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, 68 (6), 626-32

Restoring Transcription Factor HoxA5 Expression Inhibits the Growth of Experimental Hemangiomas in the Brain

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Restoring Transcription Factor HoxA5 Expression Inhibits the Growth of Experimental Hemangiomas in the Brain

Yiqian Zhu et al. J Neuropathol Exp Neurol.

Abstract

Hemangiomas are angiogenesis-dependent benign vascular tumors that can rupture and cause intracranial hemorrhages. We previously showed that the transcription factor homeobox A5 (HoxA5), which is absent in activated angiogenic endothelial cells can block angiogenesis. Here, we investigated whether restoring expression of HoxA5 blocks hemangioma growth by transplanting mouse hemangioendothelioma endothelial cells (EOMA) or HoxA5-expressing EOMA cells into the brains of mice. The EOMA cells induced brain hemangiomas characterized by large cystlike spaces lined by thin walls of endothelial cells surrounded by scant smooth muscle cells. When HoxA5-expressing EOMA cells were injected, lesion volumes were reduced between 5- and 20-fold compared with the EOMA control group (p < 0.05). Restoration of HoxA5 was associated with increased thrombospondin-2, which inhibits angiogenesis and reduced hypoxia-inducible factor 1alpha expression. These data suggest that restoring HoxA5 can attenuate experimental brain hemangioma development.

Figures

Figure 1
Figure 1
A) HoxA5 expression inhibits the growth of EOMA vascular lesions. HoxA5 (upper panels) or control transfected EOMA cells (lower panels) were stereotactically injected into the lateral caudo-putamen of adult CD1 mice and histologically examined 1, 2 and 3 weeks after injection. Photomicrographs show H&E staining of coronal sections at 1 (left side), 2 (middle) and 3 weeks (right side) following transplantation of EOMA+HoxA5 (upper panel) or EOMA cells (bottom panel) into the mouse brain. Tumors derived from HoxA5-expressing EOMA cells, were smaller than those induced by the control transfected EOMA cells. B) Bar graph shows the quantitation of the tumor volume in the EOMA+HoxA5 and EOMA groups at 1, 2, and 3 weeks after transplantation. Data are mean±SD, n=6 in each group. *p<0.05 for the difference between EOMA+HoxA5 vs. EOMA alone group for the same time point.
Figure 1
Figure 1
A) HoxA5 expression inhibits the growth of EOMA vascular lesions. HoxA5 (upper panels) or control transfected EOMA cells (lower panels) were stereotactically injected into the lateral caudo-putamen of adult CD1 mice and histologically examined 1, 2 and 3 weeks after injection. Photomicrographs show H&E staining of coronal sections at 1 (left side), 2 (middle) and 3 weeks (right side) following transplantation of EOMA+HoxA5 (upper panel) or EOMA cells (bottom panel) into the mouse brain. Tumors derived from HoxA5-expressing EOMA cells, were smaller than those induced by the control transfected EOMA cells. B) Bar graph shows the quantitation of the tumor volume in the EOMA+HoxA5 and EOMA groups at 1, 2, and 3 weeks after transplantation. Data are mean±SD, n=6 in each group. *p<0.05 for the difference between EOMA+HoxA5 vs. EOMA alone group for the same time point.
Figure 2
Figure 2
HoxA5 reduces hemorrhage and increases smooth muscle cell coverage. (A) Higher magnification of H&E staining of 3-week old lesions formed by HoxA5 (A and B) or control transfected EOMA (C and D) stereotactically injected into the lateral caudo-putamen of adult CD1 mice. The tumor edges showed acute hemorrhage (h) and chronic hemorrhage indicated by hemosiderin-laden macrophages (arrows). A,C bars=500um; B,D bar=25um. B) HoxA5 (A-C) and control transfected (D-F) EOMA cells were stereotactically injected into the caudate-putamen of adult CD1 mice and assessed by immunohistochemical staining after 14 days. Shown are photomicrographs of the edges of cavernous cystic blood-filled spaces triple labeled with FITC-lectin (A, D), Alexa 594 conjugated anti-α-SMA (B, E) and DAPI (blue in merged image C, F). Arrows point to the ECs (A, D) and arrowheads point to smooth muscle cells at the lumen (L) border. Bar=25 um.
Figure 2
Figure 2
HoxA5 reduces hemorrhage and increases smooth muscle cell coverage. (A) Higher magnification of H&E staining of 3-week old lesions formed by HoxA5 (A and B) or control transfected EOMA (C and D) stereotactically injected into the lateral caudo-putamen of adult CD1 mice. The tumor edges showed acute hemorrhage (h) and chronic hemorrhage indicated by hemosiderin-laden macrophages (arrows). A,C bars=500um; B,D bar=25um. B) HoxA5 (A-C) and control transfected (D-F) EOMA cells were stereotactically injected into the caudate-putamen of adult CD1 mice and assessed by immunohistochemical staining after 14 days. Shown are photomicrographs of the edges of cavernous cystic blood-filled spaces triple labeled with FITC-lectin (A, D), Alexa 594 conjugated anti-α-SMA (B, E) and DAPI (blue in merged image C, F). Arrows point to the ECs (A, D) and arrowheads point to smooth muscle cells at the lumen (L) border. Bar=25 um.
Figure 3
Figure 3
HoxA5 increases expression of TSP-2 in BH. A) Real-time PCR showing levels of HoxA5 (left panel) and TSP-2 mRNA (right panel) in EOMA control or HoxA5-transfected EOMA cells. B) Western blot analysis shows the levels of TSP-2 protein in BH samples from mice injected with EOMA cells and HoxA5 expressing EOMA cells (upper panel). The same membrane was stripped and re-probed with β-actin to assess total protein loading. C) Quantitative analysis of relative TSP-2 levels in the Western blots for n=3 animals. p=0.109. The densitometric analysis was performed using the Image J program (rsb.info.nih.gov/ij/).
Figure 4
Figure 4
HoxA5 reduces HIF1α in BH. A) Photomicrographs show HIF-1α (red), CD31 (green) and dapi (blue) expression within the hemangioma-like lesion in the EOMA (a, b), or HoxA5-expressing EOMA cells (c, d) transplanted into the mouse brain after 2 weeks of injection. Arrows point to representative positive nuclei staining for HIF-1α. Scale bar (a, c) = 40 μm, (b, d) =10 μm. B) Bar graph shows the average number of HIF-1α positive cells counted per field per group at each time point. Data are mean±SD, n=6 in each group. *, p<0.05 for the difference between EOMA+HoxA5 vs. EOMA group for same time point.
Figure 4
Figure 4
HoxA5 reduces HIF1α in BH. A) Photomicrographs show HIF-1α (red), CD31 (green) and dapi (blue) expression within the hemangioma-like lesion in the EOMA (a, b), or HoxA5-expressing EOMA cells (c, d) transplanted into the mouse brain after 2 weeks of injection. Arrows point to representative positive nuclei staining for HIF-1α. Scale bar (a, c) = 40 μm, (b, d) =10 μm. B) Bar graph shows the average number of HIF-1α positive cells counted per field per group at each time point. Data are mean±SD, n=6 in each group. *, p<0.05 for the difference between EOMA+HoxA5 vs. EOMA group for same time point.

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