Katharina Krenn

Differential role of TGF-beta1/bFGF and ET-1 in graft fibrosis in heart failure patients.

Collagen overproduction characteristic for dilated cardiomyopathy (DCM) is coregulated by endothelin (ET)‐1, transforming growth factor (TGF)‐β1, basic fibroblast growth factor (bFGF) and matrix metalloproteases (MMPs). Whether these molecules affect grafts transplanted to heart failure patients is unknown. In 67 idiopathic DCM patients, 31 patients with ischemic cardiomyopathy (ICM) and 16 controls, the myocardial bFGF, TGF‐β1, pro‐collagen (PrCol) type 1 (PrCol1‐α1, ‐α2) and MMP expressions were examined using real‐time RT‐PCR or Western blotting. mRNA expression was measured in grafts for 1 year. TGF‐β1/bFGF stimulation or gene silencing was used to examine their effect on collagen synthesis in cardiac tissue cultures. TGF‐β1 and PrCol1 were upregulated in DCM only, while bFGF was upregulated in both groups versus controls. TGF‐β1 downregulated MMP‐1 and upregulated collagen 1, whereas bFGF upregulated MMP‐13 in DCM tissue. Post‐transplant PrCol1‐α1, ‐α2 and ET‐1 mRNA increased over time in grafts of DCM patients only, while other factors returned to control baseline levels in DCM and ICM. These data indicate that cardiac transplantation corrects the dysregulated TGF/bFGF/MMP‐1/MMP‐13, but not the excess collagen and ET‐1 synthesis in cardiac grafts transplanted to DCM patients. ET‐1 might be a major pathologic trigger for graft fibrosis in DCM.

Donor Myocardial HIF-1α Is an Independent Predictor of Cardiac Allograft Dysfunction: A 7-Year Prospective, Exploratory Study

Knowledge on interplay between the cardiac molecular response to transplantation‐induced stress and primary graft dysfunction (PGD) is limited. A cDNA array identified HIF‐1, EGR‐1, NAB‐2, VEGF‐A and uPA as mediators of cardiac tissue response to transplantation‐induced stress. mRNA expression of these molecules was measured in left ventricular biopsies from 200 donors before and after aortic cross‐clamping and at 10‐, 30‐ and 60‐min reperfusion by real‐time RT‐PCR. HIF‐1α expression at two time points was significantly associated with PGD, as shown by univariate analysis, receiver operating characteristic curve and multivariate logistic regression. At a cut‐off level of 200 arbitrary units, HIF‐1α after aortic cross‐clamping in donors (78% sensitivity, 83% specificity) and at 10‐min reperfusion (85% sensitivity, 83% specificity) identified PGD. HIF‐1α demonstrates the potential to be a predictive marker for PGD; however, as multiple factors were tested at different time points, prospective evaluation is clearly necessary to confirm this observation.

Donor Serum SMARCAL1 Concentrations Predict Primary Graft Dysfunction in Cardiac Transplantation

Background— Primary graft dysfunction (PGD) is a life-threatening complication in cardiac transplantation. A sensitive, specific, and easily measurable predictor in donors could facilitate PGD prevention. Methods and Results— SMARCAL1 is a matrix-associated regulator of chromatin with helicase and ATPase activities, and its serum concentrations were significantly increased in a targeted protein array in donors whose grafts developed PGD. Therefore, this study analyzed SMARCAL1 serum concentrations by ELISA in 336 heart donors before and after aortic cross-clamping (ACC) and in recipients at 10, 30, and 60 minutes reperfusion. Demographic and hemodynamic parameters of donors and recipients as well as transplant procedure characteristics were documented. PGD (n=68) was defined as ventricular dilation and hypocontractility associated with systolic blood pressure <90 mm Hg, pulmonary capillary wedge pressure >20 mm Hg, and decreased mixed venous oxygen saturation necessitating mechanical circulatory support. SMARCAL1 serum protein concentration was significantly increased only before and after ACC in donors (P<0.0001) whose grafts developed PGD compared to those who did not. In receiver operating characteristic curve analysis, SMARCAL1 serum concentration at a cut-off level of ≥1.25 ng/mL before ACC in donors predicted PGD (P<0.0001, AUC=0.988, OR=17.050, 95% CI=5.200 to 55.901) with 96% sensitivity and 88% specificity. SMARCAL1 serum concentrations <1.25 ng/mL in donors before ACC resulted in 97% PGD-free outcome and SMARCAL1 concentrations ≥1.25 resulted in 83% PGD occurrence. Conclusions— Donor serum SMARCAL1 may serve as a specific, sensitive, and noninvasive predictive marker in the assessment of cardiac graft quality.

