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Mechanisms of chronic allograft injury
in a heart transplant model
Karl Lemström*, Antti Nykänen*, Jussi Tikkanen, Rainer Krebs,
Mikko Keränen, Raimo Tuuminen, Alireza Raissadati,
Simo Syrjälä, Janne Jokinen*, Jussi Ropponen*, Petri Koskinen
Transplantation Laboratory, Haartman Institute, University of Helsinki &
HUSLAB, Helsinki University Central Hospital.
*Department of Cardiothoracic Surgery, Helsinki University Central
Hospital.
Transplantation Laboratory
Leading causes of death for adult heart
transplants performed between 1992-2008
Stehlik et al. J Heart Lung Transplant 2010;29:1089
Transplantation Laboratory
Graft failure - loss of functional
microvasculature
 Primary graft failure
 immediate
loss of functional microvasculature
 brain-death
 hypothermic preservation
 warm ischemia
 “marginal donors”
 reperfusion injury
 Late graft failure
 gradual
loss of functional microvasculature
 acute rejection, cellular and antibody-mediated
 CMV infection
Transplantation Laboratory
Loss of microvascular circulation
may be fundamental cause of chronic
rejection
Organ
Chronic
Loss of
Fibrosis Vasculopathy Organ specific Prevalence
inflammation microvasculature
changes
5 y after Tx
Lung
+++
++
++
++
obliterative
bronchiolitis
50-60
Heart
++
++
++
++
cardiac fibrosis
40-50
Kidney
++
++
++
++
glomerulopathy
tubulusatrophy
20-30
Liver
+
+
+
+
vanishing bile
ducts
5-10
Transplantation Laboratory
Pre-operative ischemia-reperfusion injury
enhances acute and chronic rejection and
impairs allograft survival
 new immunosuppressive drugs effectively prevent acute
rejection
 however, they do not prevent microvascular dysfunction
and signals that mediate IRI and that bridge innate and
adaptive immune responses
 therefore, new strategies that target at other pathways
than T cell proliferation are required to prevent allograft
failure
Transplantation Laboratory
Normal microvasculature maintains tissue
homeostasis
 Quiescent endothelial cells
(EC) are tightly connected to
neighbouring EC by junction
proteins
 This prevents leakage of plasma
proteins and fluids and extravasation
of leukocytes
Transplantation Laboratory
Microvascular dysfunction - role of endothelial
cells
 loss of EC integrity
 dissociation of junctional complex protein like VEcadherin from interendothelial junctions
 relocation of actin to form cellular stress fibers, and EC
contraction
 Players: hypoxia, IRI, VEGF, thrombin, activation of
RhoA GTPase, Angiopoietin-2, LPS
 Antagonists: Angiopoietin-1
Transplantation Laboratory
Microvascular dysfunction - role of pericytes
 Pericytes

Supporting cells for ECs

Regulation of vascular tone

Pericyte – EC interactions
 Pericyte contraction impairs capillary reflow after cerebral
ischemia-reperfusion injury

Yemisci et al. Nature Medicine 2009
 Pericyte Requirement for Anti-Leak Action of Angiopoietin1 and Vascular Remodeling in Sustained Inflammation

Transplantation Laboratory
Fuxe et al. Am J Pathol 2011;178:2897
Angiogenic and Lymphangiogenic Growth
Factors
 have important roles in tissue hypoxia and injury and
acute and chronic inflammation
 strictly regulated in normal vasculogenesis
 dysregulated in
Transplantation Laboratory

