Prevalence, risk factors and evolution of diabetes mellitus after treatment in primary aldosteronism. Results from the SPAIN-ALDO registry.

PURPOSE: To evaluate the prevalence, risk factors and evolution of diabetes mellitus (DM) after targeted treatment in patients with primary aldosteronism (PA). METHODS: A retrospective multicenter study of PA patients in follow-up at 27 Spanish tertiary hospitals (SPAIN-ALDO Register). RESULTS: Overall, 646 patients with PA were included. At diagnosis, 21.2% (n = 137) had DM and 67% of them had HbA1c levels < 7%. In multivariate analysis, family history of DM (OR 4.00 [1.68-9.53]), the coexistence of dyslipidemia (OR 3.57 [1.51-8.43]) and advanced age (OR 1.04 per year of increase [1.00-1.09]) were identified as independent predictive factors of DM. Diabetic patients were on beta blockers (46.7% (n = 64) vs. 27.5% (n = 140), P < 0.001) and diuretics (51.1% (n = 70) vs. 33.2% (n = 169), p < 0.001) more frequently than non-diabetics. After a median follow-up of 22 months [IQR 7.5-63.0], 6.9% of patients developed DM, with no difference between those undergoing adrenalectomy and those treated medically (HR 1.07 [0.49-2.36], p = 0.866). There was also no significant difference in the evolution of glycemic control between DM patients who underwent surgery and those medically treated (p > 0.05). CONCLUSION: DM affects about one quarter of patients with PA and the risk factors for its development are common to those of the general population. Medical and surgical treatment provides similar benefit in glycemic control in patients with PA and DM.

The second Mexican consensus on hepatocellular carcinoma. Part II: Treatment.

Hepatocellular carcinoma (HCC) is more frequently manifesting as one of the main complications of cirrhosis of the liver, its principal risk factor. There have been modifications in its incidence over the past decade, related to an epidemiologic transition in the etiology of cirrhosis, with a decrease in the prevalence of hepatitis C and an increase in nonalcoholic fatty liver disease (NAFLD) as a cause, as well as the development of HCC in the non-cirrhotic liver due to NAFLD. Genetic markers associated with the disease have been identified, and surveillance and diagnosis have improved. Regarding treatment, surgical techniques, in both resection and transplantation, have advanced and radiologic techniques, at the curative stage of the disease, have enhanced survival in those patients. And finally, there have been radical changes in the systemic approach, with much more optimistic expectations, when compared with the options available a decade ago. Therefore, the Asociación Mexicana de Hepatología decided to carry out the Second Mexican Consensus on Hepatocellular Carcinoma, which is an updated review of the available national and international evidence on the epidemiology, risk factors, surveillance, diagnosis, and treatment of the disease, to offer the Mexican physician current information on the different topics regarding hepatocellular carcinoma. In this second part of the document, the topics related to the treatment of HCC are presented.

The second Mexican consensus on hepatocellular carcinoma. Part I: Epidemiology and diagnosis.

Hepatocellular carcinoma (HCC) is more frequently manifesting as one of the main complications of cirrhosis of the liver, its principal risk factor. There have been modifications in its incidence over the past decade, related to an epidemiologic transition in the etiology of cirrhosis, with a decrease in the prevalence of hepatitis C and an increase in nonalcoholic fatty liver disease (NAFLD) as a cause, as well as the development of HCC in the non-cirrhotic liver due to NAFLD. Genetic markers associated with the disease have been identified, and surveillance and diagnosis have improved. Regarding treatment, surgical techniques, in both resection and transplantation, have advanced and radiologic techniques, at the curative stage of the disease, have enhanced survival in those patients. And finally, there have been radical changes in the systemic approach, with much more optimistic expectations, when compared with the options available a decade ago. Therefore, the Asociación Mexicana de Hepatología decided to carry out the Second Mexican Consensus on Hepatocellular Carcinoma, which is an updated review of the available national and international evidence on the epidemiology, risk factors, surveillance, diagnosis, and treatment of the disease, to offer the Mexican physician current information on the different topics regarding hepatocellular carcinoma. In this first part of the document, the topics related to epidemiology and diagnosis are presented.

