Matrix mechanotransduction mediated bythrombospondin-1/integrin/YAP in thevascular remodelingYoshito 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 ofCardiovascular 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 thatactivate intracellular signaling through matrix–cell interactions.In blood vessels, additional mechanical cues derived from the pul-satile blood flow and pressure play a pivotal role in homeostasisand disease development. Currently, the nature of the cues fromthe ECM and their interaction with the mechanical microenviron-ment in large blood vessels to maintain the integrity of the vesselwall are not fully understood. Here, we identified the matricellularprotein thrombospondin-1 (Thbs1) as an extracellular mediator ofmatrix mechanotransduction that acts via integrin αvβ1 to estab-lish focal adhesions and promotes nuclear shuttling of Yes-associated protein (YAP) in response to high strain of cyclic stretch.Thbs1-mediated YAP activation depends on the small GTPase Rap2and Hippo pathway and is not influenced by alteration of actinfibers. Deletion of Thbs1 in mice inhibited Thbs1/integrin β1/YAPsignaling, leading to maladaptive remodeling of the aorta in re-sponse to pressure overload and inhibition of neointima formationupon carotid artery ligation, exerting context-dependent effectson the vessel wall. We thus propose a mechanism of matrixmechanotransduction centered on Thbs1, connecting mechanicalstimuli to YAP signaling during vascular remodeling in vivo.

extracellular matrix (ECM) | mechanotransduction | thrombospondin-1 |YAP | vessel remodeling

The extracellular matrix (ECM) is fundamental to cellular andtissue structural integrity and provides mechanical cues to

initiate diverse biological functions (1). The quality and quantityof the ECM determine tissue stiffness and control gene expres-sion, cell fate, and cell cycle progression in various cell types (2,3). ECM–cell interactions are mediated by focal adhesions(FAs), the main hub for mechanotransduction, connecting ECM,integrins, and cytoskeleton (1, 4). In the blood vessels, an addi-tional layer of mechanical cues derived from the pulsatile bloodflow and pressure play a pivotal role in homeostasis and diseasedevelopment. However, how cells sense and integrate differentmechanical cues to maintain the blood vessel wall is largelyunknown.Hippo effector Yes-associated protein (YAP) and transcrip-

tional coactivator with PDZ-binding motif (TAZ) serve as anon–off mechanosensing switch for ECM stiffness (5). YAP ac-tivity is regulated by a canonical Hippo pathway; MST1/2(mammalian sterile 20-like 1/2 kinases) and their downstreamkinases LATS1/2 (large tumor suppressor 1/2) control YAP in-activation via phosphorylation and retention in the cytoplasm bybinding to the 14-3-3 protein (6). YAP activity is also regulatedby Wnt/β-catenin and G protein-coupled receptor (GPCR) sig-naling pathways (7, 8). However, regulation of YAP activity is

highly context dependent, and extracellular regulators are notfully understood (6, 9).Thrombospondin-1 (Thbs1) is a homotrimeric glycoprotein

with a complex multidomain structure capable of interacting witha variety of receptors such as integrins, cluster of differentiation(CD) 36, and CD47 (10). Thbs1 is highly expressed during de-velopment and reactivated in response to injury (11), exertingdomain-specific and cell type–specific effects on cellular functions.We recently showed that Thbs1 is induced by mechanical stretchand is a driver for thoracic aortic aneurysm in mice (12, 13).In the current study, we identified Thbs1 as an extracellular

regulator of YAP, which is induced by mechanical stress and actsthrough FAs independent of actin remodeling. Mechanistically,

Significance

We propose a mechanism of matrix mechanotransduction ini-tiated by thrombospondin-1 (Thbs1), connecting mechanicalstimuli to Yes-associated protein (YAP) signaling during vas-cular remodeling. Specifically, Thbs1 is secreted from smoothmuscle cells in response to high strain of cyclic stretch andbinds to integrin αvβ1 to establish mature focal adhesions,thereby promoting the nuclear shuttling of YAP in a smallGTPase Rap2- and Hippo signaling–dependent manner. Wefurther demonstrated the biological significance of the Thbs1/integrin β1/YAP pathway using two vascular injury models.Deletion of Thbs1 in mice caused maladaptive remodeling ofthe aorta in response to pressure overload, whereas it inhibi-ted neointima formation upon carotid artery ligation, exertingcontext-dependent effects on the blood vessel wall.

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.,T.K., S. Ohata, T.A.V.N., S. Ohtsuki, K.N., and H.Y. analyzed data; and Y.Y. and H.Y.wrote the paper.

The authors declare no competing interest.

This article is a PNAS Direct Submission. K.-L.G. is a guest editor invited by theEditorial Board.

Published under the PNAS license.

