Faculty Bibliography

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.

BACKGROUND:are the most common cause of familial arrhythmogenic right ventricular cardiomyopathy. This study tests whether plakophilin-2 (PKP2) deficiency only in cardiomyocytes is sufficient to provoke premature aging and proinflammatory senescence in nonmyocyte, cardiac resident cells. METHODS:We studied mice with cardiomyocyte-specific, tamoxifen-activated loss of PKP2 (cardiomyocyte-specific conditional knockout of plakophilin-2) using conventional and multiplex imaging, cytokine arrays, epigenetic clocks, spatial transcriptomics, expansion and structured illumination microscopy, and correlative data analysis. We examined nonmyocytes and cardiomyocytes for premature aging and senescence. RESULTS:We observed senescence-associated heterochromatin foci in nonmyocytes, predominantly in cells positive for α-smooth muscle actin staining. Cytokines in media of nonmyocyte cells were consistent with senescence-associated secretory phenotype. Epigenetic clocks identified premature aging. Multiplex immunohistochemistry showed nonmyocyte cells in niches, intermingled with cardiomyocytes. Spatial transcriptomics showed overrepresentation of senescence-associated secretory phenotype-related transcripts, predominantly in myocyte-rich areas of the left ventricle. Senescence-associated heterochromatin foci and increased epigenetic age were not found in cardiomyocytes from cardiomyocyte-specific conditional knockout of plakophilin-2 hearts, although we observed structural features associated with premature aging. Cross-reference analysis showed correlation between the cardiomyocyte-specific conditional knockout of plakophilin-2 cardiac proteome and that of mice 5 or 6 times their chronological age, as well as transcriptional signatures of neurodegenerative diseases. CONCLUSIONS:Loss of PKP2 expression only in adult cardiac myocytes is sufficient to induce proinflammatory senescence in nonmyocytes, and overall premature cardiac aging. This is the first study to intersect cellular senescence and premature aging with desmosomal arrhythmogenic cardiomyopathies. We speculate that cell-agnostic molecular signatures, biomarkers, and pharmacology of senescence and of neurodegenerative diseases may be relevant to diagnose or treat PKP2 arrhythmogenic right ventricular cardiomyopathy.

OBJECTIVES/OBJECTIVE:This study evaluated the impact of prolonged glucagon-like peptide-1 (GLP-1) receptor agonist use on postoperative outcomes, including radiographic post-traumatic osteoarthritis (PTOA), fracture nonunion, and final knee range of motion-following operative management of tibial plateau fractures across multiple BMI strata. METHODS:A retrospective cohort study was conducted at an urban academic institution, including patients who underwent surgical fixation for tibial plateau fractures between 2016-2024, with a ≥6 months follow-up. The GLP-1 cohort consisted of patients with documented long-term GLP-1 use pre- and postoperatively. GLP-1 users (Group A, n=24) were compared to three non-GLP-1 cohorts stratified by BMI: Group B (BMI 18.5-25, n=150), Group C (BMI 25-30, n=150), and Group D (BMI ≥30, n=100). Outcomes included Kellgren-Lawrence osteoarthritis grade, post-reduction fracture angulation, articular step-off, Charlson Comorbidity Index (CCI), fracture complications (infection, nonunion, PTOA, revision surgery), and final knee flexion range of motion (ROM). Statistical analyses used SPSS Statistics version 29.0 (IBM Corp., Armonk, NY) with ANOVA and Chi-square tests. RESULTS:Mean follow-up was 28.83 months. Baseline age, CCI, fracture angulation, and step-off were comparable between groups. Pre-injury osteoarthritis severity was higher in Group A (0.96±0.88) than in Groups B (0.68±0.86), C (0.54 ± 0.75), and D (0.78±0.74) (p<0.001). Radiographic PTOA incidence was highest in Group D (32%, p<0.01), while Group A rates were comparable to Groups B and C (p≈0.62). Final knee flexion ROM differed significantly (p<0.01), with Group D showing the lowest mobility (119.08±16.47°). Nonunion rates were significantly higher in Group A (p<0.01). CONCLUSIONS:Among obese patients, GLP-1 receptor agonist use was associated with a lower incidence of PTOA and preserved knee ROM compared to untreated obese individuals, with outcomes similar to non-obese patients. However, GLP-1 use was also linked to increased nonunion rates. These findings suggest that while GLP-1 therapy may mitigate obesity-related joint degeneration, it may also challenge fracture healing.

