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Arteriosclerosis, Thrombosis, and Vascular Biology. 2007;27:819-825
Published online before print February 8, 2007, doi: 10.1161/01.ATV.0000260001.55955.6c
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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2007;27:819.)
© 2007 American Heart Association, Inc.

Vascular Biology

VEGFR-1 and -2 Regulate Inflammation, Myocardial Angiogenesis, and Arteriosclerosis in Chronically Rejecting Cardiac Allografts

Olivier Raisky; Antti I. Nykänen; Rainer Krebs; Maria Hollmén; Mikko A.I. Keränen; Jussi M. Tikkanen; Roope Sihvola; Leena Alhonen; Petri Salven; Yan Wu; Daniel J. Hicklin; Kari Alitalo; Petri K. Koskinen; Karl B. Lemström

From the Cardiopulmonary Research Group, Transplantation Laboratory (A.I.N., R.K., M.H., M.A.I.K., J.M.T., R.S., P.K.K, K.B.L.), University of Helsinki and Helsinki University Central Hospital; Department of Medicine, Division of Nephrology (P.K.K.), Department of Cardiothoracic Surgery (K.B.L.), Helsinki University Central Hospital; Molecular Cancer Biology Laboratory (K.A.) and Institute of Biomedicine (P.S.), Biomedicum Helsinki, University of Helsinki, Helsinki, Finland; Department of Biotechnology and Molecular Medicine, A.I. Virtanen Institute for Molecular Sciences (L.A.), University of Kuopio, Finland; Hôpital Cardiologique Louis Pradel (O.R), Lyon, France; ImClone Systems Incorporated (Y.W., D.J.H.), New York.

Correspondence to Antti Nykänen, MD, Transplantation Laboratory, Haartman Institute, P.O.Box 21 (Haartmaninkatu 3), FIN-00014 University of Helsinki, Finland. E-mail Antti.Nykanen{at}Helsinki.Fi


*    Abstract
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 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
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Objective— Interplay between inflammation and angiogenesis is important in pathological reparative processes such as arteriosclerosis. We investigated how the two vascular endothelial growth factor receptors VEGFR-1 and -2 regulate these events in chronically rejecting cardiac allografts.

Methods and Results— Chronic rejection in mouse cardiac allografts induced primitive myocardial, adventitial, and intimal angiogenesis with endothelial expression of CD31, stem cell marker c-kit, and VEGFR-2. Experiments using marker gene mice or rats as cardiac allograft recipients revealed that replacement of cardiac allograft endothelial cells with recipient bone marrow– or non–bone marrow–derived cells was rare and restricted only to sites with severe injury. Targeting VEGFR-1 with neutralizing antibodies in mice reduced allograft CD11b+ myelomonocyte infiltration and allograft arteriosclerosis. VEGFR-2 inhibition prevented myocardial c-kit+ and CD31+ angiogenesis in the allograft, and decreased allograft inflammation and arteriosclerosis.

Conclusions— These results suggest interplay of inflammation, primitive donor-derived myocardial angiogenesis, and arteriosclerosis in transplanted hearts, and that targeting VEGFR-1 and -2 differentially regulate these pathological reparative processes.

Interplay between inflammation and angiogenesis is important in pathological reparative processes such as arteriosclerosis. We investigated how the two vascular endothelial growth factor receptors VEGFR-1 and -2 regulate these events in chronically rejecting cardiac allografts. Our results suggest interplay of inflammation, primitive donor-derived myocardial angiogenesis, and arteriosclerosis in transplanted hearts, and that targeting VEGFR-1 and -2 differentially regulate these pathological reparative processes.


Key Words: angiogenesis • inflammation • transplantation • arteriosclerosis • stem cells


*    Introduction
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 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
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Normal adult vasculature is in a quiescent state,1 but growth of new blood vessels is seen in many physiological and pathological conditions involving hypoxia and inflammation.2,3 A transplanted heart faces many peri- and postoperative nonimmunologic and immunologic stimuli that may be interpreted as a need for new blood vessels. Recent knowledge indicates that angiogenic growth factors are involved in allograft inflammation4–10 and the development of cardiac allograft vasculopathy (CAV)5–7—the main reason for poor long-term survival of heart transplant patients.11

