Matrix Mechanotransduction Mediated by Thrombospondin-1/Integrin/YAP in the Vascular Remodeling

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Matrix mechanotransduction mediated by thrombospondin-1/integrin/YAP in the vascular remodeling

Yoshito Yamashiroa,1, Bui Quoc Thangb, Karina Ramireza,c, Seung Jae Shina,d, Tomohiro Kohatae, Shigeaki Ohataf, Tram Anh Vu Nguyena,c, Sumio Ohtsukie, Kazuaki Nagayamaf, and Hiromi Yanagisawaa,g,1

aLife Science Center for Survival Dynamics, Tsukuba Advanced Research Alliance, University of Tsukuba, Ibaraki 305-8577, Japan; bDepartment of Cardiovascular Surgery, University of Tsukuba, Ibaraki 305-8575, Japan; cPh.D. Program in Human Biology, School of Integrative and Global Majors, University of Tsukuba, Ibaraki 305-8577, Japan; dGraduate School of Life and Environmental Sciences, University of Tsukuba, Ibaraki 305-8577, Japan; eFaculty of Life Sciences, Kumamoto University, Kumamoto 862-0973, Japan; fGraduate School of Mechanical Systems Engineering, Ibaraki University, Ibaraki 316-8511, Japan; and gDivision of Biomedical Science, Faculty of Medicine, University of Tsukuba, Ibaraki 305-8577, Japan

Edited by Kun-Liang Guan, University of California San Diego, La Jolla, CA, and accepted by Editorial Board Member Christine E. Seidman March 15, 2020 (received for review November 9, 2019) The extracellular matrix (ECM) initiates mechanical cues that highly context dependent, and extracellular regulators are not activate intracellular signaling through matrix–cell interactions. fully understood (6, 9). In blood vessels, additional mechanical cues derived from the pul- Thrombospondin-1 (Thbs1) is a homotrimeric glycoprotein satile blood flow and pressure play a pivotal role in homeostasis with a complex multidomain structure capable of interacting with and disease development. Currently, the nature of the cues from a variety of receptors such as integrins, cluster of differentiation the ECM and their interaction with the mechanical microenviron- (CD) 36, and CD47 (10). Thbs1 is highly expressed during de- ment in large blood vessels to maintain the integrity of the vessel velopment and reactivated in response to injury (11), exerting wall are not fully understood. Here, we identified the matricellular domain-specific and cell type–specific effects on cellular functions. protein thrombospondin-1 (Thbs1) as an extracellular mediator of We recently showed that Thbs1 is induced by mechanical stretch matrix mechanotransduction that acts via integrin αvβ1 to estab- and is a driver for thoracic aortic aneurysm in mice (12, 13). lish focal adhesions and promotes nuclear shuttling of Yes- In the current study, we identified Thbs1 as an extracellular associated protein (YAP) in response to high strain of cyclic stretch. regulator of YAP, which is induced by mechanical stress and acts Thbs1-mediated YAP activation depends on the small GTPase Rap2 through FAs independent of actin remodeling. Mechanistically, and Hippo pathway and is not influenced by alteration of actin Thbs1 β fibers. Deletion of in mice inhibited Thbs1/integrin 1/YAP Significance signaling, leading to maladaptive remodeling of the aorta in response to pressure overload and inhibition of neointima formation upon carotid artery ligation, exerting context-dependent effects We propose a mechanism of matrix mechanotransduction ini- on the vessel wall. We thus propose a mechanism of matrix tiated by thrombospondin-1 (Thbs1), connecting mechanical mechanotransduction centered on Thbs1, connecting mechanical stimuli to Yes-associated protein (YAP) signaling during vas- stimuli to YAP signaling during vascular remodeling in vivo. cular remodeling. Specifically, Thbs1 is secreted from smooth muscle cells in response to high strain of cyclic stretch and binds to integrin αvβ1 to establish mature focal adhesions, extracellular matrix (ECM) | mechanotransduction | thrombospondin-1 | thereby promoting the nuclear shuttling of YAP in a small YAP | vessel remodeling GTPase Rap2- and Hippo signaling–dependent manner. We further demonstrated the biological significance of the Thbs1/ he extracellular matrix (ECM) is fundamental to cellular and integrin β1/YAP pathway using two vascular injury models. Ttissue structural integrity and provides mechanical cues to Deletion of Thbs1 in mice caused maladaptive remodeling of initiate diverse biological functions (1). The quality and quantity the aorta in response to pressure overload, whereas it inhibi- of the ECM determine tissue stiffness and control gene expres- ted neointima formation upon carotid artery ligation, exerting sion, cell fate, and cell cycle progression in various cell types (2, context-dependent effects on the blood vessel wall. 3). ECM–cell interactions are mediated by focal adhesions (FAs), the main hub for mechanotransduction, connecting ECM, Author contributions: Y.Y. and H.Y. designed research; Y.Y., B.Q.T., K.R., S.J.S., T.K., S. Ohata, T.A.V.N., S. Ohtsuki, and K.N. performed research; Y.Y., B.Q.T., K.R., S.J.S., integrins, and cytoskeleton (1, 4). In the blood vessels, an addi- T.K., S. Ohata, T.A.V.N., S. Ohtsuki, K.N., and H.Y. analyzed data; and Y.Y. and H.Y. tional layer of mechanical cues derived from the pulsatile blood wrote the paper. flow and pressure play a pivotal role in homeostasis and disease The authors declare no competing interest. development. However, how cells sense and integrate different This article is a PNAS Direct Submission. K.-L.G. is a guest editor invited by the mechanical cues to maintain the blood vessel wall is largely Editorial Board. unknown. Published under the PNAS license. Hippo effector Yes-associated protein (YAP) and transcrip- Data deposition: The secretome data have been deposited in the Japan Proteome Stan- dard Repository/Database (jPOST), http://jpostdb.org (accession no. JPST000600/ tional coactivator with PDZ-binding motif (TAZ) serve as an PXD013915) and RNA-seq data have been deposited in Gene Expression Omnibus (GEO) on–off mechanosensing switch for ECM stiffness (5). YAP ac- database, https://www.ncbi.nlm.nih.gov/geo/ (accession no. GSE131750). All data support- tivity is regulated by a canonical Hippo pathway; MST1/2 ing the findings of this study are available from the corresponding authors upon reasonable request. (mammalian sterile 20-like 1/2 kinases) and their downstream See online for related content such as Commentaries. kinases LATS1/2 (large tumor suppressor 1/2) control YAP in- 1To whom correspondence may be addressed. Email: [email protected] or activation via phosphorylation and retention in the cytoplasm by [email protected]. binding to the 14-3-3 protein (6). YAP activity is also regulated This article contains supporting information online at https://www.pnas.org/lookup/suppl/ by Wnt/β-catenin and G protein-coupled receptor (GPCR) sig- doi:10.1073/pnas.1919702117/-/DCSupplemental. naling pathways (7, 8). However, regulation of YAP activity is First published April 22, 2020.

