Angiotensin Analogs with Divergent Bias Stabilize Distinct Receptor Conformations.

"Biased" G protein-coupled receptor (GPCR) agonists preferentially activate pathways mediated by G proteins or ß-arrestins. Here, we use double electron-electron resonance spectroscopy to probe the changes that ligands induce in the conformational distribution of the angiotensin II type I receptor. Monitoring distances between 10 pairs of nitroxide labels distributed across the intracellular regions enabled mapping of four underlying sets of conformations. Ligands from different functional classes have distinct, characteristic effects on the conformational heterogeneity of the receptor. Compared to angiotensin II, the endogenous agonist, agonists with enhanced Gq coupling more strongly stabilize an "open" conformation with an accessible transducer-binding site. ß-arrestin-biased agonists deficient in Gq coupling do not stabilize this open conformation but instead favor two more occluded conformations. These data suggest a structural mechanism for biased ligand action at the angiotensin receptor that can be exploited to rationally design GPCR-targeting drugs with greater specificity of action.

The angiotensin II type 2 receptor for pain control.

All well-known deleterious effects of angiotensin (Ang) II, including vasoconstriction, inflammation, water and salt retention, and vascular remodeling, are mediated via its type 1 (AT1) receptor. This explains why AT1 receptor blockers (ARBs) and inhibitors of Ang II synthesis, such as ACE inhibitors and renin inhibitors, are beneficial for cardiovascular disease. Yet, Ang II has a second receptor, the Ang II type 2 (AT2) receptor, the function of which, even after over 20 years of research, remains largely unknown. In this issue, Marion et al. provide a new chapter to the AT2 receptor story.

Mycobacterial toxin induces analgesia in buruli ulcer by targeting the angiotensin pathways.

Mycobacterium ulcerans, the etiological agent of Buruli ulcer, causes extensive skin lesions, which despite their severity are not accompanied by pain. It was previously thought that this remarkable analgesia is ensured by direct nerve cell destruction. We demonstrate here that M. ulcerans-induced hypoesthesia is instead achieved through a specific neurological pathway triggered by the secreted mycobacterial polyketide mycolactone. We decipher this pathway at the molecular level, showing that mycolactone elicits signaling through type 2 angiotensin II receptors (AT2Rs), leading to potassium-dependent hyperpolarization of neurons. We further validate the physiological relevance of this mechanism with in vivo studies of pain sensitivity in mice infected with M. ulcerans, following the disruption of the identified pathway. Our findings shed new light on molecular mechanisms evolved by natural systems for the induction of very effective analgesia, opening up the prospect of new families of analgesics derived from such systems.

The Angiotensin AT2 Receptor: From a Binding Site to a Novel Therapeutic Target.

Discovered more than 30 years ago, the angiotensin AT2 receptor (AT2R) has evolved from a binding site with unknown function to a firmly established major effector within the protective arm of the renin-angiotensin system (RAS) and a target for new drugs in development. The AT2R represents an endogenous protective mechanism that can be manipulated in the majority of preclinical models to alleviate lung, renal, cardiovascular, metabolic, cutaneous, and neural diseases as well as cancer. This article is a comprehensive review summarizing our current knowledge of the AT2R, from its discovery to its position within the RAS and its overall functions. This is followed by an in-depth look at the characteristics of the AT2R, including its structure, intracellular signaling, homo- and heterodimerization, and expression. AT2R-selective ligands, from endogenous peptides to synthetic peptides and nonpeptide molecules that are used as research tools, are discussed. Finally, we summarize the known physiological roles of the AT2R and its abundant protective effects in multiple experimental disease models and expound on AT2R ligands that are undergoing development for clinical use. The present review highlights the controversial aspects and gaps in our knowledge of this receptor and illuminates future perspectives for AT2R research. SIGNIFICANCE STATEMENT: The angiotensin AT2 receptor (AT2R) is now regarded as a fully functional and important component of the renin-angiotensin system, with the potential of exerting protective actions in a variety of diseases. This review provides an in-depth view of the AT2R, which has progressed from being an enigma to becoming a therapeutic target.

