Biomedical subjects

Rudi Busse

Publications and source records attributed to Rudi Busse.

52 records · Page 3Linked to original sources

PubMed2003

Regulation of endothelium-derived vasoactive autacoid production by hemodynamic forces.

Endothelial cells, which are situated at the interface between blood and the vessel wall, have a crucial role in controlling vascular tone and homeostasis, particularly in determining the expression of pro-atherosclerotic and anti-atherosclerotic genes. Many of these effects are mediated by changes in the generation and release of endothelium-derived autacoids [from the Greek autos (self) and akos (remedy)], which are generally short-lived and locally acting. In vivo, endothelial cells are constantly subjected to mechanical stimulation, which in turn determines the acute production of autacoids and the levels of autacoid-producing enzymes.

Animals↗

PubMed2003

Insulin enhances the expression of the endothelial nitric oxide synthase in native endothelial cells: a dual role for Akt and AP-1.

Insulin-induced vasodilatation in vivo has been attributed to the activation of the endothelial nitric oxide (NO) synthase (eNOS). The present study addressed the effects of insulin on the activity and expression of eNOS in native and cultured endothelial cells. Insulin applied to native porcine aortic endothelial cells elicited the tyrosine phosphorylation of the insulin receptor and receptor substrate, the subsequent activation of phosphatidylinositol 3-kinase (PI 3-K), Akt (protein kinase B), and ERK1/2. Insulin did not activate eNOS in cultured endothelial cells nor relax endothelium-intact arterial segments. However, 4h after application of insulin to native endothelial cells eNOS mRNA was increased 2-fold. A comparable increase in eNOS protein was detected after 18-24h and associated with an increase in intracellular cyclic GMP. In native endothelial cells, insulin enhanced the DNA-binding activity of Sp1 and AP-1, but not that of NF-kappaB. The insulin-induced increase in eNOS expression was prevented by wortmannin as well as by AP-1 decoy oligonucleotides. The MEK1 inhibitor, PD 98059, also enhanced eNOS expression in native and cultured endothelial cells, an effect which was independent of ERK1/2 and associated with an increase in the DNA-binding activity of AP-1 and Sp1. These results demonstrate that insulin activates multiple signalling pathways in endothelial cells but does not acutely activate eNOS. Insulin however enhances eNOS mRNA and protein by a mechanism involving the combined activation of a PI 3-K- and AP-1-dependent pathway.

Animals↗

PubMed2003

Characterization of the endothelium-derived hyperpolarizing factor (EDHF) response in the human interlobar artery.

BACKGROUND: In addition to nitric oxide (NO) and prostacyclin (PGI2), the vascular endothelium can influence local vascular tone by a mechanism involving the hyperpolarization of vascular smooth muscle cells. This response is attributed to the release of an endothelium-derived hyperpolarizing factor (EDHF). The present study was performed to determine the characteristics of the EDHF that mediates the NO/PGI2-independent hyperpolarization and relaxation of human renal interlobar arteries. METHODS: Acetylcholine-induced, EDHF-mediated hyperpolarization and relaxation were assessed using sharp microelectrodes impaled into interlobar smooth muscle cells and in organ chamber experiments, respectively. All experiments were performed in the combined presence of NO synthase (NOS) and cyclooxygenase inhibitors and the thromboxane analog U46619. RESULTS: Interlobar arteries demonstrated pronounced NO/PGI2-independent relaxations and hyperpolarizations that were sensitive to the blockade of calcium-activated K+-channels (KCa+ channels) by the combination of charybdotoxin and apamin and to the inhibition of the Na-K-ATPase by ouabain. Exogenously applied KCl also exhibited relaxations and hyperpolarizations that were sensitive to ouabain but insensitive to the combined inclusion of charybdotoxin and apamin. Relaxations induced by KCl were also observed in endothelium-denuded interlobar arteries. CONCLUSION: These results indicate that in the human renal interlobar artery, EDHF-mediated responses display the pharmacologic characteristics of K+ ions released through endothelial KCa+ channels. Smooth muscle cell hyperpolarization and relaxation appear to be dependent on the activation of ouabain-sensitive subunits of the Na-K-ATPase.

Aryl Hydrocarbon Hydroxylases↗

PubMed2003

[Communication between endothelial and smooth muscle cells].

