Latrophilin mediates flow activation of Notch to control vascular endothelial phenotype and atherosclerosis

Latrophilin-2 mediates flow activation of Notch to control vascular endothelial phenotype and atherosclerosis | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Latrophilin-2 mediates flow activation of Notch to control vascular endothelial phenotype and atherosclerosis View ORCID Profile Keiichiro Tanaka , View ORCID Profile Minghao Chen , View ORCID Profile Divyesh Joshi , View ORCID Profile Manasa Chanduri , Hanming Zhang , View ORCID Profile Carlos Fernandez-Hernando , View ORCID Profile Yoo-Min Koh , View ORCID Profile Martin A. Schwartz doi: https://doi.org/10.1101/2025.07.01.662676 Keiichiro Tanaka 1 Yale Cardiovascular Research Center, Section of Cardiovascular Medicine, Department of Internal Medicine, School of Medicine, Yale University , New Haven, CT 06511, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Keiichiro Tanaka For correspondence: keiichiro.tanaka{at}yale.edu martin.schwartz{at}yale.edu Minghao Chen 1 Yale Cardiovascular Research Center, Section of Cardiovascular Medicine, Department of Internal Medicine, School of Medicine, Yale University , New Haven, CT 06511, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Minghao Chen Divyesh Joshi 1 Yale Cardiovascular Research Center, Section of Cardiovascular Medicine, Department of Internal Medicine, School of Medicine, Yale University , New Haven, CT 06511, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Divyesh Joshi Manasa Chanduri 1 Yale Cardiovascular Research Center, Section of Cardiovascular Medicine, Department of Internal Medicine, School of Medicine, Yale University , New Haven, CT 06511, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Manasa Chanduri Hanming Zhang 2 Yale Vascular Biology & Therapeutics Program Find this author on Google Scholar Find this author on PubMed Search for this author on this site Carlos Fernandez-Hernando 2 Yale Vascular Biology & Therapeutics Program Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Carlos Fernandez-Hernando Yoo-Min Koh 1 Yale Cardiovascular Research Center, Section of Cardiovascular Medicine, Department of Internal Medicine, School of Medicine, Yale University , New Haven, CT 06511, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Yoo-Min Koh Martin A. Schwartz 1 Yale Cardiovascular Research Center, Section of Cardiovascular Medicine, Department of Internal Medicine, School of Medicine, Yale University , New Haven, CT 06511, USA 3 Department of Cell Biology 4 Department of Biomedical Engineering Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Martin A. Schwartz For correspondence: keiichiro.tanaka{at}yale.edu martin.schwartz{at}yale.edu Abstract Full Text Info/History Metrics Preview PDF Abstract Fluid shear stress (FSS) is a major determinant of endothelial cell (EC) phenotype, with physio-logical laminar FSS promoting arterial identity and stability, and disturbed FSS promoting atherosclerosis. We previously identified the adhesion G protein-coupled receptor (GPCR) Latrophilin-2 (LPHN2) as a junctional protein required for FSS activation of the PECAM1/VE-cadherin/VEGFR2/PlexinD1 junctional mechanosensory pathway, which regulates EC alignment to laminar blood flow and promotes EC inflammatory activation and atherogenesis under disturbed flow. Here, we demonstrate that Lphn2 endothelial cell-specific knockout hyperlipidemic mice develop larger atherosclerotic plaques than controls—opposite from the previously reported Pecam1 knockout model. Transcriptomic analysis revealed that LPHN2 contributes more than half of flow-responsive gene expression and suppresses pro-inflammatory pathways. Critically, LPHN2 is essential for FSS-induced Notch1 activation to suppress inflammation, via a GPCR-independent mechanism. Active Notch correlates with physical association of LPHN2 with Notch1 activator, γ-secretase, and Notch1 in response to flow. Both the Notch1 intracellular domain (NICD) and transmembrane domain (NTMD) contribute to the anti-inflammatory gene program, with the non-canonical NTMD signaling specifically suppressing the pro-inflammatory YAP pathway. Together with the accompanying paper showing that LPHN2 also mediates flow activation of the Smad1/5 pathway, these data identify LPHN2 as a central mediator of EC flow responses through multiple independent mechanisms in vascular biology and disease. Introduction Atherosclerosis, a chronic inflammatory disease of arterial walls, is the leading cause of morbidity and mortality worldwide 1 , 2 . Lesions form preferentially at arterial branch points and regions of curvature where endothelial cells (ECs) are exposed to low and multidirectional flow patterns, termed disturbed FSS, whereas regions of high laminar shear stress are protected 3 , 4 . Flow-sensing mechanisms are thus crucial in vascular health and disease. Latrophilin-2 (Lphn2) is a member of the adhesion GPCR family that is well expressed in vascular endothelial cells 5 . Adhesion GPCRs have large extracellular domains that bind ligands on other cells or extracellular matrices and contains a G protein autoproteolysis (GAIN) domain that generates an internal peptide that mediates G protein activation 6 – 8 . We recently reported that Lphn2 is required for activation of the PECAM-1/VE-Cadherin/VEGFR/Plexin D1 junctional mechanosensory complex by FSS 5 , 9 , 10 . Lphn2 appears to respond to FSS-induced changes in plasma membrane composition or fluidity 5 , 11 . Lphn2 was found to be essential for activation of early flow-induced signals, for EC alignment in flow and for flow-dependent vascular remodeling in mice and zebrafish 5 . However, its roles in vascular inflammation and atherosclerosis were not