Annette Ebner

Preservation of human artery function following prolonged cold storage with a new solution

OBJECTIVE Blood vessels are an important tissue for allogenic vessel replacement surgery, which is needed for example following infection of artificial grafts. For tissue banking, European legislation requires evidence of tissue sterility with assays performed over 1 week. Currently, used cold storage solutions do not protect vascular function longer than 2 days. This does not allow completion of microbiological testing. This discrepancy has almost completely stopped vessel banking in Europe. METHODS We compared the recently developed storage solution TiProtec (Dr F Köhler Chemie, Bensheim, Germany) with traditionally used histidine-tryptophan-ketoglutarate (HTK) solution, 0.9% NaCl, and phosphate-buffered saline (physiological saline solution [PSS]) solution for extended cold (4°C) storage up to 25 days. Isolated rings of human internal mammary artery were studied with respect to several parameters of vessel function, including vessel tone development, endothelium-dependent and endothelium-independent relaxation, and tissue reductive capacity. RESULTS Vessels stored in NaCl or PSS for ≥10 hours failed to develop tone after rewarming. Mammary arteries stored in HTK for 4 hours at 4°C initially showed a well-preserved vessel function with respect to vessel tone development, as well as endothelial and smooth muscle dilatative function. However, following 4 days of cold storage, vessel tone development and dilatative responses were significantly impaired. In contrast, arteries stored in TiProtec showed full preservation of vessel tone as well as endothelial and smooth muscle function after 4 days of cold storage. Even after 10 days of cold storage, endothelium-dependent relaxation was approximately 50% of control, and smooth muscle function was fully preserved. Over 2 weeks, tissue reductive capacity was significantly better maintained after cold storage in TiProtec compared with vessels stored in NaCl. CONCLUSIONS In contrast to traditional HTK, NaCl, or PSS storage, TiProtec solution offers an excellent potential for prolonged cold storage of human arteries, which may close the existing gap between legal requirements for tissue banking and current cold preservation methods.

Preservation of endothelial vascular function of saphenous vein grafts after long-time storage with a recently developed potassium-chloride and N-acetylhistidine enriched storage solution.

OBJECTIVES Saphenous vein grafts are still commonly used in cardiac, vessel and also in transplant surgery. In cardiac surgery, a desperate graft situation could force to keep nonutilized explanted vein segments after CABG in reserve for case of early graft failure. Historically there were no options for adequate long-time graft storage protecting the endothelial layer with its important antithrombotic and immunosuppressive functional aspects. Commonly isotonic saline solution (sodium chloride [NaCl]) has been used as a storing solution in this case. We investigated the impact of long-time storage in NaCl and a recently developed potassium-chloride and N-acetylhistidine enriched storage solution (TiProtec, Dr. Köhler Chemie, Germany) on endothelial function of saphenous veins. METHODS Saphenous vein segments (n = 19) were intraoperatively isolated and stored for 24 and 96 hours. The segments were examined in a Mulvany-myograph to assess vessel function. Following preconstriction with norepinephrine, dose-response curves were assessed for relaxation with bradykinin and sodium-nitroprusside. We compared developed maximum wall tension and endothelial cell and smooth muscle cell (SMC) dependent vasodilatory function. RESULTS Maximum vessel wall tension was significantly better preserved in TiProtec-stored vessels after 24 h in comparison to segments stored in NaCl (5.11 ± 4.79 mN/mm vs. 2.48 ± 2.43 mN/mm; p = 0.033) and 96 h (4.94 ± 2.82 mN/mm vs. 2.80 ± 1.76 mN/mm; p = 0.042). Likewise endothelium-derived vasodilatory function was maintained significantly after 24 hours in TiProtec-stored vessels (36.9 ± 2.6% vs. 11.8 ± 30.9%; p = 0.005). After 96 hours, endothelium-dependent vascular function was nearly abolished in NaCl-stored vessels, but largely preserved in TiProtec-stored segments (20.6 ± 2.9% vs. 1.9 ± 4.3% in NaCl; p = 0.015). Sodium nitroprusside-mediated SMC-vasodilatory function was better maintained after 24 hours of storage in TiProtec group (88.8 ± 6.4% vs. 61.3 ± 8.2%; p = 0.009). After 96 hours of storage, SMC relaxation did not significantly differ between both storage groups which might be due to a distinct reduction of contractile function in NaCl-stored vessels (98.6 ± 5.0% and 77.9 ± 10.5% for Tiprotec and NaCl, respectively). CONCLUSION Vessel functions comprising contraction, endothelium-dependent and -independent vasodilatation are significantly reduced following 24 hours of cold storage in NaCl. After 96 hours of storage in NaCl these functions are nearly totally abolished. TiProtec is able to largely reduce this loss of function during cold storage. Therefore, TiProtec is a feasible option for longer term storage of saphenous vein grafts in CABG vessel and transplant surgery.

