Regulation of cardiovascular development and integrity by the heart of glass-cerebral cavernous malformation protein pathway

Abstract

Cerebral cavernous malformations (CCMs) are human vascular malformations caused by mutations in three genes of unknown function: KRIT1, CCM2 and PDCD10. Here we show that the heart of glass (HEG1) receptor, which in zebrafish has been linked to ccm gene function, is selectively expressed in endothelial cells. Heg1(-/-) mice showed defective integrity of the heart, blood vessels and lymphatic vessels. Heg1(-/-); Ccm2(lacZ/+) and Ccm2(lacZ/lacZ) mice had more severe cardiovascular defects and died early in development owing to a failure of nascent endothelial cells to associate into patent vessels. This endothelial cell phenotype was shared by zebrafish embryos deficient in heg, krit1 or ccm2 and reproduced in CCM2-deficient human endothelial cells in vitro. Defects in the hearts of zebrafish lacking heg or ccm2, in the aortas of early mouse embryos lacking CCM2 and in the lymphatic vessels of neonatal mice lacking HEG1 were associated with abnormal endothelial cell junctions like those observed in human CCMs. Biochemical and cellular imaging analyses identified a cell-autonomous pathway in which the HEG1 receptor couples to KRIT1 at these cell junctions. This study identifies HEG1-CCM protein signaling as a crucial regulator of heart and vessel formation and integrity.

Figure 1. Lethal cardiac and blood vessel integrity defects in HEG1-deficient embryos and neonates

(a-b) Heg1 is selectively expressed in the developing cardiovascular system. Radioactive in situ hybridization reveals Heg1 expression in the vascular endothelium, cardiac endocardium and neural tube at E10.5 (a). By E14.5 Heg1 is expressed in both endothelial and smooth muscle cells in major arteries (b). (c-f) E15 Heg1^-/- embryos exhibit deep invaginations of the ventricular chamber into the compact zone of the ventricular wall and into the septum, often associated with the presence of blood between the epicardial and myocardial cell layers of the heart (arrow; e,f show boxed regions for c,d). (g-i) Pulmonary hemorrhage in Heg1^-/- neonates (h,i) but not in Heg1^+/+ littermates (g) was manifest by the presence of erythrocytes and fibrin in alveolar air spaces. (j) Cardiac rupture in P4 Heg1^-/- neonate. Hemopericardium (left, middle panels) associated with cardiac rupture and formation of a transmural thrombus (right panel) was observed in Heg1^-/- animals. VSD, ventricular septal defect. Scale bars in a-d and j (middle panel), 200 μm, e-i and j (right panel), 20 μm.

Figure 2. Lymphatic vessel dilatation and leak in Heg1^-/- neonates

(a,b) HEG1-deficient neonates exhibit chylous ascites manifest by the accumulation of white chyle in the peritoneal space. (c-f) Lymphatic malformations in Heg1^-/- animals. Neonatal mesenteric lymphatic vessels of Heg1^-/- animals are dilated (arrows) and leak chyle into the intestinal wall as well as the peritoneum. Anti-LYVE1 immunostaining confirms the lymphatic identity of the dilated mesenteric vessels (arrows) (g,h). SMA, α-smooth muscle actin. Scale bars, 100 μm.

Figure 3. Heg1^-/-;Ccm2^+/lacZ embryos fail to establish a patent blood vascular network

Transverse sections of E9 Heg1^+/-;Ccm2^+/+ (control) and two Heg1^-/-;Ccm2^+/lacZ embryos at three levels are shown. H-E staining reveals the presence of blood-filled dorsal aortae (DA), cardinal veins (CV) and branchial arch arteries (BAA) of normal caliber in the Heg1^+/-;Ccm2^+/+ control embryo (top) but not in Heg1^-/-;Ccm2^+/lacZ littermates (below). Anti-Flk1 staining of adjacent sections at the level of the first two branchial arch arteries is shown (middle). Flk1+ endothelial cells are present at the sites of the dorsal aortae, cardinal veins and branchial arch arteries (arrows) in Heg1^-/-;Ccm2^+/lacZ embryos but these cells do not form vessels of normal caliber with patent lumens. SV, sinus venosus. Scale bars, 50 μm.

