Allosteric Activation of PP2A Inhibits Experimental Abdominal Aortic Aneurysm

Although extremely important, the molecular mechanisms that govern aortic aneurysm (AA) formation and progression are still poorly understood. This deficit represents a critical roadblock toward the development of effective pharmaceutical therapies for the treatment of AA. While dysregulation of Protein Phosphatase 2A (PP2A) is thought to play a role in cardiovascular disease, its role in aortic aneurysm is unknown. The objective of this study is to test the hypothesis that PP2A regulates abdominal aortic aneurysm (AAA) progression in a murine model. In an angiotensin II-induced AAA murine model, the PP2A inhibitor, LB-100, markedly accelerated AAA progression as demonstrated by increased abdominal aortic dilation and mortality. AAA progression was associated with elevated inflammation and extracellular matrix fragmentation, concomitant with increases in both metalloproteinase activity and reactive oxygen species production. Conversely, administration of a novel class of small molecule activators of PP2A (SMAPs) resulted in an antithetical effect. SMAPs effectively reduced AAA incidence along with the corresponding pathologies that were increased with LB-100 treatment. Mechanistically, modulation of PP2A activities in vivo functioned in part via alteration of the ERK1/2 and NFkB signaling pathways, known regulators of AAA progression. These studies, for the first time, demonstrate a role of PP2A in AAA etiology and demonstrate that PP2A activation may represent a novel strategy for the treatment of abdominal aortic aneurysms.

as summarized in a recent report [6], this paradigm could be changing. Efforts to unpack the therapeutic utility of protein phosphatases are progressing with unprecedented potential via an allosteric drug discovery approach.
In the present study, we focused on the major serine/threonine protein phosphatase 2A (PP2A) family of phosphatases, that regulates multiple key molecular pathways, including MAPK, NFkB, and Akt, all known regulators of AAA development and progression [7][8][9][10]. PP2A, a potent family of de-phosphorylating enzymes within mammalian cells, is a critical serine/threonine phosphatase that has been implicated in the regulation of many signaling pathways, including apoptosis and cell cycle progression as well as the negative regulation of many oncogenic and cell survival pathways [11][12][13]. PP2A's ability to broadly regulate diverse signaling pathways is due to its dynamic heterotrimeric composition that allows over 60 different PP2A holoenzymes to form, each with its own substrate specificity [14,15]. All three subunits of PP2A are required for its full enzymatic activity. Most PP2A exists in cells and tissues as ABC heterotrimers. The Scaffolding subunit (A Subunit) positions the complex together; the C subunit contains the catalytic site, and the B subunit (Regulatory Subunit), regulates the target specificity of PP2A.
The restoration of PP2A activity has been shown to be therapeutically effective in model systems. Several compounds have been shown to activate PP2A indirectly, mainly through inhibiting endogenous PP2A inhibitors or targeting PP2A regulators.
However, these indirect methods of PP2A activation have many additional downstream effectors, limiting their use as a tool to study PP2A activity specifically. As reported in previous publications by the Narla group, in order to improve the tolerability and Downloaded from http://portlandpress.com/clinsci/article-pdf/doi/10.1042/CS20210315/919170/cs-2021-0315.pdf by guest on 19 August 2021 purchased from the vendors were randomized into control and treatment groups (vehicle vs LB-100 or DT-1154), followed by concurrent AngII infusion and treatment (LB-100 or DT-1154). The PP2A inhibitor LB-100 (Selleck Chemicals,1.0mg/kg, dissolved in normal saline) was administered through ip injection every other day, the treatment was initiated one week prior to AngII infusion and continued until the completion of AngII infusion. SMAPs (DT-1154,100mg/kg/d, or DT-061,15mg/kg BID) were administered via oral gavage 3 days prior to AngII infusion and continued until the completion of experiments. Working solution for SMAPs were prepared in a N,N-Dimethylacetamide (DMA)/Kolliphor HS-15 (Solutol)/diH20 solution. Mice were anesthetized with ketamine (1.5mg/kg)/xylazine (0.3mg/kg), followed by mini pump implantation. To relieve pain from surgery, post-operative analgesia was administered using intra-peritoneal injection of buprenorphine (0.05-0.10mg/kg IM q8-12h) on the day of surgery and on post-operative days 1-3. At the completion of AngII infusion, transcardial perfusion in mice was accomplished via butterfly catheters to perfuse normal saline followed by 4% paraformaldehyde at 100 mmHg for 8 minutes. The aorta was exposed under a dissecting microscope (Leica, Model DM 500), peri-adventitial tissues were carefully removed from the aortic wall, and a caliper was used to measure the external diameter of suprarenal abdominal aorta. To compute for AAA incidence, an aneurysm was defined by a 50% or greater increase in the external diameter of the suprarenal aorta in comparison to aortas from saline-infused mice.

