Endothelial Cell Secretion Research Articles

Hypertension in an Animal Model of HELLP Syndrome is Associated With Activation of Endothelin 1

Women with hypertensive forms of pregnancy such as hemolysis-elevated liver enzymes-low platelet syndrome have increased circulating endothelin 1; however, the relationship between hypertension and endothelin 1 has not been studied. Using an animal model, we sought to determine whether there was an increased activation/dysfunction of endothelin 1, the effect of endothelin 1 receptor-A blockade on hypertension and other manifestations of hemolysis, elevated liver enzymes, and low platelets syndrome. On gestational day 12, timed-pregnant rats were infused with soluble fms-like tyrosine kinase 1 (sFlt-1) and soluble endoglin (sEndoglin; 4.7 and 7 µg/kg) via mini-osmotic pumps for 8 days. A subset of rats were treated with receptor-A antagonist (ABT-627, 5mg/kg) for 8 days. Rats with hemolysis-elevated liver enzymes-low platelet syndrome had significantly increased hypertension (P = .0001), circulating endothelin 1 (P = .03), and a significant 3.3- and 7.2-fold increase in preproendothelin messenger RNA (mRNA) expression in the placenta and liver (P = .01 and .04). Urinary protein:creatinine ratio was significantly increased in these animals (P = .0007), and circulating factors from these rats stimulated a significant increase in endothelial cell secretion of endothelin 1 (P = .001) in an in vitro assay. Blockade of the endothelin 1 receptor A significantly decreased hypertension (P = .001), circulating endothelin 1, and interleukin 17 (P = .004 and .003), placental preproendothelin mRNA expression (P = .016), and urinary protein:creatinine ratio (P = .007) in rats with hemolysis-elevated liver enzymes-low platelet syndrome. Blockade of the endothelin 1 receptor A significantly decreased hemolysis (P = .009), liver enzymes (P = .011), and significantly increased platelet levels (P = .03) and decreased circulating CD4+ and CD8+ T lymphocytes (P = .0004 and .0001) in rats infused with sFlt-1 and sEndoglin. These data support the hypothesis that endothelin 1 activation has a critical role in pathophysiology of as hemolysis-elevated liver enzymes-low platelet syndrome.

Functional roles of angiogenic factors and receptors on non-endothelial cells in the oropharyngeal cavity of the chukar partridge (Alectoris chukar)

The vascular endothelial growth factor (VEGF) family belong to the platelet-derived growth factor supergene family and is involved in angiogenesis and mitogenesis. The VEGF–VEGFR system regulates endothelial cell proliferation, migration, vascular permeability, secretion and other non-endothelial cells functions. To clarify the possible role of endothelial and non-endothelial cells, VEGF and its receptors, vascular endothelial cell growth inhibitor (VEGI) were immunohistochemically examined in oropharyngeal organs. Ten adult partridges were used in this study and the pharynx and larynx were dissected together with the palate and tongue. VEGI, VEGF and its receptor were highly expressed in luminal epithelial and stromal cells, when compared to glandular epithelial and muscle cells (P < 0.05). Moreover, VEGF, its receptors and VEGI were expressed rather strongly in the endothelial cells of the blood capillaries and in both the endothelial and smooth muscle cells of the large and small blood vessels. In conclusion, VEGF and its receptors (flt1/fms, flk1/KDR and flt4) and VEGI were expressed by various cell groups at varying intensity in the oropharyngeal organs. This demonstrates that they play a critical role in the regulation and maintenance of the functions in cells different from endothelial ones as well as in cell proliferation, differentiation, apoptosis and angiogenesis.

