Lipid phosphate phosphatase 3 in smooth muscle cells regulates angiotensin II-induced abdominal aortic aneurysm formation

Genetic variants that regulate lipid phosphate phosphatase 3 (LPP3) expression are risk factors for the development of atherosclerotic cardiovascular disease. LPP3 is dynamically upregulated in the context of vascular inflammation with particularly heightened expression in smooth muscle cells (SMC), however, the impact of LPP3 on vascular pathology is not fully understood. We investigated the role of LPP3 and lysophospholipid signaling in a well-defined model of pathologic aortic injury and observed Angiotensin II (Ang II) increases expression of PLPP3 in SMCs through nuclear factor kappa B (NF-κB) signaling Plpp3 global reduction (Plpp3+/−) or SMC-specific deletion (SM22-Δ) protects hyperlipidemic mice from AngII-mediated aneurysm formation. LPP3 expression regulates SMC differentiation state and lowering LPP3 levels promotes a fibroblast-like phenotype. Decreased inactivation of bioactive lysophosphatidic acid (LPA) in settings of LPP3 deficiency may underlie these phenotypes because deletion of LPA receptor 4 in mice promotes early aortic dilation and rupture in response to AngII. LPP3 expression and LPA signaling influence SMC and vessel wall responses that are important for aortic dissection and aneurysm formation. These findings could have important implications for therapeutics targeting LPA metabolism and signaling in ongoing clinical trials.

www.nature.com/scientificreports/ PCR and custom gene array (ThermoFisher Scientific) was performed on QuantStudio 7 Flex (Applied Biosystems) using TaqMan Universal PCR Master Mix and TaqMan FAM primers with 18s as an internal control.
In situ hybridization and proximity ligation assays (ISH-PLA). Dimethylation of lysine 4 on histone 3 (H3K4me2) of the smooth muscle myosin heavy chain 11 (Myh11) promoter was detected by in situ hybridization (ISH) and Proximity Ligation Assay (PLA) as previously published 24 . Briefly, the 2 kb promoter of Myh11 was amplified by PCR, cloned into pCR2.1 vector for amplification (TOPO cloning kit, ThermoFisher Scientific) and biotin labeled probes were generated by Nick Translation (Roche) using biotin-14-ATP (ThermoFisher Scientific). Probes were denatured in hybridization buffer (2X SSC, 50% high grade formamide, 10% dextran sulfate, 1 μg mouse Cot-1 DNA) for 5 min at 80 °C. Abdominal aorta sections were incubated with 0.5% pepsin at 37 °C for 15 min, followed by incubation with hybridization buffer containing biotinylated probe for 5 min at 80 °C then followed by incubation for 16-24 h at 37 °C. Following hybridization, slides were washed in 2X SSC, 0.1% NP-40 buffer. Sections were blocked, incubated with mouse H3K4dime (5 μg/mL, Millipore Sigma, clone CMA303) and rabbit Biotin (5 μg/mL, Abcam #ab53494) antibodies overnight at 4 °C, followed by incubated with secondary antibodies containing PLA probe at 37 °C for 1 h. Ligation and amplification were performed (Duolink detection kit Orange 555 nm) and mounting medium with DAPI was used. Immunofluorescence was quantified with Image J.
Aortic smooth muscle cell isolation. Aortic smooth muscle cells were isolated from fl/fl and SM22-Δ mice as previously described 17

AngII-increases PLPP3 expression in smooth muscle cells through an NF-κB dependent pathway.
