![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Previous Article | Next Article 
Molecular and Cellular Biology, November 2007, p. 7683-7692, Vol. 27, No. 21
0270-7306/07/$08.00+0 doi:10.1128/MCB.00577-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
,
Genetic Laboratory of Development and Diseases, Institute of Biotechnology, Beijing 100071,1 Department of Cell Biology, Institute of Basic Medical Sciences, Beijing 100850, People's Republic of China2
Received 2 April 2007/ Returned for modification 30 May 2007/ Accepted 13 August 2007
 |
ABSTRACT |
|---|
 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The TGF-ß superfamily members transduce their signals through transmembranous receptors (TßR) and intracellular mediator Smads (43). TGF-ß seems to regulate stabilization of vessels in multiple ways, including stimulating synthesis and deposition of extracellular matrix (ECM) components, inducing the differentiation of mesenchymal cells to mural cells, and regulating several cellular processes in both ECs and mural cells (16, 23). Genetic studies in human beings and mice have revealed pivotal roles of TGF-ß signaling during angiogenesis (7, 18, 22, 24, 29, 31, 32, 34, 48). Although mice deficient for various TGF-ß signaling components present an embryonic lethality due to angiogenetic defects, there exist certain discrepancies among the vascular phenotypes (7, 22, 24, 31, 32, 34, 48). It is presumably due to the different expression pattern and the largely distinct target genes of the signaling components (12, 33, 47).
Smad4 is a central mediator of TGF-ß signaling. Clinically, mutation in Smad4 causes a syndrome consisting of both juvenile polyposis and hereditary hemorrhagic telangiectasia phenotypes (11), emphasizing its pathological involvement in human vascular disorders. Furthermore, controlling an angiogenic switch, other than mediating TGF-ß's antiproliferative responses, has been assumed for Smad4 to serve as an alternative mechanism of tumor suppression (37). These findings strongly suggest a crucial role for Smad4 signaling in adult angiogenesis. Nevertheless, the precise roles of Smad4 in embryonic angiogenesis, in particular that as the unique common Smad, how it balances the two distinct TßRI pathways in ECs (12, 23), is by far unknown.
To address definitively the role of endothelial Smad4 in vascular development, we deleted the Smad4 gene specifically in ECs by using the Cre-LoxP system with a mouse strain that specifically expressed Cre recombinase in ECs under the control of Tie2 promoter (26). Here we show that the deletion of Smad4 in ECs results in embryonic lethality by embryonic day 10.5 (E10.5) due to cardiovascular defects, including attenuated vascular sprouting, ruptured blood vessels, and defective myocardial, as well as endocardial cushion development. Notably, Smad4-deficient ECs demonstrate an intrinsic defect in tube formation in vitro. In addition, our data also suggest that Ang2 and a gap junction component, connexin43 (Cx43), may serve as candidate targets of endothelial Smad4 signaling for maintaining intercellular interaction between ECs and surrounding cells, and therefore protecting vascular integrity and maturation.
|
MATERIALS AND METHODS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Whole-mount embryo immunostaining and LacZ staining. Whole-mount embryo immunostaining with anti-CD31 antibody (BD Pharmingen) and whole-mount LacZ staining were performed as described previously (26, 34). The whole-mount preparations were documented by using a dissecting microscope (SMZ1500; Nikon, Tokyo, Japan) with a digital camera (RT color; SPOT; Diagnostic Instruments, Sterling Heights, MI). Stained tissues were further paraffin embedded, and sections were counterstained with nuclear fast red.
Histology, immunohistology, and electron microscopy.
Mouse tissues were fixed in Bouin's fixative, embedded in paraffin, sectioned at 5 µm, and stained with hematoxylin and eosin by standard methods. Immunohistology was also performed by standard procedures. The primary antibodies used were as follows: anti-Smad4 (Santa Cruz), anti-
-smooth muscle actin (anti--SMA; Sigma), anti-CD31, antilaminin (Zymed), and anti-Cx43 (Sigma). A Nikon (Tokyo, Japan) E600 microscope with a digital camera (RT color; SPOT; Diagnostic Instruments) was used for documentation. Nikon 10x/0.30-numerical aperture (NA), 20x/0.50-NA or 40x/0.75-NA Plan Fluor objective lenses were applied. Electron microscopic analysis was performed as described previously (40). Ultrathin sections were stained in uranyl acetate and lead citrate and examined by using a Philips EM400 electron microscope.
