Intrauterine sonographic measurement of embryonic brain vesicle

Our purpose was to evaluate embryonic brain vesicle measurements using intrauterine sonography in early first-trimester pregnancy. Fifty-one women about to undergo therapeutic abortion from 7-9.9 weeks gestational age were studied with a specially developed flexible catheter-based high-resolution real-time miniature (2.4 mm in outer diameter) ultrasound transducer (20 MHz). Length, width and height of telencephalon, diencephalon, mesencephalon and rhombencephalon were measured. The normal range of embryonic brain vesicle measurements for each day of pregnancy was determined. Curvilinear relationships were found between the menstrual age and telencephalon height (r(2) = 71.2%), diencephalon length (r(2) = 39. 6%), width (r(2) = 39.4%) and height (r(2) = 48.3%) and mesencephalon length (r(2) = 59.0%) respectively. Linear relationships were found between the menstrual age and telencephalon width (r(2) = 41.4%), mesencephalon height (r(2) = 58.7%) and rhombencephalon length (r(2) = 44.9%), width (r(2) = 56.8%) and height (r(2) = 35.5%) respectively. Telencephalon length and mesencephalon width were constant throughout menstrual age. These results suggest that intrauterine sonography provides accurate embryonic brain measurements in utero. Moreover, intrauterine sonography may become an important modality in future embryological research and in detection of embryonic brain developmental disorders in early first-trimester pregnancy.     

Effects of 6-aminonicotinamide gliotoxin on blood-brain barrier differentiation in the chick embryo cerebellum

The hypothesis of astroglial cell involvement in prenatal setting up of the blood-brain barrier (BBB) has been examined by producing glial degeneration in cerebellum of chicken embryos submitted to the action of gliotoxin 6-aminonicotinamide (6-AN), which was applied onto the embryo chorioallantoic membrane during both early and late embryonic development. The effects of 6-AN on the cerebellum astroglial cells and microvessels were analysed under the light microscope by immunostaining for 3CB2 (chick-specific glial marker) and HT7 (chick-specific marker of BBB-provided brain endothelia), under the electron microscope, as well as by the vascular permeability tracer horseradish peroxidase. The results, showing good suitability of the 6-AN model also when applied in early embryonic development, demonstrated a correlation between perivascular glia depletion and endothelial barrier impairment and suggested that astroglia play a role in the BBB prenatal differentiation.     

Vascular Endothelial Growth Pattern During Demineralized Bone Matrix Induced Osteogenesis

The purpose of the current study was to determine the timely ingrowth of blood vessels associated with demineralized bone matrix (DMB) induced osteogenesis. Critical-size (10 × 5mm), full thickness bony defects in rabbit parietal bone were implanted with DBM. Histological and ultrastructural changes were examined 1, 2, 3, 4, 5, 6, 7 and 14 days later. Neovascularization was assessed by immunohistochemical staining for factor VIII antigen (marker for vascular endothelium) and also confirmed by staining using pan-endothelial antibody (CD31) (a marker for endothelium). Immunohistochemical evaluation revealed a positive staining for CD31 and Factor VIII expressed by endothelial cells by day 3 post grafting. By day 4, small blood vessels were first seen budding from host bed towards the grafted DBM. Ultrastructural identification of cells in the early stages of healing revealed the presence of macrophages. The monocyte-derived macrophage appears to play a central role in the repair process using DBM. Results of this study demonstrated a rapid vascularization during the DBM induced osteogenesis. This rapid vascularization is vital to the healing and bone induction ability of the DBM.     

Early expression of endomucin on endothelium of the mouse embryo and on putative hematopoietic clusters in the dorsal aorta.

