Vascular endothelial growth factor-C modulates proliferation and chemoresistance in acute myeloid leukemic cells through an endothelin-1-dependent induction of cyclooxygenase-2

High-level expression of vascular endothelial growth factor (VEGF)-C is associated with chemoresistance and adverse prognosis in acute myeloid leukemia (AML). Our previous study has found that VEGF-C induces cyclooxygenase-2 (COX-2) expression in AML cell lines and significant correlation of VEGF-C and COX-2 in bone marrow specimens. COX-2 has been reported to mediate the proliferation and drug resistance in several solid tumors. Herein, we demonstrated that the VEGF-C-induced proliferation of AML cells is effectively abolished by the depletion or inhibition of COX-2. The expression of endothelin-1 (ET-1) rapidly increased following treatment with VEGF-C. We found that ET-1 was also involved in the VEGF-C-mediated proliferation of AML cells, and that recombinant ET-1 induced COX-2 mRNA and protein expressions in AML cells. Treatment with the endothelin receptor A (ETRA) antagonist, BQ 123, or ET-1 shRNAs inhibited VEGF-C-induced COX-2 expression. Flow cytometry and immunoblotting revealed that VEGF-C induces S phase accumulation through the inhibition of p27 and the upregulation of cyclin E and cyclin-dependent kinase-2 expressions. The cell-cycle-related effects of VEGF-C were reversed by the depletion of COX-2 or ET-1. The depletion of COX-2 or ET-1 also suppressed VEGF-C-induced increases in the bcl-2/bax ratio and chemoresistance against etoposide and cytosine arabinoside in AML cells. We also demonstrated VEGF-C/ET-1/COX-2 axis-mediated chemoresistance in an AML xenograft mouse model. Our findings suggest that VEGF-C induces COX-2-mediated resistance to chemotherapy through the induction of ET-1 expression. Acting as a key regulator in the VEGF-C/COX-2 axis, ET-1 represents a potential target for ameliorating resistance to chemotherapy in AML patients.     

Human neutrophil peptides: a novel potential mediator of inflammatory cardiovascular diseases

The traditional view of atherosclerosis has recently been expanded from a predominantly lipid retentive disease to a coupling of inflammatory mechanisms and dyslipidemia. Studies have suggested a novel role for polymorphonuclear neutrophil (PMN)-dominant inflammation in the development of atherosclerosis. Human neutrophil peptides (HNPs), also known as alpha-defensins, are secreted and released from PMN granules upon activation and are conventionally involved in microbial killing. Current evidence suggests an important immunomodulative role for these peptides. HNP levels are markedly increased in inflammatory diseases including sepsis and acute coronary syndromes. They have been found within the intima of human atherosclerotic arteries, and their deposition in the skin correlates with the severity of coronary artery diseases. HNPs form complexes with LDL in solution and increase LDL binding to the endothelial surface. HNPs have also been shown to contribute to endothelial dysfunction, lipid metabolism disorder, and the inhibition of fibrinolysis. Given the emerging relationship between PMN-dominant inflammation and atherosclerosis, HNPs may serve as a link between them and as a biological marker and potential therapeutic target in cardiovascular diseases including coronary artery diseases and acute coronary syndromes.     

Omentin, a novel adipocytokine inhibits TNF-induced vascular inflammation in human endothelial cells