Neutrophil gelatinase-associated lipocalin is a biomarker of acute-on-chronic liver failure and prognosis in cirrhosis.

BACKGROUND & AIMS: Acute-on-chronic liver failure (ACLF) is a syndrome that occurs in cirrhosis characterized by organ failure(s) and high mortality rate. There are no biomarkers of ACLF. The LCN2 gene and its product, neutrophil gelatinase-associated lipocalin (NGAL), are upregulated in experimental models of liver injury and cultured hepatocytes as a result of injury by toxins or proinflammatory cytokines, particularly Interleukin-6. The aim of this study was to investigate whether NGAL could be a biomarker of ACLF and whether LCN2 gene may be upregulated in the liver in ACLF. METHODS: We analyzed urine and plasma NGAL levels in 716 patients hospitalized for complications of cirrhosis, 148 with ACLF. LCN2 expression was assessed in liver biopsies from 29 additional patients with decompensated cirrhosis with and without ACLF. RESULTS: Urine NGAL was markedly increased in ACLF vs. no ACLF patients (108(35-400) vs. 29(12-73)µg/g creatinine; p<0.001) and was an independent predictive factor of ACLF; the independent association persisted after adjustment for kidney function or exclusion of variables present in ACLF definition. Urine NGAL was also an independent predictive factor of 28day transplant-free mortality together with MELD score and leukocyte count (AUROC 0.88(0.83-0.92)). Urine NGAL improved significantly the accuracy of MELD in predicting prognosis. The LCN2 gene was markedly upregulated in the liver of patients with ACLF. Gene expression correlated directly with serum bilirubin and INR (r=0.79; p<0.001 and r=0.67; p<0.001), MELD (r=0.68; p<0.001) and Interleukin-6 (r=0.65; p<0.001). CONCLUSIONS: NGAL is a biomarker of ACLF and prognosis and correlates with liver failure and systemic inflammation. There is remarkable overexpression of LCN2 gene in the liver in ACLF syndrome. LAY SUMMARY: Urine NGAL is a biomarker of acute-on-chronic liver failure (ACLF). NGAL is a protein that may be expressed in several tissues in response to injury. The protein is filtered by the kidneys due to its small size and can be measured in the urine. Ariza, Graupera and colleagues found in a series of 716 patients with cirrhosis that urine NGAL was markedly increased in patients with ACLF and correlated with prognosis. Moreover, gene coding NGAL was markedly overexpressed in the liver tissue in ACLF.

Adenoviral dominant-negative soluble PDGFRß improves hepatic collagen, systemic hemodynamics, and portal pressure in fibrotic rats.

BACKGROUND & AIMS: Platelet-derived growth factor (PDGF) is the most potent stimulus for proliferation and migration of stellate cells. PDGF receptor ß (PDGFRß) expression is an important phenotypic change in myofibroblastic cells that mediates proliferation and chemotaxis. Here we analyzed the relationship between PDGFRß expression, hemodynamic deterioration, and fibrosis in CCl(4)-treated rats. Thereafter, we investigated the effects produced by an adenovirus encoding a dominant-negative soluble PDGFRß (sPDGFRß) on hemodynamic parameters, PDGFRß signaling pathway, and fibrosis. METHODS: Mean arterial pressure, portal pressure, PDGFRß mRNA expression, and hepatic collagen were assessed in 6 controls and 21 rats induced to hepatic fibrosis/cirrhosis. Next, 30 fibrotic rats were randomized into three groups receiving iv saline and an adenovirus encoding for sPDGFRß or ß-galactosidase. After 7days, mean arterial pressure, portal pressure, serum sPDGFRß, and hepatic collagen were measured. RESULTS: CCl(4)-treated animals for 18weeks showed a significantly higher increase in PDGFRß mRNA compared to those treated for 13weeks and control rats. In CCl(4)-treated rats, the fibrous tissue area ranged from moderate to severe fibrosis. A direct relationship between the degree of fibrosis, hemodynamic changes, and PDGFRß expression was observed. Fibrotic rats transduced with the adenovirus encoding sPDGFRß showed increased mean arterial pressure, decreased portal pressure, lower activation of the PDGFRß signaling pathway, and reduced hepatic collagen than fibrotic rats receiving ß-galactosidase or saline. CONCLUSIONS: PDGFRß activation closely correlates with hemodynamic disorders and increased fibrosis in CCl(4)-treated rats. Adenoviral dominant negative soluble PDGFRß improved fibrosis. As a result, the hemodynamic abnormalities were ameliorated.

