ACE2 Deficiency Enhances Angiotensin II-Mediated Aortic Profilin-1 Expression, Inflammation and Peroxynitrite Production

Inflammation and oxidative stress play a crucial role in angiotensin (Ang) II-mediated vascular injury. Angiotensin-converting enzyme 2 (ACE2) has recently been identified as a specific Ang II-degrading enzyme but its role in vascular biology remains elusive. We hypothesized that loss of ACE2 would facilitate Ang II-mediated vascular inflammation and peroxynitrite production. 10-week wildtype (WT, Ace2+/y) and ACE2 knockout (ACE2KO, Ace2−/y) mice received with mini-osmotic pumps with Ang II (1.5 mg.kg−1.d−1) or saline for 2 weeks. Aortic ACE2 protein was obviously reduced in WT mice in response to Ang II related to increases in profilin-1 protein and plasma levels of Ang II and Ang-(1–7). Loss of ACE2 resulted in greater increases in Ang II-induced mRNA expressions of inflammatory cytokines monocyte chemoattractant protein-1 (MCP-1), interleukin (IL)-1β, and IL-6 without affecting tumor necrosis factor-α in aortas of ACE2KO mice. Furthermore, ACE2 deficiency led to greater increases in Ang II-mediated profilin-1 expression, NADPH oxidase activity, and superoxide and peroxynitrite production in the aortas of ACE2KO mice associated with enhanced phosphorylated levels of Akt, p70S6 kinase, extracellular signal-regulated kinases (ERK1/2) and endothelial nitric oxide synthase (eNOS). Interestingly, daily treatment with AT1 receptor blocker irbesartan (50 mg/kg) significantly prevented Ang II-mediated aortic profilin-1 expression, inflammation, and peroxynitrite production in WT mice with enhanced ACE2 levels and the suppression of the Akt-ERK-eNOS signaling pathways. Our findings reveal that ACE2 deficiency worsens Ang II-mediated aortic inflammation and peroxynitrite production associated with the augmentation of profilin-1 expression and the activation of the Akt-ERK-eNOS signaling, suggesting potential therapeutic approaches by enhancing ACE2 action for patients with vascular diseases.


Introduction
Vascular inflammation and oxidative stress play a crucial role in the pathogenesis of vascular injury mediated by angiotensin (Ang) II, the major effector peptide of the renin-angiotensin system (RAS) [1][2][3]. Ang II has recently been shown to induce vascular injury by modulating release of inflammatory chemokines such as monocyte chemoattractant protein-1 (MCP-1), interleukin (IL)-1b, IL-6 [2,3] and promoting the NADPH oxidase activation and superoxide (O 2 -) production [4,5], contributing to endothelial nitric oxide (NO) synthase (eNOS) uncoupling and impaired NO bioavailability as well as vascular oxidative stress [1,6,7]. Within the vascular system, Ang II-mediated superoxide may drive a very fast radical-radical reaction by reacting with NO to yield the much more powerful oxidant peroxynitrite (ONOO 2 ), which causes profound vascular injury via oxidative and nitrosative reactions [8]. In addition, Ang II is a well-known activator of profilin-1, extracellular signal-regulated kinases (ERK) and phosphatidylinositol 3-kinase (PI3K)/Akt signaling cascades, leading to increased formation of nitrotyrosine, an index of peroxynitrite damage to vascular tissues [1,9,10,11,12]. The actin-binding protein profilin-1 has been shown to directly activate Akt/ERK signaling pathways, which are important contributors of eNOS uncoupling and peroxynitrite generation in the vasculature [11,13,14]. However, little is known regarding the relationship among profilin-1, inflammation and peroxynitrite production in the vascular diseases.
Pharmacological inhibition of the Ang II signaling is a key aspect of the current approach to preventing vascular inflammation and oxidative stress-related vascular dysfunction [15]. Rencently, angiotensin-converting enzyme 2 (ACE2) has been identified as a pleiotropic monocarboxypeptidase responsible for the degradation of Ang II to the vasodilatory Ang-(1-7), which has been shown to counteract the pro-inflammatory and pro-oxidative effects of Ang II via its receptor Mas, thereby functioning as a negative regulator of the RAS [5,16,17,18]. Intriguingly, the Ang II type 1 (AT1) receptor blockers may increase expression and activity of ACE2 [9,19] with attenuation of cardiovascular oxidative damage [20] and diminish the profilin-1/ERK signaling [9,10,21]. In our previously studies, we demonstrated that ACE2 deficiency mice develop impaired cardiac functions, pathological remodeling with enhanced inflammatory cytokines, oxidative stress and ERK1/2 activation in heart [20,22,23] whereas ACE2 overexpression prevents Ang II-induced inflammation, oxidative stress and ERK1/2 activation in association with an attenuation of cardiovascular remodeling and dysfunction [9,23,24], suggesting a critical role ACE2 in regulation of inflammation, oxidative stress and cardiovascular function. However, the exact role and mechanism of ACE2 in vascular biology remain largely unknown. In this work, we assessed the hypothesis that loss of ACE2 would facilitate vascular inflammation and peroxynitrite production. We randomized ACE2 knockout (ACE2KO, Ace2 2/y ) and wild-type littermates (WT, Ace2 +/y ) mice to either Ang II or saline infusion as in the previous study [23,24]. To investigate whether Ang II-induced pathological effects in the aortas of mice are mediated by AT1 receptor, we used irbesartan as a specific blocker of AT1 receptor.