determined. EC Notch signaling regulates vascular development, homeostasis, and disease 12 . Notch1 is the main Notch receptor in ECs and is activated by binding ligands such as DLL4 and Jag1 on adjacent cells 13 , which triggers Notch activation through proteolysis by ADAM10 and gamma-secretase. FSS amplifies ligand-dependent Notch activation through unknown mechanisms 14 , 15 . The released Notch intracellular domain (NICD) translocates to the nucleus, binds the transcription factor RBPJ and drives expression of target genes 16 . This canonical Notch signaling pathway inhibits VEGF receptor expression and angiogenesis, induces quiescence and promotes arterial identity 17 – 19 . Notch1 cleavage also releases its transmembrane domain (NTMD), which remains in the plasma membrane where it associates with VE-cadherin to stabilize EC cell-cell junctions and improve barrier function (non-canonical signaling) 20 . In contrast, atherogenic factors, including inflammatory cytokines, dyslipidemia and disturbed shear stress, inhibit Notch signaling 21 – 23 . Endothelial specific knockout (ECKO) of Notch1 exacerbates atherosclerosis, while its activation is protec-tive 22 , consistent with an anti-inflammatory, stabilizing function. We began this study by investigating the role of Lphn2 in atherosclerosis. Based on its position upstream of the junctional mechanosensory complex that promotes EC inflammatory activation in disturbed flow 24 , Lphn2 was expected to facilitate inflammatory gene expression. However, Lphn2 ECKO increased atherosclerotic plaque size and suppressed pro-inflammatory genes. Prompted by their co-localization at junctions and Notch’s anti-inflammatory effects, we investigated possible connections between Lphn2 and Notch1. These studies showed that Lphn2 is required for activation of Notch by FSS, and elucidate protein physical associations and functional consequences of the Lphn2-Notch axis. Results Latrophilin-2 endothelial-specific knock-out increases atherosclerotic plaques in mice Previous studies showed that deletion of PECAM-1, a component of the junctional mechanosensory complex, reduced inflammation and atherosclerosis at regions of disturbed FSS in mice 24 – 26 . To investigate the contribution of Lphn2 to atherosclerosis, we generated EC-specific Lphn2 knockout mice ( Lphn2 ECKO) using a tamoxifen-inducible Cre-loxP system, with Cre recombinase driven by the Cdh5 (VE-cadherin) promoter. To induce hyperlipidemia, mice were injected with adeno-associated virus (AAV) encoding Pcsk9 that reduces hepatic LDL receptor protein, then fed a high-fat diet ( Figure 1A ). Lphn2 ECKO significantly increased atherosclerotic plaque size in both male and female mice ( Figure 1B and 1C ) without altering circulating cholesterol or triglycerides ( Figure 1D ). Lphn2 ECKO mice also showed larger plaques in aortic root sections ( Figure 1E and 1F ). Lphn2 in ECs thus unexpectedly restrains atherosclerosis. Download figure Open in new tab Figure 1: Lphn2 endothelial-specific knockout increases atherosclerosis ( A ) Experimental timeline: Control or Lphn2 ECKO 8 week old mice were treated with tamoxifen and then subsequently injected with PCSK9-expressing AAV8 virus. Mice were fed a high-fat diet for 8 weeks then tissue harvested. ( B ) Representative images of Oil Red O-stained aortas and corresponding plaque masks from control and Lphn2 ECKO mice (male and female). ( C ) Quantification of atherosclerotic plaque area in Lphn2 ECKO mice versus control mice (plaque area ratio to total area). Data were from 7 control males, 5 Lphn2 ECKO males, 10 control females and 6 Lphn2 ECKO females. Statistics were calculated using unpaired Student ’s t-test. ( D ) Quantification of blood total cholesterol and triglyceride levels in mice from ( C ). Statistics used unpaired Student ’s t-test. ( E ) Oil Red O and hematoxylin and eosin (H&E) staining of aortic root sections from control and Lphn2 ECKO mice. Scale bar: 500µm. ( F ) Quantification of Oil Red O lesion area from control and Lphn2 ECKO mice. Statistics: unpaired Student ’s t-test. LPHN2 regulates half of flow-responsive genes To investigate these effects, we evaluated the role of LPHN2 in flow-mediated gene regulation by transcriptomic analysis of HUVECs under physiological laminar shear stress (12 dynes/cm 2 ) with control or LPHN2 knockdown ( Figure 2A ). Both laminar shear stress alone and Lphn2 knockdown alone significantly altered gene expression ( Figure 2B ). Klf2 and Klf4 were among the top flow-responsive genes that were unaffected by Lphn2 knockdown ( Figure 2C ), consistent with prior findings 5 . Notably, LPHN2 knockdown blocked approximately half of the flow-induced changes in gene expression ( Figure 2D ), identifying Lphn2 as a major determinant of EC flow transcriptomic responses. According to KEGG pathway enrichment analysis, LPHN2 knockdown in-creased multiple inflammatory pathways with the TNFα/NF-κB pathway as the top upregulated pathway. The Hippo pathway was the top downregulated pathway ( Figure 2E ), consistent with Hippo pathway kinases suppressing the Yap and Taz transcription factors that promote EC inflammatory gene expression and atherosclerosis in mice 27 – 29 . These transcriptional shifts suggest that loss of Lphn2 enhances EC pro-inflammatory phenotypes, consistent with increased atherosclerosis in Lphn2 ECKO mice. Download figure Open in new tab Figure 2: LPHN2-dependent regulation of flow-responsive transcriptome ( A ) Schematic of experimental procedure. Human umbilical vein endothelial cells (HUVECs) were transfected with control or LPHN2 siRNAs. After 3 days, cells were subjected to laminar shear stress at 12 dynes/cm 2 for 18 h or maintained under static (no flow) conditions. Total