<em>Angiotensin</em> II (AngII) mediates proinflammatory properties by activating NF-kappaB transcription factor nuclear translocation and inducing the expression of chemokines. For examination of whether AngII modulates the expression of Toll-like receptor 4 (TLR4), a key element of the innate immune system that senses LPS, mouse mesangial cells (MMC) were treated with AngII. AngII upregulated TLR4 mRNA and protein in MMC, and this effect was mediated through AngII type <em>1</em> receptors. Reporter gene experiments indicate that an activating protein-<em>1</em> (AP-<em>1</em>) as well as an E-26 specific sequence (Ets) binding site in the TLR4 promoter are responsible for the AngII-stimulated transcriptional activity of the TLR4 gene. Preincubation of MMC with AngII enhanced LPS-induced NF-kappaB activation and chemokine expression. Immunohistochemical analyses revealed that double-transgenic rats that overexpressed human renin and angiotensinogen expressed higher levels of glomerular TLR4 compared with normal Sprague-Dawley rats. In vivo, infusion with AngII but not with norepinephrine into rats for <em>7</em> d also enhanced glomerular NF-kappaB activation after systemic application of LPS, suggesting that the effects are independent of concomitantly induced hypertension. Together, these observations suggest that AngII leads to an activation of the innate immune system by a novel mechanism involving the upregulation of TLR4. Our data contribute to a better understanding of how exogenous infections may trigger renal autoimmune processes, particularly in pathophysiologic situations with high renal AngII concentrations. Because TLR4 binds endogenous ligands (e.g., extracellular matrix components) in addition to microbial products, AngII-mediated upregulation of TLR4 also could be relevant for the development of inflammation in many noninfectious renal diseases.

<em>Angiotensin</em>-converting enzyme 2 (ACE2) is an emerging cardiovascular protective target that mediates the metabolism of <em>angiotensin</em> (Ang) II into Ang (<em>1</em>-<em>7</em>). Our group has demonstrated that ACE2 overexpression enhances the function of endothelial progenitor cells (EPCs). Here, we investigated whether ACE2-primed EPCs (ACE2-EPCs) can protect cerebral microvascular endothelial cells (ECs) against injury and dysfunction in an <i>in vitro</i> model, with focusing on their exosomal and cytokine paracrine effects on endothelial mitochondria. Human EPCs were transfected with lentivirus containing null or human ACE2 cDNA (denoted as Null-EPCs and ACE2-EPCs, respectively). Their conditioned culture media, w/wo depletion of exosomes (ACE2-EPC-CM^EX-, Null-EPC-CM^EX-, ACE2-EPC-CM, and Null-EPC-CM), were used for coculture experiments. EC injury and dysfunction model was induced by Ang II before coculture. Apoptosis, angiogenic ability, mitochondrion functions (ROS production, membrane potential, fragmentation), and gene expressions (ACE2, Nox2, and Nox4) of ECs were analyzed. The supernatant was collected for measuring the levels of ACE2, Ang II/Ang-(<em>1</em>-<em>7</em>), and growth factors (VEGF and IGF). Our results showed that (<em>1</em>) ACE2-EPC-CM had higher levels of ACE2, Ang (<em>1</em>-<em>7</em>), VEGF, and IGF than that of Null-EPC-CM. (2) Ang II-injured ECs displayed an increase of apoptotic rate and reduction in tube formation and migration abilities, which were associated with ACE2 downregulation, Ang II/Ang (<em>1</em>-<em>7</em>) imbalance, Nox2/Nox4 upregulation, ROS overproduction, an increase of mitochondrion fragmentation, and a decrease of membrane potential. (3) ACE2-EPC-CM had better protective effects than Null-EPC-CM on Ang II-injured ECs, which were associated with the improvements on ACE2 expression, Ang II/Ang (<em>1</em>-<em>7</em>) balance, and mitochondrial functions. (4) ACE2-EPC-CM^EX- and Null-EPC-CM^EX- showed reduced effects as compared to ACE2-EPCs-CM and Null-EPCs-CM. In conclusion, our data demonstrate that ACE2 overexpression can enhance the protective effects of EPCs on ECs injury, majorly through the exosomal effects on mitochondrial function.

Pregnancy is a physiological condition characterized by a progressive increase of the different components of the renin-<em>angiotensin</em> system (RAS). The physiological consequences of the stimulated RAS in normal pregnancy are incompletely understood, and even less understood is the question of how this system may be altered and contribute to the hypertensive disorders of pregnancy. Findings from our group have provided novel insights into how the RAS may contribute to the physiological condition of pregnancy by showing that pregnancy increases the expression of both the vasodilator heptapeptide of the RAS, <em>angiotensin</em>-(<em>1</em>-<em>7</em>) [Ang-(<em>1</em>-<em>7</em>)], and of a newly cloned <em>angiotensin</em> converting enzyme (ACE) homolog, ACE2, that shows high catalytic efficiency for Ang II metabolism to Ang-(<em>1</em>-<em>7</em>). The discovery of ACE2 adds a new dimension to the complexity of the RAS by providing a new arm that may counter-regulate the activity of the vasoconstrictor component, while amplifying the vasodilator component. The studies reviewed in this article demonstrate that Ang-(<em>1</em>-<em>7</em>) increases in plasma and urine of normal pregnant women. In preeclamptic subjects we showed that plasma Ang-(<em>1</em>-<em>7</em>) was suppressed as compared to the levels found in normal pregnancy. In addition, kidney and urinary levels of Ang-(<em>1</em>-<em>7</em>) were increased in pregnant rats coinciding with the enhanced detection and expression of ACE2. These findings support the concept that in normal pregnancy enhanced ACE2 may counteract the elevation in tissue and circulating Ang II by increasing the rate of conversion to Ang-(<em>1</em>-<em>7</em>). These findings provide a basis for the physiological role of Ang-(<em>1</em>-<em>7</em>) and ACE2 during pregnancy.

