Abstract
Abstract: :
Purpose: To investigate molecular angiogenic mechanisms of retinoblastoma disease progression. Herein we test the hypothesis that MMP–2, MMP–9, and VEGF receptor–2 (VEGFR2) expression are upregulated in murine transgenic retinoblastoma, and that treatment with the angiostatic cortisene Anecortave Acetate (AA, a potential novel drug in treatment of retinoblastoma) downregulates their expression. Methods: To evaluate MMP–2 and –9 activity, nine 10 week old LHßTag mice (three per group) were treated with a single subconjunctival injection to the right eye of 300µg/20µl AA. Right and left eyes were enucleated at 24h, 48h, and 1 week post injection for evaluation of MMP–2 and MMP–9 activity by zymography. Three 10 week old LHßTag mice were similarly treated with 600µg/20µl AA and eyes enucleated at 1 week post injection for zymography. Four vehicle–injected 10 week old LHßTag mice we harvested as controls (one for each timepoint and dose), and three vehicle–injected 10 week old control mice negative for the LHßTag construct were used as negative controls. To evaluate VEGFR2 expression, three LHßTag mice were treated with a single subconjunctival injection to the right eye of 600 µg/20µl AA, and both eyes were enucleated at 1 week post injection for immunofluorescence analysis of VEGFR2 expression. One LHßTag mouse received vehicle injection as a control. Results: Zymography demonstrates that MMP–9 activity is strongly upregulated and MMP–2 is weakly upregulated in LHßTag eyes. Moreover, 300µg AA–treated LHßTag mice had significantly upregulated MMP–9 activity in the injected eye (but not the fellow eye) at 24 and 48 h after injection of AA compared to vehicle–injected controls. This was followed by a strong down–regulation of MMP–9 in both eyes at 1 week compared to vehicle controls. Both MMP–2 and MMP–9 activity were lowered to background levels 1 week after injection of AA. A similar downregulation of MMP–9 was observed 1 week post injection in LHßTag mice treated with 600µg AA. Finally, VEGFR2 immunofluorescence was strongly upregulated in the ganglion cell layer, inner plexiform layer, outer plexiform layer, and within the body of the tumor in LHßTag eyes with large tumors. Weaker VEGFR2 expression was detected in eyes with smaller tumors and LHßTag eyes without tumors. Conclusions: In this study we show that two important pro–angiogenic molecules: VEGFR2 and MMP–9 are both strongly upregulated in murine transgenic retinoblastoma. Treatment with AA strongly modulates MMP–9 activity, possibly a major molecular basis for the tumor–controlling mechanism of AA.
Keywords: retinoblastoma • tumors