Safrole oxide induced human umbilical vein vascular endothelial cell differentiation into neuron-like cells by depressing the reactive oxygen species level at the low concentration

Previously, we found that 5-25 microg/ml safrole oxide could inhibit apoptosis and dramatically make a morphological change in human umbilical vein vascular endothelial cells (HUVECs). But the possible mechanism by which safrole oxide function is unknown. To answer this question, in this study, we first investigated the effects of it on the activity of nitric oxide synthetase (NOS), the expressions of Fas and integrin beta4, which play important roles in HUVEC growth and apoptosis, respectively. The results showed that, at the low concentration (10 microg/ml), safrole oxide had no effects on NOS activity and the expressions of Fas and integrin beta4. Then, we investigated whether HUVECs underwent differentiation. We examined the expressions of neuron-specific enolase (NSE) and neurofilament-L (NF-L). Furthermore, we analyzed the changes of intracellular reactive oxygen species (ROS). After 10 h of treatment with 10 microg/ml safrole oxide, some HUVECs became neuron-like cells in morphology, and intensively displayed positive NSE and NF-L. Simultaneously, ROS levels dramatically decreased during HUVECs differentiation towards neuron-like cells. At the low concentration, safrole oxide induced HUVECs differentiation into neuron-like cells. Furthermore, our data suggested that safrole oxide might perform this function by depressing intracellular ROS levels instead of by affecting cell growth or apoptosis signal pathways.     

Angiotensin II induces focal adhesion kinase/paxillin phosphorylation and cell migration in human umbilical vein endothelial cells

In the present study, we demonstrated that Ang II provokes a transitory enhancement of focal adhesion kinase (FAK) and paxillin phosphorylation in human umbilical endothelial cells (HUVEC). Moreover, Ang II induces a time- and dose-dependent augmentation in cell migration, but does not affect HUVEC proliferation. The effect of Ang II on FAK and paxillin phosphorylation was markedly attenuated in cells pretreated with wortmannin and LY294002, indicating that phosphoinositide 3-kinase (PI3K) plays an important role in regulating FAK activation. Similar results were observed when HUVEC were pretreated with genistein, a non-selective tyrosine kinases inhibitor, or with the specific inhibitor PP2 for Src family kinases, demonstrating the involvement of protein tyrosine kinases, and particularly Src family of tyrosine kinases, in the downstream signalling pathway of Ang II receptors. Furthermore, FAK and paxillin phosphorylation was markedly blocked after treatment of HUVEC with AG1478, a selective inhibitor of epidermal growth factor receptor (EGFR) phosphorylation. Pretreatment of cells with inhibitors of PI3K, Src family tyrosine kinases, and EGFR also decreased HUVEC migration. In conclusion, these results suggest that Ang II mediates an increase in FAK and paxillin phosphorylation and induces HUVEC migration through signal transduction pathways dependent on PI3K and Src tyrosine kinase activation and EGFR transactivation.     

Lipopolysaccharide induces autophagy through BIRC2 in human umbilical vein endothelial cells

Lipopolysaccharide (LPS), as an important proinflammatory agent, targets the endothelium. However, almost all in vitro experiments of the effect of LPS on vascular endothelial cells (VECs) were performed under an artificially decreased concentration of serum that was not enough to maintain the cell growth for a long time. The mechanism underlying LPS action on VECs cultured in a nutrient‐rich condition is not clear. To address this question and mimic the in vivo condition, we investigated the effect of LPS on VEC autophagy, which is involved in numerous physiological processes. The effect of LPS on microtubule‐associated protein 1 light chain 3 (LC3) distribution, LC3‐II accumulation and p62 degradation showed that LPS effectively induced autophagy in VECs cultured in the presence of 20% serum. To understand the mechanism by which LPS triggers the cell autophagy, we first investigated the effects of LPS on the expression of BIRC2 (cIAP1), a well‐known apoptosis inhibitor, and on the kinase activity of mammalian target of rapamycin (mTOR) and nuclear translocation of p53. LPS increased BIRC2 expression in a dose‐ and time‐dependent manner and elevated the intranuclear level of p53 but had no effect on the mTOR pathway when it triggered VEC autophagy. Furthermore, knockdown of BIRC2 by RNA interference inhibited the autophagy and the translocation of p53 to nuclei induced by LPS. These data suggest a novel role for BIRC2 in LPS‐induced autophagy in VECs. J. Cell. Physiol. 225: 174–179, 2010. © 2010 Wiley‐Liss, Inc.     

