Manuel Rodríguez-Puyol

Nitric Oxide Regulates Transforming Growth Factor-β Signaling in Endothelial Cells

Many forms of vascular disease are characterized by increased transforming growth factor (TGF)-&bgr;1 expression and endothelial dysfunction. Smad proteins are a key step in TGF-&bgr;–initiated signal transduction. We hypothesized that NO may regulate endothelial TGF-&bgr;–dependent gene expression. We show that NO inhibits TGF-&bgr;/Smad–regulated gene transactivation in a cGMP-dependent manner. NO effects were mimicked by a soluble analogue of cGMP. Inhibition of cGMP-dependent protein kinase 1 (PKG-1) or overexpression of dominant-negative PKG-1&agr; suppressed NO/cGMP inhibition of TGF-&bgr;–induced gene expression. Inversely, overexpression of PKG-1&agr; catalytic subunit blocked TGF-&bgr;–induced gene transactivation. Furthermore NO delayed and reduced phosphorylated Smad2/3 nuclear translocation, an effect mediated by PKG-1, whereas NG-nitro-l-arginine methyl ester augmented Smad phosphorylation and gene expression in response to TGF-&bgr;. Aortas from endothelial NO synthase–deficient mice showed enhanced basal TGF-&bgr;1 and collagen type I expression; endothelial cells from these animals showed increased Smad phosphorylation and transcriptional activity. Proteasome inhibitors prevented the inhibitory effect of NO on TGF-&bgr; signaling. NO reduced the metabolic life of ectopically expressed Smad2 and enhanced its ubiquitination. Taken together, these results suggest that the endothelial NO/cGMP/PKG pathway interferes with TGF-&bgr;/Smad2 signaling by directing the proteasomal degradation of activated Smad.

Stat3 Mediates Interelukin-6 Inhibition of Human Endothelial Nitric-oxide Synthase Expression

Chronic activation of the acute phase response (APR) is associated with atherosclerosis. Elevated levels of interleukin-6, the major inducer of the APR, are associated with an increased risk of cardiovascular events. One of the clinical hallmarks of atherogenesis is endothelial dysfunction, characterized by a decrease in endothelial production of nitric oxide (NO). We hypothesized that interleukin-6 (IL-6) decreases endothelial NO synthase (eNOS) expression. We now show that IL-6 treatment of human aortic endothelial cells (HAEC) decreases steady-state levels of human eNOS mRNA and protein. This decrease in eNOS expression is caused in part by IL-6 inhibition of transactivation of the human eNOS promoter. To explore the mechanism by which IL-6 affects eNOS expression, we examined activation of signal transducer and transactivator-3 (Stat3). The IL-6 receptor (IL-6R) is expressed in HAEC, and Stat3 is phosphorylated in response to IL-6 stimulation of the IL-6R. We identified four consensus sequences for Stat3 binding (SIE) in the eNOS promoter at positions -1520, -1024, -840, and -540. Transfection of eNOS promoter mutants revealed that the SIE at -1024 mediates Stat3 inhibition of eNOS promoter activity. Gel-shift analysis of nuclear extracts from HAEC treated with IL-6 confirms that Stat3 binds to a complex containing the SIE at -1024. RNA silencing of STAT3 blocks the inhibitory effect of IL-6 on eNOS expression. Our data show that IL-6 has direct effects upon endothelial cells, inhibiting eNOS expression in part through Stat3. Decreased levels of eNOS may be an important component of the pro-atherogenic effect of the APR.

