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Abstract Calciprotein particles (CPPs) are an endogenous buffering system, clearing excessive amounts of Ca2+ and PO4 3- from the circulation and thereby preventing ectopic mineralization. CPPs circulate as primary CPPs (CPP1), which are small spherical colloidal particles, and can aggregate to form large, crystalline, secondary CPPs (CPP2). Even though it has been reported that CPPs are toxic to vascular smooth muscle cells (VSMC) in vitro, their effect(s) on the vasculature remain unclear. Here we have shown that CPP1, but not CPP2, increased arterial stiffness ex vivo. Interestingly, the effects were more pronounced in the abdominal infrarenal aorta compared to the thoracic descending aorta. Further, we demonstrated that CPP1 affected both endothelial and VSMC function, impairing vasorelaxation and contraction respectively. Concomitantly, arterial glycosaminoglycan accumulation was observed as well, which is indicative of an increased extracellular matrix stiffness. However, these effects were not observed in vivo. Hence, we concluded that CPP1 can induce vascular dysfunction.

Many genetic mutations adversely affect the structure and function of load-bearing soft tissues, with clinical sequelae often responsible for disability or death. Parallel advances in genetics and histomechanical characterization provide significant insight into these conditions, but there remains a pressing need to integrate such information. We present a novel genotype-to-biomechanical phenotype neural network (G2Φnet) for characterizing and classifying biomechanical properties of soft tissues, which serve as important functional readouts of tissue health or disease. We illustrate the utility of our approach by inferring the nonlinear, genotype-dependent constitutive behavior of the aorta for four mouse models involving defects or deficiencies in extracellular constituents. We show that G2Φnet can infer the biomechanical response while simultaneously ascribing the associated genotype by utilizing limited, noisy, and unstructured experimental data. More broadly, G2Φnet provides a powerful method and a paradigm shift for correlating genotype and biomechanical phenotype quantitatively, promising a better understanding of their interplay in biological tissues.

Abstract Ex vivo characterisation of arterial biomechanics enables detailed discrimination of the various cellular and extracellular contributions to arterial stiffness. However, ex vivo biomechanical studies are commonly performed under quasi-static conditions, whereas dynamic biomechanical behaviour (as relevant in vivo) may differ substantially. Hence, we aim to (1) develop an integrated set-up for quasi-static and dynamic biaxial biomechanical testing, (2) quantify set-up reproducibility, and (3) illustrate the differences in measured arterial stiffness between quasi-static and dynamic conditions. Twenty-two mouse carotid arteries were mounted between glass micropipettes and kept fully vasodilated. While recording pressure, axial force (F), and inner diameter, arteries were exposed to (1) quasi-static pressure inflation from 0 to 200 mmHg; (2) 300 bpm dynamic pressure inflation (peaking at 80/120/160 mmHg); and (3) axial stretch (λ z ) variation at constant pressures of 10/60/100/140/200 mmHg. Measurements were performed in duplicate. Single-point pulse wave velocities (PWV; Bramwell-Hill) and axial stiffness coefficients (c ax = dF/dλ z ) were calculated at the in vivo value of λ z . Within-subject coefficients of variation were ~ 20%. Dynamic PWVs were consistently higher than quasi-static PWVs (p

Accurate information on vascular smooth muscle cell (VSMC) content, orientation, and distribution in blood vessels is indispensable to increase understanding of arterial remodeling and to improve modeling of vascular biomechanics. We have previously proposed an analysis method to automatically characterize VSMC orientation and transmural distribution in murine carotid arteries under well-controlled biomechanical conditions. However, coincident nuclei, erroneously detected as one large nucleus, were excluded from the analysis, hampering accurate VSMC content characterization and distorting transmural distributions. In the present study, therefore, we aim to (1) improve the previous method by adding a “nucleus splitting” procedure to split coinciding nuclei, (2) evaluate the accuracy of this novel method, and (3) test this method in a mouse model of VSMC apoptosis. After euthanasia, carotid arteries from SM22α-hDTR Apoe–/– and control Apoe–/– mice were bluntly dissected, excised, mounted in a biaxial biomechanical tester and brought to in vivo axial stretch and a pressure of 100 mmHg. Nuclei and elastin fibers were then stained using Syto-41 and Eosin-Y, respectively, and imaged using 3D two-photon laser scanning microscopy. Nuclei were segmented from images and coincident nuclei were split. The nucleus splitting procedure determines the likelihood that voxel pairs within coincident nuclei belong to the same nucleus and utilizes these likelihoods to identify individual nuclei using spectral clustering. Manual nucleus counts were used as a reference to assess the performance of our splitting procedure. Before and after splitting, automatic nucleus counts differed −26.6 ± 9.90% (p < 0.001) and −1.44 ± 7.05% (p = 0.467) from the manual reference, respectively. Whereas the slope of the relative difference between the manual and automated counts as a function of the manual count was significantly negative before splitting (p = 0.008), this slope became insignificant after splitting (p = 0.653). Smooth muscle apoptosis led to a 33.7% decrease in VSMC density (p = 0.008). Nucleus splitting improves the accuracy of automated cell content quantification in murine carotid arteries and overcomes the progressively worsening problem of coincident nuclei with increasing cell content in vessels. The presented image analysis framework provides a robust tool to quantify cell content, orientation, shape, and distribution in vessels to inform experimental and advanced computational studies on vascular structure and function.

