Ezetimibe reduces plaque inflammation in a rabbit model of atherosclerosis and inhibits monocyte migration in addition to its lipid-lowering effect.

BACKGROUND AND PURPOSE: Ezetimibe, a selective inhibitor of intestinal cholesterol absorption, might also suppress inflammatory components of atherogenesis. We have studied the effects of ezetimibe on two characteristics of atherosclerotic plaques (infiltrate and fibrosis) and on expression of inflammatory genes in a rabbit model of accelerated atherosclerosis. EXPERIMENTAL APPROACH: Femoral atherosclerosis was induced by a combination of endothelial desiccation and atherogenic diet. Animals were randomized to ezetimibe (0.6 mg x kg(-1) x day(-1)), simvastatin (5 mg x kg(-1) x day(-1)), ezetimibe plus simvastatin or no treatment, still on atherogenic diet. A control group of rabbits received normolipidemic diet. KEY RESULTS: Rabbits fed the normolipidemic diet showed normal plasma lipid levels. Either the normolipidemic diet or drug treatment reduced the intima/media ratio (normolipidemic diet: 22%, ezetimibe: 13%, simvastatin: 27%, ezetimibe + simvastatin: 28%), compared with rabbits with atherosclerosis. Ezetimibe also decreased macrophage content and monocyte chemoattractant protein-1 expression in atherosclerotic lesions. Furthermore, ezetimibe reduced the increased activity of nuclear factor kappaB in peripheral blood leucocytes and plasma C-reactive protein levels in rabbits with atherosclerosis. In THP-1 cells, ezetimibe decreased monocyte chemoattractant protein-1-induced monocyte migration. Importantly, the combination of ezetimibe with simvastatin was associated with a more significant reduction in plaque monocyte/macrophage content and some proinflammatory markers than observed with each drug alone. CONCLUSIONS AND IMPLICATIONS: Ezetimibe had beneficial effects both on atherosclerosis progression and plaque stabilization and showed additional anti-atherogenic benefits when combined with simvastatin. Its effect on monocyte migration provides a potentially beneficial action, in addition to its effects on lipids.

Collagen I upregulates extracellular matrix gene expression and secretion of TGF-beta 1 by cultured human mesangial cells.

Progressive renal diseases are characterized by an increased synthesis of extracellular matrix (ECM) components. The mechanisms involved in the development of these alterations are not completely known, but a crucial role for TGF-beta 1 has been suggested. Moreover, the ability of the ECM to modulate the phenotypic expression of different cell types has been widely described. In experiments presented here, human mesangial cells (HMC) were grown on collagen type I (COL I) or IV (COL IV). ECM protein and TGF-beta 1 mRNA expression were evaluated by Northern blot analysis, and TGF-beta 1 secretion was evaluated by ELISA. The involvement of tyrosine kinase and serine-threonine kinase pathways was studied by Western blot analysis, immunofluorescence, and in vitro kinase assays. HMC cultured on COL I showed an increased mRNA expression of COL I and COL IV, fibronectin, and TGF-beta 1. Both tyrosine phosphorylation and integrin-linked kinase (ILK) activity increased when HMC were cultured on COL I, and blockade of these pathways inhibited the increased secretion of TGF-beta 1. In conclusion, the present results support a role for extracellular COL I in the regulation of TGF-beta 1 synthesis during progressive renal sclerosis and fibrosis and the subsequent increase in newly synthesized ECM proteins. In addition, ILK, along with the tyrosine kinases, participates in the genesis of this effect.

Arg-Gly-Asp-Ser (RGDS) peptide stimulates transforming growth factor beta1 transcription and secretion through integrin activation.

Extracellular matrix (ECM) components, through specific peptide motifs such as Arg-Gly-Asp (RGD), interact with integrins and can modify the behavior of cells. Transforming growth factor-beta1 (TGF-beta1) is the main cytokine involved in the synthesis of ECM proteins. We analyzed the effect of a RGD-containing peptide, as Arg-Gly-Asp-Ser (RGDS), on the regulation of TGF-beta1 secretion in cultured human mesangial cells. We found that RGDS increased mRNA expression and secretion of TGF-beta1 by stimulating the TGF-beta1 gene promoter. This effect was dependent on the interaction of RGDS with integrins. We evaluated the signaling pathways implicated in TGF-beta1 production by analyzing the effect of RGDS on kinase-related integrins. RGDS stimulated tyrosine phosphorylation as well as integrin-linked kinase (ILK) activity. However, tyrosine kinase inhibitors did not prevent the RGDS effect. In contrast, the inhibition of ILK by cell transfection with a kinase dead-ILK completely abolished the increased TGF-beta1 secretion and promoter activity in the presence of RGDS. Thus RGDS modulates the secretion of TGF-beta1, probably through increased synthesis by interacting with integrins and activating ILK. This supports a role for ECM components in the regulation of their own secretion.

Reactive oxygen species induce proliferation of bovine aortic endothelial cells.

