Browsing by Author "Fedak, Paul"

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    Acquired Mechanisms of Bicuspid Aortic Valve-Associated Aortopathy
    (2018-07-05) Guzzardi, David G.; Fedak, Paul; O'Brien, Edward; Di Martino, Elena S.
    Bicuspid aortic valve (BAV)-associated aortopathy is characterized by progressive aortic extracellular matrix (ECM) remodeling leading to aneurysm, dissection or rupture. The cause of this aortopathy is unclear; a genetically-driven basis has been favoured, but recent studies implicating an acquired valve-mediated hemodynamic mechanism have challenged this long-standing view despite no clear link between aortic hemodynamics and ECM remodeling having been delineated. We hypothesized that aortopathy in human BAV patients is influenced by valve-mediated wall shear stress (WSS) in a regionally-dependent manner. Aortic tissue specimens from BAV patients that received pre-operative 3-dimensional time-resolved phase-contrast magnetic resonance imaging (4D flow MRI) to compute regional WSS were assessed quantitatively for their expression of aortopathy. Compared to aortic tissue subjected to normal WSS, adjacent tissue from the same BAV aortas subjected to regionally-elevated WSS exhibited demonstrably worse elastic fiber histopathology, increased protease expression and elevated levels of transforming growth factor β-1 (TGFβ-1) consistent with maladaptive aortic ECM remodeling. We also observed that incremental increases in aortic WSS in the human BAV aorta correlate with increased severity of elastic fiber histopathology, and that this association is most strongly observed in BAV patients with primary stenosis and in mildly-dilated (< 4.5 cm) aortas in the earlier stages of disease. These novel data support a critical role for valve-mediated hemodynamics in coordinating the expression of BAV-associated aortopathy and dispute the assumption that aortic pathology in these patients is primarily driven by genetics. Fluoroquinolone (FQ) antibiotic use constitutes another acquired mechanism of aortopathy that may place BAV patients with pre-existing aortic pathology at risk of disease exacerbation. However, no cellular mechanism has been provided underlying this association. We hypothesized that in aortic myofibroblast cells from BAV-associated aortopathy patients, FQ exposure would alter the proteolytic profile favouring ECM dysregulation and modulate collagen expression. We observed that FQ exposure generates a functional increase in ECM degradation driven by reduced tissue inhibitors of matrix metalloproteinases (TIMPs) alongside impaired compensatory collagen-1 expression. These findings may explain the increased incidence of acquired FQ-associated aortopathy and encourage judicious use of FQ in BAV patients with pre-existing aortic pathology.
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    Bioinductive Effects of Acellular Biologic Scaffolds Promote Adaptive Cardiac Repair Following Myocardial Infarction
    (2018-09-17) Svystonyuk, Daniyil A.; Fedak, Paul; Tibbles, Lee Anne; Duff, Henry J.; Giles, Wayne R.
    Ischemic injury may lead to structural remodeling and progressive loss of function, resulting in eventual decompensation to heart failure. Acellular biologic ECM scaffolds retain their native 3-D architecture along with a profile of bioactive constituents that may be leveraged surgically to support myocardial healing. In a proof of concept study, we have previously identified FGF-2 bound to the acellular ECM scaffolds. As such, we hypothesized that FGF-2-dependent bioinductive signaling from surgically implanted acellular scaffolds may attenuate maladaptive structural remodeling and improve functional recovery post-myocardial infarction (MI). First, we observed that FGF-2 has potent anti-fibrotic properties that limited human cardiac fibroblast activation and cell-mediated ECM dysregulation in an in vitro 3-D model. Biochemical characterization showed that ECM scaffolds intact with bioactive constituents released FGF-2 under passive conditions. In a rodent model of myocardial infarction, animals that received intact ECM scaffolds following ischemic injury showed improved functional recovery with evidence of new blood vessel assembly underlying the implantation site. The functional benefits and neovascularization processes were absent in animals that received inactivated scaffolds where FGF-2 bioavailability was limited. The FGF-2-dependent bioinductive effect favorably targeted cardiac fibroblasts, who demonstrated phenotypic plasticity away from a pro-fibrotic phenotype and towards a pro-reparative vasculogenic phenotype. The anti-fibrotic effects of acellular ECM scaffold-derived FGF-2 were consistent with our in vitro studies, however the phenotypic change was unexpected. The redirection in fibroblast phenotype was associated with a modified cardiac scar characterized by a pro-vasculogenic paracrine microenvironment capable of supporting new blood vessel formation and attenuating fibrotic processes. Once again, limiting FGF-2 bioavailability from the ECM scaffolds or blocking FGF receptors in cardiac fibroblasts abolished the induced vasculogenic phenotype. We extended our observations to human subjects where biologic scaffolds were surgically implanted at the site of ischemic injury as an adjunct to standard surgical revascularization. In patients with severe microvascular obstruction and concomitant cardiac dysfunction, acellular biologic scaffolds improved global scar volume and stimulated regional recovery of resting myocardial perfusion. In summary, acellular biologic scaffolds stimulate myocardial healing following ischemic injury through FGF-2-dependent bioinductive signaling that modifies the ischemic scar to support neovascularization, adaptive remodeling, and functional recovery.
