PAR ‐3 controls endothelial planar polarity and vascular inflammation under laminar flow
2018; Springer Nature; Volume: 19; Issue: 9 Linguagem: Inglês
10.15252/embr.201745253
ISSN1469-3178
AutoresTakao Hikita, Fatemeh Mirzapourshafiyi, Pedro Barbacena, Meghan Riddell, Ayesha Pasha, Mengnan Li, Takuji Kawamura, Ralf P. Brandes, Tomonori Hirose, Shigeo Ohno, Holger Gerhardt, Michiyuki Matsuda, Cláudio A. Franco, Masanori Nakayama,
Tópico(s)Angiogenesis and VEGF in Cancer
ResumoArticle17 July 2018free access Transparent process PAR-3 controls endothelial planar polarity and vascular inflammation under laminar flow Takao Hikita Laboratory for Cell Polarity and Organogenesis, Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany Search for more papers by this author Fatemeh Mirzapourshafiyi Laboratory for Cell Polarity and Organogenesis, Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany Search for more papers by this author Pedro Barbacena Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal Search for more papers by this author Meghan Riddell Laboratory for Cell Polarity and Organogenesis, Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany Search for more papers by this author Ayesha Pasha Laboratory for Cell Polarity and Organogenesis, Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany Search for more papers by this author Mengnan Li Laboratory for Cell Polarity and Organogenesis, Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany Search for more papers by this author Takuji Kawamura Laboratory for Cell Polarity and Organogenesis, Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany Search for more papers by this author Ralf P Brandes Institute for Cardiovascular Physiology, Goethe University, Frankfurt am Main, Germany Search for more papers by this author Tomonori Hirose Department of Molecular Biology, Graduate School of Medical Science, Yokohama City University, Yokohama, Japan Search for more papers by this author Shigeo Ohno Department of Molecular Biology, Graduate School of Medical Science, Yokohama City University, Yokohama, Japan Search for more papers by this author Holger Gerhardt orcid.org/0000-0002-3030-0384 Integrative Vascular Biology Laboratory, Max-Delbrück-Center for Molecular Medicine, Berlin, Germany Search for more papers by this author Michiyuki Matsuda Department of Pathology and Biology of Diseases, Graduate School of Medicine, Kyoto University, Kyoto, Japan Search for more papers by this author Claudio A Franco orcid.org/0000-0002-2861-3883 Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal Search for more papers by this author Masanori Nakayama Corresponding Author [email protected] orcid.org/0000-0002-0153-7198 Laboratory for Cell Polarity and Organogenesis, Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany Search for more papers by this author Takao Hikita Laboratory for Cell Polarity and Organogenesis, Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany Search for more papers by this author Fatemeh Mirzapourshafiyi Laboratory for Cell Polarity and Organogenesis, Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany Search for more papers by this author Pedro Barbacena Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal Search for more papers by this author Meghan Riddell Laboratory for Cell Polarity and Organogenesis, Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany Search for more papers by this author Ayesha Pasha Laboratory for Cell Polarity and Organogenesis, Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany Search for more papers by this author Mengnan Li Laboratory for Cell Polarity and Organogenesis, Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany Search for more papers by this author Takuji Kawamura Laboratory for Cell Polarity and Organogenesis, Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany Search for more papers by this author Ralf P Brandes Institute for Cardiovascular Physiology, Goethe University, Frankfurt am Main, Germany Search for more papers by this author Tomonori Hirose Department of Molecular Biology, Graduate School of Medical Science, Yokohama City University, Yokohama, Japan Search for more papers by this