Biomedical subjects

Rudi Busse

Publications and source records attributed to Rudi Busse.

At least 37 records · Page 2Linked to original sources

PubMed2005

New fACEs to the renin-angiotensin system.

Inhibition of the angiotensin-converting enzyme (ACE) protects against the progression of several cardiovascular diseases. Recent evidence suggests that some of the beneficial effects of ACE inhibitors can be attributed to the activation of a distinct ACE signaling cascade rather than to the changes in angiotensin II and bradykinin levels. Moreover, at least one other ACE homolog (ACE2) plays a significant role in the regulation of heart and kidney function.

Amino Acid Sequence↗

PubMed2004

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.

6-Ketoprostaglandin F1 alpha↗

PubMed2004

Signaling via the angiotensin-converting enzyme enhances the expression of cyclooxygenase-2 in endothelial cells.

Angiotensin-converting enzyme (ACE) inhibitors elicit outside-in signaling via ACE in endothelial cells. This involves the CK2-mediated phosphorylation of ACE on Ser1270 and the activation of the c-Jun N-terminal kinase (JNK)/c-Jun pathway, resulting in an enhanced endothelial ACE expression. Because cyclooxygenase-2 (COX-2) expression is reported to be increased in subjects treated with ACE inhibitors, we determined the role of ACE signaling in this phenomenon and the transcription factors involved. In lungs from mice treated with the ACE inhibitor ramipril for 5 days, COX-2 expression was increased. A similar (1.5- to 2-fold) increase in COX-2 protein was detected in primary cultures of human endothelial cells treated with ramiprilat. In an endothelial cell line stably expressing human somatic ACE, ramiprilat increased COX-2 promoter activity, an effect not observed in ACE-deficient cells or cells expressing a nonphosphorylatable ACE mutant (S1270A). The ramiprilat-induced, ACE-dependent increase in COX-2 expression and promoter activity (both 1.5- to 2-fold greater than control) was prevented by the inhibition of JNK. Ramiprilat significantly enhanced the DNA binding activity of activator protein-1 in cells expressing ACE but not S1270A ACE. Activator protein-1 decoy oligonucleotides prevented the ACE inhibitor-induced increase in COX-2 promoter activity and protein expression. As a consequence of the ramiprilat-induced increase in COX-2 expression, prostacyclin and prostaglandin E2, but not thromboxane A2, production was increased and was inhibited by the COX-2 inhibitor celecoxib. These results indicate that ACE signaling may underlie the increase in COX-2 and prostacyclin levels in patients treated with ACE inhibitors.

6-Ketoprostaglandin F1 alpha↗

PubMed2004

Direct interaction of the novel Nox proteins with p22phox is required for the formation of a functionally active NADPH oxidase.

Nox1 and Nox4, homologues of the leukocyte NADPH oxidase subunit Nox2 (gp91phox) mediate superoxide anion formation in various cell types. However, their interactions with other components of the NADPH oxidase are poorly defined. We determined whether a direct interaction of Nox1 and Nox4 with the p22phox subunit of the NADPH oxidase occurs. Using confocal microscopy, co-localization of p22phox with Nox1, Nox2, and Nox4 was observed in transiently transfected vascular smooth muscle cells (VSMC) and HEK293 cells. Plasmids coding for fluorescent fusion proteins of p22phox and the Nox proteins with cyan- and yellow-fluorescent protein (cfp and yfp, respectively) were constructed and expressed in VSMC and HEK293 cells. The cfp-tagged p22phox expression level increased upon cotransfection with Nox1 or Nox4. Protein-protein interaction between the fluorescent fusion proteins of p22phox and the Nox partners was observed using the fluorescence resonance energy transfer technique. Immunoprecipitation of native Nox1 from human VSMC revealed co-precipitation of p22phox. Immunoprecipitation from transfected HEK293 cells revealed co-precipitation of native p22phox with yfp-tagged Nox1, Nox2, and Nox4. Following mutation of a histidine (corresponding to the position 115 in human Nox2) to leucine, this interaction was abolished. Transfection of rat p22phox (but not Noxo1 and Noxa1) increased the radical generation in cells expressing Nox4. We provide evidence that p22phox directly interacts with Nox1 and Nox4, to form an superoxide-generating NADPH oxidase and demonstrate that mutation of the potential heme binding site in the Nox proteins disrupts the complex formation of Nox1 and Nox4 with p22phox.

