Pharmacological Activation of NO-Sensitive Guanylyl Cyclase Ameliorates Obesity-Induced Arterial Stiffness

Pharmacological Activation of NO-Sensitive Guanylyl Cyclase Ameliorates Obesity-Induced Arterial Stiffness | 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 Pharmacological Activation of NO-Sensitive Guanylyl Cyclase Ameliorates Obesity-Induced Arterial Stiffness Enkhjargal Budbazar , Aylin Balmes , Danielle Elliott , Lisette Peres Tintin , View ORCID Profile Timo Kopp , View ORCID Profile Susanne Feil , View ORCID Profile Robert Feil , View ORCID Profile Tilman E. Schäffer , View ORCID Profile Francesca Seta doi: https://doi.org/10.1101/2025.02.17.638762 Enkhjargal Budbazar 1 Vascular Biology Section, Department of Medicine, Boston University Chobanian & Avedisian School of Medicine , Boston, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Aylin Balmes 2 Institute of Applied Physics, University of Tübingen , Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site Danielle Elliott 1 Vascular Biology Section, Department of Medicine, Boston University Chobanian & Avedisian School of Medicine , Boston, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Lisette Peres Tintin 1 Vascular Biology Section, Department of Medicine, Boston University Chobanian & Avedisian School of Medicine , Boston, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Timo Kopp 3 Interfaculty Institute of Biochemistry, University of Tübingen , Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Timo Kopp Susanne Feil 3 Interfaculty Institute of Biochemistry, University of Tübingen , Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Susanne Feil Robert Feil 3 Interfaculty Institute of Biochemistry, University of Tübingen , Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Robert Feil Tilman E. Schäffer 2 Institute of Applied Physics, University of Tübingen , Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Tilman E. Schäffer Francesca Seta 1 Vascular Biology Section, Department of Medicine, Boston University Chobanian & Avedisian School of Medicine , Boston, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Francesca Seta For correspondence: setaf{at}bu.edu Abstract Full Text Info/History Metrics Preview PDF ABSTRACT Objective Arterial stiffness, or loss of elastic compliance in large arteries, is an independent precursor of cardiovascular disease (CVD) 1 and dementia 2 . Akin to anti-hypertensive and lipid-lowering drugs, arterial de-stiffening therapies could be beneficial at decreasing CVD risk. We previously discovered that enhanced cytoskeletal actin polymerization in vascular smooth muscle cells (VSMCs) contributes to increased arterial stiffness 3 . In aortas and VSMCs, we previously found that decreased NO-sensitive guanylyl cyclase (NO-GC), the NO receptor which synthesizes cGMP, caused downregulation of cGMP-dependent protein kinase I (cGKI) and of its target vasodilator-stimulated phosphoprotein (pVASP S239 ), leading to increased cytoskeletal actin polymerization 3 . In the current study, we tested whether activating NO-GC with an NO-GC activator (cinaciguat) modulates pVASP S239 and cytoskeletal actin polymerization in VSMCs, thereby preventing obesity-induced arterial stiffness. Approach & Results Cinaciguat administration (5 mg/kg) to high fat, high sucrose diet (HFHS)-fed mice, our established model of arterial stiffness 4 , (1) decreased pulse wave velocity, the in vivo index of arterial stiffness, without affecting blood pressure, (2) increased aortic pVASP S239 levels, and (3) decreased the ratio of filamentous (F) to globular (G) actin, compared to vehicle administration. In cultured VSMCs, cinaciguat (10 μmol/L) increased pVASP S239 levels and decreased the F/G actin ratio at baseline and after stimulation with the cytokine tumor necrosis factor α (TNFα), used to mimic the inflammatory milieu of HFHS aortas. These effects were abrogated in aortas and VSMCs from mice with smooth muscle-specific cGKI deletion (cGKI SMKO ), while being mimicked by a cell-permeable cGMP analog (8-Br-cGMP, 1 μmol/L), which also decreased