Iron induces Bcl-2 expression in human dermal microvascular endothelial cells

Iron is suspected to be involved in the induction and/or progression of various human tumors. The present study was designed to investigate the effects of iron on endothelial cells, keeping in mind that the homeostasis of microvessels plays a critical role in neo-angiogenesis. Applying a model of human dermal microvascular endothelial cell terminal differentiation and death induced by serum deprivation, we found that iron salts (iron chloride and ferric nitrilotriacetate) provided a survival advantage to endothelial cells. Using immunohistochemistry and Western Blot analysis, we found that the extended cellular life span induced by iron was paralleled by an increase of Bcl-2 protein expression. Taken together, these observations suggest that iron may give a survival advantage to endothelial cells and represent a novel mechanism through which iron may contribute to tumorigenesis.     

Induction of oxidative stress by humic acid through increasing intracellular iron: a possible mechanism leading to atherothrombotic vascular disorder in blackfoot disease

Humic acid (HA), a potential toxin that has penetrated the drinking well water of blackfoot disease-endemic areas in Taiwan, has been implicated as an etiological factor of this disease. In this study, we investigated the effects of HA on the generation of reactive oxygen species (ROS) in cultured human umbilical vein endothelial cells (HUVECs). The generation of ROS was monitored by flow cytometry. Pretreatment of HUVECs with HA induced reactive oxygen species in a dose- and time-dependent manner. Xanthine oxidase inhibitor (Allopurinol), NADPH oxidase inhibitor (diphenylene iodomium) and calcium chelator (BAPTA) could not reduce the generation of ROS. Protein kinase C inhibitor (H7) could reduce the generation of ROS slightly, but the intracellular antioxidant glutathione monoethyl ester and the iron chelator desferrioxamine (DFO) could inhibit the generation of ROS completely. HA also enhanced the expression of ferritin and induced intracellular chelatable iron; however, HA reduced the expression of transferrin receptor. Pretreatment with DFO inhibited HA-mediated increases of ferritin synthesis and intracellular chelatable iron, but caused recovery of the inhibitory effect on transferrin receptor. Cotreatment with iron and HA induced more ROS and intracellular chelatable iron than iron or HA treatment alone. Furthermore, HA enhanced the accumulation of iron in endothelial cells. These data demonstrate that HA can increase the generation of ROS through enhancing the accumulation of intracellular iron. Taken together, our findings suggest that iron mediates HA-associated oxidative stress in endothelial cells, which may be a possible mechanism leading to atherothrombotic vascular injury observed for patients with blackfoot disease.     

Bcl-xL promotes metastasis of breast cancer cells by induction of cytokines resistance

Metastasis is a highly complex process involving the survival of tumor cells, both in the blood stream and within specific organs. Cell-death and survival are determined by a number of gene products from an expanding family of the Bcl-2 gene, either promoting or preventing apoptosis. Furthermore, the survival of tumor cells may favor the accumulation of additional genetic alterations causing further growth and invasive opportunities which may lead to metastasis. To examine whether the prevention of cell-death influences the metastatic behavior, we transfected a human breast cancer cell line MDA-MB-435 with the Bcl-xL cDNA and then studied metastatic ability of the selected clones in vivo. Our results show that Bcl-xL-clones had a decreased tumor growth latency and an increased metastatic ability. Apoptosis-resistance to cytokines was induced in 435 cells by Bcl-xL-expression with minor modifications in their proliferation rates. These cells also showed diminished adhesion to extracellular matrix proteins and a survival advantage in suspension over 435/Neo cells. Moreover, to determine survival in blood stream and in cells lodged in the lungs, we injected 435/Bcl-xL and 435/Neo cells at 1:3 proportion i.v., and animals were killed at intervals of 15' to 16 h after injection. Tumor cells were recovered from the lungs and Southern-blot analysis revealed the presence of exogenous Bcl-xL cDNA. These results showed that 435/Bcl-xL cells had a survival advantage in circulation over 435/Neo cells. This advantage in vivo was attributable to Bcl-xL expression. We conclude that Bcl-xL expression in breast cancer cells can increase metastatic activity. This advantage could be created by inducing resistance to apoptosis against cytokines, increasing cell survival in circulation, and enhancing anchorage-independent growth.     

