Gender-based analysis of outcome after heart transplantation.

OBJECTIVES: Gender differences between donors and recipients might have an effect on outcome after heart transplantation. Literature and registries reveal controversial results. We reviewed 1000 heart transplantations at our center focusing on the influence of gender differences on short- and long-term outcome after heart transplantation. MATERIALS AND METHODS: We performed a retrospective analysis of 1000 (960 primary and 40 redo-heart transplantations) between August 1981 and July 2008. In contrast to other studies, the data for gender differences (donor gender and recipient gender) were evaluated for recipient survival and survival conditional to early mortality. RESULTS: Female donors are significantly older than male donors (females, 36.5 ± 14.5 years; males, 31.2 ± 13.8 years). One-year survival was significantly inferior in male recipients receiving female donor hearts (mR/fD: 73.7%) compared to females receiving male donor organs (fR/mD: 90.9%) (P = .045). Univariate analysis revealed that, for recipients who survived > 1 year, survival at 10 years was significantly greater for female donors and female recipients (90%) than it was for male donors and male recipients (72%; P = .034). Multivariate analysis showed that the gender combination with female donors and female recipients was an independent indicator for greater long-term survival (P = .04). CONCLUSIONS: The gender combination of female donors and male recipients had a greater risk for early mortality after heart transplantation, and the combination of male donors and female recipients resulted in favorable short-term outcomes. In long-term follow-up, recipients of hearts from female donors had better survival, especially female recipients.

Expression of circulatory dendritic cells and regulatory T-cells in patients with different subsets of coronary artery disease.

BACKGROUND: Dendritic cells (DCs), regulators of innate and adaptive immunity, may play an important role in atherosclerosis. DC invasion was found in early atherosclerotic lesions. We aimed to characterize circulating DC gene expression in patients with different subsets of coronary artery disease (CAD). METHODS: Peripheral blood mononuclear cells were quantified using real-time polymerase chain reaction and fluorescence activated cell sorting in patients with acute coronary syndrome (ST-elevation myocardial infarction [STEMI], n = 35; non-ST-elevation myocardial infarction [NSTEMI], n = 30) and stable CAD (6 months after stent implantation without progression, n = 15) compared with control subjects (n = 15). DCs and T-cells (TCs) were characterized using specific primers for CD1a (immature), CD86 (mature), CD123 (plasmacytoid), BDCA1 (myeloid), CD178 (activated TCs), and FOXP3 (regulatory TCs). To evaluate whether serum of patients with STEMI induces DC differentiation, incubation of patient serum was performed. RESULTS: CD86 was upregulated and CD1a downregulated in all patients with CAD (P < 0.05). Patients with STEMI and NSTEMI showed a downregulation of CD1a compared with patients with stable CAD (P ≤ 0.01). In contrast, stable patients with CAD had elevated CD178 levels compared with patients with STEMI and NSTEMI (P ≤ 0.04). In patients with STEMI, FOXP3 was downregulated compared with control subjects (P < 0.0001). Incubation of STEMI serum induced an upregulation of CD1a and CD86 in a human DC cell line. Coincubation with a blocking antibody for heat shock protein 60 inhibited this upregulation. CONCLUSIONS: DCs are differentially regulated in patients with different subsets of CAD. Mature DCs are upregulated and immature DCs are downregulated in patients with CAD. Patients with STEMI show a significant downregulation of regulatory TCs. Circulating shock protein 60 induces DC differentiation in patients with STEMI.

The proton pump inhibitor pantoprazole and its interaction with enteric-coated mycophenolate sodium in transplant recipients.

