AT2R agonist NP‐6A4 mitigates aortic stiffness and proteolytic activity in mouse model of aneurysm

Abstract Clinical and experimental studies show that angiotensin II (AngII) promotes vascular pathology via activation of AngII type 1 receptors (AT1Rs). We recently reported that NP‐6A4, a selective peptide agonist for AngII type 2 receptor (AT2R), exerts protective effects on human vascular cells subjected to serum starvation or doxorubicin exposure. In this study, we investigated whether NP‐6A4–induced AT2R activation could mitigate AngII‐induced abdominal aortic aneurism (AAA) using AngII‐treated Apoe−/− mice. Male Apoe−/− mice were infused with AngII (1 µg/kg/min) by implanting osmotic pumps subcutaneously for 28 days. A subset of mice was pre‐treated subcutaneously with NP‐6A4 (2.5 mg/kg/day) or vehicle for 14 days prior to AngII, and treatments were continued for 28 days. NP‐6A4 significantly reduced aortic stiffness of the abdominal aorta induced by AngII as determined by ultrasound functional analyses and histochemical analyses. NP‐6A4 also increased nitric oxide bioavailability in aortic tissues and suppressed AngII‐induced increases in monocyte chemotactic protein‐1, osteopontin and proteolytic activity of the aorta. However, NP‐6A4 did not affect maximal intraluminal aortic diameter or AAA incidences significantly. These data suggest that the effects of AT2R agonist on vascular pathologies are selective, affecting the aortic stiffness and proteolytic activity without affecting the size of AAA.

the X-linked Agtr2 gene, is a G protein-coupled receptor and shares only 34% homology with AT1R. 7 AT2R expression is localized to vas-

cular endothelial cells (ECs), vascular smooth muscle cells (vSMCs)
and on the cells of inflammatory and immune origins such as monocytes and T cells. [8][9][10] AT2R is a reparative molecule, and its activation enhances microvascular perfusion and oxygenation and regeneration of muscle. 11,12 NP-6A4 is a patented peptide AT2R agonist (Novopyxis Inc, Boston, MA), which has acquired FDA's Orphan Drug designation for the treatment of paediatric cardiomyopathy. 8 We have recently reported that AT2R up-regulation with NP-6A4 activates protective signalling in human ECs by increasing endothelial nitric oxide synthase (eNOS) expression and bioavailability of nitric oxide (NO) and in vSMCs via suppression of reactive oxygen species. 8 Similarly, we have shown that NP-6A4 protects mouse HL-1 cardiomyocytes and human coronary artery vSMCs from acute nutrient deficiency. 13 These protective effects of AT2R stimulation have been well supported by other evidence for maintaining vascular integrity of the aorta. 14,15 The contribution of AT2R stimulation in abdominal aortic pathologies remains controversial. In an experimental elastase-induced AAA model in rat, AT2R stimulation with C21, a small molecule agonist of the AT2R, prevented aneurysm progression by preserving the functional and structural morphology of aorta. 14 However, in a mouse model, AT2R deficiency had no effects on AngII-induced AAA and atherosclerosis. 16 These conflicting reports warrant further studies to confirm the roles of AT2R stimulation in the settings of AngII-induced aortic pathologies.
The literature shows that in male Apoe −/− mice, treatment with AngII (1 µg/kg/min) results in AAA. 17 Since mouse vasculature expresses both AT1R and AT2R, the effect of AT1R seems to override any protective effects of AT2R in this model. To date, no studies have examined the effects of AT2R activation in the presence of high AngII concentrations in the context of vasculopathies including AAA. Therefore, in the present study, we aimed to determine whether activation of AT2R by NP-6A4 modulates AngII-induced vasculopathies in Apoe −/− mice. Our results show that NP-6A4-induced AT2R signalling attenuates AngII-induced aortic stiffness as a primary outcome. This protective effect involved increase in the bioavailability of NO in ECs and suppression of osteopontin and matrix metalloproteases (Mmps) in vSMCs. However, AngII-induced development of AAA as determined by maximal aortic diameter or increase in blood pressure was not suppressed by AT2R activation.