Experimental Animals and Protocols
Mutant mice have been previously described [20,22,25]. 10week male WT and ACE2KO mice received with mini-osmotic pumps (model 1002, Alza Corp, Palo Alto, CA) with Ang II (1.5 mg.kg 21 . d 21 ) or saline for 2 weeks [24]. WT mice were treated with irbesartan (50 mg.kg 21 .d 21 ; Bristol-Myers Squibb Co., Princeton, NJ) in their drinking water for 3 days before Ang II infusion and during the course of the study. Plasma levels of Ang II and Ang-(1-7) were measured by radio-immunoassay in the Hypertension and Vascular Disease Centre Core Laboratory at Wake Forest University School of Medicine as previously described [20,25]. The thoracic aorta was carefully isolated from the mice anesthetized with a mixture (100 mg/kg ketamine plus 10 mg/kg xylaxine; ip) at the end of the experiment. All experiments were approved and performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996), the Canadian Council on Animal Care and the Animal Research Ethics Committee at Shanghai Jiao Tong University School of Medicine.

Dihydroethidium and Nitrotyrosine Fluorescence
Oxidative stress is generally identified by indirect markers of the vascular oxidant damage, such as superoxide and peroxynitrite production [6,26]. The oxidative fluorescent dye dihydroethidium (DHE) and nitrotyrosine staining were used to evaluate superoxide (O 2 -) and peroxynitrite (ONOO -) levels in aorta tissues as previously described [23,24,26]. For aortic DHE staining, 20 mm fresh frozen tissue sections were washed with hanks balanced salt solution (HBSS) with magnesium and calcium and then incubated at 37uC for 30 min with DHE (10 mM) in HBSS. For nitrotyrosine staining, aortic tissue sections were immunostained using polyclonal nitrotyrosine antibodies (1:100; Upstate Biotechnology Inc, Lake Placid, NY). Fluorescent images were observed with an Olympus Fluoview laser-scanning confocal microscope mounted on an Olympus microscope.

Measurement of NADPH Oxidase Activity
The NADPH oxidase activities in aorta tissues of mice were quantified by lucigenin-enhanced chemiluminescence as previously described [20]. Briefly, the aorta homogenates were collected in 100 ml of phosphate buffer solution (PBS) mixture with protease inhibitor and phosphatase inhibitor and centrifuged at 1000 g for 10 min. The supernatants were then collected and added lucigenin (50 mM) and NADPH (1 mM) for NADPH oxidase activities assay with FB-12 luminometer in the absence or presence of diphenylene iodonium (10 mM), a selective inhibitor of NADPH oxidase. All data were calculated as the change in the rate of luminescence per minute per milligram of aorta tissue.

TaqMan Real-time PCR Analysis
The mRNA expression levels were determined by TaqMan real-time reverse transcription PCR as previously described [5,23,27]. Total RNA was extracted from flash-frozen aorta tissues using TRIzol reagent, and cDNA was synthesized from 1 mg total RNA by using random hexamers. The primers and probes for mouse ACE2, MCP-1, IL-1b, IL-6, and tumor necrosis factor-a (TNF-a) are indicated in Table 1 based on previously published reports [5,23,27]. For each gene, a standard curve was generated using known concentrations of cDNA as a function of cycle threshold (CT). The mRNA expression of the reported genes was performed by TaqMan Real-time PCR using ABI 7900 Sequence Detection System and analyzed using the SDS2.2 software (Applied. Biosystems). All samples were run in triplicates. 18S rRNA was used as an endogenous control.

Statistical Analysis
All data are shown as mean6SEM. All statistical analyses were performed with SPSS 11.5 software either by Student's t test or by ANOVA followed by the Student-Neuman-Keuls test for multiple-comparison testing as appropriate. A value of P,0.05 was considered statistically significant.

