Veronica Azcutia

Emerging mechanisms of neutrophil recruitment across endothelium.

Neutrophils are the all-terrain vehicle of the innate immune system because of their ability to gain entry into tissues and organs, and thus, play an essential role in host defense. Exactly how this marvel of nature works is still incompletely understood. In the past 2-3 years, new players and processes have been identified in the endothelial-leukocyte adhesion cascade. Novel signaling pathways have been discovered in both the endothelium and the neutrophils that regulate various steps in the recruitment process. This review focuses on these emerging pathways and the mechanisms that regulate neutrophil recruitment across endothelium.

IL-17 and TNF-α Sustain Neutrophil Recruitment during Inflammation through Synergistic Effects on Endothelial Activation

IL-17A (IL-17) is the signature cytokine produced by Th17 cells and has been implicated in host defense against infection and the pathophysiology of autoimmunity and cardiovascular disease. Little is known, however, about the influence of IL-17 on endothelial activation and leukocyte influx to sites of inflammation. We hypothesized that IL-17 would induce a distinct pattern of endothelial activation and leukocyte recruitment when compared with the Th1 cytokine IFN-γ. We found that IL-17 alone had minimal activating effects on cultured endothelium, whereas the combination of TNF-α and IL-17 produced a synergistic increase in the expression of both P-selectin and E-selectin. Using intravital microscopy of the mouse cremaster muscle, we found that TNF-α and IL-17 also led to a synergistic increase in E-selectin–dependent leukocyte rolling on microvascular endothelium in vivo. In addition, TNF-α and IL-17 enhanced endothelial expression of the neutrophilic chemokines CXCL1, CXCL2, and CXCL5 and led to a functional increase in leukocyte transmigration in vivo and CXCR2-dependent neutrophil but not T cell transmigration in a parallel-plate flow chamber system. By contrast, endothelial activation with TNF-α and IFN-γ preferentially induced the expression of the integrin ligands ICAM-1 and VCAM-1, as well as the T cell chemokines CXCL9, CXCL10, and CCL5. These effects were further associated with a functional increase in T cell but not neutrophil transmigration under laminar shear flow. Overall, these data show that IL-17 and TNF-α act in a synergistic manner to induce a distinct pattern of endothelial activation that sustains and enhances neutrophil influx to sites of inflammation.

Endothelial dysfunction through genetic deletion or inhibition of the G protein-coupled receptor Mas: a new target to improve endothelial function.

Background Endothelial dysfunction is an initial step in the pathogenesis of cardiovascular diseases. Since we previously identified the G protein-coupled receptor Mas as a receptor for angiotensin (Ang)-(1–7), a heptapeptide with endothelium-dependent vasorelaxant properties, we investigated whether alterations on the Ang-(1–7)/Mas axis alter endothelial function. Results Ang-(1–7)-mediated relaxation of murine wild-type mesenteric arteries was equally impaired in both wild-type arteries pretreated with the Ang-(1–7) receptor blocker, A779, and arteries isolated from Mas-deficient mice. Importantly, the response to the endothelium-dependent vasorelaxant, bradykinin (BK), and acetylcholine (ACh) effects were comparably inhibited, while endothelium-independent vessel relaxation by sodium nitroprusside was unaltered in these vessels. Hypothesizing endothelial dysfunction, we proved the in-vivo relevance of the ex-vivo findings investigating mesenteric properties after 1 week of minipump infusion of A779 in wild-type mice. Both BK- and ACh-induced relaxation were significantly impaired in wild-type vessels of pretreated animals. A779-induced impairment of endothelial function was confirmed in vitro, since BK-mediated nitric oxide (NO) release was increased by Ang-(1–7) and blunted by A779 pretreatment in primary human endothelial cell cultures. Conclusions Our data highlight a pivotal role for the receptor Mas in preserving normal vascular relaxation. Consequently, Mas agonists arise as a promising tool in the treatment of cardiovascular diseases characterized by endothelial dysfunction.

Statins suppress apolipoprotein CIII-induced vascular endothelial cell activation and monocyte adhesion

