DNA Synthesis In Endothelial Cells Research Articles

Rola i potencjał terapeutyczny sfingolipidowego szlaku sygnalizacyjnego w nowotworach hematologicznych

One of the key obstacles in the progress of cancer treatment is the lack of balance between the uncontrolled proliferation and cell apoptosis. It is now known that sphingolipids are essential molecules regulating the processes of growth, differentiation and death of living cells. Depending on their chemical nature, sphingolipids may have a stimulatory (S1P, sphingosine-1-phosphate) or inhibitory (ceramide) effect on cellular proliferation. A number of different studies have shown that the generation of ceramide in response to cytotoxic therapy is an important element leading to cell death. Cancer cells use different methods limiting the production of ceramides that leads to their removal. The effect of oncogenic S1P results from its stimulating effect on DNA synthesis and chemotactic mobility of the vascular endothelial cells and angiogenesis. The use of monoclonal anti-S1P antibodies is potentially a valuable therapeutic option for inhibiting angiogenesis determining the growth of tumors. It was additionally demonstrated that S1P beyond the direct and indirect by stimulating the release of vascular endothelial growth factor and basic fibroblast growth factor angiogenic action has an effect on tumor growth and its metastatic potential. Among the sphingolipids, ceramide was identified first as inducing differentiation and the death of human HL-60 promyelocytic leukemia cells. Progress in understanding the role of sphingolipids was regarded until recently as the only structural component of cell membranes allowing the use in the treatment of complex properties of this group of signaling molecules. Thus, it has become important to clarify the role of sphingolipids in the regulation of the balance between proliferation signals/ /survival rate and death of cells in order to develop new therapies for neoplastic diseases of myeloid and lymphoid origin.

Inactivation of the Human Cytomegalovirus US20 Gene Hampers Productive Viral Replication in Endothelial Cells.

The human cytomegalovirus (HCMV) US12 gene family includes a group of 10 contiguous genes (US12 to US21) encoding predicted seven-transmembrane-domain (7TMD) proteins that are nonessential for replication within cultured fibroblasts. Nevertheless, inactivation of some US12 family members affects virus replication in other cell types; e.g., deletion of US16 or US18 abrogates virus growth in endothelial and epithelial cells or in human gingival tissue, respectively, suggesting a role for some US12 proteins in HCMV cell tropism. Here, we provide evidence that another member, US20, impacts the ability of a clinical strain of HCMV to replicate in endothelial cells. Through the use of recombinant HCMV encoding tagged versions of the US20 protein, we investigated the expression pattern, localization, and topology of the US20-encoded protein (pUS20). We show that pUS20 is expressed as a partially glycosylated 7TMD protein which accumulates late in infection in endoplasmic reticulum-derived peripheral structures localized outside the cytoplasmic virus assembly compartment (cVAC). US20-deficient mutants generated in the TR clinical strain of HCMV exhibited major growth defects in different types of endothelial cells, whereas they replicated normally in fibroblasts and epithelial cells. While the attachment and entry phases in endothelial cells were not significantly affected by the absence of US20 protein, US20-null viruses failed to replicate viral DNA and express representative E and L mRNAs and proteins. Taken together, these results indicate that US20 sustains the HCMV replication cycle at a stage subsequent to entry but prior to E gene expression and viral DNA synthesis in endothelial cells. Human cytomegalovirus (HCMV) is a major pathogen in newborns and immunocompromised individuals. A hallmark of HCMV pathogenesis is its ability to productively replicate in an exceptionally broad range of target cells, including endothelial cells, which represent a key target for viral dissemination and replication in the host, and to contribute to both viral persistence and associated inflammation and vascular diseases. Replication in endothelial cells depends on the activities of a set of viral proteins that regulate different stages of the HCMV replication cycle in an endothelial cell type-specific manner and thereby act as determinants of viral tropism. Here, we report the requirement of a HCMV protein as a postentry tropism factor in endothelial cells. The identification and characterization of HCMV endotheliotropism-regulating proteins will advance our understanding of the molecular mechanisms of HCMV-related pathogenesis and help lead to the design of new antiviral strategies able to exploit these functions.

