T2*‐decay characteristics were compared with morphological MRI and histologic findings based on a region‐of‐interest analysis.All tendons were measured at 0° and 55° relative to BC), F‐test, and paired t‐test. A P value smaller than the α‐level of 0.05 was considered statistically significant.Akaike information criterion  = 1.92 msec to T2*m (55°) = 29.86 msec (15.5‐fold) in the Achilles and T2*m (0°) = 1.46 msec to T2*m (55°) = 23.33 msec (16.0‐fold) in the patellar tendons. The changes in T2*m‐values were statistically significant. For the whole tendon, according to F‐test and AICC, a bi‐exponential model was preferred for angles close to 0°, while the mono‐exponential model tended to be preferred at angles close to 55°.T2*‐decay  and tendon heterogeneity. Changes in fiber‐to‐field angle result in significant changes in T2*‐values. Thus, we conclude that awareness of T2*‐anisotropy should be noted in quantitative T2*‐mapping of tendons to avoid T2*‐misinterpretation such as a false positive detection of degeneration due to large fiber‐to‐field angles.MR‐microscopy provides a deeper insight into the relationship between T2Stage 2 Although it has been shown in recent years that T2* mapping has great potential to detect degenerations in these tissues noninvasively and even earlier than conventional morphological MRI,2 and T2*). This is due to the structural composition of collagen in which the water molecules surrounding the collagen fibers move slowly and have a preferred orientation , and the translational motion is restricted to preferred directions.2 and T2* values, often simply referred to as the “magic angle effect”.2 and T2* values differ at any angle, not just the “magic angle” of 54.7°, and inherently cannot be suppressed.Quantitative T2* mapping using ultrashort echo time (UTE) has the potential to detect degenerative changes and other tissue abnormalities in the tendon,2 and T2* anisotropy can be very considerable.Although it has been shown that T2/T2*), which is found in nonlocalized spectroscopic MR measurements,Another interesting feature of tendon imaging is the observed multiexponential character of the transverse relaxation decay  sequence2* characteristics, which we wish to demonstrate in this study.In a previous study on human menisci, it was shown that the combination of ultra‐high field strength 7 T, an MR microscopy system and a 3D variable echo time sequence meets these requirements for ultra‐short echo times and microscopic resolution2* anisotropy of tendons in their maxima and minima of dipolar interaction and additionally in 10° increments from 0° to 90°.To investigate in vitro the T2* decay characteristics in different fiber‐to‐field angles.To investigate the TThe objectives of this study are as follows:Four Achilles and four patellar tendon specimens out of four unpaired lower extremities from four body donors  were obtained from the Center for Anatomy and Cell Biology of our university (LH). Only tendons from body donors that were macroscopically intact and showed no signs of rupture or other pathological changes were used. This study was approved by the local ethics committee.For the MRI experiments, the mid‐sections of the tendons were prepared with a length of approximately 25 mm. They were each positioned in the center of a plastic sphere (30 mm diameter) on a plastic cross, where they were fixed with a surgical thread to avoid any kind of movement during the measurement. Subsequently, the sphere was filled with physiological saline solution.−1  and a 39 mm diameter volume resonator .The MR microscopy (μMRI) measurements were performed on a 7 T scanner  using a MR microscopy system with a gradient strength of 750 mT mAfter harvesting the tendons, each tendon was carefully prepared and frozen at −80°. They were then thawed before the MR experiments and measured at room temperature. All tendons were measured at fiber‐to‐field angles, which refer to the maximum and the minimum of dipolar interaction, that is, at angles of 0° and 55°, respectively. Furthermore, one Achilles and one patellar tendon were measured at a total of 11 fiber‐to‐field angles: 0°, 10°, 20°, 30°, 40°, 50°, 55°, 60°, 70°, 80°, 90°. The four Achilles and four patellar tendons are subsequently referred to as AT1, AT2, AT3, AT4, PT1, PT2, PT3, and PT4. AT4 and PT4 were the tendons measured at 11 angles.2* mapping and a morphological MRI protocol in an axial image plane were performed. For vTE T2* mapping,2; slice thickness = 400 μm; number of slices = 72; FOV = 30 × 30 mm2; matrix size = 320 × 320; flip angle = 16°; pixel bandwidth = 220 Hz; TA = 96 min (per angle).T2‐weighted fast spin echo (FSE) sequence, which was used to show different expressions of the signal intensity with different echo times: TEs = 6.6, 13, 40, 53 msec; TR = 2800 msec, in‐plane pixel size = 59 × 59 μm2; slice thickness = 400 μm; FOV = 30 × 30 mm2; matrix size = 512 × 512 and number of averages = 12.The morphological MRI protocol included a sagittal T2* decay were calculated in‐house in IDL 6.3  as described in the literature.2* values (T2*m) were calculated using a three‐parameter model fit. Bi‐exponential fitting was performed using a five‐parameter model fit. A short component of T2* (T2*s) and a long component of T2* (T2*l) were obtained. In addition, a short component fraction Fs (%) and a long component fraction Fl (%) were calculated. Only the short component fraction will be presented in the results as the long component is simply Fl = 100 ‐ Fs. The component fraction that is larger in percentage terms will be referred to as the more pronounced component below.The mono‐ and bi‐exponential T2* decays were to be considered following a bi‐exponential decay pattern. We considered those to be bi‐exponential, where the corresponding bi‐exponential fit yielded physically meaningful results.2* components must be positive and also the calculated short and long components of T2* must result in nonidentical T2* components. Furthermore, for both the F‐test and the AICC, the bi‐exponential model should be preferred over the mono‐exponential model.In Tables 2* relaxation times was performed using a manual region‐of‐interest (ROI) analysis. For each tendon, ROIs covering the entire axial slice(s) as well as ROIs containing selected substructures were placed. ROIs covering the entire axial slice of the respective tendons were used to evaluate the T2* characteristics of the whole tendon.Evaluation of TAnother ROI analysis was performed in which three ROIs were selected for the Achilles tendon and two ROIs were selected for the patellar tendon and examined at 11 different angles. For the Achilles tendon, as shown in Fig. 3c,d, the ROI (red) was drawn in a nonfascicular part of the tendon, while the other two ROIs were drawn in the polygonally shaped fascicles (blue and yellow ROIs). For the patellar tendon, as shown in Fig. 4c,d, the ROI (purple) was drawn in the nonfascicular part of the tendon and the ROI (orange) was drawn in the fascicle part. The same ROI was drawn as best as possible in the same slice but at different angles, using the contrast between the nonfascicular tissue and the fascicles as landmarks for selection.2* characteristics known from whole‐body quantitative T2* mapping.In this way, it may be possible to elucidate the contribution of partial volume effects to the bi‐exponential decay characteristics, previously observed in studies with insufficient spatial resolution,The MR measurement was followed by histological assessment of the tendon samples. The samples were processed as previously described.t‐test was performed to compare T2*m values of the 0° and 55° fiber‐to‐field angle measurement. A P value less than the α‐level of 0.05 was considered statistically significant.A paired 2* decay  a model test (F‐test) and an information criterion  were used.To answer the question which model can be regarded as the preferred one for each TF‐test and the AICC provide the means for model selection. They take into account the number of degrees of freedom and the echo times (data points) used and allow a statistical assessment of which of the two models  is the preferred one. Thus, both are useful in avoiding either overfitting or underfitting of the data.Both the C, the model with the minimum AICC value is considered the preferred model, while for the F‐test, a P < 0.05 was considered statistically significant, which in such a case indicates that the more complex model  is the statistically better model.For the AIC2* values (T2*m) of the Achilles tendons were 11‐fold to 32‐fold higher at a fiber‐to‐field angle of 55° than at a fiber‐to‐field angle of 0°  of the patellar tendons were 11‐fold to 21‐fold higher at a fiber‐to‐field angle of 55° compared with a fiber‐to‐field angle of 0°. The difference between the 0° and 55° measurement were statistically significant for all tendons  were on average T2*s = 0.86 msec and the long component (T2*l) was on average T2*l = 10.47 msec. The short component was the more pronounced and had an average short component ratio (Fs) of Fs = 87.41% at this angle  showed a preferential bi‐exponential T2* decay. The short component of T2* of these 13 slices was on average T2*s = 0.93 msec, while the long component was T2*l = 24.08 msec and the short component fraction was Fs = 6.76%.Table 2* decay and the respective mono and bi‐exponential fitting of AT1 at 0° and 55°. The slice‐wise analysis showed a clear bi‐exponential decay for 0° according to F‐test and AICC. Furthermore, for this tendon, the bi‐exponential model was found to be the preferred model for the magic angle according to AICC and F‐test. While visually it could be seen that the two models fit the data at 55° almost congruently, the bi‐exponential model was the preferred one in this example. For example, the P values of the F‐test at 55° (P < 0.001) for 29 of 30 slices and at 0° for all slices (P < 0.001) clearly showed the preference of the bi‐exponential model for AT1.In this context, it is useful to consider AT1. Figure Since the bi‐exponential fit either did not converge for angles close to the magic angle or was considered the weaker model in many cases, the table fields for which no result could be provided were left blank in Tables 2* maps of AT4 and PT4 at 11 angles to the static magnetic field are presented in Figs. 2* values are presented in Figs. 2* values with fiber‐to‐field angle with a minimum at 0° (T2*m = 2.26 msec for AT4 and T2*m = 1.25 msec for PT4 at 0°) and a maximum at angles close to the magic angle (T2*m = 37.93 msec of AT4 and T2*m = 27.74 msec of PT4 at 60°). The T2* values then decrease again to some extent up to 90° (T2*m = 7.71 msec of AT4 and T2*m = 7.17 msec of PT4 at 90°).T2*m values change on average by a factor of 16.78 when comparing angles reflecting minimum and maximum dipolar interactions  = 0.43 msec to T2*m (55°) = 34.21 msec , T2*m = 50.28 msec  and T2*m = 34.21 msec . The T2* decay of the fascicle ROIs showed a clear preference for a mono‐exponential decay according to AICC and F‐test in all angles.For fascicle ROIs, changing the angle from 0° to 55° increased the Tec Figs.  and 4c,d2* mapping with microscopic resolution provides more detailed insight into the relationship between T2* decay , T2* anisotropy and tendon heterogeneity. Our results