RNA was extracted and analyzed by bulk RNAseq. ( B ) Volcano plots of log 2 fold change (flow vs static) versus -log 10 FDR showing flow-responsive differentially expressed genes (DEGs) in LPHN2 vs control siRNAs. Top5 DEGs and KLF2/KLF4 are highlighted. ( C ) Normalized expression counts for KLF2 and LPHN2 analyzed by edgeR. Statistical significance was determined by one-way ANOVA with Tukey post-hoc analysis. ( D ) Venn diagram illustrating overlaps of flow-responsive downregulated and upregulated DEGs in control and LPHN2 knockdown cells, emphasizing LPHN2-dependent flow responsiveness. The number on the lower right indicates the number of the genes that did not change with LPHN2 KD or flow. ( E ) KEGG pathway enrichment for flow-responsive LPHN2 target genes identified in ( D ). X-axis is fold enrichment and color scale indicates FDR, with pathways upregulated by LPHN2 KD in red and downregulated pathways in blue. Lphn2 is thus required for a major fraction of flow-dependent regulation; its disruption strongly activates Yap/Taz and/or NF-kB, two major inflammatory pathways that depend on EC cell-cell junctions 27 , 30 , 31 but the well-known anti-inflammatory Klf2 pathway is Lphn2 independent 32 . Latrophilin-2 Is required for flow-induced Notch activation The inconsistency between these results and the established role of the junctional mechanosensory pathway in shear stress signaling and atherosclerosis led us to investigate whether other shear stress pathways might be regulated by Latrophilin-2. Given that Latrophilin-2 and the Notch pathway are activated by shear stress at cell-cell junctions, and Notch signaling protects against ather-osclerosis 14 , 22 , we considered whether Latrophilin2 might regulate Notch. FSS induces both canonical Notch signaling through its intracellular domain that regulates gene expression and non-canonical signaling through its transmembrane domain that stabilizes cell-cell junctions 20 . We therefore silenced LPHN2 and assessed the effect on flow-induced Notch activation. LPHN2 KD markedly reduced NICD production in response to shear stress ( Figure 3A – 3B ). By contrast, its depletion had no effect on Notch activation by its ligand DLL4 ( Figure 3C – 3D ). LPHN2 depletion also attenuated flow-induced induction of the canonical NICD direct target gene Hes1 in both HUVECs and HAECs ( Figure 3E – 3G ). Consistent with in vitro findings, en face analysis of the thoracic aorta from Lphn2 ECKO mice revealed reduced Hes1 expression ( Figure 3H – 3I ), supporting a role for Lphn2 in flow-induced Notch pathway activation in vivo . Download figure Open in new tab Figure 3: LPHN2 is required for flow-induced Notch activation ( A ) Lysates of ECs exposed to shear stress, with knockdown (KD) of control or Lphn 1-3 were assayed by Western blot for the cleaved NICD to assess Notch1 activation. ( B) Quantification of NICD in ( A ). Statistics analyzed using one-way ANOVA with Tukey post-hoc analysis. ( C ) Notch activation in response to DLL4 ligand in control and LPHN2 KD cells. (D) Quantification of NICD levels from ( C ). Data were from 3 independent experiments. Statistics used one-way ANOVA with Tukey post-hoc analysis. ( E ) Representative images of Hoechst and Hes1 staining of HUVECs with or without laminar shear stress for 16 hours. Scale bar: 50µm. ( F ) Quantification of nuclear Hes1 intensity in ( E ). n = 1146 (scrambled static), 932 (scrambled flow), 1175 ( LPHN2 KD static), 1829 ( LPHN2 KD flow) cells. Statistics used one-way ANOVA with Tukey post-hoc test. ( G ) Hes1 nuclear intensity in human aortic endothelial cells (HAECs) as in ( F) . n = 394 (scrambled static), 266 (scrambled flow), 175 ( LPHN2 KD static), 309 ( LPHN2 KD flow) cells. Statistics used one-way ANOVA with Tukey post-hoc. ( H ) En face immunostaining of thoracic aorta from Lphn2 ECKO and control mice for Hes1. ( I ) Quantification of Hes1 nuclear intensity from ( H ). N = 6 mice per condition. Statistics were analyzed using unpaired Student ’s t-test. Notch activation by flow is independent of G Proteins and beta-arrestins We next investigated the role of G proteins in Lphn2-dependent activation of Notch (Schematic in Figure 4A ). Knockdown of the G proteins involved in endothelial flow signaling had no effect on flow-induced Notch cleavage ( Figure 4B – 4C ). To independently test this result, we rescued endogenous LPHN2 knockdown with wild-type LPHN2 vs the GPCR-defective mutant (H1071A) 33 . Both constructs equally restored flow-induced Hes1 expression ( Figure 4D – 4E ), supporting G protein-independent function. We additionally assessed β-arrestins, which are key effectors in G protein-independent GPCR signaling. Knockdown of β-arrestin 1 (ARRB1), the major isoform present in ECs, had no effect on flow-induced Hes1 nuclear enrichment ( Supplemental Figure 1A-1C ). We noticed that ARRB1 KD exhibited elevated baseline Hes1 expression, likely due to the reported impairment of Notch1 degradation 34 . Lphn2-dependent Notch activation thus appears to be β-arrestin-independent. Download figure Open in new tab Figure 4: LPHN2 mediated Notch activation is G protein-independent but GAIN autopro-teolysis-dependent (A) Schematic of Latrophilin-2 (LPHN2) structure with highlighted mutations/gene perturbations. (B) EC lysates Western blotted for flow-induced NICD after knockdowns of the G proteins that couple with Latrophilin-2. ( C ) Quantification of NICD levels from ( B ). Statistical analysis used one-way ANOVA with Tukey post-hoc analysis. ( D ) Representative images of Hes1 staining following rescue with wild-type or GPCR-deficient LPHN2. ( E ) Quantification of Hes1 nuclear in-tensity from ( D ). n = 1146 (scrambled static), 932 (scrambled flow), 1175 ( LPHN2 