BACKGROUND: In this prospective study we investigated the impact of the proton pump inhibitor (PPI) pantoprazole on the bioavailability of mycophenolic acid (MPA) after oral administration of enteric-coated mycophenolate sodium (EC-MPS; Myfortic) in heart or lung transplant recipients. Previously we demonstrated that pantoprazole reduces the MPA exposure of mycophenolate mofetil (MMF; CellCept) by 34% in area under the concentration-time curve (AUC). Because gastrointestinal side-effects are common after organ transplantation, we investigated the effect of PPI on MPA levels in patients receiving EC-MPS. METHODS: MPA plasma concentrations and inosine monophosphate dehydrogenase (IMPDH) activity at baseline, 30 minutes and 1, 2, 3 and 4 hours were obtained from 21 patients. These patients were treated with pantoprazole 40 mg once daily and EC-MPS twice daily at a mean dose of 960 mg. Measurements were repeated after pantoprazole withdrawal. RESULTS: MPA concentrations and IMPDH activities did not reveal any significant difference during PPI treatment and after withdrawal. MPA AUC, MPA C(max) (maximal MPA concentration), the time until C(max) was reached (T(max)) and IMPDH activity AUC all showed no significant difference. CONCLUSION: We did not find an influence of pantoprazole on EC-MPS pharmacokinetics such as we did for MMF in our previous investigation. A further prospective, large, cross-over study is planned to support these preliminary results. Given that MPA exposure by AUC correlates with the incidence of acute rejection episodes and transplant vasculopathy, the present findings may have clinical implications.

CD34+CD140b+ cells and circulating CXCL12 correlate with the angiographically assessed severity of cardiac allograft vasculopathy.

AIMS: We sought to determine whether circulating vascular progenitor cells, such as endothelial progenitor cells (EPCs) or smooth muscle progenitor cells (SPCs), were associated with the severity of cardiac allograft vasculopathy (CAV). METHODS AND RESULTS: CD34(+)CD140b(+) SPCs and CD34(+)KDR(+) EPCs were measured in the peripheral circulation of 187 adult heart transplant recipients by flow cytometry. Cardiac allograft vasculopathy was quantified by angiography using a CAV-specific scoring system. Cardiac allograft vasculopathy was present in 84 patients (44.7%) and was classified as mild in 59 and severe in 25 cases. Circulating SPCs were more frequently detectable in CAV patients than in patients without CAV. The number of CD34(+)CD140b(+) cells showed a stepwise increase in patients with moderate and severe CAV. Smooth muscle progenitor cell counts were higher in patients with coronary stent implant compared with unstented patients with CAV. In contrast, peripheral CD34(+)KDR(+) EPC counts were not changed in CAV patients. Plasma CXCL12 levels correlated with the degree of CAV and SPC counts. None of the different immunosuppressive drug regimes was related to the SPC count or the CXCL12 levels. A multivariate regression analysis revealed that the SPC count was independently associated with the presence of CAV. CONCLUSION: Circulating SPCs, but not EPCs, and plasma CXCL12 concentrations are elevated in CAV patients, indicating that they play prominent roles in transplant arteriosclerosis.

Luminex-based virtual crossmatching facilitates combined third-time cardiac and de novo renal transplantation in a sensitized patient with sustained antibody-mediated cardiac allograft rejection.

Allosensitization represents a major obstacle to successful re-transplantation since circulating antibodies can elicit antibody-mediated rejection episodes with subsequent graft failure. Since sensitization is primarily considered a contraindication to transplantation the duration for patients waiting for a suitable donor organ to become available is considerably prolonged. Herein, we report on the successful application of a Luminex-based virtual crossmatch approach to facilitate combined third-time cardiac and de novo renal transplantation in a sensitized patient with sustained antibody-mediated cardiac allograft rejection.

Long-term outcomes after 1000 heart transplantations in six different eras of innovation in a single center.