ID: 229: Endothelial-derived IL-33 is cardioprotective after myocardial pressure overload

Hypertension increases the pressure load on the heart and is associated with a poorly understood chronic systemic inflammatory state. Interleukin-33 (IL-33) binds to membrane-bound ST2 (ST2L) and has anti-hypertrophic and anti-fibrotic effects in the myocardium. In contrast, soluble ST2 (sST2) appears to act as a decoy receptor for IL-33, blocking myocardial and vascular benefits and is a prognostic biomarker in patients with cardiovascular diseases. We found that a highly local intramyocardial IL-33/ST2 conversation regulates the heart’s response to pressure overload. Either endothelial-specific deletion of IL33 or cardiomyocyte-specific deletion of ST2 exacerbated cardiac hypertrophy with pressure overload. Furthermore, pressure overload induced systemic circulating IL-33 as well as systemic circulating IL-13 and TGF-beta1; this was abolished by endothelial-specific deletion of IL33 but not by cardiomyocyte-specific deletion of IL33. Our results demonstrated that endothelial cells are the major sources of systemic IL-33 induced by myocardial pressure overload. Our study reveals that endothelial cell secretion of IL33 is crucial for translating myocardial pressure overload into a selective systemic inflammatory state.

Heme Oxygenase-1 Is Not Decreased in Preeclamptic Placenta and Does Not Negatively Regulate Placental Soluble fms-Like Tyrosine Kinase-1 or Soluble Endoglin Secretion.

Elevated placental release of the antiangiogenic factors, soluble fms-like tyrosine kinase-1 (sFlt-1) and soluble endoglin (sENG), is central to the pathophysiology of preeclampsia. It is widely accepted that heme oxygenase-1 (HO-1) is decreased in preeclamptic placenta and negatively regulates sFlt-1 and sENG production. We set out to verify these contentions. There was no difference in HO-1 mRNA or protein levels in preterm preeclamptic placentas (n=17) compared with gestationally matched controls (n=27). In silico analysis of microarray studies did not identify decreased placental HO-1 expression in preeclamptic placenta. Silencing HO-1 in primary trophoblasts did not affect sFlt-1 protein secretion after 24 or 48 hours. Silencing nuclear factor (erythroid-derived 2)-like 2 (transcription factor that upregulates HO-1) in trophoblasts also did not affect sFlt-1 secretion. Administering tin protoporphyrin IX dichloride (HO-1 inhibitor) or cobalt protoporphyrin (HO-1 inducer) into placental explants did not affect sFlt-1 or sENG secretion. Silencing HO-1 in 2 types of primary endothelial cells (human umbilical vein endothelial and uterine microvascular endothelial cells) significantly increased sFlt-1 secretion but not sENG secretion. However, HO-1 silencing selectively increased mRNA expression of sFlt-1 i13 (generically expressed sFlt-1 variant) but not of sFlt-1 e15a (sFlt-1 variant mainly expressed in placenta). Furthermore, adding tin protoporphyrin IX dichloride decreased sFlt-1, whereas adding HO-1 inducers (cobalt protoporphyrin, dimethyl fumarate, and rosiglitazone) either had no effect or increased sFlt-1 or sENG secretion (these trends are opposite to what is expected). We conclude that HO-1 expression is not decreased in preeclamptic placenta and HO-1 does not negatively regulate placental sFlt-1 and sENG secretion in placental or endothelial cells.

Glycogen synthase kinase 3β inhibition enhanced proliferation, migration and functional re-endothelialization of endothelial progenitor cells in hypercholesterolemia microenvironment.

Hypercholesterolemia impairs the quantity and function of endothelial progenitor cell. We hypothesized that glycogen synthase kinase 3β activity is involved in regulating biological function of endothelial progenitor cells in hypercholesterolemia microenvironment. For study, endothelial progenitor cells derived from apolipoprotein E-deficient mice fed with high-fat diet were used. Glycogen synthase kinase 3β activity was interfered with glycogen synthase kinase 3β inhibitor lithium chloride or transduced with replication defective adenovirus vector expressing catalytically inactive glycogen synthase kinase 3β (GSK3β-KM). Functions of endothelial progenitor cells, proliferation, migration, secretion and network formation of endothelial progenitor cells were assessed invitro. The expression of phospho-glycogen synthase kinase 3β, β-catenin and cyclinD1 in endothelial progenitor cells was detected by Western blot. The invivo function re-endothelialization and vasodilation were also analyzed by artery injury model transplanted with glycogen synthase kinase 3β-inhibited endothelial progenitor cells. We demonstrated that while the proliferation, migration, network formation as well as VEGF and NO secretion were impaired in apolipoprotein E-deficient endothelial progenitor cells, glycogen synthase kinase 3β inhibition significantly improved all these functions. Apolipoprotein E-deficient endothelial progenitor cells showed decreased phospho-glycogen synthase kinase 3β, β-catenin and cyclinD1 expression, whereas these signals were enhanced by glycogen synthase kinase 3β inhibition and accompanied with β-catenin nuclear translocation. Our invivo model showed that glycogen synthase kinase 3β inhibition remarkably increased re-endothelial and vasodilation. Taken together, our data suggest that inhibition of glycogen synthase kinase 3β is associated with endothelial progenitor cell biological functions both invitro and invivo. It might be an important interference target in hypercholesterolemia microenvironment.