KD static), 1829 ( LPHN2 KD flow), 996 (+LPHN2-WT static), 921 (+LPHN2-WT flow), 2100 (+LPHN2(H1071A) static), and 1820 (+LPHN2(H1071A) flow) cells. Statistics used one-way ANOVA with Tukey post-hoc analysis. ( F ) Rescue of Hes1 expression by WT vs. LPHN2 GAIN domain autoproteolysis mutant. n = 149 (scrambled static), 166 (scrambled flow), 125 ( LPHN2 KD static), 118 ( LPHN2 KD flow), 208 (+LPHN2-WT static), 128 (+LPHN2- WT flow), 275 (+LPHN2(GAIN mutant) static), and 147 (+LPHN2(GAIN mutant) flow) cells. Statistics used one-way ANOVA with Tukey post-hoc analysis ( G ) Co-Immunoprecipitation of LPHN2 or its GAIN domain mutant with γ-secretase and Notch1 using isolated membrane fractions as inputs. γ-secretase was detected using an antibody targeting Presenillin-1. ( H&I ) Quantification of co-IPs of LPHN2/ɣ-secretase and LPHN2/Notch1 from ( G ). Statistics used one-way ANOVA with Tukey post-hoc test. ( J ) Co-IP validation of LPHN2-DLL4 interaction under static vs. flow conditions; no significant changes in complex formation in flow. ( K ) Quantification from ( J ). Statistics used unpaired Student ’s t-test. LPHN2-dependent Notch activation requires an intact GAIN domain and physical association To further investigate the mechanism of LPHN2-dependent Notch signaling, we examined an Lphn2 mutant lacking GAIN domain autoproteolytic activity (see Figure 4A ). This mutant failed to support flow-induced Notch pathway activation ( Figure 4F ). We next investigated whether LPHN2 physically interacts with any components of the Notch pathway. For these experiments, we expressed mClover3-tagged LPHN2 wild-type (WT) or the GAIN domain mutant and examined cells with or without FSS for 2 hours. Immunoprecipitation of isolated membrane fractions using GFP-TRAP beads followed by Western blotting showed a flow-induced increase in the as-sociation of WT LPHN2 with Notch1 and γ-secretase, the enzyme responsible for the terminal Notch cleavage that generates the NICD and TMD. By contrast, the GAIN mutant showed the opposite behavior, binding was high without flow and decreased with FSS ( Figure 4G – 4I ). As a control, we assayed the association of Notch1 with its ligand DLL4. These proteins co-immuno-precipitated but displayed no change after flow ( Figure 4J - 4K ). Lphn2-dependent Notch activation by flow thus requires an intact GAIN domain, which correlates with flow-induced physical associations. LPHN2 responds to changes in membrane fluidity to activate Notch Shear stress is reported to induce rapid increases in plasma membrane fluidity associated with decreased cholesterol content 11 , 35 . We previously reported that LPHN2 is required for EC activation of the PECAM1 pathway in response to increasing plasma membrane fluidity by depleting membrane cholesterol with methyl-β-cyclodextrin (MβCD) 5 . To test whether membrane fluidity mediates flow-induced Notch activation, we treated ECs with a low concentration of MβCD to reduce membrane cholesterol by an amount similar to FSS, demonstrated using the D4H* cholesterol reporter ( Figure 5A – 5B ). MβCD treatment activated the Notch pathway similarly to shear stress, which was blocked by LPHN2 depletion ( Figure 5C–D ). MβCD also enhanced the association of LPHN2 with γ-secretase and Notch1 ( Fig 5E-G ). These data provide further evidence that changes in membrane fluidity drive shear stress activation of Notch through LPHN2, likely through changes in intramembrane interactions. Download figure Open in new tab Figure 5: Membrane cholesterol decrease regulates LPHN2-dependent Notch activation ( A ) Representative XZ projections of the D4H-mClover3 cholesterol reporter in HUVECs under the indicated conditions. Scale bar: 10µm. ( B ) Quantification of D4H-mClover3 fluorescence in-tensity from cell membrane regions under the indicated conditions. ( C ) Immunoblot for NICD after MβCD treatment with control vs LPHN2 KD. ( D ) Quantification of NICD levels from ( C ). Statistics: one-way ANOVA with Tukey post-hoc test. ( E ) Co-immunoprecipitation of Lphn2 with ɣ-secretase and Notch1 after MβCD treatment in control vs LPHN2 KD cells. ( F&G ) Quantification of co-IPs from ( E ). Statistics used unpaired Student ’s t-test. Role of Notch in LPHN2 -dependent gene expression To further assess the role of Notch signaling downstream of LPHN2 , we evaluated the effects of rescuing Lphn2 knockdown by overexpressing either the Notch1 intracellular domain (NICD) or the transmembrane domain (TMD), which mediate canonical or non-canonical signaling, respectively ( Figure 6A - 6C and Supplemental Figure 2A ). Quantification of differentially expressed genes revealed that approximately 30% the Lphn2-dependent genes were reversed by expression of the Notch1 TMD, while 22% were reversed by expression of the NICD ( Figure 6C and 6E ). NICD overexpression upregulated multiple Notch target genes, including Hes5 by 3.3-fold, HeyL by 9.7-fold, and Hey2 by 2.1-fold ( Supplemental Figure 2B ). For genes rescued by NTMD, KEGG pathway enrichment analysis identified the Hippo pathway as the most downregulated by LPHN2 KD, while multiple inflammatory pathways were upregulated ( Figure 6D ). As Hippo suppresses inflammatory Yap/Taz signaling, these results all point toward an anti-inflammatory role for the NTMD. For NICD over-expression, the PPAR signaling and categories related to metabolism, cell cycle and inflammation were most affected ( Figure 6F ). Given the effects of Lphn2 on inflammatory gene expression ( Figure 2 ) and the paucity of information about the NTMD, we further investigated the role of the NTMD on the Hippo pathway in EC inflammation. Download figure Open in new tab Figure 6: LPHN2 downstream targets that can be rescued by Notch1 components ( A ) Schematic of experimental procedure. Control vs LPHN2 KD HUVECs were infected with lentivirus encoding Notch1 TMD, NICD or empty control as in Figure 2 . ( B ) Schematic of Notch1 rescue constructs used in this study. ( C ) Western blot for Notch1 TMD or tubulin loading control. ( D ) Venn diagrams showing overlap between LPHN2-dependent genes and those rescued by Notch1 transmembrane domain (TMD). ( E ) KEGG pathway enrichment of genes rescued by Notch1 TMD. Images are displayed as in Figure 2E . ( F ) Venn diagram of LPHN2-dependent genes and those rescued by Notch1 intracellular domain (ICD). ( G ) KEGG pathway enrichment analysis of genes rescued by Notch1 ICD. Lphn2-Notch Axis Regulates Endothelial Inflammation via YAP transcription factor The Notch1 TMD was reported to interact with the VE-cadherin TMD to stabilize endothelial junctions and enhance barrier function 20 . As cell-cell adhesions are reported to control Hippo pathway activation 31 , 36 , we examined effects of the NTMD on YAP function. LPHN2 knockdown increased YAP nuclear localization, while NTMD overexpression decreased nuclear Yap with or without LPHN2 KD ( Figure 7A ; quantified in Supplemental Figure 3A ). Consistent with the known anti-inflammatory function of Notch signaling 22 , LPHN2 KD increased basal expression of VCAM1, a marker of endothelial inflammation, which was reversed by the NTMD ( Figure 7B and 7C ). A pharmacological inhibitor (K-975) that blocks the interaction of YAP with its downstream transcription factor TEAD also reduced VCAM1 expression, confirming an inflammatory role for YAP following Lphn2 suppression ( Figure 7D and 7E ). Download figure Open in new tab Figure 7: Notch TMD regulates YAP ( A ) YAP activation assessed by nuclear/cytoplasmic ratio in control and LPHN2 KD cells with or without NTMD overexpression. ( B ) Western blot for VCAM1, an endothelial inflammation marker, in ECS with control vs LPHN2 KD, with or without NTMD overexpression. GAPDH used as a loading control ( C ) Quantification of VCAM-1 expression from ( B ). Data represent results from four independent experiments. Statistical analysis used one-way ANOVA with Tukey post-hoc analysis. ( D ) Western blot for VCAM-1 in control vs LPHN2 KD cells and treatment with 10 µM YAP-TEAD inhibitor K-975. ( E ) Quantification of VCAM-1 expression normalized to GAPDH from ( D ). Data represent results from four independent experiments. Statistical analysis used one-way ANOVA with Tukey post-hoc analysis. ( F ) Experimental timeline: Control or Lphn2 ECKO mice were injected tamoxifen at 8 weeks old and subsequently harvested for tissue analysis at 8 weeks post-injection. ( G ) En face staining of YAP and Hoechst in control mouse or Lphn2 ECKO mouse aortae. Scale bar: 50µm. ( H ) Quantification of the YAP nuclear cytoplasmic ratio in endothelial cells from ( G ). ( I ) DAPT treatment timeline. Control mice over 8 weeks old were injected with 50 mg/kg DAPT or vehicle for 5 consecutive days and tissue harvested. ( J ) En face staining of YAP and Hoechst following DAPT injections or vehicle control. ( K ) Quantification of YAP nuclear/cytoplasmic ratio from ( I ). Statistics used Student ’s t-test. n = 3 (male vehicle), 4 (male DAPT), 5 (female vehicle), and 6 (female DAPT). Lastly, we investigated the Lphn2-Notch-YAP axis in vivo . Lphn2 ECKO mice, prepared by injecting tamoxifen into Lphn2 flox/flox ; Cdh5-CreERT2 mice ( Figure 7F ) showed significantly in-creased nuclear YAP in aortic ECs ( Figure 7G and 7H ). Pharmacological inhibition of Notch1 cleavage with DAPT also strongly induced YAP nuclear translocation in vivo ( Figure 7I-K ) and in vitro ( Supplemental Figure 3B-C ). Collectively, these results demonstrate that pro-inflammatory phenotypes observed in Lphn2 ECKO mice involve diminished Notch activation, which derepresses YAP. Discussion Our study expanding the role for Latrophilin-2 in endothelial flow responses and vascular disease that go well beyond regulation of the classical junctional mechanotransduction complex. We first observed larger atherosclerotic plaques in hyperlipidemic Lphn2 ECKO mice, in contrast to PECAM-1 KO where plaque size was reduced 24 – 26 . Transcriptomic analyses revealed that knock-down of Lphn2 blocked approximately 50% of the flow-dependent changes, identifying Lphn2 as a major mediator of flow signaling. Lphn2 knockdown mainly shifted gene expression toward a pro-inflammatory signature, with upregulation of TNFα (NF-κB) and Yap/Taz pathways, which is inconsistent with blockade of the PECAM-1 junctional pathway 9 . These findings led us to consider the Notch1 pathway, which is activated by FSS, requires cell-cell contact, and promotes EC quiescence and arterial identity 12 , 14 , 15 . We found that Lphn2 is re-quired for flow activation of Notch1, which correlated with a physical association of Lphn2 with both Notch1 and its activator γ-secretase. Notch appears to control EC function through both its canonical regulation of gene expression by the NICD, and non-canonical NTMD interaction with junctional components. To our surprise, expressed NTMD had the stronger effect on EC’s transcriptomic profile, reversing ∼30% of the Lphn2-dependent genes, whereas the NICD reversed ∼22%. Analysis of DEGs identified the Hippo pathway as the strongest GO category. Hippo path-way kinases inhibit Yap/Taz activation, which promote inflammatory gene expression in ECs and atherogenesis in mouse models. Thus, preventing Notch cleavage should increase nuclear Yap/Taz and upregulate its inflammatory target genes. Indeed, overexpression of the NTMD suppressed nuclear localization of Yap and expression of the inflammatory marker VCAM-1. These effects are likely due to interaction of