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Background: Coronary allograft vasculopathy (CAV) is a devastating sequela of heart transplant in which arterial intimal thickening limits coronary blood flow. There are currently no targeted therapies to prevent or reduce this pathology that leads to transplant failure. Vascular smooth muscle cell (VSMC) phenotypic plasticity is critical in CAV neointima formation. TET2 (TET methylcytosine dioxygenase 2) is an important epigenetic regulator of VSMC phenotype, but the role of TET2 in the progression of CAV is unknown. Methods: We assessed TET2 expression and activity in human CAV and renal transplant samples. We also used the sex-mismatched murine aortic graft model of graft arteriopathy (GA) in wild-type and inducible smooth muscle–specific Tet2 knockout mice; and in vitro studies in murine and human VSMCs using knockdown, overexpression, and transcriptomic approaches to assess the role of TET2 in VSMC responses to IFNγ (interferon γ), a cytokine elaborated by T cells that drives CAV progression. Results: In the present study, we found that TET2 expression and activity are negatively regulated in human CAV and renal transplant samples and in the murine aortic graft model of GA. IFNγ was sufficient to repress TET2 and induce an activated VSMC phenotype in vitro. TET2 depletion mimicked the effects of IFNγ, and TET2 overexpression rescued IFNγ-induced dedifferentiation. VSMC-specific TET2 depletion in aortic grafts, and in the femoral wire restenosis model, resulted in increased VSMC apoptosis and medial thinning. In GA, this apoptosis was tightly correlated with proliferation. In vitro, TET2-deficient VSMCs undergo apoptosis more readily in response to IFNγ and expressed a signature of increased susceptibility to extrinsic apoptotic signaling. Enhancing TET2 enzymatic activity with high-dose ascorbic acid rescued the effect of GA-induced VSMC apoptosis and intimal thickening in a TET2-dependent manner. Conclusions: TET2 is repressed in CAV and GA, likely mediated by IFNγ. TET2 serves to protect VSMCs from apoptosis in the context of transplant vasculopathy or IFNγ stimulation. Promoting TET2 activity in vivo with systemic ascorbic acid reduces VSMC apoptosis and intimal thickening. These data suggest that promoting TET2 activity in CAV may be an effective strategy for limiting CAV progression.

Background: Up to 50% of patients with proximal deep vein thrombosis (DVT) will develop the postthrombotic syndrome characterized by limb swelling and discomfort, hyperpigmentation, skin ulcers, and impaired quality of life. Although catheter-based interventions enabling the restoration of blood flow (RBF) have demonstrated little benefit on postthrombotic syndrome, the impact on the acuity of the thrombus and mechanisms underlying this finding remain obscure. In experimental and clinical studies, we examined whether RBF has a restricted time window for improving DVT resolution. Methods: First, experimental stasis DVT was generated in C57/BL6 mice (n=291) by inferior vena cava ligation. To promote RBF, mice underwent mechanical deligation with or without intravenous recombinant tissue plasminogen activator administered 2 days after deligation. RBF was assessed over time by ultrasonography and intravital microscopy. Resected thrombosed inferior vena cava specimens underwent thrombus and vein wall histological and gene expression assays. Next, in a clinical study, we conducted a post hoc analysis of the ATTRACT (Acute Venous Thrombosis: Thrombus Removal with Adjunctive Catheter-Directed Thrombolysis) pharmacomechanical catheter-directed thrombolysis (PCDT) trial (NCT00790335) to assess the effects of PCDT on Venous Insufficiency Epidemiological and Economic Study quality-of-life and Villalta scores for specific symptom-onset-to-randomization timeframes. Results: Mice that developed RBF by day 4, but not later, exhibited reduced day 8 thrombus burden parameters and reduced day 8 vein wall fibrosis and inflammation, compared with controls. In mice without RBF, recombinant tissue plasminogen activator administered at day 4, but not later, reduced day 8 thrombus burden and vein wall fibrosis. It is notable that, in mice already exhibiting RBF by day 4, recombinant tissue plasminogen activator administration did not further reduce thrombus burden or vein wall fibrosis. In the ATTRACT trial, patients receiving PCDT in an intermediate symptom-onset-to-randomization timeframe of 4 to 8 days demonstrated maximal benefits in Venous Insufficiency Epidemiological and Economic Study quality-of-life and Villalta scores (between-group difference=8.41 and 1.68, respectively, P 8 days. Conclusions: Taken together, these data illustrate that, within a restricted therapeutic window, RBF improves DVT resolution, and PCDT may improve clinical outcomes. Further studies are warranted to examine the value of time-restricted RBF strategies to reduce postthrombotic syndrome in patients with DVT.

Background: Atherosclerotic occlusions decrease blood flow to the lower limbs, causing ischemia and tissue loss in patients with peripheral artery disease (PAD). No effective medical therapies are currently available to induce angiogenesis and promote perfusion recovery in patients with severe PAD. Clinical trials aimed at inducing vascular endothelial growth factor (VEGF)–A levels, a potent proangiogenic growth factor to induce angiogenesis, and perfusion recovery were not successful. Alternate splicing in the exon-8 of VEGF-A results in the formation of VEGFxxxa (VEGF 165 a) and VEGFxxxb (VEGF 165 b) isoforms with existing literature focusing on VEGF 165 b’s role in inhibiting vascular endothelial growth factor receptor 2–dependent angiogenesis. However, we have recently shown that VEGF 165 b blocks VEGF-A–induced endothelial vascular endothelial growth factor receptor 1 (VEGFR1) activation in ischemic muscle to impair perfusion recovery. Because macrophage-secreted VEGF 165 b has been shown to decrease angiogenesis in peripheral artery disease, and macrophages were well known to play important roles in regulating ischemic muscle vascular remodeling, we examined the role of VEGF 165 b in regulating macrophage function in PAD. Methods: Femoral artery ligation and resection were used as an in vivo preclinical PAD model, and hypoxia serum starvation was used as an in vitro model for PAD. Experiments including laser-Doppler perfusion imaging, adoptive cell transfer to ischemic muscle, immunoblot analysis, ELISAs, immunostainings, flow cytometry, quantitative polymerase chain reaction analysis, and RNA sequencing were performed to determine a role of VEGF 165 b in regulating macrophage phenotype and function in PAD. Results: First, we found increased VEGF 165 b expression with increased M1-like macrophages in PAD versus non-PAD (controls) muscle biopsies. Next, using in vitro hypoxia serum starvation, in vivo pre c linical PAD models, and adoptive transfer of VEGF 165 b-expressing bone marrow–derived macrophages or VEGFR1 +/– bone marrow–derived macrophages (M1-like phenotype), we demonstrate that VEGF 165 b inhibits VEGFR1 activation to induce an M1-like phenotype that impairs ischemic muscle neovascularization. Subsequently, we found S100A8/S100A9 as VEGFR1 downstream regulators of macrophage polarization by RNA-Seq analysis of hypoxia serum starvation-VEGFR1 +/+ versus hypoxia serum starvation-VEGFR1 +/– bone marrow–derived macrophages. Conclusions: In our current study, we demonstrate that increased VEGF 165 b expression in macrophages induces an antiangiogenic M1-like phenotype that directly impairs angiogenesis. VEGFR1 inhibition by VEGF 165 b results in S100A8/S100A9-mediated calcium influx to induce an M1-like phenotype that impairs ischemic muscle revascularization and perfusion recovery.