Binding Sites↗

PubMed2004

Insulin induces the release of vasodilator compounds from platelets by a nitric oxide-G kinase-VAMP-3-dependent pathway.

Insulin-induced vasodilatation is sensitive to nitric oxide (NO) synthase (NOS) inhibitors. However, insulin is unable to relax isolated arteries or to activate endothelial NOS in endothelial cells. Since insulin can enhance platelet endothelial NOS activity, we determined whether insulin-induced vasodilatation can be attributed to a NO-dependent, platelet-mediated process. Insulin failed to relax endothelium-intact rings of porcine coronary artery. The supernatant from insulin-stimulated human platelets induced complete relaxation, which was prevented by preincubation of platelets with a NOS inhibitor, the soluble guanylyl cyclase inhibitor, NS 2028, or the G kinase inhibitor, KT 5823, and was abolished by an adenosine A2A receptor antagonist. Insulin induced the release of adenosine trisphosphate (ATP), adenosine, and serotonin from platelet-dense granules in a NO-dependent manner. This response was not detected using insulin-stimulated platelets from endothelial NOS-/- mice, although a NO donor elicited ATP release. Insulin-induced ATP release from human platelets correlated with the association of syntaxin 2 with the vesicle-associated membrane protein 3 but was not associated with the activation of alphaIIbbeta3 integrin. Thus, insulin elicits the release of vasoactive concentrations of ATP and adenosine from human platelets via a NO-G kinase-dependent signaling cascade. The mechanism of dense granule secretion involves the G kinase-dependent association of syntaxin 2 with vesicle-associated membrane protein 3.

Adenosine↗

PubMed2004

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.

Angiogenesis Inhibitors↗

PubMed2004

From blood monocytes to adipose tissue-resident macrophages: induction of diapedesis by human mature adipocytes.

Obesity has been suggested to be a low-grade systemic inflammatory state, therefore we studied the interaction between human adipocytes and monocytes via adipose tissue (AT)-derived capillary endothelium. Cells composing the stroma-vascular fraction (SVF) of human ATs were characterized by fluorescence-activated cell sorter (FACS) analysis and two cell subsets (resident macrophages and endothelial cells [ECs]) were isolated using antibody-coupled microbeads. Media conditioned by mature adipocytes maintained in fibrin gels were applied to AT-derived ECs. Thereafter, the expression of endothelial adhesion molecules was analyzed as well as the adhesion and transmigration of human monocytes. FACS analysis showed that 11% of the SVF is composed of CD14(+)/CD31(+) cells, characterized as resident macrophages. A positive correlation was found between the BMI and the percentage of resident macrophages, suggesting that fat tissue growth is associated with a recruitment of blood monocytes. Incubation of AT-derived ECs with adipocyte-conditioned medium resulted in the upregulation of EC adhesion molecules and the increased chemotaxis of blood monocytes, an effect mimicked by recombinant human leptin. These results indicate that adipokines, such as leptin, activate ECs, leading to an enhanced diapedesis of blood monocytes, and suggesting that fat mass growth might be linked to inflammatory processes.

Adipocytes↗

PubMed2003

Inhibition of cytochrome P450 2C9 improves endothelium-dependent, nitric oxide-mediated vasodilatation in patients with coronary artery disease.

