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Biomedical subjects

Margarete Goppelt-Struebe

Publications and source records attributed to Margarete Goppelt-Struebe.

At least 19 recordsLinked to original sources
PubMed2007

Actin-dependent regulation of connective tissue growth factor.

Expression of connective tissue growth factor (CTGF) in endothelial cells is modulated by shear stress affecting the organization of the cytoskeleton. The molecular connection between alterations of actin and CTGF expression was investigated in human umbilical vein endothelial cells (HUVEC) and a microvascular endothelial cell line. Overexpression of nonpolymerizable monomeric actin R62D interfered with stress fiber formation in HUVEC and concomitantly reduced immunoreactive CTGF. In microvascular endothelial cells, flow-dependent upregulation of CTGF was prevented by this actin mutant. In contrast, overexpression of actin S14C strengthened filamentous actin and increased CTGF expression. These data indicated an inverse relationship between CTGF expression and monomeric actin. Coexpression of the mutant actins and different CTGF promoter constructs revealed an actin-sensitive site between 3 and 4.5 kb of the CTGF promoter. A CArG-like box at -3791 bp was responsible for actin-dependent CTGF induction as shown by mutagenesis. Overexpression of actin S14C activated the nonmutated promoter significantly more strongly than the mutated promoter. Actin polymerization is regulated by the small GTPase RhoA and activation of serum response factor (SRF). Overexpression of constitutively active RhoA or SRF significantly increased CTGF protein synthesis. The 4.5-kb promoter construct, but not the construct with a mutation in the CArG box, was activated by SRF or RhoA, providing evidence for a functional role of this site in CTGF induction. These findings provide novel evidence that monomeric actin is the connecting link between alterations in the cytoskeleton and CTGF gene expression and demonstrate the importance of SRF in regulating CTGF transcription.

Actins↗
PubMed2006

Mechanical regulation of the Cyr61/CCN1 and CTGF/CCN2 proteins.

Cells in various anatomical locations are constantly exposed to mechanical forces from shear, tensile and compressional forces. These forces are significantly exaggerated in a number of pathological conditions arising from various etiologies e.g., hypertension, obstruction and hemodynamic overload. Increasingly persuasive evidence suggests that altered mechanical signals induce local production of soluble factors that interfere with the physiologic properties of tissues and compromise normal functioning of organ systems. Two immediate early gene-encoded members of the family of the Cyr61/CTGF/Nov proteins referred to as cysteine-rich protein 61 (Cyr61/CCN1) and connective tissue growth factor (CTGF/CCN2), are highly expressed in several mechanical stress-related pathologies, which result from either increased externally applied or internally generated forces by the actin cytoskeleton. Both Cyr61 and CTGF are structurally related but functionally distinct multimodular proteins that are expressed in many organs and tissues only during specific developmental or pathological events. In vitro assessment of their biological activities revealed that Cyr61 expression induces a genetic reprogramming of angiogenic, adhesive and structural proteins while CTGF promotes distinctively extracellular matrix accumulation (i.e., type I collagen) which is the principal hallmark of fibrotic diseases. At the molecular level, expression of the Cyr61 and CTGF genes is regulated by alteration of cytoskeletal actin dynamics orchestrated by various components of the signaling machinery, i.e., small Rho GTPases, mitogen-activated protein kinases, and actin binding proteins. This review discusses the mechanical regulation of the Cyr61 and CTGF in various tissues and cell culture models with a special attention to the cytoskeletally based mechanisms involved in such regulation.

Bone Diseases↗
PubMed2006

STAT3-independent inhibition of lysophosphatidic acid-mediated upregulation of connective tissue growth factor (CTGF) by cucurbitacin I.