Hyperglycemia repression of miR-24 coordinately upregulates endothelial cell expression and secretion of von Willebrand factor

AbstractAn elevated level of von Willebrand factor (VWF) in diabetic patients is associated with increased risk of thrombotic cardiovascular events. The underlying mechanism of how VWF expression is upregulated in diabetes mellitus is poorly understood. We now report that hyperglycemia-induced repression of microRNA-24 (miR-24) increases VWF expression and secretion in diabetes mellitus. In diabetic patients and diabetic mouse models (streptozotocin/high-fat diet-induced and db/db mice), miR-24 is reduced in both tissues and plasma. Knockdown of miR-24 in mice leads to increased VWF mRNA and protein levels and enhanced platelet tethering (spontaneous thrombosis). miR-24 tightly controls VWF levels through pleiotropic effects, including direct binding to the 3′ untranslated region of VWF and targeting FURIN and the histamine H1 receptor, known regulators of VWF processing and secretion in endothelial cells. We present a novel mechanism for miR-24 downregulation through hyperglycemia-induced activation of aldose reductase, reactive oxygen species, and c-Myc. These findings support a critical role for hyperglycemic repression of miR-24 in VWF-induced pathology. miR-24 represents a novel therapeutic target to prevent adverse thrombotic events in patients with diabetes mellitus.

Myocardial pressure overload induces systemic inflammation through endothelial cell IL-33

Hypertension increases the pressure load on the heart and is associated with a poorly understood chronic systemic inflammatory state. Interleukin 33 (IL-33) binds to membrane-bound ST2 (ST2L) and has antihypertrophic and antifibrotic effects in the myocardium. In contrast, soluble ST2 appears to act as a decoy receptor for IL-33, blocking myocardial and vascular benefits, and is a prognostic biomarker in patients with cardiovascular diseases. Here we report that a highly local intramyocardial IL-33/ST2 conversation regulates the heart's response to pressure overload. Either endothelial-specific deletion of IL33 or cardiomyocyte-specific deletion of ST2 exacerbated cardiac hypertrophy with pressure overload. Furthermore, pressure overload induced systemic circulating IL-33 as well as systemic circulating IL-13 and TGF-beta1; this was abolished by endothelial-specific deletion of IL33 but not by cardiomyocyte-specific deletion of IL33. Our study reveals that endothelial cell secretion of IL-33 is crucial for translating myocardial pressure overload into a selective systemic inflammatory response.

Caveolin‐1 mediates tissue plasminogen activator‐induced MMP‐9 up‐regulation in cultured brain microvascular endothelial cells

