Platelet-derived growth factor activates production of reactive oxygen species by NAD(P)H oxidase in smooth muscle cells through Gi1,2.

Recent findings indicate that platelet-derived growth factor (PDGF) plays a role in the generation of reactive oxygen species (ROS) as second messengers in smooth muscle cells (SMC). To identify the source and signal transduction pathway of ROS formation in SMC, we investigated PDGF-induced ROS formation. Stimulation of SMC with PDGF resulted in a rapid increase of ROS production. Using an inactivating antibody, we identified the increase to be dependent on p22phox, a NAD(P)H-oxidase subunit. ROS release was completely inhibited by the Gi protein inhibitor PTX as well as an antibody against Galphai1,2, however, not by antibodies against Galphai3/0, Gas, and Gbeta1beta2. The effect of PDGF on ROS production in SMC membranes could likewise be mimicked by the use of a recombinant Galphai2 subunit but not by Galphai3, Galphai0, Gas, and Gbetagamma subunits. Immunoaffinity chromatography demonstrated coupling of Galphai1,2 to the PDGF a-receptor, which, after preincubation of the SMC membranes with PDGF, was increased in the absence of GTPgammaS but decreased in the presence of GTPgammaS and prevented by PTX treatment. These data define a novel G protein-dependent mechanism by which PDGF signaling is transduced through direct coupling of the Gai1,2 subunit of the trimeric G proteins to the PDGF tyrosine kinase receptor.

Interaction of transforming growth factor beta 1 with human glomerular epithelial cells in culture: opposite effects on synthesis of matrix proteins and on urokinase plasminogen activator.

The effect of transforming growth factor-beta (TGF-beta) was analyzed on the synthesis of fibronectin, collagen type IV, and urokinase plasminogen activator in human glomerular epithelial cells in culture. An increase in the abundance of specific mRNA was found for collagen type IV and fibronectin. Fibronectin protein synthesis was also increased in TGF-beta treated cells; most of the de novo synthesized fibronectin was found as an unsoluble protein associated with extracellular matrix. In the same cells the amount of plasminogen activator mRNA was found leading also to a decreased surface expression of urokinase plasminogen activator. The data support the concept that by upregulating matrix protein synthesis and downregulating the plasminogen activator system, TGF-beta favors the development of sclerosis.

Thrombin-induced MCP-1 expression involves activation of the p22phox-containing NADPH oxidase in human vascular smooth muscle cells.

UNLABELLED: Activation of vascular smooth muscle cells (VMSC) by thrombin induces the expression of the chemokine, monocyte chemoattractant protein-1 (MCP-1). We investigated in cultured human and rat VSMC whether reactive oxygen species (ROS) derived from the vascular NADPH oxidase contribute to this effect. Exposure of cultured VSMC to thrombin rapidly increased ROS formation, phosphorylation of p38 MAP kinase as well as the expression of MCP-1. Specific inhibition of the p22phox subunit of the vascular NADPH oxidase using either p22phox neutralizing antibody or p22phox antisense oligonucleotides attenuated thrombin-induced ROS generation. Furthermore, thrombin-induced p38 MAP kinase activation as well as MCP-1 expression were impaired by antioxidants as well as by p22phox antisense oligonucleotides. Inhibition of p38 MAP kinase diminished the thrombin-induced expression of MCP-1. CONCLUSION: Thrombin, by activating a p22phox-containing NADPH oxidase, elicits ROS generation and activation of p38 MAP kinase in VSMC. The subsequent induction of MCP-1 expression highligts the crucial role of the p22phox-containing NADPH oxidase in thrombin-induced signal transduction in VSMC.

Endothelin in renal diseases and cardiovascular remodeling in renal failure.

The pathogenetic mechanisms leading to progression of renal failure are only partly understood. Several studies in immune- and non-immune-mediated models of renal damage have recently implicated the endothelin (ET) system as a major player in these processes. In animal models, ET receptor antagonists have been shown to be highly effective in abrogating the progression of renal failure. Furthermore, cardiac structural alterations seen in hypertension and/or renal insufficiency, e.g. left ventricular hypertrophy, thickening of intramyocardial arterioles, and the increase in non-vascular interstitial tissue, are largely prevented by ET receptor antagonists. In this context it is of interest that these beneficial renal and cardiac effects are, at least in most studies, independent of systemic blood pressure. In addition to the specific pharmacological blockade of the renin-angiotensin system [ACE inhibitors, angiotensin II receptor (AT1) antagonists], blockade of ET receptors or ET converting enzyme (ECE) may be a new tool to interfere with progression of renal failure and cardiovascular remodeling in humans.

[Fibrinolytic therapy in thrombosis of a mitral valve prosthesis]. / Fibrinolytische Therapie bei Thrombose einer Mitralklappenprothese.