Iron loading induces cholesterol synthesis and sensitizes endothelial cells to TNFα-mediated apoptosis

In plasma, iron is normally bound to transferrin, the principal protein in blood responsible for binding and transporting iron throughout the body. However, in conditions of iron overload when the iron-binding capacity of transferrin is exceeded, non–transferrin-bound iron (NTBI) appears in plasma. NTBI is taken up by hepatocytes and other parenchymal cells via NTBI transporters and can cause cellular damage by promoting the generation of reactive oxygen species. However, how NTBI affects endothelial cells, the most proximal cell type exposed to circulating NTBI, has not been explored. We modeled in vitro the effects of systemic iron overload on endothelial cells by treating primary human umbilical vein endothelial cells (HUVECs) with NTBI (ferric ammonium citrate [FAC]). We showed by RNA-Seq that iron loading alters lipid homeostasis in HUVECs by inducing sterol regulatory element-binding protein 2–mediated cholesterol biosynthesis. We also determined that FAC increased the susceptibility of HUVECs to apoptosis induced by tumor necrosis factor-α (TNFα). Moreover, we showed that cholesterol biosynthesis contributes to iron-potentiated apoptosis. Treating HUVECs with a cholesterol chelator hydroxypropyl-β-cyclodextrin demonstrated that depletion of cholesterol was sufficient to rescue HUVECs from TNFα-induced apoptosis, even in the presence of FAC. Finally, we showed that FAC or cholesterol treatment modulated the TNFα pathway by inducing novel proteolytic processing of TNFR1 to a short isoform that localizes to lipid rafts. Our study raises the possibility that iron-mediated toxicity in human iron overload disorders is at least in part dependent on alterations in cholesterol metabolism in endothelial cells, increasing their susceptibility to apoptosis.     

Retinoic acid-inducible gene-I is induced in endothelial cells by LPS and regulates expression of COX-2

Bacterial lipopolysaccharides (LPS) induce expression of multiple genes in endothelial cells, which are critical cellular effectors in various pathologic syndromes. Using subtractive hybridization to identify genes that are differentially induced in human endothelial cells treated with LPS, we found that retinoic acid-inducible gene I (RIG-I) is induced in endothelial cells stimulated with LPS. RIG-I encodes a protein belonging to the DExH-box family which has diverse roles in regulation of gene expression and cellular functions. Cyclooxygenase-2 (COX-2) is also induced in endothelial cells by LPS. Overexpression of RIG-I selectively upregulated expression of COX-2 and also induced COX-2 promoter activity. RIG-I is an inducible gene in stimulated endothelial cells that may have important roles in vascular pathology by virtue of its ability to regulate expression of the COX-2 gene product.     

Hypothermic preconditioning of endothelial cells attenuates cold-induced injury by a ferritin-dependent process

Hypothermia for myocardial protection or storage of vascular grafts may damage the endothelium and impair vascular function upon reperfusion/rewarming. Catalytic iron pools and oxidative stress are important mediators of cold-induced endothelial injury. Because endothelial cells are highly adaptive, we hypothesized that hypothermic preconditioning (HPC) protects cells at 0°C by a heme oxygenase-1 (HO-1) and ferritin-dependent mechanism. Storage of human coronary artery endothelial cells at 0°C caused the release of lactate dehydrogenase, increases in bleomycin-detectible iron (BDI), and increases in the ratio of oxidized/reduced glutathione, signifying oxidative stress. Hypoxia increased injury at 0°C but did not increase BDI or oxidative stress further. HPC at 25°C for 15–72 h attenuated these changes by an amount achievable by pretreating cells with 10–20 μM deferoxamine, an iron chelator, and protected cell viability. Treating cells with hemin chloride at 37°C transiently increased intracellular heme, HO-1, BDI, and ferritin. Elevated heme/iron sensitized cells to 0°C but ferritin was protective. HPC increased iron maximally after 2 h at 25°C and ferritin levels peaked after 15 h. HO-1 was not induced. When HPC-mediated increases in ferritin were blocked by deferoxamine, protection at 0°C was diminished. We conclude that HPC-mediated endothelial protection from hypothermic injury is an iron- and ferritin-dependent process.     

bcl-2 Gene Prevents Apoptosis of Basic Fibroblast Growth Factor-Deprived Murine Aortic Endothelial Cells

Growth factors such as basic fibroblast growth factor (bFGF) have been found to promote the survival and proliferation of endothelial cells. However, the mechanism by which growth factors control the regeneration and degeneration of the endothelial cells remained poorly understood. In this study, we demonstrated that apoptosis of murine aortic endothelial (MAE) cells was induced by deprivation of bFGF but required new RNA and protein synthesis. Furthermore, enforced expression of bcl-2 gene in MAE cells using gene transfer techniques decreased apoptosis induced by deprivation of bFGF. These findings suggest that bcl-2 interferes with a pathway for endothelial cell death that is induced by deprivation of bFGF.     

