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Previous studies from our group and the group of the Department of Biochemistry at the University of Rheims, France [corrected] have shown that basement membrane (BM) collagen from anterior lens capsule type IV collagen (ALC-COL IV) and peptides from the noncollagenous domain (NC1) of the alpha3(IV) [corrected] chain, corresponding to residues 185-203 and 179-208, inhibit tumor cell proliferation, specifically through the interaction of the -SNS- tripeptide (residues 189-191) with the CD47/alphavbeta3 integrin receptor complex. Data presented here demonstrate that the alpha3(IV)185-203 and the alpha3(IV)179-208 peptides, from here forward [corrected] designated as oncothanin, regulate endothelial cell (EC) proliferation, adhesion, and motility which [corrected] ultimately influence angiogenesis. The data also indicate that oncothanin, when used as a chemoattractant, greatly enhanced EC chemotaxis. In contrast, pretreatment of EC with oncothanin inhibited chemotaxis toward several different chemoattractants. When oncothanin was used as a substrate, it enhanced EC adhesion that was inhibited when pretreated with same. Analysis of angiogenesis by EC differentiation (tube formation), aortic ring microvessel formation [corrected] and the chorioallantoic membrane assay, [corrected] demonstrate that oncothanin, but not the control medium or peptides, inhibits angiogenesis. In the EC differentiation assay, oncothanin completely inhibited tube formation at 25 microg/ml, whereas peptides with comparable sequences, that lacked [corrected] the -SNS- sequence, from ALC-COL IV NC1 domains alpha1 and alpha2 chains failed to inhibit tube formation. The data support the hypothesis that ALC-COL IV and oncothanin inhibit angiogenesis by modulation of EC function.

Previous reports from our laboratories showed that type IV collagen from anterior lens capsule (ALC) inhibited stimulated neutrophil function. This property was shown to reside in the region comprising residues 185-203 of the non-collagenous domain (NC1) of the alpha 3(IV) chain. We also reported that ALC-type IV collagen or the synthetic alpha 3(IV) 185-203 peptide, induced a rise in intracellular cAMP which persisted for up to 60 minutes. In the present work we extend our previous studies on signal transduction by alpha 3(IV) 185-203 and we provide new data showing the involvement of cAMP-dependent PKA and protein phosphatases. The data also show that the alpha 3(IV) peptide triggered a rise in intracellular calcium that was dependent on phospholipase C activation. Inhibitors of the Ca(2+)/calmodulin system suppressed both the alpha 3(IV) 185-203 peptide-induced cAMP increase and the inhibitory activity of the peptide on f-Met-Leu-Phe triggered O(2)(-) generation. When alpha 3(IV) 185-203 peptide-induced calcium mobilization was blocked by U-73122, an inhibitor of phospholipase C activation, or by BAPTA/AM, a chelator of intracellular calcium, the inhibitory effect of the peptide on PMA-triggered O(2)(-) production was also abolished. These findings provide evidence that signal transduction by the alpha 3(IV) peptide occurs via pathways which involve calcium. Indeed, the cAMP increase was shown to be mediated by adenosine and adenosine A2 receptors and required calcium elevation, since adenosine deaminase, theophilline, dimethylpropargylxanthine, trifluoperazine or autocamtide-2 related inhibitory peptide, suppressed the activity of the alpha 3(IV) peptide. The inhibitory effect of the peptide on f-Met-Leu-Phe-induced O(2)(-) generation was slightly affected by 1 microM KT5720 or H89, two inhibitors of cAMP-dependent PKA, but was completely suppressed by 10 nM calyculin A or 10 microM okadaic acid, two inhibitors of ser/thr phosphatases. These results suggest that Ser/Thr protein phosphatases and/or cAMP-dependent PKA are involved in signal transduction by the alpha 3(IV) 185-203 peptide and is consistent with the concept that adenosine receptor occupancy modulates neutrophil function.