VEGF functions through 2 tyrosine kinase receptors—VEGFR-1 and VEGFR-2.3 In addition to endothelial cells (ECs), VEGFR-1 is expressed in bone marrow (BM) progenitor cells,12 myelomonocytic inflammatory cells,13,14 and vascular smooth muscle cells (SMCs).15,16 VEGFR-1 in myeloid cells mediates monocyte migration13,14 and has a regulatory role in the development of inflammatory diseases such as rheumatoid arthritis17 and arteriosclerosis.18 VEGFR-1 expression in SMCs is induced by vascular injury and the receptor is involved in neointimal development.15,16,19

VEGFR-2 elicits the main mitogenic, angiogenic, and permeability effects of VEGF on ECs,3 and it has a key role in developmental angiogenesis and hematopoiesis.20 In adults, VEGFR-2 expression is downregulated in nonfenestrated vasculature such as in the heart, making these capillaries resistant to VEGF inhibition.1 In turn, VEGFR-2 is expressed at sites with active angiogenesis eg, after myocardial infarction,21 increases vascular permeability in ischemic heart,22 and participates in sepsis-related cardiac dysfunction.23 VEGF also mobilizes VEGFR-2+ endothelial progenitor cells (EPCs) from BM24 that either directly differentiate to ECs,25 or participate in angiogenesis by secreting paracrine signals to local ECs.26–28 In addition, the recruited EPCs may activate a resident cardiac pool of c-kit+ progenitor cells27—cardiac stem cells27,29,30—that preferably differentiate to ECs in injury.30

We previously showed that VEGF gene transfer enhances cardiac allograft arteriosclerosis,5 whereas VEGF inhibition with PTK7875,6—which also inhibits other receptor tyrosine kinases31—had opposite effects. In this study, we investigated the specific roles of the 2 VEGFR in cardiac allograft inflammation, angiogenesis, and arteriosclerosis, and the contribution of donor and recipient cells in allograft angiogenesis. We found that chronic rejection in transplanted hearts induced donor-derived myocardial, adventitial, and intimal angiogenesis with endothelial expression of CD31, c-kit, and VEGFR-2. VEGFR-1 inhibition reduced allograft CD11b+ myelomonocyte infiltration and allograft arteriosclerosis whereas targeting VEGFR-2 prevented c-kit+ and CD31+ angiogenesis, and decreased allograft inflammation and arteriosclerosis. These results suggest interplay of inflammation, primitive donor-derived angiogenesis, and arteriosclerosis in transplanted hearts, and that VEGFR-1 and -2 differentially regulate these pathological reparative processes.


*    Materials and Methods
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 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Experimental Design
Mouse chronic rejection heart transplantation model and immunohistochemical stainings were used to identify angiogenesis and progenitor cells in allografts. Marker gene mice and rats, and strain-specific major histocompatibility complex (MHC) class I antibodies, were used to determine whether allograft ECs originate from the donor or from the recipient. Neutralizing antibodies were used to investigate the functional role of VEGFR-1 and -2 on mouse cardiac allograft angiogenesis, inflammation, and arteriosclerosis. For the detailed methods please see the supplemental materials, available online at http://atvb.ahajournals.org.

Mouse Chronic Rejection Heterotopic Heart Transplantation Model
Heterotopic cardiac allografts were transplanted in abdominal position from Balb (B/c, H-2d) to C57 (B6, H-2b) mice (Harlan, Horst, The Netherlands). The recipients received suboptimal FK506 immunosuppression (i.m. formulation, kindly provided by Fujisawa, currently Astellas Pharma, Tokyo, Japan) and the allografts were harvested at 8 weeks.

Origin of Allograft Endothelial Cells
Tie1/LacZ rats32 were used to as allograft recipients (n=4) or donors (n=14, with or without immunosuppression) to investigate Tie1 expression, and the origin of Tie1 positive ECs in transplanted hearts. Contribution of BM-derived cells in allograft angiogenesis was investigated using recipient mice with green fluorescent protein-expressing BM cells (GFP-BM, n=3).33

VEGFR-1 and VEGFR-2 Inhibition
Cardiac allograft recipients were treated with 800 µg of rat IgG (n=8; Sigma-Aldrich), anti–VEGFR-1 antibody (n=9; MF1, ImClone), anti–VEGFR-2 antibody (n=9; DC101, ImClone), or their combination (n=10) every third day for 10 doses, starting immediately after the transplantation.