Flow cytometry, isolation of embryonic ECs, and cell culture. E9.5 embryonic cells were harvested by trypsin-EDTA digestion. For flow cytometric analysis, cells were stained with phycoerythrin-conjugated isotype or phycoerythrin-conjugated anti-mouse Tie2 antibodies (eBioscience) according to the manufacturer's instructions. For Tie2-positive cells sorting, the dissociated cells were plated on mouse embryonic fibroblasts in culture medium described previously (14). After three passages of expansion on mouse embryonic fibroblasts, ECs were isolated by using magnetic bead (MACS Anti-Biotin MicroBeads; Miltenyi Biotec) purification with biotin-conjugated anti-mouse Tie2 antibody (eBioscience) according to the manufacturer's instructions. Selective transforming of ECs with a retrovirus expressing the polyoma middle T (PmT) oncogene and maintaining of the EC culture were performed as described previously (22). PmT-transformed ECs were incubated in the presence or absence of 10 ng of TGF-ß1 (R&D Systems)/ml for 20 h prior to harvest if needed.
DiI-Ac-LDL incorporation and immunofluorescence staining. The adherent cells of the cultures were incubated with 10 µg of DiI-Ac-LDL (1,19-dioctadecyl-3,3,39,39-tetoramethylindo-carbocyanine perchlorate acetylated low-density lipoprotein; Biomedical Technologies, Inc.)/ml for 4 h at 37°C, fixed with 4% paraformaldehyde-phosphate-buffered saline, and counterstained with DAPI (4',6'-diamidino-2-phenylindole). Immunofluorescence labeling of VE-cadherin (BD Pharmingen) and Cx43 was performed as described previously (20). Phase-contrast and fluorescence images were collected by using a Nikon TE2000-U inverted microscope with Nikon 4x/0.13 NA, 10x/0.30 NA, or 20x/0.45 NA Plan Fluor objective lenses. Images were acquired by using a digital camera (Cool SNAP 5.0; Roper Scientific, Tucson, AZ). Adobe Photoshop 7 software (Adobe Systems, Munich, Germany) was used for all of the figure preparation.
In vitro tube formation assay. After cultured for another 5 days, sorted embryonic ECs were collected by trypsin-EDTA digestion, replated to 48-well plates precoated with a thin layer of Matrigel (BD Biosciences), and allowed to form tube-like structures. ECs were quantified by counting branches from each EC and the length of the tube formed at 8 h. Measurement was performed in four independent fields, and the experiment was repeated three times. Statistical analysis was applied by using two-tailed Student t test.
Reporter gene assay. PmT-transformed ECs were seeded in 24-well plates and transfected with different reporter constructs with Lipofectamine (Invitrogen) according to the manufacturer's instructions. After 24 h, the cells were incubated in the presence or absence of BMP2 (R&D Systems) or TGF-ß1 for another 16 h prior to harvest for luciferase assay. Each experiment was performed with triplicate samples and repeated three times.
RT-PCR and real-time PCR. For reverse transcription-PCR (RT-PCR) analysis, total RNA from E9.5 embryos or isolated embryonic ECs was extracted by using TRIZOL reagent (Invitrogen) and reverse transcribed by using an mRNA selective PCR kit (TaKaRa). Sequences of specific primers and amplified product sizes are listed in Table S1 in the supplemental material. Quantitative real-time PCR analysis and related primers were as described previously (41).
Western blotting. The total proteins from E9.5 embryos or cultured PmT-transformed ECs were harvested. Portions (20 µg) of the proteins were subjected to sodium dodecyl sulfate-12% polyacrylamide gel electrophoresis. Immunoblotting was performed with anti-Smad4, anti-Ang2 (Chemicon), anti-Cx43, and anti-actin (Sigma) according to the manufacturer's instructions.
|
RESULTS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
Subsequently, a total of 56 litters were examined between E8.5 and E12.5. Conditional knockout embryos were morphologically indistinguishable from their wild-type littermates at E9 but were readily identifiable at E9.5 by their avascular yolk sacs (YS) and smaller body size (Fig. 1E and 2B). Visualization of endothelial networks with whole-mount CD31 staining demonstrated the presence of major blood vessels in the mutant embryos, but the DAs in the trunk manifested a decreased diameter and were even discontinuous (Fig. 1E). At E10, Smad4Co/Co; Tie2-Cre embryos showed severe growth retardation, focal hemorrhage, and enlarged pericardium (Fig. 1F) but still had beating hearts. They died at E10.5, displaying severe distortion and diminished vasculature, and were resorbed by E11 (data not shown). Immunohistochemical staining verified the efficient disruption of Smad4 gene in the endocardium and ECs of all of the vessels, including aortas and veins (Fig. 1G). In the present study, the Smad4Co/+; Tie2-Cre mice were used as controls because they appeared no different from Smad4Co/Co, Smad4Co/+, or wild-type mice.
|
Vascular defects were also observed in the placental labyrinth. At E9.5, the labyrinth region in the control placentas contained a network of extra-embryonic capillaries interspersed with maternal sinuses, where two kinds of erythrocytes were separated (Fig. 2K). Of note, in the mutant labyrinth, clusters of embryonic nucleated erythrocytes were detected in the maternal sinuses, indicating the impaired integrity of mutant vessels (Fig. 2L). By E10, although the chorionic plate and trophoblast giant cells appeared normal, the labyrinth layer was reduced in thickness and less vascularized, as shown by LacZ staining of placentas carrying a ROSA26 allele (Fig. 2M and N).
