Loss of sphingosine kinase-1 in carcinoma cells increases formation of reactive oxygen species and sensitivity to doxorubicin-induced DNA damage

BACKGROUND AND PURPOSE

Sphingosine kinases (SK) catalyse the formation of sphingosine 1-phosphate, which is a key lipid mediator regulating cell responses such as proliferation, survival and migration. Here we have investigated the effect of targeted inhibition of SK-1 on cell damage and elucidated the mechanisms involved.

EXPERIMENTAL APPROACH

Three human carcinoma cell lines (colon HCT-116, breast MDA-MB-231, lung NCI-H358) were used, which were either transduced with shRNA constructs to deplete SK-1, or treated with a SK-1 inhibitor. Cell growth and viability were assayed by [³H]thymidine incorporation and colony formation. Reactive oxygen species (ROS) were measured by fluorescence and apoptosis by annexin V with flow cytometry. Proteins were analysed by Western blotting. DNA damage was induced by doxorubicin.

KEY RESULTS

Knock-down of SK-1 by shRNA strongly inhibited DNA synthesis and colony formation of carcinoma cells. SK-1 knock-down (SK-1kd) cells revealed dysfunctional extracellular signal-regulated protein kinase and PKB/Akt cascades, and contained increased levels of ROS. After SK-1kd, treatment with doxorubicin increased DNA damage, measured by histone-2AX phosphorylation. Similar effects were found in cells with a SK-1 inhibitor and doxorubicin. The increased damage response in SK-1kd cells was accompanied by greater reduction of DNA synthesis and colony formation, and by more pronounced apoptosis. Addition of a NADPH oxidase inhibitor reduced the increased apoptosis in doxorubicin-treated SK-1kd cells.

CONCLUSIONS AND IMPLICATIONS

SK-1kd in carcinoma cells triggered oxidative stress by increasing intracellular Ros production. Targeted inhibition of SK-1 represents a promising approach to sensitize cells to DNA damage and facilitate apoptosis upon doxorubicin treatment.     

Resveratrol dimers are novel sphingosine kinase 1 inhibitors and affect sphingosine kinase 1 expression and cancer cell growth and survival

BACKGROUND AND PURPOSE

Sphingosine kinase 1 catalyses formation of the bioactive lipid, sphingosine 1-phosphate, which protects cancer cells from apoptosis. Therefore, sphingosine kinase 1 is a novel target for intervention with anti-cancer agents. We have assessed the effect of the anti-cancer agent, resveratrol and its dimers (ampelopsin A and balanocarpol) on sphingosine kinase 1 activity and on survival of MCF-7 breast cancer cells.

EXPERIMENTAL APPROACH

Ampelopsin A and balanocarpol were purified from Hopea dryobalanoides and their effect on sphingosine kinase 1 activity and expression, [³H] thymidine incorporation, ERK-1/2 phosphorylation and PARP activity assessed in MCF-7 cells.

KEY RESULTS

Resveratrol, ampelopsin A and balanocarpol were novel inhibitors of sphingosine kinase 1 activity. Balanocarpol was a mixed inhibitor (with sphingosine) of sphingosine kinase 1 with a K_ic= 90 ± 10 µM and a K_iu of ∼500 µM. Balanocarpol and ampelopsin A also induced down-regulation of sphingosine kinase 1 expression and reduced DNA synthesis, while balanocarpol stimulated PARP cleavage in MCF-7 breast cancer cells. Resveratrol was a competitive inhibitor (with sphingosine) of sphingosine kinase 1 with a K_ic= 160 ± 40 µM, reduced sphingosine kinase 1 expression and induced PARP cleavage in MCF-7 cells.

CONCLUSIONS AND IMPLICATIONS

Each molecule of balanocarpol may bind at least two sphingosine kinase 1 catalytic molecules to reduce the activity of each simultaneously. These findings suggest that resveratrol, ampelopsin A and balanocarpol could perturb sphingosine kinase 1-mediated signalling and this might explain their activity against MCF-7 breast cancer cells.

