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Journal of Molecular and Cellular Cardiology 114 (2018) 334–344
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Peptidyl-prolyl isomerase Pin1 deficiency attenuates angiotensin II-induced abdominal aortic aneurysm formation in ApoE−/− mice
T
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Er-shun Lianga, Wen Chengb, Rui-xue Yanga, Wen-wu Baib, Xue Liua, , Yu-xia Zhaoa,b a
The Key Laboratory of Cardiovascular Remodeling and Function Research, The State and Shandong Province Joint Key Laboratory of Translational Cardiovascular Medicine, Department of Cardiology, Qilu Hospital of Shandong University, Jinan, Shandong, China b Department of Traditional Chinese Medicine, Qilu Hospital of Shandong University, Jinan, Shandong, China
A R T I C L E I N F O
A B S T R A C T
Keywords: Abdominal aortic aneurysm Pin1 Klf4 Smooth muscle Phenotype switching Ubiquitylation
Peptidyl-prolyl isomerase Pin1 has been reported to be associated with endothelial dysfunction. However, the role of smooth muscle Pin1 in the vascular system remains unclear. Here, we examined the potential function of Pin1 in smooth muscle cells (SMCs) and its contribution to abdominal aortic aneurysm (AAA) pathogenesis. The level of Pin1 expression was found to be elevated in human AAA tissues and mainly localized to SMCs. We constructed smooth muscle-specific Pin1 knockout mice to explore the role of this protein in AAA formation and to elucidate the underlying mechanisms. AAA formation and elastin degradation were hindered by Pin1 depletion in the angiotensin II-induced mouse model. Pin1 depletion reversed the angiotensin II-induced proinflammatory and synthetic SMC phenotype switching via the nuclear factor (NF)-κB p65/Klf4 axis. Moreover, Pin1 depletion inhibited the angiotensin II-induced matrix metalloprotease activities. Mechanically, Pin1 deficiency destabilized NF-κB p65 by promoting its polyubiquitylation. Further, we found STAT1/3 bound to the Pin1 promoter, revealing that activation of STAT1/3 was responsible for the increased expression of Pin1 under angiotensin II stimulation. Thus, these results suggest that Pin1 regulates pro-inflammatory and synthetic SMC phenotype switching and could be a novel therapeutic target to limit AAA pathogenesis.
1. Introduction Abdominal aortic aneurysm (AAA) is a potentially lethal disease in case of rupture [1]. Therapies are currently limited to surgical repair or interventional stent-based exclusion of the aneurysmal sac [2]. However, there is still no effective intervention for patients with small AAAs or contradictions to surgery. This is partly because the pathogenesis of AAA is not well-understood [3]. The pathology of aneurysm formation involves infiltration of inflammatory cells (T lymphocytes and macrophages) within the aortic wall [4], immune responses [5], cell apoptosis, degradation of the extracellular matrix [6], and slightly compensatory collagen deposition [7]. Studies of smooth muscle cells (SMCs) have mostly focused on SMC apoptosis and inflammation [8]. However, recent studies demonstrated that SMC phenotypic modulation is important for aortic aneurysm pathogenesis in both animal models and human subjects [9–11]. The contribution of SMCs to the development and progression of AAA maybe underestimated. In normal vessels, predominant contractile SMCs regulate vessel diameter and blood flow. In response to vascular injury, contractile SMCs lose their contractile proteins, including α-smooth muscle actin
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(SMA), smooth muscle protein-22 and calponin, and shift towards a synthetic phenotype [12]. SMCs with a synthetic phenotype can secrete numerous extracellular matrix proteins, increasing the capability for migration [13]. Studies have suggested that these alterations in SMC phenotypes play a key role in the pathogenesis of several cardiovascular disorders including atherosclerosis [14], plaque stability [15], and hypertension [16]. Because SMC proliferation and collagen synthesis are controversial in the pathogenesis of AAA, focusing on abnormal phenotypic modulation of SMCs may be a promising approach. Peptidyl-prolyl isomerase Pin1 is a unique peptidyl-prolyl cis/trans isomerase that binds to and isomerizes specific pSer/pThr-Pro motifs in specific proteins [17]. These Pin1-induced conformational changes modulate target protein function, stability, interactions, and phosphorylation status [18]. Pin1 regulates a variety of biological processes, such as gene transcription, cell proliferation, differentiation, and apoptosis [19–21]. Studies have demonstrated that Pin1 is a promising therapeutic target for several diseases including Alzheimer's disease [22], obesity [23], myocardial fibrosis [24], and vascular dysfunction [25]. However, the role of endothelial Pin1 in vascular dysfunction is controversial [26,27] and little is known about the role in SMCs.
Corresponding author at: Shandong University, Qilu Hospital, Jinan, No. 107, Wen Hua Xi Road, Jinan 250012, Shandong Province, China. E-mail address: [email protected] (X. Liu).
https://doi.org/10.1016/j.yjmcc.2017.12.006 Received 3 October 2017; Received in revised form 29 November 2017; Accepted 17 December 2017 Available online 19 December 2017 0022-2828/ © 2017 Elsevier Ltd. All rights reserved.
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Hercules, CA, USA). Relative expression was normalized to that of βactin. Primer details for real-time quantification are shown in Table S1.
In the present study, by using a conditional knockout strategy, we examined the potential function of Pin1 in SMCs and its contribution to AAA pathogenesis. The results showed that Pin1 expression was markedly upregulated in AAA tissues. Pin1 deficiency reversed angiotensin II (Ang II)-induced vascular SMC (VSMC) pro-inflammatory and synthetic phenotype switching and attenuated Ang II-induced AAA formation as well as aortic morphological damage in ApoE−/− mice. These results demonstrate that SMC-specific Pin1 plays a critical function in AAA.
2.6. Western blotting assay Total proteins were extracted from tissues or cultured cells. Equal amounts of the proteins were separated by 10% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes, followed by incubation with specific antibodies at 4 °C overnight. Horseradish peroxidase-conjugated secondary antibodies were added and incubated at room temperature for 60 min. Bound primary antibodies were detected by chemiluminescence (Millipore, Billerica, MA, USA). Bands were quantified with ImageJ software (NIH, Bethesda, MD, USA). Expression levels were normalized to the control.
2. Materials and methods 2.1. Animal models For the Ang II-induced AAA model, we used 12–14-week-old ApoE−/− Pin1sm +/+ and ApoE−/− Pin1sm −/− mice. To induce AAA formation, osmotic pumps (Alzet model 2004; Durect, Cupertino, CA, USA) containing either angiotensin II (1.44 mg/kg per day, Sigma, St. Louis, MO, USA) or saline were introduced in 12–14 weeks old ApoE−/ − background male mice as previously described [28]. n = 30 per group. At 28 days after the osmotic pumps implantation, mice were sacrificed, and aneurysms in the abdominal aorta were quantified.
2.7. Luciferase activity assay We constructed contact or mutant luciferase reporter plasmids by inserting the promoter into the pGL3-Basic cloning vector. The promoter luciferase reporter plasmids and a Renilla reporter plasmid (pRLTK) were transfected into HASMCs by electroporation. Luciferase activity was determined using the Dual Luciferase Assay Kit (Promega, Madison, WI, USA). Detailed description of Methods can be found in the Supplemental data.
2.2. Analysis and quantification of AAA Necropsy was performed to confirm aortic rupture during Ang II infusion. Abdominal aortic rupture was defined as observation of blood clots in retroperitoneal cavity. To quantify AAA incidence, the maximum width of the abdominal aorta was measured ex vivo with a vernier calipers. An aneurysm was defined as ≥ 50% enlargement of the external diameter of the suprarenal aorta compared to aortas from saline-infused control mice [29]. AAA severity was determined by measuring the wet weights of the abdominal aortas as described previously [29]. The measurement was determined by an investigator who was blinded to the experiment and mouse genotypes.
2.8. Statistical analysis All values for continuous variables are expressed as means ± SEM. Statistical tests were performed using SPSS16.0 software and GraphPad Prism software version 6.0. We tested the normality and equal variance using the Kolmogorov-Smirnov test implemented in Prism statistical software (GraphPad, San Diego) before parametric data analysis. Comparison of 2 groups of normally distributed samples was done using unpaired Student's t-tests. Comparison of multiple groups of normally distributed samples was done using ANOVA, and Tukey was performed as a post test. Nonnormally distributed data including abdominal aorta wet weights and aortic diameter data were analyzed with MannWhitney test or the Kruskal-Wallis test, as appropriate. P < 0.05 was considered statistically significant.
2.3. Histology and immunohistochemistry Pathology was present in abdominal aortas of surviving mice. Murine aortas were harvested and fixed with 4% paraformaldehyde after 4 weeks of treatment. Abdominal aortic serial cross-sections (5 μm) from the proximal to the distal region of the aneurysm were prepared for histological analysis. Aortic sections were stained with hematoxylin and eosin (H&E) for morphological assessment. VerhoeffVan Gieson Elastin (VVG, HT25A, Sigma) staining was performed to detect elastic fiber integrity of the abdominal aorta. Transmission electron microscopy (TEM) was performed to evaluate ultra-structural details.
3. Results 3.1. Pin1 is elevated in AAA tissues and mainly localizes to SMCs Pin1 is a key regulator in several disease models [31]. Therefore, we first investigated whether Pin1 was altered during AAA formation. To examine Pin1 expression in aneurysm, we stained human AAA tissues with an anti-Pin1 antibody. Compared to normal aortas, immunohistochemical staining revealed significantly higher expression of Pin1 (Fig. 1A). Furthermore, western blotting and RT-PCR analysis revealed that Pin1 is increased in AAA tissues at both the protein and mRNA levels (Fig. 1B–D). To evaluate the potential role of Pin1 in the development of AAA, we conducted confocal immunofluorescence of αSMA and Pin1 in AAA tissues, which revealed that Pin1 (red) mainly localized to the nucleus of smooth muscle cells (α-SMA, green) (Fig. 1E). However, we detected comparable levels of Pin1 expression between controls and thoracic aortic aneurysm (TAA) tissues (Fig. S1A–C). We next compared the aortic expression levels of Pin1 at early stage (7 days) in Ang II-infused ApoE−/− mice. Both thoracic (including ascending and descending aortas) and abdominal aortas have low levels of Pin1 expression in saline-infused ApoE−/− mice (Fig. S1D and E). Intriguingly, the protein level of Pin1 was significantly increased in abdominal aortas but not ascending or descending aortas in
2.4. Cell cultures Mouse primary VSMCs were isolated from mouse abdominal aortas as previously described [30]. All experiments were performed with VSMCs at passages 3–5. Human vascular smooth muscle cells (HASMCs) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Experiments were performed with VSMCs at passages 3–5. Cells were treated with Ang II (10− 7 M) or vehicle and then were harvested after 24-h stimulation. 2.5. Real-time polymerase chain reaction (RT-PCR) Total RNA was extracted using TRIzol reagent (Invitrogen) and reverse-transcribed into cDNA. The mRNA expression levels of Pin1 and Klf4 were determined by SYBR Green-based RT-PCR using a sequence detection system (IQ5 Real-Time PCR cycler, Bio-Rad Laboratories, 335
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Fig. 1. Pin1 is elevated in human AAA tissues and mainly localizes to smooth muscle cells. A, Representative immunohistochemical staining of Pin1 in human AAA and normal control aortas. B and C, Western blot analysis of Pin1 in human AAA tissues versus normal aortic tissues. D, qPCR of Pin1 in human AAA tissues versus normal aortic tissues. E, Confocal images of human AAA tissues double-stained for Pin1 and smooth muscle α-actin (α-SMA). *P < 0.05, vs. control, n = 5. (For interpretation of the references to color in this figure, the reader is referred to the web version of this article.)
7-day consecutively Ang II-infused ApoE−/− mice (Fig. S1D and E). Moreover, the increased Pin1 expression was mainly observed in the media of the suprarenal aortas (Fig. S1F and G), where AAAs most often develop in Ang II-infused mice. Therefore, we speculated that the increased Pin1 expression in AAA tissues may be linked to AAA formation.
