Priming of polymorphonuclear leukocytes: a culprit in the initiation of endothelial cell injury

Peripheral polymorphonuclear leukocytes (PMNL) in hemodialysis (HD) patients are primed, continually releasing and exposing the vascular endothelium to soluble factors such as reactive oxygen species and inflammatory mediators. To mimic the close proximity between PMNL and the endothelial monolayer and to monitor and characterize the influence of soluble mediators released from PMNL, we developed a novel cocultivation system using primary human umbilical vein endothelial cell (HUVEC) cultures and PMNL, with a sieve separating the two cell types to prevent direct adhesive effects. PMNL (10(6)) from HD patients or from healthy normal controls were cocultivated with HUVEC (10(5)) for 15 min, and endothelial cell injury was assessed by HUVEC morphology, cell detachment, and apoptosis. Proinflammatory changes were estimated by expression of HUVEC adhesion molecule P-selectin and by endothelial IL-8 and endothelial nitric oxide synthase mRNA. The levels of intracellular tissue factor reflected the procoagulant state, whereas NADPH oxidase activity served as an indicator for prooxidative changes in HUVEC. Mediators released from the primed PMNL triggered activation/dysfunction of endothelial cells, causing 1) an increase in endothelial cell detachment and apoptosis, 2) a proinflammatory state manifested by increased IL-8 mRNA expression and P-selectin on the endothelial surface, 3) activation of endothelial NADPH oxidase, 4) an increase in endothelial cell tissue factor that directly correlated with PMNL priming index, and 5) a decrease in endothelial nitric oxide synthase mRNA. Our data support a pathogenic link between PMNL priming and endothelial dysfunction, suggesting that PMNL priming is a potential new nontraditional risk factor for the development of atherosclerosis.     

Glioblastoma cells block radiation-induced programmed cell death of endothelial cells

We demonstrate that human umbilical vein endothelial cells (HUVEC) grown in co-culture (CC) with U87 glioblastoma cells transfected with green fluorescent protein (GFP-U87) exhibit resistance to radiation-mediated apoptosis. cDNA macroarray analysis reveals increases in the accumulation of RNAs for HUVEC genes encoding cell adhesion molecules, growth factor-related proteins, and cell cycle regulatory/DNA repair proteins. An increase in protein expression of integrin alphav, integrin beta1, MAPK(p42), Rad51, DNA-PK(CS), and ataxia telangiectasia gene (ATM) was detected in HUVEC grown in CC with GFP-U87 cells compared with HUVEC grown in mono-culture. Treatment with anti-VEGF antibody decreases the expression of integrin alphav, integrin beta1, DNA-PK(CS) and ATM with a corresponding increase in ionizing radiation (IR)-induced apoptosis. These data support the concept that endothelial cells growing in the tumor microenvironment may develop resistance to cytotoxic therapies due to the up-regulation by tumor cells of endothelial cells genes associated with survival.     

大鼠HO-1表达质粒构建、活力检测及抗HUVEC凋亡的作用研究

异种移植排斥反应的主要特征为内皮细胞发生Ⅱ型激活．引起黏附分子、细胞因子和前促凝分子等基因高表达．造成血管收缩、白细胞黏附、激活、聚集和血栓形成．最终导致内皮细胞凋亡。保护基因HO-1通过抑制前炎症反应及免疫调抑作用以保护异种移植器官。因此。通过构建含剪切的野生型大鼠HO-1 cDNA的表达型质粒．用DOTAP包裹转入HUVEC中表达。测定表达量及表达产物活性;采用TNF-α诱导细胞凋亡。以及Heme和SnPP分别刺激细胞。诱导和抑制细胞内HO-1表达量．流式细胞仪测定细胞凋亡率,明确HO一1的抗细胞凋亡作用。结果显示HO-1在HUVEC中高度表达。活力为对照组5倍;TNF-α诱导细胞凋亡。但Heme处理后细胞凋亡率下降至20％以下。而SnPP处理后细胞凋亡率显著上升,最高达到95％以上。并且HO-1基因表达抑制时细胞凋亡率是诱导时的5—20倍。本实验表明Heme处理后HO-1表达上调。具有显著抗细胞凋亡作用。细胞凋亡率与HO-1表达量呈负相关,提示HO-1通过抑制细胞凋亡。对细胞有保护作用。     

Human umbilical vein endothelial cells increase ex vivo expansion of human CD34(+) PBPC through IL-6 secretion