LINKED ARTICLE

This article is commented on by Hergst and Yun, pp. 1603–1604 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2012.01898.x     

Microvesicles secreted from human multiple myeloma cells promote angiogenesis

Aim： To investigate whether human multiple myeloma （MM） cells secrete microvesicles （MVs） and whether the MVs secreted from MM cells （MM-MVs） promote angiogenesis. Methods： RPMI8226 human MM cells and EA.hy926 human umbilical vein cells were used. MVs isolated from RPMI 8226 cells were characterized under laser confocal microscopy, electron microscopy and with flow cytometry. The fusion of MM-MVs and EA.hy926 cells was studied under confocal microscopy, and the transfer of CD138 to EA.hy926 cells was demonstrated with flow cytometry. The proliferation, invasion and tube formation of EA.hy926 cells in vitro were evaluated using M]-r, transwell migration and tube formation assays, respectively. The vasculization of EA.hy926 cells in vivo was studied using Matrigel plug assay. The expression of IL-6 and VEGF was analyzed with PCR and ELISA. Results： MM-MVs from the RPMI 8226 cells had the characteristic cup-shape with diameter of 100-1000 nm. Most of the MM-MVs expressed phosphatidylserine and the myeloma cell marker CD138, confirming that they were derived from myeloma cells. After added to EA.hy926 cells, the MM-MVs transferred CD138 to the endothelial cells and significantly stimulated the endothelial cells to proliferate, invade, secrete IL-6 and VEGF, two key angiogenic factors of myeloma, and form tubes in vitro and in vivo. Conclusion： Our results confirm the presence of MVs in MM cells and support the idea that MM-MVs are newfound mediators for myeloma angiogenesis and may serve as a therapeutic target to treat MM.     

N-benzyl-5-phenyl-1H-pyrazole-3-carboxamide promotes vascular endothelial cell angiogenesis and migration in the absence of serum and FGF-2

Aim:

To investigate the effect of N-benzyl-5-phenyl-1H-pyrazole-3-carboxamide (BPC) on angiogenesis in human umbilical vein endothelial cells (HUVECs).

Methods:

Capillary-like tube formation on matrigel and cell migration analyses were performed in the absence of serum and fibroblast growth factor (FGF-2). Reactive oxygen species (ROS) were measured using a fluorescent probe, 2′, 7′- dichlorodihydrofluorescein (DCHF). The nitric oxide (NO) production of HUVECs was examined using a NO detection kit. Morphological observation under a phase contrast microscope, a viability assay using 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl-tetrazolium (MTT) and a lactate dehydrogenase (LDH) activity analysis by a detection kit were performed to evaluate the toxicity of BPC on HUVECs in the presence of serum and FGF-2. The level of hypoxia-inducible factor 1α (HIF-1α) and the release of vascular endothelial growth factor (VEGF) were measured by Western blot and ELISA, respectively.

Results:

In the absence of serum and FGF-2, cells treated with BPC (5-20 μmol/L) rapidly aligned with one another and formed tube-like structures within 12 h. In the presence of serum and FGF-2, cells treated with BPC for 24, 48 and 72 h had no changes in morphology, viability or LDH release compared with the control group. Cell migration in the BPC-treated group was significantly increased compared with the control group. During this process, NO production and ROS level were elevated dramatically, and the levels of HIF-1α and VEGF were increased dependent on the generation of ROS.

Conclusion:

BPC most effectively promoted angiogenesis and migration in HUVECs in the absence of FGF-2 and serum.     

Thalidomide attenuates nitric oxide-driven angiogenesis by interacting with soluble guanylyl cyclase

Background and purpose:

Nitric oxide (NO) promotes angiogenesis by activating endothelial cells. Thalidomide arrests angiogenesis by interacting with the NO pathway, but its putative targets are not known. Here, we have attempted to identify these targets.