(Fig. 2A–C). Following the 28 days of Ang II infusion, the AAA incidence in ApoE−/− Pin1sm +/+ mice was 83.3% (25 of 30, including the mouse with aortic rupture), whereas the incidence in ApoE−/ − Pin1sm −/− was significantly reduced to 30% (9 of 30, Fig. 2A and B, Table S2). H&E staining revealed positive aortic remodeling (Fig. 2A, lower panels), accompanied by aortic wall thickening and intraluminal thrombosis. Additionally, the mortality rate due to AAA rupture was significantly higher in Ang II-infused ApoE−/− Pin1sm +/+ mice (16.7%, 5/30; at days 12, 14, 17, 23 and 25; Table S2) versus Ang IIinfused ApoE−/− Pin1sm −/− mice (0%, 0/30; Table S2). Furthermore, Ang II-infused ApoE−/− Pin1sm +/+ mice exhibited a markedly larger suprarenal aortic diameter as well as heavier abdominal aorta wet weights compared to the vehicle control mice (Fig. 2C–D). In contrast, ApoE−/− Pin1sm −/− mice attenuated the severity of Ang II-induced AAAs based on their significantly smaller aortic diameter and wet weights compared to ApoE−/− Pin1sm +/+ mice (Fig. 2C–D). Fig. 2E showed representative longitudinal and transverse images taken at the level of the suprarenal aorta by high-frequency ultrasound. Collagen and elastin are responsible for mechanical resistance, tensile strength, and elasticity of the arterial wall. Therefore, changes in their content or quality contribute to the development of AAA. We next performed Masson's trichrome staining and α-SMA staining to detect
3.2. Pin1 depletion in SMCs attenuates the incidence and severity of Ang IIinduced AAA formation in apolipoprotein E-knockout (ApoE −/−) mice As Pin1 was mainly located in SMCs (Fig. 1E), to determine whether Pin1 expression in smooth muscle contributes to AAA pathogenesis, we generate Pin1flox/flox/Acta2-Cre mice (Pin1sm −/− mice) by crossbreeding Pin1flox/flox mice with Acta2-Cre mice (Fig. S2). Littermate Pin1flox/flox/Cre− mice (Pin1sm +/+ mice) were used as controls. VSMCspecific deletion Pin1 mice on an ApoE−/− background (ApoE−/ − Pin1sm −/−) were generated by crossing Pin1sm −/− mice with ApoE−/− mice. VSMC-specific Pin1 depletion didn't alter medial SMC content or show any abnormal aortic morphology in ApoE−/− mice (Fig. S3). ApoE−/− background mice were infused subcutaneously with Ang II or vehicle (saline). No spontaneous AAAs were observed in the vehicle-infused ApoE−/− Pin1sm +/+ or ApoE−/− Pin1sm −/− mice 336
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Fig. 2. Pin1 depletion in smooth muscle cells protects ApoE−/− mice from developing AAA. A, Representative images of abdominal aortic specimens in 4 groups. B, Incidence of AAA in 4 groups (percentage). C, Maximal abdominal aortic diameters measured with vernier caliper (mm). D, Abdominal aorta wet weights in 4 groups. E, Representative longitudinal (top) and transverse (bottom) images were taken at the level of the suprarenal aorta by high-frequency ultrasound. Scale bar = 200 μm. *P < 0.05, vs. ApoE−/−/Pin1sm +/+ mice infused with vehicle. #P < 0.05, vs. ApoE−/−/Pin1sm +/+ mice infused with Ang II, n ≥ 8 per group.
phenotypes with or without Ang II treatment by western blotting. VSMC was confirmed through positive staining for smooth muscle specific α-actin antibody (α-SMA) (Fig. S4A). As shown in Fig. 4F and G, the contractile markers α-SMA and calponin were dramatically decreased following Ang II stimulation. In contrast, the levels of osteopontin and vimentin were significantly increased. Consistently, Pin1 depletion reversed the Ang II-induced VSMC synthetic phenotype switching (Fig. 4F and G) in vitro. We also found Pin1 depletion remarkably repressed the expression of MMP-2/9 in Ang II-stimulated VSMCs (Fig. 4F and H). We further tested gelatinase activity in the supernatant of treated VSMCs by zymography. Ang II stimulation markedly increased MMP-2/9 activity compared to the vehicle control (Fig. 4I and J). However, these effects induced by Ang II were ameliorated by Pin1 depletion.
alterations in collagen and SMC content. Pin1 depletion in SMCs markedly increased the relative contents of SMCs and collagen in the suprarenal aortic wall (Fig. 3A–F). Indeed, aortas from Ang II-infused ApoE−/− Pin1sm −/− mice showed densely packed parallel collagen staining in the media, which was opposite from the disrupted irregular pattern observed in ApoE−/− Pin1sm +/+ mice. Moreover, inflammatory cell infiltration as determined by CD68 immunostaining was decreased in the suprarenal aortas of Ang II-infused ApoE−/ − Pin1sm −/− mice compared with that of ApoE−/− Pin1sm +/+ mice (Fig. 2E–F). Verhoeff-Van Giessen (VVG) staining and TEM images indicated that elastin fibers were disrupted in Ang II-infused arteries (Fig. 3G–I). Further, Pin1 depletion in SMCs significantly decreased elastin fragmentation (Fig. 3G–I). These results suggest that Pin1 depletion in SMCs attenuated AAA formation and severity by ameliorating macrophage infiltration and the rupture of elastin fibers, increasing SMC content, and modulating the content and quality of collagen.
3.4. Pin1 depletion inhibits Ang II-induced expression of inflammatory cytokines in vivo and in vitro
3.3. Pin1 depletion reverses Ang II-induced VSMCs synthetic phenotype switching
As chronic inflammation plays a critical role in the formation of AAA, we examined the expression of inflammatory cytokines in aortic tissues. Our results showed that the protein expression level of inducible nitric oxide synthase (iNOS), vascular cell adhesion molecule 1 (VCAM1), intercellular adhesion molecule 1 (ICAM1), tumor necrosis factor-alpha (TNF-α) and Monocyte Chemoattractant Protein-1 (MCP1) were upregulated by Ang II stimulation but reversed by Pin1 depletion in vivo (Fig. 5A–F). Proinflammatory cytokines IL-1β and IL-6 were also inhibited in the AAA tissues by Pin1 deficiency (Fig. 5G and H). These effects were further demonstrated in vitro (Fig. 5I–M). Taken together, these data indicate that Pin1 deletion decreased a number of relevant inflammatory cytokines in AAA.
To investigate the relationship between VSMC phenotypic transition and AAA injury, we performed co-immunostaining of mouse AAA tissues. As revealed by immunofluorescence staining (Fig. 4A and B), the expression of calponin was significantly decreased, while the synthetic VSMC phenotype marker vimentin was significantly increased in aneurysm tissue compared to in the vehicle-infused control. Pin1 depletion in SMCs not only markedly increased calponin content, but also significantly decreased vimentin expression (Fig. 4A and B). As matrix metalloproteinases are responsible for elastin degradation in the vascular medium and initiate the formation of AAA [6], we next examined the effects of Pin1 depletion on MMP-2/9 expression in vivo. As depicted in Fig. 4C–E, the Ang II-induced increase in MMP-2/9 expression was suppressed by Pin1 depletion. We then isolated VSMCs from the aorta of Pin1sm +/+ and Pin1sm −/ − mice and detected the markers of contractile and synthetic VSMC
3.5. Pin1 depletion modulates VSMC phenotype switching via NF-κB/Klf4 axis Next, we evaluated why Pin1 depletion reverses the status of VSMC 337
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Fig. 3. Effect of smooth muscle Pin1 depletion on tissue components of abdominal aortic wall in Ang II-infused ApoE−/− mice. A and B, Smooth muscle cell staining shown by α-SMA staining in the suprarenal aortas of mice after AngII infusion. C and D, Representative staining with Masson's Trichrome (collagen). E and F, Macrophage staining were visualized with anti-CD68. G, Elastin fibers were shown with Verhoeff-van Gieson (VVG) staining. H, The number of elastin breaks per vessel was quantified. I, Transmission electron microscopy (TEM) was performed to evaluate ultra-structural details. Representative images are shown. Arrows indicate destroyed elastic fibers. *P < 0.05, vs. ApoE−/−/Pin1sm +/+ mice infused with Ang II, n ≥ 5 per group.
VSMC phenotype switching by regulating the NF-κB/Klf4 axis, we silenced Pin1 and NF-κB p65 in HASMCs. We observed decreased expression of both the protein and mRNA levels of Klf4 by Pin1 or NF-κB p65 knockdown (Fig. 6H and I). In HASMCs, Pin1 knockdown restrained Ang II-enhanced binding activity of NF-κB p65 with the Klf4 promoter (Fig. 6J and K). In addition, Pin1 or NF-κB p65 knockdown suppressed Klf4 promoter activity (Fig. 6L). These data suggest that Pin1 depletion reverses Ang II-induced VSMC phenotype switching via the NF-κB p65/Klf4 axis.
phenotype switching. A previous study demonstrated that NF-κB binds directly to the Klf4 promoter [15]. However, there is no evidence to date that NF-κB modulates Klf4-mediated VSMC phenotype switching during the formation of AAA. We found Pin1 depletion markedly inhibited the expression of NF-κB p65 as well as Klf4 (Fig. 6A–C), a transcription factor that directly binds to smooth muscle marker genes and regulates phenotype switching. In accordance with the study by Salmon et al. [9], we verified the effect of Klf4 inhibition on Ang IIinduced VSMC synthetic phenotype switching in vitro (Fig. S5). We hypothesized that Pin1 deletion reverses VSMC phenotype switching by suppressing the NF-κB/Klf4 axis. To determine if a Pin1-NF-κB p65 interaction occurs in VSMCs, we performed an immunoprecipitation experiment. As depicted in Fig. 6D, endogenous Pin1 and p65 show an interaction in the basal state, which was enhanced upon Ang II stimulation. Moreover, we detected polyubiquitination K48 of p65. Interestingly, Ang II stimulation did not obviously affect the general expression profile of poly-ubiquitination K48, but inhibited p65-bound poly-ubiquitination of K48. This may explain why p65 was enhanced under Ang II stimulation. Furthermore, Pin1 depletion increased p65-bound poly-ubiquitination K48 but decreased the expression of p65 (Fig. 6E). A luciferase assay and gel shift assays (EMSA) also revealed that Pin1 deficiency suppressed p65 activation (Fig. 6F and G). As it is predicted that NF-κB p65 binds directly to the Klf4 promoter in human cells [15], we cultured HASMCs to verify the binding and explore the mechanism. To further confirm that Pin1 depletion reversed
3.6. Pin1 depletion inhibited Ang II–induced VSMCs proliferation and migration Considering that activated VSMCs may become highly proliferative and migratory under stimulation of Ang II, we next evaluated the effect of Pin1 deletion on the Ang II-induced proliferative capacity. Treatment of VSMCs with Ang II but not vehicle significantly increased VSMCs proliferation (Fig. S6A and B), accompanied by elevated expression of synthetic VSMC phenotype markers including osteopontin and vimentin (Fig. 4). Importantly, Pin1 deletion did not markedly alter the basal proliferative capacity. Therefore, therapeutic strategies preventing the hyperproliferation of VSMCs, particularly VSMCs with the synthetic phenotype, is promising for the treatment of AAA. We further conducted a Transwell migration assay to evaluate migration capability. After 24 h, we found that stimulation of Ang II promoted VSMCs migration compared to in the vehicle control group, 338
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Fig. 4. Pin1 depletion modulates Ang II-induced VSMC synthetic phenotype switching in vivo and in vitro. A, Immunofluorescence staining of calponin (red) and vimentin (green) in aortic sections. Smooth muscle Pin1 depletion reverses Ang II-induced synthetic phenotype switching of VSMCs in AAA. B, Quantification of calponin and vimentin coverage. Scale bar = 50 μm. C–E, Pin1 depletion reversed Ang II-induced MMP-2/9 protein expression in vivo. F–H, Western blot analysis of contractile (calponin, α-SMA) and synthetic proteins (vimentin, osteopontin and MMP-2/9) in Pin1sm +/+ and Pin1sm −/− VSMCs treated with Ang II or vehicle for 24 h. I and J, Gelatin zymography analysis of MMP-2 and MMP-9 activity. *P < 0.05, vs. ApoE−/−/Pin1sm +/+ mice infused with vehicle or Pin1sm +/+ VSMCs treated with vehicle. #P < 0.05, vs. ApoE−/−/Pin1sm +/+ mice infused with Ang II or Pin1sm +/+ VSMCs treated with Ang II, n = 5. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
of Pin1. To determine how STAT1/3 regulates Pin1 expression, we analyzed the Pin1 promoter using the PROMO and JASPAR databases. We identified two potential binding sites for STAT1 and STAT3 recognition motifs in human cells (Fig. 7G), respectively. To verify binding of STAT1/3 to the Pin1 promoter, we performed a ChIP assay using specific primers covering −1305 to −1182 base pairs of the Pin1 promoter region. Ang II induced the binding of STAT1/3 to the Pin1 promoter (Fig. 7H and I). To confirm that the predicted binding sites on the Pin1 promoter are required for transcription activity, we constructed intact promoter-reporter plasmids and mutations of the predicted binding sites (Fig. 7G). As depicted in Fig. 7I, luciferase activity in both mutant plasmids was reduced by 50%, revealing that the core Pin1 promoter element is located in the predicted binding sites. Ang II
but reversed by Pin1 depletion (Fig. S6C and D). These results indicate that Pin1 depletion strongly inhibited Ang II-induced VSMCs proliferation and migration, which may be beneficial for decreasing abdominal aortic diameters and AAA formation.
