Cross-sections Of Arteries Research Articles

Unraveling immune checkpoint inhibitors effects on atherosclerosis: mechanisms and preventive approaches

Abstract Background Immune checkpoint inhibitors (ICIs) represent a novel and rapidly evolving field in cancer treatment. Despite increasing reports of accelerated atherosclerosis and ICI-related atherosclerotic cardiovascular (CV) events in clinical studies and case reports, there is a notable lack of data concerning the underlying mechanisms, appropriate monitoring, and potential interventions. Objectives To explore the role of ICIs in triggering and/or accelerating the atherosclerotic process, with a specific focus on the involvement of CD8+ T-cells. Additionally, we aimed to evaluate and compare the potential beneficial effects of statins and Proprotein Convertase Subtilisin/Kexin type 9 (PCSK9) inhibitors on atherosclerosis under ICIs treatment. Methods Thirty 8-week-old C57BL mice were divided into six groups as follows: (1) Control, normal diet; (2) Control, atherogenic diet; (3) Atherogenic diet + anti PD-1; (4) Atherogenic diet + statins + anti-PD1; (5) Atherogenic diet + PCSK9-i + anti PD-1; (6) Atherogenic diet + PCSK9 inhibitors. At 16 weeks, the carotid arteries and heart were removed for histological examination, and serum was withdrawn for blood work and further analysis. Results Anti-PD1 treatment resulted in extensive lymphocytic infiltration of the carotid intima, primarily constituted of CD8+ T cells. This infiltration was significantly attenuated when statins or PCSK-9 inhibitors were used in conjunction with anti-PD1 treatment. Serum analysis for CD4+ and CD8+ T cells by ELISA revealed no difference in CD4+ abundance between the groups. However, plasma CD8+ T cells were significantly increased with anti-PD1 treatment, and furthermore, when PCSK9 inhibitors were used in combination; but not when statins were used. Conclusion The induction of CD8+ T cell infiltration in the arterial carotid walls by anti-PD-1 was prevented with the use of either statins or PCSK9 inhibitors. In the plasma, while statins reduced the hyper-expression of CD8+ T cells observed under PD-1 treatment, PCSK9 inhibitors enhanced this expression. This may provide a first indication of the potential benefit of PCSK9 inhibitor treatment for both preventing arterial inflammation and enhancing the desired anti-tumor immune response under ICIs treatment. Figure legend: 1A. A cross-section of the carotid artery after 16 weeks of treatment, stained with H&E. Magnification is X100. 1B. A cross-section of the carotid artery after 16 weeks of treatment, stained with anti-CD8 Ab. Magnification is X100. 1C. quantitative CD8+ T cells staining in the carotid arteries. 1D. quantitative results of ELISA for plasma CD8+ T cells.

Coronary artery disease ameliorates extracellular vesicle lncRNA PUNISHER regulates angiogenic response and endothelial cells function via NFkB-dependent mechanism

Abstract Introduction Augmenting evidence indicates that long noncoding RNAs (lncRNAs) are playing a crucial role in diverse cellular/pathological processes. Intercellular transfer of extracellular vesicles (EVs) transmitted lncRNA regulates vascular health and diseases. However, whether lncRNA expression in EVs is regulated in patients with coronary artery disease, is unknown. A PCR-based lncRNA array analysis revealed that EV-PUNISHER was significantly upregulated in patients with CAD (n=221) compared to healthy subjects. Our data showed that PUNISHER is the most upregulated lncRNA in CAD in accordance with other highly expressed lncRNAs (FGF14-AS2, HYMAI, Gas5, Malat1, etc.). SiRNA-mediated silencing in ECs revealed that depletion of PUNISHER suppresses the migration and proliferation of ECs. Depletion of PUNISHER decreases cell survival by reducing cell viability and proliferation through increased apoptosis and cytotoxicity. To investigate EC function in PUNISHER depleted cells, sprouting and tube formation assay revealed that PUNISHER is an important mediator EC functions, which acts as an anti-angiogenic factor. In vitro atherosclerotic stimuli (OxLDL, TNF-α, IL-6) increased PUNISHER expression in EV/EC in a dose-dependent way. Microarray analysis identified a series of pro- and anti-angiogenic genes as well genes directly involved in cell viability that are differentially regulated. By using lncRNA-FISH and vesicle degradation assays, we showed that PUNISHER is incorporated into EMVs, augmented recipient EC function in vitro upon transfer via EMVs. To examine whether EMV-PUNISHER promotes the EC function, different in vitro functional experiments with ECs (tube formation, angiogenic sprouting, migration, proliferation, apoptosis, etc.) confirmed that PUNISHER is an important regulator. MS analysis has revealed EMV contains numerous proteins, including RNA binding proteins such as hnRNPU, hnRNPK, hnRNPA2/B1, etc. By using RNA-pulldown and RNA immunoprecipitation, we confirmed that PUNISHER interacts with hnRNPU, which facilitates packaging into EMV prior to transfer to recipient EC. The interaction acts as an important positive regulator of cell viability and survival of recipient cells, identified using functional assay (migration, viability, proliferation, and angiogenesis). Transcription factor array has shown that PUNISHER regulates NFkB to control cellular viability and apoptosis. A murine re-endothelialization model after injection of 1×107 EVs or bulk ncRNAs (1, 5, 10 nM) revealed that EV-PUNISHER promotes reendothelialization. Ex vivo internalization/uptake of PKH-67-labeled EVs revealed that EVs are detectable in the cross-section of EV-perfused carotid artery. Our study unveiled for the first time that EV-incorporated PUNISHER exerts its function in EC which might be beneficial in cardiovascular pathologies. Our data indicate that PUNISHER can be targeted to develop targeted therapeutics.

Genesis of Intimal Thickening Due to Hemodynamical Shear Stresses.

