FGF-2 and VEGF are potent angiogenesis inducers in vivo and in vitro. Here we show that FGF-2 induces VEGF expression in vascular endothelial cells through autocrine and paracrine mechanisms. Addition of recombinant FGF-2 to cultured endothelial cells or upregulation of endogenous FGF-2 results in increased VEGF expression. Neutralizing monoclonal antibody to VEGF inhibits FGF-2–induced endothelial cell proliferation. Endogenous 18-kD FGF-2 production upregulates VEGF expression through extracellular interaction with cell membrane receptors; high-M r FGF-2 (22–24-kD) acts via intracellular mechanism(s). During angiogenesis induced by FGF-2 in the mouse cornea, the endothelial cells of forming capillaries express VEGF mRNA and protein. Systemic administration of neutralizing VEGF antibody dramatically reduces FGF-2-induced angiogenesis. Because occasional fibroblasts or other cell types present in the corneal stroma show no significant expression of VEGF mRNA, these findings demonstrate that endothelial cell-derived VEGF is an important autocrine mediator of FGF-2-induced angiogenesis. Thus, angiogenesis in vivo can be modulated by a novel mechanism that involves the autocrine action of vascular endothelial cell-derived FGF-2 and VEGF.

An epithelial cell line (MDCK) was used to prepare monolayers which, in vitro, develop properties of transporting epithelia. Monolayers were formed by plating cells at high densities (106 cells/cm") on collagen-coated nylon cloth disks to saturate the area available for attachment, thus avoiding the need for cell division.An electrical resistance developed within 4-6 h after plating and achieved a steady-state value of 104 _ 1.8 l).cm z after 24 h. Mature monolayers were morphologically and functionally polarized. They contained junctional complexes composed of desmosomes and tight junctions with properties similar to those of "leaky" epithelia. Monolayers were capable of maintaining a spontaneous electrical potential sensitive to amiloride, produced a net water flux from the apical to basal side, and discriminated between Na § and CI-ions.The MDCK permeability barrier behaves as a "thin" membrane with negatively charged sites. It has: (a) a linear conductance/concentration relationship; (b) an asymmetric instantaneous current/voltage relationship; (c) a reduced ability to discriminate between Na § and C1-caused by lowering the pH; and (d) a characteristic pattern of ionic selectivity which suggests that the negatively charged sites are highly hydrated and of medium field strength.Measurements of Na § permeability by electrical and tracer methods ruled out exchange diffusion as a mechanism for ion permeation and the lack of current saturation in the I/A~ curves does not support the involvement of carriers. The discrimination between Na § and CI-was severely but reversibly decreased at low pH. suggesting that Na § channels which exclude C1-contain acidic groups dissociated at neutral pH.Bound Ca § ions are involved in maintaining the integrity of the junctions in MDCK monolayers as was shown by a reversible drop of resistance and opening of the junctions in Ca++-free medium containing EGTA.Several other epithelial cell lines are capable of developing a significant resistance under the conditions used to obtain MDCK monolayers.J. CELL BIOLOGY 9 The Rockefeller University Press 9

