Matthias Gmeiner

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.

Improvement of cardiac function in the failing rat heart after transfer of skeletal myoblasts engineered to overexpress placental growth factor

BACKGROUND Transplant of skeletal myoblasts is an attractive alternative to repair irreversibly damaged myocardium in ischemic heart failure. We investigated whether transplant of myoblasts overexpressing placental growth factor would stimulate angiogenesis and enhance myoblast survival in a rat heart failure model. METHODS Three weeks after myocardial infarction, Sprague-Dawley rats in heart failure received intramyocardial injections of Ringer solution (control) or autologous myoblasts, unmodified or transfected with placental growth factor expression plasmid. Sham-operated animals served as noninfarct controls. Cardiac function was assessed by echocardiography to 86 days after engraftment. Immunocytochemistry and fluorescence imaging were used to investigate vessel formation, grafted myoblast survival, infarct wall thickness, and infarct size. Quantitative real-time reverse transcriptase polymerase chain reaction and Western blotting measured tissue messenger RNA and protein expressions. RESULTS Left ventricular function significantly improved with time, and fractional shortening on day 86 was significantly enhanced in transfected myoblast group relative to control (P < .01) and unmodified myoblast (P < .05) groups. Vascular density (P < .01) and myoblast survival (P < .05) were enhanced in rats treated with transfected myoblasts relative to other groups (P < .05). Mean fraction of fibrotic scar tissue was decreased in unmodified and transfected myoblast groups relative to controls on day 86 (P < .05), and left ventricular wall thickness was significantly increased in transfected myoblast group relative to other groups (P < .05). CONCLUSIONS Intramyocardial injections of autologous myoblasts overexpressing placental growth factor improved cardiac function, attenuated adverse cardiac remodeling, induced angiogenesis, and probably enhanced survival of grafted myoblasts.

Serum Glutathione S-Transferase P1 1 in Prediction of Cardiac Function

Background Glutathione S-transferase P1 1 (GSTP1) belongs to the multigene isozyme family involved in cellular response to oxidative stress and apoptosis. Our initial retrospective proteomic analysis suggested that GSTP1 is associated with heart failure (HF). Although pro–B-type natriuretic peptide (proBNP) serves currently as a surrogate diagnostic and prognostic parameter in HF patients, its specificity remains uncertain. We hypothesized that GSTP1 might be a useful serum marker in the monitoring of HF patients. Methods and Results Serum GSTP1 and proBNP were prospectively measured in 193 patients subdivided based on their ejection fraction (EF) either in equal-sized quintiles or predefined EF groups >52%, 43%–52%, 33%–42%, 23%–32% and ≤22%. At a cutoff of ≥231 ng/mL, GSTP1 identified HF patients with EF ≤22% with 81% sensitivity and 83% specificity, and at a cutoff of ≥655 pg/mL, proBNP identified the same patient group with 84% sensitivity and 22% specificity. GSTP1 at a ≥126 ng/mL cutoff identified EF ≤42% with 90% sensitivity and 95% specificity, or proBNP at a ≥396 pg/mL cutoff had 97% sensitivity and 20% specificity. In regression analyses, GSTP1, but not proBNP, discriminated between EF ≤42% and EF >42% in HF patients. Conclusions These results suggest that GSTP1 is strongly associated with HF and could serve as a sensitive and specific marker to predict the ventricular function in HF patients.

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.