BACKGROUND: Ex vivo expansion of hematopoietic stem cells (HSC) can help reduce cytopenia following transplantation, especially in NHL patients whose BM is deficient because of extensive chemotherapy. We have previously reported that human umbilical vein endothelial cells (HUVEC) can contribute to improved PBPC expansion when used in co-culture with CD34(+) cells. METHODS: We evaluated the roles of direct HUVEC CD34(+) contact and HUVEC-produced soluble factors. We cultured CD34(+) PBPC harvested from NHL patients in four different conditions: (1) liquid culture without HUVEC; (2) co-culture in contact with HUVEC; (3) co-culture with HUVEC but without direct contact; (4) liquid culture with HUVEC-conditioned medium (CM). Thrombopoietin (Tpo), Flk2Flt3 ligand (FL) and c-kit ligand (KL) with or without rhIL-6 were added to these four culture conditions. RESULTS AND DISCUSSION: Our results showed that HUVEC co-culture or addition of HUVEC-CM to Tpo, FL and KL (TFK) improved CD34(+) PBPC expansion compared with liquid culture, as determined by total viable nucleated cells (TNC), colony-forming cell assay (CFC) and week-6 cobblestone area-forming cells (Wk-6 CAFC) expansions. Non-contact culture led to similar PBPC expansion as contact co-culture; moreover, HUVEC-CM improved PBPC expansion. However, when rhIL-6 was added to HUVEC-CM with TFK, no significant difference was observed. Finally, high quantities of IL-6 were detected in HUVEC-CM and addition of anti-IL-6 Ab inhibited the positive effect of HUVEC on PBPC expansion. Our results thus suggest that HUVEC may improve PBPC expansion, at least through IL-6 secretion.     

Paracrine or virus-mediated induction of decorin expression by endothelial cells contributes to tube formation and prevention of apoptosis in collagen lattices

Resting endothelial cells express the small proteoglycan biglycan, whereas sprouting endothelial cells also synthesize decorin, a related proteoglycan. Here we show that decorin is expressed in endothelial cells in human granulomatous tissue. For in vitro investigations, the human endothelium-derived cell line, EA.hy 926, was cultured for 6 or more days in the presence of 1% fetal calf serum on top of or within floating collagen lattices which were also populated by a small number of rat fibroblasts. Endothelial cells aligned in cord-like structures and developed cavities that were surrounded by human decorin. About 14% and 20% of endothelial cells became apoptotic after 6 and 12 days of co-culture, respectively. In the absence of fibroblasts, however, the extent of apoptosis was about 60% after 12 days, and cord-like structures were not formed nor could decorin production be induced. This was also the case when lattices populated by EA.hy 926 cells were maintained under one of the following conditions: 1) 10% fetal calf serum; 2) fibroblast-conditioned media; 3) exogenous decorin; or 4) treatment with individual growth factors known to be involved in angiogenesis. The mechanism(s) by which fibroblasts induce an angiogenic phenotype in EA.hy 926 cells is (are) not known, but a causal relationship between decorin expression and endothelial cell phenotype was suggested by transducing human decorin cDNA into EA.hy 926 cells using a replication-deficient adenovirus. When the transduced cells were cultured in collagen lattices, there was no requirement of fibroblasts for the formation of capillary-like structures and apoptosis was reduced. Thus, decorin expression seems to be of special importance for the survival of EA.hy 926 cells as well as for cord and tube formation in this angiogenesis model.     

葡萄球菌肠毒素B诱导脐静脉内皮细胞凋亡机制的探讨

目的探讨金黄色葡萄球菌(金葡菌)肠毒素B诱导脐静脉内皮细胞凋亡的机制。方法将不同浓度金葡菌肠毒素B感染脐静脉内皮细胞8 h后,用流式细胞术检测细胞凋亡率,同时用比色法检测TNF-α、caspase-3及caspase-8的产生量,并检测加入TNF-α抗体、caspase-3和caspase-8抑制剂后的细胞凋亡率。结果不同浓度肠毒素B作用脐静脉内皮细胞8 h后均可诱导细胞凋亡,且TNF-α、caspase-3和caspase-8的产生量均高于对照组(P0.01);而加入TNF-α抗体、caspase-3和caspase-8抑制剂后凋亡率明显降低。结论金葡菌肠毒素B可以诱导脐静脉内皮细胞凋亡,其凋亡机制可能是通过TNF-α介导的caspase-8及caspase-3激活的外源性死亡因子受体途径。     

Detection of measles virus-induced apoptosis of human monocytic cell line (THP-1) by DNA fragmentation ELISA