Cyclosporine a nephrotoxicity: Role of thromboxane and reactive oxygen species

The main adverse effect of cyclosporine A (CyA) is nephrotoxicity. CyA increases urinary concentrations of thromboxane A2 (TXA2), a potent vasoconstrictor that can be involved in kidney failure induced by CyA. Furthermore, it has been postulated that a relationship exists between oxygen free radicals and the synthesis of arachidonate metabolites in experimental models of CyA nephrotoxicity. We studied the effect of vitamin E (VitE), an oxygen free radical scavenger, on renal function, on glomerular synthesis of thromboxane B2 (TXB2), and on free radicals in rats treated with CyA. Four groups of male Wistar rats were studied: (1) a control group; (2) a group given VitE at 0.05 mg/dl in drinking water for 25 days; (3) a group given CyA at 50 mg/kg body weight/day orally for 10 days; and (3) a group given Vit E + CyA, in which rats were provided with drinking water containing VitE for 15 days and afterwards were treated with VitE and CyA for 10 days. Renal function parameters and glomerular synthesis of TXB2, superoxide anion (02.-), malondialdehyde (MDA), and hydrogen peroxide (H202) were evaluated. CyA decreased body weight, caused deterioration of kidney function and increased glomerular synthesis of TXB2, O2.-, MDA, and H202. Pretreatment with VitE prevented the effects of CyA on kidney function and decreased glomerular synthesis of these mediators. In conclusion, CyA induced glomerular synthesis of reactive oxygen species (ROS) and TxB2. Pretreatment with VitE inhibited acute renal failure induced by CyA, probably by scavenging free radicals and by inhibiting the synthesis of TXB2.

Cyclosporine-loaded polycaprolactone nanoparticles: immunosuppression and nephrotoxicity in rats

AIM The nephrotoxicity and immunosuppressive ability of cyclosporine (CyA) incorporated into polycaprolactone nanoparticles (CyA-NP) was assessed in vitro and in vivo and compared to the effects caused by free drug (Sandimmun. METHODS The in vivo study included four groups (12 Wistar rats each) receiving oral CyA (10 mg/kg/day for 3 days) as an emulsion of Sandimmun in whole milk or CyA-NP and equivalent doses of empty NP or cremophor in milk as controls. CyA concentrations in blood, urine, liver, spleen and kidney at 24 h post-dosing were measured by fluorescence polarization immunoassay (FPIA). The nephrotoxicity induced by each drug treatment was determined by measuring creatinine plasma levels, malonyl dialdehyde production, and H(2)O(2) and reduced glutathione contents in glomeruli. On the other hand, the immunosuppressive effect was estimated in vivo by incubating lymphocyte suspensions obtained from CyA-, CyA-NP- and control-treated rats, as well as in vitro on lymphocyte suspensions from non-treated healthy animals. RESULTS Significantly higher blood, urine and tissue levels were achieved with CyA-NP compared to free CyA. However, no changes in creatinine plasma levels were detected due to either CyA or CyA-NP treatment. Only the production of H(2)O(2) in the glomeruli exhibited a significant increase as compared to control groups, but no differences could be ascribed to the different drug treatments. In vivo, the immunosuppressive activity was also comparable for both drug treatments. In contrast, CyA-NP showed a better drug uptake in vitro at concentrations above 25 microM. No immunosuppression was detected in control groups. CONCLUSION NP improve the oral bioavailability of CyA and its uptake by lymphocytes in vitro above 25 microM. On the contrary, specific immunosuppression and adverse effects were not simultaneously increased. Further studies are needed to clarify the results.

The leukocyte-endothelial cell interactions are modulated by extracellular matrix proteins.

Background: Endothelium is supported, in normal conditions, by a basement membrane composed, among others, by collagen IV and laminin. Changes in the basement membrane composition could induce changes in endothelial cell modifying their interactions with leukocytes. Methods and Results: Isolated polymorphonuclear cells (PMN) and peripheral blood mononuclear cells (PBMC) were added to cultured human umbilical endothelial cells (HuVEC) previously seeded on collagen IV, collagen I or gelatin. Adhesion of leukocytes to HUVEC and specific cytotoxicity were analysed. PMN adhesion and cytotoxicity were lower whereas those from PBMC were higher when HuVEC were seeded on collagen I, as compared with cells seeded on collagen IV. To analyse the mechanisms involved in these phenomena, P-selectin, ICAM-1, VCAM-1 and MCP- 1 expression were evaluated in HuVEC seeded on the different ECM components. P-selectin and mRNA expression of VCAM-1 were lower in cells seeded on collagen I. By contrast, MCP-1 expression was higher in collagen I. Collagen I-dependent effects were partially prevented when collagen I was treated with pepsin. ILK activity was lower in cells seeded on collagen I, whereas ERK 1/2 activity was enhanced. ILK overexpression reduced ERK 1/2 phosphorylation and this could promote the reduction in P-selectin and the increase in MCP-1. Conclusion: Collagen I decreased ILK activity and this would induce an increase in ERK 1/2 activity in HuVEC. As a consequence, the P-selectin content is diminished and, by contrast, the MCP-1 content is increased. The final effect is a lower recruitment of PMN and a higher adhesion of PBMC.