Abnormal vasculature and impaired vascular smooth muscle cells (VSMC)/preicytes development in Smad4Co/Co; Tie2-Cre embryos. Globally, Smad4Co/Co; Tie2-Cre embryos at E10 exhibited a coarse vascular plexus and a significant reduction of capillary vessels (Fig. 3A and B). Oversized and blind-ending vessels were easily observed in the mutant heads (Fig. 3C and D). Histologically, E9.5 mutant embryos had collapsed DAs with irregularly narrowed lumens (Fig. 3E to H and K to O), while the venous system such as the ACV showed moderately increased lumen diameters (Fig. 3H). Moreover, the parallel capillary branching from DA to somites diminished, showing nonorganized EC clusters around the DA instead (Fig. 3F). Although vessels sprouting into the neural tissue could easily be seen in E9.5 control embryos (Fig. 3G), the perineural vascular plexus of the mutant embryos was dilated and failed to invade the neuroepithelium (Fig. 3H). In addition, electron microscopy demonstrated fewer junctions with numerous intervals between adjacent mutant vascular ECs (Fig. 3I and J).
|
-SMA immunostaining to illustrate the maturation of embryonic vessels. Transverse sections of DA revealed the development of VSMC/pericytes around the ECs of normal embryos at E9.5 (Fig. 3K and M). Nevertheless, -SMA-positive cell around DA was hardly detected in comparable sections of the mutant embryos and failed to make close contact with adjacent ECs if any appeared (Fig. 3L and 3N). Furthermore, -SMA expression in the somites was equivalent between the mutant and the control embryos (arrows in Fig. 3K and L), indicating that the loss of -SMA expression was specific for VSMC/pericytes. Of note, the expression of laminin, a constituent of ECM, was unaltered in the mutant embryos, although the distribution was discontinuous due to the rupture of vascular lumens (Fig. 3O). Defective myocardial development and endocardial cushion formation in Smad4Co/Co; Tie2-Cre embryos. We further determined whether Smad4 deficiency in the endocardium affected embryonic heart development. Smad4Co/Co; Tie2-Cre embryos had enlarged hearts and showed edematous pericardium (Fig. 4A and B). Dilated ventricles with reduced trabeculae of the mutant hearts were in sharp contrast to the controls (Fig. 4C to F). In some serious cases, the endocardial layer of the mutant hearts was collapsed into disorganized multiple layers and detached from myocardial trabeculae (Fig. 4G and H).
|
Reduced in vitro tube formation of ECs derived from Smad4Co/Co; Tie2-Cre mice. Since vessels of the mutant embryos were devoid of sprouting into placental labyrinth and neural tissue, we further investigated in vitro tube-forming capacity on Matrigel of mutant embryonic ECs to determine whether an intrinsic functional defect existed. Two strategies were used to obtain purified ECs from embryos. For sorted Tie2-positive cells, the purity of ECs was >90%, as evaluated by Dil-Ac-LDL incorporation (Fig. 5A). As for PmT-transformed ECs, both Dil-Ac-LDL uptake and VE-cadherin immunostaining verified their endothelial property and demonstrated unaltered expression in the Smad4-deficient ECs (Fig. 5A and data not shown). The absence of Smad4 expression in the mutant ECs was confirmed by RT-PCR and Western blot analysis (Fig. 5B). Eight hours after being plated on Matrigel, the Smad4-dificient ECs exhibited significantly attenuated capacity of forming two-dimensional tube-like structures, although they were able to change their shape and form tubes to some extent (Fig. 5C). We observed a 35% decrease in the number of tubes and a 44% reduction in the total tube length (Fig. 5D). The results strongly suggested the disrupted intrinsic function in the tube formation of Smad4-deficient ECs in vitro, corroborating the abnormal vessel sprouting and remodeling in vivo.
|
|
Given that Cx43-mediated gap junctional communication between endothelial and mesenchymal cells is crucial for mural cell differentiation (15), we examined the expression of Cx43, which is also involved in mouse vascular development (25). Cx43 expression was reduced in cultured Smad4-deficient ECs (Fig. 7A). Upon TGF-ß1 treatment, Cx43 expression was upregulated in the control ECs as previously reported (21). However, we demonstrated at both RNA and protein levels that the expression of Cx43 hardly increased but rather decreased in the mutant ECs when treated with TGF-ß1 (Fig. 7B and C). Importantly, a remarkable reduction of Cx43 expression was detected in the vascular ECs of E9.5 mutant embryos in vivo (Fig. 7D).