Reactivation of quiescent neural stem cells (NSCs) in the central nervous system (CNS) is a tightly controlled process that generates new neurons and glia to maintain homeostasis or enable repair post-injury, but it remains unclear if reactivation of distinct NSC populations is coupled. Here, we discovered that NSC quiescence exit in Drosophila follows a hierarchical sequence, whereby activation of anterior stem cells in the brain lobes precedes and is required for the timely state-transition of more posterior NSCs in the ventral nerve cord. To achieve this, quiescent NSCs transiently activate neuronal genes. This transient neuronal state is temporary and specific to NSC dormancy, as neuronal genes are switched off after stem cells resume proliferation. Blocking neuronal firing in brain lobe neurons delays the onset of posterior NSC reactivation. Our results reveal long-range communication between quiescent NSCs to coordinate reactivation across the CNS, enabled by a transient, plastic neuron-like state that allows direct interaction with neuronal axons.

Apolipoprotein B (APOB) containing lipoproteins contribute to atherosclerosis by entering the arterial wall through the endothelial cell (EC) surface receptors scavenger receptor-BI (SR-BI) and activin receptor-like kinase 1 (ALK1). We used N-terminal fragments of APOB, molecular modeling, and site-directed mutagenesis to identify and block the binding of chylomicrons and LDL to these receptors in cells and mice. We discovered that different APOB regions interact with SR-BI and ALK1 expressed on ECs APOB48 lipoproteins were only internalized by SR-BI. A fragment of APOB, comprising 18% of the N-terminal sequence, APOB18, reduced the uptake and transport of both chylomicrons and LDL by ECs, whereas a shorter fragment, APOB12, only blocked ALK1 mediated uptake of APOB100 containing lipoproteins. Importantly, overexpressing APOB18 decreased atherosclerosis in hypercholesterolemic mice. These findings identify the N-terminal region of APOB as the cause of atherosclerosis and illustrate an approach to treating or preventing vascular disease.

Donnai-Barrow Syndrome (DBS) arises from loss-of-function (LoF) variants in the endocytic receptor LRP2/megalin and is characterized by low molecular weight (LMW) proteinuria and developmental abnormalities. Urinary proteomics of nine DBS patients revealed that the urinary proteome of a DBS patient with the missense variant LRP2 p.C1400R was indistinguishable from that of patients with splice site, nonsense, or frameshift mutations. A CRISPR mouse model of the variant was generated to determine the mechanism of LoF and proteinuria. The mutant LRP2 was expressed and observed to dimerize and localize to the proximal tubule apical membrane. However, both fluid-phase and receptor-mediated endocytosis were impaired in the context of a general perturbation of endocytic flux. Immunofluorescence revealed aberrant endocytic recycling with mislocalized RAB11+ and TFR1+ compartments and enlarged lysosomes. Structural modeling showed the LRP2 assembly likely tolerates the cysteine to arginine substitution at the cell surface, but at endosomal pH the variant introduced steric clashes that may disrupt intramolecular interfaces and disturb receptor recycling. These findings point to the importance of LRP2 recycling for global endocytic flux and offer a blueprint for leveraging patient-specific alleles to dissect proximal tubule function.

Neuronal axons have traditionally been considered to be the primary mediators of functional connectivity among brain regions. However, the role of astrocyte-mediated communication has been largely underappreciated. Astrocytes communicate with one another through gap junctions, but the extent and specificity of this communication remain poorly understood. Astrocyte gap junctions are necessary for memory formation^1,2, synaptic plasticity^3-5, coordination of neuronal signalling⁶, and closing the visual and motor critical periods^7,8. These findings indicate that this form of communication is essential for proper central nervous system development and function. Despite the importance of astrocyte gap junctional networks, studying them has been challenging. Current methods such as slice electrophysiology disrupt network connectivity and introduce artefacts due to tissue damage. Here, we developed a vector-based approach that labels molecules as they are fluxed by astrocyte gap junctions in awake, behaving animals to overcome these limitations. We then used whole-brain tissue clearing^9,10 to image these intact, three-dimensional astrocyte networks. We show that multiple astrocyte networks traverse the mouse brain. These networks selectively connect specific regions, rather than diffusing indiscriminately, and vary in size and organization. We observe local networks that are confined to single brain regions and long-range networks that robustly interconnect multiple regions across hemispheres, often exhibiting patterns distinct from known neuronal networks. We also demonstrate that astrocyte networks undergo structural reorganization in the adult brain after sensory deprivation. These findings reveal a mode of communication between distant brain regions that is mediated by plastic networks of gap junction-coupled astrocytes.

