Cardiovascular effects of modulators of soluble guanylyl cyclase activity.

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

Endothelium-dependent vasoconstriction in isolated vessel grafts: a novel mechanism of vasospasm?

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

Metabolic Requirements of Blood Vessels in a Perfusion Bioreactor

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

Contact-free monitoring of vessel graft stiffness - proof of concept as a tool for vascular tissue engineering

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 (C) 2016 John Wiley & Sons, Ltd

Preservation of Adrenoceptor and Endothelin Receptor Mediated Vasoconstriction and of Endothelium-Dependent Relaxation after Cold Storage of Explanted Blood Vessels for ex vivo Analyses

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: Contractileresponses topotassium 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. (c) 2022 S. Karger AG, Base

Advantages of human umbilical vein scaffolds derived from cesarean section vs. vaginal delivery for vascular tissue engineering.

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<0.001). There was no correlation between the duration of ischemia and any of the functional parameters. This study provides evidence that vessels obtained from PCS are to be preferred for tissue engineering purposes, as they can be harvested in a sterile fashion and show superior vasoconstrictor responses and antithrombotic properties. The data also support a once-per-day pickup schedule for umbilical cords without a deterioration of the functional properties

Identification and reduction of cryoinjury in endothelial cells: a first step toward establishing a cell bank for vascular tissue engineering.

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

Cryopreservation of human endothelial cells 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

Properties of the human umbilical vein as a living scaffold for a tissue-engineered vessel graft.

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