Neutrophil microvesicles drive atherosclerosis by delivering <i>miR-155</i> to atheroprone endothelium

Neutrophils are implicated in the pathogenesis of atherosclerosis but are seldom detected in atherosclerotic plaques. We investigated whether neutrophil-derived microvesicles may influence arterial pathophysiology. Here we report that levels of circulating neutrophil microvesicles are enhanced by exposure to a high fat diet, a known risk factor for atherosclerosis. Neutrophil microvesicles accumulate at disease-prone regions of arteries exposed to disturbed flow patterns, and promote vascular inflammation and atherosclerosis in a murine model. Using cultured endothelial cells exposed to disturbed flow, we demonstrate that neutrophil microvesicles promote inflammatory gene expression by delivering miR-155, enhancing NF-κB activation. Similarly, neutrophil microvesicles increase miR-155 and enhance NF-κB at disease-prone sites of disturbed flow in vivo. Enhancement of atherosclerotic plaque formation and increase in macrophage content by neutrophil microvesicles is dependent on miR-155. We conclude that neutrophils contribute to vascular inflammation and atherogenesis through delivery of microvesicles carrying miR-155 to disease-prone regions

The prorenin receptor in the adrenal gland: expression, localization, signalling pathway and potential role in primary aldosteronism

Primary aldosteronism (PA) comprises about 11% patients referred to specialized centers for Arterial Hypertension. A considerable part of them carry a bilateral adrenocortical hyperplasia, also known as, idiopathic hypertension, which require life-long medical treatment. Notwithstanding this high prevalence rate the mechanisms leading to PA are unknown. However, anecdotal reports suggest that prorenin levels could be increased in PA, although the low or undetectable levels of active plasma renin, plasma prorenin levels could be increased. This suggests a possible role of prorenin in the pathophysiology of PA. The discovery of the prorenin receptor (PRR) in 2002, show a new way forward to the pathophysiology of the renin-angiotensin system: PRR can bind renin and prorenin inducing respectively an increase in renin catalytic activity and a non-proteolytically activation of prorenin. Till that discovery, prorenin was only seen as the inactive precursor of renin. Preprorenin is converted to prorenin in the juxtaglomerular cells of the kidney. The cleavage of the amino terminal 43-amino acid prosegment allows exposure of the active site in the secretory granules where generate renin. However the majority (75%) of prorenin is secreted constitutively; therefore The prorenin levels of human blood plasma are approximately 10-fold higher than those of renin. Further, they are increased in several conditions, including diabetes mellitus, where they could play a pathophysiologic role. PRR binding triggers activation of the mitogen activated protein (MAP) kinase–extracellular signal regulated kinase (ERK)1/2 and p38 signalling pathway independent from a possible generation of angiotensin II (Ang II). Activation of these signalling pathways, that are associated with cell proliferation and cell death has led to hypothesize that renin and prorenin could lead, via PRR, to hypertrophy and hyperplasia and ultimately to organ damage and cardiovascular events. In this study we first evaluated the gene and protein expression of PRR in adrenal gland and founded high expression of the PRR mRNA in APA, in human normal adrenal cortex and two human adrenocortical carcinoma cell lines: H295R and HAC15. Then, we sought for the expression of the PRR at the protein level with immunohystochemistry experiments on rat and human normal adrenal gland and with immunocytochemistry experiments. These experiments revealed a strong immunostaining for PRR in the adrenal gland at medulla and sub capsular level, and in aldosterone-secreting cells from the normal human ZG obtained by immunoseparation. In order to localize PRR we carried out immunoblot experiments on HAC15 and H295R cytosolic and membrane fractions and confocal microscopy experiments. These results showed that, PRR is mainly, but not exclusively, localized at the membrane level where partially colocalizes with the adhesion molecule CD56. We then investigated the subcellular localization of the PRR by immuno-gold electron microscopy experiments, and found that PRR is localized also in the nucleus, mitochondria and Golgi’s vesicles. We used H295R and HAC15 cell