Endothelial cell (EC) proliferation is a major component of angiogenesis in glioblastoma multiforme (GBM) and is regulated by several growth factors including IL-3. In this study, we have evaluated the effect of conditioned media (CM) obtained from MMP-2 knockdown xenograft 4910 and 5310 cell lines by pMMP-2 siRNA (pM.si) on the growth of ECs. Western blot analysis of CM from pM.si-treated 4910 and 5310 cells showed a significant downregulation of MMP-2 and IL-3 expression compared to mock- and pSV (scrambled vector)-CM. Proliferation decreased in ECs cultured on pM.si-CM from the xenograft cells when compared to ECs cultured on mock- or pSV-CM as determined by Ki-67 staining and MTT assays. Since Stat5 is a known target of IL-3, induces EC proliferation, and promotes neo-angiogenesis, we examined the role of pM.si in regulating IL-3-mediated Jak2/Stat5 signaling in ECs. Jak2/Stat5 suppression in ECs when cultured on pM.si-CM resulted in the decreased expression of integrin β1, Stat5, Bcl2 and Cyclin D2 at the transcription level as compared with ECs cultured on mock- and pSV-CM, as confirmed by RT-PCR analysis. Supplementation of recombinant human IL-3 counteracted and noticeably reversed pM.si-inhibited IL-3Rα, Jak2, p-Stat5 expression and proliferation of ECs, thereby confirming the role of MMP-2 in the regulation of α5β1/IL-3 mediated Jak2/Stat5 signaling. Immunoprecipitation and immunofluorescence studies revealed MMP-2 knockdown resulted in the decreased interaction of MMP-2 and integrin α5β1 in ECs, thereby implicating the downregulation of Jak2/Stat5 expression. Immunohistochemical analysis in tumor sections corroborated our in vitro studies, suggesting that the expression of p-Stat5 and Bcl2 are significantly decreased upon pM.si treatment compared to mock or pSV treatments. Our results suggest that MMP-2 knockdown by pM.si can lead to a significant inhibition of IL-3-induced proliferation of ECs and can be an effective anti-angiogenic therapy when treating patients with GBM in the future.
Citation Format: Dilip Rajasekhar Maddirela, Divya Kesanakurti, Chandramu Chetty, Jasti S. Rao. MMP-2 suppression inhibits α5β1 integrin and IL-3-mediated Stat5 activation and proliferation of endothelial cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5100. doi:10.1158/1538-7445.AM2013-5100