Impaired orthotopic glioma growth and vascularization in transgenic mouse models of Alzheimer's disease

Abstract

Alzheimer's disease (AD) is the most common form of dementia among the aging population and is characterized pathologically by the progressive intracerebral accumulation of beta-amyloid (Abeta) peptides and neurofibrillary tangles. The level of proangiogenic growth factors and inflammatory mediators with proangiogenic activity is known to be elevated in AD brains which has led to the supposition that the cerebrovasculature of AD patients is in a proangiogenic state. However, angiogenesis depends on the balance between proangiogenic and antiangiogenic factors and the brains of AD patients also show an accumulation of endostatin and Abeta peptides which have been shown to be antiangiogenic. To determine whether angiogenesis is compromised in the brains of two transgenic mouse models of AD overproducing Abeta peptides (Tg APPsw and Tg PS1/APPsw mice), we assessed the growth and vascularization of orthotopically implanted murine gliomas since they require a high degree of angiogenesis to sustain their growth. Our data reveal that intracranial tumor growth and angiogenesis is significantly reduced in Tg APPsw and Tg PS1/APPsw mice compared with their wild-type littermates. In addition, we show that Abeta inhibits the angiogenesis stimulated by glioma cells when cocultured with human brain microvascular cells on a Matrigel layer. Altogether our data suggest that the brain of transgenic mouse models of AD does not constitute a favorable environment to support neoangiogenesis and may explain why vascular insults synergistically precipitate the cognitive presentation of AD.

Figure 1.

Histologic appearance of GL261 glioma tumors in Tg APPsw, Tg PS1/APPsw and wild-type littermates (4× objective). A, Representative examples of tumor surfaces and adjacent parenchyma representing the maximum cross-sectional area of the intracranial glioma following hematoxylin and eosin staining in the different genotypes. Tumors in wild-type mice are surrounded by numerous smaller satellite tumors in contrast with tumors from Tg APPsw and Tg PS1/APPsw mice. B, Examples of tumor surfaces at higher magnification (20× objective) depicting the presence of peritumoral satellites in wild-type mice suggestive of a more invasive growth pattern than in Tg APPsw and Tg PS1/APPsw mice. C, Histogram representing the average tumor volume estimated in Tg APPsw, Tg PS1/APPsw and wild-type littermates. ANOVA revealed a significant main effect of the genotypes on tumor growth (p < 0.005). Post hoc comparisons show a significant difference in tumor volume between wild-type littermates, Tg APPsw (p < 0.005), Tg PS1/APPsw (p < 0.03) but no significant difference between the tumor volume of Tg APPsw and Tg PS1/APPsw mice (p = 0.486).

Figure 2.

A, Representative pictures (60× objective) showing VWF immunostaining depicting newly formed blood vessels in tumoral sections of Tg APPsw, Tg PS1/APPsw and wild-type littermates. VWF staining reveals higher blood vessel density in glioma tumors from wild-type mice compared with glioma tumors implanted in Tg APPsw and Tg PS1/APPsw mice. B, The histogram represents the percentage area of microscopic fields covered by VWF-immunopositive blood vessels quantified by image analysis. ANOVA revealed a significant main effect of the genotypes on the amount of VWF staining per microscopic area analyzed (p < 0.04). Post hoc analyses show that vascularization is significantly reduced in tumor sections from Tg APPsw (p < 0.03) and Tg PS1/APPsw (p < 0.02) compared with wild-type littermates but no significant difference was observed between Tg PS1/APPsw and Tg APPsw mice (p = 0.759).

Figure 3.

Effect of human recombinant Aβ1-42 on the proliferation and survival of GL261 tumor cells. A, The histogram depicts the effect of Aβ1-42 on GL261 proliferation. The NFκB inhibitor celastrol was used as a positive control and significantly inhibited the proliferation of GL261 cells (p < 0.001). ANOVA reveals no statistically significant main effect of Aβ dose (p = 0.788) showing that Aβ1-42 does not affect the proliferation of GL261 cells. B, The histogram depicts the effect of Aβ1-42 on GL261 survival estimated by the release of LDH in the culture medium. Slight toxicity of the positive control (celastrol) was observed (p < 0.01). ANOVA shows no statistically significant main effect of Aβ dose (p = 0.305) demonstrating that at the doses used, Aβ1-42 is not cytotoxic to GL261 cells.

Figure 4.

Effect of brain homogenates from Tg PS1/APPsw and wild-type mice on capillary morphogenesis. A, Representative photomicrographs (10× objective) showing the formation of tube-like structures by HBMEC when plated on a layer of Matrigel in the presence of 1% of brain homogenate from wild-type and Tg PS1/APPsw mice. B, Histogram representing the quantification of capillary network length formed by HBMEC in the presence of wild-type and Tg PS1/APPsw brain homogenates. A statistically significant inhibition of capillary formation was observed (p < 0.01) when HBMEC were cultured in the presence of Tg PS1/APPsw brain homogenate compared with wild-type brain homogenate.

Figure 5.

Effect of human recombinant Aβ1-42 on the angiogenesis stimulated by GL261 glioma cells. A, Representative photomicrographs (10× objective) showing the formation of tube-like structures by HBMEC when plated on a layer of Matrigel in regular culture conditions (AA) and in coculture with GL261 cells (AE). Disruption of capillary network formation was observed with ranging concentrations of Aβ1-42 in regular culture conditions (AB 2.5 μm, AC 5 μm, AD 10 μm Aβ1-42) and in coculture with GL261 cells (AF 2.5 μm, G 5 μm, H 10 μm Aβ1-42). B, Histogram representing the average length of capillary like structures formed by HBMEC in response to a dose range of Aβ1-42 in regular culture conditions (control) and in coculture with GL261 glioma cells (GL261). ANOVA revealed a significant main effect of GL261 cells (p < 0.001) and of Aβ doses (p < 0.001) on capillary network formation. Post hoc comparisons show statistically significant differences between the control culture conditions and the GL261 cocultures (p < 0.001) showing that GL261 glioma cells stimulate capillary like network formations by HBMEC. In addition, significant inhibition of capillary like formation was observed for all the doses of Aβ tested in regular and coculture conditions (p < 0.01). C, The histogram depicts the impact of human recombinant Aβ1-42 on LDH released in regular culture conditions (control) and in coculture with GL261 glioma cells (GL261). ANOVA revealed no significant main effect of Aβ doses (p = 0.306) showing that at the doses used, Aβ1-42 did not induce cytotoxicity to HBMEC or GL261 cells and HBMEC when cocultured.