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Aortic wall remodelling in bicuspid aortic valve (BAV) patients is heterogeneous and characterized by elastin fiber breakdown alongside impaired biomechanics. However, the relationship between aortic histopathological changes and biomechanics are incompletely understood. We clarify the influence of elastin fiber integrity on ex vivo aortic wall mechanical properties in BAV patients, and explore the influence of patient age.Aortic tissue samples (N=66) from 19 BAV patients undergoing prophylactic ascending aortic resection surgery were analyzed. Semi-quantitative histopathological analysis was conducted to assess elastin fiber integrity including elastin content and elastic fiber fragmentation.No statistically significant differences in elastin content were observed between younger and older BAV patients. Older patients showed greater elastin fiber fragmentation compared to their younger cohort (74% versus 61%). Elastin fiber histopathology was associated with differences in physiological mechanical properties: elastin fragmentation corresponded with lower LTM (P=0.005) and TZo (P=0.044) in younger BAV patients and higher LTM (P=0.049) and TZo (P=0.001) in older BAV patients. Histopathology changes were significantly associated with supraphysiological mechanical properties only in older BAV patients: decreased elastin integrity was associated with increased TZe (P=0.049) and HTM (P0.001).Elastin histopathologic changes in BAV aortopathy correspond with differences in mechanical properties and this relationship is influenced by patient age. These novel findings provide additional mechanistic insights into aortic wall remodeling and support a more nuanced stratification of BAV patients by age.

Background Empagliflozin, an SGLT2 inhibitor, has shown remarkable reductions in cardiovascular mortality and heart failure admissions (EMPA-REG OUTCOME). However, the mechanism underlying the heart failure protective effects of empagliflozin remains largely unknown. Cardiac fibroblasts play an integral role in the progression of structural cardiac remodelling and heart failure, in part, by regulating extracellular matrix (ECM) homeostasis. The objective of this study was to determine if empagliflozin has a direct effect on human cardiac myofibroblast-mediated ECM remodelling. Methods Cardiac fibroblasts were isolated via explant culture from human atrial tissue obtained at open heart surgery. Collagen gel contraction assay was used to assess myofibroblast activity. Cell morphology and cell-mediated ECM remodelling was examined with the use of confocal microscopy. Gene expression of profibrotic markers was assessed with the use of reverse-transcription quantitative polymerase chain reaction. Results Empagliflozin significantly attenuated transforming growth factor β1–induced fibroblast activation via collagen gel contraction after 72-hour exposure, with escalating concentrations (0.5 μmol/L, 1 μmol/L, and 5 μmol/L) resulting in greater attenuation. Morphologic assessment showed that myofibroblasts exposed to empagliflozin were smaller in size with shorter and fewer number of extensions, indicative of a more quiescent phenotype. Moreover, empagliflozin significantly attenuated cell-mediated ECM remodelling as measured by collagen fibre alignment index. Gene expression profiling revealed significant suppression of critical profibrotic markers by empagliflozin, including COL1A1, ACTA2, CTGF, FN1, and MMP-2. Conclusions We provide novel data showing a direct effect of empagliflozin on human cardiac myofibroblast phenotype and function by attenuation of myofibroblast activity and cell-mediated collagen remodelling. These data provide critical insights into the profound effects of empagliflozin as noted in the EMPA-REG OUTCOME study.

3-Dimensional (3D) myocardial deformation analysis (3D-MDA) enables novel descriptions of geometry-independent principal strain (PS). Applied to routine 2D cine cardiovascular magnetic resonance (CMR), this provides unique measures of myocardial biomechanics for disease diagnosis and prognostication. However, healthy reference values remain undefined. This study describes age- and sex-stratified reference values from CMR-based 3D-MDA, including 3D PS. One hundred healthy volunteers were prospectively recruited following institutional ethics approval and underwent CMR imaging. 3D-MDA was performed using validated software. Age- and sex-stratified global and segmental strain measures were derived for conventional geometry-dependent [circumferential (CS), longitudinal (LS), and radial (RS)] and geometry-independent [minimum (minPS) and maximum principal (maxPS)] directions of deformation. Layer-specific contraction angle interactions were determined using local minPS vectors. The average age was 43 ± 15 years and 55% were women. Strain measures were higher in women versus men. 3D PS-based assessment of maximum tissue shortening (minPS) and maximum tissue thickening (maxPS) were greater than corresponding geometry-dependent markers of LS and RS, consistent with improved representation of local tissue deformations. Global maxPS amplitude best discriminated both age and sex. Segmental analyses showed greater strain amplitudes in apical segments. Transmural PS contraction angles were higher in females and showed a heterogeneous distribution across segments. In this study we provided age and sex-based reference values for 3D strain from CMR imaging, demonstrating improved capacity for 3D PS to document maximal local tissue deformations and to discriminate age and sex phenotypes. Novel markers of layer-specific strain angles from 3D PS were also described.

Background The overlap between cancer and cardiovascular care continues to expand, with intersections emerging before, during, and following cancer therapies. To date, emphasis has been placed on how cancer therapeutics influence downstream cardiac health. However, whether active malignancy itself influences chamber volumes, function, or overall myocardial tissue health remains uncertain. We sought to perform a comprehensive cardiovascular magnetic resonance‐based evaluation of cardiac health in patients with chemotherapy‐naïve cancer with comparison with a healthy volunteer population. Methods and Results Three‐hundred and eighty‐one patients with active breast cancer or lymphoma before cardiotoxic chemotherapy exposure were recruited in addition to 102 healthy volunteers. Both cohorts underwent standardized cardiovascular magnetic resonance imaging with quantification of chamber volumes, ejection fraction, and native myocardial T1. Left ventricular mechanics were incrementally assessed using three‐dimensional myocardial deformation analysis, providing global longitudinal, circumferential, radial, and principal peak‐systolic strain amplitude and systolic strain rate. The mean age of patients with cancer was 53.8±13.4 years; 79% being women. Despite similar left ventricular ejection fraction, patients with cancer showed smaller chambers, increased strain amplitude, and systolic strain rate in both conventional and principal directions, and elevated native T1 versus sex‐matched healthy volunteers. Adjusting for age, sex, hypertension, and diabetes mellitus, the presence of cancer remained associated with these cardiovascular magnetic resonance parameters. Conclusions The presence of cancer is independently associated with alterations in cardiac chamber size, function, and objective markers of tissue health. Dedicated research is warranted to elucidate pathophysiologic mechanisms underlying these findings and to explore their relevance to the management of patients with cancer referred for cardiotoxic therapies.

