Dihydroartemisinin inhibits the expression of von Willebrand factor by downregulation of transcription factor ERG in endothelial cells.

Dihydroartemisinin (DHA), a semi-synthetic derivative of artemisinin, has effective antitumor and anti-inflammatory actions. von Willebrand factor (vWF), a large multifunctional glycoprotein, has a prominent function in hemostasis and is a key factor in thrombus formation. In addition, vWF has been regarded as a prospective biomarker for the diagnosis of endothelial dysfunction. In our experiment, we observed that 25 µM DHA specifically downregulated the expression of vWF mRNA and protein in human umbilical vein endothelial cells (HUVECs). Further investigations demonstrated that this DHA-decreased vWF expression was mediated by the transcription factor ERG and not GATA3. Luciferase activity assay confirmed that DHA regulated the ERG binding with the -56 ETS-binding motif on the human vWF promoter. Thus, the -56 ETS motif on the vWF promoter region regulates the expression of vWF gene which is induced by DHA. Taken together, we proved that DHA decreased the vWF transcription through the downregulation of ERG in HUVECs. As vWF plays a key role in vascular homeostasis, our findings suggest a new role of DHA in vascular diseases.

Dihydroartemisinin inhibits endothelial cell tube formation by suppression of the STAT3 signaling pathway.

AIMS: Endothelial cell (EC) tube formation is crucial for tumor angiogenesis, which becomes a target for chemotherapy. The anti-malaria agent dihydroartemisinin (DHA) inhibited tumor growth and angiogenesis. The aim of this study was to investigate the effects of DHA on EC tube formation and the underlying mechanisms. MATERIALS AND METHODS: Human umbilical vein endothelial cells (HUVECs) were cultured with different concentrations of DHA, and the tube formation was measured by in vitro angiogenesis assay. The protein levels of signal transducer and activator of transcription factor 3 (STAT3), phosphorylated STAT3 and fatty acid synthase (FASN) were detected by Western blotting. The gene expression of FASN was determined by real time-polymerase chain reaction (RT-PCR). The FASN siRNA and STAT3 (Y705D) vector were introduced into HUVECs by lipofectin transfection. KEY FINDINGS: DHA treatment inhibited tube formation, and the phosphorylation of STAT3 on Y705 of HUVECs. The expression of FASN was down-regulated by DHA and STAT3 inhibitor. The inhibitory effect of DHA on FASN expression in HUVECs was eliminated by co-treatment with the STAT3 inhibitor. Over-expression of STAT3 (Y705D) relieved the inhibitory effect of DHA on tube-formation and FASN expression. Under hypoxia condition, expression of FASN was up-regulated but inhibited by DHA treatment in HUVECs through suppression of STAT3 phosphorylation. SIGNIFICANCE: We demonstrate that DHA inhibits the protein level of FASN via attenuation of the Y705 phosphorylation of STAT3, and subsequently inhibits tube formation of HUVECs. Our results support the therapeutic potential of DHA on angiogenesis.

Endoglin-targeted contrast-enhanced ultrasound imaging in hepatoblastoma xenografts.

Angiogenesis is required for the growth of hepatoblastoma (HB). In the present study, an ultrasonic contrast agent, microbubbles (MB), was combined with an endoglin antibody, and then injected into nude mice with HB. This was conducted to detect specific binding to microvessels via non-linear harmonic imaging for tumor angiogenesis assessment. In addition, endoglin expression in experimental animals was measured using western blotting, reverse transcription-quantitative polymerase chain reaction and immunohistochemistry. In vitro, human umbilical vein endothelial cells (HUVECs) were co-cultured with conditioned media collected from HepG2 cells. Western blotting and reverse transcription-quantitative PCR was performed to detect the changes of endoglin expression. In targeted ultrasound imaging, it was determined that the differential targeted enhancement of MBendoglin was significantly higher than that of MBisotype. Over expression of endoglin was identified in the tumor of experimental nude mice; however, it was not present in the liver of the mice. Endoglin expression in HUVECs was significantly increased by co-culture with the conditioned media of HepG2 cells; therefore, the results suggest that endoglin is upregulated in angiogenic vessels in the HepG2 cell xenografts in nude mice. Thus, endoglin-targeted ultrasound imaging is presented as a potential approach for the diagnosis of liver carcinoma.

