血管内皮细胞发育及分子机制

心血管系统是胚胎发育中最先形成的器官之一, 为机体提供营养成分和氧气。血管发育包括两部分, 一是内皮祖细胞(Angioblast)聚集形成血管原基(Vasculogenesis), 二是从已有血管形成新的血管分支(Angiogenesis)。此后由初级内皮细胞管召集平滑肌细胞形成功能性血管(Vessel maturation)。内皮祖细胞起源途径包括：由Flk1阳性中胚层细胞到成血成血管细胞(Hemangioblast)到血管内皮祖细胞; 或由Flk1阳性中胚层细胞直接到血管内皮祖细胞。Flk1阳性中胚层细胞受到vegf、flk1、cloche、lycat、etsrp等关键基因或信号通路的调节, 其中核心问题是原肠期中胚层如何形成Flk1阳性中胚层细胞及进一步分化成血管内皮祖细胞和成血血管细胞。文章集中评述内皮祖细胞发育、分化及其分子遗传调控机制, 并展望本领域未来发展方向。     

The VEGF receptor Flt-1 spatially modulates Flk-1 signaling and blood vessel branching

Blood vessel formation requires the integrated regulation of endothelial cell proliferation and branching morphogenesis, but how this coordinated regulation is achieved is not well understood. Flt-1 (vascular endothelial growth factor [VEGF] receptor 1) is a high affinity VEGF-A receptor whose loss leads to vessel overgrowth and dysmorphogenesis. We examined the ability of Flt-1 isoform transgenes to rescue the vascular development of embryonic stem cell-derived flt-1-/- mutant vessels. Endothelial proliferation was equivalently rescued by both soluble (sFlt-1) and membrane-tethered (mFlt-1) isoforms, but only sFlt-1 rescued vessel branching. Flk-1 Tyr-1173 phosphorylation was increased in flt-1-/- mutant vessels and partially rescued by the Flt-1 isoform transgenes. sFlt-1-rescued vessels exhibited more heterogeneous levels of pFlk than did mFlt-1-rescued vessels, and reporter gene expression from the flt-1 locus was also heterogeneous in developing vessels. Our data support a model whereby sFlt-1 protein is more efficient than mFlt-1 at amplifying initial expression differences, and these amplified differences set up local discontinuities in VEGF-A ligand availability that are important for proper vessel branching.     

Msx genes define a population of mural cell precursors required for head blood vessel maturation

Vessels are primarily formed from an inner endothelial layer that is secondarily covered by mural cells, namely vascular smooth muscle cells (VSMCs) in arteries and veins and pericytes in capillaries and veinules. We previously showed that, in the mouse embryo, Msx1(lacZ) and Msx2(lacZ) are expressed in mural cells and in a few endothelial cells. To unravel the role of Msx genes in vascular development, we have inactivated the two Msx genes specifically in mural cells by combining the Msx1(lacZ), Msx2(lox) and Sm22α-Cre alleles. Optical projection tomography demonstrated abnormal branching of the cephalic vessels in E11.5 mutant embryos. The carotid and vertebral arteries showed an increase in caliber that was related to reduced vascular smooth muscle coverage. Taking advantage of a newly constructed Msx1(CreERT2) allele, we demonstrated by lineage tracing that the primary defect lies in a population of VSMC precursors. The abnormal phenotype that ensues is a consequence of impaired BMP signaling in the VSMC precursors that leads to downregulation of the metalloprotease 2 (Mmp2) and Mmp9 genes, which are essential for cell migration and integration into the mural layer. Improper coverage by VSMCs secondarily leads to incomplete maturation of the endothelial layer. Our results demonstrate that both Msx1 and Msx2 are required for the recruitment of a population of neural crest-derived VSMCs.     

Differential expression of KDR/VEGFR-2 and CD34 during mesoderm development of the early human embryo.

Recent findings on vertebrate embryos have provided compelling evidence for the existence of hemangioblasts, i.e. common precursors for endothelial and hematopoietic cells, characterized by expression of the VEGFR2/Flk1 receptor. We describe here a population of KDR+ CD34- mesoderm cells that emerges in early-somitic human embryos, by the beginning of the 4th week of gestation. In the developing blood vessels, KDR-expressing CD34- cells gradually coexpress increasing levels of CD34 antigen. Remarkably, as development proceeds, a KDR+ CD34- contingent persists in the paraaortic splanchnopleura until just prior to the emergence of aorta-associated hematopoietic cell clusters. These observations suggest that KDR+ CD34- mesodermal cells might represent the putative hemangioblastic precursor of human hematopoietic and endothelial lineages.     

