(Circulation. 1998;98:2248-2254.)
© 1998 American Heart Association, Inc.
Clinical Investigation and Reports |
Gene Polymorphisms for Plasminogen Activator Inhibitor-1/Tissue Plasminogen Activator and Development of Allograft Coronary Artery Disease
From the Department of Medicine (R.L.B., H.E.G., R.C.B., P.F.C., F.M.B.), Division of Cardiovascular Disease, and the Department of Surgery (J.K.K., D.C.N., D.C.M., J.F.G.), Division of Cardiothoracic Surgery, University of Alabama at Birmingham.
Correspondence to Raymond L. Benza, MD, Division of Cardiovascular Disease, Department of Medicine, University of Alabama at Birmingham, 809 BBRB, 845 19th St S, Birmingham, AL 35294-2170. E-mail rbenza{at}cardio.dom.uab.edu
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Background—Impaired fibrinolytic activity has been linked to the presence and severity of allograft vasculopathy (Tx CAD). This impairment may be associated with the presence of certain fibrinolytic protein gene polymorphisms.
Methods and Results—To investigate the relation between donor-specific fibrinolytic protein genotypes and Tx CAD, we identified donor plasminogen activator inhibitor-1 (PAI-1) HindIII and tissue plasminogen activator (TPA) EcoRI restriction fragment length polymorphisms–based genotypes by Southern blot analysis in 48 recipients of cardiac allografts and correlated these genotypes with the development of CAD. No association was found between donor TPA genotypes and the presence of Tx CAD. Among the 48 patients, 17% were homozygous for the 1/1 PAI-1 genotype, 51% for the 2/2 PAI-1 genotype, and 32% for the 1/2 PAI-1 genotype. The actuarial freedom from any CAD for the recipients with each respective donor PAI-1 genotype at 12 and 24 months was 100% and 100% for the 1/1 PAI-1 genotype, 92% and 92% for the 1/2 PAI-1 genotype, and 75% and 45% for the 2/2 PAI-1 genotype (P=0.03). Recipients with a diseased 2/2 PAI-1 genotyped allograft had longer ischemic times (P=0.02) than those recipients with a Tx CAD–free allograft.
Conclusions—These data suggest that recipients with a 2/2 PAI-1 genotype are at a significant risk of developing Tx CAD. This genotype may serve as a useful screening tool for predicting the future development of Tx CAD.
Key Words: coronary disease • fibrinolysis • genes • transplantation
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The development of an accelerated form of coronary artery disease (CAD) is a major limitation to long-term survival after heart transplantation. The development of this disease most likely involves a complicated interplay between immune and nonimmune factors. Among the nonimmune factors, impaired fibrinolytic activity and/or enhanced thrombogenicity have assumed some importance as a possible causative event. Fibrin deposition, the direct result of impaired fibrinolysis, is a common histological feature in the microcirculation of grafts with allograft vasculopathy (Tx CAD)1 2 3 and usually results from diminished levels of the tissue plasminogen activator (TPA) and/or the presence of excess PAI-1, the physiological inhibitor of TPA. Allografts with depleted levels of arteriolar TPA are at greater risk of developing earlier and more severe disease than those grafts with normal arteriolar TPA.4 Understanding the regulation of arteriolar TPA and PAI-1 may, therefore, be useful in understanding the mechanisms underlying Tx CAD.
Tissue levels of PAI-1 and TPA are reflective, in part, to the degree of gene expression within each arteriole. Transplantation of solid organs leads to a situation in which 2 sets of genes, donor and recipient, can influence the expression of fibrinolytic proteins. The specific degree of fibrinolytic protein expression within the arteriolar walls of the graft, however, most likely represents the expression of the donor's genes. The expression of these donor genes (PAI-1 and TPA) may then be influenced by the actions of various growth factors and metabolic components (ie, insulin, triglycerides)5 6 7 8 9 present in the recipient's plasma. Additionally, fibrinolytic protein expression may be regulated by the presence of specific gene polymorphisms.10 11 12
Using a HindIII restriction fragment length polymorphism (RFLP) as marker for genetic variation, 3 genotypes designated 1/1, 1/2, and 2/2 have been described for the PAI-1 gene.11 12 Analysis of these genotypes in young patients after infarction and population-based control subjects demonstrated an association between plasma PAI-1 levels, the 2/2 PAI-1 genotype and plasma very low-density lipoprotein (VLDL), and the 1/1 PAI-1 genotype and insulin.12 In vitro models with PAI-1–genotyped cultured human endothelial cells have demonstrated similar associations.13 14 15 Both hypertriglyceridemia and drug-induced hyperinsulinemia are common problems in transplantation and may induce a specific upregulation of PAI-1 expression in the arterioles of allografts with the responsive form of the PAI-1 gene. Upregulation of PAI-1 could then deplete TPA and predispose to Tx CAD.
