Salvatore V. Pizzo

Endothelial cell surface F1-FO ATP synthase is active in ATP synthesis and is inhibited by angiostatin

Angiostatin blocks tumor angiogenesis in vivo, almost certainly through its demonstrated ability to block endothelial cell migration and proliferation. Although the mechanism of angiostatin action remains unknown, identification of F1-FO ATP synthase as the major angiostatin-binding site on the endothelial cell surface suggests that ATP metabolism may play a role in the angiostatin response. Previous studies noting the presence of F1 ATP synthase subunits on endothelial cells and certain cancer cells did not determine whether this enzyme was functional in ATP synthesis. We now demonstrate that all components of the F1 ATP synthase catalytic core are present on the endothelial cell surface, where they colocalize into discrete punctate structures. The surface-associated enzyme is active in ATP synthesis as shown by dual-label TLC and bioluminescence assays. Both ATP synthase and ATPase activities of the enzyme are inhibited by angiostatin as well as by antibodies directed against the α- and β-subunits of ATP synthase in cell-based and biochemical assays. Our data suggest that angiostatin inhibits vascularization by suppression of endothelial-surface ATP metabolism, which, in turn, may regulate vascular physiology by established mechanisms. We now have shown that antibodies directed against subunits of ATP synthase exhibit endothelial cell-inhibitory activities comparable to that of angiostatin, indicating that these antibodies function as angiostatin mimetics.

Mast cell–derived tumor necrosis factor induces hypertrophy of draining lymph nodes during infection

Palpable swelling of regional lymph nodes is a common sequela of microbial infections but the mechanism responsible for the sequestration and subsequent coordination of lymphocyte responses within these dynamic structures remains poorly understood. Here we show that draining lymph nodes of mast cell–deficient mice did not demonstrate swelling after intradermal bacterial challenge. Testing of individual mast cell–derived products in this model indicated that tumor necrosis factor was the main mediator of nodal hypertrophy, whereas tryptase and histamine had no effect. After peripheral mast cell activation, both tumor necrosis factor concentrations and the recruitment of circulating T cells were increased within draining nodes. These results show a critical function for peripheral mast cell–derived tumor necrosis factor in regulating the hypertrophy of draining lymph nodes during infection.

Scavenger receptor-A mediates gp96/GRP94 and calreticulin internalization by antigen-presenting cells

gp96 (GRP94) elicits antigen‐presenting cell (APC) activation and can direct peptides into the cross‐ presentation pathways of APC. These responses arise through interactions of gp96 with Toll‐like (APC activation) and endocytic (cross‐presentation) receptors of APC. Previously, CD91, the α2‐macroglobulin receptor, was identified as the heat shock/chaperone protein receptor of APC. Recent data indicates, however, that inhibition of CD91 ligand binding does not alter gp96 recognition and uptake. Furthermore, CD91 expression is not itself sufficient for gp96 binding and internalization. We now report that scavenger receptor class‐A (SR‐A), a prominent scavenger receptor of macrophages and dendritic cells, serves a primary role in gp96 and calreticulin recognition and internalization. gp96 internalization and peptide re‐presentation are inhibited by the SR‐A inhibitory ligand fucoidin, although fucoidin was without effect on α2‐macroglobulin binding or uptake. Ectopic expression of SR‐A in HEK 293 cells yielded gp96 recognition and uptake activity. In addition, macrophages derived from SR‐A−/− mice were substantially impaired in gp96 binding and uptake. These data identify new roles for SR‐A in the regulation of cellular responses to heat shock proteins.