Vascular endothelial cells play a fundamental role in the control of vascular tone, and therefore in the control of local blood flow, by releasing various contracting (endothelin, prostaglandins) and relaxing (prostacycline, NO) factors. An additional mechanism involving the hyperpolarization of the vascular smooth muscle cells is observed mainly in the coronary vascular bed and in the periphery. This phenomenon was attributed to an elusive endothelial factor called endothelium-derived hyperpolarizing factor (EDHF). This mechanism is now better understood. It involves first an increase in the endothelial intracellular concentration of calcium, the activation of endothelial potassium channels and the resulting hyperpolarization of the endothelial cells. The hyperpolarization of the endothelial cells is transmitted to the smooth muscle cells by different pathways. This hyperpolarization propagates along the vessels not only via the smooth muscle cells but also via the endothelial cells. Therefore, the endothelial layer can also be considered as a conducting tissue. The discovery of specific inhibitors of the endothelial cell hyperpolarization allows the assessment of the contribution of EDHF-mediated responses in the control of vascular tone.

Biological Factors↗

PubMed2003

Identification of a cis-element regulating transcriptional activity in response to fluid shear stress in bovine aortic endothelial cells.

Fluid shear stress exerts numerous effects on gene expression in endothelial cells. To investigate the regulatory mechanisms involved, we designed oligonucleotides composed of a 20-bp core containing the classical shear stress response element (SSRE+) or of a 20-bp core, in which base pairs flanking the SSRE were mutated (SSRE-). Hexamers of the oligonucleotides were cloned in front of reporter genes, transfected in bovine aortic endothelial cells (BAECs), and subjected to either a continuous low shear stress (3 dynes cm(-2)) or to a stepwise increase in shear stress from 3 dynes cm(-2) to 12 dynes cm(-2) (16/4 h). Shear stress increased reporter gene activity in cells transfected with pSSRE-, but not with pSSRE+. Cyclic strain (6%, 1 Hz, 4 h) did not significantly affect reporter gene activity. In gel retardation assays, more proteins bound to SSRE- than to SSRE+ in response to high shear stress. In competition experiments, a cAMP response element-binding (CREB) protein-specific oligonucleotide suppressed protein binding to the SSRE-; however, an antibody directed against CREB itself did not affect protein binding to the SSRE+. Our results indicate that the sequence ACC(G)/(T)AGACCAG represents a novel SSRE and that a protein that binds a CREB-specific oligonucleotide is part of the complex implicated in response to shear stress.

Animals↗

PubMed2003

Molecular mechanisms involved in the regulation of the endothelial nitric oxide synthase.

The endothelial nitric oxide synthase (eNOS), the expression of which is regulated by a range of transcriptional and posttranscriptional mechanisms, generates nitric oxide (NO) in response to a number of stimuli. The physiologically most important determinants for the continuous generation of NO and thus the regulation of local blood flow are fluid shear stress and pulsatile stretch. Although eNOS activity is coupled to changes in endothelial cell Ca(2+) levels, an increase in Ca(2+) alone is not sufficient to affect enzyme activity because the binding of calmodulin (CaM) and the flow of electrons from the reductase to the oxygenase domain of the enzyme is dependent on protein phosphorylation and dephosphorylation. Two amino acids seem to be particularly important in regulating eNOS activity and these are a serine residue in the reductase domain (Ser(1177)) and a threonine residue (Thr(495)) located within the CaM-binding domain. Simultaneous alterations in the phosphorylation of Ser(1177) and Thr(495) in response to a variety of stimuli are regulated by a number of kinases and phosphatases that continuously associate with and dissociate from the eNOS signaling complex. eNOS associated proteins, such as caveolin, heat shock protein 90, eNOS interacting protein, and possibly also motor proteins provide the scaffold for the formation of the protein complex as well as its intracellular localization.

Animals↗

PubMed2003

AMP-activated protein kinase (AMPK) regulates the insulin-induced activation of the nitric oxide synthase in human platelets.

