血管内皮生长因子及其受体在斑马鱼胚胎血管发育中的作用

血管内皮生长因子(vascular endothelial growth factor,VEGF)是一种多功能的细胞因子,其主要作用是促进血管内皮细胞增殖和增加血管通透性,是肿瘤及正常组织血管生成的中心调控因素。以VEGF为靶点的肿瘤血管靶向性治疗成为近几年肿瘤治疗的新途径。斑马鱼作为一种重要的模式生物,被广泛用于胚胎的分子发育机制、疾病模型的构建以及药物筛选等研究中。文章对斑马鱼作为心血管系统研究模型的优势及其血管研究方法做一阐述,重点对斑马鱼VEGF及其受体的最新研究进展做了介绍,并展望了其发展前景。     

Def Functions as a Cell Autonomous Factor in Organogenesis of Digestive Organs in Zebrafish

Digestive organs originate from the endoderm. Morphogenesis of the digestive system is precisely controlled by multiple factors that dictate the cell fate and behavior so that the specific digestive organs are timely formed in the right place and develop into right size and structure. We showed previously that digestive organ expansion factor (def) is a gene whose expression is enriched in the liver, pancreas and intestine. Loss-of-function of def in the def^hi429 mutant confers hypoplastic digestive organs partly due to alteration of expression of genes related to the p53 pathway. However, the molecular mechanism for the involvement of Def in the organogenesis of digestive organs is still largely unknown. For example, it is not known whether Def regulates specific pathways in a specific organ. To address this question, we generated four independent Tg(fabp10a:def) transgenic fish lines which over-expressed Def specifically in the liver. We characterized Tg-I, one of the transgenic lines, in detail with genetic, molecular and histological approaches. We found that Tg-I restored the liver but not exocrine pancreas and intestine development in the def^hi429 mutant. However, Tg-I adult fish in the wild type (WT) background exhibits reduced liver-to-body ratio and all four transgenic lines conferred abnormal intrahepatic structure. Microarray data analysis showed that certain specific functional pathways were affected in the liver of Tg-I. These results demonstrate that Def functions in a cell autonomous manner during early liver development and aberrant Def protein expression might lead to disruption of the structural integrity of a normal adult liver.     

Effect of Vascular Cadherin Knockdown on Zebrafish Vasculature during Development

Background

Vascular endothelial cadherin (VE-cad) is essential for endothelial barrier integrity and vascular sprouting. However, the role of this important protein in cardiovascular development is only recently becoming apparent.

Methodology/Principal Findings

To characterize the role of VE-cadherin in cardiovascular development, we analyzed cardiovascular development in a zebrafish VE-cad knockdown model. Embryos deficient in VE-cad show profoundly impaired cardiac development despite having apparently normal peripheral vasculature. Initial formation of the heart proceeds normally in knockdown embryos, but subsequent looping morphogenesis is impaired. Consistent with these results, VE-cad knockdown embryos demonstrate impaired cardiac function and early circulatory arrest. Histologic examination of knockdown embryos shows persistent, abnormal separation of the endocardial and myocardial layers. Using transmission electron microscopy, we demonstrate that endocardial junctions form poorly in VE-cad knockdown embryos, with resulting leak across the endothelial layer and reduction in the density of the cardiac jelly.

Conclusions

Our results demonstrate a significant role for VE-cadherin in cardiac development independent of its effects on the formation of the peripheral vasculature.     

Identification of the Kelch Family Protein Nd1-L as a Novel Molecular Interactor of KRIT1

Loss-of-function mutations of the KRIT1 gene (CCM1) have been associated with the Cerebral Cavernous Malformation (CCM) disease, which is characterized by serious alterations of brain capillary architecture. The KRIT1 protein contains multiple interaction domains and motifs, suggesting that it might act as a scaffold for the assembly of functional protein complexes involved in signaling networks. In previous work, we defined structure-function relationships underlying KRIT1 intramolecular and intermolecular interactions and nucleocytoplasmic shuttling, and found that KRIT1 plays an important role in molecular mechanisms involved in the maintenance of the intracellular Reactive Oxygen Species (ROS) homeostasis to prevent oxidative cellular damage. Here we report the identification of the Kelch family protein Nd1-L as a novel molecular interactor of KRIT1. This interaction was discovered through yeast two-hybrid screening of a mouse embryo cDNA library, and confirmed by pull-down and co-immunoprecipitation assays of recombinant proteins, as well as by co-immunoprecipitation of endogenous proteins in human endothelial cells. Furthermore, using distinct KRIT1 isoforms and mutants, we defined the role of KRIT1 domains in the Nd1-L/KRIT1 interaction. Finally, functional assays showed that Nd1-L may contribute to the regulation of KRIT1 nucleocytoplasmic shuttling and cooperate with KRIT1 in modulating the expression levels of the antioxidant protein SOD2, opening a novel avenue for future mechanistic studies. The identification of Nd1-L as a novel KRIT1 interacting protein provides a novel piece of the molecular puzzle involving KRIT1 and suggests a potential functional cooperation in cellular responses to oxidative stress, thus expanding the framework of molecular complexes and mechanisms that may underlie the pathogenesis of CCM disease.     