author Shigeo Ohno Department of Molecular Biology, Graduate School of Medical Science, Yokohama City University, Yokohama, Japan Search for more papers by this author Holger Gerhardt orcid.org/0000-0002-3030-0384 Integrative Vascular Biology Laboratory, Max-Delbrück-Center for Molecular Medicine, Berlin, Germany Search for more papers by this author Michiyuki Matsuda Department of Pathology and Biology of Diseases, Graduate School of Medicine, Kyoto University, Kyoto, Japan Search for more papers by this author Claudio A Franco orcid.org/0000-0002-2861-3883 Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal Search for more papers by this author Masanori Nakayama Corresponding Author [email protected] orcid.org/0000-0002-0153-7198 Laboratory for Cell Polarity and Organogenesis, Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany Search for more papers by this author Author Information Takao Hikita1, Fatemeh Mirzapourshafiyi1, Pedro Barbacena2, Meghan Riddell1, Ayesha Pasha1, Mengnan Li1, Takuji Kawamura1, Ralf P Brandes3, Tomonori Hirose4, Shigeo Ohno4, Holger Gerhardt5, Michiyuki Matsuda6, Claudio A Franco2 and Masanori Nakayama *,1 1Laboratory for Cell Polarity and Organogenesis, Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany 2Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal 3Institute for Cardiovascular Physiology, Goethe University, Frankfurt am Main, Germany 4Department of Molecular Biology, Graduate School of Medical Science, Yokohama City University, Yokohama, Japan 5Integrative Vascular Biology Laboratory, Max-Delbrück-Center for Molecular Medicine, Berlin, Germany 6Department of Pathology and Biology of Diseases, Graduate School of Medicine, Kyoto University, Kyoto, Japan *Corresponding author. Tel: +49 6032 705 248; E-mail: [email protected] EMBO Rep (2018)19:e45253https://doi.org/10.15252/embr.201745253 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Impaired cell polarity is a hallmark of diseased tissue. In the cardiovascular system, laminar blood flow induces endothelial planar cell polarity, represented by elongated cell shape and asymmetric distribution of intracellular organelles along the axis of blood flow. Disrupted endothelial planar polarity is considered to be pro-inflammatory, suggesting that the establishment of endothelial polarity elicits an anti-inflammatory response. However, a causative relationship between polarity and inflammatory responses has not been firmly established. Here, we find that a cell polarity protein, PAR-3, is an essential gatekeeper of GSK3β activity in response to laminar blood flow. We show that flow-induced spatial distribution of PAR-3/aPKCλ and aPKCλ/GSK3β complexes controls local GSK3β activity and thereby regulates endothelial planar polarity. The spatial information for GSK3β activation is essential for flow-dependent polarity to the flow axis, but is not necessary for flow-induced anti-inflammatory response. Our results shed light on a novel relationship between endothelial polarity and vascular homeostasis highlighting avenues for novel therapeutic strategies. Synopsis Along the direction of flow, spatial activation of RhoA/Rho-kinase distributes the PAR-3/PKCλ and the GSK3β/PKCλ complexes in a mutually exclusive manner in endothelial cells. This process is important for GSK3β activation controlling inflammatory response via regulating NF-κB localization. The PAR-3/PKCλ and GSK3β/PKCλ complexes are distributed in a mutual exclusive manner in endothelial cells along the direction of flow. The spatially antagonized localization of polarity complexes is regulated by RhoA/Rho-kinase controlling GSK3β activation and thereby EC polarity. While activation of GSK3β is necessary for p65 nuclear localization, spatial distribution of active GSK3β is not essential for endothelial inflammatory responses. Introduction Cell polarity is the asymmetric organization of cellular components, such as organelles and cytoskeleton. Intrinsic cellular asymmetry is crucial for cell type-specific functions. Consequently, the establishment and maintenance of cell polarity are stringently regulated during tissue morphogenesis and in homeostasis. Conversely, loss of cell