Data deposition: The secretome data have been deposited in the Japan Proteome Stan-dard Repository/Database (jPOST), http://jpostdb.org (accession no. JPST000600/PXD013915) and RNA-seq data have been deposited in Gene Expression Omnibus (GEO)database, https://www.ncbi.nlm.nih.gov/geo/ (accession no. GSE131750). All data support-ing the findings of this study are available from the corresponding authors uponreasonable request.

See online for related content such as Commentaries.1To whom correspondence may be addressed. Email: yamayoshito@tara.tsukuba.ac.jp orhkyanagisawa@tara.tsukuba.ac.jp.

This article contains supporting information online at https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1919702117/-/DCSupplemental.

First published April 22, 2020.

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Thbs1 binds integrin αvβ1 and strengthens FAs, aiding intranslocation of YAP to the nucleus. Deletion of Thbs1 in vivoresulted in altered vascular remodeling in response to pressureoverload and flow cessation. Taken together, Thbs1 mediatesdynamic interactions between mechanical stress and the YAP-mediated transcriptional cascade in the blood vessel wall.

ResultsCyclic Stretch–Induced Secretion of ECM and Cell Adhesion MoleculesInvolved in Blood Vessel Development. Vascular ECM is synthe-sized by smooth muscle cells (SMCs) from midgestation to theearly postnatal period and determines the material and me-chanical properties of the blood vessels (14). Owing to the longhalf-life of ECM, blood vessels are generally thought to standlifelong mechanical stress without regeneration, particularly inlarge arteries. However, it is not completely known whether alocal microremodeling system exists for the maintenance of thevessel wall. To search for a potential remodeling factor(s), wefirst performed cyclic stretch experiments with high strain (1 Hz;20% strain), mimicking the pathological condition of the aorticwall. Rat SMCs were subjected to cyclic stretch for 20 h with orwithout brefeldin A (BFA), a protein transporter inhibitor, as anegative control for mass spectrometry analysis. Conditionedmedia (CM) were analyzed using quantitative mass spectrome-try, and 87 secreted proteins with more than twofold differencesin response to cyclic stretch were identified (Fig. 1 A and B andSI Appendix, Figs. S1 and S2). Gene ontology (GO) enrichmentanalysis (http://geneontology.org/) revealed that proteins in-volved in ECM organization and structural constituents werehighly enriched (Fig. 1C). Ingenuity pathway analysis (IPA) was

conducted for biological interaction network among proteins inECM–cell adhesions and blood vessel development (Fig. 1D), andThbs1 showed multiple interactions in both categories. In addition,since we reported that Thbs1 is a mechanosensitive factor involvedin aortic aneurysms (12, 13), we focused on Thbs1 for furtheranalysis. We confirmed the increased expression and secretion ofThbs1 after cyclic stretch (Fig. 1E), suggesting that Thbs1 may play animportant role in response to dynamic vessel microenvironments.

Secreted Thrombospondin-1 Binds to Integrin αvβ1 under CyclicStretch. In response to cyclic stretch, SMCs showed up-regulation of the early growth response 1 (Egr1) transcriptionfactor, phosphorylated (p) focal adhesion kinase (FAK), extra-cellular signal-regulated kinase (ERK), and stress-responsemitogen-activated protein kinase (MAP) p38 as previouslyreported (SI Appendix, Fig. S3A) (13, 15, 16). Cyclic stretch al-tered subcellular localization of Thbs1 from the perinuclear re-gion to the tip of the cell on the long axis (Fig. 2A). We examinedThbs1 localization and reorientation of SMCs as a function oftime (SI Appendix, Fig. S3 B and C). SMCs were randomly ori-entated after 30 min of cyclic stretch, but most cells were alignedin a perpendicular position to the stretch direction at 3 h andcompletely reorientated by 6 h. Thbs1 localization clearlychanged from 3 h after cyclic stretch, which coincided with thereorientation of actin fibers and elongation of cells (SI Appendix,Fig. S3D). Since FAs recruit actin stress fibers (17), we costainedSMCs with Thbs1 and p-paxillin, a FA molecule, under cyclicstretch. Thbs1 colocalized with p-paxillin independent of celldensity after cyclic stretch (Fig. 2 B and C). Since Thbs1 has well-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 testwere calculated and plotted against the fold change for all identified proteins. Each dot represents mean values (n = 3). (B) Heat map of secreted proteinsinduced 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 ofthe P value. The top enriched GO terms associated with molecular function (orange), biological process (green), and reactome pathway analysis (blue) areshown. (D) Biological network using IPA. Diagrams show the direct (solid lines) and indirect (dashed lines) interactions among proteins reported in bloodvessel 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.