Molecular Cloning of a Novel Vascular Endothelial Growth Factor, VEGF-D

We have identified and characterized a novel vascular endothelial growth factor (VEGF), VEGF-D, which is structurally related to vascular endothelial growth factor C. A full-length cDNA for human VEGF-D was cloned following the identification of an EST obtained through a TFASTA search of public EST databases. The murine VEGF-D was subsequently isolated from a mouse lung cDNA library. The human VEGF-D gene was mapped to human chromosome Xp22.31. Both human and mouse VEGF-D are strongly expressed in lung and encode the eight cysteine residues that are highly conserved among the members of this family. The high level of conservation between mouse and human VEGF-D may emphasize the biological importance of this gene. Recently the murine gene, FIGF, which is identical to mouse VEGF-D, was reported.     

Molecular Cloning of a Novel Gene ZAhi-1 and its Expression Analysis during Zebrafish Gametogenesis

Proto-oncogen Ahi-1 is closely related to a lot of human and mouse diseases. Ahi-1 mutation will lead to leukemia in mice and Joubert syndrome in human beings. We have cloned the full cDNA sequence of Ahi-1 homologous in zebrafish, and RT-PCR results of ZAhi-1 in different tissues reveal that ZAhi-1 expressed highest in the mature gonad. In situ hybridization results of zebrafish gonad show that ZAhi-1 only expressed in the early stages’ gamete cells. RT-PCR analyses of mouse Ahi-1 in different stages of spermatogenesis have been done according to the published Ahi-1 sequence, and the findings reveal that Ahi-1 is expressed in gamete of pachytene stage. It can then be safely concluded that Ahi-1 might take place in the spermatocyte from the early pachytene stage to the late pachytene stage.     

JSAP1, a Novel Jun N-Terminal Protein Kinase (JNK)-Binding Protein That Functions as a Scaffold Factor in the JNK Signaling Pathway

The major components of the mitogen-activated protein kinase (MAPK) cascades are MAPK, MAPK kinase (MAPKK), and MAPKK kinase (MAPKKK). Recent rapid progress in identifying members of MAPK cascades suggests that a number of such signaling pathways exist in cells. To date, however, how the specificity and efficiency of the MAPK cascades is maintained is poorly understood. Here, we have identified a novel mouse protein, termed Jun N-terminal protein kinase (JNK)/stress-activated protein kinase-associated protein 1 (JSAP1), by a yeast two-hybrid screen, using JNK3 MAPK as the bait. Of the mammalian MAPKs tested (JNK1, JNK2, JNK3, ERK2, and p38alpha), JSAP1 preferentially coprecipitated with the JNKs in cotransfected COS-7 cells. JNK3 showed a higher binding affinity for JSAP1, compared with JNK1 and JNK2. In similar cotransfection studies, JSAP1 also interacted with SEK1 MAPKK and MEKK1 MAPKKK, which are involved in the JNK cascades. The regions of JSAP1 that bound JNK, SEK1, and MEKK1 were distinct from one another. JNK and MEKK1 also bound JSAP1 in vitro, suggesting that these interactions are direct. In contrast, only the activated form of SEK1 associated with JSAP1 in cotransfected COS-7 cells. The unstimulated SEK1 bound to MEKK1; thus, SEK1 might indirectly associate with JSAP1 through MEKK1. Although JSAP1 coprecipitated with MEK1 MAPKK and Raf-1 MAPKKK, and not MKK6 or MKK7 MAPKK, in cotransfected COS-7 cells, MEK1 and Raf-1 do not interfere with the binding of SEK1 and MEKK1 to JSAP1, respectively. Overexpression of full-length JSAP1 in COS-7 cells led to a considerable enhancement of JNK3 activation, and modest enhancement of JNK1 and JNK2 activation, by the MEKK1-SEK1 pathway. Deletion of the JNK- or MEKK1-binding regions resulted in a significant reduction in the enhancement of the JNK3 activation in COS-7 cells. These results suggest that JSAP1 functions as a scaffold protein in the JNK3 cascade. We also discuss a scaffolding role for JSAP1 in the JNK1 and JNK2 cascades.     