Induction of cells expressing vascular endothelium markers from undifferentiated Xenopus presumptive ectoderm by co-treatment with activin and angiopoietin-2

Activin is a potent inducer of mesoderm in amphibian embryos. We previously reported that low concentrations of activin could induce the formation of blood cells from Xenopus explants (animal caps). Both hematopoietic and vascular endothelial cell lineages are believed to share a common precursor, termed hemangioblasts. In this study, we tried to induce differentiation of vascular endothelial cells in aggregates derived from Xenopus animal caps. Aggregates formed from cells that were co-treated with activin and angiopoietin-2 expressed the vascular endothelial markers, X-msr, Xtie2 and Xegfl7. However, none of these aggregates expressed the hematopoietic marker genes, globin alpha T3, alpha T5, alpha A or GATA-1. We used microarray analysis to compare the gene expression profiles of aggregates treated with activin alone or with activin and angiopoietin. The combination, but not activin alone, induced expression of vascular-related genes such as Xl-fli and VEGF. These results demonstrate that treatment of dissociated animal cap cells with activin and angiopoietin-2 can induce differentiation of endothelial cells, and provides a promising model system for the in vitro study of blood vessel induction in vertebrates.     

KLF2 transcription factor modulates blood vessel maturation through smooth muscle cell migration

Vasculogenesis, angiogenesis, and maturation are three major phases of the development of blood vessels. Although many receptors required for blood vessel formation have been defined, the intracellular signal transduction pathways involved in vascular maturation remain unclear. KLF2(-/-) embryos fail to develop beyond 13.5 days because of a lack of blood vessel stabilization. The molecular mechanism of KLF2 function in embryonic vascular vessels is still largely unknown. Here we show a normal development pattern of endothelial cells in KLF2(-/-) embryos but a defect of smooth muscle cells at the dorsal side of the aorta. This phenotype results from arrested vascular maturation characterized by the failure of mural cells to migrate around endothelial cells. This migration defect is also observed when platelet-derived growth factor-B (PDGF) controlled migration is studied in murine embryonic fibroblast (MEF) cells from KLF2(-/-) animals. In addition, KLF2(-/-) MEFs exhibit a significant growth defect, indicating that KLF2 is required to maintain the viability of MEF cells. The PDGF signal is mediated through the Src signaling pathway, and a downstream target of KLF2 is sphingosine 1-phosphate receptor 1. These studies demonstrate that KLF2 is required for smooth muscle cell migration and elucidate a novel mechanism involving communication between PDGF and KLF2 in vascular maturation.     

Wnt2 coordinates the commitment of mesoderm to hematopoietic, endothelial, and cardiac lineages in embryoid bodies

Our recent gene expression profiling analyses demonstrated that Wnt2 is highly expressed in Flk1(+) cells, which serve as common progenitors of endothelial cells, blood cells, and mural cells. In this report, we characterize the role of Wnt2 in mesoderm development during embryonic stem (ES) cell differentiation by creating ES cell lines in which Wnt2 was deleted. Wnt2(-/-) embryoid bodies (EBs) generated increased numbers of Flk1(+) cells and blast colony-forming cells compared with wild-type EBs, and had higher Flk1 expression at comparable stages of differentiation. Although Flk1(+) cells were increased, we found that endothelial cell and terminal cardiomyocyte differentiation was impaired, but hematopoietic cell differentiation was enhanced and smooth muscle cell differentiation was unchanged in Wnt2(-/-) EBs. Later stage Wnt2(-/-) EBs had either lower or undetectable expression of endothelial and cardiac genes compared with wild-type EBs. Consistently, vascular plexi were poorly formed and neither beating cardiomyocytes nor alpha-actinin-staining cells were detectable in later stage Wnt2(-/-) EBs. In contrast, hematopoietic cell gene expression was upregulated, and the number of hematopoietic progenitor colonies was significantly enhanced in Wnt2(-/-) EBs. Our data indicate that Wnt2 functions at multiple stages of development during ES cell differentiation and during the commitment and diversification of mesoderm: as a negative regulator for hemangioblast differentiation and hematopoiesis but alternatively as a positive regulator for endothelial and terminal cardiomyocyte differentiation.     

