Polarity Protein Scrib Facilitates Endothelial Inflammatory Signaling.

OBJECTIVE: The polarity protein Scrib is highly expressed in endothelial cells and is required for planar cell polarity. Scrib also facilitates recycling of integrin α5 to the plasma membrane. Because integrin α5 signals the presence of the inflammatory matrix protein fibronectin, we hypothesized that Scrib contributes to endothelial inflammatory signaling. APPROACH AND RESULTS: Cytokine treatment of human umbilical vein endothelial cells induced an inflammatory response as evident by the induction of vascular cell adhesion molecule-1 (VCAM-1). Downregulation of Scrib greatly attenuated this effect. In endothelial-specific conditional Scrib knockout mice, in vivo lipopolysaccharide treatment resulted in an impaired VCAM-1 induction. These effects were functionally relevant because Scrib small interfering RNAs in human umbilical vein endothelial cells attenuated the VCAM-1-mediated leukocyte adhesion in response to tumor necrosis factor-α. In vivo, tamoxifen-induced endothelial-specific deletion of Scrib resulted in a reduced VCAM-1-mediated leukocyte adhesion in response to tumor necrosis factor-α in the mouse cremaster model. This effect was specific for Scrib and not mediated by other polarity proteins. Moreover, it did not involve integrin α5 or classic pathways supporting inflammatory signaling, such as nuclear factor κ light chain enhancer of activated B-cells or MAP kinases. Co-immunoprecipitation/mass spectrometry identified the zinc finger transcription factor GATA-like protein-1 as a novel Scrib interacting protein. Small interfering RNA depletion of GATA-like protein-1 decreased the tumor necrosis factor-α-stimulated VCAM-1 induction to a similar extent as loss of Scrib did. Silencing of Scrib reduced GATA-like protein-1 protein, but not mRNA abundance. CONCLUSIONS: Scrib is a novel proinflammatory regulator in endothelial cells, which maintains the protein expression of GATA-like protein-1.

Reprogramming of myeloid angiogenic cells by Bartonella henselae leads to microenvironmental regulation of pathological angiogenesis.

The contribution of myeloid cells to tumour microenvironments is a decisive factor in cancer progression. Tumour-associated macrophages (TAMs) mediate tumour invasion and angiogenesis through matrix remodelling, immune modulation and release of pro-angiogenic cytokines. Nothing is known about how pathogenic bacteria affect myeloid cells in these processes. Here we show that Bartonella henselae, a bacterial pathogen causing vasculoproliferative diseases (bacillary angiomatosis), reprogrammes human myeloid angiogenic cells (MACs), a pro-angiogenic subset of circulating progenitor cells, towards a TAM-like phenotype with increased pro-angiogenic capacity. B. henselae infection resulted in inhibition of cell death, activation of angiogenic cellular programmes and induction of M2 macrophage polarization. MACs infected with B. henselae incorporated into endothelial sprouts and increased angiogenic growth. Infected MACs developed a vascular mimicry phenotype in vitro, and expression of B. henselae adhesin A was essential in inducing these angiogenic effects. Secretome analysis revealed that increased pro-angiogenic activities were associated with the creation of a tumour-like microenvironment dominated by angiogenic inflammatory cytokines and matrix remodelling compounds. Our results demonstrate that manipulation of myeloid cells by pathogenic bacteria can contribute to microenvironmental regulation of pathological tissue growth and suggest parallels underlying both bacterial infections and cancer.

Mechanisms of endothelial cell migration.

Cell migration plays a central role in a variety of physiological and pathological processes during our whole life. Cellular movement is a complex, tightly regulated multistep process. Although the principle mechanisms of migration follow a defined general motility cycle, the cell type and the context of moving influences the detailed mode of migration. Endothelial cells migrate during vasculogenesis and angiogenesis but also in a damaged vessel to restore vessel integrity. Depending on the situation they migrate individually, in chains or sheets and complex signaling, intercellular signals as well as environmental cues modulate the process. Here, the different modes of cell migration, the peculiarities of endothelial cell migration and specific guidance molecules controlling this process will be reviewed.

