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Pulsatile perfusion during extracorporeal circulation is a promising concept to improve perfusion of critical organs. Clinical benefits are limited by the amount of pulsatile energy provided by standard pumps. The present study investigated the properties of a novel positive displacement blood pump in a mock circulation.The pump was attached to an aortic model with a human-like geometry and compliance as a pseudo patient. Hemodynamic data were recorded while the pump settings were adjusted systematically.Using a regular oxygenator, maximum flow was 2.6 L/min at a pressure of 27 mm Hg and a frequency (F) of 90 bpm. Pulse pressure (PP; 28.9 mm Hg) and surplus hemodynamic energy (SHE; 26.1% of mean arterial pressure) were highest at F = 40 bpm. Flow and pressure profiles appeared sinusoid. Using a low-resistance membrane ventilator to assess the impact of back pressure, maximum flow was 4.0 L/min at a pressure of 58.6 mm Hg and F = 40 bpm. At F = 40 bpm, PP was 58.7 mm Hg with an SHE of 33.4%. SHE decreased with increasing flow, heart rate, and systolic percentage but surpassed 10% with reasonable settings.The present prototype achieved sufficient flow and pressure ranges only in the presence of a low-resistance membrane ventilator. It delivered supraphysiologic levels of pulse pressure and SHE. Further modifications are planned to establish this concept for adult pulsatile perfusion.

Background Pulsatile extracorporeal circulation (ECC) may improve perfusion of critical organs during cardiac surgery. This study analyzed the influence of the components of a minimal invasive ECC (MiECC) on the transfer of pulsatile energy into the pseudo-patient of a mock circulation. Methods An aortic model with human-like geometry and compliance was perfused by a diagonal pump. Surplus hemodynamic energy (SHE) was determined from flow and pressure data. Five adult-size oxygenator models and three sizes of cannulas were compared. Pulsatile pump settings were optimized, and parallel dual-pump configurations were evaluated. Results Oxygenator models showed up to twofold differences in pressure gradients and influenced SHE at flow rates up to 2.0 L min−1. Adjustments of frequency, systole duration, and rotational speed gain significantly improved SHE compared with empirical settings, with SHE above 21% of mean arterial pressure at flow rates of 1.0 L min−1 to 1.5 L min−1 and SHE above 5% at 3.5 L min−1. Small diameter cannula (15 Fr) limited SHE compared with larger cannula (21 Fr and 23 Fr). Two diagonal pumps did not provide higher SHE than a single pump, but permitted additional control over pulse pressure and SHE by varying the total fraction of pulsatile flow and the fraction of flow bypassing the oxygenator. Conclusions Proper selection of components and optimizations of pump settings significantly improved pulse pressure and SHE of pulsatile MiECC. Surplus hemodynamic energy depended on flow rate with a maximum at 1.0 L min−1–1.5 L min−1. Pulsatile MiECC may specifically assist organ perfusion during phases of low flow., publishedVersion

The restriction of hydroxyethylstarch (HES) necessitated changes in volume management in cardiac surgery, increasing the use of gelatin (GELA) and crystalloid (CRYS) mono strategies.This retrospective study evaluated the effects of changed volume replacement management to a GELA or CRYS mono therapy on mortality, acute kidney injury (AKI), blood loss, and transfusion in cardiac surgery patients with at least one coronary artery bypass grafting (CABG) at a university hospital. Three groups (HES n=938, GELA n=397, CRYS n=205) were derived from 1,540 patients with complete data sets. Data were analyzed by multiple regression models.Patients had similar risk profiles, comorbidities, and preoperative routine diagnostics prior to surgery. No difference was observed in mortality and AKI. HES treated patients showed highest blood loss, followed by GELA while CRYS patients had the lowest (P0.0001). Patients in the HES group had highest transfusion of packed red blood cells (PRBCs) and platelet concentrates (PCs), followed by GELA, whereas CRYS had the lowest (P0.0001). Fresh frozen plasma (FFP) transfusion, administration of fibrinogen, and prothrombin complex concentrates (PCCs) were highest in HES group. CRYS showed the shortest time of mechanical ventilation (P0.0001) and left the intensive care unit (ICU) significantly earlier (P0.0001). Multivariable regression analysis found that colloid volume significantly predicted hospital mortality and renal replacement therapy (RRT), but not AKI.Administration of crystalloids without any colloid showed no differences in mortality or AKI, but less blood loss and transfusion. Colloids should be considered critically and further studies should investigate effects of GELA in cardiac surgery.

Introduction: Adrenoceptor and endothelin (ET) receptor-mediated vasoconstriction as well as endothelium-dependent vasodilation of human saphenous veins were compared before and after 20 h of cold storage. Methods: Contractile responses to potassium chloride (KCl), norepinephrine (NE), and ET-1 as well as vasodilator responses to acetylcholine (ACh) were evaluated. Results: Storage in HEPES-supplemented Dulbecco’s modified Eagle’s medium (HDMEM) diminished KCl induced contractile forces to 71% (p = 0.002) and NE induced contractions to 80% (p = 0.037), in contrast to HEPES-supplemented Krebs-Henseleit solution (HKH) and TiProtec solution. KCl-normalized NE contractions were not affected by storage. NE EC50 values were slightly lower (7.1E−8 vs. 7.5E−8, p = 0.019) after storage in HKH, with no changes after storage in the other solutions. Endothelium-dependent responses to ACh were not affected by storage. ET-1 induced contractions were attenuated after storage in HDMEM (77%, p = 0.002), HKH (75%, p = 0.020), and TiProtec (73%, p = 0.010) with no changes in normalized constrictions. ET-1 EC50 values were not affected by storage. Conclusion: Loss of contractility after storage in HDMEM may reflect the lower content of dextrose. There was no specific attenuation of adrenoceptor, ET-receptor, or ACh receptor mediated signal transduction after storage in any of the media. HKH or TiProtec are equally suitable cold storage solutions for ex vivo measurements.

In this study, we characterize age-related phenotypes of human hematopoietic stem cells (HSC). We report increased frequencies of HSC, hematopoietic progenitor cells and lineage negative cells in the elderly but a decreased frequency of multi-lymphoid progenitors. Aged human HSC further exhibited a delay in initiating division ex vivo though without changes in their division kinetics. The activity of the small RhoGTPase Cdc42 was elevated in aged human hematopoietic cells and we identified a positive correlation between Cdc42 activity and the frequency of HSC upon aging. The frequency of human HSC polar for polarity proteins was, similar to the mouse, decreased upon aging, while inhibition of Cdc42 activity via the specific pharmacological inhibitor of Cdc42 activity, CASIN, resulted in re-polarization of aged human HSC with respect to Cdc42. Elevated activity of Cdc42 in aged HSC thus contributed to age-related changes in HSC. Xenotransplant, using NBSGW mice as recipients, showed elevated chimerism in recipients of aged compared to young HSC. Aged HSC treated with CASIN ex vivo displayed an engraftment profile similar to recipients of young HSC. Taken together, our work reveals strong evidence for a role of elevated Cdc42 activity in driving aging of human HSC, and similar to mice, this presents a likely possibility for attenuation of aging in human HSC.

