Tumor skewing of CD34+ cell differentiation from a dendritic cell pathway into endothelial cells

Patients and animals bearing tumors have increased levels of CD34⁺ progenitor cells, which are capable of developing into dendritic cells. However, addition of medium conditioned by murine Lewis lung carcinoma cells increases the cellularity of the CD34⁺ cell cultures and redirects their differentiation into endothelial cells. The resulting cells resemble endothelial cells phenotypically as well as functionally by their capacity to reorganize into cord structures. Mechanisms by which tumors induced the increased cellularity and skewing toward endothelial cells were examined. Tumor-derived VEGF contributed to the increase in cellularity, but not to the redirection of differentiation. Differentiation into endothelial cells was blocked with sTie-2, suggesting tumor-derived angiopoietins in skewing differentiation. These studies show the capacity of tumors to skew progenitor cell development toward endothelial cells and define the mediators that contribute to endothelial cell development.     

ALK2使内皮细胞转化为类干细胞的机制

内皮细胞向间充质细胞转分化（endothelial-to-mesenchymal transition,EndMT）过程是血管内皮细胞转化为类干细胞的基础,持续激活状态的ALK2在EndMT过程起到关键的促进作用,EndMT发生后内皮细胞能获得类干细胞的表现型.TGF-β2和BMP4作为特异性配体激活ALK2,下调某些内皮细胞标志物并上调某些间质细胞标志物,促进EndMT过程.此外,编码ALK2的基因序列在206号氨基酸上突变形成R206H ALK2后作用类似持续激活状态ALK2,同样也能达到促进内皮细胞向间充质细胞转分化的目的.     

Mst1 deletion reduces hyperglycemia-mediated vascular dysfunction via attenuating mitochondrial fission and modulating the JNK signaling pathway

Diabetes is a leading cause of microvascular complications, such as nephropathy and retinopathy. Recent studies have proposed that hyperglycemia-induced endothelial cell dysfunction is modulated by mitochondrial stress. Therefore, our experiment was to detect the upstream mediator of mitochondrial stress in hyperglycemia-treated endothelial cells with a focus on macrophage-stimulating 1 (Mst1) and mitochondrial fission. Our data illuminated that hyperglycemia incubation reduced cell viability, as well as increased apoptosis ratio in endothelial cell, and this alteration seemed to be associated with Mst1 upregulation. Inhibition of Mst1 via transfection of Mst1 siRNA into an endothelial cell could sustain cell viability and maintain mitochondrial function. At the molecular levels, endothelial cell death was accompanied with the activation of mitochondrial oxidative stress, mitochondrial apoptosis, and mitochondrial fission. Genetic ablation of Mst1 could reduce mitochondrial oxidative injury, block mitochondrial apoptosis, and repress mitochondrial fission. Besides, we also found Mst1 triggered mitochondrial dysfunction as well as endothelial cell damage through augmenting JNK pathway. Suppression of JNK largely ameliorated the protective actions of Mst1 silencing on hyperglycemia-treated endothelial cells and sustain mitochondrial function. The present study identifies Mst1 as a primary key mediator for hyperglycemia-induced mitochondrial damage and endothelial cell dysfunction. Increased Mst1 impairs mitochondrial function and activates endothelial cell death via opening mitochondrial death pathway through JNK.     

角膜内皮细胞再生研究新进展

人角膜内皮细胞的主要功能是维持角膜透明性,角膜内皮单层发育成熟形成细胞接触后,内皮细胞会停止分裂增殖,但并没有退出细胞周期。角膜内皮细胞的增殖有多种因素的参与和影响,接触抑制和G1期抑制使细胞增殖暂时停止;细胞因子TGF-β2抑制人角膜内皮细胞进入细胞周期S期,而EGF、FGF、NGF则能够促进细胞的增殖;ROCK抑制剂Y-27632能够促进角膜内皮细胞的粘连,有助于内皮细胞的损伤修复。体外培养角膜内皮前体细胞、诱导多潜能干细胞向角膜内皮细胞分化,为今后治疗角膜内皮失代偿提供了新方向。     

Isolation and characterization of an established endothelial cell line from transgenic mouse hemangiomas

A murine endothelial cell line was isolated from hemangiomas induced by expression of the polyoma early region gene in transgenic mice. After two cell sortings using acetylated low-density lipoprotein with a fluorescent label (Dil-Ac-LDL), a pure population of endothelial cells has been carried for more than 60 passages from the animal. The cells retain endothelial cell properties such as a characteristic cobblestone appearance at confluency, contact-inhibited growth, and active uptake of Ac-LDL. Expression analysis shows that the cells express both the polyoma transgene and the von Willebrand factor, an endothelial cell marker. Subcutaneous injection of the cultured endothelial cells into nontransgenic histocompatible mice or nude mice led to hemangioma formation, and endothelial cells were re-isolated by cell sorting from these secondary hemangiomas. This cell line represents a renewable source of murine endothelial cells derived from transgenic mice that can be studied both in vitro and by reintroduction into a host.     