Our previous work has demonstrated that LPP3 expression is dynamically upregulated in SMCs under conditions of vascular inflammation and contributes to vascular pathology 4,17,25 . We recently identified RelA and RelB responsive elements in the PLPP3 gene that regulate expression through canonical activation of NF-kB signaling 19 . We therefore sought to identify upstream regulators of LPP3 expression and observed that AngII, but not LPA or TGF-β, increased LPP3 expression in caHSMCs (Fig. 1A). As previously reported, Mβ-CD cholesterol loading of SMCs also increased LPP3 expression 22 . The AngII effect was time-dependent with highest levels of Plpp3 mRNA detected at 72 h (Fig. 1B). AngII is a known activator of NF-κB signaling 26 and p65 and IL6 expression, downstream markers of NF-κB signaling, increased at 72 h ( Fig. 1B,C). AngII receptor I inhibition blocked the AngII-mediated increase in PLPP3 gene expression (Fig. 1D), implicating Ang II receptor-mediated signaling. We previously demonstrated that C/EBPβ binding to an enhancer within PLPP3 upregulates gene expression in response to oxLDL or cholesterol loading 14,22 ; however, following AngII treatment of SMC, C/ EBPβ levels were not significantly altered (Fig. 1C). Instead, the NF-κB signaling inhibitor parthenolide attenuated AngII-induced increases in PLPP3 gene expression (Fig. 1E), suggesting a role for NF-κB signaling downstream of AngII.
Loss of Plpp3 in smooth muscle cells protects against aortic aneurysm and transmural rupture. In hyperlipidemic mice, AngII (1000 ng/kg/min) stimulates aortic aneurysm formation 27 . Therefore, we sought to determine whether AngII-upregulation of LPP3 influenced pathologic progression by using mice heterozygous for Plpp3 expression (Plpp3 +/− ). Hyperlipidemia was achieved by injecting littermate control WT or Plpp3 +/− mice with PCSK9D377Y.AAV to lower LDL receptor expression and feeding the animals Western diet. At 28 days after AngII infusion, plasma cholesterol levels were similar in Plpp3 +/− and WT littermate control animals ( Fig. 2A). Abdominal aorta expression of Plpp3 was lower in Plpp3 +/− as compared to WT mice (Fig. 2B). Interestingly, the overall abdominal aortic diameter was smaller and transmural rupture was decreased in hyperlipidemic Plpp3 +/− mice after AngII infusion (Fig. 2C,D; P < 0.05), although there was no difference in the overall incidence of aneurysm formation, as defined by a greater than 50% increase of abdominal aortic diameter from baseline to post AngII (Fig. 2E www.nature.com/scientificreports/ may normally promote aneurysm formation in the AngII model and that a reduction in LPP3 expression may be sufficient to provide protection. We next investigated whether SMC expression of LPP3 was required for aneurysm formation in response to AngII using previously characterized mice lacking SMC Plpp3 expression on the Ldlr −/− background (SM22-Δ). At 28 days after AngII infusion, systolic blood pressure (Fig. 3A) and plasma cholesterol (Fig. 3B) were similar in SM22-Δ and littermate control fl/fl animals on the Ldlr −/− background (fl/fl). No difference was observed in plasma triglycerides between fl/fl vs SM22-Δ mice (527 ± 290 vs 299 ± 246; P = 0.177). Similar to Plpp3 +/− mice, SM22-Δ mice had a significant reduction in overall abdominal aortic diameter in comparison to fl/fl mice (1.82 ± 0.53 vs 1.28 ± 0.34 mm; P < 0.001; Fig. 3C). SM22-Δ mice also had a lower overall incidence of aneurysm formation as defined by a