The objective of this study was to evaluate long-term outcomes of cardiac transplantation (HTx) in different eras of innovation at a single center during a period of 27 years. We performed a retrospective analysis of 960 cardiac allograft recipients (40 re-HTx) between 1981 and 2008. The results of six different eras based on milestones in HTx were analysed: Era 1: the early years (n = 222,1981-1992); era 2: introduction of inhalative nitric oxide, prostanoids, University of Wisconsin solution (UW) replacing Bretschneider's solution (HTK,n = 118, 1992-1994); era 3: statins (n = 102, 1994-1995); era 4: tacrolimus(n = 115, 1995-1996); era 5: mycophenolate mofetil (MMF, n = 143, 1997-2000) and era 6: sirolimus (n = 300, 2000-2008). Outcome variables weresurvival, freedom from cardiac allograft vasculopathy (CAV) and from acute rejection episodes (AREs). Differences in survival was found comparing era 1 and era 2 with era 4 and era 6 (P < 0.001). Organ preservation through UW demonstrated a significantly better survival as compared with HTK(P < 0.001). Less AREs occurred in patients receiving tacrolimus-sirolimus ortacrolimus-MMF (P < 0.001). Patients receiving tacrolimus-MMF showed less CAV than when treated with cyclosporine-MMF (P < 0.005). There were more ventricular assist device implantations and more re-HTx in era 6 (P < 0.0001)than when compared with other eras. Although the causes for improvement in survival over time are multifactorial, we believe that changes in immunosuppressive therapy have had a major impact on survival.

Proton pump inhibitor co-medication reduces mycophenolate acid drug exposure in heart transplant recipients.

BACKGROUND: Proton pump inhibitors (PPIs) are often prescribed for gastrointestinal discomfort after heart transplantation (HTx). This study investigates the impact of PPI use on mycophenolate acid (MPA) pharmacokinetics in heart transplant recipients receiving mycophenolate mofetil (MMF) and tacrolimus (Tac). METHODS: MPA post-dose plasma concentrations at 0 to 2 hours were obtained by high-performance liquid chromatography (HPLC) in 21 patients on pantoprazole 40 mg/day (PPI group) and 12 patients not on pantoprazole (control group). In a subgroup, MPA plasma concentrations at 0 to 12 hours were measured to evaluate full MPA area-under-the-curve (AUC) measurements. RESULTS: In the PPI group, the mean daily MMF dose was 1,912 +/- 1,023 mg with mean pre-dose serum concentrations (C(0)) of 1.9 +/- 1.4 mg/liter, without a significant difference from controls. Mean post-dose MPA concentrations at 30 minutes (C(0.5h)) were significantly higher in the control group (control, 17.9 +/- 11.5 mg/liter; PPI, 6.7 +/- 4.6 mg/liter; p < 0.001). One- and 2-hour (C(1h, 2h)) MPA concentrations were persistently higher in the control group (1 hour: control, 13.8 +/- 9.1 mg/liter; PPI, 7.7 +/- 4.1 mg/liter; 2 hours: control, 7.6 +/- 4.2; PPI, 5.0 +/- 2.8 mg/liter; p < 0.05). In the subgroup with full AUC measurements, the control group had higher MPA levels 12 hours after dosing (C(12h): control, 3.3 +/- 2.4 mg/liter; PPI, 1.6 +/- 1.3 mg/liter; p < 0.05) and a significantly higher dose-adjusted AUC C(0-3h) (control, 59.5 +/- 20.7 mg/liter; PPI, 41.7 +/- 23.4 mg/liter; p < 0.05). In the subgroup, the maximum plasma concentration of mycophenolic acid (MPA C(max)) in the PPI group was significantly lower (10.1 +/- 4.7 mg/liter) than in the control group (45.5 +/- 53.5 mg/liter, p < 0.0001), whereas t(max) revealed no differences (control: 1.1 +/- 0.77 hours; PPI: 1.1 +/- 0.97 hours). Furthermore, a clear trend for more acute rejection episodes (AREs) and more transplant vasculopathy (TVP) was found in the PPI group. CONCLUSIONS: Patients with PPI co-medication show significantly lower MPA plasma concentrations, possibly due to decreased absorption. Therapeutic drug monitoring allows identification of patients with decreased MMF drug exposure who might be at risk for acute rejection.