Introduction: A single bout of uninterrupted sitting impairs vascular function in the legs, which may be due to reductions in blood flow and shear stress. Participating in regular moderate-to-vigorous physical activity (MVPA) has been identified as an effective approach for improving vascular function, and recent evidence suggests meeting the physical activity (PA) guidelines may attenuate some of the negative health outcomes associated with excessive sedentary behavior; however, it is not well understood how meeting the PA guidelines may influence the acute response to sitting. Our aim was to investigate the effects of 3 h of uninterrupted sitting on hemodynamics and vascular and inflammatory biomarkers in physically inactive and active adults. Hypothesis: We hypothesized active adults would experience less detrimental physiological changes after sitting compared to inactive adults. Methods: Eleven inactive (mean±SD, age: 47.1±8.9 y, body fat: 33.1±8.5%; 78.5% women) and 16 active adults (age: 46.1±8.9 y, body fat: 25.2±7.2%; 31.1% women) completed 3 h of uninterrupted sitting. Adults self-reported their PA with the International PA Questionnaire. Adults engaging in ≥150 min·wk -1 were classified as active and -1 , inactive. Hemodynamic variables, and superficial femoral artery (SFA) diameter and blood velocity were measured each hour over 3 h of sitting. Mean arterial pressure, blood flow and shear rate were calculated. Serum vascular and inflammatory biomarkers were measured pre and post sitting. Linear mixed-effects modeling was used to assess changes in dependent variables over time and between inactive and active adults, controlling for sex. Results: Inactive and active adults self-reported 7.3±7.1 and 93.3±64.8 min·d -1 of MVPA, respectively. Endothelin-1 (baseline: 8.3±13.4 pg/mL, post: 81.1±103.0 pg/mL; p-1 ; 1 h: 55.0±27.4 s -1 ; 2 h: 45.3±24.2 s -1 ; 3 h: 40.8±25.5 s -1 ) while active participants demonstrated no change (baseline: 36.6±21.4 s -1 ; 1 h: 28.1±21.4 s -1 ; 2 h: 26.1±20.9 s -1 ; 3 h: 23.8±19.5 s -1 ). Inactive adults also had a higher oscillatory shear index compared to active adults (p Conclusion: Uninterrupted sitting induced unfavorable changes regardless of PA status; however, active adults demonstrated a more favorable shear profile. Meeting PA guidelines may attenuate some unfavorable changes within the vasculature associated with prolonged sitting.

Introduction: Acute myocardial infarction remains a leading cause of mortality. Rapid restoration of coronary blood flow is the cornerstone of treatment. Paradoxically, it leads to a condition known as ischemia/reperfusion (I/R) injury which precipitates more injuries to the heart. Erythropoietin (EPO), a hormone produced by the kidneys in response to cellular hypoxia, reportedly provides cardioprotection following cardiac I/R injury in many preclinical experiments. Unfortunately, clinical trials failed to demonstrate cardioprotection when EPO was given after reperfusion. Therefore, we aim to determine the temporal influences on administering EPO in cardiac I/R injury. Hypothesis: Administration of EPO at different time points provides varying efficacy in reducing arrhythmias, LV and cardiac mitochondrial dysfunction after cardiac I/R injury. Methods: Male Wistar rats were divided into sham-operated and cardiac I/R group. In I/R group, rats were subdivided into 4 groups (n=10/group): vehicle, EPO pretreatment (P-EPO), EPO given during ischemia (I-EPO), and EPO given at reperfusion onset (R-EPO). EPO was intravenously given to the rats (5000 unit/kg). Rats underwent 30-min LAD ligation followed by 120-min reperfusion. Arrhythmia scores and LV function were recorded throughout the protocol. Then, rats were sacrificed to determine infarct size and mitochondrial function. Results: Cardiac I/R injury induced arrhythmias, LV and mitochondrial dysfunction (Fig . Administration of EPO before or during ischemia, but not at the onset of reperfusion, significantly attenuated LV dysfunction and infarct size. However, EPO did not reduce arrhythmia scores. Although EPO given at all time points reduced mitochondrial reactive oxygen species, EPO given at reperfusion failed to prevent mitochondrial swelling (Fig) . Conclusions: Acute EPO treatment before or during ischemia, but not at the onset of reperfusion, exerted cardioprotection against I/R injury.