Background The dilation of the aorta that occurs as a consequence of a congenitally bicuspid aortic valve (BAV) is associated with a risk of dissection, aneurysm or rupture. With progressive aortopathy, surgery is often recommended, but current patient selection strategies have limitations. A blood-based assay to identify those who would most benefit from prophylactic surgery would be an important medical advance. In a proof-of-concept study, we sought to identify aorta-specific differentially methylated regions (DMRs) detectable in plasma cell-free DNA (cfDNA) obtained from patients undergoing surgery for BAV-associated aortopathy. Methods We used bioinformatics and publicly available human methylomes to identify aorta-specific DMRs. We used data from 4D-flow cardiac magnetic resonance imaging to identify regions of elevated aortic wall shear stress (WSS) in patients with BAV-associated aortopathy undergoing surgery and correlated WSS regions with aortic tissue cell death assessed using TUNEL staining. Cell-free DNA was isolated from patient plasma, and levels of candidate DMRs were correlated with aortic diameter and aortic wall cell death. Results Aortic wall cell death was not associated with maximal aortic diameter but was significantly associated with elevated WSS. We identified 24 candidate aorta-specific DMRs and selected 4 for further study. A DMR on chromosome 11 was specific for the aorta and correlated significantly with aortic wall cell death. Plasma levels of total and aorta-specific cfDNA did not correlate with aortic diameter. Conclusions In a cohort of patients undergoing surgery for BAV-associated aortopathy, elevated WSS created by abnormal flow hemodynamics was associated with increased aortic wall cell death which supports the use of aorta-specific cfDNA as a potential tool to identify aortopathy and stratify patient risk.

ObjectiveIn a proof-of-concept study we sought to identify aorta-specific differentially methylated regions (DMRs) detectable in plasma cell-free DNA (cfDNA) obtained from patients with bicuspid aortic valve (BAV)-associated aortopathy.MethodsWe used bioinformatics and publicly-available human methylomes to identify aorta-specific DMRs. We used data from 4D-flow cardiac magnetic resonance imaging to identify regions of elevated aortic wall shear stress (WSS) in patients with BAV-associated aortopathy undergoing surgery and correlated WSS regions with aortic tissue cell death assessed using TUNEL staining. Cell-free DNA was isolated from patient plasma and levels of candidate DMRs were correlated with aortic diameter and aortic wall cell death.ResultsAortic wall cell death was not associated with maximal aortic diameter but was significantly associated with elevated WSS. We identified 24 candidate aorta-specific DMRs and selected 4 for further study. A DMR on chromosome 11 showed acceptable specificity for the aorta and correlated significantly with aortic wall cell death. Plasma levels of total and aorta-specific cfDNA did not correlate with aortic diameter.ConclusionsElevated WSS created by abnormal flow hemodynamics is associated with increased aortic wall cell death which supports the use of aorta-specific cfDNA as a potential tool to identify aortopathy and stratify patient risk.Date and Number of Institutional Review Board ApprovalREB17-0207

The aortopathy associated with bicuspid aortic valve (BAV) is an epidemiologically relevant source of chronic and acute aortic disease (aneurysm and dissection). However, its pathogenesis is still the object of scientific uncertainties and debates. Indeed, the mechanisms determining the diseases of the ascending aorta in BAV patients are most likely complex and multifactorial, i.e. resulting from variable modes of interplay between genetic and hemodynamic factors. Although few scientific studies have so far taken into adequate account this complexity, leaving the precise sequence of pathogenetic events still undiscovered, the accumulated evidence from previous research approaches have at least brought about important insights. While genetic studies have so far identified variants relevant to either valve malformation or aortic complications (including those in the genes NOTCH1, TGFBR2, ACTA2, GATA5, NKX2.5, SMAD6, ROBO4), however each explaining not more than 5% of the study population, other investigations have thoroughly described both the flow features, with consequent forces acting on the arterial wall (including skewed flow jet direction, rotational flow, wall shear stress), and the main changes in the molecular and cellular wall structure (including extracellular matrix degradation, smooth muscle cell changes, oxidative stress, unbalance of TGF-beta signaling, aberrant endothelia l-tomesenchymal transition). All of this evidence, together with the recognition of the diverse phenotypes that the aortopathy can assume in BAV patients, holding possible prognostic significance, is reviewed in this chapter. The complex and multifaceted body of knowledge resulting from clinical and basic science studies on BAV aortopathy has the potential to importantly influence modes of clinical management of this disease in the near future. Crown Copyright (c) 2020 Published by Elsevier Inc. All rights reserved.

Fluroquinolone antibiotics have come under increased scrutiny given their recent association with aortic events. Although judicious use has been urged in select patient populations, such as those with Marfan and Ehlers-Danlos syndromes, that have a known predisposition for aortopathy, other at-risk patient populations may remain. We describe the atypical delayed-presentation of a type A aortic dissection in a patient with alpha-1 anti-trypsin (A1AT) deficiency and longstanding FQ exposure. This case suggests that caution in prescribing fluroquinolone antibiotics should be extended to include those with A1AT deficiency.