Visfatin exerts angiogenic effects on human umbilical vein endothelial cells through the mTOR signaling pathway

The biologically active factors known as adipocytokines are secreted primarily by adipose tissues and can act as modulators of angiogenesis. Visfatin, an adipocytokine that has recently been reported to have angiogenic properties, is upregulated in diabetes, cancer, and inflammatory diseases. Because maintenance of an angiogenic balance is critically important in the management of these diseases, understanding the molecular mechanism by which visfatin promotes angiogenesis is very important. In this report, we describe our findings demonstrating that visfatin stimulates the mammalian target of the rapamycin (mTOR) pathway, which plays important roles in angiogenesis. Visfatin induced the expression of hypoxia-inducible factor 1α (HIF1α) and vascular endothelial growth factor (VEGF) in human endothelial cells. Inhibition of the mTOR pathway by rapamycin eliminated the angiogenic and proliferative effects of visfatin. The visfatin-induced increase in VEGF expression was also eliminated by RNA interference-mediated knockdown of the 70-kDa ribosomal protein S6 kinase (p70S6K), a downstream target of mTOR. Visfatin inactivated glycogen synthase kinase 3β (GSK3β) by phosphorylating it at Ser-9, leading to the nuclear translocation of β-catenin. Both rapamycin co-treatment and p70S6K knockdown inhibited visfatin-induced GSK3β phosphorylation at Ser-9 and nuclear translocation of β-catenin. Taken together, these results indicate that mTOR signaling is involved in visfatin-induced angiogenesis, and that this signaling leads to visfatin-induced VEGF expression and nuclear translocation of β-catenin.     

Acrolein induces Hsp72 via both PKCdelta/JNK and calcium signaling pathways in human umbilical vein endothelial cells

Acrolein is a highly electrophilic alpha,beta-unsaturated aldehydes to which humans are exposed in a variety of environment situations and is also a product of lipid peroxidation. Increased levels of unsaturated aldehydes play an important role in the pathogenesis of a number of human diseases such as Alzheimer's disease, atherosclerosis and diabetes. A number of studies have reported that acrolein evokes downstream signaling via an elevation in cellular oxidative stress. Here, we report that low concentrations of acrolein induce Hsp72 in human umbilical vein endothelial cells (HUVEC) and that both the PKCdelta/JNK pathway and calcium pathway were involved in the induction. The findings confirm that the production of reactive oxygen species (ROS) is not directly involved in the pathway. The induction of Hsp72 was not observed in other cells such as smooth muscle cells (SMC) or COS-1 cells. The results suggest that HUVEC have a unique defense system against cell damage by acrolein in which Hsp72 is induced via activation of both the PKCd/JNK and the calcium pathway.     

Helicobacter pylori induces direct activation of the lymphotoxin beta receptor and non-canonical nuclear factor-kappa B signaling

The pathogen Helicobacter pylori, which infects half of the world's population, is a major risk factor for the development of gastric diseases including chronic gastritis and gastric cancer. Among H. pylori's virulence factors is the cytotoxin-associated gene pathogenicity island (cagPAI), which encodes for a type IV secretion system (T4SS). The T4SS induces fast canonical nuclear factor-kappa B (NF-κB) signaling, a major factor increasing inflammation, supressing apoptotic cell death and thereby promoting the development of neoplasia. However, H. pylori's capability to mediate fast non-canonical NF-κB signaling is unresolved, despite a contribution of non-canonical NF-κB signaling to gastric cancer has been suggested.We analyzed signaling elements within non-canonical NF-κB in response to H.?pylori in epithelial cell lines by immunoprecipitation, immunoblot, electrophoretic mobility shift assay and RNA interference knockdown. In addition, tissue samples of H. pylori-infected patients were investigated by immunohistochemistry.Here, we provide evidence for a T4SS-dependent direct activation of non-canonical NF-κB signaling. We identified the lymphotoxin beta receptor (LTβR) to elicit the fast release of NF-κB inducing kinase (NIK) from the receptor complex leading to non-canonical NF-κB signaling. Further, NIK expression was increased in human biopsies of H. pylori-associated gastritis. Thus, NIK could represent a novel target to reduce Helicobacter pylori-induced gastric inflammation and pathology.     