ACE2 Deficiency Facilitates Ang II-mediated Aortic Inflammation
We firstly evaluated the effects of Ang II on aortic expressions of ACE2 and profilin-1. Western blotting revealed that aortic profilin-1 expression ( Figure 1B) was obviously augmented at 1, and 2 weeks after Ang II infusion in WT mice compared with pre-treatment group (n = 6; P,0.01, respectively). This change was consistent with reduced protein of ACE2 ( Figure 1A) and increased levels of plasma Ang II and Ang-(1-7) ( Figure 1C) in WT mice (n = 6210; P,0.01, respectively). Chronic Ang II infusion resulted in a marked increase in plasma Ang II levels ( Figure 1C) without affecting plasma Ang-(1-7) levels in ACE2deficient mice (n = 10; P,0.01, respectively). We next investigated Ang II-induced vascular inflammation in ACE2 deficiency status. TaqMan real-time PCR analysis revealed that lack of ACE2 resulted in greater increases in Ang II-induced mRNA expressions of inflammatory cytokines MCP-1 ( Figure 1D), IL1b ( Figure 1E) and IL6 ( Figure 1F) in ACE2-dificient aortas (n = 628; P,0.01, respectively), without having a differential effect on the expression of TNF-a ( Figure 1G).

ACE2 Deficiency Worsens Ang II-mediated Aortic Peroxynitrite Production
Activation of NADPH oxidase is a central mediator of the pathological effects of Ang II, contributing to oxidative injury in the vasculature [8,28,29]. We found that chronic Ang II infusion significantly enhanced aortic NADPH oxidase activity ( Figure 3J Figure 3J) (n = 526; P,0.05, respectively). Interestingly, worsen aortic nitrotyrosine formation in ACE2dificient mice was further confirmed by Western blot analyses showing a greater increase in protein level of nitrotyrosine ( Figure 3L) in aortas in response to Ang II (n = 526; P,0.05, respectively), which indicated that loss of ACE2 facilitated Ang IImediated peroxynitrite production in the vasculature.

AT1 Blockade Suppresses Ang II-mediated Aortic Peroxynitrite Production
Blockade of AT1 receptors by irbesartan treatment largely prevented Ang II-mediated increase in aortic nitrotyrosine staining ( Figures 6F & 6G), superoxide production ( Figures 6C & 6G) and protein level of nitrotyrosine ( Figure 6I) in aorta of mice with suppression of NADPH oxidase activation ( Figure 6H) (n = 526; P,0.05, respectively), providing that Ang II mediated activation of the AT1 receptor is a key pathophysiological event in the aortic superoxide and peroxynitrite production. Collectively, our data demonstrated that chronic Ang II infusion resulted in the AT1 receptor-mediated increases in superoxide and peroxynitrite production in the vasculature via the activation of the NADPH oxidase and the Akt-ERK-eNOS signaling pathways.

