Zahra Ziaie

Lithium chloride restores host protein synthesis in herpes simplex virus-infected endothelial cells

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.

Oncothanin, a Peptide from the α 3 Chain of Type IV Collagen, Modifies Endothelial Cell Function and Inhibits Angiogenesis

Previous reports from our laboratory have shown that basement membrane (BM) collagen from anterior lens capsule type IV collagen (ALC-COL IV) and peptides from the noncollagenous domain of the α 3(IV) 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/αv β3 integrin receptor complex. Data presented here demonstrate that the α 3(IV)185-203 and the α 3(IV)179-208 peptides, designated as oncothanin, regulate endothelial cell (EC) proliferation, adhesion, and motility that 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, and the chorioallantoic membrane assay demonstrate that oncothanin, but not the control medium or peptides, inhibits angiogenesis. In the EC differentiation assay, oncothanin completely inhibited tube formation at 25 μ g/ml, whereas peptides with comparable sequences, lacking the -SNS- sequence, from ALC-COL IV NC1 domains α 1 and α 2 chains failed to inhibit tube formation. The data support the hypothesis that ALC-COL IV and oncothanin inhibit angiogenesis by modulation of EC function.

Suppression of Matrix Protein Synthesis by Herpes Simplex Virus Type 1 in Human Endothelial Cells

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.

A peptide of the α3(IV) chain of type IV collagen modulates stimulated neutrophil function via activation of cAMP-dependent protein kinase and Ser/Thr protein phosphatase

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.

Differential suppression of host cell protein synthesis and mRNA levels in herpes simplex virus-infected endothelial cells.

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.

A Peptied From the NC1 Domain of the α3 Chain of Type IV Collagen Prevents Damage to Basement Membranes by PMN

During the process of acute inflammation, circulating polymorphonuclear leukocytes (PMN) transmigrate from the vascular lumen to the site of infection or injury. This process involves the interaction of PMN with endothelial cells (EC) and subsequent diapedesis of PMN through the subendothelial basement membrane (BM). Along the path of transmigration, PMN come across and interact with numerous macromolecules of BM. Extensive research has been carried out to understand how various components of the C M e.g. type IV Collagen (COL (IV)), laminin, entactin, and proteoglycans mediate the physiological function of PMN (Matzner, Bar-Ner, Yahalom, Ishai-Michaeli, Fuks, and Vlodavsky, 1985; Matzer, Vlodavsky, Michaeli, and Eldor, 1990; Pike, Wicha, Yoon, Mayo, and Boxer, 1989; Senior, Hinek, Griffin, Pipoly, Crouch, and Mecham, 1989; Senior, Gresham, Griffin, Brown, and Chung, 1992). Studies by Huber and Weiss (1989) suggest that the transmigrating PMN cause a transient focal disruption of the BM which allows PMN to traverse it. These disruptions are rapidly repaired by the overlying EC. In our laboratory, the focus has been on the role of COL (IV), a major component of the BMs, on PMN function.