Histamine metabolism influences blood vessel branching in zebrafish reg6mutants
© Huang et al; licensee BioMed Central Ltd. 2008
Received: 26 June 2007
Accepted: 25 March 2008
Published: 25 March 2008
Vascular branching morphogenesis is responsible for the extension of blood vessels into growing tissues, a process crucial for organogenesis. However, the genetic mechanism for vessel branching is largely unknown. Zebrafish reg6 is a temperature-sensitive mutation exhibiting defects in blood vessel branching which results in the formation of swollen vessel lumina during capillary plexus formation.
We performed a screening for chemical suppressors of reg6 and identified SKF91488, an inhibitor of histamine methyltransferase (HMT), that can rescue the reg6 vessel branching defects in a dose-dependent manner. Inhibition of HMT by SKF91488 presumably blocks histamine degradation, thus causing histamine accumulation. Consistent with this idea, we found that a high level of histamine also showed significant suppression of reg6 vessel phenotypes. Interestingly, when reg6 adults that had already developed swollen vessel lumina in regenerating fins were treated with histamine or SKF91488, either treatment significantly reduced the number of swollen vessels within 12 h, suggesting a rapid and constant influence of histamine on blood vessel branching. Furthermore, the expression of HMT was significantly elevated in reg6 regenerating fins. Conversely, lowering histamine by administering urocanic acid, a histidine decarboxylase inhibitor, enhanced the reg6 phenotypes. Finally, we identified that the transcription factor, egr-1 (early growth response factor 1), was closely associated with the reg6 phenotype and chemical treatments.
Taken together, our results suggest that blood vessel branching is influenced by histamine metabolism, possibly through regulating the expression of the egr-1 transcription factor.
Blood vessels are important for transporting oxygen and nutrients to cells for survival and proper functioning. In early embryos, the pioneer trunk vessels, the dorsal aorta and cardinal vein, develop from vascular endothelial cells derived from the lateral plate mesoderm through a process called vasculogenesis. Subsequently, blood vessels grow from existing vessels, through a process referred to as angiogenesis, into peripheral tissues and developing organs . In developing embryos, increasing evidence has revealed that blood vessels also provide signals for the proper morphogenesis of developing organs . In the adult form of organisms, angiogenesis and/or vasculogenesis to regrow blood vessels in wounded tissues is needed to repair damaged and replace lost tissues [3, 4]. In humans, it has been well documented that angiogenesis plays a vital role in tumor growth, and thus the potential of antiangiogenesis drugs to inhibit tumor growth has been extensively explored worldwide .
During angiogenesis, vascular branching is one of the key morphogenetic events, which presumably is responsible for constructing a complex vascular network which assures that blood is available to every single cell. Despite its important role in sustaining organismic development throughout life, the genetic mechanism of blood vessel branching is largely unknown partly due to the intricate interactions between molecular signals and numerous cell types that are involved in angiogenesis, e.g., multiple cellular activities, including cell proliferation, migration, extension, and possibly cell fate specification all of which require signaling through multiple growth factors such as VEGF, FGF, PDGF, and EGF. Interactions between blood vessel endothelial cells and neighboring cells and even cells in the bloodstream can also regulate angiogenesis . More recently, it has been implied that immune-mediated cells and molecules can influence blood vessel growth in adults. For example, mast cells have been found to aggregate at sites of active angiogenesis under both physiological and pathological conditions . Histamine has been determined to be an angiogenic factor [8, 9] and is thought to be directly involved in tumor growth based on findings of high expression levels and enzymatic activity of the key histamine synthesis enzyme, histidine decarboxylase, in numerous growing tumors, such as small-cell lung carcinoma [10, 11]. In fact, clinical trials have shown that histamine H2 receptor antagonists, such as cimetidine and ranitidine, increase the survival of gastric and colon cancer patients [12, 13]. It has also been reported that histamine regulates angiogenesis at the level of cell proliferation and cell permeability [14, 15]. Thus, histamine seems to be involved in multiple cellular and molecular interactions during angiogenesis. It is still unclear whether histamine can influence blood vessel branching during angiogenesis, and if it does, through which intracellular signaling pathway it works.