the NTMD with the transmembrane domain of VE-cadherin to stabilize EC junctions 20 , which can control activation of Hippo pathway kinases 28 , 31 . Thus, the Notch1 TMD likely influences gene expression through effects of junctional organization on Hippo pathway kinases ( Supplemental Figure 4 ). Lphn2-dependent activation of Notch was independent of G proteins and β-arrestin. However, mutations of the Lphn2 GAIN domain that block autoproteolysis abrogated flow-dependent Notch activation. This result may indicate that the GAIN domain mediates a critical interaction, perhaps even with Notch receptors. However, molecular dynamics simulation of the non-cleavable GAIN domain from ADGRB2 predicted a rigid conformation, while the cleaved one in ADGRL1 is more flexible 37 . Furthermore, in CryoEM structures of Latrophilin-3 holoreceptor, the Latrophilin GAIN domain interacts closely with the 7-membrane spanning TMD 38 . Thus, altering the conformation of the GAIN domain may influence TMD conformations or vice versa. While the GAIN domain could mediate interactions directly, acting indirectly via effects on the transmembrane do-main seems equally likely. Indeed, this mechanism fits better with changes in membrane proper-ties as the initiating stimulus. Dissecting these interactions and functions will require a concerted structure-function analysis in future research. The accompanying paper reports that Lphn2 is also required for flow activation of the Smad1/5 pathway, which is separable from both PECAM1 and Notch pathway activation. In keeping with the major role in flow-dependent gene expression, these results demonstrate that Latrophilin is a central regulator of EC FSS signaling through three independent pathways: the PECAM-1 junctional complex, the Alk1/Endoglin-Smad1/5 pathway, and the Notch pathway, the latter acting through both canonical and non-canonical effectors. These interactions and effects of Latrophilin likely have implications for other cellular or organ systems, especially the central nervous system where both Lphn2 and Notch play important roles 39 , 40 . The unique, GPCR-independent mechanisms of Lphn2 signaling in flow may thus offer opportunities to treat disease of the cardiovascular system and elsewhere. Methods Antibodies Anti-VCAM1 (abcam) (ab134047); Anti-phospho-p65 (Cell Signaling) (3033); Anti-RFP (Invitrogen) (R10367); Anti-Tubulin (Cell Signaling) (3873); Anti-YAP (Cell signaling) (14074); Anti-cleaved Notch (Cell Signaling) (4147); Anti-Hes1 (Cell signaling) (11988); Anti-Presenillin-1 (Millipore-Sigma) (MAB1563); Anti-Gi (NewEast Biosciences)(26003); Anti-Gq/11 (BD Biosciences) (612705); Anti-Notch1 (Cell Signaling) (4380); Anti-GFP (Invitrogen) (A11122); Anti-DLL4 (Cell Signaling) (2589) Cell Culture and Treatments Primary HUVECs were obtained from the Yale Vascular Biology and Therapeutics core facility. Each batch contains cells pooled from three donors. Cells were cultured in M199 (Gibco: 11150-059) supplemented with 20% FBS, 1x Penicillin-Streptomycin (Gibco: 15140-122), 60 µg/ml heparin (Sigma: H3393), and endothelial growth cell supplement (ECGS; the total formulation termed hereafter as complete medium). HUVECs were used for experiments between passages 3 and 6. For ligand-induced Notch activation, HUVECs were seeded onto the surface coated with 10µg/mL recombinant DLL4 (R&D: 1506D4050) as described 14 . Lentiviral transduction Lenti-X 293T cells (Clontech, 632180) were cultured for at least 24 hours in DMEM supplemented with 10% FBS and lacking antibiotics, then transfected with lentiviral plasmids encoding the gene of interest and packaging plasmids (Addgene: 12259 and 12260) using Lipofectamine 2000 (Thermo Fisher Scientific: 11668-019) following the manufacturer’s protocols with Opti-MEM medium. Conditioned media from these cultures were collected 48 hours later, sterilized through 0.45µm filters and added to HUVECs together with 10µg/ml of polybrene (Sigma: 107689). After 24 hours, cells were switched to complete medium for 48 hours. siRNA transfection HUVECs were cultured in EGM TM -2 Endothelial Cell Growth Medium-2 BulletKit TM (Lonza: CC-3156 and CC-4176) for 24 hours before transfecting with RNAiMax (Thermo Fisher Scientific: 13778-150) with 20nM siRNA in Opti-MEM (Gibco: 31985-070) according to the manufacturer’s instructions. After 6 hours, cells were switched to EGM-2 medium and used for experiments 3 days later. Gα protein siRNAs were custom designed based on previous publications 41 – 43 . Latro-philin-2 knockdown used ON-TARGET plus Smartpool siRNAs from Dharmacon (L-005651-00-0005). Similarly, ARRB1 and ARRB2 knockdowns were achieved using Dharmacon SmartPools (ARRB1: L-011971-00-0005; ARRB2: L-007292-00-0005). Shear stress HUVECs were seeded on tissue culture-treated plastic slides coated with 10 µg/ml fibronectin for overnight at 37 ° C and grown to confluence. For short-term experiments, cells were starved over-night in M199 medium with 2% FBS and 1:10 of ECGS. Slides were transferred to parallel flow chambers and shear stress applied as described 44 . RNA isolation and RT-PCR RNA was isolated from HUVECs using RNeasy kit according to the manufacturer’s instructions and quantified using a nanodrop spectrophotometer. Following cDNA synthesis using Bio-Rad iScript kit, RT-PCR was performed as follows: each PCR reaction contains 42 two-step amplification cycles consisting of: (a) denaturation at 95 ° C for 30s, and (b) annealing and extension at 60 ° C for 30s. The amplification curve was recorded and the relative transcript level of the target mRNA in each sample was calculated by normalization of Ct values to the reference RNA (GAPDH or 18S). Primer sequences are shown in Table 1 . View this table: View inline