Thin-layer chromatography of hyaluronate oligosaccharides

Odd- and even-numbered hyaluronate oligosaccharides with N-acetylglucosamine, glucuronic acid, or 4,5-unsaturated glucuronic acid at their nonreducing ends were separated by thin-layer chromatography, using silica gel and a solvent system of isopropanol-water (66 : 34) containing 0.05 M NaCl. In the isopropanol system, small amounts of electrolytes were necessary for the resolution of each oligosaccharide.     

几丁聚糖和透明质酸钠对血管内皮细胞增殖的影响

目的：比较几丁聚糖和透明质酸钠对血管内皮细胞增殖的影响。方法：用含不同浓度的几丁聚糖和透明质酸钠的培养液对血管内皮细胞（EV304）进行培养,以四唑盐比色法测细胞增殖,并用流式细胞仪测定细胞周期。结果：几丁聚糖在≥0.1mg/ml时促进血管内皮细胞的增殖,透明质酸钠对血管内皮细胞的增殖有抑制作用,几丁聚糖可使细胞周期中G1期比例下降,而透明质酸钠使细胞周期中G1期比例上升。结论：几丁聚糖促进血管内皮细胞的增殖,而透明质酸钠对血管内皮细胞的增殖有抑制作用。     

Mercury-arc photolysis: a method for examining second messenger regulation of endothelial cell monolayer integrity.

Cell-cell apposition in bovine pulmonary endothelial cell monolayers was modulated by inducing transient increases in intracellular adenosine 3':5'-cyclic monophosphate (cAMP) and 1,4,5-inositol triphosphate (IP3). This was accomplished by mercury-arc flash photolysis of o-nitrobenzyl derivatives of the second messengers (caged compounds). Second messenger release by the mercury-arc lamp was determined by radioimmunoassay of cAMP to have a t1/2 of approximately 8 min. Each second messenger induced the phosphorylation of a distinct subset of cytoskeletal proteins; however, both IP3 and cAMP increased vimentin phosphorylation. Actin isoform patterns were not altered by the second messengers. Intracellular pulses of IP3 in pulmonary endothelial cells caused disruption of endothelial monolayer integrity as determined by phase-contrast microscopy and by visualization of actin stress fibers with rhodamine-phalloidin. Intracellular pulses of cAMP increased cell-cell contact, cell surface area, and apposition. IP3 appeared to have its greatest effect on the actin peripheral band. In silicone rubber contractility assays this agent caused contraction of pulmonary microvascular endothelial cells as visualized by an increase in wrinkles beneath the cells. On the other hand, cAMP appeared to effect both the peripheral band and centralized actin domains. Caged cAMP caused relaxation of endothelial cells as visualized by a disappearance of wrinkles beneath the cells.     

Anin vitro model for endothelial permeability: Assessment of monolayer integrity

Summary An essential component of anyin vitro model for endothelial permeability is a confluent cell monolayer. The model reported here utilizes primary human umbilical vein endothelial cells (HUVEC) cultured on recently developed polyethylene terephthalate micropore membranes. Using a modification of the Wright-Giemsa stain, confluent HUVEC monolayers grown on micropore membranes were routinely assessed using light microscopy. Determination of confluence using this method was confirmed by scanning electron microscopy. Transendothelial electrical resistance of HUVEC monolayers averaged 27.9±11.4 Ω · cm², 10 to 21% higher than literature values. Studies characterizing the permeability of the endothelial cell monolayer to³H-inulin demonstrated a linear relationship between the luminal concentration of³H-inulin and its flux across HUVEC monolayers. The slope of the flux versus concentration plot, which represents endothelial clearance of³H-inulin, was 2.01±0.076 × 10⁻⁴ ml/min (r²=.9957). The permeability coefficient for the HUVEC monolayer-micropore membrane barrier was 3.17±0.427×10⁻⁶ cm/s with a calculated permeability coefficient of the HUVEC monolayer alone of 4.07±0.617×10⁻⁶ cm/s. The HUVEC monolayer reduced the permeability of the micropore membrane alone to³H-inulin (1.43±0.445×10⁻⁵ cm/s) by 78%. Evans blue dye-labeled bovine serum albumin could not be detected on the abluminal side without disruption of the HUVEC monolayer. These results demonstrate a model for endothelial permeability that can be extensively assessed for monolayer integrity by direct visualization, transendothelial electrical resistance, and the permeability of indicator macromolecules.     