We recently demonstrated that renin-<em>angiotensin</em> system (RAS) overactivity during late gestation in rats is associated with increased kidney and urine levels of ANG-(<em>1</em>-<em>7</em>) and enhanced kidney immunostaining of ANG-(<em>1</em>-<em>7</em>) and <em>angiotensin</em>-converting enzyme 2 (ACE2). To understand the temporal-spatial changes in normal and hypertensive pregnancies, the renal distribution of ANG-(<em>1</em>-<em>7</em>) and ACE2 in association with kidney <em>angiotensin</em> peptides and ACE2 activity was examined in virgin, normal pregnant (NP; gestational days 5, <em>1</em>5, and <em>1</em>9) and reduced uterine perfusion pressure (RUPP at day <em>1</em>9) pregnant Sprague-Dawley rats. ANG-(<em>1</em>-<em>7</em>) and ACE2 immunocytochemical staining increased <em>1</em>.8- and <em>1</em>.9-fold and <em>1</em>.<em>7</em>- and <em>1</em>.8-fold, respectively, at days <em>1</em>5 and <em>1</em>9 of NP, compared with virgin rats. ANG-(<em>1</em>-<em>7</em>) and ANG II concentrations were increased in the kidney at <em>1</em>9 days of gestation. ACE2 activity measured using a fluorescent substrate was increased <em>1</em>.9- and <em>1</em>.9-fold in the cortex and <em>1</em>.9- and <em>1</em>.8-fold in the medulla at days <em>1</em>5 and <em>1</em>9 of NP. In the RUPP animals, ANG-(<em>1</em>-<em>7</em>) immunostaining and concentration were significantly decreased compared with <em>1</em>9-day NP rats. ACE2 activity was unchanged in the cortex and medulla of RUPP rats. In conclusion, during NP, the concurrent changes of ACE2 and ANG-(<em>1</em>-<em>7</em>) suggest that ACE2 plays an important role in regulating the renal levels of ANG-(<em>1</em>-<em>7</em>) at mid to late gestation. However, the decrease in renal ANG-(<em>1</em>-<em>7</em>) content in the absence of a concomitant decrease in ACE2 implicates the participation of other ANG-(<em>1</em>-<em>7</em>) forming or degrading enzymes during hypertensive pregnancy.

Reactive oxygen species (ROS) generated by NADPH oxidase-4 (NOX4) have been shown to initiate lung fibrosis. The migration of lung fibroblasts to the injured area is a crucial early step in lung fibrosis. The <em>angiotensin</em>-converting enzyme 2 (ACE2)/<em>angiotensin</em> (<em>1</em>-<em>7</em>) [Ang(<em>1</em>-<em>7</em>)]/Mas axis, which counteracts the ACE/<em>angiotensin</em> II (AngII)/<em>angiotensin</em> II type <em>1</em> receptor (AT<em>1</em>R) axis, has been shown to attenuate pulmonary fibrosis. Nevertheless, the exact molecular mechanism remains unclear.

OBJECTIVE

To investigate the different effects of the two axes of the renin-angiotensin system (RAS) on lung fibroblast migration and extracellular matrix accumulation by regulating the NOX4-derived ROS-mediated RhoA/Rho kinase (Rock) pathway.

RESULTS

In vitro, AngII significantly increased the NOX4 level and ROS production in lung fibroblasts, which stimulated cell migration and α-collagen I synthesis through the RhoA/Rock pathway. These effects were attenuated by N-acetylcysteine (NAC), diphenylene iodonium, and NOX4 RNA interference. Moreover, Ang(<em>1</em>-<em>7</em>) and lentivirus-mediated ACE2 (lentiACE2) suppressed AngII-induced migration and α-collagen I synthesis by inhibiting the NOX4-derived ROS-mediated RhoA/Rock pathway. However, Ang(<em>1</em>-<em>7</em>) alone exerted analogous effects on AngII. In vivo, constant infusion with Ang(<em>1</em>-<em>7</em>) or intratracheal instillation with lenti-ACE2 shifted the RAS balance toward the ACE2/Ang(<em>1</em>-<em>7</em>)/Mas axis, alleviated bleomycin-induced lung fibrosis, and inhibited the RhoA/Rock pathway by reducing NOX4-derived ROS.

METHODS

This study suggests that the ACE2/Ang(<em>1</em>-<em>7</em>)/Mas axis may be targeted by novel pharmacological antioxidant strategies to treat lung fibrosis induced by AngII-mediated ROS.

CONCLUSIONS

The ACE2/Ang(<em>1</em>-<em>7</em>)/Mas axis protects against lung fibroblast migration and lung fibrosis by inhibiting the NOX4-derived ROS-mediated RhoA/Rock pathway.