VSMC stiffness in vitro . Conclusions Collectively, our data strongly support the notion that pharmacological NO-GC activation would be beneficial in decreasing obesity-associated arterial stiffness by decreasing VSMC cytoskeletal actin hyper-polymerization. If translated to humans, NO-GC activators could become a viable approach to clinically treat arterial stiffness, which remains an unmet medical need. INTRODUCTION Cardiovascular disease (CVD) remains the leading cause of death globally, claiming more lives each year than all forms of cancer combined 5 . Arterial stiffness, or loss of elastic compliance of large arteries, which occurs with aging and obesity 6 , is an independent risk factor for CVD 1 . Compelling clinical evidence demonstrated that pulse wave velocity (PWV), the gold standard measure of arterial stiffness, strongly associates with hypertension 7 , heart failure 8 , and dementia 9 . Thus, akin to anti-hypertensive or anti-hyperlipidemic medications, aortic de-stiffening therapies may help prevent cardiovascular events. Cellular and extracellular pathogenic mechanisms of aortic wall stiffening have been identified 10 ; yet the translation of these pre-clinical findings into targeted therapies remains elusive. We previously showed that in a mouse model of dietary obesity, arterial stiffness, measured in vivo by PWV, increases after two months of high fat, high sucrose (HFHS) feeding, preceding the development of hypertension 4 , consistent with clinical findings 6 . At the cellular level, we reported that excessive cytoskeletal actin polymerization in vascular smooth muscle cells (VSMCs) is a major contributor to increased arterial stiffness 3 , 11 . We further discovered that increased cytoskeletal actin polymerization in VSMCs is dependent on the NO-sensitive guanylyl cyclase (NO-GC)/cyclic guanosine monophosphate (cGMP)-dependent protein kinase I (cGKI, aka PKGI)/phosphorylated vasodilator stimulated phosphoprotein (pVASP S239 ) signaling cascade 3 . In this study, we sought to determine whether activating NO-GC with a commercially available NO-GC activator (cinaciguat) modulates pVASP S239 and cytoskeletal actin polymerization in VSMCs, thereby preventing obesity-induced arterial stiffness. MATERIALS & METHODS Data and supporting material are available to the research community upon reasonable request. Model of diet-induced obesity and drug treatments Mice with a floxed cGMP-dependent protein kinase I allele (cGKI fl/fl ) 12 , were obtained from Dai Fukumura (Massachusetts General Hospital, Boston) under an MTA between BU and MGH. Smooth muscle-specific cGKI deletion was achieved by breeding cGKI fl/fl mice with transgenic mice expressing a smooth muscle α-actin promoter-driven tamoxifen-activated CreER T2 recombinase 13 . At six weeks of age, cGKI fl/fl / SMA-CreER T2+ mice were fed a diet containing tamoxifen (500 mg/kg, TD.130857, Teckland, Inotiv) or a standard chow for five days, to generate mice with smooth muscle-specific cGKI deletion (cGKI SMKO mice; n=12) and littermate controls (cGKI ctrl mice; n=12), respectively. At two months of age, mice received a normal diet (ND: 4.5% fat, 0% sucrose; n=6) or a high fat, high sucrose diet (HFHS: 35.5% fat (lard), 16.5% sucrose; n=18), and water ad libitum for 6 months. ND and HFHS were custom formulated to contain the same macronutrients, except for fat and sucrose (catalog # D09071702 and D09071703, Research Diets, New Brunswick, NJ, USA). After six months of HFHS, cGKI ctrl (n=9) and cGKI SMKO (n=9) mice were randomized to receive vehicle or cinaciguat (5 mg/kg body weight), daily for 3 weeks by oral gavage. Experimental timeline is illustrated in Fig. 1A . To note, this dose of cinaciguat was chosen because, reportedly, not having a significant effect on blood pressure 14 . Mice were housed in the Boston University Medical Campus Animal Facility in temperature- and humidity-controlled rooms, on a 12-hour light/dark cycle, in groups of 4-5 mice per cage. Animal procedures were approved by the institutional animal care and use committee (IACUC) at Boston