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Evidence for the Use of Multiple Mechanisms by Herpes Simplex Virus-1 R7020 to Inhibit Intimal Hyperplasia.

McCormick S, He Q, Stern J, Khodarev N, Weichselbaum R, Skelly CL - PLoS ONE (2015)

Bottom Line: The observed cytopathic effect, which included R7020 positive filopodia that extend from cell to cell and the formation of syncytia, suggests that R7020 remains cell associated after egress and spreads cell to cell instead of by diffusion through the extracellular fluid.Furthermore, R7020 inhibited the proliferative effect of TNF-α, an inflammatory cytokine associated with increased IH.Thus, the results of this study suggest that R7020 inhibits IH through multiple mechanisms.

View Article: PubMed Central - PubMed

Affiliation: Section of Vascular Surgery, Department of Surgery, University of Chicago, Chicago, Illinois, United States of America.

ABSTRACT
Intimal hyperplasia (IH) is the primary cause of vein bypass graft failure. The smooth muscle cell (SMC) is a key element of IH as it phenotypically switches from a contractile to a synthetic state which can become pathological. R7020, which is an engineered strain of Herpes Simplex Virus-1, inhibits IH in animal models. Although it has many characteristics which make it a strong candidate for use as a prophylactic agent how it inhibits IH is not well understood. The objective of this study was to identify modes of action used by R7020 to function in blood vessels that may also contribute to its inhibition of IH. The cytopathic effect of R7020 on SMCs was determined in vitro and in a rabbit IH model. In vitro assays with R7020 infected SMCs were used to quantify the effect of dose on the release kinetics of the virus as well as the effects of R7020 on cell viability and the adhesion of peripheral blood mononuclear cells (PBMCs) to SMCs in the absence and presence of tumor necrosis factor alpha (TNF-α). The observed cytopathic effect, which included R7020 positive filopodia that extend from cell to cell and the formation of syncytia, suggests that R7020 remains cell associated after egress and spreads cell to cell instead of by diffusion through the extracellular fluid. This would allow the virus to rapidly infect vascular cells while evading the immune system. The directionality of the filopodia in vivo suggests that the virus preferentially travels from the media towards the intima targeting SMCs that would lead to IH. The formation of syncytia would inhibit SMC proliferation as incorporated cells are not able to multiply. It was also observed that R7020 induced the fusion of PBMCs with syncytia suggesting the virus may limit the effect of macrophages on IH. Furthermore, R7020 inhibited the proliferative effect of TNF-α, an inflammatory cytokine associated with increased IH. Thus, the results of this study suggest that R7020 inhibits IH through multiple mechanisms.

No MeSH data available.


Related in: MedlinePlus

R7020 causes vascular SMCs to form branched protrusions in vitro.SMCS infected with R7020 (1 MOI) in the absence (R7020) and presence of TNF-α (R7020 + TNF-α) were cultured 24 h. DIC, US11 labeled and merged images are shown. The white arrows point to branch points in the protrusions. The black arrows point toward protrusions that did not stain positive for US11. The white arrow head points to a protrusion that extends from an infected cell for a short distance and then branches.
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pone.0130264.g002: R7020 causes vascular SMCs to form branched protrusions in vitro.SMCS infected with R7020 (1 MOI) in the absence (R7020) and presence of TNF-α (R7020 + TNF-α) were cultured 24 h. DIC, US11 labeled and merged images are shown. The white arrows point to branch points in the protrusions. The black arrows point toward protrusions that did not stain positive for US11. The white arrow head points to a protrusion that extends from an infected cell for a short distance and then branches.

Mentions: To determine the effect of R7020 on SMC morphology cells were infected with R7020 at 1 MOI. Viral infection was confirmed by staining the cells for HSV-1 infected cell protein 22 (ICP22) (Fig 1). ICP22 is a multifunctional protein that localizes to the nuclei of infected cells where it regulates several processes involved in HSV-1 replication including nuclear egress [27]. At 24 h p.i. the cells were undergoing morphological changes. The plasma membranes were contracting inward towards the nuclei the cells causing them to morph from their original elongated spindly shape to ones that were more ovular or circular. Syncytia were also present in the cultures at this point with cell fusion continuing to occur (Fig 1). Multiple filopodia were observed extending from cells and syncytia, some of which branched multiple times and connected with other cells and syncytia (Fig 2). We also examined the effect of R7020 on SMC morphology in the presence of TNF-α as it is produced by SMCs and immune cells at increased levels in diseased vessels [28–32]. To confirm that the cells were stimulated by TNF-α in the presence of R7020 the cells were stained for ICAM-1, a cell surface receptor known to be up regulated by TNF-α [33]. This receptor was expressed on the surfaces of R7020 infected and non-infected SMCs treated with TNF-α but not on the plasma membranes of cells cultured in the absence of TNF-α (Fig 1). TNF-α did not alter the effects of R7020 on SMC morphology for TNF-α stimulated cells also formed syncytia and filopodia (Figs 1 and 2).