3.7. STAT1/3 binds to Pin1 promoter and mediates its upregulation facilitated by Ang II stimulation As the JAK/STAT pathway has been reported to participate in Ang II-induced AAA formation [32–34], we explored the potential role of STAT signaling in Ang II stimulation-modulated Pin1. As shown in Fig. 7A–F, Ang II stimulation increased the levels of both phosphorylated and total STAT1 and STAT3 in HASMCs. Interestingly, both STAT1 and STAT3 knockdown decreased the mRNA and protein levels 339
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Fig. 5. Pin1 depletion inhibits Ang II-induced expression of inflammatory cytokines in vivo and in vitro. A–F, Western blot analysis of iNOS, VCAM1, ICAM1, TNF-α and MCP-1 expression in the suprarenal aortas. G and H, IL-1β and IL-6 expression in the suprarenal aortic tissues were detected by ELISA. I-M, Western blot analysis of iNOS, VCAM1, ICAM1, and TNF-α expression in Pin1sm +/+ and Pin1sm −/− VSMCs treated with Ang II or vehicle for 24 h. *P < 0.05, vs. ApoE−/−/Pin1sm +/+ mice infused with vehicle or Pin1sm +/+ VSMCs treated with vehicle. #P < 0.05, vs. ApoE−/−/Pin1sm +/+ mice infused with Ang II or Pin1sm +/+ VSMCs treated with Ang II, n = 5.
accompanied by a slightly compensatory collagen deposition, aortas from Ang II-infused ApoE−/− Pin1sm −/− mice showed densely packed parallel collagen in the media, whereas aortas from ApoE−/− Pin1sm +/ + mice showed the disrupted irregular pattern. Therefore, new therapeutic strategies should attempt to abrogate the synthetic hyperproliferation VSMCs to improve the content and quality of collagen. Of interest, a recent study by Shen et al. [36] revealed that Pin1 null mice expressed reduced, pulmonary expression of collagens and TIMPs but showed an increase in MMPs compared with wild-type. It may seem to conflict with our results. However, Franciosa et al. [37] reported that Pin1 silencing downmodulated MMP9 expression in human leukemic TALL-1 cells. All these results suggest tissue-specific and cell type-specific regulation of MMPs by Pin1. Therefore, study on the roles of Pin1 in AAA development with SMC-specific depletion mice is more convincing. Another major finding of this study is that Pin1 deletion reverses Ang II-induced VSMC synthetic phenotype switching via the NF-κB/Klf4 axis. Our results indicate that Pin1 mediated the Ang II-induced VSMC synthetic phenotype. We then further explored the precise mechanism of this action and found that NF-κB p65, a target protein of Pin1 [38], binds directly to the Klf4 promoter and regulates its expression. This is in accordance with the results of Ding et al. [15]. Klf4, a transcription factor that binds to smooth muscle marker genes, regulates the VSMC synthetic phenotype and aneurysm formation [9,14]. We hypothesized that Pin1 deletion reverses VSMC phenotype switching by suppressing the NF-κB/Klf4 axis. Our immunoprecipitation experiment and ChIP assay indicated that Pin1 deficiency promoted NF-κB p65 degradation via K48-linked polyubiquitination, which subsequently suppressed Klf4-mediated VSMC phenotype switching. Notably, Pin1 stabilizes NF-κB p65 proteins and thus contributes to Ang II-induced AAA. Pin1 is often associated with the ubiquitin-proteasome system, which may influence the degree of substrate polyubiquitylation and subsequent protein degradation. We found that Pin1
stimulation markedly increased luciferase activity from the WT plasmid but not mutant plasmids (Fig. 7J). Therefore, STAT1/3 binds to the Pin1 promoter and regulates its expression. 4. Discussion Here, we showed that Pin1 expression was markedly upregulated in AAA tissues and that Pin1 is mainly localized to SMCs. Pin1 depletion in SMCs significantly attenuated AAA formation in Ang II-infused ApoE−/ − mice. Histological and morphological examination revealed that Pin1 depletion decreased elastin fragmentation and increased SMC content. In the molecular mechanism, Pin1 depletion reversed Ang II-induced VSMC phenotype switching via the NF-κB/Klf4 axis and significantly suppressed the inflammatory response as well as MMP activities. Our data highlight the roles of smooth muscle Pin1 in vascular function and AAA pathogenesis. One major finding of this study is that Pin1 deletion in SMCs attenuated AAA formation. Importantly, Pin1 depletion attenuated the rupture of elastin fibers, increased SMC content, and modulated the content and quality of collagen. During the process of phenotypic switching from a contractile phenotype to a proliferative-migratory phenotype, VSMCs are prone to secrete matrix metalloproteinases (MMPs) and produce more inflammatory cytokines, which subsequently contribute to vascular remodeling [35]. Abnormal VSMCs proliferation is a hallmark of the pathogenesis of AAA [10]. Interestingly, Pin1 deletion reversed Ang II-induced hyperproliferation but did not alter the basal proliferative capacity. Furthermore, Pin1 depletion markedly reduced the activity of MMP-2/9, the major proteases involved in elastin degradation, and decreased Ang II-induced VSMCs migration. We also found that Pin1 depletion attenuated inflammation responses and macrophage infiltration. During the development of AAA, changes in collagen content are of interest as it is the ultimate structural component to prevent rupture. Although AAA was 340
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Fig. 6. Pin1 deficiency attenuates Klf4-mediated VSMC phenotype switching through NF-κB signaling. A–C, Western blot analysis of NF-κB p65 and Klf4 protein expression. VSMCs were stimulated with Ang II or vehicle for 24 h. Cell lysates were subjected to western blotting. D, Immunoprecipitation data showing Pin1 binding to NF-κB p65 in VSMCs treated with Ang II. E, Pin1 deficiency destabilized p65 by promoting p65 polyubiquitylation in vitro. F and G, Resistance of Pin1 deficiency to NF-κB activation by Ang II stimulation detected by luciferase reporter assay and EMSA. H, Western blot analysis of Klf4 protein expression with siPin1 or siNF-κB or control in HASMCs. I, Quantitative RT-PCR analysis of mRNA level of Klf4 with siPin1 or siNF-κB or control in HASMCs. J and K, ChIP assay and quantitative analysis to verify binding of NF-κB p65 to Klf4 promoter in HASMCs pretreated with siPin1 or control. L, Luciferase activity assay was performed after transfection with human Klf4 promoter reporter plasmid and pRL-TK plasmid following pretreatment with siPin1 or siNF-κB. *P < 0.05, vs. Pin1sm +/+ VSMCs treated with vehicle or VSMC treated with vehicle and control siRNA. #P < 0.05, vs. Pin1sm +/+ VSMCs treated with Ang II or VSMC treated with Ang II and control siRNA, n = 5.
it may influence the affinity of ligases for their substrates. These data illustrate that Pin1 functions as a novel switch in the ubiquitin pathway, which controls protein expression. Next, we focused on the underlying mechanisms of Ang II-stimulated upregulation of Pin1. Studies have shown that activation of the JAK/STAT pathway by Ang II is associated with AAA formation [32,33,40,41]. STAT1 showed corresponding increases in both total
deficiency leads to pronounced K48-polyubiquitylation and degradation of NF-κB p65. NF-κB signaling is activated by Ang II, but was reversed by Pin1 inhibition. In accordance with a study of Ryo et al. [39], Pin1-deficient SMCs are refractory to NF-κB activation by promoting polyubiquitin-mediated proteolysis of p65. SOCS-1 ubiquitin ligase may be involved in polyubiquitylation of p65 in Pin1 deficiency [39]. Although it is not clear how Pin1 controls the activity of ubiquitin ligases, 341
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Fig. 7. STAT1/3 mediated upregulation of Pin1 facilitated by Ang II stimulation. HASMCs were stimulated with Ang II or vehicle for 24 h. A–C, Western blotting and quantitative RT-PCR analysis of Pin1 expression with STAT1 siRNA or control. D–F, Western blotting and quantitative RT-PCR analysis of Pin1 expression with STAT3 siRNA or control. G, Predicted STAT1 or STAT3 binding site within the human Pin1 promoter; mutants with deletion of the predicted binding site (Pin1-mut1, Pin1-mut2, and Pin1-mut3) are shown. H and I, ChIP assay and quantitative analysis to verify binding of STAT1 or STAT3 to Pin1 promoter in HASMCs. J, Luciferase reporter assay was performed after transfection with Pin1 promoter vector or mutants in HASMC.*P < 0.05, vs. vehicle-stimulated HASMCs transfected with intact Pin1 promoter. #P < 0.05, vs. Ang II-stimulated HASMCs transfected with intact Pin1 promoter, n = 5.
Fig. 8. Working schematic of how Pin-1 depletion in SMC attenuated Ang II-induced AAA formation. Ang II stimulation activates STAT1/3, resulting in increased transcription activity of Pin1. Pin1 modulates VSMC phenotype switching via NF-κB/Klf4 axis. Additionally, Pin1 acts on inflammatory cytokines, and MMPs, further resulting in vascular remodeling and promoting the formation of AAA.
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protein and mRNA levels in AAA tissues [32]. Treatment with a STAT3 inhibitor decreased the incidence and severity of Ang II-induced AAA formation, which suppressed MMP activity and the ratio of M1/M2 macrophages [33]. We then evaluated whether STAT transcription factors modulate Pin1 expression in response to Ang II stimulation. Interestingly, STAT1 and STAT3 bound to distinct recognition motifs of the Pin1 promoter. We found that Ang II-induced Pin1 expression occurred via a direct interaction between STAT1/3 and the Pin1 promoter. Although STAT1/3 inhibition may prevent aneurysm formation, targeting transcription factors as potential drug targets is challenging. STAT1/3 transcription factors act on different promoters and exhibit both positive and negative interactions. Thus, Pin1 maybe a promising therapeutic target for AAA linking STAT1/3 transcription factors and aneurysm formation. We developed a mechanistic model describing the role of Pin1 in AAA formation (Fig. 8). It is interesting to observe that Pin1 expression was significantly elevated in human AAA but not TAA tissues. Simultaneously, Pin1 was markedly increased in the suprarenal abdominal aorta during the early onset of AAA in Ang II-infused ApoE−/−mice, but no obvious change was observed in the ascending or descending thoracic aorta. Although not fully understood, several possibilities may explain the differences between the abdominal and thoracic VSMC responses in aneurysm development. Firstly, AAA and TAA have different aetiologies and pathogeneses. TAA are more genetically based while AAA tends to be associated with smoking, inflammation and dyslipidemia [8,42]. AAA is associated with a Th2-predominant immune response, whereas TAA is linked with a Th1-predominant immune response [42]. Secondly, abdominal and thoracic aortas differ in embryology of VSMCs origin [43]. Medial SMCs in the abdominal aorta originate from splanchnic mesoderm [44], while those in the ascending and descending aortas arise from cardiac neural crest [45] and somites [46] respectively. SMCs of different origins may respond differently to the same exogenous stimulation such as Ang II. Moreover, differences in anatomical configuration and hemodynamic shear stress may also account for differences in Pin1 expression and aortic aneurysm development. In conclusion, we found that Ang II induces Pin1 expression, which subsequently contributes to the progression of AAA. Our study improves the understanding of the biological activities and regulatory mechanisms of smooth muscle Pin1 in AAA. These results suggest that Pin1 is a potential therapeutic target to prevent AAA formation.