Figure 4. The endothelial cells of zebrafish lacking heg or ccm2 form vessels that are normally patterned but not patent

(a) Fli1-GFP transgenic heg and ccm2 morphant embryos exhibit dilated hearts (arrows) and normal vascular patterning. (b) Angiography reveals a proximal circulatory block in heg and ccm2 morphant zebrafish. Fluorescent microspheres distribute throughout the vasculature of control but not heg or ccm2 morphant fish following venous injection. (c) FITC-dextran distributes throughout the vasculature in wild type but not ccm2 mutant fish following venous injection. Scale bars, 250 μm.

Figure 5. HEG1 is required to form normal endothelial junctions in vivo

(a-c) Dilated mesenteric lymphatic vessels in Heg1^-/- mice have severely shortened endothelial junctions and gaps between endothelial cells. (a) The mean and standard deviation of endothelial cell junction lengths in Heg1^+/+ and Heg1^-/- lymphatic vessels are shown, divided into terciles (1^st, shortest third of junctions in each group; 2^nd, middle third of junctions in each group; 3^rd, longest third of junctions in each group). N = 66 Heg1^+/+ junctions and 123 Heg1^-/- junctions. (b) The percent of endothelial junctions that were less than 1,000 nm (black), 1,000-2,500 nm (grey), and over 2,500 nm (white) in the indicated groups is shown. (c) Representative low magnification (far left) and high magnification images of endothelial cells are shown. Asterisks indicate sites of endothelial gaps, normally not present in collecting mesenteric lymphatics, and arrowheads indicate endothelial junction limits. Note the presence of endothelial gaps in Heg1^-/- but not Heg1^+/+ vessels.

Figure 6. HEG1 receptor intracellular tails associate with CCM2 through KRIT1

(a,b) Co-immunoprecipitation of HEG1 and CCM2 requires the HEG1 receptor intracellular tail. HA-CCM2 or FLAG-CCM2 and FLAG-HEG1 or FLAG-HEG1ΔC, a mutant lacking the terminal 106 amino acids of the HEG1 carboxy tail, were coexpressed in HEK293T cells and immunoprecipitations performed using anti-FLAG (a) and anti-CCM2 antibodies (b). (c) HEG1 intracellular tails interact with KRIT1 and CCM2. HA-tagged mouse CCM2 and Myc-tagged mouse KRIT1 were expressed in HEK293 cells and pulldowns performed with affinity matrices containing the intracellular tail of either the aIIb integrin subunit (aIIb) or the HEG1 receptor (HEG1). Note that the HEG1 receptor tail efficiently pulls down KRIT1 in the absence of CCM2 but not vice versa. (d) HEG1 interacts with CCM2 through KRIT1. FLAG-tagged zebrafish krit1, HA-tagged zebrafish ccm2 and HA-tagged zebrafish ccm2 L197R were expressed in HEK293 cells and pulldowns performed using aIIb or HEG1 tail affinity matrices as in c. Note that ccm2 L197R does not associate with the HEG1 tail (far right lane). (e) The HEG1 receptor tail efficiently binds endogenous KRIT1. Pulldowns using aIIb or HEG1 tail affinity matrices were performed using cell lysate from CHO cells transfected with FLAG-tagged CCM2. Endogenous KRIT1 was detected with anti-KRIT1 monoclonal antibody (top) and heterologous CCM2 detected with anti-FLAG monoclonal antibody (bottom). The far right lane shows immunoblotting of cell lysate equivalent to 5% of the input for the pull downs. (f) Molecular model of HEG1-CCM signaling at endothelial cell junctions. Shown are HEG1 receptors coupling to KRIT1 and CCM2 via HEG1 tail-KRIT1 and KRIT1-CCM2 interactions, respectively. KRIT1 also interacts with the junctional proteins VE-cadherin and beta-catenin, and HEG1-CCM signaling is proposed to regulate junction formation and function.