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    Expression and Role of Proteoglycan 4/Lubricin in the Human Pericardium: Implications as a Therapeutic to Prevent Post-Operative Pericardial Adhesions
    (2017) Park, Daniel; Fedak, Paul
    Retrosternal pericardial adhesions form post-cardiac surgery and are associated with greater mortality risks for patients undergoing re-sternotomies. Fibrous adhesion development is mediated by cardiac myofibroblasts. Expression of proteoglycan 4 (PRG4) in the human pericardial anatomy and the biological functions of recombinant human PRG4 on human cardiac myofibroblasts was investigated. Expression of PRG4 in the human pericardial fluid and localized expression of PRG4 by pericardial mesothelial cells lining the pericardial cavity was observed. Recombinant human PRG4 prevented human cardiac myofibroblast adhesion to coated surfaces, attenuated transforming growth factor-β1 mediated collagen matrix gel contraction, decreased collagen fiber alignment by activated cardiac myofibroblasts and increased cell roundness. Replacing the lost endogenous PRG4 found in pericardial fluid with recombinant human PRG4 could attenuate the formation of post-operative pericardial adhesions and increase the safety and feasibility of re-sternotomies.
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    Identification and Verification of Differentially Methylated Regions in Cell-Free DNA as a Peripheral Biomarker for Bicuspid Aortic Valve Aortopathy
    (2020-01-08) Maredia, Ashna Karimbhai; Greenway, Steven C.; Fedak, Paul; Bathe, Oliver F.; Braun, Andrew P.
    Bicuspid aortic valve (BAV) is a common congenital cardiac malformation associated with aortopathy for which the progression of aortic dilation is difficult to predict at present. BAV aortopathy has been linked to genetic factors and abnormal hemodynamic flow with regions of elevated wall shear stress (WSS) on the ascending aorta. The dying vascular smooth muscle cells release fragmented DNA into the circulation and this cell-free DNA (cfDNA) could be leveraged as a biomarker for aortopathy. Identification of tissue-specific differentially methylated regions (DMRs) in DNA provides a potential mechanism to identify cfDNA arising from the ascending aorta. The objective is to identify aorta-specific DMRs in the cfDNA of BAV patients as a biomarker for the severity of the aortopathy. We hypothesize that BAV-associated aortopathy leads to increased cell death and increased release of aorta-specific cfDNA correlating with the severity of aortopathy as defined by aortic cell death, elastin degradation and dysregulation of ECM proteins. BAV patient aortic wall samples corresponding to areas of elevated and normal WSS were collected and stained for cell death. Regions of elevated aortic WSS showed greater cell death when compared to regions of normal aortic WSS (p=0.00006). We established a bioinformatic pipeline for the identification of aorta-specific DMRs and they were verified with BAV patient cfDNA. The levels of aorta specific cfDNA of the DMRs on Chr 11, 18 and 22 of BAV patients had a significant correlation with levels of cell death in elevated aortic WSS regions. However, there was no correlation with elastin thickness, ECM concentrations of matrix metalloproteinases (MMP) types 1, 2 and 3, tissue inhibitor of metalloproteinases-1 and transforming growth factor-β1. Further work needs to be done in order to identify more specific aortic DMRs that have stronger correlation to the severity of BAV aortopathy markers with larger cohorts for biomarker validation. The identification of a peripheral biomarker that correlates with tissue disease will be an important advance in the non-invasive diagnosis of BAV-associated aortopathy and potentially help guide clinical decision-making regarding the need for surgical intervention.
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    Impacts of Aging and Hypertension on Fibrosis and Electrical Conduction in the Sinoatrial Node and Atria
    (2020-07-14) Mackasey, Martin; Rose, Robert Alan; Fedak, Paul; Nygren, Anders
    Sinoatrial node (SAN) and atrial fibrosis are major mediators of sinus node dysfunction (SND) and atrial fibrillation (AF), respectively. Both frequently co-exist in pathological states involving enhanced fibrotic remodeling. Aging and hypertension are characterized by electrical and structural remodeling and have been identified as important risk factors for the development of SND and AF. However, the underlying mechanisms facilitating these processes are incompletely understood. Natriuretic peptides (NPs) are a family of cardioprotective hormones that act partially through activation of the natriuretic peptide receptor type-C (NPR-C); although, its capacity to regulate cardiac remodeling is poorly understood. The studies presented examine the role of SAN and atrial fibrosis in regulating electrical conduction using two models associated with adverse remodeling: aging and Ang II-mediated hypertension. Aging studies also investigated the utility of assessing frailty to gain further insight into age-associated mechanisms of structural remodeling and arrhythmogenesis. Accordingly, aged and hypertensive mice exhibited enhanced SAN and atrial fibrosis as assessed by picrosirius red staining. This impacted electrical conduction as assessed by optical mapping experiments and promoted arrhythmogenesis. Frailty scores were correlated with several aspects of the remodeling process, including gene expression changes, indicating the potential for frailty score to provide additional insight into cellular and sub-cellular mechanisms. Aged and hypertensive mice exhibited distinct gene expression alterations in regulators of the cardiac extracellular matrix including collagens, transforming growth factor β1 (TGFβ1), lysyl oxidase (LOX), matrix metalloproteinases (MMPs), and tissue inhibitors of MMPs (TIMPs). In studies examining the role of NPR-C in Ang II-mediated hypertension, NPR-C-/- mice displayed substantially worse outcomes with respect to SAN and atrial fibrosis as well as electrical impairments. These alterations were associated with changes in ECM-related gene expression. Conversely, a selective NPR-C agonist cANF potently prevented Ang II-mediated fibrosis and electrical impairments. However, these changes were generally not accompanied by ECM-related gene expression alterations. Taken together, these results demonstrate a crucial role for adverse fibrotic remodeling in influencing electrical conduction and arrhythmogenesis in the SAN and atria. Additionally, these studies show that NPR-C signaling exhibits potent anti-fibrotic effects that could be utilized for future therapeutic approaches in hypertension.