Introduction: Abnormal hemodynamics with bicuspid aortic valve (BAV) is influenced by the BAV morphology. However, due to the morphological variety, the relationship between BAV morphology and hemodynamics has not been well clarified. We experimentally investigated influences of BAV morphology on hemodynamics. Methods: An MRI compatible pulsatile flow circulation system incorporating an aortic valve model and morphologically relevant aortic arch model was developed. Two types of BAV models with cusp angles of 240-120 (asymmetric BAV) and 180-180 (symmetric BAV) were prepared using bovine aorta and pericardium. The vorticity and eccentricity of jet in the aortic arch model in systolic phase were assessed using 4D-flow MRI. Streamlines at peak systole were compared among 5 BAV morphologies defined based on the symmetry and the position of leaflets (Fig). Results: Eccentric supra-valvular jets directed to the aortic wall faced to the smaller leaflet were present in the 3 asymmetric BAVs. In the ABAV-1, a markedly eccentric jet impinging on the aortic outer curvature was present. In the ABAV-2, a left-posterior directed jet shifting to the outer curvature of proximal arch was present. The asymmetric BAVs induced larger flow vorticity in the ascending aorta than the symmetric BAVs (ABAV-1 vs -2 vs -3 vs SBAV-1 vs -2, 0.018 vs 0.019 vs 0.014 vs 0.010 vs 0.011 m 2 /s). The ABAV-2 had a larger vortex in the middle arch than the ABAV-1 (ABAV-2 vs -1, 0.010 vs 0.005 m 2 /s). Conclusion: Our study indicated that the angles and orientations of the BAV impacted on the locations of jets impinging on the aortic wall and magnitudes of vorticities in systole. In the asymmetric BAVs, the direction of jet was influenced by the position of smaller leaflet. Our data suggests that the ABAV-1 morphology may be a risk factor inducing asymmetric aneurysm bulged toward the aortic outer curvature, while the ABAV-2 morphology may be a risk factor of an aortic aneurysm involving the transvers arch.

Background: Atherosclerotic lesions preferentially occur at bifurcations and branching points where blood flow exerts low and bidirectional oscillatory shear stress (OSS) to the vascular walls. This study aims to determine the role of OSS in the induction of inflammatory activation and phenotypic transition in coronary artery endothelial cells and investigate the interaction between endothelial cells and smooth muscle cells in a coronary-artery-on-a-chip. Methods and Results: A three-dimensional microengineered human coronary-artery-on-a-chip was developed and incorporated with OSS to mimic the flow patterns within the microenvironment of coronary artery in atheroprone regions. Human coronary artery endothelial cells (HCAECs) were cultured on the upper surface of a collagen I-coated membrane, and human coronary artery smooth muscle cells (HCASMCs) were seeded on the opposite side of the membrane. Single-cell RNA sequencing analysis revealed that HCAECs were segregated into four subgroups after being exposed to OSS for 24 h, and inflammatory response and EndMT-related genes were enriched simultaneously in most HCAECs. OSS-induced inflammatory response and EndMT in HCAECs were confirmed by immunoblotting and immunofluorescence, and these changes were mediated by the Notch1/p38 MAPK-NF-κB signaling pathways. Moreover, HCAECs exposed to OSS induced extracellular matrix (ECM) protein remodeling and proliferation in HCASMCs through a paracrine mechanism. Multiplex ELISA analysis identified that RANTES exhibited the greatest increase after OSS in HCAEC culture and played a major role in modulation of ECM protein remodeling in HCASMCs. Further, IL-37 was able to prevent OSS-induced inflammatory response and EndMT in HCAECs and thereby abrogated ECM protein remodeling and proliferation in HCASMCs. Conclusion: OSS provokes inflammatory response and EndMT in HCAECs through activating the Notch1/p38 MAPK-NF-κB signaling pathways. The novel findings demonstrate that OSS-induced endothelial inflammatory response and associated EndMT promote vascular adverse remodeling and that anti-inflammatory cytokine IL-37 may have therapeutic potential for suppressing endothelial changes and resultant vascular adverse remodeling.

Background: Capillary refill is an early reflection of decreased end organ perfusion resulting in shunt of blood from the extremities to vital organs. Physicians rate this as the most important factor to guide therapeutic resuscitation efforts. However, literature shows that providers have significant variability in their bedside measurement of capillary refill. Objective: The objective of this study was to develop and evaluate a novel technology to detect capillary refill in a noninvasive and rapid manner through an iterative, problem-based innovation approach. Methods: This was a prospective study at a level 1 tertiary care hospital of patients presenting to an emergency department. The research team used in a new technology that utilizes light transmission and pressure to objectively quantify extremity capillary refill time. This measurement was compared to manual refill time performed by a trained study provider. Results: Through an iterative approach the team developed a platform technology and enrolled 63 subjects in this prospective trial. 9 subjects had inadequate data and so the final cohort consisted of 54 subjects with both manual capillary refill time and the new novel technology measurement. The device measured capillary refill time showed a high degree of correlation to manual estimate of capillary refill time with a Pearson coefficient of 0.7. Conclusions: Novel technology to measure capillary refill time can significantly improve the treatment and care of multiple medical conditions that rely on timely diagnosis and initiation of resuscitation in the emergency department and other care settings. This study shows a modest correlation of technology with automated signal processing algorithms for noninvasive measurement of capillary refill. Integration of this technology into standard hospital monitors has broad applicability to make capillary refill a standard vital sign.

Introduction: Cardiac involvement determines outcome in systemic amyloidosis, but the relationship between amyloid deposits and outcomes is poorly understood. Many clinical observations are not explained by the simple concept of physical, mechanical replacement of the interstitium by amyloid material. Hypothesis: Preliminary studies in patients with cardiac amyloidosis (CA) support the hypothesis that myocardial ischaemia contributes to cell damage. This study assesses the presence and mechanisms of myocardial ischaemia using CMR with multiparametric mapping and histopathological assessment. Methods: 92 patients with CA (41 AL, 51 ATTR) and 73 without CA (26 with unobstructed coronary arteries; 47 with 3VD) underwent CMR with T1, T2, Extracellular volume (ECV) mapping and adenosine stress with myocardial blood flow (MBF) mapping. 25 cardiac biopsies and 3 explanted hearts with CA were analysed histopathologically. Results: CA patients had severe reductions in stress MBF and myocardial perfusion reserve (MPR)- (1.03ml/g/min±0.50;1.54±0.59 ) compared to patients with unobstructed coronary arteries (2.82 ml/g/min±0.53; 2.94±0.59, p Conclusion: CA is associated with severe myocardial ischaemia demonstrable by histology and CMR perfusion mapping. Histological evaluation indicates a complex pathophysiology, where direct disruption of the vessels by amyloid deposits and reduction in epicardial flow contribute to myocardial ischaemia.

