Alexandra Lucas

Virus-Encoded Serine Proteinase Inhibitor SERP-1 Inhibits Atherosclerotic Plaque Development After Balloon Angioplasty

BACKGROUNDnRecurrent atherosclerotic plaque growth, restenosis, is a significant clinical problem after interventional procedures. Initiation of restenosis involves activation of inflammatory and thrombotic cascades, which are regulated by serine proteinase enzymes and inhibitors. We have investigated the use of a viral serine proteinase inhibitor, SERP-1, to reduce plaque development after primary balloon angioplasty. This is the first experimental report of the use of a viral anti-inflammatory protein for the prevention of atherosclerosis.nnnMETHODS AND RESULTSnSeventy-four cholesterol-fed rabbits were treated with either local or systemic infusions of SERP-1 protein (or control solutions) after balloon-mediated injury. Sites of SERP-1 infusion in rabbits had dramatically reduced plaque compared with control infusions at the 4-week follow-up. At low-dose infusions (30 to 300 pg), only the primary infusion site had a demonstrable decrease in plaque, whereas at higher-dose infusions (> 3000 pg), a generalized reduction in plaque development was detected. An associated decrease in mononuclear cell infiltration of the arterial wall was detected after SERP-1 infusion within the first 24 hours. Infusion of an active-site mutant of SERP-1 (P1-P1, ala-ala) lacking serine proteinase inhibitory activity failed to prevent plaque growth.nnnCONCLUSIONSnPurified SERP-1, a virus-encoded secreted glycoprotein, reduces plaque growth after primary balloon-mediated injury. Plaque development is decreased by inhibition of serine proteinase activity and is associated with a focal reduction in macrophage infiltration immediately after injury. Investigation of serine proteinase inhibitors may provide new insight into the regulation of arterial responses to injury.

Secreted Immunomodulatory Viral Proteins as Novel Biotherapeutics

Many viruses have learned to evade or subvert the host antiviral immune responses by encoding and expressing immunomodulatory proteins that protect the virus from attack by elements of the innate and acquired immune systems. Some of these viral anti-immune regulators are expressed as secreted proteins that engage specific host immune targets in the extracellular environment, where they exhibit potent anti-immune properties. We review here viral immunomodulatory proteins that have been tested as anti-inflammatory reagents in animal models of disease caused by excessive inflammation or hyperactivated immune pathways. The potential for such viral molecules for the development of novel drugs to treat immune-based or inflammatory disorders is discussed.

Myxoma Virus M141R Expresses a Viral CD200 (vOX-2) That Is Responsible for Down-Regulation of Macrophage and T-Cell Activation In Vivo

ABSTRACT M141R is a myxoma virus gene that encodes a cell surface protein with significant amino acid similarity to the family of cellular CD200 (OX-2) proteins implicated in the regulation of myeloid lineage cell activation. The creation of an M141R deletion mutant myxoma virus strain (vMyx141KO) and its subsequent infection of European rabbits demonstrated that M141R is required for the full development of a lethal infection in vivo but is not required for efficient virus replication in susceptible cell lines in vitro. Minor secondary sites of infection were detected in the majority of rabbits infected with the M141R deletion mutant, demonstrating that the M141R protein is not required for the dissemination of virus within the host. When compared to wild-type myxoma virus-infected rabbits, vMyx141KO-infected rabbits showed higher activation levels of both monocytes/macrophages and lymphocytes in situ through assessments of inducible nitric oxide synthase-positive and CD25+ infiltrating cells in infected and lymphoid tissues. Purified peripheral blood mononuclear cells from vMyx141KO-infected rabbits demonstrated an increased ability to express gamma interferon upon activation by phorbol myristate acetate plus ionomycin compared to cells purified from wild-type myxoma virus-infected rabbits. We concluded that the M141R protein is a bona fide CD200-like immunomodulator protein which is required for the full pathogenesis of myxoma virus in the European rabbit and that its loss from the virus results in increased activation levels of macrophages in infected lesions and draining lymph nodes as well as an increased activation level of circulating T lymphocytes during infection. We propose a model whereby M141R transmits inhibitory signals to tissue macrophages, and possibly resident CD200R+ dendritic cells, that reduce their ability to antigenically prime lymphocytes and possibly provides anergic signals to T cells directly.