[5] [6]
[7]
[8] [9]
[10]
[11]
[12] [13]
[14]
[15]
[16]
[17]
[18] [19] [20]
[21]
[22]
[23]
Acknowledgments [24]
We would like to thank Dr. Wen-Cheng Zhang for providing the Acta2Cre transgenic mice. This study was supported by the National 973 Basic Research Program of China (No. 2012CB518603), and the National Natural Science Foundation of China (No. 81302939).
[25] [26]
Disclosures
[27]
None.
[28]
Appendix A. Supplementary data
[29]
Supplementary data to this article can be found online at https:// doi.org/10.1016/j.yjmcc.2017.12.006.
[30]
References
[31]
[1] N.L. Weintraub, Understanding abdominal aortic aneurysm, N. Engl. J. Med. 361 (2009) 1114–1116. [2] U. Raaz, A.M. Zollner, I.N. Schellinger, R. Toh, F. Nakagami, M. Brandt, et al., Segmental aortic stiffening contributes to experimental abdominal aortic aneurysm development, Circulation 131 (2015) 1783–1795. [3] H. Kuivaniemi, E.J. Ryer, J.R. Elmore, G. Tromp, Understanding the pathogenesis of abdominal aortic aneurysms, Expert. Rev. Cardiovasc. Ther. 13 (2015) 975–987. [4] T. Freestone, R.J. Turner, A. Coady, D.J. Higman, R.M. Greenhalgh, J.T. Powell,
[32]
[33]
343
Inflammation and matrix metalloproteinases in the enlarging abdominal aortic aneurysm, Arterioscler. Thromb. Vasc. Biol. 15 (1995) 1145–1151. K. Shimizu, R.N. Mitchell, P. Libby, Inflammation and cellular immune responses in abdominal aortic aneurysms, Arterioscler. Thromb. Vasc. Biol. 26 (2006) 987–994. G.M. Longo, W. Xiong, T.C. Greiner, Y. Zhao, N. Fiotti, B.T. Baxter, Matrix metalloproteinases 2 and 9 work in concert to produce aortic aneurysms, J. Clin. Invest. 110 (2002) 625–632. Q. Wang, Y. Ding, P. Song, H. Zhu, I.S. Okon, N.Y. Ding, et al., Tryptophan-derived 3-hydroxyanthranilic acid contributes to angiotensin II-induced abdominal aortic aneurysm formation in mice in vivo, Circulation 136 (23) (2017) 2271–2283. I.M. Nordon, R.J. Hinchliffe, I.M. Loftus, M.M. Thompson, Pathophysiology and epidemiology of abdominal aortic aneurysms, Nat. Rev. Cardiol. 8 (2011) 92–102. M. Salmon, W.F. Johnston, A. Woo, N.H. Pope, G. Su, G.R. Upchurch Jr.et al., KLF4 regulates abdominal aortic aneurysm morphology and deletion attenuates aneurysm formation, Circulation 128 (2013) S163–74. G. Ailawadi, C.W. Moehle, H. Pei, S.P. Walton, Z. Yang, I.L. Kron, et al., Smooth muscle phenotypic modulation is an early event in aortic aneurysms, J. Thorac. Cardiovasc. Surg. 138 (2009) 1392–1399. E. Crosas-Molist, T. Meirelles, J. Lopez-Luque, C. Serra-Peinado, J. Selva, L. Caja, et al., Vascular smooth muscle cell phenotypic changes in patients with Marfan syndrome, Arterioscler. Thromb. Vasc. Biol. 35 (2015) 960–972. G.K. Owens, Regulation of differentiation of vascular smooth muscle cells, Physiol. Rev. 75 (1995) 487–517. K. Riches, T.G. Angelini, G.S. Mudhar, J. Kaye, E. Clark, M.A. Bailey, et al., Exploring smooth muscle phenotype and function in a bioreactor model of abdominal aortic aneurysm, J. Transl. Med. 11 (2013) 208. L.S. Shankman, D. Gomez, O.A. Cherepanova, M. Salmon, G.F. Alencar, R.M. Haskins, et al., KLF4-dependent phenotypic modulation of smooth muscle cells has a key role in atherosclerotic plaque pathogenesis, Nat. Med. 21 (2015) 628–637. Y. Ding, M. Zhang, W. Zhang, Q. Lu, Z. Cai, P. Song, et al., AMP-activated protein kinase alpha 2 deletion induces VSMC phenotypic switching and reduces features of atherosclerotic plaque stability, Circ. Res. 119 (2016) 718–730. S.B. Zhu, J. Zhu, Z.Z. Zhou, E.P. Xi, R.P. Wang, Y. Zhang, TGF-beta1 induces human aortic vascular smooth muscle cell phenotype switch through PI3K/AKT/ID2 signaling, Am. J. Transl. Res. 7 (2015) 2764–2774. A. Tun-Kyi, G. Finn, A. Greenwood, M. Nowak, T.H. Lee, J.M. Asara, et al., Essential role for the prolyl isomerase Pin1 in Toll-like receptor signaling and type I interferon-mediated immunity, Nat. Immunol. 12 (2011) 733–741. D. Siepe, S. Jentsch, Prolyl isomerase Pin1 acts as a switch to control the degree of substrate ubiquitylation, Nat. Cell Biol. 11 (2009) 967–972. S.D. Hanes, Prolyl isomerases in gene transcription, Biochim. Biophys. Acta 2015 (1850) 2017–2034. G.L. Perrucci, A. Gowran, M. Zanobini, M.C. Capogrossi, G. Pompilio, P. Nigro, Peptidyl-prolyl isomerases: a full cast of critical actors in cardiovascular diseases, Cardiovasc. Res. 106 (2015) 353–364. H. Zheng, H. You, X.Z. Zhou, S.A. Murray, T. Uchida, G. Wulf, et al., The prolyl isomerase Pin1 is a regulator of p53 in genotoxic response, Nature 419 (2002) 849–853. L. Pastorino, A. Sun, Lu PJ, X.Z. Zhou, M. Balastik, G. Finn, et al., The prolyl isomerase Pin1 regulates amyloid precursor protein processing and amyloid-beta production, Nature 440 (2006) 528–534. Y. Nakatsu, H. Sakoda, A. Kushiyama, J. Zhang, H. Ono, M. Fujishiro, et al., Peptidyl-prolyl cis/trans isomerase NIMA-interacting 1 associates with insulin receptor substrate-1 and enhances insulin actions and adipogenesis, J. Biol. Chem. 286 (2011) 20812–20822. X. Liu, E. Liang, X. Song, Z. Du, Y. Zhang, Y. Zhao, Inhibition of Pin1 alleviates myocardial fibrosis and dysfunction in STZ-induced diabetic mice, Biochem. Biophys. Res. Commun. 479 (2016) 109–115. S. Costantino, F. Paneni, T.F. Luscher, F. Cosentino, Pin1 inhibitor Juglone prevents diabetic vascular dysfunction, Int. J. Cardiol. 203 (2016) 702–707. V.L. Chiasson, N. Munshi, P. Chatterjee, K.J. Young, B.M. Mitchell, Pin1 deficiency causes endothelial dysfunction and hypertension, Hypertension (Dallas, Tex: 1979) 58 (2011) 431–438. L. Ruan, C.M. Torres, J. Qian, F. Chen, J.D. Mintz, D.W. Stepp, et al., Pin1 prolyl isomerase regulates endothelial nitric oxide synthase, Arterioscler. Thromb. Vasc. Biol. 31 (2011) 392–398. J. Kong, K. Zhang, X. Meng, Y. Zhang, C. Zhang, Dose-dependent bidirectional effect of angiotensin IV on abdominal aortic aneurysm via variable angiotensin receptor stimulation, Hypertension (Dallas, Tex: 1979) 66 (2015) 617–626. D.B. Trivedi, C.D. Loftin, J. Clark, P. Myers, L.M. DeGraff, J. Cheng, et al., betaArrestin-2 deficiency attenuates abdominal aortic aneurysm formation in mice, Circ. Res. 112 (2013) 1219–1229. P. Zhang, S. Hou, J. Chen, J. Zhang, F. Lin, R. Ju, et al., Smad4 deficiency in smooth muscle cells initiates the formation of aortic aneurysm, Circ. Res. 118 (2016) 388–399. Y. Nakatsu, Y. Matsunaga, T. Yamamotoya, K. Ueda, Y. Inoue, K. Mori, et al., Physiological and pathogenic roles of prolyl isomerase Pin1 in metabolic regulations via multiple signal transduction pathway modulations, Int. J. Mol. Sci. 17 (2016). M. Liao, J. Xu, A.J. Clair, B. Ehrman, L.M. Graham, M.J. Eagleton, Local and systemic alterations in signal transducers and activators of transcription (STAT) associated with human abdominal aortic aneurysms, J. Surg. Res. 176 (2012) 321–328. Z. Qin, J. Bagley, G. Sukhova, W.E. Baur, H.J. Park, D. Beasley, et al., Angiotensin II-induced TLR4 mediated abdominal aortic aneurysm in apolipoprotein E knockout mice is dependent on STAT3, J. Mol. Cell. Cardiol. 87 (2015) 160–170.
Journal of Molecular and Cellular Cardiology 114 (2018) 334–344
E.-s. Liang et al.
proteolysis of p65/RelA, Mol. Cell 12 (2003) 1413–1426. [40] J. Pan, K. Fukuda, H. Kodama, S. Makino, T. Takahashi, M. Sano, et al., Role of angiotensin II in activation of the JAK/STAT pathway induced by acute pressure overload in the rat heart, Circ. Res. 81 (1997) 611–617. [41] F. Amiri, S. Shaw, X. Wang, J. Tang, J.L. Waller, D.C. Eaton, et al., Angiotensin II activation of the JAK/STAT pathway in mesangial cells is altered by high glucose, Kidney Int. 61 (2002) 1605–1616. [42] D.C. Guo, C.L. Papke, R. He, D.M. Milewicz, Pathogenesis of thoracic and abdominal aortic aneurysms, Ann. N. Y. Acad. Sci. 1085 (2006) 339–352. [43] G. Wang, L. Jacquet, E. Karamariti, Q. Xu, Origin and differentiation of vascular smooth muscle cells, J. Physiol. 593 (2015) 3013–3030. [44] M.W. Majesky, Developmental basis of vascular smooth muscle diversity, Arterioscler. Thromb. Vasc. Biol. 27 (2007) 1248–1258. [45] M.L. Kirby, K.L. Waldo, Neural crest and cardiovascular patterning, Circ. Res. 77 (1995) 211–215. [46] P. Wasteson, B.R. Johansson, T. Jukkola, S. Breuer, L.M. Akyurek, J. Partanen, et al., Developmental origin of smooth muscle cells in the descending aorta in mice, Development (Cambridge, England) 135 (2008) 1823–1832.