University. Since our previous study on the role of NO-GC/PKG/pVASP S239 signaling pathway on VSMC stiffness was conducted on male mice 3 , the current study used male but not female mice. Download figure Open in new tab Figure 1. (A) Experimental timeline: at six weeks of age, mice with floxed cGKI and smooth muscle α-actin (SMA)-Cre ER T2 recombinase (SMA-CreER T2 /cGKI fl/fl ) were treated with vehicle or tamoxifen (TXF) to generate mice with smooth muscle-specific cGKI deletion and littermate controls; at 8 weeks of age, mice received normal (ND) or high fat, high sucrose diet (HFHS); after six months, HFHS-fed mice were randomized to receive vehicle or cinaciguat (5mg/kg/d for 3 weeks). (B) Cinaciguat administration (5 mg/kg/day) for 3 weeks by oral gavage decreased pulse wave velocity (PWV, m/s), the in vivo index of arterial stiffness, in HFHS-fed mice; p=0.04 HFHS vs HFHS/CNGT by one-way ANOVA. (C) Cinaciguat administration did not significantly affect systolic and diastolic blood pressure, compared to vehicle; p=0.36 and p=0.31, respectively. (D) Filamentous (F) actin is formed by polymerization of globular (G) actin, a process mediated, among others, by VASP and inhibited by phosphorylated VASP. (E) Representative Western blot indicates that cinaciguat administration significantly decreased F/G actin ratio in aortas of HFHS-fed mice, compared to vehicle; p=0.04. (F) Cinaciguat administration significantly increased pVASP S239 levels in aortas of HFHS-fed mice, compared to vehicle; quantitation in graph indicates pVASP S239 band intensities normalized to loading control (Ponceau S-stained Western blot membranes) and expressed as fold change of vehicle; p=0.007. Levels of cGKI and NO-GCβ1 in aortic homogenates did not change significantly between vehicle- and cinaciguat-treated HFHS-fed mice. Arterial stiffness measurements Pulse wave velocity (PWV), the in vivo index of arterial stiffness, was measured on experimental mice as we previously described 3 , 4 , at three time points: (1) at baseline (cGKI Ctrl , n=12; cGKI SMKO , n=12); (2) after 6 months of ND and HFHS (cGKI Ctrl /ND, n=3; cGKI SMKO /ND, n=3; cGKI Ctrl /HFHS, n=9; cGKI SMKO /HFHS, n=9); and after randomization of HFHS-fed mice to vehicle or cinaciguat (cGKI Ctrl /HFHS/vehicle, n=5; cGKI SMKO /HFHS/vehicle, n=5; cGKI Ctrl /HFHS/cinaciguat, n=4; cGKI SMKO /HFHS/cinaciguat, n=4). Briefly, mice from the various experimental groups were kept recumbent and lightly anesthetized with 1-2% isofluorane on a heated platform to maintain body temperature and heart rate in the 400-500 bpm range during the procedure. A high-resolution Doppler ultrasound (Vevo3100, Fujifilm Visualsonics, Toronto, Canada) was used to image the aorta from the diaphragm to the iliac bifurcation (B-mode, M250 transducer), as we previously described 3 , 4 . Flow waves and simultaneous electrocardiogram (ECG) were obtained from two locations along the aorta in Power Doppler mode. Acquisitions with unstable heart rate (HR < 400bpm) or unclear QRS peaks in the ECG were excluded from further analysis (4 cGKI Ctrl and 4 cGKI SMKO ). Arrival times of the flow waves at proximal and distal locations along the aorta were measured by the foot-to-foot method using the R-wave of the ECG as a fiducial point, on at least 5 cardiac cycles for each mouse. PWV was calculated as the ratio of the distance and the difference in arrival times of flow waves at the two locations (m/s). Blood pressure measurements Mice (HFHS/vehicle, n=4 and HFHS/cinaciguat, n=4) were accustomed to the plethysmography system (BP2000, Visitech) by 10-minute training sessions for two consecutive days. On the day of the measurement, mice were carefully handled, to avoid distress, and gently restrained inside dark cassettes placed on a warm platform. Blood pressure tracings were acquired from the middle caudal artery by multiple tail cuff inflation/deflation cycles, for a total of ten minutes for each mouse. Pressure values deviating two standard deviations from the mean were excluded from further analysis. At least twenty systolic and diastolic