Purpose: Carotid artery properties can be evaluated with high accuracy and reproducibility using multiple M-line ultrasound. However, the cost of multiple M-line-based imaging modalities and the extensive operator expertise requirements hamper the large-scale application for arterial properties assessment, particularly in resource-constrained settings. This study is aimed to assess the performance of a single M-line approach as an affordable and easy-to-use alternative to multiple M-line imaging for screening purposes.Methods: We used triplicate longitudinal common carotid artery (CCA) ultrasound recordings (17 M-lines covering about 16 mm, at 500 frames per second) of 500 subjects from The Maastricht Study to assess the validity and reproducibility of a single against multiple M-line approach. The multiple M-line measures were obtained by averaging over all available 17 lines, whereas the middle M-line was used as a proxy for the single M-line approach.Results: Diameter, intima-media thickness (IMT), and Young's elastic modulus (YEM) were not significantly different between the single and multiple M-line approaches (p > 0.07). Distension and distensibility coefficient (DC) did differ significantly (p < 0.001), however, differences were technically irrelevant. Similarly, Bland-Altman analysis revealed good agreement between the two approaches. The single M-line approach, compared to multiple M-line, exhibited an acceptable reproducibility coefficient of variation (CV) for diameter (2.5 vs. 2.2%), IMT (11.9 vs. 7.9%), distension (10 vs. 9.4%), DC (10.9 vs. 10.2%), and YEM (26.5 vs. 20.5%). Furthermore, in our study population, both methods showed a similar capability to detect age-related differences in arterial stiffness.Conclusion: Single M-line ultrasound appears to be a promising tool to estimate anatomical and functional CCA properties with very acceptable validity and reproducibility. Based on our results, we might infer that image-free, single M-line tools could be suited for screening and for performing population studies in low-resource settings worldwide. Whether the comparison between single and multiple M-line devices will yield similar findings requires further study.

Background: Dysfunctional cellular mechanosensing appears central to aneurysm formation [1]. We aimed to derive material parameters of aneurysm tissue from in vivo deformations, which may increase insight into the underlying structural integrity of the pathological tissue. Methods: Videos of tracking markers (example Video in supplement, screenshot in Figure) placed on ascending aortic segments were captured alongside radial arterial blood pressure in patients undergoing open-thorax ascending thoracic aorta aneurysm (ATAA) repair (n = 5) and coronary bypass (controls; n = 2). Normalised cross-correlation was used to determine marker displacements, resulting in estimates of systolic/diastolic diameters, distensibility, and cyclic axial engineering strain. A thin-walled, cylindrical geometry was assumed, with amorphous (Neo-Hookean) and fibrous (two-family) constitutive contributions [2]. This framework was fitted to individual patient measurements, by varying parameters c (amorphous material constant), k1 and k2 (fiber stiffness and strain stiffening parameter), β (fiber angle w.r.t. circumferential direction), unloaded intact length (L), and internal radius (Ri). FigureLeft: Example of ascending aortic region of interest with tracking markers. Right: Data presented as mean ± standard deviation. SBP/DBP, systolic/diastolic blood pressure. Estimated properties are defined in the text. Results: Axial strain tended to be lower (expected) and distensibility larger (unexpected) in aneurysm than controls (Figure). However, the intrinsic pressure-dependence of distensibility must be considered when drawing conclusions related to differences in structural stiffness between both groups [3]. Material stiffness parameters (c and k1) appeared higher in aneurysm patients than in controls which is in line with previous studies in mice [4]. Conclusion: We are developing a method to determine ATAA material properties from in vivo deformations and observed increased material stiffness in ATAA.

Background: Many genetic mutations affect aortic structure and function in mice, but little is known about the influence of background strain. We compared the biomechanical, structural, and gene expression responses of C57BL/6J and 129SvEv aortas to angiotensin II (AngII)-induced hypertension. Methods: After AngII infusion (14-day, 1000 ng/kg/min) and euthanasia, excised thoracic aortas were characterized functionally using isobaric vasoactive and cyclic passive stiffness tests. Immunohistochemistry quantified medial/adventitial composition and infiltration of pan-inflammatory CD45+ cells. RNA sequencing-based gene ontology, wall stress analyses, and growth and remodeling (G&R) simulations were performed to complement our mechanical findings. Results: Baseline aortic geometry, composition, and biomechanical properties, as well as AngII-induced blood pressure increases (+34% vs. +32%, systolic), were similar across strains. Yet, AngII-induced aortic remodeling differed dramatically, with gross maladaptive, fibrotic remodeling (exuberant medial/adventitial thickening) in C57BL/6J but not in 129SvEv mice (+89% vs. +12% thickness increase, p = 0.022). CD45+ cell density was markedly higher in hypertensive C57BL/6J than 129SvEv aortas (p = 0.001), while vasoconstrictive responses to AngII (causing a wall stress decrease Δσ) were greater in 129SvEv than C57BL/6J mice, both before (Δσ = −8 vs. −24%, p = 0.023) and after (Δσ = −24 vs. −46%, p < 0.001) hypertension. Gene expression, stress analyses, and G&R simulations reinforced the emergent hypothesis that mechanical stress-mediated immune processes promote maladaptive remodeling while smooth muscle contractile processes reduce wall stress and thereby protect against fibrosis (Figure). Conclusions: Differentially expressed mechano-sensitive genes play key roles in the distinct hypertensive aortic remodeling in C57BL/6J and 129SvEv mice and must be considered when comparing studies in different background strains.