The health of the cardiovascular and pulmonary systems is inextricably linked to the renin-<em>angiotensin</em> system (RAS). Physiologically speaking, a balance between the vasodeleterious (<em>Angiotensin</em>-converting enzyme [ACE]/<em>Angiotensin</em> II [Ang II]/Ang II type <em>1</em> receptor [AT<em>1</em>R]) and vasoprotective (<em>Angiotensin</em>-converting enzyme 2 [ACE2]/<em>Angiotensin</em>-(<em>1</em>-<em>7</em>) [Ang-(<em>1</em>-<em>7</em>)]/Mas receptor [MasR]) components of the RAS is critical for cardiopulmonary homeostasis. Upregulation of the ACE/Ang II/AT<em>1</em>R axis shifts the system toward vasoconstriction, proliferation, hypertrophy, inflammation, and fibrosis, all factors that contribute to the development and progression of cardiopulmonary diseases. Conversely, stimulation of the vasoprotective ACE2/Ang-(<em>1</em>-<em>7</em>)/MasR axis produces a counter-regulatory response that promotes cardiovascular health. Current research is investigating novel strategies to augment actions of the vasoprotective RAS components, particularly ACE2, in order to treat various pathologies. Although multiple approaches to increase the activity of ACE2 have displayed beneficial effects against experimental disease models, the mechanisms behind its protective actions remain incompletely understood. Recent work demonstrating a non-catalytic role for ACE2 in amino acid transport in the gut has led us to speculate that the therapeutic effects of ACE2 can be mediated, in part, by its actions on the gastrointestinal tract and/or gut microbiome. This is consistent with emerging data which suggest that dysbiosis of the gut and lung microbiomes is associated with cardiopulmonary disease. This review highlights new developments in the protective actions of ACE2 against cardiopulmonary disorders, discusses innovative approaches to targeting ACE2 for therapy, and explores an evolving role for gut and lung microbiota in cardiopulmonary health.

Our current recognition of the renin-<em>angiotensin</em> system is more convoluted than originally thought due to the discovery of multiple novel enzymes, peptides, and receptors inherent in this interactive biochemical cascade. Over the last decade, <em>angiotensin</em>-converting enzyme 2 (ACE2) has emerged as a key player in the pathophysiology of hypertension and cardiovascular and renal disease due to its pivotal role in metabolizing vasoconstrictive/hypertrophic/proliferative <em>angiotensin</em> II into favorable <em>angiotensin</em>-(<em>1</em>-<em>7</em>). This review addresses the considerable advancement in research on the role of tissue ACE2 in the development and progression of hypertension and cardiac and renal injury. We summarize the results from recent clinical and experimental studies suggesting that serum or urine soluble ACE2 may serve as a novel biomarker or independent risk factor relevant for diagnosis and prognosis of cardiorenal disease. We also review recent proceedings on novel therapeutic approaches to enhance ACE2/<em>angiotensin</em>-(<em>1</em>-<em>7</em>) axis.

Clathrin-mediated endocytosis is a complex process regulated at many different levels. We showed previously that activation of the <em>angiotensin</em> type <em>1</em> receptor (AT<em>1</em>R), which belongs to the G protein-coupled receptor (GPCR) family, leads to c-Src-dependent tyrosine phosphorylation of beta2-adaptin, a subunit of the clathrin adaptor AP-2. The phosphorylation of beta2-adaptin on tyrosine residue <em>7</em>3<em>7</em> (Y<em>7</em>3<em>7</em>) negatively regulates its interaction with betaarrestin, another important clathrin adaptor for GPCR internalization. Here we sought to determine whether AP-2 phosphorylation represents a general mechanism for different receptors internalizing through the clathrin pathway. Using a specifically designed antibody against the phosphorylated form of Y<em>7</em>3<em>7</em> on beta2-adaptin, we demonstrate that this residue is phosphorylated by AT<em>1</em>R in different cell types like HEK293, COS-<em>7</em> and vascular smooth muscle cells. Using RNA interference approaches, we reveal that this agonist-mediated event is both betaarrestin- and c-Src-dependent, and that it occurs at the plasma membrane in clathrin-coated vesicles (CCVs). We further show that this is not only a common event employed by other GPCRs like the beta2-adrenergic, vasopressin V2, bradykinin type 2, platelet-activating factor and endothelin A receptors but that the epidermal growth factor receptor is capable of eliciting the phosphorylation of AP-2 in CCVs. Our results imply that tyrosine phosphorylation of Y<em>7</em>3<em>7</em> on beta2-adaptin is a common regulatory mechanism employed by different receptors undergoing clathrin-dependent endocytosis, and suggest a wider function for this event than originally anticipated.

<em>Angiotensin</em>-(<em>1</em>-<em>7</em>) [Ang-(<em>1</em>-<em>7</em>)] is generated in part via ACE2-dependent degradation of <em>angiotensin</em> II (ANG II). In proximal tubular cells, Ang-(<em>1</em>-<em>7</em>) inhibits ANG II-stimulated phosphorylation of the mitogen-activated protein kinases (MAPKs) p38, extracellular signal-related kinase (ERK<em>1</em>/ERK2), and c-jun N-terminal kinase (JNK), suggesting that Ang-(<em>1</em>-<em>7</em>) protects against ANG II-mediated tubulointerstitial injury. We determined the effect of Ang-(<em>1</em>-<em>7</em>) on signaling and growth responses in cultured human mesangial cells. Ang-(<em>1</em>-<em>7</em>) increased phosphorylation of p38, ERK<em>1</em>/ERK2, and JNK MAPKs, which was blocked by the Ang-(<em>1</em>-<em>7</em>) antagonist A-<em>7</em><em>7</em>9. Neither the AT(<em>1</em>) receptor antagonist losartan, nor the AT(2) antagonist PD<em>1</em>233<em>1</em>9 affected specific binding of [(<em>1</em>25)I]Ang-(<em>1</em>-<em>7</em>) or Ang-(<em>1</em>-<em>7</em>)-stimulated p38 phosphorylation. Ang-(<em>1</em>-<em>7</em>) increased cell arachidonic acid release, an effect blocked by A-<em>7</em><em>7</em>9. The p38 MAPK antagonist SB202<em>1</em>90 completely prevented Ang-(<em>1</em>-<em>7</em>)-stimulated release of arachidonic acid, whereas inhibitors of ERK or JNK had no effect. Ang-(<em>1</em>-<em>7</em>) significantly enhanced DNA synthesis and increased production of transforming growth factor-beta<em>1</em> (TGF-beta<em>1</em>), fibronectin, and collagen IV. Both A-<em>7</em><em>7</em>9 and SB202<em>1</em>90 blocked the Ang-(<em>1</em>-<em>7</em>)-stimulated increases in TGF-beta<em>1</em>, fibronectin, and collagen IV. These data indicate that Ang-(<em>1</em>-<em>7</em>) activates MAPK phosphorylation via binding to a specific receptor in human mesangial cells. Stimulation of p38 MAPK phosphorylation by Ang-(<em>1</em>-<em>7</em>) leads to release of arachidonic acid and production of TGF-beta<em>1</em> and extracellular matrix proteins. We conclude that Ang-(<em>1</em>-<em>7</em>) exerts growth-stimulatory effects in human mesangial cells.