CD109 and squamous cell carcinoma.

Squamous cell carcinoma (SCC) is well-known for its high rate of metastasis with poor prognosis. CD109 is a glycosylphosphatidylinositol-anchored cell-surface glycoprotein. Recently, CD109 emerges as a potential biomarker and a therapeutic target for SCCs. Accumulating studies have reported that CD109 is highly expressed in human SCCs of multiple organs, and may contribute to the progression of SCCs. In this review, we summarized the findings on expression pattern of CD109 in SCCs, and discussed the molecular mechanisms underlying the roles of CD109 in pathogenesis of SCCs.

Low dose cadmium upregulates the expression of von Willebrand factor in endothelial cells.

Cadmium (Cd) is a persistent and widespread environmental pollutant of continuing worldwide concern. Previous studies have suggested that Cd exposure increases the risk of cardiovascular diseases, such as atherosclerosis and hypertension. However, the underlying mechanisms are poorly understood. In this study, we observed that low dose Cd treatment induced von Willebrand factor (vWF) expression in vascular endothelial cells in mouse lung and kidney tissues. In vitro analysis showed that 1 µM Cd specifically upregulated vWF mRNA and protein expression in human umbilical vein endothelial cells (HUVECs), indicating that Cd targets vascular endothelial cells even at relatively low concentrations. Further study demonstrated that nuclear factor kappa B (NF-κB) and GATA3, two established transcription regulators of the vWF gene, were not altered in the presence of Cd. However, ETS-related gene (ERG) was significantly induced by 1 µM Cd. When ERG was knocked down by siRNA, Cd induced upregulation of vWF was totally blocked. Chromatin immunoprecipitation (ChIP) assay showed that Cd increases the binding of ERG on the -56 ETS motif on the human vWF promoter. These results indicated that ERG mediated the increased expression of vWF by Cd. Since vWF is a key regulator for vascular homeostasis, our findings may provide a novel mechanism for understanding low dose Cd induced development of vascular diseases.

Dihydroartemisinin up-regulates VE-cadherin expression in human renal glomerular endothelial cells.

The antimalarial agent dihydroartemisinin (DHA) has been shown to be anti-inflammatory. In this study, we found that DHA increased the expression of the junctional protein vascular endothelial (VE)-cadherin in human renal glomerular endothelial cells. In addition, DHA inhibited TGF-ß RI-Smad2/3 signalling and its downstream effectors SNAIL and SLUG, which repress VE-cadherin gene transcription. Correspondingly, DHA decreased the binding of SNAIL and SLUG to the VE-cadherin promoter. Together, our results suggest an effect of DHA in regulating glomerular permeability by elevation of VE-cadherin expression.

CD109 expression is upregulated in penile squamous cell carcinoma.

Cluster of differentiation (CD) 109 is a glycosylphosphatidylinositol-anchored cell surface glycoprotein and a co-receptor for transforming growth factor ß. The expression of CD109 has been detected in squamous cell carcinoma of the lung, esophagus, skin and gallbladder. The aim of the present study was to evaluate CD109 expression in penile squamous cell carcinoma (PSCC). CD109 expression in PSCC tumor and adjacent tissues from 45 specimens in tissue microarrays was examined by immunohistochemical analyses. In addition, 3 fresh surgical samples of PSCC were collected and examined for their CD109 mRNA and protein levels by reverse transcription-quantitative polymerase chain reaction, western blotting and immunofluorescence staining. CD109 transcription and expression were significantly higher in the PSCC tissues compared with adjacent normal penile tissues, and its expression was restricted to squamous cells. However, CD109 expression level was not associated with the PSCC differentiation grade. These results suggest that CD109 may be associated with the pathogenesis of PSCC, and may therefore be a potential therapeutic target.

Erratum: Dihydroartemisinin induces endothelial cell anoikis through the activation of the JNK signaling pathway.

[This corrects the article DOI: 10.3892/ol.2016.4870.].

GATA3-induced vWF upregulation in the lung adenocarcinoma vasculature.