Introduction: Peripheral Artery Disease (PAD) restricts blood flow to the legs. Its most common manifestation is claudication, a severe impairment of physical activity produced by ischemia-related leg pain and tiredness during walking. We are conducting a trial to evaluate the possibility that an ankle foot orthosis could reduce claudication symptoms and increase the physical activity of patients with PAD. The perceptions of patients with PAD on wearing the ankle foot orthosis and how they vary by baseline physical activity level are unknown. Therefore, this study explored the perceptions of patients with PAD while using an ankle foot orthosis and if these perceptions varied by level of physical activity. Hypothesis: We hypothesize that those who are more physically active will perceive more benefits as a result of using the ankle foot orthosis. Methods: Participants (n=21) wore an ankle foot orthosis for 3 months. Baseline median step count was used to divide patients into high (n=10) or low (n=11) active groups. Semi-structured interviews were conducted at midpoint and post. Data were analyzed using a summative content analysis. Results: Patients averaged 3233 ± 1523 steps/day with a median of 3137 steps/day at baseline. 45% of participants’ initial responses described a positive perception of their time wearing the ankle foot orthosis. 71.4% of participants reported an overall positive impact of wearing the ankle foot orthosis, primarily being able to walk further. Comparison by baseline physical activity level revealed 36.4% of participants with low physical activity reported seeing improvements in daily tasks (walking in the grocery store) versus 10% of those with high physical activity. Conclusions: In conclusion, our interview data demonstrate that perceived quality of physical activity in patients with PAD improved with the use of the ankle foot orthosis. Contrary to our hypothesis, minimal differences in perceptions of ankle foot orthosis use were found between patients with low versus high baseline physical activity levels.

Background: Coronary vascular function of a chronic coronary total occlusion (CTO) immediately after recanalization is known to be poor and to be partially improved by pre-treatment with loading dose of ticagrelor vs. clopidogrel. It is unknown if this vascular dysfunction is maintained at long-term follow-up and may be improved by 1-year dual antiplatelet therapy (DAPT) with ticagrelor vs. clopidogrel. Methods: The TIGER is a prospective, open-label, two parallel-group controlled clinical trial, which 1:1 randomized 50 CTO patients to pre-PCI loading dose and subsequent 1-year DAPT with ticagrelor vs. clopidogrel. Coronary blood flow (CBF) under stepwise adenosine infusion was assessed after drug loading dose and at follow-up and compared between the two drug groups, adjusting for time of follow-up. Results: Out of 50 patients with index CBF evaluation, 38 (76%) patients underwent angiographic follow-up (23 and 15 at 1 and 3-year, respectively). A higher CBF area under the curve (AUC), already observed after loading dose in ticagrelor vs. clopidogrel group (p=0.027), was maintained at follow-up (AUC 34815.22±24206.06 vs. AUC 22712.47±13768.95; p=0.071). Specifically, whereas high ticagrelor loading dose-related CBF was sustained at follow-up (p=0.933), clopidogrel loading dose-related CBF increased at follow-up (p=0.039). Conclusion: The TIGER trial showed that DAPT with ticagrelor may maintain a higher CBF in a recanalized CTO as compared to clopidogrel, whose treated patients exhibit a lower CBF immediately after PCI with slight increase at follow-up. The clinical value of such sustained higher coronary flow should be evaluated in a larger group of patients.

Introduction: Atrial fibrillation (AF) is a common cardiac rhythm disorder that is often comorbid with pulmonary hypertension (PH) and other conditions associated with abnormal atrial pressure and/or volume overload. In the setting of atrial dilation, mechanoelectric feedback has been linked to the development of ectopic beats that trigger paroxysmal AF mainly originating from pulmonary veins (PVs). However, the precise mechanisms remain poorly understood. Here, we aimed to characterize atrial wall shear stress (WSS) and vorticity in the left atrium of AF, healthy, and at-risk for AF (PH) patient models to develop predictors of AF risk. Methods: Magnetic resonance imaging and computed tomography data (10 AF, 10 PH, and 10 healthy volunteers) were obtained retrospectively. The left atria were manually segmented from each data set. Four-dimensional flow MRI was performed on one patient to derive PV flow data. The 30 atrial geometries and PV flow conditions were used to run numerical blood flow simulations, and atrial WSS and blood flow vorticity were analyzed. Results: As seen in Figure 1, wall shear stress was highest near the PV roots and on the posterior atrial wall, the most common sources of AF triggers. Average WSS and vorticity were significantly lower in PH patients than in both the healthy (p=0.003(WSS), p=0.011(vorticity)) and AF (p=0.046(WSS), p=0.069(vorticity)) groups. Both WSS (r=-0.66) and vorticity (r=-0.68) were moderately correlated to atrial volume in the PH group. Atrial volume was significantly larger in PH (p Conclusions: The larger atrial volumes of PH and AF patients lead to altered flow profiles and less frequently flow jet impingement on the atrial wall models, leading to abnormal vorticity and WSS profiles. These long term flow abnormalities may influence the development and/or localization of electrical abnormalities and AF; although further study is needed to confirm this hypothesis.