Anti-Angiogenic Effect of Metformin in Mouse Oxygen-Induced Retinopathy Is Mediated by Reducing Levels of the Vascular Endothelial Growth Factor Receptor Flk-1

Purpose

To evaluate the effect of metformin on vascular changes in oxygen-induced retinopathy (OIR) in mouse, and to elucidate the possible underlying mechanism.

Methods

OIR mice were treated with metformin by intraperitoneal injection from postnatal day 12 (P12) to P17 or P21. At P17 and P21, vessel formation and avascular areas were assessed using retinal flat mounts. Levels of vascular endothelial growth factor (VEGF) were measured by enzyme-linked immunosorbent assays, and the effects of metformin on VEGF-induced proliferation of human umbilical vein endothelial cells (HUVECs) were assessed. The effects of metformin on the levels of Flk1 (VEGF receptor-2) and phosphorylated Flk1 (pFlk1) were measured by Western blotting (HUVECs) and immunohistochemistry (retinal tissue).

Results

Retinal morphologic changes were analyzed between two groups (saline-treated OIR; metformin-treated OIR). Metformin treatment did not change the extent of avascular areas at P17. However, at P21, when OIR pathology was markedly improved in the saline-treated group, OIR pathology still remained in the metformin-treated OIR group. VEGF expression levels did not differ between metformin- and saline-treated OIR groups at P17 and P21, but Flk1 levels were significantly reduced in the metformin group compared with saline-treated OIR group. Moreover, metformin inhibited VEGF-induced cell proliferation and decreased levels of Flk1 and pFlk1, consistent with the interpretation that metformin inhibits vascular growth by reducing Flk1 levels.

Conclusion

Metformin exerts anti-angiogenesis effects and delays the normal vessel formation in the recovery phase of OIR in mice, likely by suppressing the levels of Flk1.     

Neuron-derived Neurotrophic Factor Functions as a Novel Modulator That Enhances Endothelial Cell Function and Revascularization Processes

Strategies to stimulate revascularization are valuable for cardiovascular diseases. Here we identify neuron-derived neurotrophic factor (NDNF)/epidermacan as a secreted molecule that is up-regulated in endothelial cells in ischemic limbs of mice. NDNF was secreted from cultured human endothelial cells, and its secretion was stimulated by hypoxia. NDNF promoted endothelial cell network formation and survival in vitro through activation of Akt/endothelial NOS (eNOS) signaling involving integrin αvβ3. Conversely, siRNA-mediated knockdown of NDNF in endothelial cells led to reduction of cellular responses and basal Akt signaling. Intramuscular overexpression of NDNF led to enhanced blood flow recovery and capillary density in ischemic limbs of mice, which was accompanied by enhanced phosphorylation of Akt and eNOS. The stimulatory actions of NDNF on perfusion recovery in ischemic muscles of mice were abolished by eNOS deficiency or NOS inhibition. Furthermore, siRNA-mediated reduction of NDNF in muscles of mice resulted in reduction of perfusion recovery and phosphorylation of Akt and eNOS in response to ischemia. Our data indicate that NDNF acts as an endogenous modulator that promotes endothelial cell function and ischemia-induced revascularization through eNOS-dependent mechanisms. Thus, NDNF can represent a therapeutic target for the manipulation of ischemic vascular disorders.     

