Arterial and venous endothelia display differential functional fractalkine (CX3CL1) expression by angiotensin-II.

OBJECTIVE: Angiotensin-II (Ang-II) promotes the interaction of mononuclear cells with arterioles and neutrophils with postcapillary venules. To investigate the mechanisms underlying this dissimilar response, the involvement of fractalkine (CX(3)CL1) was explored. METHODS AND RESULTS: Enhanced CX(3)CL1 expression was detected in both cremasteric arterioles and postcapillary venules 24 hours after Ang-II intrascrotal injection. Arteriolar leukocyte adhesion was the unique parameter significantly reduced (83%) in animals lacking CX(3)CL1 receptor (CX(3)CR1). Human umbilical arterial and venous endothelial cell stimulation with 1 µmol/L Ang-II increased CX(3)CL1 expression, yet neutralization of CX(3)CL1 activity only significantly inhibited Ang-II-induced mononuclear cell-human umbilical arterial endothelial cell interactions (73%) but not with human umbilical venous endothelial cells. The use of small interfering RNA revealed the involvement of tumor necrosis factor-α in Ang-II-induced CX(3)CL1 upregulation and mononuclear cell arrest. Nox5 knockdown with small interfering RNA or pharmacological inhibition of extracellular signal-regulated kinases1/2, p38 mitogen-activated protein kinase, and nuclear factor-κB also abolished these responses. Finally, when human umbilical arterial endothelial cells were costimulated with Ang-II, tumor necrosis factor-α, and interferon-γ, CX(3)CL1 expression and mononuclear cell adhesiveness were more pronounced than when each stimulus was provided alone. CONCLUSIONS: These results suggest that Ang-II induces functional CX(3)CL1 expression in arterial but not in venous endothelia. Thus, targeting endothelial CX(3)CL1-mononuclear leukocyte CX(3)CR1 interactions may constitute a new therapeutic strategy in the treatment of Ang-II-associated cardiovascular diseases.

Retinoid X receptor agonists impair arterial mononuclear cell recruitment through peroxisome proliferator-activated receptor-γ activation.

Mononuclear cell migration into the vascular subendothelium constitutes an early event of the atherogenic process. Because the effect of retinoid X receptor (RXR)α on arterial mononuclear leukocyte recruitment is poorly understood, this study investigated whether RXR agonists can affect this response and the underlying mechanisms involved. Decreased RXRα expression was detected after 4 h stimulation of human umbilical arterial endothelial cells with TNF-α. Interestingly, under physiological flow conditions, TNF-α-induced endothelial adhesion of human mononuclear cells was concentration-dependently inhibited by preincubation of the human umbilical arterial endothelial cells with RXR agonists such as bexarotene or 9-cis-retinoid acid. RXR agonists also prevented TNF-α-induced VCAM-1 and ICAM-1 expression, as well as endothelial growth-related oncogene-α and MCP-1 release. Suppression of RXRα expression with a small interfering RNA abrogated these responses. Furthermore, inhibition of MAPKs and NF-κB pathways were involved in these events. RXR agonist-induced antileukocyte adhesive effects seemed to be mediated via RXRα/peroxisome proliferator-activated receptor (PPAR)γ interaction, since endothelial PPARγ silencing abolished their inhibitory responses. Furthermore, RXR agonists increased RXR/PPARγ interaction, and combinations of suboptimal concentrations of both nuclear receptor ligands inhibited TNF-α-induced mononuclear leukocyte arrest by 60-65%. In vivo, bexarotene dose-dependently inhibited TNF-α-induced leukocyte adhesion to the murine cremasteric arterioles and decreased VCAM-1 and ICAM-1 expression. Therefore, these results reveal that RXR agonists can inhibit the initial inflammatory response that precedes the atherogenic process by targeting different steps of the mononuclear recruitment cascade. Thus, RXR agonists may constitute a new therapeutic tool in the control of the inflammatory process associated with cardiovascular disease.

Critical role of fractalkine (CX3CL1) in cigarette smoke-induced mononuclear cell adhesion to the arterial endothelium.

