MMP-2 suppression abrogates irradiation-induced microtubule formation in endothelial cells by inhibiting αvβ3-mediated SDF-1/CXCR4 signaling

Abstract

The majority of glioblastoma multiforme (GBM) tumors recur after radiation (IR) treatment due to increased angiogenesis and IR-induced signaling events in endothelial cells (ECs) that are involved in tumor neovascularization; however, these signaling events have yet to be well characterized. In the present study, we observed that IR (8 Gy) significantly elevated MMP-2 expression and gelatinolytic activity in 4910 and 5310 human GBM xenograft cells. In addition, ECs treated with tumor-conditioned media (CM) obtained from IR-treated 4910 and 5310 cells showed increased microtubule formation. In view of this finding, we investigated the possible anti-angiogenic effects of MMP-2 downregulation using siRNA (pM.si) in IR-treated cells. We also determined the effect of CM obtained from mock, pSV (scrambled vector) and pMMP-2.si on endothelial cell growth and vessel formation. pM.si-CM-treated ECs showed inhibited IR-CM-induced SDF-1, CXCR4, phospho-PI3K and phospho-AKT and αvβ3 expression levels. In vitro angiogenesis assays also showed that the pM.si+IR decreased IR-induced vessel formation in ECs. Immunofluorescence and immunoprecipitation experiments indicated the abrogation of αvβ3-SDF-1 interaction in pM.si-CM-treated ECs when compared to mock or pSV treatments. External supplementation of either rhMMP-2 or rhSDF-1 counteracted and noticeably reversed pM.si-inhibited SDF-1, CXCR4, phospho-PI3K and phospho-AKT expression levels and angiogenesis, thereby confirming the role of MMP-2 in the regulation of αvβ3-mediated SDF-1/CXCR4 signaling. In addition to the in vitro results, the in vivo mouse dorsal air sac model also showed reduced angiogenesis after injection of pM.si alone or in combination with IR-treated xenograft cells. In contrast, injection of mock or pSV-treated cells resulted in robust formation of characteristic neovascularization. Collectively, our data demonstrate the role of MMP-2 in the regulation of SDF-1/CXCR4 signaling-mediated angiogenesis in ECs and show the anti-angiogenic efficacy of combining MMP-2 downregulation and IR when treating patients with GBM in the future.

Figure 1

pM.Si-CM downregulates IR-induced angiogenesis in human xenograft cell lines 4910 and 5310. Cells (4910 and 5310) were treated with mock, pSV or pM.Si alone or in combination with IR (8 Gy) as described in Materials and methods. (A) In vitro angiogenesis. The conditioned media was added to 96-well plates that were coated with Matrigel and pre-seeded with human dermal microvascular endothelial cells (ECs) (2×10⁴ cells/well). After overnight incubation at 37°C, cells were observed under the bright field microscope for the formation of capillary-like structures. The degree of angiogenic induction by mock- and IR-CM were quantified for the numerical value of the product of the relative capillary length and number of branch points per field and indicated in a bar diagram. The data are presented as the mean ± SE of three independent replicates with significance denoted by ^*p<0.01. (B) Gelatin zymography (MMP-2) and western blot analysis (SDF-1) were performed. The experiments were carried out thrice and the data are presented as the mean ± SE of three independent replicates with significance denoted by ^*p<0.01. (C) Seventy-two hours post-transfection, xenograft cells were harvested and whole cell lysates were prepared using RIPA buffer. Whole cell lysates were subjected to western blotting for SDF-1, CXCR4, p-PI3K (Tyr 508), PI3K, AKT and p-AKT (Ser 473). GAPDH was used to confirm equal loading. The data are presented as the mean ± SE of three independent replicates with significance denoted by ^*p<0.01. (D) ECs were grown on the mock-, pSV- and pM.Si-CM for 16 h. Cells were then collected and whole cell lysates were subjected to western blotting for MMP-2, SDF-1, CXCR4, p-AKT (Ser 473), AKT, PI3K and integrin αvβ3. The blots were stripped and re-probed with GAPDH antibody as an internal control for the respective proteins. (E) In vitro angiogenesis was performed under similar conditions as described for (A). The degree of angiogenic induction was quantified for the numerical value of the product of the relative capillary length and number of branch points per field. The data are presented as the mean ± SE of three independent replicates with significance denoted by ^*p<0.05 and ^**p<0.01.