The production and localization of laminin in cultured vascular and corneal endothelial cells

The production and localization of laminin, as a function of cell density (sparse versus confluent cultures) and growth stage (actively growing versus resting cultures), has been compared on the cell surfaces of cultured vascular and corneal endothelial cells. Comparison of the abilities of the two types of cells to secrete laminin and fibronectin into their incubation medium reveals that vascular endothelial cells can secrete 20-fold as much laminin as can corneal endothelial cells. In contrast, both cell types produce comparable amounts of fibronectin. Furthermore, if one compares the secretion of laminin and fibronectin as a function of cell growth, it appears that the laminin released into the medium by either vascular or corneal endothelial cells, is a function of cell density and cell growth, since this release is most pronounced when the cells are sparse and actively growing, and decreases by 10- and 30-fold, respectively, when either vascular or corneal endothelial cell cultures become confluent. With regard to fibronectin secretion, no such variation can be seen with vascular endothelial cell cultures, regardless of whether they are sparse and actively growing or confluent and resting. Corneal endothelial cell cultures, demonstrated a twofold increase in fibronectin production when they were confluent and resting as compared to when they were sparse and actively growing. When the distribution of laminin versus fibronectin within the apical and basal cell surfaces of cultured corneal and vascular endothelial cells is compared, one can observe that unlike fibronectin, which in sparse and subconfluent cultures can be seen to be associated with both the apical cell surface. In confluent cultures, laminin can be found associated primarily with the extracellular matrix beneath the cell monolayer, where it codistributes with type IV collagen.     

Endothelial cell response to pulsed electromagnetic fields: stimulation of growth rate and angiogenesis in vitro

The effects of pulsed electromagnetic fields on the repopulation rate of denuded regions of endothelial cell monolayers and on endothelial cell reorganization into complex vessellike structures was monitored in vitro by using human umbilical vein and bovine aortic endothelial cells. A small (20-40%) but statistically significant enhancement in growth rate of partially denuded endothelial cell monolayers as determined by tritiated thymidine incorporation was observed in the presence of pulsed electromagnetic fields. Morphologically, endothelial cells entering the denuded regions were observed to be elongated, often connecting end to end to form a mycelial or "sprouting" pattern when exposed to pulsed electromagnetic fields. This was in contrast to cells outside of the field which had a more cuboidal morphology. Complete disruption of the endothelial cell monolayer by passaging the cells with EDTA-trypsin resulted in reorganization of some of the cells into three-dimensional vessellike structures after as little as 5-8 hours in the presence of the pulsed electromagnetic field. This reorganization occurred in the presence of heparin, endothelial cell growth factor, and a competent fibronectin matrix. Vascularization for comparable cultures outside of the field did not occur during the time-course of the experiments. Discrete stages of neovascularization were observed in the presence of the field that were qualitatively similar to stages of angiogenesis observed in vivo.     

Dye-coupling of melanocytes with endothelial cells and pericytes in the cochlea of gerbils

Intercellular connections via gap junctions in the stria vascularis, which constitutes the lateral wall of the cochlear duct, were investigated by the Lucifer yellow microinjection method with the aid of a confocal laser microscope. The dye injected into an intermediate cell (melanocyte) diffused into capillary endothelial cells and pericytes as well as other intermediate cells, basal cells, and fibrocytes in the spiral ligament; whereas the dye injected into a marginal cell (epithelial cell) was confined to the injected cell. The observation of dye-coupling between intermediate cells and endothelial cells and pericytes makes likely the possibility that these cells work together to play a role in the specific function of the stria vascularis (i.e., production of the positive endocochlear potential and the endolymph) and adds endothelial cells and pericytes to the current “two-cell model” of the stria vascularis.     