Objective The restriction of hydroxyethyl starch (HES) has mandated changes in volume management based on data of critically ill patients. Reliable data of structural renal damage after HES treatment in cardiac surgical patients are lacking. The influence of 6% HES 130/0.4 was investigated in this study. Design An exploratory post hoc subgroup analysis of a prospective trial was performed. Setting The study was carried out at a university hospital. Participants Forty-four low-risk cardiac surgical patients were examined. Interventions Twenty-two patients received only crystalloid solutions, and 22 were treated with balanced 6% HES 130/0.4. Measurements and Main Results Functional renal parameters and the structural biomarkers α-glutathione S-transferase, kidney injury molecule-1, liver fatty acid-binding protein, and neutrophil gelatinase-associated lipocalin were investigated. Volume balances, vasopressor dosages, blood losses, and coagulation parameters were compared. Most functional and structural renal parameters did not differ between the groups (serum creatinine p = 0.8380). Liver fatty acid-binding protein was transiently higher in the HES group only at 24 hours postoperatively (p = 0.0002). No differences in mortality, acute kidney injury, and need for renal replacement therapy were observed. Blood coagulation was significantly more compromised in the HES group at intensive care unit arrival (factor II, p = 0.0012; factor X, p = 0.0031; thrombocytes, p = 0.0010). Blood losses, and vasopressor dosages tended to be higher in HES-treated patients without significance. Conclusion Overall, the values and time courses of the biomarkers used did not indicate evidence of a mechanism for tubular injury caused by HES.

Blood oxygenized by veno-arterial extracorporeal membrane oxygenation (ECMO) can be returned to the aorta (central cannulation) or to peripheral arteries (axillar, femoral). Hemodynamic effects of these cannulation types were analyzed in a mock loop with an aortic model representative of normal anatomy and compliance under physiological pressures and flow rates. Pressures, flow rates, and contribution of ECMO flow to total flow as a measure of oxygen supply were monitored in the carotids. Steady or pulsatile ECMO flow, residual or no cardiac output, and intraaortic balloon pump counterpulsation were tested as independent factors. With residual heart function, central cannulation provided the best oxygenated flow and pressure to the carotid arteries (CA). Axillar cannulation preferentially perfused the right CA at the expense of the left CA. Femoral cannulation provided only lower amounts of oxygenated blood to both CA. Pulsation increased the surplus hemodynamic energy. Counterpulsation reduced flow with femoral cannulation but improved flow and pressure with axillar cannulation. Femoral cannulation failed to provide oxygenated blood to coronary and supraaortic arteries with residual heart function. Central cannulation provided the best hemodynamics and oxygen supply to the brain. With a resting heart but not with an ejecting heart, pulsatile ECMO flow enhanced CA hemodynamics.

OBJECTIVES Pulmonary arterial hypertension is characterized by pulmonary vascular proliferation and remodelling, leading to a progressive increase in pulmonary arterial resistance. Vasodilator properties of 3 different phosphodiesterase (PDE)-5 inhibitors alone and in combination with an endothelin (ET) receptor antagonist were compared in an ex vivo model. METHODS Segments of human pulmonary arteries (PAs) and pulmonary veins (PVs) were harvested from lobectomy specimens. Contractile forces were determined in an organ bath. Vessels were constricted with norepinephrine (NE) to determine the effects of sildenafil, tadalafil and vardenafil and with ET-1 to assess the effects of bosentan. RESULTS All 3 PDE-5 inhibitors had no relevant effect on the basal tone of the vessels. Both sildenafil and vardenafil significantly (P

Background From the results of a previous study, it remained to be investigated if a perioperative rise of few tested coagulation and inflammation markers is caused by conventional cardiopulmonary bypass (CPB) itself or rather by direct recirculation of pericardial fluids. Methods Forty-eight patients operated on with conventional CPB for myocardial revascularization were randomized either for direct recirculation of pericardial suction fluids or for cell saving (CS). Results Thrombin–antithrombin complexes showed lower values intraoperatively in the CS group (p Conclusion Direct recirculation of pericardial fluids rather than conventional CPB itself causes major intraoperative changes of some coagulation markers. Pericardial blood loss with direct recirculation should be kept to a minimum to avoid unnecessary activation of coagulation. Inflammation markers need further investigations.

Background: Minimal invasive extracorporeal circulation (MiECC) reduces the impact of cardiopulmonary bypass during cardiac surgery on inflammation and hemostasis. Pulsatile perfusion may enhance organ perfusion and help to prevent renal and neuronal damage. The present study investigated the impact of pulsatile MiECC in low-risk coronary artery bypass grafting (CABG) patients. Methods: CABG patients were prospectively randomized for non-pulsatile (np: n=19) and pulsatile (p: n=21) MiECC. Blood and urine samples were collected at several time points until 72 h post-operative and analyzed for biochemical markers of fibrinolytic capacity, renal damage, and neuronal damage. Results: Although intraoperative tissue plasminogen activator (tPA) levels tended to be higher in the p group, none of the fibrinolysis markers including plasminogen activator inhibitor (PAI-1) and the PAI-1/tPA ratio were significantly affected by pulsation. Hemolysis and markers of renal and neuronal damage were comparable between groups. Intraoperative urinary excretion [np: 400 mL (355 to 680) vs . p: 530 mL (360 to 900)] and cumulative 24 h volume intake [np: 7,090 mL (5,492 to 7,544) vs . p: 7,155 mL (6,682 to 8,710)] were increased by pulsation whereas blood losses up to 12 h post-operative [np: 365 mL (270 to 515) vs . p: 310 mL (225 to 470)] and up to 24 h post-operative [np: 760 mL (555 to 870) vs . p: 520 mL (460 to 670)] were attenuated. Conclusions: The present study did not find evidence for a beneficial effect of pulsation on markers of fibrinolysis, renal damage, and neuronal damage. However, pulsatile perfusion increased intraoperative urinary secretion and reduced post-operative blood losses.

