Impaired platelet function reduces myocardial infarct size in Galphaq knock-out mice in vivo.

Platelet aggregation and secretion play a crucial role in acute coronary syndromes. In Galpha(q) knock-out mice (Galpha(q)(-/-)) platelet function is eliminated in terms of aggregation and secretion of cytokines. We investigated whether restricted platelet aggregation and secretion reduces myocardial infarct size in vivo. Thirty minute regional myocardial ischemia was followed by 24 h reperfusion (I/R) in vivo. Infarct size was determined by counterstaining. Left ventricular function was measured by ultrasound. Infarct size to area at risk ratio was significantly smaller in Galpha(q)(-/-) mice (5.6+/-1.6%) compared to wild-type (WT) mice (27.2+/-3.0%, p<0.01). Fractional shortening was improved in Galpha(q)(-/-) mice compared to WT (42.2+/-1.4% versus 30.5+/-1.4%, respectively, p<0.01). WT mice, transplanted with Galpha(q)(-/-) bone marrow showed a significant reduction in infarct size compared to control (7.8+/-2.2% versus 18.4+/-2.7%, respectively, p<0.01). Platelets of Galpha(q)(-/-) mice had an impaired aggregation and secretion phenotype. In the in vivo model of ischemia and reperfusion, beyond impaired platelet aggregation, platelet secretion plays an additional role in myocardial infarct extension. Blocking platelet aggregation in combination with secretion might be a promising supplementary therapeutic strategy in acute myocardial infarction.

Gene transfer of the pancaspase inhibitor P35 reduces myocardial infarct size and improves cardiac function.

Myocardial infarction and subsequent reperfusion lead to the activation of apoptosis, and the final destruction of the cell. The aim of this study was to show that broad-scale inhibition of caspases, the main executioners of apoptosis, improves functional outcome after ischemia and reperfusion in an in vivo model. Twenty male Wistar rats were directly injected with an adenovirus, encoding the baculoviral protein p35. Nineteen rats served as controls, and were injected with a virus only encoding green fluorescent protein (GFP). After 3 days, 12 animals were used for Langendorff perfusion experiments, the other 27 animals were submitted to in vivo infarction. Myocardial infarction was induced by ligation of the left anterior descending artery (LAD) for 30 min, and reperfusion for 24 h. Echocardiographic and hemodynamic measurements were made 24 h after infarction. Infarct size was assessed in all animals histologically. In both, in vivo and Langendorff perfused hearts, myocardial infarct size was significantly reduced in the p35 group (for in vivo experiments: 0.11+/-0.03 vs 0.33+/-0.03 in the GFP group, p<0.01), as was the ratio of infarct size to area at risk (6 vs 17%, p<0.01). Left ventricular function was similar in both groups prior to infarction, but was significantly less compromised after infarction in the p35 group. The left ventricular systolic pressure after infarction was higher in the p35 group (107+/-5 vs 92+/-4 mmHg, p<0.05), as was the maximal rate of rise of left ventricular systolic pressure dp/dt (5,659+/-585 vs 4,634+/-256 mmHg s(-1), p<0.05). Adenoviral gene transfer of the caspase inhibitor p35 leads to a significant reduction of the myocardial infarct size after ischemia and reperfusion. Hemodynamic variables were significantly improved by treatment with p35. Cardiac restricted inhibition of apoptosis seems to be a promising approach for ameliorating the effects of ischemia and reperfusion.

Myocardial kinetics of reporter probe 124I-FIAU in isolated perfused rat hearts after in vivo adenoviral transfer of herpes simplex virus type 1 thymidine kinase reporter gene.