[34] I. Hinterseher, R. Erdman, L.A. Donoso, T.R. Vrabec, C.M. Schworer, J.H. Lillvis, et al., Role of complement cascade in abdominal aortic aneurysms, Arterioscler. Thromb. Vasc. Biol. 31 (2011) 1653–1660. [35] A.W. Orr, N.E. Hastings, B.R. Blackman, B.R. Wamhoff, Complex regulation and function of the inflammatory smooth muscle cell phenotype in atherosclerosis, J. Vasc. Res. 47 (2010) 168–180. [36] Z.J. Shen, R.K. Braun, J. Hu, Q. Xie, H. Chu, R.B. Love, et al., Pin1 protein regulates Smad protein signaling and pulmonary fibrosis, J. Biol. Chem. 287 (2012) 23294–23305. [37] G. Franciosa, G. Diluvio, F.D. Gaudio, M.V. Giuli, R. Palermo, P. Grazioli, et al., Prolyl-isomerase Pin1 controls Notch3 protein expression and regulates T-ALL progression, Oncogene 35 (2016) 4741–4751. [38] G.P. Atkinson, S.E. Nozell, D.K. Harrison, M.S. Stonecypher, D. Chen, E.N. Benveniste, The prolyl isomerase Pin1 regulates the NF-kappaB signaling pathway and interleukin-8 expression in glioblastoma, Oncogene 28 (2009) 3735–3745. [39] A. Ryo, F. Suizu, Y. Yoshida, K. Perrem, Y.C. Liou, G. Wulf, et al., Regulation of NFkappaB signaling by Pin1-dependent prolyl isomerization and ubiquitin-mediated
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Journal of Molecular and Cellular Cardiology journal homepage: www.elsevier.com/locate/yjmcc
Peptidyl-prolyl isomerase Pin1 deficiency attenuates angiotensin II-induced abdominal aortic aneurysm formation in ApoE−/− mice
T
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Er-shun Lianga, Wen Chengb, Rui-xue Yanga, Wen-wu Baib, Xue Liua, , Yu-xia Zhaoa,b a
The Key Laboratory of Cardiovascular Remodeling and Function Research, The State and Shandong Province Joint Key Laboratory of Translational Cardiovascular Medicine, Department of Cardiology, Qilu Hospital of Shandong University, Jinan, Shandong, China b Department of Traditional Chinese Medicine, Qilu Hospital of Shandong University, Jinan, Shandong, China
A R T I C L E I N F O
A B S T R A C T
Keywords: Abdominal aortic aneurysm Pin1 Klf4 Smooth muscle Phenotype switching Ubiquitylation
Peptidyl-prolyl isomerase Pin1 has been reported to be associated with endothelial dysfunction. However, the role of smooth muscle Pin1 in the vascular system remains unclear. Here, we examined the potential function of Pin1 in smooth muscle cells (SMCs) and its contribution to abdominal aortic aneurysm (AAA) pathogenesis. The level of Pin1 expression was found to be elevated in human AAA tissues and mainly localized to SMCs. We constructed smooth muscle-specific Pin1 knockout mice to explore the role of this protein in AAA formation and to elucidate the underlying mechanisms. AAA formation and elastin degradation were hindered by Pin1 depletion in the angiotensin II-induced mouse model. Pin1 depletion reversed the angiotensin II-induced proinflammatory and synthetic SMC phenotype switching via the nuclear factor (NF)-κB p65/Klf4 axis. Moreover, Pin1 depletion inhibited the angiotensin II-induced matrix metalloprotease activities. Mechanically, Pin1 deficiency destabilized NF-κB p65 by promoting its polyubiquitylation. Further, we found STAT1/3 bound to the Pin1 promoter, revealing that activation of STAT1/3 was responsible for the increased expression of Pin1 under angiotensin II stimulation. Thus, these results suggest that Pin1 regulates pro-inflammatory and synthetic SMC phenotype switching and could be a novel therapeutic target to limit AAA pathogenesis.
1. Introduction Abdominal aortic aneurysm (AAA) is a potentially lethal disease in case of rupture [1]. Therapies are currently limited to surgical repair or interventional stent-based exclusion of the aneurysmal sac [2]. However, there is still no effective intervention for patients with small AAAs or contradictions to surgery. This is partly because the pathogenesis of AAA is not well-understood [3]. The pathology of aneurysm formation involves infiltration of inflammatory cells (T lymphocytes and macrophages) within the aortic wall [4], immune responses [5], cell apoptosis, degradation of the extracellular matrix [6], and slightly compensatory collagen deposition [7]. Studies of smooth muscle cells (SMCs) have mostly focused on SMC apoptosis and inflammation [8]. However, recent studies demonstrated that SMC phenotypic modulation is important for aortic aneurysm pathogenesis in both animal models and human subjects [9–11]. The contribution of SMCs to the development and progression of AAA maybe underestimated. In normal vessels, predominant contractile SMCs regulate vessel diameter and blood flow. In response to vascular injury, contractile SMCs lose their contractile proteins, including α-smooth muscle actin
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(SMA), smooth muscle protein-22 and calponin, and shift towards a synthetic phenotype [12]. SMCs with a synthetic phenotype can secrete numerous extracellular matrix proteins, increasing the capability for migration [13]. Studies have suggested that these alterations in SMC phenotypes play a key role in the pathogenesis of several cardiovascular disorders including atherosclerosis [14], plaque stability [15], and hypertension [16]. Because SMC proliferation and collagen synthesis are controversial in the pathogenesis of AAA, focusing on abnormal phenotypic modulation of SMCs may be a promising approach. Peptidyl-prolyl isomerase Pin1 is a unique peptidyl-prolyl cis/trans isomerase that binds to and isomerizes specific pSer/pThr-Pro motifs in specific proteins [17]. These Pin1-induced conformational changes modulate target protein function, stability, interactions, and phosphorylation status [18]. Pin1 regulates a variety of biological processes, such as gene transcription, cell proliferation, differentiation, and apoptosis [19–21]. Studies have demonstrated that Pin1 is a promising therapeutic target for several diseases including Alzheimer's disease [22], obesity [23], myocardial fibrosis [24], and vascular dysfunction [25]. However, the role of endothelial Pin1 in vascular dysfunction is controversial [26,27] and little is known about the role in SMCs.
Corresponding author at: Shandong University, Qilu Hospital, Jinan, No. 107, Wen Hua Xi Road, Jinan 250012, Shandong Province, China. E-mail address: [email protected] (X. Liu).
https://doi.org/10.1016/j.yjmcc.2017.12.006 Received 3 October 2017; Received in revised form 29 November 2017; Accepted 17 December 2017 Available online 19 December 2017 0022-2828/ © 2017 Elsevier Ltd. All rights reserved.
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Hercules, CA, USA). Relative expression was normalized to that of βactin. Primer details for real-time quantification are shown in Table S1.
In the present study, by using a conditional knockout strategy, we examined the potential function of Pin1 in SMCs and its contribution to AAA pathogenesis. The results showed that Pin1 expression was markedly upregulated in AAA tissues. Pin1 deficiency reversed angiotensin II (Ang II)-induced vascular SMC (VSMC) pro-inflammatory and synthetic phenotype switching and attenuated Ang II-induced AAA formation as well as aortic morphological damage in ApoE−/− mice. These results demonstrate that SMC-specific Pin1 plays a critical function in AAA.
2.6. Western blotting assay Total proteins were extracted from tissues or cultured cells. Equal amounts of the proteins were separated by 10% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes, followed by incubation with specific antibodies at 4 °C overnight. Horseradish peroxidase-conjugated secondary antibodies were added and incubated at room temperature for 60 min. Bound primary antibodies were detected by chemiluminescence (Millipore, Billerica, MA, USA). Bands were quantified with ImageJ software (NIH, Bethesda, MD, USA). Expression levels were normalized to the control.
2. Materials and methods 2.1. Animal models For the Ang II-induced AAA model, we used 12–14-week-old ApoE−/− Pin1sm +/+ and ApoE−/− Pin1sm −/− mice. To induce AAA formation, osmotic pumps (Alzet model 2004; Durect, Cupertino, CA, USA) containing either angiotensin II (1.44 mg/kg per day, Sigma, St. Louis, MO, USA) or saline were introduced in 12–14 weeks old ApoE−/ − background male mice as previously described [28]. n = 30 per group. At 28 days after the osmotic pumps implantation, mice were sacrificed, and aneurysms in the abdominal aorta were quantified.
2.7. Luciferase activity assay We constructed contact or mutant luciferase reporter plasmids by inserting the promoter into the pGL3-Basic cloning vector. The promoter luciferase reporter plasmids and a Renilla reporter plasmid (pRLTK) were transfected into HASMCs by electroporation. Luciferase activity was determined using the Dual Luciferase Assay Kit (Promega, Madison, WI, USA). Detailed description of Methods can be found in the Supplemental data.
2.2. Analysis and quantification of AAA Necropsy was performed to confirm aortic rupture during Ang II infusion. Abdominal aortic rupture was defined as observation of blood clots in retroperitoneal cavity. To quantify AAA incidence, the maximum width of the abdominal aorta was measured ex vivo with a vernier calipers. An aneurysm was defined as ≥ 50% enlargement of the external diameter of the suprarenal aorta compared to aortas from saline-infused control mice [29]. AAA severity was determined by measuring the wet weights of the abdominal aortas as described previously [29]. The measurement was determined by an investigator who was blinded to the experiment and mouse genotypes.
2.8. Statistical analysis All values for continuous variables are expressed as means ± SEM. Statistical tests were performed using SPSS16.0 software and GraphPad Prism software version 6.0. We tested the normality and equal variance using the Kolmogorov-Smirnov test implemented in Prism statistical software (GraphPad, San Diego) before parametric data analysis. Comparison of 2 groups of normally distributed samples was done using unpaired Student's t-tests. Comparison of multiple groups of normally distributed samples was done using ANOVA, and Tukey was performed as a post test. Nonnormally distributed data including abdominal aorta wet weights and aortic diameter data were analyzed with MannWhitney test or the Kruskal-Wallis test, as appropriate. P < 0.05 was considered statistically significant.
2.3. Histology and immunohistochemistry Pathology was present in abdominal aortas of surviving mice. Murine aortas were harvested and fixed with 4% paraformaldehyde after 4 weeks of treatment. Abdominal aortic serial cross-sections (5 μm) from the proximal to the distal region of the aneurysm were prepared for histological analysis. Aortic sections were stained with hematoxylin and eosin (H&E) for morphological assessment. VerhoeffVan Gieson Elastin (VVG, HT25A, Sigma) staining was performed to detect elastic fiber integrity of the abdominal aorta. Transmission electron microscopy (TEM) was performed to evaluate ultra-structural details.
3. Results 3.1. Pin1 is elevated in AAA tissues and mainly localizes to SMCs Pin1 is a key regulator in several disease models [31]. Therefore, we first investigated whether Pin1 was altered during AAA formation. To examine Pin1 expression in aneurysm, we stained human AAA tissues with an anti-Pin1 antibody. Compared to normal aortas, immunohistochemical staining revealed significantly higher expression of Pin1 (Fig. 1A). Furthermore, western blotting and RT-PCR analysis revealed that Pin1 is increased in AAA tissues at both the protein and mRNA levels (Fig. 1B–D). To evaluate the potential role of Pin1 in the development of AAA, we conducted confocal immunofluorescence of αSMA and Pin1 in AAA tissues, which revealed that Pin1 (red) mainly localized to the nucleus of smooth muscle cells (α-SMA, green) (Fig. 1E). However, we detected comparable levels of Pin1 expression between controls and thoracic aortic aneurysm (TAA) tissues (Fig. S1A–C). We next compared the aortic expression levels of Pin1 at early stage (7 days) in Ang II-infused ApoE−/− mice. Both thoracic (including ascending and descending aortas) and abdominal aortas have low levels of Pin1 expression in saline-infused ApoE−/− mice (Fig. S1D and E). Intriguingly, the protein level of Pin1 was significantly increased in abdominal aortas but not ascending or descending aortas in
2.4. Cell cultures Mouse primary VSMCs were isolated from mouse abdominal aortas as previously described [30]. All experiments were performed with VSMCs at passages 3–5. Human vascular smooth muscle cells (HASMCs) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Experiments were performed with VSMCs at passages 3–5. Cells were treated with Ang II (10− 7 M) or vehicle and then were harvested after 24-h stimulation. 2.5. Real-time polymerase chain reaction (RT-PCR) Total RNA was extracted using TRIzol reagent (Invitrogen) and reverse-transcribed into cDNA. The mRNA expression levels of Pin1 and Klf4 were determined by SYBR Green-based RT-PCR using a sequence detection system (IQ5 Real-Time PCR cycler, Bio-Rad Laboratories, 335
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Fig. 1. Pin1 is elevated in human AAA tissues and mainly localizes to smooth muscle cells. A, Representative immunohistochemical staining of Pin1 in human AAA and normal control aortas. B and C, Western blot analysis of Pin1 in human AAA tissues versus normal aortic tissues. D, qPCR of Pin1 in human AAA tissues versus normal aortic tissues. E, Confocal images of human AAA tissues double-stained for Pin1 and smooth muscle α-actin (α-SMA). *P < 0.05, vs. control, n = 5. (For interpretation of the references to color in this figure, the reader is referred to the web version of this article.)
7-day consecutively Ang II-infused ApoE−/− mice (Fig. S1D and E). Moreover, the increased Pin1 expression was mainly observed in the media of the suprarenal aortas (Fig. S1F and G), where AAAs most often develop in Ang II-infused mice. Therefore, we speculated that the increased Pin1 expression in AAA tissues may be linked to AAA formation.
