Maria-Jesus Sanz

Angiotensin II induces leukocyte-endothelial cell interactions in vivo via AT1 and AT2 receptor-mediated P-selectin upregulation

BackgroundAngiotensin II (Ang II) plays a critical role in the development of vascular lesions in hypertension, atherosclerosis, and several renal diseases. Because Ang II may contribute to the leukocyte recruitment associated with these pathological states, the aim of the present study was to assess the role of Ang II in leukocyte–endothelial cell interactions in vivo. Methods and ResultsIntravital microscopy of the rat mesenteric postcapillary venules was used. Sixty minutes of superfusion with 1 nmol/L Ang II induced a significant increase in leukocyte rolling flux (83.8±20.7 versus 16.4±3.1 cells/min), adhesion (11.4±1.0 versus 0.8±0.5 cells/100 &mgr;m), and emigration (4.0±0.7 versus 0.2±0.2 cells/field) without any vasoconstrictor activity. These effects were not mediated by mast cell activation. Intravenous pretreatment with AT1 (losartan) or AT2 (PD123,319) receptor antagonists significantly reduced Ang II–induced responses. A combination of both receptor antagonists inhibited the leukocyte rolling flux, adhesion, and extravasation elicited by Ang II at 60 minutes. Pretreatment of animals with fucoidin or an adhesion-blocking anti–rat P-selectin monoclonal antibody abolished Ang II–induced leukocyte responses. Furthermore, rat platelet P-selectin expression was not affected by Ang II stimulation. ConclusionsAng II induces significant leukocyte rolling, adhesion, and emigration, which may contribute not only to hypertension but also to the onset and progression of the vascular damage associated with disease states in which plasma levels of this peptide are elevated.

Molecular mechanisms of NET formation and degradation revealed by intravital imaging in the liver vasculature.

Neutrophil extracellular traps (NETs) composed of DNA decorated with histones and proteases trap and kill bacteria but also injure host tissue. Here we show that during a bloodstream infection with methicillin-resistant Staphylococcus aureus, the majority of bacteria are sequestered immediately by hepatic Kupffer cells, resulting in transient increases in liver enzymes, focal ischaemic areas and a robust neutrophil infiltration into the liver. The neutrophils release NETs into the liver vasculature, which remain anchored to the vascular wall via von Willebrand factor and reveal significant neutrophil elastase (NE) proteolytic activity. Importantly, DNase although very effective at DNA removal, and somewhat effective at inhibiting NE proteolytic activity, fails to remove the majority of histones from the vessel wall and only partly reduces injury. By contrast, inhibition of NET production as modelled by PAD4-deficiency, or prevention of NET formation and proteolytic activity as modelled in NE−/− mice prevent collateral host tissue damage.

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±2.3×106 neutrophils per rat versus 0.7±0.5×106 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.

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-β-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-1α 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-1α in 1-mo ovariectomized rats. Chronic treatment of ovariectomized rats with low dose of 17-β-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.

Angiotensin II-Induced Mononuclear Leukocyte Interactions with Arteriolar and Venular Endothelium Are Mediated by the Release of Different CC Chemokines

Angiotensin II (Ang-II) is associated with atherogenesis and arterial subendothelial mononuclear leukocyte infiltration. We have demonstrated that Ang-II causes the initial attachment of mononuclear cells to the arteriolar endothelium. We now report on the contribution of CC chemokines to this response. Intraperitoneal administration of 1 nM Ang-II induced MCP-1, RANTES, and MIP-1α generation, maximal at 4 h, followed by mononuclear leukocyte recruitment at 8 and 24 h. Using intravital microscopy within the rat mesenteric microcirculation 4 h after exposure to 1 nM Ang-II, arteriolar mononuclear cell adhesion was 80–90% inhibited by pretreatment with Met-RANTES, a CCR1 and CCR5 antagonist, or an anti-MCP-1 antiserum, without affecting the increased endothelial expression of P-selectin and VCAM-1. Conversely, leukocyte interactions with the venular endothelium, although inhibited by Met-RANTES, were little affected by the anti-MCP-1. Using rat whole blood in vitro, Ang-II (100 nM) induced the expression of monocyte CD11b that was inhibited by Met-RANTES but not by anti-MCP-1. Stimulation of human endothelial cells (human umbilical arterial endothelial cells and HUVECs) with 1–1000 nM Ang-II, predominantly acting at its AT1 receptor, induced the release of MCP-1 within 1 h, RANTES within 4 h, and MCP-3 within 24 h. Eotaxin-3, a natural CCR2 antagonist, was released within 1 h and may delay mononuclear cell responses to MCP-1. Therefore, Ang-II-induced mononuclear leukocyte recruitment at arterioles and venules is mediated by the production of different CC chemokines. Thus, Ang-II may be a key molecule in the initial attachment of mononuclear cells to the arterial endothelium in cardiovascular disease states where this event is a characteristic feature.