Introduction: Pulsatile extracorporeal circulation may improve organ perfusion during cardiac surgery. Some minimally invasive extracorporeal circulation (MiECC) systems allow pulsatile perfusion. The present study investigated the influence of arterial tubing compliance on hemodynamic energy transfer into the patient. Methods: Aortic models with adult human geometry were perfused in a mock circulation. A MiECC system was connected using either high-compliance silicone tubing or standard kit tubing. Energy equivalent pressure (EEP) and surplus hemodynamic energy (SHE) were computed from flow and pressure data. Aortic models with physiological and sub-physiological compliance were tested to assess the influence of the pseudo-patient. Results: Non-pulsatile flow did not generate SHE. SHE during pulsatile flow in the compliant aortic model was significantly higher with kit tubing compared to silicone tubing. Maximum SHE was achieved at 1.6 L/min with kit tubing (7.7% of mean arterial pressure) and with silicone tubing (4.9%). Using the low-compliance aortic model, SHE with kit tubing reached a higher maximum of 14.2% at 1.8 L/min compared to silicone tubing (11.8% at 1.5 L/min). Conclusions: Flexible arterial tubing did not preserve more hemodynamic energy from a pulsatile pump compared to standard kit tubing in a model of adult extracorporeal circulation. The pseudo-patient’s compliance significantly affected the properties of the mock circulation. © In Copyright http://rightsstatements.org/vocab/InC/1.0/, publishedVersion

Purpose Medical combination therapy of pulmonary arterial hypertension (PAH) may alleviate the drawbacks of monotherapy by avoiding drug tolerance and by increasing effectiveness, as shown by the combination of ambrisentan and tadalafil (AMBITION trial). The present ex-vivo study evaluated the combination of the endothelin receptor antagonists (ERA) macitentan and bosentan with the phosphodiesterase-5 (PDE-5) inhibitor vardenafil in pulmonary arteries from patients suffering from terminal lung disease as a model of PAH. Methods Segments of the pulmonary vessels were excised from resected lungs of patients requiring lung transplantation (LTX). Contraction of pulmonary arteries (PA) was elicited by consecutive dose-response curves of endothelin-1 (ET-1) followed by norepinephrine (NE) to allow inhibition by different pathways. Forces were measured isometrically in an organ bath in the presence and absence of ERA and PDE-5 inhibitors and their combination. Results PA of 38 patients were examined between October 2016 and November 2019. Bosentan (1E-7 M) and macitentan (1E-8 M, 3E-8 M, 1E-7 M) inhibited ET-1 induced contractions, whereas vardenafil (1E-6 M, 3E-6 M, 1E-5 M) inhibited only the NE induced part of the contractions. Vardenafil enhanced bosentan-induced inhibition of vasoconstriction in a dose-dependent fashion. Combination effects exceeded single bosentan at 3E-6 M and 1E-5 M vardenafil, and they exceeded single vardenafil at the lower vardenafil concentrations. Macitentan showed a more pronounced inhibition than bosentan regardless of the lower concentrations. Accordingly, combination effects with vardenafil resembled those of macitentan alone. Conclusions Macitentan and bosentan were potent antagonists of vasoconstriction in PA of LTX patients. The benefit of drug combinations was demonstrated at selected concentrations only owing to a narrow therapeutic range of vardenafil in this ex-vivo model. These results suggest the utility of drug combinations other than the established pair of ambrisentan and tadalafil in PAH treatment but also make a case for a further assessment of vasodilator properties of drugs complementing ERA.

Background: Supposedly, minimized extracorporeal circulation or off-pump revascularization as alternatives to conventional extracorporeal circulation (ONCAB) reduce inflammation and coagulation disturbances. Methods: One hundred and twenty coronary artery bypass graft (CABG) patients were prospectively randomized for three surgical techniques. Coagulation and inflammation markers were measured up to 72 hours postoperatively. Results: Coagulation factors I, II, V, X, antithrombin III and C-reactive protein did not differ perioperatively between the groups and increased, as did several other markers, 12 to 72 hours postoperatively. Compared to its alternatives, ONCAB showed the most obvious transient increase in thrombin-antithrombin complexes (pConclusions: With similarity in pivot coagulation factors, a specific detrimental influence of ONCAB on common coagulation pathways was excluded. Higher perioperative concentrations of products from the coagulation cascade most likely indicate activation of pericardial blood – recirculated only in ONCAB. Furthermore, with only temporary differences in markers of inflammation, the alternatives to ONCAB altogether were without advantage at 72 hours postoperatively. In the general answer to surgical trauma, the part of modern extracorporeal circulation is possibly overestimated. The study is registered at the German Clinical Trial Registry. Registration number DRKS00007580. URL: https://drks-neu.uniklinik-freiburg.de/drks_web/ URL: http://apps.who.int/trialsearch/

Background Cardiopulmonary bypass procedure is associated with an increased risk of renal impairment. To which extent structural damage causes functional decline is unknown. We evaluated perioperative kidney injury and function in patients treated with conventional extracorporeal circulation (CECC), minimized extracorporeal circulation (MECC), and off-pump coronary artery bypass grafting (OPCAB). Methods Blood and urine samples, collected at baseline and up to 72 hours after surgery from patients of the HEPCON trial (DRKS00007580, 120 patients randomized for heparin management and for surgical technique), were analyzed for differences in renal injury and function. Neutrophil gelatinase-associated lipocalin, α glutathione S-transferase, liver fatty acid-binding protein, and kidney injury molecule-1 were measured as urinary protein markers of renal tubular injury. Serum creatinine, blood urea levels, and estimated glomerular filtration rate were determined to monitor renal function. Results Markers of tubular injury differed significantly between surgical technique groups early after surgery, indicating the most detrimental effect in CECC. Hemolysis and hemodilution correlated with these early changes. A late rise did not show intergroup differences. Time courses of renal function parameters, as well as the development of acute kidney injury in 15 patients (13.5%), were irrespective of surgical technique. Heparin management did not influence renal parameters. Conclusion During coronary artery bypass grafting, CECC temporarily induces more tubular injury than MECC or OPCAB. However, late changes of renal function parameters occur irrespective of extracorporeal perfusion mode and even in off-pump surgery.

Summary Background Individualized heparin management (IHM) uses heparin dose–response curves to improve hemostasis management during cardiac surgery as compared with activated clotting time-based methods. Objectives IHM was compared with conventional hemostasis management (CHM) in a randomized, prospective study (ID DRKS00007580). Methods One-hundred and twenty patients undergoing multivessel coronary artery bypass grafting (CABG) were enrolled. Heparin and protamine consumption, blood losses, blood transfusions and administration of hemostatic agents were recorded. Time courses of platelet counts and of coagulation parameters were determined. Coagulation was analyzed at intensive care unit (ICU) arrival by thromboelastometry. Results IHM patients received significantly lower initial heparin doses (289.3IU kg−1 [interquartile range (IQR) 221.5–376.2 IU kg−1] versus 350.5 IU kg−1 [IQR 346.8–353.7 IU kg−1], P