Experimental approach:

Cell-based angiogenesis assays (wound healing of monolayers and tube formation in ECV304, EAhy926 and bovine arterial endothelial cells), along with ex vivo and in vivo angiogenesis assays, were used to explore interactions between thalidomide and NO. We also carried out in silico homology modelling and docking studies to elucidate possible molecular interactions of thalidomide and soluble guanylyl cyclase (sGC).

Key results:

Thalidomide inhibited pro-angiogenic functions in endothelial cell cultures, whereas 8-bromo-cGMP, sildenafil (a phosphodiesterase inhibitor) or a NO donor [sodium nitroprusside (SNP)] increased these functions. The inhibitory effects of thalidomide were reversed by adding 8-bromo-cGMP or sildenafil, but not by SNP. Immunoassays showed a concentration-dependent decrease of cGMP in endothelial cells with thalidomide, without affecting the expression level of sGC protein. These results suggested that thalidomide inhibited the activity of sGC. Molecular modelling and docking experiments revealed that thalidomide could interact with the catalytic domain of sGC, which would explain the inhibitory effects of thalidomide on NO-dependent angiogenesis.

Conclusion and implications:

Our results showed that thalidomide interacted with sGC, suppressing cGMP levels in endothelial cells, thus exerting its anti-angiogenic effects. These results could lead to the formulation of thalidomide-based drugs to curb angiogenesis by targeting sGC.     

Cyclopeptide RA-V inhibits angiogenesis by down-regulating ERK1/2 phosphorylation in HUVEC and HMEC-1 endothelial cells

BACKGROUND AND PURPOSE

Anti-angiogenic agents have recently become one of the major adjuvants for cancer therapy. A cyclopeptide, RA-V, has been shown to have anti-tumour activities. Its in vitro anti-angiogenic activities were evaluated in the present study, and the underlying mechanisms were also assessed.

EXPERIMENTAL APPROACH

Two endothelial cell lines, human umbilical vein endothelial cells (HUVEC) and human microvascular endothelial cells (HMEC-1), were used. The effects of RA-V on the proliferation, cell cycle phase distribution, migration, tube formation and adhesion were assessed. Western blots and real-time PCR were employed to examine the protein and mRNA expression of relevant molecules.

KEY RESULTS

RA-V inhibited HUVEC and HMEC-1 proliferation dose-dependently with IC₅₀ values of 1.42 and 4.0 nM respectively. RA-V inhibited migration and tube formation of endothelial cells as well as adhesion to extracellular matrix proteins. RA-V treatment down-regulated the protein and mRNA expression of matrix metalloproteinase-2. Regarding intracellular signal transduction, RA-V interfered with the activation of ERK1/2 in both cell lines. Furthermore, RA-V significantly decreased the phosphorylation of JNK in HUVEC whereas, in HMEC-1, p38 MAPK was decreased.

CONCLUSIONS AND IMPLICATIONS

RA-V exhibited anti-angiogenic activities in HUVEC and HMEC-1 cell lines with changes in function of these endothelial cells. The underlying mechanisms of action involved the ERK1/2 signalling pathway. However, RA-V may regulate different signalling pathways in different endothelial cells. These findings suggest that RA-V has the potential to be further developed as an anti-angiogenic agent.     

Effects of angiotensin receptor blockers on endothelial nitric oxide release: the role of eNOS variants

AIM

Angiotensin II receptor blockers (ARBs) improve endothelial cell (EC)-dependent vasodilation in patients with hypertension through suppression of angiotensin II type 1 receptors but may have additional and differential effects on endothelial nitric oxide (NO) synthase (eNOS) function. To investigate this question, we tested the effects of various ARBs on NO release in ECs from multiple donors, including those with eNOS genetic variants linked to higher cardiovascular risk.

