Low-Intensity Electrostimulation Enhances Neuroregeneration and Improves Erectile Function in a Rat Model of Cavernous Nerve Injury.

BACKGROUND: Neurogenic erectile dysfunction (ED) following radical prostatectomy (RP) is a frequent complication often leading to erectile tissue remodeling and permanent ED. Low-intensity electrostimulation (LIES) has been shown to enhance peripheral nerve regeneration, however, its application on cavernous nerves (CN) has never been investigated. AIMS: To investigate whether LIES enhances CN regeneration, improves erectile function (EF) recovery, and prevents corpora cavernosal remodeling after CN injury, which is a principal factor for ED following RP. METHODS: Adult male Sprague-Dawley rats were divided into Sham, Bilateral Cavernous Nerve Injury (BCNI), and BCNI + LIES (1V, 0.1ms, 12Hz, 1h/day). After 7days, EF was assessed (ICP measurement). Penes and CN were collected for molecular analyses of TGF-ß1, Il-6, CRP, eNOS, ERK and AKT protein levels in corpus cavernosum (CC), and immunohistological analysis of DHE, total collagen and α-SMA in CC and S-100, Tub-III, DAPI, TUNEL, and nNOS in CN. OUTCOMES: Effects of LIES on EF, erectile tissue remodeling and CN structure. RESULTS: EF was decreased (P < .05) 7 days after BCNI and increased (P < .05) by LIES. Intracavernosal reactive oxygen species (DHE) was increased (P < .05) after BCNI and normalized by LIES. Protein expressions of TGF-ß1, IL-6, and CRP were increased in the penis (P < .05) after BCNI and normalized by LIES. The α-SMA and/or total collagen ratio was decreased (P < .05) after BCNI in the penis and normalized by LIES. Protein expression ratio of p-ERK/ERK and p-AKT/AKT did not change after BCNI but increased (P < .05) in LIES group. Myelination and number of nNOS positive cells in the CN were decreased (P < .05) after BCNI and normalized by LIES. The number of apoptotic nerve cells within the dorsal penile nerve was increased (P < .05) after BCNI and decreased (P < .05) by LIES compared to the BCNI group. There were no differences in eNOS expression in the penis between study groups. CLINICAL TRANSLATION: LIES may offer a potential new tool for penile rehabilitation and ED management following RP, potentially enhancing EF recovery and minimizing the side effects of this surgery. STRENGTHS & LIMITATIONS: This study provides evidence of the protective effect of LIES on EF and tissue remodeling following CN injury; nevertheless, this study has been conducted on animals and the translation to humans remains to be demonstrated. Further research to identify the underlying mechanisms of action is required. CONCLUSION: This study demonstrates that LIES of the CN after CN injury protects CN structure, enhances EF recovery, and prevents corpora cavernosal remodeling. Sturny M, Karakus S, Fraga-Silva R, et al. Low-Intensity Electrostimulation Enhances Neuroregeneration and Improves Erectile Function in a Rat Model of Cavernous Nerve Injury. J Sex Med 2022;19:686-696.

On the assessment of arterial compliance from carotid pressure waveform.

In a progressively aging population, it is of utmost importance to develop reliable, noninvasive, and cost-effective tools to estimate biomarkers that can be indicative of cardiovascular risk. Various pathophysiological conditions are associated to changes in the total arterial compliance (CT), and thus, its estimation via an accurate and simple method is valuable. Direct noninvasive measurement of CT is not feasible in the clinical practice. Previous methods exist for indirect estimation of CT, which, however, require noninvasive, yet complex and expensive, recordings of the central pressure and flow. Here, we introduce a novel, noninvasive method for estimating CT from a single carotid waveform measurement using regression analysis. Features were extracted from the carotid wave and were combined with demographic data. A prediction pipeline was adopted for estimating CT using, first, a feature-based regression analysis and, second, the raw carotid pulse wave. The proposed methodology was appraised using the large human cohort (N = 2,256) of the Asklepios study. Accurate estimates of CT were yielded for both prediction schemes, namely, r = 0.83 and normalized root mean square error (nRMSE) = 9.58% for the feature-based model, and r = 0.83 and nRSME = 9.67% for the model that used the raw signal. The major advantage of this method pertains to the simplification of the technique offering easily applicable and convenient CT monitoring. Such an approach could offer promising applications, ranging from fast and cost-efficient hemodynamical monitoring by the physician to integration in wearable technologies.NEW & NOTEWORTHY This article introduces a novel artificial intelligence method to estimate total arterial compliance (CT) via exploiting the information provided by an uncalibrated carotid blood pressure waveform as well as typical clinical variables. The major finding of this study is that CT, which is usually acquired using both pressure and flow waveforms, can be accurately derived by the use of the pressure wave alone. This method could potentially facilitate easily applicable and convenient monitoring of CT.