Human umbilical vessels have been recognized as a valuable and widely available resource for vascular tissue engineering. Whereas endothelium-denuded human umbilical veins (HUVs) have been successfully seeded with a patient-derived neoendothelium, decellularized vessels may have additional advantages, due to their lower antigenicity. The present study investigated the effects of three different decellularization procedures on the histological, mechanical and seeding properties of HUVs. Vessels were decellularized by detergent treatment (Triton X-100, sodium deoxycholate, IGEPAL-CA630), osmotic lysis (3 m NaCl, distilled water) and peroxyacetic acid treatment. In all cases, nuclease treatments were required to remove residual nucleic acids. Decellularization resulted in a partial loss of fibronectin and laminin staining in the subendothelial layer and affected the appearance of elastic fibres. In addition to removing residual nucleic acids, nuclease treatment weakened all stainings and substantially altered surface properties, as seen in scanning electron micrographs, indicating additional non-specific effects. Detergent treatment and osmotic lysis caused failure stresses to decrease significantly. Although conditioned medium prepared from decellularized HUV did not severely affect endothelial cell growth, cells seeded on decellularized HUV did not remain viable. This may be attributed to the partial removal of essential extracellular matrix components as well as to changes of surface properties. Therefore, decellularized HUVs appear to require additional modifications in order to support successful cell seeding. Replacing the vessels' endothelium may thus be a superior alternative to decellularization when creating tissue-engineered blood vessels with non-immunogenic luminal interfaces.

Tissue-engineered small-calibre vessel grafts may help to alleviate the lack of graft material for coronary and peripheral bypass grafting in an increasing number of patients. This study explored the use of endotheliumdenuded human umbilical veins (HUVs) as scaffolds for vascular tissue engineering in a perfusion bioreactor. Vessel diameter (1.2 � 0.4mm), wall thickness (0.38 � 0.09mm), uniaxial ultimate failure stress (8029 � 1714 kPa) and burst pressure (48.4 � 20.2 kPa, range 28.4–83.9 kPa) were determined in native samples. The effects of endothelium removal from HUVs by enzymatic digestion, hypotonic lysis and dehydration were assessed. Dehydration did not significantly affect contractile function, tetrazolium dye reduction, mechanical strength and vesselstructure,whereas the other methods failed in at leastone of these parameters. Denudation by dehydration retained laminin, fibronectin, collagen and elastic fibres. Denuded HUVs were seeded in a perfusion bioreactor with either allogeneic HUVs endothelial cells or with saphenous vein endothelial cells harvested from patients with coronary artery disease. Seeding in a perfusion bioreactor resulted in a confluent monolayer of endothelial cells from both sources, as judged by histology and scanning electron microscopy. Seeded cells contained von Willebrand factor and CD31. In conclusion, denuded HUVs should be considered an alternative to decellularized blood vessels, as the process keeps the smooth muscle layer intact and functional, retains proteins relevant for biomechanic properties and for cell attachment and provides a suitable scaffold for seeding an autologous and flow-resistant endothelium. Copyright © 2012 John Wiley & Sons, Ltd.

Tissue-engineered vessel grafts have to mimic the biomechanical properties of native blood vessels. Manufacturing processes often condition grafts to adapt them to the target flow conditions. Graft stiffness is influenced by material properties and dimensions and determines graft compliance. This proof-of-concept study evaluated a contact-free method to monitor biomechanical properties without compromising sterility. Forced vibration response analysis was performed on human umbilical vein (HUV) segments mounted in a buffer-filled tubing system. A linear motor and a dynamic signal analyser were used to excite the fluid by white noise (0-200 Hz). Vein responses were read out by laser triangulation and analysed by fast Fourier transformation. Modal analysis was performed by monitoring multiple positions of the vessel surface. As an inverse model of graft stiffening during conditioning, HUV were digested proteolytically, and the course of natural frequencies (NFs) was monitored over 120 min. Human umbilical vein showed up to five modes with NFs in the range of 5-100 Hz. The first natural frequencies of HUV did not alter over time while incubated in buffer (p = 0.555), whereas both collagenase (-35%, p = 0.0061) and elastase (-45%, p 0.001) treatments caused significant decreases of NF within 120 min. Decellularized HUV showed similar results, indicating that changes of the extracellular matrix were responsible for the observed shift in NF. Performing vibration response analysis on vessel grafts is feasible without compromising sterility or integrity of the samples. This technique allows direct measurement of stiffness as an important biomechanical property, obviating the need to monitor surrogate parameters. Copyright © 2016 John WileySons, Ltd.

Objectives: To compare routine activated clotting time (ACT) based hemostasis management to individualized heparin management (IHM) in terms of postoperative bleeding, platelet function, and coagulation markers. Methods: 120 CABG patients (≥ 3 distal anastomoses) were enrolled in a prospective trial and were randomized for hemostasis management (ACT versus IHM). With a target ACT of 400 second conventional hemostasis management antagonized heparin with protamine in a ratio of 1:>0.8, whereas in IHM protamine dosages were calculated from residual heparin concentrations. Hemostasis was analyzed immediately post-OP by thrombelastometry, aPTT, INR, and a range of coagulation markers. Results: A total of 112 patients (ACT: 56, IHM: 56) were included. Median heparin dosages were equivalent in both groups, whereas IHM patients received significantly less protamine. INR did not differ significantly, but aPTT values were significantly higher in HM patients. INTEM clotting times were elevated in IHM patients, whereas HEPTEM clotting times did not differ. Most coagulation markers (antithrombin III, d-dimers, fibrinogen, factors II/V/VIII/X, tissue factor pathway inhibitor, platelet counts) did not differ between groups, whereas thrombin-antithrombin complexes (TATs) were higher in ACT patients (29.69 [15.14–42.19] versus 18.50 [12.14–29.22] µg/l, p = 0.005), indicating recovered coagulation capacities. TATs correlated significantly with protamine doses (p = 0.022). Blood losses were slightly but significantly higher in IHM patients within the first 12 hour which balanced at 24 hour and which did not lead to more blood transfusions (40 PRBC in 15 patients versus 44/17, p = 0.926). Conclusions: IHM patients did not benefit from allegedly reduced coagulation factor activation mainly because heparin dosages were unexpectedly identical in both groups. On the contrary, postoperative parameters indicated an incomplete heparin antagonization in IHM patients.