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and αv; CD47; and CD36 (Fig. 2D) (10), we examined which ofthese receptors bound to Thbs1 in response to cyclic stretch.Immunostaining showed that Thbs1 colocalized with integrins αvand β1 but not with integrin β3, CD47, or CD36 (Fig. 2 E and F).Immunoprecipitation of cell lysate obtained from the static orcyclic stretch condition with anti-Thbs1 antibody followed byWestern blotting confirmed direct binding between Thbs1 andintegrins αv and β1 in the cyclic stretch condition (Fig. 2G).Consistently, proximity ligation assay (PLA) showed that integ-rins αv and β1 interacted with Thbs1 only in the cyclic stretchcondition (Fig. 2H). These data demonstrated that Thbs1 wassecreted in response to cyclic stretch and bound to integrin αvβ1.

Thrombospondin-1 Regulates Maturation of the FA–Actin Complexand Controls Cell Stiffness. We next examined the effect of de-letion of Thbs1 on cyclic stretch–induced formation of FA andreorientation of SMCs. Thbs1 knockout rat vascular SMCs(Thbs1KO) were generated using CRISPR-Cas9 genome editing(SI Appendix, Fig. S4). We evaluated possible off-target effectsand confirmed that the expression of Cpreb3 or Tet3 was not

altered in Thbs1KO cells (SI Appendix, Fig. S5 A and B). Noobvious phenotypic changes were observed in Thbs1KO cells (SIAppendix, Fig. S5 C and D). Thbs1KO cells were subjected tocyclic stretch (1 Hz; 20% strain) for 8 h and were compared withcontrol (CTRL) cells with or without 1 μM of BFA. CTRL cellsresponded to cyclic stretch and reoriented perpendicular to thedirection of stretch, whereas BFA-treated CTRL cells andThbs1KO cells neither elongated nor aligned to the perpendic-ular position (Fig. 3 A–C). Stretch-induced phosphorylation ofpaxillin and up-regulation of integrins αv and β1 in CTRL cellswere not observed in Thbs1KO cells (Fig. 3D).Mature FAs are localized at the termini of actin stress fibers

and form a FA–actin complex (18, 19). The FA–actin complex isstabilized by vinculin deposition and integrin–talin binding,which facilitates PI3K-mediated phosphatidylinositol 3,4,5-tri-phosphate (PIP3) production (20). Vinculin is present at FAs in aforce-dependent manner and increases cell stiffness (21). Toevaluate the maturation of FA–actin complex, we compared thevinculin expression and monitored PIP3 levels using a GFP-tagged PIP3 reporter (GFP-Grp1-PH) in static and stretch

Fig. 2. Thbs1 localizes to FAs and binds integrin αvβ1 under cyclic stretch. (A) Cyclic stretch alters localization of Thbs1 (n = 5). Representative immunos-taining with phalloidin (red), Thbs1 (green), and DAPI (blue). (B) Representative image of immunostaining showing Thbs1 colocalization with p-paxillin undercyclic stretch (n = 5). Immunostained with p-paxillin (red), Thbs1 (green), and DAPI (blue). (C) Immunostaining showing Thbs1 localization at FAs in confluentconditions (n = 3). Immunostained with p-paxillin (red), Thbs1 (green), phalloidin (gray), and DAPI (blue). (D) Cartoon shows various binding domains of Thbs1and 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 Thbs1with 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 ar-rowheads). Bottom shows highly magnified images of the white dashed box in Top. DAPI (blue) and phalloidin (green) are shown. In all experiments, ratvascular 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 ineach immunostaining. Two-way arrows indicate stretch directions. (Scale bars, 50 μm.)

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conditions in CTRL and Thbs1KO cells. PIP3 was enriched onstress fibers in stretched CTRL cells (Fig. 3E), and vinculin wasdeposited onto the tip of actin stress fibers which anchored FAsin stretched CTRL cells, whereas stretched Thbs1KO cells didnot show recruitment of vinculin onto the FA–actin complex(Fig. 3F). To confirm this observation, we assessed mechanicalproperties of Thbs1KO cells using atomic force microscopy(Fig. 3G). Since cell tension positively correlates with adhesionsize and its stiffness (22), we analyzed morphological changesand stiffness on the stress fibers. Stretched Thbs1KO cellsshowed a significant decrease in stiffness (Young’s modulus) onthe stress fibers compared with stretched CTRL cells, althoughthe height of stretched Thbs1KO cells was comparable to that ofstretched CTRL cells (Fig. 3H). Taken together, our findingssuggest that Thbs1 is required for maturation of the FA–actincomplex and maintenance of cellular tension, thereby correctlyorienting actin fibers in response to cyclic stretch.