Abstract Measles virus (wild strain, Toyoshima strain)-induced cell death is characterized by cell shrinkage, chromatin condensation, and nuclear fragmentation in a human monocytic cell line (THP-1). DNA fragmentation of measles virus-infected THP-1 cells was demonstrated by DNA agarose gel electrophoresis as well as by DNA fragmentation ELISA. When measles virus-infected THP-1 cells were cultured on monolayers of fibroblasts or human umbilical vein endothelial cells (HUVEC), the percentage of measles virus antigen-positive THP-1 cells and DNA fragmentation were significantly decreased. Addition of anti-intercellular adhesion molecule (ICAM)-1 (CD54) monoclonal antibody to culture of measles virus-infected THP-1 cells reduced significantly DNA fragmentation induced by measles virus. These findings suggest that inhibition of virus spread by fibroblasts and HUVEC reduces apoptosis, and ICAM-1 (CD54) may participate in the DNA fragmentation pathway.     

Origin of periendothelial cells in microvessels derived from human microvascular endothelial cells

In microvessels, periendothelial cells expressing alpha smooth muscle actin (alphaSMA) interact with the endothelial cells and are essential for vessel maturation and stabilization. In adult tissues, the cellular origin of the periendothelial cells is still not clear, in particular in humans. To determine the origin of human periendothelial cells, we used a recently developed 3D co-culture system that mimics human skin connective tissue. This system is composed of normal human dermal fibroblasts (NHDF), human dermal microvascular endothelial cells (HMEC-1), and a collagen matrix. In this system, "microvessels" composed of an endothelial lumen associated with periendothelial cells develop. Using this co-culture system, we (i) labelled fibroblasts with the vital dye CFDA-SE, cultured them with unlabelled endothelial cells, and observed that only endothelium-associated CFDA-SE-labelled cells express alphaSMA; (ii) infected endothelial cells with a retrovirus stably expressing eGFP, cultured them with unlabelled fibroblasts, and observed that cells expressing alphaSMA did not co-express eGFP, but were associated with the eGFP-expressing endothelial cells of the microvessels. Together, these results indicate that periendothelial cells arise by differentiation from fibroblasts and that they require interaction with endothelial cells to do so.     

Effect of calcitonin gene-related peptide on osteoblast differentiation in an osteoblast and endothelial cell co-culture system

We have investigated the in vitro effects and regulatory mechanism of CGRP (calcitonin gene-related peptide) on the differentiation of OB (osteoblasts) in co-culture with HUVEC (human umbilical vein endothelial cells). Primary human MOB (mandibular OB) and OB-like cells (MG-63) were either cultured directly or indirectly co-cultured with HUVEC at a 1:1 ratio. Expression of OC (osteocalcin) was measured by ELISA, and expression of ALP (alkaline phosphatase) and collagen mRNA was measured by quantitative fluorescent PCR. For mineralization nodus, OB were stained with Alizarin Red-S. When co-cultured with HUVEC, expression of OC and ALP mRNA were increased in MG-63 (P<0.01), and the expression of OC, ALP and collagen mRNA were increased in MOB (P<0.01 or 0.05). When treated with CGRP, OC and ALP mRNA and mineralization nodus numbers were increased in the MG-63 co-culture system (P<0.01 or 0.05); OC, ALP and collagen mRNA, and mineralization nodus numbers were increased in the MOB co-culture system (P<0.01 or 0.05). The effect of CGRP regulation on the differentiation of OB is not only direct but also indirect, via its effect on HUVEC and stimulation of OB.     

Apoptosis of vascular endothelial cells by fibroblast growth factor deprivation

Survival and proliferation of many types of vascular endothelial cells are influenced by fibroblast growth factor (FGF)1. Removal of FGF from the medium of human umbilical vein endothelial cells (HUVEC) in culture resulted in death of the cells. Here we show that the death caused by deprivation of FGF is active death or apoptosis, and the process of apoptosis can be inhibited by cycloheximide, an inhibitor of protein synthesis. The present study shows apoptosis occurs in endothelial cells in culture. The process of active death of vascular endothelial cells is inhibited by growth factor. This mechanism may be important for the regulation of vascular organization through the degeneration of vessels.     