indicate the impact of the substructure of the tendon as a possible source of misinterpretation concerning T2* mapping of tendons and its potential use to detect tendon degenerations. A difference of only 10° fiber‐to‐field angle can already change T2* values by about 100% .In our study, we demonstrated that quantitative T2*m values of T2*m = 3.35 msec in healthy volunteers, whereas they were T2*m = 6.56 msec in patients with degenerated Achilles tendons.2*m values as the difference caused by degeneration. This could complicate the future use of T2* mapping to detect degenerative changes in tendon tissue. Many tendons or their fibers are measured at an angle with respect to the magnetic field that can be considered relatively constant. Such a case would be, for example, an Achilles tendon in an ankle coil, where the sole of the foot is usually positioned perpendicular to the magnetic field, which then gives the long axis of the Achilles tendon a fiber‐to‐field angle of approximately 0°. This also explains, for instance, why the Achilles tendon usually provides no signal in morphological MRI and is characterized by extremely short T2* values in quantitative MRI.Juras et al reported T2* values, for example, in terms of a false positive detection of degeneration due to a large fiber‐to‐field angle, is difficult to avoid. However, in the same institution, with the same scanner and coil, the same fiber‐to‐field angle allows defining tendon degeneration in different patients with the same setup. In multicenter studies, if the tendons being compared do not have the same fiber‐to‐field angle with high accuracy, the results cannot be compared with each other since T2* anisotropy clearly appears to be a dominant influencing factor in this context.In contrast, other tendons, such as the supraspinatus tendon are not easily measured at the same angle. The particular fiber‐to‐field angle depends on both the anatomy (i.e. the course of the tendon in the human body) and the positioning of the patient in the respective coil. In any case, it is impossible to always position tendons to the same fiber‐to‐field angle and therefore a possible misinterpretation of the T2* characteristics seem to indicate some differences. In our experiments, we used Achilles tendon samples from the middle section of the Achilles tendons. In these sections, the fibers run mostly parallel to the long axis of the tendon. In contrast, the fibers of the middle section of the patellar tendon are more fan shaped.2*m values of the patellar tendon at 55° do not reach as high T2*m values as those of the Achilles tendons. When a significant portion of the fibers deviate slightly from the direction of the tendon's long axis (to which the experiments are adjusted), there is inevitably some attenuation of the anisotropy effect.While the Achilles tendon and the patellar tendon show strong structural similarities,2* values are due to the heterogeneous structure of the tissue. These regional differences have a dominant influence on the resulting T2* fiber‐to‐field angle dependence and on deciding which decay model, that is, mono‐ or bi‐exponential decay model, is the preferred one for particular angles. In this context, we would like to point out that a tendon is not a homogeneous entity, even though it may appear so even in high‐resolution MRI studies.2*‐mapping studies mainly because the spatial resolution of the respective studies was either insufficient or nonexistent  in order to visualize them. However, their possible influence on multicomponent T2* decay has been suggested previously.2* anisotropy was extremely pronounced, while in the nonfascicle regions the T2* values seem to be hardly affected by the angle, which was to be expected since the water molecules in such sub‐tissues are presumably significantly less restricted. For the whole slice, the maximum increase in T2* is less than in fascicle regions alone, because in the whole slice both sub‐tissues (fascicle and nonfascicle tissues) are combined.The results of our study also show that the regional differences in the T2* decay, while for angles closer to the magic angle, the mono‐exponential decay model was predominantly found by F‐test and AICC as the preferred model over the bi‐exponential model.For the tendons as a whole , we found that the tendons at angles 0°, 10°, 20°, and 30° show clear bi‐exponential T2* values and was thus easily distinguished from the higher T2* values of the surrounding nonfascicle portion. Therefore, the T2* decay of the entire tendon, or at voxel sizes commonly used in whole‐body MRI, appeared to be clearly better described by a bi‐exponential model at such angles.There is a relatively simple explanation for this behavior, as shown by our ROI analysis: In the range from 0° to 30°, the fascicle compartment of the tendon yielded very short to short T2* values of the fascicle component correspond to a distribution of T2* values rather than a uniform value as can be clearly seen in the presented T2* maps. In some areas, the T2* values may be lower, because the fibers there exhibit a rather low angle with the magnetic field, while in another part they are oriented somewhat closer to 55° and this part may therefore feature longer T2* values. When considering the whole tendon again, by changing the angle from 0° toward 55°, one can imagine that with each small change in angle, the short component is brought closer and closer to the long component. Our ROI analysis suggests that the fascicle component, which is predominantly responsible for the short component of T2* at 0°, changes dramatically with angle, while the nonfascicle component appears to be rather unaffected by angle changes. When the angle is shifted toward 55°, the short and the long components of T2* change as a consequence of these mechanisms. The component ratios flip over at 30°–40°. The longer component becomes more substantial than the short one. With even higher angles towards 55°, the short component approaches the values of the long component until it is no longer distinguishable and as a result, the T2* decay appears as mono‐exponential decay.An advanced model should consider that not all fascicles of a tendon run in exactly the same direction, which also means that when we look at the axial cross section of a tendon, the T2 and T2* decay in the magnetic field,Reviewing previous theoretical and molecular considerations on the molecular dynamics in tendons explaining the known properties of T2* decay of nonfascicle regions appears to be hardly angle dependent, while the fascicles themselves show a change by a factor up to 80 when measured between 0° and 55°. Minor angular T2* changes between 0° and 55° that we observed in the small ROI analyses of the nonfascicle regions were probably due to partial volume effects , after all sub‐tissue such as the endotenon is very thin. However, further measurements at even higher in‐plane resolution and thinner slices are needed in this context.This is also reflected in our results. The T2* map of the tendon cross section seem to reach the actual magic angle best at 55° , while the other side of the T2* map of the tendon cross section seems to be further away from it, but it reaches the highest T2* values at 60°, that is, the fiber bundles on this side of the tendon cross section were then obviously closer to the actual magic angle.In the Introduction, it was mentioned that the collagen fibers of the tendon are oriented approximately along the longitudinal axis of the tendon. However, this is only an approximation. In the Achilles tendon, for example, the collagen bundles run from the calcaneus toward the gastrocnemius and soleus muscles in a twisted manner.2* values and faster signal decay there. In addition, this explains why this tendon features a preferential bi‐exponential decay at this angle. However, the comparison of mono‐ and bi‐exponential fitting shows that the two fitting methods can hardly be distinguished here. Which also raises the question as to when a decay should still be considered bi‐exponential.Similarly, the Achilles tendon (AT1) does not appear to be perfectly oriented at a 55° angle. While on the left side of the tendon, the fiber bundles might have been reached the magic angle, the right side appears to be rather in the angle of approximately 30°, which also explains its shorter TC and F‐test.However, for AT2, AT3, and AT4 either a physically meaningful bi‐exponential fit could not be performed, or the bi‐exponential model proved inferior to the mono‐exponential model according to AIC2* maps. This can be explained, as already mentioned earlier, by the fact that the T2* values of the fascicle portion in the magic angle region reach extremely high T2* values closer to or even exceeding the T2* values of the endotenon, which again explains why the T2* decay of the tendons in a slice‐by‐slice analysis is preferably mono‐exponential at angles close to the magic angle. This feature has also been found in previous studies on other collagen tissue.At the magic angle, the contrast between fascicle bundles and endotenon is not well visible in microscopic TA multiexponential character with up to around four well‐defined components of the transverse relaxation decay was found in nonlocalized NMR spectroscopy.2 and T2* components to bound and free water pools has been repeatedly used in the literature,2*, whereas free water pools in the region outside the fascicles cause the long component of T2*. It is noteworthy, however, that this statement is no longer true for angles above approximately 40°, as the fascicle region obtains the same of even higher T2* values than the nonfascicle regions.The assignment of the short and long T2* compartment undetected, for example, originating from protons of the collagen itself. However, at least in a previous study by Ma et alThe argument could be made that the use of a vTE sequence compared with a radial UTE sequence could leave a very short T2* decay found in these earlier studies.The results in our study do not contradict previous results such as those of Juras et al or Liu et al,2* components. It is known from theory that residual coupling alone may cause bi‐exponential decay, as shown in the work of Kruk et al.0 have been suggested as a possible source of multicomponent T2* decay.2* characteristics and anisotropy properties, and more research is needed to reach a clear conclusion about the origin of bi‐exponential T2* decay.There are also other attempts to explain the source of the respective TIn this study, only a small sample size was used, which may lead to some bias. However, these samples were measured with extremely high accuracy and long measurement time and the results were unambiguous and statistically significant.2* component may have been sampled with only the first three or four echo times, which could have led to minimal uncertainty in the calculation of the short component.The echo time range can be seen as another limitation. At a fiber‐to‐field angle of 0°, the short T2* mapping of tendons provides a deeper understanding of the relationship between T2* decay characteristics , T2* anisotropy and the heterogeneous structure of tendons.Our study demonstrated that microscopic TAppendix S1: Supporting InformationClick here for additional data file.