Previous studies from our laboratories demonstrated that a peptide from the noncollagenous domain of the alpha3 chain of basement membrane collagen (COL IV), comprising residues 185-203, inhibits polymorphonuclear leukocyte activation and melanoma cell proliferation independently of its ability to promote cell adhesion; these properties require the presence of the triplet -SNS- at residues 189-191 (J. C. Monboisse et al., J. Biol. Chem., 269: 25475-25482, 1994; J. Han et al., J. Biol. Chem., 272: 20395-20401, 1997). More recently, we demonstrated that native COL IV and -SNS-containing synthetic peptides (10 microg/ml) added to culture medium inhibit the proliferation of not only melanoma cells but also breast, pancreas, and stomach tumor cells up to 82% and prostate tumor cells by 15%. This inhibition was shown to be dependent on a COL IV- or peptide-induced increase in intracellular cAMP (T. A. Shahan et al., Connect. Tissue Res., 40: 221-232, 1999). Attempts to identify the putative receptor(s) on tumor cells led to the isolation of five proteins (Mr 33,000, 52,000, 72,000, 95,000, and 250,000) from melanoma and prostate cells by affinity purification with the alpha3(IV)179-208 peptide. The Mr 52,000, 95,000, and 250,000 proteins were shown to be CD47/integrin-associated protein(IAP), the integrin beta3 subunit, and the alpha(v)beta3 integrin complex, respectively. The Mr 33,000 and 72,000 proteins have not yet been identified. To confirm the specificity of ligand binding to the receptors, cell membranes from either melanoma or prostate tumor cells were pretreated with the unlabeled ligand alpha3(IV)187-191 (-YYSNS-); alternatively, the peptide was pretreated with a peptide-reactive monoclonal antibody (A5D7) before receptor isolation. These treatments inhibited the purification of CD47/IAP, the integrin beta3 subunit, and the alpha(v)beta3 integrin complex from tumor cells. Furthermore, cells treated with CD47/IAP- or the alpha(v)beta3 integrin-reactive antibodies prevented the alpha3(IV)185-203 peptide from inhibiting cell proliferation and the subsequent rise in intracellular cAMP. Pretreating cells with the alpha3(IV)187-191 (-YYSNS-) peptide also inhibited their adhesion to the alpha3(IV)185-203 peptide substrate, whereas the inactive alpha1(IV)185-203 peptide, from the same region of the alpha1 chain as the alpha3(IV)185-203 peptide, had no effect. Incubation of cells with either CD47/IAP and/or alpha(v)beta3 integrin-reactive antibodies inhibited their adhesion to the alpha3(IV)185-203 peptide, whereas antibodies to the beta1 and beta2 integrin subunits were without effect. These data suggest that ALC-COL IV, through its alpha3(IV) chain, inhibits tumor cell proliferation using the receptors CD47/IAP and the alpha(v)beta3 integrin.

We have shown that basement membrane (BM) collagen (type IV), and specifically the peptide CNYYSNSYSFWLASLNPER (a.a. 185-203), from the non-collagenous domain of the alpha3 chain inhibits PMN. We examined the role of this peptide on PMN damage to BM in a vessel wall model. The presence of the endothelial monolayer as well as treatment of PMN with the alpha3(IV) 185-203 peptide reduced damage to BM by non-activated but not by activated PMN. The damage inhibition is unique to the alpha3(IV) peptide and not exhibited by comparable alpha1(IV) and alpha2(IV) chain peptides. A shorter peptide alpha3(IV) 185-191, containing the -SNS- triplet, reduced damage, whereas the one lacking the triplet, residues 194-203, was not effective. The CD47-alphavbeta3 integrin complex is the receptor for the alpha3(IV) peptide. Incubation of PMN with CD47 reactive mAb followed by the alpha3(IV) peptide abolished its protective effect on BM damage., (Copyright 1999 Academic Press.)

Previously we had shown that basement membrane collagen (COL IV), and specifically residues 185-203 of the non-collagenous domain of the alpha3(IV) chain, inhibits PMN activation. Since acetylcholinesterase (AchE) possesses collagenous and non-collagenous domains, we tested its effect on PMN activation. Whole AchE and the AchE recombinant catalytic subunit inhibited PMN superoxide anion (O2-) generation. AchE synthetic peptides, residues 139-154 and 252-266 of the catalytic subunit, with sequence homology to that of the alpha3(IV) peptide also inhibited O2- production by PMN. Reactive pAb and mAb to the alpha3(IV) 185-203 peptide abolished the inhibitory effect of the AchE. The data show that the non-collagenous domain of the AchE down-regulates O2- production by PMN. We suggest that this inhibitory activity may serve as a protective mechanism against PMN-mediated injury at the level of vessel wall and the neuromuscular junction.

Background: Patients treated with lithium salts for manic depression had a lower incidence of herpes simplex infections. Initial studies in our laboratory demonstrated that addition of LiCl in cultures of human endothelial cells infected with herpes simplex virus suppressed viral replication and allowed synthesis of host proteins., Experimental Design: Based on the above observations, we decided to study the optimal condition for the lithium effect and determine the process of inhibition of viral replication. Endothelial cell cultures infected with herpes simplex virus-1 were exposed to LiCl at various times postinfection. The levels of host and viral mRNAs were measured by Northern and slot blot hybridization. The pattern of protein synthesis was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot and replication was assessed by plaque assay., Results: LiCl inhibited virus replication in a dose- and time-dependent manner as was reflected in the sharp decrease or absence of infectious virus production. The condition for optimal effects of LiCl were the addition of the salt between 0-3 hours postinfection, and at a concentration of 30 mM. LiCl suppressed the synthesis of viral polypeptides, whereas the synthesis of host proteins was maintained. Similar results were observed with phosphonoacetic acid, an inhibitor of viral DNA polymerase. NaCl, at the same concentration as LiCl, did not prevent the virus-induced inhibition of host cell protein synthesis. The level of host mRNA for fibronectin, thrombospondin, collagen type IV, actin, and plasminogen activator inhibitor-1 were maintained in the presence of LiCl. mRNAs for viral proteins, ICP-4 and DNA polymerase were nearly undetectable when LiCl was added with the virus (0 time postinfection)., Conclusions: The data indicate that LiCl treatment results in suppression of herpes virus mRNAs, i.e., mRNAs for ICP-4 and DNA polymerase, thereby inhibiting replication. On the other hand, the levels of host mRNAs are maintained to varying degrees depending on the message. The data suggest that a very early step in the process of viral replication is affected by LiCl, since the drug is maximally effective when added with the virus.

