Faculty Bibliography

BACKGROUND:Pathogenic variants in plakophilin-2 (PKP2) cause arrhythmogenic cardiomyopathy (ACM) with intracellular calcium dysregulation as a major component of its arrhythmia phenotype. Recent adeno-associated viral (AAV) based Pkp2 (AAV-PKP2) gene therapy has shown promising results in a few different PKP2-associated ACM models. Fibroblast growth factor 21 (FGF21) has multiple cardioprotective effects and has recently emerged as a promising therapeutic agent for cardiovascular disease. OBJECTIVES/OBJECTIVE:To assess the efficacy and impact on calcium regulation of a novel AAV8-based FGF21 gene therapy on adult cardiac-specific, tamoxifen-activated PKP2 knockout (PKP2-cKO) mice. METHODS:Experiments were performed using a PKP2-cKO murine model. AAV8-FGF21 was delivered to adult mice by a single tail vein injection 7 days before tamoxifen-activated PKP2-cKO. Cardiac functions were monitored by echocardiography and electrocardiography. Intracellular calcium transients were investigated in acute isolated adult mouse cardiomyocytes and calcium fluorescent signals were acquired with the IonOptix system. RESULTS:Loss of PKP2 expression caused cardiac mechanical dysfunction and pro-arrhythmic phenotype in adult mouse models. AAV-mediated delivery of FGF21 mitigated the progression of biventricular structural changes, decreased the occurrence of adrenergic arrhythmias, and rescued intracellular calcium imbalance in the setting of PKP2 haploinsufficiency. In contrast, acute in vitro FGF21 treatment for 1 hour had no effect on intracellular calcium transients. CONCLUSIONS:These beneficial effects of AAV8-FGF21 on the PKP2-ACM phenotype suggest a therapeutic landscape for various targeted cardiomyopathies.

Ribosomes are central to protein synthesis in all organisms. In mammals, the ribosome functional core is highly conserved. Remarkably, two rodent species, the naked mole-rat (NMR) and tuco-tuco, display fragmented 28S ribosomal RNA (rRNA), coupled with high translational fidelity and long lifespan. The unusual ribosomal architecture in the NMR and tuco-tuco has been speculated to be linked to high translational fidelity. Here, we show, by single-particle cryo-electron microscopy, that despite the fragmentation of their rRNA, NMR and tuco-tuco ribosomes retain their core functional architecture. Compared to ribosomes of the guinea pig, a phylogenetically related rodent without 28S rRNA fragmentation, ribosomes of NMR and tuco-tuco exhibit poorly resolved density for certain expansion segments. In contrast, the structure of the guinea pig ribosome shows high similarity to the human ribosome. Enhanced translational fidelity in the NMR and tuco-tuco may stem from subtle, allosteric effects in dynamics, linked to rRNA fragmentation.

The discovery of non-coding RNAs has expanded our understanding of how genetic features are linked to cellular function. The illumination of this so-called dark matter of the genome has revealed new categories of RNA with essential roles in the regulation of protein-coding genes and genome organization. In particular, microRNAs and long non-coding RNAs have emerged as important regulators of cardiovascular health and disease. In this Review, we summarize our current understanding of the mechanisms and functional roles of microRNAs and long non-coding RNAs in the regulation of lipid homeostasis, vascular biology and atherosclerosis. We discuss how interruption of non-coding RNA regulatory circuits influence lipoprotein metabolism in the liver and the circulation, as well as the effects of non-coding RNAs on inflammatory processes in the artery wall that contribute to atherosclerotic plaque formation. Finally, we highlight potential opportunities to harness non-coding RNAs as biomarkers and targeted therapeutics for atherosclerotic cardiovascular disease.

An animal's survival hinges on its ability to integrate past information to modify future behavior. The nematode Caenorhabditis elegans adapts its behavior based on prior experiences with pathogen exposure, transitioning from attraction to avoidance of the pathogen. A systematic screen for the neural circuits that integrate the information of previous pathogen exposure to modify behavior has not been feasible because of the lack of tools for neuron type-specific perturbations. We overcame this challenge using methods based on compressed sensing to efficiently determine the roles of individual neuron types in learned avoidance behavior. Our screen revealed that distinct sets of neurons drive exit from lawns of pathogenic bacteria and prevent lawn re-entry. Using calcium imaging of freely behaving animals and optogenetic perturbations, we determined the neural dynamics that regulate one key behavioral transition after infection: stalled re-entry into bacterial lawns. We find that key neuron types govern pathogen lawn-specific stalling but allow the animal to enter nonpathogenic Escherichia coli lawns. Our study shows that learned pathogen avoidance requires coordinated transitions in discrete neural circuits and reveals the modular structure of this complex adaptive behavioral response to infection.