Cellular repressor of E1A stimulated genes enhances endothelial monolayer integrity

Cellular repressor of E1A stimulated genes (CREG) is a novel modulator that maintains the homeostasis of vascular cells. The present study aimed to investigate the effects of CREG on tumor necrosis factor (TNF)-α-mediated inflammatory injury of vascular endothelial cells. Human umbilical vein endothelial cells (HUVECs) were cultured and CREG overexpressing (VC), knockdown (VS) and mock-transfected (VE) HUVECs were challenged with TNF-α. We demonstrated that TNF-α prompted robust intercellular filamentous actin (F-actin) stress fiber formation as examined by rhodamin-phalloidin staining. Transwell assay and rhodamine B isothiocyanate–dextran staining indicated that TNF-α induced intercellular hyperpermeability of the HUVEC monolayers. These effects were attenuated in VC cells with forced CREG overexpression but significantly potentiated in VS cells with CREG silencing. After TNF-α stimulation, interleukin (IL)-6 and IL-8 secretions in VE cells were markedly increased and inducible nitric oxidase (iNOS) expression substantially elevated, whereas these effects were pronouncedly damped in VC cells. Conversely, in VS cells, the increase in inflammatory markers was substantially potentiated. Immunofluorescence staining demonstrated that nuclear factor κB (NF-κB) slowly and transiently translocated into the nuclei of VC cells upon TNF-α stimulation. However, a more swift and sustained nuclear translocation was observed in VS as compared to VE cells. Corresponding changes in the pattern of its protein expression was also observed. These data suggested that CREG can inhibit NF-κB activation, TNF-α-induced inflammatory responses and the hyperpermeability of endothelial cells, and may therefore represent a potential therapeutic target for pathological vascular injury.     

The intracellular pH and cell proliferation of the LS cell line and its derivative LSM adapted to monolayer growth]

The dynamics of intracellular pH (pHi) during proliferation of cells of LS line in bicarbonate-containing media and of its derivative LSM line adapted to grow in a monolayer has been studied. The contact of LS cells with a solid substrate was not accompanied by their spreading and by an increase in pHi. The pHi values of growing and resting LS cells were practically equal (7.03 and 6.97, respectively). The adhesion and spreading of LSM cells were accompanied by an increase in pHi. The proliferation of LSM cells occurred at different pHi values: at 7.32 on solid substrate with serum, at 7.18 on substrate without serum, at 7.13 in a serum-containing suspension, at 6.97 in a suspension without serum. The highest growth rate was observed at the increased pHi value. Cell proliferation on the substrate stopped at pHi values within 7.10 and 7.13 which were equal to or exceeded the pHi of growing cells in suspension. No difference was observed between LS and LSM cells in the activities of Na+/H+ exchange and transport of Cl- into cells that are involved in pHi regulation. Transport of HCO3- into the cytoplasm of LSM cells was more active than that of LS cells. The role of pHi in the anchorage dependence of cell proliferation is discussed.     

A steering model of endothelial sheet migration recapitulates monolayer integrity and directed collective migration

Cells in endothelial cell monolayers maintain a tight barrier between blood and tissue, but it is not well understood how endothelial cells move within monolayers, pass each other, migrate when stimulated with growth factor, and also retain monolayer integrity. Here, we develop a quantitative steering model based on functional classes of genes identified previously in a small interfering RNA (siRNA) screen to explain how cells locally coordinate their movement to maintain monolayer integrity and collectively migrate in response to growth factor. In the model, cells autonomously migrate within the monolayer and turn in response to mechanical cues resulting from adhesive, drag, repulsive, and directed steering interactions with neighboring cells. We show that lateral-drag steering explains the local coordination of cell movement and the maintenance of monolayer integrity by allowing closure of small lesions. We further demonstrate that directional steering of cells at monolayer boundaries, combined with adhesive steering of cells behind, can explain growth factor-triggered collective migration into open space. Together, this model provides a mechanistic explanation for the observed genetic modularity and a conceptual framework for how cells can dynamically maintain sheet integrity and undergo collective directed migration.     