Lung adenocarcinoma (LAC) is the leading cause of cancer-related death worldwide. Aberrant expression of genes expressed preferentially in the lung tumor vasculature may yield clues for prognosis and treatment. Von Willebrand factor (vWF) is a large multifunctional glycoprotein with a well-known function in hemostasis. However, vWF has been reported to exert an anti-tumor effect, independent of its role in hemostasis. We investigated the expression of vWF in LAC through immunohistochemical staining of tumor tissue microarrays (TMAs). We found that vWF was overexpressed preferentially in the tumor vasculature of LAC compared with the adjacent tissue vasculature. Consistently, elevated vWF expression was found in endothelial cells (ECs) of fresh human LAC tissues and transplanted mouse LAC tissues. To understand the mechanism underlying vWF up-regulation in LAC vessels, we established a co-culture system. In this system, conditioned media (CM) collected from A549 cells increased vWF expression in human umbilical vein endothelial cells (HUVECs), suggesting enhanced expression is regulated by the LAC secretome. Subsequent studies revealed that the transcription factor GATA3, but not ERG, a known regulator of vWF transcription in vascular cells, mediated the vWF elevation. Chromatin immunoprecipitation (ChIP) assays validated that GATA3 binds directly to the +220 GATA binding motif on the human vWF promoter and A549 conditioned media significantly increases the binding of GATA3. Taken together, we demonstrate that vWF expression in ECs of LAC is elevated by the cancer cell-derived secretome through enhanced GATA3-mediated transcription.

Dihydroartemisinin induces endothelial cell anoikis through the activation of the JNK signaling pathway.

Angiogenesis is required for the growth and metastasis of solid tumors. The anti-malarial agent dihydroartemisinin (DHA) demonstrates potent anti-angiogenic activity, but the underlying molecular mechanisms are not yet fully understood. During the process of angiogenesis, endothelial cells migrating from existing capillaries may undergo programmed cell death after detaching from the extracellular matrix, a process that is defined as anchorage-dependent apoptosis or anoikis. In the present study, DHA-induced cell death was compared in human umbilical vein endothelial cells (HUVECs) cultured in suspension and attached to culture plates. In suspended HUVECs, the cell viability was decreased and apoptosis was increased with the treatment of 50 µM DHA for 5 h, while the same treatment did not affect the attached HUVECs. In addition, 50 µM DHA increased the phosphorylation of c-Jun N-terminal kinase (JNK) in suspended HUVECs, but not in attached HUVECs, for up to 5 h of treatment. The JNK inhibitor, SP600125, reversed DHA-induced cell death in suspended HUVECs, suggesting that the JNK pathway may mediate DHA-induced endothelial cell anoikis. The data from the present study indicates a novel mechanism for understanding the anti-angiogenic effects of DHA, which may be used as a component for chemotherapy.

NF-κB signaling maintains the survival of cadmium-exposed human renal glomerular endothelial cells.

The kidney is one of the primary organs targeted by cadmium (Cd), a widely distributed environmental pollutant. The glomerular endothelium is the major component of the glomerular filtration barrier. However, the effects of Cd on glomerular endothelial cells remain largely unknown. For this purpose, we aimed to determine the effects of low dose Cd on the survival of human renal glomerular endothelial cells (HRGECs). Cultured HRGECs were exposed to 4 µM cadmium chloride (CdCl2) and examined at different time-points. We found that Cd activates the nuclear factor-κB (NF-κB) pathway without inducing the apoptosis of HRGECs. Pre-treating the cells with pyrrolidine dithiocarbamate (PDTC), a potent NF-κB inhibitor, prior to Cd exposure triggered extensive cell death (73.5%). In addition, Cd activates the c-Jun N-terminal kinase (JNK) pathway, and inhibition of the NF-κB pathway significantly elevates Cd-induced JNK phosphorylation in HRGECs (p<0.01). The combination treatment of PDTC and SP600125, a JNK pathway inhibitor, increased the survival of Cd-stimulated HRGECs compared with those cells treated with PDTC alone (p<0.05). Taken together, these findings demonstrate that the NF-κB pathway plays an essential role in maintaining the survival of Cd-exposed HRGECs.

Short-term, low-dose cadmium exposure induces hyperpermeability in human renal glomerular endothelial cells.