In vivo application and validation of a novel noninvasive method to estimate the end-systolic elastance.

Accurate assessment of the left ventricular (LV) systolic function is indispensable in the clinic. However, estimation of a precise index of cardiac contractility, i.e., the end-systolic elastance (Ees), is invasive and cannot be established as clinical routine. The aim of this work was to present and validate a methodology that allows for the estimation of Ees from simple and readily available noninvasive measurements. The method is based on a validated model of the cardiovascular system and noninvasive data from arm-cuff pressure and routine echocardiography to render the model patient-specific. Briefly, the algorithm first uses the measured aortic flow as model input and optimizes the properties of the arterial system model to achieve correct prediction of the patient's peripheral pressure. In a second step, the personalized arterial system is coupled with the cardiac model (time-varying elastance model) and the LV systolic properties, including Ees, are tuned to predict accurately the aortic flow waveform. The algorithm was validated against invasive measurements of Ees (multiple pressure-volume loop analysis) taken from n = 10 patients with heart failure with preserved ejection fraction and n = 9 patients without heart failure. Invasive measurements of Ees (median = 2.4 mmHg/mL, range = [1.0, 5.0] mmHg/mL) agreed well with method predictions (normalized root mean square error = 9%, ρ = 0.89, bias = -0.1 mmHg/mL, and limits of agreement = [-0.9, 0.6] mmHg/mL). This is a promising first step toward the development of a valuable tool that can be used by clinicians to assess systolic performance of the LV in the critically ill.NEW & NOTEWORTHY In this study, we present a novel model-based method to estimate the left ventricular (LV) end-systolic elastance (Ees) according to measurement of the patient's arm-cuff pressure and a routine echocardiography examination. The proposed method was validated in vivo against invasive multiple-loop measurements of Ees, achieving high correlation and low bias. This tool could be most valuable for clinicians to assess the cardiovascular health of critically ill patients.

Apelin-13 Protects Corpus Cavernosum Against Fibrosis Induced by High-Fat Diet in an MMP-Dependent Mechanism.

BACKGROUND: An increased fibrosis of the corpora cavernosa is a prevalent process that underlies most cases of erectile dysfunction. Apelin, an endogenous circulating peptide, has been documented as an important effector on cardiovascular homeostasis, controlling vascular function and reducing fibrosis in multiple pathological conditions. Recently, initial studies have shown that Apelin, acting through the APJ receptor, also modulates penile erection, however, the role of this system on penile structure and intracorporal collagen remodeling has not been investigated yet. AIMS: Here we sought to investigate the effect of chronic Apelin treatment on the corpus cavernosum structure of hyperchOlesterolemic mice. METHODS: Apolipoprotein gene-deleted (ApoE-/-) mice were fed with a Western diet for 11 weeks and received Apelin-13 (2 mg/kg/day) or vehicle during the last 3 weeks. Penile samples were obtained for histological and biochemical analyses to assess the intracorporal collagen content and key proteins expression. Furthermore, the effect of Apelin-13 was evaluated in cultured NIH3T3 mouse fibroblasts stimulated with TGF-ß. OUTCOME: Local expression of Apelin-13 in mouse corpus cavernosum and its protective effect against fibrosis. RESULTS: Apelin and APJ receptor were expressed (gene and protein) within the corpus cavernosum of ApoE-/- mice, indicating a local modulation of the Apelin system. Interestingly, 3 weeks of Apelin-13 treatment strongly reduced intracavernosal collagen content. In addition, Apelin-13 enhanced total matrix metalloproteinase (MMP) activity in the mice penis, which was associated with an increased protein expression of MMP-1, MMP-3, MMP-8, and MMP-9, while tissue inhibitor of metalloproteinase were unaltered. These beneficial actions were not associated with changes in nNOS or eNOS protein expression, intracavernosal reactive oxygen species content, or atherosclerotic plaque deposition. Additionally, in cultured fibroblast, Apelin-13 inhibited TGF-ß-induced fibroblast to myofibroblast differentiation and collagen production, possibly through the activation of ERK1/2 kinase. CLINICAL TRANSLATION: These results point out Apelin/APJ system as a potential target to treat intracavernosal fibrosis-related disorders. STRENGTH & LIMITATIONS: These results provide the first evidence of the Apelin system's positive role on erectile tissue structure/remodeling. Nevertheless, additional functional study addressing erectile response would bring extended validation regarding the relevance of such effect. CONCLUSION: These results suggest a local modulation of the Apelin system within the corpus cavernosum. Remarkably, Apelin-13 reduced intracavernosal fibrosis in hypercholesterolemic mice by: (i) enhancing MMPs expression and activity; and (ii) inhibiting fibroblast differentiation into myofibroblast. Altogether, these results suggest an essential protective role of Apelin, indicating Apelin/APJ system as a promising candidate for the development of fibrosis-associated erectile dysfunction treatments. Sturny M, Anguenot L Costa-Fraga FP, et al. Apelin-13 Protects Corpus Cavernosum Against Fibrosis Induced by High-Fat Diet in an MMP-Dependent Mechanism. J Sex Med 2021;18:875-888.