Earlier studies from this laboratory have shown that infection of vascular cells with herpes simplex virus 1 or 2 (HSV-1, HSV-2) results in the differential suppression of extracellular matrix proteins including fibronectin (FN), type IV collagen, thrombospondin (TSP) and Factor VIII von Willebrand protein. The present study was designed to determine whether a correlation exists between suppression of synthesis of specific proteins and their mRNA levels. We have measured the steady-state levels of mRNAs for several extracellular matrix proteins (type IV collagen, FN and TSP) and two intracellular proteins (actin and tubulin) in human endothelial cells (EC) following HSV-1 infection. The results show that during the first 5 h post-infection, when there is a rapid decrease in the synthesis of extracellular matrix proteins, the steady-state levels of the corresponding mRNAs remain relatively high, but progressively decline to levels of less than 20% by 13 h post-infection. These findings suggest that in the early hours post-infection there is an alteration in the translatability of the hybridizable message followed by degradation in the later hours.

The effects of herpes simplex virus type 1 (HSV-1) infection on proteoglycan synthesis by human endothelial cells were studied as a model of endothelial cell injury. Confluent cultures of early passage endothelial cells from human umbilical vein were infected with HSV-1 at multiplicities of infection from 0.001 to 1.0. HSV-1 infection produced a dose-dependent inhibition of total proteoglycan synthesis of up to 85%. Although there was a 2- to 3-fold increase in the quantity of virus necessary to cause 50% inhibition of heparan sulfate compared to chondroitin/dermatan sulfate proteoglycan, the inhibition was relatively parallel, even up to high virus doses. There was no inhibition of an undersulfated heparan sulfate proteoglycan that contained glycosaminoglycan chains shorter than the predominant species. The results indicate that HSV-1 infection of human endothelial cells produces complex effects on host-cell metabolism. The viral-induced changes in proteoglycan metabolism may influence cell-matrix interactions and lead to altered vessel wall function.

Herpes simplex virus type 1 produces an early decrease in protein synthesis in cultured smooth muscle cells isolated from human umbilical artery. To confirm that suppression of host protein synthesis occurs before virus protein expression, cultures were treated with actinomycin D (5 micrograms/ml) continuously from the beginning of infection. In the presence of actinomycin D, by 3.5 hours postinfection, virus-infected cultures showed much less incorporation of [35S]methionine into sodium dodecyl sulfate-polyacrylamide electrophoresis-separated products than did identically treated mock-infected cultures. In the absence of actinomycin D, there was incorporation of radiolabel into viral proteins as early as 4.5 hours postinfection. Hybridization of slot-blotted total RNA demonstrated that virus-infected cultures treated with and without actinomycin D showed a marked decrease in hybridizable RNA by 2 hours PI when compared with mock-infected controls. This decrease was seen with cDNA probes for the mRNA for secreted extracellular matrix macromolecules (fibronectin, thrombospondin and collagen chains alpha 2(I), alpha 1(III] as well as with cDNA probes for RNA of intracellular macromolecules actin and beta-tubulin. In vitro translation of total RNA isolated from infected human smooth muscle cells at 2 and 5 hours postinfection resulted in far less [35S]methionine incorporation into sodium dodecyl sulfate-polyacrylamide electrophoresis-separated products than would be predicted from the amount of hybridizable RNA available. The results suggest that host message is rapidly rendered nonfunctional as well as degraded by means of a virion-competent mechanism(s).

In previous studies we have shown that herpes simplex virus type 1 (HSV-1) infection suppresses host-cell protein synthesis in human endothelial cells (EC). It has been demonstrated that lithium salts prevent viral replication in HSV-1 infected cells. In the present study, we have measured host-cell protein synthesis in HSV-1 infected EC in the presence or absence of 20 and 30 mM LiCl. Although LiCl restored synthesis of almost all host-cell proteins, [35S]methionine incorporation was most pronounced in thrombospondin and plasminogen activator inhibitor 1 and least in fibronectin and type IV collagen. LiCl was more effective at the higher concentration (30 mM) and when the compound was added to the EC culture at the time of infection rather than after adsorption of HSV-1. Synthesis of virus proteins continued in LiCl-treated EC but at a reduced rate. The data suggest that LiCl not only interferes with virus replication, but may also, to some extent, interfere with the virion-associated inhibition of host protein synthesis.