Low disturbed blood flow (LDF) is a critical contributing factor to atherogenesis but its direct impact on the immune compartment was not well-depict. To fill this knowledge gap, we adopted scRNA-seq to capture sheer-stress induced immune responses during atherogenesis. A partial carotid artery ligation (PCAL) model was selected for its paired comparison of carotid arteries with normal flow (NF) or LDF. Indeed, we observed drastic changes in both endothelial and immune compartment. Macrophages were the most significantly increased population induced by LDF (from 4% to 12% of CD45+ cells) with two well-separated subsets (Mac-c8, Mac-c9). MacSpectrum analyses revealed that Mac-c8 displayed higher inflammatory states than the lipid-laden Mac-c9. Interestingly, three T subtypes displayed unique flow-induced enrichment patterns that were selectively enriched in LDF but not in the NF condition. Furthermore, we created an original algorithms to evaluate the impact of sheer-stress on membrane protein-mediated cell-cell interaction among all cell types in the atheroma. Several pairs of molecular interactions were identified, including multiple APP-ligands interaction pairs and those in BAG2 Signaling. Moreover, signature genes identified in these LDF-induced T cells displayed high correlation to the plaque severity in human artery-aorta samples. Collectively, our study provided a high-resolution and focused analyses of sheer-stress induced immune cell action during atherogenesis. This is also the first identification of unique T subsets, to our knowledge, that are enriched in arterial wall exposed to low and disturbed flow. Further characterization of these cells will provide valuable information to understand and therapeutically treat atherogenesis.

Introduction: Evaluating the severity of lesions in peripheral arteries is challenging. Image-based blood flow modeling from peripheral computed tomographic angiography (pCTA) may provide a non-invasive method to determine the hemodynamic significance of lesions. The objective of this study was to evaluate the diagnostic performance of a trans-lesion pressure drop computed from pCTA-based blood flow modeling in the peripheral arteries. Methods: Ten patients undergoing digital subtraction angiography (DSA) and pCTA were included. The peripheral arteries were divided into 8 segments per extremity and stenosis severity was visually graded by DSA as non-stenosed (grade 0), mild (grade I), moderate (grade II), severe (grade III), occluded (grade IV) or non-evaluable. A functionally significant lesion was defined as grade III or IV by DSA. Independent from the DSA review, a resting pressure gradient (rPG) and exercise PG (ExPG) for each segment was calculated from pCTA-based blood flow modeling (Figure), and a functionally significant lesion was defined as having an rPG > 5 mm Hg or an ExPG > 20 mm Hg. Results: Mean age was 52±16 years, 4 patients (40%) were male, 8 patients (80%) presented with critical limb ischemia, mean ankle brachial index was 0.60±0.29 and 66 arterial segments were available for both assessment methods. Twenty-two segments had functionally significant lesions by DSA. For rPG, sensitivity was 80%, specificity was 85% and accuracy was 79% with DSA as the standard; for ExPG, sensitivity was 84%, specificity was 89% and accuracy was 88%. Conclusions: Use of a resting pressure gradient > 5 mm Hg and an exercise pressure gradient > 20 mm Hg measured by peripheral computed tomography-based blood flow modeling accurately identifies functionally significant stenosis in patients with advanced peripheral vascular disease. These results support a prospective imaging trial to further validate this novel approach.

Introduction: Myocardial perfusion is enhanced during periods of elevated cardiomyocyte oxygen consumption. The coupling between blood flow and myocardial oxygen demand is mediated by redox-dependent signals, yet the influence of cardiac workload on arterial myocyte pyridine nucleotide redox status is unknown. Hypothesis: Increased cardiac workload acutely alters intramyocardial arterial myocyte [NADH] i :[NAD + ] i . Methods and Results: The genetically-encoded fluorescent biosensor Peredox-mCherry was used to monitor real-time changes in intracellular [NADH] i :[NAD] i in isolated arterial myocytes. Treatment of cells expressing Peredox-mCherry with a range of [lactate] o :[pyruvate] o resulted in significant changes in green (Ex,Em: 400, 510 nm): red (Ex,Em: 585,615 nm) fluorescence ratio (1.903 ± 0.010, 10 mM lactate, 1.072 ± 0.073, 10 mM pyruvate), affording accurate quantification of arterial myocyte [NADH] i :[NAD] i . Increased arterial myocyte [NADH] i :[NAD] i was observed in response to reductions in oxygen tension from 5% (0.0021 ± 0.0001) to 1% (0.0050 ± 0.0001; Pi :[NAD] i was not altered by direct stimulation of arterial myocytes with the beta-adrenergic agonist isoproterenol (1 μM; P = .0630). Furthermore, biosensor-expressing arterial myocytes in co-culture with human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) recapitulated the effects of elevated [lactate] o :[pyruvate] o and reduced oxygen tension, exhibiting frequency-dependent increases in [NADH] i :[NAD] I following electrical pacing (e.g., ~3.3 fold increase in 3 Hz vs. unpaced; Pi :[NAD] i , compared with those in hearts treated with vehicle (3.17 ± 0.23 vs. 2.21 ± 0.11; P Conclusions: Our results indicate that acute increases in cardiac workload promote the elevation of [NADH] i :[NAD] i in intramyocardial arterial myocytes.

Introduction: Tetralogy of Fallot (ToF) typically requires surgical repair of the right ventricular outflow tract (RVOT) and subsequent placement of an artificial pulmonary valve. Bioprosthetic valve longevity is highly variable and there is currently little understanding of what hemodynamic factors may lead to early valve dysfunction. Hypothesis: We hypothesize that cardiac output and valve orientation impact the performance of bioprosthetic valves by affecting blood flow patterns in the RVOT. Methods: We analyzed hemodynamics in a 3D printed ToF anatomy model in a physiological flow loop. A 25mm surgical valve was implanted in the model at two orientations: native and rotated 180 degrees. Full 3D, three-component, phase-averaged velocity fields were obtained over the cardiac cycle using 4D flow MRI at cardiac outputs of 2, 3.5, and 5 L/min. We acquired images of valve leaflet motion at 1500Hz. The 4D flow MRI and high-speed camera experiments were run identically, allowing us to examine the relationship between flow fields and leaflet motion. Results: The full velocity fields from the MRI scans revealed key differences among cases in flow features including location of reverse flow regions, systolic jet shape, and asymmetry local to the valve. At 2 L/min, the forward flow through the jet was more asymmetric compared to the other cases and a strong vortex formed, indicating a region of recirculation. With the rotated valve orientation, the 2 L/min case also produced a unique pattern as flow was washed from the RVOT inner curve back toward the center of the valve (Fig 1). Leaflet behavior during systole varied with cardiac output as well, as higher frequency flutter was observed at 5 L/min and the effective valve orifice area was decreased by 8.5% at 2 L/min compared to 5 L/min. Conclusions: We observed key differences in flow patterns and leaflet motion due to cardiac output and valve orientation that could impact leaflet loading and fatigue and long-term valve function.