CaMKII protects MKP-1 from proteasome degradation in endothelial cells

CaMKs are a widely distributed family of kinases with multiple and often cell specific effects on intracellular signal transduction pathway. In endothelial cells, it has been recognized a role for CamKII in several pathways such as eNOS activation and nitric oxide production. It is not clear though, whether CaMKII interfere with other endothelial cell functions such as ERK activation and cell proliferation. We explored this issue in primary cultured rat endothelial cells and we evaluated the effect on endothelial cell proliferation and DNA synthesis. CaMKII inhibition through Cantide, conducted into the cell through Antoennapedia (ANT-CN), showed positive effects on proliferation and H3-thimdine incorporation similar to insulin stimulation. Accordingly, both CaMKII pharmacological inhibition and silencing through shRNA produced activation of the p44/42 MAPK. These observations leaded to the hypothesis that CamKII could regulate p44/p42 by interfering with specific ERK phosphatases. Indeed, we found that CaMKII interacts and protect the dual specific phosphatase MKP-1 from proteasome mediated degradation while this complex is disrupted by CaMKII inhibitors. This study reveals that CaMKII, besides phosphorylation through the known ras-raf-mek pathway, can regulate also dephosphorylation of p44/p42 by modulation of MKP-1 level. This novel finding opens to a novel scenario in regulation of endothelial cell functions.

Curcumin binds tubulin, induces mitotic catastrophe, and impedes normal endothelial cell proliferation

Curcumin, a component of turmeric spice that imparts flavor and color to curry, is thought to possess anti-inflammatory and antioxidant properties in biological tissues. However, while such efficacies have been described in the context of carcinogenesis, the impact of curcumin on normal cell cycle regulation is poorly understood. Here, we provide evidence of curcumin toxicity in proliferating bovine aortic endothelial cells, at concentrations relevant to the diet and below those previously reported in cancer models. Upon confirming curcumin's ability to upregulate hemeoxygenase-1 in a dose-dependent fashion, we found the minimally efficacious curcumin concentration to also inhibit endothelial cell DNA synthesis. Moreover, curcumin concentrations below the minimum 2 μM threshold required to induce hemeoxygenase-1 bound tubulin protein in vitro and triggered hallmark evidence of mitotic catastrophe in vivo. Concentrations as low as 0.1 μM curcumin led to disproportionate DNA segregation, karyorrhexis, and micronucleation in proliferating endothelial cells. While suggesting a mechanism by which physiological curcumin concentrations inhibit cell cycle progression, these findings describe heretofore unappreciated curcumin toxicity with potential implications for endothelial growth, development, and tissue healing.

Abstract 3899: PCB153-induced angiogenesis depends on redoxsensitive Pyk2 signaling.

Abstract Estrogenic chemicals have been shown to increase the expression of the growth factor VEGF in breast cancer cells. Hence, there is some concern that environmental exposure to estrogenic chemicals such as polychlorinated biphenyls (PCBs) may excacerbate the growth of breast cancer via angiogenesis. Highly metastatic breast cancer cells exposed to PCB153 have been reported to produce a significantly greater amount of ROS compared to non-metastatic breast cancer cells. A number of paracrine effects have been reported in tumors exposed to estrogen, however, the role of estrogen-induced ROS in signaling tumor angiogenesis remains to be defined. We previously reported that PCB-induced angiogenesis can be blocked by Pyk2 siRNA. Therefore, the goal of this study was to determine whether Pyk2 small molecule inhibitors block PCB-induced vascularization in a 3-D breast tumor model. Human umbilical vein endothelial cells (HUVEC) or brain microvascular endothelial cells (hCMEC/D3) were used to study the effect of small molecule inhibitors. The effect of Pyk2 inhibition on breast tumor spheroid angiogenesis was tested in a 3-D co-culture of MDA-MB-231 breast cancer cells. MDA-MB-231 cells exposed to PCB153 produced ROS most likely H2O2; therefore we determined whether external H2O2 exposure increased DNA synthesis of vascular endothelial cells. Our results showed a significant increase in DNA synthesis from exposure to H2O2 for 18hrs. Breast tumor spheroids treated with PF431396 or Pyk2 siRNA showed a significant reduction in microvessel sprouting and length when compared to control. Confocal microscopy of endothelial cells co-cultured with smooth muscle cells and fibroblasts further showed a significant increase in endothelial cell branching when exposed to PCB153. The effects of PCB153 on endothelial cells in co-culture were inhibited by treatments with the ROS scavenger N-acetylcysteine. We have provided evidence for the role of Pyk2 redox signaling in the aggressive angiogenic phenotype of endothelial cells. Our results suggest that metastatic breast cancer cells exposed to PCBs release ROS that may directly activate the redox sensitive kinase Pyk2 in neighboring endothelial cells. Consequently, Pyk2 redox signaling in neighboring endothelial cells may promote the aggressive spread of breast cancer. Citation Format: Jayanta K. Das, Quentin H. Felty. PCB153-induced angiogenesis depends on redoxsensitive Pyk2 signaling. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3899. doi:10.1158/1538-7445.AM2013-3899

