Reprogramming Bone Marrow Stem Cells to Functional Endothelial Cells in a Mini Pig Animal Model.

BACKGROUND The aims of this study were to compare the morphological, biochemical, and functional properties of reprogrammed bone marrow stem cell (BMSC)-derived arterial endothelial cells (AECs) and venous endothelial cells (VECs), following adenosine triphosphate (ATP)-stimulation in a mini pig animal model. MATERIAL AND METHODS Bone marrow aspiration was performed in six adult mini pigs. Harvested mononuclear cells were isolated, cultured, and treated with vascular endothelial growth factor (VEGF) (16 µg/ml). Transformed cells were characterized using immunofluorescence staining for CD31 and von Willebrandt factor (vWF) and expression of endothelial nitric oxide synthase (eNOS). Cell release of nitric oxide (cNO) was measured using spectrophotometry. Matrigel assays were used to investigate angiogenesis in transformed BMSCs. RESULTS Reprogrammed BMSCs in culture showed a typical cobblestone-like pattern of growth. Immunofluorescence staining was positive for CD31 and vWF expression. Expression of eNOS, using immunofluorescence staining and Western blot, showed no difference between the reprogrammed BMSCs and VECs. Spectrophotometric examination following stimulation with 10mmol/l ATP, showed comparable cNO release for reprogrammed BMSCs (10.87±1.76 pmol/106 cells/min) and VECs (13.23±2.16 pmol/10^6 cells/min), but reduced cNO release for AECS (3.44±0.75 pmol/10^6 cells/min). Matrigel assay for angiogenesis showed vascular tube formation of differentiated BMSC endothelial cells (grade 3.25). BMSCs cultured without VEGF did not demonstrate vascular tube formation. CONCLUSIONS The findings of this study showed that eNOS expression and release of NO could be used to show that BMSCs can be reprogrammed to functional VECs and AECs.

Evaluation of conventional and frozen elephant trunk techniques on spinal cord blood flow in an animal model.

OBJECTIVES: The treatment of patients with extensive thoracic aortic disease involving the arch and descending aorta is often performed using the frozen elephant trunk technique (FET). Spinal cord blood flow (SCBF) in cervical, thoracic and lumbar sections prior, during and after aortic arch surgery were compared in conventional elephant trunk (cET) and FET technique in a pig model. METHODS: German Landrace pigs (75-85 kg) underwent aortic arch surgery using the FET (n = 8) or cET (n = 8) techniques. The E-vita Open hybrid stent graft was applied in all FET animals. Regional SCBF was measured 4 times: (i) before cardiopulmonary bypass, (ii) after 1 h, (iii) after 3 h, and (iv) after 6 h of reperfusion using fluorescence microspheres. Spinal cord segments were examined histopathologically and by immunohistochemistry. RESULTS: SCBF in FET decreased significantly from 0.13 ± 0.03 to 0.05 ± 0.02 ml/min/g after 1 h (P = 0.047). While at 3 h of reperfusion, SCBF increased and was comparable to baseline (0.09 ± 0.01 ml/min/g), beyond this time SCBF decreased again (0.05 ± 0.02 ml/min/g). A similar trend was found for SCBF in the cET group (baseline: 0.16 ± 0.04 ml/min/g, 1 h reperfusion: 0.02 ± 0.01 ml/min/g, 3 h reperfusion: 0.03 ± 0.01 ml/min/g and 6 h reperfusion: 0.02 ± 0.01 ml/min/g, P = 0.019). Cervical, thoracic and lumbar SCBF were also comparable in both groups. Histological analyses of spinal cord showed no differences in necrosis between cET and FET, while no differences were found for hypoxia-inducible factor-1α and apoptosis-inducing factor. In contrast, oxidative stress and caspase-induced apoptosis were higher in cET versus FET. CONCLUSIONS: The SCBF changed significantly during extensive aortic arch surgery with circulatory arrest and moderate hypothermia, but such changes were comparable between the FET and cET groups. The implantation of hybrid stent graft did not influence SCBF in thoracic and lumbar segments of the spinal cord. The immunohistological examination showed no differences between cET and FET regarding ischaemic damage and hypoxia-induced effects in spinal cord segments.

Effects of minocycline on parameters of cardiovascular recovery after cardioplegic arrest in a rabbit Langendorff heart model.

Pharmacological cardiac organ protection during cardiopulmonary bypass presents an opportunity for improvement. A number of different strategies have been established to minimize ischemia/reperfusion-induced damage to the heart. Among these, cardioplegia with histidine-tryptophan-ketoglutarate solution and hypothermia are the most frequently used regimens. The antibiotic minocycline has been used in this context for neuroprotection. The aim of the current study was to evaluate whether the application of minocycline prior to cardioplegia exerts a protective effect on cardiac muscle. For this purpose, this study investigated six rabbit hearts with minocycline treatment (1 µmol/L) and six without in a Langendorff model of 90 min cold cardioplegic arrest using Custodiol followed by a 30 min recovery phase. Histological analysis of cardiac muscle revealed that markers of apoptosis, oxidative and nitrosative stress were significantly lower in the minocycline group, whereas adenosine triphosphate (ATP)- and malondialdehyde (MDA)-levels and O2-consumption were not affected by minocycline. Functionally, recovery of dP/dt (max) and dP/dt (min) was significantly faster in the minocycline group than in control. This leads to the conclusion that adding minocycline to the cardioplegic solution may improve left ventricular recovery after cardioplegic arrest involving reduced pro-apoptotic effects.

Injectable tissue engineered pulmonary heart valve implantation into the pig model: A feasibility study.

BACKROUND: Transcatheter pulmonary valve replacement is currently performed in clinical trials, however limited by the use of glutaraldehyde treated bioprostheses. This feasibility study was performed to evaluate delivery-related tissue distortion during implantation of a tissue engineered (TE) heart valves. MATERIAL AND METHOD: The injectable TE heart valve was mounted on a self-expanding nitinol stent (n=7) and delivered into the pulmonary position of seven pigs, (weight 26 to 31 kg), performing a sternotomy or limited lateral thoracotomy. Prior to implantation, the injectable TE heart valve was crimped and inserted into an applicator. Positioning of the implants was guided by fluoroscopy and after carefully deployment angiographic examination was performed to evaluate the correct delivered position. Hemodynamic measurements were performed by epicardial echocardiography. Finally, the animals were sacrificed and the injectable TE heart valves were inspected by gross examination and histological examination. RESULTS: Orthotopic delivery of the injectable TE heart valves were all successful performed, expect in one were the valve migrated due to a discrepancy of pulmonary and injectable TE valve size. Angiographic evaluation (n=6) showed normal valve function, supported by epicardial echocardiography in which no increase flow velocity was measured, neither trans- nor paravalvular regurgitation. Histological evaluation demonstrated absence of tissue damage due to the delivery process. CONCLUSIONS: Transcatheter implantation of an injectable TE heart valve seems to be possible without tissue distortion due to the delivery system.