Many cells identified as macrophage-like in human and mouse atherosclerotic plaques are thought to be of vascular smooth muscle cell (VSMC) origin. We identified cholesterol-mediated down-regulation of TGFβ signaling in vitro in human (h)VSMCs by localization of TGFβ receptors in membrane lipid rafts, which was reversed by high-density lipoprotein (HDL)-mediated cholesterol efflux. This restored VSMC contractile marker (Acta2) and suppressed macrophage marker (CD68) expression by promoting TGFβ enhancement of Mir145 expression. In vivo, administration of ApoA1 (which forms HDL) to atherosclerotic mice also promoted VSMC Acta2 expression and reduced CD68 expression. Because macrophage-like VSMCs are thought to have adverse properties, our studies not only show mechanistically how cholesterol causes their transition, but also suggest that efflux-competent HDL particles may have a therapeutic role by restoring a more favorable phenotypic state of VSMCs in atherosclerotic plaques.

Ultraviolet (UV)-induced DNA mutations generate genetic drivers of cutaneous melanoma and numerous neoantigens that can trigger anti-tumor immunity. Melanoma cells must therefore rapidly evade immune detection by modulating cell-autonomous epigenetic mechanisms and tumor-microenvironment interactions. Although angiogenesis typically facilitates immune infiltration, solid tumors increase vascularization while limiting immune cell entry. By comparing transcription factor (TF) expression across early-stage melanoma, naevi, and other cancers, we found that the homeodomain TF HOXD13 drives a melanoblast-like program upregulated in melanoma and strongly correlated with angiogenesis and immune cell exclusion. Using transcriptomics, 3D chromatin profiling, and in vivo models, we show that HOXD13 promotes tumor growth by enhancing angiogenesis and suppressing T-cell infiltration. HOXD13 orchestrates 3D enhancer-promoter contacts activating VEGFA, SEMA3A, and CD73, which remodel vasculature and elevate immunosuppressive adenosine. Consistently, HOXD13-induced tumor growth is reversed by combined VEGFR and adenosine receptor (AdR) inhibition, revealing a dual pro-angiogenic and immunosuppressive HOXD13 axis with therapeutic relevance.

BACKGROUND:Inflammatory biomarkers in an injured knee have been shown to predict outcomes, yet the role of the subsequent systemic inflammatory response to injury remains poorly understood. PURPOSE/OBJECTIVE:To investigate whether synovial fluid (SF) biomarkers from the contralateral uninjured knee could predict long-term patient-reported outcomes (PROs) for the operative knee in patients undergoing arthroscopic knee surgery. STUDY DESIGN/METHODS:Case series; Level of evidence, 4. METHODS:This retrospective analysis included patients undergoing knee SF aspiration before arthroscopy with ≥8 years of follow-up. SF was aspirated from both the injured and healthy contralateral knees, and concentrations of 10 pro- and anti-inflammatory biomarkers were quantified. Patients completed visual analog scale (VAS) for pain score, Lysholm, Tegner, and Knee injury and Osteoarthritis Outcome Score Physical Function Short Form (KOOS-PS) surveys preoperatively and at the final follow-up. Stepwise linear regression was performed to identify the most significant predictor(s) of PRO scores utilizing log-normalized contralateral biomarker concentration, age, body mass index, injury type, and Outerbridge grade as covariates. Concentrations from the contralateral knee were also compared with the injured knee to assess for correlations. RESULTS:= .006) score at the final follow-up. Contralateral concentrations of monocyte chemotactic protein 1, macrophage inflammatory protein 1β, vascular endothelial growth factor, and TIMP-1 were correlated with levels in the operative knee but at significantly lower concentrations. CONCLUSION/CONCLUSIONS:SF biomarker levels in the contralateral uninjured knee at the time of arthroscopy were predictive of long-term outcomes for the operative knee. Increased levels of pro-inflammatory biomarkers were predictive of worse outcomes, while anti-inflammatory cytokines predicted improved scores. These results suggest that unilateral knee injury can result in a broader systemic inflammatory response that influences long-term outcomes in patients.