METHODS

The effects of ARBs (losartan, olmesartan, telmisartan, valsartan), at 1 µm, on NO release were measured with nanosensors in human umbilical vein ECs obtained from 18 donors. NO release was stimulated with calcium ionophore (1 µm) and its maximal concentration was correlated with eNOS variants. The eNOS variants were determined by a single nucleotide polymorphism in the promoter region (T-786C) and in the exon 7 (G894T), linked to changes in NO metabolism.

RESULTS

All of the ARBs caused an increase in NO release as compared with untreated samples (P < 0.01, n = 4–5 in all eNOS variants). However, maximal NO production was differentially influenced by eNOS genotype. Olmesartan increased maximal NO release by 30%, which was significantly greater (P < 0.01, n = 4–5 in all eNOS variants) than increases observed with other ARBs.

CONCLUSIONS

The ARBs differentially enhanced NO release in ECs in a manner influenced by eNOS single nucleotide polymorphisms. These findings provide new insights into the effects of ARBs on EC-dependent vasodilation and eNOS function.     

Protective effects of prostaglandin E1 on human umbilical vein endothelial cell injury induced by hydrogen peroxide

Aim:

To investigate the protective effects of prostaglandin E₁ (PGE₁) against H₂O₂-induced oxidative damage on human umbilical vein endothelial cells (HUVECs).

Methods:

HUVECs were pretreated with PGE₁ (0.25, 0.50, and 1.00 μmol/L) for 24 h and exposed to H₂O₂ (200 μmol/L) for 12 h, and cell viability was measured by the MTT assay. LDH, NO, SOD, GSH-Px, MDA, ROS, and apoptotic percentage were determined. eNOS expression was measured by Western blotting and real-time PCR.

Results:

PGE₁ (0.25−1.00 μmol/L) was able to markedly restore the viability of HUVECs under oxidative stress, and scavenged intracellular reactive oxygen species induced by H₂O₂. PGE₁ also suppressed the production of lipid peroxides, such as MDA, restored the activities of endogenous antioxidants including SOD and GSH-Px, and inhibited cell apoptosis. In addition, PGE₁ significantly increased NO content, eNOS protein, and mRNA expression.

Conclusion:

PGE₁ effectively protected endothelial cells against oxidative stress induced by H₂O₂, an activity that might depend on the up-regulation of NO expression.     

Chemerin/ChemR23 signaling axis is involved in the endothelial protection by K(ATP) channel opener iptakalim

Aim:

To elucidate the modulation of the chemerin/ChemR23 axis by iptakalim-induced opening of K_ATP channels and to determine the role of the chemerin/ChemR23 axis in the iptakalim-mediated endothelial protection.

Methods:

Cultured rat aortic endothelial cells (RAECs) were used. Chemerin secretion and ChemR23 protein expression were investigated using Western blot analysis. The gene expression level of ChemR23 was examined with RT-PCR. In addition, the release of nitric oxide (NO) was measured with a nitric oxide assay.

Results:

Homocysteine, uric acid, high glucose, or oxidized low-density lipoprotein (ox-LDL) down-regulated the chemerin secretion and ChemR23 gene/protein expression in RAECs as a function of concentration and time, which was reversed by pretreatment with iptakalim (1-10 μmol/L). Moreover, these effects of iptakalim were abolished in the presence of the K_ATP channel antagonist glibenclamide (1 μmol/L). Both iptakalim and recombinant chemerin restored the impaired NO production in RAECs induced by uric acid, and the effects were abolished by anti-ChemR23 antibodies.

Conclusion:

Iptakalim via opening K_ATP channels enhanced the endothelial chemerin/ChemR23 axis and NO production, thus improving endothelial function.     

