Essential role of endothelial Smad4 in vascular remodeling and integrity

Abstract

New blood vessels are formed through the assembly or sprouting of endothelial cells (ECs) and become stabilized by the formation of perivascular matrix and the association with supporting mural cells. To investigate the role of endothelial Smad4 in vascular development, we deleted the Smad4 gene specifically in ECs using the Cre-LoxP system. EC-specific Smad4 mutant mice died at embryonic day 10.5 due to cardiovascular defects, including attenuated vessels sprouting and remodeling, collapsed dorsal aortas, enlarged hearts with reduced trabeculae, and failed endocardial cushion formation. Noticeably, Smad4-deficient ECs demonstrated an intrinsic defect in tube formation in vitro. Furthermore, the mutant vascular ECs dissociated away from the surrounding cells and suffered from impaired development of vascular smooth muscle cells. The disturbed vascular integrity and maturation was associated with aberrant expression of angiopoietins and a gap junction component, connexin43. Collectively, we have provided direct functional evidence that Smad4 activity in the developing ECs is essential for blood vessel remodeling, maturation, and integrity.

FIG. 1.

EC-specific deletion of Smad4 gene results in embryonic lethality at E10.5. (A and B) Whole-mount LacZ-stained ROSA26 transgenic embryos without (A) or with (B) Tie2-Cre transgene at E9.5. (C) Cross section of LacZ-stained Tie2-Cre; ROSA26 double-transgenic mice at E9.5 shows blue-stained ECs. (D) Cross sections of LacZ-stained Tie2-Cre; ROSA26 double transgenic mice and whole-mount CD31-stained Smad4^Co/+; Tie2-Cre embryos at E9.5. The expression pattern between both stainings and the intensity of LacZ positive signals among the DAs, ACVs, and hearts are comparable. Arrowheads denote representative positive signals in the ECs. (E) Whole-mount CD31 immunostaining of E9.5 embryos shows apparent presence of major blood vessels in the mutant ones. Arrowheads denote DAs that are significantly narrowed in the mutant embryos. An open arrowhead denotes a discontinuity in the mutant DAs. (F) A whole-mount view shows growth retardation and cardiovascular defects in Smad4^Co/Co; Tie2-Cre embryos at E10. Arrowheads point to focal hemorrhage; the open arrowhead points to enlarged edematous pericardium. (G) Smad4 immunostaining of E9.5 embryos shows absent expression in the vascular ECs and endocardium of the mutant embryos. Arrowheads denote representative ECs. V, ventricles; tb, trabeculae. Scale bar: 500 μm (A, B, and E), 125 μm (C), 45 μm (D), 700 μm (F), or 25 μm (G).

FIG. 2.

Disturbed vascular remodeling in Smad4^Co/Co; Tie2-Cre YS and placentas. (A and B) Whole-mount view of E9.5 YS. (C and D) Whole-mount view of LacZ-stained YS of Smad4^Co/+; Tie2-Cre and Smad4^Co/Co; Tie2-Cre embryos carrying a ROSA26 allele. Arrowheads denote the major vessels extended from embryos proper. (E and F) Higher magnifications of panels C and D, respectively. There is proper remodeling of vitelline vessels in control mice, which is absent in the mutants. (G and H) Hematoxylin-and-eosin-stained sections of E9.5 YS show EC layers of mutant YS dissociate from surrounding cells. Red arrowheads denote the ECs away from the subjacent mesothelial layer. A black arrowhead points to an EC detached from the endodermal layer. (I and J) Electron micrographs of E9.5 YS. Abnormal deposits of ECM (arrows) separate ECs from subjacent mesothelial cells in the mutant YS. An arrowhead denotes a discontinuity in the ECs. (K and L) Hematoxylin-and-eosin-stained placental sections at E9.5. Arrowhead shows leakage of embryonic nucleated erythrocytes in the maternal blood sinuses. (M and N) Histological sections of LacZ-stained placentas of Smad4^Co/+; Tie2-Cre and Smad4^Co/Co; Tie2-Cre embryos carrying a ROSA26 allele at E10. The mutant placentas have a thinner labyrinth with fewer fetal vessels compared to the controls. ec, endothelial cells; me, mesothelial cells; lbr, labyrinth; cp, chorionic plate. Scale bar: 1,000 μm (A and B), 1,100 μm (C and D), 220 μm (E, F, M, and N), 43 μm (G and H), 2 μm (I and J), or 55 μm (K and L).

FIG. 3.

Abnormal vasculature and impaired VSMC/pericytes development in Smad4^Co/Co; Tie2-Cre embryos. (A to D) Whole-mount CD31 immunostaining of E10 embryos. The mutant embryos show a coarse vascular plexus and significantly reduced capillary vessels in the intersomitic (A and B) and head region (C and D). Arrowhead indicates blind ending vessels. (E and F) Histological sections of whole-mount CD31-stained E9.5 embryos. The mutant embryos manifest an irregularly narrowed DA lumen. An arrowhead denotes the rupture of DA. (G and H) Histological sections of LacZ-stained Smad4^Co/+; Tie2-Cre and Smad4^Co/Co; Tie2-Cre embryos carrying a ROSA26 allele at E9.5. The mutant embryos show remarkably narrowed DAs (black arrowheads) and moderately dilated ACV (arrows). A red arrowhead denotes vessels sprouted into the neural tissue of the control mice, which is absent in the mutant embryos. (I and J) Electron micrographs of inter-endothelial junctions (arrowheads) at E9.5 show a reduced junctional overlapping in the mutant vascular ECs. (K to N) α-SMA immunostaining of E9.5 embryos. Dramatically reduced expression around DAs in the mutant embryos is in contrast to the controls in which perivascular walls are lined with α-SMA-positive VSMC/pericytes. Black arrowheads denote DAs, and arrows denote the comparable α-SMA expression at the somites. A red arrowhead denotes the only α-SMA-positive cell in the field but detached from the EC layer. (O) Laminin immunofluorescence staining of E9.5 DAs shows a comparable expression level but abnormal deposition around the DAs of the mutant embryos. Scale bar: 420 μm (A through D), 100 μm (E and F), 150 μm (G, H, K, and L), 500 nm (I and J), 75 μm (M and N), or 40 μm (O).