9896–9905 | PNAS | May 5, 2020 | vol. 117 | no. 18 www.pnas.org/cgi/doi/10.1073/pnas.1919702117 Downloaded by guest on September 24, 2021 Thbs1 binds integrin αvβ1 and strengthens FAs, aiding in conducted for biological interaction network among proteins in translocation of YAP to the nucleus. Deletion of Thbs1 in vivo ECM–cell adhesions and blood vessel development (Fig. 1D), and resulted in altered vascular remodeling in response to pressure Thbs1 showed multiple interactions in both categories. In addition, overload and flow cessation. Taken together, Thbs1 mediates since we reported that Thbs1 is a mechanosensitive factor involved dynamic interactions between mechanical stress and the YAP- in aortic aneurysms (12, 13), we focused on Thbs1 for further mediated transcriptional cascade in the blood vessel wall. analysis. We confirmed the increased expression and secretion of Thbs1 after cyclic stretch (Fig. 1E), suggesting that Thbs1 may play an Results important role in response to dynamic vessel microenvironments. Cyclic Stretch–Induced Secretion of ECM and Cell Adhesion Molecules Involved in Blood Vessel Development. Vascular ECM is synthe- Secreted Thrombospondin-1 Binds to Integrin αvβ1 under Cyclic sized by smooth muscle cells (SMCs) from midgestation to the Stretch. In response to cyclic stretch, SMCs showed up- early postnatal period and determines the material and me- regulation of the early growth response 1 (Egr1) transcription chanical properties of the blood vessels (14). Owing to the long factor, phosphorylated (p) focal adhesion kinase (FAK), extra- half-life of ECM, blood vessels are generally thought to stand cellular signal-regulated kinase (ERK), and stress-response lifelong mechanical stress without regeneration, particularly in mitogen-activated protein kinase (MAP) p38 as previously large arteries. However, it is not completely known whether a reported (SI Appendix, Fig. S3A) (13, 15, 16). Cyclic stretch al- local microremodeling system exists for the maintenance of the tered subcellular localization of Thbs1 from the perinuclear re- vessel wall. To search for a potential remodeling factor(s), we gion to the tip of the cell on the long axis (Fig. 2A). We examined first performed cyclic stretch experiments with high strain (1 Hz; Thbs1 localization and reorientation of SMCs as a function of 20% strain), mimicking the pathological condition of the aortic time (SI Appendix, Fig. S3 B and C). SMCs were randomly ori- wall. Rat SMCs were subjected to cyclic stretch for 20 h with or entated after 30 min of cyclic stretch, but most cells were aligned without brefeldin A (BFA), a protein transporter inhibitor, as a in a perpendicular position to the stretch direction at 3 h and negative control for mass spectrometry analysis. Conditioned completely reorientated by 6 h. Thbs1 localization clearly media (CM) were analyzed using quantitative mass spectrome- changed from 3 h after cyclic stretch, which coincided with the try, and 87 secreted proteins with more than twofold differences reorientation of actin fibers and elongation of cells (SI Appendix, in response to cyclic stretch were identified (Fig. 1 A and B and Fig. S3D). Since FAs recruit actin stress fibers (17), we costained SI Appendix, Figs. S1 and S2). Gene ontology (GO) enrichment SMCs with Thbs1 and p-paxillin, a FA molecule, under cyclic

analysis (http://geneontology.org/) revealed that proteins in- stretch. Thbs1 colocalized with p-paxillin independent of cell CELL BIOLOGY volved in ECM organization and structural constituents were density after cyclic stretch (Fig. 2 B and C). Since Thbs1 has well- highly enriched (Fig. 1C). Ingenuity pathway analysis (IPA) was characterized cell surface receptors, including integrins β1, β3,

Fig. 1. Comprehensive secretome analysis of smooth muscle cells under cyclic stretch. (A) Volcano plots of protein secretion between the two conditions: 1) stretch vs. static, 2) stretch vs. stretch+BFA, and 3) stretch+BFA vs. static. BFA (1 μM) was used to inhibit secretion. The P values in the unpaired Student’s t test were calculated and plotted against the fold change for all identified proteins. Each dot represents mean values (n = 3). (B) Heat map of secreted proteins

induced by cyclic stretch. A list of all of the proteins is provided in SI Appendix, Fig. S2.(C) Functional enrichment analysis of 87 proteins, the negative log10 of the P value. The top enriched GO terms associated with molecular function (orange), biological process (green), and reactome pathway analysis (blue) are shown. (D) Biological network using IPA. Diagrams show the direct (solid lines) and indirect (dashed lines) interactions among proteins reported in blood vessel development (Top) and ECM and cell adhesion (Bottom). (E) Western blot for Thbs1 from CM of rat vascular SMCs in static and stretch (1 Hz, 20% strain, 20 h) with or without BFA (n = 3). The entire image of silver staining is provided in SI Appendix, Fig. S1A.

Yamashiro et al. PNAS | May 5, 2020 | vol. 117 | no. 18 | 9897 Downloaded by guest on September 24, 2021 Fig. 2. Thbs1 localizes to FAs and binds integrin αvβ1 under cyclic stretch. (A) Cyclic stretch alters localization of Thbs1 (n = 5). Representative immunostaining with phalloidin (red), Thbs1 (green), and DAPI (blue). (B) Representative image of immunostaining showing Thbs1 colocalization with p-paxillin under cyclic stretch (n = 5). Immunostained with p-paxillin (red), Thbs1 (green), and DAPI (blue). (C) Immunostaining showing Thbs1 localization at FAs in confluent conditions (n = 3). Immunostained with p-paxillin (red), Thbs1 (green), phalloidin (gray), and DAPI (blue). (D) Cartoon shows various binding domains of Thbs1 and its receptors. (E) Representative immunostaining of Thbs1 showing colocalization with integrins αv and β1 (white arrows) but not with integrin β3, CD47, or CD36 under cyclic stretch (n = 5). Immunostained for indicated antibodies (red), Thbs1 (green), and DAPI (blue). (F) Quantification of colocalization of Thbs1 with molecules shown in E using Imaris colocalization software. Bars are mean ± SEM. ***P < 0.001, two-way ANOVA. NS: not significant. (G) Immuno- precipitation (IP) with anti-Thbs1 or control immunoglobulin G (IgG) followed by Western blotting for integrins αv and β1(n = 2). Coomassie Brilliant Blue (CBB) shows the heavy chain of IgG in IP lysates. IB: immunoblot. (H) PLA shows the clusters of Thbs1/integrin αv or Thbs1/integrin β1 (red dots; white arrowheads). Bottom shows highly magnified images of the white dashed box in Top. DAPI (blue) and phalloidin (green) are shown. In all experiments, rat vascular SMCs were subjected to cyclic stretch (1.0 Hz, 20% strain for 20 h in A, C, and E or 8 h in G and H). In A, C, E, and H, 80 to 100 cells were evaluated in each immunostaining. Two-way arrows indicate stretch directions. (Scale bars, 50 μm.)