Extracellular matrix (ECM) maintains a reservoir of bioactive growth factors and matricellular proteins that provide bioinductive effects on local cells that influence phenotype and behaviors. Bioactive acellular ECM scaffolds can be used therapeutically to stimulate adaptive tissue repair. Fibroblast growth factor-2 (FGF-2) attenuates transforming growth factor-β1 (TGF-β1)-mediated cardiac fibrosis. Heparin glycosaminoglycan can influence FGF-2 bioactivity and could be leveraged to enhance tissue engineering strategies. We explored the effects of heparin on FGF-2 enhancement of bioactive ECM scaffold biomaterials for its antifibrotic effect on attenuating human cardiac myofibroblast activation. Increasing heparin concentration at a fixed concentration of FGF-2 markedly increased the amount of FGF-2 retained and eluted by ECM scaffolds. To explore synergistic bioinductive effects of heparin and FGF-2, collagen gel contraction assay using human cardiac myofibroblasts was performed in vitro. Myofibroblast activation was induced by profibrotic cytokine, TGF-β1. FGF-2 and heparin in combination reduced human cardiac myofibroblast-mediated collagen gel contraction to a greater extent than FGF-2 alone. These observations were confirmed for both human atrial and human ventricular cardiac fibroblasts. Cell death was not different between groups. In summary, heparin is an effective adjuvant to enhance FGF-2 loading and elution of acellular ECM scaffold biomaterials. Heparin increases the bioactive effects of FGF-2 in attenuating human cardiac myofibroblast activation in response to profibrotic TGF-β1. These data may inform tissue engineering strategies for myocardial repair to prevent fibrosis.

Visual Abstract, Highlights • Acellular ECM scaffolds retain bioactive properties capable of stimulating endogenous myocardial repair pathways that could be leveraged therapeutically to promote adaptive cardiac remodeling toward functional recovery after ischemic injury. • In rodents with MI, acellular bioactive ECM scaffolds surgically implanted on the epicardium stimulate adaptive cardiac repair and functional recovery with therapeutic effects highly dependent on the bioinductive properties of the biomaterial. • Interaction of human cardiac fibroblasts with bioactive ECM scaffolds can induce a robust FGF-dependent cell-mediated vasculogenic paracrine response capable of stimulating functional blood vessel assembly. • Acellular bioactive ECM scaffolds surgically implanted on the epicardium post-MI can reprogram resident fibroblasts and stimulate adaptive proreparative pathways enhancing functional recovery. • A novel surgical strategy for tissue repair is introduced that can be performed as an adjunct to conventional surgical revascularization with minimal translational challenges., Summary Structural cardiac remodeling after ischemic injury can induce a transition to heart failure from progressive loss of cardiac function. Cellular regenerative therapies are promising but face significant translational hurdles. Tissue extracellular matrix (ECM) holds the necessary environmental cues to stimulate cell-based endogenous myocardial repair pathways and promote adaptive remodeling toward functional recovery. Heart epicardium has emerged as an important anatomic niche for endogenous repair pathways including vasculogenesis and cardiogenesis. We show that acellular ECM scaffolds surgically implanted on the epicardium following myocardial infarction (MI) can attenuate structural cardiac remodeling and improve functional recovery. We assessed the efficacy of this strategy on post-MI functional recovery by comparing intact bioactive scaffolds with biologically inactivated ECM scaffolds. We confirm that bioactive properties within the acellular ECM biomaterial are essential for the observed functional benefits. We show that interaction of human cardiac fibroblasts with bioactive ECM can induce a robust cell-mediated vasculogenic paracrine response capable of functional blood vessel assembly. Fibroblast growth factor-2 is uncovered as a critical regulator of this novel bioinductive effect. Acellular bioactive ECM scaffolds surgically implanted on the epicardium post-MI can reprogram resident fibroblasts and stimulate adaptive pro-reparative pathways enhancing functional recovery. We introduce a novel surgical strategy for tissue repair that can be performed as an adjunct to conventional surgical revascularization with minimal translational challenges.

Bicuspid aortic valve (BAV) is a common congenital malformation associated with aortopathy and potential rupture. Identifying those patients at greatest risk for dissection is difficult at present. Abnormal hemodynamics in the ascending aorta creates regions of increased wall shear stress (WSS) which causes extracellular matrix dysregulation and may affect cell death. Dying cells release fragmented DNA into the blood and levels of aorta-specific cell-free DNA (cfDNA), identified by the presence of tissue-specific differentially methylated regions (DMRs), could be leveraged as a biomarker. Our objective was to determine if a relationship between elevated WSS and apoptosis in the ascending aorta existed and identify aorta-specific DMRs in the cfDNA of BAV patients. We hypothesized that areas of increased WSS would demonstrate increased cell death that would correlate with increases in aorta-specific cfDNA from patient blood. BAV patients undergoing 4D-flow cardiac magnetic resonance imaging (CMR) and surgery for ascending aorta dilation (range 36-63 mm) were recruited. Blood was collected from 23 patients at the time of CMR and used for the isolation of plasma cfDNA. Aortic wall samples (n = 30) corresponding to regions of high and low WSS were collected at surgery from 15 patients and stained for cell death using the TUNEL assay. Publicly-available methylomes and Metilene were used to identify aorta-specific DMRs in silico . Regions of elevated WSS were seen in all individuals regardless of absolute aortic dimension. These regions of elevated WSS by CMR showed significantly greater cell death when compared to the region of normal WSS in the ascending aorta (0.14 ± 0.05 vs. 0.08 ± 0.06, p=0.00006). Levels of cell death did not correlate with maximal aortic diameter. Twenty-three aorta-specific DMRs were identified. Of the four that were tested, three were found to be hypomethylated in genomic DNA from the aorta compared to 14 other tissues. Levels of aorta-specific cfDNA based on our DMRs did not correlate with maximal aortic diameter. Increased regional cell death corresponding to elevated WSS may implicate abnormal hemodynamic flow in the progression of aortopathy and provides a biological rationale for the use of cfDNA as a biomarker for aortopathy.