Aims Activation of vascular endothelial cells (ECs) contributes importantly to inflammation and atherogenesis. We previously reported that apolipoprotein CIII (apoCIII), found abundantly on circulating triglyceride-rich lipoproteins, enhances adhesion of human monocytes to ECs in vitro. Statins may exert lipid-independent anti-inflammatory effects. The present study examined whether statins suppress apoCIII-induced EC activation in vitro and in vivo. Methods and results Physiologically relevant concentrations of purified human apoCIII enhanced attachment of the monocyte-like cell line THP-1 to human saphenous vein ECs (HSVECs) or human coronary artery ECs (HCAECs) under both static and laminar shear stress conditions. This process mainly depends on vascular cell adhesion molecule-1 (VCAM-1), as a blocking VCAM-1 antibody abolished apoCIII-induced monocyte adhesion. ApoCIII significantly increased VCAM-1 expression in HSVECs and HCAECs. Pre-treatment with statins suppressed apoCIII-induced VCAM-1 expression and monocyte adhesion, with two lipophilic statins (pitavastatin and atorvastatin) exhibiting inhibitory effects at lower concentration than those of hydrophilic pravastatin. Nuclear factor κB (NF-κB) mediated apoCIII-induced VCAM-1 expression, as demonstrated via loss-of-function experiments, and pitavastatin treatment suppressed NF-κB activation. Furthermore, in the aorta of hypercholesterolaemic Ldlr−/− mice, pitavastatin administration in vivo suppressed VCAM-1 mRNA and protein, induced by apoCIII bolus injection. Similarly, in a subcutaneous dorsal air pouch mouse model of leucocyte recruitment, apoCIII injection induced F4/80+ monocyte and macrophage accumulation, whereas pitavastatin administration reduced this effect. Conclusions These findings further establish the direct role of apoCIII in atherogenesis and suggest that anti-inflammatory effects of statins could improve vascular disease in the population with elevated plasma apoCIII.

Inflammation Determines the Pro-Adhesive Properties of High Extracellular D-Glucose in Human Endothelial Cells In Vitro and Rat Microvessels In Vivo

Background Hyperglycemia is acknowledged as an independent risk factor for developing diabetes-associated atherosclerosis. At present, most therapeutic approaches are targeted at a tight glycemic control in diabetic patients, although this fails to prevent macrovascular complications of the disease. Indeed, it remains highly controversial whether or not the mere elevation of extracellular D-glucose can directly promote vascular inflammation, which favors early pro-atherosclerotic events. Methods and Findings In the present work, increasing extracellular D-glucose from 5.5 to 22 mmol/L was neither sufficient to induce intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expression, analyzed by flow cytometry, nor to promote leukocyte adhesion to human umbilical vein endothelial cells (HUVEC) in vitro, measured by flow chamber assays. Interestingly, the elevation of D-glucose levels potentiated ICAM-1 and VCAM-1 expression and leukocyte adhesion induced by a pro-inflammatory stimulus, such as interleukin (IL)-1β (5 ng/mL). In HUVEC, high D-glucose augmented the activation of extracellular signal-regulated kinase 1/2 (ERK 1/2) and nuclear transcription factor-κB (NF-κB) elicited by IL-1β, measured by Western blot and electromobility shift assay (EMSA), respectively, but had no effect by itself. Both ERK 1/2 and NF-κB were necessary for VCAM-1 expression, but not for ICAM-1 expression. In vivo, leukocyte trafficking was evaluated in the rat mesenteric microcirculation by intravital microscopy. In accordance with the in vitro data, the acute intraperitoneal injection of D-glucose increased leukocyte rolling flux, adhesion and migration, but only when IL-1β was co-administered. Conclusions These results indicate that the elevation of extracellular D-glucose levels is not sufficient to promote vascular inflammation, and they highlight the pivotal role of a pro-inflammatory environment in diabetes, as a critical factor conditioning the early pro-atherosclerotic actions of hyperglycemia.

Evidence for sodium azide as an artifact mediating the modulation of inducible nitric oxide synthase by C-reactive protein.

C-reactive protein (CRP) is an acute-phase protein identified as a cardiovascular risk marker. In recent years, an increasing number of studies have investigated the possible direct effects of CRP on the vasculature, using mainly commercial CRP. In the present work, a potential role for CRP as a modulator of inducible nitric oxide synthase (iNOS) induction was explored. Cultured human aortic vascular smooth muscle cells (HASMC) were stimulated for 18 hours with 10 ng/mL interleukin-1β (IL-1β), resulting in a marked increase of iNOS levels and NO production, as determined by Western blotting and nitrite measurement, respectively. Commercial CRP (1 to 100 μg/mL) concentration-dependently inhibited the effects elicited by IL-1β. Unexpectedly, similar results were observed when the commercial CRP solution was replaced by the corresponding vehicle medium containing growing concentrations of sodium azide. The inhibitory effects of commercial CRP or vehicle medium were lost on sodium azide removal by dialysis. In conclusion, sodium azide from the commercial CRP solution, but not CRP itself, mainly accounts for the inhibitory effect on IL-1β-evoked iNOS induction and NO release. Care should be taken before attributing any biologic role to commercial CRP containing sodium azide.

