Browsing by Author "Duff, Henry J."

Now showing 1 - 12 of 12
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    An Analysis of Dye-Mediated Photo-Oxidation Fixed Bovine Pericardium for Use in Cardiac Surgery
    (2022-04-22) Pattar, Simranjit S; Fedak, Paul WM; Duff, Henry J.; Patel, Vaibhav; Rose, Robert
    Extracellular matrix bioscaffolds have the potential to influence the cardiac microenvironment and to modulate endogenous cellular mechanisms and are therefore a promising forefront for repair and reconstruction in cardiac surgery. In this study, we investigated the biocompatibility and bioinductivity of an acellular bovine pericardium fixed via dye-mediated photo-oxidation on human cardiac fibroblasts. We compared dye-mediated photo- oxidation fixed bioscaffold to glutaraldehyde-fixed and non-fixed bioscaffolds previously characterized in the literature. Human cardiac fibroblasts were seeded on to bioscaffold materials to assess biocompatibility and bioinductivity by specifically characterizing gene expression, secretome, morphology and viability. Dye-mediated photo-oxidation fixed bovine pericardium preserved human cardiac fibroblast phenotype and viability and potentiated a pro-vasculogenic paracrine response. We also compared the tensile properties of each bioscaffold via biomechanical testing. Dye-mediated photo-oxidation fixed bovine pericardium demonstrated increased compliance compared to glutaraldehyde-fixed bioscaffold in response to tensile force. Dye-mediated photo-oxidation fixed bovine pericardium demonstrated enhanced compliance and retained bioinductivity properties which may leverage endogenous reparative pathways. The biocompatibility, bioinductivity, and biomechanical properties of dye-mediated photo-oxidation fixed bovine pericardium demonstrate its feasibility as a bioscaffold for use in cardiac surgery and warrant further investigation for its use as a tool for cardiac repair and regeneration.
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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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    Deletion of iNOS in calcineurin mice improves cardiovascular phenotypes
    (2006) Somers, Julie R.; Duff, Henry J.
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    Developmental changes in potassium currents in mouse heart
    (1996) Wang, Li; Duff, Henry J.
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    Examining the Role of Non-Canonical NOD-like Receptors and Inflammasomes in Inflammation and Disease
    (2018-03-21) Platnich, Jaye Matthew; Muruve, Daniel A.; MacDonald, Justin Anthony; Power, Christopher; Duff, Henry J.; Peters, Nathan C.
    The NOD-like Receptors (NLRs) are a family of pattern recognition receptors known to regulate a variety of immune signaling pathways. A substantial portion of NLR research focuses on the pyrin domain-containing NLRP subfamily. The canonical NLRPs are inflammasome-forming proteins responsible for the activation of caspase-1 and the maturation and secretion of the pro-inflammatory cytokines IL-1β and IL-18. In contrast, the non-canonical inflammasome-independent NLRPs regulate a variety of other pathways, including MAPK and NF-κB, through the formation of non-inflammasome complexes. Interestingly, not all inflammasomes are nucleated by NLRPs. The recently characterized non-canonical caspase-4 (caspase-11 in mice) inflammasome is known to be a key driver of the innate immune response to intracellular pathogens (and the molecules associated with them), by triggering both inflammatory cell death and the activation of canonical inflammasomes. At the outset of this PhD work, the understanding of both non-inflammasome-forming NLRPs and the non-canonical caspase-4 inflammasome was poor and the studies were sparse. It was the goal of this thesis to characterize the expression, gene regulation, and function of the non-inflammasome-forming NLR protein NLRP6, both at the cellular and biochemical level. Furthermore, using a pathogen-associated molecular pattern (PAMP)-driven model of inflammation, we sought to elucidate the function of the non-canonical caspase-4 inflammasome, particularly as it pertains to the regulation of the canonical inflammasome and cell death. By studying the fundamental biology underlying these lesser-known mediators of the innate immune system, we hoped to better understand their contribution to the early immune response and their role in driving inflammatory disease with a view to, one day, ameliorating the condition of patients suffering from these afflictions through the development of targeted therapeutics.