Genetic Analysis of Fin Development in Zebrafish Identifies Furin and Hemicentin1 as Potential Novel Fraser Syndrome Disease Genes

Using forward genetics, we have identified the genes mutated in two classes of zebrafish fin mutants. The mutants of the first class are characterized by defects in embryonic fin morphogenesis, which are due to mutations in a Laminin subunit or an Integrin alpha receptor, respectively. The mutants of the second class display characteristic blistering underneath the basement membrane of the fin epidermis. Three of them are due to mutations in zebrafish orthologues of FRAS1, FREM1, or FREM2, large basement membrane protein encoding genes that are mutated in mouse bleb mutants and in human patients suffering from Fraser Syndrome, a rare congenital condition characterized by syndactyly and cryptophthalmos. Fin blistering in a fourth group of zebrafish mutants is caused by mutations in Hemicentin1 (Hmcn1), another large extracellular matrix protein the function of which in vertebrates was hitherto unknown. Our mutant and dose-dependent interaction data suggest a potential involvement of Hmcn1 in Fraser complex-dependent basement membrane anchorage. Furthermore, we present biochemical and genetic data suggesting a role for the proprotein convertase FurinA in zebrafish fin development and cell surface shedding of Fras1 and Frem2, thereby allowing proper localization of the proteins within the basement membrane of forming fins. Finally, we identify the extracellular matrix protein Fibrillin2 as an indispensable interaction partner of Hmcn1. Thus we have defined a series of zebrafish mutants modelling Fraser Syndrome and have identified several implicated novel genes that might help to further elucidate the mechanisms of basement membrane anchorage and of the disease''s aetiology. In addition, the novel genes might prove helpful to unravel the molecular nature of thus far unresolved cases of the human disease.     

Allograft Inflammatory Factor 1 Functions as a Pro-Inflammatory Cytokine in the Oyster,Crassostrea ariakensis

The oyster Crassostrea ariakensis is an economically important bivalve species in China, unfortunately it has suffered severe mortalities in recent years caused by rickettsia-like organism (RLO) infection. Prevention and control of this disease is a priority for the development of oyster aquaculture. Allograft inflammatory factor-1 (AIF-1) was identified as a modulator of the immune response during macrophage activation and a key gene in host immune defense reaction and inflammatory response. Therefore we investigated the functions of C. ariakensis AIF-1 (Ca-AIF1) and its antibody (anti-CaAIF1) in oyster RLO/LPS-induced disease and inflammation. Ca-AIF1 encodes a 149 amino acid protein containing two typical Ca²⁺ binding EF-hand motifs and shares a 48–95% amino acid sequence identity with other animal AIF-1s. Tissue-specific expression analysis indicates that Ca-AIF1 is highly expressed in hemocytes. Significant and continuous up-regulation of Ca-AIF1 is detected when hemocytes are stimulated with RLO/LPS (RLO or LPS). Treatment with recombinant Ca-AIF1 protein significantly up-regulates the expression levels of LITAF, MyD88 and TGFβ. When anti-CaAIF1 antibody is added to RLO/LPS-challenged hemocyte monolayers, a significant reduction of RLO/LPS-induced LITAF is observed at 1.5–12 h after treatment, suggesting that interference with Ca-AIF1 can suppress the inflammatory response. Furthermore, flow cytometric analysis indicated that anti-CaAIF1 administration reduces RLO/LPS-induced apoptosis and necrosis rates of hemocytes. Collectively these findings suggest that Ca-AIF1 functions as a pro-inflammatory cytokine in the oyster immune response and is a potential target for controlling RLO infection and LPS-induced inflammation.     

ENDOSPERM DEFECTIVE1 Is a Novel Microtubule-Associated Protein Essential for Seed Development in Arabidopsis

Early endosperm development involves a series of rapid nuclear divisions in the absence of cytokinesis; thus, many endosperm mutants reveal genes whose functions are essential for mitosis. This work finds that the endosperm of Arabidopsis thaliana endosperm-defective1 (ede1) mutants never cellularizes, contains a reduced number of enlarged polyploid nuclei, and features an aberrant microtubule cytoskeleton, where the specialized radial microtubule systems and cytokinetic phragmoplasts are absent. Early embryo development is substantially normal, although occasional cytokinesis defects are observed. The EDE1 gene was cloned using a map-based approach and represents the pioneer member of a conserved plant-specific family of genes of previously unknown function. EDE1 is expressed in the endosperm and embryo of developing seeds, and its expression is tightly regulated during cell cycle progression. EDE1 protein accumulates in nuclear caps in premitotic cells, colocalizes along microtubules of the spindle and phragmoplast, and binds microtubules in vitro. We conclude that EDE1 is a novel plant-specific microtubule-associated protein essential for microtubule function during the mitotic and cytokinetic stages that generate the Arabidopsis endosperm and embryo.     