Trans- but Not Cis-Resveratrol Impairs Angiotensin-II–Mediated Vascular Inflammation through Inhibition of NF-κB Activation and Peroxisome Proliferator-Activated Receptor-γ 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-1α. In an in vitro flow chamber system, t-RESV (1–10 μM) 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-κB. Interestingly, t-RESV increased the expression of peroxisome proliferator-activated receptor-γ 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-γ 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.

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.

Activation of PPARβ/δ inhibits leukocyte recruitment, cell adhesion molecule expression, and chemokine release

The infiltration of PMNs into tissues is a prominent feature in inflammation. The mechanism underlying PMN recruitment depends on the release of chemotactic mediators and CAM expression on endothelial cells. The nuclear receptor PPARβ/δ is widely expressed in many tissues, including the vascular endothelium; however, its role in acute inflammation remains unclear. Using intravital microscopy in the mouse cremasteric microcirculation, we have shown that activation of PPARβ/δ by its selective ligand GW501516 inhibits TNF‐α‐induced leukocyte rolling flux, adhesion, and emigration in a dose‐dependant manner. Moreover, GW501516 reduced the expression of adhesion molecules such as ICAM‐1, VCAM‐1, and E‐selectin in the cremasteric postcapillary venules. Similarly, rolling and adhesion of hPMNs under physiological flow on TNF‐α‐activated HUVECs were also inhibited markedly by GW501516. These inhibitory responses of GW501516 on activated endothelium were accompanied by a reduction in TNF‐α‐induced endothelial GRO‐α release and VCAM‐1, E‐selectin, and ICAM‐1 mRNA expression. Taken together, our results show that PPARβ/δ modulates acute inflammation in vivo and in vitro under flow by targeting the neutrophil‐endothelial cell interaction.

Reactive oxygen species mediate angiotensin II-induced leukocyte-endothelial cell interactions in vivo

Chronically elevated angiotensin II (Ang‐II)‐induced hypertension is partly mediated by superoxide production. In this study, we have investigated whether the leukocyte‐endothelial cell interactions elicited by Ang‐II involve reactive oxygen species (ROS) generation. Intravital microscopy within the rat mesenteric microvessels was used. Superfusion (60 min) with Ang‐II (1 nM) induced significant increases in leukocyte rolling flux, adhesion, and emigration, which were inhibited by pretreatment with superoxide dismutase or catalase. Dihydrorhodamine‐123 oxidation indicated that ROS are primarily produced by the vessel wall. Administration of dimethylthiourea, desferrioxamine, or N‐acetylcisteine provoked significant reductions in Ang‐II‐induced leukocyte‐endothelial cell interactions. In addition, a blockade of platelet‐activating factor or leukotrienes also attenuated such responses significantly. The results presented indicate that in vivo Ang‐II‐induced leukocyte recruitment is dependent on the generation of intra‐ and extracellular ROS. Therefore, the use of anti‐oxidants might constitute an alternative therapy for the control of the subendothelial leukocyte infiltration associated with hypertension and atherosclerosis.

Accelerated communication: Effects of flavonoids on Naja Naja and human recombinant synovial phospholipases A2 and inflammatory responses in mice

Six flavonoid derivatives were tested for their influence on Naja naja and human recombinant synovial phospholipase A2. They showed a selectivity for the last enzyme with IC50 = 14.3, 17.6, 12.2 and 28.2 microM for quercetagetin, kaempferol-3-O-galactoside, scutellarein and scutellarein-7-O-glucuronide, respectively, while reduced effects were observed for hispidulin and hibifolin. After topical application all the flavonoids inhibited 12-O-tetradecanoylphorbol-13-acetate-induced ear oedema in mice with a potency comparable to that of indomethacin and they were also able to inhibit carrageenan-induced mouse paw oedema at a dose of 150 mg/kg p.o. The blockade of the free hydroxyl at C-7 or C-6 reduced the anti-inflammatory activity and also the inhibitory effect on human recombinant synovial phospholipase A2. These results are in accordance with the notion that group II phospholipases A2 may play a role in experimental inflammation, although several mechanisms seems to be involved in the anti-inflammatory effect of this group of flavonoids.