mln-4760 and Disease-Models--Animal

Angiotensin-converting enzyme 2 (ACE2) is a potent negative regulator capable of restraining overactivation of the renin-angiotensin system, which contributes to exuberant inflammation after bacterial infection. However, the mechanism through which ACE2 modulates this inflammatory response is not well understood. Accumulating evidence indicates that infectious insults perturb ACE2 activity, allowing for uncontrolled inflammation. In the current study, we demonstrate that pulmonary ACE2 levels are dynamically varied during bacterial lung infection, and the fluctuation is critical in determining the severity of bacterial pneumonia. Specifically, we found that a pre-existing and persistent deficiency of active ACE2 led to excessive neutrophil accumulation in mouse lungs subjected to bacterial infection, resulting in a hyperinflammatory response and lung damage. In contrast, pre-existing and persistent increased ACE2 activity reduces neutrophil infiltration and compromises host defense, leading to overwhelming bacterial infection. Further, we found that the interruption of pulmonary ACE2 restitution in the model of bacterial lung infection delays the recovery process from neutrophilic lung inflammation. We observed the beneficial effects of recombinant ACE2 when administered to bacterially infected mouse lungs following an initial inflammatory response. In seeking to elucidate the mechanisms involved, we discovered that ACE2 inhibits neutrophil infiltration and lung inflammation by limiting IL-17 signaling by reducing the activity of the STAT3 pathway. The results suggest that the alteration of active ACE2 is not only a consequence of bacterial lung infection but also a critical component of host defense through modulation of the innate immune response to bacterial lung infection by regulating neutrophil influx.

Topics: Angiotensin-Converting Enzyme 2; Animals; Disease Models, Animal; Female; Imidazoles; Immunity, Innate; Inflammation; Leucine; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microbial Sensitivity Tests; Neutrophils; Peptidyl-Dipeptidase A; Pseudomonas aeruginosa; Pseudomonas Infections

Previous studies have reported that lipoxin A4 (LXA4) and the angiotensin I-converting enzyme 2 (ACE2), angiotensin-(1-7) [Ang-(1-7)], and its receptor Mas [ACE2-Ang-(1-7)-Mas] axis play important protective roles in acute lung injury (ALI). However, there is still no direct evidence of LXA4-mediated protection via the ACE2-Ang-(1-7)-Mas axis during ALI. This work was performed using an LPS-induced ALI mouse model and the data indicated the following. First, the animal model was established successfully and LXA4 ameliorated LPS-induced ALI. Second, LXA4 could increase the concentration and activity of ACE2 and the levels of Ang-(1-7) and Mas markedly. Third, LXA4 decreased the levels of TNF-α, IL-1β, and reactive oxygen species while increasing IL-10 levels. Fourth, LXA4 inhibited the activation of the NF-κB signal pathway and repressed the degradation of inhibitor of NF-κB, the phosphorylation of NF-κB, and the translocation of NF-κB. Finally, and more importantly, BOC-2 (LXA4 receptor inhibitor), MLN-4760 (ACE2 inhibitor), and A779 (Mas receptor antagonist) were found to reverse all of the effects of LXA4. Our data provide evidence that LXA4 protects the lung from ALI through regulation of the ACE2-Ang-(1-7)-Mas axis.

Topics: Acute Lung Injury; Angiotensin I; Angiotensin II; Angiotensin-Converting Enzyme 2; Animals; Cell Line, Tumor; Disease Models, Animal; Humans; Imidazoles; Leucine; Lipopolysaccharides; Lipoxins; Male; Mice; NF-kappa B; Peptide Fragments; Peptidyl-Dipeptidase A; Proto-Oncogene Mas; Proto-Oncogene Proteins; Receptors, G-Protein-Coupled; Signal Transduction