Sip1, a Novel RS Domain-Containing Protein Essential for Pre-mRNA Splicing

Previous studies have shown that protein-protein interactions among splicing factors may play an important role in pre-mRNA splicing. We report here identification and functional characterization of a new splicing factor, Sip1 (SC35-interacting protein 1). Sip1 was initially identified by virtue of its interaction with SC35, a splicing factor of the SR family. Sip1 interacts with not only several SR proteins but also with U1-70K and U2AF65, proteins associated with 5′ and 3′ splice sites, respectively. The predicted Sip1 sequence contains an arginine-serine-rich (RS) domain but does not have any known RNA-binding motifs, indicating that it is not a member of the SR family. Sip1 also contains a region with weak sequence similarity to the Drosophila splicing regulator suppressor of white apricot (SWAP). An essential role for Sip1 in pre-mRNA splicing was suggested by the observation that anti-Sip1 antibodies depleted splicing activity from HeLa nuclear extract. Purified recombinant Sip1 protein, but not other RS domain-containing proteins such as SC35, ASF/SF2, and U2AF65, restored the splicing activity of the Sip1-immunodepleted extract. Addition of U2AF65 protein further enhanced the splicing reconstitution by the Sip1 protein. Deficiency in the formation of both A and B splicing complexes in the Sip1-depleted nuclear extract indicates an important role of Sip1 in spliceosome assembly. Together, these results demonstrate that Sip1 is a novel RS domain-containing protein required for pre-mRNA splicing and that the functional role of Sip1 in splicing is distinct from those of known RS domain-containing splicing factors.Pre-mRNA splicing takes place in spliceosomes, the large RNA-protein complexes containing pre-mRNA, U1, U2, U4/6, and U5 small nuclear ribonucleoprotein particles (snRNPs), and a large number of accessory protein factors (for reviews, see references 21, 22, 37, 44, and 48). It is increasingly clear that the protein factors are important for pre-mRNA splicing and that studies of these factors are essential for further understanding of molecular mechanisms of pre-mRNA splicing.Most mammalian splicing factors have been identified by biochemical fractionation and purification (3, 15, 19, 31–36, 45, 69–71, 73), by using antibodies recognizing splicing factors (8, 9, 16, 17, 61, 66, 67, 74), and by sequence homology (25, 52, 74).Splicing factors containing arginine-serine-rich (RS) domains have emerged as important players in pre-mRNA splicing. These include members of the SR family, both subunits of U2 auxiliary factor (U2AF), and the U1 snRNP protein U1-70K (for reviews, see references 18, 41, and 59). Drosophila alternative splicing regulators transformer (Tra), transformer 2 (Tra2), and suppressor of white apricot (SWAP) also contain RS domains (20, 40, 42). RS domains in these proteins play important roles in pre-mRNA splicing (7, 71, 75), in nuclear localization of these splicing proteins (23, 40), and in protein-RNA interactions (56, 60, 64). Previous studies by us and others have demonstrated that one mechanism whereby SR proteins function in splicing is to mediate specific protein-protein interactions among spliceosomal components and between general splicing factors and alternative splicing regulators (1, 1a, 6, 10, 27, 63, 74, 77). Such protein-protein interactions may play critical roles in splice site recognition and association (for reviews, see references 4, 18, 37, 41, 47 and 59). Specific interactions among the splicing factors also suggest that it is possible to identify new splicing factors by their interactions with known splicing factors.Here we report identification of a new splicing factor, Sip1, by its interaction with the essential splicing factor SC35. The predicted Sip1 protein sequence contains an RS domain and a region with sequence similarity to the Drosophila splicing regulator, SWAP. We have expressed and purified recombinant Sip1 protein and raised polyclonal antibodies against the recombinant Sip1 protein. The anti-Sip1 antibodies specifically recognize a protein migrating at a molecular mass of approximately 210 kDa in HeLa nuclear extract. The anti-Sip1 antibodies sufficiently deplete Sip1 protein from the nuclear extract, and the Sip1-depleted extract is inactive in pre-mRNA splicing. Addition of recombinant Sip1 protein can partially restore splicing activity to the Sip1-depleted nuclear extract, indicating an essential role of Sip1 in pre-mRNA splicing. Other RS domain-containing proteins, including SC35, ASF/SF2, and U2AF65, cannot substitute for Sip1 in reconstituting splicing activity of the Sip1-depleted nuclear extract. However, addition of U2AF65 further increases splicing activity of Sip1-reconstituted nuclear extract, suggesting that there may be a functional interaction between Sip1 and U2AF65 in nuclear extract.