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    Mechanical and Structural Remodeling of Cardiac Muscle Following Aerobic and Resistance Exercise Training in Rats
    (2020-05-07) Boldt, Kevin; Joumaa, Venus; Turnbull, Jeannine; Fedak, Paul; Herzog, Walter
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    Novel Approaches to the Assessment of Systemic Circulation and Ventricular Performance
    (2019-02-27) Howell, Sarah; Tyberg, John V. T.; Shrive, Nigel; Phillips, Aaron A.; Sheldon, Robert Stanley; Fedak, Paul
    The purpose of evaluating the systemic circulation using systemic vascular conductance and head-capacity curve to quantify left ventricle performance is to validate these assessment approaches instead of more commonly used cardiovascular indexes (i.e., systemic vascular resistance and ejection fraction) used for physiologic responses. Systemic vascular conductance is reciprocal of systemic vascular resistance and is defined as the flow to the systemic circulation that determines arterial pressure. Systemic vascular conductance is calculated by dividing cardiac output by arteriovenous pressure difference. Left ventricle performance is assessed using head-capacity curve. It states that left ventricle as a pump, works under a head-capacity curve. The aim of this study is to increase our understanding of systemic circulation and ventricular performance by studying how changing loading conditions using drug interventions (phenylephrine, sodium nitroprusside, and isoproterenol), proximal aortic constriction, and volume loading will affect the systemic vascular conductance and left ventricle performance. The ultimate goal of this study is to combine circulatory and ventricular properties quantitatively and define the relationship between output produced by the heart and input of the circulation. Overall, results are consistent with the hypothesized physiological changes and allow the application of both proposed indices to determine the global cardiovascular performance in a simple manner. These results further enhance our understanding that conductance of each intervention in systemic circulation determines the mean arterial pressure. Systemic vascular conductance changes markedly with changing cardiac index and these changes are mediated by left ventricle. The potential benefits of these assessment parameters will help provide insights to optimize circulatory performance in patients with cardiovascular disease.
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    The Role of Empagliflozin in Cardiac Fibroblast-Mediated Extracellular Matrix Remodeling
    (2019-01-18) Kang, Sean; Fedak, Paul; Duff, Henry J.; Tibbles, Lee Anne
    Empagliflozin, a sodium-glucose cotransporter-2 (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. Thus, in this study we sought to determine if empagliflozin has a direct effect on human cardiac fibroblast-mediated ECM remodelling. Empagliflozin attenuated myofibroblast activation as determined by collagen gel contraction and α-smooth muscle actin (α-SMA) characterization. Examination with confocal microscopy revealed cell morphology indicative of a quiescent phenotype and an attenuation of local ECM remodelling by these fibroblasts in response to empagliflozin. Furthermore, gene expression profiling indicated suppression of critical pro-fibrotic markers by empagliflozin. Taken together, we provide critical insights into the profound clinical benefits of empagliflozin on cardiac failure and mortality.
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    Role of Tissue Inhibitor of Metalloproteinase-2 in Human Cardiac Fibroblast-Mediated Extracellular Matrix Remodeling
    (2013-04-30) Ngu, Janet; Fedak, Paul
    After a myocardial infarction, extracellular matrix (ECM) dysregulation leads to maladaptive cardiac remodeling that constitutes the basis of development of heart failure. Tissue Inhibitor of Metalloproteinase-2 (TIMP-2) is an endogenous biomolecule that is critical in the maintenance of ECM architecture. Cardiac fibroblasts are the main cell type that regulates ECM homeostasis. This study employed an innovative method of three-dimensional collagen gel assay, which mimics the natural in vivo ECM. We investigated the effect of TIMP-2 on the human cardiac fibroblast-mediated ECM remodeling. TIMP-2 induced differentiation of cardiac fibroblasts into myofibroblasts that are active in collagen synthesis. Concurrently, TIMP-2 induced an increase in the total protease activity within the collagen gel microenvironment. TIMP-2 did not promote a fibrotic response, despite its ability to activate myofibroblasts. These actions appear to be independent of its MMP-inhibitory actions. In conclusion, TIMP-2 promotes ECM homeostasis via simultaneous induction of myofibroblast activation and total protease activity.