This study aims to investigate the effect of angiotensin-converting enzyme 2 (ACE2) activation on pulmonary endothelial function in the process of preventing pulmonary arterial hypertension (PAH) in rat models and to explore the underlying mechanisms. Specific pathogen free rats were randomly divided into five groups including control group, PAH group, PAH + Resorcinolnaphthalein (Res) group (ACE2 activation), PAH + Res + MLN4760 group (ACE2 inhibition), and PAH + Res + L-NAME group (endothelial nitric oxide synthase [eNOS] inhibition). Rat PAH model was constructed using combined left pneumonectomy with a single dose of monocrotaline injection 1 week after the surgery, and the rats were then given corresponding reagents. Hemodynamics, endothelial function, and pathologic changes were evaluated 3 weeks after monocrotaline injection. The concentration of nitric oxide (NO), expression of eNOS, and phosphorylation of eNOS at Ser1177 and Thr495 in the lung tissues from rats were also investigated.The Res-induced activation of ACE2 led to decreased mean pulmonary arterial pressure (mPAP) and pulmonary artery remodeling in the PAH + Res group comparing with the PAH rats (P < .05). In addition, the reduction in mPAP induced by acetylcholine (Ach) was augmented in PAH + Res group (P < .05), but this was not observed under the treatment with sodium nitroprusside (SNP) (P > .05). The ratio of decrease in mPAP caused by Ach to that caused by SNP (Ach/SNP) was also increased (P < .05) in ACE2-activated rats. However, the protective effects of ACE2 activation on PAH were counteracted by co-administration of MLN4760, an ACE2 antagonist (all P > .05). The mechanistic study showed that the concentration of NO in the lung tissues was downregulated in the PAH group but upregulated in the PAH + Res group (P < .05), whereas the NO concentration in the PAH + Res + MLN4760 group was not obviously different from that in the PAH group (P > .05). Regarding the factors regulating NO release, we found that the eNOS was upregulated in the PAH group, and Res did not affect the expression of eNOS. The phosphorylation of eNOS at Ser1177 was increased but at Thr495 was reduced after Res injection, when compared with the PAH group (P < .05). As expected, co-injection of MLN4760 eliminated these differences (P > .05). The reduction in mPAP induced by Ach was attenuated in the PAH + Res + L-NAME group compared with the PAH + Res group (P < .05), but this was not observed in rats treated wi

Topics: Angiotensin-Converting Enzyme 2; Angiotensin-Converting Enzyme Inhibitors; Animals; Disease Models, Animal; Endothelium, Vascular; Enzyme Activators; Humans; Hypertension, Pulmonary; Imidazoles; Leucine; Lung; Male; Monocrotaline; Naphthalenes; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase Type III; Peptidyl-Dipeptidase A; Phosphorylation; Pulmonary Artery; Pyrans; Rats; Rats, Sprague-Dawley; Signal Transduction; Specific Pathogen-Free Organisms; Spiro Compounds; Xanthenes

The angiotensin-converting enzyme 2/angiotensin-(1-7)/Mas axis represents a promising target for inducing stroke neuroprotection. Here, we explored stroke-induced changes in expression and activity of endogenous angiotensin-converting enzyme 2 and other system components in Sprague-Dawley rats. To evaluate the clinical feasibility of treatments that target this axis and that may act in synergy with stroke-induced changes, we also tested the neuroprotective effects of diminazene aceturate, an angiotensin-converting enzyme 2 activator, administered systemically post stroke. Among rats that underwent experimental endothelin-1-induced ischemic stroke, angiotensin-converting enzyme 2 activity in the cerebral cortex and striatum increased in the 24 hours after stroke. Serum angiotensin-converting enzyme 2 activity was decreased within 4 hours post stroke, but rebounded to reach higher than baseline levels 3 days post stroke. Treatment after stroke with systemically applied diminazene resulted in decreased infarct volume and improved neurological function without apparent increases in cerebral blood flow. Central infusion of A-779, a Mas receptor antagonist, resulted in larger infarct volumes in diminazene-treated rats, and central infusion of the angiotensin-converting enzyme 2 inhibitor MLN-4760 alone worsened neurological function. The dynamic alterations of the protective angiotensin-converting enzyme 2 pathway after stroke suggest that it may be a favorable therapeutic target. Indeed, significant neuroprotection resulted from poststroke angiotensin-converting enzyme 2 activation, likely via Mas signaling in a blood flow-independent manner. Our findings suggest that stroke therapeutics that target the angiotensin-converting enzyme 2/angiotensin-(1-7)/Mas axis may interact cooperatively with endogenous stroke-induced changes, lending promise to their further study as neuroprotective agents.

Topics: ADAM Proteins; ADAM17 Protein; Angiotensin II; Angiotensin-Converting Enzyme 2; Animals; Cerebral Cortex; Cerebral Infarction; Cerebrovascular Circulation; Corpus Striatum; Diminazene; Disease Models, Animal; Endothelin-1; Enzyme Activation; Imidazoles; Infarction, Middle Cerebral Artery; Infusions, Intraventricular; Leucine; Male; Neuroprotective Agents; Peptide Fragments; Peptidyl-Dipeptidase A; Proto-Oncogene Mas; Proto-Oncogene Proteins; Random Allocation; Rats; Rats, Sprague-Dawley; Receptors, G-Protein-Coupled; Renin-Angiotensin System; RNA, Messenger