pressure and heart rate values were obtained for each mouse. Pruned replicates for individual mice were averaged before proceeding to statistical group analysis. VSMC culture and cinaciguat treatments VSMCs were isolated from aortas of 8-week-old cGKI Ctrl (n=3) and cGKI SMKO (n=3) mice by enzymatic digestion with 1 mg/mL collagenase and 2 mg/mL elastase solutions, as we previously described 3 . Freshly isolated VSMCs were seeded on collagen-coated plates and allowed to attach, undisturbed for 3 days, with DMEM containing 1 g/L glucose, 10% fetal bovine serum (S11150, R&D Systems), and 1% antibiotic-antimycotic solution (15240062, 100x, Gibco™). At confluency, VSMCs were sub-cultured in standard culture dishes (Falcon™ 353002, Fisher Scientific) up to passage 5. VSMCs were made quiescent in FBS-free DMEM medium for 24 hours, prior to treatment with vehicle, 10 μmol/L cinaciguat (Sigma-Aldrich) or 1 μmol/L a cell membrane-permeable cGMP analog 8-Br-cGMP (SigmaAldrich) for 15 minutes or 24 hours. In a subset of experiments, VSMCs or aortas dissected from cGKI Ctrl (n=4) and cGKI SMKO (n=4) mice were pre-incubated with 10 μmol/L cinaciguat for 1 hour before stimulation with 10 ng/mL TNFα for additional 24 hours. At the end of the treatment periods, cells and aortas were collected to prepare protein homogenates for Western blot or F/G actin separation, respectively, as described below. Western Blot Aortas and VSMC homogenates or F and G actin fractions were collected in RIPA or LAS2 lysis buffer freshly supplemented with protease inhibitors, and manually homogenized on ice. Protein concentration was measured using the BCA assay (23225, PierceTM BCA Protein Assay Kit, Thermo Fisher Scientific). Equal amounts of protein (25 μg) were separated by SDS-PAGE in NuPAGE 4-12% Bis-Tris gels (NP0335PK2, Invitrogen) or 4-12% Criterion™ XT Bis-Tris (3415023/3415024, Bio-Rad Laboratories, Inc.), then transferred to 0.45-μm pore PVDF membranes. Membranes were incubated overnight at 4ºC with the following primary antibodies: phosphorylated VASP at serine 239 (3114, CST, 56 ng/mL), cGKI (3248, CST, 0.27 μg/mL), sGC isoform β1 (160897, Cayman Chemical, 0.2 μg/mL), pan-actin (AAN01, Cytoskeleton, 0.5 μg/mL), VCAM1 (14694, CST, 0.1 μg/mL), phosphorylated p65-NFκB (3033, CST, 57 ng/mL), GAPDH (2118, CST, 42 ng/mL). The membranes were then washed with TBS-T and exposed to the chemiluminescent substrate ECL (R1002, Kindle Biosciences, LLC) to visualize protein bands using the optical imager iBright (ThermoFisher) with automatic exposure settings. Protein band intensities were analyzed using ImageJ software (NIH, USA) and normalized to GAPDH or Ponceu S-stained membranes, which were used as loading controls. Band intensities from the same experimental group were averaged and the data were expressed in arbitrary units, as fold change of controls, for each replicate Western blot. F and G actin separation Separation of the filamentous (F) and globular (G) actin fractions was performed with G-actin/F-actin in vivo assay kit BK037 (Cytoskeleton, USA), following the manufacturer’s recommendations. Briefly, aortas and VSMCs were lysed in LAS2 buffer, then incubated at 37°C for 10 minutes. 100 μL lysates were centrifuged at 350 x g for 5 minutes; cleared lysates were then centrifuged at 100,000 x g (Optima Max ultracentrifuge, BeckmanCoulter) for 1 hour at 4°C. Supernatants, containing the G actin, were collected in clean tubes while pellets, containing the F actin, were dissolved in 100 μL LAS01 buffer for 1 hour on ice. F and G actin fractions were then flash-frozen in liquid nitrogen and stored at −80°C until Western blot. VSMC stiffness (Young’s modulus) was measured on freshly isolated cells using a commercial atomic force microscopy (AFM) setup (MFP3D-BIO, Asylum Research, Santa Barbara, CA) mounted on an inverted optical microscope (Ti-S, Nikon, Tokyo, Japan) and a cantilever with a pyramidal tip and a nominal spring constant of 10 pN/nm (MLCT-BIO C, Bruker; the spring constant was calibrated using thermal calibration before each measurement). Force maps of 