Impact of coronary endothelial dysfunction on adverse long-term outcome after heart transplantation.

BACKGROUND: Coronary vasomotor dysfunction is a common finding in cardiac transplant recipients and is an early marker for the development of graft atherosclerosis. The present prospective study tested whether endothelial dysfunction independently predicts cardiovascular-related events and death after heart transplantation (HTx). METHODS: Functional and structural coronary changes were evaluated in 185 consecutive patients 25+/-33 months after HTx. The following potential risk factors for graft survival were assessed at baseline: hypertension, diabetes, dyslipidemia, donor and recipient characteristics (age, gender, cytomegalovirus-infection, human leukocyte antigen-mismatch), pretransplantation diagnosis, ischemic time, treated rejection episodes, immunosuppressive regimens, and medication.The prespecified prospectively defined endpoints were cardiovascular-related events with progressive heart failure, acute myocardial infarction, coronary revascularization, retransplantation, and death. Patients were followed-up for 60+/-17 months. RESULTS: Event-free survival for the entire group was 73% (25 cardiovascular-related events, 25 deaths). Using multivariate analysis, epicardial endothelial dysfunction (relative risk [RR] 1.97; P=0.028), angiographic cardiac allograft vasculopathy (RR 2.11; P=0.023), diabetes (RR 2.32; P=0.022), high serum levels of CyA (RR 3.54; P=0.006) and Tac (RR 6.82; P=0.002), uncommon reasons for transplantation (RR 4.69; P=0.002), and the absence of statin therapy (RR 0.33; P=0.025) were detected as independent predictors of cardiovascular-related events and death. CONCLUSION: This is the first study showing that epicardial endothelial dysfunction independently predicts outcome in HTx patients providing functional and prognostic information that complete angiographic risk factor assessment.

Graft-infiltrating dendritic cells and coronary endothelial dysfunction after human heart transplantation.

BACKGROUND: Indirect allorecognition is involved in chronic transplant rejection. We prospectively characterized graft-infiltrating dendritic cells (DCs) in sequential myocardial biopsies (n = 64; 1 to 24 months after transplantation) from 16 patients after heart transplantation (HTx) and analyzed the relation between graft immune activation and structural and functional coronary changes during follow-up. METHODS: DC invasion (immunostaining) in the human myocardium was detectable early after HTx, increased further during the first year, and decreased constantly thereafter. Also, graft-infiltrating DCs expressed markers of immaturity and maturity and were time-dependently clustered with CD3-positive T cells. RESULTS: Both epicardial and microvascular endothelial dysfunction were associated with elevated CD209-positive DCs at 12 months. CD209 positivity early after HTx was an independent marker for coronary endothelial dysfunction during follow-up. Intimal hyperplasia or angiographic disease during follow-up was not associated with myocardial DC infiltration. CONCLUSIONS: DCs frequently infiltrate the cardiac allograft with a peak during the first post-operative year and time-dependently cluster with T cells. Migratory active graft-infiltrating DCs may serve as a predictor for allograft coronary endothelial dysfunction.

Dual mode of HMG-CoA reductase inhibition on dendritic cell invasion.

Atherosclerosis is a chronic disease triggered by lipid disturbances, endothelial injury and sustained by inflammation. Dendritic cells (DCs) are critical for the cell-mediated arm of an immune response and are known to initiate inflammatory immunity. We investigated the role of statins and the mevalonate pathway on DC invasion. DC incubation with atorvastatin (ATV; 0.05-1 microM) for 24h decreased DC adhesion capacity. DC invasion (adhesion/transmigration) was decreased after exposing DCs to low and moderate concentrations of statins, which was reversible by mevalonate (but not geranyl- or farnesyl-pyrophosphate) and cholesterol. Inhibition of the phosphoinositide 3-kinase (with wortmannin) and inhibition of the NO-synthase (with asymmetric dimethyl ADMA) partially reversed statin-mediated effects. High-dose statins markedly decreased DC invasion, which was reversible by adding geranyl pyrophosphate and cholesterol. Inhibition of geranylgeranyltransferase but not inhibition of farnesyltransferase significantly decreased DC invasion. Statin-mediated alteration in DC-cholesterol synthesis and subsequent activation of the Akt/NOS pathway accounts for the statin-induced decrease in DC invasion at low-moderate concentrations (0.05-0.5 microM). Additionally, at high statin concentrations (1 microM) DC invasion is reduced by inhibition of protein geranylgeranylation. As DCs control immunity, regulating DC/endothelial cell interaction by statins may have relevance to inflammation and atherogenesis.