BACKGROUND: Cigarette smoking is an important risk factor for the development of cardiovascular disease, yet the pathways through which this may operate are poorly understood. Therefore, the mechanism underlying cigarette smoke (CS)-induced arterial endothelial dysfunction and the potential link with fractalkine/CX(3)CL1 upregulation were investigated. METHODS AND RESULTS: Stimulation of human arterial umbilical endothelial cells (HUAECs) with pathophysiological concentrations of CS extract (1% CSE) increased CX(3)CL1 expression. Neutralisation of CX(3)CL1 activity under dynamic flow conditions significantly inhibited CSE-induced mononuclear cell adhesion to HUAECs (67%). The use of small interfering RNA (siRNA) revealed that nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 5 (Nox5) but not Nox2 or Nox4 is the main NADPH isoform involved in CSE-induced CX(3)CL1 upregulation and mononuclear cell arrest. Knock down of HUAEC tumour necrosis factor α expression with siRNA or pharmacological inhibition of p38 mitogen-activated protein kinase and nuclear factor κB also abolished these responses. Interestingly, circulating monocytes and lymphocytes from patients with chronic obstructive pulmonary disease (COPD) (n=29) versus age-matched controls (n=23) showed CX(3)CR1overexpression. Furthermore, CX(3)CL1 neutralisation dramatically diminished their enhanced adhesiveness to CSE-stimulated HUAECs. Finally, when animals were exposed for 3 days to CS, a mild inflammatory response in the lung was observed which was accompanied by enhanced CX(3)CL1 expression in the cremasteric arterioles, an organ distant from the lung. CS exposure resulted in increased leukocyte-arteriolar endothelial cell adhesion which was significantly reduced (51%) in animals lacking CX(3)CL1 receptor (CX(3)CR1). CONCLUSIONS: These results suggest that CS induces functional CX(3)CL1 expression in arterial endothelium and leukocytes from patients with COPD show increased CX(3)CL1-dependent adhesiveness. Therefore, targeting the CX(3)CL1/CX(3)CR1 axis might prevent COPD-associated cardiovascular disorders.

Aclidinium inhibits cigarette smoke-induced lung fibroblast-to-myofibroblast transition.

Cigarette smoking contributes to lung remodelling in chronic obstructive pulmonary disease (COPD). As part of this remodelling, peribronchiolar fibrosis is observed in the small airways of COPD patients and contributes to airway obstruction. Fibroblast-to-myofibroblast transition is a key step in peribronchiolar fibrosis formation. This in vitro study examined the effect of cigarette smoke on bronchial fibroblast-to-myofibroblast transition, and whether aclidinium bromide inhibits this process. Human bronchial fibroblasts were incubated with aclidinium bromide (10(-9)-10(-7) M) and exposed to cigarette smoke extract. Collagen type I and α-smooth muscle actin (α-SMA) expression were measured by real-time PCR and Western blotting, as myofibroblast markers. Intracellular reactive oxygen species, cyclic AMP (cAMP), extracellular signal-regulated kinase (ERK)1/2 and choline acetyltransferase were measured as intracellular signalling mediators. Cigarette smoke-induced collagen type I and α-SMA was mediated by the production of reactive oxygen species, the depletion of intracellular cAMP and the increase of ERK1/2 phosphorylation and choline acetyltransferase. These effects could be reversed by treatment with the anticholinergic aclidinium bromide, by silencing the mRNA of muscarinic receptors M1, M2 or M3, or by the depletion of extracellular acetylcholine by treatment with acetylcholinesterase. A non-neuronal cholinergic system is implicated in cigarette smoke-induced bronchial fibroblast-to-myofibroblast transition, which is inhibited by aclidinium bromide.

Trans- but not cis-resveratrol impairs angiotensin-II-mediated vascular inflammation through inhibition of NF-κB activation and peroxisome proliferator-activated receptor-gamma upregulation.

Angiotensin II (Ang-II) displays inflammatory activity and is implicated in several cardiovascular disorders. This study evaluates the effect of cis- and trans (t)-resveratrol (RESV) in two in vivo models of vascular inflammation and identifies the cardioprotective mechanisms that underlie them. In vivo, Ang-II-induced arteriolar leukocyte adhesion was inhibited by 71% by t-RESV (2.1 mg/kg, i.v.), but was not affected by cis-RESV. Because estrogens influence the rennin-angiotensin system, chronic treatment with t-RESV (15 mg/kg/day, orally) inhibited ovariectomy-induced arteriolar leukocyte adhesion by 81%, partly through a reduction of cell adhesion molecule (CAM) expression and circulating levels of cytokine-induced neutrophil chemoattractant, MCP-1, and MIP-1alpha. In an in vitro flow chamber system, t-RESV (1-10 microM) undermined the adhesion of human leukocytes under physiological flow to Ang-II-activated human endothelial cells. These effects were accompanied by reductions in monocyte and endothelial CAM expression, chemokine release, phosphorylation of p38 MAPK, and phosphorylation of the p65 subunit of NF-kappaB. Interestingly, t-RESV increased the expression of peroxisome proliferator-activated receptor-gamma in human endothelial and mononuclear cells. These results demonstrate for the first time that the in vivo anti-inflammatory activity of RESV is produced by its t-RESV, which possibly interferes with signaling pathways that cause the upregulation of CAMs and chemokine release. Upregulation of proliferator-activated receptor-gamma also appears to be involved in the cardioprotective effects of t-RESV. In this way, chronic administration of t-RESV may reduce the systemic inflammatory response associated with the activation of the rennin-angiotensin system, thereby decreasing the risk of further cardiovascular disease.