(Fig. 2A–C). Following the 28 days of Ang II infusion, the AAA incidence in ApoE−/− Pin1sm +/+ mice was 83.3% (25 of 30, including the mouse with aortic rupture), whereas the incidence in ApoE−/ − Pin1sm −/− was significantly reduced to 30% (9 of 30, Fig. 2A and B, Table S2). H&E staining revealed positive aortic remodeling (Fig. 2A, lower panels), accompanied by aortic wall thickening and intraluminal thrombosis. Additionally, the mortality rate due to AAA rupture was significantly higher in Ang II-infused ApoE−/− Pin1sm +/+ mice (16.7%, 5/30; at days 12, 14, 17, 23 and 25; Table S2) versus Ang IIinfused ApoE−/− Pin1sm −/− mice (0%, 0/30; Table S2). Furthermore, Ang II-infused ApoE−/− Pin1sm +/+ mice exhibited a markedly larger suprarenal aortic diameter as well as heavier abdominal aorta wet weights compared to the vehicle control mice (Fig. 2C–D). In contrast, ApoE−/− Pin1sm −/− mice attenuated the severity of Ang II-induced AAAs based on their significantly smaller aortic diameter and wet weights compared to ApoE−/− Pin1sm +/+ mice (Fig. 2C–D). Fig. 2E showed representative longitudinal and transverse images taken at the level of the suprarenal aorta by high-frequency ultrasound. Collagen and elastin are responsible for mechanical resistance, tensile strength, and elasticity of the arterial wall. Therefore, changes in their content or quality contribute to the development of AAA. We next performed Masson's trichrome staining and α-SMA staining to detect
3.2. Pin1 depletion in SMCs attenuates the incidence and severity of Ang IIinduced AAA formation in apolipoprotein E-knockout (ApoE −/−) mice As Pin1 was mainly located in SMCs (Fig. 1E), to determine whether Pin1 expression in smooth muscle contributes to AAA pathogenesis, we generate Pin1flox/flox/Acta2-Cre mice (Pin1sm −/− mice) by crossbreeding Pin1flox/flox mice with Acta2-Cre mice (Fig. S2). Littermate Pin1flox/flox/Cre− mice (Pin1sm +/+ mice) were used as controls. VSMCspecific deletion Pin1 mice on an ApoE−/− background (ApoE−/ − Pin1sm −/−) were generated by crossing Pin1sm −/− mice with ApoE−/− mice. VSMC-specific Pin1 depletion didn't alter medial SMC content or show any abnormal aortic morphology in ApoE−/− mice (Fig. S3). ApoE−/− background mice were infused subcutaneously with Ang II or vehicle (saline). No spontaneous AAAs were observed in the vehicle-infused ApoE−/− Pin1sm +/+ or ApoE−/− Pin1sm −/− mice 336
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Fig. 2. Pin1 depletion in smooth muscle cells protects ApoE−/− mice from developing AAA. A, Representative images of abdominal aortic specimens in 4 groups. B, Incidence of AAA in 4 groups (percentage). C, Maximal abdominal aortic diameters measured with vernier caliper (mm). D, Abdominal aorta wet weights in 4 groups. E, Representative longitudinal (top) and transverse (bottom) images were taken at the level of the suprarenal aorta by high-frequency ultrasound. Scale bar = 200 μm. *P < 0.05, vs. ApoE−/−/Pin1sm +/+ mice infused with vehicle. #P < 0.05, vs. ApoE−/−/Pin1sm +/+ mice infused with Ang II, n ≥ 8 per group.
phenotypes with or without Ang II treatment by western blotting. VSMC was confirmed through positive staining for smooth muscle specific α-actin antibody (α-SMA) (Fig. S4A). As shown in Fig. 4F and G, the contractile markers α-SMA and calponin were dramatically decreased following Ang II stimulation. In contrast, the levels of osteopontin and vimentin were significantly increased. Consistently, Pin1 depletion reversed the Ang II-induced VSMC synthetic phenotype switching (Fig. 4F and G) in vitro. We also found Pin1 depletion remarkably repressed the expression of MMP-2/9 in Ang II-stimulated VSMCs (Fig. 4F and H). We further tested gelatinase activity in the supernatant of treated VSMCs by zymography. Ang II stimulation markedly increased MMP-2/9 activity compared to the vehicle control (Fig. 4I and J). However, these effects induced by Ang II were ameliorated by Pin1 depletion.
alterations in collagen and SMC content. Pin1 depletion in SMCs markedly increased the relative contents of SMCs and collagen in the suprarenal aortic wall (Fig. 3A–F). Indeed, aortas from Ang II-infused ApoE−/− Pin1sm −/− mice showed densely packed parallel collagen staining in the media, which was opposite from the disrupted irregular pattern observed in ApoE−/− Pin1sm +/+ mice. Moreover, inflammatory cell infiltration as determined by CD68 immunostaining was decreased in the suprarenal aortas of Ang II-infused ApoE−/ − Pin1sm −/− mice compared with that of ApoE−/− Pin1sm +/+ mice (Fig. 2E–F). Verhoeff-Van Giessen (VVG) staining and TEM images indicated that elastin fibers were disrupted in Ang II-infused arteries (Fig. 3G–I). Further, Pin1 depletion in SMCs significantly decreased elastin fragmentation (Fig. 3G–I). These results suggest that Pin1 depletion in SMCs attenuated AAA formation and severity by ameliorating macrophage infiltration and the rupture of elastin fibers, increasing SMC content, and modulating the content and quality of collagen.
3.4. Pin1 depletion inhibits Ang II-induced expression of inflammatory cytokines in vivo and in vitro
3.3. Pin1 depletion reverses Ang II-induced VSMCs synthetic phenotype switching
As chronic inflammation plays a critical role in the formation of AAA, we examined the expression of inflammatory cytokines in aortic tissues. Our results showed that the protein expression level of inducible nitric oxide synthase (iNOS), vascular cell adhesion molecule 1 (VCAM1), intercellular adhesion molecule 1 (ICAM1), tumor necrosis factor-alpha (TNF-α) and Monocyte Chemoattractant Protein-1 (MCP1) were upregulated by Ang II stimulation but reversed by Pin1 depletion in vivo (Fig. 5A–F). Proinflammatory cytokines IL-1β and IL-6 were also inhibited in the AAA tissues by Pin1 deficiency (Fig. 5G and H). These effects were further demonstrated in vitro (Fig. 5I–M). Taken together, these data indicate that Pin1 deletion decreased a number of relevant inflammatory cytokines in AAA.
To investigate the relationship between VSMC phenotypic transition and AAA injury, we performed co-immunostaining of mouse AAA tissues. As revealed by immunofluorescence staining (Fig. 4A and B), the expression of calponin was significantly decreased, while the synthetic VSMC phenotype marker vimentin was significantly increased in aneurysm tissue compared to in the vehicle-infused control. Pin1 depletion in SMCs not only markedly increased calponin content, but also significantly decreased vimentin expression (Fig. 4A and B). As matrix metalloproteinases are responsible for elastin degradation in the vascular medium and initiate the formation of AAA [6], we next examined the effects of Pin1 depletion on MMP-2/9 expression in vivo. As depicted in Fig. 4C–E, the Ang II-induced increase in MMP-2/9 expression was suppressed by Pin1 depletion. We then isolated VSMCs from the aorta of Pin1sm +/+ and Pin1sm −/ − mice and detected the markers of contractile and synthetic VSMC
3.5. Pin1 depletion modulates VSMC phenotype switching via NF-κB/Klf4 axis Next, we evaluated why Pin1 depletion reverses the status of VSMC 337
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Fig. 3. Effect of smooth muscle Pin1 depletion on tissue components of abdominal aortic wall in Ang II-infused ApoE−/− mice. A and B, Smooth muscle cell staining shown by α-SMA staining in the suprarenal aortas of mice after AngII infusion. C and D, Representative staining with Masson's Trichrome (collagen). E and F, Macrophage staining were visualized with anti-CD68. G, Elastin fibers were shown with Verhoeff-van Gieson (VVG) staining. H, The number of elastin breaks per vessel was quantified. I, Transmission electron microscopy (TEM) was performed to evaluate ultra-structural details. Representative images are shown. Arrows indicate destroyed elastic fibers. *P < 0.05, vs. ApoE−/−/Pin1sm +/+ mice infused with Ang II, n ≥ 5 per group.
VSMC phenotype switching by regulating the NF-κB/Klf4 axis, we silenced Pin1 and NF-κB p65 in HASMCs. We observed decreased expression of both the protein and mRNA levels of Klf4 by Pin1 or NF-κB p65 knockdown (Fig. 6H and I). In HASMCs, Pin1 knockdown restrained Ang II-enhanced binding activity of NF-κB p65 with the Klf4 promoter (Fig. 6J and K). In addition, Pin1 or NF-κB p65 knockdown suppressed Klf4 promoter activity (Fig. 6L). These data suggest that Pin1 depletion reverses Ang II-induced VSMC phenotype switching via the NF-κB p65/Klf4 axis.
phenotype switching. A previous study demonstrated that NF-κB binds directly to the Klf4 promoter [15]. However, there is no evidence to date that NF-κB modulates Klf4-mediated VSMC phenotype switching during the formation of AAA. We found Pin1 depletion markedly inhibited the expression of NF-κB p65 as well as Klf4 (Fig. 6A–C), a transcription factor that directly binds to smooth muscle marker genes and regulates phenotype switching. In accordance with the study by Salmon et al. [9], we verified the effect of Klf4 inhibition on Ang IIinduced VSMC synthetic phenotype switching in vitro (Fig. S5). We hypothesized that Pin1 deletion reverses VSMC phenotype switching by suppressing the NF-κB/Klf4 axis. To determine if a Pin1-NF-κB p65 interaction occurs in VSMCs, we performed an immunoprecipitation experiment. As depicted in Fig. 6D, endogenous Pin1 and p65 show an interaction in the basal state, which was enhanced upon Ang II stimulation. Moreover, we detected polyubiquitination K48 of p65. Interestingly, Ang II stimulation did not obviously affect the general expression profile of poly-ubiquitination K48, but inhibited p65-bound poly-ubiquitination of K48. This may explain why p65 was enhanced under Ang II stimulation. Furthermore, Pin1 depletion increased p65-bound poly-ubiquitination K48 but decreased the expression of p65 (Fig. 6E). A luciferase assay and gel shift assays (EMSA) also revealed that Pin1 deficiency suppressed p65 activation (Fig. 6F and G). As it is predicted that NF-κB p65 binds directly to the Klf4 promoter in human cells [15], we cultured HASMCs to verify the binding and explore the mechanism. To further confirm that Pin1 depletion reversed
3.6. Pin1 depletion inhibited Ang II–induced VSMCs proliferation and migration Considering that activated VSMCs may become highly proliferative and migratory under stimulation of Ang II, we next evaluated the effect of Pin1 deletion on the Ang II-induced proliferative capacity. Treatment of VSMCs with Ang II but not vehicle significantly increased VSMCs proliferation (Fig. S6A and B), accompanied by elevated expression of synthetic VSMC phenotype markers including osteopontin and vimentin (Fig. 4). Importantly, Pin1 deletion did not markedly alter the basal proliferative capacity. Therefore, therapeutic strategies preventing the hyperproliferation of VSMCs, particularly VSMCs with the synthetic phenotype, is promising for the treatment of AAA. We further conducted a Transwell migration assay to evaluate migration capability. After 24 h, we found that stimulation of Ang II promoted VSMCs migration compared to in the vehicle control group, 338
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Fig. 4. Pin1 depletion modulates Ang II-induced VSMC synthetic phenotype switching in vivo and in vitro. A, Immunofluorescence staining of calponin (red) and vimentin (green) in aortic sections. Smooth muscle Pin1 depletion reverses Ang II-induced synthetic phenotype switching of VSMCs in AAA. B, Quantification of calponin and vimentin coverage. Scale bar = 50 μm. C–E, Pin1 depletion reversed Ang II-induced MMP-2/9 protein expression in vivo. F–H, Western blot analysis of contractile (calponin, α-SMA) and synthetic proteins (vimentin, osteopontin and MMP-2/9) in Pin1sm +/+ and Pin1sm −/− VSMCs treated with Ang II or vehicle for 24 h. I and J, Gelatin zymography analysis of MMP-2 and MMP-9 activity. *P < 0.05, vs. ApoE−/−/Pin1sm +/+ mice infused with vehicle or Pin1sm +/+ VSMCs treated with vehicle. #P < 0.05, vs. ApoE−/−/Pin1sm +/+ mice infused with Ang II or Pin1sm +/+ VSMCs treated with Ang II, n = 5. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
of Pin1. To determine how STAT1/3 regulates Pin1 expression, we analyzed the Pin1 promoter using the PROMO and JASPAR databases. We identified two potential binding sites for STAT1 and STAT3 recognition motifs in human cells (Fig. 7G), respectively. To verify binding of STAT1/3 to the Pin1 promoter, we performed a ChIP assay using specific primers covering −1305 to −1182 base pairs of the Pin1 promoter region. Ang II induced the binding of STAT1/3 to the Pin1 promoter (Fig. 7H and I). To confirm that the predicted binding sites on the Pin1 promoter are required for transcription activity, we constructed intact promoter-reporter plasmids and mutations of the predicted binding sites (Fig. 7G). As depicted in Fig. 7I, luciferase activity in both mutant plasmids was reduced by 50%, revealing that the core Pin1 promoter element is located in the predicted binding sites. Ang II
but reversed by Pin1 depletion (Fig. S6C and D). These results indicate that Pin1 depletion strongly inhibited Ang II-induced VSMCs proliferation and migration, which may be beneficial for decreasing abdominal aortic diameters and AAA formation.