Pathogen Sensing Pathways in Human Embryonic Stem Cell Derived-Endothelial Cells: Role of NOD1 Receptors

Human embryonic stem cell-derived endothelial cells (hESC-EC), as well as other stem cell derived endothelial cells, have a range of applications in cardiovascular research and disease treatment. Endothelial cells sense Gram-negative bacteria via the pattern recognition receptors (PRR) Toll-like receptor (TLR)-4 and nucleotide-binding oligomerisation domain-containing protein (NOD)-1. These pathways are important in terms of sensing infection, but TLR4 is also associated with vascular inflammation and atherosclerosis. Here, we have compared TLR4 and NOD1 responses in hESC-EC with those of endothelial cells derived from other stem cells and with human umbilical vein endothelial cells (HUVEC). HUVEC, endothelial cells derived from blood progenitors (blood outgrowth endothelial cells; BOEC), and from induced pluripotent stem cells all displayed both a TLR4 and NOD1 response. However, hESC-EC had no TLR4 function, but did have functional NOD1 receptors. In vivo conditioning in nude rats did not confer TLR4 expression in hESC-EC. Despite having no TLR4 function, hESC-EC sensed Gram-negative bacteria, a response that was found to be mediated by NOD1 and the associated RIP2 signalling pathways. Thus, hESC-EC are TLR4 deficient but respond to bacteria via NOD1. This data suggests that hESC-EC may be protected from unwanted TLR4-mediated vascular inflammation, thus offering a potential therapeutic advantage.     

Neisseria gonorrhoeae requires expression of TonB and the putative transporter TdfF to replicate within cervical epithelial cells

Neisseria gonorrhoeae has evolved a repertoire of iron acquisition systems that facilitate essential iron uptake in the human host. Acquisition of iron requires both the energy-harnessing cytoplasmic membrane protein, TonB, as well as specific outer membrane TonB-dependent transporters (TdTs.) Survival within host epithelial cells is important to the pathogenesis of gonococcal disease and may contribute to the persistence of infection. However, the mechanisms by which gonococci acquire iron within this intracellular niche are not currently understood. In this study, we investigated the survival of gonococcal strain FA1090 within ME180 human cervical epithelial cells with respect to high affinity iron acquisition. Intracellular survival was dependent upon iron supplied by the host cell. TonB was expressed in the host cell environment and this protein was critical to gonococcal intracellular survival. Furthermore, expression of the characterized outer membrane transporters TbpA, FetA and LbpA and putative transporters TdfG, TdfH and TdfJ were not necessary for intracellular survival. Conversely, intracellular survival was dependent on expression of the putative transporter, TdfF. Expression of TdfF was detected in the presence of epithelial cell culture media containing fetal bovine serum. Expression was further modulated by iron availability. To our knowledge, this study is the first to demonstrate the specific requirement for a single iron transporter in the survival of a bacterial pathogen within host epithelial cells.     

Basic fibroblast growth factor inhibits radiation-induced apoptosis of HUVECs. I. The PI3K/AKT pathway and induction of phosphorylation of BAD

Radiation-induced endothelial cell apoptosis is involved in the development of many radiation injuries, including radiation-induced skin ulcers. The proangiogenic growth factors basic fibroblast growth factor (bFGF, NUDT6) and VEGF enhance endothelial cell survival. In the present study, we used primary cultured human umbilical vein endothelial cells (HUVECs) irradiated with (60)Co gamma rays to explore the effects of bFGF on radiation-induced apoptosis of HUVECs and its signaling pathways. We found that bFGF inhibited radiation-induced apoptosis of HUVECs, and that the effect was mediated by the PI3K/AKT pathway. This pathway was activated by exposure of irradiated HUVECs to bFGF, involving phosphorylation of FGFR, PI3K and AKT. The survival-enhancing effect of bFGF was abrogated by wortmannin and LY294002. Transfection of a dominant-negative mutant of AKT completely blocked the anti-apoptosis effect of bFGF in irradiated HUVECs. We also found evidence for the first time that bFGF induced BAD phosphorylation in the gamma-irradiated HUVECs. These results showed that the PI3K/AKT pathway participated in the bFGF-induced modulation of the survival of irradiated HUVECs. Activation of the PI3K/AKT pathway plays an important role in bFGF-induced endothelial cell survival in the treatment of radiation-induced skin ulcers.     

