ACE2 Protein Levels Research Articles

Cell cycle dependence of ACE-2 explains downregulation in idiopathic pulmonary fibrosis

Alveolar epithelial type II cells, a major source of angiotensin-converting enzyme (ACE)-2 in the adult lung, are normally quiescent but actively proliferate in lung fibrosis and downregulate this protective enzyme. It was, therefore, hypothesised that ACE-2 expression might be related to cell cycle progression. To test this hypothesis, ACE-2 mRNA levels, protein levels and enzymatic activity were examined in fibrotic human lungs and in the alveolar epithelial cell lines A549 and MLE-12 studied at postconfluent (quiescent) versus subconfluent (proliferating) densities. ACE-2 mRNA, immunoreactive protein and enzymatic activity were all high in quiescent cells, but were severely downregulated or absent in actively proliferating cells. Upregulation of the enzyme in cells that were progressing to quiescence was completely inhibited by the transcription blocker actinomycin D or by SP600125, an inhibitor of c-Jun N-terminal kinase (JNK). In lung biopsy specimens obtained from patients with idiopathic pulmonary fibrosis, immunoreactive enzyme was absent in alveolar epithelia that were positive for proliferation markers, but was robustly expressed in alveolar epithelia devoid of proliferation markers. These data explain the loss of ACE-2 in lung fibrosis and demonstrate cell cycle-dependent regulation of this protective enzyme by a JNK-mediated transcriptional mechanism.

1038 GENE EXPRESSION OF CARDIAC ACE2/ACE AND MAS RECEPTOR IN EXPERIMENTAL HYPERTENSIVE RATS

Objectives: Inter-regulation between components of the RAS is common, but little is known about the direct regulatory effects between cardiac ACE2/ACE and Mas receptor. This study was to examine if the expression of the cardiac ACE2/ACE and Mas receptor changes in hypertensive rats treated with enalapril and losartan. Methods: Forty rats were divided into 4 groups: sham operated (control, n = 10), rats with the aortic ligation (AL, n = 10), AL rats fed enalapril (20 mg kg−1 d−1, n = 10), and AL rats fed losartan (30 mg kg−1 d−1, n = 10). BP was recorded using a tail-cuff method. After 4 weeks of treatment, the levels of ACE2/ACE and Mas receptor in the heart were examined by RT-PCR and western blotting. Results: Cardiac ACE mRNA and ACE protein levels were greater in AL rats than in the control (P < 0.05). In AL rats with enalapril administration, cardiac ACE mRNA and its protein levels were decreased (P < 0.01), but losartan did not affect their levels. However, cardiac ACE2 mRNA and protein levels were lower in AL rats than in the control (P < 0.01). Both enalapril and losartan increased the levels of cardiac ACE2 expression, and BP was lower (P < 0.05). Cardiac Mas mRNA level rose in AL rats treated with losartan, but not enalapril. Conclusions: Our data demonstrated that administration of enalapril and losartan increased the ratio of cardiac ACE2/ACE mRNA levels. Losartan significantly elevated the cardiac Mas expression but not enalapril. These findings suggest that AT1 blockade might increase the activity of the cardiac ACE2-Ang (1-7)-Mas axis of the RAS.

Telmisartan acts through the modulation of ACE-2/ANG 1–7/mas receptor in rats with dilated cardiomyopathy induced by experimental autoimmune myocarditis

AimRecent findings have suggested that a therapeutic approach to amplify or stimulate the angiotensin-converting enzyme-2 [ACE-2]-angiotensin 1–7 [ANG 1–7] mas axis could provide protection against the development of cardiovascular diseases. We investigated the cardioprotective effects of telmisartan in rats with dilated cardiomyopathy [DCM] after experimental autoimmune myocarditis [EAM]. Main methodsDCM was elicited in Lewis rats by immunization with cardiac myosin, and twenty-eight days after immunization, the surviving Lewis rats were divided into two groups and treated with either telmisartan (10mg/kg/day) or vehicle. Key findingsTelmisartan treatment effectively suppressed myocardial protein and mRNA expressions of inflammatory markers [CD68, iNOS, NF-kB, interleukin-1β, interferon-γ, monocyte chemotactic protein-1] in comparison to vehicle-treated rats. In contrast, myocardial protein levels of ACE-2 and ANG 1–7 mas receptor were upregulated in the telmisartan-treated group compared with vehicle-treated rats. Telmisartan treatment significantly reduced fibrosis and hypertrophy and their marker molecules [OPN, CTGF, TGF-β1 and collagens I and III and atrial natriuretic peptide and GATA-4, respectively] compared with those of vehicle-treated rats. In addition, telmisartan treatment significantly lowered the protein expressions of NADPH oxidase subunits p47phox, p67phox, and superoxide production when compared with vehicle-treated rats. Telmisartan treatment significantly decreased the expression levels of mitogen-activated protein kinase (MAPK) signaling molecules than with those of vehicle-treated rats. Also, telmisartan treatment significantly improved LV systolic and diastolic function. SignificanceThese results indicate that telmisartan treatment significantly improved LV function and ameliorated the progression of cardiac remodeling through the modulation of ACE-2/ANG 1–7/Mas receptor axis in rats with DCM after EAM.