|
|
DISCUSSION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
We revealed an indispensable function of endothelial Smad4 in angiogenesis. Smad4-deficient ECs were able to assemble into a primary vascular plexus, suggesting that vasculogenesis was intact in the mutant mice. However, the Smad4-deficient primary vessels failed to undergo remodeling and efficient spouting in vivo, indicating that angiogenesis is definitely abnormal in Smad4 mutants. The angiogenesis defects largely mimicked the phenotypes of the loss-of-function mutant mice in which a variety of other members of TGF-ß signaling pathway were globally disrupted (7, 22, 24, 31, 32, 48), a finding indicative of an essential cell-autonomous role of Smad4 signaling in ECs during vascular development. Most recently, a conditional Smad4 knockout mouse driven by a Flk1 promoter has been reported to manifest similar angiogenesis defects in the YS, whereas the influence on embryos proper is not known (34). Since Flk1 expression marks a broad spectrum of mesodermal progenitors besides ECs in the early mouse embryos (9, 30), it may further emphasize the endothelium as the predominant cell type responsible for Smad4-related vascular defect. Actually, the Smad4Co/Co; Tie2-Cre embryos also exhibited some distinct phenotypes. The DAs of Smad4Co/Co; Tie2-Cre embryos displayed irregularly narrowed lumen and were ruptured. It was much more outstanding than the changes in veins with moderate dilatation only. In the TGF-ß1, TßR2, ALK5, and endoglin knockout embryos, the lumen size and shape of the DAs are comparable to those found in wild-type mice (7, 22, 24, 32), whereas in the ALK1 and Smad5 knockout mice, the blood vessels are greatly dilated at E9.5 (31, 48). In contrast to a recent study showing that the Tie2 promoter is much more active in the arterial system than in the venous system (1), the Tie2 promoter in combination with the enhancer fragment we used in the present study ensured the equivalent Cre expression throughout the cardiovascular system (19, 36). Since Smad4 was authentically deleted in all vascular ECs (Fig. 1G and 5B), the phenotypic discrepancy between the DA and the venous system in the mutant E9.5 embryos presumably resulted from distinct roles rather than different knockout efficiency of Smad4 in arterial versus venous vessels. Notably, ALK1 is predominantly expressed in arteries over veins (12, 38), suggesting that Smad4-mediated ALK1 signals might play somewhat more significant roles in arterial ECs. It requires further clarification as to how Smad4 deficiency in ECs exerts different influences on the structure of arterial versus venous vessels.
Disrupted remodeling of embryonic vasculature in Smad4 mutants was largely due to intrinsic functional defects of Smad4 mutant ECs. Gain-of-function studies show that, whereas ALK1 via Smad1/5 stimulates EC migration and tube formation (12, 47), ALK5 via Smad2/3 inhibits EC migration and tube formation (12, 33, 47). Hence, TGF-ß exerts bifunctional effects on EC via a fine balance between ALK5 and ALK1 signaling (12). Moreover, the opposite effects on EC migration mediated by ALK1 and ALK5 are Smad4 dependent (12). Nevertheless, ALK5-deficient ECs demonstrate intact tube-forming properties in vitro (22), and up-to-date, few evidence has shown EC defect in tube formation in vitro by loss-of-function studies on TGF-ß signaling components. Here, we demonstrated the intrinsic defect in tube formation of Smad4-defecient ECs, which was a little unexpected since exogenous TGF-ß also inhibits the formation of tube-like structures of ECs on Matrigel (33). The defect presumably resulted from a disturbed balance between the ALK1 and ALK5 pathways. Except for TGF-ß1, the significance of BMP signaling in EC behavior has been elucidated in vitro and in vivo (34, 46). The Smad4-deficent ECs remarkably reduced the responsiveness to BMP2 and manifested a significant decrease in Id1 expression, which is a direct target of both TGF-ß1-ALK1 and BMP signaling. Therefore, disruption of Smad4-mediated BMP signaling may synergistically account for the attenuated tube-forming capacity of Smad4-deficient ECs and the vascular abnormalities of mutant embryos.
TGF-ß1 is thought to have a direct stimulatory effect on the synthesis and deposition of ECM components by ECs, inducing Fn and laminin production through ALK5 signaling (33, 45). Unexpectedly, the expression of Fn and laminin demonstrated hardly reduced in the Smad4-deficient ECs, although ALK5-Smad2/3 signaling was indeed attenuated. These results suggested that Smad4 is not required for the expression of some ECM components in developing ECs. Supportively, TGF-ß can modulate the expression of Fn gene in a Smad4-independent manner in some non-EC cell types (17, 44).
