Association of PDGF-BB-induced thrombomodulin with the regulation of inflammation in the corneal and scleral stroma.
OBJECTIVE
The responses of corneal and scleral stromal cells to platelet-derived growth factor (PDGF)-BB were assessed and inflammatory reactions in the cornea and sclera were investigated.


METHODS
Primary cultures of cells obtained from human subjects and strains derived from human corneal or scleral stromal cells (Cs3 and Sc1, respectively) were used. Changes in gene expression after 24 hours of exposure to 10 ng/mL PDGF-BB were analyzed with an Sc1 DNA microarray. The upregulation of several genes in Cs3 and Sc1 was confirmed by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis. The expression of bioactive factors was detected immunohistochemically in nine different clinical specimens.


RESULTS
DNA microarray analysis revealed that the gene encoding thrombomodulin (TM) was induced in Sc1 by PDGF-BB. RT-PCR confirmed that TM expression at the mRNA level was increased in both corneal and scleral stromal cells. At the protein level, TM expression was increased in scleral stromal cells, but not in corneal cells, and TM was detected in both the membrane and cytoplasmic compartments. TM was detected immunohistochemically in inflamed scleral and several corneal specimens. After TM stimulation, interleukin (IL)-18 transcription was increased in Sc1.


CONCLUSIONS
PDGF-BB induced TM mRNA expression in scleral and corneal stromal cells, but Western blot analysis revealed the increase in TM expression only in the scleral cells. TM induced IL-18 in scleral stromal cells. A cascade involving these biologically active factors may regulate scleral and corneal inflammation. The results also reveal differences in the biological response of scleral and corneal stromal cells.
Namba
Hiroyuki
H
Department of Ophthalmology and Visual Sciences, Faculty of Medicine, Yamagata University, Yamagata, Japan. nanbas2804@384.jp
Kashiwagi
Yoshiko
Y
Nishitsuka
Koichi
K
Takamura
Hiroshi
H
Yamamoto
Teiko
T
Yamashita
Hidetoshi
H
D000368:Aged; D000369:Aged, 80 and over; D043925:Angiogenesis Inducing Agents / Q000494:pharmacology; D000077214:Becaplermin; D015153:Blotting, Western; D002478:Cells, Cultured; D003315:Cornea / Q000187:drug effects* / Q000378:metabolism; D016207:Cytokines / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect; D005786:Gene Expression Regulation / Q000502:physiology*; D006801:Humans; D007124:Immunoenzyme Techniques; D007634:Keratitis / Q000235:genetics* / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D020411:Oligonucleotide Array Sequence Analysis; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology*; D020574:Proto-Oncogene Proteins c-sis; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012590:Sclera / Q000187:drug effects* / Q000378:metabolism; D015423:Scleritis / Q000235:genetics* / Q000378:metabolism; D018180:Thrombomodulin / Q000235:genetics* / Q000378:metabolism
D043925:Angiogenesis Inducing Agents; D016207:Cytokines; D010982:Platelet-Derived Growth Factor; D020574:Proto-Oncogene Proteins c-sis; D012333:RNA, Messenger; D011994:Recombinant Proteins; C546931:THBD protein, human; D018180:Thrombomodulin; D000077214:Becaplermin