This paper investigates intimal growth in arteries, induced by hemodynamical shear stress, through finite element simulation using the FEniCS computational environment. In our model, the growth of the intima depends on cross section geometry and shear stress. In this work, the arterial wall is modeled as three distinct layers: the intima, the media, and the adventitia, each with different mechanical properties. We assume that the cross section of the vessel does not change in the axial direction. We further assume that the blood flow is steady, non-turbulent, and unidirectional. Blood flow induces shear stress on the endothelium and stimulates the release of Platelet Derived Growth Factor (PDGF) which drives the growth. We simulate intimal growth for three distinct arterial cross section geometries. We show that the qualitative nature of intimal thickening varies depending on arterial geometry. For cross section geometries that are annular, the growth of the intima is uniform in the angular direction, and the endothelium stays circular as the intima grows. For non-annular cross section geometries, thicker intimas grow more compared to thinner ones, shear stress and intimal thickening are negatively correlated with the distance from the flow center, where the flow velocity is maximal. Over time, the maxima and minima of the curvature increase and decrease respectively, the PDGF concentration increases, and the lumen becomes more polygonal. The model provides a framework for coupling hemodynamics simulations to mathematical descriptions of atherosclerosis, both of which have been modeled separately in great detail.

226 Serotonin stimulates relaxation in ovine saphenous vein precontracted with ergovaline

Abstract The predominant ergot alkaloid found in wild type endophyte-infected tall fescue grass is ergovaline. Ergovaline is a potent vasoconstrictor in livestock that consume it. Previous research has shown that ergovaline can bind serotonin (5-HT) receptors and blood vessels exposed to ergovaline become unresponsive to subsequent 5-HT exposure. Serotonin can stimulate vascular contraction or relaxation depending on the receptor subtype targeted. The objective of this experiment was to evaluate 5HT as vasorelaxant in the ovine saphenous artery and vein that were precontracted with ergovaline. Blood vessels were collected from mixed breed market rams (n = 7) at slaughter, cleaned of surrounding adipose and connective tissue, sliced into 2-mm cross-sections, and mounted in a multimyograph. Artery and vein cross-sections were equilibrated to 1 g tension in continuously gassed (95% O2/5% CO2) Krebs-Henseleit buffer (pH 7.4; 37.5 °C) for 90 min, precontracted with 1.0 μM ergovaline (in a tall fescue seed extract) for 30 min, exposed to increasing concentrations of 5-HT that ranged from 1 x 10-9 to 1 x 10-4 M for a 5-min interval. Response data were normalized to the 1.0 μM ergovaline response and were analyzed as a completely randomized design in SAS. The saphenous vein was larger in diameter blood vessel than the saphenous artery. Both vessel types contracted in response to exposure to 1 μM ergovaline. The saphenous artery had a much lower maximal response to ergovaline than did the vein (P < 0.05) that could be attributed to the size difference. The lateral saphenous vein relaxed to increasing concentrations of 5-HT (P < 0.05) with 87 % relaxation occurring by 1 x 10-6 M 5-HT. Conversely, the lateral saphenous artery contracted in response to increasing concentrations of 5-HT (P < 0.05) resulting in a 43% increase from the ergovaline response by the 1 x 10-4 M addition. This response did not become significant in the artery until 1 x 10-5 M 5-HT compared with 1 x 10-8 M for the vein. This resulted in a 50% effective concentration for 5-HT in the artery of 4.5 compared with 8.1 M for the vein (P < 0.05). The differing responses across vessels are likely a result of different receptor populations and this should be verified in future research. This is the first reported observation of stimulated relaxation of a blood vessel constricted ergovaline. Additional research is needed to understand the mechanism of how 5HT can be used to stimulate vasorelaxation in livestock suffering from ergot alkaloid-induced vasoconstriction.

The morphology of internal elastic lamina corrugations in arteries under physiological conditions

Background: In elastic and resistance arteries, an elastin-rich membrane, the Internal Elastic Lamina (IEL), separates the tunica intima from the underlying tunica media. The IEL often appears wrinkled or corrugated in histological images. These corrugations are sometimes ascribed to vessel contraction ex vivo, and to fixation artifacts, and therefore regarded as not physiologically relevant. We examine whether the IEL remains corrugated even under physiological conditions.Methods: The diameters of carotid arteries of anesthetized pigs were measured by ultrasound. The arteries were then excised, inflated within a conical sleeve, fixed, and imaged by confocal microscopy. The conical sleeve allows fixing each artery across a wide range of diameters which bracket its ultrasound diameter. Thus the study was designed to quantify how corrugations change with diameter for a single artery, and test whether corrugations exist when the fixed artery matches the ultrasound diameter.Results: At diameters below the ultrasound diameter (i.e. when the artery was constricted as compared to ultrasound conditions), the IEL corrugations were found to decrease significantly with increasing diameter, but not fully flatten at the ultrasound diameter. The contour length of the IEL was found to be roughly 10% larger than the circumference of the artery measured by ultrasound. The physiological diameter is likely to be even smaller than the ultrasound diameter since ultrasound was conducted with the animal under general anesthesia which leads to vasodilation, suggesting a higher level of corrugation under physiological conditions. For arterial cross sections constricted below the ultrasound diameter, the IEL contour length decreased roughly with the square root of the diameter.Conclusion: The primary conclusions of this study are: a) the IEL is corrugated when the artery is constricted and flattens as the artery diameter increases, b) the IEL is corrugated under physiological conditions and has a contour length at least 10% more than the physiological arterial diameter, and c) the IEL despite being relatively stiffer than the surrounding arterial layers, does not behave like an inextensible membrane.

