Bei Song

Angiotensin-converting enzyme 2 attenuates oxidative stress and VSMC proliferation via the JAK2/STAT3/SOCS3 and profilin-1/MAPK signaling pathways

Angiotensin (Ang) II plays a vital role in vascular smooth muscle cell (VSMC) growth and proliferation. Angiotensin-converting enzyme 2 (ACE2) is a specific Ang II-degrading enzyme but its role in VSMC proliferation remains largely unknown. We hypothesized that ACE2 might suppress Ang II-mediated oxidative stress and VSMC proliferation. Human umbilical artery smooth muscle cells (HUASMCs) were pretreated with Ang II (100nM) for 6h and 24h, respectively. Exposure to Ang II resulted in significant increases in suppressor of cytokine signaling 3 (SOCS3) expression and phosphorylation levels of JAK2, STAT3 and ERK1/2 linked with elevated superoxide production and cell proliferation in HUASMCs. These changes were strikingly prevented by administration of ERK1/2 inhibitor PD98059 (10μM) and JAK/STAT inhibitor WP1066 (5 μM) but were largely aggravated by ACE2 inhibitor DX600 (0.5 μM). More importantly, treatment with human recombinant ACE2 (hrACE2; 1mg/ml) dramatically prevented Ang II-mediated SOCS3 expression and the JAK2-STAT3 and ERK1/2 signaling, and resulted in attenuation of superoxide production and cell proliferation in HUASMCs. Intriguingly, downregulation of profilin-1 with profilin-1 siRNA (50 nM) was able to abolish Ang II-induced upregulations of profilin-1 expression, ERK1/2 phosphorylation and superoxide production with attenuation of VSMC proliferation. In conclusion, treatment with hrACE2 prevents Ang II-mediated activation of the JAK2/STAT3/SOCS3 and profilin-1/MAPK signaling pathways, contributing to attenuation of superoxide generation and cell proliferation in HUASMCs, suggesting a protective mechanism of ACE2 against Ang II-mediated oxidative stress and VSMC proliferation. ACE2 may represent a potential candidate to prevent and treat vascular disorders.

Angiotensin-converting enzyme 2 ameliorates renal fibrosis by blocking the activation of mTOR/ERK signaling in apolipoprotein E-deficient mice

Angiotensin-converting enzyme 2 (ACE2) has been shown to prevent atherosclerotic lesions and renal inflammation. However, little was elucidated upon the effects and mechanisms of ACE2 in atherosclerotic kidney fibrosis progression. Here, we examined regulatory roles of ACE2 in renal fibrosis in the apolipoprotein E (ApoE) knockout (KO) mice. The ApoEKO mice were randomized to daily deliver either angiotensin (Ang) II (1.5mg/kg) and/or human recombinant ACE2 (rhACE2; 2mg/kg) for 2 weeks. Downregulation of ACE2 and upregulation of phosphorylated Akt, mTOR and ERK1/2 levels were observed in ApoEKO kidneys. Ang II infusion led to increased tubulointerstitial fibrosis in the ApoEKO mice with greater activation of the mTOR/ERK1/2 signaling. The Ang II-mediated renal fibrosis and structural injury were strikingly rescued by rhACE2 supplementation, associated with reduced mRNA expression of TGF-β1 and collagen I and elevated renal Ang-(1-7) levels. In cultured mouse kidney fibroblasts, exposure with Ang II (100nmolL(-1)) resulted in obvious elevations in superoxide generation, phosphorylated levels of mTOR and ERK1/2 as well as mRNA levels of TGF-β1, collagen I and fibronectin 1, which were dramatically prevented by rhACE2 (1mgmL(-1)) or mTOR inhibitor rapamycin (10μmolL(-1)). These protective effects of rhACE2 were eradicated by the Ang-(1-7)/Mas receptor antagonist A779 (1μmolL(-1)). Our results demonstrate the importance of ACE2 in amelioration of kidney fibrosis and renal injury in the ApoE-mutant mice via modulation of the mTOR/ERK signaling and renal Ang-(1-7)/Ang II balance, thus indicating potential therapeutic strategies by enhancing ACE2 action for preventing atherosclerosis and fibrosis-associated kidney disorders.