Analysis of a zebrafish VEGF receptor mutant reveals specific disruption of angiogenesis

Blood vessels form either by the assembly and differentiation of mesodermal precursor cells (vasculogenesis) or by sprouting from preexisting vessels (angiogenesis). Endothelial-specific receptor tyrosine kinases and their ligands are known to be essential for these processes. Targeted disruption of vascular endothelial growth factor (VEGF) or its receptor kdr (flk1, VEGFR2) in mouse embryos results in a severe reduction of all blood vessels, while the complete loss of flt1 (VEGFR1) leads to an increased number of hemangioblasts and a disorganized vasculature. In a large-scale forward genetic screen, we identified two allelic zebrafish mutants in which the sprouting of blood vessels is specifically disrupted without affecting the assembly and differentiation of angioblasts. Molecular cloning revealed nonsense mutations in flk1. Analysis of mRNA expression in flk1 mutant embryos showed that flk1 expression was severely downregulated, while the expression of other genes (scl, gata1, and fli1) involved in vasculogenesis or hematopoiesis was unchanged. Overexpression of vegf(121+165) led to the formation of additional vessels only in sibling larvae, not in flk1 mutants. We demonstrate that flk1 is not required for proper vasculogenesis and hematopoiesis in zebrafish embryos. However, the disruption of flk1 impairs the formation or function of vessels generated by sprouting angiogenesis.     

Stem cell-derived endothelial cells/progenitors migrate and pattern in the embryo using the VEGF signaling pathway

Endothelial precursor cells respond to molecular cues to migrate and assemble into embryonic blood vessels, but the signaling pathways involved in vascular patterning are not well understood. We recently showed that avian vascular patterning cues are recognized by mammalian angioblasts derived from somitic mesoderm through analysis of mouse-avian chimeras. To determine whether stem cell-derived endothelial cells/progenitors also recognize global patterning signals, murine ES cell-derived embryoid bodies (EBs) were grafted into avian hosts. ES cell-derived murine endothelial cells/progenitors migrated extensively and colonized the appropriate host vascular beds. They also formed mosaic vessels with avian endothelial cells. Unlike somite derived-endothelial cells, ES cell-derived endothelial cells/progenitors migrated across the host embryonic midline to the contralateral side. To determine the role of VEGF signaling in embryonic vascular patterning, EBs mutant for a VEGF receptor (flk-1(-/-)) or a signal (VEGF-A(-/-)) were grafted into quail hosts. Flk-1(-/-) EB grafts produced only rare endothelial cells that did not migrate or assemble into vessels. In contrast, VEGF-A(-/-) EB grafts produced endothelial cells that resembled wild-type and colonized host vascular beds, suggesting that host-derived signals can partially rescue mutant graft vascular patterning. VEGF-A(-/-) graft endothelial cells/progenitors crossed the host midline with much lower frequency than wild-type EB grafts, indicating that graft-derived VEGF compromised the midline barrier when present. Thus, ES cell-derived endothelial cells/progenitors respond appropriately to global vascular patterning cues, and they require the VEGF signaling pathway to pattern properly. Moreover, EB-avian chimeras provide an efficient way to screen mutations for vascular patterning defects.     

Inhibition of hepatic stellate cell contraction during activation in vitro by vascular endothelial growth factor in association with upregulation of FLT tyrosine kinase receptor family, FLT-1.

Activated hepatic stellate cells produce vascular endothelial growth factor (VEGF). VEGF has been shown to act on mesenchymal cells as well. If hepatic stellate cells can express FLT tyrosine receptor family, flt-1 and KDR/flk-1, their function might be regulated by VEGF in an autocrine manner. This hypothesis was tested using hepatic stellate cells isolated from normal rats. Northern blot analysis and immunocytochemical study revealed that hepatic stellate cells cultured for 3 days on plastic dishes expressed both flt-1 and KDR/flk-1. When the culture was prolonged to 10 days, the flt-1 mRNA expression was increased, whereas both KDR/flk-1 mRNA and protein expressions diminished. DNA and collagen syntheses were minimal in the cells cultured for 3 days, but marked in those cultured for 10 days. Addition of recombinant human VEGF to the culture medium did not change both syntheses but attenuated an increase of smooth muscle alpha-actin expression in the cells during culture on plastic dishes and also contraction of collagen gels on which the cells were cultured. We conclude that VEGF may inhibit contraction of hepatic stellate cells appearing during activation by culture, probably through attenuation of smooth muscle alpha-actin expression via upregulated VEGF receptor, flt-1.     

