Angeles Alvarez

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.

Enhanced oxidative stress and increased mitochondrial mass during Efavirenz-induced apoptosis in human hepatic cells

Efavirenz (EFV) is widely used in the treatment of HIV‐1 infection. Though highly efficient, there is growing concern about EFV‐related side effects, the molecular basis of which remains elusive.

Induction of Oxidative Stress and Human Leukocyte/Endothelial Cell Interactions in Polycystic Ovary Syndrome Patients with Insulin Resistance

CONTEXT Insulin resistance is a feature of polycystic ovary syndrome (PCOS) and is related to mitochondrial and endothelial function. OBJECTIVE We tested whether hyperandrogenic insulin-resistant women with PCOS, who have an increased risk of vascular disease, display impaired leukocyte-endothelium interactions, and mitochondrial dysfunction. DESIGN AND SETTING This was a prospective controlled study conducted in an academic medical center. PATIENTS The study population consisted of 43 lean reproductive-age women with PCOS and 39 controls subjects. MAIN OUTCOME MEASURES We evaluated anthropometric and metabolic parameters, adhesion molecules, and interactions between leukocytes and human umbilical vein endothelial cells. Mitochondrial function was studied by assessing mitochondrial oxygen consumption, membrane potential, reactive oxygen species production, glutathione levels (GSH), and the oxidized glutathione (GSSG)/GSH ratio in polymorphonuclear cells. RESULTS Impairment of mitochondrial function was observed in the PCOS patients, evident in a decrease in oxygen consumption, an increase in reactive oxygen species production, a decrease in the GSH/GSSG ratio and GSH levels, and an undermining of the membrane potential. PCOS was related to a decrease in polymorphonuclear cell rolling velocity and an increase in rolling flux and adhesion. Increases in IL-6 and TNFα and adhesion molecules (vascular cell adhesion molecule-1 and E-selectin) were also observed. CONCLUSION This study supports the hypothesis of an association between insulin resistance and an impaired endothelial and mitochondrial oxidative metabolism. The evidence obtained shows that the inflammatory state related to insulin resistance in PCOS induces a leukocyte-endothelium interaction. These findings may explain the increased risk of vascular disease in women with PCOS.

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.

Estrogens Inhibit Angiotensin II–Induced Leukocyte–Endothelial Cell Interactions In Vivo via Rapid Endothelial Nitric Oxide Synthase and Cyclooxygenase Activation

Abstract— Angiotensin II (Ang II) may be a key molecule in the development of atherosclerosis. Because the incidence of coronary atherosclerosis in premenopausal women is lower than that observed in men or postmenopausal women, we have investigated the effect of estrogens on Ang II–induced leukocyte recruitment in vivo using intravital microscopy in the rat mesenteric microcirculation. Superfusion for 60 minutes with Ang II induced a significant increase in leukocyte rolling flux, adhesion, and emigration. Administration of 17-&bgr;-estradiol (17-&bgr;-E) after 30 minutes of Ang II superfusion produced a reduction of these leukocyte responses by 55.1%, 72.7%, and 70.9%, respectively, an additional 30 minutes later. The effect observed with 17-&bgr;-E was receptor-mediated and specific. 17-&bgr;-E superfusion did not modify either L-NAME or indomethacin-induced leukocyte responses. Inhibitory responses caused by 17-&bgr;-E were not altered by either 7-nitroindazole or actinomycin D cosuperfusion. Stimulation of endothelial cells with 17-&bgr;-E caused a rapid and dose-dependent release of prostacyclin. Finally, tamoxifen or ICI 182,780 administration provoked a significant increase in leukocyte–endothelial cell interactions 90 minutes later, which were significantly attenuated by systemic preadministration with an Ang II AT1 receptor antagonist. Tamoxifen-induced leukocyte responses were also reduced by systemic pretreatment with an anti–P-selectin mAb and an anti–CD18 mAb. Hence, the antiatherogenic effects of estrogens may be mediated by inhibition of Ang II–induced leukocyte recruitment through endothelial NO and prostacyclin release. Furthermore, scarcity of estrogens resulted in decreased levels of vasodilators and the exposure of the endothelium to the deleterious action of Ang II, which may explain the higher incidence of coronary atherosclerosis in men and postmenopausal women.

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

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. Mesentery superfusion with PAF (0.1 μM) 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. Similar effects were observed when the mesentery was superfused with LPS (1 μg ml−1) for the same time period and these leukocyte parameters were nearly abrogated by rolipram pretreatment. 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. Immunohistochemistry revealed a significant increase in P‐selectin expression after 60 min superfusion with PAF which was attenuated by rolipram. 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. Significant increases in plasma cyclic AMP levels were detected at 4.5 h after rolipram administration. 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.

M2 macrophages activate WNT signaling pathway in epithelial cells: relevance in ulcerative colitis.