In this report, we took a chemical genetic approach to reveal the role of histamine in vascular branching using a zebrafish reg6 mutation which causes specific vascular branching defects without affecting endothelial cell proliferation . We show that vessel branching defects in reg6 mutants can be rescued by a high level of histamine either directly added to the water or by inhibiting histamine degradation using a histamine methyltransferase (HMT) inhibitor. On the other hand, blocking histamine synthesis by a histidine decarboxylase inhibitor enhanced the reg6 vessel phenotypes. Finally, we show that the expression level of the egr-1 transcription factor, but not the VEGF family, is strongly correlated with the reg6 phenotypes, suggesting that histamine might modulate blood vessel branching by regulating egr-1 levels.
Specific blood vessel branching defect of the zebrafish reg6mutation
Isolation of a chemical suppressor of the reg6mutation
Suppression of the reg6embryonic phenotype by a high level of histamine
As HMT is responsible for methylating histamine thus precipitating its degradation, the above results led us to hypothesize that SKF91488 might cause accumulation of histamine, which in turn would rescue reg6 phenotypes. We tested this hypothesis by treating reg6 embryos with histamine. The results showed that a high level of histamine indeed was able to rescue the embryonic phenotypes of reg6 mutants, although the concentration of histamine needed to produce significant suppression was 1000 μM (Figure 4E, green bars). Like the HMT inhibitor, a high level of histamine caused no discernible developmental defects in wild-type zebrafish embryos (data not shown).
Suppression of the reg6regenerating vessel phenotype by SKF91488 and histamine
The above results led us to hypothesize that the expression of HMT might increase in reg6 mutants, which is responsible for the histamine deficit. To test this, we analyzed the amount of HMT RNA in wild-type and reg6 regenerates by quantitative PCR (QPCR). We found that the amounts of HMT RNA were nearly 5- and 3-fold higher in reg6 2 and 3-dpa regenerates, respectively, than in wild-type ones (Figure 5D).
Reversion of reg6dilated vessels by histamine and SKF91488
We wondered whether histamine and SKF91488 are able to reduce the severity of the vessel phenotype in reg6 mutants even after significant swollen vessels had formed. To test this, we first allowed reg6 mutants to regenerate at 33°C for 3 days to develop swollen lumina, and then shifted them to either 10 μM SKF91488, 1 mM histamine, or H2O. We then compared the number of swollen vessels before and then 12 h after the shift. In the H2O group, the number of swollen vessels in reg6 fish was about the same within the 12-h period: near 20 swollen vessels/fish (Figure 5E). With SKF91488 treatment, however, the number of swollen vessels was significantly reduced to an average of 10 swollen vessels/fish (Figure 5E, P < 0.01, n = 10). Histamine also reduced the number from 20 to fewer than 15 (P = 0.03, n = 10). In the distal portions of regenerating fins of reg6 adults, the vessel defects usually caused necrosis and degeneration, which were thus greatly reduced by histamine and SKF91488 (data not shown). We concluded that a high level of histamine can reverse the swollen vessel phenotype of the reg6 mutation.
Enhancement of the reg6regenerating vessel phenotype by a histamine synthesis blocker
Histamine synthesis blocker causes blood vessel branching defect in wild-type zebrafish embryos
Association between egr-1 expression and the reg6vascular phenotype
Zebrafish angiogenesis mutation for drug discovery and chemical genetics
We utilized a zebrafish mutation to demonstrate the power of zebrafish for drug discovery and examining chemical genetics. In particular, when using the embryonic phenotype for initial screening, which only required 0.5 μg of the test compound (with a MW of 250), the adult phenotypes, if they existed, allowed us to verify the drug effect with only 5 μg of drug for each test (see also "Methods"). In conjunction with TG(fli1:EGFP)y1 transgenic fish, the screening could be specifically targeted for angiogenesis-related drugs (see also ). In addition, with zebrafish, we were able to perform toxicity tests at the same time at the whole organism level. All these factors make zebrafish a great tool for drug testing and discovery. The reg6 mutation is unique in that it is homozygously viable and displays similar blood vessel branching defects during embryogenesis and fin regeneration. More importantly, the reg6 mutation has the advantage of temperature sensitivity which allowed us to screen for suppressors and enhancers. Since reg6 mutants display specific defects in blood vessel branching, it has become a good tool for studying the genetic and pharmacological mechanisms of vessel branching which are largely unknown.