greater than 50% increase of abdominal aortic diameter from baseline to post AngII and decreased transmural rupture. (84% in fl/fl vs 20% in SM22-Δ; Fig. 3D,E). One-month mortality was similar in the groups at 14.3% in fl/fl and 10.5% in SM22-Δ male mice. Female SM22-Δ mice also displayed a smaller mean abdominal aortic diameter following AngII than their fl/fl littermates (Supplemental Figure   Results are presented as relative gene expression (mean ± SEM). *P < 0.05, One-way ANOVA, # P < 0.05 Kruskal-Wallis One-way ANOVA on Ranks. (C) caHSMCs were treated with vehicle (n = 4) or AngII (1 μM, n = 6) for 72 h. Results are presented as relative gene expression (mean ± SEM), *P < 0.05, unpaired two-tailed student t-test. (D) caHSMCs were treated with vehicle, AngII (1 μM), AngII + Irbesartan (1 μM), or AngII + PD-123,319 (1 μM) for 72 h, n = 3 for all treatment conditions. Results are presented as relative PLPP3 gene expression. (E) caHSMCs were treated with vehicle (n = 4), angiotensin II (1 μM, n = 8) or angiotensin II + parthenolide (1 μM, n = 3) for 72 h. Results are presented as relative gene expression (mean ± SEM) *P < 0.05, One-way ANOVA. www.nature.com/scientificreports/

LPP3 regulates SMC phenotypic switching and vascular inflammation. AngII treatment results
in changes in the extracellular matrix that likely result in aortic aneurysm formation. These changes are associated with phenotypic modulation of SMCs resulting in their migration, clonal proliferation, and aneurysm formation 28 . We therefore examined the effect of LPP3 on expression of genes associated with SMC phenotypic modulation, extracellular matrix, and inflammation. At 28 days after AngII infusion, a significant reduction in Acta2 expression with no change in p65, Cnn1, or Mmp9 occurred in abdominal aortas of SM22-Δ mice (Fig. 4A). In keeping with the gene expression data, ACTA2 staining ( Fig. 4B) was also significantly reduced in SM22-Δ compared to fl/fl abdominal aorta at four weeks after AngII infusion (Fig. 4C), along with reduction in calponin expression another marker of differentiated SMC (Fig. 4D), suggesting a difference in phenotypic switching in the SM22-Δ mice. In addition to phenotypic switching, SMCs undergo apoptosis during aneurysm development. To determine if the reduction in Acta2 and calponin expression reflected a lack of committed SMCs, ISH-PLA was performed on aortic sections to measure dimethylation of lysine 4 on histone 3 (H3K4me2) at the smooth muscle myosin heavy chain 11 locus, an epigenetic mark exclusive to SMC 24 that is maintained in SMC that undergo phenotypic switching. ISH-PLA for H3K4me2 in fl/fl and SM22-Δ (Fig. 4E) abdominal aorta sections revealed a similar number of positive cells per section (Fig. 4F) suggesting the number of SMCs within the abdominal aorta is similar between fl/fl and SM22-Δ mice. Together this data suggests that the loss of LPP3 in SMCs reduces expression of committed markers such as smooth muscle α-actin and calponin following AngII treatment.
To determine if LPP3 was regulating early changes in the aortic wall prior to aneurysm formation, mice were treated with AngII for one week, a time point in which abdominal diameters were the same in fl/fl and  (Fig. 5A), consistent with alterations in inflammation and extracellular matrix pathways following AngII treatment. Additionally, Acta2 expression was significantly decreased with no change in Mmp9 in SM22-Δ abdominal aortas one week after AngII treatment (Fig. 5B).