lines to investigate the functional relevance of this receptor. Stimulation with angiotensin II [100 nM], renin [50 nM] and prorenin [50 nM] induced ERK 1/2 phosphorylation. The renin and angiotensin II induced phosphorylation was abolished in presence of the angiotensin II (AT1) receptor antagonist irbesartan [5 μM] in both cell lines, whereas the phosphorylation induced by prorenin was abolished only in the H295R cells. These results suggest a role of PRR in proliferation, differentiation, and apoptosis of adrenal cortex and, therefore, a functional role of PRR in the pathophysiology of PA.L’iperaldosteronismo primario (PA) colpisce l’11.2% dei pazienti ipertesi inviati ai centri dell’Ipertensione. Una quota considerevole di essi presenta un’iperplasia bilaterale del surrene, nota anche come iperaldosteronismo idiopatico, che richiede una terapia medica per tutta la vita. Nonostante l’alta prevalenza di questa patologia i meccanismi che ne stanno alla base sono tuttora sconosciuti. Tuttavia dati aneddotici suggeriscono che nel PA, a differenza dei livelli plasmatici di renina che sono ridotti o indosabili, i livelli di prorenina potrebbero essere aumentati. Ciò suggerisce la possibilità che la prorenina giochi un ruolo nella fisiopatologia del PA. Nel 2002 la scoperta del recettore della prorenina (PRR) ha aperto una nuova finestra sulla fisiopatologia del sistema renina-angiotensina: il PRR può legare sia la renina che la prorenina inducendo, rispettivamente, un aumento nell’attività catalitica della renina e un’attivazione non proteolitica della prorenina. Fino ad allora la prorenina era considerata solo il precursore inattivo della renina. La preprorenina viene convertita a prorenina nelle cellule juxtaglomerulari renali. Il clivaggio del prosegmento amino-terminale della prorenina permette l’esposizione del sito attivo nei granuli secretori generando renina. Tuttavia, la maggior parte (75%) della prorenina viene secreta costitutivamente; pertanto i livelli plasmatici di prorenina nell’uomo sono 10 volte maggiori di quelli della renina. Essi sono inoltre aumentati in diverse condizioni, tra cui il diabete mellito, dove potrebbero svolgere un ruolo fisiopatologico. Il legame di renina e prorenina al PRR attiva vie di signalling quali MAP (mitogen activated protein) chinasi-ERK 1/2 (extracellular signal regulated kinase) e p38 indipendentemente dalla generazione di angiotensina II (Ang II). Queste vie sono implicate nella proliferazione e apoptosi, il che ha fatto ipotizzare che renina e prorenina possano indurre ipertrofia e iperplasia e quindi danno d’organo ed eventi cardiovascolari via PRR. In questa ricerca abbiamo valutato l’espressione genica e proteica del PRR nel surrene e scoperto elevati livelli di espressione di mRNA del PRR negli APA, nella corteccia surrenalica umana normale e in due linee cellulari di carcinoma corticosurrenalico umano: H295R e HAC15. Abbiamo quindi investigato la presenza e localizzazione del PRR a livello proteico mediante immunoistochimica su surrene di ratto e umano e immunocitochimica. Ciò ha evidenziato un marcato immunostaining del PRR nel surrene, a livello midollare e sub capsulare, e in cellule di ZG umana secernenti aldosterone ottenute mediante immunoseparazione. Per localizzare il PRR, sono stati condotti esperimenti di immunoblot su frazioni di cytosol e membrana di cellule HAC15 e H295R ed esperimenti di microscopia confocale. Questi risultati hanno mostrato che il PRR è principalmente, ma non esclusivamente, localizzato a livello di membrana ove colocalizza parzialmente con la molecola di adesione CD56. Attraverso esperimenti di microscopia confocale immunogold abbiamo poi indagato la localizzazione subcellulare del PRR e scoperto che il PRR è localizzato anche nel nucleo, nei mitocondri e nelle vescicole del Golgi. Abbiamo quindi utilizzato H295R e HAC15 per studiare la rilevanza funzionale di questo recettore. La stimolazione con angiotensina II [100 nM], renina [50 nM] e prorenina [50 nM] ha indotto fosforilazione di ERK 1/2. In presenza dell’antagonista del recettore dell’angiotensina II (AT1) irbesartan [5 μM], la fosforilazione indotta da renina e angiotensina II era abolita in entrambe le linee cellulari mentre quella indotta dalla prorenina era abolita solo nelle H295R. Questi risultati suggeriscono un ruolo del PRR nella proliferazione, differenziamento e apoptosi del corticosurrene e quindi un possibile ruolo funzionale di questo recettore nella fisiopatologia dell’iperaldosteronismo primario