Coronary heart disease is a leading cause of death. Tissue remodeling and fibrosis results in cardiac pump dysfunction and ischemic heart failure. Cardiac fibroblasts may rebuild damaged tissues when prompted by suitable environmental cues. Here, we use acellular biologic extracellular matrix scaffolds (bioscaffolds) to stimulate pathways of muscle repair and restore tissue function. We show that acellular bioscaffolds with bioinductive properties can redirect cardiac fibroblasts to rebuild microvascular networks and avoid tissue fibrosis. Specifically, when human cardiac fibroblasts are combined with bioactive scaffolds, gene expression is upregulated and paracrine mediators are released that promote vasculogenesis and prevent scarring. We assess these properties in rodents with myocardial infarction and observe bioscaffolds to redirect fibroblasts, reduce tissue fibrosis and prevent maladaptive structural remodeling. Our preclinical data confirms that acellular bioscaffold therapy provides an appropriate microenvironment to stimulate pathways of functional repair. We translate our observations to patients with coronary heart disease by conducting a first-in-human observational cohort study. We show that bioscaffold therapy is associated with improved perfusion of infarcted myocardium, reduced myocardial scar burden, and reverse structural remodeling. We establish that clinical use of acellular bioscaffolds is feasible and offers a new frontier to enhance surgical revascularization of ischemic heart muscle.

BackgroundInfarcted myocardium can remodel after successful reperfusion, resulting in left ventricular dilation and heart failure. Epicardial infarct repair (EIR) using a bioinductive extracellular matrix (ECM) biomaterial is a novel surgical approach to promote endogenous myocardial repair and functional recovery after myocardial infarction. Using a pre-clinical porcine model of coronary ischemia-reperfusion, we assessed the effects of EIR on regional functional recovery, safety, and possible mechanisms of benefit.MethodsAn ECM biomaterial (CorMatrix ECM) was applied to the epicardium after 75 minutes of coronary ischemia in a porcine model. Following ischemia-reperfusion injury, animals were randomly assigned in 2:1 fashion to EIR (n = 8) or sham treatment (n = 4). Serial cardiac magnetic resonance imaging was performed on normal (n = 4) and study animals at baseline (1 week) and 6 weeks after treatment. Myocardial function and tissue characteristics were assessed.ResultsFunctional myocardial recovery was significantly increased by EIR compared with sham treatment (change in regional myocardial contraction at 6 weeks, 28.6 ± 14.0% vs 4.2 ± 13.5% wall thickening, p < 0.05). Animals receiving EIR had reduced adhesions compared with animals receiving sham treatment (1.44 ± 0.51 vs 3.08 ± 0.89, p < 0.05). Myocardial fibrosis was not increased, and EIR did not cause myocardial constriction, as left ventricular compliance by passive pressure distention at matched volumes was similar between groups (13.9 ± 4.0 mm Hg in EIR group vs 16.0 ± 5.2 mm Hg in sham group, p = 0.61). Animals receiving EIR showed evidence of vasculogenesis in the region of functional recovery.ConclusionsIn addition to the beneficial effects of successful reperfusion, EIR using a bioinductive ECM enhances myocardial repair and functional recovery. Clinical translation of EIR early after myocardial infarction as an adjunct to surgical revascularization may be warranted in the future.

International audience; Objectives: The objectives of this study were to investigate an association between the magnitude of flow-mediated aortic wall shear stress (WSS) and medial wall histopathology in patients with bicuspid aortic valve (BAV) with aortopathy.Methods: Patients with BAV (n = 27; 52 ± 15 years; 3 women; proximal thoracic aorta diameter = 4.4 ± 0.7 and 4.6 ± 0.5 cm) who underwent prophylactic aortic resection received preoperative 3-dimensional time-resolved phase-contrast magnetic resonance imaging with 3-dimensional velocity encoding to quantify WSS relative to a population of healthy age- and sex-matched tricuspid aortic valve control participants (n = 20). Quantitative histopathology was conducted on BAV aorta tissue samples resected at surgery (n = 93), and correlation was performed between elastic fiber thickness and in vivo aortic WSS as continuous variables. Validation of elastic fiber thickness was achieved by correlation relative to tissue stiffness determined using biaxial biomechanical testing (n = 22 samples).Results: Elastic fibers were thinner and WSS was higher along the greater curvature compared with other circumferential regions (vs anterior wall: P = .003 and P = .0001, respectively; lesser curvature: both P = .001). Increased regional WSS was associated with decreased elastic fiber thickness (r = -0.25; P = .02). Patient stratification with subanalysis showed an increase in the correlation between WSS and histopathology with aortic valve stenosis (r = -0.36; P = .002) and smaller aortic diameters (

Background Suspected genetic causes for extracellular matrix (ECM) dysregulation in the ascending aorta in patients with bicuspid aortic valves (BAV) have influenced strategies and thresholds for surgical resection of BAV aortopathy. Using 4-dimensional (4D) flow cardiac magnetic resonance imaging (CMR), we have documented increased regional wall shear stress (WSS) in the ascending aorta of BAV patients. Objectives This study assessed the relationship between WSS and regional aortic tissue remodeling in BAV patients to determine the influence of regional WSS on the expression of ECM dysregulation. Methods BAV patients (n = 20) undergoing ascending aortic resection underwent pre-operative 4D flow CMR to regionally map WSS. Paired aortic wall samples (i.e., within-patient samples obtained from regions of elevated and normal WSS) were collected and compared for medial elastin degeneration by histology and ECM regulation by protein expression. Results Regions of increased WSS showed greater medial elastin degradation compared to adjacent areas with normal WSS: decreased total elastin (p = 0.01) with thinner fibers (p = 0.00007) that were farther apart (p = 0.001). Multiplex protein analyses of ECM regulatory molecules revealed an increase in transforming growth factor β-1 (p = 0.04), matrix metalloproteinase (MMP)-1 (p = 0.03), MMP-2 (p = 0.06), MMP-3 (p = 0.02), and tissue inhibitor of metalloproteinase-1 (p = 0.04) in elevated WSS regions, indicating ECM dysregulation in regions of high WSS. Conclusions Regions of increased WSS correspond with ECM dysregulation and elastic fiber degeneration in the ascending aorta of BAV patients, implicating valve-related hemodynamics as a contributing factor in the development of aortopathy. Further study to validate the use of 4D flow CMR as a noninvasive biomarker of disease progression and its ability to individualize resection strategies is warranted.

