Endothelial glycocalyx thickness and platelet-vessel wall interactions during atherogenesis.

The endothelial glycocalyx (EG), the luminal cover of endothelial cells, is considered to be atheroprotective. During atherogenesis, platelets adhere to the vessel wall, possibly triggered by simultaneous EG modulation. It was the objective of this study to investigate both EG thickness and platelet-vessel wall interactions during atherogenesis in the same experimental model. Intravital fluorescence microscopy was used to study platelet-vessel wall interactions in vivo in common carotid arteries and bifurcations of C57bl6/J (B6) and apolipoprotein E knock-out (ApoE-/-) mice (age 7 - 31 weeks). At the same locations, EG thickness was determined ex vivo using two-photon laser scanning microscopy. In ApoE-/- bifurcations the overall median level of adhesion was 48 platelets/mm2 (interquartile range: 16 - 80), which was significantly higher than in B6 bifurcations (0 (0 - 16), p = 0.001). This difference appeared to result from a significant age-dependent increase in ApoE-/- mice, while no such change was observed in B6 mice. At the same time, the EG in ApoE-/- bifurcations was significantly thinner than in B6 bifurcations (2.2 vs. 2.5 µm, respectively; p < 0.05). This resulted from the fact that in B6 bifurcations EG thickness increased with age (from 2.4 µm in young mice to 3.0 µm in aged ones), while in bifurcations of ApoE-/- mice this growth appeared to be absent (2.2 µm at all ages). During atherogenesis, platelet adhesion to the wall of the carotid artery bifurcation increases significantly. At the same location, EG growth with age is hampered. Therefore, glycocalyx-reinforcing strategies could possibly ameliorate atherosclerosis.

HSP70-mediated acceleration of translational recovery after stress is independent of ribosomal RNA synthesis.

HSP70 is known to protect cells against stressful events. In the present study, the hypothesis was investigated that elevated HSP70 levels protect RNA polymerase I during stress, leading to decreased inhibition of ribosomal RNA (rRNA) synthesis and accelerated recovery of protein translation after stress. To this end, transcriptional and translational activity was studied in H9c2 cells during recovery after a severe heat treatment (SHT, 1 h 45 degrees C) in the presence of elevated HSP70 levels. The latter was achieved by heat pretreatment or by adenovirus-mediated hsp70 gene transfer. Rates of transcription and translation were determined by measuring cellular 3H-labelled uridine and leucine incorporation, respectively. The two types of pretreatment did not affect basal rates of transcription and translation, immediately before SHT. During SHT, both transcriptional and translational rates dropped to less than 10% of basal levels in pretreated as well as non-pretreated cells. Two and four h after SHT, both transcriptional and translational rates were significantly higher in HSP70-overexpressing cells compared to non-pretreated cells. However, immediately after SHT, transcription rates were similarly depressed in non-pretreated and pretreated cells, showing that increased levels of HSP70 did not protect RNA polymerase I activity during SHT. Thus, the HSP70-mediated acceleration of translational recovery is not preceded in time by an enhanced recovery of rRNA synthesis. Therefore, the HSP70-mediated early recovery of protein synthesis after heat stress is independent of rRNA synthesis.

Production of arachidonic acid metabolites in adult rat cardiac myocytes, endothelial cells, and fibroblast-like cells.

Cells were incubated in the presence of the Ca2+ ionophore A23187 (10 microM) and arachidonic acid (AA, 80 microM). The release of eicosanoids from subcultivated cardiac endothelial and fibroblast-like cells amounted to 23.3 +/- 4.5 and 2.0 +/- 0.4 nmol/mg cellular protein per 30 min, respectively. The release from isolated cardiomyocytes remained below the detection limit of the high-performance liquid chromatography assay (< 0.00015 nmol/assay). When a very sensitive radioimmunoassay was applied, cardiomyocytes released 0.002 +/- 0.0001 nmol prostacyclin per milligram cellular protein per 30 min. Prostaglandin (PG) E2 and PGF2 alpha, 12-hydroxyheptadecatrienoic acid, 11- and 15-hydroxyeicosatetraenoic acid, and thromboxane B2 were the main eicosanoids released by endothelial cells. The stable product of prostacyclin, 6-keto-PGF1 alpha, contributed relatively little to the total amount of eicosanoids formed by endothelial cells. Fibroblast-like cells released predominantly PGE2 and 6-keto-PGF1 alpha and, to a lesser extent, 12-hydroxyheptadecatrienoic and 15-hydroxyeicosatetraenoic acids. Neither endothelial cells nor fibroblast-like cells released leukotrienes. A23187 stimulated eicosanoid release from endothelial cells when exogenous AA was below 40 microM. Addition of albumin reduced the amount of eicosanoids produced. Histamine and bradykinin did not influence 6-keto-PGF1 alpha and PGE2 production in cardiomyocytes. Histamine only gave rise to a slight but significantly higher release of 6-keto-PGF1 alpha in endothelial cells.

