Similarities and differences between brain and skin GNAQ p.R183Q driven capillary malformations

Abstract

Capillary malformations (CM) are congenital vascular irregularities of capillary and venous blood vessels that appear in the skin, leptomeninges of the brain, and the choroid of the eye in the disorder known as Sturge Weber Syndrome (SWS). More common are non-syndromic CM found only in the skin, without brain or ocular involvement. A somatic activating mutation in GNAQ (p.R183Q) is found in ~90% of syndromic and non-syndromic CM specimens and is present in CD31^pos endothelial cells isolated from brain and skin CM specimens. Endothelial expression of the GNAQ p.R183Q variant is sufficient to form CM-like vessels in mice. Given the distinct features and functions of blood vessels in the brain versus the skin, we examined the features of CM vessels in both tissues to gain insights into the pathogenesis of CM. Herein, we present morphologic characteristics of CM observed in specimen from brain and skin. The GNAQ p.R183Q variant allelic frequency in each specimen was determined by droplet digital PCR. Sections were stained for endothelial cells, tight junctions, mural cells, and macrophages to assess the endothelium as well as perivascular constituents. CM blood vessels in brain and skin were enlarged, exhibited fibrin leakage and reduced zona occludin-1, and were surrounded by MRC1^pos/LYVE1^pos macrophages. In contrast, the CMs from brain and skin differ in endothelial sprouting activity and localization of mural cells. These characteristics might be helpful in the development of targeted and/or tissue specific therapies to prevent or reverse non-syndromic and syndromic CM.

Keywords: GNAQ; Sturge Weber Syndrome; Vascular malformation; capillary malformation; endothelial cells; macrophages; mural cells; tight junction.

Figure 1.. Ulex europaeus agglutinin I (UEAI) staining for endothelium in capillary malformation of brain and skin.

UEAI (yellow), counter staining for nuclei by DAPI in (A) CM brain (n=4) (B) non-CM brain (n=2) (C) CM skin (n=4) (D) non-CM skin (n=2). High magnification images for CM brain (green box) and skin (red box) are presented as inserts. See Table 1 for information on tissue specimens. (E) Quantification of % sprouting vessels/total vessels. Scalebar = 100μm. The p-values were calculated by Brown-Forsythe and Welch ANOVA test followed by Dunnett T3 multi comparison test. 4–5 non-sequential sections were analyzed per tissue specimen.

Figure 2.. Movat pentachrome staining for elastin fibers and fibrin leakage in capillary malformation of brain and skin.

(A) CM brain (n=4) (B) non-CM brain (n=2) (C) CM skin (n=4) (D) non-CM skin (n=2). Elastic Fibers (black); Collagen (yellow); Fibrin (bright red); Nuclei (blue/black). Scalebar = 200μm

Figure 3.. Loss of tight junction protein, zona occludin1 (ZO1), in capillary malformation of brain and skin.

ZO1 (magenta), UEAI (yellow) and counter staining for nuclei by DAPI in (A) CM brain (n=4) (B) non-CM brain (n=2) (C) CM skin (n=4) (D) non-CM skin (n=2). Single channel images for UEAI (magenta) and ZO1 (yellow) are on the right of each merged image. Scalebar = 50 μm

Figure 4.. Mural cell environment in capillary malformation of brain and skin.

Antibody staining for calponin (left), desmin (middle) and alpha-smooth muscle actin (αSMA) (magenta) and UEAI (yellow) and nuclei staining with DAPI (blue) in (A) CM brain (n=4) and (B) CM skin (n=4). Scalebar=100μm. See Table 1 for information on tissue specimens. (C) Quantification of % number of vessels with three distinct features: mural cell layer (magenta), no mural cell coverage (black) and distant mural cell layer (purple) in CM brain, non-CM brain, CM skin, non-CM skin. (D) Quantification of vessels with distance between the mural cell layer (Calponin (left) and Desmin(right)) and UEAI^pos endothelium (μm).

Figure 5.. Mannose receptor C type 1 (MRC1^pos) and lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1^pos) cells in capillary malformation of brain and skin.

MRC1(magenta), LYVE1(grey), UEAI (yellow), and counterstaining for DAPI (blue) in (A) CM brain (n=4) (B) non-CM brain (n=2) (C) CM skin (n=4) (D) non-CM skin (n=2). See Table 1 for information on tissue specimens. (E) Quantification of colocalized MRC1^pos and LYVE1^pos cells in the CM brain and skin comparing to non-CM brain and skin. (F) Quantification of MRC1^pos cells in the CM brain and skin comparing to, non-CM brain and skin. The p-values were calculated by Brown-Forsythe and Welch ANOVA test followed by Dunnett T3 multi comparison test. 4–5 non-sequential sections were analyzed per tissue specimen. Scalebar = 200μm for panel A-C, 50μm for panel D.

Figure 6.

Summary comparing and contrasting brain and skin capillary malformation features by immunohistopathology.