Serum Matrix Metalloprotease‐1 and Vascular Endothelial Growth Factor–A Predict Cardiac Allograft Rejection

Cardiac allograft rejection is currently diagnosed from endomyocardial biopsies (EMB) that are invasive and impractical to repeat. A serological marker could facilitate rejection monitoring and minimize EMB‐associated risks. We investigated the relation of serum matrix metalloprotease (MMP)‐1 and vascular endothelial growth factor (VEGF)‐A concentrations to cardiac allograft rejection, using 1176 EMBs and serum samples obtained from 208 recipients. Acute cellular rejection was diagnosed in 186 EMBs. Mean week 1 and week 2 serum MMP‐1 concentrations predicted rejection (p = 0.001, AUC = 0.80). At the optimal cut‐off level of ≥7.5 ng/mL, MMP‐1 predicted rejection with 82% sensitivity and 72% specificity. Initial serum MMP‐1 <5.3 ng/mL (lowest quartile) was associated with rejection‐free outcome in 80% of patients. Both MMP‐1 (p < 0.001, AUC = 0.67–0.75) and VEGF‐A (p < 0.01, AUC = 0.62–0.67) predicted rejection on the next EMB, while rejection at EMB was identified only by VEGF‐A (p < 0.02, AUC = 0.70–0.77). Patients receiving combined cyclosporine‐A and everolimus had the lowest serum MMP‐1 concentrations. While serum MMP‐1 predicts rejection‐free outcome and VEGF‐A identifies rejection on EMB, both markers predict rejection in follow‐up of cardiac transplant recipients. Combination of serum MMP‐1 and VEGF‐A concentration may be a noninvasive prognostic marker of cardiac allograft rejection, and could have important implications for choice of surveillance and immunosuppression protocols.

Treatment of primary graft dysfunction after lung transplantation with orally inhaled AP301: A prospective, randomized pilot study

BACKGROUND Primary graft dysfunction (PGD) after lung transplantation (LTx) carries significant morbidity and mortality in the early post-operative period and is associated with the development of chronic lung allograft dysfunction. AP301, an activator of epithelial sodium channel-mediated Na+ uptake represents a new concept for prevention and treatment of pulmonary edema and has shown promising results in the pre-clinical setting. This pilot study investigated the clinical effect of inhaled AP301 on patients with development of PGD > 1 according to International Society of Heart and Lung Transplantation criteria after primary LTx in a high-volume center and was conducted as a randomized, placebo-controlled, single-center pilot-study including 20 patients. All consecutive patients fulfilling inclusion criteria were screened for PGD at arrival on the intensive care unit (ICU) after LTx. After randomization, inhaled AP301 or placebo was administered by nebulizer twice daily for 7 days or until extubation. Otherwise, patients were treated according to routine clinical protocol. Partial pressure of arterial oxygen (Pao2)/fraction of inspired oxygen (Fio2) values were obtained until extubation and assessed as a primary outcome parameter. Patients were monitored for 30 days within the study protocol. RESULTS From July 2013 to August 2014, 20 patients were randomized 1:1 to AP301 (Group 1) or placebo (Group 2). Both groups were comparable with regard to sex (40% women/60% men vs 50% women/50% men), mean age (55 ± 13 vs 54 ± 6 years), comorbidities, and diagnosis leading to LTx. The Pao2/Fio2 ratio at the time of inclusion was comparable in both groups, with a mean 235.65 ± 90.78 vs 214.2 ± 95.84 (p = 0.405), and there was no significant difference in the extravascular lung water index (13.88 ± 5.28 vs 16 ± 6.29 ml/kg, p = 0.476). The primary end point was mean Pao2/Fio2 ratio values between baseline and Day 3. In the AP301 group, only 1 patient was ventilated at Day 4 and no patients were ventilated after Day 4. In the placebo group, 5 patients were ventilated on Day 4 and 2 patients on Days 5, 6, and 7. The mean increase in the Pao2/Fio2 ratio was significantly higher in Group 1 patients, and the mean between baseline and at 72 hours was 365.6 ± 90.4 in Group 1 vs 335.2 ± 42.3 in Group 2 (p = 0.049). The duration of intubation was shorter in Group 1 than in Group 2 patients (2 ± 0.82 vs 3.7 ± 1.95 days; p = 0.02). ICU stay was 7.5 ± 3.13 days in Group 1 vs 10.8 ± 8.65 days in group 2 (p = 0.57). Survival at 30 days was 100%. No severe adverse events were recorded. CONCLUSIONS This study was designed as a proof-of-concept pilot study. Although it was not powered to achieve statistical significances, the study demonstrated relevant clinical effects of inhaled AP301 on patients with PGD after primary LTx. The improved gas exchange led to a significantly shorter duration of mechanical ventilation and a trend towards a shorter ICU stay. Further investigation of AP301 for treatment of PGD in larger studies is warranted. TRIAL REGISTRATION The trial is registered at https://www.clinicaltrialsregister.eu/ctr-search/trial/2013-000716-21/AT.