The Angiotensin Metabolite His-Leu Is a Strong Copper Chelator Forming Highly Redox Active Species.

His-Leu is a hydrolytic byproduct of angiotensin metabolism, whose concentration in the bloodstream could be at least micromolar. This encouraged us to investigate its Cu(II) binding properties and the concomitant redox reactivity. The Cu(II) binding constants were derived from isothermal titration calorimetry and potentiometry, while identities and structures of complexes were obtained from ultraviolet-visible, circular dichroism, and room-temperature electronic paramagnetic resonance spectroscopies. Four types of Cu(II)/His-Leu complexes were detected. The histamine-like complexes prevail at low pH. At neutral and mildly alkaline pH and low Cu(II):His-Leu ratios, they are superseded by diglycine-like complexes involving the deprotonated peptide nitrogen. At His-Leu:Cu(II) ratios of ≥2, bis-complexes are formed instead. Above pH 10.5, a diglycine-like complex containing the equatorially coordinated hydroxyl group predominates at all ratios tested. Cu(II)/His-Leu complexes are also strongly redox active, as demonstrated by voltammetric studies and the ascorbate oxidation assay. Finally, numeric competition simulations with human serum albumin, glycyl-histydyl-lysine, and histidine revealed that His-Leu might be a part of the low-molecular weight Cu(II) pool in blood if its abundance is >10 µM. These results yield further questions, such as the biological relevance of ternary complexes containing His-Leu.

Long noncoding RNA TUG1 regulates smooth muscle cell differentiation via KLF4-myocardin axis.

Abdominal aortic aneurysms (AAAs) are asymptomatic vascular diseases that have life-threatening outcomes. Smooth muscle cell (SMC) dysfunction plays an important role in AAA development. The contribution of non-coding genome, specifically the role of long non-coding RNAs (lncRNAs) in SMC dysfunction, is relatively unexplored. We investigated the role of lncRNA TUG1 in SMC dysfunction. To identify potential lncRNAs relevant to SMC functionality, lncRNA profiling was performed in angiotensin-II-treated SMCs. AAA was induced by angiotensin-II treatment in mice. Transcriptional regulation of TUG1 was studied using promoter luciferase and chromatin-immuno-precipitation experiments. Gain-or-loss-of-function experiments were performed in vitro to investigate TUG1-mediated regulation of SMC function. Immunoprecipitation experiments were conducted to elucidate the mechanism underlying TUG1-mediated SMC dysfunction. TUG1 was upregulated in SMCs following angiotensin-II treatment. Similarly, TUG1 levels were elevated in abdominal aorta in a mouse model of angiotensin-II-induced AAA. Further investigations showed that angiotensin-II-induced TUG1 expression could be suppressed by inhibiting Notch-signaling pathway, both in vitro and in mouse AAA model and that TUG1 is a direct transcriptional target of the Notch pathway. In aneurysmal tissues, TUG1 expression was inversely correlated with the expression of SMC contractile genes. Overexpression of TUG1 repressed SMC differentiation in vitro, whereas siRNA/shRNA-mediated TUG1 knockdown showed an opposite effect. Mechanistically, TUG1 interacts with transcriptional repressor KLF4 and facilitates its recruitment to myocardin promoter ultimately leading to the repression of SMC differentiation. In summary, our study uncovers a novel role for the lncRNA TUG1 wherein it modulates SMC differentiation via the KLF4-myocardin axis, which may have potential implications in AAA development.NEW & NOTEWORTHY TUG1 is an angiotensin-II-induced long noncoding RNA that mediates smooth muscle cell (SMC) dysfunction through interaction with transcriptional repressor KLF4.

Lymphatic vessels and the renin-angiotensin-system.