The polarity protein Scrib is essential for directed endothelial cell migration.

RATIONALE: Polarity proteins are involved in the apico-basal orientation of epithelial cells, but relatively little is known regarding their function in mesenchymal cells. OBJECTIVE: We hypothesized that polarity proteins also contribute to endothelial processes like angiogenesis. METHODS AND RESULTS: Screening of endothelial cells revealed high expression of the polarity protein Scribble (Scrib). On fibronectin-coated carriers Scrib siRNA (siScrib) blocked directed but not random migration of human umbilical vein endothelial cells and led to an increased number and disturbed orientation of cellular lamellipodia. Coimmunoprecipitation/mass spectrometry and glutathione S-transferase (GST) pulldown assays identified integrin α5 as a novel Scrib interacting protein. By total internal reflection fluorescence (TIRF) microscopy, Scrib and integrin α5 colocalize at the basal plasma membrane of endothelial cells. Western blot and fluorescence activated cell sorting (FACS) analysis revealed that silencing of Scrib reduced the protein amount and surface expression of integrin α5 whereas surface expression of integrin αV was unaffected. Moreover, in contrast to fibronectin, the ligand of integrin α5, directional migration on collagen mediated by collagen-binding integrins was unaffected by siScrib. Mechanistically, Scrib supported integrin α5 recycling and protein stability by blocking its interaction with Rab7a, its translocation into lysosomes, and its subsequent degradation by pepstatin-sensitive proteases. In siScrib-treated cells, reinduction of the wild-type protein but not of PSD95, Dlg, ZO-1 (PDZ), or leucine rich repeat domain mutants restored integrin α5 abundance and directional cell migration. The downregulation of Scrib function in Tg(kdrl:EGFP)(s843) transgenic zebrafish embryos delayed the angiogenesis of intersegmental vessels. CONCLUSIONS: Scrib is a novel regulator of integrin α5 turnover and sorting, which is required for oriented cell migration and sprouting angiogenesis.

Nox4 is a protective reactive oxygen species generating vascular NADPH oxidase.

RATIONALE: The function of Nox4, a source of vascular H(2)O(2), is unknown. Other Nox proteins were identified as mediators of endothelial dysfunction. OBJECTIVE: We determined the function of Nox4 in situations of increased stress induced by ischemia or angiotensin II with global and tamoxifen-inducible Nox4(-/-) mice. METHODS AND RESULTS: Nox4 was highly expressed in the endothelium and contributed to H(2)O(2) formation. Nox4(-/-) mice exhibited attenuated angiogenesis (femoral artery ligation) and PEG-catalase treatment in control mice had a similar effect. Tube formation in cultured Nox4(-/-) lung endothelial cells (LECs) was attenuated and restored by low concentrations of H(2)O(2,) whereas PEG-catalase attenuated tube formation in control LECs. Angiotensin II infusion was used as a model of oxidative stress. Compared to wild-type, aortas from inducible Nox4-deficient animals had development of increased inflammation, media hypertrophy, and endothelial dysfunction. Mechanistically, loss of Nox4 resulted in reduction of endothelial nitric oxide synthase expression, nitric oxide production, and heme oxygenase-1 (HO-1) expression, which was associated with apoptosis and inflammatory activation. HO-1 expression is controlled by Nrf-2. Accordingly, Nox4-deficient LECs exhibited reduced Nrf-2 protein level and deletion of Nox4 reduced Nrf-2 reporter gene activity. In vivo treatment with hemin, an inducer of HO-1, blocked the vascular hypertrophy induced by Nox4 deletion in the angiotensin II infusion model and carbon monoxide, the product of HO-1, blocked the Nox4-deletion-induced apoptosis in LECs. CONCLUSION: Endogenous Nox4 protects the vasculature during ischemic or inflammatory stress. Different from Nox1 and Nox2, this particular NADPH oxidase therefore may have a protective vascular function.

Chemotherapy-associated angiogenesis in neuroblastoma tumors.