An EcoRI RFLP in the exon 8 region of the TPA gene has also been described.16 Although the relation between TPA genotypes, plasma TPA levels, and CAD risk factors is not as well characterized as those for PAI-1, these TPA polymorphisms may play a determining role in the development of Tx CAD.
This cross-sectional, retrospective study was undertaken to determine whether an association exists between donor PAI-1 and/or TPA genotypes and the extent of angiographically defined Tx CAD and whether an additional correlation can be demonstrated between donor PAI-1 and/or TPA genotypes and certain risk factors for Tx CAD.
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Patients
Between June 3, 1992, and April 19, 1995, 97 patients underwent heart transplantation at the University of Alabama (UAB) at Birmingham. Donor tissue was available for genotyping 50 of these recipients. Two of these patients did not have information regarding the presence of CAD by angiography and were excluded from the analysis. Consequently, the analysis consisted of 48 patients. Follow-up for this study was complete up to June 30, 1996.
Immunosuppression between 1992 and 1995 consisted of triple drug therapy with cyclosporine, azathioprine, and prednisone.
Diagnosis and Grading of CAD
Coronary angiography was performed according to the
Judkins technique in all cases. CAD was assessed using side-by-side
comparisons with prior angiograms by 2 angiographers who were unaware
of the PAI-1 or TPA genotype status of the donor hearts.
Primary vessels were classified as proximal or mid one third of the
left anterior descending (LAD), artery, the left circumflex (LCx)
artery, or the dominant (or codominant) right coronary artery
(RCA). Branch vessels were either the diagonals, posterolateral
segmental arteries (PLSMs), marginals, or distal one third of a
primary vessel or any part of a nondominant RCA. Disease was considered
mild if the lesions were a <50% left main, <70% primary vessel,
>70% isolated single branch, or any branch stenosis <70%
(including diffuse narrowing). Disease was considered moderate if the
lesions were a 50% to 70% left main, >70% single primary vessel, or
>70% isolated single branch of 2 systems. Disease was considered
severe if the lesions were a >70% left main, or >70% in 
2 primary
vessels, >70% isolated single branch of all 3 systems. One hundred
twenty-two angiograms (2.5 per patient) were available for review over
the study period on the 48 patients (39 angiograms [32%] at <6
months, 48 [39%] at 1 year, 28 at 2 years [23%], and 8 [7%] at
3 years). In addition, each patient was assessed for fatal events
related to Tx CAD.
Donor and Recipient Demographics
Pretransplantation demographic information was collected on
donors and recipients through chart review and from data extracted from
the University of Alabama Cardiac Transplant Research Database. This
data included specific information on donor and recipient age, sex,
race, cytomegalovirus (CMV) status, presence of diabetes or
hypertension, ischemic time (minutes), pretransplantation
diagnosis, status at time of transplantation, and use of induction
therapy. Additional information was collected on recipients 12 months
after transplantation and included weight (pounds); systolic
and diastolic blood pressure (mm Hg), presence of diabetes
mellitus (insulin-dependent and non–insulin-dependent);
immunosuppressant use, dosing and levels where appropriate; length of
follow-up; CMV infection; creatinine levels; and number of
rejection episodes. Fasting cholesterol,
triglyceride, high-density lipoprotein (HDL), and
low-density lipoprotein (LDL) levels as well as lipid-lowering drug use
were also obtained by review of appropriate laboratory and clinic
data.