Biomechanics of diastolic dysfunction: a one-dimensional computational modeling approach.

Diastolic dysfunction (DD) is a major component of heart failure with preserved ejection fraction (HFpEF). Accordingly, a profound understanding of the underlying biomechanical mechanisms involved in DD is needed to elucidate all aspects of HFpEF. In this study, we have developed a computational model of DD by leveraging the power of an advanced one-dimensional arterial network coupled to a four-chambered zero-dimensional cardiac model. The two main pathologies investigated were linked to the active relaxation of the myocardium and the passive stiffness of the left ventricular wall. These pathologies were quantified through two parameters for the biphasic delay of active relaxation, which simulate the early and late-phase relaxation delay, and one parameter for passive stiffness, which simulates the increased nonlinear stiffness of the ventricular wall. A parameter sensitivity analysis was conducted on each of the three parameters to investigate their effect in isolation. The three parameters were then concurrently adjusted to produce the three main phenotypes of DD. It was found that the impaired relaxation phenotype can be replicated by mainly manipulating the active relaxation, the pseudo-normal phenotype was replicated by manipulating both the active relaxation and passive stiffness, and, finally, the restricted phenotype was replicated by mainly changing the passive stiffness. This article presents a simple model producing a holistic and comprehensive replication of the main DD phenotypes and presents novel biomechanical insights on how key parameters defining the relaxation and stiffness properties of the myocardium affect the development and manifestation of DD.NEW & NOTEWORTHY This study uses a complete and validated computational model of the cardiovascular system to simulate the two main pathologies involved in diastolic dysfunction (DD), i.e., abnormal active relaxation and increased ventricular diastolic stiffness. The three phenotypes of DD were successfully replicated according to literature data. We elucidate the biomechanical effect of the relaxation pathologies involved and how these pathologies interact to create the various phenotypes of DD.

Acute effects of transcatheter aortic valve replacement on the ventricular-aortic interaction.

Transcatheter aortic valve replacement (TAVR) is increasingly used to treat severe aortic stenosis (AS) patients. However, little is known regarding the direct effect of TAVR on the ventricular-aortic interaction. In the present study, we aimed to investigate changes in central hemodynamics after successful TAVR. We retrospectively examined 33 cases of severe AS patients (84 ± 6 yr) who underwent TAVR. Invasive measurements of left ventricular and aortic pressures as well as echocardiographic aortic flow were acquired before and after TAVR (maximum within 5 days). We examined alterations in key features of central pressure and flow waveforms, including the aortic augmentation index (AIx), and performed wave separation analysis. Arterial parameters were determined via parameter-fitting on a two-element Windkessel model. Resolution of AS resulted in direct increase in the aortic systolic pressure and maximal aortic flow (131 ± 22 vs. 157 ± 25 mmHg and 237 ± 49 vs. 302 ± 69 mL/s, P < 0.001 for all), whereas the ejection duration decreased (P < 0.001). We noted a significant decrease in the AIx (from 42 ± 12 to 19 ± 11%, P < 0.001). Of note, the arterial properties remained unchanged. There was a comparable increase in both forward (61 ± 20 vs. 77 ± 20 mmHg, P < 0.001) and backward (35 ± 14 vs. 42 ± 10 mmHg, P = 0.013) pressure wave amplitudes, while their ratio, i.e., the reflection coefficient, was preserved. Our results highlight the impact of TAVR on the ventricular-aortic interaction by affecting the amplitude, shape, and related attributes of the aortic pressure and flow pulse and challenge the interpretation of AIx as a solely vascular measure in AS patients.NEW & NOTEWORTHY Transcatheter aortic valve replacement (TAVR) is linked with an immediate increase in aortic systolic blood pressure and maximal flow, as well as steeper aortic pressure and flow wave upstrokes. After TAVR, the forward wave pumped by the heart is enhanced. Although the arterial properties remain unchanged, the central augmentation index (AIx) is markedly decreased after TAVR. This challenges the interpretation of AIx as a solely vascular measure in patients with aortic valve stenosis.

Early Morphofunctional Changes in AngII-Infused Mice Contribute to Regional Onset of Aortic Aneurysm and Dissection.