Activation and Cross-talk between Akt, NF-κB, and Unfolded Protein Response Signaling in 1-LN Prostate Cancer Cells Consequent to Ligation of Cell Surface-associated GRP78

Binding of activated forms of the proteinase inhibitor α2-macroglobulin (α2M*) to cell surface-associated GRP78 on 1-LN human prostate cancer cells causes their proliferation. We have now examined the interplay between Akt activation, regulation of apoptosis, the unfolded protein response, and activation of NF-κBin α2M*-induced proliferation of 1-LN cells. Exposure of cells to α2M* (50 pm) induced phosphatidylinositol 3-kinase-dependent activation of Akt by phosphorylation at Thr-308 and Ser-473 with a concomitant 60-80% increase in Akt-associated kinase activity. ERK1/2 and p38 MAPK were also activated, but there was only a marginal effect on JNK activation. Treatment of 1-LN cells with α2M* down-regulated apoptosis and promoted NF-κB activation as shown by increases of Bcl-2, p-BadSer-136, p-FOXO1Ser-253, p-GSK3βSer-9, XIAP, NF-κB, cyclin D1, GADD45β, p-ASK1Ser-83, and TRAF2 in a time of incubation-dependent manner. α2M* treatment of 1-LN cells, however, showed no increase in the activation of caspase -3, -9, or -12. Under these conditions, we observed increased unfolded protein response signaling as evidenced by elevated levels of GRP78, IRE1α, XBP-1, ATF4, ATF6, p-PERK, p-eIF2α, and GADD34 and reduced levels of GADD153. Silencing of GRP78 gene expression by RNAi suppressed activation of AktThr-308, AktSer-473, and IκB kinase α kinase. The effects of α2M* on the NF-κB activation, antiapoptotic signaling, unfolded protein response signaling, and proapoptotic signaling were also reversed by this treatment. In conclusion, α2M* promotes cellular proliferation of 1-LN prostate cancer cells by activating MAPK and Akt-dependent signaling, down-regulating apoptotic signaling, and activating unfolded protein response signaling.

Downregulation of Microglial Activation by Apolipoprotein E and ApoE-Mimetic Peptides

Apolipoprotein E plays an important role in recovery from acute brain injury and risk of developing Alzheimers disease. We demonstrate that biologically relevant concentrations of apoE suppress microglial activation and release of TNFalpha and NO in a dose-dependent fashion. Peptides derived from the apoE receptor-binding region mimic the effects of the intact protein, whereas deletion of apoE residues 146-149 abolishes peptide bioactivity. These results are consistent with the hypothesis that apoE modulates microglial function by binding specific cell surface receptors and that the immunomodulatory effects of apoE in the central nervous system may account for its role in acute and chronic neurological disease.

Mast cell activators: a new class of highly effective vaccine adjuvants

Mast cells (MCs) have recently received recognition as prominent effectors in the regulation of immune cell migration to draining lymph nodes and lymphocyte activation. However, their role in the development of humoral immune responses is not clear. Here, we demonstrate that subcutaneous or nasal administration of small-molecule MC activators with vaccine antigens evokes large increases in antigen-specific serum immunoglobulin G (IgG) responses. These responses were MC dependent and correlated with increased dendritic cell and lymphocyte recruitment to draining lymph nodes. Nasal instillation of these formulations also evoked antigen-specific secretory IgA and provided protection against anthrax lethal toxin challenge in vitro and against vaccinia virus infection in vivo. Collectively, these results define the MC as an integral sensory arm of the adaptive immune system. Moreover, they highlight MC activators as a new class of vaccine adjuvants, capable of inducing protective antigen-specific immune responses through needle-free routes of administration.

Physical properties of human α2-macroglobulin following reaction with methylamine and trypsin

Abstract Circular dichroism spectroscopy, sedimentation velocity and ultraviolet difference spectroscopy were used to compare α 2 -macroglobulin, α 2 -macroglobulin-trypsin complex and α 2 -macroglobulin-methylamine complex. The circular dichroic spectrum of native α 2 -macroglobulin is significantly changed in shape and magnitude following reaction with either trypsin or methylamine. The spectra of α 2 -macroglobulin-trypsin and α 2 -macroglobulin-methylamine are, however, indistinguishable. The ultraviolet difference spectrum between α 2 -macroglobulin-methylamine and native α 2 -macroglobulin displays a tyrosine blue shift consistent with the exposure of several tyrosine residues to solvent. The conformational change which occurs in α 2 -macroglobulin during reaction with methylamine follows pseudo-first-order kinetics. T 1/2 was 10.5 min for the reaction with 200 mM methylamine at pH 8.0 and 45 min for the reaction with 50 mM methylamine, also at pH 8.0. Reaction of methylamine with α 2 -macroglobulin results in loss of trypsin-binding activity which appears to be a direct consequence of the conformational change induced by methylamine. A sedimentation coefficient (s o 20 ,w) of 20.5 was determined for α 2 -macroglobulin-methylamine compared to a value of 18.5 for unreacted α 2 -macrogiobulin. This increase in sedimentation velocity is attributed to a 10% decrease in α 2 -macroglobulin Stokes radius, α 2 -Macroglobulin-trypsin complex prepared by reaction of the protease ata 2-fold molar excess with the inhibitor has a s o 20 ,w of 203. Although this sedimentation coefficient does reflect compacting of the α 2 -macroglobulin structure compared to native α 2 -macroglobulin, it is not large enough to rule out significant protrusion of the proteases from within pockets in the α 2 -macroglobulin structure.