Omentin is a recently identified adipocytokine with insulin-sensitizing effect. While lack of omentin may be related to the pathogenesis of obesity-related cardiovascular diseases, its effect in vasculature is largely unknown. We examined effects of omentin on vascular endothelial inflammatory states. Western blotting was performed to analyze inflammatory signal transduction in cultured vascular endothelia cells. The cyclic guanosine monophosphate (cGMP) content was measured using enzyme immunoassay. Treatment of human umbilical vein endothelial cells with omentin (300 ng/ml, 20 min) induced phosphorylation of 5′-AMP-activated protein kinase (AMPK) (Thr 172) and endothelial nitric oxide (NO) synthase (eNOS) (Ser 1177). Consistently, omentin increased the cGMP level. Pretreatment with omentin (300 ng/ml, 30 min) significantly inhibited the phosphorylation of JNK as well as expression of cyclooxygenase (COX)-2 by TNF-α (5 ng/ml, 20 min–24 h). An inhibitor of JNK significantly inhibited the TNF-α-induced COX-2 expression. Inhibitory effect of omentin on TNF-α-induced COX-2 was reversed by a NOS inhibitor. The present results demonstrate for the first time that omentin plays an anti-inflammatory role by preventing the TNF-α-induced COX-2 expression in vascular endothelial cells. Omentin inhibits COX-2 induction via preventing the JNK activation presumably through activation of AMPK/eNOS/NO pathways.     

Cultured Astrocytes Release a Factor that Decreases Endothelin-1 Secretion by Brain Microvessel Endothelial Cells

Abstract: Endothelin-1 (ET-1), originally characterized as a potent vasoconstrictor peptide secreted by vascular endothelial cells, has now been described to possess a wide range of biological activities within the cardiovascular system and in other organs. Brain microvessel endothelial cells, which, together with perivascular astrocytes, constitute the blood-brain barrier, have been shown to secrete ET-1, whereas specific ET-1 receptors are expressed on astrocytes. It is reported here that conditioned medium from primary cultures of mouse embryo astrocytes could significantly, and reversibly, attenuate the accumulation of both ET-1 and its precursor big ET-1 in the supernatant of rat brain microvessel endothelial cells by up to 59 and 76%, respectively, as assessed by immunometric assay. This inhibitor of ET-1 production was purified by gel-exclusion and ion-exchange chromatography as a 280-Da iron-containing molecule, able to release nitrites upon degradation. These results suggest that astrocytes, via release of an iron-nitrogen oxide complex, may be involved in a regulatory loop of ET-1 production at the level of the blood-brain barrier.     

Endothelin-1 increases expression of cyclooxygenase-2 and production of interlukin-8 in hunan pulmonary epithelial cells

Inducible cyclooxygenase (COX-2) and inflammatory cytokines play important roles in inflammatory processes of chronic obstructive pulmonary disease (COPD). Endothelin-1 (ET-1) might be also involved in the pathophysilogical processes in COPD. In the present study, we determined whether ET-1 could regulate the expression of COX-2 and alter the production of interleukin-8 (IL-8) in human pulmonary epithelial cells (A549). Induced sputum samples were collected from 13 stable COPD patients and 14 healthy subjects. The COX-2 protein, ET-1, PGE(2) and IL-8 in these sputum samples were analyzed. A549 cells were incubated with ET-1 in the presence or absence of celecoxib, a selective COX-2 inhibitor. The expression of COX-2 protein in the cell and the amounts of PGE(2) and IL-8 in the medium were measured. The levels of COX-2 protein, ET-1, PGE(2) and IL-8 were significantly increased in induced sputum from COPD patients when compared to healthy subjects. ET-1 increased the expression of COX-2 protein, as well as the production of PGE(2) in A549 cells. Increased production of PGE(2) was inhibited by celecoxib. ET-1 also increased the production of IL-8. Interestingly, ET-1-induced production of IL-8 was also inhibited by celecoxib. These findings indicate that ET-1 plays important roles in regulating COX-2 expression and production of IL-8 in A549 cells. ET-1 mediated production of IL-8 is likely through a COX-2-dependent mechanism.     