8×8 µm 2 and 10×10 pixels were recorded centrally on a VSMC with a retract distance of 2 µm, a sampling rate of 2 Hz, and a trigger deflection of 100 nm. Each cell was measured twice (before and after treatment with 8-Br-cGMP, 1 mmol/L, 1 h). Data were analyzed in Igor Pro (WaveMetrics, Lake Oswego, OR, USA). Statistical analysis Statistical analyses were performed with Graphpad Prism v.9.2 software. Datasets were first subjected to a normality test to determine whether they followed a Gaussian distribution. Means of normally distributed datasets were compared using Student’s t-test. Data that did not follow a Gaussian distribution were analyzed using non-parametric tests. Experiments with multiple treatment and genotype groups were analyzed using one-way ANOVA with appropriate post-hoc multiple comparison analysis. P values < 0.05 were considered significant. Across-test multiple test correction was not applied. For experiments with VSMCs, replicate experiments utilized individual VSMC preparations from different mice, to ensure biological, rather than technical, replication. Data are expressed as the mean ± SEM and reported as fold-change vs control for each replicate experiment before averaging by genotype or treatment group. RESULTS High fat, high sucrose diet (HFHS) feeding for six months significantly increased arterial stiffness in mice, measured in vivo by pulse wave velocity (PWV), compared to normal diet (ND) (2.7 ± 0.1 m/s in ND, n=8 vs 3.9 ± 0.20 m/s in HFHS, n=7, p=0.0001) ( Fig. 1B ), consistent with our previous reports 4 , 15 . Treatment of HFHS-fed mice with the NO-GC activator cinaciguat (CNGT; 5 mg/kg/day for 3 weeks by oral gavage) significantly decreased PWV (3.9 ± 0.2 m/s in HFHS/vehicle, n=7 vs 3.1 ± 0.1 m/s in HFHS/CNGT, n=4; p=0.04) ( Fig. 1B ). Administration of cinaciguat for three weeks did not induce statistically significant changes in systolic or diastolic blood pressures (SBP: 114.1 ± 8.1 mmHg in HFHS/vehicle, n=4 vs 104.5 ± 5.2 mmHg in HFHS/CNGT, n=4, p=0.36; DBP: 79.3 ± 4.7 mmHg in HFHS/vehicle, n=4 vs 87.8 ± 6.1 mmHg in HFHS/CNGT, n=4, p=0.31) ( Fig. 1C ). We previously reported that (1) aortic stiffness was associated with increased cytoskeletal actin polymerization, measured as filamentous (F) to globular (G) actin ratio, in aortas and VSMCs 3 , and (2) F/G actin ratios were inversely correlated with cGMP-dependent protein kinase I (cGKI)-dependent VASP phosphorylation at serine 239 3 ( Fig. 1D ). Therefore, given that NO-GC activation with cinaciguat should bring about cGKI activation, we assessed whether cinaciguat affects cytoskeletal actin polymerization and pVASP S239 levels via cGKI in aortas of HFHS-fed mice. We found that F/G actin ratio was significantly decreased (1.4 ± 0.4 A.U. in HFHS/vehicle, n=4 vs 0.3 ± 0.2 A.U. in HFHS/CNGT, n=4; p=0.04) ( Fig. 1E ), whereas pVASP S239 levels were significantly increased (1.0 ± 0.1 A.U. in HFHS/vehicle, n=4 vs 1.5 ± 0.1 A.U. in HFHS/CNGT, n=4; p=0.007) in aortas of HFHS-fed mice after cinaciguat, compared to HFHS-fed mice receiving vehicle ( Fig. 1F ). Cinaciguat administration did not affect the levels of NO-GC (subunit β1) or cGKI ( Fig. 1F ). Similarly to the aorta, treatment of cultured VSMCs with cinaciguat (10 μmol/L) decreased F/G actin ratio (1.0 ± 0.0 A.U. in vehicle, n=6 vs 0.6 ± 0.0 A.U. in CNGT, n=6; p=0.007) ( Fig. 2A ) and increased pVASP S239 levels (1.0 ± 0.0 A.U. in vehicle, n=6 vs 2.9 ± 0.6 A.U. in CNGT, n=10; p=0.009), compared to vehicle ( Fig. 2B ). The effects of cinaciguat on F/G actin and pVASP S239 were mimicked by treating VSMCs with a stable cGMP analog (8-Br-cGMP, 1μmol/L) ( Fig. 2A-B ). Likewise, cinaciguat increased pVASP S239 in VSMCs treated with TNFα (10 ng/ml), an inflammatory cytokine that we previously showed increases in aortas after HFHS feeding 4 , 16 . These pVASP S239 increases were blunted in VSMCs with cGKI deletion (cGKI SMKO ) (1.0 ± 0.0 A.U. in cGKI Ctrl /TNFα/vehicle, n=3; 9.0 ± 0.7 A.U. in cGKI Ctrl /TNFα/CNGT, n=3; 3.9 ± 0.9 A.U. in cGKI SMKO /TNFα/CNGT, n=3; p<0.0001 cGKI Ctrl /TNFα/vehicle vs cGKI