BACKGROUND: Nitric oxide (NO)- and prostacyclin-independent vasodilatation in several vascular beds has been linked to the activation of cytochrome P450 (CYP) epoxygenases expressed in endothelial cells. However, these enzymes, which generate vasodilator epoxyeicosatrienoic acids, may also produce oxygen-derived free radicals, which attenuate the bioavailability of NO. Here, we studied the involvement of CYP 2C9 in modulating endothelium-dependent and -independent changes in forearm blood flow (FBF) in healthy volunteers and in patients with manifest coronary artery disease. METHODS AND RESULTS: The effects of sulfaphenazole, a selective inhibitor of CYP 2C9, on endothelium-dependent (acetylcholine) and endothelium-independent (sodium nitroprusside, SNP) FBF responses were measured by venous occlusion plethysmography in 5 healthy subjects and in 16 patients with angiographically documented stable coronary artery disease. Sulfaphenazole did not modify FBF responses to acetylcholine or SNP in healthy subjects. In contrast, sulfaphenazole markedly and dose-dependently enhanced the FBF response to acetylcholine without affecting the response to SNP. Vitamin C also increased the FBF response to acetylcholine, but this effect was further potentiated by sulfaphenazole. In the presence of N(omega)-monomethyl-l-arginine, sulfaphenazole failed to significantly improve acetylcholine-induced vasodilatation. The oxidation of serum proteins was enhanced in patients with coronary artery disease, and this effect was significantly attenuated by sulfaphenazole. CONCLUSIONS: The CYP 2C9 inhibitor sulfaphenazole enhances endothelium-dependent vasodilator responses in patients with manifest coronary artery disease. This effect seems to be related to an increase in the bioavailability of NO, probably as a consequence of an attenuated generation of reactive oxygen species by CYP 2C9 in endothelial cells.

Acetylcholine↗

PubMed2003

Angiotensin-converting enzyme is involved in outside-in signaling in endothelial cells.

Not all of the cardiovascular effects of angiotensin-converting enzyme (ACE) inhibitors can be attributed to changes in angiotensin II and bradykinin levels. Because the cytoplasmic tail of ACE is phosphorylated, we determined whether ACE inhibitors affect the phosphorylation of ACE and whether ACE possesses the characteristics of a signal transduction molecule. The ACE inhibitors ramiprilat and perindoprilat, and the substrate bradykinin (but not angiotensin I), enhanced the activity of ACE-associated CK2 and the phosphorylation of ACE Ser1270 in cultured endothelial cells. Mitogen-activated protein kinase kinase 7 and c-Jun N-terminal kinase (JNK) coprecipitated with ACE, and stimulation of endothelial cells with ACE inhibitors increased the activity of ACE-associated JNK and elicited the accumulation of phosphorylated c-Jun in the nucleus. Ramiprilat was however unable to activate JNK or to stimulate the nuclear accumulation of c-Jun in endothelial cells expressing a S1270A ACE mutant or in ACE-deficient cells. Because the ACE inhibitor-induced increase in ACE expression has been linked to the formation of c-Jun homodimers, we investigated whether ACE signaling via JNK contributes to this response in vitro and in vivo. Prolonged ramiprilat treatment increased ACE expression in primary cultures of human endothelial cells and in vivo (mouse lung), a response that was prevented by pretreatment with the JNK inhibitor SP600125. Thus, ACE is involved in outside-in signaling in endothelial cells and "ACE signaling" may be an important cellular mechanism contributing to the beneficial effects of ACE inhibitors.

Angiotensin-Converting Enzyme Inhibitors↗

PubMed2003

Amlodipine activates the endothelial nitric oxide synthase by altering phosphorylation on Ser1177 and Thr495.

OBJECTIVE: The Ca2+ antagonist amlodipine increases the generation of nitric oxide (NO) from native and cultured endothelial cells. The aim of this investigation was to determine whether or not the activation of the endothelial NO synthase (eNOS) by this Ca2+ antagonist is related to alterations in eNOS phosphorylation. METHODS AND RESULTS: In isolated, pre-contracted, endothelium-intact porcine coronary arteries, amlodipine elicited an NO-mediated relaxation and a leftward shift in the concentration-relaxation curve to bradykinin. Moreover, the Ca2+ antagonist increased the generation of NO from native endothelial cells, as detected by electron spin resonance spectroscopy and stimulated an 8-fold increase in cyclic GMP levels in cultured endothelial cells. In unstimulated endothelial cells, eNOS was not phosphorylated on Ser1177 but was phosphorylated on Thr495. Amlodipine elicited the phosphorylation of Ser1177 and attenuated Thr495 phosphorylation, with a time course similar to that of eNOS activation. The amlodipine-induced relaxation of porcine coronary arteries was attenuated by the B2 kinin receptor antagonist, icatibant, but this antagonist did not affect amlodipine-induced changes in eNOS phosphorylation in cultured endothelial cells. Moreover, amlodipine elicited the NO-mediated relaxation of rat aortic rings which do not express the B2 receptor. Amlodipine time-dependently attenuated the phosphorylation of protein kinase C (PKC) in endothelial cells, with a time course similar to the changes in eNOS phosphorylation, and prevented the phorbol-12-myristate-13-acetate-induced activation of PKC. The PKC inhibitor, Ro 31-8220, also elicited the phosphorylation of Ser1177 and the dephosphorylation of Thr495 in cultured cells and induced a leftward shift in the concentration-relaxation curve to bradykinin in rings of porcine coronary artery. CONCLUSION: The Ca2+ antagonist, amlodipine, enhances endothelial NO generation by inducing changes in the phosphorylation of eNOS. Although the activation of eNOS was related to the activation of the B2 kinin receptor in the porcine coronary artery, a B2 receptor-independent mechanism involving the inhibition of PKC appears to account for the effects observed in the rat aorta as well as in cultured endothelial cells.