Thrombospondin-1 Regulates Nuclear Shuttling of YAP via Integrinαvβ1 in a Rap2-Dependent Manner. To understand the molecularbasis of Thbs1 contribution in mechanotransduction, we per-formed RNA sequencing (RNA-seq) using CTRL and Thbs1KO

cells in static and cyclic stretch conditions (SI Appendix, Fig. S6).In the static condition, RNA-seq revealed 38 and 47 genes thatwere differentially regulated in CTRL and Thbs1KO cells, re-spectively. Conversely, 126 genes in CTRL cells and 163 genes inThbs1KO cells were differentially expressed under cyclic stretch.Among these genes, GO enrichment analysis revealed theabundant expressions of genes involved in response to stimulus(SI Appendix, Fig. S7A). We therefore used known gene groupsinvolved in mechanical stress–induced responses, including YAP(23), Notch (24, 25), hypoxia-inducible factor-1-alpha (HIF1A)(26), endoplasmic reticulum (ER) stress response (27), and ox-idative stress response (28, 29), as keywords to further charac-terize 289 genes via IPA combined with RNA-seq analysis (SIAppendix, Fig. S7B). With the exception of gene encoding mes-othelin (Msln), the expressions of YAP target genes weremarkedly down-regulated in Thbs1KO cells, suggesting thatThbs1 strongly controls YAP target genes in the stretch condi-tion (SI Appendix, Fig. S6) and may be a critical component ofthe YAP signaling pathway.Next, to elucidate the role of Thbs1 in the mechanical regu-

lation of YAP, we examined YAP localization with or withoutThbs1 under cyclic stretch. Consistent with a previous report

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 weresubjected 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 alsoshown. 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 8h). Two-way arrows indicate stretch direction. (Scale bars, 25 μm.) Arrow heads (purple) show vinculin deposition onto the tip of actin fibers. (G) Young’smodulus of actin fibers in CTRL (n = 129) or Thbs1KO (n = 86) cells measured using atomic force microscopy. Representative topographic images and stiffnessmaps are shown. (H) Cell height and Young’s modulus are shown. Bars are means ± SEM. ***P < 0.001, unpaired t test. NS: not significant.

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(23), cyclic stretch induced nuclear shuttling of YAP in CTRLcells (Fig. 4A). Although YAP was retained in the cytoplasmafter cyclic stretch in Thbs1KO cells (Fig. 4A), exogenouslyadded recombinant human THBS1 (rhTHBS1) promoted nu-clear shuttling of YAP after cyclic stretch (Fig. 4B and SI Ap-pendix, Fig. S8). Since nuclear shuttling of YAP is regulated by akinase cascade of the Hippo pathway, we examined p-YAP andp-LATS levels using Western blotting. In CTRL cells, p-YAPand p-LATS were decreased in stretch condition, whereasp-YAP and p-LATS persisted in Thbs1KO cells (Fig. 4C). Theseobservations indicate that Thbs1 controls nuclear shuttling ofYAP in a Hippo pathway–dependent manner. The Ras-related

GTPase Rap2 was recently identified as a negative regulator ofthe Hippo pathway and as an inhibitor of YAP activation in-duced by ECM stiffness (30). To determine whether stretch- andThbs1-induced nuclear shuttling of YAP is regulated by Rap2activity, we measured active Rap2 (Rap2-GTP) levels in CTRLor Thbs1KO cells in static and stretch conditions using theRal-GDS-RBD pull-down assays. As shown in Fig. 4D, activeRap2 decreased in CTRL cells after stretch but not in Thbs1KOcells. To further determine the relationship between Rap2 ac-tivity and nuclear shuttling of YAP, CTRL or Thbs1KO cellswere transfected with myc-Rap2A (wild type [WT]), myc-Rap2A(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 cellsin the static or stretch condition with YAP (green). Phalloidin (red) and DAPI (blue) are also shown. Two-way arrows indicate stretch direction. (Scale bars, 50μm.) 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 serumstarvation, then subjected to uniaxial cyclic stretch (20% strain; 1 Hz) for 8 h. Representative immunostaining with YAP (green) and DAPI (blue). (Scale bars, 50μm.) 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-RBDagarose beads (n = 2). (E) Overexpression of Myc-Rap2A(WT), constitutively active Myc-Rap2A(G12V), or dominant-negative Myc-Rap2A(S17N) in CTRL andThbs1KO cells in the static or stretch condition (n = 3). Representative immunostaining with Myc (red), YAP (green), and DAPI (blue). Two-way arrows indicatestretch 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). YAPlocalization 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 knockdownof Itgαv, Itgβ1, and Itgβ3 are provided in SI Appendix, Fig. S10.

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dominant negative mutant. Forty-eight hours after transfection,cells were subjected to cyclic stretch (1.0 Hz, 20% strain) for 8 hand immunostained with antibodies directed against Myc andYAP and stained with DAPI to identify nuclei. Rap2 (WT)overexpression showed no effects on the localization of YAP inCTRL or Thbs1KO cells (Fig. 4E). Rap2A activation (G12V)cancelled nuclear shuttling of YAP under cyclic stretch in CTRLcells. In contrast, Rap2A inactivation (S17N) forced YAP nu-clear shuttling with or without Thbs1, overriding the regulationof Thbs1. These data suggested that Thbs1 controls nuclearshuttling of YAP in a Rap2A-dependent manner.Tension on actin fibers is also important for regulation of