Objective: We compared profiles of the numbers of circulating endothelial cells (CEC) and the apoptosis-inducing capacity of serum samples on human endothelial cells (hEC) in on-pump and off-pump coronary artery bypass grafting (CABG) patients. Methods: Blood samples from 30 patients undergoing CABG (randomly assigned to two groups: 15 patients off-pump and 15 on-pump (cardiopulmonary bypass, CPB)) were collected after induction of anesthesia (preoperatively), at weaning from CPB/end of bypass grafting (0 h), and 1, 6, 12, 24, and 48 h afterwards. CEC were isolated with immunomagnetic anti-CD146-coated Dynabeads, and counted in a Nageotte chamber. The apoptosis-inducing activity of serum samples on hEC was examined by a tissue culture assay system. Apoptotic and normal cells were identified using phase contrast/ fluorescence microscopy after DNA dye staining. Results: CEC numbers and proportions of apoptotic hEC were significantly elevated during and after surgery in both groups (p < 0.01). Compared with the on-pump group, CEC and proportions of apoptotic hEC were significantly lower (p = 0.04 and p = 0.03, respectively) in patients having CABG performed off-pump. Starting at comparable baseline levels, the mean CEC-number was highest at 6 h postoperatively with 81.9 ml -1 (range, 44-141) for on-pump patients and 63.3 ml -1 (range, 48-105) for off-pump patients. hEC apoptosis peaked also at T4: 16.5 ± 2.8% versus 11.3 ± 2.2%. In both groups, CEC numbers and proportions of endothelial apoptosis were still elevated at 48 h after surgery. Conclusion: The number of circulating endothelial cells and apoptotic endothelial cell death are markers of endothelial activation and damage during CABG. This study provides evidence that CABG with the use of CPB in comparison to OPCAB surgery is associated with a significantly more pronounced endothelial response in the immediate postoperative period.

Summary: The effects of the different types of soluble guanylate cyclase (sGC) stimulators on the phosphorylation status of vasodilator-stimulated phosphoprotein (VASP) in both human and rat platelets were studied under in vitro and in vivo conditions. sGC-dependent VASP phosphorylation (at Ser239 and Ser157) both by the new direct sGC stimulator YC-1 and by NO donors was examined by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS/PAGE) with different antibodies. One antibody, which recognizes VASP independent of its phosphorylation state, was used to detect the mobility shift of VASP caused by Ser157 phosphorylation. The other antibody was specifically directed against VASP phosphorylated at Ser239, the cGMP-dependent protein kinase (PKG) preferred phosphorylation site of VASP. In vitro YC-1 increased both VASP phosphorylation and cyclic guanosine monophosphate (cGMP) levels as did the NO donors 2-(N,N-diethylamino)-diazenolate-2-oxide (DEA/NO) and sodium nitroprusside (SNP). The combination of both types induced a synergistic effect in both VASP phosphorylation and cGMP increase. In rat platelets, similar effects could be shown in vitro. In vivo we observed a significant increase in cGMP and a distinct effect on VASP phosphorylation in rat platelets 1 h after oral administration of YC-1. These biochemical alterations are supported by a significant prolongation in rat-tail bleeding time. Direct stimulators of sGC like YC-1 are on the one hand direct potent stimulators of the cGMP/PKG/VASP pathway in platelets and on the other hand synergize with NO, the physiologic stimulator of sGC. Therefore YC-1-like substances are interesting tools for the development of new cardiovascular drugs with vasodilatory and antithrombotic properties.

A stably transfected soluble guanylate cyclase (sGC, alpha1 and beta1 subunits of the rat lung enzyme)-overexpressing CHO cell line was generated for the characterization of different types of activators of the soluble guanylate cyclase. Polyclonal antibodies directed against both subunits of the rat enzyme were used to detect both subunits in the cytosol of the transfected CHO cells. We studied the effects of different nitric oxide (NO) donors like SNP and DEA/NO and, in particular, the direct, NO-independent stimulator of the soluble guanylate cyclase 3-(5'-hydroxymethyl-2'furyl)-1-benzyl indazole (YC-1), on intracellular guanosine 3',5'-cyclic monophosphate (cGMP) production. DEA/NO (0.01-3 microM), SNP (1-10 microM), and YC-1 (1-10 microM) induced a concentration-dependent intracellular cGMP increase with maximal effects of 16-fold (3 microM DEA/NO), 8-fold (10 microM SNP), and 6-fold (10 microM YC-1) stimulation compared to controls, respectively. In addition, a synergistic effect of the combination of the NO donor and YC-1 could be observed with a maximal stimulation of 64-fold by SNP (10 microM) and YC-1 (10 microM). 1H-(1,2,4)-Oxadiazolo-(4,3-a)-6-bromo-quinoxazin-1-one (ODQ, 10 microM), a potent and selective inhibitor of sGC, inhibited both the single effects of NO donors [DEA/NO (3 microM), 77%; SNP (3 microM), 83%] and YC-1 [YC-1 (3 microM), 82%], but moreover the synergistic effects between NO donors and YC-1 [DEA/NO (3 microM) + YC-1 (3 microM), 81%; SNP (3 microM) + YC-1 (3 microM),89%] on intracellular cGMP production. In summary,we have generated a simple, sensitive, and useful bioassay method to characterize all types of sGC activators on the cellular level without the need of primary cell culture, several transfections, or purifying enzyme from biological materials.

Human umbilical vessels have been recognized as a valuable and widely available resource for vascular tissue engineering. Whereas endothelium-denuded human umbilical veins (HUVs) have been successfully seeded with a patient-derived neoendothelium, decellularized vessels may have additional advantages, due to their lower antigenicity. The present study investigated the effects of three different decellularization procedures on the histological, mechanical and seeding properties of HUVs. Vessels were decellularized by detergent treatment (Triton X-100, sodium deoxycholate, IGEPAL-CA630), osmotic lysis (3 m NaCl, distilled water) and peroxyacetic acid treatment. In all cases, nuclease treatments were required to remove residual nucleic acids. Decellularization resulted in a partial loss of fibronectin and laminin staining in the subendothelial layer and affected the appearance of elastic fibres. In addition to removing residual nucleic acids, nuclease treatment weakened all stainings and substantially altered surface properties, as seen in scanning electron micrographs, indicating additional non-specific effects. Detergent treatment and osmotic lysis caused failure stresses to decrease significantly. Although conditioned medium prepared from decellularized HUV did not severely affect endothelial cell growth, cells seeded on decellularized HUV did not remain viable. This may be attributed to the partial removal of essential extracellular matrix components as well as to changes of surface properties. Therefore, decellularized HUVs appear to require additional modifications in order to support successful cell seeding. Replacing the vessels' endothelium may thus be a superior alternative to decellularization when creating tissue-engineered blood vessels with non-immunogenic luminal interfaces.