Ontogeny of angiotensin-converting enzyme 2

This study examined the temporal expression of angiotensin (Ang)-converting enzyme 2 (ACE2) during renal, heart, lung, and brain organogenesis in the mouse. We demonstrate that kidney ACE2 mRNA levels are low on embryonic day (E) 12.5, increase fourfold during development, and decline in adulthood. In extrarenal tissues, ACE2 mRNA levels are also low during early gestation, increase in perinatal period, and peak in adulthood. The lung shows the highest age-related increase in ACE2 mRNA levels followed by the brain, kidney, and heart. ACE2 protein levels and enzymatic activity are high in all organs studied during gestation and decline postnatally. Ang II decreases ACE2 mRNA levels and enzymatic activity in kidneys grown ex vivo. These effects of Ang II are blocked by the specific Ang II AT(1) receptor (AT(1)R) antagonist candesartan, but not by the AT(2) receptor (AT(2)R) antagonist PD123319. We conclude that ACE2 gene and protein expression and enzymatic activity are developmentally regulated in a tissue-specific manner. Ang II, acting through AT(1)R, exerts a negative feedback on ACE2 during kidney development. We postulate that relatively high ACE2 protein levels and enzymatic activity observed during gestation may play a role in kidney, lung, brain, and heart organogenesis.

Antenatal Maternal Hypoxic Stress: Adaptations in Fetal Lung Renin-Angiotensin System

Antenatal maternal hypoxia (AMH) can lead to intrauterine growth restriction (IUGR), as well as idiopathic pulmonary hypertension of newborn and adult, the latter of which may be a consequence of alterations in the local pulmonary renin-angiotensin system (RAS). Little is known of these adaptations, however. Thus, we tested the hypothesis that antenatal maternal hypoxia is associated with alterations in gene and protein expression of the pulmonary renin-angiotensin system, which may play an important role in pulmonary disorders in the offspring. In FVB/NJ mice, we studied messenger RNA (mRNA) and protein expression, as well as promoter DNA methylation and microRNA (miRNA) levels in response to 48 hours hypoxia (10.5% O(2)) at 15.5 day post coitum (DPC). In response to AMH, the pulmonary mRNA levels of angiotensin-converting enzyme (ACE) 1.2, ACE-2, and angiotensin II type 1b (AT-1b) receptors were increased significantly, as compared to controls (N = 4). In response to antenatal hypoxia, pulmonary protein levels of renin and ACE-2 also were increased significantly, whereas ACE-1 protein expression was reduced. In fetal lungs, we also observed reduced expression of the miRNAs: mmu-mir -199b, -27b, -200b, and -468 that putatively increase the translation of renin, ACE-1, ACE-2, and AT-1 receptors, respectively. In response to AMH, promoter methylation of ACE was unchanged. We conclude that AMH leads to changes in expression of pulmonary RAS of fetal mice. The possible implications of these changes for the regulation of pulmonary vascular contractility in later life remain to be explored.

Cardiovascular Interactions Between Losartan and Fructose in Mice

To determine whether pharmacological blockade of angiotensin (Ang) AT1 receptors alters the cardiovascular, metabolic, and angiotensin-converting enzyme (ACE and ACE2) responses to a fructose diet in mice. C57BL male mice were fed with a 60% fructose diet for 8 weeks in combination with losartan treatment on week 9 (30 mg/kg per day). Blood pressure (BP), heart rate (HR), and autonomic balance were monitored using radiotelemetry with spectral analysis. Renal ACE and ACE2 activity and protein levels as well as Ang II and Ang 1-7 were measured. Fructose impaired glucose tolerance and increased plasma cholesterol and insulin. These effects were not corrected by losartan treatment. Fructose increased BP and HR but only during the dark period. Short-term losartan treatment decreased BP by 16% in the fructose group but had no effect in controls. This was accompanied by a decrease in BP variance and its low-frequency component. Fructose increased Ang II (plasma and kidney) and ACE 2 (renal activity and protein expression). Losartan alone increased plasma Ang II in plasma and ACE2 in kidney. There were no changes in renal Ang 1-7 levels. Losartan reversed the pressor effect of a high fructose diet, demonstrating that there are prominent interactions between a dietary regimen that produces glucose intolerance and an antihypertensive drug that antagonizes Ang signaling. The mechanism of change may be via renal Ang II rather than the ACE2/Ang 1-7 pathway because the fructose losartan combination resulted in lowered renal Ang II without changes in Ang 1-7.