Figure 2

pM.Si-CM inhibits IR-induced PI3K/AKT expression and angiogenesis in ECs and supplementation of rhMMP-/rhSDF-1 reverses inhibition. (A) ECs were grown in mock-, pSV- and pM.Si-CM with or without IR. The whole cell lysates were subjected to western blotting to check the expression levels of AKT, p-AKT, PI3K, p-PI3K, integrin αv and integrin β3 using specific antibodies. The blot was restriped and GAPDH was used as a loading control. (B) In vitro angiogenesis was done with overnight incubation of ECs at 37°C with mock-, pSV-, IR (8 Gy)-CM or pM.Si alone or in combination with IR (8 Gy). The cells were observed under a bright field microscope for the formation of capillary-like structures. The degree of angiogenic induction was quantified for the relative capillary length and number of branch points per field. The data are presented as the mean ± SE of three independent replicates with significance denoted by ^*p<0.05 and ^**p<0.01. (C) ECs from 4910 and 5310 cells were grown in the presence of mock-, pSV- or pM.Si-CM with or without IR (8 Gy) for 16 h. pSV- and pM.Si-CM were treated with 25 ng/ml rhMMP-2. Whole cell lysates of ECs were prepared at the end of 16-h treatment and subjected to western blotting to check the expression levels of SDF-1, CXCR4 and MMP-2 using specific antibodies. The blot was restriped and GAPDH was used as a loading control. The data are presented as the mean ± SE of three independent replicates with significance denoted by ^*p<0.05 and ^**p<0.01. (D) In vitro angiogenesis was carried out under similar conditions as noted in Fig. 1A and is representative of at least three independent repetitions. RhSDF-1 (25 ng/ml) was added to pSV- and pM.Si-CM and incubated for 16 h. The degree of capillary network formation is indicated in a graph. The data are presented as the mean ± SE of three independent replicates with significance denoted by ^*p<0.05 and ^**p<0.01. (E) Endothelial cells were treated with pSV-CM or pM.Si-CM supplemented with rhSDF-1 (25 ng/ml) for 16 h. The whole cell lysates were subjected to western blot analysis for the expression of SDF-1, AKT and p-AKT with their respective antibodies and GAPDH was used as the loading control.

Figure 3

Knockdown of MMP-2 by pM.Si-CM in ECs inhibits IR-induced SDF-1 expression via integrin αvβ3. Knockdown of MMP-2 inhibits integrin αvβ3-mediated SDF-1 expression in ECs. (A) ECs were plated in 4-well chamber slides (4×10³ cells/well) and grown in mock-, pSV- or pM.Si-CM of both 4910 and 5310 cells either alone or combined with IR (8 Gy) for 16 h. Cells were fixed, permeabilized and incubated with antibodies specific for integrin αvβ3 and SDF-1 (1:100 dilution) for 2 h at room temperature followed by Alexa Fluor secondary antibodies for 1 h, stained with DAPI and mounted. Randomly selected microscopic fields of three independent experimental replicates are shown. (B) EC lysates were collected and lysed after 16 h of incubation in mock-, pSV- or integrin αvβ3 blocking Ab mock- or integrin αvβ3 blocking Ab pM.Si-CM from 4910 and 5310 cells. Western blot analysis performed for SDF-1 using a specific antibody. GAPDH was used as a loading control. (C) ECs were grown on pSV-, pM.-Si-CM, with or without IR (8 Gy) for 16 h. The ECs of whole cell lystates (200 μg) were immunoprecipitated with antibodies against NSP-IgG and integrin αvβ3 using μMACS protein G microbeads and MACS separation columns. Immunoprecipitate was subjected to western blotting using SDF-1 specific antibody.

Figure 4

MMP-2 knockdown abrogated SDF-1 expression in vivo. (A) Immunohistochemistry was performed for expression of SDF-1 using specific antibody. Data shown are representative fields (×40). Also shown is the negative control where the primary antibody was replaced by non-immune serum (inserts). (B) In vivo angiogenic assay was completed using the dorsal air sac model as described in Materials and methods. Briefly, the animals were implanted with diffusion chambers in a dorsal cavity containing mock, pSV or pM.Si-transfected 4910 and 5310 cells treated with or without IR (8 Gy). Ten days after implantation, the animals were sacrificed and the number of new blood vessels covering the diffusion chamber was observed under a bright field microscope for the presence of tumor-induced neovasculature and pre-existing vasculature. Implantation of a chamber containing mock, pSV or IR (8 Gy) in 4910 and 5310 cells resulted in the development of microvessels (arrows) with curved thin structures and many tiny bleeding spots. In contrast, implantation of pM.Si with or without IR (8 Gy) resulted in a decreased number of microvessels.