valves: Rapoport et al, Biomaterials. 2007;28(4):690-9 2. Biomechanical effects of TGA crosslinking: - PowerPoint PPT PresentationTRANSCRIPT
The Joseph Stokes, Jr., Research Institute
Project 1: Biocompatible heterograft biomaterials Robert Levy, PIAims
1. Chemistry and crosslinking using triglycidyl amine (TGA) to prepare bioprosthetic heart valves:
Rapoport et al, Biomaterials. 2007;28(4):690-9
2. Biomechanical effects of TGA crosslinking:
Sacks et al, Ann Thorac Surg. 2006;82(4):1369-77
3. Extracellular matrix interactions—TGF1 related calcification mechanisms:
Clark-Greuel et al, Ann Thorac Surg. 2007;83(3):946-53
The Joseph Stokes, Jr., Research Institute
Triglycidyl Amine (TGA)
O
CH2Cl
HO
Cl
N
Cl
OHHO
Cl
NH3
i-PrOH-water
O
NO
O
aq. NaOH, toluene-THF
-HCl
TGA
Epichlorohydrin
Project 1
•Reacts irreversibly with lysine, methionine, cystine, histidine
•Results in biomechanical properties superior to glutaraldehyde
•Biocompatibility—supports cellular growth of all cardiovascular cell types
The Joseph Stokes, Jr., Research Institute
Reaction of MABP with residual epoxy groups
MABP
Collagenfiber
O
X
OH
X
HSCH2 CH2 NH CH2 CH
PO3H2
PO3H2MABP
S
NH CH2 CH
PO3H2
PO3H2
X = residue of TGA
The Joseph Stokes, Jr., Research Institute
Ca-PO4-MABP Precipitation Experiment: Prepared and Precipitated at pH=7.4
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20.00
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120.00
Ca (
ug/m
l)
2 mM MABP, 10 mM TGA
10 mM TGA
Ca-PO4-MABP Precipitation Experiment: 2 mM MABP, 10 mM TGA
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NoTreatment
2.5 mM Ca 2.5 mM Ca,2pM PO4
2.5 mM Ca,2nM PO4
2.5 mM Ca,2μM PO4
2.5 mM Ca,2mM PO4
2.5 mM Ca,20mM PO4
2.5 mM Ca,200mM PO4
Ca
(ug/
ml)
pH=7.4
pH=9.0
Ca-PO4-MABP Precipitation Experiment
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20.00
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100.00
120.00
Ca
(ug/
ml)
- 2 mM MABP
+ 2 mM MABP
MABP interactions with Ca/phosphates:
The TGA-MABP reaction product is an inhibitor of Ca-phosphate formation
The Joseph Stokes, Jr., Research Institute
Aim 3: Proposed model for 5-HT/Fenfluramine heart valve cell and/or
macrophage interactions
With resulting transcriptional effects on cytokines, proliferation, ECM
TGF1
The Joseph Stokes, Jr., Research Institute
Serotonin (5HT) Dose Response for Mitral Valve Interstitial 5HT-Receptor Signaling: ERK Phosphorylation Studies
The Joseph Stokes, Jr., Research Institute
Fenfluramine does not directly stimulate 5HTR, but by preventing transporter processing increases signaling activity
The Joseph Stokes, Jr., Research Institute
Canine Human
Fenfluramine (M):5-HT:
Serotonin transporter blockade using fenfluramine results in increased Erk1/2 phosphorylation
Erk1/pErk2/p
The Joseph Stokes, Jr., Research Institute
Fluoxetine also does not directly stimulate 5HTR, but by preventing transporter processing increases signaling activity
The Joseph Stokes, Jr., Research Institute
Human
Canine
PD-MEK inhibitorK-5HT2A antagonistSB41-5HT2B antagonistSB53-5HT2B&2C antagonistW-5HT1A antagonistGR-5HT1B antagonistBRL-5HT1D antagonist
Ketanserin, a 5HT2A receptor antagonist, inhibit signaling in both human and canine mitral valve interstitial cells
The Joseph Stokes, Jr., Research Institute
cp
m
0
200
400
600
800
1000
-8 -7 -6 -5 -405-HT (M):
ANOVA p=0.009
5HT Effects on MVIC proliferation: 3[H]Thymidine Incorporation
The Joseph Stokes, Jr., Research Institute
cp
m
0
10000
20000
30000
40000
50000
0 -9 -8 -7 -6 -5[5-HT(M)]:
ANOVA p=0.003
5HT Effects on MVIC extracellular matrix: 3[H]Proline Incorporation
The Joseph Stokes, Jr., Research Institute
cp
m
0
2000
4000
6000
8000
10000
12000
5-HT: 0 -9 -8 -7 -5-6
cpm
0
500
1000
1500
2000
2500
3000
3500
5-HT: 0 -9 -8 -7 -5-6
ANOVA (non-parametric) p=0.001 ANOVA (non-parametric) p=0.002
5HT Effects on MVIC extracellular matrix: 3[H]Glucosamine Incorporation
SecretedCellular/ECM
The Joseph Stokes, Jr., Research Institute
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50
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400
0 5 10 15 20 25
Time (min)
3H
5-H
T u
pta
ke
(pm
ol/1x10^6 c
ells)
w / 10 uM pargyline
w/o 10 uM pargyline
3H-5HT Uptake by Mitral Valve Interstitial Cells:5HTT Functionality
The Joseph Stokes, Jr., Research Institute
Work in progress
Effects of fenfluramine, fluoxetine & ketanserin on 5HT-related endpoints in cell culture
Canine clinical trial treating myxomatous mitral valve disease with ketanserin: MTA with Jannsen under negotiation
Genotyping patients with mitral valve disease: re. 5HTT polymorphisms, HUP IRB Protocol is now approved
The Joseph Stokes, Jr., Research Institute
Collaborators
Jeanne ConnollyJames FulmerJoseph Gorman, M.D., HUPRobert Gorman, M.D., HUPMark Oyama, DVM, Penn Vet. SchoolH. Scott RapoportKen Ryan, Ph.D.Michael Sacks, Ph.D., Univ.PittsburghStan Stachelek, Ph.D.