Barbara J. Ward

Attenuation of glucocorticoid functions in an Anx-A1-/- cell line.

The Ca(2+)- and phospholipid-binding protein Anx-A1 (annexin 1; lipocortin 1) has been described both as an inhibitor of phospholipase A(2) (PLA(2)) activity and as a mediator of glucocorticoid-regulated cell growth and eicosanoid generation. Here we show that, when compared with Anx-A1(+/+) cells, lung fibroblast cell lines derived from the Anx-A1(-/-) mouse exhibit an altered morphology characterized by a spindle-shaped appearance and an accumulation of intracellular organelles. Unlike their wild-type counterparts, Anx-A1(-/-) cells also overexpress cyclo-oxygenase 2 (COX 2), cytosolic PLA(2) and secretory PLA(2) and in response to fetal calf serum, exhibit an exaggerated release of eicosanoids, which is insensitive to dexamethasone (10(-8)- 10(-6) M) inhibition. Proliferation and serum-induced progression of Anx-A1(+/+) cells from G(0)/G(1) into S phase, and the associated expression of extracellular signal-regulated kinase 2 (ERK2), cyclin-dependent kinase 4 (cdk4) and COX 2, is strongly inhibited by dexamethasone, whereas Anx-A1(-/-) cells are refractory to the drug. Loss of the response to dexamethasone in Anx-A1(-/-) cells occurs against a background of no apparent change in glucocorticoid receptor expression or sensitivity to non-steroidal anti-inflammatory drugs. Taken together, these observations suggest strongly that Anx-A1 functions as an inhibitor of signal-transduction pathways that lead to cell proliferation and may help to explain how glucocorticoids regulate these processes.

A Rho-associated kinase mitigates reperfusion-induced change in the shape of cardiac capillary endothelial cells in situ

OBJECTIVE We have previously demonstrated that ischaemia and reperfusion of the myocardium alter capillary dimensions and endothelial cell shape and that these changes are likely to be effected by the actomyosin contractile system in endothelial cells. Rho GTPases are involved in the regulation of cytoskeletal re-organization and in cell contraction. Rho-associated kinase regulates the sensitivity of myosin light chain to Ca(2+) in smooth muscle but not in cardiac or skeletal muscle myocytes. This study investigated the role of Rho-associated kinase in endothelial cell shape change induced by cardiac ischaemia and reperfusion. The role of Rho proteins in endothelial cell shape change in situ in the myocardial capillary bed has to date not been investigated. METHODS Ischaemia and reperfusion were induced in Langendorff perfused rat hearts at constant flow. Electron microscopy and immunofluorescence studies localized the beta Rho-associated kinase isotype in capillary endothelial cells. Whole capillary and luminal cross-section areas, luminal and abluminal membrane lengths were measured to monitor changes in cell dimensions. We used a ROCK inhibitor, Y-27632, to investigate the role of this protein in endothelial cell shape change. RESULTS ROCK1 localized primarily to intracellular membranes in endothelial cells. Morphometric analysis and a study of capillary lumen resin casts demonstrated that inhibition of the activity of this kinase with Y-27632 ablated the change in shape of endothelial cells induced by ischaemia and reperfusion. CONCLUSION These results suggest that ROCK1 is involved in cardiac capillary endothelial cell shape change in situ and that targeting the contractile system in this way may be useful in ameliorating reperfusion injury.

Microvascular Permeability of the Isolated Rat Heart to Various Solutes in Well-Oxygenated and Hypoxic Conditions

The aim of this study was to investigate the effect of a moderate degree of hypoxia on coronary vascular permeability to various lipophobic solutes. Using the multiple indicator dilution method the permeability-surface area (PS) products were determined for 125I-albumin, 125I-insulin and 57Co-cyanocobalamin in perfused rat hearts (flow approximately 10 ml.min-1.g-1) either with well-oxygenated (pO2 approximately 96 kPa) or hypoxic (pO2 approximately 45 kPa) solutions. The PS products for albumin, insulin and cyanocobalamin during the well-oxygenated equilibration period were 0.20 +/- 0.03, 0.29 +/- 0.06 and 2.0 +/- 0.3 ml.min-1.g-1 (mean +/- SE), respectively, relative to 131I-gamma-globulin. The PS products for these solutes 15 min after the induction of hypoxia were 1.3 +/- 0.3, 0.8 +/- 0.1 (p < 0.05) and 2.1 +/- 0.2 (p < 0.05), respectively. In hearts perfused with well-oxygenated solution for 75 min, the PS products for these solutes remained stable throughout the period of the study. Electron-microscopic examination of hypoxic tissues showed the presence of endothelial gaps of approximately 1 micron which were underlined by an intact basal lamina. We conclude that a moderate degree of hypoxia produces a large increase in permeability of albumin and insulin but has no effect on the PS products for cyanocobalamin and that the endothelial gaps are the likely mechanism of the observed increase in permeability.

