Tumour metabolites regulate tissue kallikrein in human umbilical vein endothelial cells

Angiogenesis, the sprouting of new blood vessels, is tightly mediated via a myriad of endogenous factors. A pro-angiogenic alteration facilitates the formation of neovascular tumour networks, thereby providing mechanisms for uncontrolled growth. The kallikrein-kinin system is postulated to be pro-angiogenic since its components have been detected in both endothelial cells and tumour tissue. No studies have, however, focussed on the role of tissue kallikrein (TK) in human angiogenic endothelial cell-tumour interactions. This study has optimised a challenge model whereby endothelial cells are presented with neuroblastoma metabolites, and vice versa. Image analysis of immunoreactive TK revealed a dose-dependant, significant reduction of TK localisation within endothelial cells, while gene expression remained unchanged, the latter determined by in situ RT-PCR. Neuroblastoma cells, when challenged with endothelial cell metabolites, displayed no change in TK synthesis or localisation. Alterations in TK synthesis and/or storage by angiogenic endothelial cells may be mediated by tumour-released signals and possibly indicate a shift from a proteolytic to a mitogenic function of TK. The challenge model provides a relatively simple experimental system to study angiogenic factors in tumour-endothelial cell interaction, and is the first to localise both TK and its mRNA within angiogenic endothelial and tumour cells.     

KLK5 and KLK7, two members of the human tissue kallikrein family, are differentially expressed in lung cancer

Emerging data indicate that serine proteases of the kallikrein family (KLK) are implicated in various human diseases, including carcinoma; however, kallikrein gene expression has never been investigated in lung cancer. Using RT-PCR and Western blotting, we demonstrated the expression of both KLK5 and KLK7, and their respective proteins (hK5 and hK7) in tumoral and nontumoral lung tissues. Quantitative gene expression was then analyzed in a cohort of 56 patients with non-small cell lung cancer by real-time RT-PCR. KLK5 expression is significantly more expressed in squamous cell carcinoma than in matched nonmalignant lung tissue (P=0.02), whereas expression of KLK7 was decreased in adenocarcinoma (P=0.003). Multivariate analysis revealed diverse correlations between the KLK5 and KLK7 expression levels in nonmalignant and malignant tissues, and clinical parameters, including histotype, metastatic status, and grade. Our findings provide new insight into kallikrein gene expression in hormone-independent carcinoma. Altogether, our results suggest that variability in KLK5 and KLK7 gene expression might be involved in lung tumorigenesis and useful for clinical purposes.     

An optimized culture medium for human vascular endothelial cells from umbilical cord veins

Various polypeptide growth factors, culture substrates, basal media, sera and further supplements were assayed for improvement of growth of human vascular endothelial cells from umbilical cord veins. The resulting optimized medium consisted of gelatinized culture substrates, a mixture (1:1) of Iscove's MDM and Ham's F12 basal media supplemented with 20% newborn calf serum, 500 ng/ml crude fibroblast growth factor, 20 ng/ml epidermal growth factor, 5 g/ml transferrin, 5 g/ml insulin and 10 g/ml heparin. The medium allowed long term cultivation of HUVEC up to 45 generations with maximal cell densities of about 10⁵ cells per cm² and a minimal doubling time of about 14 hours at low cell densities.Abbreviations HUVEC Human Endothelial Cells From Umbilical Cord Veins - FGF Fibroblast growth factor - EGF Epidermal Growth Factor - FCS Fetal Calf Serum - NCS Newborn Calf Serum - HBS HEPES-Buffered Saline - ECM Extracellular Matrix - LHM Peptide PyroGlu-His-Ser-Phe-Thr-Ile-Lys-Ile-ThrCONH₂ - IF 1:1 mixture of Iscove's MDM and F12 basal media     

Nestin is expressed in vascular endothelial cells in the adult human pancreas.