Synthetic magnetic resonance imaging (SyMRI) is a novel quantitative and qualitative technique that permits the reconstruction of multiple image contrasts and quantitative maps from a single scan, thereby providing quantitative information and reducing scan times. The purpose of this study is to characterize intracranial meningiomas using SyMRI.The study included 35 patients with meningiomas . Using 3T MR scanners, SyMRI was performed in addition to conventional FSET2, FLAIR, DWI, T1, and T1 with gadolinium. SyMRI software was used to generate T1, T2, and PD quantitative maps. Osirix MD was used to measure quantitative values of T1, T2, and PD using a ROI.We analyzed 42 meningiomas, 8 of which were associated with edema, and 5 contained calcifications.p = .994, p = .356, and p = .221, respectively), nor between meningiomas containing and not containing calcifications . Values of T1 and T2 measured in meningiomas and the normal‐appearing white matter approximated reference values found in the literature with other quantitative methods.Mean relaxivity values of meningiomas on synthetic T1, T2, and PD maps at 3T MRI were 1382.6 ± 391.7 ms, 95.6 ± 36.5 ms, and 89.1 ± 9.7 pu, respectively. Signal intensities in terms of T1, T2, and PD did not differ significantly between meningiomas with and without edema (The presented method offers a novel approach to characterize meningiomas through their relaxation parameters measured with a SyMRI sequence.  In conventional MRI, images are generated by sequential data acquisition with specific scanner settings and the process requires a relatively long acquisition time  and informed consent was waived. A synthetic MRI was added to our usual meningioma imaging protocol in 35 patients . The patients were referred to our institution with a clinical indication for neuroimaging in the follow‐up of a previously diagnosed meningioma between October 2019 and October 2020. All patients had known meningiomas of a major axis >8 mm, to minimize partial volume effects. Patients with smaller meningiomas were not included in this study. Eleven patients underwent neurosurgery after the MRI, with histopathological confirmation of the diagnosis, and 24 patients had lesions with radiologic features and follow‐up on MRI congruent with meningiomas. All patients were 18 years old or older and had no contraindication to MR imaging.2.1.1Two sets of images were created for each patient using conventional and synthetic sequences. Synthetic MRI was performed on two different 3T MR scanners: Siemens Skyra 3.0T  and Philips Ellition X 3.0T  in addition to the conventional sequences FSET2, FLAIR, DWI, T1, and T1 with gadolinium.2, TE 23 and 100 ms, TR 4790 ms, slice thickness 4 mm with 0.4 mm gap, acquisition time 5 min 27 s for 33 slices. On the Philips scanner, the parameters were: in‐plane resolution 0.5 × 0.5 mm2, TE 13 and 100 ms, TR 4517 ms, slice thickness 4 mm with 1 mm gap, acquisition time 6 min 10 s for 30 slices.The QRAPMASTER sequence (quantification of relaxation times and proton density by the multiecho acquisition of saturation recovery with turbo‐spin‐echo readout) on the Siemens scanner and the MDME sequence (multiple‐dynamic multiple‐echo) on the Philips scanner were performed to retrieve the T1 and T2 relaxation times as well as the PD. The QRAPMASTER and the MDME sequence are multispin‐echo saturation‐recovery sequences, with 4 different saturation delays and 2 echoes. On the Siemens scanner, the technical parameters were: in‐plane resolution 0.7 × 0.7 mmQRAPMASTER and MDME data were reconstructed outside the clinical care environment with the SyMRI software, version 11.2 Ext. 2  to generate T1‐, T2‐, T2‐FLAIR‐, and PD‐weighted images . In order to determine whether the distributions were excessively nonnormal for the purposes of conducting parametric tests, we used the criteria of skewness between −2 and 2 and kurtosis between −9 and 9  and a .05 two‐tailed significance level was used for all statistical tests.3A total of 42 meningiomas found in 35 patients were included in this study . Clinical indications for MR imaging were presurgical evaluation and tumor follow‐up. The mean and standard deviation of the major axis of meningiomas were 33.52 ± 23.7 mm .–3 mm2/s. The relaxation times of Synthetic T1, T2, and PD did not differ significantly between males and females . A significant positive correlation (p < .01) was found between the mean values of T2 and ADC (r = 0.728).The mean relaxation times of all meningiomas in Synthetic T1, T2, and the values of PD at 3T MRI were 1382.6 ± 391.7 ms, 95.6 ± 36.5 ms, and 89.1 ± 9.7 pu, respectively. The ADC mean value was 0.96 ± 0.25 × 10−3 mm2/s, respectively. The relaxation times on Synthetic T1, T2, and PD of meningiomas with and without edema did not differ significantly . A statistically significant difference (p < .01) was found between the mean ADC values of meningiomas with or without perilesional edema; those with perilesional edema had a lower ADC value.Eight meningiomas had perilesional edema. The mean values of T1, T2, PD, and ADC of meningiomas with perilesional edema were 1381.7 ± 105 ms, 84.7 ± 11.13 ms, 85.3 ± 3.19 pu, and 0.81 ± 0.061 × 10−3 mm2/s, respectively. The relaxation times on Synthetic T1, T2, and PD of meningiomas with and without calcifications did not differ significantly . No significant difference (p = .155) was found between ADC values of meningiomas containing or not containing calcifications. Results are resumed in Table Five meningiomas contained calcifications. The mean values of T1, T2, PD, and ADC of meningiomas containing calcifications were 1416.40 ± 161.42 ms, 89.76 ± 21.04 ms, 86.05 ± 5.25 pu, and 0.81 ± 0.08 × 10–3 mm2/s. A statistically significant difference (p < .01) was found between the means of all values of meningiomas and normal‐appearing white matter.Concerning normal‐appearing white matter, the mean values of the relaxation times on synthetic T1, T2, and PD at 3T MRI were 801.14 ± 68.4 ms, 74.28 ± 4.5 ms, and 67.44 ± 2.6 pu, respectively. The mean ADC value of normal‐appearing white matter was 0.75 ± 0.07 × 104The clinical use of synthetic MRI (SyMRI) in neuroradiology has already been established. Several studies have focused on the diagnostic performance of this technique and on the possibility of producing images comparable with those of conventional sequences in a clinical setting  measures the magnitude of diffusion of water molecules in tissues and can be quantified by analyzing diffusion‐weighted imaging (DWI). We calculated ADC values for all meningiomas and normal‐appearing white matter because we considered ADC to be part of the quantitative description of meningiomas. We also intended to investigate whether ADC variation could correlate with other parameters. ADC mean values for meningiomas were similar to those found in the literature  between mean values of T2 and ADC (r = 0.728) for meningiomas  approved this study. This study was performed in accordance with the ethical standards as laid down in the 1964 Declaration of Helsinki and its later amendments.Patient informed consent  was waived from the local ethics committee on research involving humans.No copyrighted works owned by third parties are included in this work.https://publons.com/publon/10.1002/brb3.2769The peer review history for this article is available at


Improvement of functional capacity and mortality reduction in post-MI patients were found to be associated with regular exercise training. The cardiac magnetic resonance (CMR) is considered the most accurate non-invasive modality in quantitative assessment of left ventricular (LV) volumes and systolic functions. Our main objective was to investigate the impact of exercise training on LV systolic functions in patients post anterior MI using CMR. 32 patients on recommended medical treatment 4 week after having a successful primary PCI for an anterior MI were recruited, between May 2018 and May 2019. They were divided into two groups, training group (TG): 16 assigned to a 12 week exercise training program and control group (CG): 16 who received medical treatment without participating in the exercise training program. Treadmill exercise using modified Bruce protocol was done to TG before and after the training program in order to record the resting and maximum HR, metabolic equivalent (MET), and calculate HR reserve. CMR was performed for all patients 4 weeks after PCI and was repeated after completion of the study period to calculate ejection fraction (EF), left ventricular end-systolic volume (LVESV), left ventricular end-diastolic volume (LVEDV), and wall motion score index (WMSI).P < 0.001) and WMSI , without statistically significant change in LV volumes. Regarding CG no significant changes in EF, WMSI, LV volumes were found. There was significant improvement in EF and WMSI change before and after study in TG vs. CG  and  respectively.100% were males. 6 patients from CG dropped during follow-up, no statistically significant difference between the two groups regarding age, BMI, smoking status, hypertension, diabetes mellitus and dyslipidemia. Using the CMR, the TG showed significant improvement in EF . In post myocardial infarction (MI) patients, regular exercise training was associated with improvement of functional capacity, control of cardiovascular risk factors, and reduction of mortality . Some stAssessment of the LV volumes, global and regional systolic functions using CMR is much more accurate and reproducible than using 2D echocardiography, with no risk of cumulative hazardous radiation exposure during frequent follow-up . One of Ischemic heart diseases in most cases affect regional areas of the myocardium. This makes measure of global function insensitive to changes in regional performance, in many cases with ischemic heart disease normal EF can conceal underlying significant regional abnormalities. Moreover, since the heart during different phases of cardiac cycle moves through and rotates within any imaging plane, tomographic imaging of endocardial and epicardial motion has a complex relationship .Anatomical images provided by CMR show clearly the inner borders of endocardium and outer borders of epicardium of the heart chambers, which provide valuable data regarding LV global and regional functions .Hence, the aim of the present study was to investigate the impact of exercise training on LV systolic functions  in patients post anterior MI using CMR.n = 16 assigned to a 12 weeks exercise training program and control group (CG): 16 (six patients dropped during follow-up) who received standard guideline-directed medical treatment without participating in exercise training program. Stratified randomization was used to ensure equal numbers in the different categories of LV EF .The study included 32 patients on optimal medical treatment 4 weeks after having a successful primary PCI for anterior MI referred to our hospital cardiac rehabilitation (CR) unit between May 2018 and May 2019. The patients were randomized into two groups: training group (TG) Exclusion criteria included: patients with metallic