OBJECTIVE

Abdominal aortic aneurysms (AAAs) are characterized by chronic inflammation which contributes to the remodeling and eventual weakening of the vessel wall. Increased cyclooxygenase-2 (COX-2) expression is detected in human aneurysmal tissue and is suggested to contribute to the disease. The aim of the current study was to define the role of COX-2 expression in the development of AAAs, using a model of the disease.

METHODS

AAAs were induced in mice by chronic <em>angiotensin</em> II infusion, and were analyzed following 3, <em>7</em>, 21 or 28 days of the infusion. AAA incidence and severity, together with the expression of inflammatory markers, were compared between abdominal aortas from COX-2-deficient mice and their wild-type littermate controls.

RESULTS

The AAA incidence in COX-2 wild-type mice was 54% (13/24), whereas AAAs were not detected in COX-2-deficient mice (0/23) following 28 days of <em>angiotensin</em> II infusion. The genetic deficiency of COX-2 also resulted in a <em>7</em>3% and 90% reduction in AAA incidence following <em>7</em> and 21 days of <em>angiotensin</em> II infusion, respectively. In COX-2 wild-type mice, COX-2 mRNA expression in the abdominal aorta was induced by <em>angiotensin</em> II beginning 3 days following initiation of the infusion, which continued throughout progression of the disease. Abundant COX-2 protein expression was detected in medial smooth muscle cells adjacent to the AAAs. The deficiency of COX-2 significantly attenuated mRNA expression in the abdominal aorta of the macrophage marker CD68, and the inflammatory cell recruitment chemokines, monocyte chemotactic protein-1 and macrophage inflammatory protein-1alpha.

CONCLUSIONS

Our findings suggest that increased COX-2 expression in smooth muscle cells of the abdominal aorta contributes to AAA formation in mice by enhancing inflammatory cell infiltration.

The renin-<em>angiotensin</em>-system (RAS) is a cascade of enzymatic reactions resulting ultimately in the formation of <em>angiotensin</em> II. Recent research has expanded the knowledge about the RAS by adding new components to the pathways: <em>angiotensin</em>-(<em>1</em>-5) [Ang-<em>1</em>-5], <em>angiotensin</em>-(<em>1</em>-<em>7</em>) [Ang-(<em>1</em>-<em>7</em>)], <em>angiotensin</em>-(<em>1</em>-9) [Ang-(<em>1</em>-9)], an ACE homologous enzyme, ACE2, and the G-protein-coupled receptor mas as a molecular receptor for Ang-(<em>1</em>-<em>7</em>). Although previous studies provided some conflicting evidence about the relevance of Ang-(<em>1</em>-<em>7</em>) in the regulation of vascular and renal function, data now demonstrate that Ang-(<em>1</em>-<em>7</em>) contributes to the cardiovascular effects of ACE-inhibitors (ACE-<em>1</em>) and AT<em>1</em>-receptor-blockers (ARBs) both in experimental conditions and in humans. This review summarizes and critically discusses the currently available experimental and clinical study evidence for the role of Ang-(<em>1</em>-<em>7</em>) as a vasodilator and anti-trophic peptide in cardiovascular drug therapy. In addition, the potential therapeutic impact of currently available RAS blocking agents (ACE-<em>1</em> and ARBs) and new agents still under development (renin-inhibitors) on the RAS-effector peptides is highlighted.

This study aimed to investigate the protective effect of <em>angiotensin</em> converting enzyme 2 (ACE2) on lipopolysaccharide (LPS)-induced acute lung injury (ALI). After generating ALI mouse models by injecting LPS, the levels of ACE2, inflammatory factors, and downstream proteins of the LPS-TLR4 pathway were analyzed. LPS-challenged BEAS-2B cells were established in vitro. Next, a eukaryotic expression vector, pm-ACE2, was constructed and validated. Challenged cells were transfected with pm-ACE2 containing enhanced green fluorescent protein, or they were treated with D-Ala-Ang-(<em>1</em>-<em>7</em>), <em>angiotensin</em> converting enzyme inhibitor (ACEI), <em>angiotensin</em> receptor blocker (ARB) and the LPS-TLR4 pathway inhibitor dimethyl fumarate (DMF) for analysis of how the above factors contribute to ACE2 regulation. Expression of renin, Ang II, ACE and <em>angiotensin</em> II type <em>1</em> receptor (AT<em>1</em>R) was subsequently assessed. In the ALI model, mice exhibited decreased expression of ACE2, lung pathological injury, inflammatory injury, and abnormal activation of the LPS-TLR4 pathway. LPS-challenged BEAS-2B cells demonstrated upregulated expression of renin, Ang II, ACE and AT<em>1</em>R. After injection of ACE2, lung function and lung pathological injury were significantly improved, and that effect was accompanied by attenuated inflammation, and inactivation of the LPS-TLR4 pathway. Cell studies showed similar results. The above observations were further enhanced when there was a combined treatment with DMF and pm-ACE2. D-Ala-Ang-(<em>1</em>-<em>7</em>) treatment attenuated the protective effect of ACE2, while ACEI and ARB treatment alleviated LPS-induced pneumonic injury. In conclusion, ACE2 was expressed at low levels in response to LPS-induced ALI. Overexpression of ACE2 regulates the ACE2/Ang-(<em>1</em>-<em>7</em>)/Mas and ACE/Ang II/AT<em>1</em> axes to maintain dynamic balance of the renin-<em>angiotensin</em> system, and attenuate inflammatory response.