In this study we examined the expression of nestin in islets, the exocrine part, and the big ducts of the adult human pancreas by immunofluorescent double staining. Two different anti-nestin antisera in combination with various pancreatic and endothelial markers were employed. Nestin-immunoreactive cells were found in islets and in the exocrine portion. All nestin-positive cells co-expressed the vascular endothelial markers PECAM-1 (CD31), endoglin (CD105), and CD34 as well as vimentin. Endocrine, acinar, and duct cells did not stain for nestin. We also demonstrated that in the area of big pancreatic ducts, nestin-positive cells represent small capillaries scattered in the connective tissue surrounding the duct epithelium and do not reside between the duct cells. We detected nestin-expressing endothelial cells located adjacent to the duct epithelium where endocrine differentiation occurs. We have shown that nestin is expressed by vascular endothelial cells in human pancreas, and therefore it is unlikely that nestin specifically marks a subpopulation of cells representing endocrine progenitors in the adult pancreas.     

BRCA1在人类肿瘤细胞中的表达

人类乳癌易感基因1（BRCA1）是乳癌,卵巢癌和前列腺癌的危险因素之一,而且表现出许多的生物功能,采用Western Blotting和半定量RT－PCR的方法,我们检测了内源性BRCA1蛋白质和mRNA在从十一种人类肿瘤组织中建立的四十三种肿瘤细胞系的表达水平。在不同的肿瘤细胞中BRCA1的表达水平是各不一样的。而且并没有发现BRCA1的表达和细胞的内源性p53基因状况有明显的相关性。通过采用细胞转染乳头状瘤病毒－E6致癌基因或采用畸变的p53基因（143Ala→Val)而导致的p53基因功能失活并不对内源性BRCA1本底表达水平产生任何的影响,胆两种与p53功能有关p21(-/-)和Gadd45基因剔除则轻微地增加BRCA1蛋白质的表达。因此,虽然我们目前还不清楚BRCA1在人类肿瘤细胞中不同表达的功能意义,但本文的结果为进一步研究BRCA1在不同种瘤细胞系的生物功能提供了有价值的背景资料。     

Clec14a is specifically expressed in endothelial cells and mediates cell to cell adhesion

Clec14a is a member of the thrombomodulin (TM) family, but its function has not yet been determined. Here, we report that Clec14a is a plasma membrane protein of endothelial cells (ECs) expressed specifically in the vasculature of mice. Deletion mutant analysis revealed that Clec14a mediates cell–cell adhesion through its C-type lectin-like domain. Knockdown of Clec14a in ECs suppressed cell migratory activity and filopodial protrusion, and delayed formation of tube-like structures. These findings demonstrate that Clec14a is a novel EC-specific protein that appears to play a role in cell–cell adhesion and angiogenesis.     

Lymphatic endothelial progenitor cells contribute to de novo lymphangiogenesis in human renal transplants

De novo lymphangiogenesis influences the course of different human diseases as diverse as chronic renal transplant rejection and tumor metastasis. The cellular mechanisms of lymphangiogenesis in human diseases are currently unknown, and could involve division of local preexisting endothelial cells or incorporation of circulating progenitors. We analyzed renal tissues of individuals with gender-mismatched transplants who had transplant rejection and high rates of overall lymphatic endothelial proliferation as well as massive chronic inflammation. Donor-derived cells were detected by in situ hybridization of the Y chromosome. We compared these tissues with biopsies of essentially normal skin and intestine, and two rare carcinomas with low rates of lymphatic endothelial proliferation that were derived from individuals with gender-mismatched bone marrow transplants. Here, we provide evidence for the participation of recipient-derived lymphatic progenitor cells in renal transplants. In contrast, lymphatic vessels of normal tissues and those around post-transplant carcinomas did not incorporate donor-derived progenitors. This indicates a stepwise mechanism of inflammation-associated de novo lymphangiogenesis, implying that potential lymphatic progenitor cells derive from the circulation, transmigrate through the connective tissue stroma, presumably in the form of macrophages, and finally incorporate into the growing lymphatic vessel.     

Neurophilin-1 is a downstream target of transcription factor Ets-1 in human umbilical vein endothelial cells

Photosystem II complex (PSII) of thylakoid membranes uses light energy to oxidise extremely stable water and produce oxygen (2H(2)O-->O(2)+4H(+)+4e(-)). PSII is compared with cytochrome c oxidase that catalyses the opposite reaction coupled to proton translocation. Cytochrome c oxidase has proton and water channels, and a tentative oxygen channel. I propose that functional PSII complexes also need a specific oxygen channel to direct O(2) from the water molecules bound to specific Mn atoms of the Mn cluster within PSII out to the membrane surface. The function of this channel will be to prevent oxygen being accessible to the radical pair P680(+)Pheo(-), thereby preventing singlet oxygen generation from the triplet P680 state in functional PSII. The important role of singlet oxygen in structurally perturbed non-functional photosystem II is also discussed.     