MECHANISMS INVOLVED IN THE CONTRACTION OF ENDOTHELIAL CELLS BY HYDROGEN PEROXIDE

The importance of endothelial contraction in the genesis of inflammatory edema has been reported. ROS are metabolites synthesized in pathological conditions in that a significant intravascular fluid leak occurs, such as ischemia-reperfusion. Present experiments were designed to test the hypothesis that ROS, particularly H2O2, may elicit the contraction of endothelial cells, and to explore the mechanisms involved. Bovine aortic endothelial cells incubated with H2O2 showed a significant reduction in planar cell surface area (PCSA), and a significant increase in myosin light chain phosphorylation (MLCP), with a time- and dose-dependent pattern, without any significant toxicity. This effect of H2O2 was not blocked by sulotroban (TxA2 antagonist) or BN 52021 (PAF antagonist). Lanthanum chloride (calcium channel blocker) and EGTA partially inhibited the increase in MLCP induced by H2O2. H7 and staurosporine, PKC inhibitors, and PKC down-regulation (phorbol myristate acetate treatment, 24 h) also blocked H2O2-dependent endothelial contraction, measured as PCSA or MLCP. H2O2 increased the intracellular calcium concentration, an effect blunted by EGTA and lanthanum chloride. H2O2 also increased the phosphorylation of an 80 kD polypeptide, probably MARCKS, a PKC substrate. In summary, the present results demonstrate the ROS-dependent contraction of endothelial cells, an effect that could explain the intravascular fluid leak observed in some pathophysiological situations. Calcium and PKC may be involved in the development of this contraction.

Decreased Nitric Oxide Synthesis in Human Endothelial Cells Cultured on Type I Collagen

Endothelial dysfunction, considered as a defective vascular dilatation after certain stimuli, is characteristic of different pathological conditions, such as hypertension, atherosclerosis, or diabetes. A decreased synthesis or an increased degradation of nitric oxide (NO) has been postulated as the mechanism responsible for this alteration. The present experiments were designed to test the hypothesis that the presence of an abnormal extracellular matrix in vessel walls could be responsible for the decreased NO synthesis observed in these pathological conditions. Experiments were performed in cultured human umbilical vein endothelial cells (HUVECs) grown on type IV (Col. IV) or type I (Col. I) collagen. Cells seeded on Col. I showed decreased nitrite synthesis, nitric oxide synthase activity, eNOS protein content, and eNOS mRNA expression when compared with cells grown on Col. IV. Moreover, cells grown on Col. I failed to respond to glucose oxidase activation of the eNOS system. In both cases, the changes in the eNOS mRNA expression seemed to depend on the modulation of eNOS promoter activity. The downregulation of eNOS induced by Col. I was blocked by D6Y, a peptide that interferes with the Col. I-dependent signals through integrins, as well as by specific anti-integrin antibodies. Moreover, a decreased activation of integrin-linked kinase (ILK) may explain the effects observed in Col. I-cultured cells because the activity of this kinase was decreased in these cells and ILK modulation prevented the Col. I-induced changes in HUVECs. Taken together, these findings may contribute to explaining the basis of endothelial dysfunction in some vascular diseases.