Thrombolysis with tissue plasminogen activator (tPA) increases matrix metalloproteinase-9 (MMP-9) activity in the ischemic brain, which exacerbates blood-brain barrier injury and increases the risk of symptomatic cerebral hemorrhage. The mechanism through which tPA enhances MMP-9 activity is not well understood. Here we report an important role of caveolin-1 in mediating tPA-induced MMP-9 synthesis. Brain microvascular endothelial cell line bEnd3 cells were incubated with 5 or 20 μg/ml tPA for 24 hrs before analyzing MMP-9 levels in the conditioned media and cellular extracts by gelatin zymography. tPA at a dose of 20 μg/mL tPA, but not 5 μg/mL, significantly increased MMP-9 level in cultured media while decreasing it in cellular extracts. Concurrently, tPA treatment induced a 2.3-fold increase of caveolin-1 protein levels in endothelial cells. Interestingly, knockdown of Cav-1 with siRNA inhibited tPA-induced MMP-9 mRNA up-regulation and MMP-9 increase in the conditioned media, but did not affect MMP-9 decrease in cellular extracts. These results suggest that caveolin-1 critically contributes to tPA-mediated MMP-9 up-regulation, but may not facilitate MMP-9 secretion in endothelial cells. Thrombolysis with tissue plasminogen activator (tPA) increases matrix metalloproteinase-9 (MMP-9) activity in the ischemic brain, which exacerbates ischemic blood brain barrier (BBB) injury and increases the risk of symptomatic cerebral hemorrhage. Our results suggest a novel mechanism underlying this tPA-MMP 9 axis. In response to tPA treatment, caveolin-1 protein levels increased in endothelial cells, which mediate MMP-9 mRNA up-regulation and its secretion into extracellular space. Caveolin-1 may, however, not facilitate MMP-9 secretion in endothelial cells. Our datasuggest caveolin-1 as a novel therapeutic target for protecting the BBB against ischemic damage. The schematic outlines tPA-induced MMP-9 upreguation.

Deficiency of endothelium-specific transcription factor Sox17 induces intracranial aneurysm.

Intracranial aneurysm (IA) is a common vascular disorder that frequently leads to fatal vascular rupture. Although various acquired risk factors associated with IA have been identified, the hereditary basis of IA remains poorly understood. As a result, genetically modified animals accurately modeling IA and related pathogenesis have been lacking, and subsequent drug development has been delayed. The transcription factor Sox17 is robustly expressed in endothelial cells of normal intracerebral arteries. The combination of Sox17 deficiency and angiotensin II infusion in mice induces vascular abnormalities closely resembling the cardinal features of IA such as luminal dilation, wall thinning, tortuosity, and subarachnoid hemorrhages. This combination impairs junctional assembly, cell-matrix adhesion, regeneration capacity, and paracrine secretion in endothelial cells of intracerebral arteries, highlighting key endothelial dysfunctions that lead to IA pathogenesis. Moreover, human IA samples showed reduced Sox17 expression and impaired endothelial integrity, further strengthening the applicability of this animal model to clinical settings. Our findings demonstrate that Sox17 deficiency in mouse can induce IA under hypertensive conditions, suggesting Sox17 deficiency as a potential genetic factor for IA formation. The Sox17-deficient mouse model provides a novel platform to develop therapeutics for incurable IA.

Abstract 2983: Bisphosphonates: New strategies for targeting angiogenesis in ovarian cancer

Abstract Objectives: Bisphosphonates, known to have beneficial effects on bone and soft tissue metastases in breast cancer, are potent inhibitors of macrophages, but effects on the microenvironment are not fully understood. We investigated the biological effect of clodronate on macrophage and endothelial cell-driven angiogenesis in ovarian cancer. Methods: Using the FDA Adverse Event Reporting System (FAERS), we examined the effects of bisphosphonate use on overall cancer mortality. We examined the in vitro (endothelial cell migration, capillary formation and cytokine secretion) and in vivo (orthotopic mouse models) effects of clodronate on angiogenesis, macrophage infiltration, and tumor growth. Results: Using (FAERS) data, out of ∼17,000 patients with a cancer diagnosis co-medicated with a bisphosphonate, overall reported death rate was 36% lower (17.6% vs 27.7%, p&amp;lt;0.0001) than those not receiving bisphosphonates, independent of anti-tumor therapy. In vitro, treatment with clodronate resulted in decreased endothelial cell secretion of IL-6 (p &amp;lt; 0.001), IL-8 (p &amp;lt; 0.001), and a 2.5-fold reduction in cell migration compared to controls (p &amp;lt; 0.001), with only partial recovery after VEGF stimulation. Clodronate reduced capillary formation by 1.5-fold compared to controls. In the ID8-VEGF model, clodronate-treated mice had a 5-fold decrease in tumor weight (0.05g vs. 0.3g, p &amp;lt; 0.001) compared to controls. In the SKOV3ip1 model, clodronate treatment resulted in a 3-fold decrease in tumor weight (0.35g vs. 0.96g, p = 0.003) and tumor nodules (5.2 vs. 16, p=0.005) compared to controls. Immunohistochemical staining of clondronate-treated tumor sections showed a 2-fold and 1.5-fold (ID8-VEGF and SKOV3ip1, respectively) reduction in macrophage density (p=0.001 and p &amp;lt; 0.001, respectively) compared to controls. Clodronate-treated tumor sections showed a 3-fold decrease in capillary density (p &amp;lt; 0.001) compared to controls. Conclusions: Bisphosphonates modulate tumor angiogenesis through effects on macrophages and endothelial cells and are associated with overall decreased mortality in cancer patients, independent of chemotherapeutic agents. Bisphosphonates represent an unexplored, but attractive clinical strategy in ovarian cancer, with potential for synergistic combination with other anti-angiogenic agents. Citation Format: Heather J. Dalton, Nicole M. Reusser, Alexander Zien, David Jackson, Rebecca Previs, Rajesha Rupaimoole, Behrouz Zand, Gabriel Lopez-Berestein, Robert L. Coleman, Anil K. Sood. Bisphosphonates: New strategies for targeting angiogenesis in ovarian cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2983. doi:10.1158/1538-7445.AM2014-2983