polarity is often a hallmark of diseases including cancer and cardiovascular dysfunction 1. In vertebrates, endothelial cells (ECs), the cells forming the inner lining of the vasculature, have apical–basal polarity to establish a barrier between blood and the rest of the body 2-4. Furthermore, ECs have profound morphological adaptation to hemodynamic shear stress and possess planar cell polarity to the direction of blood flow 5, 6. To establish functional vessels, dynamic changes of microtubules and the actin cytoskeleton are observed with maintaining blood flow. Cell shape is elongated along the axis of flow, and the microtubule organization centers (MTOCs) and the Golgi apparatus are aligned in front or behind the nuclei toward the flow direction 7-10. In contrast, regions of disrupted flow in the aorta often show roundish cell shape and disorganized Golgi orientation, which is highly associated with a pro-inflammatory condition 5. Importantly, atherosclerotic plaques occur at specific sites in arteries where blood flow is slow and patterns are disturbed 6. However, a causal link between compromised endothelial polarity toward the flow direction and vascular inflammatory responses has not been firmly established. Cell polarization is achieved by integration of both extra- and intracellular signaling cascades controlling cytoskeletal dynamics. The signaling crosstalk among Rho family small GTPases, Cdc42, Rac1, and RhoA, regulates cytoskeletal reorganization mediated by key polarity regulators, the PAR polarity protein complex 11. The PAR complex is composed of PAR-3, PAR-6, and atypical PKC (aPKC) and functions in various cell polarization events across species 12. Activated Cdc42 binds to the PAR-6/aPKC complex, leading to aPKC activation and promoting association with PAR-3 13. PAR-3 directly interacts with Rac1 activation factor, Tiam1/2, and further forms a complex with aPKC, PAR-6, and Cdc42, thereby mediating Cdc42-induced Rac1 activation for actin cytoskeletal reorganization 14, 15. aPKC forms a protein complex with glycogen synthase kinase-3 beta (GSK3β) in migrating astrocytes and during epithelial cell death 16, 17. aPKC forms a complex with active form of GSK3β, whereas aPKC activity is required for GSK3β inactivation, leading to the stabilization of microtubules and MTOC reorientation. In the cardiovascular system, it has been shown that PAR-3 regulates sprouting behavior of endothelial cells (ECs) during angiogenesis 18; meanwhile, the role of PAR-3 in endothelial polarization in living organisms remains elusive. In light of these observations, we investigate the role of endothelial PAR-3 in EC polarization. EC-specific inducible PAR-3 loss-of-function mice exhibit compromised endothelial polarity to the flow axis in a flow-rate-dependent manner but do not show overt effects on apical–basal polarity. Shear stress controls the spatio-temporal antagonism of the PAR-3/aPKClambda/iota (aPKCλ), one of two isoforms of aPKC, complex versus the GSK3β/aPKCλ complex through the RhoA/Rho-kinase pathway, resulting in spatially controlled microtubule stabilization in the direction of flow. Moreover, vascular inflammatory responses are increased by regulating NF-κB, a key regulator of inflammation, nuclear localization downstream of GSK3β in PAR-3 loss-of-function conditions. Importantly, pharmacological suppression of GSK3β restored increased NF-κB nuclear localization but not endothelial polarity to the flow axis. Our results indicate an unexpected relationship between endothelial polarity to flow and vascular inflammation downstream of PAR-3. Results PAR-3 controls endothelial polarity toward the direction of flow in a shear rate dependent manner but is not essential for apical–basal polarization in the growing vasculature To gain more insight into the role of PAR-3 in endothelial polarity, we first observed the effect of PAR-3 on shear stress-dependent endothelial polarization in response to blood flow. EC nuclei and Golgi, visualized by immunostaining with ERG1/2/3, a marker of EC nucleus 19 and GM130 or GOLPH4 antibodies, respectively, showed that Golgi was regularly found in the upstream side of the nuclei in vessels at postnatal