Purpose of review This focused review summarizes key insights from the past 12 months of basic science and clinical research on bicuspid aortic valve (BAV)-associated aortopathy. Recent findings Recent studies in BAV-associated aortopathy support a heterogeneous spectrum of disease with distinct phenotypes. Basic science studies provide further support for the concept of regional differences in the severity of aortopathy within the aorta of BAV patients. Clinical studies compared outcomes of BAV patients after isolated aortic valve replacement and showed that those with primarily valvular insufficiency as compared with stenosis may be at greater risk for important aortic events over time. These novel insights will be important to optimize future aortic resection strategies and clinical practice guidelines. Summary As the most common congenital heart defect, BAV disease is a considerable health burden. Recent studies show differences in the clinical manifestation of disease patterns that may have important implications for future research and the evolution toward more patient-specific surgical practice guidelines.

Objectives Fluoroquinolone (FQ) antibiotics are associated with adverse aortic clinical events. We assessed human aortic myofibroblast-mediated extracellular matrix (ECM) dysregulation as a possible cellular mechanism underlying FQ-associated aortopathy. Methods Human aortic myofibroblasts were isolated from patients with aortopathy undergoing elective ascending aortic resection (N = 9). The capacity for extracellular matrix degradation in cells exposed to FQ was assessed by multiplex analysis of secreted matrix metalloproteinases relative to tissue inhibitors of matrix metalloproteinases (TIMPs). Direct evaluation of extracellular matrix degradation was investigated in human aortic cells using a 3-dimensional gelatin-fluorescein isothiocyanate fluorescence microgel assay. Aortic cellular collagen-1 expression following FQ exposure was determined by immunoblotting and immunofluorescent staining. Cell apoptosis, necrosis, and metabolic viability was determined by annexin-V, propidium iodide staining, and water-soluble tetrazolium salt (WST1) assay. Results FQ exposure significantly decreased aortic cell TIMP-1 (P = .004) and TIMP-2 (P = .0004) protein expression compared with vehicle control. The ratio of matrix metalloproteinase-9/TIMP-2 was increased suggesting an increased capacity for extracellular matrix degradation (P = .01). In collagen gels, we show a trend toward increased aortic myofibroblast-mediated collagen fiber degradation with FQ exposure (P = .09). Similarly, FQ exposure attenuated collagen-1 expression as assessed by immunoblotting (P = .002) and immunofluorescence (P = .02). Cell apoptosis, necrosis, and metabolic viability was not significantly influenced by FQ exposure. Conclusions For the first time, we document a putative mechanism underlying FQ-associated aortopathy whereby decreased TIMP expression with impaired compensatory collagen-1 expression results in human aortic myofibroblast-mediated extracellular matrix dysregulation. These novel data may provide a cellular and molecular mechanism to explain the established clinical association between FQ exposure and acute aortic events.

Background Tissue fibrosis and chamber remodeling is a hallmark of the failing heart and the final common pathway for heart failure of diverse etiologies. Sustained elevation of pro-fibrotic cytokine transforming growth factor-beta1 (TGFβ1) induces cardiac myofibroblast-mediated fibrosis and progressive structural tissue remodeling. Objectives We examined the effects of low molecular weight fibroblast growth factor (LMW-FGF-2) on human cardiac myofibroblast-mediated extracellular matrix (ECM) dysregulation and remodeling. Methods Human cardiac biopsies were obtained during open-heart surgery and myofibroblasts were isolated, passaged, and seeded within type I collagen matrices. To induce myofibroblast activation and ECM remodeling, myofibroblast-seeded collagen gels were exposed to TGFβ1. The extent of ECM contraction, myofibroblast activation, ECM dysregulation, and cell apoptosis was determined in the presence of LMW-FGF-2 and compared to its absence. Using a novel floating nylon-grid supported thin collagen gel culture platform system, myofibroblast activation and local ECM remodeling around isolated single cells was imaged using confocal microscopy and quantified by image analysis. Results TGFβ1 induced significant myofibroblast activation and ECM dysregulation as evidenced by collagen gel contraction, structural ECM remodeling, collagen synthesis, ECM degradation, and altered TIMP expression. LMW-FGF-2 significantly attenuated TGFβ1 induced myofibroblast-mediated ECM remodeling. These observations were similar using either ventricular or atrial-derived cardiac myofibroblasts. In addition, for the first time using individual cells, LMW-FGF-2 was observed to attenuate cardiac myofibroblast activation and prevent local cell-mediated ECM perturbations. Conclusions LMW-FGF-2 attenuates human cardiac myofibroblast-mediated ECM remodeling and may prevent progressive maladaptive chamber remodeling and tissue fibrosis for patients with diverse structural heart diseases.

Following myocardial infarction (MI), cardiac myofibroblasts remodel the extracellular matrix (ECM), preventing mechanical complications. However, prolonged myofibroblast activity leads to dysregulation of the ECM, maladaptive remodeling, fibrosis, and heart failure (HF). Chronic inflammation is believed to drive persistent myofibroblast activity; however, the mechanisms are unclear. We assessed the influence of peripheral blood monocytes on human cardiac myofibroblast activity in a three-dimensional (3D) ECM microenvironment. Human cardiac myofibroblasts isolated from surgical biopsies of the right atrium and left ventricle were seeded into 3D collagen matrices. Peripheral blood monocytes were isolated from healthy human donors and cocultured with myofibroblasts. Monocytes increased myofibroblast activity measured by collagen gel contraction (baseline: 57.6 ± 5.9% vs. coculture: 65.2 ± 7.1% contraction; P < 0.01) and increased local ECM remodeling quantified by confocal microscopy. Under coculture conditions that allow indirect cellular interaction via paracrine factors but prevent direct cell-cell contact, monocytes had minimal effects on myofibroblast activity (17.9 ± 11.1% vs. 6.4 ± 7.0% increase, respectively; P < 0.01). When cells were cultured under direct contact conditions, multiplex analysis of the coculture media revealed an increase in the paracrine factors TGF-β1 and matrix metalloproteinase 9 compared with baseline (122.9 ± 10.1 pg/ml and 3,496.0 ± 190.4 pg/ml, respectively, vs. 21.5 ± 16.3 pg/ml and 183.3 ± 43.9 pg/ml; P < 0.001). TGF-β blockade abolished the monocyte-induced increase in cardiac myofibroblast activity. These data suggest that direct cell-cell interaction between monocytes and cardiac myofibroblasts stimulates TGF-β-mediated myofibroblast activity and increases remodeling of local matrix. Peripheral blood monocyte interaction with human cardiac myofibroblasts stimulates myofibroblast activity through release of TGF-β1. These data implicate inflammation as a potential driver of cardiac fibrosis.