3.7. STAT1/3 binds to Pin1 promoter and mediates its upregulation facilitated by Ang II stimulation As the JAK/STAT pathway has been reported to participate in Ang II-induced AAA formation [32–34], we explored the potential role of STAT signaling in Ang II stimulation-modulated Pin1. As shown in Fig. 7A–F, Ang II stimulation increased the levels of both phosphorylated and total STAT1 and STAT3 in HASMCs. Interestingly, both STAT1 and STAT3 knockdown decreased the mRNA and protein levels 339
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Fig. 5. Pin1 depletion inhibits Ang II-induced expression of inflammatory cytokines in vivo and in vitro. A–F, Western blot analysis of iNOS, VCAM1, ICAM1, TNF-α and MCP-1 expression in the suprarenal aortas. G and H, IL-1β and IL-6 expression in the suprarenal aortic tissues were detected by ELISA. I-M, Western blot analysis of iNOS, VCAM1, ICAM1, and TNF-α expression in Pin1sm +/+ and Pin1sm −/− VSMCs treated with Ang II or vehicle for 24 h. *P < 0.05, vs. ApoE−/−/Pin1sm +/+ mice infused with vehicle or Pin1sm +/+ VSMCs treated with vehicle. #P < 0.05, vs. ApoE−/−/Pin1sm +/+ mice infused with Ang II or Pin1sm +/+ VSMCs treated with Ang II, n = 5.
accompanied by a slightly compensatory collagen deposition, aortas from Ang II-infused ApoE−/− Pin1sm −/− mice showed densely packed parallel collagen in the media, whereas aortas from ApoE−/− Pin1sm +/ + mice showed the disrupted irregular pattern. Therefore, new therapeutic strategies should attempt to abrogate the synthetic hyperproliferation VSMCs to improve the content and quality of collagen. Of interest, a recent study by Shen et al. [36] revealed that Pin1 null mice expressed reduced, pulmonary expression of collagens and TIMPs but showed an increase in MMPs compared with wild-type. It may seem to conflict with our results. However, Franciosa et al. [37] reported that Pin1 silencing downmodulated MMP9 expression in human leukemic TALL-1 cells. All these results suggest tissue-specific and cell type-specific regulation of MMPs by Pin1. Therefore, study on the roles of Pin1 in AAA development with SMC-specific depletion mice is more convincing. Another major finding of this study is that Pin1 deletion reverses Ang II-induced VSMC synthetic phenotype switching via the NF-κB/Klf4 axis. Our results indicate that Pin1 mediated the Ang II-induced VSMC synthetic phenotype. We then further explored the precise mechanism of this action and found that NF-κB p65, a target protein of Pin1 [38], binds directly to the Klf4 promoter and regulates its expression. This is in accordance with the results of Ding et al. [15]. Klf4, a transcription factor that binds to smooth muscle marker genes, regulates the VSMC synthetic phenotype and aneurysm formation [9,14]. We hypothesized that Pin1 deletion reverses VSMC phenotype switching by suppressing the NF-κB/Klf4 axis. Our immunoprecipitation experiment and ChIP assay indicated that Pin1 deficiency promoted NF-κB p65 degradation via K48-linked polyubiquitination, which subsequently suppressed Klf4-mediated VSMC phenotype switching. Notably, Pin1 stabilizes NF-κB p65 proteins and thus contributes to Ang II-induced AAA. Pin1 is often associated with the ubiquitin-proteasome system, which may influence the degree of substrate polyubiquitylation and subsequent protein degradation. We found that Pin1
stimulation markedly increased luciferase activity from the WT plasmid but not mutant plasmids (Fig. 7J). Therefore, STAT1/3 binds to the Pin1 promoter and regulates its expression. 4. Discussion Here, we showed that Pin1 expression was markedly upregulated in AAA tissues and that Pin1 is mainly localized to SMCs. Pin1 depletion in SMCs significantly attenuated AAA formation in Ang II-infused ApoE−/ − mice. Histological and morphological examination revealed that Pin1 depletion decreased elastin fragmentation and increased SMC content. In the molecular mechanism, Pin1 depletion reversed Ang II-induced VSMC phenotype switching via the NF-κB/Klf4 axis and significantly suppressed the inflammatory response as well as MMP activities. Our data highlight the roles of smooth muscle Pin1 in vascular function and AAA pathogenesis. One major finding of this study is that Pin1 deletion in SMCs attenuated AAA formation. Importantly, Pin1 depletion attenuated the rupture of elastin fibers, increased SMC content, and modulated the content and quality of collagen. During the process of phenotypic switching from a contractile phenotype to a proliferative-migratory phenotype, VSMCs are prone to secrete matrix metalloproteinases (MMPs) and produce more inflammatory cytokines, which subsequently contribute to vascular remodeling [35]. Abnormal VSMCs proliferation is a hallmark of the pathogenesis of AAA [10]. Interestingly, Pin1 deletion reversed Ang II-induced hyperproliferation but did not alter the basal proliferative capacity. Furthermore, Pin1 depletion markedly reduced the activity of MMP-2/9, the major proteases involved in elastin degradation, and decreased Ang II-induced VSMCs migration. We also found that Pin1 depletion attenuated inflammation responses and macrophage infiltration. During the development of AAA, changes in collagen content are of interest as it is the ultimate structural component to prevent rupture. Although AAA was 340
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Fig. 6. Pin1 deficiency attenuates Klf4-mediated VSMC phenotype switching through NF-κB signaling. A–C, Western blot analysis of NF-κB p65 and Klf4 protein expression. VSMCs were stimulated with Ang II or vehicle for 24 h. Cell lysates were subjected to western blotting. D, Immunoprecipitation data showing Pin1 binding to NF-κB p65 in VSMCs treated with Ang II. E, Pin1 deficiency destabilized p65 by promoting p65 polyubiquitylation in vitro. F and G, Resistance of Pin1 deficiency to NF-κB activation by Ang II stimulation detected by luciferase reporter assay and EMSA. H, Western blot analysis of Klf4 protein expression with siPin1 or siNF-κB or control in HASMCs. I, Quantitative RT-PCR analysis of mRNA level of Klf4 with siPin1 or siNF-κB or control in HASMCs. J and K, ChIP assay and quantitative analysis to verify binding of NF-κB p65 to Klf4 promoter in HASMCs pretreated with siPin1 or control. L, Luciferase activity assay was performed after transfection with human Klf4 promoter reporter plasmid and pRL-TK plasmid following pretreatment with siPin1 or siNF-κB. *P < 0.05, vs. Pin1sm +/+ VSMCs treated with vehicle or VSMC treated with vehicle and control siRNA. #P < 0.05, vs. Pin1sm +/+ VSMCs treated with Ang II or VSMC treated with Ang II and control siRNA, n = 5.
it may influence the affinity of ligases for their substrates. These data illustrate that Pin1 functions as a novel switch in the ubiquitin pathway, which controls protein expression. Next, we focused on the underlying mechanisms of Ang II-stimulated upregulation of Pin1. Studies have shown that activation of the JAK/STAT pathway by Ang II is associated with AAA formation [32,33,40,41]. STAT1 showed corresponding increases in both total
deficiency leads to pronounced K48-polyubiquitylation and degradation of NF-κB p65. NF-κB signaling is activated by Ang II, but was reversed by Pin1 inhibition. In accordance with a study of Ryo et al. [39], Pin1-deficient SMCs are refractory to NF-κB activation by promoting polyubiquitin-mediated proteolysis of p65. SOCS-1 ubiquitin ligase may be involved in polyubiquitylation of p65 in Pin1 deficiency [39]. Although it is not clear how Pin1 controls the activity of ubiquitin ligases, 341
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Fig. 7. STAT1/3 mediated upregulation of Pin1 facilitated by Ang II stimulation. HASMCs were stimulated with Ang II or vehicle for 24 h. A–C, Western blotting and quantitative RT-PCR analysis of Pin1 expression with STAT1 siRNA or control. D–F, Western blotting and quantitative RT-PCR analysis of Pin1 expression with STAT3 siRNA or control. G, Predicted STAT1 or STAT3 binding site within the human Pin1 promoter; mutants with deletion of the predicted binding site (Pin1-mut1, Pin1-mut2, and Pin1-mut3) are shown. H and I, ChIP assay and quantitative analysis to verify binding of STAT1 or STAT3 to Pin1 promoter in HASMCs. J, Luciferase reporter assay was performed after transfection with Pin1 promoter vector or mutants in HASMC.*P < 0.05, vs. vehicle-stimulated HASMCs transfected with intact Pin1 promoter. #P < 0.05, vs. Ang II-stimulated HASMCs transfected with intact Pin1 promoter, n = 5.
Fig. 8. Working schematic of how Pin-1 depletion in SMC attenuated Ang II-induced AAA formation. Ang II stimulation activates STAT1/3, resulting in increased transcription activity of Pin1. Pin1 modulates VSMC phenotype switching via NF-κB/Klf4 axis. Additionally, Pin1 acts on inflammatory cytokines, and MMPs, further resulting in vascular remodeling and promoting the formation of AAA.
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protein and mRNA levels in AAA tissues [32]. Treatment with a STAT3 inhibitor decreased the incidence and severity of Ang II-induced AAA formation, which suppressed MMP activity and the ratio of M1/M2 macrophages [33]. We then evaluated whether STAT transcription factors modulate Pin1 expression in response to Ang II stimulation. Interestingly, STAT1 and STAT3 bound to distinct recognition motifs of the Pin1 promoter. We found that Ang II-induced Pin1 expression occurred via a direct interaction between STAT1/3 and the Pin1 promoter. Although STAT1/3 inhibition may prevent aneurysm formation, targeting transcription factors as potential drug targets is challenging. STAT1/3 transcription factors act on different promoters and exhibit both positive and negative interactions. Thus, Pin1 maybe a promising therapeutic target for AAA linking STAT1/3 transcription factors and aneurysm formation. We developed a mechanistic model describing the role of Pin1 in AAA formation (Fig. 8). It is interesting to observe that Pin1 expression was significantly elevated in human AAA but not TAA tissues. Simultaneously, Pin1 was markedly increased in the suprarenal abdominal aorta during the early onset of AAA in Ang II-infused ApoE−/−mice, but no obvious change was observed in the ascending or descending thoracic aorta. Although not fully understood, several possibilities may explain the differences between the abdominal and thoracic VSMC responses in aneurysm development. Firstly, AAA and TAA have different aetiologies and pathogeneses. TAA are more genetically based while AAA tends to be associated with smoking, inflammation and dyslipidemia [8,42]. AAA is associated with a Th2-predominant immune response, whereas TAA is linked with a Th1-predominant immune response [42]. Secondly, abdominal and thoracic aortas differ in embryology of VSMCs origin [43]. Medial SMCs in the abdominal aorta originate from splanchnic mesoderm [44], while those in the ascending and descending aortas arise from cardiac neural crest [45] and somites [46] respectively. SMCs of different origins may respond differently to the same exogenous stimulation such as Ang II. Moreover, differences in anatomical configuration and hemodynamic shear stress may also account for differences in Pin1 expression and aortic aneurysm development. In conclusion, we found that Ang II induces Pin1 expression, which subsequently contributes to the progression of AAA. Our study improves the understanding of the biological activities and regulatory mechanisms of smooth muscle Pin1 in AAA. These results suggest that Pin1 is a potential therapeutic target to prevent AAA formation.