Angiotensin (Ang)-converting enzyme 2 (ACE2) is a key enzyme in the metabolism of Ang II. XNT (1-[(2-dimethylamino)ethylamino]-4-(hydroxymethyl)-7-[(4-methylphenyl) sulfonyl oxy]-9H-xanthene-9-one) and diminazene have been reported to exert various organ-protective effects, which are attributed to the activation of ACE2. To test the effect of these compounds, we studied Ang II degradation in vivo and in vitro as well as their effect on ACE2 activity in vivo and in vitro. In a model of Ang II-induced acute hypertension, blood pressure (BP) recovery was markedly enhanced by XNT (slope with XNT, -3.26±0.2 versus -1.6±0.2 mm Hg/min without XNT; P<0.01). After Ang II infusion, neither plasma nor kidney ACE2 activity was affected by XNT. Plasma Ang II and Ang (1-7) levels also were not significantly affected by XNT. The BP-lowering effect of XNT seen in wild-type animals was also observed in ACE2 knockout mice (slope with XNT, -3.09±0.30 versus -1.28±0.22 mm Hg/min without XNT; P<0.001). These findings show that the BP-lowering effect of XNT in Ang II-induced hypertension cannot be because of the activation of ACE2. In vitro and ex vivo experiments in both mice and rat kidney confirmed a lack of enhancement of ACE2 enzymatic activity by XNT and diminazene. Moreover, Ang II degradation in vitro and ex vivo was unaffected by XNT and diminazene. We conclude that the biological effects of these compounds are ACE2-independent and should not be attributed to the activation of this enzyme.

Topics: Angiotensin I; Angiotensin II; Angiotensin-Converting Enzyme 2; Animals; Blood Pressure; Diminazene; Disease Models, Animal; Glutamyl Aminopeptidase; Hypertension; Imidazoles; In Vitro Techniques; Kidney; Leucine; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Peptide Fragments; Peptidyl-Dipeptidase A; Rats; Xanthones

A newly produced murine recombinant angiotensin (Ang)-converting enzyme 2 (ACE2) was characterized in vivo and in vitro. The effects of available ACE2 inhibitors (MLN-4760 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 (1 mg/kg) obliterated hypertension induced by Ang II infusion by rapidly decreasing plasma Ang II. These effects were blocked by MLN-4760 but not by either form of DX600. In vitro, conversion from Ang II to Ang-(1-7) by mouse ACE2 was blocked by MLN-4760 (10(-6) m) but not by either form of DX600 (10(-5) m). Quantitative analysis of multiple Ang peptides in plasma ex vivo revealed formation of Ang-(1-9) 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 (1-7). By contrast, mouse ACE2-driven Ang-(1-7) formation from Ang II was blocked by MLN-4760 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-(1-9). There are also species differences regarding rodent and human ACE2 inhibition by known inhibitors such that MLN-4760 inhibits both human and mouse ACE2, whereas DX600 only blocks human ACE2 activity.

Topics: Angiotensin I; Angiotensin II; Angiotensin-Converting Enzyme 2; Angiotensin-Converting Enzyme Inhibitors; Animals; Blood Pressure; Disease Models, Animal; Humans; Hydrolysis; Hypertension; Imidazoles; In Vitro Techniques; Kidney; Leucine; Male; Mice; Mice, Inbred C57BL; Peptide Fragments; Peptides; Peptidyl-Dipeptidase A; Recombinant Proteins

Angiotensin-converting enzyme 2 (ACE2) is expressed at high levels in the kidney and converts angiotensin II (ANG II) to ANG-(1-7). We studied the effects of ACE2 inhibition and ANG-(1-7) in the (5/6) nephrectomy ((5/6) Nx) mouse model of chronic kidney disease (CKD). Male FVB mice underwent sham surgery (Sham) or (5/6) Nx and were administered either vehicle, the ACE2 inhibitor MLN-4760 (MLN), the AT(1) receptor antagonist losartan, MLN plus losartan, or ANG-(1-7) for 4 wk. In (5/6) Nx mice with or without MLN, kidney cortical ACE2 protein expression was significantly decreased at 4 wk, compared with Sham. Inhibition of ACE2 caused a decrease in renal cortical ACE2 activity. Kidney cortical ACE expression and activity did not differ between groups of mice. In (5/6) Nx mice treated with MLN, kidney levels of ANG II were significantly increased, compared with Sham. (5/6) Nx induced a mild but insignificant increase in blood pressure (BP), a 50% reduction in FITC-inulin clearance, and a significant increase in urinary albumin excretion. ACE2 inhibition in (5/6) Nx mice did not affect BP or FITC-inulin clearance but significantly increased albuminuria compared with (5/6) Nx alone, an effect reversed by losartan. Treatment of (5/6) Nx mice with ANG-(1-7) increased kidney and plasma levels of ANG-(1-7) but did not alter BP, FITC-inulin clearance, or urinary albumin excretion, and it increased relative mesangial area. These data indicate that kidney ACE2 is downregulated in the early period after (5/6) Nx. Inhibition of ACE2 in (5/6) Nx mice increases albuminuria via an AT(1) receptor-dependent mechanism, independent of BP. In contrast, ANG-(1-7) does not affect albuminuria after (5/6) Nx. We propose that endogenous ACE2 is renoprotective in CKD.