Menopause and ovariectomy cause a low grade of systemic inflammation that may be prevented by chronic treatment with low doses of estrogen or losartan.

The incidence of cardiovascular diseases in premenopausal women is lower than in men or postmenopausal women. This study reports the discovery of a low grade of systemic inflammation, including monocyte adhesion to arterial endothelium, elicited by menopause or estrogen depletion. Chronic treatment with low dose of 17-beta-estradiol or inhibition of the renin-angiotensin system reduced this inflammation. Using an in vitro flow chamber system with human arterial and venous endothelial cells, we found that leukocytes from healthy postmenopausal women were more adhesive to the arterial endothelium than those from premenopausal women regardless of the stimulus used on endothelial cells. Increased circulating levels of IL-8, MCP-1, RANTES, and MIP-1alpha and monocyte CD11b expression were also encountered in postmenopausal vs premenopausal subjects. This translational data led us to investigate the mechanisms in Sprague-Dawley rats. Using intravital microscopy, we imaged mesenteric arterioles and found significant increases in arteriolar leukocyte adhesion, cell adhesion molecule expression, and plasma levels of cytokine-induced neutrophil chemoattractant (CINC/KC), MCP-1, and MIP-1alpha in 1-mo ovariectomized rats. Chronic treatment of ovariectomized rats with low dose of 17-beta-estradiol, losartan, both, or benazepril inhibited ovariectomy-induced arteriolar mononuclear leukocyte adhesion by 77%, 58%, 92%, and 65% respectively, partly by inhibition of cell adhesion molecule up-regulation and the increase in circulating chemokines. These results demonstrate that menopause and ovariectomy generate a low grade of systemic inflammation. Therefore, administration of low doses of estrogens or inhibition of the renin-angiotensin system, at early stages of estrogen deficiency, might prevent the systemic inflammation associated with menopause and decrease the risk of suffering further cardiovascular diseases.

The preclinical pharmacology of roflumilast--a selective, oral phosphodiesterase 4 inhibitor in development for chronic obstructive pulmonary disease.

After more than two decades of research into phosphodiesterase 4 (PDE4) inhibitors, roflumilast (3-cyclopropylmethoxy-4-difluoromethoxy-N-[3,5-di-chloropyrid-4-yl]-benzamide) may become the first agent in this class to be approved for patient treatment worldwide. Within the PDE family of 11 known isoenzymes, roflumilast is selective for PDE4, showing balanced selectivity for subtypes A-D, and is of high subnanomolar potency. The active principle of roflumilast in man is its dichloropyridyl N-oxide metabolite, which has similar potency as a PDE4 inhibitor as the parent compound. The long half-life and high potency of this metabolite allows for once-daily, oral administration of a single, 500-microg tablet of roflumilast. The molecular mode of action of roflumilast--PDE4 inhibition and subsequent enhancement of cAMP levels--is well established. To further understand its functional mode of action in chronic obstructive pulmonary disease (COPD), for which roflumilast is being developed, a series of in vitro and in vivo preclinical studies has been performed. COPD is a progressive, devastating condition of the lung associated with an abnormal inflammatory response to noxious particles and gases, particularly tobacco smoke. In addition, according to the Global Initiative for Chronic Obstructive Lung Disease (GOLD), significant extrapulmonary effects, including comorbidities, may add to the severity of the disease in individual patients, and which may be addressed preferentially by orally administered remedies. COPD shows an increasing prevalence and mortality, and its treatment remains a high, unmet medical need. In vivo, roflumilast mitigates key COPD-related disease mechanisms such as tobacco smoke-induced lung inflammation, mucociliary malfunction, lung fibrotic and emphysematous remodelling, oxidative stress, pulmonary vascular remodelling and pulmonary hypertension. In vitro, roflumilast N-oxide has been demonstrated to affect the functions of many cell types, including neutrophils, monocytes/macrophages, CD4+ and CD8+ T-cells, endothelial cells, epithelial cells, smooth muscle cells and fibroblasts. These cellular effects are thought to be responsible for the beneficial effects of roflumilast on the disease mechanisms of COPD, which translate into reduced exacerbations and improved lung function. As a multicomponent disease, COPD requires a broad therapeutic approach that might be achieved by PDE4 inhibition. However, as a PDE4 inhibitor, roflumilast is not a direct bronchodilator. In summary, roflumilast may be the first-in-class PDE4 inhibitor for COPD therapy. In addition to being a non-steroid, anti-inflammatory drug designed to target pulmonary inflammation, the preclinical pharmacology described in this review points to a broad functional mode of action of roflumilast that putatively addresses additional COPD mechanisms. This enables roflumilast to offer effective, oral maintenance treatment for COPD, with an acceptable tolerability profile and the potential to favourably affect the extrapulmonary effects of the disease.