and αv; CD47; and CD36 (Fig. 2D) (10), we examined which of altered in Thbs1KO cells (SI Appendix, Fig. S5 A and B). No these receptors bound to Thbs1 in response to cyclic stretch. obvious phenotypic changes were observed in Thbs1KO cells (SI Immunostaining showed that Thbs1 colocalized with integrins αv Appendix, Fig. S5 C and D). Thbs1KO cells were subjected to and β1 but not with integrin β3, CD47, or CD36 (Fig. 2 E and F). cyclic stretch (1 Hz; 20% strain) for 8 h and were compared with Immunoprecipitation of cell lysate obtained from the static or control (CTRL) cells with or without 1 μM of BFA. CTRL cells cyclic stretch condition with anti-Thbs1 antibody followed by responded to cyclic stretch and reoriented perpendicular to the Western blotting confirmed direct binding between Thbs1 and direction of stretch, whereas BFA-treated CTRL cells and integrins αv and β1 in the cyclic stretch condition (Fig. 2G). Thbs1KO cells neither elongated nor aligned to the perpendic- Consistently, proximity ligation assay (PLA) showed that integ- ular position (Fig. 3 A–C). Stretch-induced phosphorylation of rins αv and β1 interacted with Thbs1 only in the cyclic stretch paxillin and up-regulation of integrins αv and β1 in CTRL cells condition (Fig. 2H). These data demonstrated that Thbs1 was were not observed in Thbs1KO cells (Fig. 3D). secreted in response to cyclic stretch and bound to integrin αvβ1. Mature FAs are localized at the termini of actin stress fibers and form a FA–actin complex (18, 19). The FA–actin complex is Thrombospondin-1 Regulates Maturation of the FA–Actin Complex stabilized by vinculin deposition and integrin–talin binding, and Controls Cell Stiffness. We next examined the effect of de- which facilitates PI3K-mediated phosphatidylinositol 3,4,5-tri- – letion of Thbs1 on cyclic stretch induced formation of FA and phosphate (PIP3) production (20). Vinculin is present at FAs in a reorientation of SMCs. Thbs1 knockout rat vascular SMCs force-dependent manner and increases cell stiffness (21). To (Thbs1KO) were generated using CRISPR-Cas9 genome editing evaluate the maturation of FA–actin complex, we compared the (SI Appendix, Fig. S4). We evaluated possible off-target effects vinculin expression and monitored PIP3 levels using a GFP- and confirmed that the expression of Cpreb3 or Tet3 was not tagged PIP3 reporter (GFP-Grp1-PH) in static and stretch

9898 | www.pnas.org/cgi/doi/10.1073/pnas.1919702117 Yamashiro et al. Downloaded by guest on September 24, 2021 CELL BIOLOGY

Fig. 3. Deletion of Thbs1 affects maturation of focal adhesion and cell stiffness. (A) Wild-type cells with or without 1 μM of BFA or Thbs1KO cells were subjected to cyclic stretch (1.0 Hz, 20% strain, 8 h). Two-way arrows indicate the stretch direction. (Scale bars, 50 μm.) Phalloidin (red) is also shown. (B and C) The orientation of each cell was analyzed by measuring the orientation angle (θ) of the long axis (yellow bars in A) of the ellipse relative to the stretch axis (n = 3). (D) Thbs1KO cells show reduced FA formation. Immunostaining with p-paxillin (red), integrin αv (red), integrin β1 (red), Thbs1 (green), and DAPI (blue) (n = 3). Phalloidin (red) is also shown. White arrows indicate colocalization with Thbs1. Two-way arrows indicate stretch direction. (Scale bars, 50 μm.) (E) Representative immunostaining of CTRL or Thbs1KO cells with vinculin (red) in static or stretch condition. GFP-GRP1-PH (green) and DAPI (blue) are also shown. White arrows show vinculin deposition onto the tip of actin fibers (n = 3). Two-way arrows indicate stretch direction. (Scale bars, 50 μm.) (F) Immunostaining of CTRL or Thbs1KO cells with vinculin (green) and phalloidin (red). Rat vascular SMCs were subjected to cyclic stretch (1.0Hz, 20% strain for 8 h). Two-way arrows indicate stretch direction. (Scale bars, 25 μm.) Arrow heads (purple) show vinculin deposition onto the tip of actin fibers. (G) Young’s modulus of actin fibers in CTRL (n = 129) or Thbs1KO (n = 86) cells measured using atomic force microscopy. Representative topographic images and stiffness maps are shown. (H) Cell height and Young’s modulus are shown. Bars are means ± SEM. ***P < 0.001, unpaired t test. NS: not significant.

conditions in CTRL and Thbs1KO cells. PIP3 was enriched on cells in static and cyclic stretch conditions (SI Appendix, Fig. S6). stress fibers in stretched CTRL cells (Fig. 3E), and vinculin was In the static condition, RNA-seq revealed 38 and 47 genes that deposited onto the tip of actin stress fibers which anchored FAs were differentially regulated in CTRL and Thbs1KO cells, re- in stretched CTRL cells, whereas stretched Thbs1KO cells did spectively. Conversely, 126 genes in CTRL cells and 163 genes in not show recruitment of vinculin onto the FA–actin complex Thbs1KO cells were differentially expressed under cyclic stretch. (Fig. 3F). To confirm this observation, we assessed mechanical Among these genes, GO enrichment analysis revealed the properties of Thbs1KO cells using atomic force microscopy abundant expressions of genes involved in response to stimulus (Fig. 3G). Since cell tension positively correlates with adhesion (SI Appendix, Fig. S7A). We therefore used known gene groups size and its stiffness (22), we analyzed morphological changes involved in mechanical stress–induced responses, including YAP and stiffness on the stress fibers. Stretched Thbs1KO cells (23), Notch (24, 25), hypoxia-inducible factor-1-alpha (HIF1A) showed a significant decrease in stiffness (Young’s modulus) on (26), endoplasmic reticulum (ER) stress response (27), and ox- the stress fibers compared with stretched CTRL cells, although idative stress response (28, 29), as keywords to further charac- the height of stretched Thbs1KO cells was comparable to that of terize 289 genes via IPA combined with RNA-seq analysis (SI stretched CTRL cells (Fig. 3H). Taken together, our findings Appendix, Fig. S7B). With the exception of gene encoding mes- suggest that Thbs1 is required for maturation of the FA–actin othelin (Msln), the expressions of YAP target genes were complex and maintenance of cellular tension, thereby correctly markedly down-regulated in Thbs1KO cells, suggesting that orienting actin fibers in response to cyclic stretch. Thbs1 strongly controls YAP target genes in the stretch condition (SI Appendix, Fig. S6) and may be a critical component of Thrombospondin-1 Regulates Nuclear Shuttling of YAP via Integrin the YAP signaling pathway. αvβ1 in a Rap2-Dependent Manner. To understand the molecular Next, to elucidate the role of Thbs1 in the mechanical regu- basis of Thbs1 contribution in mechanotransduction, we per- lation of YAP, we examined YAP localization with or without formed RNA sequencing (RNA-seq) using CTRL and Thbs1KO Thbs1 under cyclic stretch. Consistent with a previous report