Purpose: Investigating the biomechanical role of smooth muscle cells (SMCs) in arteries requires knowledge of their structural distributions. Compared to histology, 3D microscopy offers non-destructive ex vivo imaging under realistic conditions [1]. Robust 3D segmentation of SMCs, however, is challenging. We propose a method for automatic SMC quantification, and assessed its potential using a murine SMC apoptosis model. Methods: After euthanasia, carotid arteries (control and with induced SMC apoptosis: SM22α-hDTR [2]) were excised and mounted between micropipettes (Figure A). Nuclei were stained with SYTO41. Arteries were imaged using two-photon microscopy [1], while stretched to in vivo length and pressurised to 100 mmHg (Figure B). Image stacks were processed as follows: 1) deconvolution; 2) nuclei segmentation using vesselness filtering [3,4] (Figure C); 3) cylindrical coordinate system identification; 4) splitting of coincident nuclei, based on cores defined from groups of neighbouring voxels with similar orientations [3] (Figure D and E); 5) cylindrical coordinate system re-identification; and 6) cell density-distribution quantification (Figure F). Segmentation performance was assessed by comparing with manual cell counts. Figure(A) Imaging set-up illustrating acquisition of z-stack of slices. (B) Example slice of 3D stack; cell nuclei are shown in blue while elastin fibres are shown in red. (C) Segmentation results from vesselness filtering of example image stack, colours indicate separated nuclei (step 2, Methods). (D) Coinciding nuclei, corresponding with the left orange nuclei in C, shown in grey, with cell cores shown in red and blue (step 4, Methods). (E) Coinciding nuclei splitting results of nuclei shown in C. Non-split nuclei are shown in grey, while split nuclei are shown in red and blue. (F) Transmural SMC densities and cell counts for one control and one SMC apoptosis sample; manual cell counts are given between parentheses. Results: Figure E demonstrates the method’s ability to split undersegmented coinciding nuclei. Cell counts were lower in SM22α-hDTR than in control; algorithm-derived counts were comparable to manual (Figure F). The control sample showed multiple SMC layers, while the SM22α-hDTR sample showed a single SMC layer (Figure F), which was confirmed visually. Conclusion: We developed a precise tool to quantify SMC distributions in ex vivo murine arteries, to facilitate quantitative modelling of SMC biomechanics. We intend to expand the current approach to address cell orientation, shape, and size.

Background: Arterial stiffness is a strong predictor of cardiovascular diseases and all-cause mortality [1]. Increased fasting plasma concentrations of highly reactive dicarbonyl compounds – methylglyoxal (MGO), 3-deoxyglucosone (3-DG), and/or glyoxal (GO) – may cause arterial stiffening via for mation of advanced glycation endproducts, triggering maladaptive responses in vascular tissue, e.g. elastin degradation and collagen cross-linking [2]. We assessed the cross-sectional associations between MGO, 3-DG, and GO concentrations with local carotid stiffness measures (distensibility coefficient (cDC), radius-wall thickness ratio (cRWT), pulse wave velocity (cPWV), and Young’s elastic modulus (cE) using standardized main variables. Methods: Fasting dicarbonyl concentrations were determined by ultra-performance liquid chromatography tandem mass spectrometry in EDTA plasma collected from 2275 participants (age: 60 ± 8 years, mean ± SD; 49% women, 605 with type 2 diabetes mellitus) of the Maastricht Study [3], an observational, population-based cohort study. Cross-sectional associations were assessed using multivariable linear regression analysis adjusting for age, sex, mean arterial pressure (MAP), heart rate, lifestyle factors, and medication. Since arterial stiffness measures are intrinsically pressure dependent, we additionally assessed the associations with pressure-corrected counterparts [4], instead of statistically correcting for MAP. Results: Fasting dicarbonyl concentrations were associated with arterial stiffening (smaller cDC; larger cPWV and cE) in most crude models, but not in adjusted models (Table). cRWT was associated with 3-DG, but only in the crude model. The use of pressure-corrected metrics did not materially change the final models. Model cDC cRWT cPWV cE α (95% CI) α (95% CI) α (95% CI) α (95% CI) MGO 0 –0.104(–0.145;–0.063) 0.011(–0.030;0.052) 0.103(0.062;0.144) 0.099(0.059;0.140) 1 –0.032(–0.070;0.005) 0.010(–0.032;0.052) 0.032(–0.006;0.071) 0.033(–0.007;0.072) 2 0.005(–0.029;0.039) 0.000(–0.042;0.042) –0.004(–0.039;0.030) –0.006(–0.042;0.030) 3 0.003(–0.031;0.037) 0.000(–0. 043; 0.042) –0.003(–0.038;0.032) –0.005(–0.040;0.031) 3-DG 0 –0.164(–0.205;–0.123) 0.067(0.026;0.108) 0.149(0.108;0.190) 0.171(0.131;0.212) 1 –0.102(–0.154;–0.051) 0.024(–0. 033; 0.082) 0.075(0.023;0.127) 0.093(0.039;0.147) 2 –0.037(–0.084;0.009) 0.009(–0.049;0.066) 0.012(–0.036;0.060) 0.027(–0.023;0.076) 3 –0.018(–0.065;0.029) –0. 004(–0.062; 0.054) –0.007(–0.056;0.041) 0.004(–0.045;0.054) GO 0 –0.054(–0.095;–0.013) –0.032(–0.074;0.009) 0.054(0.012;0.095) 0.038(–0.003;0.079) 1 –0.015(–0.051;0.022) –0.014(–0.055; 0.026) 0.015(–0.022;0.052) 0.008(–0.030;0.046) 2 0.002(–0.031;0.035) –0.015(–0.056;0.025) –0.002(–0.035;0.032) –0.008(–0. 042;0. 027) 3 –0.009(–0.042;0.024) –0.013(–0.053; 0.028) 0.011(–0.022;0.045) 0.005(–0.030;0.040) Model 0: crude associations. Model 1: model 0 + age, sex, and glucose metabolism status. Model 2: model 1 + mean arterial pressure and mean heart rate, and anti-hypertensive drugs. Model 3: model 2 + body mass index, smoking status, physical activity, use of lipid-modifying drugs, fasting triglycerides and total-to-high-density lipoprotein cholesterol levels, alcohol use, history of cardiovascular disease, kidney function, and Dutch healthy diet score. MGO, methylglyoxal; 3DG, 3-deoxyglucosone; GO, glyoxal; cDC, carotid distensibility coefficient; cRWT, carotid radius-wall thickness ratio; cPWV, carotid pulse wave velocity; cE, carotid Young’s elastic modulus. Significant associations (p < 0.05) printed in bold. TableAssociations between fasting plasma dicarbonyls and carotid stiffness measures Conclusion: Fasting plasma concentrations of either MGO, 3-DG, or GO are not independently associated with carotid stiffness in this cross-sectional analysis.