Introduction: Left atrial appendage (LAA) is the primary source of clot formation that can cause stroke or transient ischemic attack (TIA) in patients with atrial fibrillation (AF). LAA closure devices have emerged as alternatives to traditional anticoagulation therapy for reducing stroke risk in AF patients. However, ~1-2% of AF patients undergoing LAA closure have subsequent stroke/TIA event. Hypothesis: The presence of a persistent low-flow zone in LA, distinct from LAA, explains why some patients have stroke/TIA events after LAA closure. Methods: We developed a personalized stroke risk prediction tool that uses patient’s CT image to perform computational fluid dynamics simulation and determine presence of low blood-flow in the LA before and after LAA closure. Low flow is quantified by the establishment of low velocity fraction (LVF) in the LA volume, and low wall shear stress fraction (LWSSF) on the surface of the LA wall. The tool was applied retrospectively on 4 AF patients who had undergone LAA closure: 2 patients with TIA 4 months after LAA closure, and 2 controls matched for age, gender, CHA2DS2-VASc score and LAA dimensions with no complications at follow-up up to 2 years. Results: Before LAA closure, TIA and control groups had similar LVF (0.07 and 0.06, respectively) and LWSSF (0.12 and 0.16, respectively). However, after LAA closure, the TIA group had smaller reductions as compared to controls in both LVF (67% vs 79%) and LWSSF (17% vs 52%). This suggests that the LA low-flow region (especially at LA wall) was not substantially reduced after LAA closure, explaining why these patients might have experienced TIA. Conclusion: This proof-of-concept study demonstrates that LAA closure might not substantially reduce low-flow zones for all AF patients, as some retain low-flow zones in LA. Our stroke prediction tool has the potential to identify patients at risk of future stroke/TIA, thus enabling personalized patient selection to increase efficacy of LAA closure devices.

Introduction: Atherosclerosis preferentially occurs in arterial regions exposed to disturbed blood flow ( d-flow ) while the straight regions exposed to stable flow ( s-flow ) are protected. The proatherogenic and atheroprotective effects of flow are mediated in large part by the global changes in endothelial cell gene expression, which regulate endothelial dysfunction and inflammation. Previously, we identified Kallikrein-Related Peptidase 10 (KLK10) as one of the most flow-sensitive genes in arterial endothelial cells using the partial carotid ligation model of d-flow -induced atherosclerosis. KLK10 is a secreted serine protease, but its role in endothelial function and atherosclerosis is unknown. Methods/Results: Here, we validated that KLK10 was upregulated under s-flow conditions and downregulated under d-flow conditions using the in vivo mouse models and in vitro studies using endothelial cells (ECs). Through in vitro functional studies using ECs, we found that KLK10 produced by s-flow protected against endothelial inflammation and permeability dysfunction. Furthermore, treatment with rKLK10 or overexpression of KLK10 plasmids in vivo decreased endothelial inflammation the mouse model. Further, rKLK10 injection or ultrasound-mediated transfection of KLK10 plasmids in the hind leg muscles led to inhibition of atherosclerosis in ApoE-/- mice with the partial carotid ligation surgery. Studies using the pharmacological inhibitors and siRNAs showed that the anti-inflammatory effects of KLK10 was mediated by the Protease Activated Receptors 1 and 2, but without directly cleaving them. Further studies show that KLK10’s anti-inflammatory effect was mediated by the NFκB and VCAM1 and ICAM1 expression pathway. In addition, immunostaining showed that KLK10 expression is significantly reduced in human coronary arterial sections with atherosclerotic plaques compared to the non-diseased controls. Conclusions: We found that KLK10 is a potent flow-sensitive secreted protein, which serve as a novel anti-inflammatory and anti-atherogenic factor. KLK10 may be a potential anti-atherogenic therapeutic.

Introduction: Precise analysis of aortic hemodynamics is crucial in the study of bicuspid aortic valve (BAV) disease. This study provides a comprehensive evaluation of aortic forward flow (FF), reverse flow (RF) and stasis in BAV patients using novel 3D-based techniques previously shown to be more accurate than traditional 2D analysis methods. Hypothesis: BAV patients without valve dysfunction show abnormal aortic FF, RF, and stasis compared to healthy controls. Methods: We recruited 44 BAV patients (48±15 yrs, 27% female) and 23 healthy controls (37±14 yrs, 35% female). Cardiac MRI at 3T was performed inclusive of 4D-flow imaging. Patients with any aortic stenosis (AS) or ≥mild regurgitation (AR) were excluded. Flow analysis was performed by segmented volumetric regions: left ventricular outflow tract (LVOT), ascending aorta (AAo), arch, proximal descending aorta (PDAo), and distal descending aorta (DDAo). In each region, forward flow (FF), reverse flow (RF) and stasis were averaged over the cardiac cycle on a voxel-by-voxel basis. Left ventricular (LV) end-diastolic volume, end-systolic volume and ejection fraction were also measured. T-tests (or non-parametric equivalent) compared differences in parameters between cohorts. Results: BAV patients were significantly older than controls (48±15 vs. 37±14 yrs; p=0.01) but exhibited no significant differences in LV measures. Patients showed reduced FF in the AAo (0.09±0.03 vs. 0.11±0.04 mL/cycle; p Conclusions: 3D-derived measurements of FF, RF, and stasis are significantly altered in the thoracic aorta of BAV patients in the absence of AS or AR.