The effects of reactive oxygen species (ROS) on different cellular types are variable. In some conditions they can be harmful metabolites, but they can also act as intracellular messengers that are able to activate different transcription factors. Based on previous reports in which ROS were shown to stimulate the proliferation of mesenchymal cells, this study was carried out to assess this effect on bovine aortic endothelial cells (BAECs). When cells were incubated with glucose oxidase (GO), an enzyme that generates H2O2 continuously, a significant increase in BAEC proliferation was detected. BAEC proliferation was measured by the incorporation of [3H]-thymidine in the DNA of BAECs, and also by an increase in the number of cells. The effect observed with GO was maximal at 8-24 h. Catalase abolishes proliferation. We also tested the ability of GO to phosphorylate tyrosine residues in endothelial cell proteins. A significant increase in tyrosine phosphorylation was found, which might constitute the molecular basis for proliferative effect of GO. In conclusion, these results demonstrate the ability of H2O2 to stimulate BAEC proliferation at least under certain experimental conditions. We suggest a general activation of the cascade of tyrosine phosphorylation as one of the possible cellular mechanisms responsible for GO-induced BAEC proliferation.

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.

Oxidative stress induces tyrosine phosphorylation of PDGF alpha-and beta-receptors and pp60c-src in mesangial cells.

Reactive oxygen species are autocrine and paracrine modulators of cell behavior. Hydrogen peroxide, a cellular oxidant, has been shown to stimulate mesangial cell proliferation. In the present study we analyzed the H2O2-induced early signaling events. Immunofluorescence analysis revealed a H2O2 induced dose-dependent increase in tyrosine phosphorylation. Short treatment (2 or 5 min) with 5 mM H2O2 induced a mitogenic response and a significant (P < 0.01) increase in the number of cells compared to non-treated controls. Proteins extracted from H2O2 (0.1 to 10 mM) treated cells were separated on SDS-PAGE and subjected to immunoblot analysis with anti-phosphotyrosine. A dose-dependent induction of tyrosine phosphorylation of 180 kDa, 120 kDa and 60 kDa proteins was observed within 1 to 10 minutes. By sequentially using immunoprecipitation and immunoblotting the 180 kDa tyrosine phosphorylated band was shown to represent both PDGF alpha- and beta-receptors. The tyrosine phosphorylated 60 kDa protein was identified as the cytoplasmic protein tyrosine kinase pp60c-src. The c-src phosphorylation was associated with an inhibition of c-src kinase activity, suggesting phosphorylation of tyrosine 527 in the c-src regulatory domain. Pretreatment with catalase completely abrogated the H2O2-induced PDGF receptor and c-src tyrosine phosphorylation. These data support the notion that the activation of a signaling pathway involving the PDGF receptors and c-src contributes to the mitogenic effects of reactive oxygen species.

Reactive oxygen species and platelet-activating factor synthesis in age-related glomerulosclerosis.

This work deals with the hypothesis that an increased synthesis of reactive oxygen species (ROS) or platelet-activating factor (PAF) (or both) may be implied in the genesis of age-related glomerulosclerosis. Plasma concentration and urinary excretion of both thiobarbituric acid-reactive substances (TBARS) and PAF were measured in young and old human beings and rats. Moreover, these same parameters as well as H2O2 synthesis and reduced glutathione (GSH) content were measured in isolated glomeruli of young (3 months) and old (18 months) Wistar rats. H2O2 synthesis and GSH content were also measured in cultured rat mesangial cells from young and old animals. Both human beings and rats showed a decreased glomerular filtration rate and an increased urinary protein excretion with respect to young individuals. Isolated glomeruli from old animals showed a higher protein content and a lower number of cell nuclei than those from young rats. No changes were detected in plasma concentration and urinary excretion of TBARS and PAF in either human beings or rats. Glomeruli from 18-month-old rats exhibited a higher content of TBARS and GSH and an increased synthesis of H2O2 and PAF than did those from 3-month-old rats. GSH content and H2O2 synthesis were higher in cultured cells from old rats than in those from young rats. These results point to the possibility that ROS or PAF could mediate some of the changes that characterize age-related glomerulosclerosis.

Changes in renal function in acute experimental pancreatitis in rats: a therapeutic role for somatostatin.

1. The present study was designed to determine the changes in renal function in two models of experimental pancreatitis in rats, in an attempt to assess the possible pathogenic role of reactive oxygen species and to elucidate a possible therapeutic role for somatostatin. 2. Mild pancreatitis was induced by low blockade of the biliary duct and severe pancreatitis was evoked by retrograde infusion of bile salts. Renal function was studied by clearance techniques in rats with pancreatitis, treated or not treated with somatostatin. Plasma and glomerular malonyldialdehyde levels were measured by the thiobarbituric acid method. 3. Renal function did not change in rats with low blockade of the biliary duct, but animals receiving a retrograde infusion of bile salts showed a significant decrease in glomerular filtration rate and renal plasma flow with respect to sham-operated animals. 4. Plasma malonyldialdehyde levels increased significantly in rats treated with bile salts with respect to control animals, whereas no changes were detected in glomerular malonyldialdehyde levels. Thus, the renal dysfunction does not seem to be related to an increased production of reactive oxygen metabolites at the glomerular level. 5. Somatostatin infusion significantly improved renal function in rats with severe pancreatitis (retrograde infusion of bile salts) by increasing glomerular filtration rate, renal plasma flow and filtration fraction. These results support a possible therapeutic role for somatostatin in the renal dysfunction associated with the severe forms of pancreatitis.