CXCR2 blockade impairs angiotensin II-induced CC chemokine synthesis and mononuclear leukocyte infiltration.

OBJECTIVE: Angiotensin II (Ang-II) and mononuclear leukocytes are involved in atherosclerosis. This study reports the inhibition of Ang-II-induced mononuclear cell recruitment by CXCR2 antagonism and the mechanisms involved. METHODS AND RESULTS: Ang-II (1 nmol/L, i.p. in rats) induced CXC and CC chemokines, followed by neutrophil and mononuclear cell recruitment. Administration of the CXCR2 antagonist, SB-517785-M, inhibited the infiltration of both neutrophils (98%) and mononuclear cells (60%). SB-517785-M had no effect on the increase in CXC chemokine levels but reduced MCP-1, RANTES, and MIP-1alpha release by 66%, 63%, and 80%, respectively. Intravital microscopy showed that pretreatment with SB-517785-M inhibited Ang-II-induced arteriolar mononuclear leukocyte adhesion. Stimulation of human umbilical arterial endothelial cells (HUAECs) or whole blood with 1 micromol/L Ang-II induced the synthesis of chemokines. Ang-II increased HUAEC CXCR2 expression, and its blockade caused a significant reduction of MCP-1, -3, and RANTES release, as well as mononuclear cell arrest. Ang-II-induced MIP-1alpha release from blood cells was also inhibited. CONCLUSION: Mononuclear leukocyte recruitment induced by Ang-II is, surprisingly, largely mediated by the CXC chemokines which appear to induce the release of CC chemokines. Therefore, CXC chemokine receptor antagonists may help to prevent mononuclear cell infiltration and the progression of the atherogenic process.

Changes in alpha-tocopherol and retinol levels during cardiopulmonary bypass correlate with maximal arterial partial pressure of oxygen.

Cardiopulmonary bypass (CPB) is associated with oxidative stress. This study examined antioxidant levels in adults undergoing CPB surgery and their correlation with clinical variables. Arterial blood samples were obtained from 27 patients undergoing CPB. The time-course variation of vitamin C (spectrofluorimetry), alpha-tocopherol and retinol (HPLC) levels were determined. Plasma vitamin C rose initially but gradually decayed during reperfusion until 60% reduction of baseline values post-surgery. alpha-Tocopherol and retinol were reduced along CPB with post-operative values approximately 25% lower than baseline. No significant changes were found for selenium and glutathione peroxidase. PaO(2) values rose steadily throughout CPB. A correlation existed for alpha-tocopherol and retinol depletion vs maximal PaO(2) throughout CPB but no correlation was found for antioxidant consumption vs duration of ischaemia and reperfusion and hypothermia level. In conclusion, consumption of arterial blood antioxidant vitamins occurs with CPB in relation with PaO(2) levels but not for other clinical variables measured in this study.

Is the beta3-adrenoceptor (ADRB3) a potential target for uterorelaxant drugs?

The management of premature birth still remains unsatisfactory. Since the relative lack of efficiency and/or safety of current tocolytic agents have been highlighted, it is necessary to develop new uterorelaxant drugs deprived of important maternal and foetal side effects. Our work reported in this review focuses on a potential new target for tocolytic drugs, the beta3-adrenoceptor (ADRB3). This third type of ADRB is shown to be present and functional in human myometrium. We demonstrated that ADRB3 agonists are able to inhibit in-vitro spontaneous contractions of myometrial strips, via a cyclic AMP-mediated pathway. Furthermore, we established that ADRB3 is the predominant subtype over the ADRB2 in human myometrium and that its expression is increased in near-term myometrium, compared to non-pregnant myometrium. Finally, we reported that contrary to ADRB2, the human myometrial ADRB3 is resistant to long-term agonist-induced desensitisation. These compelling data confirm the clinical potential interest of ADRB3 agonists in the pharmacological management of preterm labour.