Little is known about the signaling cascades that eventually regulate the activity of the endothelial nitric oxide synthase (eNOS) in platelets. Here, we investigated the effects of insulin on the phosphorylation and activation of eNOS in washed human platelets and in endothelial cells. Insulin activated the protein kinase Akt in cultured endothelial cells and increased the phosphorylation of eNOS on Ser(1177) but failed to increase endothelial cyclic GMP levels or to elicit the relaxation of endothelium-intact porcine coronary arteries. In platelets, insulin also elicited the activation of Akt as well as the phosphorylation of eNOS and initiated NO production which was associated with increased cyclic GMP levels and the inhibition of thrombin-induced aggregation. The insulin-induced inhibition of aggregation was accompanied by a decreased Ca(2+) response to thrombin and was also prevented by N(omega) nitro-L-arginine. In platelets, but not in endothelial cells, insulin induced the activation of the AMP-activated protein kinase (AMPK), a metabolic stress-sensing kinase which was sensitive to the phosphatidylinositol 3-kinase (PI3-K) inhibitor wortmannin and the AMPK inhibitor iodotubercidin. Moreover, the insulin-mediated inhibition of thrombin-induced aggregation was prevented by iodotubercidin. Insulin-independent activation of the AMPK using 5-aminoimidazole-4-carboxamide ribonucleoside, increased platelet eNOS phosphorylation, increased cyclic GMP levels and attenuated platelet aggregation. These results highlight the differences in the signal transduction cascade activated by insulin in endothelial cells and platelets, and demonstrate that insulin stimulates the formation of NO in human platelets, in the absence of an increase in Ca(2+), by acti-vating PI3-K and AMPK which phosphorylates eNOS on Ser(1177).

AMP-Activated Protein Kinases↗

PubMed2002

20-HETE-induced contraction of small coronary arteries depends on the activation of Rho-kinase.

20-HETE is a potent constrictor of small blood vessels and has been suggested to play a crucial role in the generation of myogenic tone and the development of hypertension. In the present study, we investigated the mechanisms by which exogenously applied 20-HETE modulates vascular tone in small porcine coronary arteries. In organ chamber experiments, 20-HETE elicited a concentration-dependent contraction of small porcine coronary artery rings that was partially inhibited by the cyclooxygenase inhibitor diclofenac, the thromboxane and endoperoxide receptor antagonist SQ29548, and the thromboxane A2 synthase inhibitor furegrelate. Removal of endothelium attenuated the response to 20-HETE, whereas preconstriction of endothelium-denuded vessels to 25% of the maximum response with KCl markedly enhanced the response to 20-HETE. This 20-HETE-induced contraction was not associated with a significant increase in the intracellular concentration of Ca2+. 20-HETE-induced contraction was also observed in beta-escin-permeabilized arteries precontracted with a submaximal concentration of Ca2+ and was abolished by the Rho-kinase inhibitor Y27632, but was insensitive to the PKC inhibitor RO 31-8220. 20-HETE elicited the phosphorylation of the myosin light chain (MLC20) in coronary artery rings, an effect that was sensitive to Y27632 and mimicked by the thromboxane analog U46619. These data suggest that in small porcine coronary arteries, 20-HETE can induce contraction by 2 mechanisms, one endothelium-dependent involving the cyclooxygenase-dependent generation of vasoconstrictor prostanoids, and the other endothelium-independent. The latter response is associated with the activation of Rho-kinase, phosphorylation of MLC20, and sensitization of the contractile apparatus to Ca2+.

Animals↗

PubMed2002

CK2 phosphorylates the angiotensin-converting enzyme and regulates its retention in the endothelial cell plasma membrane.

Soluble angiotensin-converting enzyme (ACE) is derived from the membrane-bound form by proteolytic cleavage of its C-terminal domain. Because intracellular events might be involved in the regulation of the cleavage process, we determined whether the cytoplasmic tail of ACE is phosphorylated and whether this process regulates secretion. Immunoprecipitation of ACE (180 kDa) from (32)P-labeled endothelial cells revealed that ACE is phosphorylated. Phosphorylation was not observed in endothelial cells overexpressing a mutant form of ACE (ACEDeltaS, all five cytoplasmic serine residues replaced by alanine). CK2 coprecipitated with ACE from endothelial cells, and CK2 phosphorylated both ACE and a peptide corresponding to the cytoplasmic tail. Mutation of serine(1270) within the CK2 consensus sequence almost abolished ACE phosphorylation. In ACE-overexpressing endothelial cells, ACE was mostly localized to the plasma membrane. However, no ACE was detected in the plasma membrane of ACEDeltaS-overexpressing cells, although a precursor ACE (170 kDa) was prominent in the endoplasmic reticulum and the cell supernatant contained substantial amounts of the soluble protein (175 kDa). A correlation between ACE-phosphorylation and secretion was confirmed in endothelial cells treated with the CK2-inhibitor, 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole, which time-dependently decreased the phosphorylation of ACE and increased its shedding. These results indicate that the CK2-mediated phosphorylation of ACE regulates its retention in the plasma membrane and may determine plasma ACE levels.

Amino Acid Sequence↗

PubMed2002

Epoxygenase-driven angiogenesis in human lung microvascular endothelial cells.