Evidence for the Use of Multiple Mechanisms by Herpes Simplex Virus-1 R7020 to Inhibit Intimal Hyperplasia.

McCormick S, He Q, Stern J, Khodarev N, Weichselbaum R, Skelly CL - PLoS ONE (2015)

R7020 causes vascular SMCs to form branched protrusions in vitro.SMCS infected with R7020 (1 MOI) in the absence (R7020) and presence of TNF-α (R7020 + TNF-α) were cultured 24 h. DIC, US11 labeled and merged images are shown. The white arrows point to branch points in the protrusions. The black arrows point toward protrusions that did not stain positive for US11. The white arrow head points to a protrusion that extends from an infected cell for a short distance and then branches.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4488439&req=5

pone.0130264.g002: R7020 causes vascular SMCs to form branched protrusions in vitro.SMCS infected with R7020 (1 MOI) in the absence (R7020) and presence of TNF-α (R7020 + TNF-α) were cultured 24 h. DIC, US11 labeled and merged images are shown. The white arrows point to branch points in the protrusions. The black arrows point toward protrusions that did not stain positive for US11. The white arrow head points to a protrusion that extends from an infected cell for a short distance and then branches.
Mentions: To determine the effect of R7020 on SMC morphology cells were infected with R7020 at 1 MOI. Viral infection was confirmed by staining the cells for HSV-1 infected cell protein 22 (ICP22) (Fig 1). ICP22 is a multifunctional protein that localizes to the nuclei of infected cells where it regulates several processes involved in HSV-1 replication including nuclear egress [27]. At 24 h p.i. the cells were undergoing morphological changes. The plasma membranes were contracting inward towards the nuclei the cells causing them to morph from their original elongated spindly shape to ones that were more ovular or circular. Syncytia were also present in the cultures at this point with cell fusion continuing to occur (Fig 1). Multiple filopodia were observed extending from cells and syncytia, some of which branched multiple times and connected with other cells and syncytia (Fig 2). We also examined the effect of R7020 on SMC morphology in the presence of TNF-α as it is produced by SMCs and immune cells at increased levels in diseased vessels [28–32]. To confirm that the cells were stimulated by TNF-α in the presence of R7020 the cells were stained for ICAM-1, a cell surface receptor known to be up regulated by TNF-α [33]. This receptor was expressed on the surfaces of R7020 infected and non-infected SMCs treated with TNF-α but not on the plasma membranes of cells cultured in the absence of TNF-α (Fig 1). TNF-α did not alter the effects of R7020 on SMC morphology for TNF-α stimulated cells also formed syncytia and filopodia (Figs 1 and 2).

Bottom Line: The observed cytopathic effect, which included R7020 positive filopodia that extend from cell to cell and the formation of syncytia, suggests that R7020 remains cell associated after egress and spreads cell to cell instead of by diffusion through the extracellular fluid.Furthermore, R7020 inhibited the proliferative effect of TNF-α, an inflammatory cytokine associated with increased IH.Thus, the results of this study suggest that R7020 inhibits IH through multiple mechanisms.

View Article: PubMed Central - PubMed

Affiliation: Section of Vascular Surgery, Department of Surgery, University of Chicago, Chicago, Illinois, United States of America.

ABSTRACT
Intimal hyperplasia (IH) is the primary cause of vein bypass graft failure. The smooth muscle cell (SMC) is a key element of IH as it phenotypically switches from a contractile to a synthetic state which can become pathological. R7020, which is an engineered strain of Herpes Simplex Virus-1, inhibits IH in animal models. Although it has many characteristics which make it a strong candidate for use as a prophylactic agent how it inhibits IH is not well understood. The objective of this study was to identify modes of action used by R7020 to function in blood vessels that may also contribute to its inhibition of IH. The cytopathic effect of R7020 on SMCs was determined in vitro and in a rabbit IH model. In vitro assays with R7020 infected SMCs were used to quantify the effect of dose on the release kinetics of the virus as well as the effects of R7020 on cell viability and the adhesion of peripheral blood mononuclear cells (PBMCs) to SMCs in the absence and presence of tumor necrosis factor alpha (TNF-α). The observed cytopathic effect, which included R7020 positive filopodia that extend from cell to cell and the formation of syncytia, suggests that R7020 remains cell associated after egress and spreads cell to cell instead of by diffusion through the extracellular fluid. This would allow the virus to rapidly infect vascular cells while evading the immune system. The directionality of the filopodia in vivo suggests that the virus preferentially travels from the media towards the intima targeting SMCs that would lead to IH. The formation of syncytia would inhibit SMC proliferation as incorporated cells are not able to multiply. It was also observed that R7020 induced the fusion of PBMCs with syncytia suggesting the virus may limit the effect of macrophages on IH. Furthermore, R7020 inhibited the proliferative effect of TNF-α, an inflammatory cytokine associated with increased IH. Thus, the results of this study suggest that R7020 inhibits IH through multiple mechanisms.

No MeSH data available.


Related in: MedlinePlus