Endomucin is a recently identified sialomucin that is specifically expressed on endothelium of the adult mouse. Here, we have analysed the expression of endomucin during development of the vascular system by immunohistochemistry by using three monoclonal antibodies (mAb). We demonstrate that two of the mAb, V.5C7 and V.1A7, recognize epitopes on the nonglycosylated protein, because they recognize the antigen when it is synthesized as a bacterial fusion protein and when it is in vitro translated in a membrane-free reticulocyte lysate. During in vitro differentiation of embryonic stem cells to endothelial cells, endomucin is expressed at day 6 after onset of differentiation, 1 day later than PECAM-1. During differentiation of the mouse embryo, endomucin is first detected at E8.0 in all embryonic blood vessels detectable at this stage but is absent in blood islands of the yolk sac. Analysing the paraaortic-splanchnopleura (P-SP) region and the aorta-gonad-mesonephros (AGM) region as sites of intraembryonic hematopoiesis, we found that endothelium of the dorsal aorta is brightly positive for endomucin at E8.5-9.0 and at E11.5. At later stages and in the adult aorta, endothelial staining is strongly reduced and confined to focal areas. Cell clusters associated with the luminal surface of the endothelium of the dorsal aorta could be stained for endomucin and for CD34. At a later stage (E15.5) single leukocytes in the lumen of large venules were stained for endomucin. We conclude that endomucin is an early endothelial-specific antigen that is also expressed on putative hematopoietic progenitor cells.     

Expression of the cell surface markers mAb 2F7 and PSA-NCAM in the embryonic rat brain

Cell surface markers of neuronal precursor cells are of interest since they allow the isolation of these cells from mixed populations of cells. The monoclonal antibody (mAb) 2F7, a cell surface marker, has been shown to label neuronal precursors and post-mitotic neurones in the embryonic rat spinal cord and cortex. Little is known about the expression of the epitope recognised by mAb 2F7 in other regions of the developing brain. The present study found expression of this epitope in the embryonic rat cortex, ventral mesencephalon (VM) and striatum at times at which neurogenesis is known to occur in these regions. The expression profile of mAb 2F7 was similar to that of the polysialylated form of the neural adhesion molecule (PSA-NCAM), a commonly used marker of neuronal precursor cells. The percentage of mAb 2F7-positive cells incorporating bromodeoxyuridine (BrdU) was found to be comparable to that of PSA-NCAM-positive cells in primary cell cultures of embryonic rat cortex, VM and striatum. These data demonstrate that mAb 2F7 can be used as a cell surface marker for neuronal precursor cells in several regions of the embryonic rat brain.     

Vascular, glial and neuronal effects of vascular endothelial growth factor in mesencephalic explant cultures

Vascular endothelial growth factor is a highly conserved, heparin-binding protein which mediates a number of critical developmental processes in both vertebrates and invertebrates, including angiogenesis, vasculogenesis and hematopoiesis. We employed an organotypic rat explant model (produced from embryonic day 17 fetuses) to assess the effects of vascular endothelial growth factor on brain microvasculature in general and the ventral midbrain specifically. Immunohistochemistry using antisera to rat endothelial cell antigen and laminin demonstrated a robust, dose-dependent effect of vascular endothelial growth factor, resulting in increased vessel neogenesis, branching and lumen size by three days in vitro. This effect was blocked by addition of an anti-vascular endothelial growth factor antibody. At higher doses of vascular endothelial growth factor, the effect was attenuated, though a statistically significant increase in both astrocyte, and neuronal density was observed using antisera to glial and neuronal markers. Tyrosine hydroxylase-immunoreactive (i.e. dopaminergic) neurons, particularly, exhibited increased survival in response to vascular endothelial growth factor application. Vascular endothelial growth factor had a mitogenic effect on endothelial cells and astrocytes, but not dopaminergic neurons, as demonstrated by the addition of [³H]thymidine to the cultures 2 h after the cultures were established. Similarly, results of a radioreceptor assay indicated that specific vascular endothelial growth factor binding sites were present on blood vessels and astrocytes, and were up-regulated by exposure to vascular endothelial growth factor.

We conclude that, in explants of the ventral mesencephalon, exogenously applied vascular endothelial growth factor is mitogenic for endothelial cells and astrocytes, and promotes growth/survival of neurons in general and dopaminergic neurons in particular.     