Pharmacological postconditioning by bolus injection of phosphodiesterase-5 inhibitors vardenafil and sildenafil

Postconditioning enables cardioprotection against ischemia/reperfusion injury either by application of short, repetitive ischemic periods or by pharmacological intervention prior to reperfusion. Pharmacological postconditioning has been described for phosphodiesterase-5 inhibitors when the substances were applied as a permanent infusion. For clinical purposes, application of a bolus is more convenient. In a rat heart in situ model of ischemia reperfusion vardenafil or sildenafil were applied as a bolus prior to reperfusion. Cardioprotective effects were found over a broad dosage range. In accordance with current hypotheses on pharmacological postconditioning signaling, the protective effect was mediated by extracellular signal-regulated kinase and protein kinase C pathway. Interestingly, the extent of protection was independent of the concentration applied for both substances. Full protection comparable to ischemic postconditioning was reached with half-maximal human equivalence dose. In contrast, mean arterial pressure dropped upon bolus application in a dose-dependent manner. Taken together, the current study extends previous findings obtained in a permanent infusion model to bolus application. This is an important step toward clinical application of pharmacological postconditioning with sildenafil and vardenafil, especially because the beneficial effects were proven for concentrations with reduced hemodynamic side effects compared to the dosage applied for erectile dysfunction treatment.

Heparinized blood better preserves cellular energy charge and vascular functions of intraoperatively stored saphenous vein grafts in comparison to isotonic sodium-chloride-solution

OBJECTIVE Several studies have addressed the optimal storage conditions for vascular grafts during surgery. The results remain contradictionary. This may be attributed to the fact, that the various vascular beds have a different sensitivity to storage. We analyzed the impact of storage in isotonic saline solution (NaCl) or heparinized blood the vascular functions of human saphenous vein grafts. Special care was taken to choose storage conditions which are relevant for intraoperative storage of a saphenous vein graft in a setting of coronary artery bypass grafting with vein and internal mammary artery as grafts. METHODS Intraoperatively isolated V. saphena-segments (n = 36) were stored in NaCl or heparinized blood for approximately 30 minutes at room temperature. Subsequently, the segments were examined in a Mulvany-myograph. Following preconstriction with norepinephrine, concentration-relaxation curves were assessed for bradykinin and sodium-nitroprusside to assess developed vessel-wall tension as well as endothelium- and smooth-muscle-cell dependent vasorelaxation. The availability of adenosintriphosphate (energy charge) was determined based on liquid chromatography measurements of nucleotide tissue levels. RESULTS Mean storage time was 27.4 ± 2.4 min in NaCl- and 26.3 ± 2.7 min in blood-group, respectively. After this period, receptor-dependent and-independent maximum of developed vessel wall tension was significantly reduced in NaCl-group (p = 0.05 and p = 0.045, respectively). Furthermore, the energy charge was significantly (p = 0.046) better preserved after blood storage (74 ± 1%) in comparison to NaCl-group (68 ± 2%). Endothelium-induced vasodilatation in response to bradykinin reached only 12.3 ± 2.5% in NaCl-group, but 19.3 ± 5.2% in blood-group (p = 0.033). Alike, EC50-concentration of bradykinin for half-maximal relaxation was significantly lower in blood- than in NaCl-group (log EC50 -7.08 ± 0.3 and -5.91 ± 0.4; respectively; p = 0.046). Endothelium-independent smooth muscle relaxation in response to sodium-nitroprusside was not different between both groups. CONCLUSION Heparinized blood better preserves vascular contractile and endothelial functions of the saphenous vein graft. Storage in NaCl rapidly compromises vascular functions and impaires cellular energy. NaCl should no longer be recommended for intraoperative storage of harvested V. saphena grafts.