ROS generation assay:
A subset of study mice were perfused only with ice-cold PBS (pH 7.4) without any fixatives at 100 mmHg for 5 min at 4°C. The abdominal aorta was Downloaded from http://portlandpress.com/clinsci/article-pdf/doi/10.1042/CS20210315/919170/cs-2021-0315.pdf by guest on 19 August 2021 dissected and embedded in OCT (Tissue-Tek; Miles Inc., Naperville, IL) and sectioned using a cryostat (Leica, Wetzlar, Germany). DHE (dihydroethidine hydrochloride, 5μM) was topically applied to the freshly cut frozen aortic sections (10μm) and incubated at 37°C for 30 min. The presence of ROS is shown as red. DHE fluorescence images were acquired with a Leica microscope (Model: DM2000LED) using 510 nm excitation/ 580 nm emission filters at the same photomultiplier tube voltage, gain and offset. The analyzer was blinded to the identity of sections. Images were converted to gray scale and the integrated density per image area of interest was measured using NIH ImageJ.
PP2A phosphatase activity assay -Aortic PP2A activity was measured using threonine phosphopeptide as the substrate with the PP2A immunoprecipitation phosphatase assay kit (Millipore). Tissues were lysed using a lysis buffer (20mM imidazole-HCL, 2mM EDTA, 2mM EGTA, 1mM benzamidine, pH 7.0 with 10μg/ml aprotinin and 1mM PMSF). Tissue extracts were sonicated for 10 s and centrifuged at 2000g for 5 min. 200μg of total protein extracts were incubated with anti-PP2A-C subunit antibody (4μg) 18h at 4℃ with gentle rocking. Then 40μL of Protein A agarose slurry were added and rocked 2h at 4℃. Beads were washed 3 times with 700μL TBS, and once with 500μL Ser/Thr assay buffer. The beads were then incubated with 60μL diluted phosphopeptide and 20μL Ser/Thr assay buffer at 30°C for 10min in a shaking incubator. The beads were centrifuged briefly, and the samples were analyzed in a colorimetric assay using malachite green at an absorbance of 650 nm.
Tissue lysis and western blot -Snap-frozen aorta tissues were extracted using a Total Downloaded from http://portlandpress.com/clinsci/article-pdf/doi/10.1042/CS20210315/919170/cs-2021-0315.pdf by guest on 19 August 2021 or 12 hours for protein or RNA isolation respectively. Cellular proteins were extracted with RIPA buffer supplemented with protease inhibitor tablet (Roche).
RNA extraction and Quantitative RT-PCR analysis -Cells were treated as indicated and then harvested for total RNA using Qiagen RNeasy mini kit. Abdominal aorta tissues from saline or AngII treated mice were snap-frozen in liquid nitrogen. Total RNA was isolated from cultured cells or tissues with Trizol (Invitrogen Corp., Carlsbad, CA) as described by the manufacturer. Total RNA (1µg) was DNase-treated and reversetranscribed using Bio-Rad iScript Reverse Transcription Supermix kit. The resulting cDNA was diluted to 100µL and used in subsequent real time PCR reactions. Gene expression was assessed by SYBR green on QuantStudio 6 (Applied Biosystems).
Gene expression was standardized to GAPDH or β-actin using the ΔΔCt method.
Depending on the availability of primers in the lab, GAPDH or β-actin were used interchangeably in our studies as they did not alter our data in the current study based on our experience. To ascertain our samples are free of contamination, as a standard practice for RT-PCR experiments in the lab, no template and no RT controls were always included.
MMP activity measurement in situ -In situ MMP zymography was performed on freshly cut OCT-embedded murine aorta sections. Fluorescein-conjugated gelatin substrate DQ gelatin (Invitrogen) was prepared according to the manufacturer's instructions. The substrates were then applied to sections and allowed to incubate at 37°C for 24 hours, after which green fluorescence was examined using a fluorescent microscope. Negative controls were performed on parallel sections in the presence of 5