Endothelin-1 and endothelin-converting enzyme-1 in human granulomatous pathology of eyelid: an immunohistochemical and in situ hybridization study in chalazia

Endothelin-1 (ET-1), a potent vasoconstrictor peptide, is involved in several functions of eye pathophysiology, such as regulation of intraocular tension and retinal reactive vasoconstriction. As ET-1 pro-inflammatory and fibrosing activity is emerging in different fields of pathology, we investigated the expression of ET-1 and endothelin-converting enzyme-1 (ECE-1) in chalazia, granulomatous lesions of the eyelid. ET-1 and ECE-1 were analyzed by immunohistochemistry (IHC) in twenty surgically removed chalazia, with regard to expression in eyelid structures and inflammatory infiltrate. Phenotype of ET-1 expressing inflammatory cells was established by double immunofluorescence. The cellular localization of prepro-ET-1 (pp-ET-1) mRNA and ECE-1 mRNA was studied by nonisotopic in situ hybridization (ISH). Neutrophils (PMNs), macrophages and T-lymphocytes were scattered in stroma, around alveoli and grouped in lipogranulomas. PMNs, macrophages, basal epithelium of meibomian adenomers and central ducts immunostained for ET-1. ECE-1 protein was found in meibomian adenomers, conjunctival epithelium, tarsal mucous glands and in inflammatory cells. Hybridization signals for pp-ET-1 mRNA and ECE-1 mRNA were recognized in healthy and degenerating meibomian ducts, adenomers, inflammatory cells, as well as in vessel walls. ECE-1 mRNA was also present in conjunctival epithelium and Henle's crypts. Our findings suggest that the multifunctional peptide ET-1 may have a role in molecular genesis of tissue damage in chalazia. ET-1 cytokine activity is likely to support the migration of inflammatory cells and the setting of lipogranulomas; ET-1 stimulation might contribute to proliferation of fibroblasts and collagen synthesis. ET-1 upregulation on meibomian adenomers and ducts may further enhance granulomas formation by stimulating lipid release.     

eNOS, COX-2, and prostacyclin production are impaired in endothelial cells from diabetics

The vascular endothelium is a well-recognized target of damage for factors leading to increased cardiovascular risk. Among the agents playing an important role in cardiovascular homeostasis, nitric oxide and prostacyclin represent key markers of endothelial integrity. In the present work, we report for the first time the reduced expression of both endothelial nitric oxide synthase and cyclooxygenase-2 (COX-2) proteins, as well as decreased prostacyclin production, in unstimulated human endothelial cells from insulin-dependent diabetic mothers when compared to cells from non-diabetic, control subjects. According to a major role of COX-2 as a source of prostacyclin production even in unstimulated endothelial cells, prostacyclin production was concentration-dependently inhibited by the selective COX-2 inhibitor SC236. Overall, our results suggest a possible link between reduced endothelial COX-2 and NO-synthase expression and the increased risk of cardiovascular diseases affecting diabetic patients, and point to the use of endothelial cells from diabetic patients as a tool for investigating early dysfunction in pathological endothelium.     

Enhanced secretion of endothelin-1 by elevated glucose levels from cultured bovine aortic endothelial cells

We have investigated the effect of glucose on the release of endothelin-1-like immunoreactivity (ET-1-LI) from cultured bovine aortic endothelial cells. Elevation of glucose concentrations in cultured media from 5.5 to 11.1 or 22.2 mM significantly stimulated ET-1-LI release from cultured endothelial cells. An aldose reductase inhibitor did not affect the high glucose-induced ET-1-LI release. These findings suggest the possibility that hyperglycemia in diabetic patients enhances ET-1-LI release at the local site of vascular endothelium, which might be involved in the developments of vascular complications and atherosclerosis.     