Southern Blot Analysis of PAI-1 and TPA RFLPs
Genomic DNA was isolated from pieces of donor spleen taken at
the time of allograft harvesting. DNAs (5 to 10 µg) were digested
separately for 16 hours at 37°C with the restriction enzymes
HindIII and EcoRI for PAI-1 and TPA,
respectively. Digests were separated by electrophoresis through a 0.8%
agarose gel and transferred to a nylon
membrane.17 Blots were separately hybridized with
either a 2.2-kb PAI-1 or 2-kb TPA cDNA probe, radiolabeled with
[
32P]dCTP (3000 Ci/mmol, Amersham Corp) by
random primer extension, according to the manufacturer's instructions
(Prime-It II, Stratagene). The filters were washed twice in 2x SSC,
0.5% SDS at room temperature for 30 minutes each, once in 1x SSC,
2.5% SDS at 68°C for 30 minutes and once in 0.1x SSC, 0.1 SDS at
68°C for 15 minutes.17 Phosphorimaging
autoradiography was then used to generate appropriate
hard copies of the results and to identify the presence of: 22-kb
and/or 18-kb fragments, corresponding to the PAI-1 genotypes,
designated 1/1 "homozygous" form (22-kb band only), 1/2
"heterozygous" form (22- plus 18-kb bands), and 2/2
"homozygous" form (18-kb only, contains mutation
site).12 18 Similarly, the 2.9-kb and 2.5-kb
fragments, corresponding to the TPA genotypes, were designated
1/1 "homozygous" form (2.9 band only), 1/2 "heterozygous" form
(2.9- plus 2.5-kb bands), and 2/2 "homozygous" form (2.5-kb band
only, contains mutation site).16 All Southern
blot genotyping results (hard copies) were assessed independently by 2
different investigators.
Statistical Analyses
Measures of Tx CAD were compared across the fibrinolytic protein
genotypes with the use of Kaplan-Meier estimation combined with
the log-rank test. The time-related distribution of these events was
estimated by parametric hazard analysis.
Multivariate analysis in this domain enabled
the calculation of adjusted tests and identified risk factors. Factors
included in the multivariate analysis for the
time to first disease included donor and recipient age, sex, race, CMV
status, presence of diabetes or hypertension, ischemic time
(minutes), pretransplantation diagnosis, status at time of
transplantation, use of induction therapy, recipient weight (pounds),
systolic and diastolic blood pressure (mm Hg), and
presence of diabetes mellitus (insulin-dependent and
non–insulin-dependent). These same factors were used for the
multivariate analysis for the time to first
disease in recipient's with only a 2/2 PAI-1 genotyped
allograft. The genotype groups were compared for equality of
demographic and laboratory variables with the use of basic methods
such as ANOVA, contingency table analysis, and techniques for
time trends.
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Distribution of Donor PAI-1 and TPA Genotypes in Cardiac Allograft Recipients
The distribution of donor PAI-1 (HindIII) genotypes in the study population (n=48), included 8 (17%) 1/1, 15 (32%) 1/2, and 24 (51%) 2/2. The distribution of donor TPA (EcoRI) genotypes included 13 (28%) 1/1, 24 (51%) 1/2, and 10 (21%) 2/2. A donor PAI-1 genotype was unobtainable for 1 patient and a TPA genotype for a second patient.
Relation Between Donor PAI-1 and TPA Genotypes and Presence
of Tx CAD
There was no significant difference in the development of Tx CAD
among recipients with each of the respective TPA genotypes
(Figure 1
). A significant association was
noted between recipients with a donor 2/2 PAI-1–genotyped
allograft and the development of Tx CAD (P<0.05) (Table 1). Recipients with a donor 2/2
PAI-1 genotyped allograft were significantly less likely to be
free of any Tx CAD than those recipients with a donor 1/1 or 1/2
PAI-1–genotyped allograft by 24 months (P=0.03).
The actuarial freedom from any CAD for the recipients with each
respective donor PAI-1 genotype at 12 and 24 months was 100%
and 100% for the 1/1 PAI-1 genotype, 92% and 92% for the 1/2
PAI-1 genotype, and 75% and 45% for the 2/2 PAI-1
genotype (Figure 2).
Multivariable analysis in the hazard function domain of
pretransplantation donor and recipient factors (see "Statistical
Methods") demonstrated that only the 2/2 PAI-1 donor genotype
was associated with a shorter time to Tx CAD (P=0.01). Older
donor age also appeared to be a risk factor in this evaluation
(P=0.07). A nomogram demonstrating the combined effects of
donor age and donor PAI-1 genotypes on the development of Tx
CAD at 24 months is illustrated in Figure 3. Although older donor age appeared to
increase the risk of developing Tx CAD in recipients with all 3 donor
PAI-1 genotypes, the effect was clearly more important if a
donor 2/2 PAI-1 genotype was present.
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Relation Between Donor PAI-1 Genotypes, Severity of Tx CAD,
and Death
There was no detectable relation between PAI-1 genotypes
and the overall number of allografts with severe disease
(P=0.4) or death related to Tx CAD (P=0.4) over
the study period (see Table 1
).