In December 2019, an outbreak of pneumonia caused by a novel Coronavirus (COVID-19) spread rapidly worldwide. Although the clinical manifestations of COVID-19 are dominated by respiratory symptoms, the cardiovascular system is extensively affected at multiple levels. Due to the unprecedented consequences of the COVID-19 pandemic, the ARTERY society decided to launch the Covid-19 effects on ARTErial StIffness and vascular AgeiNg (CARTESIAN) study — the first international multicentre study into the effects of COVID-19 on non-invasive biomarkers of vascular ageing. The main study objective is to evaluate the presence of Early Vascular Ageing (EVA) 6 and 12 months after COVID-19 infection. Secondary objectives are to study the effect of COVID-19 disease severity on EVA, to investigate the role of psychosocial factors in COVID-19 induced EVA, and to investigate the potential modifying effect of comorbidities and chronic treatments. In the CARTESIAN study, a broad array of cardiovascular measurements, including carotid-femoral pulse wave velocity, central blood pressure, carotid ultrasound, brachial flow-mediated dilatation, will be performed. To date, 43 centres from 21 countries have agreed to participate, with an expected study population of >2500 individuals. To our knowledge, CARTESIAN will be the first study to provide insight into the relationship between COVID-19, its severity, and early vascular ageing in a large cohort, potentially enabling future care and diagnostics to be more focused on the most vulnerable.

Abstract Background Arterial stiffness as measured by carotid-femoral pulse wave velocity (PWV) has been shown to predict cardiovascular events [1]. However, PWV is blood pressure (BP) dependent [2,3] leading to the development of cardio-ankle vascular index (CAVI) as a more blood pressure-independent index [4] that also shows predictive ability in Asian populations [5]. Recently, CAVI was further refined into CAVI0 [6], removing residual acute blood pressure dependence [7]. The present study aims to assess risk prediction by CAVI and CAVI0 in a US population. Methods We enrolled 156 subjects (94.8% male; 47.7% African-American) with and without heart failure. Subjects underwent arterial stiffness assessments (VaSera 1500 N, Fukuda Denshi Co., Tokyo, Japan). Left (L-CAVI) and right (R-CAVI) measurements were obtained from the device, CAVI0’s were converted from CAVI’s taking into account CAVI’s scale coefficients [8,9]. We prospectively followed participants for a mean of 2.56 years for the composite endpoint of death or heart failure related hospital admission. Results L-CAVI and R-CAVI did not differ significantly (9.80 ± 2.11 vs 9.66 ± 1.92, p = 0.146); neither did L-CAVI0 and R-CAVI0 (16.51 ± 5.85 vs 16.15 ± 5.34, p = 0.178). In unadjusted Cox regression, R-CAVI, L-CAVI, and R-CAVI0 but not L-CAVI0 predicted outcome (Table 1). After adjustment for age, sex, race, and systolic BP, only right-sided CAVIs and CAVI0s were predictive. Discussion We observed possible body-side differences in prediction using CAVI and CAVI0. A previous study cross-sectionally reported more pronounced body side differences in heart-to-ankle PWV related to cardiovascular disease [10]; we are unaware of published prospective studies observing this. In conclusion, both R-CAVI and R-CAVI0 predicted heart-failure related end-points. Table Cox regression results n Standardized HR [95% CI] p Unadjusted L-CAVI 155 1.33 [1.01–1.76] 0.042 R-CAVI 156 1.52 [1.10–2.11] 0.011 L-CAVI0 155 1.28 [0.97–1.68] 0.078 R-CAVI0 156 1.39 [1.04–1.87] 0.027 Adjusted for age, sex, race, and systolic BP L-CAVI 154 1.35 [0.99–1.83] 0.06 R-CAVI 155 1.55 [1.08–2.21] 0.016 L-CAVI0 154 1.30 [0.95–1.77] 0.10 R-CAVI0 155 1.39 [1.0´–1.9´] 0.044 s-HR, standardized hazard ratio; CI, confidence interval.

Background: Aging and hypertension often co-occur, which complicates distinguishing their biomechanical effects on arteries clinically. While aortic elastin (half-life ~70 years) is reduced in human aging, in mouse aging (lifespan ~2 years) it remains mostly intact. Knock-out of elastin-associated glycoprotein fibulin-5 (Fbln5−/−) in mice however yields a biomechanical phenotype that resembles human aging. Methods: Adult male and female Fbln5+/+ and Fbln5−/− mice were studied under three conditions: untreated, after 14-day 490 ng/kg/min angiotensin-II infusion (AngII), and after 13 weeks on 8%-NaCl chow + 3 g/L L-NAME drinking water (L-NAME-NaCl). After tail-cuff blood pressure (BP) measurement and euthanasia, descending thoracic (DTA) and infrarenal abdominal (IAA) aortas were dissected and tested using a computer-controlled biaxial testing device. Results: Salt+L-NAME led to adaptive remodelling (circumferential stress homeostasis) and maladaptive remodelling (lack thereof) in the IAA and DTA, respectively (figure, males), while AngII caused luminal dilatation but little remodelling of the wall. Effects of aging (Fbln5−/−) were more dramatic than those due to induced hypertension. Consequently, superimposing hypertension on aging led to modest additional changes in luminal radius and wall thickness, though increased stress and stiffness metrics due to increased pressure loading of the wall [3] Trends in females were similar to those in males. Conclusions: Effects of hypertension on aortic remodelling are modest when superimposed on aging in mice. These findings are consistent with general observations in humans [4,5], though separated here for the first time in a rodent model characterized by a severe loss of elastic fiber integrity similar to that found in the aged human aorta.

