HDAC inhibitor, MS-275, increases vascular permeability by suppressing Robo4 expression in endothelial cells

Figures & data

Figure 1. HDAC inhibitors targeting HDAC1-3 suppress Robo4 expression in endothelial cells

(A) HDAC inhibitors for various combinations of HDAC subclasses. (B) Effect of HDAC inhibitors on Robo4 expression. HUVECs were treated with the HDAC inhibitors and Robo4 expression was analyzed based on mRNA (n = 4) and protein levels (lower panel). (C) Robo4 expression in HVUECs treated with various concentrations of MS-275 (n = 4). Data are expressed as the mean ± s.e.m. Statistical significance was determined by Dunnett’s test (**p < .01; N.S., not significant).

Figure 2. Knockdown of HDAC3 downregulates Robo4 expression in endothelial cells

(A) Effect of knockdown of HDACs on Robo4 expression. HUVECs were transfected with the siRNAs against HDAC1 (A), HDAC2 (B), and HDAC3 (C), and the expression levels of HDACs and Robo4 were measured by quantitative PCR (n = 4). Data are expressed as the mean ± s.e.m. Statistical significance was determined by the unpaired t-test (**p < .01; N.S., not significant).

Figure 3. (a) Schematic illustration of the Robo4 promoter and binding sites of SP1 and GABP in the proximal region.^7,8 (b) Effect of MS-275 on Robo4 promoter activity. A reporter plasmid containing the 3 kb Robo4 promoter was transfected into HVUECs treated with or without 20 μM MS-275. Luciferase activity was measured (n = 4). (c, d) Expression of SP1 and GABP subunits in the HUVECs treated with MS-275 (c) or siRNA against HDAC3 (d) was measured by quantitative PCR. Data are expressed as the mean ± s.e.m. Statistical significance was determined by the unpaired t-test (*p < .05, **p < .01; N.S., not significant)

Figure 4. MS-275 enhances endothelial and vascular permeability

A) Vascular leakage in the lungs of mice treated with or without MS-275. Mice were administered MS-275 (30 mg/kg/day) or dimethyl sulfoxide (DMSO; Vehicle) by oral gavage for four consecutive days and intravenously injected with Evans blue dye. The extravasated dye was quantified (n = 3). (B) Robo4 expression in MS1 cells treated with or without MS-275 (n = 4). (C) Effect of MS-275 on endothelial permeability. MS1 cells were treated with MS-275 or DMSO and transendothelial electrical resistance (TEER) was measured (n = 3). (D) Effect of MS-275 on the viability of endothelial cells. WST-8 assays were performed using MS1 cells with or without MS-275 treatment (n = 6). Data are expressed as the mean ± s.e.m. Statistical significance was determined by the unpaired t-test or Bonferroni correction (*p < .05, **p < .01).

Figure 5. MS-275 promotes the transmigration of cells through the vasculature and endothelial monolayer

A) Effect of MS-275 on the extravasation of B16-F10 cells in the experimental metastasis model. A schedule for the treatment of MS-275 and injection of B16-F10 cells and for colony counting is indicated. Pictures are representative images of the lungs obtained from mice treated with the vehicle and MS-275. The graph indicates the number of colonies in the lungs from mice treated with the vehicle or MS-275 (n = 5). (B) Effect of MS-275 on the transmigration of B16-F10 cells through the MS1 cell monolayer. B16-F10 cells that transmigrated through the MS1 monolayer treated with or without MS-275 were quantified (n = 4). Data are expressed as the mean ± s.e.m. Statistical significance was determined by the unpaired t-test (**p < .01).

Figure 6. Downregulation of Robo4 promotes vascular permeability and cell transmigration through the vasculature and endothelial monolayer

A Quantification of the number of colonies in the lungs of wild-type (Robo4^+/+) and Robo4-deficient (Robo4^+/− and Robo4^−/−) mice intravenously injected with B16-F10 cells (n = 5). Pictures are representative images of lungs from the wild-type and Robo4-deficient mice. (B) Vascular leakage in the lungs of wild-type or Robo4 knockout mice injected with B16-F10 cells (n = 5). (C) Effect of the knockdown of Robo4 on the transendothelial migration of tumor cells. Migration of B16-F10 cells through MS1 cells transfected with siRNA specific for Robo4 was quantified (n = 6). (D) Effect of the knockdown of Robo4 on endothelial cell viability. WST-8 assays were performed using MS1 cells transfected with siRNA (n = 6). Data are expressed as the mean ± s.e.m. Statistical significance was determined by Dunnett’s test or the unpaired t-test (*p < .05, **p < .01; N.S., not significant).

Okada Y, Yano K, Jin E, Funahashi N, Kitayama M, Doi T, Spokes K, Beeler DL, Shih S-C, Okada H, et al. A three-kilobase fragment of the human Robo4 promoter directs cell type-specific expression in endothelium. Circ Res. 2007;100(12):1712–1722. doi:https://doi.org/10.1161/01.RES.0000269779.10644.dc.

 PubMed Web of Science ®Google Scholar

Okada Y, Jin E, Nikolova-Krstevski V, Yano K, Liu J, Beeler D, Spokes K, Kitayama M, Funahashi N, Doi T, et al. A GABP-binding element in the Robo4 promoter is necessary for endothelial expression in vivo. Blood. 2008;112(6):2336–2339. doi:https://doi.org/10.1182/blood-2008-01-135079.

 PubMed Web of Science ®Google Scholar