The force and effect of cell proliferation

EMBO J 32: 2790–2803 doi:10.1038/emboj.2013.197; published online September102013The spatiotemporal control of cell divisions is a key factor in epithelial morphogenesis and patterning. Mao et al (2013) now describe how differential rates of proliferation within the Drosophila wing disc epithelium give rise to anisotropic tissue tension in peripheral/proximal regions of the disc. Such global tissue tension anisotropy in turn determines the orientation of cell divisions by controlling epithelial cell elongation.Oriented cell divisions play important roles in the establishment of the animal body plan by both influencing tissue morphogenesis and generating cellular diversity. Generally, the direction of the cell division plane is determined by the orientation of the mitotic spindle prior to cytokinesis. The observation that the mitotic spindle in most animal cell types aligns with the cell''s longest axis has led to the formulation of the ‘long-axis-rule'', postulating that cell shape anisotropy is the main determinant of spindle orientation (Minc et al, 2011). However, cell shape anisotropy is unlikely to be the only determinant since many cell types round up during mitosis, thereby losing their shape anisotropy and others do not follow the long-axis-rule at all. In such cases, division orientation is determined by the polarizing activity of biochemical signals originating from the environment (reviewed in Morin and Bellaïche, 2011). In addition, externally applied forces have also been suggested to control division orientation of single cells in culture independently from their effect on cell shape (Fink et al, 2011).Epithelial growth implies that cells divide parallel to the tissue plane with both daughter cells remaining integrated within the tissue. Although it has been recognized that defects in apico-basal polarity lead to spindle misalignment and disruption of epithelial architecture, the molecular mechanisms underlying this regulation are still unknown. Recent work in the Drosophila wing disc epithelium uncovered that the junctional proteins Scribbled and Discs large 1 (Dlg1) are required for proper spindle alignment parallel to the tissue plane (Nakajima et al, 2013). Similarly, in the Drosophila follicular epithelium, spindle orientation is dependent on the lateral localization of Dlg1, independently of its role in apico-basal polarity (Bergstralh et al, 2013). While such mechanisms ensure that cells divide parallel to the epithelial plane, other mechanisms must still be present to determine the orientation of the mitotic spindle within this plane.In the Drosophila wing disc epithelium, symmetric cell divisions preferentially align with the proximal-distal (PD) axis, thus elongating the organ along this axis (Baena-López et al, 2005). This preferential cell division orientation is determined by the Fat-Dachsous pathway, which promotes accumulation of the atypical myosin Dachs at PD cellular junctions. The polarized activity of Dachs in turn drives cell elongation along the PD axis, leading to a preferential orientation of the mitotic spindle along this axis (Mao et al, 2011). In this issue of The EMBO Journal, Mao et al (2013) report that while mitotic cells located in central regions of the wing disc indeed elongate and divide along the PD axis, cells located in the periphery (proximal edge) elongate and divide orthogonally to the PD axis (Figure 1). These results suggested some type of global planar tissue polarization in proximal regions of the wing disc overriding the local effects of Dachs on spindle orientation. By using laser ablation to reveal tissue tension, the authors showed that in peripheral/proximal regions of the wing disc, junctions oriented orthogonal to the PD axis (PD junctions) are under higher tension than junctions oriented along this axis (lateral junctions; Figure 1). This led them to hypothesize that anisotropic tissue tension might control division orientation of proximal wing cells. Through a combination of elegant genetic experiments and theoretical modelling, the authors then demonstrated that this global tension anisotropy in the proximal wing disc arises from higher cell division rates observed in central versus proximal regions of the wing disc. Furthermore, this apparent tension anisotropy causes concentric elongation of proximal wing disc cells orienting their mitotic spindle orthogonal to the PD axis (Figure 1).Open in a separate windowFigure 1Differential rates of cell division between distal (green) and proximal (red) regions of the Drosophila wing disc epithelium (1) give rise to global patterns of tension anisotropy within the tissue (2). This tension anisotropy promotes cell elongation along the main axis of tension, thereby controlling the orientation of cell division via cell shape anisotropies in proximal regions of the wing disc (3); D, distal; P, proximal.Collectively, these results demonstrate that differential proliferation rates within a tissue can generate global tension anisotropies, which promote cell shape changes that again influence cell division orientation. Further dissection of the mechanisms by which tissue tension controls cell division orientation will clarify if anisotropic tension controls division orientation solely through cell elongation, or if additional mechanosensing mechanisms exist that more directly convey tissue tension information to the mitotic spindle. It might also be worth exploring whether cell divisions along the main axis of tension within the wing disc affect global tension anisotropy, and whether the formation of anisotropic tension around areas of cell proliferation affects the rate of cell division therein. Such interplay between tissue tension anisotropy and cell division orientation/rate will likely be critical for maintaining physiological degrees of tissue tension and growth.In general, the work by Mao et al (2013) provides compelling evidence for a functional link between tissue tension and cell division orientation in a physiological relevant context, paving the way for future studies addressing the reciprocal relationship between these two aspects in tissue morphogenesis.     