The lymphatic system is an integral part of the circulatory system and plays an important role in the fluid homeostasis of the human body. Accumulating evidence has recently suggested the involvement of lymphatic dysfunction in the pathogenesis of cardio-reno-vascular (CRV) disease. However, how the sophisticated contractile machinery of lymphatic vessels is modulated and, possibly impaired in CRV disease, remains largely unknown. In particular, little attention has been paid to the effect of the renin-angiotensin-system (RAS) on lymphatics, despite the high concentration of RAS mediators that these tissue-draining vessels are exposed to and the established role of the RAS in the development of classic microvascular dysfunction and overt CRV disease. We herein review recent studies linking RAS to lymphatic function and/or plasticity and further highlight RAS-specific signaling pathways, previously shown to drive adverse arterial remodeling and CRV organ damage that have potential for direct modulation of the lymphatic system.

CALU-3 lung cells three-dimensionally assembled onto CellFate® matrix present angiotensin-converting enzyme-2 activity.

Currently, there is a great need for the development of three-dimensional (3D) in vitro lung models. Particularly, the production of a suitable 3D model of pulmonary epithelium for understanding the pathophysiology of diseases such as the COVID-19 must consider the tissue architecture and presence, for example, of the angiotensin-converting enzyme-2 (ACE-2) in the cells. Different polymeric membranes are being used to support cell culturing, especially of lung cells, however, there is still no information about the culture of these cells onto bacterial nanocellulose (BNC) matrices. We have used the BNC matrix CellFate® as a support for the assembly of a 3D in vitro model of lung epithelium, composed of human lung fibroblasts (HLF) and lung adenocarcinoma cells (CALU-3). CellFate® matrices were made from bacterial fermentation resulting in a natural and biocompatible biopolymer. Cells were cultured onto CellFate® and maintained in a 5% CO2 humidified atmosphere at 37°C. Cell viability was assessed by the resazurin method The samples were, then, exposed to the air-liquid interface (ALI), and histologically analyzed. ACE-2 activity was verified on the hydrolyze of the fluorogenic substrate Mca-APK(Dnp)-OH, and its presence was evaluated by flow cytometry. The expression of the anionic transporter SLCO3A1 was evaluated by qPCR. Cell viability analysis indicates that CellFate® was not toxic to these cells. By flow cytometry, the presence of the ACE-2 was identified in the CALU-3 cells surface corroborating the results obtained from enzymatic activity analysis. The SLCO3A1 transporter expression was identified in cells cultured onto CellFate®, but not in cells cultured onto the transwell (control). CALU-3 cells cultivated onto CellFate® resulted in a pseudostratified organization, a typical morphology of the human respiratory tract epithelium. The current model opens perspectives for studies involving physiological characterization, improving its relevance for the understanding of the pathophysiology of diseases as well as the response to drugs.

The alternative renin-angiotensin-system (RAS) signalling pathway in prostate cancer and its link to the current COVID-19 pandemic.

BACKGROUND: The renin-angiotensin system is known to maintain blood pressure and body fluids. However, it has been found to consist of at least two major constituents, the classic and the alternative pathway, balancing and supporting each other's signalling in a very intricate way. Current research has shown that the renin-angiotensin system is involved in a broad range of biological processes and diseases, such as cancer and infectious diseases. METHODS AND RESULTS: We conducted a literature review on the interaction of the renin-angiotensin system and prostate cancer and explored the research on the possible impact of the SARS-CoV-2 virus in this context. This review provides an update on contemporary knowledge into the alternative renin-angiotensin system, its role in cancer, specifically prostate cancer, and the implications of the current COVID-19 pandemic on cancer and cancer care. CONCLUSION: In this work, we aim to demonstrate how shifting the RAS signalling pathway from the classic to the alternative axis seems to be a viable option in supporting treatment of specific cancers and at the same time demonstrating beneficial properties in supportive care. It however seems to be the case that the infection with SARS-CoV-2 and subsequent impairment of the renin-angiotensin-system could exhibit serious deleterious long-term effects even in oncology.