Human cytomegalovirus UL130 protein promotes endothelial cell infection through a producer cell modification of the virion

Human cytomegalovirus (HCMV) growth in endothelial cells (EC) requires the expression of the UL131A-128 locus proteins. In this study, the UL130 protein (pUL130), the product of the largest gene of the locus, is shown to be a luminal glycoprotein that is inefficiently secreted from infected cells but is incorporated into the virion envelope as a Golgi-matured form. To investigate the mechanism of the UL130-mediated promotion of viral growth in EC, we performed a complementation analysis of a UL130 mutant strain. To provide UL130 in trans to viral infections, we constructed human embryonic lung fibroblast (HELF) and human umbilical vein endothelial cell (HUVEC) derivative cell lines that express UL130 via a retroviral vector. When the UL130-negative virus was grown in UL130-complementing HELF, the infectivity of progeny virions for HUVEC was restored to the wild-type level. In contrast, the infectivity of the UL130-negative virus for UL130-complementing HUVEC was low and similar to that of the same virus infecting control noncomplementing HUVEC. The UL130-negative virus, regardless of whether or not it had been complemented in the prior cycle, could form plaques only on UL130-complementing HUVEC, not control HUVEC. Because (i) both wild-type and UL130-transcomplemented virions maintained their infectivity for HUVEC after purification, (ii) UL130 failed to complement in trans the UL130-negative virus when it was synthesized in a cell separate from the one that produced the virions, and (iii) pUL130 is a virion protein, models are favored in which pUL130 acquisition in the producer cell renders HCMV virions competent for a subsequent infection of EC.     

Effects of 17beta-estradiol on growth and apoptosis in human vascular endothelial cells: influence of mechanical strain and tumor necrosis factor-alpha

Vascular endothelial cell (EC) integrity is key to arterial health; endothelial dysfunction is linked to atherogenesis. Atherosclerosis shows a male preponderance, possibly related to the protective effect of estrogens in women. This study examined the effect of estrogens on growth, apoptosis and adhesion molecule expression in cultured human EC. The effects of 17beta-estradiol (E2) were studied in human umbilical vein endothelial cells (HUVEC) under normal culture conditions, and following exposure to cyclic mechanical strain or tumor necrosis factor alpha (TNFalpha). E2 enhanced HUVEC growth in serum-enriched media, in a concentration-dependent manner. This up-regulation of EC growth by E2 was associated with an increase in telomerase activity, assessed by PCR-based TRAP analysis. Cyclic strain enhanced [(3)H]-thymidine incorporation into DNA, and increased activation of mitogen-activated protein (MAP) kinase ERK1/2 and expression of early growth genes (Egr-1 and Sp-1); E2 attenuated the strain-induced ERK1/2 activation but not the early growth gene expression or DNA synthesis. TNFalpha (20 ng/mL) induced apoptosis in HUVEC, causing a decrease in DNA synthesis, increase in floating and Annexin-V-stained cell numbers, and morphological changes. TNFalpha also upregulated ERK1/2 activity and expression of adhesion molecules (ICAM-1, VCAM-1 and E-selectin). E2 significantly attenuated the effects of TNFalpha on ERK1/2 activity, apoptosis, and E-selectin expression in the cells. Thus, estradiol enhances growth and reduces TNFalpha-induced apoptosis in EC; enhanced EC growth may be mediated via upregulation of telomerase activity. These effects are possible cellular mechanisms underlying female gender-associated cardiovascular protection.     

Inhibition of p38 MAPK reduces tumor conditioned medium-induced angiogenesis in co-cultured human umbilical vein endothelial cells and fibroblasts

Tumor conditioned medium (CM) has been widely used to stimulate endothelial cells to form capillary-like structures in in vitro angiogenesis models. We report herein the effect of HT1080 and A549 CM after they were mixed with microvascular endothelial cells medium-2 (EGM-2) on angiogenesis in human umbilical vein endothelial cells (HUVECs). Both HT1080 and A549 CM decreased HUVEC proliferation, to different extents. While A549 CM significantly increased capillary-like structure formation in a co-culture system, no effect of HT1080 was apparent. Inhibition of p38 mitogen-activated protein kinase (MAPK) blocked both basal and A549 CM induced capillary-like structure formation, but inhibition of extracellular signal-regulated kinases (ERK) and that of c-Jun N-terminal protein kinases (JNK) MAPK had no such effect. Activation of ERK MAPK was inhibited by both CMs, whereas p38 MAPK was inactivated by HT1080 and activated by A549 CM and a control. Neither CM had an effect on JNK MAPK. The results suggest that p38 MAPK played a critical role in capillary-like structure formation in the co-culture, partly via promotion of apoptosis in HUVECs.     