Pro-oxidant role of heme oxygenase in mediating glucose-induced endothelial cell damage

Oxidative damage to the vascular endothelial cells may play a crucial role in mediating glucose-induced cellular dysfunction in chronic diabetic complications. The present study was aimed at elucidating the role of glucose-induced alteration of highly inducible heme oxygenase (HO) in mediating oxidative stress in the vascular endothelial cells. We have also investigated the interaction between HO and the nitric oxide (NO) system, and its possible role in alteration of other vasoactive factors. Human umbilical vein endothelial cells (HUVECs) were exposed to low (5mmol/l) and high (25mmol/l) glucose levels. In order to determine the role of HO in endothelial dysfunction and to elucidate a possible interaction between the HO and NO systems, cells were exposed to HO inducer (hemin, 10 micromol/l), HO antagonist (SnPPIX, 10 micromol/l), and NO synthase blocker (L-NAME, 200 micromol/l) with or without NO donor (arginine, 1 mmol/l). mRNA expression of HO and NO isoforms was measured by real time RT-PCR. HO activity was measured by bilirubin production and cellular oxidative stress was assessed by 8-hydroxy-2'-deoxyguanosine (8-OHdG) and nitrotyrosine staining. We also determined the expression of vasoactive factors, endothelin-1 (ET-1) and vascular endothelial growth factor (VEGF). In the endothelial cells, glucose caused upregulation of HO-1 expression and increased HO activity. A co-stimulatory relationship between HO and NO was observed. Increased HO activity also associated with oxidative DNA and protein damage in the endothelial cells. Furthermore, increased HO activity augmented mRNA expression of vasoactive factors, ET-1 and VEGF. These data suggest that HO by itself and via elaboration of other vasoactive factors may cause endothelial injury and functional alteration. These findings are of importance in the context of chronic diabetic complications.     

Activation of vascular endothelial nitric oxide synthase and heme oxygenase-1 expression by electrophilic nitro-fatty acids

Reactive oxygen species mediate a decrease in nitric oxide (NO) bioavailability and endothelial dysfunction, with secondary oxidized and nitrated by-products of these reactions contributing to the pathogenesis of numerous vascular diseases. While oxidized lipids and lipoproteins exacerbate inflammatory reactions in the vasculature, in stark contrast the nitration of polyunsaturated fatty acids and complex lipids yields electrophilic products that exhibit pluripotent anti-inflammatory signaling capabilities acting via both cGMP-dependent and -independent mechanisms. Herein we report that nitro-oleic acid (OA-NO₂) treatment increases expression of endothelial nitric oxide synthase (eNOS) and heme oxygenase 1 (HO-1) in the vasculature, thus transducing vascular protective effects associated with enhanced NO production. Administration of OA-NO₂ via osmotic pump results in a significant increase in eNOS and HO-1 mRNA in mouse aortas. Moreover, HPLC-MS/MS analysis showed that NO₂-FAs are rapidly metabolized in cultured endothelial cells (ECs) and treatment with NO₂-FAs stimulated the phosphorylation of eNOS at Ser¹¹⁷⁹. These posttranslational modifications of eNOS, in concert with elevated eNOS gene expression, contributed to an increase in endothelial NO production. In aggregate, OA-NO₂-induced eNOS and HO-1 expression by vascular cells can induce beneficial effects on endothelial function and provide a new strategy for treating various vascular inflammatory and hypertensive disorders.     

Role of caveolin-1 in EGCG-mediated protection against linoleic-acid-induced endothelial cell activation

Flavonoids can protect against inflammatory diseases such as atherosclerosis by decreasing vascular endothelial cell activation. Plasma microdomains called caveolae may be critical in regulating endothelial activation. Caveolae are particularly abundant in endothelial cells and play a major role in endothelial trafficking and the regulation of signaling pathways associated with the pathology of vascular diseases. We hypothesize that flavonoids can down-regulate endothelial inflammatory parameters by modulating caveolae-regulated cell signaling. We focused on the role of caveolae and its major protein, caveolin-1, in mechanisms of linoleic-acid-induced endothelial cell activation and protection by the catechin epigallocatechin-3-gallate (EGCG). Exposure to linoleic acid for 6 h induced expression of both caveolin-1 and cyclooxygenase (COX)-2. Pretreatment with EGCG blocked fatty-acid-induced caveolin-1 and COX-2 expression in a time- and concentration-dependent manner. Similar results were observed with nuclear factor-kappa B DNA binding activity, which was also reduced by caveolin-1 silencing. Exposure to linoleic acid rapidly increased phosphorylation of several kinases, including p38 MAPK, extracellular signal regulated kinase 1/2 (ERK1/2) and amino kinase terminal (Akt), with maximal induction at about 10 min. Inhibitors of ERK1/2 and Akt down-regulated the linoleic-acid-induced increase in COX-2 protein, which also occurred after pretreatment with EGCG. Caveolin-1 silencing blocked linoleic-acid-induced phosphorylation of ERK1/2 and protein expression of COX-2, suggesting that specific MAPK signaling is caveolae dependent. Our data provide evidence that caveolae may play a critical role in regulating vascular endothelial cell activation and protection by flavonoids such as EGCG.     