A 48-year-old woman presented with progressive dyspnea due to thrombosis of a mitral valve prosthesis. The patient had undergone mitral valve replacement (St. Jude Medical) six years prior to admission because of mitral stenosis (Class III); three years later the prosthesis had to be replaced (St. Jude Medical) because of valve thrombosis. At admission, transesophageal echocardiography showed a thrombus on the atrial side of the fixed valve leaflet and a thrombus (2.4 x 1.6 cm) floating from the left atrial roof. Because of the previous thoracotomies, thrombolysis was initiated despite the mobile thrombus with the attendant risk of embolization. Urokinase was infused in a dose to maintain the fibrinogen level around 100 mg/dl. After 24 h, the mean pressure gradient across the prosthetic mitral valve (measured by doppler echocardiography) had decreased from 23 to 11 mmHg. After 13 days of this modified thrombolytic regimen, the clinical symptoms of the patient had resolved and echocardiography showed a normal function of the prosthetic mitral valve without evidence of residual thrombosis. This patient demonstrates that prolonged cautious thrombolysis can be effective for the treatment of prosthetic valve thrombosis in hemodynamically moderately compromised patients.

Fibronectin synthesis in tubular epithelial cells: up-regulation of the EDA splice variant by transforming growth factor beta.

The influence of transforming growth factor beta 1 (TGF-beta 1) and of dexamethasone on fibronectin (FN) synthesis of human renal tubular epithelial cells in culture (TEC) was studied. Cocultivation with TGF-beta 1 increased the steady state level of FN RNA within 24 to 48 hours. By PCR and Northern blotting it was found that the EDA splice variant of FN was preferentially up-regulated. To quantitate FN protein synthesis, cells were cultivated in the presence of [35S]-methionine and FN was isolated from the cell supernatants, and the cell lysates by adsorption to gelatin-sepharose. In TGF-beta 1 treated cells, a small increase of FN in the cell supernatants was seen (1.7-fold), and a more prominent increase in the cell lysates (4.5-fold). The FN content of the extracellular matrix was also increased in TGF-beta 1 treated cells. Most of the de novo synthesized FN was identified as the EDA-variant of FN. As a further stimulus, dexamethasone was used. Again, an increase of FN-specific mRNA was seen as well as an increased FN protein synthesis. The ratio between FN and EDA-FN, however, was not altered when compared to untreated cells. Thus, an increase in EDA-FN synthesis is obviously stimulus dependent.

Adverse effects of smoking in the renal patient.

Smoking is a known risk factor for cardiovascular diseases, cancer, and several other health problems. It is the number one preventable cause of death in modern countries. The first evidence that smoking may be a renal risk factor was published in 1978. Since then, several studies documented that smoking is nephrotoxic in patients with diabetic and non-diabetic renal disease. Data from the Multiple Risk Factor Intervention Trial indicate that smoking even increases the renal risk in the general male population: an increased relative risk for end-stage renal failure (ESRF) was found for smokers as compared to non-smokers (up to 1.69 for heavy smokers). Several potential mechanisms of smoking-induced renal damage have been discussed, e.g. increase in blood pressure, alteration of intrarenal hemodynamics, as well as activation of the sympathetic nerve, the reninangiotensin and the endothelin systems. The pathomechanisms are, however, only partly understood. Once ESRF has become established, the failure to discontinue smoking adversely affects the prognosis of patients on renal replacement therapy, mainly by increasing the risk of cardiovascular complications. Discontinuation of smoking has been shown to improve both renal and cardiovascular prognosis in the renal patient and is probably the single most effective measure to retard progression of renal failure.

The terminal complement complex C5b-9 stimulates interleukin-6 production in human smooth muscle cells through activation of transcription factors NF-kappa B and AP-1.

Activation of the complement system plays an important role in the pathogenesis of atherosclerosis. The proinflammatory cytokine interleukin (IL)-6 is potentially involved in the progression of the disease. We therefore investigated whether the terminal complement complex C5b-9 affects IL-6 production from vascular smooth-muscle cells (VSMC) and set out to determine the underlying signal transduction pathway. Stimulation of human VSMC with C5b-9 resulted in an increase of IL-6 transcript and production of IL-6 protein. Pretreatment with pertussis toxin or pyrrolidine dithiocarbamate inhibited complement-dependent IL-6 mRNA expression and IL-6 release, suggesting the involvement of Gi-proteins and nuclear factor-kB (NF-kB). C5b-9 also induced formation of reactive oxygen species, which, along with IL-6 release, was inhibited by the antioxidant N-acetylcysteine. C5b-9 activated the redox-sensitive transcription factors NF-kB and activator protein-1 (AP-1), which were both involved in the induction of IL-6 by C5b-9, as demonstrated by cis element double-stranded (decoy) oligonucleotides (ODN). The results demonstrate that activation of the complement system induces IL-6 release from human VSMC by a Gi-dependent pathway involving the generation of oxidative stressand the activation of the redox sensitive transcription factors NF-kB and AP-1. Our data support a new mechanism for the proatherogenic effect of the terminal complement complex.