The role of histamine in blood vessel branching in reg6and wild-type zebrafish
Our results reveal a role of histamine in promoting vessel branching during angiogenesis using zebrafish reg6 mutants. The reg6 mutants were found to have specific defects in blood vessel branching during plexus formation . When reg6 mutants were treated with the HMT inhibitor, SKF91488, which presumably causes accumulation of histamine, or with a high level of histamine, the blood vessel branching defect of reg6 was significantly rescued. In contrast, blocking histamine synthesis enhanced the reg6 phenotype. These results were further supported by the finding that HMT expression is elevated in reg6 mutants. All these suggest a simple model for the reg6 blood vessel branching defect which results from a high level of HMT and concomitant histamine deficit.
The role of histamine in regulating blood vessel branching is also observed in wild-type zebrafish. In Figure 7, we showed that blocking histamine synthesis with urocanic acid causes blood vessel branching defect and/or a reg6-like phenotype in the caudal veins of wild-type zebrafish embryos. In this experiment, however, we learned that it really required high concentration and careful selection of time window of urocanic acid treatment in order to see its effect on blood vessel branching. Treatment of high concentration of urocanic acid for long period of time, e.g. from 6–32 hpf caused high lethality rate in developing embryos suggesting that histamine is important for other developmental processes. Furthermore, we noted that urocanic acid did not seem to suppress the branching of intersegmental vessels, suggesting that the influence of histamine on vascular branching morphogenesis is selective or more sensitive for the plexus-forming blood vessels.
Possible mechanisms of regulating blood vessel branching by histamine
As vascular branching involves complex intracellular and extracellular changes, including breakdown of the extracellular matrix and intercellular junctions, reorganization of the cytoskeleton, and induction of cell proliferation, it is difficult to predict the mechanism through which histamine rescues vessel branching defects in reg6 fish. Even though our phenotypic characterizations provide clues to the specific endothelial cellular defects underlying reg6 vascular defects, including defects in cell extension, cell junction formation, and likely permeability as well, it is still not possible at this moment to define a cellular mechanism for the rescue effect of histamine in reg6 fish due to the fact that the genetic identity of reg6 has yet to be resolved. We have mapped reg6 to the telomere region of LG 25 (CC Huang, unpublished results) where sequence information is largely lacking, and thus, it is difficult to obtain an accurate physical map. We are currently verifying a physical map of that region assembled by the Zebrafish Genome Sequencing Project at the Sanger Center with our reg6 mapping panels. We will need to build a more-accurate physical map of this region for future mapping and cloning of the reg6 gene.
Nevertheless, we have obtained more clues to reg6's function from previous and the present studies. Our previous study showed that the reg6 mutation does not cause over-proliferation of regenerating endothelial cells and that reg6 function is specifically required during plexus formation when blood vessel endothelial cells are actively branching . In this study, we further showed that the reg6 mutation does not affect the migration of regenerating endothelial cells. Instead, endothelial cells of reg6 mutants seem to be defective in extending cell processes and forming cell junctions among themselves. Our results also suggest that reg6's function might be involved in regulating histamine metabolism and egr-1 expression. Consistent with previous results, we observed no effect of histamine on endothelial cell proliferation or migration in wild-type fish, even though it has been shown that histamine is able to cause endothelial cell proliferation in vitro [11, 14]. Our studies using the zebrafish reg6 mutation provide evidence that histamine likely modulates blood vessel branching at a yet unidentified cellular level other than cell proliferation or migration.
