Oxygen-Induced Cytoskeleton
Rearrangement of Cultured Human Brain Microvascular Endothelial Cells
Minoru Tomita
1, Norio Tanahashi
1, Masahiro Kobari
2, Hidetaka Takeda
1, Masaki Takao
1, and Istvan Schiszler
1,3Key words. Oxygen, Human brain microvascular endothelial cell, Remodel- ing of cytoskeleton, Sol-gel transformation, Mobile cell
Introduction
Dynamic remodeling of endothelial cells in response to various stimuli is important for capillary flow redistribution. We previously reported that expo- sure of nonconfluent cultured human brain microvascular endothelial cells (HBEC) to oxygen induced a contraction by 14%, with formation of multiplex mesh networks consisting of star shapes, large and small polygons, and fern and brush patterns [1]. Cessation of oxygen supply induced disassembly of the network, incorporation of decomposed particles into cell components (or solation into the fluid), and recovery of the previous cell complexion.
This paper examines the reproducibility of these sequential processes, i.e., contraction–network formation in the cell–disintegration of fibers–
particulation–recovery of the original cell shape, upon repeated oxygen exposure of the same HBECs.
Materials and Methods
The materials and methods used here were the same as those reported previously [1]. Briefly, we used nonconfluent HBECs of passage 2 purchased from Cell Systems Corp. (WA, USA). During observation with video-enhanced
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1
Department of Neurology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
2
Kyosai Tachikawa Hospital, 4-2-22 Nishiki-cho, Tachikawa, Tokyo 190-0022, Japan
3
(Present address) Petofi utca 4, Torokbalint 2045, Hungary
contrast differential interference contrast (VEC) microscopy, oxygen was directly introduced repeatedly (more than three times; maximum eight times) onto the medium surface above HBEC spread on a coverslip (n = 6) for periods of approximately 30 s at intervals of approximately 30 s.
Results
Human brain microvascular endothelial cells contained a round-topped nucleus with one or two nucleoli, and a cell body surrounded by a thin, widely spread transparent lamella, as described previously. We also con- firmed that transient oxygen application produced the sequential processes of contraction–network formation in the cell–disintegration of fibers–
particulation–recovery of the original cell shape. The peripheral lamella was dragged centrally towards the nucleus and the nuclear envelope became clearly demarcated and enhanced. The plasma membrane became tightly wrapped around the cell body, which then also began to contract. When the contraction slowed down and stopped, a mesh network suddenly appeared, starting at adhesion plaques, and grew rapidly like a spider’s web. Such net- works spread from the adhesion plaques of the HBEC to the lamella, as well as to the cell body and the surface of the nucleus. We found that repeated oxygen delivery to the same cells reproduced the sequential processes described above every time when oxygen was delivered. Figure 1 shows a typical protocol of three reproducible cycles in a HBEC. It should be noted that the patterns of the mesh network were different each time, i.e., large polygonal pattern at the first exposure, fern pattern at the second exposure, and small polygonal pattern at the third exposure. After discontinuance of the oxygen blowing each time, a recovery phase followed: the network disintegrated immediately, yielding small particles of <0.5 mm in diameter which subsequently fused into the cellular structure. The HBEC completely recovered the control appearance each time. The other cells studied showed similar reproducibility. Time intervals (average of six cases) were approxi- mately 8.8 ± 4.3 s for contraction, 2.1 ± 3.1 s for network appearance, 14.4 ± 9 .1 s for network duration, and 18.8 ± 13.6 s for disappearance. The overall process was therefore rapid and, as noted above, could be reproduced in indi- vidual cells each time that oxygen gas was supplied. Thus, repeated oxygen stimuli remodeled the cytoskeleton of HBECs. The changes in the mesh network (cytoskeleton) in the same HBECs were consistent in all six cells studied. There seemed to be no rules as to pattern formation, although the patterns were tentatively categorized into fern, polygonal, star-shaped, and brush-like types.
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Discussion
The network involved could consist of stress fibers, which are believed to contain all the elements required for active contraction; myosin, ·-actin, and tropomyosin. It is likely that brain microvascular endothelial cells are remod- eling almost continuously in response to various changing stimuli. Thus, we consider that capillaries do not behave as rigid tubes, or as compliant vessels.
The remodeling upon contraction creates a capillary vasomotion not only radially, but also in a longitudinal direction, that may control the redistribu- tion of capillary flow, along with local function. The above sequential changes with development of stress fibers and adhesion plaques indicate that HBEC has many of the characteristics of mobile cells.
Conclusion
We conclude that the cytoskeleton of HBECs rapidly and reproducibly re- models after successive contraction cycles in response to multiple oxygen exposures. The appearance of a mesh network and adhesion plaques suggests that HBECs are mobile cells.
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