Verapamil Block of Large-Conductance Ca-Activated K Channels in Rat Aortic Myocytes

The effects of verapamil on the large conductance Ca-activated K (BK) channel from rat aortic smooth muscle cells were examined at the single channel level. Micromolar concentrations of verapamil produced a reversible flickering block of the BK channel activity. Kinetic analysis showed that verapamil decreased markedly the time constants of the open states, without any significant change in the time constants of the closed states. The appearance of an additional closed state — specifically, a nonconducting, open-blocked state — was also observed, whose time constant would reflect the mean residence time of verapamil on the channel. These observations are indicative of a state-dependent, open-channel block mechanism. Dedicated kinetic (group) analysis confirmed the state-dependent block exerted by verapamil. D600 (gallopamil), the methoxy derivative of verapamil, was also tested and found to exert a similar type of block, but with a higher affinity than verapamil. The permanently charged and membrane impermeant verapamil analogue D890 was used to address other important features of verapamil block, such as the sidedness of action and the location of the binding site on the channel protein. D890 induced a flickering block of BK channels similar to that observed with verapamil only when applied to the internal side of the membrane, indicating that D890 binds to a site accessible from the cytoplasmic side. Finally, the voltage dependence of D890 block was assessed. The experimental data fitted with a Langmuir equation incorporating the Woodhull model for charged blockers confirms that the D890-binding site is accessed from the internal mouth of the BK channel, and locates it approximately 40% of the membrane voltage drop along the permeation pathway. Received: 11 April 2000/Revised: 17 October 2000     

Banking of the human heart valves and the arteries at the European homograft bank (EHB) – Overview of a 14-year activity in this International Association in Brussels<Superscript>*</Superscript>

Processing of the human heart valves and arteries has been carried out at the European Homograft Bank (EHB) in Brussels since 1989 and 1991, respectively. Heart valve donors of 0–65 years were classified in (1) Beating heart donors (BHD), of which recipients of heart transplantation (RHT) and multiorgan donors (MOD) after brain death, and (2) non-beating heart donors (NBHD) with warm ischaemic time (WIT) of less then 6 h. Past history of the donors has been checked for malignant and chronic diseases, as well as biology for transmissible and infectious diseases. Perfect collaboration has been established with the transplant coordinators and transplant teams of the implanting centres. Dissection, decontamination, cryopreservation and storing in fluid nitrogen has been carried out in accordance with the Belgian and European Standards of cardiovascular allografts. During this period, a total of 2.828 hearts, 28 predissected valves and 616 batches of arteries arrived in the EHB. 3.537 valves and 1.137 different arteries were accepted for implantation. The main reasons for tissue rejection were morphology, contamination and cuts during the tissue retrieval or dissection. A huge network of different hospitals in Belgium and elsewhere in Europe and Switzerland were included in this process. Pulmonary allografts were not sent for implantation in the left ventricular outflow tract after 1998, since the early and mid-term results after 76 implantations were disappointing. The number of implanted aortic and pulmonary allografts remains stable from year to year, however the number of the allografts used for Ross operation is still increasing. Since the results of the follow up were disappointing, we still only require the implantation and immediate postoperative results, whereas the follow-up information only for specific study purposes. This revised version was published online in July 2006 with corrections to the Cover Date.     

Hydrogen sulfide attenuates ferric chloride-induced arterial thrombosis in rats

Hydrogen sulfide (H₂S) is a novel gaseous transmitter, regulating a multitude of biological processes in the cardiovascular and other systems. However, it remains unclear whether it exerts any effect on arterial thrombosis. In this study, we examined the effect of H₂S on ferric chloride (FeCl₃)-induced thrombosis in the rat common carotid artery (CCA). The results revealed a decrease of the H₂S-producing enzyme cystathionine γ-lyase (CSE) expression and H₂S production that persisted until 48?h after FeCl₃ application. Intriguingly, administration with NaHS at appropriate regimen reduced the thrombus formation and enhanced the blood flow, accompanied with the alleviation of CSE and CD31 downregulation, and endothelial cell apoptosis in the rat CCA following FeCl₃ application. Moreover, the antithrombotic effect of H₂S was also observed in Rose Bengal photochemical model in which the development of thrombosis is contributed by oxidative injury to the endothelium. The in vitro study demonstrated that the mRNA and protein expression of CSE, as well as H₂S production, was decreased in hydrogen peroxide (H₂O₂)-treated endothelial cells. Exogenous supplement of NaHS and CSE overexpression consistently alleviated the increase of cleaved caspase-3 and endothelial cell damage caused by H₂O₂. Taken together, our findings suggest that endogenous H₂S generation in the endothelium may be impaired during arterial thrombosis and that modulation of H₂S, either exogenous supplement or boost of endogenous production, may become a potential venue for arterial thrombosis therapy.     