#2115 Phase-contrast magnetic resonance imaging (PC-MRI) for quantification of volumetric flow in renal transplant arteries: an observational cohort study

Abstract Background and Aims Impaired renal transplant blood flow, a manifestation of severe transplant renal artery stenosis (TRAS), can negatively impact transplant function and survival. Phase-contrast magnetic resonance imaging (PC-MRI) is a non-invasive method of quantifying blood flow volume. Recent work has demonstrated the utility of this technique in measuring native renal blood flow, whilst validating the use of flow measurements taken on the aorta above and below the origin of the renal arteries to calculate flow in the renal arteries [1]. As part of a quality improvement study incorporating PC-MRI into renal transplant assessment, this study set out to (a) establish the normal range for transplant renal blood flow volume and (b) assess whether PC-MRI improved screening for TRAS. Method Between January 2019 and February 2021, PC-MRI sequences were included in the protocols for all magnetic resonance angiograms (MRA) performed as part of clinically-indicated renal transplant assessment. As part of the MRA study, using a coronal localizer MR image, planes were prescribed on the external iliac artery (EIA) both proximal and distal to the transplant artery anastomosis site in a perpendicular orientation (Fig. 1A). Using CV142 software [2], arterial cross-sections were highlighted manually at the pre-determined planes (Fig. 1B) to generate flow curves that coincided with the cardiac cycle (Fig. 1C). Studies were excluded if: the EIA flow waveform was incongruous with the cardiac cycle, contemporaneous heart rate data was not recorded, or the calculated flow volume was a negative value. Results During the study period 392 transplant MRAs were performed, of which 218 were included in this analysis. Potential TRAS was identified in 49 (22.5%) studies, with 19 (8.7%) having this diagnosis confirmed by intra-arterial digital subtraction angiography (IADSA). In the 169 (77.5%) studies without evidence of TRAS, the mean flow in the proximal EIA was 0.90 L/min (95% CI 0.85-0.94 L/min) and in the distal EIA 0.47 L/min (95% CI 0.44-0.50 L/min). The difference between the two measurement points, that is the flow in the transplant renal artery, was 0.42 L/min (95% CI 0.39-0.45 L/min) (Fig. 2A). In the 19 studies with TRAS confirmed by IADSA, mean flow at the time of the initial MRA was 0.34 L/min (95% CI 0.25-0.43 L/min); there was no difference when compared with the group without evidence of TRAS on MRA (p = 0.09) (Fig. 2B). Of the patients who had potential TRAS identified on MRA, 30 patients proceeded to IADSA; there was no difference in PC-MRI flow volume in the 11 who had a diagnosis of TRAS refuted on IADSA and the 19 who had diagnosis of TRAS confirmed (p = 0.97). ROC curve analysis of PC-MRI arterial flow measurement as a diagnostic test for TRAS, whereby the positive group of 19 had IADSA confirmed TRAS, the negative group of 176 had either normal MRA artery appearances or normal IADSA, and the disease prevalence is 13.7% [3], shows AUC 0.62 (95% CI 0.55-0.69) P = 0.09, sensitivity 57.9 specificity 67.1 (Fig. 2C). Conclusion This is the largest study to incorporate functional PC-MRI sequences into MRA protocols for the assessment of people with any form of kidney disease. The results suggest that the blood flow volume in healthy transplant renal arteries lies between 0.39–0.45 L/min. In comparative analysis between patients with healthy transplant arteries and those with IADSA-confirmed TRAS, there was no difference in arterial flow volume, though the study was limited by the small population in the group with IADSA-confirmed TRAS. PC-MRI does not appear to improve the screening accuracy of MRA in identifying TRAS. However, there may be a future role for PC-MRI as part of multi-parametric MR protocols for comprehensive assessment of patients with kidney transplants.

Abstract 1041: Loss Of The ETS Transcription Factor ERG Disrupts Fate-defining Programs In The Aortic Endothelium And Promotes Expansion Of Endothelial Lineage Cells In Atherosclerotic Plaque

Aim: The ETS transcription factor ERG has been identified as a principal regulator of endothelial function through its ability to repress inflammation in endothelial cells (ECs), and loss of ERG induces endothelial to mesenchymal transition (EndMT) in fibrotic disease. To investigate ERG function in atherosclerosis, we employed an EC-specific Erg knockout ( Erg EC - KO ) in PCSK9-overexpression and apoE-deficient murine atherosclerosis models. Methods: Atherosclerosis was induced in 10-week-old Erg EC - KO mice or wild-type counterparts that had a Cre-inducible EC lineage tag ( Cdh5 CreER ; R26-CAG-LSL-Sun1-sfGFP ). Atherosclerosis was modeled by Apoe deletion or AAV8-PCSK9 with 12 weeks of high-cholesterol diet (1.25%). Aortic intimal cells were assessed with flow cytometry. Plaque burden, morphology, and cellularity were characterized with brightfield, Oil Red O, H&E, Movat, and immunofluorescence imaging. Results: Erg EC-KO in PCSK9-overexpression mice led to a 1.6-fold increase in aortic plaque burden and plaque formation in regions that are usually protected (e.g., greater curvature; p≤0.03; n=9-10). Loss of ERG increased elastin breaks (1.7-fold; p=0.02; n=9) and decreased fibrous cap thickness (0.76-fold; p=0.02; n=9-10) in aortic arch cross sections. Aortic intimal digests from Erg EC- KO mice contained increased Sun1-sfGFP + EC lineage cells (1.9-fold; p=0.01; n=4), which had reduced CD31 positivity (0.79-fold; p=0.03; n=4). Notably, a 5.3-fold increase in EC lineage cells was observed in plaques from brachiocephalic artery cross sections (p=0.0001; n=6-15), which had hallmarks of EndMT (e.g., 31.5-fold increase in GFP + ACTA2 + cells, p=0.02, n=3-5). Increased aortic plaque burden was recapitulated in apoE-deficient E rg EC- KO mice (2.1-fold; p=0.02; n=4-8). Conclusion: Endothelial Erg KO results in increased plaque burden and altered plaque topography in murine atherosclerosis. Loss of ERG induces EndMT and marked ingression of ECs from the luminal endothelium to the plaque interior. These findings highlight loss of ERG as a novel mechanism by which intimal ECs acquire mesenchymal identity in atherosclerosis and implicate EC identity loss as a driver of EC migration into plaques, which is associated with a vulnerable morphology.