Inactivation of extrahepatic vascular Akt improves systemic hemodynamics and sodium excretion in cirrhotic rats.

BACKGROUND & AIMS: Increased activity of the vascular Akt/eNOS signaling pathway is involved in the hemodynamic and renal complications developed by patients and rats with cirrhosis and ascites. This occurs in the setting of impaired Akt/eNOS activity within the cirrhotic liver. Here we assessed the feasibility of selectively inhibiting vascular eNOS without further impairing the intrahepatic activity of this enzyme. Ultimately, we sought to determine whether endothelial transduction of a constitutively inactive mutant of Akt (AA-Akt) improves circulatory function and sodium excretion in cirrhotic rats with ascites. METHODS: First, we administered recombinant adenoviruses that encode the ß-galactosidase gene (ß-gal) to 5 control rats and 5 cirrhotic rats with ascites and analyzed their tissue distribution by chemiluminescence. Next, urine samples were obtained from 18 cirrhotic rats with ascites and then the animal randomly received saline or adenoviruses containing the ß-gal or the AA-Akt genes. Following a 24-h urine collection period, hemodynamic studies were performed and tissue samples were obtained to analyze Akt and eNOS expressions. RESULTS: No ß-gal activity was detected in the liver of cirrhotic rats compared to that of controls. This was paralleled by increased ß-gal activity in other territories such as the thoracic aorta. AA-Akt transduction improved systemic hemodynamics, splanchnic perfusion pressure and renal excretory function in comparison with cirrhotic rats transduced with ß-gal adenoviruses or receiving saline. Moreover, the AA-Akt transgene did not modify portal pressure. CONCLUSIONS: Inactivation of extrahepatic vascular Akt and the concomitant decrease in nitric oxide expression ameliorate systemic hemodynamics and renal excretory function in experimental cirrhosis.

Prostaglandin E2 and bone turnover markers in the evaluation of primary hypertrophic osteoarthropathy (pachydermoperiostosis): a case report.

Primary hypertrophic osteoarthropathy, or pachydermoperiostosis (PDP), is an infrequent genetic condition characterized by digital clubbing, periostosis, and pachydermia and is distinct from a more common form, secondary hypertrophic osteoarthropathy, which always associates with an underlying cause (frequently pulmonary or cardiac disease). The diagnosis of this disorder as well as its clinical evaluation can be difficult. We report a 15-year-old boy presenting with intermittent arthralgias and clubbing of fingers and toes for the previous 2 years. The ankles and knees were enlarged, and X-rays showed periosteal apposition. The search for a secondary cause was negative. The skin appearance was normal, but a skin biopsy was indicative of pachydermia, further confirming the diagnosis of PDP. Bone turnover markers were increased at diagnosis and progressively decreased during follow-up; prostaglandin E(2), a recently implicated mediator of this disorder, was markedly elevated. In the present case, carrying out a skin biopsy helped us to diagnose this condition. In addition, bone turnover markers were useful for monitoring the disease activity; whereas, increased prostaglandin E(2) levels seems to confirm the role of this mediator in the etiopathogenesis of this disorder.

Vascular endothelial growth factor and angiopoietin-2 play a major role in the pathogenesis of vascular leakage in cirrhotic rats.