Integrin-Linked Kinase Regulates Vasomotor Function by Preventing Endothelial Nitric Oxide Synthase Uncoupling Role in Atherosclerosis

Rationale: Atherosclerotic lesions develop in regions of disturbed flow, whereas laminar flow protects from atherogenesis; however, the mechanisms involved are not completely elucidated. Integrins are mechanosensors of shear stress in endothelial cells, and integrin-linked kinase (ILK) is important for blood vessel integrity and cardiovascular development. Objectives: To explore the role of ILK in vascular function by studying conditionally ILK-deficient (cKO) mice and human atherosclerotic arteries. Results: ILK expression was detected in the endothelial cell layer of nonatherosclerotic vessels but was absent from the endothelium of atherosclerotic arteries. Live ultrasound imaging revealed that acetylcholine-mediated vasodilatation was impaired in cKO mice. These mice exhibited lowered agonist-induced nitric oxide synthase (NOS) activity and decreased cyclic guanosine monophosphate and nitrite production. ILK deletion caused endothelial NOS (eNOS) uncoupling, reflected in reduced tetrahydrobiopterin (BH4) levels, increased BH2 levels, decreased dihydrofolate reductase expression, and increased eNOS-dependent generation of superoxide accompanied by extensive vascular protein nitration. ILK reexpression prevented eNOS uncoupling in cKO cells, whereas superoxide formation was unaffected by ILK depletion in eNOS-KO cells, indicating eNOS as a primary source of superoxide anion. eNOS and ILK coimmunoprecipitated in aortic lysates from control animals, and eNOS-ILK–shock protein 90 interaction was detected in human normal mammary arteries but was absent from human atherosclerotic carotid arteries. eNOS-ILK interaction in endothelial cells was prevented by geldanamycin, suggesting heat shock protein 90 as a binding partner. Conclusions: Our results identify ILK as a regulatory partner of eNOS in vivo that prevents eNOS uncoupling, and suggest ILK as a therapeutic target for prevention of endothelial dysfunction related to shear stress–induced vascular diseases.

Telomerase deficiency promotes oxidative stress by reducing catalase activity

Telomere shortening and redox imbalance have been related to the aging process. We used cultured mouse embryonic fibroblasts (MEF) isolated from mice lacking telomerase activity (Terc(-/-)) to analyze the redox balance and the functional consequences promoted by telomerase deficiency. Comparison with wild-type (WT) MEF showed that Terc(-/-) MEF had greater oxidant damage, showing higher superoxide anion and hydrogen peroxide production and lower catalase activity. Restoration of telomerase activity in Terc(-/-) MEF increased catalase expression and activity. TGF-beta1 and collagen type IV levels were higher in Terc(-/-) than in WT MEF. TGF-beta1 promoter activity decreased when Terc(-/-) MEF were incubated with exogenous catalase, suggesting that catalase deficiency is the cause of the TGF-beta1 increase. Similar results were obtained in vivo. Homogenized renal cortex from 6-month-old Terc(-/-) showed higher oxidant capacity, lower catalase activity, greater oxidative damage, and higher TGF-beta1 and fibronectin levels than that from WT mice. In summary, telomerase deficiency reduces catalase activity, determining a redox imbalance that promotes overexpression of TGF-beta1 and extracellular matrix proteins.

Oxidant/Antioxidant Balance in Isolated Glomeruli and Cultured Mesangial Cells

The mechanisms responsible for age-related glomerular sclerosis (GS) have not been clearly identified. The present experiments were aimed at assessing the importance of the oxidant/antioxidant balance in the early stages of this process. For this purpose, the renal function (biochemical and clearance studies), some characteristics of isolated glomeruli, and reactive oxygen production (superoxide anion, hydrogen peroxide) as well as the antioxidant ability (superoxide dismutase, catalase, glutathione peroxidase) of glomeruli and cultured mesangial cells were studied in 3- and 18-month-old Fischer 344 rats (YOUNG and OLD rats, respectively). OLD animals show a normal renal function, increased urine protein excretion, and augmented protein glomerular content, an indirect index of GS. Isolated glomeruli from these rats produced increased amounts of superoxide anion and hydrogen peroxide, and catalase activity was increased. The glomerular thiobarbituric acid-reactive substances (TBARS) content was higher in OLD than in YOUNG animals. Similar results were obtained in cultured mesangial cells. In summary, the present results demonstrate, at an early stage of rat GS development, an association between the functional and structural changes of this process and an increased TBARS content (likely indicative of lipid oxidative damage) at the glomerular structures as well as in cultured mesangial cells. More extensive studies are needed to confirm the nature of this association.