Humic acid induces the generation of nitric oxide in human umbilical vein endothelial cells: stimulation of nitric oxide synthase during cell injury

Humic acid (HA) has been implicated as an etiological factor in the peripheral vasculopathy of blackfoot disease (BFD). In this study, we examined the effects of HA upon the generation of nitric oxide (NO) during the process of lethal cell injury in cultured human umbilical vein endothelial cells (HUVECs). NO production was measured by the formation of nitrite (NO(2)(-)), the stable end-metabolite of NO. Cell death was assessed by measuring the release of intracellular lactate dehydrogenase (LDH). Treatment HUVECs with HA at a concentration of 50, 100, and 200 microg/ml concentration-dependently increased nitrite levels, reaching a peak at 12 h subsequent to HA treatment, with a maximal response of approximately 400 pmole nitrite (from 1 x 10(4) cells). HA-induced nitrite formation was blocked completely by N(G)-nitro-L-arginine methyl ester (L-NAME) and also by N(G)-methyl-L-arginine (L-NMA), both being specific inhibitors of NO synthase. The LDH released from endothelial cells was evoked at from 24 h after the addition of HA (50, 100, 200 microg/ml) in a concentration- and time-dependent manner. The HA-induced LDH release was also reduced by the presence of both L-NAME and L-NMA. The addition of Ca(2+) chelator (BAPTA) inhibited both nitrite formation and LDH release by HA. Moreover, the antioxidants (superoxide dismutase, vitamin C, vitamin E) and protein kinase inhibitor (H7) effectively suppressed HA-induced nitrite formation. These results suggest that HA treatment of endothelial cells stimulates NO production, which can elicit cell injury via the stimulation of Ca(2+)-dependent NO synthase activity by increasing cytosolic Ca(2+) levels. Because the destruction of endothelial cells has been implicated in triggering the onset of BFD, the induction of excessive levels of NO and consequent endothelial-cell injury may be important to the etiology of HA-induced vascular disorders associated with BFD for humans.     

Involvement of androgen receptor in nitric oxide production induced by icariin in human umbilical vein endothelial cells

Icariin, a flavonoid isolated from Epimedii herba, stimulated phosphorylation of endothelial nitric oxide synthase (eNOS) at Ser1177, Akt (Ser473) and ERK1/2 (Thr202/Tyr204). The icariin-induced eNOS phosphorylation was abolished by an androgen receptor (AR) antagonist, nilutamide in human umbilical vein endothelial cells (HUVECs). Furthermore, it was also reduced in the cells transfected with small interfering RNA in which the expression of AR was broken down. The icariin-induced eNOS phosphorylation was inhibited by wortmannin, a phosphatidylinositol 3-kinase (PI3K) inhibitor and partially attenuated by PD98059, an upstream inhibitor for ERK1/2. These data suggest that icariin stimulates release of NO by AR-dependent activation of eNOS in HUVECs. PI3K/Akt and MAPK-ERK kinase (MEK)/ERK1/2 pathways were involved in the phosphorylation of eNOS by icariin.     

Sphingosine 1-phosphate induces angiogenesis: its angiogenic action and signaling mechanism in human umbilical vein endothelial cells.

Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid metabolite abundantly stored in platelets and released upon platelet activation. Recently, S1P has been postulated for its potential roles in angiogenesis. In this study, we provided several lines of evidence showing that S1P has angiogenic activity. In vitro, S1P stimulated DNA synthesis and chemotactic motility of human umbilical vein endothelial cells (HUVECs) in a dose-dependent manner, reaching a near maximum at 1 microM. S1P also significantly induced tube formation of HUVECs on Matrigel. Matrigel plug assay in mice revealed that S1P promotes angiogenesis in vivo. In addition, exposure of HUVECs to S1P led to rapid activation of extracellular signal-regulated kinases (ERKs) and p38 mitogen-activated protein kinase (p38 MAPK) in a pertussis toxin (PTX)-sensitive manner. Notably, HUVEC migration and tube formation in response to S1P were completely blocked by pretreatment with PTX. Further, the MEK inhibitor U0126 markedly inhibited S1P-induced tube formation but S1P-induced migration was not affected by inhibition of ERK and p38 MAPK. Taken together, these results indicate that S1P induces angiogenesis predominantly via G(i) protein-coupled receptors in endothelial cells and suggest that S1P may act as an important modulator of platelet-induced angiogenesis.     