| Animals, design and aneurysmal model
Eight-week-old male Apoe −/− (B6.129P2, stock no. 002052) mice were purchased from the Jackson Laboratory. Male mice were preferred for these studies because of high incidence of AngII-induced AAA as described. 18 Mice were kept on a 12-hour/12-hour light/ dark cycle with standard chow. Aneurysmal studies were performed on these mice by infusing AngII for 28 days using published protocols. 19,20 Some animals were administered with AT2R agonist NP-6A4 (2.5 mg/kg/day, subcutaneous) until the end of the study (Supporting Figure S1). NP-6A4 was a gift from Novopyxis Inc (Boston, MA, United States). This dose of NP-6A4 was selected based on previous studies in Pulakat laboratory using mice with streptozotocin-induced diabetes that demonstrated optimal tolerance without any adverse effects (unpublished data). Animals were randomly allocated to AngII infusion, NP-6A4 treatment or control. For AngII infusion, mice were anaesthetized in a closed chamber with 1%-2% isoflurane in oxygen for 2 to 5 minutes until immobile. Each mouse was then removed and taped on a heated (37 ± 2°C) procedure board with 1.0 ± 1.5% isoflu-

| Transabdominal ultrasound imaging and quantification of aortic aneurysms
Mice were put into the anaesthesia chamber, followed by anaesthetization with oxygen and vaporized isoflurane (~2%), and ultrasound imaging was performed as described previously. 19,22 Briefly, warmed ultrasound gel was applied to the abdominal surface, and 40-MHz ultrasound transducer (Vevo MS550D, Toronto, ON) was used to collect B-mode, M-mode and ECG-based kilohertz visualization (EKV) mode images by the imaging system (Vevo 2100, VisualSonics, Toronto, ON) as described. 19,[22][23][24] Briefly, short-and long-axis scans were performed on the abdominal aorta from the level of the left renal arterial branch through to the suprarenal region. Cine loops of 100 frames were acquired throughout the renal region to determine the maximal diameters of the abdominal aorta in the suprarenal region. All the ultrasound data were collected in a blinded fashion by an experienced faculty member in the core facility at Dalton Cardiovascular Research Center for consistency. The AngII-induced AAA was defined as having at least 50% increase in NY) by an independent researcher ex vivo under a microscope. 22 MILD and MEAD were measured to depict the in vivo and ex vivo measurements of the diameter of the aorta, respectively, to complement the findings as described previously. 22 Mice were closely monitored for acute rupture incidences for first 10 days of AngII infusion. The dead mice immediately underwent autopsy, and rupture was defined by the presence of blood clot in the chest cavity and haemorrhage of abdominal aorta between the celiac artery and the left renal artery.

| Blood pressure measurement
Blood pressure was measured non-invasively on conscious mice using a CODA volume pressure recording tail-cuff system (Kent Scientific Corporation, Torrington, CT) as described previously. 19,20 Briefly, mice were acclimated for two days to restraint tubes and trial measurements. On the third day, following 5 acclimation cycles, 25 individual blood pressure measurements (technical replicates) were taken; all false readings (as determined by the diagnostic software) were excluded, and any animal failing to register at least 20 (80%) 'true' readings was excluded from analysis. Data were trimmed to exclude the lowest and highest 5% of measured values, and the mean was used to represent each animal. The measurement of blood pressure was performed blindly with respect to experimental groups.

| AAA classification
AAA complexity was determined by Daugherty's classification by measurement of the aortic diameter and histological features. 25,26 Type I represents a small single dilation (1.5-2.0 times of a normal diameter); type II denotes a large single dilation (>2 times of a normal diameter); type III is multiple dilations; and type IV is aortic rupture that leads to death because of bleeding into the peritoneal cavity.