甘油二酯激酶γ参与大鼠胚胎脑神经上皮细胞的发育

目的探讨甘油二酯激酶γ(DGKγ)在大鼠胚胎脑神经上皮发育中的作用及可能机制。方法 18只胎龄(E)11.5、E12.5、E14.5、E16.5、E18.5大鼠胚胎用于Western blotting方法检测DGKγ在大鼠胚胎脑组织的表达,E9.5~E18.5大鼠胚胎各5只用于免疫组织化学和免疫荧光染色方法检测DGKγ在大鼠胚胎脑的分布以及DGKγ与Ki67、乙酰胆碱转移酶(Ch AT)及酪氨酸羟化酶(TH)的细胞内共定位。结果 DGKγ蛋白含量在E11.5~E14.5表达逐渐增多,E16.5表达较E14.5显著减少,E18.5表达显著增多且高于E14.5水平。E10.5~E12.5,DGKγ在脑泡壁出现表达,并随着脑泡的发育表达在端脑、除大脑脚外的中脑、后脑和末脑;E13.5~E14.5,DGKγ的强阳性表达从海马及周围的新皮质、苍白球、嗅脑和大脑脚阳性延伸到除新皮质外的脑的各区域;E15.5至出生前,DGKγ在新皮质阳性表达增强,但在大脑脚和脑桥表达减弱。免疫荧光染色显示,E14.5,与海马和苍白球相比,嗅脑的DGKγ阳性细胞中Ki67阳性率较高(87%),Ch AT阳性率较低(9%)(均P0.05),中脑顶盖的DGKγ阳性细胞中62%为TH阳性;E16.5,新皮质的DGKγ阳性细胞中Ki67阳性率为26%,较E14.5时(49%)显著降低(P0.05),Ch AT阳性率为70%,较E14.5时(45%)显著增高(P0.05);DGKγ多位于神经上皮细胞的胞质,也可见于胞膜上,或者同时位于胞质和胞核。结论 DGKγ在胚胎脑的表达模式和亚细胞分布,提示其参与了胚胎脑神经上皮的增殖、迁移和分化后神经元的发育。     

Expression of the hyaluronan receptor LYVE-1 is not restricted to the lymphatic vasculature; LYVE-1 is also expressed on embryonic blood vessels

Expression of the hyaluronan receptor LYVE-1 is one of few available criteria used to discriminate lymphatic vessels from blood vessels. Until now, endothelial LYVE-1 expression was reported to be restricted to lymphatic vessels and to lymph node, liver, and spleen sinuses. Here, we provide the first evidence that LYVE-1 is expressed on blood vessels of the yolk sac during mouse embryogenesis. LYVE-1 is ubiquitously expressed in the yolk sac capillary plexus at E9.5, then becomes progressively down-regulated on arterial endothelium during vascular remodelling. LYVE-1 is also expressed on intra-embryonic arterial and venous endothelium at early embryonic stages and on endothelial cells of the lung and endocardium throughout embryogenesis. These findings have important implications for the use of LYVE-1 as a specific marker of the lymphatic vasculature during embryogenesis and neo-lymphangiogenesis. Our data are also the first demonstration, to our knowledge, that the mouse yolk sac is devoid of lymphatic vessels.     

血管活性肽在人胚胎脐血管内皮细胞中的发育

为说明人脐血管内皮细胞在胚胎发育过程中就可能合成和分泌多种血管活性物质。用免疫组织化学技术对不同发育时期人胚胎脐血管内皮细胞生长抑素、内皮素、神经肽Ｙ和５－羟色胺进行定性定位研究。结果显示：ＳＳ出现于胚胎第８周；５－ＨＴ出现于胚胎１２周；ＮＰＹ出现于胚胎第１６周；ＥＴ出现于胚胎第２０周。     

Endothelial nitric oxide synthase is differently expressed in human endometrial vessels during the menstrual cycle

Endogenous nitric oxide (NO) can be synthesized by endothelial cells and can act as a potent vasodilator. We investigated endothelial nitric oxide synthase (eNOS), one of the three different enzymes responsible for the synthesis of NO by immunohistochemical methods throughout the menstrual cycle on 34 endometrial samples and compared its detection with the von Willebrand Factor (vWF) as a reliable marker molecule of the endothelium on serial sections. Immunoreactivity for eNOS was clearly localized in various types of arterial and venous endothelial cells as well as in capillaries. In addition, in some samples there was a positive staining in endometrial glandular epithelium. There was no staining in endometrial fibroblasts or in myometrial smooth muscle cells. Whereas the endothelium was constantly stained by the monoclonal antibody against vWF, eNOS was not always expressed in the endothelial lining of the vessels during the menstrual cycle. The number of vessels positively stained for eNOS increased gradually during the proliferation phase and most of the vessels were positive in the early secretory phase. These results suggest that its markedly increased expression during the early secretory phase of the menstrual cycle indicates a physiological significance.     