Acute activation of eNOS by statins involves scavenger receptor-B1, G protein subunit Gi, phospholipase C and calcium influx

Background and purpose:

Statins (HMG CoA reductase inhibitors) have beneficial effects independent of reducing cholesterol synthesis and this includes their ability to acutely activate endothelial nitric oxide synthase (eNOS). The mechanism by which this occurs is largely unknown and thus we characterized the pathways by which statins activate NOS, including involvement of scavenger receptor-B1 (SR-B1), which is expressed in endothelial cells and maintains cholesterol concentrations.

Experimental approach:

Nitric oxide production was monitored in bovine aortic endothelial cells (BAECs) exposed to lovastatin (LOV) or pravastatin (PRA) for 10–20 min, alone or following pre-exposure to the end product of HMG-CoA reductase (mevalonate), G protein inhibitors (pertussis/cholera toxins), phospholipase C (PLC) inhibitor (U-73122), or intracellular and extracellular calcium chelators – BAPTA-AM and EGTA (respectively), or a function blocking antibody to SR-B1.

Key results:

Both statins increased NO production in a rapid, dose-dependent and HMG-CoA reductase-independent manner. Inhibiting Gi protein or PLC almost completely blocked statin-induced NO generation. Additionally, removing extracellular calcium inhibited statin-induced NO production. COS-7 cells co-transfected with eNOS and SR-B1 increased NO production when exposed to LOV or high-density lipoprotein (HDL), an agonist of SR-B1. These effects were not observed in COS-7 cells with eNOS alone or co-transfected with bradykinin receptor 2, indicating specificity for SR-B1. Further, pretreatment of BAEC with blocking antibody for SR-B1 blocked NO responses to statins and HDL.

Conclusions and implications:

LOV and PRA acutely activate eNOS through pathways that include the cell surface receptor SR-B1, Gi protein, phosholipase C and entry of extracellular calcium into endothelial cells.     

Hypoxia activates 15-PGDH and its metabolite 15-KETE to promote pulmonary artery endothelial cells proliferation via ERK1/2 signalling

BACKGROUND AND PURPOSE

Dysfunction and injury of endothelial cells in the pulmonary artery play critical roles in the hypertension induced by chronic hypoxia. One consequence of hypoxia is increased activity of 15-hydroxyprostaglandin dehydrogenase (PGDH). Here, we have explored, in detail, the effects of hypoxia on the proliferation of pulmonary artery endothelial cells.

EXPERIMENTAL APPROACH

We used bromodeoxyuridine incorporation, cell-cycle analysis, immunohistochemistry and Western blot analysis to study the effects of hypoxia, induced 15-PGDH) activity and its product, 15-keto-6Z, 8Z, 11Z, 13E-eicosatetraenoic acid (15-KETE), on endothelial cell proliferation. Scratch-wound and tube formation assays were also used to study migration of endothelial cells.

KEY RESULTS

15-KETE increased DNA synthesis and enhanced the transition from the G₀/G₁ phase to the S phase in hypoxia. Inhibition of 15-PGDH or siRNA for 15-PGDH reversed these effects. 15-KETE also activated the ERK1/2 signalling pathway. 15-KETE-induced cell migration and tube formation were reversed by blocking ERK1/2, but not the p38 MAPK pathway.

CONCLUSIONS AND IMPLICATIONS

Hypoxia-induced endothelial proliferation and migration, an important underlying mechanism contributing to hypoxic pulmonary vascular remodelling, appears to be mediated by 15-PGDH and 15-KETE, via the ERK1/2 signalling pathway.     

Atorvastatin inhibits homocysteine-induced dysfunction and apoptosis in endothelial progenitor cells

Aim:

To investigate the protective effects of atorvastatin on homocysteine (Hcy)-induced dysfunction and apoptosis in endothelial progenitor cells (EPCs) and the possible molecular mechanisms.