Yamashiro et al. PNAS | May 5, 2020 | vol. 117 | no. 18 | 9899 Downloaded by guest on September 24, 2021 (23), cyclic stretch induced nuclear shuttling of YAP in CTRL GTPase Rap2 was recently identified as a negative regulator of cells (Fig. 4A). Although YAP was retained in the cytoplasm the Hippo pathway and as an inhibitor of YAP activation in- after cyclic stretch in Thbs1KO cells (Fig. 4A), exogenously duced by ECM stiffness (30). To determine whether stretch- and added recombinant human THBS1 (rhTHBS1) promoted nu- Thbs1-induced nuclear shuttling of YAP is regulated by Rap2 clear shuttling of YAP after cyclic stretch (Fig. 4B and SI Ap- activity, we measured active Rap2 (Rap2-GTP) levels in CTRL pendix, Fig. S8). Since nuclear shuttling of YAP is regulated by a or Thbs1KO cells in static and stretch conditions using the kinase cascade of the Hippo pathway, we examined p-YAP and Ral-GDS-RBD pull-down assays. As shown in Fig. 4D, active p-LATS levels using Western blotting. In CTRL cells, p-YAP Rap2 decreased in CTRL cells after stretch but not in Thbs1KO and p-LATS were decreased in stretch condition, whereas cells. To further determine the relationship between Rap2 ac- p-YAP and p-LATS persisted in Thbs1KO cells (Fig. 4C). These tivity and nuclear shuttling of YAP, CTRL or Thbs1KO cells observations indicate that Thbs1 controls nuclear shuttling of were transfected with myc-Rap2A (wild type [WT]), myc-Rap2A YAP in a Hippo pathway–dependent manner. The Ras-related (G12V) constitutively active mutant, or myc-Rap2A (S17N)

Fig. 4. Thbs1 mediates nuclear shuttling of YAP via integrin αvβ1 in a Rap2-dependent manner. (A) Representative immunostaining of CTRL or Thbs1KO cells in the static or stretch condition with YAP (green). Phalloidin (red) and DAPI (blue) are also shown. Two-way arrows indicate stretch direction. (Scale bars, 50 μm.) Quantification of YAP localization is shown on the Right. In each experiment 150 to 200 cells were evaluated (n = 3). YAP localization in the nucleus (green) or cytoplasm (red) is shown. (B) Thbs1KO cells treated with (+) or without (−) recombinant human THBS1 (rhTHBS1; 1 μg/mL) following 24 h of serum starvation, then subjected to uniaxial cyclic stretch (20% strain; 1 Hz) for 8 h. Representative immunostaining with YAP (green) and DAPI (blue). (Scale bars, 50 μm.) Quantification of YAP localization is shown on the Right. In each experiment 80 to 120 cells were evaluated (n = 3). (C) Western blot shows Thbs1, p-YAP, and p-LATS levels in CTRL and Thbs1KO cells in the static or stretch condition (n = 3). Quantification graphs are shown on the Right. Bars are means ± SEM. **P < 0.01, ***P < 0.001, one-way ANOVA. (D) Pull down of GTP-bound Rap2 from CTRL and Thbs1KO cells in static and stretch conditions using Ral-GDS-RBD agarose beads (n = 2). (E) Overexpression of Myc-Rap2A(WT), constitutively active Myc-Rap2A(G12V), or dominant-negative Myc-Rap2A(S17N) in CTRL and Thbs1KO cells in the static or stretch condition (n = 3). Representative immunostaining with Myc (red), YAP (green), and DAPI (blue). Two-way arrows indicate stretch direction. (Scale bars, 50 μm.) Quantification of YAP localization is shown on the Right. In each experiment 50 to 100 cells were evaluated (n = 3). YAP localization in the nucleus (green) or cytoplasm (red) is shown. (F) Scramble or targeted siRNA-treated rat vascular SMCs were subjected to cyclic stretch (n = 3). Representative immunostaining with YAP (green), phalloidin (red), and DAPI (blue). Two-way arrows indicate stretch direction. (Scale bars, 50 μm.) Quantification of YAP localization is shown on the Right. In each experiment 150 to 200 cells were evaluated (n = 3). qPCR data for confirming the knockdown of Itgαv, Itgβ1, and Itgβ3 are provided in SI Appendix, Fig. S10.