The synthesis of matrix proteins was investigated in cultures of human umbilical vein endothelial cells (EC) infected with herpes simplex virus type 1 (HSV-1). EC cultures were either mock-infected or infected for 1 hour at a multiplicity of infection (MOI) of 5 or 20 infectious particles per cell. Synthesis was followed by determining [14C]-proline or [35S]-methionine incorporation into non-dialyzable proteins. Using SDS-PAGE we observed that synthesis of fibronectin (FN), type IV procollagen and thrombospondin (TSP) was inhibited in infected cultures as early as 2 hours becoming almost complete by 15 hours post-infection. The degree of inhibition of matrix protein synthesis was dependent on the dose of the virus inoculum (MOI 20 greater than MOI 5) and varied with the particular matrix protein, i.e. shut-off of type IV collagen occurred first followed by that of FN and then TSP. Pulse-chase experiments suggest that the absence of labeled matrix protein in the medium of infected cultures is not due to accumulation of protein within the infected cells since there was an equal and parallel reduction in the cell layer. Suppression of matrix proteins in infected cultures was confirmed by measuring the level of FN and TSP in uninfected and infected cultures in an enzyme-linked immunosorbent assay (ELISA). The three proteins were identified by ELISA, electroimmunoblot, immunoprecipitation and ion-exchange chromatography. The data suggest that HSV-1 infection of human EC suppresses matrix protein synthesis; the degree and time of complete shut-off varies with the protein and the virus dose.

The gene sequence for cytochrome oxidase subunit I (COI) in the ciliate Tetrahymena mitochondrial DNA has been determined and shown to be coded by the same strand as codes the genes (in order) for 14S rRNA, tRNA(trp), tRNA(glu), 21S rRNA, tRNA(leu) and tRNA(met). The predicted protein has 698 amino acids, including an NH2-terminal 57 amino acid extension and a 108 amino acid insert originally found in Paramecium COI. These extension and insert segments are not highly hydrophobic but are relatively rich in lysine, arginine and serine. In analogy with the presequence of nuclear-encoded mitochondrial proteins, they might function as a transmembrane signal. The remaining polypeptide segments show a hydrophobicity characteristic of membrane spanning proteins. TCOI shows a 64% amino acid identity with Paramecium COI but less than a 38% amino acid conservation with human COI. The Tetrahymena mitochondrial code is analogous with the mammalian mitochondrial code; but differs from the Tetrahymena nuclear genetic code; TGA is exclusively translated as tryptophan; ATA is used as an initiation codon probably for methionine, and TAA as a stop codon; the arginine codons (CGN) are not used. The use of the leucine codon TTA in TCOI is contradictory to the codon recognition pattern previously obtained from the isolated tRNA(leu) isoacceptors recognizing only the CUN codons, but consistent with the tRNA(leu) (anticodon UAA) gene encoded in the genome. The reason for this inconsistency has not been resolved.

Protein synthesis was determined in cultures of human umbilical cord vein endothelial cells (EC) infected with either herpes simplex type 1 (HSV-1) or type 2 (HSV-2). Monolayers were infected for 1 hour with either 5 or 20 infectious virus particles per EC (multiplicity of infection of 5 or 20). At different times after infection, infected and noninfected cultures were pulsed with either 5 mu Ci/ml of [14C]proline or 25 mu Ci/ml of [35S]methionine for 1 or 2 hours. Autoradiograms of sodium dodecyl sulfate-gel electrophoresis showed suppression of matrix protein synthesis by 4 hours postinfection which became almost complete at 6 hours. Multiplicity of infection of 20 was more effective than multiplicity of infection of 5 at suppressing matrix protein synthesis at 4 or 6 hours postinfection. Infection of EC with ultraviolet light-inactivated virus resulted in the shutoff of host-cell as well as virus protein synthesis. Similar results were observed when monolayers of EC were infected with intact virus in the presence of 2 micrograms/ml of actinomycin-D, an inhibitor of RNA synthesis. On the other hand, uninfected EC in the presence of actinomycin-D continued to synthesize protein from pre-existing mRNA. The time of shutoff of synthesis of specific matrix proteins varied with the protein i.e., shut-off of type IV collagen occurred first, followed by that of fibronectin, and then of thrombospondin. The data suggest that the degree of suppression of synthesis of EC matrix proteins after infection with HSV-1 or HSV-2 is dependent on the dose of the virus inoculum; it occurs at the translational level and is not dependent on new viral protein synthesis.