Cyclooxygenase-2: a therapeutic target in angiogenesis

Angiogenesis has a role in the pathogenesis of several disorders, including cancer, chronic inflammatory diseases and retinopathies. Recent evidence demonstrates that the production of prostanoids by cyclooxygenase-2 (COX-2) promotes the expression of pro-angiogenic factors. Furthermore, inhibition of COX-2 by non-steroidal anti-inflammatory drugs leads to restricted angiogenesis and downregulated production of pro-angiogenic factors, such as vascular endothelial growth factor and basic fibroblast growth factor. These findings suggest that COX enzymes could be important therapeutic targets in the treatment of pathological angiogenesis.     

Pro-oxidant Role of Heme Oxygenase in Mediating Glucose-induced Endothelial Cell Damage

Oxidative damage to the vascular endothelial cells may play a crucial role in mediating glucose-induced cellular dysfunction in chronic diabetic complications. The present study was aimed at elucidating the role of glucose-induced alteration of highly inducible heme oxygenase (HO) in mediating oxidative stress in the vascular endothelial cells. We have also investigated the interaction between HO and the nitric oxide (NO) system, and its possible role in alteration of other vasoactive factors.

Human umbilical vein endothelial cells (HUVECs) were exposed to low (5?mmol/l) and high (25?mmol/l) glucose levels. In order to determine the role of HO in endothelial dysfunction and to elucidate a possible interaction between the HO and NO systems, cells were exposed to HO inducer (hemin, 10?μmol/l), HO antagonist (SnPPIX, 10?μmol/l), and NO synthase blocker (l-NAME, 200?μmol/l) with or without NO donor (arginine, 1?mmol/l). mRNA expression of HO and NO isoforms was measured by real time RT-PCR. HO activity was measured by bilirubin production and cellular oxidative stress was assessed by 8-hydroxy-2′-deoxyguanosine (8-OHdG) and nitrotyrosine staining. We also determined the expression of vasoactive factors, endothelin-1 (ET-1) and vascular endothelial growth factor (VEGF).

In the endothelial cells, glucose caused upregulation of HO-1 expression and increased HO activity. A co-stimulatory relationship between HO and NO was observed. Increased HO activity also associated with oxidative DNA and protein damage in the endothelial cells. Furthermore, increased HO activity augmented mRNA expression of vasoactive factors, ET-1 and VEGF. These data suggest that HO by itself and via elaboration of other vasoactive factors may cause endothelial injury and functional alteration. These findings are of importance in the context of chronic diabetic complications.     

Shear stress attenuates endothelin and endothelin-converting enzyme expression through oxidative stress