9900 | www.pnas.org/cgi/doi/10.1073/pnas.1919702117 Yamashiro et al. Downloaded by guest on September 24, 2021 dominant negative mutant. Forty-eight hours after transfection, remodeling using Thbs1 null (Thbs1KO) mice. Whereas 100% of cells were subjected to cyclic stretch (1.0 Hz, 20% strain) for 8 h WT mice survived at 5 wk after TAC (n = 13), 31.9% (7/22) of and immunostained with antibodies directed against Myc and Thbs1KO mice died, with three showing aortic rupture (Fig. 5B YAP and stained with DAPI to identify nuclei. Rap2 (WT) and SI Appendix, Fig. S13A). The heart weight (HW) and body overexpression showed no effects on the localization of YAP in weight (BW) ratio (HW/BW) was significantly higher in TAC- CTRL or Thbs1KO cells (Fig. 4E). Rap2A activation (G12V) operated Thbs1KO mice than in WT mice, in accordance with a cancelled nuclear shuttling of YAP under cyclic stretch in CTRL previous report (35) (Fig. 5C). In addition, TAC-operated cells. In contrast, Rap2A inactivation (S17N) forced YAP nu- Thbs1KO aortas had enlarged aortic lumen (Fig. 5D), and dis- clear shuttling with or without Thbs1, overriding the regulation sected aortas showed severely disrupted elastic fibers (Fig. 5E). of Thbs1. These data suggested that Thbs1 controls nuclear Morphometric analysis showed a significant increase in the in- shuttling of YAP in a Rap2A-dependent manner. ternal elastic lamella (IEL) perimeter, outer perimeter, and total Tension on actin fibers is also important for regulation of vessel area in TAC-operated Thbs1KO aortas compared to TAC- YAP, dependent on or independent of the formation of FAs operated WT aortas (Fig. 5F). Immunostaining showed strong (31–33). We assessed how actin dynamics and inhibition of FAK expression of YAP and its targets, connective tissue growth affects stretch-induced nuclear shuttling of YAP. CTRL cells factor (CTGF), Serpine1, CYR61, and Caveolin-3, in TAC- were subjected to cyclic stretch for 8 h with the actin polymeri- operated WT aortas, especially in the medial layers and in the zation inhibitor cytochalasin D (CytoD) or actin de- adventitia (Fig. 5 G and H and SI Appendix, Fig. S13B). How- polymerization inhibitor jasplakinolide (Jasp) or FAK inhibitor ever, the expression of YAP and CTGF was hardly observed in PF-00562271 (FAKi). The efficiency of the treatment was eval- the medial layers of TAC-operated Thbs1KO aorta, although the uated by measuring the orientation angle (θ) of the long axis of expression in the adventitia was evident in Thbs1KO aortas. In the ellipse relative to the cyclic stretch direction (SI Appendix, contrast, the expression of Serpine1, CYR61, and Caveolin-3 was Fig. S9 A and B). Although CytoD and Jasp showed no effects on unchanged in TAC-operated Thbs1KO aortas. These data sug- nuclear shuttling of YAP under cyclic stretch, YAP remained in gest the possibility that Thbs1-dependent YAP signaling in the cytoplasm in FAKi-treated cells (SI Appendix, Fig. S9 C and SMCs plays an important role in adaptive remodeling of the E). Furthermore, although overexpression of slingshot1 (Ssh1, aortic wall in response to pressure overload. Ssh1OE), a phosphatase of cofilin, induced depolymerization of Finally, we employed carotid artery ligation injury to address actin fibers through dephosphorylation of cofilin, it did not affect the contribution of Thbs1/integrin/YAP signaling in flow YAP localization under cyclic stretch (SI Appendix, Fig. S9 D and cessation–induced neointima formation (Fig. 6A). Thbs1 has E). These observations suggest that stretch-induced nuclear been shown to be required for neointima formation (36), and CELL BIOLOGY shuttling of YAP is dependent on FAs but not on actin poly- YAP mediates phenotypic modulation of SMCs during neo- merization. To confirm this, we performed small interfering intima formation (37). Time course analyses of the ligated ar- RNA (siRNA)–mediated knockdown of integrins αv(siItgαv), β1 teries revealed that Thbs1 was up-regulated in the inner layer at (siItgβ1), and β3(siItgβ3) along with scramble siRNA (SI Ap- 1 wk after ligation when YAP expression was not evident pendix, Fig. S10) followed by cyclic stretch. As expected, (Fig. 6B and SI Appendix, Fig. S14). At 2 wk after ligation, nu- knockdown of integrin αvorβ1 was sufficient to inhibit stretch- clear YAP peaked in the developing neointima (Fig. 6C) and induced nuclear shuttling of YAP (Fig. 4F). We next investigated coexpressed with Thbs1 (Fig. 6B and SI Appendix, Fig. S15). At 3 whether matrix proteins in general serve as an extracellular wk, Thbs1 and YAP expression decreased as the neointima be- regulator of YAP under cyclic stretch. Since fibronectin has been came stabilized (Fig. 6B and SI Appendix, Fig. S16). YAP targets shown to regulate the Hippo pathway through FAK-Src-PI3K CTGF, Serpine1, CYR61, and Caveolin-3 were highly expressed during cell spreading (34) and is a ligand for integrin αvβ1, we in the neointima (SI Appendix, Fig. S17). PLA showed the in- knocked down fibronectin in SMCs and subjected them to cyclic teraction between Thbs1 and integrin β1 in the neointima (left stretch to assess its impact on YAP localization. As SI Appendix, carotid artery [LCA] in Fig. 6D) but not in the contralateral Fig. S11 shows, fibronectin knockdown had no effect on nuclear artery (right carotid artery [RCA] in Fig. 6D). To confirm the shuttling of YAP in response to stretch. We also assessed relationship between Thbs1/integrin/YAP and neointima for- whether Thbs1-mediated nuclear shuttling of YAP is observed in mation, we injected the lentivirus (LV)-encoding Thbs1 siRNA endothelial cells. Using human umbilical vein endothelial cells (siThbs1) into WT mice through tail vein injection at 2 wk after (HUVECs), we found that cyclic stretch did not induce phos- ligation and evaluated neointima at 3 wk. GFP-LV injection phorylation of paxillin or localization of Thbs1 to FAs, even from the tail vein showed clear expression of GFP in the neo- though Thbs1 was secreted into CM (SI Appendix, Fig. S12 A and intima, endothelial cells of the right carotid artery, and the liver B). Actin fibers aligned in a perpendicular position, and contact (SI Appendix, Fig. S18), confirming the delivery of siRNA into inhibition was reduced by cyclic stretch, but YAP remained in the neointima. As we expected, the neointima was not observed the cytoplasm of HUVECs (SI Appendix, Fig. S12 C and D), in siThbs1 virus-treated animals, similar to Thbs1KO mice, and suggesting that regulation of YAP by Thbs1 under cyclic stretch YAP levels and nuclear localization were markedly decreased as condition is specific to vascular SMCs. Taken together, these Thbs1 expression decreased (Fig. 6 C and E). data indicate that stretch-induced, Thbs1-mediated nuclear shuttling of YAP is dependent on integrin αvβ1 and that effect is Discussion specific to SMCs. In this study, we identified Thbs1 as a critical molecule involved in mechanotransduction and extracellular regulation of YAP, Thrombospondin-1/Integrin/YAP Signaling Is Involved in Mechanical leading to dynamic remodeling of the blood vessels. We found Stress–Induced Vascular Pathology. An in vivo model was used to that Thbs1, once secreted in response to mechanical stress, binds evaluate whether Thbs1-mediated YAP regulation is involved in to integrin αvβ1 and aids in the maturation of the FA–actin the pathogenesis of vascular diseases. We previously reported complex, downregulating Rap2 activity, thereby mediating acti- that transverse aortic constriction (TAC) induced Thbs1 ex- vation of YAP. Loss of Thbs1 decreases cell stiffness in vitro, and pression (12). Concordantly, Thbs1 was induced in endothelial deletion or down-regulation of Thbs1 in mice alters cellular be- cells (ECs), SMCs, and adventitial cells after TAC was per- havior in response to mechanical stress in a context-dependent formed in WT mice and PLA showed the interaction between manner. Taken together, we propose that the Thbs1/integrin/ Thbs1 and integrin β1 in these cells (Fig. 5A). We next evaluated YAP signaling pathway plays a critical role in vascular the effect of loss of Thbs1/integrin β1 in TAC-induced aortic remodeling in vivo (Fig. 7).

Yamashiro et al. PNAS | May 5, 2020 | vol. 117 | no. 18 | 9901 Downloaded by guest on September 24, 2021 Fig. 5. Thbs1 deficiency in mice results in aortic dissection under pressure overload. (A) Immunostaining of Thbs1 (red) in sham or TAC-operated aortas in WT mice (Top). PLA showing the interaction between Thbs1 and integrin β1 (red dots in Bottom). Right shows highly magnified images of white dashed boxes in Bottom. Arrowheads (white) show PLA cluster. DAPI (blue) and autofluorescence of elastin (green) are also shown. n = 3. (Scale bars, 50 μm.) (B) Kaplan-Meier survival curve for WT (n = 13) and Thbs1KO (n = 22) mice after TAC. *P = 0.043, long-rank test. (C) HW/BW in sham or TAC-operated mice. Bars are mean ± SEM. *P < 0.05, ***P < 0.001, quantification by the Kruskal–Wallis test with Dunn multiple comparisons is shown. The number of animals is indicated in each bar. (D) Hematoxylin and eosin staining of the ascending aortas for histological comparison between sham and TAC-operated aortas. (Scale bars, 100 μm.) (E) Aortic dissection was observed in Thbs1KO ascending aorta proximal to the constriction. (Scale bars, 100 μm.) (F) Morphometric analysis showing the IEL perimeter, outer perimeter, total vessel area, and wall thickness. Bars are mean ± SEM. *P < 0.05, ***P < 0.001, one-way ANOVA. NS: not significant. The number of animals is indicated in each bar. (G) Immunostaining of TAC-operated aortas with YAP (red) in WT and Thbs1KO mice. On the Right, highly magnified images of the white dashed boxes in the adventitial (Adv.) and medial (Med.) layers in the Left are shown. DAPI (blue) and autofluorescence of elastin (green) are also shown. n = 3. (Scale bars, 50 μm.) (H) Representative images of immunohistochemistry for CTGF in TAC-operated aortas of WT and Thbs1KO mice (n = 3 per genotype). The outer elastic lamella as a border between the medial layer and adventitia is shown by the green dashed line. Arrowheads (red) indicate CTGF-positive cells in the medial layer. (Scale bars, 100 μm.)