Endothelin-1 and smooth muscle cells: induction of jun amino-terminal kinase through an oxygen radical-sensitive mechanism.

Endothelin-1 (ET-1) has been proposed to contribute to atherogenesis and plaque rupture in coronary heart disease through activation of mitogen-activated protein kinases (MAPKs) in smooth muscle cells (SMCs). Reactive oxygen species (ROS) have been shown to be important signal transduction molecules in SMCs. Thus, the present study aimed to assess the role of ROS in ET-1-mediated activation of c-Jun amino-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) 1/2. Rat SMCs were exposed to ET-1 over time at concentrations from 10(-6) to 10(-10) mol/L, and MAPK activity was quantified. Activation of JNK and ERK was observed with a maximum stimulation at 10(-7) mol/L ET-1. JNK and ERK were activated by ET-1 binding to a single receptor (ET-1A) but differed in their downstream mechanisms: only JNK activation was sensitive to the radical scavenger N-acetylcysteine and diphenylene iodonium, an inhibitor of NADPH oxidase, indicating a role for ROS. The downstream MAPK effector and proinflammatory transcription factor, the activator protein-1 complex, was maximally activated 2 hours after the addition of ET-1. It was mainly composed of the JNK substrate c-Jun, and activation was also dependent on ROS formation. We suggest that plaque activation by ET-1 can be mediated through ROS. It can be hypothesized that the clinical benefit of antioxidants in the treatment of atherogenesis may partially depend on neutralization of ET-1-mediated ROS production.

Fibronectin synthesis by human tubular epithelial cells in culture: effects of PDGF and TGF-beta on synthesis and splicing.

BACKGROUND: Enhanced synthesis of extracellular matrix proteins including fibronectin (FN) is associated with the development of sclerosis. In this context we studied FN synthesis by tubular epithelial cells in response to transforming growth factor-beta (TGF-beta) and platelet-derived growth factor (PDGF). METHODS: FN protein synthesis by human tubular epithelial cells in culture (TEC) was measured by biosynthetic labeling and ELISA. Splicing of FN was assessed by RT-PCR and by Northern blotting. RESULTS: Cultivated TEC synthesized and released FN, the majority of which was deposited as an unsoluble protein and a minor portion (10 to 15%) was released into the supernatant. TGF-beta and, to a lesser degree, PDGF, up-regulated FN synthesis. All three FN splice variants (EDA, EDB, and IIICS) were produced. PDGF did not influence the splicing. TGF-beta preferentially up-regulated the EDA splice variant, but had no effect on the splicing of the other domains. CONCLUSIONS: PDGF and TGF-beta both up-regulate FN synthesis of TEC. TGF-beta, but not PDGF, also changed the quality of the de novo synthesized FN, and thus has a different role in the development of sclerosis.

Differential activation of mitogen-activated protein kinases in smooth muscle cells by angiotensin II: involvement of p22phox and reactive oxygen species.

The atherogenic effect of the renin-angiotensin system can be explained, in part, by the influence of its effector, angiotensin II (Ang II), on vascular smooth muscle cell (VSMC) growth. There is evidence that reactive oxygen species (ROS) play a role in the atherogenesis and activation of mitogen-activating protein (MAP) kinases, which are involved in proliferation and differentiation. The study was performed to further characterize the role of ROS in Ang II-mediated MAP kinase activation and the regulation of the transcription factor activator protein-1 (AP-1). Rat VSMCs were stimulated with Ang II. The activities of MAP kinases were assessed by Western blot analysis or by immunocomplex kinase assay. AP-1 binding was determined by using an electrophoretic mobility shift assay. Rat VSMCs were treated with Ang II-activated MAP kinases, extracellular signal-regulated kinase (ERK), c-Jun amino terminal kinase (JNK), p38 MAP kinase (p38 MAPK), and their downstream effector, AP-1. Interestingly, only the activation of ERK1/2, but not JNK or p38 MAPK, was tyrosine kinase, protein kinase C, and MEK1/2 dependent. Ang II also induced the rapid formation of ROS, which could be inhibited by a specific antibody as well as by antisense against the p22phox subunit of the NAD(P)H oxidase. JNK and p38 MAPK, but not ERK, activation was inhibited by an inhibitor of NAD(P)H oxidase. Antisense against p22phox also solely inhibited p38 MAPK but did not affect ERK. The results indicate that in VSMCs, Ang II activates MAP kinases and AP-1 through different pathways; the results further suggest that ROS, generated by p22phox, mediate Ang II-induced JNK and p38 MAPK activation, which may contribute to the pathogenesis of atherosclerosis.