PDE4 inhibitors as new anti-inflammatory drugs: effects on cell trafficking and cell adhesion molecules expression.

Phosphodiesterase 4 (PDE4) is a major cyclic AMP-hydrolyzing enzyme in inflammatory and immunomodulatory cells. The wide range of inflammatory mechanisms under control by PDE4 points to this isoenzyme as an attractive target for new anti-inflammatory drugs. Selective inhibitors of PDE4 have demonstrated a broad spectrum of anti-inflammatory activities including the inhibition of cellular trafficking and microvascular leakage, cytokine and chemokine release from inflammatory cells, reactive oxygen species production, and cell adhesion molecule expression in a variety of in vitro and in vivo experimental models. The initially detected side effects, mainly nausea and emesis, appear at least partially overcome by the 'second generation' PDE4 inhibitors, some of which like roflumilast and cilomilast are in the later stages of clinical development for treatment of chronic obstructive pulmonary disease. These new drugs may also offer opportunities for treatment of other inflammatory diseases.

A small molecule, orally active, alpha4beta1/alpha4beta7 dual antagonist reduces leukocyte infiltration and airway hyper-responsiveness in an experimental model of allergic asthma in Brown Norway rats.

alpha(4)beta(1) and alpha(4)beta(7) integrins are preferentially expressed on eosinophils and mononuclear leukocytes and play critical roles in their recruitment to inflammatory sites. We investigated the effects of TR14035, a small molecule, alpha(4)beta(1)/alpha(4)beta(7) dual antagonist, in a rat model of allergic asthma. Actively sensitized rats were challenged with aerosol antigen or saline on day 21, and the responses evaluated 24 and 48-h later. TR14035 (3 mg kg(-1), p.o.) was given 1-h before and 4-h after antigen or saline challenge. Airway hyper-responsiveness to intravenous 5-hydroxytryptamine was suppressed in TR14035-treated rats. Eosinophil, mononuclear cell and neutrophil counts, and eosinophil peroxidase and protein content in the bronchoalveolar lavage fluid (BALF) were decreased in TR14035-treated rats. Histological study showed a marked reduction of lung inflammatory lesions by TR14035. At 24-h postchallenge, antigen-induced lung interleukin (IL)-5 mRNA upregulation was suppressed in TR14035-treated rats. By contrast, IL-4 levels in BALF were not significantly affected by TR14035 treatment. IL-4 selectively upregulates vascular cell adhesion molecule-1 (VCAM-1), which is the main endothelial ligand of alpha(4) integrins. Intravital microscopy within the rat mesenteric microcirculation showed that 24-h exposure to 1 microg per rat of IL-4 induced a significant increase in leukocyte rolling flux, adhesion and emigration. These responses were decreased by 48, 100 and 99%, respectively in animals treated with TR14035. In conclusion, TR14035, by acting on alpha(4)beta(1) and alpha(4)beta(7) integrins, is an orally active inhibitor of airway leukocyte recruitment and hyper-responsiveness in animal models with potential interest for the treatment of asthma.

Olive oil-based lipid emulsion's neutral effects on neutrophil functions and leukocyte-endothelial cell interactions.

BACKGROUND: Infection remains a drawback of parenteral nutrition (PN), probably related, among other factors, to immunosuppressive effects of its lipid component. Newer preparations may have lesser immunosuppressive impact. This study examines the effects of an olive oil-based lipid emulsion (long-chain triacylglycerols-monounsaturated fatty acids [LCT-MUFA]; ClinOleic) on various functions of human neutrophils in vitro and on rat leukocyte-endothelial cell interactions in vivo compared with LCT (Intralipid) and 50% LCT-50% medium-chain triacylglycerols (MCT; Lipofundin) mixture. METHODS: Neutrophils isolated from healthy donors were incubated with concentrations (0.03-3 mmol/L) of lipid emulsions encompassing clinically relevant levels. In vivo leukocyte recruitment was studied with intravital microscopy within rat mesenteric microcirculation. RESULTS: LCT-MUFA (3 mmol/L) did not alter the N-formyl-Met-Leu-Phe (FMLP)-induced rise in [Ca2+]i, oxidative burst, chemotaxis, and elastase release, whereas LCT-MCT decreased [Ca2+]i and chemotaxis and increased oxidative burst. FMLP-induced LTB4 production was augmented by lipid emulsions. Serum-opsonized zymosan-induced phagocytosis was unaltered by lipid emulsions. Basal and FMLP-induced CD11b expression was unaffected by lipid emulsions. Lipopolysaccharide (LPS)-induced TNF-alpha, IL-1beta and IL-8 mRNA, and protein expression was unaltered by LCT-MUFA, whereas LCT and LCT-MCT decreased IL-1beta mRNA and protein. LCT-MUFA did not alter apoptosis, but LCT increased apoptosis in absence and presence of GM-CSF. LPS (1 microg/mL)-induced increase in leukocyte rolling flux, adhesion, and emigration was inhibited by LCT and LCT-MCT but unaffected in LCT-MUFA-treated rats. Immunohistochemistry showed LPS-induced increase in P-selectin expression attenuated by LCT and LCT-MCT but not LCT-MUFA. CONCLUSIONS: LCT-MUFA showed lower in vitro and in vivo impact on neutrophil function compared with LCT and LCT-MCT.