Inhibition of PP2A promotes abdominal aortic dilation and vascular inflammation
The regulation of protein phosphorylation involves the net balance of protein kinase vs protein phosphatase activity. Protein phosphatase 2A family of holoenzymes possess major serine/threonine phosphatase activity in mammalian cells [19].
Considerable experimental evidence indicates that several major signaling pathways involved in AAA progression, including MEK/ERK1/2, P38, JNK1/2 and NFkB [8-10, 20, 21], are downstream targets of PP2A. Armed with this understanding, we hypothesized that the loss of PP2A function promotes abdominal aortic dilation. To test this, we used the well characterized active catalytic site PP2A inhibitor, LB-100. The ability of LB-100 to affect AAA development was tested in an established mouse AAA model that involves the infusion of Angiotensin II into Apoe-null mice. This model successfully recapitulates many of the characteristics of human AAA, including luminal dilation, ECM fragmentation, the inflammatory response and reactive oxygen species (ROS) generation within the arterial wall [22]. Upon the completion of AngII infusion, we evaluated the AAA phenotype in Apoe-null mice. In line with the inhibition of PP2A activity (Suppl. Figure 1), in the aorta during AngII infusion, as demonstrated in Figure   1A and Supplemental Figure 2A 1.91mm; versus vehicle: 1.61mm, P =0.019) ( Figure 1A). Consistent with these observations, LB-100 treatment dramatically increased elastin degradation resulting in compromised aortic vessel wall integrity, enhanced aortic aneurysm severity ( Figure 1C and Suppl. Figure 1B&C) and increased mortality ( Figure 1D).
It is commonly believed that inflammation plays an important role in the development and progression of most AAAs. We, therefore, examined the impact of PP2A inhibition on vascular inflammation. Concordant with the enhanced severity of aortic aneurysm, LB-100 profoundly amplified vascular inflammation following AngII infusion as demonstrated by heightened AngII-elicited inflammatory cell (macrophage and T cells) infiltration in the adventitia ( Figure 1E). The expression of several key inflammatory factors (MCP-1, MMP2, and VCAM-1) was also significantly augmented ( Figure 1F, Suppl. Figure 1D). Furthermore, LB-100 substantially elevated MMP activities and reactive oxygen species generation ( Figure 1G&H), two key mediators of AAA pathogenesis.

PP2A activation strongly attenuates AAA progression
The above results support our hypothesis that loss of PP2A activity augments AngII-induced abdominal aortic dilation. To further substantiate PP2A as a critical regulator of AAA, we next sought to determine the effect of PP2A activation in the same AngII infusion model. Restoration of PP2A activity has been shown to be of significant therapeutic value, however pharmaceutically tractable approaches to directly activate PP2A remain elusive. In order to improve the tolerability and translational potential of PP2A activating compounds, the Narla lab developed a new chemical series of SMAPs We administered DT-1154 (a validated PP2A activator compound) at 100 mg/kg/day, a dose that is well tolerated and effective in murine cancer models [16,23], three days prior to AngII treatment and continued treatment throughout the ensuing 4week AngII infusion. Importantly, DT-1154 was well tolerated at the treatment dose tested in this AAA model and did not result in altered mouse behavior, weight loss or mortality. As shown in Suppl. Figure 3, SMAPs administration in mice increased PP2A activity (a newer generation of SMAPs DT-061 was used because DT-1154 has been discontinued). Following AngII infusion, we assessed the effects of SMAPs on AAA progression.
In contrast to what was found with PP2A inhibition, treatment of Apoe-null mice undergoing AngII infusion with SMAPs (DT-1154) administration markedly reduced aortic dilation ( Taken together, these data clearly demonstrate that SMAP-dependent activation of PP2A significantly inhibits experimentally induced abdominal aortic aneurysm formation and reduces the severity of abdominal aneurysm, supporting a protective role of PP2A against AAA formation.