Ctrl /TNFα/CNGT; p=0.0022 cGKI Ctrl /TNFα/CNGT vs cGKI SMKO /TNFα/CNGT) ( Fig. 2C , quantitation in 2D ). Inflammatory molecules such as VCAM1 and phosphorylated p65-NFκB remained elevated in response to TNFα stimulation, independently of vehicle or cinaciguat treatment ( Fig. 2E ). The effects of cinaciguat on pVASP S239 were abrogated by cGKI deletion in cGKI SMKO aortas treated with TNFα ex vivo , compared to TNFα-treated cGKI Ctrl aortas (1.0 ± 0.0 A.U. in cGKI Ctrl /TNFα/CNGT, n=4 vs 0.6 ± 0.0 A.U. in cGKI SMKO /TNFα/CNGT, n=4; p=0.02) ( Fig. 2F ). Moreover, smooth muscle cGKI deletion increased PWV in cGKI SMKO mice, compared to littermate controls (2.6 ± 0.1 m/s in cGKI Ctrl , n=8 vs 3.5 ± 0.3 m/s in cGKI SMKO , n=8; p=0.01) ( Fig. 2G ). Cinaciguat failed to decrease PWV in HFHS-fed cGKI SMKO mice, compared to vehicle-treated mice (4.3 ± 0.4 m/s in cGKI Ctrl /HFHS/vehicle, n=9 vs 4.6 ± 0.7 m/s in cGKI SMKO /HFHS/CNGT, n=4; p=0.74) ( Fig. 2H ). Lastly, treatment with 8-Br-cGMP (1 mmol/L) decreased VSMC stiffness on isolated VSMCs, measured by atomic force microscopy (6.7 ± 1.2 kPa in vehicle, n=25 vs 3.8 ± 1.0 kPa in 8-Br-cGMP, n=24; p=0.002) ( Fig. 2I ). Download figure Open in new tab Figure 2. (A) Representative Western blot of the F and G actin fractions in VSMCs treated with a cGMP analog (8-Br-cGMP, 1 μmol/L, 1 h) or cinaciguat (CNGT, 10 μmol/L, 1 h); quantitation of band intensities in graph; p=0.0007. (B) CNGT (10 μmol/L, 1 h) or 8-Br-cGMP (1μmol/L, 1 h) significantly increased pVASP S239 levels in VSMC, compared to vehicle; quantitation in graph indicates pVASP S239 band intensities normalized to loading control (GAPDH) and expressed as fold change of vehicle; p=0.009. (C) Western blot of pVASP S239 levels in VSMCs from control (cGKI Ctrl ) mice and mice with smooth muscle cGKI-deletion (cGKI SMKO ) stimulated with the cytokine TNFα (10 ng/ml), or vehicle; quantitation in graph (D) indicates pVASP S239 band intensities normalized to loading control (GAPDH) and expressed as fold change of vehicle; p<0.0001 cGKI Ctrl /TNFα/CNGT vs cGKI Ctrl /TNFα/vehicle; p<0.05 cGKI SMKO /TNFα/CNGT vs cGKI SMKO /TNFα/vehicle; p<0.0022 cGKI SMKO /TNFα/CNGT vs cGKI Ctrl /TNFα/CNGT. (E) CNGT (10 μmol/L) did not significantly affect the expression of inflammatory markers VCAM1 and phosphorylated p65-NFκB in cGKI Ctrl or cGKI SMKO VSMCs stimulated with the cytokine TNFα, compared to vehicle. (F) CNGT (10 μmol/L) failed to increase pVASP S239 expression in cGKI SMKO aortas stimulated ex vivo with the cytokine TNFα (10 ng/ml), compared to vehicle. (G) CNGT (10 μmol/L) failed to decrease PWV (m/s), the in vivo index of arterial stiffness, in HFHS-fed cGKISM KO mice, compared to HFHS-fed cGKI Ctrl mice; p=0.74. (H) Representative images of atomic force microscopy (AFM) measurement of stiffness (kPa) of VSMCs treated with vehicle or 8-Br-cGMP (1 mmol/L) ; bright field (BF) image shows an individual cell; heatmap indicates cell stiffness; p=0.002. Scale = 2μm. DISCUSSION When the aorta and other large arteries stiffen with aging or obesity, they lose their intrinsic capacity of buffering the pulsatility of cardiac contraction resulting in poor cardiac perfusion and elevated pulse pressures propagating to the downstream microcirculation, particularly to high flow/low resistance organs, such as the brain and kidney. Structural and functional changes in those organs, known as target organ damage, are a precursor of overt CVD and dementia 17 . Thus, anti-stiffening therapies, akin to anti-hypertensive or lipid-lowering medications, could become clinically relevant to prevent target organ damage, thereby decreasing the incidence of CVD, particularly among increasingly aging and overweight/obese populations. Here we report that pharmacological activation of NO-GC, the NO receptor in VSMCs, is effective at decreasing obesity-induced arterial stiffness in a mouse model of metabolic syndrome that, we previously showed, closely mimics the human pathology 4 . Our novel findings demonstrate that (1) the NO-GC activator cinaciguat decreases aortic