Plaque heterogeneity and the spatial distributions of its components dictate drug-coated balloon therapy

Drug-coated balloon (DCB) angioplasty is one of the potential approaches to alleviating in-stent restenosis and treating peripheral artery disease. An in-silico model has been developed for sirolimus drug eluted from an inflated balloon in a patient-specific arterial cross-section consisting of fibrous tissue, fibrofatty tissue, dense calcium, necrotic core, and healthy tissue. The convection-diffusion-reaction equation represents the transport of drug, while drug binding, both specific and non-specific, can be modelled as a reaction process. The Brinkman equations describe the interstitial flow in porous tissue. An image processing technique is leveraged for reconstructing the computational domain. The Marker and Cell, and Immersed Boundary Methods are used to solve the set of governing equations. The no-flux interface condition and convection do amplify the tissue content, and the regions of dense calcium and necrotic core limited to or extremely close to the interface pose a clinical threat to DCB therapy. Simulations predict the effects of the positioning and clustering of plaque components in the domain. This study demands extensive intravascular ultrasound-derived virtual histology (VH-IVUS) imaging to understand the plaque morphology and determine the relative positions of different plaque compositions about the lumen-tissue interface, which have a significant impact on arterial pharmacokinetics.

Deep Learning-Based Prediction of Stress and Strain Maps in Arterial Walls for Improved Cardiovascular Risk Assessment

Conducting computational stress-strain analysis using finite element methods (FEM) is a common approach when dealing with the complex geometries of atherosclerosis, which is a leading cause of global mortality and complex cardiovascular disease. The considerable expense linked to FEM analysis encourages the substitution of FEM with a considerably faster data-driven machine learning (ML) approach. This study investigated the potential of end-to-end deep learning tools as a more effective substitute for FEM in predicting stress-strain fields within 2D cross sections of arterial walls. We first proposed a U-Net-based fully convolutional neural network (CNN) to predict the von Mises stress and strain distribution based on the spatial arrangement of calcification within arterial wall cross-sections. Further, we developed a conditional generative adversarial network (cGAN) to enhance, particularly from the perceptual perspective, the prediction accuracy of stress and strain field maps for arterial walls with various calcification quantities and spatial configurations. On top of U-Net and cGAN, we also proposed their ensemble approaches to improve the prediction accuracy of field maps further. Our dataset, consisting of input and output images, was generated by implementing boundary conditions and extracting stress-strain field maps. The trained U-Net models can accurately predict von Mises stress and strain fields, with structural similarity index scores (SSIM) of 0.854 and 0.830 and mean squared errors of 0.017 and 0.018 for stress and strain, respectively, on a reserved test set. Meanwhile, the cGAN models in a combination of ensemble and transfer learning techniques demonstrate high accuracy in predicting von Mises stress and strain fields, as evidenced by SSIM scores of 0.890 for stress and 0.803 for strain. Additionally, mean squared errors of 0.008 for stress and 0.017 for strain further support the model’s performance on a designated test set. Overall, this study developed a surrogate model for finite element analysis, which can accurately and efficiently predict stress-strain fields of arterial walls regardless of complex geometries and boundary conditions.

286 Evaluation of Serotonin as a Vasorelaxant in Ovine Saphenous Artery and Vein

Abstract Herbivores that consume ergot alkaloids frequently suffer from vasoconstriction that can result in a myriad of symptoms that can range from necrosis of peripheral tissues to fetal intra-uterine growth restriction. As part of a larger study screening compounds that may mitigate the vasoconstrictive effects of ergot alkaloids, the objective of this experiment was to evaluate serotonin (5-HT) as vasorelaxant in the ovine saphenous artery and vein. Blood vessels were collected from a total mixed breed market rams (n = 16; approximate BW = 58 kg) at slaughter, cleaned of surrounding adipose and connective tissue, sliced into 2-mm cross-sections, and mounted in a multimyograph. Artery and vein cross-sections were equilibrated to 1 g tension in continuously gassed (95% O2/5% CO2) Krebs-Henseleit buffer (pH 7.4; 37.5 °C) for 90 min, precontracted with 0.0001 M phenylephrine for 15 min, exposed to concentrations of 5-HT that ranged from 1 x 10-9 to 1 x 10-4 M for a 5-min interval. Response data were normalized to the 0.0001 M phenylephrine response and were analyzed as a completely randomized design in SAS. The lateral saphenous vein (n = 5 lambs) relaxed to increasing concentrations of 5-HT (P < 0.05) with 65 to 75 % relaxation occurring at 1 x 10-7 M through 1 x 10-4 5-HT. Conversely, the lateral saphenous artery (n = 6 lambs) contracted in response to increasing concentrations of 5-HT (P < 0.05) resulting in a 255% increase from the phenylephrine response by the 1 x 10-4 M addition. In a subsequent experiment, saphenous veins from the remaining lambs (n = 5) were treated the same, but in place of increasing concentrations of 5-HT, saphenous veins were exposed to increasing concentrations of selective 5-HT receptor agonists to determine the receptor subtypes involved in the previously observed vasorelaxation. Agonists for receptors 5-HT2B (BW 723C86; α-methyl-5-(2-thienylmethoxy)-1H-indole-3-ethanamine hydrochloride), 5-HT4 (BIMU-8; 2,3-Dihydro-N-[(3-endo)-8-methyl-8-azabicyclo[3.2.1]oct-3-yl]-3-(1-methylethyl)-2-oxo-1H-benzimidazole-1-carboxamide hydrochloride), and 5-HT7 (LP44; 4-[2-(methylthio)phenyl]-N-(1,2,3,4-tetrahydro-1-naphthalenyl)-1-piperazinehexanamide, monohydrochloride) were used. These 5-HT receptor subtypes were selected based on their association with vascular smooth muscle relaxation. While all three 5-HT receptor subtypes resulted in vasorelaxation (P < 0.05), the 5-HT2B agonist only stimulated a maximal relaxation of 32% compared with the agonists for 5-HT4 and 5-HT7 that resulted in maximal relaxation of 68% and 50%, respectively. The agonist for 5-HT4 produced the greatest relaxation in the ovine lateral saphenous vein and should be targeted in subsequent research. It is likely that the combined effect of binding multiple receptor subtypes contributes to the overall effect of 5-HT. The focus on development of ergot alkaloid mitigation strategies should consider the divergent responses of arteries and veins to serotonin in peripheral tissues.