We thank Dr. Torii and colleagues for their insights regarding the combination of in vivo 4-dimensional flow magnetic resonance imaging (MRI) and in silico computational fluid dynamics (CFD) to improve risk prediction associated with bicuspid aortic valve (BAV) aortopathy. In their letter, these

We thank Dr. Murakami and colleagues for their letter regarding our recently published paper [(1)][1] in the Journal examining aortic tissue from patients with bicuspid aortic valves following pre-operative 4-dimensional (4D) flow cardiac magnetic resonance imaging. Compared with tissue from regions

BACKGROUND Empagliflozin, an SGLT2 inhibitor, has shown remarkable reductions in cardiovascular mortality and heart failure admissions (EMPA-REG OUTCOME). However, the mechanism underlying the heart failure protective effects of empagliflozin remains largely unknown. Cardiac fibroblasts play an integral role in the progression of structural cardiac remodeling and heart failure, in part, by regulating extracellular matrix (ECM) homeostasis. Thus, the objective of this study was to determine if empagliflozin has a direct effect on human cardiac myofibroblast-mediated ECM remodeling. METHODS Cardiac fibroblasts were isolated via explant culture from human atrial tissue obtained at open-heart surgery. Collagen gel contraction assay was used to assess myofibroblast activity. Cell morphology and cell-mediated ECM remodeling was examined using confocal microscopy. Gene expression of pro-fibrotic markers was assessed using RT-qPCR. RESULTS Empagliflozin significantly attenuated TGFβ1-induced fibroblast activation via collagen gel contraction after 72-hour exposure with escalating concentrations (0.5μM, 1μM, and 5μM) resulting in greater attenuation. Morphological assessment showed myofibroblasts exposed to empagliflozin were smaller in size with shorter and fewer number of extensions, indicative of a more quiescent phenotype. Moreover, empagliflozin significantly attenuated cell-mediated ECM remodeling as measured by collagen fiber alignment index. Gene expression profiling revealed significant suppression of critical pro-fibrotic markers by empagliflozin including: COL1A1, ACTA2, CTGF, FN1, and MMP-2. CONCLUSIONS We provide novel data showing a direct effect of empagliflozin on human cardiac myofibroblast phenotype and function by attenuation of myofibroblast activity and cell-mediated collagen remodeling. These data provide critical insights into the profound effects of empagliflozin as noted in the EMPA-REG OUTCOME study.

BACKGROUND: Following myocardial infarction (MI), activated cardiac myofibroblasts facilitate extracellular matrix (ECM) remodeling to prevent mechanical complications. However, prolonged myofibroblast activity leads to dysregulation of the ECM, maladaptive remodeling, fibrosis and heart failure (HF). Chronic inflammation is believed to drive persistent myofibroblast activity, however, the mechanisms are unclear. In this study, we explored the effects of peripheral blood monocytes on human cardiac fibroblast activation in a 3D ECM microenvironment. METHODS/RESULTS: Human cardiac fibroblasts isolated from surgical human heart biopsies were seeded into 3D collagen matrices. Peripheral blood monocytes isolated from healthy human donors were co-cultured with fibroblasts. Monocytes increased fibroblast activation measured by collagen ECM contraction (17.9±11.1% increase; p CONCLUSION: For the first time, we demonstrate that peripheral blood monocytes stimulate human cardiac fibroblast activation through a mechanism involving TGF-β1 release as a consequence of direct cell-cell interaction through β1-integrin. These data implicate inflammation as a driver of cardiac fibrosis post-MI, highlighting potential novel therapeutic targets for the treatment of ischemic HF.

s S75 phenylephrine (16-660mg/min, a1-agonist). By deep anaesthesia and Urapidil (a1-blockade), lower blood pressures were achieved. In a 3T MRI scanner, OS-images were acquired at 10-15mmHg increments in a mid left ventricular short axis slice and changes in signal intensity (SI) and myocardial flow response were expressed as %-change 70mmHg baseline. At each level, coronary blood flow was obtained and changes in O2er (D) were calculated (1⁄4100*[CaO2-CvO2/CaO2]), comparing the changes to the oxygenation response. RESULTS: With increasing MAP there was a strong correlation to the LAD blood flow response (red) and a moderate correlation to changes in myocardial oxygenation represented by signal intensity (blue). There was a weak correlation between myocardial OS-SI and flow response changes. Interestingly, there was a weak negative correlation between MAP and O2er changes (green), indicating that during the MAP increase there was a luxury perfusion that exceeded the myocardial oxygen demand, allowing OS-SI to increase, while O2er dropped. This was confirmed by the negative correlation of O2er with OS-SI and flow response. RPP showed the same trend as MAP (table) to flow and O2er, yet no relationship was found to SI. CONCLUSION: a1-mediated blood pressure changes induced by phenylephrine result in a luxury perfusion paralleled by an increase in myocardial oxygenation, despite increased myocardial workload. Oxygenation-sensitive cardiovascular magnetic resonance is a suitable tool to monitor the effects of vasopressors on myocardial oxygenation. Research in Anaesthesiology and Intensive Care Medicine, Inselspital Bern University Hospital 147 SURGICAL APPLICATION OF A NOVEL BIOMATERIAL ATTENUATES POST-MI REMODELING AND HEART FAILURE: IMPACT OF INTERVENTION TIMING RELATIVE TO INFARCT STAGE ON FUNCTIONAL RECOVERY HE Mewhort, JD Turnbull, G Teng, DD Belke, DA Svystonyuk, DG Guzzardi, HJ Duff, PW Fedak