Manipulation of PP2A modulates ERK1/2 and NFkB pathways
To gain additional insight into how alterations in PP2A function affect aortic dilation and aneurysm progression, we assessed several well documented signaling pathways critical in the activation of extracellular remodeling enzymes and vascular inflammation in AAA. As shown in Figure 3A, PP2A inhibition by LB-100 strongly  Figure 5A), findings in corroboration with the hypothesis that the decrease of PP2A activity may predispose aneurysm formation. Similarly, in AngII-induced murine AAA, a marked reduction of PP2A activity was seen ( Figure 5B). These findings prompted us to wonder whether PP2A subunits or holoenzyme complex might be altered in AAA. Given that there are 17 potential PP2A subunits and there are approximately 96 unique holoenzyme configurations, we recognize it is difficult to address which holoenzyme complex is involved. To gain initial insights into which specific PP2A subunits might be affected in AAA, we assessed the protein expression of several PP2A subunits in human AA tissues. As shown in Figure 5C&D, a significant reduction of the scaffolding PP2A-A subunit was seen, while no difference was observed for the regulatory PP2A-B and catalytic PP2A-C subunits. These data strongly suggest that PP2A is relevant in human aneurysm formation and, coupled with our results obtained from an experimental AAA model, suggest that activation of PP2A may serve as a novel therapeutic target for the clinical treatment of aortic aneurysms. of the current study is to determine whether SMAPs can stabilize existing aneurysms and these efforts will be the focus of future studies. Nevertheless, our proof-of-principle experimental findings here point to therapeutic reactivation of PP2A as a novel strategy for the treatment of AAA.
In our current study, we have not thoroughly explored the mechanisms by which SMAPs negatively regulate AAA progression. We showed that SMAPs inhibited vascular inflammation after AngII infusion into Apoe-null mice. Our data supported that the inhibition of two important pathways (ERK1/2 and NFkB) that promote inflammation and have been demonstrated to be required for AAA progression, likely contributed to the observed mitigation effect by SMAPs. Of note, both ERK1/2 and NFkB pathways have been demonstrated to be required for MMP and ROS generation in AAA. It would be necessary to conduct additional studies to comprehensively determine whether SMAPs also affected other major pathways that are known important regulators of inflammation and AAA, including but not limited to p38, mTOR, STAT, c-Jun N-terminal kinases. Similarly, our studies have not comprehensively explored the effects of SMAPs in different cell types that are known to be important in the inflammatory responses leading to AAA pathology. While our current data support that administration of SMAPs in macrophages recapitulated the inhibition effect on ERK1/2 and NFkB activation seen in AngII-infused aortas, we do not know the impact of SMAPs on other cell types such as endothelial cells, smooth muscle cells, fibroblasts and other immune cell types.
Lastly, whether the alteration of PP2A activity is manifested as changes in transcription regulation in our experimental conditions merits further exploration. For instance, a prior study showed that PP2A inhibition in lung epithelial cells leads to accumulation of a Downloaded from http://portlandpress.com/clinsci/article-pdf/doi/10.1042/CS20210315/919170/cs-2021-0315.pdf by guest on 19 August 2021 hyperphosphorlated inactive form of tristetraprolin (an anti-inflammatory molecule and a direct target of PP2A) that caused the augmentation of mRNA of proinflammatory molecules. Importantly, PP2A activation by FTY720 reverses this effect [24]. Whether SMAPs affect tristetraprolin function will be explored in future investigations.
In addition to the anti-inflammatory effects, SMAPs treatment also mitigated elastin degradation ( Figure 2C, Suppl Figure 4C It is conceivable that SMAPs and LB-100 could introduce "off-target" effects in our Downloaded from http://portlandpress.com/clinsci/article-pdf/doi/10.1042/CS20210315/919170/cs-2021-0315.pdf by guest on 19 August 2021 experimental animal model. We recognize that pharmacological gain-and loss-offunction approaches could be undermined by "off-target" effects. However, based on previous studies in multiple cancer (ex., lung, prostate and breast) models, all data supported SMAPs' target specificity and favorable toxicity profiles [16,23,27]. In the first study describing the use of SMAPs, SV40 small T antigen was utilized as a form of target validation to determine whether the biological effect of SMAPs was through PP2A activation. Expression of the small T antigen in lung cancer H358 cells conferred resistance to SMAP treatment in the xenograft model, indicating that SMAP-mediated growth inhibition was dependent on functional PP2A holoenzymes [16]. In another study, DT-1310 (a biologically inactive analog that lacks a N-H sulfonamide hydrogen bond donor function) is structurally similar to SMAP but is biologically inactive, was used. Treatment with DT-1310 neither induced an increase in annexin V positivity nor inhibited PP2A-regulated signaling pathways, such as AKT and MAPK signaling in human advanced lung adenocarcinoma (LUAD) cell line H1975 [28]. Recently, the 3.6 Å cryo-em structure provided insight into the unique trimeric pocket recognized by DT-061 and the molecular interactions of DT-061 with all three PP2A subunits. Specifically, these studies showed that DT-061 shifts the equilibrium of PP2A heterogeneity to favor B56⍺ ultimately [29] suggesting that B56α could play a major role in AAA.
Nevertheless, to gain in-depth insights into the requirement of specific PP2A subunit for SMAPs' effect, as well as the importance of each specific PP2A subunit in AAA, studies involving genetic manipulation of PP2A subunits via knock in or knockout approaches in vivo and in vitro are warranted. Along this line, previous studies offered several important clues. In our future investigations, we will manipulate PP2A Aα and Our studies highlight a potential new target for pharmacologic therapy and management strategies of AAA.

Data availability statement
All supporting data for this manuscript is included in the Figures and the accompanying supplemental files.