stiffness, measured in vivo by PWV, in obese mice; (2) cinaciguat increases pVASP S239 and decreases cytoskeletal actin polymerization, even in an inflammatory milieu, and (3) these effects are dependent on the cGMP-dependent protein kinase I (cGKI, aka PKGI), as demonstrated by using VSMCs and aortas from mice with smooth muscle-specific cGKI deletion (cGKI SMKO ) (illustrated in the graphical abstract ). Of note, the aortic de-stiffening effect of cinaciguat was independent of blood pressure because the cinaciguat dose we employed did not significantly affect blood pressure ( Fig. 1C ), consistent with previous reports 14 . NO-GC is a crucial enzyme for cardiovascular homeostasis 18 . Upon binding of the endogenous ligand NO to its ferrous heme moiety, NO-GC synthesizes cGMP, a second messenger required for VSMC relaxation. In the settings of oxidative stress, when NO bioavailability is diminished and/or the heme moiety is oxidized to its ferric form and unable to bind NO, NO-GC becomes inactive. Hence, NO-GC is an attractive pharmaceutical target for CVD, in which oxidative stress and low NO bioavailability are generally a major culprit. Several NO-GC modulators, classified as NO-GC stimulators and NO-GC activators based on their pharmacodynamic properties, have been developed 19 . NO-GC stimulators, of which riociguat is the lead compound, are able to activate native NO-GC, but not its oxidized/heme-free form 19 . In contrast, cinaciguat and other NO-GC activators are able to selectively activate the oxidized/heme-free enzyme, thereby stimulating cGMP synthesis even under pathological conditions in tissues containing oxidized, NO-insensitive NO-GC 19 . Because of their unique pharmacodynamic property, we posited that, in the vasculature of HFHS-fed obese mice, whose NO bioavailability is drastically decreased and oxidative stress significantly increased 4 , a NO-GC activator would sustain cGMP production more efficiently than a NO-GC stimulator. Hence cinaciguat was the drug of choice in the current study to determine the effects of NO-GC activation on obesity-induced arterial stiffness. We did not test other NO-GC modulators. Therefore, we cannot generalize our findings to other NO-GC activators or stimulators. Moreover, the translational value of cinaciguat is currently limited by its low solubility, poor bioavailability, and short half-life requiring frequent administrations 19 . The development of NO-GC activators like runcaciguat with improved bioavailability compared to cinaciguat 20 , will be crucial to translate our pre-clinical findings to the clinic. Our previous study on the role of NO-GC/cGKI/pVASP S239 cascade in VSMC actin polymerization and stiffness was conducted with male mice 3 . Therefore we did not use female mice, a limitation of the current study. Additional studies to investigate potential sex dimorphism in the effects of cinaciguat or other NO-GC modulators on arterial stiffness are essential. Polymerization of non-muscle cytoskeletal actin ¾mainly β- and g-actin, can increase VSMC stiffness and VSMC tension development in response to mechanical stimuli, thereby sustaining vessel tone and diameter 21 , independently of myosin light chain phosphorylation and actino-myosin cross-bridge cycles 22 . We 3 , 11 and others 23 demonstrated that excessive VSMC cytoskeletal actin polymerization and VSMC stiffness contribute to aortic wall stiffening suggesting regulation of actin polymerization as an appealing, albeit challenging, site for therapeutic intervention 24 , as recently suggested also by others 10 , 25 . A major mediator of actin filament polymerization, VASP interacts with vinculin, zyxin, and profilins 26 , 27 , all crucial components of actin filament assembly at the focal adhesions, which are subcellular sites of actin filament attachment to the extracellular matrix, essential for maintaining a mechanically competent aortic wall. VASP overexpression has been shown to induce F-actin assembly 28 whereas VASP phosphorylation at serine 239 is sufficient to inhibit actin filament polymerization 29 . We previously