Histology and Immunohistochemistry
Arterial occlusion percentage was determined using morphometry. Immunohistochemical stainings were performed using peroxidase ABC method or Alexa Fluor 488 (green) and 568 (red, Promega) secondary antibodies.

Analysis of Immunohistochemical Stainings
Allograft parenchymal inflammatory cells and c-kit+ capillaries were counted from 16 random sections, and are given as the mean density of positive cells or vessels. CD31 and {alpha}-SMA immunofluorescense stainings were analyzed with Axioplan 2 microscope and Axiovision 4.2 analysis software (Carl Zeiss) using a semi-automated script.

Real Time RT-PCR
Total RNA was extracted using RNeasy Mini Kit (Qiagen) (n=4 to 6 per group). RT-PCR reactions were carried out using LightCycler (Roche) and the results are given in relation to 18S rRNA molecule numbers.

Statistical Analysis
Data are mean±SEM and analyzed by parametric ANOVA with Dunnett correction to compare the treatment groups to the control group. Linear regression analysis was applied to evaluate relation of c-kit+ cells to CD11b+ cells and to CAV. P<0.05 was regarded as statistically significant.


*    Results
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 *Results
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Chronic Rejection Induces Primitive Myocardial Angiogenesis in Cardiac Allografts
We detected only occasional stem cell marker c-kit immunoreactive cells in cross sections of nontransplanted mouse hearts (Figure 1A, arrow). In contrast, numerous myocardial capillary-like c-kit+ cells (Figure 1B, arrows) and c-kit+ vein ECs (Figure 1B, arrowheads) were seen in chronically-rejecting cardiac allografts harvested 2 months after the operation. In allografts with severe arteriosclerotic changes, c-kit+ cells were also found in the adventitia and intima of coronary arteries (Figure 1C). Allograft myocardial c-kit+ cells were nearly all positive for endothelial marker CD31 (Figure 1D), and coexpressed VEGFR-2 (Figure 1E). The majority of c-kit+ capillaries did not express proliferation marker Ki67 but some c-kit+ cells with nuclear Ki67 immunoreactivity were also detected (Figure 1F, arrows). In contrast to the preferential expression of VEGFR-2 in the endothelium, VEGFR-1 was mainly expressed in allograft {alpha}-SMA+ SMCs (Figure 1G). In peripheral blood, over 50% of VEGFR-1+ cells coexpressed the myelomonocyte marker CD11b (data not shown).14


Figure 1
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  		Figure 1. Primitive myocardial angiogenesis in chronically rejecting cardiac allografts. Only few c-kit immunoreactive cells were detected in normal mouse hearts (A, arrow) whereas numerous c-kit+ cells were found in myocardium (B, arrow), vein endothelium (B, arrowheads), and arterial adventitia and intima (C) of chronically-rejecting mouse cardiac allografts under FK506 immunosuppression 2 months after transplantation. The c-kit+ cells coexpressed CD31 (D) and VEGFR-2 (E), and some expressed Ki67 (F, arrows). VEGFR-1 was mainly expressed in the media of arteries (G). The density of myocardial c-kit+ cells in cardiac allografts correlated with CD11b+ myelomonocyte density (H), incidence of arteriosclerotic changes (I) and mean occlusion of allograft arteries (J). A–C, Mayer hemalum staining; no specific immunoreactivity with IgG control (B, inset). Dashed line indicates lamina elastica interna (C). DAPI nuclear staining is shown in blue (D–G). The white box shows the area magnified for the single color images (D–G). Data analyzed by linear regression analysis (H–J). Scale bars=50 µm.

A positive correlation was verified between the density of c-kit+ capillaries in the myocardium and the number of allograft-infiltrating CD11b+ myelomonocytic inflammatory cells (Figure 1H), as well as with the incidence of arteriosclerotic changes (Figure 1I) and the mean occlusion of allograft arteries (Figure 1J). These results indicate that chronic rejection in transplanted hearts induces myocardial, adventitial, and intimal angiogenesis with endothelial expression of primitive markers c-kit and VEGFR-2.