The hepatic artery is the second most common site for aneurysms formation within the splanchnic circulation. Most hepatic artery aneurysms (HAA) are diagnosed incidentally by a computed tomography(CT) scan or a Doppler ultrasonography. We present the case of a HAA diagnosed preoperatively in a 82-year old man, who was treated with an endovascular procedure. An abdominal ultrasonography revealed by chance the presence of a HAA. The abdominal CT scan confirmed an aneurysm of the common hepatic artery, specifically at the origin of the gastroduodenal artery. The gastroduodenal artery was embolized using coils then a heparin-bonded covered stent was deployed into the common hepatic artery to exclude the aneurysm. Final arteriogram documented the regular patency of the stent and the complete exclusion of the aneurysm. No complication occurred and the patient was discharged on the second postoperative day. Six months later, a follow-up with a Duplex scan confirmed the regular patency of the stent, and the patient was in good clinical conditions.

BACKGROUND: Epicardial infarct repair (EIR) using a bioinductive extracellular matrix (ECM) biomaterial is a novel surgical approach that targets the infarcted myocardium to enhance myocardial repair and prevent ischemic HF. We previously demonstrated that EIR prevents maladaptive LV remodeling and improves post-MI functional recovery. It is unclear if these benefits are a consequence of passive biomechanical infarct restraint or secondary to active paracrine signals from within the biomaterial (bio-inductive effect). In this study we outline the mechanisms by which EIR enhances myocardial repair following ischemic injury. METHODS AND RESULTS: Epicardial infarct repair performed using a biologically active ECM-biomaterial (CorMatrix-ECM, CorMatrix Cardiovascular Inc., GA, USA) was compared to EIR performed using the same ECM-biomaterial biologically inactivated by gluteraldehyde fixation. The active (N1⁄44) or inactive (N1⁄46) ECM-biomaterial was surgically applied to the epicardial surface of the infarcted myocardium following permanent coronary artery ligation in a rat model. Using a conductance catheter, indices of cardiac performance were quantified by pressure volume loop analysis 14-weeks posttreatment. EIR with either biomaterial resulted in functional recovery above sham-treated (N1⁄44) animals (ejection fraction: 42.43 13.09% vs. 25.15 6.99%, respectively; P1⁄40.035), however we observed a trend toward greater functional improvement in active EIR-treated animals as compared to inactive EIR-treated animals (ejection fraction: 47.13 15.45% vs. 30.50 13.40%, respectively; P1⁄40.16; stroke work: 10618 1548mmHg*uL vs. 8148 2514mmHg*uL, respectively; P1⁄40.17). Increased vascularity was observed within the infarcted myocardium of active EIR-treated animals as compared to inactive EIR-treated animals (11.8 5.6 vs. 3.7 3.9 blood vessels per high power field, respectively; P1⁄40.0008) potentially resulting in increased microvascular perfusion accounting for the functional recovery observed. Increased epicardial thickness, indicative of epicardial activation, was also observed in active EIR-treated animals as compared to inactive EIR-treated animals (4.6 1.6 vs. 2.9 0.7 fold increase relative to sham-treated animals, respectively; P1⁄40.0012). An increase in the number of epicardium derived progenerator cells (EDPCs), identified by Wilms’ tumor-1 expression and nuclearization of b-catenin was also observed in active EIR-treated animals, specifically in the infarcted myocardium indicative of increased epithelial to mesenchymal transition. Vascular structures positive for both nuclear b-catenin and a-SMA identified within the infarcted myocardium of active biomaterial-treated animals indicate that EDPCs undergo EMT and differentiate into vascular smooth muscle cells (VSMCs) through vasculogenesis (Figure). CONCLUSION: These data suggest that EIR with a biologically active biomaterial enhances myocardial repair through a bio-inductive mechanism beyond passive infarct restraint. The epicardial application of ECM-biomaterial stimulates epicardial progenitor cell mobilization and increases vasculogenesis to enhance functional recovery post-MI.

BACKGROUND: Patients with congenital bicuspid aortic valves (BAV) are predisposed to progressive dilatation of the ascending aorta (“bicuspid aortopathy”). A possible inherited/genetic etiology has led to aggressive surgical resection strategies to remove fragile tissue prone to complications. We recently demonstrated patterns of altered wall shear stresses (WSS) in the BAV aorta. These observations suggest regional hemodynamics may trigger local aortic extracellular matrix (ECM) degeneration leading to aortopathy. If validated, a more patient-specific and targeted resection strategy based on preoperative regional hemodynamic assessments may be warranted. For the first time, we correlated regional aortic tissue pathology with local hemodynamics using 4D-MRI in BAVpatients undergoing ascending aortic resection. METHODS AND RESULTS: BAV patients (N1⁄411) referred for ascending aortic resection received preoperative 4D-MRI. Regional WSS differences within each patient’s aorta relative to a normal tricuspid aortic valve control population for “elevated” (>1.96 SD) and “normal” WSS were defined. Paired aortic wall samples were collected during surgical resection from the predefined “elevated” and “normal” WSS regions. Aortic tissue was examined for histologic abnormalities consistent with BAV aortopathy (cystic medial necrosis, ECM fragmentation and mucopolyssacharide deposition) and molecular mediators of ECM regulation (TGFb-1, MMP, TIMP). All BAV patients studied had adjacent aortic regions with elevated and normal areas of WSS suggesting hemodynamic heterogeneity. Within the same patients’ aorta, aortic media subjected to elevated WSS demonstrated increased mucopolyssacharide composition (Figure 1C, white arrows) and elastin fragmentation (Figure 1D, black arrows) as compared to adjacent areas with normal WSS. Multiplex protein analyses revealed a 1.49-fold ( 0.71SD) increase in the profibrotic transforming growth factor b-1 (TGFb-1) in elevated WSS regions as compared to normal (P1⁄40.045). Mean fold increases in ECM-proteases MMP-1 (1.62 0.84; P1⁄40.057), MMP-2 (1.49 1.00; P1⁄40.18), MMP-3 (1.23 0.36; P1⁄40.18), MMP-7 (1.57 0.75; P1⁄40.067), and TIMP-2 (1.26 0.33; P1⁄40.038) were observed in aortic wall subjected to elevated WSS regions as compared to normal WSS regions within the same patients’ ascending aorta. CONCLUSION: In the BAV aorta, regional WSS corresponds with local histological abnormalities and ECM dysregulation. These novel data strongly implicate local hemodynamics as a mediator of BAV aortopathy. With further validation, 4DMRI could be used to guide personalized resection strategies for patients with BAV aortopathy.