heart valves, claustrophobia, history of CABG or previous PCI, those with remaining significant coronary lesions after the primary PCI, patients with implanted cardiac pacemaker except for MRI-safe pacing system, patients with a metallic foreign body, Patient’s refusal, decompensated heart failure, uncontrolled arrhythmia, advanced heart block, musculoskeletal disease, severe systemic illness and severe valvular disease. All patients were subjected to full history and thorough physical examination.Patients on beta blockers, Renin Angiotensin Aldosteron System (RAAS) inhibiting drugs and other heart failure disease modifying treatment continued using same doses throughout the study.Both groups were subjected to formal CR program including risk factor assessment and control, psychosocial support, nutritional and physical activity counseling. Calculation of BSA was done using the following equation: Body surface area = Square root of [Height (cm) × Weight (kg)/3600] , in ordeTreadmill exercise test using modified Bruce protocol (symptom-limited) was done on all participants in the TG before and after the training program in order to record the resting and maximum HR, metabolic equivalent (MET) and calculate HR reserve (maximum HR-resting HR).Before participating in the training program, the program was explained to all participants and informed written consent was obtained. Training program done by TG was scheduled for 3 times every week, each session was designed as a moderate intensity treadmill training to achieve 40–60% of the heart rate reserve, calculated from the baseline treadmill exercise test using modified Bruce protocol (symptom-limited) done before joining the training program. Duration of each session was 30 min preceded by 5 min warm-up training such as stretching and walking. Supervision during treadmill exercise was performed by nurse or one of our study team. In order to monitor the progression of exercise intensity, Borg scale of rate of perceived exertion (RPE) was used. In absence of symptoms, the patients were exercised at 11–13 RPE. Patient monitoring included heart rate, blood pressure, continuous ECG and rating of perceived exertion (RPE) pre and post-exercise session.Non-contrast Cardiac Magnetic Resonance (CMR) using 1.5-Tesla Philips , a phased array cardiac coil of 16 channels was performed for all patients 4 weeks after PCI and was repeated after completion of the study period that lasted for 12 weeks to calculate ejection fraction (EF), left ventricular end-systolic volume (LVESV), left ventricular end-diastolic volume (LVEDV) and wall motion score index (WMSI). CMR imaging was performed with a cardiac coil positioned over the heart. Cardiac gating of the sequences was obtained by applying 4 magnetic resonance compatible electrocardiographic electrodes. Using a steady-state free precession pulse sequence (SSFP), cine images were recorded after obtaining axial, coronal and sagittal images. Short axis cine imaging of the entire left ventricle, from the base to the apex, was performed for the analysis of cardiac volumes and function. Number of slices acquired was 9–15 slices with slice thickness of 8 mm. Each cardiac phase has an acquisition window of 80–100 ms with temporal resolution of 40–50 ms. Assessment of wall motion score index (WMSI) was done according to the 16-segment model  .Regarding change in CMR parameters between TG and CG before and after the study, there was statistically significant improvement regarding EF change in TG [6.5 (2.3–9.0) vs. − 2.0 (− 6.8 to 1.3), Changes in EF and WMSI in individual patients of TG vs. CG before and after the study are shown in Figs. Demonstration of American heart association (AHA) LV 16 segments Bull’s eye plot segmental WMS by CMR for a patient from each group (TG and CG) before and after the study is represented in Figs. By using CMR, the present study showed that 12 weeks of exercise-based CR in patients post-MI resulted in a statistically significant increase in LVEF and significant improvement of WMSI, but there was no significant difference in LVEDV and LVESV mean values before and after the end of the training program, significant reductions in LV volumes might have happened with earlier initiation and longer duration of exercise training program . On the Our findings were in line with some of the previous studies regarding effect of exercise on LV EF. , 11–15P < 0.001), ejection fraction (EF), LV end-diastolic volume index  and NT-proBNP [Giallauria et al. randomized 61 post myocardial infarction (MI) patients to CG 31 and TG 30 patients. Exercise program for the TG was designed for 6 months. The assessment of LV volumes and functions for the whole study group before and after the study period was done using echocardiography. They found that trained patients showed an improvement in workload  and resting and post- dipyridamole stress LV ejection fraction (P < 0.05 in both) [In another study Giallauria et al. randomized 46 recent STEMI patients to TG (25) and CG (21), TG was subjected to 6 months exercise-based CR program. dipyridamole myocardial perfusion single photon emission computed tomography was done on the whole study group within first week after the MI and after 6 months. They found significant improvement in TG when compared to CG in resting and post- dipyridamole stress wall motion score indexes (in both) .Haddadzadeh et al. enrolled 42 patients within one month after acute coronary syndrome, the study group was randomized to 3 months training program TG (center-based exercise training 19 patients and home-based exercise training 9 patients) and CG (14 patients) with no exercise training. Significant improvement in EF was found in the TG when compared to CG, while no difference were found between center based and home-based exercise training .n = 12) and control group . Exercise program was performed over 2 months duration and it was a high-intensity training program consisting of daily walking for 2 h and 4 sessions a week of high-intensity stationary cycling (40 min at 70–80% peak capacity). [On the other hand, Dubach et al. using CMR concluded that no difference was observed in EF, LVEDV and LVESV with substantial increases in exercise capacity. 25 patients post-MI or CABG (15% of the study group were post CABG patients) were included in that study and were divided into training group . n = 12, aged 56 ± 5 years, ejection fraction 31.5% ± 7%) and control group . Exercise program was performed over 2 months duration and it was a high-intensity training program . Measures of left ventricular volumes and function were assessed by CMR before exercise program, 2 month after starting the program and 1 year later. No significant changes were found in LV volumes or ejection fraction in either training or control groups throughout the study period [In another CMR study conducted by Myers et al. on twenty-five post-MI patients with impaired LV systolic function who underwent bypass surgery. and were randomly assigned to training group (y period .n = 8) and control (n = 8), seven patients were not eligible for any revascularization strategy at time of admission and were kept only on medical treatment without revascularization. Seven patients received fibrinolytic therapy and only two patients were treated with primary percutaneous coronary intervention. The training program was moderate-intensity aerobic training. CMR was done to all participants as a baseline and after 12 weeks. It was observed that there was no significant changes in LVEF, LVEDV and LVESV mean values in both control and training group [De Santi et al. also studied the effect of exercise training in sixteen patients with anterior wall myocardial infarction who were randomized into two groups: training  and was associated with significant improvement in exercise capacity.Relatively short follow-up duration. Patients drop during follow-up.Another limitation was the exclusion of 5 patients due to claustrophobia or inability to hold breathing during CMR study. LV strain analysis was not done.Study of scar size was not done as intravenous contrast was not given.


Similarly, in lean WT mice (n = 5–6), TMAO injection prior to subjecting mice to ischemic stroke or arterial thrombosis did not increase its severity compared to vehicle treated mice. Endothelial nitric oxide synthase (eNOS) activity and intracellular stress-activated pathways remained unaltered in aorta of TMAO-injected rats, as assessed by Western Blot. Pre-incubation of human aortic endothelial cells with TMAO (10−6 M) did not alter NO release in response to Ach. Our results indicate that increased plasmatic TMAO in the near-physiological range seems to be a neutral bystander to vascular function as translationally seen in patients after bariatric surgery or in healthy lean rodent models and in endothelial cells exposed acutely to TMAO.Elevated circulating levels of nutrient-derived trimethylamine N-oxide (TMAO) have been associated with the onset and progression of cardiovascular disease by promoting athero-thrombosis. However, in conditions like bariatric surgery , stable increases of plasma TMAO are associated with improved endothelial function and reduced cardiovascular morbidity and mortality, thus questioning whether a mechanistic relationship between TMAO and endothelial dysfunction exists. Herein, we translationally assessed the effects of acute TMAO exposure on endothelial dysfunction, thrombosis and stroke. After RYGB, fasting circulating levels of TMAO increased in patients and obese rats, in parallel with an improved gluco-lipid profile and higher circulating bile acids. The latter enhanced FXR-dependent signalling in rat livers, which may lead to higher TMAO synthesis post RYGB. In lean rats, acute TMAO injection (7 mg kg Previous studies indicated that TMAO and its precursors may be correlated to the onset and progression of athero-thrombotic cardiovascular disease3 through promotion of pathologic changes including cardiac and kidney fibrosis5, enhanced platelet reactivity and thrombus formation6, tight junctions disruption7, NLRP3 inflammasome activation and senescence of endothelial cells8. However, some evidence argues against a causative role of TMAO in cardiovascular disease. Fish consumption, which is a well-known protective factor against coronary heart disease and stroke9, also increases circulating TMAO10. Further, TMAO levels were not increased nor associated with atherosclerotic disease-risk in patients with already established atherosclerosis11. Indeed, very low (i.e. < 2 μmol/L) circulating TMAO was associated with higher mortality risk in deep vein thrombosis patients with the lowest risk of death observed for TMAO around 4 μmol/L12. Interestingly, higher concentrations of plasmatic TMAO (around 10 μmol/L) are reported13 after bariatric surgery, including Roux-en-Y Gastric Bypass (RYGB)14, which significantly decreases cardiovascular morbidity and mortality15 through beneficial improvement of obesity-induced endothelial dysfunction and inflammation17. Thus, the link between TMAO levels and different patho-physiological