The mechanisms by which sympathetic function is augmented in chronic heart failure (CHF) are not well understood. A previous study from this laboratory (Circ Res. <em>1</em>998;82:496-502) indicated that blockade of nitric oxide (NO) synthesis resulted in only an increase in renal sympathetic nerve activity (RSNA) when plasma <em>angiotensin</em> II (Ang II) levels were elevated. The present study was undertaken to determine if NO reduces RSNA in rabbits with CHF when Ang II receptors are blocked. Twenty-four New Zealand White rabbits were instrumented with cardiac dimension crystals, a left ventricular pacing lead, and a pacemaker. After pacing at 360 to 380 bpm for approximately 3 weeks, a renal sympathetic nerve electrode and arterial and venous catheters were implanted. Studies were carried out in the conscious state 3 to <em>7</em> days after electrode implantation. The effects of a <em>1</em>-hour infusion of sodium nitroprusside (SNP; 3 microgram . kg-<em>1</em>. min-<em>1</em>) on RSNA and mean arterial pressure (MAP) were determined before and after Ang II blockade with losartan (5 mg/kg) in normal and CHF rabbits. Changes in MAP were readjusted to normal with phenylephrine. Before losartan, SNP evoked a decrease in MAP and an increase in RSNA in both groups that was baroreflex-mediated, because both MAP and RSNA returned to control when phenylephrine was administered. In the normal group, losartan plus SNP caused a reduction in MAP and an increase in RSNA that was <em>1</em>52.6+/-9.8% of control. Phenylephrine returned both MAP and RSNA back to the control levels. However, in the CHF group, losartan plus SNP evoked a smaller change in RSNA for equivalent changes in MAP (<em>1</em><em>1</em><em>7</em>.<em>1</em>+/-4.<em>1</em>% of control). On returning MAP to the control level with phenylephrine, RSNA was reduced to 65.2+/-2.9% of control (P<0. 000<em>1</em>). These data suggest that endogenous Ang II contributes to the sympathoexcitation in the CHF state and that blockade of Ang II receptors plus providing an exogenous source of NO reduces RSNA below the elevated baseline levels. We conclude that both a loss of NO and an increase in Ang II are necessary for sustained increases in sympathetic nerve activity in the CHF state.

A newly produced murine recombinant <em>angiotensin</em> (Ang)-converting enzyme 2 (ACE2) was characterized in vivo and in vitro. The effects of available ACE2 inhibitors (MLN-4<em>7</em>60 and 2 conformational variants of DX600, linear and cyclic) were also examined. When murine ACE2 was given to mice for 4 weeks, a marked increase in serum ACE2 activity was sustainable. In acute studies, mouse ACE2 (<em>1</em> mg/kg) obliterated hypertension induced by Ang II infusion by rapidly decreasing plasma Ang II. These effects were blocked by MLN-4<em>7</em>60 but not by either form of DX600. In vitro, conversion from Ang II to Ang-(<em>1</em>-<em>7</em>) by mouse ACE2 was blocked by MLN-4<em>7</em>60 (<em>1</em>0(-6) m) but not by either form of DX600 (<em>1</em>0(-5) m). Quantitative analysis of multiple Ang peptides in plasma ex vivo revealed formation of Ang-(<em>1</em>-9) from Ang I by human but not by mouse ACE2. Both human and mouse ACE2 led to the dissipation of Ang II with formation of Ang (<em>1</em>-<em>7</em>). By contrast, mouse ACE2-driven Ang-(<em>1</em>-<em>7</em>) formation from Ang II was blocked by MLN-4<em>7</em>60 but not by either linear or cyclic DX600. In conclusion, sustained elevations in serum ACE2 activity can be accomplished with murine ACE2 administration, thereby providing a strategy for ACE2 amplification in chronic studies using rodent models of hypertension and cardiovascular disease. Human but not mouse ACE2 degrades Ang I to form Ang-(<em>1</em>-9). There are also species differences regarding rodent and human ACE2 inhibition by known inhibitors such that MLN-4<em>7</em>60 inhibits both human and mouse ACE2, whereas DX600 only blocks human ACE2 activity.

