Angiogenic growth factor production is normal in response to hypoxia in PSMA null mice.

<p>PSMA expression was measured by qRT-PCR from RNA isolated from wild type and PSMA null retinas daily, beginning at the initiation of tissue hypoxia (P12 through P17). <b>A</b>) Wild type and PSMA mice produce similar VEGF expression levels during hypoxia treatment. <b>B</b>) PSMA null mice show similar Ang-2 levels to wild type mice over time. (n = 3 per time point).</p

Retinal vasculature develops normally in PSMA null mice.

<p>Retinas from wild-type (<b>left</b>) and PSMA-null (<b>right</b>) mice (5X) raised in room air, harvested at P17 and perfused with FITC labeled <i>ricin communis</i> agglutinin 1 (RCA-1-FITC, green). Insets: higher (40X) magnification of the same retina and show normal radial branching pattern in animals of both genotypes. (Representative image, n = 3 per group).</p

Perfusion of retinal vessels in PSMA null animals is increased.

<p>Following OIR, mice were perfused with RCA-1-FITC to detect perfused vessels immediately prior to sacrifice and harvest. Isolated retinas were then stained with Alexa 594 labeled <i>Griffonia simplicifolia</i> 1 isolectin B4 (GS Isolectin B4-A594) to stain all endothelial cells. <b>A</b>) RCA-1-FITC staining (<b>left, green</b>), GS Isolectin B4-A594 staining (<b>center, red</b>) and merged image (<b>right</b>) of representative wild type (<b>top</b>) and PSMA null (<b>bottom</b>) retinas. <b>B</b>) The double stained areas in the merged images (perfused vessels + endothelial cells) were pseudo-colored white to illustrate the area of non-perfused, GS Isolectin B4-A594 only staining endothelial cells (5X). <b>C</b>) Quantitative analysis of the relative extent of non-perfused endothelial cells per retina. (Representative image, n = 5 per group).</p

Vascular pathology is decreased in PSMA null animals undergoing OIR.

<p><b>A</b>) Retinas were harvested from OIR mice at P12 (<b>left</b>), P15 (<b>center</b>) and P17 (<b>right</b>). Vasculature was stained using RCA-1-FITC (green, P12) or GS Isolectin B4-A594 (red, P15 and P17) and <b>B</b>) the central avascular area of each retina was measured using Image J. (n = 3 per group).</p

Pathologic angiogenesis is reduced in retinas of PSMA null mice.

<p>Whole-mount retinas from mice undergoing OIR were harvested at P17 and perfused with RCA-1-FITC. Wild type retinas (<b>A</b>) show a higher degree of central avascular area than retinas from PSMA null mice (<b>B</b>). Higher magnification (40x) of <b>BC</b> wild-type and <b>D</b>) PSMA-null capillary networks at the outer edge of the retina: Retinas isolated from wild-type mice <b>C</b>) show disorganized, tortuous vessels and vascular tufts while vessels of PSMA-null retinas <b>D</b>) are less tortuous and more closely resemble normal organization. <b>E</b>) Quantification of avascular area using Image J showed wild-type mice had an average avascular area of 28.7%, compared to 18.4% in the retinas isolated from PSMA null mice, (n = 4 per group, p = 0.004). <b>F</b>) Wild type animals had an average of 8.8 vascular tufts per histologic section, compared to an average of 5.2 tufts per section in PSMA-null (n = 8 per group, p = 0.017).</p

Inhibition of PSMA decreases pathologic angiogenesis in a mouse model of OIR.