HT7, Neurothelin, Basigin, gp42 and OX-47--many names for one developmentally regulated immuno-globulin-like surface glycoprotein on blood-brain barrier endothelium, epithelial tissue barriers and neurons.

The antigens HT7 and Neurothelin were recently described as inducible membrane glycoproteins on chick blood-brain barrier endothelium. Our results demonstrate that the Neurothelin antibody 1W5 does recognize the isolated HT7 protein as well as the HT7 protein expressed by COS-1 cells transfected with the HT7 cDNA. Therefore, the HT7 and Neurothelin epitopes can be found on an unique glycoprotein. By low stringency hybridization we have isolated the murine cDNA clone encoding the homologous murine protein. The nucleotide sequence of the open reading frame is identical to that of the cDNA clones encoding the gp42 protein and the Basigin antigen expressed in F9 cells and in the early mouse embryo. The murine amino acid sequence shows 94% identity with the rat OX-47 membrane glycoprotein which was shown to be expressed in blood-brain barrier endothelium and several epithelial cell layers. These results demonstrate that HT7, Neurothelin, gp42, Basigin and OX-47 are different names for probably the same glycoprotein in different species.     

Qualitative and quantitative analysis of embryonic pulmonary vessel formation

Vessel formation in the lung has been described as occurring by two mechanisms: proximal, or branch, pulmonary arteries develop via angiogenesis; and distal, smaller vessels form by vasculogenesis. Connections between the proximal and distal vessels establish the final vascular network. The preponderance of vessel formation has been suspected to occur during the canalicular stage of lung development. To test these hypotheses, reporter gene expression under control of the regulatory domain of fetal liver kinase-1 (flk), an early endothelial cell-specific marker, was used to evaluate mouse lungs from embryonic day 10.5 (E10.5) through 2 wk postnatal age. Morphologic assessment was performed after histochemical staining, and quantification of vessel development by a chemiluminescent assay was compared with overall embryonic lung growth. LacZ expression under flk promoter control allowed: (1) early identification of differentiating endothelial cells of the branch pulmonary arteries; (2) visualization of distal vessels forming in the lung mesenchyme (primary capillary network) with subsequent remodeling; (3) recognition of early continuity between proximal and distal vessels, occurring by E10.5; and (4) assessment of developing pulmonary veins and venous confluence. Quantitative analysis revealed increased flk regulated beta-galactosidase (beta-gal) activity of 12 ng beta-gal/lung at E12.5 to 3,215 ng beta-gal/lung at 2 wk, which corresponded to overall lung growth during this period as shown by an increase in total protein content per lung from 35 microg at E12.5 to 6,456 microg at 2 wk after birth. We identified endothelial cell precursors of the developing pulmonary vasculature before vessel lumen formation. Continuity between the proximal pulmonary artery and vessels forming in the distal mesenchyme was present even at the earliest stage evaluated, suggesting endothelial cell differentiation at the site of vessel formation (i.e., vasculogenesis) as occurs with development of the aorta. Finally, we demonstrated that lung vessel development was not accentuated during the canalicular stage, but occurred at all stages and directly corresponded to overall lung growth.     