day 6 (P6) retina (Fig 1A). This was particularly obvious in ECs in the major vessels, including the artery and vein (Fig 1A). In contrast, Golgi orientation was often reversed in ECs of Pard3, the gene encoding PAR-3, EC-specific inducible KO mice (Pard3iΔEC) (Fig 1A). To further analyze the misorientation of Golgi toward the flow direction throughout the entire retinal vasculature, we employed a computational approach to calculate the efficiency of endothelial adaptation to flow by measuring the angle between EC axial polarity vectors (EC nucleus-to-Golgi axis) and the predicted blood flow vectors 10, 20, 21. All of the vascular beds in the retina were categorized into four groups: artery, vein, capillary, and sprouting front (Appendix Fig S1A); polarization of ECs toward the flow direction determined by Polarity Index was measured (Fig 1B). While the majority of the ECs showed polarized Golgi orientation in each vascular bed (Appendix Fig S1B), endothelial Golgi orientation against flow in Pard3iΔEC mutants was significantly disrupted in the artery, vein, and capillaries of the vascular plexus, but not in the sprouting front (Fig 1C). Interestingly, correlative analysis of wall shear stress (WSS) and EC polarization in the capillary vascular bed of control and Pard3iΔEC mice showed that EC polarity is compromised in low-to-medium WSS regions, but not at high levels of WSS in the retina (Fig 1D). Figure 1. PAR-3 is important for the establishment of endothelial polarity toward the flow axis Endothelial axial polarity phenotype in control and Pard3iΔEC P6 mouse retinal vasculature. Left panels show a large field of view of corresponding retinal vasculature. Right panels show Golgi orientation of ECs in designated areas, corresponding to highlighted areas in left panel (a: artery; v: vein; c: capillary). Green: ERG1/2/3; red: GM130; blue: VE-cadherin. Arrows are drawn from the center of EC nuclei to the Golgi. Scale bars: 100 μm (left columns, lower magnification) and 20 μm (right columns, higher magnification). Schema for the Polarity Index calculation. The angle of polarization (α) was calculated from the flow direction and the angle of the nucleus-to-Golgi vector. Analysis of the endothelial Polarity Index, relative to predicted blood flow for each specific vascular bed (n = 3 mice). Data are presented as mean ± SD. P-values are indicated in the figure (Student's t-test). Correlative analysis of wall shear stress and EC polarization in the capillary vascular bed. Download figure Download PowerPoint PAR-3 controls cell-to-cell contact formation and thereby epithelial polarization 22. To investigate the role of PAR-3 in EC polarity in the growing vasculature, we next examined EC-EC junction and apical–basal polarity markers in P6 mouse retinal vasculature with immunostaining. Vascular endothelial (VE)-cadherin establishes a homophilic complex at the joint point and forms an adherens junction, which defines apical–basal polarity 3. Podocalyxin is widely expressed on the apical surface of lumenized vascular endothelial cells, whereas collagen type IV is secreted by the basal region of the endothelium 23, 24. While Pard3iΔEC mutants showed the expected sprouting defects as previously reported 18 (Fig EV1A–C), immunostaining within anti-VE-cadherin antibody in Pard3iΔEC retina did not reveal any clear defects (Fig EV1A). Three-dimensional reconstituted images of the retinal vasculature stained with anti-VE-cadherin, anti-Podocalyxin, and anti-collagen type IV antibodies further confirmed equivalent apical–basal polarization between control and Pard3iΔEC mice (Fig EV1D). These results suggest that PAR-3 is important for endothelial polarity to the flow axis in vivo at low-to-moderate but not at high levels of shear stress nor apical–basal polarization during angiogenesis. Click here to expand this figure. Figure EV1. PAR-3 KO does not exhibit overt defects on adherens junction formation and apical–basal polarization in the retinal vasculature Staining of control and Pard3iΔEC P6 mice retina with adherens junction marker (VE-cadherin) and endothelial cell marker isolectin-B4 (iB4). Staining of control and Pard3iΔEC P6 retina with basement membrane marker (collagen IV), apical membrane marker (podocalyxin), and isolectin-B4 (iB4). Quantification of the number of sprouts/10 μm in angiogenic front. Data are presented as mean ± SEM (n = 3 retinas). Difference **P < 0.01, analyzed by Student's t-test. Staining of control and Pard3iΔEC P6 retina artery with basement membrane marker (collagen IV), apical membrane marker (podocalyxin), adherens junction marker (VE-cadherin), and nuclei (Hoechst 33342, Sigma). 