The sirtuin 6 prevents angiotensin II-mediated myocardial fibrosis and injury by targeting AMPK-ACE2 signaling

Sirtuin 6 (SIRT6) is an important modulator of cardiovascular functions in health and diseases. However, the exact role of SIRT6 in heart disease is poorly defined. We hypothesized that SIRT6 is a negative regulator of angiotensin II (Ang II)-mediated myocardial remodeling, fibrosis and injury. The male Sprague-Dawley rats were randomized to Ang II (200 ng/kg/min) infusion with an osmotic minipump and pretreated with recombinant plasmids adeno-associated viral vector (AAV)-SIRT6 (pAAV-SIRT6) or pAAV-GFP for 4 weeks. Ang II triggered downregulated levels of SIRT6 and angiotensin-converting enzyme 2 (ACE2) and upregulated expression of connective tissue growth factor (CTGF) and proinflammatory chemokine fractalkine (FKN), contributing to enhanced cardiac fibrosis and ultrastructural injury. Reduced levels of phosphorylated pAMPK-α, increased myocardial hypertrophy and impaired heart dysfunction were observed in both Ang II-induced hypertensive rats and ACE2 knockout rats, characterized with increases in heart weight and left ventricular (LV) posterior wall thickness and decreases in LV ejection fraction and LV fractional shortening. More importantly, pAAV-SIRT6 treatment strikingly potentiated cardiac levels of pAMPKα and ACE2 as well as decreased levels of CTGF, FKN, TGFβ1, collagen I and collagen III, resulting in alleviation of Ang II-induced pathological hypertrophy, myocardial fibrosis, cardiac dysfunction and ultrastructural injury in hypertensive rats. In conclusion, our findings confirmed cardioprotective effects of SIRT6 on pathological remodeling, fibrosis and myocardial injury through activation of AMPK-ACE2 signaling and suppression of CTGF-FKN pathway, indicating that SIRT6 functions as a partial agonist of ACE2 and targeting SIRT6 has potential therapeutic importance for cardiac fibrosis and heart disease.

Apelin Is a Negative Regulator of Angiotensin II–Mediated Adverse Myocardial Remodeling and Dysfunction

The apelin pathway has emerged as a critical regulator of cardiovascular homeostasis and disease. However, the exact role of pyr1-apelin-13 in angiotensin (Ang) II–mediated heart disease remains unclear. We used apelin-deficient (APLN−/y) and apolipoprotein E knockout mice to evaluate the regulatory roles of pyr1-apelin-13. The 1-year aged APLN−/y mice developed myocardial hypertrophy and dysfunction with reduced angiotensin-converting enzyme 2 levels. Ang II infusion (1.5 mg kg−1 d−1) for 4 weeks potentiated oxidative stress, pathological hypertrophy, and myocardial fibrosis in young APLN−/y hearts resulting in exacerbation of cardiac dysfunction. Importantly, daily administration of 100 &mgr;g/kg pyr1-apelin-13 resulted in upregulated angiotensin-converting enzyme 2 levels, decreased superoxide production and expression of hypertrophy- and fibrosis-related genes leading to attenuated myocardial hypertrophy, fibrosis, and dysfunction in the Ang II–infused apolipoprotein E knockout mice. In addition, pyr1-apelin-13 treatment largely attenuated Ang II–induced apoptosis and ultrastructural injury in the apolipoprotein E knockout mice by activating Akt and endothelial nitric oxide synthase phosphorylation signaling. In cultured neonatal rat cardiomyocytes and cardiofibroblasts, exposure of Ang II decreased angiotensin-converting enzyme 2 protein and increased superoxide generation, cellular proliferation, and migration, which were rescued by pyr1-apelin-13, and Akt and endothelial nitric oxide synthase agonist stimulation. The increased superoxide generation and apoptosis in cultured cardiofibroblasts in response to Ang II were strikingly prevented by pyr1-apelin-13 which was partially reversed by cotreatment with the Akt inhibitor MK2206. In conclusion, pyr1-apelin-13 peptide pathway is a negative regulator of aging-mediated and Ang II–mediated adverse myocardial remodeling and dysfunction and represents a potential candidate to prevent and treat heart disease.

OS 36-07 TREATMENT WITH APELIN-13 PREVENTS PRESSURE OVERLOAD-INDUCED AORTIC ADVENTITIAL REMODELING AND FIBROSIS IN HYPERTENSIVE RATS WITH TAC.