Aortic aneurysms and dissections are silent and lethal conditions, whose pathogenesis remains incompletely understood. Although angiotensin II (AngII)-infused ApoE-/- mice have been widely used to study aortic aneurysm and dissection, early morphofunctional alterations preceding the onset of these conditions remain unknown. The goal of this study was to unveil early morphofunctional changes underlying the onset of aneurysm and dissection. At 3 days post-AngII infusion, suprarenal abdominal aorta presented significant volumetric dilatation and microstructural damage. Ex vivo assessment of vascular reactivity of the suprarenal dissection-prone aorta and its side branches, showed an endothelial and contractile dysfunctions that were severe in the suprarenal aorta, moderate distally, and absent in the side branches, mirroring the susceptibility to dissection of these different vascular segments. Early and specific morphofunctional changes of the suprarenal aorta may contribute to the regional onset of aortic aneurysm and dissection by exacerbating the biomechanical burden arising from its side branches.

Characterization of the Renin-Angiotensin System in Aged Cavernosal Tissue and its Role in Penile Fibrosis.

BACKGROUND: The renin-angiotensin system (RAS) plays an important role in erectile function. The RAS contains 2 major axes: one deleterious, composed of ACE-Ang II-AT1 receptor, and another protective, composed of ACE2-Ang-(1-7)-Mas receptor. While aging is a well-known cause for development of male sexual disorders, little is known about local regulation of the RAS in age-related erectile dysfunction (ED). AIM: The present study aimed to assess regulation of the RAS in aging-associated ED rat model and evaluate possible options for disease management through pharmacological modulation of the RAS. METHODS: Penile tissues were harvested from 3-, 12-, and 24-month-old Wistar rats. Local expression of major RAS components and ED markers was measured by RT-PCR. Protein expression of RAS components was assessed by western blot. Collagen deposition was measured by Sirius Red and immunohistochemical staining. Evaluation of collagen content was also performed in penile sections of Mas-knockout mice by Sirius Red and Masson's trichrome stainings. Finally, the effect of Ang-(1-7) pretreatment on TGF-ß-induced myofibroblast activation was studied in primary cavernosal and immortalized fibroblasts. OUTCOMES: Experimental results highlighted the essential role of the RAS in modulation of cavernosal fibrosis. RESULTS: The present study demonstrates local expression of angiotensinogen mRNA alongside with major RAS components, which suggests local autonomous functioning of the RAS within penile tissue. Gene expression analysis revealed strong positive correlation between ACE-Ang II-AT1 axis with markers for inflammation and fibrosis. While corpus cavernosum from 24-month-old rats was characterized by increased collagen deposition, protein expression of ACE, AT1, and Mas was shown to be upregulated in the penile tissue of this group. At the same time, penile sections from Mas-knockout mice (FVB/N background) were also shown to have increased collagen deposition. Finally, it was demonstrated that Ang-(1-7) treatment of primary cavernosal and immortalized fibroblasts was able to alleviate TGF-ß-induced fibroblast-to-myofibroblast transition. CLINICAL TRANSLATION: The present study suggests Ang-(1-7) treatment as a possible strategy for pharmacological management of fibrosis-associated ED in aging. STRENGTHS & LIMITATIONS: The link between the RAS and penile fibrosis, indicated by a holistic screening of different ED markers, was confirmed by in vivo and in vitro data. However, results, presented in the manuscript, need to be further reinforced by human data. Important to note, the main goal of the study was to characterize RAS regulation in aging condition rather than state any causal relationships. CONCLUSION: Present study characterizes RAS regulation in aging-associated ED and indicates its important role in cavernosal fibrosis. Bragina ME, Costa-Fraga F, Sturny M, et al. Characterization of the Renin-Angiotensin System in Aged Cavernosal Tissue and its Role in Penile Fibrosis. J Sex Med 2020;17:2129-2140.

Standardization and Validation of Fluorescence-Based Quantitative Assay to Study Human Platelet Adhesion to Extracellular-Matrix in a 384-Well Plate.

Platelets play a crucial role in the immunological response and are involved in the pathological settings of vascular diseases, and their adhesion to the extracellular matrix is important to bring leukocytes close to the endothelial cells and to form and stabilize the thrombus. Currently there are several methods to study platelet adhesion; however, the optimal parameters to perform the assay vary among studies, which hinders their comparison and reproducibility. Here, a standardization and validation of a fluorescence-based quantitative adhesion assay to study platelet-ECM interaction in a high-throughput screening format is proposed. Our study confirms that fluorescence-based quantitative assays can be effectively used to detect platelet adhesion, in which BCECF-AM presents the highest sensitivity in comparison to other dyes.

On the importance of the nonuniform aortic stiffening in the hemodynamics of physiological aging.