Deletion of Smad4 exclusively in ECs might provide abnormal signals for impaired cellular interaction between endothelial and surrounding cells. Angiopoietins promote vessel stability by activating (Ang1) or antagonizing (Ang2) signaling via the Tie2 receptor (4). The disturbed balance between Ang1 and Ang2, especially the remarkable increase of Ang2, in the Smad4Co/Co; Tie2-Cre embryos was inclined to destroy the vessel stability. In mid-gestational mouse embryos, Ang2 expression was abundant in the DA and major aortic branches, specifically in the VSMC beneath the vessel endothelium (10, 27). However, its expression was induced within the ECs of small vessels at sites of vascular remodeling (8, 10). Although increased expression of Ang2 in the mutant embryos could be a secondary response to the remodeling defects in vivo, most importantly, we revealed that lack of Smad4 in ECs lead to a cell-autonomous increase of endothelial Ang2 expression. In vitro, exogenous Ang2 destabilizes quiescent endothelium and leads to prominent EC detachment in a three-dimensional coculture model (35). It is noteworthy that some of the vascular defects in Smad4Co/Co; Tie2-Cre embryos were highly reminiscent of those observed in Ang2 overexpression transgenic mice, particularly the notable vessel discontinuities and the apparent collapse of endocardium (27). Interestingly, although neither ALK1 nor ALK5 alone modulates Ang2 expression (33, 47), TGF-ß1 downregulated Ang2 expression in a Smad4-dependent manner, implying somewhat complicated regulatory mechanisms. It is possible that Smad4 signaling influences EC behavior partially through modulating angiopoietin signals or that there exists a cross talking between the two pathways, but further investigation is required.
By virtue, dissociation of ECs away from the surrounding cells in Smad4Co/Co; Tie2-Cre embryos was universal and prominent. Here we highlighted a candidate molecule, Cx43, as a structure basis for the intercellular disassociation. The decreased expression of Cx43 in the Smad4-deficient ECs was most likely cell autonomous because the results shown both in situ and in the cultured ECs were coincident. Interestingly, the responsiveness of Cx43 expression to TGF-ß1 treatment in the mutant ECs was converse to that in the controls, suggesting that Smad4-independent regulation of endothelial Cx43 expression by TGF-ß1 might exist. Functionally, Cx43-mediated gap junctional communication between endothelial and mesenchymal cells is crucial for latent TGF-ß1 activation and subsequent endothelium-induced mural cell differentiation (15). Hence, it is conceivable that cell contact-dependent activation of TGF-ß1 may be largely disturbed in the Smad4 mutant embryos (2), although TGF-ß1 transcription remained unchanged. Supportively, it is suggested that disruption of TGF-ß signaling exclusively in vascular ECs results in reduced availability of TGF-ß1 protein to adjacent cells, whereas the transcription of TGF-ß1 is unaltered (5). Thus, cell-autonomous downregulation of Cx43 in the Smad4-deficient ECs, which probably results in impaired gap junctional communication between ECs and surrounding cells, might contribute to the impaired VSMC recruitment and destabilized vasculature in the mutant embryos.
In summary, we have provided direct functional evidences that Smad4 activity in the developing ECs is essential for embryonic cardiovascular development. Further studies investigating the detailed molecular basis of Smad4 signaling in ECs, possibly including angiopoietins and gap junction molecules, are required to understand definitely the role of Smad4 signaling in vascular remodeling and integrity in embryonic development, as well as in adult tissues and tumor models.
|
ACKNOWLEDGMENTS |
|---|
We thank Chuxia Deng at the National Institutes of Health (NIH) for the Smad4 conditional gene targeting mouse, Hua Gu at the NIH for Cre plasmid, and Xiang Gao at Nanjing University for the mouse strains. We also thank Jincai Luo at Peking University for instruction on selective transformation of ECs and related retrovirus and cell lines, and we thank Yeguang Chen at Tsinghua University for reporters.
|
FOOTNOTES |
|---|
Published ahead of print on 27 August 2007.
Supplemental material for this article may be found at http://mcb.asm.org/.
|
REFERENCES |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
2. Antonelli-Orlidge, A., K. B. Saunders, S. R. Smith, and P. A. D'Amore. 1989. An activated form of transforming growth factor beta is produced by cocultures of endothelial cells and pericytes. Proc. Natl. Acad. Sci. USA 86: 4544-4548.
3. Armstrong, E. J., and J. Bischoff. 2004. Heart valve development: endothelial cell signaling and differentiation. Circ. Res. 95: 459-470.