Amlodipine↗

PubMed2003

Involvement of a cGMP-dependent pathway in the natriuretic peptide-mediated hormone-sensitive lipase phosphorylation in human adipocytes.

Our previous studies have demonstrated that natriuretic peptides (NPs), peptide hormones with natriuretic, diuretic, and vasodilating properties, exert a potent control on the lipolysis in human adipocytes via the activation of the type A guanylyl cyclase receptor (1, 2). In the current study we investigated the intracellular mechanisms involved in the NP-stimulated lipolytic effect in human preadipocytes and adipocytes. We demonstrate that the atrial NP (ANP)-induced lipolysis in human adipocytes was associated with an enhanced serine phosphorylation of the hormone-sensitive lipase (HSL). Both ANP-mediated lipolysis and HSL phosphorylation were inhibited in the presence of increasing concentrations of the guanylyl cyclase inhibitor LY-83583. ANP did not modulate the activity of the cAMP-dependent protein kinase (PKA). Moreover, H-89, a PKA inhibitor, did not affect the ANP-induced lipolysis. On primary cultures of human preadipocytes, the ANP-mediated lipolytic effect was dependent on the differentiation process. On differentiated human preadipocytes, ANP-mediated lipolysis, associated with an increased phosphorylation of HSL and of perilipin A, was strongly decreased by treatment with the inhibitor of the cGMP-dependent protein kinase I (cGKI), Rp-8-pCPT-cGMPS. Thus, ANP-induced lipolysis in human adipocytes is a cGMP-dependent pathway that induces the phosphorylation of HSL and perilipin A via the activation of cGKI. The present study shows that lipolysis in human adipocytes can be controlled by an independent cGKI-mediated signaling as well as by the classical cAMP/PKA pathway.

Adipocytes↗

PubMed2003

Phosphorylation of threonine 497 in endothelial nitric-oxide synthase coordinates the coupling of L-arginine metabolism to efficient nitric oxide production.

There is evidence that endothelial nitric-oxide synthase (eNOS) is regulated by reciprocal dephosphorylation of Thr497 and phosphorylation of Ser1179. To examine the interrelationship between these sites, cells were transfected with wild-type (WT), T497A, T497D, S1179D, and T497A/S1179D eNOS and activity, NO release and eNOS localization were assessed. Although eNOS T497A, S1179D and T497A/S1179D eNOS had greater enzymatic activity than did WT eNOS in lysates, basal production of NO from cells was markedly reduced in cells transfected with T497A and T497A/S1179D eNOS but augmented in cells transfected with S1179D eNOS. Stimulating cells with ATP or ionophore normalized the loss of function seen with T497A and T497A/S1179D eNOS to levels observed with WT and S1179D eNOS, respectively. Despite these functional differences, the localization of eNOS mutants were similar to WT. Because both T497A and T497A/S1179D eNOS exhibited higher enzyme activity but reduced production of NO, we examined whether these mutations were "uncoupling" NO synthesis. T497A and T497A/S1179D eNOS generated 2-3 times more superoxide anion than WT eNOS, and both basal and stimulated interactions of T497A/S1179D eNOS with hsp90 were reduced in co-immunoprecipitation experiments. Thus, the phosphorylation/dephosphorylation of Thr497 may be an intrinsic switch mechanism that determines whether eNOS generates NO versus superoxide in cells.