| Aortic stiffness, distensibility and radial strain measurement
In vivo aortic stiffness was measured locally in the abdominal aorta by pulse-wave velocity (PWV) technique by analysing EKV data collected at day 28 of AngII infusion using Vevo Vasc software as described previously. 19,23,27,28 Vevo Vasc software was used to calculate PWV as a ratio of the distance (d) between two locations along the aorta and time delay (∆t) of the pulse wave between both locations and is expressed in m/s. Similarly, EKV data were analysed with the Vevo Vasc software to calculate distensibility and radial strains along the two locations of suprarenal abdominal aorta. The measurements for all PWV, distensibility and radial strains were conducted blindly to the study groups. The measurement of in vitro endothelial cell stiffness was determined in the human aortic endothelial cells (hAOECs; from Cell Applications; passage 3-passage 5) by atomic force microscopy (AFM) as described. 19 The cells were pre-treated with NP-6A4 (5 μM) for 72 hours with daily NP-6A4 replenishment. During the final 24-hour incubation, cells were treated with TNF-α (10 ng/mL) in the presence or absence of NP-6A4.
Endothelial cell stiffness in the cells was measured using a standard protocol as described. 29,30

| Histology and immunohistochemistry (IHC)
After fixation, the abdominal aortae from experimental mice were rinsed with PBS and processed for paraffin embedding.
Serial sections (5 μm) were prepared by cutting abdominal aorta into two equal halves and sectioned throughout the tissue as described. 22 The sections of the abdominal aorta at regular intervals (200 μm) were subjected to haematoxylin and eosin (H&E), elastin and picrosirius red stain for histoarchitectural evaluation of aneurysm as described previously. 19

| Cell isolation, RNA extraction and quantitative real-time PCR
Bone marrow-derived macrophages (BMDMs) were isolated from eight-week-old experimental mice treated with AngII or with NP-  Supporting Table S1.

| Western blotting
Human umbilical vein endothelial cells (hUVECs; from GIBCO, Invitrogen Cell Culture; passage 3-passage 7) were pre-treated with NP-6A4 (5 μM) for 72 hours with daily media change and NP-6A4 replenishment. Some cells were exposed to TNF-α (10 ng/mL) alone or in combination with NP-6A4 for 24 hours before harvesting cells for protein extraction. Cell lysates were prepared, and 20 μg of protein was loaded on SDS-PAGE gel, electrophoresed and transferred to polyvinylidene fluoride membranes as described. 19 The primary antibodies used were eNOS (9586; 1:2000), phospho-eNOS (9575; 1:2000) and GAPDH (NB300-221; 1:5000). After washing and incubation with appropriate peroxidase-labelled secondary antibodies for 1 hour, the bands were detected using ECL reagent and the ChemiDoc imaging system. Three independent experiments were performed to ensure the reproducibility of Western blotting data and for quantification.

| Gelatine zymography (GZ) and in situ zymography (ISZ)
For GZ, the aortic tissue lysates from experimental mice with AngII infusions were used as described. 19 For ISZ, aortic tissues were cut and incubated with substrate solution containing DQ gelatine (D12054; Invitrogen) and ISZ was performed as described. 34 Negative control sections were treated without DQ gelatine. Sections were mounted with VECTASHIELD medium with DAPI (H-1800; Vector Labs). Fluorescence intensity in the medial layer of the tissue sections was quantified using Gen 5 software (BioTek).