FIG. 4.

Defective myocardial development and endocardial cushion formation in Smad4^Co/Co; Tie2-Cre embryos. (A and B) Whole-mount CD31 immunostaining of embryonic hearts at E9.5. (C and D) α-SMA immunostaining of E9.5 ventricles. (E and F) Cross sections of whole-mount CD31-stained E9.5 ventricles. Compared to the controls, the mutant embryos have enlarged hearts, dilated ventricles, and reduced trabeculae. (G and H) Cross sections of LacZ-stained hearts of Smad4^Co/+; Tie2-Cre and Smad4^Co/Co; Tie2-Cre embryos carrying a ROSA26 allele at E9.5 show the disorganized endocardial layers of the mutant hearts. (I and J) Cross sections of a whole-mount CD31-stained atrioventricular canal at E10. The mutant embryos demonstrate significantly reduced endocardial cushion cellularity, while the cushion cells have more intensive CD31 expression than that of controls. A, atria; V, ventricles; *, endocardial cushion. Scale bar: 200 μm (A and B), 105 μm (C and D), 80 μm (E and F), 140 μm (G and H), or 55 μm (I and J).

FIG. 5.

Reduced in vitro tube-forming capacity of Smad4-deficient ECs. (A) Morphology and purity of isolated embryonic ECs. The upper panels show the typical cobblestone-like shape of sorted Tie2-positive cells in culture. The middle panels show >90% EC purity as verified by DiL-Ac-LDL uptake. The lower panels show the endothelial property of PmT-transformed ECs by VE-cadherin (CD144) immunostaining. (B) RT-PCR and Western blot analysis of Smad4 expression in E9.5 YS, embryos proper, sorted Tie2-positive cells, and PmT-transformed embryonic ECs is shown from left to right in the upper part of the panel. In the lower part of the panel, “+T” indicates stimulation by 10 ng of TGF-β1/ml for 20 h. Smad4 expression is reduced in the mutant embryos and absent in the mutant ECs. (C) Tube-like structures of isolated ECs on Matrigel observed after 8 h of incubation. The mutant ECs exhibit less tube formation than that of controls. (D) Quantification shows a significant 35% reduction of tube number (left) and a remarkable 44% decrease of total tube length (right) in the mutant ECs. The data are means ± the standard error of the mean. Scale bar: 27 μm (top images of panel A), 14.5 μm (middle and lower images of panel A), or 150 μm (C).

FIG. 6.

Increased Ang2 expression in Smad4-deficient ECs. (A and C) RT-PCR analysis of molecular expression in isolated embryonic ECs. The data are representative of three repeats in sorted Tie2-positive cells and PmT-transformed embryonic ECs, respectively. (B) Reporter assays of PmT-transformed embryonic ECs. The cells were transfected with (CAGA)₁₂-luciferase or BRE-luciferase and then treated with 10 ng of TGF-β1 (+T) or 100 ng of BMP2 (+B)/ml. Inactivation of Smad4 attenuated BMP-2 and TGF-β1-stimulated luciferase activities. A representative experiment is shown. (D) Real-time PCR analysis of Ang2 and PDGFB expression using RNA extracts from PmT transformed ECs. “+T” indicates stimulation by 10 ng of TGF-β1/ml for 20 h. The mutant ECs show increased expression of Ang2 and fail to downregulate Ang2 expression upon TGF-β1 treatment. (E) RT-PCR analysis of E9.5 whole-embryo RNA extracts. The data are two representatives of four independent experiments. (F) Real-time PCR analysis of Ang1, Ang2, and PDGFB expression using E9.5 whole-embryo RNA extracts. (G) Flow cytometric analysis of Tie2 expression in whole embryos. A representative experiment is shown. (H) Western blot analysis of E9.5 whole embryos demonstrates increased Ang2 expression in the mutants. The data are expressed as means ± the standard error of the mean. *, P < 0.05; **, P < 0.01 (significantly different from the untreated controls in panels D and F).

FIG. 7.

Reduced expression of Cx43 in Smad4-deficient ECs. (A) Immunofluorescence labeling of Cx43 in PmT-transformed ECs. The Smad4-deficient ECs demonstrate notably reduced expression. (B) Representative RT-PCR analysis of Cx43 expression using RNA extracts from isolated Tie2-positive cells (left side) and PmT-transformed ECs (right side). (C) Western blotting analysis of Cx43 expression in PmT-transformed ECs. “+T” indicates stimulated by 10 ng of TGF-β1/ml for 20 h. The mutant ECs fail to upregulate the expression of Cx43 upon treatment of TGF-β1. (D) Cx43 Immunofluorescence staining of E9.5 DAs shows reduced expression in the mutant vascular ECs in vivo. Scale bar: 12 μm (A) or 25 μm (D).