Interactions of angiotensin-converting enzyme-2 (ACE2) and SARS-CoV-2 spike receptor-binding domain (RBD): a structural perspective.

BACKGROUND: Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has caused millions of infections and deaths worldwide since its discovery in late 2019 in Wuhan, China. The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein binds to the human angiotensin-converting enzyme-2 (ACE2) receptor, a critical component of the renin-angiotensin system (RAS) that initiates the viral transmission. Most of the critical mutations found in SARS-CoV-2 are associated with the RBD of the spike protein. These mutations have the potential to reduce the efficacy of vaccines and neutralizing antibodies. METHODS: In this review, the structural details of ACE2, RBD and their interactions are discussed. In addition, some critical mutations of RBD and their impact on ACE2-RBD interactions are also discussed. CONCLUSION: Preventing the interaction between Spike RBD and ACE2 is considered a viable therapeutic strategy since ACE2 binding by RBD is the first step in virus infection. Because the interactions between the two entities are critical for both viral transmission and therapeutic development, it is essential to understand their interactions in detail.

Differential behavior of Lactobacillus helveticus B1929 and ATCC 15009 on the hydrolysis and angiotensin-I-converting enzyme inhibition activity of fermented ultra-high-temperature milk and nonfat dried milk powder.

Consumers' growing interest in fermented dairy foods necessitates research on a wide array of lactic acid bacterial strains to be explored and used. This study aimed to investigate the differences in the proteolytic capacity of Lactobacillus helveticus strains B1929 and ATCC 15009 on the fermentation of commercial ultra-pasteurized (UHT) skim milk and reconstituted nonfat dried milk powder (at a comparable protein concentration, 4%). The antihypertensive properties of the fermented milk, measured by angiotensin-I-converting enzyme inhibitory (ACE-I) activity, were compared. The B1929 strain lowered the pH of the milk to 4.13 ± 0.09 at 37°C after 24 h, whereas ATCC 15009 needed 48 h to drop the pH to 4.70 ± 0.18 at 37°C. Two soluble protein fractions, one (CFS1) obtained after fermentation (acidic conditions) and the other (CFS2) after the neutralization (pH 6.70) of the pellet from CFS1 separation, were analyzed for d-/l-lactic acid production, protein concentration, the degree of protein hydrolysis, and ACE-I activity. The CFS1 fractions, dominated by whey proteins, demonstrated a greater degree of protein hydrolysis (7.9%) than CFS2. On the other hand, CFS2, mainly casein proteins, showed a higher level of ACE-I activity (33.8%) than CFS1. Significant differences were also found in the d- and l-lactic acid produced by the UHT milk between the 2 strains. These results attest that milk casein proteins possessed more detectable ACE-I activity than whey fractions, even without a measurable degree of hydrolysis. Findings from this study suggest that careful consideration must be given when selecting the bacterial strain and milk substrate for fermentation.

Microglial angiotensin type 2 receptors mediate sex-specific expression of inflammatory cytokines independently of circulating estrogen.

There are sex differences in microglia, which can maintain sex-related gene expression and functional differences in the absence of circulating sex steroids. The angiotensin type 2 (AT2) receptors mediate anti-inflammatory actions in different tissues, including brain. In mice, we performed RT-PCR analysis of microglia isolated from adult brains and RNA scope in situ hybridization from males, females, ovariectomized females, orchiectomized males and brain masculinized females. We also compared wild type and AT2 knockout mice. The expression of AT2 receptors in microglial cells showed sex differences with much higher AT2 mRNA expression in females than in males, and this was not dependent on circulating gonadal hormones, as observed using ovariectomized females, brain masculinized females and orchiectomized males. These results suggest genomic reasons, possibly related to sex chromosome complement, for sex differences in AT2 expression in microglia, as the AT2 receptor gene is located in the X chromosome. Furthermore, sex differences in expression of AT2 receptors were associated to sex differences in microglial expression of key anti-inflammatory cytokines such as interleukin-10 and pro-inflammatory cytokines such as interleukin-1ß and interleukin-6. In conclusion, sex differences in microglial AT2 receptor expression appear as a major factor contributing to sex differences in the neuroinflammatory responses beyond the effects of circulating steroids.