Background: Arterial stiffness (e.g., as measured by carotid-femoral pulse wave velocity (cfPWV)) changes acutely with blood pressure (BP). High cfPWV at high BP could be due to structural arterial changes or acute BP effects. Various parameters have been proposed as BP-independent parameters of arterial stiffness. These rely on assumptions to simplify theoretical calculations. This study evaluates a parameter quantifying BP dependency of cfPWV through BP manipulation, avoiding assumptions and mathematical derivations. Methods: Brachial BP and cfPWV (SphygmoCor XCEL, AtCor Medical, Australia) were assessed in seated and supine position. The postural change invokes a systemic and hydrostatic BP change, thus changing cfPWV. ΔcfPWV/ΔBP was calculated. Repeatability was assessed in 25 participants (28 ± 11 years (mean ± SD), 52% female) across two visits on separate days. Cross-sectional correlation with age, gender, height, weight, seated BP, and supine cfPWV was assessed by stepwise linear regression across 135 participants (49 ± 23 years, 48% female). Results: Mean ΔcfPWV/ΔBP was 0.061 ± 0.025 m/s/mmHg. Repeatability showed a between-measurement difference of 0.005 ± 0.028 m/s/mmHg (8.2% error on mean). ΔcfPWV/ΔBP was cross-sectionally correlated with seated diastolic BP (standardised β = –0.506, p < 0.001), systolic BP (β = 0.503, p < 0.001), weight (β = –0.220, p = 0.006), and heart rate (β = 0.178, p = 0.039) (model p < 0.05, r2 = 0.264). Conclusion: ΔcfPWV/ΔBP showed fair repeatability. Notwithstanding accounting for BP, it correlated with typical long-term modifiers of arterial stiffness such as chronic elevated BP and weight. Further studies are required to validate if dividing ΔcfPWV by ΔBP, following acute intervention, creates an acceptable BP-independent arterial stiffness variable before the parameter is investigated for cardiovascular risk prediction to evaluate its clinical utility.

Introduction: Acute, gravity-induced blood pressure (BP) changes during head-up tilt may generate concomitant variations in carotid-femoral pulse wave velocity (cf-PWV). We aimed to separate the pressure-dependent and -independent components of cf-PWV changes observed during head-up tilt. Methods: 30 healthy individuals (age 48 ± 18 years (mean ± SD), 38% males, BP 130/74 ± 12/8 mmHg) underwent head-up passive tilting at a = 0°, 30°, and 60°. BP was taken at the upper arm, constantly kept at heart level. Aortic BP was reconstructed from radial tonometry (SphygmoCor). Stiffness index b0 was estimated at 0°. 1 Assumptions: [1] from MRI2, the effective cf-PWV travel distance (ETD, 80% of straight carotid-to-femoral distance) begins at heart level; [2] the change in DBP along the aorta is predictable from the hydrostatic pressure gradient (0.73 mmHg/cm) 3; [3] cf-PWV and hydrostatic pressure relate linearly, hence predicted cf-PWV can be calculated as the average of aortic (PWVaorta, using b0 and aortic DBP) and femoral (PWVfem, using b0 and femoral DBP, corresponding to aortic DBP + (ETD × sin(a)*0.73)) PWVs. Results: Both young (24–48 years) and old (48–82 years) individuals showed increasing trends for peripheral SBP, DBP, PP, and central DBP with tilting; central SBP remained unchanged. Heart rate (HR) and cf-PWV increased with body tilt in both groups (Figure, left). b0 linearly correlated with age (R = 0.70, p < 0.01). After adjustment for HR4, observed-vs-predicted cf-PWV exponentially increased as a function of age (R2 = 0.38, p < 0.01 for quadratic equation, p = 0.04, vs. linear; Figure, right). Conclusion: With aging, the acute relationship between BP and cf-PWV becomes progressively nonlinear.

Background: Cardio-ankle vascular index (CAVI) has long been considered a measure of arterial stiffness independent of short-term changes in blood pressure (BP) [1]. Recently and theoretically, CAVI was found to be partially affected by actual BP, thus, a novel BP-corrected index, CAVI0, was proposed to diminish these effects [2,3]. Direct, repeated measures experimental data comparing CAVI’s and CAVI0’s acute blood pressure dependence is lacking. Hence, the aim of this study was to assess the effects of short-term changes of BP on CAVI0 in comparison with standard CAVI. Methods: 60 healthy adults were examined using vascular screening system VaSera 1500 N (Fukuda Denshi Co., Tokyo, Japan) during four examination periods lasting 5 minutes – baseline, cold pressor test (CPT), recovery period, and isometric handgrip exercise (IHE). CAVI and cardiovascular parameters for calculation of CAVI0 were measured after baseline, at the peak of pressor response to CPT, after recovery period, and at the peak of pressor response to IHE. CAVI, CAVI0, and mean BP were assessed for all periods. Results: CAVI significantly increased during CPT compared to baseline rest (p = 0.008), returned to baseline values during recovery period (p = 0.011 compared to CPT), and significantly increased during IHE compared to recovery period (p = 0.002). No significant changes of CAVI0 were found. CAVI significantly correlated with changes in mean BP (p = 0.012; multilevel regression); CAVI0 did not (p = 0.570). Conclusion: In this repeated measures, experimental, acute study, CAVI showed short-term blood pressure dependence, whereas CAVI0 did not.

Local biaxial deformation measurements are essential for the in-depth investigation of tissue properties and remodeling of the ascending thoracic aorta, particularly in aneurysm formation. Current clinical imaging modalities pose limitations around the resolution and tracking of anatomical markers. We evaluated a new intra-operative video-based method to assess local biaxial strains of the ascending thoracic aorta. In 30 patients undergoing open-chest surgery, we obtained repeated biaxial strain measurements, at low- and high-pressure conditions. Precision was very acceptable, with coefficients of variation for biaxial strains remaining below 20%. With our four-marker arrangement, we were able to detect significant local differences in the longitudinal strain as well as in circumferential strain. Overall, the magnitude of strains we obtained (range: 0.02–0.05) was in line with previous reports using other modalities. The proposed method enables the assessment of local aortic biaxial strains and may enable new, clinically informed mechanistic studies using biomechanical modeling as well as mechanobiological profiling.