states, such as atherosclerosis, obesity and aging remain controversial. Further, cut-off levels for physiological versus pathological TMAO concentrations are difficult to define and there are limited data to assess whether modulation of TMAO production may translate into a therapeutic approach to reduce atherosclerosis risk. Based on our previous experimental results showing an improvement of obesity-induced endothelial dysfunction after RYGB18, which is reported to alter circulating levels of TMAO14, we investigated in this study whether acute TMAO administration was associated with endothelial dysfunction in rats or enhanced carotid thrombotic activity and stroke severity in mice.The rising prevalence of overweight, obesity and cardio-metabolic disease highlights the need to understand better the complex interactions between the metabolic state and cardiovascular health. Meat and dairy derived L-carnitine, phosphatidylcholine and choline are catabolized by the gut microbiome into the volatile compound Trimethylamine (TMA). Through the portal vein, TMA reaches the liver where it is oxidised to Trimethylamine N-oxide (TMAO) by members of the flavin-containing monooxygenases (FMO) family, in particular isoform 3 (FMO3)p = ns) . Baseline body weight (BW) was higher in obese candidates to RYGB compared to diet, as detailed in Table TMAO levels in 29 obese patients before RYGB or diet treatment were similar to those of healthy controls . Importantly, BMI at the end of the respective follow-up was not significantly different between the two groups. Interestingly, there was no correlation between increased TMAO levels and the beneficial changes in lipid and glycaemic profile observed after RYGB or after diet  and bile acids  or down-regulated , Slc1b2 (encoding Oatp1b3), Slc22a1 (encoding Oct1)), respectively  1.5-h before sacrifice was similar to vehicle-treated rats  that resemble circulating levels measured in humans and rats after bariatric surgery and atherosclerosis  and inhibitory (Thr495) phosphorylation  followed by one hour stimulation with Ach (10−6 M). As a result, Ach-induced endothelial NO production was not impaired by the pre-incubation with TMAO  activation produces NO, which in turn induces vasodilatationion Fig. A–C. Phosion Fig. D,E. Simiion Fig. F–H were 6 M Fig.  or at thSOM Fig. . One houory Thr49 phosphorory Thr49 phosphorSOM Fig. A. To mimSOM Fig. B. SimilaSOM Fig. B. Of notSOM Fig. C,D, althSOM Fig. E was notory Thr49 phosphorSOM Fig. F. Thus, This paper investigates whether acute TMAO administration is associated with endothelial dysfunction or enhanced carotid thrombotic activity and stroke size using a combination of in vivo and in vitro methodologies. We translationally demonstrate that: (1) circulating TMAO plasma levels increased post-RYGB surgery in patients and in rats in parallel with sustained BW loss, and improved the gluco-lipid profile and endothelial function. (2) Acute TMAO injection at physiological levels was not associated with changes in endothelial vasorelaxation, arterial thrombogenicity or ischemic stroke severity in lean healthy rats and mice. (3) Acute incubation of rat aortas and HAEC with TMAO mimicking the plasma TMAO concentrations seen post-RYGB was not associated with changes in molecular targets of endothelial eNOS-NO pathway or stress-activated pro-thrombotic pathways. (4) Only pharmacological high concentrations of TMAO induced changes on eNOS phosphorylation status, which may decrease NO bioavailability. Taken together, these data indicate that TMAO per se does not alter directly and acutely endothelial NO bioavailability, arterial thrombogenicity and stroke size. Our results suggest that the interplay between TMAO and the underlying healthy or diseased metabolic constellation is a key determinant of the cardiovascular effects associated to this gut metabolite. Thus, we add to the complexity of the ongoing elucidation of the role of TMAO in cardiovascular disease.15 and increasing life expectancy compared to non-surgical obesity management21. We previously reported the early restoration of HDL-mediated vaso-protection and endothelial function along with improved T2D and dyslipidemia in patients and rodents post-RYGB18. We now show that fasting circulating TMAO levels increased concomitantly with the sustained cardio-metabolic benefits in the same patients, who were followed up to two-years after RYGB. Initial observations proposed TMAO as a causal pathogenetic player of athero-thrombotic cardiovascular disease, in particular as a predictor of coronary artery disease burden and of major adverse cardiovascular events, including stroke22. Recent evidence instead suggests that plasmatic TMAO may simply reflect a dysbiotic gut microbiota, which is often associated with metabolic disarrangements and atherosclerotic CVD23. These diseases in fact can heavily impact the quantity and quality of the gut microbiota and the microbiota-dependent processing of dietary components24. Accordingly, increased circulating levels of TMAO have been associated with glucose intolerance, T2D, NAFLD and obesity in humans and mice25. It is however important to note that circulating TMAO levels have been correlated with obesity and NAFLD only in the presence of concomitant T2D, whereas this association was lost after adjusting for HOMA2-IR in patients without T2D26. Along with this notion, increased plasmatic TMAO levels measured in patients with cardiovascular disease may be the epiphenomenon of deleterious effects of the interplay between insulin resistance and systemic inflammation on gut microbiota developing as consequence of the accompanying metabolic comorbidities of these patients. Once dysregulated, the TMA/TMAO axis will then contribute in a vicious circle to further impair glucose tolerance, dysregulate hepatic insulin signalling, and promote adipose tissue inflammation favouring the progression of atherosclerosis27. Of note, our patients were severely obese, but had a relatively healthy accompanying metabolic profile with only slightly increased pre-operative fasting glucose levels , dyslipidemia and hypertension, decreasing cardiovascular mortality28. In particular, increased circulating bile acids play a key role in BW loss along with remarkable improvements of glucose and lipid control after RYGB30. Thus, in our obese rat and human cohort, the higher TMAO concentrations after RYGB may be the epiphenomenon of a modified gut microbiota and BA-dependent hepatic FXR activation secondary to the surgical gut manipulation. Accordingly, it has been speculated that increased levels of TMAO after RYGB may occur due to the altered delivery of unabsorbed precursors due to post-operative dietary changes combined with surgical shortening of the small bowel, which creates a less anaerobic environment and thus a remodeling of the microbiome favoring TMAO-production14.In rats, we showed that RYGB increases circulating bile acid, which in turn activate hepatic FXR leading to higher plasmatic TMAO Fig. H,I. EmerTMAO levels and BA concentrations did not correlate in our bariatric cohort, as previously shown in obese patients, likely because of the low sample size at the 2 year-follow-up, being a limitation of the study  1 and 2 mRNA expression, which are key transporters for TMAO excretion in rodents(data not shown)33. In our study we, like others34, show that TMAO levels are not modified after significant diet-induced weight loss, despite an effective improvement of cardiometabolic risk parameters  as a key factor modulating TMAO levels.Reduced renal clearance can elevate TMAO levels37. On the contrary, some studies measured higher TMAO levels in healthy men35, while in mice, higher circulating TMAO levels were observed in female animals2.We did not observe sex-dependent differences in the TMAO concentration in healthy and obese patients before and after RYGB or diet-treatment SOM A,B, simi37. In this study, TMAO was tested in serum, in which concentrations of TMAO are more stable compared to citrate plasma38. In fact, different type of blood substrates and sampling practices have been implicated in the very high inter-study variability of TMAO levels38. Additional variability in TMAO plasmatic concentrations should also be considered in relation to intra-individual factors for instance the hormonal cycle, since transient trimethylaminuria has been reported in healthy women in relation to the menstruation39.We confirmed a high variability in circulating fasting TMAO levels showing a large overlap between patients and healthy controls. Since a clear definition of physiological TMAO levels is missing, there are large differences across individual studies, which make difficult to identify thresholds to distinguish TMAO levels in health and disease states40. In this study, TMAO circulating levels in healthy subjects were similar to those measured in severely obese candidate to RYGB or diet treatment . Those results assess pharmacological or overt toxic effects of TMAO rather than mimicking relevant concentrations measured in diseases, as often wrongly claimed. Notably, sustained high TMAO in serum in the mM range resulted in 50% mortality in mice43.Similarly, studies performed in endothelial cell-culture report deleterious effects of TMAO, which is used at concentrations much higher than 1044. Incubation with TMAO 300 μmol/L for 60 min increased mouse endothelial cell permeability impairing the expressions of tight junction proteins and activated TLR4- dependent inflammatory pathways7. However, deleterious endothelial effects were not observed when TMAO was used at either 10 or 30 μmol/L7. These concentrations resemble better plasmatic values observed in our RYGB rat model  and in patients . Other studies in HUVEC used TMAO at 300 μmol/L for 6 h to trigger oxidative stress and impair the eNOS-NO axis45. Interestingly, in HAEC after one hour stimulation with TMAO at 10−4 M, we saw a decrease of the eNOS pSer1177 activatory and an increase of the pThr495 inhibitory phosphorylation, although endothelial NO production was still preserved  producing circulating levels around 100 μmol/L over the first hour. Our dose of 7 mg/kg corresponds to about ¼ of the dose used by Seldin et al.44 and produced in mice mean circulating TMAO levels of 12.18 μmol/L over the first hour 22. Interestingly, patients in our study had very similar circulating TMAO of 6.50 μmol/L 2-years after RYGB, which is known to reduce major adverse cardiovascular events including ischemic stroke46.Ischemic stroke is a heterogeneous disease with variable pathogenesis. It is currently debated whether TMAO is really an atherogenic factor or whether variations in its concentrations just reflect the presence of cardiovascular and metabolic risk factors associated with ischemic stroke−6 M in HAEC. Witkowski et al.20. observed increased expression of tissue factor and VCAM1 using human microvascular endothelial cells incubated with TMAO 200 μmol/L for 2 or 6 h. In HAEC, we also observed significantly increased tissue factor protein expression when TMAO was added for one hour at 