Hyperbaric oxygen induces VEGF expression through ERK, JNK and c-Jun/AP-1 activation in human umbilical vein endothelial cells

Summary Hyperbaric oxygen (HBO) is increasingly used in a number of areas of medical practice, such as selected problem infections and wounds. The beneficial effects of HBO in treating ischemia-related wounds may be mediated by stimulating angiogenesis. We sought to investigate VEGF, the main angiogenic regulator, regulated by HBO in human umbilical vein endothelial cells (HUVECs). In this study, we found that VEGF was up regulated both at mRNA and protein levels in HUVECs treated with HBO dose- and time-dependently. Since there are several AP-1 sites in the VEGF promoter, and the c-Jun/AP-1 is activated through stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) and extracellular signal regulated kinase (ERK), we further examined the c-Jun, JNK and ERK that might be involved in the VEGF induced by HBO. The VEGF mRNA induced by HBO was blocked by both PD98059 and SP600125, the ERK and JNK inhibitors respectively. HBO induced phospho-ERK and phospho-JNK expressions within 15 min. We further demonstrated that c-Jun phosphorylation was induced within 60 min of HBO treatment. HBO also induced the nuclear AP-1 binding ability within 30–60 min, but the AP-1 induction was blocked by treatment with either the ERK or JNK inhibitor. To verify that the VEGF expression induced by HBO is through the AP-1 trans-activation and VEGF promoter, both the VEGF promoter and AP-1 driving luciferase activity were found increased by the cells treated with HBO. The c-Jun mRNA, which is also driven by AP-1, was also induced by HBO, and the induction of c-Jun was blocked by ERK and JNK inhibitors. We suggest that VEGF induced by HBO is through c-Jun/AP-1 activation, and through simultaneous activation of ERK and JNK pathways.     

Paracrine interleukin-8 affects mesenchymal stem cells through the Akt pathway and enhances human umbilical vein endothelial cell proliferation and migration

Interleukin-8 (IL-8) promotes cell homing and angiogenesis, but its effects on activating human bone marrow mesenchymal stem cells (BMSCs) and promoting angiogenesis are unclear. We used bioinformatics to predict these processes. In vitro, BMSCs were stimulated in a high-glucose (HG) environment with 50 or 100 μg/ml IL-8 was used as the IL-8 group. A total of 5 μmol/l Triciribine was added to the two IL-8 groups as the Akt inhibitor group. Cultured human umbilical vein endothelial cells (HUVECs) were cultured in BMSCs conditioned medium (CM). The changes in proliferation, apoptosis, migration ability and levels of VEGF and IL-6 in HUVECs were observed in each group. Seventy processes and 26 pathways were involved in vascular development, through which IL-8 affected BMSCs. Compared with the HG control group, HUVEC proliferation absorbance value (A value), Gap closure rate, and Transwell cell migration rate in the IL-8 50 and IL-8 100 CM groups were significantly increased (P<0.01, n=30). However, HUVEC apoptosis was significantly decreased (P<0.01, n=30). Akt and phospho-Akt (P-Akt) protein contents in lysates of BMSCs treated with IL-8, as well as VEGF and IL-6 protein contents in the supernatant of BMSCs treated with IL-8, were all highly expressed (P<0.01, n=15). These analyses confirmed that IL-8 promoted the expression of 41 core proteins in BMSCs through the PI3K Akt pathway, which could promote the proliferation and migration of vascular endothelial cells. Therefore, in an HG environment, IL-8 activated the Akt signaling pathway, promoted paracrine mechanisms of BMSCs, and improved the proliferation and migration of HUVECs.     