BACKGROUND AND AIMS: The extent and molecular mechanisms governing plasma extravasation and formation of ascites in cirrhosis are unknown. Vascular endothelial growth factor-A (VEGF-A) and angiopoietin-2 (Ang-2) are endogenous substances with powerful vascular permeability effects. We assessed regional blood flow, vascular leakage, mRNA and tissular expression of VEGF-A and Ang-2 and vascular permeability following VEGF receptor 2 blockade in control and cirrhotic rats to define the vascular territories showing altered vascular permeability in cirrhosis and to determine whether VEGF-A and Ang-2 are involved in this phenomenon. METHODS: Arterial blood flow was analysed with the coloured microsphere method. Vascular leakage was measured and visualised with the dye Evan's Blue and colloidal carbon techniques, respectively. VEGF-A and Ang-2 expression were determined by real-time polymerase chain reaction (RT-PCR), immunohistochemistry and western blot. The effect on vascular permeability induced by VEGFR(2) blockade was assessed by administration of the receptor inhibitor SU11248. RESULTS: Arterial blood flow was increased in the mesentery, pancreas and small intestine but not in the kidney and spleen of cirrhotic rats as compared to controls. Increased vascular leakage was observed in the mesentery and liver, where colloidal carbon spread from microvessels to the adjacent fibrotic tracts. Increased hepatic and mesenteric expression of VEGF-A and Ang-2 was found in cirrhotic rats as compared to controls. Blockade of VEGFR(2) markedly reduced hepatic and mesenteric vascular leakage in cirrhotic rats. CONCLUSIONS: Enhanced endothelial permeability is restricted to the hepatic and mesenteric vascular beds in cirrhotic rats with ascites and VEGF-A and Ang-2 are key factors in the signalling pathways regulating this dysfunction.

Gene transduction of an active mutant of akt exerts cytoprotection and reduces graft injury after liver transplantation.

Akt is expected to be an effective target for the treatment of ischemia-reperfusion injury (I/R) due to its anti-apoptotic properties and its ability to activate the endothelial nitric oxide synthase (eNOS) enzyme. Therefore, this study was aimed to determine the efficacy of an active mutant of Akt (myr-Akt) to decrease I/R injury in a model of orthotopic liver transplantation in pigs. In addition, we analyzed the contribution of nitric oxide in the Akt-mediated effects by using an eNOS mutant (S1179DeNOS) that mimics the phosphorylation promoted by Akt in the eNOS sequence. Donors were treated with adenoviruses codifying for myr-Akt, S1179DeNOS or beta-galactosidase 24 h before liver harvesting. Then, liver grafts were orthotopically transplanted into their corresponding recipients. Levels of transaminases and lactate dehydrogenase (LDH) increased in all recipients after 24 h of transplant. However, transaminases and LDH levels were significantly lower in the myr-Akt group compared with vehicle. The percentage of apoptotic cells and the amount of activated-caspase 3 protein were also markedly reduced in myr-Akt-treated grafts after 4 days of liver transplant compared with vehicle and S1179DeNOS groups. In conclusion, myr-Akt gene therapy effectively exerts cytoprotection against hepatic I/R injury regardless of the Akt-dependent eNOS activation.

Influence of caveolin on constitutively activated recombinant eNOS: insights into eNOS dysfunction in BDL rat liver.

Diminished endothelial nitric oxide (NO) synthase (eNOS)-derived NO production from the hepatic vascular endothelium contributes to hepatic vasoconstriction in portal hypertension. The aim of this study was to examine the mechanism of this process by testing the influence of a constitutively active form of eNOS (S1179DeNOS) in both primary and propagated liver cells in vitro and in the sham and bile duct ligated (BDL) rat liver in vivo, using an adenoviral vector encoding green fluorescent protein (AdGFP) and S1179DeNOS (AdS1179DeNOS). AdS1179DeNOS transduction augmented basal and agonist-stimulated NO generation in nonparenchymal liver cells. Sham rats transduced in vivo with AdS1179DeNOS evidenced a decreased pressor response to incremental doses of the vasoconstrictor methoxamine compared with sham rats transduced with AdGFP. However, BDL rats transduced with AdS1179DeNOS did not display improved vasodilatory responses as evidenced by similar flow-dependent pressure increases to that observed in BDL rats transduced with AdGFP, despite similar levels of viral transgene expression. We next examined the influence of the eNOS inhibitory protein caveolin on S1179DeNOS dysfunction in cirrhotic liver. Immunogold electron microscopic analysis of caveolin in BDL liver demonstrated prominent expression not only in liver endothelial cells, but also in hepatic stellate cells. In vitro studies in the LX2 hepatic stellate cell line demonstrate that caveolin precipitates recombinant S1179DeNOS in LX2 cells, that recombinant S1179DeNOS coprecipitates caveolin, and that binding is enhanced in the presence of overexpression of caveolin. Furthermore, caveolin overexpression inhibits recombinant S1179DeNOS activity. These studies indicate that recombinant S1179DeNOS protein functions appropriately in normal liver cells and tissue but evidences dysfunction in the cirrhotic rat liver and that caveolin expression and inhibition in BDL nonparenchymal cells, including hepatic stellate cells, may account for this dysfunction.