Beta-35 is a transferrin-derived inhibitor of angiogenesis and tumor growth

An angiogenesis inhibitor named Beta-35 has been identified and purified from the conditioned medium of mouse pancreatic β cells tumor cells. Beta-35 has a molecular weight of 35kDa and inhibits DNA synthesis of bovine capillary endothelial cells at a half-maximal concentration of approximately 5nM. It shows anti-angiogenic activity in the chick embryo chorioallantoic membrane at a dose of about 1μg/embryo. Amino acid microsequencing and mass spectrometric analysis of the purified protein demonstrate that Beta-35 contains the first 314 residues of the N-terminal sequence of bovine transferrin. We have cloned and expressed this protein in Escherichia coli using the corresponding gene segment of Beta-35 contained in the cDNA of human transferrin. The recombinant protein of Beta-35 shows significant anti-tumor activity at a dose of 5mg/kg/day against human pancreatic cancer or melanoma implanted subcutaneously in SCID mice.

Kupffer cells participate in 2-butoxyethanol-induced liver hemangiosarcomas

2-Butoxyethanol increases hemangiosarcomas selectively in male mouse liver after chronic inhalation through mechanisms that have not fully been elucidated. Hemolysis, a primary toxic effect associated with 2-butoxyethanol exposure in rodents, increased hemosiderin (iron) deposition in Kupffer cells in the liver. These findings, along with the induction of hepatic neoplastic lesions, led to our hypothesis that the induction hemangiosarcomas by 2-butoxyethanol is due to the activation of Kupffer cells, subsequent to hemolysis, that results in the induction of DNA synthesis in target cells (endothelial cells); allowing for the selective proliferation of preneoplastic target cells and/or the promotion of new initiated cells. The present studies were conducted to determine whether Kupffer cells contributed to 2-butoxyethanol-induced endothelial DNA synthesis in the liver, thereby determining whether a linkage exists between these events. Male B6C3F1 mice were treated with 450 and 900 mg/kg 2-butoxyethanol (via daily gavage; 5×/week) for 7 days in the presence or absence of Kupffer cell depletion (via clodronate-encapsulated liposomes). 2-Butoxyethanol (450 and 900 mg/kg/day) increased the number of F4/80 stained cells (Kupffer cells) compared to controls (∼1.3- and ∼1.6-fold over control, respectively). Clodronate liposome treatment reduced the number of Kupffer cells by >90%, as assessed by F4/80 immunohistochemistry. Increased hemolysis, measured by increases in relative spleen weights and decreased hematocrit was confirmed in 2-butoxyethanol treated mice. The percentage of iron-stained endothelial cells increased by ∼11-fold over control, and endothelial cell DNA synthesis increased ∼1.7-fold over control in 2-butoxyethanol exposed mice. Importantly, Kupffer cell depletion reduced 2-butoxyethanol-induced iron staining and hepatic endothelial cell DNA synthesis. These studies provide evidence supporting the hypothesis that the Kupffer cell modulates 2-butoxyethanol-induced endothelial cell DNA synthesis, and therefore may contribute to hemangiosarcoma induction by 2-butoxyethanol.

Involvement of the JNK activation in terbinafine‐induced p21 up‐regulation and DNA synthesis inhibition in human vascular endothelial cells

Previously, we demonstrated that the extracellular signal-regulated kinase (ERK)-mediated pathway contributes to the terbinafine (TB)-induced increases of p21 and p53 protein level as well as decrease of DNA synthesis in human umbilical venous endothelial cells (HUVEC). The aim of this study is to examine the involvement of c-Jun NH2-terminal kinase (JNK) in the TB-induced increase of p21 protein level and DNA synthesis inhibition. Western blot analysis and kinase assay demonstrated that TB treatment increased both the protein level and the kinase activity of JNK1/2 in HUVEC. Transfection of HUVEC with JNK1 dominant negative (DN-JNK1) prevented the TB-induced increases of p21 and p53 protein level and decrease of DNA synthesis, suggesting that JNK1/2 activation is involved in the TB-induced cell cycle arrest in HUVEC. Moreover, over-expression of mitogen-activated protein kinase (MEK)-1 prevented the TB-induced increase of JNK1/2 protein levels, suggesting that MEK-1 is an upstream inhibitor of JNK. Transfection of HUVEC with DN-JNK1 prevented the TB-induced inhibition of ERK phosphorylation, suggesting that JNK1/2 might serve as a negative regulator of ERK. Taken together, our results suggest that JNK activation is involved in the TB-induced inhibition of ERK phosphorylation, p53 and p21 up-regulation and DNA synthesis inhibition in HUVEC.