Tissue-engineered small-calibre vessel grafts may help to alleviate the lack of graft material for coronary and peripheral bypass grafting in an increasing number of patients. This study explored the use of endothelium-denuded human umbilical veins (HUVs) as scaffolds for vascular tissue engineering in a perfusion bioreactor. Vessel diameter (1.2 ± 0.4 mm), wall thickness (0.38 ± 0.09 mm), uniaxial ultimate failure stress (8029 ± 1714 kPa) and burst pressure (48.4 ± 20.2 kPa, range 28.4-83.9 kPa) were determined in native samples. The effects of endothelium removal from HUVs by enzymatic digestion, hypotonic lysis and dehydration were assessed. Dehydration did not significantly affect contractile function, tetrazolium dye reduction, mechanical strength and vessel structure, whereas the other methods failed in at least one of these parameters. Denudation by dehydration retained laminin, fibronectin, collagen and elastic fibres. Denuded HUVs were seeded in a perfusion bioreactor with either allogeneic HUVs endothelial cells or with saphenous vein endothelial cells harvested from patients with coronary artery disease. Seeding in a perfusion bioreactor resulted in a confluent monolayer of endothelial cells from both sources, as judged by histology and scanning electron microscopy. Seeded cells contained von Willebrand factor and CD31. In conclusion, denuded HUVs should be considered an alternative to decellularized blood vessels, as the process keeps the smooth muscle layer intact and functional, retains proteins relevant for biomechanic properties and for cell attachment and provides a suitable scaffold for seeding an autologous and flow-resistant endothelium.

Background YC-1 (3-(5′-hydroxymethyl-2′furyl)-1-benzyl-indazole) is an allosteric activator of soluble guanylyl cyclase (sGC) and a vasodilator. This study describes a paradoxical action of YC-1 in isolated vessels of patients with coronary artery disease (CAD) that appears to trigger an endothelium-dependent vasoconstrictor pathway present in vessels with endothelial dysfunction. Methods Effects of YC-1 on the tensions of isolated vessels were investigated in an organ bath. Vasoconstrictors released from the vessels were quantified through enzyme-linked immunosorbent assay. Results YC-1 elicited long-lasting constriction in saphenous veins and radial arteries from patients with CAD, but not in human umbilical veins. The half-maximal effective dose was 1.0 μmol/L. Constriction was attenuated by nifedipine (an L-type Ca 2+ -channel blocker), bosentan (an endothelin [ET] A /ET B inhibitor), BQ-788 (N-[(cis-2,6-Dimethyl-1-piperidinyl)carbonyl]-4-methyl-L-leucyl-1-(methoxycarbonyl)-D-tryptophyl-D-norleucine; an ET B inhibitor), and by denuding, but not by ODQ (1H-(1,2,4)oxadiazolo[4,3-a]quinoxalin-1-one; an inhibitor of sGC), BQ-123 (cyclo(-D-Trp-D-Asp-Pro-D-Val-Leu); an ET A inhibitor), or phosphoramidon (an endothelin converting enzyme inhibitor). Indomethacin (an inhibitor of cyclooxygenase-1 and -2) and SQ29,548 ([1S-[1α,2α(Z),3α,4α]]-7-[3-[[2-[(phenylamino)carbonyl]hydrazino]methyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid; a thromboxane receptor antagonist) suppressed YC-1–induced constriction, whereas DFU (5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulfonyl)phenyl-2(5H)-furanone; a cyclooxygenase-2 inhibitor) had no effect. Rings of saphenous vein released significantly more endothelin-1 in the presence than in the absence of YC-1. Conclusions YC-1–induced vasoconstriction demonstrates the existence of an endothelium-dependent vasoconstrictor pathway in the blood vessels of patients with CAD that to date has been described only in animal models of hypertension. Patients with CAD who have elevated plasma levels of endothelin-1 are thus prone to endothelium-dependent vasoconstriction, which may also play a role in vasospasm in vascular grafts.

Bovine saphenous veins (BSV) were incubated in a perfusion bioreactor to study vessel wall metabolism and wall structure under tissue engineering conditions. Group 1 vessels were perfused for 4 or 8 days. The viscosity of the medium was increased to that of blood in group 2. Group 3 vessels were additionally strained with luminal pressure. Groups 1-d through 3-d were similar except that BSV were endothelium-denuded before perfusion. Groups 1-a through 3-a used native vessels at elevated flow rates. Group 3 vessels responded significantly better to noradrenaline on day 4, whereas denuded vessels showed attenuated responses (p < 0.001). Tetrazolium dye reduction did not depend on perfusion conditions or time except for denuded vessels. pO(2) gradients across the vessels were independent of time and significantly higher in group 2 (p < 0.001). BSV converted glucose stoichiometrically to lactate except vessels of groups 3, 1-d, and 3-d which released more lactate than glucose could supply (p < 0.001). Group 1 vessels as well as all vessels perfused with elevated flow rates showed a loss of endothelial cells after 4 days, whereas group 2 and 3 vessels retained most of the endothelium. These data suggest that vessel metabolism was not limited by oxygen supply. Shear forces did not affect glucose metabolism but increased oxygen consumption and endothelial cell survival. Luminal pressure caused the utilization of energy sources other than glucose, as long as the endothelium was intact. Therefore, vessel metabolism needs to be monitored during tissue engineering procedures which challenge the constructs with mechanical stimuli.

We have previously shown that endothelium-denuded human umbilical veins (HUV) are suitable scaffolds for vascular tissue engineering with contractile properties and endogenous prostacyclin release. This study established efficient methods to denude and seed HUV with allogeneic endothelial cells in a perfusion bioreactor in order to create small caliber vessel grafts. Stress-strain relationships and ultimate failure stresses were determined. The effects of a gas-based denudation method on contractile function, reductive capacity, and histology were evaluated. Finally, denuded HUV were seeded in a perfusion bioreactor to create small diameter vessel grafts. HUV displayed biphasic stress-strain relationships, but higher compliances compared to autologous vessels. Burst pressures extrapolated from the ring failure stresses was approx. 1000 mm Hg. Denudation by dehydration proved to be an effective method at gas flow rates of 60-120 ml/min. Endothelial cells were removed completely, whereas neither basal membranes nor functional properties were affected. Seeding of denuded HUV with endothelial cells in a perfusion bioreactor using suitable rotation patterns resulted in an even cell distribution and a confluent endothelium which was shear-resistant at flow rates up to 40 ml/min. These results indicate that the outlined procedures are suitable components of a protocol to turn HUV into small caliber vessel grafts.

Tissue engineering protocols commonly incorporate mechanical stimuli like pulsatile flow and luminal pressure to adapt the constructs to the biomechanical requirements of the target circulation. This study investigated the effects of shear forces and of luminal pressure on blood vessel metabolism and function using bovine veins as a model. Veins were perfused for four days in M199 at 40 ml/min. Shear forces were increased by adjusting the viscosity of the medium to that of blood. Some veins were additionally treated with luminal pressure. Control groups were either denuded or perfused with 60 ml/min to assess the influences of endothelium and of elevated flow rates, respectively. Physiological shear forces were sufficient to maintain endothelial integrity, unless superphysiological flow rates were applied. Reductive capacities were not affected by any of the perfusion conditions. Responses to the receptor-dependent vasoconstrictor noradrenalin were increased after perfusing with physiological shear forces and luminal pressure. This also induced a shift of the metabolism from glycolysis and lactate fermentation to the use of alternative substrates other than glucose. Mechanically denuded control vessels were prone to apoptosis and showed an altered metabolism as well. However, denuded vessels treated with luminal pressure also showed improved responses to vasoconstrictors compared to denuded controls. These results indicate that mechanical challenges are required to maintain endothelial integrity and to improve contractile function of vessels in perfusion systems. Metabolic patterns change with mechanical challenges, and care should be taken to provide sufficient amounts of substrates other than glucose for energy metabolism.