The influences of cytotoxic drugs on endothelial cells remain incompletely understood. Herein, we examined the effects of chemotherapeutic agents in experimental angiogenesis models and analyzed vessel densities in clinical neuroblastoma tumor samples. Cisplatin (20 to 500 ng/mL), doxorubicin (4 to 100 ng/mL), and vincristine (0.5 to 4 ng/mL), drugs commonly involved in neuroblastoma therapy protocols, induced pro-angiogenic effects in different angiogenesis models. They enhanced endothelial cell tube formation, endothelial cell sprouting from spheroids, formation of tip cells in the sprouting assay, expression of αvß3 integrin, and vitronectin binding. All three drugs increased global cellular kinase phosphorylation levels, including the angiogenesis-relevant molecules protein kinase Cß and Akt. Pharmacological inhibition of protein kinase Cß or Akt upstream of phosphatidylinositol 3-kinase reduced chemotherapy-induced endothelial cell tube formation. Moreover, the investigated chemotherapeutics dose dependently induced vessel formation in the chick chorioallantoic membrane assay. Tumor samples from seven high-risk patients with neuroblastoma were analyzed for vessel density by IHC. Results revealed that neuroblastoma samples taken after chemotherapy consistently showed an enhanced microvessel density compared with the corresponding samples taken before chemotherapy. In conclusion, our data show that chemotherapy can activate endothelial cells by inducing multiple pro-angiogenic signaling pathways and exert pro-angiogenic effects in vitro and in vivo. Moreover, we report a previously unrecognized clinical phenomenon that might, in part, be explained by our experimental observations: chemotherapy-associated enhanced vessel formation in tumors from patients with neuroblastoma.

Hepatocyte growth factor induces a proangiogenic phenotype and mobilizes endothelial progenitor cells by activating Nox2.

Hepatocyte growth factor (HGF) by stimulating the receptor tyrosine kinase c-Met induces angiogenesis and tissue regeneration. HGF has been shown to antagonize the angiotensin II-induced senescence of endothelial progenitor cells (EPCs), which is mediated by NADPH oxidase-dependent reactive oxygen species (ROS) formation. As growth factors, however, usually require ROS for their signaling, we hypothesized that the proangiogenic effects of HGF require NADPH oxidases and focused on the homolog Nox2, which is most abundantly expressed in EPCs and endothelial cells. Indeed, HGF increased the H(2)O(2) formation in EPCs and human umbilical vein endothelial cells (HUVECs), and this effect was not observed in Nox2-deficient cells. HGF induced the mobilization of EPCs and vascular outgrowth from aortic explants in wild-type (WT) but not Nox2(y/-) mice. HGF also stimulated migration and tube formation in HUVECs, and antisense oligonucleotides against Nox2 prevented this effect. To identify the signal transduction underlying these effects, we focused on the kinases Jak2 and Jnk. In HUVECs, HGF increased the phosphorylation of these in a Nox2-dependent manner as demonstrated by antisense oligonucleotides. Also, the HGF-induced Jak2-dependent activation of a STAT3 reporter construct was attenuated after downregulation of Nox2. Accordingly, the HGF-stimulated tube formation of HUVEC was blocked by inhibitors of Jak2 and Jnk. In vivo treatment with the Jnk inhibitor SP600125 blocked the HGF-induced mobilization of EPCs. Ex vivo, SP600125 blocked HGF-induced migration and tube formation. We conclude that HGF-induced mobilization of EPCs and the proangiogenic effects of the growth factor require a Nox2-dependent ROS-mediated activation of Jak2 and Jnk.

Epoxyeicosatrienoic acids are part of the VEGF-activated signaling cascade leading to angiogenesis.