Protecting glomerulus: role of angiotensin-II type 2 receptor.

Podocyte injury due to either drug, toxin, infection, or metabolic abnormality is a great concern as it increases the risk of developing focal segmental glomerulosclerosis (FSGS) and proteinuric kidney diseases. The direct podocyte injury due to doxorubicin is associated with an increase in proinflammatory cytokines and induction of cathepsin L. The increased activity of cathepsin L in turn may degrade the glomerular slit diaphragm resulting in proteinuric kidney injury. The angiotensin-II type 2 receptor (AT2R) has earlier been reported to be associated with the preservation of slit diaphragm proteins and prevention of proteinuria. Recent in vivo findings by Zhang and colleagues further support the anti-proteinuric role of AT2R in preventing podocyte injury via down-regulating cytokines ccl2, and hence, cathepsin L, thereby, limiting the progression of FSGS.

The NO-cGMP-K+ Channel Pathway Participates in Diuretic and Cardioprotective Effects of Blutaparon portulacoides in Spontaneously Hypertensive Rats.

Blutaparon portulacoides is a Brazilian plant species that is widely used in folk medicine. The present study investigated the role of an aqueous extract of B. portulacoides against hypertension in spontaneously hypertensive rats. The aqueous extract of B. portulacoides was obtained from the whole plant. Its chemical profile was analyzed by ultraperformance liquid chromatography-tandem mass spectrometry. The acute toxicity of the aqueous extract of B. portulacoides was evaluated in female Wistar rats. Male 6-month-old spontaneously hypertensive rats then received the aqueous extract of B. portulacoides (30, 100, and 300 mg/kg), hydrochlorothiazide (25 mg/kg), or vehicle once daily for 28 days. On days 1, 14, and 28, the diuretic effects of the aqueous extract of B. portulacoides were evaluated. The role of prostaglandins and the nitric oxide-cyclic guanosine monophosphate-potassium channel pathway in the diuretic activity of the aqueous extract of B. portulacoides was also investigated. At the end of the treatment, hepatic and renal biochemical markers, serum nitrotyrosine, malondialdehyde, nitrite, and aldosterone levels, and angiotensin-converting enzyme activity were measured. The electrocardiographic profile, blood pressure, and renal vascular reactivity were also assessed. The heart, kidneys, and liver were collected to determine relative organ weight, histopathology, and cardiac morphometry. Caffeic acid, ferulic acid, and several flavonoids were identified in the aqueous extract of B. portulacoides. No signs of toxicity were observed. Prolonged treatment with the aqueous extract of B. portulacoides (300 mg/kg) induced significant diuretic activity by activating the nitric oxide-cyclic guanosine monophosphate-potassium channel pathway. These effects reduced blood pressure and oxidative stress and prevented renal vascular dysfunction and left ventricular hypertrophy that was induced by hypertension. Overall, the present data suggest that the aqueous extract of B. portulacoides has important diuretic and cardioprotective effects by activation of the nitric oxide-cyclic guanosine monophosphate-potassium channel pathway.

Phospholipid membranes drive abdominal aortic aneurysm development through stimulating coagulation factor activity.