Small caliber vessel grafts are one of the major challenges of vascular tissue engineering. A variety of processes have been developed to create vascular grafts from scaffolds and donor cells in bioreactors. In order to optimize such processes, this study focused on monitoring vessel metabolism under conditions typically used in perfusion protocols. Bovine veins were perfused in a bioreactor for four days. Group 1 vessels served as controls and were perfused with standard medium. Medium of group 2 was adjusted to the viscosity of blood. Group 3 vessels were additionally challenged with elevated luminal pressure. Contractile function was assessed in an organ bath. Tissue viability was determined by tetrazolium dye reduction. Oxygen gradients, dextrose consumption, and lactate production were monitored using a blood gas analyzer. KCl induced contractions did not differ between days 0 and 4. Norepinephrine dose-response curves of group 3 vessels achieved a higher maximum contraction on day 4, with no changes of EC50. Tissue viability was not altered by any of the perfusion conditions. Oxygen gradients across the vessels did not change with time but were elevated in group 2, with no signs of oxygen depletion. Dextrose consumption and lactate formation of group 1 and 2 vessels appeared to be stoichiometric. In contrast, group 3 vessels produced more lactate than dextrose could supply. These results indicate that conventional oxygenation of culture media is sufficient to meet the oxygen consumption of a functional vessel. Elevated shear forces increased the oxygen demand without affecting dextrose consumption. Elevated shear forces and luminal pressure caused the utilization of alternative energy sources. Thus online monitoring of key metabolic parameters appears to be a desirable feature of perfusion bioreactors for vascular tissue engineering.

Human umbilical veins (HUV) have recently been suggested as a starting material for vascular tissue engineering. HUV possess a functional smooth muscle layer and could be turned into an immunologically inert graft with contractile properties by creating a neoendothelium from the recipient’s own cells. This study investigated methods to remove the native endothelium without impairing the contractile function of the smooth muscle layer. These denuded HUV were then seeded with endothelial cells in a perfusion bioreactor, demonstrating the creation of a confluent, shear-resistant neoendothelium.

Soluble guanylyl cyclase (sGC) is one of the key enzymes of the nitric-oxide (NO)/cyclic 3',5'-guanosine monophosphate (cGMP) pathway. Located in virtually all mammalian cells, it controls the vessel tone, smooth muscle cell growth, platelet aggregation, and leukocyte adhesion. In vivo sGC activity is mainly regulated by NO which in turn is released from L-arginine by nitric oxide synthases. One of the main diseases of the cardiovascular system, endothelial dysfunction, leads to a diminished NO synthesis and thus increases vessel tone as well as the risk of thrombosis. The predominant therapeutic approach to this condition is a NO replacement therapy, as exemplified by organic nitrates, molsidomin, and other NO releasing substances. Recent advances in drug discovery provided a variety of other approaches to activate sGC, which may help to circumvent both the tolerance problem and some non-specific actions associated with NO donor drugs. Substances like BAY 41-2272 stimulate sGC in a heme-dependent fashion and synergize with NO, allowing to enhance the effects both of endogenous NO and of exogenous NO donors. On the other hand, heme-independent activators like BAY 58-2667 allow to activate sGC even if it is rendered unresponsive to NO due to oxidative stress or heme loss. Furthermore, a few substances have been described as specific inhibitors of sGC that allow to alleviate the effects of excess NO production as seen in shock. This review discusses the cardiovascular effects of heme-dependent and heme-independent activators as well as of inhibitors of sGC.

The current study investigated whether the mode of delivery and the mode of sample collection affect the functional properties of umbilical veins as scaffolds for vascular tissue engineering purposes. Human umbilical vein (HUV) from planned cesarean-sections (PCS) showed a 1.7-fold higher maximum contraction with potassium chloride compared to spontaneous vaginal deliveries (VDs, p=0.029). The maximum contractions with histamine were 2.0- and 2.9-fold higher in the PCS and emergency c-section (ECS) groups, respectively, compared to the VD group (p=0.003). The dose-response curves of serotonin were shifted to the right approx. 6- and 5-fold in the VD group compared to PCS and ECS, respectively (p=0.009). There were no differences between the birth groups in terms of tetrazolium dye reduction, platelet adhesion, and the structural integrity. The release of the antithrombotic compound prostacyclin from vessels of the PCS and ECS groups was 6.6- and 3.5-fold higher, respectively, than in the VD group (p

Umbilical cords are usually discarded after delivery, even though they contain a set of functional vessels. We investigated whether the human umbilical vein (HUV) is suitable as a storable scaffold for the tissue engineering of small-caliber vessel grafts. Isolated HUVs were cryopreserved by freezing or vitrification. The reaction of the vessels to vasoactive compounds and the mechanical properties were determined in an organ bath. Mitochondrial metabolism, release of antithrombotic compounds, and platelet adhesion were measured on the luminal vessel surface. Seeding with endothelial cells was tested on denuded HUVs. The vessels showed a weak response to norepinephrine but were readily contracted by serotonin and by the thromboxane A2 mimetic U46619. Endothelium-dependent vasorelaxation was weak, reaching significance only for histamine. However, the vessels relaxed to sodium nitroprusside, and to acetylcholine if sandwiched with human saphenous vein. Cryopreservation did not change the mechanical properties in the relevant tension range. Vasoconstriction to potassium chloride and serotonin were reduced after freezing (22.9+/-7.6%, 27.7+/-10.2%) and after vitrification (2.6+/-5.8%, 4.3+/-7.1%). The mitochondrial metabolism was also attenuated after freezing (57.9+/-25.9%) and after vitrification (21.7+/-6.7%). Prostacyclin release was elevated after both cryopreservation procedures (4.0-fold, 3.9-fold), whereas there was no significant change in the adhesion of platelets. Denuded HUVs could readily be seeded with isolated endothelial cells before and after freezing. We conclude that HUV is suitable as a storable living scaffold with antithrombogenic properties.