Angiogenesis is one of the most recent physiological functions attributed to products of cytochrome P-450 (CYP450) enymes. To test this at a molecular level in human cells, we used a cloned cDNA for the human endothelial enzyme CYP450 2C9 (CYP2C9) to study growth as well as differentiation of human microvascular endothelial cells from the lung (HMVEC-L). Using adenoviral vectors overexpressing mRNA for CYP2C9, we show that the presence of CYP2C9 doubles thymidine incorporation and stimulates proliferation of primary cultures of endothelial cells compared with Ad5-GFP (control) in 24 h. In addition, there is a significant increase of tube formation in Matrigel after infection of HMVEC-L with Ad5-2C9 than with Ad5-GFP. More interestingly, Ad5-2C9 expressing the antisense product of CYP2C9 (2C9AS) inhibited tube formation compared with both Ad5-GFP as well as the Ad5-2C9 constructs. Finally, we tested the most abundant arachidonic acid metabolite of CYP2C9, 14,15-epoxyeicosatrienoic acid, which induced angiogenesis in vivo when embedded in Matrigel plugs and implanted in adult rats. These data support an important role for CYP2C9 in promoting angiogenesis.

8,11,14-Eicosatrienoic Acid↗

PubMed2002

The vascular NADPH oxidase subunit p47phox is involved in redox-mediated gene expression.

An NADPH oxidase is thought to be a main source of vascular superoxide (O(2)(-)) production. The functional role of this oxidase, however, and the contribution of the different subunits of the enzyme to cellular signaling are still incompletely understood. We determined the role of the p47phox subunit of the oxidase in O(2)(-) generation and signaling in aortic rings and cultured smooth muscle cells (SMC) from wild-type (WT) and p47phox-deficient (p47phox -/-) mice. Basal O(2)(-) levels in aortae of p47phox -/- mice were lower than those in WT aortae. Infusion of [val(5)]-angiotensin II increased O(2)(-) levels in aortae from WT more than in aortae from p47phox -/- mice. O(2)(-) generation was similar in quiescent SMC from WT and p47phox -/- mice. However, exposure to thrombin selectively increased O(2)(-) generation in VSMC from WT, but not from p47phox -/- mice. Thrombin-activated redox-mediated signal transduction and gene expression was attenuated in VSMC from p47phox -/- compared to cells from WT mice as determined by p38 MAP kinase activation and VEGF gene expression. We conclude that p47phox is important for vascular ROS production and redox-modulated signaling and gene expression in VSMC.

Angiotensin II↗

PubMed2002

NADPH oxidase mediates tissue factor-dependent surface procoagulant activity by thrombin in human vascular smooth muscle cells.

BACKGROUND: Tissue factor (TF) initiates the extrinsic coagulation cascade leading to thrombin formation. Thrombin induces TF mRNA in vascular smooth muscle cells (VSMCs), thereby contributing to the prolonged procoagulant activity and enhanced thrombogenicity at sites of vascular injury. However, the signaling mechanisms mediating this thrombogenic cycle are unclear. Characteristically, vascular injury promotes the generation of reactive oxygen species (ROS). Because ROS exert signaling functions, we investigated whether the NADPH oxidase, an important source of ROS in VSMCs, contributes to upregulation of TF by thrombin. METHODS AND RESULTS: Thrombin not only stimulated TF mRNA expression, but also TF-dependent surface procoagulant activity in cultured human VSMCs. This response was attenuated by antioxidants; the flavin inhibitor diphenylene-iodonium, Clostridium difficile toxin B, which inhibits Rho GTPases, p22phox antisense oligonucleotides, or the dominant-negative RacT17N mutant. Inhibitors of p38 MAP kinase and phosphatidylinositol (PI) 3-kinase also prevented thrombin-stimulated TF mRNA expression. Furthermore, thrombin stimulated the phosphorylation of the PI 3-kinase target protein kinase B/Akt in a redox-sensitive and NADPH oxidase-dependent manner. CONCLUSION: These findings indicate that the NADPH oxidase is essentially involved in the redox-sensitive induction of TF mRNA expression and surface procoagulant activity by thrombin. This response is mediated by NADPH oxidase-dependent activation of p38 MAP kinase and the PI 3-kinase/protein kinase B/Akt pathway. Given that active TF promotes thrombin formation, the NADPH oxidase may play a crucial role in perpetuating the thrombogenic cycle in the injured vessel wall.

Bacterial Proteins↗

PubMed2002

Dynamic modulation of interendothelial gap junctional communication by 11,12-epoxyeicosatrienoic acid.