To understand whether these differences reflected alterations in the phenotypic state of SMCs, aortic SMCs were isolated from fl/fl and SM22-Δ mice and treated with AngII in culture. As expected, Plpp3 expression was significantly lower in SM22-Δ than fl/fl cells (Fig. 5C) following AngII treatment. SMCs from SM22-Δ mice had significantly reduced levels of Myh11 and Col1a1 with a trend of decreasing Acta2 gene expression (Fig. 5C). While vimentin gene expression was not increased in isolated SMCs from SM22-Δ mice following AngII treatment, vimentin staining (Fig. 5D) was increased within the medial layer of aortas from SM22-Δ mice following 1 week AngII infusion, suggesting a fibroblast-like phenotype. In keeping with these observations, FSP-1 (Fig. 5E) staining was higher within the medial layer of aortas from SM22-Δ mice. Recently, the pluripotency factor Oct4 has been demonstrated to regulate SMC function and phenotypic switching 29 . We observed significant www.nature.com/scientificreports/ upregulation of Oct4 mRNA in abdominal aorta of control SM22-Δ mice (Fig. 5F) and isolated aortic SMC from SM22-Δ mice (Fig. 5G). These findings suggest that in the absence of LPP3, SMC have lower expression of SMC markers and higher vimentin, FSP-1 and Oct4, suggesting changes in regulation of SMC differentiation state. LPP3 is known to regulate LPA signaling 22,30-32 , which in turn promotes SMC de-differentiation 33 and fibrosis [34][35][36] . Indeed, a reduction in LPP3 expression will lower LPA degradation and could potentiate the effect of LPA on de-differentiation and fibrosis, consistent with the phenotype observed in the SM22-Δ mice. If LPP3 is normally limiting the effects of LPA, then we would expect that LPA signaling may provide protection against the development of dissecting aneurysm in response to AngII. In mice, LPAR4 has been implicated in vascular development and inflammation in the context of atherosclerosis 21 , and therefore, we examined the phenotype of mice lacking LPAR4 signaling in the AngII-aneurysm model. For these experiments, LPAR4 deficient mice (LPAR4 Y/− ) or WT controls were treated with PCSK9D377Y.AAV and fed Western diet to elicit hyperlipidemia. Plasma cholesterol levels were similar at 1 week after AngII infusion (Fig. 6A). Abdominal aortic diameter was significantly greater in LPAR4 Y/− mice compared to WT (Fig. 6B). Additionally, the LPAR4 Y/− mice were more prone to death due to rupture with an odds ratio of > 9 ( Fig. 6C; P = 0.026). In the surviving mice, plasma cholesterol, abdominal aortic dimensions, Plpp3 and Acta2 gene expression were similar between WT and LPAR4 Y/− mice at four weeks (Supplemental Figure IX). The early increase in abdominal diameters and increased rupture rate in mice lacking LPAR4 is consistent with the hypothesis that heightened LPA signaling in context of LPP3 deficiency contributes to protection from aneurysm formation. www.nature.com/scientificreports/

Discussion
Abdominal aortic aneurysm (AAA) is a potentially fatal condition due to aortic rupture, with a 4-7% prevalence in men over 55 years old 37 . Currently, monitoring aneurysm growth followed by surgical intervention represents the only treatment option for AAA 38 . The pathophysiology of AAA is complex and not completely understood but includes macrophage accumulation, alterations in inflammatory mediators, changes in extracellular matrix integrity, and smooth muscle cell phenotypic switching 38 .
In this study, we provide the first evidence that lysophospholipid signaling regulates the development of dissecting aortic aneurysm elicited by AngII in hyperlipidemic mice and suggest novel targets for therapeutic intervention. Specifically, the loss of LPP3 in SMC confers protection against the formation of dissecting abdominal aortic in association with significant reductions in SMC makers, inflammatory markers and a corresponding increase in fibroblast markers. We previously demonstrated that LPP3 regulates SMC differentiation following vascular injury in carotid arteries or in the context of atherosclerosis. Somewhat surprisingly, while the loss of LPP3 in SMC increased the development of atherosclerosis in mice, it protects from AngII-mediated aneurysm formation. The role of LPP3 in regulating the development of dissecting aortic aneurysm may reflect a role in influencing phenotypic switching within SMC and the associated changes in the SMC contractile apparatus. www.nature.com/scientificreports/ LPP3 degrades and thereby inactivates the bioactive lipid LPA, which is a potent inducer of SMC de-differentiation 17 and a pro-fibrotic mediator. We have demonstrated that LPP3 controls LPA levels, LPA signaling 17,39 and that the absence of LPP3 increases LPA receptor mediated signaling in SMC. Thus, it is possible that the effects of LPP3 on SMC differentiation markers are mediated by regulating local levels of bioactive LPA in the context of aneurysm formation. In the absence of LPP3, excessive LPA signaling could result in exaggerated dedifferentiation of SMC and their conversion to a more fibroblast-like phenotype that provides protection against aneurysm formation. In support of this hypothesis, the loss of the LPA specific LPAR4 receptor worsens AngIIinduced abdominal aortic expansion and increases overall mortality associated with rupture. These findings indicate that LPA signaling plays a protective role in this model of AAA, which is in contrast to the permissive effect of LPA signaling on experimental atherosclerosis.
