Programmed cell death occurs in
ischemia when cell surface
death receptors (DRs) are stimulated by death-inducing
ligands (DILs).
Matrix metalloproteinases are extracellular matrix-degrading
enzymes involved in the shedding of DRs and DILs from the cell surface.
Tissue inhibitor of metalloproteinase-3 (TIMP-3), which is bound to the extracellular matrix, has been shown to promote apoptosis in
cancer cell lines by inhibiting cell surface sheddases. Since apoptosis is an important mechanism of cell death in
ischemia, the authors hypothesized that
TIMP-3 would be expressed in ischemic neurons that are undergoing programmed cell death. Spontaneously hypertensive rats had a 90-minute
middle cerebral artery occlusion with reperfusion. Transcription of
TIMP-3 mRNA was measured by quantitative reverse transcription-polymerase chain reaction at 2, 6, 24 and 48 hours after reperfusion. Western blots were used to measure
TIMP-3 protein expression. Spatial distribution and production of
TIMP-3 was studied by immunohistochemistry at 3, 24, and 48 hours, 5 days, and 3 weeks. DNA fragmentation in cells dying by
necrosis and apoptosis was identified with
terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate-biotin nick end labeling (TUNEL). After 2 hours of reperfusion,
TIMP-3 mRNA increased significantly in both ischemic and nonischemic hemispheres. Western blot analysis confirmed the identity of the
TIMP-3, which appeared to be increased on the ischemic side. After 3 hours of reperfusion,
TIMP-3 immunostaining was increased in neurons on the ischemic side, and by 24 hours the majority of the ischemic neurons were TIMP-3-positive. Dual-fluorescence staining for TUNEL and
TIMP-3 showed that they were coexpressed in many neurons. The results suggest that ischemic neurons express
TIMP-3, which may be inhibiting sheddases. The authors propose that
TIMP-3 facilitates cell death in ischemic neurons. Further studies are needed to identify the sheddases inhibited by the
TIMP-3, and on the effect of inhibition of
matrix metalloproteinases on cell death mechanisms.