In vivo and in vitro models of demyelinating diseases: Tropism of the JHM strain of murine hepatitis virus for cells of glial origin

n Abstractn n Infection of mice with the neurotropic JHM strain of murine hepatitis virus causes demyelinating lesions resulting from an infection of the oligodendroglia. This was most evident in mice inoculated intraperitoneally with JHM. Such CNS lesions were not observed in mice inoculated intraperitoneally with the MHV3 strain. An in vitro system is described in which the rat glial RN2 cell line functions as a discriminating host for the JHM virus. Shortly after inoculation, this virus establishes a persistent infection in which there is a cyclical rise and fall in titer with an accompanying cytopathology. Furthermore, this host cell confers a thermal lability which the virus does not demonstrate in the fully permissive host cell, L-2. By comparison, infection of RN2 cells with the prototype MHV3 is aborted immediately. In the persistent infection of RN2 cells with measles virus, Hallé strain, the cell again confers a temperature sensitivity which the virus does not possess when replicating in Vero cells.n This appears to be the first instance in which a cloned cell line of glial origin determines the outcome of the infectious process, discriminating in favor of a neurotropic variant which possesses a tropism for the glia in vivo. Systems such as the one described here may now offer a specific screening procedure for selecting, identifying and characterizing the nature of neurotropic viruses.n n

In vivo and in vitro models of demyelinating diseases II. Persistence and host-regulated thermosensitivity in cells of neural derivation infected with mouse hepatitis and measles viruses

n Abstractn n Following inoculation of continuous cell lines of neural and other derivations, persistent infections are established with facility by mouse hepatitis and measles viruses. This occurs equally with the prototype MHV3 and its neurotropic variant JHM as well as with the Edmonston vaccine and SSPE Hallé measles variants. In almost every instance that the infection becomes persistent at 32.5°, virus replication is found to be thermosensitive at 39.5°; however, progeny virus derived from such infections at 32.5° is itself thermostable when replicating in the indicator, fully permissive cell lines. The new data, therefore, reveal the existence of a host-conferred interrelationship between persistence and virus restriction at elevated temperature. They indicate that the two agents with neurotropic potential, when they become established as pathogens in the nervous system, could be under close host cell regulation involving as yet unknown mechanisms.n n

Serp-1, a viral anti-inflammatory serpin, regulates cellular serine proteinase and serpin responses to vascular injury.

Complex DNA viruses have tapped into cellular serpin responses that act as key regulatory steps in coagulation and inflammatory cascades. Serp-1 is one such viral serpin that effectively protects virus-infected tissues from host inflammatory responses. When given as purified protein, Serp-1 markedly inhibits vascular monocyte invasion and plaque growth in animal models. We have investigated mechanisms of viral serpin inhibition of vascular inflammatory responses. In vascular injury models, Serp-1 altered early cellular plasminogen activator (tissue plasminogen activator), inhibitor (PAI-1), and receptor (urokinase-type plasminogen activator) expression (p < 0.01). Serp-1, but not a reactive center loop mutant, up-regulated PAI-1 serpin expression in human endothelial cells. Treatment of endothelial cells with antibody to urokinase-type plasminogen activator and vitronectin blocked Serp-1-induced changes. Significantly, Serp-1 blocked intimal hyperplasia (p< 0.0001) after aortic allograft transplant (p < 0.0001) in PAI-1-deficient mice. Serp-1 also blocked plaque growth after aortic isograft transplant and after wire-induced injury (p < 0.05) in PAI-1-deficient mice indicating that increase in PAI-1 expression is not required for Serp-1 to block vasculopathy development. Serp-1 did not inhibit plaque growth in uPAR-deficient mice after aortic allograft transplant. We conclude that the poxviral serpin, Serp-1, attenuates vascular inflammatory responses to injury through a pathway mediated by native uPA receptors and vitronectin.

Transplant vasculopathy: viral anti-inflammatory serpin regulation of atherogenesis

BACKGROUNDnSurgical and ischemic injury to the artery wall initiates vascular wound-healing responses that stimulate atherosclerotic plaque growth. The plasminogen activators have cellular chemotactic, adhesion, and proteolytic activity. Serp-1 is a secreted myxoma virus glycoprotein serpin that binds and inhibits plasminogen activators. We have examined the effects of Serp-1 on plaque growth and inflammatory cell invasion in animal models after balloon injury and after aortic allograft transplant.nnnMETHODSnWe used histologic analysis to assess 4 animal models of angioplasty-mediated injury and 2 models of aortic allograft transplant for intimal hyperplasia and cellular invasion. We assessed plasminogen activator (uPA and tPA) and inhibitor (PAI-1) expression in rat iliofemoral arteries after balloon injury using Western blot, enzyme activity, and quantitative reverse transcriptase-polymerase chain reaction (RT-PCR).nnnRESULTSnPlaque growth after balloon injury decreased after Serp-1 treatment in all balloon-injury models tested. Transplant vasculopathy also significantly decreased in 2 rat models of aortic allograft transplant. Infusion of a Serp-1 active site mutant, that lacked plasminogen activator inhibiting activity, did not inhibit plaque growth. Quantitative RT-PCR detected increased transcription of PAI-1 mRNA. Increased PAI-1 protein and enzyme-inhibitory activity was also detected in Serp-1-treated arteries by activity assay and Western blot.nnnCONCLUSIONSnThrombolytic serpins are central regulatory agents in vascular wound-healing responses. Investigation of the inhibitory mechanisms of viral serpins may provide new insights into atherogenesis.

