Sonic hedgehog is expressed in human brain arteriovenous malformations and induces arteriovenous malformations in vivo

Abstract

Abnormalities in arterial versus venous endothelial cell identity and dysregulation of angiogenesis are deemed important in the pathophysiology of brain arteriovenous malformations (AVMs). The Sonic hedgehog (Shh) pathway is crucial for both angiogenesis and arterial versus venous differentiation of endothelial cells, through its dual role on the vascular endothelial growth factor/Notch signaling and the nuclear orphan receptor COUP-TFII. In this study, we show that Shh, Gli1 (the main transcription factor of the Shh pathway), and COUP-TFII (a target of the non-canonical Shh pathway) are aberrantly expressed in human brain AVMs. We also show that implantation of pellets containing Shh in the cornea of Efnb2/LacZ mice induces growth of distinct arteries and veins, interconnected by complex sets of arteriovenous shunts, without an interposed capillary bed, as seen in AVMs. We also demonstrate that injection in the rat brain of a plasmid containing the human Shh gene induces the growth of tangles of tortuous and dilated vessels, in part positive and in part negative for the arterial marker αSMA, with direct connections between αSMA-positive and -negative vessels. In summary, we show that the Shh pathway is active in human brain AVMs and that Shh-induced angiogenesis has characteristics reminiscent of those seen in AVMs in humans.

Keywords: Arteriovenous malformation; Sonic hedgehog; VEGF; angiogenesis; growth factors.

Figure 1.

Immunohistochemical analysis of Shh pathway expression in human brain AVMs. Representative images of human brain AVMs and control brain vessels. Cells in the intimal layer of AVMs are strongly immunopositive for Shh and Gli1. COUP-TFII immunopositive cells are detectable in the intimal and medial layers of AVMs. Control brain vessels are negative in terms of Shh, Gli1, and COUP-TFII expression. Shh: Sonic hedgehog.

Figure 2.

Characterization of the arterial and venous phenotype of Shh-induced corneal neovessels. (a) When pellets containing Shh are implanted in the corneal of Efnb2/LacZ mice, the resulting angiogenic process consists of large Efnb2-positive arterial vessels (red/yellow staining) that grow directly from the main limbus artery toward the pellet and large and tortuous Efnb2-negative, CD31-positive veins (green staining) directly connected to the main limbus vein. (b) When pellets containing VEGF are implanted in the cornea of Efnb2/LacZ mice, the resulting angiogenic process consists of short Efnb2-positive arterial vessels (red/yellow staining) that grow directly from the main limbus artery and are orderly distributed along the corneal circumference. (c) X-gal staining of the corneas of Efnb2/LacZ mice implanted with Shh-containing pellets shows the concomitant presence of X-gal positive arteries (blue staining) and X-gal negative veins. (d) In the corneas of Efnb2/LacZ mice, Shh-induced angiogenesis is characterized by the presence of direct connections (white arrowheads) between Efnb2-positive arteries (red/yellow staining) and Efnb2-negative, CD31-positive veins (green staining).

Figure 3.

Quantification and characterization of Shh- and VEGF-induced corneal angiogenesis. (a) Vessel length (P < 0.0001), lumen diameter (P < 0.0001), and circumferential extent of neovascularity (P < 0.001) are significantly greater in Shh rather than VEGF-induced corneal angiogenesis, while the ratio between arteries and veins is greater in VEGF-induced angiogenesis compared to Shh-induced angiogenesis (P < 0.01). (b) In the setting of Shh-induced corneal angiogenesis, veins are significantly longer than arteries (P < 0.01) and have a significantly greater lumen diameter compared to arteries (P < 0.001). Shh: Sonic hedgehog; VEGF: vascular endothelial growth factor.

Figure 4.

Effects of phShh injection in rat brain. In five rats, one brain hemisphere was treated with an intracerebral injection of phShh, while the contralateral hemisphere received an injection of empty plasmid. A robust neoangiogenic process developed in the hemispheres injected with phShh (right panels, 5×, 10×, and 20× magnification), while no changes were observed in the vascularization of the brain hemisphere treated with the empty plasmid (left panels, 5×, 10×, and 20× magnification).

Figure 5.

Presence of αSMA-positive and αSMA-negative vessels in the context of Shh-induced brain neoangiogenesis. (a) Sections of brain hemispheres treated with phShh were stained for CD31 and αSMA. In the context of phShh-induced brain neoangiogenesis, it was possible to distinguish vessels that were αSMA-positive (red/yellow staining) and vessels that were αSMA-negative (green staining). (b) Quantification of the percentage of αSMA-positive and αSMA-negative vessels within the vascular tangles grown in response to phShh intracerebral injection. (c) Quantification of the mean lumen diameter of αSMA-positive and αSMA-negative vessels within the vascular tangles grown in response to phShh intracerebral injection.

Figure 6.

Presence of direct arteriovenous connections in the context of Shh-induced brain neoangiogenesis. (a) Representative image of direct connections (white arrows) between two αSMA-negative vessels (green staining) and one αSMA-positive vessel (red/yellow staining) within phShh-induced brain neoangiogenesis. (b) Rarefaction of the brain vasculature in the area surrounding phShh-induced neoangiogenesis compared to the same brain area in the contralateral hemisphere injected with the empty plasmid.

Figure 7.

Expression of COUP-TFII in phShh-induced brain neovessels. Sections of brain hemispheres injected with phShh and empty plasmid were stained for DAPI (blue staining) to identify cell nuclei, lectin (red staining) to identify blood vessels, and COUP-TFII (green staining) to identify cells expressing this vascular differentiation marker. (a) COUP-TFII-positive cells were found in the neovessels grown in the brain in response to phShh injection, both at the level of the intimal layer and the vascular wall. (b) COUP-TFII-positive cells were virtually absent in brain hemispheres injected with the empty plasmid.