9902 | www.pnas.org/cgi/doi/10.1073/pnas.1919702117 Yamashiro et al. Downloaded by guest on September 24, 2021 Fig. 6. Thbs1 regulates YAP expression during neointima formation upon carotid artery ligation. (A) Diagram of LCA ligation. (B) Immunostaining of cross sections of ligated arteries with YAP (red) and Thbs1 (white) at 1 wk (n = 5), 2 wk (n = 5), and 3 wk (n = 4) after ligation in WT mice. DAPI (blue) and autofluorescence of elastin (green) are also shown. Quantification of colocalization with YAP and DAPI using Imaris colocalization software is indicated in yellow (number of nuclear YAP/total number of nuclei). (Scale bars, 50 μm.) (C) Quantification of colocalization between YAP and DAPI in B and E using Imaris CELL BIOLOGY colocalization software. Bars are mean ± SEM. **P < 0.01, ***P < 0.001, one-way ANOVA. (D) PLA showing cluster of Thbs1/integrin β1 (red dots, yellow arrowheads) at 3 wk after ligation in RCA and LCA. DAPI (blue) and autofluorescence of elastin (green) are also shown. (Scale bars, 50 μm.) n = 3. (E) Cross sections of ligated arteries at 3 wk postinjury in WT (n = 4), siThbs1-treated WT mice (n = 4), and Thbs1KO (n = 5) mice. Immunostaining with YAP (red), Thbs1 (white). DAPI (blue), and autofluorescence of elastin (green). Note neointima formation in WT arteries. Yellow arrowheads show nuclear localization of YAP. (Scale bars, 50 μm.)

Mechanical Stress and Micro- and Macroremodeling of the Vessel Wall mechanism by which secreted Thbs1 is recruited to integrin αvβ1 by ECM. Vascular ECM, a major component of the vessel wall, is is unclear. However, we speculate that Thbs1 ligation to integrins subjected to repeated mechanical stress throughout life. Due to activates downstream molecules, leading to maturation of FAs its long half-life [for example, 50 to 70 y for elastic fibers of the and strengthening of actin fibers. Interestingly, a loss of Thbs1 human artery (38)], the vascular ECM is considered to be diffi- decreased cell stiffness and altered YAP target gene expression cult to regenerate once it is damaged by physical and/or bio- after cyclic stretch. We further showed that stretch down- chemical insults. Although ECM synthesis via mechanical stretch regulated Rap2 activity in CTRL cells, whereas Rap2 activity was previously described (39, 40), its biological significance, remained high in the absence of Thbs1, which forced YAP re- whether it contributes to microremodeling or dynamic remod- tention in the cytoplasm. Our observation agrees with the recent eling of the vessel wall, and how it is connected to the mecha- study by Meng et al. demonstrating that Rap2 negatively regu- notransduction cascade are not completely understood. Using lates nuclear shuttling of YAP in a Hippo pathway–dependent mass spectrometry and GO analysis, we found that high strain of manner. These authors proposed a cascade of signaling triggered cyclic stretch induced secretion of various ECM proteins in- by FAs → PLCγ1 (phospholipase C gamma 1) → PtdIns(4,5)P2 volved in cell adhesions, elastic fiber formation, and TGF-β (phosphatidylinositol 4, 5-bisphosphate) → PA (phosphatidic signaling. Using IPA, Thbs1 was identified as one of the key acid) → PDZGEF (RAPGEF) → Rap2 → MAP4K → LATS → molecules involved in these biological processes. Interestingly, YAP (30) and showed that increased matrix stiffness led to in- mechanical stress and secretary pathway are interconnected activation of Rap2 and nuclear shuttling of YAP. It was also since BFA treatment or deletion of Thbs1 led to the alteration of proposed that this signaling axis works in parallel with RhoA, the mechanoresponse in SMCs and disrupted alignment of cells which mediates cytoskeletal tension during cell spreading (5, 32). in response to cyclic stretch. Our observations indicate that Our data showed that stretch-induced YAP activation is Rap2 mechanical stress induces secretion of Thbs1, which initiates dependent and is not influenced by altered actin fibers. There- mechanotransduction and activates the integrin/YAP pathway, fore, it is possible that Thbs1 mediates nuclear shuttling of YAP thereby remodeling the vessel wall to accommodate the changes by inactivating Rap2 through altering the composition of FA or in mechanical microenvironment. Thbs1 and integrins are es- by controlling FA assembly (41). A previous study described that sential components of matrix mechanotransduction, serving as a mutant cell line lacking YAP showed decreased Thbs1 levels biomechanical ligand and sensors of the microenvironment. along with down-regulation of YAP target genes and FA molecules, indicating that Thbs1 may act as a feedforward driver for Thbs1 as an Extracellular Mediator of YAP Signaling Pathway. We YAP signaling (41). Interestingly, the proteolytic remodeling of showed that mechanical stretch–induced, secreted Thbs1 directly laminin-1 has been shown to modulate awakening of dormant bound to integrin αvβ1 and colocalized with p-paxillin, and fa- cancer cells through integrin/FAK/ERK/MLCK (myosin light- cilitated maturation of FA complex by inducing deposition of chain kinase)/YAP signaling (42), and fibroblast-derived peri- vinculin at the tip of actin fibers. Currently, the precise ostin (encoded by Postn) has been shown to mediate colorectal

Yamashiro et al. PNAS | May 5, 2020 | vol. 117 | no. 18 | 9903 Downloaded by guest on September 24, 2021 Fig. 7. A model illustrating the Thbs1/integrin/YAP signaling pathway in remodeling of vessel walls. Cyclic stretch induces secretion of Thbs1, which bindsto integrin αvβ1 and aids in the maturation of the FA–actin complex, thereby mediating nuclear shuttling of YAP via inactivation of Rap2 and the Hippo pathway. In vivo, Thbs1/integrins/YAP signaling may lead to maturation of FA after TAC-induced pressure overload to protect the aortic wall. In contrast, Thbs1/integrins/YAP signaling leads to neointima (NI) formation upon flow cessation by carotid artery ligation. EL: elastic lamella.