10−4 M .Our study is limited to a short-term TMAO exposure in healthy rodent models as attempt to dissect direct and immediate mechanistic TMAO effects on the vasculature with focus on endothelial function. Although we followed up a rather small cohort of patients undergoing RYGB, our and other observationsOur results indicate that increased plasmatic TMAO in the near-physiological range may be a neutral bystander to vascular function as seen in patients after bariatric surgery or in healthy lean rodent models and in endothelial cells exposed acutely to TMAO. Therefore, the role exerted by TMAO in the development of cardiovascular disease seems to be modulated by multifactorial metabolic constellations, as graphically summarized in Fig. The surgery group consisted of 29 patients undergoing primary laparoscopic proximal RYGB. The diet group consisted of 21 obese patients matched for BMI, age and sex to the RYGB group 2 years after surgery (n = 14). Moreover, 33 normal-weight volunteers age- and sex-matched were enrolled.The local Research and Ethic Committees in Zurich, Switzerland and in Padova, Italy approved the study. All patients gave written informed consent. Studies were performed according to the principles of the Declaration of Helsinki.47. Bile acids were determined by enzymatic assay (Roche Cobas® Integra 8000 device). Plasmatic lipids  and fasting glycemia were measured using nuclear magnetic resonance at Numares, AG using their Axinon system.Blood samples were obtained after overnight fasting in healthy subjects or in patients before RYGB and starting of diet and at follow-up appointments. Serum was collected in non-additive vacutainers  that were kept at + 4 °C before and after blood collection. TMAO and choline were determined using liquid chromatography coupled to mass spectrometry, as describedhttps://arriveguidelines.org).All methods were carried out in accordance with relevant guidelines and regulations and the animal study protocol were approved by the Zurich Cantonal Veterinary office . All methods are reported in accordance with ARRIVE guidelines  or RYGB (n = 8), one-month post-surgery, as published18.Male Wistar rats  were fed a high-fat, high-cholesterol diet containing 60% kcal fat and 1.25% cholesterol for 7 weeks to achieve diet-induced obesity. Obese rats underwent sham (n = 12) or RYGB (n = 12) surgery and were assessed 8-days post-surgery−1) or vehicle PBS (n = 6), injected 1.5 h before sacrifice. The concentration was adapted from the first article testing43 the serum concentration of TMAO following its injection i.p. in mice. A single injection of 7 g/kg gave a peak serum average concentration around 50 mM in the first hours. We therefore injected in our rodent experiments a thousand times lower concentration of TMAO.For the acute TMAO injection experiment, adult male Wistar rats obtained from Janvier, France, weighed 250–300 g at arrival (approx. 12 weeks of age). Animals were randomized to either TMAO injection (n = 6) (7 mg kg18. Cumulative relaxation responses of the thoracic aorta were performed to acetylcholine (10−12–10−6 mol/L), insulin (10−11–10−6 mol/L), and GLP-1 (10−11–10−6 mol/L) after submaximal contraction with norepinephrine (10−6 mol/L)18.Whole thoracic aorta was excised and immediately placed in physiological Krebs buffer (in mmol/L: NaCl (118.6), KCl (4.7), CaCl2 (2.5), KH2PO4 (1.2), MgSO4 (1.2), NaHCO3 (25.1), glucose (11.1), calcium EDTA (0.026)) on ice, before being cut into ≈ 2 mm thick rings (8 aortic rings per each rat) and mounted on isometric force transducers  within 1 h of sacrifice. Isometric tension was recorded continuously. After a 30-min equilibration period, rings were gradually stretched to the optimal point of their length-tension curve as determined by the contraction in response to potassium chloride (100 mmol). Concentration–response curves were obtained in a cumulative fashion. Several rings (range 2–4 rings) cut from the same artery were studied in parallel for each mediator47.Before proceeding with the in-vivo Carotid Artery Thrombosis Model TMAO circulating concentrations were assessed in a pilot group of n = 4 PBS-treated controls and n = 5 TMAO-treated mice receiving the treatment as described, below. TMAO was measured by liquid chromatography coupled to mass spectrometry, as described48. TMAO treatment (7 mg kg−1) was performed by intra-peritoneal injection every 24-h starting 49-h before surgery. 1-h after the second injection, mice were anaesthetized by i.p. injection of sodium pentobarbital . The depth of anaesthesia was confirmed by the absence of twitch reflex. Rose Bengal  was diluted to 12 mg/mL in phosphate-buffered saline and then injected through the tail vein at a concentration of 63 mg/kg. Mice were placed in supine position under a dissecting microscope and the right common carotid artery was exposed by a midline cervical incision. A Doppler flow probe  was applied and connected to a flowmeter . Five to ten min after Rose Bengal injection, a 1.5 mW green light laser  was applied to the site of injury at a distance of 6 cm from the artery for 60 min or until thrombosis occurred. From the onset of injury, carotid blood flow and heart rate were continuously monitored up to 120 min. Occlusion was defined as blood flow below 0.1 mL/min for at least 1 min.After the pilot test, 3 months old C57BL/6 male mice (n = 5–11) underwent photochemical injury of the common carotid artery (CCA) as previously described49. TMAO treatment (7 mg kg−1) was performed by intra-peritoneal injection every 24-h starting 49-h before surgery. 1-h after the second injection, 3 months-old C57BL/6 male mice (n = 5–11) were anaesthetized using isoflurane 3% and 1.5% for induction and maintenance respectively. Body temperature was kept at 37 °C using a heating pad, which was controlled by an anal probe. For analgesia, 0.5% bupivacaine was infiltrated at the incision side. Ischemia was induced by inserting a 6–0 silicone-coated filament  into the common carotid artery until the origin of the left MCA after the dissection of common, internal and external carotid arteries. After 45 min, reperfusion was allowed by removing of the threat. Neurological status was assessed 2, and 24 h after t-MCAO by a four-point scale neurological score based on Bederson et al.61 as follows: grade 0, normal neurological function; grade 1, forelimb and torso flexion to the contralateral side upon lifting the animal by the tail; grade 2, circling to the contralateral side; grade 3, leaning to the contralateral side at rest; grade 4, no spontaneous motor activity, as previously described29. Neurological performance was determined by the RotaRod test 24 h after t-MCAO: animals were placed on a rotating rod at increasing speeds (4–44 revolutions/min) and latency to fall was recorded. After the experiments, animals were euthanized with carbon dioxide. For determination of stroke volumes, brains were cut into 5 (2-mm thick) coronal sections and immersed in a 2% solution of 2,3,5-triphenyltetrazolium chloride (TTC)  at 37 °C for 20 min. Stroke areas were quantified using ImageJ Software . The following formula was applied in order to compensate for cerebral swelling (edema) and subsequent overestimation of the infarct volume: corrected infarct volume = contralateral hemisphere volume − .To induce I/R brain injury, tMCAO was performed as previously described in TMAO- or vehicle-treated mice50. In brief, after 4-h starvation, HAEC were incubated for 1- or 2-h at 37 °C with DAF-2 diacetate , that forms the fluorescent triazolofluorescein upon reaction with cellular NO. Following incubation, cells were transferred to a black microplate and fluorescence was measured on a Tecan Infinite M200 PRO reader  with excitation and emission wavelenghts of 485 nm and 535 nm, respectively.Primary human aortic endothelial cells (HAECs) were obtained from Lonza, Basel, Switzerland, and cultured in endothelial cell basal medium-2 (Lonza) supplemented with endothelial growth medium–SingleQuots as indicated by the manufacturer . HAECs were grown to sub-confluency and rendered quiescent before experiments by incubation in medium containing 0.5% foetal bovine serum. Thereafter, cells were treated for 1-h with 10-4 M or 10-6 M TMAO  dissolved in DMSO. In a separate experiment, cells were co-incubated with 10-6 M of acetylcholine, to account for pathway cross-interactions. A total of 4 independent cell-culture experiments were performed, with 1–2 replicates per experiment per condition. After incubation, cells were washed with ice-cold PBS, 150uL of lysis buffer  containing protease- and phosphatase-inhibitors was added to each well and plates were immediately frozen. We use a combination of freeze–thaw cycles and plate scratching to prepare samples for Western Blotting. Measurement of endothelial cell NO production by 4,5-diaminofluorescein diacetate (DAF-2) staining. Endothelial cell NO production was determined as describedFrozen aortae or frozen stimulated cells were dissolved in lysis buffer  for immunoblotting. The samples (30 µg) were subjected to SDS-PAGE gel for electrophoresis and incubated with SAPK/JNK and phospho-SAPK/JNK (Thr183/Tyr185) antibodies , eNOS/NOS Type III, eNOS (pS1177) antibodies , eNOS (pT495) (BD Biosciences), Akt and phospho-Akt (Ser473) antibodies , PKCßII and phospho-PKCßII (threonine 638/641) antibodies , PKA and phospho-PKA (threonine 197) antibodies  and anti-GAPDH antibody . The immunoreactive bands were detected by chemiluminescence  and quantified densitometrically with Image J software .Total RNA was isolated from frozen liver specimens by TRIzol method  and quantified at 260 nm by Nanodrop. cDNA was synthesized from two micrograms of total RNA using oligo(dT) primers and Superscript II retrotranscriptase . The cDNA was amplified by real time PCR analysis with the TaqMan Universal PCR Master Mix  for the following targets: Nr0b2 (encoding Shp) (Rn00589173_m1), Cyp7a1 (Rn00564065_m1), Cyp8b1 (Rn01445029_s1), Slc10a1 (encoding Ntcp) (Rn00566894_m1), Slco1b2 (encoding Oatp1b2) (Rn01492634_m1), Slc22a1 (encoding Oct1) (Rn00562250_m1) and 18S rRNA (4318839).p < 0.05, Dunnett corrections are applied as indicated in the figure legends. All data were analysed and graphed in GraphPad Prism 8.4.2.Continuous variables were presented as mean ± SEM. Discrete variables were summarized as frequencies and percentages. The distribution of the data was analysed with a 1-sample Shapiro–Wilk test. Logarithmic transformation was performed to achieve normal distribution for skewed variables. Continuous data were compared by use of the 2-tailed paired or unpaired t test  or the Mann–Whitney U or Wilcoxon signed-rank test (for skewed variables). We used a 2-way ANOVA with repeated measures to compare repeated measurements on the same animals. For studying the complete outcome of each variable over time, we applied the linear mixed model using an unstructured covariance matrix for quantitative variables. All tests were 2 sided, and statistical significance was accepted if the null hypothesis could be rejected at Supplementary Information.