Fibulin-5 antagonizes vascular endothelial growth factor (VEGF) signaling and angiogenic sprouting by endothelial cells

Fibulin-5 (FBLN-5) is a widely expressed, integrin-binding extracellular matrix protein that mediates endothelial cell adhesion and scaffolds cells to elastic fibers. It is also a gene target of TGF-beta in fibroblasts and endothelial cells that regulates cell proliferation and motility in a context-specific manner. Whereas FBLN-5 expression is low in adult vasculature, its expression is high in developing and injured vasculature, implicating FBLN-5 in regulating angiogenesis and endothelial cell function. We show here that TGF-beta stimulates FBLN-5 expression in endothelial cells, and that this response was inhibited by coadministration of the proangiogenic factor, VEGF. FBLN-5 expression was downregulated significantly during endothelial cell tubulogenesis, implying that FBLN-5 expression antagonizes angiogenesis. Accordingly, FBLN-5 overexpression in or recombinant FBLN-5 treatment of endothelial cells abrogated their ability to undergo angiogenic sprouting, doing so by inhibiting endothelial cell proliferation and invasion through Matrigel matrices. Moreover, FBLN-5 antagonized VEGF signaling in endothelial cells, as well as enhanced their expression of the antiangiogenic factor, thrombospondin-1. Finally, the ability of FBLN-5 to antagonize angiogenic processes was determined to be independent of its integrin-binding RGD motif. Collectively, our findings establish FBLN-5 as a novel antagonist of angiogenesis and endothelial cell activities, and offer new insights into why tumorigenesis downregulates FBLN-5 expression.     

Production of immortal human endothelial cell lines by strontium phosphate transfection and electroporation of SV40 sequences

Summary Eleven human endothelial cell lines have been produced by introducing sequences from the DNA tumor virus SV40 into human umbilical vein endothelial cells either by strontium phosphate coprecipitation or electroporation. The resultant lines were confirmed as being endothelial in origin by their production of endothelial-specific von Willebrand factor. The growth characteristics of the different lines in normal and reduced levels of serum was determined, as was their cellular response to endothelial cell growth supplement in combination with heparin, basic fibroblast growth factor, transforming growth factor-alpha, and epidermal growth factor.     

Soluble epithin/PRSS14 secreted from cancer cells contains active angiogenic potential

Epithin (PRSS14/matriptase/ST14), a type II membrane protein, is involved in progression of epithelial cancers and metastasis as well as in the normal epidermal barrier function. When activated, it translocates into the cell-cell contacts and sheds into media. In order to understand the specific mechanism during tumor progression, we tested the angiogenic potential of secreted form of epithin. Epithin produced from the cancer cells shed more in hypoxia and induced motility of endothelial cells. Epithin enhanced the migration and invasion of mouse and bovine endothelial cells without cell proliferation. Furthermore, soluble epithin induced endothelial differentiation in the assay of the human endothelial microvessel-like tube formation and in that of the chicken chorioallantoic membrane. The knock-down of epithin in the 427 thymoma cell line abolished the protease activity of secreted epithin fraction, reduced the invasion of endothelial cells through matrigel, and tube formation activity. Only specific antibodies abolished the migration of endothelial cell and the vessel morphogenesis, suggesting that epithin specifically functions in these systems. Therefore, we propose that the secreted epithin in the hypoxic cancer microenvironment plays a role as a proangiogenic factor, and can be modulated with specific antibodies.     

Use of high throughput genomic tools for the study of endothelial cell biology

The endothelium is an active, dynamic and heterogeneous organ. It lines the vessels in every organ system and regulates diverse and important biological functions. Over the past several years researchers have gained enormous insights into endothelial cell function in physiological processes such as coagulation and vascular reactivity, and pathophysiological disease states such as inflammation and atherosclerosis. Despite our expanding knowledge of endothelial cell biology, the molecular mechanisms underlying these functions remain largely unknown. The newly developed high throughput genomic tools and accompanying analytical methods provide powerful approaches for identifying new endothelial cell genes and characterizing their role in health and disease. Here, we review some of the recent genomics and proteomic advances that are providing new methodologies for endothelial cell and vascular biology research.     

Minimal contribution of marrow-derived endothelial precursors to tumor vasculature