Abdominal aortic aneurysm (AAA) is an inflammatory vascular disease with high mortality and limited treatment options. How blood lipids regulate AAA development is unknown. Here lipidomics and genetic models demonstrate a central role for procoagulant enzymatically oxidized phospholipids (eoxPL) in regulating AAA. Specifically, through activating coagulation, eoxPL either promoted or inhibited AAA depending on tissue localization. Ang II administration to ApoE-/- mice increased intravascular coagulation during AAA development. Lipidomics revealed large numbers of eoxPL formed within mouse and human AAA lesions. Deletion of eoxPL-generating enzymes (Alox12 or Alox15) or administration of the factor Xa inhibitor rivaroxaban significantly reduced AAA. Alox-deficient mice displayed constitutively dysregulated hemostasis, including a consumptive coagulopathy, characterized by compensatory increase in prothrombotic aminophospholipids (aPL) in circulating cell membranes. Intravenously administered procoagulant PL caused clotting factor activation and depletion, induced a bleeding defect, and significantly reduced AAA development. These data suggest that Alox deletion reduces AAA through diverting coagulation away from the vessel wall due to eoxPL deficiency, instead activating clotting factor consumption and depletion in the circulation. In mouse whole blood, ∼44 eoxPL molecular species formed within minutes of clot initiation. These were significantly elevated with ApoE-/- deletion, and many were absent in Alox-/- mice, identifying specific eoxPL that modulate AAA. Correlation networks demonstrated eoxPL belonged to subfamilies defined by oxylipin composition. Thus, procoagulant PL regulate AAA development through complex interactions with clotting factors. Modulation of the delicate balance between bleeding and thrombosis within either the vessel wall or circulation was revealed that can either drive or prevent disease development.

Antibodies to the N-Terminal Domain of Angiotensin-Converting Enzyme (ACE2) That Block Its Interaction with SARS-CoV-2 S Protein.

SARS-CoV-2 is a new coronavirus that is the cause of COVID-19 pandemic. To enter the cell, the virus interacts via its surface S protein with angiotensin-converting enzyme 2 (ACE2), the main entry receptor on the cell membrane. Most of protective antibodies, including those induced by vaccinations, target the S protein, preventing its interaction with the ACE2 receptor. We have evaluated an alternative strategy for blocking the S-ACE2 interaction using new antipeptide antibodies to the N-terminus of the ACE2 molecule. These antibodies allow detection of human ACE2 in vitro and ex vivo.

Dipeptidyl peptidase 3 modulates the renin-angiotensin system in mice.

Dipeptidyl peptidase 3 (DPP3) is a zinc-dependent hydrolase involved in degrading oligopeptides with 4-12 amino acid residues. It has been associated with several pathophysiological processes, including blood pressure regulation, pain signaling, and cancer cell defense against oxidative stress. However, the physiological substrates and the cellular pathways that are potentially targeted by DPP3 to mediate these effects remain unknown. Here, we show that global DPP3 deficiency in mice (DPP3-/-) affects the renin-angiotensin system (RAS). LC-MS-based profiling of circulating angiotensin peptides revealed elevated levels of angiotensin II, III, IV, and 1-5 in DPP3-/- mice, whereas blood pressure, renin activity, and aldosterone levels remained unchanged. Activity assays using the purified enzyme confirmed that angiotensin peptides are substrates for DPP3. Aberrant angiotensin signaling was associated with substantially higher water intake and increased renal reactive oxygen species formation in the kidneys of DPP3-/- mice. The metabolic changes and altered angiotensin levels observed in male DPP3-/- mice were either absent or attenuated in female DPP3-/- mice, indicating sex-specific differences. Taken together, our observations suggest that DPP3 regulates the RAS pathway and water homeostasis by degrading circulating angiotensin peptides.

Polyphenolic Composition and in Vitro Antihypertensive and Anti-Inflammatory Effects of Cuphea lindmaniana and Cuphea urbaniana.

The present study investigates the chemical composition, anti-inflammatory, and antihypertensive activities, in vitro, from extracts of Cuphea lindmaniana and Cuphea urbaniana leaves. The extraction was performed ultrasound-assisted, and UHPLC/MS analysis was in positive mode ionization. The anti-inflammatory activity of the extracts and miquelianin were assayed at concentrations 0.001-10 µg/mL by chemotaxis on rat polymorphonuclear neutrophils. The antihypertensive activity was performed by angiotensin-converting enzyme (ACE) inhibition. From the nineteen proposed compounds, six of them are described for the first time in this genus. The extracts displayed antichemotactic effect with a reduction of 100 % of the neutrophil migration, in vitro, in most concentrations. The ACE-inhibition presented results ranging from 19.58 to 22.82 %. In conclusion, C. lindmaniana and C. urbaniana extracts contain a rich diversity of flavonoids and display in vitro anti-inflammatory and antihypertensive potential. Thus, this study could serve as a scientific baseline for further investigation, on developmental novel products with therapeutic actions.