tumorigenesis through integrin/FAK/Src/YAP signaling (43), different mechanical stresses induce a distinct subset of YAP further supporting the idea that an ECM can regulate YAP target genes and lead to vascular remodeling, although a full signaling in various biological conditions. characterization of Thbs1-dependent YAP targets in different injury models is warranted. Taken together, our observations Role of Matrix Mechanotransduction in Vessel Wall Remodeling. emphasize the highly context dependent action of Thbs1/integ- Recent studies have shown that ECM mechanical cues mediate rins/YAP in remodeling of the vessel wall. cell proliferation, differentiation, autophagy, and lipid metabo- – lisms by regulating focal adhesions (1, 44 47). Thbs1 is present at Materials and Methods low levels in the postnatal vessels and up-regulated in various vascular diseases such as atherosclerosis (48), ischemia reperfu- Detailed descriptions are provided in SI Appendix. sion injury (49), aortic aneurysm (13), and pulmonary arterial Cell Culture and Reagents. Rat vascular SMCs (Lonza, R-ASM-580, isolated hypertension (50). YAP is also known to play a role in an from the aorta of 150 to 200 g adult male Sprague-Dawley rats) were grown emergency stress response to maintain tissue homeostasis (51, in Dulbecco’s modified Eagle medium (DMEM) with 20% (vol/vol) fetal bo- 52). As we describe here, Thbs1KO mice promoted maladaptive vine serum (FBS) and 1× Antibiotic-Antimycotic (ThermoFisher Scientific). remodeling in response to pressure overload and showed higher HUVECs were grown in HuMedia-MvG (KURABO) supplemented with 5% mortality after TAC, increased occurrence of aortic dissection, (vol/vol) FBS and growth factors. The cell lines were used between passages 7 and lack of up-regulation of YAP in medial SMCs. In WT mice, and 12 and tested for mycoplasma contamination using a mycoplasma de- YAP was induced and activated in adventitial cells and SMCs, tection set (TaKaRa, #6601) following the manufacturer’s protocol (the re- leading to proliferation and hypertrophy, respectively, and sult is provided in SI Appendix, Fig. S19). Brefeldin A (B5936) was purchased maintenance of wall tension during aortic wall dilatation. Since from Sigma-Aldrich. FAK inhibitor PF-00562271 (S2672) was purchased from Ctgf null mice exhibit higher mortality after TAC (53), CTGF Selleck. Cytochalasin D (037-17561) and latrunculin A (125-04363) were may be one of the YAP target genes crucial for response to purchased from Wako. Jasplakinolide (AG-CN2-0037-C050) was purchased pressure overload. Although Thbs1-dependent YAP nuclear from AdipoGen Life Sciences. Recombinant human thrombospondin-1 was shuttling in the medial SMCs was not as clear as that of in vitro purchased from R&D Systems (3074-TH-050). assays, our data suggest that the formation of FAs by Thbs1 and induction of Thbs1/integrin/YAP signaling play a protective role Mice. C57BL/6J (wild-type) mice were purchased from Charles River, and Thbs1 in response to pressure overload, presumably by increasing cell null mice were purchased from Jackson Laboratory (B6.129S2-Thbs1tm1Hyn/J) stiffness. Suppression of Thbs1/integrin/YAP signaling reduced and crossed with C57BL/6 mice for line maintenance. Male mice were used proliferation of neointimal cells upon carotid artery ligation and for experiments to exclude hormonal regulation by estrogen. All mice were thus had a beneficial effect on flow cessation–induced malad- kept on a 12 h/12 h light/dark cycle under specific pathogen-free conditions, aptive vascular remodeling. The reasons for this discrepancy and all animal protocols were approved by the Institutional Animal Experi- between the TAC model and flow cessation model may be that ment Committee of the University of Tsukuba.

9904 | www.pnas.org/cgi/doi/10.1073/pnas.1919702117 Yamashiro et al. Downloaded by guest on September 24, 2021 Statistical Analysis. All experiments are presented as means ± SEM. Statistical geo/); the GEO accession number is GSE131750 (https://www.ncbi.nlm.nih.gov/geo/ analysis was performed using Prism 7 (Graph Pad, version 7.0e). A Shapiro– query/acc.cgi?acc=GSE131750). Wilk test was used for the normality test. Statistical significance was de- termined by either unpaired t test, one-way ANOVA, or two-way ANOVA ACKNOWLEDGMENTS. We thank S. Sato, K. Sugiyama, and H. T. Hang for followed by Tukey’s multiple comparison test. If the normality assumption was technical assistance. We acknowledge technical support from J. D. Kim, violated, nonparametric tests were conducted. A Kruskal–Wallis test with M. Muratani, and Tsukuba i-Laboratory LLP for RNA-seq analysis. We Dunn’s multiple comparisons was used in Fig. 5C. To estimate the cumulative appreciate Z.-P. Liu, K. Ohashi, and T. Mayers for critical reading of the survivals, Kaplan–Meier survival curves and a long-rank test were used in manuscript. We thank Mayumi Mori for assistance with graphics. This work Fig. 5B. P < 0.05 denotes statistical significance. was supported in part by grants from a Grant-in-Aid for Young Scientists (Grant 18K15057), a Japan Heart Foundation Research Grant, the Inamori Foundation, the Japan Foundation for Applied Enzymology and Takeda Data Availability. The secretome data were deposited in the Japan Proteome Science Foundation, the MSD Life Science Foundation, the Uehara Memorial Standard Repository/Database (jPOST) (http://jpostdb.org); the accession number is Foundation, and the Japan Heart Foundation Dr. Hiroshi Irisawa & Dr. Aya JPST000600/PXD013915 (https://repository.jpostdb.org/entry/JPST000600). The RNA- Irisawa Memorial Research Grant (all to Y.Y.) and the MEXT KAKENHI (Grant seq data were deposited in National Center for Biotechnology Information’s JP 17H04289), the Naito Foundation, and the Astellas Foundation for (NCBI’s) Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/ Research on Metabolic Disorders (all to H.Y.).