Akt-mediated phosphorylation of the G protein-coupled receptor EDG-1 is required for endothelial cell chemotaxis.

The role of the protein kinase Akt in cell migration is incompletely understood. Here we show that sphingosine-1-phosphate (S1P)-induced endothelial cell migration requires the Akt-mediated phosphorylation of the G protein-coupled receptor (GPCR) EDG-1. Activated Akt binds to EDG-1 and phosphorylates the third intracellular loop at the T(236) residue. Transactivation of EDG-1 by Akt is not required for G(i)-dependent signaling but is indispensable for Rac activation, cortical actin assembly, and chemotaxis. Indeed, T236AEDG-1 mutant sequestered Akt and acted as a dominant-negative GPCR to inhibit S1P-induced Rac activation, chemotaxis, and angiogenesis. Transactivation of GPCRs by Akt may constitute a specificity switch to integrate rapid G protein-dependent signals into long-term cellular phenomena such as cell migration.

Akt down-regulation of p38 signaling provides a novel mechanism of vascular endothelial growth factor-mediated cytoprotection in endothelial cells.

Vascular endothelial growth factor (VEGF) utilizes a phosphoinositide 3-kinase (PI 3-kinase)/Akt signaling pathway to protect endothelial cells from apoptotic death. Here we show that PI 3-kinase/Akt signaling promotes endothelial cell survival by inhibiting p38 mitogen-activated protein kinase (MAPK)-dependent apoptosis. Blockade of the PI 3-kinase or Akt pathways in conjunction with serum withdrawal stimulates p38-dependent apoptosis. Blockade of PI 3-kinase/Akt also led to enhanced VEGF activation of p38 and apoptosis. In this context, the pro-apoptotic effect of VEGF is attenuated by the p38 MAPK inhibitor SB203580. VEGF stimulation of endothelial cells or infection with an adenovirus expressing constitutively active Akt causes MEKK3 phosphorylation, which is associated with decreased MEKK3 kinase activity and down-regulation of MKK3/6 and p38 MAPK activation. Conversely, activation-deficient Akt decreases VEGF-stimulated MEKK3 phosphorylation and increases MKK/p38 activation. Activation of MKK3/6 is not dependent on Rac activation since dominant negative Rac does not decrease p38 activation triggered by inhibition of PI 3-kinase. Thus, cross-talk between the Akt and p38 MAPK pathways may regulate the level of cytoprotection versus apoptosis and is a new mechanism to explain the cytoprotective actions of Akt.

Suppression of vascular endothelial growth factor-mediated endothelial cell protection by survivin targeting.

The protective genes that mediate endothelial cell (EC) survival during angiogenesis have not been completely characterized. Here, we show that an antisense oligonucleotide to the apoptosis inhibitor survivin suppressed de novo expression of survivin in ECs by vascular endothelial cell growth factor (VEGF). In contrast, the survivin antisense oligonucleotide did not affect anti-apoptotic bcl-2 levels in endothelium. When assessed in cell death assays, antisense targeting of survivin abolished the anti-apoptotic function of VEGF against tumor necrosis factor-alpha- or ceramide-induced cell death, enhanced caspase-3 activity, promoted the generation of a approximately 17-kd active caspase-3 subunit, and increased cleavage of the caspase substrate, polyADP ribose polymerase. In contrast, the survivin antisense oligonucleotide had no effect on EC viability in the absence of VEGF. Antisense oligonucleotides to platelet-endothelial cell adhesion molecule-1 (PECAM-1, CD31), lymphocyte function-associated molecule-3 (LFA-3, CD58), or intercellular adhesion molecule-1 (ICAM-1, CD54) did not reduce the anti-apoptotic function of VEGF in endothelium. When tested on other angiogenic activities mediated by VEGF, survivin antisense treatment induced rapid regression of three-dimensional vascular capillary networks, but did not affect EC migration/chemotaxis. These data suggest that the anti-apoptotic properties of VEGF during angiogenesis are primarily mediated by the induced expression of survivin in ECS: Manipulation of this pathway may increase EC viability in compensatory angiogenesis or facilitate EC apoptosis and promote vascular regression during tumor angiogenesis.