Transient expression of osteopontin mRNA and protein in amoeboid microglia in developing rat brain

To investigate a potential role of osteopontin (OPN) in developing rat brain, the expression of OPN mRNA and protein in the developing rat brain relative to the distribution of brain macrophages was investigated using in situ hybridization and immunohistochemistry, and the phagocytic capability of OPN-expressing cells was accessed using rhodamine isothiocyanate (RhIc) as a tracer. OPN-expressing cells appeared from embryonic day 16. During the first week of postnatal life, OPN-labeled cells increased markedly, and peaked around P7, then declined and had completely disappeared by the end of the second postnatal week. The spatiotemporal distribution pattern of OPN mRNA closely matched that of OPN protein. Their morphology and localization were compared with those of cells expressing the established microglial marker OX-42 in adjacent sections, and double-labeling studies demonstrated that OPN was localized to the amoeboid microglia which stain with the lectin GSI-B₄, another marker for microglia. Furthermore, OPN-labeled cells were confirmed to be active phagocytes emitting RhIc fluorescence indicating that the tracer into the brain tissues was engulfed by phagocytosis. Therefore, these results provide the first evidence that OPN is transiently expressed in active brain macrophages in the embryonic and early postnatal brain, and suggest that OPN may contribute to the migration and phagocytic function of brain macrophages in the developing brain.This work was supported by a Korea Research Foundation grant (KRF-2002-015-EP0106)     

Regulation of aminopeptidase A in human brain tumor vasculature: evidence for a role of transforming growth factor-beta

Angiotensin peptides are potent vasoconstrictors, cell growth factors, and neuromodulators in normal and pathological situations. To assess the potential role of the angiotensins in brain tumor-associated vessels, the expression of the enzymes of the angiotensin cascade were evaluated in these tumors. The production of these bioactive peptides is dependent on the activities of exopeptidases, including several aminopeptidases and carboxypeptidases, producing angiotensin (Ang) I, II, III, IV and Ang 1-7. Human cerebral parenchymal and glioblastoma cells expressed renin, and tumor vasculature, but not glioblastoma cells, expressed angiotensin-converting enzyme. High aminopeptidase A (APA) activity, but no aminopeptidase N/B activity, was observed in human brain tumor vasculature, suggesting a predominant production of Ang III. Grafting of rat glioma cells in rat brains yielded tumors with high APA and low aminopeptidase N/B activities in tumor vessels, confirming human results. Tumor growth and APA activity in tumor vessels were not affected by chronic angiotensin-converting enzyme inhibition. The brain-derived EC219 endothelial cells expressed high APA activity, which was not involved in endothelial cell proliferation, but was down-regulated by exposure of cells to transforming growth factor-beta (TGF beta) or to TGF beta-secreting tumor cells, suggesting a role for this peptide in the control of APA activity in cerebral vasculature. Thus, APA is a potential marker of chronic dysfunction, involving loss of TGF beta function, of the metabolic blood-brain barrier, but not of neovascularization.     

MDR1 P-glycoprotein is expressed by endothelial cells of newly formed capillaries in human gliomas but is not expressed in the neovasculature of other primary tumors.

The expression of human MDR1 P-glycoprotein (Pgp) in the capillary endothelial cells of the central nervous system has been demonstrated. The brain capillary endothelial cells maintain the structure and function of the blood-brain barrier. Recently, the human MDR1 Pgp (and its mouse homologue MDR1a Pgp) has been shown to function as an important part of this barrier, pumping out xenobiotics from endothelial cells into the lumen of capillaries resulting in the protection of the brain parenchyma. To examine whether the endothelial cells of the newly formed capillaries during neoangiogenesis within malignant human brain tumors express MDR1 Pgp, 35 adult surgical brain tumor specimens (29 gliomas and 6 tumors metastatic to the brain) were obtained from previously untreated patients and studied by a new immunohistochemical sandwich method developed in our laboratory using the JSB-1 monoclonal antibody. JSB-1 is specific for the Pgp product of the human MDR1 (and not MDR3) gene. This sensitive method allows the detection of Pgp in capillary endothelial cells of normal brain in conventional paraffin sections after formalin fixation. The endothelial cells of the newly formed capillaries in 25 of 29 gliomas (86%) and 3 of 6 metastatic tumors, immunostained positive for MDR1 Pgp. The tumor cells in 7 of 35 cases were also positive for Pgp. In the 35 brain tumor cases investigated, the endothelial cells were Pgp positive in the tumor-brain border and in the brain further from the tumor. Capillary endothelial cells of neovasculature in 137 malignant tumors (non-brain) obtained from previously untreated patients showed no MDR1 Pgp expression. These results demonstrated that MDR1 Pgp is expressed not only in the capillaries of normal brain but also in the majority of the newly formed capillaries of brain tumors. Multidrug resistance of brain tumors may result not only from the expression of resistance markers in neoplastic cells but also from the MDR1 Pgp expression in endothelial cells of tumor capillaries. Pgp in this special localization can exclude chemotherapeutic agents from tumor cells that are located around the capillaries. The therapeutic benefit and selectivity of chemotherapeutic agents in combination with a Pgp-reversing agent should be evaluated.     

