Suppression of thrombospondin 1 and 2 production by herpes simplex virus 1 infection in cultured keratocytes.
OBJECTIVE
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.
Choudhary
Anshoo
A
Unit of Ophthalmology, Department of Medicine, UCD, University of Liverpool, Liverpool, United Kingdom. a.choudhary@liverpool.ac.uk
Hiscott
Paul
P
Hart
Charles A
CA
Kaye
Stephen B
SB
Batterbury
Mark
M
Grierson
Ian
I
D015153:Blotting, Western; D002478:Cells, Cultured; D003319:Corneal Stroma / Q000166:cytology*; D015536:Down-Regulation; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts / Q000378:metabolism* / Q000821:virology*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism; D018259:Herpesvirus 1, Human / Q000502:physiology*; D006801:Humans; D007124:Immunoenzyme Techniques; D019700:Thrombospondin 1 / Q000378:metabolism*; D019699:Thrombospondins / Q000378:metabolism*; D014759:Viral Envelope Proteins / Q000378:metabolism; D000099262:Thrombospondin 2
D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D019700:Thrombospondin 1; D019699:Thrombospondins; D014759:Viral Envelope Proteins; D000099262:Thrombospondin 2; C039447:glycoprotein D, Human herpesvirus 1; C038535:glycoprotein gC, herpes simplex virus type 1