Abstract. Human endothelial cells are induced toform an anastomosing network of capillary tubes on a gel of collagen I in the presence of PMA. We show here that the addition of mAbs, AK7, or RMAC11 directed to the ~ chain of the major collagen receptor on endothelial cells, the integrin ct2/3t, enhance the number, length, and width of capillary tubes formed by endothelial cells derived from umbilical vein or neonatal foreskins. The anti-ot~31 antibodies maintained the endothelial cells in a rounded morphology and inhibited both their attachment to and proliferation on collagen but not on fibronectin, laminin, or gelatin matrices. Furthermore, RMAC11 promoted tube formation in collagen gels of increased density which in the absence of RMAC11 did not allow tube formation. Neither RMACll or AK7 enhanced capillary formation in the absence of PMA. Lumen structure and size were also altered by antibody RMACll. In the absence of antibody the majority of lumina were formed intracellularly from single cells, but in the presence of RMACll, multiple cells were involved and the lumen size was correspondingly increased. Endothelial cells were also induced to undergo capillary formation in fibrin gels after PMA stimulation. The addition of anti-otv/33 antibodies promoted tube formation in fibrin gels and inhibited EC adhesion to and proliferation on a fibrinogen matrix. The enhancement of capillary formation by the anti-integrin antibodies was matrix specific; that is, anti-txv/33 antibodies only enhanced tube formation on fibrin gels and not on collagen gels while anti-o~v/31 antibodies only enhanced tubes on collagen and not on fibrin gels. Thus we postulate that changes in the adhesive nature of endothelial cells for their extracellular matrix can profoundly effect their function. Anti-integrin antibodies which inhibit cell-matrix interactions convert endothelial cells from a proliferative phenotype towards differentiation which results in enhanced capillary tube formation.
Thus, in the formation of a mature multicellular lumen we have identified a number of key events. First, cell-cell contact is essential in order to define the intercellular space. Second, at least three morphologically distinct subpopulations of ECs are involved. Third, vacuole formation and programmed cell death are required for expansion of the intercellular space which ultimately becomes the lumen.
The cellular pattern of corpus luteal (CL) growth was studied in rats a t Days 6, 10, 12, 14, 16, 17 and 22 of pregnancy: term is Day 23. Measurements were taken of the percentage of the CL occupied by luteal cells, connective tissue and vascular space, luteal cell and nuclear volumes, and the number of luteal and endothelial cells in each of three CL in both ovaries of five rats a t each stage of pregnancy. Total CL volume increased from 1.08-3.23 pl over Days 10-17. This was mainly due to an increase in luteal cell volume from 3.72 pl to 9.30 pl. Neither the number of luteal cells per CL (range 212,000-287,000) nor the percentage of the CL occupied by luteal cells (range 85-90%) had much influence on growth. Nuclear volume increased roughly in proportion to cytoplasmic volume but near term it decreased despite little change in cytoplasmic volume. The number of endothelial cells per CL increased steadily from 398,000 a t Day 6 to 1,545,000 at Day 22. There was a strong negative correlation (r = -0.78) between the number of luteal cells per CL and mean luteal cell volume that was evident at all stages of pregnancy. There was a positive, but weaker correlation (r = 0.35) between number of luteal cells and CL volume. Thus, CL volume seems to be partly determined by the number of luteal cells at Day 6 but this effect is moderated by local control of luteal cell volume.The corpora lutea are the major source of progesterone during pregnancy in the rat. The rate of progesterone production assessed from the collection of ovarian venous effluent (Fajer and Barraclough, '67; Hashimoto et al., '68; Uchida et al., '70) and indirectly from peripheral plasma progesterone levels (Wiest et al., '68; Morishige et al., '73; Bartholomeusz et al., '76) rises during the first five days of pregnancy, plateaus, or falls slightly until about Day 10, increases to a peak at about Day 16 then falls to very low levels about 24 hours prior to parturition on Day 23. Although this temporal pattern of secretion has now been well-established, little is known of its control or indeed of the limitation to progesterone production in any one rat.We are interested in morphological factors which may be of significance and whether morphology can be used as an index of function in the corpus luteum. Changes in the total mass of secreting tissue (Uchida et al., '70) and in some ultrastructural features of luteal cells (Long, '73) have already been reported. However, other than some relatively qualitative studies (Bassett, '49; Long, '73) there is little information on changes in the number and size of luteal cells, particularly during mid-gestation when corpus luteal volume and rate of progesterone synthesis reach maximum levels. This forms the major subject of the present study. We also carried out statistical analyses to identify cellular differences between corpora lutea and whether these might be related to the number of corpora lutea per rat. MATERIALS A N D METHODSThirty-five nulliparous Albino Wistar rats with a mean weight at mating of...
The synthesis and secretion of progesterone by the corpus luteum (CL) may be limited or controlled by transport mechanisms operating between circulating blood and luteal cell cytoplasm. To examine this possibility, the structural features involved in transport, including membrane surface areas and diffusion distances, were quantitated in the CL of 16-day pregnant rats. One ovary from each of eight rats was fixed by perfusion via a cannula inserted into the parametrial artery, and two CL from each ovary were processed for electron microscopy and examined with standard morphometric techniques. For comparison, one CL from each of a further eight ovaries was diced into small cubes, fixed by immersion, and analyzed similarly. In perfusion-fixed CL, there was a substantial volume of vascular space (20% of the total) and interstitial space (5%) and an extensive surface area of capillaries (441 mm2 per CL). The luteal-cell membrane had numerous projections which increased its surface area by a factor of 3.08. Almost 60% of the luteal-cell surface directly faced a capillary, and a further 37% faced interstitial space which probably extended to a capillary surface. Only 3% was in direct contact with a neighboring luteal cell. Despite the extensive interstitial space the harmonic mean thickness, an estimate of likely effective diffusion distance between luteal cell cytoplasm and blood, was only 0.42 micron. This was less than half of the calculated arithmetic mean thickness owing to the presence of surface projections and an uneven capillary endothelium. Results from immersion-fixed CL were qualitatively similar; but the proportion of interstitial space was only 59% of that in perfusion-fixed CL, and the contribution of surface projections to the total area of luteal-cell membranes was significantly reduced. Collectively, these results suggest that membranes and spaces between blood and luteal-cell cytoplasm are structured so as to minimize transport distances.
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