In Vivo Optical Analysis of Quantitative Changes in Collagen and Elastin During Arterial Remodeling

Abstract Altered collagen and elastin content correlates closely with remodeling of the arterial wall after injury. Optical analytical approaches have been shown to detect qualitative changes in plaque composition, but the capacity for detection of quantitative changes in arterial collagen and elastin content in vivo is not known. We have assessed fluorescence spectroscopy for detection of quantitative changes in arterial composition in situ, in rabbit models of angioplasty and stent implant. Fluorescence emission intensity (FEI) recorded at sites remote from the primary implant site was correlated with immunohistochemical (IH) analysis and extracted elastin and collagen. FEI was significantly decreased (P < 0.05) after treatment with anti-inflammatory agents, and plaque area decreased on comparison with saline-treated rabbits after stent implant or angioplasty (P ≤ 0.013). Excellent correlations for FEI with elastin and collagen I, III and IV content measured by IH (R2 ≥ 0.961) analysis were detected by multiple regression (MR) analysis. Good correlations also were found for FEI with elastin and collagen measured by high-performance liquid chromatography; MR analysis provided highly predictive values for collagen and elastin (R2 ≥ 0.994). Fluorescence spectroscopic analysis detects quantitative compositional changes in arterial connective tissue in vivo, demonstrating changes at sites remote from primary angioplasty and stent implant sites.

Optical detection of triggered atherosclerotic plaque disruption by fluorescence emission analysis.

Fluorescence emission analysis (FEA) has proven to be very sensitive for the detection of elastin, collagen and lipids, which are recognized as the major sources of autofluorescence in vascular tissues. FEA has also been reported to detect venous thromboemboli. In this paper we have tested the hypothesis that FEA can reproducibly detect in vivo and in vitro triggered plaque disruption and thrombosis in a rabbit model. Fluorescence emission (FE) spectra, recorded in vivo, detected Russells viper venom (RVV)–induced transformation of atherosclerotic plaque. FE intensity at 410–490 nm 4 weeks after angioplasty was significantly lower (P < 0.0033 by analysis of variance) in RVV‐treated rabbits when compared to control animals with stable plaque. FE spectral profile analyses also demonstrated a significant change in curve shape as demonstrated by polynomial regression analysis (R2 from 0.980 to 0.997). We have also demonstrated an excellent correlation between changes in FE intensity and the structural characteristics detected at different stages of “unstable atherosclerotic plaque” development using multiple regression analysis (R2= 0.989). Thus, FEA applied in vivo is a sensitive and highly informative diagnostic technique for detection of triggered atherosclerotic plaque disruption and related structural changes, associated with plaque transformation, in a rabbit model.

Fluorescence spectroscopy and birefringence of molecular changes in maturing rat tail tendon

Tissue remodeling during maturation, wound healing, and response to vascular stress involves molecular changes of collagen and elastin in the extracellular matrix (ECM). Two optical techniques are effective for investigating these changes--laser-induced fluorescence (LIF) spectroscopy and polarizing microscopy. LIF spectroscopy integrates the signal from both elastin and collagen cross-linked structure, whereas birefringence is a measure of only collagen. Our purpose is (1) to evaluate the rat tail tendon (RTT) spectroscopy against data from purified extracted protein standards and (2) to correlate the two optical techniques in the study of RTT and skin. Spectra from tissue samples from 27 male rats and from extracted elastin and collagen were obtained using LIF spectroscopy (357 nm). Birefringence was measured on 5-mum histological sections of the same tissue. Morphometric analysis reveals that elastin represents approximately 10% of tendon volume and contributes to RTT fluorescence. RTT maximum fluorescence emission intensity (FEI(max)), which includes collagen and elastin, increases with animal weight (R(2)=0.64). Birefringence, when plotted against weight, increases to a plateau (nonlinear correlation: R(2)=0.90), tendon having greater birefringence than skin. LIF spectroscopy and collagen fiber birefringence are shown to provide complementary measurements of molecular structure (tendon birefringence versus FEI(max) at R(2)=0.60).