Cucurbitacins are recognised as anti-tumour agents because of their interference with STAT3 signalling, but may also affect the integrity of the actin cytoskeleton. In the present study the effect of cucurbitacin I was investigated in fibroblasts. In these cells, cucurbitacin I interfered with lysophosphatidic acid (LPA) signalling. It inhibited tyrosine phosphorylation of focal adhesion proteins and induction of connective tissue growth factor (CTGF), a potent profibrotic protein. Inhibition of Src family kinases with PP2, but not the inactive analogue PP3, also interfered with LPA-mediated tyrosine phosphorylation and induction of CTGF. Jak2-STAT3 signalling seemed to be the connecting link, because CTGF induction was sensitive to AG490, an inhibitor of Jak2, and cucurbitacin I, an inhibitor of Jak2 and STAT3. However, LPA did not activate tyrosine phosphorylation of STAT3. Furthermore, cucurbitacin I was as effective in STAT3 knock out cells as in control cells. Therefore, the inhibitory effect of cucurbitacin I was not related to inhibition of STAT3. Immunocytochemical analysis of cucurbitacin I-treated cells revealed disassembly of F-actin fibres, reorganisation into F-actin patches and resolution of focal adhesions. The phenotypic changes resembled changes observed after treatment of the cells with cytochalasin D, which has been shown to interfere with CTGF induction. Concentrations of cucurbitacin I, which have been shown to target Jak2-STAT3 signalling, thus, profoundly affect the actin cytoskeleton and may therefore modulate cell morphology, migration, adherence and gene expression also in non-tumour cells.

Actins↗
PubMed2006

Connective tissue growth factor is released from platelets under high shear stress and is differentially expressed in endothelium along atherosclerotic plaques.

Connective tissue growth factor (CTGF) is overexpressed in atherosclerotic blood vessels. To further investigate the role of CTGF in atherosclerosis, we examined whether CTGF is released from platelets by high shear stress, and whether the expression of CTGF along the atherosclerotic lesions depends on local hemodynamic conditions. Human platelets were subjected to 10 dyn/cm2 or 120 dyn/cm2 and analysed by Western blotting. Furthermore, longitudinal sections of 25 carotid plaques were immunohistochemically analysed for the endothelial expression of CTGF. A very low CTGF amount was secreted from platelets at low shear stress (11.4 +/- 3.9% of total CTGF in platelets). On the contrary, high shear stress caused a markedly increased CTGF release from platelets (29 +/- 13.8%, p = 0.07 vs low shear stress, n = 4). Immunohistochemical analyses showed that the mean numbers of CTGF-positive endothelial cells were significantly higher up-stream as compared with down-stream regions of the luminal surface of atherosclerotic vessels (21.3 +/- 3.6 vs 13.9 +/- 2.8 down-stream, p < 0.001). Moreover, in plaques undergoing intimal neovascularization, newly formed vessels accumulated particularly in up-stream parts of the lesions. In conclusion, this study demonstrated that CTGF is released from platelets by high shear stress. Furthermore, disturbed flow along atherosclerotic vessels may induce endothelial CTGF expression and contribute to the progress of atherosclerotic lesions.

Atherosclerosis↗
PubMed2005

Differential involvement of the integrin-linked kinase (ILK) in RhoA-dependent rearrangement of F-actin fibers and induction of connective tissue growth factor (CTGF).