Discussion
It is generally considered that peroxynitrite triggers cellular responses ranging from subtle modulations of cell signaling to overwhelming vascular oxidative injury and represents a crucial pathogenic mechanism of vascular diseases such as hypertension in experimental animal models and patients [1,6,8]. However, the molecular changes and signaling mechanisms still remain unclear in the peroxynitrite generation underlying Ang II-mediated vascular diseases. Within the vascular system, peroxynitrite is produced in all layers of the vascular wall by all vascular cell types, including endothelial, smooth muscle, and adventitial cells. They are formed by various enzyme systems, including uncoupled eNOS and NADPH oxidase, and others [1,7,8,26,29]. We have previously shown that loss of NADPH oxidase reduced Ang IImediated superoxide production while enhancement of NADPH oxidase markedly augmented superoxide production in hearts of Ang II-infused ACE2 mutant mice [24,28]. In the present study, we demonstrated that chronic Ang II infusion results in marked increases in aortic peroxynitrite production in both WT and ACE2 mutant mice with enhancement of superoxide production derived by NADPH oxidase activation. It has become clear that eNOS normally generates NO from L-arginine by utilizing NADPH [1,29,30]. Peroxynitrite may oxidize and disrupt the zinc thiolate center of eNOS by releasing zinc and oxidizing the thiols. This modification of eNOS (eNOS uncoupling) leads to diminishment of NO bioactivity and elevation of vascular superoxide production, which reacts with NO, further generating ONOO 2 [8]. The present data suggest that the enhancement of NADPH-derived superoxide may act as a ''kindling radical'' to cause eNOS activation and peroxynitrite production in Ang IIinfused mice. Ang II infusion increases, rather than decreases, the phosphorylation and protein levels of eNOS in both WT and ACE2-deficient mice. These findings are in line with previous observations in the rat model of oxidative stress mediated by Ang II [30] where eNOS expression was significantly upregulated in aorta tissues. More importantly, our current report exhibited that AT1 receptor blockade by irbesartan significantly attenuated Ang II-mediated aortic peroxynitrite production in WT mice, along with a marked reversal of Ang II-induced activation of NADPH oxidase and eNOS, confirming a critical contribution of Ang II/ AT1 receptor-mediated activation of NADPH oxidase and eNOS to the peroxynitrite production.
The pivotal role of ACE2 as a negative regulator of Ang IImediated signaling in the vascular system implies that ACE2 deficiency could facilitate the adverse effects of Ang II [15,16,17,24,28]. In the present study, aortic ACE2 protein is obviously reduced in WT mice in response to Ang II. This downregulation of ACE2 by Ang II in WT mice is related to marked increases in aortic profilin-1 protein. Moreover, absence of ACE2 leads to greater Ang II and lowered Ang-(1-7) levels in ACE2KO mice. In the Ang II-treated ACE2KO mice, loss of ACE2 triggers a greater increase in aortic profilin-1 expression linked with higher plasma Ang II levels. Ang II is critically involved in the activation of inflammatory cytokines, which may in turn trigger oxidative stress and cardiovascular dysfunction [2,3,27,31,32]. The key peptidase action of ACE2 is degradation of Ang II to Ang-(1-7), turning the balance within the RAS cascade from pro-inflammatory and pro-oxidative actions to antiinflammatory and anti-oxidative actions [4,12,28,31]. TaqMan real-time PCR analysis in this work revealed that lack of ACE2 results in greater increases in Ang II-induced mRNA expression of inflammatory cytokines MCP-1, IL1b and IL6 in ACE2-dificient aortas, without having a differential effect on TNF-a expression. The Ang II-induced aortic inflammation is significantly prevented by the AT1 receptor blocker irbesartan along with the suppression of peroxynitrite production and the enhancement of ACE2 expression. This is in agreement with other reports demonstrating that the enhanced ACE2 is associated with attenuation of Ang IIinduced MCP-1 expression in cultured human monocyte cell line macrophages [33] and in aortas of the hypertensive rats [17]. Taken together, ACE2 deficiency facilitates Ang II-induced aortic inflammation and peroxynitrite production while enhanced ACE2 by AT1 receptor blockade is, at least in part, responsible for the attenuation of aortic inflammation and peroxynitrite generation.
There is growing evidence that profilin-1, a commonly recognized intracellular actin-binding protein, plays a prominent role in vascular pathology and vascular diseases [13,14,34]. Our current report for the first time demonstrated that ACE2 deficiency leads to greater increases in aortic profilin-1 expression in response to Ang II with the upregulation of eNOS in both phosphorylation and protein levels. It is not yet known whether this increase of profilin-1 is of pathophysiological significance in Ang II-mediated vascular diseases, but it has been revealed that profilin-1 overexpression leads to vascular inflammation [35] and vascular remodeling [14,21]. Profilin-1 gene may represent a new therapeutic target in the treatment of vascular diseases such as hypertension [9,10,21]. Interestingly, profilin-1 has been shown to regulate eNOS phosphorylation and activity under atherogenic dietary and profilin-1 transgenic or mutant conditions [21,34,35]. In addition, profilin-1 interacts with signaling molecules including vasodilator-stimulated phosphoprotein (VASP), a validated marker for the activity of the eNOS/NO signaling pathway in vascular tissues [30]. Furthermore, profilin-1 triggers the activation of PI3K/Akt, which is an important regulator of vascular eNOS activation and peroxynitrite production through p70S6 kinase and ERK signaling pathways [2,11,13,21]. In this work, Ang IImediated increase in aortic profilin-1 expression is linked with augmented phosphorylation level of Akt at both regulatory sites, Ser 473 and Thr 308 . In agreement with the pattern of Akt activation, phosphorylation levels of p70S6 kinase, ERK1/2, and eNOS are markedly augmented in WT and ACE2 mutant mice in response to Ang II, when compared with their corresponding control mice. Long-term blockade of AT1 receptors by irbesartan significantly augments the mRNA and protein expression of ACE2. More intriguingly, irbesartan provides protection against Ang II-mediated aortic peroxynitrite production via the suppression of profilin-1 expression and the Akt-ERK-eNOS signaling pathways in the present study. Consistent with our previous studies [9], these findings suggesting that Ang IImediated upregulation of profilin-1 might, in part, contribute to the vascular peroxynitrite production.
Consequently, our findings demonstrate that ACE2 deficiency worsens Ang II-induced aortic inflammation and peroxynitrite production with the augmentation of profilin-1 expression and the activation of the Akt-ERK-eNOS signaling pathways, suggesting a critical role of ACE2 in the suppression of Ang II-mediated vascular injury and potential therapeutic approaches by enhancing ACE2 action for patients with vascular diseases. New roles for ACE2 may yet remain to be discovered and elucidated in the vascular biology. As highlighted in this work, further studies are required to better define the changes in vascular profilin-1 levels and the relative contribution and precise mechanism of profilin-1 in the inflammation and peroxynitrite production in the vasculature.