p38 MAPK activation coupled to endocytosis is a determinant of endothelial monolayer integrity

We show in rat lung microvessel endothelial cells (RLMVEC) that endocytosis is a critical determinant of activation of mitogen-activated protein kinase (MAPK) and thereby regulates endothelial monolayer integrity. In RLMVEC grown in serum-free medium, we observed that albumin supplementation induced the phosphorylation of p38 MAPK within 30 min, which persisted for up to 2 h. Engagement of the endocytic machinery regulated the activation of p38 MAPK that contributed to endothelial cell proliferation and reduction of apoptosis. We also observed an interaction between the caveolar protein caveolin-1 and p38 MAPK with reciprocal coimmunoprecipitation assays and colocalization using double-label immunofluorescence staining. Knockdown of caveolin-1 expression with small interfering RNA significantly reduced endocytosis and activation of p38 MAPK and interfered with the ability of endothelial cells to form a confluent monolayer. Thus caveolae-mediated endocytosis and concomitant activation of p38 MAPK may help to maintain endothelial monolayer integrity by signaling proliferation and survival of endothelial cells.     

Endogenous regulation of endothelial cell proliferation

Bovine aortic endothelial cells (BAEC) in culture have the ability to regulate their own proliferation. We have found that a fraction below 100,000 daltons obtained from the media of confluent cultures of BAEC inhibits tritiated thymidine [3H]TdR incorporation as well as their proliferation. The inhibition is dose- and time-dependent; maximum inhibition of [3H]TdR incorporation occurs 8 hr after cells are released from synchronization and the inhibitory fraction is added. Inhibition is evident at concentrations as low as 50 micrograms/ml and reaches a maximum at 600 micrograms/ml. The blockage of [3H]TdR incorporation is reflected in the inhibition of cell proliferation. In the presence of 400 micrograms of endogenous inhibitor per ml of media, added at the time of plating, the average population doubling time increases from 19 to 41 hr. These findings indicate that, in culture, BAEC can regulate their own proliferation by synthesizing an endogenous inhibitor(s) of proliferation.     

Vacuum-ultraviolet circular dichroism of sodium hyaluronate oligosaccharides and polymer segments

The CD spectrum of an enzymatically derived sodium hyaluronate (NaHA) segment preparation with chain length 18 ± 3 disaccharide units [NaHAseg, ( NaGlcUA GlcNAc)_15–20°. NaGlcUA, sodium D -glucuronate; GlcNAc, 2-acetamido-2-deoxy-D -glucose] in H₂O was recorded to 180 nm using a computer-controlled vacuum-uv CD instrument. Near 190 nm the spectrum is of low intensity, similar to the sum of the free monosaccharide contributios, attributed to the π–π* transitions of the acetamido and carboxylate substituents. In contrast, much smaller oligosaccharides, also derived from high-molecular-weight NaHA by enzymatic digestions, show CD spectra in H₂O with prominent bands centered near 190 nm. The oligosaccharide spectra can be matched as linear combinations of interior sugar residue (? NaHAseg) and end sugar residue CD contributions. End residues from oligosaccharides of the type (NaGlcUA-GlcNAc)_n show a negative CD band near 190 nm. End residues from oligosaccharides of the reverse sequence (GlcNAc-NaGlcUA)_n show a positive CD band near 190 nm. Averaging of the two end-residue spectral contributions yields an approximate match for the spectrum of NAHAseg below 200 nm. It is proposed that the low intensity CD of NaHA in the π–π* region is the result of large-magnitude, oppositely signed contributions, which can be visulized by studying oligosaccharides.     