Role of HIF-1α in the regulation ACE and ACE2 expression in hypoxic human pulmonary artery smooth muscle cells

Angiotensin-converting enzyme (ACE) enhances the proliferation and migration of pulmonary artery smooth muscle cells (PASMCs), which contribute to the pathogenesis of hypoxic pulmonary hypertension (HPH). Previous reports have demonstrated that hypoxia upregulates ACE expression, but the underlying mechanism is unknown. Here, we found that ACE is persistently upregulated in PASMCs on the transcriptional level during hypoxia. Hypoxia-inducible factor 1alpha (HIF-1alpha), a key transcription factor activated during hypoxia, was able to upregulate ACE protein expression under normoxia, whereas knockdown of HIF-1alpha expression in PASMCs inhibited hypoxia-induced ACE upregulation. Furthermore, HIF-1alpha can bind and transactivate the ACE promoter directly. Therefore, we report that ACE is a novel target of HIF-1alpha. Recently, a homolog of ACE, ACE2, was reported to counterbalance the function of ACE. In contrast to ACE, we found that ACE2 mRNA and protein levels increased during the early stages of hypoxia and decreased to near-baseline levels at the later stages after HIF-1alpha accumulation. Thus HIF-1alpha inhibited ACE2 expression, and the accumulated ANG II catalyzed by ACE is a key mediator in the downregulation of ACE2 by HIF-1alpha. Moreover, a reduction of ACE2 expression in PASMCs by RNA interference was accompanied by significantly enhanced proliferation and migration during hypoxia. We conclude that ACE is directly regulated by HIF-1alpha, whereas ACE2 is regulated in a bidirectional way during hypoxia and may play a protective role during the development of HPH. In sum, these findings contribute to the understanding of the pathogenesis of HPH.

Discrepancy between Intrarenal Messenger RNA and Protein Expression of ACE and ACE2 in Human Diabetic Nephropathy

Background: The intrarenal angiotensin-converting enzyme (ACE) and type 2 ACE (ACE2) play important roles in the pathogenesis of diabetic nephropathy, but human data are limited. We studied glomerular and tubulointerstitial mRNA and the protein expression of ACE and ACE2 in patients with diabetic nephropathy. Methods: We studied renal biopsy specimens of 22 patients with diabetic nephropathy and 11 transplant donors as normal controls. Intrarenal mRNA expression of ACE and ACE2 was measured by laser microdissection and real-time quantitative polymerase chain reaction; expression at the protein level was determined by immunostaining. Results: Glomerular and tubulointerstitial mRNA expression levels of ACE and ACE2 were significantly higher in patients with diabetic nephropathy than in normal controls (p < 0.001 for all comparisons). Glomerular ACE and ACE2 protein levels of patients with diabetic nephropathy were significantly higher than those of kidney donors (4.90 ± 2.55% vs. 2.64 ± 0.98%, p = 0.022, and 7.40 ± 3.36% vs. 4.37 ± 2.36%, p = 0.017, respectively). The tubulointerstitial ACE at the protein level, however, was similar between diabetic patients and controls (8.76 ± 4.18% vs. 10.44 ± 6.61%, p = 0.453), and the tubulointerstitial ACE2 at the protein level was significantly lower in diabetic nephropathy (16.48 ± 7.68% vs. 23.23 ± 7.65%, p = 0.025). Conclusion: The mRNA expression of ACE and ACE2 increased in both the glomerular and tubulointerstitial area of diabetic nephropathy. However, the tubulointerstitial ACE expression at the protein level remained unchanged, while that of ACE2 actually decreased. Our results suggest a posttranscriptional modulation of tubulointerstitial ACE and ACE2 expression. Experimental data of intrarenal mRNA expression of ACE and ACE2 should be interpreted with caution.

High glucose-induced Nox1-derived superoxides downregulate PKC-βII, which subsequently decreases ACE2 expression and ANG(1-7) formation in rat VSMCs