Donor and Recipient Demographics by Donor PAI-1 Genotypes
Univariate analyses of donor and recipient
demographics are summarized in Table 2
and Table 3. No differences were noted in
baseline donor or recipient characteristics among recipients with each
of the 3 PAI-1 genotypes. Serum cholesterol and
azathioprine doses were significantly greater at 12 months in
recipients with a 2/2 PAI-1 genotyped allograft (Table 3). No
other significant differences were noted at that time interval.
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2/2 PAI-1 Genotyped Allografts: Donor and Recipient
Demographics by CAD Status
Univariate analyses of donor and recipient
demographics were also performed to detect differences in donor and
recipient demographics among recipients with a 2/2 PAI-1
genotyped allograft with and those without Tx CAD in Table 4 and Table 5. Recipients with a diseased 2/2 PAI-1
genotyped allograft had significantly longer ischemic
times and tended to have higher donor rate of CMV exposure and
lipoprotein (Lp)(a) levels at 12 months than those recipients with a Tx
CAD–free allograft. Multivariable analysis in the hazard
function domain of pretransplantation donor and recipient factors (see
"Statistical Methods") identified longer ischemic times was
a risk factor for the development of Tx CAD in those recipients with a
2/2 PAI-1 genotyped allograft (P=0.02). The
actuarial freedom from any CAD for the recipients with a donor 2/2
PAI-1 genotype at 12 and 24 months with 
120 minutes of
ischemic time was 98% and 70% and 50% and 28% with >120
minutes of ischemic time (Figure 4).
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Impaired fibrinolytic activity is known to play an integral role in the development of vascular disease.19 20 The degree of fibrinolytic dysfunction may be determined in vivo by analyzing levels and/or activity of fibrinolytic proteins (PAI-1, TPA) in plasma samples or by in situ analysis of tissue samples taken from diseased vessels. When detected in the plasma, impaired fibrinolytic activity correlates best with the occurrence of clinical events such as myocardial infarction and stroke but not clearly with angiographic or histological extent of atherosclerosis.19 21 In contrast, in situ evidence of impaired fibrinolytic activity clearly correlates with the extent of histological atherosclerosis. This may also be true for Tx CAD. In a recent study, allografts with depleted levels of arteriolar TPA (possibly as a result of complex formation with PAI-1) were at greater risk of developing earlier and more severe disease than those grafts with normal arteriolar TPA.4 In the case of the transplanted heart, arteriolar levels of fibrinolytic proteins most likely represent activation of donor genes, whereas plasma levels most likely reflect expression of the recipient's genes. For this reason, only donor genotypes were analyzed in this initial study.
The genetic regulation PAI-1 is complex. Small changes in the gene sequence, such as the HindIII polymorphism, appear to play an important regulatory role in eventual antigen expression12 13 14 15 Furthermore, specific external factors may also selectively enhance the regulation of each allelic form of the gene.12 13 14 15 For example, in vitro studies using pure 2/2 or 1/1 PAI-1 genotyped endothelial cell cultures demonstrated that triglycerides and Lp(a) selectively regulate the 2/2 PAI-1 genotype, whereas insulin regulates the 1/1 PAI-1 genotype.13 14 15 These interactions between "atherogenic-like" factors and PAI-1 genotypes may, therefore, adversely effect fibrinolytic function and promote atherogenesis. This concept, supported by a cross-sectional study, correlates severity of native atherosclerosis with both the 2/2 and 1/1 PAI-1 genotypes.22 Given that these metabolic factors are present in most recipients' plasma after transplantation, it is understandable why allografts with a 1/1 or 2/2 PAI-1 genotype may be more prone to develop Tx CAD.
The 2/2 PAI-1 genotyped allografts were, as hypothesized, more likely to develop disease in this study and, as noted earlier, this form of the PAI-1 gene appears to be highly regulated by certain lipid fractions. Hyperlipidemia, in particular hypertriglyceridemia, is prevalent in the posttransplantation environment and is related to the development of Tx CAD. Serum cholesterol was significantly higher in the group of recipients with 2/2 PAI-1 genotyped allografts. Serum triglyceride levels, although not significantly different between groups, were at least 3 times higher than normal serum values. Elevation of serum triglycerides to this level may be a particularly important in the group of recipients with a 2/2 PAI-1 genotyped allograft for reasons noted earlier. Upregulation of the PAI-1 gene through this lipid/gene interaction could lead to enhanced PAI-1 expression, depleted TPA, and the promotion of vasculopathy. Other lipoproteins known to regulate PAI-1 expression include the well-characterized LDL-like particle (Lp)(a).23 We have recently shown that Lp(a), like triglycerides (VLDL), upregulate PAI-1 expression, primarily in 2/2 PAI-1 genotyped cells.15 Interestingly, Lp(a) levels tended to be higher in those recipients with a 2/2 PAI-1 genotyped allograft that eventually developed vasculopathy. This may represent an additional regulatory mechanism, accounting for the vasculopathy that developed in the 2/2 PAI-1 grafts.