View popup Download powerpoint Table 1: Primer sequences used for quantitative PCR in this study RNA Sequencing RNA was extracted from HUVECs subjected to Lphn2 knockdown and/or Notch1 domain over-expression using QIAGEN RNEasy kit following the manufacturer’s protocol. RT-PCR con-firmed efficient Lphn2 knockdown, successful overexpression of Notch1 TMD (this study) or Notch1 ICD (addgene; 64622), and robust flow-mediated induction of KLF2, a previously vali-dated Lphn2-independent flow-responsive gene 5 . Libraries were prepared based on poly A enrichment and sequenced using standard protocols of Yale center of Genomic Analysis. Data analysis of RNA Sequencing Raw RNAseq data in FASTQ format were first assessed for quality using FastQC (v0.12.1). Adapter sequences and low-quality bases were trimmed from the reads using fastp (v0.23.2) with default parameters. The resulting high-quality reads were aligned to the human reference genome (GRCh38) using STAR aligner (v2.7.11a). PCR duplicates were identified and removed using Pi-card MarkDuplicates (v2.25.6) to ensure accurate quantification. Gene-level read counts were generated with the featureCounts function from the Subread package (v2.0.3) using GENCODE gene annotation. To correct for potential batch effects, count matrices were processed with ComBat-seq in Bioconductor sva package (v.3.56.0). Count normalization was conducted using edgeR (v.4.6.2) and differential gene expression analysis was performed using DESeq2 (v1.44.0) with significance thresholds set at false discovery rate (FDR) 1. Gene Ontology (GO) and pathway enrichment analyses were conducted using the genekitr package (v1.0.5) to identify significantly enriched biological processes and pathways among differentially expressed genes. Western Blotting Cell lysates or immunoprecipitation samples extracted in Laemmli sample buffer were separated by SDS-PAGE and transferred onto nitrocellulose membranes. Membranes were blocked with 5% milk in TBS-T and probed with primary antibodies at 4 ° C for overnight. The targeting proteins were visualized using HRP-conjugated secondary antibodies and the HRP-luminol reaction. Immunofluorescence HUVECs were washed with PBS, fixed for 10 minutes with 4% PFA in PBS and permeabilized with 0.5% Triton X-100 in PBS for 10 minutes, and blocked with 3% BSA in PBS for 1 hour. Slides were then incubated with primary antibodies at 4 ° C for overnight, washed with PBS and incubated with secondary antibodies and Hoechst for 1 hour. Samples were then washed 4 times with PBS and mounted. Image analysis Images were acquired on a PerkinElmer spinning disk microscope using a 20x objective. Hes1 and YAP1 nuclear translocation was quantified using a custom ImageJ macro code. The code first creates nuclear masks derived from Hoechst-stained images, then generates cytoplasmic masks by enlarging these nucleus masks using ImageJ’s built-in ENLARGE function, performing this expansion five times and subsequently subtracting the original nucleus masks to isolate the cytoplasmic region. The code quantifies the intensity of Hes1 or YAP1 images within both the nucleus and the cytoplasm areas, reporting the nuclear intensity for Hes1/YAP1 or calculating the ratio of nu-clear to cytoplasmic intensity of YAP1. Coimmunoprecipitation of Lphn2 for detection of interaction with Notch components Cells were infected with lentivirus encoding Lphn2-mClover3, at 3 days after infection washed with cold PBS, scraped, and homogenized in 200 μl of buffer containing 10 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 1 mM DTT, 0.25 M sucrose, and a cocktail of protease inhibitors (Thermo Fisher Scientific, 78440) per 1 well of 6 well plate. Nuclei and unbroken cells were removed by centrifugation at 500 × g for 5 min at 4°C, and the postnuclear supernatant was centrifuged at 100,000 × g for 1 hour at 4°C to separate the membrane and cytoplasmic fractions. Membrane fractions were solubilized overnight at 4°C in PIPES buffer with 1% CHAPSO (10 mM PIPES, pH 7.0, 5 mM CaCl 2 , 5mM MgCl 2 , 1% CHAPSO, and protease inhibitor cocktail). After solubilization and centrifugation to remove undissolved fraction, membrane fraction lysates were incubated with GFP-Trap TM beads (Chromotek, gta) for 2 hours at 4°C with gentle rotation. Beads were washed three times with PIPES buffer containing 0.25% CHAPSO, eluted with 1x Laemmli buffer and analyzed by SDS-PAGE and immunoblotting as described above. Animals All mice used in this study were on the C57BL/6J background. B6;129S6-Adgrl2 tm1Sud /J mice (Jackson number 023401, hereafter Lphn2 flox/flox ) and B6;129-Tg (Cdh5-Cre)1Spe/J mice (Jackson number 017968, hereafter CDH5-Cre ERT2 ). Lphn2 flox/flox mice were crossed with CDH5-Cre ERT2 to get Lphn2 flox/flox ; CDH5-Cre ERT2 mice (hereafter Lphn2 iECKO). Lphn2 flox/flox mice (also treated with tamoxifen) served as controls. Mice were maintained in a light- and temperature-controlled environment with free access to food and water. Consistent with federal guidelines, all animal usage was approved by the Yale University Institutional Care and Use Committee. Immunostaining and imaging of mouse aorta Aortae were perfusion-fixed in situ with 4% paraformaldehyde (overnight) prior to staining with primary Hes1 antibody (Cell signaling: 11988) or primary Yap antibody (Cell signaling: 14074) and fluorophore-conjugated secondary antibody (Invitrogen, donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 647, A31573) in Claudio buffer (1% FBS, 3% BSA, 0.5% Triton X-100, 0.01% Sodium deoxycholate, 0.02% sodium azide in PBS pH7.4) 45 . Cells were stained with Hoechst (Invitrogen: H1399) to label nuclei. Images of stained vessels were acquired using a Leica SP8 confocal microscope