Lipopolysaccharide-induced apoptosis of endothelial cells and its inhibition by vascular endothelial growth factor

Endothelial injury is a major manifestation of septic shock induced by LPS. Recently, LPS was shown to induce apoptosis in different types of endothelial cells. In this study, we observed that pretreatment with vascular endothelial growth factor (VEGF), a known cell survival factor, blocked LPS-induced apoptosis in endothelial cells. We then further defined this LPS-induced apoptotic pathway and its inhibition by VEGF. We found that LPS treatment increased caspase-3 and caspase-1 activities and induced the cleavage of focal adhesion kinase. LPS also augmented expression of the pro-apoptotic protein Bax and the tumor suppressor gene p53. The pro-apoptotic Bax was found to translocate to the mitochondria from the cytosol following stimulation with LPS. Pretreatment of endothelial cells with VEGF inhibited the induction of both Bax and p53 as well as the activation of caspase-3. These data suggest that VEGF inhibits LPS-induced endothelial apoptosis by blocking pathways that lead to caspase activation.     

Transferrin: a potential source of iron for oxygen free radical-mediated endothelial cell injury.

The ability of transferrin to potentiate oxygen free radical-mediated endothelial cell injury was assessed. 51Cr-labeled endothelial cells derived from rat pulmonary arteries (RPAECs) were incubated with hydrogen peroxide (H2O2) in the presence and absence of holosaturated human transferrin, and the effect of transferrin on H2O2-mediated endothelial cell toxicity was determined. Addition of holosaturated transferrin potentiated H2O2-mediated RPAEC cytotoxicity at concentrations of H2O2 greater than 10 microM, suggesting that transferrin may provide a source of iron for free radical-mediated endothelial cell injury. Free radical-mediated injury is dependent on non-protein-bound iron. The ability of RPAECs to facilitate the release of iron from transferrin was assessed. We determined that RPAECs facilitate the release of transferrin-derived iron by reduction of transferrin-bound ferric iron (Fe3+) to ferrous iron (Fe2+). The reduction and release of transferrin-derived Fe2+ were inhibited by apotransferrin and chloroquine, indicating a dependence on receptor-specific binding of transferrin to the RPAEC cell surface, with subsequent endocytosis, acidification, and reduction of transferrin-bound Fe3+ to Fe2+. The release of transferrin-derived Fe2+ was potentiated by diethyldithiocarbamate, an inhibitor of intracellular superoxide dismutase (SOD). In contrast, exogenous SOD did not alter iron release, suggesting that intracellular superoxide anion (O2-) may play an important role in mediating the reduction and release of transferrin-derived iron. Results of this study suggest that transferrin may provide a source of iron for oxygen free radical-mediated endothelial cell injury and identify a novel mechanism by which endothelial cells may mediate the reduction and release of transferrin-derived iron.     

Iron overload induces cerebral endothelial senescence in aged mice and in primary culture in a sex-dependent manner

Iron imbalance in the brain negatively affects brain function. With aging, iron levels increase in the brain and contribute to brain damage and neurological disorders. Changes in the cerebral vasculature with aging may enhance iron entry into the brain parenchyma, leading to iron overload and its deleterious consequences. Endothelial senescence has emerged as an important contributor to age-related changes in the cerebral vasculature. Evidence indicates that iron overload may induce senescence in cultured cell lines. Importantly, cells derived from female human and mice generally show enhanced senescence-associated phenotype, compared with males. Thus, we hypothesize that cerebral endothelial cells (CEC) derived from aged female mice are more susceptible to iron-induced senescence, compared with CEC from aged males. We found that aged female mice, but not males, showed cognitive deficits when chronically treated with ferric citrate (FC), and their brains and the brain vasculature showed senescence-associated phenotype. We also found that primary culture of CEC derived from aged female mice, but not male-derived CEC, exhibited senescence-associated phenotype when treated with FC. We identified that the transmembrane receptor Robo4 was downregulated in the brain vasculature and in cultured primary CEC derived from aged female mice, compared with those from male mice. We discovered that Robo4 downregulation contributed to enhanced vulnerability to FC-induced senescence. Thus, our study identifies Robo4 downregulation as a driver of senescence induced by iron overload in primary culture of CEC and a potential risk factor of brain vasculature impairment and brain dysfunction.     