vascular leakage

angiogenesis

diabetic retinopathy

tumour growth

arteriosclerosis
Angiogenic growth factors regulate endothelial
cell and pericyte assembling in vasculogenesis
Ang1
VEGF
PDGFR-ß
Tie2
VEGFR
PDGF-B
Angioblast
Endothelial cells
form patent tubes
TGF-ß
PDGF-B
Ang1
Recruitment of
pericytes/SMC
Blood vessel
maturation
Modified from Cleaver&Melton Nature Medicine 2003;9:661
Transplantation Laboratory
HIF and angiogenic and lymphangiogenic growth
factors have an important role in chronic rejection
in cardiac allografts without ischemia
Adenovirus
EC Injury and
Inflammation
TxCAD
Early response
SMC
proliferation
Late response
 Ang1
 VEGF,
 VEGFR3-Ig
AAV
 Ang1/2
 PDGF-A/C/
 HIF
Abs, RTKIs
 VEGFR-1/-2
 PTK797
 imatinib
Circulation 2002;105:2524; Circulation 2003;107:1308; Circ Res 2006;98:1373; Arterioscler Thromb Vasc Biol, 2007;27:819;
J Am Coll Cardiol 2002;39:710; Transplantation 2003;75:334; Transplantation 2005;79:182; Arterioscler Thromb Vasc Biol,
2009;29:691
Transplantation Laboratory
Effect of imatinib on allograft arteriosclerosis
in cardiac allografts without any ischemia and
with 4-hour cold ischemia
4-h ischemia
Billingham score 0-4
Billingham score 0-4
No ischemia
Ctrl
Imatinib
Sihvola et al. Circulation 1999;99:2895
Transplantation Laboratory
Ctrl
Imatinib
Tuuminen et al. Unpublished
Modification of cardiac allograft model
Analysis
P hase
Ischemic time
Recovery time
point
1. Preser vation
0- and 4-h cold
ischemia,
± 1h warm ischemia
Preservation
immediate or 4hour preservation, injury
± 1-hour warm
ischemia
2. Permeabilit y
Re perfusion
0- and 4-h cold
ischemia,
± 1-h warm ischemia
30 min,
6 hours
IRI
Innate immune
response
3. Acute RX
0- and 2-h cold
ischemia,
± 1-h warm ischemia
10 days
Alloimmune
response
Early fibrosis
4. C hronic RX
0- and 2-h cold
ischemia,
± 1-h warm ischemia
8 weeks
Late fibrosis
Allograft
vasculpathy
Transplantation Laboratory
Hypothermic preservation increases innate
immune ligands and receptors during IRI
Transplantation Laboratory
Syrjala et al. J Heart Lung Transplant 2010;29;1047
Hypothermic preservation increases DC
activation and proinflammatory cytokine
production during IRI
Transplantation Laboratory
Syrjala et al. J Heart Lung Transplant 2010;29;1047
Preoperative ischemia enhances cardiac
fibrosis and arteriosclerosis
10 days after Tx
2 months after Tx
Syrjala et al. J Heart Lung Transplant 2010;29;1047
Transplantation Laboratory
Hypothesis:
HIF, angiogenic, and lymphangiogenic growth factors may
have a critical role microvascular dysfunction
Transplantation Laboratory
Statins and
microvascular dysfunction
Transplantation Laboratory
Pleiotropic effects of statins through RhoA
inhibition - cytoskeleton and intracellular signalling
Transplantation Laboratory
Microvascular endothelial cells and pericytes
express HMG-CoA reductase in normal hearts
EC
Transplantation Laboratory
Pericyte
SMC
Cardiomyocyte
Tuuminen et al Circulation 2011;124
Effect of donor simvastatin treatment on
preservation injury of cardiac allografts
DA rat
Simvastatin (5mg/kg p.o.) to donor
2 hours before graft removal
WF rat
4-h cold and 1-h warm ischemia
0
VascuIar integrity
Graft mRNA
Transplantation Laboratory
30 min
Permeability
Perfusion
6h
IRI
10 d
2 months
Alloimmunity
Fibrotic cascades
Chronic rejection
Tuuminen et al Circulation 2011;124
During preservation donor simvastatin treatment
prevents phosphorylation of adducin downstream of RhoA/ROCK activation
Transplantation Laboratory
Tuuminen et al Circulation 2011;124
Donor simvastatin treatment prevents EC-EC
gap formation during preservation
Transmission electron microscopy