Endothelial Replacement With Recipient-Derived Cells Is Rare in Cardiac Allografts
As recipient-derived circulating EPCs could differentiate to ECs in the transplanted heart, we next determined the origin of cardiac allograft ECs by using marker gene rats (Tie1/LacZ) or mice (GFP-BM) as allograft recipients. When Tie1/LacZ allografts were transplanted to wild-type (WT) recipients, areas with abundant X-gal reactivity in allograft endothelium was detected (Figure 2A and 2B) indicating Tie1 expression in the donor ECs. Next, WT cardiac allografts were transplanted to Tie1/LacZ recipients to detect recipient-derived ECs in the transplanted hearts. Only few donor-derived X-gal+ ECs (Figure 2C and 2D, arrows) localizing to severely fibrotic areas were seen in cross sections in a total of 14 WT cardiac allografts.


Figure 2
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  		Figure 2. Endothelial replacement with recipient-derived cells in chronically rejecting cardiac allografts. Areas with abundant X-gal reactivity were detected in vein (A, arrow) and capillary endothelium (A and B) of the Tie1/LacZ cardiac allografts transplanted to WT recipients (n=4). Only few recipient-derived Tie1+ ECs (C and D, arrows) were detected in WT cardiac allografts (n=14) transplanted into Tie1/LacZ recipients. No BM-derived GFP+ cells colocalized with CD31+ EC (E) or c-kit+ capillaries (F) in WT allografts transplanted to GFP-BM recipients (n=3). Anti-C57 (G) and anti-Balb (H) MHC class I immunohistochemical stainings revealed numerous recipient cells around occluded arteries (G, arrows) but only few in the intima (G). In contrast, numerous donor cells were found in the neointimal (H, arrows). A–D, Nuclear fast red staining. DAPI nuclear staining is shown in blue (E and F). G and H, Mayer hemalum staining. Transplantation indicated by TX. Scale bars=50 µm.

Additionally, GFP-BM mice were used as cardiac allograft recipients allowing the detection of BM-derived cells in the allografts. The majority of allograft-infiltrating CD11b+ myelomonocytic cells expressed GFP (data not shown). Although GFP+ cells often surrounded allograft blood vessels, no colocalization with allograft CD31+ (Figure 2E) or c-kit+ capillaries (Figure 2E) was detected.

Furthermore, donor- and recipient-specific MHC class I antibodies were used to identify the source of ECs in allograft arteriosclerotic arteries. Numerous recipient MHC class I+ cells were found around occluded arteries (Figure 2G, arrows) whereas only few positive cells were detected in the intima (Figure 2G). In contrast, abundant donor MHC Class I immunoreactivity was found in the neointima (Figure 2H, arrows). The contribution of recipient-derived SMCs to neointimal formation34 was not assessed as MHC Class I expression was low in SMCs34 (Figure 2G and 2H).

VEGFR-2 Inhibition Normalizes C-Kit+ and CD31+ Capillary Density in Chronically Rejecting Cardiac Allografts
To investigate the functional role of VEGFR-1 and -2, mouse cardiac allograft recipients were treated with rat IgG, or antibodies against VEGFR-1, VEGFR-2, or both. Two months after heart transplantation, targeting VEGFR-2 reduced the density of myocardial c-kit+ capillaries (Figure 3A) and CD31+ capillaries (Figure 3B) in the allograft to the level found in nontransplanted mouse hearts (Figure 3A and 3B, dashed lines). VEGFR-1 inhibition also resulted in a smaller decrease in c-kit+ capillary density (P=NS with Dunnett correction, P<0.05 with LSD correction). VEGFR-1 or -2 inhibition did not change the density of SMC coated vessels (Figure 3C) indicating that VEGFR-2 inhibition specifically regulated angiogenesis at microvascular level.


Figure 3
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  		Figure 3. The effect of VEGFR inhibition on capillary angiogenesis and arteriosclerosis in chronically rejecting mouse cardiac allografts. Chronically rejecting mouse cardiac allograft recipients with suboptimal FK506 immunosuppression received IgG (n=8), or monoclonal antibodies against VEGFR-1 (MF1, n=9), VEGFR-2 (DC101, n=9), or both (n=10) for 30 days. At 8 weeks, VEGFR-2 inhibition decreased allograft myocardial c-kit+ (A) and CD31+ capillary density (B). VEGFR inhibition did not change {alpha}-SMA+ vessel density (C). A, Mayer hemalum staining. The dashed red line indicates nontransplanted mouse heart values (A and B). Data are given as mean±SEM, by ANOVA with Dunnett correction, comparing the treatment groups with the control group. Scale bars=50 µm.