Amadori adducts activate nuclear factor‐κB‐related proinflammatory genes in cultured human peritoneal mesothelial cells

Diabetes mellitus leads to a high incidence of several so‐called complications, sharing similar pathophysiological features in several territories. Previous reports points at early nonenzymatic glycosylation products (Amadori adducts) as mediators of diabetic vascular complications. In the present study, we analysed a possible role for Amadori adducts as stimulators of proinflammatory pathways in human peritoneal mesothelial cells (HPMCs). Cultured HPMCs isolated from 13 different patients (mean age 38.7±16 years) were exposed to different Amadori adducts, that is, highly glycated haemoglobin (10 nM) and glycated bovine serum albumin (0.25 mg ml−1), as well as to their respective low glycosylation controls. Amadori adducts, but not their respective controls, elicited a marked increase of NF‐κB activation, as determined by electromobility shift assays and transient transfection experiments. Additionally, Amadori adducts significantly increased the production of NF‐κB‐related proinflammatory molecules, including cytokines, such as TNF‐α, IL‐1β or IL‐6, and enzymes, such as cyclooxygenase‐2 and inducible nitric oxide (NO) synthase, this latter leading to the release of NO by HPMCs. The effects of Amadori adducts were mediated by different reactive oxygen and nitrosative species (e.g. superoxide anions, hydroxyl radicals, and peroxynitrite), as they were blunted by coincubation with the appropriate scavengers. Furthermore, NO generated upon exposure to Amadori adducts further stimulated NF‐κB activation, either directly or after combination with superoxide anions to form peroxynitrite. We conclude that Amadori adducts can favour peritoneal inflammation by exacerbating changes in NO synthesis pathway and triggering NF‐κB‐related proinflammatory signals in human mesothelial cells.

Glycosylated human oxyhaemoglobin activates nuclear factor‐κB and activator protein‐1 in cultured human aortic smooth muscle

Diabetic vessels undergo structural changes that are linked to a high incidence of cardiovascular diseases. Reactive oxygen species (ROS) mediate cell signalling in the vasculature, where they can promote cell growth and activate redox‐regulated transcription factors, like activator protein‐1 (AP‐1) or nuclear factor‐κB (NF‐κB), which are involved in remodelling and inflammation processes. Amadori adducts, formed through nonenzymatic glycosylation, can contribute to ROS formation in diabetes. In this study, we analysed whether Amadori‐modified human oxyhaemoglobin, glycosylated at either normal (N‐Hb) or elevated (E‐Hb) levels, can induce cell growth and activate AP‐1 and NF‐κB in cultured human aortic smooth muscle cells (HASMC). E‐Hb (1 nM–1 μM), but not N‐Hb, promoted a concentration‐dependent increase in cell size from nanomolar concentrations, although it failed to stimulate HASMC proliferation. At 10 nM, E‐Hb stimulated both AP‐1 and NF‐κB activity, as assessed by transient transfection, electromobility shift assays or immunofluorescence staining. The effects of E‐Hb resembled those of the proinflammatory cytokine tumour necrosis factor‐α (TNF‐α). E‐Hb enhanced intracellular superoxide anions content and its effects on HASMC were abolished by different ROS scavengers. In conclusion, E‐Hb stimulates growth and activates AP‐1 and NF‐κB in human vascular smooth muscle by redox‐sensitive pathways, thus suggesting a possible direct role for Amadori adducts in diabetic vasculopathy.

CD47 plays a critical role in T-cell recruitment by regulation of LFA-1 and VLA-4 integrin adhesive functions

CD47 plays an important but incompletely understood role in innate and adaptive immune responses. CD47 associates in cis with T-cell LFA-1 integrins and regulates expression of high-affinity conformations of both LFA-1 and VLA-4.

Endothelial CD47 Promotes Vascular Endothelial-Cadherin Tyrosine Phosphorylation and Participates in T Cell Recruitment at Sites of Inflammation In Vivo

At sites of inflammation, endothelial adhesion molecules bind leukocytes and transmit signals required for transendothelial migration (TEM). We previously reported that adhesive interactions between endothelial cell CD47 and leukocyte signal regulatory protein γ (SIRPγ) regulate human T cell TEM. The role of endothelial CD47 in T cell TEM in vivo, however, has not been explored. In this study, CD47−/− mice showed reduced recruitment of blood T cells as well as neutrophils and monocytes in a dermal air pouch model of TNF-α–induced inflammation. Reconstitution of CD47−/− mice with wild-type bone marrow cells did not restore leukocyte recruitment to the air pouch, indicating a role for endothelial CD47. The defect in leukocyte TEM in the CD47−/− endothelium was corroborated by intravital microscopy of inflamed cremaster muscle microcirculation in bone marrow chimera mice. In an in vitro human system, CD47 on both HUVEC and T cells was required for TEM. Although previous studies showed CD47-dependent signaling required Gαi-coupled pathways, this was not the case for endothelial CD47 because pertussis toxin, which inactivates Gαi, had no inhibitory effect, whereas Gαi was required by the T cell for TEM. We next investigated the endothelial CD47-dependent signaling events that accompany leukocyte TEM. Ab-induced cross-linking of CD47 revealed robust actin cytoskeleton reorganization and Src- and Pyk-2–kinase dependent tyrosine phosphorylation of the vascular endothelial-cadherin cytoplasmic tail. This signaling was pertussis toxin insensitive, suggesting that endothelial CD47 signaling is independent of Gαi. These findings suggest that engagement of endothelial CD47 by its ligands triggers outside-in signals in endothelium that facilitate leukocyte TEM.