Sulforaphane inhibits TNF-α-induced adhesion molecule expression through the Rho A/ROCK/NF-κB signaling pathway.

Endothelial dysfunction is an early indicator of cardiovascular diseases. Increased stimulation of tumor necrosis factor-α (TNF-α) triggers the inflammatory mediator secretion of endothelial cells, leading to atherosclerotic risk. In this study, we investigated whether sulforaphane (SFN) affected the expression of intracellular adhesion molecule-1 (ICAM-1) in TNF-α-induced ECV 304 endothelial cells. Our data showed that SFN attenuated TNF-α-induced expression of ICAM-1 in ECV 304 cells. Pretreatment of ECV 304 cells with SFN inhibited dose-dependently the secretion of proinflammatory cytokines, such as interleukin (IL)-1β, IL-6, and IL-8. SFN inhibited TNF-α-induced nuclear factor-κB (NF-κB) DNA binding activity. Furthermore, SFN decreased TNF-α-mediated phosphorylation of IκB kinase (IKK) and IκBα, Rho A, ROCK, ERK1/2, and plasminogen activator inhibitor-1 (PAI-1) levels. Collectively, SFN inhibited the NF-κB DNA binding activity and downregulated the TNF-α-mediated induction of ICAM-1 in endothelial cells by inhibiting the Rho A/ROCK/NF-κB signaling pathway, suggesting the beneficial effects of SFN on suppression of inflammation within the atherosclerotic lesion.

HO-1 expression is not reduced in preeclamptic placenta, and does not regulate sFlt-1 or soluble endoglin secretion in either primary trophoblast or endothelial cells

Results: Histological abnormalities of the three villi populations were observed in the normotensive, mild, moderate and severe hypertensive groups. The occurrence of syncytial knots were observed more frequently in the severely hypertensive than the normotensive group and the moderately hypertensive groups. Fibrinoid necrosis of villi occurred in all villous types in the normotensive group as well as in the hypertensive groups. These areas appeared acidophilic, showing strongly PAS positive necrotic areas of the placental stromal tissue. Complete necrosis of villous stroma was also observed. Hypovascular villi were occasionally observed in all study groups. Thickening of the trophoblast basement membrane was observed in 9 cases of the severely hypertensive group. A semiquantitative increase in the number of villous cytotrophoblast cells were noted in the severely hypertensive group compared to the normotensive group. A nuclear thinned-out area of villous syncytium bulging over the surface of stromal capillaries into the intervillous space was observed in all study groups. Discussion: The histopathological features noted are consistent with hypoxia which emanates from the defective placentation associated with the lack of transformation of the spiral arteries in pre-eclampsia.

Fully human monoclonal antibodies to phosphorylcholine inhibit tnf-induced IL-6 and VCAM-1 in human umbilical venous endothelial cells

Objectives: Anti-inflammatory properties of human polyclonal antibodies to phosphorylcholine (anti-PC) have been described previously by our group. We aimed at investigating effect of a panel of fully human monoclonal antibodies to phosphorylcholine (anti-PC mAbs) on TNF-induced IL-6 secretion and ICAM-1 expression in human umbilical vein endothelial cells (HUVECs) in vitro.