| Statistical analysis
Statistical analyses were performed with GraphPad Prism version 7.0 (GraphPad Software, Inc, San Diego, CA, USA). All the data were assessed for normality and equal variance using Shapiro-Wilk test and Levene's test, respectively. Unpaired two-tailed Student's t test was used to determine statistical difference between two groups for normally distributed continuous variables. For comparison of multiple groups, ANOVA followed by Tukey's multiple comparison analysis was used. Normally distributed data were analysed by nonparametric Mann-Whitney test or Kruskal-Wallis test. For survival graphs and incidence of AAA, log-rank test and Fisher's exact test were applied, respectively. Data are presented as median ± interquartile range for the PWV, MILD, MEAD, distensibility and radial strain. For rest of the quantitation, mean ± SEM was calculated. P < 0.05 was considered statistically significant for all tests.
Another cohort of Apoe −/− mice were pre-treated with NP-6A4 (2.5 mg/kg/day) for 14 days before starting AngII exposure. NP-6A4 treatment reduced early death events marginally to 29.4% (5 out of 17) ( Figure 1A). The overall AAA incidence in the AngII groups was  Figure S4A,B).

Next, we investigated whether NP-6A4 treatment mitigated
AngII-induced aortic wall dysfunction and damage. Aortic stiffness, as measured by pulse-wave velocity (PWV), was significantly higher at day 28 with AngII infusion compared with controls ( Figure 1E).
NP-6A4 significantly decreased the aortic stiffness (1.99 ± 0.26 m/s vs 1.68 ± 0.23 m/s, P = 0.0278) in these mice. AngII infusion significantly decreased the aortic wall distensibility and radial strain measured at the suprarenal aorta compared with saline controls ( Figure 1F,G). NP-6A4 treatment with AngII infusion significantly improved the distensibility, whereas radial strain was slightly improved. These functional parameters remain unchanged in the control groups with saline or NP-6A4. AngII infusion alone or with NP-6A4 significantly increased systolic arterial blood pressure as compared to their respective controls such that no significant differences were observed in these two groups at day 28 (Supporting Figure S2). These results demonstrate that AT2R stimulation with NP-6A4 mitigates aortic stiffness. We also examined whether NP-6A4 increases the expression of AT2R in the aortic tissues of a mouse model of angiotensin II (AngII)-induced AAA. Immunohistochemical staining showed increased expression of AT2R in the adventitial region of abdominal aorta in response to AngII, which was further increased with NP-6A4 pre-treatment, without significant change at mRNA level (Supporting Figure S3).

| NP-6A4 treatment modulates AngII-induced changes in collagen location and expression in the aortic tissues of Apoe −/− mice
Aortic tissue cross sections from the experimental mice were subjected to histological analysis to characterize the vascular lesions. No noticeable difference in the adventitial thickening and F I G U R E 1 NP-6A4 attenuates AngII-induced aortic stiffness in Apoe −/− mice. A, Graph showing the survival rate of Apoe −/− mice in response to NP-6A4 and AngII. Five out of 17 mice died of abdominal aortic rupture in NP-6A4-treated AngII animals, whereas 10 out of 23 mice died in the AngII group. B, Aneurysm severity (type I to type IV) scored using a classification system at day 28. C, Quantification for maximal intraluminal diameter (MILD) as measured by ultrasound at day 28 of AngII infusion. D, Quantification of ex vivo maximal external aortic diameter (MEAD) of suprarenal aorta (mm) by microscopy at day 28. E, Pulse-wave velocity (PWV) calculated from measurements of abdominal aortic pulse pressure as determined by EKV in response to AngII at day 28. F and G, Distensibility and radial strain at day 28 of AngII and NP-6A4 treatments as measured by Vevo Vasc analysis. Log-rank test was used for comparing survival in A. Fisher's exact test was used for analysis in B. Kruskal-Wallis test was applied for statistics in C and D. Tukey's multiple comparisons test was used for data analysis in E-G. *P < 0.05; **P < 0.01; ***P < 0.001, ns = non-significant  Figure 2S). In NP-6A4 + AngII-treated mice, collagen III (green fluorescence) seemed to be minimally expressed ( Figure 2T). Additionally, IHC staining of the experimental tissues with antibody specific to collagen I showed decreased staining in the adventitial region of NP-6A4treated mice (Supporting Figure S5). These data showed that the protective effects of NP-6A4 on aortic stiffness may partly be associated with NP-6A4-mediated modulation of collagen turnover with the AngII treatment.

