HMVEC cells treated with vascular endothelial growth factor, anthrax edema toxin, and an Epac activator
ABSTRACT: Human microvascular endothelial cells (HMVEC) treated with vascular endothelial growth factor (VEGF), Antrhax Edema Toxin (ET), or the Epac activator, 8-pCPT-2'-O-Me-cAMP (8CPT) Human microvascular endothelial cells (HMVEC) were treated with VEGF alone or VEGF in combination with either the the Epac-specific cAMP-mimetic, 8-pCPT-2'-O-Me-cAMP (8CPT), or anthrax edema toxin (ET), an adenylyl cyclase. ET or 8CPT can inhibit VEGF-mediated chemotaxis and angiogenesis. The goal of the study was to identify genes regulated by cAMP production (ET) or by activation of Epac/Rap (8CPT) that may mitigate the effects of VEGF treatment. Overall design: Gene expression was measured 4 hours after treatment with VEGF, VEGF + 8CPT, VEGF + ET or mock treatment. Each sample contained one replicate.
INSTRUMENT(S): [HG-U133_Plus_2] Affymetrix Human Genome U133 Plus 2.0 Array
Project description:Human microvascular endothelial cells (HMVEC) treated with vascular endothelial growth factor (VEGF), Antrhax Edema Toxin (ET), or the Epac activator, 8-pCPT-2'-O-Me-cAMP (8CPT); Human microvascular endothelial cells (HMVEC) were treated with VEGF alone or VEGF in combination with either the the Epac-specific cAMP-mimetic, 8-pCPT-2'-O-Me-cAMP (8CPT), or anthrax edema toxin (ET), an adenylyl cyclase. ET or 8CPT can inhibit VEGF-mediated chemotaxis and angiogenesis. The goal of the study was to identify genes regulated by cAMP production (ET) or by activation of Epac/Rap (8CPT) that may mitigate the effects of VEGF treatment. Experiment Overall Design: Gene expression was measured 4 hours after treatment with VEGF, VEGF + 8CPT, VEGF + ET or mock treatment. Each sample contained one replicate.
Project description:A common finding amongst patients with inhalational anthrax is a paucity of polymorphonuclear leukocytes (PMNs) in infected tissues in the face of abundant circulating PMNs. A major virulence determinant of anthrax is edema toxin (ET), which is formed by the combination of two proteins produced by the organism, edema factor (EF), which is an adenyl cyclase, and protective antigen (PA). Since cAMP, a product of adenyl cyclase, is known to enhance endothelial barrier integrity, we asked whether ET might decrease extravasation of PMNs into tissues through closure of the paracellular pathway through which PMNs traverse.Pretreatment of human microvascular endothelial cell(EC)s of the lung (HMVEC-L) with ET decreased interleukin (IL)-8-driven transendothelial migration (TEM) of PMNs with a maximal reduction of nearly 60%. This effect required the presence of both EF and PA. Conversely, ET did not diminish PMN chemotaxis in an EC-free system. Pretreatment of subconfluent HMVEC-Ls decreased transendothelial 14 C-albumin flux by ~ 50% compared to medium controls. Coadministration of ET with either tumor necrosis factor-? or bacterial lipopolysaccharide, each at 100 ng/mL, attenuated the increase of transendothelial 14 C-albumin flux caused by either agent alone. The inhibitory effect of ET on TEM paralleled increases in protein kinase A (PKA) activity, but could not be blocked by inhibition of PKA with either H-89 or KT-5720. Finally, we were unable to replicate the ET effect with either forskolin or 3-isobutyl-1-methylxanthine, two agents known to increase cAMP.We conclude that ET decreases IL-8-driven TEM of PMNs across HMVEC-L monolayers independent of cAMP/PKA activity.