Changes in mast cell number and stem cell factor expression in human skin after radiotherapy for breast cancer

Background and purposeMast cells are involved in the pathogenesis of radiation fibrosis and may be a therapeutic target. The mechanism of increased mast cell number in relation to acute and late tissue responses in human skin was investigated. Materials and methodsPunch biopsies of skin 1 and 15–18months after breast radiotherapy and a contralateral control biopsy were collected. Mast cells were quantified by immunohistochemistry using the markers c-Kit and tryptase. Stem cell factor (SCF) and collagen-1 expression was analysed by qRT-PCR. Clinical photographic scores were performed at post-surgical baseline and 18months and 5years post-radiotherapy. Primary human dermal microvascular endothelial cell (HDMEC) cultures were exposed to 2Gy ionising radiation and p53 and SCF expression was analysed by Western blotting and ELISA. ResultsDermal mast cell numbers were increased at 1 (p=0.047) and 18months (p=0.040) using c-Kit, and at 18months (p=0.024) using tryptase immunostaining. Collagen-1 mRNA in skin was increased at 1month (p=0.047) and 18months (p=0.032) and SCF mRNA increased at 1month (p=0.003). None of 16 cases scored had a change in photographic appearance at 5years, compared to baseline. SCF expression was not increased in HDMECs irradiated in vitro. ConclusionsIncreased mast cell number was associated with up-regulated collagen-1 expression in human skin at early and late time points. This increase could be secondary to elevated SCF expression at 1month after radiotherapy. Although mast cells accumulate around blood vessels, no endothelial cell secretion of SCF was seen after in vitro irradiation. Modification of mast cell number and collagen-1 expression may be observed in skin at 1 and 18months after radiotherapy in breast cancer patients with no change in photographic breast appearance at 5years.

Total polyphenol of Anemarrhena asphodeloides ameliorates advanced glycation end products‐induced endothelial dysfunction by regulation of AMP‐Kinase (知母多酚通过激活AMP‐激酶来缓解糖基化终末产物引起的内皮损伤)

AbstractObjectiveAnemarrhena asphodeloides Bunge is widely used in China for the treatment of diabetes and the polyphenol components are responsible for its anti‐diabetic action. This study aimed to investigate the effect of total polyphenol of Anemarrhena asphodeloides (TPAA) on endothelial dysfunction and to elucidate underlying mechanisms.MethodsWe stimulated endothelial cells with advanced glycation end products (AGEs) to establish the model of endothelial dysfunction in vitro and observed the effect of TPAA (10, 30, or 100 μg/mL) on AMP‐Kinase (AMPK) activation implicated in regulation of nitric oxide (NO) and endothelin‐1 (ET‐1) production. Meanwhile, nuclear factor‐κB (NF‐κB) activation, intracellular reactive oxygen species (ROS) production, mitochondrial membrane potential (ΔΨm) and eNOS expression were investigated by western blot, fluorescence microscopy and real time‐quantitative PCR analysis, respectively.ResultsTotal polyphenol of Anemarrhena asphodeloides enhanced AMPK phosphorylation and promoted the basal NO production along with the inhibition of ET‐1 secretion in endothelial cells. TPAA inhibited NF‐κB activation by attenuating p65 phosphorylation and suppressed ROS production, well demonstrating its action in inhibition of ROS‐associated inflammation in the endothelium. Meanwhile, TPAA protected mitochondrial function and endothelial homeostasis against AGEs insult by restoring ΔΨm and mRNA expression of eNOS. AGEs stimulation inhibited AMPK activation and induced the loss of NO production together with increased secretion of ET‐1, but these changes were reversed by TPAA in a concentration‐dependent manner. Compound C, an AMPK inhibitor, attenuated the effects of TPAA mentioned above, indicating the involvement of AMPK.ConclusionsTotal polyphenol of Anemarrhena asphodeloides inhibited AGEs‐induced ROS‐associated inflammation and ameliorated endothelial dysfunction through beneficial regulation of AMPK activation.