4. Armulik, A., A. Abramsson, and C. Betsholtz. 2005. Endothelial/pericyte interactions. Circ. Res. 97: 512-523.
5. Carvalho, R. L., L. Jonker, M. J. Goumans, J. Larsson, P. Bouwman, S. Karlsson, P. T. Dijke, H. M. Arthur, and C. L. Mummery. 2004. Defective paracrine signaling by TGFß in yolk sac vasculature of endoglin mutant mice: a paradigm for hereditary haemorrhagic telangiectasia. Development 131: 6237-6247.
6. Dennler, S., S. Itoh, D. Vivien, P. ten Dijke, S. Huet, and J. M. Gauthier. 1998. Direct binding of Smad3 and Smad4 to critical TGFß-inducible elements in the promoter of human plasminogen activator inhibitor-type 1 gene. EMBO J. 17: 3091-3100.[CrossRef][Medline]
7. Dickson, M. C., J. S. Martin, F. M. Cousins, A. B. Kulkarni, S. Karlsson, and R. J. Akhurst. 1995. Defective hematopoiesis and vasculogenesis in transforming growth factor-ß1 knockout mice. Development 121: 1845-1854.[Abstract]
8. Eklund, L., and B. R. Olsen. 2006. Tie receptors and their angiopoietin ligands are context-dependent regulators of vascular remodeling. Exp. Cell Res. 312: 630-641.[CrossRef][Medline]
9. Ema, M., S. Takahashi, and J. Rossant. 2006. Deletion of the selection cassette, but not cis-acting elements, in targeted Flk1-lacZ allele reveals Flk1 expression in multipotent mesodermal progenitors. Blood 107: 111-117.
10. Gale, N. W., G. Thurston, S. F. Hackett, R. Renard, Q. Wang, J. McClain, C. Martin, C. Witte, M. H. Witte, D. Jackson, C. Suri, P. A. Campochiaro, S. J. Wiegand, and G. D. Yancopoulos. 2002. Angiopoietin-2 is required for postnatal angiogenesis and lymphatic patterning, and only the latter role is rescued by Angiopoietin-1. Dev. Cell 3: 411-423.[CrossRef][Medline]
11. Gallione, C. J., G. M. Repetto, E. Legius, A. K. Rustgi, S. L. Schelley, S. Tejpar, G. Mitchell, E. Drouin, C. J. Westermann, and D. A. Marchuk. 2004. A combined syndrome of juvenile polyposis and hereditary haemorrhagic telangiectasia associated with mutations in MADH4 (SMAD4). Lancet 363: 852-859.[CrossRef][Medline]
12. Goumans, M. J., G. Valdimarsdottir, S. Itoh, A. Rosendahl, P. Sideras, and P. ten Dijke. 2002. Balancing the activation state of the endothelium via two distinct TGF-ß type I receptors. EMBO J. 21: 1743-1753.[CrossRef][Medline]
13. Hata, A., J. Seoane, G. Lagna, E. Montalvo, A. Hemmati-Brivanlou, and J. Massague. 2000. OAZ uses distinct DNA- and protein-binding zinc fingers in separate BMP-Smad and Olf signaling pathways. Cell 100: 229-240.[CrossRef][Medline]
14. Hatzopoulos, A. K., J. Folkman, E. Vasile, G. K. Eiselen, and R. D. Rosenberg. 1998. Isolation and characterization of endothelial progenitor cells from mouse embryos. Development 125: 1457-1468.[Abstract]
15. Hirschi, K. K., J. M. Burt, K. D. Hirschi, and C. Dai. 2003. Gap junction communication mediates transforming growth factor-beta activation and endothelial-induced mural cell differentiation. Circ. Res. 93: 429-437.
16. Hirschi, K. K., S. A. Rohovsky, and P. A. D'Amore. 1998. PDGF, TGF-beta, and heterotypic cell-cell interactions mediate endothelial cell-induced recruitment of 10T1/2 cells and their differentiation to a smooth muscle fate. J. Cell Biol. 141: 805-814.
17. Hocevar, B. A., T. L. Brown, and P. H. Howe. 1999. TGF-beta induces fibronectin synthesis through a c-Jun N-terminal kinase-dependent, Smad4-independent pathway. EMBO J. 18: 1345-1356.[CrossRef][Medline]
18. Johnson, D. W., J. N. Berg, M. A. Baldwin, C. J. Gallione, I. Marondel, S. J. Yoon, T. T. Stenzel, M. Speer, M. A. Pericak-Vance, A. Diamond, A. E. Guttmacher, C. E. Jackson, L. Attisano, R. Kucherlapati, M. E. Porteous, and D. A. Marchuk. 1996. Mutations in the activin receptor-like kinase 1 gene in hereditary haemorrhagic telangiectasia type 2. Nat. Genet. 13: 189-195.[CrossRef][Medline]
19. Kisanuki, Y. Y., R. E. Hammer, J. Miyazaki, S. C. Williams, J. A. Richardson, and M. Yanagisawa. 2001. Tie2-Cre transgenic mice: a new model for endothelial cell-lineage analysis in vivo. Dev. Biol. 230: 230-242.[CrossRef][Medline]
20. Kwak, B. R., M. S. Pepper, D. B. Gros, and P. Meda. 2001. Inhibition of endothelial wound repair by dominant negative connexin inhibitors. Mol. Biol. Cell 12: 831-845.