Objective: The Apelin/APJ system has recently been implicated in pathologies of hypertension. However, little was elucidated upon effects of Apelin on vascular adventitial remodeling progression. Here, we examined regulatory roles of Apelin in pressure overload-induced adventitial remodeling and fibrosis in hypertensive rats. Design and Method: The male Sprague–Dawley rats were performed with transverse aortic constriction (TAC). The rats with TAC were randomized to daily deliver either pyroglutamyl Apelin-13 (50 &mgr;g/kg) or saline for 4 weeks. Results: Histomorphometric analysis by HE and Masson trichrome staining revealed increased medial and adventitial thicknesses, especially in the adventitia, in ascending aortas in rats with TAC when compared with the sham-operated rats. Downregulation of APJ receptor and elevations in systolic blood pressure, phosphorylated mTOR and ERK1/2 levels were observed in hypertensive rats with TAC. Pressure overload-mediated by TAC led to marked increases in heart weight (HW), HW/body weight ratio, malonyldialdehyde (MDA) contents and aortic fibrosis in the hypertensive rats with enhanced activation of NADPH oxidase activity. The pressure overload-mediated pathological adventitial remodeling was strikingly rescued by Apelin-13 supplementation, associated with attenuation of aortic fibrosis and reduced mRNA expression of TGF-ß1 and collagen I. In cultured rat adventitial fibroblasts, exposure with Ang II (100 nmol L−1) resulted in obvious increases in MDA, phosphorylated levels of mTOR and ERK1/2 and mRNA levels of TGF-ß1, collagen I and fibronectin 1, which were dramatically prevented by Apelin-13 (100 nmol L−1) or mTOR inhibitor rapamycin (10 &mgr;mol L−1). However, Apelin-13 had no effect on collagen III levels. Conclusions: Our results demonstrate the importance of Apelin-13 in amelioration of aortic oxidative stress, adventitial remodeling and fibrosis in hypertensive rats with TAC via modulation of the mTOR/ERK signaling, thus indicating potential therapeutic strategies by enhancing Apelin/APJ action for preventing hypertensive vascular adventitial remodeling and fibrosis and pressure overload-associated cardiovascular disorders.

Ascending aortic adventitial remodeling and fibrosis are ameliorated with Apelin-13 in rats after TAC via suppression of the miRNA-122 and LGR4-β-catenin signaling

Apelin has been proved to be a critical mediator of vascular function and homeostasis. Here, we investigated roles of Apelin in aortic remodeling and fibrosis in rats with transverse aortic constriction (TAC). Male Sprague-Dawley rats were subjected to TAC and then randomized to daily deliver Apelin-13 (50μg/kg) or angiotensin type 1 receptor (AT1) blocker Irbesartan (50mg/kg) for 4 weeks. Pressure overload resulted in myocardial hypertrophy, systolic dysfunction, aortic remodeling and adventitial fibrosis with reduced levels of Apelin in ascending aortas of rat after TAC compared with sham-operated group. These changes were associated with marked increases in levels of miRNA-122, TGFβ1, CTGF, NFAT5, LGR4, and β-catenin. More importantly, Apelin and Irbesartan treatment strikingly prevented TAC-mediated aortic remodeling and adventitial fibrosis in pressure overloaded rats by blocking AT1 receptor and miRNA-122 levels and repressing activation of the CTGF-NFAT5 and LGR4-β-catenin signaling. In cultured primary rat adventitial fibroblasts, exposure to angiotensin II (100nmolL-1) led to significant increases in cellular migration and levels of TGFβ1, CTGF, NFAT5, LGR4 and β-catenin, which were effectively reversed by pre-treatment with Apelin (100nmolL-1) and miRNA-122 inhibitor (50nmolL-1). In conclusion, Apelin counterregulated against TAC-mediated ascending aortic remodeling and angiotensin II-induced promotion of cellular migration by blocking AT1 receptor and miRNA-122 levels and preventing activation of the TGFβ1-CTGF-NFAT5 and LGR4-β-catenin signaling, ultimately contributing to attenuation of aortic adventitial fibrosis. Our data point to Apelin as an important regulator of aortic remodeling and adventitial fibrosis and a promising target for vasoprotective therapies.