Cytochrome P-450 (CYP) epoxygenases metabolize arachidonic acid to epoxyeicosatrienoic acid (EET) regioisomers, which activate several signaling pathways to promote endothelial cell proliferation, migration, and angiogenesis. Since vascular endothelial growth factor (VEGF) plays a key role in angiogenesis, we assessed a possible role of EETs in the VEGF-activated signal transduction cascade. Stimulation with VEGF increased CYP2C promoter activity in endothelial cells and enhanced CYP2C8 mRNA and protein expression resulting in increased intracellular EET levels. VEGF-induced endothelial cell tube formation was inhibited by the EET antagonist 14,15-epoxyeicosa-5(Z)-enoicacid (14,15-EEZE), which did not affect the VEGF-induced phosphorylation of its receptor or basic fibroblast growth factor (bFGF)-stimulated tube formation. Moreover, VEGF-stimulated endothelial cell sprouting in a modified spheroid assay was reduced by CYP2C antisense oligonucleotides. Mechanistically, VEGF stimulated the phosphorylation of the AMP-activated protein kinase (AMPK), which has also been linked to CYP induction, and the overexpression of a constitutively active AMPK mutant increased CYP2C expression. On the other hand, a dominant-negative AMPK mutant prevented the VEGF-induced increase in CYP2C RNA and protein expression in human endothelial cells. In vivo (Matrigel plug assay) in mice, endothelial cells were recruited into VEGF-impregnated plugs; an effect that was sensitive to 14,15-EEZE and the inclusion of small interfering RNA directed against the AMPK. The EET antagonist did not affect responses observed in plugs containing bFGF. Taken together, our data indicate that CYP2C-derived EETs participate as second messengers in the angiogenic response initiated by VEGF and that preventing the increase in CYP expression curtails the angiogenic response to VEGF.

Cytochrome P450 2C9-induced angiogenesis is dependent on EphB4.

OBJECTIVE: Cytochrome P450 (CYP) epoxygenase-derived epoxyeicosatrienoic acids (EETs) are known to stimulate angiogenesis, but the mechanisms involved are incompletely understood. Because EphB4 is involved in vascular development, the aim of this study was to investigate whether, and to what extent, EphB4 is part of the signaling cascade that results in CYP2C9-mediated angiogenesis. METHODS AND RESULTS: CYP2C9 overexpression as well as stimulation with 11,12-EET (up to 48 hours) time-dependently increased EphB4 expression in endothelial cells. This effect and the activation of the EphB4 promoter were mediated by the phosphatidylinositol-3-kinase (P13-K)/Akt pathway and sensitive to the P13-K inhibitor LY 294002 as well as to simultaneous transfection with dominant-negative Akt. 11,12-EET treatment also increased EphB4 expression in isolated mouse mesenteric arteries as well as in the vessels that developed in 11,12-EET-impregnated Matrigel plugs. Moreover, the CYP2C9-stimulated formation of capillary-like structures in a modified spheroid assay was markedly attenuated by EphB4 downregulation (antisense oligonucleotides). Using a parallel approach in vivo, the inclusion of siRNA directed against EphB4 in EET-impregnated Matrigel plugs prevented endothelial cell invasion and vascularization. CONCLUSIONS: Our data indicate that EphB4 is a critical component of the CYP2C9- and 11,12-EET-activated signaling cascade that promotes angiogenesis in vitro as well as in vivo.

Role of cytochrome P450 2C epoxygenases in hypoxia-induced cell migration and angiogenesis in retinal endothelial cells.

PURPOSE: Cytochrome P450 (CYP) epoxygenase-derived epoxyeicosatrienoic acids (EETs) elicit cell proliferation and promote angiogenesis. The aim of this study was to determine the expression of CYP epoxygenases in the bovine retina and the potential role of EETs in hypoxia-induced angiogenesis in bovine retinal endothelial cells. METHODS: Bovine retinal endothelial cells were cultured under normoxic (21% O(2)) or hypoxic (1% O(2)) conditions, and CYP2C expression was determined by Western blot analysis. The effect of hypoxia on EET levels was determined by LC-MS/MS. Cell migration (Transwell filter assays) and endothelial cell tube formation (on basement membrane matrix) were assessed in vitro in the absence and presence of pharmacologic inhibitors and CYP2C antisense oligonucleotides. RESULTS: Bovine retinal endothelial cells expressed CYP2C protein in culture and generated detectable levels of EETs under basal conditions. Hypoxia (6-48 hours) enhanced CYP2C protein expression (2-fold) and EET formation (1.5-fold). Moreover, endothelial cells preexposed to hypoxia demonstrated an increase in serum-induced cell migration that was sensitive to the CYP2C inhibitors sulfaphenazole and MS-PPOH and the EET antagonist 14,15-epoxyeicosa-5(Z)-enoic acid. Furthermore, preventing the hypoxia-induced expression of CYP2C (antisense oligonucleotides) suppressed hypoxia-induced cell migration. In an in vitro angiogenesis model, the preexposure of endothelial cells to hypoxia increased CYP2C expression and enhanced endothelial tube formation, which was blocked by the EET antagonist and by the CYP2C antisense oligonucleotides. CONCLUSIONS: Taken together, these data indicate that CYP2C-derived EETs are implicated in angiogenesis by retinal endothelial cells, especially under hypoxic conditions.

