Abstract
Purpose::
To investigate the role of heat shock protein 70 (Hsp70) in cell death after photodynamic therapy (PDT) in a laser induced model of choroidal neovascularization (CNV).
Methods::
CNV was induced in the eyes of 31 adult Brown-Norway rats using 532nm laser (532nm, 150mW, 100µm, 100ms). Two weeks after laser injury, PDT (689nm, 600mW/cm2, 25J/cm2, verteporfin 6mg/m2) was performed. Expression of Hsps 25, 70 and 90 was examined by immunohistochemistry and RT-PCR in combination with laser-capture microdissection (LCM). To study the role of Hsp70 level, either quercetin (100mg/kg), an Hsp70 inhibitor, or vehicle was injected intraperitoneally 1 hour before PDT. Animals were sacrificed 6 hours or 7 days after PDT. Photoreceptor apoptosis was assessed by TUNEL. CNV was evaluated by fluorescein angiography and choroidal flatmount perfused with high molecular weight FITC.
Results::
Immunohistochemistry and RT-PCR with LCM showed that Hsp70 was increased after PDT in CNV, but not Hsp90. Hsp25 was shown to be increased only by RT-PCR with LCM, not by immunohistochemistry. Immunohistochemistry showed that Hsp70 was not colocalized with TUNEL (+) cells in the CNV lesions. Six hours after PDT, significantly more TUNEL (+) cells were found in the CNV lesions of quercetin-treated animals (3535±53/mm2, n=6) when compared to those of the vehicle-treated animals (2434±159/mm2, n=6, p<0.0001). One week after PDT, the quercetin-treated group had a higher rate of CNV closure (57%; 40/70 lesions in 6 rats) compared to the control group (10%; 5/48 lesions in 4 rats). The size of CNV was also smaller in the quercetin-treated group (0.013±0.003 mm2, n=6) measured on choroidal flatmount compared to the control group (0.021±0.002 mm2, n=5, p<0.05).
Conclusions::
Hsp70 plays an important role in the survival of cells in CNV after PDT. Hsp70 inhibition may be used as an adjunctive treatment with PDT for CNV.
Keywords: photodynamic therapy • choroid: neovascularization • apoptosis/cell death