Herpes Simplex Virus–1 Infection Selectively Suppresses Expression of Thrombospondin 1 and 2 in Human Keratocytes

Abstract

Abstract: : Purpose: Stromal vascularization is a frequent occurrence in Herpes simplex keratitis (HSK) and carries a poor prognosis for penetrating keratoplasty. The pathogenesis may involve disruption of the normal equilibrium between angiogenic and anti–angiogenic factors in and around the cornea. Thrombospondin (TSP) 1 and 2 are multifunctional matricellular glycoproteins with potent anti–angiogenic properties and are expressed by human keratocytes in a stromal wound repair model. We hypothesise that the synthesis of these anti–angiogenic proteins by keratocytes is inhibited by Herpes simplex virus–1 (HSV–1) and that such a mechanism may contribute to stromal vascularization in HSK. Methods: Non–confluent monolayers of human keratocytes were infected with HSV–1 at a multiplicity of infection of 5 plaque forming units / keratocyte. Expression of TSP 1 and 2 was determined by immunohistochemistry and SDS–polyacrylamide gel electrophoresis at 0, 2, 4, 6, 8, 24, 48 and 72 hours post–infection (p.i). Expression of glyceraldehyde 3 phosphate dehydrogenase (GAPDH) served as a control. Expressions of immediate early and late viral proteins were also determined. Protein expressions were quantified by densitometric analysis of the immunoblot bands. Results: Human keratocytes supported the growth of HSV–1 at all times p.i. TSP 1 and 2 were down–regulated as early as 2 hours p.i to a 50% reduction by 8 hours (p< .001), and were absent from 24 hours p.i (p< .001). There was no change in the level of expression of GAPDH throughout the duration of the experiment. Immediate early viral proteins (HSV1 (ICP27)) could be detected from 6 hours p.i reaching maximum intensity 24 hours p.i and late proteins (HSV–1gD) were expressed from 24 – 72 hours. Conclusions: The synthesis of TSP 1 and 2 is selectively down–regulated by HSV–1 infection in human keratocytes. Addition of these proteins or their angio–active peptides in early stage HSK therapy may be an important adjuvant in controlling HSV–1 induced corneal vascularization.

Keywords: cornea: stroma and keratocytes • herpes simplex virus • neovascularization