YAP, dependent on or independent of the formation of FAs(31–33). We assessed how actin dynamics and inhibition of FAKaffects stretch-induced nuclear shuttling of YAP. CTRL cellswere subjected to cyclic stretch for 8 h with the actin polymeri-zation inhibitor cytochalasin D (CytoD) or actin de-polymerization inhibitor jasplakinolide (Jasp) or FAK inhibitorPF-00562271 (FAKi). The efficiency of the treatment was eval-uated by measuring the orientation angle (θ) of the long axis ofthe ellipse relative to the cyclic stretch direction (SI Appendix,Fig. S9 A and B). Although CytoD and Jasp showed no effects onnuclear shuttling of YAP under cyclic stretch, YAP remained inthe cytoplasm in FAKi-treated cells (SI Appendix, Fig. S9 C andE). Furthermore, although overexpression of slingshot1 (Ssh1,Ssh1OE), a phosphatase of cofilin, induced depolymerization ofactin fibers through dephosphorylation of cofilin, it did not affectYAP localization under cyclic stretch (SI Appendix, Fig. S9 D andE). These observations suggest that stretch-induced nuclearshuttling of YAP is dependent on FAs but not on actin poly-merization. To confirm this, we performed small interferingRNA (siRNA)–mediated knockdown of integrins αv (siItgαv), β1(siItgβ1), and β3 (siItgβ3) along with scramble siRNA (SI Ap-pendix, Fig. S10) followed by cyclic stretch. As expected,knockdown of integrin αv or β1 was sufficient to inhibit stretch-induced nuclear shuttling of YAP (Fig. 4F). We next investigatedwhether matrix proteins in general serve as an extracellularregulator of YAP under cyclic stretch. Since fibronectin has beenshown to regulate the Hippo pathway through FAK-Src-PI3Kduring cell spreading (34) and is a ligand for integrin αvβ1, weknocked down fibronectin in SMCs and subjected them to cyclicstretch to assess its impact on YAP localization. As SI Appendix,Fig. S11 shows, fibronectin knockdown had no effect on nuclearshuttling of YAP in response to stretch. We also assessedwhether Thbs1-mediated nuclear shuttling of YAP is observed inendothelial cells. Using human umbilical vein endothelial cells(HUVECs), we found that cyclic stretch did not induce phos-phorylation of paxillin or localization of Thbs1 to FAs, eventhough Thbs1 was secreted into CM (SI Appendix, Fig. S12 A andB). Actin fibers aligned in a perpendicular position, and contactinhibition was reduced by cyclic stretch, but YAP remained inthe cytoplasm of HUVECs (SI Appendix, Fig. S12 C and D),suggesting that regulation of YAP by Thbs1 under cyclic stretchcondition is specific to vascular SMCs. Taken together, thesedata indicate that stretch-induced, Thbs1-mediated nuclearshuttling of YAP is dependent on integrin αvβ1 and that effect isspecific to SMCs.

Thrombospondin-1/Integrin/YAP Signaling Is Involved in MechanicalStress–Induced Vascular Pathology. An in vivo model was used toevaluate whether Thbs1-mediated YAP regulation is involved inthe pathogenesis of vascular diseases. We previously reportedthat transverse aortic constriction (TAC) induced Thbs1 ex-pression (12). Concordantly, Thbs1 was induced in endothelialcells (ECs), SMCs, and adventitial cells after TAC was per-formed in WT mice and PLA showed the interaction betweenThbs1 and integrin β1 in these cells (Fig. 5A). We next evaluatedthe effect of loss of Thbs1/integrin β1 in TAC-induced aortic

remodeling using Thbs1 null (Thbs1KO) mice. Whereas 100% ofWT mice survived at 5 wk after TAC (n = 13), 31.9% (7/22) ofThbs1KO mice died, with three showing aortic rupture (Fig. 5Band SI Appendix, Fig. S13A). The heart weight (HW) and bodyweight (BW) ratio (HW/BW) was significantly higher in TAC-operated Thbs1KO mice than in WT mice, in accordance with aprevious report (35) (Fig. 5C). In addition, TAC-operatedThbs1KO aortas had enlarged aortic lumen (Fig. 5D), and dis-sected aortas showed severely disrupted elastic fibers (Fig. 5E).Morphometric analysis showed a significant increase in the in-ternal elastic lamella (IEL) perimeter, outer perimeter, and totalvessel area in TAC-operated Thbs1KO aortas compared to TAC-operated WT aortas (Fig. 5F). Immunostaining showed strongexpression of YAP and its targets, connective tissue growthfactor (CTGF), Serpine1, CYR61, and Caveolin-3, in TAC-operated WT aortas, especially in the medial layers and in theadventitia (Fig. 5 G and H and SI Appendix, Fig. S13B). How-ever, the expression of YAP and CTGF was hardly observed inthe medial layers of TAC-operated Thbs1KO aorta, although theexpression in the adventitia was evident in Thbs1KO aortas. Incontrast, the expression of Serpine1, CYR61, and Caveolin-3 wasunchanged in TAC-operated Thbs1KO aortas. These data sug-gest the possibility that Thbs1-dependent YAP signaling inSMCs plays an important role in adaptive remodeling of theaortic wall in response to pressure overload.Finally, we employed carotid artery ligation injury to address