Monitoring of the sublingual microcirculation in cardiac surgery using orthogonal polarization spectral imaging: preliminary results.

BACKGROUND: The recent introduction of orthogonal polarization spectral imaging enables the direct visualization of the microcirculation of man without imaging enhancing dyes. The authors studied the changes in microvascular perfusion of sublingual mucosa during cardiac surgery with the use of cardiopulmonary bypass (CPB) using this optical method. METHOD: Orthogonal polarization spectral images were recorded in 47 patients after skin incision (T1), after the start of CPB (T2), in the late phase of CPB (T3), and 1 h after the discontinuation of CPB (T4). The images were analyzed for microvascular diameter, erythrocyte velocity, and functional capillary density using an established analysis routine for intravital microscopy studies. In a subpopulation (n = 8), the expression of the adhesion molecules CD18 on circulation leukocytes was compared with the number of visualized rolling leukocytes. RESULTS: Preoperatively, no significant changes of the microvascular diameter and erythrocyte velocity were seen. The functional capillary density was significantly reduced at T3 to 90% of the values observed before CPB but recovered at T4 and showed a weak but significant correlation with body temperature (r = 0.38, P < 0.01) and hemoglobin concentration (r = 0.20, P < 0.05). Expression of CD18 was significantly increased in the late phase of CPB (T3) only, whereas the numbers of rolling leukocytes increased during CPB and revealed a significant threefold increase 1 h after termination of CPB. CONCLUSIONS: Orthogonal polarization spectral imaging revealed no major changes of microvascular perfusion during uncomplicated hypothermic CPB. The slightly reduced functional capillary density during CPB may be caused by several factors all present during CPB, including hypothermia, the artificial extracorporeal perfusion, surgical trauma, hemodilution, and inflammatory reaction. The current data do not allow differentiation between the effects of those possible causes.

Expansion of circulating Toll-like receptor 4-positive monocytes in patients with acute coronary syndrome.

BACKGROUND: Atherosclerosis is an inflammatory disease in which monocytes and macrophages have been suggested to play an essential role. The underlying signaling mechanisms are unknown thus far. We hypothesized that the human isoform of Toll-like receptor (hTLR)-4 is involved in monocyte activation of patients with accelerated forms of atherosclerosis. METHODS AND RESULTS: Expression of hTLR4 on circulating monocytes from 30 controls, 20 patients with stable angina (SA), 40 patients with unstable angina (UA), and 28 patients with acute myocardial infarction (AMI) was compared with the use of flow-cytometry and reverse transcription-polymerase chain reaction. Regulation of interleukin (IL)-12 and B7-1 as downstream events of TLR4 activation was analyzed after lipopolysaccharide stimulation of monocytes. TLR4-transfected Chinese hamster ovary (CHO) cells were used to identify potential hTLR4 ligands in the serum of patients with UA or AMI. Circulating hTLR4+/CD14+ monocytes were approximately 2.5-fold increased above controls and patients with SA in the UA and AMI groups (P<0.0001). This was paralleled by enhanced transcript levels of TLR4 and Myd88 in patients with UA and AMI (P<0.0001) and increased expression of IL-12 (UA 35.5+/-7.8, AMI 31.8+/-7.7 versus SA 2.2+/-0.5, controls 2.1+/-0.3 pg/mL; P<0.0002) and B7-1 (UA 27.3+/-14.4, AMI 22.6+/-11.1 versus SA 3.4+/-2.5, controls 2.4+/-2.3%; P<0.0001). Compared with serum from patients with UA and AMI, challenging TLR4-transfected CHO cells with serum from SA patients yielded only a weak response (P<0.0001). Coincubation with anti-heat shock protein 60 inhibited CHO cell activation. CONCLUSIONS: UA and AMI are associated with enhanced expression and signaling events downstream of hTLR4 in circulating monocytes. These observations suggest hTLR4 activation as a signaling mechanism in immune-mediated progression of atherosclerosis.