Macrophages, which exhibit great plasticity, are important components of the inflamed tissue and constitute an essential element of regenerative responses. Epithelial Wnt signalling is involved in mechanisms of proliferation and differentiation and expression of Wnt ligands by macrophages has been reported. We aim to determine whether the macrophage phenotype determines the expression of Wnt ligands, the influence of the macrophage phenotype in epithelial activation of Wnt signalling and the relevance of this pathway in ulcerative colitis. Human monocyte-derived macrophages and U937-derived macrophages were polarized towards M1 or M2 phenotypes and the expression of Wnt1 and Wnt3a was analyzed by qPCR. The effects of macrophages and the role of Wnt1 were analyzed on the expression of β-catenin, Tcf-4, c-Myc and markers of cell differentiation in a co-culture system with Caco-2 cells. Immunohistochemical staining of CD68, CD206, CD86, Wnt1, β-catenin and c-Myc were evaluated in the damaged and non-damaged mucosa of patients with UC. We also determined the mRNA expression of Lgr5 and c-Myc by qPCR and protein levels of β-catenin by western blot. Results show that M2, and no M1, activated the Wnt signaling pathway in co-culture epithelial cells through Wnt1 which impaired enterocyte differentiation. A significant increase in the number of CD206+ macrophages was observed in the damaged mucosa of chronic vs newly diagnosed patients. CD206 immunostaining co-localized with Wnt1 in the mucosa and these cells were associated with activation of canonical Wnt signalling pathway in epithelial cells and diminution of alkaline phosphatase activity. Our results show that M2 macrophages, and not M1, activate Wnt signalling pathways and decrease enterocyte differentiation in co-cultured epithelial cells. In the mucosa of UC patients, M2 macrophages increase with chronicity and are associated with activation of epithelial Wnt signalling and diminution in enterocyte differentiation.

Neuronal bioenergetics and acute mitochondrial dysfunction: a clue to understanding the central nervous system side effects of Efavirenz

BACKGROUND Neurological pathogenesis is associated with mitochondrial dysfunction and differences in neuronal/glial handling of oxygen and glucose. The main side effects attributed to efavirenz involve the CNS, but the underlying mechanisms are unclear. METHODS Human cell lines and rat primary cultures of neurons and astrocytes were treated with clinically relevant efavirenz concentration. RESULTS Efavirenz alters mitochondrial respiration, enhances reactive oxygen species generation, undermines mitochondrial membrane potential, and reduces adenosine triphosphate (ATP) levels in a concentration-dependent fashion in both neurons and glial cells. However, it activates adenosine monophosphate-activated protein kinase only in glial cells, upregulating glycolysis and increasing intracellular ATP levels, which do not occur in neurons. To reproduce the conditions that often exist in human immunodeficiency virus-related neuroinflammatory disorders, the effects of efavirenz were evaluated in the presence of exogenous nitric oxide, an inflammatory mediator and mitochondrial inhibitor. The combination potentiated the effects on mitochondrial parameters in both neurons and glial cells, but ATP generation and lactate production were enhanced only in glial cells. CONCLUSIONS Efavirenz affects the bioenergetics of neurons through a mechanism involving acute mitochondrial inhibition, an action exacerbated in neuroinflammatory conditions. A similar scenario of glial cells survival and degeneration of neurons with signs of mitochondrial dysfunction and oxidative stress has been associated with neurocognitive disorders.

Efavirenz and the CNS: what we already know and questions that need to be answered

The NNRTI efavirenz has long been one of the most frequently employed antiretroviral drugs in the multidrug regimens used to treat HIV infection, in accordance with its well-demonstrated antiretroviral efficacy and favourable pharmacokinetics. However, growing concern about its adverse effects has sometimes led to efavirenz being replaced by other drugs in the initial treatment selection or to switching of therapy to efavirenz-free regimens in experienced patients. Neurological and neuropsychiatric reactions are the manifestations most frequently experienced by efavirenz-treated patients and range from transitory effects, such as nightmares, dizziness, insomnia, nervousness and lack of concentration, to more severe symptoms including depression, suicidal ideation or even psychosis. In addition, efavirenz has recently been associated with mild/moderate neurocognitive impairment, which is of specific relevance given that half of the patients receiving ART eventually suffer some form of HIV-associated neurocognitive disorder. The mechanisms responsible for efavirenz-induced neurotoxicity are unclear, although growing evidence points to disturbances in brain mitochondrial function and bioenergetics. This review offers a comprehensive overview of the current evidence on the interaction that efavirenz displays with the CNS, including the penetration and concentration of the drug in the brain. We discuss the prevalence, types and specificities of its side effects and recently uncovered cellular mechanisms that may be involved in their development.

Abacavir and didanosine induce the interaction between human leukocytes and endothelial cells through Mac-1 upregulation.

Objective:Abacavir and didanosine are nucleoside reverse transcriptase inhibitors (NRTI) widely used in therapy for HIV-infection but which have been linked to cardiovascular complications. The objective of this study was to analyze the effects of clinically relevant doses of abacavir and didanosine on human leukocyte–endothelium interactions and to compare them with those of other NRTIs. Design and methods:The interactions between human leukocytes – specifically peripheral blood polymorphonuclear (PMN) or mononuclear (PBMC) cells – and human umbilical vein endothelial cells were evaluated in a flow chamber system that reproduces conditions in vivo. The expression of adhesion molecules was analyzed by flow cytometry. Results:Abacavir induced a dose-dependent increase in PMN and PBMC rolling and adhesion. This was reproduced by didanosine but not by lamivudine or zidovudine. Both abacavir and didanosine increased Mac-1 expression in neutrophils and monocytes, but produced no effects on either lymphocytes or the expression of endothelial adhesion molecules. The PMN/PBMC rolling and adhesion induced by abacavir or didanosine did not occur when antibodies against Mac-1 or its ligand ICAM-1 were blocked. Conclusion:Abacavir induces significant human leukocyte accumulation through the activation of Mac-1, which in turn interacts with its endothelial ligand ICAM-1. The fact that didanosine exhibits similar effects and that lamivudine and zidovudine do not points to a relationship between the chemical structure of NRTIs and the induction of leukocyte/endothelial cell interactions. This mechanism may be especially relevant to the progression of the vascular damage associated with atherosclerosis and myocardial infarction in abacavir and didanosine-treated patients.