Methods:

EPCs were divided into six groups: Hcy treatment groups (0, 50, and 500 μmol/L) and atorvastatin pretreatment groups (0.1, 1, and 10 μmol/L). EPC proliferation, migration, in vitro vasculogenesis activity, and apoptosis rate were assayed by the MTT assay, modified Boyden chamber assay, in vitro vasculogenesis kit, and AnnexinV-FITC apoptosis detection kit, respectively. The level of reactive oxygen species (ROS) in cells was measured using H₂DCF-DA as a fluorescence probe. The activity of NADPH oxidase was evaluated with lucigenin-enhanced chemiluminescence. NO in the supernatant was detected by the nitrate reductase assay. The eNOS mRNA expression and p-eNOS, p-Akt, p-p38MAPK protein expression were measured by RT-PCR and Western blotting analysis, respectively. Caspase-3 activity was determined by colorimetric assay.

Results:

Hcy does-dependently impaired the proliferation, migration and in vitro vasculogenesis capacity of EPCs, induced cell apoptosis, increased ROS accumulation and NADPH oxidase activation, and decreased the secretion of NO compared with the control group (P<0.05 or P<0.01). The detrimental effects of Hcy were attenuated by atorvastatin pretreatment. Furthermore, Hcy caused a significant downregulation of eNOS mRNA, p-eNOS, and p-Akt protein expression as well as an upregulation of p-p38MAPK protein expression and caspase-3 activity. These effects of Hcy on EPCs were reversed by atorvastatin in a does-dependent manner.

Conclusion:

Atorvastatin inhibited homocysteine-induced dysfunction and apoptosis in endothelial progenitor cells, which may be related to its effects on suppressing oxidative stress, up-regulating Akt/eNOS and down-regulating the p38MAPK/caspase-3 signaling pathway.     

Activation of the ERK signaling pathway is involved in CD151-induced angiogenic effects on the formation of CD151-integrin complexes

Aim:

To assess the roles of extracellular signal-regulated kinase (ERK), p38, and CD151-integrin complexes on proliferation, migration, and tube formation activities of CD151-induced human umbilical vein endothelial cells (HUVECs).

Methods:

CD151, anti-CD151 and CD151-AAA mutant were inserted into recombinant adeno-associated virus (rAAV) vectors and used to transfect HUVECs. After transfection, the expression of CD151 was measured. Proliferation was assessed using the 3-[4,5-dimethylthiazol- 2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay. Cell migration was evaluated in Boyden transwell chambers using FBS as the chemotactic stimulus. The tube formation assay was performed on matrigel. The potential involvement of various signaling pathways was explored using selective inhibitors.

Results:

CD151 gene delivery increased the expression of CD151 at both the mRNA and protein levels. Overexpression of CD151 promoted cell proliferation, migration and tube formation in vitro, and phosphorylation of ERK was also increased. Further, CD151-induced cell proliferation, migration, and tube formation were attenuated by the ERK inhibitor PD98059 (20 μmol/L) but not by a p38 inhibitor (SB203580, 20 μmol/L). Moreover, there was no significant difference in CD151 protein expression between the CD151 group and the CD151-AAA group, but the CD151-AAA mutant abrogated cellular proliferation, migration, and tube formation and decreased the phosphorylation of ERK.

Conclusion:

This study suggests that activation of the ERK signaling pathway may be involved in the angiogenic effects of CD151. Activation of ERK was dependent on the formation of CD151-integrin complexes. Therefore modulation of CD151 may be as a novel therapeutic strategy for regulating angiogenesis.     

Downregulation of lentivirus-mediated ILK RNAi on tractional force generation in human retinal Muller cells

Aim:

To investigate the effect of lentivirus-mediated integrin-linked kinase (ILK) RNA interference (RNAi) on human retinal Müller cells transdifferentiation into contractile myofibroblasts.

Methods:

A lentiviral vector expressing ILK-specific shRNA was constructed and introduced into cultured retinal Müller cells. Silencing of the ILK gene was identified by real time RT-PCR and Western blot. The Müller cell phenotype change was confirmed by immunodetection of α-smooth muscle actin (α-SMA) stress fiber formation. The generation of tractional force was assessed using a tissue culture assay with cells incubated in three-dimensional collagen gels; cell migration was determined by the Boyden chamber method, using 10% FBS as a chemotactic factor.