From endothelium-derived hyperpolarizing factor (EDHF) to angiogenesis: Epoxyeicosatrienoic acids (EETs) and cell signaling.

Epoxyeicosatrienoic acids (EETs) are generated from arachidonic acid by cytochrome P450 (CYP) epoxygenases. The expression of CYP epoxygenases in endothelial cells is determined by a number of physical (fluid shear stress and cyclic stretch) and pharmacological stimuli as well as by hypoxia. The activation of CYP epoxygenases in endothelial cells is an important step in the nitric oxide and prostacyclin (PGI2)-independent vasodilatation of several vascular beds and EETs have been identified as endothelium-derived hyperpolarizing factors (EDHFs). However, in addition to regulating vascular tone, EETs modulate several signaling cascades and affect cell proliferation, cell migration, and angiogenesis. Signaling molecules modulated by EETs include tyrosine kinases and phosphatases, mitogen-activated protein kinases, protein kinase A (PKA), cyclooxygenase (COX)-2, and several transcription factors. This review summarizes the role of CYP-derived EETs in cell signaling and focuses particularly on their role as intracellular amplifiers of endothelial cell hyperpolarization as well as in cell proliferation and angiogenesis. The angiogenic properties of CYP epoxygenases and CYP-derived EETs implicate that these enzymes may well be accessible targets for anti-angiogenic as well as angiogenic therapies.

Cytochrome P450 epoxygenases 2C8 and 2C9 are implicated in hypoxia-induced endothelial cell migration and angiogenesis.

Recent studies suggest that cytochrome P450 (CYP) epoxygenase-derived epoxyeicosatrienoic acids (EETs) elicit cell proliferation and promote angiogenesis. The aim of this study was to determine the role of CYP 2C8/9-derived EETs in the process of angiogenesis under hypoxic conditions. In human endothelial cells, hypoxia enhanced the activity of the CYP 2C9 promoter, increased the expression of CYP 2C mRNA and protein and augmented 11,12-EET production. In Transwell assays, the migration of endothelial cells pre-exposed to hypoxia to increase CYP expression was abolished by CYP 2C antisense oligonucleotides as well as by the CYP inhibitor MS-PPOH and the EET antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (EEZE). Similar findings were obtained in porcine coronary artery endothelial cells. CYP 2C9 overexpression in endothelial cells increased the association of PAK-1 with Rac, a response also elicited by the CYP 2C9 product 11,12-EET. Matrix metalloprotease (MMP) activity was increased in CYP-2C9-overexpressing cells and correlated with increased invasion through Matrigel-coated Transwell chambers: an effect sensitive to the CYP 2C9 inhibitor sulfaphenazole as well as to EEZE and the MMP inhibitor GM6001. In in vitro angiogenesis models, the EET antagonist inhibited tube formation induced by CYP 2C9 overexpression as well as that in endothelial cells exposed to hypoxia to increase CYP 2C expression. Furthermore, in the chick chorioallantoic membrane assay, EEZE abolished hypoxia-induced angiogenesis. Taken together, these data indicate that CYP 2C-derived EETs significantly affect the sequence of angiogenic events under hypoxic conditions.

Cytochrome P4502C9-derived epoxyeicosatrienoic acids induce the expression of cyclooxygenase-2 in endothelial cells.