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    Mapping Ligand Binding Sites in hERG1 Channel with Biomolecular Simulations
    (2021-03-01) Mousaei, Mahdi; Noskov, Sergei Yu; Duff, Henry J.; Tieleman, Dirk Peter
    The human ether-a-go-go related gene 1 (hERG1) K+ ion channel generates the rapid delayed rectifier potassium current, or IKr, which is essential for the normal repolarization phase in the ventricular action potential. The drug-induced blockade of the channel is a frequent side effect of various classes of drugs which can cause QT interval prolongation. The consequent cardiac arrhythmia, known as acquired long QT syndrome, has led to the withdrawal of several approved drugs and a mandatory preclinical stage safety assessment for hERG1 blockade. However, cardiotoxicity detection due to the hERG1 blockade remains a challenging task because of the variability of the methodologies employed. In vitro studies are costly, labor-intensive, and technically demanding. Recent development of various in-silico predictive tools has paved the way for a cost-effective cardiotoxicity determination. The availability of the cryo-EM structure of hERG1 at a 3.8 Å resolution provides a unique opportunity for the application of rapidly evolving fragment-based approaches for mapping of potential drug binding sites in hERG1 and rapid assessment of drug blockade. In this thesis, first, we used free energy sampling methods to establish a connection between the cryo-EM structure of the hERG1 channel and its functionality. Furthermore, using the hERG1 cryo-EM structure, we studied the putative druggable binding pockets of hERG1 with the site identification by ligand competitive saturation (SILCS) simulation method. The generated affinity maps from SILCS account for protein flexibility, solutes desolvation effects, and protein-fragments interaction. Using SILCS, we mapped the binding sites of the hERG1 channel including the intracellular cavity, lipid phasing domains, and voltage sensor domains. Our SILCS- Hotspots model showed the existence of two distinct regions inside the IC in agreement with the previously proposed “deep” and “shallow” binding pockets in this region. Finally, using the optimized SILCS Monte-Carlo, SILCS-based docking method, we designed a protocol for rapid prediction of the ligands binding affinity to hERG1. The outcome of this research will be used for rapid and cost-effective computer-aided drug design.
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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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    The Role of RVLM and PACAP in Sympathetic Long-Term Facilitation after Exposure to Acute Intermittent Hypoxia Hypercapnia
    (2018-02-21) Derakhshan, Fatemeh; Wilson, Richard J. A.; Duff, Henry J.; Thompson, Roger J.
    Intermittent hypoxia (IHx) and hypercapnia (Hc) episodes are typically a consequence of obstructive sleep apnea (OSA) in adults and immature respiratory control in pre-term infants. IHxHc contributes to immediate and long-term co-morbidities including increased sympathetic output, hypertension, long-term cardiorespiratory instability and stroke. Exposure to an acute phase of IHxHc results in sympathetic long-term facilitation (LTF). Despite intensive investigation, the mechanisms linking IHxHc to increased sympathetic activity and cardiorespiratory instability remain poorly understood. In my thesis project, I explored the role of rostral ventrolateral medulla (RVLM) neurons in development of sympathetic LTF after exposure to IHxHc. I report that PACAP, a highly conserved excitatory neuropeptide, which can function as an "emergency response" co-transmitter in the sympathoadrenal axis, plays a significant role in activating the sympathetic responses to IHxHc, with a prominent role in the RVLM. First, I showed that PACAP plays a critical role during IHx and can save the life of PACAP-KO mice exposed to acute IHx. To the best of our knowledge, PACAP is the first neuropeptide, which is required to survive acute IHx. Second, intermittent stimulation of RVLM area, mirrors the effect of IHxHc with inducing LTF and is sufficient for development of sympathetic LTF, emphasizing on an important role of RVLM neurons in the induction and maintenance of a sympathetic surge after IHxHc. Third, I showed that PACAP action at the RVLM level is necessary for the maintenance of induced sympathetic LTF after exposure to IHxHc. Fourth, I demonstrated that carotid sinus denervation decreases the baseline sympathetic nerve activity but does not suppress the sympathetic nerve response to hypoxia. This finding was explained with the consecutive discovery of spinal cord oxygen sensors. The spinal cord oxygen sensors (SOS) are active over the physiological range and have several qualities of primary oxygen sensors, including a highly-sensitive and rapid physiologic response to changes in oxygen levels. The discovery of PACAP’s role in the maintenance of sympathetic LTF and the existence of SOS introduces a new chapter in current cardiorespiratory research. This new realm has implications for translational studies, such as those investigating sustained sympathetic nerve activity in heart failure,aiming to help patients with chronic obstructive pulmonary disease (COPD), OSA, paroxysmal sympathetic hyperactivity (PSH), and neonates at risks for sudden infant death syndrome (SIDS).