Results:

Significant decreases in ILK mRNA and protein expression were detected in Müller cells carrying lentiviral ILK-shRNA vector. Cells treated with anti-ILK siRNA showed less α-SMA stress fiber formation under hypoxic conditions or cell subcultivation. Lentiviral ILK-shRNA vector transfection also significantly reduced cell migration and cell-mediated gel contraction.

Conclusion:

Lentivirus-mediated ILK RNAi decreased cell migration and contractile force generation by inhibiting α-SMA stress fiber formation in human retinal Müller cells. This tool might be useful to treat ocular fibroproliferative diseases associated with transdifferentiated Müller cells.     

Glucagon-like peptide-1 activates endothelial nitric oxide synthase in human umbilical vein endothelial cells

Aim:

To investigate the effects of glucagon-like peptide-1 (GLP-1) on endothelial NO synthase (eNOS) in human umbilical vein endothelial cells (HUVECs), and elucidate whether GLP-1 receptor (GLP-1R) and GLP-1(9–36) are involved in these effects.

Methods:

HUVECs were used. The activity of eNOS was measured with NOS assay kit. Phosphorylated and total eNOS proteins were detected using Western blot analysis. The level of eNOS mRNA was quantified with real-time RT-PCR.

Results:

Incubation of HUVECs with GLP-1 (50–5000 pmol/L) for 30 min significantly increased the activity of eNOS. Incubation of HUVECs with GLP-1 (500–5000 pmol/L) for 5 or 10 min increased eNOS phosphorylated at ser-1177. Incubation with GLP-1 (5000 pmol/L) for 48 h elevated the level of eNOS protein, did not affect the level of eNOS mRNA. GLP-1R agonists exenatide and GLP-1(9–36) at the concentration of 5000 pmol/L increased the activity, phosphorylation and protein level of eNOS. GLP-1R antagonist exendin(9–39) or DPP-4 inhibitor sitagliptin, which abolished GLP-1(9–36) formation, at the concentration of 5000 pmol/L partially blocked the effects of GLP-1 on eNOS.

Conclusion:

GLP-1 upregulated the activity and protein expression of eNOS in HUVECs through the GLP-1R-dependent and GLP-1(9–36)-related pathways. GLP-1 may prevent or delay the formation of atherosclerosis in diabetes mellitus by improving the function of eNOS.     

Role of endothelin-1 in the hyper-responsiveness to nitrovasodilators following acute NOS inhibition

BACKGROUND AND PURPOSE

Acute NOS inhibition in humans and animals is associated with hypersensitivity to NO donors. The mechanisms underlying this phenomenon have not been fully elucidated. The purpose of the present study was to assess whether hypersensitivity to NOS-blockade is linked to endothelin-1 (ET-1) signalling.

EXPERIMENTAL APPROACH

Sprague Dawley rats were instrumented with indwelling arterial and venous catheters for continuous assessments of haemodynamic parameters and drug delivery, respectively. Mesenteric arteries were isolated and tested for reactivity by wire myography.

KEY RESULTS

NOS blockade with L-N^G-nitroarginine methyl ester (L-NAME) caused a pronounced increase in arterial blood pressure (BP) (∼40 mmHg). In L-NAME-treated animals, the dose of sodium nitroprusside (SNP) required to cause a significant reduction in arterial BP was lower than in vehicle-treated rats (P < 0.001), and the magnitude of the reduction in BP was greater. Similar results were obtained with other NO mimetics, but not isoprenaline; moreover, decreasing the BP back to baseline levels with prazosin after L-NAME treatment did not attenuate the hyper-responsiveness to NO donors. The increased responsiveness to NO donors was abolished by pretreatment with the ET_A/B receptor antagonist, PD145065, or the ET_A receptor-specific antagonist ABT627. Ex vivo, L-NAME treatment potentiated the constriction induced by big endothelin-1 (bET-1), the precursor to active ET-1, but had no effect on the ET-1-mediated constriction.