| NP-6A4 affects phenotypic changes in aortic tissues
Monocyte chemoattractant protein-1 (MCP1), a chemotactic cytokine, is reportedly regulated by AT2Rs. 35,36 Therefore, we examined whether NP-6A4-AT2R signalling affects Mcp1 expression of aortic tissues in mice exposed to AngII. As shown in Figure 3A,E, a significant increase in Mcp1 expression in AngII-treated mice was observed, which was diminished in mice treated with NP-6A4. In contrast, expression of F4/80, a marker for macrophage contents, was not significantly decreased with NP-6A4 treatment in response to AngII ( Figure 3B,F). In addition, no change in the mRNA expression of inflammatory genes (iNos, Il6 and Tnfα) was observed in macrophages isolated from experimental mice (Supporting Figure S6). Next, to understand whether NP-

| NP-6A4 treatment suppresses AngII-induced osteopontin and proteolytic activity
Next, we examined the expression of osteopontin (OPN), a matrix protein that modulates SMC proliferation and apoptosis. 37 Specifically, matrix metalloproteinases (MMP)-2 and MMP-9 are OPN-dependent molecules that promote vascular instability. 38 Increased expression of OPN was observed in the adventitial layer of mice aorta exposed to AngII. This AngII-induced increase in OPN was suppressed by NP-6A4 treatment ( Figure 3D,H). A significant reduction in Mmp2 and Mmp9 gene expression was observed with NP6-A4 treatment in the abdominal aortae of experimental mice ( Figure 4A,B). We further confirmed the NP-6A4-mediated reduction in MMP activity in the aortic tissues by in situ zymography and gelatine zymography ( Figure 4C-F). These data suggest that NP-6A4 may attenuate aortic stiffness via decreasing MMP-mediated proteolytic activity.

| NP-6A4 attenuates endothelial dysfunction
Endothelial cells (ECs) play a critical role in vascular stiffness by maintaining bioavailability of NO and proper vasodilation. We previously reported that AT2R activation with NP-6A4 increases NO bioavailability in human coronary artery endothelial cells. 8 Figure 5E). Fresh aortic tissue sections also demonstrated slightly increased NO production in response to NP-6A4 compared with AngII controls (P = 0.1932) ( Figure 5F,G). Importantly, AFM of hAOECs treated with TNF-α in the presence or absence of NP-6A4 revealed significant attenuation of endothelial cell stiffness with NP-6A4 treatment ( Figure 5H). Collectively, these data suggest that AT2R activation by NP-6A4 is upstream of NO release and is crucial for decreasing endothelial cell-mediated aortic stiffness. Moreover, since NO is known to regulate MMP activity, 39 these data suggest that NP-6A4 may decrease proteolytic activity possibly via regulation of NO.