the contribution of Thbs1/integrin/YAP signaling in flowcessation–induced neointima formation (Fig. 6A). Thbs1 hasbeen shown to be required for neointima formation (36), andYAP mediates phenotypic modulation of SMCs during neo-intima formation (37). Time course analyses of the ligated ar-teries revealed that Thbs1 was up-regulated in the inner layer at1 wk after ligation when YAP expression was not evident(Fig. 6B and SI Appendix, Fig. S14). At 2 wk after ligation, nu-clear YAP peaked in the developing neointima (Fig. 6C) andcoexpressed with Thbs1 (Fig. 6B and SI Appendix, Fig. S15). At 3wk, Thbs1 and YAP expression decreased as the neointima be-came stabilized (Fig. 6B and SI Appendix, Fig. S16). YAP targetsCTGF, Serpine1, CYR61, and Caveolin-3 were highly expressedin the neointima (SI Appendix, Fig. S17). PLA showed the in-teraction between Thbs1 and integrin β1 in the neointima (leftcarotid artery [LCA] in Fig. 6D) but not in the contralateralartery (right carotid artery [RCA] in Fig. 6D). To confirm therelationship between Thbs1/integrin/YAP and neointima for-mation, we injected the lentivirus (LV)-encoding Thbs1 siRNA(siThbs1) into WT mice through tail vein injection at 2 wk afterligation and evaluated neointima at 3 wk. GFP-LV injectionfrom the tail vein showed clear expression of GFP in the neo-intima, endothelial cells of the right carotid artery, and the liver(SI Appendix, Fig. S18), confirming the delivery of siRNA intothe neointima. As we expected, the neointima was not observedin siThbs1 virus-treated animals, similar to Thbs1KO mice, andYAP levels and nuclear localization were markedly decreased asThbs1 expression decreased (Fig. 6 C and E).

DiscussionIn this study, we identified Thbs1 as a critical molecule involvedin mechanotransduction and extracellular regulation of YAP,leading to dynamic remodeling of the blood vessels. We foundthat Thbs1, once secreted in response to mechanical stress, bindsto integrin αvβ1 and aids in the maturation of the FA–actincomplex, downregulating Rap2 activity, thereby mediating acti-vation of YAP. Loss of Thbs1 decreases cell stiffness in vitro, anddeletion or down-regulation of Thbs1 in mice alters cellular be-havior in response to mechanical stress in a context-dependentmanner. Taken together, we propose that the Thbs1/integrin/YAP signaling pathway plays a critical role in vascularremodeling in vivo (Fig. 7).

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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 WTmice (Top). PLA showing the interaction between Thbs1 and integrin β1 (red dots in Bottom). Right shows highly magnified images of white dashed boxes inBottom. Arrowheads (white) show PLA cluster. DAPI (blue) and autofluorescence of elastin (green) are also shown. n = 3. (Scale bars, 50 μm.) (B) Kaplan-Meiersurvival 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 eachbar. (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 IELperimeter, outer perimeter, total vessel area, and wall thickness. Bars are mean ± SEM. *P < 0.05, ***P < 0.001, one-way ANOVA. NS: not significant. Thenumber of animals is indicated in each bar. (G) Immunostaining of TAC-operated aortas with YAP (red) in WT and Thbs1KO mice. On the Right, highlymagnified images of the white dashed boxes in the adventitial (Adv.) and medial (Med.) layers in the Left are shown. DAPI (blue) and autofluorescence ofelastin (green) are also shown. n = 3. (Scale bars, 50 μm.) (H) Representative images of immunohistochemistry for CTGF in TAC-operated aortas of WT andThbs1KO 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.)

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Mechanical Stress and Micro- and Macroremodeling of the Vessel Wallby ECM. Vascular ECM, a major component of the vessel wall, issubjected to repeated mechanical stress throughout life. Due toits long half-life [for example, 50 to 70 y for elastic fibers of thehuman artery (38)], the vascular ECM is considered to be diffi-cult to regenerate once it is damaged by physical and/or bio-chemical insults. Although ECM synthesis via mechanical stretchwas previously described (39, 40), its biological significance,whether it contributes to microremodeling or dynamic remod-eling of the vessel wall, and how it is connected to the mecha-notransduction cascade are not completely understood. Usingmass spectrometry and GO analysis, we found that high strain ofcyclic stretch induced secretion of various ECM proteins in-volved in cell adhesions, elastic fiber formation, and TGF-βsignaling. Using IPA, Thbs1 was identified as one of the keymolecules involved in these biological processes. Interestingly,mechanical stress and secretary pathway are interconnectedsince BFA treatment or deletion of Thbs1 led to the alteration ofthe mechanoresponse in SMCs and disrupted alignment of cellsin response to cyclic stretch. Our observations indicate thatmechanical stress induces secretion of Thbs1, which initiatesmechanotransduction and activates the integrin/YAP pathway,thereby remodeling the vessel wall to accommodate the changesin mechanical microenvironment. Thbs1 and integrins are es-sential components of matrix mechanotransduction, serving asbiomechanical ligand and sensors of the microenvironment.