CONCLUSIONS AND IMPLICATIONS

These data suggest that the increased sensitivity to NO donors is mediated, at least in part, by ET-1 in vivo, and the mechanism may involve the conversion of bET-1 to ET-1.     

Endocannabinoid-like N-arachidonoyl serine is a novel pro-angiogenic mediator

BACKGROUND AND PURPOSE

N-arachidonoyl serine (ARA-S) is a recently identified endocannabinoid-like lipid with weak affinity for the fully characterized cannabinoid receptors (CB₁ and CB₂) and the transient receptor potential vanilloid receptor 1 (TRPV-1). ARA-S induces vasodilatation and shows vasoprotective potential via activation of key signalling pathways in endothelial cells. Based on these findings, the effect of ARA-S on endothelial functions was further studied.

EXPERIMENTAL APPROACH

Primary human dermal microvascular endothelial cells (HMVEC) were used to investigate effects of ARA-S (0–10 µM) on certain endothelial functions, using cell proliferation, migration and wound repair models in vitro, and angiogenesis assays in vitro and ex vivo. Selective CB receptor antagonists and specific siRNAs were deployed to block individual CB receptors.

KEY RESULTS

We found that ARA-S stimulated angiogenesis and endothelial wound healing through induction of vascular endothelial growth factor C and its cognate receptor expression in primary HMVEC. Moreover, knock-down of G protein-coupled receptor 55 (GPR55) partly inhibited ARA-S-induced signal transduction and endothelial functions.

CONCLUSIONS AND IMPLICATIONS

Our results indicate that ARA-S is a pro-angiogenic factor in addition to a vessel dilator. The GPR55 receptor may serve as one target of ARA-S.     

Methylglyoxal scavengers attenuate endothelial dysfunction induced by methylglyoxal and high concentrations of glucose

BACKGROUND AND PURPOSE

Endothelial dysfunction is a feature of hypertension and diabetes. Methylglyoxal (MG) is a reactive dicarbonyl metabolite of glucose and its levels are elevated in spontaneously hypertensive rats and in diabetic patients. We investigated if MG induces endothelial dysfunction and whether MG scavengers can prevent endothelial dysfunction induced by MG and high glucose concentrations.

EXPERIMENTAL APPROACH

Endothelium-dependent relaxation was studied in aortic rings from Sprague-Dawley rats. We also used cultured rat aortic and human umbilical vein endothelial cells. The MG was measured by HPLC and Western blotting and assay kits were used.

KEY RESULTS

Incubation of aortic rings with MG (30 µM) or high glucose (25 mM) attenuated endothelium-dependent, acetylcholine-induced relaxation, which was restored by two different MG scavengers, aminoguanidine (100 µM) and N-acetyl cysteine (NAC) (600 µM). Treatment of cultured endothelial cells with MG or high glucose increased cellular MG levels, effects prevented by aminoguanidine and NAC. In cultured endothelial cells, MG and high glucose reduced basal and bradykinin-stimulated nitric oxide (NO) production, cGMP levels, and serine-1177 phosphorylation and activity of endothelial NO synthase (eNOS), without affecting threonine-495 and Akt phosphorylation or total eNOS protein. These effects of MG and high glucose were attenuated by aminoguanidine or NAC.

CONCLUSIONS AND IMPLICATIONS

Our results show for the first time that MG reduced serine-1177 phosphorylation, activity of eNOS and NO production. MG caused endothelial dysfunction similar to that induced by high glucose. Specific and safe MG scavengers have potential to prevent endothelial dysfunction induced by MG and high glucose concentrations.