Effect of boldine, secoboldine, and boldine methine on angiotensin II-induced neutrophil recruitment in vivo.

Angiotensin-II (Ang-II) has inflammatory activity and is involved in different diseases associated with the cardiovascular system. This study has evaluated the effect of boldine (B), and two phenanthrene alkaloids semisynthesized by us, secoboldine (SB) and boldine methine (BM), on Ang-II-induced neutrophil recruitment. Intraperitoneal administration of 1 nM Ang-II induced significant neutrophil accumulation, which was maximal at 4-8 h. BM inhibited neutrophil infiltration into the peritoneal cavity at 4 h and 8 h by 73% and 77%, respectively, SB at 8 h by 55%, and B had no effect on this response. Although BM inhibited the release of cytokine-inducible neutrophil chemoattractant/keratinocyte-derived chemokine, macrophage inflammatory protein-2 (MIP-2), and platelet-activating factor (PAF) elicited by Ang-II, SB only reduced the release of MIP-2 after 4 h of its administration. Sixty-minute superfusion of the rat mesentery with 1 nM Ang-II induced a significant increase in the leukocyte-endothelial cell interactions and P-selectin up-regulation, which were inhibited by 1 microM BM and SB. The generation of reactive oxygen species (ROS) in endothelial cells stimulated with Ang-II was inhibited significantly by the three alkaloids tested. BM also diminished Ang-II-induced interleukin-8 release from endothelial cells and blocked the PAF receptor on human neutrophils (concentration of the compound needed to produce 50% inhibition value: 28.2 microM). Therefore, BM is a potent inhibitor of Ang-II-induced neutrophil accumulation in vivo. This effect appears to be mediated through inhibition of CXC chemokine and PAF release, ROS scavenging activity, and blockade of the PAF receptor. Thus, it may have potential therapeutic interest for the control of neutrophil recruitment that occurs in inflammation associated with elevated levels of Ang-II.

Angiotensin II induces neutrophil accumulation in vivo through generation and release of CXC chemokines.

BACKGROUND: Angiotensin II (Ang II) is implicated in the development of cardiac ischemic disorders in which prominent neutrophil accumulation occurs. Ang II can be generated intravascularly by the renin-angiotensin system or extravascularly by mast cell chymase. In this study, we characterized the ability of Ang II to induce neutrophil accumulation. METHODS AND RESULTS: Intraperitoneal administration of Ang II (1 nmol/L) induced significant neutrophil recruitment within 4 hours (13.3+/-2.3x10(6) neutrophils per rat versus 0.7+/-0.5x10(6) in control animals), which disappeared by 24 hours. Maximal levels of CXC chemokines were detected 1 hour after Ang II injection (577+/-224 pmol/L cytokine-inducible neutrophil chemoattractant [CINC]/keratinocyte-derived chemokine [KC] versus 5+/-3, and 281+/-120 pmol/L macrophage inflammatory protein [MIP-2] versus 14+/-6). Intravital microscopy within the rat mesenteric microcirculation showed that the short-term (30 to 60 minutes) leukocyte-endothelial cell interactions induced by Ang II were attenuated by an anti-rat CINC/KC antibody and nearly abolished by the CXCR2 antagonist SB-517785-M. In human umbilical vein endothelial cells (HUVECs) or human pulmonary artery media in culture, Ang II induced interleukin (IL)-8 mRNA expression at 1, 4, and 24 hours and the release of IL-8 at 4 hours through interaction with Ang II type 1 receptors. When HUVECs were pretreated with IL-1 for 24 hours to promote IL-8 storage in Weibel-Palade bodies, the Ang II-induced IL-8 release was more rapid and of greater magnitude. CONCLUSIONS: Ang II provokes rapid neutrophil recruitment, mediated through the release of CXC chemokines such as CINC/KC and MIP-2 in rats and IL-8 in humans, and may contribute to the infiltration of neutrophils observed in acute myocardial infarction.