Angioinhibitory Action of NK4 Involves Impaired Extracellular Assembly of Fibronectin Mediated by Perlecan-NK4 Association

NK4, a fragment of hepatocyte growth factor (HGF), exerts bifunctional action as a competitive antagonist against HGF and its receptor c-Met and an angiogenesis inhibitor. Here we studied the anti-angiogenic mechanism of NK4. In cultured human endothelial cells, NK4 inhibited DNA synthesis induced not only by HGF but also by either basic fibroblast growth factor or vascular endothelial growth factor. Even if c-Met expression was diminished by small interference RNA, NK4 inhibited basic fibroblast growth factor-induced DNA synthesis, indicating that anti-angiogenic action of NK4 is c-Met-independent. Affinity purification with NK4-immobilized beads revealed that NK4 binds to perlecan. Consistent with this, NK4 colocalized with perlecan in endothelial cells. Perlecan is a multidomain heparan sulfate proteoglycan that interacts with basement membrane components such as fibronectin. NK4 inhibited extracellular assembly of fibronectin, by which fibronectin-dependent endothelial cell spreading was inhibited by NK4. Knockdown of perlecan expression by small interference RNA significantly abrogated the inhibitory effect of NK4 on fibronectin assembly and cell spreading. In NK4-treated endothelial cells, tyrosine phosphorylation of focal adhesion kinase and Rac activation were reduced, whereas overexpression of activated Rac recovered the DNA synthesis in NK4-treated endothelial cells. These results indicate that the association between NK4 and perlecan impairs fibronectin assembly, thereby inhibiting anchorage-dependent signaling. The identified mechanism for angiostatic action provides further proof of significance for NK4 in the treatment of cancer and potentially for vascular regulation as well.

The Role of IGF-system in Vascular Insulin Resistance

Insulin and IGF-I are closely related peptides, which interact by several mechanisms. In high supraphysiological concentrations (> or = 10 (-8) M), they cross-react with each other's receptors with 100- to 1000-fold lower affinity than with their cognate receptors. This can cause confusion, since in many in vitro studies, insulin has been used in high unphysiological concentrations, which activate IGF-I receptors. Due to the differences in affinity, insulin and IGF-I probably do not activate each other's receptors in vivo. IGF-I receptors are several-fold more abundant than insulin receptors in human micro- and macrovascular endothelial cells and in human vascular smooth muscle cells. Both insulin and IGF-I receptor protein can be demonstrated and they are activated by their cognate ligand at physiological concentrations of 10 (-9)-10 (-10) M. In vascular smooth muscle cells, IGF-I but not insulin stimulates metabolism and growth. IGF-I stimulates DNA-synthesis and growth in microvascular endothelial cells, but neither insulin nor IGF-I have any effect on macrovascular endothelial cells. Both insulin and IGF-I have been shown to stimulate nitric oxide production in endothelial cells, but only the effect of IGF-I was obtained at a physiological concentration. In both endothelial and vascular smooth muscle cells, insulin and IGF-I receptors occur as insulin/IGF-I hybrid receptors with high affinity to IGF-I and low for insulin. Due to the low number of insulin receptors and the presence of hybrid receptors the insulin receptor signal is probably too attenuated to elicit biological effects, explaining the insulin resistance of vascular cells in vitro. In vivo both insulin and IGF-I have been reported to increase muscle blood flow in physiological concentrations. Whether this is due to direct effects on endothelial cells or indirectly induced is not clear. The effect of insulin is attenuated by insulin resistance. In conclusion, the in vitro data suggest that endothelial cells and vascular smooth muscle cells are sensitive to IGF-I, but insensitive to insulin, and this is due to a preponderance of IGF-I receptors and the presence of insulin/IGF-I hybrid receptors.