Introduction: NHLBI supported STICHES trial (The Surgical Treatment for Ischemic Heart Failure Extended Study) (NCT00023595) was conducted to test whether blood flow restoration by coronary revascularization recovers chronic left ventricular dysfunction and improves survival, as compared to medical therapy alone in patients with congestive heart failure and coronary artery disease amenable to surgical revascularization. We reused publicly available individual patient-level STICHES trial data from NHLBI Data Repository (BioLINCC) to perform hypothesis-generating secondary analyses by machine learning (ML) using random survival forest (RSF) to identify gender, race and ethnicity, and age specific predictors for all-cause mortality (ACM). Methods: The population was sub-grouped by gender (male vs. female), race (white vs. Hispanic/Latinos/non-white), and age (< 55, 55-60, 61-69, and ≥70). RSF was performed on 48 baseline variables from 1212 patients to identify predictors of ACM. Top 10 RSF predictors for each subgroup were included in a multivariate analysis using a Cox proportional hazards model. Results: Top 10 predictors of ACM are shown in Table 1. While known cardiometabolic and vascular predictors were among the top predictors, RSF uniquely identified renal function related biomarkers and plasma sodium among important top predictors across the subgroups. Age was an important predictor for male and female, Hispanics/Latinos/non-whites, and patient groups ≥70 years old. Also, top predictors of ACM were current smoking status among age groups of Conclusions: Using ML, we uncovered in an unbiased fashion, gender, age, race and ethnicity specific, unanticipated top predictors of ACM in STICHES trial. This highlights the value of ML for analyzing disease and therapeutic intervention outcomes to help implement precision medicine.

Introduction: Stroke remains a leading cause of disease disability worldwide. Rapid restoration of blood flow after occlusion is required to minimize brain damage. However, with the reperfusion there is upregulation to oxidative stress pathways that stimulates interleukin-1 activates kinase type I (IRAK-1) within endothelial cells leading to proinflammatory genes expression (ICAM-1/VCAM-1). We previously showed that the Nck family of adaptor proteins promotes oxidative stress-induced inflammation following ischemia reperfusion (IRI). Nck1 and Nck2 are two highly similar proteins but have inherent functional differences. Hypothesis: We hypothesized that Nck1 is required for IRAK-1 activation in response to IRI. Methods: Human brain endothelial cells (ECs) were genetically silenced for Nck1, Nck2, or interleukin-1 receptor type I kinase-1 (IRAK-1) and exposed to hydrogen peroxide (exogenous oxidative stress) or hypoxia re-oxygenation (endogenous oxidative stress). Additionally, adult male mice were subjected to 1 hour of middle cerebral artery occlusion (MCAO) followed by 24h reperfusion. Neurological deficits were assessed using the 24 sensorimotor scoring system. Infarct volume, blood brain barrier (BBB) integrity, and brain inflammatory profiles were assessed using immunostaining and immunoblotting. Results: Knock down of endothelial Nck1 or Nck2 of cells markedly reduced VCAM-1 and ICAM-1 expression and NF-κB activation in response to H 2 O 2 and hypoxia re-oxygenation. In ECs treated with the proinflammatory mediator interleukin-1 (IL-1β), there was upregulation of IRAK-1 and NF-κB activation. However, deletion of Nck1, but not Nck2, significantly decreased IRAK-1/ NF-κB activation by IL-1β. Moreover, Nck1 inhibition decreased endothelial permeability-induced by oxidative stress. In response to oxidative stress, Nck1 directly binds to IRAK-1. Nck1 KO mice had significantly less infarct volume and neurological deficits after MCAO/reperfusion. BBB disruption measured by Evans Blue extravasation and Fibrinogen staining was remarkably reduced in Nck1 KO mice. Conclusions: Nck1 promotes ischemia/reperfusion-induced inflammation, and brain injury, therefore, Nck1 inhibition is a promising therapy to improve IRI.

Background: Excessive peripheral microvascular constriction during acute psychological stress, measured using peripheral arterial tonometry reflects similar changes in coronary blood flow and is a predictor of adverse cardiovascular outcomes. The ratio of digital pulse wave amplitude during stress compared to rest (sPAT) is used to estimate the degree of microvascular response to stress. We sought to determine if genetic factors contribute to the degree of microvascular constriction during mental stress. Methods: A total of 642 post-MI and stable CAD subjects from two prospective cohort studies underwent mental stress testing with a standardized public speaking stressor. Digital pulse wave amplitude was continuously measured using PAT and the stress/rest PAT ratio (sPAT) of pulse wave amplitude during mental stress/baseline was calculated. Genotyping was performed using Illumina’s Multi-Ethnic Genotyping Array (MEGA) platform and imputed to the 1000 Genome reference panel. Race stratified genome-wide association studies (GWAS) of sPAT were conducted using linear regression of additive genetic mode adjusted for age, sex and population stratification in the two cohorts. A trans-ethnic meta-analysis integrated the four sets GWAS results. Results: Mean age was 63±9; 65% male, 35% Black. We identified two SNPs in linkage disequilibrium on chr4:185503564 and chr4:185491706 rs13353812 (with 35% and 28% allele frequency, respectively) that were associated with greater sPAT ratio ( P = 1.42E-08). The mean sPAT ratio during mental stress for subjects with none, one and two AT insertion alleles of SNP chr4:185503564 were 0.67, 0.76 and 0.93, respectively, an average of 12% (P < 0.001), per allele. Results were similar for G insertion alleles of SNP rs13353812. The nearest gene of the sPAT-associated locus is CASP3 which encodes caspase, an essential protein for apoptosis signaling and brain and hematopoietic stem cell development. Conclusion: We have identified a genetic basis for stress-induced vasomotion. Presence of the chr4:185503564 allele is associated with less vasoconstriction during mental stress, and thus may be protective against long term cardiovascular risk. These findings need further exploration.

Insufficient sleep, defined as chronic short sleep duration (A receptor antagonist) and ACh + BQ-123 in both groups and after the exercise intervention in the short sleepers. As expected, forearm vasodilator responses to ACh were lower (20%; PA receptor blockade. These results indicate that regular aerobic exercise can reverse the negative influence of insufficient sleep on endothelial vasomotor function, independent of changes in nightly sleep duration.