Kaposi's Sarcoma-Associated Herpesvirus-Positive Primary Effusion Lymphoma Tumor Formation in NOD/SCID Mice Is Inhibited by Neomycin and Neamine Blocking Angiogenin's Nuclear Translocation

Angiogenin (ANG) is a 14-kDa multifunctional proangiogenic secreted protein whose expression level correlates with the aggressiveness of several tumors. We observed increased ANG expression and secretion in endothelial cells during de novo infection with Kaposi's sarcoma-associated herpesvirus (KSHV), in cells expressing only latency-associated nuclear antigen 1 (LANA-1) protein, and in KSHV latently infected primary effusion lymphoma (PEL) BCBL-1 and BC-3 cells. Inhibition of phospholipase Cγ (PLCγ) mediated ANG's nuclear translocation by neomycin, an aminoglycoside antibiotic (not G418-neomicin), resulted in reduced KSHV latent gene expression, increased lytic gene expression, and increased cell death of KSHV(+) PEL and endothelial cells. ANG detection in significant levels in KS and PEL lesions highlights its importance in KSHV pathogenesis. To assess the in vivo antitumor activity of neomycin and neamine (a nontoxic derivative of neomycin), BCBL-1 cells were injected intraperitoneally into NOD/SCID mice. We observed significant extended survival of mice treated with neomycin or neamine. Markers of lymphoma establishment, such as increases in animal body weight, spleen size, tumor cell spleen infiltration, and ascites volume, were observed in nontreated animals and were significantly diminished by neomycin or neamine treatments. A significant decrease in LANA-1 expression, an increase in lytic gene expression, and an increase in cleaved caspase-3 were also observed in neomycin- or neamine-treated animal ascitic cells. These studies demonstrated that ANG played an essential role in KSHV latency maintenance and BCBL-1 cell survival in vivo, and targeting ANG function by neomycin/neamine to induce the apoptosis of cells latently infected with KSHV is an attractive therapeutic strategy against KSHV-associated malignancies.

Quercetin suppresses inflammation by reducing ERK1/2 phosphorylation and NF kappa B activation in Leptin-induced Human Umbilical Vein Endothelial Cells (HUVECs)

BackgroundHigh concentrations of plasma leptin and the release of pro-inflammatory cytokines in leptin-resistance in obesity have been reported to trigger endothelial dysfunction. The objective of this study was to elucidate the role of quercetin in modulating leptin-induced inflammation as assessed by the levels of Ob-Ra expression, ERK1/2 phosphorylation, NF-kappa B activation and TNF-alpha secretion in umbilical vein endothelial cells (HUVECs) in vitro.FindingsHUVECs were exposed to either control levels (0 ng/ml) or 500 ng/mL leptin (L) for 48 hours, followed by control or 125 uM quercetin (Q) for another 6 h. The experimental groups were as follows: L0Q0, L0Q125, L500Q0, L500Q125. The presence of the short chain leptin receptor isoform Ob-Ra in HUVECs was determined by Western blot and immunocytochemistry analyses. Ob-Ra expression, ERK1/2 phosphorylation, NF-kappa B activation and TNF-alpha secretion were quantified by ELISA, and NF-kappa B activationby immunofluorescence staining. Our results showed that Ob-Ra expression, ERK1/2 phosphorylation and NF-kappa B activation increased significantly after 500 ng/mL leptin exposure (1.8x, 1.5x, 6.2x for Ob-Ra, ERK1/2 and NF-kappa B, respectively), but were reduced by addition of 125 uM quercetin (0.7x, 0.3x and 0.4x for Ob-Ra, ERK1/2 and NF-kappa B, respectively), and that quercetin could also partially suppress leptin-induced TNF-alpha secretion (3.8x) by 0.8x.ConclusionExposure of HUVECs to leptin up-regulated Ob-Ra expression and elevated ERK1/2 phosphorylation and NFkB activation, and increased TNF-alpha secretion. These effects strongly suppressed by quercetin, with the exception of TNF-alpha which was partially suppressed. The findings might be of clinical significance, as endothelial dysfunction that could lead to cardiovascular disease is preventable, and quercetin is a natural compound found in various plants and fruits.