with the Leica Application Suite (LAS) software. DAPT injections DAPT dissolved in 5% DMSO, 40% PEG300, 5% Tween 80, and 50% deionized water was injected intraperitoneally into 8-week-old mice at a dose of 50 mg/kg daily for 5 consecutive days. Mouse atherosclerosis model Mice at 8-9 weeks of age were injected intraperitoneally with murine Pcsk9 adeno-associated virus (pAAV/D377Y-mPcsk9; 1011 copies, produced by the Gene Therapy Program Vector Core at the University of Pennsylvania School of Medicine). Mice were then fed a high fat diet (Clinton/Cybulsky high fat rodent diet with regular casein and 1.25% added cholesterol, D12108c, Research Diet) for 8 weeks before sacrifice. Mouse whole aorta en face oil red O staining Mice were euthanized with an overdose of isoflurane (Henry Schein) and perfused through the left ventricle with PBS followed by 3.7% formaldehyde. The whole aorta was carefully dissected and fixed in 3.7% formaldehyde overnight at 4℃, then washed 3x with PBS. For whole aorta Oil Red O staining, the formaldehyde fixed whole aorta was opened longitudinally, pinned en face on a soft bottom dish, briefly rinsed with 78% methanol, then incubated with Oil Red O solution (0.2% Oil Red O in 7:2 methanol:1M NaOH). Stained tissue was washed 3x in 78% methanol and photographed using a Leica DFC295 digital microscopic camera. Mouse blood collection and measurement Blood samples were collected from mice by heart puncture at sacrifice. Blood was mixed with EDTA to prevent coagulation and was then centrifuged at 14000rpm at 4℃ for 10 minutes and the supernatant serum removed carefully by pipet. Total serum cholesterol was determined using a cholesterol oxidase/colorimetric assay (Abcam; #65390). Plasma TAG levels were measured using a commercially available kit (Wako Pure Chemicals). Statistical Analysis Data are presented as means ± SEM. Statistical significance was determined using Student’s t-test or ANOVA as indicated in the figure legends. P < 0.05 was considered significant. Author Contributions KT and MAS designed the project. KT performed in vitro experiments, in vivo mouse experiments and bioinformatics analysis. MChen performed in vivo mouse experiments. DJ helped with mouse dissections. MChanduri helped with in vitro experiments. HZ performed measurements of blood lipid levels from atherosclerosis mouse experiments. CFH provided equipment and advice for atherosclerosis experiments. YMK provided technical support for carrying out the experiments. KT wrote the first draft of the manuscript. KT and MAS wrote and reviewed the manuscript with the input of all authors. Disclosure & competing interests statement The authors declare no conflict of interest. Figure Legends Download figure Open in new tab Supplemental Figure 1: Beta-arrestins do not regulate flow-induced Notch signaling ( A ) Quantitative PCR for ARRB1 (top) and ARRB2 (bottom) mRNA in Scrambled, ARRB1 KD, ARRB2 KD and ARRB1/2 KD in HUVECs . Values are fold change relative to Scrambled control. ( B ) Representative images of Hes1 and Hoechst staining in HUVECs following knockdown of beta-arrestin isoforms with or without flow. Scale bar: 50µm. ( C ) Quantification of Hes1 nuclear intensity from ( B ). n = 1513 (Scrambled static), 1746 (Scrambled flow), 917 ( ARRB1 KD static), 738 ( ARRB1 KD flow), 1204 ( ARRB2 KD static), 1411 ( ARRB2 KD flow), 1032 ( ARRB1/2 KD static), 1174 ( ARRB1/2 KD flow). Statistics assessed using one-way ANOVA with Tukey post-hoc tests. Download figure Open in new tab Supplemental Figure 2: Validation of Notch1 fragment expression ( A ) Quantitative RT-PCR of Notch1 TMD and Notch1 ICD in samples analyzed in Figure 6 . ( B ) RNA-seq read counts for Notch1 and Notch signaling target genes following NICD overexpression. Download figure Open in new tab Supplemental Figure 3: LPHN2-Notch axis induces vascular inflammation through YAP ( A ) Quantification of the YAP nuclear:cytoplasmic ratio from Figure 7A . Statistical analysis used one-way ANOVA with Tukey post-hoc test. ( B ) Representative immunofluorescence image showing increased YAP nuclear translocation with and without flow at 12 dynes/cm2 for 16 hours, with DAPT at the indicated doses. Scale bar: 50µm. ( C ) Quantification of YAP nuclear intensity from ( B ). Statistics: one-way ANOVA with Tukey post-hoc tests. Download figure Open in new tab Supplemental Figure 4 : Latrophilin-Notch axis suppresses vascular inflammation Acknowledgements This work was supported by NIH grant R01 HL155543 and RO1 HL169510 to MAS. Funder Information Declared National Institutes of Health, https://ror.org/01cwqze88 , R01HL155543 , R01HL169510 Footnotes All figures and main text updated; author list updated References ↵ Libby , P . Inflammation in atherosclerosis . Arterioscler Thromb Vasc Biol 32 , 2045 – 2051 ( 2012 ). doi: 10.1161/ATVBAHA.108.179705 OpenUrl Abstract / FREE Full Text ↵ Herrington , W. , Lacey , B. , Sherliker , P. , Armitage , J. & Lewington , S . Epidemiology of Atherosclerosis and the Potential to Reduce the Global Burden of Atherothrombotic Disease . Circ Res 118 , 535 – 546 ( 2016 ). doi: 10.1161/CIRCRESAHA.115.307611 OpenUrl Abstract / FREE Full Text ↵ Davies , P. F . Hemodynamic shear stress and the endothelium in cardiovascular pathophysiology . 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Schwartz bioRxiv 2025.07.01.662676; doi: https://doi.org/10.1101/2025.07.01.662676 Share This Article: Copy Citation Tools Latrophilin-2 mediates flow activation of Notch to control vascular endothelial phenotype and atherosclerosis Keiichiro Tanaka , Minghao Chen , Divyesh Joshi , Manasa Chanduri , Hanming Zhang , Carlos Fernandez-Hernando , Yoo-Min Koh , Martin A. 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