Adenosine Triphosphate↗

PubMed2003

Extracellular superoxide dismutase is a major determinant of nitric oxide bioavailability: in vivo and ex vivo evidence from ecSOD-deficient mice.

The bioavailability of nitric oxide (NO) within the vascular wall is limited by superoxide anions (O2.-). The relevance of extracellular superoxide dismutase (ecSOD) for the detoxification of vascular O2.- is unknown. We determined the involvement of ecSOD in the control of blood pressure and endothelium-dependent responses in angiotensin II-induced hypertension and renovascular hypertension induced by the two-kidney, one-clip model in wild-type mice and mice lacking the ecSOD gene. Blood pressure was identical in sham-operated ecSOD+/+ and ecSOD-/- mice. After 6 days of angiotensin II-treatment and 2 and 4 weeks after renal artery clipping, blood pressure was significantly higher in ecSOD-/- than ecSOD+/+ mice. Recombinant ecSOD selectively decreased blood pressure in hypertensive ecSOD-/- mice, whereas ecSOD had no effect in normotensive and hypertensive ecSOD+/+ mice. Compared with sham-operated ecSOD+/+ mice, sham-operated ecSOD-/- mice exhibited attenuated acetylcholine-induced relaxations. These responses were further depressed in vessels from clipped animals. Vascular O2.-, as measured by lucigenin chemiluminescence, was higher in ecSOD-/- compared with ecSOD+/+ mice and was increased by clipping. The antioxidant tiron normalized relaxations in vessels from sham-operated and clipped ecSOD-/-, as well as from clipped ecSOD+/+ mice. In contrast, in vivo application of ecSOD selectively enhanced endothelium-dependent relaxation in vessels from ecSOD-/- mice. These data reveal that endogenous ecSOD is a major antagonistic principle to vascular O2.-, controlling blood pressure and vascular function in angiotensin II-dependent models of hypertension. ecSOD is expressed in such an abundance that even in situations of high oxidative stress no relative lack of enzyme activity occurs.

Acetylcholine↗

PubMed2003

Aged spontaneously hypertensive rats exhibit a selective loss of EDHF-mediated relaxation in the renal artery.

Endothelium-dependent relaxation is frequently attenuated in hypertension. We hypothesized that the contribution of the endothelium-derived hyperpolarizing factor (EDHF) to the acetylcholine (ACh)-induced, endothelium-dependent relaxation is attenuated with aging in the renal artery of spontaneously hypertensive rats (SHR) compared with age-matched Wistar-Kyoto (WKY) rats. ACh-induced, NO-mediated relaxation was identical in young (8-week-old) WKY and SHR, whereas EDHF-mediated relaxations (assessed in the presence of Nomega-nitro-l-arginine and diclofenac) were much more pronounced in SHR than WKY. KCl-induced relaxations were more pronounced in vessels from young WKY rats than from young SHR. The cytochrome P450 inhibitor sulfaphenazole significantly inhibited EDHF-mediated relaxation in vessels from young SHR but not WKY. Vessels from old (22 months) SHR exhibited a slightly reduced NO-mediated relaxation but a complete loss of EDHF-mediated responses. In contrast, aging did not affect EDHF-mediated responses in WKY. Moreover, ACh-induced hyperpolarization and resting membrane potential were decreased in old SHR but not in WKY. KCl-induced relaxation increased with age in WKY, whereas no response to KCl was recorded in arteries from aged SHR. In vessels from old WKY but not old SHR, mRNA expression of the Na-K-ATPase subunit alpha2 was increased by 2-fold compared with young animals. These data indicate that the increase in EDHF responses in renal arteries from aged WKY can be attributed to the release of K+ ions from the endothelium, whereas increased EDHF responses in renal arteries from young SHR can be attributed to a sulfaphenazole-sensitive cytochrome P450-dependent EDHF.

Acetylcholine↗

PubMed2003

11,12-Epoxyeicosatrienoic acid-induced inhibition of FOXO factors promotes endothelial proliferation by down-regulating p27Kip1.