Functional gap junctional communication between vascular cells has been implicated in ascending dilatation and the cytochrome P-450 (CYP) inhibitor-sensitive and NO- and prostacyclin-independent dilatation of many vascular beds. Here, we assessed the mechanisms by which the epoxyeicosatrienoic acids (EETs) generated by a CYP 2C enzyme control interendothelial gap junctional communication. In CYP 2C-expressing porcine coronary endothelial cells, bradykinin, which enhances EET formation, elicited a biphasic effect on the electrical coupling and transfer of Lucifer yellow between endothelial cells, consisting of a transient increase in coupling followed by a sustained uncoupling. The initial phase was sensitive to the CYP 2C9 inhibitor sulfaphenazole and the protein kinase A (PKA) inhibitors Rp-cAMPS and KT5720 and could be mimicked by forskolin and caged cAMP as well as by the PKA activators 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole 3',5'-cyclic monophosphorothioate sodium salt and Sp-cAMPS. Gap junction uncoupling in bradykinin-stimulated porcine coronary endothelial cells was prevented by inhibiting the activation of extracellular signal-regulated kinase (ERK)1/2. In human endothelial cells, which express little CYP 2C, bradykinin elicited only an ERK1/2-mediated inhibition of intercellular communication. The CYP 2C9 product, 11,12-EET, also exerted a dual effect on the electrical and dye coupling of human endothelial cells, which was sensitive to PKA inhibition. These results demonstrate that an agonist-activated CYP-dependent pathway as well as 11,12-EET can positively regulate interendothelial gap junctional communication, most probably via the activation of PKA, an effect that is curtailed by the subsequent activation of ERK1/2.

8,11,14-Eicosatrienoic Acid↗

PubMed2002

Vascular gene transfer of phosphomimetic endothelial nitric oxide synthase (S1177D) using ultrasound-enhanced destruction of plasmid-loaded microbubbles improves vasoreactivity.

BACKGROUND: Local gene therapy has enormous potential for the treatment of vascular disease. We determined whether diagnostic ultrasound-mediated destruction of plasmid-loaded albumin microbubbles is a feasible and efficient technique for local vascular gene delivery. For gene transfer, we used a phosphomimetic, active endothelial nitric oxide synthase (eNOS) construct in which Ser1177 was replaced by aspartic acid (S1177D) and exhibits a 2-fold higher basal activity than the wild-type enzyme. METHODS AND RESULTS: Gas-filled microbubbles (3.0 +/- 1.2 microm) were created by sonication of 5% human albumin in the presence of plasmid DNA encoding for LacZ or eNOS S1177D. Porcine coronary arteries were perfused with DNA-loaded albumin microbubbles in vitro, exposed to diagnostic ultrasound (5 seconds), and incubated for a further 24 hours. Detection of the beta-galactosidase in LacZ-transfected vessels revealed a predominant staining of endothelial cells without any functional impairment of vasoreactivity. Western blotting demonstrated the expression of the eNOS S1177D construct in extracts from the transfected segments. Vascular responsiveness was tested with prostaglandin F(2alpha) and the NOS inhibitor N(omega)nitro-L-arginine. Compared with segments treated with the expression plasmid alone, the contractile response to prostaglandin F(2alpha) was impaired in segments transfected with eNOS S1177D, whereas the contractile response to the administration of N(omega)nitro-L-arginine was markedly enhanced. CONCLUSIONS: Ultrasound-mediated destruction of eNOS S1177D DNA-loaded albumin microbubbles is a feasible and efficient method for vascular gene transfection. Transfection resulted in significant protein expression and enhanced NO-mediated relaxation of bradykinin-stimulated porcine coronary arteries.

Amino Acid Substitution↗

PubMed2002

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.

8,11,14-Eicosatrienoic Acid↗

PubMed2002

EDHF: bringing the concepts together.

Endothelial cells synthesize and release vasoactive mediators in response to various neurohumoural substances (e.g. bradykinin or acetylcholine) and physical stimuli (e.g. cyclic stretch or fluid shear stress). The best-characterized endothelium-derived relaxing factors are nitric oxide and prostacyclin. However, an additional relaxant pathway associated with smooth muscle hyperpolarization also exists. This hyperpolarization was originally attributed to the release of an endothelium-derived hyperpolarizing factor (EDHF) that diffuses to and activates smooth muscle K(+) channels. More recent evidence suggests that endothelial cell receptor activation by these neurohumoural substances opens endothelial cell K(+) channels. Several mechanisms have been proposed to link this pivotal step to the subsequent smooth muscle hyperpolarization. The main concepts are considered in detail in this review.

Animals↗