Arachidonic acid incorporation in cardiomyocytes, endothelial cells and fibroblast-like cells isolated from adult rat heart.

The incorporation of radiolabeled arachidonic acid (3[H]-AA) in normoxic cardiomyocytes (MC), cardiac endothelial cells (EC) and fibroblast-like cells (FL) isolated from adult rat heart was studied. Deposition of 3[H]-AA in the cellular lipid pool was assessed with biochemical and autoradiographic techniques. Extraction and subsequent analysis of lipids from the three different cell types revealed that MC contained significantly more triacylglycerols than EC and FL. The proportion of (unlabeled) AA was also higher in MC triacylglycerols than in EC and FL. The quantity of phospholipids did not differ among the three cell types studied. However, the content of (unlabeled) AA in the MC phospholipid pool was twice as high as in EC and FL. The amount of 3[H]-AA incorporated in the cellular lipid pool of MC, EC and FL depended on the concentration of AA in the incubation medium and the incubation time. In EC and FL incorporation of 3[H]-AA was highest in the cellular phospholipid pool (0.01 microM AA, 30 min incubation). With increased concentration of AA and longer incubation times, the cellular triacylglycerol pool became more important as site of 3[H]-AA incorporation. In MC, comparable amounts of 3[H]-AA were incorporated in the cellular triacylglycerol and phospholipid pools (0.01 and 1 microM AA). At higher AA concentrations (10 microM) the triacylglycerol pool was the preferred site of 3[H]-AA deposition. Autoradiographic analysis at the light microscopic level revealed that the extra-nuclear space was readily stained when the three cell types were incubated with 3[H]-AA. These findings indicate that all cellular lipid pools and membranes are most likely site of deposition of radiolabeled arachidonic acid.

Ischemia and reperfusion induced multilamellar vesicles in isolated rabbit hearts: time correlation between morphometric data and metabolic alterations.

In normoxic hearts a limited number of multilamellar vesicles was found in both endothelial cells and myocytes. The total number of multilamellar vesicles observed in myocytes, particularly those extruded from mitochondria, significantly increased in hearts rendered ischemic for at least 60 mins. The number of multilamellar vesicles extruded from sarcolemma was increased in hearts reperfused after this period of ischemia. The number of multilamellar vesicles in or adjacent to lipid droplets was independent of the duration of ischemia. Multilamellar vesicles were similar in size and periodicity of the lamellae. It is proposed that the number of multilamellar vesicles can be used to quantitate ischemic membrane injury. The formation of multilamellar vesicles was significantly related in time to (a) the accumulation of arachidonic acid and total fatty acids; (b) a decrease in the tissue content of ATP and (c) the release of lactate dehydrogenase (LDH). No significant alterations in the total tissue content of triacylglycerols and phospholipids were detected. The amount of arachidonic acid accumulated in the hearts reflects the degradation of only a minor fraction of the phospholipid pool. Assuming a close relationship between phospholipid degradation, induction of multilamellar vesicles and loss of cellular integrity, the present findings might indicate that the loss of a small part of phospholipids might have serious pathophysiological consequences, as indicated by the morphological changes in cellular membranes and the release of cytoplasmic macromolecules.

Formation of multilamellar vesicles by addition of tannic acid to phosphatidylcholine-containing small unilamellar vesicles.

Tannic acid induces aggregation and formation of multilamellar vesicles when added to preparations of small unilamellar vesicles, specifically those containing phosphatidylcholine. Aggregation and clustering of vesicles was demonstrated by cryo-electron microscopy of thin films and by freeze-fracture technique. Turbidity measurements revealed an approximately one-to-one molar ratio between tannic acid and phosphatidylcholine necessary for a fast and massive aggregation of the small unilamellar vesicles. When tannic acid-induced aggregates were dehydrated and embedded for conventional thin-section electron microscopy, multilamellar vesicles were retrieved in thin sections. It is concluded from morphological studies, as well as previous tracer studies, that tannic acid, at least to a great extent, prevents the extraction of phosphatidylcholine. Multilamellar vesicles were also observed in tannic acid-treated vesicles prepared from total lipid extracts from either rabbit or rat hearts. Substantially more multilamellar vesicles were retrieved in the rabbit vesicle preparation. This difference can probably be explained by the difference in the proportion of the plasmalogen phosphatidylcholine, and possibly the content of sphingomyelin, in lipid extracts of rabbit and rat hearts. It is concluded that the dual effect (reduced extraction and aggregation) of tannic acid on phosphatidylcholines should be taken into consideration when tannic acid is used in tissue preparation.