Prevention of Coronary Artery Disease: Recent Advances in the Management of Hypertension

As coronary artery disease (CAD) still represents the leading cause of death worldwide, more efforts should be made to improve CAD prevention with both lifestyle changes and medical treatment. Elevated blood pressure has been identified as a risk factor for CAD; however, recent evidence suggests that lowering blood pressure too much could be harmful in patients at high cardiovascular risk. Despite the availability of a wide selection of antihypertensive drugs, new strategies and treatments are needed to improve blood pressure control and reduce cardiovascular risk factors associated with elevated blood pressure. New fixed-dose combinations have been recently approved; they usually contain an inhibitor of the renin-angiotensin system, a calcium antagonist and/or a diuretic. Although research and development related to new antihypertensive drugs has slowed in recent years, some new antihypertensive compounds with novel mechanisms of action or dual activity are currently in clinical development

AT(2) Receptor and Tissue Injury: Therapeutic Implications

The renin-angiotensin system (RAS) plays an important role in the initiation and progression of tissue injuries in the cardiovascular and nervous systems. The detrimental actions of the AT(1) receptor (AT(1)R) in hypertension and vascular injury, myocardial infarction and brain ischemia are well established. In the past twenty years, protective actions of the RAS, not only in the cardiovascular, but also in the nervous system, have been demonstrated. The so-called protective arm of the RAS includes AT(2)-receptors and Mas receptors (AT(2)R and MasR) and is characterized by effects different from and often opposing those of the AT(1)R. These include anti-inflammation, anti-fibrosis, anti-apoptosis and neuroregeneration that can counterbalance pathological processes and enable recovery from disease. The recent development of novel, small-molecule AT(2)R agonists offers a therapeutic potential in humans with a variety of clinical indications

Combined Angiotensin Receptor Modulation in the Management of Cardio-Metabolic Disorders

Cardiovascular and metabolic disorders, such as hypertension, insulin resistance, dyslipidemia or obesity are linked with chronic low-grade inflammation and dysregulation of the renin–angiotensin system (RAS). Consequently, RAS inhibition by ACE inhibitors or angiotensin AT1 receptor (AT1R) blockers is the evidence-based standard for cardiovascular risk reduction in high-risk patients, including diabetics with albuminuria. In addition, RAS inhibition reduces the new onset of diabetes mellitus. Yet, the high and increasing prevalence of metabolic disorders, and the high residual risk even in properly treated patients, calls for additional means of pharmacological intervention. In the past decade, the stimulation of the angiotensin AT2 receptor (AT2R) has been shown to reduce inflammation, improve cardiac and vascular remodeling, enhance insulin sensitivity and increase adiponectin production. Therefore, a concept of dual AT1R/AT2R modulation emerges as a putative means for risk reduction in cardio-metabolic diseases. The approach employing simultaneous RAS blockade (AT1R) and RAS stimulation (AT2R) is distinct from previous attempts of double intervention in the RAS by dual blockade. Dual blockade abolishes the AT1R-linked RAS almost completely with subsequent risk of hypotension and hypotension-related events, i.e. syncope or renal dysfunction. Such complications might be especially prominent in patients with renal impairment or patients with isolated systolic hypertension and normal-to-low diastolic blood pressure values. In contrast to dual RAS blockade, the add-on of AT2R stimulation does not exert significant blood pressure effects, but it may complement and enhance the anti-inflammatory and antifibrotic/de-stiffening effects of the AT1R blockade and improve the metabolic profile. Further studies will have to investigate these putative effects in particular for settings in which blood pressure reduction is not primarily desired