Adaptive Induction of Growth Differentiation Factor 15 Attenuates Endothelial Cell Apoptosis in Response to High Glucose Stimulus

Growth differentiation factor 15 (GDF15), a direct target gene of p53, is a multifunctional member of the TGF-β/BMP superfamily. GDF15 can be induced and is implicated as a key secretory cytokine in response to multiple cellular stimuli. Accumulating evidence indicates that GDF15 is associated with the development and prognosis of diabetes mellitus, while whether GDF15 can be induced by high glucose is unknown. In the present study, we revealed that high glucose could induce GDF15 expression and secretion in cultured human umbilical vein endothelial cells in a ROS- and p53-dependent manner. Inhibition of high glucose-induced GDF15 expression by siRNA demonstrated that adaptively induced GDF15 played a protective role against high glucose-induced human umbilical vein endothelial cell apoptosis via maintaining the active state of PI3K/Akt/eNOS pathway and attenuating NF-κB/JNK pathway activation. The protective effects of GDF15 were probably achieved by inhibiting ROS overproduction in high glucose-treated human umbilical vein endothelial cells in a negative feedback manner. Our results suggest that high glucose can promote GDF15 expression and secretion in human umbilical vein endothelial cells, which in turn attenuates high glucose-induced endothelial cell apoptosis.

Differential effects of pravastatin on secretion of pro‐ and anti‐angiogenic factors from trophoblast and endothelial cells

We recently reported pravastatin (Prav) lowers blood pressure but does not improve circulating angiogenic balance (↑soluble fms‐like tyrosine kinase‐1, sFlt‐1; ↑soluble endoglin, sEng; and ↓vascular endothelial growth factor, VEGF) in rats with placental ischemia induced hypertension. Since the placenta is thought to be the primary source of sFlt‐1 and sEng, we hypothesized Prav would have pro‐angiogenic effects in human umbilical vein endothelial cells (HUVEC) but anti‐angiogenic effects in trophoblast cells. Cells were cultured in atmospheric O2 (19%), physiologic normoxic (8%) and hypoxic (1.5%) conditions and treated with Prav (0, 10, 20 μmol/L) for 12 hours. Data analysis was by two‐way ANOVA and significance accepted when P&lt;0.05. Hypoxia stimulated (P&lt;0.05) VEGF and sEng but not sFlt‐1 concentrations in the media of BeWo trophoblast cells. An interaction between O2 and Prav was observed such that sEng decreased (P&lt;0.05) with increasing doses of Prav. In Jar cells, hypoxia did not affect VEGF or sFlt‐1 but decreased sEng secretion into culture media. Prav increased (P&lt;0.05) sEng secretion from both normoxic and hypoxic Jar cells in a dose dependent manner. Hypoxia and Prav had no effect on VEGF, sFlt‐1 or sEng in HUVECs. These data indicate Prav has cell‐type, O2 concentration and dose‐dependent effects on VEGF and sEng secretion in placental and endothelial cells that may not be beneficial in preeclampsia.

TNF‐a induced eNOS uncoupling mediates endothelial dysfunction through elevated reactive oxygen species

Oxidative stress is a known feature of calcific aortic valve disease (CAVD), but upstream activators of reactive oxygen species (ROS) are not well understood. We examined the role of inflammation in stimulating aortic valve endothelial cell (VEC) secretion of ROS. Using 3D culture, we determined that inflammatory cytokine TNF- α significantly increases VEC superoxide (223+/− 4% of control) and hydrogen peroxide (165 +/− 2% of control) within 5 minutes, blocked by the NOS inhibitor L-NAME, implicating eNOS uncoupling in ROS generation. Increased H2O2 production has long-term (48 hr) effects on VEC, causing increased ICAM-1, VCAM-1, and MMP-9, and decreased VE-cadherin and nitric oxide compared to untreated VEC. Similarly, we find that endothelial cells in calcified human aortic valves have elevated levels of superoxide, both near and distal to the primary nodules. Thus, the inflammatory phase of valve disease increases VEC oxidative stress, with long-term consequences including endothelial dysfunction. These results offer new paths for clinical intervention in early CAVD, such as using BH4 to promote eNOS coupling and reduce the downstream effects of inflammatory ROS. Funding: NIH 1R01HL110328–01