Background: Matrix metalloproteinase-12 (MMP12) may modulate arterial stiffening with age [1]. We aimed to study the effect of aging on biaxial arterial stiffness in wild-type (WT) and MMP12 knock-out (MMP12-/-) mice. Methods and Results: After euthanasia, descending thoracic (DTA) and suprarenal abdominal (SAA) aortas of young and old, WT (ages 21 ± 0 and 103 ± 1 weeks; mean ± SE) and MMP12-/- (13 ± 0 and 52 ± 0 weeks) male mice were dissected and cannulated on glass pipettes in a computer-controlled biaxial testing device. Pressure-diameter tests were performed at 95%/100%/105% of estimated in vivo stretch; axial force-length tests at pressures of 10/60/100/140 mmHg. Data were fitted using a four-fiber constitutive model [2]. WT and MMP12-/- blood pressures were comparable (133/88 vs. 126/93 mmHg; SBP/DBP; telemetry); WT aging did not influence blood pressure [3]. All metrics are therefore presented at a common pressure (figure). At first sight, MMP12-/- aging resembles WT aging: increased wall thickness (figure, panel A) leading to decreased circumferential stress (B) and decreased stored strain energy (C) [3–5]. However, in WT aging, circumferential material stiffness decreased, which did not occur in MMP12-/- (D). Structural stiffness and pulse wave velocity remained constant in WT mice but increased in MMP12-/- (E-F). Discussion: Our findings suggest that in both WT and MMP12-/-, mechanical homeostasis with aging was compromised, a finding that was exacerbated with MMP12-/-. MMP12-/- was previously reported to reduce age-associated stiffening [1]. This contradictory finding may be explained by the use of atomic force microscopy in [1] (measuring compressive stiffness) versus our use of tensile biaxial testing.

We read with great interest Alizargar et al [...]

Background: Pulse wave velocity (PWV) is quantified by time difference of arrival of the blood pressure (BP) wave at two sites along the arterial bed (transit time; TT-PWV), or by combining measured arterial pressure and diameter using the Bramwell-Hill equation (BH-PWV). Besides the dependence of PWV on BP, TT-PWV also depends on heart rate (HR). The present study aimed to also quantify the dependence of BH-PWV’s on HR, as a function of diastolic BP (DBP). Methods: Adult anaesthetised rats (n = 24) were randomly paced at 300–500 bpm, at 50-bpm steps. At each step, aortic TT-PWV (two pressure-tip catheters) and BH-PWV (pressure-tip catheter and ultrasound wall-tracking; abdominal aorta) were measured simultaneously, across a pharmacologically induced DBP range of 60–110 mmHg. Data from 9142 heart beats was analysed using mixed-effects modelling. Results: HR dependence of TT-PWV increased from 0.03 m/s/100 bpm at DBP = 60 mmHg to 0.06 m/s/100 bpm at DBP = 110 mmHg (both p ≤ 0.023). HR dependence of BH-PWV was 0.11 m/s/100 bpm at DBP = 60 and 85 mmHg, but paradoxically decreased to 0 at DBP = 110 mmHg (p = 0.686). This decrease in dependence is explicable in that standard BH-PWV uses an approximate derivative of pressure to diameter, which overestimates PWV with increasing pulse pressure (PP). PP decreases as HR increases, potentially causing a BH-PWV decrease with HR. This effect can be overcome by estimating the full pressure-diameter curve for each HR, and calculating the true derivative at DBP, yielding a BH-PWV that no longer shows significant HR dependence (p ≥ 0.076 at all DBPs). Conclusions: BH-PWV and TT-PWV show a different HR dependence, affected by DBP.

Purpose/ Background/ Objectives: We have shown that aortic stiffness improves as early as 3 months post-kidney transplantation (KTx). Aortic stiffness index β0, a blood pressure independent parameter, has been proposed to be a better indicator of vascular wall property. This study was designed to examine 1) the early versus late changes in aortic stiffness index β0 and 2) to define the characteristics of patients with favourable and unfavourable trajectories of aortic stiffness index β0 after KTx. Methods: In 79 patients who underwent KTx, aortic stiffness was assessed before, 3, 6 and 24 months after KTx. Aortic stiffness was determined by carotid-femoral pulse wave velocity (cf-PWV), while aortic stiffness index β0 was obtained using a formulae proposed by Spronck and colleagues. Cytokines profile was measured in plasma by ELISA. Results: There was a reduction of β0 3 months after KTx (29.0±2.0 to 25.8±1.2, P = 0.033). Then, aortic stiffness index β0 gradually increased at 6 (28.0±1.4, P = 0.005 vs 3 months) and 24 months (28.3±1.3, P = 0.003 vs 3 months). Unfavourable progression of β0 was not related to renal function, age, comorbidities or kidney donor characteristics. However, the unfavourable progression of β0 was associated with higher levels of interleukin-6 (P = 0.029). Conclusions: The improvement of aortic stiffness index β0 3 months after KTx suggests that KTx leads to an early improvement of the intrinsic mechanical properties of aorta. However, this improvement is followed by a late progression of β0, which is associated with increased pro-inflammatory cytokine, suggesting that activation of immune system may be involved in arterial wall remodeling in kidney transplant recipients.