Store-operated calcium entry in thrombosis and thrombo-inflammation

Cytosolic free calcium (Ca²⁺) is a second messenger regulating a wide variety of functions in blood cells, including adhesion, activation, proliferation and migration. Store-operated Ca²⁺ entry (SOCE), triggered by depletion of Ca²⁺ from the endoplasmic reticulum, provides a main mechanism of regulated Ca²⁺ influx in blood cells. SOCE is mediated and regulated by isoforms of the ion channel proteins ORAI and TRP, and the transmembrane Ca²⁺ sensors stromal interaction molecules (STIMs), respectively. This report provides an overview of the (patho)physiological importance of SOCE in blood cells implicated in thrombosis and thrombo-inflammation, i.e. platelets and immune cells. We also discuss the physiological consequences of dysregulated SOCE in platelets and immune cells and the potential of SOCE inhibition as a therapeutic option to prevent or treat arterial thrombosis as well as thrombo-inflammatory disease states such as ischemic stroke.     

Thigmomorphogenesis: The role of ethylene in the response of Pinus taeda and Abies fraseri to mechanical perturbation

Ethylene production was monitored for 48 h in two half-sibs of Pinus taeda L. grown in the greenhouse and given mechanical perturbation (MP) by flexing; and for 22 h in Abies fraseri (Pursh) Poir, grown in the field and exposed to wind-mediated MP. Both species produced a peak of ethylene 18 h after MP. Seedlings of P. taeda exposed to MP for the duration of the growing season (preconditioned) produced less ethylene compared to non-MP controls, with a peak production at 8 h. One half-sib which responded to MP by an increase in radial growth produced 16 times more ethylene than another half-sib which had no significant change in radial growth. Preconditioned A. fraseri produced no significant quantities of ethylene after MP. The production of wound ethylene appears to be different from MP-induced ethylene. When an ethylene-generating solution was applied to P. taeda seedlings, it mimicked many of the morphological and mechanical characteristics of MP seedlings. The putative role of ethylene in the thigmomorphogenetic response is addressed.     

Gelatin interpenetration in poly N-isopropylacrylamide network reduces the compressive modulus of the scaffold: A property employed to mimic hepatic matrix stiffness

The progression of liver disease from normal to cirrhotic state is characterized by modulation of the stiffness of the extracellular matrix (ECM). Mimicking this modulation in vitro scaffold could provide a better insight into hepatic cell behavior. In this study, interpenetrating poly(N-isopropylacrylamide-co-gelatin) cryogels were synthesized in 48 different compositions to yield scaffolds of different properties. It was observed that a high concentration of N-isopropylacrylamide (NIPAAm) leads to the formation of small pores while gelatin interpenetration on poly-NIPAAm framework renders porous structure. Swelling properties and porosity of the gels decreased with an increase in NIPAAm concentration owing to the increased compactness of the gels. Gelatin interpenetration relaxed the gels and enhanced these properties. An increase in gelatin concentration led to a reduction in compressive moduli indicating that gelatin interpenetration in the poly-NIPAAm network softens the cryogel. With the increase in NIPAAm concentration, the effect of gelatin interpenetration in reducing the compressive moduli expanded. The cytocompatibility studies indicated that the gels are cell-adherent and compatible with HepG2. Furthermore, biochemical and real-time polymerase chain reaction studies revealed that HepG2 and Huh-7 cells cultured on scaffolds mimicking the ECM stiffness of normal liver (1.5–2.5 kPa) exhibited optimum liver-specific functionalities. Increasing the stiffness to fibrotic (4–9 kPa) and cirrhotic (10–20 kPa) ECM decreases the functionality.     