EC-EC gaps
Intracoronary lectin perfusion: EC + exposed BM
Transplantation Laboratory
Donor simvastatin decreases HIF-1, iNOS and
ET-1 and increases HO-1 mRNA during
preservation
Transplantation Laboratory
Donor simvastatin prevents vascular leakage
and maintains capillary perfusion 30 min after
re-perfusion
Extravasation of
Evans blue dye
L. Esculentum
lectin
Transplantation Laboratory
Tuuminen et al Circulation 2011;124
Donor simvastatin treatment restores
immediate microvascular circulation and
prevents cardiac edema after reperfusion
laser doppler
MRI
Transplantation Laboratory
Tuuminen et al Circulation 2011;124
Effect of donor simvastatin treatment on
ischemia-reperfusion injury
DA rat
Simvastatin to donor, recipient, or
both
WF rat
4-h cold and 1-h warm ischemia
0
VascuIar integrity
Graft mRNA
Transplantation Laboratory
30 min
Permeability
Perfusion
6h
IRI
10 d
2 months
Alloimmunity
Fibrotic cascades
Chronic rejection
Tuuminen et al Circulation 2011;124
Donor but not recipient simvastatin treatment
prevents ischemia-reperfusion injury
Troponin-t
Macrophages
Neutrophils
D – Donor simvastatin treatment
R – Recipient simvastatin treatment
D/R – Donor and Recipient simvastatin treatment
Transplantation Laboratory
Tuuminen et al Circulation 2011;124
Donor simvastatin treatment increases Ang-1
and Hsp27 and decreases HIF-1, bFGF and
TGF-ß during IRI
Transplantation Laboratory
Tuuminen et al Circulation 2011;124
Beneficial effects of donor simvastatin treatment
on microvascular stability and perfusion are
mediated through NO and RhoA
Vascular leakage
Macrophages
Transplantation Laboratory
Neutrophils
Perfused vessels
CD4+ T cells
L-NAME - NOS inhibition
ZnPP - HO-1 inhibition
GGPP - RhoA pathway supplementation
Troponin T
CD8+ T cells
OX62+ DC
Tuuminen et al Circulation 2011;124
Donor simvastatin treatment has sustained
anti-inflammatory effects 10 days after TX
VCAM-1
Macrophages
Transplantation Laboratory
Neutrophils
CD4+ T cells
CD8+ T cells
OX62+ DC
Tuuminen et al Circulation 2011;124
Donor simvastatin treatment decreases
fibrosis and TGF-b signaling 10 days after TX
Fibroblasts
TGF-ß activation
Transplantation Laboratory
Tuuminen et al Circulation 2011;124
Simvastatin inhibits TGF-b-induced
endothelial-mesenchymal transition of human
cardiac microvascular endothelial cells
Transplantation Laboratory
Tuuminen et al Circulation 2011;124
Simvastatin treatment of both donor and recipient maintains
microvascular density and reduces fibrosis and arteriosclerosis in
cardiac allografts
Capillary
density
Fibrosis
Transplantation Laboratory
Arterial
occlusion
Acknowledgments
Transplantation Laboratory
Molecular Cancer Biology Laboratory,
 Antti Nykänen, MD
Biomedicum Helsinki, Helsinki, Finland
 Jussi Tikkanen, MD, PhD
 Kari Alitalo, MD, PhD
 Petri Koskinen, MD, PhD
 Rainer Krebs, MSc
A.I. Virtanen Institute for Molecular Sciences,
 Mikko Keränen, MD
University of Kuopio, Kuopio, Finland
 Raimo Tuuminen, MD
 Seppo Ylä-Herttuala, MD, PhD
 Simo Syrjälä, MB
 Alireza Raissadati, MB
Imclone, New York
 Janne Jokinen, MD, PhD
 Jan Wy, PhD
 Jussi Ropponen, MD
 Eeva Rouvinen, RN
Novartis Pharma, Basle, Switzerland
 Eriika Wasenius, RN
 Elisabeth Buchdunger, PhD
 Jeanette Wood, PhD
 Risto Renkonen, MD
 Heikki Helin, MD
Transplantation Laboratory
Acknowledgments - Sources of funding
 Academy of Finland
 Sigrid Juselius Foundation
 Helsinki University Central Hospital Research Funds
 Finnish Foundation for Cardiovascular Research
 Päivikki and Sakari Sohlberg Foundation
 University of Helsinki
Transplantation Laboratory
Thank you!
Transplantation Laboratory