Tele-ultrasound in community with limited resources: The association of hypertension on reduced carotid artery circumferential strain

Introduction: Stroke is one of the leading causes of death worldwide, in majority triggered by modifiable risk factors such as hypertension. AIM: to assess relations between hypertension and carotid arteries radial and circumferential deformation, assessing a population of participants after a first ischemic stroke and healthy controls. Methods: Retrospective study, from 2016 to 2017, with a sample of the PAI project control group, an urban population of the São Francisco Valley in the Northeast of Brazil, 105 people between 40-80 years-old, including patients after a first episode of ischemic stroke in the absence of known cardiovascular disease; and a control group of healthy volunteers, matched by age, sex, and presence of hypertension. Asynchronous analysis was performed by tele-ultrasonography of carotid artery ultrasound with images acquisition of EKG-synchronized videos in cross-section of the common carotid artery bilaterally and then strain values were acquired by Speckle Tracking technique, computing the mean circumferential and radial peaks of both carotids. Univariate and multivariable analyses assessed the association between carotid strain and hypertension. Results: 84 participants had analyzable images (29 patients after a first episode of stroke). The mean age was 60.5 + 11.6 years, 46.5% were women, 70.2% had hypertension. Radial deformation was lower but non-significant in participants with hypertension compared to those without hypertension (-3.13% vs. -3.35%, respectively, p = 0.54). There was significantly less circumferential deformation among participants who had hypertension compared to those without hypertension (4.16% vs. 5.3%, respectively, p = 0.003). Hypertension maintained association with lower circumferential strain in multivariate analysis (coef = -1.13; p = 0.019). Conclusion: hypertension was independently associated with the decrease in the carotid artery circumferential deformation. Tele-ultrasound associated with carotid strain analysis shows promising results for the evaluation of subclinical changes in carotid arteries in populations with limited resources in remote regions such as the Northeast of Brazil.

An agent-based model of cardiac allograft vasculopathy: toward a better understanding of chronic rejection dynamics.

Cardiac allograft vasculopathy (CAV) is a coronary artery disease affecting 50% of heart transplant (HTx) recipients, and it is the major cause of graft loss. CAV is driven by the interplay of immunological and non-immunological factors, setting off a cascade of events promoting endothelial damage and vascular dysfunction. The etiology and evolution of tissue pathology are largely unknown, making disease management challenging. So far, in vivo models, mostly mouse-based, have been widely used to study CAV, but they are resource-consuming, pose many ethical issues, and allow limited investigation of time points and important biomechanical measurements. Recently, agent-based models (ABMs) proved to be valid computational tools for deciphering mechanobiological mechanisms driving vascular adaptation processes at the cell/tissue level, augmenting cost-effective in vivo lab-based experiments, at the same time guaranteeing richness in observation time points and low consumption of resources. We hypothesize that integrating ABMs with lab-based experiments can aid in vivo research by overcoming those limitations. Accordingly, this work proposes a bidimensional ABM of CAV in a mouse coronary artery cross-section, simulating the arterial wall response to two distinct stimuli: inflammation and hemodynamic disturbances, the latter considered in terms of low wall shear stress (WSS). These stimuli trigger i) inflammatory cell activation and ii) exacerbated vascular cell activities. Moreover, an extensive analysis was performed to investigate the ABM sensitivity to the driving parameters and inputs and gain insights into the ABM working mechanisms. The ABM was able to effectively replicate a 4-week CAV initiation and progression, characterized by lumen area decrease due to progressive intimal thickening in regions exposed to high inflammation and low WSS. Moreover, the parameter and input sensitivity analysis highlighted that the inflammatory-related events rather than the WSS predominantly drive CAV, corroborating the inflammatory nature of the vasculopathy. The proof-of-concept model proposed herein demonstrated its potential in deepening the pathology knowledge and supporting the in vivo analysis of CAV.

PAR4 Inhibition Reduces Coronary Artery Atherosclerosis and Myocardial Fibrosis in SR-B1/LDLR Double Knockout Mice.