VEGFR-1 and -2 Inhibition Reduces Inflammation in Chronically Rejecting Cardiac Allografts
Immunohistochemical analysis showed that targeting VEGFR-1, VEGFR-2, or both profoundly reduced the density of allograft-infiltrating CD11b+ myelomonocytic cells (supplemental Figure IA, available online at http://atvb.ahajournals.org). VEGFR-2 inhibition also resulted in a similar reduction in CD8+ (Figure IB) and CD4+ (supplemental Figure IC) lymphocyte density in the allograft (for the combination group: P=NS with Dunnett correction and P<0.05 with LSD correction).

VEGFR-1 and -2 Inhibition Reduces Arteriosclerosis in Chronically Rejecting Cardiac Allografts
Morphometric analysis of allograft arteries revealed that targeting VEGFR-1, VEGFR-2, or both decreased the incidence of allograft arteries with intimal changes (Figure 4A). A similar result was also obtained on the mean occlusion of allograft arteries (Figure 4B). These results indicate that both VEGFR-1 and -2 are involved in events leading to CAV.


Figure 4
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  		Figure 4. The effect of VEGFR inhibition on arteriosclerosis in chronically rejecting mouse cardiac allografts. VEGFR inhibition reduced the incidence (A) and severity (B) of allograft arteriosclerosis. Representative photomicrographs from IgG (C), VEGFR1-Ab (D), VEGFR2-Ab (E), and VEGFR1-Ab and VEGFR2-Ab (F) treated animals stained with hematoxylin-eosin and Resorcin fuchsin for internal elastic lamina. Data as in Figure 3. Scale bars=50 µm.

Effect of VEGFR-1 and -2 Inhibition on Allograft Cytokine mRNA Levels
Finally, we used real time RT-PCR to determine the mRNA levels of inflammatory cytokines IFN-inducible protein-10 (IP-10) and monocyte chemotactic protein-1 (MCP-1) that are potentially regulated by VEGF in cardiac allografts,8,35 and the mRNA levels of stem cell factor (SCF) that is the ligand for c-kit. VEGFR-2 inhibition decreased allograft IP-10 mRNA by approximately 50% alone and by 75% in combination with VEGFR-1 inhibition (Figure 5A), and MCP-1 mRNA by 50% (Figure 5B). In contrast, allograft tumor necrosis factor (TNF)-{alpha} (Figure 5C) and SCF (Figure 5D) mRNA levels were similar in the control and treatment groups. These results indicate that the VEGFR-2 inhibition regulated at least in part the T cell and monocyte recruitment by decreasing IP-10 and MCP-1 production,8,35 respectively. Also, the effect of VEGFR-2-inhibition on c-kit+ capillaries was not associated with changes in SCF production.


Figure 5
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  		Figure 5. The effect of VEGFR inhibition on allograft cytokine mRNA levels. Shown are the real time RT-PCR results (n=4 to 6) for IP-10 (A), MCP-1 (B), TNF-{alpha} (C), and SCF (D). Results are in relation to 108 (A–C) or 106 (D) 18S rRNA molecules. Data as in Figure 3.


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
Angiogenesis is a prominent feature in the intima36–38 and adventitia39 of cardiac allograft coronary arteries and it may be a driving force for the development of CAV.5,40 Here we show that in addition to intimal and adventitial angiogenesis, chronic rejection induces the expression of primitive markers c-kit and VEGFR-2 in allograft myocardial capillaries. As the density of myocardial c-kit+ capillaries correlated with the severity of cardiac allograft inflammation and arteriosclerosis, alloimmune and ischemic stimuli may be important regulators of the myocardial angiogenesis we observed. This primitive c-kit+ angiogenic response probably represents a repair process that, interestingly, in light of the present VEGF intervention results, may in fact aggravate inflammation and arteriosclerosis in transplanted hearts. Importantly, there may be a balance between early capillary formation and later destruction of allograft capillaries as seen in skin transplants.41