Erythromycin exerts in vivo anti-inflammatory activity downregulating cell adhesion molecule expression.

1. Macrolides have long been used as anti-bacterial agents; however, there is some evidence that may exert anti-inflammatory activity. Therefore, erythromycin was used to characterize the mechanisms involved in their in vivo anti-inflammatory activity. 2. Erythromycin pretreatment (30 mg kg(-1) day(-1) for 1 week) reduced the lipopolysaccharide (LPS; intratracheal, 0.4 mg kg(-1))-induced increase in neutrophil count and elastase activity in the bronchoalveolar lavage fluid (BALF) and lung tissue myeloperoxidase activity, but failed to decrease tumor necrosis factor-alpha and macrophage-inflammatory protein-2 augmented levels in BALF. Erythromycin pretreatment also prevented lung P-selectin, E-selectin, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) mRNA upregulation in response to airway challenge with LPS. 3. Mesentery superfusion with LPS (1 mug ml(-1)) induced a significant increase in leukocyte-endothelial cell interactions at 60 min. Erythromycin pretreatment abolished the increases in these parameters. 4. LPS exposure of the mesentery for 4 h caused a significant increase in leukocyte rolling flux, adhesion and emigration, which were inhibited by erythromycin by 100, 93 and 95%, respectively. 5. Immunohistochemical analysis showed that LPS exposure of the mesentery for 4 h caused a significant enhancement in P-selectin, E-selectin, ICAM-1 and VCAM-1 expression that was downregulated by erythromycin pretreatment. 6. Flow cytometry analysis indicated that erythromycin pretreatment inhibited LPS-induced CD11b augmented expression in rat neutrophils. 7. In conclusion, erythromycin inhibits leukocyte recruitment in the lung and this effect appears mediated through downregulation of CAM expression. Therefore, macrolides may be useful in the control of neutrophilic pulmonary diseases.

Effects of bemiparin on airway responses to antigen in sensitized Brown-Norway rats.

Heparins have demonstrated activity in asthma. The effects of bemiparin, a low molecular weight heparin, were examined on antigen-induced responses in sensitized Brown-Norway rats. Inhaled bemiparin (1 mg/ml) reduced the acute bronchospasm produced by aerosol antigen, prevented airway hyperresponsiveness to 5-hydroxytryptamine postantigen exposure, and reduced the eosinophil count (from 0.205+/-0.062 to 0.054+/-0.016 x 10(6) cells/ml in antigen and antigen+bemiparin groups, respectively; P<0.05), eosinophil peroxidase activity, and proteins in the bronchoalveolar lavage fluid (BALF), as well as the transiently augmented mucin Muc5ac expression. Hyperresponsiveness to adenosine was not affected by bemiparin. In similar experiments, inhaled fondaparinux (1 mg/ml) did not affect the antigen-induced responses, while a low-anticoagulant low molecular weight heparin was effective. In conclusion, bemiparin showed beneficial effects in experimental asthma, probably unrelated to its anticoagulant activity, which extends the previous positive findings obtained with other heparins.

[Monitoring protocol of native vascular accesses for haemodialysis]. / Programa de monitorización de accesos vasculares nativos para hemodiálisis.

Vascular access failure is an important cause of morbidity and mortality for patients on haemodialysis. We have prospectively studied, with a 5 years follow up, a monitoring protocol of native vascular accesses, using the available methods in every haemodialysis unit. All the native vascular accesses, created from 1- 1998 to XII-2001, with a posterior follow up until XII-2002, were monitored. Monitoring was based on physical examination, dificulty for blood flow greater than 300 ml/min, and in a monthly basis: dynamic venous pressure, urea recirculation and urea kinetic model. Abnormalities suggestive of fistulogram were a priori defined. During the recruitment period, a total of 164 accesses were created in 144 patients. Of these only 3 were grafts, 28 native vascular accesses were never functioning (primary failure rate 17. 1%), and 127 native accesses created in 113 patients (age 63.3 +/- 12.4 years; 18 % diabetics), were monitored (83% cephalic vein). Monitoring findings indicated realization of fistulogram in 35% and percutaneus angioplasty in 25% of the accesses. In order to maintain patency, the surgical intervention rate was 0.03 procedures/patient/year, the radiological 0.10 and the total 0.13. During the 5 years of the study occurred 41 thrombosis episodes in 40 accesses (0.07 thrombosis/patient/year), with percutaneus repermeabilization in 30%. Primary (unassisted) survival was 30.3 months (Confidence Interval 95% 25.6, 35.0) and secondary (assisted) survival 42.8 months (Confidence Interval 95%: 38.7, 46.9). Logistic regression analysis showed that presence of a central catheter at the time of creating the vascular access posses a greater risk for thrombosis (Relative Risk 5.6 if in subclavian vein), whereas age, diabetes, time to canulation, number of previous accesses and anatomic type do not increase that risk. In conclusion, in an old haemodialysis population, with an important diabetes prevalence, it is possible to create functioning native vascular accesses in most of them. The monitoring protocol allowed the detection and posterior correction of stenosis in a great number of accesses. The main risk of thrombosis is the presence of a central catheter at the time of creating a vascular access.