Project description:Endothelin-1 (ET-1) is increased in patients with high-altitude pulmonary edema and acute respiratory distress syndrome, and these patients have decreased alveolar fluid reabsorption (AFR).To determine whether ET-1 impairs AFR via activation of endothelial cells and nitric oxide (NO) generation.Isolated perfused rat lung, transgenic rats deficient in ETB receptors, coincubation of lung human microvascular endothelial cells (HMVEC-L) with rat alveolar epithelial type II cells or A549 cells, ouabain-sensitive 86Rb+ uptake.The ET-1-induced decrease in AFR was prevented by blocking the endothelin receptor ETB, but not ETA. Endothelial-epithelial cell interaction is required, as direct exposure of alveolar epithelial cells (AECs) to ET-1 did not affect Na,K-ATPase function or protein abundance at the plasma membrane, whereas coincubation of HMVEC-L and AECs with ET-1 decreased Na,K-ATPase activity and protein abundance at the plasma membrane. Exposing transgenic rats deficient in ETB receptors in the pulmonary vasculature (ET-B(-/-)) to ET-1 did not decrease AFR or Na,K-ATPase protein abundance at the plasma membrane of AECs. Exposing HMVEC-L to ET-1 led to increased NO, and the ET-1-induced down-regulation of Na,K-ATPase was prevented by the NO synthase inhibitor l-NAME, but not by a guanylate cyclase inhibitor.We provide the first evidence that ET-1, via an endothelial-epithelial interaction, leads to decreased AFR by a mechanism involving activation of endothelial ETB receptors and NO generation leading to alveolar epithelial Na,K-ATPase down-regulation in a cGMP-independent manner.
Project description:Bacillus anthracis, the causative agent of anthrax, secretes three toxin proteins: protective antigen (PA), lethal factor (LF), and edema factor (EF). PA is a transporter of LF and EF into host cells by receptor-mediated endocytosis. LF is a metalloprotease that cleaves mitogen-activated protein kinase (MAPK) kinases (MKK), while EF is an adenylate cyclase, which converts ATP to cAMP. We used microarrays to decipher the specific gene regulation in edema toxin (ET), the complex of EF and PA, treated mouse bone marrow derived macrophages. Keywords: Time course Overall design: BMDM were treated with 1 mg/ml of ET and the RNAs were purified at 0, 2, and 4h after toxin treatment.
Project description:Bacillus anthracis, the causative agent of anthrax, secretes three toxin proteins: protective antigen (PA), lethal factor (LF), and edema factor (EF). PA is a transporter of LF and EF into host cells by receptor-mediated endocytosis. LF is a metalloprotease that cleaves mitogen-activated protein kinase (MAPK) kinases (MKK), while EF is an adenylate cyclase, which converts ATP to cAMP. We used microarrays to decipher the specific gene regulation in edema toxin (ET), the complex of EF and PA, treated mouse bone marrow derived macrophages. Experiment Overall Design: BMDM were treated with 1 mg/ml of ET and the RNAs were purified at 0, 2, and 4h after toxin treatment.
Project description:Anthrax is a highly lethal disease caused by the Gram-(+) bacteria Bacillus anthracis. Edema toxin (ET) is a major contributor to the pathogenesis of disease in humans exposed to B. anthracis. ET is a bipartite toxin composed of two proteins secreted by the vegetative bacteria, edema factor (EF) and protective antigen (PA). Our work towards identifying a small molecule inhibitor of anthrax edema factor is the subject of this letter. First we demonstrate that the small molecule probe 5'-Fluorosulfonylbenzoyl 5'-adenosine (FSBA) reacts irreversibly with EF and blocks enzymatic activity. We then show that the adenosine portion of FSBA can be replaced to provide more drug-like molecules which are up to 1000-fold more potent against EF relative to FSBA, display low cross reactivity when tested against a panel of kinases, and are nanomolar inhibitors of EF in a cell-based assay of cAMP production.
Project description:Vascular endothelial growth factor (VEGF) has previously been demonstrated to accelerate compensatory lung growth (CLG) in mice and may be a useful therapy for pulmonary hypoplasia. Systemic administration of VEGF can result in side effects such as hypotension and edema. The aim of this study was to explore nasal delivery as a route for intrapulmonary VEGF administration. Eight-week-old C57BL/6 male mice underwent left pneumonectomy, followed by daily nasal instillation of VEGF at 0.5 mg/kg or isovolumetric saline. Lung volume measurement, morphometric analysis, and protein expression studies were performed on lung tissues harvested on postoperative day (POD) 4. To understand the mechanism by which VEGF accelerates lung growth, proliferation of human bronchial epithelial cells (HBEC) was assessed in a co-culture model with lung microvascular endothelial cells (HMVEC-L) treated with and without VEGF (10 ng/mL). The assay was then repeated with a heparin-binding EGF-like growth factor (HB-EGF) neutralizing antibody ranging from 0.5-50 ?g/mL. Compared to control mice, the VEGF-treated group displayed significantly higher lung volume (P = 0.001) and alveolar count (P = 0.005) on POD 4. VEGF treatment resulted in increased pulmonary expression of HB-EGF (P = 0.02). VEGF-treated HMVEC-L increased HBEC proliferation (P = 0.002) while the addition of an HB-EGF neutralizing antibody at 5 and 50 ?g/mL abolished this effect (P = 0.01 and 0.002, respectively). These findings demonstrate that nasal delivery of VEGF enhanced CLG. These effects could be mediated by a paracrine mechanism through upregulation of HB-EGF, an epithelial cell mitogen.