Topics: Albuminuria; Angiotensin I; Angiotensin II; Angiotensin II Type 1 Receptor Blockers; Angiotensin-Converting Enzyme 2; Angiotensin-Converting Enzyme Inhibitors; Animals; Blood Pressure; Body Weight; Chronic Disease; Disease Models, Animal; Glomerular Filtration Rate; Hematocrit; Imidazoles; Infusion Pumps; Injections, Subcutaneous; Inulin; Kidney; Kidney Diseases; Leucine; Losartan; Male; Mice; Nephrectomy; Organ Size; Peptide Fragments; Peptidyl-Dipeptidase A; Proto-Oncogene Mas; Proto-Oncogene Proteins; Receptor, Angiotensin, Type 1; Receptors, G-Protein-Coupled; Time Factors

Angiotensin-converting enzyme 2 (ACE2) is expressed in gastrointestinal tissue. Previous studies of GL1001, a potent and selective ACE2 inhibitor, have revealed anti-inflammatory activity in the mouse digestive tract. We hypothesized that GL1001 might also produce beneficial effects in a mouse DSS model of inflammatory bowel disease.. Female mice were used for study.. Animals were treated for 5 days with 5% DSS in the drinking water to induce colitis. For the following 9 days, animals were treated twice daily with GL1001 (30, 100, 300 mg/kg, s.c.), sulfasalazine (150 mg/kg, p.o.), or vehicle.. Throughout the experiment, body weight, rectal prolapse, stool consistency, and fecal occult blood were monitored. At termination, colon length, histopathology, and myeloperoxidase activity were assessed.. High-dose GL1001 ameliorated DSS-induced disease activity, including rectal prolapse and intestinal bleeding. The most robust effect of GL1001 was observed 48-96 h post DSS treatment and was comparable in magnitude to that of sulfasalazine. Colon pathology and myeloperoxidase activity were also markedly attenuated by high-dose GL1001 treatment, with the most profound effects observed in the distal segment.. The findings support the previously observed anti-inflammatory effects of ACE2 inhibition in gastrointestinal tissue and suggest that GL1001 may have therapeutic utility for inflammatory bowel disease.

Topics: Angiotensin-Converting Enzyme 2; Angiotensin-Converting Enzyme Inhibitors; Animals; Body Weight; Colitis; Colon; Dextran Sulfate; Disease Models, Animal; Female; Humans; Imidazoles; Inflammatory Bowel Diseases; Leucine; Mice; Mice, Inbred BALB C; Peptidyl-Dipeptidase A; Peroxidase; Random Allocation

Angiotensin-converting enzyme-2 (ACE2) converts angiotensin II (ANG II) to angiotensin-(1-7) [ANG-(1-7)], and this enzyme may serve as a key regulatory juncture in various tissues. Although the heart expresses ACE2, the extent that the enzyme participates in the cardiac processing of ANG II and ANG-(1-7) is equivocal. Therefore, we utilized the Langendorff preparation to characterize the ACE2 pathway in isolated hearts from male normotensive Sprague-Dawley [Tg((-))] and hypertensive [mRen2]27 [Tg((+))] rats. During a 60-min recirculation period with 10 nM ANG II, the presence of ANG-(1-7) was assessed in the cardiac effluent. ANG-(1-7) generation from ANG II was similar in both the normal and hypertensive hearts [Tg((-)): 510 +/- 55 pM, n=20 vs. Tg((+)): 497 +/- 63 pM, n=14] with peak levels occurring at 30 min after administration of the peptide. ACE2 inhibition (MLN-4760, 1 microM) significantly reduced ANG-(1-7) production by 83% (57 +/- 19 pM, P<0.01, n=7) in the Tg((+)) rats, whereas the inhibitor had no significant effect in the Tg((-)) rats (285 +/- 53 pM, P>0.05, n=10). ACE2 activity was found in the effluent of perfused Tg((-)) and Tg((+)) hearts, and it was highly associated with ACE2 protein expression (r=0.78). This study is the first demonstration for a direct role of ACE2 in the metabolism of cardiac ANG II in the hypertrophic heart of hypertensive rats. We conclude that predominant expression of cardiac ACE2 activity in the Tg((+)) may be a compensatory response to the extensive cardiac remodeling in this strain.

Topics: Angiotensin I; Angiotensin II; Angiotensin-Converting Enzyme 2; Angiotensin-Converting Enzyme Inhibitors; Animals; Animals, Genetically Modified; Cardiomegaly; Disease Models, Animal; Half-Life; Hypertension; Imidazoles; Kinetics; Leucine; Male; Mice; Myocardium; Peptide Fragments; Peptidyl-Dipeptidase A; Rats; Rats, Sprague-Dawley; Renin