Rolipram inhibits leukocyte-endothelial cell interactions in vivo through P- and E-selectin downregulation.

1. Rolipram, a selective phosphodiesterase (PDE) type 4 inhibitor, was used to characterize leukocyte recruitment mechanisms in models of acute and subacute inflammation. Intravital microscopy within the rat mesenteric microcirculation was employed. 2. Mesentery superfusion with PAF (0.1 microM) induced a significant increase in leukocyte rolling flux, adhesion and emigration at 60 min. Rolipram pretreatment, markedly inhibited these parameters by 100, 95 and 95% respectively. 3. Similar effects were observed when the mesentery was superfused with LPS (1 microg ml(-1)) for the same time period and these leukocyte parameters were nearly abrogated by rolipram pretreatment. 4. LPS exposure of the mesentery for 4 h caused a greater increase in leukocyte rolling flux, adhesion and emigration which were inhibited by rolipram administration by 51, 71 and 81% respectively. 5. Immunohistochemistry revealed a significant increase in P-selectin expression after 60 min superfusion with PAF which was attenuated by rolipram. 6. LPS exposure of the mesentery for 4 h caused a significant increase in P- and E-selectin, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expression. Rolipram pretreatment down-regulated both P- and E-selectin expression but had no effect on ICAM-1 and VCAM-1 expression. 7. Significant increases in plasma cyclic AMP levels were detected at 4.5 h after rolipram administration. 8. In conclusion, we have demonstrated that rolipram is a potent in vivo inhibitor of leukocyte-endothelial cell interactions. The effects observed are mediated through endothelial P- and E-selectin downregulation. Therefore, selective PDE-4 inhibitors may be useful in the control of different inflammatory disorders.

Effect of two phenanthrene alkaloids on angiotensin II-induced leukocyte-endothelial cell interactions in vivo.

1. The present study has evaluated the effect of two phenanthrene alkaloids, uvariopsine and stephenanthrine, on angiotensin II (Ang-II)-induced leukocyte-endothelial cell interactions in vivo and the mechanisms involved in their activity. Intravital microscopy within the rat mesenteric microcirculation was used. 2. A 60 min superfusion with 1 nm Ang-II induced a significant increase in the leukocyte-endothelial cell interactions that were completely inhibited by 1 microm uvariopsine cosuperfusion. A lower dose of 0.1 microm significantly reduced Ang-II-induced leukocyte adhesion by 75%. 3. When Ang-II was cosuperfused with 1 and 0.1 microm stephenanthrine, Ang-II-induced leukocyte responses were significantly diminished. A lower dose of 0.01 microm only affected Ang-II-induced leukocyte adhesion. 4. Both alkaloids inhibited Ang-II-induced endothelial P-selectin upregulation and the generation of reactive oxygen species (ROS) in endothelial cells stimulated with Ang-II, in fMLP-stimulated human neutrophils (PMNs) and in the hypoxanthine-xanthine oxidase system. However, cyclic AMP levels in PMNs stimulated with fMLP were not affected. 5. Uvariopsine and stephenanthrine inhibited PAF-induced elevations in intracellular calcium levels in PMNs (IC50 values: 15.1 and 6.1 microm respectively) and blocked the binding of [3H]PAF to these leukocytes. They also reduced PAF-induced increases in intracellular levels of superoxide anion and hydrogen peroxide. 6. In conclusion, stephenanthrine and uvariopsine are potent inhibitors of Ang-II-induced leukocyte accumulation in vivo. This effect appears to be mediated through ROS scavenging activity and blockade of PAF receptor. Thus, they have potential therapeutic interest for the control of leukocyte recruitment that occurs in cardiovascular disease states in which Ang-II is involved.