<p><b>A</b>) Wild type mice undergoing OIR were treated systemically with either a single dose of 100mg/kg 2-PMPA on P14 (n = 3, <b>center</b>) or three daily doses from P14 through P16 (n = 6, <b>right</b>), controls received vehicle (PBS, <b>left</b>). Retinas were isolated on P17, vasculature was stained using GS Isolectin B4–A594 and the avascular area calculated using Image J software. <b>B</b>) Mice receiving a single dose on P14 showed a slight but not statistically significant decrease in avascular area, whereas mice treated from P14–16 showed a significant decrease (23%) in avascular area compared to vehicle treated controls (p = 0.0055). <b>C</b>) Wild type mice on the OIR protocol were treated once on P14 with 10mg intravitreal 2-PMPA (10mg/ml, 1mL) in one eye and 1mL Vehicle (PBS) in the contralateral eye. Retinas treated with intravitreal 2-PMPA showed a significant decrease (16.66%) in avascular area compared to control retinas.</p

Retinal vasculature of wild type mice undergoing OIR expresses PSMA.

<p><b>A</b>) A time line depicting the stages of retinal layer vascularization with diagrams of approximate vascular morphology of the retina at various time points. <b>B</b>) Conventional RT-PCR of RNA isolated from wild-type OIR retinas from P17 mice are positive for PSMA; PSMA wild-type kidney and TRAMP-C1 cells- PSMA positive controls, PSMA null retina- negative control. <b>C</b>) qRT-PCR for PSMA over time from retinal RNA isolated at the indicated time points relative to P12 levels. Paraffin embedded OIR retinas were immunostained for PSMA protein using the 3E2 antibody. Staining (red-brown) was observed on vascular tufts (arrows) of wild-type (<b>D</b>) but not PSMA-null (<b>E</b>) retinas. (n = 3 per group), **p,0.05, ***p,0.001.</p

Hypoxia level of spheroids was positively correlated with E-cadherin level in pancreatic cancer cell lines.

<p>(A) Representative phase contrast images of four types of pancreatic cancer cell lines cultured on 2D condition. Scale bar, 100 μm. (B) Western blotting analysis of E-cadherin in the 4 types of pancreatic cancer cell lines cultured on NCPs for 7 days. β-actin was examined as the loading control. Signal intensities were quantified, and relative values were shown on the graph. (C) Representative hypoxic images of pancreatic cancer cell lines cultured on NCPs. Hypoxia Probe was added to the culture medium and visualized under fluorescence microscopy, shown in red. The fluorescence and bright field images were merged. Scale bar, 200 μm. (D) Hypoxia levels among the cell lines. Integrated fluorescent intensities (IFI) of the Hypoxia Probe taken in spheroids were calculated as described in Materials and Methods. N = 3. Data are mean ± SD.</p

Hypoxia level of A549 spheroid was declined with TGF-β2 and elevated with SB431542.

<p>(A) A549 spheroids cultured on NCP for 3 days were stimulated with TGF-β2 for 4 days in indicated concentrations. Hypoxia Probe was then added. Integrated fluorescent intensities (IFI) of Hypoxia Probe were reduced in a dose depending manner without affecting viability. (B) A549 spheroids were treated with TGF-β2 (5 ng/mL) and indicated concentration of SB431542 for 4 days. IFI of Hypoxia Probe were increased in a dose depending manner without affecting viability. N = 3. Data are mean ± SD. (C) Representative images of hypoxic spheroids restituted by SB431542. A549 spheroids were treated with 5 ng/mL TGF-β2 and SB431542 at indicated concentrations. Hypoxia levels were visualized with Hypoxia Probe in red color Scale bars, 200 μm.</p

Dose dependency of SEMTIN activity of screened compounds.

<p>(A) Representative images of spheroids that was altered by drugs. Spheroids were treated with 5 ng/mL TGF-β2 and each drug at indicated concentrations. Hypoxia levels of spheroids were measured and shown in red. Scale bars, 200 μm. (B) Dose-dependent SEMTIN activities of the compounds. SB431542 is a positive control. SEMTIN activity IC₅₀ of SB-525334, SU9516, and SB431542 were 0.31 μM, 1.21 μM, and 2.38 μM, respectively. N = 4. Data are mean ± SD. (C) Cellular viability under the exposure of compounds.</p