SR-B1 (scavenger receptor class B type 1)/LDLR (low-density lipoprotein receptor) double knockout mice fed a high-fat, high-cholesterol diet containing cholate exhibit coronary artery disease characterized by occlusive coronary artery atherosclerosis, platelet accumulation in coronary arteries, and myocardial fibrosis. Platelets are involved in atherosclerosis development, and PAR (protease-activated receptor) 4 has a prominent role in platelet function in mice. However, the role of PAR4 on coronary artery disease in mice has not been tested. We tested the effects of a PAR4 inhibitory pepducin (RAG8) on diet-induced aortic sinus and coronary artery atherosclerosis, platelet accumulation in atherosclerotic coronary arteries, and myocardial fibrosis in SR-B1/LDLR double knockout mice. SR-B1/LDLR double knockout mice were fed a high-fat, high-cholesterol diet containing cholate and injected daily with 20 mg/kg of either the RAG8 pepducin or a control reverse-sequence pepducin (SRQ8) for 20 days. Platelets from the RAG8-treated mice exhibited reduced thrombin and PAR4 agonist peptide-mediated activation compared with those from control SRQ8-treated mice when tested ex vivo. Although aortic sinus atherosclerosis levels did not differ, RAG8-treated mice exhibited reduced coronary artery atherosclerosis, reduced platelet accumulation in atherosclerotic coronary arteries, and reduced myocardial fibrosis. These protective effects were not accompanied by changes in circulating lipids, inflammatory cytokines, or immune cells. However, RAG8-treated mice exhibited reduced VCAM-1 (vascular cell adhesion molecule 1) protein levels in nonatherosclerotic coronary artery cross sections and reduced leukocyte accumulation in atherosclerotic coronary artery cross sections compared with those from SRQ8-treated mice. The PAR4 inhibitory RAG8 pepducin reduced coronary artery atherosclerosis and myocardial fibrosis in SR-B1/LDLR double knockout mice fed a high-fat, high-cholesterol diet containing cholate. Furthermore, RAG8 reduced VCAM-1 in nonatherosclerotic coronary arteries and reduced leukocyte and platelet accumulation in atherosclerotic coronary arteries. These findings identify PAR4 as an attractive target in reducing coronary artery disease development, and the use of RAG8 may potentially be beneficial in cardiovascular disease.

Numerical investigation of the effect of carotid bifurcation stenosis degree on pulsatility characteristics.

Arterial bifurcations are regions that are susceptible to hemodynamic effects and thrombus formation. In the current study, the hemodynamic effects of a simplified 3D model of an arterial bifurcation were simulated using the commercial computational fluid dynamics software FLUENT. The non-Newtonian properties of blood were modeled using the Carreau model, and the pulsation dynamics and heat transfer characteristics of blood at different degrees of stenosis in the arterial bifurcation were analyzed. The results indicate that arterial stenosis caused by a thrombus when the pulsation velocity reaches its peak has an essential impact on blood transport. The stenosis of the bifurcation increases the peak pulsatile flow pressure drop, and each 0.5mm stenosis of the arterial bifurcation increases the mean wall shear stress of the bifurcated segment by approximately 0.25Pa. From the heat transfer perspective, arterial stenosis has little effect on the heat transfer coefficient. The heat transfer coefficient measured inside the bifurcation is much larger than that measured outside the bifurcation. The stenosis of the arterial bifurcation causes an increase in the mean velocity of the arterial cross-section, and the volume-averaged absolute vorticity is introduced to quantify the secondary flow effect during the pulsation cycle, where the arterial stenosis causes an increase in the mean absolute vorticity at pulsation velocity and accelerates the decay of the vorticity at uniform velocity. In this paper, the hemodynamics of carotid bifurcation pulsation is analyzed in conjunction with flow field properties to reveal the flow field dynamics factors and heat transfer characteristics of local stenosis of the carotid bifurcation and to conduct an exploratory study for the diagnosis and treatment of carotid bifurcation thrombosis.

Effect of Cilostazol in Animal Models of Cerebral Ischemia and Subarachnoid Hemorrhage: A Systematic Review and Meta-Analysis.

Cilostazol, a phosphodiesterase III inhibitor, appears to be a promising agent for preventing cerebral ischemia in patients with aneurysmal subarachnoid hemorrhage. Here, the authors perform a systematic review and meta-analysis to quantitatively assess the effects of cilostazol on brain structural and functional outcomes in animal models of cerebral ischemia and subarachnoid hemorrhage-induced cerebral vasospasm. By using the PRISMA guidelines, a search of the PubMed, Scopus, and Web of Science was conducted to identify relevant studies. Study quality of each included study for both systematic reviews were scored by using an adapted 15-item checklist from the Collaborative Approach to Meta-Analysis of Animal Data from Experimental Studies. We calculated a standardized mean difference as effect size for each comparison. For each outcome, comparisons were combined by using random-effects modeling to account for heterogeneity, with a restricted maximum likelihood estimate of between-study variance. A total of 22 (median [Q1, Q3] quality score of 7 [5, 8]) and 6 (median [Q1, Q3] quality score of 6 [6, 6]) studies were identified for cerebral ischemia and subarachnoid hemorrhage-induced cerebral vasospasm, respectively. Cilostazol significantly reduced the infarct volume in cerebral ischemia models with a pooled standardized mean difference estimate of - 0.88 (95% confidence interval [CI] [- 1.07 to - 0.70], p < 0.0001). Cilostazol significantly reduced neurofunctional deficits in cerebral ischemia models with a pooled standardized mean difference estimate of - 0.66 (95% CI [- 1.06 to - 0.28], p < 0.0001). Cilostazol significantly improved the basilar artery diameter in subarachnoid hemorrhage-induced cerebral vasospasm with a pooled standardized mean difference estimate of 2.30 (95% CI [0.94 to 3.67], p = 0.001). Cilostazol also significantly improved the basilar artery cross-section area with a pooled standardized mean estimate of 1.88 (95% CI [0.33 to 3.43], p < 0.05). Overall, there was between-study heterogeneity and asymmetry in the funnel plot observed in all comparisons. Published animal data support the overall efficacy of cilostazol in reducing infarct volume and neurofunctional deficits in cerebral ischemia models and cerebral vasospasm in subarachnoid hemorrhage models.

Deep learning-based detection of functionally significant stenosis in coronary CT angiography.