The effect of fibrin-stabilizing factor on the subunit structure of human fibrin

The formation of human fibrin from fibrinogen has been examined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate, a method which separates a mixture of proteins on the basis of differences in molecular weight. It has been found that the plasma from a patient with a congenital deficiency of fibrin-stabilizing factor forms clots lacking the cross links among the alpha- and gammachains found in normal, cross-linked human fibrin. The addition of purified fibrin-stabilizing factor or normal plasma to the deficient plasma results in extensive cross-linking of the chains. Thus, the fibrinogen in the fibrin-stabilizing factor deficient plasma appears to be normal and forms fibrin which contains dimeric, cross-linked gamma-chains and polymeric, high molecular weight forms of alpha-chains. By the use of these electrophoretic methods, it has also been possible to develop a highly sensitive method for measuring the content of fibrin-stabilizing factor in plasma. This method depends upon the use of urea-treated fibrinogen, which is completely devoid of fibrin-stabilizing factor, but which forms the usual cross-linked subunits after conversion to fibrin by thrombin in the presence of fibrin-stabilizing factor.

Physical Conditioning Augments the Fibrinolytic Response to Venous Occlusion in Healthy Adults

The effects of a 10-week physical-conditioning program on fibrinolytic activity at rest and after stimulation by venous occlusion were studied in 69 healthy adults 25 to 69 years old. Physical conditioning was documented by treadmill performance, and fibrinolysis was measured with a newly developed radioenzymatic assay. Whereas fibrinolysis declined at rest from 16.2 +/- 1.3 to 11.4 +/- 0.8 units (mean +/- S.E.M.) (P = 0.0017), the increment in fibrinolysis produced by venous occlusion was increased from 21.7 +/- 2.9 to 33.8 +/- 4.7 units (P = 0.0037). This augmentation was most marked in women, persons with low initial levels of stimulated fibrinolysis, and persons with low initial physical fitness. We conclude that physical conditioning can enhance the augmentation of fibrinolytic activity that occurs in response to venous occlusion. Enhanced fibrinolysis in response to thrombotic stimuli could be an important mechanism in the beneficial effect of habitual physical activity on the risk of cardiovascular disease.

Demonstration and characterization of specific binding sites for factor VIII/von Willebrand factor on human platelets.

The presence of specific Factor VIII/von Willebrand factor (FVIII/vWF) binding sites on human platelets has been demonstrated by using 125I-FVIII/vWF and washed human platelets. Binding is ristocetin-dependent and increases in proportion to the concentration of ristocetin from 0.2 to 1 mg/ml. Binding of 125I-FVIII/vWF to platelets can be competitively inhibited by unlabeled human or bovine FVIII/vWF, but not by human thrombin, fibrinogen, alpha 2-macroglobulin, equine collagen, or a lectin of Ricinus communis. Scatchard analysis of binding data indicated that the dissociation constant of FVIII/vWF receptors is 0.45--0.5 nM. There are 31,000 binding sites per platelet at 1 mg/ml of ristocetin concentration. The optimal pH range for binding is from 7.0 to 7.5. At a concentration of 2 mM, EGTA inhibits 86% of the binding; however, 20 mM of Ca++, Mg++, or EDTA have little effect. Binding sites for FVIII/vWF were found only on platelets, and no significant binding was detected with human erythrocytes or polymorphonuclear leukocytes.