demonstrated that aortic VASP ser239 phosphorylation is decreased in models of arterial stiffness 3 , 15 . Overall, our findings that cinaciguat decreases F/G actin ratios in aortas and VSMCs, and arterial stiffness in vivo , are consistent with cinaciguat increasing levels of pVASP ser239 , as we similarly reported in platelets 30 . Moreover, our study highlights a crucial role of smooth muscle cGKI in regulating pVASP ser239 and arterial stiffness, as demonstrated by the fact that smooth muscle cGKI deletion increases PWV in young cGKI SMKO mice ( Fig. 2G ). Our study also demostrates that cGKI is a downstream effector of cinaciguat since cinaciguat failed to decrease PWV in HFHS-fed cGKI SMKO mice ( Fig. 2H ). Our results do not exclude that cGKI may regulate actin polymerization via additional mechanisms, other than pVASP. For instance, cGKI is able to phosphorylate cofilin1, an actin depolymerizing factor, resulting in cofilin1 inhibition and decreased filamentous (F) actin 31 . Further studies are warranted to explore these cGKI-dependent mechanisms in VSMCs and how they affect VSMC stiffness. Nonetheless, our brief report demonstrates a beneficial effect of NO-GC activation against arterial stiffness and encourages further studies on the therapeutic potential of the NO-GC/cGKI/pVASP signaling pathway to tackle arterial stiffness, which remains an unmet medical need. FUNDING SOURCES This work was supported by NIH R01 grant HL136311 to FS; BU CTSI Integrated Pilot grant 1UL1TR001430 to FS; a BU CTSI Voucher Award 1UL1TR001430 to EB; by the Deutsche Forschungsgemeinschaft (German Research Foundation) – Projektnummer 335549539 – GRK 2381 RF/SF/TS; by the Reinhard Frank-Stiftung; and by an Add-on Fellowship of the Joachim Herz Foundation to AB. ACKNOWLEDGEMENTS We would like to thank the Boston University Medical Campus Analytical Instrumentation (Drs. Lynn Deng and Matthew Au) and Cellular Imaging Cores (Dr. Michael Kirber) for their expert technical support. Figures 1A, 1D , and the graphical abstract were created with BioRender.com. AUTHORS CONTRIBUTIONS AB, EB, DE, and LPT performed experiments and reviewed the manuscript; TK provided cells for the AFM measurement; SF, RF, and TS provided constructive criticism to the manuscript; FS designed and coordinated the study, performed PWV measurements, analyzed and interpreted the data, and wrote the manuscript. DISCLOSURES The authors have no conflicts of interest to report. Non-standard Abbreviations and Acronyms AFM atomic force microscopy CNGT cinaciguat CVD cardiovascular disease CreER T2 tamoxifen-activated Cre recombinase cGMP cyclic guanosine 3’,5’-monophosphate 8-Br-cGMP 8-bromo cyclic guanosine 3’,5’-monophosphate cGKI cGMP-dependent protein kinase I cGKI SMKO mice with smooth muscle-specific cGKI deletion ND normal diet NO nitric oxide HFHS high fat, high sucrose diet pNFκB phosphorylated nuclear factor κB pVASP phosphorylated vasodilator-stimulated phosphoprotein PWV pulse wave velocity NO-GC NO-sensitive guanylyl cyclase SMMHC smooth muscle myosin heavy chain TNFα tumor necrosis factor α VASP vasodilator-stimulated phosphoprotein VCAM1 vascular cell adhesion molecule 1 VSMCs vascular smooth muscle cells REFERENCES 1. ↵ Mitchell GF , Hwang SJ , Vasan RS , Larson MG , Pencina MJ , Hamburg NM , Vita JA , Levy D , Benjamin EJ . Arterial stiffness and cardiovascular events: the Framingham Heart Study . Circulation . 2010 ; 121 : 505 – 511 . 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Platelets . 2024 ; 35 . doi: 10.1080/09537104.2024.2313359 . OpenUrl CrossRef 31. ↵ Negash S , Narasimhan SR , Zhou W , Liu J , Wei FL , Tian J , Usha Raj J. Role of cGMP-dependent protein kinase in regulation of pulmonary vascular smooth muscle cell adhesion and migration: effect of hypoxia . Am J Physiol Heart Circ Physiol . 2009 ; 297 . doi: 10.1152/AJPHEART.00077.2008 . OpenUrl CrossRef View the discussion thread. Back to top Previous Next Posted February 23, 2025. Download PDF Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. 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