Patients with intermediate anatomical degree of coronary artery stenosis require determination of its functional significance. Currently, the reference standard for determining the functional significance of a stenosis is invasive measurement of the fractional flow reserve (FFR), which is associated with high cost and patient burden. To address these drawbacks, FFR can be predicted non-invasively from a coronary CT angiography (CCTA) scan. Hence, we propose a deep learning method for predicting the invasively measured FFR of an artery using a CCTA scan. The study includes CCTA scans of 569 patients from three hospitals. As reference for the functional significance of stenosis, FFR was measured in 514 arteries in 369 patients, and in the remaining 200 patients, obstructive coronary artery disease was ruled out by Coronary Artery Disease-Reporting and Data System (CAD-RADS) category 0 or 1. For prediction, the coronary tree is first extracted and used to reconstruct an MPR for the artery at hand. Thereafter, the coronary artery is characterized by its lumen, its attenuation and the area of the coronary artery calcium in each artery cross-section extracted from the MPR using a CNN. Additionally, characteristics indicating the presence of bifurcations and information indicating whether the artery is a main branch or a side-branch of a main artery are derived from the coronary artery tree. All characteristics are fed to a second network that predicts the FFR value and classifies the presence of functionally significant stenosis. The final result is obtained by merging the two predictions. Performance of our method is evaluated on held out test sets from multiple centers and vendors. The method achieves an area under the receiver operating characteristics curve (AUC) of 0.78, outperforming other works that do not require manual correction of the segmentation of the artery. This demonstrates that our method may reduce the number of patients that unnecessarily undergo invasive measurements.

Abstract 15088: Cholesterol-Induced Suppression of Kir2.1 Contributes to the Development of Atherosclerosis

Introduction: Our recent studies demonstrated that cholesterol sensitivity of inwardly-rectifying K + channels (Kir2.1) contributes to the impairment of flow-induced vasodilation (FIV) in resistance arteries. Methods: In this study we developed a new CRISPR mouse model, originated from our molecular dynamics research for the Kir 2.1 channel. This model Kir2.1 L222I , has a point mutation (L222I) that renders the channel cholesterol insensitive. We also crossed our new CRISPR model with a dyslipidemic ApoE -/- mice resulting in a ApoE -/- Kir2.1 L222I . We evaluated Kir2.1 channel function with patch clamp for both models. We then placed our mice on a high fat (42% kcal from fat) diet (HFD) from 12 -14 weeks and analyzed atherosclerotic lesions with Oil Red O in En Face aortas and cross sections of carotid arteries for both female and male mice. We also documented weight gain, food consumption and blood biochemistry. Results: A lack of cholesterol sensitivity of Kir2.11 L222I was functionally confirmed in endothelial cells freshly isolated from mesenteric arteries of Kir2.1 L222I and ApoE -/- Kir2.1 L222I mice. We found that ApoE -/- Kir2.1 L222I mice have significant reduction in the area of atherosclerotic lesions in their aortas, as compared to ApoE -/- mice. This phenomenon was observed both in the aortic arch and in the descending aorta. Specifically, the lesion areas in the arch were 37.2± 1.87% (n:8) vs. 20.3 ±2.3% (n:8) in ApoE -/- vs. ApoE -/- Kir2.1 L222I mice respectively. The effect on the lesion area in the descending aorta was even more pronounced: 16.5 ±2.1% (n:8) in ApoE -/- to 5.6± 1.3% (n:8) in ApoE -/- Kir2.1 L222I . The same effect was observed in males and females. In addition, we also analyzed the lesion areas and stenosis in carotid arteries of the same mice and found that ApoE -/- Kir2.1 L222I mice are also partially protected, as compared to ApoE -/- . However, no differences were found in the levels of LDL, HDL and total cholesterol between these mice. Conclusion: Our data suggest that cholesterol-induced suppression of Kir2.1 is an important contributor to the development of atherosclerosis.

Abstract 15731: Rpl13a Small Nucleolar RNAs Promote Atherosclerosis and Oxidative Stress

Reactive oxygen species (ROS) exacerbate atherosclerosis (athero). ROS levels are elevated by specific non-coding, small nucleolar (sno) RNAs encoded within introns of the Rpl13a gene. We therefore tested the hypothesis that these snoRNAs promote athero, using “snoKO” mice deficient in Rpl13a snoRNAs, but not in Rpl13a itself. ROS levels assessed by CellROX Orange were 35% lower in snoKO than snoRNA +/+ aorta frozen sections ( p &lt;0.01). After 14 wk on Western diet, female snoKO/ Apoe -/- unexpectedly showed total cholesterol levels 20% higher than Apoe -/- mice in (1,046 vs 869 mg/dl, p &lt;0.05). Despite this, neointimal lesions in brachiocephalic artery (BCA) cross-sections were 50% smaller in snoKO/ Apoe -/- than in Apoe -/- mice, and lumen size was 45% larger (both p &lt;0.01, n=8/group). Similar data were obtained in males: snoKO/ Apoe -/- mice had 40% smaller BCA lesion areas ( p &lt;0.02, n=8/group). After being stained for cholesteryl ester with BODIPY, for ACTA2 by immunofluorescence and for DNA (Hoechst), BCAs from female snoKO/ Apoe -/- mice (n=9) demonstrated 50% less foam cell-positive and 95% more ACTA2 + area, 40% less necrotic core area, and a 60% lower prevalence of ACTA2 + foam cells ( p &lt;0.05 for each). Thus, Rpl13a snoRNAs promote vascular ROS and athero. Assessed by MitoSOX Red, ROS levels were 25% lower in snoKO than WT M1-polarized bone marrow-derived Mϕs in vitro (n=3, p&lt;0.05). To identify mechanisms linking the Rpl13a snoRNAs to ROS and athero, we performed LC-MS/MS on WT and snoKO aortic SMCs. COX4I2 was expressed 5.7-fold higher in snoKO than WT SMCs by MS/MS, and 2.5-fold higher in snoKO SMCs by immunoblot (n=3/group, p &lt;0.05). As part of mitochondrial complex IV, COX4I2 lowers cellular ROS levels. We used CRISPR/Cas9 to create 293T cells lacking either the RPL13a -snoRNA U34A or the irrelevant snoRNA U25 . With mRNA from these cells we performed reverse transcription at low [dNTP] followed by qPCR (RTL-P) for COX4I2 . Inversely proportional to the degree of mRNA 2’- O -methylation (mediated by snoRNAs), the RTL-P efficiency was 4-fold higher in U34A -knockout than control cells (3 clones/genotype, p &lt;0.01). Thus, COX4I2 mRNA appears to be regulated by RPL13A -snoRNA-guided 2’- O -methylation in a manner that could link Rpl13a snoRNAs, vascular ROS, and athero.