OBJECTIVE: Cytochrome P450 (CYP) epoxygenases metabolize arachidonic acid to epoxyeicosatrienoic acids (EETs). CYP2C9-derived EETs elicit endothelial cell proliferation and angiogenesis, but the signaling pathways involved are incompletely understood. Because cyclooxygenase-2 (COX-2) is involved in angiogenesis, we determined whether a link exists between CYP2C9 and COX-2 expression. METHODS AND RESULTS: Human umbilical vein endothelial cells were infected with CYP2C9 sense or antisense adenoviral constructs. Overexpression of CYP2C9 increased COX-2 promoter activity, an effect accompanied by a significant increase in COX-2 protein expression and elevated prostacyclin production. The CYP2C9-induced expression of COX-2 was inhibited by the CYP2C9 inhibitor, sulfaphenazole, whereas 11,12-EET increased COX-2 expression. Overexpression of CYP2C9 and stimulation with 11,12-EET increased intracellular cAMP levels and stimulated DNA-binding of the cAMP-response element-binding protein. The protein kinase A inhibitor, KT5720, attenuated the CYP2C9-induced increase in COX-2 promoter activity and protein expression. Overexpression of CYP2C9 stimulated endothelial tube formation, an effect that was attenuated by the COX-2 inhibitor celecoxib. Identical responses were observed in cells preconditioned by cyclic strain to increase CYP2C expression. CONCLUSIONS: These data indicate that CYP2C9-derived EETs induce the expression of COX-2 in endothelial cells via a cAMP-dependent pathway and that this mechanism contributes to CYP2C9-induced angiogenesis. Overexpression of cytochrome P450 (CYP) 2C9 in endothelial cells increased cAMP levels, stimulated the cAMP-response element-binding protein, and enhanced cyclooxygenase-2 (COX-2) promoter activity, protein expression, and prostacyclin production. CYP2C9 overexpression stimulated endothelial tube formation, which was attenuated by the COX-2 inhibitor celecoxib. Thus, COX-2 contributes to CYP2C9-induced angiogenesis.

Valproic acid inhibits angiogenesis in vitro and in vivo.

Valproic acid (VPA) is a widely used antiepileptic agent that is undergoing clinical evaluation for anticancer therapy. We assessed the effects of VPA on angiogenesis in vitro and in vivo. In human umbilical vein endothelial cells, therapeutically relevant concentrations of VPA (0.25 to 1 mM) inhibited proliferation, migration, and tube formation. VPA 1 mM inhibited endothelial cell proliferation by 51 +/- 5%, migration by 86 +/- 11%, and tube formation by 82 +/- 3%. These changes were preceded by the hyperacetylation of histone H4, indicating the inhibition of histone deacetylase (HDAC), and a decreased expression of the endothelial nitric-oxide synthase (eNOS). The inhibition of endothelial cell tube formation by VPA was prevented by addition of the nitric oxide donor (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA NONOate). The anticonvulsive active VPA derivative 2-ethyl-4-methylpentanoic acid, which does not inhibit HDAC, did not affect endothelial cell proliferation, tube formation, or eNOS expression. VPA was also found to inhibit angiogenesis in vivo in the chicken chorioallantoic membrane assay and in a Matrigel plug assay in mice. Embryos from VPA-treated mice showed disturbed vessel formation. These results indicate that therapeutic plasma levels of VPA inhibit angiogenesis by a mechanism involving a decrease in eNOS expression preceded by HDAC inhibition.

Cytochrome P450 epoxygenases and vascular tone: novel role for HMG-CoA reductase inhibitors in the regulation of CYP 2C expression.