Mathematical models of the arterial tree constitute a valuable tool to investigate the hemodynamics of aging and pathology. Rendering such models as patient specific could allow for the assessment of central hemodynamic variables of clinical interest. However, this task is challenging, particularly with respect to the tuning of the local area compliance that varies significantly along the arterial tree. Accordingly, in this study, we demonstrate the importance of taking into account the differential effects of aging on the stiffness of central and peripheral arteries when simulating a person's hemodynamic profile. More specifically, we propose a simple method for effectively adapting the properties of a generic one-dimensional model of the arterial tree based on the subject's age and noninvasive measurements of aortic flow and brachial pressure. A key element for the success of the method is the implementation of different mechanisms of arterial stiffening for young and old individuals. The designed methodology was tested and validated against in vivo data from a population of n = 20 adults. Carotid-to-femoral pulse wave velocity was accurately predicted by the model (mean error = 0.14 m/s, SD = 0.77 m/s), with the greatest deviations being observed for older subjects. In regard to aortic pressure, model-derived systolic blood pressure and augmentation index were both in good agreement (mean difference of 2.3 mmHg and 4.25%, respectively) with the predictions of a widely used commercial device (Mobil-O-Graph). These preliminary results encourage us to further validate the method in larger samples and consider its potential as a noninvasive tool for hemodynamic monitoring.NEW & NOTEWORTHY We propose a technique for adapting the parameters of a validated one-dimensional model of the arterial tree using noninvasive measurements of aortic flow and brachial pressure. Emphasis is given on the adjustment of the arterial tree distensibility, which incorporates the nonuniform effects of aging on central and peripheral vessel elasticity. Our method could find application in the derivation of important hemodynamic indices, paving the way for novel diagnostic tools.

Α systematic review and meta-analysis of the efficacy of aortic anastomotic devices.

OBJECTIVE: One of the factors contributing to complications related to open repair of the aorta is the construction of a hand-sewn anastomosis. Aortic anastomotic devices (AADs), such as the intraluminal ringed graft (IRG), and the anastomotic stenting technique have been developed to perform a sutureless and less complicated anastomosis. This study performed a systematic review and meta-analysis of the literature reporting clinical use of AADs and aimed to assess, primarily, the effect of each device on 30-day overall and operation-related mortality and aortic cross-clamping time and, secondarily, the rate of successful two-sided application of the IRG device and the operation-related morbidity for each device. METHODS: An electronic search was performed using MEDLINE, Scopus, ScienceDirect, and Cochrane Library by two independent authors. Our exclusion criteria included studies incorporating fewer than three patients and studies reporting results solely from animals or in vitro testing, results solely from end-to-side anastomosis, and results solely from endarterectomy procedures. The last search date was February 1, 2018. RESULTS: A total of 41 studies were identified that reported outcomes for the use of three different device types: IRG, anastomotic stenting technique, and surgical staplers. The last two types were classified together as the non-IRG group. The meta-analysis included 27 studies with 50 cohorts incorporating 1260 patients. The median age of the incorporated patients was 61.4 years (range, 51-73 years), and 68.9% were male. The operations were performed for the treatment of acute aortic dissection in 82.3%. The pooled overall 30-day mortality rate varied by device type; IRG devices had a mean rate of all-cause mortality of 9.71%, whereas non-IRG devices were associated with a significantly (I2 = 15.78%; P for Cochrane Q test < .19) lower rate of death (1.47%). The pooled mean aortic cross-clamping time was 35.83 minutes. Metaregression showed that the performance of two-sided anastomosis with the IRG device significantly decreased the aortic cross-clamping time. However, a successful two-sided ringed anastomosis was performed in approximately half of the cases. CONCLUSIONS: Taking into account that the majority of operations were performed for the treatment of acute aortic dissection, AADs had a relatively low rate of 30-day mortality. Despite the observed heterogeneity in study protocols and the small sample size in the non-IRG group, the non-IRG group presented with the lowest 30-day mortality rate. Specific device-related complications between the different device types need further investigation.

Experimental Drainage Device to Reduce Lymphoedema in a Rat Model.

OBJECTIVE: Despite recent advances in pharmacological research and microsurgery, lymphoedema remains an incurable disease that deeply affects quality of life. There is an urgent need for innovative approaches to restore continuous lymph flow in affected tissues. To this end, the efficacy of a subcutaneously implanted draining device in reducing lymphoedema volume in a rat hindlimb lymphoedema model was tested. METHODS: A rat model of chronic lymphoedema was developed by surgical removal of popliteal and inguinal lymph nodes, followed by irradiation. The model was characterised by monitoring limb volume via tape measure, skin water content via dielectric constant measurement, and lymphatic drainage via lymphofluoroscopy. After lymphoedema establishment in 16 Wistar rats, a device made of fenestrated tubing equipped with a miniaturised pumping system, was implanted subcutaneously in the affected limb to restore continuous recirculation of interstitial fluid. RESULTS: Lymphofluoroscopy imaging showed impaired lymphatic drainage following lymphadenectomy and irradiation. Affected limb volume and skin water content increased significantly compared with the untreated limb, with a median (interquartile range) of 3.85 (0.38) cm3 versus 3.03 (0.43) cm3 for volume (n = 16, p = .001) and 26.6 (9.1) versus 16.6 (3.7) cm3 for skin dielectric constant (n = 16, p = .001). Treatment of lymphoedema with the implanted drainage device showed that 5 weeks post-implant excess volume was significantly reduced by 51 ± 18% compared with the pre-implant situation (n = 9 sham group, n = 7 pump group). CONCLUSION: Lymphoedema volume in the rat model was significantly reduced by restoring continuous drainage of excess fluid using a novel subcutaneously implanted device, opening the way to the development of an artificial lymphatic vessel.