We analyzed a cryopreservation protocol which improves long-term storage of endothelial cells (EC) for tissue engineering purposes. Human umbilical vein EC were frozen in a high-potassium solution containing 10% dimethyl sulfoxide using 3 different cooling rates. After a storage time in liquid nitrogen of 1, 4, or 12 months, samples were thawed and compared to fresh cells in terms of growth rates, anti-inflammatory, and anticoagulant functions. Independent of cooling rate and storage time, the retrieval after cryopreservation ranged between 60% and 80%. However, viability of the cells cryopreserved at 10 degrees C/min decreased significantly from 78 +/- 5% to 64 +/-3% with storage. Storage time of 4 months resulted in a decreased cell multiplication factor over 4 and 12 days in culture. The lag phases returned to normal in the next passage. Thawed cells showed increased metabolic activity, reduced expression of thrombomodulin, and unchanged basal expression of adhesion molecules. However, the tumor necrosis factor-induced expression of adhesion molecules was significantly increased after long-term storage. This effect was partially compensated after expansion of the cells, whereas the prostacyclin release increased. Expansion of cryopreserved/thawed EC resulted in highly proliferative cells with antithrombotic properties and a capacity for inflammatory reactions, which makes them suitable for vascular tissue engineering.

Objectives: The mechanical properties and seeding with endothelial cells were investigated in fresh and cryopreserved human umbilical vein. Methods: Human umbilical veins (HUV) were frozen in Euro-Collins/1M DMSO at –1°C/min and stored in liquid nitrogen. Stress-strain relationships of fresh and thawed veins were determined in an uniaxial tension-testing rig. HUV endothelial cells (HUVEC) were seeded onto denuded HUV under static conditions and grown for 3d. Luminal surfaces were analyzed by scanning electron microscopy. Calcein-stained cells were seeded hyperconfluently to determine the cell retention capacity of fresh and cryopreserved veins. Results: The stress-strain relationships of HUV followed a biphasic pattern typical for natural vessels. Neither the failure stress (2.71±0.36 vs. 3.25±0.97 N, n=3) nor the displacement required to achieve failure (9.73±0.9 vs. 7.43±2.07mm, n=3) were altered by cryopreservation. The burst pressure was estimated as approx. 1000mm Hg within the limitations of the uniaxial model. HUVEC seeded onto denuded HUV formed patches (at 9E3 cells per cm2) or an almost confluent endothelium (at 3E4 cells per cm2) within three days. The capacity to retain seeded HUVEC of denuded HUV was not altered by cryopreservation (1.15±0.08E5 vs. 1.26±0.14E5 cells per cm2, n=6). Conclusions: The burst pressure of HUV seems to be sufficiently high for the human arterial circulation and is not altered by cryopreservation. HUVEC can establish a confluent endothelium on denuded HUV. Therefore HUV appears to be a suitable storable scaffold for vascular tissue engineering.

To investigate the influence of cryopreservation on endothelial cell growth, morphology, and function human umbilical vein endothelial cells (HUVECs) were frozen following a standard protocol. Cell suspensions were exposed to 10% dimethyl sulfoxide in a high-potassium solution, cooled to -80 degrees C at 1 degrees C/min and stored in liquid nitrogen for 7-36 days. Samples were thawed in a 37 degrees C water bath and the cryoprotectant was removed by serial dilution. The growth of cell suspensions was assayed by culturing 7300 cells/cm2 for 3-5 days in order to determine the cell multiplication factor. Fresh and cryopreserved/thawed cells were analyzed for their growth, and their anti-inflammatory and anti-coagulant function by using cellular ELISA. Cryopreservation resulted in a retrieval of 66 +/- 5% and a viability of 79 +/- 3%. Cryopreserved/thawed and fresh cells showed identical doubling times and identical cell counts in the confluent monolayers. However, the lag phase of thawed HUVECs was approximately 36 h longer, resulting in significant differences in the cell multiplication factor at 3 and 5 days after seeding. After expansion to a sufficient cell count the lag phases were identical. Fresh and cryopreserved/thawed cells showed comparable anti-inflammatory and anti-coagulant activity, as judged by the basal and TNF-induced VCAM-1, ICAM-1, E-selectin, and thrombomodulin expression. Cryopreserved/thawed and recultivated endothelial cells are suitable for endothelialization of autologous allograft veins. Such tissue-engineered grafts will offer the necessary clinical safety for those patients who lack autologous material.

Soluble guanylyl cyclase (sGC) is the main receptor for nitric oxide, a messenger molecule with multiple clinical implications. Understanding the activation of sGC is an important step for establishing new therapeutic principles. We have now overexpressed sGC in a baculovirus/Sf9 system optimized for high protein yields to facilitate spectral and kinetic studies of the activation mechanisms of this enzyme. It was expressed in a batch fermenter using a defined mixture of viruses encoding the alpha and beta1 subunits of the rat lung enzyme. The expressed enzyme was purified from the cytosolic fraction by anion exchange chromatography, hydroxyapatite chromatography, and size exclusion chromatography. By use of this new method 2.5 l culture yielded about 1 mg of apparently homogeneous sGC with a content of about one heme per heterodimer without the need of a heme reconstitution step. The enzyme did not contain stoichiometric amounts of copper. The basal activities of the purified enzyme were 153 and 1259 nmol min(-1) mg(-1) in the presence of Mg2+ and Mn2+, respectively. The nitric oxide releasing agent 2-(N,N-diethylamino)-diazenolate-2-oxide (DEA/NO) stimulated the enzyme 160-fold with Mg2+, whereas the NO-independent activator 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1) induced an increase in the activity of 101-fold at a concentration of 300 microM. The combination of DEA/NO (10 microM) and YC-1 (100 microM) elicited a dose-dependent synergistic stimulation with a maximum of a 792-fold increase over the basal activity in the presence of Mg2+, resulting in a specific activity of 121 micromol min(-1) mg(-1). The synergistic stimulation of DEA/NO and YC-1 was attenuated by the sGC inhibitor 1H-(1,2,4)oxadiazole(4,3-a)quinoxalin-1-one (ODQ) (10 microM) by 94%. In a different experimental setup a saturated carbon monoxide solution in the absence of ambient oxygen or NO stimulated the enzyme 15-fold in the absence and 1260-fold in the presence of YC-1 compared to an argon control. The heme spectra of the enzyme showed a shift of the Soret peak from 432 to 399 and 424 nm in the presence of DEA/NO or carbon monoxide, respectively. The heme spectra were not affected by YC-1 in the absence or in the presence of DEA/NO or of carbon monoxide, which reflects the fact that YC-1 does not interact directly with the heme group of the enzyme. In summary, this study shows that our expression/purification procedure is suitable for producing large amounts of highly pure sGC which contains one heme per heterodimer without a reconstitution step. The activator experiments show that in a synergistic stimulation with YC-1 sGC can be activated maximally both by nitric oxide and by carbon monoxide and that YC-1 does not directly act via heme. The described method should help to facilitate the investigation of the new therapeutic principle of NO-independent guanylyl cyclase activators.