Rho GTPase/Rho kinase negatively regulates endothelial nitric oxide synthase phosphorylation through the inhibition of protein kinase B/Akt in human endothelial cells

Endothelial nitric oxide synthase (eNOS) is an important regulator of cardiovascular homeostasis by production of nitric oxide (NO) from vascular endothelial cells. It can be activated by protein kinase B (PKB)/Akt via phosphorylation at Ser-1177. We are interested in the role of Rho GTPase/Rho kinase (ROCK) pathway in regulation of eNOS expression and activation. Using adenovirus-mediated gene transfer in human umbilical vein endothelial cells (HUVECs), we show here that both active RhoA and ROCK not only downregulate eNOS gene expression as reported previously but also inhibit eNOS phosphorylation at Ser-1177 and cellular NO production with concomitant suppression of PKB activation. Moreover, coexpression of a constitutive active form of PKB restores the phosphorylation but not gene expression of eNOS in the presence of active RhoA. Furthermore, we show that thrombin inhibits eNOS phosphorylation, as well as expression via Rho/ROCK pathway. Expression of the active PKB reverses eNOS phosphorylation but has no effect on downregulation of eNOS expression induced by thrombin. Taken together, these data demonstrate that Rho/ROCK pathway negatively regulates eNOS phosphorylation through inhibition of PKB, whereas it downregulates eNOS expression independent of PKB.     

Interleukin-6 impairs the insulin signaling pathway, promoting production of nitric oxide in human umbilical vein endothelial cells

Interleukin 6 (IL-6) is an independent predictor of type 2 diabetes and cardiovascular disease and is correlated with insulin resistance. Insulin stimulates nitric oxide (NO) production through the IRS-1/PI3-kinase/Akt/eNOS pathway (where IRS-1 is insulin receptor substrate 1, PI3-kinase is phosphatidylinositol 3-kinase, and eNOS is endothelial NO synthase). We asked if IL-6 affects insulin vasodilator action both in human umbilical vein endothelial cells (HUVEC) and in the aortas of C57BL/6J mice and whether this inhibitory effect was caused by increased Ser phosphorylation of IRS-1. We observed that IL-6 increased IRS-1 phosphorylation at Ser(312) and Ser(616); these effects were paralleled by increased Jun N-terminal protein kinase (JNK) and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation and reversed by JNK and ERK1/2 inhibition. In addition, IL-6 treatment resulted in impaired IRS-1 phosphorylation at Tyr(612), a site essential for engaging PI3-kinase. Furthermore, IL-6 treatment reduced insulin-stimulated phosphorylation of eNOS at the stimulatory Ser(1177) site and impaired insulin-stimulated eNOS dephosphorylation at the inhibitory Thr(495) site. Insulin-stimulated eNOS activation and NO production were also inhibited by IL-6; these effects were reversed by inhibition of JNK and ERK1/2. Treatment of C57BL/6J mice with IL-6 resulted in impaired insulin-dependent activation of the Akt/eNOS pathway in the aorta as a result of JNK and ERK1/2 activation. Our data suggest that IL-6 impairs the vasodilator effects of insulin that are mediated by the IRS-1/PI3-kinase/Akt/eNOS pathway through activation of JNK and ERK1/2.     

Stem cell factor/c-kit signaling promotes the survival, migration, and capillary tube formation of human umbilical vein endothelial cells

c-kit receptor tyrosine kinase is a marker of progenitor cells, which differentiate into blood and/or vascular endothelial cells, and has an important role in the amplification/mobilization of progenitor cells. c-kit is expressed in mature endothelial cells, but its role there is unclear. Stem cell factor, a c-kit ligand, dose-dependently promoted survival, migration, and capillary tube formation of human umbilical vein endothelial cells. These effects mimicked those of vascular endothelial growth factor, except that stem cell factor did not sufficiently support proliferation of these cells. After exposing cells to this factor, Akt, Erk1/2, and c-kit were immediately (相似文献   

Sphingosine 1-phosphate induces membrane ruffling and increases motility of human umbilical vein endothelial cells via vascular endothelial growth factor receptor and CrkII

Sphingosine 1-phosphate (S1P), a ligand for endothelial differentiation gene family proteins, is one of the most potent signal mediators released from activated platelets. Here, we report that S1P induces membrane ruffling of human umbilical vein endothelial cells (HUVECs) via the vascular endothelial growth factor receptor (VEGFR), Src family tyrosine kinase(s), and the CrkII adaptor protein. S1P induced prominent phosphorylation of CrkII in HUVECs, indicating that CrkII was involved in the S1P-induced signaling pathway. S1P-induced CrkII phosphorylation was blocked by pertussis toxin and overexpression of the carboxyl terminus of beta-adrenergic receptor kinase, indicating that the betagamma subunit of G(i) was required for the phosphorylation. Notably, the S1P-induced CrkII phosphorylation was also abolished by inhibitors of VEGFR or Src family tyrosine kinases. By using Picchu, a real time monitoring protein for CrkII phosphorylation, we found that S1P induced rapid CrkII phosphorylation at membrane ruffles. Finally, we observed that expression of a dominant negative mutant of CrkII inhibited the S1P-induced membrane ruffling and cell migration. These results delineated a novel S1P signaling pathway that involves sequential activation of G(i)-coupled receptor(s), VEGFR, Src family tyrosine kinase(s), and the CrkII adaptor protein, and which is responsible for both the induction of membrane ruffling and the increase in cell motility.     