During embryogenesis, vascular and hemopoietic cells originate from a common precursor, the hemangioblast. Recent evidence suggests the existence of endothelial precursors in adult bone marrow cells, but it is unclear whether those precursors have a role in tumor neovascularization. In this report, we demonstrate that murine bone marrow contains endothelial progenitors, which arise from a cell with self-renewing capacity, and can integrate into tumor microvasculature, albeit at a very low frequency. A transgenic double-reporter strategy allowed us to demonstrate definitively that tumor bone marrow-derived endothelial cells arise by transdifferentiation of marrow progenitors rather than by cell fusion. Single cell transplants showed that a common precursor contributes to both the hemopoietic and endothelial lineages, thus demonstrating the presence of an adult hemangioblast. Furthermore, we demonstrate that increased vascular endothelial growth factor (VEGF)-A secretion by tumor cells, as well as activation of VEGF receptor-2 in bone marrow cells does not alter the mobilization and incorporation of marrow-derived endothelial progenitors into tumor vasculature. Finally, in human umbilical cord blood cells, we show that endothelial precursors make up only approximately 1 in 10(7) mononuclear cells but are highly enriched in the CD133+ cell population. By ruling out cell fusion, we clearly demonstrate the existence of an adult hemangioblast, but the differentiation of marrow stem cells toward the endothelial lineage is an extremely rare event. Furthermore, we show that VEGF-A stimulation of hemopoietic cells does not significantly alter this process.     

Regulation of endothelial cell differentiation and transformation by H-Ras

Angiogenesis is regulated by growth factors which activate tyrosine kinase receptors leading to the activation of a number of intracellular signaling pathways. The specific function of H-Ras during FGF-2 stimulated endothelial cell differentiation, defined as invasive growth and formation of branching networks in fibrin gels, was investigated by using conditionally immortalized endothelial cell lines induced to express H-Ras mutants. Expression of inhibitory N17Ras did not impair differentiation in response to FGF-2 and TNF-alpha. The farnesyltransferase inhibitor FTI-277 inhibited farnesylation of Ras but did not inhibit differentiation of human microvascular endothelial cells or mouse brain endothelial cells. In contrast, activated V12Ras inhibited endothelial cell differentiation and cells displayed a transformed phenotype with an increased rate of proliferation and loss of contact inhibited growth. Furthermore, V12Ras expressing endothelial cells grew as solid tumors when injected subcutaneously into mice. Our data suggest that, in endothelial cells, H-Ras activity is not required for differentiation. However, this activity must be tightly regulated as aberrant activity can disturb the ability of endothelial cells to undergo differentiation.     

SURZB/Kir6.1通道开放剂纳他卡林对低氧致大鼠主动脉内皮细胞损伤的保护作用

目的:探讨纳他卡林对低氧引起大鼠主动脉内皮细胞损伤的保护作用及其机制。方法:选取大鼠主动脉内皮细胞作为体外低氧损伤的细胞模型,分为正常对照组、低氧模型组、纳他卡林低、中、高剂量组,利用MTT法测定细胞生存率,硝酸还原酶法检测一氧化氮(NO)释放,RT-PCR法检测细胞间粘附因子-1(ICAM-1)、内皮素-1(ET-1)、血管内皮生长因子(VEGF)mRNA水平。结果:纳他卡林三个剂量组均可逆转低氧所致的血管内皮细胞功能改变,包括提高内皮细胞生存活力和NO的释放水平,显著抑制低氧引发的内皮细胞ICAM-1,ET-1,VEGF mRNA表达量的上调。结论:纳他卡林对低氧诱发的血管内皮细胞分泌功能改变、细胞通透性增加及炎性因子的分泌均具有保护作用。     

Brain Endothelial Cell Death: Modes,Signaling Pathways,and Relevance to Neural Development,Homeostasis, and Disease

Emerging evidence indicates that brain microvascular endothelial cells play a critical role in brain development, maturation, and homeostasis. Acute or chronic insults, including oxidative stress, oxygen–glucose deprivation, trauma, infections, inflammatory cytokines, DNA damaging agents, β-amyloid deposition, and endoplasmic reticulum stress induce brain endothelial cell dysfunction and damage, which can result in cell death. The homeostatic balance between endothelial cell survival and endothelial cell death is critical for brain development, remodeling, and repair. On the other hand, dysregulation of brain endothelial cell death exacerbates, or even initiates, several inflammatory, ischemic, and degenerative disorders of the central nervous system. In here, the morphological, biochemical, and functional characteristics of the brain endothelium and its contribution to brain homeostasis will be reviewed. Recent insights into modalities and regulatory pathways involved in brain endothelial cell death will be described. The effects of regulated and dysregulated endothelial cell death leading to angiogenesis will be outlined. The relevance of brain endothelial cell dysfunction and death to disease processes will be discussed with special reference to recent findings that could help translate current knowledge on brain endothelial cell apoptosis into new therapeutic strategies for the treatment of certain neurological disorders.     