072 Wound Healing Properties of Reconstituted Freeze-Dried Platelets

The use of fresh platelets has gained value in medicine as an essential part of wound treatments. This is not surprising since platelets contain a number of bioactive factors that contribute to the process of wound healing, such as platelet-derived growth factor (PDGF) and transforming growth factor (TGF). Fresh platelets’ short shelf life limits platelet-based therapies. If platelets can be stabilized in freeze-dried form (FDP) then long-term storage as well as pathogen inactivation methods become possible. Adlyfe and Oregon Freeze-Dry have been developing technology to stabilize freeze-dried human platelets that can be subjected to gamma irradiation and stored for a long duration. Upon reconstitution, irradiated FDP retained growth factors PDGF-AB, PDGF-BB, and TGF-B1 in quantities similar to fresh platelets as judged by capture ELISA. The rehydrated FDP promoted new DNA synthesis and cellular proliferation of primary human dermal fibroblasts and endothelial cells (HUVECs) similar to fresh platelets. The FDP also promoted remodeling of extracellular matrix by accelerating fibroblast-mediated contraction of collagen gels and stimulated HUVECs to undergo angiogenesis and form capillary structures in vitro. Therefore, we conclude that FDP and fresh platelets have comparable in vitro wound healing potential. Preclinical wound healing studies in diabetic mice are under way and further development will allow FDP to be a safe and well-suited alternative to fresh platelets for wound healing applications.

Endothelial α1‐adrenoceptors regulate neo‐angiogenesis

Intact endothelium plays a pivotal role in post-ischaemic angiogenesis. It is a phenomenon finely tuned by activation and inhibition of several endothelial receptors. The presence of alpha(1)-adrenoceptors on the endothelium suggests that these receptors may participate in regenerative phenomena by regulating the responses of endothelial cells involved in neo-angiogenesis. We evaluated the expression of the subtypes of the alpha(1)-adrenoceptor in isolated endothelial cells harvested from Wistar-Kyoto (WKY) rats. We explored the possibility these alpha(1)-adrenoceptors may influence the pro-angiogenic phenotype of endothelial cells in vitro. In vivo, we used a model of hindlimb ischaemia in WKY rats, to assess the effects of alpha(1) adrenoceptor agonist or antagonist on angiogenesis in the ischaemic hindlimb by laser Doppler blood flow measurements, digital angiographies, hindlimb perfusion with dyed beads and histological evaluation. In vitro, pharmacological antagonism of alpha(1)-adrenoceptors in endothelial cells from WKY rats by doxazosin enhanced, while stimulation of these adrenoceptors with phenylephrine, inhibited endothelial cell proliferation and DNA synthesis, ERK and retinoblastoma protein (Rb) phosphorylation, cell migration and tubule formation. In vivo, we found increased alpha(1)-adrenoceptor density in the ischaemic hindlimb, compared to non-ischaemic hindlimb, suggesting an enhanced alpha(1)-adrenoceptor tone in the ischaemic tissue. Treatment with doxazosin (0.06 mg kg(-1) day(-1) for 14 days) did not alter systemic blood pressure but enhanced neo-angiogenesis in the ischaemic hindlimb, as measured by all our assays. Our findings support the hypothesis that the alpha(1)-adrenoceptors in endothelial cells provide a negative regulation of angiogenesis.

Inhibition of Tumor Angiogenesis by Targeting Endothelial Surface ATP Synthase with Sangivamycin

Sangivamycin, an antibiotic with anti-tumor and anti-herpes virus activities by inhibiting both DNA/RNA synthesis and protein kinase C activity, was reported to suppress selectively DNA synthesis and growth of human umbilical vein endothelial cells and their tube formation in vitro. Here, to address the potential clinical use of sangivamycin in future, we investigated its anti-angiogenic effect in in vivo chicken chorioallantoic membrane (CAM) and mouse dorsal air sac (DAS) assays, and investigated underlying mechanism. The effect of sangivamycin on blood vessel formation in CAM was observed under the microscope after treating for two days. For DAS assays, chambers fulfilled with tumor cells were implanted beneath mouse dorsal skin. After the mice were administered with sangivamycin, tumor-induced angiogenesis was observed under the microscope. The effect of sangivamycin on ATP synthesis on the endothelial cell surface was assayed by measuring ATP production with bioluminescence assay. Sangivamycin suppressed angiogenesis within CAM down to 94-71%, which was partially blocked by simultaneous addition of a 40-fold excess of adenosine. Sangivamycin also inhibited tumor-angiogenesis in the DAS assay by 61%, and suppressed ATP production on the endothelial cell surface by 75%. Sangivamycin inhibits the in vivo angiogenesis within CAM and tumor-induced angiogenesis within mouse dorsal skin, at least in part via inhibiting endothelial cell surface ATP metabolism in addition to inhibition of DNA/RNA synthesis and/or protein kinase C activity, suggesting a potential clinical use of sangivamycin as a novel anti-cancer reagent capable of targeting not only cancer cells but also endothelial cells.