N-Cadherin is expressed on human hematopoietic progenitor cells and mediates interaction with human mesenchymal stromal cells

Specific cell–cell junctions between hematopoietic stem cells (HSC) and their niche have been shown to regulate stem cell function. N-cadherin was suggested to play a central role in this process, whereas other studies indicated that it did not play an essential role in the murine model. We have analyzed the role of N-cadherin for interaction between hematopoietic progenitor cells (HPC) and supportive mesenchymal stromal cells (MSC) in a human–human setting. Expression of N-cadherin and of cadherin-11 (osteoblast cadherin) was analyzed in HPC by quantitative RT-PCR, Western blot, and flow cytometry. N-cadherin and cadherin-11 were expressed in HPC at a moderate level, whereas they were not detectable in differentiated cells. Confocal laser scanning microscopy revealed that N-cadherin and β-catenin are colocalized at the junction of HPC and MSC. siRNA knockdown of N-cadherin or cadherin-11 as well as treatment with the blocking function antibody decreased adhesive interaction of HPC to MSC. Furthermore, knockdown of N-cadherin or blocking function antibody impaired maintenance of long-term culture-initiating cells (LTC-IC) on coculture of HPC and MSC. These results indicate that N-cadherin is involved in the bidirectional interaction of human HPC with their cellular determinants in the niche.     

Pleiotrophin stimulates fibroblasts and endothelial and epithelial cells and is expressed in human cancer.

Previously we reported the purification of the heparin-binding growth factor pleiotrophin (PTN) from supernatants of the human breast cancer cell line MDA-MB-231. To investigate further the biological activities of PTN and its potential role in cancer, we cloned a PTN cDNA and expressed the gene in a human kidney and in a human adrenal carcinoma cell line (SW-13). The supernatants harvested from cells transfected with PTN contained a heparin-binding specific protein of an apparent molecular mass of 18 kDa. These supernatants stimulated the proliferation of endothelial cells as well as the anchorage-independent growth of SW-13 cells and of normal rat kidney fibroblasts. Furthermore, SW-13 cells transfected with PTN acquired autonomous growth in soft agar and were tumorigenic in athymic nude mice. In contrast to these results with PTN from human cells, PTN obtained from insect cells (Sf9) using recombinant baculovirus as a vector was biologically inactive. We detected high levels of PTN mRNA in 16 of 27 primary human breast cancer samples (62%) as well as in 8 of 8 carcinogen-induced rat mammary tumors. Furthermore, 9 of 34 human tumor cell lines of different origin showed detectable PTN mRNA. We conclude that PTN may function as a tumor growth and angiogenesis factor in addition to its role during embryonic development.     

Prostacyclin synthesis by endothelial cells from human umbilical veins: effect of cumulative population doublings

There is a wide range of reported values for prostacyclin (PGI2) synthesis by cultured endothelial cells from human umbilical veins (HUVE). Part of this variation may be due to differences in isolation and culture conditions, but part may be due to previously unstudied variation in the number of population doublings (PDs) which the cells have undergone in vitro. Attention is now shifting to arachidonic acid (AA) metabolism by cells from adult human vessels and these cells may require increased PDs to obtain confluent cultures for testing. Therefore, we have examined the effect of number of cell population doublings as well as number of subcultivations on PGI2 synthesis using HUVE as a model system. Primary and first subcultivation cultures inoculated at high density, so that PDs at confluence were less than 4, synthesized 10 times as much PGI2 as the same isolates inoculated at low density with PDs greater than 4. Isolates inoculated and subcultivated so that the PDs at confluence after the fourth subcultivation were less than 6, showed 50% less PGI2 synthesis between the primary and first subcultivation and between the first and second subcultivations. Isolates with less than 4 PDs after the fourth subcultivation were carried further to determine the effect of extensive subcultivation. Four of six isolates showed a sudden increase in PGI2 synthesis which occurred between subcultivations 5 and 12 (PDs 4-6). These results demonstrate that AA metabolism is markedly affected by growth in culture and serial subcultivation.     