3D-reconstituted cross-section images from the region indicated with white dashed lines are shown in the images. Data information: Scale bars: 100 μm (A and B, lower magnification), 25 μm (A and B, higher magnification), and 10 μm (D). Download figure Download PowerPoint PAR-3 controls endothelial polarity to the flow axis and inflammatory responses in established vessels The inner curvature of the aorta is an area where ECs experience low-to-moderate WSS and disturbed flow, and which is prone to atheroma plaque formation 6. To examine whether the role of PAR-3 in endothelial polarity toward the flow axis is restricted only to developmental processes, we next investigated endothelial polarity toward the flow axis in the aortic arch in the established vessels of adult Pard3iΔEC mice. En face confocal microscopy analysis of ECs in the inner curvature to sidewall at the proximal arch showed a gradient of morphological diversity (Figs 2A and EV2A). To quantify endothelial polarity toward the flow axis at the arterial arch, we measured the angle of the EC axial polarity vector with the proximal–distal axis and a Polarity Index was calculated (Figs EV2B and 1B). To evaluate EC orientation, we measured the angle between the primary axis of the best fitting ellipse to the EC shape and the orthogonal axis of the direction of flow (Fig EV2C). In control mice, ECs showed roundish shape at the inner curvature, whereas those at the sidewall were aligned and showed a polarized morphology (Fig 2A–C). Consistent with our observation in the retinal vasculature (Fig 1), endothelial Golgi in the sidewall exhibited polarized localization against the flow direction; however, ECs in the inner curvature did not (Fig 2A–C). At the marginal zone, the region in between the sidewall and the inner curvature, endothelial orientation, and Golgi axis were also polarized even though cell shapes were roundish (Fig 2A–C). In Pard3iΔEC mice, Golgi orientation was significantly compromised only in ECs at the marginal zone, whereas cell elongation and orientation were not affected (Fig 2A–C). These observations further suggest that PAR-3 controls endothelial polarity toward the flow axis in response to moderate shear stress. Figure 2. Loss of endothelial PAR-3 disrupts regional axial cell polarity and increases pro-inflammatory response of ECs En face confocal microscopy of the aortic arch from the inner curvature to sidewall, stained with anti-VE-cadherin (gray), GOLPH4 (red), NF-κB p65 subunit (green), and ERG1/2/3 (blue) from P56 male mice. Axis of the aorta is indicated on right side of images. Higher magnification images from (A). Lower panels show p65 signal (gray). Regional quantification of the cell elongation, cell orientation, Golgi orientation (Polarity Index), and nuclear p65 intensity of ECs. En face confocal microscopy of the inner curvature of the aortic arch and descending aorta from P56 control and Pard3iΔEC mice. VCAM-1 is green, and EC nuclei (ERG1/2/3) are red. Quantification of the relative intensity of VCAM-1 signal in the ECs of inner curvature of the aorta and descending aorta from control and Pard3iΔEC mice. Data information: In (C and E), data are presented as mean ± SEM (n = 4 mice). ns: not significant; P ≥ 0.05; differences *P < 0.05, **P < 0.01, analyzed with two-way ANOVA with Tukey's multiple comparison post hoc analysis (C) or Student's t-test (E). Scale bars: 50 μm (A), 20 μm (B and higher magnification images in D), and 100 μm (lower magnification images in D). Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Analysis of ECs in the aorta To examine the effect of loss of PAR-3, the highlighted region of the aorta within the red dotted lines was dissected and analyzed. To quantify endothelial Golgi orientation toward flow, the angle between the axial polarity vector and the proximal–distal vector (−1 to 1) was analyzed. White arrows indicate the axial polarity vectors from each EC. Left panel shows the VE-cadherin signal of aorta ECs. Right panel shows the ellipse fitted to each of the EC, generated by ImageJ software. Red arrows indicate the vectors of the major axis of each ellipse. Distribution of the sin(θ) of the angle between the axial polarity vector and the proximal–distal vector was analyzed (0 to 1). Data information: Scale bars: 20 μm (B, C). Download figure Download PowerPoint The inner curvature of the aorta is an area prone to atherosclerosis. Disrupted endothelial polarity toward the flow axis is considered to be pro-inflammatory, which increases the susceptibility for atherosclerotic plaque formation 25-27. To clarify the relationship between endothelial polarity toward the flow axis and endothelial inflammatory responses, we examined the NF-κB complex nuclear localization, a key regulator of inflammation. p65, a major component of the NF-κB complex, immunoreactivity was gradually increased in the ECs from the sidewall to the inner curvature in both control and Pard3iΔEC mice. Suggestive of a molecular connection, p65 staining intensity inversely related to an increased endothelial polarization (Fig 2B and C). However, significantly nuclear-localized p65 protein was found at the marginal and the inner curvature in Pard3iΔEC aortas but control mice showed diffuse localization (Fig 2B and C). On the other hand, Golgi orientation at the inner curvature was not affected in mutants (Fig 2B and C). Active NF-κB induces expression of vascular cell adhesion molecule-1 (VCAM-1), controlling the adhesion of leukocytes to the endothelium 28. In accordance, Pard3iΔEC mice showed a dramatic increase in VCAM-1 protein expression in the inner curvature of the aorta compared to control mice (Fig 2D and E). Interestingly, ECs of the descending aorta in Pard3iΔEC mice did not show VCAM-1 upregulation, where ECs are subjected to high shear stress (Fig 2D and E), confirming the specific requirement of PAR-3 signaling in low-to-moderate WSS regions. These results were further confirmed with primary cultured aortic ECs. When ECs from control mice were subjected to flow, the VCAM-1 mRNA level was downregulated. However, this was compromised in ECs isolated from PAR-3 KO mice (Appendix Fig S2). Taken together, these results suggest that PAR-3 simultaneously negatively regulates NF-κB activation and positively controls Golgi orientation in response to low-to-moderate blood flow. Atherosclerosis formation was increased in PAR-3 EC-specific inducible KO mice As disrupted endothelial polarity toward the flow axis and inflammation is linked to atherosclerosis, we hypothesized that Pard3iΔEC mice would be more susceptible to the development of atherosclerotic lesions. Thus, we next bred Pard3iΔEC mice with apolipoprotein (Apo) E KO mice, a well-established model to study atherosclerosis. To induce PAR-3 gene knockout, tamoxifen was injected daily from P42 to P46, then control and Pard3iΔEC mice were placed on high-fat diet. To analyze the effect of PAR-3 KO on the onset of atherosclerotic plaque formation, mice were sacrificed after 10 weeks of high-fat diet feeding. Then, aortas were collected and stained with Oil Red O to highlight lipid accumulation. Control ApoE−/−mutant animals showed Oil Red O staining at the inner curvature and branching points of aortic arches but not in the descending aorta (Fig 3A and B). However, under these conditions, Pard3iΔEC/ApoE−/− mutants showed a significant expansion of the region stained with Oil Red O (Fig 3A and B), while neither serum cholesterol levels nor body weight was affected (Fig 3C and D). To further confirm the role of PAR-3 in atherosclerosis formation, en face staining of the aortic arch was performed with an antibody against MOMA-2, a marker of monocytes/macrophages. MOMA-2-positive area was increased in Pard3iΔEC/ApoE−/− mice in the neointima of aortic arch when compared to control mice, indicating an increase in macrophage infiltration in endothelial-specific