8D.01: EFFECTS OF ANGIOTENSIN-CONVERTING ENZYME 2 ON RENAL OXIDATIVE STRESS LEVELS IN APOLIPOPROTEIN E-DEFICIENT MICE.

Objective: The renin-angiotensin system (RAS) has been known for more than a century as a cascade that regulates body fluid balance, renal functions and blood pressure. Angiotensin-converting enzyme 2 (ACE2) is now known as a negative regulator of RAS, and activation of the ACE2 is a possible alternative target for new drugs, since some protective influences on renal and cardiovascular function have been revealed. We hypothesized that ACE2 would exert beneficial effects on oxidative stress levels and renal injury in apolipoprotein E (ApoE) -knockout (KO) mice. Design and method: In this study, we used 12-week-old wild-type, ApoEKO, and ACE2/ApoE double KO mice. The ApoEKO mice were treated with recombinant human ACE2 (hrACE2) with the daily dose of 2 mg/kg. We characterized the functional, structural and molecular signaling changes in mice kidneys. Results: Compared with the ApoEKO mice, ACE2 deficiency led to greater increases in renal oxidative stress levels and expression of oxidative stress-inducible proteins NADPH oxidase 4 (NOX4) in the ACE2/ApoE double KO mice. These changes were associated with exacerbation of renal tubule ultrastructure injury and greater activation of Akt and ERK1/2 phosphorylated signaling. Conversely, treatment with hrACE2 significantly attenuated renal oxidative stress levels and ultrastructure injury, and prevented the expression of NOX4 and phosphorylated level of Akt and ERK1/2 in ApoEKO mouse kidneys. However, there were no changes in renal expression of NOX2 and Mas receptor among groups. Conclusions: Deletion of ACE2 triggers greater increases in renal oxidative stress and tubular ultrastructure injury in the ACE2/ApoE double mutant mice with greater activation of Akt-ERK1/2 phosphorylated signaling. While ACE2 overexpression alleviates renal tubular injury in ApoE-mutant mice with suppression of superoxide generation and downregulation of the Akt-ERK phosphorylated signaling. Strategies aimed at enhancing ACE2 action may have important therapeutic potential for atherosclerosis and renal diseases.

GW24-e3625 Effects of apelin on the phosphodiesterase 1 expression and oxidative stress levels in mouse kidney fibroblast cells

Objectives Apelin is a recently-discovered cardiovascular bioactive polypeptide with multiple biological effects, which is the endogenous ligand of the orphan G protein-coupled receptor-APJ receptor and may cause vasodilation and lower blood pressure levels. This study focused on the effects of Apelin on the phosphodiesterase 1 (PDE1) expression and oxidative stress levels in mouse kidney fibroblast cells. Methods The mouse kidney fibroblast cells were primary cultured using the routine method. The angiotensin II (Ang II; 100 nmol/L) was used to stimulate the cells for 1 h in the presence and absence of Aplein (100 nmol/L), PDE1 inhibitor vinpocetine (10 μmol/L) and extracellular regulated protein kinase (ERK) inhibitor PD98059 (10 mmol/L). The specific fluorescent probe-dihydropyridinum (DHE) staining and colorimetric method were used for the determination of superoxide generation and malondialdehyde (MDA) content in cells, respectively. Results In the cultured mouse kidney fibroblast cells, exposure to Ang II (100 nmol/L) obviously promoted protein expressions of PDE1A and PDE1B (n = 5-6; P < 0.01, respectively), without having a differential effect on the expression of PDE1C. These changes were associated with marked increases in phosphorylated expression of ERK1/2, superoxide generation and MDA levels (n = 5-6; P < 0.01, respectively). In addition, treatment with Apelin (100 nmol/L) and PDE1 inhibition with vinpocetine (10 μmol/L) significantly prevented expressions of PDE1A and PDE1B mediated by Ang II in cells (n = 5; P < 0.05, respectively). More importantly, intervention with Apelin (100 nmol/L) and vinpocetine (10 μmol/L) strikingly reduced the cellular oxidative stress in response to Ang II, as evidenced by decreases in MDA levels and superoxide production, linked with a marked downregulation in phosphorylated ERK1/2 levels (n = 5-6; P < 0.05 or P < 0.01, respectively). Conclusions Treatment withApelin significantly prevents the oxidative stress formation in cultured mouse kidney fibroblast cells in response to Ang II through the modulation of PDE1-ERK1/2 signalling pathway, suggesting the potential anti-oxidative stress effect of Apelin. The enhanced activities and/or expression of Apelin may have potential therapeutic benefits on renal diseases associated with oxidative stress. This work was supported by National Natural Science Foundation of China (81170246 & 30973522) and Shanghai Pujiang Talents Program (11PJ1408300).

