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

Kleaveland B, Zheng X, Liu JJ, Blum Y, Tung JJ, Zou Z, Sweeney SM, Chen M, Guo L, Lu MM, Zhou D, Kitajewski J, Affolter M, Ginsberg MH, Kahn ML - Nat. Med. (2009)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine and Cardiovascular Institute, University of Pennsylvania, 421 Curie Blvd., Philadelphia, Pennsylvania 19104, USA.

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.

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

Mentions: Identical big heart phenotypes arise in zebrafish embryos lacking heg, krit1 (santa) or ccm2 (valentine), and co-morpholino experiments demonstrate strong interactions between these genes in the fish12. In contrast, human CCMs have been linked to loss of function mutations in KRIT1 and CCM2 but not HEG1, and HEG1-deficient mice do not experience the early embryonic lethality reported for KRIT1-deficient mice18. To determine whether and to what extent HEG1 receptors interact with CCM signaling proteins in mammals we intercrossed Heg1+/-;Ccm2lacZ/+ animals. The Ccm2lacZ allele is predicted to be a allele because the Ccm2lacZ mRNA transcript lacks the 3′ half of the Ccm2 mRNA (exons 6-10, Supp. Fig. 6), and Ccm2lacZ/lacZ embryos experience early embryonic lethality that phenocopies Krit1-/- embryos (see below and18). In contrast to Heg1-/-;Ccm2+/+ animals that exhibit no phenotype prior to midgestation, both Heg1-/-;Ccm2lacZ/+ and Ccm2lacZ/lacZ embryos died prior to E10 (Supp. Table 2). Heg1-/-;Ccm2lacZ/+ and Ccm2lacZ/lacZ embryos were indistinguishable from littermates until E9 when they exhibited identical phenotypes of growth retardation and marked pericardial edema despite the presence of a visibly normal heartbeat (data not shown). Histologic examination revealed normal development of the ventricular chamber and bulbis cordis (future right ventricle), but formation of a dilated aortic sac (Fig. 3 and Supp. Fig. 7). In contrast to control embryos, the rostral paired dorsal aortae of E9 Heg1-/-;Ccm2lacZ/+ and Ccm2lacZ/lacZ embryos appeared small or undetectable and completely lacked luminal blood cells (Fig. 3 and Supp. Fig. 7). Analysis of serial transverse sections revealed that the aortic sac failed to connect to a lumenized first, second or third branchial arch artery in Heg1-/-;Ccm2lacZ/+ and Ccm2lacZ/lacZ embryos, suggesting that blood in the heart did not enter the dorsal aortae despite the presence of a normal heartbeat (Fig. 3 and Supp. Fig. 7). When visible, the cardinal veins of E9 Heg1-/-;Ccm2lacZ/+ and Ccm2lacZ/lacZ embryos were also devoid of blood except at the point where they connected to the sinus venosus of the heart (Fig. 3, right panels). In contrast to the lack of blood in the vessels, extravasated blood cells were frequently present in the dilated pericardial cavity (Fig. 3 and data not shown), a finding that may reflect a primary defect in the integrity of the heart like that observed in deficient zebrafish embryos11,12, or may arise secondary to the heart beating against a closed circulatory system and/or reduced embryonic viability at that timepoint.


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

Kleaveland B, Zheng X, Liu JJ, Blum Y, Tung JJ, Zou Z, Sweeney SM, Chen M, Guo L, Lu MM, Zhou D, Kitajewski J, Affolter M, Ginsberg MH, Kahn ML - Nat. Med. (2009)

Heg1-/-;Ccm2+/lacZ embryos fail to establish a patent blood vascular networkTransverse 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.
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Related In: Results  -  Collection

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Figure 3: Heg1-/-;Ccm2+/lacZ embryos fail to establish a patent blood vascular networkTransverse 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.
Mentions: Identical big heart phenotypes arise in zebrafish embryos lacking heg, krit1 (santa) or ccm2 (valentine), and co-morpholino experiments demonstrate strong interactions between these genes in the fish12. In contrast, human CCMs have been linked to loss of function mutations in KRIT1 and CCM2 but not HEG1, and HEG1-deficient mice do not experience the early embryonic lethality reported for KRIT1-deficient mice18. To determine whether and to what extent HEG1 receptors interact with CCM signaling proteins in mammals we intercrossed Heg1+/-;Ccm2lacZ/+ animals. The Ccm2lacZ allele is predicted to be a allele because the Ccm2lacZ mRNA transcript lacks the 3′ half of the Ccm2 mRNA (exons 6-10, Supp. Fig. 6), and Ccm2lacZ/lacZ embryos experience early embryonic lethality that phenocopies Krit1-/- embryos (see below and18). In contrast to Heg1-/-;Ccm2+/+ animals that exhibit no phenotype prior to midgestation, both Heg1-/-;Ccm2lacZ/+ and Ccm2lacZ/lacZ embryos died prior to E10 (Supp. Table 2). Heg1-/-;Ccm2lacZ/+ and Ccm2lacZ/lacZ embryos were indistinguishable from littermates until E9 when they exhibited identical phenotypes of growth retardation and marked pericardial edema despite the presence of a visibly normal heartbeat (data not shown). Histologic examination revealed normal development of the ventricular chamber and bulbis cordis (future right ventricle), but formation of a dilated aortic sac (Fig. 3 and Supp. Fig. 7). In contrast to control embryos, the rostral paired dorsal aortae of E9 Heg1-/-;Ccm2lacZ/+ and Ccm2lacZ/lacZ embryos appeared small or undetectable and completely lacked luminal blood cells (Fig. 3 and Supp. Fig. 7). Analysis of serial transverse sections revealed that the aortic sac failed to connect to a lumenized first, second or third branchial arch artery in Heg1-/-;Ccm2lacZ/+ and Ccm2lacZ/lacZ embryos, suggesting that blood in the heart did not enter the dorsal aortae despite the presence of a normal heartbeat (Fig. 3 and Supp. Fig. 7). When visible, the cardinal veins of E9 Heg1-/-;Ccm2lacZ/+ and Ccm2lacZ/lacZ embryos were also devoid of blood except at the point where they connected to the sinus venosus of the heart (Fig. 3, right panels). In contrast to the lack of blood in the vessels, extravasated blood cells were frequently present in the dilated pericardial cavity (Fig. 3 and data not shown), a finding that may reflect a primary defect in the integrity of the heart like that observed in deficient zebrafish embryos11,12, or may arise secondary to the heart beating against a closed circulatory system and/or reduced embryonic viability at that timepoint.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine and Cardiovascular Institute, University of Pennsylvania, 421 Curie Blvd., Philadelphia, Pennsylvania 19104, USA.

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.

Show MeSH
Related in: MedlinePlus