[5] [6]
[7]
[8] [9]
[10]
[11]
[12] [13]
[14]
[15]
[16]
[17]
[18] [19] [20]
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[23]
Acknowledgments [24]
We would like to thank Dr. Wen-Cheng Zhang for providing the Acta2Cre transgenic mice. This study was supported by the National 973 Basic Research Program of China (No. 2012CB518603), and the National Natural Science Foundation of China (No. 81302939).
[25] [26]
Disclosures
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None.
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Appendix A. Supplementary data
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Supplementary data to this article can be found online at https:// doi.org/10.1016/j.yjmcc.2017.12.006.
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References
[31]
[1] N.L. Weintraub, Understanding abdominal aortic aneurysm, N. Engl. J. Med. 361 (2009) 1114–1116. [2] U. Raaz, A.M. Zollner, I.N. Schellinger, R. Toh, F. Nakagami, M. Brandt, et al., Segmental aortic stiffening contributes to experimental abdominal aortic aneurysm development, Circulation 131 (2015) 1783–1795. [3] H. Kuivaniemi, E.J. Ryer, J.R. Elmore, G. Tromp, Understanding the pathogenesis of abdominal aortic aneurysms, Expert. Rev. Cardiovasc. Ther. 13 (2015) 975–987. [4] T. Freestone, R.J. Turner, A. Coady, D.J. Higman, R.M. Greenhalgh, J.T. Powell,
[32]
[33]
343
Inflammation and matrix metalloproteinases in the enlarging abdominal aortic aneurysm, Arterioscler. Thromb. Vasc. Biol. 15 (1995) 1145–1151. K. Shimizu, R.N. Mitchell, P. Libby, Inflammation and cellular immune responses in abdominal aortic aneurysms, Arterioscler. Thromb. Vasc. Biol. 26 (2006) 987–994. G.M. Longo, W. Xiong, T.C. Greiner, Y. Zhao, N. Fiotti, B.T. Baxter, Matrix metalloproteinases 2 and 9 work in concert to produce aortic aneurysms, J. Clin. Invest. 110 (2002) 625–632. Q. Wang, Y. Ding, P. Song, H. Zhu, I.S. Okon, N.Y. Ding, et al., Tryptophan-derived 3-hydroxyanthranilic acid contributes to angiotensin II-induced abdominal aortic aneurysm formation in mice in vivo, Circulation 136 (23) (2017) 2271–2283. I.M. Nordon, R.J. Hinchliffe, I.M. Loftus, M.M. Thompson, Pathophysiology and epidemiology of abdominal aortic aneurysms, Nat. Rev. Cardiol. 8 (2011) 92–102. M. Salmon, W.F. Johnston, A. Woo, N.H. Pope, G. Su, G.R. Upchurch Jr.et al., KLF4 regulates abdominal aortic aneurysm morphology and deletion attenuates aneurysm formation, Circulation 128 (2013) S163–74. G. Ailawadi, C.W. Moehle, H. Pei, S.P. Walton, Z. Yang, I.L. Kron, et al., Smooth muscle phenotypic modulation is an early event in aortic aneurysms, J. Thorac. Cardiovasc. Surg. 138 (2009) 1392–1399. E. Crosas-Molist, T. Meirelles, J. Lopez-Luque, C. Serra-Peinado, J. Selva, L. Caja, et al., Vascular smooth muscle cell phenotypic changes in patients with Marfan syndrome, Arterioscler. Thromb. Vasc. Biol. 35 (2015) 960–972. G.K. Owens, Regulation of differentiation of vascular smooth muscle cells, Physiol. Rev. 75 (1995) 487–517. K. Riches, T.G. Angelini, G.S. Mudhar, J. Kaye, E. Clark, M.A. Bailey, et al., Exploring smooth muscle phenotype and function in a bioreactor model of abdominal aortic aneurysm, J. Transl. Med. 11 (2013) 208. L.S. Shankman, D. Gomez, O.A. Cherepanova, M. Salmon, G.F. Alencar, R.M. Haskins, et al., KLF4-dependent phenotypic modulation of smooth muscle cells has a key role in atherosclerotic plaque pathogenesis, Nat. Med. 21 (2015) 628–637. Y. Ding, M. Zhang, W. Zhang, Q. Lu, Z. Cai, P. Song, et al., AMP-activated protein kinase alpha 2 deletion induces VSMC phenotypic switching and reduces features of atherosclerotic plaque stability, Circ. Res. 119 (2016) 718–730. S.B. Zhu, J. Zhu, Z.Z. Zhou, E.P. Xi, R.P. Wang, Y. Zhang, TGF-beta1 induces human aortic vascular smooth muscle cell phenotype switch through PI3K/AKT/ID2 signaling, Am. J. Transl. Res. 7 (2015) 2764–2774. A. Tun-Kyi, G. Finn, A. Greenwood, M. Nowak, T.H. Lee, J.M. Asara, et al., Essential role for the prolyl isomerase Pin1 in Toll-like receptor signaling and type I interferon-mediated immunity, Nat. Immunol. 12 (2011) 733–741. D. Siepe, S. Jentsch, Prolyl isomerase Pin1 acts as a switch to control the degree of substrate ubiquitylation, Nat. Cell Biol. 11 (2009) 967–972. S.D. Hanes, Prolyl isomerases in gene transcription, Biochim. Biophys. Acta 2015 (1850) 2017–2034. G.L. Perrucci, A. Gowran, M. Zanobini, M.C. Capogrossi, G. Pompilio, P. Nigro, Peptidyl-prolyl isomerases: a full cast of critical actors in cardiovascular diseases, Cardiovasc. Res. 106 (2015) 353–364. H. Zheng, H. You, X.Z. Zhou, S.A. Murray, T. Uchida, G. Wulf, et al., The prolyl isomerase Pin1 is a regulator of p53 in genotoxic response, Nature 419 (2002) 849–853. L. Pastorino, A. Sun, Lu PJ, X.Z. Zhou, M. Balastik, G. Finn, et al., The prolyl isomerase Pin1 regulates amyloid precursor protein processing and amyloid-beta production, Nature 440 (2006) 528–534. Y. Nakatsu, H. Sakoda, A. Kushiyama, J. Zhang, H. Ono, M. Fujishiro, et al., Peptidyl-prolyl cis/trans isomerase NIMA-interacting 1 associates with insulin receptor substrate-1 and enhances insulin actions and adipogenesis, J. Biol. Chem. 286 (2011) 20812–20822. X. Liu, E. Liang, X. Song, Z. Du, Y. Zhang, Y. Zhao, Inhibition of Pin1 alleviates myocardial fibrosis and dysfunction in STZ-induced diabetic mice, Biochem. Biophys. Res. Commun. 479 (2016) 109–115. S. Costantino, F. Paneni, T.F. Luscher, F. Cosentino, Pin1 inhibitor Juglone prevents diabetic vascular dysfunction, Int. J. Cardiol. 203 (2016) 702–707. V.L. Chiasson, N. Munshi, P. Chatterjee, K.J. Young, B.M. Mitchell, Pin1 deficiency causes endothelial dysfunction and hypertension, Hypertension (Dallas, Tex: 1979) 58 (2011) 431–438. L. Ruan, C.M. Torres, J. Qian, F. Chen, J.D. Mintz, D.W. Stepp, et al., Pin1 prolyl isomerase regulates endothelial nitric oxide synthase, Arterioscler. Thromb. Vasc. Biol. 31 (2011) 392–398. J. Kong, K. Zhang, X. Meng, Y. Zhang, C. Zhang, Dose-dependent bidirectional effect of angiotensin IV on abdominal aortic aneurysm via variable angiotensin receptor stimulation, Hypertension (Dallas, Tex: 1979) 66 (2015) 617–626. D.B. Trivedi, C.D. Loftin, J. Clark, P. Myers, L.M. DeGraff, J. Cheng, et al., betaArrestin-2 deficiency attenuates abdominal aortic aneurysm formation in mice, Circ. Res. 112 (2013) 1219–1229. P. Zhang, S. Hou, J. Chen, J. Zhang, F. Lin, R. Ju, et al., Smad4 deficiency in smooth muscle cells initiates the formation of aortic aneurysm, Circ. Res. 118 (2016) 388–399. Y. Nakatsu, Y. Matsunaga, T. Yamamotoya, K. Ueda, Y. Inoue, K. Mori, et al., Physiological and pathogenic roles of prolyl isomerase Pin1 in metabolic regulations via multiple signal transduction pathway modulations, Int. J. Mol. Sci. 17 (2016). M. Liao, J. Xu, A.J. Clair, B. Ehrman, L.M. Graham, M.J. Eagleton, Local and systemic alterations in signal transducers and activators of transcription (STAT) associated with human abdominal aortic aneurysms, J. Surg. Res. 176 (2012) 321–328. Z. Qin, J. Bagley, G. Sukhova, W.E. Baur, H.J. Park, D. Beasley, et al., Angiotensin II-induced TLR4 mediated abdominal aortic aneurysm in apolipoprotein E knockout mice is dependent on STAT3, J. Mol. Cell. Cardiol. 87 (2015) 160–170.
Journal of Molecular and Cellular Cardiology 114 (2018) 334–344
E.-s. Liang et al.
proteolysis of p65/RelA, Mol. Cell 12 (2003) 1413–1426. [40] J. Pan, K. Fukuda, H. Kodama, S. Makino, T. Takahashi, M. Sano, et al., Role of angiotensin II in activation of the JAK/STAT pathway induced by acute pressure overload in the rat heart, Circ. Res. 81 (1997) 611–617. [41] F. Amiri, S. Shaw, X. Wang, J. Tang, J.L. Waller, D.C. Eaton, et al., Angiotensin II activation of the JAK/STAT pathway in mesangial cells is altered by high glucose, Kidney Int. 61 (2002) 1605–1616. [42] D.C. Guo, C.L. Papke, R. He, D.M. Milewicz, Pathogenesis of thoracic and abdominal aortic aneurysms, Ann. N. Y. Acad. Sci. 1085 (2006) 339–352. [43] G. Wang, L. Jacquet, E. Karamariti, Q. Xu, Origin and differentiation of vascular smooth muscle cells, J. Physiol. 593 (2015) 3013–3030. [44] M.W. Majesky, Developmental basis of vascular smooth muscle diversity, Arterioscler. Thromb. Vasc. Biol. 27 (2007) 1248–1258. [45] M.L. Kirby, K.L. Waldo, Neural crest and cardiovascular patterning, Circ. Res. 77 (1995) 211–215. [46] P. Wasteson, B.R. Johansson, T. Jukkola, S. Breuer, L.M. Akyurek, J. Partanen, et al., Developmental origin of smooth muscle cells in the descending aorta in mice, Development (Cambridge, England) 135 (2008) 1823–1832.
[34] I. Hinterseher, R. Erdman, L.A. Donoso, T.R. Vrabec, C.M. Schworer, J.H. Lillvis, et al., Role of complement cascade in abdominal aortic aneurysms, Arterioscler. Thromb. Vasc. Biol. 31 (2011) 1653–1660. [35] A.W. Orr, N.E. Hastings, B.R. Blackman, B.R. Wamhoff, Complex regulation and function of the inflammatory smooth muscle cell phenotype in atherosclerosis, J. Vasc. Res. 47 (2010) 168–180. [36] Z.J. Shen, R.K. Braun, J. Hu, Q. Xie, H. Chu, R.B. Love, et al., Pin1 protein regulates Smad protein signaling and pulmonary fibrosis, J. Biol. Chem. 287 (2012) 23294–23305. [37] G. Franciosa, G. Diluvio, F.D. Gaudio, M.V. Giuli, R. Palermo, P. Grazioli, et al., Prolyl-isomerase Pin1 controls Notch3 protein expression and regulates T-ALL progression, Oncogene 35 (2016) 4741–4751. [38] G.P. Atkinson, S.E. Nozell, D.K. Harrison, M.S. Stonecypher, D. Chen, E.N. Benveniste, The prolyl isomerase Pin1 regulates the NF-kappaB signaling pathway and interleukin-8 expression in glioblastoma, Oncogene 28 (2009) 3735–3745. [39] A. Ryo, F. Suizu, Y. Yoshida, K. Perrem, Y.C. Liou, G. Wulf, et al., Regulation of NFkappaB signaling by Pin1-dependent prolyl isomerization and ubiquitin-mediated
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