Statins decrease Toll-like receptor 4 expression and downstream signaling in human CD14+ monocytes.

OBJECTIVE: Anti-inflammatory effects of statins contribute to their clinical benefit. Molecular mechanisms underlying these effects have not been well explored. Because statins attenuate lipopolysaccharide (LPS) responsiveness, we hypothesized that part of the pleiotropic effects are mediated through innate immunity. METHODS AND RESULTS: Toll-like receptor (TLR) 4 expression and downstream signaling in CD14+ monocytes after incubation with simvastatin and atorvastatin were quantified via flow-cytometry, quantitative RT-PCR, kinase assay, and enzyme-linked immunosorbent assay. Incubation with intermediates/ inhibitors of the mevalonate pathway was used to identify the mode of statin action. Statin incubation resulted in a dose-dependent reduction of TLR4 expression (53+/-7.6% reduction compared with untreated monocytes; P<0.005), transcript levels (68+/-6.3%; P<0.002), decreased IRAK phosphorylation (37+/-8.3%; P<0.05), and LPS-induced IL-6, IL-12, tumor necrosis factor (TNF)-alpha, and B7-1 expression (P<0.05). Four weeks of treatment with atorvastatin significantly reduced TLR4 expression on circulating CD14+ monocytes by 36.2+/-4.2% (P<0.05). Effects of statins were reversed by mevalonate (P=0.57). Incubation with specific inhibitors of geranylgeranyltransferase (54+/-4.3%), farnesyltransferase (57+/-5.1%), or with clostridium-difficile toxin B (58+/-6.1%, P<0.01) imitated the statin effects. Whereas wortmannin and LY294002 inhibited the statin effect (P=0.27), incubation with a specific RhoA kinase inhibitor had no effect (P=0.57). CONCLUSIONS: Statins influence TLR4 expression and signaling via inhibition of protein geranylgeranylation and farnesylation. These observations imply interactions with innate immunity as one pleiotropic mechanism.

Role of cytokines in cardiovascular diseases: a focus on endothelial responses to inflammation.

Complex cellular and inflammatory interactions are involved in the progress of vascular diseases. Endothelial cells, upon exposure to cytokines, undergo profound alterations of function that involve gene expression and de novo protein synthesis. The functional reprogramming of endothelial cells by cytokines is of importance especially in patients with chronic vascular inflammation. The intercellular network of dendritic cells, T-lymphocytes, macrophages and smooth muscle cells generates a variety of stimulatory cytokines [e.g. TNF-alpha (tumour necrosis factor-alpha), IL (interleukin)-1, IL-6 and IFN-gamma (interferon-gamma)] and growth factors that promote the development of functional and structural vascular changes. High concentrations of proinflammatory cytokines increase oxidative stress, down-regulate eNOS (endothelial nitric oxide synthase) bioactivity and induce endothelial cell apoptosis. Chemoattractant cytokines [e.g. VEGF (vascular endothelial growth factor), TGF-beta1 (transforming growth factor-beta1) and IL-8] are important regulators of inflammation-induced angiogenesis and are directly modulated by nitric oxide. This review will focus on the vascular mechanisms orchestrated by cytokines and summarizes the current knowledge concerning the contribution of cytokines to cardiovascular diseases.