Breast cancer remains the second leading cause of cancer-related mortality among women, with triple-negative breast cancer (TNBC) exhibiting a particularly poor five-year prognosis. Here, we demonstrated that, among genetic and pharmacological perturbations targeting DNA replication, suppression of DNA polymerase epsilon (POLE) induced a potent, TNBC-specific gene expression signature enriched in inflammatory cytokines that are transcriptional targets of NF-κB. TNBC cells exhibited markedly higher levels of DNA damage and canonical NF-κB activation compared to luminal breast cancer cells. Notably, NF-κB activation in this context depended on the canonical component RELA but not the non-canonical component RELB. Mechanistically, ATM, STING, and RIG-I each contributed to NF-κB activation following POLE suppression. POLE suppression in an in vivo murine TNBC model led to cancer cell-intrinsic elimination of tumor burden and increased immune cell infiltration. Together, these findings support a model in which replication stress from POLE inhibition triggers robust NF-κB-mediated inflammation and immune microenvironment remodeling in TNBC and can independently trigger tumor eradication. These results suggest a potential therapeutic avenue for targeting POLE in TNBC.

UNLABELLED:LKB1 mutations in lung cancer promote an immunosuppressive tumor microenvironment, but the underlying mechanisms remain unknown. Using genetically engineered mouse models and human tumor samples, we demonstrate that LKB1 loss leads to high expression of the cytokine leukemia-inhibitory factor (LIF), which through a cancer cell-autonomous autocrine loop, orchestrates the infiltration of immunosuppressive SiglecFHi neutrophils and Arg1+ interstitial macrophages. Genetic deletion of Lifr, the receptor for LIF, on Lkb1-mutant lung tumors revealed that autocrine LIF signaling induces tumor plasticity and the emergence of a Sox17+ dedifferentiated inflammatory cell state. Antibody-mediated LIF neutralization selectively eliminates the Sox17+ tumor cell state, reduces immunosuppressive myeloid cells, and enhances antitumor T-cell responses. Our study uncovers a novel LKB1-LIF axis driving immune evasion and identifies LIF as a potential therapeutic target in LKB1-mutant lung cancer. This work highlights the interplay between tumor genetics, cellular plasticity, and immune regulation in lung cancer progression. SIGNIFICANCE/UNASSIGNED:LKB1-mutant lung cancers express LIF, which induces an immunosuppressive Sox17+ tumor state. Anti-LIF therapy eliminates this state and restores antitumor immunity, revealing a novel vulnerability in this aggressive cancer subtype lacking effective targeted therapies.

Cerebral malaria is a major complication of Plasmodium falciparum infection that occurs upon the sequestration of infected red blood cells (iRBCs) in brain capillaries, resulting in the loss of endothelial barrier integrity, brain swelling, and frequently long-term sequelae or death. P. falciparum-iRBCs cause the disruption of human brain microvascular endothelial cell barrier integrity in vitro, mimicking the microenvironment of cerebral malaria, yet the specific mechanisms mediating this process remain unknown. Upon infection of the host RBCs, P. falciparum produces hemozoin, a crystal form of heme generated following the degradation of hemoglobin by the parasite. Here, we show that the endothelial barrier-disrupting activity is found entirely in the hemozoin fraction of P. falciparum-iRBCs. This activity is not caused by the hemozoin crystal itself, which is not able to induce barrier disruption, but by the biomolecules that are associated with it. Treatment of purified P. falciparum hemozoin with proteases inhibits the disruption of endothelial barrier integrity caused by the hemozoin, indicating an important role for proteins in the disruption of the barrier. Conversely, treatment with nucleases did not affect hemozoin barrier-disrupting activity. These results identify a key molecular mechanism in the P. falciparum-mediated brain endothelial barrier disruption during cerebral malaria and may open new avenues for the treatment of this complication.IMPORTANCEWhile several specific biomolecules have been proposed to contribute to the disruption of endothelial barrier integrity in cerebral malaria, no single Plasmodium falciparum- or host-derived factor has been definitively identified as the primary driver of this disruption. Here, we identify the brain endothelial barrier-disruptive P. falciparum-infected red blood cell (iRBC)-derived activity to be caused by biomolecules bound to hemozoin, identifying a key, novel mechanism in the pathogenesis of cerebral malaria. The finding that P. falciparum hemozoin also disrupts a pulmonary endothelial cell barrier opens the possibility that this mechanism underlies other severe malaria complications. The implication of P. falciparum-iRBC-derived proteins in this mechanism is in line with previous reports, providing a novel interpretation of these findings in the context of hemozoin-binding. This knowledge provides a new perspective in the search for specific molecules and mechanisms involved in barrier disruption, which may lead to the development of much-needed specific treatments for cerebral malaria.