21. Larson, D. M., T. G. Christensen, G. D. Sagar, and E. C. Beyer. 2001. TGF-beta1 induces an accumulation of connexin43 in a lysosomal compartment in endothelial cells. Endothelium 8: 255-260.[Medline]
22. Larsson, J., M. J. Goumans, L. J. Sjostrand, M. A. van Rooijen, D. Ward, P. Leveen, X. Xu, P. ten Dijke, C. L. Mummery, and S. Karlsson. 2001. Abnormal angiogenesis but intact hematopoietic potential in TGF-ß type I receptor-deficient mice. EMBO J. 20: 1663-1673.[CrossRef][Medline]
23. Lebrin, F., M. Deckers, P. Bertolino, and P. Ten Dijke. 2005. TGF-beta receptor function in the endothelium. Cardiovasc. Res. 65: 599-608.
24. Li, D. Y., L. K. Sorensen, B. S. Brooke, L. D. Urness, E. C. Davis, D. G. Taylor, B. B. Boak, and D. P. Wendel. 1999. Defective angiogenesis in mice lacking endoglin. Science 284: 1534-1537.
25. Li, W. E., K. Waldo, K. L. Linask, T. Chen, A. Wessels, M. S. Parmacek, M. L. Kirby, and C. W. Lo. 2002. An essential role for connexin43 gap junctions in mouse coronary artery development. Development 129: 2031-2042.[Medline]
26. Li, W. L., X. Cheng, X. H. Tan, J. S. Zhang, Y. S. Sun, L. Chen, and X. Yang. 2005. Endothelial cell-specific expression of Cre recombinase in transgenic mice. Yi Chuan Xue Bao 32: 909-915.[Medline]
27. Maisonpierre, P. C., C. Suri, P. F. Jones, S. Bartunkova, S. J. Wiegand, C. Radziejewski, D. Compton, J. McClain, T. H. Aldrich, N. Papadopoulos, T. J. Daly, S. Davis, T. N. Sato, and G. D. Yancopoulos. 1997. Angiopoietin-2, a natural antagonist for Tie2 that disrupts in vivo angiogenesis. Science 277: 55-60.
28. Mandriota, S. J., and M. S. Pepper. 1998. Regulation of angiopoietin-2 mRNA levels in bovine microvascular endothelial cells by cytokines and hypoxia. Circ. Res. 83: 852-859.[Medline]
29. McAllister, K. A., K. M. Grogg, D. W. Johnson, C. J. Gallione, M. A. Baldwin, C. E. Jackson, E. A. Helmbold, D. S. Markel, W. C. McKinnon, J. Murrell, et al. 1994. Endoglin, a TGF-ß binding protein of endothelial cells, is the gene for hereditary haemorrhagic telangiectasia type 1. Nat. Genet. 8: 345-351.[CrossRef][Medline]
30. Motoike, T., D. W. Markham, J. Rossant, and T. N. Sato. 2003. Evidence for novel fate of Flk1+ progenitor: contribution to muscle lineage. Genesis 35: 153-159.[CrossRef][Medline]
31. Oh, S. P., T. Seki, K. A. Goss, T. Imamura, Y. Yi, P. K. Donahoe, L. Li, K. Miyazono, P. ten Dijke, S. Kim, and E. Li. 2000. Activin receptor-like kinase 1 modulates transforming growth factor-ß1 signaling in the regulation of angiogenesis. Proc. Natl. Acad. Sci. USA 97: 2626-2631.
32. Oshima, M., H. Oshima, and M. M. Taketo. 1996. TGF-beta receptor type II deficiency results in defects of yolk sac hematopoiesis and vasculogenesis. Dev. Biol. 179: 297-302.[CrossRef][Medline]
33. Ota, T., M. Fujii, T. Sugizaki, M. Ishii, K. Miyazawa, H. Aburatani, and K. Miyazono. 2002. Targets of transcriptional regulation by two distinct type I receptors for transforming growth factor-beta in human umbilical vein endothelial cells. J. Cell Physiol. 193: 299-318.[CrossRef][Medline]
34. Park, C., K. Lavine, Y. Mishina, C. X. Deng, D. M. Ornitz, and K. Choi. 2006. Bone morphogenetic protein receptor 1A signaling is dispensable for hematopoietic development but essential for vessel and atrioventricular endocardial cushion formation. Development 133: 3473-3484.