Over the last 10 years it has become increasingly clear that cytochrome P450 (CYP) enzymes expressed within endothelial and vascular smooth muscle cells play a crucial role in the modulation of vascular homeostasis. There is strong evidence suggesting that the activation of a CYP 2C epoxygenase in endothelial cells is an essential step in nitric oxide (NO)- and prostacyclin (PGI(2))-independent vasodilatation of several vascular beds, particularly in the heart and kidney. Moreover, CYP epoxygenase products as well as CYP-derived reactive oxygen species are intracellular signal transduction molecules involved in several signaling cascades affecting numerous cellular processes, including vascular cell proliferation and angiogenesis. Various pharmacological compounds enhance vascular CYP 2C expression. One group of substances which highlight the possible effects of CYP induction in endothelial cells on vascular function are the HMG-CoA reductase inhibitors (statins). Cerivastatin and fluvastatin increase CYP 2C mRNA and protein in native and cultured endothelial cells, and enhance the bradykinin-induced NO/PGI(2)-independent relaxation of arterial segments as well as the generation of reactive oxygen species. However, statins also increase the expression of the endothelial NO synthase by approximately twofold. As a consequence, the probability that NO and reactive oxygen species react to generate peroxynitrite is increased and the treatment of vascular segments with statins resulted in enhanced protein tyrosine nitration. These data highlight the role played by CYP 2C in vascular homeostasis and its potential regulation by cardiovascular drugs.

Cytochrome P450 2C9-derived epoxyeicosatrienoic acids induce angiogenesis via cross-talk with the epidermal growth factor receptor (EGFR).

Cytochrome P450 (CYP) epoxygenase products, such as 11,12-epoxyeicosatrienoic acid (EET), stimulate endothelial cell proliferation. We set out to identify the signal transduction cascade linking EET generation to enhanced proliferation and angiogenesis. In human endothelial cells overexpressing CYP 2C9, cell number was increased compared with control cells and was inhibited by the CYP 2C9 inhibitor, sulfaphenazole. CYP 2C9 overexpression was associated with the activation of Akt and an increase in cyclin D1 expression, effects that were abolished by the epidermal growth factor (EGF) receptor inhibitor, AG1478, which also prevented the CYP 2C9-induced increase in cell proliferation. Stimulation of EGF receptor overexpressing cells with 11,12-EET or transfection of these cells with CYP 2C9 enhanced the tyrosine phosphorylation of the EGF receptor. Endothelial tube formation in a fibrin gel was significantly enhanced (6-fold) in CYP 2C9 overexpressing cells and was comparable with the tube formation induced by EGF. In the chick chorioallantoic membrane, 11,12-EET stimulated vessel formation (3.5-fold) and induced vessel convergence, an effect that was abolished by cotreatment with either an EGF receptor-neutralizing antibody or AG1478. These results indicate that CYP 2C9-derived EETs stimulate angiogenesis by a mechanism involving the activation of the EGF receptor.

Cytochrome P450 2C9-induced endothelial cell proliferation involves induction of mitogen-activated protein (MAP) kinase phosphatase-1, inhibition of the c-Jun N-terminal kinase, and up-regulation of cyclin D1.

Cytochrome P450 (CYP)-derived epoxyeicosatrienoic acids (EETs) are important modulators of endothelial cell homeostasis. We investigated the signaling pathway linking the activation of CYP 2C9 to enhanced endothelial cell proliferation. Overexpression of CYP 2C9 in cultured human endothelial cells markedly increased proliferation. This effect was paralleled by an up-regulation of the G(1) phase regulatory protein, cyclin D1. The specific CYP 2C9 inhibitor, sulfaphenazole, prevented both the enhanced cell proliferation and up-regulation of cyclin D1. CYP 2C9 overexpression also decreased the activity of the c-Jun N-terminal kinase (JNK). Coexpression of wild type JNK with CYP 2C9 attenuated the CYP 2C9-induced increase in cyclin D1 expression and abolished the CYP 2C9-induced proliferation response. In contrast, cotransfecting dominant negative JNK with CYP 2C9 restored the CYP 2C9-mediated up-regulation of cyclin D1 and proliferation. The inactivation of JNK is linked to its dephosphorylation by dual specificity mitogen-activated protein (MAP) kinase phosphatases (MKPs). Overexpression of CYP 2C9 significantly increased the expression of MKP-1, as did incubation with 11,12-EET. These data demonstrate that the mitogenic effect of CYP 2C9 is due to the generation of EETs, which promote the MKP-1-mediated dephosphorylation and inactivation of JNK, effects ultimately culminating in the expression of cyclin D1 and endothelial cell proliferation.