| D ISCUSS I ON
AngII-induced vasculopathies, particularly AAA, has been studied extensively in male mice. Although AngII binds to both AT1R and AT2R in the vascular cells, the AngII-AT1R-induced inflammation The partial effects of NP-6A4 on AAA can be attributed to several factors in this study. (a) We used to a relatively lower dose of NP-6A4 for a longer duration to minimize adverse effects. It will be interesting to examine whether higher dose of NP-6A4 (up to 20 mg/ kg/day can be tolerable to mice without any side-effects; unpublished data) extends its protective role to luminal diameter and inflammatory response. (b) In the present study, we examined prophylactic effects of NP-6A4 on AAA. Our recent findings indicate that aortic stiffness rather than size of AAA is a determinant factor of stability in the small/actively growing AAAs. 22 Hence, it is highly conceivable that attenuating aortic stiffness and improving distensibility of the aorta with NP-6A4 may stabilize the AAAs as therapeutic treatment.
(c) The study is also limited by the fact that as we are utilizing a competitive AngII-induced AAA model, AT1R might be overriding the protective effects of AT2R agonist. We observed a significant reduction in Agtr1b (AT1Rb subtype) in response to NP-6A4, but not in Agtr1a (AT1Ra subtype). It is conceivable that reduction in AT1Rb partially contributes to NP-6A4's protective effects in the aorta; however, further studies are needed to verify this possibility. It will be very intriguing to examine the effects of NP-6A4 in the elastase or calcium chloride models of aneurysm. Overall, our studies highlight previously unknown protective effects of AT2R agonist on aortic stiffness and proteolytic activity, which may have potential implications to preserve the aortic integrity of the aorta in vascular pathologies.
The formation of AAA includes the degradation of medial extracellular matrix and subsequent medial rupture, followed by complex cellular changes in the intima, media and adventitia. 25,40 Additionally, the progression and rupture of aneurysms are known to be influenced by aortic stiffness as well as the blood pressure (BP). Reduced severity of AAA observed in response to NP-6A4 pre-and co-treatments correlated with decreased aortic stiffness, and this result is in agreement with the previous report. 14 Moreover, the effect of NP-6A4 treatment on the collagen content and location in the aortic tissues from mice with AngII-induced AAA suggests that protective effect of NP-6A4 on AngII-induced vascular pathology is in part mediated via attenuating collagen disorganization. We speculate that there is a higher turnover of the collagens in the aorta because of increased proteolytic activity in response to AngII and NP-6A4 co-treatment that suppresses proteolytic activity of MMPs reduces collagen degradation and improves vascular stability.  42 In addition, endothelial dysfunctions also influence arterial remodelling by causing phenotypic changes in the vSMC-enriched medial layer including proliferation, migration and apoptosis. 43,44 it is conceivable that NP-6A4-AT2R-induced increase in phospho-eNOS and bioavailability of NO could have improved endothelial function and thus mitigated AngII-induced aortic stiffness. 8 Although AT2R stimulation is shown to be anti-inflammatory in macrophages, 45 our data indicate that decreased Mcp1 and proteolytic activity observed in our model could be contributed from cell types other than macrophages such as CD4 + T cells. However, it is also true that inflammation plays an active role in the progression of AAA. 25,31 In the future, it will be interesting to dissect the protective cell types associated with NP-6A4-mediated protection in the AngII-induced mouse model of AAA.

AngII treatment increased BP in
In conclusion, AT2R stimulation with NP-6A4 attenuated only aortic stiffness in response to AngII in mouse model of AAA.
Increased levels of phospho-eNOS and NO availability that can result in restoration of endothelial dysfunction, and inhibition of proteolytic activity were the major pathways found to be involved in protective effects of NP-6A4. Increased RAS activity and elevation of AngII are one of the mechanisms for the development of AAA. As AngII can activate AT2R, the fact that AngII infusion causes AAA in male mice indicates that AngII-induced AT2R activation that can happen at the same time as AngII-induced AT1R activation in male aortic tissue is not sufficient to prevent development of AAA. Conversely, female mice that express higher levels of AT2R in their vasculature are better protected from AngII-induced AAA. 46 Importantly, our data show that NP-6A4-AT2R signalling mitigates AngII-induced aortic stiffness in male mice suggesting that NP-6A4 treatment could be effective in restoring vascular protective AT2R signalling in male mice that have less AT2R expression compared with their female counterparts.
Thus, this study can be of clinical significance because of potential implication of this AT2R agonist in the treatment options for AAA, a male predominant disease.

ACK N OWLED G EM ENTS
The authors thank Dr Zhe Sun at imaging core facility at Dalton Cardiovascular Research Center for capturing the ultrasound images and assistance with AFM. This work was supported by Grants R01HL124155 (CPH), 1R01HL118376 (LP) and 1R01HL138988 (LP), and funding from the Research Institute at the University of Missouri to CPH. NP-6A4 was a gift from Novopyxis Inc (Boston, MA, United States).

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data are available in the manuscript. The data that support the findings of this study are available on request from the corresponding authors.