The integrin-linked kinase (ILK) serves as an adapter protein to link the cytoplasmic domains of integrins with cytoskeletal components. Organization of the actin cytoskeleton is strongly influenced by the small GTPase RhoA, which also regulates gene expression. To investigate the impact of ILK deficiency on RhoA-mediated signaling we used ILK-deficient fibroblasts. The cytoskeleton of ILK (-/-) cells was characterized by less organized F-actin fibers, compared to wild type mouse fibroblasts. Stimulation of the cells with lysophosphatidic acid (LPA) or the microtubule disrupting agent colchicine increased polymerization of F-actin stress fibers in ILK (+/+) cells, whereas ILK (-/-) cells showed a network of short thin cortical actin fibers, cell rounding and finally detachment from the surface of the culture plates. The structural changes were primarily attributable to the activation of RhoA in both cell types. ILK deficiency also affected gene expression. The basal levels of several proteins related to fibroblast differentiation, such as connective tissue growth factor (CTGF), thrombospondin 1 and alpha smooth muscle actin, were reduced in ILK (-/-) cells. However, induction of CTGF expression by LPA or colchicine was comparable in ILK (+/+) and ILK (-/-) cells. Furthermore, stimulation of CTGF or thrombospondin by TGFbeta was not reduced by ILK deficiency. Inhibition of the RhoA-associated kinase or overexpression of dominant negative RhoA reduced the stimulated CTGF expression indicative of a role for RhoA signaling in CTGF expression. Taken together, ILK is involved in RhoA-dependent reorganization of the actin cytoskeleton, whereas activation of RhoA and RhoA-mediated gene expression is independent of ILK.

Actins↗
PubMed2005

Connective tissue growth factor is overexpressed in complicated atherosclerotic plaques and induces mononuclear cell chemotaxis in vitro.

OBJECTIVE: Atherosclerotic blood vessels overexpress connective tissue growth factor (CTGF) mRNA, but the role of CTGF in atherosclerosis remains controversial. To assess the hypothesis that CTGF is involved in atherosclerotic plaque progression, we investigated CTGF protein expression and distribution in the different types of plaque morphology. METHODS AND RESULTS: Serial cross-sections of 45 human carotid plaques were immunohistochemically analyzed for the presence of CTGF protein, neovascularization (von Willebrand factor), macrophages (CD68), and T cells (CD3). The lesions were categorized according to American Heart Association (AHA) classification as fibrous (type IV and V) or complicated plaques (type VI). The levels of CTGF were significantly higher in complicated compared with fibrous plaques (P=0.002). CTGF accumulated particularly in the rupture-prone plaque shoulder and in the areas of neovascularization or infiltration with inflammatory cells. Macrophage-like cells stained positive for CTGF protein in plaques. Subsequent in vitro studies showed that although monocyte-derived macrophages do not produce CTGF on stimulation with transforming growth factor-beta, lipopolysaccharide, or thrombin, they take it up from culture medium. Furthermore, CTGF induces mononuclear cell chemotaxis in a dose-dependent manner. CONCLUSIONS: CTGF protein is significantly increased in complicated compared with fibrous plaques and may enhance monocyte migration into atherosclerotic lesions, thus contributing to atherogenesis.

Aged↗
PubMed2004

Monitoring the cellular effects of HMG-CoA reductase inhibitors in vitro and ex vivo.

OBJECTIVE: Inhibition of 3hydroxy3methylglutaryl-coenzyme A (HMG-CoA) reductase by statins and the subsequent reduction in Rho protein isoprenylation inactivates these important signaling molecules. The purpose of this study was to directly monitor statin effects on Rho proteins. METHODS AND RESULTS: We used biphasic Triton X-114 system, 1-dimensional isoelectric focusing, and 2D-electrophoresis for the separation of modified and nonmodified Rho proteins. These methods were evaluated in human fibroblasts treated with simvastatin. 2D-electrophoresis, which proved to be the most sensitive method, revealed 2 major spots of identical molecular weight but different isoelectric points, with the more basic spot representing the carboxymethylated form of RhoA. In control cells, 90% of RhoA was fully modified (carboxymethylated). After treatment with simvastatin, a significant shift toward the unmethylated form was observed, representing inhibition of isoprenylation, which is a prerequisite to further modification. Similar shifts were observed for Rac1 and Cdc42. In freshly isolated peripheral blood mononuclear cells, a shift toward nonmodified RhoA was observed after treatment with atorvastatin in vitro and in vivo. There was a significant increase in unmethylated RhoA in statin-treated individuals versus control individuals. CONCLUSIONS: 2D-electrophoresis is a sensitive method for detecting changes in the amount of nonisoprenylated Rho proteins, allowing monitoring the direct cellular effects of statins.