Project description:The objectives of this study were to determine the effects of deoxyshikonin on lymphangiogenesis. Deoxyshikonin enhanced the ability of human dermal lymphatic microvascular endothelial cells (HMVEC-dLy) to undergo time-dependent in vitro cord formation. Interestingly, an opposite result was observed in cells treated with shikonin. The increased cord formation ability following deoxyshikonin treatment correlated with increased VEGF-C mRNA expression to higher levels than seen for VEGF-A and VEGF-D mRNA expression. We also found that deoxyshikonin regulated cord formation of HMVEC-dLy by increasing the HIF-1 ? mRNA level, HIF-1 ? protein level, and the accumulation of HIF-1 ? in the nucleus. Knockdown of the HIF-1 ? gene by transfection with siHIF-1 ? decreased VEGF-C mRNA expression and cord formation ability in HMVEC-dLy. Deoxyshikonin treatment could not recover VEGF-C mRNA expression and cord formation ability in HIF-1 ? knockdown cells. This indicated that deoxyshikonin induction of VEGF-C mRNA expression and cord formation in HMVEC-dLy on Matrigel occurred mainly via HIF-1 ? regulation. We also found that deoxyshikonin promoted wound healing in vitro by the induction of HMVEC-dLy migration into the wound gap. This study describes a new effect of deoxyshikonin, namely, the promotion of cord formation by human endothelial cells via the regulation of HIF-1 ? . The findings suggest that deoxyshikonin may be a new drug candidate for wound healing and treatment of lymphatic diseases.
Project description:Increased retinal vascular permeability contributes to macular edema, a leading cause of vision loss in eye pathologies such as diabetic retinopathy, age-related macular degeneration, and central retinal vein occlusions. Pathological changes in vascular permeability are driven by growth factors such as VEGF and pro-inflammatory cytokines such as TNF-?. Identifying the pro-barrier mechanisms that block vascular permeability and restore the blood-retinal barrier (BRB) may lead to new therapies. The cAMP-dependent guanine nucleotide exchange factor (EPAC) exchange-protein directly activated by cAMP promotes exchange of GTP in the small GTPase Rap1. Rap1 enhances barrier properties in human umbilical endothelial cells by promoting adherens junction assembly. We hypothesized that the EPAC-Rap1 signaling pathway may regulate the tight junction complex of the BRB and may restore barrier properties after cytokine-induced permeability. Here, we show that stimulating EPAC or Rap1 activation can prevent or reverse VEGF- or TNF-?-induced permeability in cell culture and in vivo Moreover, EPAC activation inhibited VEGF receptor (VEGFR) signaling through the Ras/MEK/ERK pathway. We also found that Rap1B knockdown or an EPAC antagonist increases endothelial permeability and that VEGF has no additive effect, suggesting a common pathway. Furthermore, GTP-bound Rap1 promoted tight junction assembly, and loss of Rap1B led to loss of junctional border organization. Collectively, our results indicate that the EPAC-Rap1 pathway helps maintain basal barrier properties in the retinal vascular endothelium and activation of the EPAC-Rap1 pathway may therefore represent a potential therapeutic strategy to restore the BRB.
Project description:We found that intrathecal injection of anthrax edema toxin (ET), causes analgesia in adult B6J mice. In order to determine if DRG neuron transcriptional responses play a role in pain blockade, we performed a bulk RNA-seq experiment on dissected mouse DRG neurons. We report that anthrax edema toxin causes transcriptional responses in DRG neurons 2h after in vivo administration Overall design: We injected 6 mice (intrathecally) with PBS (control) and 6 mice with ET. After waiting 2h and observing pain blockade in the ET injected animals, we dissected the lumber DRGs and proceeded to RNA-extraction