s S155 BACKGROUND: Allo-reactive memory T cells are a recognized threat to solid-organ transplants, and yet there is limited information regarding their development and activation following solid-organ transplantation. Using an animal model of cardiac transplantation we previously demonstrated that CD8+ T cells were critical for the development of chronic rejection in the presence of calcineurin inhibitor (CNI) immunosuppression. Clinically, CNI initiation is often delayed post-transplant in an attempt to prevent the development of renal insufficiency. Based on recent reports concerning the development of memory CD8+ T cells we hypothesized that the degree of CNI delay could strongly influence the development of allo-reactive memory CD8+ T cells post-transplant. This was investigated in the current study using a murine model of allogeneic cell priming. METHODS: Allo-reactive memory T cells are a recognized threat to solid-organ transplants, and yet there is limited information regarding their development and activation following solid-organ transplantation. Using an animal model of cardiac transplantation we previously demonstrated that CD8+ T cells were critical for the development of chronic rejection in the presence of calcineurin inhibitor (CNI) immunosuppression. Clinically, CNI initiation is often delayed post-transplant in an attempt to prevent the development of renal insufficiency. Based on recent reports concerning the development of memory CD8+ T cells we hypothesized that the degree of CNI delay could strongly influence the development of allo-reactive memory CD8+ T cells post-transplant. This was investigated in the current study using a murine model of allogeneic cell priming. RESULTS: Effector and memory CD8+ T cell development was prevented when CyA was initiated at 0 or 2d post-prime (p

s S153 CONCLUSION: In this pilot study there were no observable histological differences in human right atrial tissue from individuals at highand low-risk for OSA. Further investigation would be required for more definitive results. Canadian Cardiovascular Society (CCS) Oral

The aims of this study were: 1) to assess the relative prognostic value of predischarge dobutamine echocardiography (DE) and exercise electrocardiography (EE) in patients after a first uncomplicated acute myocardial infarction (AMI), and 2) to evaluate the optimal prognostic strategy by using the two tests in different combinations.DE (dobutamine infusion 5 to 40 micrograms/kg/min plus atropine 0.25 to 1 mg, if needed) and symptom-limited bicycle EE were performed in 208 patients (mean age 58 +/- 9 years, 90% males), on different days and in random order, 12 +/- 4 days after a first uncomplicated AMI and after pharmacological washout. A stress-induced dyssynergy and ST segment depression1 mm were considered criteria of positivity for DE and EE, respectively. Only spontaneous cardiac events were considered: cardiac death, reinfarction (= hard events), and unstable angina requiring hospitalization (= soft events).Thirty-eight events occurred during follow-up (16 +/- 13 months; range: 1-44 months); 5 cardiac deaths, 6 reinfarctions and 27 unstable angina. Patients with a positive DE had a twofold increase in all event rates (26 vs 12%, p0.01) and a fourfold increase in the rate of hard events (9 vs 2%, p0.05). In contrast, no statistically significant difference was observed in the distribution of the same events between patients with positive and negative EE. Both tests showed similar negative (DE 88%, EE 85%) and positive (DE 26%, EE 24%) predictive values. Among six different strategies (performing either DE or EE only in all patients; EE in all patients; EE in all patients and DE only in those with a positive EE; and DE only in those with a negative EE; EE in all patients and DE only in those with anterior AMI), EE only in patients with inferior or non-Q AMI and DE only in those with anterior AMI), performing DE only in patients with a positive EE gave the highest predictive accuracy-74% (95% confidence intervals 68 to 80) for all events and 77% (95% confidence intervals 71 to 83) for hard events.In patients with a first uncomplicated AMI, DE is useful in identifying patients at high and low risk of future spontaneous cardiac events. The optimal strategy for prognostication of these patients is to perform EE in all and DE only in the ones with a positive EE.

To assess the clinical significance of the reaction to blood pressure measurement, 1,013 (889 men) borderline to severe hypertensive patients, enrolled between 1984 and 1993, were studied. Their mean age (+/- SE) was 33.6 +/- 0.46 (range 16-75 years) and their mean office blood pressure (+/- SE) was 152.3 +/- 0.56/95.5 +/- 0.39 mmHg. All subjects underwent ambulatory blood pressure monitoring, ECG and fundoscopy. On the basis of the latter two tests a target organ damage score was calculated, from 0 (no abnormalities) to 5 (maximum severity). In 731 patients an echocardiogram was also performed. The white-coat effect was assessed by measuring the regression of office blood pressure on ambulatory blood pressure and calculating the residual office blood pressure. The subjects with high residual blood pressure showed a greater degree of age-adjusted target organ damage compared to those with intermediate or low residual blood pressure (systolic p0.0001; diastolic p = 0.04). Age-adjusted left ventricular mass was influenced by residual diastolic blood pressure (p0.0001). In a stepwise multiple regression analysis, where age, ambulatory blood pressure levels, sex and duration of hypertension were added to the model, residual blood pressure showed a relationship with the degree of target organ damage and left ventricular mass. In conclusion, the present results show that the white-coat-effect is not innocent, as it is associated with a high degree of cardiovascular abnormalities.