Cell Line↗
PubMed2004

Induction of connective tissue growth factor (CTGF) in human endothelial cells by lysophosphatidic acid, sphingosine-1-phosphate, and platelets.

Endothelial dysfunction is characterized by multiple interactions between endothelial cells and components of the blood. This study focussed on the induction of the pro-atherogenic connective tissue growth factor (CTGF) in endothelial cells by bioactive lipids and platelets. Lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) led to a time- and concentration-dependent increase in CTGF mRNA and protein expression in the human endothelial cell line EAHY 926 and in primary cultures of human umbilical vein endothelial cells (HUVEC). As both cell types expressed various receptors for LPA and S1P, signaling pathways were further characterized by pharmacological means: induction of CTGF was pertussis toxin-insensitive and inhibition of activation of p42/44 MAP kinases only partially reduced CTGF expression. On the contrary, interference with the RhoA signaling pathway by simvastatin, an inhibitor of geranylgeranyltransferases, or the Rho-kinase inhibitor Y27632 prevented induction of CTGF. Co-incubation of endothelial cells with freshly isolated human platelets significantly increased the expression of CTGF mRNA in endothelial cells, which was also sensitive to simvastatin. Up-regulation of CTGF in endothelial cells, induced by LPA, S1P, or platelets, may contribute to the initiation and progression of atherosclerosis. Interference of simvastatin with the synthesis of this pro-atherogenic factor further supports the anti-atherogenic role of statins.

Blood Platelets↗
PubMed2004

Differential effects of simvastatin on mesangial cells.

BACKGROUND: Statins are increasingly recognized as mediators of direct cellular effects independent of their lipid lowering capacity. Therefore, the time and concentration dependence of various statin-mediated cellular alterations was compared in renal mesangial cells. METHODS: The effects of statins on cell proliferation, gene expression, cytoskeletal alterations, apoptosis, and cytotoxicity were analyzed in cultured mesangial cells using standard techniques. RESULTS: Simvastatin and lovastatin decreased proliferation and cell number of rat mesangial cells concentration-dependently. Concurrently, the expression of the fibrogenic protein connective tissue growth factor (CTGF) was impaired and actin stress fibers, which are typical of mesangial cells in culture, became disassembled by simvastatin. A decrease of the posttranslational modification of RhoA by geranylgeranyl moieties was detected, supporting a role for RhoA as mediator of statin effects. Induction of apoptosis, determined by activation of caspase-3 and DNA fragmentation, and necrosis only occurred at later time points, when the morphology of the cells was strongly altered and the cells detached from the surface due to changes in the actin cytoskeleton. Basically, the same results were obtained with a human mesangial cell line. Furthermore, statin effects were mimicked by inhibition of the geranylgeranyltransferase. CONCLUSION: Most of the cellular effects of the lipophilic statins occurred within the same time and concentration range, suggesting a common molecular mechanism. Only apoptosis and necrosis were observed at later time points or with higher concentrations of simvastatin and thus seem to be secondary to the changes in gene expression and alterations of the actin cytoskeleton.

Actins↗
PubMed2004

Activated human platelets release connective tissue growth factor.