Thbs1 as an Extracellular Mediator of YAP Signaling Pathway. Weshowed that mechanical stretch–induced, secreted Thbs1 directlybound to integrin αvβ1 and colocalized with p-paxillin, and fa-cilitated maturation of FA complex by inducing deposition ofvinculin at the tip of actin fibers. Currently, the precise

mechanism by which secreted Thbs1 is recruited to integrin αvβ1is unclear. However, we speculate that Thbs1 ligation to integrinsactivates downstream molecules, leading to maturation of FAsand strengthening of actin fibers. Interestingly, a loss of Thbs1decreased cell stiffness and altered YAP target gene expressionafter cyclic stretch. We further showed that stretch down-regulated Rap2 activity in CTRL cells, whereas Rap2 activityremained high in the absence of Thbs1, which forced YAP re-tention in the cytoplasm. Our observation agrees with the recentstudy by Meng et al. demonstrating that Rap2 negatively regu-lates nuclear shuttling of YAP in a Hippo pathway–dependentmanner. These authors proposed a cascade of signaling triggeredby FAs → PLCγ1 (phospholipase C gamma 1) → PtdIns(4,5)P2(phosphatidylinositol 4, 5-bisphosphate) → PA (phosphatidicacid) → PDZGEF (RAPGEF) → Rap2 → MAP4K → LATS →YAP (30) and showed that increased matrix stiffness led to in-activation of Rap2 and nuclear shuttling of YAP. It was alsoproposed that this signaling axis works in parallel with RhoA,which mediates cytoskeletal tension during cell spreading (5, 32).Our data showed that stretch-induced YAP activation is Rap2dependent and is not influenced by altered actin fibers. There-fore, it is possible that Thbs1 mediates nuclear shuttling of YAPby inactivating Rap2 through altering the composition of FA orby controlling FA assembly (41). A previous study described thata mutant cell line lacking YAP showed decreased Thbs1 levelsalong with down-regulation of YAP target genes and FA mole-cules, indicating that Thbs1 may act as a feedforward driver forYAP signaling (41). Interestingly, the proteolytic remodeling oflaminin-1 has been shown to modulate awakening of dormantcancer cells through integrin/FAK/ERK/MLCK (myosin light-chain kinase)/YAP signaling (42), and fibroblast-derived peri-ostin (encoded by Postn) has been shown to mediate colorectal

Fig. 6. Thbs1 regulates YAP expression during neointima formation upon carotid artery ligation. (A) Diagram of LCA ligation. (B) Immunostaining of crosssections 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) andautofluorescence of elastin (green) are also shown. Quantification of colocalization with YAP and DAPI using Imaris colocalization software is indicated inyellow (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 Imariscolocalization software. Bars are mean ± SEM. **P < 0.01, ***P < 0.001, one-way ANOVA. (D) PLA showing cluster of Thbs1/integrin β1 (red dots, yellowarrowheads) 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) Crosssections 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.)

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tumorigenesis through integrin/FAK/Src/YAP signaling (43),further supporting the idea that an ECM can regulate YAPsignaling in various biological conditions.

Role of Matrix Mechanotransduction in Vessel Wall Remodeling.Recent studies have shown that ECM mechanical cues mediatecell proliferation, differentiation, autophagy, and lipid metabo-lisms by regulating focal adhesions (1, 44–47). Thbs1 is present atlow levels in the postnatal vessels and up-regulated in variousvascular diseases such as atherosclerosis (48), ischemia reperfu-sion injury (49), aortic aneurysm (13), and pulmonary arterialhypertension (50). YAP is also known to play a role in anemergency stress response to maintain tissue homeostasis (51,52). As we describe here, Thbs1KO mice promoted maladaptiveremodeling in response to pressure overload and showed highermortality after TAC, increased occurrence of aortic dissection,and lack of up-regulation of YAP in medial SMCs. In WT mice,YAP was induced and activated in adventitial cells and SMCs,leading to proliferation and hypertrophy, respectively, andmaintenance of wall tension during aortic wall dilatation. SinceCtgf null mice exhibit higher mortality after TAC (53), CTGFmay be one of the YAP target genes crucial for response topressure overload. Although Thbs1-dependent YAP nuclearshuttling in the medial SMCs was not as clear as that of in vitroassays, our data suggest that the formation of FAs by Thbs1 andinduction of Thbs1/integrin/YAP signaling play a protective rolein response to pressure overload, presumably by increasing cellstiffness. Suppression of Thbs1/integrin/YAP signaling reducedproliferation of neointimal cells upon carotid artery ligation andthus had a beneficial effect on flow cessation–induced malad-aptive vascular remodeling. The reasons for this discrepancybetween the TAC model and flow cessation model may be that

different mechanical stresses induce a distinct subset of YAPtarget genes and lead to vascular remodeling, although a fullcharacterization of Thbs1-dependent YAP targets in differentinjury models is warranted. Taken together, our observationsemphasize the highly context dependent action of Thbs1/integ-rins/YAP in remodeling of the vessel wall.