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Monoclonal antibodies were prepared to anti-HLA-DR cytolytic T lymphocytes (CTLs) and screened for inhibition of CTL-mediated killing. Binding of monoclonal antibodies to four types of molecules, LFA-1, LFA-2, LFA-3, and HLA-DR, inhibited killing, suggesting that these molecules participate in the CTL-target cell interaction. The antigens were characterized by immunoprecipitation, crosslinking, NaDodSO4/polyacrylamide gel electrophoresis, and immunofluorescence flow cytometry. The LFA-1 antigen contains a and ,B polypeptide chains of Mr 177,000 and 95,000 that are noncovalently associated in an alp, structure. It is present on both B and T lymphocytes and marks subpopulations that differ in quantitative expression. Human LFA-1 appears to be the homologue of mouse LFA-1. Human LFA-2 is of Mr 49,000 with a minor component of Mr 36,000. It is expressed on CTL lines but not on a B-cell line and in peripheral blood preferentially on T lymphocytes. Human LFA-3 is of Mr 60,000 and is expressed on both B and T lymphocytes.Antigen-specific T-lymphocyte-mediated killing is a multistep process involving antigen recognition and adhesion of the cytolytic T lymphocyte (CTL) to the target cell, delivery of the lethal hit, and target cell lysis (1)(2)(3). Bystander cells and the CTL itself are unharmed in the killing reaction, and the CTL can detach and engage in further killing encounters. The molecular basis ofCTL-mediated killing is ofconsiderable interest, both in itself and for our understanding of T lymphocyte-mediated immunity in general. In previous studies designed to identify the molecules involved in mouse CTL-mediated killing, rat monoclonal antibodies (MAb) were prepared to mouse CTLs and screened for their ability to inhibit killing (4)(5)(6)(7)(8)(9). MAb to the Lyt-2,3 and lymphocyte function-associated 1 (LFA-1) antigens, but not MAb to many other types ofsurface structures, were found to inhibit killing. The LFA-1 antigen appears generally important in mouse T-lymphocyte immune interactions, since MAb to it block not only allogeneic and xenogeneic CTLmediated killing but also T-helper-cell antigen-specific proliferative responses and T-cell-dependent B-cell responses (4). Anti-LFA-1 MAb exert their effect on killing by binding to the CTL rather than to the target cell and block the formation of CTL-target conjugates and the Mg2+-dependent adhesion step but not the Ca2+-dependent lethal hit stage (4, 7). The mouse LFA-1 antigen contains two noncovalently associated polypeptide chains of Mr 180,000 and 95,000 (10, 11). No human homologue of mouse LFA-1 has been defined, but is the human homologue of Lyt-2,3 (12).We have probed the molecular mechanisms of human T-lymphocyte-mediated killing by preparing MAb to human anti- (14,15) are associated with CTL-mediated killing; however, none of these antigens has thus far been shown to be the T-cell antigen receptor. Direct screening for MAb blocking killing should be a more efficient method ofidentifying the antigen receptor and other molecules involved in C...

With a technique of preselecting the mitotic cell in the living state for subsequent electron microscopy, it has been possible to examine the ultrastructure of the various stages of mitosis with greater precision than has been reported previously. The early dissolution of the nuclear envelope has been found to be preceded by a marked undulation of this structure within the nuclear "hof." This undulation appears to be intimately related to the spindle-forming activity of the centriole at this time. Marked pericentriolar osmiophilia and extensive arrays of vesicles are also prominent at this stage, the former continuing into anaphase. Progression of the cell through prophase is accompanied by a disappearance of these vesicles. A complex that first makes its appearance in prophase but becomes most prominent in metaphase is a partially membrane-bounded cluster of dense osmiophilic bodies. These clusters which have a circumferential distribution in the mitotic cell are shown to be derived from multivesicular bodies and are acid phosphatase-positive. The precise selection of cells during the various stages of anaphase has made it possible to follow chronologically the morphological features of the initiation of nuclear membrane reformation. The nuclear membrane appears to be derived from polar aggregates of endoplasmic reticulum, and the process begins less than 2 minutes after the onset of karyokinesis. While formation of the nuclear envelope is initiated on the polar aspects of the chromatin mass, envelope elements appear on the equatorial aspect long before the polar elements fuse. Apparently interfering with this fusion are continuous spindle tubules which traverse the chromatin mass in striking density at characteristic points. Several cortical changes, also most pronounced in anaphase, have been described, as has the kinetochore which is seen to good advantage only in this stage. The Golgi complex has been found to disappear both morphologically and histochemically during mitosis and to reappear rapidly in telophase. Evidence is presented which implicates the continuous spindle tubules in certain phases of chromosome movement.