UNLABELLED: Reporter gene imaging holds promise for noninvasive monitoring of cardiac gene therapy. We recently demonstrated that (124)I-labeled 2'-fluoro-2'-deoxy-5'-iodo-1beta-d-arabinofuranosyluracil ((124)I-FIAU) is suitable for PET of myocardial expression of herpes simplex virus type 1 thymidine kinase reporter gene (HSV1-tk). In contrast to previous studies in tumors, early specific uptake was followed by rapid washout. Myocardial kinetics of (124)I-FIAU are still poorly understood. This study aimed at a further investigation under controlled conditions using an isolated heart perfusion model. METHODS: Male Wistar rats underwent transthoracic regional injection of replication-defective adenovirus (2.5 x 10(9) plaque-forming units) containing either HSV1-tk (n = 16) or LacZ reporter gene (n = 15) into the inferior wall. Nonmanipulated rats (n = 5) served as further controls. Hearts were excised 2 d later and perfused according to the Langendorff technique with (124)I-FIAU-containing buffer (15 min, followed by 30 min of nonradioactive perfusion). Experiments were performed under baseline conditions and in the presence of thymidine (competitive substrate) or fludarabine (in vitro inhibitor of 5'-nucleotidase). Time-activity curves were acquired by external coincident detectors. The myocardial rate of (124)I-FIAU uptake (K(i)), clearance rate (K(o)), and volume of distribution (V(d) = K(i)/K(o)) were calculated. Subsequently, hearts were subjected to gamma-counting, followed by microtome slicing and autoradiography. RESULTS: The V(d) from Langendorff perfusion significantly correlated with final whole-heart tracer retention (r = 0.88, P = 0.019) and the autoradiographic area of regional myocardial activity (r = 0.89, P = 0.016). HSV1-tk hearts showed higher K(i) and V(d) of (124)I-FIAU compared with that of controls (P < 0.001) and detectable but slower washout compared with that of the LacZ group (P < 0.01). Addition of thymidine to the perfusate inhibited myocardial uptake of (124)I-FIAU by reducing V(d) and K(i) in HSV1-tk and LacZ hearts compared with the baseline. Addition of fludarabine did not result in the expected reduction of washout in HSV1-tk hearts due to inhibition of 5'-nucleotidases (which may dephosphorylate (124)I-FIAU monophosphate). It acted as an uptake inhibitor similar to thymidine, reducing V(d) in HSV1-tk hearts. CONCLUSION: Assessment of specific reporter probe kinetics after regional in vivo reporter gene transfer is feasible using the isolated perfused rat heart preparation. This model allows one to study the effects of pharmacologic interventions and may refine understanding of the reporter probe signal for in vivo imaging. Different nucleoside analogs significantly inhibit (124)I-FIAU uptake, emphasizing the importance of transporter mechanisms for reporter probe kinetics.

PET of cardiac transgene expression: comparison of 2 approaches based on herpesviral thymidine kinase reporter gene.

UNLABELLED: PET of reporter gene expression holds promise for noninvasive monitoring of gene therapy. Previously, 2 approaches based on the herpes simplex virus type 1 thymidine kinase gene (HSV1-tk) have been successfully applied to the heart. Wild-type HSV1-tk was imaged with (124)I-labeled 2'-fluoro-2'-deoxy-5-iodo-1-beta-D-arabinofuranosyl-5-iodouracil (FIAU), and a mutant HSV1-tk (HSV1-sr39tk) was imaged with (18)F-labeled 9-[4-fluoro-3-(hydroxymethyl)butyl]guanine (FHBG). The aim of this study was to compare these 2 combinations with regard to specificity, imaging contrast, and reporter probe kinetics using dynamic PET in small and large animals. METHODS: Similar titers of adenovirus-expressing wild-type HSV1-tk (Ad(tk)), mutant HSV1-sr39tk (Ad(sr39tk)), or control genes were directly injected into the myocardium of 24 rats and 8 pigs. Two days later, dynamic PET was performed with a clinical scanner during the 120 min after injection of (124)I-FIAU (Ad(tk) animals and controls) or (18)F-FHBG (Ad(sr39tk) animals and controls). Imaging with (13)N-ammonia was performed to identify cardiac regions of interest. RESULTS: In rats, significant cardiac (124)I-FIAU accumulation occurred in images obtained early (10-30 min) after Ad(tk) injection. Because of tracer washout, however, no difference between Ad(tk)-injected animals and controls was seen in the images obtained later. For (18)F-FHBG, specific myocardial accumulation greater than background levels was detected in Ad(sr39tk)-injected animals at early imaging and, in contrast to (124)I-FIAU accumulation, increased over time until the latest imaging (105-120 min). At maximum, cardiac (18)F-FHBG concentration showed a 4.15 +/- 1.65-fold increase compared with controls (105-120 min), and cardiac (124)I-FIAU concentration reached a maximal increase of 1.34 +/- 0.38-fold compared with controls (10-30 min, P = 0.0014). Global cardiac reporter probe kinetics in rats were confirmed by regional myocardial analysis in pig hearts. Transgene expression was specifically visualized by both approaches. The highest target-to-background ratio of (124)I-FIAU in Ad(tk)-infected pig myocardium was 1.50 +/- 0.20, versus 2.64 +/- 0.49 for (18)F-FHBG in Ad(sr39tk)-infected areas (P = 0.01). In vivo results were confirmed by ex vivo counting and autoradiography. CONCLUSION: Both reporter gene/probe combinations were feasible for noninvasive imaging of cardiac transgene expression in different species. Specific probe kinetics suggest different myocardial handling of pyrimidine (FIAU) and acycloguanosine (FHBG) derivatives. The results favor (18)F-FHBG with mutant HSV1-sr39tk because of continuous accumulation over time and higher imaging contrast.