It is important to note that Sm22 expression is not limited to smooth muscle cells 40 so it is possible other cell types could be responsible for the reduction in AAA seen in the SM22-Δ mice. CD68 staining, a macrophage marker, revealed no difference between fl/fl and SM22-Δ mice indicating any potential loss of LPP3 from myeloid cells due to off targeted effects of the SM22 Cre did not impact macrophage migration into aortic tissue.
In other models, de-differentiation of SMC promotes aneurysm formation 41,42 and atherosclerosis burden 43 . Indeed, our own results demonstrate that the loss of LPP3 in SMC promotes atherosclerosis at the aortic root and arch. It is possible that the protection from aneurysm despite SMC de-differentiation observed in this study reflects a difference in the mechanism of formation (elastase vs AngII) and/or differences in embryonic lineage of SMCs within the aorta. Aortic root and aortic arch SMCs derive from the secondary heart field and cardiac neural crest, respectively, while abdominal aortic SMCs arise from somites 44 . Differences in embryonic origin of the SMC may also contribute to phenotypic differences in the atherosclerosis and aneurysm models. Finally, while the pathophysiology of atherosclerosis and aneurysm are similar there are notable examples of discordance in their development, such as occurs in diabetics who are predisposed to atherosclerotic vascular disease and protected from AAA; likewise, testosterone 45 and the interferon gamma system 46 have discordant effects on development of atherosclerosis and aneurysm.
Our previous work 17 and supplemental Figure III, IV show no change in baseline vasculature vessel function in SM22-Δ, despite consistent gene expression indicating SMC phenotyping switching at baseline. Recent single cell RNA-Seq work has shown that SMC phenotypic switching lies on a continuum during disease progression 47 and we hypothesize that the loss of Lpp3 in SMCs promotes phenotypic switching but prevents progression of SMC phenotyping switching that is detrimental in disease states. This may reflect the ischemic pre-conditioning model in cardiac injury in which small amounts of ischemia protect against subsequent ischemic events 48 . Future work examining the progression of SMC phenotyping switching during AAA and single cell RNA-Seq cell clustering will aid in our understanding of how SMC phenotyping switching in SM22-Δ mice may differ to provide protection against AAA.
Of important clinical relevance, if heightened LPA signaling contributes to the protection from dissecting aortic aneurysm, then inhibitors of autotaxin, the enzyme responsible for bioactive LPA generation, could promote the pathology. At present, autotaxin inhibitors are in phase 3 clinical trials for efficacy in treating idiopathic pulmonary fibrosis 49 and should be monitored cautiously for an increase in risk for aortic aneurysm rupture.
A limitation of our study is the use of AngII to promote the development of aneurysm formation. While this model shares some features with AAA development in humans, it does not faithfully recapitulate pathology that occurs in humans. Indeed, some have proposed that it is a better model of aortic dissection than aneurysm 27 . www.nature.com/scientificreports/ Additionally, small samples size and wide variation in gene expression profiles for some genes limit the conclusions that can be made; however, consistent changes in Acta2 in smooth muscle deficient Lpp3 mice highlight a role for smooth muscle Lpp3 in AngII mediated abdominal aortic aneurysm. Further studies will need to address the exact mechanism by which Lpp3 loss in smooth muscle protects against abdominal aortic aneurysm. In summary, we demonstrate a novel pathway for regulation of LPP3 during vascular inflammation involving a NF-κB dependent pathway. SMC LPP3 regulates phenotypic modulation, and in the absence of LPP3, SMCs assume a more de-differentiated fibroblast-like phenotype that is associated with protection from the development of abdominal aortic aneurysm following AngII infusion. Loss of LPAR4 promotes early aneurysmal dilation and rupture, indicating that LPA signaling normally protects against AngII-induced pathology. These findings provide important information about novel pathways that regulate SMC phenotype in the context of pathologically important processes.