Activation of intracellular angiotensin AT2 receptors induces rapid cell death in human uterine leiomyosarcoma cells

Abstract The presence of angiotensin type 2 (AT 2 ) receptors in mitochondria and their role in NO generation and cell aging were recently demonstrated in various human and mouse non-tumour cells. We investigated the intracellular distribution of AT 2 receptors including their presence in mitochondria and their role in the induction of apoptosis and cell death in cultured human uterine leiomyosarcoma (SK-UT-1) cells and control human uterine smooth muscle cells (HutSMC). The intracellular levels of the AT 2 receptor are low in proliferating SK-UT-1 cells but the receptor is substantially up-regulated in quiescent SK-UT-1 cells with high densities in mitochondria. Activation of the cell membrane AT 2 receptors by a concomitant treatment with angiotensin II and the AT 1 receptor antagonist, losartan, induces apoptosis but does not affect the rate of cell death. We demonstrate for the first time that the high-affinity, non-peptide AT 2 receptor agonist, Compound 21 (C21), penetrates the cell membrane of quiescent SK-UT-1 cells, activates intracellular AT 2 receptors and induces rapid cell death; approximately 70 % of cells died within 24 h. The cells, which escaped cell death, displayed activation of the mitochondrial apoptotic pathway, i.e. down-regulation of the Bcl-2 protein, induction of the Bax protein and activation of caspase-3. All quiescent SK-UT-1 cells died within 5 days after treatment with a single dose of C21. C21 was devoid of cytotoxic effects in proliferating SK-UT-1 cells and in quiescent HutSMC. Our results point to a new, unique approach enabling the elimination non-cycling uterine leiomyosarcoma cells providing that they over-express the AT 2 receptor

Expression and functional role of the prorenin receptor in the human adrenocortical zona glomerulosa and in primary aldosteronism

OBJECTIVES: Prorenin can be detected in plasma of hypertensive patients. If detected in patients with primary aldosteronism could implicate prorenin in the development of primary aldosteronism. To address this issue, we measured the plasma prorenin levels in primary aldosteronism patients, the expression of the prorenin receptor (PRR) in the normal human adrenocortical zona glomerulosa and aldosterone-producing adenoma (APA), and we investigated the functional effects of PRR activation in human adrenocortical cells. METHOD: Plasma renin activity, aldosterone, and active and total trypsin-activated renin were measured in primary aldosteronism patients, essential hypertensive patients, and healthy individuals, and then prorenin levels were calculated. Localization and functional role of PRR were investigated in human and rat tissues, and aldosterone-producing cells. RESULTS: Primary aldosteronism patients had detectable plasma levels of prorenin. Using digital-droplet real-time PCR, we found a high PRR-to-porphobilinogen deaminase ratio in both the normal adrenal cortex and APAs. Marked expression of the PRR gene and protein was also found in HAC15 cells. Immunoblotting, confocal, and immunogold electron microscopy demonstrated PRR at the cell membrane and intracellularly. Renin and prorenin significantly triggered both CYP11B2 expression (aldosterone synthase) and ERK1/2 phosphorylation, but only CYP11B2 transcription was prevented by aliskiren. CONCLUSION: The presence of detectable plasma prorenin in primary aldosteronism patients, and the high expression of PRR in the normal human adrenal cortex, APA tissue, CD56+ aldosterone-producing cells, along with activation of CYP11B2 synthesis and ERK1/2 phosphorylation, suggest that the circulating and locally produced prorenin may contribute to the development or maintenance of human primary aldosteronism