Cardiac shock wave therapy (CSWT) is a novel therapeutic procedure for patients with angina that is refractory to conventional therapy. We investigated the potential mechanism and therapeutic efficacy of non-R-wave-triggered CSWT to attenuate myocardial dysfunction in a large animal model of hypertensive cardiomyopathy. Sustained elevated blood pressure (BP) was induced in adult pigs using a combination of angiotensin-II and deoxycorticosterone acetate (DOCA). Two sessions of non-R-wave-triggered CSWT were performed at 11 and 16 weeks. At 10 weeks, systolic and diastolic blood pressure, LV posterior wall thickness and intraventricular septum thickness significantly increased in both the hypertension and CSWT groups. At 20 weeks, +dP/dt and end-systolic pressure-volume relationship (ESPVR) decreased significantly in the hypertension group but not the CSWT group, as compared with week 10. A significant improvement in end-diastolic pressure-volume relationship (EDPVR) was observed in the CSWT group. The CSWT group exhibited significantly increased microvascular density and vascular endothelial growth factor (VEGF) expression in the myocardium. Cytokine array demonstrated that the CSWT group had significantly reduced inflammation compared with the hypertension group. Our results demonstrate that non-R-wave-triggered CSWT is safe and can attenuate LV systolic and diastolic dysfunction via enhancement of myocardial neovascularization and anti-inflammatory effect in a large animal model of hypertensive cardiomyopathy. Heart failure (HF) represents a major socioeconomic health burden around the world, and approximately half of affected patients suffer from HF with preserved ejection fraction (HFpEF). In China, up to 1.3% of the population suffers from HF. The prevalence of HF in the United States and Europe varies from 1% to 12% based on different studies or reports . Indeed,Ultrasound-guided cardiac shock wave therapy (CSWT) has been proposed as a therapeutic option for patients with angina that is refractory to conventional medical treatment ,7. In coWe hypothesize that non-R-wave-triggered CSWT can be delivered without proarrhythmias and can improve CMD via neovascularization and anti-inflammatory effect, with consequent attenuation of myocardial dysfunction in hypertensive cardiomyopathy.No significant differences in ambulatory systolic or diastolic BP were observed from baseline to 10- and 20-week follow-up in the control group A,B. AfteLV pressure volume loop analysis was used to perform hemodynamic assessments to evaluate cardiac function at baseline, 10 weeks and 20 weeks in the control, hypertension and CSWT groups . MaximumEchocardiographic measurement was performed at baseline, 10 weeks and 20 weeks to evaluate cardiac function and ventricular wall thickness . There wTaken together, our study showed that CSWT neither decreased systolic nor diastolic BP nor did it reduce LVH, but it significantly attenuated cardiac dysfunction in hypertensive cardiomyopathy.p > 0.05).In the CSWT group, all animals underwent non-R-wave-triggered CSWT without any complications. Continuous ECG monitoring during CSWT revealed only 8 single premature ventricular contractions out of a total 80,000 applications of CSWT, and no sustained arrhythmias were noted during CSWT. Moreover, there was no significant change in serum cardiac troponin level after CSWT  antibodies was performed to determine vessel formation in the endocardium, mid-myocardium and epicardium A. ComparThe level of circulating cytokines was detected using cytokine array in the control, hypertension and CSWT groups at the 20th week . After iTo the best of our knowledge, this is the first pre-clinical study to investigate the safety and efficacy of CSWT in a clinically relevant large animal model of hypertensive cardiomyopathy. First, we demonstrated that non-R-wave-triggered CSWT can be safely delivered without any proarrhythmias or myocardial injury. Second, although there were no significant therapeutic effects on BP, LVH or myocardial fibrosis, our results showed that CSWT could significantly preserve LV systolic function and attenuate LV diastolic dysfunction in hypertensive cardiomyopathy. Third, CSWT significantly increased microvascular density via local VEGF expression and exerted systemic anti-inflammatory effects. These findings provide important proof-of-principle data to support the application of non-R-wave-triggered CSWT as a novel non-invasive therapy for hypertensive cardiomyopathy.Hypertensive cardiomyopathy with left ventricular hypertrophy is the most common etiology of HFpEF . UnfortuShockwaves have been applied to clinical patients during lithotripsy as a way to disintegrate kidney and urethral stones and enhance healing of soft-tissue defects of non-healing wounds, but also as an effective and non-invasive therapeutic strategy for ischemia heart disease, such as refractory angina and ischemic cardiomyopathy ,8,20,21.Our findings have several potential clinical implications. First, our results provide novel evidence that treatment of CMD via myocardial neovascularization using CSWT, independent of the persistence of hemodynamic stress with elevated BP and LVH, can still improve LV function. Second, the findings in this preclinical large animal study provide important proof-of-principle data to support future clinical trials of CSWT in patients with HFpEF. Third, we also demonstrated that non-R-wave-triggered CSWT was safe, with no proarrhythmia or myocardial injury in the presence of HF. Previous preclinical studies demonstrated that CSWT was a new therapy method for patients who suffered from chronic myocardial ischemia or refractory angina unresponsive to conventional treatments such as percutaneous coronary intervention (PCI) or coronary artery bypass grafting (CABG) . AccordiOur study has several limitations. First, we did not include a treatment arm with anti-hypertensive therapy and CSWT in this study. It remains unclear whether CSWT can provide an incremental benefit on top of BP lowering in our animal model of hypertensive cardiomyopathy. Our recent study showed that BP lowering with splanchnic denervation could also result in regression of LVH and improved LV diastolic function in the same animal model of hypertensive cardiomyopathy . Second,In conclusion, non-invasive, non-R-wave-triggered CSWT significantly improved the microvascular density in the myocardium, ameliorated systemic inflammation and improved cardiac performance without any effect on BP and LV hypertrophy or fibrosis in a large animal model of hypertensive cardiomyopathy. The safety and efficacy of CSWT in hypertensive cardiomyopathy with HFpEF should be further investigated in clinical trials.All procedures that involved animals were approved by the Committee on the Use of Live Animals in Teaching and Research (CULATR) at the University of Hong Kong. (CULATR number 4662-18). Adult female pigs weighing 35–45 kg (9–12 months old) were used in this study to create a large animal model of hypertensive cardiomyopathy as described in our previous studies . In brieTelemetry recording of ambulatory BP  was performed every 2 weeks in conscious animals. Serial echocardiogram and invasive hemodynamic assessment were performed at baseline, 10 weeks and 20 weeks in all three groups to assess LV dimension and function, respectively. All animals were sacrificed for histological and immunohistochemical assessments at 20 weeks.At 10 weeks following Ang-II infusion and DOCA implantation, animals with hypertensive cardiomyopathy were assigned to the no-therapy group  or CSWT group (n = 8). Six pigs without hypertension induction served as the control group (n = 6). Animals in the CSWT group received two sessions of CSWT, in the 11th and 16th weeks, using a non-R-wave-triggered fixed frequency at a constant frequency of 4 Hz, with the guidance of echocardiography instruments mounted to the basal septal, anterior, inferior and lateral wall. In each session, 400 shots/spot were applied to 10 spots at energy level 3 . During the procedure, the safety of CSWT, including the incidence of premature atrial and ventricular contractions and their temporal correlation to the shockwave pulse were analyzed, and pigs were monitored for the occurrence of severe arrhythmia such as ventricular fibrillation.All animals underwent transthoracic echocardiograms using a commercially available echocardiographic system with a 3–9 MHz transducer  at baseline and in the 10th and 20th weeks . Standarmax, end-systolic pressure–volume relationship (ESPVR) and end-diastolic pressure–volume relationship (EDPVR) were recorded to assess LV performance.Invasive hemodynamic assessment was performed to evaluate cardiac function at baseline and in the 10th and 20th weeks ,24. In bDetailed histological assessments were performed to evaluate changes in cardiac structure and the myocardial vascular density ,27. In bTotal protein samples were extracted from hearts and loaded on SDS-PAGE gel and then transferred to PVDF membranes. After blocking with BSA for 2 h, the membranes were incubated overnight with VEGF monoclonal antibody  at 4 °C. Membranes were then incubated with appropriate fluorescence conjugate secondary antibody for 2 h at room temperature. Finally, the membranes were visualized by enhanced chemiluminescence and protein bands were quantified using Image J 1.8.0 .g for 15 min to collect plasma. Level of plasma porcine cytokines, including interleukin (IL)-10, IL-8, IL-1β, IL-6, IL-4, IL-12, transforming growth factor β1 (TGFβ1), tumor necrosis factor (TNF-α), interferon gamma (INFg) and granulocyte–macrophage colony-stimulation factor (GM-CSF) were determined using a cytokine array kit  following a standard protocol. In brief, 100 μL of plasma was added to each well and incubated at room temperature for 2 h. After washing, the slide was incubated with biotinylated antibody cocktail for 2 h, and then further incubated with Cy3 equivalent dye-labeled streptavidin for 1 h. Finally, a laser scanner equipped with a Cy3 wavelength was used to scan the signals.At 20 weeks, blood samples were collected from the femoral vein and centrifuged at 6000× In the CSWT group, plasma porcine cardiac Troponin T  level was evaluated before and 1 day after the first session of CSWT using an enzyme-linked immunosorbent assay (ELISA) kit  to determine myocardial injury.t-tests. Statistical significance was defined as a p-value less than 0.05. All statistical analyses were performed using SPSS 23, .All data are expressed as mean ± SEM. Comparison of serial changes in blood pressure, echocardiographic and invasive hemodynamic parameters at different time points between groups was made using a two-way repeated ANOVA with Tukey’s test as appropriate. Histology and immunohistochemical staining results of different groups were compared by independent Student