Functional, morphologic, and molecular characterization of cold storage injury.

OBJECTIVE Cold storage is used to preserve tissue for later transplantation. There is particular interest in prolonging cold storage time for transplantation purposes. To date, the mechanisms that contribute to vascular dysfunction in response to cold storage are poorly understood. The present study aims to characterize cold storage injury of blood vessels on functional and molecular levels. METHODS To assess vessel function of mouse aorta, isometric force measurements were performed in a Mulvany myograph after cold storage at 4°C for various intervals. Morphologic changes were judged by histologic analysis of aortic cross-sections. To characterize cold storage-induced alterations on RNA levels, microarray analysis with subsequent polymerase chain reaction analysis was performed. RESULTS Cold storage for 2 days revealed significant impairment of vessel function with respect to potassium-induced vessel tone development and acetylcholine-induced vessel relaxation. Detailed analysis of acetylcholine-mediated vascular response using specific pharmacologic blockers revealed that calcium-activated potassium channels seem to be impaired after 2 days of cold storage. At this point, no severe histologic changes (eg, elastic fiber disruption) were visible. RNA expression of 24 genes was significantly changed due to cold storage even after 2 hours. These include genes associated with vessel tone development (prostaglandin E(3) receptor), cardiovascular function (adiponectin), electron transport chain (uncoupling protein 1), or calcium signaling (protein kinase A regulatory subunit 2b). CONCLUSIONS Long-term cold storage impairs vascular function, especially with respect to potassium signaling by calcium-dependent potassium channels. Microarray analysis confirmed impairment of pathways that are involved in calcium signaling and vascular function. Furthermore, various genes were significantly altered even after 2 hours, significantly before functional impairment was observed.

In situ postconditioning with neuregulin-1β is mediated by a PI3K/Akt-dependent pathway.

BACKGROUND The myocardial infarct size can be reduced by pharmacological postconditioning using cardioprotective agents. Neuregulin-1β is a potential candidate, but previous studies in an isolated heart model of ischemia and reperfusion displayed controversial results. An in situ model of ischemia/reperfusion was used to clarify whether the remote application of neuregulin-1β can reduce the reperfusion injury. A second aim was to evaluate, if the effects are specific for reperfused tissue or if this is a general antiapoptotic effect. In addition, the contributing molecular mechanisms were investigated. METHODS In an open chest model, mouse hearts were subjected to a regional ischemia (45-minute) using ligature of the left anterior descending artery. Neuregulin-1β (80 ng/kg) was given using an intraperitoneal bolus injection 5 minutes before reopening of the ligature followed by a 30-minute reperfusion. RESULTS Remote application of recombinant neuregulin-1β protected the heart from reperfusion injury without influencing hemodynamics. This beneficial effect specifically targets reperfusion injury. In contrast, nonreperfused needle trauma was not reduced by neuregulin-1β when applied remotely. Pharmacological blocking experiments and enzyme activation analysis using Western blot analysis revealed a crucial involvement of the antiapoptotic reperfusion injury salvage kinase cascade. In contrast, contribution of the survivor activating factor enhancement pathways to this early cardioprotection was not observed. CONCLUSIONS Remote application of neuregulin-1β protects hearts from early reperfusion injury by activation of the reperfusion injury salvage kinase pathway without relevant effects on intracardiac pressures in myocardial infarction. Besides its potential pharmacological application, neuregulin-1β might act as an endogenously produced mediator in remote postconditioning.