Correlation of egr-1 and the vessel branching defects of reg6
It is very intriguing that we found a close relationship between the egr-1 transcription factor and reg6 phenotypes. The level of egr-1 was low in reg6 mutant fish during regenerative angiogenesis, was elevated by histamine and an HMT inhibitor, and was further reduced upon treatment with a histamine synthesis blocker (Figure 8D). Although the role of egr-1 and its relationship with histamine in vascular branching awaits further studies, we found an interesting feature of egr-1 in our studies that seem to echo previous finding. Fahmy et al  has shown that egr-1 is required for angiogenesis and tumor growth through an FGF but not a VEGF signaling pathway. Our QPCR analyses showed that the transcription levels of VEGFs and VEGFRs remained the same in wild-type and reg6 fish with or without histamine manipulations suggesting that the mechanism(s) of blood vessel branching mediated by histamine that involves egr-1 is independent of the VEGF signaling. It would be exciting to know whether histamine modulates blood vessel branching directly through egr-1 and/or a FGF pathway.
Direct and indirect actions of histamine on blood vessel endothelial cells
Histamine has been shown to increase vessel permeability by regulating the expression of cell adhesion molecules such as VCAM  and the composition of the extracellular matrix . Presumably, these actions together can promote vessel branching. Our result that histamine can prevent the formation of swollen vessel lumina suggests its influence on actively branching vascular cells. We also showed that histamine can reverse the swelling of vessels and return them to normal, suggesting a role of histamine during vascular plexus remodeling. One possible explanation for these results is that histamine affects a cellular mechanism that is common to both vascular activities, for example cell adhesion or extracellular matrix remodeling. In fact, the histamine receptor inhibitor, cimitidine, has been shown to downregulate the endothelial expression of E-selectin adhesion molecules and prevent tumor metastasis , which raises the possibility that histamine might directly modulate vessel branching on endothelial cells by regulating E-selectin expression. However, the fact that histamine does not change endothelial cell proliferation or cause over-branching or any other morphological changes in wild-type fish suggests that endothelial cells are not the direct target of histamine. Thus, it is possible that histamine might both directly and indirectly influence vascular branching.
We demonstrate the power of using a zebrafish mutation in pharmacogenetic studies. Our studies using the zebrafish reg6 mutation revealed the role of histamine in regulating blood vessel branching and the potential linkage of angiogenic factor egr-1 to this process.
Fish maintenance and breeding were performed according to standard procedures .
Chemical screening with reg6embryos
reg6 homozygous embryos were obtained by in vitro fertilization for chemical screening of clones due to the concerns that reg6 embryos from different clutches might show variable degrees of phenotypic penetrance. Embryos from different pairs of parents were kept separate, and 20 embryos from each individual clutch were set aside to monitor the phenotypic penetrance. The protocol for chemical treatment of zebrafish embryos was adapted from Peterson et al.  with minor modifications. These embryos were allowed to develop at 28.5°C until 6 h post-fertilization (hpf) and then were arrayed into 96-well plates, at 5 embryos/well with 200 μl of egg water (distilled water containing 60 μg/ml sea salt) without antibiotics. The LOPAC chemical library (Sigma, St. Louis, MO, USA) was prepared at 2 mM in DMSO, and 1 μl of each was added to each well to make 10 μM for our initial screening. These plates were kept in a 33°C incubator and examined at around 32 hpf with a fluorescent stereomicroscope. Chemicals used in this study were purchased from Sigma unless otherwise signified.
Chemical treatment of adult fish
Adult fish were briefly anesthetized with 0.01% tricaine, and the caudal fins were amputated vertically at 50% of the proximal-distal axis. Five amputated fish were put into a 250-ml beaker containing 100 ml of aquatic water with or without chemicals. These fish were then kept in a 33°C incubator for 3 days without feeding. With this protocol, the wild-type fish could regenerate normally, while reg6 fish developed swollen vessel lumina. To quantify the reg6 phenotype, the number of vessels that developed obviously swollen lumina was determined. Since each fin ray except for the two lateral fin rays contains three vessels , 48 vessels of the 16 fin rays were examined and scored for a typical caudal fin with 18 fin rays.