Out of 68 patients, 43 patients were further examined after initial treatment. At follow‐up, the stiffness of the proximal arteries, given as characteristic impedance, decreased from 0.12 to 0.08 mmHg s mL–1. Wave speed was attenuated from 13.3 to 9.1 m s–1, and the return of reflected waves was delayed from 64% to 71% of systole. In conclusion, reflected waves arrive at variable times in PAH. Early return of reflected waves was associated with more RV hypertrophy. PAH treatment not only decreased PVR, but also delayed the timing of reflected waves.Right ventricular (RV) wall tension in pulmonary arterial hypertension (PAH) is determined not only by pressure, but also by RV volume. A larger volume at a given pressure generates more wall tension. Return of reflected waves early after the onset of contraction, when RV volume is larger, may augment RV load. We aimed to elucidate: (1) the distribution of arrival times of peak reflected waves in treatment‐naïve PAH patients; (2) the relationship between time of arrival of reflected waves and RV morphology; and (3) the effect of PAH treatment on the arrival time of reflected waves. Wave separation analysis was conducted in 68 treatment‐naïve PAH patients. In the treatment‐naïve condition, 54% of patients had mid‐systolic return of reflected waves (defined as 34–66% of systole). Despite similar pulmonary vascular resistance (PVR), patients with mid‐systolic return had more pronounced RV hypertrophy compared to those with late‐systolic or diastolic return  wall tension in pulmonary arterial hypertension (PAH) is determined not only by pressure, but also by RV volume. Larger volume at a given pressure causes larger RV wall tension.Early return of reflected waves adds RV pressure in early systole, when RV volume is relatively large. Thus, early return of reflected waves may increase RV wall tension. Wave reflection can provide a description of RV load.In PAH, reflected waves arrive back at variable times. In over half of PAH patients, the RV is exposed to mid‐systolic return of reflected waves. Mid‐systolic return of reflected waves is related to RV hypertrophy.PAH treatment acts favourably on the RV not only by reducing resistance, but also by delaying the return of reflected waves.Arrival timing of reflected waves is an important parameter for understanding the relationship between RV load and its function in PAH. Abstract figure legend Return of reflected waves augments right ventricular pressure in early systole, when the ventricular volume is relatively large. This increases ventricular wall tension, resulting in hypertrophy. Right ventricular (RV) dysfunction is the major determinant of clinical outcome in pulmonary arterial hypertension (PAH)  to investigate the prevalence of early‐ or mid‐systolic return of reflected waves in PAH patients; (2) to elucidate the relation between reflected wave arrival time with RV morphology in treatment‐naïve PAH patients; and (3) to assess the effect of PAH treatment on the arrival time of reflected waves.Declaration of Helsinki, except for registration in a database. Informed consent was given by all patients included after 2010 to use their data for scientific purposes. For patients included before 2010, the medical ethical committee waived the requirement of consenting the patients in retrospect and allowed using the clinical data collected in that period for scientific purposes .The present study was conducted retrospectively on a registry of patients with diagnosed PAH who routinely underwent right‐sided heart catheterization (RHC), cardiac magnetic resonance imaging (MRI), a 6 min walk test and blood sampling. This study conformed to the standards set by the We screened 324 patients who were diagnosed as PAH from February 1996 until December 2018 at Amsterdam University Medical Centre, Vrije Universiteit Amsterdam . Wave separation analysis requires the complete digital data of both pulmonary artery (PA) pressure and flow waveforms. Thus, enrolment criteria were: (1) to have digital data of both PA and RV pressure curves at the initial diagnosis before treatment and (2) to have PA flow curves obtained by cardiac MRI in treatment‐naïve condition within 28 days before or after diagnostic RHC [a median interval between RHC and MRI was 1 day: interquartile range (IQR) 0–6 days]. In the present study, 68 treatment‐naïve patients were enrolled and separated into three groups according to the timing of peak reflected waves returning to the RV: a group with reflected waves during mid‐systole (referred to as mid‐systolic returners), a group with reflected waves during late‐systole (late‐systolic returners) and a group with reflected waves during diastole (diastolic returners). Early‐systole was defined by 0–33% of systole, mid‐systole was 34–66% of systole and late‐systole was 67–100% of systole. In our study, no patients had reflected waves during early systole.Out of 68 treatment‐naïve patients, 43 patients who had digital data of PA flow and pressure waves at both baseline (under treatment‐naïve condition) and 1 year of follow‐up (under treatment condition) were examined to assess the effect of PAH treatment on timing of reflected waves . Images of the main PA cross section were obtained with velocity encoding perpendicular to the imaging plane . The circumference of the main PA was contoured semi‐automatically, and the vertebrate or the fat tissue was also contoured manually for the background correction. After subtracting velocity of the background from that in the main PA, velocity of the main PA was multiplied with area of the contour. The sampling period of volumetric flow signals was 45 Hz (22 ms). Volumetric flow signals were interpolated to acquire the signals at 1000 Hz using Mathematica software   . Phase‐contrast velocity quantification was performed to obtain volumetric flow curves in the main PA Fig. A. Imageane Fig. A. The cfollow‐up‐RVmass/BSAbaseline)/RVmass/BSAbaseline × 100. PA flow waves during a cardiac cycle were obtained , end‐systolic volume (RVESV) and RV mass were obtained by contouring endocardial and epicardial borders of the RV on short‐axis at the end‐diastolic and end‐systolic phase using a medical imaging software  . A change in the area of the main PA during the cardiac cycle (∆area) was given as difference between the largest and the smallest area. By multiplying ∆ area with 2.0 cm, which is assumed as the length of the main PA for all patients, local compliance of the main PA was given  calculated as the averaged modulus between the third and tenth harmonics of PA input impedance  .Continuous variables are presented as the mean ± SD or median (IQR), as appropriate. Normality was tested by visual assessment of a histogram and a quantile‐quantile plot and further confirmed by a Shapiro–Wilk test. One‐way ANOVA using Bonferroni's multiple comparison was performed to compare three groups . Variables were log‐transformed in case of non‐normal distribution. When log‐transformed variables were distributed non‐normally, a Kruskal–Wallis test with Dunn's multiple comparisons was conducted to compare three groups. To compare variables between baseline and 1 year of follow up, a paired A demonstrates the distribution of reflected wave arrival times in the treatment‐naïve condition. The peak of the reflected wave appeared in mid‐systole in thirty‐seven patients (54%), in late systole in 20 patients (29%) and in diastole in 11 patients (16%). When the peak of the reflected waves returned during mid‐systole or late‐systole, the reflected waves affected the shape of PA flow waves, which was apparent as notched flow waves . By contrast, when peak reflected waves arrive back during diastole, reflected waves little affected the shape of flow waveform, and flow waves almost maintained a triangular shape.Sixty‐eight patients were included in the present study: 84% with idiopathic PAH and 16% with heritable PAH . Figure Zc than diastolic returners  Table . Althougers Fig. . There wers Fig. . RV systers Fig. Zc, wave transmission in mid‐systolic returners was slower than in diastolic returners    Fig. .Zc from 0.12 to 0.08 mmHg s  mL–1, increased compliance of the main PA from 6.8 to 9.1 mL mmHg–1, and delayed wave speed from 13.3 to 9.1 m s–1 .Out of 68 treatment‐naïve patients, 43 patients were further examined to perform wave separation analysis at 1 year of follow‐up. Duration from baseline to follow‐up RHC was 11.9 months (6.1–13.5 months)   Fig. .We found that, in over half of treatment‐naïve PAH patients, the peak reflected wave returned to the RV during mid‐systole. Although PVR was similar, patients exhibiting mid‐systolic return of the reflected wave had more pronounced RV hypertrophy. PAH treatment attenuated the stiffness of the proximal arteries and decreased wave speed, which delayed the arrival of peak reflected waves. Delayed return of reflected waves was associated with attenuation of RV hypertrophy after treatment.–1 for wall tension and dyne cm–2 for wall stress) , females had faster wave transmission  as a consequence of higher Zc . There was no sex difference in the mean area of the main PA, suggesting that the main PA was stiffer in female. Although PVR, total arterial compliance, the magnitude and timing of reflected waves, and RV volume and mass were similar, RVEDP was higher in female than male group [female, 19 (14–23) mmHg vs. male 16 (12–18) mmHg, P = 0.032). These sex differences may explain the difference of RV adaptation to increased afterload among diseases.So far, PAH was considered to be associated with a later return of reflected waves than CTEPH . Furthermore, the geometry of the distal part of the proximal PA may be different from the mid‐systolic returners and late‐systolic/diastolic returners (see section below for details). A wider proximal PA with less tapering but abrupt narrowing may induce early return of reflected waves even at a low wave speed. The initial development of PAH may involve the acceleration of wave speed  when the speed of wave transmission is high or (2) when the distance from the RV to the reflection sites is short  and wave speed, which delayed the arrival of peak reflected waves. As PAH treatment decreased PVR  do not differ  but stiffer vessels at distal part of elastic arteries . All authors contributed to interpretation of the data. All authors were involved in drafting the work and approved the final version of the manuscript submitted for publication. All authors agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All those who qualify for the authorship are listed.Dr M. Fukumitsu was supported by European Respiratory Society Short‐Term Research Fellowship 2019 (STRF201904‐00595) and Japan Heart Foundation/Bayer Yakuhin Research Grant Abroad. Drs B. E. Westerhof and J. A. Groeneveldt were supported by NWO‐VICI (918.16.610). Dr A. Bayoumy was supported by the Egyptian ministry of Higher education. Dr H‐J Bogaard was supported by The Netherlands CardioVascular Research Initiative . Drs A. Vonk Noordegraaf and F. S. de Man were supported by The Netherlands CardioVascular Research Initiative  and The Netherlands Organization for Scientific Research .Statistical Summary DocumentClick here for additional data file.Peer Review HistoryClick here for additional data file.