Olmesartan blocks advanced glycation end products (AGEs)-induced angiogenesis in vitro by suppressing receptor for AGEs (RAGE) expression

We have previously shown that advanced glycation end products (AGEs)–their receptor (RAGE) interaction elicits angiogenesis through autocrine production of vascular endothelial growth factor (VEGF), thus suggesting the active involvement of the AGEs–RAGE system in proliferative diabetic retinopathy (PDR). Since the crosstalk between the AGEs–RAGE and the renin–angiotensin system has also been proposed in the pathogenesis of PDR, we investigated here whether olmesartan, an angiotensin II type 1 receptor blocker, inhibited the AGEs-elicited angiogenesis in vitro by suppressing the NF-κB-mediated RAGE expression. Olmesartan significantly inhibited the AGEs-induced NF-κB promoter activity and RAGE gene expression in cultured microvascular endothelial cells (ECs). Further, olmesartan was found to block the AGEs-induced up-regulation of VEGF mRNA levels and consequent increase in DNA synthesis in ECs. These results demonstrated for the first time that olmesartan inhibited the AGEs signaling to angiogenesis by suppressing RAGE expression in ECs. Our present study suggests that blockade of the renin–angiotensin system by olmesartan may play a protective role against PDR by attenuating the deleterious effects of AGEs via down-regulation of RAGE.

Synergistic Antitumor Effect of an Angiogenesis Inhibitor (TNP-470) and Tumor Necrosis Factor in Mice

We investigated the potentiation of combination therapy using tumor necrosis factor (TNF) with TNP-470, a potent angiogenesis inhibitor. We evaluated the antitumor effect in vivo against subcutaneous (s.c.) MC38 mouse colon adenocarcinoma tumors in C57BL/6 mice. The mice were treated with a single bolus injection via the tail vein of 3 or 8 microg rhTNF in 0.5% bovine serum albumin/normal saline (BSA/NS), or with 0.5% BSA/NS alone as a control, with or without TNP-470 pretreatment, given as 30 or 60 mg/kg x 2 days, s.c. DNA synthesis in human umbilical endothelial cells (HUVEC) was assessed by [(3)H]thymidine uptake after incubation with TNF, with or without TNP-470. The antitumor effect of TNP-470 pretreatment combined with 3 microg recombinant human (rh) TNF injection resulted in an 80% reduction of tumor volume compared with the control. This was significantly better than that induced by 3 microg rhTNF alone (P < 0.005). DNA synthesis in HUVEC was inhibited by TNF with TNP-470 in a dose-dependent manner, but there was no enhanced effect against MC38 in vitro. These results suggest that the combination of the angiogenesis inhibitor TNP-470 and TNF might have a synergistic antitumor effect on solid tumors in vivo.

Group VIA Calcium-independent Phospholipase A2 Mediates Endothelial Cell S Phase Progression

Arachidonic acid and its metabolites have been previously implicated in the regulation of endothelial cell proliferation. Arachidonic acid may be liberated from cellular phospholipids by the action of group VIA calcium-independent phospholipase A2 (iPLA2-VIA). Consequently, we tested the hypothesis that iPLA2-VIA activity is linked to the regulation of endothelial cell proliferation. Inhibition of iPLA2 activity by bromoenol lactone (BEL) was sufficient to entirely block endothelial cell growth. BEL dose-dependently inhibited endothelial cell DNA synthesis in a manner that was reversed upon the exogenous addition of arachidonic acid. DNA synthesis was inhibited by the S-isomer and not by the R-isomer of BEL, demonstrating that endothelial cell proliferation is mediated specifically by iPLA2-VIA. iPLA2-VIA activity was critical to the progression of endothelial cells through S phase and is required for the expression of the cyclin A/cdk2 complex. Thus, inhibition of iPLA2-VIA blocks S phase progression and results in exit from the cell cycle. Inhibition of iPLA2-VIA-mediated endothelial cell proliferation is sufficient to block angiogenic tubule formation in co-culture assays. Consequently, iPLA2-VIA is a novel regulator of endothelial cell S phase progression, cell cycle residence, and angiogenesis.