Connective tissue growth factor (CTGF) is overexpressed in wound healing, fibrosis and advanced atherosclerotic lesions. Platelets adhere to CTGF, suggesting that this protein may be involved in the formation of platelet-rich thrombi at the sites of tissue injury or atherosclerotic plaque rupture. Since platelets contain a wide array of biologically active proteins, we investigated the presence, localization and release of CTGF from these cells. For this purpose, human platelets from healthy donors were washed and stimulated with thrombin or ADP. Following incubation, proteins from unstimulated and stimulated cell lysates and the supernatants were analysed by Western blotting. The experiments showed that unstimulated platelets contain considerable amounts of CTGF, whereas no CTGF was detectable in platelet-poor plasma. To elucidate the origin of CTGF in platelets, we performed immunohistochemical analysis of human bone marrow sections. The analysis showed that although CTGF protein is widely expressed in bone marrow cells, it is not expressed by platelet-producing megakaryocytes, suggesting that CTGF presence in platelets is a result of endocytosis from extracellular environment in bone marrow. Agonist-stimulation of platelets resulted in a significant release of CTGF from the storage granules, with thrombin at 0.1 U/mL being a more potent activator than ADP at 20 micro mol/L. The agonist-dependent CTGF secretion was significantly inhibited by aspirin. In conclusion, CTGF is stored in normal human platelets, and can be released upon platelet activation. Aspirin treatment prevents CTGF release, suggesting that clinical benefits of this drug may involve the inhibition of CTGF secretion.

Adenosine Diphosphate↗
PubMed2003

Modulation of the expression of connective tissue growth factor by alterations of the cytoskeleton.

Modulation of the cytoskeletal architecture was shown to regulate the expression of CTGF (connective tissue growth factor, CCN2). The microtubule disrupting agents nocodazole and colchicine strongly up-regulated CTGF expression, which was prevented upon stabilization of the microtubules by paclitaxel. As a consequence of microtubule disruption, RhoA was activated and the actin stress fibers were stabilized. Both effects were related to CTGF induction. Overexpression of constitutively active RhoA induced CTGF synthesis. Interference with RhoA signaling by simvastatin, toxinB, C3 toxin, and Y27632 prevented up-regulation of CTGF. Likewise, direct disintegration of the actin cytoskeleton by latrunculin B interfered with nocodazole-mediated up-regulation of CTGF expression. Disassembly of actin fibers by cytochalasin D, however, unexpectedly increased CTGF expression indicating that the content of F-actin per se was not the major determinant for CTGF gene expression. Given the fact that cytochalasin D sequesters G-actin, a decrease in G-actin increased CTGF, while increased levels of G-actin corresponded to reduced CTGF expression. These data link alterations in the microtubule and actin cytoskeleton to the expression of CTGF and provide a molecular basis for the observation that CTGF is up-regulated in cells exposed to mechanical stress.

Actins↗
PubMed2003

Induction of connective tissue growth factor by angiotensin II: integration of signaling pathways.

OBJECTIVE: Angiotensin II is recognized as one of the major mediators of cardiovascular pathology. Because connective tissue growth factor (CTGF) is involved in the pathophysiologic processes underlying fibrotic diseases, its regulation by angiotensin II was investigated. METHODS AND RESULTS: In the 2-kidney, 1-clip model of renovascular hypertension, increased expression of CTGF was detectable in the hypertrophic left ventricle. By activation of angiotensin II type 1 receptors, angiotensin II caused rapid expression of CTGF mRNA and protein in a human fibroblast cell line. Activation of the p42/44 mitogen-activated protein (MAP) kinase signaling pathway proved to be essential for angiotensin II-stimulated CTGF expression. Inhibition of MAP kinase activation by forskolin prevented CTGF induction. Inhibition of the isoprenylation of small GTPases by simvastatin or pretreatment of the cells with toxin B reduced basal CTGF expression below detection limits and prevented induction by angiotensin II. Specific interference with RhoA signaling by Y27632 primarily reduced basal CTGF expression. There was no significant reduction of expression of angiotensin II type 1 receptors by simvastatin. These data indicate cooperation between the Rho signaling and the angiotensin II-activated MAP kinase pathways. CONCLUSIONS: Direct induction of CTGF by angiotensin II is indicative of a role for CTGF in angiotensin II-mediated fibrosis and might be a target of antifibrotic interventions.

Angiotensin II↗
PubMed2003

Expression of cyclooxygenase-2 in biopsies obtained from human transplanted kidneys undergoing rejection.