Dexrazoxane prevents the development of the impaired cardiac phenotype in caveolin-1-disrupted mice.

Abstract: Caveolin-1-deficient (cav1−/−) mice display a severely diseased cardiac phenotype with systolic and diastolic heart failure. Accumulating evidence supports a causative role of uncoupled endothelial nitric oxide synthase in the development of these abnormalities. Interestingly, a similar molecular mechanism was proposed for anthracycline-induced cardiomyopathy. Currently, dexrazoxane is approved for the prevention of anthracycline-induced cardiomyopathy. Given the molecular similarities between the anthracycline-induced cardiomyopathy and the cardiomyopathy in cav1−/− mice, we questioned whether dexrazoxane may also prevent the evolution of the cardiac pathologies in cav1−/− mice. We evaluated dexrazoxane treatment for 6 weeks in cav1−/− mice and wild-type controls. This study provides the first evidence for a reduced reactive oxygen species formation in the vessels of dexrazoxane-treated cav1−/− mice. This reduced oxidative stress resulted in a markedly reduced rate of apoptosis, which finally was translated into a significantly improved heart function in dexrazoxane-treated cav1−/− mice. These hemodynamic improvements were accompanied by significantly lowered proatrial natriuretic peptide levels. Notably, these protective properties of dexrazoxane were not evident in wild-type animals. Taken together, these novel findings indicate that dexrazoxane significantly reduces vascular reactive oxygen species formation cav1−/−. Because this is paralleled by an improved cardiac performance in cav1−/− mice, our data suggest dexrazoxane as a novel therapeutic strategy in this specific cardiomyopathy.

Evaluation of cold storage conditions for vessels obtained from donor rats after cardiac death

OBJECTIVE To improve the availability of vessel grafts for allotransplantation, the current experimental study was designed to first investigate the function of vessels obtained from non-heartbeating donor rats at various time points postmortem; second, to assess the sensitivity of vessels recovered after circulatory arrest toward prolonged cold storage; and third, to determine vessel function following cold storage with antimicrobial additives. METHODS We investigated vessel tone development and endothelium-dependent and endothelium-independent relaxations in a Mulvany myograph of aorta and saphenous artery sampled up to 24 hours after circulatory arrest. Additionally, tissue reductive capacity and lactate dehydrogenase release were measured. RESULTS Vessels recovered 2 hours postmortem showed similar results as controls recovered without delay. Vessels recovered 6 hours or more after circulatory arrest showed reduced vessel tone development (ie, aorta): response to potassium <15% and response to norepinephrine <25% of vessels recovered without delay; A. saphena response to potassium: <12% and response to norepinephrine <10% of control vessels recovered without delay. All vessels recovered after circulatory arrest showed a similar cold storage sensitivity as controls, with exception of a decreased endothelial function of A. saphena harvested 6 hours postmortem (one-third response of non-stored control vessels). Treatment of vessels recovered immediately or after circulatory arrest with gentamycin, piperacillin, and metronidazole as additives to the optimized cold storage solution did not alter vessel function. Flucloxacillin as a cold storage additive reduced vessel tone development in aorta but not in A. saphena. Addition of amphotericin B to the storage solution completely abolished any vessel function and impaired tissue reductive capacity despite presence of radical scavengers. CONCLUSIONS The use of vessels from non-heartbeating donors in general and subsequent prolonged cold storage seems feasible when vessels are recovered within 2 hours. The use of antibiotics needs to be carefully assessed for each intended-to-use tissue. For vessels tested, a combination of gentamycin, piperacillin, and metronidazole supported the maintenance of vessel function.