Volume changes of an endothelial cell monolayer on exposure to anisotonic media

The osmotic process plays an important role in controlling the distribution of water across cell membranes and thus the cell volume. A system was designed to detect the volume changes of an endothelial cell monolayer when cells were exposed to media with altered osmolalities. Electrodes housed in a flow chamber measured the resistance of ionic media flowing over a cultured cell layer. Assuming the cell membrane acts as an electrical insulator, volume changes of the cell layer can be calculated from the corresponding changes in chamber resistance. The media used in the experiments had osmolalities in the range 120-630 mmol/kg. When cells were exposed to hypertonic media, there was rapid shrinkage with an approximate 30% reduction in total cell volume for a twofold increase in osmolality. On exposure to hypotonic media, the cells initially swelled with an approximate 20% volume increase for a decrease in osmolality by half. With sustained exposure to low osmolality media, there was a gradual and partial return of cell volume towards isotonic values that started 10 minutes after and was complete within 30 minutes of the osmolality alteration. This finding suggests regulatory volume decrease (RVD); however, no regulatory volume increase (RVI) was observed with the continued exposure to hypertonic media over 45 minutes.     

Heparin-derived oligosaccharides: affinity for acidic fibroblast growth factor and effect on its growth-promoting activity for human endothelial cells

The minimal structural requirements for the interaction of heparin with acidic fibroblast growth factor (aFGF) were investigated. Oligosaccharides (tetra- to decasaccharides) obtained by nitrous acid depolymerisation of standard heparin were separated by affinity chromatography on Sepharose-immobilised aFGF. The shortest fragment retained by the affinity column at 0.2 M NaCl and eluted at 1 M NaCl was a "regular" hexasaccharide, a trimer of the most abundant disaccharide sequence in heparin. More complex octa- and decasaccharides were also retained by the column. The oligosaccharides eluted by 1 M NaCl from the affinity column ("high-affinity" oligosaccharides) and those washed from the column at 0.2 M NaCl ("low-affinity" oligosaccharides) were compared for their capacity to protect aFGF from proteolysis and to potentiate its mitogenic activity. At a low ionic strength, all oligosaccharides tested, except the "regular" disaccharide, protected aFGF against trypsin and collagenase digestion. At higher ionic strength (greater than 0.2 M NaCl), only high-affinity oligosaccharides showed a protective effect. The high-affinity oligosaccharides (hexa- to decasaccharides) potentiated the mitogenic activity of aFGF, as measured by [3H]thymidine incorporation into DNA of human fibroblasts. The effect of the oligosaccharides on human endothelial cell proliferation was more complex: inhibition of proliferation was observed in the presence of serum and low concentrations of aFGF (1-5 ng/ml) and potentiation in the presence of higher concentrations of aFGF. The potentiating effect increased as a function of molecular size of the heparin fragments and, for a given size, as a function of the anionic charge of the oligosaccharide. Our results suggest that inhibition of cell proliferation by heparin may result from interference with an autocrine basic FGF-like activity.     

Fibrinogen alters mouse brain endothelial cell layer integrity affecting vascular endothelial cadherin

Many inflammatory diseases are associated with elevated blood concentration of fibrinogen (Fg) leading to vascular dysfunction. We showed that pathologically high (4 mg/ml) content of Fg disrupts integrity of endothelial cell (EC) layer and causes macromolecular leakage affecting tight junction proteins. However, role of adherence junction proteins, particularly vascular endothelial cadherin (VE-cadherin) and matrix metalloproteinase-9 (MMP-9) in this process is not clear. We tested the hypothesis that at high levels Fg affects integrity of mouse brain endothelial cell (MBEC) monolayer through activation of MMP-9 and downregulation of VE-cadherin expression and in part its translocation to the cytosol.The effect of Fg on cultured MBEC layer integrity was assessed by measuring transendothelial electrical resistance. Cellular expression and translocation of VE-cadherin were assessed by Western blot and immunohistochemical analyses, (respectively). Our results suggest that high content of Fg decreased VE-cadherin expression at protein and mRNA levels. Fg induced translocation of VE-cadherin to cytosol, which led to disruption of cell-to-cell interaction and cell to subendothelial matrix attachment. Fg-induced alterations in cell layer integrity and their attachment were diminished during inhibition of MMP-9 activity.Thus Fg compromises EC layer integrity causing downregulation and translocation of VE-cadherin and through MMP-9 activation. These results suggest that increased level of Fg could play a significant role in vascular dysfunction and remodeling.     