Shear stress is known to dilate blood vessels and exert an antiproliferative effect on vascular walls. These effects have partly been ascribed to shear stress-induced regulation of the secretion of endothelium-derived vasoactive substances. In this study, to elucidate the role of shear stress in endothelin production by endothelial cells, we examined the effect of physiological shear stress on the mRNA expression of endothelin-converting enzyme-1 (ECE-1) as well as endothelin-1 (ET-1) in cultured bovine carotid artery endothelial cells (BAECs) and human umbilical vein endothelial cells (HUVECs), using a parallel plate-type flow chamber. ECE-1 mRNA expression was significantly down-regulated by shear stress in an intensity- and time-dependent manner within the physiological range (1.5 to 15 dyn/cm(2)). ET-1 mRNA expression decreased together with ECE-1 mRNA expression. Shear stress at 15 dyn/cm(2) for 30 min induced a significant increase in the intracellular peroxide concentration, and the down-regulation of ECE-1 and ET-1 mRNA expression by shear stress was attenuated almost completely on treatment with N-acetyl cysteine (NAC), an antioxidant (20 mM). Furthermore, when H(2)O(2) (0.5 to 2 mM) was added to BAECs in static culture, the ECE-1 as well as ET-1 mRNA expression was attenuated in proportion to the concentration of H(2)O(2). It is suggested that endothelial cells sense shear stress as oxidative stress and transduce signal for the regulation of the gene expression of ECE as well as ET to attenuate vascular tone and inhibit the proliferation of vascular smooth muscle cells.     

Inhibition of cyclooxygenase (COX)-2 affects endothelial progenitor cell proliferation

Growing evidence indicates that inducible cyclooxygenase-2 (COX-2) is involved in the pathogenesis of inflammatory disorders and various types of cancer. Endothelial progenitor cells recruited from the bone marrow have been shown to be involved in the formation of new vessels in malignancies and discussed for being a key point in tumour progression and metastasis. However, until now, nothing is known about an interaction between COX and endothelial progenitor cells (EPC). Expression of COX-1 and COX-2 was detected by semiquantitative RT-PCR and Western blot. Proliferation kinetics, cell cycle distribution and rate of apoptosis were analysed by MTT test and FACS analysis. Further analyses revealed an implication of Akt phosphorylation and caspase-3 activation. Both COX-1 and COX-2 expression can be found in bone-marrow-derived endothelial progenitor cells in vitro. COX-2 inhibition leads to a significant reduction in proliferation of endothelial progenitor cells by an increase in apoptosis and cell cycle arrest. COX-2 inhibition leads further to an increased cleavage of caspase-3 protein and inversely to inhibition of Akt activation. Highly proliferating endothelial progenitor cells can be targeted by selective COX-2 inhibition in vitro. These results indicate that upcoming therapy strategies in cancer patients targeting COX-2 may be effective in inhibiting tumour vasculogenesis as well as angiogenic processes.     

An involvement of SR-B1 mediated PI3K-Akt-eNOS signaling in HDL-induced cyclooxygenase 2 expression and prostacyclin production in endothelial cells

It is well-known that sphingosine-1-phosphate (S1P), the phospholipid content of HDL, binding to S1P receptors can raise COX-2 expression and PGI(2) release through p38MAPK/CREB pathway. In the present study we assess the action of SR-B1 initiated PI3K-Akt-eNOS signaling in the regulation of COX-2 expression and PGI(2) production in response to HDL. We found that apoA1 could increase PGI(2) release and COX-2 expression in ECV 304 endothelial cells. Furthermore, SR-B1 was found to be involved in HDL induced up-regulation of COX-2 and PGI(2). Over-expressed SR-B1 did not significantly increase the expression of COX-2 and the PGI(2) levels, but knock-down of SR-B1 by siRNA could significantly attenuate COX-2 expression and PGI(2) release together with p38MAPK and CREB phosphorylation. Consistently, the declines of p-p38MAPK, p-CREB, COX-2 and PGI(2) were also observed after incubation with LY294002 (25μmol/L; PI3K special inhibitor) or L-NAME (50μmol/L; eNOS special inhibitor). In addition, we demonstrated the increases of PGI(2) release, COX-2 expression and p38MAPK phosphorylation, when nitric oxide level was raised through the incubation of L-arginine (10 or 20nmol/L) in endothelial cells. Taking together, our data support that SR-B1 mediated PI3K-Akt-eNOS signaling was involved in HDL-induced COX-2 expression and PGI(2) release in endothelial cells.