An essential role for Prox1 in the induction of the lymphatic endothelial cell phenotype

The process of angiogenesis has been well documented, but little is known about the biology of lymphatic endothelial cells and the molecular mechanisms controlling lymphangiogenesis. The homeobox gene Prox1 is expressed in a subpopulation of endothelial cells that, after budding from veins, gives rise to the mammalian lymphatic system. In Prox1(-)(/-) embryos, this budding becomes arrested at around embryonic day (E)11.5, resulting in embryos without lymphatic vasculature. Unlike the endothelial cells that bud off in E11.5 wild-type embryos, those of Prox1-null embryos did not co-express any lymphatic markers such as VEGFR-3, LYVE-1 or SLC. Instead, the mutant cells appeared to have a blood vascular phenotype, as determined by their expression of laminin and CD34. These results suggest that Prox1 activity is required for both maintenance of the budding of the venous endothelial cells and differentiation toward the lymphatic phenotype. On the basis of our findings, we propose that a blood vascular phenotype is the default fate of budding embryonic venous endothelial cells; upon expression of Prox1, these budding cells adopt a lymphatic vasculature phenotype.     

Cell-autonomous requirement for beta1 integrin in endothelial cell adhesion, migration and survival during angiogenesis in mice

beta1 integrin (encoded by Itgb1) is established as a regulator of angiogenesis based upon the phenotypes of complete knockouts of beta1 heterodimer partners or ligands and upon antibody inhibition studies in mice. Its direct function in endothelial cells (ECs) in vivo has not been determined because Itgb1(-/-) embryos die before vascular development. Excision of Itgb1 from ECs and a subset of hematopoietic cells, using Tie2-Cre, resulted in abnormal vascular development by embryonic day (e) 8.5 and lethality by e10.5. Tie1-Cre mediated a more restricted excision of Itgb1 from ECs and hematopoietic cells and resulted in embryonic lethal vascular defects by e11.5. Capillaries of the yolk sacs were disorganized, and the endothelium of major blood vessels and of the heart was frequently discontinuous in mutant embryos. We also found similar vascular morphogenesis defects characterized by EC disorganization in embryonic explants and isolated ECs. Itgb1-null ECs were deficient in adhesion and migration in a ligand-specific fashion, with impaired responses to laminin and collagens, but not to fibronectin. Deletion of Itgb1 reduced EC survival, but did not affect proliferation. Our findings demonstrate that beta1 integrin is essential for EC adhesion, migration and survival during angiogenesis, and further validate that therapies targeting beta1 integrins may effectively impair neovascularization.     

Endothelial function and vascular oxidative stress in long-lived GH/IGF-deficient Ames dwarf mice

Hypopituitary Ames dwarf mice have low circulating growth hormone (GH)/IGF-I levels, and they have extended longevity and exhibit many symptoms of delayed aging. To elucidate the vascular consequences of Ames dwarfism we compared endothelial O2(-) and H2O2 production, mitochondrial reactive oxygen species (ROS) generation, expression of antioxidant enzymes, and nitric oxide (NO) production in aortas of Ames dwarf and wild-type control mice. In Ames dwarf aortas endothelial O2(-) and H2O2 production and ROS generation by mitochondria were enhanced compared with those in vessels of wild-type mice. In Ames dwarf aortas there was a less abundant expression of Mn-SOD, Cu,Zn-SOD, glutathione peroxidase (GPx)-1, and endothelial nitric oxide synthase (eNOS). NO production and acetylcholine-induced relaxation were also decreased in aortas of Ames dwarf mice. In cultured wild-type mouse aortas and in human coronary arterial endothelial cells treatment with GH and IGF significantly reduced cellular O2(-) and H2O2 production and ROS generation by mitochondria and upregulated expression of Mn-SOD, Cu,Zn-SOD, GPx-1, and eNOS. Thus GH and IGF-I promote antioxidant phenotypic changes in the endothelial cells, whereas Ames dwarfism leads to vascular oxidative stress.