Apollon/RNF41 myocardial messenger RNA diagnoses cardiac allograft apoptosis in rejection.

Background. Endomyocardial biopsy (EMB) remains the gold standard for acute cellular rejection (ACR) diagnosis in cardiac transplantation yet is subject to interobserver variability. A method that could avoid discordant EMB analysis would be desireable. The apoptosis rate in EMB correlates with ACR severity. Apollon inhibits apoptosis, and RNF41 catalyzes its degradation. Whether tissue Apollon/RNF41 could diagnose ACR is not known. This study addressed this issue. Methods. Apollon/RNF41 messenger RNA (mRNA) was measured by real time reverse-transcriptase polymerase chain reaction and apoptosis was quantified with TUNEL assays in EMBs of 268 transplant recipients. EMBs were obtained at 1, 2, 3, 4, 7, 12, 24, and 52 posttransplant weeks. Results. At all time points posttransplant, Apollon mRNA decreased significantly in EMBs with ACR grades 2R/3R combined (P≤0.0010) compared with 0/1R combined, although RNF41 mRNA significantly increased in EMBs with ACR grade 1R (P<0.0001) or 2R/3R combined (P<0.0001) compared with 0. At the identified cut-off level of less than or equal to 168.2 arbitrary units, Apollon mRNA identified ACR grades 2R/3R with 100% sensitivity and 84% specificity, whereas RNF41 mRNA at the cut-off level of more than or equal to 51.8 identified ACR grades 1R-3R with 99% sensitivity and 95% specificity. Increased RNF41 (rs, 0.728; P<0.0001) and decreased Apollon (rs, −0.562; P<0.0001) expression correlated significantly with the degree of apoptosis in EMBs. Conclusions. Combined Apollon/RNF41 mRNA quantitatively and specifically identifies ACR associated with apoptosis in cardiac allografts and could validate ACR grading variability associated with histologic EMB analysis.

Persistent plasminogen activator inhibitor 1 gene expression in cardiac transplant recipients with idiopathic dilated cardiomyopathy.

OBJECTIVES Plasminogen activator inhibitor 1 is the primary regulator of urokinase plasminogen activator and tissue plasminogen activator. Plasminogen activator inhibitor 1 is essential in the control of the thrombotic/fibrinolytic balance and is a marker of endothelial cell injury. Idiopathic dilated cardiomyopathy is reportedly associated with endothelial cell dysfunction. Whether endothelial cell damage plays a role in patients with dilated cardiomyopathy after cardiac transplantation remains unknown. METHODS In this study explanted hearts of cardiac transplant recipients with ischemic cardiomyopathy and dilated cardiomyopathy, as well as control myocardial tissue, were investigated for expression of urokinase plasminogen activator, tissue plasminogen activator, urokinase plasminogen activator receptor, and plasminogen activator inhibitor 1 and 2. Furthermore, plasminogen activator inhibitor 1 expression was examined in endomyocardial biopsy specimens and sera of patients with ischemic cardiomyopathy and those with dilated cardiomyopathy during the first posttransplantation year. The effect of the patients serum on endothelial cells was assessed in vitro to examine the role of circulating endothelial cell damage-related factors. RESULTS Plasminogen activator inhibitor 1 expression was upregulated in ischemic cardiomyopathy and dilated cardiomyopathy myocardial tissue versus that seen in control tissue. After transplantation, plasminogen activator inhibitor 1 expression returned to control levels in patients with ischemic cardiomyopathy. In patients with dilated cardiomyopathy, plasminogen activator inhibitor 1 expression increased at 24 weeks after transplantation in both biopsy specimens and sera versus that seen in control tissue. Sera of patients with dilated cardiomyopathy, but not that of patients with ischemic cardiomyopathy, inhibited vascular endothelial growth factor A-induced proliferation of endothelial cells, although downstream target gene activation of early growth response factor 1 and NGFI-A binding protein 2 was not affected. CONCLUSIONS These data suggest for the first time that the endothelial cell damage-related process recurs in patients with dilated cardiomyopathy after transplantation, which, independently of vascular endothelial growth factor, is associated with increased plasminogen activator inhibitor 1 expression, and that this pathology might play a role in allograft remodeling in patients with dilated cardiomyopathy.