Background: Carotid-femoral pulse wave velocity (cfPWV) is predictive of cardiovascular outcomes but clinical interpretation is confounded by blood pressure (BP) interaction. This study proposes a method for PWV pressure dependency measurement suitable for routine clinical or research use. Methods: Carotid tonometry and thigh-cuff volumetric displacement allowed cfPWV measurement in the seated and supine position. Brachial oscillometry gave systemic BP. Solving simultaneous equations describing the seated and supine measurement gave hydrostatic BP change across the carotid-femoral arterial path and the pressure dependency of cfPWV. Stepwise multiple linear regression quantified the association of pressure dependency of cfPWV with demographic and cardiovascular parameters. Results: Of 88 subjects (19 to 91 years, 41 female), 4 (4.5%) had an unexpected increase in cfPWV with decreased BP from seated to supine position. Cross-sectional analysis in the remaining cohort showed blood pressure dependency of cfPWV correlated with brachial pulse pressure (β = 0.40, p < 0.001), diastolic pressure (β = −0.33, p < 0.001), gender (β = 0.25 for female/male = 1/0, p = 0.010), and heart rate (β = 0.23, p = 0.033). There was no correlation with supine cfPWV nor age. Average pressure dependency of cfPWV was 0.6±0.3 m/s per 10 mmHg (range of 0.09 to 1.5 m/s per 10 mmHg). Average change in transmural BP across the carotid-femoral arterial path was 20±7 mmHg (diastolic BP change 4±7 mmHg; hydrostatic BP change 16±2 mmHg). Conclusions: Changing from seated to supine position imparts a BP change across the carotid- femoral arterial path, the majority of the effect being hydrostatic. Measuring cfPWV in these two stable BP positions allows calculation of the BP dependency of cfPWV.

Background: We anticipate that in vascular outpatients followed over time, measured changes in arterial stiffness will be the multifactorial result of pressure-dependence, ageing- related degeneration, wall stress homeostasis, and medical treatment. Carotid ultrasound enables assessment of carotid pulse wave velocity (cPWV, via Bramwell- Hill), geometry (relative wall thickness, RWT = 2*IMT/diameter), and intrinsic material stiffness (Young’s-modulus, via Moens-Korteweg). We investigated changes in these carotid properties over time, and their interrelationship. To check whether the change in measured cPWV could be merely due to pressure-dependence, we calculated based on the stiffness index β0 [2] the theoretical pressure-dependent change in cPWV [3]. Methods: Hypertensive outpatients (n = 147) were assessed at baseline and 3.5±1.1 year follow- up, and were stratified according to baseline-to-follow-up change in diastolic blood pressure (ΔDBP) into three groups: decreasedDBP (ΔDBP < −7 mmHg), constantDBP (−7 mmHg ≤ ΔDBP≤7 mmHg) and increasedDBP (ΔDBP > 7 mmHg), with the cut-off being twice the typical DBP measurement error [1]. Results: The theoretical pressure-dependent change in cPWV was 0.4±1.3 m/s lower (p < 0.001, n = 147), corroborating the anticipated multifactorial conditions. Table 1 shows no changes in cPWV, RWT and Young’s-modulus for decreasedDBP. For constantDBP, both cPWV and Young’s-modulus were increased at follow-up. IncreasedDBP showed increases in cPWV and Young’s-modulus and a decreased RWT. The latter implies a 9.2±10.7 kPa increase in circumferential wall stress (p < 0.001), in contrast to a 5.3±6.9 kPa decrease (p < 0.001) in decreasedDBP (p < 0.05 for between groups). n = 147 ΔcPWV [m/s] ΔRWT [−] ΔYoung’s-modulus [MPa] Mean±sd p Mean±sd p Mean±sd p DecreasedDBP (n = 53) −0.1±1.4 0.72 0.00±0.04 0.38 −0.01±0.32 0.87 ConstantDBP (n = 67) +0.6*±1.4

Introduction: Although pulse wave velocity (PWV), a proxy of arterial stiffness, is a strong predictor of cardiovascular complications, it is confounded by blood pressure (BP) and heart rate at the time of examination. Furthermore, establishing whether an artery behaves stiffer or less stiff does not inform a clinician on the cause of the stiffening. Quantification of arterial stiffness: This paper focuses on BP as a confounder of PWV. We developed a method to patient-specifically determine the dependence of PWV on BP — on average 1 m/s per 10 mmHg diastolic BP — and used it to disentangle BP-dependent and -independent stiffening in hypertension and cancer patients. We furthermore showed that the so-called cardio-ankle vascular index (CAVI) — a measure deemed BP-independent — shows a residual BP-dependence that is readily correctable using a modified equation (CAVI0). Both developed methods are directly applicable to clinical measurements in individual patients. Interpretation of arterial stiffness: We developed a computer modelling procedure to disentangle contributions of the individual wall components — collagen, elastin, and smooth muscle — to arterial stiffening as observed in patients. Our model-based approach shows that with ageing, the biomechanical phenotype shifts from elastin-dominated to collagen-dominated load bearing. Model-based assessment of arterial wall mechanics provides a promising tool to further improve interpretation of arterial stiffness measurements in patients. Further development of such methodology applied to various mouse models may improve the understanding and interpretation of arterial stiffening in ageing and disease.