Dysregulation of the Renin-Angiotensin-Aldosterone System (RAA) in Patients Infected with SARS-CoV-2-Possible Clinical Consequences.

SARS-CoV-2 impairs the renin-angiotensin-aledosterone system via binding ACE2 enzyme. ACE2 plays a key role in the biosynthesis of angiotensin (1-7), catalyzing the conversion of angiotensin 2 into angiotensin (1-7) and the reaction of angiotensin synthesis (1-9), from which angiotensin is (1-7) produced under the influence of ACE (Angiotensin-Converting Enzyme). Angiotensin 2 is a potent vasoconstrictor and atherogenic molecule converted by ACE2 to reducing inflammation and vasodilating in action angiotensin (1-7). Angiotensin (1-9), that is a product of angiotensin 1 metabolism and precursor of angiotensin (1-7), also exerts cell protective properties. Balance between angiotensin 2 and angiotensin (1-7) regulates blood pressure and ACE2 plays a critical role in this balance. ACE2, unlike ACE, is not inhibited by ACE inhibitors at the doses used in humans during the treatment of arterial hypertension. Membrane ACE2 is one of the receptors that allows SARS-CoV-2 to enter the host cells. ACE2 after SARS-CoV-2 binding is internalized and degraded. Hence ACE2 activity on the cell surface is reduced leading to increase the concentration of angiotensin 2 and decrease the concentration of angiotensin (1-7). Disturbed angiotensins metabolism, changes in ratio between angiotensins with distinct biological activities leading to domination of atherogenic angiotensin 2 can increase the damage to the lungs.

Significance of neutrophil microparticles in ischaemia-reperfusion: Pro-inflammatory effectors of endothelial senescence and vascular dysfunction.

Endothelial senescence is an emerging cause of vascular dysfunction. Because microparticles are effectors of endothelial inflammation and vascular injury after ischaemia-reperfusion, we examined leucocyte-derived microparticles of spleen origin as possible contributors. Microparticles were generated from primary rat splenocytes by either lipopolysaccharide or phorbol-myristate-acetate/calcium ionophore, under conditions mimicking innate and adaptive immune responses. Incubation of primary porcine coronary endothelial cells with either type of microparticles, but not with those from unstimulated splenocytes, leads to a similar threefold raise in senescence-associated ß-galactosidase activity within 48 hours, indicating accelerated senescence, to endothelial oxidative stress, and a fivefold and threefold increase in p21 and p16 senescence markers after 24 hours. After 12-hour incubation, the endothelial-dependent relaxation of coronary artery rings was reduced by 50%, at distinct optimal microparticle concentration. In vitro, microparticles were pro-thrombotic by up-regulating the local angiotensin system, by prompting tissue factor activity and a secondary generation of pro-coagulant endothelial microparticles. They initiated an early pro-inflammatory response by inducing phosphorylation of NF-κB, MAP kinases and Akt after 1 hour, and up-regulated VCAM-1 and ICAM-1 at 24 hours. Accordingly, VCAM-1 and COX-2 were also up-regulated in the coronary artery endothelium and eNOS down-regulated. Lipopolysaccharide specifically favoured the shedding of neutrophil- and monocyte-derived microparticles. A 80% immuno-depletion of neutrophil microparticles reduced endothelial senescence by 55%, indicating a key role. Altogether, data suggest that microparticles from activated splenocytes prompt early pro-inflammatory, pro-coagulant and pro-senescent responses in endothelial cells through redox-sensitive pathways. The control of neutrophil shedding could preserve the endothelium at site of ischaemia-reperfusion-driven inflammation and delay its dysfunction.