<em>Angiotensin</em>-converting enzyme 2 (ACE2) is a negative regulator of the renin-<em>angiotensin</em> system, and functions as the key SARS coronavirus receptor and stabilizer of neutral amino acid transporters. ACE2 catalyzes the conversion of <em>angiotensin</em> II to <em>angiotensin</em> <em>1</em>-<em>7</em>, thereby counterbalancing ACE activity. Accumulating evidence indicates that the enzymatic activity of ACE2 has a protective role in cardiovascular diseases. Loss of ACE2 can be detrimental, as it leads to functional deterioration of the heart and progression of cardiac, renal, and vascular pathologies. Recombinant soluble human ACE2 protein has been demonstrated to exhibit beneficial effects in various animal models, including cardiovascular diseases. ACE2 is a multifunctional enzyme and thus potentially acts on other vasoactive peptides, such as Apelin, a vital regulator of blood pressure and myocardium contractility. In addition, ACE2 is structurally a chimeric protein that has emerged from the duplication of 2 genes: homology with ACE at the carboxypeptidase domain and homology with Collectrin in the transmembrane C-terminal domain. ACE2 has been implicated in the pathology of Hartnup's disease, a disorder of amino acid homeostasis, and, via its function in amino acid transport, it has been recently revealed that ACE2 controls intestinal inflammation and diarrhea, thus regulating the gut microbiome. This review summarizes and discusses the structure and multiple functions of ACE2 and the relevance of this key enzyme in disease pathogenesis.

The effect of a synthetic analogue of atrial natriuretic peptide (Ileu-ANP) on haemodynamic, hormonal, and electrolyte excretion indices was studied in <em>7</em> patients with chronic congestive heart failure. Patients received in random order placebo or Ileu-ANP infusions (5 micrograms/min) for 4 h on 2 separate occasions, at least <em>1</em> week apart. Compared with placebo, Ileu-ANP caused significant reductions in mean systemic arterial pressure, mean pulmonary artery pressure, pulmonary diastolic pressure, and right atrial pressure. These changes were sustained for at least 2 h after infusion. Cardiac output increased from 6.2 to <em>7</em>.4 l/min at 60 min, then returned to pre-infusion levels. Despite considerable falls in systemic pressure there was no significant increase in heart rate or plasma noradrenaline. With Ileu-ANP infusion, plasma renin activity, <em>angiotensin</em>, arginine vasopressin, aldosterone, and cortisol values were not significantly different from placebo values. Plasma cortisol and aldosterone increased after stopping Ileu-ANP. Neither urine volume nor sodium excretion rate was significantly increased by Ileu-ANP.

Mutational analysis of the tachykinin NK-<em>1</em> (refs <em>1</em>-<em>7</em>), NK-2 (ref. 8) and <em>angiotensin</em> AT-<em>1</em> (refs 9, <em>1</em>0) receptors indicates that non-peptide antagonists act through residues located between the seven transmembrane segments, whereas natural peptide agonists bind mainly to residues scattered in the exterior part of the receptor. The presumed contact points for the prototype NK-<em>1</em> antagonist CP96,345 cluster on opposing faces of the outer portions of transmembrane helices V and VI (refs <em>1</em>-5). Here we show that systematic introduction of histidyl residues at this antagonist-binding site in the human NK-<em>1</em> receptor gradually converts it into a high-affinity metal-ion-binding site without affecting agonist binding. In a double mutant with histidine residues substituted at the top of transmembrane segments V and VI, respectively, Zn2+ inhibits binding of radiolabelled agonist peptide and efficiently blocks phosphoinositol turnover induced by substance P. We propose that Zn2+ and CP96,345 act as 'allosteric competitive' antagonists by stabilizing inactive conformations of the mutant and the wild-type receptor respectively. Introduction of metal-ion-binding sites could be used as a general tool in the structural and functional characterization of helix-helix interactions in G-protein-coupled receptors, as well as in other membrane proteins.

The rapid rise in pharmaceutical costs in France has been driven by new technologies and the growing prevalence of chronic diseases as well as considerable prescribing freedom and choice of physician among patients. This has led to the introduction of a number of reforms and initiatives in an attempt to moderate expenditure whilst ensuring universal coverage and rewarding innovation. These reforms include accelerating access to and granting average European prices for new innovative drugs, delisting drugs where there are concerns over their value and instigating rebates for excessive prescribing. Alongside this, ongoing initiatives to improve the quality and efficiency of prescribing include programmes to enhance generic prescribing and dispensing as well as to reduce antibacterial and anxiolytic/hypnotic prescribing. However, there have been few publications documenting the impact of specific reforms on the overall costs and quality of care, which have been exacerbated by compartmentalization of budgets. Estimates suggest savings of over 2<em>7</em> million euro/year by decreasing antibacterial prescribing, 450 million euro/year by not reimbursing ineffective drugs, 6<em>7</em>0 million euro/year from pharmaceutical company rebates and approximately <em>1</em> billion euro/year from increased prescribing and dispensing of generics (year 2003-<em>7</em> values). Additional savings of at least <em>1</em>.5 billion euro/year are seen as being possible from increased use of generics such as generic proton pump inhibitors, statins (HMG-CoA reductase inhibitors) and ACE inhibitors instead of current branded products such as <em>angiotensin</em> II type <em>1</em> receptor antagonists (<em>angiotensin</em> receptor blockers [ARBs]). Delisting drugs when there are concerns about their value provides an example to other countries with currently limited demand-side measures. Other possible examples include price : volume agreements and multifaceted campaigns to enhance generic prescribing and dispensing and reduce antibacterial prescribing. Possible future initiatives could include adopting more stringent criteria for categorizing new drugs as innovative as well as further reductions in the prices of generics. Other initiatives could include further enhancement of the quality and efficiency of prescribing, including formal auditing of physician prescribing, as well as increasing efforts to monitor the risk : benefit ratio of new drugs post-launch in real-world practice.