In experiments using marker gene animals and donor or recipient specific antibodies, we found only few recipient-derived ECs in the transplanted hearts and they were restricted to severely fibrotic areas. These observations—together with earlier findings using heterotopic heart transplantation models34,42—suggest that recipient-derived circulating cells do not differentiate into allograft EC unless the injury to the allograft extensive. Although this argues against direct involvement of recipient-derived EPCs in allograft angiogenesis, these circulating cells may have important paracrine effects as seen in nontransplantation situations.26–28 Our results on the origin and c-kit+ phenotype of allograft EC further indicates that donor-derived progenitor cells—such as resident cardiac stem cells29,30,43 or adventitial stem cells44–46—directly participate in allograft angiogenesis. Alternatively, the hypoxic and inflammatory signals related to the transplantation may have induced dedifferentiation of allograft ECs to a more primitive phenotype. Interestingly, EPC-derived soluble factors such as VEGF, VEGF-B, stromal cell derived factor-1, and insulin-like growth factor-1,27 and also hepatocyte growth factor47 may regulate the functions of c-kit+ cardiac progenitor cells, and the present results suggest important role for VEGFR-2.

VEGF is perhaps the most important angiogenic cytokine and it has also many proinflammatory properties.3 The present findings support the regulatory role of VEGF in the pathogenesis of alloimmune responses8,9 and CAV5,6 in transplanted hearts, and shed light to the mechanisms, and the 2 VEGFR involved. In transplanted hearts VEGFR-2 inhibition reduced myocardial angiogenesis to the level seen in normal hearts consistent with the important angiogenic role for VEGFR-2.3 In addition, targeting VEGFR-2 decreased inflammatory cell infiltration, and production of IP-10 and MCP-1 in the allograft similarly to previous reports with anti-VEGF therapies.8 Our results thus suggest that VEGFR-2 in cardiac allografts functions mainly at the endothelial level and regulates both pathological capillary angiogenesis and inflammation. Involvement of VEGFR-2 in cardiac inflammation may be a more general phenomenon as the receptor participates in cardiac dysfunction during sepsis,23 and also in vascular permeability following myocardial infarction.22

In contrast to VEGFR-2, VEGFR-1 was primarily found in allograft SMCs and in peripheral blood myelomonocytic cells. As VEGFR-1 directly regulates SMCs during arterial injury,15,16,19 VEGFR-1 inhibition in the current study may have directly decreased SMC recruitment to the intima. VEGFR-1 inhibition also profoundly reduced myelomonocyte recruitment to the allograft consistent with its role in monocytes13,14,48 and inflammatory diseases.17,18 Although VEGFR-2 inhibition prominently decreased the density of myocardial c-kit+ cells, also VEGFR-1 inhibition had a similar but more subtle effect. This indicates that also VEGFR-1 may in part regulate the capillary angiogenesis, and possibly involves cross-talk with VEGFR-249 or indirect inflammation-mediated effects. The reason why combined VEGFR-1 and -2 inhibition did not have a beneficial additive effect may be explained by the moderate injury in the current experimental setting. Supporting this, our unpublished trachea transplantation findings (Krebs et al, 2007) show additive beneficial effect after severe but not after moderate tracheal injury.

In conclusion, we found that chronic rejection in cardiac allografts induced donor-derived capillary angiogenesis. Also, selective VEGFR-inhibition prevented allograft angiogenesis and had beneficial effects on inflammation and arteriosclerosis. These results suggest therapeutic applications for anti-VEGF strategies during pathological angiogenesis and inflammation in transplanted hearts.


*    Acknowledgments
 
We thank Ms Eeva Rouvinen, RN, for her excellent technical assistance, and Dr Anita Friedrich (Astellas Pharma GmbH, Munich, Germany) for providing FK506.

Sources of Funding

This study was supported by grants from University of Helsinki, The Academy of Finland, The Sigrid Juselius Foundation, Helsinki University Central Hospital Research Funds, The Finnish Life and Pension Insurance Companies, The Finnish Foundation for Cardiovascular Research, Research and Science Foundation of Farmos, The Finnish Medical Foundation, Ida Montin Foundation, Maud Kuistila Memorial Foundation, Aarne Koskelo Foundation and, Aarne and Aili Turunen Foundation, Biomedicum Helsinki Foundation and The Finnish Society of Transplant Surgeons.

Disclosure

D.J. Hicklin and Y. Wu are employees of ImClone Systems Inc.


*    Footnotes
 
O.R. and A.I.N. contributed equally to this study.

Original received December 11, 2006; final version accepted January 19, 2007.


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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 up arrowDiscussion
 *References
 

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