Heme oxygenase-1 is an important modulator in limiting glucose-induced apoptosis in human umbilical vein endothelial cells

Hyperglycaemia is associated with oxidative stress. The inducible isoform of heme oxygenase (HO-1) is an effective system to counteract oxidative stress, yet it is unclear how hyperglycaemia affects HO-1. In this study, we explored: 1) the HO-1 protein content and HO activity in human umbilical vein endothelial cells (HUVECs) exposed to different glucose concentrations, and 2) the mechanisms which account for the high glucose-induced effects on HO-1. We evaluated HO-1 protein expression, HO activity, apoptosis and reactive oxygen species (ROS) in HUVECs treated for 48 h with 5.5, 10 and 20 mM glucose. A dose-dependent production of reactive oxygen species was observed. At 10 mM glucose, an increase of HO-1 protein expression and HO activity was observed, whereas at 20 mM, there was no change in protein content and activity relative to at 5.5 mM glucose. HO-1 protein expression in HUVECs exposed to 20 mM of glucose was increased in the presence of 20 U/ml superoxide dismutase (SOD). HO-1 gene silencing augments ROS production both at 5.5 and 10 mM glucose, leading to an increased apoptosis. We conclude that, in endothelial cells, the regulation of HO-1 by glucose is dependent upon levels of glucose itself. Lack of homeostatic HO-1 upregulation fails to protect from oxidative damage and results in a higher rate of apoptotic cell death.     

Synthesis of protein C in human umbilical vein endothelial cells.

By monitoring the activation of protein C and the regulation of factor Xa-catalyzed thrombin formation by the activated protein C (APC) on the surface of human umbilical vein endothelial cells (HUVEC), we found that functional protein C was synthesized in cultured HUVEC and expressed thereon in the presence of vitamin K. Furthermore, without exogenously added protein S, time-dependent and saturable accumulation of APC (20 fmol APC/10(5) cells) on the surface of HUVEC was observed. During prothrombin activation by the complex of membrane-bound factor Xa and endogenous factor Va formed on the surface of HUVEC, APC was generated, and the rate of thrombin formation decreased. Treatment of HUVEC with an antibody that inhibits the APC-catalyzed inactivation of endogenous factor Va clearly quenched the activity of surface-associated APC. Immunostaining of HUVEC with a horseradish peroxidase (HRP)-conjugated antibody that solely recognizes human protein C confirmed the presence of protein C on the surface of HUVEC. Northern blot analysis revealed that an about 1.8 kb mRNA species derived from HUVEC was hybridized with 32P-labeled protein C cDNA, as in the case of those from HepG2, which are known to synthesize normal protein C. The increase in the amount of protein C mRNA in HUVEC in parallel with cell growth provided supporting evidence for the synthesis of protein C during the culture of HUVEC. These results indicate that blood coagulation is regulated by endogenously generated and activated protein C, together with or without protein S, through inactivation of factor Va on the surface of endothelial cells.     

B7-H1 is expressed by human endothelial cells and suppresses T cell cytokine synthesis

Human endothelial cells (ECs) provide costimulatory signals sufficient to activate resting memory T cells to produce IL-2 and IFN-gamma, at least in part through CD58-CD2 interactions. Recently, the B7-like molecule, B7-H1 (PD-L1), was described and shown to regulate T cell activation; however, there are conflicting reports on whether it stimulates or inhibits T cell cytokine synthesis. B7-H1 is not expressed constitutively by ECs; however, it is rapidly induced by IFN-gamma, and synergistically by IFN-gamma and TNF. In inflamed skin, B7-H1 is expressed by a subset of microvessels, and by keratinocytes, but is barely detectable in normal skin. Blocking the interaction of EC-expressed B7-H1 with its T cell ligand, programmed death-1 (PD-1), using a PD-1-Fc fusion protein, or by blocking B7-H1 expression with morpholino antisense oligonucleotides, augments expression of IL-2 and IFN-gamma, implicating B7-H1 as a negative regulator of cytokine synthesis. However, signaling through PD-1 does not affect induction of the activation markers CD25 or CD69 on T cells, suggesting that its effects are specific to cytokine synthesis. The suppressive effects of B7-H1 on cytokine expression are proportional to the strength of the primary stimulus, allowing for B7-H1 to determine the level of T cell activation in response to ECs. Our results demonstrate that B7-H1 negatively regulates cytokine synthesis in T cells activated by ECs.