Coronary artery disease ameliorates extracellular vesicle lncRNA PUNISHER regulates angiogenic response and endothelial cells function via NFkB-dependent mechanism

Abstract Augmenting evidence indicates that long noncoding RNAs are playing a crucial role in diverse cellular/pathological processes. Intercellular transfer of extracellular vesicles transmitted lncRNA regulates vascular health and diseases. However, whether lncRNA expression in EVs is regulated in patients with coronary artery disease, is unknown. A PCR-based lncRNA array analysis revealed that EV-PUNISHER was significantly upregulated in patients with CAD (n=221) compared to healthy subjects. Our data showed that PUNISHER is the most upregulated lncRNA in CAD in accordance with other highly expressed lncRNAs. To examine the specific role of PUNISHER in EC phenotypic regulation, siRNA-mediated silencing in ECs revealed that depletion of PUNISHER suppresses the migration and proliferation of ECs. Moreover, depletion of PUNISHER decreases cell survival by reducing cell viability and proliferation through increased apoptosis and cytotoxicity. In order to investigate EC function in PUNISHER depleted cells, in vitro sprouting and tube formation assay revealed that PUNISHER is an important mediator EC functions, which acts as an anti-angiogenic factor. In vitro atherosclerotic stimuli increased PUNISHER expression in EV/EC in a dose-dependent way. Microarray analysis identified a series of pro- and anti-angiogenic genes as well genes directly involved in cell viability that are differentially regulated. We confirmed that PUNISHER is incorporated into endothelial microvesicles (EMVs) and transferred to recipient cells by using different experiments. By using lncRNA-FISH and vesicle degradation assays, we showed that PUNISHER is incorporated into EMVs, augmented recipient EC function in vitro upon transfer via EMVs. To examine whether EMV-PUNISHER promotes the EC function, different in vitro functional experiments with ECs confirmed that PUNISHER is an important regulator. Mass spectrometry analysis has revealed EMV contains numerous proteins, including RNA binding proteins such as hnRNPU, hnRNPK, hnRNPA2/B1, etc. By using RNA-pulldown and RNA immunoprecipitation (RIP), we confirmed that PUNISHER interacts with hnRNPU, which facilitates packaging into EMV prior to transfer to recipient EC. The interaction acts as an important positive regulator of cell viability and survival of recipient cells, identified using functional assay (migration, viability, proliferation, and angiogenesis). Transcription factor array has shown that PUNISHER regulates NFkB to control cellular viability and apoptosis. A murine re-endothelialization model revealed that EV-PUNISHER promotes reendothelialization. Ex vivo internalization/uptake of PKH-67-labeled EVs revealed that EVs are detectable in the cross-section of EV-perfused carotid artery. Our study unveiled for the first time that EV-incorporated PUNISHER exerts its function in endothelial cells which might be beneficial in cardiovascular pathologies where the endothelial function is dysregulated. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): German Research Foundation (DFG)German Society of Cardiology (DGK)

Role of Cholesterol in the Regulation of Hydrogen Sulfide Signaling within the Vascular Endothelium.

H2S is a gaseous signaling molecule enzymatically produced in mammals and H2S-producing enzymes are expressed throughout the vascular wall. We previously reported that H2S-induced vasodilation is mediated through transient receptor potential cation channel subfamily V member 4 (TRPV4) and large conductance (BKCa) potassium channels; however, regulators of this pathway have not been defined. Previous reports have shown that membrane cholesterol limits activity of TRPV4 and BKCa potassium channels. The current study examined the ability of endothelial cell (EC) plasma membrane (PM) cholesterol to regulate H2S-induced vasodilation. We hypothesized that EC PM cholesterol hinders H2S-mediated vasodilation in large mesenteric arteries. In pressurized, U46619 pre-constricted mesenteric arteries, decreasing EC PM cholesterol in large arteries using methyl-β-cyclodextrin (MBCD, 100 µM) increased H2S-induced dilation (NaHS 10, 100 µM) but MBCD treatment had no effect in small arteries. Enface fluorescence showed EC PM cholesterol content is higher in large mesenteric arteries than in smaller arteries. The NaHS-induced vasodilation following MBCD treatment in large arteries was blocked by TRPV4 and BKCa channel inhibitors (GSK219384A, 300 nM and iberiotoxin, 100 nM, respectively). Immunohistochemistry of mesenteric artery cross-sections show that TRPV4 and BKCa are both present in EC of large and small arteries. Cholesterol supplementation into EC PM of small arteries abolished NaHS-induced vasodilation but the cholesterol enantiomer, epicholesterol, had no effect. Proximity ligation assay studies did not show a correlation between EC PM cholesterol content and the association of TRPV4 and BK. Collectively, these results demonstrate that EC PM cholesterol limits H2S-induced vasodilation through effects on EC TRPV4 and BKCa channels.