Matthew C. P. Glyn

Lymphocyte transcellular migration occurs through recruitment of endothelial ICAM-1 to caveola- and F-actin-rich domains

During inflammation, leukocytes bind to the adhesion receptors ICAM-1 and VCAM-1 on the endothelial surface before undergoing transendothelial migration, also called diapedesis. ICAM-1 is also involved in transendothelial migration, independently of its role in adhesion, but the molecular basis of this function is poorly understood. Here we demonstrate that, following clustering, apical ICAM-1 translocated to caveolin-rich membrane domains close to the ends of actin stress fibres. In these F-actin-rich areas, ICAM-1 was internalized and transcytosed to the basal plasma membrane through caveolae. Human T-lymphocytes extended pseudopodia into endothelial cells in caveolin- and F-actin-enriched areas, induced local translocation of ICAM-1 and caveolin-1 to the endothelial basal membrane and transmigrated through transcellular passages formed by a ring of F-actin and caveolae. Reduction of caveolin-1 levels using RNA interference (RNAi) specifically decreased lymphocyte transcellular transmigration. We propose that the translocation of ICAM-1 to caveola- and F-actin-rich domains links the sequential steps of lymphocyte adhesion and transendothelial migration and facilitates lymphocyte migration through endothelial cells from capillaries into surrounding tissue.

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.

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.

The influence of 5-azacytidine on the condensation of the short arm of rye chromosome 1R in Triticum aestivum L. root tip meristematic nuclei.

Abstract. This paper describes the effects of 5-azacytidine on the condensation state of rye (Secale cereale L.) chromatin introduced into the wheat genome (Triticum aestivum L. cv. Beaver). The wheat cultivar Beaver carries a translocation between the short arm of rye chromosome 1R (1RS) and the long arm of wheat chromosome 1B (1BL/1RS). 1RS can be detected using genomic in situ hybridisation and carries a ribosomal DNA (rDNA) locus that can be simultaneously detected using multiple labelling strategies. The rDNA locus divides 1RS into a distal region that is gene rich and a proximal region that is gene poor and highly methylated. 1RS also carries a large block of subtelomeric heterochromatin. The drug, which acts to inhibit DNA methylation in plants, has three pronounced effects on interphase nuclei. (1) It induces aberrant condensation of the rye subtelomeric heterochromatin and in many cases induces sister chromatid separation in the subtelomeric heterochromatin of G2 nuclei. (2) Nuclei trisomic for 1RS are observed at low frequency in treated material and are probably a consequence of aberrant sister chromatid separation or condensation. (3) The drug alters normal condensation of 1RS euchromatin. However, contrary to expectation the effect is not simply to induce decondensation. The proximal region of the arm actually condenses at low levels of drug administration while the distal region remains unaltered or increases its decondensation state. Increasing the concentration of 5-azacytidine induces a biphasic response and at the highest concentration used all regions of the arm show signs of decondensation. Thus the influence of the drug on chromatin condensation depends on the genomic structure.

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.