Vascular Endothelial Growth Factor Signaling to Endothelial Nitric Oxide Synthase

See related article, pages 715–722 There is substantial evidence supporting the idea that the process of angiogenesis requires the synthesis of endothelium-derived nitric oxide (NO). NO, characterized as a major endothelial-derived relaxing factor, exerts paracrine and autocrine roles in maintaining cardiovascular homeostasis, vascular tone, and microvascular permeability. During the early steps of angiogenesis in which new blood vessels sprout from existing vascular beds, there is a persistence of vasodilation and increase in vascular permeability, suggesting that these hemodynamic changes in the existing vasculature are indispensable during an angiogenic process. A number of angiogenic factors can triggers the release of NO, synthesized by the endothelial isoform of NO synthase (eNOS). One such factor is the vascular endothelial growth factor (VEGF), which as its name implies, is a critical mitogenic, chemoattractant, and survival factor for endothelial cells1 in addition to being characterized as a potent vascular permeability factor.2 Early studies have demonstrated that VEGF can readily stimulate NO production in cultured cells and isolated blood vessels and that NO is essential in mediating the VEGF-induced endothelial cell proliferation and organization into tubes in 3D cultures.3,4 Subsequent in vivo studies have also demonstrated that eNOS knockout mice had significantly attenuated VEGF and ischemia induced angiogenesis and vascular permeability.5,6 These studies place eNOS derived NO as a key mediator of VEGF induced angiogenesis in postnatal mice. Since the discovery of VEGF, or VEGF-A, and subsequent members of the VEGF family (VEGF-B, -C, -D, -E, and placenta growth factor, PlGF), intense research has been performed to elucidate their modes of action. These growth factor ligands bind to 3 receptor tyrosine kinases (RTKs), namely VEGF receptor-1, -2, and -3 as well as to coreceptors such as heparan sulfate proteoglycans or neuropilins. Like many other RTKs, these receptors are able to form …

N-Glycosyl-thiophene-2-carboxamides: synthesis, structure and effects on the growth of diverse cell types

A range of N-glycosyl-thiophene-2-carboxamides, including a 6 H-thieno[2,3- c]pyridin-7-one and a bivalent compound, have been synthesised and assayed for their effects on DNA synthesis in bovine aortic endothelial cells or on the growth of synoviocytes. Per-O-acetylated analogues of the glycoconjugates were significantly more effective inhibitors when compared to their corresponding non-acetylated analogues, indicating that the lower potency observed for hydroxylated derivatives is due to less efficient transport of these compounds across the cell membrane. Thiophene-2-carboxamide was inactive as an inhibitor of bFGF induced proliferation, confirming the requirement of the carbohydrate residue for the observed biological properties. Glucose, mannose, galactose and 2-amino-2-deoxy-glucose analogues were active as were a variety of substituted thiophene derivatives; the 6 H-thieno[2,3- c]pyridin-7-one conjugate was inactive. Conformational analysis of the title compounds was investigated. X-ray crystal structural analysis of four N-glucosyl-thiophene-2-carboxamides showed that the pyranose rings adopted the expected 4 C 1 conformations and that Z- anti structures were predominant (H1–C1–N–H anomeric torsion angle varied from −168.2° to −175.0°) and that the carbonyl oxygen and sulfur of the thiophene adopted an s-cis conformation in three of the isomers. In a crystal structure of a 3-alkynyl derivative, the hydrogen atom of the NH group was directed toward the acetylene group. The distance between the hydrogen atom and acetylene carbons and angles between nitrogen, hydrogen and carbon atoms were consistent with hydrogen bonding and this was supported by IR and NMR spectroscopic studies. The geometries of thiophene-2-carboxamides were explored by density functional theory (DFT) and Møller-Plesset (MP2) calculations and the s-cis conformer of thiophene-2-carboxamide was found to be more stable than its s-trans isomer by 0.83 kcal mol −1. The s-cis conformer of 3-ethynyl-thiophene-2-carboxamide was 5.32 kcal mol −1 more stable than the s-trans isomer. The larger stabilisation for the s-cis conformer in the 3-alkynyl derivatives is explained to be due to a moderate hydrogen bonding interaction between the alkyne and NH group.