An optimized and validated 384-well plate assay to test platelet function in a high-throughput screening format.

Despite significant advances in the treatment of cardiovascular diseases, antiplatelet therapies are still associated with a high risk of hemorrhage. In order to develop new drugs, methods to measure platelet function must be adapted for the high-throughput screening (HTS) format. Currently, all assays capable of assessing platelet function are either expensive, complex, or not validated, which makes them unsuitable for drug discovery. Here, we propose a simple, low-cost, and high-throughput-compatible platelet function assay, validated for the 384-well plate. In the proposed assay, agonist-induced platelet activity was assessed by three different methods: (i) measurement of light absorbance, which decreases with platelet aggregation; (ii) luminescence measurement, based on ATP release from activated platelets and luciferin-luciferase reaction; and (iii) automated bright-field microscopy of the wells and further quantification of platelet image area, described here for the first time. Brightfield imaging results were validated by demonstrating the similarity of dose-response curves obtained with absorbance and luminescence measurements after stimulating platelets, pre-incubated with prostaglandin E1 or tirofiban, and demonstrating the similarity of dose-response curves obtained with agonists. Assay quality was confirmed using the Z'-factor, a statistical parameter used to validate the robustness and suitability of an HTS assay. The results showed that, under high rotations per minute (1200 RPM), an acceptable Z'-factor score is reached for absorbance measurements (Z'-factor - 0.58) and automated brightfield imaging (Z'-factor - 0.52), without the need of replicates, while triplicates must be used to achieve an acceptable Z'-factor score (0.54) for luminescence measurements. Using low platelet concentration (4 × 104/µl - 10 µl), the brightfield imaging test was further validated using washed platelets. Furthermore, drug screening was performed with compounds selected by structure-based virtual screening. Taken together, this study presents an optimized and validated assay for HTS to be used as a tool for antiplatelet drug discovery.

Apelin-13 treatment enhances the stability of atherosclerotic plaques.

BACKGROUND: Apelin is an endogenous peptidergic system which modulates cardiovascular function. Recent studies pointed out a fundamental contribution of apelin on atherosclerosis development; however, such reports revealed contradictory data, and to date, it is difficult to accurately define a beneficial or deleterious role. To better understand apelin function on atherosclerosis, we aimed to investigate apelin-13 treatment effects on atherosclerotic plaques composition. DESIGN: Apolipoprotein E gene-deleted mice were fed on Western-type diet for 11 weeks. Atherosclerotic plaque formation was induced in the carotid artery by a shear stress modifier device, which exposes the same vessel to distinct patterns of shear stress enabling the formation of plaques with different composition. Mice were treated with apelin-13 (2 mg kg-1 day-1 ) or vehicle for the last 3 weeks. RESULTS: Apelin-13 treatment did not alter the lipid content of low shear stress- and oscillatory shear stress-induced plaques in the carotid. However, apelin-13 greatly ameliorated plaque stability by increasing intraplaque collagen content and reducing MMP-9 expression. Furthermore, apelin-13 decreased the infiltration of inflammatory cells (neutrophil and macrophage) and intraplaque reactive oxygen species content. Interestingly, apelin-13 treatment reduced total cholesterol, LDL levels and free fatty acid serum levels, while HDL, triglycerides serum levels were not significantly changed. CONCLUSIONS: Apelin-13 treatment for 3 weeks did not alter the lesion size, but it significantly enhanced the stable phenotype of atherosclerotic plaques and improved serum lipid profile. These results indicate that activation of apelin system decreases plaque vulnerability.

TGFß (Transforming Growth Factor-ß) Blockade Induces a Human-Like Disease in a Nondissecting Mouse Model of Abdominal Aortic Aneurysm.

OBJECTIVE: Current experimental models of abdominal aortic aneurysm (AAA) do not accurately reproduce the major features of human AAA. We hypothesized that blockade of TGFß (transforming growth factor-ß) activity-a guardian of vascular integrity and immune homeostasis-would impair vascular healing in models of nondissecting AAA and would lead to sustained aneurysmal growth until rupture. APPROACH AND RESULTS: Here, we test this hypothesis in the elastase-induced AAA model in mice. We analyze AAA development and progression using ultrasound in vivo, synchrotron-based ultrahigh resolution imaging ex vivo, and a combination of biological, histological, and flow cytometry-based cellular and molecular approaches in vitro. Systemic blockade of TGFß using a monoclonal antibody induces a transition from a self-contained aortic dilatation to a model of sustained aneurysmal growth, associated with the formation of an intraluminal thrombus. AAA growth is associated with wall disruption but no medial dissection and culminates in fatal transmural aortic wall rupture. TGFß blockade enhances leukocyte infiltration both in the aortic wall and the intraluminal thrombus and aggravates extracellular matrix degradation. Early blockade of IL-1ß or monocyte-dependent responses substantially limits AAA severity. However, blockade of IL-1ß after disease initiation has no effect on AAA progression to rupture. CONCLUSIONS: Endogenous TGFß activity is required for the healing of AAA. TGFß blockade may be harnessed to generate new models of AAA with better relevance to the human disease. We expect that the new models will improve our understanding of the pathophysiology of AAA and will be useful in the identification of new therapeutic targets.