The endothelial nitric oxide synthase cofactor tetrahydrobiopterin shields the remote myocardium from apoptosis after experimental myocardial infarction in vivo

BACKGROUND Following myocardial infarction (MI), apoptosis occurs early in the remote myocardium and contributes to the processes of myocardial remodelling. Increased nitrosative stress is a well-known and potent inductor of myocardial apopto¬sis. Excess activation of endothelial nitric oxide synthase (eNOS) increases its uncoupling potential and results in nitrosative stress via formation of peroxynitrite. However, the pathophysiological role of eNOS signalling in the remote myocardium after MI is as yet undefined. AIM The impact of eNOS activation on pro- and anti-apoptotic signalling in the remote myocardium and the influence of pretreatment with the eNOS cofactor tetrahydrobiopterin (BH4) on eNOS activation, nitrosative stress level, and apoptosis induction and execution were studied in a rat MI model in vivo. RESULTS Twenty-four hours after anterior MI, eNOS activity in animals treated with left anterior descending coronary artery ligation (LIG) significantly increased in the posterior left ventricular (LV) myocardium as did protein nitrosylation when com¬pared to sham treatment. This was paralleled by induction of apoptosis via the extrinsic and intrinsic pathways. Moreover, anti-apoptotic signalling via protein kinase B/Akt and glycogen synthase-kinase 3 beta was suppressed. Notably, pretreatment with the eNOS cofactor BH4 reduced eNOS activation, prevented excess protein nitrosylation, blunted apoptosis induction, facilitated anti-apoptotic signalling, and eventually prevented apoptosis execution. CONCLUSIONS Here we showed that 24 h after experimental MI in rats in vivo, apoptosis was induced in the posterior non-in¬farcted LV wall. Evidence is presented that pretreatment with the eNOS cofactor BH4 resulted in less nitrosative stress and weakened apoptotic processes, although the stabilisers contained did participate in this phenomenon. Because apoptosis is a crucial component of myocardial remodelling, influencing eNOS signalling might be an interesting pharmacological target for the development of novel anti-remodelling therapies.

Endothelial Nitric Oxide Synthase-Induced Hypertrophy and Vascular Dysfunction Contribute to the Left Ventricular Dysfunction in Caveolin-1−/− Mice

BACKGROUND Caveolin-1 (Cav1)-/- mice display impaired development of left ventricular pressure and increased left ventricular wall thickness but no dilated ventricle; these are typical findings in patients with heart failure with preserved ejection fraction (HfpEF). Aiming to clarify if dysfunctional endothelial nitric oxide synthase (eNOS) influences cardiomyocyte contractility, cardiac conduction system, or afterload/vascular resistance, we studied Cav1-/-/eNOS-/- mice. METHODS Cardiac function was assessed in vivo by pressure-volume-catheterization of the left ventricle, echocardiography and electrocardiography. In addition, isolated tissue experiments were performed to evaluate cardiomyocyte contractility (atria) and vessel morphology and function (aorta). Histology, immunoblotting and quantitative polymerase chain reaction were applied to characterise radical formation and oxidative stress in the heart. RESULTS Cardiac hypertrophy was completely reversed in Cav1-/-/eNOS-/- mice. The impaired pump function in Cav1-/- mice was significantly improved in Cav1-/-/eNOS-/- mice, but no complete alignment with eNOS-/- controls was achieved, indicating an additional eNOS-independent mechanism contributing to HFpEF in Cav1-/- mice. It is unlikely that frequently occurring arrhythmias contributed to HFpEF in Cav1-/- mice. In contrast, numerous eNOS-dependent and eNOS-independent vascular abnomalities could explain the cardiac phenotypes of Cav1-/- mice. CONCLUSIONS Synergistic effects between eNOS-related cardiac hypertrophy and vascular hypercontractility appear to underlie the left ventricular dysfunction in Cav1-/-mice. These findings provide insights relevant to the poorly understood pathophysiology of HFpEF.