1040 ANGIOTENSIN II-MEDIATED VASCULAR INFLAMMATION IS ACCELERATED IN ACE2 NULL MICE WITH ENHANCED PROFILIN-1 EXPRESSION

Objectives: We hypothesized that angiotensin-converting enzyme 2 (ACE2) deficiency would facilitate angiotensin (Ang) II-mediated vascular inflammation and the actin-binding protein profilin-1 signaling. Design and methods: We randomized 10-week ACE2 knockout (ACE2KO, Ace2–/y) and wild-type littermates (WT, Ace2+/y) mice to Ang II (1.5 mg.kg−1.d−1) infusion with mini-osmotic pumps and treated daily with irbesartan (50 mg/kg) for 2 weeks. Results: Aortic ACE2 protein was obviously reduced in WT mice in response to Ang II related to increases in profilin-1 protein. Loss of ACE2 resulted in greater increases in Ang II-induced mRNA expressions of inflammatory cytokines MCP-1, IL-1&bgr;, and IL-6 in aortas of ACE2KO mice, along with enhanced profilin-1 expression and phosphorylated ERK1/2 levels. Interestingly, irbesartan significantly attenuated Ang II-mediated aortic inflammation in WT mice with enhanced ACE2 levels and suppression of the profilin-1/ERK signaling. Conclusions: Our findings reveal that ACE2 deficiency worsens Ang II-mediated aortic inflammation and profilin-1 signaling, suggesting a potential therapeutic approach by enhancing ACE2 action for patients with vascular diseases. Supported by National Natural Science Foundation of China (30973522&81170246), Shanghai Pujiang Talents Program (11PJ1408300), and CIHR (86602&84279).

1039 Ace2 Deficiency Faciliates Angiotensin II-Mediated Vascular Peroxynitrite Production With Activation of the Akt/Enos Signaling

Objectives: Angiotensin (Ang) II is major causes of vascular oxidative-nitrosative stress in hypertension. We hypothesized that loss of angiotensin-converting enzyme 2 (ACE2), a specific Ang II-degrading enzyme, would facilitate Ang II-mediated vascular superoxide and peroxynitrite production. Design and methods: 10-week ACE2 knockout (ACE2KO, Ace2–/y) and wild-type littermates (WT, Ace2+/y) mice received with mini-osmotic pumps with Ang II (1.5 mg.kg−1.d−1) or saline for 2 weeks. The Ang II-infused WT mice were treated daily with an AT1 receptor blocker irbesartan (50 mg/kg). The fluorescent dye dihydroethidium and nitrotyrosine staining were used to evaluate superoxide (O2–) and peroxynitrite (ONOO–) levels in mice aorta. Results: Loss of ACE2 led to greater increases in Ang II-mediated NADPH oxidase activity and superoxide and peroxynitrite production in the aortas of ACE2KO mice associated with enhanced phosphorylated levels of Akt, p70S6 kinase, and endothelial nitric oxide synthase (eNOS). Interestingly, treatment with irbesartan significantly prevented Ang II-mediated aortic NADPH oxidase activity, superoxide and peroxynitrite production in WT mice with increased ACE2 protein and suppression of the Akt/eNOS signaling pathways. Conclusions: Collectively, loss of ACE2 accelerates Ang II-induced aortic superoxide and peroxynitrite production with the augmented NADPH oxidase activity, implicating the potential role of ACE2 gene as a novel therapeutic target for oxidative-nitrosative stress related vascular diseases such as hypertension. This work was supported by National Natural Science Foundation of China (Nos. 81170246 & 30973522), Shanghai Pujiang Talents Program (11PJ1408300), and the Canadian Institute for Health Research (Nos. 86602 & 84279).