Transmission electron microscopy
Embryos were fixed in 2% glutaraldehyde, 2% paraformaldehyde, and 0.1 M sodium phosphate (pH 7.4) overnight at 4°C. Fixed embryos were washed with 0.1 M sodium phosphate buffer 3 times for 5 min each, and post-fixed with 1% OsO4 for 1 h at room temperature. The fixed embryos were dehydrated by the following ethanol series: 30% for 10 min, 50% for 10 min, 70% for 20 min, 80% for 20 min, 90% for 20 min, 95% for 20 min, and 100% for 60 min, and then twice in acetone for 30 min each. Infiltration was carried out first with 1:1 resin: acetone for 1 h, 2:1 resin: acetone for 1 h, pure resin for 1 h, and finally pure resin overnight. Embryos were placed in a 70°C oven overnight for embedding. The plastic blocks were then trimmed and sectioned to obtain 1 μm or ultrathin section of 70~90 nm which were collected on 200-mesh copper grids and stained with uranyl acetate for 50 min and lead citrate for 10 min. Sections were analyzed under a Hitachi H-7000 transmission electron microscope equipped with AMT XR40 CCD camera (Advanced Microscopy Techniques, Danvers, MA USA).
Whole mount in situhybridization
Regenerative fins were harvested and fixed in 4% paraformaldehyde overnight at 4°C. The whole-mount in situ hybridization was done with the InsituPro VS robot by Intavis Bioanalytical Instruments AG (Koln, Germany) following standard procedures of the manufacturer.
Quantitative polymerase chain reaction (QPCR)
Wild-type or reg6 homozygous embryos were treated with chemicals for 26 h (from 6 to 32 hpf) or 8 h (from 24 to 32 hpf) at 33°C and then manually dechorionated at the end of treatment. Fifty embryos from each treatment were subjected to RNA extraction with Trizol following instructions of the manufacturer. Around 5 μg of total RNA was then used to generate cDNAs with Superscript II (Invitrogen). Quantitative PCR was performed with a Power Cybergreen (ABI) labeling kit in an ABI7000 thermocycler. The data were analyzed with the software provided by ABI. When using adult fish, five 3-dpa regenerative fins from treated or untreated wild-type or reg6 fish were collected.
Primers used for quantitative PCR were egr-1F, AGTTTGATCACCTTGCTGGAGATAC; egr-1R, AGGGTGAAACGGCCTGTGT; HMT-F, AATGAAGTGGTGGAACCA AGTAAT G; HMT-R, AAGATCGGGAGATGTTGACACTCT; VEGF-A-F, GCTGTAAAGGCTGCCCACATAC; VEGF-A-R, ACCAGCAGCTCTCGGGTCTT; VEGF-C-F, GCGGACCACACCATTACC; VEGF-C-R, TGCGGTTGAGAGGTTGAC; VEGF-D-F, AAAGAGGGAGTTACCTGCCGTAAT; VEGF-D-R, AGCACAGGCTCTGGTCCAGATA; VEGFR1-F, GTCACTAACCCAGACGCCAAAG; VEGFR1-R, ATGAATCCCTGCCTGCTGTT; VEGFR2-F, TCTTCACTCTTCACGTGCTTTTTAG; VEGFR2-R, GAAGGTGTGTATCTCCATCAGGAA; VEGFR3-F, ACTGTCGGCCGTGTGGTTA; and VEGFR3-R, CGAATCCTTCAGGGATAGTGGTT.
We thank Dr. Jeff Lee at National Taiwan University for providing the VEGFC primers for the QPCR and the Core facility of the Institute of Cellular and Organismic Biology at Academia Sinica for their assistance with in vitro hybridization, QPCR, and TEM. C.C.H. was a distinguished postdoctoral fellow, Academia Sinica. This work was supported by grant 94M011-1 from the Genomic Research Center of Academia Sinica to J.Y.
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