Saikosaponin C induces endothelial cells growth, migration and capillary tube formation

Saikosaponin C is one of the saikosaponins that are consisted in a Chinese herb, Radix Bupleuri. Recently, saikosaponins have been reported to have properties of cell growth inhibition, inducing cancer cells differentiation and apoptosis. However, saikosaponin C had no correlation with cell growth inhibition. In this study, we investigated the role of saikosaponin C on the growth of endothelial cells and angiogenesis. We found that saikosaponin C yielded a potent effect on inducing human umbilical vein endothelial cells (HUVECs) viability and growth. In addition to inducing endothelial cells growth, saikosaponin C also induced endothelial cells migration and capillary tube formation. The gene expression or activation of matrix metalloproteinase-2 (MMP-2), vascular endothelial growth factor (VEGF) and the p42/p44 mitogen-activated protein kinase (MAPK, ERK) that correlated with endothelial cells growth, migration and angiogenesis were also induced by saikosaponin C. From these results, we suggest that saikosaponin C may have the potential for therapeutic angiogenesis but is not suitable for cancer therapy.     

MiR-93 enhances angiogenesis and metastasis by targeting LATS2

Here we report that miR-93, a miRNA in the miR-106B~25 cluster, a paralog of the miR-17–92 cluster, was significantly upregulated in human breast carcinoma tissues. We stably expressed miR-93 in the MT-1 human breast carcinoma cell line and found that tumors formed by the miR-93 cells contained more blood vessels than those formed by the control cells. Co-culture experiments indicated that the MT-1 cells displayed a high activity of adhesion with endothelial cells and could form larger and more tube-like structures with endothelial cells. Lung metastasis assays were performed in a mouse metastatic model, and it was found that expression of miR-93 promoted tumor cell metastasis to lung tissue. In cell culture, expression of miR-93 enhanced cell survival and invasion. We examined the potential target that mediated miR-93’s effects and found that the large tumor suppressor, homology 2 (LATS2) was a target of miR-93. Higher levels of LATS2 were associated with cell death in the tumor mass. Silencing LATS2 expression promoted cell survival, tube formation and invasion, while ectopic expression of LATS2 decreased cell survival and invasion. These findings demonstrated that miR-93 promoted tumor angiogenesis and metastasis by suppressing LATS2 expression. Our results suggest that the inhibition of miR-93 function may be a feasible approach to repress tumor metastasis.     

The effects of markedly raised intracellular sphingosine kinase-1 activity in endothelial cells

The enzyme sphingosine kinase-1 (SK1) promotes the formation of sphingosine-1-phosphate (S1P), which is an important survival factor for endothelial cells (EC). Modest increases in intracellular SK1 activity in the EC are known to confer a survival advantage upon the cells. Here, we investigated the effects of more dramatic increases in intracellular SK1 in the EC. We found that these cells show reduced cell survival under conditions of stress, enhanced caspase-3 activity, cell cycle inhibition, and cell-cell junction disruption. We propose that alterations in the phosphorylation state of the enzyme may explain the differential effects on the phenotype with modest versus high levels of enforced expression of SK1. Our results suggest that SK1 activity is subject to control in the EC, and that this control may be lost in conditions involving vascular regression.     

Protein kinase B inhibits endostatin-induced apoptosis in HUVECs

Endostatin is a tumor-derived angiogenesis inhibitor, and the endogenous 20 kDa carboxyl-terminal fragment of collagen XVIII. In addition to inhibiting angiogenesis,endostatin inhibits tumor growth and the induction of apoptosis in several endothelial cell types. However, the mechanisms that regulate endostatin-induced apoptotic cell death are unclear. Here, we investigated apoptotic cell death and the underlying regulatory mechanisms elicited of endostatin in human umbilical vein endothelial cells (HUVECs). Endostatin was found to induce typical apoptotic features, such as, chromatin condensation and DNA fragmentation in these cells. Thus, as the phosphoinositide 3-OH kinase (PI3K)/protein kinase B (PKB) signaling pathway has been shown to prevent apoptosis in various cell types, we investigated whether this pathway could protect cells against endostatin induced apoptosis. It was found that the inhibition of PI3K/PKB significantly increased endostatin-induced apoptosis, and that endostatininduced cell death is physiologically linked to PKB-mediated cell survival through caspase-8.