Calcium channel blockers inhibit hydrogen peroxide-induced proliferation of cultured rat mesangial cells.

The present experiments were designed to assess a possible role of H2O2 in the proliferation of cultured rat mesangial cells, as well as to evaluate the effect of different calcium channel blockers and a platelet-activating factor antagonist on this proliferation. Cultured rat mesangial cells were plated at two densities (10,000 and 25,000 cells/well) in 24-well dishes, and proliferation was measured by analyzing [3H]thymidine incorporation and by directly counting the cells. Hydrogen peroxide, 100 microM, increased [3H]thymidine incorporation at the two densities tested (85 and 59%, respectively), as well as the number of cells (53 and 23%, respectively). This effect was dose dependent and it was blocked completely by verapamil and diltiazem, 10 microM, but not by TMB-8 [3,4,5-trimethoxybenzoic acid 8-(diethylamino) octyl ester] at the same concentration. BN 52021, a competitive antagonist of platelet-activating factor, only slightly blocked the H2O2-dependent proliferation. The inhibitory action of the two calcium antagonists tested started at concentrations as low as 1 nM, and inhibited completely the H2O2 stimulated proliferation at concentrations between 0.1 and 1 microM. These results establish that H2O2 is able to induce proliferation of mesangial cells. Although the pathophysiological implications of this finding remain to be proven, these data suggest a potential therapeutic action of calcium antagonist in inflammatory conditions such as glomerulonephritis.

A dual effect of somatostatin on the proliferation of cultured rat mesangial cells.

The present experiments were designed to analyze the ability of somatostatin to modulate the proliferation of cultured rat mesangial cells. In the absence of fetal calf serum, somatostatin stimulated cell proliferation in a dose-dependent manner. In contrast, in proliferating cells, somatostatin inhibited cell proliferation, also in a dose-dependent fashion. Zaprinast, a rather specific cyclic GMP phosphodiesterase blocker, inhibited the somatostatin-dependent proliferation in the absence of growth factors. However, it potentiated the inhibitory effect on proliferating cells. These results support a dual role for somatostatin in the regulation of mesangial cell proliferation. The inhibitory effect of the peptide may be mediated by cyclic GMP.

Somatostatin antagonizes angiotensin II effects on mesangial cell contraction and glomerular filtration.

The effects of somatostatin (ST) on the regulation of the glomerular filtration rate have not been extensively studied. The present experiments were designed to analyze this possible relationship. ST alone did not modify the planar cell surface area (PCSA) of cultured rat mesangial cells (CRMC), but it prevented and reversed the reduction in PCSA induced by 10 nM angiotensin II (Ang II) in a dose- and time-dependent manner. ST (1 microM) completely prevented and reversed the increase in the myosin light chain phosphorylation induced by 10 nM Ang II. Incubation with pertussis toxin (PT, 0.5 micrograms/ml) inhibited the effect of ST on the Ang II-dependent changes in PCSA, but this effect was not inhibited by the blockade of the vasodilatory prostaglandins (indomethacin, 10 microM) or nitric oxide (L-N-methyl-arginine, 0.2 mM) synthesis. 2',5'-dideoxyadenosine (DDA, 0.1 mM), an adenylate cyclase blocker, and methylene blue (MB, 30 microM), a soluble guanylate cyclase blocker, did not interfere with the ST inhibitory effect on the Ang II-dependent reduction in PCSA of rat mesangial cells. ST also blocked the reduction in PCSA induced by phorbol myristate acetate (PMA, 300 nM). ST was also able to prevent and revert the Ang II dependent reduction in glomerular cross-sectional area of isolated rat glomeruli, also in a dose- and time-dependent fashion. Finally, intravenous administration of ST (200 ng/kg body wt as a bolus plus a continuous injection of 25 ng/min/kg body wt) partially blocked the reduction in GFR (measured as CIn) and RPF (measured as CPAH) and the increase in filtration fraction induced by the intravenous administration of Ang II (1.7 micrograms/min/kg body wt) in anesthetized rats. In summary, these results suggest that ST could antagonize the renal actions of Ang II, increasing the GFR and RPF decreased by Ang II, and this effect could be dependent, at least partially, on a direct relaxing effect of ST on mesangial cells.

[Hypersensitivity syndrome caused by allopurinol: report of 2 cases and review of the literature]. / Síndrome de hipersensibilidad por alopurinol: a propósito de dos casos y revisión de la literatura.

A severe adverse drug effect secondary to allopurinol ingestion has been described, characterized by fever, eosinophilia, cutaneous rash, hepatic lesion and renal failure, with a high mortality (21-26%) and unknown ethiopathogenicity. In many cases patients had a previous disorder on their renal function (53%) and more than half received allopurinol due to asymptomatic hyperuricemia. We present two new cases and review other 18 patients diagnosed in the last nine years, analyzing the ethiopathogenicity, epidemiological, clinical, therapeutical and preventive aspects.