We postulate that an orchestrated network composed of various vasodilatory systems participates in the systemic and local hemodynamic adaptations in pregnancy. The temporal patterns of increase in the circulating and urinary levels of five vasodilator factors/systems, prostacyclin, nitric oxide, kallikrein, <em>angiotensin</em>-(<em>1</em>-<em>7</em>) and VEGF, in normal pregnant women and animals, as well as the changes observed in preeclamptic pregnancies support their functional role in maintaining normotension by opposing the vasoconstrictor systems. In addition, the expression of these vasodilators in the different trophoblastic subtypes in various species supports their role in the transformation of the uterine arteries. Moreover, their expression in the fetal endothelium and in the syncytiotrophoblast in humans, rats and guinea-pigs, favour their participation in maintaining the uteroplacental circulation. The findings that sustain the functional associations of the various vasodilators, and their participation by endocrine, paracrine and autocrine regulation of the systemic and local vasoactive changes of pregnancy are abundant and compelling. However, further elucidation of the role of the various players is hampered by methodological problems. Among these difficulties is the complexity of the interactions between the different factors, the likelihood that experimental alterations induced in one system may be compensated by the other players of the network, and the possibility that data obtained by manipulating single factors in vitro or in animal studies may be difficult to translate to the human. In addition, the impossibility of sampling the uteroplacental interface along normal pregnancy precludes obtaining longitudinal profiles of the various players. Nevertheless, the possibility of improving maternal blood pressure regulation, trophoblast invasion and uteroplacental flow by enhancing vasodilation (e.g. L-arginine, NO donors, VEGF transfection) deserves unravelling the intricate association of vasoactive factors and the systemic and local adaptations to pregnancy.

BACKGROUND

Endothelial dysfunction plays an integral role in pulmonary hypertension (PH). AMPK (AMP-activated protein kinase) and ACE2 (angiotensin-converting enzyme 2) are crucial in endothelial homeostasis. The mechanism by which AMPK regulates ACE2 in the pulmonary endothelium and its protective role in PH remain elusive.

OBJECTIVE

We investigated the role of AMPK phosphorylation of ACE2 Ser680 in ACE2 stability and deciphered the functional consequences of this post-translational modification of ACE2 in endothelial homeostasis and PH.

METHODS

Bioinformatics prediction, kinase assay, and antibody against phospho-ACE2 Ser680 (p-ACE2 S680) were used to investigate AMPK phosphorylation of ACE2 Ser680 in endothelial cells. Using CRISPR-Cas9 genomic editing, we created gain-of-function ACE2 S680D knock-in and loss-of-function ACE2 knockout (ACE2-/-) mouse lines to address the involvement of p-ACE2 S680 and ACE2 in PH. The AMPK-p-ACE2 S680 axis was also validated in lung tissue from humans with idiopathic pulmonary arterial hypertension.

RESULTS

Phosphorylation of ACE2 by AMPK enhanced the stability of ACE2, which increased Ang (<em>angiotensin</em>) <em>1</em>-<em>7</em> and endothelial nitric oxide synthase-derived NO bioavailability. ACE2 S680D knock-in mice were resistant to PH as compared with wild-type littermates. In contrast, ACE2-knockout mice exacerbated PH, a similar phenotype found in mice with endothelial cell-specific deletion of AMPKα2. Consistently, the concentrations of phosphorylated AMPK, p-ACE2 S680, and ACE2 were decreased in human lungs with idiopathic pulmonary arterial hypertension.

CONCLUSIONS

Impaired phosphorylation of ACE2 Ser680 by AMPK in pulmonary endothelium leads to a labile ACE2 and hence is associated with the pathogenesis of PH. Thus, AMPK regulation of the vasoprotective ACE2 is a potential target for PH treatment.

OBJECTIVE

To investigate whether <em>angiotensin</em> (Ang)-(<em>1</em>-<em>7</em>), its receptor Mas, and <em>angiotensin</em>-converting enzyme type 2 (ACE2) are present in human ovary.

METHODS

Cross-sectional study.

METHODS

Academic hospital.

METHODS

Twelve reproductive-age women and five postmenopausal women undergoing oophorectomy for nonovarian diseases and seven women having controlled ovarian hyperstimulation for IVF.

METHODS

Ovarian tissue was obtained from the reproductive-age women and postmenopausal women undergoing oophorectomy for nonovarian diseases. Follicular fluid (FF) samples were obtained from the women having controlled ovarian hyperstimulation for IVF.

METHODS

Localization of Ang-(<em>1</em>-<em>7</em>) and Mas by immunohistochemistry; measurement of Ang-(<em>1</em>-<em>7</em>) in ovarian FF by RIA; detection of messenger RNAs encoding Mas and ACE2 with use of real-time polymerase chain reaction; assessment of <em>1</em>25I-labeled Ang-(<em>1</em>-<em>7</em>) binding to ovarian sections with use of autoradiographic binding assay.

RESULTS

<em>Angiotensin</em>-(<em>1</em>-<em>7</em>) and the receptor Mas were localized to primordial, primary, secondary, and antral follicles, stroma, and corpora lutea of reproductive-age ovaries. Postmenopausal women expressed both the peptide and its receptor in the ovarian stroma. <em>Angiotensin</em>-(<em>1</em>-<em>7</em>) was detectable in FF (mean±SE: <em>1</em>9<em>1</em>±54 pg/mL). Both Mas and ACE2 messenger RNAs were expressed in ovarian tissue, as revealed by real-time polymerase chain reaction, and ovarian binding sites for <em>1</em>25I-labeled Ang-(<em>1</em>-<em>7</em>) were identified by autoradiography.

CONCLUSIONS

<em>Angiotensin</em>-(<em>1</em>-<em>7</em>), its receptor Mas, and ACE2 are expressed in the human ovary. The peptide is present in several ovarian compartments and can be quantified in FF.