Alamandine abrogates neutrophil degranulation in atherosclerotic mice.

BACKGROUND: Neutrophil-mediated inflammation was recently identified as an active contributor to athero-progression. Therapeutic strategies inhibiting neutrophil degranulation or recruitment were hypothesized to positively impact on plaque vulnerability. In this study, we investigated whether treatment with the recently discovered agonist of the Mas-related G-coupled receptor type D (MrgD) alamandine would impact on neutrophil degranulation in vivo and in vitro. MATERIALS AND METHODS: Fifteen-week-old ApoE-/- mice were fed with a Western-type diet for an additional 11 weeks. After the first 2 weeks of diet, mice were surgically implanted with a carotid 'cast' device that alters the blood shear stress and induces different carotid plaque phenotypes. During the last 4 weeks before euthanasia, mice were randomly assigned to subcutaneously receive vehicle (NaCl 0·15 M) or alamandine (24 µg/kg/h) by micropump. For in vitro experiments, neutrophils were obtained after thioglycollate intraperitoneal injection in ApoE-/- mice. RESULTS: Treatment with alamandine was well-tolerated, but failed to affect lipid, macrophage, neutrophil or collagen content within carotid and aortic root plaques. Also, treatment with alamandine did not affect Th-cell polarization in lymphoid organs. Conversely, alamandine administration was associated with a reduction in serum levels of neutrophil granule enzymes, such as MMP-9 and MPO as well as MMP-9 content within aortic root plaques. In vitro, preincubation with alamandine dose-dependently abrogated PMA-induced neutrophil degranulation of MMP-9 and MPO. CONCLUSION: These results suggest that treatment with the MrgD agonist alamandine led to a reduced release of neutrophil granule products, potentially interfering with pro-atherosclerotic neutrophil activation.

Ascending Aortic Aneurysm in Angiotensin II-Infused Mice: Formation, Progression, and the Role of Focal Dissections.

OBJECTIVE: To understand the anatomy and physiology of ascending aortic aneurysms in angiotensin II-infused ApoE(-/-) mice. APPROACH AND RESULTS: We combined an extensive in vivo imaging protocol (high-frequency ultrasound and contrast-enhanced microcomputed tomography at baseline and after 3, 10, 18, and 28 days of angiotensin II infusion) with synchrotron-based ultrahigh resolution ex vivo imaging (phase contrast X-ray tomographic microscopy) in n=47 angiotensin II-infused mice and 6 controls. Aortic regurgitation increased significantly over time, as did the luminal volume of the ascending aorta. In the samples that were scanned ex vivo, we observed one or several focal dissections, with the largest located in the outer convex aspect of the ascending aorta. The volume of the dissections moderately correlated to the volume of the aneurysm as measured in vivo (r(2)=0.46). After 3 days of angiotensin II infusion, we found an interlaminar hematoma in 7/12 animals, which could be linked to an intimal tear. There was also a significant increase in single laminar ruptures, which may have facilitated a progressive enlargement of the focal dissections over time. At later time points, the hematoma was resorbed and the medial and adventitial thickness increased. Fatal transmural dissection occurred in 8/47 mice at an early stage of the disease, before adventita remodeling. CONCLUSIONS: We visualized and quantified the dissections that lead to ascending aortic aneurysms in angiotensin II-infused mice and provided unique insight into the temporal evolution of these lesions.

Total arterial compliance, estimated by a novel method, is better related to left ventricular mass compared to aortic pulse wave velocity: The SAFAR study.