BACKGROUND: The inducible cyclooxygenase (COX)-2 is a target of immunosuppressive drugs routinely administered to patients after transplantation. This study investigates a potential involvement of COX-2 in transplant rejection. Therefore, we examined the expression of COX-2 in biopsies obtained for diagnostic purposes. METHODS: COX-2 was detected by immunohistochemistry and in situ hybridization. Congruent staining was obtained by both methods: in specimens of a kidney explanted as the result of vascular rejection, tubular epithelial cells and endothelial cells stained positively for COX-2. Furthermore, in appendiceal specimens obtained at surgery, epithelial cells of the crypts, interstitial cells, and mesothelial cells were positive by both methods, affirming the specificity of the antibody. RESULTS: Compared with healthy control subjects, intensive staining of COX-2 was observed in most of the 28 biopsies obtained from patients diagnosed with vascular rejection combined with cellular interstitial rejection and tubulitis. Glomeruli and the macula densa area were essentially negative compared with prominent staining in cortical and medullary epithelial cells of the tubuli. Staining was distinct with individual positive cells in the tubular cross sections. Few arteries expressed COX-2 in intimal cells. Less prominent expression of COX-2 was detected in the biopsies of six kidneys obtained from patients diagnosed with acute tubular necrosis. CONCLUSION: This is the first report to show the up-regulation of COX-2 in human transplanted kidneys, despite ongoing immunosuppressive treatment. It remains to be established whether the up-regulation of COX-2 is part of the rejection process or has to be considered implicated in renal preservative mechanisms.

Appendicitis↗
PubMed2003

Failure of cyclosporin A to induce transforming growth factor beta (TGF-beta) synthesis in activated peripheral blood lymphocytes.

Induction of transforming growth factor beta (TGF-beta) by the immunosuppressive drug cyclosporin A (CsA) in activated lymphocytes has been claimed to add to the renal pro-fibrotic effects of CsA. The aim of this study was to evaluate CsA-mediated TGF-beta induction in a larger number of lymphocyte preparations from different donors. Peripheral blood lymphocytes (PBL) were obtained from healthy blood donors. The cells were stimulated with phytohemagglutinin E (PHA) and phorbol ester (tetradecanoyl phorbol acetate, TPA) in the presence or absence of CsA. TGF-beta, interleukin-2 (IL-2) and cyclooxygenase-2 (Cox-2) mRNA were detected by Northern blot analysis or by real time reverse transcriptase-polymerase chain reaction (RT-PCR). TGF-beta and IL-2 protein were determined in the cellular supernatants by enzyme-linked immunosorbent assay. TGF-beta mRNA and protein were up-regulated when the cells were stimulated with PHA/TPA. Cyclosporin A at high concentrations (500 ng/mL) caused a transient increase in TGF-beta mRNA which was significant after 2 h. CsA did not induce sustained TGF-beta protein expression (24-72 h) in any of the preparations (n = 14), whereas the up-regulation of IL-2 mRNA and protein was prevented by CsA in the same preparations. Furthermore, up-regulation of Cox-2 mRNA was inhibited by CsA. Taken together, there was no evidence for TGF-beta as a clinically relevant mediator being induced by CsA in activated peripheral blood T-lymphocytes.

Adult↗
PubMed2002

Role of macula densa cyclooxygenase-2 in renovascular hypertension.