1. J. D. Humphrey, E. R. Dufresne, M. A. Schwartz, Mechanotransduction and extracel- 28. A. B. Howard, R. W. Alexander, R. M. Nerem, K. K. Griendling, W. R. Taylor, Cyclic lular matrix homeostasis. Nat. Rev. Mol. Cell Biol. 15, 802–812 (2014). strain induces an oxidative stress in endothelial cells. Am. J. Physiol. 272, C421–C427 2. A. J. Engler, S. Sen, H. L. Sweeney, D. E. Discher, Matrix elasticity directs stem cell (1997). lineage specification. Cell 126, 677–689 (2006). 29. P. R. Standley, A. Cammarata, B. P. Nolan, C. T. Purgason, M. A. Stanley, Cyclic stretch 3. E. A. Klein et al., Cell-cycle control by physiological matrix elasticity and in vivo tissue induces vascular smooth muscle cell alignment via NO signaling. Am. J. Physiol. Heart – stiffening. Curr. Biol. 19, 1511 1518 (2009). Circ. Physiol. 283, H1907–H1914 (2002). 4. Z. Sun, S. S. Guo, R. Fässler, Integrin-mediated mechanotransduction. J. Cell Biol. 215, 30. Z. Meng et al., RAP2 mediates mechanoresponses of the Hippo pathway. Nature 560, – 445 456 (2016). 655–660 (2018). – 5. S. Dupont et al., Role of YAP/TAZ in mechanotransduction. Nature 474, 179 183 31. B. Zhao et al., Cell detachment activates the Hippo pathway via cytoskeleton re- (2011). organization to induce anoikis. Genes Dev. 26,54–68 (2012). 6. A. Totaro, T. Panciera, S. Piccolo, YAP/TAZ upstream signals and downstream re- 32. M. Aragona et al., A mechanical checkpoint controls multicellular growth through sponses. Nat. Cell Biol. 20, 888–899 (2018). YAP/TAZ regulation by actin-processing factors. Cell 154, 1047–1059 (2013). 7. L. Azzolin et al., Role of TAZ as mediator of Wnt signaling. Cell 151, 1443–1456 (2012). 33. Y. Tang et al., MT1-MMP-dependent control of skeletal stem cell commitment via a 8. F. X. Yu et al., Regulation of the Hippo-YAP pathway by G-protein-coupled receptor β1-integrin/YAP/TAZ signaling axis. Dev. Cell 25, 402–416 (2013). signaling. Cell 150, 780–791 (2012). 34. N. G. Kim, B. M. Gumbiner, Adhesion to fibronectin regulates Hippo signaling via the 9. Z. Meng, T. Moroishi, K. L. Guan, Mechanisms of Hippo pathway regulation. Genes FAK-Src-PI3K pathway. J. Cell Biol. 210, 503–515 (2015). Dev. 30,1–17 (2016). CELL BIOLOGY 35. Y. Xia et al., Endogenous thrombospondin 1 protects the pressure-overloaded myo- 10. A. Resovi, D. Pinessi, G. Chiorino, G. Taraboletti, Current understanding of the cardium by modulating fibroblast phenotype and matrix metabolism. Hypertension thrombospondin-1 interactome. Matrix Biol. 37,83–91 (2014). – 11. J. M. Sipes, J. E. Murphy-Ullrich, D. D. Roberts, Thrombospondins: Purification of 58, 902 911 (2011). human platelet thrombospondin-1. Methods Cell Biol. 143, 347–369 (2018). 36. R. Moura, M. Tjwa, P. Vandervoort, K. Cludts, M. F. Hoylaerts, Thrombospondin-1 12. Y. Yamashiro et al., Abnormal mechanosensing and cofilin activation promote the activates medial smooth muscle cells and triggers neointima formation upon mouse – progression of ascending aortic aneurysms in mice. Sci. Signal. 8, ra105 (2015). carotid artery ligation. Arterioscler. Thromb. Vasc. Biol. 27, 2163 2169 (2007). 13. Y. Yamashiro et al., Role of thrombospondin-1 in mechanotransduction and devel- 37. X. Wang et al., The induction of yes-associated protein expression after arterial injury opment of thoracic aortic aneurysm in mouse and humans. Circ. Res. 123, 660–672 is crucial for smooth muscle phenotypic modulation and neointima formation. Ar- (2018). terioscler. Thromb. Vasc. Biol. 32, 2662–2669 (2012). 14. H. Yanagisawa, J. Wagenseil, Elastic fibers and biomechanics of the aorta: Insights 38. S. M. Arribas, A. Hinek, M. C. González, Elastic fibres and vascular structure in hy- from mouse studies. Matrix Biol. 85–86, 160–172 (2019). pertension. Pharmacol. Ther. 111, 771–791 (2006). 15. J. G. Wang, M. Miyazu, E. Matsushita, M. Sokabe, K. Naruse, Uniaxial cyclic stretch 39. D. Y. Leung, S. Glagov, M. B. Mathews, Cyclic stretching stimulates synthesis of matrix induces focal adhesion kinase (FAK) tyrosine phosphorylation followed by mitogen- components by arterial smooth muscle cells in vitro. Science 191, 475–477 (1976). activated protein kinase (MAPK) activation. Biochem. Biophys. Res. Commun. 288, 40. Q. Li, Y. Muragaki, I. Hatamura, H. Ueno, A. Ooshima, Stretch-induced collagen 356–361 (2001). synthesis in cultured smooth muscle cells from rabbit aortic media and a possible 16. H. T. Nguyen et al., Cyclic stretch activates p38 SAPK2-, ErbB2-, and AT1-dependent involvement of angiotensin II and transforming growth factor-beta. J. Vasc. Res. 35, signaling in bladder smooth muscle cells. Am. J. Physiol. Cell Physiol. 279, 93–103 (1998). C1155–C1167 (2000). 41. G. Nardone et al., YAP regulates cell mechanics by controlling focal adhesion as- 17. S. Na et al., Time-dependent changes in smooth muscle cell stiffness and focal ad- sembly. Nat. Commun. 8, 15321 (2017). – hesion area in response to cyclic equibiaxial stretch. Ann. Biomed. Eng. 36, 369 380 42. J. Albrengues et al., Neutrophil extracellular traps produced during inflammation (2008). awaken dormant cancer cells in mice. Science 361, eaao4227 (2018). 18. S. K. Mitra, D. A. Hanson, D. D. Schlaepfer, Focal adhesion kinase: In command and 43. H. Ma et al., Periostin promotes colorectal tumorigenesis through integrin-FAK-Src – control of cell motility. Nat. Rev. Mol. Cell Biol. 6,56 68 (2005). pathway-mediated YAP/TAZ activation. Cell Rep. 30, 793–806.e6 (2020). 19. B. Geiger, J. P. Spatz, A. D. Bershadsky, Environmental sensing through focal adhe- 44. P. Romani et al., Extracellular matrix mechanical cues regulate lipid metabolism sions. Nat. Rev. Mol. Cell Biol. 10,21–33 (2009). through Lipin-1 and SREBP. Nat. Cell Biol. 21, 338–347 (2019). 20. M. G. Rubashkin et al., Force engages vinculin and promotes tumor progression by 45. A. Mamidi et al., Mechanosignalling via integrins directs fate decisions of pancreatic enhancing PI3K activation of phosphatidylinositol (3,4,5)-triphosphate. Cancer Res. progenitors. Nature 564, 114–118 (2018). 74, 4597–4611 (2014). 46. A. Totaro et al., Cell phenotypic plasticity requires autophagic flux driven by YAP/TAZ 21. A. Carisey et al., Vinculin regulates the recruitment and release of core focal adhesion mechanotransduction. Proc. Natl. Acad. Sci. U.S.A. 116, 17848–17857 (2019). proteins in a force-dependent manner. Curr. Biol. 23, 271–281 (2013). 47. J. Lu et al., Basement membrane regulates fibronectin organization using sliding 22. N. Perez Gonzalez et al., Cell tension and mechanical regulation of cell volume. Mol. focal adhesions driven by a contractile winch. Dev. Cell 52, 631–646.e4 (2020). Biol. Cell 29, 2509–2601 (2018). 48. R. Moura et al., Thrombospondin-1 deficiency accelerates atherosclerotic plaque 23. V. A. Codelia, G. Sun, K. D. Irvine, Regulation of YAP by mechanical strain through Jnk – and Hippo signaling. Curr. Biol. 24, 2012–2017 (2014). maturation in ApoE-/- mice. Circ. Res. 103, 1181 1189 (2008). 24. J. H. Zhu et al., Cyclic stretch stimulates vascular smooth muscle cell alignment by 49. J. Favier, S. Germain, J. Emmerich, P. Corvol, J. M. Gasc, Critical overexpression of – redox-dependent activation of Notch3. Am. J. Physiol. Heart Circ. Physiol. 300, thrombospondin 1 in chronic leg ischaemia. J. Pathol. 207 , 358 366 (2005). β H1770–H1780 (2011). 50. R. Kumar et al., TGF- activation by bone marrow-derived thrombospondin-1 causes 25. D. Morrow et al., Cyclic strain inhibits Notch receptor signaling in vascular smooth Schistosoma- and hypoxia-induced pulmonary hypertension. Nat. Commun. 8, 15494 muscle cells in vitro. Circ. Res. 96, 567–575 (2005). (2017). 26. H. Chang, K. G. Shyu, B. W. Wang, P. Kuan, Regulation of hypoxia-inducible factor- 51. N. Miyamura et al., YAP determines the cell fate of injured mouse hepatocytes in vivo. 1alpha by cyclical mechanical stretch in rat vascular smooth muscle cells. Clin. Sci. Nat. Commun. 8, 16017 (2017). (Lond.) 105, 447–456 (2003). 52. S. Yui et al., YAP/TAZ-dependent reprogramming of colonic epithelium links ECM 27. L. X. Jia et al., Mechanical stretch-induced endoplasmic reticulum stress, apoptosis and remodeling to tissue regeneration. Cell Stem Cell 22,35–49.e37 (2018). inflammation contribute to thoracic aortic aneurysm and dissection. J. Pathol. 236, 53. M. S. Fontes et al., CTGF knockout does not affect cardiac hypertrophy and fibrosis 373–383 (2015). formation upon chronic pressure overload. J. Mol. Cell. Cardiol. 88,82–90 (2015).

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