miR-181a/b-5p regulates human umbilical vein endothelial cell angiogenesis by targeting PDGFRA

Type 2 diabetes mellitus (T2DM) is a growing burden in low-and middle-income countries. Changing lifestyles and lack of physical activity are some of the reasons contributing to this epidemic increase. Co-morbidities associated with T2DM are largely due to the complications which arise as a consequence of endothelial dysfunction. Platelet derived growth factor-alpha (PDGFRA) is a protein responsible for cell proliferation, angiogenesis, migration and invasion. Increased levels of PDGFRA have been reported in T2DM. This study assessed the epigenetic regulation of PDGFRA through microRNAs (miR-181a/b-5p).Using a bioinformatics-based approach, we assessed the binding of miR-181a/b-5p to PDGFRA. Experimentally, this binding was confirmed using a dual luciferase reporter assay. Further, we overexpressed miR-181a/b-5p in Human umbilical vein endothelial cells (HUVECs) and the influence of over-expression on cell proliferation, migration and angiogenesis was assessed using in-vitro approaches. The influence of miR-181a/b-5p over expression on cellular apoptosis was ascertained using a TUNEL assay with concomitant changes being observed in the levels of Bcl-2 and cleaved Caspase-3.In HUVECs, PDGFRA is a direct target for miR-181a/b-5p. Over expression of miR-181a/b-5p decreased cellular proliferation, migration, invasion, and tube formation—a surrogate marker for angiogenesis. miR-181a/b-5p may be used as a therapeutic intervention to restrict uncontrolled levels of PDGFRA and thereby rescue the phenotypes of increased cell proliferation, migration, invasion and tube formation. miR-181a/b negatively regulates PDGFRA levels. Significance of the study : T2DM and its associated complications emerge from endothelial dysfunction. The associated phenotypes are regulated by a number of proteins, one such member being, PDGFRA. PDGFRA is in turn regulated by miR-181a/b-5p. Complementation with miR-181a/b-5p resulted in reversion of phenotypes. Thus, miR-181a/b-5p-mediated suppression of PDGFRA may be used as a therapeutic intervention in the management of type 2 diabetes.     

Lithium reverses the effect of opioids on eNOS/nitric oxide pathway in human umbilical vein endothelial cells

The main challenge of pain management with opioids is development of acute and chronic analgesic tolerance. Several studies on neuronal cells have focused on the molecular mechanisms involved in tolerance such as cyclic AMP (cAMP) activation, and nitric oxide (NO) pathway. However, the effects of opioids on non-neuronal cells and tolerance development have been poorly investigated. Lithium chloride is a glycogen synthase kinase 3β (GSK-3β) inhibitor and exert its effects through modulation of nitric oxide pathway. In this study we examined the effect of lithium on acute/chronic morphine and methadone administration in endothelial cells which express mu opioid receptors. Human umbilical vein endothelial cells (HUVECs) were treated with different doses of morphine, methadone, and lithium for six and 48 h. Then we evaluated cell viability, nitrite and cyclic AMP levels, as well as the expression of endothelial nitric oxide synthase (eNOS) protein using Immunocytochemistry (ICC) assay and phosphorylated GSK-3β enzyme by western blot analysis in cells. Both chronic morphine and methadone treatment increased NO level and eNOS expression in HUVECs. Morphine induced cAMP overproduction after 48 h exposure with cells. Lithium pretreatment (10 mM) in both morphine and methadone received groups significantly reduced nitrite and cAMP levels as well as eNOS expression as compared to the control. The decreased amount of phospho GSK-3β due to the opioid exposure was increased following lithium treatment. Tolerance like pattern may occur in non-neuronal cells with opioid receptors and this study clearly revealed the attenuation of morphine and methadone tolerance like behavior by lithium treatment in HUVECs.