Anti-apoptotic and anti-senescence effects of Klotho on vascular endothelial cells

Klotho-mutated mice manifest multiple age-related disorders that are observed in humans. A recent study suggested that Klotho protein might function as an anti-aging hormone in mammals. Because it has been reported that apoptosis and senescence in vascular endothelial cells are closely related to the progression of atherosclerosis, we investigated Klotho's ability to interfere with apoptosis and cellular senescence in human umbilical vascular endothelial cells (HUVEC). Klotho overexpression decreased H(2)O(2)-induced apoptosis in COS-1 cells and Jurkat cells. Klotho protein also reduced H(2)O(2)- and etoposide-induced apoptosis in HUVEC. Caspase-3 and caspase-9 activity was lower in Klotho-treated HUVEC than in control cells. Senescence-associated beta-gal staining showed that Klotho protein interferes with H(2)O(2)-induced premature cellular senescence. The expression of p53 and p21 was lower in Klotho-treated cells. Our study suggests that Klotho acts as a humoral factor to reduce H(2)O(2)-induced apoptosis and cellular senescence in vascular cells.     

Magnolol suppresses proliferation of cultured human colon and liver cancer cells by inhibiting DNA synthesis and activating apoptosis

Magnolol, a hydroxylated biphenyl compound isolated from the Chinese herb Hou p'u of Magnolia officinalis, has been reported to have anti-cancer activity. In the present study, magnolol at very low concentrations of 3-10 microM inhibited DNA synthesis and decreased cell number in cultured human cancer cells (COLO-205 and Hep-G2) in a dose-dependent manner, but not in human untransformed cells such as keratinocytes, fibroblasts, and human umbilical vein endothelial cells (HUVEC). Magnolol was not cytotoxic at these concentrations and this indicates that it may have an inhibitory effect on cell proliferation in the subcultured cancer cell lines. [(3)H] thymidine incorporation and flow cytometry analyses revealed that magnolol treatment decreased DNA synthesis and arrested the cells at the G0/G1 phase of the cell cycle. Moreover, the magnolol-induced cell cycle arrest occurred when the cyclin-CDK system was inhibited, just as p21 protein expression was augmented. When magnolol concentration was increased to 100 microM, apoptosis was observed in COLO-205 and Hep-G2 cells, but not in cultured human fibroblasts and HUVEC. COLO-205 cells implanted subcutaneously in nude mice formed solid tumors; subsequent daily i.p.-injections of magnolol led to profound regression of these tumors of up to 85%. In these tumors, an increase in the expression of p21 protein level and the occurrence of apoptosis were observed. These findings demonstrate for the first time that magnolol can inhibit the proliferation of tumor cells in vitro and in vivo.     

Hypoxic Preconditioning Increases Survival and Pro-Angiogenic Capacity of Human Cord Blood Mesenchymal Stromal Cells In Vitro

Hypoxic preconditioning was shown to improve the therapeutic efficacy of bone marrow-derived multipotent mesenchymal stromal cells (MSCs) upon transplantation in ischemic tissue. Given the interest in clinical applications of umbilical cord blood-derived MSCs, we developed a specific hypoxic preconditioning protocol and investigated its anti-apoptotic and pro-angiogenic effects on cord blood MSCs undergoing simulated ischemia in vitro by subjecting them to hypoxia and nutrient deprivation with or without preceding hypoxic preconditioning. Cell number, metabolic activity, surface marker expression, chromosomal stability, apoptosis (caspases-3/7 activity) and necrosis were determined, and phosphorylation, mRNA expression and protein secretion of selected apoptosis and angiogenesis-regulating factors were quantified. Then, human umbilical vein endothelial cells (HUVEC) were subjected to simulated ischemia in co-culture with hypoxically preconditioned or naïve cord blood MSCs, and HUVEC proliferation was measured. Migration, proliferation and nitric oxide production of HUVECs were determined in presence of cord blood MSC-conditioned medium. Cord blood MSCs proved least sensitive to simulated ischemia when they were preconditioned for 24 h, while their basic behavior, immunophenotype and karyotype in culture remained unchanged. Here, “post-ischemic” cell number and metabolic activity were enhanced and caspase-3/7 activity and lactate dehydrogenase release were reduced as compared to non-preconditioned cells. Phosphorylation of AKT and BAD, mRNA expression of BCL-XL, BAG1 and VEGF, and VEGF protein secretion were higher in preconditioned cells. Hypoxically preconditioned cord blood MSCs enhanced HUVEC proliferation and migration, while nitric oxide production remained unchanged. We conclude that hypoxic preconditioning protects cord blood MSCs by activation of anti-apoptotic signaling mechanisms and enhances their angiogenic potential. Hence, hypoxic preconditioning might be a translationally relevant strategy to increase the tolerance of cord blood MSCs to ischemia and improve their therapeutic efficacy in clinical applications.