The kidney is the principal organ targeted by exposure to cadmium (Cd), a well-known toxic metal. Even at a low level, Cd damages glomerular filtration. However, little is known about the effects of Cd on the glomerular endothelium, which performs the filtration function and directly interacts with Cd in blood plasma. In this study, we cultured human renal glomerular endothelial cells (HRGECs) in the presence of serum with treatment of a short term (1 h) and low concentration (1 µm) of Cd, which mimics the pattern of glomerular endothelium exposure to Cd in vivo. We found that this short-term, low-dose Cd exposure does not induce cytotoxicity, but increases permeability in HRGECs monolayers and redistributes adherens junction proteins vascular endothelial-cadherin and ß-catenin. Though short-term, low-dose Cd exposure activates all three major mitogen activated protein kinases, only the inhibitor of p38 mitogen activated protein kinase partially prevents Cd-induced hyperpermeability in HRGECs. Our data indicate that the presence of Cd in blood circulation might directly disrupt the glomerular endothelial cell barrier and contribute to the development of clinical symptoms of glomerular diseases.

Catalpol downregulates vascular endothelial­cadherin expression and induces vascular hyperpermeability.

Catalpol, an iridiod glucoside isolated from Rehmannia glutinosa, has been reported to possess anti­inflammatory properties. However, the molecular mechanisms underlying this effect have not been fully elucidated. This study aimed to investigate the effects of catalpol on vascular permeability. Using Transwell permeability assays and measurements of trans­endothelial electrical resistance (TEER), it was demonstrated that 1 mM catalpol induces a significant increase in the permeability of the monolayers of human umbilical vein endothelial cells (HUVECs). Western blotting and immunofluorescence demonstrated that catalpol inhibits the expression of vascular endothelial (VE)­cadherin, the key component of adherens junctions, but not occludin, the major constituent of tight junctions. In addition, catalpol inhibits the ETS transcription factor ERG, a positive regulator of VE­cadherin. Knockdown of ERG expression compromised the catalpol­induced reduction of TEER in HUVECs. The present study revealed a novel effect of catalpol on vascular permeability and gave insight into the multifaceted roles of catalpol in inflammation.

Dihydroartemisinin transiently activates the JNK/SAPK signaling pathway in endothelial cells.

Artemisinin and its derivatives are well-known anti-malaria drugs and in the early stages of research for cancer treatment. Dihydroartemisinin (DHA), a more water-soluble derivative of artemisinin, has demonstrated strong anti-angiogenic activity. The purpose of the present study was to investigate the underlying molecular mechanisms of the effect of DHA on angiogenesis. Human umbilical vein endothelial cells (HUVECs) treated with DHA were examined for apoptosis and activation of the c-Jun N-terminal kinase (JNK) signaling pathway, one of the major mitogen-activated protein kinase cascades. It was observed that 20 µM DHA induces transient activation of JNK in HUVECs. DHA also elevates the expression of cyclooxygenase-2 and matrix metalloproteinase-13, which is abolished by treatment with the JNK inhibitor SP600125. Although DHA persistently increases inhibitor of κB-α protein and thus inhibits nuclear factor-κB signaling, it does not affect apoptosis or caspase 3/9 activities in HUVECs. The present study provides key information for understanding the effects of DHA on endothelial cells, which is required for investigating its potential for clinic application as a chemotherapeutic agent.

Plasma von Willebrand factor level is transiently elevated in a rat model of acute myocardial infarction.

The von Willebrand factor (vWF) is a plasma glycoprotein that plays an essential role in hemostasis by supporting platelet adhesion and thrombus formation in response to vascular injury. Plasma levels of vWF are an independent risk factor for patients with acute myocardial infarction (AMI); however, clinical data have demonstrated a marked variation of vWF levels in patients with AMI, the reason for which has not yet been identified. In the present study, a rat model of ST-segment elevation AMI was established, and cardiac and peripheral blood was collected for a time-course examination of the plasma levels of vWF and tumor necrosis factor-α (TNF-α). The level of vWF in the blood plasma increased, peaked at 1 h and decreased to normal levels by day 7 following AMI, while the level of TNF-α peaked at 24 h and remained elevated until day 7. The effects of TNF-α on vWF secretion and expression were examined in cultured human umbilical vascular endothelial cells (HUVECs). TNF-α treatment increased vWF secretion from the HUVECs but inhibited the mRNA and protein expression of vWF in the HUVECs. These results indicate that vWF secretion from endothelial cells is transiently elevated following AMI, and then decreases as the expression of vWF is inhibited by TNF-α. The present study increases the understanding of the pathophysiology of vWF and indicates that the determination of vWF levels may be useful in the clinical evaluation of AMI.

