Suppression of thrombospondin 1 and 2 production by herpes simplex virus 1 infection in cultured keratocytes

Mol Vis. 2005 Mar 2;11:163-8.

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 hypothesize that the synthesis of these anti-angiogenic proteins by keratocytes is inhibited by HSV1 and that such a mechanism may contribute to stromal vascularization in HSK.

Methods: Nonconfluent monolayers of human keratocytes were infected with HSV1 at a multiplicity of infection of 5 virus particles/keratocyte. Expression of TSP1 and TSP2 was determined by immunohistochemistry and SDS-polyacrylamide gel electrophoresis at 0, 2, 4, 6, 8, 24, 48, and 72 h after infection (ai). Expression of glyceraldehyde 3 phosphate dehydrogenase (GAPDH) served as a control. Expression of immediate early and late viral proteins was also determined. Protein expression was quantified by densitometric analysis of the immunoblot bands.

Results: Human keratocytes supported the growth of HSV1 at all times ai. TSP1 and TSP2 were downregulated as early as 4 h ai to a 50% reduction by 8 h (p<0.002), and were absent from 24 h ai (p<0.001). 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 h ai reaching maximum intensity 24 h ai and late proteins (HSV:1gD) were expressed from 24 h.

Conclusions: The synthesis of TSP1 and TSP2 is selectively downregulated by HSV1 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 HSV1 induced corneal vascularization.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Blotting, Western
  • Cells, Cultured
  • Corneal Stroma / cytology*
  • Down-Regulation
  • Electrophoresis, Polyacrylamide Gel
  • Fibroblasts / metabolism*
  • Fibroblasts / virology*
  • Glyceraldehyde-3-Phosphate Dehydrogenases / metabolism
  • Herpesvirus 1, Human / physiology*
  • Humans
  • Immunoenzyme Techniques
  • Thrombospondin 1 / metabolism*
  • Thrombospondins / metabolism*
  • Viral Envelope Proteins / metabolism

Substances

  • Thrombospondin 1
  • Thrombospondins
  • Viral Envelope Proteins
  • glycoprotein D, Human herpesvirus 1
  • glycoprotein gC, herpes simplex virus type 1
  • thrombospondin 2
  • Glyceraldehyde-3-Phosphate Dehydrogenases