AIM: The investigation of the association between total arterial compliance (CT)-estimated by a novel technique-with left ventricular mass (LVM) and hypertrophy (LVH). Our hypothesis was that CT may be better related to LVM compared to the gold-standard regional aortic stiffness. Within the frame of the ongoing cross-sectional study "SAFAR," 226 subjects with established hypertension or with suspected hypertension underwent blood pressure (BP) assessment, carotid-to-femoral pulse wave velocity (cf-PWV), and echocardiographic measurement of LVM. LVM index (LVMI) was calculated by the ratio of LVM to body surface area. CT was estimated by a previously proposed and validated formula: CT = 36.7 /cf-PWV2 [ml/mmHg]. LVMI was related to age (r = 0.207, p = 0.002), systolic BP (r = 0.248, p < 0.001), diastolic BP (r = 0.139, p = 0.04), mean BP (r = 0.212, p = 0.002), pulse pressure (r = 0.212, p = 0.002), heart rate (r = -0.172, p = 0.011), cf-PWV (r = 0.268, p < 0.001), and CT (r = -0.317, p < 0.001). The highest correlation was observed for CT that was significantly stronger than the respective correlation of cf-PWV (p < 0.001). In multivariate analysis, CT was a stronger determinant, compared to cf-PWV, of LVMI and LVH. It remains to be further explored whether CT has also a superior prognostic value beyond and above local or regional (segmental) estimates of pulse wave velocity.

Update on the role of angiotensin in the pathophysiology of coronary atherothrombosis.

BACKGROUND: Coronary atherothrombosis due to atherosclerotic plaque rupture or erosion is frequently associated with acute coronary syndromes (ACS). Significant efforts have been made to elucidate the pathophysiological mechanisms underlying acute coronary events. MATERIALS AND METHODS: This narrative review is based on the material searched for and obtained via PubMed up to August 2014. The search terms we used were as follows: 'angiotensin, acute coronary syndromes, acute myocardial infarction' in combination with 'atherosclerosis, vulnerability, clinical trial, ACE inhibitors, inflammation'. RESULTS: Among several regulatory components, the renin-angiotensin system (RAS) was shown as a key pathway modulating coronary atherosclerotic plaque vulnerability. Indeed, these molecules are involved in all stages of atherogenesis. Classically, the RAS is composed by a series of enzymatic reactions leading to the angiotensin (Ang) II generation and activity. However, the knowledge of RAS has expanded and become more complex. The discovery of novel components and their functions has revealed additional pathways that contribute to or counterbalance the actions of Ang II. In this review, we discussed on recent findings concerning the role of different angiotensin peptides in the pathophysiology of ACS and coronary atherothrombosis, exploring the link between these molecules and atherosclerotic plaque vulnerability. CONCLUSIONS: Treatments selectively targeting angiotensins (including Mas and AT2 agonists, ACE2 recombinant, or Ang-(1-7) and almandine in oral formulations) have been tested in animal studies or in small human subgroups, expanding the perspective in the ACS prevention. These novel strategies, especially in the counter-regulatory axis ACE2/Ang-(1-7)/Mas, might be promising to reduce plaque vulnerability and inflammation.

Diminazene protects corpus cavernosum against hypercholesterolemia-induced injury.

INTRODUCTION: Angiotensin-converting enzyme 2 (ACE2) is a key enzyme of the renin angiotensin system, which breaks down angiotensin II and forms angiotensin-(1-7). In erectile tissues, it has been documented that angiotensin II contributes to the development of erectile dysfunction (ED), while treatment with angiotensin-(1-7) improves penile erection. However, the expression and function of ACE2 in erectile tissues have never been investigated. AIM: Here, we examined the expression of ACE2 in erectile tissues and its actions against hypercholesterolemia-induced corpus cavernosum (CC) injury. METHODS: Hypercholesterolemic apolipoprotein E knockout (ApoE(-/-) ) mice, a well-known model of ED, were treated with diminazene aceturate (DIZE), an ACE2 activator compound, or vehicle for 3 weeks. Reactive oxygen species (ROS), collagen content, and protein expression of ACE2, neuronal nitric oxide synthase (nNOS), endothelial nitric oxide synthase (eNOS), nicotinamide adenine dinucleotide phosphate-oxidase (NADPH) subunits were evaluated in the penis of DIZE-treated and untreated ApoE(-/-) mice. Functional studies were performed in CC strips. MAIN OUTCOME MEASURES: ACE2 expression and its role in modulating nitric oxide (NO)/ROS production and fibrosis within the CC of hypercholesterolemic mice were the main outcome measures. RESULTS: ACE2 was expressed in smooth muscle and endothelial cells of mouse CC. Interestingly, ACE2 was downregulated in penis of hypercholesterolemic mice with ED, suggesting a protective role of ACE2 on the CC homeostasis. In accordance with that, pharmacological ACE2 activation by DIZE treatment reduced ROS production and NADPH oxidase expression, and elevated nNOS and eNOS expression and NO bioavailability in the penis of ApoE(-/-) mice. Additionally, DIZE decreased collagen content within the CC. These beneficial actions of DIZE on the CC were not accompanied by improvements in atherosclerotic plaque size or serum lipid profile. CONCLUSION: ACE2 is expressed in erectile tissue and its reduction is associated with hypercholesterolemia-induced ED. Additionally, treatment with DIZE improved hypercholesterolemia-induced CC injury, suggesting ACE2 as a potential target for treating ED. .