Objectives: Arterial stiffness index β and cardio-ankle vascular index (CAVI) are widely accepted to quantify the blood pressure (BP)-independent, intrinsic exponent (β0) of the BP-diameter relationship. CAVI and β assume an exponential relationship between pressure (P) and diameter (d). We aim (1) to demonstrate that, under this assumption, β and CAVI as currently implemented are inherently BP-dependent and (2) to provide corrected, BP-independent forms of CAVI and β. Methods and results: In P=Pref*exp[β0(d/dref−1)], usually reference pressure (Pref) and reference diameter (dref) are substituted with diastolic BP and diameter to accommodate measurements. Consequently, the resulting exponent is not equal to the pressure-independent β0. CAVI does not only suffer from this reference pressure effect, but also from the approximation of dP/dd. For example, assuming β0=7, an increase of systolic/diastolic BP from 110/70 to 170/120 mmHg increased β by 8.1% and CAVI by 14.3%. We derived corrected forms of β and of CAVI (CAVI0) that did not change with BP and represent the pressure-independent β0. To substantiate the BP effect on CAVI in a typical follow-up study, we realistically simulated patients (n=161) before and following BP-lowering treatment (assuming no follow-up change in intrinsic β0 and therefore in actual P-d relationship). Lowering BP from 160±14/111±11 to 120±15/79±11 mmHg (p

Objective: This study examines effects of high salt diet on arterial blood pressure and aortic function in rats. Methods: Sprague-Dawley rats were fed either high salt content chow or normal chow from weaning. Weight, tail-cuff systolic blood pressure (SBP), water and food intake, and urine output were measured with age. At 16 weeks, rats were anaesthetised and thoracic and abdominal aortic blood pressure measured across a mean arterial pressure range of 60 to 150 mmHg, induced via intra-venous infusion of phenylephrine and sodium nitroprusside. Aortic pulse wave velocity (aPWV) and thoracic to abdominal aortic pulse pressure amplification (PPA) were calculated. Post-mortem weights of the left ventricle and kidneys were recorded. Statistical comparison between groups across the blood pressure range was by robust analysis of covariance. Results: Rats on a high salt diet had lower weights (p=0.04) but similar body mass index. Food intake was similar whilst water intake was greater on a high salt diet, with correspondingly greater urine output. Tail-cuff SBP was higher in rats on a high salt diet. There was no left ventricular hypertrophy (p=0.16) but greater kidney mass in high salt rats (p=0.01). High salt diet resulted in higher aPWV (p

Cervical cancer is associated with a causative role of human papillomavirus (HPV), which is a highly prevalent infection. Recently, women with a genital HPV infection were found to have increased incidence of cardiovascular diseases (CVD), including severe cardiovascular events such as myocardial infarction and stroke. The pathomechanisms of this relation are not yet fully understood, and may significantly affect the health of a large part of the population. Accelerated atherosclerosis is assumed to play a key role in the pathophysiology of this relationship. To identify high-risk groups of the population, it is necessary to stratify the CVD risk. Current algorithms, as widely used for the estimation of CVD risk, seem to be limited by the individual misclassification of high-risk subjects. However, personalised prediction of cardiovascular events is missing. Regarding HPV-related CVD, identification of novel sensitive biomarkers reflecting early atherosclerotic changes could be of major importance for such personalised cardiovascular risk prediction. Therefore, this review focuses on the pathomechanisms leading to HPV-related cardiovascular diseases with respect to atherosclerosis, and the description of potential novel biomarkers to detect the earliest atherosclerotic changes important for the prevention of CVD in HPV infection and cervical cancer.

Background: The relative content and loading of collagen and elastin in the arterial wall determine in vivo longitudinal pre-stretch. Assessment of arterial distensibility at varying longitudinal pre-stretch could improve characterisation of arterial collagen-elastin matrix properties. We introduce a technique to impose extra longitudinal pre-stretch (Δstretch) to the carotid artery in vivo and investigate whether we can predict Δstretch from ultrasound distensibility measurements using a constitutive model. Methods: In 11 healthy volunteers (22±3 yrs, mean±SD, 6m/5f) we obtained right common carotid artery bifurcation-to-bifurcation length by phase-contrast MR-angiography in two positions: head facing forward (relaxed, R) and head facing up and rotated to the left (stretched, S). We estimated Δstretch from the MR images by two independent operators (Bland-Altman inter-operator bias = 0.5±3%, mean±2SD). Additionally, we obtained brachial blood pressures and carotid diameter-distension (considered as cross-sectional area) by ultrasound echo-tracking. We fitted a constitutive model [Spronck et al., AJP-Heart 2015] to average single-exponential pressure-area curves for both R and S states. We predicted Δstretch using the model, assuming constant axial force over the cardiac cycle. Results: MRI-estimated Δstretch was +2% while constitutive model-predicted Δstretch was +6%. To check these estimates, we additionally predicted Δstretch based on echo-derived intima-media-thickness measurements, yielding +6%. Conclusion: Measured and predicted Δstretch values were of the same order of magnitude. Assessment of carotid artery distensibility at varying longitudinal pre-stretch could improve model-based in vivo assessment of arterial wall collagen-elastin matrix properties, which are relevant in age- and disease-related arterial remodelling.

In ureter peristalsis, the orientation of the contracting smooth muscle cells is essential, yet current descriptions of orientation and composition of the smooth muscle layer in human as well as in rat ureter are inconsistent. The present study aims to improve quantification of smooth muscle orientation in rat ureters as a basis for mechanistic understanding of peristalsis. A crucial step in our approach is to use two-photon laser scanning microscopy and image analysis providing objective, quantitative data on smooth muscle cell orientation in intact ureters, avoiding the usual sectioning artifacts. In 36 rat ureter segments, originating from a proximal, middle or distal site and from a left or right ureter, we found close to the adventitia a well-defined longitudinal smooth muscle orientation. Towards the lamina propria, the orientation gradually became slightly more disperse, yet the main orientation remained longitudinal. We conclude that smooth muscle cell orientation in rat ureter is predominantly longitudinal, though the orientation gradually becomes more disperse towards the proprial side. These findings do not support identification of separate layers. The observed longitudinal orientation suggests that smooth muscle contraction would rather cause local shortening of the ureter, than cause luminal constriction. However, the net-like connective tissue of the ureter wall may translate local longitudinal shortening into co-local luminal constriction, facilitating peristalsis. Our quantitative, minimally invasive approach is a crucial step towards more mechanistic insight into ureter peristalsis, and may also be used to study smooth muscle cell orientation in other tube-like structures like gut and blood vessels.