Hypoxia and loss of PHD2 inactivate stromal fibroblasts to decrease tumour stiffness and metastasis

Cancer‐associated fibroblasts (CAFs) interact with tumour cells and promote growth and metastasis. Here, we show that CAF activation is reversible: chronic hypoxia deactivates CAFs, resulting in the loss of contractile force, reduced remodelling of the surrounding extracellular matrix and, ultimately, impaired CAF‐mediated cancer cell invasion. Hypoxia inhibits prolyl hydroxylase domain protein 2 (PHD2), leading to hypoxia‐inducible factor (HIF)‐1α stabilisation, reduced expression of αSMA and periostin, and reduced myosin II activity. Loss of PHD2 in CAFs phenocopies the effects of hypoxia, which can be prevented by simultaneous depletion of HIF‐1α. Treatment with the PHD inhibitor DMOG in an orthotopic breast cancer model significantly decreases spontaneous metastases to the lungs and liver, associated with decreased tumour stiffness and fibroblast activation. PHD2 depletion in CAFs co‐injected with tumour cells similarly prevents CAF‐induced metastasis to lungs and liver. Our data argue that reversion of CAFs towards a less active state is possible and could have important clinical implications.     

Combination of CD34‐positive cell subsets with infarcted myocardium‐like matrix stiffness: a potential solution to cell‐based cardiac repair

Detection of the optimal cell transplantation strategy for myocardial infarction (MI) has attracted a great deal of attention. Commitment of engrafted cells to angiogenesis within damaged myocardium is regarded as one of the major targets in cell‐based cardiac repair. Bone marrow–derived CD34‐positive cells, a well‐characterized population of stem cells, might represent highly functional endothelial progenitor cells and result in the formation of new blood vessels. Recently, physical microenvironment (extracellular matrix stiffness) around the engrafted cells was found to exert an essential impact on their fate. Stem cells are able to feel and respond to the tissue‐like matrix stiffness to commit to a relevant lineage. Notably, the infarct area after MI experiences a time‐dependent stiffness change from flexible to rigid. Our previous observations demonstrated myocardial stiffness‐dependent differentiation of the unselected bone marrow–derived mononuclear cells (BMMNCs) along endothelial lineage cells. Myocardial stiffness (~42 kPa) within the optimal time domain of cell engraftment (at week 1 to 2) after MI provided a more favourable physical microenvironment for cell specification and cell‐based cardiac repair. However, the difference in tissue stiffness‐dependent cell differentiation between the specific cell subsets expressing and no expressing CD34 phenotype remains uncertain. We presumed that CD34‐positive cell subsets facilitated angiogenesis and subsequently resulted in cardiac repair under induction of infarcted myocardium‐like matrix stiffness compared with CD34‐negative cells. If the hypothesis were true, it would contribute greatly to detect the optimal cell subsets for cell therapy and to establish an optimized therapy strategy for cell‐based cardiac repair.     

Influence of muscle activity on musculotendinous stiffness quantification in stunted,prepubertal children

The quick-release technique to estimate musculotendinous (MT) stiffness has been extensively used over the last years, in both animals and humans, to gain insights in the adaptive process of the series elastic component (SEC). Recently, MT stiffness quantification, i.e., SEC behavior, has been revisited for subjects not able to fully activate their muscles (effects of long-term spaceflight or non-mature muscles). Such a phenomenon can also be encountered in stunted children. So, the aim of the present study was to analyze the effect of stunting on MT stiffness taking into account possible defect in muscle activation. For this study, 20 eutrophic children (EU) with an average age of 9 years ± 4 months were compared to 11age matched stunted children (S) evaluated by the height-to-age index. The MT stiffness index was obtained with regard to stiffness–torque and stiffness–soleus EMG relationships. The children of the S group presented a significantly lower Maximal Voluntary Contraction (MVC) in plantar flexion in comparison with children of the EU group (?37.8%). The significantly lower MT stiffness index for S children (?42.6%) was evidenced only when quantified with regard to the stiffness–soleus EMG relationship (66.5 ± 42.8 vs. 38.2 ± 19.9 Nm rad^?1%^?1). Possible delay in fiber type differentiation or tendinous structure maturation can account for the lower MT stiffness index in S children. In conclusion, stunting during early childhood delays the differentiation and maturation processes of musculotendinous structures as shown by the lower MT stiffness quantified with regards to muscle activity, also altered for stunted prepubertal children.