OBJECTIVE

<em>Angiotensin</em> II produces oxidative stress and endothelial dysfunction in cerebral arteries, and <em>angiotensin</em> II type I receptors may play a role in longevity and vascular aging. <em>Angiotensin</em>-converting enzyme type 2 (ACE2) converts <em>angiotensin</em> II to <em>angiotensin</em> (<em>1</em>-<em>7</em>) and thus, may protect against effects of <em>angiotensin</em> II. We hypothesized that ACE2 deficiency increases oxidative stress and endothelial dysfunction in cerebral arteries and examined the role of ACE2 in age-related cerebrovascular dysfunction.

METHODS

Endothelial function, expression of <em>angiotensin</em> system components, NADPH oxidase subunits, and proinflammatory cytokines were examined in cerebral arteries from adult (<em>1</em>2 months old) and old (24 months old) ACE2 knockout (KO) and wild-type (WT) mice. The superoxide scavenger tempol was used to examine the role of oxidative stress on endothelial function.

RESULTS

Vasodilatation to acetylcholine was impaired in adult ACE2 KO (24±6% [mean±SE]) compared with WT mice (52±<em>7</em>%; P<0.05). In old mice, vasodilatation to acetylcholine was impaired in WT mice (29±6%) and severely impaired in ACE2 KO mice (<em>7</em>±5%). Tempol improved endothelial function in adult and old ACE2 KO and WT mice. Aging increased mRNA for tumor necrosis factor-α in WT mice, and significantly increased mRNA levels of NAPDH oxidase 2, p4<em>7</em>(phox), and Regulator of calcineurin <em>1</em> in both ACE2 KO and WT mice. mRNA levels of <em>angiotensin</em> system components did not change during aging.

CONCLUSIONS

ACE2 deficiency impaired endothelial function in cerebral arteries from adult mice and augmented endothelial dysfunction during aging. Oxidative stress plays a critical role in cerebrovascular dysfunction induced by ACE2 deficiency and aging.

The endothelium exerts multiple actions involving regulation of vascular permeability and tone, coagulation and fibrinolysis, inflammatory and immunological reactions and cell growth. Alterations of one or more such actions may cause vascular endothelial dysfunction. Different risk factors such as hypercholesterolemia, homocystinemia, hyperglycemia, hypertension, smoking, inflammation, and aging contribute to the development of endothelial dysfunction. Mechanisms underlying endothelial dysfunction are multiple, including impaired endothelium-derived vasodilators, enhanced endothelium-derived vasoconstrictors, over production of reactive oxygen species and reactive nitrogen species, activation of inflammatory and immune reactions, and imbalance of coagulation and fibrinolysis. Endothelial dysfunction occurs in many cardiovascular diseases, which involves different mechanisms, depending on specific risk factors affecting the disease. Among these mechanisms, a reduction in nitric oxide (NO) bioavailability plays a central role in the development of endothelial dysfunction because NO exerts diverse physiological actions, including vasodilation, anti-inflammation, antiplatelet, antiproliferation and antimigration. Experimental and clinical studies have demonstrated that a variety of currently used or investigational drugs, such as <em>angiotensin</em>-converting enzyme inhibitors, <em>angiotensin</em> AT<em>1</em> receptors blockers, <em>angiotensin</em>-(<em>1</em>-<em>7</em>), antioxidants, beta-blockers, calcium channel blockers, endothelial NO synthase enhancers, phosphodiesterase 5 inhibitors, sphingosine-<em>1</em>-phosphate and statins, exert endothelial protective effects. Due to the difference in mechanisms of action, these drugs need to be used according to specific mechanisms underlying endothelial dysfunction of the disease.

Recent epidemiological studies associate health effects and particulate matter in ambient air. Exacerbation of the particle-induced inflammation can be a mechanism responsible for increased hospitalization and death due to cardiopulmonary events in high-risk groups of the population. Systems regulating blood pressure that depend on lung integrity can be involved in progression of cardiovascular diseases. This study focused on the expression levels of various genes involved in cardiovascular and pulmonary diseases to assess their role in the onset of cardiovascular problems due to ambient particulate matter and compared these with the corresponding products. Rats with ozone-induced (<em>1</em>600 microg/m(3); 8 h) pulmonary inflammation were exposed to 0.5 mg, <em>1</em>.5 mg, or 5 mg of particulate matter (PM) from Ottawa Canada (EHC-93) by intratracheal instillation. mRNA levels of various genes and their products were measured 2, 4, and <em>7</em> d after instillation. At 2 d after exposures to PM, tumor necrosis factor (TNF)-alpha levels in bronchoalveolar lavage fluid (BALF) were elevated approximately 4 times for the highest EHC-93 dose. MIP-2 protein levels in BALF were elevated approximately three times during the entire time period studied, whereas IL-6 levels were not affected compared to control groups. The MIP-2 mRNA levels revealed a similar pattern of induction. A twofold increase in endothelin (ET)-<em>1</em> levels at d 2 and a 20% decrease in <em>angiotensin</em>-converting enzyme (ACE) activity at d <em>7</em> were measured in plasma. A 60% decrease of ACE and ET-<em>1</em> mRNA levels suggested a possible endothelial damage in the lung blood vessels. Inducible nitric oxide synthase (iNOS) mRNA was found to be increased 3.5 times 2 d after instillation of the particles. Therefore, the endothelial damage could have been caused by large amounts of the free radical NO. Also, plasma levels of fibrinogen were elevated (20%), which could presumably increase blood viscosity, leading to decreased tissue blood flow. These changes in hematological and hemodynamic parameters observed in our study are in line with heart failure in high-risk groups of the population after high air pollution episodes.