Elevated blood pressure (BP ≥130/80 mmHg) is associated with increased risk for myocardial infarction, heart failure, stroke and vascular disease. Insufficient nightly sleep (G -monomethyl-L-arginine (L-NMMA) and the antioxidant vitamin C were determined by venous occlusion plethysmography. The FBF response to ACh was significantly lower (~20%) in the short sleep (from 3.8±0.2 to 11.0±0.6 ml/100 ml tissue/min) compared with the normal sleep duration group (from 4.2±0.2 to 13.6±0.6 ml/100 ml tissue/min). L-NMMA significantly reduced (~25%) the FBF response to ACh in the normal sleep but not the short sleep group. Vitamin C markedly increased (~35%; P

Background: Myxomatous valve degeneration (MVD) involves the progressive thickening and degeneration of the heart valves, leading to valve prolapse, regurgitant blood flow, and impaired cardiac function. Leukocytes composed primarily of macrophages have recently been detected in myxomatous valves, but the timing of the presence and the contributions of these cells in MVD progression are not known. Methods: We examined MVD progression, macrophages, and the valve microenvironment in the context of Marfan syndrome (MFS) using mitral valves from MFS mice ( Fbn1 C1039G/+ ), gene-edited MFS pigs ( FBN1 Glu433AsnfsX98/+ ), and patients with MFS. Additional histological and transcriptomic evaluation was performed by using nonsyndromic human and canine myxomatous valves, respectively. Macrophage ontogeny was determined using MFS mice transplanted with mTomato+ bone marrow or MFS mice harboring RFP (red fluorescent protein)–tagged C-C chemokine receptor type 2 (CCR2) monocytes. Mice deficient in recruited macrophages ( Fbn1 C1039G/+ ;Ccr2 RFP/RFP ) were generated to determine the requirements of recruited macrophages to MVD progression. Results: MFS mice recapitulated histopathological features of myxomatous valve disease by 2 months of age, including mitral valve thickening, increased leaflet cellularity, and extracellular matrix abnormalities characterized by proteoglycan accumulation and collagen fragmentation. Diseased mitral valves of MFS mice concurrently exhibited a marked increase of infiltrating (MHCII+, CCR2+) and resident macrophages (CD206+, CCR2–), along with increased chemokine activity and inflammatory extracellular matrix modification. Likewise, mitral valve specimens obtained from gene-edited MFS pigs and human patients with MFS exhibited increased monocytes and macrophages (CD14+, CD64+, CD68+, CD163+) detected by immunofluorescence. In addition, comparative transcriptomic evaluation of both genetic (MFS mice) and acquired forms of MVD (humans and dogs) unveiled a shared upregulated inflammatory response in diseased valves. Remarkably, the deficiency of monocytes was protective against MVD progression, resulting in a significant reduction of MHCII macrophages, minimal leaflet thickening, and preserved mitral valve integrity. Conclusions: All together, our results suggest sterile inflammation as a novel paradigm to disease progression, and we identify, for the first time, monocytes as a viable candidate for targeted therapy in MVD.

Article, see p 148 Heart failure (HF) with preserved ejection fraction (HFpEF) is a huge public health problem.1 There is no proven effective treatment. Patients with HFpEF are not able to engage in physical activity without developing symptoms of dyspnea and fatigue. This diminishes quality of life and is associated with increased mortality.1,2 To perform physical activity, we increase O2 consumption (Vo2). Oxygen transport is accomplished through convective and diffusive processes. Convective transport involves 2 steps: introduction of O2 into the lungs (alveolar ventilation) and transport from the lungs to the periphery in the circulation (cardiac output [CO]). Diffusive O2 transport describes 2 additional steps: movement of O2 across the alveolar-pulmonary capillary interface in the lung and unloading of O2 from hemoglobin in skeletal muscle capillaries (DM), where mitochondria consume it to make ATP. Impairments in any or several of these steps can constrain the ability of the body to increase Vo2 and thus perform activity. When patients with HF display low peak Vo2, it is often assumed that this reflects a deficit in CO. However, according to the Fick principle, Vo2 is equal to the product of CO and the arterial-venous O2 content difference (AVO2diff). More than 20 years ago, Wilson and colleagues3 made the remarkable observation that 1/4 of patients with severe HF and reduced ejection fraction (HFrEF) actually display normal leg blood flow during exercise. Despite adequate convective O2 delivery, these patients developed profound leg fatigue and an accelerated increase in venous lactate, indicating a switch to anaerobic glycolysis. The patients were less able to increase AVO2diff during exercise, and this drove their low peak Vo2 rather than poor …

Visualization and quantification of the adverse effects of distorted blood flow are important emerging fields in cardiology. Abnormal blood flow patterns can be seen in various cardiovascular diseases and are associated with increased energy loss. These adverse energetics can be measured and quantified using 3-dimensional blood flow data, derived from computational fluid dynamics and 4-dimensional flow magnetic resonance imaging, and provide new, promising hemodynamic markers. In patients with palliated single-ventricular heart defects, the Fontan circulation passively directs systemic venous return to the pulmonary circulation in the absence of a functional subpulmonary ventricle. Therefore, the Fontan circulation is highly dependent on favorable flow and energetics, and minimal energy loss is of great importance. A focus on reducing energy loss led to the introduction of the total cavopulmonary connection (TCPC) as an alternative to the classical Fontan connection. Subsequently, many studies have investigated energy loss in the TCPC, and energy-saving geometric factors have been implemented in clinical care. Great advances have been made in computational fluid dynamics modeling and can now be done in 3-dimensional patient-specific models with increasingly accurate boundary conditions. Furthermore, the implementation of 4-dimensional flow magnetic resonance imaging is promising and can be of complementary value to these models. Recently, correlations between energy loss in the TCPC and cardiac parameters and exercise intolerance have been reported. Furthermore, efficiency of blood flow through the TCPC is highly variable, and inefficient blood flow is of clinical importance by reducing cardiac output and increasing central venous pressure, thereby increasing the risk of experiencing the well-known Fontan complications. Energy loss in the TCPC will be an important new hemodynamic parameter in addition to other well-known risk factors such as pulmonary vascular resistance and can possibly be improved by patient-specific surgical design. This article describes the theoretical background of mechanical energy of blood flow in the cardiovascular system and the methods of calculating energy loss, and it gives an overview of geometric factors associated with energy efficiency in the TCPC and its implications on clinical outcome. Furthermore, the role of 4-dimensional flow magnetic resonance imaging and areas of future research are discussed.