Incorporation and distribution of 3H oleic acid in the isolated, perfused guinea-pig heart made hypoxic

Improved methods of autoradiography and lipid extraction have been used to study the influence of hypoxia on the fate of radioactive fatty acids in the isolated guinea pig heart. Evidence is provided that hypoxia causes a shift of the rate-limiting step from transport into the cell to oxidation in the mitochondria. This leads to an increased radioactivity in myocardial free fatty acid and in the cytosol. Radioactivity in the mitochondria is decreased and disappears more slowly. At the same time, there is an increased radioactivity in lipid droplets and in triacylglycerol. There is no evidence of a specific location of radioactivity in the sacroplasmic reticulum.

Changes in the Actin Cytoskeleton of Cardiac Capillary Endothelial Cells during Ischaemia and Reperfusion: The Effect of Phalloidin on Cell Shape

A reduction in capillary dimensions has been demonstrated in postischaemic reperfusion in the heart. The aim of this study was to demonstrate that in ischaemia and ischaemia followed by reperfusion, the change in shape of the constituent endothelial cells can be inhibited by phalloidin which stabilises the actin microfilament system. Isolated, perfused rat hearts were made globally ischaemic both with and without reperfusion and in the presence or absence of phalloidin. Changes in ischaemic endothelial cell dimensions were quantified by measuring whole capillary and luminal cross-sectional areas, abluminal and luminal membrane lengths. The distribution of β-actin within the endothelial cells was determined by immunocytochemistry. In control hearts, β-actin is distributed throughout the endothelium with a slight increase towards the luminal membrane. In ischaemia, this was more marked and other patterns of actin distribution were also observed. After reperfusion, a ‘double ring’ of actin could be distinguished. With phalloidin, the actin staining was more regular and the ring pattern was not observed. Morphometry showed that phalloidin was more effective in reducing endothelial cell shape change after reperfusion than after ischaemia alone. We conclude that endothelial cell shape change on reperfusion can be modified by agents which target the contractile proteins.

A β-Actin Isotype Is Present in Rat Cardiac Endothelial Cells But Not in Cardiac Myocytes

Objective: Active constriction in cardiac capillary endothelial cells (CCECs) is controversial. It is thought by many researchers that CCECs are not actively involved in constriction; others believe that these cells do contribute some of the force required for capillary constriction. Because actin is a major component of most contractile mechanisms responsible for changing cell shape, we compared two probes as potential monitors of actin distribution in CCECs in situ.

Structural changes in the cardiac microvasculature of the rat in response to acute high glucose levels: a comparison with diabetes.

Objective: Isolated rat hearts were used to determine whether structural changes in the coronary microcirculation caused by diabetes mellitus occur soon after the onset of hyperglycemia and whether early changes might be reversed by return to normoglycemic conditions.

Rho Kinase-Mediated Reduction in Cardiac Capillary Endothelial Cell Dimensions, In Situ, Against Flow.

OBJECTIVE We previously showed that ischemia, induced by interrupting vascular flow, reduced cardiac capillary caliber. This was accomplished by a reduction in endothelial cell dimensions which was sensitive to Rho kinase (ROK) inhibition and stabilization of the actin cytoskeleton. Here, we investigated whether similar changes in endothelial cells, in situ, could be elicited in the presence of flow through the capillary bed. METHODS Langendorffs perfused rat hearts were subjected to vasoactive agents, ischemia, and reperfusion. Luminal and abluminal perimeters of capillary cross-sections and their areas were measured from electron micrographs to monitor changes in endothelial cell dimensions. RESULTS Histamine (100 microM) reduced capillary endothelial cell dimensions, in situ, without endothelial injury. While cross-sectional areas of endothelial cells were not altered by histamine, all other parameters measured were significantly reduced in comparison to controls. These changes were pre- vented by ROK inhibition. CONCLUSIONS Cardiac capillary endothelial cells, in situ, are able to change shape against continuous flow. One hundred (100) microM histamine induces morphometric changes in these endothelial cells, in situ, without cell damage. These cell-shape changes require ROK and mimic those observed following myocardial ischemia. Targeting the actomyosin contractile system may be useful in ameliorating effects of ischemia on the myocardium

Change in endothelial cell shape in response to ischaemia in the cardiac and retinal capillary beds

The change in shape of cardiac capillary endothelial cells in response to ischaemia and ischaemia followed by reperfusion is well established. We investigated the possibility that this process is an active rather than a passive process and that the change might be prevented or attenuated in order to minimise reperfusion injury and ‘no‐reflow’. We have also compared the change in shape of cardiac capillaries with those of the retina. We used an isolated heart preparation in which global ischaemia was induced by cessation of flow. Retinal capillaries were made ischaemic by one of 2 methods, using firstly superfusion with an oxygen depleted medium, and secondly by occlusion of upstream vessels with starch spheres. Ultrastructural and morphometric analysis of cardiac and retinal capillaries demonstrated that the change in shape seen in cardiac capillaries in response to ischaemia does not occur in retinal capillaries reflecting the different metabolic activities of the 2 tissues. Immunocytochemical localisation of _‐actin showed no apparent difference in the retinal capillaries between control and ischaemic capillaries. Cardiac capillaries, however, showed an irregular redistribution of _‐actin in ischaemic vessels which could be attenuated by exposure to phalloidin. Resin casts of ischaemic capillaries showed focal narrowings which were not present in controls. From these results we conclude that the change in endothelial cell shape in response to ischaemia is not a passive process and can be modulated by agents which target the microfilament system.