Pard3 loss-of-function mice (Fig 3E and F). Thus, PAR-3 inhibits atherosclerosis onset by blocking endothelial inflammation. Figure 3. Loss of endothelial PAR-3 accelerates regional atherosclerosis development Representative aorta of mice fed with high-fat diet for 10 weeks (18-week-old male mice) stained en face with Oil Red O. Smaller panels show higher magnification images from the aortic arch (1) and descending aorta (2). Quantification of Oil Red O-positive regions in aortic arch (upper panel) and descending aorta (lower panel). Quantification of serum cholesterol level of control (ApoE−/−) and ApoE−/−; Pard3iΔEC animals after 10 weeks of high-fat diet feeding. Body weights of control (ApoE−/−) and ApoE−/−; Pard3iΔEC animals after 10 weeks of high-fat diet feeding. Representative aorta of mice fed with high-fat diet for 10 weeks (18-week-old male mice) stained en face with monocyte/macrophage marker (MOMA-2, green) and EC marker (EGR, red). White arrowheads indicate MOMA-2-positive areas. Smaller panels (a, b, c, d) show higher magnification images from the aortic arch. Quantification of MOMA-2-positive areas in the aortic arch. Data information: In (B–D and F), data are presented as mean ± SEM. (B–D): n = 5 mice, (F): n = 3 mice. ns: not significant; P ≥ 0.05; differences *P < 0.05, analyzed with Student's t-test (B, C, F) or two-way ANOVA with Bonferroni multiple comparison post hoc test (D). Scale bars: 1 mm (A and E, larger panels), 500 μm (A and E, smaller panels), and 50 μm (E, bottom panels). Download figure Download PowerPoint The antagonism between the PAR-3/aPKC complex and the aPKC/GSK3β complex regulates GSK3β activation To gain mechanistic insight into the role of PAR-3 in endothelial polarity toward the flow axis in response to shear stress, we established an in vitro culture system. We confirmed efficient knocked down (KD) of PAR-3 in HUVECs with two different siRNAs (siPAR-3#1 and #2) (Appendix Fig S3A), and these siRNAs were used to examine the function of PAR-3 in flow-mediated polarity establishment. Confluent HUVECs were seeded in flow chambers coated with fibronectin and exposed to a range of shear stress. Consistent with the in vivo observations, Golgi polarization was compromised in PAR-3 KD cells in the presence of low-to-moderate flow but not when exposed to high flow (Fig EV3A–C). Moreover, ECs isolated from Pard3iΔEC and control mice aorta were subjected to the same range of shear stress. We confirmed the flow-rate-dependent compromised Golgi orientation in Pard3iΔEC aortic ECs (Appendix Fig S3B). However, the amount of ECs isolated from mice was limited. Thus, we decided to use HUVEC for further analysis. Click here to expand this figure. Figure EV3. PAR-3 regulates EC Golgi reorientation toward flow in vitro A. Representative images of flow chamber-cultured ECs transfected with control (Scrambled) or PARD3-specific siRNA (siPAR-3#1) and exposed to the indicated value of shear stress for 60 min. The cells were stained for nuclei (DAPI, blue) and Golgi apparatus (GOLPH4, red). Black arrows indicate corresponding axial polarity vectors. Flow direction is indicated on the right. Scale bar, 35 μm. B, C. Axial polarity of ECs treated with control (Scrambled) or PARD3-specific siRNAs (siPAR-3#1, B; or siPAR-3#2, C) in response to 12, 18, and 30 dyn/cm2 laminar flow for the indicated time. C. Western blotting of EC lysates treated with 18 dyn/cm2 laminar flow for indicated times, with 1 μM of 6BIO (+) or control (−) containing growth medium. Upper panels show the blot of acetylated α-tubulin (Ac α-tubulin), and lower panels show total tubulin. Quantitation of relative intensity of Ac α-tubulin is shown. Data information: In (B and C), data are means ± SEM (n = 3 independent experiments and n = 100 cells for each experiment). In (D), data are means ± SEM (n = 3 experiments). Statistical significance (*P < 0.05; **P < 0.01) was evaluated with two-way ANOVA and Bonferroni multiple comparisons post hoc analysis. Download figure Download PowerPoint GSK3β is a ubiquitously expressed and constitutively active protein kinase, which was implicated in cytoskeletal
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