The decreased 2/2 PAI-1 genotyped allografts in this study had longer ischemic times. This factor has been implicated in the development of Tx CAD through endothelial cell injury.24 25 26 Ischemia in the form of reduced oxygen tension has been demonstrated to both induce synthesis of PAI-1 mRNA and antigen and reduce TPA antigen release from cultured endothelial cells.27 It is possible that endothelial cells with the 2/2 PAI-1 genotype may be more susceptible to the effects of ischemia than other forms of the PAI-1 gene, resulting in a greater expression of PAI-1. This coupled with the reduction in TPA levels would significantly reduce fibrinolytic activity and possibly promote the early development of vasculopathy.
Immunosuppressive agents are well-known perturbants of fibrinolytic dysfunction in transplantation recipients and some have been implicated in the development of Tx CAD.28 Both cyclosporine and prednisone promote the expression of PAI-1 both in vitro and in vivo and could theoretically contribute to the development of Tx CAD through this mechanism.29 30 Azathioprine, although considered to have procoagulant activity, has not been shown to induce PAI-1 expression to date.29 Similarly, there are no studies demonstrating a genotype-specific relation between any of these drugs and PAI-1 expression. Whether these drugs do indeed promote the development of Tx CAD through a genotype-specific regulation of PAI-1 has yet to be determined and are the focus of ongoing research in this laboratory.
In summary, this study suggests that heart transplantation recipients with a 2/2 PAI-1 genotyped allograft may be at increased risk for the development of Tx CAD. The mechanism through which this genotype lends to the development of disease may be through the genotype-specific overexpression of PAI-1, leading to localized depletion of TPA. Further identification of potential regulatory factors affecting the expression of this genotype and confirmation of these results in a large multi-institutional trial are under way. Future identification of donor PAI-1 genotypes may be a useful early screening tool to identify patients at risk for developing Tx CAD. Identification and modification of regulatory factors might help attenuate the development of Tx CAD by suppressing aberrant fibrinolytic gene activation.
Limitations
Tx CAD was not seen in the 1/1 PAI-1 genotyped allografts,
as might have been expected. The reason for this is not readily
apparent but may be related to the relatively small number of
allografts with the 1/1 PAI-1 genotype. In addition, because
serum insulin levels were not obtained in this study, it is unclear
whether insulin levels were in the range necessary to activate
the 1/1 form of the PAI-1 gene. This notion is supported by the
relatively low prevalence of posttransplantation diabetes (
25%) and
low prednisone dosing used (10 mg/d) in this group.
A larger-than-expected population of 2/2 PAI-1 genotyped
allografts was noted in this study. The prevalence of the 1/1 and 2/2
PAI-1 genotypes is usually between 17% to 30%, with the 1/2
genotype 47% to 57%.12 31 32 The
higher prevalence of PAI-1 2/2 allografts in the study may be related
to and reflect the relatively small sample size examined. A study with
a larger patient size and appropriately drawn insulin levels is
currently under way that should clarify both of these issues.
The limitations of angiography and the potential greater sensitivity of intravascular ultrasound as a means to determine the presence Tx CAD are well recognized and acknowledged. Unfortunately, intravascular ultrasound remains primarily a research tool and was not used diagnostically at this institution during the collection phase of this study. Future studies will attempt to incorporate this technique.
The decision to study only the donor's genetic profile may be viewed as a limitation. This initial direction was supported, in part, by literature based on studies of fibrinolytic dysfunction in native CAD, as outlined in the introduction and "Discussion." Additionally, the retrospective nature of this study, by design, made it difficult and costly to arrange for patients now several years after transplantation to come in for genetic analysis. A carefully designed, prospective trial is currently under way that will incorporate and analyze the relation between the recipient's genetic profile and Tx CAD and thus address this important issue.
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Acknowledgments |
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This work was supported in part by National Institutes of Health grants HL-49764 and HL-52791. We wish to thank the Alabama Organ Center, Birmingham, for providing the blood from donors for genotyping.
Received February 9, 1998; revision received July 15, 1998; accepted July 24, 1998.
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