Structural characterization of neovascularization in adenovirus-12-induced retinal tumor

In order to elucidate a relationship between newly formed micro-blood vessels in retinal tumor and blood-retina barrier, we studied light and electron microscopic and immunohistochemical findings of newly-formed micro-blood vessels in human adenovirus 12-induced retinoblastoma-like tumors in rats. In contrast to other experimentally induced tumors of the central nervous system, there was no evidence of formation of Weibel-Palade bodies and endothelial gaps in this experimental model of retinoblastoma, but factor VIII related antigen was evidently demonstrated. Increased pinocytotic vesicles suggested that the vast majority of these blood vessels did not retain the normal physiological barrier functions. The basement membrane of the micro-blood vessels was poorly developed. The morphological characteristics of the micro-blood vessels of this retinoblastoma-like tumor were compared with those of human retinoblastoma and other experimentally induced tumors of the central nervous system.     

Immunolocalization of occludin and claudin-1 to tight junctions in intact CNS vessels of mammalian retina

The distributions of occludin and claudin-1, two tight junction–associated integral membrane proteins were investigated by immunohistochemical analysis of whole-mount preparations of the blood vessels in the myelinated streak of the rabbit retina. Light microscopy revealed that occludin and claudin-1 immunoreactivities were abundant along the interface of adjacent endothelial cells of all blood vessels. Electron microscopy revealed that both proteins were distributed in a regular pattern (at regular intervals of approximately 80 nm) along the length of tight junctions, probably in the regions of tight junction strands. No other structures or cell types expressed either of these two proteins in the myelinated streak. Whereas occludin immunoreactivity was concentrated only at the tight junction interface, claudin-1 immunoreactivity also extended into the cytoplasm of the endothelial cells, suggesting a different structural role for claudin-1 than for occludin at tight junctions. Retinal pigment epithelial cells expressed occludin around their entire circumference, consistent with the function of these cells as a barrier separating the retina from the leaky vessels of the choroid. Also consistent with the association of occludin expression with vessels that exhibit functional tight junctions, this protein was expressed at only a low level in, and showed an irregular distribution along, the vessels of the choroid, a vascular bed that lacks blood-barrier properties. Further, the distribution of occludin was examined during formation and remodelling of the rat retinal vasculature. Occludin expression was evident at the leading edge of vessel formation and was found on all vessels in both the inner and outer vascular plexus. Numerous vascular segments at the early stage of vascular formation and regression lost occludin expression. The biological significance of this transient loss of occludin expression in terms of barrier function remains to be elucidated.     

STRUCTURAL CHARACTERIZATION OF NEOVASCULARIZATION IN ADENOVIRUS-12-INDUCED RETINAL TUMOR

In order to elucidate a relationship between newly formed micro-blood vessels in retinal tumor and blood-retina barrier, we studied light and electron microscopic and immunohistochemical findings of newly-formed micro-blood vessels in human adenovirus 12-induced retlnoblastoma-Hke tumors in rats. In contrast to other experimentally induced tumors of the central nervous system, there was no evidence of formation of Weibel-Palade bodies and endothelial gaps in this experimental model of retinoblastoma, but factor VIII related antigen was evidently demonstrated. Increased pinocytotic vesicles suggested that the vast majority of these blood vessels did not retain the normal physiological barrier functions. The basement membrane of the micro-blood vessels was poorly developed. The morphological characteristics of the micro-blood vessels of this retinoblastoma-like tumor were compared with those of human retinoblastoma and other experimentally induced tumors of the central nervous system.