Platelet-activating factor receptor and innate immunity: uptake of gram-positive bacterial cell wall into host cells and cell-specific pathophysiology

The current model of innate immune recognition of Gram-positive bacteria suggests that the bacterial cell wall interacts with host recognition proteins such as TLRs and Nod proteins. We describe an additional recognition system mediated by the platelet-activating factor receptor (PAFr) and directed to the pathogen-associated molecular pattern phosphorylcholine that results in the uptake of bacterial components into host cells. Intravascular choline-containing cell walls bound to endothelial cells and caused rapid lethality in wild-type, Tlr2(-/-), and Nod2(-/-) mice but not in Pafr(-/-) mice. The cell wall exited the vasculature into the heart and brain, accumulating within endothelial cells, cardiomyocytes, and neurons in a PAFr-dependent way. Physiological consequences of the cell wall/PAFr interaction were cell specific, being noninflammatory in endothelial cells and neurons but causing a rapid loss of cardiomyocyte contractility that contributed to death. Thus, PAFr shepherds phosphorylcholine-containing bacterial components such as the cell wall into host cells from where the response ranges from quiescence to severe pathophysiology.     

Tumor necrosis factor reduces proteoglycan synthesis in cultured endothelial cells

Tumor necrosis factor (TNF)-induced disruption of vascular endothelial barrier function may be due in part to alterations in proteoglycan metabolism. To test this hypothesis, confluent endothelial cell monolayers were exposed for 24 h to 500 or 1,000 U of TNF per mililiter of culture medium together with 20 μCi Na₂ ³⁵SO₄. HPLC anion-exchange separation of proteoglycans secreted into media of control as well as TNF-treated cultures revealed one major peak (representing 95% of total radioactivity) and one minor peak (representing 5% of total radioactivity), which eluted at 0.6 and 0.9 M NaCl, respectively. One single peak was obtained from control as well as TNF-treated endothelial cell monolayers and eluted at 1.2 M NaCl. TNF treatment did not change the total quantity of radioactive proteoglycans secreted into the media but significantly decreased the amount of proteoglycans in endothelial cell monolayers. However, TNF treatment did not alter the size or glycosaminoglycan (GAG) composition of the proteoglycans either in the media or in the cell monolayers. In addition, mRNA levels of specific proteoglycans, perlecan and biglycan, were measured upon TNF treatment, using Northern analysis. TNF treatment caused a dose-dependent decrease in mRNA levels for the biglycan in endothelial cultures. These results suggest that TNF decreases production of proteoglycans and alters normal endothelial cell proteoglycan metabolism which may be sufficient to impair endothelial barrier function. © 1995 Wiley-Liss, Inc.     

Covalently grafted VEGF(165) in hydrogel models upregulates the cellular pathways associated with angiogenesis

Angiogenesis is an important biological response known to be involved in many physiological and pathophysiological situations. Cellular responses involved in the formation of new blood vessels, such as increases in endothelial cell proliferation, cell migration, and the survival of apoptosis-inducing events, have been associated with vascular endothelial growth factor isoform 165 (VEGF(165)). Current research in the areas of bioengineering and biomedical science has focused on developing polyethylene glycol (PEG)-based systems capable of initiating and sustaining angiogenesis in vitro. However, a thorough understanding of how endothelial cells respond at the molecular level to VEGF(165) incorporated into these systems has not yet been established in the literature. The goal of the current study was to compare the upregulation of key intracellular proteins involved in angiogenesis in human umbilical vein endothelial cells (HUVEC) and human microvascular endothelial cells (HMEC) seeded on PEG hydrogels containing grafted VEGF(165) and adhesion peptides Arg-Gly-Asp-Ser (RGDS). Our data suggest that the covalent incorporation of VEGF(165) into PEG hydrogels encourages the upregulation of signaling proteins responsible for increases in endothelial cell proliferation, cell migration, and the survival after apoptosis-inducing events.     

alphavbeta3 integrin-dependent endothelial cell dynamics in vivo

A major challenge confronting developmental cell biologists is to understand how individual cell behaviors lead to global tissue organization. Taking advantage of an endothelial cell-specific marker and scanning time-lapse microscopy, we have examined the formation of the primary vascular pattern during avian vasculogenesis. Five types of distinguishable endothelial cell motion are observed during formation of a vascular plexus: (1) global tissue deformations that passively convect endothelial cells; (2) vascular drift, a sheet-like medial translocation of the entire vascular plexus; (3) structural rearrangements, such as vascular fusion; (4) individual cell migration along existing endothelial structures; and (5) cell process extension into avascular areas, resulting in new links within the plexus. The last four types of motion are quantified and found to be reduced in the presence of an alphavbeta3 integrin inhibitor. These dynamic cell motility data result in new hypotheses regarding primordial endothelial cell behavior during embryonic vasculogenesis.