Low-Dose Cadmium Upregulates VEGF Expression in Lung Adenocarcinoma Cells.

Cadmium (Cd) is a heavy metal and environmental toxin. Exposure to Cd has been associated with a variety of human cancers. In this study, we performed in vitro assays to examine the effects of cadmium chloride (CdCl2) on A549 cells, a human lung adenocarcinoma cell line. Cd does not affect proliferation, migration, or apoptosis of A549 cells at concentrations of 0.1-10 µM. At 0.5 and 1 µM, Cd increases the expression of vascular endothelial growth factor (VEGF) (p < 0.05, p < 0.01, respectively), but not basic fibroblast growth factor (b-FGF) in A549 cells. The conditioned media were collected from the A549 cells treated with 1 µM Cd and were co-cultured with human umbilical vein endothelial cells (HUVECs). Upon treatment with the conditioned media, the proliferation and migration of HUVECs significantly increased (p < 0.01, p < 0.05, respectively), while apoptosis remained unchanged. In addition, 1 µM Cd increases the level of hypoxia inducible factor 1-α (HIF1-α), which is a positive regulator of VEGF expression. Although low-dose Cd does not directly affect the growth of lung adenocarcinoma cells, it might facilitate the development of tumors through its pro-angiogenic effects.

CD109 is a novel marker for squamous cell/adenosquamous carcinomas of the gallbladder.

Gallbladder cancer is the most common biliary tract malignancy with the worst overall prognosis. CD109 is a co-receptor of TGF-ß1 and suppresses TGF-ß signaling. In this study, CD109 protein expression in three subtypes of gallbladder cancer was examined by immunohistochemistry on human tissue samples and tissue microarrays. We found that CD109 is specifically expressed in malignant squamous cells in squamous cell carcinomas (86.7%) and adenosquamous carcinomas (91.7%), but not in adenocarcinomas or normal gallbladder tissues. Thus, CD109 may be a potential pathology marker for gallbladder squamous cell/adenosquamous carcinomas.

Trichostatin A suppresses lung adenocarcinoma development in Grg1 overexpressing transgenic mice.

Trichostatin A (TSA) is a histone deacetylase inhibitor and a potential therapeutic for various malignancies. The in vivo effect of TSA, however, has not been investigated in a transgenic lung cancer model. Previously, we generated transgenic mice with overexpression of Groucho-related-gene 1 (Grg1) and these mice all developed mucinous lung adenocarcinoma. Grg1 is a transcriptional co-repressor protein, the function of which is thought to depend on HDAC activity. However, functions outside the nucleus have also been proposed. We tested the supposition that Grg1-induced tumorigenesis is HDAC-dependent by assaying the therapeutic effect of TSA in the Grg1 transgenic mouse model. We found that TSA significantly inhibited lung tumorigenesis in Grg1 transgenic mice (p < 0.01). TSA did not affect overall Grg1 protein levels, but instead reduced ErbB1 and ErbB2 expression, which are upregulated by Grg1 in the absence of TSA. We confirmed this effect in A549 cells. Furthermore, lapatinib, an inhibitor of both ErbB1 and ErbB2, effectively masked the effect of TSA on the inhibition of A549 cell proliferation and migration, suggesting TSA does work, at least in part, by downregulating ErbB receptors. We additionally found that TSA reduced the expression of VEGF and VEGFR2, but not basic FGF and FGFR1. Our findings indicate that TSA effectively inhibits Grg1-induced lung tumorigenesis through the down-regulation of ErbB1 and ErbB2, as well as reduced VEGF signaling. This suggests TSA and other HDAC inhibitors could have therapeutic value in the treatment of lung cancers with Grg1 overexpression.