Materials and MethodsDetailed descriptions are provided in SI Appendix.

Cell Culture and Reagents. Rat vascular SMCs (Lonza, R-ASM-580, isolatedfrom the aorta of 150 to 200 g adult male Sprague-Dawley rats) were grownin Dulbecco’s modified Eagle medium (DMEM) with 20% (vol/vol) fetal bo-vine serum (FBS) and 1× Antibiotic-Antimycotic (ThermoFisher Scientific).HUVECs were grown in HuMedia-MvG (KURABO) supplemented with 5%(vol/vol) FBS and growth factors. The cell lines were used between passages 7and 12 and tested for mycoplasma contamination using a mycoplasma de-tection set (TaKaRa, #6601) following the manufacturer’s protocol (the re-sult is provided in SI Appendix, Fig. S19). Brefeldin A (B5936) was purchasedfrom Sigma-Aldrich. FAK inhibitor PF-00562271 (S2672) was purchased fromSelleck. Cytochalasin D (037-17561) and latrunculin A (125-04363) werepurchased from Wako. Jasplakinolide (AG-CN2-0037-C050) was purchasedfrom AdipoGen Life Sciences. Recombinant human thrombospondin-1 waspurchased from R&D Systems (3074-TH-050).

Mice. C57BL/6J (wild-type)micewere purchased from Charles River, and Thbs1null mice were purchased from Jackson Laboratory (B6.129S2-Thbs1tm1Hyn/J)and crossed with C57BL/6 mice for line maintenance. Male mice were usedfor experiments to exclude hormonal regulation by estrogen. All mice werekept on a 12 h/12 h light/dark cycle under specific pathogen-free conditions,and all animal protocols were approved by the Institutional Animal Experi-ment Committee of the University of Tsukuba.

Fig. 7. A model illustrating the Thbs1/integrin/YAP signaling pathway in remodeling of vessel walls. Cyclic stretch induces secretion of Thbs1, which binds tointegrin αvβ1 and aids in the maturation of the FA–actin complex, thereby mediating nuclear shuttling of YAP via inactivation of Rap2 and the Hippopathway. 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.

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Statistical Analysis. All experiments are presented as means ± SEM. Statisticalanalysis was performed using Prism 7 (Graph Pad, version 7.0e). A Shapiro–Wilk test was used for the normality test. Statistical significance was de-termined by either unpaired t test, one-way ANOVA, or two-way ANOVAfollowed by Tukey’s multiple comparison test. If the normality assumption wasviolated, nonparametric tests were conducted. A Kruskal–Wallis test withDunn’s multiple comparisons was used in Fig. 5C. To estimate the cumulativesurvivals, Kaplan–Meier survival curves and a long-rank test were used inFig. 5B. P < 0.05 denotes statistical significance.

Data Availability. The secretome data were deposited in the Japan ProteomeStandard Repository/Database (jPOST) (http://jpostdb.org); the accession number isJPST000600/PXD013915 (https://repository.jpostdb.org/entry/JPST000600). The RNA-seq data were deposited in National Center for Biotechnology Information’s(NCBI’s) Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/

geo/); the GEO accession number is GSE131750 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE131750).

ACKNOWLEDGMENTS. We thank S. Sato, K. Sugiyama, and H. T. Hang fortechnical assistance. We acknowledge technical support from J. D. Kim,M. Muratani, and Tsukuba i-Laboratory LLP for RNA-seq analysis. Weappreciate Z.-P. Liu, K. Ohashi, and T. Mayers for critical reading of themanuscript. We thank Mayumi Mori for assistance with graphics. This workwas supported in part by grants from a Grant-in-Aid for Young Scientists(Grant 18K15057), a Japan Heart Foundation Research Grant, the InamoriFoundation, the Japan Foundation for Applied Enzymology and TakedaScience Foundation, the MSD Life Science Foundation, the Uehara MemorialFoundation, and the Japan Heart Foundation Dr. Hiroshi Irisawa & Dr. AyaIrisawa Memorial Research Grant (all to Y.Y.) and the MEXT KAKENHI (GrantJP 17H04289), the Naito Foundation, and the Astellas Foundation forResearch on Metabolic Disorders (all to H.Y.).

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