35. Scharpfenecker, M., U. Fiedler, Y. Reiss, and H. G. Augustin. 2005. The Tie-2 ligand angiopoietin-2 destabilizes quiescent endothelium through an internal autocrine loop mechanism. J. Cell Sci. 118: 771-780.
36. Schlaeger, T. M., S. Bartunkova, J. A. Lawitts, G. Teichmann, W. Risau, U. Deutsch, and T. N. Sato. 1997. Uniform vascular-endothelial-cell-specific gene expression in both embryonic and adult transgenic mice. Proc. Natl. Acad. Sci. USA 94: 3058-3063.
37. Schwarte-Waldhoff, I., O. V. Volpert, N. P. Bouck, B. Sipos, S. A. Hahn, S. Klein-Scory, J. Luttges, G. Kloppel, U. Graeven, C. Eilert-Micus, A. Hintelmann, and W. Schmiegel. 2000. Smad4/DPC4-mediated tumor suppression through suppression of angiogenesis. Proc. Natl. Acad. Sci. USA 97: 9624-9629.
38. Seki, T., J. Yun, and S. P. Oh. 2003. Arterial endothelium-specific activin receptor-like kinase 1 expression suggests its role in arterialization and vascular remodeling. Circ. Res. 93: 682-689.
39. Soriano, P. 1999. Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat. Genet. 21: 70-71.[CrossRef][Medline]
40. Sun, Y., J. Zhou, X. Liao, Y. Lu, C. Deng, P. Huang, Q. Chen, and X. Yang. 2005. Disruption of Smad5 gene induces mitochondria-dependent apoptosis in cardiomyocytes. Exp. Cell Res. 306: 85-93.[CrossRef][Medline]
41. Tang, N., F. Mack, V. H. Haase, M. C. Simon, and R. S. Johnson. 2006. pVHL function is essential for endothelial extracellular matrix deposition. Mol. Cell. Biol. 26: 2519-2530.
42. Taylor, L. M., and L. M. Khachigian. 2000. Induction of platelet-derived growth factor B-chain expression by transforming growth factor-beta involves transactivation by Smads. J. Biol. Chem. 275: 16709-16716.
43. ten Dijke, P., and C. S. Hill. 2004. New insights into TGF-ß-Smad signaling. Trends Biochem. Sci. 29: 265-273.[CrossRef][Medline]
44. Tsuchida, K., Y. Zhu, S. Siva, S. R. Dunn, and K. Sharma. 2003. Role of Smad4 on TGF-ß-induced extracellular matrix stimulation in mesangial cells. Kidney Int. 63: 2000-2009.[CrossRef][Medline]
45. Usui, T., M. Takase, Y. Kaji, K. Suzuki, K. Ishida, T. Tsuru, K. Miyata, M. Kawabata, and H. Yamashita. 1998. Extracellular matrix production regulation by TGF-ß in corneal endothelial cells. Investig. Ophthalmol. Vis. Sci. 39: 1981-1989.
46. Valdimarsdottir, G., M. J. Goumans, A. Rosendahl, M. Brugman, S. Itoh, F. Lebrin, P. Sideras, and P. ten Dijke. 2002. Stimulation of Id1 expression by bone morphogenetic protein is sufficient and necessary for bone morphogenetic protein-induced activation of endothelial cells. Circulation 106: 2263-2270.
47. Wu, X., J. Ma, J. D. Han, N. Wang, and Y. G. Chen. 2006. Distinct regulation of gene expression in human endothelial cells by TGF-ß and its receptors. Microvasc. Res. 71: 12-19.[CrossRef][Medline]
48. Yang, X., L. H. Castilla, X. Xu, C. Li, J. Gotay, M. Weinstein, P. P. Liu, and C. X. Deng. 1999. Angiogenesis defects and mesenchymal apoptosis in mice lacking SMAD5. Development 126: 1571-1580.[Abstract]
49. Yang, X., C. Li, P. L. Herrera, and C. X. Deng. 2002. Generation of Smad4/Dpc4 conditional knockout mice. Genesis 32: 80-81.[CrossRef][Medline]
50. Yang, X., C. Li, X. Xu, and C. Deng. 1998. The tumor suppressor SMAD4/DPC4 is essential for epiblast proliferation and mesoderm induction in mice. Proc. Natl. Acad. Sci. USA 95: 3667-3672.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| J. Bacteriol. | J. Virol. | Eukaryot. Cell |
|---|
| Microbiol. Mol. Biol. Rev. | Clin. Vaccine Immunol. | All ASM Journals |
|---|