Ascorbic acid stimulates barrier function of cultured endothelial cell monolayer

The macromolecular permeability of cultured bovine aortic, bovine venous, and human umbilical vein endothelial cell monolayers was decreased significantly in culture medium containing L-ascorbic acid (Asc Acid; 0.01–0.1 mM) and L-ascorbic acid 2-phosphate (Asc 2-P). Dithiothreitol, which shows reducing activity equivalent to that of Asc Acid, did not affect endothelial permeability. Asc Acid induced a sixfold increase in collagen synthesis by the endothelial cells. The coexistence of L-azetidine 2-carboxylic acid, an inhibitor of collagen synthesis, attenuated the effect of Asc 2-P in a dose-dependent manner. Another collagen synthesis inhibitor, ethyl-3,4-dihydroxybenzoate, also inhibited collagen synthesis and increased endothelial permeability. The decrease in permeability of the endothelial monolayer was dependent on a reduction of the permeability coefficient of the endothelial monolayer. These findings indicate that endothelial barrier function is stimulated by Asc Acid via an increase in collagen synthesis. © 1995 Wiley-Liss, Inc.     

The effect of selenium on cell proliferation in liver and colon

Epidemiologic and experimental evidence support a chemoprotective role for selenium (Se) in malignancy. Many mechanisms have been proposed to explain this phenomenon. In this study, the effect of Se intake on proliferation of hepatocytes and normal colonic epithelial cells in rats was determined using autoradiographic analysis of thymidine incorporation into DNA. Hepatocyte proliferation was measured 24 h after partial hepatectomy. Selenium-dosed animals demonstrated a significant reduction in hepatocyte labeling compared to the control group (6.1±2.6 vs 29.2±15.6,p=0.003). However, Se dosing did not affect the thymidine-labeling indices or distribution of labeling in colonic epithelium. Selenium may inhibit cell proliferation when it is the result of an unusually intense stimulus. This finding could explain in part the inhibitory effect of Se in some experimental cancer models. Dr. Tempero is a recipient of a Junior Clinical Faculty Fellowship from the American Cancer Society.     

The effect of human follicular fluid on endothelial cells: proliferation and DNA synthesis

Human follicular fluid has been reported to cause angiogenesis. Although endothelial cell mitogenesis is a major component of the process of angiogenesis, the findings in the literature regarding the effects of human follicular fluid in in vitro endothelial cell growth assays are equivocal. In the present study, we examined the effect of human follicular fluid from preovulatory follicles on fetal bovine aortic endothelial cell proliferation. Human serum was used as a control since follicular fluid is largely a transudate of serum and could contain serum-derived endothelial cell mitogens. Neither human follicular fluid nor serum directly caused endothelial cell proliferation. However, follicular fluid, as well as serum, caused an increase in thymidine incorporation by endothelial cells, and resulted in an increased proportion of cells in the DNA synthesis and G2 phases of the cell cycle. Although follicular fluid was not directly mitogenic, it, in contrast to human serum, together with fetal bovine serum markedly enhanced endothelial cell proliferation beyond that caused by fetal bovine serum alone. These results suggest that a combination of factors, some of ovarian origin present in follicular fluid, and others from as yet unidentified sources, govern the mitogenic component of new blood vessel growth in the ovary.     

Inhibition of endothelial cell proliferation by gamma-interferon

Endothelial cell growth factor (ECGF) is a potent polypeptide mitogen for endothelial cells and fibroblasts. The mitogenic effects of ECGF are inhibited by the lymphokine gamma-interferon (gamma-IFN) in a dose- dependent manner. Gamma-IFN also induces a unique change in endothelial cell morphology which is maximally expressed in the presence of ECGF. The antiproliferative and phenotypic modulatory effects of gamma-IFN on endothelial cells are reversible. Inhibition of ECGF-induced endothelial cell proliferation by gamma-IFN is accompanied by a concentration- and time-dependent decrease in binding of 125I-ECGF to the endothelial cell surface. Scatchard analyses of the binding data in the presence and absence of gamma-IFN demonstrate a decrease in the number of ECGF-binding sites rather than a decrease in ligand affinity for the receptor. Cross-linking experiments with disuccinimidyl suberate demonstrate a decrease in the 170,000 Mr cross-linked receptor- ligand complex. These data suggest that gamma-IFN inhibits endothelial cell proliferation by a mechanism which involves growth factor receptor modulation.