Mechanisms of 2-Butoxyethanol–Induced Hemangiosarcomas

Chronic exposure to 2-butoxyethanol increased liver hemangiosarcomas in male mice. The mechanism for the selective induction of hemangiosarcomas by 2-butoxyethanol is unknown but has been suggested to occur through non-DNA-reactive mechanisms. The occurrence of liver hemangiosarcomas in male mice has been linked to oxidative damage subsequent to RBC hemolysis and iron deposition and activation of macrophages (Kupffer cells) in the liver, events that exhibit a threshold in both animals and humans. 2-Butoxyethanol is metabolized to 2-butoxyacetaldehyde and 2-butoxyacetic acid, and although the aldehyde metabolite is short lived, the potential exists for this metabolite to cause DNA damage. The present study examined whether 2-butoxyethanol and its metabolites, 2-butoxyacetaldehyde and 2-butoxyacetic acid, damaged mouse endothelial cell DNA using the comet assay. No increase in DNA damage was observed following 2-butoxyethanol (1-10mM), 2-butoxyacetaldehyde (0.1-1.0mM), or 2-butoxyacetic acid (1-10mM) in endothelial cells after 2, 4, or 24 h of exposure. Additional studies examined the involvement of hemolysis and macrophage activation in 2-butoxyethanol carcinogenesis. DNA damage was produced by hemolyzed RBCs (10 x 10(6), 4 h), ferrous sulfate (0.1-1.0 microM; 2-24 h), and hydrogen peroxide (50-100 microM; 1-4 h) in endothelial cells. Hemolyzed RBCs also activated macrophages, as evidenced by increased tumor necrosis factor (TNF) alpha, while neither 2-butoxyethanol nor butoxyacetic acid increased TNF-alpha from macrophages. The effect of activated macrophages on endothelial cell DNA damage and DNA synthesis was also studied. Coculture of endothelial cells with activated macrophages increased endothelial cell DNA damage after 4 or 24 h and increased endothelial cell DNA synthesis after 24 h. These data demonstrate that 2-butoxyethanol and related metabolites do not directly cause DNA damage. Supportive evidence also demonstrated that damaged RBCs, iron, and/or products from macrophage activation (possibly reactive oxygen species) produce DNA damage in endothelial cells and that activated macrophages stimulate endothelial cell proliferation. These events coupled together provide the events necessary for the induction of hemangiosarcomas by 2-butoxyethanol.

Estrogen-induced DNA synthesis in vascular endothelial cells is mediated by ROS signaling

BackgroundSince estrogen is known to increase vascular endothelial cell growth, elevated estrogen exposure from hormone replacement therapy or oral contraceptives has the potential to contribute in the development of abnormal proliferative vascular lesions and subsequent thickening of the vasculature. How estrogen may support or promote vascular lesions is not clear. We have examined in this study whether estrogen exposure to vascular endothelial cells increase the formation of reactive oxygen species (ROS), and estrogen-induced ROS is involved in the growth of endothelial cells.MethodsThe effect of estrogen on the production of intracellular oxidants and the role of estrogen-induced ROS on cell growth was studied in human umbilical vein endothelial cells. ROS were measured by monitoring the oxidation of 2'7'-dichlorofluorescin by spectrofluorometry. Endothelial cell growth was measured by a colorimetric immunoassay based on BrdU incorporation into DNA.ResultsPhysiological concentrations of estrogen (367 fmol and 3.67 pmol) triggered a rapid 2-fold increase in intracellular oxidants in endothelial cells. E2-induced ROS formation was inhibited to basal levels by cotreatment with the mitochondrial inhibitor rotenone (2 μM) and xanthine oxidase inhibitor allopurinol (50 μM). Inhibitors of NAD(P)H oxidase, apocynin and DPI, did not block E2-induced ROS formation. Furthermore, the NOS inhibitor, L-NAME, did not prevent the increase in E2-induced ROS. These findings indicate both mitochondria and xanthine oxidase are the source of ROS in estrogen treated vascular endothelial cells. E2 treated cells showed a 2-fold induction of BrdU incorporation at 18 h which was not observed in cells exposed to vehicle alone. Cotreatment with ebselen (20 μM) and NAC (1 mM) inhibited E2-induced BrdU incorporation without affecting the basal levels of DNA synthesis. The observed inhibitory effect of NAC and ebselen on E2-induced DNA synthesis was also shown to be dose dependent.ConclusionWe have shown that estrogen exposure stimulates the rapid production of intracellular ROS and they are involved in growth signaling of endothelial cells. It appears that the early estrogen signaling does not require estrogen receptor genomic signaling because we can inhibit estrogen-induced DNA synthesis by antioxidants. Findings of this study may further expand research defining the underlying mechanism of how estrogen may promote vascular lesions. It also provides important information for the design of new antioxidant-based drugs or new antioxidant gene therapy to protect the cardiovascular health of individuals sensitive to estrogen.