In rat diabetic animal models, ANG(1-7) treatment prevents the development of cardiovascular complications. Angiotensin-converting enzyme (ACE)2 is a major ANG(1-7)-generating enzyme in vascular smooth muscle cells (VSMCs), and its expression is decreased by a prolonged exposure to high glucose (HG), which is reflected by lower ANG(1-7) levels. However, the underlying mechanism of its downregulation is unknown and was the subject of this study. Rat aortic VSMCs were maintained in normal glucose (NG) or HG ( approximately 4.1 and approximately 23.1 mmol/l, respectively) for up to 72 h. Several PKC and NADPH oxidase inhibitors and short interfering (si)RNAs were used to determine the mechanism of HG-induced ACE2 downregulation. Cell lysates were subjected to Western blot analysis, real-time quantitative PCR, and ANG(1-7) radioimmunodetection. At 72 h of HG exposure, ACE2 mRNA, protein, and ANG(1-7) levels were decreased (0.17 +/- 0.01-, 0.47 +/- 0.03-, and 0.16 +/- 0.01-fold, respectively), and the expression of NADPH oxidase subunit Nox1 was increased (1.70 +/- 0.2-fold). The HG-induced ACE2 decrease was reversed by antioxidants and Nox1 siRNA as well as by inhibitors of glycotoxin formation. ACE2 expression was PKC-betaII dependent, and PKC-betaII protein levels were reduced in the presence of HG (0.32 +/- 0.03-fold); however, the PKC-betaII inhibitor CG-53353 prevented the HG-induced ACE2 loss and Nox1 induction, suggesting a nonspecific effect of the inhibitor. Our data suggest that glycotoxin-induced Nox1 expression is regulated by conventional PKCs. ACE2 expression is PKC-betaII dependent. Nox1-derived superoxides reduce PKC-betaII expression, which lowers ACE2 mRNA and protein levels and consequently decreases ANG(1-7) formation.

Angiotensin-Converting Enzyme II in the Heart and the Kidney

The renin-angiotensin system (RAS) has been recognized for many years as critical pathway for blood pressure control and kidney functions. Although most of the well-known cardiovascular and renal effects of RAS are attributed to angiotensin-converting enzyme (ACE), much less is known about the function of ACE2. Experiments using genetically modified mice and inhibitor studies have shown that ACE2 counterbalances the functions of ACE and that the balance between these two proteases determines local and systemic levels of RAS peptides such as angiotensin II and angiotensin1-7. Ace2 mutant mice exhibit progressive impairment of heart contractility at advanced ages, a phenotype that can be reverted by loss of ACE, suggesting that these enzymes directly control heart function. Moreover, ACE2 is also found to be upregulated in failing hearts. In the kidney, ACE2 protein levels are significantly decreased in hypertensive rats, suggesting a negative regulatory role of ACE2 in blood pressure control. Moreover, ACE2 expression is downregulated in the kidneys of diabetic and pregnant rats and ACE2 mutant mice develop late onset glomerulonephritis resembling diabetic nephropathy. Importantly, ACE2 not only controls angiotensin II levels but functions as a protease on additional molecular targets that could contribute to the observed in vivo phenotypes of ACE2 mutant mice. Thus, ACE2 seems to be a molecule that has protective roles in heart and kidney. The development of drugs that could activate ACE2 function would allow extending our treatment options in diabetic nephropathy, heart failure, or hypertension.

Effects of spironolactone and eprosartan on cardiac remodeling and angiotensin-converting enzyme isoforms in rats with experimental heart failure

Angiotensin-converting enzyme (ACE)-2 is a newly described enzyme with antagonistic effects to those of the classical ACE (ACE-1). Both ANG II and aldosterone play an important role in the pathophysiology of congestive heart failure (CHF) and in the adverse cardiac remodeling during its development. In this study, we examined the effects of experimental CHF induced by an aortocaval fistula (ACF) and of its treatment with ANG II and aldosterone inhibitors on the relative levels of ACE-1 and ACE-2. We also compared the effects of spironolactone, an aldosterone antagonist, and eprosartan, an ANG II receptor antagonist, on heart hypertrophy and fibrosis in rats with ACF. Spironolactone (15 mg x kg(-1) x day(-1) ip, via minipump) or eprosartan (5 mg x kg(-1) x day(-1) ip, via minipump) was administered into rats with ACF for 14 and 28 days. Specific antibodies were used to determine the protein levels of myocardial ACE-1 and ACE-2. ACF increased the cardiac levels of ACE-1 and decreased those of ACE-2. Heart-to-body weight ratio significantly increased from 0.30 +/- 0.004% in sham-operated controls to 0.50 +/- 0.018% and 0.56 +/- 0.044% (P < 0.001) in rats with ACF, 2 and 4 wk after surgery, respectively, in association with increased plasma levels of aldosterone. The area occupied by collagen increased from 2.33 +/- 0.27% to 6.85 +/- 0.65% and 8.03 +/- 0.93% (P < 0.01), 2 and 4 wk after ACF, respectively. Both spironolactone and eprosartan decreased cardiac mass and collagen content and reversed the shift in ACE isoforms. ACF alters the ratio between ACE isoforms in a manner that increases local ANG II and aldosterone levels. Early treatment with both ANG II and aldosterone antagonists is effective in reducing this effect. Thus ACE isoform shift may represent an important component of the development of cardiac remodeling in response to hemodynamic overload, and its correction may contribute to the beneficial therapeutic effects of renin-angiotensin-aldosterone system inhibitors.