Sphingosine 1-phosphate activates Akt, nitric oxide production, and chemotaxis through a Gi protein/phosphoinositide 3-kinase pathway in endothelial cells.

Sphingosine 1-phosphate (SPP) binds to members of the endothelial differentiation gene family (EDG) of receptors and leads to diverse signaling events including cell survival, growth, migration and differentiation. However, the mechanisms of how SPP activates these proangiogenic pathways are poorly understood. Here we show that SPP signals through the EDG-1 receptor to the heterotrimeric G protein G(i), leading to activation of the serine/threonine kinase Akt and phosphorylation of the Akt substrate, endothelial nitric-oxide synthase (eNOS). Inhibition of G(i) signaling, and phosphoinositide 3-kinase (PI 3-kinase) activity resulted in a decrease in SPP-induced endothelial cell chemotaxis. SPP also stimulates eNOS phosphorylation and NO release and these effects are also attenuated by inhibition of G(i) signaling, PI 3-kinase, and Akt. However, inhibition of NO production did not influence SPP-induced chemotaxis but effectively blocked the chemotactic actions of vascular endothelial growth factor. Thus, SPP signals through G(i) and PI 3-kinase leading to Akt activation and eNOS phosphorylation.

Membrane estrogen receptor engagement activates endothelial nitric oxide synthase via the PI3-kinase-Akt pathway in human endothelial cells.

17beta-Estradiol (E(2)) is a rapid activator of endothelial nitric oxide synthase (eNOS). The product of this activation event, NO, is a fundamental determinant of cardiovascular homeostasis. We previously demonstrated that E(2)-stimulated endothelial NO release can occur without an increase in cytosolic Ca(2+). Here we demonstrate for the first time, to our knowledge, that E(2) rapidly induces phosphorylation and activation of eNOS through the phosphatidylinositol 3 (PI3)-kinase-Akt pathway. E(2) treatment (10 ng/mL) of the human endothelial cell line, EA.hy926, resulted in increased NO production, which was abrogated by the PI3-kinase inhibitor, LY294002, and the estrogen receptor antagonist ICI 182, 780. E(2) stimulated rapid Akt phosphorylation on serine 473. As has been shown for vascular endothelial growth factor, eNOS is an E(2)-activated Akt substrate, demonstrated by rapid eNOS phosphorylation on serine 1177, a critical residue for eNOS activation and enhanced sensitivity to resting cellular Ca(2+) levels. Adenoviral-mediated EA.hy926 transduction confirmed functional involvement of Akt, because a kinase-deficient, dominant-negative Akt abolished E(2)-stimulated NO release. The membrane-impermeant E(2)BSA conjugate, shown to bind endothelial cell membrane sites, also induced rapid Akt and consequent eNOS phosphorylation. Thus, engagement of membrane estrogen receptors results in rapid endothelial NO release through a PI3-kinase-Akt-dependent pathway. This explains, in part, the reduced requirement for cytosolic Ca(2+) fluxes and describes an important pathway relevant to cardiovascular pathophysiology.

Vascular endothelial growth factor-stimulated actin reorganization and migration of endothelial cells is regulated via the serine/threonine kinase Akt.