Cytochrome P450-derived epoxyeicosatrienoic acids (EETs) stimulate endothelial cell proliferation and angiogenesis. In this study, we investigated the involvement of the forkhead box, class O (FOXO) family of transcription factors and their downstream target p27Kip1 in EET-induced endothelial cell proliferation. Incubation of human umbilical vein endothelial cells with 11,12-EET induced a time- and dose-dependent decrease in p27Kip1 protein expression, whereas p21Cip1 was not significantly affected. This effect on p27Kip1 protein was associated with decreased mRNA levels as well as p27Kip1 promoter activity. 11,12-EET also stimulated the time-dependent phosphorylation of Akt and of the forkhead factors FOXO1 and FOXO3a, effects prevented by the phosphatidylinositol 3-kinase inhibitor LY 294002. Transfection of endothelial cells with either a dominant-negative or an "Akt-resistant"/constitutively active FOXO3a mutant reversed the 11,12-EET-induced down-regulation of p27Kip1, whereas transfection of a constitutive active Akt decreased p27Kip1 expression independently of the presence or absence of 11,12-EET. To determine whether these effects are involved in EET-induced proliferation, endothelial cells were transfected with the 11,12-EET-generating epoxygenase CYP2C9. Transfection of CYP2C9 elicited endothelial cell proliferation and this effect was inhibited in cells co-transfected with CYP2C9 and either a dominant-negative Akt or constitutively active FOXO3a. Reducing FOXO expression using RNA interference, on the other hand, attenuated p27Kip1 expression and stimulated endothelial cell proliferation. These results indicate that EET-induced endothelial cell proliferation is associated with the phosphatidylinositol 3-kinase/Akt-dependent phosphorylation and inactivation of FOXO factors and the subsequent decrease in expression of the cyclin-dependent kinase inhibitor p27Kip1.

8,11,14-Eicosatrienoic Acid↗

PubMed2003

Transdifferentiation of blood-derived human adult endothelial progenitor cells into functionally active cardiomyocytes.

BACKGROUND: Further to promoting angiogenesis, cell therapy may be an approach for cardiac regeneration. Recent studies suggest that progenitor cells can transdifferentiate into other lineages. However, the transdifferentiation potential of endothelial progenitor cells (EPCs) is unknown. METHODS AND RESULTS: EPCs were obtained from peripheral blood mononuclear cells of healthy adults or coronary artery disease (CAD) patients by cultivating with endothelial cell medium and growth factors. After 3 days, >95% of adherent cells were functionally and phenotypically EPCs. Diacetylated LDL-labeled EPCs were then cocultivated with rat cardiomyocytes for 6 days, resulting in significant increases of EPC cell length and size to a cardiomyocyte-like morphology. Biochemically, 9.94+/-1.39% and 5.04+/-1.09% of EPCs from healthy adults (n=15) or CAD patients (n=14, P<0.01 versus healthy adults), respectively, expressed alpha-sarcomeric actinin as measured by flow cytometry. Immunocytochemistry showed that human EPCs expressed alpha-sarcomeric actinin, cardiac troponin I (both with partial sarcomeric organization), atrial natriuretic peptide, and myocyte enhancer factor 2. Fluo 4 imaging demonstrated calcium transients synchronized with adjacent rat cardiomyocytes in transdifferentiated human EPCs. Single-cell microinjection of Lucifer yellow and calcein-AM labeling of cardiomyocytes demonstrated gap junctional communication between 51+/-7% of EPCs (16 hours after labeling, n=4) and cardiomyocytes. EPC transdifferentiation into cardiomyocytes was not observed with conditioned medium but in coculture with paraformaldehyde-fixed cardiomyocytes. CONCLUSIONS: EPCs from healthy volunteers and CAD patients can transdifferentiate in vitro into functionally active cardiomyocytes when cocultivated with rat cardiomyocytes. Cell-to-cell contact but not cellular fusion mediates EPC transdifferentiation. The therapeutic use of autologous EPCs may aid cardiomyocyte regeneration in patients with ischemic heart disease.

Adult↗

PubMed2003

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.

Animals↗

PubMed2003

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.

8,11,14-Eicosatrienoic Acid↗