Upregulation of the inducible cyclooxygenase (COX-2) in the macula densa accompanies the activation of the juxtaglomerular apparatus in many high-renin conditions. The functional role of COX-2 in these disease states is poorly understood. We tested whether COX-2 is required to increase renin in renovascular hypertension. Rats with established two-kidney, one-clip (2K1C) hypertension were treated for 2 wk with two different inhibitors of COX-2, NS-398 and rofecoxib, respectively. Hypertension in 2K1C rats was not affected or slightly enhanced by COX-2 inhibition, as measured intra-arterially in conscious animals. The increase in plasma renin activity was also unchanged by both rofecoxib and NS-398. The number of glomeruli with a renin-positive juxtaglomerular apparatus was elevated in clipped kidneys and decreased in contralateral kidneys of 2K1C rats. This pattern was unaltered by COX-2 inhibition. To test the effects of COX-2 blockade on a primarily macula densa-mediated stimulus, we studied salt depletion for comparison. A low-salt diet induced a significant increase in plasma renin activity, which was partially inhibited by treatment with NS-398. We conclude that inhibition of COX-2 in established renovascular hypertension does not affect renin synthesis or release. Thus either COX-2 is not necessary for the macula densa mechanism or the macula densa is not important for maintaining high renin in renovascular hypertension.

Animals↗
PubMed2002

Role of Rac and Cdc42 in lysophosphatidic acid-mediated cyclo-oxygenase-2 gene expression.

The role of Rho proteins in lysophosphatidic acid (LPA)-mediated induction of cyclo-oxygenase-2 (Cox-2) was investigated in renal mesangial cells. Previous studies had shown that toxin B, an inhibitor of Rho, Rac and Cdc42, suppressed Cox-2 induction. A role for RhoA in pertussis toxin-sensitive LPA signalling was excluded with C3 transferase from Clostridium limosum, used as the fusion toxin C2IN-C3 (where C2IN is part of the C2I toxin of C. botulinum). Incubation of the cells with C2IN-C3 disrupted cytosolic actin stress fibres, but had no effect on Cox-2 induction. Similarly, activation of p42/44 mitogen-activated protein kinase (MAP kinase), an upstream step in Cox-2 induction, was inhibited by toxin B, but not affected by C2IN-C3. Upon treatment with toxin B, focal adhesion kinase and paxillin were dephosphorylated at tyrosine residues and the actin cytoskeleton was completely destroyed. An intact cytoskeleton, however, was not required for p42/44 MAP-kinase activation or Cox-2 induction, as shown by the actin-depolymerizing agent cytochalasin D. Toxin B did not influence functionality of LPA receptors, because G(i)-mediated Ca(2+) release from intracellular stores remained unchanged. Within 1 h, toxin B inactivated and translocated RhoA and Cdc42 to the cellular membranes. Within the same time frame, monoglucosylated Rac1 was degraded. Direct stimulation of Rho proteins by cytotoxic necrotizing factor type 1 (CNF1) induced Cox-2 expression, which was sensitive to inhibition of the MAP-kinase pathway by PD98059, but not to an inhibitor of RhoA kinase. By exclusion of RhoA and non-specific cytoskeletal effects, the results in the present study indicate an important role for Rac and/or Cdc42 in pertussis toxin-sensitive LPA-mediated Cox-2 induction.

Animals↗
PubMed2002

Induction of cyclooxygenase-2 and enhanced release of prostaglandin E(2) and I(2) in human endothelial cells by engagement of CD40.

OBJECTIVES: The hypothesis was tested that CD40-CD154 interaction is involved in the induction of cyclooxygenase-2 and the release of prostanoids in human endothelial cells. METHODS AND RESULTS: In a coculture model of human endothelial cells and a transfected CD154 positive cell line, engagement of CD40 on endothelial cells dramatically increased the synthesis of prostacyclin, prostaglandin E(2) and thromboxane A(2). This upregulation was mediated through an induction of cyclooxygenase-2 (Cox-2), as it was blocked by Cox-2-selective inhibitors. Western blot analysis demonstrated that Cox-2 protein was markedly increased in endothelial cells following CD40 engagement, an effect that was inhibited by pretreatment of cells with an anti-CD154 antibody. CONCLUSION: The data indicate that signaling via CD40 constitutes a major pathway in human endothelial cells for the induction of Cox-2 and release of prostanoids. The CD40-Cox-2 axis thus may represent an important pathway for initiating or maintaining an inflammatory process at the vessel wall.

Blotting, Western↗