Vascular endothelial growth factor (VEGF) induces endothelial cell proliferation, migration, and actin reorganization, all necessary components of an angiogenic response. However, the distinct signal transduction mechanisms leading to each angiogenic phenotype are not known. In this study, we examined the ability of VEGF to stimulate cell migration and actin rearrangement in microvascular endothelial cells infected with adenoviruses encoding beta-galactosidase (beta-gal), activation-deficient Akt (AA-Akt), or constitutively active Akt (myr-Akt). VEGF increased cell migration in cells transduced with beta-gal, whereas AA-Akt blocked VEGF-induced cell locomotion. Interestingly, myr-Akt transduction of bovine lung microvascular endothelial cells stimulated cytokinesis in the absence of VEGF, suggesting that constitutively active Akt, per se, can initiate the process of cell migration. Treatment of beta-gal-infected endothelial cells with an inhibitor of NO synthesis blocked VEGF-induced migration but did not influence migration initiated by myr-Akt. In addition, VEGF stimulated remodeling of the actin cytoskeleton into stress fibers, a response abrogated by infection with dominant-negative Akt, whereas transduction with myr-Akt alone caused profound reorganization of F-actin. Collectively, these data demonstrate that Akt is critically involved in endothelial cell signal transduction mechanisms leading to migration and that the Akt/endothelial NO synthase pathway is necessary for VEGF-stimulated cell migration.

Vascular endothelial growth factor production in peritoneal macrophages of cirrhotic patients: regulation by cytokines and bacterial lipopolysaccharide.

Vascular endothelial growth factor (VEGF) is an angiogenic peptide with vascular permeability and relaxing properties. This study assessed whether peritoneal macrophages of cirrhotic patients can be up-regulated to produce VEGF under proper stimulatory conditions. Macrophages were isolated from ascites. VEGF protein secretion and mRNA expression were measured in basal conditions and after stimulation with lipopolysaccharide (LPS), tumor necrosis factor alpha (TNF-alpha), and interleukin-1 (IL-1). These substances induced a time- and dose-dependent increase in both VEGF production and transcript expression. Assays with actinomycin D showed that VEGF mRNA induction is secondary to both higher VEGF gene transcription and mRNA stability. Ascites and plasma concentration of VEGF was also measured in cirrhotic patients with (n = 15) and without (n = 10) spontaneous bacterial peritonitis (SBP). Plasma values did not differ between both groups of patients. However, ascites VEGF levels were higher in SBP patients than in noninfected cirrhotic patients (710 +/- 183 vs. 94 +/- 15 pg/mL; P <.025). These results indicate that cytokines and LPS markedly increase VEGF protein secretion and mRNA expression in macrophages of cirrhotic patients, and suggest that this substance could be an important mediator of the pronounced arterial vasodilation frequently occurring in SBP patients.

Nitric oxide production by peritoneal macrophages of cirrhotic rats: a host response against bacterial peritonitis.

BACKGROUND & AIMS: Patients and rats with cirrhosis and ascites are prone to develop peritonitis. The aim of this study was to assess whether peritoneal macrophages of cirrhotic rats without peritoneal infection produce nitric oxide and express inducible NO synthase (iNOS). METHODS: NO2- accumulation produced by macrophages from control rats and cirrhotic rats with ascites was determined. iNOS messenger RNA and protein expression were analyzed by Northern and Western blot and immunocytochemical analysis. The in vivo effects of inhibiting iNOS were investigated by giving the specific iNOS inhibitor L-N-(1-iminoethyl)-lysine (L-NIL) or sterile saline to 9 and 7 cirrhotic rats with ascites, respectively. RESULTS: Cirrhotic macrophages produced NO2- that was around fourfold greater than that of control macrophages after 30 hours in culture. Northern and Western blot and immunocytochemical analysis showed the presence of iNOS messenger RNA and protein in macrophages of cirrhotic rats. Ascites cultures were positive in all rats administered L-NIL and negative in those administered saline. CONCLUSIONS: Macrophages of cirrhotic rats produce NO and express iNOS messenger RNA and protein, and these changes are not a consequence of overt bacterial infection. Because iNOS inhibition results in peritoneal infection, these results suggest that iNOS induction in macrophages of cirrhotic rats is a host defense response to prevent bacterial peritonitis.